Rapid Screening And Evaluation Of Key Antioxidant Phenolic Compounds Of Longan Seeds By Online Knockout And Real-time Cell-based Impedance Analysis

Currently, about 17% of the fresh weight of longan fruit remains as waste in the form of seeds during processing, which are abundant in antioxidant phenolic compounds. Therefore, efficient utilization of longan seed polyphenols is an effective way to promote the comprehensive utilization with high value of longan fruit. However, the current information on key antioxidant compounds of longan seeds are unavailable. Based on this, the present study screened out the key phenolic compounds accounting for the antioxidant effects of longan seeds using a combination of online knockout, and in vitro chemical-based antioxidant strategies. The detailed structures of key phenolic compounds were elucidated for the first time from longan seeds by high-performance liquid chromatography electrospray ionization mass spectrometry(HPLC-ESI-MS), fourier transform ion cyclotron resonance mass spectrometry(FT-ICR–MS), nuclear magnetic resonance(NMR) and other spectroscopic methods. Meanwhile, their excellent antioxidant activities in different chemical-based and cell-based assays were further confirmed. We then attempted to employ microcalorimetry and real-time cell-based impedance analysis(RTCA) to investigate the protective effects of key phenolic compounds of longan seeds and 12 kinds of representive phenolic compounds in H2O2-induced NIH-3T3 cells, and the results indicated that RTCA model was more suitable for the rapid screening and accurate evaluation of antixodant constituents in cell physiological level. The main results were showed as follows: 1. Separation and purification of phenols from longan seedsOrthogonal design was used to optimize the extraction conditions of phenols from longan seeds. Orthogonal design was employed to evaluate the significance of four crucial variables including alcohol concentration, solid-to-solvent ratio, extraction temperature and extraction time, and to determine the effect of prime factors on the yield of phenols. The optimum conditions were ethanol concentration 40%, solid-to-solvent ratio 1:20(g/m L), extraction temperature 70℃, and extraction time 90 min. D3520 resin was selected to purify longan seed phenols by comparing static absorption and dynamic desorption capacities of four macroporous resins, and 40% ethanol was chosen as the best elution solvent. 2. Online screening of key antioxidant phenolic compounds of longan seedsA combination of online knockout method, FT-ICR–MS and activity evaluation assays(determination of the reducing power, DPPH-scavenging activity and oxygen radical absorbance capacity) was used to successfully screen out the bioactive group(fraction 2-2) and key antioxidant phenolic compounds(peaks 4 and 6) of longan seeds. While gallic acid,(S)-flavogallonic acid, methyl-ellagic acid glucopyranoside, and ethyl gallate did not affect the potent antioxidant activity of longan seed polyphenols notably, showing a small contribution to the total bioactivity. The structural properties of peaks 4 and 6 were preliminarily analyzed by HPLC-ESI-MS, and the results showed that peak 4 was unknown and peak 6 might be the derivative of ellagic acid. 3. Structural elucidation and antioxidant activity confirmation of key phenolic compounds isolated from longan seedsWe further elucidated the detailed structures of the key phenolic compounds responsible for the antioxidant activity of longan seeds by 1H-NMR, 13C-NMR, heteronuclear multiple bond correlation(HMBC), and the detailed structures of three phenolic compounds, namely 9-O-(3-carboxymethyl-4-(p-formylstyryl))hydroxybutanoic acid(compound 2), 2-hydroxy-3-methoxycaffeic acid 5-O-β-D-glucopyranoside(compound 4) and 3′-O-methyl-4′-O-(4-O-galloyl-α-L-rhamnopyranosyl)ellagic acid(compound 6), were identified for the first time from longan seeds. The new compounds 4 and 6 were proved to be the main contributors to the potent activity of longan seeds by four different assays including the trolox equivalent antioxidant capacity(TEAC) assay, the oxygen radical absorbance capacity(ORAC) assay, β-carotene–linoleate model assay and the assay of inhibiting the activity of xanthine oxidase. We further used cell-based models to investigate the antioxidant effects of key phenolic compounds of longan seeds on H2O2-induced NIH-3T3 cells. The results showed that 2-hydroxy-3-methoxycaffeic acid 5-O-β-D-glucopyranoside(P4) and 3′-O-methyl-4′-O-(4-O-galloyl-α-L-rhamnopyranosyl)ellagic acid(P6) showed more potent antioxidant activities, which can effectively improve the activities of SOD, CAT and GSH-Px, reduce the excessive ROS level, decrease the damage degree of mitochondria and DNA, and inhibit apoptosis in H2O2-induced NIH-3T3 cells, when compared with 3′-O-methylellagic acid 4′-O-β-D-glucopyranoside(P7) and ethyl gallate(P8) which showed the poorest antioxidant effects in chemical-based assays. The above chemical-based and cell-based results fully validated the feasibility of online knockout method. 4. Microcalorimetry was preliminary used for the screening of antioxidant phenolsIn order to seek for a rapid, efficient and more physiological relevant method for the screening of antioxidant phenols, microcalorimetry was used to investigate the effects of four longan seed polyphenols(P4, P6, P7 and P8) on H2O2-induced NIH-3T3 cells. The results confirmed that P4 and P6 showed the strongest antioxidant effects from the perspective of thermochemistry, however, the relationship of microcalorimetric with conventional cell-based results was poor, indicating that the microcalorimetric assay might not be an optimal model for evaluating the antioxidant properties of phenolic compounds. We further selected 12 kinds of representive phenolic compounds to validate the feasibility of microcalorimetric assay, and the results showed that there were some discrepancies between the microcalorimetric and previously chemical-based results, suggesting that the enthalpy change of the cells was not an accurate reflection of antioxidant effects of phenolic compounds. Therefore, microcalorimetric assay was not suitable for the screening and evaluating of antioxidant compounds. 5. Impedance-based RTCA was used for the rapid screening of antioxidant phenolsRTCA was used to investigate the effects of four longan seed polyphenols(P4, P6, P7 and P8) on H2O2-induced NIH-3T3 cells. The results confirmed that P4 and P6 showed the strongest antioxidant effects from the perspective of electrochemistry, and the RTCA and conventional cell-based results was highly correlated, indicating that the RTCA assay might be an optimal model for evaluating the antioxidant properties of phenolic compounds. We further selected 12 kinds of representive phenolic compounds to validate the feasibility of RTCA model, and the results showed that RTCA results were consistent with previously chemical-based results, suggesting that the impedance change in RTCA system was an accurate reflection of antioxidant effects of phenolic compounds. Therefore, RTCA model was suitable for the rapid screening and accurate evaluation of antioxidant compounds in cell physiological level.