Preparation And Bioactivity Evaluation Of Aglycone By Combined Method

The structure of soybean isoflavones includes free type and bound type.The free aglycone mainly plays a role in biological activity.At present,the content of aglycone in soybean isoflavone products which are used as industrial raw materials is low.Therefore,in this study,we choose the commercial soybean isoflavone powder as raw material,and then the combined techniques of frying,alkali,acid,ultrasound and enzymatic hydrolysis were used to hydrolyze the soybean isoflavone powder to improve the conversion rate of aglycone.After the enzymatic hydrolysate was purified by activated carbon and macroporous resin,the solvent was removed by evaporation,and then vacuum freeze-drying was performed to prepare high purified aglycone.The prepared aglycone was used to intervene ovariectomized rats with osteoporosis to evaluate the biological potency and dose-effect relationship of aglycone.(1)We chose the commercial soybean isoflavone powder as raw material,and the combined teniques of frying,alkaline hydrolysis and acid hydrolysis were performed to hydrolyze the soybean isoflavone powder.The optimal conditions of acid hydrolysis were p H4.1,4 h and 78℃,which were determined by the response surface analysis.The total conversion rate of aglycone after acid hydrolysis reached 45.04%.Subsequently,the ultrasonic method was used to continue the hydrolysis.The optimal conditions of ultrasonic hydrolysis were 200 W,2 h and 60℃,determined by response surface analysis.The total conversion rate of aglycone based on acid-ultrasonic hydrolysis reached 54.11%.Our data demonstrated that acid hydrolysis is an effective way to transform glucoside into aglycone.Furthermore,ultrasound hydrolysis could improve the conversion rate of aglycone.(2)β-glucosidase was used to further hydrolyze aglycone after frying,alkali,acid,and ultrasonic hydrolysis.The optimal conditions of enzymatic hydrolysis were enzyme 1%(97.13U),40℃ and 4 h,determined by orthogonal experiment.The total conversion rate of aglycone by enzymatic hydrolysis was 67.43%.The results showed that enzymatic hydrolysis could effectively increase the conversion rate of aglycone.The total conversion rate of aglycone by the above 5 steps was much higher than that of pre-treatment.(3)The enzymatic hydrolysate obtained from the above five-step hydrolysis was decolorized with activated carbon.The optimal conditions for decolorization were 2 h,35℃and 9 g,determined by orthogonal experiment.The decolorization rate was 54.49%.Afterwards,the macroporous resin which was the most suitable for the purification of aglycone was screened by dynamic adsorption-elution test.The optimal purification conditions were as follows: sample concentration 1.0 mg/m L,sample solution p H 5,adsorption time 3 h,eluent flow rate 1 min/m L,eluent p H 5,desorption solution dosage 80 m L,desorption solution concentration 70%,desorption solution p H 5.5 and desorption time 2 h.Under this situation,the adsorption rate,elution rate and desorption rate of AB-8 macroporous adsorption resin was72.83%,94.53% and 78.45%,respectively.The results showed that the weak polar AB-8macroporous resin was the most suitable for purification of aglycone.After purification by AB-8 macroporous resin,the purity of aglycone was increased from 34.69% to 61.84%.(4)Aglycone was used to intervene ovariectomized rats with osteoporosis to evaluate its effect on osteoporosis.The ovariectomized rats were intragastric administrated with low,middle and high dose aglycone for 60 days.The results showed that the body weight of rats with weight gain was significantly decreased in aglycone middle and high dose groups.And the uterine atrophy of rats,serum bone markers,blood lipids and trabecular microstructure parameters were also significantly improved.The levels of serum TC,TG and serum bone markers(Str ACP,BGP and NTX-I)decreased significantly(P<0.05),while the levels of serum bone markers(Ca,P and BAP)increased significantly(P<0.05).Meanwhile,the microstructure parameters of bone trabeculae(BMD,Tb.Th,BV/TV and Tb.N)increased significantly(P<0.05),and the level of trabecular separation(Tb.Sp)decreased significantly(P<0.05).Furthermore,the effects of aglycone on osteoporosis were all dose-dependent.The biological potency of aglycone was significantly higher than that of mixed soybean isoflavones.