Study On Extraction And Glucosamine Modification Of Glycyrrhetinic Acid And Its Hypoglycemic Activity

Glycyrrhetinic acid,a triterpenoid compound,has been drawn more and more attention by biochemists and pharmacologists for its anti-inflammatory,anti-tumor,anti-virus,anti-oxidation,hypoglycemic and hypolipidemic activities.In recent years,structural modification of glycyrrhetinic acid to improve its low aqueous solubility and improve its activity has become a heated-discussed research topic.This project introduced hydrophilic glucosamine into the glycyrrhetinic acid structure to study its aqueous solubility and hypoglycemic activity.This project mainly including:The content of glycyrrhiza offal was determined according to national standard and literature method.The results are as follows:water content was 6.23%±0.02%,fat content was 1.25%±0.02%,glycyrrhizic acid was 2.15%±0.09%,ash content was 3.97%±0.01%,protein content was 3.23%±0.18%,crude fiber content was 13.70%±0.08%,total flavonoids content was 2.23%±0.04%.Analysis showed that glycyrrhizin,flavone and other components were still contained in glycyrrhizin offal,which could be further processed in order to extract active components.The optimum extraction conditions of glycyrrhetinic acid were determined by single factor and orthogonal test as follows:acidolysis temperature was 90℃,solid-liquid ratio was1:30（g/m L）,dilute sulfuric acid concentration was 12.5%,acidolysis time was 8.5 h.Under these conditions,the yield of glycyrrhetinic acid was 1.45%±0.02%.Glycyrrhetinic acid was purified by column chromatography using cyclohexane:ethyl acetate（3:2）as eluent.The purity of glycyrrhetinic acid was 94.00%.The purified glycyrrhetinic acid was identified as glycyrrhetinic acid by thin layer chromatography（TLC）,nuclear magnetic resonance spectroscopy（NMR）,high resolution mass spectrometry（MS）and specific rotation.The 3-hydroxyl groups of glycyrrhetinic acid and the 1,3,4,6 hydroxyl groups of glucosamine were protected by acetylation.Then,the 3-acetyl glycyrrhetinic acid was combined with 1,3,4,6-tetra-o-acetyl-2-deoxy-2-amino-β-D-glucopyranose.Finally,the hydroxyl protection was removed to obtain 2-（N-3-acetylglycyrrhetinoyl）-N-glucopyranosyl-2-acetamide（compound 9）in 20.33%overall yield.The structure of the compound was characterized by MS,NMR.The specific rotation of compound 9 was determined as+85°.The log P of compound 9 was 3.29±0.65,lower than the predicted value of 6.28 for glycyrrhetinic acid.And its water solubility was obviously better than that of glycyrrhetinic acid.The study on inhibitory effects of glycyrrhetinic acid and compound 9 onα-glucosidase activity was by enzyme inhibition method.The IC₅₀ of compound 9 and glycyrrhetinic acid were 0.160mmol/L and 0.381mmol/L,respectively.The results demonstrated that compound9 had better inhibitory activity than glycyrrhetinic acid.And both compounds exhibited mixed inhibition of reversible and non-competitive.By establishing insulin resistance（Hep G2-IR）model,it was found that both glycyrrhetinic acid and compound 9 could increase glucose consumption of Hep G2-IR cells.And the effect of compound 9 was better than glycyrrhetinic acid.