Preliminary Study On The Antiirritant Effect Of Sojae Sojae Preparatum And Its GABA Formation Mechanism

ObjectiveStudy the relationship between the antidepressant effect of Sojae Semen Praeparatum(SSP)and the content of?-aminobutyric acid(GABA),and to analyze the influencing factors of GABA enrichment during the processing of SSP,so as to scientifically explain the“eliminating effect”of SSP.And to lay the foundation for revealing the mechanism of GABA formation in the processing of SSP.Method1.In the early stage of the laboratory,the SSP processing method has been established according to the Chinese Pharmacopoeia.In this study,the SSP was prepared according to the processing method,and samples of different time points were obtained.2.Chronic unpredictable mild stress(CUMS)depression model was used to study the relationship between antidepressant effect and GABA content of SSP.(1)Determination of GABA content:The GABA content of samples at different time points during the processing of the SSP was determined by pre-column on-line derivatization high performance liquid chromatography(HPLC-OPA)established by our group.(2)Preparation of CUMS depression model:100 Kunming mice(KM)were randomly divided into blank group,model group,fluoxetine hydrochloride group,GABA group,black soybean group,and fermentation.6 days group,then boring 6days group,and then boring 15 days high,medium and low dose groups.10 in each group,half male and half female.Feed in each single cage.CUMS depression mouse model was randomly established by one or two stimuli per day for 28 consecutive days..Except the blank control group,the other groups were fed with single cage and the following chronic stress stimulation:fasting for 24 h,water for 24 h,moist litter,tail 1 Min(make the mouse whistling sound is appropriate),swimming at 45? for 3min,incline cage for 24 h,4? cold water stimulation for 3 min,shaking for 2 min,overnight illumination for 24 h,a total of 9 kinds of stimulation randomly selected one day Stress,sustained stress for 28 days,prevented the mice from predicting the stress to be administered.The specific arrangements are as follows:1st,11th,20th day is water-free;2nd,10th,21st,28th tail;3,12,19 days is wet padding;4th,13th,24th day is heat stimulation;5th 15,22,22 days for fasting;days 6,16,and 23 are oblique cages;days 7,14,and 26 are cold stimulation;days 8,18,and 27 are overnight illumination;and days 9,17,and 25 are oscillations.At the same time,the model was administered by continuous intragastric administration for 28 days,and the changes of behavioral indexes such as body weight,tail suspension,forced swimming,open box and sugar water preference were observed.3.Preliminary analysis of primary and secondary factors influencing GABA enrichment during the processing of SSPApplication of correlation analysis,multiple regression analysis and other statistical analysis methods to analyze the pH,temperature,moisture,protease(acid,medium and alkaline)and Glutamic acid decarboxylase(GAD)indicators in the processing of SSP.The primary and secondary status in the formation of GABA.(1)Detection of related indicators in the processing of SSP:pH,temperature,moisture,protease and glutamic acid were determined by using pH meter,thermometer,dry weight loss method,forinol method and Berthelot colorimetric method respectively.Dynamic changes of decarboxylase and other indicators were carried out.The dynamic changes of GABA during the processing of SSP were determined by pre-column on-line HPLC-OPA method.(2)Analysis of statistical methods:The GABA content of the samples at different time points in the processing process was taken as the ordinate,and the scatter plots were taken at the corresponding time points for pH,temperature,moisture,GAD and protease content as the abscissa,using SPSS21.0.The statistical software performs a correlation analysis between the two variables and performs linear regression.The Pearson correlation coefficient was used to evaluate the correlation between each index and GABA content,and the two-tailed test was used to evaluate the significant level.The linear regression model was evaluated by R~2,and the significance of the regression equation was evaluated by the F-test.On this basis,a multivariate regression equation is established.By comparing the absolute values of the coefficients of each factor,the primary and secondary status of each index in the formation of GABA is clarified.4.GAD Capability Analysis of GABA Microorganisms Produced by SSP.According to the main factors affecting the formation of GABA during the processing of the SSP,the ability of each dominant microorganism to produce GAD at different pH and temperature was studied.Result1.The SSP was prepared according to the SSP processing method established in the previous study,and the samples were obtained at different time points:black soybean(H)and“yellow clothes on the top”stage for fermentation for 0,3,and 6 days(respectively labeled as F0,F3,F6)“Re-stuffing”stage is 3,6,9,12,15 days(respectively recorded as Z3,Z6,Z9,Z12,Z15),finished product traits:black surface,uneven,soft,brown Black,full of gas,slightly sweet.The sample traits and physical and chemical indicators are in line with the requirements of the 2015 edition of the Chinese Pharmacopoeia.2.Antidepressant effect of SSP and its relationship with GABA content(1)GABA content in each drug group:According to the pre-established fermentation processing technology,the samples of different time points were obtained from the SSP,and the GABA content in the decoction liquid of some samples was determined.The pre-column on-line HPLC-OPA method established in the previous stage was used in black soybeans and fermentation for 6 days.GABA was not detected in the middle,and the GABA content of suffocating for 6 days and then boring for 15 days was 5.559 mg/g and 8.421 mg/g,respectively.(2)The relationship between antidepressant effect and GABA of SSP:The single-cage feeding plus chronic mild and unpredictable stress-replicating depression model was used.After 28 days of continuous administration,the behavioral indicators changed as follows:compared with the blank group,the body weight,syrup preference,cross-grid number and erect number of the model group were Decreased(p<0.01);the time of suspension and swimming immobility increase(p<0.05);indicating that the CUMS depression model was successfully constructed.Compared with the model group,body weight:there was no difference between the groups before the model establishment(p>0.05).After the model was established,the fluoxetine hydrochloride group,the GABA group,the boring group for 6 days,the nausea for 15 days,and the medium dose increased.There was no difference(p<0.05)between the black soybean group,the 6-day fermentation group and the 15-day low-dose group(p>0.05).The saccharification water preference was no difference between the groups before the model establishment(p>0.05).After the model was established,the positive drug group,GABA group,and the 6-day group(p<0.01)and the 15-day high and medium dose(p<0.05)increased.The black soybean group,the6-day fermentation group and the boring 15 There was no difference in the low-dose group(p>0.05).The open-box experiment showed no difference in the number of cross-grids and erects between the groups before the model establishment(p>0.05).After the model establishment,the positive drug group,the GABA group,the boring 6d group,the boring 15 d high,the middle dose cross-grid number increased There was no difference between the black-soy group,the 6-day fermentation group and the15-day low-dose group(p>0.05).The number of erects in the positive drug group,the GABA group(p<0.01),and the high-dose group(p<0.05)increased.The black soybean group,the 6-day fermentation group,the 6-day group,and the 15-day boring group Medium and low dose There were no differences in the groups(p>0.05).The tail suspension experiment:the positive drug group,the GABA group,the nausea group for 6 days,and the 15 days high and medium dose group were reduced(p<0.01),the black soybean group,the fermentation 6 days group and the boring 15 There was no difference in the low-dose group(p>0.05).Forced swimming experiment:the swimming time of each group was shortened(p<0.01).3.Primary and secondary factors affecting GABA enrichment during the processing of SSP.(1)Dynamic changes of various indexes during the processing of SSP:(1)GABA content:Before the processing,the raw black soybeans contained trace amounts of GABA,and GABA was not detected in the“Yellow Clothes Up”stage.After washing the yellow clothes into the“re-suffocation”stage,the GABA content gradually increased,and the content was stable until 15 days.(2)pH value:During the processing of the SSP,the pH value is generally weaker and the acidity increases first and then decreases.(3)temperature:The temperature of the processing system is gradually decreasing,and the temperature is the highest in the initial stage of“Yellow Clothes Up”.(4)moisture:The water gradually decreases in the"Yellow clothing over"stage,and gradually increases after entering the boring stage.(5)protease:During the processing of the SSP,the acid,medium and alkaline proteases showed a trend of decreasing first,then increasing and then decreasing.the acid protease is relatively weak,acidic,neutral protease and alkaline The highest proteases were 11.88 U/g,116.56 U/g and 69.20 U/g,respectively.(6)GAD:GAD activity showed a decrease first and then increased,and the activity was as high as 17.64 U/g when it was further boring for 15 days.(2)The primary and secondary factors of GABA enrichment in the processing of SSP:the correlation coefficient between water and acid protease and GABA in the processing of SSP was 0.211 and-0.340,respectively,and the P values were 0.324and 0.228,respectively,with little correlation and no correlation.Statistical differences;comparing the absolute values of regression coefficients,the primary and secondary status of other indicators in GABA formation is pH(-0.375)>temperature(-0.284)>GAD(0.140)>alkaline protease(0.047)>Protease(-0.030),in which pH,temperature and neutral protease have a negative correlation with GABA,GAD activity and alkaline protease have a positive correlation with GABA.4.GAD ability of GABA microbes produced by SSP:Bacillus subtilis Xd3(Bacillus subtilis)and Enterococcus faecium15R3(Enterococcus avium)produced the highest GAD activity at pH=6.0 and 34?,and the GAD activity reached 19.43 U./h and11.18 U/h;while Enterococcus faecium15r1(Enterococcus faecium)had the highest GAD ability at pH 5 and 31?,and the enzyme activity reached 3.41 U/h;The Mycobacterium elegans ME4(Cladosporium cladosporioides)produced a strong GAD at a pH of 4 and a temperature of 28?,and the enzyme activity was 11.50 U/h;both Aspergillus flavus MB2(Aspergillus flavus)and Penicillium citrinumME3(Penicillium citrinum)were at pH 7.When the temperature was 28?,the highest yield of GAD was obtained,and the enzyme activities were 41.97 and 3.44 U/h,respectively.The Aspergillus niger JD2(Aspergillus niger)was the best for GAD at pH 7 and 31?,and the enzyme activity was 25.78 U/h.Aspergillus MA3is the most suitable for GAD at pH 8 and 28?,and the enzyme activity is 12.49 U/h.White rot fungus MG1(Aspergillus tamarii)is the most suitable for GAD at pH 6 and 25?.Up to 15.74 U/h.Conclusion1.The finished product traits and physical and chemical indicators of the SSP prepared according to the previous establishment of the light soybean meal processing method are in line with the requirements of the 2015 edition of the Chinese Pharmacopoeia.2.The results showed that the SSP had a better effect on the depression behavior of the model mice.The antidepressant effect was related to the GABA content of the sample,which provided a basis for further clarifying the material basis and mechanism of the effect of removing the anesthetic effect of the SSP.3.The pH,temperature,GAD,neutral and alkaline proteases in the proces sing of the SSP have an important effect on the formation of GABA.The ord er of the primary and secondary is pH>temperature>GAD>alkaline protease>neu tral protease.4.The conditions for the optimal production of GAD in different dominant strains during the processing of the SSP are also different.The optimum conditions for GAD of each strain are consistent with the natural processing conditions of SSP.,in order to further clarify the processing of the SSP.The mechanism for the dynamic change of GABA in the process lays the foundation.