Phytomedicine 17 (2010) 11021105

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Phytomedicine
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Short communication

Anthelmintic activity of steroidal saponins from Paris polyphylla

G.-X. Wang , J. Han, L.-W. Zhao, D.-X. Jiang, Y.-T. Liu, X.-L. Liu
Northwest A&F University, Yangling 712100, China

a r t i c l e

i n f o

a b s t r a c t
The present study was undertaken to investigate the anthelmintic activity of crude extracts and pure compounds from the rhizomes of Paris polyphylla. The methanol extract showed a promising anthelmintic activity against Dactylogyrus intermedius (EC50 value = 18.06 mg l1 ). Based on these nding, the methanol extract was fractionated on silica gel column chromatography in a bioassay-guided fractionation affording two known steroidal saponins showing potent activity, dioscin (1) and polyphyllin D (2). Both dioscin and polyphyllin D exhibited signicant activity against D. intermedius with EC50 values of 0.44 and 0.70 mg l1 , respectively, which were more effective than the positive control, mebendazole (EC50 value = 1.25 mg l1 ). The acute toxicities (LC50 ) of dioscin and polyphyllin D for goldsh were 1.37 and 1.08 mg l1 , respectively. These results indicated that P. polyphylla extract and the isolated compounds are potential natural agents for the control of Dactylogyrus infestation. This is the rst report on in vivo anthelmintic investigation for P. polyphylla. Crown Copyright 2010 Published by Elsevier GmbH. All rights reserved.

Keywords: Paris polyphylla Anthelmintic activity Steroidal saponins Dactylogyrus intermedius Polyphyllin D Dioscin

Introduction The continuing growth of sh aquaculture worldwide has been accompanied by the increasing importance of parasites as agents of disease. Monogenean parasite, Dactylogyrus intermedius is endemic ectoparasite in Asia, Central Europe, Middle East and North America (Paperna 1964), cause serious economic damage in aquaculture industry in these regions. They are usually attached to the gills of freshwater sh causing irritation, excessive mucus production, accelerated respiration and mixed infection with other pathogen (Dove and Ernst 1998; Reed et al. 2009), leading to a serious damage to the host such as loss of appetite, lower growth performance and high mortalities. Use of traditional parasiticides including praziquantel (Schmahl and Mehlhorn 1985), mebendazole (Buchmann et al. 1993) and trichlorphon (Prost and Studnicka 1966) in the control of Dactylogyrus is well known. Another synthetic symmetric triazinone, toltrazuril, which had signicant efcacy in treating Dactylogyrus infestation by bath treatment (Schmahl and Mehlhorn 1988). However, the frequent use of these chemical-based agents for the control of the parasites can be problematic, resulting in the potential deleterious effects on both the environment and the human consumers of their products due to public health implications of residues (Committee on Mutagenicity of Chemicals in Food, Consumer Products and the Environment 1999) as well as the rise

Corresponding authors. Tel.: +86 29 87092102. E-mail addresses: wanggaoxue@126.com, wanggaoxue@nwsuaf.edu.cn (G.-X. Wang).

in drug-resistant parasites (Goven et al. 1980), which has led to an increasing search for new antiparasitic for the control of Dactylogyrus infestation. Plant secondary metabolites have been used for centuries in traditional medicine and therefore represent a source of potentially active compounds (Bourgaud et al. 2001). Antiparasitic plant-derived compounds have been used as leads to develop semi-synthetic or synthetic drugs with better efcacy and safety (Tagboto and Townson 2001). Rhizoma Paridis (Chonglou in Chinese), refers to the dried rhizomes of Paris polyphylla Smith var. chinensis (Franch.) Hara (PPY) of Lilaceae family, mostly distributed in the southwest of China, is widely used in traditional Chinese medicine for antifebrile, alexipharmic, detumescent, demulcent, hemostatic, and the treatment of hepatopathy. It is the main component of Yun-nan-bai-yao and Ji-desheng-she-yao-pian, which are well-known Chinese medicine preparations. Previous phytochemical research revealed that steroidal saponins constitute the major bioactive components in P. polyphylla (Zhang 2007), which have been shown to have signicant biological activities including antitumor (Wu et al. 2004; Lee et al. 2005; Sun et al. 2007), antifungal (Deng et al. 2008), and inhibitory activities against abnormal uterine bleeding (Tian et al. 1986; Fu et al. 2008). However, there is little information available on the use of this plant for the control of monogenean parasite D. intermedius. The principal objective of this study was to assess the anthelmintic properties of P. polyphylla and isolate the active constituents responsible for the activity using in vivo anthelmintic assay associated with bioassay-guided fractionation. Additionally, the acute toxicity for the goldsh of the methanol extract and the active compounds were evaluated. We also speculate on the mode of action of the active isolated saponins.

0944-7113/$ see front matter. Crown Copyright 2010 Published by Elsevier GmbH. All rights reserved. doi:10.1016/j.phymed.2010.04.012

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Materials and methods Plant material Rhizomes of P. polyphylla var. yunnanensis (PPY) were collected in Yunnan province, China, in October 2007 and authenticated by Prof. X.L. He in Northwest A&F University, China. A voucher specimen has been deposited in the Herbarium of College of Life Science of the university. They were cleaned and air dried for a week at 3540 C and pulverized in electric grinder. The powdered plant samples were stored at 20 C until further use. Extraction and isolation procedure The plant samples were extracted with petroleum ether, chloroform, ethyl acetate, methanol, and water, respectively. Portion of each extract was dried under vacuum and assayed for anthelmintic activity in vivo. Results revealed activity in the methanol extract, a large-scale of plant sample (3.0 kg) were extracted with methanol (15 l 3 times) at room temperature for 24 h. The extract was evaporated to dryness under reduced pressure in a rotary evaporator to yield the crude extract (343.0 g). This extract was subjected to column chromatograph and successively eluted with petroleum ether/ethyl acetate/methanol gradients. Repetition of the chromatographic separations and recrystallization led to the isolation of two active compounds, dioscin (1, 235.0 mg) and polyphyllin D (2, 175.0 mg). The structures of dioscin and polyphyllin D (PD) were established by nuclear magnetic resonance (NMR) data by comparison with literature values (Deng et al. 1999; Hu et al. 1996; Yoon and Kim 2008), are shown in Fig. 1. In vivo bioassays For the in vivo bioassays, the test samples were dissolved in DMSO at a concentration of 10 mg ml1 for the pure compounds, 0.5 g ml1 (sample/solvent) for the crude extracts and stored at 20 C until further required. The highest concentration of DMSO in the treatment was less than 1% (preliminary analysis with 1% DMSO showed no anthelmintic activity). Parasites and hosts Healthy goldsh, with a mean mass of 3.7 g (0.3) were obtained from Changxing sh farm (Xian yang city, Shaanxi province, China) and maintained in 180-l glass aquarium receiving ltered groundwater. The sh were acclimatized under laboratory conditions for 7 days and cohabitated with the ones infected with D. intermedius

which were reared in the laboratory. The parasitized sh were prepared following the protocol described in our previous research (Wang et al. 2008). Briey, this procedure included the collection of parasite eggs, hatching of eggs, and re-infection of the parasites. Water parameters such as dissolved oxygen (saturation 7084%) and pH (7.78.4) were monitored daily, and water was heated (2224 C) to favour parasite transmission. After three weeks of post-infestation, ve sh were randomly sampled, killed by spinal severance, and eight gill laments of each sh were biopsied to determine the adult D. intermedius infestation level and intensity under a light microscope (Olympus BX41, Tokyo, Japan) at 10 4 magnication before they were used for the assays. Fish were chosen for the experiments when the infestation rate reached 100% and moderate infestation intensity was obtained (> 30 per sh). For acute toxicity tests, parasite-free goldsh were also obtained from the commercial sh farm and maintained in a 180-l glass aquarium supplied with ltered groundwater under the same conditions as parasitized sh. On arrival, the absence of the parasites was carefully checked by examining ten sh randomly selected. Anthelmintic study The anthelmintic efcacy of the crude extracts and fractions derived from the methanol extract as well as the pure compounds were determined following the protocol described in a previous work (Wang et al. 2008). Briey, the assays were conducted in 5 l plastic pot containing 2 l of the working solutions and ve infected goldsh. Initial tests were undertaken to determine the concentration boundaries for the efcacy tests, and the nal tests for conducted with three replicates. The test samples were assayed at a different series of concentrations which were prepared from the stock solutions. Control groups with no chemical were set under the same experimental conditions as the test groups. Mebendazole was used as the positive control. After 48 h treatment, the sh mortality was recorded and the surviving goldsh in all the treatment and control groups were biopsied under a light microscope at 4 10 magnication as described above. The anthelmintic efcacy of tested samples was determined by comparison of the number of parasites in the treatments with those in the control groups, and calculated using the following equation described in our previous work (Wang et al. 2008): AE = [B T (P) 100%/B (1)

where AE is the anthelmintic efcacy, B is the mean number of the surviving parasites in the negative group and P in the positive control and T is the treatment.

Fig. 1. Structures of polyphyllin D and dioscin from methanol extract of P. polyphylla.

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Acute toxicity Acute toxicities of the methanol extract and pure compounds were performed as described in our previous work (Wang et al. 2008). Each sample was assayed at a different series of concentrations ranged between 30.0 and 45.0 mg l1 for methanol extract, 1.31.5 mg l1 for compound 1, and 1.01.2 mg l1 for compound 2 (based on initial tests). The sh were carefully observed for any signs of distress indicative of toxic insult such as increased respiration frequency and erratic behavior. Under there circumstances, the experiments were stopped and sh were transferred to freshwater. Mortality response was noted after 48 h of exposure, during which no food was offered to the sh. Statistical analysis The standard error was calculated by using the software developed by Reddy et al. (1992). Probit analysis (Finney 1971) was used for the calculation of the EC50 responding to the active fractions or compounds at the 95% condence level with upper condence limit and lower condence limit, while LC50 for the methanol extract and active compounds with 95% condence level. Results and discussion Application of plants or their extracts to control monogenean parasites has been extensively studied during recent decades. Compounds from plant extracts provide a potential alternative to existing parasiticides, based on promising results to control parasites with various plant species, such as methanol extracts of the seeds of Piper guineense (Piperaceae), showed promising activity against monogenean parasites (Ekanem et al. 2004). In our continuous efforts to develop sustainable control against Dactylogyrus infestation in sh aquaculture, attempts were made to identify plants showing promising anthelmintic properties, such as osthol and isopimpinellin from Fructus Cnidii and two lignan compounds, arctigenin and arctiin from Fructus Arctii were identied (Wang et al. 2008, 2009), with high efcacy and low toxicity. In the present study, we described the pharmacological activity obtained from the methanol extract of the rhizomes of P. polyphylla and the novel pharmacological activity of steroidal saponins, polyphyllin D (PD) and dioscin, against monogenean parasite, D. intermedius. In order to make fully evaluation of the plant, ve extracts by different solvents (petroleum ether, chloroform, ethyl acetate, methanol, and water) of increasing polarity were assayed for the anthelmintic activity. Among them, only the methanol extract exhibited the most signicant activity with EC50 value of 18.06 mg l1 , which prompted us to perform a bioassay-guided fractionation of the anthelmintic effect. The bioactive methanol extract was performed on further isolation and purication using various chromatographic techniques and two active compounds were obtained. The anthelmintic efcacy of the isolated compounds was compared with the original methanol extract, as shown in Table 1.

The two active steroidal saponins, PD and dioscin, showed better activity than the methanol extract, which indicated that the two active saponins were responsible for the anthelmintic activity. In our assay conditions, PD and dioscin exhibited a remarkable anthelmintic activity, with calculated EC50 values of 0.44 and 0.70 mg l1 , respectively, and they were 41 and 26-fold more effective than methanol extract (EC50 = 18.06 mg l1 ). These two compounds showed extraordinary activity when compared with mebendazole, which is widely used for the control of D. intermedius in practice (EC50 value = 1.25 mg l1 ) (Buchmann et al. 1993). More interestingly, dioscin displayed higher activity then PD did, which may contribute to the different sugar moiety in the two compounds. This was in accordance with the previous report on steroidal saponins that the activity of rhamnopyanoside was stronger than that of arabinofuranoside-containing compound, although the number of glycosides was the same; the varieties of glycosides were different (Yan et al. 2009). The two diosgenyl saponins, dioscin and PD, are steroidal glycosides, bear the diosgenin as the aglyone and they are often found as major components in many traditional medicines. Various biological activities have been observed for these two saponins, including antibacterial (Hufford et al. 1988), antiviral (Ikeda et al. 2000), antifungal (Sautour et al. 2004; Takechi et al. 1991), and antiinammatory activities (Kim et al. 1999). Recently, they were found to exhibit cytotoxic properties against several strains of human cancer cells (Cai et al. 2002; Cheung et al. 2005; Mimaki et al. 2001), with their IC50 values being at the M level. The high cytotoxic potency of the two saponins may responsible for the signicant anthelmintic activity and high toxicity for the goldsh regarding the two compounds (dioscin and PD) with LC50 values of 1.31 and 1.08 mg l1 in the present study. Recent research addressed that the main biologic activity ascribed to saponins was their membrane permeabilizing property (Menin et al. 2001; Plock et al. 2001). The main actions were considered changes in membrane permeability and pore formation (Melzig et al. 2001; Seeman et al. 1973), which is similar with two conventional anthelmintic drugs such as praziquantel (Schmahl and Taraschewski 1987), and toltrazuril (Schmahl et al. 1988); That is, they would affect the permeability of the cell membrane of the parasites and cause causes vacuolization and disintegration of monogenea teguments. As recent researches indicated, mitochondria were the major cellular target of dioscin in its induction of apoptosis in HL-60 cells and that dioscin exerted its cytotoxicity through multiple apoptosis-inducing pathways (Wang et al. 2006). Cheung et al. (2005) showed that PD elicited cell apoptosis through mitochondrial dysfunction and cell cycle arrest. As is well known, mitochondria are the key players that control and regulate apoptosis; a direct action on mitochondria might be involved in the eradication of the parasites. However, the detailed mechanism of action regarding the anthelmintic activity of the two steroidal saponins should be further addressed. Our results revealed that steroidal saponins, dioscin and PD showed remarkable activity against monogenean parasite, D. intermedius. Both compounds can be chosen as lead compounds for

Table 1 Anthelmintic activity and acute toxicity of the methanol extract and compounds after 48 h. Test samples Anthelmintic efcacy Slope SE Methanol extract Dioscin Polyphyllin D Mebendazolea 4.87 6.77 9.27 2.74 0.58 1.33 1.31 0.50 EC50 (mg l1 ) (95% CL) 18.06 (15.7720.26) 0.44 (0.370.49) 0.70 (0.650.74) 1.25 (1.001.46) Acute toxicity Slope SE 16.21 2.42 44.63 6.35 29.18 4.57 ND LC50 (mg l1 ) (95% CL) 34.60 (30.6237.70) 1.37 (1.351.39) 1.08 (0.991.15) ND

CL, condence level; ND, not determined. a Positive control.

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the development of new antiparasitic agents against Dactylogyrus. Also, a further study about the steroidal saponins structureactivity relationship and structure modication with strong antiparasitic activity and low toxicity will need to be performed. Acknowledgements This work was supported by post-doctoral programs and we thank Doctor J. Yang for structure identication assistance. References
Buchmann, K., Slotved, H.C., Dana, D., 1993. Epidemiology of gill parasite infections in Cyprinus carpio in Indonesia and possible control methods. Aquaculture 118, 921. Bourgaud, F., Gravot, A., Milesi, E., Gontier, E., 2001. Production of plant secondary metabolites: an historical perspective. Plant Sci. 161, 839851. Cai, J., Liu, M.J., Wang, Z., Ju, Y., 2002. Apoptosis induced by dioscin in HeLa cells. Biol. Pharm. Bull. 25 (2), 193196. Cheung, J.Y., Ong, R.C., Suen, Y.K., Ooi, V., Wong, H.N., Mak, T.C., et al., 2005. Polyphyllin D is a potent apoptosis inducer in drug-resistant HepG2 cells. Cancer Lett. 217 (2), 203211. Committee on Mutagenicity of Chemicals in Food, Consumer Products and the Environment, 1999. Statement for COT: Malachite Green and Leucomalachite Green. Department of Health, London, http://archive.food.gov.uk/dept health/ pdf/mala.pdf. Deng, D.W., Lauren, D.R., Cooney, J.M., Jensen, D.J., Wurms, K.V., Upritchard, J.E., Cannon, R.D., Wang, M.Z., Li, M.Z., 2008. Antifungal saponins from Paris polyphylla Smith. Planta Med. 74, 13971402. Dove, A., Ernst, I., 1998. Concurrent invadersfour exotic species of Monogenea now established on exotic freshwater shes in Australia. Int. J. Parasitol. 28, 17551764. Deng, S.J., Yu, Biao., Hui, Y.Z., Yu, Hai., Han, X.W., 1999. Synthesis of three diosgenyl saponins: dioscin, polyphyllin D, and balanitin 7. Carbohyd. Res. 317, 5362. Ekanem, A., Wang, M., Simon, J., Obiekezie, A., Morah, F., 2004. In vivo and in vitro activities of the seed extract of Piper guineense Schum. and Thonn. against skin and gill monogenean parasites of goldsh (Carassius auratus auratus). Phytother. Res. 18, 793797. Finney, D.J., 1971. Probit Analysis, 3rd edn. Cambridge University Press, Cambridge. Fu, Y.L., Yu, Z.Y., Tang, X.M., Zhao, Y., Yuan, X.L., Wang, S., Ma, B.P., Cong, Y.W., 2008. Pennogenin glycosides with a spirostanol structure are strong platelet agonists: structural requirement for activity and mode of platelet agonist synergism. J. Thromb. Haemost. 6, 524533. Goven, B., Gilbert, J., Gratzek, J., 1980. Apparent drug resistance to the organophosphate dimethyl (2,2,2-trichloro-1-hydroxyethyl) phosphonate by monogenetic trematodes. J. Wildlife Dis. 16 (3), 343346. Hufford, C.D., Liu, S., Clark, M.A., 1988. Antifungal activity of Trillium grandiorum constituents. J. Nat. Prod. 51, 9498. Hu, K., Dong, A., Yao, X., Kobayashi, H., Iwasaki, S., 1996. Antineoplastic agents. I. Three spirostanol glycosides from rhizomes of Dioscorea collettii var. hypoglauca. Planta Med. 62 (6), 573575. Ikeda, T., Ando, J., Miyazono, A., Zhu, X.H., Tsumagari, H., Nohara, T., et al., 2000. Anti-herpes virus activity of steroidal glycosides. Biol. Pharm. Bull. 23, 363 364. Kim, S.Y., Son, K.H., Chang, H.W., Kang, S.S., Kim, H.P., 1999. Inhibition of mouse ear edema by steroidal and triterpenoid saponins. Arch. Pharm. Res. 22, 313316. Lee, M.S., Yuet-Wa, J.C., Kong, S.K., Yu, B., Eng-Choon, V.O., Nai-Ching, H.W., et al., 2005. Effects of polyphyllin D, a steroidal saponin in Paris polyphylla, in growth inhibition of human breast cancer cells and in xenograft. Cancer Biol. Ther. 4, 12481254. Melzig, M.F., Bader, G., Loose, R., 2001. Investigations of the mechanism of membrane activity of selected triterpenoid saponins. Planta Med. 67 (1), 4348.

Mimaki, Y., Yokosuka, A., Kuroda, M., Sashida, Y., 2001. Cytotoxic activities and structure-cytotoxic relationships of steroidal saponins. Biol. Pharm. Bull. 24 (11), 12861289. Menin, L., Panchichkina, M., Keriel, C., Olivares, J., Braun, U., Seppet, E.K., et al., 2001. Macrocompartmentation of total creatine in cardiomyocytes revisited. Mol. Cell Biochem. 220 (12), 149159. Prost, M., Studnicka, M., 1966. Investigations on the use of organic esters of phosphoric acid in the control of external parasites of farmed sh. II. Control of the invasion of parasites of Dactylogyrus and Gyrodactylus. Med. Weter. 22, 644650. Paperna, I., 1964. Adaptation of Dactylogyrus extensus (Muller and Van, 1932) to ecological conditions of articial ponds in Israel. J. Parasitol. 50, 9093. Plock, A., Sokolowska-Kohler, W., Presber, W., 2001. Application of ow cytometry and microscopical methods to characterize the effect of herbal drugs on Leishmania spp. Exp. Parasitol. 97 (3), 141153. Reed, P.A., Francis-Floyd, R., Klinger, R.C., 2009. FA28/FA033: Monogenean parasites of sh. EDIS-Electronic Data Information Source-UF/IFAS Extension. University of Florida. http://edis.ifas.u.edu/FA033. 17 May 2009. Reddy, P.J., Krishna, D., Murthy, U.S., Jamil, K., 1992. A microcomputer FORTRAN program for rapid determination of lethal concentration of biocides in mosquito control. CABIOS 8, 209213. Schmahl, G., Mehlhorn, H., 1985. Treatment of sh parasites. 1. Praziquantel effective against Monogenea (Dactylogyrus vastator, Dactylogyrus extensus, Diplozoon paradoxum). Z. Parasitenkd. 71, 727737. Schmahl, G., Mehlhorn, H., 1988. Treatment of sh parasites. 4. Effects of sym. Triazinone (toltrazuril) on monogenea. Parasitol Res. 75, 132143. Sun, J., Liu, B.R., Hu, W.J., Yu, L.X., Qian, X.P., 2007. In vitro anticancer activity of aqueous extracts and ethanol extracts of fteen traditional Chinese medicines on human digestive tumor cell lines. Phytother. Res. 21, 11021104. Sautour, M., Mitaine-Offer, A.C., Miyamoto, T., et al., 2004. Antifungal steroid saponins from Dioscorea cayenensis. Planta Med. 70, 9092. Seeman, P., Cheng, D., Iles, G.H., 1973. Structure of membrane holes in osmotic and saponin hemolysis. J. Cell Biol. 56 (2), 519527. Schmahl, G., Mehlhorn, H., Haberkorn, A., 1988. Sym. Triazinone (toltrazuril) effective against sh-parasitizing Monogenea. Parasitol. Res. 75 (1), 6768. Schmahl, G., Taraschewski, H., 1987. Treatment of sh parasites. 2. Effects of praziquantel, miclosamide, levamisole hydrochloride and metrifonate on Monogenea (Gyrodactylus aculeati, Diplozoon paradoxum). Parasitol. Res. 73, 341351. Tagboto, S., Townson, S., 2001. Antiparasitic properties of medicinal plants and other naturally occurring products. Adv. Parasitol. 50, 199295. Tian, Y., Zheng, L.H., Xu, Z.Y., Sun, L.Q., Gao, C.K., Zheng, Q.Z., Zhang, Z.H., Shu, Y.J., 1986. Clinical and pharmacological study of the hemostatic action of Rhizoma Paridis by contraction of uterus. J. Trad. Chin. Med. 6, 178182. Takechi, M., Shimada, S., Tanaka, Y., 1991. Structureactivity relationships of the saponins dioscin and dioscinin. Phytochemistry 30, 39433944. Wang, Y., Cheung, Y.H., Yang, Z.Q., et al., 2006. Proteomic approach to study the cytotoxicity of dioscin (saponin). Proteomics 6, 24222432. Wu, S.S., Gao, W.Y., Duan, H.Q., Jia, W., 2004. Advances in studies on chemical constituents and pharmacological activities of Rhizoma Paridis. Chin. Trad. Herba Drugs. 35, 344347. Wang, G.X., Zhou, Z., Cheng, C., Yao, J.Y., Yang, Z.W., 2008. Osthol and isopimpinellin from Fructus cnidii for the control of Dactylogyrus intermedius in Carassius auratus. Vet. Parasitol. 158, 144151. Wang, G.X., Han, J., Feng, T.T., Li, F.Y., Zhu, B., 2009. Bioassay-guided isolation and identication of active compounds from Fructus Arctii against Dactylogyrus intermedius (Monogenea) in goldsh (Carassius auratus). Parasitol. Res. 106, 247255. Yoon, K., Kim, J., 2008. Preparative separation of dioscin derivatives from Dioscorea villosa by centrifugal partition chromatography coupled with evaporative light scattering detection. J. Sep. Sci. 31, 24862491. Yan, L.L., Zhang, Y.L., Gao, W.Y., Man, S.L., Wang, Y., 2009. In vitro and in vivo anticancer activity of steroid saponins of Paris polyphylla var. yunnanensis. Exp. Oncol. 31 (1), 2732. Zhang, S.P., 2007. Research progression chemical constituents and pharmacological effect of Genus Paris. Strait Pharm. J. 19 (16), 47.