| Objectives Liquorice flavonoids and saponins were enriched by macroporous resin and polyamide resin, different ingredient fingerprint of it were established by HPLC. The spectrum effect relationship study of liquorice flavonoids and saponins was carried out by using the method of grey correlation to determine the relevancy between chemical constituents of each chromatographic peak from liquorice flavonoids and antitussive function, as well as the relevancy among chemical constituents of each chromatographic peak from liquorice saponins and antitussive function and hepatoprotective effect. The effective ingredients which may have a good relationship were determined, which give a scientific basis of regularity for Chinese herbal medicine quality control and provide a certain amount of experimental data for Extensive cultivation and planting of Liquorice.Methods1. Liquorice flavonoids and saponins of ten different regions were enriched by macroporous resin and polyamide resin and enrichment fraction of liquorice flavonoids and saponins of ten different regions were obtained.2. Fingerprint of liquorice flavonoids and saponins of ten different regions were established by the method of HPLC/DAD, and fingerprint similarity of liquorice flavonoids and saponins were evaluated by "Chinese medicine chromatographic fingerprint similarity evaluation software".3. Enrichment fraction of pharmacodynamic study(1) Flavonoids and saponins with ammonia water induced cough in mouse recording the cough incubation period and 2 min in the cough times to evaluate the liquorice flavonoids and saponins antitussive effect.(2)Using carbon tetrachloride induced liver injury in mice experimental determination of serum alanine aminotransferase(ALT), aspartate aminotransferase(AST) enzyme activity to evaluate protect liver function of liquorice saponins.4. To study the relationship between liquorice flavonoids and saponins class composition of the chemical composition of the fingerprint characteristic peak represents information and the cough and liver efficacy, using grey correlation analysis method. According to the correlation between the size to determine the chemical composition of the fingerprint characteristic peak represents the contribution degree of effect on its effectivenessResults1. Ten different origin of liquorice herbs was enriched by AB-8 macroporous adsorption resin and polyamide column chromatography technology, obtained the extract of flavonoids ingredients. Respectively to the Yuanzhou district 7.20 g, Xinjiang 11.60 g, Hongsibu 8.40 g, Gansu 8.20 g, Yanchi power stations 10.90 g, Qingyang 4.80 g, Yanchi medicinal materials base of 7.00 g, Suide 12.00 g, Inner Mongolia 8.20 g, Shizuishan 6.80 g, extract rate respectively is 3.60%,5.80%,4.20%,4.10%,5.45%,2.40%,3.50%,6.60%,4.10% 〠3.40%. 10 different origin of licorice herbs by D101 macroporous adsorption resin enrichment from licorice extract saponins, they respectively are Hongsipu area 7.50 g, Suide 8.80 g, Longde 7.50 g, Xinjiang 6.20 g, Qingyang 6.80 g, Inner Mongolia 7.20 g, Shizuishan 5.00 g, Yuanzhou District 6.80 g, Yanchi 6.30 g, commercially available 5.40 g, extract rate respectively is 15%, 17.60%, 15%, 12.40%, 13.60%, 14.40%, 10%, 13.60%, 12.60% and 10.80%.2. Fingerprint of licorice flavonoids and saponins of the different origin was seted up by the "traditional Chinese medicine(TCM) chromatographic fingerprint similarity evaluation software", the fingerprint similarity of liquorice flavonoids and saponins were evaluated, and the fingerprint of liquorice flavonoids of 11 common peaks were determined, the retention time in 11.550 min is liquiritin apioside, the retention time in 12.919 min is liquiritin, the retention time in 34.837 min is apioside isoliquiritin, the retention time in 44.512 min is glycyrrhizin, the retention time in 80.844 min is isoliquiritigenin. Determine the liquorice saponin fingerprint of 13 common peak, the retention time in 37.355 min is glycyrrhizic acid, the retention time in 74.370 min is glycyrrhetinic acid.3. The experiment of mice caused by ammonia, compared with the blank and positive control group, 10 different origin liquorice flavonoids and saponins class substances had statistical significance(P < 0.05, P < 0.01), Cause liver injury in mice experiment, compared with model group, 10 different property liquorice saponin class material also statistically significant(P < 0.05, P < 0.01)4. Fingerprint and pharmacodynamics studies of analysis to processed by grey correlation, the following are obtained results:(1)10 characteristic peaks and antitussive effect correlation is greater than 0.6 in liquorice flavonoids fingerprint, there are five characteristic peaks of antitussive effect correlation is greater than 0.8, and the correlation is greater than 0.8 of the substance order for retention time was 36.823 min substances > retention time was 41.845 min substances > apioside isoliquiritin> retention time was 24.266 min substances > liquiritin.(2) 12 characteristic peaks and antitussive effect correlation is greater than 0.6 in saponins fingerprint, there are two characteristic peaks of antitussive effect correlation is greater than 0.8, and the correlation is greater than 0.8 of the substance order for retention time was glycyrrhizic acid > retention time was 38.102 min substances.(3) 12 characteristic peaks and AST enzyme activity effect correlation is greater than 0.6 in saponins fingerprint, there are three characteristic peaks of the serum AST enzyme effects activity is greater than 0.8, and the correlation is greater than 0.8 of the substance order for retention time was 38.102 min substance > retention time was 72.643 min > retention time was 11.935 min substance, 11 peaks with ALT activity the role of the association degree greater than 0.6, there are four characteristic peaks correlation with the effects of the serum ALT enzyme activity is greater than 0.8, the correlation degree greater than 0.8 substance order for retention time was 38.102 min substance > retention time was 72.643 min substance > retention time was 11.935 min substance > glycyrrhizic acid substances.Conclusion1. In this experiment, AB-8, D101 macroporous adsorption resin and polyamide resin were used for enrichment of licorice flavonoids and saponins of Radix glycyrrhizae. The method is convenient and the result is reliable.2. The chromatographic fingerprint of licorice flavonoids and saponins generated by 10 batches of Radix glycyrrhizae from different plant habitats were established under the optimized HPLC conditions, respectively. A total of 11 common characteristic peaks were found in licorice flavonoids fingerprint, whereas licorice 13 common characteristic peaks were shared in saponins fingerprint. Our established HPLC method is accurate, simple, rapid, precise and reproducible, which could be used for the quality control of Radix glycyrrhizae.3. The results of pharmacodynamics experiment exhibited licorice flavonoids from different habitats displayed different degree in antitussive effect, whereas licorice saponins all have certain effect on antitussive and liver.4. Using the grey correlation analysis method of fingerprint and pharmacodynamics, the following results are obtained:(1) 10 characteristic peaks were relevant with antitussive effect in licorice flavonoids, among which, 5 characteristic peaks had a high correlation with antitussive effect;(2) 12 characteristic peaks were relevant with antitussive effect in licorice saponins, among which, 2 characteristic peaks had a high correlation with antitussive effect; A total of 12 characteristic peak has correlation with serum AST enzyme activity, among which, 3 characteristic peaks is highly related to ALT enzyme activity and AST enzyme activity in serum; A total of 11 characteristic peaks were relevant with serum, among which, 4 characteristic peaks had a high correlation with ALT enzyme activity in serum. All the results revealed that therapeutic effects of licorice concentration of flavonoids and saponins on antussive effect and Hepatoprotective effect were contributed by multiple bioactive constituents. |
