A new 5,6-dihydro-2-pyrone derivative from Phomopsis amygdali,an endophytic fungus isolated from hazelnut (Corylus avellana)

Aims of this study were to isolate endophytes from different parts of hazelnut – Corylus avellana L. to obtain bioactive secondary metabolites and search for the presence of gene region of taxadiene synthase (Ts), a key enzyme in taxol biosynthesis, on selected fungi. Fourteen fungal species were isolated and cultured for the screening studies. The cell-free fermentation broths were extracted with chloroform. The chloroform extracts were tested for cytotoxic activity by MTT method. Based on the activity results and chemical profiles, the isolate identified as Phomopsis amygdali by internal transcribed spaces (ITS) sequence analysis using ITS1 primer was selected for further studies. After large-scale fermentation and purification studies, two major compounds, one of which turned out to be a new secondary metabolite, were isolated and characterized. Structure of the new metabolite was elucidated as (S)-4-butoxy-6-((S)-1-hydroxypentyl)-5,6-dihydro-2H-pyran-2-one by the extensive use of 1D and 2D NMR, and HR-MS, whereas the known compound was identified as (−)pestalotin. Additionally, to evaluate taxol-producing potential of the selected isolate, a PCR amplification study followed by gel electrophoresis analysis was carried out revealing no Ts gene region.     

Bioactive metabolites produced by Chaetomium globosum,an endophytic fungus isolated from Ginkgo biloba

A novel cytotoxic chlorinated azaphilone derivative named chaetomugilin D (1), together with three known metabolites, chaetomugilin A (2), chaetoglobosins A (3) and C (4), has been isolated by a bioassay-guided fractionation from the EtOAc extract of the cultures of Chaetomium globosum, an endophytic fungus found in the leaves of Ginkgo biloba. Structure of 1 was established by analyses of spectroscopic methods, including 2D-NMR experiments (COSY, NOESY, HMQC, and HMBC). Compounds 1–4 displayed significant growth inhibitory activity against the brine shrimp (Artemia salina) and Mucor miehei.     

Cytotoxic metabolites from Botryotinia fuckeliana A-S-3: An endophytic fungus from Ajuga decumbens

Available online from an cytotoxic endophytic fungus Botryotinia fuckeliana A-S-3, three cytochalasans phenochalasin B (4), [12]-cytochalasin (5) and one [1,3] dioxacyclotridecino (6), along with two new ent-eudesmane sesquiterpenes, 1-keto-4α,15-epoxyeudesm-11-ol (1), and ent-4(15)-eudesmen-5,6-ol-1-one (2), and one known ent-eudesmane sesquiterpene ent-4(15)-eudesmen-11-ol-1-one (3) were isolated. The structures of these compounds were elucidated by interpretation of spectroscopic data. Among these compounds, two cytotoxic constituents were identified.     

Bioactive metabolites from Alternaria brassicicola ML-P08, an endophytic fungus residing in Malus halliana

A total of 48 strains were isolated from the normal tissues of Malus halliana and the EtOAc extracts of their cultures were subjected to primary antimicrobial screening against four test bacteria and three fungi. As a result, 22 strains exhibited antimicrobial activity against at least one test microbe. Among them, Alternaria brassicicola ML-P08 showing strong activity (MICs: 0.31–2.50 mg/ml) was selected for further investigation on its secondary metabolites. Bioassay-guided fractionation of the EtOAc extract of its liquid culture afforded seven compounds, which were identified as alternariol (1), alternariol 9-methyl ether (2), altechromone A (3), herbarin A (4), cerevisterol (5), 3β,5α-dihydroxy-(22E,24R)-ergosta-7,22-dien-6-one (6) and 3β-hydroxy-(22E,24R)-ergosta-5,8,22-trien-7-one (7), respectively, by spectral means (MS, IR, ¹H- and ¹³C-NMR). In vitro antimicrobial assay showed that compound 3 was substantially active against Bacillus subtilis, Escherichia coli, Pseudomonas fluorescens and Candida albicans with the MICs of 3.9, 3.9, 1.8, and 3.9 μg/ml, respectively. Compound 4 also showed pronounced antifungal activity against Trichophyton rubrum and C. albicans with MICs of both 15.6 μg/ml. In addition, compound 1 exhibited strong xanthine oxidase inhibitory activity with the IC₅₀ of 15.5 μM, comparable to that of positive control, allopurinol (IC₅₀: 10.7 μM).     

Two new alkaloid metabolites produced by endophytic fungus Stagonosporopsis oculihominis isolated from Dendrobium huoshanense

Two new alkaloids (1–2), together with six known compounds (3–8), were isolated from cultures of the endophytic fungus Stagonosporopsis oculihominis. Their structures were elucidated through extensive spectroscopic methods including 2D NMR and HRMS analyses. The absolute configurations of 1 was determined by the comparison of experimental and theoretical electronic circular dichroism spectra. The new compounds were investigated for their cytotoxicity against five human cancer cell lines.     

内生真菌X1-2次级代谢产物研究

在从神农架地区特有植物内生真菌中寻找各种活性次级代谢产物的过程中,从濒危植物珙桐叶片中分离得到一株内生真菌X1-2。经过高效液相色谱(HPLC)和薄板色谱(TLC)分析其固体发酵产物多样性,通过正相硅胶柱层析、重结晶、高效液相色谱制备等手段纯化次级代谢产物,从该真菌中分离获得四个次级代谢产物。经核磁共振波谱(NMR),质谱(MS)等波谱学方法鉴定其结构,四个化合物分别为icosalide A1(1),militarinone A(2),(+)-N-deoxymilitarinone A(3),β-hydroxytetradecanoyl-β-hydroxyl tetraecanoyl-Rha-Rha-C_(14)-C_(14)(4),其中icosalide A1(1),militarinone A(2),(+)-N-deoxymilitarinone A(3)为三个复杂的生物碱类化合物,而化合物4是首次从真菌中获得。     

Bioactive metabolites from Chaetomium globosum L18, an endophytic fungus in the medicinal plant Curcuma wenyujin

An endophytic fungus, strain L18, isolated from the medicinal plant Curcuma wenyujin Y.H. Chen et C. Ling was identified as Chaetomium globosum Kunze based on morphological characteristics and sequence data for the internal transcribed spacer (ITS-5.8S-ITS2) of the nuclear ribosomal DNA. A new metabolite named chaetoglobosin X (1), together with three known compounds erogosterol (2), ergosterol 5α,8-peroside (3) and 2-methyl-3-hydroxy indole (4), were isolated from C. globosum L18. Their structures were elucidated by spectroscopic methods including NMR, UV, IR and MS data and comparison with published data. Chaetoglobosin X (1) is hitherto unknown, whereas 2-methyl-3-hydroxy indole (4) is reported for the first time as a fungal metabolite, and erogosterol (2) and ergosterol 5α,8-peroside (3) are known fungal metabolites previously identified in other genera. Chaetoglobosin X (1) exhibited a broader antifungal spectrum and showed the strongest cytotoxic activity against H22 and MFC cancer cell lines.     

Bioactive metabolites from the endophytic fungus Ampelomyces sp. isolated from the medicinal plant Urospermum picroides

Extracts of cultures grown in liquid or on solid rice media of the fungal endophyte Ampelomyces sp. isolated from the medicinal plant Urospermum picroides exhibited considerable cytotoxic activity when tested in vitro against L5178Y cells. Chromatographic separation yielded 14 natural products that were unequivocally identified based on their ¹H and ¹³C NMR as well as mass spectra and comparison with previously published data. Six compounds (2, 4, 5, 7, 9 and 11) were natural products. Both fungal extracts differed considerably in their secondary metabolites. The extract obtained from liquid cultures afforded a pyrone (2) and sulfated anthraquinones (7 and 9) along with the known compounds 1, 3, 6 and 8. When grown on solid rice medium the fungus yielded three compounds 4, 5 and 11 in addition to several known metabolites including 6, 8, 10, 12, 13 and 14. Compounds 4, 8 and 10 showed the strongest cytotoxic activity against L5178Y cells with EC₅₀ values ranging from 0.2–7.3 μg/ml. Furthermore, 8 and 10 displayed antimicrobial activity against the Gram-positive pathogens, Staphylococcus aureus, S. epidermidis and Enterococcus faecalis at minimal inhibitory concentrations (MIC) of 12.5 μg/ml and 12.5–25 μg/ml, respectively. Interestingly, 6 and 8 were also identified as constituents of an extract derived from a healthy plant sample of the host plant U. picroides thereby indicating that the production of bioactive natural products by the endophyte proceeds also under in situ conditions within the host plant.     

Myrotheciumones: Bicyclic cytotoxic lactones isolated from an endophytic fungus of Ajuga decumbens

Two new bicyclic lactones, myrotheciumones A (1) and B (2) which possessed a rare ring-fusion system were isolated from Myrothecium roridum (M. roridum), an endophytic fungus of the medicinal herb plant Ajuga decumbens (A. decumbens) via an in vitro cytotoxicity assay. Structures were deduced from 1D and 2D NMR (Nuclear magnetic resonance) data. Myrotheciumone A’s in vitro cytotoxicity and apoptotic activity were evaluated and myrotheciumone A was shown to exert cytotoxicity via inducing apoptosis in cancer cell line.     

Anti-Candida metabolites from endophytic fungi

Submerged cultures of some 1500 Ascomycota and Basidiomycota isolated from their fruit-bodies or as soil-borne, coprophilous or endophytic fungi were screened for activity against Candida albicans and a range of other pathogenic and saprotrophic fungi. Considerably more Ascomycota (11-16%) than Basidiomycota (3.5%) produced metabolites with activity against C. albicans. From five species of endophytes, six bioactive compounds were isolated and identified, viz. cerulenin (1), arundifungin (2), sphaeropsidin A (3), 5-(1,3-butadiene-1-yl)-3-(propene-1-yl)-2-(5H)-furanone (4), ascosteroside A (formerly called ascosteroside; 5) and a derivative of 5, ascosteroside B (6). 1, 3 and 5 were isolated from fungi belonging to different orders than previously described producers. Antifungal activities of 2 and 4-6 in the agar diffusion test were comparable with those of amphotericin B. Compound 6 exhibited a similar antifungal activity as 5 but its cytotoxicity towards Hep G2 cells was considerably lower. This study points to endophytic fungi related to hemibiotrophic or latent plant pathogens as an important source of bio- and chemodiversity.     

Isolation, structure elucidation and biological activity of metabolites from Sch-642305-producing endophytic fungus Phomopsis sp. CMU-LMA

Eight polyketide compounds were isolated from the cultivation broth of Phomopsis sp. CMU-LMA. We have recently described LMA-P1, a bicyclic 10-membered macrolide, obtained as a bioconversion derivative of Sch-642305, the major compound isolated in this study. Benquinol is the ethyl ester derivative of the 13-dihydroxytetradeca-2,4,8-trienoic acid produced by Valsa ambiens. This compound is concomitantly produced with the 6,13-dihydroxytetradeca-2,4,8-trienoic acid (DHTTA) previously isolated from Mycosphaerellarubella. The absolute configuration of the new compound, (2R,3R,4S,5R)-3-hydroxy-2,4-dimethyl-5-[(S,Z)-3-methylpentenyl]-tetrahydro-pyranone LMA-P2 was confirmed by X-ray crystallography. The δ-lactone 2,3-dihydroxytetradecan-5-olide (DHTO) was previously isolated from Seiridium unicorne. This compound may form through the cyclization of the methyl-2,3,5-trihydroxytridecanoate LMA-P3, a new linear polyketide isolated in this study. Benquoine, a new 14-membered lactone generated from the cyclization of benquinol, is proposed as the key precursor for the biosynthesis of Sch-642305. Antimicrobial activity and cancer cell viability inhibition by the new compounds were investigated. Benquoine exhibits antimicrobial activity against Gram positive bacteria, and cytotoxicity against HCT-116 cancer cell line.     

Bioactive endophytic streptomycetes from the Malay Peninsula

Three novel endophytic streptomycetes have been isolated and characterized from plants with ethnobotanical uses on the Malay Peninsula including: Thottea grandiflora (family -Aristolochiaceae), Polyalthia spp. (family -Annonaceae), and Mapania sp. (family -Cyperaceae). Each isolate, as studied by scanning electron microscopy, has small hyphae, and produces typical barrel-shaped spores arising by hyphal fragmentation. Interestingly, although none has any detectable antibacterial killing properties, each has demonstrable killing activity against one or more pathogenic fungi including organisms such as Phytophthora erythroseptica, Pythium ultimum, Sclerotinia sclerotiorum, Mycosphaerella fijiensis and Rhizoctonia solani. Molecular biological studies on the rRNA gene sequence of each isolate revealed that it is distinct from all other genetic accessions of streptomyectes in GenBank, and each bears some genetic similarity to other streptomycetes. The bioactivity of each microbe was extractable in various organic solvents.     

Penicidones A-C, three cytotoxic alkaloidal metabolites of an endophytic Penicillium sp

Along with the known secondary metabolites lumichrome, physcion, and emodin-1,6-dimethyl ether, three alkaloids named penicidones A-C (1-3) were isolated from the culture of Penicillium sp. IFB-E022, an endophytic fungal strain residing in the stem of Quercus variabilis (Fagaceae). The structures of penicidones A-C were established by a correlative interpretation of spectroscopic data including IR, UV and HR-ESI-MS, as well as by analysis of a set of 1D and 2D NMR experiments. The stereochemistry of compounds 1 and 2 was obtained by comparison of the optical rotation with those of vermistatin and its analogues. Penicidones A-C were the first group of natural products possessing a penicidone framework. Compounds 1-3 exhibited moderate cytotoxicity against four cancer cell lines.     

Metabolites from the endophytic fungus Phomopsis sp. PSU-D15

From the endophytic fungus Phomopsis sp. PSU-D15, three metabolites named as phomoenamide (1), phomonitroester (2) and deacetylphomoxanthone B (3), were isolated together with three known compounds, dicerandrol A (4), (1S,2S,4S)-p-menthane-1,2,4-triol (5) and uridine. Their structures were elucidated by spectroscopic methods. Phomoenamide (1) exhibited moderate in vitro antimycobacterial activity against Mycobacterium tuberculosis H37Ra.     

Three new napthalene derivatives from the endophytic fungus Phomopsis fukushii

An endophytic Phomopsis fukushii was isolated from the rhizome of Paris polyphylla var. yunnanensis, Diaporthaceae family, and three new (1–3) and four known napthalene derivatives (4–7) were isolated from the fermentation products of this fungus. Their structures were elucidated by spectroscopic methods including extensive 1D- and 2D-NMR techniques. Compounds 1–3 were evaluated for their anti-methicillin-resistant Staphylococcus aureus (anti-MRSA) activity with vancomycin as positive control. The results showed that compounds 1–3 showed good inhibitions with MCI valves of 4, 4 and 6 mg/mL, respectively.     

Two new cytochalasins from an endophytic fungus,KL-1.1 isolated from Psidium guajava leaves

Chemical investigation of the endophytic fungus, KL-1.1, isolated from the leaves of Psidium guajava (Linn) led to the isolation of two new cytochalasin derivatives, 18-desoxy-19,20-epoxycytochalasin C and 18-desoxycytochalasin C, together with five other known derivatives. The structures of the isolated compounds were elucidated by one and two dimensional nuclear magnetic resonance spectroscopy as well as by mass spectrometry. These compounds represent novel chemical scaffold with potential for development into anticancer agents.     

Bioactive benzylisoquinoline alkaloids from Artabotrys hexapetalus

Two new benzylisoquinoline alkaloids, namely, hexapetalines A (1) and B (2), along with 11 known alkaloids were isolated from the stems of Artabotrys hexapetalus. The structures of 1 and 2 with their absolute configurations were elucidated by extensive spectroscopic methods and the known compounds were identified by comparisons with data in the literature. All new compounds were evaluated for their cytotoxicities against five human cancer cell lines: HL-60, SMMC-7721, A-549, MCF-7 and SW480 in vitro. Alkaloids 1 and 2 exhibited inhibitory effects with IC₅₀ values comparable to those of cisplatin.     

Cytosporone derivatives from the endophytic fungus Phomopsis sp. PSU-H188

Two new cytosporone derivatives (1 and 2) were isolated from the endophytic fungus Phomopsis sp. PSU-H188 together with 19 known compounds. Their structures were elucidated by analysis of spectroscopic data. Known mycoepoxydiene showed potent cytotoxic activity towards both MCF-7 and noncancerous Vero cell lines with the respective IC₅₀ values of 9.27 and 4.06 μM. It exhibited inhibition on glucose output in mouse primary hepatocytes with the IC₅₀ value of 16.06 μM, but did not show cytotoxicity on primary mouse hepatocytes. Additionally, known cytosporone B displayed protective activity against INS-1 832/13 pancreatic β-cells by an EC₅₀ value of 11.08 μM whereas known diaporthalasin displayed antibacterial activity against methicillin-resistant Staphylococcus aureus with an MIC value of 4 μg/mL. Both of them were noncytotoxic to Vero cells.