| The applications of quantitative analysis of multi-component with single marker and high-speed countercurrent chromatography in quality control of two traditional Chinese medicinal herbs,including Rehmanniae Radix and Cassiae Semen,were mainly investigated in the present work.The quantitative analysis of multi-component with single marker was established for determination of contents of chemical constituents in traditional Chinese medicinal herb based on an internal substance reference.The relative correction factors were achieved by the relationship between an internal standard substance and other components.The content of these components were calculated by the relative correction factors.The content of active components in traditional Chinese medicinal herb could be determined directly with a lower cost.At the same time,simultaneous determination of various components was obtained.Countercurrent chromatography is a liquid-liquid partition chromatographic technology without irreversible adsorption and high peak resolution could be obtained as that of column chromatography.Countercurrent chromatography was used to prepare the main active substances isolated from Rehmanniae Radix and Cassiae Semen,and to provide an effective way for the preparation of the reference substance involved in this article,which could provide the substance references for determination of contents of components in the traditional Chinese herbs.Two kinds of elution modes,including conventional and pH-zone-refining countercurrent chromatography,were employed in the present work.The main contents of this paper are as follows:(1)Acteoside,an active component of the traditional Chinese medicinal herb Rehmanniae Radix,was separated with high purity by a combination of macroporous resin column chromatography and countercurrent chromatography.The static adsorption and desorption of acteoside in the crude extract were investigated using four kinds of macroporous resin,among which the D101 macroporous resin presented the best adsorption and desorption rates toward the compound.The crude extract was then gradient-eluted with increasing concentrations of ethanol,where it was found that the highest content of acteoside was obtained when using 10%(v/v)ethanol as eluent,in which the purity of acteoside was increased from 4.9%to 32.6%.Then,the partially purified crude extract(165 mg)was further purified by high speed countercurrent chromatography using a two-phase solvent system consisting of ethyl acetate-n-butanol-water(1:4:5,v/v),yielding 45 mg of acteoside with 96%purity.(2)Seven hydroxyanthraquinones were successfully separated and purified from the traditional Chinese medicinal herb Cassiae Semen by conventional and pH-zone-refining countercurrent chromatography with an environmental-friendly biphasic solvent system,in which elution-extrusion mode was employed to reduce the separation time.Elution-extrusion mode was investigated for pH-zone-refining countercurrent chromatography for the first time.A two-phase solvent system composed of n-hexane:ethyl acetate:ethanol:water(5:3:4:4,v/v)was used for the conventional countercurrent chromatography while the same two-phase solvent system composed of n-hexane:ethyl acetate:ethanol:water(3:5:2:6,v/v)was used for pH-zone-refining countercurrent chromatography,in which 20 mmol/L trifluoroacetic acid was added in the organic phase as a retainer and 15 mmol/L ammonia was added to the aqueous phase as an eluter.100 mg of crude sample was separated by conventional countercurrent chromatography while over 400 mg of crude sample could be well separated by pH-zone-refining countercurrent chromatography,yielding 53 mg of aurantio-obtusin,40 mg of chryso-obtusin,18 mg of obtusin,24 mg of obtusifolin,10 mg of emodin and 105 mg of the mixture of chrysophanol and physcion with a purity of over 95.8%,95.7%,96.9%,93.5%,97.4%,77.1%and 19.8%,as determined by HPLC.Furthermore,the difference in elution sequence between conventional and pH-zone-refining mode was discussed.(3)Determination of seven hydroxyanthraquinones in Cassiae Semen by quantitative analysis of multi-components by single marker method was investigated.The relative correction factors between the internal standard substance and other six components were established under detection wavelength of 280 nm,in which the aurantio-obtusin was used as the internal standard substance.The relative correction factors for six components were 1.4523,1.4945,1.8053,3.1003,3.8456,3.1547,respectively.The calculated content of seven hydroxyanthraquinones were found to be 0.118%,0.030%,0.024%,0.050%,0.016%,0.169%and 0.026%,respectively.Experiment results showed that RSD was less than 5%in comparison with that of external standard method and no significant difference in contents of above chemical constituents was found for the two kinds of methods.UV absorption of each hydroxyanthraquinone was determined.According to UV absorption curve concerning relative difference or ratio of six components when aurantio-obtusin was used as the internal standard substance,12 absorption peaks and valleys of the relative difference,including 206,225,240,254,280,285,300,325,340,345,365 and 370 nm were selected as detection wavelengths.The relative correction factors of the other six components under different wavelength were achieved,and it was found that the relative correction factors of the hydroxyanthraquinones were changed with different detection wavelengths,where a trend of positive correlation between detection wavelength and relative correction factors was found.(4)Determination of three components in Rehmanniae Radix by quantitative analysis of multi-components by single marker.The relative correction factors for two components,rehmannioside D and acteoside,were obtained under the detection wavelength of 200 nm when the catalpol was used as the internal standard substance.The relative correction factors for two components were 0.7132 and 2.3743 when catalpol was used as the internal standard substance.The calculated content of three components were 1.0342%,0.2105%and 0.1033%,respectively.Experiment results showed that RSD was less than 5%in comparison with that of external standard method and no significant difference in contents of the two chemical constituents was found for the two kinds of methods.According to UV absorption curve concerning relative difference or ratio of two components when catalpol was used as the internal standard substance,4 absorption peaks and valleys of the relative difference values,200,218,290 and 334 nm,were selected as detection wavelengths.The relative correction factors of the two components under different wavelength were achieved,and it was found that the relative correction factors were changed with different detection wavelengths,where a trend of positive correlation between detection wavelength and relative correction factors was found.The research topic on how to select a suitable UV detection wavelength was mainly proposed and verified according to the above experimental studies. |
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