A new dimeric anthraquinone from endophytic Talaromyces sp. YE3016

Abstract A new unsymmetrical dimeric anthraquinone, 3-demethyl-3-(2-hydroxypropyl)-skyrin (1) was isolated from the solid-state fermentation extract of an endophytic fungal strain Talaromyces sp. YE 3016, together with five known compounds, skyrin (2), oxyskyrin (3), emodin (4), 1,3,6-trihydroxy-8-methyl-anthraquinone (5) and ergosterol (6). The structure of the new compound was elucidated on the basis of spectroscopic analysis. Compounds 1–3 exhibited moderate cytotoxic activities against MCF-7 cell line.


Introduction
Endophytes can be found in virtually all terrestrial plants and play an important role for the growth of hosts. They have been recognised as important sources of a variety of structurally novel active secondary metabolites with anticancer, antimicrobial and other biological activities. (Strobel 2003;Qin et al. 2009). The genus Talaromyces not only plays a positive role with benefit enhance host growth (Mickael et al. 2015), but also is a potential source of secondary metabolites for drug discovery. There are some bioactive natural products that had been reported from this genus, such as diphenyl ether derivatives (Chen et al. 2015), isocoumarin (Miao et al. 2012), talaroconvolutins (Suzuki et al. 2000), oxaphenalenone dimmers (Mikio & Emi 1980), and xanthone dimer (Koolen et al. 2013). Talaromyces sp. YE 3016, an endophytic fungal strain isolated from Aconitum carmichaeli, was selected for further chemical investigation due to the antitumor activity of its fermentation extracts. The follow-up study led to the isolation of a new unsymmetrical dimeric anthraquinone, named 3-demethyl-3-(2-hydroxypropyl)-skyrin (1), together with five known compounds, skyrin (2) (liu et al. 2010), oxyskyrin (3) , emodin (4) (Choi et al. 2000), 1,3,6-trihydroxy-8-methyl-anthraquinone (5) (Bunluepuech et al. 2011) and ergosterol (6) (liu et al. 2012) (Figure 1). Here, we report the isolation and structure elucidation of these metabolites, as well as the cytotoxic activities of compounds 1-3.

Results and discussion
The molecular formula of compound 1 was determined as C 32 H 22 o 11 by HR-ESIMS at m/z 581.1075 [M−H] − (Calcd 581.1084). The 1 H NMR spectrum (Table S1) revealed the presence of six aromatic singlet protons at δ H 7.38, 7.31, 7.10, 7.04, 6.69 and 6.68, one oxygenated methine group at δ H 3.96 (m), one doublet methylene group at δ H 2.72, one singlet methyl group at δ H 2.34 and one doublet methyl group at δ H 1.16. These spectroscopic features combined with HRESIMS and 13 C NMR data suggested that compound 1 was an unsymmetrical dimeric anthraquinone. The NMR spectra of compound 1 showed similarities to those of the known compound skyrin (Gill et al. 1988). The significant differences between them are the presence of an additional methylene group (δ H 2.72, d, J = 4.8 Hz; δ C 46.4) and an oxygenated methine group (δ H 3.96, m; δ C 68.8), as well as a doublet methyl group (δ H 1.16; δ C 23.2) in 1 instead of a singlet methyl group as in the known compound. In addition, the molecular ion of 1 was 44 mass units greater than that of skyrin. All these data suggested that 1 had an additional hydroxypropyl group instead of a methyl group compared with skyrin. The 1 H-1 H CoSY spectrum showed the couplings of the oxygenated methine proton at δ H 3.96 to the methylene protons at δ H 2.72 and the methyl protons at δ H 1.16, indicating the 2-hydroxypropyl group in 1. The HMBC spectrum displayed correlations from the methylene group at δ H 2.72 to the quaternary carbon at δ C 150.5 and the methine carbons at δ C 125.1 and 121.7. Further comparison these 13 C NMR data with those of skyrin revealed that the 2-hydroxypropyl group was attached to C-3. Therefore, the structure of compound 1 was determined to be 3-demethyl-3-(2-hydroxypropyl)-skyrin.
Skyrin is chiral by virtue of restricted rotation about the biaryl linkage. The absolute configuration of the axial chirality was determined to be S, as depicted in formula 1, according to its positive optical rotation (Gill et al. 1988) and biogenetic consideration , compared with the known compounds 2 and 3. The absolute configuration of C-12 was not determined tentatively due to the limited amounts of the compound.
The cytotoxic activities against MCF-7 of compounds 1-3 were evaluated by the MTT method. The results showed that compounds 1-3 exhibited cytotoxicities against MCF-7 with IC 50 values of 20.76 ± 3.41, 19.21 ± 3.85 and 29.83 ± 4.21 μg/ml, respectively. The paclitaxel was used as the positive control with IC 50 value of 0.43 μg/ml.
Anthraquinones have been isolated from various fungal genus, such as Alternaria, Nigrospora, Aspergillus, Penicillium and Xylaria (Huang et al. 2014;Hussain et al. 2015). They are also commonly produced by the genus Talaromyces. However, their dimers have been rarely reported from Talaromyces previously. The only example is antibiotic anthraquinone dimers isolated from Talaromyces wortmannii, an endophyte of Aloe vera Bara, Zerfass et al. 2013). To the best of our knowledge, the current study is the second time to obtain these dimers from the genus Talaromyces.
Anthraquinone dimers from endophytic fungi have been evaluated and demonstrated good biological properties, such as antibacterial, antidiabetic, antiviral, anti-HCV, antifibrotic, fungicidal, anti-inflammatory and anticancer activities (Hussain et al. 2015). It had been reported that the inhibition of glucagon stimulation was a specific property displayed by skyrin and oxyskyrin but not shared by other bisanthroquinones (Parker et al. 2000). Skyrin exhibited strong cytotoxic activity against the insect cell line Sf9 with Id 50 value of 9.6 μg/ ml (Patricia et al. 2003), and significantly suppressed the growth of Hela, Vero, K562, Raji, Wish and Calu-1 tumor cell lines (lin et al. 2001). In this paper, we report the isolated anthraquinone dimers (1-3) exhibiting moderate cytotoxic activity against MCF-7 cell line for the first time.

General experimental procedures
Precoated silica-gel G plates (Sio 2 ; Qingdao Marine Chemical Factory, Qingdao, China) were used for analytical TlC analyses. Silica gel (Sio 2 ; 200-300 mesh; Qingdao Marine Chemical Factory), Sephadex lH-20 (Pharmacia) and Reversed-phase C 18 (RP-18) silica gel (Merck) were used for column chromatography. optical rotation was measured on a Jasco dIP-1020 polarimeter. uV spectra were recorded on a Shimadzu double-beam 210A spectrophotometer; λ max (log ε) in nm. IR was determined in Bio-Rad FTS spectrometer, with KBr pellets; ν max in cm −1 . 1d and 2d NMR spectra were obtained on a Bruker dRX-500 spectrometer; δ in ppm rel. to Me 4 Si as internal standard, J in Hz. HRESI-MS spectra were recorded with an Agilent 3250AA lC/MSd ToF spectrometer in m/z.

Fungal material
The fungal strain YE 3016 was isolated from surface-sterilised fresh roots of an apparently healthy A. carmichaeli collected in August 2011 in lijiang County, Yunnan Province, P.R. China. It was identified as a Talaromyces sp. on the basis of the sequence data of the ITS genes that gave a 99% sequence similarity to that accessible at the BlAST of T. wortmannii (GenBank accession No. KF984829.1).

Cytotoxicity test
The cytotoxic activities of compounds 1-3 towards human breast adenocarcinoma cell line MCF-7 were tested in vitro according to the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) method (Mosmann 1983) in 96-well microplates. Briefly, 100 μl of adherent cells was seeded into each well of 96-well cell culture plates and allowed to adhere for 12 h before drug addition, while suspended cells were seeded just before drug addition with an initial density of 1 × 10 5 cells/ml. MCF-7 cell line was exposed to the test compounds dissolved in dMSo at various concentrations in triplicates for 48 h, with paclitaxel as a positive control. After compound treatment, cell viability was detected and the cell growth curve was graphed. The IC 50 value was calculated by Reed and Muench's method (Reed & Muench 1938).

Supplementary material
Supplementary material relating to this article is available online, alongside Table S1 and Figures S1-S5.

Funding
This work was financially supported by the National Natural Science Foundation of China [grant number 81460545].