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.Fitoterapia 70 1999 451471

Review

/Schisandra chinensis Turcz. Baill

J.L. Hanckea,U, R.A. Burgosb, F. AhumadabaLaboratorios Garden House, A . Presidente J. Alessandri 12310, San Bernardo, Santiago, Chile

bInstituto de Farmacologa, Facultad de Ciencias Veterinarias, Uni ersidad Austral de Chile, P.O.

Box 567, Valdi ia, Chile

Received 16 March 1999; accepted 1 June 1999

Abstract

Different aspects of the pharmacology of Schisandra chinensis fruit and dibenzo-w xa,c cyclooctene lignans from this plant are reviewed focusing in particular on the antihepa-totoxic, antioxidant and antitumoural activities, and on the effects on physical performanceand on the central nervous system. 1999 Elsevier Science B.V. All rights reserved.

Keywords: Schisandra chinensis; Lignans; Antioxidant activity; Antihepatotoxic activity; Antitumoralactivity; Physical performance; CNS

1. Botany

. .Schisandra chinensis Turcz. Baill Schisandraceae grows wild in the most .Eastern parts of Russia Primorsk and Chabarowsk regions , the Kuril islands,w xsouthern Sachalin and also north-eastern China, Korea and Japan 1 . Schisandra

species grow mainly in China, Japan, the Himalayas and Jawa. The seeds and thew xfruit are the parts used in medicine 24 . S. chinensis is a monoecius liana withattractive leaves and a woody stem. The winding stem, reaching 10 15 m in lengthand 1.21.5 cm in diameter, is twisting around the trunks of trees, climbing to their

top. The leaves are alternate, elliptic, cuspidate, with a wedge-shaped base. Theflowers are white or slightly cream-coloured, wax-like, unisexual with a pleasant

UCorresponding author. Tel.: q 56-2-5281411; Fax: q 56-5293646.

.E-mail address: [email protected] J.L. Hancke

0367-326Xr 99r $ - see front matter 1999 Elsevier Science B.V. All rights reserved. .PII: S 0 3 6 7 - 3 2 6 X 9 9 0 0 1 0 2 - 1

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( )J.L. Hancke et al. r Fitoterapia 70 1999 451471452smell. The flowers are in clusters of 25. When the fruit is ripening, the receptacleis substantially lengthened and turned into a pedicle with the appearance of agrape cluster, 68 cm long with several bright red fruits. The fruits, ripen in

SeptemberOctober, have an almost spherical shape and contain 12 yellow seeds.The skin and pulp taste sour and sweet. The kernel is pungent, bitter and overall

salty. It is called in mandarin wu-wei-zi, literal English translation: five-taste. .fruit , in Japanese Gomishi, in Korean Omicha Chinese Materia Medica .w xExperiences of its cultivation were reported 1 .

In western botany the Chinese wu-wei-zi was first named Kadsura chinensis in an1832 publication of the Russian botanist Nikolai S. Turczaninov. In 1856, to honour

.his most famous colleague K.J. Maximowicz 18271891 , the Russian botanist .Franz J. Ruprecht 18141870 created a new genus called Maximowiczia and

called the plant Maximowiczia chinensis. In 1866, the French botanist H.E. Baillon .18271895 transferred the plant to the genus Schisandra and since that time the

. w xplant has been known as S. chinensis Turcz. Baill. 3 . The generic nameSchisandra is derived from the Greek schizein, meaning to cleave and andros,man, referring to the cleft or separate anther cells on the stamens of S. coccinea.

.Fructus schisandrae, of the Chinese Pharmacopoeia, consists of two members: 1 S. . . .chinensis Turcz. Baill. Northern Schisandra and 2 S. sphenanthera Rehd. et

. w xWils. Southern Schisandra 2 .

2. Chemistry

w x Many dibenzo a,c cyclooctene derivatives, present in different quantities fruit.and seeds: 7.219.2%; stems: 1.310% have been isolated from S. chinensisw x520 . Some of the main structures are shown in Table 1. Biosynthetic precursors

to the dibenzocyclooctene derivatives, such as pregomisin and epigalbacin, havew xbeen also isolated 20 . The fruits also contain about 1.5% sugars, tannins, coloursubstances and about 3% of essential oils citral, -chamigrene, -camigrenol,

. . -bisabolene, sesquicarene , organic acids citric, malic, fumaric and tartaric acid ,2 w xvitamin C and E, and metals such as copper, manganese, nickel and zinc 21 .

3. Pharmacology

w xS. chinensis is officially listed in the Chinese Pharmacopoeia 22 and indexed asa tonic and sedative. It is also listed in the Shen Nong Ben Tsao Ching book, year

.1596 2697 BC as a superior drug that helps in coughs and prevents asthma. It wasw xfirst reported in Divine Husbandmans Classic of the Materia Medica 3 . Accord- .ing to Chinese philosophy the drug has sour and warm properties. It: a enters the

.lung and kidney channels and the stomach meridians; b contains the leakage of

lung Qi and stops coughing used for deficient lung and kidney patterns with. . cough and wheezing ; c restrains the Essence and stops diarrhoea used for

. .nocturnal emission, spermatorrhea, deficiency of the spleen and kidneys ; d stops

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( )J.L. Hancke et al. r Fitoterapia 70 1999 451471 455excessive sweating used for deficient Yang spontaneous sweating or deficient

. . . w xYin night sweat ; e calms the spirit used for forgetfulness and insomnia 4,10 .S. chinensis fruit has been used for a long time in the Far East as a stimulating

and fortifying agent in cases of physical exhaustion, and to inhibit fatigue. TheNanajs tribals used S. chinensis dried berries to combat fatigue in their huntingw xtrips 1 .

A monograph on S. chinensis preparations was introduced officially to thew xRussian Pharmacopoeia in 1961 23 .3.1. Antihepatotoxic effect

.Several reports indicate that S. chinensis dried fructus and seed is an effectivew xliver protective drug 2426 . In experimental models, Glutamic Piruvic Transami- . .nase GPT activities induced by carbon tetrachloride CCl or paracetamol in4

mice, thiacetamide in rats, and ethinylestradiol 3-cyclopentylether in rabbits werereduced by oral administration of the ethanol extract of the seed of S. chinensis . w x110 gr kg prior to and after the administration of the hepatotoxic agents 24,25 .The alcoholic extract of S. chinensis kernels reduced elevated GPT levels in micetreated with CCl or thioacetamide, while a water extract of the kernels and an4 w xethanol extract of the shells of the seed were ineffective 21 . Primary cultured rathepatocytes treated with 0.11 mgr ml of either an ether, ethyl acetate, methanolor water extract of S. chinensis fruit were effective in reducing the galactosaminew xand CCl -induced cytotoxicity 20 . Different lignans isolated from S. chinensis4 w xhave been associated to this antihepatotoxic effect 27,28 . Seven lignans isolatedfrom S. chinensis kernels and tested for antihepatotoxic activity have been shownw xeffective liver protecting drugs 23 . Most of them prevented the elevation of serumGPT levels and the morphological changes of the liver, such as inflammatory

.infiltration and liver cell necrosis induced by CCl . Gomisin B 50 mgr kg, p.o ,4 . .gomisin A, 50 mgr kg, p.o. , schisandrin C 50100 mgr kg, p.o. , schisandrin B

. . 50100 mgr kg, p.o. deoxyschisandrin 200 mgr kg, i.p. , -schisandrin 50100. .mgr kg, p.o. and gomisin C 200 mgr kg, i.p. decreased the GPT levels after CCl4w x .27 . Gomisin B, gomisin A and schisandrin at doses of 100 mgr kg, p.o. were also

w xeffective against thiacethamide-induced liver damage in mice 21,27 .In fasting mice, the lignans stimulated the glycogen synthesis, the order of theactivity being gomisin A) deoxyschisandrin s -schisandrin. The activity of

.gomisin A was comparable to that of cortisone 100 mgr kg, p.o. . Since similarresults were obtained in adrenalectomized mice, the effect of these lignans onw xglycogenesis seems not mediated by the adrenals 21,27 .

.Pretreatment of male rats with gomisin A 50 mgr kg, i.p. prevented the rise in .GPT and Glutamic Oxaloacetic Transaminase GOT and hepatic necrosis of cellsw x induced by acetaminophen 29 . The repeated administration of gomisin A 30 or

.100 mgr kg, p.o., daily for 4 days induced an apparent increase of liver weight inw xliver-injured and normal rats 30 . Gomisin A suppressed the increase in serum

transaminase activity and the appearance of histological changes such as hepato-cyte degeneration and necrosis, inflammatory cell infiltration and fatty deposition

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( )J.L. Hancke et al. r Fitoterapia 70 1999 451471456w xinduced in liver by CCl , D-galactosamine or D-,L-ethionine 30 . Gomisin A4

decreased serum triglycerides and lipid contents of the liver. It also increasedmicrosomal cytochrome b , P-450, NADPH cytochrome C reductase, aminopyrine5

N-demethylase and 7-ethoxycoumarin O-dethylase and decreased 3,4-benzo-w x w xa pyrene hydroxylase 30 .A hepatoprotective effect of deoxyschisandrin, -schisandrin, schisandrin C,

gomisin A, and schisandrin has been associated to their inhibitory effect onCCl -induced lipid peroxidation and the binding of CCl metabolites to lipids of4 4w xthe liver microsomes 3133 .w x w xSchisandrin B 34 and schisanhenol 35 under oxidative stress, and gomisin A inw ximmunologic liver injury 36 increased the membrane stability of hepatocytemembrane. This effect can be related to a stimulating effect on the hepatic-gluta-w xtione antioxidant system 37 and may involve the enhancement of mitochondrial

w xglutathione redox status in rats 38 . .It is also suggested that gomisin A 50 mgr kg, i.p. possesses a liver function

enhancing property in normal and injured liver, and that its preventive action onCCl -induced cholestasis is sustained by the secretory function of the bile acids4 w xindependent fraction 39 .

Gomisin A and schisandrin B induced hypertrophy and mild hyperplasia, aug-w14 xmenting the liver weight. C Phenylalanine incorporation, protein content, andw xhepatic microsomal cytochrome P-450 content were enhanced 40,41 . Gomisin A .10100 mgr kg, p.o. for 4 days also increased the liver regeneration in rats afterpartial hepatectomy, increased the regeneration rate of the liver cells, and im- .proved the serum retention rate of the foreign dye sulfobromophtalein BSP ,w xwhich was dose-dependent 42 . In addition, gomisin A also enhanced the incor-w14 xporation of C phenylalanine into liver protein and shortened the hexobarbital-induced sleeping time. These changes caused by gomisin A are similar to those ofw xphenobarbital 42 . However, gomisin A is distinctly different from phenobarbitalin the finding that phenobarbital diminished the survival of CCl -intoxicated mice,4w xbut gomisin A did not 42 .

Ultrastructural studies of liver tissue using the transmission electron microscoperevealed an increase in rough and smooth endoplasmic reticulum in the groups

.receiving gomisin A 100 and 300 mgr kg per day . Gomisin A accelerated both theproliferation of hepatocytes and the recovery of liver function after partial hepatec-tomy and increased hepatic blood flow. It is thought that the liver enlargementcaused by repeated administration of gomisin A is associated with the proliferationw xof endoplasmic reticulum 42 .

.Gomisin A 10 or 30 mgr kg, p.o. for 3 or 6 weeks suppressed the fibrosisproliferation and accelerated both the liver regeneration and the recovery of liverw xfunction after partial hepatectomy in CCl -induced chronic liver injury in rats 43 .4Also, gomisin A regenerated the liver tissue after partial hepatectomy by enhanc-ing ornithine decarboxylase activity, which is an important biochemical event in the

w x early stages of liver regeneration in rats 44 . Gomisin A 100 mgr kg, p.o. daily for.14 days promoted hepatocyte growth after mitosis during regeneration of partially

resected rat liver, inducing directly or indirectly an enhanced activation of the

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( )J.L. Hancke et al. r Fitoterapia 70 1999 451471 457proliferative processes of non-parenchimal cells that involved an increase in c-mycw xproduct, a gene that precedes DNA replication in proliferating cells 45 .

The effects of gomisin A on immunologically induced liver injuries have been

investigated in vivo and in vitro. Following injection of a small dose of lipopolysac-charide in mice previously treated with heat-killed Propionibacterium acnes, most of

.the animals died with acute hepatic failure. Gomisin A 550 mgr kg, p.o reduceddose-dependently the mortality of mice with acute hepatic failure. Histologically,necrosis was suppressed by gomisin A, but infiltration of non-specific inflammatorycells was not affected. In in vitro experiments, the liver cells were injured by

.antibody-dependent cell-mediated cytotoxicity ADCC reaction or activation ofmacrophage in vitro. Inhibition of the isolated liver cell injuries induced by ADCCreaction or activation of macrophages in vitro, suggested that gomisin A can bew xmarkedly protective against immunological liver injuries 46 . In guinea pigs sensi-

tised with trinitrophenylated liver macromolecular protein fraction, gomisin A 50. w xmgr kg, p.o. was effective in reducing the acute hepatic failure 47 . Also the acute

hepatic failure induced by heat-killed Propionibacterium acnes followed by a smallamount of Gram-negative lipopolysaccharide was prevented after 4 weeks of

. w xgomisin A 60 mgr kg per day for 410 weeks administration 48 . The survival ratewas 80% as compared to 5% of the control group. In Long Evans Cinnamon rats,spontaneously developing hepatitis, treatment with gomisin A did not modify thedeath rate, but the time of survival was increased by 710 weeks as compared withw xthe control group 49 .

Leukotrienes are potent inflammatory agents that are thought to play a role inw xinflammatory liver diseases 50 . In inmunological hepatic failure, mononuclearcells are the predominant cells producing leukotrienes. Gomisin A 0.1 mgr ml,

7 .added to 10 macrophage cellsr ml suspension produced on the biosynthesis ofleukotrienes stimulated in rat peritoneal macrophages by Ca2q ionophore A2318an inhibitory effect which may be partially associated with its antihepatotoxic effectw x51 .3.2. Antioxidant and detoxificant effect

w xThe antioxidant effect of S. chinensis is attributed to the dibenzo a,c cyclooctenew xlignan constituents 52,53 . In in vitro studies, induction of antioxidative enzymeshas been observed with S. chinensis lignans which inhibited the lipid peroxidation

.measured by means of malondialdehyde MDA formation induced by ironr cy- .steine in rat liver microsomes: at 1 mM concentration, schisanhenol, S y -schi-

.sandrin C and S y -schisandrin B were shown to be more potent than vitamin Ew x . .35 . Schisanhenol 1 mM and schisandrin B 1 mM also inhibited gossypol-in-duced superoxide anion generation in rat liver microsomes. The preventive oral

.administration 200 mgr kg, once daily for 3 days of either schisanhenol or .schisandrin B reduced liver MDA formation induced by 50% ethanol 15 mlr kg

w x54 . In vitro, schisanhenol demonstrated an oxygen scavenging activity in ironr cy-w x .steine and NADPHr ascorbic acid method, 55 and CCl -OH and -CCl -induced4 3w xlipid peroxidation in hepatocytes 34,56 . The release of GPT and lactate dehydro-

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( )J.L. Hancke et al. r Fitoterapia 70 1999 451471458 .genase LDH was also reduced. As a consequence, the hepatocyte viability wasw xincreased as well as the integrity of the hepatocyte membrane 35 . Moreover, the

hepatoprotective effects of Schisandra lignans may be attributed to the enhance-

ment of the hepatic antioxidant system. In fact, schisandrin B and schisanhenolwere also able to increase superoxide dismutase, catalase activities in rat liverw x .cytosol, 54 and the function of the hepatic reduced glutathione GSH anti-oxi-w xdant system 37 . Intragastric pre-treatment of female Balbr c mice with schisan-

.drin B 416 mgr kg per day for 3 days caused dose-dependent increases in . .hepatic glutathione S-transferase GST and glutathione reductase GRD activi-

ties. In other experiments, 24 h after the last dose of schisandrin B, all rats were .treated with CCl 0.1 mlr kg . The activities of glucose-6-phosphate dehydroge-4

. .nase G6PDH , Se-glutathione peroxidase GPX , and gamma-glutamylcysteine .synthetase GCS are down-regulated to varying degrees in a dose-dependent

w xmanner by schisandrin B 37 . The beneficial effect of schisandrin B on the hepaticGSH antioxidant system is more evident after CCl challenge. The hepatoprotec-4tion was associated with significant enhancement in hepatic GSH, as indicated bythe substantial increase in tissue GSH levels and the corresponding decrease in thew xsusceptibility of tissue homogenates to GSH depletion 37 . The hepatoprotective

. .effect of schisandrin B 8 mgr kg, i.p. was not affected by 1,3-bis 2-chloroethyl -1- .nitrosourea, an inhibitor of GRD, at a dose of 2 mmolr kg i.p. in female Balbr c

mice. The mechanism of hepatoprotection by schisandrin B may involve theenhancement of mitochondrial glutathione redox status greatly impaired by CCl -4w xintoxication 38 .Interestingly, while well known antioxidant agents such as -tocopherol acetatedid not protect against hepatic damage induced by other hepatotoxins such asaflatoxin B or Cd, S. chinensis reduced the hepatotoxic effect of these agents in a1

. w xnon-selective manner Tables 2 and 3 57 . In fact, pre-treatment with a lignanenriched extract of S. chinensis fruit stimulated the hepatic antioxidantr detoxifica-tion system, as shown by increased hepatic GSH levels as well as hepatic GRD andw xGST activities in rats 57 .

Some comparative studies in female Balbr c mice have shown that schisandrin B .12 mgr kg per day, p.o. for 3 days increased the hepatic mitochondrial-GSH level,

.whereas butylated hydroxytoluene BHT decreased it. However, both schisandrinB and BHT increased, albeit to a different extent, the activity of mitochondrialw x GRD, particularly after CCl challenge 58 . Pre-treatment with schisandrin B 124

.mgr kgr day, p.o. for 3 days sustained the hepatic mitochondrial GSH level inCCl -intoxicated mice and protected against CCl -induced hepatotoxicity, while4 4BHT pre-treatment did not. Moreover, while both schisandrin B and BHT in-

.creased hepatic ascorbic acid vitamin C level in control animals, only schisandrinB pre-treatment sustained a high hepatic vitamin C level in CCl -intoxicated mice.4Also, schisandrin B pre-treatment prevented the CCl -induced decrease in the4hepatic vitamin E level. However, schisandrin B inhibited NADPH oxidation in

.mouse liver microsomes incubated with CCl 10 mM in vitro, whereas, BHT4stimulated this oxidation. The ability to sustain the hepatic mitochondrial GSHlevel and the hepatic vitamin C and vitamin E levels may represent a crucial

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Table2

a

.

TheeffectofS.chinensisandvitaminEtrea

tmentonaflatoxinB

A-B

-inducedhepatotoxicityinrats

1

1

Treatment

PlasmaGOT

Hepatictissue

.

U

l

Malondialdehyde

GSH

GSHreductase

GSH

S-transferase

.

.

nmolmgwettissue

mU

mgwettissue

Control

0.0

37

0.0

02

5.8

9

0.1

4

4.8

4

0.1

0

77.5

3.3

17.9

1.6

b

b

b

A-B

0.0

48

0.0

03

5.6

2

0.1

5

5.5

7

0.2

3

82.2

2.9

164.3

24.5

1

c

b,c

b,c

b,c

c

S.chinensis

A-B

0.0

38

0.0

01

7.4

5

0.4

2

10.1

7

0.1

4

171.0

10.3

46.2

5.6

1

b

c

d

d

b,c,d

Vit.E

A-B

0.0

47

0.0

03

6.0

8

0.1

9

5.1

6

0.1

0

74.8

3.3

622.6

274.2

1

aValues

aremean

S.E.M.,n

5;

bP

-0.0

5vs.control;cP-0.0

5vs.

A-B

;dP

-0.0

5.vs.

S.chinensis

A-B.

One-wayANOVAfollowedby

Duncan

1

1

wx.

smultiplerangetest.

Reproducedwithperm

issionofPharmacologyandToxic

ology57.

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( )J.L. Hancke et al. r Fitoterapia 70 1999 451471460Table 3

aThe effect of S. chinensis and vitamin E pre-treatment on Cd-induced hepatotoxicity in rats

PlasmaHepatic tissueGOTMalondialdehyde GSH GSH reductase GSH S-transferase .Ur l nmolr mg wet mUr mg wet

. .tissue tissue

Control 0.044 " 0.002 7.04 " 0.22 4.90 " 0.26 98.4 " 11.3 37.4 " 1.8b bCd 0.054 " 0.005 6.55 " 0.24 4.34 " 6.32 54.6 " 11.1 96.6 " 16.2

S. chinensisc b,c b,c cq Cd 0.042 " 0.003 7.07 " 0.48 7.60 " 0.16 193.0 " 3.9 57.5 " 7.4

d c,d b,dVit. E q Cd 0.049 " 0.003 6.03 " 0.59 4.55 " 0.24 91.8 " 9.5 85.6 " 7.3a Values are mean " S.E.M., n s 5; bP- 0.05 vs. control; cP- 0.05 vs. Cd; dP- 0.05 vs. S.

chinensisq

Cd. Data were analysed using one-way ANOVA followed by Duncan s multiple range test. w x .Reproduced from Ip S.P. et al. 57 , with permission of Pharmacology and Toxicology .

antioxidant property of schisandrin B in protection against CCl hepatotoxicity4w x58 .The effects of schisandrin B and vitamin E have been compared on ferric

3q .chloride Fe -induced oxidation of erythrocyte membrane lipids in vitro andCCl -induced lipid peroxidation in vivo. Vitamin E produced a pro-oxidant effect4at 110 M and a biphasic effect at 1.0 mM on the Fe3q -induced TBARS .thiobarbituric acid reactive substances in human erythrocyte membranes; thepro-oxidant effect, lasting 20 min, was followed by a complete suppression of

.TBARS antioxidant effect. Schisandrin B 110 M was capable to inhibit TBARSw x .formation 59 . Pre-treatment with vitamin E 3 mmolr kg per day, p.o. for 3 daysdid not protect against CCl -induced lipid peroxidation and hepatocellular damage4

in mice, whereas schisandrin B pre-treatment 0.3 3.0 mmolr kgr day, equivalent.to 1.212 mgr kg per day, p.o. for 3 days produced a dose-dependent protective . w xeffect on the CCl -induced hepatotoxicity Table 4 59 .4

The scavenging effects of different structures and configurations of schisandrins

on active oxygen radicals have been demonstrated using active oxygen radicalsfrom human polymorphonuclear leukocytes stimulated with phorbol myristateacetate. The scavenging effects of schisandrins depend on the stereoconfigurations,

. .the effect of S y -schisandrin B being stronger than that of R q -schisandrin andw xthat of schisandrin C stronger than that of schisandrin B 60 . This difference maybe explained by the dioxymethyl group that captures electrons facilitating radicalw x .attack 60,61 . Surprisingly, the scavenging effect of S,R " -schisandrin B was

. .stronger than that of either S y - or R q -schisandrin B. The reason for thisw xeffect is unknown 61 . .Another lignan, schisanhenol 1 mmolr l was able to scavenge oxygen radicals

produced by human neutrophils stimulated by tetradecanoylphorbol acetate. InFenton reaction system, the inhibitory rate of hydroxyl radical by schisanhenol was34.4%. In xanthinexanthine oxidase and UV-irradiation of riboflavin systems,

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( )J.L. Hancke et al. r Fitoterapia 70 1999 451471 461Table 4

a .Effects of Schisandrin B S-B and vitamin E pre-treatment on CCl -induced hepatotoxicity in mice4

MDA Plasma ALT . .pmolr mg tissue Ur l

Control 60 " 1 14.4 " 0.7b cCCl 87 " 6 12 526 " 7964

S-B q CCl4d e( )0.5 mmolr kg 68 " 3 1008.4 " 447.4 92d e( )3.0 mmolr kg 56 " 2 24.1 " 4.6 99

Vit. E q CCl43.0 mmolr kg 89 " 9 12 904 " 873

a Values are mean " S.E.M., n s 5; bP- 0.05; cP- 0.001 vs. control; dP- 0.05; eP- 0.001 vs.CCl Students t-test. The italic number in parentheses is the percent of protection. Reproduced from4,w x .59 , with permission of Molecular and Cellular Biochemistry .

schisanhenol scavenged superoxidase anion radical by 26.1% and 21.9%, respec-w xtively. In all these systems, schisanhenol was more potent than vitamin E 55 .3.3. Anticarcinogenic effect

w x .Benzo a pyrenes BPs are well known carcinogens, widely distributed in thew xenvironment 62,63 . The elimination of these polycyclic aromatic hydrocarbonsw xfrom the body requires their conversion to water-soluble metabolites 64 . Some of

the enzymes involved in BP metabolism, such as cytochrome P-450, epoxide . .hydratase EH , and arylhydrocarbonhydroxylase AHH are induced by various

substances found in edible plants. There is some evidence that consumption ofvegetables like sprouts, cabbages, broccoli, alfalfa and fibres may reduce thew xincidence of stomach and colon cancers 65,66 .

The effect of deoxyschisandrin, -schisandrin, schisandrin C, gomisin A, B and C .orally given 100200 mgr kg per day for 3 days to male rats has been studied in

vitro on liver microsomal monooxygenases and epoxide hydrolase. Among thesecompounds, schisandrin B, schisandrin C, gomisin A and biphenyl dimethyl dicar-

.boxylate BDD , a synthetic derivative of gomisin C, significantly increased rat livercytochrome P-450 concentration, NADPH-cytochrome C reductase, benzo-

phetamine and aminopyrene demethylase activities. Four compounds -schi-.zandrin, schizandrin C, gomisin A and BDD also markedly stimulated prolifera-

tion of smooth endoplasmic reticulum of liver cells from rats treated with schisan-w xdrin B 32 . .It is known that phenobarbital PB; 80100 mgr kg, p.o. for 3 days -induced

microsomal monooxygenase activities are preferentially inhibited by metyrapone,

an enzymatic inhibitor of cytochrome P-450 enzymes involved in the synthesis ofadrenocorticosteroids. Schisandrin B, schisandrin C, gomisin A, and BDD inhibited

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( )J.L. Hancke et al. r Fitoterapia 70 1999 451471462aminopyrene demethylase activity of liver microsomes in a similar manner. Dual

.induction by gomisin A 100200 mgr kg, p.o. and PB decreased the mutagenicityof BP via a decreased covalent binding of BP metabolites to DNA. Gomisin A also

decreased the capacity of BP-induced rat microsomes to activate BP to its muta-w xgenic metabolites 31,32 .Using male mice of the strain C57B16 that responded with a marked induction

of hepatic microsomal benzopyrene hydroxylase activity, S. chinensis fructus fine.powder, 5% in diet for 14 days induced a threefold increase in cytochrome P-450.

EH was stimulated significantly by S. chinensis. It is known that the addition ofpurified EH to the Salmonella mutagenecity Ames test reduces BP mutagenicity by

.3050%. Total BP metabolism was significantly increased 1.6-fold in the S. .chinensis 1 mgr ml group. Phenol II formation relative to total metabolites was

significantly increased in the S. chinensis group as compared to the control group

w x .67 . Both 7-ethoxycoumarin O-de-ethylase ECD and aryl hydrocarbon hydroxy- . w xlase AHH activities were also increased significantly 68 . The binding of aflatoxinw xto DNA was diminished by S. chinensis 68 .

The effect of gomisin A on hepatocarcinogenesis caused by 3-methyl-4-dimeth- .ylaminoazobenzene 3-MeDAB in male Donryu rats has been investigated.

.Gomisin A 30 mgr kg per day, p.o. for 5 weeks significantly inhibited the .appearance in the liver of foci for glutathione-S-transferase placental form GST-P ,

a marker enzyme of preneoplasm. Gomisin A decreased the number of hepaticaltered foci such as the clear cell and basophilic cell type foci in the early stagesw x .69,70 . Gomisin A 30 mg

rkg per day, p.o. decreased the concentration of3-MeDAB-related azo dyes in the liver, and increased their excretion in the bile.

After the withdrawal of 3-MeDAB, carcinogen related azo dyes were not detectedeither in the liver or the bile, but the proportion of diploid nuclei, thoughdiminished, remained high. It seems that gomisin A improved liver function byw xreversing abnormal ploidization 71 .

.Gomisin A 0.03% in diet for 10 weeks inhibited the development of preneopla-sic liver lesions. In fact, gomisin A inhibited the level of GST-P, and the numberand size of GST-P positive foci increased in the liver after treatment with3-MeDAB. Moreover, although the population of diploid nuclei was increased and

that of tetraploid nuclei was decreased by pre-treatment with 3-MeDAB, gomisinw xA reverted this effect to near the normal ploidy pattern 71 . This suggests thatgomisin A may inhibit the hepatocarcinogenesis induced by 3-MeDAB by enhanc-ing the excretion of the carcinogen from the liver and by reversing the normalw xcytokinesis 72 .

.Gomisin A 30 mgr kg, p.o. daily for 5 weeks inhibited the increase in serum bileacid concentration induced by the administration of other tumour promotors such

. w xas deoxycholic acid DCA 73 . Although hepatocarcinogenesis has been reportedto be promoted by exogenous administration of bile acids, the relation of en-w xdogenous bile acids to hepatocarcinogenesis is not completely understood 74,75 .

.The oral administration of gomisin A 30 mgr kg significantly inhibited theincrease of serum bile acids, especially DCA, and the appearance of preneoplastic

.lesions number and area of GST-P-positive foci in the liver , induced by 3-

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Fig. 1. Inhibitory effect of gomisin A on the promotion of skin papillomas by 12-O-tetrade- .canoylphorbol-13-acetate TPA in DMBA-initiated mice. From 1 week after initiation by a topical

application of 50 g of DMBA, 2.5 g of TPA was applied twice weekly. Topical application of gomisin .A 5 mol and vehicle was performed 30 min before each TPA treatment. Data are expressed as

. .percentage of mice bearing papillomas A and average number of papillomas per mouse B . w x .Reproduced from Yasukawa K. et al. 77 with permission of S. Karger AG, Basel .

MeDAB. These results confirm that DCA is an endogenous risk factor for

hepatocarcinogenesis and suggest that the anticarcinogenic effect of gomisin Aw xmay be based on improving metabolism of bile acids 76 . . .Application 1 gr ear of 12-O-tetradecanoylphorbol-13-acetate TPA , a tu-

mour-promoting agent, to mice induces inflammation. Local application 0.6.mgr ear of gomisin A inhibited TPA-induced inflammation in mice. Also gomisin J

and schisandrin C inhibited the inflammation induced by TPA in mice. The ED50of these compounds ranged between 1.4 and 4.4 mol, gomisin A showing thestrongest inhibitory effect. Furthermore, at 5 molr mouse, it markedly suppressed

.the promotion effect of TPA 2.5 gr mouse on skin tumour formation in micew x . w x following initiation with 7,12-dimethylbenz a antracene 50 gr mouse 77 Fig..1 .

3.4. Effects on physical performance

A number of Russian reports indicate that S. chinensis is able to counteract theeffect of fatigue, increase endurance, and improve the physical performance ofw xsportsmen 78 , but no controlled studies were done in the western world until thelate 1980s. To validate the hypothesis that this plant can improve the physicalrecovery, in a first series of trials, 50 g of S. chinensis fructus dried extract wereadministered to thoroughbred horses prior to a 800-m race at maximum speed, and

to polo horses submitted to a 12-min gallop at a speed of 400 mr min, or a 5-minw xgallop at a speed of 700 mr min 79 . S. chinensis counteracted significantly theanticipatory respiratory and cardiac frequency as compared to the control group.

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. .Fig. 2. Effect of S. chinensis treatment on lactate a and glucose b plasmatic profile in race horses .subjected to effort. Arrows represent the first record after the exercise 7 min . Values are mean "

U UU . S.E.M., n s 5; P- 0.05; P- 0.01 vs. control saline ; Students t- test. Reproduced withw x.permission of Fitoterapia 80 .

Also, the seric lactic acid was reduced and the plasmatic glucose increased in S.chinensis treated horses. Interestingly, the horses treated with S. chinensis wereable to complete the race at an average of 1.8 s faster, indicating an improvementw xin the physical performance of the horses 7981 . On a second series of studies,

.the effect S. chinensis fructus dried extract single dose of 6 g, p.o. was studied inrace and spring horses in order to asses whether the type and intensity of thew xexercise is critical for the effect 80 . S. chinensis was capable of reducing signifi-cantly the heart rate and respiratory frequency at different time intervals after thetrial, particularly in race horses. Plasmatic glucose concentration increased signifi-

cantly in both types of exercise. The plasmatic concentration of lactic acid wasreduced in S. chinensis treated horses as compared to the controls, this decreasew x .being again more evident in race horses 80 Fig. 2 .The liver accomplishes important functions in the metabolisation of lactic acid,

and its functionality determines to a great extent the performance level of horses.Accordingly, an increase of the transaminase activity results in an impairment ofw xthe horses physical performance 82 . As it is known that S. chinensis decreases thew xhepatic transaminases activity 20,21 , the hypothesis was made that S. chinensiscould lower the seric levels of transaminases and, thus, reverse the impairedw xperformance of horses 83 . Indeed, an association between poor performance and

w xhigh seric levels of hepatic enzymes in sport horses was shown 82 . Moreover, . w xtraining leads to an increment of creatinine phosphokinase CPK 82 , an enzyme

present in the striated and heart muscle, and intense anaerobic exercise can lead to

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. .Fig. 3. Effect of S. chinensis on GOT a and CPK b serum levels in poorly performing horses.U UU .Values are mean " S.E.M., n s 12; P- 0.05; P- 0.01 vs. control saline , Student s t- test.

w x.Reproduced with permission of Phytomedicine 83 .

muscle skeletal damage, with increase in seric level of CPK and transaminases.Poorly performing sport horses with long lasting high levels of -gluta-

.myltransferase GGT , GOT and CPK were orally administered 3 g of S. chinensisdried extract, during 14 days. S. chinensis reduced the levels of GGT and GOT inw xthe serum at day 7 and 14 after administration 83 . Surprisingly, the CPK levelswere also reduced by day 7 and 14 after administration indicating that thesew xanimals presented a muscular damage that could be reverted with S. chinensis 83 . w xFig. 3 . Simultaneously, Ko et al. 84 reported a protective effect against physicalexercise-induced muscle damage and a myocardial protective effect in rats pre-

treated with a lignan enriched extract of S. chinensis fruit 0.8 gr kg day, p.o for 3. w xdays 85 . Protection was associated with a significant enhancement in the hepaticw xantioxidant status, as assessed by GSH and MDA concentrations 8487 . Fig. 4

summarises the possible effects of S. chinensis on the metabolic pathways duringmaximum physical effort. S. chinensis reduced the hepatic damage leading to a

.decrease in transaminases GOT, GGT . As a consequence, gluconeogenesis char-acterised by an increase in seric glucose level was improved. On the other hand, S.chinensis reduced the striated muscle damage via a decrease of seric CPK level andreduced the seric lactate levels, probably by an antioxidant effect.

( )3.5. Acti ity on central ner ous system CNSActivation of the CNS by S. chinensis has been evidenced during electroen-

cephalographic examination. In particular, S. chinensis, antagonised the effect ofsubstances supressing the CNS such as barbiturates, chloral hydrate, aminazine,

w x .and halothane 88,89 . Schisandra lignans 15 mgr kg, i.p. antagonised the effectw xof hexenal and chloral hydrate in rats 88 . The CNS activating effect of S. chinensiswas observed even under the presence of antagonist to dopamine receptors DA2

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Fig. 4. Antioxidant effect of S. chinensis on liver and striated muscle during exercise.

w x w x88 . However, potentiation of phenobarbital sleeping time 90 would indicate thatS. chinensis has a depressing action on the CNS. This could be explained by thepresence in the tested extracts of different concentrations of schisandrol A which is

known to prolongue the sleeping time induced by phenobarbital and decrease thew xspontaneous motor activity in mice 91 .The cholinergic system is also significantly influenced by S. chinensis. A crude

.petrol ether fruit extract 1030 mgr kg, p.o. decreased the convulsant thresholdand potentiated the antidiuretic action of nicotine and potentiated the excitatoryaction of carbachol on the intestine in rats. This petrol ether extract potentiated

. w xthe action of reserpine only at higher doses 1.5 gr kg 92,93 . This extract affectedmostly the cholinergic system, with a biphasic response. At dose of 280 mgr kg p.o.,it showed an indirect nicotinomimetic action potentiating the carbachol intestinal

. w xmotility, whereas, a higher dose 840 mgr kg, p.o. had a cholinolytic effect 93 .Schisanhenol and schisandrin B have been shown to protect peroxidative damagew x y 4 .of aging and ischemic rat brain 94 . Schisanhenol and schisandrin 10 M

completely inhibited the swelling and disintegration of brain mitochondria, as well2q . w xas the reduction of brain membrane fluidity induced by Fe cysteine 94 . In vitro

experiments on mitochondria and membrane from ischemic and reperfusion brainindicate that both lignans significantly inhibited production of MDA and loss ofATPase activity induced by reoxygenation following anoxia. Oral administration .150 mgr kg of schisanhenol or schisandrin B induced increase of cytosol glu-tathione-peroxidase of brain in mice under the condition of reoxygenation fol-w xlowing anoxia 94 .

Human intellectual activity can be enhanced by S. chinensis so that work .efficiency is also increased. Schisandrin 510 mgr day, p.o. improved certain

activities requiring concentration, fine coordination, sensitivity and endurance, as

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( )J.L. Hancke et al. r Fitoterapia 70 1999 451471 467demonstrated in healthy young male adults in the following experiments: insertionof thread into needle within 5 min; error rate in telegraphist reception and

.transmission; running marathon. S. chinensis seed powder 3 g daily, p.o. could

improve vision, enlarge the visual field, improve hearing power, and increase thew xdiscriminating ability of skin receptors 95 .3.6. Pharmacokinetics and metabolism

Until now, there are no reports on the pharmacokinetics of S. chinensis extracts.After oral administration to healthy male subjects of 15 mg of schisandrin thew xmean value of maximum plasma concentration was 96.1 " 14.1 ngr ml 96 .Schisandrin was metabolised by rat liver microsomes to give three main first phasicmetabolites. Several oxidation routes appear to be involved: hydroxylation of analkyl substituent at first and then demethylation of the -OCH groups on the3 w xaromatic rings. The metabolites were found in urine and bile of rats 97 .

After oral administration of 10 mgr kg to rats, the maximum serum concentra- .tion of gomisin A 1446.1 " 131.8 ngr ml was reached at 1530 min, over 80%w xbeing bound to serum proteins 98100 . The rapid metabolisation of gomisin A,w xhas been attributed to a first pass effect, producing demethylated metabolites 98w xand glucuronic and arylsulfate conjugates 101 .

After oral administration to rats of schisandrol A, this compound was absorbedfrom the gastrointestinal tract with a half-life of 58 min. After i.v. injection of

schisandrol A, the blood level showed a biphasic decline, with a half-life of theelimination phase of 42 min. Schisandrol A was detected in urine 1 h after oralw xadministration 10 . Five minutes after i.v. administration, high levels of schisandrolA were found in the lungs, moderate amounts in the liver, heart, brain, and kidneysand low amounts in the ileum and spleen. In the brain, the higher amounts werefound in the hypothalamus, corpus striatum and hippocampus, and moderateamounts in the cerebral cortex and cerebellum. These differences may be relevantw xto the neuroleptic and anticonvulsant properties of schisandrol A 102 .3.7. Toxicology

.The acute toxicity for S. chinensis fructus dried extract 4:1 , standardised to a . w xconcentration of 2% of schisandrin was low LD ) 21 gr kg, p.o. in rats 103 .50

Other authors reported the absence of lethal effects following intragastric adminis-w xtration of 5 gr kg to mice 95 . The oral and i.p. LD values in mice for a50 .petroleum ether extract of S. chinensis fruit 10% schisandrins were 10.5 and 4.4

gr kg, respectively. The oral LD of a petroleum ether extract standardised to 40%50 w xof schisandrins was 2.8 gr kg in mice 93 .An ethanol extract of S. chinensis orally given to mice at doses of 0.6 and 1.2

gr kg for 10 days resulted in only mild toxic effects, such as decrease in activity,piloerection and apathy, while body weight increase, blood picture and main organsw xwere not significantly altered 95 .

The p.o. toxicity of S. chinensis fructus dried extract standardised to a minimum

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( )J.L. Hancke et al. r Fitoterapia 70 1999 451471468of 2% of schisandrins was studied in Landrace piglets for 90 days at daily doses of0.07, 0.36 and 0.72 gr kg. The body weight and food intake, were not affectedduring the whole experimental period. No changes in the red blood cells, white

blood cells, haemoglobin and hematocrit were found. The glycemia, urea andprotein concentrations did not show any significant variations with respect to thecontrol. Triglycerides, GOT and GGT were also not modified by S. chinensis

administration. In the tissues examined liver, heart, kidneys, intestine, lungs,. w xspleen and gonads no toxic effect was observed 103 .w xIn other studies 103 and using the same standardised dried extract of S.

.chinensis fruit 0.1050.5 gr kg per day, p.o. , the potential toxic effects on thereproductive function was studied in rats and mice. No foetotoxicity in theseexperimental models was found. No changes in the implantation efficiency or otherinvestigated parameters were observed.

Information on the toxicity of S. chinensis lignans is very limited. With schisan-drin B, no death was observed following a single intragastric dose of 2 gr kg to rats.Intragastric dosing of 200 mg for 30 days caused no significant effect on bodyw xweight, haemoglobin and histology of the major organs in mice 95 . Schisandrin B,given intragastrically to dogs at 10 mgr kg daily for 4 weeks, did not affect appetite,body weight, blood picture, liver and kidney functions, as well as the histology ofw xthe liver 95 .

4. Conclusions

S. chinensis has been used in traditional Chinese medicine for thousands ofyears. In the last decades, the pharmacology and chemistry of this drug has beenextensively studied. Much evidence shows that S. chinensis and its dibenzocy-clootene lignans may act on the function of the liver. The findings are useful forfurther understanding the pharmacological basis of S. chinensis as an antioxidant,anticancer, tonic and antiaging drug. Furthermore, S. chinensis might also be usefulin the treatment of other diseases related to oxygen free radical injury andmetabolic disturbances, such as radiation injury, inflammations and reperfusion of

ischemic organs, as well as in stress conditions and sport medicine.Schisandra lignans seem also a potential source of new synthetic drugs, as is thew xcase of BDD 104,105 . Recently, halogenated gomisin J derivatives have been

.shown to possess anti-human immunodeficiency virus HIV activities, by inhibitingthe activity of the enzyme reverse transcriptase as well as expressing cytoprotectivew xactivity in HIV-1-infected H9 cells. 106

Nevertheless, despite the numerous pharmacological studies available, clinicaltrials are necessary to support the use of S. chinensis in the medical practice.

Acknowledgements

This work was financed in part by a grant from the Swedish Herbal Institute,

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( )J.L. Hancke et al. r Fitoterapia 70 1999 451471 469Gothenburg, Sweden. The authors wish to thank Christina Gomzi for secretarialassistance.

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A new acyclic monoterpene glucoside fromthe capitula of Tagetes patula

S.N. GargU, Reena Charles, Sushil KumarDepartment of Phytochemical Technology, Central Institute of Medicinal and Aromatic Plants,

Lucknow 226015, India

Received 2 November 1998; accepted 15 January 1999

Abstract

A new acyclic monoterpene glucoside, 2-methyl-6-methylen-2,7-octadiene-1-O--D-gluco-( )pyranoside 1 was isolated from Tagetes patula flowers in addition to known compounds

helenien, xanthophyll, patuletin and patuletrin. 1999 Elsevier Science B.V. All rightsreserved.

Keywords: Tagetes patula; Monoterpenoids; 2-methyl-6-methylen-2,7-octadiene 1-O--D-glucopyranoside

1. Introduction

.Tagetes patula L. Asteraceae a bushy annual with centre of origin in Mexico, iswidely cultivated as a garden plant in temperate and semitemperate regions ofAsia, Europe and America. This ornamental species is grown all over India up tow xthe height of approximately 2000 m 1 . Tagetes patula is a source of commerciallyimportant carotene compounds, helenien, xanthophyll, and essential oil. Helenienis used in pharmaceuticals especially in eye care formulations. Xanthophyll is usedas a direct and indirect colouring. Flavonoids have also been characterised fromw xthe capitula of T. patula 25 . Here we report on the isolation of a new acyclicmonoterpene glucoside, 2-methyl-6-methylen-2,7-octadiene 1-O--D-glucopyrano-

( )side 1 .

U

Corresponding author. Tel.: q 91-342676; fax: q 91-522342666. .E-mail address: [email protected] S.N. Garg


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( )S.N. Garg et al. r Fitoterapia 70 1999 472474 4732. Experimental

2.1. Plant material

Tagetes patula seeds were sown in November 1997 in raised nursery beds at the .Institute farm. The flowers inflorescence-capitula were harvested in February

1998. A voucher specimen has been deposited at the Institute herbarium.

2.2. Extraction and isolation

.Dried flowers 500 g were Soxhlet extracted in MeOH and the extract was .concentrated to a viscous mass 60 g . Si gel CC of 50 g of this material using

. .CHCl MeOH mixtures provided helenien 800 mg , xanthophyll 100 mg , pat-3 . . .uletin 60 mg , patuletrin 40 mg and the new compound 1 30 mg .

( ) w x2-Methyl-6-methylene-2,7-octadiene 1-O--D-glucopyranoside 1 . Viscous oil -D . . y 121.5 c 0.25, MeOH ; IR bands Neat 3390, 1655, 1630, 1590, 990 and 900 cm ;

1 . . H-NMR 400 MHz, Py-d : : 6.28 1H, dd, J 18 and 10 Hz, H-7 , 5.41 1H, br t, J5. . 7 Hz, H-3 , 5.28 1H, dd, J 18 and 1.3 Hz, H-8a , 5.15 1H, dd, J 10.5 and 1.3 Hz,

. . . .H-8b , 5.09 1H, br s, H-9a , 5.05 1H, br s, H-9b , 3.95 2H, d, J 12 Hz, H-1 , 1.62 . . 3H, s, H-10 , 2.102.24 4H, overlapping signals, H-4 to H-5 -sugar unit: 4.20 1H,

. . d, J 8 Hz, H-1 , 3.71 1H, dd, J 6 and 12 Hz, H-6a , 3.85 1H, dd, J 12 and 2 Hz,. . 13H-6b , 3.23.60 4H, overlapping signals, H-2 to H-5 ; C-NMR 100 MHz Py-d :5 . . . . . .144.5 C-6 , 137.7 C-7 , 132.8 C-2 , 130.4 C-3 , 115.8 C-8 , 114.2 C-9 , 102.5

. . . . . . .C-1 , 77.9 C-3 , 77.4 C-5 , 75.2 C-2 , 74.6 C-1 , 71.8 C-4 , 62.4 C-6 , 30.4 . . . . q . . .C-5 , 26.2 C-4 , 16.2 C-10 , El-MS 70 eV m r z: 314 M 10 , 152 85 .

3. Results and discussion

The methanolic extract of the capitula of T. patula, on chromatography oversilica gel, afforded a new compound 1, C H O , IR absorption bands at 339016 26 6 . y 1 1OH , 1665, 1630, 1590, 990 and 900 cm for olefinic double bands. H-NMRspectrum revealed the presence of six olefinic protons including an ABX system of

. .a vinyl group at 6.28 dd, Js 18 and 10 Hz , 5.28 dd, Js 18 and 1.3 Hz , and . .5.15 dd, Js 10.5 and 1.3 Hz , two methylene olefinic protons at 5.09 1H, brs

. .and 5.05 1H, br s and a separate olefinic proton at 5.41 br t, J 7 Hz . Inaddition, one methyl proton at 1.62 as a singlet, two oxymethylene proton at

. 13.95 d, Js 12 Hz and seven sugar protons were also observed. These H-NMRsignals were in agreement with the attribution of structure 1 to the compound. The1 13 .H- and C-NMR values see Section 2 were also in agreement with thosew xreported for similar compounds 610 .

The linkage of the sugar unit with oxymethylene carbon C was confirmed by the1

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( )S.N. Garg et al. r Fitoterapia 70 1999 472474474

1

C carbon downfield shift by 7 ppm in comparison to oxymethylene carbon of18-hydroxy-3,7-dimethyl-1,6-octadiene. The anomeric proton in position was indi-

. 13cated by the proton doublet at 4.20 1H, d, Js 8 Hz supported by C-NMRsignal at 102.5.

Acknowledgements

The authors are grateful to the Regional Sophisticated Instrument Centre,

Central Drug Research Institute Lucknow for 400 MHz

1

H-NMR, 100 MHz13 C-NMR and Mass spectra of the compound. Thanks are also due to the BotanyDepartment, Central Institute of Medicinal and Aromatic Plants, Lucknow forestablishing the identity of plant material.

References

w x1 The wealth of India, raw materials, vol X. New Delhi: CSIR, 1976:109111.w x2 Rodriguez E, Mabry JJ. In: Heywood VH, Harborne JB, Turner BL, editors. The biology andchemistry of the compositae. London: Academic Press, 1977:786797.w x

3 Bhardwaj DK, Bisht MS, Jain SC, Mehta CK, Sharma GS. Phytochemistry 1980;19:713.w x4 Tripathi AK, Paliwal MK, Singh J. J. Indian Chem. Soc. 1991;68:674.w x5 Ivancheva S, Zdranvekova M. Fitoterapia 1993;64:555.w x6 Otsuka H. Phytochemistry 1994;37:461.w x7 Gering B, Wichtl M. J. Nat. Prod. 1987;50:1048.w x8 Takeda Y, Takechi A, Masuda T, Otsuka H. Planta Med. 1998;64.w x9 Byers JA, Schlyter F, Birgesson G, Francke W. Experientia 1990;46:78.w x10 Rucker G, Mayer R, Mans D. J. Nat. Prod. 1987;50:287.

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A novel isoflavone from the stems ofAgeratum conyzoides

R.N. YadavaU, Saurabh KumarNatural Products Laboratory, Department of Chemistry, Dr. H.S. Gour Uni ersity, C-101,

Uni ersity Campus, Gour Nagar, Sagar 470 003, M.P., India

Received 2 September 1998; accepted in revised form 7 December 1998

Abstract

.A novel isoflavone glycoside, 5,7,2,4-tetrahydroxy-6,3-di- 3,3-dimethylallyl -isoflavone . ( )5-O--L-rhamnopyranosyl- 1 4 --L-rhamnopyranoside 1 , was isolated from the stems of

Ageratum conyzoides. 1999 Published by Elsevier Science B.V. All rights reserved.

Keywords: Ageratum conyzoides; Isoflavonoids

1. Introduction

.Ageratum conyzoides L. Asteraceae , commonly known as Kubhi in Hindi andw xdistributed throughout India up to 1500 m, is useful in fever 1 . The ayurvedicsystem of medicine describes that the roots of this plant possess anthelmintic andw xantidysenteric properties 2,3 . The isolation of a new isoflavonoid, 5,7,2,4-tetrahy-

. .droxy-6,3-di- 3,3-dimethylallyl -isoflavone 5-O--L-rhamnopyranosyl- 1 4 --L- .rhamnopyranoside 1 , from the stems of this plant is here reported.

2. Experimental

2.1. Plant material

A. conyzoides stems, collected in Sagar region in July 1997 and identified by the

U

Corresponding author. Tel.: q 91-7582-26465; fax: q 91-7582-23236.0367-326Xr 99r $ - see front matter 1999 Published by Elsevier Science B.V. All rights reserved.

.PII: S 0 3 6 7 - 3 2 6 X 9 9 0 0 0 5 6 - 8

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( )R.N. Yada a, S. Kumar r Fitoterapia 70 1999 475477476

.Department of Botany of this University. A voucher specimen No. X has beendeposited in the Natural Product Laboratory, Department of Chemistry, Dr. Hari

.Singh Gour University, Sagar M.P. , India.


.Air-dried and powdered stems 5 kg were extracted with 95% MeOH. The .concentrated extract was successively partitioned with petrol 4060C , benzene,

.CHCl , acetone, EtOAc and MeOH. The concentrated acetone-soluble part 56 g3was Si-gel CC eluting with benzeneEtOAc mixtures. Elution with benzeneEtOAc

.3:7 yielded compound 1 150 mg as light brown needles from Et O, mp 174175C,2 . . . . .C H O ; UVmax MeOH : 214 log 4.60 , 267 4.40 ; q NaOMe 224 sh ,37 46 14

. . .278, 344; q NaOAc 274, 348; q AlCl 214, 269 nm; IRmax KBr : 3365, 13483y 1 1 . . .cm . Acetate: H-NMR 300 MHz, CDCl : 6.22 1H, s, H-2 , 6.61 1H, s, H-8 ,3 . . 6.52 1H, d, J 8.5 Hz, H-5 , 7.01 1H, d, J 8.5, Hz H-6 , 3.41 2H, d, J 6.5 Hz,

. . . H-1 5.34 2H, m, H-2, H-2 , 1.69 6H, s, Me-4, Me-5 , 3.48 2H, d, J 6.5 Hz,. . .H-1 , 1.81 6H, s, Me-4, Me-5 , 2.322.48 9H, s, phenolic acetoxyls sugar

. .region: 2.022.14 18H, m, sugar acetoxyls , 4.84 1H, d, J 1.5 Hz, H-1 rham , 5.35 . .1H, d, J 1.5 Hz, H-1 rham , 4.815.57 10H, m, sugar protons , 0.91 and 1.14 . 13 .each 3H, d, J 6 Hz, Me-6, Me-6 rham ; C-NMR 400 MHz, DMSO-d : 152.86 . . . . . . .C-2 , 124.2 C-3 , 181.4 C-4 , 166.6 C-5 , 104.4 C-6 , 164.1 C-7 , 94.5 C-8 , 153.1 . . . . . . .C-9 , 106.2 C-10 , 125.9 C-1 , 98.6 C-2 , 148.2 C-3 160.1 C-4 , 118.2 C-5 ,

. . . 121.4 C-6 , 21.421.5 C-1, C-1 , 121.4, 121.6 C-2, C-2 , 132.1, 132.8 C-3,. . .C-3 , 25.6, 19.2 C-4, C-4 , 21.7, 17.8 C-5, C-5 sugar region: 101.1

. . . . . . .C-1 , 70.4 C-2 , 71.4 C-3 , 74.6 C-4 , 69.6 C-5 , 17.4 C-6 , 101.4 C-1

, 70.6 . . . . . . w q xC-2 , 71.3 C-3 , 71.1 C-4 , 68.3 C-5 , 17.1 C-6 ; EIMS mr z rel. int.% : M . w q .x . w q x . .absent , 423 M - acetylated di-rhamnoside 14 , 422 M 100 , 407 1.6 , 405 . . . . . . . . .1.1 , 379 32 , 367 53 , 366 20 , 351 50 , 323 63 , 311 41 , 165 25 , 147 4.5 .

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( )R.N. Yada a, S. Kumar r Fitoterapia 70 1999 475477 477 .Acid hydrolysis 10% HCl, 2 h at 100C of compound 1 yielded 5,7,2,4-tetrahy-

. w xdroxy-6,3-di- 3,3-dimethylallyl -isoflavone 4 and rhamnose.

Acknowledgements

.Thanks are due to the Director of the CDRI, Lucknow U.P. for the recordingof various spectra and Prof. V.K. Saxena, Department of Chemistry, Dr H.S. GourUniversity, Sagar for critical suggestions.

References

w x1 Kirtikar KR, Basu BD, Indian medicinal plants, II. Allahabad: Lalit Mohan Basu and CompanyPublication, 1935:1330.w x2 The Wealth of India. A dictionary of raw materials and industrial products, I. New Delhi: CSIRPublication, 1948:40.w x3 Chopra RN, Nayar SL, Chopra IC. Glossary of Indian Medicinal Plants. New Delhi: CSIRPublication, 1956:9.w x4 Tahara V, Inyham JL, Nakahara S, Mizutani J, Harborne JB. Phytochemistry 1984;23:1889.

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Flavone glycosides from Mentha longifolia

M. SharafU

, M.A. El-Ansari, N.A.M. SalehPhytochemistry and Plant Systematics Department, National Research Centre, Dokki-12311,

Cairo, Egypt

Received 12 November 1998; accepted 15 January 1999

Abstract

In addition to isoorientin, vicenin-2, hypolaetin, lucenin-1, luteolin 7-O-glucoside and7-O-neohesperidoside, the aerial parts of Mentha longifolia yielded three new flavonoids,

.identified as tricetin 7-O-methylether 3-O-glucoside 5-O-rhamnoside 1 , tricetin 3-O-glu- . . ( )coside 5-O-rhamnoside 2 and tricetin 3-O-rhamnosyl- 1 4 -rhamnoside 3 . 1999

Elsevier Science B.V. All rights reserved.

Keywords: Mentha longifolia; Flavonoids; Tricetin glycosides

1. Introduction

13 Little has been reported on the C-NMR of the glycosylation at ring-B 3. 13andr or 4 of flavones. The available reports are those dealing with the C-NMRw xof luteolin 3-glucoside 1 , 7-neohesperidoside-4-sophoroside, 7-neohesperidoside-w x4-glucoside and 7,4-dineohesperidoside 2 , in which the chemical shift values

reported for C-3 and C-4, under glycosylation, showed unexpected values inw xcomparison with C-7 when glycosylated 3,4 . To the best of our knowledge nothinghas been reported on the 13 C-NMR of 3- or 3,5-glycosylation of 5,7,3,4,5-pen-tahydroxyflavone.

The present communication describes the isolation and structural elucidation of

U

Corresponding author.


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( )M. Sharaf et al. r Fitoterapia 70 1999 478483 479 . .three new glycosides isolated from M. longifolia L. Hudson Lamiaceae grown in

Saudi Arabia.

2. Results and discussion

The methanolic extract of the aerial parts of M. longifolia was fractionated on apolyamide column. Purification of the compounds was achieved by a combinationof silica gel TLC and Sephadex LH-20. The flavonoids 1 3 were isolated.

Acid hydrolysis of 1 released glucose, rhamnose and the new aglycone, tricetin . 17-methyl ether 1a which was identified by UV and H-NMR. The UV spectralw xdata of 1 with diagnostic shift reagents 5 indicated a flavone substituted at

7-position, a free 4-hydroxyl group and absence of a free ortho-dihydroxy patternat B-ring. The 1 H-NMR spectrum of 1 showed that it is a tricetin diglycoside onthe basis of the two sugar C-1 proton doublets at 5.20 and 5.10. The doublets at

. . 5.10 J 2 Hz and 1.10 J 5.5 Hz, Me-rha. indicate the presence of onew xrhamnose unit in 1 5 . The other C-1 proton doublet at 5.20 must derive from .glucose, and the coupling constant J 7 Hz is characteristic for a -linked glucosew x .6 . Furthermore, the chemical shifts 5.20, 5.10 indicated that both glucose andw x 1rhamnose moieties are directly attached to the aglycone 7 . The H-NMR spec-

trum of1 also showed a singlet at 7.30 assigned to H-2 and H-6, confirming the . .presence of tri-substituted pattern 3, 4 and 5- at ring-B. Two doublets J 2 Hz

at 6.80 and 6.35 were assigned to the H-6 and H-8, while H-3 appeared as asinglet at 6.95 and the methoxy group as a singlet at 4.10.

13

.The C-NMR spectrum Table 1 confirmed that 1 is a diglycoside of tricetin on .the basis of the signals of C-6 of glucose and rhamnose 61.12 and 18.00 . The13 C-NMR shifts of the aglycone part of 1 correspond well to the shifts of tricetinw x3 , the only difference being a downfield shift of the signal assigned to C-7 by

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( )M. Sharaf et al. r Fitoterapia 70 1999 478483480 .approximately 3.7 ppm 164.0167.7 and upfield shift of the ortho-related carbons

. 13see Table 1 , confirming the location of the OCH group at C-7. The C-NMR3

spectrum of 1 showed unexpected values for carbons 3, 4 and 5, which are inw xagreement with a previous report 2 . The absence of a signal at 138.00 was .characteristic for C-4 in tricetin 3,4,5-tri-OH and the presence of only one

signal at 148.71 confirmed the 3,5-disubstitution pattern. This signal was assignedto C-3, C-4 and C-5, the upfield shift of C-3 and C-5 being caused byglycosylation, and the downfield shift of C-4 as being ortho related carbon to C-3and C-5. From the above data compound 1 is identified as tricetin 7-O-methyl-ether 3-O--D-glucoside 5-O--L-rhamnoside.

Compound 2 gave glucose, rhamnose and tricetin after acid hydrolysis. The UVspectra indicated a flavone with a free 5,7,4-trihydroxy pattern and absence of a

free o-dihydroxy pattern. The1

H-NMR and13

C-NMR spectra of 2 showed a veryclose similarity with those of 1, the only differences being the elimination of thesignals assigned for the OCH from the spectra of 2, while C-3, C-4 and C-53resonated at 146.20. The rest of the spectra indicated a tricetin 3,5-disubstituted

.pattern Table 1 . Thus, compound 2 is identified as tricetin 3-O--D-glucoside5-O--L-rhamnoside.

It was expected for both compounds 1 and 2, as they have different glycosylation .pattern glucose at C-3 and rhamnose at C-5 , that different chemical shifts

should be obtained for C-3 and C-5 in their 13 C-NMR spectra. Unexpectedly, the .same chemical shift for C-3 and C-5 148.71 for 1 and 146.20 for 2 was observed.

Rhamnose and tricetin were the only products released after acid hydrolysis of3.The UV spectral data of 3 indicated a flavone with 7,4-dihydroxyl groups, and thepresence of a free o-dihydroxy pattern at ring-B. Two rhamnose moieties were

.present in 3 as indicated by the presence of two doublets J 5.5 Hz for tworhamnose methyl groups at 0.50 and 0.70 ppm in the 1 H-NMR spectrum. Thespectrum also showed the aromatic protons as a singlet at 7.40 ppm assigned to

.H-2 and H-6 confirming the tri-substitution pattern 3, 4 and 5 , and expectedsignals for H-3, H-6 and H-8.

13 .The C-NMR spectrum of3 Table 1 showed the expected signals for tricetinsubstituted in 3-position indicated by the presence of a signal at 151 ppm, and afree OH group at C-4 and C-5 indicated by the presence of a signal at 147.00 ppmassigned for both carbons. Comparison of the rhamnose carbon chemical shifts inthe spectrum of 3 and those of unsubstituted methyl-4-O-glycosylated--L-rhamnoside showed similarities. Thus, the chemical shift for the carbon atomlinked to the second sugar in the case of 2-O-glycosylation is at 79.0, that of aw x3-O-glycosylation is at 78.8 and in the case of a 4-O-glycosylation is at 80.8 8 .The corresponding atom in 3 resonates at 81.0 indicating that rhamnose must belinked to the 4-hydroxyl group of the rhamnosyl moiety. From the above data

.compound 3 is identified as tricetin 3-O--L-rhamnosyl- 1 4 -rhamnoside.

Comparison of the13

C-NMR assignments of compounds 13 with those reportedw x w xfor tricetin 3 , luteolin 3-glucoside 1 , and luteolin 7-O-neohesperidoside 4-O-w x .sophoroside 2 Table 1 , supported the proposed structures for 1, 2 and 3.As previously reported, the effect of glycosylation of the 7-hydroxyl on the C-7

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( )M. Sharaf et al. r Fitoterapia 70 1999 478483 481Table 113 ( ) ( ) ( )C-NMR of tricetin I , luteolin 3-glucoside II , luteolin 7-neohesperidoside 4-sophoroside III and

acompounds 1, 2, and 3

Carbon I II III 1 2 3

2 164.2 163.2 163.8 164.83 164.43 164.193 103.2 103.9 104.4 104.81 103.07 103.94 181.6 181.7 181.8 182.51 182.07 182.055 161.6 161.5 161.0 161.42 161.39 161.426 99.0 99.0 100.6 98.49 99.41 101.07 164.2 163.5 163.0 167.70 164.03 164.038 93.9 94.2 94.2 95.29 93.59 94.09 157.9 157.5 157.1 157.53 157.09 157.0910 104.0 103.4 105.7 104.81 103.65 104.0

1 120.9 122.1 124.9 120.43 121.42 121.572 106.0 115.2 113.9 105.99 107.5 109.503 146.5 145.7 147.3 148.71 146.20 151.04 137.9 150.9 148.7 148.71 146.20 147.05 146.5 116.6 116.7 148.71 146.20 147.06 106.0 122.1 118.4 105.99 107.51 109.01-G 102.4 100.7 101.04 102.82

101.62 73.5 79.7 72.82 72.69

74.73 77.4 76.0 77.56 78.36

76.94 70.3 70.4 79.91 70.65

72.05 76.3 77.0 77.15 78.36

77.16 61.2 60.7 61.12 61.01

60.41-R 98.1 98.50 99.41 102.50

101.502 70.0 70.23 70.51 70.50

70.103 70.6 70.80 70.65 70.50

70.104 73.1 72.82 73.62 81.00

72.505 68.2 68.88 69.23 69.00

69.006 17.7 18.50 19.0 18.00

18.50OCH 56.823

a 13 w x w xC-NMR of compounds I, II and III were obtained from Markham and Chari 4 , Markham 1 w xand Osterdahl 2 , respectively. G, glucose; R, rhamnose.

signal is approximately 1.5 ppm upfield shift, accompanied by downfield shift ofw xapproximately 2 ppm in the para related carbon 4 . In our study, glycosylation at3 andr or 5 showed shifts qualitatively similar but quantitatively different. These

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( )M. Sharaf et al. r Fitoterapia 70 1999 478483482w xresults are in agreement with previous data for 3- and 4-substituted flavones 1,2 .

In conclusion, this is the first report of the natural occurrence of compounds 1, 2

and 3. Also, from the chemosystematic point of view, it is interesting to note thatflavones with a trisubstituted B-ring have not been reported before in the familyLamiaceae.

3. Experimental

3.1. Plant material

M. longifolia aerial parts, collected from El-Madina, Saudi Arabia, in March 1997and authenticated by Prof. L. Boulos. A voucher specimen is deposited in theHerbarium of NRC, Cairo.


.The air-dried plant 200 g was extracted with 80% MeOH. The concentrated . .extract 11 g was subjected to polyamide CC. The major components 1 21 mg , 2

. . .28 mg and 3 31 mg were isolated by PTLC on Si-gel GF Merck eluting with254CHCl MeOHH O 65:45:12, and further purified using Sephadex LH-20 eluting3 2

with MeOH.

3.3. Acid hydrolysis

Glycosides 13 were hydrolyzed with 2 N HCl at 100C for 60 min.

.3.4. Tricetin 7-O-methylether-3-O--D-glucoside-5-O--L-rhamnoside 1

.UVmax MeOH : 250, 270, 350; q NaOMe 262, 417; q AlCl 250, 270, 350;3q AlCl q HCl 250, 270, 350; q NaOAc 250, 270, 350, 425; q NaOAc q H BO ,3 3 31 . . 250, 270, 350 nm; H-NMR 270 MHz, DMSO-d : 7.30 2H, s, H-2,6 , 6.95 1H,6

. . . s, H-3 , 6.80 1H, d, J 2 Hz, H-8 , 6.35 1H, d, J 2 Hz, H-6 , 5.20 1H, d, J 7 Hz,. . H-1 , 5.10 1H, d, J 2 Hz, H-1 , 4.003.60 8H, m, sugar protons, hidden by

. . . 13 hydroxyl groups , 4.10 3H, s, OCH , 1.1 3H, d, J 5.5 Hz, Me-rha ; C-NMR see3.Table 1 .

.3.5. Tricetin 7-methyl ether 1a

.UVmax MeOH : 255, 265sh, 349; q NaOMe 263, 299sh, 394; q AlCl 272,3296sh, 331, 434;

qAlCl

qHCl 272, 295, 359, 390;

qNaOAc 295, 267sh, 366, 403;3 1 . NaOAc q H BO , 258, 371 nm; H-NMR 270 MHz, DMSO-d : 7.28 2H, s,3 3 6

. . . .H-2,6 , 6.91 1H, s, H-3 , 6.77 1H, d, J 2 Hz, H-8 , 6.33 1H, d, J 2 Hz, H-6 , 3.98 .3H, s, OCH .3

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( )M. Sharaf et al. r Fitoterapia 70 1999 478483 483 .3.6. Tricetin 3-O--D-glucoside 5-O--L-rhamnoside 2

.UVmax MeOH : 255, 270, 347;q

NaOMe 267, 325sh, 405;q

AlCl 267, 300sh,3380; q AlCl q HCl 260, 295sh, 353; q NaOAc 273, 315sh, 380; q NaOAc q31 . .H BO 262, 375 nm; H-NMR 270 MHz, DMSO-d : 7.40 2H, s, H-2,6 , 6.903 3 6

. . . 1H, d, J 2 Hz, H-8 , 6.65 1H, s, H-3 , 6.50 1H, d, J 2 Hz, H-6 , 5.30 1H, d, J 7. . Hz, H-1 , 4.65 1H, s, H-1 , 4.10 y 3.65 8H, m, sugar protons, hidden by

. . 13 .hydroxyl groups , 0.70 3H, d, J 5.5 Hz, Me-rha ; C-NMR see Table 1 .

.3.7. Tricetin 3-di-O--L-rhamnoside 3

.UVmax MeOH : 255, 270, 347; q NaOMe 267, 325sh, 405; q AlCl , 267, 300sh,3380; q AlCl q HCl 260, 295sh, 353; q NaOAc, 273, 315sh, 380; q NaOAc q3 1 . .H BO 262, 375 nm; H-NMR 270 MHz, DMSO-d : 7.40 2H, s, H-2,6 , 6.903 3 6 . . . 1H, d, J 2 Hz, H-8 , 6.65 1H, s, H-3 , 6.45 1H, d, J 2 Hz, H-6 , 5.15 1H, d, J 2

. . Hz, H-1 , 5.00 1H, d, J 2 Hz, H-1 , 4.704.20 8H, m, sugar protons, hidden by. . .hydroxyl groups , 0.70 3H, d, J 5.5 Hz, Me-rha. , 0.50 3H, d, J 5.5 Hz, Me-rha. ;

13 .C-NMR see Table 1 .

References

w x1 Markham KR, Ternai B, Stanly R, Geiger H, Mabry TJ. Tetrahedron 1978;34:1389.x2 Osterdahl BO. Acta Chem Scand 1979;B33:119.w x3 Markham KR, Chari VM. The flavonoids: advances in research, In: Harborne JB, Mabry TJ,editors. Chapman and Hall, London, 1982:spectrum No. 38.w x4 Markham KR, Chari VM. The flavonoids: advances in research, In: Harborne JB, Mabry TJ,editors. Chapman and Hall, London, 1982:39.w x5 Mabry TJ, Markham KR, Thomas MB. The systematic identification of flavonoids. Berlin:Springer-Verlag, 1970:269.w x6 Kamerling JP, Bie MJA, Vliegenthart JFG. Tetrahedron 1972;28:3037.x7 Osterdahl BO, Lindberg G. Acta Chem Scand 1979;B31:293.w x8 Liptak D, Nansai P, Neszmelyi A, Wagner H. Tetrahedron 1980;36:1261.

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Effect of Semecarpus anacardium nutextract against aflatoxin B -induced1

hepatocellular carcinomaB. Premalatha, P. SachdanandamU

Department of Medical Biochemistry, Dr.A.L.M. P-G.I.B.M.S., Uni ersity of Madras,Taramani campus, Chennai 600 113, India

Received 12 November 1997; accepted in revised form 30 March 1999

Abstract

The antitumour activity of Semecarpus anacardium nut extract against aflatoxin B -in-1 .duced experimental hepatocellular carcinoma HC was investigated. The adverse changes

induced by aflatoxin B were reversed to near normal and the histological pattern was1almost normal in treated rats. These results suggest that S. anacardium nut extract haspotential anticarcinogenic activity against aflatoxin B -mediated HC. 1999 Published by1Elsevier Science B.V. All rights reserved.

Keywords: Semecarpus anacardium; Antitumour activity; Hepatocellular carcinoma; Aflatoxin B 1

1. Introduction

How cancer develops from apparently normal tissue is one of the unresolved .biological problems of our time. Hepatocellular carcinoma HC , a fatal malig-

nancy, represents 4% of all malignant tumours and is the seventh most commonw xcancer in man worldwide 1 . In China, where the predominance of HC is high, thetraditional medicinal herbs are used as an effective treatment against primary liverw xcancers 2 .

.Semecarpus anacardium L. Anacardiaceae is a deciduous tree, distributed in thew xsub-Himalayan tract and in tropical parts of India 3 . It is commonly known asU

Corresponding author. Tel.: q 91-44-4925548; fax: q 91-44-4926709.0367-326Xr 99r $ - see front matter 1999 Published by Elsevier Science B.V. All rights reserved.

.PII: S 0 3 6 7 - 3 2 6 X 9 9 0 0 0 7 0 - 2

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( )B. Premalatha, P. Sachdanandam r Fitoterapia 70 1999 484492 485marking nut in English and has high priority and applicability in indigenousw x w xsystems of medicine. Many compounds, mainly biflavonoids 4 , phenolics 5 andw xglycosides 6 have been identified as constituents of S. anacardium nut extract.Many medicinal properties such as antimicrobial, anti-inflammatory, anthelminthicw xand anti-amoebic have been attributed to the nut of the plant 7 . It is claimed tobe effective against a variety of ailments, such as lepranodules, warts and rheuma-w xtism 8 . In traditional medicine, it is highly valued for the treatment of tumoursand malignant growth. Recent studies carried out on an Ayurveda marking nutpreparation have also shown promising results in the treatment of cancers of thew xoesophagus, urinary bladder, liver, and leukaemia 9 . Earlier studies from ourlaboratory have proved the anticancer and hepatoprotective activity of S. anac-

. w xardium nut milk extract against aflatoxin B AFB -induced HC in rats 10 and1 1established its protective role on deranged cell membranes in AFB -induced HC1w x11 .

The aim of this work was to further evaluate the antitumour activity of S.anacardium nut extract, correlating biochemical and histological changes in AFB -1induced HC-bearing rats.

2. Experimental

2.1. Drugs

Semecarpus anacardium nut milk extract commercially named Serankottai nei.in the Siddha system of medicine was obtained from Indian Medical Practitioners

.Co-operative Pharmacy and Stores IMPCOPS , Chennai, India. The formulationw xwas prepared according to a recipe of the Formulary of Siddha Medicine 12 as . .follows. Boil S. anacardium purified nuts 200 g with milk 500 ml . Decant the

.decoction, add milk 500 ml to the boiled nuts and boil again for some time. .Recover the decoction and repeat the process again with milk 500 ml . Combine

.the three portions of milk nut decoction, mix with ghee 1.5 kg and boil tillw xdehydrated 12 .2.2. Animals

Wistar male rats weighing 100120 g, obtained from Tamilnadu Veterinary andAnimal Sciences University, Chennai, India, were used in the experiments. They

were maintained under standard experimental conditions temperature 27 " 1C;.relative humidity 60 " 5% and 12 h lightr dark cycle and fed with pelleted diet

.Gold Mohur rat feed, Mr s. Hindustan Lever Limited, Mumbai and water adlibitum.

2.3. Experimental protocol

The animals were divided into four groups of six animals each:

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( )B. Premalatha, P. Sachdanandam r Fitoterapia 70 1999 484492486 .Group I: Normal control, received a single intraperitoneal i.p. dose of dimethyl

.sulphoxide DMSO; 0.5 ml

. Group II: HC was induced by single dose of AFB Sigma in DMSO 2 mgr

kg,1. w xi.p. 13 .Group III: HC-induced animals as in Group II were treated with S. anacardium

. .nut extract 200 mgr kgr day for 14 days in sunflower oil 2.5 mlr kgby gavage

Group IV: Animals received only the same dosage of S. anacardium nut extractas Group III

2.4. Biochemical analysis

At the end of the experimental period, all animals were fasted for 18 h beforebeing killed by cervical decapitation. Liver and kidney tissues were immediately

.excised, weighed and then homogenized in TrisHCl buffer 0.1 M pH 7.4 . Thetissue homogenates were used for the estimation of proteins by the method ofw xLowry et al. 14 . The nucleic acids from the tissues were extracted by the methodw x .of Schneider 15 with trichloroacetic acid TCA and DNA was estimated by thew x w xmethod of Burton 16 and RNA according to Rawal et al. 17 .2.5. Statistical analysis

Statistical evaluation was carried out using one way analysis of variance .ANOVA and F-ratio was computed to detect the significant changes between thegroups. The Students NeumannKeul test was used to compare Group I withGroups II, III and IV, and Group II with Group III.

2.6. Tissue processing

Immediately after killing, liver and kidneys were rapidly excised, serially sec-tioned and macroscopically examined. Microscopic samples from liver were taken

from right portion of the median lobe since this portion of the rat liver has beenw xfound to be a site of more apparent histological lesions 18 . The tissues were fixedin 10% buffered formalin. Consecutive sections were stained with haemotoxylin

.and eosin H & E .

3. Results

3.1. Body and li er weightsBody and liver weights were significantly decreased in AFB -induced HC condi-1

.tions Group II . The rat body wt. regularly increased in controls and progressivelydeclined in tumour hosts. These changes were reversed to near normal in S.

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( )B. Premalatha, P. Sachdanandam r Fitoterapia 70 1999 484492 487Table 1

aEffect of Semecarpus anacardium nut extract on aflatoxin B -induced hepatocellular carcinoma in rats1

Parameters Group I Group II Group III Group IV F-ratiomgr g

.wet tissue

U ULiver DNA 6.36 " 0.48 9.41 " 0.89a 7.02 " 0.68b 6.50 " 0.64 20.86UU UUURNA 4.10 " 0.36 5.35 " 0.54a 4.52 " 0.47b 4.21 " 0.41 7.19U UProtein 159.32 " 11.63 96.52 " 9.63a 151.37 " 14.12b 155.49 " 14.47 26.98

U UUKidney DNA 4.99 " 0.46 6.86 " 0.68a 5.29 " 0.53b 5.07 " 0.48 12.94UU UUURNA 3.14 " 0.28 4.09 " 0.42a 3.48 " 0.32b 3.26 " 0.31 7.72

UU UUProtein 138.16 " 10.05 110.16 " 10.21a 132.83 " 10.24b 136.29 " 9.97 8.23

a Values are expressed as mean " S.D.; n s 6. Group I: normal control; group II: HC-induced rats; .group III: HC-induced treated with S. anacardium extract 200 mgr kgr day = 14 days ; group IV:

.control rats receiving only S. anacardium extract as in group III. Comparisons were made: a group I vs. . U UUgroups II, III and IV; b group II vs. group III. Statistical significance: P- 0.001, P- 0.01,

UUUP- 0.05; ANOVA and Students Neuman Keul test.

.anacardium nut extract treated animals Group III . Extract control animals .Group IV did not show any significant variation in body and liver weights.

3.2. Biochemical changes

. .Increased level of DNA P- 0.001 with subsequent increase in RNA P- 0.01 .was observed in Group II carcinoma-bearing animals Table 1 . These levels were

decreased to near normal values in extract treated Group III animals. No signifi- .cant alteration of nucleic acids was observed in extract control animals Group IV .

In contrast to nucleic acids, the protein content was significantly decreased inHC-bearing animals. The recoupment of protein level to near normal liver

.P- 0.001, kidney P- 0.01 was observed in S. anacardium administered animals .Group III . Extract control animals showed no significant alteration in proteincontent.

3.3. Gross morphology

In Group II animals, the liver of three rats showed marked congestion while inthe remaining mottling was evident. Kidney did not reveal any gross changeabnormalities except in two rats where the cortical surfaces revealed congestion. InGroup III animals, although the liver showed a slight congestion, the architecturewas almost normal. Kidneys from these animals did not reveal any appreciablechange when compared with normal controls.

3.4. Histology

Histologically, the liver from control animals revealed a normal architecture

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( )B. Premalatha, P. Sachdanandam r Fitoterapia 70 1999 484492488 . .Fig. 1 . In HC-bearing animals Group II , the liver showed marked congestion of

.central vein along with intense cytoplasmic granularity of the hepatocytes Fig. 2 .

T

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