Study On The Preparation,Characterization And Antibacterial Properties Of Artemisia Argyi Var.Argyi ’Qiai’ Silver Nanoparticles

Objective:To establish a method for rapid screening of antioxidant active ingredients in Qiai based on ultra-performance liquid chromatography post-column derivatization,and provide basis for Qiai green reduction method to prepare Silver nanoparticles（Ag NPs）.Single factor and orthogonal experiment were used to optimize the preparation process of silver nanoparticles.Ag NPs were characterized by UV-Vis,Malvern Laser Particle sizer,Transmission Electron Microscope（TEM）,etc.Compareing the influence of adding different types of cyclodextrin on Qiai AGNPs,prepare good stability and small diameter Ag NPs,and then develop Qiai related products,and provide a certain theoretical basis for the deep use of Qiai resources.Method:1.Establish an online UPLC-DAD-DPPH analysis method,use gallic acid as a positive control,investigate the stability and reproducibility of the online method,and quickly screen the antioxidant active ingredients in the extract of Qi Ai,using UHPLC-LTQ/Orbitrap high-resolution mass spectrometry identified the active ingredients and screened the active ingredients.2.Study on the preparation process of Ag NPs:Single factor and orthogonal test were used to optimize the reaction time,reaction temperature,Qiai extract dosage and Ag NO₃ concentration.At the same time,the stability and dispersion of Ag NPs prepared under the optimal conditions were investigated.3.The Ag NPs were prepared with Qiai extract as a green reducing agent,andβ-cyclodextrin,HP-β-cyclodextrin,γ-cyclodextrin and a cationic cyclodextrin were added separately to explore its influence on the formation of Ag NPs.The TEM and Malvin particle size analyzer were used to characterize the particle size,morphology and crystal structure of Ag NPs composites.The antimicrobial activity of different Ag NPs compounds against Gram-negative bacteria（Escherichia coli）and Gram-positive bacteria（Staphylococcus aureus）was investigated by modified edge azuria staining.Result:1.Establish an online UPLC-UV-DPPH detection method,use gallic acid as a positive control,and confirm that the delay time between online monitoring PDA detector and DAD detector is about 3 min;verify the online detection method with the same concentration and different concentrations of gallic acid.The stability and reproducibility of the instrument,the stability and reproducibility of the instrument are good.Using UPLC-UV-DPPH online detection method,7 antioxidant active substances were screened from the extract of Qiai,and 17 compounds were identified from Qiai extract by UHPLC-LTQ/Orbitrap high-resolution mass spectrometry,of which 6 had the antioxidant activities are neochlorogenic acid,cryptochlorogenic acid,isochlorogenic acid B,isochlorogenic acid A,isochlorogenic acid C and 3,4,5-tricaffeoylquinic acid.2.Through single factor experiment combined with orthogonal design,it is concluded that the order of the influence of various factors on the synthesis of Ag NPs is:reaction temperature>Qiai extract dosage>reaction time.The optimized preparation process of Qiai Ag NPs is as follows:temperature 95℃,Qiai extract amount 10.0 m L,Ag NO₃ concentration 1.0mmol/L,reaction time 90 min.The resulting Ag NPs have good stability and dispersion in aqueous solution.3.After addingβ-CD,HP-β-CD,γ-CD and a cationic cyclodextrin under the optimal conditions of the preparation process,the average particle diameters of the Ag NPs complexes obtained were 46.28 nm,49.86nm,46.76 nm and 580.40 nm,respectively.nm.Characterized by TEM and UV-vis and other characterization methods.Stability investigation,after addingβ-cyclodextrin,HP-β-cyclodextrin,γ-cyclodextrin,the stability of Ag NPs solution increases,but after adding cationic cyclodextrin,there is basically no detection of Ag NPs,which may be a package Sufficient,but its stability performance is poor.The modified resazurin staining method was used to investigate the concentration of resazurin and the positive drug（ciprofloxacin）,and finally determined that the concentration of resazurin was 7.5 m M and the concentration of ciprofloxacin was 5×10^-5 mg/m L,Ag NPs complex has a strong inhibitory effect on both Gram-negative bacteria（E.coli）and Gram-positive bacteria（Staphylococcus aureus）.The addition of cyclodextrin has reduced the antibacterial effect due to some of the Ag NPs being encapsulated.Among them,γ-CD-Ag NPs have good stability and antibacterial properties.Conclusion:In this experiment,the online UPLC-UV-DPPH method could quickly screen the antioxidant active ingredients in traditional Chinese medicine.The method has good stability and reproducibility.It can be widely used to screen active substances in traditional Chinese medicine and other complex matrices.Antioxidant active ingredients were screened out to provide a basis for the preparation of nano silver from Qiai;Based on the requirements of green chemistry,the green reduction method of Qiai extract was used to successfully prepare Ag NPs with particle size.By adding different types of cyclodextrins,their stability can be increased.The antibacterial test shows that Qiai Ag NPs have good antibacterial activity,which provides a reference for the development and utilization of Qiai resources in the future.