Stackebrandtia endophytica sp. nov., an actinobacterium isolated from Tripterygium wilfordii.

A novel endophytic actinobacterium, designated strain YIM 64602T, was isolated from healthy stems of Tripterygium wilfordii. It grew at 15-40 °C, pH 6.0-9.0 and in the presence of 0-3 % (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequence showed that strain YIM 64602T belongs to the genus Stackebrandtia. Whole-cell hydrolysates of strain YIM 64602T contained the amino acid meso-diaminopimelic acid with the sugars mannose, rhamnose and glucose, and a trace of ribose. The major polar lipids were diphosphatidylglycerol, phosphatidylmethylethanolamine and phosphatidylethanolamine. MK-10(H6), MK-10(H4) and MK-11(H4) were the predominant components in the quinone system. The fatty-acid pattern was mainly composed of the saturated branched-chain acids iso-C16 : 0, anteiso-C17 : 0, iso-C15 : 0 and iso-C17 : 0. The DNA G+C content was 72.4 mol%. 16S rRNA gene sequence analysis showed the highest pairwise sequence identity (96.0-98.5 %) with the members of the genus Stackebrandtia. Strain YIM 64602T displayed a DNA-DNA relatedness of 43.9±0.4 % with the type strain Stackebrandtia albiflava YIM 45751T. Based on evidence from this polyphasic study, strain YIM 64602T ( = BCRC 16954T = DSM 45928T) is considered to represent a novel species of the genus Stackebrandtia, for which the name Stackebrandtia endophytica is proposed.

Effects of plant stress signal molecules on the production of wilforgine in an endophytic actinomycete isolated from Tripterygium wilfordii Hook.f.

The endophytic actinomycete F4-20 was isolated from Tripterygium wilfordii Hook.f. and was confirmed to produce wilforgine, a secondary metabolite discovered in its host. F4-20 showed a close phylogenetic relationship to Streptomyces species. To seek elicitors that may enhance the production of wilforgine in F4-20, four plant stress molecules were applied to the in vitro liquid cultures. Results showed that methyl jasmonate (MeJA), salicylic acid (SA), and hydrogen peroxide (H2O2) inhibited bacterial growth, whereas glutathione (GSH) treatment significantly increased bacterial growth. The wilforgine contents in the mycelia of F4-20 were reduced by MeJA and GSH but were induced by SA and H2O2. When added in the end of the culture period (7 day), 1 mM SA and 5 mM H2O2 resulted in 69.35 ± 1.71 and 71.80 ± 3.35 µg/g DW of wilforgine production, 1.55 and 1.60 fold to that of control (44.83 ± 1.35 µg/g DW), respectively. Though this improved production was about 6.5 times lower than that of the natural root (454.00 µg/g dry root bark), it provided an alternative method for the production of valuable plant secondary metabolites.

Nonomuraea rhizophila sp. nov., an actinomycete isolated from rhizosphere soil.

A novel actinomycete, designated strain YIM 67092(T), was isolated from rhizosphere soil of the perennial vine Tripterygium wilfordii Hook. f. collected from Yunnan province, South-west China. The strain formed well differentiated aerial and substrate mycelia and grew in the presence of up to 7 % (w/v) NaCl. Phylogenetic analysis based on the 16S rRNA gene showed that strain YIM 67092(T) belonged to the genus Nonomuraea, with highest sequence similarity to Nonomuraea rosea GW 12687(T) (99.0 %). Sequence similarities between strain YIM 67092(T) and other species of the genus Nonomuraea ranged from 97.8 % (Nonomuraea dietziae DSM 44320(T)) to 93.8 % (Nonomuraea kuesteri GW 14-1925(T)). Key morphological, physiological and chemotaxonomic characteristics of strain YIM 67092(T) were congruent with the description of the genus Nonomuraea. The G+C content of the genomic DNA was 69.3 mol%. Based on comparative analysis of physiological, biochemical and chemotaxonomic data, including low DNA-DNA hybridization results, strain YIM 67092(T) represents a novel species of the genus Nonomuraea, for which the name Nonomuraea rhizophila sp. nov. is proposed. The type strain is YIM 67092(T) ( = CCTCC AA 209044(T)  = DSM 45382(T)).

New immunosuppressive secondary metabolites from the endophytic fungus Aspergillus sp.

Six new metabolites, including two diphenolic derivatives (1 and 2), one pseurotin (3), one butenolide derivative (4), one benzopyran (5) and one isochromane lactone (6), together with ten known compounds (7-16) were isolated from an endophytic fungus Aspergillus sp. Their planar structures and absolute configurations were established based on techniques of MS, NMR, IR, UV, [Rh2(OCOCF3)4] complex-induced ECD, quantum chemical electronic circular dichroism (ECD) calculations, and single crystal X-ray diffraction. Structurally, compound 2 represents the first example of diphenolic derivative possessing an unusual 1-oxaspiro[2.4]heptane core bearing a 5/3 bicyclic skeleton; compound 3 represents the first example of pseurotin type natural products that only one hydroxy group is substituted at side chain. In bioassay, compounds 3, 7 and 8 exhibited potential inhibitory effect on the proliferation of anti-CD3/anti-CD28 monoclonal antibodies (mAbs) induced murine T cells, with IC50 values of (7.81 ± 0.71), (8.25 ± 0.78) and (8.84 ± 0.81) µM, respectively.

Terrusnolides A-D, new butenolides with anti-inflammatory activities from an endophytic Aspergillus from Tripterygium wilfordii.

Terrusnolides A-D (1-4), four butenolides were isolated from an endophytic Aspergillus from Tripterygium wilfordii. The structures of 1-4 were established by comprehensive spectroscopic analyses and electronic circular dichroism (ECD) calculation. It is interesting that 1 was a butenolide derived by a triple decarboxylation, while 2-4 were the metabolites with 4-benzyl-3-phenyl-5H-furan-2-one motif possessing an isopentene group fused to the benzene ring. In vitro anti-inflammatory effects of these isolates were evaluated in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. 1-4 exhibited excellent inhibitory effects on the production of interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α), and nitric oxide (NO) in LPS-induced macrophages, comparable with the positive control (indomethacin). Those results indicated that, terrusnolides A-D might serve as new potential natural remedies for the treatment of inflammation.

A new wortmannine derivative from a Tripterygium wilfordii endophytic fungus Talaromyces wortmannii LGT-4.

A new wortmannine derivative named wortmannine E (1) was isolated from Talaromyces wortmannii LGT-4, an endophytic fungus of Tripterygium wilfordii. Its structure was established by 1D and 2D NMR spectra.

A new furanosteroid from Talaromyces sp. lgt-4, a fungal endophyte isolated from Tripterygium wilfordii.

Wortmannolol (1), a new furanosteroid, along with five known compounds, wortmannolone (2), ergosterol (3), p-hydroxyphenyl ethanol (4), trans-6-dodecene (5), (2Z, 4E) -5-(8-hydroxy-1,5-dimethyl-3-oxo-6-oxabicyclo [3.2.1] octan-8-yl) -3-methylpenta-2,4-dienoic acid (6) were isolated from a fungal endophyte Talaromyces sp. lgt-4. Their structures were elucidated by IR, MS, 1D and 2D NMR spectra. Compound 1 show weak monoamine oxidase inhibitory activity.

Immunomodulatory compounds from Pestalotiopsis leucothës, an endophytic fungus from Tripterygium wilfordii.

The immunomodulatory effects of three compounds designated BS, GS, and YS produced by Pestalotiopsis leucothës, an endophytic fungus isolated from Tripterygium wilfordii, were evaluated. The 50% inhibition concentration (IC50) value of BS in the proliferative assay with various stimulating agents such as phytohemagglutinin-M (PHA-M), phorbol myristate acetate (PMA)/ionomycin, mixed lymphocyte reaction (MLR) and poke weed mitogen (PWM) was 0.35, 1.6, 0.8 and 5.4 microg/ml, respectively. In addition, BS significantly inhibited the production of cytokines such as interleukin (IL)-1beta, IL-2, interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha, by peripheral blood mononuclear cells (PBMNC) and soluble IL-2 receptor expression at concentrations greater than 1 microg/ml. Inhibition of PHA stimulated PBMNC proliferation and IL-2 and sIL-2R production by BS indicates that it is a T-cell specific immunosuppressant. However, BS also moderately inhibited immunoglobulin (Ig) G and M at concentrations greater than 1 mug/ml suggesting that it also has B cell immunosuppressive effects. YS was 10% less active than BS in all assay systems. In contrast, GS exhibited both suppression and enhancement of PBMNC proliferation in the presence of various stimulants. However, GS inhibited PWM stimulated PBMNC proliferation and IL-4 and IgG and IgM production at concentrations above 1 mug/ml. All three fungal compounds altered the percentage of T-lymphocyte subpopulations only at high concentrations. Cell viability was not affected at the immunosuppressive concentrations of these compounds. In conclusion, work from our laboratory has identified three potentially potent immunomodulatory compounds from P. leucothës. These compounds have variable effects on T- and B-cells and monocytes. They may partially explain the immunosuppressive activity of T. wilfordii. In addition, they may represent a new source of immunomodulatory compounds for the treatment of human immune mediated diseases.

Penifupyrone, a new cytotoxic funicone derivative from the endophytic fungus Penicillium sp. HSZ-43.

Penifupyrone (1), a new funicone derivative, has been isolated from the endophytic fungus Penicillium sp. HSZ-43, along with three known analogues, funicone (2), deoxyfunicone (3) and 3-O-methylfunicone (4). These structures were identified by using spectroscopic methods, including UV, MS, 1D and 2D NMR experiments. The structure of 1 was confirmed by single-crystal X-ray diffraction analysis. All the isolated compounds were evaluated for cytotoxicity against human oral epidermoid carcinoma KB cells, and compound 1 exhibited moderate cytotoxic activity with IC50 value of 4.7 µM.

Two new cyclic dipeptides from Rhinocladiella sp. lgt-3, a fungal endophyte isolated from Tripterygium wilfordii Hook.

Two new cyclic dipeptides, rhinocladin A (1) and rhinocladin B (2), were isolated from a fungal endophyte (Rhinocladiella sp. lgt-3) of Tripterygium wilfordii Hook. Their structures were elucidated by 1D and 2D NMR spectra. The monoamine oxidase inhibitory activity of 1 and 2 was also evaluated.

Kineosporia mesophila sp. nov., isolated from surface-sterilized stems of Tripterygium wilfordii.

An endophytic actinomycete strain, designated YIM 65293(T), was isolated from a surface-sterilized stem sample of Tripterygium wilfordii collected from Yunnan province, south-west China, and its taxonomic position was investigated. The chemical and morphological properties of the organism were consistent with those of the genus Kineosporia. Phylogenetic analysis indicated that the levels of 16S rRNA gene sequence similarity between strain YIM 65293(T) and other type strains of recognized members of the genus Kineosporia were 97.0-98.2 %. However, the DNA-DNA hybridization values, in combination with differences in phenotypic characteristics, revealed that the strain differed from recognized species of the genus Kineosporia. Therefore, strain YIM 65293(T) represents a novel species of the genus Kineosporia, for which the name Kineosporia mesophila sp. nov. is proposed. The type strain is YIM 65293(T) (=CCTCC AA 208061(T)=DSM 45271(T)).

Saccharopolyspora tripterygii sp. nov., an endophytic actinomycete isolated from the stem of Tripterygium hypoglaucum.

An endophytic actinomycete, designated strain YIM 65359(T), was isolated from a surface-sterilized stem sample of Tripterygium hypoglaucum collected from Yunnan province, south-west China. The morphological and chemotaxonomic properties of the new isolate were consistent with those of members of the genus Saccharopolyspora. Analysis of 16S rRNA gene sequences revealed that the new isolate was most closely related to 'Saccharopolyspora endophytica' YIM 61095 (98.6 %), Saccharopolyspora flava AS4.1520(T) (97.6 %) and Saccharopolyspora spinosa DSM 44228(T) (97.0 %). The results of DNA-DNA hybridizations (57.5 %, 44.9 % and 48.5 %, respectively) with the above micro-organisms, in combination with differences in the biochemical and physiological characteristics, suggested that strain YIM 65359(T) should be classified as a novel species of the genus Saccharopolyspora. The name Saccharopolyspora tripterygii sp. nov. is proposed for this novel species, with YIM 65359(T) (=CCTCC AA 208062(T)=DSM 45269(T)) as the type strain.