Research On Preparation,Analyses Of Phenolic Acid From Agaricus Bitorquis(Quél.) Sacc.

Agaricus bitorquis(Quél.)Sacc.is one of the rare edible fungi endemic to theQinghai-Tibet Plateau.It has the characteristics of low fat,high carbohydrate,protein and rich amino acid composition,which has high research value.At the present stage,the research on Agaricus bitorquis(Quél.)Sacc.mainly focuses on polysaccharides,while the study on phenolic acid has not been reported in relevant literature,and phenolic acid has good antioxidant,anti-cancer,anti-inflammatory and other activities.In this paper,phenolic acids were extracted from fruiting bodies,intracellular and extracellular of Agaricus bitorquis(Quél.)Sacc.,and the extraction and purification processes were optimized,after separation and purification,the mixture system of fruiting body phenolic acid,intracellular phenolic acid and extracellular phenolic acid were obtained,and the structure characteristics of the three mixed systems were identified,the antioxidant,bacteriostatic and anti-anoxic activities of the mixed systems of the three phenolic acids were studied in vitro.The main contents of the study are as follows:(1)The ultrasonic-assisted extraction of phenolic acid from fruiting body,intracellul ar and extracellular broth of Agaricus bitorquis(Quél.)Sacc.was studied.The optimum conditions were determined by single-factor and response surface tests.The optimum extraction conditions of phenolic acid from fruit body were determined:ethanol concentration 80%,ultrasonic power 130W,liquid/solid ratio 1:50,ultrasonic temperature 50°C.Under these conditions,the maximum predicted yield of phenolic acid was 19.59 mg/g,the yield of phenolic acid was(19.51±2.14)mg/g,which was close to the predicted value.The optimum extraction conditions of phenolic acid from intracellular were ethanol concentration 90%,ultrasonic power130W and extraction temperature 61°C.Under these conditions,the maximum yield of phenolic acid was predicted to be 39.02 mg/g,the yield of phenolic acid was(39.09±2.14)mg/g,which was close to the predicted value,and the optimum extraction conditions of phenolic acid from extracellular were determined:ethanol concentration80%,ultrasonic power 110W,extraction temperature 50°C.Under these conditions,the maximum yield of phenolic acid was predicted to be 36.93 mg/m L.The yield of phenolic acid was(36.52±1.14)mg/m L,which was close to the predicted value.Therefore,it is reasonable and reliable to optimize the extraction process of these three phenolic acids by single factor and response surface methodology.(2)The adsorption capacity and resolution of D101,X-5,PHD101 and AB-8macroporous resins were compared by static adsorption-resolution test.D101macroporous resin was selected as the packing material for phenolic acid column chromatography of fruiting body and intracellular,X-5 macroporous resin was selected as packing material for phenolic acid column chromatography of extracellular,and the purification process of these three phenolic acids was optimized,to determine the best purification process.The optimum purification conditions of fruit body phenolic acid were determined:loading concentration 20 mg/m L,loading volume 0.3BV,loading flow rate 3 m L/min.Under these conditions,the recovery rate of fruit body phenolic acid was increased from(13.26±0.46)%to(26.26±0.01)%.The optimum purification conditions of intracellular phenolic acid were determined:loading concentration 20 mg/m L,loading volume 0.2 BV,loading flow rate 4m L/min.Under these conditions,the recovery rate of intracellular phenolic acid was(14.16±4.62)%to(40.26±0.01)%.The optimum purification conditions of extracellular phenolic acid were determined:loading concentration 40 mg/m L,loading volume 0.2BV,loading flow rate 4 m L/min.Under these conditions,the recovery rate of phenolic acid in fermentation broth was increased from(46.75±6.83)%to(70.66±0.48)%.(3)The structure characteristics of the mixed system of intracellular,extracellular and fruiting body phenolic acid were analyzed by using UPLC-Q-TOF-MS,the results showed that the extracellular phenolic acid contained(-)-epicatechin gallate,arabelline,yunnan acid D,2'-o-o-p-hydroxybenzoyl-6'-o-trans-caffeine,4'-O-methylgallocatechin-(4->8)-4'-O-methylepigarabellineallocatechin;The intracellular phenolic acid contains baicalein-7-O-sulfate,ynnan acid D,rhaponticin-2"-gallate,4'-O-methylgallocatechin-(4->8)-4'-O-methylepigallocatechin,6-methoxykaempferol-3-O-(6-E-feruloyl)-?-D-glucopyranoside,2'-O-p-hydroxybenzoyl-6'-O-trans-caffeoyl gardoside and arabelline.The composition of phenolic acid was not detected in the mixture of extraction and purification of fruiting body.(4)The antioxidant activities of the three purified phenolic acid mixtures were evaluated by using 5 in vitro antioxidant systems including reducing capacity,DPPH radical scavenging capacity,ABTS radical scavenging capacity,hydroxyl radical scavenging capacity and superoxide anion radical scavenging capacity.Using Escherichia coli,Staphylococcus aureus and Bacillus subtilis to perform in vitr o antibacterial tests on the three purified phenolic acid mixed systems.The results showed that only phenolic acid had bacteriostatic activity.The scavenging capacity of DPPH free radical in intracellular phenolic acid(IPA)was(79.84±0.76)%.It was higher than Vc group(4.31%),fruiting body phenolic acid(FBPA)group(9.46%)and extracellular phenolic acid(EPA)group 26.27%.And the half inhibitory concentrations in IPA group was 0.61 mg/m L.The half inhibitory concentrations in FBPA group was 0.93 mg/m L.The IC₅₀ values in EPA group was 1.04 mg/m L.The results of ABTS free radical scavenging rate showed that the highest scavenging rate was(75.82±0.69)%in IPA group at high concentration.It was higher than Vc group(3.69%),FBPA group(19.37%)and EPA group 15.73%.the IC₅₀ values were 0.73mg/m L,0.77 mg/m L,1.02 mg/m L and 0.98 mg/m L.The scavenging rate of hydroxyl radical in IPA group was(78.60±0.55)%.It was higher than that FBPA group(39.86%)and EPA(47.74%).The IC₅₀ values were 0.67 mg/m L,0.49 mg/m L,1.27 mg/m L and 1.42 mg/m L.The results showed that the scavenging rate of superoxide radical in IPA group was(84.30±2.74)%.It was higher than that FBPA group(15.01)and EPA(2.93%).The IC₅₀values were 0.33 mg/m L,0.33 mg/m L,0.98 mg/m L and 0.79 mg/m L.The reduction ability results showed that the reduction ability of the IPA group was close to that of the Vc group,with a reduction ability of 1.23±0.01,which was higher than the FBPA group(304.59%)and EPA group(66.53%).When the concentration of extracellular phenolic acid is 1200mg/m L.Its antibacterial effect on Bacillus subtilis is the most ob vious,and the inhibition zone is also(24.17±0.17)mm;the minimum inhibitory conce ntration of extracellular phenolic acid against Bacillus subtilis(MIC)and minimum bactericidal concentration(MBC)are the lowest,respectively 18.75mg/m L and 75.00mg/m L.In summary,the three phenolic acids after purification have obvious antioxidant effects.(5)In this experiment,the best hypoxic conditions,best drug treatment conditions a nd best drug activity were determined by CCK-8 detection.Apoptosis rate,Western bl ot,and ELISA methods were used to analyze the purified fruit body phenolic acid,intracellular phenolic acid and extracellular phenolic acid's anti-hypoxia activity.The CCK-8 test results show that the conditions for drug treatment are the best when the oxygen concentration is 4%and the treatment is 24 hours,and the activity of mycelial phenolic acid is the best,and its optimal concentration is 200?g/ml;In the cell apoptosis experiment,the structure characterization and anti-hypoxic activity analysis of the extracellular phenolic acid on PC-12 cells showed that:under 4% hypoxia conditions,intracellular phenolic acid concentration was 250?g/m L,The cell survival rate is the best;when the intracellular phenolic acid concentration was 250?g/m L,the cell morphology was uniform,the cells were evenly distributed,and the cell protection was the best;when the intracellular phenolic acid was 250?g/m L.It was significantly reduced hypoxia-induced PC-12 cell damage by reducing the excessive secretion of ROS in the cell,increased??m and reduced the rate of apoptosis;Western blot technology was used to analyze the relative expression of intracellular phenolic acid on certain proteins in PC-12 cells,such as EPO,FIH,HIF-1?,PHD-1,i NOS,VEGF and VHL,the results showed that:Under conditions,intracellular phenolic acid has high expression of FIHP,HD-1,VHL,EPO,and VEGF protein in PC-12 cells,and the expression of i NOS and HIF-1?decreases,indicating that intracellular phenolic acid can regulate related proteins.Improve the effect of hypoxia capacity of PC-12 cells;ELISA method was used to analyze the changes in LDH content of PC-12 cells treated with mycelial phenolic acid.The results showed that:under hypoxic conditions,after PC-12 cells were treated with intracellular phenolic acid group,their LDH content decreased significantly,indicating that the intracellular phenolic acid can enhance the anti-hypoxia ability of PC-12 cells.In conclusion,the phenolic acid Agaricus bitorquis(Quél.)Sacc.in has good anti-hypoxia activity.