CHEMICAL INVESTIGATION OF NEEM LEAF GLYCOPROTEIN USED AS IMMUNOPROPHYLACTIC AGENT FOR TUMOR GROWTH RESTRICTION
Objective: Unique immune modulatory function of an aqueous preparation of neem leaf (NLP) in relation to cancer has already been reported. The objective of this present study is to find out the active component present in NLP.
Methods: NLP was exposed to a gradient of temperature, pH and enzymes to use for mice immunization before tumor inoculation. Glycoprotein extracted from NLP was purified and analyzed by using Folinâ€™s phenol reagent, polyacrylamide gel electrophoresis, scanning electron microscopy and amino acid analysis. Carbohydrate moiety of this protein was analysed by GLC-MS. Immunogenicity was checked by ELISA and immunoblotting.
Results: Exposure of NLP to adverse temperature and pH causes significant reduction in tumor growth restricting function of NLP. Treatment of NLP with proteolytic enzymes results abolition of the tumor growth restriction in mice. Biochemical assays indicated the presence of a glycoprotein in NLP, designated as neem leaf glycoprotein (NLGP) which appeared in non-denatured PAGE as a single band, and as three bands in SDS-PAGE having molecular weights 48 Kda, 24 Kda and 15 Kda. NLGP constitutes the carbohydrate moiety of about 33% consisting of arabinose, galactose and glucose. This glycoprotein consisted of seventeen amino acids of which nine were essential. Immunogenecity of this protein was defined by strong reaction of the ant-NLGP sera with NLGP by ELISA and immunoblot.
Conclusion: Overall results demonstrated the immense potential of newly identified NLGP, present in NLP as an immunoprophylactic agent for tumor growth restriction.
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