Study On Preparation And Biological Activity Of Glucuronic Acid From Tremella Fuciformis Berk

Tremella fuciformis Berk,also known as snow ear,is a precious edible and medicinal fungus.Tremella fuciformis polysaccharide is one of the main components of Tremella fuciformis fruiting body,the main components are:mannose,fucose,xylose,glucose and glucuronic acid,among which the content of glucuronic acid accounts for about 10%.Glucuronic acid is widely used in medicine,cosmetics,health food and other fields because of its functions of participating in body metabolism,cell protection and immune regulation.In general,glucuronic acid in polysaccharides is often bound together with glycoside bonds.Glucuronic acid in polysaccharides can be free or produce small molecular structures with glucuronic acid by hydrolysis and other methods,which can be further separated and purified.Extraction of glucuronic acid by polysaccharide hydrolysis has the advantages of natural diversity and wide availability of raw materials,but there are few reports on the preparation of glucuronic acid from polysaccharides at present.In this thesis,tremella polysaccharides were used as raw materials to prepare,isolate,identify and purify Tremella glucuronic acid,and its antioxidant activity and protective activity against alcoholic liver injury in vitro were studied.The main contents and results of this research are as follows:（1）Optimization of the preparation process of Tremella glucuronic acidThe effects of temperature,time,substrate concentration,acid concentration and other single factors on the extraction rate of glucuronic acid from Tremella fuciformis polysaccharides during hydrolysis were studied by single factor test.Based on the results of single factor experiment,the process was optimized by orthogonal test design,and the optimum process parameters were obtained as follows:temperature 80 C,hydrolysis for 5 h,hydrochloric acid concentration 1 mol/L,and substrate concentration 0.6 mg/m L.After the hydrolysate was condensed and cooled,the tremella glucuronic acid was precipitated by 4 times anhydrous ethanol for 4 h.Under the optimal extraction conditions,the results were verified by three parallel experiments,and the average extraction rate was 32.03%±1.70%,which was basically consistent with the predicted results.（2）Separation and Purification of Glucuronic Acid from Tremella fuciformis BerkHigh performance liquid chromatography（HPLC）was used to detect gluconic acid in Tremella fuciformis extract.Through optimization of conditions,the selected chromatographic conditions were determined as follows:Inertsil ODS-3 C18（4.6 mm*250 mm）column,column temperature 30 C;mobile phase:acetonitrile:water（0.1%H₃PO₄）=10:90（volume ratio）,detection wavelength 220 nm,flow rate 1.0m L/min,injection volume 20μL.Under this chromatographic condition,the obtained standard curve equation of glucuronic acid is y=2475.9x-77.669,R²=0.9992,and the linear correlation is good.RSD of repeatability test peak area was 1.28%and 1.25%,average recovery was100.16%,RSD was 1.18%,RSD of stability test was 1.69%and 2.23%,respectively.It can be seen that this method is sensitive,accurate,reliable and suitable for quantitative analysis of glucuronic acid.The content of glucuronic acid in Tremella fuciformis accounted for about 28.57%by this method.The absorption peak of glucuronic acid in Tremella fuciformis was collected to obtain the product of glucuronic acid with higher purity.（3）Antioxidant effect of Tremella glucuronic acid in vitroIn vitro antioxidant test showed that the antioxidant activity of Tremella glucuronic acid was superior to that of Tremella Polysaccharide and glucuronic acid.When the concentration of Tremella glucuronic acid reached 5 mg/m L,its scavenging rates of hydroxyl radical（-OH）,ABTS radical and DPPH radical were 30.51%,99.40%and 82.01%,respectively.Combining the antioxidant test results of Tremella Polysaccharide and glucuronic acid,it can be seen that Tremella glucuronic acid has a slightly worse scavenging effect on hydroxyl radical（-OH）（p>0.05）,but very excellent scavenging effect on ABTS radical and DPPH radical（p<0.01）.（4）Protective effect of Tremella glucuronic acid on alcoholic liver injury in miceThe experiment of acute liver injury induced by alcohol in mice showed that TGA could significantly prolong the intoxication time,shorten the awake time（0.01<p<0.05）,inhibit the increase of liver index caused by alcohol,and have potential protective effects on alcoholism.Compared with the model group,the TGA high dose group could significantly reduce the levels of ALT,AST,MDA and ADH in the serum of mice（p<0.01）,and increase the levels of SOD,CAT and GSH-PX in the liver（p<0.01）,which was consistent with the pharmacological direction of biphenyl diester.The above results showed that TGA is similar to glucaldolactone on acute alcoholic liver injury（p>0.05）.