Inhibition Activity Of Malus Flavonoids To Helicobacter Pylori

The natural extracts of many plants have bactericidal effects.For example,the flavonoids extracted from apples have abundant activities of biological.Antibacterial is one of the most important activities.However,its inhibitory activity and mechanism for different strains are still lacking.Helicobacter pylori is a potential cause of stomach cancer in human.Most current therapies are dependent on antibiotics.However,the lack of antibiotic use will cause strain variation which leading to incurable disease,and antibiotic overdose will cause microbial disorders in the body which affecting health.It is clearly stated that the antibacterial effect of H.pylori is still unclear.Therefore,the contents of this experiment first systematically clarified and quantified the antibacterial activity and inhibition ratio of flavonoids,phenols,terpenoids and organic acids isolated and purified from Malus in a macro perspective on H.pylori.Kaempferol with the best antibacterial effect was selected as the main research object.The antibacterial mechanism of kaempferol was explored from the perspective of structure-activity relationship by comparing the inhibition ratio,hydrophobicity parameters and the minimum energy configuration of kaempferol with other four flavonoid compounds.Furthermore,by comparing the antibacterial activity between kaempferol and clarithromycin or amoxicillin,the subplaceability and availability of kaempferol in Malus extracts to some antibiotics were determined.The effects on cell structures were observed using electron microscopes(scanning and transmission models),and the related intracellular contents were determined.Combined with transcriptome analysis,the bacteriostatic mechanism and pathway of kaempferol were explored from different perspectives.This study provides a theoretical basis for the development of high-content kaempferol diet and drugs,and provides a new idea for the prevention and inhibition of H.pylori from the perspective of diet.The key results were detailed as follows:1.In vitro antibacterial experiment showed that kaempferol(0.1 mM)had the strongest antibacterial activity against H.pylori among 18 kinds of flavonoids,phenols and terpenoids(at the same concentration)from Malus,and the inhibition ratio reached 53.95 %.2.The hydroxyl at 2’ position in the B ring of flavonoids is the key site that affects the antibacterial effect.The flavonoids lacking the hydroxyl at 2’ position reduce the inhibition ratio of H.pylori by about 30%,while the increase of the hydroxyl at 1’ position has no promoting effect on the antibacterial effect.The flavonoids with hydroxyl groups at the 5’and 7’ sites of ring A have higher activity,while the hydroxyl groups at the 3’ sites of ring C play a vital role in the antibacterial effect.Minimum-energy configuration of those flavonoid compounds showed that the presence of hydroxyl groups in the A and B ring of flavonoids would affect the hydrophobicity of the compounds and the minimum energy configuration of the molecules,this observation also verified by their related hydrophobicity traits.Therefore,within a certain range,the hydrophobicity of flavonoids was negatively correlated with the antimicrobial activity.3.H.pylori was further treated with the same concentration of antibiotics(0.1 mM),in which the inhibition ratio of clarithromycin was 58.6 %,and that of amoxicillin was only41.8 %.When different concentrations of kaempferol were used in combination with clarithromycin or amoxicillin,the antimicrobial effect was not as good as that of using alone,indicating that kaempferol could replace antibiotics when using alone.4.Through microscopic observation,it was found that kaempferol could destroy the cell membrane structure of H.pylori and disorganize it,leading to block bacterial growth.Propidium iodide(PI)and fluorescein diacetate(FDA)staining showed that the cell membrane permeability of H.pylori was increased by kaempferol.The leakage rate of intracellular phosphorus(inorganic)and protein showed that H.pylori inclusions exudated and cell membrane was damaged.5.Through transcriptome analysis,it was clear that kaempferol was different from clarithromycin and amoxicillin in the antibacterial mechanism of H.pylori.Both antibiotics affected the metabolic pathway of H.pylori.However,kaempferol mainly affected the cell membrane pathway,enzyme activity and flagellin expression of H.pylori,and greatly inhibited the synthesis of ribosomes in H.pylori.