COMPARATIVE IN VITRO ANTHELMINTIC ACTIVITY OF THREE PLANTS FROM THE AMARANTHACEAE FAMILY

The three plants presented in this study are Amaranthus spinosus, Amaranthus caudatus and Amaranthus viridis L. They belong to the Amaranthaceae family and are traditionally used as vermicides. Our aim was to investigate the anthelmintic activity of the three plants using earthworms (Pheretima posthuma). Methanol extracts of the three plants at different concentrations (10, 20, 40, 60, 80, 100 mg/ml) showed dose-dependent vermicidal activities. At concentrations of 80 and 100 mg/ml all three plant extracts caused paralysis (8.18, 5, 12.16, 5.75, 10.2, 7.8 min) and death (14.65, 9.12, 18.6, 8.5, 18.6, 12.7 min), respectively. Piperazine was used as a reference standard at a concentration of 10 mg/ml. Our study found that the three plants possess potent anthelmintic activity when compared to Piperazine.


INTRODUCTION
The World Health Organization estimates that a staggering two billion people harbor parasitic worm infections.Parasitic worms also infect livestock and crops, affecting food production with a resultant economic impact.Despite this prevalence of parasitic infections, the research on the anthelmintic drug is sparse.According to the WHO, only a few drugs are used in treatment of helminthes in humans.Anthelmintics from natural sources could play a key role in the treatment of these parasite infections.In view of this, attempts have been made to study the anthelmintic activity of traditional medicinal plants.

Collection of Plant Material and Extraction
Fresh A. spinosus, A. caudatus, and A. viridis plants were collected from Chickballapur and were authenticated by Dr. Rajan from the Department of Botany, Government Arts College, Ootcamund, Tamilnadu.A voucher specimen (SKVCP 11, 12 and 13) was deposited in the college herbarium.The whole plant was shade-dried and coarsely powdered.The coarse powder was extracted with methanol using the soxhlet apparatus.The extract was concentrated to dryness in vacuum.

Phytochemical screening
The methanol extracts of three plants were screened for the presence of various phytoconstituents such as steroids, alkaloids, terpenoids, glycosides, flavonoids, phenolic compounds, and carbohydrates (Kokate, 1986).

Animals
Indian adult earthworms (Pheretima posthuma) were used to study anthelmintic activity.The earthworms were collected from moist soil and washed with normal saline to remove all fecal matter.Earthworms 3-5 cm in length and 0.1-0.2cm in width were used for all experimental protocol.

Drugs and Chemicals
Piperazine citrate (Glaxo Smithkline), methanol (Karnataka chemicals) was used during experimental protocol.

Anthelmintic Activity
The anthelmintic assay was carried out as per the method of Ajaiyeoba et al. (2001) with minor modifications.The assay was performed on the adult Indian earthworm Pheretima posthuma due to its anatomical and physiological resemblance to the human intestinal roundworm parasite (Vidyarthi, 1967;Chatterjee, 1967).Due to their ready availability, earthworms have been used widely for the initial evaluation of anthelmintic compounds in vitro (Sollmann, 1918;Jain et al., 1972;Dash et al., 2002).
The earthworms were divided into different groups, each group containing six worms.Fifty ml formulations containing four different concentrations of methanolic extracts of A. spinosus, A. caudatus and A. viridis (10, 20, 50, 80 and 100 mg/ml in distilled water) were prepared.The time of paralysis was noted when no movement of any sort could be observed except when the worms were shaken vigorously.The times of death of the worms were recorded after ascertaining that worms neither moved when shaken vigorously or when dipped in warm water (50°C).Piperazine citrate (10mg/ml) was used as reference standard while water served as a control.

RESULTS AND DISCUSSION
By preliminary phytochemical screening it was found that all the three plants' extracts contain flavonoids, glycosides, steroids, alkaloids, phenolic compounds, carbohydrates, proteins and tannins.
The earthworm selected for the antihelmintic activity was most sensitive to the methanol extract of A. spinosus as can be seen in Fig. 1.The graph revealed dose-dependent paralysis ranging from loss of motility to loss of response to external stimuli, which eventually progressed to death.At 60, 80 and 100 mg/ml concentrations paralysis was observed respectively at 17.6, 8.18 and 5 min and death at 24.03, 14.65 and 9.12 min post-exposure.
The methanol extracts of A. caudatus and A.viridis also exhibited dose-dependent anthelmintic activities that caused paralysis at 19.21, 14.33 min (at 60 mg/ml); 12.16, 10.2 min (at 80 mg/ml); Figure 1.In vitro anthelmintic activity of three plants belonging to family Amaranthaceae and 5.75, 7.8 min (at 100 mg/ml), and death at 27.7, 26.6 min (at 60 mg/ml); 18.6, 18.6 min (80 mg/ml); and 8.5, 12.7 min (100 mg/ml) post-treatment (Fig. 1).The earthworms were more sensitive to the extracts of A. spinosus, A. caudatus, and A.viridis at 60, 80 and 100 mg/ml concentrations as compared to the reference drug piperazine citrate (10 mg/ml).All the three plants' methanol extracts were more effective in causing the death of the worms as well as promoting paralysis.
Most worm expellers like piperazine citrate cause paralysis of the worms so that they are expelled in the feces.The methanol extracts of the three plants not only demonstrated this property but also killed the worms.
The anthelmintic activity of the methanol extracts of A. spinosus, A. caudatus and A. viridis may be due to the presence of polyphenolic compounds (Bate-Smith, 1962).The wormicidal activity of the methanol extract as described herein against earthworms suggests that it could be effective against parasitic infections of humans.