| Type 2 diabetic liver disease(2-DLD)is a chronic complication characterized by abnormal liver histology/liver function during the development and treatment of type 2diabetes.Symptomatic treatment is the main treatment in clinic,with no cure yet.Edgeworthia gardneri(Wall.)Meisn.is the dry flower bud of the genus Edgeworthia gardneri.(Wall.)Meisn..It is often used in the prevention and treatment of diabetes,hypertension,hyperlipidemia,coronary heart disease,vasculitis,vasculitis and so on.The flower bud of Edgeworthia gardneri is often used as "Mimenghua" in the Han people,which has the effect of nourishing the liver and improving the eyes.Based on the different medicinal uses used by Tibetan and Han ethnic groups on Edgeworthia gardneri,and in previous studies,it was proved that the flower of Malus microcarpa not only has the effect of reducing blood sugar(type 2 diabetes),but also has the effect of protecting liver(liver injury caused by CCl4),suggesting that the flower of Edgeworthia gardneri may have the effect of anti type 2 diabetes liver disease.In this study,pharmacodynamic validation,material basis,and mechanism of action were conducted for it,Specific research methods and results are as follows:1.Pharmacodynamic verification of Tibetan medicine Edgeworthia gardneri D60 in anti-type 2 diabetic liver disease:1)A rat model of type 2 diabetic liver injury induced by STZ combined with high sugar and fat was established,continuous intragastric administration for 42 days,the liver tissue lesions of type 2 diabetic liver disease rats were observed by pathological section,and serum indexes TC,TG,ALT,AST,TBA and TBIL were detected by ELISA kit.The results showed that the 60% ethanol macroporous resin elution site of 70% ethanol extract(D60)of Tibetan medicine can reduce the fasting blood glucose level,reduce the liver weight index and improve the pathological changes of liver tissue in rats with type 2 diabetic liver disease.Meanwhile,it can significantly reduce the serum levels of TC,TG,ALT,AST,TBA and TBIL in rats with type 2 diabetic liver disease in a dosedependent manner.2)To explore the mechanism of anti-type 2 diabetic liver disease by using western blotting technology.The results showed that the D60 site could significantly reduce the expression level of AMPK and PPAR-γ protein,and significantly increase the expression level of NF-κB protein.Therefore,it was speculated that the anti-type 2diabetic liver disease by D60 site could be realized through the regulation of AMPK/PPAR-γ/NF-κB pathway.2.Study on the spectral effect relationship of the effective parts of Edgeworthia gardneri anti-type 2 diabetic liver disease:1)The UPLC-QTOF-MS technique was used to construct the fingerprint of D60 parts of 10 batches of Tibetan medicine Edgeworthia gardneri.A total of 22 common peaks were revealed,and 18 monomer compounds were inferred according to the mass spectrum fragment ion information.They were umbellifolactone(1),edgeworoside C(2),rutin(3),gardnerol(4),isoquercitrin(5),salicylic acid(6),gardnerol C(7),kaempferol-3-O-rutanoside(8),daphnoretin-5-O-β-D-glucopyranoside-(1→2)-β-Dglucopyranoside(9),astragalin(10),(-)-secoisola-riciresinol(11),edgeworic acid(12),quercetin(13),rutarensin(14),tiliroside(15),edgeworoside A(16),kaempferol(17),daphnoretin(18).Further compared with existing standard substances,a total of 11 monomer compounds were identified,including umbellifolactone(1),edgeworoside C(2),rutin(3),isoquercitrin(4),salicylic acid(5),kaempferol-3-O-rutanoside(6),astragalin(7),quercetin(8),tiliroside(9),kaempferol(10)and daphnoretin(11).2)STZ combined with high sugar and fat was used to establish a rat model of type 2diabetic liver disease and to evaluate anti-type 2 diabetic liver disease effect of 10 batches of Tibetan medicine D60 parts.Efficacy study results showed that 10 batches of Tibetan medicine D60 can reduce the fasting blood glucose level,improve the liver histopathological changes,and reduce the serum levels of TC,TG,ALT,AST,TBA and TBIL in rats with liver injury of type 2 diabetes.3)With the 22 common peak-peak areas in 10 batches of D60 part fingerprint of Tibetan medicine Edgeworthia gardneri and the pharmacodynamics indexes of inhibition rate of TC,TG,ALT,AST,TBA and TBIL as correlation variables,Pearson correlation analysis was used to establish the spectral effect relationship of D60 part of Tibetan medicine Edgeworthia gardneri against type 2 diabetic liver disease.The results of the study on the spectral effect relationship showed that the correlation between 18 common components and inhibition rate of TC ranked from large to small was quercetin,rutarensin,rutin,kaempferol,gardnerol C,tiliroside,daphnoretin-5-O-β-Dglucopyranosyl-(1→2)-β-D-glucopyranoside,(-)-secoisola-riciresinol,edgeworoside A,daphnoretin,edgeworic acid,isoquercetin,gardnerol B,salicylic acid,kaempferol-3-O-rutin,edgeworoside C,umbellifolactone,astragalin;Ranking the correlation with inhibition rate of TG from greatest to smallest,they were gardnerol C,kaempferol,quercetin,tiliroside,rutin,rutarensin,(-)-secoisola-riciresinol,daphnoretin-5-O-β-Dglucopyranosyl-(1→2)-β-D-glucopyranoside,daphnoretin,edgeworic acid,edgeworoside A,isoquercitrin,salicylic acid,edgeworoside C,gardnerol B,kaempferol-3-O-rutanoside,astragalin and umbellifolactone;Ranking the correlation with inhibition rate of ALT in descending order,they were gardnerol C,gardnerol B,salicylic acid,isoquercitrin,umbellifolactone,rutin,kaempferol-3-O-rutanoside,daphnoretin-5-O-β-D-glucopyranosyl-(1→2)-β-D-glucopyranoside,rutarensin,(-)-secoisola-riciresinol,kaempferol,edgeworic acid,tiliroside,edgeworoside A,daphnoretin,astragalin,edgeworoside C and quercetin;Ranking inhibition rate of AST correlation from most to least,they were salicylic acid,edgeworoside A,gardnerol B,rutin,rutarensin,isoquercitrin,umbelliferone,edgeworic acid,daphnoretin,edgeworoside C,edgeworthia gardneri glycosides C,tiliroside,kaempferol,(-)-secoisola-riciresinol,quercetin,daphnoretin-5-O-β-D-glucopyranosyl-(1→2)-β-Dglucopyranoside,astragalin and kaempferol-3-O-rutanoside;Ranking from most to least relevant to inhibition rate of TBA,They are gardnerol C,edgeworoside C,rutin,rutarensin,quercetin,isoquercetin,kaempferol-3-O-rutanoside,daphnoretin-5-O-β-Dglucopyranosyl(1→)-β-D-glucopyranoside,salicylic acid,gardnerol B,umbelliferone,(-)-secoisola-riciresinol,edgeworoside A,edgeworic acid,kaempferol,tiliroside,astragalin,daphnoretin;Ranking the correlation with inhibition rate of TBIL from greatest to smallest,gardnerol B,kaempferol-3-O-rutanoside,daphnoretin-5-O-β-Dglucopyranosyl(1→2)-β-D-glucopyranoside,salicylic acid,daphnoretin,kaempferol,isoquercitrin,astragalin,(-)-secoisola-riciresinol,gardnerol C,umbelliferone,rutarensin,tiliroside,edgeworic acid,rutin,edgeworoside A,quercetin and edgeworoside C.3.In vitro pharmacodynamic validation of 11 monomer components from D60 part of Tibetan medicine Edgeworthia gardneri on type 2 diabetic liver disease:1)LO2 cells were induced by oleic acid to establish the cell model of type 2 diabetic liver disease,and 11 monomer components of D60 part of Tibetan medicine Edgeworthia gardneri were explored: umbelliferone(1),edgeworoside C(2),rutin(3),isoquercitrin(4),salicylic acid(5),kaempferol-3-O-rutanoside(6),astragalin(7),quercetin(8),tiliroside(9),kaempferol(10)and daphnoretin(11).Effects of TC,TG,ALT,AST on pharmacodynamic indexes of liver injury in type 2 diabetes mellitus.The results showed that among the 11 monomeric components,salicylic acid,umbelliferone,edgeworoside C,rutin,isoquercetin,kaempferol-3-O-rutanoside,astragalin,quercetin,tiliroside,kaempferol and daphnoretin could significantly reduce the TC level of LO2 cells.Edgeworoside C,rutin,isoquercetin,astragalin,quercetin and tiliroside significantly decreased the TG level of LO2 cells,while salicylic acid,umbelliferone,edgeworoside C,rutin,isoquercetin,kaempferol-3-O-rutanoside,astragalin,quercetin,tiliroside,kaempferol and daphnoretin significantly decreased the ALT level of LO2 cells.Salicylic acid,umbelliferone,isoquercetin,astragalin,quercetin,kaempferol and daphnoretin significantly reduced AST levels in LO2 cells.2)Hep G2 cells induced by oleic acid were used to establish cell model of type 2 diabetic liver disease,and 11 monomer components of D60 part of Tibetan medicine Edgeworthia gardneri were explored: umbelliferone(1),edgeworoside C(2),rutin(3),isoquercitrin(4),salicylic acid(5),kaempferol-3-O-rutanoside(6),astragalin(7),quercetin(8),tiliroside(9),kaempferol(10)and daphnoretin(11).Effects of TC,TG,ALT,AST on pharmacodynamic indexes of liver injury in type 2 diabetes mellitus.The results showed that among the 11 monomeric components,edgeworoside C,rutin,isoquercetin,kaempferol-3-O-rutanoside,astragalin,kaempferol and daphnoretin could significantly reduce the TC level of Hep G2 cells.Salicylic acid,umbelliferone,edgeworoside C,rutin,isoquercetin,astragalin,quercetin,tiliroside,kaempferol and daphnoretin could significantly reduce the TG level of Hep G2 cells.Salicylic acid,umbelliferone,edgeworoside C,rutin,isoquercetin,kaempferol-3-O-rutanoside,kaempferol,quercetin and kaempferol can significantly reduce the ALT level of Hep G2 cells.The AST level of Hep G2 cells was significantly decreased by salicylic acid,umbelliferone,edgeworoside C,rutin,isoquercetin,kaempferol-3-O-rutanoside,astragalin,quercetin and kaempferol.The above research results showed that the part D60 of the Tibetan medicine Edgeworthia gardneri has the effect of anti-type 2 diabetic liver disease.It is preliminarily speculated that the material basis of its efficacy may be related to umbellifolactone,edgeworoside C,rutin,isoquercitrin,salicylic acid,kaempferol 3-Orutanoside,astragalin,quercetin,tiliroside,kaempferol and daphnoretin,which may be through the regulation of AMPK/PPAR-γ/ NF-κ B path implementation. |
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