Studies On Pharmacodynamic Material Basis Of Ginsenosides In Xuesaitong Injection

Xuesaitong injection is a sterile water solution which contains Panax Notoginseng Saponins（PNS）, the refined effective components extracted from Panax notoginseng（Burk.） F. H. Chen. The injection is mainly used for the clinical treatment of stroke hemiplegia, cerebral infarction, cerebral thrombosis, cerebral hemorrhage sequelae, and retinal vein occlusion. Studying on pharmacodynamic material basis is important to control the quality of injection and the safety of clinical medication. So the analysis of components in the injection and the inhibiting effect of Xuesaitong injection, the mixture of major saponins, the mixture of rare saponins, and PNS to platelet aggregation induced by ADP had been developed in this paper.According to the preparation process of the Xuesaitong injection, the Panax notoginseng extract were preparated and the chemical constituents were isolated. Twenty compounds including 10 major ginsenosides（Ginsenoside F₁, Rf, Rg₁, Rg₂, Re, Rd, Rb₁, Rb₂, Rb₃, and Notoginsenoside R₁） and 10 rare ginsenosides（Ginsenoside Rk₃, 20（S）-Rh₁, 20（R）-Rh₁, Rg₆, F₄, Rk₁, Rg₅, 20（S）-Rg₃, 20（R）-Rg₃, and Notoginsenoside R₂） were identified.In order to know the contents of gisenosides in Xuesaitong injection, different methods had been developed for the analysis of major ginsenosides, rare ginsenosides, and Panax Notoginseng Saponins in Xuesaitong injection. It is the first time that preliminary treatment of Xuesaitong injection and Panax notoginseng（Burk.） F. H. Chen had been established for determining the contents of rare ginsenosides.A method of ionic liquid salt aqueous two-phase extraction coupled with high-performance liquid chromatography had been developed for the analysis of seven rare ginsenosides including Ginsenoside Rk₃, 20（S）-Rg₃, 20（R）-Rg₃, Rg₆, F₄, Rk₁, Rg₅ in Xuesaitong injection. The experimental parameters including the volume of ionic liquid, the amount of K₂HPO₄, the p H value of system, and the time of ultrasonic treatment were optimized. The optimal conditions established were as follows. When 1.00 m L [Bmim]Br and 3.50 g K₂HPO₄ were used as extraction solvent,the analytes could be extracted from 3.0 m L injection, the ultrasonic time was 5 min. The LODs of seven rare ginsenosides were in the range of 0.17⁰.48μg m L-1. The adding standard recoveries were in the range of 89.05%¹11.29%（n=6） and the RSDs were in the range of 0.47².47 %（n=6）. The present method is simple, rapid, accurate, and helpful for the determination of trace component in Xuesaitong injections.The results showed that the contents of the rare ginsenosides in Xuesaitong injections were summarized in a manufacturer from different batchs. The contents of the total rare ginsenosides（the summation of seven rare ginsenosides） in Xuesaitong injection have little difference in same specification from different manufacturers, and they were 714.08 and 713.06 μg/m L respectively. The contents of the single rare ginsenosides in Xuesaitong injection from different manufacturers in same specification have a little difference, they were as follows: 86.01⁹5.10（Rg₆）, 48.30⁶0.81（F₄）, 132.89¹56.76（Rk₃）, 199.90²23.83（20（S）-Rg₃）, 84.39¹05.25（20（R）-Rg₃）, 52.64⁶0.22（Rk1）, and 51.60⁷0.12（Rg5） μg/m L, respectively.And seven major ginsenosides including Ginsenoside Rg1, Re, Rb₁, Rd, Rb₂, F₁ and Notoginsenoside R1 in Xuesaitong injection were detected by HPLC. The results showed that the contents of the major ginsenosides were larger than the rare ones. The contents of the major ginsenosides in Xuesaitong injection had very small difference from different manufacturers, and the ranges were as follows: 4.44⁵.09（R1）, 12.59¹3.84（Rg1）, 1.81¹.91（Re）, 13.37¹4.35（Rb1）, 0.74⁰.96（Rb2）, 0.085⁰.132（F1）, and 3.46³.90（Rd） mg/m L, respectively.For the purpose of exploring the source of rare ginsenosides, a method of ultrasonic-assisted ionic liquid-salt aqueous two-phase extraction coupled with high-performance liquid chromatography（HPLC） had been developed for determining the contents of rare ginsenosides in Panax notoginseng（Burk.） F.H.Chen（Sanqi）. The factors influencing on ultrasonic-assisted extraction was investigated in detail. The optimal conditions established were as follows. When 1.0 m L [Bmim]Br, 1.0 m L water and 7.0 g K₂HPO₄ were used as extraction solvent,the analytes could be extracted from 0.6 g Sanqi, the ultrasonic time was 20 min. The LODs of five rare ginsenosides were in the range of 0.18⁰.48 μg m L-1; the adding standard recoveries were in the range of 92.07 %¹10.55 %（n=6） and the RSDs were in the range of 2.38⁵.43 %（n=6）. The present method is simple, accurate, and helpful for the determination of rare ginsenosides in Panax notoginseng（Burk.） F.H.Chen（Sanqi）.The results showed that rare ginsenosides including 20（R）-Rg₃, Rg₆, F₄, Rg₅, which were not detected in Panax notoginseng（Burk.） F.H.Chen（Sanqi）, were detected in Xuesaitong injection. The reason may be that some hydrolysis and elimination reactions exist in the process of dissolving, heating, and refining which leads to the produce of rare ginsenosides.Xuesaitong injection was proved to be effective to treatment of stroke hemiplegia, cerebral infarction, cerebral thrombosis, cerebral hemorrhage sequelae, and retinal vein occlusion. In order to explore the material basis and the mechanism of this effect, we studied the inhibiting effect of Xuesaitong injection, major ginsenosides, rare ginsenosides, and PNS（major ginsenosides + rare ginsenosides） to platelet aggregation induced by adenosine diphosphate（ADP）. The results showed that Xuesaitong injection, the mixture of major ginsenosides, the mixture of rare ginsenosides, and PNS had obvious inhibiting effect. The orders of the inhibiting activity are as follows: PNS > major ginsenosides > rare ginsenosides.The inhibiting effect of 15 ginsenosides to platelet aggregation induced by ADP was studied. The results showed that 14 ginsenosides of PNS had obvious inhibiting effect in a concentration-dependent manner, which indicated that both major ginsenosides and rare ginsenosides are main active ingredients of inhibiting effect to platelet aggregation induced by ADP. The orders of the inhibiting activity are as follows: Rg₅>Rk₁^Rg₆>F₄>Rg₃>Rd^Re^Rb₃^Rb₂>Rk₃>Rh₁ >Rb₁>R₁>Rg₁.The results showed that rare ginsenosides played an important role in inhibiting effect of platelet aggregation induced by ADP. Under the same concentration, the rare ginsenosides had better inhibiting effects than major ones. Improving the content of rare ginsenosides in the injection may further enhance its antithrombotic therapeutic effects.All in all, the analysis of components in the injection and biological activity of PNS developed in this paper is helpful for studying on pharmacodynamic material basis of Xuesaitong injection and improving its quality standard. This is significant for improving QC and secondary development of Xuesaitong injection.