Structure-activity relations of rosmarinic acid derivatives for the amyloid β aggregation inhibition and antioxidant properties

Amyloid-β aggregation inhibitors are expected to be therapeutic or prophylactic agents for Alzheimer\u27s disease. Rosmarinic acid, which is one of the main aggregation inhibitors derived from Lamiaceae, was employed as a lead compound and its 25 derivatives were synthesized. In this study,the structure-activity relations of rosmarinic acid derivatives for the amyloid-β aggregation inhibitory effect (MSHTS assay), antioxidant properties, and xanthine oxidase inhibition were evaluated. Among the tested compounds, compounds 16d and 19 were found to the most potent amyloid aggregation inhibitors. The SAR revealed that the necessity of the presence of the phenolic hydroxyl on one side of the molecule as well as the lipophilicity of the entire molecule. The importance of these structural properties was also supported by docking simulations

Rosmarinic acid is a novel inhibitor for Hepatitis B virus replication targeting viral epsilon RNA-polymerase interaction

Current therapeutics for hepatitis B virus (HBV) patients such as nucleoside analogs (NAs) are effective; however, new antiviral drugs against HBV are still desired. Since the interaction between the epsilon (c) sequence of HBV pregenomic RNA and viral polymerase (Pot) is a key step in the HBV replication cycle, we aimed to identify small compounds for its inhibition, and established a pull-down assay system for the detection of c-RNA-binding-Pol. Screening showed that 5 out of 3,965 compounds inhibited c-Pol binding, and we identified rosmarinic acid, which exhibited specificity, as a potential antiviral agent. In order to examine the anti-HBV effects of rosmarinic acid, HBV-infected primary human hepatocytes from a humanized mouse liver were treated with rosmarinic acid. The rosmarinic acid treatment decreased HBV components including the amounts of extracellular HBV DNA with negligible cytotoxicity. We also investigated the combined effects of rosmarinic acid and the NA, lamivudine. rosmarinic acid slightly enhanced the anti-HBV activity of lamivudine, suggesting that the HBV replication step targeted by rosmarinic acid is distinct from that of NA. We analyzed an additional 25 rosmarinic acid derivatives, and found that 5 also inhibited c-Pol. Structural comparisons between these derivatives implied that the "two phenolic hydroxyl groups at both ends" and the "caffeic acid-like structure" of rosmarinic acid are critical for the inhibition of c-Pol binding. Collectively, our results demonstrate that rosmarinic acid inhibits HBV replication in HBV-infected cells by specifically targeting c-Pol binding

Rosmarinic acid is a novel inhibitor for Hepatitis B virus replication targeting viral epsilon RNA-polymerase interaction

Current therapeutics for hepatitis B virus (HBV) patients such as nucleoside analogs (NAs) are effective; however, new antiviral drugs against HBV are still desired. Since the interaction between the epsilon (ε) sequence of HBV pregenomic RNA and viral polymerase (Pol) is a key step in the HBV replication cycle, we aimed to identify small compounds for its inhibition, and established a pull-down assay system for the detection of ε-RNA-binding-Pol. Screening showed that 5 out of 3, 965 compounds inhibited ε-Pol binding, and we identified rosmarinic acid, which exhibited specificity, as a potential antiviral agent. In order to examine the anti-HBV effects of rosmarinic acid, HBV-infected primary human hepatocytes from a humanized mouse liver were treated with rosmarinic acid. The rosmarinic acid treatment decreased HBV components including the amounts of extracellular HBV DNA with negligible cytotoxicity. We also investigated the combined effects of rosmarinic acid and the NA, lamivudine. rosmarinic acid slightly enhanced the anti-HBV activity of lamivudine, suggesting that the HBV replication step targeted by rosmarinic acid is distinct from that of NA. We analyzed an additional 25 rosmarinic acid derivatives, and found that 5 also inhibited ε-Pol. Structural comparisons between these derivatives implied that the “two phenolic hydroxyl groups at both ends” and the “caffeic acid-like structure” of rosmarinic acid are critical for the inhibition of ε-Pol binding. Collectively, our results demonstrate that rosmarinic acid inhibits HBV replication in HBV-infected cells by specifically targeting ε-Pol binding

Analysis of functional ingredients of tea and verification of relaxation effects of theanine and green tea

Tea originated from China and is a beverage with the longest history in the world. Earlier people used to drink tea for its benefits, but is now a regular beverage that is a globe favorite due to its distinctive flavor. Various physiologically active substances are present in tea, and in recent years,various physiological effects of tea leaf ingredients linked to prevention of cancer and adult diseases have been proved, and there are high expectations from the functionality of tea. 　We analyzed the functional ingredients present in tea extracts using several various tea samples such as green tea, black tea, and Chinese tea. Tannin content was high in black tea, while in green tea and Chinese tea, tannin content was almost the same. 　Healthy young women （n=6） were asked to consume green tea, 200 mg theanine, and water, and relaxation effect before and after consumption was measured. A significant decreasing trend was observed in the heart rate and cortisol value for women who consumed theanine. There was no change observed in the physiological index of women who consumed green tea, but significant induction of relaxation was observed in the subjective index obtained with a questionnaire.departmental bulletin pape

Rosmarinic acid suppresses HBV replication in HBV-infected primary human hepatocytes.

<p>(A-C) PXB-cells were infected with HBV, and treated with 30 μM rosmarinic acid or 500 nM lamivudine. Seven to 12 days post-infection, extracellular HBV DNA was quantified by qPCR, intracellular HBV 3.5 kb RNA was quantified by RT-qPCR, and SHBs were measured by ELISA. (D-F) PXB-cells were infected with HBV, and were treated with 30 μM rosmarinic acid and/or 20 nM lamivudine. Extracellular HBV DNA, intracellular HBV 3.5-kb RNA, and SHBs were measured as in (A-C). Data are from one representative of at least two independent experiments; the means and S.D. of duplicate or triplicate experiments are shown (* <i>p</i> < 0.05, ** <i>p</i> < 0.01). N.D.: not detected, n.s.: not significant.</p

Citotoxicity of the compounds.

<p>Citotoxicity of the compounds.</p

Five ε-Pol binding inhibitors selected by pull-down assay-based screening.

<p>(A) Scheme of the detection of ε-Pol binding by pull-down assays. The lysate of HEK-293T cells expressing 3×FLAG-Pol was incubated with ε-biotin, and pulled down using streptavidin sepharose. Precipitated 3×FLAG-Pol was detected by a Western blot analysis using an anti-FLAG antibody. (B) A Western blot analysis for Pol pulled-down by the indicated RNAs (ε RNA with biotin, ε RNA without biotin, and control RNA with biotin). (C and D) A Western blot analysis for Pol pulled-down by 10 pmol ε-biotin in the presence of the indicated compounds. Lamivudine was used as a negative control. Arrows: bands detected at a position of the estimated mass of full-length 3×FLAG-Pol.</p

Chemical structures.

<p>Chemical structures of compounds with ε-Pol inhibitory activity (A), and without ε-Pol inhibitory activity (B).</p