Sung-Yuan Hsieh

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.

Secondary Metabolites from the Endophytic Fungus Annulohypoxylon stygium BCRC 34024

A new γ-butyrolactone, annulostygilactone (1), along with nine known compounds consisting of seven benzenoids [p-hydroxybenzaldehyde (2), methylparaben (3), p-hydroxybenzoic acid (4), syringaldehyde (5), eugenol (6), vanillic acid (7), and ferulic acid (8)], and two triterpenoids [ursolic acid (9) and _-daucosterol (10)] were isolated from the endophytic fungus Annulohypoxylon stygium BCRC 34024. Their structures were determined based on their 1D and 2D NMR and HR-ESI-MS spectral data as well as comparison with previous literature data. All compounds were found for the first time in this species. It is worthy of note that this is the first report of secondary metabolites isolated from this fungus. The inhibitory effects of some isolates (compounds 1, 9, and 10) on nitric oxide (NO) production in lipopolysaccharide (LPS)-activated macrophages were evaluated. Compound 1 showed stronger inhibition of NO production than the positive control quercetin.

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.

Secondary metabolites isolated from the fungus Biscogniauxia cylindrospora BCRC 33717

One new dihydroisocoumarin, (3S)-5-hydroxy-8-O-methylmellein (1), as well as nine known compounds, 5-formylmellein (2), mellein-5-carboxylic acid (3), 5-hydroxymethylmellein (4), 3β-hydroxystigmast-5- en-7-one (5), N-trans-feruloyltyramine (6), N-cis-feruloyltyramine (7), vanillic acid (8), methyl paraben (9), and syringaldehyde (10), were isolated from the n-BuOH-soluble fraction of the 70% EtOH extract of rice fermented with the endophytic fungus Biscogniauxia cylindrospora (BCRC 33717). Their structures were elucidated by 1D and 2D NMR spectroscopy together with HR-ESI-MS analysis and comparison of the spectroscopic data with those reported for structurally related compounds.

Secondary metabolites from the endophytic fungus Annulohypoxylon boveri var. microspora BCRC 34012

A chemical study on the n-BuOH-soluble fraction of the 95% EtOH extract of long-grain rice (Oryza sativa) fermented with the endophytic fungus Annulohypoxylon boveri var. microspora (BCRC 34012) has resulted in the isolation of one new natural azaphilone derivative, designated as annulohypoxyboverin (1) together with 12 known compounds, (3R,6R,7E)-3-hydroxy-4,7-megstigma-dien-9-one (2), α-tocopheryl quinone (3), isofraxidin (4), coumarin (5), cinnamic acid (6), a mixture of palmitic acid (7) and stearic acid (8), N-cis-feruloyltyramine (9), luteolin (10), kaempferol (11), kaempferitrin (12), and 4,5,4′,5′-tetrahydroxy-1,1′-binaphthyl (13). Annulohypoxyboverin (1) contains a dihydrobenzofuran-2,4-dione backbone, 1-hydroxyhexyl side chain, and one γ-lactone ring. Their structures were elucidated by spectroscopic analyses including 1D and 2D NMR experiments, and by HR-ESI-MS. The relative configuration of 1 was confirmed by NOESY experiment. Other known compounds were identified by comparing their spectral data with those of literature data.

A New Pyrrole Metabolite from the Endophytic Fungus of Xylaria papulis

One new pyrrole derivative, papupyrrolal (1), together with seven known metabolites consisting of three flavonoids [myricitrin (2), quercitrin (3), and myricetin (4)], and four triterpenoids [3β-trans-p-coumaroyloxy-2α,23-dihydroxyolean-12-en-28-oic acid (5), chebuloside I (6), arjunolic acid (7), and taraxerol (8)] were isolated from the endophytic fungus Xylaria papulis BCRC 09F0222. Structures were established on the basis of their spectroscopic data, including 1H NMR, 13C NMR, HSQC, HMBC, as well as by HR-ESI-MS experiments and comparison with literature data. It is worthy to mention that this is the first report on secondary metabolites from this fungus.

Chemical Constituents of the Fungus of Eurotium chevalieri BCRC 07F0022

Eurotium species are frequently isolated from dry foodstuff. So far, 19 species are recognized [1], and all species are xerophilic and can produce anamorphic stages that belong to the Aspergillus glacus group [2, 3]. Most of the species produce versatile pigments and second metabolites [4]. They are not clearly pathogenic, but a few species have been reported to produce toxic substrates, including E. chevalieri, E. repens, and E. rubrum [2]. Eurotium sp. produce a variety of metabolites, including meroterpenoids, sesquiterpene alkaloids, sesquiterpene, benzaldehyde derivatives, phenolic compounds, and diketopiperazines [5–7]. However, investigations on the chemical constituents and biological activities of the endophytic fungus E. chevalieri have not been conducted. The 95% ethanolic extract of the title fungus showed NO inhibitory activity (approximately 90% inhibition at a concentration of 10 g/mL). It is worth investigating its bioactive compounds. In this study, the fungus of E. chevalieri BCRC 07F0022 was fermented on rice for 30 days. The fermented rice was extracted with 95% ethanol and partitioned between EtOAc and H2O. The EtOAc layer was separated by column chromatography to afford seven pure substances. Six compounds, including 2-(2 ,3-epoxy-1 ,3 -heptadienyl)-6-hydroxy-5-(3-methyl2-butenyl)benzaldehyde (1) [8], 2-(2 ,3-epoxy-1 ,3 ,5 -heptatrienyl)-6-hydroxy-5-(3-methyl-2-butenyl)benzaldehyde (2) [9], flavoglaucin (3) [9], dihydroauroglaucin (4) [10], ergosterol (5) [11], and tardioxopiperazine A (6) [12], were isolated from the fermented rice of E. chevalieri. All of these compounds were analyzed using 1D and 2D NMR spectroscopy and mass spectrometry. We have surveyed past studies on the secondary metabolites of rice, and all the structures found in this study were not found in our previous reports. Compounds 3 and 4 were evaluated for their antiinflammatory activity using RAW264.7 cells supplemented with LPS, which induce cell inflammation and cause nitrite accumulation in the medium. Among the tested compounds, compounds 3 and 4 showed strong inhibition of NO production of macrophages consistent with antiinflammatory activity (Table 1). The high cell viability (> 90% Table 1) showed that the inhibitory activity of LPS-induced nitrite production by compounds 3 and 4 did not resulted from its cytotoxicity. The fungus of E. chevalieri was collected from mangrove soil in Tainan City, Taiwan in March 2005. The fungus was identified by Dr. Sung-Yuan Hsieh and deposited (BCRC 07F0022) at the Bioresource Collection and Research Center (BCRC) of the Food Industry Research and Development Institute (FIRDI). The E. chevalieri fermented rice (1.0 kg) was extracted with 95% EtOH at room temperature. The crude ethanol syrup was evaporated under reduced pressure and partitioned between EtOAc–H2O (1:1) to give EtOAcand H2O-soluble fractions.

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.

Secondary metabolites from the endophytic fungus of Annulohypoxylon ilanense

In the course of screening for biologically and chemically novel agents from Formosan fungal materials, A. ilanense was chosen for further phytochemical investigation. This is also being studied and published for the first time. The n-BuOH-soluble fraction of the fungus was subjected to solvent partitioning and chromatographic separation to afford 25 pure substances. The compounds, including eight coumarins, angelicin (1) [1], jatamansinol (2) [2], columbianetin (3) [3], umbelliferone (4) [4], seselin (5) [5], osthenol (6) [5], 7-O-prenylumbelliferone (7) [5], and brosiparin (8) [6], nine flavonoids, kaempferol (9) [7], quercetin (10) [8], genkwanin (11) [9], (+)-catechin (12) [10], (–)-catechin (13) [11], (–)-4 -hydroxy5,7,3 -trimethoxyflavan-3-ol (14) [12], 5-hydroxy-7-methoxyflavone (15) [13], 3,7-dimethoxy-5-hydroxyflavone (16) [14], and 5-hydroxy-3,6,7-trimethoxyflavone (17) [15], three lignans, syringaresinol (18) [16], (–)-sesamin (19) [17], and (+)-4-ketopinoresinol (20) [18], three benzenoids, 4-hydroxybenzaldehyde (21) [19], protocatechuic acid (22) [20], trans-ferulic acid (23) [21], and two others, 1-O-hexadecanoyl glycerol (24) [22] and balansenate I (25) [23], were isolated from the endophytic fungus A. ilanense. All of these compounds were found for the first time in this fungus. The structures of the isolated compounds were determined by extensive analysis of their spectroscopic data as well as by comparison with literature reports. In summary, the chemical characteristics, as well as the biological activities, of many Annulohypoxylon metabolites remain unclear. Thus, the different biological activities of Annulohypoxylon spp. and related diverse secondary metabolites seem worthy of further exploration. General Experimental Procedures. Optical rotations were measured on a Jasco P-1020 digital polarimeter, UV spectra were obtained on a Jasco UV-240 spectrophotometer in MeOH, and IR spectra (KBr or neat) were taken on a Perkin–Elmer System 2000 FT-IR spectrometer. 1D (1H, 13C, DEPT) and 2D (COSY, NOESY, HSQC, HMBC) NMR spectra using CDCl3 as solvent were recorded on a Varian Mercury-400 (400 MHz for 1H NMR, 100 MHz for 13C NMR) spectrometer. Chemical shifts were internally referenced to the solvent signals in CDCl3 ( H 7.26; C 77.0) with TMS as the internal standard. Low-resolution ESI-MS spectra were obtained on an API 3000 (Applied Biosystems) spectrometer, and highresolution ESI-MS spectra on a Bruker Daltonics APEX II 30e spectrometer. Low-resolution EI-MS spectra were recorded on a Quattro GC/MS spectrometer having a direct inlet system. Silica gel (70–230, 230–400 mesh) (Merck) was used for column chromatography, and silica gel 60 F-254 (Merck) was used for TLC and preparative TLC. Fungus Material. Annulohypoxylon ilanense was used throughout this study, and specimens were deposited at the Bioresource Collection and Research Center (BCRC) of the Food Industry Research and Development Institute (FIRDI). A. ilanense was maintained on potato dextrose agar (PDA), and the strain was cultured on potato dextrose agar slants at 25 C for 7 days and the spores harvested by sterile water. The spores (5 105) were seeded into 300 mL shake flasks containing 50 mL RGY medium (3% rice starch, 7% glycerol, 1.1% polypeptone, 3% soybean powder, 0.1 % MgSO4, 0.2 % NaNO3) and cultivated with shaking (150 rpm) at 25 C for 3 days. After the mycelium enrichment step, an inoculum mixing 100 mL mycelium broth and 100 mL RGY medium was inoculated into plastic boxes (25 cm 30 cm) containing 1 kg sterile rice and cultivated at 25 C to produce the rice, and the above-mentioned RGY medium was added for maintaining growth. After 21 days of cultivation, the rice was harvested and used as a sample for further extraction.

A new furan-3-one derivative from the endophytic fungus Annulohypoxylon sp.

Investigation of the EtOAc-soluble fraction of the 95% EtOH extract of rice fermented with the endophytic fungus Annulohypoxylon sp. led to the isolation of one new furan-3-one derivative, designated as annulofuranone (1), together with five known compounds, 2-methylcardol (2), 2-hydroxy-5-methoxy-3-tridecyl-1,4-benzoquinone (3), 3-methoxy-5-pentylphenol (4), 3-methoxy-5-hexylphenol (5), and ergosta-4,6,8(14),22-tetraen-3-one (6). Their structures were elucidated by 1D and 2D NMR spectroscopy together with MS analysis, and comparison of the spectroscopic data with those reported for structurally related compounds. All compounds were found in this fungus for the first time.