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Objective: To establish a determination method for kinds of chemical components of iridoids in the roots of Gentiana crassicaulis in transdermal absorption liquid, and research its transdermal permeability, so as to provide scientific basis for transdermal delivery system, clinical medication and reform of the traditional forms of G. crassicaulis. Methods: Based on the results of the previous investigation, in this paper, using the roots of G. crassicaulis as the research subject, a certain concentration solution of G. crassicaulis extract was prepared by the alcohol extraction method. Three kinds of common penetration enhancers, azone, borneol and propylene glycol were used. The effects of single penetration enhancer and dual compound penetration enhancers on the transdermal penetration of five kinds of chemical components of loganic acid, shanzhiside methyl ester, swertimarin, gentiopicroside and sweroside in vitro and the three kinds of chemical components of gentiopicroside, loganic acid and swertimarin in vivo were quantitatively studied by the method of HPLC to investigate the transdermal permeability of G. crassicaulis extract in mice skin model. Results: According to the experimental results, compared to the control group, penetration enhancers significantly increased the absorption of five chemical components of G. crassicaulis in vitro. Transdermal absorption rates (J) of loganic acid, shanzhiside methyl ester, swertimarin, gentiopicroside and sweroside were 12.306 0, 1.248 8, 4.187 5, 153.030 0 and 5.012 6 μg/(cm2∙h), respectively. The transdermal enhancer effects of A (5% azone), B (5% borneol), C (5% propylene glycol), A + B (2.5% azone and 2.5% borneol), A + C (2.5% azone and 2.5% propylene glycol), A + C (2.5% borneol and 2.5% propylene glycol) were 9.73, 2.57, 13.94, 15.92 and 8.08 times faster than the control group, respectively. Among these, the group of A + C had a marked osmotic enhancer effect in vitro. In comparison with the control group, the in vivo percutaneous penetration test indicated that the dual compound penetration enhancers of 2.5% azone and 2.5% propylene glycol had a marked effect for the permeability enhancement. Conclusion: This study showed azone and propylene glycol significantly promoted the percutaneous penetration effect of loganic acid, shanzhiside methyl ester, swertimarin, gentiopicroside and sweroside of G. crassicaulis, and this study laid a foundation for the quality control of percutaneous drug delivery preparation of G. crassicaulis.
RESUMO
Objective: To develop and validate a ultra-high performance liquid chromatography-quadrupole/electrostatic field orbitrap high-resolution mass spectrometry (UPLC-Q-Orbitrap HRMS) Methods: for simultaneously qualitative and quantitative determination of eight chemical components (gentiopicroside, loganic acid, swertiamarin, sweroside, luteolin, isovitexin, apigenin, and kaempferol) in raw and processed products of Gentiana crassicaulis. Methods Waters Acquity UPLC BEH C18 (100 mm × 2.1 mm, 1.7 μm) column was used for gradient elution with methanol-0.1% formic acid in mobile phase. The volume flow was 0.3 mL/min and the column temperature was 35 ℃. The mass spectrometer was detected using negative ion detection mode. Results: After frying and wine frying, the content of iridoid in G. crassicaulis was significantly increased. Statistically, the differences between gentiopicroside and loganic acid after frying were significant. The content of flavonoids changed little after frying and wine frying. The total content changes of the two components were as follows: the content of iridoids: frying > wine frying > raw products; flavonoids content: raw products > wine frying > frying. Conclusion: Based on UPLC-Q-Orbitrap HRMS, the quantitative analysis method of eight components of raw and processed products of G. crassicaulis was established, which provided a basis for optimizing the processing technology of G. crassicaulis and improving the clinical curative effect of G. crassicaulis.
RESUMO
Objective The effects of far infrared, vacuum, freezing, hot air, dry in the shade and “sweating” drying methods on the quality of Gentiana crassicaulis were evaluated through chemical component content determination. Methods A UPLC-ESI-HRMSn method was used to determine quality score of four iridoids (swertiamarin, gentiopicroside, loganic acid, and sweroside) and four flavonoids (luteolin, isovitexin, apigenin, and kaempferol) of G. crassicaulis. In order to determine the best drying method, the results were combined with analysis of variance, cluster analysis and TOPSIS analysis, which could comprehensively evaluate the G. crassicaulis obtained with different drying methods. Results The contents of swertiamarin in samples dried in different methods were similar. The contents of gentiopicroside, loganic acid, sweroside, luteolin, isovitexin, apigenin, and kaempferol in samples dried in different methods were obviously different. Among them, the four iridoids were best preserved in the freezing drying samples, and the four flavonoids were best preserved in the “sweating” drying samples. All compounds significantly degraded in the sample dried in the shade. Cluster analysis and TOPSIS analysis showed that the samples of “sweating” and freezing drying methods had a similar and high quality, follow by the samples of hot air and far infrared ray. The samples dried in the shade had the worst quality. Conclusion It was noteworthy that the effects of different drying methods on the quality of G. crassicaulis. This study showed that the “sweating” drying method as official method listed in Chinese Pharmacopoeia was verified to be scientific. However, the traditional “sweating” method needed a long time for drying. In order to enhance the drying efficiency, further research should focus on the combination of “sweating” method and modern drying techniques, such as hot air, freeze, vacuum drying methods, which could shorten the drying time on the basis of the stable quality of G. crasicaulis herbs.
RESUMO
A rapid method combining microwave-assisted extraction (MAE) and high-speed counter-current chromatography (HSCCC) was applied for preparative separation of six bioactive compounds including loganic acid (I), isoorientin-4'-O-glucoside (II), 6'-O-ß-d-glucopyranosyl gentiopicroside (III), swertiamarin (IV), gentiopicroside (V), sweroside (VI) from traditional Tibetan medicine Gentiana crassicaulis Duthie ex Burk. MAE parameters were predicted by central composite design response surface methodology. That is, 5.0 g dried roots of G. crassicaulis were extracted with 50 mL 57.5% aqueous ethanol under 630 W for 3.39 min. The extract (gentian total glycosides) was separated by HSCCC with n-butanol/ethyl acetate/methanol/1% acetic acid water (7.5:0.5:0.5:3.5, v/v/v/v) using upper phase mobile in tail-to-head elution mode. 16.3, 8.8, 12., 25.1, 40.7, and 21.8 mg of compounds I-VI were obtained with high purities in one run from 500 mg of original sample. The purities and identities of separated components were confirmed using HPLC with photo diode array detection and quadrupole TOF-MS and NMR spectroscopy. The study reveals that response surface methodology is convenient and highly predictive for optimizing extraction process, MAE coupled with HSCCC could be an expeditious method for extraction and separation of phytochemicals from ethnomedicine.
