Isolation And Characterization Of Hyaluronic Acid Of Skin Mucus From Andrias Davidianus

Chinese giant salamander?Andrias davidianus?is one of the class II national protected animals in our country.The cultured giant salamander had appeared everywhere in China.The skin mucus from A.davidianus was mainly used to produce glycopeptides,but there was little research about hyaluronic acid?HA?contained in the skin mucus.In this paper,the purification,properties and toxicological safety of HA of skin mucus from A.davidianus were investigated,as well as the face pack was prepared.The data were theoretical basis for comprehensive utilization of skin mucus from giant salamander.The main results were as follows:The skin mucus from A.davidianus was used as materials for purifying HA.Precipitation at isoelectric point combined with ethanol and Sevag reagent was used to remove proteins.Enzyme dosage,time and temperature of hydrolysis as single factor assay were designed to investigate the effects on the yield of hyaluronic acid,and then orthogonal experiment were designed according to the results of the single factor assay to find the optimized levels.The crude HA from A.davidianus was purified by ion-exchange on Cellulose DE-52,eluted with 0-1 mol/L NaCl.HA molecular weight was calculated according to the standard curve of molecular weight with standard dextran on TSK-gel G4000 PW_XL column by HPLC.Characteristic group of the purified HA were determined by FI-IR.The purification results showed that the optimized levels were 1.5%of trypsin,pH8,4 h at 37?,with 1.7041 mg/g of yield.The molecular weight of purified HA was 4.85×10⁶ Da.The FT-IR of HA obtained with the purification method from the skin mucus was aligned with the standard hyaluronate obtained from Aladdin biochemical polytron technologies Inc.The antioxidant activities in vitro were investigated by scavenging free radicals and reducing Fe³⁺.The results of scavenging DPPH free radicals showed that the HA of skin mucus from A.davidianus had the activity of scavenging DPPH free radicals.The clearance rate of DPPH free radicals was 89.16%at the HA concentration of 17 mg/mL.And the IC₅₀ of scavenging DPPH free radicals was 8.13 mg/mL.The results of scavenging hydroxyl free radicals showed that the activity of scavenging hydroxyl free radicals increased with the HA concentration rising.The clearance rate of hydroxyl free radicals was 73.50%at the HA concentration of 20 mg/mL.And the IC₅₀ of scavenging hydroxyl free radicals was 12.10 mg/mL.The results of scavenging ABTS⁺free radicals showed that the ability of scavenging ABTS⁺free radicals of HA of skin mucus from A.davidianus increased with the rising concentration of HA.The clearance rate of ABTS⁺free radicals was 67.73%at the HA concentration of 15 mg/mL.And the IC₅₀ of scavenging ABTS⁺free radicals was 7.00 mg/mL.The results of reducing Fe³⁺showed that with the increase of HA concentration,the reducing power increased.The reducing power was 0.64 at 11 mg/mL of HA.Therefore,HA of skin mucus from A.davidianus had antioxidation somewhat in vitro.On the basis of"Hygienic standard for cosmetics",acute toxicity test on mice and acute percutaneous test on rats were conducted.During the period of acute oral toxicity test on mice,no obvious toxic symptoms were observed.The continuous observation for two weeks,no animal died in the whole process,and abnormalities were not found in the control group.The results of acute percutaneous test on rats showed that there were no obvious symptoms of poisoning when the rat back was covered with the HA.No rats died during the continuous observation of two weeks,and the control group was also normal.No skin irritation was observed in the skin.At the end of the experiment,foraging and breathing were normal.The fur was smooth,and no edema,erythema and desquamation were found in the skin.These results indicate that the effect of the HA is harmless to human body.Carboxymethyl cellulose,sodium alginate,polyethylene glycol 1500 as mask substrate,the recipe of HA mask was investigated by adding glycerol,Mitilan and essence.The results showed that,the mask with 6%HA has good moisturizing effect,stability and strong antioxidant activity in vitro,i.e.the ability of scavenging DPPH free radical,hydroxyl free radical,ABTS⁺free radical.