Development Of Antioxidant Evaluation Models Based On Cell Electrochemical Biosensor And Caenorhabditis Elegans And Their Applications On Anthocyanins

Rapid,efficient,and objective evaluation of active antioxidant components is difficult in the basic research of natural products and food quality.Traditional in vitro methods to evaluate antioxidant capacity mainly focus on free radical scavenging,and it is difficult to elucidate the valence of exerting antioxidant activity in real organisms as well as the comprehensive effect with other internal factors.However,the traditional animal model for antioxidant evaluation is costly and time-consuming for the preliminary screening and grading evaluation of abundant potential antioxidant components in natural plant resources.Therefore,it is necessary to develop new methods for antioxidant activity evaluation based on the physiological environment,and to evaluate the efficacy of potential active components in food by multiple means,tiers,and in a rapid manner,so as to provide data for the quality control of plant food with high active components and the development of related products.In our study,a cell line that is sensitive to oxidative stress was selected as the bio-recognition element to deveplop new methods for antioxidant activity evaluation based on cell electrochemical biosensors.We then applied them to evaluate the activity of 16 anthocyanins and determine their classification.Furthermore,Caenorhabditis elegans was used as an in vivo antioxidant evaluation model,and was combined with a metabonomics analysis to study the metabolite levels and potential effect mechanisms of anthocyanins in in vitro cells and in C.elegans.A summary of our work is as follows:1.Development of a novel A549 cell-based electrochemical biosensor based on impedance signal response and its application for the antioxidant evaluation of 16anthocyanins and their glycoside.In order to achieve a sensitive and objective evaluation of the antioxidant activity of anthocyanins with a rapid response,the A549 human non-small cell lung cancer cell line was used to develop an oxidative stress damage model.Sodium alginate?Alg?gel was selected to construct a three-dimensional cell culture system.A mixture of Alg and cells was immobilized on the surface of a glass carbon electrode?GCE?that had been electroplated with gold nanoparticles?Au NPs?and self-assembled?-cysteine??-Cys?to successfully develop a cell-based electrochemical biosensors based on Alg/A549 cell/?-Cys/Au NPs-modified GCE.Under the optimized electrode modification parameters,a satisfactory linear response was obtained between the electrical impedance signal and the concentrations of generated hydrogen peroxide?H₂O₂?.The biosensor's response was linear response for phloretin as an antioxidant from 20?M to 100?M.The linear equation was y=-0.648x+206.93?R²=0.9944?,and a 1.96?M detection limit was obtained.According to the changes in the impedance signal,16 anthocyanins and their glycoside derivatives were divided into 3 groups with significant difference in antioxidant activity.Compared with the traditional cell antioxidant activity method,the detection time for this cell electrochemical biosensor was short,within 2 min,and without additional pretreatment.This method has high sensitivity and can be effectively used for the rapid antioxidant evaluation of active compounds.2.Development of a cell electrochemical biosensor based on the current signal response to H₂O₂ release level and its application for the antioxidant evaluation of anthocyanins.To further increase the analytical selectivity and response specificity of the electrochemical biosensor,an A549 cells/hydrogel@carbon nanofibers?CNFs?/manganese dioxide nanowires?Mn O₂NWs?/Au NPs-modified electrochemical biosensor was successfully prepared based on the catalytic reaction between H₂O₂ and Mn O₂ respond to the current signal.The biosensor was sensitive under the optimized conditions of 0.1%Mn O₂NWs,10%hydrogel,0.5 mg/m L CNFs,and 1×10⁶ A549 cells/m L.When A549 cells were tested,a good linear correlation was found between oxidation peak current Ip and H₂O₂ concentration induced by paraquat?PQ?.The linear equation is Ip??A?=58.199C_H2O2+5.825(C_H2O2 for H₂O₂concentration)with R²=0.993,and the detection limit of H₂O₂ is 0.02?M,which indicates high sensitivity,and satisfactory reproducibility and stability of this method.The antioxidant activities of the 16 anthocyanins and their glycosides were rapidly and effectively evaluated using this constructed biosensor.Combined with the verified results of extracellular H₂O₂release levels,the biosensor method exhibited great potential to effectively evaluate the antioxidant activity of anthocyanins and other natural products.3.Different metabolites and enrichment metabolic pathways related to intracellular oxidative stress were screened by cellular metabolomics analysis,and the antioxidant activity as well as the potential mechanism of the anthocyanins from a metabolomics perspective were investigated.On the basis of previous quantification of the antioxidant activity of anthocyanins,cellular metabolomics analysis was conducted to further comprehensively and systematically determine the antioxidant activity and the potential mechanism of metabolic regulation of anthocyanins in cells.Multivariate statistical results showed that there were significant differences in principal components between the control group,anthocyanin pre-treated groups,and PQ-induced group,indicating that metabolite levels were changed after treatment.The change levels of metabolites through cluster analysis from high to low were:PQ<malvidin-3-arabinoside?Mal-3-ara?<cyanidin?Cya?<delphinidin?Del?.After screening the differential metabolites in the PQ-,Cya-,Del-,and Mal-3-ara-treated groups?metabolites were 12,8,6,and 7,respectively?,metabolic biomarker combinations related to the antioxidant activity of the Cya,Del,and Mal-3-ara anthocyanins were obtained by further regression model screening:alanine-oleic acid-threonine,alanine-galactose-glucose,and alanine-valine-threonine,respectively.The metabolism of amino acids,lipids and energy was disordered by PQ induction.Anthocyanins pre-treatment partially reversed the levels of metabolites,indicating that anthocyanins exerted a protective effect on PQ-induced oxidative stress injury in cells,and the antioxidant activities of anthocyanins were correlated with the maintenance of metabolite levels.The cellular metabolome data provide the characteristic fingerprint of the antioxidant activity of anthocyanins,and cellular metabolomics technology can be used as a powerful tool for antioxidant evaluation and mechanism elucidation of active components.4.C.elegans was selected as an in vivo biological model of oxidative stress to analyze the antioxidant activity of anthocyanins.On the basis of the antioxidant results at the in vitro cellular level,an oxidative stress model of C.elegans induced by PQ was developed to evaluate the in vivo antioxidant activity of anthocyanins.The results showed that at the high dose of 50?M,Del,Cya,and Mal-3-ara effectively reduced the mild oxidative stress induced by PQ and thermal stress injuries in nematodes,and extended their lifespan under oxidative stress.The strength of the antioxidant activities from high to low is ordered as Del>Cya>Mal-3-ara.Moreover,high-dose anthocyanin treatment significantly delayed the paralysis of CL4176 nematodes induced by?-amyloid 1-42.The paralysis time in the Del,Cya and Mal-3-ara pre-treated groups was extended by 16.27%,9.16%,and 7.87%,respectively,indicating that anthocyanins have a neuroprotective effect that was positively correlated with their antioxidant activities.The three anthocyanins did not exert any nonprotective effect on C.elegans when the worms were subjucted to acute oxidative stress injury induced by PQ,and was unable to prolong their lifespan.The in vivo antioxidant evaluation method based on the C.elegans model organism provides objective results regarding the comprehensive antioxidant activity of components,and exhibited great advantages in the development of the evaluation system for antioxidant activity.5.The regulatory effect and mechanism of anthocyanins on the disturbance of metabolite levels in nematodes under PQ exposure were investigated by biogenic metabolome analysis.In order to further reveal the potential antioxidant mechanisms of anthocyanins in C.elegans and the correlation between antioxidant activity and metabolite levels,the effects of anthocyanins pre-treatment on the whole metabolite levels and metabolic pathways in nematodes were systematically analyzed by a metabonomics method based on nematodes.The results of multivariate statistics and cluster analysis showed that,compared with the control group,the metabolism in nematodes was disordered under 0.3 m M PQ exposure,and the levels of multiple metabolites were significantly upregulated or downregulated,indicating that the oxidative stress model of C.elegans was successfully developed.13,7,4,and 10 differencial metabolites were screened in the PQ-,Cya-,Del-,and Mal-3-ara-treated groups,respectively.Fumaric acid and phenylalanine were further screened as a combination of common metabolic biomarkers for the evaluation of antioxidant activity of the three anthocyanins.Metabolite pathway enrichment indicated that PQ exposure significantly affected the tricarboxylic acid cycle,and the metabolism of pyruvate,glycerophospholipid,glutathione,and various amino acids.Anthocyanins reduced the metabolic disorders in nematodes under oxidative stress by mediating a variety of metabolic pathways associated with redox balance.Del exhibited the highest ability to maintain nematode metabolites,followed by Cya and Mal-3-ara.The maintenance of metabolic homeostasis reflects the oxidative stress resistance of the three anthocyanins,which was consistent with the results of the previous biochemical results.The antioxidant evaluation of the analytes based on metabonomics provides more abundant bioinformatic information.In conclusion,the development of antioxidant activity evaluation methods and models based on two cell electrochemical biosensors and C.elegans provides the antioxidant activity information regarding biological effect of active compounds for objective and comprehensive evaluation of the antioxidant ability.Metabonomics analysis technology,which is integrated with in vitro cell culture and the entire organism,was used to develop and optimize the efficient,accurate evaluation and quality screening methods of natural antioxidant products.New methods and ideas will be provided for further rapid and efficient antioxidant preliminary screening of multi-component complex extracts and elucidation of action mechanism as well as product quality grading by analyzing the efficacy activity of monomer components.