Steroids and Sesquiterpenes From Cultures of the Fungus Phellinus igniarius

Two new steroids, 3α,17α,19,20-tetrahydroxy-4α-methylpregn-8-ene (1) and 3α,12α,17α,20-tetrahydroxy-4α-methylpregn-8-ene (2) and three new sesquiterpenoids, 12-hydroxy-α-cadinol (3), 3α,12-dihydroxy-δ-cadinol (4), and 3α,6α-dihydroxyspiroax-4-ene (5), have been isolated from cultures of the fungus Phellinus igniarius. Their structures were characterized based on extensive spectroscopic data. In preliminary in vitro assays, compounds 3 and 4 exhibited the vascular-activities against phenylephrine-induced vasoconstriction with the relaxing rates of 11.0 % and 7.0 % at 3 × 10−4 M, respectively. Electronic supplementary material The online version of this article (doi:10.1007/s13659-014-0045-z) contains supplementary material, which is available to authorized users.


Introduction
Fungi are biosynthetically talented organisms capable of producing a wide range of chemically diverse and biologically intriguing small molecules. Phellinus igniarius, belonging to Polyporaceae family, is widely distributed in Yunnan and Sichuan Provinces of China [1]. It preferably grows on stems of aspen, robur, and birch. Its fruiting body was used to treat fester, abdominalgia, bloody gonorrhea and antidiarrheal in traditional Chinese medicine [2]. Previous chemical investigations on both fruiting bodies and cultures of this fungus reported various secondary metabolites with interesting structures and significant bioactivities [3][4][5][6][7]. Phelligridins D and E showed selective cytotoxicity against a human lung cancer cell line (A 549) and a liver cancer cell line (Bel 7402) [3], while phelligridins H-J, being pyrano [4,3-c] [2] benzopyran-1,6-dione and furo [3,2-c]pyran-4-one derivatives, showed cytotoxic activity against human cancer cell lines and protein tyrosine phosphatase 1B inhibition [4]. A pyrano [4,3-c] [2] benzopyran-1,6-dione derivative and a novel 26-membered macrocyclic metabolite phelligridimer A with antioxidant activities were also isolated from the fruiting bodies [5,6]. Moreover, several tremulane sesquiterpenes were obtained from the cultures of this fungus, some of which showed significant vascular-relaxing activities against phennylephrine-induced vasoconstriction [7]. To seek for more active molecules, further investigation of this fungus has resulted in the isolation of two pregnene steroids (1 and 2) and three sesquiterpenes (3)(4)(5) (Fig. 1). Compounds 1 and 2 are unusual 4-methyl homopregnane derivatives [8], and compound 5 is a rare spiroaxane sesquiterpene which was firstly isolated from the marine sponge Axinella cannabina in 1973 [9]. Based on the results of previous biological assays [10,11], compounds 1, 2, and 5 were tested for their cytotoxicity in vitro against five human tumor cell lines, while compounds 3 and 4 were tested for their vascularactivities against phenylephrine-induced vasoconstriction. This paper describes the isolation, structure elucidation and results of biological activities.

Results and Discussion
Compound 1 was isolated as a white amorphous powder. Apart from one double bond, the remaining four degrees of unsaturation indicated that 1 possessed a four-ring system. Inspection of 1 H-1 H COSY correlations resulted in the fragments as shown (Fig. 2). In the HMBC spectrum (     (Table 1) spectroscopic data were similar to those of compound 1. The main differences were the missing of an oxygenated methylene and the presence of an oxygenated methine in 2, which revealed the change of substitution of a hydroxy group. It was further confirmed by the HMBC correlations of d H 0.63 (3H, s, Me-18) with the oxygenated methine carbon. The mentioned information suggested that the hydroxy group was located at C-12. On the basis of the ROESY experiment, H-12 was elucidated to be b oriented by correlations of H-12 with H-18. Therefore, compound 2 was established to be 3a,12a,17a,20-tetrahydroxy-4a-methylpregn-8-ene, as shown.
Compound 3, a colorless oil, had a molecular formula of C 15 H 26 O 2 on the basis of HREIMS at m/z 238.1931 ([M] ? ). The 13 C NMR ( Table 2) and DEPT spectra of 3 indicated 15 carbon resonances, including three methyls, five methylenes (one oxygenated at d C 67.1), five methines (one sp 2 carbon at d C 123.3), and two quaternary carbon (one oxygenated at d C 72.9 and one sp 2 carbon at d C 136.0). Detailed analysis of NMR data suggested that compound 3 should be a cadinane-type sesquiterpene with a similar structure to that of 15-hydroxy-a-cadinol [12]. The main difference between the two compounds was that the hydroxy should be placed at C-12 in 3 rather than at C-15 in 15-hydroxy-a-cadinol, which was confirmed by the correlations of d  parts of the structure of 3 were the same as those of 15-hydroxy-a-cadinol [12]. Therefore, compound 3 was deduced to be 12-hydroxy-a-cadinol. Compound 4, a white amorphous powder, possessed the molecular formula C 15 H 26 O 3 , on the basis of its HREIMS at m/z 254.1904 ([M] ? ), 16 mass units higher than that of 3. The 1D NMR spectroscopic data ( Table 2) Table 2) and DEPT spectra indicated 15 carbon resonances, classified as four methyls, three methlyenes, six methines (two oxygenated at d C 76.3 and 78.2; one sp 2 carbon at d C 130.7), and two quaternary carbons (one sp 2 carbon at d C 144.9; one sp 3 quaternary carbon at d C 57.8).
Compounds 1, 2, and 5 were evaluated for their cytotoxicity against five human cancer cell lines. None was found to possess significant activity with IC 50 values less than 40 lM.
Compounds 3 and 4 were tested for the vascular-activities against phenylephrine-induced vasoconstriction. They exhibited the vascular-activities with the relaxing rates of 11.0 % and 7.0 % at 3 9 10 -4 M, respectively. It's worth mentioning that this is the first time to report the vascular-activity of cadinane-type sesquiterpenes.

General Experimental Procedures
Optical rotations were measured on a Jasco-P-1020 polarimeter. IR spectra were obtained using a Bruker Tensor 27 FT-IR spectrometer with KBr pellets. NMR spectra were acquired with instrument of a Bruker Avance I 600 with deuterated solvent signals as internal standards. HREIMS were measured on a Waters Autospec Premier P776 spectrometer. HRESIMS were recorded on an API QSTAR Pulsar spectrometer. Silica gel (200-300 mesh and 80-100 mesh, Qingdao Marine Chemical Inc., China), Sephadex LH-20 and RP-18 gel (20-45 lM, Fuji Silysia Chemical Ltd., Japan) were used for column chromatography (CC). Preparative HPLC was performed on an Agilent 1100 series with a Zorbax SB-C18 (5 lM, 9.4 9 150 mm) column. Fractions were monitored by thin layer chromatography (TLC) (Qingdao Marine Chemical Inc., China) and spots were visualized by heating silica gel plates immersed in H 2 SO 4 in EtOH, in combination with the Agilent 1200 series HPLC system (Eclipse XDB-C18 column, 5 lM, 4.6 9 150 mm).

Fungal Material and Cultivation Conditions
Fruiting bodies of Phellinus igniarius were collected at Changbai Mountain National Nature Reserve, Antu, Jilin Province, China in 2008 and identified by Prof. Yu-Cheng Dai (Beijing Forestry University). A specimen (No. KIB20081017) was deposited at Kunming Institute of Botany, Chinese Academy of Sciences. The culture medium was composed of glucose (5 %), pork peptone (0.15 %), yeast powder (0.5 %), KH 2 PO 4 (0.05 %) and MgSO 4 (0.05 %). The initial PH was adjusted to 6.0 and the fermentation was carried out on a shaker at 150 rpm for 25 days.

Extraction and Isolation
The cultures (20 L) were filtered through cheesecloth to separate broth and mycelium. The broth was extracted four times with ethyl acetate, while the mycelium was extracted three times with CHCl 3 -MeOH (1:1). The organic layer of both parts were evaporated together to yield a crude extract (9 g). Then this residue was subjected on reverse-phased C18 column eluted with gradient mixture of MeOH and H 2 O (30:70-100:0, v/v). Fractions were collected and monitored by TLC. Similar fractions were pooled to give twelve sub-fractions (A-L). Sub-fraction L was isolated by reverse-phased C18 column eluted with mixture of MeOH and H 2 O (45:55, v/v), then purified by Sephadex LH-20 CC (Me 2 CO) to yield compound 1 (1.4 mg). Sub-fraction G was subjected to Sephadex LH-20 CC (MeOH) and silica gel CC eluted with a petroleum ether-acetone gradient system (6:1, v/v) to give compound 2 (0.8 mg). Sub-fraction I was separated by repeated CC on silica gel and purified by preparative HPLC (MeCN/H 2 O, from 0:100 to 40:60, 10 mL/min, 40 min) to obtain compound 3 (1.8 mg). Sub-fraction A, isolated by Sephadex LH-20 CC (MeOH) and reverse-phased C18 column eluted with mixture of MeOH and H 2 O (30:70, v/v) to yield compound 4 (0.7 mg). Sub-fraction J was subjected to silica gel CC eluted with a petroleum ether-acetone gradient system (4:1, v/v) and Sephadex LH-20 CC (Me 2 CO) to yield compound 5 (1.4 mg).