Photoreaction products of extract from the fruiting bodies of Polyozellus multiplex.

Photochemical reactions are powerful tools for synthesizing organic molecules. The input of energy provided by light offers a means to produce strained and unique molecules that cannot be assembled using thermal protocols, allowing for the production of immense molecular complexity in a single chemical step. Furthermore, unlike thermal reactions, photochemical reactions do not require active reagents such as acids, bases, metals, or enzymes. Photochemical reactions play a central role in green chemistry. This article reports the isolation and structure determination of four new compounds (1-4) from the photoreaction products of the Polyozellus multiplex MeOH ext. The structures of the new compounds were elucidated using MS, IR, comprehensive NMR measurements and microED. The four compounds were formed by deacetylation of polyozellin, the main secondary metabolite of P. multiplex, and addition of singlet oxygen generated by sunlight. To develop drugs for treating Alzheimer's disease (AD) on the basis of the amyloid cascade hypothesis, the compounds (1-4) obtained by photoreaction were evaluated for BACE1 inhibitory activity. The hydrolysates (5 and 6) of polyozellin, the main secondary metabolites of P. multiplex, were also evaluated. The photoreaction products (3 and 4) and hydrolysates (5 and 6) of polyozellin showed BACE1 inhibitory activity (IC50: 2.2, 16.4, 23.3, and 5.3 µM, respectively).

Inhibition of Aß aggregation by naphtho-γ-pyrone derivatives from a marine-derived fungus, Aspergillus sp. MPUC239.

Alzheimer's disease (AD) is an important human disease that mainly causes cognitive impairments. Growing evidence has shown that amyloid-ß (Aß) peptide plays a key role in AD pathogenesis in what is known as the Aß cascade hypothesis. This hypothesis suggests the importance of suppressing Aß aggregation and Aß production. The latter process is governed by ß-site APP Cleaving Enzyme1 (BACE1) and γ-secretase. We, therefore, focused on Aß aggregation inhibitory activity, initially assessing numerous extracts derived from our marine-derived fungus collections. One EtOAc extract derived from an Aspergillus sp. exhibited Aß aggregation inhibitory activity. Eleven known compounds (1-11) were isolated from CHCl3 and EtOAc extracts derived from the fungus, and the structures were identified based on MS, NMR, and ECD spectra. Compounds 2, 6, and 10 inhibited Aß aggregation with IC50 values of 2.8, 3.9, and 8.1 µM, respectively. The protective effect on SH-SY5Y cells against Aß toxicity was also evaluated, and compounds 6 and 10 significantly alleviated Aß toxicity. BACE1 inhibitory activity was also examined, and compounds 4, 5, 7, 10, and 11 inhibited BACE1 activity with IC50 values of 14.9, 70.0, 36.5, 28.0, and 72.8 µM, respectively. These data suggest that compound 10 could be useful in AD treatment.

Enniatins from a marine-derived fungus Fusarium sp. inhibit biofilm formation by the pathogenic fungus Candida albicans.

Candidemia is a life-threatening disease common in immunocompromised patients, and is generally caused by the pathogenic fungus Candida albicans. C. albicans can change morphology from yeast to hyphae, forming biofilms on medical devices. Biofilm formation contributes to the virulence and drug tolerance of C. albicans, and thus compounds that suppress this morphological change and biofilm formation are effective for treating and preventing candidemia. Marine organisms produce biologically active and structurally diverse secondary metabolites that are promising lead compounds for treating numerous diseases. In this study, we explored marine-derived fungus metabolites that can inhibit morphological change and biofilm formation by C. albicans. Enniatin B (1), B1 (2), A1 (3), D (4), and E (5), visoltricin (6), ergosterol peroxide (7), 9,11-dehydroergosterol peroxide (8), and 3ß,5α,9α-trihydroxyergosta-7,22-dien-6-one (9) were isolated from the marine-derived fungus Fusarium sp. Compounds 1-5 and 8 exhibited inhibitory activity against hyphal formation by C. albicans, and compounds 1-3 and 8 inhibited biofilm formation by C. albicans. Furthermore, compounds 1-3 decreased cell surface hydrophobicity and expression of the hypha-specific gene HWP1 in C. albicans. Compound 1 was obtained in the highest yield. An in vivo evaluation system using silkworms pierced with polyurethane fibers (a medical device substrate) showed that compound 1 inhibited biofilm formation by C. albicans in vivo. These results indicate that enniatins could be lead compounds for therapeutic agents for biofilm infections by C. albicans.

Inhibition of BACE1 and Amyloid-ß Aggregation by Meroterpenoids from the Mushroom Albatrellus yasudae.

To develop drugs to treat Alzheimer's disease (AD) on the basis of the amyloid cascade hypothesis, the amyloid-ß (Aß) aggregation inhibitory activities of 110 extracts from mushrooms were evaluated by thioflavin T (Th-T) assays. The MeOH extract of Albatrellus yasudae inhibited Aß aggregation, and the bioactivity-guided fractionation of the extract afforded four novel meroterpenoids, named scutigeric acid (1), albatrelactone methyl ester (2), albatrelactone (3), and 10',11'-dihydroxygrifolic acid (4), together with two known compounds, grifolin (5) and grifolic acid (6). The structures of 1-4 were elucidated using NMR, MS, UV, IR, and induced ECD spectral data. The structure of 1 was determined as a methyl ester (1a) by 2D NMR spectroscopy. Th-T assays showed that compounds 1-4 and 1a possessed inhibitory activities against Aß aggregation, with IC50 values of 6.6, 40.7, 51.4, 53.3, and 50.3 µM, respectively. Notably, 1 possessed an inhibitory activity against Aß aggregation comparable to that of myricetin as a positive control. Moreover, 1-6 exhibited inhibitory activities against BACE1, with IC50 values of 1.6, 10.9, 10.5, 34.4, 6.1, and 1.4 µM, respectively.

Amyloid ß aggregation inhibitory activity of triterpene saponins from the cactus Stenocereus pruinosus.

Six new triterpene saponins (1-5,7) and 3 known saponins (6,8,9) were isolated from MeOH extracts of the cactus Stenocereus pruinosus. The structures of the isolated saponins were elucidated using MS, IR, and comprehensive NMR measurements. To develop drugs for treating Alzheimer's disease (AD) on the basis of the amyloid cascade hypothesis, the isolated saponins were evaluated for inhibition of BACE1 activity and amyloid beta (Aß) aggregation using thioflavin-T assay, and triterpenes as an aglycone moiety and an alkaline hydrolysate of the saponins were also evaluated. One saponin, stenoside A (7), exhibited inhibitory activity related to Aß aggregation and its degree of Aß aggregation was 40.6% at 100 µM.

Isolation of three new meroterpenoids and seven known compounds from Albatrellus yasudae and their Aß-aggregation inhibitory activity.

Alzheimer's disease is a serious neurologic disorder that cannot be cured completely. In this study, we targeted compounds that inhibit amyloid-beta (Aß) aggregation, based on the amyloid cascade hypothesis. Ten compounds (1-10) were isolated from CHCl3 extracts of the mushroom Albatrellus yasudae using Aß-aggregation inhibitory activity-guided separation. The structures of these compounds were elucidated from 1D and 2D NMR and MS spectral data. Compounds 1-3 were novel, whereas 4-10 were identified as the known compounds grifolin, grifolic acid, neogrifolin, confluentin, 2-hydroxyneogrifolin, daurichromenic acid, and a cerebroside derivative. Compounds 1-10 were tested for Aß-aggregation inhibitory activity. Compounds 1, 3, 5, 6, 8, and 9 have potential as Aß-aggregation inhibitory activity.

Meroterpenoids with BACE1 Inhibitory Activity from the Fruiting Body of Boletinus asiaticus.

BACE1 inhibitory activity-guided fractionation of an extract of the fruiting body of Boletinus asiaticus yielded five novel meroterpenoids (1-5) and one known compound (6; asiaticusin A). The structures of these compounds were determined by interpretation of NMR, MS, and IR spectral data. The five new compounds contain 4-hydroxybenzoic acid and geranylgeranoic acid units. Compounds 4-6 possessed BACE1 inhibitory activity (IC50 values: 14.7, 11.4, and 2.0 µM, respectively).

Naturally occurring biflavonoids with amyloid ß aggregation inhibitory activity for development of anti-Alzheimer agents.

Amyloid ß (Aß) aggregation plays an essential role in promoting the progression of Alzheimer's disease (AD). Therefore, the inhibition of Aß aggregation is a potential therapeutic approach for AD. Herein, twenty-seven biflavonoids with different inter-flavonyl linkages and methoxy substitution patterns were isolated from several plants, and their Aß40 aggregation inhibitory activity was evaluated by the thioflavin-T fluorescence assay. Amentoflavone (1) and its monomethoxy derivatives (2, 3, and 5) exhibited the most potent inhibitory activity, with IC50 values of approximately 5 µM. It was clarified that increasing the number of methoxy substituents on the biflavonoid structures attenuated the inhibitory activity. Moreover, the linkage and the methoxy substitution pattern had a marked influence on the inhibitory activity. Our investigation strongly supports that biflavonoids can be considered a new type of anti-Alzheimer agents that may be successfully developed for AD patients.

Isoindolinones, Phthalides, and a Naphthoquinone from the Fruiting Body of Daldinia concentrica.

A chemical investigation of the ascomycetes of Daldinia concentrica was performed using silica gel column chromatography, ODS column chromatography, and preparative HPLC. Two new isoindolinone compounds, daldinans B (1) and C (2), two new phthalide compounds, daldinolides A (3) and B (4), and a new naphthoquinone, daldiquinone (5), were isolated together with two known compounds (6 and 7). The structures of 1, 2, and 5 were established using NMR, MS, and IR methods, and the structures of 3 and 4 were determined by derivatization from known compounds (6 and 7). 5 exhibited antiangiogenesis activity against HUVECs (IC50 = 7.5 µM).

Synthesis and anti-influenza virus evaluation of triterpene-sialic acid conjugates.

We are interested in new non-natural glycosides with sialic acid conjugates and their biological activities. We report the synthesis of eleven non-natural occurring glycosides, which are triterpene (glycyrrhetinic acid and its derivatives)-sialic acid conjugates, and their inhibitory activities against influenza virus sialidases and influenza virus multiplication in MDCK host cells. Deoxoglycyrrhetol-sialic acid conjugates (6d and 6e) and oleanolic acid-sialic acid conjugates (7d and 7e) showed strong inhibitory activities against three subtypes of influenza virus sialidases. These four compounds (6d, 6e, 7d and 7e) showed clear inhibition to influenza virus multiplication but not to MDCK host cell survival.

Triterpenoid saponins from Polaskia chichipe Backbg. and their inhibitory or promotional effects on the melanogenesis of B16 melanoma cells.

Five new oleanane-type saponins 1-5 together with a known saponin 6 and a steroidal glycoside 7 were isolated from Polaskia chichipe Backbg., and their structures were determined from their 1D and 2D NMR and HRFABMS spectral data. The six isolated saponins 1-6 were tested for their effects on the melanogenesis of B16 melanoma 4A5 cells. Compound 1 exerted an inhibitory effect at 100 µM whereas compound 3 promoted melanogenesis at the same concentration, even though these two compounds contain the same aglycon structure. The dose-dependent activities of compounds 1 and 3 on melanin synthesis were investigated.

Inhibition of amyloid ß aggregation and protective effect on SH-SY5Y cells by triterpenoid saponins from the cactus Polaskia chichipe.

Alzheimer's disease (AD) destroys brain function, especially in the hippocampus, and is a social problem worldwide. A major pathogenesis of AD is related to the accumulation of amyloid beta (Aß) peptides, resulting in neuronal cell death in the brain. Here, we isolated four saponins (1-4) and elucidated their structures from 1D and 2D NMR and HRFABMS spectral data. The structures of 1 and 2 were determined as new saponins which have cochalic acid as the aglycon, and 3 was determined as a new saponin with oleanolic acid as the aglycon. Compound 4 was confirmed as the known saponin chikusetsusaponin V (=ginsenoside R0). Isolated saponins (1-4) and six previously reported saponins (5-10) were tested for their inhibitory effects of Aß aggregation and their protective effects on SH-SY5Y cells against Aß-associated toxicity. As the results, compounds 3 and 4 showed inhibitory effect of Aß aggregation and compounds 5-8 exerted the protective effects on SH-SY5Y cells against Aß-associated toxicity.

Conditional changes enhanced production of bioactive metabolites of marine derived fungus Eurotium rubrum.

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, 9.1µM 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.

Macrolides from a Marine-Derived Fungus, Penicillium meleagrinum var. viridiflavum, Showing Synergistic Effects with Fluconazole against Azole-Resistant Candida albicans.

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.

Biosynthesis of Panaefluoroline B from the Cultured Mycobiont of Amygdalaria panaeola.

Panaefluoroline B (2) is a fluorescent yellowish-green pigment produced by the cultured mycobiont of a lichen, Amygdalaria panaeola. Panaefluoroline B (2) has an isoquinoline skeleton, a C5 unit, and an amino acid, glycine, in its structure. The biosynthetic pathway of 2 was revealed by feeding experiments using [1-(13)C]-sodium acetate and [1,2-(13)C2][(15)N]-glycine. The analysis of labeling patterns of 2 and its mass spectrum suggested the isoquinoline part is biosynthesized via the acetate-malonate pathway with glycine as the nitrogen source and that the C5 unit originates from the mevalonate pathway.

A novel nucleoside from the edible mushroom, Tricholoma japonicum.

A novel nucleoside, 9-ß-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-ß-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-ß-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.

Inhibitory activities of biflavonoids against amyloid-ß peptide 42 cytotoxicity in PC-12 cells.

A major hallmark of Alzheimer's disease is the cerebral accumulation and resulting cytotoxicity of amyloid-ß peptides, particularly Aß42. In this study, we used an MTT assay to investigate the inhibitory activity of biflavonoids 1-22 against Aß42 cytotoxicity in PC-12 cell cultures. Cytoprotective effects were observed for the following amentoflavone type biflavonoids: podocarpusflavone B 8, isoginkgetin 10, sciadopitysin 13, and kayaflavone 15. These biflavonoids exhibited strong activity in tested compounds, with EC50 values of 5.18, 10.77, 9.84, and 5.29 µM, respectively. Cell viability tests of PC-12 cells revealed that biflavonoids 13 and 15 had stronger inhibitory activities than apigenin 23 and (-)-epigallocatechin gallate 24.

Suppression of adipocyte hypertrophy by polymethoxyflavonoids isolated from Kaempferia parviflora.

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.

Antiangiogenic activity of hypoxylonol C.

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).