Study On The Method Development For The Assessment Of The Bioactivity Of Natural Products Based On Static Headspace Analysis

The growing public concerns caused by microbial infections and cancers year by year,haveseriously threaten human health and quality of life,and meanwhile broughtgreat challenge to modern medical research.Natural products derived from plants,animals and microorganisms are rich in resources,with complex chemical structures and high biocompatibility,havealso playedan important role in the research and development of new drugs or lead compounds.A wide variety ofof the skeleton structuresof natural products arean ecological defense system generated by the long-term interaction between organisms and the environment during the growth process.Therefore,new drug cues obtained from natural products are generally of better quality,and more bio-friendly.Antibacterial susceptibility testing and the determination of the drug's antitumor activity in vitro are the general assays for drug screening and the subsequent evaluation of the bioactivities of new drugs,as well as the important basis for epidemiological research and clinical use.During the activity screening of natural products,some medicinal extracts,especially crude extracts of plants with relatively complex components,also contain various pigments and insoluble components.Based on the traditional counting or optical methods,there aremore or less interferencesoruncertainties,which cannot meet the needs of accurate evaluation of the bioactivity of drugs and the comparisons of results between different laboratories.Therefore,it is desired to develop new methods for the assessment of drug bioactivity based on modern analytical techniques.The determination of growth-dependent metabolites is considered to be an indirect method for monitoring microbial growth.These metabolites havebeen widely used as a biomarker for microbial research according to the metabolic characteristics of microorganisms.In these studies,carbon dioxide?CO₂?is one of the regular end-product of microbial respiratioin,that is,the activity of microbial cells can be reflected by detecting the amount of CO₂ released during microbial metabolism.Compared with traditional disc diffusion method,plate counting method and turbidity method,the headspace gas chromatography method based on modern analytical instruments has higher detection sensitivity and wider linear range,which can detect low-concentration carbon dioxide produced by microbial metabolism,and sensitively reflect the cell activity.In the field of drug screening fromnatural products,no method for drug screening based upon headspace gas chromatography has been established,so it is of great significance to establish a better method to determinethe antibacterial activity and antiproliferativeactivity of drugs,especially natural drugs,with headspacegas chromatography.1.A new method was established to determine the antimicrobial activity of drug compounds or crude extracts from natural products by measuring the amount of metabolic carbon dioxide produced fromthe drug-bacteria incubation system.2 mL of medium containing bacteria and drug of interest was incubated at 37°C for 24 h.The amount of metabolic carbon dioxide partitioned in the headspace was measured to evaluate the drug antimicrobial activity andthe minimum inhibitory concentration using headspace gas chromatography?HS-GC?coupled with thermal conductive detector?TCD?.The principle and the standard procedures of the present method have been developed and verified.As a result,the precision of the present method was less than 4%?expressed as relative standard deviation?,and an excellent agreement was found on the determination of both inhibition rate?R²=0.935?and the half inhibition concentration?R²=0.994?between the present method and a reference method?optical density method?.Because of the in-situ culture and detection in the closed system,this method is simpler and safer than the traditional methods in microbial detection,and it is also suitable for routine analysis of antibacterial activity of natural products with high flexibility in bacterial strains and sample properties.2.A new method for oxygen removal for atmospheric pressure was developed using an easily-made laboratory device,which can provide different oxygen concentrations for atmosphere-controlled culture through experimental settings.By accquiring a dataset containing the volume of anaerobic bottle,aeration time and oxygen removal rate,a mathematical model of oxygen removal in anaerobic bottle based on gas displacement kinetics was established and verified.The results show that the predicted results are in good agreement with the measured results?R²=0.98?,which indicates that the method can be used as a reliable tool for oxygen removal in anaerobic atmosphere,and can provide a mathematical approximation of the final oxygen concentration or oxygen removal rate.In short,this method can quickly achieve the purpose of deaeration,itis simple and easy to operate,and can be achieved by common laboratory equipments.Thepressure has not changed in the oxygen removal process,which provides the feasibility for thesubsequent anaerobic experiments.3.A novel method for the determination of the anti-anaerobic activity of drug candidates by automated headspace-gas chromatography?HS-GC-TCD?was established.Anaerobic bacteria were inoculated in an anaerobic atmosphere or rapidly using sterile syringe in an air-tight manner,and incubated with and without drugs for48h.The metabolic acidities of the cultured media were used as an indicator of cell activities and measured as end products in place by HS-GC-TCD after being completely converted to CO₂ with sodium bicarbonate.The present method is precise?relative standard deviation is below 5%?and validated by excellent agreements with a reference method on the determinations of the inhibition rates?root-mean-square error=10%?and half maximal inhibitory concentrations?R²=0.996?of both pure drug compounds and plant extracts.Advantageously,the present method is sensitive in response to cell activity,safe with regard to cross contamination,and suitable for routine screening of diversified drug candidates for anti-anaerobic activity.4.A new method for measuring cell activity using headspace gas chromatography was established and validated.The cells were cultured in the headspace vial,and then the amount of CO₂ produced by cell aerobic repirationwas measured by gas chromatography to reflect the number of cells,and the accuracy and precision?relative standard deviation less than 5%?of the method were verified.By comparing with the traditional Sulforhodamine B?SRB?method to detect the number of cultured cells in 96-well plates,it was proved that HepG2 cells could grow well in the headspace vials?R²=0.933?.The reliability of this method in determining cell proliferativeactivity was proved by comparison with the SRB method?R²=0.948?.Compared with the traditional 96-well-plate culture staining method,thismethod avoids the error caused by human operation factors during the measurement process,reduces the contamination problems in the cell culture,and saves time with fast determination speed.