Effects of ammonium feeding on the production of bioactive metabolites (cordycepin and exopolysaccharides) in mycelial culture of a Cordyceps sinensis fungus

AIMS: To examine the effects of ammonium feeding on the production of cordycepin (3'-deoxyadenosine, a nucleoside analogue) and exopolysaccharides (EPS) in mycelial culture of a new Cordyceps sinensis fungus Cs-HK1. METHODS AND RESULTS: Cs-HK1 fungus was cultivated in a liquid medium containing glucose, yeast extract, peptone and a few major inorganic salts. NH(4)Cl was fed to the mycelial culture at various concentrations from 5 to 40 mmol l(-1) on day 3 (during exponential phase). NH(4)Cl, fed at 10 mmol l(-1), stimulated the cordycepin production most significantly, with nearly fourfold increase in the cordycepin content of mycelia (from 28.5 to 117 microg g(-1)), and also increased the EPS production by 40% (from 2.6 to 3.7 g l(-1)). The ammonium feeding had a slightly positive effect at 5-10 mmol l(-1), but a negative effect at higher concentrations on the mycelium growth. Ammonium feeding also caused a sharp drop of the medium pH, owing perhaps to the uptake of NH(3) and the release of H(+) by the fungal cells. CONCLUSIONS: Ammonium feeding to the mycelial culture of Cs-HK1 fungus enhanced the intracellular cordycepin accumulation and the EPS production. The enhanced cordycepin production may be attributed to the uptake of ammonia for nucleoside synthesis, and the enhanced EPS to the increased uptake of glucose for EPS biosynthesis. SIGNIFICANCE AND IMPACT OF THE STUDY: It is useful for the production of bioactive metabolites and for understanding ammonium metabolism and its relationship to the biosynthesis of nucleosides in a precious medicinal fungus.     

Genetic variation of Cordyceps sinensis, a fruit-body-producing entomopathogenic species from different geographical regions in China

Cordyceps sinensis is one of the most valuable medicinal fungi in the Orient. It is naturally distributed in the eastern extension area of the Qinghai-Tibet plateau, at an altitude over 4000 m high. In order to investigate genetic variation and evolutionary relationships of C. sinensis from different geographical regions, 17 isolates of C. sinensis were collected from different provinces and the complete sequences of rDNA ITS were determined. On the basis of 5.8S rDNA and ITS region analysis, it was clearly shown that the ITS sequences within C. sinensis are highly homologous regardless of geographical origin. The distance values between the sequences in this study were lower than 0.03. This implied that C. sinensis from different geographic regions are the same species; they are not different species or a species complex. The results also showed that distance values between C. sinensis and Hirsutella sinensis are of the same order as those within C. sinensis from different geographic regions. This confirmed our previous results that C. sinensis should only have H. sinensis as its asexual stage whatever the geographic region from which the samples were collected. An rDNA ITS clone library was established to obtain further evidence for the interpretation of the fungal community structure from C. sinensis and to confirm the accuracy of the taxonomic identities produced by directly sequencing the rDNA ITS region. The discrimination between intraspecies of C. sinensis might provide additional data for the authentication of medicinal material at the species or intraspecies level.     

南海软海绵Halichondria sp.化学成分研究

为了寻找有生物活性的次生代谢产物,对从采集自中国南海的软海绵(Halichondria sp.)进行了化学成分研究,从中共分离得到了9个化合物,并对部分化合物进行了抗菌活性测试。根据现代波谱技术并结合文献数据,鉴定化合物的结构为:1,2,3,4-四氢-3-羧基-2-卡波林(1),色氨酸(2),环(异亮氨酸-脯氨酸)(3),开环(脯氨酸-缬氨酸)(4),环(丙氨酸-脯氨酸)(5),胆甾醇(6),二-(2-乙基己基)邻苯二甲酸酯(7),邻苯二甲酸正丁异丁酯(8)和邻苯二甲酸二异丁酯(9)。     

蝉虫草（蝉花）来源的化合物鉴定与抗菌活性检测

蝉虫草（蝉花）是我国重要的传统中药材之一,用于治疗慢性肾炎等,但对其活性成分的研究仍不充分。本文采用硅胶柱、凝胶柱、ODS柱分离,以及HPLC等色谱技术,对蝉虫草子实体样品的乙酸乙酯提取物进行分离纯化,得到3个化合物。根据波谱学数据,化合物1-3分别鉴定为(E,E)-9-酮-10,12-十八碳二烯酸(E,E)-9-oxooctadeca-10,12-dienoic acid（1）、麦角甾醇ergosterol（2）和白僵菌酮bassiatin（3）,其中化合物1为首次从蝉虫草中分离获得。抑菌实验表明,3个化合物均对革兰氏阴性大肠杆菌Escherichia coli和革兰氏阳性枯草芽孢杆菌Bacillus subtilis有显著的抑菌活性,化合物1和3还对条件致病真菌白色念珠菌Candida albicans有明显的抑菌活性。本文的研究结果丰富了蝉虫草的活性化合物组成。     

Description of a marine peritrichous ciliate, Pseudovorticella sinensis n. sp. (Ciliophora, Peritrichia) from China

A new marine peritrich ciliate, Pseudovorticella sinensis n. sp. was isolated from a shrimp-farming pond in the littoral area of Qingdao, China. The morphology, infraciliature, and silverline system were studied based on living and silver-impregnated specimens. This species is characterized by (1) an elongated bell-shaped body that measures 50-60 x 35-45 microm in vivo, (2) one large, ventrally located contractile vacuole, and (3) a pellicle covered by a layer of transparent, cortical vesicles. The number of transverse silverlines from the peristomial area to the aboral ciliary wreath is 26-32, and from the aboral ciliary wreath to the scopula is 12-15. The stalk measures about 160-250 microm long x 5-6 microm wide. The spasmoneme has one row of conspicuous thecoplasmic granules, which are about 0.8 microm in diameter.     

Structural elucidation of an exopolysaccharide from mycelial fermentation of a Tolypocladium sp. fungus isolated from wild Cordyceps sinensis

A novel polysaccharide designated EPS-1A with an average molecular weight around 40 kDa was fractionated and purified by anion-exchange and gel-filtration chromatography from the crude exopolysaccharide (EPS) isolated from fermentation broth of Cs-HK1, a Tolypocladium sp. fungus isolated from wild Cordyceps sinensis. The structural characteristics of EPS-1A were determined with various methods (e.g. GC, GC–MS, FT-IR, ¹H NMR and ¹³C NMR) and through acid hydrolysis, methylation, periodate-oxidation and Smith degradation. The results suggested that EPS-1A was composed of glucose, mannose and galactose at 15.2:3.6:1.0 M ratio. EPS-1A was a slightly branched polysaccharide and its backbone was composed of (1 → 6)-α-d-glucose residues (77%) and (1 → 6)-α-d-mannose residues (23%). Branching occurred at O-3 position of (1 → 6)-α-d-mannose residues of the backbone with (1 → 6)-α-d-mannose residues and (1 → 6)-α-d-glucose residues, and terminated with β-d-galactose residues.     

不同产地冬虫夏草及不同厂家虫草发酵菌丝的可溶性蛋白比较

冬虫夏草〔Ｃｏｒｄｙｃｅｐｓｓｉｎｅｎｓｉｓ (Ｂｅｒｋ .)Ｓａｃｃ.〕为我国滋补名贵出口中药 ,主产于青海、西藏、四川、云南、甘肃等省。因野生资源有限 ,严重脱销。为解决药源问题 ,近年来我国已开发出了虫草菌丝体发酵产品[1] 。由于虫草系真菌寄生在昆虫体上的复合体     

刺孢吸水链霉菌发酵液的化学成分及抗炎活性研究

研究一株刺孢吸水链霉菌(Streptosporangium hygroscopicus n.sp.)发酵液的化学成分及其抗炎活性。应用多种色谱方法进行分离和纯化,并用NMR、IR和MS等方法解析其结构;然后采用叉菜胶所致大鼠足趾肿胀模型对所得化合物的抗炎活性进行了考察。最终从刺孢吸水链霉菌代谢产物中分离得到5个单体化合物,它们的结构分别为芒柄花素(1),N-乙酰基-2-(4-羟苯基)乙胺(2),阿魏酸(3),3,4,5-三甲基-1,2-苯二酚(4),7,3’-二羟基-4’-甲氧基黄酮(5)。化合物1、2、4和5在15 mg/(kg·d)时,对交叉菜胶所致大鼠足趾肿胀的抑制率分别为47.3%、16.8%、13.6%和50.4%。化合物1、4和5首次从该菌中分离得到;化合物1和5具有较强的抗炎作用。     

Hypocreaterpenes A and B,cadinane-type sesquiterpenes from a marine-derived fungus,Hypocreales sp.

Two new cadinane-type sesquiterpenes, hypocreaterpenes A (1) and B (2), along with five known compounds (3–7) were isolated from a marine-derived fungus Hypocreales sp. strain HLS-104 isolated from a sponge Gelliodes carnosa. Their structures were determined by a combination of spectroscopic methods. All compounds were tested for the inhibitory effects on the nitric oxide (NO) production in lipopolysaccharide (LPS)-treated RAW264.7 cells. Among them, compounds 3 and 6 showed moderate anti-inflammatory activity with average maximum inhibition (E_max) values of 10.22% and 26.46% at 1 μM, respectively.     

Fractionation,characterization and antioxidant activity of exopolysaccharides from fermentation broth of a Cordyceps sinensis fungus

Exopolysaccharide (EPS) fractions P_1/5, P_2/5, P₁, P₂ and P₅ were isolated from the fermentation medium of a medicinal fungus Cordyceps sinensis by gradient precipitation with ethanol at 1/5, 2/5, 1, 2, and 5 volume ratios to the liquid medium. P_1/5 and P_2/5 were mainly composed of polysaccharides with negligible protein content and a large molecular or particle size (intrinsic viscosity [η] 2025 mL/g; hydrodynamic radius R_h 905 nm). The fraction attained at a higher ethanol volume ratio had a much higher protein and lower carbohydrate content, and a smaller molecular size. In particular, P₅ was composed of mainly protein with an average molecular weight 16 kDa, [η] 4.3 mL/g and R_h 23.5 nm. The antioxidant activities of EPS fractions showed a significant dependence on the protein content, being negligible of P_1/5 and P_2/5, low to moderate of P₁ and P₂, and very strong of P₅. Gradient ethanol precipitation was proven a simple and workable method for initial fractionation of polysaccharides, proteins and their complexes with different molecular sizes and for further identification of the bioactive components.     

Isolation and characterization of a mannoglucan from edible Cordyceps sinensis mycelium

Cordyceps sinensis is a well known tonic food or invigorant with broad-spectrum medicinal properties that is widely used in the People's Republic of China. A neutral mannoglucan 1 with a molar mass of approximately 7.7x10(3) Da was obtained from the 0.05 M acetate buffer extract of C. sinensis mycelium. It had [alpha](D)(20)+126 (c 0.2, H(2)O) and consisted of Man and Glc units in the molar ratio of 1:9. A combination of chemical analysis and NMR and IR spectroscopy together with digestion with alpha-D-amylase showed 1 to have a alpha-D-glucan backbone with (1-->4)- and (1-->3)-linkages, and the side chains of alpha-D-(1-->6)-Manp were attached to the backbone via O-6 of alpha-(1-->3)-Glcp residues. Compound 1 showed weak cytotoxicity activity against SPC-I (IC(50)=63 microg/mL) cancer line, and no obvious cytotoxicity activities against BCAP37 (IC(50)>100 microg/mL) and SW480 (IC(50)>100 microg/mL) cancer lines.     

A novel endophytic Huperzine A–producing fungus,Shiraia sp. Slf14, isolated from Huperzia serrata

Aims: To characterize and identify a novel Huperzine A (HupA)‐producing fungal strain Slf14 isolated from Huperzia serrata (Thunb. ex Murray) Trev. in China. Methods and Results: The isolation, identification and characterization of a novel endophytic fungus producing HupA specifically and consistently from the leaves of H. serrata were investigated. The fungus was identified as Shiraia sp. Slf14 by molecular and morphological methods. The HupA produced by this endophytic fungus was shown to be identical to authentic HupA analysed by thin layer chromatographic, High‐performance liquid chromatography (HPLC), LC‐MS, ¹H NMR and acetylcholinesterase (AChE) inhibition activity in vitro. The amount of HupA produced by Shiraia sp. Slf14 was quantified to be 327·8 μg l^?1 by HPLC, which was far higher than that of the reported endophytic fungi, Acremonium sp., Blastomyces sp. and Botrytis sp. Conclusions: The production of HupA by endophyte Shiraia sp. Slf14 is an enigmatic observation. It would be interesting to further study the HupA production and regulation by the cultured endophyte in H. serrata and in axenic cultures. Significance and Impact of the Study: Although the current accumulation of HupA by the endophyte is not very high, it could provide a promising alterative approach for large‐scale production of HupA. However, further strain improvement and the fermentation process optimization are required to result in the consistent and dependable production.     

飞机草地上部分化学成分及其抑菌活性研究

为探究飞机草地上部分化学成分及其抑菌作用.采用正相硅胶、Sephadex LH-20等色谱方法,从飞机草乙酸乙酯提取物中得到10个化合物,根据1H NMR、13C NMR、MS波谱数据,分别鉴定为:5,6,7,4'-四甲氧基黄烷酮(1)、圣草素-7,4'-二甲醚(2)、5,7-二羟基-6,4'-二甲氧基黄酮(3)、二氢...     

土壤真菌 Aspergillus fumigatus固体发酵产物的化学成分及抗氧化活性研究

为研究来源于哀牢山国家级自然保护区河谷的土壤真菌Aspergillus fumigatus固体发酵产物的化学成分及抗氧化活性研究,采用正相硅胶柱层析、反相硅胶柱层析和Sephadex LH-20分离纯化,借助核磁共振波谱等方法鉴定化合物结构,从中共得到13个化合物,分别鉴定为rubrofusarin B(1)、rubrofusarin A(2)、carbonarone A(3)、aspernigrin A(4)、flavasperone(5)、(22 E,24 R)-5α,8α-过氧麦角甾-6,22-二烯-3β-醇(6)、(22 E,24 R)-5α,8α-过氧麦角甾-6,9(11),22-三烯-3β-醇(7)、ourosperone A(8)、(22 E,24 R)-麦角甾-5,7,22-三烯-3β-醇(9)、stigmast-1,5-dien-3β-ol(10)、fonsecinones A(11)、asperpyrone C(12)、asperpyrones B(13)。其中,化合物1~5和7~13为从该菌种中首次分离。抗氧化活性结果显示,化合物8对DPPH(IC₅₀=3.453 mg/mL)、ABTS+(IC₅₀=0.155 mg/mL)、OH(IC₅₀=0.019 mg/mL)自由基都有一定的清除效果。     

Isolation,structural identification and herbicidal activity of N-phenylpropanamide from Streptomyces sp. KA1-3

The secondary metabolites produced by Streptomyces sp. KA₁-3, cultured on starch casein broth, was extracted by ethyl acetate and concentrated. Purification of the compound by thin layer chromatography lead to isolation of N-phenylpropanamide from one polar fraction. The structure of the herbicidal compound was elucidated on the basis of UV, FT-IR, mass and H¹ NMR spectroscopy. The herbicidal activity of the isolate was tested against the weeds Cassia occidentalis L. and rhizome Cyperus rotundus L. by moist chamber technique and rolled towel paper assay method. Herbicidal activity of the bioactive compound N-phenylpropanamide was further evaluated under in vitro condition. The herbicidal compound showed 80% of seed germination inhibition in C. occidentalis L. and rhizome C. rotundus L. weed. The actinobacterium can be used as a source for bioherbicidal agent.     

番石榴叶化学成分分离鉴定及psiguadial D的抗癌活性研究

本文对番石榴Psidium guajava叶的化学成分进行分离鉴定并对部分化合物抗肿瘤活性进行评价。采用正相硅胶、Sephadex LH-20、MCI柱色谱和制备液相色谱等方法对番石榴叶甲醇提取物的化学成分进行分离纯化,运用现代波谱技术鉴定了13个化合物,分别为4,5-diepipsidial A(1)、psidial A(2)、guajadial(3)、psiguadial A(4)、psiguadial D(5)、α-生育酚(6)、亚油酸(7)、槲皮素-3-O-β-D-吡喃木糖苷(8)、槲皮素-3-O-α-L-吡喃阿拉伯糖苷(9)、槲皮素-3-O-α-L-呋喃阿拉伯糖苷(10)、β-谷甾醇(11)、儿茶素(12)、没食子酸(13)。其中化合物1为首次从天然产物中分离得到。化合物5对人体肿瘤细胞株HL-60、SMMC-7721、A-549、MCF-7、SW-480均显示出较为明显的抑制活性,半数抑制浓度为4.08~11.23μmol/L。     

土壤和植物对冬虫夏草寄主昆虫规模化饲养的影响

冬虫夏草大量人工培植的关键之一是寄主昆虫的规模化饲养,本文对西藏林芝地区冬虫夏草生境中不同土壤基质在室内对蝙蝠蛾幼虫生长的影响以及寄主幼虫对5种植物的取食行为、嗅觉反应进行了系统研究。结果表明蝙蝠蛾幼虫偏好土质疏松、渗透性好的土壤,土壤因子中湿度和有机质含量与蝙蝠蛾幼虫存活率成正相关。蝙蝠蛾3龄幼虫对适生地植物的取食选择性与嗅觉反应趋性顺序为鹅绒委陵菜>珠芽蓼>小大黄>圆穗蓼>羊角天麻;进一步Spearman相关分析显示,幼虫对各植物的选择系数与可溶性糖（R=0.850,P<0.05）和粗蛋白（R=0.898,P<0.05）都存在显著的正相关关系,而与粗纤维（R=-0.952,P<0.05）有着显著的负相关关系,与粗灰分（R=-0.391,P=0.516）之间没有显著的相关关系;取食鹅绒委陵菜、珠芽蓼后的幼虫单头虫重显著大于取食其他植物的幼虫。这一研究结果为冬虫夏草寄主昆虫规模化饲养提供了理论依据。     

Pullulanases of alkaline and broad pH range from a newly isolated alkalophilicBacillus sp. S-1 and aMicrococcus sp. Y-1

Summary Two highly alkalophilic bacteria, and potent producers of alkaline pullulanase, were isolated from Korean soils. The two isolates, identified asBacillus sp. S-1 andMicrococcus sp. Y-1, grow on starch under alkaline conditions and effectively secrete extracellular pullulanases. The two isolates were extremely alkalophilic since bacterial growth and enzyme production occurred at pH values ranging from pH 6.0 to 12.0 forMicrococcus sp. Y-1 and pH 6.0 to 10.0 forBacillus sp. S-1. Both strains secrete enzymes that possess amylolytic and pullulanolytic acitivities. Extracellular crude enzymes of both isolates gave maltotriose as the major product formed from soluble starch and pullulan hydrolysis. Compared to other alkalophilic microbes such asMicrococcus sp. (0.57 units ml^–1),Bacillus sp. KSM-1876 (0.56 units ml^–1) andBacillus No. 202-1 (1.89 units ml^–1) these isolates secreted extremely high concentrations (7.0 units ml^–1 forBacillus sp. S-1 and 7.6 units ml^–1 forMicrococcus sp. Y-1) of pullulanases in batch culture. The pullulanase activities from both strains were mostly found in the culture medium (85–90%). The extracellular enzymes of both bacteria were alkalophilic and moderately thermoactive; optimal activity was detected at pH 8.0–10.0 and between 50 and 60°C. Even at pH 12.0, 65% of original Y-1 pullulanase activity and 10% of S-1 pullulanase activity remained. The two newly isolated strains had broad pH ranges and moderate thermostability for their enzyme activities. These result strongly indicate that these new bacterial isolates have potential as producers of pullulanases for use in the starch industry.     

The specificity of 1-naphthol oxygenases from three bacterial isolates, Pseudomonas spp. (NCIB 12042 and 12043) and Rhodococcus sp. (NCIB 12038) isolated from garden oil

Abstract Three bacterial isolates which appeared to use the insecticide, carbaryl (1-naphthyl, N -methyl-carbamate) as their sole carbon and nitrogen sources were originally selected from garden soil. Only one isolate, Pseudomonas sp. (NCIB 12043) could metabolise carbaryl rapidly to 1-naphthol and methylamine. The other two isolates, Pseudomonas sp. (NCIB 12042) and Rhodococcus sp. (NCIB 12038) relied on slow chemical hydrolysis of carbaryl to 1-naphthol and methylamine. All three isolates used 1-naphthol as their sole carbon source; however, their ability to use naphthalene and a range of mono- and dihydroxy-substituted naphthalene compounds varied. NCIB 12038 and NCIB 12043 showed little or no growth on naphthalene, 2,3-dihydroxynaphthalene or 1,3-dihydroxynaphthalene as sole carbon sources and their 1-naphthol oxygenases had little activity with these substrates. In contrast, NCIB 12042 could use these compounds as sole carbon sources and its 1-naphthol oxygenase also showed activity with them. We conclude that 1-naphthol oxygenase from NCIB 12042 is a relatively non-specific dioxygenase, whereas the 1-naphthol oxygenases from NCIB 12038 and NCIB 12043 are relatively specific monooxygenases requiring hydroxylated naphthalene compounds as substrates.