Anti Head Lice Essay Example for Free

Anti Head Lice EssayAbstract. The present sketch focused on the separation and acknowledgment of the active fuses against head lice from the hexane straighten up of Annona squamosa L seed. Chromatographic and spectroscopic techniques revealed that two major rises of the hexane seed pluck were oleic acid and triglyceride with one oleate ester. The yields of these compounds were 13.25% and 7.74% dry weight, respectively. The compounds were time-tested in vitro against head lice, comparing to the crude hexane extract of the seed. The triglyceride with one oleate ester and the crude hexane extract reduce with coconut palm anele 11. These compounds were found to polish all in all tested head lice in 49, 11 and 30 minutes, respectively. The triglyceride ester can be used as a marker for quantitative analysis of the active compound for quality control of the raw material A. squamosa seed and its extract. This first finding will be useful for quality assessment and the chem ical stability of the antihead lice provision from this plant.INTRODUCTIONAnnona squamosa L. (Custard apple) is a plant belonging to the family Annonaceae. It is popularly cultivated in all parts of Thailand, especially in the northeast, as a sweetened fruit. The seed of this plant is well known for killing head lice in many countries (Boonyaprapasara, 1998). The human head louse (Pediculus humanus capitis) is a small insect cause a public health problem, especially in poor sanitary conditions. In Thailand, research has shown the anti-head lice activity of A. squamosa. Puapatanakul (1980) reported that the extract of custard apple seeds in coconut oil at the ratio of 12 can kill 98% of head lice deep down two hours, while the leaf extractshows slight potency. Gritsanapan et al (1996) found that the petroleum ether extract of the leaves and seeds dissolved in coconut oil at a ratio of 11, kill 90% of head lice in vitro by 53 and 26 minutes, respectively. A 20% cream (oil/ water) preparation of petroleum ether extract of custard apple seeds can kill 93% of head lice Correspondence Wandee Gritsanapan, Department of Pharmacognosy, Faculty of Pharmacy, Mahidol University, 447 Si Ayutthaya Road, Ratchathewi, Bangkok 10400, Thailand. Tel +66 (0) 2644-8677 ext 1500, 5530 Fax +66 (0) 2644-8701 E-mail emailprotected 532within 3 hours (Areekul and Chaikledkaew, 1994, personal communication). Gritsanapan et al (1996), reported that 20 g of 20% freshly prepared cream can kill 94.59.1% of head lice within 3 hours of application to school girls hair. Tiangda et al (2000) found the cream preparation of custard apple seed is biologically stable for at least 12 months. However, it is easier to control the quality and stability of the preparation by quantitative analysis of the active chemical components. The active compounds of A. squamosa seed extract have not been reported elsewhere. The present study, therefore, is focused on the isolation and designation of the anti-h ead lice components in the seeds of A. squamosa.MATERIALS AND METHODSPreparation of plant extracts A. squamosa seeds were purchased from Pak Chong District, Nakhon Ratchasima Province, Thailand in October 2004. The samples were identified by comparison with the herbarium at Forest Herbarium, Department of National Parks, Wild-life and Plant Conservation, Ministry of Natural Resources and Environment, Bangkok. The voucher exemplar (WAS 0704) has been deposited at the Department of Pharmacognosy, Faculty of Pharmacy, Mahidol UniVol 37 no 3 May 2006A NTI-HEAD L ICE EFFECTOFA.SQUAMOSAversity, Bangkok, Thailand. The seeds were washed and dried in a hot air oven at 55C for 24 hours. The dried seeds were ground in an electric mill. Extraction and separation of major compounds The powdered seeds of A. squamosa (1.1 kg) were macerated with hexane (23 l) for third days at room temperature. The mixture was filtered and the filtrate was concentrated by a rotary evaporator and evaporated in a hot water bath until a constant weight (282.4 g) was obtained. The extract (75 g) was separated using silica gel column chromatography (400 g silica). Hexane (CH2Cl2 (11) 100% CH 2Cl 2 CH 2Cl2 MeOH (11)) was used as an eluent. Fifty mile fractions were collected and the fractions with the same tender loving care pattern (SiGF 254 hexane ethyl acetate 103) were combined. The fractions containing two major spots (R f 0.20 and 0.72) were eluted in 100% CH2Cl2 fractions. To isolate utter(a) compounds, the fractions containing major compounds were combined and concentrated. The mixture was further fractionated using silica gel column chromatography (200 g). Isocratic elution by hexane ethyl acetate (103) was performed (approximately 25 ml per fraction).The fractions with the same TLC pattern were combined to yield five fractions. The second and quaternth fractions gave compound AS1 (38.7 g) and compound AS2 (22.6 g), respectively. Compounds AS1 and AS2 were purified to give pure c ompounds. Testing for anti-head lice activity of pure compounds and crude extract The hexane extract and the two major pure compounds were tested for anti-head lice activity according to the method of McCage (2002). The extract and pure compounds were separately dissolved in coconut oil at dilutions of 11 to 18 ww. The same amount of each solution (0.05 ml) was put in a Petri dish and dissemination in a thin layer over a 2 cm2 area. Seven equal sized head lice collected from school girls hair were position in the Petri dish containing solutions of the extract and the two major pure compounds. Non-moving head lice, which were determined as dead lice, were counted every 5 minutes until all the lice were dead. A commercial anti-head Vol 37 No. 3 May 2006lice cream, Hexin, which is gamma benzol hexachloride (1% w/w) and coconut oil were used as a positive and minus controls, respectively.RESULTSCompound AS1 was a pale yellow oil, yielded 13.25% w/w of dried seeds. TLC (SiGF254, hexa neethyl acetate 103) had an Rf value of 0.20 ( common fig 1). The EI mass spectrum had a molecular ion peak at m/z 283.2 M+1 and a prominent peak at m/z 264.3. The IR spectrum of compound AS1 revealed absorption peaks at 3000-2930 (O-H stretch), 2850 (C-H stretch), and 1700 (C=O stretch, carboxylic) cm-1. The 1H NMR spectrum of compound AS1 indicated the presence of one methyl proton at 0.90 (3H, t, H-18) a methylene proton group at 1.26 (20H, m, H-4-7 and H-12-17) two methylene proton groups at 1.65 (2H, m, H-3) four methylene proton groups at 2.00 (4H, m, H-8, 11) two methylene proton groups at 2.35 (2H, t, H-2) two olefinic methane proton groups at 5.35 (2H, m, H-9,10) and the broad peak of a hydroxyl proton at 10.15 (1H).The 13C NMR spectrum and Distortionless Enhancement by Polarization Transfer (DEPT) exhibited 16 carbon resonances, revealing the presence of thirteen methylene carbons, one methyl carbon, two olefinic methine carbons and one carbonyl carbon. These spectra l data suggested that compound AS1 was a fatty acid. Comparing the NMR spectra of compound AS1 with Aldrich Library (1993) of 13C and 1H FT NMR spectra, confirmed the molecular structure of AS1 was an oleic acid (Fig 2). Compound AS2 was in any case pale yellow oil, yielded 7.74% w/w of dried seeds. The Rf value (SiGF254, hexane ethyl acetate 103) was 0.72 (Fig 1).The 1H NMR spectrum looked similer to the AS1 spectrum, with additional signals at 4.15 and 4.30. The IR spectrum of compound AS2 showed bands at 2925 (C-H stretch) and 1746 (C=O stretch, ester) cm-1. 533S OUTHEAST ASIAN J T ROP MED P UBLIC H EALTHTable 1 Head lice killing time of the crude extract and pure compounds from Annona squamosa seeds (n=3). Test sample Dilution (ww) Killing time (min) 30.674.04 34.334.04 41.003.61 55.005.00 49.333.06 54.675.51 59.006.56 61.334.16 11.001.00 12.002.00 16.001.00 22.332.52 180 180 1 2 3Hexane crude extract11 12 14 18 Oleic acid (AS1) 11 12 14 18 Triglyceride with one oleateester ( AS2) 11 12 14 18 Coconut oil (-ve control) Not diluted Hexin (+ve control) Not diluted1 = AS1, 2 = AS2, 3 = hexane crude extract. Fig 1TLC chromatogram of AS1, AS2 and hexane crude extract.HAfter comparing the NMR spectra of compound AS2 with Aldrich Library (1913) of 13C and 1 H FT NMR spectra, compound AS2 was felt to be a triglyceride with one oleate ester (Fig 2). The hexane crude extract of compound AS1 (oleic acid) and compound AS2 (triglyceride with one oleate ester) from Annona squamosa seeds showed in vitro anti-head lice activity as summarized in the Table 1. The data show that the triglyceride with one oleate ester was the most active compound against head lice. It killed all tested head lice within 11 minutes when diluted with coconut oil to a ratio of 11. Both the Hexin and the coconut oil killed all the head lice within a period of 180 minutes.H COOH (CH2) 7H3C(CH2) 7Oleic acidO CH2O CHO CH2O C R1 R2 OleateTriglyceride with one oleate ester R 1, R2 = H/ other fatty aci d Fig 2Structure of separated compounds from Annona squamosa seed.DISCUSSIONThe triglyceride with one oleate ester in coconut oil (11) was significantly more active against head lice than gamma benzene hexachloride 1% cream and the hexane crude extract. These data are supported by previous reports(Gritsanapan et al, 1996 Tiangda et al. 5342000). This result is useful for the standardization of Annona squamosa seed and its extract. The active compound may be used for the qualitative assessment of the chemical stability of the custard apple cream preparation. This is the a first report of the active anti-head lice components from A. squamosa seeds. Vol 37 No. 3 May 2006A NTI-HEAD L ICE EFFECTOFA.SQUAMOSAREFERENCESAldrich Chemical Co, Inc. The Aldrich Library of 13C and 1H FT NMR Spectra. 1st ed, 1993. Boonyaprapasara N, Chokchaicharoenporn O, eds. Samunprai Maipuenbaan. Vol. 2. Bangkok Faculty of Pharmacy, Mahidol University, 1998 454 (in Thai). Gritsanapan W, Somanabandhu A, Titirung ruang C, Lertchaiporn M. A study on the antiparasitic activities and chemical constituents of extracts from the leaves and seeds of custard apple (Annona squamosa Linn.). Proceedings of Third NRCT-JSPS Joint Seminar, 1996 209-15. McCage CM, Ward SM, Paling CA, Fisher DA, Flynn PJ, McLaughlin. Development of a paw paw herbal shampoo for the removal of head lice. Phytomedicine 2002 9 743-8. Puapatanakul O. Clinical studies of Annona squamosa seeds and leaves for the treatment of head lice. 1980 37 pp (in Thai). T iangda C, Gritsanapan W, Sookvanichsilp N, Limchalearn A. Antihead-lice activity of Annona squamosa seed extract. Southeast Asian ledger of Tropical Medicine and Public Health 200031 (suppl 1) 174-7.Vol 37 No. 3 May 2006