Secondary metabolites of Xylaria sp., an endophytic fungus from Taxus mairei

Abstract One new metabolite 3,7-dimethyl-9-(-2,2,5,5-tetramethyl-1,3-dioxolan-4-yl)nona-1,6-dien-3-ol, together with nine known compounds, were isolated from the strain Xylaria sp., an endophytic fungus of Taxus mairei. Their structures were deduced from 1D and 2D NMR data. In vitro cytotoxicity and antibacterial activity of these compounds were evaluated. Some of them exhibited substantial activity.


Introduction
Paclitaxel was first isolated from the bark of the pacific yew tree in 1970s (Ketchum et al. 1999). Taxol (paclitaxel) appears to be one of the most promising antineoplastic agents of the last decade, with demonstrated activity in advanced and refractory ovarian, breast, lung, and head and neck cancers (Rowinsky et al. 1993). The discovery of paclitaxel (taxol) as a potent anticancer drug, initially isolated from Taxus brevifolia, has spurred several groups all over the world to conduct research work on other Taxus species, to isolate potentially more effective paclitaxel derivatives or as starting materials for semisynthesis (Sun et al. 2015).
endophytes are a rich source of natural products displaying a broad spectrum of biological activities (Tan & Zou 2001;Strobel 2003;Strobel et al. 2004). after an initial report of the production of paclitaxel from an endophyte of the North-west Pacific yew (Stierle et al. 1993), isolation of anticancer agents from fungal endophytes has gained increased interest. Taxus chinensis var. mairei is a valuable plant that belongs to the Taxaceae family (Wu et al. 2015). During our screening for bioactive endophytic fungus from this medicinal plant, one strain (Xylaria sp.) was fermented according to its cytotoxic activity. Xylaria have received attention due to their potential as a source of novel and bioactive secondary metabolites (huang et al. 2014;Rivera-chávez et al. 2015;Sawadsitang et al. 2015).Isolation of the chemical constituents of this strain led to the discovery of one new and nine known compounds. This article illustrated the isolation, structure elucidation of the new compound and the bioactivities of these compounds.

General experimental procedures
The 1 h (600 Mhz), 13 c (125 Mhz) and 2D NMR spectra were recorded in c 3 D 6 O on a Bruker DRX2500 instrument using TMS as an internal reference. eSI-MS was acquired using Finnigan LcQ-advantage.

Fungal material
The strain was preserved at School of Life Sciences, Xiamen university. a BLaST search result showed that the internal transcribed spaces sequence of this fungus was highly homologous (99% similarity) to that of Xylaria sp. JK50 (accession number: JX624289.1), indicating that the fungus belongs to this genus.

Culture conditions, extraction and isolation
The strain was grown on PDa plates (10 L) at 28 °c for 15 days. The cultured agar was extracted with etOac-MeOh-acOh (80:15:5, v/v/v, 10 L) at room temperature overnight for three times. The organic filtrations were concentrated under vacuum and the remaining aqueous solution was extracted with etOac to afford etOac extract (3.81 g).

Biological assay
hundred microlitre of medium (test microorganism concentration:10 6 cfu) and 100 μL of compound solution (50 μg/mL final concentration) were incubated at 30 °c for 18 h. Growth was evaluated by the percentage decrease in the OD at 560 nm in comparison to that of the negative control with the formula: [OD (negative control) -OD (sample)]/OD (negative control The cytotoxic activities of the compounds were measured by MTT method (Solis et al. 1993) in heLa cell line. compound 10 exhibited prominent bioactivity of 94.1% inhibition at concentration of 10 μg/mL.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
This work was supported by the Fundamental Research Funds for the central universities [grant number 0250-ZK1027].