Study On Extraction,purification And Antioxidant And Antibacterial Activities Of Flavonoids From Brasenia Schreberi

Brasenia schreberi is a perennial aquatic vegetable of the Cabombaceae family.It was originally widely distributed in most regions of the northern hemisphere.It is now mainly distributed in China,Japan,North America and eastern Oceania.The wild species is endangered,and it is mainly distributed in Chongqing Shizhu,Hubei Lichuan,Sichuan Leibo and Zhejiang West Lake in China.Brasenia schreberi is a traditional Chinese medicinal and edible plant.It is rich in polysaccharides,proteins,polyphenols,trace elements and vitamins and other active ingredients.It has antibacterial,anti-inflammatory,anti-tumor,and hypoglycemic effects.In recent years,researchers at home and abroad have done more scattered and not deep studies on Brasenia schreberi,mainly focusing on cultivation management,morphology,anatomy,and eating,and various aspects of research have not yet formed a system.There are few basic researches on the active substances of Brasenia schreberi,while Brasenia schreberi flavonoids research on similar substances is even less.In this study,the four major producing areas of Brasenia schreberi as the test materials,the extraction solvents with the best extraction effect of Brasenia schreberi flavonoids were screened first,and then the extraction and purification process of Brasenia schreberi flavonoids was optimized,and then high performance liquid chromatography（HPLC）was used to qualitative and quantitative analysis of the composition and content of flavonoids in Brasenia schreberi in each producing area,and the antioxidant and antibacterial activities of flavonoids in Brasenia schreberi in each production area were determined.Finally,analyze the correlation between the composition and content of Brasenia schreberi flavonoids and the antioxidant and antibacterial activities,and determine the main antioxidant and antibacterial components in the flavonoids.The main research results are as follows:（1）By comparing the extraction effect of ethyl acetate,acetone,anhydrous methanol,anhydrous ethanol and distilled water on the extraction of flavonoids from Brasenia schreberi,it is found that the extraction effect of methanol is better than that of the other four extraction solvents.Therefore,methanol is used as the extraction solvent.On the basis of single-factor experiments,the total flavonoids extraction process of Brasenia schreberi was optimized through the response surface Box-Behnken design.The results were:methanol volume fraction 90%,liquid-to-material ratio 40:1.0（mL:g）,ultrasonic time 54 min,ultrasonic temperature58℃,under this process,the extracted content of total flavonoids of Brasenia schreberi is 46.56±0.030 mg/g.（2）By comparing the adsorption rate and desorption rate of 10 kinds of macroporous resins to the total flavonoids of Brasenia schreberi,it is found that the AB-8 type macroporous resin has the best overall effect.Therefore,the AB-8 type macroporous resin was selected,and the purification process of the total flavonoids of Brasenia schreberi was optimized through the orthogonal experiment design.The result of the purification process is:loading concentration 0.5 mg/mL,loading flow rate 1.0mL/min,loading p H=2,eluent dosage 40 mL,eluent concentration 70%,elution rate 1.0mL/min.Under this process,the total flavonoid content in the crude extract of Brasenia schreberi flavonoids increased from 6.27%before purification to 22.65%,which is about 3.6 times that before purification.（3）The total flavonoids content of Brasenia schreberi in the four major producing areas is between（20.137±1.658 mg/g）and（46.593±1.642 mg/g）.Among them,the total flavonoid content of Shizhu Brasenia schreberi is the highest,Lichuan（43.586±1.397mg/g）and Leibo（28.525±1.741 mg/g）followed by,the West Lake Brasenia schreberi the lowest content.Using 12 standard flavonoids,a total of 7 flavonoids were detected from the methanol extract of Brasenia schreberi by HPLC,which were isorhamnetin,quercetin,kaempferol,eriodictyol,luteolin,orientin and apigenin.The content was（0.36±0.01 mg/kg）～（367.53±3.82 mg/kg）,the main components are isorhamnetin and quercetin.（4）Brasenia schreberi flavonoids in the four major producing areas have certain antioxidant activities,and the scavenging rates of DPPH free radicals and ABTS free radicals are above 85%and 95%,respectively.The corresponding IC₅₀ values of the corresponding semi-scavenging concentrations are below（0.1055±0.001 mg/mL）and（0.0543±0.006 mg/mL）respectively,and the scavenging rates of these two free radicals are greater than ascorbic acid;The scavenging rates of hydroxyl free radicals and superoxide anion free radicals are above 55%and 59%,respectively.The IC₅₀ values of the half-scavenging concentration are below（0.3233±0.008 mg/mL）and（0.1824±0.004mg/mL）respectively,and the scavenging rates for these two types of free radicals were less than ascorbic acid.The antioxidant APC index indicated that the antioxidant activity of Brasenia schreberi flavonoids differed in production areas,and the order of their sizes was:Shizhu>Lichuan>Leibo>West Lake.（5）Brasenia schreberi flavonoids in the four major producing areas have different levels of inhibitory activity against 5 bacteria and 4 fungi.The antibacterial APC index shows that the order of the difference in antibacterial activity is:Shizhu>Lichuan>Leibo>West Lake.On the whole,in the process of bacterial inhibition,Shizhu Brasenia schreberi is moderately sensitive to Escherichia coli（15.66±0.803 mm）and Proteus（13.23±1.358 mm）and Lichuan Brasenia schreberi is moderately sensitive to Escherichia coli（13.75±1.894mm）.In addition,the others are all low sensitivity;In the process of fungi inhibition,it was found that the antibacterial activity against Mucor and Aspergillus niger was strong,and the antibacterial rate was over 72%and 55%,respectively.The antibacterial activity against Penicillium Secondly,the antibacterial activity against Fusarium is the weakest,with the antibacterial rate below 41%.The minimum inhibitory concentration（MIC）showed that the MIC value of Brasenia schreberi in Shizhu,Lichuan and Leibo 3 producing areas had no difference in the production area,but all were less than the MIC value of West Lake,and the MIC value of bacteria was less than that of fungi as a whole.It shows that the bacteria are more sensitive to Brasenia schreberi flavonoids than fungi.（6）The analysis of the correlation between Brasenia schreberi flavonoids and antioxidant and antibacterial activities showed that the total flavonoids and 7 flavonoids all have different degrees of promotion in the antioxidant and antibacterial activities,and the correlation coefficient between the antioxidant activity is between 0.518 and0.997,the correlation coefficient with antibacterial activity is between 0.407 and 0.999.In the antioxidant process,total flavonoids（r=0.986）,isorhamnetin（r=0.966）and orientin（r=0.979）play an important role in the scavenging process of DPPH free radicals.Luteolin（r=0.997）is a key factor in the scavenging process of hydroxyl free radicals.Total flavonoids（r=0.970）and orientin（r=0.952）play an important role in the scavenging process of superoxide anion free radicals.In the antibacterial process,total flavonoids,isorhamnetin and orientin play a major role in the antibacterial process of various bacteria and fungi.In addition,quercetin（r=0.999）and kaempferol（r=0.998）and luteolin（r=0.991）play a key role in the bacteriostatic process of Proteus,quercetin（r=0.992）and luteolin（r=0.999）play a key role in the bacteriostatic process of Bacillus subtilis.Kaempferol（r=0.958）plays an important role in the antibacterial process of Fusarium.