Uniform Design-high Throughput Pharmacodynamic Screening And Mechanism Of Radix Salviae Miltiorrhizae Hydrosoluble Constituents

With the social development, changed environment and improvement of living standards and lifestyle changed, the incidence of cardiovascular disease have increased year by year. It seriously threats to the human health.The exactly pathophysiological mechanisms of cardiovascular disease have not been clear yet. Many studies show that cardiovascular disease is caused by many different things. There are many views and hypothesis about the cause of cardiovascular disease, including the oxidative stress, oxidized low-density lipoprotein, cytokine, homocysteine, uric acid, endothelial microparticles, blood rheology, etc. In recent years, a growing number of studies demonstrated that oxidative stress may be a major cardiovascular disease triggers. High cholesterol, high blood pressure, high blood sugar,ischemic injury and so on, can cause oxidative stress, with leading to the occurrence of cardiovascular diseases. Traditional Chinese medicine Radix Salviae Miltiorrhizae(RMX) have a significant effect on cardiovascular diseases.The study was guided by chinese traditional medicine memory, and inspired by thought of traditional chinese medicine-compatibility- monarch,minister, assistant and guide. The bioactive ingredient compatibility of RXM hydrosoluble constituents(protocatechuic aldehyde(Y1),protocatechuic acid(Y2), rosmarinic acid(Y3), caffeic acid(Y4), sodium danshensu(Y5), salvianolic acid A(Y6) and salvianolic acid B(Y7)) was taken through uniform design-high throughput screening method which was a totally new method. Hydrosoluble constituents of RXM was made up in seven concentrations By reduction ability, DPPH oxidative method,vascular endothelial cells induced by H2O2 and vascular endothelial cells serum deprived model, we take uniform design-high throughput screen. we got the best combination samples. The efficacy and mechanism of the optimal combination samples was further confirmed by cerebral ischemia and reperfusion mice.1 Screening of optimal RSM hydrosoluble combination in vitro1.1 Anti-DPPH oxidativeWith establishment of anti-DPPH radical experiment, bioactive ingredient of RSM hydrosolube constituents were combined to be a new compatible group by uniform design(A1~A14). And the effects were detected by DPPH oxidative methods. Hydrosolube constituents of RSM had a significantly effect on anti-DPPH. After high-throughput screening and rescreening, optimal combination [A11(Y1:10-6 mol·L-1, Y2:10-9mol·L-1, Y3:10-9 mol·L-1, Y4:10-4 mol·L-1, Y5:10-3 mol·L-1, Y6:10-3mol·L-1, Y7 : 10-6 mol·L-1),(56.9±1.3)%] compatibility of RXM were obtained.1.2 Reduction abilityWith establishment of reduction ability experiment, bioactive ingredients of RSM hydrosolube constituents were mixed to be a new compatible group by uniform design(B1~B14). And the effects were detected by reduction ability method. Water-soluble components of RSM had a significantly effect on reduction capacity. After high-throughput screening and rescreening, optimal combination [B11(Y1:10-6 mol·L-1,Y2:10-9 mol·L-1,Y3:10-9 mol·L-1,Y4:10-4 mol·L-1,Y5:10-3 mol·L-1,Y6:10-3 mol·L-1,Y7:10-6 mol·L-1),(75.3±2.6)%] compatibility of RSM were obtained.1.3 The protective effects of vascular endothelial cell induced by H2O2With establishment of the model which vascular endothelial cells was induced by H2O2, CCK-8 assay was performed to select non-toxic dose of RSM hydrosoluble in treating vascular endothelial cell. vascularendothelial cell were subjected to oxidative damage with H2O2 in the combination which was through uniform design(C1~C14). Y2, Y5, Y4 and Y7 had protective effects on vascular endothelial cell induced by H2O2.After high-throughput screening and rescreening, optimal combination [C2(Y1:10-5 mol·L-1, Y2:10-7 mol·L-1, Y3:10-4 mol·L-1, Y4:10-10 mol·L-1, Y5:10-10 mol·L-1, Y6 : 10-7 mol·L-1, Y7 : 10-9 mol·L-1),(70.2±4.3)%]compatibility of RSM hydrosoluble were obtained.1.4 The protective effect of serum deprived vascular endothelial cellWith establishment of the model which vascular endothelial cell was deprived by serum, CCK-8 assay was performed to select non-toxic dose of RSM hydrosoluble in treating vascular endothelial cell. vascular endothelial cell were subjected to serum deprived in the combination which was through uniform design(D1~D14). Y1, Y2, Y5, Y6 and Y7 had protective effects on serum deprived vascular endothelial cell. After high-throughput screening and rescreening, optimal combination [D4(Y1:10-7 mol·L-1,Y2:10-4 mol·L-1,Y3:10-4 mol·L-1,Y4:10-9 mol·L-1,Y5:10-10 mol·L-1, Y6 : 10-10 mol·L-1, Y7 : 10-7 mol·L-1),(76.0±3.2)%]compatibility of RXM hydrosoluble were obtained.2 The Influence of RSM Hydrosoluble combinations on the memory dysfunction induced by ischemia-reperfusion in miceThe protective effects of optimal combination(A11, C2, D4) on cerebral ischemia and reperfusion injury and its mechanism were studied.Cerebral ischemia and reperfusion model was conducted referring to improved Himori method in which bilateral common carotid arteries were temporarily obstructed. The effect of combination on learning and memory were examined in Morris water maze task. Anoxia survival time of decapitated mice, the activities of SOD(superoxide dismutase, SOD),CAT(catalase, CAT), TCh E(acetylcholine esterase, TCh E) and the content of MDA(malondialdehyde, MDA) were measured. The influences of combination were studied on the neurological function and the expressionof apoptosis related protein caspase-3 in hippocampal neuron and nerve growth factor in cortex using immunohistochemistry. Combinations(C2,A11) could obviously improve memory impairment induced by cerebral ischemia and reperfusion in mice, enhance hypoxia tolerance, increase the activities of SOD and CAT, with decreasing the activities of Ach E and the content of MDA. At the same time, the expression of capase-3 were inhibited, increasing NGF. Effect of combination were most obviously in group(D4 1.4μmol·L-1, A11 30μmol·L-1), C2 combination has no significant effect each dose. Combinations(D4, A11) have a significantly protective effect on hippocampal neurons after cerebral ischemia and reperfusion injury. The mechanism may be related to romoving free radicals, inhibiting apoptosis of nerve cells, and promoting nerve regeneration.