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.

Heart failure is one of the most common causes of death and disability and is among the syndromes most commonly encountered in clinical practice. Despite important advances in the pharmacotherapy of heart failure over the past decade, the mortality rate still approaches 50%.

The cardiac glycosides have been used for centuries as therapeutic agents. Compounds containing the molecular motifs common to these agents- a steroid nucleus containing an unsaturated lactone at the C-17 position a~d one or more glycosidic residues at C-3 are found in many plants and several toad species, usually acting as venoms or toxins that serve as protection against predators. The steroidal cardenolides comprise one of the most interesting group of naturally occurring substances which are efficacious in the treatment of heart diseases. As components of arrow poisons, primitive people had used these substances from time immemorial.

The first comprehensive description of Digitalis glycosides in the treatment of congestive heart failure, as well as other ailments, are recorded in William Withering's 1785 monograph on the therapeutic efficacy and toxicities of the leaves of the common foxglove plant, Digitalis purpurea. Eversince, a dozen families of cardenolide- bearing plants have been recognised which include Digitalis lanata, Strophanthus gratus, S. divaricatus, S. sarmentosus, S. thollonii, Nerium oleander, Thevetia neriifolia, Cerbera j1oribunda, C. dilatata, Beaumontia grandiflora, Acocanthera longiflora, A. oppositifolia

*BECPRG, National Physical Laboratory, New Delhi, 11001 2, India

and Roxpellina boivini1'2 .The compounds are found in

almost all parts of the plant, but the total amount or relative distrilbution in any given plant varies with ecological factors, stage of development, time of harvest and mode of drying etc.

In the 1990s, digoxin has become by far the most commonly prescribed cardiac glycoside because of its convenient pharmacokinetics, alternative routes of administration, and the widespread availability of techniques for its measurement.

A number of cardiac glycosides have been isolated from Thevetia neriifolia, one of which, the peruvoside is more potent than digoxin.

Pharmacological Evaluation Thevetia neriifolia Juss was used in ancient India

(I 000 BC) in various skin diseases by Charak. It was classified under poisons by Sushruta in 1000 BC and was known to be a horse poison, hence named Ashwamarak or Ashwahan- the horse killer in Sanskrie. It was only in 1863 that its cardiotonic activity was discovered. Subsequently, the ouabain­like action of glycoside present in the kernels of this plant was noticed4

. The kernels are very bitter and when chewed produce numbness in the tongue. A decoction of the seeds acts as a violent emetic, hinders respiration and causes paralysis of the heart. However, with caution it can be used for the treatment of haemorrhoids . The seeds are also employed in criminal poisoning of ~he cattle. The animals fed with it, show salivation, expectoration and drowsiness5

. Kernels are also used as an insecticide, mashed with soap solution6

· 8

. Bactericidal activity has also been detected in seed oil distillates9-~ 1. Antifungal principle was detected in

GULA TI el a/.: EXPERIMENTAL STUDIES ON THE VET/A NERIJFOUA JUSS- A REVIEW 809

h fl al 12 . 8 . l . 13 E t e or extracts aga.tnst tpo ans oryzae . vans and Kaleysa8 reported larvicidal activity and Qamar et al. 14 nematicidal activity. Recently Vohora and Mishra15 reported its utility in skin diseases. All parts of the plant produce latex, which is used for healing sores and too_thache.

Every part of the plant, however, is· poisonous, the kernels being the most toxic. The toxic manifestations of yellow oleander- poisoning mainly involve the

d. I d h . . 1 4 !&-car wvascu ar system an t e gastromtestma tract · 20

. Jaundice and renal failure is also reported in yellow oleander poisoning21

'22

. The toxic principles in yellow oleander are the cardiac glycosides; kernels contain nearly seven times as much glycosides as the leaves, stem, flowers or fruit pulp23

• A number of cardiac glycosides have been isolated from this laticifer, some of which are thevetin cerberin, peruvoside and nerrifoside24·25

. A number of experiments conclude ouabain or digoxin-like action on heart muscles. Positive inotropic effect followed by cardiac arrest in isolated frog, guinea pig or albino rats26 and emesis and musculotropic activity was produced in cats and pigeons. Perfusion of thevetin, peruvoside or ruvoside (Thevetia glycosides) produces positive inotropic effect on hypodynamic myocardium and electrocardiographic changes. It also exhibits marked cardiotonic effect in isolated denervated heart lung preparation of dog. All cardiac glycosides, thevetin, peruvoside, neriifolin are as potent as Digitalis glycosides and appear to be promising drugs for congestive heart failure27

•30

• One of these, peruvoside is even more potent than digoxin and has been marketed in Germany under the trade name Encordin25

.

Thevetia Glycosides Thevetin was the first glycoside to be isolated31

from kernels. Since then as many as 15 glycosides are reported in the fresh seeds32

. The natural glycosides of Thevetia are triosides as they contain an aglycone unit combined with three units of sugar. The aglycone of these glycosides is digitoxigenin (or the related cannogenin and cannogenol) which is one of the aglycones of the glycosides of Digitalis purpurea. Cannogenin is the 19-oxo form and cannogenol is the 19-oxy form of digitoxigenin. The sugars are D-glucose and L-thevetose (6-deoxy-3-0-methyl-L-arabinose). An analysis of thevetioside (a mixture of cardenolides) showed presence of peruvoside, neriifolin and cerberin33

.

Thevetin, the first trioside to be isolated34, is the

major component of the seeds31• It was subsequently

found to be a mixture of two triosides, cerberoside (thevetin-B) and thevetin A. A small proportion of another trioside, 2'-0-acetyl cerberoside is also associated with thevetin35

• The separation of thevetin into pure components, thevetin A and cerberoside was achieved by partial chromatographic and counter current techniques by several workers. By the counter current technique, thevetin-A and cerberoside were obtained in a ratio of 1:236

.37

• 5-Methyl ether of apigenin is also reported from seeds38

. The monosides separated from the seeds include neriifolin, cerberin (2'-0-acetyl neriifolin), peruvoside, theveneriin (ruvoside) and peruvosidic acid (perusitin)39

. These glycosides do not occur as such in the seeds and are formed as a result of enzymic hydrolysis of the triosides. Peruvoside is obtained by partial hydrolysis of thevetin A; upon reduction, peruvoside yields theveneriin (ruvoside). Peruvoside and theveneriin are related as aldehyde and alcohol to each other40

•

Peruvosidic acid is obtained upon oxidation of peruvoside by Cr03.

During the isolation of cerberoside (thevetin-B), part of the trioside is partially hydrolyzed, losing two molecules of glucose, yielding a mixture of cerberoside, and neriifolin41

• The enzyme thevetinase is responsible for the hydrolysis. If incipient enzymic action is carefully prevented, as high as 4.5% trioside is obtained, with correspondingly insignificant amount of monoside. In contrast, if the enzymic action is deliberately promoted such as by fermenting the seeds, almost the entire quantity of the trioside disappears,

. 1 'd be h' h3436404243 concomttant y monost e comes tg · · · · . Fermented seeds additionally yield acetyl glycosides (neriifolin monoacetate, peruvoside monoacetate). Partial acetylation of neriifolin yields cerberin and isomeric neriifolin- 4"-monoacetate 35

•

Thevetia glycosides are chemically closely related to each other as represented in Chart 1.

The aglycones as well as the glycosides undergo isomeric changes in the presence of bases.

A number of known and new glycosides have also been isolated from various parts of this plant, albeit in small amounts44

. Abe et a/. 45 isolated several new cardiac glycosides from air dried leaves including neriifolin (C:mll3404), solanoside, thevetioside (A-G). A new cardenolide, neriifoside 3~-0-a.-L-enolide was

isolated from fresh uncrushed leaves along with peruvoside46

. Besides, a steroid, 4,16-pregnadien-126-

810 INDIAN 1 CHEM, SEC B, -NOVEMBER 2000

Thevetin (Crude)

! . C erberos1de (ThevetinB)

enzyme action

-2 mots glucose

J

Special

enzymic -2 mols oct1on glucose

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: oleanolic acid, ursolic acid, a-amyrin acetate, hitherto unreported from this source, h~J.Ve been isolated.

Amongst irridoids, theviridoside 47 was the first to be isolated and characterized in T. neriifolia. Irridoids are more stable in flowers and fruits as compared to leaves. Theveside is completely destructed in yellow senescent leaves due to blocked chromogenic groups48

. Two minor irridoids, 1 0-0-P-D-fructofuranosyl theviri­doside and 6'-0-B-D-glucopyranosyl theviridoside have been reported recently from root s49

·50

.

Leaves ofT. neriifolia also contain flavonol sinapoyl glycosides. Abe et al 51 reported few of these, kaempferol 3-glucosyl (1-4) [6"-sinapoyl glucosyl] (1-2) galactoside and 3-[2"-sinapoyl glucosyl] (1-4) [6"­sinapoyl glucosyl] ( 1-2) galactoside, and quercetin 3-[6"-sinapoyl glucosyl] ( 1-2) galactoside along with known compounds kaempferol and quercetin 3-glucosyl ( 1-2) galactoside. Dinormonoterpenoids and their apiosyl glycosides have recently been reported52

.

All parts of the plant except the flowers are known to contain cardiac glycosides. However, Gunasegaran and Nai r53 reported flavol glycoside, thevefolin along with 3-galactosides of quercetin and tamarixetin and 3-digdlactoside of yuercetin extracted by exhaustive

extraction of fresh flowers with 90% ethanol. Later they also reported two phenyl propanoids from flowers54

. Apigenin-5-methyl ether occurs in seed shells ofT. neriifolia55

.

Detection For detection and quantification of Tltevetia cardiac

glycosides various techniques have been used, which include radioimmunoassal6

, HPLC determination57,

enzyme linked immunosorbent assal8•59 and

fluorescence polarization 60.

In vitro Studies Owing to short life of seeds, dorrnacy, poor

germination and difficulty in propagation through cuttings, alternative methods of multiplication become imperative. Besides, poor stability of cardenolides towards acids and bases, the presence of rare sugar residues hinders their chemical synthesis and restrict their extractio.J and isolation from natural sources. Since glycosides are prone to hydrolytic enzymes, they are found in very low amounts in plants. Hence, pharmaceutical industry looks forward to undertake a

biotechnological approach for the synthesis of such 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 genetic homogeneity of desirable characters can be maintained in the resultant plantlets. Also, predictable yield of glycosides can be ensured. A number of cardenolides have been reported in in vitro cultures of D

. . 1. 61-64 tglta IS .

In vitro regeneration of T. neriifolia has been attempted by a few workers. Initially, regeneration and rooting from stem cuttings was attempted with several growth regulators. Kumar and Sharma65 reported positive role of IAA in the induction of roots and regeneration of shoots, while it was adversely affected by GA3. Lower concentrations of coumarin promoted root formation and inhibited shoot regeneration, whereas higher concentration retarded both. In vitro proliferation and good growth with rooting could be achieved with NAA and kinetin66 in cotyledonary callus raised on WB medium with NAA + kinetin67·68

•

They could detect thevetin in callus cultures by TLC. However, the percentage of thevetin decreased with age of callus and disappeared completely after six months. Yield of peruvoside and neriifolin could be successfully modulated using various stress causing agents under in vitro conditions69

. The yield obtained was however explant dependent.

Modulation of cardenolide biosynthesis failed in such cultures when tried using different sugars ( 4% sucrose, glucose and maltose), yeast extract, casein hydrolysate, malt extract, coconut milk, amino acids or growth hormones with Wood and Brown (WB) medium. Callus tissues grown for I month under different light conditions and with precursors (cholesterol + progesterone) also failed to produce thevetin. Thevetin-A and thevetin-B were detected in cultures raised on Murashige and Skoog's medium67.

Effect of exogenous precursors on in vitro secondary metabolism was also studied in T. neriifolia. Transformation of cholesterol to pregnenolone and progesterone was observed in suspension cultures within 24 hr. Increase in progesterone concurred with decrease of cholesterol and pregnenolone and vice­versa70. Later Dantas - Barros el a/.71 reported cardenolide formation in cell suspension cultures. In vitro cultures opened up the possibility Df using plant cells for the transformation of steroids as is I commonly done with microorganisms. Results of biotransfor­mation with plant cells, of different substances, in particular of steroids are compiled in several reviews72

-

75 . A comparison of biotransformation potential of Thevetia neri~folia and Digitalis lanata cell cultures

revealed that cardenolides common to the plant species were metabolized by plant cell cultures more rapidly than cardenolides uncommon to that species . Both glucosylation and deacetylation could be observed Thevetia and Digitalis cell cultures, while deglucosylation and oxidation only occurred with the Thevetia and 12'13-hydroxylation only with Digitalis cell cultures. Furthermore, the Theve~ia cultures were not capable of transforming methyl digitoxin into

h I d. . 76 77 met y 1goxm · .

Barros et a/.78 reported biotransformation of the synthetic substrate, ethyl 2-acetyl amino 2-carbethoxy-4-(phenyl sulphinyl)butanoate by cell suspension cultures of Catharanthus roseus and Thevetia neriifolia. It was found that only three of the six cell lines of T. neriifolia tested, biotransformed the 2-test substrate into a new product, ethyl 2-acetyl amino-2-carbethoxy-4-(phenylsulphonyl)-butanoate, through a selective oxidizing process not previously described . Cardenolide content also varies in different celllines79

.

Such a biotechnological process could be of great interest for the production of new chemical compound involving stereospecific chemical reactions hitherto unreported through chemical synthesis.

In view of the ever increasing demand for novel and more potent cardenolides, additional efforts need to be made to ensure constant and stable supply of these drugs. Owing to the presence of highly stereospecific sugar moieties, chemical synthesis is hindered hence, attempts should be made to explore the possibility of detecting cardenolides in in vitro cultures.

Acknowledgements AG is grateful to CSIR for a fellowship. Thanks are

also due to all the colleagues in PTC Lab, Jamia Harndard for help in various ways.

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