Extraction And Separation Of Active Constituents From Garlic And Study On Their Antibacterial And Antibiofilm Activities

Objective: In order to solve the increasingly serious problem of bacterial resistance,especially the multi-drug resistance caused by the formation of biofilm(BF)in vivo and in vitro,and to develop new natural components with high efficiency and low toxicity against drug-resistant bacteria,the extraction and separation of garlic polysaccharide and alliin and their antibacterial and anti-BF activities were studied in this paper,which provided a theoretical basis for further development of the application value of garlic polysaccharide and alliin.Methods: In this study,the crude polysaccharide of garlic was extracted by water extraction and alcohol precipitation,and the components were analyzed by visible spectrophotometer.Alliin was separated by 732 strong acid cation exchange resin,and the components of alliin extract were detected by high performance liquid chromatography(HPLC).Taking Staphylococcus aureus,Escherichia coli and KA as the research objects,the growth curves of the three bacteria were detected,the logarithmic growth phase of the strains was determined,and the corresponding relationship between the bacterial concentration and the absorbance(OD)value was determined.The minimum inhibitory concentration(MIC)of garlic polysaccharide and alliin on the three strains was studied by micro broth dilution method and paper method.The effect of garlic polysaccharide on E.coli BF was detected by crystal violet staining.The effect of garlic polysaccharide on the phenotype and structure of E.coli BF was detected by scanning electron microscopy and fluorescence inverted microscope.The key targets and mechanism of the possible active ingredient structure of garlic polysaccharide against BF were screened by network pharmacology and molecular docking technology.Results:1.The yield of garlic crude polysaccharide extracted and isolated in this study was 36.31 %,and the composition analysis showed that the neutral sugar content was 68.40 %,the uronic acid content was 41.59 %,the sulfate sugar content was 5.57 %,and the protein content was 3.24 %.The yield of alliin was 2.04 %,and the purity was 44.43-51.03 %.2.The results of antibacterial experiments showed that garlic polysaccharide had no inhibitory effect on Escherichia coli,Staphylococcus aureus and Klebsiella pneumoniae;the MIC of alliin against Escherichia coli was 40 mg / m L,the MIC of alliin against Staphylococcus aureus was 20 mg /m L,and the MIC of alliin against multidrug-resistant Klebsiella pneumoniae was 40 mg / m L.3.The results of garlic polysaccharide anti-biofilm activity showed that garlic polysaccharide had inhibitory effect on E.coli BF by crystal violet staining.The results detected by scanning electron microscopy and Fluorescence inverted microscope were consistent with those of crystal violet staining.4.It was predicted by network pharmacology that garlic polysaccharide inhibited BF by acting on lactotransferrin(LTF),interleukin 2(IL2),epidermal growth factor receptor(EGFR),albumin(ALB),catalase(CAT)and other targets.It is related to response to hormone,response to peptide and positive regulation of transferase activity.KEGG pathway enrichment analysis showed that the mechanism of garlic polysaccharide on BF was closely related to Fox O signaling pathway,Endocrine resistance,Prolactin signaling pathway and Relaxin signaling pathway.Molecular docking results showed that the monosaccharide active ingredient mannitol of garlic polysaccharide could bind to HSP90AA1 and EGFR gene targets through hydrogen bonds.Conclusions: Alliin not only has an inhibitory effect on the standard strains of Gram-negative bacteria and positive bacteria,but also has an inhibitory effect on multidrug-resistant Klebsiella aerogenes.It is the first time to find the antibacterial activity of alliin against drug-resistant Klebsiella aerogenes,which provides an important theoretical basis for clinical application.Garlic polysaccharide had no significant bacteriostatic effect,but this study found for the first time that garlic polysaccharide could inhibit bacterial biofilm,which was of great significance for the study of bacterial resistance and the deep development of garlic polysaccharide.