Untransformed root cultures of Nothapodytes foetida and production of camptothecin

Untransformed root cultures of Nothapodytes foetida were established from immature zygotic embryos on MS basal medium supplemented with different concentration of growth regulators. Alkaloid contents in untransformed root cultures showed that root elongation and growth regulators accompanied product synthesis. Basal medium supplemented with NAA and BA achieved maximum number of elongated roots. Maximum concentration of camptothecin (0.01% DW) and 9-methoxy camptothecin (0.0016% DW) were synthesised by untransformed root cultures incubated on MS medium supplemented with NAA (71.36 M) and BA (8.87 M). Culture medium containing NAA (71.36 M) and Kn (9.29 M) proliferated callus interspersed with roots which synthesised camptothecin (0.00017% DW) and 9-methoxy-camptothecin (0.000058% DW).     

Camptothecine from callus cultures of Nothapodytes foetida

Callus cultures were established from excised embryos of Nothapodytes foetida on Murashige and Skoog medium supplemented with picloram (2 mg l^–1) and 3% (w/v) sucrose. The embryos developed into callus after 4 weeks of incubation at 25 ± 2°C in dark. The cultures produced camptothecine and 9-methoxy-camptothecine as determined by TLC, UV, HPLC, electron spray mass spectral analysis.     

Heterologous overexpression of Nothapodytes foetida strictosidine synthase enhances levels of anti-cancer compound camptothecin in Ophiorrhiza rugosa

Plant Cell, Tissue and Organ Culture (PCTOC) - Nothapodytes foetida, an endangered tree of Indian origin, is a major source of the anti-cancer monoterpenoid indole alkaloid, camptothecin (CPT)....     

Influence of medium composition on callus induction and camptothecin(s) accumulation in Nothapodytes foetida

Camptothecin(s) production was examined in callus cultures derived from cotyledons of Nothapodytes foetida (Weigh) Sleumer. The calluses were grown on various combinations of Murashige and Skoog's basal media supplemented with auxins 2,4-dichlorophenoxyacetic acid (2,4-D), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), a-napthalene acetic acid (NAA) and indole-3-acetic acid (IAA) with 6-benzyl aminopurine (BA)/kinetin in different concentrations. The presence of camptothecin (CPT) and 9-methoxycamptothecin (9-OMeCPT) were analyzed by HPLC in relation to the media composition. Hyper production of CPT(1.306% on dry wt. basis) was observed with a combination of 2,4-D with BA and 2,4,5-T and NAA in 1-month-old callus.     

An endophytic <Emphasis Type="Italic">Neurospora</Emphasis> sp. from <Emphasis Type="Italic">Nothapodytes foetida</Emphasis> producing camptothecin

The medicinal plant, Nothapodytes foetida, contains a number of important alkaloids like camptothecin (an anticancer drug molecule), but its concentration is less to meet the existing demand of this important molecule, in an effort for accessible availability of camptothecin. An endophyte (designated ZP5SE) was isolated from the seed of Nothapodytes foetida and was examined as a potential source of anticancer drug lead compound, i.e., camptothecin, when grown in Sabouraud liquid culture media under shake flask conditions. The presence of an anticancer compound (camptothecin) in this fungus was confirmed by chromatographic and spectroscopic methods in comparison with authentic camptothecin. Isolated endophyte (Neurospora crassa) producing camptothecin may become an easily accessible source for the production of a precursor anticancer drug molecule in the future at a large scale. Published in Russian in Prikladnaya Biokhimiya i Mikrobiologiya, 2008, Vol. 44, No. 2, pp. 225–231. The text was submitted by the authors in English.     

An endophytic/pathogenic Phoma sp. from creosote bush producing biologically active volatile compounds having fuel potential

A Phoma sp. was isolated and characterized as endophytic and as a pathogen of Larrea tridentata (creosote bush) growing in the desert region of southern Utah, USA. This fungus produces a unique mixture of volatile organic compounds (VOCs), including a series of sesquiterpenoids, some alcohols and several reduced naphthalene derivatives. Trans-caryophyllene, a product in the fungal VOCs, was also noted in the VOCs of this pungent plant. The gases of Phoma sp. possess antifungal properties and is markedly similar to that of a methanolic extract of the host plant. Some of the test organisms with the greatest sensitivity to the Phoma sp. VOCs were Verticillium, Ceratocystis, Cercospora and Sclerotinia while those being the least sensitive were Trichoderma, Colletotrichum and Aspergillus. We discuss the possible involvement of VOC production by the fungus and its role in the biology/ecology of the fungus/plant/environmental relationship with implications for utilization as an energy source.     

贵州马比木内生真菌的多样性研究

为了解贵州省马比木内生真菌的多样性,通过采集贵阳修文、遵义播州、铜仁万山3个县区的不同季节的健康马比木植物不同组织部位样本进行内生真菌的分离,分析分离菌ITS rDNA序列,采用分子系统学方法,对3个地区的不同季节马比木植物不同组织部位的内生真菌进行鉴定、归类,并进行多样性评价。结果表明,从1 444个马比木组织块中分离出1 037株内生真菌,分别隶属于30个属,其中间壳座属Diaporthe为优势属,分离率与分离频率分别为40.24%与57.58%;夏季内生真菌的多样性指数最高,4个季节的相似度在0.40-0.71之间,贵阳的相似性指数最高,三地的相似性指数在0.42-0.55之间,果实的多样性指数最高,各个组织部位的相似性指数在0.00-0.54之间。表面植株生长环境及部位对内生真菌的组成和多样性均有影响。     

Effect of thidiazuron on adventitious shoot regeneration from seedling explants of Nothapodytes foetida

Summary Regeneration of adventitious shoots from the medicinal plant Nothapodytes foetida (Weight) Sleumer Syn. Mappia foetida (family Ieacinaceceae) has been achieved using different seedling explants. Direct, regeneration of shoot buds was observed in Murashige and Skoog's (MS) basal medium supplemented with various concentrations of thidiazuron. The optimum levels of thidiazuron concentrations were 0.91–4.45 μM. Leaf explants formed more shoots followed by hypocotyls or cotyledons. The shoot buds elongated and rooted on MS basal medium with N⁶-benzyladenine (0.88–2.22 μM) and indole-3-butyric acid (0.49 μM).     

9-methoxycamptothecin from Nothapodytes foetida induces apoptosis in murine sarcoma S180 cells

9-Methoxycamptothecin (MCPT) was found to have antitumour activities through topoisomerase inhibition. However, the type of cell death induced in the tumour cells treated with MCPT was not elucidated. In this study, MCPT and camptothecin were isolated from Nothapodytes foetida distributed in Hubei Province, China and identified by NMR spectroscopy. MCPT was tested by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay using camptothecin as reference. Annexin V-FITC/propidium iodide double staining and real-time PCR were also performed. The IC50 value was (0.385 +/- 0.08) microM. The apoptosis rates increased from 9.5% to 17.27%, 30.14%, and 66.46% with an increase in MCPT concentrations from 0, 0.19, 0.38, to 0.95 microM, respectively. The ratio of Bax/Bcl-2 also increased from 1 to 1.61, 2.43, and 4.57, respectively. Bax and Bcl-2 are crucial to the mitochondria pathway. The results indicate that the mitochondria pathway may be involed in MCPT-induced murine sarcoma S180 apoptosis.     

Secondary metabolites from endophytic Streptomyces sp. Lz531

From the branch tissue of Maytenus hookeri, the endophytic strain Lz531 was isolated, and determined to belong to Streptomyces, according to its 16S rRNA sequence. From the extracts of the fermentation broth of Streptomyces sp. Lz531, two new and four known compounds were isolated. The two new compounds were identified as cyclo(L-Pro-L-Val-L-Val) (1) and 13-methyl-N-(2-phenylethyl)tetradecanamide (2).     

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.     

Furoic acid derivatives from the endophytic fungus Coniothyrium sp.

The endophytic fungus Coniothyrium sp. was isolated from leaves of Quercus robur. Fermentation of this fungus on solid rice medium yielded two new furoic acid derivatives ( 1 and 2 ) and two additional known compounds. The structures of the new compounds were determined by extensive analysis of 1D and 2D nuclear magnetic resonance spectra as well as high-resolution mass spectrometry data. Compound 1 , containing three aromatic chromophores attached by rotatable sigma bonds and a chirality center in benzylic position, was found to be a scalemic mixture with an excess of the (S) enantiomer, the absolute configuration of which was elucidated as by the solution time-dependent density functional theory-electronic circular dichroism approach. The ωB97X/TZVP PCM/MeCN and SOGGA11-X/TZVP SMD/MeCN methods were used for geometry reoptimization to reproduce the solution conformational ensemble. All isolated compounds were tested for their cytotoxicity but proved to be inactive.     

Metabolites from the endophytic mitosporic Dothideomycete sp. LRUB20

The endophytic mitosporic Dothideomycete sp. LRUB20 was found to produce pyrone derivatives, dothideopyrones A-D (1, 3, 4, and 5), together with seven known compounds, including questin (9), asterric acid (10), methyl asterrate (11), sulochrin (12), and eugenitin (13), 6-hydroxymethyleugenitin (14), and cis, trans-muconic acid (15). Dothideopyrone D (5) and its acetate derivative 6 exhibited moderate cytotoxic activity. This is the first report on a naturally occurring muconic acid, which is commonly known as a biomarker in environments after exposure to benzene and phenol (or derivatives). Interestingly, the LRUB20 fungus could produce muconic acid in relatively high yield (47.8 mg/L). The utility of endophytic fungi in the field of white biotechnology is discussed.     

Antifungal secondary metabolites from endophytic Verticillium sp.

Endophytes were isolated from roots of wild Rehmannia glutinosa to screen the strains with antifungal metabolites. A strain identified as Verticillium sp. was selected for chemical and biological investigations because of the strong antifungal activity of the crude extract against Pyricularia oryzae P-2b. Chemical investigations of culture broth afforded three compounds: 2,6-dihydroxy-2-methyl-7-(prop-1E-enyl)-1-benzofuran-3(2H)-one, massariphenone and ergosterol peroxide. The metabolites were isolated by silica gel and Sephadex LH-20, 2,6-dihydroxy-2-methyl-7-(prop-1E-enyl)-1-benzofuran-3(2H)-one was reported for the first time and the chemical structure was established following the analysis of NMR, UV, IR, MS data. 2,6-Dihydroxy-2-methyl-7-(prop-1E-enyl)-1-benzofuran-3(2H)-one and ergosterol peroxide displayed clear inhibition of the growth of three pathogens as well as Verticillium sp.     

Dihydronaphthalenones from the endophytic fungus Botryosphaeria sp. BCC 8200

Five new dihydronaphthalenones (1, 2a/2b, 3a/3b), four of which were isolated as two inseparable mixtures of isomers, together with two known compounds, 7-hydroxy-2-(2-hydroxypropyl)-5-methylchromone (4) and siderin (5), were identified as secondary metabolites of the endophytic fungus Botryosphaeria sp. BCC 8200. The structures were elucidated by interpretation of NMR spectroscopic and mass spectrometry data. Compounds 2a/2b, 3a/3b, and 5 exhibited weak cytotoxic activities against cancer cell lines.     

Metabolites from the endophytic fungus Xylaria sp. PSU-D14

Glucoside derivatives, xylarosides A (1) and B (2), were isolated from the broth extract of the endophytic fungus Xylaria sp. PSU-D14 along with two known compounds, sordaricin (3) and 2,3-dihydro-5-hydroxy-2-methyl-4H-1-benzopyran-4-one (4). The structures were assigned by spectroscopic methods. Sordaricin (3), one of the known metabolites, exhibited moderate antifungal activity against Candida albicans ATCC90028 with a MIC value of 32 microg/ml.     

Metabolites from the endophytic fungus Phomopsis sp. PSU-D15

From the endophytic fungus Phomopsis sp. PSU-D15, three metabolites named as phomoenamide (1), phomonitroester (2) and deacetylphomoxanthone B (3), were isolated together with three known compounds, dicerandrol A (4), (1S,2S,4S)-p-menthane-1,2,4-triol (5) and uridine. Their structures were elucidated by spectroscopic methods. Phomoenamide (1) exhibited moderate in vitro antimycobacterial activity against Mycobacterium tuberculosis H37Ra.     

Benzophenone derivatives from the plant endophytic fungus,Pestalotiopsis sp.

Two new (1–2) and one known (3) benzophenone derivatives, along with five known ambuic acid analogues (4–8) were isolated from the solid cultures of a Pestalotiopsis sp. Compound 2 represented both enantiomeric and atropisomeric isomers, and the absolute configurations of enantiomers [(−)-2 and (+)-2] were determined by electronic circular dichroism (ECD) calculations. All the isolates were evaluated for their antimicrobial and cytotoxic activities. Chlorinated compounds 2 and 3 showed potent antimicrobial activities against four pathogenic bacteria, and compound 3 also displayed strong antifungal activity against Candida glabrata (ATCC 90030) with an MIC₅₀ value of 2.6 ± 0.1 μg/mL. Compound 1 exhibited moderate cytotoxicity against U2OS and MCF-7 with IC₅₀ values of 11.6 and 16.8 μM, respectively.     

Antitumor anthraquinones from an endophytic actinomycete Micromonospora lupini sp. nov

Two novel anthraquinones, lupinacidins A (1) and B (2), have been isolated from the culture broth of a new endophytic actinomycete belonging to the genus Micromonospora. Lupinacidins were found to show significant inhibitory effects on the invasion of murine colon 26-L5 carcinoma cells without inhibiting cell growth.     

New p-terphenyls from the endophytic fungus Aspergillus sp. YXf3

Five new p-terphenyls named prenylterphenyllin D (1), prenylterphenyllin E (2), 2′-O-methylprenylterphenyllin (3), 4-O-methylprenylterphenyllin (4) and 3′-O-methylterphenyllin (5) together with seven known compounds (6–12), were isolated from cultures of Aspergillus sp. YXf3. The structures of the new compounds were elucidated by extensive MS and NMR analyses. The NMR and MS data of 5 is reported for the first time, as its structure was listed in SciFinder Scholar with no associated reference. Compounds 6 and 7 were distinguished from each other on the basis of 2D NMR experiments. Compounds 1, 2, 3 and 8 showed antibacterial activities against X. oryzae pv. oryzicola Swings and E. amylovora with the same MIC values of 20 μg/mL while 10 exhibited activities against E. amylovora with an MIC value of 10 μg/mL.