Study On The Separation And Determination Of Polyphenol From Sanguisorba Officinalis L.,and Its Antibacterial Action

Sanguisorba officinalis L.is widely distributed in China.It is a substance with the same origin as medicine and food.It has coagulation,anti-ulcer,anti-oxidation,anti-allergy,anti-tumor,antidiarrheal and antibacterial abilities.Polyphenolic compounds are the main active ingredients in S.officinalis L.,and its antibacterial ability has high potential application value in the pharmaceutical industry.On the purpose of applying S.officinalis L.polyphenol extracts on the preservation of food,this study aimed at the extraction and purification process of S.officinalis L.polyphenols,identifying the polyphenolic components,and comparing the inhibitory effects of S.officinalis L.polyphenols before and after purification.At the same time,the mechanism of the inhibition on Bacillus subtilis by purified polyphenolic extract(PPE)was further explored.The results provide the theoretical support for the research and application of S.officinalis L.polyphenols as natural preservatives,and further improve S.officinalis L.economic and application value in the food industry.The main research results are as follows:1.After the pretreatment of S.officinalis L.,the crude polyphenolic extract(CPE)are attained by ultrasound-assisted method and purified by macroporous resin method.Purification results showed that Under the conditions of a loading concentration 1 mg/mL,a loading volume 380 mL,a loading flow rate 2 mL/min,an eluent 70%ethanol,an eluent volume 160 mL,and an elution flow rate 2 mL/min.,the purity of S.officinalis L.polyphenols increased from 10.8%to 33.89%.2.Use HPLC-ESI-QTOF-MS/MS technology to identify the components of PPE.The mobile phase consists of 0.2%formic acid aqueous solution(A)and methanol(B).A total of 44 compounds were identified,including 23 phenolic acids,21 flavonoids and their derivatives.The main components were 10 gallic acids,8 ellagic acids,11 catechins and their derivatives.3.The inhibition zone(DIZ),the minimum inhibitory concentration(MIC)and the minimum bactericidal concentration(MBC)experiments were conducted to determine the CPE and PPE on Gram-positive bacteria(Staphylococcus aureus,B.subtilis,and Listeria monocytogenes)and Gram-negative bacteria(Escherichia coli,Salmonella typhimurium).The results showed that the DIZs of the PPE for all three Gram-positive bacteria were greater than 24 mm,while the maximum DIZ of CPE was only 15.18 ±0.29 mm.For two Gram-negative bacteria,PPE also significantly increased the DIZs(10.90 ± 0.33 mm and 14.73 ± 0.27 mm).Similarly,the MICs of PPE against three Gram-positive bacteria were less than 1.56 mg/mL,the MBCs were less than 6.25 mg/mL,while the MIC and MBC of CPE are up to 6.25 mg/mL and 25 mg/mL,respectively.Therefore,this study demonstrated that the antibacterial effect of PPE was significantly higher than that of CPE(P<0.05),and the inhibitory effect on Gram-positive bacteria was greater than that of Gram-negative bacteria.4.This research further studied the antibacterial mechanism of PPE against B.subtilis.The growth curve experiment showed that PPE at MIC could shorten the rapid growth period of B.subtilis,while at MBC,the growth of B.subtilis was completely inhibited.The apoptosis experiment qualitatively proven the antibacterial ability of PPE.At MIC and MBC,the apoptosis rate reached 60.75±0.03%and 96.54 ± 0.02%respectively.Fourier transform infrared spectroscopy experiment analyzed the changes of fatty acids,peptides,proteins,polysaccharides and intracellular nucleic acids on the cell wall membrane of B.subtilis.Compared with the control group,the spectrum of the characteristic absorption regions of these five substances in the treated group changed significantly,thus suggesting that PPE changed the characteristics of the cell wall membrane of B.subtilis.The results of cell membrane integrity and permeability experiments further showed that,at the MIC,PPE caused more serious damage to the B.subtilis membrane integrity than at the MBC.On the contrary,the fatty acid profile measurement results showed that the PPE at the MBC significantly changed the ratio of anteiso-and iso-branched phospholipid fatty acid(PLFA)from 64.17±0.28%(control group)and 27.23±0.03%(control group)to 5.57±1.06%and 6.00±1.40(P<0.001),which reduced the cells fluidity.However,the PLFA composition of the MIC group was not significantly different from that of the control group,indicating that PPE can inhibit B.subtilis in different ways at different concentrations.Finally,scanning electron microscopy directly confirmed that PPE could destroy the integrity of the cell membrane and inhibit cell growth at a lower concentration while at a higher concentration,it could change the membrane fluidity,which hindered the cell membrane from exerting the normal function.