Anti-hepatitis C Virus Activity of Juglorubin Derivatives.

Juglorubin is a natural dye isolated from the culture of Streptomyces sp. 3094, 815, and GW4184. It has been previously synthesized via the biomimetic dimerization of juglomycin C, a plausible genetic precursor. In this study, the derivatives of juglorubin, 1-O-acetyljuglorubin dimethyl ester and juglorubin dimethyl ester, were found to exhibit antiviral activity against hepatitis C virus (HCV) without exerting any remarkable cytotoxicity against host Huh-7 cells. They also inhibited liver X receptor α activation and lipid droplet accumulation in Huh-7 cells. These findings suggest that 1-O-acetyljuglorubin dimethyl ester and juglorubin dimethyl ester targeted the host factors required for HCV production.

Identification of Methylsulochrin as a Partial Agonist for Aryl Hydrocarbon Receptors and Its Antiviral and Anti-inflammatory Activities.

Although aryl hydrocarbon receptors (AhRs) are related to the metabolic pathway of xenobiotics, recent studies have revealed that this receptor is also associated with the life cycle of viruses and inflammatory reactions. For example, flutamide, used to treat prostate cancer, inhibits hepatitis C virus proliferation by acting as an AhR antagonist, and methylated-pelargonidin, an AhR agonist, suppresses pro-inflammatory cytokine production. To discover a novel class of AhR ligands, we screened 1000 compounds derived from fungal metabolites using a reporter assay and identified methylsulochrin as a partial agonist of the aryl hydrocarbon receptor. Methylsulochrin was found to inhibit the production of hepatitis C virus (HCV) in Huh-7.5.1 cells. Methylsulochrin also suppressed the production of interleukin-6 in RAW264.7 cells. Furthermore, a preliminary structure-activity relationship study using sulochrin derivatives was performed. Our findings suggest the use of methylsulochrin derivatives as anti-HCV compounds with anti-inflammatory activity.

Cyclic Phthalate Esters as Liver X Receptor Antagonists with Anti-hepatitis C Virus and Anti-severe Acute Respiratory Syndrome Coronavirus 2 Properties.

The liver X receptor is a nuclear hormone receptor that regulates lipid metabolism. Previously, we had demonstrated the antiviral properties of a liver X receptor antagonist associated with the hepatitis C virus and severe acute respiratory syndrome coronavirus 2. In this study, we screened a chemical library and identified two potential liver X receptor antagonists. Spectroscopic analysis revealed that the structures of both antagonists (compounds 1 and 2) were cyclic dimer and trimer of esters, respectively, that consisted of phthalate and 1,6-hexane diol. This study is the first to report the structure of the cyclic trimer of phthalate ester. Further experiments revealed that the compounds were impurities of solvents used for purification, although their source could not be traced. Both phthalate esters exhibited anti-hepatitis C virus activity, whereas the cyclic dimer showed anti-severe acute respiratory syndrome coronavirus 2 activity. Cyclic phthalate derivatives may constitute a novel class of liver X receptor antagonists and broad-spectrum antivirals.

[A Rapid and Simple Method for Detection of Colchicum autumnale Using PCR].

Colchicum autumnale is a perennial, toxic plant that originated in Europe and North Africa. Although inedible, it is occasionally consumed accidentally because it resembles the edible Allium victorialis and other related species. This misidentification has led to episodes of food poisoning in Japan. However, determining the causative agent of a food poisoning outbreak by observing the sample visually or analyzing the chemical composition is challenging when dealing with small samples. Therefore, we developed a novel set of PCR primers that anneal to the internal transcribed spacer (ITS) region of C. autumnale ribosomal DNA, designed to detect the presence of C. autumnale in small samples. These primers successfully detected C. autumnale in all samples in which it was present, and did not give a positive PCR band in the 48 other distinct crop species tested, in which it was not present. Further, our method could amplify DNA from samples of C. autumnale that had been heat-treated and digested using artificial gastric fluids. Thus, this PCR strategy is highly specific and can be used to distinguish C. autumnale simply and rapidly from various other crops.

Improved On-Site Protocol for the DNA-Based Species Identification of Cannabis sativa by Loop-Mediated Isothermal Amplification.

Cannabis sativa L. is cultivated worldwide for a variety of purposes, but its cultivation and possession are regulated by law in many countries, necessitating accurate detection methods. We previously reported a DNA-based C. sativa identification method using the loop-mediated isothermal amplification (LAMP) assay. Although the LAMP technique can be used for on-site detection, our previous protocol took about 90 min from sampling to detection. In this study, we report an on-site protocol that can be completed in 30 min for C. sativa identification based on a modified LAMP system. Under optimal conditions, the LAMP reaction started at approximately 10 min and was completed within 20 min at 63°C. It had high sensitivity (10 pg of purified DNA). Its specificity for C. sativa was confirmed by examining 20 strains of C. sativa and 50 other species samples. With a simple DNA extraction method, the entire procedure from DNA extraction to detection required only 30 min. Using the protocol, we were able to identify C. sativa from various plant parts, such as the leaf, stem, root, seed, and resin derived from C. sativa extracts. As the entire procedure was completed using a single portable device and the results could be evaluated by visual detection, the protocol could be used for on-site detection and is expected to contribute to the regulation of C. sativa.

Rapid identification of drug-type strains in Cannabis sativa using loop-mediated isothermal amplification assay.

In Cannabis sativa L., tetrahydrocannabinol (THC) is the primary psychoactive compound and exists as the carboxylated form, tetrahydrocannabinolic acid (THCA). C. sativa is divided into two strains based on THCA content-THCA-rich (drug-type) strains and THCA-poor (fiber-type) strains. Both strains are prohibited by law in many countries including Japan, whereas the drug-type strains are regulated in Canada and some European countries. As the two strains cannot be discriminated by morphological analysis, a simple method for identifying the drug-type strains is required for quality control in legal cultivation and forensic investigation. We have developed a novel loop-mediated isothermal amplification (LAMP) assay for identifying the drug-type strains of C. sativa. We designed two selective LAMP primer sets for on-site or laboratory use, which target the drug-type THCA synthase gene. The LAMP assay was accomplished within approximately 40 min. The assay showed high specificity for the drug-type strains and its sensitivity was the same as or higher than that of conventional polymerase chain reaction. We also showed the effectiveness of melting curve analysis that was conducted after the LAMP assay. The melting temperature values of the drug-type strains corresponded to those of the cloned drug-type THCA synthase gene, and were clearly different from those of the cloned fiber-type THCA synthase gene. Moreover, the LAMP assay with simple sample preparation could be accomplished within 1 h from sample treatment to identification without the need for special devices or techniques. Our rapid, sensitive, specific, and simple assay is expected to be applicable to laboratory and on-site detection.

Development of Loop-Mediated Isothermal Amplification (LAMP) Assay for Rapid Detection of Cannabis sativa.

In many parts of the world, the possession and cultivation of Cannabis sativa L. are restricted by law. As chemical or morphological analyses cannot identify the plant in some cases, a simple yet accurate DNA-based method for identifying C. sativa is desired. We have developed a loop-mediated isothermal amplification (LAMP) assay for the rapid identification of C. sativa. By optimizing the conditions for the LAMP reaction that targets a highly conserved region of tetrahydrocannabinolic acid (THCA) synthase gene, C. sativa was identified within 50 min at 60-66°C. The detection limit was the same as or higher than that of conventional PCR. The LAMP assay detected all 21 specimens of C. sativa, showing high specificity. Using a simple protocol, the identification of C. sativa could be accomplished within 90 min from sample treatment to detection without use of special equipment. A rapid, sensitive, highly specific, and convenient method for detecting and identifying C. sativa has been developed and is applicable to forensic investigations and industrial quality control.