Antibacterial Activity Components From The Aerial Parts Of Allium Sativum

Objective:This project intends to study the antibacterial activity of the aerial part of A.sativum,explore the antibacterial mechanism of the activity part on the tested bacteria,isolate the chemical components and identify the structure of the activity part,and screen the isolated monomer compounds for in vitro antibacterialactivity.In ordertoclarify the material basis andmechanism ofantibacterial efficacy.Methods:1.The 30 kg the aerial parts of A.sativum was extracted twice with 70 % ethanol heated and reflow for 2 h each time,filtered,combined with the filtrate,and recovered the solvent under reduced pressure to obtain the total extract.The total extract was suspended in an appropriate amount of distilled water and extracted with petroleum ether,ethyl acetate and water-saturated n-butyl alcohol in turn to obtain four extraction parts: petroleum ether layer,ethyl acetate layer,n-butyl alcohol layer and water layer,andthe yield wascalculated.2.The Oxford cup method and microdilution method were used tostudy the antibacterial activity oftotal extract of the aerial parts of A.sativum and its extract parts against Staphylococcus epidermidis,Staphylococcus aureus,Escherichia coli and Pseudomonas aeruginosa invitro.3.The growth curves of Staphylococcus epidermidis and Staphylococcus aureus were measured,and the effects of the active site on the biofilm,cell wall and intracellular proteins of Staphylococcus epidermidis and Staphylococcus aureus were studied.4.Silica gel chromatography,ODS(Octadecylsilyl)and HPLC were used to separate the active part,nuclear magnetic resonance(NMR)and mass spectrometry(MS)were used to identify the structure,andthe antibacterial activity of the isolated monomeric compounds werescreened.Results:1.The total extract of the aerial parts of A.sativum was 2723 g with a yield of 9.1 % by heating reflux extraction.In each extraction part,the petroleum ether layer(257 g),ethyl acetate layer(117 g),n-butanol layer(396 g)and water layer(1935 g)were obtained with the yield of 9.3 %,4.3 %,14.5 %and 71.0 %,respectively.2.The results of in vitro antibacterial test showed that the total extract of A.sativum aboveground part had antibacterial activity against Staphylococcus epidermidis,Staphylococcus aureus,Escherichia coli and Pseudomonas aeruginosa,and the inhibition zone diameters were 15.67 ± 0.58 mm,11.33 ± 0.33 mm,14.5 ± 0.75 mm and 14.66 ± 0.58 mm,respectively.Among the four extraction fractions,the ethyl acetate layer,n-butanol layer and water layer showed different degrees of antibacterial activity against Staphylococcus epidermidis,Staphylococcus aureus,Escherichia coli and Pseudomonas aeruginosa.However,thepetroleum ether layerdid not showantibacterial activity.3.The results showed that the ethyl acetate layer had a certain inhibitory effect on the growth process of Staphylococcus epidermidis and Staphylococcus aureus.The results of crystal violet staining showed that the concentration of 0.5 MIC and 1 MIC of ethyl acetate layer had obvious inhibitory effect on the biofilm of Staphylococcus epidermidis and Staphylococcus aureus,and the inhibition ratio of Staphylococcus epidermidis was 15.2 % and 34.6 %,respectively.The inhibition ratio of Staphylococcus aureus biofilm was 12.6 % and 47.8 %,respectively,and the inhibition ratio increased with the increase of drug concentration in a dose-dependent manner.The results of bio-microscope further confirmed the inhibitory effect of the ethyl acetate layer on the biofilm of S.epidermidis and S.aureus.By measuring the alkaline phosphatase(AKP)and protein content in the bacterial culture medium,the ethyl acetate layer had destructive effects on the cell wall and membrane of S.epidermidis and S.aureus.4.The Silica gel column chromatography,polyamide chromatography,ODS and HPLC were used to separate and purify the ethyl acetate layer,and NMR and MS spectroscopic methods were used to analyze and identify the 12 compounds.The monomeric compounds were identified as3,5,3’-trihydroxy-7,4’-dimethoxyfla-vone(1),3′,4′,5,7-tetrahydroxy-3-methoxyflavone(2),isorhamnetin-3-O-β-D-glucopyranoside(3),kaempferol-3-O-β-D-xylopyranoside(4),Dihydroquercetin-7,4’-dimethylether(5),Vanillic acid(6),3-hydroxyl-4-methoxyl benzoic acid(7),Ferulic acid(8),Dehydrovomifoliol(9),N-trans-feruloyl 3′-O-methyldopamine(10),Paprazine(11)and Dibutyphthalate(12).Compounds 1,2,10 and 12 showed antibacterial activity against Staphylococcus epidermidis.Compounds 1 to 5 and 10 to 12 showed antibacterial activity against Staphylococcus aureus.Conclusion:A.sativum aboveground part and its extract parts had different degrees of antibacterial activity against Staphylococcus epidermidis,Staphylococcus aureus,Escherichia coli and Pseudomonas aeruginosa.The 12 compounds were isolated and identified from the ethyl acetate layer,including 5flavonoids,2 alkaloids,2 phenols,1 penylpropanoids1 benzoic acid and 1 β-ionone.In vitro experiments showed that compounds 1,2,10 and 12 had antibacterial activity against Staphylococcus epidermidis and Staphylococcus aureus.Compounds 3 to 5 and 11 showed antibacterial activity only against Staphylococcus aureus.