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Poria, the dried sclerotium of Wolfiporia cocos , is a medicinal mushroom that is widely used in traditional Japanese medicine. The fruit body of W. cocos is rarely found in the natural environment in Japan, therefore an optimized technique for fruit body formation is essential for producing new strains through crossbreeding and for biological research. Here, we developed a cultivation technique for fruit body formation of W. cocos using three strains collected from different areas of Japan. When mycelia were cultured on sawdust-based medium after liquid medium culture, all strains successfully formed fruit bodies as a brown honeycomb-like structure. Furthermore, we analyzed single nucleotide polymorphisms of the three strains using the STE3-like pheromone receptor protein gene, STE3.2, and found a genetic marker for discriminating one strain from the others. The results are expected to promote extensive studies on crossbreeding and domestic production of W. cocos .

Maoto () is a traditional Japanese Kampo formula composed of four crude drugs-Ephedrae Herba, Armeniacae Semen, Cinnamomi Cortex, and Glycyrrhizae Radix-and has been used to treat febrile viral illnesses. Among these, Ephedrae Herba has been associated with antiviral activity in previous studies. However, little is known about the effects of Ephedrae Herba on viral infection, spike (S) protein-mediated syncytium formation (cell-cell fusion), and viral enzymes, or the specific metabolites underlying these activities. In this study, we focused on Ephedrae Herba and comparatively evaluated the effects of the four crude drugs of Maoto on SARS-CoV-2 infection, S protein-mediated cell-cell fusion, and virus replication-related processes. We evaluated the inhibitory effects of the four crude drugs of Maoto on SARS-CoV-2 infection in VeroE6/TMPRSS2 cells. S protein-mediated cell-cell fusion was evaluated using a split-luciferase assay in HEK293T cells. The inhibitory effects on viral enzymes, including 3C-like protease, papain-like protease, and RNA-dependent RNA polymerase (RdRp), were assessed using enzymatic assays. Comparative analyses of the four crude drugs showed that Ephedrae Herba, Armeniacae Semen, and Cinnamomi Cortex suppressed SARS-CoV-2 infection in VeroE6/TMPRSS2 cells, whereas Glycyrrhizae Radix inhibited S protein-mediated cell-cell fusion in HEK293T cells. In the biochemical assays, Ephedrae Herba and Cinnamomi Cortex showed inhibitory activity against SARS-CoV-2 RdRp. Further analysis of Ephedrae Herba showed that its high-polarity fraction exhibited concentration-dependent inhibitory activity against both SARS-CoV-2 and norovirus RdRp with IC values of 8.6 and 16.5 μg/mL, respectively. Ephedrae Herba contains high-polarity metabolites associated with RdRp-related inhibitory activity in biochemical assays. Nevertheless, further studies are needed to determine the extent to which this activity contributes to the effects observed in cells.

Cell adhesion plays a crucial role in candidiasis through invasion of the human body and obtaining resistance to drugs by forming biofilms. Cell adhesion thus is a critical target for combating candidiasis by preventing the entry of fungal hyphae into the epithelium. We report here that dehydrocurvularin (1), isolated from the marine-derived fungus Curvularia aeria, exhibited anti-fungal activities for Candida albicans and Candida auris. This compound also prevented the adherence of C. albicans to human adenocarcinoma cells. Real-time RT-PCR analysis showed that exposure to 1 results in decreased expression of HWP1, EFG1, and ECE1, genes involved in Candida adhesion to epithelial cells and hyphal morphogenesis.

The coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged as a pandemic and has inflicted enormous damage on the lives of the people and economy of many countries worldwide. However, therapeutic agents against SARS-CoV-2 remain unclear. SARS-CoV-2 has a spike protein (S protein), and cleavage of the S protein is essential for viral entry. Nattokinase is produced by Bacillus subtilis var. natto and is beneficial to human health. In this study, we examined the effect of nattokinase on the S protein of SARS-CoV-2. When cell lysates transfected with S protein were incubated with nattokinase, the S protein was degraded in a dose- and time-dependent manner. Immunofluorescence analysis showed that S protein on the cell surface was degraded when nattokinase was added to the culture medium. Thus, our findings suggest that nattokinase exhibits potential for the inhibition of SARS-CoV-2 infection via S protein degradation.

Two novel aromatic polyketides, penicanesins J and K ( 1 , 2 ), were isolated from the marine-derived fungus Didymella aeria , along with the known compound integrastatin B ( 3 ). The structures of the new compounds were determined by NMR spectroscopy and synthetic methods. The isolated compounds were tested for monoamine oxidase (MAO) B inhibition, anti-amyloid beta (Aβ) aggregation, and protective activity against H 2 O 2 -induced cell death in human neuroblastoma SH-SY5Y cells. Integrastatin B ( 3 ) showed potential activity for inhibition of Aβ aggregation and protection against H 2 O 2 -induced cell death. Graphical Abstract

Papain-like protease (PLpro) enzyme plays a vital role in viral replication as it breaks down polyproteins and disrupts the host's immune response. There are few reports on Kampo formulas that focus on PLpro activity. In this study, we evaluated the inhibitory effects of senkyuchachosan, a traditional Japanese medicine, on PLpro of SARS-CoV-2, the virus responsible for causing COVID-19. We purified the PLpro enzyme and conducted in vitro enzymatic assays using specific substrates. Among the nine crude drugs present in senkyuchachosan, four (Cyperi Rhizoma, Schizonepetae Spica, Menthae Herba, and Camelliae sinensis Folium [CsF]) strongly inhibited PLpro activity. CsF, derived from Camellia sinensis (green tea), contains polyphenols, including catechins and tannins. To confirm that the PLpro inhibitory effects of senkyuchachosan predominantly stem from tannins, the tannins were removed from the decoction using polyvinylpolypyrrolidone (PVPP). The inhibitory effect of senkyuchachosan on PLpro activity was reduced by the removal of PVPP. In addition, the tannin fraction obtained from the CsF extracts showed significant PLpro inhibitory effects. These findings lay the groundwork for the potential development of therapeutic agents that target SARS-CoV-2 infection by intervening in proteolytic cleavage of the virus. Graphical Abstract

The spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) contains a cleavage motif R-X-X-R for furin-like enzymes at the boundary of the S1/S2 subunits. The cleavage of the site by cellular proteases is essential for S protein activation and virus entry. We screened the inhibitory effects of crude drugs on in vitro furin-like enzymatic activities using a fluorogenic substrate with whole-cell lysates. Of the 124 crude drugs listed in the Japanese Pharmacopeia, aqueous ethanolic extract of Cnidii Monnieris Fructus, which is the dried fruit of Cnidium monnieri Cussion, significantly inhibited the furin-like enzymatic activities. We further fractionated the plant extract and isolated the two active compounds with the inhibitory activity, namely, imperatorin and osthole, whose IC 50 values were 1.45 mM and 9.45 µM, respectively. Our results indicated that Cnidii Monnieris Fructus might exert inhibitory effects on furin-like enzymatic activities, and that imperatorin and osthole of the crude drug could be potential inhibitors of the motif cleavage.

PurposeColchicum autumnale (Colchicaceae) exhibits toxicity, and severe poisoning cases due to ingestion of this plant have been reported in Japan. Identifying the cause of poisoning is important for emergency medical treatment, and a rapid and simple detection technique is required for the identification of the cause of poisoning. In the present study, we developed a rapid and simple method for the detection of C. autumnale based on a loop-mediated isothermal amplification (LAMP) assay, which is 100- to 1000-fold higher than the conventional polymerase chain reaction method.MethodsSpecific LAMP primers for C. autumnale were designed based on the trnL-trnF intergenic spacer region. Using the LAMP primers, the LAMP assay was performed at an isothermal reaction temperature of 63 ℃.ResultsThe LAMP reaction was shown to be specific and highly sensitive to C. autumnale, given that the assay can be used for 10 pg of purified DNA. Using a simple protocol for on-site detection, the entire procedure from pretreatment to evaluation required approximately 1 h. Moreover, the LAMP method using a microfluidic device detected multiple genes, including C. autumnale-specific DNA regions, at a time.ConclusionsThe currently proposed protocol exhibits good potential as a screening method for C. autumnale poisoning in emergency medical care.

Background Zingiber officinale Roscoe (ginger) has been traditionally used not only as a culinary ingredient, but also for its medicinal properties. In Japan, ginger-derived crude drugs are categorized into dried ginger (Zingiberis Rhizoma [ZR]) or steamed ginger (Zingiberis Rhizoma processum [ZR-P]). Heating ginger converts gingerol, the primary component of ZR, into shogaol. In this study, we aimed to evaluate the inhibitory effects of ginger and its constituents on SARS-CoV-2 infection. Methods We assessed the inhibitory effects of ZR, ZR-P, [6]-gingerol, and [6]-shogaol on SARS-CoV-2 infection in VeroE6/TMPRSS2 cells. To understand the molecular mechanism underlying this inhibitory effect, we evaluated the activity of [6]-shogaol against the viral proteases 3 C-like protease (3CLpro) and papain-like protease (PLpro). Results We observed that ZR-P (66.1% suppression at 50 µg/ml) and [6]-shogaol (70.0% suppression at 25 µM) exhibited significant inhibitory effects against SARS-CoV-2 infection. We found that [6]-shogaol effectively inhibited 3CLpro activities both in vitro (20.4% inhibition at 40 µM) and in FlipGFP reporter system (19.7% inhibition at 25 µM). Conclusions This study highlights [6]-shogaol and ZR-P as promising natural inhibitors of SARS-CoV-2, providing new insights into the pharmacological potential of traditional ginger preparations and their active components against COVID-19.