Pharmacodynamic Studies Of Auricularia Auricula Melanin On Alcohol-induced Liver Injury In Vivo And In Vitro

Liver is the main organ for alcohol metabolism,therefore a large number of harmful substances would be produced in the process of alcohol metabolism,resulting in liver injuries.Finding natural active substances from edible and medicinal fungi is a research hotspot in the prevention and treatment of alcoholic liver injury.As a traditional edible and medicinal mushroom,Auricularia auricula has a wealth of nutritional value and variety of biologically active.Auricularia auricula contains a lot of melanin,but there are few reports about the biological activity of melanin in Auricularia auricula,which has greatly limied the development and application of this natural pigment.In order to exploit Auricularia auricula melanin,the melanin from Auricularia auricula was identified and the cellulase-ultrasonic synergistic extraction process was explored.The intervention effect and preliminary pharmacological mechanism of Auricularia auricula melanin on alcoholic liver injury were explored through in vitro and in vivo experiments.The contents and results are as follows:(1)The structural characterization of melanin was analyzed through elemental analysis,gel permeation chromatography(GPC),UV-visible spectroscopy(UV-visible),infrared spectrum(IR)and nuclear magneticresonance spectra(NMR).Furthermore,the extraction technology combinded with cellulase-ultrasound for melanin was studied.The results showed that Auricularia auricula melanin belonged to eumelanin,the average molecular weight was estimated to be 48.99 kDa,the maximum absorption peak was 215 nm,the indole structure exists in its molecule,which accords with the typical characteristics of melanin.Cellulase-ultrasonic synergistic extraction can significantly improve the extraction rate of melanin from Auricularia auricula.Under the optimal conditions,the extraction rate of melanin from Auricularia auricula could reach 10.48%,which was about 47% more than that of traditional extraction method.(2)The effect of Auricularia auricula melanin on alcoholic liver injury was investigated at the cellular level.MTT assay was used to test the cells viability,and the levels of reactive oxygen species(ROS)and the ratio of GSH/GSSG in L02 hepatocytes were analyzed to estimate the intervention effects of Auricularia auricula melanin on alcohol-induced oxidative stress.The results showed that Auricularia auricula melanin significantly elevated the survival rate of L02 hepatocytes(p < 0.01),ameliorated the cell morphology,reduced the ROS level of(p < 0.01)and increased the ratio of GSH/GSSG(p < 0.01)of ethanol-pretreated L02 cells.(3)The intervention effects of Auricularia auricula melanin on alcoholic liver injury were further studied by animal experiments.The model of liver injury was established by intragastric administration of alcohol to mice.The therapeutic and preventive effects of Auricularia auricula melanin on alcoholic liver injury were evaluated by the level indexes of serum and liver function,intercomparison of the pathological sections and the expression of related antioxidant enzyme mRNA.The results showed that Auricularia auricula melanin had a good preventive and therapeutic effect on alcoholic liver injury in mice,because it could significantly decrease the levels of ALT,AST and MDA and increase the levels of ADH,SOD and CAT in mice(p < 0.01).Moreover,Auricularia auricula melanin also obviously ameliorated the morphological changes of liver tissue induced by alcohol in mice.The fluorescence quantitative PCR test indicated that Auricularia auricula melanin could up-regulate the expression level of Nrf2 and that of its downstream antioxidant enzyme mRNA(p < 0.01).In this thesis,the therapeutic and preventive effects of Auricularia auricula melanin on alcoholic liver injury in mice and its preliminary pharmacological mechanism were studied.The pharmacological efficacy of Auricularia auricula melanin,and provided a new idea to develope its further related products.This study also provides a new method for the efficient extraction of melanin from Auricularia auricula.