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Fitoterapia 70 1999.

451 471

Review

Schisandra chinensis Turcz./ Baill


J.L. Hancke a,U , R.A. Burgos b, F. Ahumadab
a b

Laboratorios Garden House, A . Presidente J. Alessandri 12310, San Bernardo, Santiago, Chile Instituto 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 dibenzow a,c xcyclooctene lignans from this plant are reviewed focusing in particular on the antihepatotoxic, antioxidant and antitumoural activities, and on the effects on physical performance and on the central nervous system. 1999 Elsevier Science B.V. All rights reserved.
Keywords: Schisandra chinensis; Lignans; Antioxidant activity; Antihepatotoxic activity; Antitumoral activity; 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, southern Sachalin and also north-eastern China, Korea and Japan w1x. Schisandra species grow mainly in China, Japan, the Himalayas and Jawa. The seeds and the fruit are the parts used in medicine w2 4x. S. chinensis is a monoecius liana with attractive leaves and a woody stem. The winding stem, reaching 10 15 m in length and 1.2 1.5 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. The flowers are white or slightly cream-coloured, wax-like, unisexual with a pleasant
U

Corresponding author. Tel.: q56-2-5281411; Fax: q56-5293646. E-mail address: gardenh@netline.cl J.L. Hancke.

0367-326Xr99r$ - 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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smell. The flowers are in clusters of 2 5. When the fruit is ripening, the receptacle is substantially lengthened and turned into a pedicle with the appearance of a grape cluster, 6 8 cm long with several bright red fruits. The fruits, ripen in September October, have an almost spherical shape and contain 1 2 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.. Experiences of its cultivation were reported w1x. In western botany the Chinese wu-wei-zi was first named Kadsura chinensis in an 1832 publication of the Russian botanist Nikolai S. Turczaninov. In 1856, to honour his most famous colleague K.J. Maximowicz 1827 1891., the Russian botanist Franz J. Ruprecht 1814 1870. created a new genus called Maximowiczia and called the plant Maximowiczia chinensis. In 1866, the French botanist H.E. Baillon 1827 1895. transferred the plant to the genus Schisandra and since that time the plant has been known as S. chinensis Turcz.. Baill. w3x. The generic name Schisandra 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 Wils. Southern Schisandra. w2x.

2. Chemistry Many dibenzow a,c xcyclooctene derivatives, present in different quantities fruit and seeds: 7.2 19.2%; stems: 1.3 10%. have been isolated from S. chinensis w5 20x. Some of the main structures are shown in Table 1. Biosynthetic precursors to the dibenzocyclooctene derivatives, such as pregomisin and epigalbacin, have been also isolated w20x. The fruits also contain about 1.5% sugars, tannins, colour substances and about 3% of essential oils citral, -chamigrene, -camigrenol, 2-bisabolene, sesquicarene ., organic acids citric, malic, fumaric and tartaric acid., vitamin C and E, and metals such as copper, manganese, nickel and zinc w21x.

3. Pharmacology S. chinensis is officially listed in the Chinese Pharmacopoeia w22x and indexed as a 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 was first reported in Divine Husbandmans Classic of the Materia Medica w3x. According 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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excessive sweating used for deficient Yang spontaneous sweating or deficient Yin night sweat.; e. calms the spirit used for forgetfulness and insomnia. w4,10x. 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. The Nanajs tribals used S. chinensis dried berries to combat fatigue in their hunting trips w1x. A monograph on S. chinensis preparations was introduced officially to the Russian Pharmacopoeia in 1961 w23x. 3.1. Antihepatotoxic effect Several reports indicate that S. chinensis dried fructus and seed. is an effective liver protective drug w24 26x. In experimental models, Glutamic Piruvic Transaminase GPT. activities induced by carbon tetrachloride CCl 4 . or paracetamol in mice, thiacetamide in rats, and ethinylestradiol 3-cyclopentylether in rabbits were reduced by oral administration of the ethanol extract of the seed of S. chinensis 1 10 grkg. prior to and after the administration of the hepatotoxic agents w24,25x. The alcoholic extract of S. chinensis kernels reduced elevated GPT levels in mice treated with CCl 4 or thioacetamide, while a water extract of the kernels and an ethanol extract of the shells of the seed were ineffective w21x. Primary cultured rat hepatocytes treated with 0.1 1 mgrml of either an ether, ethyl acetate, methanol or water extract of S. chinensis fruit were effective in reducing the galactosamine and CCl 4-induced cytotoxicity w20x. Different lignans isolated from S. chinensis have been associated to this antihepatotoxic effect w27,28x. Seven lignans isolated from S. chinensis kernels and tested for antihepatotoxic activity have been shown effective liver protecting drugs w23x. Most of them prevented the elevation of serum GPT levels and the morphological changes of the liver, such as inflammatory infiltration and liver cell necrosis induced by CCl 4 . Gomisin B 50 mgrkg, p.o., gomisin A, 50 mgrkg, p.o.., schisandrin C 50 100 mgrkg, p.o.., schisandrin B 50 100 mgrkg, p.o.. deoxyschisandrin 200 mgrkg, i.p.., -schisandrin 50 100 mgrkg, p.o.. and gomisin C 200 mgrkg, i.p.. decreased the GPT levels after CCl 4 w27x. Gomisin B, gomisin A and schisandrin at doses of 100 mgrkg, p.o.. were also effective against thiacethamide-induced liver damage in mice w21,27x. In fasting mice, the lignans stimulated the glycogen synthesis, the order of the activity being gomisin A ) deoxyschisandrin s -schisandrin. The activity of gomisin A was comparable to that of cortisone 100 mgrkg, p.o... Since similar results were obtained in adrenalectomized mice, the effect of these lignans on glycogenesis seems not mediated by the adrenals w21,27x. Pretreatment of male rats with gomisin A 50 mgrkg, i.p.. prevented the rise in GPT and Glutamic Oxaloacetic Transaminase GOT. and hepatic necrosis of cells induced by acetaminophen w29x. The repeated administration of gomisin A 30 or 100 mgrkg, p.o., daily for 4 days. induced an apparent increase of liver weight in liver-injured and normal rats w30x. Gomisin A suppressed the increase in serum transaminase activity and the appearance of histological changes such as hepatocyte degeneration and necrosis, inflammatory cell infiltration and fatty deposition

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induced in liver by CCl 4 , D-galactosamine or D-,L-ethionine w30x. Gomisin A decreased serum triglycerides and lipid contents of the liver. It also increased microsomal cytochrome b5 , P-450, NADPH cytochrome C reductase, aminopyrine N-demethylase and 7-ethoxycoumarin O-dethylase and decreased 3,4-benzow axpyrene hydroxylase w30x. A hepatoprotective effect of deoxyschisandrin, -schisandrin, schisandrin C, gomisin A, and schisandrin has been associated to their inhibitory effect on CCl 4-induced lipid peroxidation and the binding of CCl 4 metabolites to lipids of the liver microsomes w31 33x. Schisandrin B w34x and schisanhenol w35x under oxidative stress, and gomisin A in immunologic liver injury w36x increased the membrane stability of hepatocyte membrane. This effect can be related to a stimulating effect on the hepatic-glutatione antioxidant system w37x and may involve the enhancement of mitochondrial glutathione redox status in rats w38x. It is also suggested that gomisin A 50 mgrkg, i.p.. possesses a liver function enhancing property in normal and injured liver, and that its preventive action on CCl 4-induced cholestasis is sustained by the secretory function of the bile acids independent fraction w39x. Gomisin A and schisandrin B induced hypertrophy and mild hyperplasia, augmenting the liver weight. w 14 CxPhenylalanine incorporation, protein content, and hepatic microsomal cytochrome P-450 content were enhanced w40,41x. Gomisin A 10 100 mgrkg, p.o. for 4 days. also increased the liver regeneration in rats after partial hepatectomy, increased the regeneration rate of the liver cells, and improved the serum retention rate of the foreign dye sulfobromophtalein BSP., which was dose-dependent w42x. In addition, gomisin A also enhanced the incorporation of w 14 Cxphenylalanine into liver protein and shortened the hexobarbitalinduced sleeping time. These changes caused by gomisin A are similar to those of phenobarbital w42x. However, gomisin A is distinctly different from phenobarbital in the finding that phenobarbital diminished the survival of CCl 4 -intoxicated mice, but gomisin A did not w42x. Ultrastructural studies of liver tissue using the transmission electron microscope revealed an increase in rough and smooth endoplasmic reticulum in the groups receiving gomisin A 100 and 300 mgrkg per day.. Gomisin A accelerated both the proliferation of hepatocytes and the recovery of liver function after partial hepatectomy and increased hepatic blood flow. It is thought that the liver enlargement caused by repeated administration of gomisin A is associated with the proliferation of endoplasmic reticulum w42x. Gomisin A 10 or 30 mgrkg, p.o. for 3 or 6 weeks. suppressed the fibrosis proliferation and accelerated both the liver regeneration and the recovery of liver function after partial hepatectomy in CCl 4-induced chronic liver injury in rats w43x. Also, gomisin A regenerated the liver tissue after partial hepatectomy by enhancing ornithine decarboxylase activity, which is an important biochemical event in the early stages of liver regeneration in rats w44x. Gomisin A 100 mgrkg, 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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proliferative processes of non-parenchimal cells that involved an increase in c-myc product, a gene that precedes DNA replication in proliferating cells w45x. 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 lipopolysaccharide in mice previously treated with heat-killed Propionibacterium acnes, most of the animals died with acute hepatic failure. Gomisin A 5 50 mgrkg, p.o. reduced dose-dependently the mortality of mice with acute hepatic failure. Histologically, necrosis was suppressed by gomisin A, but infiltration of non-specific inflammatory cells was not affected. In in vitro experiments, the liver cells were injured by antibody-dependent cell-mediated cytotoxicity ADCC. reaction or activation of macrophage in vitro . Inhibition of the isolated liver cell injuries induced by ADCC reaction or activation of macrophages in vitro, suggested that gomisin A can be markedly protective against immunological liver injuries w46x. In guinea pigs sensitised with trinitrophenylated liver macromolecular protein fraction, gomisin A 50 mgrkg, p.o.. was effective in reducing the acute hepatic failure w47x. Also the acute hepatic failure induced by heat-killed Propionibacterium acnes followed by a small amount of Gram-negative lipopolysaccharide was prevented after 4 weeks of gomisin A 60 mgrkg per day for 4 10 weeks. administration w48x. The survival rate was 80% as compared to 5% of the control group. In Long Evans Cinnamon rats, spontaneously developing hepatitis, treatment with gomisin A did not modify the death rate, but the time of survival was increased by 7 10 weeks as compared with the control group w49x. Leukotrienes are potent inflammatory agents that are thought to play a role in inflammatory liver diseases w50x. In inmunological hepatic failure, mononuclear cells are the predominant cells producing leukotrienes. Gomisin A 0.1 mgrml, added to 10 7 macrophage cellsrml suspension. produced on the biosynthesis of leukotrienes stimulated in rat peritoneal macrophages by Ca2q ionophore A2318 an inhibitory effect which may be partially associated with its antihepatotoxic effect w51x. 3.2. Antioxidant and detoxificant effect The antioxidant effect of S. chinensis is attributed to the dibenzow a,c xcyclooctene lignan constituents w52,53x. In in vitro studies, induction of antioxidative enzymes has been observed with S. chinensis lignans which inhibited the lipid peroxidation measured by means of malondialdehyde MDA. formation induced by ironrcysteine in rat liver microsomes: at 1 mM concentration, schisanhenol, Sy.-schisandrin C and Sy.-schisandrin B were shown to be more potent than vitamin E w35x. Schisanhenol 1 mM. and schisandrin B 1 mM. also inhibited gossypol-induced superoxide anion generation in rat liver microsomes. The preventive oral administration 200 mgrkg, once daily for 3 days. of either schisanhenol or schisandrin B reduced liver MDA formation induced by 50% ethanol 15 mlrkg. w54x. In vitro, schisanhenol demonstrated an oxygen scavenging activity in ironrcysteine and NADPHrascorbic acid method, w55x and CCl 4 -OH and -CCl 3 .-induced lipid peroxidation in hepatocytes w34,56x. The release of GPT and lactate dehydro-

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genase LDH. was also reduced. As a consequence, the hepatocyte viability was increased as well as the integrity of the hepatocyte membrane w35x. Moreover, the hepatoprotective effects of Schisandra lignans may be attributed to the enhancement of the hepatic antioxidant system. In fact, schisandrin B and schisanhenol were also able to increase superoxide dismutase, catalase activities in rat liver cytosol, w54x and the function of the hepatic reduced glutathione GSH. anti-oxidant system w37x. Intragastric pre-treatment of female Balbrc mice with schisandrin B 4 16 mgrkg per day for 3 days. caused dose-dependent increases in hepatic glutathione S-transferase GST. and glutathione reductase GRD. activities. In other experiments, 24 h after the last dose of schisandrin B, all rats were treated with CCl 4 0.1 mlrkg.. The activities of glucose-6-phosphate dehydrogenase G6PDH., Se-glutathione peroxidase GPX., and gamma-glutamylcysteine synthetase GCS. are down-regulated to varying degrees in a dose-dependent manner by schisandrin B w37x. The beneficial effect of schisandrin B on the hepatic GSH antioxidant system is more evident after CCl 4 challenge. The hepatoprotection was associated with significant enhancement in hepatic GSH, as indicated by the substantial increase in tissue GSH levels and the corresponding decrease in the susceptibility of tissue homogenates to GSH depletion w37x. The hepatoprotective effect of schisandrin B 8 mgrkg, i.p.. was not affected by 1,3-bis2-chloroethyl.-1nitrosourea, an inhibitor of GRD, at a dose of 2 mmolrkg i.p.. in female Balbrc mice. The mechanism of hepatoprotection by schisandrin B may involve the enhancement of mitochondrial glutathione redox status greatly impaired by CCl 4intoxication w38x. Interestingly, while well known antioxidant agents such as -tocopherol acetate did not protect against hepatic damage induced by other hepatotoxins such as aflatoxin B 1 or Cd, S. chinensis reduced the hepatotoxic effect of these agents in a non-selective manner Tables 2 and 3. w57x. In fact, pre-treatment with a lignan enriched extract of S. chinensis fruit stimulated the hepatic antioxidantrdetoxification system, as shown by increased hepatic GSH levels as well as hepatic GRD and GST activities in rats w57x. Some comparative studies in female Balbrc mice have shown that schisandrin B 12 mgrkg per day, p.o. for 3 days. increased the hepatic mitochondrial-GSH level, whereas butylated hydroxytoluene BHT. decreased it. However, both schisandrin B and BHT increased, albeit to a different extent, the activity of mitochondrial GRD, particularly after CCl 4 challenge w58x. Pre-treatment with schisandrin B 12 mgrkgrday, p.o. for 3 days. sustained the hepatic mitochondrial GSH level in CCl 4-intoxicated mice and protected against CCl 4-induced hepatotoxicity, while BHT pre-treatment did not. Moreover, while both schisandrin B and BHT increased hepatic ascorbic acid vitamin C. level in control animals, only schisandrin B pre-treatment sustained a high hepatic vitamin C level in CCl 4 -intoxicated mice. Also, schisandrin B pre-treatment prevented the CCl 4-induced decrease in the hepatic vitamin E level. However, schisandrin B inhibited NADPH oxidation in mouse liver microsomes incubated with CCl 4 10 mM. in vitro, whereas, BHT stimulated this oxidation. The ability to sustain the hepatic mitochondrial GSH level and the hepatic vitamin C and vitamin E levels may represent a crucial

Table 2 The effect of S. chinensis and vitamin E treatment on aflatoxin B1 A-B1 .-induced hepatotoxicity in rats a Plasma GOT Url.

Treatment GSH 5.89 " 0.14 5.62 " 0.15 7.45 " 0.42b,c 6.08 " 0.19c 4.84 " 0.10 5.57 " 0.23b 10.17 " 0.14b,c 5.16 " 0.10d GSH reductase mUrmg wet tissue.

Hepatic tissue GSH S-transferase 77.5 " 3.3 82.2 " 2.9 171.0 " 10.3b,c 74.8 " 3.3d

Malondialdehyde nmolrmg wet tissue.

Control A-B1 S. chinensis q A-B1 Vit.E q A-B1

0.037 " 0.002 0.048 " 0.003b 0.038 " 0.001c 0.047 " 0.003b

17.9 " 1.6 164.3 " 24.5b 46.2 " 5.6c 622.6 " 274.2b,c,d

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a Values are mean "S.E.M., n s 5; b P - 0.05 vs. control; c P - 0.05 vs. A-B1 ; d P - 0.05. vs. S. chinensis q A-B1 . One-way ANOVA followed by Duncan s multiple range test. Reproduced with permission of Pharmacology and Toxicology w57x..

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Table 3 The effect of S. chinensis and vitamin E pre-treatment on Cd-induced hepatotoxicity in rats a Hepatic tissue Malondialdehyde nmolrmg wet tissue. Control Cd S. chinensis qCd Vit. E q Cd
a

GSH

GSH reductase mUrmg wet tissue. 4.90 " 0.26 4.34 " 6.32 7.60 " 0.16b,c 4.55 " 0.24d

GSH S-transferase

Plasma GOT Url.

0.044 " 0.002 0.054 " 0.005 0.042 " 0.003c 0.049 " 0.003

7.04 " 0.22 6.55 " 0.24 7.07 " 0.48 6.03 " 0.59

98.4 " 11.3 54.6 " 11.1b 193.0 " 3.9b,c 91.8 " 9.5c,d

37.4 " 1.8 96.6 " 16.2b 57.5 " 7.4c 85.6 " 7.3b,d

Values are mean "S.E.M., n s 5; b P - 0.05 vs. control; c P - 0.05 vs. Cd; d P - 0.05 vs. S. chinensis q Cd. Data were analysed using one-way ANOVA followed by Duncan s multiple range test. Reproduced from Ip S.P. et al. w57x, with permission of Pharmacology and Toxicology ..

antioxidant property of schisandrin B in protection against CCl 4 hepatotoxicity w58x. The effects of schisandrin B and vitamin E have been compared on ferric chloride Fe 3q .-induced oxidation of erythrocyte membrane lipids in vitro and CCl 4-induced lipid peroxidation in vivo. Vitamin E produced a pro-oxidant effect at 110 M and a biphasic effect at 1.0 mM on the Fe 3q-induced TBARS thiobarbituric acid reactive substances . in human erythrocyte membranes; the pro-oxidant effect, lasting 20 min, was followed by a complete suppression of TBARS antioxidant effect. Schisandrin B 110 M. was capable to inhibit TBARS formation w59x. Pre-treatment with vitamin E 3 mmolrkg per day, p.o. for 3 days. did not protect against CCl 4-induced lipid peroxidation and hepatocellular damage in mice, whereas schisandrin B pre-treatment 0.3 3.0 mmolrkgrday, equivalent to 1.2 12 mgrkg per day, p.o. for 3 days. produced a dose-dependent protective effect on the CCl 4-induced hepatotoxicity Table 4. w59x. The scavenging effects of different structures and configurations of schisandrins on active oxygen radicals have been demonstrated using active oxygen radicals from human polymorphonuclear leukocytes stimulated with phorbol myristate acetate. The scavenging effects of schisandrins depend on the stereoconfigurations, the effect of Sy.-schisandrin B being stronger than that of Rq.-schisandrin and that of schisandrin C stronger than that of schisandrin B w60x. This difference may be explained by the dioxymethyl group that captures electrons facilitating radical attack w60,61x. Surprisingly, the scavenging effect of S, R ".-schisandrin B was stronger than that of either S y.- or Rq.-schisandrin B. The reason for this effect is unknown w61x. Another lignan, schisanhenol 1 mmolrl. was able to scavenge oxygen radicals produced by human neutrophils stimulated by tetradecanoylphorbol acetate. In Fenton reaction system, the inhibitory rate of hydroxyl radical by schisanhenol was 34.4%. In xanthine xanthine oxidase and UV-irradiation of riboflavin systems,

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Table 4 Effects of Schisandrin B S-B. and vitamin E pre-treatment on CCl 4 -induced hepatotoxicity in mice a MDA pmolrmg tissue. Control CCl4 S-B q CCl4 0.5 mmolrkg 3.0 mmolrkg Vit. E q CCl4 3.0 mmolrkg
a

Plasma ALT Url. 14.4 " 0.7 12 526 " 796c 1008.4 " 447.4 (92)e 24.1 " 4.6 (99)e 12 904 " 873

60 " 1 87 " 6b 68 " 3d 56 " 2d 89 " 9


b c

Values are mean " S.E.M., n s 5; P - 0.05; P - 0.001 vs. control; d P - 0.05; e P - 0.001 vs. CCl 4, Students t-test. The italic number in parentheses is the percent of protection. Reproduced from w59x, with permission of Molecular and Cellular Biochemistry..

schisanhenol scavenged superoxidase anion radical by 26.1% and 21.9%, respectively. In all these systems, schisanhenol was more potent than vitamin E w55x. 3.3. Anticarcinogenic effect Benzow axpyrenes BPs. are well known carcinogens, widely distributed in the environment w62,63x. The elimination of these polycyclic aromatic hydrocarbons from the body requires their conversion to water-soluble metabolites w64x. 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 of vegetables like sprouts, cabbages, broccoli, alfalfa and fibres may reduce the incidence of stomach and colon cancers w65,66x. The effect of deoxyschisandrin, -schisandrin, schisandrin C, gomisin A, B and C orally given 100 200 mgrkg per day for 3 days. to male rats has been studied in vitro on liver microsomal monooxygenases and epoxide hydrolase. Among these compounds, schisandrin B, schisandrin C, gomisin A and biphenyl dimethyl dicarboxylate BDD., a synthetic derivative of gomisin C, significantly increased rat liver cytochrome P-450 concentration, NADPH-cytochrome C reductase, benzophetamine and aminopyrene demethylase activities. Four compounds -schizandrin, schizandrin C, gomisin A and BDD. also markedly stimulated proliferation of smooth endoplasmic reticulum of liver cells from rats treated with schisandrin B w32x. It is known that phenobarbital PB; 80 100 mgrkg, 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 of adrenocorticosteroids. Schisandrin B, schisandrin C, gomisin A, and BDD inhibited

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aminopyrene demethylase activity of liver microsomes in a similar manner. Dual induction by gomisin A 100 200 mgrkg, p.o.. and PB decreased the mutagenicity of 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 mutagenic metabolites w31,32x. 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 of purified EH to the Salmonella mutagenecity Ames test reduces BP mutagenicity by 30 50%. Total BP metabolism was significantly increased 1.6-fold. in the S. chinensis 1 mgrml. group. Phenol II formation relative to total metabolites was significantly increased in the S. chinensis group as compared to the control group w67x. Both 7-ethoxycoumarin O-de-ethylase ECD. and aryl hydrocarbon hydroxylase AHH. activities were also increased significantly w68x. The binding of aflatoxin to DNA was diminished by S. chinensis w68x. The effect of gomisin A on hepatocarcinogenesis caused by 3-methyl-4-dimethylaminoazobenzene 3-MeDAB. in male Donryu rats has been investigated. Gomisin A 30 mgrkg 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 hepatic altered foci such as the clear cell and basophilic cell type foci in the early stages w69,70x. Gomisin A 30 mgrkg per day, p.o.. decreased the concentration of 3-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 detected either in the liver or the bile, but the proportion of diploid nuclei, though diminished, remained high. It seems that gomisin A improved liver function by reversing abnormal ploidization w71x. Gomisin A 0.03% in diet for 10 weeks. inhibited the development of preneoplasic liver lesions. In fact, gomisin A inhibited the level of GST-P, and the number and size of GST-P positive foci increased in the liver after treatment with 3-MeDAB. Moreover, although the population of diploid nuclei was increased and that of tetraploid nuclei was decreased by pre-treatment with 3-MeDAB, gomisin A reverted this effect to near the normal ploidy pattern w71x. This suggests that gomisin A may inhibit the hepatocarcinogenesis induced by 3-MeDAB by enhancing the excretion of the carcinogen from the liver and by reversing the normal cytokinesis w72x. Gomisin A 30 mgrkg, p.o. daily for 5 weeks. inhibited the increase in serum bile acid concentration induced by the administration of other tumour promotors such as deoxycholic acid DCA. w73x. Although hepatocarcinogenesis has been reported to be promoted by exogenous administration of bile acids, the relation of endogenous bile acids to hepatocarcinogenesis is not completely understood w74,75x. The oral administration of gomisin A 30 mgrkg. significantly inhibited the increase 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-tetradecanoylphorbol-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.. Reproduced from Yasukawa K. et al. w77x 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 A may be based on improving metabolism of bile acids w76x. Application 1 grear. of 12-O-tetradecanoylphorbol-13-acetate TPA., a tumour-promoting agent, to mice induces inflammation. Local application 0.6 mgrear. of gomisin A inhibited TPA-induced inflammation in mice. Also gomisin J and schisandrin C inhibited the inflammation induced by TPA in mice. The ED50 of these compounds ranged between 1.4 and 4.4 mol, gomisin A showing the strongest inhibitory effect. Furthermore, at 5 molrmouse, it markedly suppressed the promotion effect of TPA 2.5 grmouse. on skin tumour formation in mice following initiation with 7,12-dimethylbenzw axantracene 50 grmouse. w77x Fig. 1.. 3.4. Effects on physical performance A number of Russian reports indicate that S. chinensis is able to counteract the effect of fatigue, increase endurance, and improve the physical performance of sportsmen w78x, but no controlled studies were done in the western world until the late 1980s. To validate the hypothesis that this plant can improve the physical recovery, in a first series of trials, 50 g of S. chinensis fructus dried extract were administered 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 mrmin, or a 5-min gallop at a speed of 700 mrmin w79x. S. chinensis counteracted significantly the anticipatory 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 " S.E.M., n s 5; U P - 0.05; UU P - 0.01 vs. control saline.; Students t- test. Reproduced with permission of Fitoterapia w80x..

Also, the seric lactic acid was reduced and the plasmatic glucose increased in S. chinensis treated horses. Interestingly, the horses treated with S. chinensis were able to complete the race at an average of 1.8 s faster, indicating an improvement in the physical performance of the horses w79 81x. On a second series of studies, the effect S. chinensis fructus dried extract single dose of 6 g, p.o.. was studied in race and spring horses in order to asses whether the type and intensity of the exercise is critical for the effect w80x. S. chinensis was capable of reducing significantly the heart rate and respiratory frequency at different time intervals after the trial, particularly in race horses. Plasmatic glucose concentration increased significantly in both types of exercise. The plasmatic concentration of lactic acid was reduced in S. chinensis treated horses as compared to the controls, this decrease being again more evident in race horses w80x 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 of the horses physical performance w82x. As it is known that S. chinensis decreases the hepatic transaminases activity w20,21x, the hypothesis was made that S. chinensis could lower the seric levels of transaminases and, thus, reverse the impaired performance of horses w83x. Indeed, an association between poor performance and high seric levels of hepatic enzymes in sport horses was shown w82x. Moreover, training leads to an increment of creatinine phosphokinase CPK. w82x, 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. Values are mean " S.E.M., n s 12; U P - 0.05; UU P - 0.01 vs. control saline., Student s t- test. Reproduced with permission of Phytomedicine w83x..

muscle skeletal damage, with increase in seric level of CPK and transaminases. Poorly performing sport horses with long lasting high levels of -glutamyltransferase GGT., GOT and CPK were orally administered 3 g of S. chinensis dried extract, during 14 days. S. chinensis reduced the levels of GGT and GOT in the serum at day 7 and 14 after administration w83x. Surprisingly, the CPK levels were also reduced by day 7 and 14 after administration indicating that these animals presented a muscular damage that could be reverted with S. chinensis w83x Fig. 3.. Simultaneously, Ko et al .w84x reported a protective effect against physical exercise-induced muscle damage and a myocardial protective effect in rats pretreated with a lignan enriched extract of S. chinensis fruit 0.8 grkg day, p.o for 3 days. w85x. Protection was associated with a significant enhancement in the hepatic antioxidant status, as assessed by GSH and MDA concentrations w84 87x. Fig. 4 summarises the possible effects of S. chinensis on the metabolic pathways during maximum physical effort. S. chinensis reduced the hepatic damage leading to a decrease in transaminases GOT, GGT.. As a consequence, gluconeogenesis characterised 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 and reduced the seric lactate levels, probably by an antioxidant effect. 3.5. Acti ity on central ner ous system (CNS) Activation of the CNS by S. chinensis has been evidenced during electroencephalographic examination. In particular, S. chinensis, antagonised the effect of substances supressing the CNS such as barbiturates, chloral hydrate, aminazine, and halothane w88,89x. Schisandra lignans 1 5 mgrkg, i.p.. antagonised the effect of hexenal and chloral hydrate in rats w88x. The CNS activating effect of S. chinensis was observed even under the presence of antagonist to dopamine receptors DA 2

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

w88x. However, potentiation of phenobarbital sleeping time w90x would indicate that S. chinensis has a depressing action on the CNS. This could be explained by the presence in the tested extracts of different concentrations of schisandrol A which is known to prolongue the sleeping time induced by phenobarbital and decrease the spontaneous motor activity in mice w91x. The cholinergic system is also significantly influenced by S. chinensis. A crude petrol ether fruit extract 10 30 mgrkg, p.o.. decreased the convulsant threshold and potentiated the antidiuretic action of nicotine and potentiated the excitatory action of carbachol on the intestine in rats. This petrol ether extract potentiated the action of reserpine only at higher doses 1.5 grkg. w92,93x. This extract affected mostly the cholinergic system, with a biphasic response. At dose of 280 mgrkg p.o., it showed an indirect nicotinomimetic action potentiating the carbachol intestinal motility, whereas, a higher dose 840 mgrkg, p.o.. had a cholinolytic effect w93x. Schisanhenol and schisandrin B have been shown to protect peroxidative damage of aging and ischemic rat brain w94x. Schisanhenol and schisandrin 10y4 M. completely inhibited the swelling and disintegration of brain mitochondria, as well as the reduction of brain membrane fluidity induced by Fe2q . cysteine w94x. In vitro experiments on mitochondria and membrane from ischemic and reperfusion brain indicate that both lignans significantly inhibited production of MDA and loss of ATPase activity induced by reoxygenation following anoxia. Oral administration 150 mgrkg. of schisanhenol or schisandrin B induced increase of cytosol glutathione-peroxidase of brain in mice under the condition of reoxygenation following anoxia w94x. Human intellectual activity can be enhanced by S. chinensis so that work efficiency is also increased. Schisandrin 5 10 mgrday, p.o.. improved certain activities requiring concentration, fine coordination, sensitivity and endurance, as

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demonstrated in healthy young male adults in the following experiments: insertion of 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 the discriminating ability of skin receptors w95x. 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 the mean value of maximum plasma concentration was 96.1 " 14.1 ngrml w96x. Schisandrin was metabolised by rat liver microsomes to give three main first phasic metabolites. Several oxidation routes appear to be involved: hydroxylation of an alkyl substituent at first and then demethylation of the -OCH 3 groups on the aromatic rings. The metabolites were found in urine and bile of rats w97x. After oral administration of 10 mgrkg to rats, the maximum serum concentration of gomisin A 1446.1 " 131.8 ngrml. was reached at 15 30 min, over 80% being bound to serum proteins w98 100x. The rapid metabolisation of gomisin A, has been attributed to a first pass effect, producing demethylated metabolites w98x and glucuronic and arylsulfate conjugates w101x. After oral administration to rats of schisandrol A, this compound was absorbed from 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 the elimination phase of 42 min. Schisandrol A was detected in urine 1 h after oral administration w10x. Five minutes after i.v. administration, high levels of schisandrol A were found in the lungs, moderate amounts in the liver, heart, brain, and kidneys and low amounts in the ileum and spleen. In the brain, the higher amounts were found in the hypothalamus, corpus striatum and hippocampus, and moderate amounts in the cerebral cortex and cerebellum. These differences may be relevant to the neuroleptic and anticonvulsant properties of schisandrol A w102x. 3.7. Toxicology The acute toxicity for S. chinensis fructus dried extract 4:1., standardised to a concentration of 2% of schisandrin was low LD50 ) 21 grkg, p.o.. in rats w103x. Other authors reported the absence of lethal effects following intragastric administration of 5 grkg to mice w95x. The oral and i.p. LD50 values in mice for a petroleum ether extract of S. chinensis fruit 10% schisandrins . were 10.5 and 4.4 grkg, respectively. The oral LD50 of a petroleum ether extract standardised to 40% of schisandrins was 2.8 grkg in mice w93x. An ethanol extract of S. chinensis orally given to mice at doses of 0.6 and 1.2 grkg 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 organs were not significantly altered w95x. The p.o. toxicity of S. chinensis fructus dried extract standardised to a minimum

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of 2% of schisandrins was studied in Landrace piglets for 90 days at daily doses of 0.07, 0.36 and 0.72 grkg. The body weight and food intake, were not affected during the whole experimental period. No changes in the red blood cells, white blood cells, haemoglobin and hematocrit were found. The glycemia, urea and protein concentrations did not show any significant variations with respect to the control. Triglycerides, GOT and GGT were also not modified by S. chinensis administration. In the tissues examined liver, heart, kidneys, intestine, lungs, spleen and gonads. no toxic effect was observed w103x. In other studies w103x and using the same standardised dried extract of S. chinensis fruit 0.105 0.5 grkg per day, p.o.., the potential toxic effects on the reproductive function was studied in rats and mice. No foetotoxicity in these experimental models was found. No changes in the implantation efficiency or other investigated parameters were observed. Information on the toxicity of S. chinensis lignans is very limited. With schisandrin B, no death was observed following a single intragastric dose of 2 grkg to rats. Intragastric dosing of 200 mg for 30 days caused no significant effect on body weight, haemoglobin and histology of the major organs in mice w95x. Schisandrin B, given intragastrically to dogs at 10 mgrkg daily for 4 weeks, did not affect appetite, body weight, blood picture, liver and kidney functions, as well as the histology of the liver w95x.

4. Conclusions S. chinensis has been used in traditional Chinese medicine for thousands of years. In the last decades, the pharmacology and chemistry of this drug has been extensively studied. Much evidence shows that S. chinensis and its dibenzocyclootene lignans may act on the function of the liver. The findings are useful for further understanding the pharmacological basis of S. chinensis as an antioxidant, anticancer, tonic and antiaging drug. Furthermore, S. chinensis might also be useful in the treatment of other diseases related to oxygen free radical injury and metabolic 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 the case of BDD w104,105x. Recently, halogenated gomisin J derivatives have been shown to possess anti-human immunodeficiency virus HIV. activities, by inhibiting the activity of the enzyme reverse transcriptase as well as expressing cytoprotective activity in HIV-1-infected H9 cells. w106x Nevertheless, despite the numerous pharmacological studies available, clinical trials 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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Gothenburg, Sweden. The authors wish to thank Christina Gomzi for secretarial assistance.

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Fitoterapia 70 1999. 472 474

A new acyclic monoterpene glucoside from the capitula of Tagetes patula


S.N. GargU , Reena Charles, Sushil Kumar
Department 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-glucopyranoside (1) was isolated from Tagetes patula flowers in addition to known compounds helenien, xanthophyll, patuletin and patuletrin. 1999 Elsevier Science B.V. All rights reserved.
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, is widely cultivated as a garden plant in temperate and semitemperate regions of Asia, Europe and America. This ornamental species is grown all over India up to the height of approximately 2000 m w1x . Tagetes patula is a source of commercially important carotene compounds, helenien, xanthophyll, and essential oil. Helenien is used in pharmaceuticals especially in eye care formulations. Xanthophyll is used as a direct and indirect colouring. Flavonoids have also been characterised from the capitula of T. patula w2 5x. Here we report on the isolation of a new acyclic monoterpene glucoside, 2-methyl-6-methylen-2,7-octadiene 1-O--D-glucopyranoside (1).
U

Corresponding author. Tel.: q91-342676; fax: q91-522342666. E-mail address: root@cimap.sirnetd.ernet.in S.N. Garg.

0367-326Xr99r$ - 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 0 4 4 - 1

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2. 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 3 MeOH mixtures provided helenien 800 mg., xanthophyll 100 mg., patuletin 60 mg., patuletrin 40 mg. and the new compound 1 30 mg.. 2-Methyl-6-methylene-2,7-octadiene 1-O--D-glucopyranoside (1). Viscous oil w x D 21.5 c 0.25, MeOH.; IR bands Neat. 3390, 1655, 1630, 1590, 990 and 900 cmy1 ; 1 H-NMR 400 MHz, Py-d 5 :.: 6.28 1H, dd, J 18 and 10 Hz, H-7., 5.41 1H, br t , J 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., 2.10 2.24 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, H-6b., 3.2 3.60 4H, overlapping signals, H-2 to H-5.; 13 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 C-5., 26.2 C-4., 16.2 C-10., El-MS 70 eV. m r z : 314 Mq. 10., 152 85..

3. Results and discussion The methanolic extract of the capitula of T. patula, on chromatography over silica gel, afforded a new compound 1, C 16 H 26 O6 , IR absorption bands at 3390 OH., 1665, 1630, 1590, 990 and 900 cmy1 for olefinic double bands. 1 H-NMR spectrum revealed the presence of six olefinic protons including an ABX system of a vinyl group at 6.28 dd, J s 18 and 10 Hz., 5.28 dd, J s 18 and 1.3 Hz., and 5.15 dd, J s 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.. In addition, one methyl proton at 1.62 as a singlet, two oxymethylene proton at 3.95 d, J s 12 Hz. and seven sugar protons were also observed. These 1 H-NMR signals were in agreement with the attribution of structure 1 to the compound. The 1 H- and 13 C-NMR values see Section 2. were also in agreement with those reported for similar compounds w6 10x. The linkage of the sugar unit with oxymethylene carbon C 1 was confirmed by the

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C 1 carbon downfield shift by 7 ppm in comparison to oxymethylene carbon of 8-hydroxy-3,7-dimethyl-1,6-octadiene. The anomeric proton in position was indicated by the proton doublet at 4.20 1H, d, J s 8 Hz. supported by 13 C-NMR signal 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 MHz 13 C-NMR and Mass spectra of the compound. Thanks are also due to the Botany Department, Central Institute of Medicinal and Aromatic Plants, Lucknow for establishing the identity of plant material.

References
w1x The wealth of India, raw materials, vol X. New Delhi: CSIR, 1976:109 111. w2x Rodriguez E, Mabry JJ. In: Heywood VH, Harborne JB, Turner BL, editors. The biology and chemistry of the compositae. London: Academic Press, 1977:786 797. w3x Bhardwaj DK, Bisht MS, Jain SC, Mehta CK, Sharma GS. Phytochemistry 1980;19:713. w4x Tripathi AK, Paliwal MK, Singh J. J. Indian Chem. Soc. 1991;68:674. w5x Ivancheva S, Zdranvekova M. Fitoterapia 1993;64:555. w6x Otsuka H. Phytochemistry 1994;37:461. w7x Gering B, Wichtl M. J. Nat. Prod. 1987;50:1048. w8x Takeda Y, Takechi A, Masuda T, Otsuka H. Planta Med. 1998;64. w9x Byers JA, Schlyter F, Birgesson G, Francke W. Experientia 1990;46:78. w10x Rucker G, Mayer R, Mans D. J. Nat. Prod. 1987;50:287.

Fitoterapia 70 1999. 475 477

A novel isoflavone from the stems of Ageratum conyzoides


R.N. YadavaU , Saurabh Kumar
Natural 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 and distributed throughout India up to 1500 m, is useful in fever w1x. The ayurvedic system of medicine describes that the roots of this plant possess anthelmintic and antidysenteric properties w2,3x. The isolation of a new isoflavonoid, 5,7,2,4-tetrahydroxy-6,3-di-3,3-dimethylallyl.-isoflavone 5-O--L-rhamnopyranosyl-1 4.--Lrhamnopyranoside 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.: q91-7582-26465; fax: q91-7582-23236.

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Department of Botany of this University. A voucher specimen No. X. has been deposited in the Natural Product Laboratory, Department of Chemistry, Dr. Hari Singh Gour University, Sagar M.P.., India. 2.2. Extraction and isolation Air-dried and powdered stems 5 kg. were extracted with 95% MeOH. The concentrated extract was successively partitioned with petrol 40 60C., benzene, CHCl 3 , acetone, EtOAc and MeOH. The concentrated acetone-soluble part 56 g. was Si-gel CC eluting with benzene EtOAc mixtures. Elution with benzene EtOAc 3:7 yielded compound 1 150 mg. as light brown needles from Et 2 O, mp 174 175C, C 37 H 46 O 14 ; UVmax MeOH.: 214 log 4.60., 267 4.40.; qNaOMe. 224 sh., 278, 344; qNaOAc. 274, 348; qAlCl 3 . 214, 269 nm; IRmax KBr.: 3365, 1348 cmy1 . Acetate: 1 H-NMR 300 MHz, CDCl 3 .: 6.22 1H, s, H-2., 6.61 1H, s, H-8., 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 ., 2.32 2.48 9H, s, phenolic acetoxyls. sugar region: 2.02 2.14 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., 4.81 5.57 10H, m, sugar protons., 0.91 and 1.14 each 3H, d, J 6 Hz, Me-6, Me-6 rham.; 13 C-NMR 400 MHz, DMSO-d6 .: 152.8 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., 21.4 21.5 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 C-2., 71.3 C-3., 71.1 C-4., 68.3 C-5., 17.1 C-6.; EIMS mrz rel. int.%.: wMqx absent., 423 wMq- acetylated di-rhamnoside.x 14., 422 wMqx 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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Acid hydrolysis 10% HCl, 2 h at 100C. of compound 1 yielded 5,7,2,4-tetrahydroxy-6,3-di-3,3-dimethylallyl.-isoflavone w4x and rhamnose.

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

References
w1x Kirtikar KR, Basu BD, Indian medicinal plants, II. Allahabad: Lalit Mohan Basu and Company Publication, 1935:1330. w2x The Wealth of India. A dictionary of raw materials and industrial products, I. New Delhi: CSIR Publication, 1948:40. w3x Chopra RN, Nayar SL, Chopra IC. Glossary of Indian Medicinal Plants. New Delhi: CSIR Publication, 1956:9. w4x Tahara V, Inyham JL, Nakahara S, Mizutani J, Harborne JB. Phytochemistry 1984;23:1889.

Fitoterapia 70 1999. 478 483

Flavone glycosides from Mentha longifolia


M. Sharaf U , M.A. El-Ansari, N.A.M. Saleh
Phytochemistry 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 and 7-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-glucoside 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 Little has been reported on the 13 C-NMR of the glycosylation at ring-B 3 andror 4. of flavones. The available reports are those dealing with the 13 C-NMR of luteolin 3-glucoside w1x, 7-neohesperidoside-4-sophoroside, 7-neohesperidoside4-glucoside and 7,4-dineohesperidoside w2x, in which the chemical shift values reported for C-3 and C-4, under glycosylation, showed unexpected values in comparison with C-7 when glycosylated w3,4x. To the best of our knowledge nothing has been reported on the 13 C-NMR of 3- or 3,5-glycosylation of 5,7,3,4,5-pentahydroxyflavone. The present communication describes the isolation and structural elucidation of
U

Corresponding author.

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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 a polyamide column. Purification of the compounds was achieved by a combination of silica gel TLC and Sephadex LH-20. The flavonoids 13 were isolated. Acid hydrolysis of 1 released glucose, rhamnose and the new aglycone, tricetin 7-methyl ether 1a. which was identified by UV and 1 H-NMR. The UV spectral data of 1 with diagnostic shift reagents w5x indicated a flavone substituted at 7-position, a free 4-hydroxyl group and absence of a free ortho-dihydroxy pattern at B-ring. The 1 H-NMR spectrum of 1 showed that it is a tricetin diglycoside on the 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 one rhamnose unit in 1 w5x. 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 glucose w6x. Furthermore, the chemical shifts 5.20, 5.10. indicated that both glucose and rhamnose moieties are directly attached to the aglycone w7x. The 1 H-NMR spectrum of 1 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 a singlet at 6.95 and the methoxy group as a singlet at 4.10. The 13 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.. The 13 C-NMR shifts of the aglycone part of 1 correspond well to the shifts of tricetin w3x, the only difference being a downfield shift of the signal assigned to C-7 by

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approximately 3.7 ppm 164.0 167.7. and upfield shift of the ortho-related carbons see Table 1., confirming the location of the OCH 3 group at C-7. The 13 C-NMR spectrum of 1 showed unexpected values for carbons 3, 4 and 5, which are in agreement with a previous report w2x. 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 assigned to C-3, C-4 and C-5, the upfield shift of C-3 and C-5 being caused by glycosylation, and the downfield shift of C-4 as being ortho related carbon to C-3 and C-5. From the above data compound 1 is identified as tricetin 7-O-methylether 3-O--D-glucoside 5-O--L-rhamnoside. Compound 2 gave glucose, rhamnose and tricetin after acid hydrolysis. The UV spectra indicated a flavone with a free 5,7,4-trihydroxy pattern and absence of a free o-dihydroxy pattern. The 1 H-NMR and 13 C-NMR spectra of 2 showed a very close similarity with those of 1, the only differences being the elimination of the signals assigned for the OCH 3 from the spectra of 2, while C-3, C-4 and C-5 resonated 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-glucoside 5-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 of 3. The UV spectral data of 3 indicated a flavone with 7,4-dihydroxyl groups, and the presence 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 two rhamnose methyl groups at 0.50 and 0.70 ppm in the 1 H-NMR spectrum. The spectrum 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 expected signals for H-3, H-6 and H-8. The 13 C-NMR spectrum of 3 Table 1. showed the expected signals for tricetin substituted in 3-position indicated by the presence of a signal at 151 ppm, and a free OH group at C-4 and C-5 indicated by the presence of a signal at 147.00 ppm assigned for both carbons. Comparison of the rhamnose carbon chemical shifts in the spectrum of 3 and those of unsubstituted methyl-4-O-glycosylated--Lrhamnoside showed similarities. Thus, the chemical shift for the carbon atom linked to the second sugar in the case of 2-O-glycosylation is at 79.0, that of a 3-O-glycosylation is at 78.8 and in the case of a 4-O-glycosylation is at 80.8 w8x. The corresponding atom in 3 resonates at 81.0 indicating that rhamnose must be linked 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 the 13 C-NMR assignments of compounds 1 3 with those reported for tricetin w3x, luteolin 3-glucoside w1x, and luteolin 7-O-neohesperidoside 4-Osophoroside w2x 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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Table 1 13 C-NMR of tricetin (I), luteolin 3-glucoside (II), luteolin 7-neohesperidoside 4-sophoroside (III) and compounds 1, 2, and 3 a Carbon 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 1-G 2 3 4 5 6 1-R 2 3 4 5 6 OCH3
a 13

I 164.2 103.2 181.6 161.6 99.0 164.2 93.9 157.9 104.0 120.9 106.0 146.5 137.9 146.5 106.0

II 163.2 103.9 181.7 161.5 99.0 163.5 94.2 157.5 103.4 122.1 115.2 145.7 150.9 116.6 122.1 102.4 73.5 77.4 70.3 76.3 61.2

III 163.8 104.4 181.8 161.0 100.6 163.0 94.2 157.1 105.7 124.9 113.9 147.3 148.7 116.7 118.4 100.7 101.6 79.7 74.7 76.0 76.9 70.4 72.0 77.0 77.1 60.7 60.4 98.1 70.0 70.6 73.1 68.2 17.7

1 164.83 104.81 182.51 161.42 98.49 167.70 95.29 157.53 104.81 120.43 105.99 148.71 148.71 148.71 105.99 101.04 72.82 77.56 79.91 77.15 61.12 98.50 70.23 70.80 72.82 68.88 18.50 56.82

2 164.43 103.07 182.07 161.39 99.41 164.03 93.59 157.09 103.65 121.42 107.5 146.20 146.20 146.20 107.51 102.82 72.69 78.36 70.65 78.36 61.01 99.41 70.51 70.65 73.62 69.23 19.0

3 164.19 103.9 182.05 161.42 101.0 164.03 94.0 157.09 104.0 121.57 109.50 151.0 147.0 147.0 109.0

102.50 101.50 70.50 70.10 70.50 70.10 81.00 72.50 69.00 69.00 18.00 18.50

C-NMR of compounds I, II and III were obtained from Markham and Chari w4x, Markham w1x w2x, respectively. G, glucose; R, rhamnose. and Osterdahl

signal is approximately 1.5 ppm upfield shift, accompanied by downfield shift of approximately 2 ppm in the para related carbon w4x. In our study, glycosylation at 3 andror 5 showed shifts qualitatively similar but quantitatively different. These

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M. Sharaf et al. r Fitoterapia 70 (1999) 478 483

results are in agreement with previous data for 3- and 4-substituted flavones w1,2x. 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 that flavones with a trisubstituted B-ring have not been reported before in the family Lamiaceae.

3. Experimental 3.1. Plant material M. longifolia aerial parts, collected from El-Madina, Saudi Arabia, in March 1997 and authenticated by Prof. L. Boulos. A voucher specimen is deposited in the Herbarium of NRC, Cairo. 3.2. Extraction and isolation 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 GF254 Merck. eluting with CHCl 3 MeOH H 2 O 65:45:12, and further purified using Sephadex LH-20 eluting with MeOH. 3.3. Acid hydrolysis Glycosides 1 3 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; qNaOMe 262, 417; qAlCl 3 250, 270, 350; qAlCl 3 q HCl 250, 270, 350; qNaOAc 250, 270, 350, 425; qNaOAc q H 3 BO 3 , 250, 270, 350 nm; 1 H-NMR 270 MHz, DMSO-d6 .: 7.30 2H, s, H-2,6., 6.95 1H, 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 ., 4.00 3.60 8H, m, sugar protons, hidden by hydroxyl groups., 4.10 3H, s, OCH 3 ., 1.1 3H, d, J 5.5 Hz, Me-rha.; 13 C-NMR see Table 1.. 3.5. Tricetin 7-methyl ether 1a. UVmax MeOH.: 255, 265sh, 349; qNaOMe 263, 299sh, 394; qAlCl 3 272, 296sh, 331, 434; qAlCl 3 q HCl 272, 295, 359, 390; qNaOAc 295, 267sh, 366, 403; qNaOAc q H 3 BO 3 , 258, 371 nm; 1 H-NMR 270 MHz, DMSO-d6 .: 7.28 2H, s, 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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3.6. Tricetin 3 -O--D-glucoside 5-O--L-rhamnoside 2. UVmax MeOH.: 255, 270, 347; qNaOMe 267, 325sh, 405; qAlCl 3 267, 300sh, 380; qAlCl 3 q HCl 260, 295sh, 353; qNaOAc 273, 315sh, 380; qNaOAc q H 3 BO 3 262, 375 nm; 1 H-NMR 270 MHz, DMSO-d6 .: 7.40 2H, s, H-2,6., 6.90 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 hydroxyl groups., 0.70 3H, d, J 5.5 Hz, Me-rha.; 13 C-NMR see Table 1.. 3.7. Tricetin 3 -di-O--L-rhamnoside 3. UVmax MeOH.: 255, 270, 347; qNaOMe 267, 325sh, 405; qAlCl 3 , 267, 300sh, 380; qAlCl 3 q HCl 260, 295sh, 353; qNaOAc, 273, 315sh, 380; qNaOAc q H 3 BO 3 262, 375 nm; 1 H-NMR 270 MHz, DMSO-d6 .: 7.40 2H, s, H-2,6., 6.90 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 ., 4.70 4.20 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
w1x Markham KR, Ternai B, Stanly R, Geiger H, Mabry TJ. Tetrahedron 1978;34:1389. w2x Osterdahl BO. Acta Chem Scand 1979;B33:119. w3x Markham KR, Chari VM. The flavonoids: advances in research, In: Harborne JB, Mabry TJ, editors. Chapman and Hall, London, 1982:spectrum No. 38. w4x Markham KR, Chari VM. The flavonoids: advances in research, In: Harborne JB, Mabry TJ, editors. Chapman and Hall, London, 1982:39. w5x Mabry TJ, Markham KR, Thomas MB. The systematic identification of flavonoids. Berlin: Springer-Verlag, 1970:269. w6x Kamerling JP, Bie MJA, Vliegenthart JFG. Tetrahedron 1972;28:3037. w7x Osterdahl BO, Lindberg G. Acta Chem Scand 1979;B31:293. w8x Liptak D, Nansai P, Neszmelyi A, Wagner H. Tetrahedron 1980;36:1261.

Fitoterapia 70 1999. 484 492

Effect of Semecarpus anacardium nut extract against aflatoxin B 1-induced hepatocellular carcinoma
B. 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 1-induced experimental hepatocellular carcinoma HC. was investigated. The adverse changes induced by aflatoxin B 1 were reversed to near normal and the histological pattern was almost normal in treated rats. These results suggest that S. anacardium nut extract has potential anticarcinogenic activity against aflatoxin B 1-mediated HC. 1999 Published by Elsevier Science B.V. All rights reserved.
Keywords: Semecarpus anacardium; Antitumour activity; Hepatocellular carcinoma; Aflatoxin B1

1. Introduction How cancer develops from apparently normal tissue is one of the unresolved biological problems of our time. Hepatocellular carcinoma HC., a fatal malignancy, represents 4% of all malignant tumours and is the seventh most common cancer in man worldwide w1x. In China, where the predominance of HC is high, the traditional medicinal herbs are used as an effective treatment against primary liver cancers w2x. Semecarpus anacardium L. Anacardiaceae. is a deciduous tree, distributed in the sub-Himalayan tract and in tropical parts of India w3x. It is commonly known as
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Corresponding author. Tel.: q91-44-4925548; fax: q91-44-4926709.

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marking nut in English and has high priority and applicability in indigenous systems of medicine. Many compounds, mainly biflavonoids w4x, phenolics w5x and glycosides w6x have been identified as constituents of S. anacardium nut extract. Many medicinal properties such as antimicrobial, anti-inflammatory, anthelminthic and anti-amoebic have been attributed to the nut of the plant w7x. It is claimed to be effective against a variety of ailments, such as lepranodules, warts and rheumatism w8x. In traditional medicine, it is highly valued for the treatment of tumours and malignant growth. Recent studies carried out on an Ayurveda marking nut preparation have also shown promising results in the treatment of cancers of the oesophagus, urinary bladder, liver, and leukaemia w9x. Earlier studies from our laboratory have proved the anticancer and hepatoprotective activity of S. anacardium nut milk extract against aflatoxin B 1 AFB1 .-induced HC in rats w10x and established its protective role on deranged cell membranes in AFB1-induced HC w11x. The aim of this work was to further evaluate the antitumour activity of S. anacardium nut extract, correlating biochemical and histological changes in AFB1induced 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 formulation was prepared according to a recipe of the Formulary of Siddha Medicine w12x 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 till dehydrated w12x. 2.2. Animals Wistar male rats weighing 100 120 g, obtained from Tamilnadu Veterinary and Animal 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 lightrdark cycle. and fed with pelleted diet Gold Mohur rat feed, Mrs. Hindustan Lever Limited, Mumbai. and water ad libitum. 2.3. Experimental protocol The animals were divided into four groups of six animals each:

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Group I: Group II: Group III:

Group IV:

Normal control, received a single intraperitoneal i.p.. dose of dimethyl sulphoxide DMSO; 0.5 ml. HC was induced by single dose of AFB1 Sigma. in DMSO 2 mgrkg, i.p.. w13x HC-induced animals as in Group II. were treated with S. anacardium nut extract 200 mgrkgrday for 14 days. in sunflower oil 2.5 mlrkg. by gavage Animals received only the same dosage of S. anacardium nut extract as Group III

2.4. Biochemical analysis At the end of the experimental period, all animals were fasted for 18 h before being killed by cervical decapitation. Liver and kidney tissues were immediately excised, weighed and then homogenized in Tris HCl buffer 0.1 M pH 7.4.. The tissue homogenates were used for the estimation of proteins by the method of Lowry et al. w14x. The nucleic acids from the tissues were extracted by the method of Schneider w15x with trichloroacetic acid TCA. and DNA was estimated by the method of Burton w16x and RNA according to Rawal et al. w17x. 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 the groups. The Students Neumann Keul test was used to compare Group I with Groups II, III and IV, and Group II with Group III. 2.6. Tissue processing Immediately after killing, liver and kidneys were rapidly excised, serially sectioned and macroscopically examined. Microscopic samples from liver were taken from right portion of the median lobe since this portion of the rat liver has been found to be a site of more apparent histological lesions w18x. The tissues were fixed in 10% buffered formalin. Consecutive sections were stained with haemotoxylin and eosin H & E..

3. Results 3.1. Body and li er weights Body and liver weights were significantly decreased in AFB1-induced HC conditions Group II.. The rat body wt. regularly increased in controls and progressively declined in tumour hosts. These changes were reversed to near normal in S.

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Table 1 Effect of Semecarpus anacardium nut extract on aflatoxin B1 -induced hepatocellular carcinoma in rats a Parameters mgrg wet tissue. Liver Group I Group II Group III Group IV F-ratio

DNA 6.36 " 0.48 RNA 4.10 " 0.36 Protein 159.32 " 11.63

9.41 " 0.89aU 5.35 " 0.54aUU 96.52 " 9.63aU

7.02 " 0.68bU 6.50 " 0.64 20.86 4.52 " 0.47bUUU 4.21 " 0.41 7.19 151.37 " 14.12bU 155.49 " 14.47 26.98 12.94 7.72 8.23

Kidney

DNA 4.99 " 0.46 6.86 " 0.68aU 5.29 " 0.53bUU 5.07 " 0.48 UU RNA 3.14 " 0.28 4.09 " 0.42a 3.48 " 0.32bUUU 3.26 " 0.31 Protein 138.16 " 10.05 110.16 " 10.21aUU 132.83 " 10.24bUU 136.29 " 9.97

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 mgrkgrday = 14 days.; group IV: control rats receiving only S. anacardium extract as in group III. Comparisons were made: a. group I vs. groups II, III and IV; b. group II vs. group III. Statistical significance: U P - 0.001, UU P - 0.01, UUU P - 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 significant alteration of nucleic acids was observed in extract control animals Group IV.. In contrast to nucleic acids, the protein content was significantly decreased in HC-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 protein content. 3.3. Gross morphology In Group II animals, the liver of three rats showed marked congestion while in the remaining mottling was evident. Kidney did not reveal any gross change abnormalities except in two rats where the cortical surfaces revealed congestion. In Group III animals, although the liver showed a slight congestion, the architecture was almost normal. Kidneys from these animals did not reveal any appreciable change when compared with normal controls. 3.4. Histology Histologically, the liver from control animals revealed a normal architecture

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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.. There was a slight increase in Kupffer cell activity. In one rat, hepatocytomegaly was prominent in a few hepatocytes. Single cell necrosis was also found in these conditions. In two rats there was diffuse hydropic degeneration of the hepatocytes. Attempted nodular formation was seen in one rat. Kupffer cell hyperplasia and total necrosis of hepatocytes with mononuclear cell infiltration were also observed. The liver from Group III animals showed almost normal architecture Fig. 3. and no abnormality was detected. One rat showed mild congestion of the central vein. In two animals, hepatocytes revealed hydropic degenerative changes along with Kupffer cell activity. Rat liver and kidney excised from S. anacardium control Group IV. animals showed no appreciable changes or histological abnormalities. Microscopically, kidneys from Group II animals revealed congestion of the glomerular tuft and granular degenerative changes in the proximal convoluted tubule. In three cases, eosinophilic, albuminous casts were also seen within the lumen of the tubules. In two animals, there was atrophy of isolated glomerulus along with mild periglomerular cell infiltration. In one animal, tubular epithelial cells revealed degenerative and necrotic changes. The kidneys from S. anacardium treated animals did not reveal any appreciable adverse change.

4. Discussion Malignancies may engender complex metabolic and histologic disturbances which

Fig. 1. Liver from control rats showing normal architecture. Haematoxylin and eosin 400 = ..

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Fig. 2. Liver from Group II HC-bearing rats showing marked congestion of central vein along with intense cytoplasmic granularity of the hepatocytes. Haematoxylin and eosin =400..

Fig. 3. Liver from Group III drug-treated rats showing almost normal architecture. Haematoxylin and eosin =400..

result in rapid body weight loss and tissue wasting. The decreased body weight in HC conditions may be due to decreased food intake andror absorption, which contribute to muscle waste in tumour cachexia w19x. The steadily increased body weight in extract treated rats may be due to the anticancer potency of the S. anacardium nut.

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In neoplasms, the size of the tumour often correlates with its DNA content and hence it is found to be an independent indicator of prognosis w20x. The increased DNA content in HC liver may be due to the increased expression of enzymes necessary for DNA synthesis in tumour cells with repression of many enzymes related to differentiated cell function w21x. The RNA level in liver and kidney of cancer-bearing animals was also increased but not as significantly as DNA. The increased DNA content may lead to an increased transcription, which might have resulted in the moderately elevated RNA content in tumour cells. Decreased levels of DNA and RNA content to near normal levels were observed in S. anacardium nut extract treated animals. This indigenous drug imparts a significant inhibition on the rate of development of tumour. Reports from the in vitro studies on acetylated oil of S. anacardium nuts indicated the significant inhibition of incorporation of radiolabelled precursors in DNA and RNA and also their biosynthesis at a low concentration of 40 75 grml within 2 h w22x. Also, flavonoids present in S. anacardium nut extract may contribute to its tumour growth inhibiting property. Flavonoids are potent inhibitors of tumour cell proliferation and they are known to inhibit several biochemical events associated with abnormal cellular growth w23x. The decreased protein content in HC animals implies the underlying metabolic imbalance which is being expressed by an elevation in the apparent protein-degradation rate with no changes in the apparent synthesis rate w24x. Reduced liver protein in Morris hepatoma-bearing rats and Walker 256 carcinoma in other reports, have also suggested the protein degradation w25x. Semecarpus anacardium nut extract, due to its anticancer potency, may prevent protein degradation rate and hence the total protein content was almost normalised in treated animals. The liver from Group II animals showed histologic features of development of pure, well-differentiated liver cell carcinoma. Biliary proliferation was not seen in any of the rat livers. These lesions observed after AFB1 treatment are in agreement with previous report on the administration of AFB1 w13x. The basic nature of this toxin is perhaps to disturb the internal mileu of the sensitive cells in such a manner that their biosynthetic pathways get disturbed. As a consequence, many cells fail to complete their division cycle and may even get damaged and destroyed. Many of the cells that escape destruction resistant cells. start dividing more rapidly to compensate the loss. This compensatory division probably becomes unbridled of all the possible control mechanism, resulting in neoplasia w26x. The toxicity of aflatoxin with the accompanying regenerative hyperplasia of parenchymal cells, in Group II animals contributes to the development of HC in at least two ways. First, cell replication is essential for carcinogenesis. Second, the carcinogen-altered hepatocytes appear better, able to survive and grow in the face of a general hepatotoxin w27x. There have been reports which have described the presence of Kupffer cells within these areas of hyperplasia and concluded that these cells represent earliest evidence of malignancy w28x. Hyperplastic areas progress into larger neoplastic nodules and finally into HC. Hepatocytes undergo a sequence of alterations recognized morphologically by a progression from altered foci to nodules and from nodules to cancer w29x.

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The nodules appearing in Group II animals represent transitional stages from normal liver parenchyma to HC and may be considered as the direct forerunners of the latter. Kalengavi and Desmet w30x have also reported the development of liver cell hyperplastic foci and nodules in AFB1-induced carcinogenesis. Microscopically, associated adverse changes were observed in kidney in HC conditions. It is probable that when the normal liver cells transform into malignant cells, the transformation may not be possible without these changes in other organs like kidney. Reports have suggested the presence of epithelial cell proliferation, anaplastic cells in corticomedullary region and nuclear enlargement of tubular epithelium in kidney in AFB1-induced hepatocellular carcinoma w31x. The normalization of adverse histologic patterns observed in Group III animals may be due to the administration of S. anacardium nut extract which augments the nodular formation in cancer-bearing animals. In one animal, the liver pattern was very normal and practically no abnormality was detected. Positive changes of tumour marker enzymes were also observed due to the anticancer potency of S. anacardium nut extract against AFB1-induced HC conditions w32x. No toxic symptom was ever noticed in Group IV drug control animals. On the contrary, a marked improvement in general overall condition of these animals was observed. There were no significant histopathological changes in any of the vital organs. Reports from histopathological studies with liver, lung, kidney and heart did not reveal any significant detrimental pathological lesions even when the nut extract was administered in the high dose level of 1000 mgrkg w33x. In conclusion, it can be stated that S. anacardium nut extract has definite beneficial role against AFB1-induced HC and may bestow some protective mechanism against cell growth by increasing intrinsic body resistance.

Acknowledgements The authors are thankful to Dr A. Albert and Dr P. Jayaprakash Narayanan for their valuable suggestions. We are also grateful to Dr Sunderrajan and Dr Murali Manohar for their invaluable help in the histopathology part of the work. The financial assistance rendered by University Grants Commission to one of the authors B.P. is gratefully acknowledged.

References
w1x Parkin DM, Stiernward J, Mujr CS. Bull WHO 1984;62:163. w2x Campbell PN. Arogya J. Health Sci. 1984;X:1. w3x Kirthikar KR, Basu BD. In: Basu LM, editor. Indian medicinal plants, vol. I. India: Allahabad, 1933:667. w4x Ishartulla K, Ansari WH, Rahman W, Okigawa M, Kawano N. Indian J Chem 1977;15B:617. w5x Prakasha Rao NS, Ramachandra Rao L, Brown RT. Phytochemistry 1973;12:671. w6x Indap K, Ambaye RY, Gokhale SV. Indian J Physiol Pharmacol 1983;27:83. w7x Patwardan B, Ghoo RB, David SB. Indian J Pharm Sci 1988;50:130.

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w8x Chopra RN, Nayar SL, Chopra IS. In: Glossary of Indian medicinal plants. New Delhi, India: CSIR, 1956:225. w9x Vad BG. Indian Practitioner 1973;26:253. w10x Premalatha B, Muthulakshmi V, Vijayalakshmi T, Sachdanandam P. Int J Pharmacognosy 1997;35:1. w11x Premalatha B, Muthulakshmi V, Vijayalakshmi T, Sachdanandam P. Indian Drugs 1997;34:384. w12x Formulary of Siddha medicine, 2nd ed. India: The Indian Medical Practitioners Co-operative Pharmacy and Stores Ltd, 1972:197. w13x Angsubhakorn S, Get Ngern P, Miyamoto M, Bharmapravati N. Int J Cancer 1990;46:664. w14x Lowry OH, Rosenbrough WJ, Farren AI, Randal RJ. J Biol Chem. 1951;193:265. w15x Schneider WC. In: Colowick SP, Kaplan NO, editors. Methods in enzymology, Vol. III. New York: Academic Press, 1957:680. w16x Burton K. Biochem J 1956;62:315. w17x Rawal VM, Patel VS, Rao GN, Desai RR. Arogya J Health Sci 1977;3:69. w18x Kalengavi MMR, Desmet VI. Cancer Res 1975;35:2836. w19x Pain VM, Randall DP, Garlick PJ. Cancer Res 1984;44:1054. w20x Ellis EN, Burnette JJ, Sedlack R, Dyas C, Blackemore WSA. Surgery 1991;173:329. w21x Gallagher RE. In: Moosa AR, Robson MC, Schimpff SC, editors. Comprehensive textbook of oncology. Baltimore: Williams and Wilkins, 1986:36 45. w22x Phatak MK, Ambaye RY, Indap MA, Bhatia KG. Indian J Physiol Pharmacol 1983;27:166. w23x Kandaswami C, Perkins E, Soloniuk DS, Drzewiecki G, Middleton E. Cancer Lett 1991;56:147. w24x Tessitore L, Bonelli G, Baccina FM. Biochem J 1987;241:153. w25x Landel AM, Hammond WG, Meguid MM. Cancer 1985;55:230. w26x Bilgrami KS, Sinha SP, Ranjan KS. Curr Sci 1986;55:1092. w27x Roebuck BD, Maxuitenko YY. In: Eaton DL, Groopman JD, editors. Toxicology of aflatoxins. San Diego: Academic Press, 1994:27 43. w28x Teebor G. In: Becker FF, editor. Cancer, vol. I. Etiology chemical and physical carcinogenesis. New York: Plenum, 1977:345 351. w29x Sell S, Leffert HL. Hepatology 1982;2:77. w30x Kalengavi MMR, Desmet VJ. Cancer Res 1975;35:2845. w31x Rati ER, Shantha T, Ramesh HP. Indian J Exp Biol 1991;29:813. w32x Premalatha B, Muthulakshmi V, Sachdanandam P. Phytother Res 1999;13:183. w33x Ghosh D, Thejomoorthy P, Shetty BMV, Veluchamy G. J Res Ayur Siddha 1981;2:150.

Fitoterapia 70 1999. 493 497

Folk medicinal plants of riverside forests of the Southern Blue Nile district, Sudan
H.H. El-Kamali a,U , K.F. El-Khalifa b
b a Department of Botany, Omdurman Islamic Uni ersity, P.O. Box 382, Omdurman, Sudan Department of Sil iculture, Faculty of Forestry, Uni ersity of Khartoum, P.O. Box 32, Shambat, Sudan

Received 31 August 1998; accepted in revised form 6 April 1999

Abstract The therapeutic uses of 34 species of the most commonly prescribed medicinal plants in the riverside forests of Southern Blue Nile, central eastern Sudan are described. 1999 Elsevier Science B.V. All rights reserved.
Keywords: Folk medicine; Southern Blue Nile; Central eastern Sudan

1. Introduction The present communication is part of our efforts to record the uses of medicinal plants in various parts of Sudan that have been handed down to the present time by oral tradition w1 3x. This work was carried out to document the contemporary folk medicine of Southern Blue Nile, central eastern Sudan. The study area, centred around 1236 N latitude and 349 E longitude, covers an area of 5838 km2 The region is inhabited by several tribes who live in villages in the vicinity of the forests area Guraan, Rigeba, Um dungo, Radona, Kebaishab, Taaisha and Shamia omar constituting the bulk of the population.. The average annual rainfall is approximately 620 mm. The temperature ranges between 16 and 30C in winter and 24 41C in summer. The soil can be differentiated in three different types, namely Gerf , Mayaa and Karab Dahra.. Gerf soil is rich and
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contains high amounts of silt and clay. Mayaa is the cracked clay soil, dark in colour. Karab soil is characterised by the presence of high amounts of sand and gravel. The population is composed of villagers who usually depend on the forests for their needs of fuel wood and local building material. For this purpose, they usually get licences from the nearest forest office. Most of the inhabitants are herdsmen and farmers, while few people work in the forests. The type of livestock depends on the tribe but generally they have sheep as the main livestock. However, Taaisha and Rigeba tribes possess considerable numbers of cattle. 2. Methodology Several ethnobotanic field trips were carried out between March 1995 and June 1997 in villages around the riverside forests of Southern Blue Nile. The various data local name, medicinal uses, parts of plant used, method of preparation and administration. were collected from local inhabitants who had knowledge of the curative properties of plants by personal interview. Voucher specimens are deposited in the Department of Silviculture, Faculty of Forestry, Khartoum University. Identification of plant species was done according to the works of El-Amin w4x and El-Khalifa w5x. 3. Results The plants are listed in alphabetical order of their botanical name, followed by family and vernacular local. name, brief notes on the therapeutic uses and methods of preparation. Acacia nilotica L.. Del. ssp. nilotica Mimosaceae., Garad Abulamaa. The powder of the leaves and bark is used externally to treat eye diseases. The powder of the gum is taken to treat diarrhoea. A decoction prepared from the bark is used as a stimulant, to treat fever and indigestion. Acacia nubica Benth. Mimosaceae., La ot. The powder of the leaves is used externally as a poultice to treat swellings. Acacia polyacantha Willd. ssp. camplyacantha Hochst. ex. A. Rich.. Brenan Mimosaceae., Abu Sineina. An infusion prepared from the stem bark is used to treat jaundice. The powdered root bark mixed with honey is used for cough and asthma. Acacia senegal L.. Willd, var. senegal Brenan Mimosaceae., Hashab. Fresh or dried powder of the gum is taken to treat stomach disorders and diarrhoea and it is used as an emollient. Adansonia digitata L. Bombacaceae., Tebaldi Gongolaz.. A decoction or suspension in milk prepared from the fruit pulp is used to treat dysentery and diarrhoea. As fumigation, the bark is used to treat fever.

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Ailanthus excelsa Roxb. Simaroubaceae., Ilansus. A decoction prepared from the bark is used to treat dysentery. A decoction prepared from the leaflets is used to treat malaria. Albizzia lebbeck L.. Benth. Mimosaceae., Dign el Basha. An infusion prepared from the bark is used to treat diarrhoea and dysentery. A decoction prepared from the bark is used as a mouthwash to treat gum disease. Azadirachta indica A. Juss. Meliaceae., Neem. A decoction prepared from the leaves is used to treat fever, headache and malaria. The powder of the leaves is used externally to treat skin diseases and wounds. Balanites aegyptiaca L.. Del. Balanitaceae .. Hejleej Lalob.. The fruit is sucked as a purgative. A paint is prepared from the seed oil for burns. A decoction prepared from the bark is used to treat jaundice. As fumigation, branches are used externally to treat rheumatism. Borassus aethiopium Mart. Palmaceae., Doleib. The fruits are eaten to treat stomachache, skin and respiratory troubles. Calotropis procera Ait.. Ait. f. Asclepiadaceae., Ushar. Fresh flowers are eaten as a digestive. Capparis decidua Forsk.. Edgew. Capparidaceae.. Tundub. A decoction prepared from the stem bark is used to treat diarrhoea. A decoction prepared from the roots is used to relieve fever and is also used for jaundice. As fumigation, root is used to treat fever and rheumatism. Cassia obtusifolia L. Caesalpiniaceae ., Kawal. An infusion prepared from the upper aerial parts is used to treat stomach troubles. Ceiba pentandra L.. Gaertn. Bombacaceae. Gotton Harrery A decoction prepared from the branches is used as an emetic. Corchorus trilocularis L. Tiliaceace., Molukhiat al-Khalla. Cooked leaves are eaten as demulcent. The powder of the seeds is used externally as a poultice to treat dropsy. Cymbopogon ner atus Hochst.. Chiov. Poaceae., Nal. An infusion prepared from the leaves is used to treat indigestion and also as a carminative and tonic. Cyperus rotundus L. Cyperaceae., Seida. A decoction prepared from the tubers is used to treat stomach troubles and as an anthelmintic. Dalbergia melanoxylon Guill. et. Perr. Papilionaceae., Abanos. The stems are applied by fumigation for rheumatism. Dalbergia sissoides DC. Papilionaceae., El-Sarsu. A decoction prepared from the tender leaves is used to treat diarrhoea and dysentery. Eclipta alba L.. Hassk. Asteraceae ., Tamr al-Ghaanm. The powder of the leaves is used externally as a poultice to treat scorpion sting.

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Eucalyptus camaldulensis Dehn. Myrtaceae., Kaffor Ahmer Baladi. The leaves are inhaled fumigation. to treat respiratory diseases. The fresh leaves are applied on the affected parts to treat rheumatism. Faidherbia albida Del.. Chev. Mimosaceae., Haraz. A decoction prepared from the stem bark is used to treat cough and diarrhoea. Grewia tenax Forsk.. Fiori Tiliaceae., Goddaim. The fruits are eaten to treat anaemia and chest diseases. A decoction prepared from the bark is used as an anthelmintic. Hydnora abyssinica A. Braun Hydnoraceae., Tartooth. A decoction prepared from the stem is used as astringent in dysentery and also for stomach troubles. Khaya senegalensis Desr.. A. Juss. Meliaceae., Mahogany. A decoction prepared from the stem bark is used to treat malaria. The fresh internal stem bark is smeared on the tooth to relieve toothache. Ipomoea eriocarpa R. Br. Convolvulaceae., Tibr. A paint prepared from the herb boiled in oil. is used for rheumatism. Lonchocarpus laxiflorus Guill. et. Perr. Papilionaceae., Khash-khash Azrag. A decoction prepared from the bark is used for indigestion. An infusion prepared from the root is used to treat backache. Ricinus communis L. Euphorbiaceae., Khirwe. The fresh leaves are rubbed on the head to relieve headache and also applied on the legs to relieve swellings. Sclerocarya birrea A. Rich.. Hochst. Anacardiaceae., Houmad. A decoction prepared from the bark is used to treat dysentery. Solanum nigrum L. Solanaceae.. Einab al Deeb. Berries are eaten to treat diarrhoea. Sterculia setigera Del. Sterculiaceae ., Tirtir. An infusion prepared from the bark is used to treat jaundice. Tamarindus indica L. Caesalpiniaceae ., Aradaeb. An infusion or decoction prepared from the fruit is used as laxative and also to treat malaria. Ziziphus abyssinica Hochst. ex A. Rich. Rhamnaceae., Nabag al Feil. The powder of the flowers is used externally to treat wounds. A decoction prepared from the flowers is used to treat bilharzia. Ziziphus spina-christi L.. Desf. Rhamnaceae., Nabag. Fruits are eaten to treat diarrhoea and malaria and also used as antispasmodic. The powder of the twigs is used externally to treat rheumatism and scorpion sting.

4. Discussion The present study identified 34 plant species, belonging to 22 families, currently

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used in villages around the riverside forests of Southern Blue Nile for curing 36 types of ailments. The acceptability of these plants as claimed effective remedies is quite high among the population of this area. The medicinal uses of 24 plant species recorded in this study have not been previously mentioned in the ethnobotanical literature of central and northern Sudan w1 3x and for the other ones new therapeutic uses are here reported.

Acknowledgements The authors wish to express their profound appreciation to all the people who participated in this survey by providing information on medicinal plants used in the treatment of various diseases.

References
w1x w2x w3x w4x w5x El-Kamali HH, Khalid SA. Fitoterapia 1996;67:301. El-Kamali HH, Khalifa KF. Fitoterapia 1997;68:527. El-Kamali HH, Khalid SA. Fitoterapia 1998;69:118. El-Amin HM. Trees and Shrubs of the Sudan. Ithaca Press Exeter, 1990. El-Khalifa KF. Forest Botany. Sudan: Khartoum University Press, 1996.

Fitoterapia 70 1999. 498 501

Protective effects of Picrorrhiza kurroa against HClrethanol-induced ulceration in rats


Rangasamy AnandanU , Ravikumar Deepa Rekha, Natarajan Saravanan, Thiruvengadam Devaki
Department of Biochemistry and Molecular Biology, Uni ersity of Madras, Guindy Campus, Chennai 600 025, India

Received 19 October 1998; accepted in revised form 13 April 1999

Abstract The antiulcerogenic effects of the ethanol extract of Picrorrhiza kurroa rhizomes and roots PK. on HClrethanol-induced ulceration in rats with respect to acidrpepsin, lipid peroxidation, antioxidant status, proteins and glycoproteins in the gastric mucosa have been investigated. Oral pre-treatment with PK 100 mgrkgrday for 10 days. significantly prevented the induced ulceration and maintained the rats at near normal status. 1999 Elsevier Science B.V. All rights reserved.
Keywords: Picrorrhiza kurroa; Antiulcer activity

1. Introduction The dried rhizomes and roots of Picrorrhiza kurroa Royle ex Benth. Scrophulariaceae., Indian name kutki, have been used in Indian ayurvedic medicine for the treatment of ulcers w1x. In traditional medicine, the plant has also been used to cure hepatitis, lung diseases, abdominal pain, stomach disorders, anemia, jaundice and to promote bile secretion w2x. We have now attempted to
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assess the antiulcerogenic potential of the ethanol extract of P. kurroa rhizomes and roots PK. on HClrethanol-induced gastric lesions in rats. Peptic ulcer refers to the group of ulcerative disorders of upper gastrointestinal tract involving principally the most proximal portion of the duodenum and the stomach, which have in common the participation of acidrpepsin w3x. It occurs as a result of disturbance of the natural balance between aggressive acidrpepsin and mucosal defensermucosal turnover w4x. Administration of HCl and ethanol produces ulcerative lesions and increases lipid peroxidation in the gastric mucosa, which plays a significant part in the pathogenesis of the mucosal lesions w5x. A significant depletion of gastric glutathione GSH. has been reported in gastric ulceration in rats w6x. Depletion of GSH is known to result in enhanced lipid peroxidation and excessive lipid peroxidation can cause increased GSH consumption and increase the susceptibility of the gastric mucosal cells to oxygen metabolites and acid mediated cell damage w7 9x. GSH and GSH-dependent enzyme systems are directly related to the pathogenic mechanism of gastric ulcer formation w10x. Free radicals created by HClrethanol injury attack the protein in the gastric mucosa and lead to a reduction in the protein levels w11x. The structural organisation of glycoproteins with their protruding oligosaccharide chains makes them available for the HClrethanol attack, resulting in decreased N-acetylneuraminic acid levels in the gastric mucosa w12x.

2. Experimental 2.1. Drugs and chemicals Ethanol was obtained from Bengal Chemicals and Pharmaceuticals Ltd., Mumbai, India; bovine serum albumin, tetramethoxypropanol, epinephrine from the Sigma Chemical Co., St. Louis, MO, USA. All other chemicals were of analytical grade. PK was obtained from TTK Pharmaceuticals, Chennai, India w13 15x. 2.2. Animals Male Wistar rats weighing 120 150 g. were allowed standard pelleted diet Mrs Hindustan Lever Foods, Bangalore, India. and water ad libitum and housed under standard environmental conditions. Animals were deprived of food for 24 h prior to ulcer induction. 2.3. Antiulcerogenic acti ity The experimental animals were divided into four groups of six animals each. Rats in Group I normal control. received only the standard diet. In Group II, normal rats were treated with PK 100 mgrkgrday, p.o. for 10 days.. In Group III, ulcer was induced by oral administration of 1.5 ml of HClrethanol mixture containing 0.15 N HCl in 70% vrv ethanol w16x. Group IV was pre-treated with PK

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100 mgrkgrday, p.o. for 10 days. before induction of ulcer as described for Group III. At the end of the experiment, all four groups underwent surgery according to Takeuchi et al. w17x and gastric juice was collected for 4 h. Rats were then killed by an overdose of chloroform and the stomach was removed after the esophagus had been damped. The gastric juice was centrifuged and the volume was noted. The stomach was inflated with normal saline and then incised through the greater curvature and examined under a dissecting microscope for the number of lesions. The total acidity was determined by titrating with 0.1 N NaOH with phenolphthaleine used as indicator. The mucosal tissue was scraped from the stomach and the gastric mucosa was estimated for its peptic activity w18x, lipid peroxides LPO. w19x, GSH w20x and the activities of superoxide dismutase SOD. w21x catalase CAT. w22x, glutathione peroxidase GSH-px. w23x and glutathione-S-transferase GST. w24x. The protein w25x content, hexose w26x and hexosamine w26x and sialic acid w27x contents were also estimated. 2.4. Statistical analysis Results are expressed as mean " S.D., and Students t-test was used to assess statistical significance.
Table 1 Effect of Picrorrhiza kurroa ethanol extract on HClrethanol-induced ulcer in rats a Parameters b Group I Normal control 1.98 " 0.09 189.4 " 16.3 664.9 " 56.1 3.42 " 0.18 4.63 " 0.16 3.78 " 0.29 3.86 " 0.24 214.4 " 23.7 4.32 " 0.54 19.2 " 1.33 14.3 " 1.44 9.32 " 0.68 1.63 " 0.18 Group II PK pretreated A. 2.03 " 0.12 176.9 " 5.6 673.2 " 55.4 3.50 " 0.23 4.68 " 0.14 3.91 " 0.31 3.74 " 0.16 223.3 " 21.5 4.18 " 0.41 18.6 " 1.16 14.8 " 1.53 8.86 " 0.54 1.68 " 0.16 Group III HClrethanolinduced ulcer B. 15.36 " 1.4UUU 3.41 " 0.21UUU 278.8 " 26.4UUU 607.6 " 53.2UUU 7.98 " 0.74UUU 2.08 " 0.14UUU 1.66 " 0.30UUU 1.34 " 0.18UUU 142.6 " 20.8UUU 3.27 " 0.45UUU 9.82 " 0.98UUU 7.63 " 1.12UUU 4.36 " 0.51UUU 0.64 " 0.15UUU Group IV A q B. 4.38 " 0.91UUU 2.32 " 0.14UUU 194.7 " 16.4UUU 652.4 " 54.7UUU 4.02 " 0.31UUU 4.48 " 0.15UUU 3.43 " 0.34UUU 3.51 " 0.19UUU 203.1 " 18.5UUU 3.98 " 0.33UUU 17.68 " 1.12UUU 14.5 " 1.38UUU 8.53 " 0.61UUU 1.54 " 0.14UUU

Number of lesions Volume of gastric juice Acid output Pepsin LPO SOD CAT GSH GSH-px GST Proteins Hexose Hexosamine Sialic acid
a

Values are expressed as mean " S.D.; n s 6; UUU P - 0.001; Group III vs. Group I; Group IV vs. Group III; Students t-test. A.: PK, 100 mgrkgrday, p.o., for 10 days. B.: 1.5 ml of HClrethanol mixture 0.15 N HCl in 70% vrv ethanol.. b Volume of gastric juice s mlr4 h; acid output s eq.r4 h; pepsin s mol tyrosiner4 h; LPO s nmolrmg protein; SOD s one unit of the SOD activity is the amount of protein required to give 50% inhibition of epinephrine autoxidation; CAT s mol of H 2 O 2 consumedrminrmg protein; GSH s nmolrg tissue; GSH-px s nmol GSH oxidisedrminrmg protein; GST s mol of 1-chloro-2,4 dinitrobenzene conjugate formedrminrmg protein; proteins, hexose, hexosamine and sialic acid s mgrg.

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3. Results and discussion Oral administration of HCl and ethanol 0.15 N HCl in 70% vrv ethanol. caused a significant increase in the number of lesions in the gastric mucosa, increases in volume of gastric juice and acidity, and the decreased activity of pepsin. Activities of the antiperoxidative enzymes SOD and CAT., GSH-dependent enzymes GSH-px and GST. and the levels of GSH, proteins and glycoproteins in gastric mucosa significantly decreased in ulcerated rats, while an increase in LPO level was observed Table 1.. Oral pre-treatment with PK 100 mgrkgrday for 10 days. significantly prevented these adverse changes and maintained the rats at nearly normal status. The antiulcerogenic effect of PK is probably related to its ability to maintain near to the normal status the activities of free radical scavenging enzymes and the level of GSH which protects mucosa against oxidative damage, by decreasing lipid peroxidation and strengthening the mucosal barrier, which is the first line of defense against exogenous and endogenous ulcerogenic agents.

References
w1x Saraswat B, Visens PKS, Patnaik GK, Dhawan BN. Ind J Exp Biol 1993;31:316. w2x Anandan R, Devaki T. Med Sci Res 1998;26:349. w3x Mc Guigan JE. In: Kurt JI, Eugene B, Margin JB, Willson JD, Karper D, editors. Harrisons principles of internal medicine, 13th ed., vol. 2, 1994:1363 1364. w4x Piper DW, Stiel D. Med Proc 1986;2:7. w5x Ito M, Shii D, Segami T, Kojima R, Suzuki Y. Jpn J Pharmacol 1992;59:267. w6x Boyd SC, Sesame HA, Boyd MR. Life Sci 1981;28:2987. w7x Younes M, Soegers CP. Chem Biol Interact 1981;34:257. w8x Comporti M. Lab Invest 1985;53:599. w9x Mutoh H, Ota S, Hiraishi H, Ivey KJ, Terano A, Sugimoto T. Am J Physiol 1991;261:65. w10x Morenkova SA, Tabutsadze TU, Fedorova LM, Masenko NP. Biull. Eksp. Biol Med 1987;104:570. w11x Pihan G, Rigello C, Szabo S. Dig Dis Sci 1987;32:1395. w12x Gottschalk A. In: Glycoproteins: their compositions, strucutre and function, 2nd ed. Amsterdam: Elsevier, 1972:136 172. w13x Anand N. In: Hansch C, Sammes PG, Taylor JB, Kennewell PD, editors. Comprehensive medicinal chemistry, vol. 1. Oxford: Pergamon Press, 1990:113 131. w14x Anandan R, Devaki T. Fitoterapia 1999;70:54. w15x Anandan R, Pandimadevi K, Devaki T, Govindaraju P. J Clin Biochem Nutr 1998;25:87. w16x Hara N, Okabe S. Folia Pharmocol Jpn 1985;85:443. w17x Takeuchi K, Okabe S, Takagi K. Am J Dig Dis 1976;21:782. w18x Anson ML. J Gen Physiol 1938;22:79. w19x Ohkawa H, Ohishi N, Yagi K. Anal Biochem 1979;95:351. w20x Ellman GL. Arch Biochem Biophys 1959;82:70. w21x Misra HP, Fridovich I. J Biol Chem 3170 1967;247:158. w22x Nell JV, Kobra TY, Ogura Y, Nishimura ET. J Clin Invest 1960;29:610. w23x Pagila DE, Valentine WN. J Lab Clin Med 1967;70:158. w24x Habig WH, Pabst MJ, Jakoby WB. J Biol Chem 1974;249:7130. w25x Lowry OH, Rosenborough NJ, Farr AL, Randall RJ. J Biol Chem 1951;193:265. w26x Winzler RJ. Methods Biochem Anal 1958;2:279. w27x Warren L. J Biol Chem 1971;234:1959.

Fitoterapia 70 1999. 502 513

Malay ethno-medico botany in Machang, Kelantan, Malaysia


H.C. OngU , M. Nordiana
Institute of Biological Sciences, Faculty of Science, Uni ersity of Malaya, 50603 Kuala Lumpur, Malaysia Received 9 October 1998; accepted in revised form 8 April 1999

Abstract A survey of the Malay ethno-medico botany in the Machang district, Kelantan state, Malaysia, identified 146 species used by the villagers to treat various ailments. 1999 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Malaysia; Ethnomedicine

1. Introduction Machang is a district in the central region of the state of Kelantan which is situated in the north-eastern part of peninsular Malaysia. Machang district, which is made up of six villages, covers an area of 15 km2 within 544 and 546 N latitude and 10213 and 10215 E longitude w1x. The altitude of the study area varies from sea level to 290 m above sea level. The villages are remote and traditional medicine is widely practiced. The main source of income in this area is agriculture with activities such as tapping rubber, planting rice, vegetables and fruits. The Malay community makes up more than 90% of the total population of approximately 2500 people living in the study area. Ethno-medico botanical knowledge is still in the form of unwritten state in the study area, thus the interest in recording it. This study is part of a larger programme to conduct ethnobotanical
U

Corresponding author. Tel.: q60-3-7594-356; fax: q60-3-7594-178. E-mail address: george@botany.um.edu.my H.C. Ong.

0367-326Xr99r$ - see front matter 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 6 7 - 3 2 6 X 9 9 . 0 0 0 7 7 - 5

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studies of medicinal plants in all parts of Malaysia where most of the knowledge on traditional medicine is still unwritten and variations and differences of knowledge and practices exist between different communities and sites.

2. Methodology Field work was carried out at various times for periods of 1 4 weeks duration each trip in 1996 and 1997. Information was obtained through interviews with traditional healers, herbalists, shamans, midwives, collectors, traders and other villagers in the study area. Data obtained from a total of 24 informants are presented in this paper. Only data coming from at least three informants were retained. The plants were identified by the authors with the help of related literature w2 8x. Voucher specimens are deposited in the Institute of Biological Sciences, Faculty of Science, University of Malaya.

3. Results The plants are listed in alphabetical order of the botanical name, followed by family, vernacular local Malay. name and brief notes on methods of preparation and administration. Acalypha indica L. Euphorbiaceae., Ceka emas. A decoction of the leaves is drunk daily after breakfast to cure aching bones. Acorus calamus L. Araceae., Jerangau. A paste of the roots pounded finely together with a little ginger Zingiber officinale Rosc.. is applied externally to cure bone aches. Ageratum conyzoides L. Asteraceae ., Rumput tahi ayam. Decoction of the whole plant is drunk three times daily to cure diarrhoea. Allium ascolanicum L. Liliaceae ., Bawang merah kecil. Juice extracted from the bulbs is applied on the feet to treat cracks and improve skin. Pounded bulbs mixed with egg yolk are applied on the body to treat fever. An infusion of the bulbs is drunk to treat menstrual problems. Heated bulb is applied on sores as a cure. Allium cepa L. Liliaceae ., Bawang besar. Decoction of the bulb is dropped into the nose to induce sneezing, expel phlegm and treat nose ulcers. Allium sati um L. Liliaceae ., Bawang putih. The peeled bulb is dipped in coconut oil and heated before placing on skin where a splint or thorn is embedded in the flesh. After some hours the splint or thorn can easily be removed. Finely chopped bulbs mixed with coconut sugar or fresh lime w Citrus aurantifolia Christm.. Swinglex with water are drunk to treat loss of appetite. Pounded bulbs mixed with coconut oil are applied on the scalp to get rid of head lice. Allomorphia malaccensis Ridl. Melastomataceae ., Senduduk gajah. Pounded leaves mixed with salt are applied on sores. Aloe era L. Liliaceae ., Lidah buaya. Roasted glutinous rice Oryza sati a L.. is

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pounded finely and mixed with mucilage obtained from the leaves of this plant and applied on the face to prevent pimples and to smooth the skin. Alpinia conchigera Griff. Zingiberaceae., Lengkuas genting. A paste of the rhizome is applied on skin to treat bone aches and pains. The paste mixed with kerosene is rubbed on skin with fungal infection. Alpinia galanga L.. Willd. Zingiberaceae., Lengkuas. The leaves are part of a compound mixture used to make a decoction drunk to treat diarrhoea. Alstonia angustifolia Wall. ex A.DC. Apocynaceae., Pulai. A decoction of the roots is drunk three times per week to cure asthma. Amomum kepulaga Sprague et Burkill ex Hooper Zingiberaceae., Buah pelaga. The fruits are eaten together with cloves to alleviate addiction to cigarettes. The fruits are also eaten to counter the feeling of wanting to vomit. Anacardium occidentale L. Anacardiaceae., Ketereh. Decoction of the mature leaves is drunk three times per day to treat diarrhoea. Ananas comosus L.. Merr. Bromeliaceae., Nenas. Juice expressed from the young fruits is applied on the head and left there for 15 min before washing off. This is repeated for several days as a cure for dandruff. Andrographis paniculata Nees Acanthaceae ., Hempedu bumi. The whole plant is used but the leaves are preferred. Decoction of the whole plant or leaves, or fresh leaves pounded and rolled into pills are taken to treat high blood pressure. Bananas are then eaten in order to counter the bitter taste. The treatment is repeated three to four times a week. Annona squamosa L. Annonaceae., Buah nona. Ten leaves are pounded, mixed with water in a bowl and then rubbed on the head of children with fever to prevent attacks of fits. Ardisia crenata Sims Myrsinaceae ., Mata ayam. Pounded leaves are applied on sores. A decoction of the leaves is drunk to treat diarrhoea. Areca catechu L. Arecaceae., Pinang. Young nuts are pounded with leaves of Mimosa pudica L. and a little salt, mixed with some warm water and applied on the breasts for firmness. Arundina graminifolia D.Don. Hochr. Orchidaceae., Ubi bemban. Juice extracted from the swollen stem is drunk to treat stomachache. Asplenium nidus L. Polypodiaceae., Semum. Pounded leaves of this plant are mixed with grated coconut and the juice expressed is used as hair wash for healthier hair, free of dandruff and split ends. A errhoa bilimbi L. Oxalidaceae., Belimbing buluh. Grated fruits, with a little salt added, are applied on the face to treat pimples. Juice expressed from one large fruit or two small fruits is taken once per day to treat diabetes. Baccaurea par iflora Muell.Arg.. Muell.Arg. Euphorbiaceae., Setambun putih. A decoction of the leaves is drunk three times per day as a diuretic. Barringtonia racemosa L.. Sprengel Lecythidaceae ., Putat kendul. A paste of the pounded leaves is placed on itching skin. Barringtonia scortechinii King Lecythidaceae ., Putat. A decoction of the leaves is drunk after childbirth to cleanse the blood and stimulate uterus contraction. Blumea balsamifera DC. Asteraceae ., Sembong. Decoction of the leaves is drunk

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to cure coughs and throat itch. Smoke from the burning roots is inhaled to treat nose ulcers. Bryophyllum pinnatum Kurz Crassulaceae ., Setawar. Juice obtained from pounded leaves is drunk to treat constipation. Leaves are pounded and mixed with a little water, and the obtained liquid is applied on the forehead to relieve from fever. Canthium hirtellum Ridl. Rubiaceae., Bunga pekan. The flower, placed in a bowl of water, kept in an open place overnight and then boiled, is eaten as appetizer. Capparis microcantha DC. Capparidaceae., Melada. The roots are part of a mixture used to make a decoction drunk after childbirth. A decoction of the roots is applied on the skin as a remedy against jaundice in newborns. Carica papaya L. Caricaceae ., Betik. Two or three drops of the latex are dropped into a glass of warm water, mixed and drunk after breakfast to alleviate gastric problems due to hyperacidity. Juice obtained from pounded leaves is mixed with water and used as a bath against malaria. Decoction of the leaves is drunk daily for slimming. The latex is applied within decayed teeth causing toothaches. Ripe fruits are eaten to stimulate bowel movements. Carmona retusa Vahl.. Musamune Boraginaceae., Pokok pagar. A decoction of the leaves is drunk three times per day to treat diarrhoea. Casuarina equisetifolia J.R. et G. Forst. Casuarinaceae ., Ru. The fruits, pounded together with galls from Croton caudatum Geisel. and added with a little vinegar, are rubbed on the gums to strengthen the teeth. Catharanthus roseus L.. G.Don Apocynaceae., Kemuning Cina. An infusion of the leaves is taken to treat painful menstruation. Cibotium barometz L.. J.Sim Dicksoniaceae., Penawar jambi. The hairs are applied on cuts and wounds to stop bleeding. Cinnamomum iners Reinw. Lauraceae ., Pokok teja. A decoction of the roots is drunk against fever. A decoction of the bark is drunk after childbirth as post-partum medicine. Citrus aurantifolia Christm.. Swingle Rutaceae., Limau nipis. Juice extracted from the fruit is applied on the forehead against headache. The fruit juice is rubbed on the scalp to treat dandruff. The fruit juice mixed with turmeric Curcuma domestica Valeton., salt and brown sugar is drunk to treat stomachache. The juice mixed with pounded pomegranate Punica granatum L.. fruit peel, sugar and warm water is drunk to treat white vaginal discharges. Citrus grandis L.. Osbeck Rutaceae., Limau besar. Flesh of the fruit is eaten to treat stomachache. Citrus hystrix DC. Rutaceae., Limau purut. Fruit juice is rubbed on the skin to soften it. Fruit juice is mixed with bath water to get rid of body smell. Cnestis palala Lour.. Merr. Connaraceae., Terkilir. The climbing stems and roots form part of a mixture of plants used to prepare a decoction drunk for general health and cure all. Cocos nucifera L. Arecaceae., Kelapa coconut.. Coconut oil is applied on skin for 20 min and then rubbed with grated coconut before washing with warm water for smoother skin. Grated coconut is rubbed on hair before bath to darken the hair

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and promote hair growth. Ash obtained from burning the shells is applied on cuts and wounds as a cure. The milk is drunk and bread dipped in the milk is eaten to promote bowel movement. The water is drunk to treat measles. Coelostegia griffithii Benth. Bombacaceae., Punggai. Paste of fresh, finely pounded leaves is applied on swollen breasts mastitis. as a remedy. Coleus amboinicus Lour. Lamiaceae., Hati-hati. Juice obtained from pounded leaves is drunk to cure constipation. Coleus blumei Benth. Lamiaceae., Ati-ati. A decoction of the leaves is drunk to reduce high blood pressure. A paste of the pounded leaves is placed on cuts and wounds to stop bleeding. Commelina nudiflora L. Commelinaceae., Rumput aur. A paste of the finely pounded leaves is applied on sores as a cure. Connarus planchonianus Schellenb. Connaraceae., Sembelit angin. Decoction of the climbing stems and roots is taken to treat flatulence. Coscinum blumeanum Miers. Menispermaceae., Tuba kupak. Pounded roots and stems are made into paste and applied on cuts, wounds and sores. Costus speciosus Smith Zingiberaceae., Setawar hutan. A paste of the pounded stem is placed on skin with rashes. Cratoxylon cochinchinense Lour.. Bl. Bignoniaceae., Bonglai kayu. The roots form part of a mixture of plants used to prepare a decoction drunk for general health and cure all. Croton caudatum Geisel. Euphorbiaceae., Manjakani. The galls, pounded together with fruits of Casuarina equisetifolia J.R. et G. Forst. and added of a little vinegar, are rubbed on the gums to strengthen the teeth. A paste of the pounded gall is applied on burns and blisters. Cucumis sati us L. Cucurbitaceae ., Timun. A mixture of the fruits with ripe fruits of Diospyros kaki L.f. is left overnight and then drunk to treat sore throats and hoarse voice. Curcuma domestica Valeton Zingiberaceae., Kunyit turmeric.. The rhizome is pounded with betle leaves and rice to form a paste which is applied on the abdomen to treat stomachache and stomach swellings. Young rhizomes are eaten to improve complexion. Curcuma xanthoriza Roxb. Zingiberaceae., Temulawak. Juice obtained from the rhizome is applied on the face to cure pimples. Cymbopogon citratus Stapf. Poaceae., Serai. A paste of the pounded leaves is placed on the forehead to cure headache or rubbed on aches and pains as a remedy. A decoction of the leaves is drunk to cure stomachache. Decoction of the roots added of a little honey is drunk to cure cracks in the feet. Cymbopogon nardus L. Poaceae., Serai wangi. The plant is used to make hair shampoo preventing dandruff. Crushed leaves are placed in the bath water to invigorate the body and get rid of body smell. Datura metel L. Solanaceae., Kecubung. The leaves are dried and smoked like cigarettes against asthma. A paste of the pounded leaves is applied on the skin to treat aching bones. Smoke obtained by burning the fruits is directed into the mouth to relieve toothache.

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Derris elliptica Benth. Papilionaceae., Tuba. Roots are pounded and rubbed on the scalp to kill head lice. Pounded leaves are applied on itching skin. Dioscorea daemona Roxb. Dioscoreaceae., Ubi gadong. A paste of the pounded tuber is applied into the nose to stop nose bleeding. This paste is also applied on the face to treat pimples. Dioscorea hispida Dennst. Dioscoreaceae., Ubi arak. A decoction of the leaves is applied on the feet frequently to treat foot cracks. Diospyros kaki L.f. Ebenaceae ., Pisang kaki. The ripe fruits are blended with cucumbers Cucumis sati us ., the mixture is left overnight and then drunk to treat sore throats and hoarse voice. The fruits are eaten as an antidote for snake-bites and insect-stings. Elephantopus scaber L. Asteraceae., Tutup bumi. The plant is boiled together with red beans and the obtained decoction drunk to treat flatulence. Entada phaseoloides Merr. Mimosaceae., Beluru. The stem is pounded, water is added and the resulting soapy liquid is used as body shampoo to kill lice. Epipremnum giganteum Schott. Araceae., Rengot. The smoke of burning roots is inhaled to treat nose ulcers, twice a week. Eriocaulon sexangulare L. Eriocaulaceae ., Sepulih. A decoction of the leaves and roots is drunk as contraceptive before or within a day after sexual intercourse. Eupatorium odoratum L. Asteraceae., Pokok kapal terbang. Pounded leaves are applied on cuts and wounds to stop bleeding. Euphorbia tirucallii L. Euphorbiaceae., Tulang-tulang. The plant, pounded and mixed with mud, is applied onto cuts and wounds as a cure. Eurycoma longifolia Jack Simaroubaceae., Tongkat ali. A decoction of the roots is drunk as aphrodisiac for men. Garcinia atro iridis Griff. Guttiferae ., Gelugur. To soften the skin, juice obtained from the pounded fruits is mixed with honey, applied on the face and left to dry; when dry, the face is washed. A decoction obtained by boiling dried slices of the fruits is drunk to cure cracks on the feet. Garcinia mangostana L. Guttiferae ., Manggis. Hot sand is placed on the leaves, which are then tied with cloth onto sprains. Gastrochilus panduratum Ridl. Zingiberaceae., Tepus sehelai setahun. The rhizome is used in a mixture to prepare a decoction drunk after childbirth to promote physical recovery. Glycosmis puberula Lindl. Rutaceae., Terapai. The roots form part of a mixture used to make a compound decoction drunk after childbirth to promote physical recovery. Goniothalamus scortechinii King Annonaceae., Bunga Cenang. A decoction of the whole plant is drunk daily for three days after childbirth to promote physical recovery. Hedyotis capitellata Wall. Rubiaceae., Meroyan putih. A decoction of the roots is drunk after childbirth to promote physical recovery. Helmintostachys zeylanica Hk.f. Ophioglosaceae., Tunjuk langit. Pounded leaves are applied on sores. A decoction of the leaves is drunk against diarrhoea.

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Hibiscus rosa-sinensis L. Malvaceae., Bunga raya putih. A decoction of the flowers is drunk to reduce high blood pressure. Hoya di ersifolia Bl. Asclepiadaceae., Seraput. The roots are pounded finely and eaten by women after childbirth to promote physical recovery. Hydnocarpus castanea Hk.f. Flacourtiaceae ., Setumpul. Juice obtained from pounded leaves is taken daily to increase the amount of male ejaculate. Hydrocotyle asiatica L. Umbelliferae., Pegaga. A paste made by pounding leaves of this plant with rice Oryza sati a. and turmeric is applied on the face to treat pimples. Hyptis sua eolens Poit. Labiatae ., Kemangi. A paste of the pounded leaves is applied on sores and against skin fungal infection. Illicium erum Hk.f. Winteraceae ., Bunga lawang. A mixture of the dried fruits with clove flower buds Syzygium aromaticum L.. Merr. et Perry., lime and coconut oil is applied on itching skin. Impatiens balsamina L. Balsaminaceae .. A decoction of the plant is drunk every morning as a cure for high blood pressure. Imperata cylindrica Beauv. Poaceae., Lalang. A decoction of the rhizomes added with sugar is drunk to treat headache. Ipomoea aquatica Forsskal Convolvulaceae., Kangkung. A paste of the pounded leaves is applied on boils to speed up the expulsion of pus and healing. Jasminum sambac Ait. Oleaceae ., Melur. Roots of this plant mixed with rice O. sati a. are used as facial powder to treat pimples and smooth skin. Jatropha gossypifolia L. Euphorbiaceae., Jarak. The pounded leaves are applied on swollen breasts mastitis., itching skin and boils. Kyllingia bre ifolia Rottb. Cyperaceae., Kancing baju. The leaves are eaten to treat diarrhoea and stomachache. A paste of the pounded roots is applied on sores as a cure. Lantana camara L. Verbenaceae ., Bunga busuk. Juice extracted from the leaves is rubbed on the aching part to treat bone aches and pains. A paste obtained by pounding the leaves is applied on cuts and wounds. Lasianthus oblongus King et Gamble Rubiaceae., Sekentut pokok. The roots form part of a compound mixture drunk after childbirth to promote physical recovery. Lawsonia inermis L. Lythraceae ., Inai. A decoction of the leaves is drunk to smooth face skin. A paste of the pounded leaves is placed on cuts and wounds. Infusion of the leaves in hot tea is applied on the hair and left overnight before washing for healthier hair. Lepidagathis longifolia Wight Acanthaceae ., Segugur. A decoction of the whole plant or only of the leaves is drunk to induce abortion. Leucaena leucocephala Lamk.. de Wit Mimosaceae., Petai belalang. Leaf ash is mixed with oil and applied on sores. Roasted seeds are pounded, mixed with warm water and drunk to cure diabetes. Lophatherum gracile Brongn. Poaceae., Rumput kelurut. A decoction of the whole plant is drunk after childbirth to promote physical recovery.

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Lycopodiun carinatum Desv. Lycopodiaceae., Akar ru. A decoction of the whole plant is used as hair wash to promote healthy hair, free of dandruff and split ends. Lycopodium cernuum L. Lycopodiaceae., Rumput serani. A decoction of the whole plant is drunk to treat cough and asthma. Mangifera indica L. Anacardiaceae., Mempelam. A decoction of the tree bark with a little salt added is used as a gargle to treat swollen gums. Melastoma malabathricum L. Melastomataceae ., Senduduk. A decoction of the leaves is drunk to treat diarrhoea. Juice from the fruits is rubbed on dry lips. Mentha ar ensis L. Lamiaceae., Daun pudina. Juice expressed from the leaves is mixed with a little fresh lime juice and drunk to treat sore throats. Mimosa pudica L. Mimosaceae., Semalu. The leaves are pounded with young betle nuts Areca catechu. and a little salt, mixed with some warm water and applied on the breasts for firmness. Mimusops elengi L. Sapotaceae., Bunga tanjung. A decoction of the bark and leaves is drunk to promote healthy skin and as a general tonic. Mitragyna speciosa Korth. Rubiaceae., Pokok biak. A decoction of the leaves is drunk to overcome drug addiction. Momordica charantia L. Cucurbitaceae ., Peria. The seeds are eaten to treat high blood pressure. A paste of the pounded leaves is applied on the head of babies to promote hair growth. The fruits are eaten raw to cure diabetes. Juice obtained from the leaves is drunk for three days to treat coughs expelling sputum mixed with blood. Morinda citrifolia L. Rubiaceae., Mengkudu. A decoction of the leaves is drunk to treat diabetes. Morinda elliptica Ridl. Rubiaceae., Mengkudu hutan. A decoction of the leaves is drunk to treat diarrhoea. Moringa oleifera Lamk Moringaceae., Remunggal. Pounded leaves are applied on swollen breasts, especially of mothers after childbirth. Cooked leaves or fruits are eaten to prevent constipation. Pounded roots are placed on the abdomen of women after childbirth to promote contraction of the uterus. Musa paradisiaca L. Musaceae., Pisang tanduk. Sap from the petiole is applied on skin with blisters. Two leaves are heated over fire and placed one under the patient and the other one on top to treat fever. Juice obtained from a section of the petiole heated over hot ash is dropped into infected ear to induce expulsion of pus and recovery. Nigella sati a L. Ranunculaceae ., Jintan hitam. A mixture of pounded seeds with rice water is drunk to treat internal swellings. A mixture of pounded seeds with water is drunk in case of blood-containing stool. Nothopanax scutellarium Merr. Araliaceae., Pokok puding mangkuk. Decoction of the leaves is taken three times per day to treat constipation. Ocimum gratissimum L. Lamiaceae., Selasih. A paste obtained by pounding the leaves is applied on skin with measles. Oryza sati a L. Poaceae., Padi. A decoction of the grains is taken to cure diarrhoea. Water used to wash the grains is used to wash the face to cure pimples,

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prevent greasiness or oilyness and improve the facial skin. A decoction of the grains mixed with a little sugar and salt is drunk as a tonic. Pachyrrhizus erosus L.. Urban Papilionaceae., Sengkuang. Flesh of the tuber is pounded and applied on skin with rashes. Paederia foetida L. Rubiaceae., Sekentut akar bukit. A decoction of the roots is drunk after childbirth to promote physical recovery. Pandanus odorus Ridl. Pandanaceae ., Pandan. Pounded leaves mixed with water are applied on hair and kept for 15 min before washing to treat dandruff. Peperomia pellucida H.B. et K. Piperaceae., Ketumpangan air. A decoction of the plant is drunk to treat bone aches and pains. Phyllagathis rotundifolia Bl. Melastomataceae ., Tapak sulaiman. A decoction of the roots and leaves is drunk to treat malaria. Phyllanthus niruri L. Euphorbiaceae., Pokok dukung anak. A decoction of the whole plant is drunk three times per day against diarrhoea and also used as bath to treat jaundice. Crushed leaves of the plant together with leaves of Eupatorium odoratum and lime are applied on boils. Piper betle L. Piperaceae., Sireh. The petiole is dipped in mucilage from Aloe era and applied on the eye brows to promote thickening of the eye brows. Finely sliced leaves, soaked in water and added of salt, are used as gargle to treat bad breath. A rolled leaf is placed into the nose to stop nose bleeding. A paste of pounded leaves is placed on sores to promote pus discharge. Piper nigrum L. Piperaceae., Lada hitam black pepper.. The seeds with garlic, wrapped in banana leaves and heated over fire, are eaten to treat asthma. Piper sarmentosum Roxb. Piperaceae., Kadok. A decoction of the leaves is drunk to treat malarial fever. Plantago major L. Plantaginaceae ., Daun sejumbok. A decoction of the leaves and roots is drunk to treat coughs, diabetes and to cleanse the blood. Plumbago zeylanica L. Plumbaginaceae ., Pokok ceraka. A decoction of the roots is drunk to induce abortion. Polyalthia bullata King Annonaceae., Tongkat Ali Hitam. A decoction of the roots is drunk regularly to treat kidney infections. Flowers, leaves and the roots are finely pounded and taken to reduce high blood pressure and treat diabetes. A decotion of the roots mixed with Eurycoma longifolia is drunk as an aphrodisiac for men. Polygonum minus Hudson Polygonaceae., Daun kesum. A paste of the pounded leaves mixed with a little kerosene is rubbed on the skin to get rid of fungal infections. Porterandia anisophylla Jack ex Roxb.. Ridl. Rubiaceae., Tinjau belukar. The roots are part of a mixture used to prepare a decoction drunk to induce abortion. Pouzolzia zeylanica L.. Benth. Urticaceae ., Ubai-ubai. A decoction of the leaves is drunk or the leaves are eaten to kill worms. Psidium guaja a L. Myrtaceae., Jambu batu. A decoction of the leaves mixed with a little salt is applied on sores. A mixture of pounded young leaves, a little salt

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and water is drunk by pregnant women to alleviate morning sickness. A decoction of the leaves is drunk to treat diarrhoea. Pternandra coerulescens Jack Melastomataceae ., Sial menaung. Juice obtained from pounded leaves or a paste of the leaves are rubbed on the skin treat chest pains. Punica granatum L. Lythraceae ., Delima. A mixture of dried fruit peel pounded finely, tea leaves, sugar, fresh lime and hot water is drunk to treat vaginal white discharges. Juice from the fruits is mixed with a little salt and drunk regularly for slimming the body. Rhodomyrtus tomentosa W.Ait.. Hassk. Myrtaceae., Kemunting. Ripe fruits are eaten raw to treat diarrhoea. Ricinus communis L. Euphorbiaceae., Jarak besar. Oil extracted from pounded seeds is applied topically to treat piles. Senna alata L.. Roxb. Caesalpiniaceae ., Gelenggang besar. Cut leaves soaked in kerosene or dipped in sulphur are rubbed onto skin with ringworm or other fungal infections. A paste obtained by pounding the leaves is applied on sores. Young leaves are eaten to alleviate constipation. Senna obtusifolia L.. Irwin et Barneby Caesalpiniaceae ., Gelenggang kecil. The young leaves are eaten raw to alleviate constipation. Pounded leaves are applied on the skin to cure itches. Sida acuta Burm. Malvaceae., Kelulut. A paste of the pounded roots is placed on boils. Sindora coriacea Baker. Prain Caesalpiniaceae ., Sepetir daun licin. A decoction of the fruits is drunk to reduce high blood pressure, treat diabetes and sore throat. Sindora wallichii Benth. Caesalpiniaceae ., Sepetir daun tebal. A decoction of the fruits is drunk to treat diabetes and sore throat. Smilax calophylla Wall. Liliaceae ., Tembaga suasa. Leaves heated over fire are wrapped on swollen limbs as a cure. Solanum tor um Swartz Solanaceae., Terung pipit. The fruits are eaten to reduce high blood pressure. A paste of the pounded roots is placed in tooth cavity to relieve pain. Spilanthes acmella Murr. Asteraceae ., Pokok getang. Pounded flowers are placed in tooth cavities to relieve pain. Syzygium aromaticum L.. Merr. et Perry Myrtaceae., Bunga cengkih. A mixture of the flower buds with star anise Illicium erum., sulphur powder, lime and coconut oil is applied on itching skin. The buds pounded with garlic are rubbed on the teeth and also used as gargle to treat bad breath. Tabernaemontana malaccensis Hk.f. Apocynaceae., Lada-lada. Pounded root paste of the whole plant is applied on aching and painful parts. Talauma mutabilis Bl. Magnoliaceae., Kepala landak. A decoction of the roots is drunk to stimulate bowel movement. The root decoction is mixed in bath water to promote general health. Tamarindus indica L. Caesalpiniaceae ., Asam celagi. Infusion of the fruits is drunk and applied on the body against fever. The fruit is fried together with rice and rubbed on the skin to get rid of body smell. Infusion of the fruits is used to

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H.C. Ong, M. Nordiana r Fitoterapia 70 (1999) 502 513

soak the feet to improve the skin. A decoction of the fruits mixed with ginger is drunk to treat asthma. Tetracera indica Merr. Dilleniaceae ., Mempelas. Decoction of the climbing stems and roots is drunk to reduce high blood pressure. The leaves, crushed and mixed with water, are applied on the whole body to treat fever. Tharacostachyum bancanum Kurz. Cyperaceae., Senayan. The fruits are eaten three times per day as a diuretic. Tinospora crispa Diels. Menispermaceae., Patawali. Dried stems, pounded finely, are eaten with bananas or betle leaves to treat high blood pressure. A decoction of the dried stems can be used for the same purpose and also taken to prevent malaria. In fact, it is claimed that mosquitoes and other insects will not bite a person consuming this plant regularly. A paste of the stem is topically applied on aches and pains. Vernonia cinerea Less. Asteraceae ., Ekor kuda. The juice of the finely pounded whole plant is drunk to treat cancer. Vitex pubescens Vahl. Verbenaceae ., Halban. A paste obtained by pounding the leaves is applied on cuts and wounds. Zea mays L. Poaceae., Jagung. Juice obtained from grated grains is applied on the face everyday as skincare and to get rid of pimples. A decoction of corn silk is drunk regularly to cure high blood pressure. Zingiber officinale Rosc. Zingiberaceae., Halia. A decoction of the rhizome mixed with dried chillies and garlic is drunk to treat abdominal swellings and stomachache. Pounded rhizome is wrapped in a piece of cloth and rubbed on skin with fungal infection. Young rhizomes are eaten raw for healthier skin. Juice obtained from the rhizome mixed with egg yolk and honey is drunk to treat chest pains. Pounded rhizome mixed with a little sugar and vinegar is rubbed on the abdomen and hips of women after childbirth to promote physical recovery.

4. Discussion This study shows that knowledge and usage of herbal medicine for the treatment of various ailments among Machang villagers is still a major part of their life and culture. They use forest plants, weeds, fruit plants, vegetables, spices, ornamental plants, ferns and many other plants as traditional medicine. Although many of these species are known as medicinal plants, others are mainly used for nonmedicinal purposes. Even among the known medicinal plant species, there are variations and differences in the methods of utilisation and ailments treated. The data collected show many plant species used for external complaints, i.e. skin ailments. Medicinal plants used after childbirth are also numerous and this conforms with the importance placed on post-natal physical recovery among the Malay communities. Numerous species are used for problems associated with the gastrointestinal system and high blood pressure, which are common medical problems encountered in most communities.

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Acknowledgements The authors are very grateful to the Institute of Biological Sciences, University of Malaya, for providing research fund for this project and the people of Machang for sharing their knowledge on herbal medicine.

References
w1x Pemetaan J. Atlas Progresif Fajar Bakti, Penerbit Fajar Bakti Mapping Department. Progressive atlas of Fajar Bakti. Fajar Bakti Publishers .. Malaysia: Petaling Jaya, 1986. w2x Henderson MR. Malayan wild flowers. Dicotyledons. Kuala Lumpur, Malaysia: The Malayan Nature Society, 1974. w3x Henderson MR. Malayan wild flowers. Monocotyledons. Kuala Lumpur, Malaysia: The Malayan Nature Society, 1974. w4x Holttum RE. The Gardens Bulletin XIII 1950:1. w5x Hou D, Larsen K, Larsen SS. Flora Malesiana Ser. I 1996;12:409. w6x Ng FSP. Tree flora of Malaya. A manual for foresters, vol. 3. Malaysia: Longman, 1978. w7x Ng FSP. Tree flora of Malaya. A manual for foresters, vol. 4. Malaysia: Longman, 1989. w8x Whitmore TC. Tree flora of Malaya. A manual for foresters, vol. 1. Malaysia: Longman, 1972.

Fitoterapia 70 1999. 514 516

Short report

Cleome hirta essential oil as livestock tick Rhipicephalus appendiculatus/ and maize weevil Sitophilus zeamais/ repellent
M.W. Ndungua , S.C. Chhabraa,U , W. Lwande b
b

Chemistry Department, Kenyatta Uni ersity, P.O. Box 43844, Nairobi, Kenya Beha ioural and Chemical Ecology Department, International Centre of Insect Physiology and Ecology, P.O. Box 30772, Nairobi, Kenya

Received 23 November 1998; accepted in revised form 30 March 1999

Abstract The repellency of the essential oil of the shrub Cleome hirta and of three identified constituents phytol, q.-cedrol, n-octacosane. was evaluated against the livestock tick, Rhipicephalus appendiculatus and the maize weevil, Sitophillus zeamais. In a tick climbing repellency bioassay, the oil exhibited repellency which, at the highest dose, was comparable to that of the commercial arthropod repellent N, N-diethyltoluamide DEET.. In a Y-tube olfactometer bioassay, the oil showed higher or comparable repellency against S. zeamais relative to DEET at all the doses tested. The potential of C. hirta in livestock tick and maize weevil control is discussed. 1999 Elsevier Science B.V. All rights reserved.
Keywords: Cleome hirta; Essential oil; Rhipicephalus appendiculatus; Sitophillus zeamais; Repellent activity

Plant. Cleome hirta Oliv. Capparidaceae., fresh aerial parts collected from Konza, Machakos District, Kenya in August 1992 and authenticated by Mr Simon Mathenge. A voucher specimen No. 92r2. is deposited in the University of Nairobi Herbarium.
U

Corresponding author. Tel.: q254-2-8109-01; fax: q254-2-8115-75 E-mail address: ku-chem@thorntree.com S.C. Chhabra.

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Uses in traditional medicine. The leaf is used to treat stomachache and eaten as a vegetable in east and west Africa w1x. Previously isolated constituents. No report. New-isolated constituents. q.-Cedrol relative %, by GC-MS: 2.3., phytol 8.9., pulegone 8.7., n-octacosane 1.2. and terpinen-4-ol 1.1.. Tested material. Essential oil obtained by hydrodistillation yield: 0.43%., therein identified GC-MS. constituents, q.-cedrol, phytol, n-octacosane Aldrich Chemical Co., England. and N, N-diethyl-m-toluamide DEET. as active reference substance w2x. Pulegone was not tested since not available in sufficient amount. Studied activity. a. Maize weevil repellency determined by Y-tube olfactometer w3x. b. Livestock tick repellency determined by tick climbing repellency w4x. Used microorganisms. a. Maize weevils Sitophilus zeamais. obtained from a laboratory colony and reared under ambient conditions with natural photoperiods on untreated insecticide-free . maize seeds. Weevils of mixed age and sex were used in the bioassays w3x. b. Livestock ticks Rhipicephalus appendiculatus. reared at the International Centre of Insect Physiology and Ecology, Nairobi, Kenya. The ticks were maintained by feeding on the ears of rabbits w5x. Results. Reported in Table 1 maize weevil repellency. and Table 2 livestock tick repellency..

Conclusions The oil of C leome hirta aerial parts is repellent to livestock tick and maize weevil. Also in view of previous observations made with Gynandropsis gynandra, C. hirta could represent an attractive candidate for field evaluation as an anti-tick

Table 1 Maize weevil repellency of the essential oil of Cleome hirta aerial parts and its identified constituents a Sample % Repellency at mlrdisc 0.1 C. hirta q.-Cedrol Phytol n-Octacosane DEET reference .
a

0.01 43.0 " 0.4 59.9 " 0.8 9.1 " 0.1 39.3 " 0.3 40.5 " 0.2

0.001 37.6 " 9.4 41.8 " 0.5 y7.3 " 0.8 34.4 " 0.6 33.8 " 0.2

0.0001 37.1 " 0.3 30.9 " 0.7 y9.9 " 4.4 28.6 " 0.7 25.6 " 0.1

75.7 " 1.4 65.7 " 1.2 38.2 " 0.1 41.0 " 0.4 52.2 " 0.3

Values are mean " S.E., n s 5.

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M.W. Ndungu et al. r Fitoterapia 70 (1999) 514 516

Table 2 Livestock tick repellency of the essential oil of Cleome hirta aerial parts and its identified constituents a Sample % Repellency at mlrdisc 0.1 C. hirta q.-Cedrol Phytol n-Octacosane DEET reference .
a

0.01 84.0 " 3.9 21.1 " 1.4 39.9 " 2.9 41.1 " 2.9 82.8 " 3.6

0.001 24.1 " 0.0 18.0 " 1.3 34.8 " 2.6 26.7 " 2.9 75.6 " 4.5

0.0001 18.2 " 1.2 19.1 " 1.0 23.0 " 2.5 70.5 " 3.6

89.9 " 0.0 29.7 " 5.0 48.4 " 1.8 53.7 " 4.3 84.0 " 3.9

Values are mean " S.E., n " 5.

pasture plant w6,7x. In addition, C. hirta oil could find use in post-harvest protection of grain in traditional silos or storage pots. Examination of the repellency activities of C. hirta constituents against both R. appendiculatus and S. zeamais seems to indicate that the repellent action of the oil is due to an additive effect of its constituents. Moreover, the general pattern of repellency activities of the identified components Tables 1 and 2. suggests that structural requirements for repellency are different in the two organisms. This means that the whole essential oil, rather than isolated constituents, might constitute an agent with a sufficiently broad spectrum of activity for use as a general purpose arthropod repellent.

Acknowledgements The authors are grateful to Mr S. Mathenge of Botany Department, Nairobi University, Nairobi for plant identification, and to Mr O. Wanyama of International Centre for Insect Physiology and Ecology, Nairobi for the help rendered in obtaining GC-MS spectra of the oils.

References
w1x Watt JM, Breyer-Brandwijk MG. Medicinal and poisonous plants of southern and eastern Africa. 2 Edinburgh: E.& S. Livingstone Ltd, 1962:161. w2x Hassanali A, Lwande W, Ole-Sitayo N, Moreka L, Nokoe S, Chapya A. Discov Innov 1990;2:91. w3x Smith CN, Gilbert IH, Gouck HK, Bowman MC. USDA Ars Tech Bull 1963:36. w4x Ndungu M, Lwande W, Hassanali A, Moreka L, Chhabra SC. Entomol Exp Appl 1995;76:217. w5x Bailey KP. Bull Epizoot Dis Afr 1960;8:33. w6x Malonza MM, Dipeolu OO, Amoo AO, Hassan SM. Vet Parasitol 1992;42:123. w7x Dipeolu OO, Mongi AO, Punyua DK, Latif AA, Amoo OA, Odhiambo TR. Discov Innov 1992;4:35.

Fitoterapia 70 1999. 517 520

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Antimicrobial properties of Indigofera dendroides leaves


C.O. Esimone a,U , M.U. Adikwua , K.N. Muko b
a

Department of Pharmaceutics, Pharmaceutical Microbiology Unit, Uni ersity of Nigeria, Nsukka, Nigeria b Department of Pharmacognosy, Uni ersity of Nigeria, Nsukka, Nigeria

Received 5 October 1998; accepted in revised form 30 March 1999

Abstract The antibacterial and antifungal activities of the aqueous, petroleum ether, methanol extracts and eight TLC fractions of the methanol extract of Indigofera dendroides leaves have been evaluated. 1999 Elsevier Science B.V. All rights reserved.
Keywords: Indigofera dendroides; Antibacterial activity; Antifungal activity

Plant. Indigofera dendroides fresh leaves Papilionaceae., collected around Nsukka, Nigeria, in September 1997 and identified by Mr A.O. Ozioko of the Botany Department, University of Nigeria, Nsukka. Voucher specimens have been deposited at the herbarium of the Department of Pharmacognosy, University of Nigeria, Nsukka. Use in traditional medicine. In skin diseases, severe inflammations and for gargles w1x. Tested material. Petroleum ether, methanol and aqueous extracts and eight TLC
U

Corresponding author. Fax: q234-42-770644

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Table 1 Phytochemical screening of Indigofera dendroides leaf extracts a Tested material Petroleum ether extract Aqueous extract Methanol extract Mn1 Mn2 Mn3 Mn4 Mn5 Mn6 Mn7 Mn8
a

Yield %. 4.03 6.25 15.12 0.21 4.68 2.27 2.10 0.98 1.12 4.05 0.76

Positive tests for Tannins, flavonoids, cyanogenic glycosides Carbohydrates, saponins Flavonoids, carbohydrates, cyanogenic glycosides, tannins, saponins n.t. Saponins Tannins Tannins n.t. Carbohydrates Flavonoids, cyanogenic glycosides n.t.

Mn 1 8: TLC fractions of methanol extract yields are % of the methanol extract.; n.t., not tested.

fractions Mn 1 8. of the methanol extract. The relevant yields and results of phytochemical screening w2,3x are given in Table 1. Previously isolated classes of constituents. No report. Studied activity. Antibacterial and antifungal activity by disc diffusion technique w4x and minimum inhibitory concentration MIC. determination by agar diffusion method w5x. Used microorganisms. Listed in Tables 2 and 3. Results. Reported in Table 1 yields and phytochemical screening., Table 2 antibacterial and antifungal screening. and Table 3 MICs of fractions Mn3 and Mn 7.. Conclusions. All tested extractives showed a wide spectrum of activity against bacteria and fungi. Most of the activity of methanol extract was concentrated in fractions Mn3 and Mn7. The obtained results may provide a support to some uses of the plant in traditional medicine.

Acknowledgements The authors acknowledge the help of Mr Kalu Ogboso of the Pharmaceutical Microbiology Unit of the University of Nigeria, Nsukka.

C.O. Esimone et al. r Fitoterapia 70 (1999) 517 520

Table 2 Antibacterial and antifungal activity of I. dendroides extractives a Microorganisms Extracts 40 mgrdisc. P.E. Staphylococcus aureus Bacillus subtilis Escherichia coli Salmonella typhimurium Klebsiella pneumoniae Pseudomonas aeruginosa Candida albicans Aspergillus niger qqq qqqq qqq y qqq q qq q M.E. qq qq qq q qq y qqq qq A.E. qq qq qq y qq y q q TLC fractions 14 mgrdisc. Mn1 q y q y y y y y Mn2 q q q y qq q y y Mn3 qqq qqq qqq qq qq qq q q Mn4 y y y y y y qqq qqq Mn5 y y y y q y y y Mn6 q y y y y y y y Mn7 qqq qq qq qq qq q q y Mn8 q y y q y y y y Gentamycin 50 grdisc. qqq qqqqq qqq qqq qqqq qq n.t. n.t. Clotrimazole 20 grdisc. n.t. n.t. n.t. n.t. n.t. n.t. qqqqq qqq

a P.E., petroleum ether extract; M.E., methanol extract; A.E., aqueous extract; Mn1 8, TLC fractions of M.E. Inhibition zone diameter: q, 3 6 mm; qq, 7 11 mm; qqq, 12 17 mm; qqqq, 18 24 mm; qqqqq, ) 25 mm; n.t., not tested; and , no inhibition.

519

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C.O. Esimone et al. r Fitoterapia 70 (1999) 517 520

Table 3 Minimum inhibitory concentration MIC. of TLC fractions Mn3 and Mn7 from the methanol extract of Indigofera dendroides leaves Microorganism MIC grml. Mn3 Staphylococcus aureus Bacillus subtilis Escherichia coli Klebsiella pneumoniae Pseudomonas aeruginosa 1400 1200 2200 3400 4200 Mn7 1600 1200 2000 3800 4600 Gentamycin 15.9 15.0 13.2 18.4 17.8

References
w1x Watt JM, Breyer-Brandwijk MC. Medicinal and poisonous plants of southern and east Africa. Edinburgh: E. and S. Livingstone, 1962. w2x Iwu MM, Chiori CO. Fitoterapia 1984;6:355. w3x Trease GE, Evans WC. Pharmacognosy, XII ed. London: Baillere Tindall, 1989. w4x Bauer AW, Kirby WMM, Sherris JC, Turck M. Am J Clin Pathol 1966;45:493. w5x Ebi GC, Ofoefule SI. Phytother Res 1997;11:150.

Fitoterapia 70 1999. 521 522

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Anti-inflammatory activity of Butea frondosa leaves


S.A. MengiU , S.G. Deshpande
C.U. Shah College of Pharmacy, S.N.D.T. Womens Uni ersity, Santacruz (W), Mumbai-400 049, India

Received 27 May 1998; accepted in revised form 8 April 1999

Abstract In the carrageenan-induced rat paw oedema, the aqueous extract of Butea frondosa leaves showed dose-dependent 25 100 mgrkg, p.o.. anti-inflammatory activity which at the highest dose was almost comparable to ibuprofen 25 mgrkg, p.o...The LD50 was higher than 5 grkg, p.o. in rats. 1999 Elsevier Science B.V. All rights reserved.
Keywords: Butea frondosa; Anti-inflammatory activity

Plant. Butea frondosa Koen. ex Roxb. Papilionaceae., leaves collected in May 1995, from a village in Maharashtra, India and authenticated at St. Xaviers Blatters Herbarium, Mumbai, India. Leaves were shade-dried for 48 h and coarsely powdered. Uses in traditional medicine. In inflammatory conditions, skin diseases, worm infestation and haemorrhoids w1,2x. Previously isolated constituents. No report. Tested material. Water extract yield: 14%.. Phytochemical screening w3,4x gave positive results for flavonoid glycosides, proteins and amino acids.
U

Corresponding author. Fax: q91-22-6162-040

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Studied activity. Anti-inflammatory activity determined by carrageenan induced rat paw oedema w5x. Acute toxicity determined by Weils method w6x. Animals. Wistar rats of either sex weighing 180 220 g., maintained in standard environmental conditions and fed with standard diet and water ad libitum, were used. Results. Reported in Table 1 anti-inflammatory activity.. LD50 was found to be ) 5 grkg, p.o.
Table 1 Effect of Butea frondosa leaf water extract on carrageenan-induced rat paw oedema Treatment Dose mgrkg, p.o.. Paw volumea ml. 4.5 " 0.09 Inhibition %. 0

Control saline 1 mlrkg, p.o.. Ibuprofen Butea frondosa

25 25 50 100
U

3.0 " 0.07U 3.9 " 0.02U 3.5 " 0.06U 3.1 " 0.03U

100 39 66 93

Values are mean q S.E.M., n q 6; P - 0.001 vs. control; Students t-test.

Conclusions. Butea frondosa leaf water extract showed dose-dependent antioedematogenic activity which supports the traditional use of the plant as an anti-inflammatory.

References
w1x Jain SK. Dictionary of Indian folk medicine and ethnobotany. New Delhi: Deep Publications, 1991:340. w2x Sivrajan VV, Balachandran J. Ayurvedic drugs and their plant sources. New Delhi: Oxford & IBH publishing Co. Pvt. Ltd, 1994:340. w3x Kokate CK, Parikh KM. Practical pharmacognosy. 1 Delhi, India: Vallabh Prakashan, 1989:111. w4x Trease EG, Evans WC. Pharmacognosy. London: ELBS, 1992:132. w5x Winter LA, Risley EA, Nuss GW. Proc Soc Biol Med 1962;111:544. w6x Weil CS. Biometrics 1952;8:249.

Fitoterapia 70 1999. 523 525

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Potamogeton nodosus: an aquatic source for bioactive compounds


Zenefar Alam, M. Mostaqul Huq, A. Jabbar, C.M. HasanU
Department of Pharmacy, Faculty of Pharmacy, Uni ersity of Dhaka, Dhaka-1000, Bangladesh

Received 8 March 1999; accepted 25 March 1999

Abstract 2-Hydroxyheptane-3,5-dione 1. was obtained from the petrol-soluble fraction PE. of the ethanol extract of Potamogeton nodosus. Biological screening of PE revealed antibacterial activity and cytotoxicity. Compound 1 showed only antibacterial activity. 1998 Elsevier Science B.V. All rights reserved.
Keywords: Potamogeton nodosus; 2-Hydroxyheptane-3,5-dione; Aliphatics; Antibacterial activity; Cytotoxicity

Plant. Potamogeton nodosus Poir. Potamogetonaceae., fresh submerged aquatic herb, collected from a dead river of Chuadanga District, Bangladesh, in April 1997 and identified by Bangladesh National Herbarium, Dhaka. Uses in traditional medicine. The plant has been reported to be active against cancer, tuberculosis, acne, common cough and cold, wounds and abdominal discomfort w1x.
U

Corresponding author. Tel.: q880-2-505420; fax: q880-2-865583 E-mail address: cmhasan@citechco.net C.M. Hasan.

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Table 1 In vitro antibacterial activity of extractives from Potamogeton nodosusa Microorganisms Diameter of zone of inhibition mm. PE 250 grdisc. Gram-positive Bacillus megaterium QL38 Bacillus cereus QL29 Bacillus subtilis QL40 Sarcina lutea QL166 Staphylococcus aureus ATCC25923 Gram-negative Escherichia coli FPFC281 Klebsiella sp. Pseudomonas aeruginosa CRL Shigella flexneri AL30372 Shigella boydii AL17313 Shigella sonnei AJ8992 Salmonella paratyphi-B AG1407 Vibrio mimicus CRL Vibrio parahaemophylia CRL Vibrio cholerae AD66
a

1 200 grdisc. 14 nd nd nd 12

Kanamycin 30 grdisc. 32 29 30 31 30

12 7 13 10

12 10 12 8 10 8 11

12 nd nd 10 9 7 nd nd

29 35 34 27 29 25 30 29 30 32

, No inhibition; nd, not done due scarcity of sample; PE, petrol-soluble fraction of ethanol extract; 1, 2-hydroxyheptane-3,5-dione.

Previously isolated constituents. An antibacterial furanoid diterpene, 15,16-epoxy12-oxo-817.,1316.,14-labdatrien-19,20-olide w2x. New-isolated constituent. 2-Hydroxyheptane-3,5-dione 1., yield 0.01%, as colorless, fragrant oil, IR bands KBr.: 3450, 2900, 1710, 1410, 1260, 1150 cmy1 ; 1 H-NMR 500 MHz, CDCl 3 .: 4.12 1H, q, J 7 Hz, H-2., 3.66 2H, s, H-4., 2.31 2H, q, J 7.32 Hz, H-6., 1.6 3H, d, J 7 Hz, H-1., 0.88 3H, t , J 7.32 Hz, H-7.. Tested material. Petrol-soluble fraction of the ethanol extract and therefrom isolated compound 1. Studied activity. Antibacterial activity by disc diffusion method w3x and cytotoxicity by brine shrimp lethality bioassay w4,5x. Used microorganisms. The test microorganisms see Table 1. were obtained from the Institute of Nutrition and Food Science INFS., University of Dhaka, Bangladesh. Results. Reported in Table 1 antibacterial activity.. The LC 50 median lethal concentration . of the brine shrimp lethality bioassay was 35.36 and 150 grml for PE and compound 1, respectively.

Z. Alam et al. r Fitoterapia 70 (1999) 523 525

525

Conclusions. Both PE and compound 1 showed antibacterial activity against most test microorganisms. PE exhibited also moderate cytotoxicity, not observed with compound 1. The obtained results may provide a support to some uses of the plants in traditional medicine.

Acknowledgements The authors wish to thank Md. Shawkat Ali, on Ph.D. study in Japan, for recording the NMR spectrum of the compound.

References
w1x Bisaws SK, Ghosh SE. Bharatio Banaushadhi, vol. II, 2nd ed. Calcutta: Calcutta University Press, 1977:1457 1459. w2x Qais N, Mandal MR, Rashid MA et al. J Nat Prod 1998;61:156. w3x Baurer AW, Kirby WMM, Sherris JC, Truck M. Am J Clin Pathol 1966;45:493. w4x Meyer BN, Ferrigni NR, Jacobsen LB, Nicholos DE, Mclaughlin JL. Planta Med 1982;45:31. w5x Goldstein AL, Arnold L, Kalkan SM. Principles of drug action. 2nd ed. Wiley Biomedical Publication, 1974:376 381.

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Antimicrobial activity of the essential oils of five Eucalyptus species growing in Nigeria
Adebola O. Oyedeji a , Olusegun Ekundayo a,U , Olayide N. Olawore b, Bolanle A. Adeniyi c , Wilfried A. Koenig d
Department of Chemistry, Uni ersity of Ibadan, Ibadan, Nigeria Department of Chemistry, Ladoke Akintola Uni ersity of Technology, Ogbomoso, Nigeria c Department of Pharmaceutical Microbiology and Clinical Pharmacy, College of Medicine, Uni ersity of Ibadan, Ibadan, Nigeria d Institut fur Organische Chemie, Uni ersitat Hamburg, D-20146, Hamburg, Germany
b a

Received 4 November 1998; accepted in revised form 19 April 1999

Abstract At 5 mgrml concentration, the volatile oils from the leaves of five Eucalyptus spp growing in Nigeria exhibited considerable antibacterial activity against Gram-positive and Gramnegative microorganisms and antifungal activity against C. albicans. 1999 Elsevier Science B.V. All rights reserved.
Keywords: Eucalyptus spp; Essential oils; Antimicrobial activity

Plants. The fresh leaves matured. of Eucalyptus alba, E. deglupta, E. saligna, E. camaldulensis var. catharine and E. camaldulensis var. mysore Myrtaceae. were collected in April 1997 from Agodi Gardens, Ibadan and authenticated at Forest Research Institute of Nigeria FRIN., Ibadan, Nigeria by Mr T.K. Odewo. Voucher specimens were deposited at the FRIN Herbarium.
U

Corresponding author.

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Uses in traditional medicine. Eucalyptus leaf essential oils have been used in the treatment of lung diseases, as expectorant and cough stimulants w1 3x. They have also been reported to possess antitubercular properties w4x. Previously isolated classes of constituents. Tannins, flavonoids, and terpenes w5 9x. Tested material. Essential oils obtained by hydrodistillation according to British pharmacopoeia w10x: E. alba yield: 0.28%; main components m.c.,%.: -thujene 32.9, p-cymene 12.9, 1,8-cineole 13.3, -carophyllene 7.8.; E. deglupta 0.20%; m.c.: -pinene 24.7, E--nerolidol 34.8.; E. saligna 0.30%: m.c.: -thujene 63.8, 1,8cineole 12.3.; E. camaldulensis var. catharine 0.26%: m.c.: -pinene 8.8, -pinene 9.0, 1,8-cineole 70.4. and E. camaldulensis var. mysore -pinene 17.5%, 1,8-cineole 32.8%, Z--ocimene 11.6%.. Studied activity. Antibacterial and antifungal activities by agar disc diffusion method w11x. Used microorganisms. Listed in Table 1. Results. Reported in Table 1.

Table 1 Antimicrobial activity of essential oils from Eucalyptus spp growing in Nigeria Microorganisms Inhibition zone mm.a E1 Gram positive Staphylococcus aureus UCH560 S. aureus UCH 681 S. aureus UCH 511 Bacillus cereus Gram negative Escherichia coli NCTC 7001 E. coli UCH 307 E. coli UCH 270 P. aeruginosa NCTC 675 P. aeruginosa UCH 655 Fungus Candida albicans E2 10 11 10 11 E3 10 11 12 10 E4 10 12 12 E5 12 11 11 12 Amp Gen Tio

R R

14 10 12 14

n.t. n.t. n.t. n.t.

10 18 10

10 16 11 9 12

18 14

10 24 10 12

10 22 18 11

9 12 9

14 14 10

n.t. n.t. n.t. n.t. n.t.

12

12

14

13

n.t.

n.t.

16

a Values are the mean of three replicates. E 1 5 s essential oil of Eucalyptus alba, E. deglupta, E. saligna, E. camaldulensis var. catharine, E. camaldulensis var mysore, respectively in 10% DMSO dilution. 5 mgrml; Amp, ampicillin 2.5 grml; Gen, gentamycin 1 grml; Tio, tioconazole 5 grml; R, resistant; n.t., not tested; , no inhibition. NCTC, National Collection Type Cultures; UCH, clinical isolates, University of Ibadan, College Hospital Collection.

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A.O. Oyedeji et al. r Fitoterapia 70 (1999) 526 528

Conclusions. All the five essential oils were active against E. coli UCH 307 while at least four showed activity against B. cereus, S. aureus UCH 560 and UCH 511, E. coli NCTC 7001, and P. aeruginosa UCH 655. With the exception of E. alba, they also showed activity against C. albicans. The obtained results show a spectrum of antimicrobial activities which provides a support to some traditional uses of Eucalyptus essential oils.

Acknowledgements AOO is indebted to DAAD for a fellowship award. Financial support from the University of Ibadan Senate Research Grant is gratefully acknowledged.

References
w1x Goldstein M, Simonetti G, Watschinger M. Complete guide to trees and their identification. London: Macdonald & Co., Ltd, 1990:219. w2x Sticher ONew natural products and plant drugs with pharmacological, biological or theurapeutical activity. Berlin: Springer-Verlag, 1977:138. w3x Ngugen TT, Lai TV, Neguyen TTH. Tap Chi Duoc Hoc 1994;1:18. w4x Low D, Rawal BD, Griffin WJ. Planta Med 1974;26:184. w5x Ma Z, Porler LJ. Zhiwu Xuebao 1989;30:534. w6x Shen Z, Yu Q, Wang Y, Lin G. Linchan Huaxue Yu Gongye 1987;7:35. w7x Shen Z, Yu Q. Linchan Huaxue Yu Gongye 1987;7:28. w8x Franich RA. Phytochemistry 1986;25:245. w9x Weston RJ. Phytochemistry 1943;23:1984. w10x British Pharmacopoeia. London: HMS Stationary Office, vol. 2, 1980:A109. w11x Kavanagh F. Analytical microbiology. New York: Academic Press, 1972.

Fitoterapia 70 1999. 529 531

Phytochemical communication

Constituents from the roots of Acanthopanax setchuenensis


W.M. Zhao a,U , G.W. Qin a , R.S. Xua , X.Y. Li a , J.S. Liu b, Y. Wang b, M. Feng b
a

Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 200031, China b Sichuan Institute of Forestry, Chengdu 610061, China

Received 28 January 1999; accepted 30 March 1999

Keywords: Acanthopanax setchuenensis; Phenylpropanoids; Lignans; Flavonoids

Plant. Acanthopanax setchuenensis Harms roots, collected in Baoxing County, Sichuan Province, in May 1990 and identified by Dr Jiansheng Liu. A voucher specimen was deposited in the herbarium of Shanghai Institute of Materia Medica, Chinese Academy of Sciences. Uses in traditional medicine. The root bark of A. setchuenensis has been used, instead of other widely used Acanthopanax sp. in Shanxi and other provinces of China, as tonic, for treatment of rheumatism and to improve blood circulation w1x. Previously isolated constituents. No report. New-isolated constituents. Eugenyl -rutinoside 1. w2,3x 0.0013%., sasanquin 2. w4x 0.001., hesperidin 3. w5x 0.00053., q.-syringaresinol di-O--D-glucoside 0.00037., q.-syringaresinol O--D-glucoside 0.00067., q.-syringaresinol 0.00047., -sitosterol 0.00083. and daucosterol 0.00067..
U

Corresponding author. E-mail address: wmzhao@mail.scnc.ac.cn W.M. Zhao.

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W.M. Zhao et al. r Fitoterapia 70 (1999) 529 531

. w xq Eugenyl -rutinoside 1.. w x16 D y45.6 py, c 0.15 ; FAB-MS m r z : 495 M q Na q 1 and 511wM q Kx ; H-NMR 400 MHz, C 5 D5 N.: 7.60 1H, d, 8.1, H-6., 6.89 1H, br d, 8.1, H-5., 6.28 1H, s, H R-1 ., 5.95 1H, ddt, 16.7, 12.0, 6.4, H-8., 5.50 1H, d, 6.3, H G-1 ., 5.44 1H, dd, 16.7, 1.8, H-9a., 5.00 1H, dd, 12.0, 1.8, H-9b., 4.56 1H, br s, H r-2 ., 3.66 3H, s, OCH 3 ., 3.23 2H, br d, 6.4, H-7., 1.58 3H, d, 5.7, H R-6 .; 13 C-NMR 75 MHz, C 5 D5 N.: 146.7 C-1., 150.6 C-2., 114.1 C-3., 135.0 C-4., 121.7 C-5. , 118.1 C-6., 40.1 C-7., 138.5 C-8., 115.7 C-9., 56.3 OCH 3 ., 103.4 C G-1 ., 75.1 C G-2 ., 78.8 C G-3 ., 71.8 C G-4 ., 77.5 C G-5 ., 68.2 C G-6 ., 102.6 C R-1 ., 73.0 C R-2 ., 72.4 C R-3 ., 74.3 C R-4 ., 70.0 C R-5 ., 18.7 C R-6 .. . w xq and 497 Sasanquin 2.. w x16 D y49.5 py, c 0.08 ; FAB-MS m r z : 481 M q Na wM q Kxq; 1 H-NMR 400 MHz, C 5 D5 N.: 7.69 1H, d, 8.1, H-6., 6.88 1H, br d, 8.1, H-5., 6.85 1H, br s, H-3., 5.96 1H, ddt, 16.7, 12.1, 6.4, H-8., 5.57 1H, d, 6.9, H G-1 ., 5.04 1H, br d, 16.7, H-9a., 4.99 1H, br d, 12.1, H-9b., 4.98 1H, d, 7.5, H X-1 ., 3.69 3H, s, OCH 3 ., 3.26 2H, br d, 6.4, H-7.; 13 C-NMR 75 MHz, C 5 D5 N.: 146.3 C-1., 150.1 C-2., 113.4 C-3., 134.4 C-4., 121.5 C-5., 117.2 C-6., 39.9 C-7., 138.2 C-8., 115.5 C-9., 55.8 OCH 3 ., 102.7 C G-1 ., 74.7 C G-2 .U , 78.4 C G-3 ., 71.1 C G-4 ., 77.6 C G-5 ., 69.5 C G-6 ., 105.8 C X-1 ., 75.0 C X-2 .U , 78.1 C X-3 ., 71.1 C X-4 ., 67.0 C X-5 .. Data with U may be interchangeable w4x. Hesperidin 3.. IRmax KBr.: 3420, 1650, 1610, 1520 cmy1 ; 1 H-NMR 400 MHz, C 5 D5 N.: 7.51 1H, d, 1.6, H-2., 7.10 1H, dd, 8.2, 1.6, H-6., 6.98 1H, d, 8.2, H-5., 6.60 1H, d, 2.1, H-8., 6.48 1H, d, 2.1, H-6., 5.60 1H, d, 7.4, H G-1 ., 5.45 1H, br s, H R-1 ., 3.72 3H, s, 4-OCH 3 ., 3.20 1H, dd, 17.0, 12.8, H-3a., 2.84 1H, dd, 17.0, 2.8, H-3b., 1.56 3H, d, 5.8, H R-6 . w5x.

Acknowledgements We would like to thank the State Key Laboratory of New Drug Research for partial support of the work.

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References
w1x Jiangsu new Medical College, Dictionary of Chinese traditional medicine. Shanghai: Shanghai Science and Technology Publisher, 1986:381. w2x Orihara Y, Furuya T, Hashimoto N, Deguchi Y, Tokoro K, Kanasawa T. Phytochemistry 1992;31:827. w3x Sashida Y, Ori K, Mimaki Y. Chem Pharm Bull 1991;39:2362. w4x Yamada T, Aoki H, Tamura T, Sakamoto Y. Agr Biol Chem 1967;31:85. w5x Sadtler Research Laboratories Inc. Sadtler Standard Infrared Spectra. Philadelphia: Sadtler Research Laboratories, Inc., 1975;35:34600K.

Fitoterapia 70 1999. 532 535

Phytochemical communication

Constituents from the roots of Bowdichia irgilioides


L.S.M. Velozo, B.P. Da Silva, E.M.B. Da Silva, J.P. ParenteU
Nucleo de Pesquisas de Produtos Naturais, Uni ersidade Federal do Rio de Janeiro, 21941 590 Rio de Janeiro, Brazil Received 16 March 1999; accepted 16 April 1999

Abstract The isolation and 13 C-NMR data of isoflavone derivatives from Bowdichia irgilioides roots are reported. 1999 Elsevier Science B.V. All rights reserved.
Keywords: Bowdichia irgilioides; Isoflavones

Plant. Bowdichia irgilioides Kunt. Papilionoideae. roots, collected at Pac o do Lumiar, Maranhao, Brazil, in March 1997 and identified by Dr T.J.A.S. Rego. A voucher specimen no. 1137. is deposited at Atico Seabra Herbarium, Sao Luiz, MA, Brazil. Uses in traditional medicine. The plant, known in folk medicine as sucupira is popularly used to treat rheumatism, arthritis and diabetes w1x. Previously isolated constituents. Terpenoids, phenolics, ketones w2 6x. New-isolated constituents. Odoratin (1) w7x 0.003%., afromosin (2) w8,9x 0.002., wistin (3) w10,11x 0.004., cladrastin (4) w12x 0.002., cladrastin 7-O--D-glucoside (5)
U

Corresponding author. Tel.: q55-270-2683; fax: q55-270-2683. E-mail address: adm@nppn.ufrj.br J.P. Parente .

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w13x 0.003., fujikinetin (6) w14,15x 0.002., fujikinin (7) w13x 0.001., isohemiphloin (8) w16x 0.008.. 13 C-NMR data on compounds 2, 3 and 6 are available in the literature w9,11,16x. Odoratin (1). 13 C-NMR 50 MHz, DMSO-d 6 .: 152.65 C-2., 122.89 C-3., 174.23 C-4., 116.32 C-4a., 104.80 C-5., 146.98 C-6., 55.83 O C H 3-6., 151.69 C-7.,

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L.S.M. Velozo et al. r Fitoterapia 70 (1999) 532 535

102.85 C-8., 152.86 C-8a., 125.02 C-1., 116.59 C-2., 146.01 C-3., 147.55 C-4., 55.68 OCH 3-4., 111.92 C-5., 119.75 C-6.. Cladrastin (4). 13 C-NMR 50 MHz, DMSO-d 6 .: 152.84 C-2., 123.33 C-3., 174.57 C-4., 116.54 C-4a., 104.42 C-5., 147.16 C-6., 55.85 O C H 3-6., 151.75 C-7., 102.68 C-8., 152.98 C-8a., 124.91 C-1., 112.95 C-2., 148.80 C-3., 55.64 OCH 3-3., 148.50 C-4., 55.68 OCH 3-4., 111.52 C-5., 121.21 C-6.. Cladrastin 7-O--D-glucoside (5). 13 C-NMR 50 MHz, DMSO-d 6 .: 153.46 C-2., 122.88 C-3., 174.29 C-4., 117.85 C-4a., 104.82 C-5., 147.52 C-6., 55.88 OCH 3-6., 151.24 C-7., 103.46 C-8., 151.60 C-8a., 124.28 C-1., 112.86 C-2., 148.63 C-3., 55.64 OCH 3-3., 148.32 C-4., 55.68 OCH 3-4., 111.64 C-5., 121.19 C-6., 99.96 C-1., 73.28 C-2., 77.37 C-3., 69.85 C-4., 76.89 C-5., 60.91 C-6.. Fujikinin (7). 13 C-NMR 50 MHz, DMSO-d 6 .: 153.28 C-2., 122.80 C-3., 174.25 C-4., 117.79 C-4a., 104.80 C-5., 147.50 C-6., 55.86 OCH 3-6., 155.18 C-7., 103.48 C-8., 151.58 C-8a., 124.60 C-1., 109.38 C-2., 146.98 C-3., 146.90 C-4., 101.03 O-CH 2-O., 108.09 C-5., 122.35 C-6., 99.69 C-1., 73.05 C-2., 77.22 C-3., 69.63 C-4., 76.76 C-5., 60.67 C-6.. Isohemiphloin (8).13 C-NMR 50 MHz, DMSO-d 6 .: 78.45 C-2., 42.10 C-3., 196.60 C-4., 101.64 C-4a., 161.84 C-5., 95.00 C-6., 166.16 C-7., 105.93 C-8., 162.90 C-8a., 129.00 C-1., 128.45 C-2 and C-6., 115.38 C-3 and C-5., 157.84 C-4., 73.14 C-1., 70.46 C-2., 79.18 C-3., 70.79 C-4., 81.59 C-5., 61.67 C-6..

Acknowledgements The authors are grateful to CNPq, CAPES, FUJB, PADCT and UFMA for financial support.

References
w1x Friese FW. Plantas medicinais brasileiras. Sao do Estado de Sao Paulo: Instituto Agronomico Paulo, 1934:252. w2x Calle AJ, Rivera UA, Moreno E. Rev Colomb Cienc Quim Farm 1983;4:93. w3x Torrenegra GR, Escarria RS, Bauereiss P, Achenbach H. Planta Med 1985;51:276. w4x Torrenegra R, Bauereiss P, Achenbach H. Phytochemistry 1989;28:2219. w5x Marinho LC, Da Cunha CMTM, Thomas G, Barbosa Filho JM. Fitoterapia 1994;65:475. w6x Arriaga AMC, Machado MIL, Gomes GA, Craveiro AA. J Essent Oil Res 1998;10:205. w7x Hayashi T, Thomson RH. Phytochemistry 1943;13:1974. w8x McMurry TBH, Theng CY. J Chem Soc 1960:1491. w9x Jha HC, Zilliken F, Breitmaier E. Can J Chem 1980;58:1211. w10x Shibata S, Nakahara M. Chem. Pharm. Bull. 1963;11:372.

L.S.M. Velozo et al. r Fitoterapia 70 (1999) 532 535 w11x w12x w13x w14x w15x w16x Tostes JBF, Silva AJR, Parente JP. Phytochemistry 1999;50:1087. Shamma M, Stiver LD. Tetrahedron 1969;25:3887. Ohashi H, Goto M, Imamura H. Phytochemistry 1976;15:354. Imamura H, Hibino Y, Ohashi H. Mokuzai Gakkaishi 1972;18:325. Rao EV, Murthy MSR, Ward RS. Phytochemistry 1984;23:1493. Hillis WE, Horn DHS. Aust J Chem 1965;18:531.

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Phytochemical communication

Constituents of Haplopappus sonorensis


R. Encarnacion Dimayugaa , J.I. Murillo a , G. Malmstrmb, C. Christophersen b,U
a

Departamento de Agronoma, de Baja California Sur, A. P. 19-B, La Uni ersidad Autonoma Paz, B.C.S. 23080, Mexico b Marine Chemistry Section, The H. C. rsted Institute, Uni ersity of Copenhagen, Uni ersitetsparken 5, DK-2100 Copenhagen, Denmark

Received 25 February 1999; accepted 23 March 1999

Abstract The isolation and NMR spectra of 5,7-dihydroxy-3,4X-dimethoxyflavone from Haplopappus sonorensis are reported. 1999 Elsevier Science B.V. All rights reserved.
Keywords: Haplopappus sonorensis; 5,7-Dihydroxy-3,4 -dimethoxyflavone; Flavonoids; Triterpenoids
X

Plant. Haplopappus sonorensis A. Gray. S. F. Blake Asteraceae ., known locally as Hierba del Pasmo, was collected in Cabo Pulmo, B.C.S. Mexico, in August 1988 and identified by Ing. Jorge Agundez Espinoza from the Botany Laboratory, Universidad Autonoma de Baja California Sur, Mexico. A duplicate voucher specimen is deposited in the Botanical Department of the Biology Institute of Universidad Nacional Autonoma de Mexico. Uses in traditional medicine. Used against skin ulcer, cold, general infections, heart troubles, headache, toothache, cough, tetanus, wounds, stings of poisonous animals and badly smelling feet w1,2x.
U

Corresponding author. Tel.: q45-35-32-01-57, fax: q45-35-32-02-12. E-mail address: carsten@kiku.dk C. Christophersen.

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537

Previously isolated compounds. 5-Hydroxy-3,7,4X-trimethoxyflavone w3x. New-isolated constituents. 5,7-Dihydroxy-3,4X-dimethoxyflavone w4x 1. yield: 0.035%., friedelin w5x 0.0015. and friedelan-3 -ol w5x 0.0011.. The yields are based on dry plant weight. 5,7-Dihydroxy-3,4X-dimethoxyfla one 1.. 1 H-NMR 400 MHz, DMSO-d6 .: 12.6 1H, s, OH., 10.8 1H, s, OH., 8.02 2H, dd, J 9.0r3.0., 7.13 2H, dd, J 8.8r3.0., 6.45 1H, d, J 2.0., 6.21 1H, d,J 2.0., 3.86 3H, s, OMe., 3.78 3H, s, OMe.; 13 C-NMR 100.6 MHz, DMSO-d6 .: 178.0 C-4., 164.3 C-7., 161.4 C-9., 161.3 C-4X ., 156.5 C-5., 155.2 C-2., 138.0 C-3., 130.0 C-6X ., 122.2 C-1X ., 114.3 C-3X ., 104.3 C-10., 98.7 C-6., 93.8 C-8., 59.8 C-3-OMe., 55.5 C-4X-OMe.; EIMS mrz : 314 Mq, 100., 28524., 27182..

Acknowledgements Financial support was obtained from FOMES 97 and UABCS. We are grateful to Juan Gonzales Velasco and Elodia Ruiz for technical assistance.

References
w1x Encarnacion DR. Medicina tradicional y popular de Baja California Sur. 1a ed. Artes graficas. Mexico: UABCS, 1996. w2x Christophersen C, Larsen C, Encarnacion DR. Traditional medicine a potential resource. Exploitation of natural products. Copenhagen: The HC rsted Institute, 1991. w3x Gajhede M, Encarnacion DR, Christophersen C, Nielsen PH, Leal GC, Patino JC. Acta Cryst. C 1990;45:2012. w4x Calvert DJ, Cambie RC, Davis BR. Org Magn Res 1979;12:583. w5x Ho L-K, Chang C-R, Chang Y-S. J Chinese Chem Soc 1995;42:93.