Secondary Metabolites of the Endophytic Fungus Lachnum abnorme from Ardisia cornudentata.

Fractionation of an EtOAc-soluble fraction of the solid fermentate of an endophytic fungus, Lachnum abnorme Mont. BCRC 09F0006, derived from the endemic plant, Ardisia cornudentata Mez. (Myrsinaceae), resulted in the isolation of three new chromones, lachnochromonins D-F (1-3), one novel compound, lachabnormic acid (4), along with nine known compounds (5-13). Their structures were elucidated by spectroscopic analyses. Alternariol-3,9-dimethyl ether (6) was given the correct data as well as 2D spectral analyses for the first time. This is the first report of the isolation of one unprecedented compound 4 from Lachnum genus, while all known compounds were also found for the first time from Lachnum. The effects of some isolates (3, 4, 7-9, 10, and 13) on the inhibition of nitric oxide (NO) production in lipopolysaccharide (LPS)-activated RAW 264.7 murine macrophages were also evaluated. Several compounds exhibited weak inhibitory activity on lipopolysaccharide (LPS)-stimulated NO production in RAW 264.7 macrophages.

Inhibitory effects of constituents of an endophytic fungus Hypoxylon investiens on nitric oxide and interleukin-6 production in RAW264.7 macrophages.

Three new compounds, hypoxyloamide (1), 8-methoxynaphthalene-1,7-diol (2), and hypoxylonol (3), together with seven compounds isolated from nature for the first time, investiamide (4), hypoxypropanamide (5), hypoxylonol A (6), investienol (7), 2-heptylfuran (8), (3S)-5-methyl-8-O-methylmellein (9), (4R)-O-methylsclerone (10), along with 19 known compounds, 11-29, were isolated from the culture broth of Hypoxylon investiens BCRC 10F0115, a fungal endophyte residing in the stems of an endemic Formosan plant Litsea akoensis var. chitouchiaoensis. The structures of the new compounds were established by spectroscopic methods, including UV, IR, HR-ESI-MS, and extensive 1D- and 2D-NMR techniques. Of these isolates, 2, 8-methoxynaphthalen-1-ol (15), and 1,8-dimethoxynaphthalene (16) showed nitric oxide (NO) inhibitory activity with IC50 values of 11.8±0.9, 17.8±1.1, and 13.3±0.5 µM, respectively, stronger than the positive control quercetin (IC50 36.8±1.3 µM). Compounds 2, 15, and 16 also showed interleukin-6 (IL-6) inhibitory activity with IC50 values of 9.2±1.7, 18.0±0.6, and 2.0±0.1 µM, stronger than the positive control quercetin (IC50 31.3±1.6 µM). To the best of our knowledge, this is the first report on guaiane sesquiterpene metabolites, 3, 6, and 7, from the genus Hypoxylon.

Inhibitory effects of maleimide derivatives from the mycelia of the fungus Antrodia cinnamomea BCRC 36799 on nitric oxide production in lipopolysaccharide (LPS)-activated RAW264.7 macrophages.

Four new maleimide derivatives, antrocinnamomins E-H (1-4, resp.), together with (3S,4R)-1-hydroxy-3-(4-hydroxyphenyl)-4-(2-methylpropyl)pyrrolidine-2,5-dione (5) and ergosterol were isolated from the mycelia of Antrodia cinnamomea BCRC 36799. The structures were elucidated by 1D- and 2D-NMR spectroscopy, and mass spectrometry. Compounds 1-5 were evaluated for their inhibitory effects on nitric oxide (NO) production by macrophages. Compounds 2 and 4 showed stronger inhibition of NO production than the positive control quercetin.

Chemical constituents from a soil-derived actinomycete, Actinomadura miaoliensis BCRC 16873, and their inhibitory activities on lipopolysaccharide-induced tumor necrosis factor production.

An investigation on the secondary metabolites from the BuOH extract of the fermentation broth of the thermotolerant polyester-degrading actinomycete Actinomadura miaoliensis BCRC 16873 was carried out. One previously undescribed α-pyrone (=pyran-2-one) derivative, designated as miaolienone (1), and a new butanolide, miaolinolide (2), together with 13 known compounds, 3-15, were obtained. Their structures were established on the basis of extensive 1D- and 2D-NMR analyses in combination with HR-MS experiments. In addition, the isolated compounds 1-15 were evaluated for the inhibitory effects of the isolates on the production of tumor necrosis factor (TNF-α) induced by lipopolysaccharide (LPS). Among the isolates, 1 and 2 significantly inhibited TNF-α production in U937 cells in vitro, and the IC(50) values were 0.59 and 0.76 µM, respectively. Compounds 3-5 displayed moderate inhibitory activities on LPS-induced TNF-α production.

Phytochemical investigation of Annulohypoxylon ilanense, an endophytic fungus derived from Cinnamomum species.

Cultivation of the fungal strain Annulohypoxylon ilanense, an endophytic fungus isolated from the wood of medicinal plant Cinnamomum species, resulted in the isolation of one new furanoid derivative, ilanefuranone (1), one new pyrrole alkaloid, ilanepyrrolal (2), and one new biarylpropanoid derivative, ilanenoid (3), together with 22 known compounds, of which one α-tetralone analog, (-)-(4R)-3,4-dihydro-4,6-dihydroxynaphthalen-1(2H)-one (4) was isolated for the first time from a natural source. The structures were elucidated on the basis of physicochemical evidence, in-depth NMR spectroscopic analysis, and high-resolution mass spectrometry, and the antimycobacterial activities were also evaluated.

Deriving the phylogenetic information from some physicochemical properties of protein sequences computed.

The evolutionary relationships of organisms are traditionally delineated by the alignment-based methods using some DNA or protein sequences. In the post-genome era, the phylogenetics of life could be inferred from many sources such as genomic features, not just from comparison of one or several genes. To investigate the possibility that the physicochemical properties of protein sequences might reflect the phylogenetic ones, an alignment-free method using a support vector machine (SVM) classifier is implemented to establish the phylogenetic relationships between some protein sequences. There are two types of datasets, namely, the "Enzymatic" (assigned by an EC accession) and "Proteins" used to train the SVM classifiers. By computing the F-score for feature selection, we find that the classification accuracies of trained SVM classifiers could be significantly enhanced to 84% and 80%, respectively, for the enzymatic and "proteins" datasets classified if the protein sequences are represented with some top 255 features selected. These show that some physicochemical features of amino acid sequences selected are sufficient for inferring the phylogenetic properties of the protein sequences. Moreover, we find that the selected physicochemical features appear to correlate with the physiological characteristic of the taxonomic classes classified.

Monasnicotinates A-D, four new pyridine alkaloids from the fungal strain Monascus pilosus BCRC 38093.

Four new pyridine derivatives, monasnicotinates A-D (1-4) were isolated from the red yeast rice of Monascus pilosus BCRC 38093. Their structures were elucidated on the basis of physicochemical evidence, in-depth NMR spectroscopic analysis, and high-resolution mass spectrometry. Their inhibitory effects on NO production was also evaluated.

A new pyrrole derivative from the extracts of the fungus monascus pilosus-fermented rice.

A chemical study on the n-BuOH-soluble fraction of the 95% EtOH extract of red yeast rice fermented with the fungus Monascus pilosus BCRC 38093 (Eurotiaceae) has resulted in the isolation of one new natural pyrrole derivative, designated as monascuspyrrole (1) together with nine known compounds, 3ß-hydroxystigmast-5-en-7-one (2), ß-sitostenone (3), monascin (4), ankaflavin (5), N-trans-feruloyltyramine (6), N-cis-feruloyltyramine (7), vanillic acid (8), methyl paraben (9), and syringaldehyde (10). The structure of the new compound 1 was identified by 1D and 2D NMR spectroscopy, as well as by high-resolution mass spectrometry. Other known compounds were identified by comparison of their spectral data with the literature data of authentic samples. Compounds 1 and 4 displayed mild inhibitory effect of nitric oxide production. Among the nine known isolates, compounds 2, 3, 6, and 7 were found for the first time in this species.

Maleimide and maleic anhydride derivatives from the mycelia of Antrodia cinnamomea and their nitric oxide inhibitory activities in macrophages.

On cultivation of the fungus Antrodia cinnamomea (BCRC 36799) on a medium, the mycelium was extracted and evaluated for nitric oxide (NO) inhibitory activity. Bioactivity-directed fractionation led to the isolation of two new maleimide derivatives, antrocinnamomins A (1) and B (2), and two new maleic anhydride derivatives, antrocinnamomins C (3) and D (4), along with three known compounds, 3-isobutyl-4-[4-(3-methyl-2-butenyloxy)phenyl]furan-2,5-dione (5), 3-isobutyl-4-[4-(3-methyl-2-butenyloxy)phenyl]-1H-pyrrole-2,5-dione (6), and 3-isobutyl-4-[4-(3-methyl-2-butenyloxy)phenyl]-1H-pyrrol-1-ol-2,5-dione (7). Structural elucidation of compounds 1-4 was carried out by spectroscopic data. Compound 1 displayed significant inhibitory effect on nitric oxide (NO) production.

Selection of an effective red-pigment producing Monascus pilosus by efficient transformation with aurintricarboxylic acid.

The filamentous fungus Monascus pilosus was genetically transformed with a reporter plasmid, pMS-1.5hp, by aurintricarboxylic acid (ATA) treatment to obtain an efficient red-pigment producing mutant. The transformation efficiency of Monascus pilosus was higher with the ATA-treatment than with either a non-restriction-enzyme-mediated integration (REMI) or a REMI method. This valid and convenient random mutagenesis method shows that ATA can be applied in fungi for efficient genetic transformation.

Evaluating the urate-lowering effects of different microbial fermented extracts in hyperuricemic models accompanied with a safety study.

Uric acid (UA) is an end product of purine metabolism by the enzyme xanthine oxidase (XOD). Hyperuricemia is characterized by the accumulation of serum UA and is an important risk factor for gout and many chronic disorders. XOD inhibitors or uricase (catalyzes UA to the more soluble end product) can prevent these chronic diseases. However, currently available hypouricemic agents induce severe side effects. Therefore, we developed new microbial fermented extracts (MFEs) with substantial XOD inhibition activity from Lactobacillus (MFE-21) and Acetobacter (MFE-25), and MFE-120 with high uricase activity from Aspergillus. The urate-lowering effects and safety of these MFEs were evaluated. Our results showed that MFE-25 exerts superior urate-lowering effects in the therapeutic model. In the preventive model, both MFE-120 and MFE-25 significantly reduced UA. The results of the safety study showed that no organ toxicity and no treatment-related adverse effects were observed in mice treated with high doses of MFEs. Taken together, the results showed the effectiveness of MFEs in reducing hyperuricemia without systemic toxicity in mice at high doses, suggesting that they are safe for use in the treatment and prevention of hyperuricemia.

Association algorithm to mine the rules that govern enzyme definition and to classify protein sequences.

BACKGROUND: The number of sequences compiled in many genome projects is growing exponentially, but most of them have not been characterized experimentally. An automatic annotation scheme must be in an urgent need to reduce the gap between the amount of new sequences produced and reliable functional annotation. This work proposes rules for automatically classifying the fungus genes. The approach involves elucidating the enzyme classifying rule that is hidden in UniProt protein knowledgebase and then applying it for classification. The association algorithm, Apriori, is utilized to mine the relationship between the enzyme class and significant InterPro entries. The candidate rules are evaluated for their classificatory capacity. RESULTS: There were five datasets collected from the Swiss-Prot for establishing the annotation rules. These were treated as the training sets. The TrEMBL entries were treated as the testing set. A correct enzyme classification rate of 70% was obtained for the prokaryote datasets and a similar rate of about 80% was obtained for the eukaryote datasets. The fungus training dataset which lacks an enzyme class description was also used to evaluate the fungus candidate rules. A total of 88 out of 5085 test entries were matched with the fungus rule set. These were otherwise poorly annotated using their functional descriptions. CONCLUSION: The feasibility of using the method presented here to classify enzyme classes based on the enzyme domain rules is evident. The rules may be also employed by the protein annotators in manual annotation or implemented in an automatic annotation flowchart.

Six new metabolites produced by Colletotrichum aotearoa 09F0161, an endophytic fungus isolated from Bredia oldhamii.

Six new compounds, colletobredins A-D (1-4) and colletomelleins A and B (5 and 6), along with 12 previously identified compounds, were isolated from the culture broth of Colletotrichum aotearoa BCRC 09F0161, a fungal endophyte residing in the leaves of an endemic Formosan plant Bredia oldhamii Hook. f. (Melastomataceae). The structures of the new compounds were established by spectroscopic methods, including UV, IR, HR-ESIMS and extensive 1D and 2D NMR techniques. The effects of some isolates on the inhibition of nitric oxide (NO) production in lipopolysaccharide-activated murine macrophage RAW264.7 cells were evaluated. All these compounds inhibited NO production in activated macrophages without any cytotoxicity at a concentration of 100 µM. Of these isolates, 1 showed weak NO inhibitory activity with IC50 value of 182.2 µM. To the best of our knowledge, this is the first report on isochroman glycoside metabolites (1-4) from the genus Colletotrichum.

Analysis of Taiwan patents for the medicinal mushroom "Niu-Chang- Chih".

"Niu-Chang-Chih" (Antrodia cinnanomea) is a medicinal mushroom that has only been collected from the aromatic tree, Cinnamomum kanehirai, which is native to Taiwan. A total of 105 Taiwan patent applications and patents for "Niu-Chang-Chih" were collected and analyzed. Patent applications and granted patents claiming newly identified functional components from "Niu-Chang-Chih," biologically pure cultures of the mushroom strain, and cultivation of "Niu-Chang-Chih" were examined. Several applications and patents claim identified active compounds from "Niu-Chang- Chih," which provide better patent protection. These newly identified functional compounds include cyclohexanones, maleic and succinic acid derivatives, labdane diterpenoids, and benzenoids. Newly identified functional proteins include a glutathione-dependent formaldehyde dehydrogenase (GFD), a glycoprotein named ACA1, and a laccase. Newly identified functional polysaccharides include ACP1, ACP2, and ACP3. The number of patents for newly identified compounds and their uses are expected to continue growing.

Monascusazaphilones A-C, three new azaphilone analogues isolated from the fungus Monascus purpureus BCRC 38108.

Investigation of the 95% EtOH extract of red yeast rice fermented with the pink mutant of the fungus Monascus purpureus BCRC 38108 led to the isolation of three new azaphilone derivatives, namely monascusazaphilones A-C (1-3), together with two known compounds. Compounds 1-3 were isolated from this species for the first time. Their structures were elucidated by 1-D and 2-D nuclear magnetic resonance spectroscopy together with HR-ESI-MS analysis and comparison of the spectroscopic data with those reported in the literatures. All isolates were evaluated for their inhibitory effects on nitric oxide (NO) production by macrophages. Among the isolates, compound 1 demonstrated stronger inhibition on NO production.

Preventive effects of Monascus on androgen-related diseases: androgenetic alopecia, benign prostatic hyperplasia, and prostate cancer.

Androgen-related diseases impair the well-being of many aging men. Unfortunately, the medications used to treat these diseases have many side effects. Therefore, there is a significant need for the development of novel drugs to treat androgen-related diseases. In this study, we investigated the effects of Monascus cursory extraction (M-CE) on androgen-related diseases, including androgenetic alopecia (AGA), benign prostatic hyperplasia (BPH) and prostate cancer. We found that M-CE suppressed baldness in male B6CBAF1/j mice. Furthermore, M-CE decreased PSA levels, indicating a protective effect of M-CE on testosterone-induced hyperplasia. M-CE also significantly decreased tumor volume and tumor incidence in an N-methyl-N-nitrosourea (MNU)/testosterone-induced rat prostate cancer model and markedly decreased dihydrotestosterone (DHT) but not testosterone. Additionally, PCNA expression was decreased in the prostate of rats treated with M-CE. These results suggest that M-CE could be a new potential therapeutic candidate for the treatment of androgen-related diseases.

Monascuspiloin induces apoptosis and autophagic cell death in human prostate cancer cells via the Akt and AMPK signaling pathways.

Monascus pigments have been reported to possess anticancer effects in various cancer cells; however, the molecular mechanisms of their anticancer properties remain largely unknown. Monascuspiloin is an analogue of the Monascus pigment monascin, and its anticancer growth activity against human prostate cancer cells was evaluated using in vitro and in vivo models. Monascuspiloin effectively inhibits the growth of both androgen-dependent LNCaP and androgen-independent PC-3 human prostate cancer cells. Monascuspiloin preferentially induces apoptosis in LNCaP cells by attenuating the PI3K/Akt/mTOR pathway. In androgen-independent PC-3 cells, monascuspiloin induces G2/M arrest and autophagic cell death by an AMPK-dependent pathway. Induction of autophagy in PC-3 cells further sensitizes cells to apoptosis induced by monascuspiloin. Monascuspiloin inhibits tumor growth in nude mice bearing PC-3 xenografts through induction of apoptosis and autophagy. This study is the first to demonstrate that monascuspiloin has therapeutic potential for the treatment of both androgen-dependent and -independent human prostate cancers.

Monascuspiloin enhances the radiation sensitivity of human prostate cancer cells by stimulating endoplasmic reticulum stress and inducing autophagy.

Prostate cancer is a very common cancer among males. Traditional treatments for prostate cancer have limited efficacy; therefore, new therapeutic strategies and/or new adjuvant drugs must be explored. Red yeast rice (RYR) is a traditional food spice made in Asia by fermenting white rice with Monascus purpureus Went yeast. Accumulating evidence indicates that RYR has antitumor activity. In this study, PC-3 cells (human prostate cancer cells) were used to investigate the anti-cancer effects of ionizing radiation (IR) combined with monascuspiloin (MP, a yellow pigment isolated from Monascus pilosus M93-fermented rice) and to determine the underlying mechanisms of these effects in vitro and in vivo. We found that IR combined with MP showed increased therapeutic efficacy when compared with either treatment alone in PC-3 cells. In addition, the combined treatment enhanced DNA damage and endoplasmic reticulum (ER) stress. The combined treatment induced primarily autophagy in PC-3 cells, and the cell death that was induced by the combined treatment was chiefly the result of inhibition of the Akt/mTOR signaling pathways. In an in vivo study, the combination treatment showed greater anti-tumor growth effects. These novel findings suggest that the combined treatment could be a potential therapeutic strategy for prostate cancer.

Antioxidant activities of extracts and metabolites isolated from the fungus Antrodia cinnamomea.

Three different solvent partitions (n-hexane, ethyl acetate [EtOAc] and n-BuOH) of the culture broth from Antrodia cinnamomea were assayed with two different radical scavenging methods: 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging and superoxide radical scavenging (SOD) assay. The EtOAc layer exhibited the best antioxidant activity. Two major antioxidant metabolites were isolated from the active EtOAc layer. The antioxidant activities of compounds 1-6 were further evaluated by DPPH, SOD and trolox equivalent antioxidant capacity (TEAC) assays. Compounds 3 and 5 showed stronger free radical scavenging than the reference BHA, ED50 = 1.36 and 34.24 µM. Compound 5 displayed moderate SOD activity (ED50 = 310.0 µM), and its antioxidant capacity of TEAC value was 2.2 mM trolox equivalency.

A new sesquiterpene isolated from the extracts of the fungus Monascus pilosus-fermented rice.

A new sesquiterpene, monaspilosuslin, as well as seven known compounds 3beta-hydroxystigmast-5-en-7-one, beta-sitostenone, monascin, ankaflavin, N-trans-feruloyltyramine, vanillic acid and alpha-tocopheryl quinone, were isolated from the n-BuOH-soluble fraction of the 70% ethanolic extract of red yeast rice fermented with the fungus Monascus pilosus BCRC 38072 (Eurotiaceae). The structures of these compounds were determined by analyses of spectroscopic data, mainly 2D nuclear magnetic resonance (NMR) experiments. All of the isolates were also evaluated for their scavenging properties toward the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical in thin layer chromatography (TLC) autographic and spectroscopic methods.