In Vitro and in Silico Studies of Potential Coronavirus-Specific 3C-Like Protease Inhibitors.

We investigated similar compounds to ebselen and tideglusib, which exhibit strong activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), using Molecular ACCess System (MACCS) keys. Four candidate compounds were identified. One of them, phenyl-benzothiazol-3-one, showed coronavirus-specific 3C-like (3CL) protease inhibitory activity. The results indicated that a similarity score above 0.81 is a good indicator of activity for ebselen-and-tideglusib-like compounds. Subsequently, we simulated the ring-cleavage Michael reaction of ebselen at the Se center, which is responsible for its 3CL protease inhibitory activity, and determined the activation free energy of the reaction. The results showed that reaction simulation is a useful tool for estimating the activity of inhibitory compounds that undergo Michael addition reactions with the relevant cysteine S atom of 3CL proteases.

Curcuma sp.-derived dehydrocurdione induces heme oxygenase-1 through a Michael reaction between its α, ß-unsaturated carbonyl and Keap1.

To elucidate the anti-inflammatory mechanism of Curcuma sp., we investigated whether dehydrocurdione, a sesquiterpene contained in Curcuma sp., induces heme oxygenase (HO)-1, an antioxidative enzyme, in RAW 264.7 macrophages. Dehydrocurdione was extracted from the rhizome of Curcuma sp., and its purity was verified by high performance liquid chromatography. Treatment with 10-100 µM dehydrocurdione transiently and concentration-dependently increased HO-1 mRNA and protein levels. Docking simulation suggested the presence of the Michael reaction between dehydrocurdione and Kelch-like ECH-associated protein (Keap)1 keeping nuclear factor-erythroid2-related-factor (Nrf)2, a transcription factor, in the cytoplasm. Nrf2 that was definitely free from Keap1 was detected in the nuclei after dehydrocurdione treatment. Subsequently, the HO-1 E2 enhancer, a target of Nrf2, was activated, resulting in HO-1 expression. Also, an investigation using 6-shogaol and 6-gingerol supported the concept that the α, ß-unsaturated carbonyl structure plays an important role in the interaction with Keap1. Dehydrocurdione suppressed lipopolysaccharide-induced NO release, a marker of inflammation. Clarification of the HO-1 synthesis increase mechanism revealed in this study will help contribute to the development of novel phytotherapeutic strategies against inflammation-associated diseases.

Anti-SARS-CoV-2 Agents in Artemisia Endophytic Fungi and Their Abundance in Artemisia vulgaris Tissue.

Coronavirus disease 2019 is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Therapeutic agents for the disease are being developed. Endophytes are diverse and produce various secondary metabolites and bioactive substances. We isolated 13 endophytes from the leaves and stems of Artemisia vulgaris. Antiviral testing using the culture extracts of these endophytic fungi revealed that five isolates effectively inhibited the replication of SARS-CoV-2. These extracts were used to study the inhibitory effect of SARS-CoV-2 on 3C-like protease, and two isolates proved useful. Both isolates were from the genus Colletotrichum; therefore, the percentage of Artemisia endophytic fungi in the plant tissue was observed to be an important factor in plant site selection. Thus, we conducted a macroanalysis using next-generation sequencing to analyze the percentage of endophytes in the stems (whole, skin, and inner), leaves, roots, and cultivating soil, as well as to determine the location of each genus. To the best of our knowledge, this study is the first to report that Colletotrichum spp. are abundant in stems and that stem-based methods are the most efficient for isolating endophytes targeting Colletotrichum spp.

Indoleamine 2, 3-dioxygenase is responsible for low stress tolerance after intracerebral hemorrhage.

In the chronic phase after intracerebral hemorrhage (ICH), the aftereffect-associated lowering of motivation burdens many patients; however, the pathogenic mechanism is unclear. Here, we revealed for the first time that indoleamine 2, 3-dioxygenase (IDO) expression and enzyme activity are increased in the collagenase-induced murine ICH model. IDO is a rate-limiting enzyme situated at the beginning of the kynurenine pathway and converts tryptophan, a source of serotonin (5-hydroxytryptamine; 5-HT), to kynurenine. In this study, we showed that IDO is localized in 5-HTergic neurons. After ICH, the synaptosomal 5-HT level decreased, but this effect was neutralized by subcutaneous injections of 1-methyl tryptophan (MT), a specific IDO inhibitor. These results suggest that ICH-induced IDO weakens the activity of 5-HTergic neurons. Accordingly, we next investigated whether the IDO increase contributes to the depression-like behaviors of ICH mice. The immobility times of tail suspension and forced swimming tests were significantly prolonged after ICH but shortened by the administration of 1-MT. In conclusion, the increased IDO after ICH was found to decrease 5-HT levels and subsequently reduce stress tolerance. These findings indicate that IDO is a novel therapeutic target for the ICH aftereffect-associated lowering of motivation.

Environmental differences between Japan and Indonesia provide endophyte diversity associated with Artemisia plant and variety of artemisinin derivatives in microbial conversion.

We compared the endophytic compositions of Artemisia plant from different environments (Japan and Indonesia) to demonstrate that the endophytic filamentous fungi in both species differed based on their environments. To prove that the species were identical, both Artemisia plants were identified by comparing the scanning electron micrographs of their pollens, as well as the nucleotide sequences (ribosomal internal transcribed spacer and mitochondrial maturase K) of the two gene regions. After isolating the endophytic filamentous fungi from each plant, we observed that those from Japan and Indonesia comprised 14 and 6 genera, respectively. We assumed that the genera, Arthrinium and Colletotrichum, which exist in both Artemisia species, were species-specific filamentous fungi, while the other genera were environment-dependent. In the microbial-conversion reaction with artemisinin as a substrate using Colletotrichum sp., the peroxy bridge of artemisinin, which is an active site for achieving antimalarial effect, was converted into an ether bond. However, the reaction using the environment-dependent endophyte did not eliminate the peroxy bridge. These endophytic reactions indicated the different roles of endophytes within Artemisia plants.

Bioproduction of Cinchona alkaloids by the endophytic fungus Diaporthe sp. associated with Cinchona ledgeriana.

We report that an endophytic filamentous fungus species of the genus Diaporthe isolated from Cinchona ledgeriana (Rubiaceae) produces Cinchona alkaloids (quinine, quinidine, cinchonidine, and cinchonine) upon cultivation in a synthetic liquid medium. This study provides evidence that Cinchona alkaloids are produced not only in Cinchona plant cells, but also in the endophytic microbe cells, and will help to elucidate the relationship between endophytic microbes and their host plants.

Cinchona alkaloids are also produced by an endophytic filamentous fungus living in cinchona plant.

We report that the endophytic filamentous fungus Diaporthe sp., isolated from Cinchona ledgeriana and cultivated in a synthetic liquid medium, produces Cinchona alkaloids (quinine, quinidine, cinchonidine, and cinchonine). This shows that Cinchona alkaloids are produced not only in Cinchona plant cells, but also in endophytic microbe cells.

Microbial conversion of curcumin into colorless hydroderivatives by the endophytic fungus Diaporthe sp. associated with Curcuma longa.

We investigated the microbial conversion of curcumin (1) using endophytic fungi associated with the rhizome of Curcuma longa (Zingiberaceae). We found that Diaporthe sp., an endophytic filamentous fungus, converts curcumin (1) into four colorless derivatives, namely (3R,5R)-tetrahydrocurcumin (2), a novel (3R,5S)-hexahydrocurcumin (3) named neohexahydrocurcumin, (3S,5S)-octahydrocurcumin (4) and meso-octahydrocurcumin (5).

Anthraquinone-containing compound in rhubarb prevents indole production via functional changes in gut microbiota.

Indole is produced from dietary tryptophan by tryptophanase in intestinal bacteria, such as Escherichia coli. In the liver, indole is converted into indoxyl sulfate, a uremic toxin and risk factor for chronic kidney disease (CKD). Probiotics and prebiotics are currently used for suppressing CKD, but there are no drugs that directly suppress indole production. In this study, we developed an optimized HPLC method for analyzing indole production and evaluated the effect of diets and rhubarb on indole production via the changes of gut microbiota. In high-carbohydrate and high-fat diet-fed mice, the indole production was significantly higher than in high-fiber diet-fed mice. We further used the high-carbohydrate diet-fed mice as a model for examining the effect of rhubarb on indole production. The 20% methanol-eluted fraction of aqueous rhubarb extract significantly suppressed indole production, and the eluate constituent rhein 8-O-ß-D-glucopyranoside (RG) contributed to this effect in a concentration-dependent manner. The effect of RG depended on the anthraquinone core substructure, i.e., the aglycone moiety (rhein) of RG, which appeared to inhibit the tryptophanase function in gut microbiota. Thus, in addition to earlier reports that rhubarb is an effective CKD treatment, our study demonstrated that the anthraquinone moiety in rhubarb prevents uremic toxin production via functional changes in gut microbiota, which suppresses CKD progression.

High ophiobolin A production in endophytic fungus Bipolaris sp. associated with Datura metel.

Ophiobolin A, a metabolite of fungi, is known to induce cell death and have anticancer activity. Therefore, obtaining ophiobolin A has become an important aspect in studying activity with medicinal properties that are affected by it. Ophiobolin A-producing filamentous fungi are endophytic or infectious microbes that attack annual and short-cycle plants. Here we isolated the endophyte of Datura metel, which is an annual plant that produces ophiobolin A. Results of this study have led to the identification of an endophytic filamentous fungus Bipolaris sp. with high ophiobolin A production (235 mg/L) in liquid culture after 21 days. Our findings further indicate that ophiobolin A-producing fungi live in short-cycle plants, and a method of finding the fungus is described.

Endophyte composition and Cinchona alkaloid production abilities of Cinchona ledgeriana cultivated in Japan.

New eight endophytic filamentous fungi were isolated from the young stems of Cinchona ledgeriana (Rubiaceae) cultivated in Japan. They were classified into four genera based on phylogenetic analysis of the nucleotide sequences of the internal transcribed spacers (ITS1 and ITS2), including the 5.8S ribosomal DNA region. Of the eight fungi isolated, there were five genera Cladosporium, one Meira sp., one Diaporthe sp. and one Penicillium sp. Genus of Cladosporium and Meira were first isolated fungi from Cinchona plant. In a previous study, we applied the same process to the same plant cultivated in Indonesia. The endophyte compositions for the two cultivation regions were found to differ at the genera level. The ability of Cinchona endophytes cultivated in Japan to produce Cinchona alkaloids was also assessed. We found that three isolates have producing ability of Cinchona alkaloids. However, the amount produced was very small compared to that produced by the endophytes of Indonesian Cinchona ledgeriana. In addition, the total content amount of Cinchona alkaloids, especially quinine, produced by the extract of Cinchona cultivated in Japan was much smaller than that from Indonesia. These finding indicate that endophyte composition has an influence on the Cinchona alkaloid content amount in the Cinchona ledgeriana host.

Shogaol but not gingerol has a neuroprotective effect on hemorrhagic brain injury: Contribution of the α, ß-unsaturated carbonyl to heme oxygenase-1 expression.

We investigated the effects of shogaol, which has an α, ß-unsaturated carbonyl group, and gingerol, which does not, on primary-cultured microglia to understand how the α, ß-unsaturated carbonyl interacts with Kelch-like ECH-associated protein (Keap)1. Shogaol (1 µM) but not the same concentration of gingerol significantly increased heme oxygenase (HO)-1 protein levels in cultured microglia without cytotoxicity. In addition, shogaol suppressed the release of the inflammation marker nitric oxide induced by 30 U/ml thrombin treatment. A docking simulation suggested that the α, ß-unsaturated carbonyl of shogaol but not gingerol interacts with Keap1. Nuclear import of nuclear factor E2-related factor 2 and increased binding of the HO-1 E2 enhancer support the docking-simulation prediction. The transcription inhibitor actinomycin D (0.1 µg/ml) markedly blocked the increase of HO-1 mRNA levels by shogaol. To evaluate whether the α, ß-unsaturated carbonyl can be used for intracerebral hemorrhage (ICH) therapy, we investigated the effect of shogaol on an in vivo mouse ICH model. Intracerebroventricular injection of 0.2 nmol shogaol increased striatal HO-1 protein levels and rescued ICH-induced neuron loss. Thus, the α, ß-unsaturated carbonyl is necessary for the interaction of compounds, such as shogaol, with Keap1, and these findings may be useful for screening novel ICH therapeutic agents that increase HO-1 expression.

Composition of the endophytic filamentous fungi associated with Cinchona ledgeriana seeds and production of Cinchona alkaloids.

Four kinds of endophytic filamentous fungi (code names: CLS-1, CLS-2, CLS-3, and CLS-4) associated with the seeds of Cinchona ledgeriana (Rubiaceae) from West Java, Indonesia, were isolated. All of the isolates were classified into Diaporthe spp. based on phylogenetic analysis of the nucleotide sequences of the internal transcribed spacers (ITS1 and ITS2) including the 5.8S ribosomal DNA region. All four of these endophytic fungi produce Cinchona alkaloids, mainly quinine and quinidine, in synthetic liquid medium.

Ability of endophytic filamentous fungi associated with Cinchona ledgeriana to produce Cinchona alkaloids.

We have investigated the ability of endophytic filamentous fungi associated with Cinchona ledgeriana (Rubiaceae) to produce Cinchona alkaloids on potato dextrose agar medium and in a synthetic liquid medium. It was found that all twenty-one endophytic fungi produce Cinchona alkaloids, despite their genetic differences.

Composition of endophytic fungi living in Cinchona ledgeriana (Rubiaceae).

A total of 21 endophytic filamentous fungi were isolated from the young stems of Cinchona ledgeriana (Rubiaceae) cultivated in West Java, Indonesia. They were classified into six genera, namely nine Phomopsis spp., six Diaporthe spp., two Schizophyllum spp., two Penicillium spp., one Fomitopsis sp., and one Arthrinium sp. by using nucleotide sequence analysis of the internal transcribed spacers (ITS1 and ITS2) including 5.8S ribosomal DNA region and phylogenetic analysis.

Stereoselective oxidation at C-4 of flavans by the endophytic fungus Diaporthe sp. isolated from a tea plant.

The microbial transformation of five flavans (1-5) by endophytic fungi isolated from the tea plant Camellia sinensis was investigated. It was found that the endophytic filamentous fungus Diaporthe sp. oxidized stereoselectively at C-4 position of (+)-catechin (1) and (-)-epicatechin (2) to give the correspondent 3,4-cis-dihydroxyflavan derivatives (6, 10), respectively. (-)-Epicatechin 3-O-gallate (3) and (-)-epigallocatechin 3-O-gallate (4) were also oxidized by the fungus into 3,4-dihydroxyflavan derivatives (10, 12) via (-)-epicatechin (2) and (-)-epigallocatechin (11), respectively. Meanwhile, (-)-gallocatechin 3-O-gallate (5), (-)-catechin (ent-1) and (+)-epicatechin (ent-2), which possess a 2S-phenyl substitution, resisted the biotransformation.

Biooxidation of (+)-catechin and (-)-epicatechin into 3,4-dihydroxyflavan derivatives by the endophytic fungus Diaporthe sp. isolated from a tea plant.

The microbial transformation of (+)-catechin (1) and (-)-epicatechin (2) by endophytic fungi isolated from a tea plant was investigated. It was found that the endophytic filamentous fungus Diaporthe sp. transformed them (1, 2) into the 3,4-cis-dihydroxyflavan derivatives, (+)-(2R,3S,4S)-3,4,5,7,3',4'-hexahydroxyflavan (3) and (-)-(2R,3R,4R)-3,4,5,7,3',4'-hexahydroxyflavan (7), respectively, whereas (-)-catechin (ent-1) and (+)-epicatechin (ent-2) with a 2S-phenyl group resisted the biooxidation.

Transformation of Cinchona alkaloids into 1-N-oxide derivatives by endophytic Xylaria sp isolated from Cinchona pubescens.

The microbial transformation of four Cinchona alkaloids (quinine, quinidine, cinchonidine, and cinchonine) by endophytic fungi isolated from Cinchona pubescens was investigated. The endophytic filamentous fungus Xylaria sp. was found to transform the Cinchona alkaloids into their 1-N-oxide derivatives.