| Objective:1.To clarify the chemical basis of Cyclocarya paliurus and provide guideline for the quality control of Cyclocarya paliurus,the HPLC fingerprints of 10 batches of water extract of Cyclocarya paliurus from different habitats were established.Also,the main components in the water extract of Cyclocarya paliurus were analyzed.Furthermore,the quality of Cyclocarya paliurus was evaluated by similarity analysis,chemical module analysis and content determination,so as to clarify the chemical material basis of Cyclocarya paliurus and provide basis for the quality evaluation of Cyclocarya paliurus.2.Based on the four index components calibrated by the fingerprint of Cyclocarya paliurus,the pharmacological evaluation of the four index components was carried out by using the network pharmacological database.Furthermore,the active components with inhibition ofα-glucosidase were screened by molecular docking and molecular dynamics simulation,and the virtual screening results were verified by enzyme activity experiments in vitro to explore the possibility of inhibition ofα-glucosidase activity by the active components of Cyclocarya paliurus.3.The insulin resistant HepG2（IR-HepG2）cell model was established to study active components’effects on glucose metabolism,glycogen synthesis and key enzymes of liver glucose metabolism in IR-HepG2 cells.Furthermore,to explore its effect on IR-HepG2,we investigated potential effects on the key factors of IRS-1/PI3K/Akt insulin signal pathway in IR-HepG2 cells.Methods:1.The characteristic fingerprints of 10 batches of water extracts of Cyclocarya paliurus from different habitats were established by HPLC.The chromatographic column was X-peonyx C18 column,the mobile phase was 0.2%phosphoric acid aqueous solution（A）-acetonitrile（B）,and the gradient elution procedure was 0～15 min,92%～92%A;15～50 min,92%～82%A;50～60 min,82%～81%A;Gradient elution,flow rate 1.0 m L/min.The column temperature is 30℃,and the detection wavelength is 280 nm.Taking protocatechuic acid,chlorogenic acid,caffeic acid and rutin as quality control indexes,chemical module analysis and content determination were carried out to evaluate the quality of Cyclocarya paliurus from different producing areas,so as to provide a theoretical basis for further clarifying the material basis and quality evaluation of Cyclocarya paliurus.2.The pharmacological safety of four index components was evaluated by network pharmacological database.The affinity of four PCA,CGA,CFA and Rutin compounds toα-glucosidase was virtually screened by molecular docking and molecular dynamics simulation,and their molecular mechanism was analyzed.Furthermore,the inhibitory effect and mechanism of Cyclocarya paliurus and its active substances CGA and Rutin onα-glucosidase activity were studied by p-nitrophenyl-α-D-glucopyranoside（PNPG）method and acarbose as control.3.After the cells were treated with different concentrations of CP,PCA,CGA,CFA and Rutin for 24 hours,the cell survival rate was measured by MTT assay.4.HepG2 cells were stimulated with 10-5,10-6,10-7,10-8mol/L insulin for36 hours to screen the optimal concentration of IR-HepG2 cells.Furthermore,the best concentration of insulin was used to induce HepG2 cells at different time,the best conditions for insulin stimulation were determined.A stable and reliable IR-HepG2 cell model was established.5.Glucose oxidase method was used to detect the glucose consumpti on of cell culture medium after CP（25,50,100μg/m L）、PCA（125,250,500μmol/L）、CGA（31.25,62.5,125μmol/L）、CFA（35,70,140μmol/L）、R utin（1.563,3.125,6.25μmol/L）intervened in IR-HepG2 cells for 24 hour s.6.Glycogen,pyruvate kinase and T-SOD kits were used to determine the contents of liver glycogen,pyruvate kinase and total SOD in IR-HepG2 cells treated with CP,CGA and Rutin for 24 hours.7.RT-qPCR was used to detect the mRNA expressions of INSR,IRS-1,PI3K,Akt2 and GSK-3βgenes in the IRS-1/PI3K/Akt insulin signaling pathway after the high-dose groups of CP,CGA and Rutin intervened in IR-HepG2 cells for 24 hours.Results:1.The fingerprints of 10 batches of water extracts of Cyclocarya paliurus were established,9 common peaks were matched,and 4 of them were identified as PCA,CGA,CFA and Rutin.The results of fingerprint similarity showed that the similarity values of 10 batches of Cyclocarya paliurus from different producing areas were all more than 0.9,and the similarity was good,which met the requirements of establishing fingerprint.The results of chemical module analysis showed that 10 batches of Cyclocarya paliurus from different producing areas were divided into 3 categories.The results of content determination showed that there were some differences in the quality of Cyclocarya paliurus from different areas.2.The screening results of network pharmacological database showed that PCA,CGA,CFA and Rutin had good pharmacological parameters and safety.The results of molecular docking showed that the binding energies of the four compounds toα-glucosidase were all less than-5.0 kcal/mol.Molecular dynamics simulation results show that CGA and Rutin are most similar to acarbose in binding energy and molecular dynamics parameters,indicating that these two active compounds may have the effect of inhibitingα-glucosidase.The in vitro enzyme activity test showed that the IC50 ofα-glucosidase by Cyclocarya paliurus and its main active substances CGA and Rutin were3599μg/m L,398.9μg/m L and 351.8μg/m L,respectively,in a concentration-dependent manner.The kinetic results of enzyme activity showed that both CGA and Rutin belonged to mixed inhibition type.3.From MTT assay,we screenned the relavantly appropriate concentration of five components.CP concentration was 25,50,100μg/m L,PCA concentration was 125,250,500μmol/L,CGA concentration was the 31.25,62.5,125μmol/L,CFA concentration was 35,70,140μmol/L and Rutin concentration was 1.563,3.125,6.25μmol/L,The intervention of HepG2 cells had no obvious toxicity to HepG2 cells（P>0.05）.4.The results of different concentrations and time of action of high concentration of insulin showed that the best modeling conditions of IR-HepG2cells were listed as follows:10-7mol/L insulin stimulated HepG2 cells for 36hours,IR-HepG2 cells with good stability could be induced successfully.5.After IR-HepG2 cells were treated with different doses of CP,PCA,CGA,CFA and Rutin for 24 hours,compared with the model group,PCA and CFA had no significant effect on the glucose consumption of IR-HepG2 cells（P>0.05）.CP,CGA and Rutin could improve the glucose consumption of IR-HepG2 cells（P<0.05）,and the improvement was enhanced with the increase of concentration.6.After inducing HepG2 cells with 10-7mol/L insulin for 36h,the glycogen content,pyruvate kinase（PK）activity and total SOD content in HepG2 cells were significantly decreased（P<0.01）.Compared with the model group（MOD）,the high-dose CP,CGA and Rutin groups could significantly increase the intracellular glycogen content,PK activity and total SOD content of IR-HepG2cells after 24 hours of intervention in IR-HepG2 cells（P<0.05 or P<0.01）.7.After the establishment of IR-HepG2 cell model,compared with the normal group,the mRNA expression of INSR,IRS-1,PI3K,Akt2 and GSK-3βin the IRS-1/PI3K/Akt signal pathway of HepG2 cells in the model group was significantly down-regulated.After 24 hours of intervention with high-dose CP,CGA and Rutin,the expressions of INSR mRNA,IRS-1 mRNA,PI3K mRNA and GSK3-βgenes in the model group were significantly up-regulated compared with the model group.Compared with the model group,Akt2 gene CGA group did not significantly up-regulate the expression of Akt2 mRNA,but the expression was increased to some extent compared with the model group.CP group and high-dose Rutin group could significantly up-regulate the expression of Akt2 mRNA（P<0.05）.Conclusion:The results of HPLC fingerprints showed that the fingerprints of 10 batches of Cyclocarya paliurus from different areas had good similarity,and the chemical fingerprints of samples were all more than 0.9.10 batches of Cyclocarya paliurus tea from different regions were divided into three categories by chemical module analysis,and the contents of PCA,CGA,CFA and Rutin were different among different areas.The network pharmacological parameters of PCA,CGA,CFA and Rutin were evaluated.All of them showed good pharmacological safety.The results of molecular docking showed that the four active components have good binding activity toα-glucosidase and the binding energy is less than-5.0 kcal/mol.Molecular dynamics showed that CGA and Rutin were similar to the positive drug acarbose in the binding process ofα-glucosidase.It may be a potentialα-glucosidase inhibitor.The enzyme activity test in vitro showed that CP and its main active components CGA and Rutin could inhibitα-glucosidase to a certain extent,and both CGA and Rutin were mixed inhibition types.CP,CGA and Rutin significantly improved glucose consumption,hepatic glycogen synthesis,pyruvate kinase（competitive）activity and total SOD activity in IR-HepG2 cells,which may be related to the regulation of INSR,IRS-1,PI3K,Akt2 and GSK-3βmRNAs in IRS/PI3K/Akt signaling pathway. |
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