| Research background:Diseases caused by bacterial and fungal infections are still the most common and most harmful diseases in human daily life.With the large number and long-term cross-use of various types of antibacterial drugs,the widespread and irrational use of drugs in clinical treatment,and the increase in the types of pathogenic bacteria,resulting in growing fungus infection disease,bacterial,fungal resistance is also escalating.At present,the treatment of bacterial and fungal infectious diseases at home and abroad is still dominated by antibiotics,but the clinically common pathogenic bacteria are generally resistant to commonly used antibacterial drugs.Now faced with the pathogen drug resistance rate continues to accelerate,and the development of new antibiotics is growing slowly.In order to research and develop more and more efficient antibacterial drugs,countries all over the world invest a lot of manpower,financial and material resources in the research and development of antibacterial drugs every year,but the results are not significant.The development of new antibacterial drugs is still a difficult problem faced by the world today.In recent years,with the continuous development of modern Chinese medicine research and technology,research antibacterial medicine has made great progress.Chinese medicine has been used for the prevention and treatment of diseases for thousands of years of,which has a multi-component,multi-target and multi-role characteristics,Clinical studies and numerous prescriptions and prescriptions have shown that Chinese medicines for clearing away heat and detoxifying and clearing away heat and dampness have antibacterial activity.Studies have shown that,combined with the infection of pathogenic bacteria,people with low immunity and low metabolism are more susceptible to infection by bacterial species.In clinical treatment,doctors often prescribe certain vitamin B and C supplements to require patients to take.This article relies on the active ingredients of traditional Chinese medicine with many antibacterial advantages,By chemical synthesis,vitamin compounds and effective antibacterial ingredients are combined together to develop a new drug that has both antibacterial activity and can enhance the body’s immunity,providing a certain research idea for the research and development of antibacterial drugs.Research purposes:In this study,the chlorogenic acid structure was modified from the raw materials to improve its stability and lipid-water partition coefficient,synthesize chlorogenic acid analogs,and then based on the drug combination principle,the vitamin B family of nicotinamide was introduced into chlorogenic acid analogs,a brand new target compound(NACA-I)is synthesized,After that,in vitro antibacterial activity evaluation and toxicity evaluation were conducted to evaluate whether the target compound has the advantages of high efficiency and low toxicity.And preliminary study on its antibacterial mechanism,to provide a reference for further research and development of the target compoundResearch content:(1)Based on the existing research on the synthesis of chlorogenic acid and the combination principle of medicinal chemistry,Starting with quinic acid and caffeic acid,the two components that make up chlorogenic acid,protected by the ortho hydroxyl group,quinic acid Oxidation and reductive amination of 5-position hydroxyl group,acyl chloride of caffeic acid,condensation reaction of 5-hydroxyl-group quinic acid with caffeic acid chloride,synthesis of new chlorogenic acid analogs with amide bond instead of ester bond,Except for the amide bond.it retains the same structural characteristics as chlorogenic acid.After that,the 1st carboxyl group of the new chlorogenic acid analog is combined with nicotinamide to synthesize the final target compound.The structural identification of the synthesized compounds was carried out by spectroscopy methods such as IR,1H-NMR,1 3C-NMR,and HRMS elemental analysis.(2)The in vitro antibacterial activity of the newly synthesized target compound was studied,and the in vitro antibacterial effect of the target compound on Candida albicans and Staphylococcus aureus was evaluated by the filter paper diffusion method and the diameter of the inhibition circle.,using chlorogenic acid test solution,nicotinamide test solution,and the mixed test solution of chlorogenic acid and nicotinamide serves as a control.Using the microdilution method to detect the minimum inhibitory concentration(MIC)and minimum bactericidal concentration(MBC)of the target compound And draw a time killing curve for Candida albicans and Staphylococcus aureus,comprehensively evaluate whether it has a fast and efficient bacteriostatic effect.(3)In vitro cytotoxicity tests were conducted on newly synthesized target compounds to initially assess its safe concentration thresholds.CCK-8 reagent was used to detect the change of OD value of the target compound on L-02 human normal liver cells,and compared with the control group to comprehensively evaluate the cytotoxicity of the target compound,and screen out the the safe concentration range of the target compound.(4)Under the premise of determining that the target compound has good antibacterial activity,we carry out preliminary exploration on the antibacterial mechanism of the target compound,Detection of bacterial electrolytes by conductivity meter,detection of bacterial proteins by Coomassie brilliant blue kit,detection of related enzymes by alkaline phosphatase(AKP)kit,and study of the antibacterial mechanism of target compounds by microplate reader and scanning electron microscope,etc,it can provide some reference data for follow-up research.Research result:(1)The expected target compound was successfully synthesized,and its structure was initially identified,and several key intermediate structures were identified during the synthesis process.(2)Through the filter paper diffusion test,it was found that when the concentration of the target compound NACA-I test solution was 40 mg·mL-1,it was extremely sensitive to Candida albicans and Staphylococcus aureus.It belongs to the range of low sensitivity to drug-resistant of Staphylococcus aureus.When the concentration of the target compound NACA-I test solution was diluted to 20 mg·mL-1,the diameter of its inhibitory circle was greater than 15 mm and less than 20 mm after acting on Candida albicans and Staphylococcus aureus.The sensitivity evaluation result shows that it is highly sensitive to these two bacteria at this concentration,and it is still highly sensitive to drug-resistant Staphylococcus aureus.And until it is diluted until the concentration of the target compound NACA-I test solution is 1.25 mg·mL-1,it still has low sensitivity to Candida albicans and Staphylococcus aureus.At this time,drug-resistant Staphylococcus aureus has no bacteriostatic effect.The MIC values of the target compound against Candida albicans and Staphylococcus aureus were determined to be 40 μg·mL-1 and 160 μg·mL-1 by microdilution method,respectively.The MBC values for Candida albicans and Staphylococcus aureus were 160μg·mL-1 and 320 μg·mL-1,respectively.The time kill curve proves that the target compound has the advantage of rapid and efficient sterilization.(3)Cytotoxicity results show that when the target compound NACA-I concentration is in the range of 5～40 μg·mL-1,it can promote the proliferation of L-02 cells,and at the concentration of 40 μg·mL-1,the cells survival rate is the largest.When the concentration of the target compound NACA-I is in the range of 80～320 μg·mL-1,it may have certain toxicity to L-02 cells.If the safe concentration of NACA-I related activity of the target compound is determined only by the survival rate of L-02 cells,the optimal concentration is 40～160 μg·mL-1.(4)The preliminary study on the antibacterial mechanism of the target compound against Candida albicans shows that its antibacterial effect is to destroy the cell wall and cell membrane system of the cell body,make the cell wall concave and rupture,and the permeability of the cell membrane increases,which affects the normal organism of Candida albicans Features.Research conclusion:After modifying the structural defects of chlorogenic acid,the introduction of nicotinamide not only makes the target compound more stable overall,but also has significantly improved antibacterial activity,which has the advantages of rapid and efficient sterilization.The cytotoxicity of the target compound is similar to that before the structural modification,which achieves the intended purpose of this study and provides a certain reference value for subsequent further research and the development of new antibacterial drugs. |
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