Establishment And Application Of Approach For Antioxidant Activity Based On Celluar Metabolomics

The excessive free radicals induced by oxidative stress can damage DNA, proteins and lipids directly and indirectly, and then the balance of oxidation and antioxidation system in body is broken. The use of exogenous antioxidants is the main means against excessive free radicals in the field of medicine. Due to the respective technical defects, the traditional evaluation approaches of antioxidant activity restrict the discovery of antioxidant substances seriously. Therefore, it is urgent to establish a new approach for the evaluation of antioxidant activity. Celluar metabolomics is a branch of system biology and it can be developed as a new approach for the evaluation of antioxidant activity because of its high sensitivity, good selectivity, high speed, high accuracy, strong controllability and so on. Compared with the chemical evaluation approach, the approach of antioxidant activity based on celluar metabolomics was not out of the organism and was closer to the biological oxidation process; compared with the cytologic evaluation approach, the approach of antioxidant activity based on celluar metabolomics could reflect the information of intracellular metabolites systematically. The approach of celluar metabolomics, HepG2 cells and HPLC-DAD-MS technology platform were used in this study. Combing with a variety of pattern recognition technology, the biomarkers which were relevant to antioxidant activity were indentified. Approach of antioxidant activity based on celluar metabolomics was estanblished and applicated in evaluating the extracts of Cynomorium songaricum Rupr.. The main contents were as follows:(1) Optimization of sample preparation methods and HPLC methods. In order to get more and more stable metabolites and obtain reliable analysis results, sample preparation methods and HPLC methods were optimized systematically. Sample preparation methods were that cells were quenched by﹣20°C 60% methanol and disrupted by ultrasonic processor, then metabolites were extracted with 60% methanol. HPLC chromatographic conditions: a flow rate was 1.0 mL/min; column temperature was 30°C; chromatographic column was performed on an Agilent ZORBAX SB-Aq(250 mm x 4.6 mm, 5 μm); the mobile phase was 1.93 g/L ammonium acetate(pH = 4.81) and acetonitrile; the solvent gradient elution was 0.5- 5% acetonitrile(0- 10 min) and 5- 95% acetonitrile(10- 60 min); the wavelength was 254 nm.(2) The discovery of biomarkers for oxidative stress based on celluar metabolomics. In order to establish the model of oxidative stress, cells were treated with 5 mg/L H2O2 for 4 h, 100 mg/L VE for 24 h and 250 mg/L GSH for 24 h. In order to verify the model of oxidative stress, the level of ROS and the activities of enzyme were determined. The metabolites were analysed by HPLC and 28 common peaks from typical HPLC-DAD spectra were analysed. According to the plot of S-plot and the value of VIP based on OPLS-DA model, 11 potential biomarkers of oxidative stress were discovered. The peaks were analysed qualitatively by LC-MS spectrometry, UV spectra of metabolites and control article, and addition technique. The p1, p2, p3 and p9 were identified as malic acid, VC, GSH and tryptophan.(3) Establishment of approach for antioxidant activity based on celluar metabolomics. On the basis of OPLS-DA model, the qualitative approach for antioxidant activity based on celluar metabolomics was established. According to value of CMAA unit for VE or GSH, the antioxidant acitivities of substances were evaluated quantitavely and quantitative approach of antioxidant activity based on celluar metabolomics was established.(4) Application of approach for antioxidant activity based on celluar metabolomics. Using the established approach of antioxidant activity based on celluar metabolomics to evaluate the extracts of Cynomorium songaricum Rupr., and then using chemical evaluation approach and cytologic evaluation approach to verify the new approach. The results showed that the approach of antioxidant activity based on celluar metabolomics was reliable, and the results were consistent with the results of traditional vitro evaluation approaches for antioxidant activity. The results of all evaluation approaches showed that polysaccharide and flavonoids of Cynomorium songaricum Rupr. had antioxidant activity and the antioxidant activity for flavonoids of Cynomorium songaricum Rupr. was stronger.