Contents Preface ix
11. Plantcell andtissueculture;biological conversions; clonalpropagation j2
Contributors xi
12. Phytochemicalvariationwithin a species 80 Pqrt I Introducfion
13. Deteriorationof storeddrugs 9l I
l. Plantsin medicine:the originsof pharmacognosy 3 2. The scopeandpracticeof pharmacognosy 5 3. Plant nomenclatureand taxonomv 8 Pqrl2 The plont qnd onimql kingdoms os
sourcesof drugs
ll
4. Biologicalandgeographical sourcesof drugs 13 5. A taxonomicapproachto the studyof medicinalplantsandanimal-derived drugs 15 6. Pharmacologicalactivitiesof natural products 4I 7. Synergyin relationto the pharmacological actionof phytomedicinals 49 E. M. Williamszn
14. Quality control 95 Pqrt 4 Some currenl frends
107
15. Plant productsand High Throughput Screening 109 M. J. O'Neill, J. A. Lewis 16. Biologically activecompoundsfrom marine organisms 115 G. Blttnden 17. Traditionalplant medicinesas a sourceof new drugs 125 P. J. Houghton Pqrt 5
Phyrochemisrry 135 18. Generalmethodsassociatedwith the phytochemicalinvestigationof herbal products 137 19. Basicmetabolicpathwaysandthe origin of secondarymetabolites150
Port 3 Pqrt 6 Principlesrelqted to the commerciqt qnd relqted drugs of Phqrmqcopoeiol production, quolity qnd stqndqrdizqtion biologicol origin l7l of nolurql products 55 Introduction 173
8. Commercein crudedruss 5i R. Baker
20. Hydrocarbonsand derivatives 174
9. Productionofcrude drugs 6l
21. Carbohydrates 191
10. Plantgrowthregulators 6l
22. Phenolsand phenolicglycosides 214
Id..:e6Nif 23. Volatile oils and resins 253 24. Saponins,cardioactivedrugsand other steroids 289 25. Miscellaneousisoprenoids 315 26. Cy anageneticglycosides,glucosinolate compoundsand miscellaneous glycosides 321 27. Alkaloids
37. Aspectsof Asian medicineand its practicern the West 461 M. Aslam
38. Chineseherbsin the West 482 S. Y.Mills
39. Plantsin African traditional medicine-an overview 488 A. Sofowora
333
28. Tumour inhibitors from plants 394 P. M. Dewick 29. Antiprotozoalnatural products 401 C. W. Wright 30. An overview of drugs with antihepatotoxic and oral hypoglycaemic activities 414
Port 8 Nonmedicinol toxic plonts ond pesticides 497 40. Hallucinogenic, allergenic,teratogenicand other toxic plants 499 4L. Pesticidesof naturalorigin
509
3L. Vitamins and hormones 42I
33. Colouringandflavouringagents 435
Pcrrt9 Morphologicol qnd microscoPicql exqminqtion of drugs 513
34. Miscellaneousproducts 440
I n t r o d u c t i o n5 1 5
32. Antibacterial and antiviral drugs 429
Porl7 Plqnts in complementqry ond troditionql syslems of medicine 445 Introduction 441 35. Herbal medicine in Britain and Europe: regulation and practice 449 S. Y.Mills 36. Homoeopathicmedicine and aromatherapy 460 M. A. Healy, M. Aslam
42. Plant description,morphology and anatomy 516 43. Cell differentiation and ergasticcell contents 526 44. Techntquesin microscoPY 538 Index
549
E
Plonts in medicine: theorigins of phormocognosy
The universal role of plants in the treatment of diseaseis exemplified by their employment in all the major systemsof medicine irrespective of the underlying philosophical premise. As examples, we have Western medicine with origins in Mesopotamia and Egypt, the Unani (Islamic) and Ayurvedic (Hindu) systemscentred in western Asia and the Indian subcontinent and those of the Orient (China, Japan,Tibet, etc.). How and when such medicinal plants were first used is, in many cases, lost in pre-history, indeed animals, other than man, appear to have their own materia medica. Following the oral transmission of medical information came the use of writing (e.g. the Egyptian Papyrus Ebers c.1600 nc), baked clay tablets(some 660 cuneiform tablets c. 650 ec from Ashurbanipal's library at Nineveh, now in the British Museum, refer to drugs well-known today), parchments and manuscript herbals, printed herbals (invention of printing l44O AD), pharmacopoeias and other works of reference (ftrst London Pharmacopoeia, 1618; first British Pharmacopoeia 1864), and most recently electronic storage of data. Similar records exist for Chinese medicinal plants (texts from the 4th century ec), Ayurvedic medicine (Ayurveda 2500-600 nc) and Unani medicine (Kitab-Al-ShiJa, rhe Magnum Opus of Avicenna,980-1037 ao). In addition to the above recorded information there is a great wealth of knowledge concerning the medicinal, narcotic and other properties ofplants that is still ffansmitted orally from generationto generationby tribal societies,particularly those of tropical Africa, North and South America and the Pacific countries. These are areas containing the world's greatest number of plant species, not found elsewhere, and with the westernization of so many of the peoples of these zones there is a pressing needto record local knowledge before it is lost forever. In addition, with the extermination of plant species progressing at an alarming rate in certain regions, even before plants have been botanically recorded, much less studied chemically and pharmacologically, the need arises for increasedefforts directed towards the conservation of genepools. A complete understandingof medicinal plants involves a number of disciplinesincluding commerce,botany,horticulture, chemistry enzymology, genetics, quality control and pharmacology.Pharmacognosyis not any one of theseper sebut seeksto embracethem in a unified whole for the betterunderstandingand utilization of medicinal plants.Aperusal ofthe monographson crude drugs in a modem pharmacopoeiaat once illusfates the necessityfor a multidisciplinary approach.Unlike those who laid the foundations of pharmacognosy,no one person can now expectto be an expert in all areasand, asis illustratedin the next chapter, pharmacognosycan be independently approachedfrom a number of viewpoints. The word 'pharmacognosy'had its debut in the early 19th century to designatethe discipline related to medicinal plants; it is derived from the Greek pharmakon, 'a drug', and gignosco, 'to acquire a knowledge of' and, as recordedby Dr K. Ganzinger(Sci. Pharm.1982, 50, 351), the terms 'pharmacognosy' and 'pharmacodynamics' were probably first coined by JohannAdam Schmidt (1759-1809) in his hand-written manuscript Lehrbuch der Materia Medica, which was posthumously published in Vienna in 18I 1. Schmidt was, unril his death,professor at the medico-surgical JosephAcademy in Vienna; interestingly he was also Beethoven's physician. Shortly after the above publication, 'pharrnacognosy' appears again in 1815 in a small work by Chr. Aenotheus Seydler entitled Analecta Pharmacognostica. Pharmacognosyis closely related to botany and plant chemistry and, indeed, both originated from the eartier scientific studieson medicinal plants. As late as the beginning of the 20th century, the subject had developed mainly on the botanical side, being concemed with the description and identification of drugs, both in the whole state and in powdeq and with their history, commerce, collection, preparation and
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storage. Such branches of pharmacognosy are still of fundamental importance, particularly for pharmacopoeialidentification and quality control purposes, but rapid developments in other areas have enormously expandedthe subject. The use of modern isolation techniquesand pharmacologicaitesting proceduresmeansthat new plant drugs usually find their way into medicine as purified substancesrather than in the fbrm of galenical preparations.Preparationis usually confined to one or a few companieswho process all the raw material; thus, few pharmacists have occasion to handle dried Catharanthus roseils although they are familiar with formulations ofthe isolated alkaloids vinblastine and vincristine. For these new drugs it is important that the pharmacist, rather than being fully conversant with the macroscopical and histological charactersof the dried plant, is able to cary out the chromatographic and other proceduresnecessaryfor the identification and determination of purity of the preparation supplied. Similar remarks apply to such drugs as Rauwolfia, the modem preparationsof ergot, and the cardioactive and purgative drugs. When specific plants,including thoseusedin traditional medicine, suddenly become of interest to the world at large, the local wild sourcessoon become exhausted.This necessitates,as in the caseof Catharanthusroseus,Coleus forskohlii, Arnica montana and Taxus brevfolia, research into the cultivation or a.rtificial propagation by cell culture, etc., of such species.In order to avert the type of supply crisis that arose at the clinical trial stage with the anticancer drug taxol, isolated from 7. brevfolia, the US National Cancer Institute has initiated plans for future action when a similar situation again arises (see G. M. Cragg et al., J. Nat. Prod., 1993, 56, 165'7).
However, it has been repofied that as a result of demandfor the new drug galanthamine(qv) for the treatmentof Alzheimer's disease,the native sourceof Leucojum aestivumis now in danger. The use of single pure compounds,including synthetic drugs, is not without its limitations, and in recent years there has been an immenserevival in interestin the herbal and homoeopathicsystems of medicine, both of which rely heavily on plant sources,At the 9th Congress of the Italian Society of Pharmacognosy(1998) it was stated that the current return of phytotherapy was clearly reflected by the increased market of such products. In 1995 the latter, for Europe, reached a figure of $6 billion, with consumption for Germany $2.5 billion, France $1.6 billion and ltaly 600 million. In the US, where the use ofherbal products has never been as strong as in continental Europe, the increase in recent years has also been unprecedentedwith the market for all herb salesreaching a peak in 1998 approaching$700 million. Again, illustrating the same trend, the editor of Journal of Natural Products, 1999, writes that in response to the increasing prominence of herbal remedies, additional contributions describingscientific investigationsof a rigorous nature are welcomed. Undoubtedly, the plant kingdom still holds many speciesof plants containingsubstances of medicinalvalue which haveyet to be discovered; large numbers of plants are constantly being screenedfor their possiblepharmacological value (particularly for their anti-inflammatory, hypotensive, hypoglycaemic, amoebicidal, anti-fertility, cytotoxic, antibiotic and anti-Parkinsonismproperties). Pharmacognosistswith a multidisciplinary background are able to make valuable contributions to theserapidly developing fields of study.
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Thescopeond proctice of phormocognosy
Until relatively recently pharmacognosywas regarded, almost exclusively, as a subject in the pharmaceuticalcurriculum focused on those natural products employed in the allopathic system of medicine. Coincident with the increasing attractivenessof alternative (complementary) therapies and the tremendous range of herbal products now generally available to the public, regulatory requirements covering medicinal herbs are being introduced by many countries in order to control the quality ofthese products.Monographs are now available on a large number of such drugs giving descriptions,testsfor identity and purity and assaysof active constituents.These monographs are being compiled by a number of bodies (see below). In this respect recognition should be given to the pioneering production ofthe Brirlsh Herbal Pharmacopoeia, first produced in 1974with subsequenteditions, and a new one pending. Pharmacognosyis also important in those countries having their own systems of medicine in which plants are important components. Although pharmacognosyis principally concernedwith plant materials, there are a small number of animal products which are traditionally encompassedwithin the subject; these include such items as beeswax,gelatin, woolfat, vitamins, etc. Other natural products such as the antibiotics, hormones and others may or may not be involved, depending on the teaching practice of a particular institution. As is shown in Chapter 16 m4rine organisms,many of the animal kingdom, are receiving increasing attention. Materials having no pharmacological action which are of interest to pharmacognosistsare natural fibres, flavouring and suspendingagents,colourants,disintegrants,stabilizers and filtering and support media. Other areasthat have natural associations with the subject are poisonous and hallucinogenic plants, allergens,herbicides,insecticidesand yrolluscicides. Vegetabledrugs can be arrangetlfor study under the following headings.
t . Alphabetical. Either Latin or vernacular names may be used. This arrangement is employed fol dictionaries, pharmacopoeias, etc. Although suitable for quick referenceit gives no indication ofinterrelationshipsbetween drugs. 2. Taxonomic.On the basis ofan acceptedsystemofbotanical classification (Chapter 3), the drugs are arranged according to the plants from which they are obtained, in classes,orders, families, genera and species.It allows for a precise and ordered arrangement and accommodatesany drug without ambiguity. As the basic botanical knowledge of pharmacy studentsdecreasesover the years this system is becoming less popular for teaching purposes. Morphological. The drugs are divided into groups such as the following: leaves, flowers, fruits, seeds,herbs and entire organisms, woods, barks, rhizomes and roots (known as organized drugs), and dried latices, extracts, gums, resins, oils, fats and waxes (unorganized drugs). These groupings have some advantagesfor the practical study of crude drugs; the identification of powdered drugs (see Chapter 44) is often basedon micro-morphological characters. 4 . Pharmacological or Therapeutic. This classification involves the grouping of drugs according to the pharmacological action of their most important constituent or their therapeutic use. R. Pratt and H. W. Youngken Jr. were, in 1956, the first to use this approach for an English languagetextbook and now, with so many plant materials being screenedfor specific pharmacological activity, this type of listing is found increasingly in the literature. Its use is illustrated in Chapters28-32.However, it is important to appreciatethat the constituents of any one drug may fall into different pharmacological groups. Chemical or Biogenetic. The important constituents,e.g. alkaloids, glycosides, volatile oils, etc., or their biosynthetic pathways, form
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Until relatively recently pharmacognosywas regarded,almost exclusively, as a subjectin the pharmaceuticalcumiculumfocusedon those natural products employed in the allopathic system of medicine. Coincident with the increasingattractivenessof alternative(complementary)therapiesand the tremendousrange of herbal productsnow generally available to the public, regulatory requirementscovering medicinal herbs are being introducedby many countries in order to controlthe quality o1'theseproducts.Monographsarenow availablcon a largenurnberof suchdrugsgiving descriptions,testsfor identity and purity and assaysof active constituents.Thesemonographsare being compiled by a number of bodies(seebelow). In this respectrecognition shouldbe given to the pioneeringproductionof theBriti,shHerbal Phormacopoeia,first producedin 1974with subsequent editions,and a new one pending.Pharmacognosyis also importantin thosecountries having their own systemsof medicine in which plants are important components. Although pharmacognosyis principally concemedwith planrmaterials, there are a small number of animal productswhich are traditionally encompassedwithin the subject; these include such items as beeswax,gelatin,woolfat. vitamins,etc.Othernaturalproductssuchas the antibiotics, hormones and others may or may not be involved, dependingon the teaching practice of a particular institution.As is shown in Chapter l6 marine organisms,many of the animal kingdorn, are receiving increasingattention.Materials having no pharmacological actionwhich are of interestto pharmacognosists are natr.rral fibres, llavouring and suspendingagents.colourants.disintegrants,stabilizers ir:r.li" iiiilt;..,,r and filtering and supportmedia.Other areasthat have naturalassocia- ,t:tltiiut:ti : tions with the subjectare poisonousand haliucinogenicplants. aller- ititlu:itau itit.ti]illlrl: gens,herbicides,insecticidesand molluscicides. tit,lri1; Vegetabledrugscan be arrangedfor studyunderthe fbllowing head:tiiril rliiit ings. l . Alphabetical.Either Latin or vernacularnamesmay be used.This
arrangementis employed for dictionaries, pharmacopoeias,etc. Although suitablefbr quick referenceit gives no indicationof interrelationshipsbetweendrugs. Taronomic.On the basisof an acceptedsystemofbotanical classification (Chapter3), the drugs are arrangedaccordingto the plants from which they are obtained,in classes,orders,families, genera and species.It allows for a preciseand ordered arrangementand accommodatesany drug without ambiguity.As the basic botanical knowledgeof pharmacystudentsdecreases over the yearsthis system is becominglesspopuiarfor teachingpurposes. 3 . Morphological. The drugs are divided into groups such as the following: leaves.flowers, fruits, seeds,herbs and entire organisms. woods.barks,rhizomesand roots (known as organizeddrugs).and dried latices,extracts,gums, resins,oi1s,fats and waxes (unorganized drugs).Thesegroupingshave some advantagesfbr the practical study of crude drugs;the identitlcationof powdereddrr.rgs(see Chapterzlzl)is often basedon micro-morphologicalcharacters. 4 . PharmacoktgicaLor Therupeutic.This classificationinvolves the grouping of drugs accordingto the pharmacologicalaction of their most impoftant constituentor their therapeuticuse. R. Pratt and H. W YoungkenJr. were, in 1956.the first to use this approachfor an English languagetextbook and now. with so many plant materials being screenedfbr speciticpharmacologicalactivity.this type of listing is found increasinglyin the literature.Its use is illr-rstrated in Chapters28-32. However,it is importantto appreciatethat the constituentsof any one drug rnay fall into different pharmacological groups. Chemicalor Biogenetic.The importantconstituents,e.g. alkaloids. glycosides.volatile oils, etc., or their biosyntheticpathways.form
IE
INTRODUCTION As rnentioncdpreviously,a nllrnberof bodieshave implernented the basis of classificationof the drugs.This is a popular approach on ncdicinal herbs.The aint has is phytochernicallybiased. researchand publishednlonogrilphs when the teaching of pl.rarmacognosy Anbiguities alise when particulardrugs possess a numberof active been to sct standardsfix' cluality.etlicacy and sat'etyin order that the principlesbelongingto diflerent phiitochenricalgroups.as illustrat- many tl'aditionalherbs mcct legal requirernents.The follou'ing are of note: ed b1,liquorice.ginseng.r'aleliart.ctc. The sclremeis ernployedin drugs. Chaptels20 27 tbr anangin-tthe cstablishedphartnacopoeial 'I'hese 'nvet'c cleveloped German Commission E monographs. 'I'he *'ill fbllou'ing list of works. arrangedin the abcxe five gror.rps. fbr the Gelrnan FccleralHealth Authoritl' betwccn 1978-1994and andalsoproviclea usefullist oftcxtbooksandt'orks serveascxanrples involr,e32.1herbsused in Gelrnantladitionalmedicine.The monoof reftlence:tlroseno longer in print may be fbund in established graphs give sollrces.constitllentsand considerablcphannacological pharnracer,rticirl liblaries. They havc nou' beentranslatedinto English and clinicalintbrrnation. b1'theArnericiinBotanicalCtiuncilin 1999as tr single and pLrblishcd l. Alphobeticol a handbookfbr practiceon a sciBissetN G (cd). Wichtl N'l 1996Herbal cltr.rgs. u'ork. errtific basis.Meclphanr ScientificPublishcrs,StLtttgat'l r . l i t i s h H c r b a lM c d i c i n e B r a c l l e l ' PR ( e d ) 1 9 9 2B r i t i s hh e r b a lc o n t p c n d i u n B A s s o c i a t i o nB. o u r n e m o u t hU. K Press.London Vols I-II Blitish PhalmacopoeiaI 999. Pharrnaceutical B r i t i s hH c r b a lP h ; r r m a c o p o e1i a9 9 6 .B r i t i s hH e r b a lM e c l i c i n eA s s o c i a t i o t r . Exeter.UK Niartindale:the Extr-aPharmacopoeia.32nd ccln. | 999. PharntaccuticalPress. Londou l999 2-llNationalFornrr-rlary LinitedStatesPhamracopoeia Wren R Cl I988 Potter'sneu'c1'clopocidaol botanrcaldrugs and pfeparations. reuritten b1'WilliamsonE NI and Er'ansF J. C W Daniel Co. Safll'orr \\alden. UK Tl-re national pharmacopocias of niarry countries and the Europeau Pharmacopoeia; the relevant crude drug mono-qraphs of the latter are i r r c l u t l e tilr t t h e B l i t i r h P h r r t n l r c o p o e i r
2. Toxonomic . a r i s . 3r ' o l s . a s s o nP P a l i sR R . N I o l ' s eH 1 9 6 5 .1 9 6 7 .1 9 7I M a t i d r cr n d d i c a l eM ThornasH 1929 Handbuchdet'Phalmacie.Urban and Schu arzcnbcrg.Berlin. Band V 2 r'ols, Phannacogrtosr' . t he d n .B a i l l i i r e T i n d a l la n d T r e a s eG E , E r , a n sW C 1 9 7 2P h a r n a c o g n o s l l' O Casscll.London
3. Morphologicol f, .-1 !
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Mauclt'ich.Vienna.Vol I. Barks and Belger F Hanclbuchder Dt'ogenkunclc. f l o u ' e l s .1 9 . 1 9V: o l I I . L e a r e s .1 9 5 0 :V o l I I I . F f u i t sa n d u ' o o d s ,1 9 5 2 1V o l I V . Vol V. Roots. I 960r Vol Vl. Rcsinsetc and seeds.196.{:Vol Herbs. I 9-5,1: V I I . I n d e x .1 9 6 7 JacksonB P. Snou don D W I 990 Atlas o 1'nticrtrscopl'of nredicinalplants. culinar.vherbsand spices.BelhavenPrcss.l-ondon N'lortonJ F 1977Mixor medicinalplants:botany'.culturc and uses.Thomas. S p l i n g f i e l dI.L . U S A 5th edn. Churchill Livingstone. \\allis T E, I 967 Tertbook of Pharmacognosl'. l"ondon
Coopelativefbr Scier.rtific ESCOPmonographs. ESCOP(European Phytotherapy)is an alllliation of Europeanassociatioltswhich has ploducecl60 monographson helbal clrugs.pttblishedin loose-leaffbrm in six thscicr-rle s. harmonizingthe standaldsfbr thesedrugsthror-rghout thc Er.rlopeanUnion. Intbrrration is given on approved therapcutic uses, ancl unlike the ComrnissionE nono-graphs.plovidcs ref'elerrces. AHP monographs. The American Herbal Pharntacopoeiaplanned by the end of 2000.'nvithsorne30 to havepublishedI l- I 3 t.nonographs of American traditional more pending.Thele is naturally a selectiot-t herbs n'ith somc overlap l''ith thc European monographs.The as trncxamplethe St John'sWort monograph tleatmentis exhaustir,e: rvith ovcr publishedin HelbalGraml99T. No .l extendsto -i2 pa-ees andchemicalfbrmttlae. I50 ref'erences. colourphotographs WHO monographs. The World Health Organization published MerlicirutlPluttts irt 1999.It ttrtSeleL'tetl Volunre I of its MottogrtLplt.s contaiusstandardsfbr clualityof dlr.rgstogethelwith a thelapeuticsccin the tion: 3l plant species.the majcx'ityol u'hich at'ealso inclLrded Vcrlume2. containinga further29 rnonoabovelists.are consiclered. graphs.is cluelbr pLrblicationin 2000. is alsoproducUSPmonographs. The United StatesPharr-nacopocia all involving ing helbnl monographs.Elevcn har"ebeertpr"rblished. drugstteateclabove.and twelve more are expecteclduring 2000.
wishing to read ttliginnl resealchwill Current awareness. Str.rdents in this book and shouldlearnhow to find sin-rilar find rnar-ry ret'erences A, Libcrti t- E I 988 Natural ploduct nredicinc.G F Stickley. Del i\'lalclerosian As no onecanhopeto readall the scientificliteraonesfbl themselves. P h i l a d c l p h i aP. A . U S A specialior.rrnllsare clevotedto the publicationof ture that is pLrblished. PrattR, YoungkenH W. Jr 19-56Pharrlacognosy.2nd edn. Lippincott. P h i l a d e l p h i aP.A . U S A brief abstlacts from the oliginal papers. Such abstractsgive the Ross N4S F. Brain K R 1977An intfoductionto phytopharmacy.Pitnlall author's name.the sub.iectoi the rcscarch.the ref'elencencccssaryto N{edical.Tunbridge\\iells a blief outlineof the locatethe papelin the oliginaljoLrrnalanclusr-rally work it contains. Most phat'nracydepartntentlibraries contain 5. Chemicol phy'tochcrnistry'. medicinarlplants.Intefcept BnrnetonJ 1999Pharnracognos-v. Cltenitul Abstrrr't.r'and Biolo,gital Abstrccls,which in the appropriate Scicntific. N'ledicaland TechnicalPublications EVen st'. the :)':tetrllttr scctionscover all aleasof pharntaco-[nosy. Deu,ick P N{ I 997 \Iedicinal naturalproducts.a biosl'nthcticiipproach.John canilselfbe ofthe absuactsto covera broacllielclof intcrests searching W i l e y .C h i c h e s t e r lrtles and publicatictns as and such ClrcntittLl rnost tinre-consunrin-9. 6 t l r i n e g g e l E 1 9 9 9 P h a r m a k o g n o s i c P h y t o p h a r m a z i e . H i i n s eR l . S t i c h c rO . S t e CurrentCottentscan be usedto give a rrore rapidindicationof recent edn. Springer.Berlin (|ir C)t'ntrcnt1 bibRobbersJ E. SpeedicM K. Tl ler V E 1996 Pharmacognosyand pharmacoregularlyincludesa selected publications. Phltothenq1'Re.search b i o t e c h n o l o g lW ' . i l l i a m s& W i l k i n s .B a l t i m o r e lioglaphy relating to plant dlugs. Inlbrntatioti stot'ageanclretrieval is Tauchnitz.Leipz-iglttro editiort.s TschirchA Handbuchder Pl.tilrnako-unosic. occupiedby sucnow itself a scicnce.anc'la glanceat the shelf:space t oltnttt'strltto l 9-l-i1 tutd nunterou.s is sutlicierrtto indicatethatbetbre ceedingyearsof CftcrnlL:trlAb.strtLcts long. if not already'.manual searchesof the litcrature u'ill become With the increase in interest in rnedicinal plants world-wide there are Inevitablyit will bc necessalyto rely on inrpossiblylon-eprocedures. n o \ \ ' n r a n y p u b l i c a t i o n s c o \ ' e f i n g r c g i o n a l a l e a so f t h e g l o b e . T r e a t m e n t databasesfbr litelature scanniug.PltunnucogttostTitlesis a cornputcr of the plants in thcse wolks rna)' be on an1, of the abovc lines. Some researchpublicationsup to 1974 of phytochenrical abstl'act co\rerage cxamples are given fbllowing the Introduction to Paft VII.
4. Phormqcologicol or Theropeutic
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T H ES C O P EA N D P R A C T I COEF P H A R M A C O G N O S Y tl0 vols) producedundel the directionof Prof-essol N. Farnsr','or.th.as anv book can). Symposiall,hich cor,ervaLiolrsaspectsof pharnraI i n i ' " ' e r s i t y o f I l l i n o i s . S u b s e q u c n t l y .F a r n s w o r t h i n t r . o d u c e c lcognosyarc tiequently held in variouspartsofthe world and scientists \APRALERT. a NaturalProcluctDarabasewhich is rrainly. br.rtnor can easilvbecomeacquninted u'ith othershar,inglike interests. Often cntirely.post-ltl75and is vieu.'ed bv rrany as a logicaland inclispens- the infbn-r-ral discussionswhich invariably ariseat sr.rch meetingscanbe able collectiono1'pharmacognostic information.The NAPRALERT an extremelyuselulnteansof disseminating infbrmation.ln addition, database is availableon a schedulcd-fee basisft) scientists.industrial the lecturesplesentedat such nteetingsare often sr.rbsequentlv pubfirns. -qovemment agenciesand acadenticinstitutions. Among othcr lished in book fbrm. Modern comntunicationsystemsmake u'orlduselirl databaseshaving a reler,'ance to pharntaco-tnosy and published '"videcontactbetweenresealchers much sintpler. on the Web arc MEDLINE. conrpiledby the US NarionalLibrarl,of Now al'ailableto Westernscientists interestcd in orientalrnedicineis Medicineand EMBASE. producedby ExccrptitMedica. the qualtelly joumal Abstnrc:t.s o.l'CltineseMeditirrc, publishedby the S o m e j o u l r r a l s f o r e r a r - n p l e .P l r t r t t u M e d i t ; u . . l o u r n o l o f ' ChineseUniversityol HorrgKong. This givesabstractsin Englishof Ltltrtophunrrut'ologv Plntocltetnisn'y nnd Jountttl of' NtttunLl significantChineseresearch papersfrorn morethanonehundredscienPt'o(luLt.\-periodicallycolttuin reviL-\\s on \oll(. i.rs|ectof medicinal titic journalsnot feadilyar,ailable outsideChina. plants.Otlrel pcliodical publicationsappearingin bor-rndfbr-m are Usefirl dictionaliesto be lbund in most Unir.ersitylibrtrriesinclude devoteclto reviewson certainaspectsof plar-rtconstituclttsand are use- DicticrttLryof Organic Contpountlsconsisting of 7 r'olurnesand 10 ful fbr updating;ofien the rcr,ielvscor,eronly the adrrncesin I parti- sLrpplements (to 1992).Dittiotmry ol Alkaloitl.s12 r,olunies)(1989). culal tlc-lclsincethe pler,iousr,olunte.Examplesare NtfturtLl Product Dictionar-t o.f Tbrpertritls( 19911and Dit:tionurt' of Natuml pnxlucts Reports(six issuespcl vear) .andAlkaloitl.y(AcadenricPrcss).Books (1994) all publisheclbv Chapmanand Hall and also phttot'hemictrl thatarenot partofa seliesbut. like the above,rrultiauthorand dealing Dictiornrt': A Hundbookol Bioactit'eCompountls.frcmplutfi.t ( 1993), \\'ithccrtainspeciaiized areas(c.g.alkaloids.flavonoids.isoprenoids). publishedby Taylol and Francis.Sorneof thesemore expensivevolcorrtinuallyappearand generallvgive up-to-dateinfolmation (in so flr Lrmesare availableon CD-ROM.
z
IE BOTANICAT NOMENCTATURE Beforethe time of Linnaeus( 1707 1778)many plantswere known by a double Latin title; however,it is to this great Swedishbiologist that we owe the generaladoptionof the presentbinon-rialsystem.in which the flrst narrc dcnotcs the genus, while the second(specific) name denotesthe species.All specificnamesmay be written with small initial letters although tirmerly capitals were used where specieswere named after persons. Thus the species of Cinchona named after CharlesLedger,who broughtits seedsliorn Brazil in 1865.is now writtcn Clrrry'rono Ietlgerionaratherthan CinchonuLedgerianu. The specificname is usually chosento indicatesome striking characteristicof the plant fbr example.the hemlock with the spottedstem is namedConiunrntaculuturtt(.mutulutus.-d. -1u11. spotted).Sometimes the reasonfbr the name is not as obvious as in the examplejust mentioned.but onceit is discoveredit will serveas a reminderofa characteristicof the plant-fbr example,Stl c/rnos potatorum (potator,-oris, a drinker) bearsa namewhich is only intelligiblewhen it is known that the seedsof this speciesareusedin India lbr clearingwater. The modernrules governingthe terrninologyof plant taxonomy are laid down in the Internroioncl Codeof BotanicalNomenclature. Unlike the names of chernical substances,which are subject to changeswhich confolm to evolving systemsof nomenclature. systematic plant namesare strictly controlledby ruleswhich give precedence to that name used by the botanist who first described the species. Nevertheless, this seeminglystraightfbrwardapproachcan give rise to variousquilks in spelling.The following are threeexamplesinvolving rnedicinal plants Rauvolfia vis ir vis RauwoLfia;the former name was given to this Apocynaceousgenusby Plumier in 1703,honouringthe botanistLeonard Rauwolf. This spelling ovelsight causednuch contentionover the yearscentringon whetherPlumier's obvious intention shouldbe adoptedin the nameRaLntollia.Both spellingsarecommonly fbund but the rules dictate that Rauvolfia has priority. In anotherexample the downy thornapplemay be encounteredas eilher Datura innoria or Datttra irnxia. The fbrmer. as Datura irutoxia Miller. was used in 1768(Gurd.Dlo.. edn.8, Dutttru no. 5) and this spellingwas invariably employedfor some200 years:howeverin Miller's original description. the plant was characterizedas: 'Datura (lnoxitt) pericarpiisspinosis inoxiis ovatis propendentibusfoliis cordatis pubescentibus'(W. E. Saflbrd,J. \Wrsh.Acud. Sci.. 1921,11,173) and taxonomistsnow considerD. irro-rlaMiller to havepriolity. Both versionsarestill commonly encountered. A third exarnpleconcernsthe genusofthe cocaplantwhich may appearas Er1,1fup.ry1u^, or in older literature as Erythrox:-lon.
Plont nomencloture ondtoxonomy
3. i
r
SUBDIVISIONS OF THEPHYTA The branchesofthe genealogicaltreediffer so much in sizethat it is not easyto decidewhich are of equalsystematicimportance,and what one biologist may consideras a lamily anothermay regardas a subfamily. Similarly. the speciesof one botanistmay be the subspeciesor variety of another.The main hierarchicalsubdivisionsof a division. aranged according to Engler's scheme.may be illustrated by the lollowing exampleshowingthe systematicpositionof peppermint.
h !: I
l
BOTANICAT NOMENCLATURE8 SUBDIVISIONSOF THE PHYLA
8
BOTANICAT SYSTEMS OF CTASSIFICATION9 TAXONOMICCHARACTERS9 PTANTTAXONOMY CHEMICAT
ro
Division Class Subclass Order Suborder Family Subfamily Tribe
Anglospermae Dicotyledoneae Sympetalae Tubiflorae Velbenineae Labiatae (Lamiaceae) Stachydoideae Satureieae
.q.#{
PLANTNOMENCLATURE AND TAXONOMY Genus Species Varieties
Mentha MenthapiperitaLinnaeus(Peppermint) Menthupiperita var.olrtcinalis Sole (White Peppermint) Menthupiperita var.vul,qari,s Sole(Black Peppermint)
It will be notedthat in pharn-racopoeias and in researchpublications botanicalnamesarefbllowed by the nar-nes of personsor their accepted (e.g.Linnaeusand Sole in the caseofpeppermintgiren abbreviations above).Theserefer to the botanistwho first describedthc speciesor variety.Studentsneednot attemptto memorizethesenames.and in the fbllowing pagesthey areusuallyomittedexceptin caseswheredif-fbrent botanicalnameshave at differenttimes beenappliedto the sameplant and thereis possibilityof confusion.The sourceof cloves,fbl example. is now usuallygiven asS,t';.r'glum aromaticltm(L.) Men. et Pen'y:prior to 1980 the B.P. used the name Eugetict ----
Me
Me
\W furan(31) Sesquiterpene
r
TT RENDS S O M EC U R R E N Fig. l6.l (continued)
cooH
cooH
Me
Me OH OH E2 (PGE2)(32) Prostaglandin (33) F2" (PGF2q) Prostaglandin Me \+ Me-N-
S-S
YI N
Mel
(CH2)n-COOH2N\*.'o tl
Md 'Me
H-N
(35) n = I = glycinebetaine
Pfrs,
acidbetaine(36) n - 2 = y-aminobutyric Nereistoxin(34)
H H , H
. rN'
,rfiA*
N-,,
r -OH OH
acidbetaine(37) n = 3 = 6-aminovaleric
Saxitoxin(38)
OH H
cHo
Me .H HH
o
H
BrevetoxinA (39)
HOOC Me OH
Me
1 (40) Dinophysistoxin-
Me
cHo
t"-*
Y" -Y
-
H
I
N
OH
o
;=^ (41) Lophotoxin
(42) Lyngbyatoxin
-
|F-
BIOLOGICATLY ACTIVE COMPOUNDS FROMMARINE ORGANISMS Fig. l6.I
(confinued)
Debromoaplysiatoxin (43)
O
M
e
H
A^-]{> .
S q ,
{.=
!t= a 7
+ 9 l s q
;
(
:
:
h
C
x ' ; . q . :
tr !
.3:
u
d
=
o ) g I 5 ! 9 c < + ! ; , i > V Y c
5 : E ;
€ . X
h I o c ' N i E : x o v * L . - = c o
c h
i
o=
!.i
c Y s =
F
s E i Y i E
d- =
72
e u cg
a o .E ,9 'u
[|
U a)
E o n
E E c
.c
,o
s o_ -E
.c o s :z
L
.9
-
I
! E E !
c t
€
ul E' t o
I
o
o E
E o
n
L
!
r
Y
^
/-v
I
o
!,
o o
(
o o ? a
o
5 'g
c
+ (E
z(\
E l ry
=
Q O
o ;-r
- t ; . :
o o9
o
o
o
!
6
O
Y R E . X
:
n
;sF
o
2tHl+]02
ATP is a coenzymeand the high energy of the terminal phosphate bond is availableto the organismfbr the supplyof the energynecessary fbr endergonicreactions.The Hill reactionproducesfree oxygen and hydrogenions which bring aboutthe conversionof the electroncarrier, 'Coenzymes'). NADP, to its reducedform NADPH (see In this complicatedprocess,two systems,PhotosystenrI and PhotosystemIl (alsoknown as pigment systemsI and lI) arecommonPHOTOSYNTHESIS ly ref-enedto; they involve two chlorophyll complexeswhich absorb light at diff-erent wavelengths (above and below )" = 685 nm). Phuosynthesis,by which the carbondioxide of the trtn'rosphere is con- PhotosystemII producesAIP and PhotosystemI supplies all the vertedinto sugarsby the greenplant. is one of the fundamentalcycles reducedNADP and someAIP. In theselight reactionsthe chlorophyll on rvhich life on Earth, as we know it, depends.Until 1940. when molecule captures solar enelgy and electrons become excited and investigationsinvolving isotopeswereundertaken,the detailedmecha- move to higher energy levels; on returning to the normal low-energy 'carbon nisrnofthis reduction'wasunknown.althoushthe basicover- state.the electronsgive up their excessenergy,which is passedthrough all reiiction. a seriesof carriers (including in the case of PhotosystemII plastolirht quinone and several cytochromes)to generateATP PhotosystemI - rcH.or+ 02 co, + H.o involves an electron acceptorand the subsequentreduction of feredoxin in the production of NADPH. Ref'erenceto the current literature had beenacceptedlbr many years. Photosynthesisoccurs in the chloroplasts:green, disc-shaped indicatesthat the natureand organizationof the photosystemsremains organellesof the cytoplasrnwhich areboundedby a definite membrane a very active resealcharea.Studentswill have observedthat an alcoand which are autoreproductive. Separatedfrom the rest ofthe cell, the holic solution of chlorophyli possesses,in sunlight, a red fluoreschloroplastscan carry out the complete processof photosynthesis. cence-no carriersare availableto utilize the capturedenergyand it is Bodies with similal propertiesare fbund in cells of the red algaebut re-emiftedas light. Hill first demonstratedrn 1931that isolatedchloro-
BASICMETABOTIC PATHWAYS AND THEORIGIN OF SECONDARY METABOLITES plasts,when exposedto light, were capableofproducing oxygen,provided that a suitablehydrogen acceptorwas present.Work with isotopeshas sinceproved that the oxygen liberatedduring photosynthesis is derivedtiom water and not liom carbondioxide. Following the light reactions,a seriesof dark reactionsthen utilize NADPH in the reduction of carbon dioxide to carbohydrate. Current researchsuggeststhat terrestrialplants can be classifiedas C:, C+, intermediateCj-Ca and CAM plants in relation to photosynthesis.
cular bundle cells, where it is oxidatively decarboxylatedto pyruvate again,carbondioxide and NADPH, which areusedin the Calvin cycle. Pyruvatepresumablyreturnsto the mesophyllcells. Plantspossessing this facility exhibit two types of photosyntheticcells which difl'er in their chloroplasttype.IntermediateCj-C.1 pathwaysare alsoknolvn.
CAM plonts This term standslbr 'crassulacean acid metabolism'.so calledbecause it was in the Crassulaceaefamily that the distinctive characterof a build-up of malic acid during hours of darknesswas first observed. C3 plonts Other large families, including the Liliaceae, Cactaceaeand possessmembersexhibiting a similar biochemistry.As The elucidationof the carbon reduction cycle (Fig. 19.3), largely by Euphorbiaceae. Calvin and his colleagues,was in large measuredeterminedby meth- with Ca plants, this is an adaptationof the photosyntheticcycle of ods dependenton exposing living plants (Chlorella) to 1'+C-labelled plants which can exist under drought conditions.When water is not carbon dioxide for precise periods of time, some very short and available,respiratorycarbon dioxide is recycled,under conditionsof amountingto a liaction of a second.The radioactivecornpoundspro- darkness.with the formation of rnalic acid as an intermediate.Carbon ducedwere then isolatedand identified. In this way a sequencefor the dioxide and water loss to the atmospherear"eeliminated,a condition formation of compoundswas obtained.3-Phosphoglycericacid, a C3 which would be fatal for normal Cj plants. compound,was the compoundfirst fbrmed in a iabelledcondition but From fi-nctose,producedin the Calvin cycle, other important conit was only later in the investigation.after a number of 4-, 5-, 6- and stituents,suchas glucose.sucroseand starch,irrederived:erythroseis a was precursor in the synthesisof some aromatic compounds.Glucose7-carbonsystemshad beenisolated,that ribulose-1,5-diphosphate shownto be the moleculewith which carbondioxide first reactsto give 6-phosphateis amongthe early productsfbrmed by photosynthesis and it is an importantintermediatein the oxidativepentosephosphatecycle two moleculesof phosphoglycericacid. An unstableintermediatein this reactionis 2-carboxy-3-ketopentinol. and for conversionto glucose-I -phosphatein polysaccharidesynthesis.
Caplonts Some plants which grow in semi-aridregionsin a high light intensity possessan additionalcarbon-fixationsystemwhich, althoughlesseffiin its use of carcient in terms of energyutilization, is more e1I'ective bon dioxide, so cutting down on photorespirationand ioss of water. Such plants are known as C4 plants.becausethey synthesize,in the presenceof light, oxaloaceticand other C4 acids.Carbonassimilation is basedon a modified leaf anatomyand biochemisffy.Its presencein plantsappearshaphazardand hasbeenlinked with the so-calledKranz syndrome of associatedanatomicalfeatures.In the mesophyll cells pyruvateis convertedvia oxaloacetate to malate,utilizing carbondioxide, and the rnalate,or in somecasesaspafiate,is transportedto the vas-
UTiLtl{IlON,, ,],,CARBOHYDI{TE Storagecarbohydratesuch as the starchof plants or the glycogen of animals is made availablefor energy production by a process'"vhich involves conversion to pyruvate and then acetate,actually acetylcoenzymeA, the latter then passinginto the tricarboxylic acid cycle (Fig. 19.a).As a result of this. the energy-richcarbohydrateis eventually oxidized to carbon dioxide and water. During the process,the hydrogen atoms liberated are carried by coenzymes into the cytochromesystem,in which energyis releasedin stages,with the possibleformation of ATP from ADP and inorganicphosphate.Eventually the hydrogencombineswith oxygento form water. A number of pathways for the initial metabolism of glucose are known for various Iiving tissues.One involves compoundswhich are found in the photosyntheticcycle, and it appealsas a reversalof this cycle but the mechanismis apparentlyquite different.Another pathway is the Embden-Meyerhoffschemeof glycolysis(Fig. 19.5).
Acetyl-CoA *^
-/"roroloocetore
Citrote
Mobte
^"o1 Erythrose4-P
,.oI__-^_ tumorore
-'"'Yi"o
CisoC6nibte
^ Y'*
Succirrfe
lsocitrote
,A
Orobswcinote
),.. @"'
Fig. 19.3 (ofter theformuloe Thepothof corbonin photosynthesis Colvin); of the involved 21. sugors oregivenin Chopter
Fp'
:":h:^
Srccjnrl CoA
M4z,
\o-oxogbto.r"7@2
F i g .1 9 . 4 Thetricorboxylic cycle. ocidcycle(TCA) or Krebs
E
PHYTOCHEMISTRY
cHo
cHo
I H-C-OH I H O- C - H I H-c-oH I H- C - O H
I
H-c-oH I HO-C-H I
ATP llerolinose
--
Phosphogh,cose iso|nero3e +
H-c-oH I H-c-oH
-
I CH2OP Glucosc-6-P
I I
CH2OH 0- Glucose
cHroP t C:O I HO-C-H I H-C-OH I H-C-OH I CH2OP Fruclosc| '6-diP
tnor*gtycero- CH2OP IHrOP CHOH '-* CHOH =i-t l
cooP
cooH
1'3-OiPhosPhoglycericocid
3-Phosphoglycericbcid
1l tl
cl'lzoPOlHz 3Glyceroldehyde
It
cH2oH I
F i g .1 9 . 5 Outline of theEmbden-Meyerhoff scheme of glycolysis.
CH"OH t C=O I HO- C-H I H-C-OH I H-C-OH I CH2OP Fructose6- P
ph06ghole
-Hao
CHOP =!ttl"*I c@H 2- Ph6phoglyc.ricocid
The kinetics and enzymesystemsinvolved in the abovepathwayshave beenstudiedextensively. It will be notedthatonemoleculeofglucosecan give rise to two moleculesof pyruvate,each of which is convertedto acetate,and one molecule of carbon dioxide. One tur"nof the TCA cycle represents the oxidationof one acetateto two moleculesof carbondioxide. giving rise to twelve moleculesof ATP The overall reaction fbr the metabolismof one moleculeof glucosein termsof ADP andAIP is C6HI2O6+ 6CO2 + 38ADP + 38P(inorganic) ---> 6H2O + 6COr + 38AIP The above schemes,given in barest outline, are fundamentalnot only fbr the building up and breakingdown of reservefoodstufl\. but also in that the intermediatesare availablefor the biosynthesisof all other groupsof compoundsfound in plants.The levels at which some of thc importantgroupsariseare indicatedin Fig. 19.2.
i". dop I
Pyiuvole
--- limse
coo|'l
io' i" ----'ito P co
cooH Pyruric
PhosPhocnolpyruvicocid
l-aoo AcclylcocnzymcA
The fbrmation and hydrolysisof an O-glycosidesuchas salicinmay be represented:
EFE
" Sugar
o.x.+ R.ox+Hzo Aglycone ot
Glycoside
!:= C6H'q.O.C6H6.CH2OH C6H11O5OH + FlO.C6H..Cl-'lzOH + l-l2O Salicin Glucosc Salicylalcobol (saligenin)
It will be notedthat suchreactionsarereversibleand in plantsglycosidesareboth synthesizedand hydrolysedunderthe influenceof more or less specific enzymes.While glycosidesdo not themselvesreduce Fehling'ssoh.rtion. the simplesugarswhich they produceon hydrolysis will do so with precipitationof red cr.rprous oxide. The sugarsfound in glycosidesmay be monosaccharides such as glucose,rhamnoseand fucoseor, more rarely.deoxysugarssuchas the ' , GIYCOSIDES i cymarosefound in cardiacglycosides.More than one moleculeof such sugarsmay be attachedto the aglycone eithel by separatelinkages, Glycosiclesare tblrned in nature by the interactionof the nucleotide which is rare.or, more commonly,as a di-. tri- or tetrasaccharide. Such glycosides-fbl erample. Lrridine diphosphate glucose (UDP- complex glycosidesare fbrmed by the stepwiseaddition of sugarsto glucose)-with thc alcoholicor phenolicgroupof a secondcompound. the aglyconemo1ecule. Such glycosides.sometimescalled O-glycosides.are the most numerSince sugarsexist in isomerica- and B-forms,both types are theoous onesfound in natr.rre. Other glycosidesdo, however.occurin which retically possible.Practicallyall naturalglycosides,however,areof the the linkage is through sulphur(S-glycosides).nitrogen (N-glycosides) B-type, although the o-linkage is lbund in nature in some carbohyor carbon( C-glycosides). drates such as sucrose,glycogen and starch.In ft-strophanthoside. a
BASICMETABOLIC PATHWAYS AND THEORIGINOF SECONDARY METABOLITES glycoside formed from the aglycone strophanthidin and strophanthotriose(cymarose+ glucose+ glucose)the outer glucosemolecule has the cr-linkageand the inner glucosethe B-linkage.Isomericglycosides may be preparedsynthetically;for example,from glucose and methyl alcohol one obtains both the cr- and B-methyl glucosidesby introducing methyl groups into the OH groups printed in heavy type in the formulae of glucose below.
Hoa
H O H
rH
?-r cH.oH HO.CH
I
O
lr.or Il t
cH-r I cH2oH
p-Glycosideforming structur€
c--r t
l
HO.CH
O
cH.oH I
6r,o, Il r cH---r
cruoH tr-Gtycoside formrng structure
The term 'glycoside'is a very generalone which embracesall the many and varied combinationsof sugarsand aglycones.More precise terms are availableto describepafiicular classes.Someof theseterms refer to the sugar part of the molecule, others to the aglycone, while othersindicate some well-defined physical or pharmacologicalproperty. Thus, a gLucosideis a glycosidehaving glucoseas its sole sugar component; a pentoside yields a sugar such as arabinose;rharnnosicles yield the methyl-pentoserhamnose;and rhamnoglttcosides yield both rhamnoseand glucose.Terms used for aglyconesare generally selfexplanatory(e.g., phenol, anthraquinoneand sterol glycosides).The names'saponin'(soap-like),'cyanogenetic' (producinghydrocyanic acid) and 'cardiac'(havingan action on the heart),althoughappliedto thesesubstanceswhen little was known about them, are useful terms which do in fact bring together glycosides of similar chemical structure. The older systemof naming glycosidesusing the termination '-in' (e.g. senegin,salicin, aloin, sffophanthin)is too well establishedto be easily changed.It is not ideal, however, since many non-glycosidal (e.g. inulin and pectin)have the sametermination.Modern substances workers more frequentlyuse the termination '-oside' (e.g. sennoside) and attempt to drop the older forms by writing sinigroside for sinigrin, salicosidefor salicin and so on. Although glycosides form a natural group in that they all contain a sugar unit, the aglyconesare of such varied nature and complexity that glycosidesvary very much in their physical and chemical propertiesand in their pharmacological action. In Part 6 they are classilled according to that aglyconefragment with which they often occur in the plant.
anda thiolysisIiberatinga two-carbonunit of acetyl-CoA.A11thesereactionscan be shownby isolatedenzymestudiesto be reversible.anduntil about 1953it was generally assumedthat the biosynthetic roLrteof fafty acids operatedin the reversedirection, stafting from acetyl-CoA. This, however,is not preciselyso,asindicatedbelow.
Biosynthesisof soturoted fotty ocids From the aboveit was generallyassumedthat the biosyntheticroutefor fatty acidsoperatedin the reversedirectionto the B-oxidativesequence for the degradation of these acids and starred wirh acetyl-CoA. However,the unfavourableequilibrium of the initial thiolasereaction in sucha pathway:
o
o
o
l l l l l 2CH3-C-SCoA r= CH3-C-CH2-C-SCoA
+ CoA
&o=1'6x10-5 and the poor yield of long-chain fatty acids obtained with purified enzymesof the B-oxidationpathway togetherwith other observations suggestedthe possibility that this was nor necessarilythe principal biosyntheticpathway.It was then shown,with animaltissues,that malonyl coenzyme A was necessaryfor the initial condensationwith acetate.In fact, in palmitic acid (C16,)only one of the eight C7 units (i.e.the C15 + Cl6 carbons)is deriveddirectlyfiom acetyl-CoA;the other sevenC, units are attachedby malonl'l-CoA. which is lbrmed by carboxylationof acetyl-CoA. One featureof this anabolicpathwayis the involvementof the acyi canier protein (ACP) to producefatty acyl thioestersof ACP.This conversionserves(1) to activatethe relativelyunreactivefatty acid and (2) to provide a carrier for the fatty acid acyl group. Thus. theseACP thioestersappearto be obligatoryintermediatesin latty acid synthesis, whereasit is the fatty acid thioestersof coenzymeA which operatein the catabolic oxidation pathway. The synthesisis explained by the reactionsshown in Fig. 19.7.Reactions4-7 arethen repeated,lengthening the fatty acid chain by two carbons (derived from malonylS-ACP) at a time. Eight enzymes have been identified as being involved in the synthesisof palmitoyl-ACP and stearoyl-ACPfiom acetyl-CoA. In purified enzyme systems,when propionyl-CoA replacedacetylCoA, the productwas a C17acid and, similarly,butyryl-CoA gaveprimadly a C1s (stearic)acid. Thus, the formation of theseC16,C17and C13 acids may well dependon the availability of acetyl-, propionyland butyryl-CoA. The system of biosynthesis of fatty acids in plants appears to operatein the sameway as in animaltissues,but whereasin the latF.t +
Fattyecid
-CFfr-Clt-Cl{2-CO.SCoA
Fats,consideredin more detail in Chapter20, are triglyceridesinvolving long-chainsaturatedor unsaturatedacidsand,as such,constitutean important food reservefor animals and plants (particularlyin seeds). Relatedto the simple fats are the complex lipids, most of which are diesters of orthophosphoric acid; they have the status of fundamental cellular constituents. The fatty acids,on liberation from the fat, are availablefor the production of acetyl-CoA by the removal of C2 units. The elucidation of this spiral (Fig. 19.6) owes much to the work of Lynen and it was primarily investigatedwith animal tissues.In this B-oxidationsequenceone tum of Fig. 19.6 the spiral involves four reactions-two dehydrogenations,one hydration Degrodotion of fottyocids.Reoctions involved in oneturnof thespirol.
rfrFrn
ffi*
PHYTOCHEMISTRY
HOOCCH2CO'$CoA+ ADP + Pi ".ffi; acetyl-SACP + CoASH acetyl-S'CoA + ACP-SH-) malonyl-$CoA + ACP-SH------r malonyl-S'ACP + CoASH
CHgCO-S-CoA+ CO2 + lrf
1.
2. 3. 4. 5. Fig. r9.7 o[ in thebiosynthesis stoges Initiol fottyocids.
malonyl-9AcP + acetyl-SAcP ---') + NADPH* H* ---+ acetoacetyl-S-ACP
6. 7.
acetoacetyl-S-AcP+ CO, + ACP'SH + NADP* o(-)-3-hydroxybutyryl-9ACP
c rotonyl-SACP + HOH o(- )-3-hydroxybutyryl-S-ACP----------+ butyryl-S-ACP+ NADP* crotonyl-S-AcP + NADPH * H* --"----------+
ter the enzymesarelocatedin the cytoplasm,in plantsthey aretbund oration of Ll-l4Clacetateinto 6-methylsalicylicacid by Penicillium in the rnitochondria and chloroplasts. The mitochondria derived griseofulvumtakesplaceas below: from the mesocarpof the avocado fruit have been used to demoncHl strate the incorporationof Il4Clacetateinto esterifiedlong-chain fatty acids.
Biosynthesisof unsoturoted fotty ocids As a numberof unsaturatedfatty acidshave a particularsignificancein pharmaceuticalproducts, a treatment of their formation is deferred until Chapter20.
.cooH
!", z{-€oox
cHr-rcooH --------+ll
cHl
I .cooH
I
\.4o"
tcooH Hr
and the production of the mould anthraquinonemetabolite endocrocin, from eight C2 units, is representedin Fig. 19.9.Decarboxylationof endocrocin affords emodin. The original chain lengthening,which can The shikimicocid pothwoy be representedas a condensationof acetateunits, is the sameas in fatty This appears to be an inlportant route from carbohydrate fbr the acidproduction(q.v.above)but doesnot requirethe reductionof =CO to biosynthesisof the C6-Cj units (phenylpropanederivatives).of which =CH:. Thus, malonic acid plays a similar role in this aromatic synthesis phenylalanineand tyrosineareboth examples.A schemeof biogenesis to that in latty acid formation and the chain is built up from the combinfor thesearomaticamino acids,as elucidatedin various organisms.is ation of malonyl units with a terminal acetyl (the starter)unit. Sometimes given in Fig. 19.8;for higher plants,the presenceof the enzymesystem the stafterunit is not acetate.as indicatedwith the formation of the tetraresponsiblefor the synthesisof shikimic acid has beenconfirmed.An cycline antibioticsfrom nine units; here malonamide-SCoAis the starter important branchingpoint arisesat chorismic acidl anthranilatesyn- unit and by invoking standard biochemical reactions tetracycline is thaseuseschorismic acid as a substrateto give anthranilicacid which formed (Fig. 19.10).Higher plantsalsor"rtilize the polyacetate-malonate is a precursorof tryptophan.The synthesisis controlledby the latter pathway for the biosynthesisof emodin-typeanthraquinones,suchcomacting as a feedbackinhibitor: chorismatemutaseconvertschorismic poundsall having substituentson both outer rings. acid to prephenate,the preculsorof phenylalanineand tyrosine,and a listed in Chapter22, it will be seenthat the From the anthraquinones varietyof controlmechanismsappearto operateat the branchingpoint. stluctures of some anthraquinones are not fully explained by the The opium alkaloidsare synthesizedvia this pathway(Fig. 27. I 5) and acetate-malonatepathway and these are discussedbelow. However, two isofbrms of chorismatemutasehave beenisolatedand character- thereappearto be many exceptionsto the rules in applyingthe general ized liom poppy seedlings (M. Benesova and R. Bode, P/t1'to- pathwaysto specil'icanthrrquinones. 3f , 2983). clrcmistry'.1992. Although there are only smali difl'erencesin the sequenceof reac- Compounds confoining qromqtic rings of different tions lbr the shikimatepathwayin bacteria.fungi and plants there are origin considerabledifferencesin the molecularorganizationof the pathway. The structuresof anthraquinones suchas dlizarin,rubiadin,pseudopur(For a review (123 ref.s),see J. Schmid and N. Amrhetn' Phyto- purin and morindadiol,pigmentsof the Rubiaceaeand other families, chemistrv,1995.39, 13'7.) cannotbe readily explainedon the acetatehypothesis. The shikimic acid pathway is also important in the genesisof the o o g O H aromaticbuilding blocks of lignin and in the formation of some tanZr.-A.r.-S #o' Z\,;{r'\G and coumarins nins, vanillin and phenylpropaneunits of the 1-lavones f' ll rl I l' ll ll I l^ll.ll.l 'FurtherReading'' \,.\ufcoo" \-/W\cn, \-Al^V/'* (seeChapter22). For a teview see
AROMATICBIOSYNTHESIS
nlo*,""'
i"""ll
p*uoao*ilurin
The qcetote hypothesis Circumstantialevidence that these compounds might be formed The centralposition oi iicetatein relationto the generalmetabolismof with the participationof mevalonicacid (a key plantshas alreadybeenindicatedand it is possibleto devisemany pos- from naphthoquinones of isopentenylunits, q'v.) was provided by vxrformation gl\e a precursor in the to occur could sible routesby which acetatecondensation haviety of aromatic compounds.The generalvalidity of the mechanism their co-occurrencein teak and otherplantswith naphthoquinones hasbeenestablishedby the useof labelledcompoundsbut the detaiied ing an isopentenyl residue (e.g. formula below). That mevalonate stepsfor many compoundsremain to be established.Thus. the incotp- (itself derived from acetate)is involved in this formation has been
bASICMETABOLIC PATHWAYS ANDTHEORIGINOF SECONDARY METABOLITES @oH
cooH
I
I
C=O
Q-O-POsFh
Qru
cooH
CHOH
-L
I
tl CHa Phosphoenolpyruvic acid + -
I
I
I H-C-OH I H-C-OH I
cHo I
H-C-OH I H-C-OH I cH?oPo3H2 Erythrose-4-phosphate
t o
E ' " OH acid 3-Dehydroquinic
cruoPo3H2
2-Keto 3-deoxy-7-phosphoo-glucoheptonicacid
l ^oH ; "
6n
3-Dehydroshikimicacid
cooH I
co I
cooH
Ho@aoocHe
fn
i l t l \,/
cooH
Shikimic acid-3-phosphate
ry;"
9H' I t
t
l
Y
Anthranilic acid I RtroeptraiUosyt lrrytahosPhat€
I
Phenylpyruvic acid
OH p Hydroxyphenyl pyruvicacid
n"oorirr" "rin"tionf J cooH cooH I
cH-NH2
?Fh h, \./ Phenylalanine
I
cH-NH2
_
OH
Chorismicacid
I C=O I
?*.
I
H2o3Pos'YoH -
cooH
I C=O I
\,,/
fit.
OH
Prephenic acid
(\
+'.:i.:coo" -Hzo a
?o't-c@H :
fz
H O H
cooH
oPosHa
I
Is",in"
I
rfut-cH2-cH-cooH Nllz \Ap,c-*
H Tryptophan
?"'
h
I OH Tyrosine
Fig. I9.8 vio theshikimic Biosynthesis of oromoticcompounds ocid pothwoy.
shown by tracerexperimentswith Rubia tinctorum;ring C and carbon side-chain of pseudopurpurin and rubiadin are derived from mevalonate, and in the sameplant shikimic acid has been shown to be incorporatedinto ring A of alizarin.
The aromaticrings of somecompoundscan be derived from both the shikimic acid andthe aceticacid pathways.Thus,a phenylpropaneformed by the former route may undergo chain lengtheningby the addition of 'Fatty acetateunits (via malonyl; see Acids', above)to give a polyketide andthen,by ring closure,give a flavonoidderivative(Fig. 19.11). Isoflavones are formed in the samemanner,with a rearrangementof the aryl-B ring in relation to the three carbons (Fig. 19.11). The flavonoids,in addition to their importancein plants as pigments,have interestingmedicinal propertiesand are discussedin more detail in Chapter 22.
PHYTOCHEM!STRY
-
.{. .1. .f.
Cl-l;CoOH CooH CO0HCOOH
fH. ,.r, f^,
HOOC-CH3COOHCOOHCOOH
i i
,rc{t,rc{t rc{' cH3-co co co P Enzsoc-\ )\" z"^" )* co co co
Hsc HOOC O H O O H Endocrocin
A I I I I
-ooc-cH+-cH2-coScoA
Acetyl-CoA
ix,
+
H ydroxymethylglutarate
CHTCOCH2COSCoA Acctoacetyl-CoA
oH I
HOCH2-CH2-C,-CH2_COOI cHr Mcvalonate
I ArP I
l OH
I - - -o5,P2ocHz-cHrt-cH2coo-
OH
I - -o3PocH2-cH2-c-cH2-coo-
ATP f--
I
I
cHr
CHr 5 Pyrophosphomevalonate
5-Phosphomevalonate
^rP - cor J - - -O6,P2OCH2-CH2-C:CH2 cHl Isopentenylpyrophosphatc
1l t l
--* a"t-i
t,
- cH3-c:cH-cH2oP2o6I cHr pyrophosphate Dimethylallyl
CH-CH2-CH2-C:CH-CH2OP2O6cHr
cHr Geranylpyrophosphatc
CH3-C:CH-CH2-CH2-C:CH-CH2-CHrC:CH-4 t t cHt CHI
| +l.op"nt"nyl lrroehosehate J
H2OP2O6,-- l CHI
Farnesylpyrophosphate Fig.I9.18 Preliminory stogesin the biosynthesis of isoprenoid compounds.
hasbeenextensivelystudiedin animalsand more recentlyin plants.As with many enzymesthe situation is complicatedby the existenceof more than one speciesof enzyme and a plant may possessmultiple lbrms each having a separatesubcellularlocation associatedwith the biosynthesisof different classesof terpenoids.For a review of the functions and propertiesof the important isomeraseenzyme isopentenyl diphosphateisomerasesee 'Further Reading'. It shouldbe notedthat somemetabolitesof mixed biogeneticorigin involve the mevalonicacid pathway;prenylationfor exampleis common, with Cs, Cro and C15 units associatedwith flavonoids, coumarins,benzoquinones, cannabinoids,alkaloids,etc. The validity of the mevalonatepathway in the formation of all the major groupsnotedin Fig. 19.I t hasbeenshown,and until recentlyno other biosyntheticroute to isoprenoidshad beendiscovered.
precursorfor all isoprenoidsyntheses.However, in 1993M. Rohmer et al., (.Biochem.J., 1993,295,517) showed that a non mevalonate pathway existed for the formation of hopane-typetriterpenoidsin bacteria. The novel putative precursor was identified as 1deoxy-o-xylulose-5-phosphate, formed from glucose via condensation of pyruvate and glyceraldehyde-3-phosphate. Subsequentsteps including a skeletal reaffangement afford isopentenyl pyrophosphate-the same methyl-branched isoprenoid building block as formed by the MVA route. It was soondemonstratedthat this novel route to IPP was also operative in the formation of monoterpenes(Mentha piperita, Thymus vulgcrls), diteryenes (Ginkgo biloba, Taxus chinensis) and carotenoids (Daucus carota). This raised the question of to what extent the two pathwaysco-existedin the plant and it was hypothesizedthat the classical acetate/mevalonatepathway was a feature of cytoplasmic reacThe 1-deoxy-o-xylulose(triose/pyruvate) pathway. Followingits tions whereas the triose/pyruvate sequencewas a characteristic of the discoveryin 1956mevalonicacid cameto be consideredthe essential plastids.This did not exclude either the movementof plastid-synthe-
h.
r;itc-.j!l.
4CJ'
"tI[
BASICMETABOLIC PATHWAYS AND THEORIGINOF SECONDARY METABOLITES A Me. -gH
c"""nyr!|fprrospr,"e -/ Monoterpenes, C16 (straightchain, cyclic and bicyclic)
|
Isopentenyl PYroPhosPhate
,--
TiXi'"" MVA-SPP
I Crspp Farnesyl pyrophoephatc
-/ / Sasquiterpenes,Cl5 / (openchain, / monocyclic / ,Z and bicyclic) / t caoPP
/
\ \
r \ Triterpencs Steroids
IPP
-\L*
Y" u"z\,cFLoen il.
Y \
DMAPP
\ C2oPP
/
|
"
t',,"*"*',
tim" sq,*J.", o,H,:"i", l,r
/ \
,df:{ftoPP H.' -F{o
,/
#a
H";\o;*'one
rf H".
i"
Ircpentenyr pyrophosphate
\
T
Me
Al
t""\
l t Carotenoids Gutta
,r,
FL. (.u. .)"Td.r."r{2oPP_
"' l,i. ^/r^'H"
x"^Tu cf .ue lc-cl^,cxropp
- "-fr'""ioi
--Ff,'
I
PP.J
x. $
Fig.19.19 Biosynthesis of isoprenoid compounds.
Me
Jk\.cHppp \H["
c sized IPP and DMAPP from the organelle to the cytoplasm or the translocationof a suitableCs-acceptorto the plastid. Evidenceaccumulating indicates a cooperative involvement of both pathways. Indeed recent work on the biosynthesisof the isoprene units of (K.-P.Adam and J. Zapp, Phttochemistry, chamomile sesquiterpenes 1998,48, 953) has shown that for the threeC5 units ofboth bisaboloxide A and chamazulene,two were mainly formed by the nonmevalonatepathwayand the third was of mixed origin. The deoxyxylulose(DOX) pathway has helped explain the previously reported rather poor incorporations of MVA into certain isoprenoids.Thus V. Stanjeket al. (Phytochemistry,l999, 50, 1l4l) have obtained a good incorporationof labelled deoxy-o-xyluloseinto the prenylated segment of furanocoumarins of Apium graveolens leaves, suggestingthis to be the preferred intermediate. Fig. 19.21illustrateshow [1-t:C]-*lucose, when fed to plants,can be used to differentiate IPP and subsequentmetabolites,formed either by the MVA pathway or the DOX route.
Hb.
/rx \.Me
"t"fc;d;,
e1/ ucH"oPe
@
x.jr cf..ue ;c-c.^.Hc
,
).1r"
Ft "e cl+oPP
I
t
PPOl"rHl
x".f ^..ue n-c"-\--s
['t'"
s;;t
Fig. 19.2O Stereospecific reoclions in lerpenoidbiogenesis. A, Formotion from mevolonicocid (MVA)of isopentenyl pyrophosphote {lPP)by trons eliminotion; isomerizotion to dimethylollyl (DMAPP). pyrophosphole B, Associolion of thetwo 5-C unitswilh ironseliminotion of hydrogen. C, As B but involvingformotion of cisdoublebondsos foundin rubber.
convertedto male pheromones.The literature concerning chemical ecology is regularly reviewed (J. B. Harborne.Nat. Prod. Rep., 1993, As indicatedearlier in Fig. 19.2,the basic metabolicpathwaysconsti- 10,327: 1997,14,83 1999,16, 509) and the volatile isoprenoidsthat tute the origins of secondaryplant metabolismand give rise to a vast control insectbehaviourand developmenthavebeenreportedon (J.A. array of compounds;someof theseare responsiblefor the characteris- Pickett, Nat. Prod. Rep., 1999, f6, 39). The enzymology associated tic odours,pungenciei and colours of plants, others give a particular with secondarymetabolism is now receiving considerableattention plant its culinary, medicinal or poisonousvirtues and by far the greatest and, with respectto alkaloid formation, a number of enzymesassocinumberare,on currentknowledge,of obscurevalue to the plant (andto ated with the biosynthesisof the tropane, isoquinoline and indole the human race). However, a number of modern authors suggestthat groupshave beenprepared.The biosyntheticorigins of thosemetabosecondary metabolites, rather than constituting waste products of lites of medicinalinterestareconsideredin more detail in Part 6. which metabolism,arebiosynthesizedto aid the producer'ssurvival.The pos- is arrangedprincipally accordingto biogeneticgroups. sible functions in the plant of one large group of secondary metaAlthough a number of the biogenetic groups are characterizedby bolites,alkaloids,are discussedin Chapter27. particularskeletalsffuctures,the actualchemicalpropertiesofparticuRecently, considerable attention has been directed to the possible lar compoundsare determinedby the acquisitionof functionalgroups. ecologicalimplicationsof secondarymetabolitesnot only in relationto Thus, terpenesmay occur as alcohols (menthol), ethers (cineole), plant-plant interaction but also concerning the inteffelationshipof ketones(carvone),etc.,and as suchhave similar chemicalpropefiiesto plants and animals.Various insects sequesterspecific alkaloids, irinonteryenoidcompoundspossessingthe samegroup; aldehydes,as an doids, lactonesand flavonoidswhich serveas defensiveagentsor are exampleof a functionalgroup, may be of aliphaticorigin (citronellal),
PHY1OCHTMISTRY
ro:
Ho-)!\-oH nv
oH
-Gtucose 11-13c1
\\ 'CHq l
^-n v-v
-
I SCoA Acetyl-CoA
II
t
CH3-CO-_CHz -CO-SCoA
cHo
i-
3oo-
Glyceraldehyde-3-P
\ \ \
Pyruvate
/ /
Thiaminediphosphateparticipation
\ ,\.// I Y -cH. t nn
Acetylacetyl-CoA
CHe l -CH2 HOOC C -.CH2- CH2OH
? *. CO
HO-CH I .cH2oP
I H-C-OH I HO_CH I -cH20P
1-Deoxyxylulose-5-P
I
OH Mevalonic acid
HaC ' \
//,c-cH2-cH2oPP
Fig. 19.21 Theincorporotion into of It-13C1-nlucose isopentenyl diphosphote: left,vio the mevolonic ocid pothwoy;right,vio the pothwoy. deoxyxyulose
HeC
HzC
.c-cH2-cH2oPP HzC
lsopentenyldiphosphate
lsopentenyldiphosphate
aromatic (cinnamic aldehyde), steroidal (some cardioactive glycosides);and resultingfrom the introductionof a heterocyclicsystemone biogenetic group may possess some of the chemical properties of another(e.g.steroidaialkaloids). A particular group of compounds may also involve different biogenetic entities; thus, the complex indole alkaloids contain moieties derived from both the shikimate and isoprenoid pathways. In contrast, the same structure,as it occurs in different compounds,may arise from different pathways, as has been previously indicated with the formation of aromaticsystems.
Stresscompounds These are compounds which accumulate in the plant to a higher than normal level as a result of some form of injury, or disturbance to the metabolism; they may be products of either primary or secondary metabolism. Common reactions involved in their formation are the polymerization, oxidation or hydrolysis of naturally occurring substances;many however, are entirely secondary in their formation. A
,ry
number of environmental and biological factors promote the synthesis of stresscompoundsand these include mechanicalwounding of the plant, exposure to frost, ultraviolet irradiation, dehydration, treatment with chemicals,and microbial infection (seephytoalexinsbelow). The productionof suchcompoundshas also beenobservedin cell cultures subjectedto antibiotic treatmentand in cells immobilized or brought into contact with calcium alginate. Examples of the latter include the formation of acridone alkaloid epoxides by Ruta graveolens, and the increasedproduction of echinatin and the novel formation of a prenylated compound by Glycyrrhiza echinata cultures. Stresscompoundsare of pharmaceuticalinterest in that they may be involved in various crude drugs formed pathologically (e.g. some gums and oleoresins)and potential drugs (gossypol);they are implicated in the toxicity of some diseasedfoodstuffs and they play a role in the defensive mechanismof the plant. In the latter area,the phytoalexins have received considerableattention in recent years and can be regarded as antifungal compounds synthesized by a plant in greatly increasedamountsafter infection. The antifunsal isoflavonoid otero-
[email protected] BASICMETABOLIC PATHWAYS AND THEORIGINOF SECONDARY METABOLITES carpansproducedby many speciesof the Leguminosaeare well known. Other phytoalexins produced in the same famity are hydroxyflavanones,stilbenoids,benzofurans,chromonesand furanoacetylenes. Sesquiterpenephytoalexins have been isolated from infected U/nus glabra and Gossypium.In the vine (Vitis vinfera) the fungus Botntis cinerea acts as an elicitor for the production ofthe stilbenesresveratrol (q.v.) and pterostilbene. Chemically, stresscompounds are, in general,of extreme variability and include phenols, resins, carbohydrates,hydroxycinnamic acid derivatives, coumarins, bicyclic sesquiterpenes,triterpenes and steroidalcompounds.For the promotion of stresscompoundsin cell culturesseeChapterI 1 and Table 11.1.
Furtherreoding Cordell G A 1995Changingstrategiesin naturalproductschemistry. Phytochemistry40: 1585-16 12 Dey P M, Harbome J B (eds) 1997Plant Biochemistry.AcademicPress, London
GraysonD H 2000 Monoterpenoids(a review coveringmid- 1997 to mid-1999).Natural ProductReporrs17(4):385 Ikan R (ed) 1999Naturally occurringglycosides.JohnWiley, Chichester,UK KnaggsA R I 999, 2000 The biosynthesisof shikimatemetabolites.Natural ProductReports 16(4):525-560; 11(3): 269-292 Kruger N T, Hill S A, Ratcliffe R G (eds) 1999 Regulation of primary metabolic pathways in plants. Kluwer, Dordrecht, Netherlands Ramos-Valdivia A C, Van der Heijden R, Verpoorte R 1997 Isopentenyl diphosphateisomerase:a core enzymein isoprenoidbiosynthesis.Natural ProductReports14(6):591-604 RawlingsB J 1998Biosynthesisof fatty acidsand reiatedmetabolites.Natural ProductReports15(3):275-308 Rohmer M 1999 The discovery of a mevalonate-independentpathway for isoprenoidbiosynthesisin bacteria,algaeand higher plants.Natural Producl Reports l6(5): 565-57,1 SeiglerD S 1998Plant secondarymetabolism.Kluwer, Dordrecht, Netherlands Singh B K 1999(ed) Plant amino acids-biochemistry and biotechnology. Marcel Dekker Inc., New York Wink M (ed) 1999Biochemistryof plant secondarymetabolism.Sheffield AcademicPress
E PART
6
Phqrmqcopoeiolq n d relqted drugs of biologicql orrgrn o
o
I !.
(
f ' i {,.
I
t
\
\ {
I
p structurallyand stereochernically spccific enzyr.nes has now received (stearo) I-S-ACP) experimental support.By this means(Fig.20..+) therangeof acetylenes CH3(CHr)7CH:CH(CH,)7-CO-S-ACP + HzO + NADP lbund in Basidiomycetesand in the Contpositae.Araliaceae and Urnbelliferaecan be derivedtiom linolcic acid via its acetylenic12,I 3loleoyl-S-ACP.1 dehydloderivative, crepenynicacid. an acid first isolatedfrom seeds The positionofthe introduceddoLrblebond in respectto thc carboxyl oils of CreTrrs spp. group is governedby the enzyme;hence.chain length ofthe substrate acid is most important.The hydrogeneliminationis specilicallycis but Aromotic ocids a ferv unusualfatty acids such as that in the seedoll of PLutir-tt gruno- Two commonaromaticacidsare benzoicacid and cinnamicacid tum with the structurel8:3 (9c.I lt.l3c) hayetrartsbonds.As illr.rstrat- (unsaturated side-chain), whicharcwiclclidistribLrted in natureand
Acetrte + malonate
I I
Y
CHI(CHilTTCOOH
Palmitic
II
+ CH3(CHilt6COOH
Stearic
I l-2H
Y
cH3(cHrTcHlcxlcxrlrcoox oleic l-2H CH3(CHjaCHacx-cxz-cHS Fig.20.l S e q u e n c eo f f o r m o t i o no f o l e f i n i c fottyocids in plonts.
6g16gt7cooH
Linoteic
I l-2H cH!-cH2-cHgcH-cH2-cHgcH-cH2-cHgcH(cHJTcooH
OH C H 3 ( C H 2h C H 2 - C H - C H 2 C O- S - C o A Intemediolep-hydrcry ocid of fotly ocid synthesis
/
./-,,o\ -
cH3(cHahCHzCH:CHCO-S-CoA
Fig.2O.2 Alternotive pothwoys for synthesis of unsoluroted fottyocids.
I n"ar'i- onc
lfurlherodditio.t of 5 r cr units { Sleoricocid
\ CH3(CH2l7Cx:CHCH2CO-S-CoA | *on,on o,
I3rczunirs Oleicocid
Linotenic
@
PHARMACOPOEIAL AND RELATED DRUGS OF BIOTOGICAT ORIGIN CHr(CH2}CH:CH(CHtTCOOH
Oleicacid
I I
Mcthionine
cH3(cHt5cH(oH)cHr-cH:cH(cHtTcooH Ricinoleic acid
, [H3C-S-CH2-CH2-CH(NHdFCOOH]
I
CH3(c Hj7-C\H -c H-( CH|TCOOH Dihydrosrerculic acid V cHz | -.*
c Hr(cHzh_\:t{cxrhcoox Fig.2O.3 Oleicocidostheprecursor of
Srcrculicacid
V
cHr
r i c i n o l e i co n d s t e r c u l i co c i d s .
CHr(CHJiCH:CH-CH2-CH:CH(CHjTCOOH
Linoleicacid
| _,* CH3(CH2)aC-C-CH2-CH:CH(CHTTCOOH
Crepenynicacid
l'" CH3-CH2-CH2-C
H:CH-C:C-C
Hz-CH:
C H(CHTTCOOH
I I
D e h y d r o c r e p e n y n iacc i d
Range of rcet)rlenet formcd by furthcr introduction of acetylenic bonds et the 'distal'
Fis.20.4
of morecules and bv chainshortenins in lr'l,fi:.l:Jl;1.,ti:Jr.crrboxvl sroup)
Formotion of ocetulenic fottuocids. oflen occur liee and combinedin considerableamountsin drugs such as balsams.Truxillic acid. a polymer of cinnamic acid. occursin coca leaves. Other related acids of fairly common occllrrenceare those having phenolic or other groupings in addition to a carboxyl group; suchare: salicyclic acid (o-hydroxybenzoicacid),protocatechuicacid (3,4-dihydroxybenzoic acid), veratric acid (3.,1-dimethoxybenzoic acid), gallic acid (3,4,5-trihydroxybenzoic acid) and 3,,1.5trimethoxybenzoicacid. Similarly. derived from cinnamic acid, one finds p-coumaric acid Q;-hydroxycinnamicacid), ferulic acid (hydroxymethoxycinnamic acid), catTeic acid (hydroxycinnamic acid) and 3,4,5-trimethoxycinnamicacid. Unbelliferone, which occurs in combination in asafbetida,is the lactone of dihydroxycinnamic acid. Acids having an alcoholgroup arequinic acid (tetrahydroxyhexahydrobenzoicacid), which occurs in cinchonabark and in some gymnosperms:shikimic acid. which is lound in Japanesestar-aniseand is an important intermediate metabolite; and mandelic acid, C6HsCHOHCOOH,which occursin combinationin cyanogeneticglycosidessnch as thoseof bitter almondsand other speciesof Prunus. Tropic acid and phenyllactic acid are two aromatic hydroxy acids which occur as estersin tropanealkaloids (q.v.).For examplesof the aboveseeFig. 20.5. Chlorogenicor cafleotannicacid is a condensationproductof caffeic acid and quinic acid. It occurs in matd, coffee, elder flowers, lime flowers. hops and nux vomica and is converted into a green compound, which serves for its detection, when an aqueousextract is treated with ammonia and exposedto air. See also 'Pseudotannins' (Chapter22). The biogenesis of the aromatic ring has been discussedin Chapter19.
DlBASlc AND rii,6As'ie'.l,Aci,ni.,'..'',.,,'..'.ll:'..,,..',l... Oxalic acid, (COOH)2,forms the first of a seriesof dicarboxylicacids which includes malonic acid, CH2(COOH)1,and sr.rccinicacid, (CH2)2(COOH)2.Closely related to malonic acid is the unsaturated acid fumaric acid. COOH-CH=CH-COOH. Malic acid contains an alcohol group and has the forrnula COOH CH2-CHOH-COOH. It is found in fruits suchas applesand tamarinds.A high percentageoitartaric acid, COOH-(CHOH)2-COOH. and its potassiumsalt occursin tamarindsand other fruits. The tribasic acids, citric, isocitric and aconitic are closely related to one another.The Krebs'citric acid cycle,which is discussedin Chapter 19,is very important.Citric acid is abundantin fruitjuices, and aconitic acid,which occursin Aconitumspp..is anhydrocitricacid.It forms part of the Krebs'cycle andthe glyoxalatecycle in microorganisms.
cH2.cooH I
I
HOC.COOH
I
cH2.cooH Gric acid
H.C.COOH -H,O
--€
tl il +H,O c'cooH -+ II cH2.cooH
cis -Aconitic acid
HO.C.COOH
I I cH2.cooH
H.C.COOH
lsocitric acid
Of recentinterestareopines.a group of substances formed by a host plant after infection with Agrobacterium spp.; a number of these compoundsaredi- and tri-carboxylicacids.For furlher detailsseeChapters 1 1a n d 1 2 .
HYDROCARBONS ANDDERIVATIVES @
cooH
I
I
I \
Z
t i l Y\ot"
Yot
I OMe
I OH
\-)
Veratric acid
Protocatechuic acrd
Benzoic acrd
Lower members of the series are found principally combined as esters e.g. methyl salicylate in oil of wintergreen and methyl and ethyl estersresponsiblefor some liuit aromas.Esterified long-chain alcoholsareconstituents of somepharmaceutically importantanimal waxes and include cetyl alcohol (Ct6Hi3OH), ceryl alcohol (C26H51OH)and myricyl alcohol (Cr0H61OH).Such alcoholsalso participate in the forn-rationof esterswhich are constituentsof leaf cuticular waxes: an example is CarnaubaWax BP which contains myricyl cerotate.
z\
t t l
cHz-o (B)
Fig.2O.7 Formotion of ocylglycerols; Rl, R2
R2cooK
+
I
lr,o.o*' cH2-oH _C_X XO
Rt cooK
cH2oH
1,2-Diocylglycerol
neutralizethe aceticacidfleedby the hr drolr rrr ol I g of the acetylated fat. The oil is tirst acetylatedwith acetic.rr)h\ilride. r.vhichcombines with any hydroxyl groupspresent.Becau:ethe:c iirr-absentlronr most fatty acids.the smallacetylvaluesLrsuall)ohlilinedlrc clueto relatively small amountsof sterols.In an oil suchas e.r\t()r()il. honcvcr.the acetyl value is high (146-150),ouing to thc lirfrc rinrountsof the hydroxy acid ricinoleic acid. Certainphysicalconstants of flxed oils and fut' urc'significant:specific gravity,melting point, refractiveinder and .onrctinre:optical rotation(e.g.castoroil). Table20.6 showshorr chcnricul.tilndlrrd\are relatedto chemicalcomposition.The gas chrunrat,rslilnirie.c'prlation and quantiticationof the acidsproducedby thc hrdlolr.i. ot :pc'cific fixed oils is an ollicial methodfor their identitlcationrindrlualitr control; type chromatogramsare included in the BP. Sonrc eranrplesof this applicationarementionedunderthe oils dcscribedbclou. Wqxes The term'wax', althoughsometimesapplicdto thc'hrclroearbon nrixturehardparaflin,is bestcontlnedto thosenaturalnrirturc'\e()ntuining appreciablequantitiesof estersderivedtiorn hichcr nronohrdlic alco-
Numericol prcperties of fixed oils. Approximotefo\ composition
Fotor oil
Meltingpoint, "C
Soponification volue
lodine value
Soturoted(%)
Almond Costor
-tB -tB
I 83-208 1Z 5 - t8 3
99-l 03 B4
0.3-2.s
t
I B s - t9 6 18 8 - r9 6 250-264 190-r 98 248 19 3 - t 9 5 192-198 I B0-190
79_BB 86-t 06 7-1 1 1 0 9 - tt 6 r3 . 5
^l \JIIVC
Arochis Coconut Coffonseed rolm
Theobromo Lord Cod liver
-5 to +3 23-26 0 (Before winterizing) 30 31-34 34-41 ^l
L l e o r O TU
*Ol. = oleic;Ln.= linoleic;tdepositspolmitinot 2'C.
aa
ta
s0-66 I 55-tB0
t l
7-20 IB 92 27 50 59 60 U n d elr5
(%) Unsoturoted B BO l . L n . 88-94 Ricinoleic; 5-,l5 Ol. Ln. 75-93 Ol. Ln. 82 Ol. Ln. B Ol. 2 3 L n .O l . 50 Ol. Ln. 4l Ol.Ln. 40 01. Over 85 with up to six unsoturoted bonds
LR I G I N P H A R M A C O P O E IA N L D R E L A T EDDR U G SO F B I O L O G I C AO hols of the methyl alcohol seriescombined with fatty acids. In this seriesof alcoholsthe uembers chan-eefr"omliquids to solids.become less soluble in water and have highel ntelting points with increasein molecular wei-qht.The fi'st solid of the series is dodecyl alcohol, Cl2HrsOH. Waxes include vegetableproductssuch as carnaubawax 'wooland animalproductssuchas spermaceti.beeswaxand so-called fat'. Although waxesareabundantin nature(e.g.on epidermalsurfaces). a limited numberonly areof commercialimportance:someof the bestknown and theil chief alcoholsare given at the end of the chapter. An impoltant practicaldiff-erenccbetweenfats and waxesis that fats may be saponifiedby meansof either aqueousor alcoholic aikali but waxesare only saponifiedby alcoholic alkali. This fact is usedfor the detectionof fats when addedas adulterantsto waxes(e.g.fbr detecting 'Japanwax' as an adulterantin beeswax).Saponificationof the the fat wax estercetyl pahnitatemay be representedas: * alcoholicKOH --> Cl6HrsOH C15H.1.COOCrr,H3-l CetYlalcohol Cetyl palmitate + cl5Hl3.cooK Potassiumpalmitate While fats consist almost entirely of esters.waxes. in addition to estersof the cetyl palmitatetype. often contain appreciablequantities of fiee acids.hydrocarbons,fiee alcoholsand sterols.The hydrocarbons and sterolsare unsaponifiableand both spermacetiand rvool fat. of these,have high saponificawhich contain considerableqr.rantities tion values.If analyticaldata for fats and waxes are cornpared,it will of waxestend to be higher-for example. be notedthat the acid vah-res beeswaxcontainsabout l57r of fi"eecerotic acid. C26H5jCOOH.In most waxes,iodine valuesare relativelylorv and unsaponifiablematter is hish (Table20.7).
N IN G ACIDS, AtC O HO t5 P FU_G_S__C_O_NTAI AND ESTERS Tomorindpulp The drug consists of the fruit of the tree Tamarindus indica (Leguminosae)deprived of the blittle. outer part of the pericarp and preservedwith sugar'.The fiuits are about 5-15 cm long. They have a brittle ep:icarp.a pulpy mesocarp.through which run from the stalk aboutfive to nine blanchedfibres. and a leatheryendocarp.The latter forms from four to twelve chambers.in eachof which is a singleseed. ln the WestIndies the fiuits ripen in June,July and August.The epicarpsale removed,the fiuits arepackedin layersin barrels.andboiling syrup is poured over thern; alternatively,each layer of fiuits is sprinkled with powderedsugar.
Toble 2O.7
Tamarind pulp occurs as a reddish-brown.moist. sticky mass, in which the yellowish-brownfibres mentionedabove are readily seen. Odour'.pleasantand fmity; taste.sweetand acid. The seeds,eachenclosedin a leatheryendocarp,are obscurelyfbursided or ovate and abor-rt15 mm long. They have a rich brown testa marked with a large patch or oreole. Within the testa,which is very thick and hard. lies the embryo. The large cotyledonsare composed very largely of hemicellulosewhich stainsblue with iodine. The pulp containsfiee organic acids (about l}c/c of tartaric,citric and rnalic),their salts(about8% ofpotassiumhydrogentartrate).a litof invert sugar.It is reportedthat t1enicotinic acid and about 30--:l0clc the tartaric acid is synthesizedin the actively metabolizingleavesof the plant and then translocatedto the fruits as they develop.The addition of sugarto the manufacturedpulp, to act as a preservatlve,somewhat lowersthe naturalproportionof acids. Flavonoid C-glycosides (vitexin, isovitexin, orientin and isoorientin) occur in the leaves.The fixed oil ofthe seedscontainsa mixture of glvceridesol saturatedand unsaturated(oleic, linoleic) acids. Tamarind pulp is a mild laxative and was lormerly used in Conf'ectionof Senna:it has tladitional medicinalusesin the W Indies and in China and the leaves have been suggestedas a commercial sourceof tartaricacid. Mqnnq 'manna' is applied to a number of different plant products. The name The biblical mannawas probablythe lichen Lecarutraesculenta,which can be carriedlong distancesby wind. The only mannaof commercial importance is ash manna, derived frorn Fraxinus ornus (Oleaceae) The drug is collectedin Sicily. When the treesare about l0 yearsold. tlansversecuts are made in the trunk. A sugaryexudationtakesplace and when sufficiently dried is picked off (f1akemanna)or is collected on leavesor tiles. Manna occursin yellowish-whitepiecesup to 15 cm long and 2 cm wide or in agglutinatedmassesof brokenf'lakes,with a pleasantodour and sweettaste.It containsabout557cof the hexahydricalcohol mannitol, relatively small amountsof hexosesugarsbut larger amountsof the more complex sugarsmannotrioseand mannotetrose(stachyose). The tliose on hydrolysisyields glucose(1 mol) and galactose(2 mol). while the tetroseyields glucose( I mol), fructose( I mol) and galactose (2 mol). Manna has a mild laxativeaction.
BENZOIN Of the two commercial varieties of benzoir-r-Siam Benzoin and SumatraBenzoin-only the lattet is now includedin the BP. Sumatra Benzoin (Gum Benjamin)is a balsamicresin obtainedfrom the incised poralleloneurtrsPerkins stem of StyraxbenzoirtDryand, and Sry'ra-:r (Styracaceae). It is producedalmost exclusivelyfrom cultivatedtrees grown in Sumatra.althoughthe tree is also native to Javaand Borneo Siam benzoin.derived from Slr,rzr tonkinensis,is producedin a rela-
Chemicol ctandords of woxes.
Wox
Acid volue
Soponificotion volue
lodine value
Importontconstituents
Spermoceti Beeswox
Belowl t8-24
12 0 - l 3 6 volue) 70-80 (ester
below )
Cetylpolmitoteond cetylmyristote 72o/oesters,moinlymyricylpolmitote;free ceroficocid; sterylesfers
Cornoubo Wool fqt
4-7 Belowl
79-95 90-t 06
t0-14 r B-32
B-tI
Sterylesters,estersof otheroliphoticolcohols, fottyocidsond hydrocorbons
H Y D R O C A R B O NASN D D E R I V A T I V E S tively small areain the Thai provinceof Luang Probangand is mainly usedin perfumery. History. The drug was noted by Ibn BatLrta.who visited Sumatrain the fburteenthcentury,but was not regulally in-rported into Eulope until the sixteenthcentury. Collection and preparation. Sumatrabenzoinis a purely pathological product and there is sonte evidenceto show that its fbrmation is brought about not only b1' the incisions made. but also by lungi (see 'Stress Cornpounds'.Chapterl9). In Sumatrathe seedsare sou,nin rice fields,the rice shadingthe young treesduring theil first year.After the harvestingof the rice the treesare allowed to grow until they are about7 yearsold. Tapping, The rather complicatedprocessconsistsof making in eachtrunk threelines of incisionswhich aregradLralli'lengthened.The first triangular wounds are made in a vertical row about 40 cm apalt. the bark between the wounds being then sclaped smooth. The first secretionis verv sticky and is rejected.After rnakingfurther cuts.each about 4 cm above the precedingones,a hardel secretionis obtained. Further incisions are made at 3-rnonthly intervals and the secretion insteadof being amorphousbecontescrystalline.About 6 weeks after each fiesh tapping the product is scrapedofT, the outer layer (flnest quality) being kept separatefr"omthe next layer (intermediatequality). About 2 weeks later the strip is scrapedagain, giving a lower quality darker in color.rrand containingfragmentsof bark. Freshincisionsare then made and the above processis repeated.After a time the line of incisionsis continuedfurther up the tlunk. Grades. The above three qualities are not sold as such but ale blendedin Palembangto give the benzoin gradesof comr.nerce. The best grade containsthe most 'almonds' and the worst containsa few almondsbut abr.rndant resinousmatrix. The blendingis doneby breaking up the drug, mixing difl'erentproportionsof the threequalitiesand softeningin the sun. It was fbrn-rerlyerported aftel stampinginto tins but now the commelcial drug arrivesin plaited containerswith a plastic wrapping. Characters. Sumotrubenzoinoccursin brittle massesconsistingof opaque,whitish or reddish tearsembeddedin a tlanslucent.reddishbrown or greyish-brown,resinousmatrix. Odour. agreeableand balsamic but not very rnarked; taste, slightly acrid. Sianresebenz.oin occursin tearsor in blocks.The tearsare ofvariable sizeand flattened: they are yellowish-brown or reddish-brown externally, but milkywhite and opaque internally.The block folm consistsof snrall tears ernbeddedin a somewhatglassy,reddish-brown,resinousrnatrix.It has a vanilla-like odour and a balsamictaste. When graduallyheated,benzoinevolveswhite tumes of cinnamic and benzoic acidswhich readily condenseon a cool surfaceas a crystalline sublimate.On warming a little powderedbenzoinwith solr:tion of potassium permanganate,a faint odour of benzalclehycleis notedwith Sumatrabenzoinbut not with the Siamese.When an alcoholic solution of f-erricchloride is added to an alcoholic extract of Siamesebenzoin,a greencolour is produced.Sumatrabenzoindoes not give this test. The BP inclLrdesa TLC test tbl the absenceof Dammar gum.
Up to about 20c/rof fiee acids n-raybe plescnt. High-glrde nraterial Irom S. purullelorrcLtrwncontainsbenzoic acid3%, r.anillin 0.-5(Zancl cinnanricacict20-30%. The more expensiveSiamesebenzoindifl'ersfront the above irr that it containsinsufTicientcinnamicaciclto give an odour of benzaldehl,de rvhenwarmeclwith potassiurrlpermanganatesolution.The mnjor corrstitLrent(aboLrt7-5%) secms 1() be the ester coniferyl benzoate. Conif'erylalcohol.3-metl.roxy-4-hydroxycinnamyl alcohol.is fbund in the carnbial strp of both gvmnospermsand an-tiosperms.Other constituentsare fice benzoic acid. tritcrpenoid acids arrd vanillin. Sou're rccent (20001parcels of Siam benzoirremanatinglionr Hong Kong have been fbund to be heavily adrllteratedrvith Surnatlarnaterialand applicationof thepotassiumpermanganate test(seeabove)is essential. Alfied drug. Pulenthcutgben:.oitt.an infelior vadety ploduced in Sumatra.rnay be collectedfiom isolatecltreesfiom which the resinhas not beenstrippedfbr sometime. It is easiiydrstinguished, beingvery light in weight and breaking u,ith an irregulal porous fracture.It consistsalmostentirelyof reddish-broun re-sin.with only a f-ewvely small tearsenbeddcdin it. Palembang bcnzoinis r.rsed as a soufceof natural benzoicacid. Uses. Benzoin. r'n'hentaken intelnalll,. acts as an expectorantand antiseptic.It is mainly useclas an ingledient of friar's balsam.or as a cosmeticlotion preparedliom a simple tincture. It finds considerablc useworld-wiciein the fbod. drinks.perfiulelv rnd toiletrv industries:it is a componentof incense.
TOIU BAISAM Tolr.rBirlsamis obtainedby incision flom the trunk o1'Mlro,rtlon bulsomLrnt (L.) Hanr-rs. (a widely sprcacl polvurorphic species) (Lcguminosae),a large tree which cliltersbut little f}om that yielding balsamof Peru.Wild treesoccurin ColombiaandVenezuela irndin the former countly lalge quantitiesofbalsant lvereproducedin the neighbourhoodof the Magdalenaand Caucl rivers.The treesare cultivated in the West Indies.particularlyin CLrba. History. Balsamof Tolu i,vasdescribedbv Monardesin l-574and its collectionwasobserve clb-vWeir in 1863. Collection. The drug is collectedby making V-shapcdincisionsirr the bark. the secletionbeing reccived in a calabashplaced in the angleof the V. Many suchreceiversare tixed on eachtree.rhr')'ield per tree bcing B-10 k-q.Periodically.the balsarnis transf'erredto larger containers.It is exportedin tins fiont Cartagc.na. Sabanillaand Sta.Marta. Characters. When fieshly imported. tolu is a soti. yellow sernisolid. On keepingit trults to a bro$'lt. brittle solid. Jt sottenson warming. and if a little is then plessed betr.r'eentwo glass slides, microscopicalexaminationshou'scr'1-stals ol cinnamic acid. arnorphous resin and vegetabledebris.Oclouris arorratic and fra-erant:taste. aromaticlthe drug forms a plasticmasswhen chewecl. It is almostentirely solublein alu^olrol. the solutionbeing acid to litmus. and giving a greencolour with lerric chloride (the latter possibly owing to the presenceof resinotannol). Like other drugs containing cinnamic acid, it yields an odour of benzaldehydewhen a filtered decoctionis oxidizedwith potnrsiurnpennanganrtL' solution.
Constituents. Sumatra benzoin contains fiee balsamic acids (cinnamic and benzoic) and estersderir,'edfiom them. Also presentare triterpenoidacidssuch as siaresinolicacid (19-hydroxyoleanolicacid.) Constituents. Tolu containsabor-rt80% of resin derived from resin and sumaresinolicacid (6-hydroxyoleanolicacid). For the formula of alcoholscombinedwith cinnamicand benzoicacids.The drug is rich oleanolicacid seeunder 'TriterpenoidSaponins'.The contentof total in free aromaticacidsand containsabout l2-157c of fi"eecinnamicand balsamicacids (calculatedas cinnamic acid) is at least 20%. and the about 8clcof free benzoic acid (acid value fiom l 00- I 60). Other conarnountof cinnamic acid is usuallv about double that of benzoicacid. stituentsareesterssuchasbenzylbenzoateand benzylcinnamateand a
E
LR I G I N P H A R M A C O P O E IA N L D R E L A T EDDR U G SO F B I O L O G I C AO little vanillin. Recentinvestigationshavc shown the presenceof other cally. The chief balsamic estcrs present are benzyl cinnamate(cinesters.styrene,eugenol.vanillin. l'erulic acid. 1,2-diphenylethane.namein) C6H5CH=CHCOOCH2C6H5(sap. value 23zl). benzyl benhydrocarbonsand alcohols.The balsamalso zoate(sap.value 264.3)and cinamyl cinnamate(styracin).The drug mono- and sesquiterpene Tolu containsfi'om 3-5to 50% of total alsocontainsabout287cof resin,which is saidto consistof perulesinotritcrpenoids. containsnllmerous tannolcombinedwith cinnamicandbenzoicacids.alcohois(nelolidol. balsamicacidscalculatedon the dry alcohol-solublematter. thrnesoland benzyl alcohol) and small quantitiesof vanillin and frcc Uses. GenuineTolu balsarnis difTicultto obtain and the BP now uses cinnarnicacid. Tolu-flavourSolution.containingfive aromatics,fbr the preparationof Work on the isoflavonoidscontainedin the trunk-wood has indiTolu Syrup.Balsarnof Tolu has antisepticproperties.It was a common cated that considerablechemical diff'erencescharacterizethe various ingr"edientof cou-ehmixtures, to which it was added in the fbrrn of gtt:tup. fbrms or speciesof the M. bcLlsantLun syftlpor tlncture. Uses. Balsam of Peru is used as an antisepticdressingfbr"wounds and as a parasiticide.Now that it is no longer usedin Tr-rllegras dressPERUBAIsAM ings, it is of less currentinterestin Westerrrmedicine.Taken internally it is used to treat catarrh and diarrhoea.Allelgic responsesrre balsantumvar. of M.r'rc-n'lon frorn the trunk is obtained of Peru Balsarn pereirae (Leguminosae),after it has been beatenand scorched.The drug is ploduced in Central Arnerica (San Salvador,Honduras and Guaterrala)and is now includedin the EtrropettnPharnurct4toeiaand the BP (2000). History. The drug clerivesits name fiom the tact that when first imported into Spain it came via Callao in Peru. It was known to Monardes and the nethod of preparationwas describedas early as 1576. although aftelwards fbrgotten. In 1860 the coilection was describedand illustratedby Dorat.
possible. Prepored sforox PreparedStorax is a balsam obtained from the wor-rndedtrunk of purified. and sr.rbsequently Licluidamburorientalis (Hamamelidaceae) fbund srnall tlee fi'om a and is obtained Levant storax This is known as in the south-westof Turkey.
Collection and preparation, In the early summerthe bark is injured by bruising or by making incisions.After a time the outerbark may be pared olT, or the whole bark may be lefi until the autumn.when it is Collection and preparation. ln Novernber ol December strips of removed.The piecesof bark are pressedin horse-hairbags.lirst in the bark, nreasuringabout 30 x l5 cm. are beatenwith the back of an axe cold and againafter steepingin hot water.Sometimesthe bark is boiled or other blunt instrument.The bark soon cracksand may be pulled off in the Eastfor with water and againpressed.The exhaustedbark is r-rsed aiter 2 weeks.As in the caseof Tolu balsam.the secretionis purely fumigation.The crudeor liquid storaxis exportedin casksfiom Izmir. pathologicalin origin and r,ery little balsan can be obtainedfrom the Storaxis obtainedby dissolvingthe crudebalsarnin alcohol,filterbark unlessit is chan'edu'ith a torch about 1 week after the beating. ing and recoveringthe solventat as low a temperatureas possibleso as The balsarnproducedin the bark is obtainedby boiling the bark in not to lose any of the volatile constituents.The alcohol-insolublemat(preparedwithout fire) ot halstuttct water and is known as t(lcLtusottte ter consistsof vegetabledebrisand a resin. (balsamof the bark). de cctst:ard The greater part of the balsam. ho*ever. is prepared, after the Characters. Crtrdestorttr ts a gt'eyish,viscousliquid with a pleasant removal ofthe bark, by the secondmethod.The balsamwhich exudes odour and bitter taste. It usually contains about 20-30% of water. is soakedup with rags.which. aftel somedays. are cleanedby gently About 82 879t is alcohol soluble. Purified.s/orz,rfbrms a brown. viscous.semisolidmasswhich loses boiling u'ith water and squeezingin a rope press.The balsarnsinks to (.balsamo not more than5c/aof its weight when dried on a water-bathfor t h. It is the balsam decanled. been watel having and, the the bottom completelysolublein alcohol and partially in either.It has a characterde trapo) is pouredoffand strained. and istic balsamicodour and taste. Less destructivemethodsof preparationhave beeninvesti-qated replacement of include the removal of narrow strips of bark and the Constituents. Storax is very rich in free and combined cinnamic recovthe tree With treatment this of a hot iron. with the use scorching acid.After purilicationit yields 30-,47Vcof total balsamicacids. els in 6 months, comparedwith 8 years aller the drastic traditional By steam distiltation storax yields an oily liquid containing method.The drug is chielly exportedfrornAcajutla (SanSalvador)and pbenylethylene(styrene),C6-H5CH=CHr.cinnamicestels,vanillin and Belize (British Honduras)in tin canistersholding about27 kg. portion of the drug consistsof resin fiee cinnamic acid. The resit.tot-ts with cinnamicacid.The presand combined present both fiee alcohols Characters. Balsam of Peru is a viscid liquid of a somewhatoily is shown by the odour of benzaldeir-r thc drug acid of cinnamic it is ence seen in bulk. When stringiness. nature,but fiee fi'om stickinessand when the drug is mixed with sandand warmed dark blown or nearly black in colour. but in thin layers it is reddish- hyde which is prodr.rced The original containershave a u'hitish scum on with a solutionof potassiumpermanganate. blown and transparent. Recent researchcarried out in Turkey has shown the presenceof the surface.The balsam has a pleasant,somewhatvanilla-like odour compounds not previously reported; these include monotermany taste. and an acrid, slightly bitter penes.phenylpropanesand aliphaticacids. The drug is almostinsolublein water.It is solublein one I'olume of alcohol (90%), but the solutionbecomesturbid on the addition of furAlfied drug. American storax obtainedfrom L. s1'rociJlua,alarge ther solvent.The relativedensity,l.14-l.l'7, is a good indicationof tree found near the Atlantic coast from Centlal America to purity, and if abnorrnalindicatesadulterationwith fixed oils, alcohol, Connecticut.is also used in the USA. This balsarn resemblesthe kerosene.etc. The BP includestestsfor the absenceof artificial bal- Levant storaxin constituents.Thirty-six comporLnds havebeenidentiin petroleumspirit). fixed oils (solubilsams(solubility characteristics fied in the leaf'-oil of the plant and tannins and related phenolics ity in chloral hydratesolution)and turpentine(odourtest). obtainedfiorn cell cultures. Constituents. The ofllcial drug is requiredto contain not less than Uses. Storax is chiefly used in the preparationof friars' balsamand '70Vc wlw of esters,assayedgravimetri- benzoininhalation. 45.jVawlw and not more than
H Y D R O C A R B O NASN D D E R I V A T I V E S
PHARMACEUTICATFIXEDOILS AND FATS ATMONDOIt Alrnond oil is a llxed oil obtained by expressionfiom the seedsof (Rosaceae)yar.tlulcis (sweetalmonds),or P all.1gPnuuts turtygdalrr^s drtlus var. unnru (.biIteralmonds).The oil is mainly ploduced liom almonds grown in the cor.rntriesbordering the Mediterranean(ltaly, France,Spainand North Africa). Characters of plants and seeds. Al-r-rondtrees are about 5 m in height and the varieties,exceptlbr difI'erencesin the seeds,are ahrost indistinguishable.The young fiuits have a soft, t-elrlike pelicarp, the inner part of which gradually becomessclerenchymatous as the tiuit ripensto forn'ra pitted endocarpor"shell.The shells,consistin,emainlv of sclerenchymatous cells. are sometimesground and usedtt'radulteratepowdereddr-r,rgs. The sweetalmondis 2-3 cnr in len-qth.roundedat one end and pointed at the ofher.The bitter almonclis 1.5-2 cm in length but of similar breadthto the sweet almond. Both varietieshave a tl-rin.cinnamonbrown testa which is easily removed after soaking in warm water. a processwhich is known as blanching.The oily kernelconsistsof two large. oily planoconvexcotyledons.and a small plumule and radicle, the latter lying at the pointed end of the seed.Sorre almonds have cotyledonsof unequalsizesand are iffegularly tblded. Bitter almonds al'e sometimesfbund in samplesof su'eetalmonds.particularlythose ofAfrican origin: their plesencemay be detectedby the sodiumpicrate test fbr cyanogeneticglycosides. Constituents. Both varietiesof almondcontain40 55c/cof fixed oil. about207cof proteins,mucilageand emulsin.The bitter almondscontain in addition2.5-1.}c/cof the colourless.crystalline.cyrnocenetic -qlycosideamygdalin(seeChapter'26). Alnrond oil is obtainedby grinding the seedsand expressingthernin canvasbags betweensli-ehtlyheatediron plates.They are bometilnes blanchedbefore-erindir-rg, but this doesnot appearto be of any particlllar advantage.The oil is clarified by subsidenceand filtration. It is a pale yellow liquid with a slight odoLu'andbland,nutty taste.It contains a considerableamount of olein. with smaller quantitiesof the glycosidesof linoleic and other acids.The usualstandardsfbr fixed oils (glc of fatty acids)are includedin the -BPto-qether u'ith testsfbr absenceof variousother oils and sterols. EssentiaLor yolatile oil o.falmotds is obtainedfron'rthe cake left afier expressingbitter ahnonds.This is maceratedwith water for some hor.rrsto allow hydrolysis of the amygdalin to take place. The benzaldehydeand hydrocyanicacid are then separatedby steamdistillatlon. Bitter ahnond oil containsbenzaldehydeand 21% of hydrocyanic acid. Purified volatile oil ofbitter almondshas had all its hydrocyanic acid removedand thereforeconsistsmainly of benzaldehyde. Ht'drogenatedAlmond Ol1is alsoincludedin the ,BP Uses. Almond oil is used in the preparationof rnany toilet articles and as a vehicle fbr oily injections. When taken internally. it has a mild, laxativeaction.The volatilealmondoils are usedas flavouring agents.
ARACHISOIL Arachis oil is obtained by expressionfiom the seeds of Arachi.s Itt'pctguea(Legun-rinosae)(earth-nut, g,rrnnd-nut.peanut) a small annualplant cultivatedthroughouttropicalAfrica and in India, Brazil, southernUSA and Australia.Various senotvDesexist which show dif-
f-erenccsin the relative amounts of fatty acids contained in the oil. Enormousqr.rantities of the fiuits and seedsare shippedto Marseilles and other Er.rropean ports lbr expression.Ground-nutsare the w'orlcl's fourth iargestsor.rrce of fixed oi1. Preparation, During ripening the fruits buly themselves in thc sandy soil in which the plants grow. Each fruit containsflom one to three reddish-brownseeds.The fruits are shelledby a machine.The kernelscontain-10-.50%of oil. Owing to the high oil contenrthe seeds. when clushed. are somcwhat difllcult to express.Atter the initial 'cooking'.part ofthe oil is lcmovedin a low-pressure expellerandthe cake is solvcnt extncted. The two oil fiactions are then rnixed before purification.The presscake firrrnsan excellentcattlefbod. The ground per"icarps havebeenuscclrs ur.tadr-rlterant of powdeleddru-us. Constituents. Arachisoil consistsofthe glycelidesofoleic. linoleic, palmitic,arachidic,steanc.lignocerrcanclothel acids.When saponified with alcoholicpotassiunr hr droricle.crt'stalsof impurepotassitur arachidateseparateon standing.-\rachis oil is one of the most likely adulterants of otherfixed oils (e.g.olivc oil). The BP examinationof the oil is similarto that mentione(lunclcr'C)livcOil' below: the temperatureat which the cooling.h1'dlolrsecl oil be-contes cloLrdyshould not be below 36"C. As with olive oil nrore strinsentstandardsare requiredfor oil to be usedparenterally. The hydrogenatedoi1is alsoofTicial. Uses. Arachisoil hassimilarpropertiesto olir e oil. It is rn insredient of camphoratedoil but is usedmairrlf in the-produetion of ntargarine. cookingfats,etc.
CASTOROtt Castoroil (c'old-drawncastoroil) is a fixed oil obtainetltronr 1l.rc sceds of RicinLtsconununis (Euphorbiaceae).Thc ll'r-ritir r rhrcc ccllecl thornycapsule.The castoris a nativeof India:thc prineipalpnrducing countries are Brazil. India. China. the fbrnrcr Sor iet L'niorr and Thailand.There ale about l 7 varieties.which nrar be rouqhlr _llouped into shrubsand treesproducinglargeseeds.and annuulhcrbsproducing smallerseeds.It is mainlv the smallervarietie:thul .rrc.nou cultivated and these have been developed. br br-ecdtng.to give high-yieldingseed plants.Mechanicalharrestingrs norr replacing hand-picking. Characters of seeds. The seedsshou consiclcrable clifflrencesin sizeand colour.They areova1.somewhatconrprL-\\cd. 8 l8 mm long and4 12 mm broad.The testais vcrl'sntooth. thin and brittle.The colourmay be a more or lessunifbrm grL-\.[]r'o\\n ol black.or may be variously mottled with brown or black. .\ small. ofien yellowish. caruncleis usuallypresentat one cnd. lronr nlrich luns the rapheto terminatein a slightly raisedchalazaat thL-oppositeend of the seed. The testa is easily remor,edto disclosethe papery remainsof the nucellussurroundinga largeoill'cndosperm.Within the latterlies the embryo,with two thin. flat cotl'leclons and a radicledirectedtowards the caruncle.Castor seeds,if in sood condition, have ver:ylittle odour: taste,somewhatacrid. ll'the testasare broken.ranciditvwill develop. Preparation and characters of oil. Ninety per cent of the wolld's castoroil is extractedin Blazil nnd India. Relativelysmall amountsof the whole seedsale now exported.The various pl'ocesses involved in the preparationof castor oil are describedin the 8th edltion of this book. Briefly, the seedsare deprived of their testasand the kernels cold-expressedin suitable hydraulic presses.The oil is refined by stearning,filtration and bleaching.Cold expressionyields about 337c
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P H A R M A C O P O E IA N L D R E L A T EDDR U G SO F B I O L O G I C AO LR I G I N of r-r-redicinal oil and t'urthcrquantiiiesof oil of lorver quality may be obtainedby other methods Medicinal castoroil is a colourlessor pale yellow liquid. with a slightodourandtaintll,acridtaste.For its chenricalandphysrcalconstants.see the phalrnacopoeias. The acid value increasessomewhat with age and an initiall,v high r,alue indicates the use of darr.raged seedsor carelessextractionor storage.Castoroil has an extremely high viscosity. Constituents. Castorseedscontain46-53c/cof lixed oil. which consists of thc glycosides of ricinoleic, isoticinoleic. stearic and dihydroxystearic acids.Thc purgativeactionofthe oil is saidto be due lo free ricinoleic acid and its stereoisor-ner, which are produced by h1'drolysis in the duodenum.Theseacidshavethe formula.
cHrlcH,lscH(oH)cH.cH:cH IcH l,lTcooH For thebiogenesis ofricinoleicacid.seeFig. 20.3.Castoroil andthe oil from Ricinu,s:.cutT.ibariuus are remarkablelbr their hi-ghricinoleic acid content,which is about887cand92'/c,respectively. The cake lelt afier expressioncontainsextremelypoisonoustoxins knou'n asricins,which rnakeit unfit fbl useasa cattlefood. ln the body they prodrlcean antitoxin (antiricin).Ricin D is a sugarprotein wtth a stronglethal toxicity; it contains.193arnino acids and 23 sr.rgars. Two otherricins.acidicricin and basicricin, havesimilarproperties. Ricin 'Abrr-rs and abrin (see Seeds'below)exhibit antitumou properties.The seedsalso contain lipasesand a crystallinealkaloid.r'icinine,which is not rnarkedlytoxic and is stluctr.rallyrelatedto nicotinamide. Uses. Castoroil. oncervidell'usedasa domesticpurgatire. is noir more restrictedto hospital use lbr adninistration after fbod poisoning and as a prelirninary to intestinalexamination.Owing to the presenceof ricin, the seedshiive a nruch more violent action than the oil and ale not usedas a purgativein the West. The oil is a constituentof llexible collodion. HydlogenatedCastorOil USP/NF (19951is usedas a stifl'eningagent. Undecylenicacid.which is prepaledlrom castoroil, is usedin lungistatic preparations. (polyethoxylated asis alsoits zinc salt.Nonionicsurfactants castoroils) of variablecompositionareprodr.rced by thereactionof castor oil with etl-rylene oxide and are useclin certainintravenouspreparations whicl-rcontaindrugswith low aqr.reous solubility.The oil and its derivatives flnd many nonpharnraceutical usesinchrdingthe nanufactureof plasticizers TurkeyRed O11.soaps,paints.r,'iunishes. and lubricants. Allied drugs. Crctut .seetlsare obtained fron Croton tiglitun (ELrphorbiaceae), a small trce producing similar capsulesto those of castorbu1devoid of spines.The seedsresemblecastorsecdsin sizeand shapebut have a dull. cinnarron-browncolour and readily lose their carunclcs.They contain about 50% of fixed oil which uontainscloton resin;also 'crotin', ii mixtureof croton-globulinand croton-albumin compalablewith ricin. The oil also containsdiestersof the tetracyclic diterpenephorbol (esterifyingacid at Rl and Rl. Rl = H in the fbrmula below):acidsinvolvedareaceticas a short-chain acid.andcapric,lauric and palmiticas long-chainacids.Thesecompoundsarecocarcinogens and :rlso possessinflammatory ancl vesicant properties (see 'Ditelpenes'). Also presentare phorbol-12.13,20-triesters 1Rl. Rr and R3 are all acyl groups in the fbrmula shown).These are 'cryptic irritants', so callcd becausethey are not biologicallyactiveas suchbut becomeso by removal of the C-20 acyl group by hydrolysis.Rotation locular counter-cuffentchromatography(q.v.) has been used to separate thesetwo groLlpsof esters.A number of the phorbol estershave been tested for anti-HIV-I activity (S. El-Mekkawy et al., Phytochemistry'.2000. 53. .157).The plant also contains alkaloids. Croton oil shor-rlcl be handlcd with extremecaution: it is not used in Westernmeclicine.but if takeninten-rally,it actsas a violent cathartic.
Phorbolestersof croton oil Plttsic nttts or Purging nuts Llrethe seedsof Jatropha cLtrcos,another menrberof the Eupholbiaceere. The seedsare black, oval and 15-20 mm in length.They contain about 40% offixed oil and a substancecompalable with ricin, called culcin. Both seedsand oil ale powerful purgatives. (prayerbeads)are the attractivered and black. but poiAhrus .seed.s sonous,seedsof Abrus precaktrils (Leguminosae).They contain a toxic glycoprotein(abrin) resemblingricin togetherwith anothernontoxic peptidehaving haemagglutinatingproperties.Various alkaloids (abrine.hyaphorine.precatorine)of the indole type havebeenreported. also various sterolsand lectins.The seedshave been r.rsedin folklore medicinein Asia.Africa and S. America to treatmany ailments,alsoto procureabortionand to hastenlabour.In lndia they areemployedas an oral contraceptiveand asthey areremarkablyuniform. and eachweighs about I carat(c. 200 rrg), havebeenusedtraditionallyas weights.
ouvEorr Olive oil (sulad oil, n'eet oil) is a fixed oil which is expressedfrom the ripe lruits oI OIea eurq)oeo (Oleaceae).The olive is an evergreentree, which lives to a greatagebut seldomexceeds12 m in height.It produces drupaceousfruits about 2-3 crn in length. The var. ltttifolitt bearsIarger f'ruitsthan the var.ktngifolln, but the latteris saidto yield the bestoi1.The oil is expressedin all the Mediten'aneancountriesand in Califbrnia. haly. Spain,France,GreeceandTunisiaproduce90clcofthe world's production. Olive oil has been ranked sixth in the world's production of vegetable oils (F. D. Gr.rnstone et ul.. 1994,The Lipid Handbooft,Chapman andHall). History. The olive appearsto be a native of Palestine.It was known in Egypt in the seventeenthcenturyBC, and was introducedinto Spain at an early period. Collection and preparation. The methodsusedforthe preparationof the oil naturally vary somewhataccordingto local conditions.In the modern factorieshydraulic pressesare widely used but in the more remotedistrictstheprocedureis cssentiallythat which hasbeenfollowcd for hundredsof yealsandis describedin earliereditionsof this book.The first oil to be expressed from thegror.rnd fruitsis known asvirgin oil; subsequentlythe marc may be solvent extractedto obtain the lower quality oil. The superiorgradesof oil areextravirgin. virgin andpure or reflned. Characters. Olive oil is a pale yellow liquid, which sometimeshasa greenishtint. The amountof color-rring matterpresent,whetherchlorophyll or carotene,appearsto detenninethe naturalfluorescenceof the oil in ultravioletlight. The oil has a slight odour and a bland taste.If the fruits used have beenallowed to ferment.the acid value of the oil will be higher than is officially permitted. It should comply with the tests for absenceof arachisoil, cotton-seedoil. sesameoil and tea-seedoil. The latter oil, which is obtainedfrom China. is not from the ordinarv tea plant but liom a tree, Cantelliuso.t(tnqua.
HYDROCARBONS AND DERIVATIVES Constituents. Olive oils fiom different sourcesdiffer somewhatin composition.This may be due eitherto the useof the dilferent varieties of olive or to climatic differences.Two types of oil may be distinguished:(a) that producedin Italy, Spain,Asia Minor and California. which containsmore olein and lesslinolein than type (b), producedin the Dodecaneseand Tunisia.Typical analysesofthese typesare:
Oleic acid Linoleic acid Palmitic acid'l Stearicacid J
Tj'pe(a)
T'pe (b)
BP limits
Vc
Vc
ch
78-86 o-1
65-'70 10-15
9-12
1)
56-85 3.520.0 [7.s-20.0
io.s-s.o
triglyceridescontainingonly the shortand mediumchain-lengthfatty acids(e.9.octanoic,decanoic;seeTable20.2).It maintainsits lor.',, r'iscosity until near the solidillcation point (about 0'C) and is a useful nonaqucousmedium fol the oral adrninistrationof somemedicaments. Metliurtt-chairtTrigbceritles BP, EP, svnonymouswith the abovc, may also be obtainedflorn the dried endospermof Elaeis guineen.si.s (Palmae).The fatt1'acidcornpositionof the hydrolysedoil, determined by GC, has the fbllou'ing specifications:caproic acid ), 2c/c,caprylic acid 50-80%, caplic acid 20-5OC/o,lauric acid > 3.\ch, n.ryristic a c i d) 1 . 0 % .
COD.LIVER OIt AND HAIIBUT.IIVER OIt SeeChapter3 I
COTTONSEED OIt
The characteristicodour of olive oil, particularlythe virgin oils. arises from the presenceof volatile C,.. alcohols (hexanol. E-2-hexenol, Z-3-hexenol),C6 aldehydesand acetylatedesters.Oils arising from different chemotypescan be distinguishedby headspacegas-liquid chromatography(M. Williams et al., P ht,tochemi str l-, 1998,47, 1253). The dehydrogenaseswhich produce the unsaturatedalcohois have beenstudiedin the pulp of developingolive pericalps(J. J. Salasand J. Sdnchez,Pht'tochemistn',1998, 48, 35). The BP examinationof the oil includesa TLC testfol identity and.to detect foreign oils, the GC determinationof the individual methyl estersof the acidsproducedby hydrolysis.Limits of the principal acids are given above: other percentagelimits, which exclude foreign oils, include saturatedfatty acids of chain length less than C16(#0.1), linolenic(> 1.2),arachidic(>0.7). behenic(>0.2). Therearealsolimits for a numberof sterols.The USP/NF ( I 995) includesspecifictests for the absenceof sesameoil (furfural test),cottonseedoi1,peanutoil and teaseedoil. For the biosynthesisof the acids.seethe introductionto this chapter. The saturatedglyceridestend to separatefrom the oil in cold weather; at I OoCthe oil beginsto becomecloudy and at aboutOoCforms a soft mass.
Cottonseedoil is expressedfr-unr the seeds of varior.rsspecies of (Malvaceae)in Anicrica and Europe.In the UK, Egyptian Gossy1tium and Indian cottonseed.which do not rccluilc delinting on arrival, are largelyused.Seeunder 'Cotton'. The preparationof cottonseedoil i s one of hot crpressionand a pressureof about 10 000 kPa is used.The cnrdeoil is thick and tulbid and is retinedin variousways.that knour.ras 'uinter blcached'beingthe bestofthe refinedgrades.Cottonseed oil is a sentidr1,'ing oil andhasa fairly high iodine value.When usedto adulterateother oils its presence may be detectedby the test for sernidrl,ingoils describedin the ,BP monoglaphfor ArachisOil. The hydrogenatedoil. only, is of1icial.
Uses. Olive oil is usedin the preparationof soaps.plasters,etc., and is widely employedas a saladoil. Oil for usein the manufactureof parenteralpreparationsis requiredto havea lower acid value andperoxide value than that normally required, and to be almost fiee of water' (0.1%) as determinedby the specifiedKarl Fischerrnethod. Recent researchhas suggestedthat olive oil may protect against colonic carcinogenesisby virtue of its action on prostaglandins:rals fed on a diet containingolive oil, as distinct from thosereceiving safflower oil. were protected(R. Bartoli et ttl., Gut,2000, 46, 191).
Macroscopical characters. The seeds are o\ate. flattened and obliquely pointedat one end; about4-6 mm lor.rgand I 1.5 mrn broad. The testais brown, glossyand finely pitted.OdoLrrless: tastc.mucilaginous and oily. If cruciferousseedsare present.a pungentodour and taste may develop on crushing and moistening.A transvelsesection showsa narow endospermand two iar-ec.planoconrex cotyledons.
cocoNuTott The expressedoil of the dried solid part of the endospermof the coconut, Cocos nucifera L. (Palmae)is a semisolid.melting at about 24'C and consistingof the triglyceridesof mainly lauric and myristic acids, together with smaller quantities of caproic. caprylic. oleic, palmitic and stearicacids.This constitutiongives it a very low iodine value (7.0-1 1.0)and a high saponificationvalue. The particularly high proportion of medium chain-length acids means that the oil is easily absorbedliom the gastrointestinaltract, which rnakesit of value to patientswith fat absorptionproblems. Fractionated coconut oil. Fractionatedand purified endospermoil of the coconutC. nut:ifern,or Thin VegetableOil of the BPC, contains
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LINSEED AND LINSEED OIt Linseed (flaxseed)is the dlied ripe seed of Littttnt Lr.:ir,tti.:sirnutt (Linaceae),an annualherb abotrt0.7 m high qith blue lloriers and a globular capsule.The flax has long been cr-rltivatcdlbr its pericyclic fibres and seeds.Suppliesof the latterarederivedflonr SouthArnerica. India, the USA and Canada.Large quantitiesof oil are erpressedin England.particularlyat Hull. and on the Continent.
Microscopical characters. Microscopicalexaminationof the testa showsa mucilage-containing outer epidcrnris:one or two layersofcollenchyrna or 'round cells': a single la1'erof longitudinally directed elongatedsclerenchyma; the hyalinelal"ersor'cross-cells'composed in the ripe seedof partially or conpletelv obliteratedparenchymatous cells with their long axis at right anglesto thoseof the sclerenchymatous layer; and an innermostlayer of pigment ceils. The outer epidermis is composedof cells, rectangularor five-sided in surfaceview, which swell up in water and becorne mucilaginous.The outer cell walls, when swollen in water. show an or-rtersolid stratifiedlayer and an inner part yielding mucilage. itself faintly stratified. The radial layersor 'round cells'are c.vlindricalin shapeand show distinct triangular intercellularair spaces.The sclerenchyrnatous layer is composed of elongatedcells, up to 250 tim in length,with lignified pitted walls. The hyaline layersoften remain attachedto portionsof the sclerenchymatouslayer in the powdcreddrug (Fig. 12.71).Thepignrentlayer is composedof cells with thickenedpitted walls and containing arnor-
P H A R M A C O P O E IA N L D R E L A T EDDR U G SO F B I O L O G I C AO LR I G I N phous reddish-brown contents (Fig. zl2.7H). The ce1ls of the endospermand cotyledonsare polygonal with somewhat thickened walls, and containnumerousaleuronegrainsand -globulesof fixed oil. Starchis presentin unripe scedsonly.
RAPESEED OIt
Refined RapeseedOil EP, BP is obtainedby mechanicalexpressionor by extractionliom the seedsof Bzrssicanapus andB. canpestris. The crop is now extensivelycultivatedin Europeand oils with various propertiesdependingon glyceridecornpositionare commercially Constituents. Linseed containsabout 30-40c/cof fixed oi]r.6Vc of mucilage(^BPswellingindextbr whole seeds< 4.0),257cof proteinand available.As was indicatedon p.8l not all varietiesyield an oil suitsmall quantitiesof the cyanogeneticglucosideslinamarin and lotaus- able fbr medicinalpurposesso that the specifiedpharmacopoeiallimits tralin.Otherconstituents arephenylpropanoid glycosides(L. Luyengi er of the various esterityingacids are important standardsas are relative ul..J. Nat.Protl..1993.56.2012). flavonoids. thelignan(-)-pinoresinol density(c. 0.917)andrefractiveindex (r.. L473). As with someother oils, the additionof a suitableantioxidantis perdiglucoside(a tetrahydrofurofuran-type lignan-see Table22.7) andthe cancer chemoprotectivemammalian lignan precursor secoisolarici- mitted,the nameand concentrationof which must be statedon the label together with a statementas to whether the oil was obtained by (S.-X.Qil et ol., Phamt.Biol..1999,37.l). resinoldiglucoside Cell culturesof Linun albtonareableto synthesize andaccumulate the m e c h a n i c ae l\ p r e \ s i o no r b 1 e x t r a c t i o n . (T. Smollnyara1., lignanspodophyllotoxinand5-methylpodophyllotoxin Pht'tochenisrn,1998.48. 975). SESAME OIt Sesameotl(Gingelly'oil, Teeloil) is obtainedby refining the expressed or extractedoil from the seedsof Sesantumindicum (Pedaliaceae), a herb which is widely cultivatedin India, China.Japanand many tropical countries.The oil is otficial in the EP and BP. The seedscontain about 507c of lixed oil which closely resembles olive oil in its propertiesand which it has, in somemeasure.replaced. It is a pale yellow. bland oil which on cooling to about-zl'C solidifies to a buttery mass:it hasa saponificationvaluethe sameasthat tbr olive oil and a somewhathigher iodine value ( I 04-120). Principal componentsofthe oil are the glyceridesofoleic and linoleic acids with small proportions of palmitic, stearic and arachidic acids.It alsocontainsabott lVc of the lignan sesamin.and the related sesamolin.The characteristic phenoliccomponentis the basisof the BP test for identity and also the test fol the detectionof sesameoi1 in other oils. The originaltest involvedthe productionof a pink colour when the oil was shaken with half its volume of concentrated Uses. Crushedlinseed is used in the folm of a poultice and whole hydrochloric acid containing l7c of sucrose (Baudouin's test). seedsare employedto make demulcentpreparations.The oil is usedin However, some commerciallylefined oils may not give a positive liniments.and researchhas suggested that hydrolysedlinseedoil has Baudouin's test. With the current BP test for the absenceof sesame potentially useful antibacterialpropertiesas a topical preparationin oil in other oils. e.g. olive oil, the reagentsare aceticanhydride,a that it is efl'ectiveagainstSlapftt'lococt:tts drrclr strainsresisrantio solution of furfuraldehydeand sulphuric acid; a bluish-greencolour antibiotics.Linseedcake is a valuablecattlefood. is a positiveresult.The compositionof triglyceridesin sesameoil is determinedin the BP by liquid chr"omatography. those triglycerides having as acid radicalsoleic I part and linoleic 2 parts.and thosehavPAIM OIL AND PALMKERNET OIL ing oieic 2 parts and linoleic I part being among the most predomiPalm oil is obtainedby steamingand expressionof the mesocatpof the nant. fruits of E/aelsguine?nsis(Palmae).World prodLrction amountsto over As statedabove,sesameseedsand oil also containlignans:these I I .5 rnillion tonnes.over half of which is producedin Malaysia.In terms are antioxidants of the tetrahydrofurofuran-type(Table 22.7) and of world consumptionit hasnow overtakensunflowerandrapeseedoils. include sesamin. sesamolinol and sesamolin. Another lignan, Pahn oil is yellowish-brown in colour. of a buttery consistency sesaminol.is formed during industrialbleachingof the oil. The bio(m.p. 30'C) and of agreeabieodour. Palmitic and oleic acids are the logical activities of these compoundsinclr-rdereduction in selum principalestedfyingacids. cholesterollevelsand increasedvitamin E activities.For the biogenPalm kernel oil. Pahn kerneloil is obtainedby heatingthe separated e s i so f s u c hl i g n a n ss e eM . J . K a t o e t a l . , P h y t o c h e n i s t r y . 1 9 9 8 , 4 7 . seedsfbr 4 6 houls to shrink the shell. which is then crackedand the 5 8 3 . kernelsremovedwhole. The oil is then obtainedby expression.It diff'erschemicallyliom palm oil (above)in containinga high proportion SOYAOIL (50%) of the triglyceridesof lauric acid, a saturated.medir-rmchainlength fatty acid (Table 20.2). The mixture of other acids resembles Soya oil is derived from the seedsof Glycine na,r (Leguminosae). that found in coconnt oil. The oi1 is a source of Medium-chain After refining. it is deodorizedand clarified by filtration at about0oC. It shouldremainbright when kept at OoCfor l6 h. The principal esteriTriglyceridesEPIBP. fying fatty acids are iinoleic (41-62%), oleic (19-307c), palmitic Fractionated Palm Kernel Oil BP is pahn oi1which has undergone ('7 14%),linolenic(1-11%)and stearic(1.1-5.55i). selectivesolventfiactionationand hydrogenation.It is a white, brittle Thereis an official limit test for the amounrpf 1[e ,\s.22-24B-methyl solid,odourlessor almostso. with m.p. 31'-36'C making it suitable plant sterol. brassicasterol,which as the name implies occurs in for useas a suppositorybase. Brassicaspp.including B. raptr (c1.v.). Linseed oil. The extractionof linseedoil is one of hot expressionof a linseed meal and the pressis adjustedto leave sufficient oil in the caketo make it suitableas a cattlefood. Linseed oil of BP quality is a yellowish-brown dryin-e oil with a characteristicodou' and bland tastel much commercial oil has a markedodour and act'idtaste.On exposureto air it graduallythickens and torms a hard varnish.It hasa high iodinevalue({ 175)as it contains considerablequantitiesof the glycosidesof unsaturatedacids. Analysesshow linolenicacid,CITH,eCOOH(36-50c/o). linoleic acid (23-21c/().oleic acidClTHr3CooH (10-18%).togethCTTH3TCOOH er with some saturatedacids-myristic, stearicand palmitic (5-ll%). For the fonnation of the unsaturatedacids.seeFigs. 20.1 and20.2. For usein paint.linseedoil was boiledwith 'driers'sr.rch as lithargeor rnanganese resinatewhicl-r.by fonning metaliic salts.causedthe oil to dry morerapidly.Such 'boiledoils'must not be usedfbr medicinalpurposes.
HYDROCARBONS AND DERIVATIVES
THEOBROMA OIt Oil of theobromaor cocoabuttermay be obtainedfrorn the groundkernels of Theobromacocao (Sterculiaceae) by hot expression.The oil is filtered and allowed to set in moulds. Much is refined in Holland. Cocoa butter.as it is commonly termed.consistsof the glyceridesof steadc,palmitic. arachidic,oleic and other acids.Theseacidsare combined with glycerol partly in the usual way as triglyceridesand partly as mixed glyceridesin which the glycerol is attachedto more than one of the acids.It is the most expensiveof the commercialfixed oils and rnay be adr.rlterated with waxes, stearin(e.g. coconut stearin).animal tallows or vegetabletallows (e.g. from seedsof Barsia Longfolia and Stillingitt sebiJera).For the characterand testsfbr purity of oil of theobroma, see the pharmacopoeias. Its melting point (31-3zl') makes it ideal fbr the preparationof suppositories.
The oil should be protectedliom light and oxygen and srorcdar a temperaturenot exceeding25oC.
Eveningprimrose oil The fixed oil frorn the seedsof Oenotheruspp. (Onagraceae) contains substantial amounts of esterified y-linolenic aeid (GLA). a C 1s6,9.12-triene.
ueffioox acid {GLAI 7-Linolenic
cooH Me
WOOL FAT Wool fat (anhydrottslanolin) is a purified fat-like substanceprepared fiom the wool of the sheep,Oulsarles (Bovidae). Raw wool containsconsiderablequantitiesof 'woo1 grease'or crude lanolin. the potassium salts of fatty acids and earthy matter. Raw lanolin is separatedby 'cracking' with sulphuric acid fiom the washingsof the scouringprocessand purilied to fit it lbr medicinal use. Purification may be done by centrifuging with water and by bleaching. Wool fat is a pale yellow, tenacionssubstancewith a faint but characteristicodour.It is insolublein water and a high proportion of water may be incorporatedwith it by melting (m.p. 36-42'C) and stirring. Soluble in ether and chloroform. Like other waxes, it is not readily saponifiedby aqueousalkali,but an alcoholicsolutionofalkali causes saponification. Saponilicationvalue 90-105: iodine value 18-32; acid valuenot morethan l. Hydrouswool fat or lanolincontains25% water. The chief constituentsof wool fat arecholesteroland isocholesterol. unsaturatedmonohydricalcoholsof the formula C27H.+5OH. both tiee and combined with lanoceric,lanopalmitic.carnaubicand other fatty acids.Wool fat also containsaliphaticalcoholssuchas cetyl, ceryl and carnaubl,lalcohols.BLrtylatedhydroxytoluene,up to 200 p.p.m., may be addedas an antioxidant, WoolAlcohoLsEP, BP are preparedby the saponificationof crude lanolin and the separationof the alcohol fraction.The productconsists of steroidand triterpenealcohols,including cholesterol(not less than 30%) and isocholestelol.As for wool fat, an antioxidantmay be added. To testfor cholesteroldissolve0.5 g in 5 ml of chloroform,add I ml aceticanhydrideand two dropsof sulphuricacidl a deep-greencolour is produced. Hy'drogeruttedWooLFat EP, BP is obtained by the high pressure/ high temperaturehydrogenationof anhydrouswool f-at.It containsa mixture of higher aliphaticalcoholsand sterols. Wool fat is usedas an emollientbasefbr creamsand ointments.
Arachidonicacid
Ez(PGEz) E Prostaglandin z(PGEz)
The principal speciescultivateclin thr-UK is O. bieruis which yields an oil containingl-9c/aGLA. althoughrnorclecent work showshigher yields for the oils of someother species.nantelv O. acen,iphilla novct (15.68%),O. porttdo-ra(.l4.4lVc)anclan ccotvpeof O. rubricaulis (13.15Vc).Researchhas involved breedingneu varietiesfor high yields of oil and reducingthe lif'ecyclcof the plant f rorn 14 to 7 months (Pltarm. J., 1994, 252. 189). The sequencefor the formation of such acids in the plant via cislinoleicacidhasalreadybeenindicatedin Fig. 10.l. In animaltissuesit appearsthat the prostaglandinsareformed from dictarl' linoleic acid by conversionto GLA which undergoesCr addition and further desaturation to give acids such as arachidonicacid an inrmediateprecursorof someprostaglandins. The beneficialeffectsof eveningprimroseoil ntar nell be relatedto affording a precunor of the prostaglandinsfbr those individr,ralswhose enzymicconversionof linoleic acid to GLA is delicient.Thc oil is norv widely marketedasa dietarysupplement.for cosnreticpurposes.andmore specifically for the treatment of atopic eczemil and premcnstrualsyndrome(prostaglandin E may be depletedin this condition).Furlherpossibilitiesincludeits usein diabeticneuroDathv andrheumatoidafthritis. Other sourcesof GLA. A number of other seedoil: contain appreciable quantitiesof GLA; theseinclude thoseof Ribesnigrunt andR. rubrum (the black and red currant).Borago of]it'irnli.s(starflower)and S1-mphytum oJlicinale(comfrey).B. offitirtulis is now cultivatedcommercially in the UK to yield an oil containing 25c/cGLA; details of commercial seed production. plant breeding programmesand husbandry will be found in a generalarticle on the crop by A. Fieldsend (Biolo gisr, 1995,42, 203).
Hydnocorpusoil Corn oil
This is the fixed oil obtainedby cold expressionfrom the fresh ripe Corn oil USP/NF (1995),or maize oil, is obtainedby expressionof seedsof Hl,dnocarpuswightiuttt, H. anthelmintica,H. heterophylla the fixed oil from the embryos oI Zea mays L. (Graminae) and and other speciesof Hy,dnocarpus.and also ol TttrtLktogenos kurz,ii. refined. These plants are lbund in India. Bulma. Siam and Indo-China and For the separationof the embryos from maize see 'Preparationof belongto the Flacourtiaceae. Maize Starch'. The refined light golden yellow oil consistslargely of The oil of H. wightianT contains hydnocarpic acid (about 2187c), triglyceridesof the unsaturatedoleic and linoleic acids with smaller chaulmoogricacid (about 21c/c),gorlic and other acids (formulae.see proportionsofpalmitic and steadcacids.Becauseofthe high unsatura- Table 20.4); the structuresof severalnew cyclopentenylfatty acids tion (iodinevalue I l0-128), it is regardedasofvalue in dietsdesigned haverecentlybeenelucidated.Theseacidsdo not appearto be fbrmed to limit blood cholesterollevels. from straightchain acids and they accumulateduring the last 3-4
P H A R M A C O P O E IA N L D R E L A T EDDR U G SO F B I O L O G I C AO LR I G I N months of matul'ation of the fluit. They are strongly bactericidal towardsthe leprosymicrococclrs.but the oil has now to a largeextent beenreplacedby the ethyl esterstrndsaltsof hydnocarpicand chaulmoogric acid. The esterifiedoil of H. n'ig,htittna is plefelableto that of other species.in that it yields when fiactionatedalmost pure ethyl hydnocarpate. This was includedin the BPC ( 1965) but has now been deleted.as mol'cef1'e ctivc rcmediesale availablc. Lord Lard (.preparedlartll is the purilied internal fat of the hog, Sus scrofc t o r J e l U n - r : t r l l rS t ru. i t l a rc. For medicinal purposeslard is preparedfi'om the abdominal fat known as 'flare'.from which it is obtainedby treatmentwith hot water at a temperaturenot exceeding57'C. Lard is a soft. white tat with a nonrancidodour.Acid v:rluenot more than I .2.Lard hasa lowermeltingpoint (3,1.11"C) anda higheriodine value (52*66) than suet.Saponification value 192-198.It shouldbe lree fiom moisture.beef'-lat.sesame-seed and cotton-seedoils. alkalis andchlorides. Larcl contains approximately 40c/t.of solid glycerides such as myristin. stearin and palmitin. and 60%, of mixed liquid glycerides such as olein. These fiactions arc somewhatseparatedby pressureat 'stearin'and'lard OoCand soldas oil'respectively.Lard is usedas an ointmentbasebut is no longer of1icialin Britain. It is sornewhatliable to becomerancid. but this may be retardedby benzoination,Siamese benzoinbeing more efl'ectivethan the Sumatravariety.
slow bleachingactionof light. air and moisture.In the lattermethod the melted wax is allowed to tall on a revolving cylinder which is kept moist. Ribbon-like str"ipsof wax are thus formed which are exposedon clothsto the actionof light and air. being moistenedand turned at intervals until the outer surface is bleached.The whole proccssis repeatcdat least once, and the wax is linally cast into circular cakes. Characters. Beeswax is a yellowish-brown or yellowish-white solid. It breakswith a granularfractureand has a characteristicodour. It is insolublein water and sparinglysoluble in cold alcohol,but dissolvesin chlorofbnn and in warm fixed and volatile oils (e.s.oil ofturpentlne). Constituents. Beeswax is a true wax. consistingof about 80% of myricyl pahnitate(myricin),CtsH3ICOOC39H6,. with possiblya little myricyl stearate.It also contains about I5Vc of free cerotic acid, C.6H5rCOOH, an aromatic substancecerolein, hydrocarbons,lactones.cholesterylestersand po1lenpigments. 'drop point' Standards. T'heseincludea of 61-65' for both the white 'ester and yellow wax, value' (q.v.) and 'ratio number' (ratio of the ester value to the acid value) which fbr both waxes lies between 3.3 4.3.
Adulterants, The most likely adulterantsandtheir methodsof detection will be lbund rnlhe Pfurrnrucopoeia. Japanwax, which is there mentioned.is not a true wax but a fat and may be saponifiedby means Suet. Sr.retis the purified intelnal fat of the abdomenof the sheep. ofboiling aqueoussodiumhydroxide.Waxesare saponifiedby strong by aqueousalkali. Ovi.suries.It containsabout 50 60% of solid glyceridesand melts at alcolnLic potash, but are practically r-rnaffected about zl,5"C.It is used as an ointment basc in tropical and subtropical Japan wax is preparedfi'om the tiuits of various speciesof R/rls (Anacardiaceae). countnes. Uses. Beeswaxis usedin the preparationof plasters.ointmentsand polishes.
YELLOWBEESWAX,WHITEBEESWAX Bees',vaxis obtainedby rneltingand puritying the honeycornbof Api,i mellifica and other bees.The wax is imported from the West Indies, Califbrnia. Chile. Africa. Madagascarand India. The EP and BP include separatemono-eraphs for the yellow and the white wax. Preparation, Wax is secretedby worker beesin cells on tht: ventral surfaceof the last four segmentsof their abdomen.The wax passesout throughporesin the chitinousplatesofthe sternumand is used.particularly by the yor.rngworkers.to fbrm the comb. Yellon'beeswa,ris prepared.after removal of the honey.by melting the comb underwater (residualhoney dissolvin-gin the water and solid irnpuritiessinking).straining.and allowing the wax to solidify in suitablernor.rlds. Wltite beesv,uris preparedfrom the above by treatmentwith charcoal.potassiumpermanganate. chromicacid.chlorine.ctc..or by the
CARNAUBAWAX Carnar.rba wax, included tn the EP/BP (2000) and USP/NF (1995), is derived from the leaves of Copernicia cerifera (Palmae). It is removed from the leavesby shaking and purified to remove foreign mattel'. The wax is hard.light brown to paleyellow in colour and is supplied as a moderatelycoarsepowder.as f'lakesor irregularlumps; it is usually tastelesswith a slight characteristicodour liee from rancidity. Esters,chietly myricyl cerotate,are the principal components,with somefree alcoholsand other minor constituents.The acid value is low (BP, USP/NF.2-'7)" Ihe saponificationvalue 78-95. lt has an iodine value of 7-14. Carnaubawax is Lrsedin pharmacyas a tablet-coating agentand in otherindustriesfor the manufactureofcandlesand leather polish. It has been suggestedas a replacementfor beeswax in the p r e p a r r l i oo nf p h l t o c o s r n c t i c s .
PHARMACOPOEIAL AND RELATED DRUGS OF BIOLOGICAT ORIGIN sucrose;it is formed in photosynthesis by the reactionof UDPG with (Fig. 21.3).Control mechanismsfor the build-up fructose-6-phosphate of sucrosein leaves,and its breakdown for tlanspofi to storageorgans, areachievedby metaboliteeffectorcontrolof the appropriateenzymes. The reverse process, hydrolysis. is brought about by suitable enzymes or by boiling with dilute acid. The same sugars may be linked to one anotherin variousways.Thus,the disaccharides maltose, cellobiose,sophoroseand trehaloseareall composedof two molecules of glucosejoined by c,-1,,1-,B-1,4-, B-1,2- and cr,cr-1,1-(nonreducing) linkages,respectively.
HO.^rH
cHo
Y-t
I
HcoH I
HCOH I
HOCH I HCOH
HOCH O
xEox Il t
I HCOH I
CH I
cl-tzoH
cH2oH
Aldohexosc (uglucose)
Aldohcxose (F-p-glucose,showing pyranose ring and the OH taking part in glycoside formrtion)
cH2oH I C=O
Polysocchorides By condensationinvolving sugar phosphatesand sugar nucleotides, polysaccharides are derivedfrom monosaccharides in an exactly similar mannerto the formation of di-. tri-and tetrasaccharides. The name 'oligosaccharide'(Greek oligo, tew) is often applied to saccharides containing from two to l0 units. In polysaccharidesthe number of sugar r.rnitsis much larger and the number lbrming the molecule is often only approximatelyknown. The hydrolysis of polysaccharides, by enzymesor reagents,ofien resultsin a successionof cleavages,but the final productsarehexosesor pentosesor their derivatives.The term 'polysaccharide'may usefullybe takento includepolysaccharidecomplexes which yield in addition to monosaccharidestheir sulphate esters,uronic acidsor amino sugars. Table21.2 indicatesthe characterof someofthe polysaccharides. In addition to the well-establishedpolysaccharide-containing pharmaceuticalmaterialsdescribedlater in this chapterthere is now considerable interest in a number of polysaccharideswith other pharmacologicalactivities.These include immuno-modulating,antitumour, anti-inflammatory,anticoagulant,hypoglycaemicand antiviral properties.Specificexamplesare the glycyrrhizansof Gl.1'cyrrlti:a uralensis and G. gkhra and the glycans of ginseng and Eleutherococurs(q.v.).In generalpolysaccharides from lungi exhibit antitumour activity, those from higher plants are immunostimulatory
6
I
H?O3POCH2^
HOCH I HCOH I
OH
t-l"\^
ffi'"-
HCOH I
cH2oH
O H H uFructose-6-phosphate (furanose ring)
Kctohexose (o-fructose)
_TTN{V* c.H2oH
cr-D-Glucosc (pyranosering)
P->Glucose
OH p-o-Glucose (anotherreprosentation)
F i g .2 l . l Hexose structuresond representotion
cHo
cHo F-oH
cHo Hoi
l-or OH
t -or
-
o-Ribose (pentose)
cHo
Fo'
io* cH2oH
fon
l
f--oH
D-Xylose (pentose)
o-Ribulose (pentose)
H O--l
-i
HO
CHg
Hamamelose r-Rhamnose pentose) (hydroxymethylribose) (methyl
fo' -ot cH2oH o-Mannose (hexose)
D-Xylulose (penrose)
CHeOH I -
cHo
HO
HO
Fig.21,2 Exomples of Cato Cz monosocchorides structures). {Fischer
l-oH
I cH2oH
cHo -.1
cHo
uon,c-f oH
Ho--] I -ot cH2oH
r-oH
cH2oH
o-Arabinose (pentose)
n-n
I
l -ot
cH2oH
CH,OH t -
CH,OH l -
Ho--]
l-o*
t l-on
cH2oH D-Erythrose (tetrose)
cHo lot
HO
l-o'
lI
HO
l-ot
CH2OH o-Galactose (hexose)
Ho---] I -I o H
l-ot -ot
cH20H o-Sedoheptulose (heptose)
CARBOHYDRATES Toble 2l. I
Typ" Di-
Telro'
Some di-, tri- ond ietrosocchorides. Nome
Productsof hydrolysis
Occurrence
Sucrose Glucose,fructose Sugorcone,sugorbeet,efc. Moltose Glucose,glucose Eniymichydrofisisof ,rorch Loctose Glucose,goloctose Mil[ Cellobiose Glucose,glucose Enzymicbreokdownof cellulose Treholose Glucose,glucose Ergot,Rhodophy."o",y"oro Sophorose Glucose,glucose SJphoroiaplnico,6vil.fvri, of srevioside Primeverose Glucose,xylose Fitipendu'to'ulmrrr. fiyarjyri, of spiroein Gentionose Glucose,glucose,fruclose Genfionospp. Melezitose Glucose,fructose,glucose Monno from lorx Plonteose Glucose,fruclose,goloctose Seedsof psylliun spp. Roffinose Goloctose,glucoselfrucfose Mony seeds(e.g.cotton_seed) Monneotriose Goloctose,goloctose,glucose Monno of orh, Fror.irrcornm Rhomninose Rhomnose, rhomnose, goloctose Rhomnusinfectorio Sciliotriose Rhomnose, glucose,glucose Glycosideof squill .. other exomplesof irisocchorides ore omongthe glycosidesol DigitolisondstropAonthus (q.v.) Stochyose or monneoietrose Goloctose,goloctose,glucose,fiuctose Tub-ers'of'Stochys ioponicoondmonnoof rroxrnuSornus Olher exomplesof tekosocchorides ore omongthe glycosidesof Drgilol,s{q.v.)
Uridinc diphosphate glucose (UDp-glucose)
pouredin to form a layerbelow the aqueoussolution.With an insol_ uble carbohydratesuch as cotton-wool (celluiose)the colour will not appearuntil the acid layer is shakento bring it in contactwith phosphstc synlhase I Sucrose the material. (ECz.l.t.tl) [ 3. OsaT.one formation. Osazonesare sugarderivativesfbrmed by heat_ ing a sugar solution with phenylhydrazinehydrochloride,sodium SucroseGphosphate acetateand aceticacid.If the yellow crystalswhich form areexam_ ined under the microscopethey are sutTicientlycharacteristicfor I phosphaore(EC 3.1.3.2a) lSrcrosc certain sugarsto be identified. It should be noted that glucoseand t fructoseform the sameosazone(glucosazone.m.p. 205oC).Before melting points are taken,osazonesshouldbe purified by recrystal_ lization from alcohol.Sucrosedoesnot form an osazone.but under the conditionsof the abovetest sufficienthydrolysistakesplacefbr Fig. 2 | .3 the productionof glucosazone. Biosynthesis of sucrose. 4. Resorcinoltestfor ketones.This is known as Selivanoff's test. A crystal of resorcinol is added to the soiution and warmed on a water-bathwith an equalvolume of concentratedhydrochloricacid. and the algal polysaccharides, which often contain sulphate,are good A rosecolour is producedif a ketoneis present(e.g.fructose,honey anticoagulants. or hydrolysedinulin). 5. Test.forpenloses.Heat a solutionof the substancein a test-tubewith Testsfor corbohydrotes an equal volume ofhydrochloric acid containinga little phloroglu_ The following are some of the more useful testsfor sugarsand other cinol. Formationof a red colour indicatespentoses. carbohydrates. 6. Keller-Kiliani testfor deontszgars.Deoxysugarsare found in car_ 7. Reductiond Fehling's solution. To a heatedsolution of the sub_ diac glycosides such as those of Digitalis and Strophantlra.sspp. stanceadd drop by drop a mixture of equal partsof Fehling,ssolu_ (seeChapter24).The sugaris dissolvedin aceticacid containinga tion No. I and No. 2. In certaincasesreductiontakesplacenearthe trace of ferric chloride and transferred to the surface of concen_ boiling point and is shown by a brick-red precipitateof cuprous tratedsulphuricacid.At thejunction ofthe liquids a reddish_brown oxide. Reducing sugarsinclude all monosaccharides, many disaccolour is producedwhich graduallybecomesblue. charides(e.g. lactose,maltose,cellobioseand gentiobiose).Non_ 1. Enzyme reactions.Since certain carbohydratereactionsare only reducing substancesinclude some disaccharides(sucrose and brought about by certain specific enzymes,such enzymesmay be trehalose,the latter a sugarfound in somefungi) and polysacchar_ usedfor identification. ides. Non-reducing carbohydrateswill on boiling with acids be 8. Chromntograpfty.Chromatographic methodsare particularly suitedto convertedinto reducing sugars,but studentsare remindedto neu_ the examination of drug extracts, which may contain a number of tralize any acid used for hydrolysis befbre testing with Fehling's carbohydratesoften in very small amounts.Not only are they applica_ solution,or cuprousoxide will fail to precipitate. ble to carbohydratesoriginally present in the plant, but also 2. Molisch's te.st.All carbohydratesgive a purple colour when treated they may be used to study the products of hydrolysis of polysaccha_ with o-naphthol and concentratedsulphuric acid. With a soluble ride complexessuchasgums and mucilages.As standardsfor compar_ carbohydratethis appearsas a ring if the sulphuric acid is gently ison many pure sugars,uronic acids and other sugar derivatives are
Fructose Gphosnnate !
N uop I
l\- n su.L."
ORIGIN OF BIOLOGICAL DRUGS AND RELATED PHARMACOPOEIAL
Tqble 2I.2
The 21.0%)(mainly solublein dilute hydrochloric acid) and loss sacor crop' The nectar''which consistslargell' of suclose'is n-rixedwith the swelling index ({ 6.0) and salivary secretioncontaining the enzyme invertaseand u'hile in the on drying (* | 5.0%). Other standardsare (sodium titration)' (0.03 thiosulphate 0.2%) bee content at the hive the iodine total On arrival honey-sacis hydrolysedinto invert sugar. has, in the past, been and propenies has thyroactive prevla in Bladderwrack them deposits and the honey-sac of brings back the contents with a more preparations other employedin iodine therapyl however ousll preprretluell of the honcycotnb from its arising preferred. Again now are content heather' iodine consistent The besthoney is that derivedfiom f-lowelssuchasclover and as a slimming in teas promoted been has bladderwrack cut fiom the content, iodine and separated obtainedfrom hives that havenever swarmed. agent.In suitabledosesit servesas a dietary supplementfor traceelecornbeitherby drainingor by meansof a centrifuge.Honey obtainedby with the wax.The nectarof certain mentsand as a bulk laxative. expressionis liableto be contatninated may give the honey a flowers (e.g.of speciesof Eucabptusor Bcrnksia) Dutunt slt.tt]lottilult, from nectar Odourand taste; somewlratUnpleasant CETRARIA poisonous honey' give to known spp. are ragwofis andRhododendl?/r is a foliaCetraliaor Iceland moss,Cetruria islandica(Parmeliaceae). Siberia Europe, grass in central and moss growing amidst Characters. Honey, when tieshly prepared,is a c1ear,syrLrpyliquid ceouslichen central of slopes mountain lower the on and to crysAmerica. it tends North and keeping. of a pale yellow or reddish-browncolour.On collected in it is usually purposes medicinal For Spain. depend and and taste Europe odour The granular. and opaque tallize lnd become l u to P e . S c r n d i n a v iaan de e n t r r E vcrv lar'-telyon the llowers usedin its preparation The BP/EP drug consistsof irregularly lobed, leafy thalli' about 5-10 crn long and about 0.5 mm thick. The upper surfaceis greenishConstituents. Honey consistsmainly of invert sugar and water' It oii, volatile anclsometimescovered with reddish points, while the lower brown acid, fbrmic dextrin, of sucrose. quantities containssmall markedwith white. irreguwax and pollcn grains.Micloscopical examinationof the latter atlbrd surfaceis pale brown or greyish-greenand margin of the thallus are the along intervals frequent At The most likely adulterantsale artiti- lar spots. valuableer idcnceof the sour.ce. but becomescartilaginous is brittle drug The dried tot' plojections. The tests glucose. rninute liquid cial invcrt sLlgar.sucroseand commercial mucilaginousand bitter' taste, slightl Odour. water. with tor The limit tests moistening on be noted. should purified honey purity of the BP is stainedblue by which cooling. chloride and sulphateare important, as starch and sucrosemay he A 5clcdecoction tbms a ielly on swelling value of has an offlcial It (distinction carrageen). fi'om hydrolysedwith acidsto give commercialliquid glucoseand artilicial iodine drug reveal the of the drug. Sections powdered on the furfiLral, determinecl contains 4.5 sugar invert sugar. respectively.Artificial invert alga unicellular cells ofthe rounded plant. sn-rall the but this ofthe acid, nature clual hydrochlolic in resorcinol r.r'ith red colour which gives a closely or less rlore the by hunticolctbeing enclosed C1'stococ'cLts may be formed in genuinehoney b.vprolongedheatingor lengthystorwoven hyphaeof the tungus. age.
CARBOHYDRATES The drug containscarbohydrates, known as licheninand isolichenin. Licheninis only solublein hot waterand is nor colouredblue by iodine, while isolicheninis solublein cold water and gives a blue colour with iodine. Both on hydrolysisgive glucose.Cetrariaalso containsa very bitter depsidone,cetraricacid, and other acidssuch as lichestearicand usnic acids.The latter compound is an antibiotic which may not be unimportantin contributingto the efficacyof Icelandmossas a demulcentfbr the treatmentof cough involving throatirritation.Icelandmoss has beenusedas a bitter tonic and for disguisingthe tasteof nauseous medicines.
Prunes Prunes(Rosaceae)are dried plums derived from prunus domestir:u L. Of the many cultivated forms, yar. Juliana de Candolle, which is
largely -urown in France. produces those commonll, uscd in Europeanmedicine.Prunesof excellentquality are alsoproducedin Calilbrnia. The appearance ofprunes requiresno description.The pulp contains about4'1%of suglrs (rnainly glucose),malic acid and water.The kernel contains-1591of flxed oil. and small quantitiesof amygclalinancl benzoicacid. Prunesareusedin Confectionof Senna. Further reoding Weymouth-Wilson A C I 997Theroleof carbohydrates in biologically active naturalproducls. NalurrlProductReports1.1(2): 99-l l0 Whistler R L, BeMillerJ N (edsr I 991Indusrriai gums:polysaccharides and theirderivatives. 3rdedn.Acadentic Press. London, UK WhistlerR L. BeMillcrJ N 1997Carbohvdrate chcr.nistry tbr foodscientists. EaganPress. StPaul.Minncsota
.,
ait.
Phenols probabli, constitute the lalgest gloup of plant secondarr rnetabolites.Widespreadin Nature.and to be for-urdin most classcso1' ruaturalcor.r'rpourrds having aromaticrnoieties.they lange tiorn simplc structureswith one aronatic ring to highly complexpolymericsubstancessuchastanninsand lignins.Phenolsareimpoltantconstituents of some medicinal plants and in the fbod industr-vthey are utilized as colouringagents.flavourings.aromatizelsand antioxidants.This chapter mainlydealswith thosephenolicclasses of pharmaceutical interest. (2) tannins.(3) coumalins namely:(l) simple phenoliccompouncls, (.1)anlhraquinones (-5)naphandtheil gll,cosides, ancitheirglycctsides, (6) flavoncand lciatcdflavonoidglycosides.(7) anthothoquinones. (8) lignansand lignin. The biosynthetic cyanidinsand anthocyanins, origin of someof thesecompoundsinvolr,ingthe shikimicacid pathr v a v i s s h o n ' ni n F i g . 2 2 . l . P h e n o l sr n a y a l s o h a v e a r o m r t i er i n g s (Fi-q.19.9.). derivedby acetateconderrsation
Phenols ond phenolic Alycosides
SIMPLEPHENOLICCOMPOUNDS Catechol (o-dihydroxybenzene) occurs ftee in kola seedsancl in thc leaveso1'Carrltheriaspp.and its deri'n'ativcs arc the urushiolphenolso1' the poison oak ancl poison ivv (q.v.). Dcrivativcs of rcsorcinol (rn constitutcthc nalcoticprinciplcsofcannabisandthc dihydroxybenzene) glucosidearbr"rtin involr,csquinol (hl,droquinone.p-dihydroxybenzene). The taenicidalconstitucntsof malef'ern.the bitterprinciplesof hopsancl thc lipophilic components ol hypelicurn (q.r'.) are phloro-elr.rcinol derivatives.
ol{
OH I a/Y-" I
OH
I
d'
50); E' frondboses(x1);C, entireromentum; sectionof rhizomeond surrounding Mole fern.A, Whole drug (r0.5);B, tronsverse g't' o, storchgronules; filixioemino. Athyrium of bose leofy oio section (x65); F, tronsverse glonds sectionthroughrhizomerr,.*i.g'i.i.r."llulor m, meristele. glondulortrichome;
PHENOTA SND PHENOLIC GLYCOSIDES
HrC
HOYYOH l
-
r
HrcQ
CH.
xo-))rru$lsopipcritonol
(-llsopipedtenore
(-|cir-lcopubgone
I
I
A
A
+"-q"
r"' r"
(+|Ncoisomenlhol
(+)-PulogoD€
+)rlsom€ohone
II
I
I
I
monoterpenoids ol Mentho x piperito.
V"ox
X..OH (+)"lsomcnlhol
I
I
I
^
Fig.23.3 A possible biogenetic scheme forsome
Mathofunn
i
(+)-Neomontbol
t
\ i
o
(-FMenthone
\
l o i
H
(-!Menthol
V O T A T I LOEI L SA N D R E S I N S later in the seasonand is absentliorn plantletsproducedby shoot-tip is influencedby the age cuiture.Again like pepperrrint.oil prodr.rction of plant,time of collection,chemicalvarietiesand hybridization.
SAGEIEAF
reported in 1989 have deodorantpropertics.7-. .rrr7l11lrontuild thyme),inclr.rded inLheBHP. is not admittedb1'theBP and is detected long trichomesat the baseof leavesu'hichareri eaklr by chalacteristic pubescentin otherparts.The volatile oil of T. serprlliu//r conti,rius mure linalool and7r-cymolthan do the official species.
The ofTicialdrug consistsof whole or cut leavesof Salvia o.ffic'inuLis (Labiatae)containingnot lessrhan 1.5%(whole leaf) or 1.jc/c(cut leaf) of essentialoil which is determinedby steamdistillation.The plant is indigenousto Mediterraneanareasbut is now cultivated world-wide. principally for its use as a culinary herb.
Thyme oil BP. Thyrne oil BP is obtainedby stean.r distillation fl'onr the fresh aerialpartsand containsthymol 36-557c,carvacrol | c/r.pcymene 15-287c, lterpinene 5- I 0olctogetherwith linalol. B-rny rcene ancl terpinen-2l-ol as dctermined by gas chromatography. Spain accountsfor some90% of the world oroductionof thvme oil.
Macroscopical characters. The petiolateoblong-lanceolateleaves are up to 10 cm length and 2 cm in breadth,greenish-greyon the upper surfaceand tomentoseon the lower with a markedly reticulatevenation. The leaf apexis lounded,the baseroundedor cordateandthe margin crenulate.The odour and tasteare characteristicallypungent. Microscopical characters. The upper epidermalcells have beaded anticlinal walls, the lower onesare thin-walled and sinuous;both epidelmi possessdiacytic stomata.Glandular trichomes of the typical labiatetype occur on both surtaceswith rarer uniseriateglandulartrichomeshaving a double-or single-celledhead.Clothing trichomesare numerous! particularly on the lower surface, composed ol a short thickenedbasalcell with articulatedand bent terminalcells.A few single-celledwarty-walledtrichomesare present.The long protectivetrichomes serve to distinguish S. officinalis liom S. sclareu and S. pratensis(M. Then et aL.,Acta Pharm.Hung.,1998,63,163). Constituents. The volatile oil of sagecontainsabout 50% of a- and B(Fig. 23.2). thujonetogetherwith cineole,bomeol and otherconstituents Varietiesand other speciesof sagecontain differing amountsof thujone. Non-volatile componentsof the leaf include diterpenes,phenolic glycosidesbased on caffeic and p-hydroxybenzoicacids (fbr recent isolationsseeM. Wanget aL..J. Nat. Prod., 1999,62,454),and tannins. Action and uses. Sageas an infusion is usedas a nouthwash and garg1efor its antisepticand astringentaction. Recent attention has fbcused on the cholinergic activity of the drug and its possiblerole in the treatmentofAlzheimer'sdiseaseandmemoryloss.It is interestingto notethat long before recentadvancesin the understandingof the neurobiolo-eyof Alzheimer'sdisease,plant materialsincluding sageand balm (Mellssa oJficinalis'\ were recornmendedin old ref'erencebooks as possessing memory-improving properties (see E. K. Perry et ul., J. Phann. Pharmttcol.,1999,51, 527). The phenolicglycosidesof sagetogether with those of MeLissaolJtcinalis and Lot,tuttlulu Llngustifoliu possess antioxidantproperlies(J. Hohmannet al., PlantaMedica,1999,65.576).
THYMEAND THYMEOIt Thymus v'uLgarisand f. l.r.gisor mixtures of both are otficial in the EP and BP. The drug consistsof whole leaves and f-lowerswith stems exceedinga specifiedsize limited b 10"/".The minimum requirement fbr volatile oil contentis 1.27av/w with a phenolvalue of not lessthan 0.57cexpressedas thymol and calculatedwith referenceto the anhydrous dr-r.rg. The phenolsin the isolatedoil are determinedby reaction with aminopyrazoloneand potassium ferricyanide in ammoniacal solutionwith subsequentmeasurementof absorbenceat 450 nm. 'thymol' 'carvacrol' A numberof chemicalraces,e.g. and types are known and it is thesephenolsthat are held largely responsiblefor the antiseptic,antitussiveand expectorantpropertiesof thyme. The spasmolytic effect may be due to the flavonoids of the leaves.Biphenyls
LAVENDER OIt After some 20 years lavenderoil as a result of its EP statusis again included in the BP. The sourceis Lat'rtntlulu ungLtstit'bliuMlller (L. ofticlnalls Chaix).Originally (BP 1980)oil from this specieswas referredto as 'foreign oil' to distinguishit flom that of L. intermediaLoisel.which 'Englishoil'. The latterhas a much flner fiagrancethan the was temred Continentaloil and therewere separatepharmacopoeialstandardsfor the two oils. Unfbrtunatelythe sffagglyhabit of the Englishlavenderdoes not lend itself to mechanicalharvestingand oil producedcommerciaily is now ofthe Continentaltype. France,oncethe principalproducer,has beensuperseded by Bulgaria,with smallerquantitiesof oil comingfrom the lbrmer USSR.Auslraliaand othercountries. Lavender oil types. The taxonomyofthe lavendersis confusingand Continentaloils difl'el amongtherrselvesowing to the fact that a number of diff-erentspecies.varieties and hybrids ale distilled. The true lavender,L. offi-
HOHpC
d
Fi1.27,2 S o m e o r n i t h i n e - d e r i v e do l k o l o i d s .
|
t*--Necine
----------- senecio alkaloids
ALKALOIDS Me
BIOGENESIS OF TROPANEAIKALOIDS
Nr)---l
As the charactelisticalkaloidsofthe groupareestersofhydroxytropanes and variolrsacids(tropic.tiglic. etc.)thereare.tbr cachalkaloid.two distinct biogeneticmoietieswhich warrantconsideration.Most studiesin Ph oco this connectit'rrr hal'e r-rtilizecl various speciest'f Datura because,fcrra numbero1'reasons. they ar"eoneof the mostcorl'enientof the Solanaceae I cHzoH cH2oH with rvhich to work. However.with the adventof isolatedroot culture technique s the stuclyol alkaloid fbnration in other generahas become Hyoscyamine, Alropine H y o s c i n e{ S c o p o l a m i n e } more eviclentand Japanese vn'orkers in particulalhavecmployedspecies of Hlo.r'rrzzrrrr.r ind Duboisiuwith considerable success. 'l'ropane enzymatic hydrolysis but the chemical treatment convefts it to thc moiety. The availableevidencesuggeststhat the forrnamore stablegeometricisomer.oscine). tiorr of the tropancring svstemis generallysimilar for all Solanaceae These thlee specitic alkaloids are confined to the Solanaceae.in studiedbr-rttherealc still apparentI'ariationsbetwecnspecies.farticuwhich sonre,10difl'erentesterbasesof the tropanet-vpchavenow beerr larly in the steleospecificincolpolationof sonreprecursors. discovered;they constitutean interestingchemotaxonomicstudyrvithin Early work with isotopcsindicatedthat ornithine and acetatewere the fanrily.Exarrplesof tropanolestersare-eivenin Table27.2.Dimeric precursolsof the troparrerruclcr-tsl later'.the incorporationof ornithine and trirneric tropanol ester alkaloids involving the dicarboxylic was shownto be stereospecific. H1'glinccan alsoserveasa prccursorof acidsmesaconicand ittrconicacidsiile tor.rndin Schi:.anthLrs. For recent the tropancring but is not novuconsiclered to lie on the principalpathisofationsfrorri S. ltorrigens see O. Muiioz and S. Cort6s. Phunn. way. The N-rnethyl group of the tropanesystemcan be suppliedby Biol.. 1998,36. 162.Other tropanebasesoccur in the Erythroxylaceae methionineanclcan be incorporatecl at a very early stageof biosynthe(see cocaine in coca leaves). Convolvulaceae.Dioscoreaceae. sis. as demonstratedby the intact incorporationof ly'-nethylornithine Rhizophoraceae. Crucif-erae and Euphorbiaceae. into hyoscineand hyoscyamineof l)utlo'tt trtetel .:.ndD. strunoritutt. Early involvementof thc N-methyl group was reinfbrcedby the isolation in l98l of natulall-voccurring6-N-nethl"lolnithinefrorn bellaclon*.CHs nl plants. Also supportingthe stereospeciticityof the olnithine ,cH, Hot /l incorporationu,asthe work of McGaw and Woolley (.Phvochenistry, | 982. 21. 26-53) which shou'edthat fbl D. neteloitleslhe C-2 of hygrine was specificallyincorporatedinto the C-3 of the tropine moiety of the tl isolatedalkaloid. Putrescine(the symmetricaldian.rinefbrmed b1' the o ^.\ \-H decarboxylationof ornithine)and its N-n'rethyldclivative also serveas l l precursols,u'hich.takenin conjunctionwith the stereospecific incorporation of ornithine.makesit difTicultto constrlrcta singlepathway for i l l tropancrin-t formatii'rn.Arrotherproblern involved the fbnnation of a o cHc derivative of hvgrine by the condensationreaction between the Nrnethyl-A1-pyrroliniumion and an trcetate-derived precursor.Fol recent Schizanthine Z- a tropandiol mesaconic aciddiester work on this aspectseeR. Duran-Patl'on(,/ ul., Pltytot'henrisar'. 2000. 53.117.A schemefbr thc biogenesis of the tropanentoiery.consistent Altogethelover 200 tropanealkaloidshave now been recorded. u'ith the abovefindings.is shor.vnin Fig. 27.3. (Buscopan). Semisl'nthetic delivatives. e.g.hyoscinebLrtylbrornide are Studieson the enzymc putrescineN-rrethyltransf'elase in cultr.u'ed of medicinalirnportance. loots of llr'osct untu.s ulbus sLrpportthe rolc of this enzynreas the f irst
r-> /
\
\
Y
? " ntc-l\y">l\
x Z \ \i
\
d
o>r\-\o
lable 27.2
Exomples of ester components of tropone olkoloids of the Solqnoceoe.
G e n e r o o f p h o r m o c e u t i c o li n t e r e s l
Atropo,Acnistus, Scopolio,Physochloino, Przewolskio, Hyoscyomus, Physolis, Mandragoro,Doturo, Solondro.Duboisio.Anthocercis
T r o p o n o l c o m p o n e n l so f e s t e r s
N-R'
N-CH
OH
OH Rl=HorCH: R2=HorOH R3=HorOH (Tropine:Rl = CHr, R2=R3=H) Esterifying ocids
*.tF!h^..,.
Scopine:Rl = CHr Norscopine: Rt = H (Esterifiedwith tropic or atropicacid only)
H q-Tropine (Esterifiedwith tiglic acidonly)
Acetic,propionic,isobutyric, isovoleric, 2-methylbutyric, tiglic,nononoic,tropic,otropic,2-hydroxy3-phenylpropionic, 2,3-dihydroxy-2-phenylproponoic, p- methoxyphenylocetic, onisic
@
P H A R M A C O P O E IA N L D R E L A T EDDR U G SO F B I O L O G I C AOL R I G I N
onic acid(phenyllacticacid)of thc tropanealkaloidlittorine.The specitic incorporationsobtainedwith phenylalanineare given in Fi-1. of the side-chainin involvedin the rearrangement 21.4.The sequencc the conversionofphenylalanineto tropic acidhas beenthe subjectot' longstanding debate. Rccently. Ansarin and Wooller (Pht'rothentisrn'.199,1.35. 935). by f'eedingphenyl f l.-jlrC2llaetie acid to D. struntoniuntanelexnnriningthe llC-NMR spectraof thrsubsequently isolatedhyoscineand hyoscyarrine.have substantiatecl the hypothesisthat tlopic acid is formed by an intramolecular Furthcrmore.it hasbeendernonstratrearangementof phenyllactate. fiorr littorine by a process ed that hyoscyamineis biosynthesized involving the intramolecularrearan-eementof the phcnyllactatemoiety of the alkaloid(R. J. Robinsat ul.,J. Chem.Sot. PerkinTruts l. : . A n s a r i na n d J . G . W o o l l e y .J . C h e m .S o c . 1 9 9 4 .6 1 5 : 1 9 9 5 . ' 1 8 1M Perk. Trutts1. I 995. 487). I n concordancewith this. transformedroots u'ill convert exogenouslyadded littorine to of Daturu strtu'rtonit.urt hyoscyamine(357crnetabolismrecolded)but, in contrast,exogcltis not rnetabolized to littorine(I. Zabetakis oLrslyaddcdhyoscyanrine et al., Pltutt Cell.Rep.,1998,18, 3,11).The abovepathwayhast'ecentworkers (R. Duran-Palron et al.. Esterification. The next stagein the biosynthcsisofhyoscyamine,the ly been confilmed by other l1 1 Phrtochen i stn, 2000. 53. ). by esterilicationof tropine and tropic acid. has been demonstrated lsoleucineservesas a precursorof the tigloyl and 2-methylbutanoyl feeding experimentsand isoiatedenzyntes.It was over 35 years ago moieties of varior.rsrrono- and di-estersof the hydroxytropanes. of a hyoscyrmineesterase thatKaqzkowskifirst recoldedthe presence more recentlyRobins at al. (.FEBSLett.. 1991,292, Biogenesisof hyoscine(scopolamine). Wolk initiatcd by Romeikein in D. stramctnirrT?; 293) demonstratedthe involvernent of two acetyl-CoA-dependent 1962 showedthat hyoscineappealedto be fbnned in the leavesof D. acyltransferasesin the respective forn'ration of 39.-and 3B-ace- .feror frorn hyoscyaminevia 6-hydrox,vhyoscyamine and 6.7-dehydroroot cultures. toxytropanesin D. .stramortii,rt-transfoLmed as hyoscyamine.The fbrmer intermediatehas been well substantiated, Acid moiety. The tropic acid iiagrnent of hyoscineand hyoscyamine indicatedbelow,and occursin quantityin someother genera(Scopoliu. Przev'ttlskia)but the latter, although incorporatedinto is derived from phenylalanine,as is the u-hydroxy-B-phenylpropi- Phl'soc'hluina.
committed enzyme specific to the biosynthesisof tropanealkaloids. (N. Hibi er al.. Plant Physiol.,1992,100. 826.) It will be observedfrom Fig. 27.3thatthe reductionof tropinoneyielcls both tropine (3c-hydroxytropane)and pscuclotropine(38-hydroxl'tropane). These reductions are brought about by two independent (EC 1.I . I .236),oftenref'erredto asTR-l andTR-l I. tropinonereductases researchinvolrwhich acceptNADPH ascoenzyme.Alter considerable ing principally D. .strunoniumroot culturesboth enzynreshave nor.vbeen (A. Portstefl-en et ol., Plntotltern., separatelypurilied and characterized 1994,37,391). Furtherrnorc.cDNA clorrescoding fbr the two sepai'ate enzymesTR-l and TR-II have been isolatcd and shown to involve polypeptides containing 212 and 260 amino acids respectively (K. Nakajimaet al.. Pntc. Natl Acud. Scl. USA. 1993.90. 9591).These clones were expressedin Escherit:hiut'oli anclthe samereductive specificity demonstratedasfor the naturalTRs isolatedfiom plant rnaterial. alkaloids.hydroxyls and As indicatedin Table 27.2 for solanaceous estergroupsarealsocommonat C-6 andC-7 (Rl anclR3) of the tropane ring system.Cunent evidencesuggeststhat hydloxylationof thesecarbonsprobablyoccursafterthe C-3 hydroxyl hasbeenestelified.
cooH
cooH
D*t, \
l\4ethylation D*n, \
Decarboxylation NHCH3
NHCH3
NHz
N-methylputrescine
6-N-methylornithine
Ornithine
I I
oxioation
I /
COSCoA
\
f-\
z
.-
x
Acetyt-coA
|,-CHz/ N - C H 3 / C : O NHCH3
N-Methyl-A'-pyrrolinium sall
Fig.27.3 P o s s i b l eb i o g e n e t i cr o u l e sf o r t r o p i n e o n d p s e u d o t r o p i n e( s e et e x t f o r o d d i t i o n o lc o m m e n t s ) .
Eo Tropinone
Reduction F.-)
"t
Tropine(and'li-tropine)
4-Methylaminobutanal
ALKALOIDS
Doturo stromonium
i l l Y + l H O H,C-"'C-H
P h e n y l a l a nn ie
I .cooH
'CH, II H-C-OH
I
.cooH o - H y d r o x y - p - p h e n y l p r o p i o n i ca c i d
Tropic acid
Fig.27.4 Demonstroted incorporotions of phenylolonine intothetropicocidondcr-hydroxy-B-phenylpropionic ocid(phenylloctic ocid)moieties of hyoscyomine o n d l i t t o r i n e ,r e s p e c t i v e l yF. o r m o r e r e c e n tw o r k c o n c e r n i n gt h e i n l o c t i n c o r p o r o t i o no f ( R ) - ( + ) - 3 - p h e n y l l o cot icci d i n t o t h e h o p o y l m o i e f i e so f t r o p o n e o l k o l o i d s s e e r e f e r e n c e si n t e x t .
hyoscinewhen fed asa precursorb D. ferox,has neverbeenisoiatedfrom normal plants. Some 25 years later Hashimoto's group, using Hyoscyamus niger cultured roots, isolated and pafiially purified the enzyme responsiblefor the conversion of hyoscyamine to 6-hydroxyhyoscyamine.They used this enzyme to prepare 1$-taOl-hydroxyhyoscyamine from hyoscyamine and showed that when the labelled compound, fedto Duboisia myoporoides,was convefied to hyoscinethe l80 was retained, thus eliminating 6,7-dehydrohyoscyaminefiom the pathway (Fig. 27.5). For this reaction to proceedthe epoxidaseenzyme requires2-oxo-glutarate,fenous ions and ascorbateascofactors,together with molecular oxygen (fbr full details see T. Hashimoto et al., Plant Physiol., I 987, 84, 144;Phyrochernisrry,1989,28, 1077). The elucidation of the above pathway, which has spannedmany years,aptly illustratesthe value of biotechnologyand enzymologyin contributingto the resolutionof someuncertaintiesresultingfrom traditionallabelled-precursor experiments. Ontogenesis. In some plants of the Solanaceae(e.g. belladonnaand scopolia) hyoscyamineis the dominant alkaloid throughout the life cycle of the plant. In D. stramoniumhyoscyamineis the principal alkaloid at the time of flowering and after, whereas young plants contain principally hyoscine;in many other speciesof Datura (e.g. D. ferox)
H rbt-c.-:
Hr?-B.-i |
I
,N-M" i
l-lrc-fi-i
i
,N-M"i
HOC-C-: H H
i
6-Hydroxyhyoscyamine
Hyoscyamine
_-/ ->s
I
hyoscineis the principalalkaloid of the leavesat all times.The relative proportionsof hyoscine and hyoscyaminein a particular speciesnot only vary with ageof the plant,but alsoare susceptibleto other factors, including day length, light intensity. general climatic conditions, chemical sprays,hormones,debudding and chemical races. Isolated organ cultures of belladonna,stramonium and hyoscyamusindicate that the root is the principal site of alkaloid synthesis;however, secondary modifications of the alkaloids may occur in the aerial parts, lbr example,the epoxidationof hyoscyamineto give hyoscine. and the formation of meteloidinefrom the corresponding3,6-ditigloyl ester.
Further reoding Griffin W J, Lin G D 2000 Chemotaxonomy and geographical distribution of tropane alkaloids. Phytochemistry 53: 623-637 LounasmaaM, TamminenT, 1993Tropanealkaloids.In: Cordell G A (ed) The alkaloids. Chemistry and pharmacology.Vo1zl4.Academic Press,London. A review with 184 referenceslisting all known tropane alkaloids. Robins R J, Walton N J 1993The biosynthesisof tropanealkaloids.In: The alkaloids. Chemistry and pharmacology.A review with 191 reJerences. Woolley J G 1983Tropanealkaloids.In: WatermanP G (ed) Methods in plant biochemistry.Vol 8. Academic Press,London,p 133
STRAMONIUMLEAF Stramonium Leaf BP (Thornapple Leaves: Jimson or Jamestown Weefl consistsof the dried leavesor dried leavesand flowering tops of Datura stramoniumand its varieties(Solanaceae). The drug is required to containnot less than025% of alkaloidscalculatedas hyoscyamine. The plant is widespreadin both the Old and New Worlds. British supplies are derived mainly from the Continent (Germany, France, Hungary,etc.).
Plant. D. Stramoniumis a bushy annualattaininga height of about 1.5 m and having a whitish root and numerousrootlets.The erectaerii H H al stem shows dichasial branching with leaf adnation. The stem and Feedngof c-c-! branchesareround, smoothand green.The flowers (Fig. 27.6) aresoliPrecuraor tary, axillary and short-stalked.They have a sweet scent.Each has a HC-C-l tubular,five-toothedcalyx about 4.5 cm long, a white, funnel-shaped H H i H i corolla about 8 cm long, five stamensand a bicarpellaryovary. The 6,7-Dehydrohyoscyardne Hyoscine plant flowers in the summerand early autumn.The fruit is originally Fig.27.5 bilocular but as it maturesa false septumarises,exceptnear the apex, Route forfheformotion (portiol of hyoscine fromhyoscyomine lormuloe] so that the maturefruit is almostcompletelyfour-celled.The ripe fruit
Hc_g_i
ll)'-*i
I
dl 'N-u"i t-c'-l
@
AL RIGIN S FB I O L O G I CO P H A R M A C O P O EAIN AD L R T I A T EDDR U G O
Fig.27,6 (theorrow B,corollo cutopenC, pistil(therestof theflowerhosbeencutowoy);D,floroldiogrom shoot; A, Endof flowering DJturo stromonium. i n d i c o t e st h e p l o n e o f s y m m e t r y ) ;E , c o p s u l eo p e n i n g ; F , c o p s u l e i n t r o n s v e r s es e c t i o n ;G , s e e d i n t r o n s v e r s es e c t i o n ,s h o w i n g c u r v e d e m b r y o . A l l s l i g h t l yr e d u c e d e x c e p t G , w h i c h i s e n l o r g e d . ( F r o mR e n d l e ' sC l o s s i f t c a f i o no f F l o w e r i n g P l o n t s . )
seeds(Fig. 27.6G) is a thornycapsuleabout3 z1crn long.Stt'amonium in or-rtline and about 3 reniform blackish in colour. are dark brown or mm long. The testais reticulatedand finely pitted.A coiled embryo is embeddedin an oily endospernr. var.tatula closely resemblesthe abo\e: it\ stenlsxre D. stram.oniurn reddishand the leaveshave purplish veins. as also have the lavender'colouredcoroilas.Varietiesof both the abovetblms occur with spinelesscapsules.
tened.longitudinallywrinkled. somewhathairy and vary in colour fronr light olive brown (D. stamoniurl) to purplish-brown(var. tut' a/a). Stramoniumhas a slight but unpleasantodour, and a bitter taste.
Microscopical characters. A transversesectionof a leaf (Fig. 27.7t showsthat it has a bitacial sffucture.Both surlacesare coveredwith a smoothcuticle and possessboth stomataand hairs.Cluster crystalsol' calcium oxalate are abundantin the mesophyll (Fig. 27.7F, G), and the microsphcnoidaland prismaticcrystalsare alsofound. The stomataarLhy to*'alds grown in England Gerarde was History. Stramonium end of the sixteenthcenturyfiom seedsobtainedfiom Conslantinople. of the anisocyticand anomocytictypes.The epidermalcells havewar'1 The genericnarne.Datura. is derivedfront the nanteof the poison.riftrit. walls, particularlythoseof the lower epidermis.The uniseriateclothing hairs are three- to five-celled, slightly curved. and have thin. wartl which is preparcdfiom Indianspeciesand was usedby the Thugs. wal1s(Fig. 21.78). The basal cell is usually more than 50pm long Macroscopical characters. Fresh stramonittmleavesor herbarium (distinctionfrom D. rzclel). Small glandularhairs with a one- or twospecimensshould tirst be examined.since the commercialleavesare celledpediceland otherswith a two-celledpediceland an oval headof two to sevencells arealsofound. If poltions of the leaf areclearedwith much shrunkenand twisted.and their shapccan only be ascertainedby aftet'soakingthem in water. chloral hydrate solution. the abundanceof the cluster crystals ot carefuln-ranipulation The dlied leaves are greyish-greenin colour. thin, brittle. twisted calcium oxalateand their distt'ibutionwith regardto the veins may be and ofien broken.Whole leaves(Fig. 27.6) are 8-25 cm long and 7-I-5 noted. The midrib showsa bicollateralstluctureand characteristicsubepicm widel they are shortly petiolate.o\rateor triangular-ovatein shape. nlargin.They are dermal mzrsses of collenchymaon both surfaces.The xylem forms a ale acuminateat the apex and have a sinuate-dentate stronglycurvedarc. Sclerenchymais absent. distinguishedfrom the leaves of the Indian species.D. innctxia.D. teeth dividing Stemsare present,but few of theseshould exceed5 mm diameter. metel and D. .fustuostt.by the margin. u'hich possesses They possessepiderrnal hairs up to 800 pm long and have perithe sinuses.and by the lateralveins which run into the marginalteeth. The commercialdrug containsoccasionalflowers and young cap- medullary phloern. The stem parenchymacontains calcium oxalate sules. which have been desclibed above. The stems al'e ofien f-lat- sirnilarto that fbund in the leaf.
ALKALOIDS UEP
-,\,
c J
.j;,: i
. \
J.i^" '"-J
':if:!*
Fig.27.7 Doturostromonium leof.A, Tronsverse sections of midrib(x15);B, tronsverse sectionportionof lomino;C, lowerepidermis with stomotoond glondulortrichome; D, glondulorhichomeovervein;E,clothinghichomes{ollx200}; F,orrongement of colciumoxololecrystols in crystolloyer, surfoceview (x50);G, colciumoxolotecrystols in cells;H, upperepidermis showingcicotrixond stomoto(G ond H, x20O).l, J, Sconning electron microgrophs of {l)clothingtrichome ond (J)glondulortrichome. c, Collenchymo; cic,cicotrix;c.l, crystolloyer;e, endodermis; id, idioblostcontoining micro-crystols; i.ph,introxylory phloem;l.ep,lowerepidermis with stomo;m, mesophyll; ox, colciumoxolotecrystol;p, polisodeloyer;ph, pntoem; u.ep,upperepidermis with stomo;vt, veinlel;xy, xylem.(Phologrophs: L.Seedond R.Worsley.) Constituents. Stramoniumusually contains0.2-0.45c/cof alkaloids. the chief of r."'hichare hyoscyamineand hyoscine,but a little atropine may be formed from the hyoscyarr-rine by racemization.At the time of collectionthesealkaloidsare usually presentin the ploportionof about two pafis of hyoscyamineto one part of hyoscine,but in young plants hyoscineis the predominantalkaloid.The TLC testfor identitygiven in the BP enablesolher Datura speciescontaining diff'erentproportions of alkaloidsto be detected.The largerstemscontainlittle alkaloid and the official drug should contain not more Iha'n37a stem with a diameter exceeding 5 mm. Stramonium seedscontain about O.2Vr:of mydriatic alkaloids and about | 5-307c of tixed oil. The roots contain.in addition to hyoscineandhyoscyamine,ditigloyl estersof 3.6-dihydroxytropane and 3,6.7{rihydroxytropane. respectively,and a higher propofiion of alkamines than the aerial portions. D. stramonituttcell and root cultureshave been considerablyutitized in biogeneticstudies(seeabove and M. G. Hilton and M. J. C. Rhodes.Plunta Med., 1993.59, 340).
thn
PreparedStranton.iunt BP is the finely powdereddrug adjustedto an alkaloidcontentof 0.23-0.21Vo. Alfied species. All Dlturo speciesexaminedto date contain those alkaloids fbund in stramonium.but fi'equentlyhyoscine.rather than hyoscyamine,is the principal alkaloid. A number of alkaloids not reported in D. stramoniunt have also been isolated from vadous species:these include apohyoscine.norhyoscine,norhyoscyamine. tigloidine. 3a-tigloyloxytropane,3o-acetoxytropane.hetero diesters of di- and trihydroxytropane,meteloidineand cuscohygrine. ' Commercial 'daturaleaf consistsof the dried leavesand flowering tops of D. innoxia and D. ntetel:it is obtainedprincipally from India. Like thoseof stramonium.the dried leavesare curled and twisted.but :Lreusually sonrewhatbrowner in colour.with entiremarginsand with difTerences in venationand trichomes.The leavescontain about 0.57c of alkaloids.Variationsin hvoscineand atropinecontentsin different
E
ORIGIN AND RELATED DRUGSOF BIOLOGICAL PHARMACOPOEIAT organs of D. metel during development have been studied (S. Af.sharypuoret trL..Plttnta Med.. 1995,61, 383). Over 30 alkaloids have beencharacterizedfiom D. inno.ria by capillary GLC-mass spectrometry.For studieson the anatomyof the leaf of D. meteL,seeY. C. Anozie, Int. J. Crude Drug Res.,1986,24,206;and for the isolationof see S. Siddiqut et al., J. Nat Prod., 1986,49, 3cx-anisoyloxytropane 'Datura seeds' are derived from D. metel and possibly other 511. species.Each seedis light brown in colour and ear-shaped.They are largerand more flattenedthan stramoniumseedsbut resemblethe latter in internal structure.The alkaloid content,hyoscinewith tracesof hyoscyamineand atropine,is about O.2Vc.D..feror, a specieshaving very large spines on its capsules,contains as its major alkaloids hyoscineand meteloidine. 'ffee-daturas'constituteSectionBrugmansiaof the genus;these The alboraceous,perennialspeciesare indigenousto South America and are widely cultivated as ornamentals.They produce large, white or coloured trumpet-shapedflowers and pendantunarmedfruits. Some speciesconstitutea potentialsourceof hyoscine(W. C. Evans,Pharm. in particular,hasproveda most J).,1990,244,651)andD. sanguinea, interestingplant with respectto its wide rangeof tropanealkaloidsand has been cultivated commercially in Ecuador.lt yields about 0.8% hyoscine. Plantationshave an economically useful lif'e of about l0 years. Chemical races of D. sanguineaare evident. particularly one producing relatively large amounts of 6B-acetoxy-34-tigloyloxytropane. Various tree datura hybrids developed at Nottingham University.UK, have been used by a number of workers for alkaloid studiesinvolving hairy root and root cultures; as an example see P. et al., Plant CeLlRep.,1998,17,405. Nussbaumer The South American Indianshave long cultivated various racesof theseplants for medicinal and psychotropicuse (for a comparisonof of their potencywith alkaloid content,seeBristol er native assessment al ., Lkt ,-dia, 1969, 32, 123). Withanolides (q.v.) are also found in some speciesof the genus.(For the fbnnulae of I 1 compoundsof the daturametelintype seeK. Shingu er ttl., Chem.Pharm.Bull., 1990,38. 2866.)Recentreportsfeaturetheisolation of a new pentahydroxywithanolidefrom D. fastuosa (M. Manickam et al., Phl'tctcherr.,1998, 47, 1421)and the 7-hydroxywithanolidesfrom D. ferox (A. S. Veleiro et al., J. Nat. Prod., 1999,62, 1010). A classicalwork of referenceon the geneticsof the genusis that of Averyet al., Blakeslee.The GenusDatura (1959);this was updatedby anotherpublication(No. I 2 in a seriesof Monographsin Developmental Biology) and had a wider scope:Conklin, Geneticsand BiochemicaLAspectsof the Developmentof Datura(1976).
Uses. Atropine has a stimulantaction on the centralnervouss\ \telr: and depressesthe nerve endings to the secretoryglands and plaLr: muscle. Hyoscine lacks the central stimulant action of atropine: rtsedativeproperliesenableit to be usedin the control of motion sick' ness.Hyoscinehydrobromideis employedin preoperativemedication. usually with papaveretum,some 30-60 min before the induction Atropine and hyoscine are used to a large extent in of anaesthesia. ophthalmicpracticeto dilate the pupil of the eye.
HYOSCYAMUSLEAF HyoscyamusLeaf (Henbane)consistsof the dried leavesot the dned leaves and flowering tops of Hyoscyatnusnlger (Solanaceae).It i. requiredto containnot lessthan 0.057cof total alkaloidscalculatedat hyoscyamine.The EP (BP) descriptionrefersto petiolateaswell as sessile leaves,the first-yearbiennialleavesbeing thus admitted.Henbane is no longercultivatedcommerciallyin Britain and suppliesareimported from central Europe. The plant is also cultivated in the USA.
Plant. Henbaneis a biennial(.var.a-biennis)or annual(var.B-anruutt plant.It is found wild, chiefly nearold buildings.both in the UK and in the restof Europe,and is widely cultivated.Beforeexaminingcommercial henbaneleavesit is advisableto studygrowing plantsor herbariunt specimens.The differencestabulatedin Table27.3 shouldbe noted. HenbaneJknver.shave the formula K(5), C(5), 45, G(2). The hair1. five-lobed calyx is persistent.The fruit is a small, two-celled pyxis (Fig. 42.68), which containsnumerousseeds. Henbaneseedsaredark grey in colour, somewhatreniform in shape and about 1.5mm long. They have a minutely reticulatedtestaand an internal structure closely resembling that of stramonium seeds. Henbaneseedscontain about 0.06-0.i07o of alkaloids (hvoscvamine with a little hyoscineand atropine). History. Henbane,probablythe ContinentalH. albus, was known ttr Dioskurides and was used by the ancients.Henbane was used in Englandduring the Middle Ages.After a period of disusein the eighteenth century the drug was restored tothe London Pharmacopoeia of 1809,Iargelyowing to the work of Stcjrck.
Collection and preparation. Biennial henbanewas the variety traditionally grown in England,but much of the imported drug is now of the annual variety or is derived from the allied speciesH. albus.The germination of henbaneseedsis slow and often erratic and may often be assistedby specialtreatments(e.g.concentratedsulphuricacid,gibberellic acid or splitting of the testa).The plant may be attackedby the Adulterants cited are the leaves of species of Adulteration. potato beetle, and spraying with denis or pyrethrum may be necessary. Xanthium (Compositae),Carthamus (Compositae)and Chenopoditrm The annualplant usually flowers in July or August and the biennial (Chenopodiaceae). which are, however,easily distinguishedfrom the in May or June.The leavesshouldbe dried rapidly,preferablyby artigenuinedrug. ficiai heat at a temDeratureof about 40-50"C.
Table 27.3
Comporison of commerciol vorieties of hyoscyomus.
First-yearbienniol
Second-yearbiennial
Annual
Stemvery short Leovesin o rosetteneorthe ground. Ovote-lonceolote ond petiolote, up to 30 cm long,thelomino beingup to 25 cm long.Hoiry Doesnot normollyflowerin the first yeor
Stembronchedond up lo 1.5 m high to triongulor-ovote. Leovessessile,ovote-oblong lO-20 cm long.Morgindeeplydentofe in the or pinnotifid. Veryhoiry,especiolly neighbourhood of themidribond veins FlowersMqy or June.Corolloyellowishwith deep purpleveins
Stemsimpleond obout0.5 m high Smollerthonthoseof the bienniol Leoves sessile, plont,with o lessincisedmorginond fewer hoirs Flowers Julyor August.Corollopolerin colour ond lessdeeplyveined
{
ALKALOIDS Macroscopical characters, Commelcial henbane consists of the leavesand flowering tops describedabove.The leavesaremore or less broken but are characterizedby their greyish-greencolour,very broad midrib and greathairiness.If not perf-ectlydry, they are clammy to the touch, owing to the secretionproduced by the glandular hairs. The stems are mostly less than 5 mm diameter and are also very hairy. The flowers arecompressedor brokenbut their yellowish corollaswith purple veins are often seenin the drug. Henbanehas a characteristic, heavy odour and a bitter, slightly acrid taste. Microscopical characters. A transversesection of a henbaneleaf shows a bifacial structure(Fig. 27.8A). Both surfaceshave a smooth cuticle.epidermalcellswith wavy walls. stomataof both anisocyticand anomocytictypes. and a large number of hairs. which are particularly abundanton the midrib and veins. The hairs are up to 500 pm long; some are uniseriateand two to six cells long, while others have a uniseriatestalk and a large,ovoid, glandularhead,the cuticle of which is often raisedby the secretion(Fig. 27.8E).Similar hairs are found on the stems.The spongymesophyllcontainscalcium oxalate,mainly in the form of single and twin prisms,but clustersand microsphenoidal crystalsare also present(Fig. 27.8B,D). The broad midrib containsa vascularbundle, distinctly broaderthan that of stramonium,showing the usualbicollateralarrangement, which is alsoto be seenin the stems. The mesophyllof the midrib is madeup of two thin zonesof collenchy-
ma immediatelywithin the epidermi and a ground massof colourless parenchymashowing large, intercellular air spacesand containing prismsor, occasionally,microsphenoidalcrystalsof calcium oxalate. The calyx possesses trichomesand stomata,as in the leaf.The corolla is glabrous on the inner surlace but exhibits trichomes on the outer' surface,particularly over the veins (Fig. 27.8G). Those cells of the corolla which contain bluish anthocyaninsturn red with chloral hydrate solution.Numerouspollen grains are present,about 50 pm diameter, tricolpate with three wide pores and an irregularly, finely pitted exine (Fig. 27.8F).The testaof the seedshas an epidermiswith lignified and wavy anticlinalwalls, and sclereidsarepresentin the pericarp. Constituents. Henbaneleavescontain about 0.045-0.14% of alkaloids and yield about 8-727a of acid-insolubleash (BP not more than l2clc).Hyoscyamineand hyoscinearethe principal alkaloids.The petiole appearsto containmore alkaloid than the lamina or stem. PreparedHyosclantusBP ts the drug in fine powderadjustedto con'loss tain 0.05-0.077cof total alkaloids.It has a on drying' requirement of not more lhan 5.07a. Allied drugs. H1,-oscl'amus albus rs grown on the Continent.particularly in France,and in the Indian subcontinent.It has petiolate stemleaves and the flowers have pale yellow, nonveined corollas. Quantitativelyand qualitativelyits alkaloidsappearsimilar to thoseof H. niger. It hasbeenusedin biogeneticstudies(q.v.)andthe hairy roots
i--c.l t
m
31."p
$tr. &
#& D
@
Fig.27.8 Hyoscyomus niger.A, Tronsverse sectionof midribof leof (x40);B, tronsverse sectionof portionof leoflomino;C, portionof leofupperepidermis, E, trichomes; F,pollengroins;G, portionof epidermis of corollowith ottochedglondulortrichome(oll surfoceview;D, colciumoxolotecrystols; x20O).b, Boseof trichome; g.t, glondulorhichomeor portionof; id, idioblost;i.ph, c, collenchymo; cic, cicotrix;c.l, crystolloyer;e, endodermis; phloem;l.ep,lowerepidermis; m, mesophyll; p, polisodeloyer;ph, phloem;st,slomo;tr1,tr2,wholeond brokenclothingtrichomes, introxylory respectively; vl, veinlet;xy, xylem. u.epiupperepidermis;
at;h*
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PHARMACOPOEIAL AND RELATED DRUGSOF BIOLOGICAL ORIGIN (transtbrmedwith Agrobacteriumrhizogenes)have beenanalysedfbr littorine.hyoscineandhyoscyamine 7B- and6B-hydroxyhyoscyamine. (M. Sauerweirr and K. Shimomura.Ph;'tochemistrt.1991,30.3217). ln traditionalmedicineof the Tr.rscan archipelagothe seedsarepressed into the cavities of decayedteeth to obtain pain relief (R. E. Uncini 181). ManganelliandP.E. Tornei,./.EthnopluLrmutologl',1999,65, H1'oscyamus muticus is indigenousto India and Upper Egypt; it has beenintroducedinto Algiers. For further details,seebelow. Indian henbane.Under this name considerablequantitiesof drug into Britain during World War Il. Although H. niger is were irr-rported grown in hidia and Pakistan,much of the drug came liotn a closely hyoscineand hyoscyamine relatedplant. H. reticultttus.This cor-rtains and microscopicallyit is alr-nostidenticalro H. niger. HyoscyarnusaureusandH. pusillus aretwo specieswhich produced hyoscineas the principal alkaloid.
HERB BETLADONNA BelladonnaHerb BP (.Belladutnalerfl consistsof the dried leavesand tlowering tops of Atropa belladonna (Solanaceae);it contains not less than 0.307coftotal alkaloidscalculatedashyoscyamine.Traditionally the BP drug has consistedof all the aerial parts(BelladonnaHerb) but under thereis a limit (37c)of stemwith a diameter the Eulopeanrequirements exceeding5 mm. The USP.which requires0.35%alkaloid,alsoadmitsA. rt.t:uminatt(seebelow)in the BelladonnaLeaf monograph. A. belladonn is cultivatedin Europeand the USA.
Plant. The deadly nightshade,A. belladonna, is a perennial herb which attainsa height of about 1.5 m. Owing to adnation,the leaveson the upperbranchesarein pairs,a largeleaf and a smallerone. The f'lowers appear about the beginning of June. They are solitarl'. shortlystalked,droopingandabout2.5 cm long.The corollais campanupurplish colour.The five-lobed calyx is perUses, Henbaneresemblesbelladonnaand stramoniumin action but late, five-Iobed and of a dull to the purplish-black beny. The latter is sistent, remaining attached is somewhatweaker.The higher relativeproportionof hyoscinein the conttrins numerous seedsandis aboutthe sizeof a cheny (Fig. bilocular, give rise to cerebralexcitement alkaloid mixture makesit lesslikely to plant is often known as the 'Poison Black 42.6C). In the USA the than does belladonna.lt is often used to relieve spasmo1'the urinary 'Tollkirschen'(i.e. Mad Cheny). A Cherry', while the Germannameis t | a c tu r r dw i t h \ t r o n gp u r g a t r v ct :o p r e \ c n tg r i p i n g . yellow variety ofthe plant lacksthe anthocyaninpigmentation;the leaves and stemsare a yellowish-greenand the tlowers and berriesyellow. Egyption henbqne Egyptian henbaneconsistsol the dried leavesand llowering tops of History. Belladonnawas probablyknown to the ancientsbut it is not Hyoscyamusnnticus (Solanaceae).The plant is a perentrialabout clearly recorded until the beginning of the sixteenth century. Thc 30-60 cm in height.It is indigenousto desertregionsin Egypt, Arabia, leaveswere introducedinto the Lontlon Pharmacopoeiaof 1809,but lran, Baluchistan.Sind, westernPunjab.and has beenintroducedinto the root was not used in Britain until a liniment preparedfrom it was Algiers and is cultivatedin southernCalitbrnia.In Egypt it is collected introducedby Squirein 1860. fiom wild plantsby Arab shepherds. Macroscopicalcharacters. The drug consistsof leaves.stems,flowers and fiuits. The leavesare usually matted and lbrm a lower propotlion of the drug than in the caseof Europeanhenbane.The leavesarepubescent,pale greento yellowish,rhomboidalor broadlyellipticaland up to about15 cm long. Midrib broad,venationpinnate,marginentireor with aboutfive largeteethon eachside.Petiolealmostabsentor up to 9 cm long. The stemsare greyish-yellow,striated,slightly hairy and hollow. The flowers are shorlly stalked, with large hairy bracts, a tubular fivetoothedcalyx and a yellowish-brown corolla which in the dry drug rnay show deeppurplepatches.The fruit is a cylindricalpyxidium sunounded by a persistentcalyx and containing numerousyellowish-greyto brown seeds.Odour,slightly foetid;taste,bitterand acrid. Microscopical characters. Egyptianhenbaneis easily distinguished and unbranchedglandulartrifrorn H. nigerby the nunterousbr"anched chomes.which have a one- to four-celledstalk and unicellularheads. Additional characters are the striated cuticle, the prisrns of oxalate ,15-110ptm,twin prismsand occasionalclustersand microsphenoids.
Cultivation, collection and preparation. Belladonnais grown fi'om seed.The leavesare said to be richestin alkaloid at the end ofJune or in July, and a sunny position is said to give more active leavesthan a shadyone.Plantsabout3 yearsold are sufficientlylargeto give a good yield of leavesand,if the rootsarebeing collected.it would seemto be 'BelladonnaRoot'). Two bestto replantabouteverythird year (seealso or more crops of leavesmay be collectedannually.Leavesleft in an imperfectly dry statedeteriorateand give off ammonia.They should therefbrebe dried immediatelyafter collectionand be carefully stored. Leavesof a good colour may be obtainedby drying in thin layersstarting with a moderateheat which is gradually increasedto about 60oC and then graduallydecreased. Sometimesthe leavesarebadly attacked by insectsand the rootsby a fungus.
Macroscopical characters. The drug consists of leaves and the smaller stems,the latter seldom exceeding5 mm diameter,together with flowers and fruits as describedabove.If the drug is little broken. the arrangementofthe leavesin unequalpairsmay be seen.The leaves aredull greenor yellowish-greenin colour.the uppersidebeing somewhat darkerthanthe lower. Eachhasa petioleabout0.5 -:l cm long and Constituents. Ahmed and Fahmy fbund about 1.1% of alkaloidsin a broadiy ovate, slightly decunent lamina about 5 25 crr long and the leaves.0.57cin the stemsand 2.0"/cin the flowers. The chief alka- 2.5-12 cm wide. The margin is entire and the apex acuminate.A feu loid is hyoscyaminefbr the isolationof which (as atropine)the plant is llowers and fiuits may be found. If the leavesarebroken,the most useprincipally used. The alkaloidal mixture of plants grown in Af'- ful diagnosticcharactersare the venationand roughnessofthe surface. ghanistanhad the following composition:hyoscyamine75Vc,apoatro- The latter is due to the presenceof calcium oxalate in certain of thc pine 157o,hyoscine 5clr, with smaller quantitiesof noratropineand mesophyllcells which causesminute points on the surfaceof the leaf norhyoscine.A number of non-alkaloidalketones.an acid and sito- as the other cells contractmore on drying. stelol have been characterizedfiom plants raised in Lucknow. India. The formation of alkaioidsin suspensioncell cultureshasbeenwidely Microscopical characters. A transversesectionof the leal ol A. belThe epiderladonnais shownin Fig. 27.94. lt has a bifacial stt'ttcture. investigatedwith variableresults;with callus culturesthe addition of phenylalanineto the medium producedmaximum alkaloid production mal cells have wavy walls and a striatedcuticle (Fig. 27.98). Stomata (3.97ck)whereasisoleucinegavethe greatestgrowth (M. K. El-Bahr et of the characteristicanisocytictype and also some of the anomocytic type are presenton both surfacesbut are most common on the lower. al., Fitoterapia, 1997, 68, 423).
ALKALOIDS
xy ph
a
fr[
Fig.27.9 section of portion of lomino(x200);C, dishibution of idioblosts, surfoce Atropo bellodonno leof.A, Tronsverse section of midrib(x40);B,tronsverse (G)of (ollx2O0);G ond H, Sconningelectronmicrogrophs E, lowerepidermis; F,trichomes viewof leofcleoredin chlorol(x50);D, upperepidermis; of cuficle;c, collenchymo; e, endodermis; glondulortrichomeond epidermolcellswith striotedcuticleond (H)stomoond striotedcuticle.o, Shiotions phloem;m, mesophyll; p, polisode id, idioblostcontoining crystols of colciumoxolote;i.ph,introxylory ox, colciumoxolotecrystols; ep, epidermis; L.Seedond R.Worsley.) loyer;ph, phloem;st,stomo;vt, veinlet;xy, xylem.(Photogrophs, Hairs aremost numerouson young leaves.Someof the hairs areuniseriate,two-to four-celledclothing hairsl othersresemblethesebut have a unicellularglandularhead;while a third kind has a shortpediceland a multicellular glandularhead (Fig. 27.9F).Certain of the cells of the ('sandy') crystalsof spongymesophyllarefilled rvith n.ricrosphenoidal calcium oxalate (Fig. 27.98, C). The midrib is convex above and shows the usual bicollateralvascularbundle. A zone of collenchvma underliesboth epidermiin the region of the midrib.
cium oxalate.They yield aboLrtl,l% of ash and not more than 4clr of acid-insol-rbleash. PrepuretlBelladonnaHerh is the fincl5' porvdereddrug adjustedto 'loss on drying' contain 0.28-0.32% of total alkaloids.Note the fequlrement.
Allied drugs. lndian helladonna front A. acuminattt Royle ex Lindley difl'ersfiorn that delived fiorn A. belladonnain that its f-lowers are yellowish-brownand its leavesbrownish-green, oblong-elliptical and tapering towards both base and apex. It grows wild in the Constituents. The drug lrom A. belladonnucontains0.3-0.60% of Himalayanregionsof northernIndia (1800-3,100m) and is cultivated alkaloids.the chief of which is hyoscyamine.Srnall quantitiesof volatile bases,such as pyridine and N-methylpyrroline,are present, in the Kashmir valley. Atropu haetica Willk. is a speciesnltive to southernSpain and and if not removedduring the assayof the drug by heating,increasethe titration and appearin the result as hyoscyamine.The leavesalso con- northernMorocco: it producesyellow flowers and black berriesand is (scopoletin),and ca1- re-eardedas an endangeredspecies.R. Zitrareet c1. have describeda tain a fluorescentsubstance.B-methylaesculetin
E
PHARMACOPOEIAL AND RELATED DRUGSOF BIOLOGICAT ORIGIN rapid ir?vilro propagationmethod for the plant, and from hairy root cultureshave isolatedtigloylpseudotropine-a new alkaloid not found in the matureplant(PlantCell Rep.,1999,18,4i8). Adulterants. Of the numerous recorded adulterants of belladonna leaves, those of Ph1'trsluccadecandra (Phytolaccaceae)and Ailanthus glandulosa (Simaroubaceae)are perhaps the inost important. In Phltolacca the lamina is denserand less decurent than in belladonna; the epidermalcells have straightwalls, the stomataare of the anomocytic type and some of the mesophyll celis contain bundles of needleshapedcrystalsof calcium oxalate.Ailanthus leavesaretriangular-ovate, have straighfwalled epidermalcells showing a strongly striatedcuticle, cluster crystalsof calcium oxalate,and on both surfaceswhite, unicellular clothinghairswhich arelignified (Fig. 43.3F). Uses. Belladonna leaves are mainly used for internal preparations which areusedas sedativesand to check secretion.Preparationsof the root are mainly used externally. Bellqdonno roof Belladonnaroot consistsof the dried roots or rootstock and roots of At ropa belladonna (Solanaceae). Collection and preparation. Much of the A. belladonLradrug is of small size and poor quality. The firstyear roots are not profitable to collect from the commercial point of view, although they contain a high proportionof alkaloids.The autumnof the third year would seem to be a suitabletime for collection.The rootsareCugup, washed,sliced and dried.
ertiesto other samplesof the enzyme previously described,has been isolatedfrom transformedbelladonnaroot cultures.The pseudotropine could have implications for the formation of calystegines (q.v). Littorine hasbeendetectedin both non-transformedand hairy root-cultures(F. Nakanishiet al., Plant Cell Rep., 1998,18,249). Allied drug. Indian belladonna root from Atropa acuminata (see 'Belladonna under Herb') consistsof brownish-grey roots, stolons. rootstockand stem bases.lt has been describedin detail by Melville. The roots are cylindrical, Iongitudinally wrinkled, occasionally branched,and 0.5-3 cm diameter.Young roots resemblethose of A. belladonnabut older ones show concentriczonationof the secondary xylem. The rootstockis 3-9 cm diameterat the top and bearsthe bases of 1-12 aerial stems.The rootstock,stembasesand stolonsall possess a pith which becomeshoilow in the stem bases.The constituentsare similar to thoseof Europeanbelladonna. Adulterant. The root of Phytolacca decandra (Phytolaccaceae)is sometimesslicedand mixed with samplesof belladonna.It bearslinle resemblanceto belladonnaroot, but a casualandinexperiencedobserver might perhapsmistakeit for piecesof an old belladonnacrown. The transversesectionshowsa numberof concentriccambia,eachproducing a ring of wood bundles.The parenchymacontainsabundantacicular crystalsof calcium oxalate.
DUBOISIAIEAVES
Threespeciesof Duboisia areindigenoustoAustralia and two of these. D. myoporoides and D. leichhardtii, have for over 50 years been a Macroscopical characters. Atropa belladonna r^pidly develops a major world sourceof tropanealkaloids.The third species,D hoplarge branching root. The aerial stemsdie back eachyear and new ones woodii, containsprincipally nicotine and related alkaloids and was 'pituri' by mixarise independently from the large crown. Dried roots of 3-year-old usedby theAustralianaboriginesfor the preparationof plantsareabout3 cm diameterandrootsover 4 cm diameterareexcep- ing powderedleaveswith an alkaline wood ash to form a quid which was held in the cheekpouch. tional. Most commercialdrug is abouthalf this thickness. D. myoporoides,discovered by Robert Brown, naturalist to the The drug is usuallycut into shortlengths,which are sometimessplit Flinders expeditionof 1802,occursalong the east coastofAustralia. longitudinally. The outer surface is a pale greyish-brown.The root where the rainfall exceedsa monthly mean of 5 cm for l1 months of breaks with a short fracture and then shows a whitish or. if overheated during drying, brownish interior. A yellowish-green colour in the the year and where frosts rarely occur.D. leichhardtii was described by Mueller in 1877 and is named after the explorer Ludwig region of the cambiumis often seen. A transversesectionof the bark is nonfibrousand the wood doesnot Leichhardt,who originally collectedthe plant; it occursnaturally in a limited area of south-eastQueenslandknown locally as the South show a radiateappearance. The wood consistsof scatteredgroups of vessels,tracheidsand fibres which are most abundantnear the camb- West Burnett. D. hop-*oodii is of wide distribution in Western and ium; thereis a centralmassof primary xylem (Fig. 42.8G).The exten- CentralAustralia. Of the two tropanealkaloid-containingspeciesD. myoporoidesis sive parenchymaof bark and wood containssandycrystalsof calcium the larger and more densely leaved; both, however, are bushy treesand oxalate and abundantsimple and compound starch grains. The structure gradually changesas the roots pass into rhizome, the have the advantage that in one year repeated harvests can often be wood becomingdenserand exhibiting a distinctlyradiatestructure;the taken from the same plants. For collection, the small branchesare rhizome also shows a distinct pith and internal phloem. The aerial removed, tied in bundles and stood in shedsto dry; the leaves are then easilyremovedby beating. stemsfound on the upper surfaceof the crown are hollow In additionto hyoscineandhyoscyamine,minor alkaloidsoccur in variable amounts and include norhyoscyamine,6B-hydroxyhyoscyamine, Constituents. Atropa belladonna toot contains about 0.4-0.87o of valeroidine, tigloidine, poroidine, isoporoidine, valtropine, 3cx-tigloylalkaloidscalculatedas hyoscyamine(BPC requiresO.4Vo). Samplesof belladonnaroot examinedby Kuhn and Schdfershowed oxytropane,3u-acetoxytropane,3o-nonanoyloxytropane,butropine and 0.3-1.0Vc of alkaloids, of which 82.8-97.3Vo was hyoscyamine, apohyoscine.Two discopine esterswere identified in 1980 in D. leich2.7-1 5 .2Voatropine,and0.0-2.6Voscopolamine.Capillary GLC-mass hardtii and the greenhouseleaveshave yielded calysteginesB,, 82, Ba, spectrometrydata has revealedthe presenceof hygrine, hygroline, cus- C1, and C2 (A. Kato et al., Phytochemistry-, 1997,45,425). Other concohygrine, tropinone, tropine, pseudotropineand nine tropanol esters stituentsinclude the triterpenoidsursolic acid and betulonic acid and a (F. Oprach et al., Planta Med., 1986,513). Other constituentsprevi- numberof recentlyreportedaliphaticconstituents. A number of chemical races occur, parlicularly in D. myoporoides, ously reportedinclude belladonninetogetherwith B-methylaesculetin, and include the well-established'northern'and'southern'raceswhich calcium oxalate and starch. differ in their relative contentsof hyoscine and hyoscyamine,and a A pseudotropine-forming,tropinone reductase(see biogenesisof tropaneaikaloids),not entirely similar in chemicaland catalyticprop- race which containsnicotine and anabasineas principal bases.
il
ALKALOIDS For a numberof years,growershavealsobeencultivatinga hybrid of the collection of the root was formerly accompaniedby specialrites. the two species,the origin of which is doubtful,but which Griffin con- The drug, like belladonna,has long been known to contaln atropine sidersmay derive from the experimentalwork of the CSIRO caried out and the fluorescentsubstancescopoletin.Recent investigationshave in the early I 950s.Establishedpiantationsof the hybrid exhibit no mor- establishedthe presenceof severalother solanaceous alkaloids. phological difTerencesand propagationis carried out vegetatively. In a series of experiments on the hybrid, Griffin and Luanratana (J. Nat. COCAIEAFAND COCAINE Prod.. 1980,43,552:.1982,45,270)have shown that the total alkaloid contentof the leavesdoes not vary throughoutthe year but there is a Coca leavesare derived from shrubsof the Erythroxylaceae,namely decreasein hyoscinecontentfrom Januaryto June(summerto autumn) Erythrorylum coca (Bolivian or Huanuco)andE. truxillense(Perr"rvian or Truxillo), cultivatedin Peru,Bolivia, Colombia and Indonesia. and a gradual increasefrom June to September;the reverseis true for hyoscyamine.Repeatedsprayingsof plants with cytokinin solulion (which also has a beneficial elTecton plant growth), in the lbrm of a sea- History. Cocaleaveshavebeenusedin SouthAmericaasa masticatory weed extract,preventedthe hyoscinedecline.Such treatmentof plants from very e:Lrlytimes.They were formerly reservedfor the soleuseof the could possibly enhancethe hyoscine yield from all-year harvesting. native chieli and Incas.Coca was introducedinto Europeabout 1688and Thereis evidence(Y. Kitamura et al., Phv-tochentistry^,1996,42,1331)cocaine was isolated in 1860.By employing the alkaloid in ophthalmic surgeryin I 884 Carl Koller was the first to introduceit into clinical practhat in the plant the tropic acid moiety of atropinemay be recycled. For reviewscovering tice so heraldingthe era of modem anaesthetics. Addition of putrescineand spermidineto the culture medium of D. m-toporoidesroot cultures has been shown to increasethe hyoscine both the historical and other aspectsof coca seethe specialissueof l/ze (1981),Vol. 3: CocaonclCocaine. Joumal of Ethnophannacoktg,v content(T. Yoskiokaet al., Planta Med.. 1989,55,573). Most of the Australiancrop (some 1200tonnes)is exportedto West Germany,Switzerlandand Japanfor processing.Plantationshave also Collection. In Bolivia and Peru coca is cultivated at an altitude of 500-2000 m. The cultivated plants are usually pruned so as not to beenestablishedin Ecuador. exceed2 m in height. Three harvestsare collectedannually,the first from the pruned twigs, the secondin June and the third in November. Scopolio All speciesof Scopolictinvestigatedappearto contain tropane alka- The leavesare aftificially or sun-driedand packedin bags. loids similar to thosefound in belladonna(q.v.).Although little usedin westel'nEurope,theseplantsconstitutea useful sourceof hyoscyamine and galenicalsin regionswhere the plant is availablelocallry.Scopolia caniolica is a centraland easternEuropeanspeciessomewhatsmaller than belladonna.In shape the leaves resemblethose of belladonna, althoughthey are more lanceolateand translucent.The cuticle is striated but less markedly so than in belladonna,sandy crystals are less numerous,hairsarerareor absent,and stomataarepresenton the lower surfaceonly. The fruit, which is a pyxis, may often be found in the drug. The rhizomes(BPC 1934),which are nearly black in colour and bear numerousdepressedstem scars,are used similarly to belladonna root. In additionto hyoscyamineandhyoscine,otheralkaloidsreported in this speciesare cuscohygrine,3o-tigloyloxytropane.pseudotropine and tropine.S. caucasia,S. lurida andS. tanguticcall appearto be suitable as sourcesof hyoscyamine;the last two also contain 6-hydroxyhyoscyamineand an alkaloid nameddaturamine(anisodine)which is a 'hydroxyhyoscine'.Both thesealkaloidsareproducedcommerciallyin China. The dried rhizomes of S. Japonlca ('JapaneseBelladonna Root') were official in rhe JapanesePharmacopoeia196l; the isolahas beenreportedfrom this tion of steroidalglycosides(scopolosides) species(S. Okamuraet aL.,Chem.Phurnt.Bull., 1992,40,2981). Przen'alskiutonguticais a relatedtropanealkaloid-containingplant and is usedin Tibetantraditionalmedicine.The roots have a high content of hyoscyaminewith total alkaloidsamountingto 1.7-3.87c;6$hydroxyhyoscyamineand small amountsof hyoscineare alsopresent. Mondroke The true mandrake, Mandragora o.fficinarum, is one of several Mediteranean species.It was well known to Dioskurides(see R. T. Gunther'sEnglish edition of The GreekHerbal of Dioscorides,1934, Oxford. UK: OUP). B. P.Jackson,in an investigationof the botanical source of the drug, found that the speciesM. autumnalis ts also involved.The leavesand roots were official in France(1818-1883) and in Spain.The roots occur in fusiform or two-branchedpiecesand their microscopicalstructureand distinctionfrom belladonnaroot has beendescribedby Berry and Jackson(PlantaMed..1976,30,281). The plant is surroundedwith rnuch folklore and superstitionand even
Macroscopical characters. 7. Huanuco or Boliviat't coca leavesare shortly petiolate,oval, 2.5-7.5 cm long and 1.5-4 cm wide. The laminais greenishbrown to brown and glabrous;margin entire.The midrib is prominenton the lower surface, bearsa ridge on its uppel surface,and projects slightly beyond the lamina as an apiculus.The latter is often broken in the commercial drug but the leaves are otherwise fairly entire. The lower surface showstwo, very characteristic,curved lines, one on either side ofthe midrib. Odour, characteristic;taste,at first bitter and slightly aromatic, the alkaloidsafterwardscausingnumbnessofthe tongueand lips. 2. Trttxillo or Peruvian coca leaves are pale green in colour, are mote papery in texture than the Huanuco and are usually broken. Lamina about 1.6*5 cm long; lines on the lower surface usually indistinct. Flowers of a speciesof lnga (Leguminosae,subfamily Mimosoideae) aresometimesaddedto the leaves. 3. Javanesecocc leavesof the Truxillo type were formerly exported for the manufacture of cocaine. Microscopical characters. A transvetsesectionof a cocaleaf shows upper epidermis,palisadeparenchymacontaining prisms of calcium oxalate,spongyparenchymaand a very characteristiclower papillose epidermiswith numerousstomata.The midrib is partly surroundedby an arc of pericyclic fibres, above and below which is a considerable amountof collenchyma.A surfacepreparationof the lower epidermis showsthe papillaeas well-markedcircies,and numerousstomata(Fig. 43.2J1,eachwith four subsidiarycells, two of which have their long axesparallelto the pore. Constituents. Cocaleavescontainabott O.'7-1.57aof total alkaloids, of which cocaine, cinnamylcocaineand q,-truxilline are the most important.They occur in difTerentproportionsin differentcommercial varieties. Javaneseleaves are usually richest in total alkaloids, of which the chief is cinnamylcocaine,while the Bolivian and Peruvian Ieavescontain less total alkaloid but a higher proportion of cocaine. Other substancesisolated from various varieties of the leaves are hygrine, hygroline, cuscohygrine,dihydrocuscohygrine.tropacocaine
PHARMACOPOEIAT AND RELATED DRUGSOT BIQLOGICALORIGIN
:l:rrail::iirll.lirirril,,iall i rllillrlll:ii
(3B-benzoyloxytropane), crystalline glycosidesand cocatannicacid. l-Hydroxytropacocaine(free hydroxyl situatedat a bridgeheadcarE. bon) has beenisolatedas a major alkaloid of greenhouse-cultivated noyogran(fiensevar. noNogranatense:much lower amounts were detectedrn var. truxillenseand in field cultivatedcoca from Colombia 1994,36,351). and Bolivia (J. M. Moore et al.. Phytochemistry-, The leaves also contain essentialoil and as early as 1894 Van Romburghidentified methyl salicylateas a component;this was confirmed (13.67c) in a recent study, together with N-methylpynole (3.77o)and possiblyN,N-dimethylbenzylamine(0.57o)and two dihy(38.9%).The grassyodour ofthe leavesis explained drobenzaldehydes to a large extent by the presencein the oil of trans-2-hexenal(.10.4Va') were and cls-3-hexen-1-ol(.16.1%):no mono- or sesquiterpenes detected(M. Novdk et al., Planta Med., 198'7,53, 113). Although it had been generally assumedthat ecgonine,the basic moiety of the cocaines,was ornithine-derived(Fig.27 .2), the practical demonstrationof the incorporationof the usualprecursorsproved difficult. Then Leete (./. Am. Chem. Soc., 1982, 104, 1403) obtained a significant level of radioactivity in cocaine isolated from Erythroxylumcoca, lhe leavesof which were paintedwith an aqueous solution of Dl-[5-14c]ornithineHCl. The pathway to ecgonine appearsto be similar to that for tropine except that the carboxyl is retainedand the different stereospecificities needto be accommodated. The benzoyl moiety of cocaineis derivedfrom phenylalanine.For work on the incorporationof labelled I -methyl-AI -pyrrolinium chloride into cuscohygrine,indicating the alkaloid to be a mixture of its meso and optically active diastereomers, see E. Leete et al., Phytochemistry,1988,27, 401. Manufacture of cocaine. The crude alkaioids may be extractedwith dilute sulphuric acid or by treatment with lime and petroleum or other organic solvents.Non-alkaloidalmatter is roughly separatedby transferring the alkaloids from one solvent to another.The crude alkaloids are obtained in solid form either as free bases by precipitation with alkali, or as hydrochloridesby concentratingan acidified solution. Pure cocaine is prepared from the leaves, the crude bases or the crude hydrochlorides.The processdependson the fact that cocaine, cinnamyl-cocaineand o-truxilline are closely related derivativesof ecgonine (Fig. 21.2), which is produced by hydrolysing them with boiling dilute hydrochloricacid. Cocaine+ ecgonine+ methyl alcohol + benzoicacid Cinnamyl-cocaine-+ ecgonine+ methyl alcohol+ cinnamicacid -+ ecgonine+ methyl a-Truxilline (y-isoatropyl-cocaine) alcohol+ cx-truxillicacid
The ecgonine hydrochloride is purified and converted into the free base.This is benzoylatedby interaction with benzoic anhydride and the benzoylecgoninepurified. The benzoylecgonineis methylated with methyl iodide and sodium methoxide in methyl alcohol solution, to give methylbenzoylecgonine or cocaine. The latter is converted into the hydrochloride and purified by recrystallization. Much illicit cocaine is extracted locally in South America and despite the unsophisticatedmethods employed a high degreeof puritl, can be attained. In view of the importance of quantitatively determining cocaine and its metabolite,benzoylecgonine,in body fluids, etc., many assaysare availablefor thesealkaloids. Allied species. There are over 200 species of Erythro4tlun found throughout the tropical and pantropicalregions of the world. Few of the non-cocaine-producingspecieshave been systematicallyexamined but the majority of thosethat havecontaina rangeof tropanealkaloids(W. C. Evans,"/.Ethnopharmacol.,1981,3, 265 and for Pt. 12 of a further series 1053).Recent of papersseeP.Christenet al., Phytochemistry,7995,38, studieshave revealednew alkaloids from E lucidum (A. Brachet et al.. Phytochemistry,1997,46,1439) and from E. moonii (Atta-ur-Rahmaner al., Phlttochemistry,1998, 48,311). Three new alkaloids have recently been characterizedfrom E. 4eylanicum,the one speciesendernicto Sri Lanka (G. Bringmannet al., Phytochemistry,2000,53,409). Uses. Cocaineand its saltswere the earliestof the modern local anaesthetics but, becauseof their toxic and addictive properties,their use is now almost entirely confined to ophthalmic,ear,noseand throat surgery.
Colystegines These relatively new alkaloids are trihydroxy-, tetrahydroxy- or pentahydroxy derivatives of nortropane.They were originally isolated from the roots of the bindweed Calystegia sepium and given the names (Fig. 21.2) and calystecalystegineA'3 (a 1,2,3-trihydroxynortropane) gine 82 @ 1,2,3,4-tetrahydroxynortropane). By 1998 the structuresof nine such alkaloids had been elucidated including the 3-O-B-oglucopyranosideof calystegineB 1. In a chemotaxonomicstudy of the Convolvulaceaeinvolving the GC-MS analysesof 65 species,T. Schimminget al. (Phytochemistry,1998,49,1989)reportedthe occurrence of 1 to 5 calysteginesin 30 speciesbelonging to 15 genera. Calystegineshave also beenreportedin the Solanaceaeand Moraceae including belladonnaand hyoscyamusroot cultures (B. Driiger and A. Schaal, Phytochemistry, 1994, 35, 1441). R. J. Nash et a/., (Phytochemistrr-,1993,34,1281) found thesecompoundsto be present in the tubers and leavesof potato plants and that thesealkaloids can be
coocH3 oco.c6H' c-Truxillicacid
Cocaine
p.r,'co'cH3 p.r,'cH'cH3 f,}.r,'co.HJ] oH 8", 1". 3r. Hygrine
Hygroline
Cuscohygrine
1".
ALKALOIDS isolatedfrom certainmoths and butterflies.the larvaeof which t-eedon the plant. Pharmaceutically.interestlies in the calysteginesbecausethey are potentinhibitorsof glycosidases(R. J. Molyneux et al.. Arch. Biochent Biophl'sics,1993,304, 8l) making them possiblecandidatesfor the developmentof a drug for the treatmentof AIDS (cf the tlihydroxyindolizidine alkaloidscastanospermine and swainsonineand the tetfahydroxypynolizidine alkaloid australine).
Toboccoolkoloids The principal alkaloidsof the -eenusNicoriana have a pyridine moiety associatedwith eithera pyrrolidinering (ornithine-derived)or a piperidine ring (lysine-derived).The fbrmer group is represented by nicotine (Flg. 27.2) and the latterby anabasine(Fig 27.1I ). Although, with the exceptionindicatedbelow, no drugs are derived from thesealkaloids,they have beenextensivelystudiedin relationto tobacco manufactureand smoking, and as insecticides(see Chapter 4l). Consequently,much is known of their plant biochemistry and geneticsof formation. A recent pharmaceuticalintroduction is that of nicotine chewinggum, nasalsprayor patch.intendedto help smokerswho want to give up smokingbut who experiencegreatdilTicultyin so doing becauseof their nicotinedependence.
frequentlyoccur as esters,being linked with characteristicmono- or dibasic acids called the necic acids. They are biosynthesizedfrom omithinevia a symmetricalintermediateandlabellingexperimentshave shownthe involvementof putrescincand homospermidine.Two molecules of putrescineare requiredto form one of homospermidine.This pathway has been supportedby the isolation,partial purification and characterization of the NAD+-dependentenzymehomospermidinesynthase, the first pathway-specificenzyme in pyrrolizidine alkaloid biosynthesis(F. Bijttcher et ul.. Ph-,-tochentistl',1993,32,679). The componentsof senecionineareillustratedin Fig. 27.10.The hepatotoxic propertiesare believedto ariseby breakdownofthe alkaloidsin the liver to stronglyalkylatingpyrrole esters. For reportson pyrrolizidine alkaloidssee:T. Hartmannand L. Witte (1995). Alkaloids, Chemical and BiologicaLPerspectives(ed. S. W Pelletier).Vol.9. New York: Wiley. p 155. A generalreview: K. Ndjoko et ol., Pl(tntaMed.. 1999,65,562. Determinationin Senecio soecies.
As the next homologueto ornithine.Iysineand its associatedcompoundsgive rise to a numberof alkaloids.someof which are analogous to the ornithine group (see Fig. 27.11). The lycopodium alkaloidsare also derivedfiom lysine.Although in somecases.such PYRROLIZIDINE ALKATOIDS as the quinolizidinelupin alkaloids,Iysineis incorporatedvia a symmetrical precursor,e.g. cadaverine.in the majority of examples Althoughthesealkaloidshaveat presentno greatmedicinalsignificance (anabasine.sedamine,N-rrethylpelletierine)the incorporationis they areimportantin thatthey constitutethe poisonoushepatotoxiccon- asymmetric.In general.fbr the simple cx,-subsrituted piperidines,the stituentsof plantsof the genusSenecio(Compositae),well-known fbr C-2 of lysine becomesthe point of attachmentof the cxside-chain. their toxicity to livestock.Someofthe alkaloidsalsoshow crrcinogenic (For researchin this area and references.seeE. Leete, Nat. Prod., "/. and mutagenicproper-ties and havecausedconcernin that they occur in 1982.45. 197: T. Hemscheidtand L D. Spenser,J. Ant Chen. Soc., quantities small in some herbal products such as comfiey 1 9 9 0 , 1 1 2 . 6 3 6 0 . ) (Boraginaceae) and coltsfoot(Compositae).Thesealkaloidsare known The wide distributionof thesebasesthroughoutthe plant kingdom is to havean ecologicalrole in somespeciesofbutterlly affbrdingprotec- illustratedby the drugswhich fbllow. tion to someandconvertingto femaleflight arrestants in others.Indicine N-oxide hasantitumourproperlies(q.v.).Australine.recentlycharacterLOBETIA izedfrom the seedsofthe legurninoustreeCastanosperniltm ttustrale,is a tetrahydroxypyrrolizidine alkaloid.It was obtainedby useolrepeated Lobelia BHP; BP 1988(Lobelia Herb. lndian Tobacco)consistsof the preparativecentrifugalTLC. Like the polyhydroxyindolizidinealka- dried aerial parts of Lobelin inflata (Campanulaceae), an annualherb loids it exhibits glycosidaseinhibitory activity (for lurther detailssee indigenousto the easternUSA and Canada.It is cultivatedin the USA R. J. Molyneux et ol., J. Nat Prod., 1988,51, ll98). The alkaloids and Holland.
O r n i t h i n(eq y . )
lsolcucine( q.v.)
HsL\a..H.
cfrzoH
il
. 1 t
ar. o,
t-l
HOOC-C-CH2-C-C-COOH
t
H Rclronecinc HYr C .\cz . H
Senecicocid a cHr oH
,,o ll l - t -.lo o_c-c-crni-l *i( "$ i, Eig,27.rO Formotion ondlivermetobolism of pyrrolizidi ne-ester olkoloids.
"*hs"'.,
l
CH3
Ul,-,i
Senecionine
t'o\,--r== oripavine + morphinone -; morphine(Fig. 27.15). Two alkaloids which arise as branchesof the principal biogenetic pathwayare neopine,the presenceof which in opium is explainedas a reduction product of neopinone, and papaverine,which arises b1 methylation of norreticuline (Fig. 27.17) followed by dehydrogenation. The presenceof some of the other minor alkaloidsof opium can be explained by various methylationsand dehydrogenationsof laudanosoline.reticulineand their nor-derivatives.Variousoxidativecouplings ofreticuline accountfor otherminor alkaloids(e.g.corytuberine and isoboldine). Role ofreticuline in alkaloid biosynthesis. The alkaloidsinvolved in Fig. 27.15 are derived from (-)-reticuline, and the enzymic racemization of reticuline,which is essentialfor the biosynthesisof the principal opium alkaloids, is very substratespecific. Thus the N-ethyl. 6-ethoxyand 4'-ethoxyanaloguesof reticulinearecompletelyresistant to racemization.(+)-Reticulinealso gives rise to a number of bases: narcotine(noscopine)and narceineof opium; canadine,berberineand hydrastineof Hydrastis (Berberidaceae);and sinomenine(the enantiomer of the opium alkaloid salutaridine,Fig. 27.15, of Sinomeniunt With the exceptionof sinomenine.these acutum (Menispermaceae). 'berberinebridged'alkaioidsand they arisefrom alkaloidsare termed norlaudanosolineand a one-carbonunit, which is derived from the N-methyl group of (+)-reticuline.The methylenedioxygroup of these alkaloids is formed by oxidative cyclization of an o-methoxyphenol function.A schemeindicating the origin of some of thesealkaloidsis given in Fig. 27.18. For berberine, 13 enzymes are involved in its biosynthesisfrom two moleculesof tyrosine.The enzyme associated with the final reaction,the formationof the methylenedioxybridge,has now been detected in microsomal preparations from diff'erent
M"o)z),^.1
TNQ"'"4?=l' 4a
*.d
L-Tyrosine
\
ffcrc HO,JV
(SlNorcochurine
p-HydroryphenylacchHchyde
,.|o'\./ (SlC.oclaurirc
II
(5)N-Mcthylcoclaurine
Fig.27.16 routefrom A reiisedbiogenetic
(I}Reticuline [(+fReticuline]
to (S)+eticuline. LLiyrosine
(S)-5'-Hydroxy-IV-mcthylcoclaurine
cH.o HO
NH
+ HO
Fig.27.17 in the A moiorpofhwoy biogenesis of popoverine.
cHto Norreticuline
( - ) Norloudonine
(-)Tetrohydropcpoverine Popoverine
'il
ALKALOIDS
( +).Rcti(uline
/
\
ocH3
Fig.27.r8 from Someolkoloids derived (+)+eticuline.
Ranunculaceae cell cultures(M. Rueffer and M. H. and Berberidaceae Zenk, P hytochemist try,1994, 36, 1219). Enzyme studies. As indicatedin Chapter 1l cell culturesof P. sontnferum do not producemorphine-typealkaloidsbut accumulatelarge amountsof sanguinarineand other alkaloids.However, some of the enzymesof the morphinanpathwayare presentin suchcell culturesas shown by the reductionof codeinoneto codeineby immobilized cells of P. sontnferum andby the isolation fiom cell cultures of the enzyme which reducessalutaridineto (7,5)-salutaridinol. This enzymehasbeen fully characterized (R. Gerardy and M. H. Zenk, Phl'tochemistry, reductaseisolated from 1993,34, 125) as has 1,2-dehydroreticuline poppy seedlings.The samegroup has shown (R. Wilhelm and M. H. Zenk, Phytochernistry^, 1991, 46, 701) that the enzyme necessaryfor the enol cleavagein the conversionof thebaine to neopinone(Fig. 27.15) is presentin cell cultures,as evidencedby the formation of a new alkaloid, thebainone,as a result of feeding labelled thebaineto low-thebaineproducing P. somniferumcultures. For work on genes and codeinone encoding (S)-N-methylcoclaurine-5'-hydroxylase reductaseseeF.-C. Huang and T. M. Kutchan,Phytochemisnl, 2000, 5 3 .5 5 5 .
ocH3 (-!Stylopioe
Uses. The alkaloidspresentin opium in greatestproportiondecrease in narcotic properties in the order morphine, codeine, noscopine. Opium and morphine are widely used to relieve pain and are particularly valuableas hypnotics,as, unlike many other hypnotics,they act mainly on the sensorynervecells of the cerebrum.Codeineis a milder sedativethan morphine and is useful fbr allaying coughing.Both morphine and codeine decreasemetabolism, and the latter, particularly before the introduction of insulin. was used fbr the treatmentof diabetes. Opium, while closely resembling morphine, exerts its action more slowly and is therefore preferablein many cases(e.g. in the treatment of diarrhoea).Opium is also used as a diaphoretic.The habitual use of codeinemay, in some individuals. produce constipatron. Manufacture of opium alkaloids. The majority of legal opium is usedfor the isolationof its constituentalkaloids.and in Britain some 90Vaof the morphine produced is convertedinto other basessuch as codeine, ethylmorphine and pholcodine. In recent years attempts have been made to reduce the illicit traffic in opium either by banning the cultivation of the opium poppy or by cultivation under strict licences.However,two methodsby which the opium stageis eliminated are by extraction of the whole poppy capsule and by the use of other species(e.g. P. bracteatum) which do not contain morphine.
Tests for opium alkaloids. Tests for morphine and other alkaloids aregiven in the pharmacopoeias. The solubility of morphinein sodium hydroxide solution is explained by its phenolic nature. Conversely, Extraction of poppy capsules and straw. The feasibility of codeineis precipitatedby sodiumhydroxide. extracting poppy straw has long been known and utilized in Europe and recently there has been a world-wide trend towards the extracStorage. Opium requirescareful storageif the morphine content is to be maintained.In the past, Indian opium appearsto have sufferedmore tion of the dried poppy capsules (e.g. in Hungary, former USSR. morphine Iossduring preparationand storagethan the other varieties,but Tasmania).In a study of poppy capsule drying and storage under when it is dried at 100'C and stored out of contact with air. the loss of commercial conditions in Tasmaniait has been shown that kiln-drymorphine is small.Abraham and Rae ( I 926) ascribethe loss of morphine ing of immature capsules (,12 days old) at various temperatures to a peroxidasewhich they called opiase.More recently a phenoloxidase (40-100"C) resultedin a loss of morphine contentof up to about 11% without effect on codeine and thebaine.However. this morwhich actson morphine has beenisolatedfrom poppy capsules. phine loss was significantly less than with the field-dried material. Adulteration. Opium has been adulteratedwith sugary fiuits, gum, To avoid deterioration of the dried product the moisture content powderedpoppy capsulesand other substances too numerousto men- should not exceed 167o. Processeshave also been developed in tion. As far as legitimate cornmerceis concerned,such adulteration is France and in the UK for the harvestingand processingof the green now pointlessbecausethe product is analysedand the price paid is capsule, one technical difficulty being the separationof the seed governedby the contentof morphineand other alkaloids. from the fruit at this stageof development.
iilhrr,
E
DRUGSOF BIOLOGICAT ORIGIN PHARMACOPOEIAT AND RELATED Use of morphine-free species of Papaver. The increasing abuse of opiates has stimulated the searchfor raw materials other than Papaver somniferumwhich would meetthe requirementsof the pharmaceutical industry. Thus, plants containing nonaddictivethebaine as principal alkaloid could be usedlbr the manufactureof codeine,naloxone(a narcotic antagonistprescribedfor babiesof heroin addicts)and etorphine (a 'Bentley' compoundusedfor sedatinglargewild animals). In this respect attention focused on three closely related perennial speciesof Papaverof the sectionO4,-tonaBetnh.of the family, namely,P bracteatum,P. orientale andP.pseudo-orientale.Theseareindigenousto the mountainousdistricts of Iran, eastemTurkey and the Transcaucasian former USSR. Confusion concerning the identity of the characteristic alkaloids for each specieshad arisen from various factors, including incorrect identification. variations of chromosome number within a species,the existenceof chemicalracesand geographicalinfluences. Papaver brqctealum Thebaineis the predominantalkaloid of this speciesand in a UN-backed programmelarge-scalecultivation trials were organizedin various countries. High yielding strainswere introduced,for example,Ayra II. a race obtainedfrom west Iran in 1974 which gives 3.57othebainein the dried capsules.Problems associatedwith the development were the insufficiency of seed of high yielding strains and the poorer crops obtained when the piantswere removedfrom their normal environment.However, political decisionsalsojeopardizedthe continuationof the programme. P. bracteatumproducessome 27 alkaloids belonging to l0 of the 14 alkaloid groups describedfor Papaver. The biogenesisof thebainefollows the samepathway as tn P. somniferum.Feeding experimentswith labelled intermediateshave shown that the plant is capableofconverling codeinone to codeine but cannot perform either of the demethylations leading to codeineor directly to morphine.Thebainedoes not appearto be entirely an end-product and undergoesfurther metabolism to unknown products(for a report, seeH. G. Theunset aI., Phytochemisu.'v, new alkaloid is salutaridine1985,24,58 1). A more recently-described N-oxide (G. Sariyaret a l., PlantaMed., 1992,58, 368). Papaver orienlqle This speciesis commonly cultivated as the ornamentalpoppy and a number of alkaloid chemotypeshave been described.Generally,oripavine(formed by demethylationof the aromaticring of thebaine,Fig. 27.15) is the principal alkaloid, but Phillipson et al. (Planta Med., I 98 1, 43. 26 1) describedone chemotypewith mecambridine(a berberine type alkaloid)as principal constituent.
cH3o
baine, mecambridineand orientalidineas major alkaloids,two contained principally salutaridineand thebaine,and one samplepossessedsalutaridine as the major component.(For other aikaloids and biogenetictrans1986,25,2403.) formations seeG. S2ariyaet al., Ph,u-tochemislry.', (For a comprehensivereview of the morphine alkaloids covering general chemistry, biogenesis,occurrenceand structure elucidation (391refs)seeC. S2antayet al.,TheAlkaktids,1991,45,128.)
SERPENTARY Serpentaryconsistsof the dried rhizome and roots of Aristolochin reticulota(Aristolochiaceae). This is known in commerceas Texanor Red River snake-root and is collected in the woods of Texas. Louisiana.Arkansasand Oklahoma. Macroscopical characters. The drug has a yellowish colour when fresh,becomingbrown on keeping.It consistsof small rhizomesbearing the remains of subaerialstemsand numerouswiry roots. The rhizomes areabout 1-2 cm Iong and 2-3 mm diameter,while the roots areabout 10 cm long and0.2-1.2 mm diameter.The drug containsup to l07c of subaerial stems.Odour, camphoraceous;taste,camphoraceousand bitter. A transversesectionof the rhizomesshowsa starchy,eccentricpith (nearer the upper surface of the rhizome than the lower), wedgeshaped,yellowish vascularbundlesseparatedby wide medullaryrays. and a narrow bark. Allied drugs. \4rginian snake-root, from Aristolochiaserpentaria,was formerly official but its regular importation has now ceased.It closely resemblesthe Texan drug, but has smallerrhizome and more wiry roots. Indian aristolochia or Indian birthwort consistsof the roots and rhizome of Aristolochia indica; it contains aristolochic acid together with other phenanthrenederivatives,an N-glycosideand steroids.A. heteropl'tyllais used in China, andA. constricta, recentlyinvestigated(L. Pastrelliet al.. J. Nat. Prod., 1997,60, 1065),is widely employedin folk medicinein S. America. A. clematis (bithwort) is European and has been used both intemally and extemally.It containssimilar constituentsto other species. Constituents. Many speciesof Aristolochia including A. reticulata contain aristolochicacid and the tumour-inhibitingpropertiesof this compound are of interest.However, in experimentalanimals it can causetumour fbrmation and has been associatedwith casesof renal failure. Aristolochic acid is not alkaloidal,belongingto a small group of naturally occurring nitro-compounds,but is included here because of its direct derivationfrom isothebainederivativesin the plant.
'/) \-: +
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lsothebaineiiomer
Popaver pseudo-orienlale The plant (2n = 42') is intermediatein many of its charactersbetween those ofthe above two speciesand may have arisenas an allohexaploid from them. Phillipson (above reference)divided 16 Turkish samplesin threecytological and alkaloid groups.Thirleen samplescontainedisothe-
Aristolochic acid
(For a review on the stmctureof the aristolochicacidsand their correspondinglactams(aristolactams)seeD. B. Mix et al., J. Nat. Prod., 1982, 45, 657 and for the isolationof aristolochicacidsI-IV and aristolactams I-III from A. auricularis see P. J. Houshton and M. Ogutveren,Phytochemistry1991,30, 253.)
{#qI
ATKALOIDS Uses and dangers. Snakeroothas been traditionally employed as an aromatic bitter but other Aristolochia spp. have also been employed in Chineseherbalmedicinesfor varioustreatments.As from 28 July 1999an emergencyban on the import, saleand supply of medicinalproductscontaining Aristolochia spp. came into force for the UK. This arosein part from two UK casesof end-stagerenal failure in patientsusing Chinese medicinescontainingAristolochia and from many casesof renal failure in Belgium when Aristolochia was substitutedfor Stephaniain a herbal preparation.Apparently in Chineseherbal preparationsit is liable to be substitutedfor other innocuouscomponentssuchasStephania,Akebia or Clematis (MCA / CSM, UK, Current Problems in Phatmacovigilance, 1996,22,10;seealsotherepot, Pharm.J.,2000,265, 10).
Constituents. Hydrastiscontainsthe alkaloidshydrastine,berberine and canadine(seeFig. 27.l8) and other minor ones.Commercialsampies yield l.54Va of hydrastineand0.5-6.07oof berberine.The latter, as a constituentof an extract of the root, is responsiblefor activity againstmultiple drug resistantMycobacteriumtuberculosls(seeE. J. GenIryet al., J. Nat. Prod., 1998,61, 1187)
BOTDOTEAVES
Cqlumbq root Calumba is the dried, sliced root of Jileorhiz,a palmata (J. coltmba) (Menispermaceae),a dioecious climbing plant indigenous to the forests of Mozambique and Madagascar(Malagasy Republic) and other eastAfrican countries.It is exported to Europe from Tanzaniaand the name derives from the fact that it was at one time exoorted from Colombo (Sri Lanka).
Boldo leavesBP|EP are derived from Peumus boldus (Monimiaceae): they contain aporphine-type alkaloids, chiefly boldine. The drug has antihepatoxic activity and is describedin Chapter 30.
Hydrostis Hydrastis (Golden Seal Root BHP, Yellow Roof) consists of the dried rhizome and roots of Hydrastis canadensis (Berberidaceae),a small perennialplant indigenousto the woodsofeasternCanadaand the eastem USA. The wild plantshavebeenexterminatedin many districtsand the speciesnow hasCITES listing (q.v.)for the USA and Canada.Most of the commercial drug is now obtained from cultivated plants grown in America and in Europe. The use of hydrastis,both as a drug and as a dye, was learned by the early European settlers from the Cherokee Indians. Characters. The drug consistsof almostcylindrical rhizomesabout 1-5 cm long and 2-10 mm diameter.The rhizomesgrow more or less obliquely and bear numerous!shortbranches,which terminatein cupshapedscars and bear encircling cataphyllary leaves. Similar scale leaves are found on the rhizome, the outer surface of which is yellowish-brownor greyish-brown.The roots, which originateon the ventral and lateral surfaces,are long and wiry, and in the commercial drug are often broken at a distanceof a centimetre or so from the rhizome. The drug breakswith a short,waxy fracture.It has a slight but distinctive odour and bitter taste. A transversesection of the rhizome shows a fairly thick, yellow or yellowish-brownbark; 12-20 radially elongated,bright yellow wood bundles, separatedby wide medullary rays; a large pith.
Uses. The use of hydrastisto check uterinehaemorrhage,as a bitter stomachicand locally in the treatmentof catarrhalconditions of the genito-urinary tract is largely based on empirical observations. Hydrastine hydrochloride and hydrastinine hydrochloride have been used in various forms to control uterine haemorrhage.
Collection. Attempts to cultivate the drug in various areasdo not appearto have been successfuland collection is from the wild. The plant possessesa somewhat slender rhizome from which numerous large fusiform roots arise. The older reports statethat these are dug up during dry weather (March), the rhizomes are rejected and the roots cut into transverseor oblique slicesand dried in the shade.The imported 'naturalcaiumba'is frequentlywashed, brushedand graded,the product being known as 'washedcalumba'. Macroscopical characters. Calumba occurs in circular or oblique slices.Theseareusually 2-8 cm diameterand 3- I 2 mm thick. The cork is thin, greyish-brownor reddish-brownin colour and longitudinally wrinkled. Within it lies a broad, greenish-yellowzone which extendsto the cambium and containsin its outer part isolated sclerenchymatouscellswithin which aredark-grey,sinuousstrandsof sieve tissue. The greyish wood, which is separatedfrom the bark by a dark cambium line, shows numerous nalrow, radiating lines of yellor.vvesselsseparatedby abundantparenchyma.The vesselsare closetogether in the region near the cambium and again in the extreme centre of the root, but they are less numerousin the intermediatezone, which therefore shrinks considerablyand becomesdepressedon drying. Some
'/l \-l
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P a l m a t i n eR: 1 = R 2 = C H s J a t r o r r h i z i n eR : 1= H , R 2 = C H r C o l u m b a m i n :eR r= C H a ,R 2 : H
oR2 C o l u m b i nR : r : { H , R 2= H l s o c o l u m b i nR : r=- H,R2=H : r= { 5, fz=glycosyl C o l u m b i n ygl l u c o s i d eR
Other diterpenesaresimilarisomersdifferingin the positionsof the epoxidering and in the stereochemistry of the C-12{-13 bond.
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DRUGSOF BIOLOGICAL ORIGIN AND RFLATED PHARMACOPOEIAL piecesshowtwo or more concentriczonesof wood.The fractureis short and starchy;odour,slight and somewhatmustyl taste,bitter. Calumbafrequentlycontainsoccasionalslicesof calllll ba rhiz.ome. Theseaverageabout 2-3 cm diameter.The structureis markedlyradiate and, owing to its greaterwoodinessin that region. is not depressed in the centre.
MeN
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Microscopical characters. The drug is characterizedby the sclereids which have unevenly thickened, yellow walls and contain a number of cellscontaining prismsof calciumoxalate,by abundantparenchymatous starchgrains.eachgrain measuringabout 20-85 pLmlong and having an eccentric,very distinct radiateor cleft hilum, and by the yellow reticulate cellsandvesselson ffeatThe walls ofboth the sclerenchymatous vessels. ment with 667cv/r' sulphuricacid changecolour from yellow to green. Constituents. Calumba contains about 2-3% of isoquinoline alkaloids, palmatine,jatrorrhizine and columbamine.Bisjatrorhizine is a quatemarydimeric alkaloid formed by orrlro-oxidativecoupling of the phenolic group of jatrorrhizine. Other constituentsare the nonalkaIoidal furanoditerpenescolumbin, isocolumbin, palmarin, chasmanthin, jateorin and isojateorin.Some of theseoccur as glucosidesand havebeennamedpalmatosidesA to G. Uses. Calumba is used as a bitter tonic and, as it containsno tannin, may be prescribedwith iron salts.In theBHP it is specificallyindicar ed lbr anorexiaand flatulentdyspepsia.
CURARE 'curare' is generic one applied to various South American The term a anow poisons.Theseextractsaremadefiom a numberof different plants, particularly membersof the Menispetmaceae(.e.g.Chondrodendron.)and the Loganiaceae.From the fomer, tubocurarineis obtainedand the (+)hydrochlorideofthis is includedin the BP asa musclerelaxant. Curaresfrom the upperAmazon (Brazil and Peru) seemto be mainin origin. The original genusChondrodendronhas Iy menispermaceous beendivided into two by Barnebyand Krukoff (Mem. N. Y. Bot. Gdn, 1911,22, l); Chondrodentlronrlsell with three species(Cft. tomentosum, Ch. plaryphyllum, Ch. microphl'llum) and Curarea with four species(Cu. toricoJbra.Cu. cnndit:ans,Cu. tectrnaruntandCu. cttarte(rhe casasii).The principal ones used in curare are Ch. tomentosLLm only plant known to contain tubocurarine),and the first three of the Curarea species.Severalother generaof Menispermaceaeare known to have been. or to be, ingredientsof curares including speciesof Sciadotenio,Abuta, Telitoxicumand Cissampelos,but little is known aboutthe muscle-relaxantactivity.if any, of their alkaloids. The curaresfrom Guyana,Venezuelaand Colombia owe much of their activity to speciesof Strl-chnos(Loganiaceae);around20 species are known to have beenincorporatedinto curaresand theseinclude S. toxifera,S.joberticma,S.peckii andS. guianensis. (For reviews covering the history, uses,botany and chemistry of l;Alkalnids, curareseeN. G. Bisset.J. Ethnopharmacologt',1992,36, Chenl Biol. Perspec't..1992,8, 1.) History. There is a close botanical relationship between Pareira Brava Radix of the seventeenth-and eighteenth-centurypharmacopoeias and the menispermaceousplants yielding curare. In both casesthe botanicalsourceof the drugshaslong beenin doubt.It is now known that Pereira Brava is obtained from two species of Chondrodendron, namely, C. nticroph,,-lhln.which contains (+)bebeerine,and C. platyphllftln. which contains(-)-bebeerine.A similar case is the so-called C. tomentosurz.which sometimes yields
f+)-Tubocu rarine (+)-tubocurarineand sometimesthe less active (-)-tubocurarine.This 'It seemsvery probablethereforethat twt'r led King (1948) to write: speciesare involved underthe nameC. tonlentosum'. Boehm ( 1895) distinguishedthreekinds of curaredifferentiatedfirst b1' theil containers,and secondby their different chemicalcharacteristics. (l) Tube-curare,packedin bambootubes.This came from Brazii and in origin and containedthe alkaPeru.was mainly menisperrnaceous 'tubocurarine'and loid which Boehm (1895)isolatedas amorphous which King (l 935) first preparedcrystallineastubocurarinechloride. (2) Calabash-curareis packed in gourds and comes mainly from Guiana,Venezuelaand Columbia. It was formerly the type of curare most commonly found in commerce.Chemical investigationsof Wieland er al. (193141). King (1949)and Karrer (1946-54) shou thal Str.,-chnos speciesfirrni sh impofi ant constitLrents. (3) Pot-curare,which is no longera commercialarticle,was packedin earthenwarepots. ThesevaLriedin size and were glazed,unglazedor omamentedwith paint.King's examinationof one smallpot led him in origin and conto the conclusionthat it was menispermaceous tainednoStnchnosspp.However.Bauer(i969,1981)showedinan extensive investigalion of museum samplesof curare that pot origin. curareswere usuallyof mixed Loganiaceae/Menispermaceae Curare in tins. Much curare has been imported in tins containing about I kg of a viscous dark-brown or blackish extract. It has little odour but a very bitter taste. This drug is similar to the specimenexaminedby King ( I 948), who receivedit from Asher Kates y Cia S.A. of Lima, Peru, togetherwith the leavesof the plant fiom which it was prepared.Theseleaveswere indistinguishablefrom those of C. tomentosumand its menispermaceousnature,and similarity to the old tube-curare,was confirmed by the isolationof (+)-tubocurarinechloride and four other alkaloids. Constituents. (l) Menispermaceoustube-ctrare and the form now imporled in tins containtubocurarine.From the tin-curareKing (1948) isolated (+)-tubocurarinechloride and four non-quatemarybases(isochondrodendrinedimethyl ether, (-)-curine (bebeerine),(+)-chondrocurine and (+)-isochondrodendrine). (2) Loganiaceous species.parderivesits activity largelyfrom Stryc/rrros calabash-curare ticularly S. toriftra. King (1949)has shownthat the bark of this species contains 12 crystalline quaternary alkaloids, the toxiferines I-XII, of which two had previouslybeen isolatedby Wieland et al. (193141). These latter authors examined calabash-curare.probably fronr Venezuela, andisolatedseveralalkaloidsknown asC-curarines(C signifies calabash).Toxiferines I and II have beenfound in calabash-curare. spp.havebeenexamand S/r'],cftt?o.s More recentlycalabash-curares ined by Karrer and his colleagueswith the isolationof a large number of new alkaloids.(+)-Tubocurarineis a bisbenzylisoquinolinealkaloid and as such is derived from dopamine, whereas the Loganiaceous curaresare indolic and contain C,1gcompoundsof the dimeric strychnine type. In a review of 1979, Guha et al. Iisted 186 bisbenzylisoquinoiine alkaloids occurring in 13 tamilies, principally the
ALKALOIDS Menispermaceae,and divisible into 24 structuralgroups.The group has since been three times reviewed by SchifT(ref. p. 354) who, in 1991.recordedalmost400 alkaloids. Uses. Curare is now little used except as a source of alkaloids. Tubocurarinechloride, official in rhe BP. is used to securemuscular relaxationin surgicaloperationsand in certainneurologicalconditions. Annons squqmosq (Annonoceoe) Variouspartsof this plant havefeaturedin the folk medicineof Africa, India and the Far East for the treatmentof a number of conditions including heartdisorders.The cardioactiveeffecthasbeenattributedto the alkaloid higenamine,an importantprecursorof a number of other isoquinolines(Pie. 27.16).
Higcnamine (Norcoclaurine)
Chelerythrine;Rl = R2 = Me Sanguinarine; Rl + R2=CHz (forms methylenedioxy goup)
Bloodroot Bloodroot consistsof the dried rhizomes and roots of Sanguinaria canadensis(Papaveraceae), a perennialherb widely distributedin the woodsof North America.The drug consistsof dark brown, more or less cylindrical pieces of rhizome, 2-7 cm long and 5-15 mm diameter. Someof the piecesare branchedand someshow numerouswiry roots. The latter, however, are usually broken off in the commercial drug. The rhizome breaks with a short fracture and, if not overheatedduring drying, showsnumerousred dots (secretioncells) distributedthroughout the starch-containingparenchymaof the bark and large pith. If dried at too high a temperature,the secretionescapesfrom its containingcells and the whole sectionassumesa deep red or brownish-redcolour.A ring of small, yellow, librovascularbundleslies about I mm from the outside.Odour, slight; taste,acrid and bitter. Bloodroot containsthe benzophenanthridine alkaloids sanguinarine,chelerythrine,allocryptopine,protopine and dihydrosanguilutine.Sanguinarineand chelerythrine, although themselvescolourless,form red and yellow salts, respectively.The drug alsocontainsred resin and starch. In a report by Rho et al. (Appl. Microbiol. Biorechnol., 1992,36, 611) S. canadensiscultured cells produced sanguinarine(about 807c of the total alkaloid) together with chelirubine and chelerythrine.In contrast, in the normal rhizome sanguinarine and sanguirubine '/0Vo together account for some of the total alkaloids. (For elicitor studies see G. B. Mahady and C. W. W. Beecher,Phytochemistry, 1994.37. 115.\ Bloodroot is used mainly in the USA, where it is an ingredientof Compound White Pine Syrup. Sanguinarine,like colchicine, causes the doublins of the chromosomesin cells.
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TETRAHYDROISOGIU INOLINE MONOTERPENOID AIKAIOIDS AND GLYCOSIDES Thesealkaloidsand alkaloid-glycosidesderive from the condensation of dopaminewith secologanin(a C 19monoterpene)to give two series of compounds.The bestknown examplesof their limited occurrence are in speciesof Cephadlis(Rubiaceae)andALangium(Alangiaceae). The former givesipecacuanha root, and the root, bark,fruits and leaves of A. Lamarckiiare used in Ayurvedic medicinefbr the treatmentof a numberof conditions.
IPECACUANHA Ipecacuanha(lpecacuanhaRoot) of the BP is the dried root or rhizome and root of Cephatlis ipecactnnha (Brotero)A. Richard (Rubiaceae), known in commerceas Matto Grosso Ipecac.or of Cephaiilisacumir1dl,rKarsten,known in commerceas CostaRica lpecac.It shouldcontain a minimum of 27cof ether-solublealkaloids. C. ipecctcuanhais a shrub 20--'10cm high found over a large area in Brazil, particularly in the moist and shady forests of Matto Grosso and Minis Geraes;plantationshave been establishedin the Matto Grosso area.It is cultivatedto someextentin Malaya,Burma andthe Darjeeling Hills of West Bengal. C. acuminuta is exported from Colombia, Nicaraguaand CostaRica; CostaRica is at presentthe principalsource of the drlg. HoweverIndia is now in tull productionof CostaRicantype root which is of high quality (in excess of 3.5c/ototal alkaloid) and extremelycompetitivein price;extractsofthe Indian root arenow being produced and exported. History. What appearsto have been ipecacuanhawas mentioned, underthe nameof Igpeca.ta,bya Porluguesefiiar around 1600.It was introducedinto Europein 1672. Collection and preparation. In the Matto Grossodistrict of Brazil the drug is collectedfrom wild plants.The collector,using a pointedstick, levers the plant from the ground and, having removed most of the roots, replacesit in the ground, where it usually lives to producefurther crops. The roots are dried in the sun or by fires and transporteddown river to ports such as Rio de Janeiro, Bahia and Pernambucofrom which they are exportedin bales.Other SouthAmerican ipecacuanhasare collected in a sirnilar way. The supply of ipecacuanhahas been very eratic for many years and the high price favours cultivation and stimulatesefforts to producethe alkaloidsby cell culture (seebelow). Only modestsuccesshas resultedfrom the effofts to grow the drug in Malaya. Macroscopical characters. The undergroundportion consists of thin. horizontal rhizomes from the lower surfaceof which roots are given ofT. Some of the latter remain thin, while others develop an abnormallythick bark and becomeannuiated. The Matto Grossodrug occursin tortuouspiecesup to I 5 cm long and 6 mm diameter,but it is usually smaller.The colour of the outer surfacevaries from a deepbrick-red to a very dark brown, the colour being very largely dependenton the type of soil in which the plant has been grown. Most of the roots are more or less annulatedexternally, and somehave a portion of the rhizome attached(Fig. 27.19A), while separatepoftions of rhizome and non-annulatedroots are also found. Generally,the drug of present-daycommerceis lessmarkedly annulated than was formerly the case,a fact that points to earlier collection. The ridgesareroundedand completelyencirclethe root; hereand there the bark has completely separatedfrom the wood.
E
DRUGSOT BIOLOGICAL ORIGIN PHARMACOPOEIAL AND RELATED
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The root breakswith a shortliacture and showsa thick greyishbark and a small,densewood, but no pith. The rhizomes,on the otherhand. havea much thinnerbark and a definitepith (Fig. 21.19C,D). The drug has little odour, but is irritating and sternutatory when in fine powder, and has a bitter taste. The CostaRica drug (Fig. 27.198) is exportedfiom Cartagenaand betweenthe Rio and Cartagenadrugs Savanilla.The main ditTerences are listed in Table27.5. Ipecac'uanhu.rler?s. although containing the same alkaloids as the roots,usuallycontainthem in smallerpropoftion.An excessiveamount of stemmust. thereiore,be regardedas an adulteration. Microscopical characters. A transverse section of the root (Fig. 21.l9C) showsa thin, brown cork, the cells of which containbrown, granular material. Within this is a wide secondarycortex (phelloderm). the cells of which :Lreparenchymatousand contain starch,usually in compound grains with from two to eight components,or raphidesof calcium and up to l5 or 20 oxaiate.The individual starchgrainsare mr.rller-shaped pm diameter.The phloem is entirely parenchymatous,containingno sclerenchymatouscells or fibles. The compactcentralmassof xylem is com-
loble27.5
Comporison of ipecocuonho (Cepho6lis) roors. C. acuminolo
C. ipecocuanho
4-6.5 mm Greyish-brown Lesscrowdedond lessproiecting groins groinsup to 15 prm lndividuol Individuol up to 20 prm
Usuoldiometer l-4 mm Brick-redto brown Colour Annulotions Verycrowded Storch
posedof small tracheidalvessels,tracheids,substitutefibres,xylem fibres and xylem parenchyma.Starch is presentin the xylem parenchymaand substitutefibres contain starch(Fig. 27.198-H). The transversesectionof ipecacuanharhizome (Fig. 27.19D) shous the presenceof a ring of xylem and a large pith. The pericyclecontains cells.Spiralvesselsoccurin the protocharacteristicsclerenchymatous xylem. The pith is composedof pitted parenchymawhich showssonre lignification. 'ipecacuanhas' were regularll Adulterants. At one time other imported,the namebeing appliedin SouthAmerica to a numberof dit: ferent roots which were reputed to have emetic properties. Most of these, briefly describedin the 10th edition, are very easily distinguishedfiom the genuinedrug and arenow rarely imported. Constituents. Ipecacuanhacontainsthe alkaloidsemetine(Pelletier and Magendie, 1817), cephadline(Paul and Cownley, 189,1),ps1 chotrine,psychotrinemethyletherand emetamine.Theseareisoquino line derivativesof a group only known with cefiainty to occur in the lamilies Alangiaceae,Icacinaceaeand Rubiaceae.However, emetinr-type alkaloids are not necessarily a characteristic of the genus 33, 1117t Cephadlisas a whole as Soliset al. (.Phytochemistm,l993, recordedanew indole alkaloid and four other known indole alkaloids from the aerial parts of C. dichroa irom WesternPanama. In a review (411 refs) ofthe ipecacuanhaand relatedbases(T. Fujii and M. Ohba, The Alkaloids, 1998, 51, 21 l) 39 new alkaloids frorn ipecacuanhaandAlangium arerecordedfor the 14 yearsto 1997. Other constituentsof the official drug are monoterpenoidisoquinoline glucosidesincluding ipecoside(Fig.27.21),alangisideand a number of recently-isolatednovel glucosides(A. Itoh et al., Chem.Pharnt. 8u11., 1989,37, ll37 ; Phytochemisrtl', 199 1, 30, 3 117; 1993,32, 761).
tll
rdf O
o,A
ll)
.-)
"9";%-a";
?ao@s";,s"
" G ,
fa-;*- N
/il M H
Fig.27.19 ;f , n sev(ebrostehsxel )c; t iroonootD , C. e p h o i l i s i p e c o c u o n h o r o o t s w i t h r h i z o m e ; B , C . o c u m i n o t o r o o t s w i t h rCh,ilzr o m l p e c o c u o nA ho colcium in cold loctophenol); G, idioblostcontoining tronsverse sectionof rhizome{bothx4); E, corkcellsin surfoceview;F,storchgronules(mounted onnulotion of o2, incomplete of C. ipecocuonho; fromSchultze mocerotion of wood (ollx200). o1, Completeonnulolion H, elements oxololecrystols; ph, phloem;rh, rhizome;tr.v, colciumoxolote;p, pith;pd, phelloderm; f, fibrouscell;id, ldiobloslcontoining ck, cork;e, endodermis; C. ocuminoto; trocheidvessel; xy, xylem;xy.p,xylemporenchymo.
ALKALOIDS The iridoid glucosidesswerosideand 7-dehydrologanintogetherwith starchand calcium oxalateare alsofbund in the root. Ipecacuanhinand ipecacuanhicacid, originally designatedglycosidal tannins, ale now thoughtto havebeenimpure mixturesol ipecosideand sucrose. As rnay be seenfrom Fig. 21.20, the principal alkaloidsare closely relatedto one another;emetineand psychotrinemethyletherare nonphenolic,whereascephadlineand psychotrineare phenolic.
Thus, emetine,which is the alkaloid usually requiredin medicine, may be preparedby methylatingthe cepha6lineoriginally presentin the drug. Thesealkaloidsmay be legardedasbeing formed in the plant from two phenylethylamineunits and a C9 terpenoid precursor.The latter is provided in the plant by secologaninand is incorporated via desacetylisoipecoside into the emetine alkaloids (Fig. 21.21'1. The glucosidic compound ipecoside is formed fiom the epimer
crrHrrorNr.oH
.t".r.
Cephaeline \
cH30
cH3o cH30
cH3O cH2,cH3
cHr.cH3
cH3o
cHsO cH3o
HO
Emetine
Psychotrine
Fig,27.20 Relotionship between theprincipol olkoloids
\
^f i^^.^",,^^h^
.,F czeH3,o3Nz.oc Psychotrinemethylether
ilon,. + ffiu,,. cHrozc&o
Secologonin
Doponine q-egime? fonnalion | | F-epit"t
formolion
,/\
DesocelyIisoipecoside
Desocelylipecoside
Y Y
I lpecoside
Fig,27.2r Proposed biosynthetic sequence for thebiosynihesis of cephoeiline, emetine ipecoside ond theolkoloidolglucoside {seeN. Nogokuroet ol., PlontoMed., 1978,34,3811.
rffir*.,
R. H ' CePhoeline R =CH3,Emetine ocH! OR
@
PHARMACOPOEIAT AND RELATED DRUGSOF BIOLOGICAL ORIGIN
'i:,t1.'tt
l:.:
(cf. Fig.27 .27, which illustratesthe involvementof desacetylipecoside secologaninin the fbrmation of some indole alkaloids; in this case, however,only the ct-epimer,strictosodine,is formed,but rt can serveas a precursorfor alkaloidswith B-configuration).The recentisolationby lloh et ol., Chem.Pharm. Bull.,1994,l7,1460) of tetrahydroisoquinoline-monoterpenoidglucosides,from Alangium lamarckii fiuits, with the samestereo-configuration as desacetylisoipecoside has supported the role of the latter as an intermediatein the formation of emetine-type alkaloids. Furthermorecell-free extracts of A. lamarckil have been shownto containtwo enzymeactivitiespromotingthe condensationof dopamineand secologaninto give the (S)- and (R)-productsrespectively (W. De-Eknamkulet aL.,Ph.t:tochemistr\', 1991,45,477). Variousvarietiesof ipecacuanhacontaindifferentproportionsof the principalalkaloids.Thus,the Rio drug, which is now difficult to obtain commerciallyand is the most esteemed,contains2-2.4Vaalkaloids.of which 60-757c is emetine.Thosevarietiesderivedfrom C. acuminattt yreld2-3.57c alkaloids,of which emetinemay constitute30-50Vc. Cell and root cultures. Production of the ipecacuanhaalkaloids by artificial culture would be commercially highly desirable and some researchin this area has been repofted. The composition of the cr.rlture medium and the natureofthe addedhormonesgreatlyinfluencesalkaloid productionbut generallyroot culturesappearto be more satisfactorythan callus or suspensioncultures.Increasedgrowth of roots is obtainedby the induction of hairy roots. In contrastto whole roots, cell culturesproduce more cephaelinethan emetine,and immobilized cell systemsgive higher amounts of cephadlinecomparedwith static cell cultures. (For original researchpapersseeK. Yoshimatsuet aL.,Phybchemistry,1991.30,507; S. Jhaet al., ibid.,30,3999;C.Veeresham e/ al., ibid.,1994,35,947.) A simple one-stepmethodfor the productionof ipecacuanhaplants fiom root cultures has been described (K. Yoshimatsu and K. Shimomura,Plant Cell Rep..1994,14,98). Test for emetine. Mix 0.5 g of the powdered drug with 20 ml of hydrochloric acid and 5 ml of water; filter, and to 2 ml of the filtrate add0.01 g ofpotassiumchlorate;ifemetineis present,a yellow colour appears,which, on standingfor about I h. graduallychangesto red. Prepared lpecacuanhais the drug reduced to a fine powder and adjustedto contain 1.90-2.10%of total alkaloids. Uses. Ipecacuanhais used as an expectorantand emetic and in the treatment of amoebic dysentery (Chapter 29). Emetine has a more expectorantand lessemetic action than cephaeline,a fact that accounts for the preference shorvn for the Brazilian drug. In the treatment of amoebicdysenteryemetinehydrochlorideis frequentlygiven by injection, and emetineand bismuth iodide by mouth. Psychotrineand its Omethyl etherare selectiveinhibitorsof humanimmunodeficiencyvirus and their study could lead to the developmentof therapeuticallyuseful agents(G. J.Tan et al., J. Biol. Chem.,1,991,266,23529).
coctrrANA CocillanaBP (GrapeBark, Guapi Bark) is the dried bark of Guarearusbyi (Meliaceae)and other closely related species.The treesare native to the SouthAmerican Andes and the bark is collectedin Bolivia and Haiti. Macroscopical characters. The commercialbark which has a slight aromatic odour, occurs in lairly large flattish or curved pieces,up to 60 cm long and 5-20 mm thick. Externally,the cork may be quite extensive and fissured,but the outer layers are missing in some areasand covered by lichen patchesin others.The inner surfaceshows Iongitudinal striations and is lighter in colour than the grey-brown or orange brown outer tissues(Fig. 27.224,8).
Microscopical characters. The lignified cork cells occur in bands alternatingwith layers of lignified parenchymaand sclereids.In the phloemthe narrow medullaryrays,somecells of which aresclerenchymatous,run betweennumerousfibre groups,each containinga prisnr sheath.Many cells contain pigmentedcontentsand a little starch is present(Fig.27 .22 C-H'). Constituents. There appearsto be no recentwork which delineates the active constituentsof the drug and it is placed in this position becauseof its associationwith ipecacuanha. Arising from work carried out at the end of the nineteenthcentury,the bark is describedas contarntng237oresins,2.57cfixed oil, tannin.a small quantityof alkaloid and possiblya glycoside.A studycarriedout in I 966, which duplicated the earlier methods of extraction and fractionation, gave diiferent resultsand addedcredenceto the widespreadbelief that presentcommercial suppliesmay not be identicalwith the earlierones. Uses. A liquid extract,with other ingredients,is usedin the form of a linctus as an expectorantgiving an alternativeto ipecacuanhain the treatmentof coughs.
n rK A t o l D s , , ' , , , ' , ' ' , : , : r , ,'.',{ *vltibAC,EA The bulbs of this family are well-known for their toxic properties,rt leastone fatality in the UK being recordedin 1999 as a result of mistakenconsumptionof dafTodilbulbs for onions. The alkaloidsare derived from one molecule of phenylalanineand one of tyramine and biochemicallyfall into three seriesdependingon the type of oxidativephenoliccouplingundergoneby the precursorp'(fbrmulaFig. 27.13).TheseseriesarerepresentO-methylnorbelladine ed by the alkaloids haemanthamine(p,7:r'coupling,lycorine (o,p' coupling)and galanthamine(.o',p coupling)(Fig. 27.23),all threetypes often co-occurring in one species.Some 20 genera of the family produce these alkaloids but in the past they have been of minor significance in Western medicine. However Narcissustazetta \tar. chinensis,the bulbs of which contain lycorine, pseudolycorineand tazettine,is describedin Chinesetraditionaland herbalremedytexts. O. Hoshino has reviewed (203 refs) the Amaryllidaceaealkaloids considering ten types plus miscellaneousalkaloids (The AlkaLoid.s. 1998.51.323). Currentlythereis much interestin galanthaminewhich hasemerged as a useful drug fbr the treatmentof Alzheimer's disease. Golonthomine This base was first isolated from the Caucasiansnowdrop Galanthus woronovtiby Russianworkersin 1952and subsequently by other scientistsfrom the commonsnowdropG. nival.i,s.Itwasrapidly shownto be a metaboliteof many speciesof the family (J.J. Willamanand H.-L. Li, J. Nttt.Prod.,19'70,33,17)liom which Leucojwnaestivumhasemergedas a commercial source with the consequencethat wild plants are. like many other medicinalplants,becomingan endangered species. A. Poulev et til. (Planta Medica, 1993,59, 4.12)have used ELISA techniques(Ch. 14) for the quantitativedeterminationof galanthamine in plant materialsand obtainedyields of from 0.1 to 2.0% for the dried bulbs of L. aestivumwith Phaedranassanegistrophl'llafrom Perugivtng 7.47o. Various Narcissus cultivars have been examined for the alkaloid and it has been shown that planting depth, planting density, bulb size and flower bud removal did not affect the galanthaminecontent (R. M. Moraes-Cerderaet al., Planta Medica, 199'7,63,4'72). There is a considerablebody of literatureon the pharmacologyof galanthamine(seereview by A. L. Harvey,Phurmac. Ther., 1995,68.
ALKALOIDS
ckz
tph
p.f g.s
Co oeod 6H Fi1.27,22 section;E, fibre sectionof bork (x1);D, corkcellsin tronsverse surfoceof bork;B, internolsurfoce{bothxO.5);C, lronsverse Cocillono.A, Externol medulloryroy in tronsverse longitudinol section; G, porenchymo ond with porlionof crystolsheoth;F,portof prismsheothof fibrewithossocioted with outercorkremoved; ck, corkcells;ck1,outercork;ck2,newlyformedcork;f.ph,fibrous H, storchgronules(ollx2OO).o, Exferior sclereids; strioe;m.r,medulloryroy cells;ox, prismof colciumoxolote;p.c,cellwith pigmented phloem;g.s,gronulorsurfoce;l, lichenpotch;l.s,longitudinol roy; s.f,splintery frocture. fibres;sc1,sclereidloyer;sc2,sclereid of medullory conlents; p.f, protruding
I l3) and readerswill note a possibleconfusion in nomenclaturein a number of the more recent publications by use of the designation 'galantamine',a name also employedby the manufacturerto describe the new productmentionedbelow. inhibitor and as such has Galanthamineis an acetylcholinesterase been used extensiveiysince the late 1950sin EasternEurope and the former Soviet Union in anaesthesiaas a curare reversal agent. Secondly, the alkaloid also acts centrally by penetrating the blood-brain barrier to serve as a modulator of nicotinic cholinergic It is receptors,thus augmentingcentralcholinergicneurotransmission. the latter which has invoked investigationinto its use in the palliative treatmentof Alzheimer'sdrsease. Clinical trials with the isolatedalkaloid involving patientspresenting with mild to moderate symptoms of Alzheimer's diseasehave given positivefindings for the improvementof memory and intellectual functioning.The drug, as Reminyl@,was grantedEuropeanmarketing approvalin Juiy 2000.
ALKALOIDS PHENETHYTISOGIUINOLINE This group of alkaloidswas flrst recognizedin 1966during investigations on the biosynthetic origin of colchicine, the principal alkaloid of the
m,
autumn crocus. They representanaloguesof the benzyltetrahydroisoquinoline alkaloidsand are found in a number of generaof the Liliaceae. Colchicine-typealkaloidsare presentin many speciesof Colchicum (e.g. C. Iuteum and C. speciosum).Also. the generaAndrocvmbium. Bulbocodium, Camptorrhis, Dipidax, Gloriosa, Iphigenia, Littonia, Merendera, Ornithoglo,ssumand Sandersoniapossesssimilar constltuents.
COTCHICUM SEEDAND CORM Colchicum seed and corm are derived fiom the autumn crocus or meadow saffron,Colchicuntauttunnale(Liliaceae).The plant, whose life cycle is describedbelow, is found in Britain and in many other parts of Europe. Commercial supplies come from Poland, Czechoslovakia,former Yugoslaviaand The Netherlands.Colchicum luteumis usedin Indian medicine. History. Drugs believed to have been derived from species of 'colchicum', Colchicum have long been known under the names of 'hermodactyl','surinjan'and 'ephemeron',and somehavebeenidentified as C. ctutumnale.Dioskurideswas aware of the poisonousnature of a Colchicumwhich may or may not havebeenthe speciesnow used in medicine.The genus derives its name irom Colchis on the Black
@
PHARMACOPOEIAL AND RELATED DRUGS OF BIOLOGICAL ORIGIN
::::
Haemanthamine
,.ll
:i
Lycoflne
Fig.27.23 (+r"r+"rnl
t',noc nf
Amoryllidoceoe olkoloids
Galanthamine
Sea,one ofthe placeswhere this plant is found. The drug was recommendedin Arabian writings for use in gout, but it was little employed in either classicalor mediaevaltimes. owing to the wholesomef'ear inspiredby its poisonousproperties.Colchicum corm appearedin the LondonPhannacopoeia,i of 1618,1627,1632and 1639.It was then deletedbut reappearedin the edition of 1788.The uncertainaction of the corm led Dr W. H. Williams, of Ipswich, to introducethe useof the seedsabout 1820. and thesewere admitted lo the Pharmacopoeiaof 1824.Colchicinewas isolatedby Pelletierand Caventouin 1820. Life cycle. The corm consistsofan enlargedundergroundstembearing foliage leaves,sheathingleavesand fibrous roots. If the plants are examined in the latter pafi of the summer it will be found that a new corm is developingin theaxil ofa scaleleafnearthebaseofthe old corm,the new plant occupying an infolding in the sideof the parentcorm. In September the parentcorm bearsthe remainsof recentlywithered leavesand is very much larger than the daughtercorm. For medicinal pulposes the cotm would havebeencollectedshortly after the withering ofthe leaves('early summer') and befbre the enlargementof its axial bud. The corms are surroundedby a dark. membranouscoat. The young corm developsfibrous roots at its base.and in Au-eustor Septembertwo to six flowers emerge tiom it. but its foliage leavesdo not appearaboveground until the following spring.The flowers are 10-12 cm long. Each has six stamensand a perianthconsistingof six lilac or pale-purplesegmentswhich fuse into an exceptionally long perianth tube, at the base of which lies the superior ovary. More than half the length of the flower is below ground. and the fruit lies protectedthroughout the winter by the surrounding com and earth.The truit is a threelobed.three-celled, septicidalcapsule,which is canied above ground in the spring by the expanding leaves.The fully grown leavesare radical,linear-lanceolateand about l2 cm long. During these changesthe daughter corrn grows at the expenseof the parent, which now graduallyperishes.Beforedoing so.howeveqit may produce in its secondspring one or more small cormsby meansof which the number of plantsmay be increased. Characters ofseed. The seedsare collectedwhen ripe, usually in July or August, and dried. They are ovoid or globular in shapeand 2 3 mm in
diameter.They are extremely hard and have a reddish-brown.minutely pitted testa.During drying the seedsdarkenin colour andbecomecovered with a sugaryexudation.The seed,asin mostLiliaceae.developsfrom an amphitropousovule. From a slight projection at the hilum there extends for aboutone-quarterof the circumf-erence a well-marked strophiole.Thc smallembryolies embeddedin holny endosperm. Microscopical examinationshows that the testa consistsof somewhat thick-walled reddish-brown parenchyma;that the endosperm cells havepitted walls and containfixed oil and aleuronegrainsup to 5 pm in diameter;and that the strophiolecontainsstarch. Colchicum seedscontain0.6-l .2c/oof colchicine.a numberof other colchicine-typealkaloids.a resin.fixed oil and reducingsugars. Characters of corm. The corms are collected about July. cut into transverseslicesand dried at a temperaturenot exceeding65'C. The outer membranesare rejected. The whole corms are 2-3 cm diameter. but the dried drug consistsof somewhatrenifbrm,transverseslicesand occasionalmore ovate longitudinal slices, about 2--5 mm thick. The epidermalsurfaceis cinnamon-brownand slightly wrinkled. The interior is white and starchyand. if carefully smoothed.shows scattered fibrovascularbundles.The drug breaks with a short mealy tiacture. The odour is much less marked than in the fi'esh drug. Taste,bitter. Microscopicalexaminationshowsnumerousstarchgrainscontained in parenchyma,some simple but the majority consisting of two to sevencomponents.Individual grainsarefrom 6 to 30 ptmdiameter.and hilum. Their shapevariesfiom sphershow a triangularor star-shaped ical or ovoid to polygonal.Vesselswith a spiral or annularthickening and portions of brownish epidermis with very occasional circular stomatamay aisobe seen. On treatingthe drug with 60-70% sulphuricacid or with concentrated hydrochloric acid, a yellow colour, due to colchicine. is produced.The corms contain up to about 0.67c colchicine, other related alkaloids and starch. Colchicine This is an amorphous,yellowish-white alkaloid. which darkens on exposureto light and gives a yellow coloration with strong mineral acids.Colchicineleadily dissolvesin water.alcohol or chloroformbut is only slightly solublein etheror petroleumspirit. It is a weak baseand may be extractedfrom either acid or alkaline solution by means of chloroform.ColchicineBP is assayedby non-aqueoustiuution. The rather unusualchemical structureof colchicine meant that its probablebiogeneticorigin fiom simpler moleculescould not be easily predicted.Examplesof the occurrenceof the tropolone ring (ring C) are rare in higher plants, although it featuresin mould metabolisml also,the position of the nitrogenatom is unusual.Owing mainly to the work of Battersby,Leete and their coworkersinvolving tracer studies on C. autumnaleand C. byzantium.the principal pathwayfbr the biogenesisofcolchicine has now beenestablished. Ring A and carbons5, 6 and 7 are derivedfrom phenylalanine;the from tyrosineby ring cleavagefollowed by clotropolonemoiety :Lrises ring. In contrastto mould metabolism. sureto give a seven-membered acetatedoes not contribute directly to the tropolone ring but is merely eff'ectivein supplying the N-acetyl group. An intermediateformed early in the pathway as the result of union of the two amino acids is a 1phenylethylisoquinoline derivative.This is a memberof a classof alkaloids first reported in 1966, the tirst two representativesbeing androcymbineand the dimer melanthioidine,alkaloidsof Androcymbium melanthioides,a closerelativeof colchicum.Demecolcine,also a constituentof Colchicumspp..is a more immediateprecursorof colchicine. The sequentialformation of thesecompoundsis indicated rnFtg.21 .24. (For a studieson the early stagesof colchicinebiosynthesisleadingto the
ALKALOIDS
Z:\\t/'\\.cozH
I
ll
Ju*. +
Hol\.,"
Tyrosine
COzH
o
I
H
)
Phenvlalanine
@H:
l
A phenylethylisoquinoline derivative
cH30 Ring cleavege
cH30
and expansion (mechanism not indicated) OCH:
Fig.27.24 Steps in thebiogenesis of colchicine.
R = CHr; Demecolcine R : COCHr: Colchicine
formation of phenethylisoquinolineintermediatesseeR. B. Herbefi er a1.. Tetrnhedrctn.f990, 46,7 I I 9 and for recentrefinementsto the biosynthetic sequence seeA. Nasreenet ol., Phy-tochemistry, 1997,46. l1i). The richest sourcesof colchicine are the corms and seeds.but the difficulty of obtainingadequatesuppliesof thesehas 1edSantavj'and coworkers(.PlantaMed., 1979,36, 1l9l 1981,43, 153)to investigate the possibility of using leavesand llowers fbr extractionpurposes.In colchicinecontentthe flowers comparewith the seeds.The leavescontain only one-fifteenththe alkaloid contentofthe seedsbut, compared with the corms,they containhalf the amount of 2-demethyl-demecolcine. The latter alkaloid can be chemically convertedto demecolcine. On slow drying of the leaves,the proportionof 2- and 3-demethylated derivativesof colchicineincreases;thesearenot glycosidicbreakdown productsbut arisefrom unknown compoundsas a result of enzyrnatic liberation.Sr"rspension and callus culturesof C. autumnalehave been shownto producecolchicine.
cH30
Dienonerelatedto methvl androcymbine
reactioninvolving the c-carbon atom of the indole nucleusaffordsa Bcarbolinederivative;reactioninvolving the B-positiongires rise to rn indolenine(Fig. 27.25). A number of simple tryptarninederivativesand B-carbolineshave psychomimeticproperliesand are illustratedon p. 499. For a review of their phytochemistry,chemotaxonomyand phalmacology,see Allen and Holmstedt(.Phytochemisrn,, 1980,19, 1573). Some examples of alkaloids of pharmaceuticalintelest. derived from tryptamine,are given in Ftg. 27.26.It will be notedthat the more complex indole alkaloidscontain a nontryptophan-derived portion of the moleculeand this is suppliedby rnevalonicacid. which in the case of the ergot alkaloidsis a C5-isopentenylunit and with the alkaloidsof the Apocynaceae,Loganiaceae.Rubiaceae etc.. a C16-ger"aniol (monoterpenoid)contribution. Some 2000 monoterpenoidalkaloids are known and Fig. 27.21 tll:ustrates how a number of alkaloid types n i t h i nt h i sg r o u pc a na r i : e . A key intermediatein the biogenesisof the monoterpeneindole Uses. Colchicum preparationsare used to relieve gout. but must be alkaloids is 3cr(S)-strictosidine; it was first isolatedin 1968by G. N. employed with caution. Colchicine is frequently prescribedin tablet Smith from Rhaz-t:a stricta and until 1997 its structurewas basedon form and transdermalpreparationscontainingcolchicine are the sub- compoundsof known stereochemistry, then.direct instrumentalmeasject of a Japanesepatent( I 99 I ). The alkaloid is alsousedin biological urements furnished its lirst detailed stereochemical analysis experimentsto produce polyploidy or multiplication of the chromo- (A. PatthyLukats.,/. Nnt.Prod., 1991 ,60,69). It is fbrmed by the enzysomesin a cell nucleus(seeChapter12). matic condensationof tryptamine and secologanin(Fig. 27.28). The enzymeresponsible for this importantreaction.strictosidine synthase. Further reoding has been isolatedand characterizedflom cell culturesof a number of LettelloC 2000Thepharmacology andtherapeutic aspects of colchicine. species including Rauu'olfia serpentina. Cittchona robu.stu and Alkaloids 53:287-352 Catharanthtrsroseusand a number of isofolms have been described. The R. serpentina generelating to this enzyme has been cloned and heterologouslyexpressedin microorganismsincluding Escherit:hia c'r.tliand Saccharomy'ces cerey'isiue(baker'syeast).This examplerepWith a few minor exceptions,tryptophanand its decarboxylationprod- resentedthe first cloning of cDNA fbr an enzymeof alkaloid biosynuct, tryptamine.give rise to the large classof indole alkaloids.These thesis.The geneis a singlepolypeptideM, about 34 000, possessinga basesusually contain two niffogen atoms;one is the indolic nitrogen 5.3% carbohydratecontent.An investigationof 10 spp. of Rautrolfia and the secondis generallytwo carbonsremovedfrom the B-position using a polymerasechain reactioncomparisonshowedthe geneto be of the indole ring. Of the several alkaloid groups within the indole highly conserved.which was unexpectedconsideringthe geographical class,two may be produced.dependingon the type of condensation rangeof the speciesand the fact that it would be conventionallyconoccurringbetweentryptamineand an aldehydeor ketoacid.A Mannich sideredas an unimportantgeneof secondarymetabolism.
'tu,
DRUGS ORIGIN AND RELATED OF BIOLOGICAL PHARMACOPOEIAL
rffi \z^ilY
c-condensation
f-cerbolinc
G-..Y-..,, H
Tryptamine
/i-condcnsation
Fig.27.25 Formotion of theindoleolkoloids
An indoleninc
oPo!H
4ffi li I \. v /
/
/
/
/
PrrlocYbrn
cx,xx coo-,41-, tH'.--| t t l * \.4n,\N.'/
-
/
?
-/
ll
ll
\/.-x.
r
r cH' u,"f,li
cooH
Zr-.,>___-1.-\.a.cooK
I
+NH ./ \ cH, cH'
ll rl H
C)"-.",
n,.
ltSot altdcids
Trypto,Phm
ffi VA'-J
t*l*.
H
iH'
,V
LY*6ic
rid
"**nI atid
rttin*on
I
J
Fig.27,26 os Tryplophon ond tryptomine precursors of indoleolkoloids.
Strictosidineglucosidasewas reportedin 1996 from a suspension divaricata (T. J. C. Luijendijk et al., cell culture of Tabenruemontana P hytochemist n-, 1996,41, 145 1). Prior to 1977, 3F(R)-vincoside(Fte. 27.28), the epimer of 3o(S)strictosidine was acceptedas the naturally occurring precursor of the monoterpenoidindole alkaloidsand elaboratebiomimetric modelshad to be conceivedto accommodatethe necessaryinversion of configuration at C-3 to give the naturalalkaloids.Quinine is also derivedfrom tryptophanbut this is not immediatelyobviousby inspectionof its for'Cinchona'. The antileukaemic mula; its biogenesisis outlined under alkaloids ol Catharanthus roseus, vinblastine and vincristine are
Stychnine
dimeric alkaloidsof this group (seeChapter28) and their biogenesis production in artificial culture and enzymic aspects remain a most active areaof research. (For articleswhich encompasssome aspectsof the above seeT. M. Kutchan, Phytochemistt!, 1993, 32, 493; A. I. Scott, Chemistry in Britain,1993, p. 681, Tetrahedron1992,48, 2559.)
ERGOTAND ERGOTAIKAIOIDS
ErgoI (Ergot of Rye) rs the dried sclerotium of a fungus, Cluvicepspurpurea Ttlasne (Clavicipitaceae),arising in the ovary of the rye, Secale
ALKALOIDS
Z-
(see'troptenoid BiotYnth€rit')
\ OH
-1( 'Vo" \,
O-Gluc.
--+ l'teOOC- \z Loganin
Genniol Mevilonic acid
II
J
(4-",".
bt
'-""oo.4r,,6
SerPcntinc (ring C reduced : aimalicinc)
S€cologanin
Corynanhe'tYPc !lk!loids
lt
l
r l l b
a..A.* Iiogo-tyPe alkaloids
Fig.27,27 ocidinlo of mevolonic Incorporotion s o m ei n d o l oe l k o l o i d s .
)9
l i
/
\
dkaloids AspidosPetma-ryPc
/
N H
l,le Cathrrsnthane
Vindoline
Life history and collection. The fungus C. purpurea and other speciessuch as C. nticrocephalaWallr.,C. nigricans Tul. and C. pasprzliproduceergotson many membersof the Gramineae(including the genera Triticutn, At'ena. Festuca, Poa, Loliuttt, Moliniu and Nurdus) and Cyperaceae(including the genera Scirpus and Ampelctdesma). Many of theseergotsappearto be extremelytoxic and to producetypical ergotism. In the caseof the rye, the plant becomesinfected in the spring or of the fungus.Thesearecarriedby the early summerby the ascospores base of the young ovary, where in damp insects to the and ergotism wind or by whether ergot doubt as to There is considerable History. 'ignis weather they find sufficient moisture to germinate, forming filamentwere known to the ancients,and it is impossibleto say whetherthe sacer' of the Romans refered to ergotism. The outbreaksof ignis St ous hyphae which enter the wall of the ovary by enzyme action and Antonii', or St Antony's fire, which occurredduring the Middle Ages, do, form a soft,white massover its surface.During this stagethe sphacelia, however, appear to have been of ergot origin, Outbreaks of ergotism as the white massis called,producesa yellowish, reducingsaccharine 'honeydew'. At the same time chains of small oval conioccurredin Germanyin 1581,1587and 1596and at intervalsin Europe secretion, until recent years. Ergotism was never common in England, probably diosporesare abstrictedfrom the ends of some of the hyphae. The owing to the fact that rye is little grown, and the only seriousoutbreak honeydew attractsants, weevils and other insects,which carry the conidiosporesto otherplantsand so spreadthe disease. recorded,which took place rn 1762,was causedby wheat. During the sphaceliastagethe hyphaeonly penetratethe outer pafi World-wide, sporadicreportsof ergot poisoning still appearin the literatureand in 1992 an analysisofrye flour sold in Canadashowed of the ovary, but as developmentproceedsthey penetratedeeperand that low-level contaminationby the fungus still exists;of 128 samples deeper,f'eedingon the ovariantissueand finally replacingit by a cornwhich fbrms the scierotiumor resting pact tissue(pseudoparenchyma), tested 118 proved positive for ergot alkaloids at concentrationsof 70414 ng g-1 whereaswith wheatflour the incidenceand levelswere stageof the fungus.The sclerotiumincreasesin sizeduring the summer and projects,bearingthe sphacelialremainsat its apex,from the ear of much lower. The obstetricuse of ergot was known in the sixteenthcentury,but the rye. The number and size of the ergotsproducedon eachspike of cereal the drug was not widely employeduntil the nineteenthcentury.It was first introducedinto the London Pharmacopoeiaof I 836. The fungoid by C. purpurerrvaries,rye usually bearing a considerablenumber ot sclerotia,while wheatbearsvery few. Ergot is eithercollectedby hand origin of ergot was recognizedby Miinchausenin 1764,while the life history ofthe funguswas worked out and the nameClavicepspurpurea or separatedfrom the rye by specialmachines,that which is collected beforethe rye harvestbeing saidto be the more active. siven to it bv Tulasnein 1853.
cereale.Controlled field cultivation on rye is the main sourceof the crude drug. The most important producers are Czechoslovakia. Hungary, Switzerland and former Yugoslavia.With modem tarming the 'natural' ergot is decreasingand fields of rye are devotedto its supply of cultivation. Different selectedstrains of C. purpurea are used fbr the production of the alkaloids ergotamine,ergocristine,or ergocomineand ergokryptine.Commercially,ergotof rye is becominglessimportantand currently(1994)UK dealerstrademainly in (c.907a)ergotofwheat.
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AND RELATED DRUGS OF BIOLOGICAL ORIGIN PHARMACOPOEIAL
I . l I
I
,
:,:
.
Oqn'' I H Tryptamin€
t':
MeOzC
l , : I . :
Camptothecin (seeChapter28) Secologanin
3o(,S)-Striclosidine I synthase I Cathenamine
I
-
Fig.27,28 Formotion ondmetobolism of strictosidine
Other types of monoterpene indole slkaloids(FiS.27 .27)
Cathenamine (Corywfla-type alkaloid)
Any ergotwhich is not collectedfalls to the groundand in the following spring puts out a number of stalkedprojectionsknown as stromata.These have globular headsin the surfaceof which are a large number of flaskshapedpocketscalled perithecia.Each peritheciumcontainsseveralsacs which, or asci, eachof which contain eight of the thread-likeascospores, aspreviouslymentioned,infect the young rye ovariesin the spring. Ergotswill usuallygerminatein soil if they areexposedto frost.The ascosporesobtainedrnay be also germinatedon nutritive media and culturesobtained.A suspenlarge quantitiesof conidiospore-bearing sion of conidiosporesmay be used as a spray to infect rye plants and machinesare usedfol treatinglarge areasfor the commercialproduction of ergot.In this way too, chemicalstrainsof ergot,selectedfor particular alkaloids,can be produced. The vegetativephaseof the funguscan, like that of other moulds.be cultivated artificially-if necessary,in large fermenters.Under such conditions the typical sclerotia do not develop. The commercial importanceand the phytochemicalaspectsof artificial culture are discussedunder'Constituents'.
Powderedergotwhen treatedwith sodiumhydroxide solutiondevelops a strongodour of trimethylamine.In filtered ultraviolet light it hasa strong reddishcolour by meansof which its presencein flour may be detected.
Microscopical characters. Ergot shows an outer zone of purplishbrown, rectangularcells, which are often more or lessobliterated.The pseudoparenchyma consistsof oval or roundedcells containingfixed oil and protein, and possessinghighly refractive walls which give a reactionfor chitin. Celluloseand lignin are absent.
Constituents. The ergot alkaloids (ergolines)can be divided into two classes:(1) the clavinetype alkaloids,which are derivativesof 6,8dimethylergoline and have been extensively studied in cultures of the mycelium of the ergot fungus; and (2) the lysergic acid derivatives which arepeptidealkaloids. It is the latter classthat containsthe pharmacologically active alkaloids that characterizethe ergot sclerotium(ergot). Eachactivealkaloidoccurswith an inactiveisomerinvolving isolysergi acid. These alkaloids have been studied over many years and were not 'ergotoxine',which sinceits isolationin 1906 Thus easyto characterize. (by Barger and Carr and independentiyby Kraft) had beenacceptedas a Macroscopical characters. The drug consists almost entirely of sclerotia,the amountof other organicmatterbeing generallylimited to pure substance,and in the form of ergotoxineethanosulphonatewas formerly used as a standard,was shown to be a mixture of the three alkanot more than 7Vc.Each sclerotiumis about 1.0-4 cm long and 2-7 mm broad; fusiform in shapeand usually slightly curved. The outer loids ergocristine,ergocornineand ergocryptine. Six pairs of alkaloidspredominatein the sclerotiumand fall into either surface,which is of a dark, violerblack colour,is often longitudinally furrowed and may bear small transversecracks.Ergot breakswith a the water-solubleergometrine(or ergonovine)group or the water-insolu 'ergotoxine'groups.Table2'7.6gives the more physible ergotamineand short fracture and shows within the thin, dark outer layer a whitish or in which darkerlines ologically active memberof eachpair first. Alkaloids of groupsII and III pinkish-whitecentralzoneof pseudoparenchyma radiating from the centre may be visible. Ergot has a characteristic are polypeptidesin which lysergic acid or isolysergic acid is linked to other amino acids.In the ergometrinealkaloidslysergic acid or its isomer odour and an unpleasanttaste.
ALKATOIDS
loble 27.6
Alkoloids of ergoi. Alkoloid
l. Ergometrine Group ll. Ergotomine group
ErgomelrineJ Ergomelrinine I Ergoiominel Ergotominine J Frnnqina
lll. Ergotoxine group
I I
Ergosinine J Ergocristinel Ergocristinine I Ergocryptine I Ergocryptinine I Ergocornine J Ergocornrnrne,,
is linked to an amino alcohol. Ergometrinewas synthesizedby Stoll and Hofmann in 1943.Other, neq peptidealkaloidshave beenisolatedfrom submergedcultvresol C. purpLLreaandfromlhe field-glowing fungus(L. Cv ak et al ., Phyrochemistrt^, I99 6, 42, 23 1; 1997, 44. 3 65'). Among the less important constituentsof ergot may be mentioned histamine,tyramine and other aminesand amino acids;acetylcholine; colouring matters;sterols(ergosteroland fungisterol);and about 307o fat. The cell walls arechitinous. Variation in alkaloid constituents.Not only are chemical races very evidentin C. purpureu with respectto alkaloid productionbut alsothe host plant is not without influence.Thus a new commercial strain of ergot adapted fiom a wild grass (Anthraxon lancifolius) to rye gave sclerotiacontaining0.57ototal alkaloidsinvolving ergometrine(33%), ergotamine( l7 .6%), ergocornine( 18.77c) and ergocryptine(22.7Va'). However,sclerotiaproducedon the grassasa resultof naturalinf'ection did not contain ergometrine(K. K. Janardhananet aL.,PLantaMed., 1982,44, 166). The applicationof specific amino acids to maturing sclerotiacan alsobe usedto influencethe type of alkaloidsproduced(a techniquealso usedwith saprophyticcultures).
Formula
Discovered
C1eH2202N3
Dudleyond Moir (1935)
C33H35O5N5
Spiroond Stoll{1920}
C3sH3705N5
S m i t ho n dT i m m i {s1 9 3 7 )
C35H39O5N5
(1932) Stollond Burckhordt
c 3 2 H a 1 o 5 Nt 5 calH3eosNsI
,1943) S t o lol n d H o f f m o n (n1 9 3 8 ,
and unnatural alkaloids from submergedcr.rlturesol C. pLtrpttreaseeN. C. Perellinoet al., J. Nat. Prod., 1992,55, 424:1993.56,489.)
Biosynthesis. The majority of biosynthetic studieswere at first directed to the clavine alkaloids,which could be easilyproducedin cultures but, until recently,their biologicalrelationshipto the lysergicacidderivatives remained obscure.The ergoline nucleus is derived from tryptophan and mevalonate, and current work has involved elucidating the biosynthetic relationship between the various clavine alkaloids, determining which of theseintermediatesis the true naturalprecursorof lysergic acid, and studying the initial hydrogen elimination fiom the C-4 of mevalonateto yield the stereo-configuration of chanoclavine-1. Possible biosynthetic routes for lysergic acid involving two isomeric intermediatesare given in Frg. 27.29.A major problemhas beennot so much in discovering which reactionsthe fungus can effect when suppliedwith a given substrateas which route is actually involved in its normal metabolism. As a resultof work with cell-fieesystems,Abe assigneda primary role to 4-isopentenyltryptophan and lysergene rather than to the dimethylallyl compound, agroclavine or chanoclavine.Later work by Grogeret al. (PlantaMed., 1980,40, 109)appearedto favour a scheme Alkaloid production in artificial cultures. The artificial culture of involving the latter compoundsand this has now beengenerallysubstanthe ergot fungus has receivedconsiderableattention,and, obviously, tiated with the intermediatesinvolved with the ring closure between large-scalesubmergedfermentationwith selectedstrainsto give alkal- dimethylallyltryptophan and chanoclavinehaving beeninvestigated(A. oids of choicehascommercialpossibilities.Abe's initial work in Japan P.Kozikowski et ttl., J. Amer Chem.Soc..1993,115,2482). showedthat submergedculturesdid not producethe typical alkaloids Work on the origin ofthe nitrogenofthe peptideportionsofthe ergot associatedwith the sclerotiumbut, rather,a seriesof new nonpeptide alkaloidsindicatesthat appropriateamino acids are specilicallyincorbases(clavines)which unfbrtunatelypossessed no significantpharma- porated.Abe's schemeis shown in Fig. 27.30 and he has obtained cological action.Attempts were made by many workers to influence intact incorporationof the units shown, in certainstrains,but workers alkaloidproductionby modificationof the culturemedium and the fun- in Germany were unable to confirm these results (see D. Groger, gus strain.As a result of successfulexperimentsin 1960,the commer- Planta Med., l9'75,28,37; see also P. A. Stadler,Planta Med., 1982, cial manufactureof simple lysergic acid derivativesby fermentative 46, 131). As hasbeenmentionedearlier,unnaturalamino acidscan also paspall becamefeasible.The alkaloids be incorporatedinto the alkaloids. growth of a sffain of CLaviceps producedare convertedto lysergic acid which is usedlbr the part-synthesisofergometrineandrelatedalkaloids.Other strainsarenow avail- Varieties. Commercial ergot varies considerably in activity liom able which produce the peptide alkaloids in culture; not only can batchto batch,and the differencescannotbe fully explainedby differdifferent chemical races of the fungus be used to produce specific encesin storage;further, it is often found that inf'erior-looking ergot is groups of alkaloids but synthesiscan also be directed by the addition of highly active. Such variations are apparently due to the fact that there certain amino acids or their analoguesto the fermentation liquid. In this are a number of different chemical racesof C. purpurea and in cultivatway new unnatural alkaloids can be produced. ing ergot by modern methods it is obviously important to prepare the Flieger et al. (J. Nat Prod., 1989,52, 1003) found that with sub- spore-cuitures used for infecting the rye from a race of the fungus mergedculturesin the postproductionstageboth the alkaloid concentra- known to develop ergots having a high content of the required alkaltion and the composition of the alkaloid mixtures underwent dramatic oids. Cultivated ergot may contain up to 0.57cof total alkaloids,and changesincluding the production oftwo new alkaloids,8-hydroxyergine 0.15Vais a minimum commercial value. In addition, there may be a and 8-hydroxyerginine. (For papers pertaining to the isolation of new minimal requirementfor water-solublealkaloids.The alkaloidscan be
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OF BIOLOGICAL PHARMACOPOEIAT AND RELATED DRUGS ORIGIN
'Y
NHz
cooH
\
N H
@@{
4-(d.&Dimethylallyl)tryptophan
Isopentcnylpyrophosphate (scc Chaptcr19)
NHr
cooH
4-lsopcntenyltryptophan H
N-CH3
Chanoclavine
cHloH
N H N-CH!
Agroclavine
Lysergene
H Lysergol
I
I
r.!oo* -)N-cH, :
PePtide , alkaloids Fig,27.29 Possible biogenesis of lysergic ocid.
lsolysergic acid Lysergicacid
determinedby colorimetry,asthey give a blue colour with a solutionof p-dimethylaminobenzaldehyde.
The sclerotiamay attain as much as 9 cm in length and are spiralll twisted.
Substitutes. Ergot of wheat is now imported into Britain and has been used medicinally in France. The sclerotia are shorter and thicker than thoseofrye. Instancesofergot on barley and rye in Britain, and on wheat and rye in the USA and Canadahavebeenreported. Ergot of oats hasbeenusedmedicinallyin Algiers. The sclerotiaare black in colour, 10-12 mm long and 3-4 mm diameter. Ergot of diss,which is producedon theAlgerian reedAmpelodesma tenax, has appearedin commerce and is said to be highly active.
Storage. Ergot is particularly liable to attack by insects, moulds and bacteria. After collection it should be thoroughly dried, kept entire, and stored in a cool, dry place. If powdered and not immediately defatted, the activity decreases,but if defatted and carefulll' storedin an air-tight container,it will remain active for a long period. Any sample of ergot which shows worm holes or a considerable amount of insect debris will almost certainly deterioratefurther on storage.
ALKALOIDS
>__-t
#-"' (A,.,JI
.n\ ./)cHcHcooH CH,' i
o-Lysergic rcid
NH. L.V!line
/
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l
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H
!
I
)*-c*, I
D.LysertylL..lenine
!
|
f
i
*
o:.\*/ l l HN
x
C-O
, , \'cn. -{
*-/ ) L-PhenylalanylD-prolyl.lact.m
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l h a O=C !
N.
H
/N-
cH3
o-Lysergyt. L-valine
I \
HN
cooH
,f-A
j
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,^, .",' \-/-
i l i H CO-.NH-CH l \ t I
loo"i
)---^
(
( |
l
t', I
CHI-CH cHa
L-kucyl-Dprolyl-lactam
c H-,
c ,4, H, _,4
_
c,"^+ fH co--NH--.P'JA..)
Y..-. (
Fig.27.3O Biogenesis of ergofpeplide (ofter Abe). olkoloids
'N-c", o=j_-'i \.-,/
, l t \CHI--CHf ' '
\-1--'
-t--->--
Er3otrminc - ErSotamininc
Ergocryptine
i'=o
H -
cH' Ergaryptininc
Uses. Although whole ergot preparationswere traditionally used in what wdnkled. On either sideof the grooveis a well-markedridge and labour to assistdelivery and to reduce post-partumhaemorrhage,ergot in the groove itself are the greyish,paperyremainsof the funiculus.A itself has been largely replaced in the pharmacopoeiasby the isolated ffansversesection shows a large central cavity and two, very hard, alkaloids.Only ergometrineproducesan oxytocic (literally 'quick delivconcavo-convexcotyledons. ery') effect, ergotoxine and ergotaminehaving quite a different action. Ergometrineis solublein water or in dilute alcohol.It is often known, par- Constituents. The seeds contain the alkaloids physostigmine or ticularly in the USA, as ergonovine.Ergotamine and the semisynthetic eserine,eseramine,isophysostigmine,physovenine,geneserine,N-8dihydroergotaminesaltsare employedas specificanalgesicsfor the treat- norphysostigmine, calabatineand calabacine.The structureof geneserment of migraine.Lysergicacid diethylamide(LSD-25), preparedby par- ine, long regarded as an N-oxide, has been revised to include the tial synthesisfrom lysergic acid, is a potent specificpsychotomimetic. oxygen in a ring system.The chief alkaloid,physostigmine.is present to the extent of about 0.15%. h is derived from tryptophan(see Fig. Colobor beon ond physostigmine 27.26). Onexposureto air it oxidizesinto a red compound,rubreserine, Calabar beans (Ordeal beans) are the dried ripe seedsof Physostigma and shouldthereforebe protectedfrom air and light. The official salt, venenosum(Leguminosae),a perennialwoody climber found on the the salicylate,is more stablethan the sulphateand is non-deliquescent. banks of streamsin West Africa. The plant bearstypical papilionaceous flowers, and legumesabout 15 cm long, each containingtwo or three Uses. Physostigminesalicylateis used for contractingthe pupil of seeds. the eye, often to combatthe effectof mydriatics.It hasalsobeeninvestigatedas an intravenousinjectionfor reversingthe effectsof a number History. The seedswere formerly used by the west African tribes as of sedatives.With Alzheimer's diseaseit has shown someevidenceof 'ordeal poison'. They were first known in England in 1840. The inducing an a slight improvementin intellectualand cognitive performmyotic effect of the drug was noted in 1862 by Fraser,and physostig- ance(Pharm. J., 1992,249,376) but galanthamine(q.v.) may prove mine was isolatedin 1864bv Jobstand Hesse. superior. Physoveninehas the same order of activity but that of eseramineis much lower. Characters. Calabar beans have a somewhat flattened. reniform shape.They are 15-30 mm long, 10-15 mm wide and up to 15 mm Nux vomicq thick. The seedsare extremely hard. The dark brown testa is smooth, Nux vomica consistsof the dried, ripe seedsof Strychnosnux-vomica except in the neighbourhood of the grooved hilum, which runs the (Loganiaceae),a ffee 10-13 m high with a distribution including whole length of the convex side and round one end, where it is some- Ceylon, India, East Bengal, Burma, Thailand, Laos, Cambodia and
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PHARMACOPOEIAL AND RETATED DRUGSOF BIOLOGICAT ORIGIN S.Vietnam.The drug is mainiy collectedin India and exportedfrom Bombay,Madras,Cochin, Cocanadaand Calcutta. History. Nux vomica was known in Europe in the sixteenth century and was soldin Englandin the time of Parkinson(1640),mainly for poisoninganimals.Strychninewas isolatedin 1817and brucinein 1819. Collection and preparation. The fruit is a beny about the size of a small orange.When ripe it has a ratherhard orange-yellowepicarpand a white, pulpy.interior in which 1-5 seedsareembedded.The seedsare washedfree from pulp and dried. They are exportedin small sacks, known as 'pockets',holding about l8-25 kg. I
Macroscopical characters, Nux vomica seedsare extremelyhard and shouldbe boiled in water for at leastan hour in order to softenthem sufficiently ibr dissection.The seedsare greenish-grey.disc-shaped,10-30 mm diameterand zl-6 mm thick. Most of the seedsarenearly flat andregular in shape,but a few areirregularly bent and somewhatoval in outline. The edge is rounded or acute. The testa is covered with silky, closely appressed,radiating hairs. In the centre ofone ofthe f'lattenedsidesis a distinct hilum, and a small prominenceon the circumferencemarks the position of the micropyle, which is joined to the hilum by a radial ridge. To examinefurlher, a boiled seedshould be cut transverselyand another oneopenedlike an oysterby inseningthebladeof a smallknife or scalpel at a point on the circumference opposite the micropyle. The small embryo with two cordatecotyledonsand a cylindrical radicle, the latter directedtowards the micropyle, will be seenembeddedin a grey, homy endosperm(Fig. 27.3I ). In the centreof the seedis a slit-likecavity.The seedsare odourlesswhen dry; but if soakedin water and left for a day or two, they develop a very unpleasantodour.They have a very bitter taste. Microscopical characters. A radial sectionshowsa very thin testaconsistingof collapsedparenchymaand an epidermallayer of very characteristic lignified hairs (Fig. 42.1M). The latter have a very large. thick-walled basewith slit-like pits. Surfaceiregularities in the basesof the hails causethem to interlock with one another.The upperportions of
A
ffi^
Ku;
@ h
B i
t i
I
ffi
b
the hairs are set at almost a right angle to the basesand all radiate ou towards the margin of the seed,giving the testa its characteristicsilkr appearance.On the ridge connectinghilum and micropyle, however thr hairs are irregularly arranged.The upper part of the wall of the hair ir composedof about 10 longitudinal ridgeJike thickeningsunited by a thir wall so that the lignified ribs readily separatefrom one anotheron po\\ dering.The lumen is circular in the upperparl, but in the basehasbranch es conesponding with the oblique pits in the wall. Fragmentsof testa removedfrom a soakedseed,may be disintegratedby treatmentwith 50i nitric acid and a little potassiumchlorate;the hairs can then be separate The endospermconsistsof large.thick-walledcells. which are non lignified and yield galactoseand mannoseon hydrolysis.When mount ed in solution of iodine, they show well-markedprotoplasmicthread (plasmodesma)passing through the walls (Fig. 43.1G) and an oil, plasmacontaininga few aleuronegrains and the alkaloids strychnin and brucine. Strychnine is most abundant in the inner part of th( endospermand brucinein the outer layers.The presenceof strychnin is shownby mountinga sectionin a solutionof ammoniumvanadateir sulphuricacid,when a violet colour is produced;of brucine,by mount ing in nitric acid, when a cdmson colour is observed. The lengthof lignified ribs of the hairsper milligram of nux vomic seedhasbeenusedfor the determinationof the contentof seedsin vet erinary medicines!see'QuantitativeMicroscopy'.
Constituents. Nux vomica usually containsabout 1.8-5.3% of tht indole alkaloids strychnine and brucine. Strychnine (formula Fig 27.26) is physiologicallymuch more activethan brucineand the seed are therefore assayedfor strychnine and not for total alkaloids (see p 102).They usuallycontainabott 1.237oof strychnineand about I .557 of brucine. Minor related alkaloids include s-colubrine. B-colubrin icajine, 3-methoxyicajine,protostrychnine,vomicrne, novacine N-oxystrychnine,pseudo-strychnine and isostrychnine.The seedsals contain chlorogenicacid (Fig. 20.5), a glycoside (loganin) and abou 3% of fixed oil. Loganin,althoughpresentonly in small amountsin th( seed, occurs to the extent of about 57o in the fiuit pulp together witl secologanin;thesecompoundsare intermediatesin the biogenesiso the strychnine-typealkaloids(Fig.27 .27'1. Seasonalvariations in alkaloid content of S. nux-vomica have bee studied(K. H. C. BaserandN. G. Bisset,Phytochemistrl, 1982,21, 1423
Uses. The action of the whole drug closely resemblesthat of strych nine. The alkaloid was formerly used as a circulatory stimulant in suc casesas surgicalshock,but its useis now more limited to that of a resp ratory stimulant in certain casesof poisoning. Like other bitters, strych nine improves the appetite and digestion, but it has been considerab 'general misusedasa tonic'. Nux vomicais usedin Chinesemedicinefo much the samepurposesas in Westernmedicine and the seedsare usua ly processedto reducetheir toxicity. Heat-treatmentof the seedsreduce the normal levels of the principal alkaloids and the amountsof isostrych nine, isobrucine,strychnineN-oxide and brucine N-oxide are increase (B-C.Cai et al., Chem.Pharm.8u11.,1990, 38, 1295).
Aflied drugs. The genusStrychnoscontinuesto attractconsiderab attention. (For extensive reviews on the taxonomy, chemistry and eth nobotanyof the American,African and Asian speciesseeN. G. Bisse er al., Lloydia, 33, 20 1; 34, I ; 35, 95, 193; 36, 1'79 ; 37, 62; 39, 263; als Fig.27.3r Bisset's review on alkaloids of the Loganiaceae in Indole an A, Strychnos nux-vomico seed;B, S. nux-blondoseed.Surfoceond BiogeneticallyRelatedAlkaloids, 1980(edsJ. D. Phillipsonand M. K lolerolviewsof entireseedond innersurfoceof horizontollv solitseeds. Zenk) p.27, London:AcademicPress;and J. Quetin-Leclercqetal., t All x0.8. m, Micropyle;r, ridge;h, hilum;b, loterolridge;ep, epidermis; Ethnopharm, 1990,28, 1, review with c. 150 refs.) f, oreoof fusionof fwo endosperm holves;c, oreoof cenlrolcovity;em, Ignatius beans arethe seedsof Strychnosignatii,aplant occuning ir embryo.{Drownby Dr.T.D. Turner. Forfurtherdeloils,seeJ.Phorm. the Philippines, Vietnam and elsewhere.The fruits are larger than thos Phormocol., 1963, I 5, 594.)
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ALKALOIDS of nux vomica and may contain as many as 30 seeds.Theseare about 25 mm long. dark grey in colour and irregularly ovoid in shape.The structure closely resemblesthat of nux vomica, but the testa, which bears irregularly aranged greyish hairs. is easily rubbed off and is almost entirely absent in the commercial drug. The seeds contain about 2.5-3.07oof total alkaloids, of wbich about16-62Vc is strychnine.They are mainly usedfor the preparationof strychnineand brucine.The seeds of S. ignatii from Java(S. tieute) contatn 1.57cstrychnineand no brucine and from Hainen (S. hainanensis'lmainly bmcine with little strychnine. In addition to the seeds,other parts of the plants of Stry'c/rilosspp. may contain alkaloids including strychnine(B. De Datta and N. G. Brsset,Planta Med., 1990,56, 133; G. Massiot et al., Ph-ttochernisnl. 1992.31.2813). S.potatorLtm,from India, andS. nur-blanda, liom Burma, have been substitutedfor nux vomica; although they contain no strychnine or brucine,seedsof the former havebeenreportedto containthe alkaloid diaboline and its acetyl derivative, triterpenesand sterols.They are best distinguishedby meansof the ammonium vanadatereagent.The seedsof.S.potatorum are usedin India for clearingwater,whencethe specificname.They will also flocculateheavy metal contaminantsin water and arecapableof moppingup radioactiveisotopesfrom nuclear waste.The protein responsiblefor this propertyhas now beenisolated (Pharm. J., 1994, 252, 238).
Gelsemium Geisemiumconsistsof the dried rhizomesand roots of the American yellow jasmine, GeLsemium sempervirens (.C. nitidum) family Loganiaceae.indigenousto southernUSA. It is a climbing plant and producesscentedyellow flowers; it should not be confusedwith the yellow-flowering jasmine (Jasminum nudiflorunt, family Oleaceae) cultivatedas an omamentalin Europe. The drug occurs in cylindrical pieces 3-20 cm long and 3-30 mm diameter.The outer cork cells of the rhizome are reddish-brownand the inner onesyellowish.As growth takesplace,the outercork cells crack and the inner cork shows itself as a yellowish-brown reticulation.The roots are somewhat smaller than the rhizome and have a uniform yellowishbrown cork. Gelsemiumbreakswith an inegular splinteryfracture.It has a slightly aromaticodour and a bitter taste.A transversesectionof the rhi-
CHr
I H Gelsemine
./\
\-! t I
r
ocH3 Gelsedine: R = H G e l s e m i c i n eR: = O C H r
^-i|||.r-ilTrtrr
zome showsa thick cork, a cortexcontaininggroupsof sclerenchyma. a densewood, internalas well as externalphloem and a small pith. The roots,on the other hand.have no sclerenchymain the cortex and no pith. Gelsemium contains extremely toxic alkaloids of unique skeletal type. Gelsemineis the principalalkaloidand is the one most studied althoughit is not as toxic as gelsemicine.Other oxindolebasescharacterized are sempervirine,I-methoxy- and 2l-oxo-gelsemine,l.lhydroxygelsemicine.gelsedineand I 4-hydroxy-gelsedine. In a review (i29 rcfs) H. Takayamaand S. Sakai list,15 alkaloids derivedfrom G. eleguts. G. .sentpen'irens and G. rankhii; they areclassified into five groupsaccordingto structure(TheAlkaloids,1997,49, l). Scopoletin is responsiblefbr the blue fluorescenceof the broken drug in ultraviolet light. Iridoids and glucoiridoidshave been isolated fiom the aerial parts. Gelsemiumis used(BHP. 1983i in the treatmentof trigeminal neuralgia and migraine,but its userequrre:Srcutcare.as dangeroussideeffectsmay develop.It has beenstudiedfor its anticancerproperties. G. elegansis usedin Oriental fblk rnedicinefor mr,rchthe samepurposesas G. nititlum. Rouwolfio (Rouvolfio) Rauwolfia consistsof the dried rhizome and roots of RuLotoffiuserpentina (Rauvolfiaserpentina),Apocynaceae.a small shrub found in India, Pakistan.Burma, Thailand and Javn.The geographicalsource appearsto influencethe alkaloidal content.and mairuiactu,crstend to pref'er drug obtained from India or Pakistan. Reserpine.the most impofiant constituent,is containedin many other speciesof Ruuttolfict (see'African Rauwolfla' below). History. Although used in India from time immemorial. it was not until I 942 that favourablereportswere publishedof the useof the drug in powderedform. Sincethen researchworkers have studiedthe pharmacognosy, chemistry, pharmacology and clinical uses of many speciesof Rauwolfia andof the alkaloidsobtainedfrom them. Collection and preparation. The drug is collected mainly fiom wild plants, but cultivation of the drug will probably increaseas wild plants become more scarce;in parts of India collectors are required to leave someroot from eachplant in the ground for future growth. Neverlheless, and coupledwith the low seedviability, the plant is regardedas an endangeredspeciesin India. Consequently,the potentialfor the regenerationof plants from cell culturesand the possibleutilization of nodal culture has receivedsome attention(seeC. M. Ruyter er al., Planta Med., 1991, 57, 328;N. Sharmaand K. P.S. Chandel,Plant Cell Rep.,1992,11,200). As other speciesof Rarwoffia are found in India, care is neededto identify the correctplant.When first imported,many commercialsamples were found to be adulterated;this was due in many casesto lack of knowledge,and substitutionof, or adulterationwith, other specieshas become much rarer in recent years.After collection the drug is cut transverselyinto convenientsizedpiecesand dried. Macroscopical characters. The first detaileddescriptionofthe drug was madeby Wallis and Rohatgi in 1949.It usually occursin cylindrical or slightly tapering,tortuouspiecesabout 2-i0 cm long and 5-22 mm in diameter (Fig. 27.324), The roots are rarely branched and rootlets, 0.5-1 mm in diameter,are rare. Pieces of rhizome closely resemblethe root but may be identified by a small central pith; they occasionallyhave attachedto them small piecesof aerial stem. The outer surfaceis greyish-yellow,light brown or brown with slight wrinkles (young pieces) or longitudinal ridges (older pieces): occasionalcircular scarsofrootlets. In this speciesthe bark exfoliatesreadily, particularlyin the older pieces,and may leavepatchesof exposed
PHARMACOPOEIAL AND RELATED DRUGS OF BIOLOGICAL ORIGIN
'
N
cd',\
'Fril F ) W
J
f
Fig.27.32 R o u w o l f i o s e r p e n t i n o o n d R . v o m i f o r i o r o o t s . A ,RR.os oe tropfe n f i n o ( r l ) ; o , t r o n s v e r s e s e c t i oonf s( ToS m)e ( x l ) ;B , r o o t o l R . v o m i t o r i o , x l ; b , T S o f s o m e { x 1 }C; , d i o g r o m m o t i c T SR o f. s e r p e n t i nr o o t ( x l 5 ) ;D , d i o g r o m m o t i c T SRo .fv o m i t o r irao o t { x 1 5 )E; , T S o fc o r k o fR . s e r p e n t i n o ; F , T S t hoef s e c o n d o r y w o o d oRf. s e r p e n t i n o ; G , f i b r e s o n d v eosf sRe.ls e r p e n t i n o , i s o l o t e d b y m o c e r o no ; fc o r k o fR .v o m i t o r i a ; l , T S o Hf,iToS t hf e s e c o n d o r y wood of R. vomitorio, E, F,G, H ond l, (ollxl50). ck, cork;f, fibre;g.r,growthring;m.r,medullory roy; pd, phelloderm; ph, phloem;r, resinous moterioi; s, slorch;sc,groupof sclereids; v, wood vessel; xy,xylem(J.D. Kulkorni,portlyofierT. E. Wollisond S. Rohotgi(R.serpenfino) ond W. C. Evons(R.vomitorioJl wood. The drug breaks readily with a short fracture.The smoothed transversesurfaceshowsa narrow.yellowish-brownbark and a dense pale yellow wood. which occupiesabout three-quartersof the diameter.Both bark and wood contain abundantstarch.Some commercial samplesshow mould. The recentlydried drug has a slight odour which seemsto decreasewith age.Taste,bitter. Microscopical characters. The cork is stratified into about two to eight zones(Fig.21 .328), which consistof smallerand radially narrower suberizedbut unlignified cells altemating with larger radially broader cells which are lignified. In many piecesmuch of the cork is exfoliated, and for sectioncutting it may be best to selectpieceswith little exfoliation, separatethesefrom the wood and cut sectionsof the bark and wood
separately.Most of the cells of the secondarycortex are parenchymatous and containstarch;isolatedlatex cells may occur in this region,particularly in the Dehra Dun variety.The phloem is narrow and consistsmainly of parenchymawith scatteredsieve tissue.Sclerenchymais absent(distinction from many other speciessuchasR. tetraphylla (R, canescens),R. micrantha, R. densiflora, R. perakensisandR. vomitoria; seeFig.21 .32 C, D). Most of the parenchymatouscel1sof the bark contain starchgrains, and othersprisms or conglomeratecrystalsof calcium oxalate. The xylem is entirely lignified and usually showsthreeto six annual rings. The medullary rays, which are one to five cells wide, contain starchand alternatewith the rays of the secondaryxylem, which consist of vessels,fibres and xylem parenchyma.Comparedwith many other speciesof Raw,offia. the vesselsof R. serpentinaare small (up to
ALKALOIDS 57 ptm) and are less numerousthan in most of the likely adr-rltcrants. R. tetraphrlltt (R. ctmescens,R. hirsuttt) is a speciesot u ide distriThe starchgrains are larger in the rvood than in the bark and measure br.rtion-tropical Sor"rthAmerica. the Caribbean.lndia. Australia (Queensland). fiom 5-8 to 12-20 um. The root was i,rtone time occasionally substituted fbr R. serpettitlo and could be recognizedby its nonstratifiedcork. and sclerConstituents. Rauwolfia containsat least 30 alkaloids.which total eid groupsin the phloern.It hasservedasa commercialsourceofrcsersome 0.7 2.1c/c.Other substancespresentinclude phytosterols.tatty pine and the alkaloid deserpidinc.possiblyparticularlyimporrantas R. acids,unsaturated alcoholsand sugars. serpentitutis now classedas an endangeredspecies.Micropropagation ln 1931Siddiquiand Siddiquiisolatedajmaline(rauwolfine),ajma- protocolshave been describedtor in vitro ntassrnultiplicationof thc linine, ajmalicine.scrpentineand serpentinine. The chief therapeutical- plant(D. Sarmaer a1..Plarttt Metlicu,1999,65,211). ly importantalkaloidsare reserpine(isolatedin 1952;fbrmula Fig. R. nitida is a West Indian speciesfrom the root-bark of which 33 21.26) and rescinnarnine(isolatcdin 195,1).These are estersdcrived indole alkaloidshavebeenisolated. fi'om methyl reserpateand trimethoxybenzoicacid in the caseof reserpine and trimethoxycinnamicacid in the caseof lcscinnantine.New Uses. Rauwolfla preparationsand reserpine;u'eused in the managealkaloidscontinueto be isolated. mentof essentialhypeltcnsionand in certainneuropsychiatric disolders. Ajmaline. which has pharmacologicalpropertiessimilar to those of Cell and root cultures. R. serpentincL cell suspensioncultureshave quinidine.is marketedin Japanfor the lreatmentof cardiacarrhythmias. provedan importanttool in theelucidationof monoterpenoid indolealkaAn estirnated3500 kg of ajmalicineis isolatedannuailyfrorn either loid biogenesisand in this connectionthe significance,and isolation,of Rtttrtt'olfiaor Cathanrnthusspp. by pharmaceuticalindustlies for the strictosidinesynthasehasbeencon:idered(p. 37l). Bv theuseolcell cul- treatmentof circulatorydiscases. turcsStrickigtin 1988was ableto clalify a l0-stepbiosyntheticpathway Conflicting reports on the possible involvement of thc rauwolfia tiom strictosidineto the typical lauwolfia alkaloid,ajmaline.His group alkaloidsin breastcancerhavc engendereda naturalhesitationin their' hasalsoshownthattheprincipalalkaloidof cell suspension culturesol R. use.A report in tlte Luncet (1976) suggestedthat the alkaloidsdo not serpentitutis raucaftiicineoccuning in anrountsof up to 1-6 g I I in the initiate the carcinogenicprocessbut that they promote brcast cancer nutlient medium,represcnting2.3% of the dried cells,a value some67 fiom previouslyinitiatedcells. times higher than fbund lbr the loots. Ajmalicine. an anti-anhythmic drug, is alsoprodr.rced to the extentof 0.6c/c(cell dry wt.) togetherwith Africon Rouwolfio (Africon Rouvolfio) over'30 different nronoterpenoidalkaloidsin trace amountsincluding African lar-rwolfiaconsistsof the dried roots of Rauvrolfiavonitoria five glucoalkaloids. Addition ofhigh levelsofajrrralineto the cell culture Afz. The plant is a bush or tree widely distributedin tropical Africa medium promotedthe fbmation of a new group of alkaloids.the rau- fiom the west coastto Mozanrbiqrle.It is the most importantAfiican maclines,not fbund in the roots(for fiuther detailsseeS. Endresseral., rauwolfla lor the commercialpreparationof reserpine Ph.y-tochemistry', 1993,32, 725 andreferences citedtherein). Hairy root cultures ol R. serpentina,producedby Agrobacterimt Collection and preparation. As the treemay attaina heightof l0 m. rhiT.ogene.s transfbrmation,synthesizedajmaline(O.O45c/( dry wt.) and the roots are largerthan thoseof R. serpentina.Before drying they are serpentine(0.001%dly wt.) (8. D. Benjaminet al.. Pltttochentistn', cut transversely,bLrtare rarely sliced longitudinally.Roots up to 5 cm 1 9 9 4 , 3 5 . 3 8 1 ) l m i n o r a l k a l o i d s h a v e a l s o b e e n r e c o r d c d ( H . or lnore in diameterare sometimesfbLrndbut thc commercialdrug usuIralkenhagenet ol., Cat. J. Chent., 1993. 71, 2201). R. r,erti(illut(l ally consistsof much smallerpieces.Occasionalshipnrentshave been haily rootshavebeenshownto producereserpineand aimaline. madeconsistingof the bark only. Two monoterpenoidindole alkaloids and four B-carbolineshave been isolatedfrom cultured hyblid cells of Rouwolfia .terpetilinaand Macroscopical characters. The first detaileddescriptionofthe drug Rhaz,t'trstricta, rTorall of the compoundsbeing found in the parent was publishedby Evans in 1956. lt occurs in cylindrical or flattcned plants(N. Aini er al., Chem.Phann.8u11.,1996.44, 1637). pieces,usually0. I5-L5 cm diameterand up to 30 crn long.The roots taperslightly and areoccasionallybranched.The outer surfaceis greyStandardization. An assayfbr total alkaloids is not a true measurcof ish-brown,longitudinaily funowed or rubbed smooth.since the outer' therapeuticactivity, sincc only some of the alkaloids have the desired cork easily flakes ofT. Piecesdo not break easily,but the ft'actureis pharmacological action.The BPC 1988and USP/NF1995determinethe short in the bark and splintery in the wood. The smoothedtransverse reserpineJikealkaloidsby utilizing the coloul reactionbetweenan acid surfaceshowsa narrow brown bark and a bufTor yellowish.linelir radisoluticlnof reserpine(zurdrescinnamine) and sodiumnitritesolution. ate wood. Odourless:taste. bitter. Piecesof rootstock with attached stem-bases are sometimesfbund in the drug. Allied drugs. The I l0 Rauwolfiaspeciesclassifiedby Pichonrn 1941 were reducedby Woodsonin 1957to 86. Someol theseoccur in more Microscopical characters. The drug is easily distinguishedfrom R. than one geographicareabut their approximategeographicareasare as serpentinaby the groupsof sclereidsin the bark arrangedin up to live follows: Centlaland SouthAmerica3zl.Africa 20. Far East2zl,India and discontinr.rous bandsand by the largevcsselsof the wood which are up Burma 7, Hawaii,New Guineaand New Caledoriia6. A largenumberof (Fig.27.32 D andI). to 180pm in diarnerer" thesespecieshavebeenexaminedfor reserpineandrelatedalkaloids. In the identification of the roots of speciesof Rauwoffic useful Constituents. African rauwolfla contains reserpine and rescincharactersto be seerrin transversesectionsare: cork (whether stlati- namine and alkaloidsofthe sametype suchas reserpoxidineand serefied or lignified); cortex erndphloem (presenceor absenceof sclereids dine. Many other alkaloidssuch as ajn'raline.alstonineand l,ohimbine or fibres); wood (relativenur.nber. distributionand sizeof vessels).As are also present.Court'sgroup (seePlunta Med..1982,45. 105)has the speciesvary liom herbsto large trees,the roots vary considerably isolated42 indole alkaloids1i'omthe stem-barkand identified 39. The in size.Somesanrples of drug containaerialstems,which usuallycon- major alkaloids were heteroyohimbines(especiallyreserpiline) and tain less reserpine than the roots and have unlignified pericyclic No-demethyldihydroindoles. The interelationship of these alkaloids fibres. with thosein the root of the plant is discussedin the samepaper.New
-r|Nil'*ilF
E
PHARMACOPOEIAL AND RELATED DRUGSOF BIOTOGICAT ORIGIN indole alkaloidsfiom R. vonitorio cxtractscontinueto be reoorted(M. Lov ati et ol., Fitote rapi a. 1996,67, 422).
Uncoriq species In additionto Unt oria ganbir. the sourceof catechu(q.v.),the genusi. notablefor its alkaloids.which resemblethoseof Mitrttgrnri. Uncari:r Allied drugs. Other Afi1can rauwolfias containingreserpineare R. hooks. the dried climbing hooks and stems of U..rlrierrsls,harc caffra (R. ntrtalensis),R. montbnsitutct, R. oreogiton,R. obsc'ura.R. sedative and antispasmodicproperties.They are used in Chinerc R. t'olkensiiandR. roseo. Court and coworkershave nrade medicinefor the relief of headachesand dizzinesscausedby hypertcn ctrtttrttirtsii. a systematicstudy of the microscopyand chemistly of Afiican species sion and fbr the treatmentof convulsionsin children.The drug contain. (for a report seeW. E. CourI.PlontctMed.. 1983,48,228).The rootsof indole alkaloids.e.g. rhyncophyllineand indole alkaloid glycosidc' R. c'affrocloselyresemblethoseof R. vonitoria but the cork hasnot the which exhibit a long-lastinghypotensiveefiect (K. Endo el al., Plottt,i sametendencyto flake ofT.In sectionsthe main difl'erenceis that in R. Med.. 1983.49.188;S. Kawazoeer al.. ibitl., 1991,57,47). vomitoria there are alternatinglignified and unlignified cork cells, Callus cultures of Uncaria rhtttchopht'lla,a speciesalso used in while in R. cuJJictall the cork cells ale lignified. R. caffra containsthe Chinesernedicine.producehirsuteine.hursutine,3a-dihydrocadambinc alkaloid raucafTricine-one of lelatively few examplesof monoter- andursolicacid(H. Kohdaet al.. Chem.Phcum.Bull., 1996,44,352). penoid indole glucoalkaloidswithin the group.R. monbasianctdillers U. tonrcntosu, one of two specieslound in S. America.featuresin thc from R. vomitoriu in the structureof the wood of the root; R. rosea,R. traditionalmedicineof Peru. It producessimilar hooks to U. sinerr.ir' yolkensii andR. obscuralack sclereiddevelooment. beingknown locally by the Spanishas 'una degato'(tomcat'sclaw).Th.' planthasa potentialimmr.rnostimulant actionandhasbeenexaminedtirr Alstonia borks its pharmacologicaland toxicologicalproperlies(K. Keplingerer al...l. SeveralAlstunia species(Apocynaceae)have been usedin the past as Ethnopharnutcologt'. L999.64.23 l. Lemaireer a\.. ibid..64, I09). antimalarials.and the barks of the Indian Alstonitt sthtiaris and the AustralianA. constricto were included in the 1914 edition of the Brill,i/r Vinca maiorond V minor Phannacopoeia.At least I I speciesare krown to contain alkaloids,such The greaterper"iwinkle(.Vincanru.jor)togetherwith the lesserperiir inasalstonine,alstoniline,cillastonineand echitamine.Interestin them was kle (V.ntinor) arethe only rnembersof the essentiallytropical and suhagainawakenedby the isolationin 1955of reselpinein moderateyield tropical family Apocynaceaefound wild in the British Isles. Thc from the root-barkofA. constricttt;reserpinehas sincebeenisolatedfiorr-r lbrmer is listedin IheBHP ( 1983)for the treatmentof rnenorrhagiaantl A. venenota.Descriptionsof Al.stoniabarkswill be foundin the olderedi- topically as an applicationfbr haemorrhoidsbut is now seldom used. tions olref'erencebooks (e.g.the 22nd edition of the USD. p. 1221). The following indole alkaloids have been characterized:reserpine-
Yohimbebork Yohimbe bark is derived from Parsln-r'staliat'ohimbe(Rubiaceae).a tree growing in the CameroonRepublic. It occurs in f-lator slightly quilled piecesup to 75 cm long and 2 cm thick. The grey-blown cork has furrows and cracksand patchesof lichen.The inner sur'laceis reddish-blown and striated.Taste.bitter. It containsthe indole alkaloid yohimbine,which is structulallyrelatedto reserpine. The bark is well-recognizedfor its aphlodisiacpropertyand yohimbine is eff'ectivein the symptomatictreatmentof erectiledysfunction, producing f'ewer side-efI'ectsthan invasive treatments(M. H. Pittler, I ortschritteder Medi"in, 1998,f 16, 32). Aspidospermq borks (Apocynaceae)containsmany alkaloidThe large genusA,spido,spenna containirrgSouth American trees.The alkaloidsare of various indole types fbrmed by a number of diff'erentbiogeneticpathways.Among the many investigatedare yohimbine, which is structurallyrelated to reserpine.andaspidospenline,which hasthe samegeneralstructureasvindoline (see Fig.27.27). The Aspidospenrrrrbarks are therefbre potential sourcesof alkaloids, becausethe treesare large and the barks would be commerciallyavailablecheaplyandin almostunlimitedquantities. Catharonthus roseus For an accountofthis importantplant. seeChapter28. Milragyna leqves The genus Mitragtnu (Rubiaceae)occurs in West and East Afr"ica. India and S. E. Asia. More than 30 different alkaloidshave beencharacterized,and the majority of theseare indole or oxindole sffuctures with an open or closed rin-eEl they exist in various isomeric forms. One alkaloid.mitragynine.isolatedfrom Mitragltta ,speciostt hasanalgesic and antitussivepropertiessimilar to those of codeine.Shellard and coworkers published extensivelyon the genus during the 1960s and 1970s (tbr more recent work on M. spec'ioscf}om the same DepartmentseeP.J. Houghtonet al., Phytochemistry,1991,30, 3,17).
majdine. akuammicine, strictosodine.pseudoakuammigine.akuanrmine and possibly l0-hydroxycathofoline.New alkaloidscontinuet() be reported e.g. Atta-ur-Rahmane/ al., Phttocheri'rlstn,.1995. 38. 1057.More than 50 alkaloidshave been isolatedfrom the leavesof \. minor, afew of them quatemary(for a recentreportsee D. Uhrin cl a1.. Vincamine,lirstisolatedfromV.minorin J. Nat. Prod., 1989,52,637). I 953. is availableas a vasodilatorvdrus.
CINCHONA
Cinchona bark consists of various species,races and hybrids ot' Cinchona (Rubiaceae),large trees indigenousto Colombia, Ecuador. Peru and Bolivia. The BP and EP recognizeC. succirubra Pavon.(C-. pubescensVahl.) and its varietiesand hybrids containingnot lessthan 6.57c of total alkaloids, 30-607a of which consistsof quinine-type alkaloids.The former"importanceof cinchonabark and its alkaloidsin the treatmentof malariahas beenlessenedby the introductionof synthetic drugs,but it remainsof greateconomicimportance.and saltsof quinine and quinidineare includedin most pharnracopoeias. Collection fiom wild trees was soon leplaced by cultivation, and most researchwas undertakenby the Dutch in Java and the British in India to obtain hybrids which are rich in alkaloids.While Indonesia and India remain important prodr.rcers of cinchona.a high percentage of the total crop is now grown on plantationsin Tanzania,Kenya. Guatemalaand Bolivia. History. The natives of South America do not appear to have been acquaintedwith the medicinal properties of cinchona bark. the bitter tasteof which inspired them with f'ear.Although Peru was discoveredin I 5 I 3. the bark was first usedfor the cure of f'eversabout 1630. The narne 'Cinchona'is saidto be derivedfrom a Countessof Chinchon.wif'eof a viceroyof Pem who it was long believedwascuredin 1638liom a f'ever by the useof the bark.Accordingto recentstudyof the Count'sdiary.it appearsthat the Countessnever sufI'eredfiom malaria or other fever during her stayin Peru,and althoughthe Count himself did so, thereis no
-".-**-qril
ALKALOIDS recordofhis havingbeentreatedwith cinchonabark.The remedy,which 'Pulvo becameklown as de la Condesa'.acquireda considerable reputation andwasknown in Spainin 1639.The lurtherdistributionof thebark was largely dr"reto the Jesuitpriests,and the drug becameknown as Jesuit'sPowder or PeruvianPowder.It filst appearedin the London Pharnttrcopoeia in l61l underthe nameof'Cortex Peruanus'. The bark was originally obtainedby felling the wild trees.which were exterminatedin many districts.Ruiz (1792) and Royle (1839) suggestedthe cultivation of cinchonasin other parts of the wor'ld. Weddell germinatedseedsin Paris in 1848,and the plants were introduced into Algiers in the following year but without much success.A further attemptby the Dutch was madein 185,1,seedsand plantsbeing obtainedfrom Peru by Hasskarland introducedinto Java.An English expeditionunder Markham in 1860 led to the introductionof C. succirttbra (the most hardy species),C. c'alisat'u,and C. micrantha into India. Seedsof C. ledgeriana were obtained in Bolivia by Charles Ledger in I 865 and were bought by the Dutch for their Javaneseplantations.A fascinatingbook covering Ledger's exploits is The Life of Charles Ledger (1818-1905)by G. Gramrnicia,Macmillan Press. London, 1988.World War II and subsequentfighting in Malaya and Vietnamincreasedthe demandfor cinchonaand stimulatedcultivation in Afiica and Centraland SouthAmerica.
Special characters. In view ofthe numberofhybrids r'"hichare cultivated. the distinctionof the various commercialcinchonabarks is a matter of some difliculty. In Table 2'7.1the notes on fbur importanr specieshavebeenmadeasconciseaspossibleto facilitatee()mplri\on. Microscopical characters. Cinchonabarks have the generalmicroscopicalstmctureshownin Fig.27 .33.The cork is composedof several layers of thin-walled cort cells, arrangedin legular radial rows and appearingpolygonalin surfaceview.Their cell contentsaredark reddish in colour.Within the cork cambiumis a phellodermof severallayersof regular cells with dark walls. The cortex is composedof tangentially elongated,thin-walledcells containingarnorphousreddish-brownrnatter or small starch grains 6-10 pm diameter.Scatteredin the cortex are idioblastscontaining microcrystalsof calcium oxalate and secretion cells. The phloem consistsof nanow sieve-tubesshowing transverse sieveplates,phloemparenchymaresemblingthatolthe coftex andlarge phloem fibres with thick conspicuously charactelisticspindle-shaped pits. The phloem fibres occur striatedwalls traversedby funnel-shaped isolated or in inegular radial rows. The distribution and size of the phloem fibres diff'erin the various species(thoseof C. succirubra are 350-600-700-1400,um long and 30-40-70-100 trrm diameter; for other speciessee I Othedition).The medullaryrays aretwo or threecells wide. the cellsbeing thin-walledand somewhatradially elongated.
Cultivation, collection and preparation. The ploduction of cinchona bark is a highly specializedsectionof tropical agriculture.An acid soil, rainlall and altitudeare all importantfactorsin cinchonaproduction.Selectionof high-yieldingstrainsis of paramountimportance, and graftingtechniqueswith C. succirubrcras stock may be employed. Seedlingsneed careful treatment and propagationto avoid disease attack.etc. Sincethe mid- I 970s a diseaseof the cinchonatree.known as stripecanker,has poseda threatto the plantationsof CentralAfrica. The disease.also known in Central America. is causedby the phytopathogenicfungus Pft.rroplthoracinnamoirrl,which causessunken necroticstripesin the balk and kills thousandsoftrees a year.
Constituents. Cinchonabark containsquinoline alkaloids (see Fig. 21.34).The principalalkaloidsare the stereoisomers quinine and quinidine and their respective6ldemethoxy derivatives.cinchonidineand cinchonine.The quinine serieshas the conti_euration 8.S,9Rand the quinidine8R. 95 (Fig. 27.3,1);other alkaloidsof lesserimportancehave beenisolated.Someofthese(e.g.quinicineand cinchonicine)areamorphous.The amountof alkaloidspr"esent andthe ratiosbetweenthemvary considerablyin the difI'erentspeciesand hybrids,also accordingto the environmentof the tree and the age and methodof collection of the bark. The alkaloidsappearto be presentin the parenchymatous tissuesof the bark in combinationwith quinic acid and cinchotannicacid.Quinic General characters acid (seeFig. 20.5) is presentto the extentof 5-8%. Cinchotannicacid 'druggist's'quills 1. Stem-bark.The commercial are up to 30 cm long is a phlobatanninand a considerableamountof its decompositionprod'cinchona red', is also found in the bark. and usually 2-6 mm thick. Bark for manutacturingpurposesis fre- uct, Other constituentsare quently in small curved pieces.The outer surtacefrequentlybears quinovin (up to 2%'), which is a glycosideyielding on hydrolysis mossor lichen.The cork may or may not be longitudinallywrinkled, quinovaicacid and quinovose(isorhodeose). and usually bearslongitudinaland transversecracks.which valy in Anthraquinones,which as a group of compoundsare associatedwith frequencyand distinctnessin the different varieties.The inner sur- the family Rubiaceae(seeTable 22.3'1.arenot nonnally found in quantity lace is striatedand varies in colour from yellowish-brownto deep in thebark of cinchonaasindicatedby theisolationof norsolorinicacid,a reddish-blown.The fi'actureis short in the outer paft but somewhat tetrahydroxyanthraquinone. in 0.0008%yield from the balk ol C. ledgerifibrousin the inner part.Odour,slightl taste,bitter and astringent. arza.However.they areproducedin cell culturesofthe plant and by infec2. Root-bark. Root-bark occurs in channelled.often twisted pieces tion of the bark with Pbtophthora cinnumomi. The latter case may be with a phytoalexindefencemechanism;in infectedmaterial, about 2-7 cm long. Both surfacesare of similar colour, the outer. associated however.being somewhatscaly,while the inner surfaceis striated. alkaloid production is lowered. In connection with the production of
loble 27,7
l-pc,tFF
€omparison
of Ginchonospecies.
C. succirubra(Fig.27.33)
C. colisaya
C. ledgeriano
C. officinalis
Frequently 20-40 mmdiomeler, ond 2-6 mm thick
Diometerl2-25 mm ond 2-5 mmthick
Similorto C. colisoya
Up to l2 mmdiometer, ond I m mt h i c k
Well-morked longitudi nolwrinkles, relotivelyfew tronsverse crocks. Somepieces,but by no meonsoll, show reddishworts
Broodlongitudinol fissures; Similorto C. calisaya,but crocks Tronsverse crocks,very tronsverse crocksoboul 6-12 mm morenumerous ond lessdeep. numerous, oftenlesslhon oporl Somepiecesshowlongitudinol 6 mmoport wrinklesond reddishworts
Powderreddish-brown
Powdercinnomon-brown
Powdercinnomon-brown
Powderyellowish
E
DD R U GO S FB I O L O G I CO AL PHARMACOPOE AIN AD LR E L A T E RIGIN
id
t.f Li
ffifiu zr A€ ooo. ^"^=6 t2 v/o o,O \t o"o
Fig.27,33 (x 0.5); B, tronsverse sectionof bork (x25);C, isolotedphloemfibres(x50);D, portionof of Cinchonosuccirubro Cinchonobork.A, specimen with colciumoxolote;G, storch{ollx 200). ck, Cork;ct, cortex; phloemfibrewith surrounding porenchymo; E, corkcellsin surfoceview;F,idioblost m.r,medulloryroy; pd, phelloderm; pg, phellogen; p.f, l.f, longitudinol fissure; f, fibresprotruding fromfrocture;id, idioblosi;l, lichenpotches; phloemfibres;s.c,secretory fissure. cell;t.f,tronsverse
in cell cultures,enzymesassociated with thelaterstagesof direction.IsoenzymeII has a broad specificityacting on all the quinoanthraquinones glycosideformationhavebeenisolated.This work involvedglr.rcosidasesline alkaloidsof cinchonatested. (For studieson alkaloid synthesisin C. ledgeriana seedlingssce cell culturesand the isolationof llve distinctglucosylin C. .sucr:irubra (EC2,4,1,-) which catalysethe transferof theglucosylrr.roiety R. J. Aertset al.. Phl'tochemi.rtry', 1991,30, 3571.) transferases from UDP-glucoseto the hydroxyl groupsof thoseanthraqllinonesfbllnd Chemical tests. The colour reactionfbr quinine and quinidine alkaquinizarin,etc.). in cinchonacultures(e.g.emodin,anthrapurpr.rrin, loids (see the BP) using bromine and ammonia is known as the thalleioquintest.This testcan be appliedto cinchonabark, by the techBiosynthesis of alkaloids. Grafting experimentsand organ culture nique describedby Wagg (Y.B. Pharm., 1938). suggestthat the alkaloidsare formed principally in the aerial parts of If powderedcinchonais slightly moistenedwith glacial aceticacid the plant. Although the quinoline alkaloids have structureswhich by andheatedin an ignition tube,blood-reddropscondenseon the sidesof inspection rnight suggestanthranilic acid as a biological precursor. the tube.Cinchonabark gives reactionsfor phlobatannrn. they are.in tact, as originally suggestedby Janotet al. in I 950. delived assayof the BP for quinine and The two-point spectrophotometric from indolic precursors.This has been demonstratedby the specrfic cinchonine-typealkaloids should be noted. Thermosprayliquid chroincorporationoftryptophan (indole moiety). loganin and geraniol(termatography-massspectrometryis a sophisticatedmethod of analysis penoidmoiety) into the quinine of Cinchonaspp.The pathway,largely which has been used for studying Cinchona alkaloids rn in vitro culestablishedby Battersby'sand Leete'sgroups,involvesalkaloidsofthe tures(C. Giroud et al., Pkutta Med., 1991.57, 142). serpentinetype as illustlatedin Fig. 27.321. The proposedbiosyntheticroute has beensupportedand elaboriited Alfied drugs. The barks of certain speciesof Remiiia (Rubiaceae by recenl enzymestudies.The importantrole of strictosidinesynthase contain alkaloids.That of R. pedunculatais quoted (USP) as a source in the initial stagesofthe biogenesisof sometryptophan-derivedalka- of quinidine. It also containscupreine,an alkaloid which respondsto loids and its isolationfrom C. robu,stawas mentionedat the beginning the thalleioquintest and by methylationforms quinine. False cuprea of this section.The enzyrnetryptophandecarboxylase(EC 4. L L28). bark (R.putdiena) containsno quininebut an alkaloid cusconidineand which providestryptamine,is alsoinvolved in theseearly leactions.At small proportionsof cinchonineand cinchonamine. the AFRC Institute of Food Research,Norwich, UK an enzyme (cinchoninone:NADPH oxidoreductase)associatedwith the pathway has Cinchona leaf alkaloids. Alkaloids of the indole type (e.g. cinbeen isolatedfiom cells of a suspensionculture of Cinchona ledgeri- chophylline)are generallyconsideredto typify the leaves,so that it is ana: it catalysesthe reductionof cinchoninoneto an unequalmixture of interestthat Phillipsonet al. (J. Phttrm.Phttrmacol.,1981,33, 15P) of cinchonine and cinchonicline.The enzyme was resolved (by ion have also isolated quinine from leaves of C. succirubra grown in exchange)into two isoenzynticforms both of which had an absolute Thailand.Thirteenalkaloidshavebeenseoaratedbv HPLC. requirement for NADPH and catalysed reversible reactions. IsoenzymeI actsspecificallyon cinchoninonein the lbrward direction Uses. Galenicalsof cinchonahavelong beenusedasbitter tonicsand ol the oathwav and on cinchonidine and cinchonine in the reverse stomachics.On accountof the asffinsentaction. a decoctionand acid
ALKALOIDS EEl
COOH
Tryptophan
r"'
Corynantheal
Strictosidine
/
[2-14C]Geraniol
Cinchoninone
Cinchonidinone
Q u i n i d i n eR: = O C H g C i n c h o n i nR e := H
Quinine:R = OCFt R= H Cinchonidine:
Fig.27.34 chonges. illustrote thestructurol Morkedotoms olkoloids. forCinchono pothwoy biogenetic of possible Outline
orginfusion are sometimesusedas gargles.Before World War II, quinine erties and like quinine is ef-fectiveagainstchloloqrrine-resistant largely anlsms. but became malaria was the drug of choicefor the treatmentof supersededby the adventof syntheticantimalarialsdevelopedduring that period. It has, however,remainedof importancein Third World countries and has re-emerged as suitable for the treatment of Plasmodiumfalciparum inf'ections(talciparum malalia) in the many areaswhere the organism is now resistantto chloroquineand other There are a number of relatively small groups of alkaloids.some trt whose biosynthetic rclationshipsto partictllar amino acids hare not antimalarials. does not involve direet Quinidine is employed for the prophylaxis of cardiac arrhythmias been lirrnly establishedor whose formation participation. propamino acid has antin.ralariai and for the treatment of atrial fibrillation: it also
-lnq|n
JFafr:J,rlF
PHARMACOPOE AD LR E L A T E AIN DD R U GO S FB I O L O G I CO AL RIGIN
INDOLIZIDINEALKAIOIDS Only a small numberof indolizidine alkaloidsarecurrentlyknown but they have recentlybecomeof pharmaceuticalinterestthroughthe discovery of the tetrahydroxy alkaloids castanospermineand 6-epicastanospermine, which are possible lead compoundsin the search for anti-AIDS drugs (seeChapter32). Also, like the above,swainsonine. the toxic constituentof locoweeds and Austrahan Sv,ainsonu spp.,is a powerful glycosidaseinhibitor; this alkaloid is a trihydroxyindolizidine. (For a review of the simple indolizidine alkaloids (154 refs) seeJ. Takahataand T. Momose.Alkctloitls.1993.44. 189.)
IMIDAZOLEALKATOIDS The most important pharmaceuticalexamplesof this group are the Pilocarpusalkaloids,pilocarpinelinding useas an ophthalmiccholinergic drug. Possiblebiosynthetic routes to pilocarpine (see formula 'Jaborandi under Leaf and Pilocarpine',below) could involve eitherof the amino acidshistidineor threonine.
JABORANDIIEAFAND PITOCARPINE 'jaborandi' The name is now appliedto the leafletsof various species of Pilocarpus(Rutaceae),a genusof treesand shrubswell represented in SouthAmerica and lbund to a lesserextent in the West Indies and Central America. The principal jaborandi now imported, Maranham jaborandi. is that derived from the Brazilian planl Pilocarpus rnicrophyllus. The state of Maranhio accounts for abor.rt90% of the Brazilian leaf. Traditionally the crop is collected from wild plants but over the years production has fallen due to non-sustainabilityof the supply and attemptsto cultivatethe plant commerciallyhave beenundertaken.As with a number of medicinalplants.transfbrminga wild speciesinto a cultivablecrop is not necessarilyeasyand a balanced collection between wild and cultivated plants may be desirable. A comprehensiveaccountrelating to jaborandi productionis given by C. U. B. Pinheiro(.EcononicBotanl.199'7,51,49). Jaborandiwas tormerly official but is now used mainly as a source of the medicinallyimportantalkaloid pilocarpine.
Characters of principal sources
7. Maranham jobortutdi. The plant P. mir:rc4th,vllus, which prol11.-. the Maranhamdrug, bearsimparipinnatecompoundlcare: rr;::: about sevenleaflets(Fig.27.35). The leafletsare altachcdrt,.. somewhat winged rachis which is almost glabrous (Sllr/r/.-i(,:. hairy). The drug consistsof separatedleaflets.a certaintnt()unr, : rachisand an occasionalfruit. The leafletsare 2-5 cm long. I -1i:: broad, and emarginateat the apex. The terminal leafletsare or.,. symmetricaland have a petiolule5-15 mm long, with a rrinlc: margin which passesimperceptiblyinto the lamina.The remainin: leaflets are obovate,asymrnetricaland sessile.Leaflets of thc lc:: and right sidesof the leaf may be distinguishedfrom one anorhcrh.. the fact that the broader side of each leaflet lies away front rh. rachis.The veins are pinnateand anastomosenearthe rnargin.Thc drug is greyish-greento greenish-brownand brittle in terturc Numeroussmall oil cells may be seenby transmittedlight. Odou:. when crushed.slightly aromatic;taste,bitterish and aromaticsiri: inductionof salivation. 2. Pernambuco .jctborondi. Pernambuco jaborandi consisrs ('l the leaflets oI Pilocnrpus jaborutcli Holmes. which are obtainci from a compound imparipinnate leaf with I 9 leaflets. Leal'lcr. 4 - 1 2 c m l o n g a n d 2 4 c m b r o a d ( F i g . 2 7 . 3 5 ) .p e t i o l u l e ss h o r r . apex emarginate;base usually asymmetric;margin enlire an.1 slightly revolute. Upper surlace glabror"rsand greyish to brou n ish-green;lower surfaceyellowish- or greenish-brownand sli-!rhr ly pubescent.Midvein not prominent on the upper surfaccbur very prominent on the lower suriace (in the midveins of thc Maranhamleafletsthe reverseis the case).A transversesecnon()l the midveinsis often useful for distinguishingbetweenthe various speciesof Pilocarpus-tor exarnple,the Pernambucovarietr shows a completering of pericvclic sclerenchyma,the Maranhani a brokenring. A compound. l-phenyl-5-vinyl-5,9-dimethyldecane has been obtained from the foliar epicuticularwax of P. jaborantli and b1 TLC it can be used to distinguish this species from others of Pilocarpu.s(G. Negri et al., Phy-tr.tcltemistry-, 1998.49,127). 3. Paragtray.jaborandi.Paraguayjaborandi,derived from P. pennati folius Lemale. Greyish-greenlpapery in texture; usually equal at base;veins not prominenton the uppersurfaceand the anastomoses not marked. Pericyclic sclerenchymamore broken than in Maranhamor Pernambuco.The abovethree varietieshave a single palisade layer, a point which distinguishes them from thc Guadeloupe andAracativarieties.
Pilocarpine, Pilosine,
R = CHe-CHeR = CoHs-?HOH
Fig.27.35 A, Leofof Pilocorpus microphyllus; B,leofletof P yoborondi Joborondi. {x0.5)
{fl;
ALKALOIDS 4. Cearajaborantli. Cearajaborandi is derived fiom P trachtktphus Holmes and is exportedfrom the Brazilian provincesof Cearaand Maranhio. Leafletsare smallerthan thoseof P.jaborantll; oblong or elliptical; coriaceous;both surfaces bearing short cr.rrvedhairs, which are particLllarlyabundanton the lower surf'ace.
way. Theophyllinehas generallysimilar propertiesto the above.with a shorter.though n-rorepowerful diuretic action than caf-teinelit relaxes rnvoluntary muscles more eff-ectivelythan either caffeine or theobromine.The threeaikaloidsare official in the CP and BP.
Biogenesis. The ring fbrmationof the purinealkaloidsappearsto follow the classicalschemelor the biosynthesisofpurine nucleotideswith C1-moietiesarising from such compoundsas formatesand fbrmaldehyde.Methylamineis alsocfl-ectivelyincorporatedinto the ring system; studies by Suzuki et a1. indicated that methylamine is oxidized to formaldehydeand then metabolizedas a C, compound.For cafl'eine, the purine basessuch as hypoxanthine,adenineand guanine.and the nucleosidescan alsobe incorporatedby the plant into the molecule. In both tea and cotl'eeplants and in suspensionc:uhtxesof Collea arabica rthas beenclearlydemonstrated that theobromineis methylatedto cafl'eine.In 1919,as a result of work involving N-methyltransferases, -+ 7Robertsand Waller sr"Lggested the pathway 7-methylxanthosine -+ 3.7-dimethylxanthine methylxanthine(heteroxanthin) (theobromine) (caffeine)which has been substantiated + 1.3,7-trimethylxanthine by Uses. Salts of pilocarpine (e.g. Pilocarpine Hydrochloride and later work. S-Adenosylmethionine is utilized as a donor of the methyl Nitrate BP) are usedin ophthalmicpractice,as they causecontracrion gror.rps. Attemptsto isolatethe individualN-methyltransf'erase enzymes of the pupil of the eye, their action being antagonisticto that of do not yet appearto have been successfuidue partly to their extreme atropine.In early glaucomatreatmentthey serveto increasethe irriga- lability. Howeverprogresshas beenmadeon their biochemicalcharaction of the eye and relieve pressure.A study in the USA involving 207 terizationandtheir tirnecourseduringleafdevelopmentofCoJfeacLrabipatientssuft-eringfrom dry mouth resulting from radiation treatment czr(S. S. Mcisli Waldhar.rser et ol., Phttochemi.sul',1991. 44,853). The for head or neck cancerindicated that oral pilocarpinecan possibly origin of the caffeinemolecr"rle is shownin Fig. 27.36. o1I'errelief (Pharn. J., 1993.251,215).In 1994its use was approved (For a review on this aspectsee T. Sr-rzukiet nl., Ph,-tochemistr-t, tbr this purposeby the US Food and Drug Administratron. 1992.31.2515.\ Constituents. Maranhamleavescontainabout0.7-0.87cof the alkaloids, pilocarpine.isopilocarpine.pilosine and isopilosineand about 0.57cof volatileoil. An examinationof the volatileoil compositionof a number of speciesindicated a total of 22 componentsoccurring throughoutthe samples.Theseincludedmonoterpenes(e.g.limonene, (e.g. caryophyllene) sabiene,c-pinene) sesquiterpenes but not in P jaborutndi.and 2-undecanone or 2-tridecanone. Pilocarpine.the lactone of pilocarpic acid. contains a glyoxaline nucleus and with heat or alkalis is convertedinto its isomer isopilocarpine.Isopilocarpineoccursin small quantity in the leaf but more is fbrmed during the extractionprocess.The dried leavessoon lose their activity on storage.
Aspartic ' \ acid
/ FO, - -SAM / y.?/
PURINEALKALOIDS
sAM....- \ u"\zc-6t{.Frcmgrycine The purinenucleotides, togetherwith thepyrimidinenucleotides, constitute vital structuralunits ofthe nucleicacids:they alsoft-rnction ascoenC1-fragment I^ ill i -CH i^i // -. C1-fragment zymes (Chapter l9) and as porlions of complex substratemolecules. o-'-:*/'YFf('";:il;' 'foimatel Adenineand guanineare the purinesmost commonly involved in these sAM+,1"\ roles,but xanthineand hypoxanthinefeaturein their biosynthesis. Amide-Nof glulamine 'Purine alkaloids'constitutesecondarymetabolitesand are derivatives of xanthine;three well-known examplesare cafieine (1,3,7- Fig.27.36 trimethylxanthine). theophylline (1,3-dimethylaxanthine) and Biogenetic originof thecoffeinemolecule (SAM= Sodenosylmethionine). theobromine(3,7-dimethylxanthine). Beveragessuch as tea and coffee owe their stimulantpropertiesto thesesubstances. Caff'einestimuiatesthe central nervoussystemand COCOASEED has a weak diuretic action. whereastheobromineacts in the reverse Cocoa seeds (.CoconBettns) are obtained from Theobrcnla cacao (Sterculiaceae), a tree usually4-6 m high. Cocoais producedin South OH America (Ecuador,Colombia,Brazil, Venezuelaand Guiana),Central America. the West Indies, West Afiica (Nigeria and Ghana),Ceylon *t-c-c-N\ OZc-.g,-N1 and Java. c H l l l
T"' I
I
Hc.--".-i--r*/
HrN-
I I I c\*--c--*/
H
n
Adenlnc
Guanlne
o
il
"Tihi-\ ozci-;-li'--1/ H
Xenthine
) -ru'IilF
F H
H
History. Cocoa has long been used in Mexico and was known to Columbusand Cortez.Cocoabutter was preparedas early as 169-5. Collection and preparation. Cocoafruits are 15-25 cm long and are borne on the trunk as well as on the branches.Cocoa plantationsare very vulnerableto pestattackand recentlymodernpheromonetechnology has been usedto control the cocoa pod borer, also known as the cocoa moth (Conopontorphacrcunerella),the most serior.rspest of the crop in S.E.Asia. Collectioncontinuesthroughoutthe year.but the largestquantitiesareobtainedin the springand autumn.The fiuits have a thick, coriaceousrind and whitish pulp in which 40 50 seedsare embedded. In different countries the seeds are prepared in different
P H A R M A C O P O E IA N L D R E L A T EDDR U G SO F B I O L O G I C AO LR I G I N ways, but the following may be taken as typical: the fruits are opened and the seeds,embeddedin the whole pulp or roirghly separatedfi-om it. are allowed to f'erment.Fermentationocculs in tubs. boxesor cavities in the earth;the processlasts3 9 days.and the temperatureis not allowedto rise above60"C. In Jamaicafermentationis allowed to proceedfor 3 daysat a temperatLrre of 30 43'C. During this processa liquid drainsfiom the seeds.which changein colour fiom white or red to purple, and also acquirea difl-erentodour and tastc.After.t-ermentation the seeds may or may not be washed. They are then roasted at 100-140'C, when they losc water and acetic acid and acqr.riretheir characteristicodour and taste.Roastin-e facilitatesremoval of the testa. The seedsare cooled as rapidly as possibleanclthe testaremovedby a 'nibbling' machine.The nibs or kernelsare separatedfrom the husk by winnowing. Sometimesthe seedsare simply dried in the sun but these are not as highly regardedowin-eto their astringentand bitter taste. Plctin or bitter chocolule is a mixture of ground cocoa nibs with sucrose.cocoabutter and flavouring.Milk chocolatecontainsin addition nilk powder. Macroscopicalcharacters. Cocoaseedsareflattenedovoid in shtrpe, 2-3 cm long and 1.5cm wide.The thin testais easilyremovedfrom preparedcocoa beans,but is difiicult to remove front thosethat have not beent'ermentedand roasted.The embryo is surror.rnded by a thin membraneof endosperm.The cotyledonsform the greaterpart of the kemel and are planoconvexand irregularly fbtded. Each shows on its plane lace threelargefurrows.which accountfbr the readinesswith which the kernelbreaksinto angularfrugrnents.Both testaund kernelareof a reddish-browncolour,which varies,however,in differentcommercialvarietiesand dependson the lbrmationof'cacao-red'duringprocessing. Constituents. Cocoa kernelscontain 0.9-3.0% of theobromineand the huskscontain0.19-2.987cof this alkaloid.The seedsalsocontain 0.0-50.36% cafi'eine,cocoa firt or butter (nibs zl-5-53%.husk 4-8%). During the fermentationand loasting.rnuch of the theobromineorigirrallypresentin the kernel passesinto the husk. The constituentsother than fat and theobronrineare exlremely complex and have been intenselystudiedin recentyears.The fresh seedscontainabout5-l 0% of water-soluble polyphenols (epicatechol.leucoanthocyaninsand anthocvanins)which are largelvdecomposedduring processin-e, forming the colouredcompiex fbrn-rerlyknown as 'cocoa-red'.Condensed tannins are also prescnt,and some 84 diffelent volatilc compounds. includingglucosinolates. areresponsible for the aroma(seeM. S. Gill et ul.. P l:lr' tocltentis n-t',1984.23, 1937). Tlrcctbronineis producedon the commcrcialscetlefrom cocoahusks. The processconsistsofdecoctingthe huskswith water.filtering,precipitatinc 'tannin' with lead acetate,filtering, removingexcessof lead and e'n'apolating to dryness.Theobrorlineis extractedfi'om the residLreby mcansof alcoholand pLrriliedby recrystallization fiom water" Theobromineis 3.7-dirrethylxanthine (seep. 387),the lower homoIogue of caffeirre(trimethylxanthine).It is isomericwith theophylline which occurs in small qualtiries in rea. 11,-3-dimethylxanthine), Theobrominccrystallizesin white rhombicneedles.It givesthe murexide reaction(seep. 336). and may be distingLrished fi'om caffeineby the fact that it is precipitatedfrom a dilute nitric acid solution by silver nitrate.Theobrominesr.rblirnes at 220"C. caff-eineat 178- I 80'C. Callr.rsand suspension culturesof cocoaboth producecaff'eine,theobromineandtheophyllineand areconsideredusefulfbr studyingsecondary metabolisrnin t'iuo (K.A. Gurneyet ul., J. Exp.Bot..1992,43,161)). Uses. Cocoa has nutritive, stimulant and diuretic properties. Thcobromineis usedasa diuretic.lt haslessactionon thecentralnervous system than caffeine br-rtis more diuretic. In its isomer. theo-
phylline. the cliureticeffect is even more marked. Oil of theobroma (q.v.)is r.rsed in phzrrmacychiefly as a suppositolybase. Allied drugs. Kolu seeds(bi.rst or goot'oonut.\').Contmercialkola consistsof the dried cotyledonsof the seedsof variousspeciesof Co1rl (Sterculiaceae), treesfbund in WestAfrica. the WestIndies.Brazit and Java.The colour of the fiesh seedsvaries,thoseof C. ucuntinatobeing white or crimson, C. ostrophoftzred, C. alba white antl C. t,era (C. niti.1d)(which is possiblya hybrid of the two latter species)eithcr red or white. The dried cotyledonsarc usuallyof a dull. reddish-browncolour anclmore or lessbrokcn.They areusuallygradedzis'halvcs'trnd'quarters'.The whole seedsarc 2-5 cm long, and in the seedsr-rsually imported therearetwo cotyledons.Odor.rrless: taste.slightly astringr-nt. Kola seedscontain cafl'eine(l-2.5%) and a little rheobromine. which appearto be partly in the free stateand pafily combined.Kola also containsabout 5-10% of tannoids(the 'kolatin' of earlier workers).particulally catecholand epicatechol.During preparation,oxidation and polymerizationof theseproducesthc insolublephlobaphene 'kola-recl'. (C. Maillald et al.. PkrntttMed.,1985, It hasbeensuggested 515) that the difl'erencesin the stimulatoryaction betweenfiesh and dricd seeds may be due to thc formation of a cafTeine-catechin complex in the latter. For a historical comntentary. see R.W.D. Nickalls.Pharnt J., 1986,236.401. Gtrurtma (PtrstaGuuruta or Bru:.iliut cocori) is a dried pasteprepared.mainlyfrom the seedsof Puullirin cup(Ltio(Sapindaccae). The seedsarecollectedflont wild or cultivatedplantsin the upperAmazon basinby membersof the Gr.raranis tribe. The kernelsareroughly separated fiom the shell. brokcn and rrade into a pastewith water. starch and other substancesbeing fiequently added.The paste is then made into suitableshapesand dried in the sun or over fires. The drug usually occurs in cylindrical rolls 10-30 cm long and 2.-5-4cm diameter. Portions of broken seed project liorn the dart chocolate-brownouter surface.When broken. similal fiagments prG ject liom the fracturedsurface.The drug has no marked odour but an astringentbitter taste. Guaranacontains2.5-1.)c/aof caffeine.other xanthinederivatives" tanninsabout l2% ('guaranared') and otherconstituents resenthling. as far as is known, thoseof cola and cocoa.Guaranarcsemblestea and coffee in its action and the powder gratedfiom the massesis used in South Arnerica with water to make a drink. In the West it is now a popularrernedyfbl combatingtatigue,for slimming. and fbr the treatment of diarrhoea.The fat contentof the drug is statedto efI'ecta slow but steadyreleaseofthe alkaloids(fbr a shortarticle on guaranasce P Houghton.Plwnn. J., I 995. 254, 435). Cof?e consistsof the seedsoI Co(feaarubico and othcr speciesof (Rtbiaceae).It containscafTeine(I 2Vc),tannin and chloroCttJJbcr genic(cafl'eotannic) acid(seeFig. 20.5).fat, sugarsand penrosans. PreparedcofI'eeis the kernel of the dried ripe seeds of various species.inclLrdingC. urubiccr (Arabica coffee), C. libericu and C. canephoru(Robustacofi'ec)(Rubiaceae).deprivedof most of the seed coat androasted.The kernelsaredark brown, hard and blittle. elliptical or planoconvexand about l.0cm long. Coffee has a characteristic odour and taste.A decoctionis usedas a flavouring agentin Caffeine Iodide Elixil BPC ( 1979).Preparedcof'feecontainsabout l-27c of caff'eine,plobably combinedwith chlorogenicacid and potassiurr.Other constituentsinclude nicotinic acid, fixed oil and carbohydrates caramelizedduring roasting. C. arubico,both as wholc plantsand as cell suspensioncultr.rres, has been considerablyemploycd to study purine alkaloid variations and (q.v.). biosynthesis Ierr consistsof the preparedleaves of Carlr:lliu sinen.sis (T'heusinenslsJ (Theaceae),a shrub cultivated in lndia, Sri Lanka. East Aflica.
,fi
ALKALOIDS Mauritius,China and Japan.The lcavescontaintheuse.un cnzlmic mixture containingan oxidase,which partly convertsthc phlobatannin into phlobaphene.This oxidasemay be destroyedby steamingfor 30 s. TeacontainsI -5 7cof cat-fei ne and 1,0-21c/a of tanninI also small q r.rantitiesof theobromine, theophyllineand volatileoil. l'he alkaloidcontentofthe leavesis very muchdependent on ageand season. Callus and root suspensionculturesof C. sinensishave beenshown to accumulatecaft-eineand theobrorr-rine (A. Shclvinglonet ul.. Phtto. c:henti.str-t. l99lJ,47. 1535) The possiblebeneficial cfl'ectsof drinking black or grcen tea have receivedconsidcrablecoveragcin the rredical and nationalpress.An infr-rsion of teacontainsin additionto caffeinea mixture of polyphenols includin-r epigallocatechin-3-gallate possessingstrong antioxidant ancltrce-radicalscavcngingpropertics.Possiblebencficialef-fectsar.e: inhibitionof angiogenesis" a processinvolving thc growth of bloocl vesselsnecessaryfbr tumorlr growth and metastasis:the treatmentol genetic haemochromatosis bi' the inhibition of absorptionof iron bv tannatesand othel li-eands:treatmentof blindnesscausedby diabctes (an nngiogenic related coridition)l and a lowering of the risk of ischcmicheartdiseasein older men (a f inding not substantiated with tea witl-rmilk added)seeN,l.C. L. Hertoget ul.. Amer. J. Clirt. Nutr... lL)97.65, 1489;J. P Kaltwasser. E. Wcrner,K. Schalka a/.. Grrr.1998, 4 3 . 6 4 9 lY. C a oa n dR . C a o .N a t u r e , 1 9 9 9 . 3 9 8 . 3 8 1 .
toin (0.6 0.8cli in the roots) which can be regardedas a breakdown pr:ocluct of ulic ircid.Allarrtoinstimulatestissueregencrationand thereforc the clru-ehas been usedfbr externalinjuries and gastr.iculcers.A neu' snponininr,olvingoleanicacid glycosylatedat C-3 with arabinose-giucose-glucosc has beenrcported(V. U. Ahmad et al.. J. Nat. Prod.. 1993.56. 329). (I;or a reccntl'eporton the isolationof other bidesmosicUc triteryrenoiclal saponinssee F. V Mohammed er a/., -fhe PlarttttMed.. 1995.61. 9,1.) rclativelyrecentdiscoveryof a range of pyrr"olizidinc rlkaloids in corrfrey and in Russiancomfiey (5'.x uplundi 4Me.cooH
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of them number amongstthe most cytotoxic compoundsknown. This high cytotoxicity carriesover to ln ulr.'osystems,and the resultantrelatively narrow therapeuticindex makesthe compoundsunpromisingas therapeutic agents. The observation of antitumour activity in colchicineand its derivativesis particularlyinteresting.Colchicine is widely used in plant breedingfor its property as a mitotic poison (see Chapter12),and it is known that this and its rumour-inhibitoryactivity are closely connected.Podophyllotoxin, vinblastine and vincristine also act asmitotic poisonsand,indeed,major successes in drug control of cancershave been mainly in the area oi rapidly-growing tumours; highly activeanticancerdrugs suchas vincristineaffectcells only during the division cycle. Slow-growing tumours generally seem to respondpoorly to drugs. Susceptibilityto cancerdrugs may be determined by the fraction of cells dividing at any one time, A largenumber of naturaland semi-syntheticderivativesof colchicinehave beentested, severalin clinical trials, but the resultshave been disappointing. Nevertheless,colchicinederivatives,e.g. demecolcine,are being used in cancertreatmentin somecountries. Furtherexamplesof known compoundsbeing establishedas tumour inhibitors only at much later datesare the alkaloidsacronycine,ellipticine, emetineand nitidine. The antitumouractivity of acronycine,an acridonealkaloid first isolatedfrom Achronychiabaueri in 1948,was reportedin 1966,andthis compoundexhibiteda very broadantitumour spectrum,possessingsignificantactivity against 12 of the l7 tumour systemstested.In markedcontrast,however,phaseI clinical trials displayed no significantantitumouractivity and the obvious discrepancy is attributedto the sensitivityofthe preliminary screensapplied.Such observationspromptedchangesin the preliminary screensemptoyedin the NCI studiesduring the lifetime of the programme.Clinically useful antitumour agentsbased on the pyridocarbazolealkaloids ellipticine
Triptolide, R= H Tripdiolide. R = OH
and 9-methoxyellipticinefrom Ocfuo,siaelliptica (Apocynaceae)and other plants of this family, may well result liom clinical trials with thesealkaloids and a number of syntheticanalogues.Thesealkaloids are potent inhibitors of several cancerousdisorders,but preclinical toxicology indicated a number of side-ef1'ects, including haemoiysis and cardiovascularefl'ects. Ellipticines are planar molecules that intercalatebetweenthe basepairs of DNA and causea partial unwinding of the helicalarray.Thereis somecorrelationbetweenthe degreeof unwinding and the biological properties,those showing the largest unwinding inhibiting the greatestnumber of cancerouscells. Recent researchsuggeststhere may be more than one mechanismof action, however.Ellipticine is oxidizedin vivo matnlyto 9-hydroxyellipticine, which has an increasedactivity,and it is believedthat this may in fact be the activeagent.Poor water-solubilityofellipticine and derivatives gaveproblemsin formulation for clinical use,but quaternizationof 9hydroxyellipticineto give the water-soluble9-hydroxy-2-N-methylellipticinium acetate(elliptinium acetate)(Fig. 28.2) has produced a highly active material, of value in some forms of breastcancer,and perhapsalso in renal cell cancer.A variety of suchquaternizedderivatives is being tested,and some water-solubleN-giycosidesalso show high activity. Cephaelisipecacuanha(see p. 365) has been used for many years as an emetic and expectorantand the principal alkaloid, emetine,is of value in the treatmentof amoebicdysentery.The antitumour propertiesof emetine were realized following plant screening, and this alkaloid and somesemi-syntheticderivativeswere also tested clinically. Clinical usefulnesswas marginai,however,and sometoxic effectswere noted.Nitidine is a benzophenanthridine alkaloid isolated from Zanthoxylum nitidum, and has more recently been obtained from screensof Fagara species(Rutaceae).Nitidine was selectedfor development based on its exceptional antiieukaemic activity, but was
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PHARMACOPOEIAL AND RETATED DRUGSOF B I O L O G I C O AL RIGIN NHz
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ris,28.2 Somedrugsbosedon plontnoturolproducts. droppedowing to eratic toxicity. The closely relatedalkaloid fagaronine is less toxic than nitidine. Similar benzophenanthridine derivatives are presentin Chelicloniurnmajus (Papaveraceae), a plant with substantialfolklore history of use in the treatmentof cancers.More recently,NK 109 (Fig. 28.2), a syntheticisomer of fagaridinefound in Fagara ranthoryloides. has been found to have greater antitumour activity than any of the natural benzophenanthridine structures!coupled with excellentstability,and has enteredclinical trials in Japan.
:,,i ill\llW:N{fUn*ll,pn60UCig,WltH',,,,.,,,,, ANTITUMOUR ACTIVITY The systematicstudieshave also resultedin the isolationof many new natural products exhibiting antitumour activity, and a number of these havebeenconsideredsufficientlyactivefor clinical studiesto be commenced (Fig. 28.1 and Table 28.1). Tylocrebine, a phenanthroindolizidine alkaloid from \'lophora crebiJTorawas sufficiently active for further development. but in a clinical trial unmanageableCNS effectsprecludedcontinuationof the studies.Two bis-benzylisoquinoline alkaloids. thalicarpine from Thalictrum dasycarptmt and tetrandrine from C1'cleapeltata appearedparticularly promising and were selectedfor development.Clinical trials showedno antitumouractivity. and these compounds have been dropped from further study. Once again,theseare examplesof compoundsisolatedbecausethe screens employed at that time were too sensitive.On the other hand, tetrandrine is curently of interestfor its ability to block calciumchannels.and may yet have applicationsin the treatment of cardiovasculardisorders. Camptothecin and derivatives, alkaloids from the Chinese tree Camptothecaacuminata, showed broad-spectrumactivity and produceda fair responsein limited clinical trials,but toxicity andpoor solubility were problems. The natural l0-hydroxycamptothecinis more active than camptothecin,and is used in China againstcancersof the neck and head. Synthetic analogues9-aminocamptothecinand particular1ythe water-solublederivativestopotecanand irinotecan(Fig. 28.2)
showedgood responsesin a numberof cancers;topotecanand irinotecan are now available for the treatmentof ovarian cancerand colorectal cancer,respectively.Irinotecan is a carbamatepro-drug of lO-hydroxy7-ethylcamptothecin,and is convertedinto the active drug by liver enzymes. The quassinoidsor simaroubolidesare a group of terpenoid-related (see compoundsisolatedfrom a variety of plantsin the Simaroubaceae p.32;l). Many of theseplants have folk-medicinehistory,particularly for antiamoebicactivity, and a number of the isolatedquassinoidsare cuffently of interest for their antitumour properties.Thus, Brucea antidysentericais usedin Ethiopia in the treatmentof cancer,and systematic ftactionation of this plant has led to the isolation of bruceantin, which shows high antileukaemicactivity at 1ow dosages,and over a wide doserange.Bruceantinactsthroughinhibition ofprotein synthesis,and hasundergoneclinical trials in man. No significanttherapeutic activity has beennoted in theseearly studies,but researchon a whole rangeofquassinoidsrelatedto bruceantincontinues.Maytenusserrata (Celastraceae) and other speciesof Ma\tenus contain maytansine,an ansamacrolide,which was regardedas an antitumouragentof exceptional promise. It is active against severalof the experimentalneoplasmsat very low dosage(microgramsper kilogram of animal body weight) over a 50- to 10O-folddosagerange,and shows a favourable therapeuticindex.It actsthroughinhibition of mitosis.In clinical trials, maytansinewas a big disappointment,and showed few beneficial effects. Synthetic or semi-synthetic derivatives may offer more hope. Other maytansinoidsisolated from Maytenus are also highly active; maytansinewas chosenfor study simply becauseof its relativelyhigher concentrationin the plants.Thereis now the opportunityof producing maytansinoidsvia microorganisms.A speciesof Nocardiahasbeen shown to produce ansamitocin,which is a mixture of estersof maytansinol,the parentalcohol of maytansine.This compoundcould then serveas starting material for semi-synthesisof a whole range of derivatives, and thereis still potentialfor developingthesecompounds. Severalother natural products have also proved sufficiently interesting tojustify clinical trials, or toxicological testingprior to further study. The diterpenestriptolide and tripdiolide isolatedfrom Tripterygiumwil-
T U M O U RI N H I B I T O RFSR O MP L A N T S fordii arepotent antileukaemicagentsthat contain a reactive triepoxide system.The plant is not readily accessibleand containsoniy small amountsof thesecompounds.Large-scaleisolationthus delayedtheir evaluation, and no furlher development occuffed. Neverlheless, in China crude extractsof Triptery-giumn*ilfotdii areusedin inflammatory and immune disorders,and triptolide is currentlyundergoingextensive evaluationin the treatmentof thesedisorders.Of many sesquiterpene lactonestested,few show useful in ylvo antitumour activity, but several of the bestin vivo activecompounds,e.g. the germanacrolide elephantopin from Elephantopuselalas, have beenevaluated.Baccharinis a trichothecenesesquiterpeneisolated from the Brazilian plant Baccharis megapotamicaand is closely related to the fungal trichothecenetoxins (seep. 507).This and someofthe fungal-derivedcompoundsareundergoing thorough evaluation.A seriesof novel alkaloidal estersfrom Cephalotaxusspeciesare cuffently being isolated on a large scale for toxicologicalstudiespreliminary to clinical trials. The parentalkaloid cephalotaxineis inactive. but the estersharingtonine and homoharringtonine from C. harringtonia show good activity in a number of systems. Chineseresearchershave reported favourable results in clinical studies using alkaloidalfractionsof C. hatingtonic, and homohaningtoninein particular is active in patients with leukaemia resistantto existing chemotherapies.Homoharringtonineis only a minor constituentin CephaLotaxus,but can be obtained by semi-synthesisfrom the more abundantcephalotaxine.Tissueculturesof Cephnlotanisalso synthesize cephalotaxineand the activeestersand may ofTerpotentialaccess to these alkaloids in useful quantities. The Central American tree Phyllanthusdcuminatuscontainsin its roots a complex mixture of glycosides,two of which, phyllanthostatinI and phyllanthosidehave demonstratedmarked antitumour properties.Phyllanthoside is in early clinical trials. The cls-stilbenecombretastatinA-4 is one of the mosr potent antimitotic agentsfrom about 20 active substancesisolated f}om the African tree Combretum caJJi'um.A water-soluble phosphateprodrug is now in clinical development. One of the most promising planrderived anticanceragentsto be identified in recent yearsis the diterpenoidtaxol (paclitaxel)isolated fiom the bark ofthe Pacific yew, Taxusbrevfolia (seep. 405). Taxol is structurallyrelatedto the taxanediterpenoids,long known as the toxic constituentsof various Za-rasspecies,including the common yew I baccata. Clinical trials have demonstratedencouragingresponsesin ovarian and breastcancers!and taxol has become an important anticancerdrug. The taxol contentof T. brevfolia tissuesis relatively low (about 0.01-0.03Va)and the bark from about three treesis neededto produceone gram. This restrictsavailability of taxol for drug use and luture suppliesare not assured.However, considerableprogresshas been made in producing taxol by semi-synthesisfiom other taxane derivativessuchas lO-deacetylbaccatin III which can be isolatedfrom leavesof Thxusbaccataand other laxls speciesin much higher yields (0.1-0.2Va).A side-chainanalogueof taxol, known as taxotere,has alsobeendevelopedby semi-synthesis from 10-deacetylbaccatin III. It has improved water solubility and better activity than taxol in some assays,and is aiso currently available for drug use againstresistant breastcancer.Undoubtedly,othertaxol analogueswill be preparedand developedinto evenbetterdrugs. The fiactionation schemesemployed in the NCI programme are based on the rationale that a balance between hydrophilicity and lipophilicity is important for substancesto reachbiological receptors. Thus, extremelypolar plant extracts(aqueous)or extremelynon-polar extracts(petrol) are unlikely to show as much activity as extractsof intermediate polarity (ethanol, chloroform, etc.) which are normally screened.Recently,many peptidesand proteinsfrom microorganisms have shownhigh antitumouractivity,ashavesomewater-solublepolysaccharides,and considerationis thus being given to the testing of
nFrII:r
ru
aqueousextractsfrom plants.Already. screeningof the Mexican plant Bott,ardia ternijolia has yielded the bicyclic hexapeptidesbouvardin and deoxybouvardinas active principles.Bouvardin was selectedfor further development.but was subsequentlydroppedbecauseofits narrow spectrum of activity. The mistletoe proteins mentioned earlier ofl'era furtherexarnple.Futurework on plant peptidescould thusprove mostvaluable.
MECHANISTAS OF ACTION The activity of many of the drugscurrentlyin usein cancerchemotherapy can probablybe ascribedto inhibition ofnucleic acid synthesis,but mechanismsof action differ widely. The examplesquoted above and the many other natural products which have exhibited antitumour activity spana very wide rangeof structuraltypes,yet thereis no common f'eatureto explain their biological activity. Some compoundsare mitotic inhibitors, e.g. colchicine,podophyllotoxin,vincristine.maytansine.and combretastatinA-4, and they act by binding to the protein tubulin in the mitotic spindle,preventingpolymerizationand assembly into microtubules.During mitosis,the chromosomesseparatewith the assistanceof these microtubules.and after cell division. the microtubulesare transformedback to tubulin. Taxol, on the other hand, is a completelydifferent type of antimitotic agent in that it promotesthe assemblyof microtubulesand stabilizesthem againstdepolymerization. Although podophyllotoxinis a tubulin binder.it is intriguing rhar the semi-syntheticanticancerdrugs etoposideand teniposidederived from it have a diff-erentmode of action.Thesedrugs inhibit DNA synthesisand replicationvia the enzymetopoisomerase II. Camptothecin, on the other hand, is known to have specific activity in the topoisomeraseI system.Topoisomerases areenzymesinvolved in DNA replication by their ability to break and resealthe DNA strands,and are classifiedas type I or II accordingto their ability to cleaveone or"both strands,respectively.of DNA. Interactionof ellipticineswith DNA is rnainly by intercalation.and this relatesto their planargeometry. A large number of the natural tumour inhibitors probably act as alkylating agents,and this can be linked to their possessionof highly electrophilic centres,such as o,B-unsaturatedester or lactone tlnctions.The cucurbitacins,bruceantin,triptolide,elephantopin,and baccharin offer examples.Someofthese also containhighly electrophilic epoxidefunctions.There is growing evidenceto supportthe view that these compounds may act via selective alkylation of nucleophilic groups!perhapsthiols, in enzymesthat control cell division (contrast the low specilicity of alkylation by the nitrogen mustards).Chemical modification of the compounds,destroying these electrophilic centres, results in products having negligible antitumour activity. Most agents are cytotoxic, killing normal and cancerouscells alike. Selectivity may result fiom many factors,among which are the transport of the tumour inhibitor into the cell, and the chemicalnatureand steric environment of the specific nucleophile to be alkylated. Specific featuresof the active molecule could be involved in the formation of molecular complexes with growth-regulatorybiological macromolecules. The developmentof taxol and camptothecininto clinically effective agentsis almost entirely due to subsequentrecognitionof their novel mechanismsof action.Initial clinical testsdid not show any significant advantagesin activity over other materialstested,and in fact, lurther developmentalmostceased.Interestwas renewedasthe cellularaction becameknown, and it was appreciatedthat theseagentscomplemented other drugs,ratherthan replacedthem. Likewise, the successofetoposide and its novel mode of action has stimulated a whole area of researchinto other inhibitorsof toooisomerase II.
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PHARMACOPOEIAL AND RELATED DRUGS OF BIOLOGICAL ORIGIN
,.fLIu'IE,DIEVELOPMENTS
-
The searchlbr new antitumourcompoundsin natureis by no meansconfined to plants.Microorganisms,widely employedas sourcesof antibiotics (seeChapter32), producea considerablenumber of metabolites havingantitumouractivity.Many of thesealsohaveantibioticpropefiies. Amongstmaterialsin generaluseagainstcancersaredactinomycin(acti nomycin D). bleomycin. doxorubicin (adriamycin), daunorubicin, mithramycin and mitomycin C. Microorg:mismshave peuticularadvantagesover plants as tar as easeof culture and the opporlunity lbr genetic manipulationare concerned.The recent identificationof epothilones from the bacterium Sorangiutttcellulosum has thus generatedconsiderableinterest,sincetheseagentsmimic the effectsof taxol,and someanalogues are much more potent than taxol, especially towards multidrug-resistant cell lines. Marine animals.e.g. corals and stalfish. may also be a fiuitful sourceof potentially uselul anticanceragentsand this areaof researchis also producing a number of natural productswith antitumour activity. Compounds of pafiicular note are the depsipeptide didemnin B, isolated from the Caribbean sea-squirt Trididemnun't solidtrm,Ihebryostatins,a group of macrocycliclactonesfiom the marine bryozoanBugula neritina, and dolastatin10,a linezLrpeptidefrom the sea hare, Dolabella auricularia. Didemnin B shows activity againstseveral human cancers (including prostate. lung and brain cancers and lymphomas)and bryostatin I has been lbund to bind to and activateprotein kinaseC which mediatesgrowth of cancercells.Dolastatin10 is a very potent inhibitor of microtubule assembly,and synthetic material is now availablefor testing.All three compoundsare in clinical trials. However.despitethe successof the screeningprogrammesin identifying cytotoxic and antitumouragents,thereis somemeasureof disappointment at the small number of clinical agentsproduced.This has causedthe NCI to rethink its luture policy. The major successareafor anticancerdrugsin generalis againstrapidly proliferatingtumoursand there are now effective treatmentsfor cancers such as childhood leukaemias,testiculartumoursand non-Hodgkin'slymphoma that formerly were soon fatal. Adult solid tumours such as lung, breastand colon tumours still respondpoorly to chemotherapy.There are large differencesamongsttumour types in heterogeneity,accessibility,size anddiffuseness,aswell asgrowth rate,and theseleadto significantdifferencesin sensitivityto drugs. It is highly unlikely that broad-spectrum anticancerdrugs will actually be found. Of necessity,the assay methodsusedin the NCI screenshad to be fairly rapid to copewith the large throughput of samples, and consequently rapidly-dividing tumour lines were employed. Thus, the antitumour agents detected were thoseeffectiveagainstfast-growingtumoursand it is not sulpdsing then that they have little activity againstslow-growingtumoursin human studies.In addition, these slow-growing tumours grow and divide more slowly than rapidly proliferating nolmal human tissues such as bone marrow and gastrointestinalepithelium. These tissues thus su1I'ertoxic eff-ectsfrom the administered drug, and the major bonemarrow repressionplus nauside-effectsof cancerchemotherapy, seaand vomiting, tend to limit the dosagetoleratedby the patient. There is obviously a needlbr more specificity,and ideally the drug should selectivelyand specificallyaffect tumour tissuewithout damaging any normal tissue.If drugscan be targetedto specificorgans,or alternatively,if they are specifically bioactivatedonly in the target organ,then more effective and bettertoleratedtreatmentswill result. A natural product of high interestto the NCI is 4-ipomeanol,a toxin isolated from mould-infected sweet potatoes (Ipomoea batatas) which, when ingested,affectsthe lungs and kidneys of mammals.4Ipomeanolis selectivelyoxidized by enzymesin certainlung cells to give a reactive dialdehyde,and this provides a powerful alkylating
agentonly in thosecells containingthe enzyme system.4-lpomeanol is undergoingclinical trials againstlung cancers.There is a high possibility of discoveringother selectiveagentsamongstnatural toxins. Targeting of cytotoxic agents or toxins to cancer cells or tumours using monoclonalantibodiesis also receiving considerableattention, and somesuccesshas beenachievedin animal studiesusing the castor seed toxin ricin (see p. 186) bound to monoclonal antibodies. Knowledge of the basic biological mechanismsby which antitumour agentsact is also aiding the screeningprogrammes.Assayshave now been developed to observe the effects of compounds on specific enzymesystems,whetherthey bind to nucleic acidsor other cell components,and so on. The currentNCI screensare being designedto overcomethe shortcomings of the random mass screeningapproachand to direct more effort towards discoveryof agentsof value in treatinghuman cancers spanninga wider range of types. It currently screensagainstabout 60 different cell lines representinga variety of human tumour types spanning leukaemias,melanomas,and tumoursof the lung, colon, kidney. ovaly. breast, prostate and brain. It is also gradually introducing screensdevelopedliom knowledgeof the basicbiological mechanism of action of known clinically effective anticancer agents. These include tubulin binding (vincristine,vinblastine),tubulin stabilization (taxol), topoisomeraseI (camptothecin),and topoisomeraseII (etoposide).In this way, it is hoped to discoveragentsthat are tissue-specific or highly tissue-selectiveand so producemore useful and effective drugs. Natural product extractsfor the testing programme are to be selected from plants, microorganisms and marine animals with emphasison plants used locally as medicinals and on speciesand groups which have not beenextensivelystudiedin the past.The continued effort to isolate and identify tumour-inhibitory compounds fiom natural sourcesmay well result in the introduction of new anticancer drugs in the future. Even if no natural products themselves attain this status,the benefitsof this researchwill probably be measured in terms of our lurther understandingof the biochemical phenomenaof cancerand the developmentof useful drugs using natural productsas the chemicaltemplates.
ROsEUs CATHARANTHUS The Madagascanperiwinkle, Catharunthusroseus,has beenvariously designated Vinca rosea and Lochnera rosea (.Apocynaceae).It is indigenousto Madagascarbut is now widely distributedthroughout warm regions and is much cultivatedas an ornamental;it grows profusely in southern Florida. Commercial supplies of the drug are obtained from both wild and cultivated plants produced in various locations,including Africa, India, Thailand, Taiwan, easternEurope. Spain,USA andAustralia. Characters. C. roseusis a herbaceoussubshrub,40-80 cm high, becorning woody at the base. The leaves are oppositely arranged, oblong with a petiolateacutebase,a roundedor mucronateapexand an entiremargin.In form the flowers resemblethoseof the common periwinkle Vlncamujor andare colouredviolet, rose,white (var.albus) or white with a red eye (.var.oceLlatus).The fruit is a divergentfollicle. Tetraploid plants are reported to have a more vigorous growth habit and largerflowers than diploid ones. History, Although the plant has a certain reputationin fblk medicine for the treatment of diabetes,modern investigatorshave been unable to confirm this property. Instead Canadian workers, during 1955-1960, discovered that extracts of the leaves produced
""'af,n
TUMOURINHIBITORS FROMPLANTS leukopenic actions in rats. These observationsled researchersat Eli Lilly to undertakean intensive phytochemical investigation of the plant with a view to the isolation of constituentsof value in cancer chemotherapy.Six alkaloidsproved active in this respectand two are now available commercially (see R. L. Noble, Bioclzem.Cell Biol., 1990,68,1344-13s1). Catharanthusis an example of a drug plant which has been introducedinto medicineduring recentyears,and it is usedfor the isolation of pure substancesrather than for galenicalpreparation.Indeed,simple galenicals,preparedfrom the dried plant materialand containinga wide spectrumof alkaloids, would be quite uselesstherapeuticalty. Hence, in normal circumstances,the raw material is handled by the manuf'acturerand doesnot reachthe oharmacistas such. Constituents. About 150 alkaloids have now been isolated from C. roseus;some,for example,ajmalicine, lochnerine,serpentineand tetrahydroalstonine,occur in other generaof the family. Of particular interestis a group of about 20 bisindole alkaloids which contains those having antineoplastic activity, including leurocristine (vincristine) and vincaleukoblastine (vinblastine). Vinblastine is produced by coupling of the indole alkaloids catharanthine and vindoline, both of which occur liee in the plant. Formation of 3',4'anhydrovinblastine from these monomers has been effected with peroxidase isozymes isolated from C. ros€ls suspensioncultures and with commercial horseradishperoxidase(see J. P. Kutney, Nar. Prod. Rep., 1990,7,85-103). Vincristine is structurallysimilar to vinblastine, but has a formyl group rather than a methyl on the indole nitrogen in the vindoline derived portion. Because these alkaloids are only minor constituents of the plant (vincristine is obtained in about 0.0002Vcyield from the crude drug), large quantities ofraw material are required and chromatographicfractionations
are extensively employed in the isolation procedures.In addition, there is a growing demandfor vincristine rather than vinblastine,but the plant produces a much higher proportion of vinblastine. Fortunately,it is now possibleto convert vinblastine into vincristine either chemically, or via a microbiological N-demethylation using Streptomyces al bog ri seol us. Cell cultures. In efforts to improve the productionof alkaloids,cell cultures of C. roseus have received considerableattention (Chapter ll). Successhas been achieved in obtaining total alkaloid yields conesponding to 0.1-1.5% dry weight culturedcells, but culturesproduced catharanthineand tabersonine,and not vindoline, so lacked one of the essentialprecursorsfor formation of the bisindole alkaloids. Researchis still necessaryto find meansofinducing the productionofthe useful alkaloids. Uses. Mnblastine is used mainly for the treatmentof generalized Hodgkin's disease,and non-Hodgkin'slymphomas.Vincristineis used principallyin the treatmentof acutelymphocyticleukaemiain children. It hasotherapplicationsfor lymphomas,small cell lung cancer,cervical and breast cancers.The semi-syntheticvindesineis also used in the treatmentof acute lymphoid leukaemiain children. Mncristine has a superiorantitumouractivity comparedto vinblastine,but is moreneurotoxic. Vinorelbine is a newer, orally active, semi-syntheticanhydro derivative of 8'-norvinblastine with a broader anticancer activity and lower neurotoxic side-effectsthan the other Catharanrlru.s. alkaloi ds. Other species. A number of other Catharanthusspecieshave been investigatedand found to contain vindoline-typealkaloids;some(e.g. C. longifolius, C. tichophl-llus and C. lanceus) contain dimeric alkaloids similar to vincristineand vinblastine.
/\,--f^*'1
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PHARMACOPOEIAL AND RELATED DRUGS OF BIOLOGICAL ORIGIN
RESIN PODOPHYTTUM AND PODOPHYTTUM Podophyllum (Podophyllum Rhizome. May-apple Root, Wild Mandrake) consistsof the dried rhizome and roots oI Podopbllunt peltatum (Berberidaceae,sometimes Podophyllaceae),a perennial herb common in moist shadysituationsin the easternpartsof Canada and the USA. The drug is collected in Virginia, Kentucky, North and Indiana. Carolina.Tennessee
zomesbreakingwith a homy fracturethe starchshowsgelatinization.The rootshavea centralwood occupyingaboutone-sixthofthe total diameter
Constituents. The activeprinciplesofpodophyllum arecontainedin 'podophyllin', the resin, podophyllum resin or which is preparedby pouring an alcoholic extractof the drug into water and collecting and dlying the plecipitate.American podophyllumyields about 2-87c and lndian podophyllum (seebelow) about 6-127c ofresin. Podophyllum Resinof the [/SP was obtainedsolely from the American drug but that Collection. The rhizome.which is abouta metrein length,is dug up. of the BP may be eitherArnericanor Indian, althoughthe resinsfrom cut into piecesabout 10 cm in length,and dried. thesetwo sourcesare not identical. The chief constituentsof the root belong to the group of lignans. History. The drug haslong beenusedby the North AmericanIndians which are C1scompoundsderivedbiosyntheticallyby dimerizationof was introduced into the 186;l as a vermifuge and emetic, and two C6-Cj units (e.g. coniferyl alcohol) at the B-carbonof the sidePharmacopoeia.Podophyllin, a crude resin obtained from the rhichains.The most important ones presentare podophyllotoxin (about zomesand roots.was subsequentlyemployedas a purgative.but Lrsage 0.255;). B-peltatin(about 0.337c)and o-peltatin (about 0.257o)(see declined,until in 19112podophyllin was recommendedfor the treatD. E. Jacksonand P M. Dewick, Phytochemistrl-,1984, 23, 114'7has led to an ment of venerealwarts. Since then. extensiveresearch 1L52).In the root, all of theseoccurboth free and as glucosides.Preparappreciation of podophyllum's antitumourproperties,and the develation of the resin resultsin considerablelossesof the glucosides.The opmentof successfulanticanceragents. root also containssmalleramountsof the closely related4'-demethylMacroscopical characters. Podophyllum occurs in subcylindrical podophyllotoxinand its glucoside.desoxypodophyllotoxinand podoreddish-brownpieces about 5-20 cm long and -5-6 mm thick. The phyllotoxone.These compoundsall possesscytotoxic or antitumour outer surfaceis smooth (autumn r"hizome)or wrinkled (sumrnerrhi- activity. but activity is lost on mild basetreatment.Epimerization.cr zome).The nodesare enlargedto from two to threetirnesthe diameter' to the carbonyl, resultsin the formation of the thermodynamically more of the internodes.On theseswellingsthe remainsof the aerialstemsare stable cl,i-fusedlactone ring, rather than the severelystrained/rarr.r arrangementof the naturalcompounds. visible on the uppel surfaceas large cup-shapedscarssurroundedby \.)-21
OH
OH I
,"='rsr\-" i l t
(
t.'r""\,-(
t
o
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ocHa
OH R = CH3 Podophyllotoxin, R= H Demethylpodophyllotoxin.
R = CHs o-Peltatin, s-Peltatin,R = H
the remains of the cataphyllaryleaves,some of which have buds in their axils. On the lower sideofeach node are about5-12 root scarsor portionsof roots. The latter,if entire, are 2-'l cm in length and about 1.5 mm in diameter.The drug breakswith a shortfiacture and showsa starchyor horny interior.The transversesectionof the internodeshows a starchybark and pith and a ring of 20-30 small flbrovascularbundles. The latter are not radially elongated(cf. lndian podophyllum).A section of the nodeis similar but showsbranchesfrom the ring of bundles running upwards to the cup-shapedscar of the aerial stem or downwardsto the roots.Odour, slight; taste,disagreeablybitter and acrid. Microscopical characters. A transversesectionof the rhizonteshowsa dark-colouredepidermis,one or two layers of cork cells. a large collenchymatousand parenchyrnatouscoftex and pith. and a ring of srnall vascularbundles.The smallvesselsof thelatterhavesimpleporesor reticulate thickenin-u.Many of the cells of the ground tissuecontain reddishbrown massesof resin.clustercrystalsof calciumoxalate"andstarch.The cluster crystals are 30-60 100 ptm in diameter,many exceeding60 pm (cf. Indianpodophyllum).The starchoccursin simplegrains3-15-25 pm in diameterand in compoundgrains with 2- I 5 components.In thoserhi-
Etoposide
Uses. Podophyllumresin has long been usedas a purgativebut has largelybeenreplacedby lessdrasticdrugs.It hasa cytotoxic actionand is used as a paint in the treatmentof soft venerealand other warts. Podophyllotoxin is also used for this purpose. Etoposide (4'is a lignan derivademethylepipodophyllotoxinethylideneglucoside) tive obtainedsemi-syntheticallyfrom podophyllotoxinand usedin the treatmentof small-celllung cancerandtesticularcanceraswell aslymphomas and leukaemias. The water-soluble pro-drug etopophos (etoposide4'-phosphate)is also available. The related thenylidene derivativeteniposidehas similar anticancerpropertiesand though not as widely used as etoposidehas value in paediatricneuroblastoma. lymphocyticleukaernia,and brain tumoursin childlen (seeH. Stiihelin andA. von Wartburg,CancerRes.,1991,51, 5-15).
INDIAN PODOPHYTTUM Indian podophyllum consists of the dlied rhizome and roots of a perennial Podophyllumherandrum, syn. P. emodi (Berberidaceae), herb found in Tibet, Afghanistanand the Himalayanareasof Pakistan and India. The drug is collectedin India, Pakistanand China.
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T U M O U RI N H I B I T O RFSR O MP L A N T S Macroscopical characters. The dr-Lrg,at tlrst glance. shows little resemblanceto American podophyllum.The rootsfiequently breakoff and some samplesconsist almost entirely of rhizomes.while others consistlargelyof roots. The rhizomesoccur in much contortedpiecesof an earthy brown colour.about21 cn long and I 2 crr in diameter.The internodesare much shorterthan in the Americandrug. with thc rcsult that eachpiece bearsthe remainsofabout 3 6 branchesendingin cup-shapedscarsand about20-'10rootsor root scars.The rhizomeis hard and somewhatdifllcult to break.Internallyit is pale blown in colout and horny (usually) or starchy.The generalaffangementof the tissuesresemblesthat fbund in Americanpodophyllum.but the vascularbundlesa.remore elongated radiaily.The odour and tasteresemblethoseof theAmericandrug. Microscopical characters. The calciurn oxalateclllster crystalsare fewer and smaller,20-30-60 ;tm. The starchgrainsare simpleor 2-20 compound;individual glains 2-7-34 pm (cf. Americanpodophyllum). Constituents. Indian podophylium containsrnorc resin (about6-12%) than the Amedcan drLrgand the percentageof podophyllotoxin in the resin (up to 40%) is much higher.The root contains aboutlVr: podophyllotoxin. 0.45c/c4'-demethylpodophyllotoxin and smaller amounts of relatedlignans(see D. E. Jacksonand P M. Dewick. Phvtochentistrl'. 1984.23. 1141-1152).Only tracesof the peltatinsare present.but the rangeof corlstituents is much the sameasin theArnericauresin. Uses. Indian podophyilumis usedlbr the preparationof the resinand isolationof podophyllotoxinfor drr.rgr.rse and semi-synthesis of etoposide.Other lesscommon speciesof Podophvllun (.e.g.P. pleianthuml and relatedgenela(e.g. Diphl"lleial also containpodophyllotoxinand structurallyrelated lignans (seeA. J. Broomheadand P. M. Dewick, Phytochemistrl', 1990,29, 383I 3837).
TAXUSBREVIFOLIA AND TAXOL A nctteon nornenclantre'. the nametaxol was given to a diterpeneester with anticancerpropertieswhen it was flrst isolatedin 1971 ftom Zrrrl,i brevijblia.When this compoundwas subsequentlyexploitedcorlmer-
cially as a drug, Taxol was registeredas a trademark.Accordingly.the genericnamepaclitaxelhasbeenassignedto the compound.The literalure now containsan unhappy r-uixtureof the two names.thollgh the originalnametaxolis mostoftenemployed. The Pacific yew. Ia.ras bret,ifrlio (Taxaceae)is a slow-growing shrub/treefoLrndin the forestsof Nolth-WeslCanada(BritishColumbia) and the USA (Washington,Oregon.Montana.Idaho and N. Caliibmia). Although the plant is not rare. it docs not lbrm thick populations.and needsto be mature(aboutI 00 yearsold) to be largeenoughfbr exploitation of its bark.At this agc.the treewill be some6-9 rn high, and havea trunk of about25 crl in clian-rctcr. The balk is removedflorn mxturetrees during the periodMay-August.The wood of I l:trct'ifoliais not sLritable for timber.and in somealeas.plantshavebeensysternatically destloyed to allow cLlltivationof faster'-growiu-e commerciallyexploitableconifels. Hari'estingis now stlictly regulated.Although taxol is crrrently extracted from the dried bark. it is rcalizedthat this cannotbe expectedto provide a satisfactorylong-termsupplyof the drug. It requiresthe bark tiom aboutthleenratLlreI OO-year-old treesto providconegramof taxol.anda courseof treatmcntmay nccd2 glarnsof taxol.Currentdemandfor taxol is in the re-qionof 100-200kg per annum. Constituents. All parts of ?l.urs 11vp1,ifolia contain a wide range of diterpenoidderivativestermed taxanes.which arc structurallyrelated to the toxic constituentsfounclin other Zl.rHsspecies.e.g. the common yew. Zr,ursltur:catu.Over a hundredtaxaneshave beencharacterized fi'om various ?r-ru.sspecies,and taxol is a memberof a small group of compoundspossessinga four-memberedoxetanering and a complex esterside-chair-r in their structures.both of which are essentialfor antitumolrr activity.Taxol is fbund predominantlyin the bark of I brevr,fo1ia.but in relativelylow amounts(0.0l-0.027.). Up to 0.0337cof taxol has been recordedin somc samplesof letrvesand twigs (see N. C. Wheeler et al., J. Nar. Prod.. 1992,55. 432 140), br:t genelally the taxol contentis much lower than in the bark. Significant r aliation in taxol contentdependingon season.-qeogrilphical location,and environmental factors as well as individual populationsof tlees have been noted.The contentof someothel taxanederivativesin the bark is considerablyhigher',e.g. up to 0.27cbaccatinIII. Othel taxanederivalives characterizedinclude 10-deacetyltaxol.10-deacetylbaccatin III,
n Y
oAruH
(cH3)3c\o
I oAttH
o
r=-A
o-
ococH3
OH
Taxotere (Docetaxel)
Taxol (Paclitaxel)
1O-Deacetylbaccatin lll
4m--
*filP
*!*ihqilD
Etr
PHARMACOPOEIAL AND RELATED DRUGSOF BIOLOGICAL ORIGIN cephalomannineand 10-deacetylcephalomannine. A more satisfactory solution now employedfor the long-term supply of taxol and derivatives for drug use is to produce thesecompoundsby semi-synthesis from more accessiblestructurallyrelated materials.Both baccatinIII and l0-deacetylbaccatinIII may be efficiently transformedinto taxol. lO-Deacetylbaccatin III is readily extractedfrom the leavesand twigs of Taxusbaccata,and althoughthe contentis variable,it is generally presentat much higher levels(up to 0.2%) than taxol can be found in Z brevifolia. Thxusbaccatu, the common yew, is widely planted as an ornamentaltree in Europe and the USA and is much faster growing than the Pacilic yew. Cell culturesof Taxusspeciesalsooffer excellent potential for production of taxol of lO-deacetylbaccatinIII; taxol yields of up to 0.27c dry weight cultured cells have been reported. Taxus cuspidalc (Japaneseyew): large-scalecell cultures have produced approximately3 mg/l of taxol and 74mgll total taxanesafter 27 daysof growth(S. H. Son et al., Planr CeLlRep.,2000,19.628). For a review listing the variousconstituents(374) of yew treessee V S. Parmaret al., Phytochemisny.1999,50, 1267.
Uses. Taxol@(paclitaxel)is being usedclinically in the treatrlrentof ovariancancers,breastcancersand non-smallcell lung cancer.It ma1' alsohave potentialvalue againstother cancers.Taxotere@(docetaxel) is a side-chainanalogueof taxol. which has also been producedby' semi-synthesisfrom l0-deacetylbaccatinIII. It has improved watersolubility and is usedin treatmentofbreast cancers.
Furfher reoding CordellG A 1993Thediscovery of plantanticancer agents. Chernistry and lndustry8,+l-84.+ KinghornA D, Balandrin M F (eds)1993Humanmedicinal agents fiom plarrts (ACSSymposinrn Series, 53,1). AmericanChemical Society. Washington SinhaS.JainS I 994.Naturalproducts asanticancer agents. Progress in Drue Research 42:53-132:13:211282 CraggG M, NewmanD J.WeissR B 1997.Coralreefs.fbrests, andthermal vents:theworldwideexploration of naturefor novelantitumor agents. Seminars in Oncology 24: 156-163 ShuY-Z I 998Recentnaturalproducts based drugdevelopment: a pharmaceutperspective. icalindustry Journal ofNaturalProducts 6l: 1053-1071
@t
Antiprolozool noturol products C. W.Wright
Diseases causedby protozoaarercsponsible lbr consiclerable rnortalitl. anclmorbidity.especiallyin the developingwor.ld.Many plantspecics areusedin thepreparation of traditionalnedicinestbr the treatmentof plotozoaldiseases and plantsarethe sourceofthe clinicallyusedanti(l) (fiom Cirtchontrspp.).and afiemisinin(7) rrralalialdlu-ssqr-rinine (trom Arlcllrsru oruludl.Quininehasbeenusedas a templatefbr the der,elopment of a nuntberof svntheticantirnalarials such as chloroquine,u'hileI numberof scnti-synthetic artemisininderivatives(9, l0) which are theraper-rticallv superiorto artcmisininitself are in clinical use. Other examplesof natural product-dcrivedantiprotozoalagents arethe rritroimidazole s u'hicharebasedon the antibioticazomycin(2) producedby a speciesof Strelttontteswhich was collectedon the Islandof Rdunion.Azornycirru,aslbundto be activeagainstthe protozoan Tricltontonusrogirtulis. the causativca-9entof trichomoniasis. and the syntheticanalo-eucmetronidaroleu'as the first efl'ectivetrear mentfbr this disease. Laterit w,aslbund that the latterwas alsohighly efI'ectir,ein the treatmentof amoebiasisand siar.diasis as well as in the trcirtmentof infections car.rsed by anacrobicbacteria.A more recent example of a natulal prodnct-delivedantiprotozoaldrug is the antimalarial atovaqlronewhich was developedfiom lapachol(13) a naphthoquinonefound in S. American speciesof the Bignoniaceae.Natural productshave maclea sisnificant contlibution to the chernother-apy of protozoaldiseasesand it is possiblethat the continuedinvestigationof natural producfderived compounds will provide new anliprotozoal drugs in the future.As shown in the tbllowing section.rnanyof the availableantiprotozoalagcntshave serior-rs lirr-ritations due to their toxicity and/or the dcveloprnentof clrug-resistantparasitesso that new drugsale urr:entlynecded.
DISEASES CAUSEDBY PROTOZOA
DISEASES CAUSEDBY PROTOZOA 407 METHODSOF INVESTIGATION 408
Moloriq In the mid-1950sit was confidentlyanticipated that malariawould be eradicatedbut by the | 990s the diseasehad reachedeprdemicpt.oprrrr" tions throughout the tropical world. The tailure to er.adicatemalaria ,,,,'i,:. was due to a numbel'of f actorsincluding the entergenceof malariiiparI ..t asitesresistantto antimalarialdrugs. resistanceof the vector female ,, ,:,,., I r' : anophelinemosquitoesto insecticidessr.rchas DDT and the avoidance :1rr,1 of insecticideuse becauseof bxicological and ecologicalconsider- .,1.,.. ations.It is estimatedthat betweenone ancltwo rnillion people.mainly ,rl.,,,: children.die fiorn thc diseaseeachyeal and that some.100 800 million peoplecontractma1ariaannuiilly.MaladacausedbyPI(|'\t|1()(liIt|n'f.tlL. parrnt is the most serioustype becauseit otten provesfatal due to the complicationof cerebralmalariaunlessprompt trettment is given. Therc is now widespteadresistanceof P. .fulcipttrtLrrr to chloroqr.rine 11,,,,', "''l''"1 and to someothel antimalarialdrugs.P liL'a,ris also a common cause of malafiabut it is usually sensitiveto trettnrentr.vithchloroqLrine (fbl,.t, ,.. lowedby a courseof prirnaquineto eradicateparasileswhich lie dorrnant in the liver and may later cause relapses). although chloroquine-resistant strainshave beenrepor.ted.The other spcciesof malariaparasitewhich (lessconrmonly)inftct man arep. mtrlttritrt uttl P. otttIt'.
MODESOF ACTIONOF NATURAT ANTIPROTOZOATAGENTS4O9 EXAMPLES OF ANTIPROTOZOATNATURAT PRODUCTS 409
coNcrusroNs4t3
Tryponosomiosis A.fiicansleepingsickness(Aftican trvpanosomiasis) is causedeither by Tn'punosornubrutei gombiense(W. Aflican fbrm) or by T. brucei rltotlesiense(E. Afiican form). The diseaseis transmittedby the Tsetse f1yandinitially causesa f'everish illness.Later.stases ol thetlisease are characterizedby effects on the central nervous system, including movementdisordersandconvulsions. cxcessivesleepiness anclfinally
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LRIGIN P H A R M A C O P O E IA N L D R E L A T EDDR U G SO F B I O L O G I C AO coma. About 50 million people live in areas where the diseaseis Anfimoloriol ossoys testsarecommonly employedutilizing P endemicand in addition to its threatto humansit is also a major prob- 1n liro assays. Two diff-er"ent lem for livestock. Current drug treatmentis limited as suramin and berghei in mice. In the 4-day suppressivetest (Peters'test),mice are pentamidineare only efTectivein the early stagesof the diseasewhile inocrrlatedwith red blood cells infected wtth P. berghei. The plant the arsenicaldrug rnelarsoprol.which is usedin the late stages.is toxic. extractor compoundundertestis administereddaily for 4 days,stafiing or Treatment has improved with the developmentof eflornithine (uon the day of int-ection,and may be given orally.or by subcutaneous difluoromethylornithine),a polyamine synthesisinhibitor, bLrtthis is intraperitonealinjection. Different dose levels are administeredto groupsof live rnice and on the fifth day a blood sampleis taken from not eff'ectiveagainstthe E. Afiican fbrm of the disease. In SouthAmerica.Chagas'diseaseresultsfrom inf-ectionwith T. cnrzi eachmouse.Blood films are preparedand stained(e.g. using Giemsa which is transmittedby housebugs living in the cracksof mud-walled stain) so that the rralaria parasitesmay be observedmicroscopically. causesheartfailure and othercomplications;it of parasitizedred blood cells in the testgroupsand conhouses.Chagas'disease The percentage is estimatedthat some20 million peopleareinfectedwith 7. crur:i.Only trol group of mice are determinedand the ED59value (i.e. the doseof is calwhich causesa 50t/creductionin parasitaemia) the acutestagesof the diseaseare amenableto ffeatmentbut the available extract/compound arepoorly tolerated. lf any test animalsdie beforethe end of the assaydeathis condrugs,nifurtimox andbenznidazole. cr-rlated. sideredto be due to the toxicity of the substanceundertest. Leishmoniosis The Rane test utilizes an alternativeprocedurein which mice are which afTectsmore than 20 million peopleworldwide.is given a standaldinoculationol P. berg,heiwhich would normally be Leishmaniasis, causedby various speciesof Leishmunia which are ffansmittedby expectedto kill thc animalswithin 6 days. On day 4. a single doseof leishma- the exffact/compoundunder lsst is given at I seriesof different dose f'emalesandfliesof Phlebottn,rlsspp.In S.America,clrtaneous niasis(inf'ectionsof the skin and mucousmembranes)is causedby L levels.The tcst malerial is consideredto be active if the mice survive for l2 daysor more.The maximurnefl'ectivedoseis comparedwith the mexicana and L. braziliensls and in the Old World by L. tntpica and L. (kala maximum tolerateddose(i.e. the dosethat producesno more than one rnalor.Anotherform of the diseaseknown asvisceralleishmaniasis azar) which occurs in northem India is causedby L. drnovani which in five toxic deaths).In this way a measureof the diff-erencebetween infects the reticuloendothelialsystem:unlesstreatedthis condition is the efTectivedoseand the toxic doseis obtained. patientssuchasthosewith AIDS are rapidlyfatal.Immunocompromised susceptibleto inf'ection with L. infcutturl in Meditenanean countries. In vitro assays. Plttsntodiumfalc'ipurum is cultured in human red Antimonial drugs, diamidines and arnphotericinB are used in the treat- blood cells in 96-wel1 microtitrc plates. The inhibition of parasite growth in the presenceof drugs may be assessedby measuringthe rnent of severeforms of leishmaniasisbut toxic ef-fectsare common. incorporationof [3H]-hypoxanthineinto the parasite.Recently,a new diseoses methodhas beendevelopedwhich doesnot requirethe useofradiolaGqstrointestinol Diarrhoealdiseaseis often the resultof protozoalinfectionsin the gas- belled compounds.Parasitelactatedehydrogenase(LDHI activity is trointestinal tract. Giardiasis, caused by Giardiu intesthali.s (al'so measuredby addinga reagentcontainingan analogueof NAD (APAD, known as G. tluodenalisand G. Iamblia) is thoughtto infect 200 milacetylpyridine adenine dinucleotide) and a tetrazolium compound. lion peopleeachyear and is responsiblefol an estimatedI 0 000 deaths. APAD is reducedby parasiteLDH (but not by red cell LDH) and then the reducedAPAD in turn reducestetrazoliumto give a blue colour Amoebic dysentery is caused by Entamoeba histob'tit:tt which, if the intensityof the colour untreated, may give rise to serious complications inclLrding liver which is measuredspectrophotometrically; There are sorne212million casesannuallyand an estimated is proportionalto parasitegrowth. abscesses. 75 000 deaths.Both diseasesaretreatablewith metronidazolealthough ossoys Antiomoebic this drug is poorly toleratedby somepatients.As a result of the AIDS epidemic the importance ol Cn'ptosporiditrmpan-unt as a common In vivo assay. Rats are used for deterrninationof activity against causeof dianhoea in both normal and immunocomplomisedpatients intestinal infections and hamstersare used for assessingactivit) has beenrecognized;currentlythereis no effectivetreatmentfbr cryp- againsthepaticinfections.E. histol,rticais introducedinto the caecum via the rectum and the intestine is examinedfor the presenceof tosporidiosis. amoebaeand ulceration.Liver infections ale initiated by injection of amoebaeinto the lobes. In practice, such in vlv., tests are extremely METHODSOF INVESTIGATION ditficult to perform, are time-consuming and are unpleasantfor the animals. The study of antiprotozoalcompoundsfiom plants has required the developmentofbioassaytechniques.especiallylt lilro methodswhich In vitro assays. The developrnentof axenic culturesof E. histolytica allow largenumbersofplant extractsto be screenedfor activity against has enabledthe developmentof ln I'llro assays.Before the development of axenicmedia it was only possibleto grow amoebaein the prespathogenicspeciesof protozoa.In vitrctassaysare particularlyusefll fractionationof plant extracts.It is not alwayspos- enceof bacteria(polyxenicculture),which madeinteryretationof test for bioassay-guided sible to test againstthe speciesor stagesofthe lif'e cycle which actual- resultsextremelydifticult. E. histol,tticais grown in 96-well microtitre At the ly int'ectman becausethey cannotbe culturedor will not infect animal platesin the presenceof serialdilutions of extracts/compounds. by visual end of the test time the growth of amoebaemay be assessed models; for example, i? l,i//? tests againsl Trypanosomaspp. are olten observationwith a microscope.Alternatively a colorimetric method which arefound in the insectvector.An canied out using epirnastigotes in vitro assay for activity against Plasmotlitmtfalcipurzar?was not may be usedin which the culturemedium is removed,leaving healthy developeduntil 1979and it hasnot provedpossibleto cultureit in ani- amoebaeattachedto the bottom of the wells while dead amoebaeare mal models. hence the most common in f iro testsutilize the rodent washed;rway.A measureoi the number of amoebaeremainingin the malariaP. berg,heiinmice. Brief descriptionsof antimalarialand anti- wells is obtainedby fixing and stainingthe parasites.The quantity of amoebic tests are given here in order to illustrate some of the tech- stain taken up is pr"oportionalto the number of amoebaeand is detercally. n'rined spectrophotometri niqueswhich are ernployed.
ANTIPROTOZOAL PRODUCTS NATURAL
E
ilmrlstigeousleishmaniasisand hasbeenshown to eliminateL. tttrt.jt,r althoughit was lesseft-ectivein animal models otesfrorn macrophages of cutaneouslcishmaniasis.Belberine(3) and a numberof rclatedalkaloids including palmatineand jattrorhizinehave potent in vitro actit'ities againstP..fttlciparuntbut they havelittle activity againstP berghei in micel in contrast.belberinehasbeenreportedto be effectiveagainst intestinalalnoebiasisin mice but it is only weakly activeagainstE. ftrstoh'ticu irt t'itro. Thesefindin-ssillustratethe limitationsof ln r'ilro tests tbr the prediction of activitf irt t'it'o as compoundsmay need to be n-retabolicallyactivatedirr lrlo lnd thus may be inactive in in t,itro tests.Conversely,drugs which alc active ir vitro may be metabolized to inactive metabolitesur ln rr and lack of activity ln rllo may also be due to problemsrelatedto the absolptionand distributionof the drug.
MODESOF ACTIONOF NATURAT ANTIPROTOZOALAGENTS
trgentis dependentupon a The effectivenessof any chemotherapeutic favor"rrable therapeuticratio, i.e. the dlug must kill or inhibit the parasitebut havelittle or no toxicity to the host.Although a lar-tenumberof natural productshave been shown to be able to inhibit the growth of one or nore speciesof protozoa.very few havebeenshownto be selectively toxic to the parasite.Selectivitydependsupon difl'erencesin biochemistrybetweenthe parasiteand the host suchthat a dru-qcan act on a biochemicaltargetin the parasitewhich is eitherabsentin, or significantiy diff'erentfrom, that in the host. Marked differencesin metabolism from that in mammaliancells havebeenlbund in somespeciesof pathogenicprotozoa.For example, Trlpanosonn spp. are unique in Conessine that glycolysis takes place in an organelleknown as the glycosome (Apocynaceae)known as In India. the bark of Holurrhenu Ttttbe.scert.s ratherthan in the cytosol as in all other organisms.The unusualpathkurchi bark has been used traditionalh l'or thc treatmentof amoebic ways found in the glycosomemay provide viable biochernicaltargets. dysentely.A numberof steroidalalkLrloidshavebeenisolatedfrom the In the following sectionssomeof the biochemicaldifl'erencesbetween bark (see p. 393) and a few of these. incluciing the rnajor alkaloid parasitesand host cells which are exploitedby antiprotozoaldrugs are conessine(4) have been shown to have rrr lrtrzr activity againstE /rl.iproductswith described.However,the modesof actionof many natr"rral toly'tica. antiprotozoalactivitiesare, at present,unknown and it is possiblethat someof thesemay act on biochemicaltargetsuniqueto protozoa.
Cryptolepine
EIA$PIES OF ANilPROTOZOAINArURAI PRODUCTS ALKALOIDS Berberine This benzylisoquinolinealkaloid, comrnon in members of the has been used clinically in the treatmentof cutanMenispermaceae,
A decoction of the roots of Cryptolepis sungLtirtolertl./(Asclepiadaceae),a climbing plant, is usedin WestAfiica fbr the treatmentof malalia and various other infectious diseases.The indoloquinoline alkaloid cryptolepine (5) is the main alkaloid present and this has potenti/7r.'ltroactivity againstP.fiLlcipurttntbulit is alsocytotoxic as a resultof intelcalationinto DNA as well as inhibition of DNA synthesis II. Crypblepine itself is too toxic for use as an and of topoisomer"ase antimalarialbut a number of derivativeshave been made which have sood antinlasmodialactivitiesbut with reducedinteractionswith DNA
fi-N
il- N ^ Ntlo " t H
ocH3 ocH3 berberinechloride(3)
azomycin(2)
q u i n i n e( 1 )
Un"
H
CHc I -
NI
H I I
I
H
H
CH3\N
cHs/ (4) conessine
emetine(6)
cryptolepine (5)
Fi1.29.1 properties withontiprotozool Exomples of someolkoloids
q-
ielt:.._
.!4@.\-:il
P H A R M A C O P O E IA N L D R F L A T EDDR U G SO F B I O L O G I C AO LR I G I N and lower cytotoxicity.Theseare cllrrentl) bcing assessed fbr ili llr,o antimalarialactivity.
antiplasrnodialactivitiesand tubulosineu'as actil'e tgainst malaria in mice. Several alkaloids isoli,rtedfl'om S/n.ry'irosLtsomlttrrensi.s *crc fbr-rndto be active againstE. listolt'ticu. P. .frtlc:iltarumancl(jittnli,i Emefine inte.stirtulis in yitro br"rtin contrastto emetinethey were lesstoxic tr, This alkaloid was discoveredas a resultof investigatingCeltlnelis cells.An interesting seriesof 2-substitllted quinolinealkaloidsisolatctl (Rubiaceae). ipec'ut'uartltcr a plant usedby S. ArnericanIndiansas a from Galipeulougifloru(Rutaceae). a speciesr.rsed in Bolivia to trc-ar treatmentfor d),sentery(see p.365). Er.netine(6) is highly active cutaneousleishmaniasisand f-everin Bolivia. havebeenassessed fol iir againstE. histolttitu in vitrc and is effectivein the treatmentof both lr/r'o and ir ynzr antileishrnanial activities.ChimanineD (2-ll'.1'hepaticand intestinalarroebiasisbut has toxic ef-fects, espcciallyon transcpoxypropyllquinoline) was more potentthan meslumineanrithe heart.It inhibits proteinsynthesiswhich is probabll,responsiblefbr m o n a t e ( G l u c a n t i r l e ) a g a i n s t c u t a n e o L l sd i s e a s ec a u s e d b 1 , L . its antiamoebicaction and the toxrc eff-ectsseenin man. The relatcd t u n t t : t , nt(r s i \r n d u l . e f l e c t i e r i n : t r p p l c r . i r t rp: i r r u : i t l r c n t i rl t r l ] i e , . (synthetic)compounddehyc'lroemetine is lesstoxic. probablybecause with visceralleishmaniasis duc to l-. dortoytuti. AnotherconstitLlent. lit is eliminatedfrorn the bodv morc raoidlv than emetine. n-pentylquinolinewas not active against LelsllruuirL sp. br.rthas il r.'llr:o and rrr r..lloantimalalialactivity.The B-carbolinealkaloid harnra Quinine line. a constituentof PeganLtntltamuiltt and some relatedtryptaminc The barksof variousspeciesol Cirttltonu oliginating from S. Amcrica derivatives,have been testedagainstL. untaa.onertsis: harmalineha. were usedtbr the trcatmentof feversincludingmalariafiont aboLrt1630 potenti/l r,Ilrc activity but a-ethyltryptaminewas orallv activein micc (seep. 382) until they were superseded by quinine (1) (isolatedtion infectedwith the sameorganism.The relatedcontpoundharmineua. Cintlnna balk in I f320).For more than a centuryqr.rininewas the only tbr-rndto havc activity n-qainst I crrr;r epimzrsti-esotes. Ellipticine. a coneft-ectiveantimalarialavailablebut the del,elopmentof syntheticanti- stituentof OchrosictelliptictL.and a number of dcrivativeshave bcr.n malarials-somc of which wele basedon the quininc moleculesuchas investigatedfor their etl'ectsagtrinstI cra:/. Thesccompoundsinterncr quinacline.chlorocprine and more recentlymefloquine led to a decline with DNA but it appearsthat in trypanosornes the kinetoplastDNA i: in the useof qLrinine.However.with the cleveloprnent ol P.JLLlcipttn.tnt more susceptiblethan the nuclcal DNA to thesecompoundstrnd ntar resistantto chloroquineand other antimalarialsthere has bcen a resur- thercfbre be a potential biochernicaltarget. Several alkaloids of thc gencein the r-rse of quininewhich is usr.rally efTectivein the treatmentof acridonetype havebeenshownto haye itt y'itroantiplasmodialactivit\ : chloroquine-resistant rnalaria.Although cluinine is a relatively toxic atalaphillinine isolated from Ataluttitr ntorutphtlla (Rutaceae)r'"a: drug. it has a selcctiveaction againstPlusnuttliuduc to difl'erences alsoactivein rniceinftcted with P. berglrci.The aporphinealkaloid(-t betr"'een the hostandthe parasite:trvo distinctmechanisms areinvolved. roemrefidinefront SporattultlrcliLondnloaotlutil(Hernandiaceae) llso Firstly. the drLrgis able to iiccumulatein the parasiteto concentrations has in r.'rrizantiplasmodial activity(againstchloroquine-sensitive ancl higherthanthosein thehostcells.Malariapirrasites stfains)and is acti\,eirr lluo againstP. bergheiin -elowingin redblood chloloquine-resistant cellsdi-9est h;remoglobinin an acidicfbod vtrcuole:qr-rinine beinga basic mice. dru-{is concentratedin the acidic viicnoleby an 'ion trapping'mechaSpeciesof Alstoniu (Apocynaceae) are widely used in traditional nism. Secondly.in the food vacuolehaerroglobinis broken down into medicine lbr the treatrnentof rlalaria bllt the rnonoterpenoidindole amino acids (which may be utilized by the parasite)leaving the l.raem alkaloidsthey containhavewith f'ewexceptionsbeenfor.rndto havc litring as an Llnwantecl lesidue. Haem is toxic and in rnarrmaliancells it is tle or no activity against malzrriaparasites.Those which have bccn brokendorvnby the enzyne haenroridasebut this enzynreis trotpresent shown to haye in y'itroantiplasmodialacti\,ity suchas villastonineancl in malariaparasites.In ordel to detoxify haem the parusiteconrertsit macralstonine(presentin A. angustifollrr)aredirnericalkaloiclswhereinto a polyn-reric substance, haemozoin,alsoknown asmalariiipi-qment. as monomericAlstctnirL alkaloidssuchas alstonerine. echitamineand QLrinineand some other antimalarialssuch as chloroquineand meflo- pleiocarparnineare only weakly activc. It is possiblethat thc major quine bind to haem, thus preventingthe fbrrnationof haemozoin;the alkaloidspresentin Alstonia specieshaveother ef-fects. e._s. untipyrerie haern drug complexis toxic andcausesparasitedeath(Fi-e.29.2). propefiieswhich may explain the rvidespreaduse of thesespeciesin malariatreatment.In this eontcrt it ir intelestingto ltote that the.lOther olkoloids quinazole derivative. t-eblifirgine.found in the Chinese antimalarial In addition to the above.many othel alkaloidshave becn investigated plant Dichnta febrifuga (Saxifra-qaceae) is ef1'ective againstmaiaria in for their antiprotozoal properties. Bisbenzylisoquinolinealkaloids rnice and also enhances nitric oxide nroduction in activated have been isolatedliom a nurrber of Menispermaceous plant species usedtraditionallyfbr malaliatleatmentand someof them.e.g.tetfanHaemozoin dline and phaeanthine, haveir r.'itrzr antiplasmodial acti\ities;rnterestpigment) (malaria ingly. these compounds were fbund to be more active against l chloroquine-resistant than againstchloroquine-sensitive strains.The Blockedby ..... ..t above alkaloids har,e also been shown to reverse chloroquinequrnrne Digestion rcsistancein P.falciparurT?. an action rvhich rnaybe relatedto their calHaemoglobin+ H a e m+ A m i n o cium channcl-blockingactivity. while others such as glrorarpine, : ?CldS daphnandrincand obaberinehave actir,itiesagainstpromastigotesof Leislururtiu sp. The African antirnalarialrnedicinal plants Arrcl"ilrY oclatlLts ubbreyittttts (Ancistrocladaceae)and Triphtophvllunt peltaR-X Activation Alkylation llm (Dioncophvllaccae) containnaphthylisocluinoline alkaloicls which Artemisinin+ R- -----------> drug-protein have ln urlrp and in y'it'o antimalarial activityl dioncophylline C adduct appearsto be a promisinglead fbr lurther investigations. A numberof alkaloidsrelatedto emetinehave beenshown to have t19,29.2 antiprotozoal activitics:two alkaloidsisolatedftom Pogonopustubu- S c h e m iel l u s t r o t i n t hgeo n t i m o l o r i m o lo d e so f o c t i o no f q u i n i n eo n d /osas (Rubiaceae).cephaelineand tr"rbulosine. showedpotent lt r..'ltrc o r t e m i s i n i n .
I I
ANTIPROTOZOAL NATURALPRODUCTS macrophages.It is suggestedthat the antimalarialactivity of febrifugine is due to enhancedhost def-ence mechanisms.
risk of artemisinin-resistant parasitesdeveloping.In the body,all of the artemisininderivativesare metabolizedto dihydroartemisininwhich is more active againstmalaria parasitesthan aftemisinin.Some derivatives suchas sodiumartesunateare so rapidly hydrolysedthat the latter TERPENES may be consideredto be a 'pro-drug' while artemetheris metabolized Artemisinin more slowly so that both the parentand the metabolitecontributeto the For hundredsof yearsthe Chinesehave usedthe herb known as Qing antimalarialaction. for the treatmentof fevers includHao (Artenisia annua, Asteraceae) Artemisinin and its derivatives are the most rapidly acting antiing malariabut it was not until 1971 that Chinesescientistsisolatedthe malarialsknown and are highly selectiveagainstmalariaparasites.As sesquiterpene lactoneaftemisinin (7) (seep. 319) and showedthat it with quinine,haem is involved in their mode of action but the mechawas highly active againstP.falciparunt. Clinical trials showedthat the nism is quite different (Fig. 29.2). Artemisinin is unusual in that the drug was efl'ectivein treatingmalaria including chloroquine-resistant moleculecontainsan endoperoxidegroup and this reactswith the iron malariaand the ofien fatal complicationof cerebralmalaria.However, in haem, giving rise to highly reactivefree radicals.Parasitedeathis after one month many patientshad a recunenceof malaria(known as believedto result from the reactionof thesefree radicalswith parasite recrudescence) becausenot all of the parasiteshad been killed by the molecules such as proteins and nucleic acids.Artemisinin does not drug. In an attemptto overcomethis problemandto developdrugswith react with the iron in haemoglobin so that uninf'ectedred cells are unafimproved formulation characteristics,a number of derivativeshave fected.In the clinic, artemisininderivativesare remarkablynon-toxic been prepared by reduction of the lactone carbonyl to give dihy- and althoughanimal experimentsraisedfearsthat neurotoxicitymight (8) followed by the preparationof ether(9) or ester( 10) be a problem, this has not yet been shown in malaria patients. droarten.risinin derivatives.The methyl ether.aftemether,is solublein oil and is given Artemisinin may, however, be embryotoxic so that the use of these by intramuscularinjection while esterssuch as sodium artesunateand drugs is not recommendedin early pregnancy.One interestingfeature sodiumartelinatearewater solubleand may be given orally or by intra- of the artemisininderivativesis that they areactiveagainstthe gametois still a problem with thesederiva- cyte form of the malaria parasiteswhich is responsiblefor the transvenousinjection. Recrr.rdescence tives and it is recommendedby the World Health Organizationthat a mission of the diseasefiom man to the mosquitoduring feeding;thus second drug, e.g. mefloquine, be given fbllowing a course of an thesedrugsnot only cure the patientbut may help to reducetransmisand also to reducethe sion ofthe disease. artemisininderivativeto Dreventrecrudescence
artemisinin(7)
i
i
o__.,\ t OH
(8) dihydroartemisinin /a
i l o>.\ I
OR ETHERS(9)
Fig.29.3 Artemisinin o n d s o m ec l i n i c o l l y usedderivolives.
artemether; R = CHs arteether R = CzHs
\
A
")"\
I
ocoR ESTERS(10) sodium artesunate;R = CHzCHzCOONa sodium artelinate;R = CH21
l-\ )-COONa
E
PHARMACOPOEIAL AND RELATED DRUGS OF BIOLOGICAT ORIGIN AlthoLrghartemisininhas been synthesized.the processis cornplex which werefbund to be activeagainstavian malariain a screeningpro_ and not eco'omically 'irible. therefbreartemisininis extr"acted fiom,,r. grammein the 1940s.Quassinitself (seep.3211 is not active but a unntruherband then derivatizedas required.The amou't of ar-temisinin number of quassinoidspossessingan unsaturated A_ring,a lactonering presentin the plant is about0.5% of the dry weight of rheherbbut some and a rnethyleneoxygenbridge in the C ring suchas in brusatol(12) (a strains of A. ttnnutr havc higher amounts and plant breeding pro_ constituent of Brucect.javcutica)havevery potentantiprotozoalproper_ grammesare bein_q carriedout in order to increasethe yicld turther.In ties againstseveralspeciesincluding p. fatciparum. E. histolyticnand addition, plants contain ra'ger amountsof the compounclartemisinic G. i,testirtalis.Unfort.nately. thesecompoundsare also very toxic to acid which may be con'erted chemicallyinto artemisinin.thr-rsincreas- mammalian cells and atremptsto improve their selectivityby making ing the yield. cu.rently thcreis interestin someArncan countriesin structural changeshave not so far been very successful.Both the growingA. unnuulbr local use as a herbalantimalarialbut studiesto antlprotozoaland cytotoxic activitiesare due to the inhibition of prodemonstrateits eftlcacvwlrenusedin this way havenot yet beencarriecr tein synthesisand as theseprocessesappearto be similar in protozoa out. In china. a clinical trial in which malariap.tientsreceivedcapsules and mammaliancells it is likely to be difficult to improve selectivity. containingc.r-rdealcoholic extractsof A. annua rbu'd thar uhile However it is of interestto note that one quassinoid,glaucarubinone. patientsdid respondto the tl'eatmentinitially. recrudescence rateswere fbund in Simorcubaglout:tt,was formerly usedin Francetbr the treat_ verirhigh. Mala.ia pa'asitesresistantto artemisininha'e beenorocluced ment of amoebicdysentery.The Meliaceaeis a plant family relatedto in the laborato.ybut at the time of writing therehaveheenno repor.tsof the Simaror-rbaceae which producesbitter tppenoids chemicallyrelat_ reslstancein the lield. lt is irrperativethat stepsbe takento pre'ent the ed to the quassinoids,known as limonoids./Theneemtree,Aaadirru:htu emergenceof parasitesresistantto artemisininderivativesand it woLrld indictt' is wideiy used in Asia for mararia.treatmentand contains a seemto be unwiseto use herbalprepar.ations which may resultin para_ number of limonoids such as gedunin which have in viu.o antiDlas_ sitesbeing exposedto sr-rb-lethal cutcentrationsof clrug,thusencourag_ modial activitiesbut they are lesspotent than the quassinoicls. i n gt h ed e re l o p m c r o r lf r e s i : l : r n c e . Other terpenoids which have reported antiprotozoal'activities As afiemisininis a con-rplex molecule,much effon has beenput into include the triterpenetingenone.a red-orange pigmenrpresenrln some synthesizingcompoundsbasedon the 1,2.4-trioxane ring of arternisinin. speciesof the Celastraceaeand Hypocrataceae.It is highly active Many compoundshavebeenmade.someof which havepr.orrisinginvivo against I Cnr;l epimastigotesir vitro and it appearsto interact with activities in animal models. Endopc'oxide-containing sesqurterpenes DNA but is also an inhibitor of ntitochondrialelectrontransport and have also been tbund in anotherChinesespecies.Ar.tabotns nncitrtus has antineoplasticproperties. Jatrophone is a diterpene found in (Annonaceae), alsoknown asyin-uzHao.yingzhaosuA (11) andC were Jcttrcplta isabelli (ELrphorbiaceae) which has activity against active againstP. bergheiin mice althoughthey were less potent than Leishmaniapromastigotes andT.c.ruziepimastigotes and was eft-ective al'temisinin.A syntheticderivativeknown as arteflenewas evaluatedin in treating mice infected with l-. nmaT.onensis. The polyoxygenated clinicaltrialsbut wasabandoned dueto high ratesofrecruciescence. monoterpeneiridoid. arbortristosideA, fiom N,,-c anthes arhortristis (Oleaceae)was as effectiveagainstL. tlonovaniinfection in mice as a Quossinoids standarddrug althoughit was only weakly active againstl. donovttni The quassinoidsare derivedbiosyntheticallyfiom triterpenoidprecur_ amastlgotes in vitro. Interestin naturalproductsisolatedfiom 'rarine sors and are bitter principlespresentin speciesof Ihe Sinttrutubttt:e'e sollrces growing is and a number of diterpenes(isocyanoisocycloant_
OH
yingzhaosuA (11)
HO
brusatol(12)
o
lapachol(13)
o
I GI
p l u m b a g i n( 1 4 )
licochalconeA (15)
Fig.29.4 E x o m p l e so f s o m e n o n - o l k o l o i d o o l n t i p r o t o z o o lc o m p o u n d s
ffi
o
phloridzin('t6)
ANTIPROTOZOAL NATURALPRODUCTS philectanes) have been repofted liom the spon-eeClnrbcstela hooperi potentin vilro activitiesagainstchloroquine-sensitive and which possess P..falciparum with relarivelylow cytotoxicity. chloroquine-resistant
QUINONES Lopochol derivqtives Lapachol (13) is a naphthoquinonefound in the heartwoodof S. American speciesof Bignoniaceae,for example Tabebuiarosect. Although lapacholitself has only weak antiprotozoalproperties,a numberof syntheticderivativesare more potcnt. B-Lapachoneand allyl-B-lapachone were found to be activeagainstT. cruzi epimastigotes and the latter was shown to reduce the infectivity of trypomastigotesinoculatedinto mice. B-Lapachonewas not effectivelr vlvo as it reactswith haemoglobinwith the formation of methaernoglobin.The action of thesecompoundsappearsto dependupon their intracellular reduction to fr"eeradical specieswhich stimulate the production of hydrogen per"oxideby acting as electron carriers betweenNADH or NADPH and oxygen.Hydrogenperoxideis especially toxic to trypanosomesas they have unusualantioxidantpathways; Z cruzi is particularly vulnerableas it lacks the cnzyme superoxidedismutase.Researchon lapacholderivativeshas led to the developmentof atovaquone,a syntheticnaphthoquinonenow Iicensedfbr use(in combinationwith proguanil)fbr the treatrnentof falciparum malaria.
ceralfbrns of leishmaniasis as well as in mice infectedwith P loelli. have shoun that this compor.rndalters the ultrastructureand Str.rdies functicrnol' nritochondliain Leisltmurlaparasites. LicochalconeA is considelcdto bc a promising lcad compoundfor the developmentof new anti-leishmanial agents.
Phloridzin This naturallyoccurrin-u tlavonoidglycosideis of intcrestls it was it hasa bittertaste. onceusedasa treatmenttirl nrnlariaas.like cluinine. It inhibits tl.reglowth of r.r.ralrrirparasitesin vitro by inhibiting the increasein pelrneabilitl'ol the-rccl ccll membranewhich occurs in infectedcells.thus deprivin-u tlre palasiteof glucoseand other nutrients.Unfortunately,phlolidzin {16) also hlocks the reabsorptionof glucoseby the kidneytubulesso thatit is not sLlitable for clinicaluse. Exarnplesof someothcr phcnoliccolr.rl.rounds actir-ererinst protozoa inclr-rde oil the polyphenolgossl'pol.a constituentof cottonseed r.vhichis well known fbr its r-rse as a male contraceptivein China,but is T. crrr:l and E. histoh'ticain vitrct.In also activc againstP..fLlc:ipcLrtutt. T. cruz,igossypolinhibits thc oxiclolcductasccnr\mcs u-hydroxyacid dehydrogenaseand malate dehydrogenase.In the body. gossypol is concentratedin the liver and colon as a result ol bein-sexcretedin the bilc and thercfble may be woilh investigatingfbr the treatment of amocbiasiswherc iimoebaerral' be presentboth in the liver and in the gut lunren.A 1-ewflavonoidshave in vitro activit)' atainstP..ftrlciparunt including the exiguaflavones isolated frorn Arterni.yittindict.r (Asteraceae).The rlrethoxylatedflavonesartemetinand casticinhave beenshownto act synergistically with artemisininand it hasbeensuggestedthat tlavonoidspresentin Artentisia anrruamighl contributeto the antimalarial action of extractsor hcrbal teas preparedfiorn this species.
Plumbogin is used The S. American speciesPlunba,gobertensis(Euphorbiaceae) traditionally fbr the treatmentof leishmaniasisand containsthe naphthoquinonesplumbagin (14) as well as the dimers 3.3'-plurnbaginand 8,8'-plumbagin. These compounds trre active againsr Leishmutitt species ln vitro and some activity irt tit,o has also been shown. Plumbaginhas also beenreportedto have in vitro acri,'iies againstP faLciparumand epimastigotesof I crl:/. Diospyrin. a constitucntof havebecomeimportantantiis anothernaphthoquinone dirnerand In recentyearsthe artemisininderivatives Diosp\ros ntontonu(Ebenaceae), malarial drugs and this illustratesvery u'ell the potential of natural has beenshown to inhibit the -erowthof L. donovrmipromastigotesir productsto provide highly efl'ectiveantiprotozoalagents.The above rllro. However. the tetraacetoxy and tetrahydloxy derivatives of has illustrated tl-rewidc range of compoundsfbund in plant species diospyrinwere much more activethan the parentcompoundagainstP (multi-drug generally rnore which possessantiprotozoalactivitiesand it is likely that naturalprodresistant strain Kl) and were .falciparum active againstL. donovani amastigotes.T. b. brucei trypomastigotes uctswill continueto providenor,elcompourrdsu'hich may lead to new and T. cruzi amastigotes.It is likely that the abovc compoundshave a antiprotozoaldrugs. Horvevel. many of thc people who are alllicted with protozoaldiscasesdo not havethe resourcesto aflbrd pharmaceusimilar mode of actionto that of the lapacholderivatives. Other quinoneswith antiprotozoalpropertiesinclude the prenylated ticals and it is impotant to assesslocally usedtraditionalremediesto preanthraquinones, vismiones H and D. constituentsof Vl,inria determine their efficacy and sat-ety.One initiative which has been guineensis(Guttiferaceae)which have potent in vitro antiplasmodial setup to encouragethis is the Global Initiative For TraditionalSystems activities. The quinones. 2-( 1-hydroxyethyl)naphtho[2,30]fiu'an-4.9- (GIFTS of Health) basedin Oxtbrd. UK. It is to be hoped that further quinoneand isopinnatalisolatedfrom Kigelia pinnata (Bignoniaceae) lesearch will provicle new medicines based not only on pure were found to possesspotent i? r'lrro activitiesagainstI b. hrut:ei and compoundsbut alsohelbal productslbr the tl'eatmentof protozoaldiseases. T. h. rhotle.ti e tt\(,tr) pomilstiSotes.
coNcLuSroNs
Furtherreoding
PHENOTIC COMPOUNDS Licocholcone A The Chineseliquoricespecies(G.r'c-rrrhiz,a urulensis,glabra anditfla/a) are the source of an oxygenatedchalcone.IicochalconeA (15) which has in vito activities againstP ftrlciparum as well as against amastigotesand promastigotesof L. major and L. dottottuti. Activity has also been shown in animal models of both the cutaneousand vis-
Rayo CamachoCorona N'ldel. Cloti S L. PhillipsonJ D 2000 Natural products as sourcesof antiprotozoaldrugs.Currenl Opinion in Anti infective 2: .17 62 lnvcstigationalDrr-rgs PhillipsonJ D. Wright C W. Kirby G C. \\'arhurstD C 1995Phytochemistryof someplantsusedin traditionalnredicinefor the treatmentofprotozoal diseascs.ln: HostettmanK. MarstonA (eds)Ph)'tochcmistryof plants usedin . x f b r d .U K . p p 9 5 1 3 5 t r a d i t i o n am l e d i c i n eO . x f b r d U n i v e r s i t )P l e s s O Wright C W. PhillipsonJ D 1990Naturalproductsand the developnrentof selectiveantiprotozoaldrugs.PhytotherapyRescarch.l: 121 139
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The botanicalsconsideredin this chapterare oneswhich, althoughnot normarllyencounteredin orthodox medicine.have important roles in traditional rnedicine fbr the treatment of two widespr.eadand life_ threateningconditions.In recent years new technologieshave lecl to rapidadvances in thebiolo-uical testingandche'ical elircidatio'olthe active constituentsof many of theseplants.Much. but not all. of the researchis attributableto workersin Asian institutions.In the space availablehereit is possibleto provide a few examplesonly to illustrate the wide variety of chemical structur.es involvecl.For.ft-rrtherinforma_ tion readersareref'en'edto the variousreviewsand referencesouoted.
An overview of drugs with ontihepototoxic ond orolhypoglycoemic l '
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ocltvtttes
PLANTSIN THETREATMENTOF TIVERAND BITIARYTRACTDISEASES 414 PTANTSWITH ORAL HYPOGTYCAEMIC ACTTV|TY 419
The liver. the principal organof metabolismand excretionis sr.rbject to a number of diseaseswhich may be classedas liver cirrhosis (cell destruction and increasein tlbr.ous tissuc). acllte chronic hepatitis (inflammatory disease)and hepatitis (noninflammatory condition). Jaundice,a yellow discolorationof the skin and eyescausedby bile in the blood is a symptom of blockageof the bile duct, or diseasewithin the tissueofthe liver itself. The predominanttype of liver diseasevaries accordingto country and may be influencedby local factors.For 1989it was estimatedthat therewere sonte200 million chronic carriersof the hepatitisB virr-rsof which ,10%were expectedeventuallyto die of hepatocellularcarcino_ ma and l-5%,of cirrhosis.Causativefactorsof liver disordersinclude: virus infection;exposureto. or consumptionof. certainchemicals.e.g. the excessiveinhalation of chlorinated hydrocarbonsor overindul_ gencein alcohol:medicationwith antibiotics.chemotherapeutic agents and possibly plant materials such as those containing pyrrolizidine alkaloids(q.v.):contaminated fbod containingtoxinssuchasaflatoxins (q.v.)or peroxidesin oxidizededibleoils:ingestionof industrialpollLr_ tants. including radioactivemater.ial.Drug abusein Wester.nsociety and poor"sanitaryconditionsin Third World cor-rntries are contributing fireto|s t0 the iibove. Except for the use of the appropriatevaccine for the treatmentof hepatitiscausedby viral infection. there are few eff-ectivecures tbr liver diseases.It is not surprisingtherefbrethat there has clevelopeda considerableinterestin the examinationof thosenumerousworldwide traditionalplant remedieswhich areusedfbr suchtreatmentand that in recentyears in yivo and in yitro test modelshave beendevelopedfor the evaluationof piantstbr their antihepatotoxicactivities.Thesesys_ tel.nsmeasurethe ability of the test plant extractto preventor cure in rats or mice liver toxicity inducedby varioushepatotoxins.The latter include carbon tetrachloride (the rrlost commonly used), o_galac_ tosamine(this producesliver lesionscomparableto thoscfound in viral hepatitis)and peroxides.In such liver darnagethe serum level of the liver enzymes,particularly serum glutamic-pyruvic transaminase,is raised and the extent of its control by the antihepatotoxicdru-eunder test is used as a basis for estimation.Other etl'ectsof induced liver damagewhich can also be usedin the evalr.ration of plant extractsare: the prolongedlengtheningof the tin.reof lost r.eflexinducedby short_ acting barbiturates;reduction of prothrombin synthesis giving an extended prothron-rbintime; reduction in clearanceof certain sub_ stancessuchas bromosulphalein. In order to reduce the number of anirnal experimentsinvolved in thesesurveysH. Hikino and colleaguescleveloped (19g3-g5) assay methodsfor antihepatotoxicactivity using hepatotoxin-induced cyto_ toxicity in prirnarycultuled hepatocl,tes. The fl"actionated plant extract under test and the hepatotoxin(e.g.CCla) are addedto the hepatocyte medium and incr-rbated: the activity of the transaminases releasedinto
AN OVERVIEWOF DRUGSWITH ANTIHEPATOTOXIC AND ORALHYPOGLYCAEMIC ACTIVITIES the medium are then determined.The resultsobtainedby this rnethod were comparablewith the ln llyr-rassays.(For furtherdetailsand ret'erencesreadersshouldconsr-rlt H. Hikino in Biologicullt'Actit,eNatu'ttl ProdLrcts,eds K. Hostettmanand P J. Lea, Proc. Pht'tochenticolSoc. E u r o p e . 1 9 8 7N. o . 2 1 . p . 1 4 3 ) . Handa and his grotp (F i toterap ia. | 99 l, 62. 229) reportedthat about 170 phytoconstituents isolatedfrom I l0 plantsbelongingto 55 farnilies were statedto possessliver-protectiveactivity: about 600 commercialherbalfbnnulationswith claimedhepatoplotectire acti\ ity are being marketedworld-wide. The activeconstituentselucidatedto date involve erwide lange of componentsincluding terpenoids,curcuminoids. lignoids,flavonoids,cyanogeneticglycosidesetc.. and some examplesare given in Fig.30. l. The terminaleventsin the attackon the liver by carbon tetrachlorideinvolve the production of a highly reactiveradical leadingto lipid oxidation and the inhibition of the ca1cium pump of the microsomegiving rise to liver lesions.Glycyrrhizin, glycyrhetinic acid (Table 2,1.5)and wuweizisu C (Table 22.7'1and gomisin A (lignoid constituentsof Schizandruchinen.sisfruits) exert their activityas antioxidants. A numberof plant dr-ugsusedfbr treatingbiliary disordersare cholagogues(they promote the flow of bile). Herbalists prescribesr-rch drugs either singly or more commonly as mixtures;a cholagoguetea, for example.may consist of a mixture of Peppennintleaves 50.0% (principal cholagogue),Melissa leaves 20.07c (sedative adjuvant). Fennel liuits 20.07c (complementarycarminative). Frangula bark I 0.0olc(gentlelaxative). In Europethe most widely usedplant hepatoprotective agentis silymarin. a pulified extract from Siltbunt nturitutum.Silymarin is also usedasa standardagainstwhich to testotherdrugs.This and otherantihepatotoxicand chola-uogue dru-esare listed below.
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