Chemical constituents from the fruiting bodies of Phellinus igniarius

Abstract A new tirucallane-type triterpenoid igniarine (1), and four known compounds meshimakobnol A (2), meshimakobnol B (3), ergosterol (4) and ergosterol peroxide (5), were purified from the methanol extracts of the fruiting bodies of Phellinus igniarius (DC. ex Fr.) Quél. The structure of 1 was elucidated using a combination of 1D and 2D NMR techniques and HR-ESI-MS analyses. In addition, the isolated compounds were examined for their cytotoxicity against several tumour cell lines and part of the tested compounds demonstrated weak cytotoxicity.


Introduction
Many medically significant metabolites had been reported from various edible fungi (Vamanu 2017). Phellinus is a genus of fungi in the family Hymenochaetaceae. Wood-rotting fungi of the genus Phellinus are rich sources of aromatics, polysaccharides, sterols and triterpenes. They have been described in Asian herbal medicine literature to be effective on a diversity of diseases, including improving blood circulation, enhancing detoxification and hepatoprotection of human body, combating allergy and diabetes, curing oral ulcer, alleviating gastroenteric disorder or lymphatic disease, diarrhoea, haemorrhage and cancer Yun 2007, 2011;Zhu et al. 2008). The fruiting bodies of the fungus Phellinus igniarius had historically been used for the treatment of fester, bellyache and bloody gonorrhoea in China (Jiangsu New Medical College 1977). In Vietnam, P. igniarius was natively distributed in the area around Nghean and Hue. In the course of our phytochemical studies on Vietnamese fungi, the fruiting bodies of P. igniarius were investigated to search for the natural lead drugs.
In the downfield region of the 13 C-NMR spectrum, there was an oxygenated methine at δ 78.9 (C-3), one set of tetrasubstituted double bonds at δ 134.3 (C-9) and 134.4 (C-8) and one conjugated carbonyl group at δ 193.8 (C-22). The location of hydroxy group at C-3 was determined by 2 J, 3 J-HMBC correlations from CH 3 -28 to C-3, from CH 3 -29 to C-3 and from H-3 to C-2, respectively. The C-8/C-9 double bond was also verified by HMBC correlations between CH 3 -19 and C-1/C-5/C-9/C-10, and between CH 3 -30 and C-8/C-13/C-14/C-15. The carbonyl group was located at C-22 evidenced by HMBC analytical results which exhibited correlations from H-20 to C-17, -21 and C-22. The NOESY spectroscopic analysis also displayed similar results as described in the literature (James et al. 1997) and therefore the stereochemical configurations of 1 were constructed. The other proton and carbon assignments of 1 (Table  S1) were accomplished by the combination of 2D NMR experiments. Consequently, the structure of 1 was established as shown ( Figure 1). In addition, four known compounds were characterised as meshimakobnol A (2) (Akito et al. 2004), meshimakobnol B (3) (Akito et al. 2004), ergosterol (4) (Li et al. 2012) and ergosterol peroxide (5) (Fangkrathok et al. 2013). Their chemical structures were identified by comparison of their physical and spectroscopic data with the reported values in the cited literature. These isolates were screened for in vitro cytotoxicity against MCF-7, HepG2 and Lu tumour cell lines as described previously  and the data are given in Table  S2. Compounds 1-4 showed effective inhibition against HepG2 cells with the IC 50 values of 18.4 ± 2.6, 19.2 ± 2.0, 16.7 ± 1.6 and 21.5 ± 5.1 µM. In contrast, compound 5 showed only weak cytotoxic activity against HepG2 cells with an IC 50 value of 46.9 ± 3.7 µM. Except for the weak inhibitory effects of compounds 4 and 5 (IC 50 > 50 µM), the other compounds 1-3 exhibited cytotoxic activity against Lu cells with IC 50 values of 29.1 ± 3.6, 35.2 ± 3.4 and 21.7 ± 2.8 µM. Among the examination of cytotoxicity against MCF-7 cancer cell lines, only compound 4 showed weak inhibitory effect with an IC 50 value of 43.6 ± 5.1 µM.

General experimental procedures
Melting points were determined using Yanagimoto MP-S3 apparatus without corrections. Optical rotations were measured using a JASCO DIP-370 polarimeter. The UV spectra were obtained on a Hitachi UV-3210 spectrophotometer, and IR spectra were recorded on a Shimadzu FTIR-8501 spectrophotometer. 1 H-and 13 C-NMR, COSY, NOESY, HMQC and HMBC spectra were obtained on the Bruker AV-III 400 NMR spectrometer, with tetramethylsilane (TMS) as the internal standard and chemical shifts were reported in δ values (ppm). The electrospray ionisation (ESI) and high resolution electrospray ionisation (HR-ESI) mass spectra were determined using an Agilent 1200 LC-MSD Trap spectrometer. Column chromatography (CC) was performed on silica gel (Kieselgel 60, 70-230 mesh and 230-400 mesh, E. Merck). Thin layer chromatography (TLC) was conducted on precoated Kieselgel 60 F 254 plates (Merck) and the compounds were visualised by spraying with 10% (v/v) H 2 SO 4 followed by heating at 110 °C for 10 min.

Fungal material
The basidiomycetes of P. igniarius were collected at the Puhuong National Park of Nghean Province, Vietnam, in August 2015 and identified by Prof. Dr Ngo Anh, Department of Biology, Hue University. A voucher specimen (Vinh-TSWu 20150815) was deposited at the herbarium of the Department of Chemistry, Vinh University.

Extraction and isolation
The fruiting bodies of P. igniarius (4.5 kg) were air-dried, powdered and extracted with methanol (10 L × 3) at ambient temperature, and the combined extracts were concentrated under reduced pressure to give a deep brown syrup (154 g). The crude extracts were suspended in water and partitioned with ethyl acetate and butanol to afford ethyl acetate (42 g) and butanol fractions (67 g), respectively.

Cell lines and culture
MCF-7 (human breast adenocarcinoma), HepG2 (liver hepatocellular cells) and Lu (Lung cancer) cells were obtained from the American Type Culture Collection (ATCC). The cells were maintained in RPMI or IMDM (GibcoBRL, NY, USA) with 10% foetal bovine serum (FBS) supplemented with 2% penicillin and 100 µg/mL of streptomycin at 37 °C in a humidified atmosphere containing 5% CO 2 .

Cytotoxic activity
The cytotoxicity against three human tumour cell lines (MCF-7, HepG2 and Lu) was maintained in RPMI 1640 or IMDM that included L-glutamine (GIBCO) with 10% FBS (GIBCO) and 2% penicillin-streptomycin (GIBCO). Cells were cultured at 37 °C in a 5% CO 2 incubator. Cytotoxic activity was measured using a modified MTT assay . Viable cells were seeded in the growth medium into 96-well microtiter plates (1 × 10 4 cells/well) and were incubated at 37 °C in a 5% CO 2 incubator. A test sample was dissolved in DMSO and was adjusted to final sample concentration ranging from 5 to 100 µg/mL by diluting with the growth medium. Each sample was prepared in triplicate. The final DMSO concentration was adjusted to < 0.1%. After standing for 24 h, the test sample was added to each well. The same volume of medium with 0.1% DMSO was added to the control wells. 48 h after the addition of the test sample, MTT reagent was added to each well (final concentration: 5 µg/ mL). 4 h later, the plate was centrifuged for 5 min at 1500 rpm, the medium was removed and the resulting formazan crystals were dissolved in DMSO. The optical density (OD) was measured at 570 nm using a Titertek microplate reader (Multiskan MCC/340, Flow). The cytotoxicity was expressed as IC 50 values (50% inhibitory concentration).