Study On The Preparation And Antioxidant Activities Of Proteins And Enzymatic Peptides From Oviductus Ranae

Oviductus ranae is a dried product of oviduct of female chensinensis.It is a Chinese medicinal material that has been included in PRC Pharmacopoeia.Protein is the main active ingredient in Oviductus ranae,accounting for more than 50%.Protein extracts from Oviductus ranae?PEOR?has antioxidant,anti-fatigue,anti-aging and enhancing immunity activities.Therefore,in order to systematically study the PEOR,based on active retention,combining with antioxidant activity tracking,the extraction technology of PEOR was studied and optimized firstly,and its antioxidant activity and antifatigue effect were further investigated both in vitro and in vivo.It was found that PEOR exerts higher antioxidant activity in vivo than that of in vitro,which indated that the digestive products of PEOR may be the real antioxidant substances in vivo,thus we simulated the process and procedure of protein digestion in vivo to prepare the enzymatic peptide from Oviductus ranae by sequential hydrolysis of pepsin and trypsin,the amino acid composition and antioxidant activity were also studied.PEOR was extracted by water extraction combined with the monitoring of DPPH clearance,five single-factor indexes including pH value,ratio of material to liquid,extraction time,extraction times and salt content of medium were determined to optimize the extraction process and obtain the optimum extraction conditions as follows:phosphoric acid buffer solution?containing 0.5 mol/L NaCl?at pH 6.5 was extracted twice at the ratio of material to liquid 1:150 for 12 hours each time;Under these conditions,the extraction rate of PEOR was 12.5%,and the protein content was60.7%by Coomassie brilliant blue method;The molecular weight distribution of PEOR was determined by SDS-polyacrylamide gel electrophoresis.It was found that PEOR contained three monomeric proteins in the range of 44.3 kDa¹16 kDa.The composition of amino acids in PEOR was also analyzed.The results showed that there are 17 amino acids present in PEOR,7 of which are essential amino acids,accounting for 41.9%.The total content of amino acids was 668.3 mg/g.The antioxidant activity of PEOR in vitro was evaluated by using hydroxyl radical scavenging rate,DPPH radical scavenging rate,superoxide anion scavenging rate and total reducing power as the in vitro antioxidant evaluation models and VC as positive control.The results showed that the IC₅₀0 values of PEOR scavenging hydroxyl radicals,DPPH radicals and superoxide anions were 4.85 mg/mL,4.98mg/mL and 2.58 mg/mL,respectively.The reduction capacity of Fe³⁺was 11.8mg/mL when the A_{700 nm}00 nm value was 0.2.Malondialdehyde?MDA?,glutathione?GSH?,superoxide dismutase?SOD?and protein carbonyl?PC?were used as antioxidant biomarkers and VC as positive control to evaluate the in vivo antioxidant activity of PEOR.The results showed that PEOR can decrease the contents of MDA and PC in mice,and increase the levels of GSH and SOD in a dose-dependent manner.Among them,400 mg/kg of PEOR has significant anti-fatigue effect in weight-bearing swimming mice.It can prolong the swimming time of mice with weight-bearing fatigue and reduce the increase of blood urea nitrogen?BUN?and blood lactic acid?BLA?caused by strenuous exercise.The preparation conditions of enzymatic peptide from Oviductus ranae by sequential hydrolysis of pepsin and trypsin were as follows:defated Oviductus ranae was added 50 times of artificial gastric juice,hydrolyzed for 2 hours,adjusted pH to8.5,hydrolyzed for 4 hours again,and quickly inactivated by high temperature in water bath.Under these conditions,the extraction rate of enzymatic hydrolysis of Oviductus ranae was 31.5%.The antioxidant activity of enzymatic hydrolysate of Oviductus ranae was studied by using hydroxyl radical scavenging rate,DPPH scavenging rate,superoxide anion scavenging rate and total reducing power and VC as positive control.The results showed that the IC₅₀0 values of hydroxyl radical scavenging,DPPH scavenging and superoxide anion scavenging were 3.96 mg/mL,3.25 mg/mL and 2.58 mg/mL respectively.The reduction ability of the enzymatic hydrolysate of Rana Rana oil to Fe³⁺was 11.8 mg/mL at the A_{700 nm}00 nm value of 0.2.In conclusion,the antioxidant effect of PEOR in vivo is better than that in vitro,and the antioxidant effect of enzymatic peptid es from Oviductus ranae is better than that of PEOR.