鹿衔草内生真菌Penicillium solitum次生代谢产物的分离鉴定与神经保护活性筛选

【背景】鹿衔草(Pyrola incarnata Fisch)化学成分丰富,而且具有多种药理活性,在食品、医药领域得到广泛应用,但尚未见其内生真菌次生代谢产物的相关报道。【目的】对鹿衔草内生真菌Penicillium solitum (P.solitum)的次生代谢产物进行分离鉴定,筛选出具有抗神经炎症的活性成分。【方法】采用柱层析和制备型高效液相色谱对鹿衔草内生真菌P.solitum次生代谢产物进行分离和纯化,通过核磁共振和高分辨质谱进行结构鉴定,利用反向分子对接技术筛选出抗神经炎症活性的化合物,采用脂多糖(lipopolysaccharide,LPS)诱导的BV-2细胞炎症模型进行验证。【结果】从鹿衔草内生真菌P.solitum次生代谢产物中分离鉴定出12个化合物,分别为cyclopenin (1)、dehydrocyclopeptin (2)、viridicatin (3)、对羟基苯甲酸(4)、吲哚乙酸(5)、methyl compactin (6)、cyclopeptine (7)、cyclopenol (8)、原儿茶酸(9)、viridicatol (10)、N-[2-(3-indolyl) ethyl]malonamic acid (11)和solitumidine A (12)。分子对接结果显示化合物11与炎症相关的丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)信号通路中p38丝裂原活化蛋白激酶(p38 mitogen-activated protein kinase,简称p38)和细胞外信号调节激酶1(extracellular regulated protein kinase 1,ERK1)有较高的结合活性。BV-2细胞实验表明,化合物11 (10、20μmol/L)能显著提高BV-2细胞存活率,并有效抑制炎症因子(肿瘤坏死因子-α、白介素-1β、白介素-6)和一氧化氮的释放。【结论】化合物11是新天然产物,具有抗神经炎症活性,其作用机制可能与抑制MAPK信号通路中p38和ERK蛋白的表达有关。     

南美蟛蜞菊中二萜化学成分的定性与定量分析研究

为了解南美蟛蜞菊[Sphagneticola trilobata (L.) Pruski]植物的化学成分,从其全株乙醇提取物中分离得到8个二萜类化合物。经光谱分析,分别鉴定为ent-16β,17-dihydroxy-9(11)-kauren-19-oic acid (1)、cussovantonin B (2)、ent-16-kauren-19-oic acid(3)、ent-3β-hydroxy-16-kauren-19-oic acid (4)、16α-hydroxy-ent-kauran-19-oic acid (5)、2β,16α-dihydroxy-ent-kauran-19-oic acid (6)、3α-angeloyloxypterokaurene L₃ (7)和pterokaurene L₃ (8)。化合物4为首次从蟛蜞菊属植物中分离获得,化合物1、2 和6为首次从南美蟛蜞菊中分离得到。化合物3具有显著的抗菌活性和显著抑制α-葡萄糖苷酶活性。采用五氟苄溴进行底物五氟苄基衍生化并结合GC-MS 分析表明,化合物3在南美蟛蜞菊茎和叶中的含量分别为(1.00±0.21) mg g^-1 FW和(0.78±0.04) mg g^-1 FW。     

光枝勾儿茶内生真菌多样性及抑菌活性研究

为探索贵州苗药光枝勾儿茶内生真菌类群特征、分布部位及其抑菌活性,该研究采用传统方法对贵州省贵阳市和黔西市光枝勾儿茶内生真菌进行分离,并基于分子生物学及统计学对其分类地位进行鉴定及多样性评价,最后通过微量肉汤倍比稀释法筛选具有抑菌活性的菌株。结果表明:(1)从光枝勾儿茶中分离到191 株内生真菌,隶属于3 个门5 个纲10 个目15 个科19 个属,优势属为叶点霉属(Phyllosticta)、间座壳属(Diaporthe)、葡萄座腔菌属(Botryosphaeria)和刺盘孢属(Colletotrichum)。(2)黔西光枝勾儿茶内生真菌香农-维纳多样性指数(H''_Q=2.112)较贵阳(H''_G=1.801)高,索伦森相似性指数Cs_G-Q为0.923,不同组织香农-维纳多样性指数为茎(H''_S=2.004)>根(H''_R=1.764)>叶(H''_L=1.654)>果实(H''_F=1.473),茎和叶内生真菌的索伦森相似性最高(Cs_S-L=0.667)。(3)筛选出的21株内生真菌对供试菌大肠杆菌(Escherichia coli)、金黄色葡萄球菌(Staphylococcus aureus)和沙门氏菌(Salmonella enterica)具有抑菌效果,其中Diaporthe sp. QX4G6对大肠杆菌、金黄色葡萄球菌和沙门氏菌的最小抑菌浓度分别为12.5、6.25、12.5 mg·mL^-1,最小杀菌浓度分别为12.5、6.25、12.5 mg·mL^-1。以上研究结果揭示了光枝勾儿茶蕴藏丰富的内生真菌资源,不同地区及组织内生真菌类群组成有差异,多个分离菌株具有抗菌活性,为光枝勾儿茶内生真菌天然抗菌药物或药源研发奠定了基础。     

滇重楼内生真菌Aspergillus fumigatus代谢产物的研究

为研究滇重楼内生真菌Aspergillus fumigatus的代谢产物,采用多种色谱分离技术对其菌丝体和发酵液进行分离纯化,得到31个化合物。根据理化性质及波谱数据,结构分别鉴定为tryprostatin B (1)、tryprostatin A (2)、12,13-dihydroxyfumitre- morgin C (3)、cyclotryprostatin B (4)、14-norpseurotin A (5)、pseurotine F1 (6)、pseurotine F2 (7)、azaspirofuran A (8)、pseurotin D (9)、spirotryprostatin K (10)、6-methoxyspirotryprostatin B (11)、deacetylpyripyropene A (12)、烟曲霉素 (13)、fuma- gillene A (14)、5,9-dihydroxy-β-trans-bergamotene (15)、对羟基苯乙酸 (16)、demethoxyfumitremorgin C (17)、fumiquinazoline J (18)、烟曲霉酸 (19)、麦角甾醇 (20)、过氧麦角甾醇 (21)、4,4-dimethyl-5α-ergosta-8,24(28)-dien-3β-ol (22)、羊毛甾醇 (23)、亚油酸 (24)、油酸 (25) 、烟曲霉毒素C (26)、烟曲霉毒素B (27)、震颤真菌毒素 (28)、pseurotin A (29)、fumiquinazoline C (30)和questin (31)。其中,化合物1~16仅从发酵液中分离得到;化合物17~25仅从菌丝体中分离得到;其余化合物在发酵液和菌丝体中均分离得到。化合物15和23为首次从烟曲霉属真菌中分离得到。此外,化合物26~29的乙酰胆碱酯酶抑制活性和烟草黑胫病菌抑制活性评价表明,化合物26和27对烟草黑胫病菌具有微弱的抑制作用,抑菌率分别为19.64%和17.86%。     

贵州鼠尾草化学成分研究

为了解贵州鼠尾草(Salvia cavaleriei)的化学成分,运用色谱分离技术,从其95%乙醇提取物中分离得到16个化合物,经波谱数据分析分别鉴定为：β-倍半水芹烯 (1)、6R-(7R,11-二甲基-10-乙烯亚基)-环己烯酮 (2)、β-没药烯 (3)、α-姜黄烯 (4)、3,4-二羟基苯乙酸甲酯 (5)、香草醛 (6)、姜油酮 (7)、6-姜烯酚 (8)、阿魏酸十八烷酯 (9)、维生素E醌 (10)、5-羟甲基-2-糠醛 (11)、硫代乙酸酐 (12)、吲唑 (13)、棕榈酸 (14)、角鲨烯 (15)和二十六烷 (16)。其中,化合物2为新天然产物,除化合物3、4和6外,所有化合物均为首次从贵州鼠尾草中分离得到,此外,化合物2、5、9和12为首次从唇形科(Lamiaceae)植物中分离得到,化合物1和15为首次从鼠尾草属植物中分离得到。     

紫茎泽兰的化学成分研究

为了解紫茎泽兰(Eupatorium adenophorum Spreng.)的化学成分,从其乙醇提取物中分离得到7 个化合物。通过波谱分析,分别鉴定为万寿菊苷(1)、7-O-(6-methoxykaempferol)-β-D-glucopranoside (2)、4'-甲基醚万寿菊苷(3)、3-O-(6-methoxykaempferol)-β-D-glucopranoside (4)、邻苯二甲酸二丁酯 (5)、邻苯二甲酸二(2-乙基)己酯 (6)、1,4-bis(2-benzoxazolyl)naphthalene (7)。其中化合物1~4 为首次从紫茎泽兰中分离得到。     

一株内生真菌Talaromyces sp.次级代谢产物及其活性

为了研究红树杨叶肖槿来源内生真菌Talaromyces sp. YX-001的次级代谢产物及其乙酰胆碱酯酶(acetylcholinesterase, AChE)抑制活性,该文运用硅胶柱层析和HPLC等手段从菌株YX-001的PDB培养基发酵提取物中分离得到单体化合物,而后结合1/2D NMR和MS等现代波谱技术并与文献数据对比鉴定其结构,最后通过比色法测定各单体化合物的AChE抑制活性。结果表明:(1)共分离得到9个单体化合物,分别为asterrelenin(1),aszonalenin(2),cladosporisteroid C(3),sitosterol(4),ergosterol(5),cyclo-Ile-Pro-diketopiperazine(6),cyclo(-Pro-Val)(7),4-methoxy-2-methylisoquinolin-1-one(8)和allantoin(9)。(2)其中化合物1和2显示出一定的AChE抑制活性,IC₅₀值分别为81.5、105.8 μmol·L^-1。该文首次对杨叶肖槿来源内生真菌次级代谢产物及其AChE抑制活性进行了研究,为后续红树植物杨叶肖槿来源内生真菌资源的开发与再利用奠定了基础。     

异叶三宝木的二萜成分及抗菌活性研究

为研究异叶三宝木(Trigonostemon heterophyllus)的二萜成分及其抗菌活性,采用硅胶柱层析、凝胶柱层析、高效液相色谱对萃取物进行分离纯化,结合现代波谱技术对所得化合物进行结构鉴定,并通过牛津杯法和2倍稀释法检测化合物对革兰氏阴性菌大肠杆菌(Escherichia coli)以及革兰氏阳性菌肺炎双球菌(Pneumococcus)的生长抑制活性和MIC值。从异叶三宝木中分离得到了6个化合物,包括5个二萜类, 1个木脂素类,分别鉴定为trigonochinene E (1)、neoboutomannin (2)、6,9-O- dedimethyltrigonostemone (3)、stelltian B (4)、3,4-secosonderianol (5)、biondinin A (6)。化合物1、2、3、5对大肠杆菌有抑制作用,MIC值分别为9.375、18.75、18.75、18.75 μg/mL。化合物2、3、4、6为首次从该种植物中分离得到,化合物4为首次从该属植物中分离得到。除化合物4外,其他化合物都有一定的抗菌活性,且化合物1、2、3对大肠杆菌抑制作用强于阳性对照硫酸卡那霉素。     

姜状三七根茎的皂苷类化学成分研究

为了解姜状三七(Panax zingiberensis)根茎的皂苷类化学成分,采用正相硅胶、反相硅胶和凝胶等色谱方法从其根茎的乙醇提取物中得到9个皂苷类化合物,根据理化性质和波谱数据,其结构分别鉴定为人参皂苷SL₁ (1)、人参皂苷Rh₁ (2)、三七皂苷R₈ (3)、竹节参皂苷IVa (4)、越南人参皂苷R₁₀ (5)、人参皂苷Rg₁ (6)、菠菜皂苷A 28-O-β-d-葡萄糖苷 (7)、齐墩果酸28-O-β-d-葡萄糖苷 (8)和姜状三七苷R₁ (9)。化合物1、3、5、7和8为首次从该植物中分离得到, 其中化合物5为奥克梯隆醇型皂苷,此类皂苷在该植物中未见报道。     

桐花树活性内生真菌筛选及其抗菌化学成分的研究

为了探究桐花树内生真菌在抑菌方面的价值,该文以内生真菌发酵产物的抑菌作用为评价指标筛选活性菌株,采用生物活性跟踪方法结合多种色谱技术分离活性菌株的化学成分,通过波谱与文献数据比对鉴定单体化合物结构,并利用微孔板法测定单体化合物的抑菌活性。结果表明:(1)从桐花树分离得到的16株内生真菌分属2纲7目10科10属,镰刀菌属(Fusarium)为优势菌属。内生真菌GXIMD02029和GXIMD02039的发酵产物对枯草芽孢杆菌、表皮葡萄球菌、耐甲氧西林金黄色葡萄球菌、藤黄微球菌、粘性放线菌和金黄色葡萄球菌有不同程度的抑制作用,GXIMD02038发酵产物对耐甲氧西林金黄色葡萄球菌、藤黄微球菌和金黄色葡萄球菌有抑制作用。(2)7个化合物从内生真菌Phomopsis sp. GXIMD02029中被分离并鉴定为(15R)-acetoxydothiorelone A(1)、cytosporone B(2)、pestalotiopsone H(3)、pestalotiopsone B(4)、4-Hydroxybenzaldehyde(5)、p-Hydroxybenzoic acid(6)、N-(2-phenylethyl)acetamide(7)。(3)化合物1和2有不同程度的抑菌作用,化合物1对枯草芽孢杆菌、表皮葡萄球菌、耐甲氧西林金黄色葡萄球菌的MIC值为16.25 SymbolmA@ g·mL^-1,对藤黄微球菌和粘性放线菌的MIC值为7.812 5 SymbolmA@ g·mL^-1,对金黄色葡萄球菌的MIC值为31.25 SymbolmA@ g·mL^-1。化合物2对藤黄微球菌的MIC值为62.5 SymbolmA@ g·mL^-1,对枯草芽孢杆菌、表皮葡萄球菌、耐甲氧西林金黄色葡萄球菌、粘性放线菌的MIC值为125 SymbolmA@ g·mL^-1,对金黄色葡萄球菌的MIC值为250 SymbolmA@ g·mL^-1。该文筛选了3株活性菌株,首次报道化合物1具有抗菌活性,为桐花树内生真菌在抗菌价值方面提供了依据。     

Effects of MULTIFOLIATE-PINNA, AFILA, TENDRIL-LESS and UNIFOLIATA genes on leafblade architecture in Pisum sativum

In order to dissect the genetic regulation of leafblade morphogenesis, 16 genotypes of pea, constructed by combining the wild-type and mutant alleles of MFP, AF, TL and UNI genes, were quantitatively phenotyped. The morphological features of the three domains of leafblades of four genotypes, unknown earlier, were described. All the genotypes were found to differ in leafblade morphology. It was evident that MFP and TL functions acted as repressor of pinna ramification, in the distal domain. These functions, with and without interaction with UNI, also repressed the ramification of proximal pinnae in the absence of AF function. The expression of MFP and TL required UNI function. AF function was found to control leafblade architecture multifariously. The earlier identified role of AF as a repressor of UNI in the proximal domain was confirmed. Negative control of AF on the UNI-dependent pinna ramification in the distal domain was revealed. It was found that AF establishes a boundary between proximal and distal domains and activates formation of leaflet pinnae in the proximal domain.     

Presence of Rhizobium Etli bv . Phaseoli and Rhizobium Gallicum bv . Gallicum in Egyptian Soils

Seven bean rhizobial strains EBRI 2, 3, 21, 24, 26, 27 and 29 identified as Rhizobium etli, and EBRI 32 identified as Rhizobium gallicum, isolated from Egyptian soils and which nodulated Phaseolus vulgaris efficiently, were subjected to hybridization with a nifH probe in order to estimate the copy number of this gene. Seven strains (EBRI 2, 3, 21, 24, 26, 27 and 29) which were only able to nodulate Phaseolus vulgaris, contained three copies of the nifH gene, consistent with their identification as Rhizobium etli bv. phaseoli. Only one strain (EBRI 32) which nodulated both Phaseolus vulgaris and Leucaena leucocephala, had one copy of nifH gene. This confirmed the classification of this strain as Rhizobium gallicum bv. gallicum.     

Prosthecium species with Stegonsporium anamorphs on Acer

Data from microscopic morphology, single-spore cultures, and DNA analyses of teleomorphs and anamorphs support the recognition of five species of Prosthecium with Stegonsporium anamorphs on Acer: P. acerinum sp. nov., the teleomorph of S. acerinum; P. acerophilum comb. nov., formerly known as Dictyoporthe acerophila; P. galeatum comb. nov., originally described as Massaria galeata; P. opalus sp. nov.; and P. pyriforme sp. nov., the teleomorph of S. pyriforme s. str. The morphology of both type specimens and freshly collected material was investigated. The teleomorphs have brown ellipsoidal ascospores with five distosepta and often a longitudinal distoseptum. The anamorphs of all species described here belong to Stegonsporium; their connection to the Prosthecium teleomorphs was demonstrated by morphology and DNA sequences of single spore cultures derived from both ascospores and conidia. The anamorphs and teleomorphs of all five Prosthecium species are described and illustrated by LM images, and a key to these species is provided. As perceived from this work, S. pyriforme is restricted to Europe and does not occur in North America, whereas S. acerinum is restricted to North America, not found in Europe. The host associations given in the literature are revised and evidence is provided that only A. opalus, A. pseudoplatanus, and A. saccharum are confirmed hosts of Prosthecium with Stegonsporium anamorphs. Molecular phylogenetic analyses of tef1, ITS rDNA, and partial nuLSU rDNA sequences confirm that the species with Stegonsporium anamorphs are closely related to P. ellipsosporum, the generic type species. Stilbospora macrosperma is confirmed as the anamorph of P. ellipsosporum by DNA data of single spore isolates obtained from both ascospores and conidia.     

Characterisation of cell wall polysaccharides, arabinogalactans-proteins (AGPs) and phenolics of Cola nitida, Cola acuminata and Garcinia kola seeds

Many Cola plant species are endemic to West and Central Africa. Cola acuminata and Cola nitida are used as masticatory when fresh, while the dried nuts are used for beverages and pharmaceutical purposes in Europe and North America. Garcinia kola seeds, that serve as a substitute for the true kola nuts, are used in African traditional medicine for the treatment of various diseases, including colic, headache and liver cirrhosis. Seeds extracts of G. kola are also known for their anti-inflammatory, antimicrobial and antiviral properties. To gain information on the chemical properties of the kolas, we have isolated and analyzed cell wall polysaccharides, arabinogalactan-proteins and phenolic substances from the seeds of the three kola species. The sugar composition of cell wall material of C. acuminata, C. nitida and G. kola revealed that Gal (up to 30%), Ara, GalA and Glc as the predominant monosaccharides, representing approximately 90% by mol of the total hydrolysable sugar present in this material. In Ammonium oxalate cell wall fraction, GalA was found to be the major sugar present in all kola species. In the alkali-soluble fraction, there were significant differences in the level of Glc and Gal. The level of Glc was high in C. acuminata and C. nitida while the level of Gal and Xyl were high in C. nitida and G. cola. Isolation and quantification of arabinogalactan-proteins demonstrate that G. kola seeds contained four to eight times more of these proteoglycans than the seeds of the other two species. Finally, analysis of soluble phenolic substances shows that caffeine and catechin were largely represented in C. acumina and C. nitida seeds, with caffeine accounting for 50% of all soluble phenolics. These findings indicate that the three Kola seeds are highly enriched in pectins and proteoglycans and that C. acuminata and C. nitida can be used as a possible source of caffeine and catechin.     

中国黑粉菌属和利罗黑粉菌属

黑粉菌属是Roussel 1806年建立的,全世界记载有三百余种,主要寄生于禾本科,是经济作物及牧草的重要致病菌·长期以来,对黑粉菌的邢子使用过各种名称,如厚垣孢子,冬孢子及黑粉孢子等.本文采用黑粉孢子以区别锈菌的冬孢子. 芳’(1979)在《中国真菌总汇》中列出黑粉菌属五十种及一个变型.作者经过显微结构和超显微结构的研究,承认其中二十九种为正确名称,八种及一变型为异名,顶黑粉菌(Ustilago acrearus Berk.)由于错拼而被废弃.埃地黑粉菌(Ustilago emodensis Berk.)被转移至利罗粉菌属(Liroa).另有十一种黑粉菌因缺少标本留待今后订正.自1979年以后,杨信东(1983)增加黑粉菌属二种我国新纪录,K.范基和郭林(1986)描述一新种,四种新纪录.在本文中,作者描述一新种:鸢尾蒜黑粉(Ustilago ixiolirii Guo L) ,孢子堆生在蒴果内,不开裂,黑色,粉末状.黑粉孢子球形,近球形,稀椭圆形, 12.5-21×10-21μm,黑褐色,壁厚1-1.Sμm,纹饰脑状.是迄今生在石蒜科植物上唯一黑粉菌的种,其它几种黑粉菌均属条黑粉菌属.本文增加七种我国新纪录.共计四十九种,寄生于六科四十四属植物,主要是禾本科和蓼科.这仅是黑粉菌属研究的初步报告,在全国范围内大量采集黑粉菌标本后,作者相信会有更多新种和我国新纪录被发现.利罗黑粉菌属(Liroa)是从黑粉菌属(Ustaligo)分出的,此属为单种属.     

Comparative study of Bifidobacteriumanimalis, Escherichiacoli, Lactobacilluscasei and Saccharomycesboulardii probiotic properties

The present work investigates some probiotic properties of four different microorganisms (Bifidobacterium animalis var. lactis BB-12, Escherichia coli EMO, Lactobacillus casei and Saccharomyces boulardii). In vitro and in vivo tests were carried out to compare cell wall hydrophobicity, production of antagonistic substances, survival capacity in the gastrointestinal tract of germ-free mice without pathological consequence, and immune modulation by stimulation of Küpffer cells, intestinal sIgA and IL-10 levels. In vitro antagonism against pathogenic bacteria and yeast was only observed for the probiotic bacteria B. animalis and L. casei. The hydrophobic property of the cell wall was higher for B. animalis and E. coli EMO, and this property could be responsible for a better ability to colonize the gastrointestinal tract of germ-free mice. Higher levels of sIgA were observed mainly for S. boulardii, followed by E. coli EMO and B. animalis, and only S. boulardii induced a significant higher level of IL-10. In conclusion, for a probiotic use, S. boulardii presented better characteristics in terms of immunomodulation, and B. animalis and L. casei for antagonistic substance production. The knowledge of the different probiotic properties could be used to choice the better microorganism depending on the therapeutic or prophylactic application.     

Nomenclatural changes resulting from the transfer of tropical African Sarcostemma to Cynanchum ( Apocynaceae : Asclepiadoideae )

Summary  Four species of tropical African Sarcostemma are transferred to Cynanchum together with two subspecies of S. viminale. In addition, Sarcostemma mulanjense is reduced to subspecific rank under C. viminale.     

Probing the Drosophila retinal determination gene network in Tribolium (II): The Pax6 genes eyeless and twin of eyeless

The Pax6 genes eyeless (ey) and twin of eyeless (toy) are upstream regulators in the retinal determination gene network (RDGN), which instructs the formation of the adult eye primordium in Drosophila. Most animals possess a singleton Pax6 ortholog, but the dependence of eye development on Pax6 is widely conserved. A rare exception is given by the larval eyes of Drosophila, which develop independently of ey and toy. To obtain insight into the origin of differential larval and adult eye regulation, we studied the function of toy and ey in the red flour beetle Tribolium castaneum. We find that single and combinatorial knockdown of toy and ey affect larval eye development strongly but adult eye development only mildly in this primitive hemimetabolous species. Compound eye-loss, however, was provoked when ey and toy were RNAi-silenced in combination with the early retinal gene dachshund (dac). We propose that these data reflect a role of Pax6 during regional specification in the developing head and that the subsequent maintenance and growth of the adult eye primordium is regulated partly by redundant and partly by specific functions of toy, ey and dac in Tribolium. The results from embryonic knockdown and comparative protein sequence analysis lead us further to conclude that Tribolium represents an ancestral state of redundant control by ey and toy.