Cytotoxic metabolites from Perenniporia tephropora, an endophytic fungus from Taxus chinensis var. mairei

Based on bioactivity-oriented isolation, the EtOAc extract of a culture broth of the endophytic fungus Perenniporia tephropora Z41 from Taxus chinensis var. mairei, with strong anti-Pyricularia oryzae activity, afforded a new sesquiterpenoid, perenniporin A (1), together with three known compounds, ergosterol (2), rel-(+)-(2aR,5R,5aR,8S,8aS,8bR)-decahydro-2,2,5,8-tetramethyl-2H-naphtho[1,8-bc]genfuran-5-ol (3), and albicanol (4). Their structures were elucidated by means of spectroscopic methods. All the isolated compounds and the EtOAc extract of P. tephropora Z41 (EPT) were evaluated for their cytotoxic activity against three human cancer cell lines (HeLa, SMMC-7721, and PANC-1). EPT demonstrated significant cytotoxicity with IC₅₀ values ranging from 2 to 15 μg/mL. Compound 2 was the most cytotoxic constituent against the tested cell lines with IC₅₀ values of 1.16, 11.63, and 11.80 μg/mL, respectively, while compounds 1, 3, and 4 exhibited moderate cytotoxicity with IC₅₀ values ranging from 6 to 58 μg/mL. We conclude that the endophytic fungus P. tephropora is a promising source of novel and cytotoxic metabolites.     

南方红豆杉产IAA内生芽胞杆菌的分离、鉴定及产脂肽类化合物研究

通过组织研磨法对南方红豆杉(Taxus wallichiana var. mairei)新鲜组织进行内生细菌的分离，利用16S rRNA测序技术对分离的内生细菌进行鉴定分析，使用高效液相色谱-高分辨飞行质谱测定菌液提取物IAA含量，利用HPLC-TOF-MS对提取物脂肽类化合物进行分离、鉴定，结合数据库筛选菌株产脂肽类化合物种类及含量差异。结果表明，从南方红豆杉根茎叶中共分离鉴定出内生细菌31株，筛选出产IAA菌株20株，其中编号为KLBMPTC10的芽胞杆菌属菌株产IAA含量高达210.3955 μg·L^-1。在对产IAA菌株所产脂肽类化合物的研究中发现，菌株KLBMPTC10是所有产IAA内生细菌中脂肽类化合物含量最丰富的，其产Iturin A2相对含量为26.06%，明显优于其他产IAA菌株。芽胞杆菌属菌株KLBMPTC10同时具备高产IAA和脂肽类化合物的能力，可作为研究菌株进一步开发。     

Screening of taxol-producing endophytic fungi from Taxus chinensis var. mairei

A total of 38 endophytic fungus strains were isolated from Taxus chinensis var. mairei by aseptic technique. Genomic DNA was extracted from isolated endophytic fungi and subjected to polymerase chain reaction (PCR) analysis for the presence of Taxus taxadiene synthase (TS) gene, a rate-limiting enzyme gene in the taxol biosynthetic pathway. Twelve out of 38 isolated endophytic fungus strains showed PCR positive for the ts gene. Subsequently, taxol and its related compounds were extracted from culture filtrates and mycelia of the PCR positive strains, separated by column chromatography and analyzed by High Performance Liquid Chromatography and Mass Spectrum. The analysis result showed that 3 strains could produce taxol and its related compounds at the detectible level. This study indicates that molecular detection of the ts gene is an efficient method for primary screening of taxol or its related compounds-producing endophytic fungi which can improve prominently screening efficiency.     

南方红豆杉内生真菌的抗肿瘤产物震颤毒素的研究

从红豆杉内生真菌青霉菌Penicillum sp. H⑥.1发酵产物中自然析出一种化合物,经图谱检测和文献比对,鉴定为震颤毒素Penitrem A。四唑盐（MTT）比色法研究表明Penitrem A对恶性黑色素瘤细胞A375有一定的抑制作用,且抑制作用随浓度的增大而增强,IC₅₀为27.24μg/mL。本研究是首次从红豆杉内生真菌发酵产物中分离得到震颤毒素Penitrem A,为抗肿瘤药物开发提供了理论参考。     

Taxol from Tubercularia sp. strain TF5, an endophytic fungus of Taxus mairei

The diterpenoid taxol is an important anticancer agent used widely in the clinic. The purpose of this work was to identify a taxol-producing endophytic fungus (strain TF5) isolated from Taxus mairei and study its anticancer activities. Strain TF5 was identified as a Tubercularia sp. according to the morphology of the fungal culture, the mechanism of spore production and the characteristics of the spores. Strain TF5 produced taxol, when grown in potato dextrose liquid medium and analyzed by thin layer chromatography, high performance liquid chromatography, ultraviolet and mass spectrometry. The fungal taxol, which was isolated from the organic extract of the TF5 culture, had strong cytotoxic activity towards KB and P388 cancer cells in vitro, tested by the MTT assay. Observed with immunofluorescence and electron microscopy, the fungal taxol enhanced microtubule stability and bundling in culture cells and induced tubulin polymerization in vitro similar to the authentic taxol.     

不同立地条件下南方红豆杉幼苗的生长和生理特性

为阐明不同立地条件下南方红豆杉的生长发育、光合特性、活性氧代谢及其与环境的关系,本试验以2年生南方红豆杉幼苗为材料,在福建省泉州地区开展桉树林、樟树林和空地3种生境下南方红豆杉幼苗的生长和生理特性研究,探索适宜南方红豆杉幼苗生长的环境特点.结果表明:桉树林和樟树林下南方红豆杉幼苗的株高和新梢长度显著高于空地,桉树林和樟树林有利于南方红豆杉幼苗的生长;桉树林和樟树林下南方红豆杉幼苗叶绿素b含量和类胡萝卜素含量显著高于空地,气孔导度和蒸腾速率显著提高,净光合速率也高于空地;桉树林和樟树林下南方红豆杉幼苗可溶性糖和氨基酸含量高于空地,蛋白质含量低于空地.桉树林和樟树林下南方红豆杉幼苗超氧化物歧化酶和过氧化氢酶活性显著低于空地,而过氧化物酶活性高于空地;这可能是空地生境对南方红豆杉幼苗产生一定的光胁迫效应,桉树林和樟树林可为南方红豆杉幼苗生长提供必要的遮荫环境,有利于南方红豆杉幼苗的生长.     

南方红豆杉产紫杉醇内生真菌的分离鉴定

从南方红豆杉(Taxus chinensisvar.mairei)植株分离到内生真菌63株,其中24株经鉴定分属于19个种(属).真子囊菌纲的2个属2株,半知菌纲的14个属22株.真子囊菌纲的无毛毛壳属(Achaetomiumsp.)的1株为新发现种.半知菌纲的葡萄孢属(Botrytissp.)1株和无孢菌群(Mycelia sterlia)2株的培养液,经浓缩萃取,薄层层析检测,Rf值分别为0.404、0.404、0.401,与紫杉醇对照品Rf值一致,为南方红豆杉产紫杉醇内生真菌.     

不同生境及采样部位对南方红豆杉活性成分含量的影响

采用HPLC法,对采自浙江、福建和江西7个产地以及不同坡向(阴坡和阳坡)、不同坡位(高、中、低坡位)的野生南方红豆杉[Taxus wallichiana var.mairei (Lemée et Lévl.)L.K.Fu et Nan Li]枝叶中紫杉醇、7-木糖紫杉醇和三尖杉宁碱含量进行了分析,并比较了南方红豆杉不同部位(茎皮和枝叶)和不同冠层中3种成分的含量;在此基础上对3种成分含量与环境因子(包括地理、气候及土壤因子)的相关性进行了分析.结果表明:采自阳坡的枝叶中3种成分的含量明显高于阴坡,不同坡向间紫杉醇和7-木糖紫杉醇含量差异显著(P＜0.05).采自不同坡位的枝叶中3种成分的含量均有一定差异,其中采自低坡位的枝叶中3种成分的含量均最高.不同产地间紫杉醇含量有极显著差异(P＜0.01)、7-木糖紫杉醇和三尖杉宁碱含量则有显著差异(P＜0.05);总体上看,采自福建建阳和江西景德镇的枝叶中3种成分的含量相对较高.南方红豆杉茎皮中3种成分的含量显著高于其枝叶;高冠层枝叶中3种成分的含量均高于低冠层枝叶,不同冠层间紫杉醇和7-木糖紫杉醇含量差异显著(P＜0.05).相关性分析结果表明:南方红豆杉中三尖杉宁碱含量与土壤中全N和土壤有机质含量呈极显著或显著的负相关性,与其他因子的相关性均不显著;而紫杉醇和7-木糖紫杉醇含量与所有因子均无显著的相关性;海拔与3种成分的含量均呈不显著的负相关性,提示低海拔可能有利于南方红豆杉中3种成分的合成及积累.     

南方红豆杉内生真菌的分离鉴定及其细胞毒活性研究

采用平板培养法对采自广东省的南方红豆杉进行内生真菌的分离,并通过形态学和分子生物学方法进行分类鉴定;共分离鉴定内生真菌22株,主要为刺盘孢属(Colletotrichum)、球座菌属(Guignardia)、座腔菌属(Botryosphaeria)、节菱孢属(Arthrinium)、炭角菌属(Xylaria)、毛壳菌属(Chaetomium)、交链孢属(Alternaria)、拟茎点霉属(Phomopsis)、长蠕孢属(Helminthosporium)等属。还采用MTT法测定了这些内生真菌发酵粗提物的细胞毒活性,活性研究发现有6个菌株的粗提物在作用浓度为100μg/mL时,对至少1种受试肿瘤细胞株的抑制率在50%以上,其中菌株A223和A240对人乳腺癌细胞MCF-7的抑制率在90%以上。表明南方红豆杉内生真菌多样性丰富,有些菌株具有良好的细胞毒活性,值得进一步深入研究。     

不同种源南方红豆杉生长差异分析及早期速生优良种源筛选

在浙江龙泉和安吉试验点,对来源于10省(区)24个种源的南方红豆杉〔Taxus wallichiana var.mairei(Lemée et Lévl.)L.K.Fu et Nan Li〕4年生幼苗的7个生长指标进行了测定及方差分析,并对生长指标间以及生长指标与地理-气候因子间的相关性进行了分析;此外,以树高和地径为标准初步筛选出一些速生优良种源。测量结果显示:2个试验点不同种源4年生幼苗的树高、地径、当年抽梢长和侧枝数以及侧枝的最大长度及最大直径的变化幅度以及安吉试验点各种源幼苗冠幅的变化幅度均在25%以上,说明各种源间生长指标的差异较大。方差分析结果表明:龙泉试验点各种源间幼苗侧枝的最大直径以及安吉试验点各种源间幼苗的树高无显著差异,其他生长指标在种源间均有显著或极显著差异,且冠幅、当年抽梢长和侧枝的最大长度具有显著或极显著的种源-地点互作效应。相关性分析结果表明:幼苗的冠幅与地径和树高呈显著或极显著的正相关,且侧枝的最大长度与树高和冠幅也呈显著或极显著的正相关;虽然2个试验点间各种源幼苗的7个生长指标与地理-气候因子的相关性有一定差异,但总体上未表现出明显的地理变异模式。根据2个试验点幼苗树高和地径的综合表现,初步筛选出湖南靖州、江西井冈山、福建沙县、江西武宁、云南石屏和贵州梵净山6个种源为南方红豆杉的早期速生优良种源。     

南方红豆杉内生真菌分离时的表面消毒方法探索

表面消毒是植物内生菌分离的关键环节,在南方红豆杉(Taxus chinensis var. mairei)内生真菌分离的过程中,使用75％乙醇、2％次氯酸钠（NaClO）代替升汞对外植体进行了表面消毒摸索。结果显示：红豆杉叶、茎、皮经预处理后,先用75％乙醇浸泡3 min,再用2％次氯酸钠浸泡2 min,然后红豆杉叶、茎、皮分别用75％乙醇浸泡3 min、5 min、7 min,可取得满意的表面消毒效果。     

一株产白藜芦醇虎杖内生真菌的分离和鉴定

从药食同源植物虎杖根中分离得到15株内生真菌,在马铃薯葡萄糖液体培养基(PDB)中发酵后对所有内生真菌发酵液进行HPLC检测,有5株菌的发酵液中相关成分与白藜芦醇标准品保留时间一致,确定5株菌能产生白藜芦醇,在此基础上测定了5株菌的生长曲线。其中菌株H8生长最旺盛,白藜芦醇产量最高。应用形态学及ITS序列分析方法对其进行鉴定,确定为拟轮枝镰孢菌Fusarium verticillioides。     

Structural diversity of naturally regenerating Chinese yew (Taxus wallichiana var. mairei) populations in ex situ conservation

The Chinese yew (Taxus wallichiana var. mairei) is ranked in the first class of important wild endangered plants in China. Due to overexploitation, it now occurs scattered only in the forest undergrowth along the Yangtze River Valley. To improve the conservation management of the species, we applied structural indices to investigate the structural diversity of naturally regenerating yew populations that have established via ex situ conservation. The results show that most yews had larger non‐yew tree neighbors; these were 30–70% larger than their reference trees. Collectively, the average distances between the yews and the three nearest‐neighboring trees were short ( 3 m), suggesting that the yews face strong interspecific competition from neighbors. In these two forest stands, most of the pole‐sized yews were found beneath a single tall neighboring tree (height  10 m), and their growth was enhanced under a single neighboring tree but not under two, three or zero neighboring trees. Finally, we recommend simple silvicultural measures to reduce interspecific competition and improve the vitality of the yew population; specifically, the cutting or pruning of branches of large neighboring trees in tandem with the thinning of canopy trees growing next to the mother yews.     

蛇足石杉中产石杉碱甲内生真菌的分离和鉴定

【目的】从野生蛇足石杉(Huperzia serrata)中分离筛选产石杉碱甲的内生真菌。【方法】采用薄层层析及高效液相色谱法对内生真菌代谢产物进行测定和分析以期分离获得产石杉碱甲菌株,运用形态及ITS序列分析方法对产石杉碱甲菌株进行鉴定,并利用连续传代方法考察菌株遗传稳定性。【结果】经筛选获得一株产石杉碱甲内生真菌NSH-5,经形态学鉴定及ITS序列分析鉴定为轮枝镰孢菌(Fusarium verticillioides),其石杉碱甲产量为11.76 mg/100 m L,菌株经20次连续传代后遗传稳定。【结论】NSH-5菌株为一株具有产石杉碱甲能力的轮枝镰孢菌,该菌株的发现为生物合成石杉碱甲提供了新的菌种资源。     

南方红豆杉天然林群落结构特征研究

本文对福建三明陈大镇南方红豆杉天然林群落进行研究,测定了该群落11种主要树种的重要值,分析了该群落的种类组成、物种多样性、群落结构及其动态。结果表明,人为干扰较轻的南方红豆杉天然林群落比较稳定,天然更新良好,处于旺盛生长阶段。     

云南红豆杉(Taxus Yunnanensis)内生真菌中产紫杉醇真菌的筛选

报道了从云南红豆杉（Taxus Yunnanensis)树皮和枝条中分离得到的52株内生真菌中,有19株菌的发酵产物经TLC分析和HPLC检测验证后产紫杉醇或紫杉烷类物质。其中紫杉醇含量在1‰以上的菌株有8株。TAX-47（茎点霉属）和TAX-49（组丝核菌属）的紫杉醇得率分别为47.302μg/L和31.06μg/L,TAX-25（茎叶核菌属）巴可亭Ⅲ得率为744.2μg/L,这些菌株值得进一步研究。     

南方红豆杉丛枝菌根(AM)的研究

研究了南方红豆杉根部丛枝菌根真菌(AMF)侵染情况、菌根形态结构以及根际土中AMF孢子的种类与数量.结果显示:南方红豆杉可与AMF形成典型的丛枝-泡囊型菌根,侵染率在71.2%~94.4%,但是历山、蟒河自然保护区的侵染强度优于人工栽培区;在南方红豆杉根际土中共分离鉴定出5种AMF,无梗囊霉属1种、球囊霉属4种,分别是:光壁无梗囊霉、地表球囊霉、地球囊霉、缩球囊霉、明球囊霉,其中光壁无梗囊霉为优势种;南方红豆杉的根由表皮、外皮层、内皮层、中柱组成,AMF只侵染表皮层、内皮层,不能侵染中柱.这为将来利用AMF接种技术进行南方红豆杉的繁殖、移植栽培以及紫杉醇的积累等研究提供了理论依据.     

Isolation of taxol producing endophytic fungus Alternaria brassicicola from non-Taxus medicinal plant Terminalia arjuna

World Journal of Microbiology and Biotechnology - Shigella type III effector OspF, a nuclear translocation protein, has been showed to have an essential role in Shigella flexneri infection and...     

RAPD Analysis on High-taxane-contained Accessions of Taxus chinensis var. mairei

Taxus chinensis var. mairei (kemee et Levi. ) Cheng et L.K. Fu is one of the resource plants in China. The taxot contents from different localities are variable. RAPD and HPLC analysis were carried out with 24 samples from 12_localities. A total of 256 loci were amplified among which 224 loci were polymorphic (87.8%). Based on the HPLC and morphological analysis, the relationship among RAPD markers, morphology and habitat was explored. According to the results, high-taxane-contained accessions are present in three localities. This study might be of benefit to the resource conservation and species improvement.     

Taxines from the needles of Taxus wallichiana.

A taxine, 5 alpha O-(3'-dimethylamino-3'-phenylpropionyl) taxinine M (1) together with two known compounds 7-O-acetyltaxine A (2) and 2 alpha-acetoxy-2' beta-deacetylaustrospicatine (3) were isolated from the needles of the Himalayan yew, Taxus wallichiana Zucc. Their structures were elucidated on the basis of the NMR spectral data, ESI-MS/MS analysis and chemical methods. Compounds 1 and 3 showed moderate cytotoxic activity against the lung cancer cell line A549 in vitro.