New 1, 2-naphthoquinone-derived pigments from the mycobiont of lichen Trypethelium eluteriae Sprengel

A new yellow pigment trypethelonamide A (1), and a new dark violetred pigment 5′-hydroxytrypethelone (2), as well as three known dark violet-red pigments (+)-8-hydroxy-7-methoxytrypethelone (3), (+)-trypethelone (4) and (-)-trypethelone (5) were isolated from the cultured lichenized fungus Trypethelium eluteriae. The structures of 1 and 2 were determined to be 1, 2-naphthoquinone derivatives by extensive spectroscopic analysis. The absolute configurations of 1 and 2 were assigned by quantum electronic circular dichroism (ECD) calculation. All isolated compounds were evaluated for cytotoxicities against A549, HepG2 and RKO cell lines, and antioxidant effects on DPPH. Compounds 1–5 showed moderate and weak cytotoxicities against RKO cell line with IC50 from 22.6 to 113.5 μM. © 2018 Informa uK limited, trading as taylor & Francis Group


Introduction
Lichen is a stable fungal-algal or fungal-cyanobacterial extracellularly mutualistic community in the ecosystem of the Earth's biosphere. Each mutualistic community is composed of a lichenized fungus (mycobiont) as a constructive species together with a corresponding alga or cyanobacterium as a companion species. Most of the lichens have been found to produce an array of secondary metabolites, such as polyketides, terpenes, steroids (Feige et al. 1993;Huneck and Yoshimura 1996;Oksanen 2006;Moreira et al. 2015). The reported bioactivities for these secondary metabolites include antioxidation, antivirus, anti-inflammation, antitumor, etc. (Bézivin et al. 2003;De Carvalho et al. 2005;Segatore et al. 2012;Shrestha and Clair 2013).
Pigments have been widely used in food, textile, painting, cosmetics, pharmaceutics, and plastics. In recent years, the demand for natural pigments is increasing due to the harmful effects of some synthetic dyes. Natural pigments can be obtained from ores, insects, plants, and microorganisms. In contrast to other natural pigments, bacterial and fungal pigments possess many biological properties such as antioxidant, antimicrobial, and anticancer activities (Dufossé et al. 2014;Rao et al. 2017). With the aim to explore new pigments from fungi, the mycobiont of lichen Trypethelium eluteriae Sprengl that has been reported to produce pigments (Mathey et al. 1980(Mathey et al. , 2002 was put into the chemical investigation. In this paper, we mainly described the isolation, structural elucidation and cytotoxicities of two new compounds trypethelonamide A (1), and 5′-hydroxytrypethelone (2) from the crude extract of mycobiont of lichen T. eluteriae.
5′-Hydroxytrypethelone (2) was obtained as a dark violet-red amorphous powder. Its molecular formula was confirmed as C 16 H 16 O 5 with 9 degrees of unsaturation on the basis of HRESIMS data at m/z [M + H] + 289.1073. The 1 H and 13 C NMR data of 2 showed similarity with that of 4 and 5 except for the loss of a single methyl group, and the presence of hydroxylmethyl group [δ H 3.80 (d, J = 11.4, H 2 -5′), 3.70 (d, J = 11.4, H 2 -5′); δ C 64.1 (C-5′)]. The hydroxylmethyl group was attached at C-3′ by HMBC correlations ( Figure S1) of H 2 -5′ to C-2′, C-3′, and C-4′. The NOE correlation ( Figure S1) of H-2′ with H 3 -4′ indicated that H-2′ and H 3 -4′ were on the same side. Computer simulation of the ECD spectra for 2a and 2b was conducted by using the TDDFT at B3LYP/6-31G(d) basis set level in methanol as described for 1. A systematic conformational analysis for 2a and 2b yielded four lowest energy conformers for enantiomers 2a and 2b. The ECD spectrum for 2b matched well with the experimental ECD for 2 ( Figure S3). Therefore, the absolute configuration of 2 was assigned as 2′R, 3′S.
Compounds 1-5 are polyketide class of natural product and are supposed to be originated from heptaketide precursor (6) (Elsebai et al. 2011;Nazir et al. 2015) (Scheme S1). Cyclization of heptaketide precursor gave a major structure phenalenone (7), which was transformed into compounds 4 and 5 by oxidation, prenylation, and cyclization. The oxidation of C-5′ and methylation at C-7, hydroxylation at C-8 of 5 afforded 2 and 3, respectively, which were further transformed into intermediate 8 via oxidation, methylation, and cyclization. Compound 1 was derived from the reaction between intermediate 9 and 4-methylpentan-2-one.

General experimental procedures
HRTOFMS data were acquired with an Agilent Accurate-Mass-Q-TOF LC/MS 6520 spectrometer. UV and IR data were obtained on a Thermo Genesys-10S UV/Vis and Nicolet IS5FT-IR spectrophotometer, respectively. NMR spectra were recorded on a Bruker Avance-500 spectrometer. TLC was conducted on plates percolated with Silica gel HSGF 254 and the spots were detected under UV lights or by heating after spraying with 10% H 2 SO 4 . Silica gel (200-300 mesh, Qingdao Haiyang Chemical Co., Ltd., People′s Republic of China), and Sephadex LH-20 (Amersham Biosciences) were used for column chromatography. Semi-preparative HPLC was performed with a Waters 2489 HPLC system using an octadecylsilyl (ODS) column (YMC-Pack, ODS-A, 10 × 250 mm, 5 μm).

Fungal material
The voucher specimen (coll. no. M265) was collected from Hainan Province of China and deposited in Lichen Section of the Herbarium Mycologicum Academiae Sinicae (HMAS-L, No. 300252). The lichen mycobiont was isolated by ascospores discharging method (Yoshimura et al. 2002) as follows: The small lichen tissue piece with perithecia was washed by tap water, 75% alcohol, and sterile water for once in order, each taking 2 min, then adhered to sterile vaseline piece situated inside the cover of petri dish, discharging the ascospores on the surface of water agar medium, and cultured in the incubator under 16 °C.
The identification of lichen mycobiont of Trypethelium eluteriae has been done combining both morphological characters and ITS sequence by the co-author, Dr. Xinli Wei, who is a lichenologist. Firstly, the morphology of ascospores of T. eluteriae has been checked, after finishing the ascospores discharging step, the process of ascospores germination of T. eluteriae on the surface of water agar medium has been tracked, ensuring the fungal colony indeed germinated from the ascospores of T. eluteriae; secondly, both lichen thallus and the isolation of mycobiont were isolated the DNA and sequenced the ITS sequence then compared the similarity, and at the same time constructed the phylogenetic tree, in order to confirm its phylogenetic position and the isolation is just the lichen mycobiont of T. eluteriae.
T. eluteriae was cultured on Potato Dextrose agar plates at 25 °C for 7 days. Agar plugs were inoculated into a 250 mL Erlenmeyer flask containing 100 mL Potato Dextrose broth medium cultured at 25 °C for 15 days on a rotary shaker at 180 rpm. Mass scale fermentation was carried out in 20 × 500 mL Fernbach culture flasks each containing 80 g of rice and 100 mL of distilled water. Each flask was inoculated with 5 mL of seed culture medium and incubated at 25 °C for 60 days in dark.

Computation section
The ECD calculations were conducted by the same program as described in our early report (Tao et al. 2016). Compound 1 was simplified to be 1 J for CD calculation. A systematic conformational analysis was performed for 1 J and 2 by the Confab at MMFF94 force filed, followed by re-oprimized at B3LYP/6-31G(d) level to yield two lowest energy conformers for enantiomers 1Ja and 1Jb, and four lowest energy conformers for enantiomers 2a and 2b.
The overall ECD spectra were generated according to Boltzmann weighting of each conformer.

Cell proliferation assays
A549 (human lung carcinoma), HepG2 (human hepatocellular carcinoma) and RKO (Human colorectal cancers) cell lines were purchased from National Infrastructure of Cell Line Resource (Beijing, China). Roswell Park Memorial Institute (1640), Dulbecco's modified eagle medium (DMEM) and PBS were purchased from hyclone (Greeley, CO, USA). Fetal bovine serum was purchased from Gibco (Life Technologies, Carlsbad, CA, USA).The cytotoxicities of compounds 1-5 against A549, HepG2 and RKO cell lines were investigated with the Cell Counting Kit 8 cell viability assay (Liu et al. 2017). The inhibition rate was calculated and plotted control vs. test concentrations to afford the IC 50 in triplicate micro plate reader.

DPPH scavenging assay
As described in our earlier work (Ma et al. 2014), the assay was conducted in a 96-well plate, and the absorbance was measured at 517 nm using a Spectra Max 190 micro plate reader (Molecular Devices Inc.). The scavenging ability was calculated as follows: scavenging ability (%) = A 517 of control − A 517 of sample ∕A 517 of control × 100. Ascorbic acid was used as positive control with an IC 50 of 18.6 ± 0.7 μM.

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