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Anthelmintic activity of steroidal saponins from Paris polyphylla.

ABSTRACT

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 ([EC.sub.50] value = 18.06 mg [l.sup.-1]). Based on these finding, 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 significant activity against D. intermedius with [EC.sub.50] values of 0.44 and 0.70 mg [l.sub.-1], respectively, which were more effective than the positive control, mebendazole ([EC.sub.50] value = 1.25 mg [l.sup.-1]). The acute toxicities ([LC.sub.50]) of dioscin and polyphyllin D for goldfish were 1.37 and 1.08 mg [l.sup.-1], 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 first report on in vivo anthelmintic investigation for P. polyphylla.

ARTICLE INFO

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

Crown Copyright [C] 2010 Published by Elsevier GmbH. All rights reserved.

Introduction

The continuing growth of fish 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 fish 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 significant efficacy 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 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 efficacy 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 significant 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 goldfish of the methanol extract and the active compounds were evaluated. We also speculate on the mode of action of the active isolated saponins.

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 35-40 [degrees]C and pulverized in electric grinder. The powdered plant samples were stored at -20 [degrees]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 (151 x3 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.

[FIGURE OMITTED]

In vivo bioassays

For the in vivo bioassays, the test samples were dissolved in DMSO at a concentration of 10 mg [ml.sup.-1] for the pure compounds, 0.5 g[ml.sup.-1](sample/solvent) for the crude extracts and stored at -20[degrees]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 goldfish, with a mean mass of 3.7 g ([+ or -]0.3) were obtained from Changxing fish farm (Xian yang city, Shaanxi province, China) and maintained in 180-1 glass aquarium receiving filtered groundwater. The fish 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 fish were prepared following the protocol described in our previous research (Wang et al. 2008). Briefly, this procedure included the collection of parasite eggs, hatching of eggs, and re-infection of the parasites. Water parameters such as dissolved oxygen (saturation 70-84%) and pH (7.7-8.4) were monitored daily, and water was heated (22-24 [degrees]C) to favour parasite transmission. After three weeks of post-infestation, five fish were randomly sampled, killed by spinal severance, and eight gill filaments of each fish were biopsied to determine the adult D. intermedius infestation level and intensity under a light microscope (Olympus BX41, Tokyo, Japan) at 10x4 magnification 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 fish).

For acute toxicity tests, parasite-free goldfish were also obtained from the commercial fish farm and maintained in a 180-1 glass aquarium supplied with filtered groundwater under the same conditions as parasitized fish. On arrival, the absence of the parasites was carefully checked by examining ten fish randomly selected.

Anthelmintic study

The anthelmintic efficacy 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). Briefly, the assays were conducted in 51 plastic pot containing 21 of the working solutions and five infected goldfish. Initial tests were undertaken to determine the concentration boundaries for the efficacy tests, and the final 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 fish mortality was recorded and the surviving goldfish in all the treatment and control groups were biopsied under a light microscope at 4x10 magnification as described above. The anthelmintic efficacy 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) x 100%/B] (1)

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

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 [l.sup.-1] for methanol extract, 1.3-1.5 mg [l.sup.-1] for compound 1, and 1.0-1.2 mg [l.sup.-1] for compound 2 (based on initial tests). The fish 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 fish were transferred to freshwater. Mortality response was noted after 48 h of exposure, during which no food was offered to the fish.

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 [EC.sub.50] responding to the active fractions or compounds at the 95% confidence level with upper confidence limit and lower confidence limit, while [LC.sub.50] for the methanol extract and active compounds with 95% confidence 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 fish 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 identified (Wang et al. 2008, 2009), with high efficacy 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, five 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 significant activity with [EC.sub.50] value of 18.06 mg [l.sup.-1], which prompted us to perform a bioassay-guided fractionation of the anthelmintic effect. The bioactive methanol extract was performed on further isolation and purification using various chromatographic techniques and two active compounds were obtained.

The anthelmintic efficacy 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 [EC.sub.50] values of 0.44 and 0.70 mg [l.sup.-1], respectively, and they were 41 and 26-fold more effective than methanol extract ([EC.sub.50] = 18.06 mg [l.sup.-1]). These two compounds showed extraordinary activity when compared with mebendazole, which is widely used for the control of D. intermedins in practice ([EC.sub.50] value = 1.25 mg [l.sup.-1]) (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).
Table 1
Anthelmintic activity and acute toxicity of the methanol extract and
compounds after 48 h.

  Test samples             Anthelmintic efficacy

                  Slope [+ or -] SE   [EC.sub.50] (mg[l.sup.-1])
                                                (95% CL)

Methanol extract  4.87 [+ or -] 0.58      18.06 (15.77-20.26)
Dioscin           6.77 [+ or -] 1.33       0.44 (0.37-0.49)
Polyphyllin D     9.27 [+ or -] 1.31       0.70 (0.65-0.74)
Mebendazole (a)   2.74 [+ or -] 0.50       1.25 (1.00-1.46)

  Test samples               Acute toxicity

                  Slope [+ or -] SE    [LC.sub.50] (mg [l.sup.-1])
                                                 (95% CL)

Methanol extract  16.21 [+ or -] 2.42      34.60 (30.62-37.70)
Dioscin           44.63 [+ or -] 6.35      1.37 (1.35-1.39)
Polyphyllin D     29.18 [+ or -] 4.57      1.08 (0.99-1.15)
Mebendazole (a)   ND                       ND

CL, confidence level; ND, not determined.
(a) Positive control.


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 antiinflammatory 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 [IC.sub.50] values being at the [micro]M level. The high cytotoxic potency of the two saponins may responsible for the significant anthelmintic activity and high toxicity for the goldfish regarding the two compounds (dioscin and PD) with [LC.sub.50] values of 1.31 and 1.08 mg [l.sup.-1] 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. intermedins. Both compounds can be chosen as lead compounds for the development of new antiparasitic agents against Dactylogyrus. Also, a further study about the steroidal saponins structure-activity relationship and structure modification 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 identification assistance.

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* Corresponding authors. Tel.: +86 29 87092102.

E-mail addresses: wanggaoxue@126.com, wanggaoxue@nwsuaf.edu.cn (G.-X. Wang).

0944-7113/$-see front mailer. Crown Copyright [C]2010 Published by Elsevier GmbH. All rights reserved.

G.-X. Wang *, J. Han, L.-W. Zhao, D.-X. Jiang, Y.-T. Liu, X.-L. Liu *

Northwest A&F University, Yangling 712100, China

doi:10.1016/j.phymed.2010.04.012
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Author:Wang, G.-X.; Han, J.; Zhao, L.-W.; Jiang, D.-X.; Liu, Y.-T.; Liu, X.-L.
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