Introduction

Mushrooms in the genus of Panus belonging to the family of Polyporaceae are featured with thin-fleshed but tough basidiomata. They live on a wide range of broadleaf trees and distribute in tropical regions worldwide ^[1]. The Panus mushrooms producing diverse bioactive polyketone ^[2], alkaloid ^[3], and sesquiterpene ^[4] were reported to have wide pharmacological properties, such as dispersing cold, relaxing tendons, activating collaterals, clearing heat, detoxification, anti-inflammatory ^[5]. Specifically, hexacyclinol, isolated from Panus rudis HKI 0254, showed antiproliferative activity ^[6] ; Panepophenanthrin, isolated from mushroom Panus rudis Fr. IFO 8994, was a novel inhibitor of the ubiquitin-activating enzyme ^[7]; Panepoxydon, produced by Panus mushroom, exhibited cytotoxic activity ^{[2], [8]}. Agmatine, isolated from fruit-bodies of Panus tigrinus, displayed hypoglycemic activity ^[3]; Two sesquiterpenes, neamatolon and panudial isolated from Panus sp. 9096, showed inhibitory activity against platelet aggregation ^[9].

As a continuation of our investigation on structurally novel and biologically active metabolites from mushrooms ^[10,11] , two new meroterpenoid compounds (1 and 2) together with five known meroterpenoid derivatives (3–7) were isolated from the EtOAc extract of mushroom Panus lecomtei. Structurally, compound 1 possessed an unprecedented spiroketal. Herein, we report the isolation, structure elucidation, biological activity and the plausible biosynthetic pathway of 1–7.

Results and Discussion

Compound 1 was obtained as an orange red oil and its molecular formula, C₁₂H₁₂O₄, was deduced from HR-ESI-MS m/z 221.0822 [M + H]⁺ (Calcd. for C₁₂H₁₃O₄, 221.0808), corresponding to 7 indices of hydrogen deficiency. (Fig. 1) The ¹H NMR data of 1 (Table 1) exhibited characteristic signals for two methyls at δ_H 1.25 (s, H₃-12) and 1.50 (s, H₃-11), one methoxyl at δ_H 3.79 (s, H₃-10) and three aromatic protons at δ_H 7.26 (d, J = 3.1 Hz, H-6), 7.16 (dd, J = 3.2, 9.1 Hz, H-7) and 6.84 (d, J = 9.1 Hz, H-8). The ¹³C NMR (Table 1) and HSQC spectra of 1 showed the presence of 12 carbons, including a ketocarbonyl group, six aromatic carbons, two oxygenated quaternary carbons (one ketal at δ_C 95.7), one oxygenated methyl, and two methyls.

Figure  1.  Chemical structures of compounds 1–7

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Table  1.  ¹H and ¹³C NMR data of 1 and 2 (500, 125 MHz, TMS, δ ppm) in CD₃OD.

No.	1		2
	δC	δH (J, Hz)	δC	δH (J, Hz)
1	84.5		178.0
2	95.7		82.9
3	191.3		169.7
4	119.4		126.6
5	155.3		156.5
6	108.5	7.26 (d, J = 3.1)	116.7	7.37 (d, J = 3.1)
7	126.6	7.16 (dd, J = 3.2, 9.1)	120.1	7.06 (dd, J = 3.2, 9.1)
8	120.7	6.84 (d, J = 9.1)	123.6	7.09 (t, J = 9.1)
9	155.3		149.6
10	56.2	3.79 (3H, s)	56.2	3.80 (1H, s)
11	20.9	1.50 (3H, s)	25.1	1.56 (3H, s)
12	20.7	1.25 (3H, s)	25.1	1.56 (3H, s)

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Analysis of the ¹H NMR and ¹³C NMR data (Table 1) of 1 with those of benzofuran-3(2H)-one ^[12] showed almost superimposable structural relationships. The remarkable distinction was presence of an epoxy ethane group with two methyls linked with benzofuranone through the C-2 (δ_C 95.7) based on the HMBC correlations (Fig. 2) from H₃-11 to C-1, C-2, H₃-12 to C-1, C-2 and degrees of unsaturation of 1. Additionally, there was a methyoxyl group in 1 (δ_C/H 56.2/3.79), which was connected with C-5 (δ_C 155.3) through the HMBC correlation (Fig. 2) from H₃-10 to C-5. Thus, the planar structure of 1 was established to be a meroterpenoid. The small optical rotation ($\left[ {\text{α}} \right]_{\rm{D}}^{25}$+2 (c 0.5 MeOH)) of 1 and the absence of Cotton effects in the CD spectrum of 1 suggested it might be a racemic mixture.

Figure  2.  Key ¹H–¹H COSY and HMBC correlations of 1 and 2

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Compound 2, yellow oil, showed the molecular formula of C₁₂H₁₄O₆ (m/z 255.0987 [M + H]⁺, Calcd. for [M + H]⁺ ion at m/z 255.0863) via HR-ESI-MS, corresponding to six degrees of unsaturation. The ¹H NMR spectrum (Table 1) of 2 revealed the presence of two methyls at δ_H 1.56 (s, H₆-11, 12), one methoxyl at δ_H 3.80 (s, H₃-10) and three aromatic protons at δ_H 7.37 (d, J = 3.1 Hz, H-6), 7.06 (dd, J = 3.2, 9.1 Hz, H-7) and 7.09 (t, J = 9.1 Hz, H-8). The ¹³C NMR spectrum (Table 1) of 2 displayed 12 carbon signals, including two carboxyl carbons, six aromatic carbons, an oxygenated quaternary carbon, a methoxyl and two methyls. The aforementioned NMR data of 2 showed great similarity to that of 2-(2-acetyl-4-methoxyphenoxy)acetic acid ^[13]. The main differences were the appearance of oxydimethylacetic acid and carboxyl group in 2, instead of oxyacetic acid and acetyl in 2-(2-acetyl-4-methoxyphenoxy)acetic acid, which was verified by the HMBC correlations (Fig. 2) from H-6 to C-3, C-7 and C-9, H₃-11 to C-1, C-2 and H₃-12 to C-1, C-2 together with the chemical shift of C-3 (carboxyl group, δ_C 169.7). Taken together, the complete structure of 2 was resolved as 2-((2-carboxypropan-2-yl)oxy)-5-methoxybenzioc acid.

In addition, on the basis of spectral data and compared with those reported in the literatures, five known meroterpenoid derivatives (3−7) were identified as (3S, 4S)-3,4-dihydroxy-6-methoxy-2,2-dimethylchromom (3) ^[14], (3R, 4R)-3,4-dihydroxy-6-methoxy-2,2-dimethylchromom (4) ^[14], (3S, 4R)-3,4-dihydroxy-6-methoxy-2,2-dimethylchromom (5) ^[14], 3-hydroxy-6-methoxy-2,2-dimethylchroman-4-one (6) ^[15], 7-desoxypanepoxydol (7) ^[2]. Among of them, compounds 3 and 4 were a pair of enantiomers, which were isolated from different fractions due to their difference in solubility.

A plausible biosynthetic pathway for compounds 1−7 is presented in Scheme 1. Briefly, compound 7 was derived from prenylhydroquinone (i) via reduction and oxidation. What is more, intermediate i would be cyclized, methylated and hydroxylated to generate compounds 3, 4 and 5 which would be transformed into compounds 2 and 6 via oxidation ^[16]. Then, compound 1 was generated via carbocation rearrangement and oxidation of 6 ^[17].

  1.  Plausible biosynthetic pathway of 1–7

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All isolated compounds were evaluated for antibacterial activities against Staphylococcus aureus, Escherichia coli, Bacillus subtilis, Pseudomonas aeruginosa and Bacillus Calmette–Guérin. Compound 3 displayed weak antibacterial activity against Bacillus Calmette–Guérin with the inhibition radio of 83.6% at 100. Other compounds showed no antibacterial activities at 100 μmol·L⁻¹. In particular, compound 3 showed antibacterial activity against Bacillus Calmette –Guérin. However, compounds 4 and 5 exhibited no antimicrobial activity, which interestingly proved that the chirality of compounds could influence the antimicrobial activity.

Experimental

General

Optical rotations (OR) were recorded on an Anton Paar MCP 200 Automatic Polarimeter. UV spectra were obtained on a Thermo Genesys-10S UV-vis spectrophotometer, respectively. NMR spectra were recorded with a Bruker Avance-500 spectrometer in CD₃OD (δ_H 3.31/δ_C 49.00) or CDCl₃, (δ_H 7.26/δ_C 77.16). HSQC and HMBC experiments were optimized for 145.0 and 8.0 Hz, respectively. HR-ESI-MS data were obtained on an Agilent Accurate Mass-Q-TOF LC/MS 6520 instrument. OD absorbance data were tested on a Spectra Max 190 microplate reader.

Solvents including methanol, dichloromethane, and ethyl acetate used for extraction and chromatographic separation were of analytical grade. TLC was carried out on Silica gel HSGF₂₅₄ plates and the spots were visualized by UV at 254 nm or sprayed with 10% H₂SO₄ followed by heating. Silica gel (150–250 μm, Qingdao Haiyang Chemical Co., Ltd.) and octadecylsilyl (ODS, 50 μm, YMC Co., LTD) were used for column chromatography (CC). HPLC separation was performed on an Agilent 1200 HPLC system with a DAD (Diode Array Detector) detector using an ODS column (C₁₈, 250 mm × 9.4 mm, YMC Pak, 5 μm) at a flow rate of 2.0 mL·min⁻¹

Cultivation, fermentation and isolation

The fruiting body of mushroom Panus lecomtei was collected in Daocheng Yading Natural Reserve, Sichun of China. The strain of P. lecomtei was isolated from the fruiting bodies of mushroom P. lecomtei and identified by Prof. ZHAO Rui-Lin from State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences.

The strain was cultured on PDA plates and incubated at 28 ºC for 7 days. Then agar plugs were inoculated in a 500 mL Erlenmeyer flask containing 200 mL PDB, and incubated at 28 ºC on a rotary shaker at 180 rpm for 7 days as seed medium. Large scale cultivation was carried out in 500 mL Fernbach flasks each containing 80 g of rice and 100 mL of distilled H₂O. Each flask was inoculated with 5 mL seed medium and incubated at 28 ºC for 28 days. The fermented rice substrate was extracted with EtOAc by exhaustive maceration (4 × 4 L), and the organic solvent was evaporated to dryness under vacuum to afford the crude extract (17.28 g).

The crude extract (17.28 g) was subjected to a silica gel eluted with a gradient of mineral ether–EtOAc (V/V 100∶0, 100∶1, 100∶2, 100∶4 and 100∶5, each 500 mL), CH₂Cl₂–acetone (V/V 50∶1, 20∶1 and 10∶1, each 500 mL) and CH₂Cl₂–MeOH (V/V 50∶1, 20∶1, 10∶1, 5∶1, 2∶1 and 0∶100 mL, each 500 mL) to give 12 fractions (Fr. 1 to Fr. 12). Fr. 2 (1.4 g) and Fr. 3 (2.1 g) were merged named Fr. A which was further separated to ODS CC by elution with MeOH–H₂O (V/V 10%–100%) to give 11 subfractions (Fr. A-1 to Fr. A-11). Compound 2 (5.3 mg, t_R = 34.5 min) were isolated from Fr. A-3 (37.0 mg) by semi-preparative HPLC (20% MeCN in H₂O). Fr. A-4 (225.3 mg) was purified by semi-preparative HPLC (35% MeCN in H₂O) to give 1 (4.28 mg, t_R = 7.6 min) and 6 (2.1 mg, t_R = 32.5 min). Fr. 4 (0.9 g) was separated by ODS CC eluted with MeOH-H₂O (V/V 20%–100%) to give 6 subfractions (Fr. 4-1 to Fr. 4-6). Compound 3 (735.3 mg) were obtained from Fr. 4-1 (735.3 mg). Fr. 5 (1.2 g) was subjected to ODS CC eluted with MeOH–H₂O (V/V 20%–100%) to give 11 subfractions (Fr. 5-1 to Fr. 5-11). Fr. 5-6 (961.6 mg) was purified by semi-preparative HPLC (28% MeCN in H₂O) to 4 (551.11 mg, t_R = 19.3 min) and 5 (403.5 mg, t_R = 23.5 min). Fr. 6 (3.0 g), Fr. 7 (0.8 g) and Fr. 8 (2.4 g) were merged named Fr. B, which was separated by ODS CC eluted with MeOH−H₂O (V/V 20%–100%) to give 20 subfractions (Fr. B-1 to Fr. B-20). Fr. B-8 (132.1 mg) was separated by HPLC (22% MeCN in H₂O) to give 7 (4.5 mg, t_R = 71.2 min).

5-Methoxy-3′,3′-dimethyl-3H-spiro[benzofuran-2,2′-oxiran]-3-one (1). orange red oil; ¹H NMR data (500 MHz, CD₃OD), Table 1; ¹³C NMR data (125 MHz, CD₃OD), Table 1; $\left[ {\text{α}} \right]_{\rm{D}}^{25}$+2 (c 0.5 MeOH); HR-ESI-MS m/z 221.0822 [M + H]⁺ (Calcd. for C₁₂H₁₃O₄, 221.0808).

2-((2-Carboxypropan-2-yl)oxy)-5-methoxybenzioc acid (2): yellow oil. UV (MeOH) λ_max (log ε) 214 (0.26), 240 (0.06), 300 (0.03) nm; ¹H NMR (500 MHz, CD₃OD) data, Table 1; ¹³C NMR data (125 MHz, CD₃OD), Table 1; HR-ESI-MS m/z 255.0987 [M + H]⁺ (Calcd. for C₁₂H₁₅O₆, 255.0863).

(3S,4S)-3,4-Dihydroxy-6-methoxy-2,2-dimethylchromom (3). colorless needle. The NMR data were identical with the reported previously ^[14]; $\left[ {\text{α}} \right]_{\rm{D}}^{25}$–10 (c 0.5 MeOH); HR-ESI-MS m/z 247.0952 [M + Na]⁺ (Calcd. for C₁₂H₁₆O₄Na, 247.0941).

(3R,4R)-3,4-Dihydroxy-6-methoxy-2,2-dimethylchromom (4). colorless needle. The NMR data were identical with the reported previously ^[14]; $\left[ {\text{α}} \right]_{\rm{D}}^{25}$+8 (c 0.5 MeOH); HR-ESI-MS m/z 247.0944 [M + Na]⁺ (Calcd. for C₁₂H₁₆O₄Na, 247.0941).

(3S,4R)-3,4-Dihydroxy-6-methoxy-2,2-dimethylchromom (5). colorless needle. The NMR data were identical with the reported previously ^[14]; $\left[ {\text{α}} \right]_{\rm{D}}^{25}$ = –26 (c 0.5 MeOH); UV (MeOH) λ_max (log ε) 227 (0.25), 297 (0.10) nm; HR-ESI-MS m/z 247.0955 [M + Na]⁺ (Calcd. for C₁₂H₁₆O₄Na, 247.0941).

3-Hydroxy-6-methoxy-2,2-dimethylchroman-4-one (6). yellow oil. The NMR data were identical with the reported previously ^[15]; HR-ESI-MS m/z 223.0945 [M + H]⁺ (Calcd. for C₁₂H₁₅O₄, 223.0965).

7-Desoxypanepoxydol (7). white crystal. The NMR data were identical with the reported previously ^[2]; HR-ESI-MS m/z 197.1189 [M + H]⁺ (Calcd. for C₁₁H₁₇O₃, 197.1172).

Antibacterial assay

Five pathogenic bacteria: Assay for antibacterial activities including Staphylococcus aureus CGMCC 1.2465, Escherichia coli CGMCC 1.2340, Bacillus subtilis ATCC 6633, Pseudomonas aeruginosa PAO1 and Bacillus Calmette–Guérin Pasteur 1173P2, were carried out as previously reported methods ^[18-21]. MIC values for compounds that displayed inhibition zones were then tested in the 96-well plates with corresponding broth. The influence of these compounds on the bacterial growth was assessed after incubation at 37 °C for 12 h, and the MIC values was defined as the minimum concentration of compounds that inhibited visible microbial growth compared with the blank group.