Effects And Mechanisms Of Flavonoids Monomer From Total Flavonoids Of Litsea Coreana L (tflc) On The Proliferation Of Hepatic Stellate Cells Induced By TGF-β1

Liver fibrosis is a serious public health problem of disease, which mainly result from the unbalance between synthesis and degradation of extracellular matrix (ECM), lead to excessive ECM deposition in the liver. Hepatic stellate cells (HSC) activation, proliferation; transformation is the central link of liver fibrosis, which leads to the important cause of sedimentary ECM is the activation of the proliferation and HSC collagen excessive formation.Laoying tea(also called Litsea coreana )is leaf of Litsea Coreana Leve which belongs to Neolitsea Lauraceae department,flavonoids is the main component of Laoying tea. It has been reported that the extract of Litsea Coreana L. exerts various pharmacological effects including antioxygen, antihyperlipidemic, antihypertensive, anti-inflammatory anti-hyperlipidemic and immunomodulation. In the pre-study, four effective flavonoids have been separated from the plant. Their structures are identified as quercetin-3-O-β-D-glucoside (I), quercetin-3-O-β-D-galactoside (II), kaempferol-3-O-β- D-glucoside (Ⅲ) and kaempferol-3-O-β-D-galactose (IV). Therefore, this study is to investigate whether the flavonoid monomers through regulating TGF-β1 to improve liver fibrosis, thus further understanding of total flavonids of litsea coreana of hepatic fibrosis antioxidant mechanism. Specific content includes the following two parts:1. Studies on the extraction and separation of four flavonoids monomer from Litsea Coreana L.With the flavonoids content as a evaluation index, orthogonal design L9(33) is applied to optimize the water extraction technique for total flavonoids of Litsea Coreana L. The best extraction technology is that 12BV water, 1 hour of reflux, and 3 times of extraction. The D101 macroporous resin (non-polar type) is the suitable medium to prepare TFLC. After adsorption on macroporous resin and desorption by ethanol, the content of total flavonoids in Litsea Coreana L is 48.59%, and the recovery rate is > 90 %. In this paper, four flavonoids are separated from TFLC by the normal-phase and reversed-phase silica gel column chromatography. Compared with the known compounds, they are identified as: quercetin-3-O-β-D-galactoside, quercetin-3-O-β-D-glucoside, kaempferol-3-O-β-D-galactoside, and kaempferol-3-O-β-D-glucoside.2. Studies on the effects and mechanisms of flavonoids monomer on the proliferation of hepatic stellate cells induced by TGF-β1The article adopts the liver perfusion to isolate the normal rat hepatic stellate cells, and culture it. This separation of hepatic stellate cell ratio of 2 x 107 / liver, the cell vitality and purity is more than 90%. By MTT method for detecting four flavonoids monomer proliferation of inhibition on HSC, the result shows that, after cultivating 24,48,72hour, four kinds of flavonoids monomer have varying levels of inhibition on the proliferation of HSC, but kaempferol-3-O-β-D-glucoside have the strongest effective at 48h. Reverse transcription - polymerase chain reaction (RT-PCR) analysis of kaempferol-3-O-β-D-glucoside on HSC which was induced by TGF-β1 was detected in collagenⅠ,collagenⅢ,Smad2, Smad3 and Smad7 receptor mRNA expression in the impact. it can obviously down-regulate collagenⅠ, collagenⅢ, Smad2 and Smad3 receptor mRNA expression,and it can up-regulate Smad7 mRNA expression.