| As we all know, cardiovascular disease has been recognized as the world’s first killer, atherosclerosis is one of the most common cardiovascular disease in China the disease’s incidence increased year by year, which has become a major cause of death among adults. Atherosclerosis is a kind of chronic nonspecific vascular disease which resulting from lipid metabolism disorder, together with the influence of a variety of other common chronic stress factors, leading to endothelial dysfunction damage.A large number of clinical statistics show that:there is numerous sphingomyelin accumulating in human and animal’s atherosclerotic plaque, at the same time it has been confirmed that human plasma sphingomyelin level and the proportion of sphingomyelin/lecithin is closely related to the incidence of coronary heart disease (CHD). Therefore, sphingomyelin regulation is expected to achieve the treatment of atherosclerosis. The plasma level of sphingomyelin and lipoprotein metabolism is closely related with the incidence of atherosclerosis. Sphingomyelin synthase (SMS) is the last enzyme involved in the SM biosynthesis that transfers the phosphorylcholine moiety from phosphatidylcholine onto the primary hydroxyl of ceramide-producing SM and diglyceride, which can control the content level of cell sphingomyelin, ceramide, diglyceride and the lecithin, and directly affect the pathological process of atherosclerosis. Therefore, the research based on the target of sphingomyelin synthase and its inhibitors has become one of research hotspots in the field of new.Looking for drug with anti-atherosclerosis activity from herbal medicine has drawn much attention, so the research of Chinese medicine’s anti-atherosclerotic mechanisms on the basis of cellular and molecular levels is expected to bring breakthrough in atherosclerosis-related diseases’traditional Chinese medicine treatment.Modern pharmacological studies have shown that combining with some receptors or channels on the cell membrane is the first step for drug action. Therefore, the ability of a drug to interact with cell membranes is very important for the behavior of the drug in the organism. Based on this principle, some approaches including cell membrane chromatography (CMC) and frontal affinity chromatography, in which entrapped cell membranes and immobilized receptors or antibodies were used as the stationary phase of LC, were developed for on-line pharmacological studies and as rapid screens for the isolation and identification of lead drug candidates from complex biological or chemical mixtures.Rhizoma Polygoni Cuspidati is the dried rhizome of Polygonum cuspidatum Sieb. et Zucc. (Polygonaceae) and is one of the most famous and important traditional Chinese medicines (TCMs). It has been widely used for the treatment of atherosclerosis, hypertension, cough, suppurative dermatitis, and gonorrhea in Chinese clinical practice. This study combined with liver cell membrane model and overexpress sphingomyelin synthases cells model to preliminary screening potential anti-atherosclerosis activities in traditional medicine Rhizoma Polygoni Cuspidati. In chapter 1, the HPLC method for detection of Rhizoma Polygoni Cuspidati extract was establisheded. HPLC analysis was performed using a Y MC-Pack ODS-A column (250 x 4.6 mm I.D.,5 microns) at 30℃. The mobile phase consisted of 0.5% v/v aqueous acetic acid and acetonitrile with gradient elution procedure. The detection wavelength was set at 290 nm. Under this chromatographic condition,9 kinds of components in Rhizoma Polygoni Cuspidati extract show good chromatographic peak and were totally separated. The above chromatography method was validated for those items:linear range, detection limit, quantitative limit, precision, accuracy, repeatability and stability, the results show that this method is of high sensitivity and specificity. A method of ultrahigh pressure liquid chromatography combined with secondary mass spectrometry was adopted for further structure qualitative of related components in Rhizoma Polygoni Cuspidati extract, as a result,14 kinds of components were identified. As a result of the limitation of reference substance and drug concentration,11 kinds of components were studied detailedlyIn chapter 2, rat hepatocyte membranes was chose as screening model to screen the potential components in Rhizoma Polygoni Cuspidati extract. By comparing chromatograms of samples prepared before and after interaction with hepatocyte membranes, determine the potential components which can combine with the cell membranes. The degree of interaction between the drug and hepatocyte membranes can be judged by variation in related components’chromatogram peak area. With the above method,11 permeable compounds were identified which can combine with cell specifically:3,5,4’-Trihydroxystilbene-3-O-(6"-galloyl)-glucoside, Emodin-1-O-glucoside,Emodin-8-O-(6’-malonyl)-glucoside,Physcion-8-O-(6’-acetyl)-glucoside, piceid, resveratrol, emodin-8-β-D-glucoside, physcion-8-β-Dglucoside, aloe-emodin, emodin, and physcion, respectively.In chapter 3, in order to improve screening specificity and further confirmed the potential of the active ingredient of Rhizoma Polygoni Cuspidati, we chose overexpress sphingomyelin synthases cells model combined with live cell extraction method to screen potential active ingredients. Living cells under suitable conditions interacted with Rhizoma Polygoni Cuspidati extract, the active ingredient will combine with or enter into the cell. After interaction, the cells were washed repeatedly〠denatured and extracted with methanol. Eight compounds were identified as piceid, 3,5,4’-Trihydroxystilbene-3-O-(6"-galloyl)-glucoside, Emodin-1-O-glucoside, resveratrol, emodin-8-p-D-glucoside, physcion-8-β-Dglucoside, emodin, and physcion, respectively. As the circumstances of live cells are more similar to that of cells in organisms than that of lysed cell membranes, so that the cell-compound interactions more resemble that in organisms, leading to the bioactive candidates with high probability. The above results further verify the results of cell membrane model. The compounds piceid, resveratrol, emodin-8-β-D-glucoside, emodin and physcion have been reported to be active in inhibiting atherosclerosis, which, was consistent with our experimental results... |
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