full issue - Association of Biotechnology and Pharmacy

Current Trends in Biotechnology and PharmacyVol. 5 (1) 1029-1037 January 2011. ISSN 0973-8916 (Print), 2230-7303 (Online)1030traditional medicines, used in treatment of a widerange of afflictions. Use of Neem oil for humanuse has an extensive history in India and itssurrounding regions for a variety of therapeuticpurposes (10).Formulations made of neem oil are beingused as biopesticide for organic farming, as itrepels a wide variety of pests (11). Neem oil canalso control the black spot, powdery mildew,anthracnose and rust. The oil extraction usingsolvents has several advantages. It gives highyield and less turbid oil than the mechanicalextraction whereas operating cost is also low whencompared to supercritical fluid extraction.Materials and MethodsMaterial preparation: Neem seeds, used in thisstudy, were collected from trees of 13 to 20years old growing in Black, Red and Sandy soilsof Guntur district. The disease free, mature, ripe(deep yellow to light yellow) fruits were collected.The seeds were then manually depulped, cleanedunder running tap water and then air dried. Themesocarp adhering to the hard epicarp wasremoved by scrubbing the seed on a roughsurface, by using a sand paper and subsequentlydried in oven at 50 0 C until the seed reached to aconstant moisture content.Oil extraction by solvent extractionprocedure, using two solvents i.e.,petroleum etherand ethanol at three temperature levels (30 0 C,40 0 C and 50 0 C) was carried out by takingneem seed powder and solvent in a ratio of 1:5 insoxhlet apparatus (AOAC, 1975). The extractionwas continued for about 6 hours and the filtrateobtained was heated and evaporated at 70 0 C toobtain solvent free neem oil. Further solventtraces were removed by putting the oil in a roundbottomed flask and placed on a water bath for 12hours at 60 0 C -70 0 C. Then the oil was weighedand its percentage was calculated.The acid and peroxide values weredetermined using titrimetric method of Cox andPearson (18). Acid value was expressed intermsof free fatty acid in oil. The peroxides weredetermined by titration against thiosulphate inpresence of potassium iodide with starch as anindicator. The iodine value was determined bytitrimetric method described in AOAC (1975).The neem oil contains both saturated andunsaturated fatty acids. Iodine gets incorporatedinto the fatty acid chain wherever the double bondexist.Seed protein was estimated adopting themicrokjeldhal method described in AOAC (1970).Seed weight of 100 completely depulped seedsalong with seed coat taken from each tree wasmeasured. The amount of azadirachtin -A ofneem seed kernels was estimated through HPLC(12). The seed kernels were made into finepowder and subjected to repeated extraction withn- hexane through soxhlet extraction apparatusfor complete removal of fat. The residue wasair-dried and the azadirachtin content wasextracted by methanol. The precipitate obtainedthrough methanol extraction was ground againwith methanol and 25 ìl of this extract was utilizedfor quantifying azadirachtin- A (13). C8 columnswere used with a mobile phase containing water,methanol and acetonitrile in the ratio of 50:35:15with a flow rate of 1ml/min and the azadirachtin-A was detected at 215 nm.Results and DiscussionExtracts isolated using both the solventsfrom the trees growing in black soil differedsignificantly in oil yield, acid value, peroxide valueand iodine value and at all temperatures (Table1- 4). However peroxide value with ethanol at30 0 C differed non significantly. Neem oil yieldwas high during extraction with both petroleumether and ethanol at 50 0 C compared to other twotemperatures and it was more conspicuous withSatyanandam et al