剑叶龙血树内生真菌的分离及体外抑菌活性筛选

为研究剑叶龙血树内生真菌资源多样性,初步探讨和筛选具有抑菌活性的特异性菌株以及进一步开发剑叶龙血树内生真菌的抗菌活性化合物。该文采用植物组织分离法从剑叶龙血树茎和叶中分离内生真菌,对内生真菌进行液体发酵7 d,经乙酸乙酯萃取后制得粗提物,并采用牛津杯扩散法,以10种常见病原菌和5种临床耐药菌为靶标检测其发酵粗提物的抑菌活性,对有较好抑菌活性的内生真菌进行分子鉴定。结果表明:(1)从剑叶龙血树茎、叶中共分离得到345株内生真菌,294株对一种以上指示菌有抑制活性;(2)其中84株内生真菌对5株临床耐药菌均有不同程度的抑制活性,占所分离菌株总数的24.35%,75%的内生真菌对金黄色葡萄球菌有抑制活性。这说明剑叶龙血树中存在多种有抑菌活性的内生真菌,为剑叶龙血树内生菌抗菌活性成分挖掘及新型抗菌药物筛选奠定了基础。     

蚁巢膝束霉次级代谢产物及生物活性

菌株L342分离自中国四川省的废弃白蚁巢,rDNA序列分析表明L342为蚁巢膝束霉Geniculisynnema termiticola。抗氧化活性评价结果显示其PDB发酵产物的乙酸乙酯提取物具有很强的还原能力和中等强度的DPPH清除能力。采用硅胶柱色谱、ODS柱色谱、Sephadex LH-20柱色谱、HPLC等分离方法,从G. termiticola的PDB发酵粗提物中分离鉴定了6个化合物：3,4-Dihydro-6,8-dihydroxy-3-methylisocoumarin (1), regiolone (2), (3S, 4S)-3,4,6,8-tetradydroxy-3,4-Dihydronaphthalen-1(2H)-one (3), ergosta-4,6,8(14),22-tetraen-3-one (4), cerebroside A (5), and cerebroside C (6)。其中化合物4-6具有中等强度的铁离子还原能力,化合物4还具有一定的HeLa细胞毒活性（IC50=98.3μmol/L）。G. termiticola发酵产物具有产生有生物活性次级代谢产物的潜力,有望开发成为一种新型的功能性食品。     

Metabolites from Two Dominant Thermophilic Fungal Species Thermomyces lanuginosus and Scytalidium thermophilum

Thermomyces lanuginosus and Scytalidium thermophilum are among the most ubiquitous thermophilic fungi in compost and soil. Chemical study on these two prevalent strains collected from Yunnan led to isolation of 23 metabolites, including one new metabolite, therlanubutanolide, and 15 known compounds, isolated from the YGP culture broth of Thermomyces lanuginosus and 7 known compounds isolated from Scytalidium thermophilum, respectively. Therlanubutanolide shared the quite similar features of the same carbon skeleton and saturation as natural hexadecanoic acids. This was the first reported discovery of such a lactone as natural occurring metabolite. All the compounds were reported for the first time from thermophilic fungi. Among them, N‐[(2S,3R,4E,8E)‐1,3‐dihydroxy‐9‐methyloctadeca‐4,8‐dien‐2‐yl]acetamide was for the first time reported to be a naturally occurring metabolite and its NMR data was first provided in this study. A type of PKS‐derived metabolites, three 3,4‐dihydronaphthalen‐1(2H)‐ones, which were widely found in plant pathogenic fungi as phytotoxins and reported to have antimicrobial activity, were obtained from both dominant thermophilic fungi. The frequent occurrence of such PKS phytotoxins in these two thermophilic fungi might suggest particular ecological interest.     

Bioactive stilbenes from a Bacillus sp. N strain associated with a novel rhabditid entomopathogenic nematode

Aims: The aim of the present study was to purify and characterize a natural antimicrobial compound from Bacillus sp. strain N associated with a novel rhabditid entomopathogenic nematode. Methods and Results: The cell‐free culture filtrate of a bacterium associated with a novel entomopathogenic nematode (EPN), Rhabditis (Oscheius) sp. exhibited strong antimicrobial activity. The ethyl acetate extract of the bacterial culture filtrate was purified by column chromatography, and two bioactive compounds were isolated and their chemical structures were established based on spectral analysis. The compounds were identified as 3,4′,5‐trihydroxystilbene (1) and 3,5‐dihydroxy‐4‐isopropylstilbene (2). The presence of 3,4′,5‐trihydroxystilbene (resveratrol) is reported for the first time in bacteria. Compound 1 showed antibacterial activity against all the four test bacteria, whereas compound 2 was effective against the Gram‐positive bacteria only. Compounds 1 and 2 were active against all the five fungi tested and are more effective than bavistin, the standard fungicide. The antifungal activity of the compounds against the plant pathogenic fungi, Rhizoctonia solani is reported for the first time. Conclusions: Cell‐free extract of the bacterium and isolated stilbenes demonstrated high antibacterial activity against bacteria and fungi especially against plant pathogenic fungi. We conclude that the bacterium‐associated EPN are promising sources of natural bioactive secondary metabolites. Significance and Impact of the Study: Stilbene compounds can be used for the control of fungi and bacteria.     

Terpenes with antimicrobial activity from Cretan propolis

Five terpenes, the diterpenes: 14,15-dinor-13-oxo-8(17)-labden-19-oic acid and a mixture of labda-8(17),13E-dien-19-carboxy-15-yl oleate and palmitate as well as the triterpenes, 3,4-seco-cycloart-12-hydroxy-4(28),24-dien-3-oic acid and cycloart-3,7-dihydroxy-24-en-28-oic acid were isolated from Cretan propolis. Moreover, 18 known compounds were also isolated, seven of them for the first time as propolis components. All structures were established on the basis of spectroscopic analysis and chemical evidence. All isolated compounds were tested for antimicrobial activity against some Gram-positive and Gram-negative bacteria as well as against some human pathogenic fungi showing a broad spectrum of antimicrobial activity.     

Studies on the Compounds Produced by Molds

Three isocoumarin compounds (BV 1, 2 and 3) were isolated from the cultural broth of Aspergillus oniki 1784. BV 1 was mellein (3-methyl-8-hydroxy-3,4-dihydroisocoumarin). BV 2 and 3 were assigned to be 3-methyl-4,8-dihydroxy-3,4-dihydroisocoumarin, 3-methyl-3,8-dihydroxy-3,4-dihydroisocoumarin, respectively. These two compounds (BV 2, 3) were newly isolated. Also another component named BV 4 was proved to be 6-methylsalicylic acid.     

Biologically active secondary metabolites from Actinomycetes

Secondary metabolites obtained from Actinomycetales provide a potential source of many novel compounds with antibacterial, antitumour, antifungal, antiviral, antiparasitic and other properties. The majority of these compounds are widely used as medicines for combating multidrug-resistant Gram-positive and Gram-negative bacterial strains. Members of the genus Streptomyces are profile producers of previously-known secondary metabolites. Actinomycetes have been isolated from terrestrial soils, from the rhizospheres of plant roots, and recently from marine sediments. This review demonstrates the diversity of secondary metabolites produced by actinomycete strains with respect to their chemical structure, biological activity and origin. On the basis of this diversity, this review concludes that the discovery of new bioactive compounds will continue to pose a great challenge for scientists.     

Chemical analyses of wasp-associated streptomyces bacteria reveal a prolific potential for natural products discovery

Identifying new sources for small molecule discovery is necessary to help mitigate the continuous emergence of antibiotic-resistance in pathogenic microbes. Recent studies indicate that one potentially rich source of novel natural products is Actinobacterial symbionts associated with social and solitary Hymenoptera. Here we test this possibility by examining two species of solitary mud dauber wasps, Sceliphron caementarium and Chalybion californicum. We performed enrichment isolations from 33 wasps and obtained more than 200 isolates of Streptomyces Actinobacteria. Chemical analyses of 15 of these isolates identified 11 distinct and structurally diverse secondary metabolites, including a novel polyunsaturated and polyoxygenated macrocyclic lactam, which we name sceliphrolactam. By pairing the 15 Streptomyces strains against a collection of fungi and bacteria, we document their antifungal and antibacterial activity. The prevalence and anti-microbial properties of Actinobacteria associated with these two solitary wasp species suggest the potential role of these Streptomyces as antibiotic-producing symbionts, potentially helping defend their wasp hosts from pathogenic microbes. Finding phylogenetically diverse and chemically prolific Actinobacteria from solitary wasps suggests that insect-associated Actinobacteria can provide a valuable source of novel natural products of pharmaceutical interest.     

Diversity and pharmaceutical screening of fungi from benthic mats of Antarctic lakes

During the MICROMAT project, the fungal diversity of microbial mats growing in the benthic environment of Antarctic lakes was accessed for the discovery of novel antibiotics and anticancers. In all, 160 filamentous fungi belonging to fifteen different genera and 171 yeasts were isolated from 11 lakes, classified and cultivated in different media and at different temperatures. Filamentous fungi were then screened to discover novel antimicrobial and cytotoxic compounds. A total of 1422 extracts were prepared by solid phase extraction of the culture broths or by biomass solvent extraction. 47 (29%) filamentous fungi showed antimicrobial activity; most of them inhibited the growth of gram-positive Staphyloccus aureus (14%), gram-negative E. coli (10%), and of yeasts Candida albicans (11%) and Cryptococcus neoformans (8%). Less activity was detected against representatives of enterobacteria and filamentous fungi. The most productive in terms of bioactivities were cold-tolerant cosmopolitan hyphomycetes such as Penicillium, Aspergillus, Beauveria and Cladosporium. Two bioactive bis-anthraquinones (rugulosin and skyrin) were identified by LC–MS as the main products in a strain of Penicillium chrysogenum isolated from a saline lake in the Vestfold Hills. LC–MS fractionation of extracts from two diverse species of Aspergillus, that exhibited relatively potent antimicrobial activities, evidenced a chemical novelty that was further investigated. To our knowledge, this is the first report of new antibiotics produced by fungi from benthic microbial mats from Antarctic lakes. It can be concluded that these microbial assemblages represent an extremely rich source for the isolation of new strains producing novel bioactive metabolites with the potential to be developed as drugs.     

低温型真菌抗肿瘤活性次级代谢产物研究进展

真菌栖息生境及物种的多样性使其次级代谢产物化学结构和生物活性呈多样性特点,结构与功能多样的真菌次级代谢产物已成为新药先导化合物发现的重要资源。低温型真菌因其独特而恶劣的生存环境进化出特有的适冷特性及代谢途径,使其产生结构新颖、活性显著的次级代谢产物的几率大大提升,已成为药物研发的热点领域。近年来,研究者陆续从高海拔地区、南北极地和深海地带等低温生境分离鉴定出上百种结构新颖的次级代谢产物,并显示出抗肿瘤、抗菌、抗病毒和免疫调节等多种活性。本文对近10年来源于上述生境的低温型真菌抗肿瘤活性次级代谢产物的研究进展进行综述,为其用于创制抗肿瘤新药提供新思路。     

Insecticidal and insect growth regulatory activities of secondary metabolites from entomopathogenic fungi,Lecanicillium attenuatum

Insect growth regulators (IGRs) are effective alternatives to chemical insecticides because of their specificity and low environmental toxicity. Entomopathogenic fungi are an important natural pathogen of insects and have been developed as biological control agents. They produce a wide range of secondary metabolites such as antibiotics, pesticides, growth-promoting or inhibiting compounds and insect attracting agents. In this study, to explore novel IGR substances from entomopathogenic fungi, culture extracts of 189 entomopathogenic fungi isolated from Korean soil samples were investigated for their juvenile hormone (JH)-based IGR activities. Whereas none of the culture extracts exhibited JH agonist (JHA) activity, 14 extracts showed high levels of JH antagonist (JHAN) activity. Among them, culture extract of JEF-145 strain, which was identified as Lecanicillium attenuatum, showed the highest insecticidal against Aedes albopictus and Plutella xylostella. At liquid culture condition, JHAN activity was observed in culture soup rather than mycelial cake, indicating that substances with JHAN activity are released from the JEF-145 strain during culture. Furthermore, while extract from solid cultured JEF-145 strain showed insecticidal activities against both A. albopictus and P. xylostella, that from liquid cultured fungi showed insecticidal activity only against A. albopictus, indicating that L. attenuatum JEF-145 strain produces different kinds of secondary metabolites with JHAN activity depending on culture conditions. These results suggested that JHAN substances derived from entomopathogenic fungi could be usefully exploited to develop novel eco-friendly IGR insecticides.     

地衣型真菌次级代谢产物抗菌活性初步研究

地衣是一种传统的民族药物,能产生多种具有活性的物质。该研究对地衣型真菌(Xanthoria elegans,Myelochroa indica,Ramalina peruviana,Cladonia macilenta,Nephromopsis pallescens,Cladonia coccifera)进行液体培养,2个月后,培养液用乙酸乙酯萃取后获得初提物。该研究采用抑菌圈法评价地衣型真菌初提物对7种致病细菌(Bacillus subtilis,Bacillus cereus,Vibrio parahaemolyticus,Straphylococcus haemolyticus,Pseudomonas aeruginosa,Staphylococcus aureus,Micrococcus luteus)的抗菌活性,并测定最低抑菌浓度(MIC)。结果表明:6种地衣型真菌的初提物均具有一定的抗菌活性,且不同培养基对地衣型真菌产生抗菌物质有显著影响。其中,R.peruviana在MY液体培养基中所产生的次级代谢产物对金黄色葡萄球菌、藤黄微球菌、溶血性葡萄球菌、铜尿假单胞菌具有抑制效果,但在YMG培养基中所得初提物对供试7种致病细菌不具有抑菌效果。X.elegans在YMG培养基中所得初提物对枯草芽孢杆菌具有明显抗菌活性,其抑菌圈直径可达17.77 mm。该研究证实不同地衣型真菌液体培养初提物具有抗菌活性,不同的培养基也直接影响地衣型真菌抗菌效果。该研究结果为地衣型真菌的进一步研究及民族药的开发利用奠定了基础。     

Isolation and identification of antimicrobial secondary metabolites from Bacillus cereus associated with a rhabditid entomopathogenic nematode

The cell-free culture filtrate of Bacillus cereus associated with an entomopathogenic nematode, Rhabditis (Oscheius) sp., exhibited strong antimicrobial activity. The ethyl acetate extract of the bacterial culture filtrate was purified by silica gel column chromatography to obtain six bioactive compounds. The structure and absolute stereochemistry of these compounds were determined based on extensive spectroscopic analyses (LCMS, FABMS, ¹H NMR, ¹³C NMR, ¹H ?¹H COSY, ¹H ?¹³C HMBC) and Marfey’s method. The compounds were identified as cyclo(D-Pro-D-Leu), cyclo(L-Pro-D-Met), cyclo (L-Pro-D-Phe), cyclo (L-Pro-L-Val), 3,5-dihydroxy-4-ethyl-trans-stilbene, and 3,5-dihydroxy-4-isopropylstilbene, respectively. Compounds recorded antibacterial activity against all four tested bacteria strains of Bacillus subtilis, Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. 3,5-dihydroxy-4-isopropylstilbene recorded activity only against Gram-positive bacteria while cyclo(L-Pro-L-Val) recorded no antibacterial activity. Best antibacterial activity was recorded by 3,5-dihydroxy-4-ethyl-trans-stilbene (4 μg/ml) against Escherichia coli. The six compounds recorded significant antifungal activities against five fungal strains tested (Aspergillus flavus, Candida albicans, Fusarium oxysporum, Rhizoctonia solani and Penicillium expansum) and they were more effective than bavistin, the standard fungicide. The activity of cyclo(D-Pro-D-Leu), cyclo(L-Pro-D-Met), 3,5-dihydroxy-4-ethyl-trans-stilbene, and 3,5-dihydroxy-4-isopropylstilbene against Candida albicans was better than amphotericin B. To the best of our knowledge, this is the first report of antifungal activity of the bioactive compounds against the plant pathogenic fungi Fusarium oxysporum, Rhizoctonia solani, and Penicillium expansum. We conclude that the Bacillus cereus strain associated with entomopathogenic nematode is a promising source of natural bioactive secondary metabolites which may receive great benefit as potential sources of new drugs in the agricultural and pharmacological industry.     

Isolation and characterization of a co-producer of fengycins and surfactins, endophytic Bacillus amyloliquefaciens ES-2, from Scutellaria baicalensis Georgi

An endophytic bacterium was isolated from Chinese medicinal plant Scutellaria baicalensis Georgi. The phylogenetic and physiological characzterization indicated that the isolate, strain ES-2, was Bacillus amyloliquefaciens, which produced two families of secondary metabolites with broad-spectrum antibacterial and antifungal activities. Culture filtrate of ES-2 displayed antagonism against some phytopathogenic, food-borne pathogenic and spoilage bacteria and fungi owing to the existence of antimicrobial compounds. A HPLC-MS analysis showed two series of ion peaks from the culture filtrate. A further electrospray ionization/collision-induced dissociation spectrum revealed that the two series ion peaks represented different fengycin homologues and surfactin homologues, respectively, which had a potential for food preservation and the control of several fungal plant diseases.     

Isosclerone,a New Metabolite of Sclerotinia sclerotiorum (Lib.) De Bary

Isosclerone, mp 74~76°C, , a new metabolite of Sclerotinia sclerotiorum, was isolated and its absolute structure was determined as (4S)-3,4-dihydro-4,8-dihydroxy-1(2H)-naphthalenone. It stimulated the root elongation of rice seedlings by ca. 30% at concentrations of 1~10 ppm, and inhibited the growth of shoots and roots at high concentrations above 50 ppm.     

黄河三角洲耐盐真菌Penicillium chrysogenum HK14-01的次生代谢产物

【目的】从黄河三角洲耐盐微生物的代谢产物中寻找具有抗菌和抗肿瘤活性的化合物。【方法】应用化学与生物活性相集成的筛选方法,从耐盐微生物中筛选获得代谢产物丰富并具有生物活性的目标菌株;通过高盐胁迫目标菌株,利用硅胶柱色谱、凝胶柱色谱和高效液相色谱等方法对发酵产物进行分离、纯化,运用波谱解析、钼靶X-射线单晶衍射分析、圆二色散谱(CD)和密度泛函数(DFT)-ECD的计算鉴定化合物的结构。【结果】从采自黄河三角洲的泥土样品中得到一株耐盐真菌HK14-01,鉴定为产黄青霉Penicilliumchrysogenum;从其发酵产物中分离鉴定了8个化合物:(2S,3R)-oxaline(1,主产物)、(3R,4R)-3,4,8-trihydroxy-3,4-dihydronaphthalen-1(2H)-one(2)、(Z)-N-(4-hydroxystyryl)formamide(3)、(E)-N-(4-hydroxystyryl)formamide(4)、emodin(5)、4-(2-hydroxyethyl)benzene-1,2-diol(6)、methyl 2-(4-hydroxyphenyl)acetate(7)和2-(4-hydroxy phenyl)acetonitrile(8);化合物1、3和4对大肠杆菌以及化合物1和5对金黄色葡萄球菌表现出抑菌活性,化合物5对P388细胞表现出微弱的细胞增殖抑制活性。【结论】首次确定了化合物2的绝对构型、首次报道了化合物1和2的CD数据;从黄河三角洲的耐盐微生物的次生代谢产物中可以得到活性化合物,这一区域的耐盐微生物资源值得深入研究。     

Cis-2′, 3′-Dihydrodiol production on flavone B-Ring by Biphenyl Dioxygenase from Pseudomonas pseudoalcaligenes KF707 expressed in Escherichia coli

Escherichia coli JM109 strains expressing either toluene dioxygenase from Pseudomonas putida F1 or biphenyl dioxygenase from Pseudomonas pseudoalcaligenes KF707 were examined for their ability to catalyze flavones. Biphenyl dioxygenase produced metabolites from flavone and 5,7-dihydroxyflavone which were not found in the control experiments. The absorption maxima of UV-visible spectra for the metabolites from flavone and 5,7-dihydroxyflavone were found at 337 and 348 nm respectively by using a photodiode array detector in the HPLC. Liquid chromatography/mass spectroscopy (LC/MS) showed molecular weights 256 and 288 for the metabolites, respectively. The metabolite of flavone, which was isolated and purified from the bacterial culture, was further subjected to analysis by ¹H and ¹³C nuclear magnetic resonance (NMR) spectroscopy. Based on the LC/MS and NMR results, biphenyl dioxygenase inserted oxygen at C2′ and C3′ on the B-ring of flavone, resulting in the formation of flavone cis-2′, 3′-dihydrodiol (2-[3,4-dihydroxy-1.5-cyclohexadienyl]-4H-chromen-4-one). Since this product is not found in Chemical Abstracts, this compound is considered a novel one. In addition, biotransformation of flavones by biphenyl dioxygenase suggested a potential role of bacterial dioxygenase to synthesize novel compounds from plant secondary metabolites. This revised version was published online in June 2006 with corrections to the Cover Date.     

Isolation and Analysis of Bacteria with Antimicrobial Activities from the Marine Sponge Haliclona simulans Collected from Irish Waters

Samples of the marine sponge Haliclona simulans were collected from Irish coastal waters, and bacteria were isolated from these samples. Phylogenetic analyses of the cultured isolates showed that four different bacterial phyla were represented; Bacteriodetes, Actinobacteria, Proteobacteria, and Firmicutes. The sponge bacterial isolates were assayed for the production of antimicrobial substances, and biological activities against Gram-positive and Gram-negative bacteria and fungi were demonstrated, with 50% of isolates showing antimicrobial activity against at least one of the test strains. Further testing showed that the antimicrobial activities extended to the important pathogens Pseudomonas aeruginosa, Clostridium difficile, multi-drug-resistant Staphylococcus aureus, and pathogenic yeast strains. The Actinomycetes were numerically the most abundant producers of antimicrobial activities, although activities were also noted from Bacilli and Pseudovibrio isolates. Surveys for the presence of potential antibiotic encoding polyketide synthase and nonribosomal peptide synthetase genes also revealed that genes for the biosynthesis of these secondary metabolites were present in most bacterial phyla but were particularly prevalent among the Actinobacteria and Proteobacteria. This study demonstrates that the culturable fraction of bacteria from the sponge H. simulans is diverse and appears to possess much potential as a source for the discovery of new medically relevant biological active agents.     

The chemical constituents of the fungus Stereum sp

Four new compounds, including a sesquiterpene and three aromatic compounds, and a known compound were isolated from a culture broth of the fungus Stereum sp. The novel sesquiterpene was determined to be stereumone A ((+)-2,3,4a,5,6,7,8a,9-octahydro-5-hydroxy-6,6,9-trimethyl-4,8a-epoxynaphtho[2,3-b]furan-8(8H)-one; 1), and the three new aromatic compounds were elucidated as 3,5-dihydroxy-4-(3-methylbut-2-enyl)benzene-1,2-dicarbaldehyde (2), 5,7-dihydroxy-6-(3-methylbut-2-enyl)isobenzofuran-1(3H)-one (3), butyl 2,4-dihydroxy-6-methylbenzoate (4), together with the known compound methyl 2,4-dihydroxy-6-methylbenzoate (5). The structures were established by spectroscopic methods including 2D-NMR techniques. Compounds 2 and 4 showed evident nematicidal activity against nematode Panagrellus redivivus.     

Secondary metabolites from endophytic fungi isolated from the Chilean gymnosperm Prumnopitys andina (Lleuque)

Two endophytic fungi isolated from the phloem of the Chilean gymnosperm Prumnopitys andinawere cultured in liquid potato-dextrose medium. The secondary metabolites were isolated and identified by spectroscopic methods. The fungus E-3 which could not be identified yielded 4-(2-hydroxyethyl)phenol, p-hydroxybenzaldehyde and the isochromanone mellein. The second fungus, identified as Penicillium janczewskii K.M. Zalessky yielded peniprequinolone and gliovictin, reported for the first time for this species. This is the first report on secondary metabolites produced by endophytic fungi from Chilean gymnosperms.