Study On The Material Basis And Mechanism Of Action Of Danshen In Treating Diabetic Complications Based On Spectrum-effect Relationshi

ObjectiveSalvia miltiorrhiza(Danshen in Chinese)is the dried root and rhizome of Salvia miltiorrhiza.Bge.It can invigorate the circulation of blood,induce menstruation to relieve menalgia,clear away the heart-fire and so on.Modern pharmacological studies showed that Danshen has significant effect on the treatment of cardiovascular diseases,such as atherosclerosis,hypertension,hyperlipidemia,stroke.In addition,it can also improve the hemorheology,which plays a significant role in the microcirculation disorders.According to the study on literatures,Salvia miltiorrhiza is one of the most common used herb for treating diabetes and its complications.Nevertheless,research on the material basis and mechanism of Danshen and its related preparations for the treatment of diabetic complications is very limited until to now.Therefore,this research was to reveal the material basis and mechanisms of Danshen for the treatment of diabetic complications by using the plasma pharmacology and plasma pharmacochemistry.Specifically,we studied the spectrum-effect relationship between pharmacochemistry and the polyol pathway,aldose reductase and oxidative stress effect of Danshen containing plasma.It can not only clarify the material basis and mechanisms of Danshen for the prevention and treatment of diabetic complications,but also provide some new ideas and methods for the study of Chinese herbal medicine.Methods1.In the present study,an HPLC LTQ-Orbitrap method was established by using a Waters XBridge C18 column under the gradient elution of acetonitrile and water containing 0.1%formic acid.Fistly,we analyzed the chemical components of Danshen extracts.Then,the prototype and metabolic components in rat plasma,urine,fece and bile after oral administration of Danshen were analyzed,which laid the foundation for the further study of the metarial basis and pharmacological study of Danshen.2.An UPLC Q-TOF MS method was established by using a Waters ACQUITY UPLC BEH C18 column under the gradient elution of acetonitrile and water containing 0.1%formic acid,which has been applied for relative quantification of the prototype and metabolic components in rat plasma at different time points after oral administration of Danshen extract.3.At the cellular level,the inhibitory effect of drug containing plasmas at different time points on the increase of sorbitol induced by high glucose was investigated;the inhibitory effect of drug containing plasmas at different time points on the increase of ROS caused by high glucose was also investigated.At the molecular level,aldose reductase,the key enzyme of polyol pathway,was further studied.We extracted aldose reductase from rat lens,and then investigated the inhibitory effect of drug containing plasmas at different time points on AR activity.4.The spectrum-effect relationship between the chemical profiles of rat plasmas at different time points and pharmacological effects of plasma samples was investigated by using partial least squares(PLS)method.Several groups of chemical components closely related to the separate pharmacological effects were obtained.Results1.The chemical and metabolic profiles of Danshen were analyzed by HPLC LTQ-Orbitrap under positive and negative ion modes.As a result,69 chemical constituents were identified from Danshen extracts.Among which,56 components were known components,including 23 salvianolic acids and 33 tanshinone.16 chemical constituents were confirmed by the standard compounds.In addition,13 unknown components were also found.Furthue more,118 components were identified in rat plasma,urine,feces and bile samples after oral administration of Danshen.35 components,including 17 prototype components and 18 metabolites were identified in rat plasma.63 compounds were identified in rat urine,of which there were 23 prototype components and 40 metabolic components.62 compounds were identified in rat feces,among which,34 were prototype components and 28 were metabolic components.18 compounds were identified in rat bile,the prototype and metabolic components were 7 and 11,respectively.2.By using UPLC Q-TOF MS method,24 components were identified in rat plasma in positive and negative ion mode.Relative quantifications of these components in rat plasma were performed by using extracted ion chromatography(EICs)and peak area.From this way,we got the chemical spectrums of the plasma at different time points.3.Effects of plasmas at different time points were investigated at the cellular level and molecular level,including decrease of sorbitol in cells,inhibition of AR activity,and suppress of oxidative stress.4.The PLS method was used to analyze the correlation between the plasma chemical spectrums and pharmacological effects.Results showed that Danshensu,Salvianolic acid A,Demethyl neocryptotanshinone,Hydroxyl cryptotanshinone,Hydroxyl and dehydro tanshinone Ⅱ A,Hydroxyl tanshinone Ⅰ,Hydrated tanshinone Ⅰ,Demethyl and hydroxyl miltirone,Tanshinone ⅡB,Iso hydroxyl and dehydro tanshinone Ⅱ A,Hydroxyl tanshinone Ⅱ A,Hydrated cryptotanshinone,Iso Hydroxyl cryptotanshinone,Cryptotanshinone were positive related to the decrease of sorbitol in cells,among which,Danshensu,Salvianolic acid A,Hydroxyl and dehydro tanshinone Ⅱ A,Hydroxyl tanshinone ⅡA maybe the main bioactive components with larger positive correlation coefficients;Danshensu,Demethyl neocryptotanshinone,Hydroxyl cryptotanshinone,Hydroxyl tanshinone Ⅰ,Demethyl and hydroxyl miltirone,Tanshinone Ⅱ B,Decarbonylated neocryptotanshinone,Tanshinol B,Iso Dihydrotanshinone I,Hydroxyl tanshinone Ⅱ A,Dihydrotanshinone Ⅰ,Hydrated cryptotanshinone,Iso hydroxyl cryptotanshinone,Cryptotanshinone,Tanshinone ⅡA were positive related to the inhibitory effects on AR activity,among which,Danshensu,Demethyl neocryptotanshinone,Demethyl and hydroxyl miltirone,Tanshinol B,Hydroxyl tanshinone ⅡA maybe the main bioactive components with larger positive correlation coefficients;Danshensu,Salvianolic acid A,Hydroxyl and dehydro tanshinone Ⅱ A,Hydroxyl tanshinone Ⅰ,Hydrated tanshinone Ⅰ,Demethyl and hydroxyl miltirone,Salvianonol,Tanshinone ⅡB,Hydroxyl tanshinone Ⅱ A,Iso hydroxyl cryptotanshinone,Cryptotanshinone,Tanshinone ⅡA were positive related to the decrease of ROS,among which,Danshensu,Hydroxyl and dehydro tanshinone Ⅱ A,Salvianonol,Tanshinone ⅡB maybe the main bioactive components with larger positive correlation coefficients.Conclusions:1.The chemical and metabolic profiles of Danshen were comprehensively studied in vitro and in vivo,which laid foundation for the material basis study and pharmacological study of Danshen.2.Danshen containing plasma could inhibit the activation of polyol pathway induced by high glucose in vascular endothelial cells,and reduce the content of sorbitol in cells.A group of effective components were obtained by spectrum-effect relationships.Among which,Danshensu,Salvianolic acid A,Hydroxyl and dehydro tanshinone Ⅱ A,Hydroxyl tanshinone ⅡA maybe the main bioactive components.3.Danshen containing plasma could inhibit the activity of aldose reductase in vitro.A group of effective components were obtained by spectrum-effect relationships.Among which,Danshensu,Demethyl neocryptotanshinone,Demethyl and hydroxyl miltirone,Tanshinol B,Hydroxyl tanshinone ⅡA maybe the main bioactive components.4.Danshen containing plasma could inhibit the production of ROS induced by high glucose in vascular endothelial cells.A group of effective components were obtained by spectrum-effect relationships.Among which,Danshensu,Hydroxyl and dehydro tanshinone Ⅱ A,Salvianonol,Tanshinone ⅡB maybe the main bioactive components.