An efficient method for the purification and characterization of nematicidal azadirachtins A, B, and H, using MPLC and ESIMS.
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 (EC(50) 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 (EC(50) 63.7 ppm) and Sclerotium rolfsii (EC(50) 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.
Sharma
Vandana
V
Division of Agricultural Chemicals, Indian Agricultural Research Institute, New Delhi-110 012, India.
Walia
Suresh
S
Kumar
Jitendra
J
Nair
Muraleedharan G
MG
Parmar
Balraj S
BS
D000935:Antifungal Agents / Q000494:pharmacology; D000969:Antinematodal Agents / Q000494:pharmacology; D031601:Azadirachta / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D036701:Limonins / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D021241:Spectrometry, Mass, Electrospray Ionization
D000935:Antifungal Agents; D000969:Antinematodal Agents; D036701:Limonins; C476491:azadirachtin H; C010329:azadirachtin; C476490:azadirachtin B