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Objective To develop a systematic chromatography separation method for flavonoids from Glycyrrhiza uralensis Fisch. (GU). Methods A new method for the separation of effective parts and monomers of flavonoids from GU by two-dimensional reversed-phase liquid chromatography was developed using the self-developed preparation chromatography plant system with independent intellectual property rights. Flavonoids compounds were enriched with specific adsorption materials. The separation conditions of the chromatography were optimized by the chromatographic separation expert system software, and the loading weight of samples and the enrichment times of a separation were investigated. Results The process of chromatography separation of flavonoids from GU had good precision and reproducibility with C18 as separation and enrichment solid phase and the methanol/water and acetonitrile/water as mobile phase of one-dimensional and two-dimensional chromatography system. The dilution solution which used for one-dimensional and two-dimensional enrichment chromatography was water. The flow rate of gradient elution and dilution enrichment solution was 21 mL/min. The sample loading amount of chromatography separation was 300 mg each time. A total of 16 flavonoids parts contained stable chemical composition were obtained by the repeatable separation method after three times of enrichment. Nine pure compounds were obtained and identified by NMR and MS, which were liquiritin, liquiritigenin, formononetin, echinatin, 7,4'-dihydroxyflavone, 4'-O-[β-D-apio-D-furanosyl-(1→2)-β-D-glucopyranosyl] liquiritigenin, isoliquiritigenin, glycyrol, and glycycoumarin. Conclusion The study can provide a certain reference value for the systematic separation and cognition for flavonoids from GU.
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Objective To establish a quantitative analysis of multi-components by single marker (QAMS) for determination of five active components in Draconis Resina and discuss application of QAMS in quality control of ethnic medicines. Methods Using the method of HPLC, the Fortis Xi C18 column (250 mm × 4.6 mm, 5 μm) was used. The mobile phase was composed of acetonitrile (A)-1.0% acetic acid (B) with gradient elution (0-10 min, A: 25%→30%; 10-60 min, A: 30%→50%) at the flow rate of 1.0 mL/min. The detection wavelength was 278 nm, the column temperature was 30 ℃ and the sample size was 10 μL. Pterostilbene was selected as an internal standard to establish the relative correction factors (RCFs) of 7, 4’-dihydroxyflavone, resveratrol, loureirin A, and loureirin B with reference to pterostilbene so as to achieve simultaneous determination of multi-indexed components. The contents of five active components were determined by both external standard method (ESM) and QAMS. Meanwhile, relative error (RE) between QAMS and ESM was analyzed to evaluate QAMS method. Results There were good linearities in the range of 10.23-102.27 μg/mL for 7,4’-dihydroxyflavone, 11.01-110.14 μg/mL for resveratrol, 9.47-94.72 μg/mL for loureirin A, 11.59-115.90 μg/mL for loureirin B and 24.35-243.52 μg/mL for pterostilbene, RCFs of 7,4’-dihydroxyflavone, resveratrol, loureirin A and loureirin B with reference to pterostilbene were 0.626, 1.064, 1.154, and 0.837 respectively, and repeatability was good in different experimental conditions (RSD < 3.0%).There were no significant difference between the quantitative results of the two methods. Conclusion QAMS method is feasible, credible, and can be used to determine multiple components in Draconis Resina. QAMS can be adopted as a novel strategy for quality control of ethnic medicines.
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Objective To study the effects of different transport protein on the transport of 7,4'-dihydroxyflavone (7,4'-DHF) and its metabolite (7,4'-DHF-S) in Caco-2 cell model.Methods Ultra performance liquid chromatography was employed to determinethe content of 7,4'-DHF and 7,4'-DHF-S incubation buffer,their structures were identified by LC-MS/MS.Bidirectional transport of Caco-2 cells model was used to investigate the influence of ko143 (the inhibitor of BCRP) and MK571 (the inhibitor of MRP2) on the transport of 7,4'-DHF and 7,4'-DHF-S,respectively.Results Metabolic product of 7,4'-DHF in Caco-2 monolayer cell was identified as one monosulfate;PDR of 7,4'-DHF was (1.43 ± 0.11),PDR of ko143 and MK571 on the apparent permeability of 7,4'-DHF was (1.59 ± 0.04) and (1.48 ± 0.07) (P ＞ 0.05);PDR of 7,4'-DHF-S was (1.60 ± 0.06);ko143 could significantly reduce the apparent permeability of 7,4'-DHF-S,and the PDR was (0.23 ±0.03) (P ＜ 0.01);MK571 had no significant effect on the apparent permeability of the 7,4'-DHF-S,and the PDR was (1.51±0.04) (P ＞ 0.05).Conclusion Caco-2 cells can mediate the suffonated reaction of 7,4'-DHF;7,4'-dihydroxyflavone sulfonated combination product may be a substrate for BCRP.
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Objective: To evaluate the antioxidant capacity of the main components from Dracaena cochinchinensis by HPLC-DPPH and provide a foundation for "spectrum-effect" quality control standard. Methods: The determination was developed on Phenomenex lura C18 column (250 mm × 4.6 mm, 5 μm) with gradient elution of methanol-acetonitril-0.2% H3PO4 and the detective wavelength was set at 306 nm. The column temperature was 35℃. Results: The DPPH free radical scavenging abilities of these five antioxidants were as follows: pterstilbene > resveratrol > luoreirin B > luoreirin A > 7,4'-dihydroxyflavone. The analysis on the structures of the five compounds showed that the hydroxymethylation didn't decrease the antioxidant activities of stilbenes. The symmetry of B ring of dihydrochalcone has the significant effect on the antioxidant activities. However, 7,4'-dihydroxyflavone didn't show the related activity in D. cochinchinensis. Conclusion: HPLC-DPPH can not only be used for screening the components with antioxidant potency but also for the purpose of quality evaluation of D. cochinchinensis, and the method proves to be selective, simple, and reproducible.