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Simultaneous determination of five azadirachtins in the seed and leaf extracts of Azadirachta indica by automated online solid-phase extraction coupled with LC-Q-TOF-MS.

ABSTRACT: Neem (Azadirachta indica) extract is well-known as a natural pesticide for the control of agricultural pests. Azadirachtin A and its structural analogues are considered as active compounds. However, the amounts of azadirachtins varies in neem extracts, providing a variety of insecticidal activities. In this study, a novel method of automated online solid-phase extraction coupled with liquid chromatography/quadrupole-time-of-flight mass spectrometry (SPE-LC-Q-TOF-MS) was developed and validated for simultaneous quantification of five azadirachtins (azadirachtins A, B, D, H and I) in seed and leaf extracts of A. indica. Different experimental parameters (such as SPE cartridge, injection volume and washing step) were optimized. The optimized SPE-LC-Q-TOF-MS method showed good recovery (82.0-102.8%), linearity (r²???0.9991) and precision (0.83-4.83%). The limit of detections (LODs) for the five analytes ranged from 0.34 to 0.76 ng mL^-1. The validated method was successfully applied for determination of the analytes in the neem leaves and seeds from different locations and a neem formulation. The online SPE-LC-Q-TOF-MS method was found to be a simple, precise and accurate and can be used as a powerful tool for quality control of neem extracts or its formulations.

SUBMITTER: Song L

PROVIDER: S-EPMC6053346 | biostudies-literature | 2018 Jul

REPOSITORIES: biostudies-literature

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Simultaneous determination of five azadirachtins in the seed and leaf extracts of Azadirachta indica by automated online solid-phase extraction coupled with LC-Q-TOF-MS.

Song Li L Wang Jin J Gao Quan Q Ma Xiaojiang X Wang Yuwei Y Zhang Yaoyao Y Xun Hang H Yao Xi X Tang Feng F

Chemistry Central journal 20180719 1

Neem (Azadirachta indica) extract is well-known as a natural pesticide for the control of agricultural pests. Azadirachtin A and its structural analogues are considered as active compounds. However, the amounts of azadirachtins varies in neem extracts, providing a variety of insecticidal activities. In this study, a novel method of automated online solid-phase extraction coupled with liquid chromatography/quadrupole-time-of-flight mass spectrometry (SPE-LC-Q-TOF-MS) was developed and validated f ...[more]

PMID: 30027362

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Project description:BackgroundAntibiotic resistances of pathogens and breast cancer warrant the search for new alternative strategies. Phytoextracts can eradicate microbe-borne diseases as well as cancer with lower side effects compared to conventional antibiotics.AimUnripe and ripe Azadirachta indica (neem) seed extracts were explored as potential antibiofilm and anticancer agents in combating multidrug-resistant infectious bacteria as well as anticancer agents against the MDR breast cancer cell lines.MethodsShed-dried neem seeds (both unripe and ripe) were pulverized and extracted using methanol. The chemical components were identified with FTIR and gas chromatography - mass spectrometry. Antibiofilm activity of neem seed extracts were assessed in terms of minimum biofilm inhibitory concentration (MBIC), minimum biofilm eradication concentration (MBEC), and fluorescence microscopic studies on Staphylococcus aureus and Vibrio cholerae. Bacterial cells were studied by fluorescence microscopy using acridine orange/ethidium bromide as the staining agents. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values were evaluated to observe the antibacterial activities. Cytotoxicity of the extracts against human blood lymphocytes and the anticancer activity against drug-resistant breast cancer cell lines were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and fluorescence-activated cell sorting (FACS) studies.Results4-Ethyl-2-hydroxy-2-cyclopentene-1-one, phthalic acid, and 2-hexyl-tetrahydro thiophane were the major compounds in unripe neem seed, whereas 3,5-dihydroxy-6-methyl-2,3-dihydro-4-H-pyran-4-one and 4-ethylbenzamide were predominant in ripe neem seed. Triazine derivatives were also common for both the extracts. MBIC values of unripe and ripe neem seed extracts for S. aureus are 75 and 100 µg/mL, respectively, and for V. cholerae, they are 100 and 300 µg/mL, respectively. MBEC values of unripe and ripe seed extracts are 500 and 300 µg/mL, respectively for S. aureus and for V. cholerae the values are 700 and 500 µg/mL, respectively. Fluorescence microscopic studies at 16 and 24 h, after bacterial culture, demonstrate enhanced antibiofilm activity for the ripe seed extract than that of the unripe seeds for both the bacteria. MTT assay reveals lower cytotoxicity of both the extracts towards normal blood lymphocytes, and anticancer activity against breast cancer cell line (MDA-MB-231) with superior activity of ripe seed extract. FACS studies further supported higher anticancer activity for ripe seed extract.ConclusionsMethanolic extract of neem seeds could substantially inhibit and eradicate biofilm along with their potent antibacterial and anticancer activities. Both the extracts showed higher antibiofilm and antibacterial activity against S. aureus (gram-positive) than V. cholerae (gram-negative). Moreover, ripe seed extract showed higher antibiofilm and anticancer activity than unripe extracts.

Dataset Information

Simultaneous determination of five azadirachtins in the seed and leaf extracts of Azadirachta indica by automated online solid-phase extraction coupled with LC-Q-TOF-MS.

Publications

Simultaneous determination of five azadirachtins in the seed and leaf extracts of Azadirachta indica by automated online solid-phase extraction coupled with LC-Q-TOF-MS.

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