Nrf2-inducing and HMG-CoA reductase inhibitory activities of a polyphenol-rich fraction of Guazuma ulmifolia leaves

To fractionate and identify polyphenols from Guazuma ulmifolia Lam. leaves, and to explore their antioxidant, 5-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitory, and Nrf2 modulatory activities. Methods: The 1,1-diphenyl-2-picrylhydrazyl assay was used to evaluate the antioxidant activity of a polyphenolic fraction of the extract of Guazuma ulmifolia Lam. leaves. THP-1 gene reporter cell lines constructed with a transcriptional response element specific for Nrf2 and a minimal promoter for the firefly luciferase–green fluorescent protein transgene were used to determine the effect of the polyphenolic fraction on the Nrf2 signaling pathway. Furthermore, an assay of HMG-CoA reductase inhibitory activity was performed by using a commercial enzyme kit. Polyphenolic compounds were identified by liquid chromatography-tandem mass spectrometry. Results: The polyphenolic fraction showed fairly strong antioxidant activity [IC50 = (14.90 ± 4.70) μg/mL] and inhibited HMG-CoA reductase activity by 69.10％, which was slightly lower than that by pravastatin (84.37％) and quercetin (84.25％). Additionally, the polyphenolic fraction activated the Nrf2 antioxidant signaling pathway at 500 μg/mL. Eleven subfractions resulting from the column chromatography separation of the polyphenolic fraction also showed relatively strong antioxidant activities (IC50: 17.46–217.14 μg/mL). The subfraction (F6) stimulated the Nrf2 signaling pathway and had HMG-CoA reductase inhibitory activity (65.43％). Moreover, the subfraction contained two main flavonoids: quercetin and quercimeritrin. Conclusions: The polyphenolic fraction of Guazuma ulmifolia could induce antioxidant genes via the Nrf2/antioxidant regulatory elements pathway, and is a promising candidate for an inhibitor of HMG-CoA reductase.

Dissolution kinetics of Compound Danshen Tablets based on cell bioelectrical sensing effect / 中草药

Objective: Establishing the model of cell bioelectrical sensing effect of Compound Danshen Tablets to study its dissolution kinetics. Methods: By means of real-time cell-based assay, the in vitro dissolution of Compound Danshen Tablets can be investigated, and then the dissolution kinetics model can also be established. In addition, the result was compared and verified by UV-Vis. Results: The cell line with specific dependence on Compound Danshen Tablets was screened by CCK-8 experiment and RTCA experiment. The dissolution kinetics model of Compound Danshen Tablets based on RTCA technology was established, and the best fitting model was obtained: Weibull model ln{ln[1/(1-Q)] =1.071 4 lnt-3.736 7; Establish a dissolution kinetic model of Compound Danshen Tablets based on UV spectrophotometry to obtain the best fitting model, Weibull model ln{ln[1/(1-Q)]}=1.080 4 lnt-3.723 4; Comparing the two Weibull models, the RTCA fitted model worked better. Conclusion: The application of RTCA in the dissolution kinetics of traditional Chinese medicine compound solid preparations is feasible, Which provides new ideas for traditional Chinese medicines and the quality evaluation of traditional Chinese medicine compunds.

High-content analysis of in vitro hepatocyte injury induced by various hepatotoxicants

In vitro prediction of hepatotoxicity can enhance the performance of non-clinical animal testing for identifying chemical hazards. In this study, we assessed high-content analysis (HCA) using multi-parameter cell-based assays as an in vitro hepatotoxicity testing model using various hepatotoxicants and human hepatocytes such as HepG2 cells and human primary hepatocytes (hPHs). Both hepatocyte types were exposed separately to multiple doses of ten hepatotoxicants associated with liver injury whose mechanisms of action have been described. HCA data were obtained using fluorescence probes for nuclear size (Hoechst), mitochondrial membrane potential (TMRM), cytosolic free calcium (Fluo-4AM), and lipid peroxidation (BODIPY). Cellular alterations were observed in response to all hepatotoxicants tested. The most sensitive parameter was TMRM, with high sensitivity at a low dose, next was BODIPY, followed by Fluo-4AM. HCA data from HepG2 cells and hPHs were generally concordant, although some inconsistencies were noted. Both hepatocyte types showed mild or severe mitochondrial impairment and lipid peroxidation in response to several hepatotoxicants. The results demonstrate that the application of HCA to in vitro hepatotoxicity testing enables more efficient hazard identification, and further, they suggest that certain parameters could serve as sensitive endpoints for predicting the hepatotoxic potential of chemical compounds.

Molecular and functional analysis of monoclonal antibodies in support of biologics development

Monoclonal antibody (mAb)-based therapeutics are playing an increasingly important role in the treatment or prevention of many important diseases such as cancers, autoimmune disorders, and infectious diseases. Multi-domain mAbs are far more complex than small molecule drugs with intrinsic heterogeneities. The critical quality attributes of a given mAb, including structure, post-translational modifications, and functions at biomolecular and cellular levels, need to be defined and profiled in details during the developmental phases of a biologics. These critical quality attributes, outlined in this review, serve an important database for defining the drug properties during commercial production phase as well as post licensure life cycle management. Specially, the molecular characterization, functional assessment, and effector function analysis of mAbs, are reviewed with respect to the critical parameters and the methods used for obtaining them. The three groups of analytical methods are three essential and integral facets making up the whole analytical package for a mAb-based drug. Such a package is critically important for the licensure and the post-licensure life cycle management of a therapeutic or prophylactic biologics. In addition, the basic principles on the evaluation of biosimilar mAbs were discussed briefly based on the recommendations by the World Health Organization.

A cell-based high-throughput approach to identify inhibitors of influenza A virus

Influenza is one of the most common infections threatening public health worldwide and is caused by the influenza virus. Rapid emergence of drug resistance has led to an urgent need to develop new anti-influenza inhibitors. In this study we established a 293T cell line that constitutively synthesizes a virus-based negative strand RNA, which expresses Gaussia luciferase upon influenza A virus infection. Using this cell line, an assay was developed and optimized to search for inhibitors of influenza virus replication. Biochemical studies and statistical analyses presented herein demonstrate the sensitivity and reproducibility of the assay in a high-throughput format (Z' factor value>0.8). A pilot screening provides further evidence for validation of the assay. Taken together, this work provides a simple, convenient, and reliable HTS assay to identify compounds with anti-influenza activity.