The Study Of The Efficacy And Mechanism Of Foodborne Hepatoprotective Active Ingredients

The liver is the largest substantive organ of the body,which is involved in the physiological processes of metabolism,detoxification,immunity and coagulation,and plays a crucial role in maintaining the health of the body.However,unfavorable factors such as environmental pollution,drug abuse,high life pressure and alcoholism continue to increase,and the incidence of liver injury-related diseases continues to rise,seriously threatening people's normal lives.The commonly used liver-protecting drugs are mostly chemical synthetic drugs,which have the disadvantages of long development cycle and high cost,and are often accompanied by adverse reactions and drug resistance.In recent years,it has been found that foodborne natural plant medicine has shown unique advantages in the treatment of liver injury,such as safe,low toxicity,economical and diversified treatment.Therefore,in this paper,the active ingredients with liver protection effect were explored from the natural food product Xm,and the mechanism of action was preliminarily studied.Chapter?ReviewThis chapter summarizes the liver injury and its pathogenic factors,mainly summarizes the mechanism of liver injury,expounds the research progress of foodborne active ingredients in the treatment of liver injury,and prospects the new research methods of liver injury.In recent years,with the emergence and development of the global"return to nature"wave,it has become a new trend for the research and development of anti-liver injury to develop the liver protective active ingredients from foodborne natural products.Chapter?Screening and identification of foodbornehepatoprotective active ingredientsThis chapter adopts the pharmacodynamic tracing method to extract Xm water extract with various polar solvents?methylene chloride,ethyl acetate,n-butanol?.A liver injury model induced by CCl₄ was used to assess the hepatoprotective effect of each extraction site.The results showed that n-butanol could significantly reduce the level of transaminase in the liver damaged mice,and it had a good protective effect on the liver.The active parts of n-butanol were separated and purified by silica gel chromatography column and ODS column in turn,and then analyzed by HPLC-DAD to obtain Compound 1.The structure of the compound was identified as 1-[O-?4-hydroxymethylphenyl?]-?-L-rhamno-pyranoside?Gastrodigenin Rhamnopyranoside,GR?by MS and NMR.Pharmacodynamics experiment proved that GR was the liver protecting active ingredient of Xm.Chapter?Experimental study on acute toxicity of GRIn this chapter,the acute oral toxicity of GR was studied using a fixed dose method?FDP?.Different doses of GR?50,500,1000 and 2000 mg/kg?was administered at one time,followed by continuous observation for 14 days.In the meantime,there was no death,and all the mice had smooth fur color,strong activity,normal diet,and no loose stools or diarrhea.The body weight and organ coefficient of both male and female mice in GR treatment group were not statistically different from those in the normal group?p>0.05?.In addition,HE staining showed that GR did not cause pathological damage to the heart,liver,spleen,lung,kidney and brain.Therefore,GR had no effect on the behavioral response,weight change and tissue morphology of mice.No significant organ changes and death were observed in the GR treatment group at the dose of 2000mg/kg,indicating that the lethal dose of GR(LD₅₀)was higher than 2000 mg/kg.According to the globally harmonized system?GHS?,GR was a class 5 chemical substance,which was non-toxic and had high safety.Chapter?Establishment and verification of in vivoanalysis method of GRIn this chapter,the UPLC-MS/MS in vivo analysis method of GR was established and its methodology was verified.The separation was achieved with a Shim-pack XR-ODS?C₁₈ column?2.0×75 mm,1.6?m?at 40°C,while the mobile phase?acetonitrile/0.1%formic acid=12:82,v/v?was at an eluting rate of 0.2 mL/min.The injection volume and detection wavelength were 2?L and 222 nm,respectively,and the operation time was 5 min.The anionic-multiple reaction monitoring?MRM?mode was used to detect the ion pairs.The change results of the ion pairs of GR and the internal standard?tyrosol?were:GR was m/z[M+HCOO]^-314.9?[M-H]^-269.0,and the collision energy CE was 17 eV.The internal standard is m/z[M+HCOO]^-182.85?[M-H]^-137.0,and the collision energy CE is 25 eV.The results showed that the linear range of GR in biological samples was 10-2500 ng/mL?r?0.999?,and the quantitative lower limit?LLOQ?was 10 ng/mL.The precisions?intra-and inter-day?were within 5%,and the accuracy was-11.4%to 8.33%.The extraction recovery and matrix effect were 80.32¹01.31%and 90.36¹03.76%,respectively.GR biological samples were placed at room temperature and-20?for 24 h and 15 d,respectively,without significant concentration change and showed good stability.It shows that the method meets the requirements of methodology and can be applied to the qualitative and quantitative analysis of GR and internal standard.Chapter?Pharmacokinetic and tissue distribution of GR invivoIn this chapter,the pharmacokinetic and tissue distribution of GR after oral and caudal administration were studied by using the UPLC-MS/MS method.Pharmacokinetic results indicated that the plasma concentration of GR in the oral?i.g?and tail vein?i.v?groups decreased rapidly from C_max?558.14±86.91 ng/mL and2329.47±659.70 ng/mL?within 3 h,and was not detected after 3 h.In addition,the half-life(t_1/2)of the oral and intravenous groups were 32.76±5.39?i.g.?min and 27.98±5.81?i.v.?min,respectively,furthermore the mean retention time?MRT?was 46.35±4.97?i.g.?min and 24.84±4.78?i.v.?min,respectively.This suggests that GR stays in the body for a short time and can be quickly cleared from the circulatory system.The results of tissue distribution showed that GR was rapidly distributed in all tissues in a short time after oral and intravenous administration,and was mainly distributed in tissues with abundant blood flow,such as heart,spleen,kidney,etc.,indicating that organ blood flow and perfusion rate may affect the distribution of GR in vivo.Meanwhile,the distribution of GR was also detected in brain tissue,suggesting that the compound had the ability to penetrate the blood-brain barrier.In addition,GR has a consistent and stable distribution in the liver,suggesting that it may have some liver protection potential in vivo,which needs further in-depth study..However,the retention time of GR in most tissues was relatively short,and the drug content decreased by nearly 90%within 30 minutes after administration in the tail vein,suggesting that the compound could be rapidly cleared in the body.Chapter?The study of hepatoprotective activity andmechanism of GRIn this chapter,mouse acute liver injury model was induced by intraperitoneal injection of 0.2%CCl₄ to evaluate the liver protective effect of GR,and the liver protective mechanism of GR was explored from the antioxidant stress and anti-inflammatory approaches.The results found that:compared with the model group,GR could significantly alleviate the abnormal liver hypertrophy and growth caused by CCl₄,also can significantly reduce the levels of alanine transaminase?ALT?,aspartate aminotransferase?AST?and alkaline phosphatase?AKP?in the serum of mice(^#p<0.05,^##p<0.01,^###p<0.001),and its recovery rate is close to silibinin group.At the same time,the GR groups of mice liver enzymes of antioxidant index?superoxide disproportionation enzyme?SOD?,glutathione peroxidase?GSH-Px?and catalase?CAT??,the antioxidant enzyme index?glutathione?GSH?and total antioxidant capacity?T-AOC??content significantly increased,the levels of lipid peroxide malondialdehyde?MDA?level decreased significantly(^#p<0.05,^##p<0.01,^###p<0.001),and present a dose of correlation,explain GR can effectively inhibit oxidative liver injury caused by CCl₄.In addition,tests of inflammatory cytokines showed that the GR treatment group significantly reduced the levels of inflammatory cytokines(Tumor necrosis factor?TNF-??,interleukin-1?IL-1??and monocyte chemotacticprotein 1?MCP-1?and regulated the levels of anti-inflammatory cytokines interleukin-10?IL-10?,thereby inhibiting CCl₄-induced inflammatory liver injury.The histopathological results of the mouse liver showed that the degree of liver damage in the mice with different doses of GR was significantly improved,including the structure of the central venule,reduced infiltration of inflammatory cells,and no obvious degeneration,swelling and necrosis of liver cells,indicating that GR could effectively repair the liver tissue damage caused by CCl₄.