| In this paper,loquat leaves in "Santan" area of She County were studied.The content of corosolic acid in loquat leaves was determined and purified.And its pilot scale production and systemic hypoglycemic activity were studied.Through HepG2 cells,zebrafish glycogen transport mechanism and blood glucose and lipid metabolism of diabetic rats,its hypoglycemic effect was studied to clarify medicinal effective components and action mechanism of hypoglycemic effect of corosolic acid.The main research contents are divided into the following four aspects:1.Preparation of corosolic acid,component analysis and content determination of triterpene acid in loquat leavesThe triterpenes were extracted from loquat leaves by ethanol extraction,followed by decolorization of activated charcoal.The product A and product B were prepared by semi-preparative chromatography.The structural difference of isomeride corosolic acid and maslinic acid was analyzed by positive ion fragmentation pathway by electron ionization mass spectrometry(EI-MS).IR,MS and NMR were used in corosolic acid structural analysis.The product A was identified as maslinic acid and product B was identified as corosolic acid.The HPLC method for determination of four triterpene acid content in loquat leaves was established.With 0.814% corosolic acid,loquat leaves in Santan area could be used as high quality raw materials for development of related products.2.Study on technology to extract corosolic acid from loquat leavesFirst,the optimal extraction parameters were determined by orthogonal test and single factor test with content of corosolic acid as an indicator.The extraction conditions were as follows: 80% ethanol was used as extraction solvent at 80 ℃ for 3 h,solid-liquid ratio was 1:14(m/V),extraction was performed twice.Under this condition,the extraction rate was 90% or more.Secondly,the content of corosolic acid in the extract of loquat leaves was increased to 12.2% by adjusting polarity of the extract,and the problem of difficult filtration and solvent recovery was solved.Under this condition,content of triterpene acid was improved: 5.56% for maslinic acid,3.83% for oleanolic acid,20.1% for ursolic acid.Pretreatment sample was prepared for further preparation of chromatographic separation monomer.Further,with the content of corosolic acid as an indicator,pretreatment,mobile phase,stationary phase and loading amount were investigated for the sample for preparative chromatography.In this paper,the C18,10μm filler was used to fill 250 × 40 mm preparative chromatographic column,and methanol solution with volume fraction of 1% acetic acid aqueous solution at 0.14 was sued as eluant.At a flow rate of 20 m L / min and with loading amount at 5m L,separation factors of critical pair of corosolic acid and maslinic acid were above 0.8,which was able to meet the requirement for monomer preparation.The corosolic acid after recrystallization of methyl alcohol was≥97%.3.Study on pilot process of loquat leaf productionUnder determined pre-laboratory process parameters,pilot-scale study of a total of 100 Kg raw materials with 25 Kg each time was carried out in a local factory in She County.Under determined process,with extraction temperature at 70℃,extraction period for 3h and ethanol concentration at 80%,the loquat leaves were coarsely crushed(1cm × 1cm)and concentrated to 20.0% of the volume of the extract before natural settlement.Product could then be obtained after filtration and drying.The average yield of the product was 3.46%,and the average content of corosolic acid in the product was 10.57%,which confirmed the results of the previous laboratory test.4.Study on hypoglycemic activity and mechanism of corosolic acidStudy on the hypoglycemic activity and mechanism of corosolic acid was carried out on the basis of the above obtained corosolic acid monomer.(1)The expression of PEPCK gene in HepG2 cells treated with cAMP and DEX was significantly different from that of the control group,which could be used as a model for study on hypoglycemic activity of corosolic acid.Secondly,the effects of different concentrations of corosolic acid on consumption of glucose in HepG2 cells were investigated.The expression of PEPCK gene in the corosolic acid treated group was decreased gradually with the increase of concentration when the concentration of corosolic acid was 0.1 ～ 10μM.The effect of 10μM corosolic acid on reversal of PEPCK gene overexpression was similar to that of 10μM positive drug metformin.The results showed that corosolic acid could increase the content of glycogen in HepG2 cells induced by cAMP and DEX,and could improve degradation of intracellular glycogen.(2)The zebrafish juvenile animal model with PEPCK gene overexpression and insulin resistance was first established by cAMP and 1000 n M DEX treatment.The relative expression of PEPCK gene was approximately 4.638 times that of the control group.Based on RT-q PCR detection,the model could cause overexpression of insulin and GLUT2 gene,lead to significantly increased expression of insulin receptor,GP,GYS1,G6 P and PFKFB3 genes.Moreover,the model can not only cause hyperglycemia but also cause insulin resistance.Secondly,according to the zebrafish toxicity experiment,it was found that 0.1 ～ 10μM of corosolic acid was almost non-toxic to zebrafish juveniles.The effect of reducing PEPCK gene expression in zebrafish juvenile animal model was better than that of positive drug 10μM metformin,indicating that 0.1 ～ 10μM corosolic acid has a good hypoglycemic effect.(3)Next,normal control group,model control group(cAMP + DEX),positive drug control group(metformin + cAMP + DEX)and corosolic acid treatment group were established.The method of RT-q PCR was used to determine the expression of m RNA in PEPCK,GLUT2,GP,GYS1,G6 Pase,PFKFB3,INSa,INSR genes of zebrafish of PEPCK overexpression model.The results showed that 0.1 ～ 10μM corosolic acid decreased the level of gluconeogenesis in the liver by inhibiting expression of PEPCK gene,reduced glycogen degradation and increased glycogen accumulation by inhibiting GP gene and increasing GYS1 gene expression.Corosolic acid reduced the amount of glycogen output via these two means.Corosolic acid increased glucose consumption caused by elevated levels of glycolysis by increasing expression of G6 Pase and PFKFB3 genes;Corosolic acid increased sensitivity of insulin receptor by increasing the expression of INSR gene,and meanwhile,increasedexpression of GLUT2,improved transport of glucose by peripheral tissues,and alleviated hyperinsulinemia by reducing INSa overexpression.(4)High fat diet and STZ induced type II diabetic rat models were established in normal control group,model control group,positive control group(metformin)and corosolic acid treatment group.According to the investigation,corosolic acid(50 ～ 100 mg / kg)showed good hypoglycemic effect,which could inhibit the weight loss of diabetic rats,improve OGTT,significantly reduce fasting blood glucose concentration and inhibit rate of increase in GSP concentration.(5)Through continuous intragastric administration for 6 weeks,corosolic acid(50 or 100 mg / kg)significantly reduced TC,TG,LDL-c,FFA in rat serum,increased HDL-c,and insulin levels were significantly decreased,insulin resistance index decreased;Corosolic acid(50 or 100 mg / kg)significantly reduced MDA,ICAM-1 levels,and SOD activity increased significantly.The results indicate that corosolic acid may improve insulin sensitivity of diabetic rat to enhance the effect of insulin,reduce postprandial blood glucose and improve lipid metabolism;it can have hypoglycemic effect through various channels such as anti-oxidation,reduction of damage of free radicals to β cells.The results show that loquat leaves from "Santan" can used an effective plant origin that can be prepared corosolic acid.Research results can be implemented in local area.It is effective to raise the farmers income and change the local loquat farmers income structure.and has important significance to protect the interest of loquat growers.The project has been support from the Department of Science &Technology of Anhui Provinces and Shexian Science and Technology Bureau,and play a good demonstration effect for the deep processing of plant resources in Anhui province. |
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