Physicochemical and Chemical Variation in Neem Oils and Some Bioactivity Leads against Spodoptera litura F. Jitendra Kumar and Balraj S. Parmar* Division of Agricultural Chemicals, Indian Agricultural Research Institute, New Delhi 110012, India Forty-two neem ecotypes of India have been found to show a wide variation in the content of oil, and their physicochemical characteristics (color, specific gravity, refractive index, iodine value, acid value, and saponification value), total fatty acid, fatty acid composition (oleic, stearic, palmitic, linoleic, myristic, arachidic, and behenic acids), and the key meliacins (azadirachtin, nimbin, and salannin). The azadirachtin content did not correlate with any of the physicochemical and chemical parameters, but the nimbin and salannin contents correlated significantly with each other. The refractive index (+ve correlation) and iodine value (-ve correlation) showed weak but significant correlation with the contents of nimbin and salannin. Insect growth inhibition of Spodoptera litura revealed a wide variation in the EC 50 of the oils. The salannin and azadirachtin contents of the oils correlated the most with bioactivity. The iodine and acid values correlated weakly but significantly with bioactivity. Keywords: Neem; neem oil; physicochemical variation in neem; chemical variation in neem; neem bioactivity INTRODUCTION The ever increasing emphasis on developing environ- mentally benign pest control agents has brought neem, Azadirachta indica A. Juss, to the fore. Several reports confirming the effectiveness of neem-based products against various insect pests have appeared in the literature and can be referred to in the reviews by Saxena (1989), Schmutterer (1990), Singh (1993), Singh and Kataria (1991), and others. Neem oil is a key derivative that finds application as an important pest control agent. In India, the oil-based formulations have been prescribed to contain a mini- mum of 300 ppm of azadirachtin (Parmar and Ketkar, 1993). It is, however, known that besides azadirachtin, there are several other constituents of the oil that influence its bioactivity (Schmutterer, 1990). Neem kernels contain 30-45% (w/w) oil (Koul et al., 1990; Parmar and Ketkar, 1993). Variation in its physicochemical characteristics such as color, specific gravity, refractive index, saponification, iodine and acid values, and chemical constituents, namely the fatty acid composition, is reported (Roy and Dutt, 1929; Child and Ramanathan, 1936; Hilditch and Murti, 1939; Dasa Rao and Seshadari, 1942; Skellon et al., 1962). The variation in content of the key meliacin, azadirachtin, is also well documented (Ermel et al., 1987; Rengasamy et al., 1993). Despite the variation, specifications for neem oil are already prescribed (ISI, 1975; The Indian Pharmaco- poeia, 1966), and the samples for various uses are required to conform to these. Even though variation in the various physicochemical and chemical parameters of the oils has been reported, it is not known if the content of the meliacins correlates with any of these parameters. This paper reports information on this aspect. Additionally, preliminary information on the effect of these parameters on bio- activity against Spodoptera litura F. has been gener- ated. MATERIALS AND METHODS Neem Oils. Forty-two neem seed samples from different areas of India were procured through M/s Neem Mission, Pune, India. The dry, cleaned seeds were decorticated manually to obtain kernels which were crushed in a Waring blender and extracted with n-hexane (60-80 °C) in a Soxhlet extractor for 8 h (at this stage, a drop of hexane extract when evaporated on a filter paper left no residual oily spot). Hexane was removed in a rotavapor under reduced pressure at 60 °C to yield oil. Chemicals. Standard azadirachtin (purity 95%, HPLC), M/s Trifolio-M GMBH, Germany, was obtained through the courtesy of M/s Neem Mission. Reference salannin (purity 85%, HPLC) was obtained from Dr. E. D. Morgan, Keele University, Staffordshire, U.K., and reference nimbin (purity 85%, HPLC) was obtained from Dr. C. Devakumar of this Institute. Reference samples of methyl linoleate, methyl palmitate, and methyl oleate (Aldrich Chemical Co., Milwau- kee, WI) were obtained through the courtesy of Dr. T. R. Madan of the Biochemistry Department of this Institute. Methyl myristate and methyl stearate were prepared in the laboratory by esterification of their respective acids (Aldrich) with diazomethane in diethyl ether (AOAC, 1975). For routine laboratory work, laboratory grade chemicals and solvents were used. For HPLC analysis, the solvents were of analytical grade. Experimental Insect. Twelve-hour-old neonate first in- star larvae of S. litura F. were used. The culture was maintained at 27 ( 1 °C, 70-75% relative humidity, and 16:8 h light-dark (LD) and was reared on castor leaves until transferred to experimental diet (no. 9795, BioServ, Inc., French Town, NJ). Physicochemical Properties. Specific gravity (25 °C), refractive index (27 °C, M/s Toshniwal India Ltd. refractome- ter), and iodine, acid, and saponification values (AOAC, 1975) of oils were determined. The color was examined using Munsell color charts (1975 edition) by holding 1 mL of the oil sample in a 3.5 cm × 1 cm diameter glass tube directly behind the aperture separating the closest chip. Methyl Esters of Fatty Acids in Neem Oils. Accurate weights of oils (around 1 g) were saponified with alcoholic KOH. Alcohol was removed under reduced pressure in a rotavapor, and the saponified oil was hydrolyzed with dilute HCl to obtain free fatty acids. The whole content was taken in a separatory funnel and partitioned with diethyl ether (2 × 5 mL). The ether extracts were esterified with diazo- methane. The methyl esters were determined by gas-liquid chromatography (Hewlett-Packard, Model 5890 A, fitted with coiled glass column, 2 m × 2 mm i.d., packed with 3% SE-30 on 2137 J. Agric. Food Chem. 1996, 44, 2137-2143 S0021-8561(95)00283-4 CCC: $12.00 © 1996 American Chemical Society