Anti-diabetic Activity Screening Of Traditional Chinese Medicines And The Molecular Mechanism

Diabetes mellitus(DM)is a chronic metabolic disease characterized by high blood glucose,which has become the third-largest disease following cardiovascular disease and cancer,posing great threat to the health of human being.Furthermore,with the improvement of living standards,the number of patients suffered from DM in China is increased year by year and China has become the country with the largest number of DM patients in the world.Therefore,to develop safety and effective anti-diabetic drugs has been a hotspot of research area recently.China has a long history of using Traditional Chinese Medicines(TCMs)to treat DM.To find new anti-diabetic drugs from TCMs is extremely promising strategy.In this thesis,we developed two new screening assays at molecular level for the discovery of anti-diabetic activities of TCMs and/or their ingredients.Besides,we also screened the anti-diabetic activities of constituents from Garcinia xanthychymus and extracts of TCMs at cellular level in order to find out new type of candidates against DM.Main contents are as follows:1)Magnetic nanoparticles grafted with amino groups were synthesized and characterized.a-Amylase was immobilized on the surface of magnetic nanoparticles(MNPs)using co-precipitation method.The enzyme-immobilized MNPs combined with ligand fishing and high performance liquid chromatography(HPLC)techniques were employed to develop a new screening method for the discovery of a-amylase inhibitors.In this method,the immobilized enzyme was found stable.After 30 days’storage at 4 ℃ and 16 assays,the activity of immobilized a-amylase merely decreased by 11.6%.Then,this method was applied to find out 3 ligands of a-amylase from the extracts of G.xanthychymus.The ligands were isolated and purified by column chromatography and semi-preparation HPLC.Their structures were elucidated according to the spectra of ultra violet(UV),mass spectra(MS)and 1H NMR.Finally,they were identified as GB2a glucoside,GB2a and Fukugetin,respectively.The a-amylase inhibition activities were verified.The results showed that GB2a and Fukugetin had higher activities than acarbose,a positive control,with IC50 values being 3.46 ± 0.32 μg/mL and 0.97 ± 0.09 μg/mL respectively,while the activity of GB2a glucoside was lower than that of acarbose with an IC50 value of 44.59 ±1.45 μg/mL.2)Based on affinity theory,GSK3β was immobilized on the surface of magnetic beads.A synthesized peptide,ILSRRRSpYR,was used as the substrate of the enzyme.After incubation,HPLC was employed as a tool for the separation of substrate and product.The peak area of product detected at 214 nn wavelength was used for the assessment of the activity of GSK3β.This new method established herein was environmental-friendly,simple,economical and reliable.The immobilized-enzyme was characterized and its performance was investigated.The enzyme immobilized at 4 ℃ had higher stability with the RSD value of 10 assays maintained at 5.7%.A known inhibitor of GSK3P,indirubin,was used to verify the reliability of the method for inhibitor screening.The Z’ factor of indirubin was 0.72,indicating the screening method was reliable.Then,the method was applied to screening the activities of 15 extracts of TCMs.It was found out that the extracts of G.xanthychymus possessed the highest activity at the concentration of 24 μg/mL,with the inhibitory rate being as high as 99.4%.Fukugetin,a main ingredient from G.xanthychymus,was also found with higher GSK3β inhibitory activity.The IC50 of Fukugetin was 3.18 ± 0.066 μM.Its mechanism was revealed by enzyme kinetics and molecular docking studies.The results shown Fukugetin may compete with the peptide substrate at the same binding site on the enzyme and it was a non-ATP competitive inhibitor of GSK3β.3)The anti-diabetic activities of 24 compounds from G.xanthychymus was evaluated using L6 myotubes.The cells were incubated with the compounds,respectively;then,glucose assay kit was used to determine the consumption of glucose by the cells.As a result,three compounds were found be able to significantly stimulate the glucose uptake of L6 cells(p<0.01).They are 12b-hydroxy-des-D-garcin A(compound 19),1,2,5,6-tretrahydroxy-4-(1,1-dimethy-2-propenyl)-7-(3-methyl-2-butenyl)xanthone(compound 21)and 1,5,6-trihydroxy-7,8-di(3-methyl-2-butenyl)-6’,6’-dimethylpyrano(2’,3’,3,4)xanthone(compound 24).Compared with the control group(as 100%),the glucose uptake rates of compound 19,21 and 24 were 208.4 ± 14.3%,166.2 ± 22.9%and 195.28 ± 3.2%,respectively.Compound 19 and 21 were abundant in this plant,so their molecular mechanisms were investigated after optimization of their concentration and incubation time.Finally,the concentration of 25 μM and the incubation time of 4 h were selected as the best conditions for both compound 19 and 21.The results of signaling pathway inhibition assays,western blot assays and GLUT4 translocation experiments displayed that both of the compounds can induce the translation of GLUT4 through activating of PI3K/Akt and AMPK signaling pathways,thus,promoting the glucose uptake by L6 cells.4)The anti-diabetic activities of 27 extracts of TCMs were screened by L6 myotubes.The extracts of Scrophularia ningpoensis,Wikstroemia indica,Glycosmis pentaphylla,etc,were found be able to significantly promote the glucose uptake of L6 cells.Among them,S.ningpoensis had the highest activity(126.8 ± 8.9%and 127.3 ±6.3%of control for concentrations of 50 μg/mL and 25 μg/mL,respectively,p<0.01).Subsequently,the concentration and the incubation time of S.ningpoensis extracts were optimized as 50 μg/mL and 4 h.A preliminary study of the molecular mechanism was carried out,finding that S.ningpoensis extracts may activated both PI3K/Akt and AMPK signaling pathways without affecting the translocation and expression of GLUT4.