Chinese Prickly Ash To Hepg2 Cells And C57bl Mice In The Condition Of High Cholesterol And Inflammation Cholesterol Metabolism And The Influence Of The Separation And Purification Of Active Ingredients

Aim:To determine the effects and mechanisms on cholesterol metabolism of different fractions from zanthoxylum (PE, EtOAc and BuOH) induced by sterols and LPS in vitro and in vivo. In addition, isolate the active compounds by means of chromatography and HPLC.Methods:1. The air-dried fruit (15kg) of zanthoxylum bungeanum were extracted with Ethanol (25L) at50℃by heating reflux method for3hours and decocted for three times. The decoctions were dried by vacuum rotary evaporator to yield an ethanol extraction (5kg). The ethanol extraction was suspended in a solution of ethanol/H2O (1:1) and partitioned with PE, EtOAc and BuOH respectively to yield PE extraction (400g), EtOAc extraction (2.5kg) and BuOH extraction (1.8kg). Measure total phenolic contents using Folin&Ciocalteu’s phenol and anti-oxidative capacity using DPPH scavenging activity assay.2. HepG2Cells were plated in10-cm cell culture clusters at a density of1×105cells/dish in DMEM. After24h cells were washed with PBS and incubated in serum-free DMEM medium with0.05mg/ml PE, EtOAc and BuOH fractions for12h. Cells were then added1μg/ml25-hydroxycholesterol and10μg/ml cholesterol or solvent and incubated for another24h. To validate this model, simvastatin (1μM), which is well-established drug to treat atherosclerosis, was used as positive control in these experiments. Determine cell viability using MTT; Measure cellular total cholesterol content and ApoB secretion; Total RNA was extracted and mRNA expression of SREBP-2, LDLR, HMGCR, CYP27A1, ABCA1was determined by realtime PCR; Protein expression of ACAT and LDLR were determined by western blotting.3. HepG2Cells were plated in10-cm cell culture clusters at a density of1×105cells/dish in DMEM. After24h cells were washed with PBS and incubated in serum-free DMEM medium with0.05mg/ml,0.1mg/ml or0.2mg/ml BuOH fraction for12h. Cells were then added1μg/ml25-hydroxycholesterol and10μg/ml cholesterol or solvent and incubated for another24h. To validate this model, simvastatin (1μM), which is well-established drug to treat atherosclerosis, was used as positive control in these experiments. Determine cell viability using MTT; Measure cellular total cholesterol content, free cholesterol content, free fatty acid and TC using enzymatic methods. ApoB and ApoA1secretion were measured by ELISA; Total RNA was extracted and mRNA expression of SREBP-2, LDLR, HMGCR, CYP27A1, LXR-α and ABCG1was determined by realtime PCR; Protein expression of SREBP-1, LDLR, HMGCR, AC AT and LXR-α were determined by western blotting.4.23-25g male C57BL/6mice were divided into6groups:(1) control group,(2) LPS group,(3) PE group,(4) EtOAc group,(5) BuOH group,(6) dexamethasone group. Mice were fed400mg/kg PE, EtOAc or BuOH fractions of zanthoxylum bungeanum for7days. For the other series of experiments, the mice were divided into control group, LPS groups,125mg/mg BuOH group,250mg/kg BuOH group,500mg/kg group and dexamethasone group, and fed with indicated concentration of BuOH fraction for7days.On the7th day, mice were challenged with LPS (0.2mg/kg) in0.5ml saline or saline alone by i.p. Positive control mice were treated with dexamethasone (5mg/kg) by i.p.2h before LPS injection. Following2hours recovery, blood sample were drawn from orbit, then mice were sacrificed and livers were collected. Serum TNF-α, IL-6levels were measured by ELISA; hepatic TC was determined by enzymatic methods; gene expression of TNF-α、COX-2、iNOS、 IL-6、 ABCA1and LXR-α were measured by realtime PCR; hepatic protein expression of SREBP-1and PPAR-γ were measured by western blotting.5. The EtOAc extract (200g) was chromatographed over a reverse phase silica gel column and eluted with a gradient of PE:EtOAc (10:1to0:1) to afford16fractions (A1-A16). Fraction A2was eluted on Sephadex LH-20with CHCl3: CH3OH (1:1) to afford compound1(60mg). Fraction A4was eluted on normal phase silica gel column and eluted with CH3OH:water (3:1) to afford compound2(15mg). Fraction A13was passed through a silica gel with CHCl3:CH3OH (100:1to10:1) to give13subfractions (A13-1to A13-13). A13-13was repeatedly chromatographed on a silica gel and eluted with CHCl3:CH3OH (50:1to10:1) to yield compound3(55mg).6. The BuOH extract (50mg) was chromatographed on a silica gel column eluting with gradient of acetone:CH3OH (50:1to0:1) to give19fractions (Fraction B1-B19). Fr.17was subjected into Sephadex LH-20and eluted with CHCl3:CH3OH (1:1) to afford5subfractions (Fr.17-1to Fr.17-5). Fr.17-2was further chromatographed on a silica gel and then subjected into semiprepared HPLC (waters, USA)(MeOH/H2O20:80,3mL/min, tR=6min) to obtain compound4(3mg). Results:1. The anti-oxidative capacity of BuOH fraction is highest (134.3mg/g) followed by EtOAc (75.7mg/g)2. Experiment results on HepG2cells show for the first time that PE, EtOAc and BuOH fractions can reverse cholesterol accumulation and inhibit ApoB secretion induced by sterols. Results from C57BL/6mice also show that EtOAc and BuOH fractions can regulate hepatic cholesterol efflux accompanied by anti-inflammatory effects.3. By means of chromatography and HPLC, four compounds isolated from EtOAc and BuOH fractions may partly afford the activity. All of them were demonstrated to have beneficial effects on lipid metabolism or anti-inflammation. Syringaresinol-β-D-glucoside was first time to be isolated from zanthoxylum.