The Study For The Hypoglycemic Mechanism On Type 2 Diabetes Mellitus Model Rats And Chemical Constituents Of Cyclocarya Paliurus (Batal.) Ijinskaja From Guizhou Province

Objective:Cyclocarya paliurus?Batal.?Iljinskaja is the species belong genus Cyclocarya of Juglandaceae,which has been reported to have a variety of health effects such as hypoglycemic,hypolipidemic and anti-oxidation.According to the"Chinese traditional medicine resources",cyclocarya paliurus has certain curative effect on diabetes.However,there was a lack of in-depth systematic research on the effective hypoglycemic active chemical constituents of cyclocarya paliurus,which is not conducive to the further development and utilization of cyclocarya paliurus at present.Therefore,this study researched the in vitro and in vivo treating mechanism and chemical constituents of cyclocarya paliurus on type II diabetes rats from the aspects of whole animal,in vitro model and chemical composition.Thus,the mechanism of cyclocarya paliurus active fraction to improve type 2 diabetes mellitus and the chemical constituents of hypoglycemic of cyclocarya paliurus was clarified,which provided basis for the basic analysis and clinical application of cyclocarya paliurus.Methods:1.To study mechanism of hypoglycemic effect of cyclocarya paliurus on type 2diabetes mellitus rats.The model rats of type 2 diabetes mellitus were induced by high-fat diet feeding combined with intraperitoneal injection of low-dose streptozocin?STZ?.Metformin Hydrochloride and Rosiglitazone Maleate Tablets as positive drug and low dose?1.5g/kg?,medium dose?3g/kg?,high dose?6g/kg?of water extract of cyclocarya paliurus from Guizhou province were treated with the model rats 12 weeks on time.The fasting blood glucose of each group of rats was measured by tail vein blood at week 2,wek 4,week 8,and week 12 after administration.The last fasting blood glucose and sugar tolerance of each group of rats were measured after 12 weeks of administration.And the GSP,TC,TG,HDL-C and LDL-C were detected in rat serum.Pancreatic and liver tissue of each group of rats were treated into paraffin section and HE staining,then their histopathological morphology was analyzed by microscopy.2.To study active chemical constituents of cyclocarya paliurus.The protocatechuic acid,isoquercitrin,quercetin,kaempferol,oleanolic acid and ursolic acid as components of quality evaluation of cyclocarya paliurus were analyzed.Established HPLC wavelength switching method to analyze the above components in different cyclocarya paliurus samples by the chromatographic conditions,including Thermo Syncronis C₁₈ column?5?m,250mm×4.6mm?,the methanol?A?-0.1%phosphoric acid water?B?as mobile phase,gradient elution;flow rate 1.0 mL/min,column temperature 30?;detection wavelengths were 260 nm,360 nm,210 nm,etc.And the total polysaccharide content was quantitatively determined by colorimetric method to judge the quality of cyclocarya paliurus from Guizhou province.The polar eluted fraction s of cyclocarya paliurus were separated by macroporous resin column chromatography,which separation situation were determined by HPLC.And the total flavonoids and total saponins content of each eluted fractions were determined by UV spectrophotometry.Further,each eluted fractions were determined inhibition effect of?-glucosidase in vitro to select the active fraction of hypoglycemic.3.To study Mass spectrometric analysis of active fraction of hypoglycemic of cyclocarya paliurus.In order to clarify the compounds of the active fraction of hypoglycemic of cyclocarya paliurus,the compounds of the 60%ethanol elution of the ethyl acetate?EtOAc-60%-EtOH?fraction of cyclocarya paliurus was qualitatively analyzed by Q Exactive Orbitrap LC-MS/MS,which conditions including Column Thermo Accucore C₁₈ column?2.6?m,100mm×2.1mm?;acetonitrile?A?-0.1%formic acid water?B?as mobile phase,gradient elution;flow rate of 0.3mL/min,column temperature 35?;the ESI source,the detection mode is positive mode and negative mode;the scan mode is Full MS/dd-MS2,the scan range is m/z 200¹600,and the collision energy in MS/MS mode is NCE 20eV,40eV and 60eV,etc.The mass spectral data of each chromatographic peaks were analyzed,the cleavage laws of known compounds were compared,and the retention time,MS data and reference report data were used to identify each MS chromatographic peaks.The petroleum ether fraction of cyclocarya paliurus with certain hypoglycemic effect was qualitatively analyzed by Gas Chromatography-Mass Spectrometer?GC-MS?,which conditions including 5HP quartz capillary column?30m×0.25mm,0.2?m?,carrier gas flow rate of 0.9mL/min,gasification chamber temperature of 280?,gradient heating;ion source is EI,ion source temperature 250?;electron energy 70eV,Scanning range of30⁶00 m/z,etc.The mass spectral data of each chromatographic peak was compared and analyzed by the NIST database.The Results showed as follows:1.The water extract of cyclocarya paliurus from Guizhou province has significant hypoglycemic effect,which can significantly reduce the fasting blood glucose of Type 2diabetes mellitus rats,and enhance its tolerance to glucose,and reduce the level of GSP and TC,TG,HDL-C,LDL-C.HE staining showed that the islet cells and hepatocytes in the cyclocarya paliurus treating group were normal and the hepaticcord was repaired,which proved that cyclocarya paliurus could inhibit islet cell atrophy,improve islet damage,reduce fat content of liver and improve liver damage.Therefore,it is concluded that the mechanism of hypoglycemic effect in cyclocarya paliurus from Guizhou province may be:Repairing damaged islet?cells,increasing insulin levels while avoiding further damage of islet function;inhibiting the vicious circle caused by the production of glycosylation end products;Protecting liver tissue and improving the lipid metabolism disorder.2.The qualitative and quantitative methods for the cyclocarya paliurus chemical constituents were established by HPLC.The most of cyclocarya paliurus samples contained quercetin and kaempferol,and the polysaccharide was the main component in the water extract of cyclocarya paliurus,which can be used as the iconic constituents of the cyclocarya paliurus.In cyclocarya paliurus from Guizhou province,the content of quercetin is 0.1232mg/g,the content of kaempferol is 0.0883mg and the total polysaccharide content is 70.11mg/g,which is higher than other experimental samples and it belongs to the quality top grade.The eluted fractions of cyclocarya paliurus were analyzed by UV spectrophotometry.The content of total flavonoids and total saponins in the EtOAc-60%-EtOH fraction respectively were 111.8?g/mL and 4401?g/mL.Further?-glucosidase inhibition experiments in vitro showed that the EtOAc-60%-EtOH fraction had the best inhibitory effect,which IC₅₀ is 20.40 mg/mL.3.According to LC-MS/MS analysis,the EtOAc-60%-EtOH fraction may contain3 flavonoid glycosides and 19 triterpenoid saponins,which may be the main hypoglycemic constituents of cyclocarya paliurus,such as Rhamnazin?Kaempferitrin?cyclocarioside C?cyclocarioside??secodammara-25,26-dien-3-oic acid-24-O-D-xylopyranoside,etc.GC-MS analysis showed that the petroleum ether fraction of cyclocarya paliurus with certain hypoglycemic effect was mainly composed of eugenol and phytol,and also contained caryophyllene,eugenol acetate,methyl ester Octadecatrienoic acid,neophytadiene,Squalene,?-elemene,?-curcumene,atc.The volatile fraction of cyclocarya paliurus were mainly composed of?-ionone and?-sitosterol.Eugenol,phytol,?-ionone and?-sitosterol can also be used as the active constituents of cyclocarya paliurus.Preliminary clarify the chemical constituents basis of the hypoglycemic effect of cyclocarya paliurus.