Altenuene derivatives produced by an endophyte Alternaria alternata.

Two novel aromatic polyketides 1 and 3 and five known compounds, (4S,10S)-talaroflavone (2), altenuene (4), isoaltenuene (5), alternariol (6), and altenusin (7), were isolated from an endophytic strain of Alternaria alternata SI-694. The structures of the new compounds, including their absolute configurations, were elucidated by NMR, IR, UV, and ECD spectroscopies, and the phytotoxicities of the isolated compounds were also evaluated. Altenusin (7) showed moderate cytotoxicity against HL-60 cells, with an IC50 of 6.65 µM, whereas 5, 6, and 7 were phytotoxic against Lactuca sativa, Brassica campestris L., Stellaria aquatica (L.) Scop. and Digitaria ciliaris.

Aspergillus oryzae α-l-rhamnosidase: Crystal structure and insight into the substrate specificity.

The subsequent biochemical and structural investigations of the purified recombinant α-l-rhamnosidase from Aspergillus oryzae expressed in Pichia pastoris, designated as rAoRhaA, were performed. The specific activity of the rAoRhaA wild-type was higher toward hesperidin and narirutin, where the l-rhamnose residue was α-1,6-linked to ß-d-glucoside, than toward neohesperidin and naringin with an α-1,2-linkage to ß-d-glucoside. However, no activity was detected toward quercitrin, myricitrin, and epimedin C. rAoRhaA kinetic analysis indicated that Km values for neohesperidin, naringin, and rutin were lower compared to those for hesperidin and narirutin. kcat values for hesperidin and narirutin were higher than those for neohesperidin, naringin, and rutin. High catalytic efficiency (kcat /Km ) toward hesperidin and narirutin was a result of a considerably high kcat value, while Km values for hesperidin and narirutin were higher than those for naringin, neohesperidin, and rutin. The crystal structure of rAoRhaA revealed that the catalytic domain was represented by an (α/α)6 -barrel with the active site located in a deep cleft and two ß-sheet domains were also present in the N- and C-terminal sites of the catalytic domain. Additionally, five asparagine-attached N-acetylglucosamine molecules were observed. The catalytic residues of AoRhaA were suggested to be Asp254 and Glu524, and their catalytic roles were confirmed by mutational studies of D254N and E524Q variants, which lost their activity completely. Notably, three aspartic acids (Asp117, Asp249, and Asp261) located at the catalytic pocket were replaced with asparagine. D117N variant showed reduced activity. D249N and D261N variants activities drastically decreased.

Cytotoxicity and induction of G1 phase cell cycle arrest in human acute promyelocytic leukemia HL60 cells by indole alkaloids isolated from Dialium corbisieri seeds.

The phytochemical investigation of Dialium corbisieri seeds led to the isolation of five monoterpenoid indole alkaloids along with a phytoserotonin, 1-6 and among the known compounds, the spectroscopic data of (5S)-methoxy-akuammiline (1) was reported for the first time. The structures were elucidated based on nuclear magnetic resonance spectroscopic techniques such as ultraviolet, infrared, high-resolution electrospray ionization time-of-flight mass spectrometry, and electron-capture dissociation spectrum calculations. The isolated compounds were evaluated for their cytotoxicity and cell progression in the human acute promyelocytic leukemia HL60 cell line.

Integracide derivatives produced by endophyte Fusarium armeniacum M-3 isolated from Digitaria ciliaris.

Two new integracides, dedimethyl integracide B (1) and integracide K (2), were isolated from rice cultures of Fusarium armeniacum M-3, an endophyte isolated from Digitaria ciliaris, together with the known terpenoids, integracide B (3) and 2-deoxy-integracide B (4). Assignment of the signals in the 1H and 13C NMR spectra of 1 and 2 was achieved using DEPT, HMQC, HMBC, and NOESY spectra. Compound 2 exhibited moderate cytotoxicity against HeLa cells.

Two new octahydronaphthalene derivatives, trichodermic acids C and D produced by Trichoderma sp. HN-1.1.

Two new octahydronaphthalene derivatives, trichodermic acid C (1) and trichodermic acid D (2), along with known analogs, trichodermic acid (3), trichodermic acid A (4) and trichodermic acid B (5), were isolated from an ethyl acetate extract of endophytic strain Trichoderma sp. HN-1.1. The structures of compounds 1 and 2 were elucidated using spectroscopic methods including UV, IR, HRESITOFMS, ECD, 1 D and 2 D NMR. The cytotoxic activity of the isolated compounds was evaluated on the rat hepatoma cell line H4IIE, using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. According to the results, only compound 3 showed a significant reduction of H4IIE cells from 75 to 21% (p < 0.01) with an IC50 value of 50% cell survival estimated as 143.1 µM, which indicated weak cytotoxic activity.

The 2,3-epoxy naphthoquinol produced by endophyte Arthrinium marii M-211.

A novel 2,3-epoxy naphthoquinol, named (6R,7R,8R)-theissenone A (1), possessing an oxatricyclo[5.4.0.03,5]undeca-trien-2-one skeleton, together with two known compounds, (6S,7R,8R)-theissenone (2) and arthrinone (3), were produced by an endophytic fungus, Arthrinium marii M-211, which was isolated from mangrove plants. The structure of 1, including the absolute stereochemistry, was elucidated by analysis of nuclear magnetic resonance (NMR) and mass spectrometry (MS) data and time-dependent density functional theory (TDDFT) calculations of electronic circular dichroism (ECD) spectra. Additionally, the absolute structure of 2 was deduced as a diastereomer of 1 using ECD spectral data analysis. Compounds 1, 2 and 3 exhibited cytotoxic activity against the H4IIE rat hepatoma cells, with IC50 values of 67.5, 46.6 and 13.4 µM, respectively.

Tryptamine, a Microbial Metabolite in Fermented Rice Bran Suppressed Lipopolysaccharide-Induced Inflammation in a Murine Macrophage Model.

Fermentation is thought to alter the composition and bioavailability of bioactive compounds in rice bran. However, how this process affects the anti-inflammatory effects of rice bran and the bioactive compounds that might participate in this function is yet to be elucidated. This study aimed to isolate bioactive compounds in fermented rice bran that play a key role in its anti-inflammatory function. The fermented rice bran was fractionated using a succession of solvent and solid-phase extractions. The fermented rice bran fractions were then applied to lipopolysaccharide (LPS)-activated murine macrophages to evaluate their anti-inflammatory activity. The hot water fractions (FRBA), 50% ethanol fractions (FRBB), and n-hexane fractions (FRBC) were all shown to be able to suppress the pro-inflammatory cytokine expression from LPS-stimulated RAW 264.7 cells. Subsequent fractions from the hot water fraction (FRBF and FRBE) were also able to reduce the inflammatory response of these cells to LPS. Further investigation revealed that tryptamine, a bacterial metabolite of tryptophan, was abundantly present in these extracts. These results indicate that tryptamine may play an important role in the anti-inflammatory effects of fermented rice bran. Furthermore, the anti-inflammatory effects of FRBE and tryptamine may depend on the activity of the aryl hydrocarbon receptor.

Mutational analysis of the effects of N-glycosylation sites on the activity and thermal stability of rutinosidase from Aspergillus oryzae.

Purified recombinant rutinosidase from Aspergillus oryzae expressed in Pichia pastoris (rAoRutM) exhibits increase in thermal stability after treatment with endo-ß-N-acetylglucosaminidase H (endo-H). In this study, the role of N-glycosylation in the activity and thermal stability of rAoRutM was analyzed via site-directed mutagenesis. Based on the crystal structure of AoRutM, five N-glycosylation sites (N32, N128, N176, N288, and N359) were identified in the AoRut protein. Among five single variants constructed for these sites, the N128D, N176D, and N359D variants exhibited similar mobility bands compared to that of the wild-type enzyme based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, whereas the N32D and N288D variants exhibited slightly and considerably increasing mobility bands, respectively. The N128D and N288D variants showed increasing and decreasing rutinosidase activity, respectively, compared to the case for the wild-type, without and with endo-H treatments. While the N128D and N176D variants had lower Km values, the N288D and N359D variants had higher Km values, compared to the wild-type, without and with endo-H treatments. Surprisingly, the N32D and N176D variants exhibited considerably greater thermal stability than the wild-type, without or with the endo-H treatments, whereas the N128D and N359D variants exhibited drastically decreased thermal stability. Circular dichroism (CD) spectra of the N128D and N359D variants showed a similar CD profile to that of the wild-type treated with endo-H; however, the molar ellipticity values of the peaks at 208 nm and 212 nm in the above variants varied from those of the intact wild-type and other variants.

Structure elucidation of a new bicoumarin derivative from the leaves of Dysoxylum parasiticum (Osbeck) Kosterm.

A new bicoumarin derivative, bidysoxyletine (1), was isolated from the leaves of Dysoxylum parasiticum (Osbeck) Kosterm. The structure of 1 was elucidated by analysis of NMR, UV, IR, HR-ESITOFMS, and DDFT approach using the B3LYP exchange-correlation function for 13 C NMR and UV spectroscopic data. The results indicated that the structure of 1 possessed a dibenzonapthyrone skeleton.

Bidysoxyphenols A-C, dimeric sesquiterpene phenols from the leaves of Dysoxylum parasiticum (Osbeck) Kosterm.

Three new sesquiterpene phenol dimers, bidysoxyphenols A-C (2-4), along with two known compounds, namely sesquiterpene phenol (1) and ionone derivatives (5), were isolated from the leaves of Dysoxylum parasiticum (Osbeck) Kosterm. The structures of these new compounds, including their absolute configurations, were elucidated by nuclear magnetic resonance spectroscopy, ultraviolet spectroscopy, infrared spectroscopy, high-resolution electrospray ionization time-of-flight mass spectrometry, and electronic circular dichroism. Compounds 1 and 2 showed cytotoxicity against human promyelocytic leukemia cells, with IC50 values of 18.25 ± 1.52 and 39.04 ± 3.12 µM, respectively.

Fermented rice bran supplementation attenuates chronic colitis-associated extraintestinal manifestations in female C57BL/6N mice.

Patients with inflammatory bowel disease (IBD) have higher incidence of extraintestinal manifestations (EIM), including liver disorders, sarcopenia, and neuroinflammation. Fermented rice bran (FRB), generated from rice bran (RB), is rich in bioactive compounds, and exhibits anti-colitis activity. However, its role in EIM prevention is still unclear. Here, for the first time, we investigated whether EIM in female C57Bl/6N mice is attenuated by FRB supplementation. EIM was induced by repeated administration of 1.5% dextran sulfate sodium (DSS) in drinking water (4 d) followed by drinking water (12 d). Mice were divided into 3 groups-control (AIN93M), 10% RB, and 10% FRB. FRB ameliorated relapsing colitis and inflammation in muscle by significantly lowering proinflammatory cytokines Tnf-α and Il-6 in serum and advanced glycation end product-specific receptor (Ager) in serum and muscle when compared with the RB and control groups. As FRB reduced aspartate aminotransferase levels and oxidative stress, it might prevent liver disorders. FRB downregulated proinflammatory cytokine and chemokine transcripts responsible for neuroinflammation in the hippocampus and upregulated mRNA expression of G protein coupled receptors (GPRs), Gpr41 and Gpr43, in small and large intestines, which may explain the FRB-mediated protective mechanism. Hence, FRB can be used as a supplement to prevent IBD-associated EIM.

Antioxidant p-terphenyl compound, isolated from edible mushroom, Boletopsis leucomelas.

A new acetyl p-terphenyl derivative, boletopsin 15, was isolated from the ethyl acetate extract of fruit bodies of the Basidiomycete Boletopsis leucomelas, together with 4 known compounds. The structures of these compounds were elucidated by spectral analysis as well as by directly comparing the spectral data of the new compound with those of known compounds. The free radical-scavenging activity of the compounds was assayed using the 2,2-diphenyl-1-picrylhydrazyl scavenging method. The results showed that compounds 1 and 2 exhibited significant antioxidant activity (1: EC50 = 2.1 µm and 2: EC50 = 6.6 µm).

Cochlioquinone derivatives produced by coculture of endophytes, Clonostachys rosea and Nectria pseudotrichia.

Three new meroterpenoid derivatives, furanocochlioquinol (1) and furanocochlioquinone (2), as well as nectrianolin D (3), together with two known biogenetically related compounds 4 and 5 were isolated from a mixed culture of two mangrove-derived fungi, Clonostachys rosea B5-2 and Nectria pseudotrichia B69-1. The structures of 1-3 were deduced based on the interpretation of HRMS and NMR data. Compounds 1-5 exhibited cytotoxicity against human promyelocytic leukemia (HL60) cells with IC50 values ranging from 0.47 to 10.16 µM.

Identification and characterization of an acetyl xylan esterase from Aspergillus oryzae.

In this study, we report the identification and characterization of an acetyl xylan esterase, designated as AoAXEC, which was previously annotated as a hypothetical protein encoded by AO090023000158 in the Aspergillus oryzae genomic database. Based on its amino acid sequence, a low sequence identity to known acetyl xylan esterases was observed in the sequence of characterized acetyl xylan esterase. The gene fused with α-factor signal sequence of Saccharomyces cerevisiae instead of the native signal sequence was cloned into a vector, pPICZαC, and expressed successfully in Pichia pastoris as an active extracellular protein. The purified recombinant protein had pH and temperature optima of 7.0 and 50 °C, respectively, and was stable up to 50 °C. The optimal substrate for hydrolysis by the purified recombinant AoAXEC, among a panel of α-naphthyl esters (C2-C16), was α-naphthyl propionate (C3), with an activity of 0.35 ± 0.006 units/mg protein. No significant difference of the Km value was observed between C3 (2.3 ± 0.7 mM) and C2 (1.9 ± 0.4 mM). In contrast, kcat value for C3 (18 ± 3.9 s-1) was higher compared to C2 (4.5 ± 0.7 s-1). The purified recombinant enzyme displayed a low activity toward acyl chain substrates containing eight or more carbon atoms. Recombinant AoAXEC catalyzed the release of acetic acid from wheat arabinoxylan. However, no activity was detected on methyl esters of ferulic, p-coumaric, caffeic, or sinapic acids. Additionally, the liberation of phenolic acids, such as ferulic acid, from wheat arabinoxylan was not exhibited by the recombinant protein.

Fermented Rice Bran Supplementation Prevents the Development of Intestinal Fibrosis Due to DSS-Induced Inflammation in Mice.

Fermented rice bran (FRB) is known to protect mice intestines against dextran sodium sulfate (DSS)-induced inflammation; however, the restoration of post-colitis intestinal homeostasis using FRB supplementation is currently undocumented. In this study, we observed the effects of dietary FRB supplementation on intestinal restoration and the development of fibrosis after DSS-induced colitis. DSS (1.5%) was introduced in the drinking water of mice for 5 days. Eight mice were sacrificed immediately after the DSS treatment ended. The remaining mice were divided into three groups, comprising the following diets: control, 10% rice bran (RB), and 10% FRB-supplemented. Diet treatment was continued for 2 weeks, after which half the population of mice from each group was sacrificed. The experiment was continued for another 3 weeks before the remaining mice were sacrificed. FRB supplementation could reduce the general observation of colitis and production of intestinal pro-inflammatory cytokines. FRB also increased intestinal mRNA levels of anti-inflammatory cytokine, tight junction, and anti-microbial proteins. Furthermore, FRB supplementation suppressed markers of intestinal fibrosis. This effect might have been achieved via the canonical Smad2/3 activation and the non-canonical pathway of Tgf-ß activity. These results suggest that FRB may be an alternative therapeutic agent against inflammation-induced intestinal fibrosis.

ß-Resorcylic Acid Derivatives, with Their Phytotoxic Activities, from the Endophytic Fungus Lasiodiplodia theobromae in the Mangrove Plant Xylocarpus granatum.

Nine new ß-resorcylic acid derivatives, (15S)-de-O-methyllasiodiplodin (1), (13S,15S)-13-hydroxy-de-O-methyllasiodiplodin (2), (14S,15S)-14-hydroxy-de-O-methyllasiodiplodin (3), (13R,14S,15S)-13,14-dihydroxy-de-O-methyllasiodiplodin (4), ethyl (S)-2,4-dihydroxy-6-(8-hydroxynonyl)benzoate (5), ethyl 2,4-dihydroxy-6-(8-hydroxyheptyl)benzoate (6), ethyl 2,4-dihydroxy-6-(4-methoxycarbonylbutyl)benzoate (7), 3-(2-ethoxycarbonyl-3,5-dihydroxyphenyl)propionic acid (8), and isobutyl (S)-2,4-dihydroxy-6-(8-hydroxynonyl)benzoate (9), together with a known ethyl 2,4-dihydroxy-6-(8-oxononyl)benzoate (10) were obtained from Lasiodiplodia theobromae GC-22. The structures of these compounds were elucidated by extensive spectroscopic analyses. Compounds 1, 3, and 6 showed growth inhibitory effects against Digitaria ciliaris. Conversely, treatment with compounds 5, 6, 7, 9, and 10 stimulated elongation activity toward the root of Lactuca sativa. These data expand the repertoire of new ß-resorcylic acid derivatives that may function as lead compounds in the synthesis of new agrochemical agents.

New naphthoquinone derivatives from Fusarium napiforme of a mangrove plant.

Two new naphthoquinone derivatives, 6-hydroxy-astropaquinone B (1) and astropaquinone D (2) as well as the known compound 3-O-methyl-9-O-methylfusarubin (3) were isolated from Fusarium napiforme, an endophytic fungus isolated from the mangrove plant, Rhizophora mucronata. The structures of 1 and 2 were determined by 1D and 2D NMR spectroscopic analyses. Compounds 1, 2 and 3 exhibited moderate antibacterial activity against Staphylococcus aureus and Pseudomonas aeruginosa. Furthermore, 1, 2 and 3 were phytotoxic action in lettuce seeding at a concentration of 30 µg · mL-1.

New metabolites produced by endophyte Clonostachys rosea B5 - 2.

The endophytic fungus, Clonostachys rosea B5-2 was isolated from mangrove plants and subjected to the one strain many compounds (OSMAC) methodology. By this approach, it was found that modification of the culture media enhanced the production of secondary metabolites by C. rosea B5-2. The apple juice supplemented solid rice media led to significant changes in the secondary metabolism of the fungus, C. rosea B5-2, and induced the production of four new compounds, (-)-dihydrovertinolide (2), and clonostach acids A (3), B (4), and C (5) together with the known compound, (-)-vertinolide (1). The new compound (-)-dihydrovertinolide (2) exhibited phytotoxicity against lettuce seedlings at a concentration of 50 mg L-1.

New compounds from Japanese oak wilt disease-associated fungus Raffaelea quercivora.

Two new compounds, a new lactone, quercilactone A (1), and (17R)-hydroxynafuredin (5), as well as five known compounds, scytalone (2), 3S,4R-hydoxy-scytalone (3), nafuredin (4), (+)-(3R,5R)-3-hydroxy-5-decanolide (6) and 3-ethyl-4-hydroxy-6-methyl-2H-pyran-2-one (7), were isolated from Raffaelea quercivora, a fungus that causes Japanese oak wilt disease. The structures of these compounds were determined by 1D and 2D NMR spectroscopic analyses. The absolute configuration at C-17 of 5 was determined to be R by the modified Mosher's method. Compounds 1, 2, and 7 exhibited weak phytotoxic activity in lettuce seedlings at a concentration of 100 µg mL-1.

Aspergillus oryzae Rutinosidase: Biochemical and Structural Investigation.

The rutinosidase (Rut)-encoding gene Aorut has been expressed in Pichia pastoris with its native signal sequence from Aspergillus oryzae Biochemical and structural investigation of the purified recombinant mature A. oryzae Rut (AoRut), designated rAoRutM, was performed in this study. A 1.7-Å resolution crystal structure of rAoRutM was determined, which is an essential step forward in the utilization of AoRut as a potential catalyst. The crystal structure of rAoRutM was represented by a (ß/α)8 TIM barrel fold with structural similarity to that of rutinosidase from Aspergillus niger (AnRut) and an exo-ß-(1,3)-glucanase from Candida albicans The crystal structure revealed that the catalytic site was located in a deep cleft, similarly to AnRut, and that internal cavities and water molecules were also present. Purified rAoRutM hydrolyzed not only 7-O-linked and 3-O-linked flavonoid rutinosides but also 7-O-linked and 3-O-linked flavonoid glucosides. rAoRutM displayed high catalytic activity toward quercetin 3-O-linked substrates such as rutin and isoquercitrin, rather than to the 7-O-linked substrate, quercetin-7-O-glucoside. Unexpectedly, purified rAoRutM exhibited increased thermostability after treatment with endo-ß-N-acetylglucosaminidase H. Circular dichroism (CD) spectra of purified intact rAoRutM and of the enzyme after N-deglycosylation showed a typical α-helical CD profile; however, the molar ellipticity values of the peaks at 208 nm and 212 nm differed. The Km and kcat values for the substrates modified by rutinose were higher than those for the substrates modified by ß-d-glucose.IMPORTANCE Flavonoid glycosides constitute a class of secondary metabolites widely distributed in nature. These compounds are involved in bitter taste or clouding in plant-based foods or beverages, respectively. Flavonoid glycoside degradation can proceed through two alternative enzymatic pathways: one that is mediated by monoglycosidases and another that is catalyzed by a diglycosidase. The present report on the biochemical and structural investigation of A. oryzae rutinosidase provides a potential biocatalyst for industrial applications of flavonoids.