| Yupingfengsan (YPF, Jade Windscreen Powder in English, Gyokuheifusan in Japan), a classical traditional Chinese medicine (TCM) formula, contains three herbal medicines: root of Astragalus membranaceus (Fisch.) Bge. var. mongholicus (Bge.) Hsiao, Rhizoma Atractylodis Macrocephalae (baked), Saposhnikovia divaricata (Turez.) Schischk. Our previous research demonstrated a fractioned polysaccharide (YPF-P) from YPF total polysaccharide was active in immunological regulation and had protection effect on actue liver injury in mice. Nowadays, the active components from the whole TCM formula are considered more reasonable to be regards as the active origin of formula pharmacological action. Therefore, in this paper, we isolated YPF-P from the whole YPF formula, purified its component polysaccharides, and investigated the chemical features of YPF-P1, the main constituent polysaccharide of YPF-P. On the other hand, the prevention effect of YPF-P on carbon tetrachloride-induced hepatic fibrosis in rats and the inhibition effect of YPF-P and YPF-P1 on HSC-T6 proliferation induced by TGF-β1 were investigated.1. Preparation of YPF-P and investigations on composition and structureTo select the optimum extraction process of YPF-P, orthogonal test was employed. Polysaccharide was extracted by deionized water and precipitated by ethanol. The content of sugar was determined by phenol-sulfuric acid method. As a result, the optimum extraction condition was 15 times amount of deionized water, refluxing 3 times in waterbath under 90°Сand 2 hours each time. In this part, the analytical method of sugar content of YPF-P is established. The verification test shows that the optimum extraction process is simple, stable and feasible.For further studies on its constituents and structure features, YPF-P was isolated and purified by anion-exchange chromatography and gel filtration successively. Three purified polysaccharides YPF-P1, YPF-P2, YPF-P3 from YPF-P were obtained. The sugar content and UV absorption were detected. Their molecular weights as well as homogeneity were determined using high performance liquid chromatography coupled with gel permeation chromatography (HPLC-GPC). Among the three, structural features of YPF-P1 was investigated by monosaccharide composition analysis, periodic acid oxidation, IR, 1H-NMR and 13C-NMR spectroscopy. As a result, the weight average molecular weights of YPF-P1, YPF-P2, and YPF-P3 were 488,500Da, 232,500Da and 1,863,000Da respectively. It is proposed that YPF-P1 is a highly branched heteroglycan composed of Glc, Gal, Fru and Ara in a molar ratio of 3: 2.5: 1.75: 1 along with a trace amount of Rha, and about half amount of Gal is in form of galacturonic acid. The main chain is composed of 1,6-linkedα-D-glucopyranoside and 1,6-linkedβ-D-galactopyranose in a molar ratio of 6: 5, to which side chains consisting of 1, 3-linkedβ-D-fructofuranose, methyl-β-arabino furanoside, and/or methyl-α-rhamnoside is attached at position O-6 of the glucosyl residues.2. Prevention effects of YPF-P on experimental hepatic fibrosis in vivo and in vitroIn vivo, it was aimed to investigate the prevention effects and its possible mechanism of YPF-P on carbon tetrachloride (CCl4)-induced liver fibrosis in rats. Hepatic fibrosis was induced by subcutaneous injection with CCl4 twice weekly for 12 weeks in Sprague-Dawley rats. YPF-P (30, 60, 120 mg·kg?1), polysaccharide fraction from root of Astragalus membranaceus (AP, 60 mg·kg?1) and the positive control colchicine (0.1 mg·kg?1) was administered intragastrically daily to CCl4-treated rats. Histopathological changes of liver and hepatic stellate cells (HSC) were evaluated by masson staining and transmission electron microscope, respectively. Serum levels of hyaluronic acid (HA), laminin (LN), type IV collagen (CIV) and type III procollgen (PCIII) were determined by radioimmunoassay (RIA). TGF-β1 in serum and tissue was assayed separatedly by ELISA and immunohistochemistry. The protein and mRNA ofα-SMA was determined by immunohistochemistry and RT-PCR. The mRNA expressions of tissue inhibitor of metalloproteinase-1 (TIMP-1), matrix metalloproteinases-13 (MMP-13), procollagen ? and collagenШwere detected by reverse transcription-polymerase chain reaction (RT-PCR). As a result, YPF-P dose-dependently alleviated the degree of liver fibrosis and inhibited HSC transformation into myofibroblast-like cells, reduced the elevated levels of serum HA, LN, CIV, PCIII,TGF-β1,α-SMA, markedly suppressed procollagen ? and collagen III expression, and improved the TIMP-1/MMP-13 ratio due to decrease on TIMP-1 expression. MMP-13 expression was only promoted moderately by YPF-P (120 mg·kg?1). Although AP possessed most effects on above markers except MMP-13 expression, YPF-P showed more potency than AP used individually on PCШand collagenⅢmRNA (p<0.01) as well as HA, TGF-β1, TIMP-1 mRNA, TIMP-1/MMP-13 ratio and procollagen ? mRNA (p<0.05) with no apparent differences in LN, CIV and MMP-13 mRNA. These findings suggested that YPF-P prevented the progress of rat liver fibrosis induced by CCl4 and presented the superiority of compatibility on the prevention effects. The prevention effect might be associated with its ability to inhibit the synthesis of matrix collagen and balance the TIMP/MMP system.In vitro, it was aimed to investigate the effect of YPF-P and YPF-P1 on HSC-T6 growth curve and TGF-β1-induced HSC-T6 proliferation. The results showed that YPF-P and YPF-P1 had obvious impact on HSC-T6 growth. YPF-P(50,100,200mg/L)and YPF-P1(100,200mg/L)inhibit the cell numbers and growth rate of normal proliferation. Both YPF-P 50-200mg/L(p<0.01)and YPF-P1 12.5-25 mg/L(p<0.05),50-200 mg/L(p<0.01)had the inhibition effect on TGF-β1-induced HSC-T6 proliferation. The effect of YPF-P1 with different doses was generally more powerful than YPF-P except for 100 mg/L. Therefore, YPF-P and YPF-P1 could inhibit HSC-T6 proliferation induced by TGF-β1, and YPF-P exert its effect faster while YPF-P1was more powful。...
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