Simultaneous determination of thioridazine and its S-oxidized and N-demethylated metabolites using high-performance liquid chromatography on radially compressed silica

A method for simultaneously quantifying thioridazine, northioridazine, thioridazine-2-sulfoxide, thioridazine-2-sulfone and thioridazine-5-oxide in serum and plasma is described. Following solvent extraction these compounds were separated by high-performance liquid chromatography on radially compressed silica gel and detected by UV absorbance at 254 nm. Chromatography time is less than 7 min. The relative retention of these compounds as a function of the methanol and methylamine content of the mobile phase is discussed. Practical limits of detection, based upon an assayed plasma or serum volume of 1 ml, were 20 ng/ml for thioridazine-5-oxide and 10 ng/ml for the other compounds. The coefficient of variation for all compounds was less than 13%. The method is compared with more conventional high-performance liquid chromatographic and gas chromatographic methodology.     

Biotransformation of artemisinic acid by the fungus Trichothecium roseum and anti-candidal activity of its metabolites

The microbial transformation of artemisinic acid (1) using cell culture of endophytic fungus Trichothecium roseum was investigated. Previously, we have reported two major metabolites, 3β-hydroxyartemisinic acid (2) and 3β,15-dihydroxyartemisinic acid (3) from the biotransformation of artemisinic acid by the fungus T. roseum CIMAPN1. Here in the present paper, we obtained a new minor compound 4 (5.2% in yield) along with compounds 2 and 3 through scale-up of biotransformation process of artemisinic acid using the same fungus. The structure of compound 4 was established as 3-oxoartemisinic acid on the basis of its IR, ESI-MS, HRMS, 1?D (¹H and ¹³C, DEPT), and 2?D (COSY, HSQC, HMBC) NMR spectral data analysis. The possible reaction mechanism of the formation of 3-oxoartemisinic acid from artemisinic acid was proposed. Furthermore, all the three metabolites along with the artemisinic acid were evaluated for their antifungal activity against the three fungal strains Candida albicans (ATCC 14053), Candida albicans clinical isolates and Candida kefyr (ATCC 204093). 3-Oxoartemisinic acid was the most active (4 to 16 times more potent than artemisinic acid) with MIC ranges from 125 to 500?µg/mL among all tested compounds. This study suggested that the artemisinic acid molecule has a great potential to be exploited for further biotransformation by the different fungi and can produce chemically diverse molecules with better biological activity.     

Endophytic fungi from a pharmaceutical plant, Camptotheca acuminata: isolation, identification and bioactivity

About 174 endophytic fungi were isolated from the pharmaceutical plant, Camptotheca acuminata. Of the 18 taxa obtained, non-sporulating fungi (48.9%), Alternaria (12.6%), Phomopsis (6.9%), Sporidesmium (6.3%), Paecilomyces (4.6%) and Fusarium (4.6%) were dominant. ITS rDNA assay indicated that most of the non-sporulating fungi belonged to the Pyrenomycetes and Loculoascomycetes ascomycetes or their anamorph Coelomycetes. The results of the bioactivity test showed that 27.6% of the endophytic fungi displayed inhibition against more than one indicator microorganism. 4.0% and 2.3% of the endophytic fungi showed cytotoxicity and protease inhibition, respectively. The endophytic fungi with bioactivities were distributed in more than 12 taxa including non-sporulating fungi, which are reliable sources for bioactive agents.     

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.     

盐角草内生真菌分离鉴定及抑制水产腐败细菌菌株的筛选

盐角草是一种耐盐植物,有重要的食用、药用价值.为研究盐角草的内生真菌及其活性次生代谢产物的多样性,该文对采集于广西北部湾沿海盐角草的内生真菌进行分离纯化,采用RAPD对内生真菌多样性进行分析,采用ITS基因序列对内生真菌进行鉴定,并对内生真菌提取物抑制等3种水产腐败细菌的活性进行筛选.结果表明:(1)从北部湾盐角草植物...     

Study on the communities of endophytic fungi and endophytic actinomycetes from rice and their antipathogenic activities in vitro

The populations of endophytic fungi and actinomycetes from four rice cultivars in the Panyu district (Site 1) and Wushan district (Site 2) in Guangdong province, South China, were studied. The preponderant endophytic fungi and actinomycetes isolated belonged to Fusarium and Streptomyces respectively. The incidence of Streptomycetes griseofuscus ranged from 36.1 to 69% out of all the different rice cultivars from the two sites. It is the commonest population of endophytic actinomycetes, and constituted the greatest part of all the antagonistic communities. The distributions of endophytic fungi and actinomycetes in roots and leaves were different, endophytic fungi from leaves were diverse, some were organ-specific. More diverse endophytic actinomycetes were isolated from roots than from leaves. The endophytic fungi isolated from rice in Site 2 were more diverse than that in Site 1. The diversity of the endophytic actinomycetes, however, was less than that in Site 1. Acid soil in Site 2 is ideal for the growth and colonization of fungi while the alkaline soil in Site 1 is better for the growth and colonization of actinomycetes. The results suggested that differences in the chemical composition of soil could influence the endophytic microbial communities of rice plants. The endophytic fungi and actinomycetes isolated from poor-growing seedlings and susceptible rice cultivars were more abundant than that the disease-resistant counterparts. In the dual culture and activity detection of the metabolites, 41.2% of all the isolated endophytic fungi showed antagonism to rice pathogens. Fifty percent of all the isolated endophytic actinomycetes were antagonistic to those pathogens. The percentage of Streptomyces griseofuscus and hygroscopicus reached 55.4 and 21.4% of all the active actinomycetes. This revised version was published online in August 2006 with corrections to the Cover Date.     

Microbial hydroxylation of imidacloprid for the synthesis of highly insecticidal olefin imidacloprid

Microorganisms that bring about the aerobic transformation of imidacloprid (IMI) were isolated and screened, and the microbial regio- and stereoselective hydroxylation of IMI was studied. Some bacteria and fungi transformed IMI to 5-hydroxyl IMI. Bacterium Stenotrophomonas maltophilia CGMCC 1.1788 resting cells transformed IMI into R-5-hydroxyl IMI at the highest conversion rate. The enzyme catalyzed the stereoselective hydroxylation at position C12 of IMI in the imidazolidine ring. Under acidic conditions, 5-hydroxyl IMI was converted into olefin IMI in high molar conversion yield. The olefin IMI exhibited about 19 and 2.2 times more insecticidal efficacy than IMI against horsebean aphid imago and nymph, respectively, and about 1.4 times more active than IMI against brown planthopper imago. The transformation rate of IMI by resting cells of S. maltophilia CGMCC 1.1788 was promoted significantly by some carbohydrates and organic acids. The reaction medium with 5% sucrose resulted in 8.3 times greater biotransformation yield as compared with that without sucrose.     

北细辛不同部位内生真菌的多样性与抑菌活性

为探讨北细辛(Asarum heterotropoides)不同部位可培养内生真菌种群结构的差异,采用植物组织平板分离法分离北细辛根、根状茎、越冬芽三个部位内生真菌,结合形态学和分子生物学方法鉴定菌株,并系统分析和比较其种类和多样性。从北细辛三个部位共分离得到117株内生真菌,其中97株归属于6纲6目8科9属17种。相似性和多样性分析表明,北细辛不同部位内生真菌的组成结构上存在差异,根部内生真菌的多样性指数最高,越冬芽最低。采用菌饼法检测其抑菌活性,G 3-3、G 4-10-1、G 2-2-1、GZJ 2-12-2对金黄色葡萄球菌(Staphylococcus aureus)有抑制作用,G 3-3、G 2-2-1、GZJ 2-12-2、GZJ 1-6、G 3-8对枯草芽胞杆菌(Bacillus subtilis)有抑制作用。GZJ 2-12-2对金黄色葡萄球菌、大肠埃希菌(Escherichia coli)、枯草芽胞杆菌均有抑制作用。     

Lapachol biotransformation by filamentous fungi yields bioactive quinone derivatives and lapachol-stimulated secondary metabolites

Abstract

Lapachol is a natural naphthoquinone with a range of biological effects, including anticancer activity. Microbial transformations of lapachol can lead to the formation of new biologically active compounds. In addition, fungi can produce secondary metabolites that are also important for drug discovery. The goal of this study was to evaluate the ability of filamentous fungi to biotransform lapachol into biologically active compounds and identify secondary metabolites produced in the presence of lapachol. Seven out of nine strains of filamentous fungi tested exhibited the ability to biotransform or biodegrade lapachol. The bioactive derivatives norlapachol and isolapachol were identified among biotransformation products. Moreover, lapachol stimulated the production of pyrrolo-[1,2-a] pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl) and phenol-2,4-bis-(1,1-dimethylethyl), secondary metabolites already known to have antimicrobial and antioxidant activities. These results open the perspective of using these strains of filamentous fungi for lapachol biotransformation and efficient production of several biologically active compounds.     

Diversity and antimicrobial activity of endophytic fungi isolated from Nyctanthes arbor-tristis, a well-known medicinal plant of India

Endophytic fungi from Nyctanthes arbor-tristis were isolated and evaluated for their antimicrobial activity. A total of 19 endophytic fungi were isolated from 400 segments of healthy leaf and stem tissues of N. arbor-tristis. Eighteen endophytic fungi were obtained from leaf, while only ten from stem. Alternaria alternata had the highest colonization frequency (15.0%) in leaf, whereas Cladosporium cladosporioides ranked first in stem with a colonization frequency of 12%. The diversity and species richness were found higher in leaf tissues than in stem. The similarity indices between leaf and stem were 0.473 for Jaccard’s and 0.642 for the Sorenson index, respectively. Of 16, 12 (75%) endophytic fungal extracts showed antibacterial activity against either one or more pathogenic bacteria. The endophytic Nigrospora oryzae showed maximum inhibition against Shigella sp. and Pseudomonas aeruginosa. The leaf endophytes Colletotrichum dematium and Chaetomium globosum exhibited a broad range of anibacterial activity and were active against Shigella flexnii, Shigella boydii, Salmonella enteritidis, Salmonella paratyphi, and P. aeruginosa. Nine out of 16 (56.25%) endophytic fungi exhibited antifungal activity to one or more fungal pathogens. Colletotrichum dematium inhibited 55.87% of the radial growth of the phytopathogen Curvularia lunata. The antimicrobial activity of these endophytic microorganisms could be exploited in the biotechnological, medicinal, and agricultural industries.     

Metabolomic Analysis of Combretum lanceolatum Plants Interaction with Diaporthe phaseolorum and Trichoderma spirale Endophytic Fungi through 1H-NMR

Endophytic fungi are an important class of microorganisms, able to interact with a host plant via a mutualistic mechanism without visible symptoms of the fungal colonization. The synergy between endophytic fungi and their host plant can promote morphological, physiological and biochemical changes through the expression of bioactive metabolites. This work aims to correlate metabolic changes in the Combretum lanceolatum plant metabolome with its endophytic fungi Diaporthe phaseolorum (Dp) and Trichoderma spirale (Ts), and to discover corresponding metabolite-biomarkers, with the principal focus being on its primary metabolism. The ¹H-NMR metabolomic analysis of qualitative and quantitative changes was performed through multivariate statistical analysis and the identification of primary metabolites was achieved on the Madison Metabolomics Consortium Database. The presence of Dp significantly impacted the plant's metabolic pathways, improving the biosynthesis of primary metabolites such as threonine, malic acid and N-acetyl-mannosamine, which are precursors of special metabolites involved in plant self-defence. This work represents a valuable contribution to advanced studies on the metabolic profiles of the interaction of plants with endophytes.     

青檀叶片内生和附生真菌组成及生态分布

对我国古老特有植物青檀叶片进行内生和附生真菌的研究,以了解青檀叶片内生和附生真菌的组成特点和探讨内生和附生真菌菌群之间的可能联系,为研究真菌资源多样性、植物附生和内生真菌的相互演化关系及真菌与宿主植物协同进化等提供有益参考资料。研究结果表明,从健康的青檀叶片获得可培养内生真菌839株,附生真菌1857株,共计2696株,鉴定其分属于4目,5科,43属。在目的分类水平上,内生和附生真菌均以丛梗孢目Moniliales为优势菌群,分别占90.23%和92.51%;在科的水平上,内生真菌以暗梗孢科Dematiaceae和丛梗孢科Moniliaceae为优势菌群,分别占47.56%和42.67%,附生真菌以丛梗孢科Moniliaceae和暗梗孢科Dematiaceae为优势菌群,分别占67.04%和25.47%;在属的水平上,内生真菌以黑团孢属Periconia和青霉属Penicillium为优势菌群,分别占31.47%和10.73%,附生真菌以小球霉属Glomerularia、膝葡孢属Gonatobotrys和青霉属Penicillium为优势菌群,分别占20.03%、13.95%和12.22%。青檀叶片内生真菌和附生真菌均存在的菌群数量达到23个属,占53.49%。内生真菌特有的属有6个,共分离19株,占0.70%,附生真菌特有的属有14个,共分离120株,占4.45%。内生真菌的Shannon-Wiener index(H')多样性指数(2.44)和Margalef index(R)丰富度指数(2.88)分别小于附生真菌ShannonWiener index(H')多样性指数(2.57)和Margalef index(R)丰富度指数(3.32),但两者的Evenness index(E)均匀度指数几乎相等。青檀叶片内生和附生真菌菌群组成具有较高的相似性,相似性系数达0.70。通过Fisher's exact test分析表明青檀叶片内生和附生真菌菌群组成无明显差异(P=0.072)。     

黄花倒水莲内生真菌生物活性评价及HNLF-44菌株鉴定

为充分开发黄花倒水莲(Polygala fallax)的内生真菌资源，获得具有抗植物病原真菌、抗氧化活性的内生真菌，该文以黄花倒水莲内生真菌为研究对象，使用平板对峙法检测内生真菌对6种植物病原真菌的抑菌活性，测定内生真菌发酵液的DPPH清除自由基能力和总还原能力，评价内生真菌的抗氧化活性，并对具有强抑菌活性和抗氧化活性的菌株进行形态和ITS鉴定。结果表明：(1)黄花倒水莲内生真菌中有2株内生真菌对香蕉专化尖孢镰刀菌、柑橘树脂病菌、叶点霉菌、香蕉具条叶斑病菌、茄病镰刀菌、三七根腐病菌具有明显的抑菌活性，抑菌率在50.3%～91.4%之间，其中HNLF-5对柑橘树脂病菌的抑菌率为73.2%,HNLF-44对香蕉专化尖孢镰刀菌抑菌率为91.4%。(2)内生真菌发酵液具有良好的抗氧化活性，DPPH清除率均在80%以上，总还原能力吸光值范围为0.279 2～0.748 8。(3)HNLF-44菌株为链格孢属真菌。该研究表明，药用植物黄花倒水莲内生真菌具有较好的生物活性，为后续从黄花倒水莲内生真菌中挖掘潜在新型抑菌活性和抗氧化活性物质奠定了基础。     

Synthesis of optically pure <Emphasis Type="Italic">S</Emphasis>-sulfoxide by <Emphasis Type="Italic">Escherichia coli</Emphasis> transformant cells coexpressing the P450 monooxygenase and glucose dehydrogenase genes

A cytochrome P450 monooxygenase (P450SMO) from Rhodococcus sp. can catalyze asymmetric oxygenation of sulfides to S-sulfoxides. However, P450SMO-catalyzed biotransformations require a constant supply of NAD(P)H, the expense of which constitutes a great hindrance for this enzyme application. In this study, we investigated the asymmetric oxygenation of sulfide to S-sulfoxide using E. coli cells, which co-express both the P450SMO gene from Rhodococcus sp. and the glucose dehydrogenase (GDH) gene from Bacillus subtilis, as a catalyst. The results showed that the catalytic performance of co-expression systems was markedly improved compared to the system lacking GDH. When using recombinant E. coli BL21 (pET28a-P450-GDH) whole cell as a biocatalyst, NADPH was efficiently regenerated when glucose was supplemented in the reaction system. A total conversion of 100% was achieved within 12 h with 2 mM p-chlorothioanisole substrate, affording 317.3 mg/L S-sulfoxide obtained. When the initial sulfide concentration was increased to 5 mM, the substrate conversion was also increased nearly fivefold: S-sulfoxide amounted to 2.5 mM (396.6 mg/L) and the ee value of sulfoxide product exceeded 98%. In this system, the effects of glucose concentration and substrate concentration were further investigated for efficient biotransformation. This system is highly advantageous for the synthesis of optically pure S-sulfoxide.     

云南三个地区马铃薯内生真菌多样性研究

为研究云南马铃薯(Solanum tuberosum)内生真菌的多样性,该文以采自云南省德宏芒市、大理喜洲和临沧双江3个地区的马铃薯植株为研究对象,采用组织块分离法、尖端菌丝挑取法对马铃薯根、茎及块茎中的内生真菌进行分离纯化,并采用形态学鉴定方法和ITS序列分析法对分离得到的内生真菌进行鉴定,对内生真菌的定殖率、分离率及多样性指数进行计算和分析。结果表明:(1)共分离得到内生真菌98株,其中从德宏芒市的样品中获得40株,从大理喜洲的样品中获得27株,从临沧双江的样品中获得31株。(2)经鉴定,分离得到的马铃薯内生真菌共涵盖10目10科13属,大多为子囊菌门和担子菌门,优势菌为镰刀菌属(Fusarium)和青霉属(Penicillium),褶皱裸孢壳(Emericella rugulosa)、接骨木镰刀菌(Fusarium sambucinum)、毛韧革菌(Stereum hirsutum)、Psathyrella sulcatotuberculosa和Epicoccum catenisporum 5种真菌均为首次从马铃薯植株中分离得到。(3)马铃薯块茎内生真菌的定殖率最高,根部内生真菌定殖率最低; 内生真菌的分离率以马铃薯根部为最高,而茎部最低; 不同组织中内生真菌的多样性指数趋势均为根>块茎>茎。从综合来看,云南马铃薯植株中的内生真菌具有较高的多样性,不同地区的马铃薯样品中内生真菌优势菌不同,马铃薯根部具有最丰富的内生真菌种群和最高的分离率,是最适合进行内生真菌分离的材料。该研究结果为后期探究马铃薯内生真菌对病原菌的拮抗作用奠定了基础,也为马铃薯内生真菌多样性研究提供了参考数据。     

黑老虎内生真菌及根际土壤真菌的群落结构与生态功能分析

为探讨黑老虎(Kadsura coccinea)根际土壤和组织内生真菌菌群的组成及其生态功能，该研究采用ITS高通量测序技术对成熟黑老虎(根、茎、叶)内生真菌及根际土壤真菌群落结构、多样性和生态功能进行了分析。结果表明：(1)从12个样品中共获得2 241个可操作分类单元(OTU),涉及10门、41纲、95目、212科、367属，内生真菌(根、茎、叶)和根际土壤真菌OTU数分别为386、536、258、1 435个，其中共有的OTU为18个。在门水平上，黑老虎内生真菌及根际土壤真菌优势群落均为子囊菌门和担子菌门，其中子囊菌门在叶和茎中占比分别高达96.99%和95.37%;在属水平上，黑老虎根际土壤真菌中腐生真菌被孢霉属占比较高(为13.5%),叶和茎等生长旺盛的组织中子囊菌门未分类属和痂囊腔菌属占比较高。(2)α多样性分析结果显示，黑老虎根际土壤真菌群落的丰度和多样性明显高于内生真菌，茎中内生真菌丰度显著高于根和叶，而根、茎和叶组织间内生真菌多样性差异不显著；PCoA分析结果显示，叶和茎的真菌群落结构相似性更高。(3)利用FUNGuild数据库进行的功能预测分析结果显示，黑老虎根际土...     

Microbial transformation of hydrocortisone by two fungal species Fusarium fujikuroi PTCC 5144 and Rhizomucor pusillus PTCC 5134

Abstract

This paper examines the biotransformation of hydrocortisone (1) by Fusarium fujikuroi and Rhizomucor pusillus. These species have not previously been tested for hydrocortisone biotransformation. The metabolites produced during hydrocortisone biotransformation by these two fungi were 11β,17α,20β,21-tetrahydroxypregn-4-en-3-one (2) and 11 β-hydroxyandrost-4-en-3,17-dione (3). Chemical structures were determined by spectroscopic methods. A time course study revealed that the disappearance of hydrocortisone was accompanied by the formation of metabolites 2 and 3. Metabolite 2 was produced as the major metabolite with high yield but the transformation to metabolite 3 was considerably lower, as determined by HPLC.     

罗汉果内生真菌多样性研究

该文采用传统形态学方法结合r DNA-ITS序列分析,对我国重要药用植物罗汉果中的内生真菌进行了鉴定并研究其多样性。结果表明:采用组织培养法从罗汉果健康植株中共分离得到150株内生真菌,包括罗汉果中雌株的内生真菌96株、雄株的内生真菌54株。122株内生真菌经形态学结合r DNA-ITS序列分析鉴定为9个属,均归属为子囊菌门,包含座囊菌纲(Dothideomycetes)和子囊菌纲(Sordariomycetes)。其中,座囊菌纲(Dothideomycetes)真菌包含3科、3属;子囊菌纲(Sordariomycetes)真菌包含6科、6属。优势属为刺盘孢属(Colletotrichum)和镰刀菌属(Fusarium)。罗汉果雌、雄植株不同组织中内生真菌的定殖率及分离率的变化规律均不相同,雌株中以根中内生真菌的定殖率和分离率最高,叶片中的最低;在雄株中以叶片中的定殖率和分离率最高,根中的最低。不同菌株在雌、雄两种植株的不同组织中的分布均不同,结合内生真菌群落组成的相似性比较结果,表明部分内生真菌对罗汉果雌株和雄株,以及同一植株中的不同组织均具有偏好性。不同组织中内生真菌的多样性指数在...     

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...     

蒜头果内生真菌次生代谢产物抑制人类致病菌活性的研究

蒜头果是我国特有的单种属稀有树种,为了进一步开发利用蒜头果树皮内生真菌的抗菌活性化合物,该研究对来自蒜头果的植物内生真菌(白黄笋顶孢霉、哈茨木霉、大棘黑团孢、枝状枝孢菌、斑污拟盘多毛孢、赭绿青霉、淡紫紫孢菌、朱黄青霉、Xenoacremonium recifei、Xylaria feejeensis)进行液体培养,10 d后回收培养液并用乙酸乙酯萃取获得初提物,采用抑菌圈法检测蒜头果内生真菌初提物抑菌活性,同时测定了最低抑菌浓度(MIC)。结果表明:白黄笋顶孢霉、大棘黑团孢、枝状枝孢菌、斑污拟盘多毛孢、赭绿青霉、淡紫紫孢菌均有抑菌活性,大棘黑团孢、斑污拟盘多毛孢、淡紫紫孢菌的初提物均对缓慢芽孢杆菌、无乳链球菌和藤黄微球菌有明显抗菌活性,最低抑菌浓度在1.562 5~6.25 mg·m L~(-1)之间。这说明蒜头果树皮内生真菌的次生代谢产物具有抗菌活性,各内生真菌次生代谢产物的抗菌效果不同。