IN VITRO EVALUATION OF ANTHELMINTIC ACTIVITY OF ETHANOLIC EXTRACT OF CENTELLA ASIATICA LINN. IN INDIAN ADULT EARTHWORMS

Objective: Helminthiasis is one of the most prevalent parasitic infestations worldwide posing a major threat to public health. The control of these nematodes has relied largely on the use of a limited number of anthelmintics. However, emerging resistance and side effects to the currently available anthelmintic drugs is a major concern and discovery of newer anthelmintics with a novel mode of action is the need of the hour. The present study is aimed to evaluate the anthelmintic activity of ethanolic extract of Centella asiatica Linn. (EECA) on Indian earthworms ( Pheretima posthuma ). Methods: The earthworms were divided into 4 groups with 6 worms in each group. The anthelmintic activity of EECA at two different concentrations (25 mg/ml and 50 mg/ml) was evaluated by assessing the time of paralysis and time of death of the worms. Albendazole was used as standard and 2% gum acacia as control. Results: Albendazole at 25 mg/ml showed the highest anthelmintic activity and had significant difference (p<0.001) with EECA at both 25 mg/ml and 50 mg/ml. Conclusion: Both doses of the test drug showed anthelmintic activity but the extract at either dose was found to be less effective than the standard drug. Further studies with higher doses of the extract should be done to evaluate the anthelmintic activity in a dose-dependent manner.


INTRODUCTION
Helminthiasis also known as worm infestation is one of the most prevalent parasitic infestations both in developed and developing countries affecting around 1/3 rd of the world's population. As per WHO, around 1.5 billion people are infested with soil-transmitted helminths (STH) worldwide [1]. India has the highest burden of STH among all the countries contributing to one-fourth of total global cases which is prevalent among children of age group 1-14 y [2]. Overall prevalence of STH in India ranges from 13% to 66% [3]. Parasites causing infections can be broadly classified into tapeworms, flukes, and roundworms. Helminths cause infection by faeco-oral route or by direct entry through the skin of the host and then lodging in the intestine [4]. Intestinal worms cause decreased absorption, blood loss, intestinal or lymphatic obstruction and secretion of toxins which leads to health hazards like undernourishment, anaemia, eosinophilia and pneumonia [5]. In extreme cases of intestinal infestation, the mass and volume of the worms may cause tear of the muscular layer leading to peritonitis, volvulus and gangrene of the intestine [6].
A large number of anthelmintic drugs (AHDs) are now available in the market which include benzimidazole class like albendazole and mebendazole, macrocyclic lactones like ivermectin or pyrazinoisoquinoline derivative like praziquantel [7,8]. The current anthelmintic therapies exert their action by incapacitating the parasite by causing damage and paralysis of the worm so that the immune system can eliminate it, or by altering its metabolic processes. But the metabolic requirements of these parasites vary greatly from one species to another and because of that drugs that are highly effective against one type of worm may be ineffective or less effective against others [9]. But the emergence of resistance to the currently available AHDs is a major concern and newer anthelmintics with a novel mode of action has become necessary [10].
Herbal drugs in recent years have become popular and also gained importance due to their safety, efficacy and cost effectiveness in treating various diseases. Centella asiatica Linn. known as 'Gotu Kola' in Hindi, 'Thanakuni' in Bengali, 'Peruk' in Manipuri, 'Indian Pennywort' in English is a medicinal plant that has been in use since prehistoric times in folk medicine for the treatment of a wide range of diseases. The herb is used for the treatment of wound healing, various skin conditions such as leprosy, varicose ulcers, eczema, psoriasis. It is also used in diarrhoea, liver diseases, fever, amenorrhoea, diseases of the female genitourinary tract. The plant also has analgesic, anticonvulsant, antidiabetic, antispasmodic, antiulcer, anxiolytic and nootropic actions [11][12][13]. Centella asiatica Linn. contains phytochemical constituents like tannins such as phlobatannins, saponins, flavonoids, terpenoids, alkaloids and cardiac glycosides etc [14].
As such, considering its use in gastro-intestinal ailments by locals, the present study was aimed at the evaluation of in vitro anthelmintic effect of ethanolic extract of leaves of Centella asiatica Linn. (EECA) on Indian earthworms (Pheretima posthuma).

Collection of plant materials
The present study was conducted in the Department of Pharmacology, Regional Institute of Medical Sciences, Imphal, Manipur. The plant Centella asiatica Linn. was collected from Lamphel local market, Imphal, Manipur and was authenticated by the Department of Botany, D. M. College, Imphal having Acc. No. DMH 09.15.

Preparation of plant extract
The leaves of the plant Centella asiatica Linn. were shade dried and powdered using a mixer grinder. Powdered dry plant material (50 gm) was extracted with 100 ml ethanol for 24 h and thus ethanolic extract was obtained using soxhlet apparatus manufactured by Jain Scientific Glass Works, Ambala Cantt, Haryana, India. The extract was filtered and concentrated under vacuum-sounding apparatus for 30 min and then stored at 4 °C [15]. The percentage yield was 21 % and the extract thus obtained was used for the anthelmintic study.

Worm collection and authentication
The study was done on adult Indian earthworm Pheretima posthuma because of its similar anatomical and physiological features to Ascaris lumbricoides [16,17]. The earthworms (5-8 cm in length) were collected from moist soil and authenticated by the Department of Life Sciences, Manipur University. They were cleaned with normal saline (NS 0.9%) to remove soil and faecal matter.

Phytochemical analysis
The preliminary phytochemical analysis of the plant extract was carried out using a standard procedure to identify various constituents [18].

Experimental design
The earthworms were divided into 4 groups with six worms in each group. Standard and test drugs were dissolved in 2 % gum acacia and 10 ml of the desired formulation were poured in separate petri dishes and were kept under room temperature. The earthworms were placed in the petri dishes containing the extract solutions or the standard drug as mentioned below in table 1. EECA at a dose of 50 mg/ml

Evaluation of anthelmintic activity
The living or viable worms were kept under close observation. External stimuli was frequently applied to each worm which stimulated and induced movement in live earthworms. Time taken for paralysis of the worms were noted when no spontaneous movement could be observed except when shaken vigorously. Time taken for death of worms were recorded when worms did not move even after vigorous shaking or when dipped in warm water (50 °C) which was followed by gradual fading of their body color [19].

Statistical analysis
Data obtained was analysed using One way ANOVA followed by Bonferroni test post hoc. Results were expressed in mean±SEM.  1). However, the extract at either dose was found to be less effective than the standard drug.

DISCUSSION
The emergence of resistance and toxicity to the conventional anthelmintic drugs and the increasing concern over the presence of drug residues in animal products has led to a renewal of interest in the use of plant-based drugs. Evaluation of anthelmintic activity of new plant compounds by in vitro methods using free living stages of parasitic nematodes have become popular [20]. Because of their low-cost effectiveness, simplicity and rapid turnover in vitro techniques are preferred over in vivo methods [21].
The preliminary phytochemical analysis shows presence of polyphenolic compound tannins. Tannins present in the extract possibly can interfere with energy generation in helminths by uncoupling oxidative phosphorylation which is similar to some synthetic phenolic anthelmintics e. g niclosamide, oxyclozanide, bithionol etc. Tannins can also exert anthelmintic action by binding to free proteins in the GIT of host or glycoprotein on the cuticle of the parasite and causes death [22]. Phytochemical investigation of the extract also reveals presence of saponin which has membrane permeabilizing and pore forming property. This is similar to two conventional anthelmintic drugs such as praziquantel and toltrazuril. Thus, permeability of the cell membrane of the parasites is affected which forms vacuoles, resulting in disintegration of monogenea teguments [23]. Presence of alkaloids can block intake of acetylcholine from the host which expels out the worms by peristaltic movement of intestine [24].
Conventional anthelmintic drugs like albendazole, piperazine citrate and mebendazole are being used in these studies. Albendazole is a safe, highly effective therapy for parasitic infections and an established standard drug in anthelmintic studies. Possible mechanism of action is thought to be inhibition of microtubule polymerization by binding to β-tubulin. The selective toxicity of this agent against helminths results from higher affinity for parasite βtubulin than for the same target in higher eukaryotes [7]. The least time taken for paralysis and death of worms by albendazole correlates with previous studies [19].
Time taken for paralysis and death of the worms by EECA can correspond with observations of the previous anthelmintic studies on the plant extracts [25]. The extract is more effective at 50 mg/ml but the extract at either dose was found to be less effective than the standard drug.

CONCLUSION
The discovery of a potent remedy from plant origin will be a great advancement in the treatment of helminthic infections. Use of these plants in folk medicine suggests that they represent an economic and safe alternative to treat infectious diseases. The current study evidenced that the ethanolic extracts of Centella asiatica has a promising in vitro anthelmintic activity. However further studies with higher doses of the plant are required to evaluate the dose dependent anthelmintic activity and also to determine the active principle responsible for exact mechanism of anthelmintic activity.