Two new 13-membered macrolides (1, 7), along with known 13-membered macrolides PF1163A, B, D, H, and F (2–6), were isolated from a strain of a marine-derived fungus, Penicillium meleagrinum var. viridiflavum. The structures of 1 and 7 were elucidated from spectroscopic data (NMR, MS, IR). Compounds 1–7 showed synergistic effects with fluconazole against azole-resistant Candida albicans by a checkerboard assay.

•Different cultivation showed different anti melanogenesis activity.•Diketopiperazine compounds were isolated from mycelium by using wheat medium.•The difference of activities were the presence of diketopiperazine compounds.Metabolites of marine derived fungus Eurotium rubrum MPUC136 differed between cultivation on wheat medium and Czapek-Dox agar medium. Melanin synthesis inhibitory activity of crude extract of culture on wheat medium showed stronger activity than that of crude extract of culture on Czapek-Dox agar medium. A new diketopiperazine compound isoechinulin D (1) and eight reported diketopiperazines (2–9) were isolated from the crude extract of wheat medium. The structure of 1 was established using NMR, MS and IR methods. 2–5 inhibited melanogenesis using B16 melanoma cells (IC50 = 68, 2.4, 83, 9.1 μM each). Structure–Activity-Relationships of diketopiperazines (1–10) indicated the importance of the prenyl groups at C-2, C-5 and C-7, the vinyl group at C-12 to C-25 and the sp2 carbons at C-8 and C-9. Isolated compounds (1–9) were not or slightly observed from the extracts of Czapek-Dox agar medium by HPLC analysis, suggesting that different cultivation processes could affect metabolism and enhance bioactivities.

Five new labdane-type diterpenoids, sterebins Q1 (1), Q2 (2), Q3 (3), Q4 (4), and R (5), were isolated from Stevia rebaudiana fermented by Saccharomyces cerevisiae. The structures of these compounds were established using NMR, MS, and IR methods and their absolute configurations were determined by the ECD spectra of their benzoate derivatives. Through the structures of these new compounds, it was established that the fermentation process caused selective epoxidation and nucleophilic addition of water. Thus, fermentation is an effective way to develop new natural resources for use as drug candidates by enhancing the diversity of natural compounds.

The diversity of natural compounds is extensive but not unlimited. Fermentation of rhizomes of Atractylodes lancea by marine fungus (Cladosporium cladosporioides) enhanced the diversity of natural compounds. From the fermented rhizomes of A. lancea, two new spirovetivane-type sesquiterpenoids, 2-oxo-12-hydroxy-hinesol (1) and 2-oxo-15-hydroxy-hinesol (2); a new guaiane-type sesquiterpenoid atractylol A (3); and three new eudesmane-type sesquiterpenoids, 14-hydroxy-isopterocarpolone (4), 3α-hydroxy-pterocarpol (5), and (11R)-2, 11, 12-trihydroxy-β-selinene (6), were isolated along with known sesquiterpenoids (7–10). The structures of these compounds were established using NMR, MS, and IR methods. These compounds (1–6) were not observed in A. lancea extracts by TLC and HPLC analysis, suggesting that these compounds were generated during the fermentation process.

A novel nucleoside, 9-β-d-ribopyranosylpurine (2), along with three known nucleosides, adenosine (1), uridine (3) and nebularine (4), were isolated from the edible mushroom, Tricholoma japonicum. The structure of 2 was determined as 9-β-d-ribopyranosylpurine by comparing the reported spectral data of 2 with that of a synthetic compound. Isolation of the glycoside, which contains the sugar ribopyranose, from natural resources is very unusual. There are reports on the synthesis of 9-β-d-ribopyranosylpurine (2), but this is the first report on the isolation from natural resources. The antiproliferative activity of compounds 1–4 was evaluated using human umbilical vein endothelial cells. Compound 4 showed the highest inhibitory activity.

Hypoxylonol C (1), isolated from the inedible mushroom Hypoxylon truncatum, exhibited inhibitory activities against the migration and tube formation of HUVECs. A cDNA microarray analysis was performed to investigate the target of hypoxylonol C (1) in HUVECs, and it was found that the genes related to cell cycle and adhesion were down-regulated. The down-regulation of mRNA levels of cell cycle and adhesion genes was confirmed by real-time RT-PCR. Cell cycle arrest and suppression of adhesion molecule expression might be plausible mechanisms of actions for the antiangiogenic activity of hypoxylonol C (1).

One novel p-terphenyl compound, polyozellic acid (1), and its acetone adduct (3), along with a known p-terphenyl compound, thelephoric acid (2), were isolated from the mushroom Polyozellus multiplex. Their molecular structures were determined by spectroscopic analysis, X-ray crystallographic analysis, and chemical modification. In some assays related to angiogenesis, compounds 1 and 2 in particular showed inhibitory effects on proliferation, tubule formation, and invasion of human umbilical vein endothelial cells. The quinone moiety within these molecules possibly contributes to their antiangiogenesis activity.

The diversity of natural compounds is extensive but not unlimited. We therefore studied the feasibility of fermenting plant extracts to increase the diversity of natural compounds for use as drug candidates. Three new terpenoids, sterebins O (1), P1 (2), and P2 (3), were isolated from an extract of Stevia rebaudiana fermented by Saccharomyces cerevisiae. The structures of these compounds were established using NMR, MS, and IR methods. The absolute configuration of 1 was determined by X-ray diffraction analysis, and that of 2 and 3 from the ECD spectrum of their benzoate derivatives. These three compounds were not observed in S. rebaudiana extracts by TLC, suggesting that the compounds were generated during the fermentation process. Compounds 1, 2, and 3 all inhibited melanogenesis in theophylline-stimulated B16 melanoma cells, with 1 exhibiting the lowest IC50 value (9.8 μM). The results indicate that fermentation of plant extracts may provide a route for generating many useful compounds.

Four new 4-hydroxy-2-pyridone alkaloids, didymellamides A–D (1–4), were isolated from the marine-derived fungus Stagonosporopsis cucurbitacearum. The structures of 1–4 were elucidated from spectroscopic data (NMR, MS, and IR), and the absolute configuration of 1 was determined by X-ray diffraction analysis. Didymellamide A (1) exhibited antifungal activity against azole-resistant Candida albicans.

Four novel benzo[j]fluoranthene derivatives, hypoxylonols C (3), D (4), E (5), and F (6), have been isolated from the mushroom Hypoxylon truncatum, together with two known benzo[j]fluoranthene derivatives, hypoxylonols A (1) and B (2). The structures were established by analysis of NMR spectroscopic data and X-ray diffraction data. Compounds 4 and 5 showed antiproliferative activity against HUVECs (human umbilical vein endothelial cells) and HUAECs (human umbilical artery endothelial cells).

Three new diterpenes, myrocin D (1), libertellenone E (2), and libertellenone F (3), and a new isocoumarin, decarboxyhydroxycitrinone (4), were isolated from the marine fungus Arthrinium sacchari, together with three known compounds (5–7). The structures of 1–4 were elucidated from spectroscopic data (NMR, MS, IR), and the absolute configurations of 1–3 were determined by X-ray diffraction analysis. The antiangiogenic activity of these compounds was evaluated by measuring their antiproliferation effects on human umbilical vein endothelial cells (HUVECs) and human umbilical artery endothelial cells (HUAECs). Compounds 4–7 showed inhibitory activity.

The aims of this study were to investigate the role of tyrosine kinase in intracellular signaling and to search for lead compounds with tyrosine kinase inhibitory activity from metabolites of marine-derived fungi. We initially prepared 400 extracts from 200 species of marine fungi and then subjected them to a tyrosine kinase screening assay using human umbilical vein endothelial cell lysate. Tyrosine kinase inhibitory activity was observed among certain metabolites of Hypocrea vinosa. We isolated one known compound, SC2051 (1), as well as two new compounds, hypochromins A (2) and B (3), which have a bis(naphtho-γ-pyrone) skeleton. Compounds 1−3 showed tyrosine kinase inhibitory activity, with IC50 values of 42.1, 58.7, and 18.0 μΜ, respectively. Furthermore, compounds 1−3 exhibited inhibitory effects on proliferation, migration, and tubule formation.

Three novel p-terphenyl compounds, named boletopsins A (1), B (2), and C (3), and four known analogues (4−7) were isolated from fruiting bodies of the mushroom Boletopsis leucomelas. Compounds 1−7 were tested for KDR kinase inhibitory activity, and boletopsin C (3) was found to have an IC50 value of 70.7 μΜ. Compound 3 also showed inhibition of proliferation of human umbilical vein endothelial cells, with an IC50 value of 9.04 μM.

We previously demonstrated that ethyl acetate extracts of Kaempferia parviflora Wall. Ex Baker (KPE) improve insulin resistance in TSOD mice and showed that its components induce differentiation and adipogenesis in 3T3-L1 preadipocytes. The present study was undertaken to examine whether KPE and its isolated twelve components suppress further lipid accumulation in 3T3-L1 mature adipocytes. KPE reduced intracellular triglycerides in mature adipocytes, as did two of its components, 3,5,7,3′,4′-pentamethoxyflavone and 5,7,4′-trimethoxyflavone. Shrinkage of lipid droplets in mature adipocytes was observed, and mRNA expression levels of adipose tissue triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) were up-regulated by these two polymethoxyflavonoids (PMFs). Furthermore, the protein expression level of ATGL and the release level of glycerol into the cell culture medium increased. In contrast, the peroxisome proliferator-activated receptor γ (PPARγ) agonist, troglitazone, did not decrease intracellular triglycerides in mature adipocytes, and the mRNA expression level of PPARγ was not up-regulated in mature adipocytes treated with the two active PMFs. Therefore, suppression of lipid accumulation in mature adipocytes is unlikely to be enhanced by transcriptional activation of PPARγ. These results suggest that KPE and its active components enhance lipolysis in mature adipocytes by activation of ATGL and HSL independent of PPARγ transcription, thus preventing adipocyte hypertrophy. On the other hand, the full hydroxylated flavonoid quercetin did not show the suppressive effects of lipid accumulation in mature adipocyte in the same conditions. Consequently, methoxy groups in the flavones are important for the activity.Download high-res image (215KB)Download full-size image