Detection of Various Traditional Chinese Medicinal Metabolites as Angiotensin-Converting Enzyme Inhibitors: Molecular Docking, Activity Testing, and Surface Plasmon Resonance Approaches.

Angiotensin-converting enzyme 1 (ACE1) is a peptide involved in fluid and blood pressure management. It regulates blood pressure by converting angiotensin I to angiotensin II, which has vasoconstrictive effects. Previous studies have shown that certain compounds of natural origin can inhibit the activity of angiotensin-converting enzymes and exert blood pressure-regulating effects. Surface Plasmon Resonance (SPR) biosensor technology is the industry standard method for observing biomolecule interactions. In our study, we used molecular simulation methods to investigate the docking energies of various herbal metabolites with ACE1 proteins, tested the real-time binding affinities between various herbal metabolites and sACE1 by SPR, and analyzed the relationship between real-time binding affinity and docking energy. In addition, to further explore the connection between inhibitor activity and real-time binding affinity, several herbal metabolites' in vitro inhibitory activities were tested using an ACE1 activity test kit. The molecular docking simulation technique's results and the real-time affinity tested by the SPR technique were found to be negatively correlated, and the virtual docking technique still has some drawbacks as a tool for forecasting proteins' affinities to the metabolites of Chinese herbal metabolites. There may be a positive correlation between the enzyme inhibitory activity and the real-time affinity detected by the SPR technique, and the results from the SPR technique may provide convincing evidence to prove the interaction between herbal metabolites and ACE1 target proteins.

[Construction of IL-6/STAT3 reporter gene system for screening and validation of traditional Chinese medicine monomers inhibiting IL-6/STAT3 signaling pathway].

It is important to find monomers of traditional Chinese medicine that can inhibit the activation of IL-6/STAT3 signaling pathway to suppress the growth and deterioration of tumors. A novel expression vector containing STAT3 enhancer sequence and NanoLuc (NLuc) reporter gene sequence was constructed by gene recombination technology, then a cell line expressing NLuc luciferase and regulated by STAT3 was further established. The cell line was used to quantitatively detect the regulatory effects of various traditional Chinese medicine monomers on the IL-6/STAT3 signal pathway. The effects of the traditional Chinese medicine monomers inhibiting the IL-6/STAT3 signal pathway were verified by Western blotting immunoassay and Real-time PCR analysis. By enzyme digestion and DNA sequencing analysis, it showed that the reporter gene expression vector pQCXIP-STAT3-Nluc was constructed successfully. The addition of Interleukin-6 (IL-6), a stimulator of STAT3 transcription factor, to the constructed cell line specifically enhanced the luciferase enzymatic reaction in a dose dependent manner. These results demonstrated that the cell line expressing NLuc luciferase and regulated by STAT3 has been constructed successfully. We used the the cell line screened out dendrobiine and tetrandrine which could significantly inhibit IL-6/STAT3 signal pathway and down-regulated the expression of Bcl-2 and Bcl-x in a dose-dependent manner. In conclusion, we have constructed an efficient reporter gene system to detect the transcriptional activity of STAT3, by which the Chinese medicine monomers inhibiting IL-6/STAT3 signaling pathway were successfully screened out, which has some potential theoretical and practical values.

[Progress of gene editing technologies and prospect in traditional Chinese medicine].

Gene editing is a kind of technologies that makes precise modification to the genome. It can be used to knock out/in and replace the specific DNA fragment, and make accurate gene editing on the genome level. The essence of the technique is the DNA sequence change with use of non homologous end link repair and homologous recombination repair, combined with specific DNA target recognition and endonuclease.This technology has wide range of development prospects and high application value in terms of scientific research, agriculture, medical treatment and other fields. In the field of gene therapy, gene editing technology has achieved cross-time success in cancers such as leukemia, genetic disorders such as hemophilia, thalassemia, multiple muscle nutritional disorders and retrovirus associated infectious diseases such as AIDS and other diseases. The preparation work for new experimental methods and animal models combined with gene editing technology is under rapid development and improvement. Laboratories around the world have also applied gene editing technique in prevention of malaria, organ transplantation, biological pharmaceuticals, agricultural breeding improvement, resurrection of extinct species, and other research areas. This paper summarizes the application and development status of gene editing technique in the above fields, and also preliminarily explores the potential application prospect of the technology in the field of traditional Chinese medicine, and discusses the present controversy and thoughts.

[Study on compatibility of traditional Chinese medicine active constituents with antioxidant activity].

OBJECTIVE: To find a research method suitable to multi-component multi-index composition-activity relationship of traditional Chinese medicines (TCMs) with the antioxidant activity of four compatible active constituents of TCMs as the subject. METHOD: LARS-based regression algorithm and comprehensive weight coefficient method were adopted to study in vitro clearance of DPPH and polyaromatic hydrocarbon with different doses of four compatible active constituents--total flavonoids of Glycyrrhizae Radix et Rhizoma (TFG), Ginkgo Folium extract (GBE), total flavonoids of Epimedii Folium (TFE) and astragaloside (AST) according to the comprehensive efficacy assessment procedures, that is test design, efficacy test and mathematical modeling (model verification). RESULT: LARS and comprehensive weight coefficient method was adopted to assess the optimal comprehensive efficacy of clearing DPPH and polyaromatic hydrocarbon. The results showed that the optimal dose combination of compatible constituents is TFG-GBE-TFE-AST 1: 0.2545: 0.007 6:0.011 5. CONCLUSION: The above compatibility of TCMs constituents can effectively clear polyaromatic hydrocarbon and DPPH with a good antioxidation. The uniform design adopted in this experiment, in combination of synthetic weight method, is suitable to the data analysis of "non-linear and small-sample" biotic experiment and the study on screening and assessment of compatibility of TCMs constituents.

[Research of herb components on scavenging harmful components and reducing cytotoxicity of cigarette smoke].

OBJECTIVE: To study the in vitro effect of herb components on scavenging harmful components of cigarette smoke such as radicals, polycyclic aromatic hydrocarbons, nitrosamines in vitro, and its reducing effect on cytotoxicity of cigarette smoke. METHOD: spectrophotometry was used to examine the scavenging effect of herb components on DPPH free radicals, superoxide anion radical, and hydroxyl radical, and the results were compared with the anti-oxidation of ascorbic acid. Fluorescence spectroscopy was used to examine the scavenging effect of herb components on polycyclic aromatic hydrocarbons. UV spectrophotometry was used to examine the scavenging effect of herb components on volatile nitrosamines. MTT assay was used to examine cytotoxicity of cigarette smoke. RESULT: All the herb components showed a certain scavenging effect on DPPH free radicals, superoxide anion radical, hydroxyl radical, polycyclic aromatic hydrocarbons and volatile nitrosamines, espacially the ginkgo biloba extract (GBE), flavonoids of glycyrrhiza, procyanidine, total saponins in ophiopogonin, total saponins of astragalus and epimediun flavonoids. After these six herb components were added to cigarette, the cytotoxicity of cigarette smoke on BEP2D cells was remarkably reduced, by increasing cell survival fraction (SF, %) and mean lethal dose (DML). CONCLUSION: The herb components can scavenge harmful components of cigarette smoke such as radicals, polycyclic aromatic hydrocarbons and nitrosamines, which reduce the damage of cigarette smoke on human being.