Study On The Therapeutic Basis Of Total Saponins From Panax Notoginseng In Combination With Total Flavonoids From Puerariae Radix On Acute Blood Stasis

Blood stasis is an important underlying pathology of many cardiovascular and cerebrovascular diseases,such as coronary artery disease,angina pectoris and cerebral apoplexy.During the change of diet structure and aged tendency of population,it has become a major threat to human health and should be paid more attention.Over the past few years,Chinese patent medicine has attracted a great deal of global attention and ratification in cardiovascular and cerebrovascular diseases prevention.Because of the characteristics of Chinese patent medicine including multiple components and multi-target actions,the study of therapeutic basis of Chinese medicine formula becomes more and more important for evaluating Chinese medicines on a scientific basis and promoting the modernization of Chinese materia medica.In the present study,using a combined method of serum pharmacology,serum pharmacochemistry,metabonomics and pharmacokineticspharmacodynamics modeling based on high-pressure liquid chromatography-mass spectrometry,we investigated the therapeutic basis of total saponins from Panax notoginseng and total flavonoids from Puerariae radix on hemorheological disorders in rat model of acute blood stasis made by injecting with adrenaline hydrochloride and soaking in ice-water.The variance in hemorheological(whole blood viscosity at different shear rates of 200/s、30/s and 1/s,and plasma viscosity)parameter,the injured degree of cardiac muscle cell and the continuity of the sticking and pooling state of blood were used as indexes to compare the two most commonly used methods of duplicating acute blood stasis model,one was made by injecting with high-molecular weight dextran,the other was made by injecting with adrenaline hydrochloride and soaking in ice-water.Of the two methods,we demonstrated the latter was more appropriate for our study.A ultra-HPLC-ESI-MS metabonomic method was developed for metabolites profile analysis of urine from control rats and acute blood stasis model rats,and 24 metabolites were obtained in positive ion and negative ion mode.According to the variation trend of 15 metabolites obtained in positive ion mode,the urinary metabolite profiles from rats on the 1st day,the 2nd day,the 3rd day and the 5th day after model duplicating,were compared with those from control rats,which was the premise and reference of drawing up a reasonable dose regimen.Healthy rats were respectively administered total saponins from Panax notoginseng(13.5,27 and 54 mg/kg)and total flavonoids from Puerariae radix(27,54 and 108 mg/kg)of different doses via the caudal vein once each day for continuous seven days before model duplicating,and puerarin injection(54 mg/kg)was used as a positive control in this pharmacodynamic study.According to the variation trend of whole blood viscosity at different shear rates(200/s,30/s and 1/s)and plasma viscosity,we demonstrated the effective doses of total saponins from Panax notoginseng and total flavonoids from Puerariae radix were 27 and 54 mg/kg,respectively.A ultra-HPLC-ESI-MS metabonomic method was developed for metabolites profile analysis of plasma and heart tissue from control rats and acute blood stasis model rats.Twelve metabolites and sixteen metabolites were obtained from plasma and heart tissue in positive ion mode,respectively.According to the variation trend of these metabolites in positive ion mode,the metabolite profiles of plasma and heart from rats,which were respectively administered puerarin(54 mg/kg),total saponins from Panax notoginseng(27 mg/kg)and total flavonoids from Puerariae radix(54 mg/kg)seven days before model duplicating,were compared with those from control rats and model rats,and then,the mechanism of action of total saponins from Panax notoginseng and total flavonoids from Puerariae radix on acute blood stasis rats was revealed.In the present study,we found that there were defferences in target and mechanism of action between total saponins from Panax notoginseng and total flavonoids from Puerariae radix,and besides puerarin,there might be other active components in total flavonoids from Puerariae radix.Acute blood stasis rats were administered total saponins from Panax notoginseng(27 mg/kg)in combination with total flavonoids from Puerariae radix(54 mg/kg)via the caudal vein,and physiological saline was used as a negative control.To obtain the pharmacological effect-time curve of total saponins from Panax notoginseng in combination with total flavonoids from Puerariae radix on acute blood stasis rats,the rate of descent of hemorheological parameter was measured at 1.0,1.5,2.0,2.5,3.0,3.5,4.0,5.0 and 6.0 h after administration.Acute blood stasis rats were administered the extract of 2.0 h medicated serum via the caudal vein,and the extract of blank serum was used as a negative control.The rate of descent of hemorheological parameter was measured at 0.5,1.0,1.5,2.0,3.0 and 4.0 h after administration to obtain the pharmacological effect-time curve of the extract of 2.0 h medicated serum on acute blood stasis rats.Compared with the two pharmacological effect-time curves,there was a "time lag" existed in the pharmacodynamic response of hemorheological parameter after administration,so that,it is necessary to correct the pharmacological effect-time curve along the time axis during the correlation analysis between pharmacokinetics and pharmacodynamics.A ultra-HPLC-ESI-MS/MS method was developed for the simultaneous determination of six major isoflavonoids in total flavonoids from Puerariae radix in rat plasma,including 3 ’-hydroxypuerarin,6”-O-xylosylpuerarin,mirificin,puerarin,3 ’-methoxypuerarin and daidzin,using rutoside as an internal standard after protein precipitation with methanol containing 0.1%formic acid.The simple,sensitive,specific,and handy method met the requirements of biological sample analysis,and was successfully applied to pharmacokinetic studies of the six isoflavonoids in heathy rats.After intravenous administration of total flavonoids from Puerariae radix at a dose of 16 mg/kg,the elimination of the six isoflavonoids in rat plasma was rapid with an elimination half-life of less than 1.5 h,and these isoflavonoids could be detected up to 8.0 h.In this study,we identified the major metabolites in rat urine and plasma after intravenous administration of total flavonoids from Puerariae radix by HPLC-ESI-MS in positive ion mode.Nine metabolites of the major isoflavonoids in total flavonoids from Puerariae radix were detected in urine,which correlated well with the metabolites in plasma.In this paper,we reported for the first time both the pharmacokinetic study and the metabolic study of the major isoflavonoids,except for puerarin,in total flavonoids from Puerariae radix,which would provide important information for the further research on total flavonoids from Puerariae radix.A HPLC-ESI-MS method both in positive ion and in negative ion mode was established to identify multi-components of Xuesaitong injection and extract of Puerariae radix used in our study,and 64 peaks were detected,including 26 saponin peaks and 38 flavonoid peaks.Associated with the major metabolites we detected before,there were 63 peaks detected in the extract of 2 h medicated serum in positive ion and selected ion monitoring acquisition mode,including 63 peaks of unchanged drugs and 10 peaks of metabolites.The 44 major peaks of them were selected as "marker components",and a HPLC-ESI-MS/MS method in positive ion and selective reaction monitoring acquisition mode was developed for the internal reference analysis without calibration factor of these“marker components”in rat plasma after protein precipitation with acetonitrile containing 0.1%formic acid,using baicalin and digoxin as internal standards.The simple,sensitive and specific method met the requirements of biological sample analysis,and was successfully applied to analysis of these“marker components”in acute blood stasis rat plasma at 1.0,1.5,2.0,2.5,3.0,3.5,4.0,5.0 and 6.0 h after administration of total saponins from Panax notoginseng(27 mg/kg)in combination with total flavonoids from Puerariae radix(54 mg/kg)via the caudal vein,which would provide accurate and dependable data for the correlation analysis between pharmacokinetics and pharmacodynamics.During the interval of hemorheological properties-variation,a linear correlation analysis was performed on the natural logarithm of plasma concentration of“marker components”and the data of pharmacodynamics corrected along the time axis.A "combinatorial plasma concentration”was calculated by combining the natural logarithm of plasma concentration of“marker components",using the slopes obtained above as weight factors.And then,a linear correlation analysis was performed on the“combinatorial plasma concentrations" and the corrected data of pharmacodynamics.As a conclusion,the "combinatorial plasma concentrations”showed significant positive correlations with the rate of descent of whole blood viscosity at shear rate of 30/s and plasma viscosity.Thus,after the correlation analysis of pharmacokinetics and pharmacodynamics,it was feasible to use coefficients and significance as the degree of correlation with pharmacological effect,and to use slopes as the contribution to pharmacological effect,including synergism and antagonism.When total saponins from Panax notoginseng used in combination with total flavonoids from Puerariae radix,28 component,including puerarin,daidzin,3’-hydroxypuerarin,notoginsenoside R1 and ginsenoside Rg1,acted as the therapeutic basis for the descent of whole blood viscosity of acute blood stasis rats,while 34 component,including 3’-hydroxypuerarin,mirificin,6”-O-xylosylpuerarin,notoginsenoside R1 and ginsenoside Rg1,acted as the therapeutic basis for the descent of plasma viscosity of acute blood stasis rats.In the present study,a“combinatorial plasma concentrations”-pharmacodynamics modeling was established to investigate the therapeutic basis of total saponins from Panax notoginseng and total flavonoids from Puerariae radix on hemorheological disorders in rat model of acute blood stasis,which is designed to construct a demonstration platform of the system research on the therapeutic basis of Chinese herbal medicine and Chinese medicine formula and to solve the bottleneck problem on the study of therapeutic basis.