Antioxidative diarylheptanoid derivatives from Alnus japonica by high-speed counter-current chromatography, and HPLC method development and validation of isolated compounds
- Antioxidative diarylheptanoid derivatives from Alnus japonica by high-speed counter-current chromatography, and HPLC method development and validation of isolated compounds
- 최순정; 임순성; 이민영; 안홍열; 이재용; 정상훈
- Alnus japonica; Diarylheptanoids; High-speed counter-current chromatography; Online HPLC-ABTS+ bioassay; Method validation
- Issue Date
- International Biotechnology Symposium (IBS) and Exhibition
- Antioxidant compounds are now of considerable interest due to their effect of preventing or delaying aging and various diseases. In the present study, the antioxidant constituents in the A. japonica were identified and their individual antioxidant capacities were evaluated by on-line HPLC-2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS)+ bioassay system. The ABTS+-based antioxidant activity profile showed that three negative peaks exhibited antioxidant activity. We isolated 3 diarylheptanoid derivative compounds from the extract by using high-speed counter-current chromatography (HSCCC) with a two-phase solvent systems composed of n-hexane/ethylacetate/methanol/water (4:6:4:6, v/v) and ethyl acetate/methanol/water (1:0.1:1, v/v). From 2.0 g of A. japonica, a total of 527 mg of hirsutanonol 5-O-β-D-glucopyranoside, 80.04 mg of 3-deoxohirsutenonol 5-O-β-D-glucopyranoside, and 91.0 mg of hirsutenone were obtained with purity of 98.2%, 91.7%, and 98.5%, respectively. The purity of the isolated compounds was analyzed by HPLC and their structures were identified by 1H- and 13C-nuclear magnetic resonance spectrometry (NMR), heteronuclear multiple bond correlation (HMBC), and heteronuclear single quantum correlation (HSQC). The developed method was validated in terms of specificity, linearity, range, limit of detection and quantification, accuracy, precision, following the ICH guideline. These studies conclude therefore massive isolation of compounds from A. japonica by HSCCC is a powerful and fast technique. Moreover, developed HPLC method for standardization is simple and reproducible which might be applied for quantitative analysis of A. japonica.
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