Isolation and Structure Determination of New Pyrones from Dictyostelium spp. Cellular Slime Molds Coincubated with Pseudomonas spp.

Cellular slime molds are excellent model organisms in the field of cell and developmental biology because of their simple developmental patterns. During our studies on the identification of bioactive molecules from secondary metabolites of cellular slime molds toward the development of novel pharmaceuticals, we revealed the structural diversity of secondary metabolites. Cellular slime molds grow by feeding on bacteria, such as Klebsiella aerogenes and Escherichia coli, without using medium components. Although changing the feeding bacteria is expected to affect dramatically the secondary metabolite production, the effect of the feeding bacteria on the production of secondary metabolites is not known. Herein, we report the isolation and structure elucidation of clavapyrone (1) from Dictyostelium clavatum, intermedipyrone (2) from D. magnum, and magnumiol (3) from D. intermedium. These compounds are not obtained from usual cultural conditions with Klebsiella aerogenes but obtained from coincubated conditions with Pseudomonas spp. The results demonstrate the diversity of the secondary metabolites of cellular slime molds and suggest that widening the range of feeding bacteria for cellular slime molds would increase their application potential in drug discovery.

Anti-trypanosomal Lignans Isolated from Salvadoran Peperomia pseudopereskiifolia.

A search for anti-trypanosomal natural compounds from plants collected in El Salvador, a country particularly endemic for Chagas disease, resulted in the isolation of five lignan-type compounds (1-5) from Peperomia pseudopereskiifolia. The lignan derivatives 1, 2, and 4 are new. Their absolute configuration was determined by chemical derivatization. Compounds 1, 5, 6, and 8 exhibited anti-trypanosomal activity against the amastigote form of T. cruzi comparable to that of the existing drug benznidazole.

Compound-compound interaction analysis of baicalin and berberine derivatives in aqueous solution.

Baicalin and berberine are biologically active constituents of the crude drugs Scutellaria root and Coptis rhizome/Phellodendron bark, respectively. Baicalin and berberine are reported to combine together as a 1:1 complex that forms yellow precipitates by electrostatic interaction in decoctions of Kampo formulae containing these crude drugs. However, the structural basis and mechanism for the precipitate formation of this compound-compound interaction in aqueous solution remains unclarified. Herein, we searched for berberine derivatives in the Coptis rhizome that interact with baicalin and identified the chemical structures involved in the precipitation formation. Precipitation assays showed that baicalin formed precipitates with berberine and coptisine but not with palmatine and epiberberine. Thus, the 2,3-methylenedioxy structure may be crucial to the formation of the precipitates, and electrostatic interaction is necessary but is not sufficient. In this multicomponent system experiment, palmatine formed a dissociable complex with baicalin and may competitively inhibit the formation of berberine and coptisine precipitation with baicalin. Therefore, the precipitation formed by berberine and baicalin was considered to be caused by the aggregation of the berberine-baicalin complex, and the 2,3-methylenedioxy structure is likely crucial to the aggregation of the complex.

The inhibitory effect of DIF-3 on polyinosinic-polycytidylic acid-induced innate immunity activation in human cerebral microvascular endothelial cells.

For the treatment and prevention of autoinflammatory diseases, it is essential to develop the drug, regulating the innate immune system. Although differentiation-inducing factor (DIF) derivatives, extracted from the cellular slime mold, Dictyostelium discoideum, exhibit immunomodulatory effects, their effects on the regulation of innate immunity in brain are unknown. In this study, we used the human cerebral microvascular endothelial cell line, hCMEC/D3, to investigate the effects of DIF derivatives on the generation of C-X-C motif chemokine (CXCL) 10 and interferon (IFN)-ß induced by polyinosinic-polycytidylic acid (poly IC). DIF-3 (1-10 µM), but not DIF-1 and DIF-2, dose-dependently inhibited the biosynthesis of not only CXCL10 but also CXCL16 and C-C motif chemokine 2 induced by poly IC. DIF-3 also strongly decreased IFN-ß mRNA expression and protein release from the cells induced by poly IC through the prohibition of p65, a subtype of NF-ĸB, not interferon regulatory transcription factor 3 phosphorylation. In the docking simulation study, we confirmed that DIF-3 had a high affinity to p65. These results suggest that DIF-3 regulates the innate immune system by inhibiting TLR3/IFN-ß signaling axis through the NF-ĸB phosphorylation inhibition.

Elucidation of the inhibitory effect of (+)-hopeaphenol on polyinosinic-polycytidylic acid-induced innate immunity activation in human cerebral microvascular endothelial cells.

Drug development for regulating the innate immune system is important for the prevention and treatment of autoinflammatory and autoimmune diseases. In this context, we investigated the effect of resveratrol derivatives on the inflammatory reactions in the brain. Resveratrol, which can be found in Vitis plants in the form of oligomers, exhibits neuroprotective effects; however, its regulatory effects on innate immunity are still unclear. We examined the effects of (+)-hopeaphenol, a resveratrol tetramer, and its derivatives on the polyinosinic-polycytidylic acid (poly IC)-induced production of interferon (IFN)-ß and C-X-C motif chemokine 10 (CXCL10) in the cultured human cerebral microvascular endothelial cell line hCMEC/D3. (+)-Hopeaphenol (1-10 µM) inhibited the poly IC-induced production of not only CXCL10 but also retinoic acid-inducible gene-I in a dose-dependent manner and significantly reduced the poly IC-induced IFN-ß gene expression and protein release from hCMEC/D3 cells by inhibiting the phosphorylation of p65 but not that of the interferon regulatory transcription factor IRF3. A docking study indicated a high affinity of (+)-hopeaphenol for p65. These results suggest that (+)-hopeaphenol can regulate the innate immune system by inhibiting the poly IC/IFN-ß/CXCL10 signaling axis via suppression of the phosphorylation of the transcription factor NF-ĸB.

Oxidation of methylophiopogonanone A on the surface of TLC plate.

Methylophiopogonanone A (MOPNA, 1) is a characteristic homoisoflavonoid, having two methyl groups on the A ring, isolated from Ophiopogon Root (enlarged part of the root of Ophiopogon japonicus Ker-Gawler, Liliaceae). Although MOPNA is chemically stable in various organic solvents, such as acetone, chloroform, methanol and dimethyl sulfoxide, it gave a spot of higher polarity in addition to the spot of MOPNA on the surface of TLC plate. The spot was isolated and the structure of the major compound was revealed to be a compound with an oxygen atom at C-6 of the A ring. This oxidation on the surface of TLC plate was observed for synthetic intermediates of MOPNA having two methyl groups on a phloroacetophenone-type ring, suggesting that the methyl groups enhanced susceptibility to air oxidation. The structure of the major oxidation product was similar to those of humulone and sufflomin A. Humulone has two prenyl units and sufflomin A has two C-glucosyl moieties instead of the two methyl groups, respectively. As dialkylated phloroacetophenone derivatives seem susceptible to oxidation, air oxidation might be involved in the formation of these compounds.

Anti-trypanosomal screening of Salvadoran flora.

Chagas disease is caused by the protozoan parasite Trypanosoma cruzi, and in Central America, it is considered one of the four most infectious diseases. This study aimed to screen the anti-trypanosomal activity of plant species from Salvadoran flora. Plants were selected through literature search for plants ethnobotanically used for antiparasitic and Chagas disease symptomatology, and reported in Museo de Historia Natural de El Salvador (MUHNES) database. T. cruzi was incubated for 72 h with 2 different concentrations of methanolic extracts of 38 species, among which four species, Piper jacquemontianum, Piper lacunosum, Trichilia havanensis, and Peperomia pseudopereskiifolia, showed the activity (≤ 52.0% viability) at 100 µg/mL. Separation of the methanolic extract of aerial parts from Piper jacquemontianum afforded a new flavanone (4) and four known compounds, 2,2-dimethyl-6-carboxymethoxychroman-4-one (1), 2,2-dimethyl-6-carboxychroman-4-one (2), cardamomin (3), and pinocembrin (5), among which cardamomin exhibited the highest anti-trypanosomal activity (IC50 = 66 µM). Detailed analyses of the spectral data revealed that the new compound 4, named as jaqueflavanone A, was a derivative of pinocembrin having a prenylated benzoate moiety at the 8-position of the A ring.

Solubility enhancement of berberine-baicalin complex by the constituents of Gardenia Fruit.

A Kampo prescription usually consists of several crude drugs and contains many kinds of compounds. Physicochemical interactions between the compounds may occur in the process of decoction, by which Kampo prescriptions are usually prepared for ingestion, and the interactions may change the extraction yields of the constituents. Berberine and baicalin have been reported to form precipitates. Orengedokuto, which consist of Coptis Rhizome, Gardenia Fruit, Phellodendron Bark and Scutellaria Root, has been a representative Kampo prescription used to treat inflammatory diseases. In our previous papers, we revealed that the precipitates formed in the decoction of orengedokuto without Gardenia Fruit mainly consists of berberine-baicalin complex and that Gardenia Fruit reduced the amount of the precipitates in orengedokuto decoction. In this report, through solubility-enhancement assay based on HPLC, we identified crocins as the constituents of Gardenia Fruits, which enhanced the solubility of berberine-baicalin complex. All-trans crocin-1 (1) and 13-cis crocin-1 (5) showed high activities among the isolated crocins, and the number of glucosyl groups in the molecule seemed correlated with the activity. As berberine and baicalin were reported as the anti-inflammatory constituents of Coptis Rhizome and Phellodendron Bark, and Scutellaria Root, respectively, Gardenia Fruit contributes anti-inflammatory activity of orengedokuto by increasing solubilities of anti-inflammatory constituents of the other component crude drugs in the prescription. Our result will add a scientific basis to the understanding of the effectiveness of orengedokuto as a whole.

Natural Products with Potential to Treat RNA Virus Pathogens Including SARS-CoV-2.

Three families of RNA viruses, the Coronaviridae, Flaviviridae, and Filoviridae, collectively have great potential to cause epidemic disease in human populations. The current SARS-CoV-2 (Coronaviridae) responsible for the COVID-19 pandemic underscores the lack of effective medications currently available to treat these classes of viral pathogens. Similarly, the Flaviviridae, which includes such viruses as Dengue, West Nile, and Zika, and the Filoviridae, with the Ebola-type viruses, as examples, all lack effective therapeutics. In this review, we present fundamental information concerning the biology of these three virus families, including their genomic makeup, mode of infection of human cells, and key proteins that may offer targeted therapies. Further, we present the natural products and their derivatives that have documented activities to these viral and host proteins, offering hope for future mechanism-based antiviral therapeutics. By arranging these potential protein targets and their natural product inhibitors by target type across these three families of virus, new insights are developed, and crossover treatment strategies are suggested. Hence, natural products, as is the case for other therapeutic areas, continue to be a promising source of structurally diverse new anti-RNA virus therapeutics.

Citrus Fruit-Derived Flavanone Glycoside Narirutin is a Novel Potent Inhibitor of Organic Anion-Transporting Polypeptides.

Organic anion-transporting polypeptides (OATPs) 1A2 and OATP2B1 are expressed in the small intestine and are involved in drug absorption. We identified narirutin, which is present in grapefruit juice, as a novel OATP inhibitor. The citrus fruit jabara also contains high levels of narirutin; therefore, we investigated the inhibitory potency of jabara juice against OATPs. The inhibitory effects of various related compounds on the transport activity of OATPs were evaluated using OATP-expressing HEK293 cells. The IC50 values of narirutin for OATP1A2- and OATP2B1-mediated transport were 22.6 and 18.2 µM, respectively. Other flavanone derivatives from grapefruit juice also inhibited OATP1A2/OATP2B1-mediated transport (order of inhibitory potency: naringenin > narirutin > naringin). Five percent jabara juice significantly inhibited OATP1A2- and OATP2B1-mediated transport by 67 ± 11 and 81 ± 5.5%, respectively (p < 0.05). Based on their inhibitory potency and levels in grapefruit juice, the inhibition of OATPs by grapefruit juice is attributable to both naringin and narirutin. Citrus × jabara, which contains narirutin, potently inhibits OATP-mediated transport.

Five new 2-(2-phenylethyl)chromone derivatives from agarwood.

Agarwood has been used as an incense and in traditional medicines as aphrodisiac, sedative, cardiotonic, and carminative. In this study, five new 2-(2-phenylethyl)chromones (2, 13-16) and eleven known compounds (1, 3-12) were isolated from the agarwood. The structures of the new compounds were determined by 1H-, 13C-, and two-dimensional NMR together with electronic circular dichroism (ECD) spectroscopy. All isolated compounds were evaluated for the phosphodiesterase (PDE) 3A and 5A1 inhibitory activity by the fluorescence polarization method. Dimeric 2-(2-phenylehyl)chromones (13, 14, 16) had potent inhibitory activity to PDE 5A1 with IC50 values of micro molar range (13: 4.2 µM, 14: 7.9 µM, 16: 4.3 µM), whereas they had weak activity to PDE 3A. In contrast, compound (15), which has a phenylpropionic acid moiety instead of the 2-(2-phenylethyl)chromone moiety in the dimers, showed moderate inhibition of both PDE 3A (IC50: 42.6 µM) and PDE 5A1 (IC50: 15.1 µM).

Continuous Estimation of Annual Committed Effective Dose of Radioactive Cesium by Market Basket Study in Japan from 2013 to 2019 after Fukushima Daiichi Nuclear Power Plant Accident.

Radionuclide contamination in foods has been a great concern after the Fukushima Daiichi Nuclear Power Plant (FDNPP) accident. To estimate time trends of daily intake and annual committed effective dose of radionuclides after the accident, radioactive cesium (r-Cs; 134Cs and 137Cs) and potassium-40 (40K) in market basket (MB) samples prepared at 6-month intervals in periods from September 2013 to March 2019 in 15 regions of Japan were analyzed using γ-ray spectrometry. The annual committed effective dose of r-Cs, calculated at non-detected radionuclide levels assumed to be half the limit of detection (LOD), appeared to decrease gradually in 11 regions close to the FDNPP that were more likely to be affected by the accident. Differences in doses among the 15 regions were large just after the accident, but gradually decreased. In particular, 134Cs has not been detected in any MB sample in any region since September 2018, and annual committed effective dose from 134Cs in all regions was mostly constant at around 0.3 µSv/year (given the respective LODs). The maximum annual committed effective dose of r-Cs in this study was decreased from 2.7 µSv/year in September 2013 to 1.0 µSv/year in March 2019. In contrast, the range of annual committed effective dose of 40K varied from approximately 150 to 200 µSv/year during that time frame and did not change much throughout the period of this study. Although annual committed effective doses of r-Cs in regions close to the FDNPP appeared to be higher than in regions far from the FDNPP, doses in all regions are remaining at a much lower levels than the intervention exemption level, 1 mSv/year, in foods in Japan.

Preparation of menisdaurigenin and related compounds.

Menisdaurin (1), a cyano glucoside, was first isolated in 1978 from Menispermum dauricum (Menispermaceae) and named after the plant. It has been also isolated from several plant sources. The stereochemistry of the aglycone part was first reported as (Z,4R,6S)-enantiomer of (4,6-dihydroxy-2-cyclohexen-1-ylidene)acetonitrile based on the CD spectrum of menisdaurilide (2), the α,ß-unsaturated γ-lactone obtained by an acid hydrolysis of menisdaurin. Later, the absolute stereochemistry was revised as (Z,4S,6R) by X-ray crystal analysis of 1 isolated from Saniculiphyllum guangxiens. The aglycone part of menisdaurin (1) has not been obtained from 1, because an acid hydrolysis of 1 gave menisdaurilide (2), and enzymatic hydrolysis with emulsin did not give the aglycone. On the other hand, a compound named coculauril (3) was isolated from Cocculus lauriforius. This compound has the same planner structure corresponding to the aglycone of 1, but the stereochemistry was reported to be (E,4R,6S). Here, we confirmed the absolute stereochemistry of 1 by Mosher's method to be (Z,4S,6R), and prepared the aglycone of 1, i.e., menisdaurigenin (4) by an enzymatic hydrolysis of 1. We also revealed that 4 is a different compound from 3 and unstable in water and MeOH.

Efficient Synthesis of Theaflavin 3-Gallate by a Tyrosinase-Catalyzed Reaction with (-)-Epicatechin and (-)-Epigallocatechin Gallate in a 1-Octanol/Buffer Biphasic System.

Theaflavins, the orange-red pigments contained in black tea, have attracted attention as a result of their health-promoting effects. However, their synthetic preparation, in which the enzymatic oxidation of catechol-type catechin is followed by the quinone-induced oxidative dimerization of selectively combined catechol- and pyrogallol-type catechins, provides only a low yield. In the present study, we found that a 1-octanol/buffer biphasic system improved the yield of theaflavin 3-gallate in a tyrosinase-catalyzed synthetic reaction with (-)-epicatechin and (-)-epigallocatechin gallate. When the enzymatic reaction proceeded in a buffer solution, oxidized (-)-epigallocatechin gallate was preferentially used for self-dimerization. However, self-dimerization was suppressed in the octanol phase, allowing oxidized (-)-epigallocatechin gallate to participate in coupling with (-)-epicatechin quinone, leading to effective production of theaflavin 3-gallate. Furthermore, the preferential localization of theaflavin 3-gallate in the octanol phase prevented (-)-epicatechin-quinone-induced degradation.

[Concentration of Radioactive Cesium in Domestic Foods Collected from the Japanese Market in Fiscal Years 2014-2016].

Between fiscal years 2014 and 2016, we surveyed the concentration of radioactive cesium in commercial foods produced in areas where there is a risk of radiation contamination due to the Fukushima Daiichi nuclear disaster. The number of samples with a concentration of radioactive cesium that exceeded the regulatory limit （100 Bq/kg for general foods） was 9 out of 1,516 （0.6％） in fiscal 2014, 12 out of 900 （1.3％） in fiscal 2015, and 10 out of 654 （1.5％） in fiscal 2016. Even though some samples were expected to be contaminated with radioactive cesium, because wild mushrooms and edible wild plants were intentionally included in this survey, the percentage of samples that exceeded the regulatory limit was only around 1％. The surveillance results confirmed that the pre-shipment food monitoring conducted by local governments was properly and efficiently performed, although continuous monitoring of the concentration of radioactive cesium in cultivated and wild mushrooms, edible wild plants, and wild animal meats is still required.

Determination of polychlorinated biphenyls in marine fish obtained from tsunami-stricken areas of Japan.

We determined the polychlorinated biphenyl (PCB) congeners in 101 marine fish obtained from tsunami-stricken areas following the Great East Japan Earthquake in 2011. In particular, to determine the degree of PCB contamination in the fish, we investigated the concentration of total PCB (∑PCB) and the proportions of 209 individual PCB congeners by high-resolution gas chromatography/high-resolution mass spectrometry. The ∑PCB concentration was 1.7-33 ng/g in fat greenling (n = 29), 0.44-25 ng/g in flounder (n = 36), and 1.6-86 ng/g in mackerel (n = 36), all values being much lower than the provisional regulatory limit in Japan. In the congener analysis, tetra-, penta-, hexa-, and hepta-chlorinated PCB congeners dominated in all samples (comprising over 86% of the ∑PCB). The proportions of the chlorinated PCB congeners were similar to the contamination patterns derived from Kanechlor in the environment, implying that the marine fish were not contaminated with fresh PCBs.

Surveillance of Strontium-90 in Foods after the Fukushima Daiichi Nuclear Power Plant Accident.

As a result of the Fukushima Daiichi nuclear power plant (NPP) accident, various radionuclides were released into the environment. In this study, we surveyed strontium-90 ((90)Sr) concentrations in several foodstuffs. Strontium-90 is thought to be the third most important residual radionuclide in food collected after the Fukushima Daiichi, NPP accident after following cesium-137 ((137)Cs) and cesium-134 ((134)Cs). Results of (90)Sr analyses indicated that (90)Sr was detect in 25 of the 40 radioactive cesium (r-Cs) positive samples collected in areas around the Fukushima Daiichi NPP, ranging in distance from 50 to 250 km. R-Cs positive samples were defined as containing both (134)Cs and (137)Cs which are considered to be indicators of the after-effects of the Fukushima Daiichi NPP accident. We also detected (90)Sr in 8 of 13 r-Cs negative samples, in which (134)Cs was not detected. Strontium-90 concentrations in the r-Cs positive samples did not significantly exceed the (90)Sr concentrations in r-Cs negative samples or the (90)Sr concentration ranges in comparable food groups found in previous surveys before the Fukushima Daiichi NPP accident. Thus, (90)Sr concentrations in r-Cs positive samples were indistinguishable from the background (90)Sr concentrations arising from global fallout prior to the Fukushima accident, suggesting that no marked increase of (90)Sr concentrations has occurred in r-Cs positive samples as a result of the Fukushima Daiichi NPP accident.

Redox-coupled structural changes of the catalytic a' domain of protein disulfide isomerase.

Protein disulfide isomerase functions as a folding catalyst in the endoplasmic reticulum. Its b' and a' domains provide substrate-binding sites and undergo a redox-dependent domain rearrangement coupled to an open-closed structural change. Here we determined the first solution structure of the a' domain in its oxidized form and thereby demonstrate that oxidation of the a' domain induces significant conformational changes not only in the vicinity of the active site but also in the distal b'-interfacial segment. Based on these findings, we propose that this conformational transition triggers the domain segregation coupled with the exposure of the hydrophobic surface.