INTRODUCTION:

Fruit of Citrus medica is known as citron. The plant of Citrus medica was probably originated in Northern India and has long been cultivated in South-Eastern Asia. The citron especially sounds to be native to India bordering Myanmar, where it is found in valleys at the foot of the Himalaya Mountains, and in the Western Ghats. It is a small thorny tree with attractive purple and white flowers and a fruit resembling a large lemon. Fruit of Citrus medica have several medicinal properties. It is vata and pitta suppressant, a good pain relief anti-inflammatory agent as well as excellent appetizer and improves digestive condition in body. It also prevents dehydration in the body. It stimulates liver for its proper functioning. It is effective in wormal infestation. It also acts as a good heart toner and helps in improving the blood circulation (1). Phytochemical analysis shows the presence of steroids, flavonoids and carbohydrates in the methanolic extract of irradiated and unirradiated Citrus medica fruit bio-mass. The present study was therefore undertaken to evaluate In-vitro anthelmintic activity of methanol extract of unirradiated and gamma irradiated Citrus medica fruit bio-mass.

MATERIALS AND METHODS:

Plant Material:

Fruits of Citrus medica were collected from National Research Center for Citrus (NRCC), Nagpur and authenticated by Department of Botany, Rashtrasant Tukadoji Maharaj Nagpur University, Nagpur.

Preparation of Extract:

The peels, seeds and juice of the fruit were removed. The solid bio-mass was washed with distilled water filtered at suction pump and dried at room temperature. The dried solid bio-mass was then powdered using grinder to have uniform size. It was divided into two parts 1) In this bio-mass was irradiated with gamma radiations (⁶⁰Co γ source GC-900 at a dose of ~ 0.7 kGy/hour) for 48 hrs. 2) The bio-mass was used without exposure to radiation. Both of these materials were defatted with petroleum ether (60-80 ⁰C) followed by methanol using Soxhlet extraction process. The solvent was evaporated under reduced pressure at 50 ⁰C and the extract thus obtained was used directly for the assessment of anthelmintic activity through in vitro method.

Anthelmintic Bioassays:

Indian earthworm pheretima posthuma (Annelida) was used for evaluating anthelmintic activity of crude extract using the reference substance for comparison. These were collected from the water logged areas of soil and identified at the Department of Zoology, Rashtrasant Tukadoji Maharaj Nagpur University, Nagpur, Maharashtra.

Preparation of test sample:

Samples for in-vitro study were prepared by dissolving and suspending 2.5 g of crude methanolic extract in 25 ml of distilled water to obtain a stock solution of 100 mg/ml. From this stock solution different working dilutions were prepared to get concentration range of 10, 25 and 50 mg/ml.

Anthelmintic activity:

The anthelmintic assay was carried as per the method of Ajayieoba E. O. et al² with minor modifications. The assay was performed on adult Indian earthworm Pheretima posthuma, due to its anatomical and physiological resemblance with the intestinal roundworm parasites of human beings^3-5. Pheretima posthuma worms are easily available and used as a suitable model for screening of anthelmintic drug was advocated earlier^6-11. 50 ml formulations containing three different concentrations, each of crude alcoholic extract and its various fractions (10, 25 and 50 mg/ml in distilled water) were prepared and five worms (same type) were placed in it. Time for paralysis was noted when no movement of any sort could be observed except the worms were shaken vigorously. Time for death of worms were recorded after ascertaining that the worms neither moved when shaken vigorously nor when dipped in warm water at 50 ⁰C^12-13. Piperazine citrate (10 mg/ml) was used as reference standard while distilled water as the control.

RESULTS AND DISCUSSION:

Preliminary phytochemical screening of methanol extract of unirradiated and Gamma

irradiated Citrus medica fruit bio-mass shows the presence of steroids, flavonoids and carbohydrate. Polyphenolic compounds are generally responsible for anthelmintic activity.

Table 1: Anthelmintic activity of alcoholic extract of Citrus medica

Sr. No	Extract	Conc (mg/ml)	Time taken for paralysis (P) and for death of *Pheretima posthuma* worms (D) in min.
Sr. No	Extract	Conc (mg/ml)	P	D
1)	MEUCM	10	36.39±0.24	43.05±0.81
		25	18.03±0.13	35.50±0.71
		50	12.44±0.29	31.20±0.21
2)	MERCM	10	42.13±0.95	60.48±0.55
		25	40.75±0.82	53.15±0.16
		50	24.78±0.37	38.12±0.37
3)	Piperazine citrate	10	20.16±0.16	65.25 ±0.32

(Results expressed as Mean ± SEM from four observations)

MEUCM – methanolic extract of unirradiated Citrus medica

MERCM – methanolic extract of radiated Citrus medica

As shown in table 1 extract of Citrus medica and its different fractions exhibited anthelmintic activity in dose-dependant manner giving shortest time of paralysis (P) and death (D) with 50 mg/ml concentration. The methanolic extract of unirrdiated Citrus medica caused paralysis in 12.44 min. and death in 31.20 min. while radiated methanol fractions revealed paralysis in 24.78 min and death in 38.12 min. respectively against the earthworm Pheretima posthuma. The reference drug Piperazine citrate showed the same at 20.16 and 65.25 min. respectively. Piperazine citrate by increasing chloride ion conductance of worm muscle membrane produces hyper polarization and reduced excitability that leads to muscle relaxation and flaccid paralysis¹⁴.

In conclusion, the study has shown that unirradiated methanolic extract of biomass of Citrus medica caused paralysis and death of worms in shorter time as compared to radiated methanolic extract. Further studies are in process to identify the possible phytoconstituents responsible for anthelmintic activity.

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Received on 01.08.2010 Modified on 02.09.2010

Research J. Pharm. and Tech. 4(3): March 2011; Page 411-412