In Vitro Screening of Anti-lice Activity of Pongamia pinnata Leaves

Article information

Korean J Parasitol. 2009;47(4):377-380
Publication date (electronic) : 2009 December 02
doi : https://doi.org/10.3347/kjp.2009.47.4.377
1School of Pharmacy, Masterskill University College of Health Sciences, Taman Kemacahaya 11, Jalan Kemacahaya, Cheras, Selangor, Malaysia.
2Honorary Associate, School of Pharmacy, La Trobe University, Bendigo, Victoria 3552, Australia.
3School of Biomedicine and Health, Masterskill University College of Health Sciences, Taman Kemacahaya 11, Jalan Kemacahaya, Cheras, Selangor, Malaysia.
Corresponding author (anbujsunil@yahoo.co.in)
Received 2009 July 16; Revised 2009 August 20; Accepted 2009 August 31.

Abstract

Growing patterns of pediculocidal drug resistance towards head louse laid the foundation for research in exploring novel anti-lice agents from medicinal plants. In the present study, various extracts of Pongamia pinnata leaves were tested against the head louse Pediculus humanus capitis. A filter paper diffusion method was conducted for determining the potential pediculocidal and ovicidal activity of chloroform, petroleum ether, methanol, and water extracts of P. pinnata leaves. The findings revealed that petroleum ether extracts possess excellent anti-lice activity with values ranging between 50.3% and 100% where as chloroform and methanol extracts showed moderate pediculocidal effects. The chloroform and methanol extracts were also successful in inhibiting nymph emergence and the petroleum ether extract was the most effective with a complete inhibition of emergence. Water extract was devoid of both pediculocidal and ovicidal activities. All the results were well comparable with benzoyl benzoate (25% w/v). These results showed the prospect of using P. pinnata leave extracts against P. humanus capitis in difficult situations of emergence of resistance to synthetic anti-lice agents.

INTRODUCTION

Pediculus humanus capitis, otherwise called as the human head louse, infestation is a major concern in public health-associated problem. Head lice are ectoparasites and its infestation due to unhygienic conditions has negatively affected the society for decades, back to the earliest Homo sapiens. The condition is distributed around the world invading various ethnic groups with no restrictions of sex and socioeconomic status [1]. In Malaysia, people buy costly products in combating head lice and the money they plough into annually seems greater when they realize that the products they used were apparently ineffective [2]. This lack of efficacy is due to the emergence of resistance by the head louse to synthetic compounds and researchers were aimed on the search of new substitutes to synthetic ingredients, such as phytoconstituents obtained from plant sources [3,4].

Pongamia pinnata Linnaeus (Fabacae) is a medium-sized glabrous tree distributed along the coasts and river banks in India and Myanmar. Native to the Asian subcontinent, this species has been introduced to humid tropical lowlands in the Philippines, Malaysia, Australia, Seychelles, United States, and Indonesia [5]. Pongamol and karanjin isolated from fruits of P. pinnata were reported to have antihyperglycemic activity [6]. The oil obtained from this plant is used for the treatment of rheumatism, scabies as well as human and animal skin diseases in folk medicine [7]. Juices of leaves are used in curing cold, coughs, diarrhea, dyspepsia, flatulence, and gonorrhea. Dried leaves are used as an insect repellent in stored grains, and also used as a pesticide [8]. The ethanomedical claim by local tribes in Malayasia (Orang Asli) uncovered that P. pinnata is a potential source for controlling pediculosis. On these grounds of traditional claim, we have decided to screen various extracts, such as chloroform, petroleum ether, methanol, and water of P. pinnata leaves on adults, nymphs, and nits for its potential anti-lice and ovicidal activities.

MATERIALS AND METHODS

Plant material

Fresh leaves of P. pinnata were collected from Taman Alam Jaya, Selangor, Malaysia in April 2008. The samples were identified and authenticated by Dr. Sani Miran, botanist, National University Malaysia, Selangor, Malaysia. A voucher specimen (MUCHH/PH12/PP03) was deposited at the herbarium, School of Pharmacy, Masterskill University College of Health Sciences, Cheras, Selangor, Malaysia. The leaves were washed under tap water to remove debris and dried under shade for 10 days. The dried leaves were size-reduced to coarse powder in a mill.

Extraction

The coarse powdered leaves of P. pinnata (1,000 g) were extracted successively with petroleum ether, chloroform, methanol, and water by Soxhlet extraction technique [9]. All the extracts were concentrated using rotary vaccum evaporator and kept in a dessicator until further studies. The color, consistency, and percentage yield were observed.

Collection of head lice

Adults, nymphs, and nits of P. humanus capitis were collected from children between the age group of 8-12 by combing through sections of the scalp using a clean comb. After combing, the lice were carefully removed from the teeth of the comb into plastic boxes. All the subjects had not been treated with any anti-lice products for the preceding 3 months.

Anti-lice activity

Petroleum ether, chloroform, methanol, and water extracts of P. pinnata were tested for pediculocidal activity by filter paper diffusion method [10]. All the extracts were dissolved in distilled water to obtain 3 different concentrations (5%, 10%, and 20%). After careful selection under a dissecting microscope, the adults and nymphs were identified and separated. All the test organisms in a ratio of 3.6/1.4 (adult/nymph) were divided into 16 groups (5 lice each) and were placed on a filter paper at the bottom of petri dish and kept open. A 0.5 ml of each test samples was poured on the test organisms and allowed to spread as a thin layer of 4 cm2. Group 1 was treated with 0.5 ml distilled water and served as control. Group 2 to group 13 received 0.5 ml of various concentrations of aqueous, petroleum ether, chloroform, and methanol extracts respectively. Group 14 to group 16 were treated with 0.5 ml of 5%, 10%, and 20% of benzyl benzoate 25% (w/v) (RidPed). All the Petri dishes were set aside for 1 hr in a dark chamber at 26 ± 0.5℃ and 70 ± 1% humidity [11]. At the end of 1 hr, the dishes were taken out and applied 0.5 ml of distilled water and further placed in the chamber under the condition mentioned above. After 18 hr, the dishes were observed under a dissecting microscope for any possible movement of lice and absence of any movement were considered dead [12]. All the treatment was triplicate.

Ovicidal effects

The ovicidal activity was tested by placing 5 brownish oval eggs with an unbroken operculum on the filter paper (Whatmann No. 1; 6 cm diameter) placed in the bottom of each petri dish. Then, 0.5 ml of each test solution and control were applied on the nits. All the dishes were then incubated in a dark chamber at 26 ± 0.5℃ for 14 days. To maintain the moisture, 0.1 ml of distilled water was added at 48 hr interval. Hatching of eggs was monitored under a microscope and the percentage of emergence, i.e., partially hatched nits, was observed, and the findings were recorded [11]. Each treatment was replicated 3 times.

RESULTS

The color, consistency, and percentage yield of petroleum ether, chloroform, methanol, and water extracts of P. pinnata were recorded in Table 1. All the extracts displayed concentration (5%, 10%, and 20%) dependent activity among which petroleum ether extract showed higher mortality followed by methanol and chloroform extracts, respectively and was well comparable with the standard. Water extract in various concentrations showed minimal anti-lice activity (Table 2).

The color, consistency, and percentage yield of extracts

Effects of Pongamia pinnata leaf extracts against Pediculus humanus capitis adults and nymphs

The findings of ovicidal activity of P. pinnata extracts were tabulated in Table 3. Petroleum ether extract showed pronounced ovicidal activity by delaying the emergence of nymph on the 6th day and 14th day and completely inhibited the emergence similar to that of the standard. Methanol and chloroform extracts moderately delayed emergence of nymph, whereas water extract was devoid of ovicidal activity.

Effects of Pongamia pinnata leaf extracts against Pediculus humanus capitis nits

DISCUSSION

The use of P. pinnata extracts for controlling lice infestations has been authenticated from the excellent results obtained after screening various extracts for potential anti-lice and ovicidal activity. Oils from natural sources, such as eucalyptus, marjoram, spearmint, peppermint, sage, rosewood, clove bud, and cinnamon bark have exhibited significant pediculocidal activity in filter paper bioassays [13-16]. Another study carried out on school children revealed that 20% petroleum ether extract of custard apple seeds killed 95.3% of head lice [17]. Previous literatures on phytochemistry of P. pinnata seeds reported the presence of sterol and its derivatives together with saturated and unsaturated fatty acids (monoenoic, dienoic, and trienoic acids) establishing its lipophilic nature [13].

The findings of this study showed excellent anti-lice and ovicidal activities of petroleum ether extract of P. pinnata which may be due to the presence of these sterol derivatives responsible for the enhanced penetration and bioavailability of oil components into the body of louse. Penetration of extracts into the alimentary tract of lice could be ignored since all the extracts was applied on lice placed on the filter paper which also subsequently avoided immense dissemination of active constituents into the cuticle when the compound is directly applied to the insect skin [18]. Additionally, the lice was not exposed in an enclosed environment with the petri dish kept open which limits the possibility of volatile agents getting absorbed through the spiracles. For synthetic pediculocidal agents, the residue which remains in the head even after rinsing with water gives an enhanced control against lice but also noted for the development of resistance for lice [19,20]. Natural extracts from medicinal plants has been noticed for its safe and effective use, and appearance of resistance patterns were minimal due to its different mode of action [21,22] which greatly supports the safe use of P. pinnata extracts as a potent anti-lice agent.

Eradication of lice would be complete if the products used for pediculocidal activity also delays nymph emergence and potentially kill the nymph. Extracts of P. pinnata succeeded in delaying the emergence of nymphs, and its oily nature may help to detach nits from the hair before hatching. Among all the extracts, petroleum ether exhibited the maximum pediculocidal effects and completely inhibited nymph emergence at 2 different concentrations (10% and 20%). Hence, the results obtained from this research present a promising scenario for using P. pinnata leaf extract as an effective alternative for treating human head lice.

ACKNOWLEDGEMENTS

The authors are thankful to the management of Dato' Prof. Dr. Ishak Bin Tambi Kechik, Vice-Chancellor, and Dato' Edmund Santhara, CEO, Masterskill University College of Health Sciences, Malaysia, for their funding, continuous encouragement and support.

References

1. Burkhart CG, Burkhart CN, Burkhart KM. An assessment of topical and oral prescription and over-the-counter treatments for head lice. J Am Acad Dermatol 1998;38:979–982. 9632008.
2. Meinking TL, Serrano L, Hard B, Entzel P, Lemard G, Rivera E, Villar ME. Comparative in vitro pediculicidal efficacy of treatments in resistant head lice populations in the United States. Arch Dermatol 2002;138:220–224. 11843643.
3. Mumcuoglu KY, Miller J, Gofin R, Adler B, Ben-Ishai F, Almog R, Kafka D, Klaus S. Epidemiological studies on head lice infestation in Israel. I. Parasitological examination of children. Int J Dermatol 1990;29:502–506. 2228380.
4. Mumcuoglu KY. Prevention and treatment of head lice in children. Paediatr Drugs 1999;1:211–218. 10937452.
5. NFT Highlights. A quick guide to useful nitrogen fixing trees from around the world 1997. NFTA.
6. Tamrakar AK, Yadav PP, Tiwari P, Maurya R, Srivastava AK. Identification of pongamol and karanjin as lead compounds with antihyperglycemic activity from Pongamia pinnata fruits. J Ethnopharmacol 2008;118:435–439. 18572336.
7. Chopade VV, Tankar AN, Pande VV, Tekade AR, Gowekar NM, Bhandari SR, Khandake SN. Pongamia pinnata: Phytochemical constituents, traditional uses and pharmacological properties: A review. Int J Green Pharm 2008;2:72–75.
8. Warrier PK, Nambiar VPK, Ramankutty C. Indian Medicinal Plants: A Compendium of 500 Species 1995. Vol 4India: Universities Press.
9. Anbu Jeba Sunilson J, Suraj R, Anandarajagopal K, Rejitha G, Vignesh M, Promwichit P. Preliminary phytochemical analysis, elemental determination and antibacterial screening of Codium decorticatum-A marine green algae. Int J Biol Chem 2009;3:84–89.
10. Picollo MI, Vassena CV, Mougabure Cueto GA, Vernetti M, Zerba EN. Resistance to insecticides and effect of synergists on permethrin toxicity in Pediculus capitis (Anoplura: Pediculidae) from Buenos Aires. J Med Entomol 2000;37:721–725. 11004784.
11. Carpinella MC, Miranda M, Almirón WR, Ferrayoli CG, Almeida FL, Palacios SM. In vitro pediculicidal and ovicidal activity of an extract and oil from fruits of Melia azedarach L. J Am Acad Dermatol 2007;56:250–256. 17147968.
12. Meinking TL, Taplin D, Kalter DC, Eberle MW. Comparative efficacy of treatments for pediculosis capitis infection. Arch Dermatol 1986;122:267–271. 2420284.
13. Yang YC, Lee SH, Lee WJ, Choi DH, Ahn YJ. Ovicidal and adulticidal effects of Eugenia caryophyllata bud and leaf oil compounds on Pediculus capitis. J Agric Food Chem 2003;51:4884–4888. 12903940.
14. Yang YC, Choi HY, Choi WS, Clark JM, Ahn YJ. Ovicidal and adulticidal activity of Eucaliptus globulus leaf oil terpenoids against Pediculus humanus capitis (Anoplura: Pediculidae). J Agric Food Chem 2004;52:2507–2511. 15113148.
15. Yang YC, Lee HS, Clark JM, Ahn YJ. Insecticidal activity of plant essential oils against Pediculus humanus capitis (Anoplura: Pediculidae). J Med Entomol 2004;41:699–704. 15311463.
16. Yang YC, Lee HS, Lee SH, Clark JM, Ahn YJ. Ovicidal and adulticidal activities of Cinnamomum zeylanicum bark essential oil compounds and related compounds against Pediculus humanus capitis (Anoplura: Pediculicidae). Int J Parasitol 2005;35:1595–1600. 16188263.
17. Tiangda CH, Gritsanapan W, Sookvanichsilp N, Limchalearn A. Anti-headlice activity of preparation of Annona squamosa seed extract. Southeast Asian J Trop Med Public Health 2000;31:174–177. 11414452.
18. Burkhart CN, Burkhart CG. Recommendation to standardize pediculicidal and ovicidal testing for head lice (Anoplura; Pediculidae). J Med Entomol 2001;38:127–129. 11296812.
19. Meinking TL, Taplin D. Infestations: Pediculosis. Curr Probl Dermatol 1996;24:157–163. 8743266.
20. Mumcuoglu KY. Prevention and treatment of head lice in children. Paediatr Drugs 1999;1:211–218. 10937452.
21. Breuer M, Hoste B, De Loof A, Naqvi SNH. Effect of Melia azedarach extract on the activity of NADPH-cytochrome c reductase and cholinesterase in insects. Pestic Biochem Physiol 2003;76:99–103.
22. Isman MB. Barriers to the commercialization of new botanical insecticides. Biopesticides for crop protection. Proceedings of the '96 Agricultural Biotechnology Symposium 1996. Suwon, Korea:

Article information Continued

Table 1.

The color, consistency, and percentage yield of extracts

Extracts Color Consistency % yield
Petroleum ether Pale greenish Sticky mass 6.0
Chloroform Greenish Sticky mass 9.0
Methanol Greenish brown Semi solid 18.0
Water Dark green Semi solid 23.0

Table 2.

Effects of Pongamia pinnata leaf extracts against Pediculus humanus capitis adults and nymphs

Test sample Concentration (%) Average mortalitya (%)
Distilled water (0.5 ml) - 9.6
Petroleum ether extract (0.5 ml) 5 50.3
10 88.7
20 100
Chloroform extract (0.5 ml) 5 31.4
10 43.1
20 76.9
Methanol extract (0.5 ml) 5 17.3
10 32.6
20 82.9
Water extract (0.5 ml) 5 10.3
10 10.7
20 18.7
Benzoyl benzoate 25% w/v (0.5 ml) 5 60.7
10 100
20 100
a

n = 3.

Table 3.

Effects of Pongamia pinnata leaf extracts against Pediculus humanus capitis nits

Test sample Concentration (%) Emergencea (%)
Day 6 Day 14
Distilled water (0.5 ml) - 81.3 91.6
Petroleum ether extract (0.5 ml) 5 20.8 6.9
10 0 0
20 0 0
Chloroform extract (0.5ml) 5 56.2 41.3
10 52.1 28.7
20 21.6 11.4
Methanol extract (0.5 ml) 5 61.8 51.9
10 47.1 24.4
20 38.9 9.3
Water extract (0.5 ml) 5 62.3 45.5
10 29.4 14.8
20 18.6 10.4
Benzoyl benzoate (25% w/v) (0.5 ml) 5 6.1 0
10 0 0
20 0 0
a

n = 3.