Study On Component Analysis,Biological Activity And Antibacterial Mechanism Of Different Extracts From Hawthorn(Crataegus Pinnatifida)

Hawthorn(Crataegus pinnatifida),also known as the mountain fruit,mountain red,red fruit,is one kind of medicinal and edible fruit.It has the following efficacy such as lowering blood pressure,promoting good digestion,dissipating blood stasis,promoting circulation of blood&phlegm,and etc.Taking the local hawthorn as experimental materials,the chemical compositions,biological activities of different solvent extracts from hawthorn and its antibacterial mechanism were investigated in this paper,which provide a reference for the development of natural antioxidants and bacteriostatic agents,and has very important significance to promote economic development of hawthorn and related industry.The main results are as follows:(1)There were significant differences in contents of total phenolics and flavonoids among hawthorn extracts with different polar solvents.The content of total phenolics and flavonoids in methanol extracts was the highest with 61.84 mg RE/g and 38.40 mg GAE/g respectively,followed by EE and AE,while the EAE,TE and PE had a low level of total phenolics and flavonoids.(2)The 10 compounds including catechin,terephthalic acid,chlorogenic acid,quercetin,hyperin,epicatechin,proanthocyanidin B2,isoquercitrin,caffeic acid and luteolin glucoside were identified in hawthorn.The result indicated that the compounds and concentrations in six extracts were significantly different.Among ten compounds,all compounds were found in ME,EE and AE,and exhibited the higher contents compared to other extracts.Other compounds except isoquercitrin and luteolin glucoside were found in EAE.Quercetin,isoquercitrin and luteolin glucoside were not detected in TE while PE had proanthocyanidin B2,epicatechin and hyperin.(3)The antioxidant effects of different solvent extracts were significantly different.ME and EE exhibited good antioxidant activity in different antioxidant systems such as Fe³⁺reducing power system,total reduction capacity,DPPH&ABTS free radical scavenging activity,protective capability by·OH mediated protein and DNA damage.Antioxidant ability of other extracts showed that the order from high to low was AE,EAE,TE and PE.(4)The antibacterial activity of the extracts varied greatly with the extraction solvent.ME,EE and AE had a broad spectrum of antimicrobial activity,all of them had antibacterial effects on seven tested bacterial.With increase in the concentrations of extracts,its effects were obvious.EAE exhibited antibacterial effects on Staphylococcus aureus,Bacillus subtilis,Escherichia coli,Salmonella typhimurium,while no inhibitory effect was found against other tested trains.The result also showed that TE and PE had no inhibitory effect on tested strains.ME of all extracts exhibited the best antibacterial activity against tested bacterial,especially S.aureus(G⁺)and S.typhimurium(G^-).(5)After the treatment with ME,the cell membrane potential of S.aureus and S.typhimurium were increased,it means that ME caused membrane depolarization;Results from SEM showed that ME altered the morphology of S.aureus and S.typhimurium,changed the surface of the bacteria and eventually led to the death of the bacteria;The results of TEM showed that the cell wall and membrane,intercellular substance of the bacteria were damaged,the cytoplasm was non-uniformity,leakage of intracellular substances,and the vacuoles and clefts structure were discovered in cytoplasm of S.aureus and S.typhimurium;The relative electrical conductivity and macromolecules content of bacterial suspensions increased.(6)ME exhibited a certain suppressive effect on intracellular enzyme activity such as ATPase,SOD and CAT.ATP contents decreased after ME treatment,which significantly disturbed normal energy metabolism in cells.The content of ROS and MDA increased,Apoptotic peak was detected by flow cytometry and it also had some dose-effect relation.In addition to this,ME can affect the synthesis of intracellular protein and DNA.After treated with ME at the concentration of MIC,the expression of sig B and din F was increased,while the expression of clp P?mtg A?ica A was inhibited in S.aureus;The expression of rpo S and ybi J was increased and the expression of atp A?rfb S and omp F was inhibited in S.typhimurium.(7)A total of 1085 proteins were detected by label-free quantitative proteomic analysis.There were 136 proteins showing significant changes in expression between the ME-treated and the control group.Among those proteins,48 proteins were up-regulated while 88 proteins were down-regulated.Many metabolic pathways were affected by ME,especially glycolysis metabolism,tricarboxylic acid cycle and purine metabolism.The protein interaction of these differential proteins was closely,leading to the suppression of the material synthesis and energy production,thereby inhibiting the growth of bacteria.