| Objective:Through the study on the extraction process of total saponins of American ginseng,Quantitative study of the main components of American ginseng to determine the content of prototype of several main components in the rat blood and cerebrospinal fluid,and determination of the content of prototype components in cerebrospinal fluid,to clarify the absorption of drug prototype components in blood and cerebrospinal fluid and the correlation between the two,and to provide experimental basis for the further determination of American ginseng in the compatibility of other traditional Chinese medicine.Method:①Using the colorimetric method.Taking the content of total saponins of American ginseng as evaluation index,the extraction process of total saponins of American ginseng was optimized by orthogonal design.D101 macroporous adsorption resin was used to optimize the enrichment process of total saponins of American ginseng.②Utilization UPLC-MS/MS was used to detect the main saponins in American ginseng.Chromatographic column is Acquity BEH C18(2.1 mm×100mm,1.7μm)chromatography column.0.1%formic acid in water(A)and acetonitrile(B)as the mobile phase,gradient elution,flow rate 0.25ml/min-1,column temperature 35℃;using electrospray ionization(ESI),mode of multiple reaction monitoring(MRM)contents were determined.③Wistar rats given ginseng extract(6g crude drug·kg-1·d-1)was administered for three days,two times a day.In the last administered after 1.5h,gather jugular vein blood 3ml and CSF 0.2ml,preparation containing serum and cerebrospinal fluid(CSF),sample was pretreated by solid phase extraction,diosgenin as standard.Quantitative analysis was carried out using UPLC-MS/MS.Result:①the optimal extraction and enrichment process was:adding 70%alcohol into crude ginseng(8:1 in weight)and extracting 2 times;for 3h each extraction.0.5BV sample liquid(0.5g·mL-1)passed D101 resin,eluted by 5BV water and 3BV 20%ethanol,eluted by 6BV 70%ethanol,collected eluate.Determination of the transfer rate of total saponins of American ginseng saponins was 81.04%,and the purity was 73.75%.②Determining the main component of American ginseng enrichment is ginseng saponin Rb1(43.90 mg·g-1)、ginseng saponin Re(14.54mg·g-1)、ginseng saponin Rc(2.72mg·g-1)、ginseng saponin Rd(7.71mg·g-1)、ginseng saponin Rb2(6.24 mg·g-1)、ginseng saponin Rb3(4.34 mg·g-1)、ginseng saponin Rg1(1.71 mg·g-1)、pseudoginsenoside F11(2.03 mg·g-1).③Identify the chemical constituents of rat plasma samples obtained after oral administration of American ginseng is ginseng saponin Rb1(167.27ng·mL-1)·ginseng saponin Rb2(31.27ng·mL-1)、ginseng saponin Rb3(13.54 ng·mL-1)、ginseng saponin Re(2.14 ng·mL-1)、ginseng saponin Rc(9.81ng·mL-1)、ginseng saponin Rd(21.25 ng·mL-1).Identify the chemical constituents of rat cerebrospinal fluid samples obtained after oral administration of American ginseng is ginseng saponin Rb1(32.01ng·mL-1).Conclusion:①This method had a better extraction process and purification capacity for total saponins for American ginseng.②The method is stable,reliable and reproducible,and the content of American ginseng extracted and enriched by UPLC-MS/MS is stable ③The research method for the determination of the prototype components in the blood and cerebrospinal fluid of American ginseng in rats is suitable for the study of the material basis of American ginseng.The method is stable,reliable and good reproducibility.Analysis of direct action of American ginseng at vivo substance is ginseng saponin Rb1、ginseng saponin Rb2、ginseng saponin Rb3、ginseng saponin Re、ginseng saponin Rc、ginseng saponin Rd.American ginseng in the central role of the direct substance is ginseng saponin Rb1. |
