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An Efficient Method for the Purification and Characterization of Nematicidal Azadirachtins A, B, and H, Using MPLC and ESIMS

Sharma, Vandana, Walia, Suresh, Kumar, Jitendra, Nair, Muraleedharan G., Parmar, Balraj S.
Journal of agricultural and food chemistry 2003 v.51 no.14 pp. 3966-3972
Azadirachta indica, Caenorhabditis elegans, Corticium rolfsii, Rotylenchulus reniformis, Thanatephorus cucumeris, active ingredients, antifungal properties, azadirachtin, liquid chromatography, purification methods, seeds
Azadirachtin A enriched concentrate containing 60% active ingredient (a.i.) was prepared from the methanolic extract of the de-fatted neem (Azadirachta indica A. Juss) seed kernels. Azadirachtins A, B, and H, the three major bioactive constituents of neem seed kernel, were purified from this methanolic concentrate by employing reverse phase medium-pressure liquid chromatography (MPLC), using methanol−water solvent system as an eluant. The three pure azadirachtin congeners thus obtained were characterized by their unique mass spectral fragmentation, using electrospray probe in positive ion mode (ESI). All three azadirachtins exhibited nematicidal and antifungal activities. Azadirachtin B was the most effective against the reniform nematode Rotylenchulus reniformis (EC50 96.6 ppm), followed by Azadirachtin A (119.1 ppm) and H (141.2 ppm). At 200-ppm concentration, the test compounds caused 50−65% mortality of Caenorhabditis elegans nematode. Azadirachtin H showed the highest activity against the phytophagous fungi Rhizoctonia solani (EC50 63.7 ppm) and Sclerotium rolfsii (EC50 43.9 ppm), followed by B and A. The isolation of pure azadirachtins A, B, and H directly by MPLC purification from its concentrate and their characterization by ESIMS are unique and less time-consuming.