EVALUATION OF ANTHELMINTIC ACTIVITY OF ETHANOLIC ROOT EXTRACT OF ALLOPHYLUS SERRATUS.

About Authors:
A.SRILAKSHMI^*, AMRUTHA R.E, N.KRISHNA SREE
Department of Pharmacology
P. Rami Reddy Memorial College of Pharmacy,
Andhra Pradesh, India.
*amrutharamapuram@gmail.com

Abstract:
Present study was aimed to evaluate the anthelmentic activity of ethanolic extract of Allophylus serratus against Indian earthworms Pheritima posthuma. The various concentrations of ehanolic extract of Allophylus serratus were tested for paralysis time and death time of the worms. Results revealed that all the concentrations of Allophylus serratus possess anthelmentic activity in a dose dependant manner. Potency of the test samples was found to be inversely proportional to the time taken for paralysis/death of the worms. The activities were comparable with the reference drug Albendazole(20mg/ml). Among the various concentrations of extracts 400mg/ml dose was found to possess promising anthelmentic activity in comparison to other extracts like 100mg/ml,200mg/ml. Phyto constituents of ethanolic extract of Allophylus serratus were found to be Flavonoids, Phenolic compounds,steroids,tri-terpenoids, Glycosides. The present study therefore justifies its use in the folklore remedies as an anthelmentic drug of natural origin.

Reference Id: PHARMATUTOR-ART-1423

INTRODUCTION
Among the most widespread of all chronic infections are those caused by various species of parasitic helminths (worms).Helmintic infections are now being recognized as cause of much chronic ill health and sluggishness amongst the tropical people. Diseases caused by helminth parasites in livestock continue to be a major productivity constraint, especially in small ruminants in the tropics and sub tropic ¹. In the developing world, the greatest impact of parasitic diseases indirect and potential productivity losses ².Infections by gastrointestinal helminth parasites of livestock are among the most common and economically important diseases ³.

Allophylus serratus [Family- Sapindaceae ] is a deciduous tree and frequently found in the hilly area of Tirumala. The purpose of the present study was to find out the antihelminthic activity of ethanolic leaf extract of  Allophylus serratus.

Materials and Methods:
a)      Collection of Plant material:
The leaves were collected from the tirumala forest, Andhra pradesh, India during month of January 2011 and its identity as Allophylus serratus was authenticated by Assistant professor in the department of botany, Sri venkateswara University, Tirupati. The leaves were shade-dried, pulverized in a mechanical grinder and stored in room temperature in a closed container for further use.

b)     Preparation of plant extract:
In the present work, the authenticated shade dried leaves of the plant, approximately (500 g), were  powdered to coarse particle size no. (#) 40 and subjected to continuous hot extraction with 90 % ethanol in a soxhlet extractor for 72 h. The total ethanol extract was filtered and concentrated to dryness at 40°C under reduced pressure in a rota evaporator. The yield of ethanol extract was found to be 100 gm (20% w/w). The extract was kept in a dessicator till the experiment.The extract was tested for presence of various phytoconstituents as per standard procedures.

c)Worms’s collection and authentification:
Indian earthworm Pheretima posthuma (Annelida) were obtained from water logged area of soil and identified at department of applied zoology. S.V.University, Tirupathi, Andhrapradesh, India.

Anthelmintic assay
The anthelmintic assay was carried as per the method of Ajaiyeoba et al., with necessary modifications. The assay was performed on Indian adult earth worm, Pheretima posthuma due to its anatomical and physiological resemblance with the intestinal round worm parasite of human being ^4,5,6,7.Because of easy availability earth worms have been widely used for the initial evaluation of anthelmintic compounds In vitro ^8,9,10,11. 50 ml of formulation containing different concentrations of crude extract and test standard (10, 20, 50 mg/ml) were prepared by triturating the samples with 15% tween80 and the resultant mixture were stirred using a mechanical stirrer for 30 min.Five groups of worms with five worms in each group were taken in a petridish. The time of paralysis was noted when no movement of any sort could be observed except when the worm were shaken vigorously not when dipped in warm water (45^oC).
Group 1- Distilled water which served as control.
Group2- Received Albendazole at the dose of 10 mg/ml as the standard.
Group3- Received alcoholic extract at the dose of 10 mg/ml
Group4- Received alcoholic extract at the dose of 20 mg/ml
Group5- Received alcoholic extract at the dose of 50 mg/ml

METHODLOGY: Treatment sehedule of animals ,

Statistical Analysis
All the values were expressed as mean±standard error of mean (S.E.M.) and analyzed for ANOVA and post student’s t testfor n= 6 animals  Differences and  were considered significant at P < 0.001 levels Vs standard groups.

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RESLUTS
a) Extraction
The dried powder of Allophylus serratus leaves (50 gms) was extracted with 90% aqueous ethanol by soxhelation. The yield of aqueous ethanolic extract is 5.89g

b) Identification of Phyto chemical constituents
All extracts obtaining during successive extraction ofAllophylus serratus leaves were examined for the presence of various phytoconstituents by performing qualitative phytochemical tests and the results are recorded in table no:2

+VePositive,-Ve: Negative.

c) Results of pharmacological investigations:
Alcohol extract from the leaves of Allophylus serratus were investigated for their anthelmintic, activity against Pheretima  posthuma. Variousconcentrations (1%, 2.%, and 5%) of extract,were tested in the bioassay, which involveddetermination of time of paralysis and time ofdeath of the worms. Albendazole  wasincluded as standard reference and distilled water as control. The anthelmintic assay wascarried as per the method of Ajaiyeoba et al. with minor modifications.The assay was performed on adult Indian earthworm, Pheretima  posthuma, collected from moist soil and washed with normal saline to remove all faecal matter were used for the anthelmintic study. The earthworms of 3-5 cm inlength and 0.1-0.2 cm in width were used for all the experimental protocol. In the first set of experiment, five groups of six earthworms were released in to 20 ml of solutions of  Albendazole  ethanol extracts of leaves of Allophylus serratus  (1%, 2.% and 5% each) in distilled water. Observations were made for the timetaken to paralysis and death of individual worms. Time for paralysis was noted when no movement of any sort could be observed except when the worms were shaken vigorously. Death was concluded when the worms lost their motility followed with fading away of their body colours.

Anthelmintic activity of alcoholic extract of leaves of Allophylus serratus Test  substance concentration mg/ml Time taken for Paralysis (P) and Death (D) of worms in min (Pheretima posthuma)  .

Value are mean SEM = Standard error mean;** P < 0.01 V/S Standard groups  calculated by ANAOVA followed by student’s t test. n=6

DISCUSSION
The alcoholic extract of ethanolic extract of Allophylus serratus exhibit anthelmintic activity dose dependent manner. The alcoholic extract at dose 50mg/ml caused paralysis in 40 min and death in 68 min against Pheretima posthuma compared to the reference standard Albendazole (20 mg/ml) showed the same at paralysis time 34 min and death time 63 min.

The alcoholic extract of the plant not only demonstrated paralysis, but also caused death in shorter time as compared to reference drug Albendazole. The anthelmintic potency of the prepared samples and marketed available standard drug  (Albendazole).

CONCLUSION
The phytochemical screening of the ethanolic extract has revealed the presence of glycosides and steroids in alkaloids and flavonoids, tannins, carbohydrates, Antihelmintic effect due to presence of flavonoids . The ethanolic extract showed significant anthelmintic activity when compared to reference drug Albendazole. Further, it would be interesting to isolate the possible phyto constituents which may be responsible for the activity and the mechanism of action.

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