Indian Journal of Chemistry Vol. 398, November 2000, pp. 808-812

Review Article

Experimental studies on Thevetia neriifolia Juss-A review

Anarnika Gulati*t, S K Jain & P S Srivastava Department of Biotechnology, Hamdard University, New Delhi II 0062, India Received 18 August 1999; accepted (revised) 16 August 2000

'fhis review article provides a comprehensive description of various studies, carried out on Thevetia neriifoiia which is a source of several cardioactive glycosides, since early 20th century. The article provides an overview of various other medicinal herbs containing cardiac glycosides and discusses in depth various com[pounds isolated so far from this medicinal herb and their pharmacology. Additionally, various in vitro and biotransformation studies carried out on Thevetia and yield of glycosides in cultures is also detailed.

1 2 is one of the most common causes of and Roxpellina boivini ' .The compounds are found in death and disability and is among the syndromes most almost all parts of the plant, but the total amount or commonly encountered in clinical practice. Despite relative distrilbution in any given plant varies with important advances in the pharmacotherapy of heart ecological factors, stage of development, time of failure over the past decade, the mortality rate still harvest and mode of drying etc. approaches 50%. In the 1990s, has become by far the most The cardiac glycosides have been used for centuries commonly prescribed because of its as therapeutic agents. Compounds containing the convenient pharmacokinetics, alternative routes of molecular motifs common to these agents- a steroid administration, and the widespread availability of nucleus containing an unsaturated lactone at the C-17 techniques for its measurement. position a~d one or more glycosidic residues at C-3 are A number of cardiac glycosides have been isolated found in many plants and several toad species, usually from Thevetia neriifolia, one of which, the peruvoside acting as venoms or toxins that serve as protection is more potent than digoxin. against predators. The steroidal comprise one of the most interesting group of naturally occurring Pharmacological Evaluation substances which are efficacious in the treatment of Thevetia neriifolia Juss was used in ancient India heart diseases. As components of arrow poisons, (I 000 BC) in various skin diseases by Charak. It was primitive people had used these substances from time classified under poisons by Sushruta in 1000 BC and immemorial. was known to be a horse poison, hence named The first comprehensive description of Digitalis Ashwamarak or Ashwahan- the horse killer in glycosides in the treatment of congestive heart failure, Sanskrie. It was only in 1863 that its cardiotonic as well as other ailments, are recorded in William activity was discovered. Subsequently, the ­ Withering's 1785 monograph on the therapeutic like action of glycoside present in the kernels of this 4 efficacy and toxicities of the leaves of the common plant was noticed . The kernels are very bitter and foxglove plant, Digitalis purpurea. Eversince, a dozen when chewed produce numbness in the tongue. A families of - bearing plants have been decoction of the seeds acts as a violent emetic, hinders recognised which include Digitalis lanata, respiration and causes paralysis of the heart. However, Strophanthus gratus, S. divaricatus, S. sarmentosus, S. with caution it can be used for the treatment of thollonii, Nerium oleander, Thevetia neriifolia, haemorrhoids. The seeds are also employed in criminal Cerbera j1oribunda, C. dilatata, Beaumontia poisoning of ~ he cattle. The animals fed with it, show 5 grandiflora, Acocanthera longiflora, A. oppositifolia salivation, expectoration and drowsiness . Kernels are 6 also used as an insecticide, mashed with soap solution · 8 *BECPRG, National Physical Laboratory, New Delhi, 11001 2, . Bactericidal activity has also been detected in seed 9 1 India oil distillates -~ . Antifungal principle was detected in GULA TI el a/.: EXPERIMENTAL STUDIES ON THE VET/A NERIJFOUA JUSS- A REVIEW 809

12 . . l . 13 E 31 t h e fl oral extracts aga.tnst 8tpo ans oryzae . vans major component of the seeds • It was subsequently and Kaleysa8 reported larvicidal activity and Qamar et found to be a mixture of two triosides, cerberoside al. 14 nematicidal activity. Recently Vohora and (thevetin-B) and thevetin A. A small proportion of Mishra15 reported its utility in skin diseases. All parts another trioside, 2'-0-acetyl cerberoside is also 35 of the plant produce latex, which is used for healing associated with thevetin • The separation of thevetin sores and too_thache. into pure components, thevetin A and cerberoside was Every part of the plant, however, is· poisonous, the achieved by partial chromatographic and counter kernels being the most toxic. The toxic manifestations current techniques by several workers. By the counter of yellow oleander- poisoning mainly involve the current technique, thevetin-A and cerberoside were 4 36 7 card wva. scu I ar system an d t he gastromte. stma. 1 tract ·!&- obtained in a ratio of 1:2 .3 • 5-Methyl ether of 20 38 . Jaundice and renal failure is also reported in yellow apigenin is also reported from seeds . The monosides 2122 oleander poisoning ' . The toxic principles in yellow separated from the seeds include neriifolin, cerberin oleander are the cardiac glycosides; kernels contain (2'-0-acetyl neriifolin), peruvoside, theveneriin 39 nearly seven ti mes as much glycosides as the leaves, (ruvoside) and peruvosidic acid (perusitin) . These 23 stem, flowers or fruit pulp • A number of cardiac glycosides do not occur as such in the seeds and are glycosides have been isolated from this laticifer, some formed as a result of enzymic hydrolysis of the of which are thevetin cerberin, peruvoside and triosides. Peruvoside is obtained by partial hydrolysis 2425 nerrifoside · . A number of experiments conclude of thevetin A; upon reduction, peruvoside yields ouabain or digoxin-like action on heart muscles. theveneriin (ruvoside). Peruvoside and theveneriin are 40 Positive inotropic effect followed by cardiac arrest in related as aldehyde and alcohol to each other • isolated frog, guinea pig or albino rats26 and emesis and Peruvosidic acid is obtained upon oxidation of

musculotropic activity was produced in cats and peruvoside by Cr03. pigeons. Perfusion of thevetin, peruvoside or ruvoside During the isolation of cerberoside (thevetin-B), part (Thevetia glycosides) produces positive inotropic effect of the trioside is partially hydrolyzed, losing two on hypodynamic myocardium and electrocardiographic molecules of glucose, yielding a mixture of 41 changes. It also exhibits marked cardiotonic effect in cerberoside, and neriifolin • The enzyme thevetinase is isolated denervated heart lung preparation of dog. All responsible for the hydrolysis. If incipient enzymic cardiac glycosides, thevetin, peruvoside, neriifolin are action is carefully prevented, as high as 4.5% trioside is as potent as Digitalis glycosides and appear to be obtained, with correspondingly insignificant amount of 27 30 promising drugs for congestive heart failure • • One of monoside. In contrast, if the enzymic action is these, peruvoside is even more potent than digoxin and deliberately promoted such as by fermenting the seeds, has been marketed in Germany under the trade name almost the entire quantity of the trioside disappears, 25 Encordin . concomttant. 1y monost'd e becomes htg' h3436404243· · · · . Fermented seeds additionally yield acetyl glycosides Thevetia Glycosides (neriifolin monoacetate, peruvoside monoacetate). Thevetin was the first glycoside to be isolated31 Partial acetylation of neriifolin yields cerberin and 35 from kernels. Since then as many as 15 glycosides are isomeric neriifolin- 4"-monoacetate • 32 reported in the fresh seeds . The natural glycosides of Thevetia glycosides are chemically closely related to Thevetia are triosides as they contain an aglycone unit each other as represented in Chart 1. combined with three units of sugar. The aglycone of these glycosides is digitoxigenin (or the related The aglycones as well as the glycosides undergo cannogenin and cannogenol) which is one of the isomeric changes in the presence of bases. aglycones of the glycosides of Digitalis purpurea. A number of known and new glycosides have also Cannogenin is the 19-oxo form and cannogenol is the been isolated from various parts of this plant, albeit in 44 45 19-oxy form of digitoxigenin. The sugars are D-glucose small amounts . Abe et a/. isolated several new and L-thevetose (6-deoxy-3-0-methyl-L-arabinose). An cardiac glycosides from air dried leaves including analysis of thevetioside (a mixture of cardenolides) neriifolin (C:mll3404), solanoside, thevetioside (A-G). A showed presence of peruvoside, neriifolin and new cardenolide, neriifoside 3~-0-a.-L-enolide was 33 cerberin . isolated from fresh uncrushed leaves along with 34 46 Thevetin, the first trioside to be isolated , is the peruvoside . Besides, a steroid, 4,16-pregnadien-126- 810 INDIAN 1 CHEM, SEC B, -NOVEMBER 2000

Thevetin (Crude) J C erberos1de! . (ThevetinB)

enzyme -2 mots enzymic -2 mols action glucose oct1on glucose

Special reduction process oxidation Neriifolin ______:. ______Peruvoside -----Peruvo acid Reduction acetylation

Cerberin 4'-0-acetylneriifolin ---- Theveneriin(rlM>side) (2'-0-acetylneriifolin)

Source: Wealth of India Chart 1

hydroxy-3,20-dione and four pentacyclic triterpenes: extraction of fresh flowers with 90% ethanol. Later oleanolic acid, ursolic acid, a-amyrin acetate, hitherto they also reported two phenyl propanoids from 54 unreported from this source, h~J.Ve been isolated. flowers . Apigenin-5-methyl ether occurs in seed 47 55 Amongst irridoids, theviridoside was the first to be shells ofT. neriifolia . isolated and characterized in T. neriifolia. Irridoids are more stable in flowers and fruits as compared to leaves. Detection Theveside is completely destructed in yellow senescent For detection and quantification of Tltevetia cardiac 48 leaves due to blocked chromogenic groups . Two glycosides various techniques have been used, which 6 57 minor irridoids, 10-0-P-D-fructofuranosyl theviri­ include radioimmunoassal , HPLC determination , 8 59 doside and 6'-0-B-D-glucopyranosyl theviridoside have enzyme linked immunosorbent assal • and 49 50 60 been reported recently from root s · . fluorescence polarization . Leaves ofT. neriifolia also contain flavonol sinapoyl glycosides. Abe et al 51 reported few of these, In vitro Studies kaempferol 3-glucosyl (1-4) [6"-sinapoyl glucosyl] (1- Owing to short life of seeds, dorrnacy, poor 2) galactoside and 3-[2"-sinapoyl glucosyl] (1-4) [6"­ germination and difficulty in propagation through sinapoyl glucosyl] ( 1-2) galactoside, and quercetin 3- cuttings, alternative methods of multiplication become [6"-sinapoyl glucosyl] ( 1-2) galactoside along with imperative. Besides, poor stability of cardenolides known compounds kaempferol and quercetin 3- towards acids and bases, the presence of rare sugar glucosyl ( 1-2) galactoside. Dinormonoterpenoids and residues hinders their chemical synthesis and restrict 52 their apiosyl glycosides have recently been reported . their extractio.J and isolation from natural sources. All parts of the plant except the flowers are known to Since glycosides are prone to hydrolytic enzymes, they contain cardiac glycosides. However, Gunasegaran and are fo und in very low amounts in plants. Hence, Nai r53 reported flavol glycoside, thevefolin along with pharmaceutical industry looks fo rward to undertake a 3-galactosides of quercetin and tamarixetin and 3- biotechnological approach for the synthesis of such digdlactoside of yuercetin extracted by exhaustive important cardenolides. In vitro raised cultures woulc GULATI eta!.: EXPERIMENTAL STUDIES ON THEVETIA NERIIFOLIA JUSS- A REVIEW 811 prove profitable, as true to type individuals with revealed that cardenolides common to the plant speci es genetic homogeneity of desirable characters can be were metabolized by plant cell cultures more rapidly maintained in the resultant plantlets. Also, predictable than cardenolides uncommon to that species. Both yield of glycosides can be ensured. A number of glucosylation and deacetylation could be observed cardenolides have been reported in in vitro cultures of Thevetia and Digitalis cell cultures, while . . . 61-64 Dtglta 1IS . deglucosylation and oxidation only occurred with the In vitro regeneration of T. neriifolia has been Thevetia and 12'13-hydroxylation only with Digitalis attempted by a few workers. Initially, regeneration and cell cultures. Furthermore, the Theve~ia cultures were rooting from stem cuttings was attempted with several not capable of transforming methyl into 65 . . 76 77 growth regulators. Kumar and Sharma reported met hy I d1goxm · . positive role of IAA in the induction of roots and Barros et a/.78 reported biotransformation of the regeneration of shoots, while it was adversely affected synthetic substrate, ethyl 2-acetyl amino 2-carbethoxy- by GA3. Lower concentrations of coumarin promoted 4-(phenyl sulphinyl)butanoate by cell suspension root formation and inhibited shoot regeneration, cultures of Catharanthus roseus and Thevetia whereas higher concentration retarded both. In vitro neriifolia. It was found that only three of the six cell proliferation and good growth with rooting could be lines of T. neriifolia tested, biotransformed the 2-test achieved with NAA and kinetin66 in cotyledonary substrate into a new product, ethyl 2-acetyl amino-2- 67 68 callus raised on WB medium with NAA + kinetin · • carbethoxy-4-(phenylsulphonyl)-butanoate, through a They could detect thevetin in callus cultures by TLC. selective oxidizing process not previously described. 79 However, the percentage of thevetin decreased with Cardenolide content also varies in different celllines . age of callus and disappeared completely after six Such a biotechnological process could be of great months. Yield of peruvoside and neriifolin could be interest for the production of new chemical compound successfully modulated using various stress causing involving stereospecific chemical reactions hitherto 69 agents under in vitro conditions . The yield obtained unreported through chemical synthesis. was however explant dependent. In view of the ever increasing demand for novel and Modulation of cardenolide biosynthesis failed in more potent cardenolides, additional efforts need to be such cultures when tried using different sugars ( 4% made to ensure constant and stable supply of these sucrose, glucose and maltose), yeast extract, casein drugs. Owing to the presence of highly stereospecific hydrolysate, malt extract, coconut milk, amino acids or sugar moieties, chemical synthesis is hindered hence, growth hormones with Wood and Brown (WB) attempts should be made to explore the possibility of medium. Callus tissues grown for I month under detecting cardenolides in in vitro cultures. different light conditions and with precursors (cholesterol + progesterone) also failed to produce Acknowledgements thevetin. Thevetin-A and thevetin-B were detected in 67 AG is grateful to CSIR for a fellowship. Thanks are cultures raised on Murashige and Skoog's medium . also due to all the colleagues in PTC Lab, Jamia Effect of exogenous precursors on in vitro secondary Harndard for help in various ways. metabolism was also studied in T. neriifolia. Transformation of cholesterol to pregnenolone and References progesterone was observed in suspension cultures I Venkata Rao E, Indian 1 Phann, 35, 1973, 107. within 24 hr. Increase in progesterone concurred with 2 Radford D J, Gillies A D, Hinds J A & Duffy P, Mcd 1 Aust, decrease of cholesterol and pregnenolone and vice­ 144, 1986, 540. versa70. Later Dantas - Barros el a/. 71 reported 3 .Chopra R N, Sci Cult, 24, 1958, 59. cardenolide formation in cell suspension cultures. In 4 Arora R B, lnr Symp Medicinal Plants, Kandy, Ceylon, 15-18 Dec, 1964, 21 3. vitro cultures opened up the possibility Df using plant 5 Christopher J, Indian Farm , 16, 1966, 43. cells for the transformation of steroids as is I commonly 6 Cherian M C & Ramachandran S, Madras Agric 1, 30, 1942, done with microorganisms. Results of biotransfor­ 260. mation with plant cells, of different substances, in 7 Pandey N D, Mathur K K, Pandey S & ·Tripathi R A, Indian 1 72 Ent, 48, 1986, 85. particular of steroids are compiled in several reviews - 8 Evans D A & Kaleysa R R, Indian 1 Med Res, 88, 1988, 38. 75 . A comparison of bi otransformation potential of 9 Bushnell A 0 , Pacific Sci 4, 1950, 167. Thevetia neri~fo lia and Digitalis lanata cell cultures I 0 Saxena V K & Jai n S K, Fitoterapia, 6 1, 1990, 348. 812 INDIAN J CHEM, SEC B, NOVEMBER 2000

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