| 1AIMShuangdan oral liquid has been recorded as a traditional Chinese medicinestandard preparation of Chinese Pharmacopoeia (ChP)(2005and2010). It wascomposed of the root of red-rooted salvia and moutan bark, and has featured theirefficacy of promoting blood circulation, removing blood stasis, dredgingcollaterals and relieving pain. This preparation was used for chest numbnesscaused by blood stasis congesting which usually represents symptom of chesttightness and cardiodynia. As a common used clinical preparation, the rate oftherapeutic efficiency achieved more than90%and significantly improved TCMsymptoms. However, the compounds of Shuangdanfang oral liquid are toocomplicated and the therapeutic mechanism is unclear. Therefore, it has greatsignificance to clarify the mechanism of Shuang Dan oral liquid and to re-exploit.Salvianolic acid B (salvianolic acid B, Sal B) and paeonol (paeonol, Pae) was used as main component of Shuangdanfangoral liquid to composed a MolecularChinese medicine，and play synergistic role,, according to design of Chinesemedicine molecules with the content and ratio in the Shuangdanfang oral liquid.In vivo animal model, tissue culture in vitro of coronary artery, and humanumbilical vein endothelial cell model approach were used to explore and revealthe mechanism of molecular traditional Chinese medicine salvianolic acid B withpaeonol anti-atherosclerosis, expansion of coronary flow, effect and mechanism ofendothelial cells’ protection. This study was aimed to provide experimental basisfor the secondary development of molecular medicine new drugs from ShuangDan oral solution.2Methods2.1Establishment on content determination method of SalB and Pae inShuangdan oral liquid: Content of SalB and Pae in Shuang Dan oral solutionwas determined by HPLC method. Column SinoChrom ODS-BP (4.6mm×250mm,5μm, Yilite) with column temperature30℃was used as stationary phase,detection wavelength was280nm, and flow rate was1ml/min. Methanol and2%glacial acetic acid gradient elution was used as mobile phase. The elutionconditions were as follows: methanol-2%glacial acetic acid (12:88) was used inthe first12min, methanol-2%glacial acetic acid (50:50) in the following1min,and methanol-2%glacial acetic acid (70:30) was set as elution condition till30min. The sample volume was10μl. And the methodological study was alsoinvestigated in this study, including the linear relationship between theinspection, precision experiments, repeated experiments, stability experiment,and recovery experiments.2.2Study of SalB with Pae on fat-induced atherosclerosis in rats: High-fatdiet with gavage propyl thiouracil was used to set up the rat atherosclerosis model. After grouping administration for4weeks, rat’s heart rate, blood pressure,respiration were determined; lipid parameters was set as follows: total serumcholesterol (TC), triglyceride (TGoxide), high density lipoprotein cholesterol(HDL-C), low-density lipoprotein cholesterol (LDL-C), apolipoprotein a(APOA), apolipoprotein B (of APOB); serum enzymes: nitric oxide (NO), nitricoxide synthase (NOS), superoxide dismutase (SOD) and content ofmalondialdehyde (MDA); and pathology morphological observation of aortawas also studied.2.3In vitro influence of SalB with Pae on rat coronary artery contraction:high K+solution were used to make coronary depolarize. PDC channels thenopened with calcium influx and induced sarcoplasmic reticulum calciumreleasing in cell intracellular so that concentration of calcium increased.Coronary artery smooth muscle was contracted caused by high calciumconcentration. So KCl and CaCl2were added, respectively, to observe influenceof SalB with Pae on rat coronary artery contraction. Hist could combinate withthe histamine receptor on coronary smooth muscle cells, and start ROC channelsto cause calcium influx and induce vasoconstriction. So after adding Hist,inhibition effect of contraction caused by salvianolic acid B with paeonol wasobserved; and inhibition effect of contraction with adding the Hist withoutCa2+solution was also investigated. Then Ca2+solution was added into solutionto examine its blocking effect of contraction caused by the external calciuminflux.2.4Protective effect and mechanism of SalB with Pae on H2O2-inducedoxidative damage model of human umbilical vein endothelial cells:H2O2-induced oxidative damage model of human umbilical vein endothelialcells was used in this study. Morphological observation, MTT assay, Hoechst 33342staining and electron microscopy examination was employed toinvestigate protective effect of salvianolic acid B with paeonol’s on endothelialcells, SYBR Green RT-PCR was used to detect expression change of relatedgenes Bcl-2, Bax and caspase-3mRNA on endothelial cells apoptosis afteradministration; Western blot was used to detect expression of proteins Bcl-2,Bax, caspase-3related to endothelial cell apoptosis.3Results3.1The content of salvianolic acid B and paeonol in Shuang Dan oralsolution: The contents of salvianolic acid B and paeonol in Shuang Dan oralsolution were approximately12mg mL-1and0.20mg mL-1. Thedetermination method is simple and reliable with well peak shape, good stability,and reproducibility.3.2Study of SalB with Pae on fat-induced atherosclerosis in rats: Nosignificant influence of Salvianolic acid B with paeonol on atherosclerotic heartrate, blood pressure, and breathing were observed (P>0.05) while Salvianolicacid B with paeonol could significantly reduce serum total cholesterol,triglycerides, low density lipoprotein levels (P<0.05or P <0.01), andsignificantly increase serum high-density lipoprotein, MDA and NOS (P <0.05or P <0.01). At the meanwhile, Salvianolic acid B with paeonol has a clearregulatory effect on Serum apolipoprotein A and apolipoprotein B (P <0.05or P<0.01). It was found by histopathology observation that arterial intimal plaque,intimal thickening, and connective tissue components has been significantlyimproved in treatment group.3.3In vitro influence of SalB with Pae on rat coronary artery contraction:While different cumulative concentration of KCl solution and Hist solution wereadded to K-H solution, different cumulative concentration of CaCl2was added to K-H solution without calcium and with high concentration of K+respectively,the rat coronary ring would show dependently contract. After incubation withdrug (salvianolic acid B with paeonol), given the same degree of KCl, CaCl2andHist stimulate, the dose-response curve moved toward the bottom right of thepeak time and reduced the maximum response. Hist make specimens contractionreached its peak, CaCl2make the further contraction in the coronary ring,salvianolic acid B with paeonol could significantly inhibited the two-phasecontraction. Results are statistically significant ((P <0.05or P <0.01)3.4Salvianolic acid B with paeonol’s protective effect and mechanism onH2O2-induced oxidative damage model of human umbilical vein endothelialcells: Salvianolic acid B with paeonol’s serum group could improve the status ofH2O2damage cells, and it’s dose-dependent; the activity of endothelial cell wasincreased with the increase of the concentration of Salvianolic acid B withpaeonol，2%serum containing compared with model group, the data wan’tstatistically significant (P>0.05), and4%,6%serum containing the data wasstatistically significant (P <0.01); cell proliferation rate is50.25%when theconcentration of drug is2%,67.37%when the concentration is4%, and67.14%when the concentration is6%, the data had statistically significant (P <0.01).The results of Hoechst33342staining and transmission electron microscopystudy indicated that the situation of Apoptosis improved with the increase of thedrug’s concentration, the cells survived in good condition of Medium dose andhigh dose group. Bcl-2mRNA levels increased in the cells, while the Bax gene,caspase-3expression decreased, it’s more consistent with the results of Westernblot analysis of protein content, and data was statistically significant (P <0.01).4ConclusionsHPLC method was employed to determine the content and ratio of salvianolic acid B and paeonol in Shuangdan oral solution. And take the resultas the instruction for the prescription of molecular medicine (salvianolic acid Bwith paeonol). In vivo animal models, blood vessels in vitro model, humanumbilical vein endothelial cell model were used to confirm that salvianolic acidB with paeonol has effect of anti-atherosclerosis, Dilating coronary artery, andprotecting of the vascular endothelial cells. The mechanism might be as follows,through reducing the serum TC, TG and LDL, MDA, increasing HDL, SOD,and NO level to form antioxidant and regulate the lipid metabolism, delay ormitigate the AS formation. Coronary artery blood vessels were expanded byinhibiting PDC and ROC channels and inhibiting intracellular calcium insmooth muscle cells release. The vascular endothelial cells were protected byregulating the expression of Bcl-2and Bax mRNA and protein expression andinhibiting the caspase-3activation of the downstream molecules. In summary,salvianolic acid B with Wu Danpi phenol treatment of atherosclerosis has goodapplication prospects. |
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