An Efficient Method for the Purification and Characterization of Nematicidal Azadirachtins A, B, and H, Using MPLC and ESIMS VANDANA SHARMA, † SURESH WALIA, † JITENDRA KUMAR, † MURALEEDHARAN G. NAIR,* ,‡ AND BALRAJ S. PARMAR † Division of Agricultural Chemicals, Indian Agricultural Research Institute, New Delhi-110 012, India and Department of Horticulture and National Food Safety and Toxicology Center, Michigan State University, East Lansing, Michigan 48824 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. KEYWORDS: Azadirachta indica; azadirachtin; Caenorhabditis elegans; Rotylenchulus reniformis; Rhizoctonia solani; Sclerotium rolfsii; antifungal; nematicidal; ESIMS INTRODUCTION Azadirachtin-A, the major bioactive secondary metabolite of Azadirachta indica A Juss (neem), is well-known for its excellent insecticidal, antifungal, and growth disruptive activity against a variety of insect pests (1-6). An increased under- standing of its molecular structure, as well as that of its natural and synthetic congeners, revealed interesting structure-activity relationships (7-10). Among the large number of tetranortri- terpenoids isolated from various parts of A. indica, azadirachtin A and its several congeners have been found to exhibit significant biological activity. Several reports are now available about the extraction and isolation of neem azadirachtinoids (1, 11-13). The tedious process of the isolation of these constitu- ents involves extraction, partitioning of extractives between different solvents, column chromatography, preparative thin- layer chromatography, and preparative high performance liquid chromatography. Cost intensive and time-consuming high performance liquid chromatography has been employed in the past for the separation of azadirachtins A, B, D, H, and I from the seed kernel concentrate (14-16). In these studies, partially purified compounds obtained from the combined peaks for azadirachtins A + D and azadirachtins H + I in the first preparative LC run on one column were further subjected to another preparative LC run on RP-8/RP-18 columns to obtain azadirachtins A and D as well as H and I, respectively. Recently, minor meliacin constituents such as 11-epi-Azadirachtin H and 11-epi-azadirachtin D, the epimers of azadirachtins H and D (17-19), and three other azadirachtin congeners, namely azadirachtin K (20), 13,14-desepoxyazadirachtin A (21), and 1-tigloyl-3-acetyl-11-hydroxy-4-methyl meliacarpin (22) have been separated and identified from neem seed extracts. While azadirachtin A was present in the extract to the extent of 85%, azadirachtins B and H, the other two major components, were present only at concentrations up to 15% (23). Other azadirachtin congeners occurred as minor constituents in neem seed extracts. Despite the considerable progress that has been made in neem research so far, only a few methods are available for the separation of major azadirachtins. To determine activity profile and content of major azadirachtins in azadirachtin-based neem formulations and technical materials, azadirachtin congeners A, B, and H are required both as test materials and as reference standards. Because these materials are not available com- mercially, a fast and economical method for their separation and characterization from neem extractives in adequate quanti- * To whom correspondence should be addressed. Telephone: (517) 353- 2915. Fax: (517) 432-2310. Email: nairm@msu.edu. † Indian Agricultural Research Institute. ‡ Michigan State University. 3966 J. Agric. Food Chem. 2003, 51, 3966-3972 10.1021/jf0342167 CCC: $25.00 © 2003 American Chemical Society Published on Web 05/31/2003