Rationale for Water Extraction of Lipophilic Azadirachtin ( Tetranor Triterpenoid) from Neem Seeds - A Conundrum

Objectives: To extract lipophilic Azadirachtin from seed kernels of Azadirecta indica (Neem) with water and the rationale behind it as its solubility in water is very negligible (0.26 gm/lit of water).Methods: Neem seeds obtained from local market were dehulled to obtain kernels which were cold pressed to remove oil and extracted with four volumes of water at room temperature. Azadirachtin content in cold pressed neem kernel was determined using High Performance Liquid Chromatography (HPLC) with reverse phase silica column (RP18).Proteincontentinaqueousextractwasdeterminedby micro-Kjeldahl’s method and sugars by acid hydrolysis, permethylation and Gas Chromatography (GC). Findings: Aqueous extract of neem seed kernel was found to contain about 670 ppm of azadirachtin (AZA). A small portion of this aqueous extract when lyophilized yielded a hygroscopic material showing the presence of 4% AZA (HPLC method), 25% proteins (micro-Kjeldahl’s method) and about 50% of carbohydrates and other aliphatic compounds. The aqueous solution when re-extracted into ethyl acetate followed by precipitation using n-hexane yielded azadirachtin of 40% purity.Application: Lipophilic azadirachtins get extracted into water may be due to micelle formation of polysaccharides and proteins that cooccur in neem seed kernel.This hypothesis has been substantiated by addition of external azadirachtin to aqueous extract of neem seed kernel thereby enhancing its solubility from initial 670 ppm by more than two fold to a level of 1800 ppm. To obtain azadirachtin of about 40% purity directly from neem seed kernels would be possible only when first extracted with water.