Processes of High-Yield Isolation and Flash Chromatographic Purification of Azadiradione from Neem Fruits

Neem (Azadirachta indica) is a well-known medicinal tree uniquely producing more than 300 diversified limonoids (tetra-nor-triterpenoids). While azadirachtin is well studied from amongst them due to its abundance in the plant. Other limonoids like azadiradione, nimbocinol, salannin, nimbin, etc., are also being increasingly recognized for their valuable biological activities. Availability of their sufficient quantities is often a critical limitation in expanding our understanding of their bioactive properties. This also limits applications, warranting development of appropriate processes for their high-yield isolation and/or production by chemical or biological transformation. Therefore, in this study, we have developed a process of high-yield extraction and flash chromatographic purification of azadiradione from neem fruit as well as developed a chemical transformation-aided efficient process for production of nimbocinol from azadiradione. The solvent extraction process provided 3.5% crude limonoid extract, which on flash chromatographic purification provided 8.13% of the limonoid extract enriched as azadiradione. Overall, extraction and purification process provided 0.05% yield of azadiradione isolated from neem fruit on fresh weight basis. This study also provided an efficient (> 85% yield) process for transformation of azadiradione into nimbocinol by avoiding the problem of reaction byproducts, unlike previous processes and approaches. Identity and purity of the isolated azadiradione and transformation products (nimbocinol) were established by multiple analytical and spectral analyses such as HPLC, LC-MS and NMR. Overall, the results of the study provided an efficient  protocol  for large-scale production of the neem phytochemical. This would pave the way for the evaluation of their biological studies using in vitro and in vivo tests for their efficacy, stability, toxicity, etc., as well as other diversified applications in agriculture and other products including nutraceutical formulations.