Study On Hypoglycemic Active Components Of Cyclocarya Paliurus And Coptis Chinensis

Cyclocarya paliurus（C.paliurus）is a plant of the Juglans family and the genus Cyclocarya paliurus.It has the effects of dispelling wind and arresting itching,decreasing blood glucose,decreasing blood pressure and so on.Coptis chinensis（C.chinensis）is the dried rhizome of a plant in the genus Coptis,which has certain effects in relieving fire,detoxifying,relieving thirst and so on.C.paliurus and C.chinensis are often used in combination for diabetes treatment.In order to better understand the active ingredients and the best ratio between active ingredients in C.paliurus and C.chinensis of hypoglycemic activity,this article has carried out corresponding investigations.The specific results were as follows:1.The crushed leaves of C.paliurus were extracted with 70%ethanol,the ethanol extract of C.paliurus was obtained and the yield was 24.18%.The water extract of C.paliurus was obtained by adding water after ethanol extraction and the yield was 6.02%.The ethanol extract and the water extract of C.paliurus were mixed to obtain the C.paliurus extract,which was separated by macroporous resin AB-8 to obtain four components.The streptozotocin（STZ）-induced diabetic mice models were used to study the hypoglycemic activity.It was found that the 30%ethanol eluted fraction had significant effects on fasting blood glucose（FBG）and glucose tolerance test（OGTT）（P<0.05）.MCI column and ODS-A column were used to separate and purify to obtain two monomer compounds.The structures of two compounds were identified by nuclear magnetic resonance（NMR）.Compound 1 was quercetin-3-O-β-D-glucuronide-6"-methyl ester and compound 2 was kaempferol-3-O-β-D-glucuronide-6"-methyl ester.Compound 1 and compound 2 could reduce FBG and area under the curve（AUC）of OGTT in diabetic mice（P<0.05）,and compound 1 had better hypoglycemic activity than compound 2.The enzymology of compound 1 and compound 2 was studied in vitro.Compound1 and compound 2 could inhibit the activity ofα-amylase andα-glucosidase,and half maximal inhibitory concentrations(IC₅₀)of compound 1 to two enzymes were smaller than that of compound 2.Compound 1 and compound 2 were non-competitive inhibitors of two enzymes,and could produce static quenching with only one binding site between them.Compound 1 and compound 2 could change the secondary structures of two enzymes.Hydrogen bonding was the main binding force between compound 1,compound 2 and two enzymes.2.The extraction process of C.paliurus was optimized with the yield of total flavonoids as the evaluation index.According to the results of single factor experiments,the volume ratio of water to ethanol in the solvent used for extraction was 1:1,the ratio of the mass of C.paliurus to the extraction solvent was 1:20 kg/L,the optimal temperature for extraction was 70°C,and the extraction time was 3 h.According to the results of response surface test,the extraction conditions of C.paliurus were:the ethanol concentration was 55%,the temperature of the system was 70°C,the extraction time was 3.5 h,and the yield of total flavonoid was（11.46±0.31）%.The optimized process conditions could increase the high performance liquid chromatography（HPLC）peak area of compound 1 and compound 2 with hypoglycemic activity,indicating that the optimized process conditions were reliable.3.The crushed C.chinensis was extracted with 70%ethanol to obtain the ethanol extract of C.chinensis with a yield of 16.38%.Then C.chinensis was extracted with water to obtain the water extract of C.chinensis and the yield was 8.72%.The ethanol extract and the water extract of C.chinensis were mixed to obtain the extract of C.chinensis.After separation by MCI column and screening of hypoglycemic activity through diabetic mice,95%ethanol elution had excellent hypoglycemic activity（P<0.05）.After separation by ODS-D column,compound 3 was obtained,which was identified as berberine by NMR.Compound 3 performed excellent hypoglycemic activity in animal experiments（P<0.01）.Compound 3 showed different types of inhibition on two enzymes,which were non competitive inhibition（α-amylase）and competitive inhibition（α-glucosidase）.The quenching mode of compound 3 was static and there was a binding site between compound 3 and two enzymes,respectively.The primary force of compound 3 was hydrophobic force,which was different from the primary force of compound 1 and compound 2.It provided references for the study of the interaction between enzymes and hydrophobic compounds.4.Compound 1 with better hypoglycemic activity in C.paliurus and compound 3 with hypoglycemic activity in C.chinensis were selected to explore the optimal ratio.FBG and AUC of diabetic mice were used as the main indexes to evaluate hypoglycemic activity.The applicable ratio ranges of hypoglycemic activity between compound 1 and compound 3 were0.4:0.6～0.2:0.8,which could reduce OGTT and FBG of diabetic mice after three weeks of medication treatment.In this range,hypoglycemic activity was better than the group which was only given compound 1 or compound 3,respectively.When the ratio between compound 1 and compound 3 ranged from 0.4:0.6 to 0.2:0.8,the lipid levels,liver antioxidant enzymes and oxidative stress index,hemoglobin A1c（Hb A1c）and insulin resistance of diabetic mice were improved.In summary,compound 1 and compound 2 were extracted from C.paliurus,and compound 3 was extracted from C.chinensis,compounds 1～3 showed hypoglycemic activity.The combination of compound 1 and compound 3 performed better hypoglycemic activity than single compound groups.This study could provide references for the separation of hypoglycemic active ingredients from natural resources and the compatibility of C.paliurus and C.chinensis.