In Vitro. Screening of Cleome Viscosa. Extract for Anthelmintic Activity

Abstract

The crude alcohol and aqueous extracts of the seeds of Cleome viscosa. Linn. (Capparidaceae) were investigated for their anthelmintic activity against Pheretima posthuma. and Ascardia galli.. Various concentrations (10–100 mg/mL) of each extract were tested in the bioassay, which involved determination of time of paralysis and time of death of the worms. Both extracts exhibited considerable anthelmintic activity in a dose-dependent manner. The most significant activity was observed at the highest concentration of 100 mg/mL against both types of worms. Piperazine citrate (10 mg/mL) was included as standard reference and distilled water as control.

Keywords:

• Anthelmintic activity
• Ascardia galli
• Cleome viscose
• Pheretima posthuma
• piperazine citrate

Introduction

Cleome viscosa. Linn (Capparidaceae), commonly known as “hul-hul,” is an annual common weed found all over the plains of India and throughout the tropics of the world. In the Ayurvedic system of medicine, this plant is believed to have several medicinal properties such as stomachic, laxative, diuretic, anthelmintic, and it is considered useful in skin diseases, itching, ulcers, leprosy, and malarial fevers (Chatterjee & Prakashi, 1991). Several therapeutic uses such as carminative, anthelmintic, and antiseptic have been ascribed to the seeds of C. viscosa. in the Unani system of medicine. The seeds are also given in cases of fevers and diarrhea (Kirtikar & Basu, 1935; Nadkarni, 1982). Subsequent to a large number of claims about the wide range of traditional medicinal properties of the plant, considerable efforts have been made to verify its efficacy as a curative agent through pharmacological investigations. The plant is reported to possess antipyretic (Devi et al., 2003), antidiarrheal (Devi et al., 2002), antimicrobial (Devi et al., 2006; Sudhakar et al., 2006), anti-inflammatory (Parimala et al., 2003a), immunomodulatory (Tiwari et al., 2004), analgesic (Parimala et al., 2003b), and local anesthetic activities (Singh & West, 1991).

Traditional medicines hold a great promise as sources of easily available, effective anthelmintic agents to the people, particularly in tropical developing countries, including India. It is in this context that the people consume several plants or plant-derived preparations to cure helminthic infections (Satyavati, 1990). Continuing our studies of the Capparidaceae plants for anthelmintic activity (Mali et al., 20042005), in the current study we evaluated anthelmintic potential of alcohol and aqueous extracts of seeds of C. viscosa..

Materials and Methods

Plant collection and authentication

Dried seeds of C. viscosa.were collected from the local market. The seeds were identified and authenticated by Prof. D.A. Patil, Head, PG Department of Botany, SSVPS'S LK Dr. P.R. Ghogrey Science College (Dhule, India), and a voucher specimen (RGM/05/10) was deposited.

Plant extraction

The seeds were cleaned and ground with the help of an electric grinder. The powder obtained was filtered through fine muslin cloth and was transferred to thimble of Whatman filter paper no. 1 in a Soxhlet apparatus. Ethyl alcohol was used as a solvent for alcohol extract, whereas distilled water was used for aqueous extract. The solvent was allowed to evaporate in a rotary vacuum evaporator, and after complete removal of solvent, percentage yields were estimated and plant extracts were stored in a refrigerator until further use.

Worm collection and authentication

Indian earthworms Pheretima posthuma. L. Vaill (Annelida) were collected from the waterlogged areas of soil, and Ascardia galli. Schrank (Nematode) worms were obtained from freshly slaughtered fowls Gallus gallus. Spadiceus (Phasianidae). Both worm types were identified at the P.G. Department of Zoology, Pratap College (Amalner, Maharashtra, India).

Anthelmintic assay

The anthelmintic assay was carried as per the method of Ajaiyeoba et al. (2001) with necessary modifications. Formulations (50 mL) containing different concentrations of crude extracts (10, 50, and 100 mg/mL in distilled water) were prepared, and six worms (same type) were placed in it. This was done for both types of worms. Time for paralysis was noted when no movement of any sort could be observed except when the worms were shaken vigorously. Time for death of worms were recorded after ascertaining that worms neither moved when shaken vigorously nor when dipped in warm water (50°C). Piperazine citrate (10 mg/mL) was used as reference standard and distilled water served as the control.

Statistical analysis

Results were analyzed using one-way analysis of variance (ANOVA) followed by Dunnett's test. The value of p < 0.05 was considered as significant.

Results and Discussion

The percentage yields of extracts and the results of the anthelmintic studies of alcohol and aqueous extracts of seeds of C. viscosa. are presented in Table 1. The assay was performed on adult Indian earthworm, Pheretima posthuma., due to its anatomic and physiologic resemblance to the intestinal roundworm parasite of human beings (Chatterjee, 1967; Vidyarthi, 1967; Thorn et al., 1977; Vigar, 1984). Because of easy availability, earthworms have been used widely for the initial evaluation of anthelmintic compounds in vitro. (Sollmann, 1918; Jain & Jain, 1972; Dash et al., 2002; Shivkar & Kumar, 2003; Szewezuk et al., 2003). Ascardia galli. worms are easily and plentily available from freshly slaughtered fowls, and its use as a suitable model for screening of anthelmintic drug was advocated earlier (Kaushik et al., 1974; Lal et al., 1976; Tandon et al., 1997).

Table 1.. Anthelmintic activity of alcohol and aqueous extract of C. viscose. seeds.

			Time taken for paralysis (P) and death (D) of worms in minutes (±SEM)
			P. posthuma.		A. galli.
Test substrate	Yield (%)	Concentration (mg/mL)	P	D	P	D
Control	—	—	—	—	—	—
Alcohol extract	10.21	10	39.63 ± 0.29	78.29 ± 0.19	27.27 ± 0.23	68 ± 0.33
		50	23.84 ± 0.23	63.14 ± 0.28	19.9 ± 0.27	44.7 ± 0.33
		100	11.35 ± 0.20***	23.97 ± 0.28***	9.18 ± 0.27*	22.6 ± 0.31***
Aqueous extract	6.32	10	23.57 ± 0.31	57.81 ± 0.22	32 ± 0.30	62.7 ± 0.31
		50	16.03 ± 0.20**	43.80 ± 0.21**	22.6 ± 0.30	47.1 ± 0.27
		100	5.30 ± 0.24***	22 ± 0.28***	6.8 ± 0.20**	22.8 ± 0.31***
Piperazine citrate	—	10	20.70 ± 0.31	59.94 ± 0.31	12.79 ± 0.22	40.76 ± 0.17

Values are expressed as mean ± SEM (n = 6). Significantly different from standard at *p < 0.05; **p < 0.01; ***p < 0.001.

As shown in Table 1, the alcohol and aqueous extracts of seeds of C. viscosa. displayed significant anthelmintic properties at higher concentrations. Both the extracts showed anthelmintic activities in dose-dependent manner giving shortest time of paralysis (P) and death (D) with 100 mg/mL concentration, for both types of worms. The alcohol extract of C. viscosa. caused paralysis of 11 min and time of death of 23 min, and aqueous extract showed P and D of 5 and 22 min against the earthworm P. posthuma.. The reference drug piperazine citrate showed the same at 20 and 59 min.

Ascardia galli. worms also showed sensitivity to the alcohol and aqueous extracts of C. viscosa. (Table 1). The alcohol extract caused paralysis of 9 min and time of death of 22 min, and aqueous extract displayed P and D of 6 and 22 min. Piperazine citrate did the same in 12 and 40 min.

The predominant effect of piperazine citrate on the worm is to cause a flaccid paralysis that results in expulsion of the worm by peristalsis. Piperazine citrate by increasing chloride ion conductance of worm muscle membrane produces hyperpolarization and reduced excitability that leads to muscle relaxation and flaccid paralysis (Martin, 1985). The seed extracts of C. viscosa. not only demonstrated paralysis but also caused death of worms especially at the higher concentration of 100 mg/mL.

In conclusion, the traditional use of seeds of the plant Cleome viscosa. as an anthelmintic has been confirmed, as the seed extracts displayed anthelmintic properties against the worms used in the study. Further, it would be interesting to isolate the possible constituents that are responsible for anthelmintic activity and establish the possible mechanism(s) of action. We are working on isolation of anthelmintic compounds from these extracts, and this will be reported at a later date.