ABSTRACT
Objective::To establish an UPLC method for the simultaneous determination of 6 flavonoids, and to research for the effect of Astragali Radix directional processed with four enzymes (complex enzyme, plant cellulase, snail enzyme, and β-glucosidase) on the contents of flavonoid glycosides and their aglycones in this herb. Method::Chromatographic separation was carried out on ACQUITY UPLC HSS T3 column (2.1 mm×100 mm, 1.8 μm) with the mobile phase of 0.1 mol·L-1 formic acid solution-0.1 mol·L-1 formic acid acetonitrile solution for gradient elution. The detection wavelength was set at 260 nm, the flow rate was 0.3 mL·min-1, the column temperature was 30 ℃, and the injection volume was 2 μL. Result::Calycosin-glucoside, calycosin, ononin, formononetin, 9, 10-dimethoxy-pterocarpan-3-O-β-D-glucoside and 3-hydroxy-9, 10-dimethoxy-pterocarpan showed good linear relationships within their own ranges (R2≥0.998 5), the relative standard deviations (RSDs) of precision, stability and repeatability were all <5.0%, and the average recovery was 97.62%-101.13% with RSDs of 1.4%-2.7%. In 0.5 g·L-1 level of enzyme solution, the contents of calycosin-glucoside, calycosin, ononin, formononetin, 9, 10-dimethoxy-pterocarpan-3-O-β-D-glucoside and 3-hydroxy-9, 10-dimethoxy-pterocarpan in Astragali Radix processed with complex enzyme were 0.082 0, 0.335 9, 0.055 9, 0.104 9, 0.015 0, 0.009 7 mg·g-1, the contents of them in Astragali Radix processed with plant cellulase were 0.105 7, 0.364 2, 0.070 2, 0.117 4, 0.020 8, 0.012 5 mg·g-1, their contents in Astragali Radix processed with snail enzyme were 0.031 4, 0.510 0, 0.043 5, 0.210 9, 0.013 0, 0.013 0 mg·g-1, and their contents in Astragali Radix processed with β-glucosidase were 0.085 3, 0.312 4, 0.061 5, 0.110 8, 0.005 8, 0.009 6 mg·g-1, respectively. Conclusion::After the processing of Astragali Radix by four enzymes, in addition to 9, 10-dimethoxy-pterocarpan-3-O-β-D-glucoside, the contents of calycosin-glucoside and ononin are reduced, but the contents of their three corresponding aglycones are significantly increased. The established method is simple, accurate and reproducible, and is suitable for the simultaneous determination of 6 flavonoids in Astragali Radix, which can provide a reference for this herb directional processed with enzymes.
ABSTRACT
OBJECTIVE：To establish a method for the determination of 8 glycosides（astragaloside Ⅰ，Ⅱ，Ⅲ，Ⅳ and calycosin glucopyranoside ，2′-hydroxy-3′，4′-dimethoxy-isoflavan-glucoside and 9，10-dimethoxy-pterocarpan-glucoside） and 4 aglycones（calycosin，formononetin，7，2′-dihydroxy-3′，4′-dimethoxy-isoflavan and 3-hydroxy-9，10-dimethoxy-pterocarpan） in Astragalus membranaceus ，and to investigate the effects of different processing temperatures on the contents of above 12 components. METHODS ：The contents of 12 components in A. membranaceus and samples processed under different temperatures（120，140，160，180，200 ℃）were determined by UPLC-MS/MS. The determination was performed on ACQUITY UPLC HSS T 3 column with mobile phase consisted of 0.1 mol/L formic acid water solution -0.1 mol/L formic acid acetonitrile solution （gradient elution ）at the flow rate of 0.5 mL/min. The column temperature was 30 ℃. The detection wavel ength was 260 nm，and sample size was 2 μL. Electrospray ion source（ESI）was used under positive ion mode （ESI+）. The mass scanning range was mass ratio （m/z）of 50-1 500，with capillary voltage of 2 000 V and ion source temperature of 100 ℃. The desolvation temperature was 400 ℃；flow rate of atomizing gas （N2） was 40 L/h，and that of desolvation was 800 L/h；collision energy （CE）was 20-30 V；data acquisition rate was 0.5 s/scan. RESULTS：The linear range of astragaloside Ⅰ，astragaloside Ⅱ，astragaloside Ⅲ，astragaloside Ⅳ，calycosin-glucopyranoside， calycosin，ononin，formononetin，2′-hydroxy-3′，4′-dimethoxy-isoflavan-glucoside，7，2′-dihydroxy-3′，4′-dimethoxy-isoflavan，9， 10-dimethoxy-pterocarpan-glucoside and 3-hydroxy-9，10-dimethoxy-pterocarpan were 0.001 16-0.232 0，0.000 276-0.055 2， 0.000 22-0.044 0，0.000 225-0.045 0，0.000 734-0.587 0，0.001 17-0.234 0，0.000 742- 0.148 0，0.001 30-0.260，0.003 98-0.795 0， 0.000 476-0.476 0，0.001 89-0.378 0，0.000 336-0.336 0 μg（all R2≥0.999 2），respectively. The limits of detection were 6.2×10－6， 4.8×10－6，3.8×10－6，3.4×10－6，5.8×10－6，4.8×10－6，4.2×10－6，3.2×10－6，5.8×10－6，2.6×10－6，4.2×10－6，6.4×10－6 μg，respectively. The limits of quantitation were 12.6×10－6，16.2×10－6，14.4×10－6，14.8×10－6，18.8×10－6，16.4×10－6，15.4×10－6，10.8×10－6，20.2×10－6， 12.4×10－6，14.6×10－6，23.4×10－6 μg，respectively. RSDs of precision ，stability（24 h）and repetition tests were all lower than 3.0％（n＝6）. The average recoveries were 99.1％，100.2％，98.7％，101.9％，98.6％，102.1％，99.2％，100.3％，98.7％， 99.2％，99.3％ and 100.8％，with the RSDs of 1.9％，2.2％，2.4％，1.8％，2.1％，1.7％，2.3％，1.9％，2.4％，1.8％，2.2％ and 1.9％（n＝6），respectively. The results showed that the contents of astragaloside Ⅰ，Ⅱ and Ⅲ decreased gradually with the increase of processing temperature ；the content of astragaloside Ⅳ increased gradually with the increase of temperature. The content of flavonoid glycosides ，such as calycosin glucopyranoside ，ononin，2′-hydroxy-3′，4′-dimethoxy-isoflavan-glucoside and 9， 10-dimethoxy-pterocarpan-glucoside decreased with the increase of temperature ；the corresponding aglycone components as flavonoid glycosides ，formononetin，3-hydroxy-9，10-dimethoxy- pterocarpan increased firstly and then decreased with the increase ； the content of 7，2′-dihydroxy-3′，4′- dimethoxy-isoflavan decreased with the increase of temperature. CONCLUSIONS ：Established UPLC-MS/MS method can be used for determination of 12 components in A. membranaceus . After processed under different temperature，the contents of glycosides decreased in general ，while the contents of aglycones increased in general.