INTRODUCTION:

Annona squamosa L. commanly known as “Sitaphal” is found throughout India, the evergreen small tree belong to family Annonaceae¹ . Pharmacological activities reported with A. squamosa include Anti-inflammatory², Antidiabetic³, Antioxidant⁴, Antibacterial⁵,Cytotoxic⁶, Antitumour⁷, Antifeedant⁸, Larvicidal⁹, Molluscicidal¹⁰ and Insectcidal¹¹. Isecticidal property of A. squamosa is attributed to a novel class of secondary metabolites called Acetogenins^{12, 13.}Ethanobotanical literatures mention that seeds of A. squamosa are used by tribal people in India to treat lice infestation^14.

The aim of present work was to evaluate licicidal activity of different fractions obtained from leaves of A squamosa by using in vitro method. Feeding habit and Anatomy of Human-Headlice resemble Goat-lice Damalinia caprae¹⁵ hence latter species was used as experimental organism in the study^.Lindane 1 % is commanly used synthetic insecticide for topical application to treat lice infestation¹⁶ hence it was used as standard drug for comparison in the study.

MATERIALS AND METHODS:

Plant Material:

Annona squamosa Linn. (Annonaceae) leaves were collected from tree located at Amravati (MS), and

authenticated by botanist Dr. Mrs. Prabha Bhogaonkar, Director Govt. Vidarbha Institute of Science and Humanities, Amravati (MS). The leaves were dried for 3 days under shade and ground to coarse particle size.

Extraction:

100 Gm. coarse powder of A. squamosa leaves was exhaustively extracted in Soxhelet apparatus successively by using n-hexane, chloroform and methanol. Each time the marc was dried at 45^oC and solvent was evaporated at 45^oC to get respective fraction. Finally the dried marc was boiled with distilled water for 30 minutes, filtered and filtrate was evaporated at 45^oC to get dry aqueous fraction.

Preparation of Test Solutions:

0.1, 1, 10 % w/w Test solutions of n-Hexane fraction and Chloroform fraction were separately prepared in coconut oil.

0.1, 1, 10 % w/w Test solutions of Methanol fraction and aqueous fraction were separately prepared in Distilled water.

Standard Solution:

1 % w/w Lindane solution

Experimental organism:

Goat-lice Damalinia caprae (Trichodectidae) were collected from healthy goat located in Amravati (MS).

In an earlier observation independent of present study the lice were found to remain live for 24-48 hours when removed and kept away from host body.

Experimental Procedure

This was carried out by modifying a method used by Pollack et al.(1999)¹⁷.

A 25 Ml. capacity glass beaker was taken. A filter paper disc coinciding with internal diameter of the beaker was cut. 0.15 Gm. test solution was applied as thin layer on bottom of beaker and on the disc by using brush, then the disc was placed in beaker. A group of 5 lice was placed over the disc and observed through magnifying glass.The time in minutes at which each louse became immobile was noted down.

The immobilized lice were taken out, placed on fresh dry filter paper and observed for 6 hours at interval of 30 minutes. The lice which did not show movement during this period were considered dead at the time when these were observed immobile. Same procedure was carried out for each test solution and standard solution.

Statistical Analysis:

Separate group of 5 lice was assigned to each test solution and standard drug hence n = 5. All the values expressed as Mean + S.E.M. The data was analysed by Student’s t test. p value < 0.01 was considered statistically significant.

Table 1: Effect of A.squamosa leaf fractions on Goat-lice.

Group	Treatment	Mean Time in Minutes +S.E.M.
Group 1	1 % w/w n-Hexane fraction Test solution	126 * + 4.86
Group 2	10 % w/w n-Hexane fraction Test sol.	90 + 6.14
Group 3	10 % w/w Chloroform fraction Test sol.	108 + 4.64
Group 4	1 % w/w Lindane solution ( standard )	95 + 5.25

n = 5 in each group. Values are Mean time in minutes at which lice were considered dead + S.E.M. *p value < 0.01

RESULTS AND DISCUSSION:

Methanol and Aqueous fraction have not showed activity.

Values of the mean time in minutes at which lice were immobilized and considered dead on treatment with n-Hexane and Chloroform fractions are shown in Table 1.

Significant increase in the mean time (p value < 0.01) was observed with 1 % n-Hexane fraction when compared to lindane i.e. potency significantly less than Lindane 1 %.

Licicidal activity with 10 % n-hexane fraction and 10 % Chloroform fraction was found comparable to Lindane 1 %.

CONCLUSION:

The study confirmed that A. squamosa leaves have licicidal activity and the active constituents are predominately soluble in n-Hexane.

REFERENCES:

1. The Wealth Of India (Raw Materials), NISCAIR, CSIR, New Delhi, 2003, I:A, 286-295.

2. Dash GK et al. Analgesic and Anti-inflammatory activity of Annona squamosa Leaves. Indian Journal of Natural roducts. 2001; 17(2): 32-36.

3. Shirwaikar A. et al. Oral antidiabetic activity of Annona squamosa leaf alcohol extract in NIDDM rats. Pharmaceutical Biology. 2004; 42(1): 30-35.

4. Shirwaikar A. et al. In vitro antioxidant studies of Annona squamosa Linn. Leaves. Indian Journal of Experimental Biology. 2004; 42(8): 803-807.

5. Joy B. and Sobhana D. Antibacterial activity of Kauranes from Annona squamosa L. pericarp. Asian Journal of Chemistry. 2007; 19(4): 2773-2778.

6. Hop DC et al. Novel Mono-tetrahydrofuran ring acetogenins from bark of Annona squamosa showing cytotoxic selectivities for the human pancreatic carcinoma cell line PACA-2. Journal of Natural Products. 1997; 60(6): 581-586.

7. Pardhasarathi BVV et al. Antitumour activity of Annona squamosa seed extracts is through the generation of free radicals and induction of apoptosis. Indian Journal of Biochemistry and Biophysics. 2004; 41(4): 167-172.

8. Soni AK et al. Tobacco caterpillar antifeedants from seeds and leaves of Annona squamosa. IUPAC International Conference on Biodiversity and Natural Products Chemistry and Medical Applications. New Delhi. 2004; 338

9. Prasad KD et al. Effect of Annona Squamosa Seed on Culex Larvae. Indian Journal of Indigenous Medicine. 1995; 17(1): 83-87.

10. Dos Santos AF and sant’Ana AEG. Molluscicidal properties of some species of Annona. Phytomedicine. 2001; 8(2): 115-120.

11. Harborne JB and Baxter H. Chemical Dictionary of Economic Plants. Jhon Weily And Sons. Ltd., England. 2001, 133.

12. Bruneton J. Pharmacognosy Phytochemistry Medicinal Plants. Lavoisier Publishing, Paris, France. 2^nd Edition,.1999, 180.

13. Johnson HA et al. Thwarting Resistance:Annonaceous Acetogenins as New Pesticidal and Antitumor Agents.In: Biologicaly Active Natural Products: Pharmaceuticals, Edited by Cutler SJ, Cutler HG , CRC Press LLC U.S.A. 2000. pp.173-181.

14. Govil JN et al. Recent Progress in Medicinal Plants: Search for Natural Drugs. Volume 13. Studium Press LLC, USA. 2006. 27.

15. Chandler AC. Introduction to Parasitology. John Wiley and Sons Inc., New York.1955, 597-617.

16. Sweetman SC. Martindale The Complete Drug Reference. Pharmaceutical Press, London, UK. 33 Edition. 2002. 1434.

17. Pollack RJ et al. Differential Permethrin Susceptibility of head lice sampled in the United States and Borneo. Arch Pediatr adolesc Med. 1999; 153: 969-973.

Received on 21.10.2008 Modified on 15.12.2008

Research J. Pharm. and Tech. 2(1): Jan.-Mar. 2009; Page 218-219