Preliminary Study On The Separation And Purification Of 1,4-naphthoquinone Components In Impatiens And Its Antibacterial Mechanism

Impatiens balsamina L.is a plant in Balsaminaceae,widely distributed in Chinese resources,and has multiple functions in ornamental and medicinal use.Flavonoids,naphthoquinones,coumarins and sterols are the main components in impatiens.Flavonoids have good antioxidant activity,and naphthoquinones have good antibacterial effects.Studies at home and abroad have shown that 1,4-naphthoquinones in Impatiens are the main active ingredients of antibacterial,which can fight against various skin fungi and treat various skin diseases caused by fungi.However,there were few reports on the antibacterial dosage of Impatiens as an antibacterial drug.Firstly,the extraction process of 1,4-naphthoquinones from Impatiens was optimized,and the active ingredients were further enriched and purified.The in vitro antimicrobial activity of naphthoquinones from Impatiens was discussed,and the antimicrobial mechanism was preliminarily discussed,which provided a theoretical basis for expanding the combined application of Chinese and Western medicines and developing new antimicrobial preparations of traditional Chinese medicine.Objective:(1)Establish the optimum extraction process of naphthoquinones from Impatiens and optimize the process.(2)To determine the process of enrichment and purification of two naphthoquinones in Impatiens by macroporous resin.(3)To determine the optimum conditions for the purification of lawsone and 2-methoxy-1,4-naphthoquinone by silica gel.(4)To study the antifungal activities of lawsone and 2-methoxy-1,4-naphthoquinone in vitro.(5)To explore the combination of naphthoquinones and commonly used antifungal drugs.(6)Preliminary study on the bacteriostatic mechanism of naphthoquinones.Methods:(1)Balsamina as raw material,the extraction solvent,solid-liquid ratio and extraction time were optimized through single factor experiment,and the content was de termined by HPLC.By using the Box-Behnken design of the response surface with the Design Expert software,multiple regression simulation equations and response surface graphs of HNQ and MeONQ extraction rates were obtained.The extraction rate of HNQ and MeONQ was improved and the antibacterial activity of 2 kinds of naphthoquinone constituents in vitro was investigated.(2)The adsorption and desorption properties of Lawsone and 2-methoxy-1,4-naphthoquinone on 8 types of macroporous resins were investigated by HPLC,and the adsorption and desorption rates of Lawsone and 2-methoxy-1,4-naphthoquinone were investigated,and the model of resin was screened.A single factor test was used to investigate the effects of the sample solution concentration,pH,diameter-high ratio of resin bed,the sample flow rate,sample luquid volume,the concentration and the amount of eluent,and the elution flow rate on the purification of macroporous resin.(3)The eluate of silica gel column chromatography was selected by thin layer chromatography to purify the primary purified macroporous resin,and the obtained monomer compound was identified.(4)The in vitro antimicrobial activity of HNQ and MeONQ against Candida albicans and Trichophyton mentagrophytes was investigated by filter paper dilution method,and the minimal inhibitory concentration(MIC)and minimal bactericidal concentration(MBC)of fungi were determined by microdilution method,as well as the combination of MeONQ with commonly used antifungal drugs in clinic.(5)The inhibitory effect of MeONQ on the status of albicans dental tubes was investigated by dental tube generation test.(6)The bacteriostasis mechanism was preliminarily studied by conductivity meter,alkaline phosphatase(AKP)assay kit,Coomassie blue kit,inverted fluorescence microscope,scanning electron microscope and microplate reader.Results:(1)The optimal extraction conditions of HNQ and MeONQ from balsamine were as follows:the extraction solvent of 40%ethanol,solid-liquid ratio of 1:24,extraction time of 3.6h.Under these conditions,the extraction rates of HNQ and MeONQ were the highest,which were 7.6111 and 2.3785 mg.g-1,respectively.(2)The optimum purification process parameters of X-5 type macroporous resin are as follows:sample solution concentration of Lawsone and 2-methoxy-1,4-naphthoquinone of 0.35 and 0.21 g.L-1,pH of 4.47(original sample liquid),sample flow rate of 3 mL.min-1,diameter-high ratio of resin bed 1:4,washed impurity with 4BV of 20%ethanol,eluted with 9 BV of 95%ethanol at 2mL.min-1.The eluent was collected.The purity of Lawsone increased from 0.72%to 7.07%,and the purity of 2-methoxy-1,4-naphthoquinone increased from 0.23%to 4.77%,which was 9.8 times and 20.7 times higher than that before purification.(3)After silica gel purification,the monomer compounds of HNQ and MeONQ were obtained,and other impurities were preliminarily purified and segmented.The structure was identified by HPLC,UV,IR,NMR?etc.,and the obtained compounds were confirmed to be HNQ and MeONQ,respectively.The purity of HNQ was 98.1%,and the purity of MeONQ was 98.3%?(4)It was found through the filter paper test that when the concentration of HNQ was 20 mg.mL-1,there was no inhibition zone,indicating that HNQ had no obvious antibacterial activity against Candida albicans at this concentration.However,MeONQ had good antibacterial activity against it.When the concentration of antibacterial solution was 1.5 mg.mL-1,MeONQ was highly sensitive to candida albicans.Both naphthoquinones had inhibitory effects on Trichophyton mentagrophytes,but the bacteriostatic effect of HNQ was not as good as that of MeONQ.When the concentration of HNQ less than 10mg.mL-1,it had no inhibitory effect on Trichophyton mentagrophytes.when MeONQ concentration was 4 mg.mL-1,MeONQ was extremely sensitive to Candida albicans.It was preliminarily proved that MeONQ had stronger antifungal activity than HNQ and was the main antifungal active ingredient in impatiens.(5)The MIC of the MeONQ to Candida albicans was 500?g.mL-1,MBC was 1000?g.mL-1 and the combined MIC was 250 ?g.mL-1 by microdilution method.It was proved that MeONQ had good antibacterial activity and additive effect when used in combination with common clinical antifungal drugs.(6)Dental canal formation test showed that Candida albicans canal formation was completely inhibited when MeONQ concentration was more than 1/2 MIC.When MeONQ was 1/4MIC,the canal formation rate was 3%,and most of the canals were inhibited;when the concentration was 1/8 MIC,the canal formation rate was 38%,which still inhibited most of the canal growth;when MeONQ concentration was 250?g.mL-1,the canal formation rate was completely inhibited and prevent its changing growth form from invading cells and causing infection.(7)Preliminary studies on the bacteriostasis mechanism showed that the bacteriostasis effect of MeONQ did not affect the nucleic acid and protein of macromolecule bioactive substance in Candida albicans,but it could cause the leakage of electrolyte in Candida albicans and the change of membrane permeability.Scanning electron microscopy showed that the cell wall was defective to a certain extent,which led to the death of Candida albicans and played an antimicrobial role.Conclusion:Extraction of HNQ and MeONQ from Impatiens by leaching method is a stable and scientific extraction method,which is convenient,simple and efficient.X-5 macroporous resin has a good separation and purification effect on HNQ and MeONQ.The process is simple,stable and feasible,and the enrichment efficiency is high.It provides theoretical guidance for the development and utilization of HNQ and MeONQ.Silica gel purification is a relatively stable purification method,which does not destroy the stability of naphthoquinones in impatiens,and has a good purification effect.It can obtain the monomer compounds of HNQ and MeONQ.MIC and MBC of MeONQ were determined by in vitro bacteriostasis circle test and microdilution method,which provided the basis of in vitro drug sensitivity test for clinical using of MeONQ.At the same time,MeONQ and the commonly used antifungal drugs amphotericin B and fluconazole were investigated.It was found that MeONQ and Western medicine had good combined effect of antimicrobial activity.The combined using of Chinese and Western medicines was more effective in treating Candida albicans-induced infections than the single use of Chinese or Western medicines.At the same time,the study of bacteriostasis mechanism started with cell and ultrastructure,which provided a deeper mechanism study for the antimicrobial effect of MeONQ.It was found that the targets of MeONQ were cell wall and cell membrane system,which could make cell wall depressed and defective,and increased cell membrane permeability,thus playing a bacteriostatic role.