| In recent years, with the drastic increasing of aging problem in our society, the incidence of the ischemic cerebrovascular disease (ICVD) has shown an upward trend, and it has become one of the prime aims in the prevention and treatment of senile diseases. Recent studies have shown that Radix Salviae Miltiorrhizae (RXM), one of the traditional Chinese medicine, has curative effect on ICVD by preventing the development of the disease at different aspects.In this study, the evaluation of the bioactive components group of RXM was taken through uniform design-high throughput screening method which was a totally new method. The RXM effective monomers were named as follows:Tanshinone I (Y1), Tanshinone IIA (Y2), Cryptotanshinone (Y3), Dihydrotanshinone (Y4), Protocatechuic aldehyde (Y5), Protocatechic acid (Y6), Rosmarinic acid (Y7), Caffeic acid (Y8), Sodium Danshensu (Y9), Salvianolic acid A (Y10), Salvianolic acid B (Y11), Notoginsenoside R1(Y12), Ginsenoside Rb1(Y13), Ginsenoside Rgl(Y14), and Borneol (Y15). The groups were divided like this, A and a groups contained the same7kinds of monomers (Y5、Y6、Y7、Y8、Y9、 Y10、Y11), the7concentration of the monomers in the A group are ranging from1×10-3to1×10-9mol·L-1, and in the a group, the7concentration levels are1×10-3,0.5×10-3,1×10-4,0.5×10-4,1×10-5,0.5×10-5, and1×10-6mol·L-1, the B and the b groups also contained the same7kinds of monomers (Y2、Y3、Y11、Y12、Y13、Y14、Y15), and in the B group, the7concentration of the monomers are ranging from 1×10-3to1×10-9mol·L-1, and in the b group, the7concentration levels are1×10-3,0.5×10-3,1×10-4,0.5×10-4,1×10-5,0.5×10-5, and1×10-6mol·L-1, The C and the c groups contained the same eleven monomers (Y1、Y2、 Y3、Y4、Y5、Y6、Y7、Y8、Y9、Y10、Y11), in the C group, the6concentration levels are ranging from1x10-3to1×10-8mol·L-1, and in the c group, the6concentration levels are1×10-3,0.5×10-3,1×10-4,0.5×10-4,1×10-5, and0.5×10-5mol·L-1, each group set the compatibility combinations on the the concentration leves of the monomers.The compatible combination samples were made according to the corresponding uniform design table. The104kinds of combination samples, named A1～A14、al～a14、B1～B14、b1～b14、C1～C24、c1～c24respectively, were obtained from the6groups. To obtain the better samples, the high throughput screening was taken to detect the capability of anti-DPPH oxidation and reduction ability of the compatible combination samples. The data were analyzed by the WUST package of uniform design and parameter optimization, wich can obtain the optimal combination samples and then repeated the experiment to obtain the better composite samples in the preliminary Pharmacodynamic exploration (screening). The injury model of hippocampal neuronal cells established by hydrogen peroxide (H2O2) was used as cell target to investigate the cell protection of the composite samples (re-screening). Finally, the optimal composite samples were obtained.1The result of antioxidant experiment in vitroThe result indicated that the highest activity of the combination samples on anti-DPPH (the clearance ratio) was48.92%and the reduction ability was62.26%. In the B and the b groups which were higher in fat soluble monomers indicated lower ability on anti-DPPH (lower than38.80%) and reduction ability (lower than33.74%)2The result of cytology experimentThe result indicated that the combination samples have significant effect on the cell protection (the cell survival ratio was higher than72%and up to94.7%) which was substantially higher than the positive group of Vitamin E (the cell survival ratio were75.4%) on the whole.After screening and re-screening, three combination samples, named all, P1and P4, were obtained, their cell survival ratio were all higher than90%. The cell protection was also significantly higher than that of the positive control of Vitamin E group and their respective monomers which are at the same concentration. The results indicated that the optimal compatibility samples on anti-oxidation and cell protective effect have no obvious correlation with the total molar concentration, this result may be related to the pathway role of multi-factor and multi-target. This study shows that UD-HTS is suitable for the research of the multi-factor and multi-lever of the traditional Chinese medicine, and it can sharply improve the research efficiency. By the studying of the RXS on pharmacodynamic about the effective components and different levels of the compatibility combinations, we have preliminarily explored the mechanisms on several kinds of effective monomers and different concentration ratio. It also provided a theoretical basis and data support for the effective prevention and treatment of ICVD and the cerebral vascular dementia diseases. |
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