Research On Anti-atherosclerosis Mechanism And Relative Contribution Of Hydroxysafflor Yellow A In Guanxin â…¡

Object:Atherosclerosis(As) is a chronic inflammation inside the arterial wall caused by abnormal lipids metabolism and it is also the basic pathogenesis of cornary heart disease(CHD), myocardial infarction, hypertension and other cardiovascular diseases. GuanxinⅡ was created by the late famous TCM physician-Pro. Guo Shikui and has been wildly used in therapy of CHD. This formula was composed of five herbs, such as Radix Salviae Miltiorrhizae, Rhizoma Ligustici Chuanxiong, Radix Paeoniae Rubra, Flos Carthami and Lignum Dalbergiae Odoriferae, of which the common ratio was2:1:1:1:1in weight Chinese medicine formula is considered to be crucial for treating diseases in clinical practice. The objective of this study are as follows:①To determine five compounds in GuanxinⅡ by ultra performance liquid chromatography (UPLC) technologies. Five compounds include Ferulic acid, Hydroxysafflor yellow A(HsYA), Tanshinol, Paeoniflorin and Protocatechuic aldehyde;②To develop ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) technology for qualitative and quantitative analysis of three compounds(Ferulic acid, Hydroxysafflor yellow A and Tanshinol) in plasma of C57BL/6J mice with after oral administration of GuanxinⅡ decoction;③To study anti-atherosclerosis mechanisms of Hydroxysafflor yellow A and preliminary quantitative analysis of its relative contribution to Guanxin Ⅱ in ApoE-/- mice with As.Method:①The analysis was performance on UPLC. The mobile phase comprising methanol (A) and1%acetic acid (B) was used to elute the targets in gradient elution mode(0min97%B;1min97%B;6min85%B;11min80%B;15min55%B)O②The plasma were collected from C57BL/6J mice with after oral administration of Guanxin Ⅱ decoction. Three compounds(Ferulic acid, Hydroxysafflor yellow A and Tanshinol) were qualitatively and quantitatively analysied by ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) technology. Electrospray ionization(ESI) source was applied and operated in the positive ion mode. Mutiple reaction monitoring(MRM) mode with the transition of m/z192.8-m/z133.96, m/z611-m/z491.04and m/z196.8-m/z178.96were used to detect Ferulic acid, Hydroxysafflor yellow A and Tanshinol.③Hydroxysafflor yellow A was chosen to explore and define its anti-atherosclerosis mechanism and compared therapeutic effects with Guanxin Ⅱ. With the computational formula(relative contribution=HsYA effectiveness/Guanxin Ⅱ effectiveness×100%), we made preliminary quantitative analysis of HsYA’s relative contribution to Guanxin Ⅱ. ApoE-/-mice were fed with high fat diet to develop atherosclerosis. Detected the effects of Guanxin Ⅱ and HsYA on mice surum lipid levels of triglyceride(TG), total cholesterol(TC), high density lipid Cholesterol(HDL-C) and low density lipid Cholesterol(LDL-C). Tested the development of atherosclerosis in mice arcus aortae with H&E staining and Oil-Red O staining. Determined the surum levels of malondialdehyde(MDA), superoxidedismutase(SOD), glutathione(GSH) and catalase(CAT). Analyzed the mRNA levels of tumor necrosis factor-α(TNF-α), vascular cell adhesion molecule-1(VCAM-1) and interleukin-1β (IL-1β) in thoraco-abdominal aorta by Real Time-Q PCR and measured protein expression of TNF-α,VCAM-1and IL-1β with Western Blot.Result:①Ultra performance liquid chromatography (UPLC) technology were used for simultaneous determination of five compounds in Guanxin Ⅱ. The five marker constituents were sufficiently separated successfully in less than15min. There were no interference peaks in the vicinity of the target compounds in the chromatograms of Guanxin Ⅱ decoction. All data indicated good linear regression, precision, recoveries and reproducibility of UPLC. Quantitative determination of five marker constituents in Guanxin Ⅱ were as follow:Ferulic acid(128.84±0.47ng/mg), Hydroxysafflor yellow A(1949.61±1.56ng/mg), Tanshinol(270.38±1.95ng/mg), Protocatechuic aldehyde (184.84±0.80ng/mg) and Paeoniflorin(389.67±0.91ng/mg).②According to the ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) technologies, three compounds(Ferulic acid, Hydroxysafflor yellow A and Tanshinol) were qualitatively and quantitatively analyzed in plasma of C57BL/6J mice after oral administration of Guanxin Ⅱ decoction. Quantitative determination of three compounds in plasma were as follow:Ferulic acid(20.4ng/ml), Hydroxysafflor yellow A(154.0ng/ml) and Tanshinol(6.9ng/ml).③Data showed:a). The serum levels of TC, LDL-C and HDL-C in ApoE-/-mice were significantly higher than those in C57BL/6J mice, but there were no differences in TG. While after oral administration of Guanxin Ⅱ decoction and HsYA, the serum levels of TC, LDL-C and HDL-C decreased and the relative contribution of HsYA to Guanxin Ⅱ were:46.42%in TC,64.01%in HDL-C and53.89%in LDL-C; b). There were apparent atherosclerotic plaque in arcus aortae with H&E staining and the plaque and fat were stained red by Oil-Red O staining. Atherosclerotic lesions area in vehicle group, Guanxin Ⅱ group and HsYA group respectively were:(43.25±3.70)%,(30.20±1.48)%and (35.63±2.06)%. Relative contribution of HsYA to decreasing atherosclerotic lesions area was58.39%; c). Compared to C57BL/6J mice, ApoE-/-mice showed lower activities of serum SOD, GSH and CAT, but higher levels of MDA. Administration of Guanxin Ⅱ and HsYA could decrease levels of MDA while increase concentrations of SOD, GSH and CAT. Relative contribution of HsYA to indexes about oxidative stress were:55.79%in MDA,56.00%in SOD,32.61%in GSH and28.34%in CAT; d). Determined by Real Time-Q PCR and Western Blot, compared to C57BL/6J mice, ApoE-/-mice showed higher mRNA and protein levels in TNF-α,VCAM-1and IL-1β at thoraco-abdominal aorta. While administration of Guanxin Ⅱ and HsYA could decrease those mRNA and protein levels. Relative contribution of HsYA to mRNA level were:81.83%in TNF-α,85.96%in VCAM-1and82.67%in IL-1β, as well as58.88%in TNF-α,61.83%in VCAM-1and52.17%in IL-1β, to protein level.Conclusion:︰ltra performance liquid chromatography (UPLC) technology were used for simultaneous determination of five compounds in Guanxin Ⅱ.This technology was rapid, sensitive, accurate and reliable.㏕hree compounds(Ferulic acid, Hydroxysafflor yellow A and Tanshinol) in plasma of C57BL/6J mice after oral administration of Guanxin Ⅱ decoction were successfully qualitatively and quantitatively analyzed with ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) technology. Data showed that the concentration of HsYA was highest, which laid a foundation for the research on HsYA anti-atherosclerosis mechanism and relative contribution to Guanxin Ⅱ.㏕he atherosclerotic animal model was successfully established by ApoE"’" mice fed with high fat die for8weeks, while administration of Guanxin Ⅱ and HsYA could modulate blood lipid and inhibit atherosclerotic plaque progression.(4)The oxidative stress was enhanced in ApoE-/-mice with athrosclerosis, while administration of Guanxin II and HsYA could reduce oxidative stress and inhibit the development of atherosclerosis. ⑤According to anti-oxidative stress, HsYA and GuanxinⅡ played roles on anti-atherosclerosis. Through effectiveness were compared with HsYA and Guanxin Ⅱ, we could make a preliminary quantative analysis on relative contributation of HsYA in the anti-atherosclerosis process of Guanxin Ⅱ.