A coacervate extraction method, based on hexafluoroisopropanol (HFIP)-induced catanionic surfactants and coupled with a back-extraction procedure, was developed for separation and purification of proteins, using sodium dodecyl sulfate (SDS) and dodecyltrimethyl ammonium bromide (DTAB) as representative catanionic surfactants and lysozyme as a model protein. After the coacervate extraction and back extraction, the obtained lysozyme solutions were examined in terms of quantitative analysis by capillary electrophoresis, bacteriolytic activity, and circular dichroism (CD). The effects of several parameters including back-extraction solvent, HFIP content, total surfactant concentration, and SDS/DTAB molar ratio were investigated in detail on the extraction efficiency and activity of lysozyme. Under the optimized extraction conditions (66 mM KH2PO4 buffer with pH 6.2 as back-extraction solvent, SDS/DTAB molar ratio = 1:1 mol/mol, total surfactant concentration = 30 mM, HIFP concentration = 8 % v/v), the extraction recovery was 89.8 % (±4.7, n = 3), limit of detection was 2.2 (±0.3, n = 3) μg mL−1, and meanwhile nearly 65 % of native lysozyme activity was retained. In addition, the activity and CD assays showed that SDS/DTAB molar ratio had a significant influence on the activity and structure of lysozyme after extraction. The DTAB-rich extraction systems, in which the DTAB mole fraction was equal to or larger than 70 %, could keep the activity and structure of lysozyme almost in the native state.

Garcinia xanthochymus is a widely used folk medicine in southwestern China. Previous studies indicated it possesses potential anti-diabetic activities both in vitro (Fu et al., 2014; Nguyen et al., 2017) and in vivo (Shivanand et al., 2017). To discover bioactive ingredients from it and unveil their mechanism of action against diabetes, the present study was designed to isolate constituents from extract of G. xanthochymus, determine their structures, screen their activities and investigate mechanism of action of the active substances. Twenty compounds including a new depsidone named garciniadepsidone A (20) and 19 known xanthones were obtained. All of them were screened to discover the active compounds with anti-diabetic activities. Finally, three xanthones including 12b-hydroxy-des-d-garcigerrin (5), 1,2,5,6-tretrahydroxy-4-(1,1-dimethyl-2-propenyl)-7-(3-methyl-2-butenyl) xanthone (13) and 1,5,6-trihydroxy-7,8-di(3-methyl-2-butenyl)-6′,6′-dimethylpyrano (2′,3′:3,4) xanthone (18) were found to be able to significantly stimulate the glucose uptake in the skeleton muscle cells. The effects of the three compounds were comparable to those of insulin and metformin. Based on molecular mechanistic study, it was found that both of compound 5 and 13 promoted glucose uptake by activating phosphatidylinositol-3 kinase (PI3K)/the serine/threonine kinase protein kinase B (PKB/Akt) signaling pathway and AMP-activated protein kinase (AMPK) signaling pathway, resulting in the translocation of GLUT4 in L6 myotubes without affecting the expression of GLUT4. Compound 5 and 13 have great potential to be developed as promising leads to target diabetes.Download high-res image (154KB)Download full-size image

•Gemini pyrrolidinium surfactants were used as carriers in sweeping-MEKC.•C16-4-C16PB is the most suitable sweeping carrier.•Nine organic water pollutants were simultaneously concentrated and separated.•Sensitivity enhancement factors for nine analytes are in range of 310–580.•C16-4-C16PB-based sweeping–MEKC is suited for the analysis of real water samples.A series of novel cationic gemini surfactants with pyrrolidinium head groups, 1,1′-(butane-1,s-alkyl) bis (1-alkylpyrrolidinium) (Cn-4-CnPB, n = 12, 14, 16), were employed as carriers in sweeping–micellar electrokinetic chromatography (sweeping-MEKC) for simultaneous detection of nine organic water pollutants, including polycyclic aromatic hydrocarbons, sulfonamides and steroids. The sweeping and separation conditions were optimized. Cn-4-CnPB (n = 12, 14, 16) were compared with cetyltrimethylammonium bromide (CTAB) in terms of their abilities to preconcentrate and separate the nine analytes. Under the optimized conditions, the sensitivity enhancement factors based on the peak height (SEFsHeight) were ca. 310–580 of C16-4-C16PB, which were higher than those of C14-4-C14PB (120–290) and C12-4-C12PB (110–160). Meanwhile, the SEFsHeight of C16-4-C16PB were higher than those of 30% (v/v) methanol-modified CTAB (140–320). The C16-4-C16PB based sweeping-MEKC, coupled with offline solid phase extraction and UV detection (228 nm), was used to analyze spiked environmental water samples. The nine analytes were successfully separated and detected. The limit of detection (S/N = 3) was in range of 2.79–3.76 ng/mL, and the recovery ranged from 70.8% to 95.5% with the RSDs less than 9.89%. This study confirms that the C16-4-C16PB based sweeping-MEKC has significant advantages over the CTAB based sweeping-MEKC and it is a promising method for sensitive and simultaneous detection of polycyclic aromatic hydrocarbons, sulfonamides and steroids in environmental water samples.

•Gemini surfactants (Cn-4-CnPB) were used as novel dynamic coating additives in CE.•HFIP can improve the effect of Cn-4-CnPB on the adsorption and separation of proteins.•Neutral and charged proteins were well separated with 115,000–318,000 plates/m.The separation of acidic and basic proteins using CE has been limited in part due to the adsorption of proteins onto the capillary wall. In this work, the efficient control of EOF and the simultaneous separation of acidic and basic proteins are achieved by use of C18-4-C18PB as a dynamic coating additive, which is a representative surfactant for 1,1′-(butane-1,s-alkyl)bis(1-alkylpyrrolidinium) bromide (Cn-4-CnPB, n = 10, 12, 14, 16 and 18). C18-4-C18PB exhibits a powerful capability in the reversal of EOF, and a low concentration even less than 0.001 mM is sufficient to reverse EOF at the tested pH values (3.0–9.0). Baseline separation of eight proteins with sharp peaks and high efficiencies (54,000–297,000 plates/m) is obtained with 30 mM NaH2PO4 buffer (pH 5.0) containing 4 mM C18-4-C18PB. At the same buffer condition, the Cn-4-CnPB with shorter alkyl chain (n = 10, 12, 14, 16) cannot achieve the same effective protein separation as C18-4-C18PB. However, the combined use of small amounts (≤0.5%, v/v) of hexafluoroisopropanol (HFIP) and Cn-4-CnPB (n = 10, 12, 14, 16) as additives can completely separate all eight proteins with high efficiencies of 81,000–318,000 plates/m. The RSDs of migration time are less than 0.80% and 5.84% for run-to-run and day-to-day assays (n = 5), respectively, and the protein recoveries are larger than 90.15%. To the best of our knowledge, this is the first report on the simultaneous separation of acidic and basic proteins using Cn-4-CnPB surfactants or Cn-4-CnPB surfactants combined with HFIP as dynamic coating additives.

•A screening method using GSK-3β-immobilized MBs coupled with HPLC-UV was developed.•This method was used to screen the inhibitory activity of extracts of 15 TCMs.•A new inhibitor of GSK-3β, fukugetin, was found from Garcinia xanthochymus.•Fukugetin was a non-ATP competitive inhibitor and its IC50 was 3.18 ± 0.07 μM.Glycogen synthase kinase-3β (GSK-3β) was immobilized on magnetic beads (MBs) by affinity method for the first time. The enzyme-immobilized MBs were coupled with high-performance liquid chromatography-ultraviolet (HPLC-UV) technique to establish a cost-effective and reliable method for screening of inhibitors of GSK-3β. A peptide substrate of GSK-3β containing a tyrosine residue was employed since it can be sensitively detected by UV detector at 214 nm. The substrate and its phosphorylated product were separated by baseline within 10 min. The enzyme activity was determined by the quantification of peak area of the product. Parameters including enzyme immobilization, enzyme reaction and the performance of immobilized-enzyme were investigated. The immobilized enzyme can be reused for 10 times and remain stable for 4 days at 4 °C. The inhibitory activities of extracts of 15 traditional Chinese medicines (TCMs) were screened. As a result, three of them including Euonymus fortunei, Amygdalus communis and Garcinia xanthochymus were found possessing high inhibitory activities (inhibition rate >90%). From G. xanthochymus, a new inhibitor of GSK-3β, fukugetin, was discovered with an IC50 value of 3.18 ± 0.07 μM. Enzyme kinetics and molecular docking experiments further revealed the inhibitory mechanism, indicating fukugetin was a non-ATP competitive inhibitor interacting with the phosphate recognizing substrate binding site of GSK-3β.

•A novel method, CE with ILIF detection, was developed for the determination of alditols.•CE separation parameters were optimised and the method was validated.•The applicability of this method was demonstrated by the analysis of real samples.•The results obtained by CE-ILIF method were similar to those by CE-IUV method.A novel method was developed based on capillary electrophoresis with indirect laser-induced fluorescence detection for the determination of alditols including xylitol, sorbitol, arabinitol and mannitol. The baseline separation was achieved with a capillary with 30 μm inner diameter and running buffer (pH 9.6) composed of 40 mM sodium tetraborate, 5% (v/v) methanol, 10−5 M sodium fluorescein as background fluorescence. Calibration curves (0.2–2.0 mg mL−1) showed good linear correlations (r > 0.999). The detection limits of the alditols were in the range of 19.0–24.4 μg mL−1. The intra-day and inter-day RSDs for migration times and peak areas were less than 1.6% and 4.3%, respectively. The average recoveries were in the range of 99.6–105.5%. The developed method was successfully applied to determination of alditols in different real samples, and the results were compared with those of capillary electrophoresis with indirect ultraviolet detection method.

The doxorubicin (DOX) uptake in single human leukemia K562 cells with changes in both drug dosage and exposure period was studied using capillary electrophoresis (CE) coupled with laser-induced fluorescence (LIF) detection. The cells were treated with DOX at different concentrations (1, 3, 10, 20, 30, and 50 μM) and for different exposure times (1, 3, 5, 24, and 48 h). At least 20 cells were analyzed for each DOX-treated cell population. A marked heterogeneity in DOX uptake among single cells was observed, because the relative standard deviation of the uptake of DOX by single cells ranged from 24.0% to 61.1% within each cell population. The cell-to-cell heterogeneity in DOX uptake first decreased and then became constant with increasing drug concentration, but it did not exhibit regular variation with increasing exposure time. The mean DOX uptake was a linear function of drug concentration (r ≥ 0.9667). In terms of the correlation with exposure time, the mean DOX uptake reached its maximum at 3 h for the cell populations treated with 1–10 μM DOX, while it kept increasing during 48 h exposure of cell populations to 20–50 μM DOX. Because it eliminates DOX fluorescence quenching and sample loss, the CE-LIF method directly detects the true DOX uptake by single cells, and thus presents accurate information on both the cell-to-cell heterogeneity in DOX uptake and the patterns of DOX uptake in K562 cells as functions of drug concentration and exposure time.

Hexafluoroisopropanol (HFIP)-induced sodium dodecyl sulfate/dodecyltrimethylammonium bromide (SDS/DTAB) catanionic surfactant coacervate extraction method coupled with high performance liquid chromatography (HPLC) was used to detect the migration of phthalates from disposable tablewares to drinking water. The concentration factors are larger than 82 and extraction recoveries over 53% for water samples spiked with 100 or 200 ng/mL phthalates. Limit of detection is in the range of 1.0–2.6 ng/mL. Good linearity with correlation coefficients larger than 0.9985 is obtained in the concentration of 20–1500 or 40–3000 ng/mL. Relative recoveries are from 82.4% to 123.6% for water samples spiked with 30/60, 250/500, and 1500/3000 ng/mL phthalates, respectively. Relative standard deviations (RSDs) are 0.4%–7.4% for intraday precision (n=5) and 0.6%–7.8% for interday precision (n=3). Four of studied phthalates are found in the drinking water samples prepared from four kinds of tablewares.