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Objective Based on the main morphological traits and chemical constituents of 42 Glycyrrhiza uralensis germplasm in China, the genetic diversity of them was analyzed comprehensively. Methods Twelve morphological traits and five kinds of chemical components of G. uralensis germplasm transplanted in the same place were collected. The genetic diversity index and coefficient of variation were calculated, using cluster analysis and principal component analysis for statistical analysis. Results Among the five chemical constituents, the highest genetic diversity index of liquiritin content was 2.05; The maximum coefficient of variation of isoliquiritin content was 99.50%; The content of liquiritin was moderately correlated with the content of isoliquiritin. Among 12 morphological traits, the highest genetic diversity index of plant height was 2.08, and the maximum coefficient of variation of actual fruit sequence was 37.09%. The variation of fruit type and plant type was greater than that of leaf type. Cluster analysis divided 42 germplasms into three types, and the second group had better germplasm quality. The principal component analysis reduced 17 indicators to six factors, with a cumulative contribution rate of 72.96%. Factor 6 was a factor that represents glycyrrhizic acid, liquiritin, and isoliquiritin. Conclusion The genetic diversity of the main morphological traits and chemical constituents of 42 G. uralensis germplasms is rich, and six excellent germplasms are V08, V10, V17, V34, V36, and V38.
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Objective: To establish the quality control method for high-quality Glycyrrhizae Radix et Rhizoma Praeparata Cum Melle (GRRPM) in Xinshenghua Granules (XG). Methods: The HPLC fingerprint analysis method for high-quality GRRPM was developed. The method of quantitative analysis of multi-components by single marker (QAMS) for simultaneously determining the six active constituents (liquiritin, isoliquiritin, glycyrrhizic acid, liquiritigenin, liquiritin apioside, and isoliquiritin apioside) was developed and evaluated by comparison of the quantitative results with external standard method. Results: The fingerprints of GRRPM were established by HPLC from 30 batches. Fourteen peaks were acquired as common fingerprint peaks and seven peaks among them were identified with chemical reference. The relative retention time of common peaks and the peak area ratio of some common peaks were used to differentiate high-quality products from general products as indicators for fingerprint evaluation. With liquiritin and glycyrrhizic acid as internal standards, QAMS was developed and the mean relative correlation factors (RCFs) of isoliquiritin, liquiritigenin, liquiritin apioside, and isoliquiritin apioside were 0.502, 0.578, 0.252, and 0.257, respectively. The specifications of high-quality GRRPM were established for XG. Conclusion: These methods could be used for quality control of high-quality GRRPM of XG.
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Objective: To investigate the effect of extraction and reverse extraction conditions on the transfer of isoliquiritin. Methods: The extraction rate of isoliquiritin was used as the index to determine the best composition and concentration of complexing extractant. Taking the reverse extraction rate of isoliquiritin as the index, the species and concentration of the reverse extraction agent were investigated, and finally the technological conditions for the extraction and reverse extraction of isoliquiritin from glycyrrhizin ultrafiltration were obtained. Results: The best complexation extraction condition was: the ratio of TRPO to sulfonated kerosene was 7:93, and the extraction rate of isoliquiritin reached 97.60%. The best reverse extraction agent was 0.26% NaOH aqueous solution, and the reverse extraction rate of isoliquiritin reached 95.40%. Conclusion: Under the optimal conditions of extraction and reverse extraction obtained in this experiment, isoliquiritin can be transferred from glycyrrhizin ultrafiltration to complexing extractant and then to alkaline reverse extraction agent, and finally isoliquiritin can be obtained by extraction and reverse extraction.
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Objective: To study the chemical constituents of flavonoids from Glycyrrhizae Radix et Rhizoma. Methods: The compounds were isolated and purified by column chromatography over HP-20 macroporous resin, silica gel, Sephadex LH-20, and preparative RP-HPLC. Their structures were elucidated by physicochemical properties and spectral analyses. Results: Ten flavonoids were isolated and identified as 4’,6,7-trihydroxy-2’-methoxyl-chalcone (1), 3’,4’,5,7-tetrahydroxy-8-(3-hydroxy-3- methylbutyl)-isoflavone (2), isoliquiritigenin (3), isoliquiritin (4), echinatin (5), orobol (6), ononin (7), 2(S)-3’,5’,7-trihydroxy- flavanone (8), 2(S)-naringenin-4’-O-β-D-glucopyranoside (9), and 4’,7-dihydroxyflavone (10). Conclusion: Compounds 1 and 2 are new compounds named isolicochalcone B and licoisoflavone G, while compound 9 is isolated from the genus for the first time.
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OBJECTIVE： To optimize the extraction technology of the flavonoids from Glycyrrhiza uralensis. METHODS： Using total contents of four flavonoids， liquiritinapioside， glycyrrhizin， isoliquiritin apioside and formononetin as indexes， types and volume fractions of extraction solvents （water， ethanol）， volume of addition and extraction time as factors， based on single factor experiment， Box-Behnken design-response surface method was used to optimize the extraction technology of flavonoids from G. uralensis. Validation test was also conducted. RESULTS： The optimal extraction technology was 50 mL 50％ ethanol as extraction solvent， 0.200 g G. uralensis， ultrasonic extraction for 50 min. In validation test， the extraction amounts of liquiritinapioside， glycyrrhizin， isoliquiritin apioside and formononetin were 10.733 0， 27.784 9， 3.441 9， 0.429 1 mg/g， respectively （all RSDs＜3.0％， n＝3）. The average total extraction amount of four flavonoids was obtained was 42.388 9 mg/g， the relative error of which to predicted value （42.173 2 mg/g） was 0.52％ （n＝3）. CONCLUSIONS： The optimized extraction technology is simple， rapid and stable， and can be used for the extraction of flavonoids from G. uralensis.
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This study was aimed to establish an HPLC method for the determination of liquiritin, isoliquiritin, liquiritigenin and glycyrrhizic acid in roots and knotty rhizome of Glycyrrhiza uralensis. The analysis was performed on a Diamonsil C18 column (250 mm í 4.6 mm, 5 μm) by using a gradient elution with mobile phase of water, phosphoric acid, acetonitrile at the flow rate of 1.0 mL·min-1. The detection wavelength was 276 nm (0~18 min), 360 nm (18~24 min), 276 nm(24~30 min), and 250 nm (30~65 min). The column temperature was set at 30℃. The results showed that the linear range of iquiritin, isoliquiritin, liquiritigenin, glycyrrhizic acid was 0 . 108 5~1 . 085、0 . 016 8~0 . 168、0 . 0049 4~0 . 049 4、0 . 407~4 . 07μg , respectively . The average recoveries of four constituents were 96.61%~100.89%, with RSD ≤ 0.81%. The contents of four constituents in roots of five batches were 0.513%, 0.072 9%, 0.048 4%, and 1.945%, respectively. Contents of four constituents in knotty rhizome from two batches were 0.456%, 0.063 6%, 0.036 2%, and 1.630%, respectively. It was concluded that there was good linear relationship between the response and concentration. Contents of four constituents in knotty rhizome were basically the same as those in the roots. The knotty rhizome can be used as raw material for the extraction of active components.
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Objective: To study the effects of the flavonoids from Glycyrrhizae Radix (FGR) on the function and expression of P-glycoprotein (P-gp) in Caco-2 cells, and to further explore the detoxification mechanisms of FGR. Methods: Flow cytometry was used to study the effects of FGR on the uptake of Rhodamine 123, which showed the toxic efflux function of P-gp; Western blotting method was used to detect the expression level of P-gp in Caco-2 cells. Results: Compared with the control group, after treated with the total flavonoids from Glycyrrhizae Radix (TFGR) at the different concentration (5, 10, 50, and 100 μg/mL) for 1 h, the uptake of Rhodamine 123 in Caco-2 cells was decreased by 61.1%, 56.3%, 49.2%, and 45.4%; liquiritin, isoliquiritin, liquiritigenin, isoliquiritigenin, liquiritin apioside, and isoliquiritin apioside with the same dose significantly decreased the fluorescence intensity of Rhodamine 123 by 19.3%-37.9%. The expression levels of P-gp in Caco-2 cells were significantly increased (P < 0.01) after the co-incubation of TFGR (10-400 μg/mL) for 72 h, and liquiritin, isoliquiritin, liquiritigenin, and liquiritin apioside (5-400 μmol/L) had the similar functions, so did isoliquiritin and isoliquiritin apioside (10-400 μg/mL, P < 0.01). Conclusion: FGR could strengthen the function, up-regulate the expression of P-gp in Caco-2 cells, promote the efflux of toxic substances, and decrease the absorption of toxic substances, which could be one of the new mechanisms for Glycyrrhizae Radix detoxification.
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OBJECTIVE: To establish a determination method of five index components to control the quality of Glycyrrhizae Radix et Rhizoma and Longchai Decoction. METHODS: An ODS C18 column (4.6 mm × 250 mm, 5 μm) was adopted; the mobile phase consisted of acetonitrile -0.05% phosphoric acid with gradient elution at the flow rate of 1 mL · min-1; the detection wavelength was 237 nm, and the column temperature was 30°C. RESULTS: The five index components achieved baseline separation, and the negative sample showed no interference. The linear ranges were 0.156 8-1.568 μg for liquiritin (r=0.999 7), 0.157 1-1.571 μg for isoliquiritin (r=0.999 8), 0.155 8-1.558 μg for liquiritigenin (r=0.999 6), 0.187 1-1.871 μg for glycyrrhizic (r=0.996 9) and 0.158 1-1.581 μg for isoliquiritigenin (r=0.999 5), respectively. The average recoveries were 101.47% (RSD=1.272%), 101.09% (RSD=1.937%), 101.14% (RSD=2.388%), 100.38% (RSD=1.448%) and 100.86% (RSD=1.759%), respectively (n=5). CONCLUSION: This method has good resolution and high precision, and can be used for the quality control of Glycyrrhizae Radix et Rhizoma and Longchai decoction.