Studies On Anthocyanin Composition And Antioxidant Activity Of Black Soybean Seed Coat And Its Anti-Proliferative Effect Of Vascular Smooth Muscle Cells

It was proved that oxidative stress was interrelated with atherosclerosis and other chronic diseases, so selection of nature resources with strong antioxidative activities for the prevention and treatment of these diseases have become the hot topic of medical science and chemical plants. Black soybean could be used as food and medicinal materials in China, and is an important sourance for the development of natural antioxidants due to rich phenolics compounds in its seed coat. In this paper, accessions with strongest activities and highest phenolics contents were selected after comparing the differences of antioxidative action and phenolics contents of different black soybean varieties, and the correlation between antioxidant activity and phenolics was analysed to determine the main components that contribute to the antioxidative activity, then the qualitative and quantitative assays of active components were analysised by HPLC. In order to research the mechanism by which black soybean seed coat exerted antiatherogenic effiency. The antioxidative activity and the proliferation inhibitory effects of the athocyanins components in seed coats on vascular smooth muscle cells(VSMC) stimulated by ox-LDL was investigates was also evaluated. The main foundings are as follows:(1) The correlations between antioxidantions, congtents of total phenolics and procyanidins and anthocyanin in 60 accessions of black soybean were studied. The results show that the total antioxidative activity (TAC), oxygen radical scavenging capative (ORAC), DPPH, total phenolics, proanthocyanidins and anthocyanin content of the 60 black soybean seed coats have greater amplitude and coefficient of variation, showing significant genotypic differences. And the anthocyanin content of black soybean germplasm shows a big difference in different ecological zones:Yunnan has the highest anthocyanin content, followed by the ones from Shanxi, and those from Heilongjiang province has the lowest in general. The possible reason is that-the average duration of sunshine in Yunnan Province is longer, with more ultraviolet radiation intensity, which causes the differences in anthocyanin content. Black soybean 17501 and daheiyang exhibited the greatest antioxidant activity and the highest phenolics contents in all of the black soybean germplasms. Correlative analysis shows that procyanidin and anthocyanin are the main fundamental substances of antioxidant activity in seed coat and germplasm of black soybeans. Fast clustering method can be used to cluster 60 black soybean germplasms into four categories:Ⅰ—black soybean germplasm with high-fat,Ⅱ—black soybean germplasm with high-protein, high anthocyanin,Ⅲ—black soybean germplasm with high-fat, high activity,Ⅳ—black soybean germplasm with low-nutrient, low activity.(2) The identifications and peak assignments of anthocyanins were primarily based on comparision of their retention times with those of standards, a blueberry refrence sample, and literatures. The quantitative analysis of anthocyanins was performed by HPLC according to Baj with a slight modification by using five standards. Separations were conducted with a Waters XBridge C18 reversed-phase column(5.0μm, 4.6mm×250mm). Gradient elution was performed with a mobile phase of solvent A (10% formic acid/H2O) and solvent B(methanol/acetonitrile/water/formic acid (25:25:40:10, v/v):0min,16%B; 8min,20%B;llmin,38%B;20min,46%B. Chromatography was performed at 25℃with a flow rate of 1mL/min and injection volume of 20μL. Six anthocyanins(Dp-3-glu, Cy-3-gal, Cy-3-glu, Pt-3-glu, Pn-3-glu, Mv-3-glu) were detected in seed coats of black soybeans. All the 6 components were detected in 44 varieties of the black soybean, and only 4 or 5 anthocyanins were detectable in the other 16 varieties. the dominant component was Cy-3-glu whose contents in seed coats of 60 black soybean varieties were in the ranges 61.82-1617.44mg/100g. The content of anthocyanins and Cy-3-glu of Daheiyang were higher than other black soybean varieties in our current study(3) By antioxidative activities assay, the anthocyanin monomer with the strongest activities was selected out by the chemcial and cellular testes, and the inhibition on the proliferation of VSMC induced by ox-LDL was determined. The results show that the DPPH-scavenging capabilitis of 5 anthocyanin monomers display as: Dp-3-glu>Cy-3-glu> Pt-3-glu>Mv-3-glu>Pn-3-glu. The DPPH·scavenging capability of Dp-3-glu and Cy-3-glu were found 3.13 and 2.06 times to Vc respectively. All of the 5 anthocyanin monomers could inhibite the proliferation of VSMC induced by ox-LDL at different degrees, and exhibited significant dose-effect relationships (P<0.05). Dose related increased SOD acitivity and reduced production of MDA and NO were found in Cy-3-glu pretreated VSMCs than those treated only with ox-LDL. Cy-3-glu and Pn-3-glu can keep higher rate of cells staying in the in the G0/G1 stage to repress the proliferation of VSMC, and Cy-3-glu showed stronger activity.