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

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Proceedings of the National Academy of Sciences, India Section B: Biological Sciences Aims and scope Submit manuscript

Abstract

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.

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Acknowledgements

NSS acknowledges financial support under network project BSC0203-CSIR, RSS & NSS acknowledges financial support CSIR-HCP. LKN is thankful to UGC (India) for fellowship.

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Authors and Affiliations

  1. Department of Metabolic and Structural Biology, CSIR-Central Institute of Medicinal and Aromatic Plants (CSIR-CIMAP), Lucknow, 226015, UP, India

    Lokesh Kumar Narnoliya, Laxminarain Misra & Rajender Singh Sangwan

  2. Academy of Scientific and Innovative Research (AcSIR), CSIR- HRDC Campus, Sector 19, Kamla Nehru Nagar, Ghaziabad, UP, 201002, India

    Rajender Singh Sangwan

  3. Department of Biochemistry, Central University of Haryana, Mahendergarh, Haryana, 123029, India

    Neelam S. Sangwan

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  1. Lokesh Kumar Narnoliya
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  2. Neelam S. Sangwan
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  3. Laxminarain Misra
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  4. Rajender Singh Sangwan
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Correspondence to Neelam S. Sangwan or Rajender Singh Sangwan.

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Significance Statement: A method for azadiradione (limonoid) purification and estimation was established by using flash chromatography followed by HPLC/LC–MS analyses. Further, methanolic NaOH/KOH based, an efficient chemical transformation process for conversion of azadiradione into nimbocinol was developed.

Supplementary Information

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Supplementary file 1 (TIFF 3942 kb) Fig. S1. TLC profiling of selected purified fractions from flash chromatography

40011_2021_1254_MOESM2_ESM.tiff

Supplementary file 2 (TIFF 260 kb) Fig. S2. HPLC chromatogram of purified pure compound fractions from flash chromatography

40011_2021_1254_MOESM3_ESM.tiff

Supplementary file 3 (TIFF 308 kb) Fig. S3. NMR analysis of azadiradione and nimbocinol. (A) 1H NMR of azadiradione (B) 13C NMR of azadiradione and (C) 1H NMR of nimbocinol

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Narnoliya, L.K., Sangwan, N.S., Misra, L. et al. Processes of High-Yield Isolation and Flash Chromatographic Purification of Azadiradione from Neem Fruits. Proc. Natl. Acad. Sci., India, Sect. B Biol. Sci. 91, 847–853 (2021). https://doi.org/10.1007/s40011-021-01254-x

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  • DOI: https://doi.org/10.1007/s40011-021-01254-x

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