| Glycyrrhetinic acid(GA)is a natural product of pentacyclic triterpenes with an oleanolane skeleton.It is an aglycone of glycyrrhizic acid and one of the important active ingredients of licorice.Although G A has a variety of pharmacological effects,it is severely limited in clinical applications due to its strong lipophilicity and low water solubility caused by its structure.In addition,the strong lipophilicity and low water solubility caused by the structure of GA make it severely limited in clinical application.In order to improve the performance of GA and develop its application potential,based on the optimized extraction method,this paper modified its structure and studied the hypoglycemic activity of GA and its derivatives.The main research work is as follows:Firstly,the best process for extracting GA by heating acid hydrolysis was determined by single factor and orthogonal experiments:the ratio of material to liquid was 1:20(g/mL),the concentration of dilute sulfuric acid was 5%(m/m),the acid hydrolysis temperature was 80℃,and the acid hydrolysis time was 8 h.The extraction yield of GA under this process was 3.26 ± 0.09%.Steam blasting technology was used to assist in the extraction of GA by heating and acid hydrolysis.The steam was exploded at 1.5 MPa for 3 min,and GA was extracted by optimized heating and acid hydrolysis process.At this time,the yield was 4.57±0.18%,an increase of 40.53%.The crude extract was purified by silica gel column chromatography,and the collected GA purity was 91.78%.The structure of the purified GA was identified by thin-layer chromatography,nuclear magnetic resonance spectroscopy,and high-resolution mass spectrometry.With GA as the parent,glycine,aspartic acid,and arginine were introduced into its C-30 carboxyl group,respectively.The N-glycyrrhetic acid glycine,N-glycyrrhetic acid aspartic acid and N-glycyrrhetic acid arginine were successfully synthesized with total yields of 43.60%,39.33%,and 35,10%respectively.The structure of the new derivatives was characterized by thiN-layer chromatography,nuclear magnetic resonance spectroscopy,and high-resolution mass spectrometry.The specific optical rotations of GA,N-glycyrrhetic acid glycine,N-glycyrrhetic acid aspartic acid,and N-glycyrrhetic acid arginine were determined using an automatic polarimeter as+165°,+25°,+20°,+5°.By measuring their N-octanol/water partition coefficient(log P),it was found that the log P values of the new derivatives were significantly lower than GA,indicating that the water solubility of GA was significantly improved after access to amino acids.Finally,the hypoglycemic activity of GA and its derivatives was investigated using in vitro enzyme inhibition and building HepG2 cell insulin model.In vitro α-glucosidase activity inhibition test results showed that GA had a good inhibitory effect on α-glucosidase with an IC50 value of 0.628 mmol/L.The inhibitory effect of N-glycyrrhetic acid arginine was better than GA and IC50 at 0.513 mmol/L,N-glycyrrhetic acid glycine and N-glycyrrhetic acid aspartic acid were the worst,with IC50 values of 2.190 mmol/L and 3.540 mmol/L,respectively.The α-glucosidase inhibition types of derivatives were mixed reversible competitive and non-competitive inhibition.To investigate the effects of GA and N-glycyrrhetic acid arginine on PI3K/Akt signaling pathway protein,it was found that compared with the model group,in the administration group,PI3K and Akt proteins expression were significantly increased,and GSK-3β protein expression was decreased,the amount of glycogen synthesis was increased,and the state of insulin resistance was improved.The above results indicated that the effect of lowering blood glucose changed when the amino acid was inserted into the carboxyl group of GA,and the activity of lowering blood glucose increased when the arginine was inserted. |
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