The effect of α-tocopherol and dithiothreitol in ameliorating emamectin benzoate cytotoxicity in human K562 cells involving the modulation of ROS accumulation and NF-κB signaling.

Emamectin benzoate (EMB) toxicity contributes a potential risk to environment and human health. To investigate the effect of α-tocopherol (VitE) and dithiothreitol (DTT) in ameliorating EMB-induced cytotoxicity in human K562 cells, in vitro cultured human K562 cells were incubated with different concentrations of EMB in supplement with VitE and DTT when the cells were in the logarithmic phase. Next, the cell growth inhibition was evaluated using the MTT assay and cellular morphology observation. Reactive oxygen species (ROS) production was monitored using DCFH-DA probe and NF-κB signaling was determined using Western blotting. The results demonstrated that treatment with EMB (time- and concentration-dependent) showed significantly greater inhibition on K562 cell viability, heavier chromatin condensation and DNA fragmentation, and stronger suppression of NF-κB/p105 and p65/RelA expression of K562 cells than the control group (p < 0.01). The supplementation of VitE or DTT could help protect K562 cells against EMB-induced cytotoxicity by improving cell viability, preventing ROS accumulation and up-regulating NF-κB signaling through their ameliorating effects against oxidative stress induced by EMB. VitE had a stronger synergistic effect in limiting EMB cytotoxicity than DTT. Our findings indicate that VitE and DTT are potent antioxidants for human K562 cells, offering a promising means of ameliorating EMB cytotoxicity.

[Fast determination of betaine in Lycii Fructus by ultra-performance convergence chromatography-tandem mass spectrometry].

Over the past 2500 years, Lycii Fructus has been widely used as a functional food and tonic in Chinese herbal medicine. It can nourish the liver and kidneys, moisten the lungs, and improve eyesight. In this study, a new rapid and sensitive method has been developed for the quantitative determination of betaine (index compound) in Lycii Fructus by using ultra-performance convergence chromatography-tandem mass spectrometry (UPC2-MS). The separation of betaine was successfully achieved on an ACQUITY UPC2 BEH 2-EP column (150 mm×2.1 mm, 1.7 µm), with isocratic elution by a CO2-methanol (80:20, v/v) solvent at a flow rate of 0.7 mL/min. The conditions used in the separation process were as follows:modifier, 0.1% (v/v) formic acid in methanol; column temperature, 40℃; backpressure, 1.31×107 Pa; injection volume, 1 µL; and retention time, 3 min. The MS system was equipped with an electrospray ionization (ESI) ion source and operated in the selected ion recording (SIR) and positive ion mode. Under the abovementioned conditions, the calibration curve was obtained. The linear range of detection was 0.5-50.0 µg/mL, with a correlation coefficient of 0.9992, and the limit of detection (LOD) was found to be 0.013 µg/mL. The validity of the method was tested by analyses of precision, repeatability, stability, and accuracy (average recovery:96.3%). Finally, the developed method was applied to analyze 11 batches of samples. The results indicated that this method was suitable for evaluating the quality of Lycii Fructus.

UPLC-MS/MS analysis for antioxidant components of Lycii Fructus based on spectrum-effect relationship.

Lycii Fructus is widely cultivated in the Northwest China. It is well-known for its antiaging effect in traditional Chinese medicines (TCMs), but the effective components are not clear. In this work, the ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) was used to study the antioxidant components of Lycii Fructus through analyzing the spectrum-effect relationship, and the positive correlation components with antioxidant activity were partially identified. The extractums of Lycii Fructus were adsorbed with macroporous resin, and then eluted with water and 30%, 60%, 90% ethanol in turn. The extract fraction eluted with 60% ethanol was determined as the best, and was taken for subsequent experiments. With the above separation method, UPLC fingerprints of thirty batches of Lycii Fructus (from different areas) were obtained, and thirty common peaks were selected through similarity analysis (SA). Combined with the data of the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) assays, the spectrum-effect relationship was studied. The results showed that the main peaks with antioxidant activity were P14, P26, P8, and P21 for DPPH, and P26, P14, P21, and P19 for ABTS. Using the UPLC-MS/MS data, peaks P14, P19, P21, and P30 were respectively identified as chlorogenic acid, quercetin, kaempferol, and isorhamnetin, and then the results were confirmed through comparison with the standards and other references. Finally, their strong antioxidant activities were validated experimentally.

Optimization of a Decoction Process for an Herbal Formula Using a Response Surface Methodology.

An herbal formula, Huang-Qi-Liu-Yi Tang, is a prescription medicine that has been commonly used for the treatment of prostatitis and condyloma acuminatum over the last thousand years. In this study, response surface methodology, with a Box-Behnken design (BBD), was used to optimize the decoction conditions of an herbal formula. Astragaloside, calycosin-7-glucoside, glycyrrhizic acid, liquiritin, polysaccharide, and extractum were used as multiple evaluation markers. Soak time, water-to-medicinal herb ratio, and extraction time were determined as the three main variables by single-factor experiments and further optimized to obtain the maximum yields for the six marker compounds. Data from well-designed experiments were fitted to obtain second-order polynomial equations using multiple regression analysis, and the accuracies of these equations were evaluated and verified using statistical methods. By solving the regression equations and analyzing the three-dimensional response surfaces, optimum conditions were obtained and are summarized as follows: soak time of 57 min, water-to-medicinal herb ratio of 9:1 (mL/g), and extraction time of 48 min. Under optimized conditions, the experimental yields of astragaloside, calycosin-7-glucoside, glycyrrhizic acid, liquiritin, polysaccharide, and extractum were 0.13, 0.085, 1.64, 1.56, 72.19, and 25.64%, respectively, which was in good agreement with the values predicted by the BBD.