Competitive Protein Binding Assay of Naproxen by Human Serum Albumin Functionalized Silicon Dioxide Nanoparticles.

We have developed a new competitive protein binding assay (CPBA) based on human serum albumin functionalized silicon dioxide nanoparticles (nano-SiO2-HSA) that can be used for naproxen determination in urine. Compared with a conventional multi-well reaction plate, nano-SiO2 with a high surface-area-to-volume ratio could be introduced as a stationary phase, markedly improving the analytical performance. Nano-SiO2-HSA and horseradish peroxidase-labeled-naproxen (HRP-naproxen) were prepared for the present CPBA method. In this study, a direct competitive binding to nano-SiO2-HSAwas performed between the free naproxen in the sample and HRP-naproxen. Thus, the catalytic color reactions were investigated on an HRP/3,3'5,5'-tetramethylbenzidine (TMB)/H2O2 system by the HRP-naproxen/nano-SiO2-HSA composite for quantitative measurement via an ultraviolet spectrophotometer. A series of validation experiments indicated that our proposed methods can be applied satisfactorily to the determination of naproxen in urine samples. As a proof of principle, the newly developed nano-CPBA method for the quantification of naproxen in urine can be expected to have the advantages of low costs, fast speed, high accuracy, and relatively simple instrument requirements. Our method could be capable of expanding the analytical applications of nanomaterials and of determining other small-molecule compounds from various biological samples.

Panax ginseng Polysaccharide Protected H9c2 Cardiomyocyte From Hypoxia/Reoxygenation Injury Through Regulating Mitochondrial Metabolism and RISK Pathway.

Background and Objective: Ischemic heart disease (IHD) has been the major issue of public health. Panax ginseng (ginseng) has been verified as an effective traditional Chinese medicines and exerted cardioprotective effect. This study aimed to investigate the polysaccharide fraction of ginseng on hypoxia/reoxygenation (H/R) injury in cardiomyocytes and the underlying mechanisms. Methods: Ginseng was extracted by ethanol and fractionated by high-speed counter current chromatography (HSCCC) and column separation. The cardioprotective effect was evaluated in H9c2 cardiomyocytes underwent H/R treatment. The cell viability, apoptosis and mitochondrial respiration were examined. Results: An acid polysaccharides fraction of ginseng (AP1) was identified the most effective fraction in protecting cardiomyocytes from H/R injury. AP1 restored the mitochondrial function by maintaining mitochondrial membrane potential (MMP), blocking the release of cytochrome C, and increasing the ATP generation and oxygen consumption rate (OCR) of cardiomyocytes. Meanwhile, AP1 induced the expression of glucocorticoid receptor (GR) and estrogen receptor (ER) which further activated reperfusion injury salvage kinase (RISK) pathway. Finally, AP1 increased nitric oxide (NO) production and regulated endothelial function by increasing endothelial NO synthase (eNOS) expression and decreasing inducible NOS (iNOS) expression in H/R injury. Conclusion: The results suggested that AP1 exerted a protective effect in myocardial H/R injury mainly through maintaining myocardial mitochondrial function, thereby inhibiting myocardial H/R caused apoptosis and increasing the expressions of GR and ER, which in turn mediated the activation of RISK pathway and eNOS-dependent mechanism to resist the reperfusion injury.

An integrated strategy for rapid discovery and identification of quality markers in Guanxin Kangtai preparation using UHPLC-TOF/MS and multivariate statistical analysis.

BACKGROUND: Guanxin Kangtai preparation (GXKT), consisting of Panax ginseng, Panax notoginseng and Ilex pubescens, is a new proprietary Chinese medicines under development for treating coronary heart disease. Like other Chinese medicines, the components of GXKT were complex and the bioactive compounds remained unclear. PURPOSE: To discover bioactive compounds as quality markers (Q-markers) for better quality control of GXKT. STUDY DESIGN: Chinese medicines was separated into fractions. The correlation between chemical information and bioactivity of these fractions were analyzed with multivariate statistical methods to discover bioactive compounds responsible for the actions of Chinese medicine. METHOD: GXKT was separated into fractions by using high-performance liquid chromatography (HPLC). Ultra HPLC coupled with time-of-flight mass spectrometer (UHPLC-TOF/MS) was applied to detect compound information from these fractions to form a chemical database. The bioactivity of these fractions in protecting cardiomyocytes from ischemia/reperfusion injury was examined in H9c2 cells that were exposed to hypoxia followed by reoxygenation (H/R). Then, partial least square model and orthogonal projections to latent structures discriminant analysis were employed to discover bioactive compounds from the chemical database that were positively correlated with the bioactivity of GXKT fractions. Finally, the bioactivity of these compounds was confirmed by bioassay in H9c2 cells. RESULTS: The chemical information of 120 fractions separated from GXKT was detected and extracted by UHPLC-TOF/MS, and a chemical database including 61 high abundance compounds were formed from all fractions. These fractions produced different extent of protective effect to H9c2 cell underwent H/R treatment with cell viability ranging from 33.43% to 74.91%, demonstrating the separation of bioactive compounds among different fractions. The multivariate analysis discovered 16 compounds from GXKT positively correlated with the bioactivity of GXKT. Of these compounds, 6 compounds, i.e.: ginsenoside Rg1, Rb1, Rh1, Rc, ilexsaponin A1, and chikusetsusaponin IVa were chemical identified and also confirmed for their responsibility to the action of GXKT by bioassay. CONCLUSION: Ginsenoside Rg1, Rb1, Rh1, Rc, ilexsaponin A1, and chikusetsusaponin IVa were bioactive compounds and qualified as Q-markers for quality control of GXKT. This research provided a useful reference for the quality research of Chinese medicines.