Mitochondrial proteomic analysis of ecdysterone protection against oxidative damage in human lens epithelial cells

AIM: To investigate the protective effects of the natural medicinal monomer ecdysterone(ECR) with estrogenic activity against oxidative damage in human lens epithelial cells B3(HLE-B3) caused by hydrogen peroxide 21(H2 O2) and to pursue the possible mitochondrial proteomic regularity of the protective effects. · METHODS: HLE-B3 cells were treated with H2O2(300μmol/L),β-estuarial(E2; 10-8mol/L) and H2 O2,ECR(10-6mol/L) and H2 O2,or left untreated. Altered expression of all mitochondrial proteins was analyzed by protein array and surface-enhanced laser desorption ionization time of flight mass spectrometry(SELDI-TOF-MS). The mass/charge(M/Z) ratios of each peak were tested by the Kruskal-Wallis rank sum test,and the protein peak value of the M/Z ratio for each treatment by pair comparison was analyzed with the Nemenyi test. ·RESULTS: H2O2 up-regulated expression of two protein spots(with M/Z of 6 532 and 6 809). When E2 mitigated the oxidative damage,the expression of one protein spot(M/Z 6 532) was down-regulated. In contrast,ECR downregulated both of protein spots(M/Z 6 532 and 6 809). · CONCLUSION: ECR could effectively inhibite H2O2 induced oxidative damage in HLE-B3 cells. The protein spot at M/Z of 6 532 might be the target spot of ECR against oxidative damage induced by H2 O2.

Study on the Flow Injection Micro-Column Pre-Separation System Coupled With High Performance Liquid Chromatography for the Determination of Ecdysterone in Traditional Chinese Medicine

A flow injection （FI） micro-column system coupled with high performance liquid chromatography （HPLC） was proposed for the pre-separation and determination of active organic component （ecdysterone） in traditional Chinese medicine, Loulu. The factors influencing separation performance were investigated and optimized. Under the optimal conditions, the contents of ecdysterone in Loulu were determined by HPLC system using MeOH-H2O （40: 60,V/V） as the mobile phase at a flow rate of 1. 0 mL/min. The calibration curve was linear in the range of 0. 5 100 mg/L of ecdysterone concentrations. The detection limit of the analyte was 0. 11mol/L（3） with a precision of 0. 38% RSD （n=7 f c= 10. 0 mg/L）. The average recovery of the method was 98. 7%. The proposed method has been applied to determine ecdysterone in practical samples, and the determined values by both external standard method and standard addition method were in good agreement. Compared to the traditional solid extraction method, the system proposed has the advantages of simple procedure, good reproducibility, minimum volume requirement, reduction of matrix interference and low contamination risk.

Phytoecdysteroids from <i>Silene praemixta</i>

The waste products of the production of ecdysterone (3) from Silene praemixta M. Pop. (Caryophyllaceae) were investigated. The column chromatography of the technological waste has allowed to isolating 2-de- soxyecdysone (1), 2-deoxyecdysterone (2), ecdysterone (3) and 22-O-β-D-glucopyranoside of 2-deoxyecdy- sone (4). Glucoside 4 was found for the first time in the plant Silene praemixta. Ecdysteroids 1, 2 prevailed in the sum. Compounds 3 and 4 were minor components of the waste products. The identification of com- pounds 1-4 was carried out by using the modern me- thods of 1D and 2D NMR spectroscopy: 1H, 13C, DEPT, 1H-1H COSY, HSQC, NOESY, IR spectroscopy and ESI MS.