A New Benzoquinone and a New Benzofuran from the Edible

Food Chemistry 141 (2013) 1614–1618

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Food Chemistry

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A new benzoquinone and a new benzofuran from the edible mushroom Neolentinus lepideus and their inhibitory activity in NO production inhibition assay ⇑ ⇑ Yongxia Li a,b,1, Li Bao a,1, Bin Song c, Junjie Han b, Heran Li b, , Feng Zhao d, Hongwei Liu a, a State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, No. 9, Beiertiao, Zhongguancun, Haidian District, Beijing 100190, People’s Republic of China b College of Pharmacy, Soochow University, No. 199, Ren Ai Rd., Suzhou Industrial Park, Suzhou, People’s Republic of China c Guangdong Institute of Microbiology, Guangdong Academy of Sciences, No. 100, Xianlie Road, Yuexiu District, Guangdong 510070, People’s Republic of China d School of Pharmacy, Yantai University, No. 32, Qingquan Road, Laishan District, Yantai 264005, People’s Republic of China article info abstract

Article history: The fruiting bodies or mycelia of mushrooms have been used as food and food-ﬂavoring material for cen- Received 16 October 2012 turies due to their nutritional and medicinal value and the diversity of their bioactive components. The Received in revised form 21 February 2013 present research is the ﬁrst to investigate the bioactive secondary metabolites from the solid culture of Accepted 30 April 2013 the edible mushroom Neolentinus lepideus. Two new secondary metabolites, 5-methoxyisobenzofuran- Available online 23 May 2013 4,7(1H,3H)-dione (1) and 1,3-dihydroisobenzofuran-4,6-diol (2), as well as seven known compounds including one benzoquinone derivative (3) and six cinnamic acid derivatives (4–9) were obtained. Their Keywords: structures were established by means of spectroscopic methods, including 1D and 2D NMR. The bioactiv- Neolentinus lepideus ity on the nitric oxide production in lipopolysaccharide-induced macrophages was evaluated for all Structure elucidation NO inhibition metabolites (1–9) isolated. Compound 1 showed strong NO inhibitory activity with the IC50 value of Antioxidation 6.2 lM. Compound 2 displayed moderate NO inhibitory activity with the IC50 value of 88.8 lM. In the DPPH scavenging assay, compound 2 displayed antioxidant activity with IC50 of 68.6 lM. The discovery of new NO production inhibitors from N. lepideus expands its usage as a functional food. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction extracts from the fruiting bodies of L. lepideus has been conﬁrmed to contain antihyperlipidemic (Yoon, Lee, et al., 2011), antioxidant Mushrooms have attracted much more attention as functional (Yoon, Alam, et al., 2011), antityrosinase (Yoon, Alam, et al., 2011), foods or physiologically beneﬁcial medicine due to their nutritional and immunomodulating activities (Jung et al., 2008). A number of and medicinal value and the diversity of their bioactive secondary secondary metabolites including a-copaene, a-elemene, metabolites (Barro, Baptista, Estevinho, & Ferreira, 2007; Barros b-farnesene, a-and c-muurolene, d-cadinene, cadina-1,4-diene, et al., 2007). The technique of solid state fermentation (SSF) refers a-calacorene, ()-torreyol, ()-T-muurolol, (+)-Tcadinol, a-cadinol, to microbial growth on moist solid medium without free water. cubenol, epicubenol, farneso and drimenol, lentideusether, The traditional Japanese food ‘‘natto’’ (Bacillus-fermented soybean) isolentideusether and 10-hydroxy-lentideusether have been and the Chinese food ‘‘anka’’ (Monascus-fermented rice) were isolated from the fruiting bodies of the basidiomycete L. lepideus produced by this method. Our early studies on edible mushroom (Hanssen, 1982, 1985). In the current research, we reported the iso- showed that mushrooms fermented with this technique could lation of one new benzoquinone (1), one new benzofuran (2), as well enhance their ability in production of the bioactive secondary as seven known secondary metabolites including benzoquinone metabolites (Wang, Bao, Liu, et al., 2012; Wang, Bao, Yang, Dai, derivative (3) and six known cinnamic acid derivatives from the et al., 2012; Wang, Bao, Yang, Li, et al., 2012; Wang, Li, et al., solid culture of N. lepideus fermented on cooked rice. Their chemical 2012; Wang et al., 2013). structures (Fig. 1) were elucidated by their physicochemical proper- Neolentinus lepideus is a basidiomycete mushroom of the genus ties and spectral data, including 1D, 2D NMR and HRESI-MS. Neolentinus, previously well known as Lentinus lepideus. It is one of the popular edible mushroom in China, Japan and Korea. The 2. Materials and methods

2.1. General methods ⇑ Corresponding authors. Tel.: +86 512 65882541 (H. Li), tel.: +86 10 62566577 (H. Liu). E-mail addresses: [email protected] (H. Li), [email protected] (H. Liu). UV data were recorded on a Shimadzu Biospec-1601 spectro- 1 These authors contributed equally to this work. photometer. IR data were recorded using a Nicolet Magna-IR 750

(Fr. 2–1–Fr. 2–10). Fr. 2–3 (50 mg) was separated by ODS column using a gradient of methanol–water (20–100%) to afford 9 (22 mg). Fr. 2–5 (80 mg) was separated by ODS column eluted with 200 ml of methanol–water (50%, v/v) followed by HPLC using 29%

acetonitrile in water to afford 3 (12 mg, tR 14.92 min). Compounds 4 (2 mg, tR 15.65 min), 7 (2 mg, tR 18.61 min), and 8 (40 mg, tR 15.23 min) were obtained from subfraction 2–8 (70 mg) by HPLC puriﬁcation using 49% methanol in water. Fr. 3 (100 mg) eluted with dichloromethane–acetone (75:1, v/v) was separated by ODS column using a gradient of methanol–water (20–100%) to give seven subfractions (Fr. 3–1–Fr. 3–7). Fr. 3–4 (33 mg) was puriﬁed by

HPLC using 40% methanol in water to afford 5 (3.8 mg, tR 23.78 min) and 6 (5 mg, tR 28.25 min). Fr. 5 (556 mg) eluted with dichloromethane–acetone (20:1, v/v) was separated by ODS column using a gradient methanol (20–100%) in water to afford thir- teen subfractions (Fr. 5–1–Fr. 5–13). Fr. 5–3 (35 mg) was subjected to a silica gel column eluted with 200 ml of dichloromethane– Fig. 1. Secondary metabolites isolated from Neolentinus lepideus. acetone (90:1, v/v) to give compound 1 (12 mg). Fr. 10 (460 mg) spectrophotometer. ESIMS and HRESIMS data were obtained using eluted with dichloromethane–methanol (20:1, v/v) was ﬁrst a Bruker APEX III 7.0 T spectrometer. 1H and 13C NMR, along with separated by ODS column using a gradient of methanol–water 2D NMR spectra were acquired with Bruker AV-500 NMR spec- (20–100%) to afford 11 subfractions (Fr. 10–1–Fr. 10–11). Compound 2 (3.2 mg, t 22.1 min) was isolated from subfraction trometer, using (methanol-d4: dH 3.33/dC 49.9; deuterated chloro- R 10–4 (18 mg) by HPLC using 22% methanol in water. The other form: dH 7.26/dC 77.7) as references. The HMQC and HMBC experiments were optimised for 145.0 and 8.0 Hz, respectively. fractions were all separated during the isolation process. The de- tails of the isolation procedure for other fractions are not described TLC was carried out on Silica gel 60F254 and the spots were visual- since we did not get secondary metabolites from these fractions in ised by spraying with 10% H2SO4 and heating. LH-20 (Amersham Biosciences) and ODS (Lobar, 40–63 lm, Merck) were used for col- this research. umn chromatography. Preparative HPLC was performed on an Agi- lent 1200 HPLC system using an ODS column (RP-8, 250 10 mm, 2.3.2. 5-Methoxyisobenzofuran-4,7(1H,3H)-dione (1)

YMC Pak, 5 lm; detector: UV) with a flow rate of 2.5 ml/min. Yellow needle crystalline; UV (methanol) kmax nm (log e): 200 (4.02), 255(3.66), 295 (2.82); IR (neat) mmax: 3413, 2931, 1726, 1641, 1589, 1440, 1408, 1236, 1120, 1039, 892 cm1; Positive HRE- 2.2. Fungal material + 1 SIMS: m/z [M+H] 181.0495 (calcd. for C9H8O4H, 181.0529); H 13 The fungal strain used in this study was isolated from fruiting NMR and C NMR data see Table 1. body of mushroom N. lepideus in Guangdong Province, in October 2004, and was identified by Professor Bin Song. The sequence data 2.3.3. 1,3-Dihydroisobenzofuran-4,6-diol (2) derived from the fungal strain has been submitted and deposited at Colourless oil; UV (methanol) kmax nm (log e): 230 (4.22), 280 GenBank with accession number JX401542. BLAST search result (4.16); IR (neat) mmax: 3260, 1606, 1459, 1340, 1140, 1018, 1 + showed that the sequence was similar (99%) to the sequence of 885 cm ; Positive HRESIMS: m/z [M+H] 153.0548 (calcd. for 1 13 N. lepideus (HM536076.1). The strain is preserved with Guangdong C8H8O3H, 153.0546). H NMR and C NMR data see Table 1. Culture Collection Center, Guangdong Institute of Microbiology with accession number GIMCC 5.195. The fungal strain was cul- 2.4. NO inhibition assay (Qiu et al., 2008) tured on slants of potato dextrose agar at 25 °C for 10 d. Agar plugs were inoculated in 500 ml Erlenmeyer flask containing 120 ml of Mouse monocyte-macrophages RAW 264.7 (ATCC TIB-71) were media (0.4% glucose, 1% malt extract, and 0.4% yeast extract), and purchased from the Chinese Academy of Science. RPMI 1640 the final pH of the media was adjusted to 6.5 before sterilisation, medium, penicillin, streptomycin, and foetal bovine serum were and incubated at 25 °C on a rotary shaker at 170 rpm for 7 d. Large purchased from Invitrogen (NY, USA). Lipopolysaccharide, DMSO, scale cultivation was carried out in thirty 500 ml Fernbach flasks 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bro- each containing 80 g of rice and 120 ml of distilled water. Each mide (MTT), and hydrocortisone were obtained from Sigma flask was inoculated with 5.0 ml of the culture medium and incu- Chemical Co. (St. Louis, MO, USA). RAW 264.7 cells were bated at 25 °C for 40 d. maintained in RPMI 1640 medium supplemented with penicillin

Table 1 2.3. Extraction and isolation NMR spectral data for 1 and 2 (500 MHz).

Position 1 (CDCl ) 2 (CD OD) 2.3.1. General 3 3 The fermented rice substrate was extracted with ethyl acetate dC dH (J in Hz) dC dH (J in Hz) by exhaustive maceration (3 1 l), and the organic solvent was 1 73.7 a 5. 00 (s) 75.3 4.94 (s) a evaporated to dryness under vacuum to afford the crude extract 3 73.3 5. 00 (s) 73.1 4.94 (s) 4 178.0 153.9 (21 g). The ethyl acetated extract (20 g) was subjected to a silica 4a 142.1 117.7 gel column chromatography eluted with dichloromethane–ace- 5 160.1 102.9 6.18 (d, 2.0) tone followed by dichloromethane–methanol in a stepwise man- 6 107.8 5.84 (s) 160.4 ner. Twelve fractions (Fr. 1–Fr. 12) were obtained on the basis of 7 183.5 100.5 6.19 (d, 2.0) TLC analysis. Fr. 2 (825 mg) eluted with dichloromethane–acetone 7a 146.1 143.3 CH3O-5 57.0 3.83 (s) (100:1, v/v) was further separated on a silica gel column eluted with a gradient of n-hexane–ethyl acetate to afford 10 subfractions a Signals might be interchangeable. 1616 Y. Li et al. / Food Chemistry 141 (2013) 1614–1618

(100 U/ml), streptomycin (100 lg/ml), and 10% heat-inactivated radicals, resulting in a ﬁnal concentration of 0.2 mM DPPH. The foetal bovine serum at 37 °C in a humidiﬁed incubator with 5% mixture was shaken vigorously and left to stand for 30 min in

CO2 and 95% air. The medium was routinely changed every 2 d. the dark, and the absorbance was then measured at 517 nm. Ascor- RAW 264.7 cells were passaged by trypsinisation until they at- bic acid was used as a positive control. tained confluence and were used for assays during the exponential growth phase. 3. Results and discussion Compounds 1–9 were dissolved in DMSO and were further di- luted with the culture medium to give a final DMSO concentration 3.1. Structure elucidation of 0.2% in the assay. This concentration of DMSO had no significant effect on the growth of the cell line tested. Cell concentration was ad- Compound 1 was obtained as yellow needle crystal. The molec- justed to 5 105 cells/ml, and 200 ll were seeded in every well of a ular formula was established as C H O on the basis of HRESIMS 96-well plate. After 1 h incubation, the cells were treated with 1 lg/ 9 8 4 data (m/z 181.0495 ([M+H]+)), requiring 6° of unsaturation. The ml of LPS and various concentrations of test compounds for 24 h. 1H NMR spectrum of 1 exhibited signals due to one olefinic proton Control groups received an equal amount of DMSO. As a parameter at d 5.84 (1H, s), one oxymethyl at d 3.83 (3H, s), and two oxygen- of NO release, the nitrite concentration was measured in the super- ated methylenes at d 5.00 (4H, s). The 13C NMR spectrum evidenced natant of RAW 264.7 cells by the Griess reaction. Briefly, 100 llof a benzoquinone moiety at d 107.8, 142.1, 146.1, 160.1, 178.0 and culture medium in each well was transferred to another plate, and 183.5, two oxygen atoms at d 73.3 and 73.7, and one methoxyl the level of NO was assessed by measuring the accumulation of group at d 57.0. The complete assignment was performed by anal- nitrite (NO) using 100 llofGriess agent (mixture of 0.1% N-[naph- 2 yses of HMBC data (Fig. 2). The long-range correlations from H-1 to thalen-1-yl]ethylenediamine in 5% phosphoric acid and 1% sulfanil- C-3, C-4a, C-7, and C-7a, from H-3 to C-1, C-4, C-4a, and C-7a, from amide). The concentration of NO was calculated by a working curve 2 H-6 to C-4, C-5, C-7, and C-7a, from MeO-5 to C-5 observed in the from 0, 1, 2, 5, 10, 20, 50, 100 lM NaNO solutions. The inhibitory 2 HMBC spectrum confirmed the structure of 1 as 5-meth- rate of the compounds on NO production induced by LPS was calcu- oxyisobenzofuran-4,7(1H,3H)-dione. lated by the NO levels as follows. The inhibition rate was calculated 2 Compound 2 was obtained as colorless oil. The molecular for- and plotted versus test concentrations to afford the IC50. mula was determined to be C8H8O3 on the basis of HRESIMS data + 1 ½NO2 LPS ½NO2 LPSþsample (m/z [M+H] 153.0548), requiring 5° of unsaturation. In the H Inhibitory rate ½%¼100 NMR spectrum of 2, the resonances corresponding to two aromatic ½NO2 LPS ½NO2 untreated protons at d 6.18 (d, J = 2.0 Hz, H-5) and 6.19 (d, J = 2.0 Hz, H-7) were observed, which together with the 13C NMR signals at d 2.5. MTT assay (Wang et al., 2013) 100.5, 102.9, 117.7, 143.3, 153.9, 160.4 indicated the presence of tetrasubstituted benzene ring. Two oxygenated methylenes were Cytotoxic activity for cancer cell lines HepG2 and K562 was eval- also revealed by the NMR signals at d 4.94 (4H, s, H-1/H-3) and d uated with the MTT assay. Cytotoxic activity for the HeLa cancer cell 73.1 and 75.3. The skeleton of benzofuran ring was finally assigned line was evaluated with the MTT assay. After treating cells with the by analyses of HMBC data (Fig. 2). In the HMBC spectrum, the long- compounds tested (DMSO as solvent) for 72 h, 50 ll of MTT/med- range correlations from H-1 to C-3, C-4a, C-7, and C-7a, from H-3 to ium solution (0.5 mg/ml) was added to each well, and the tumor C-1, C-4, C-4a, and C-7a, from H-5 to C-4, C-4a, C-7, and C-6, and cells were incubated at 37 °C in a humidified atmosphere of 5% from H-7 to C-1, C-4a, C-5, and C-6 were observed. The bigger

CO2 air for 4 h. Upon removal of MTT/medium, 100 ll of DMSO chemical shift of C-4 (dC 153.9) and C-6 (dC 160.4), as well as the was added to each well, and the plate was agitated at 60 rpm for molecular formula of 2 indicated that C-4 and C-6 all bear a hydro- 5 min to dissolve the precipitate. The assay plate was read at xyl group. Thus, the structure of 2 was identified as 1, 540 nm using a microplate reader. IC50 (±SD) values were calcu- 3-dihydroisobenzofuran-4,6-diol. lated using linear interpolation of inhibition curves for three inde- Compounds 3–9 were identified as 5-methoxy-2,3-dimethylcy- pendent experiments, each carried out in triplicate. The reference clohexa-2,5-diene-1,4-dione (3)(Ried & Schaefer, 1971), (E)-3-(3- substance used was etoposide that showed cytotoxicity against methoxyphenyl)acrylic acid (4)(Aung, Furukawa, Nikai, Niwa, & HepG2 and K562 with IC50 of 3.68 and 2.66 lM, respectively. Takaya, 2011), 3-(4-methoxyphenyl)propan-1-ol (5)(Duarte et al., 2004), (E)-3-(4-methoxyphenyl)acrylic acid (6)(Aung et al., 2.6. DPPH scavenging assay (Hatano, Kagawa, Yasuhara, & Okuta, 2011), (E)-methyl 3-(2-methoxyphenyl)acrylate (7)(Wheeler & 1988) Covarrubias, 1963), (E)-methyl 3-(3-hydroxy-4-methoxyphenyl) acrylate (8)(Lan, 2010), and (E)-methyl 3-(4-methoxyphenyl) Each compound in DMSO (final concentration 200 lM, 100 ll) acrylate (9)(Lan, 2010) by comparison of their NMR and MS data was mixed with 100 ll ethanolic solution containing DPPH (sigma) with literature values, respectively. The benzoquinone derivative

H O H H H H HO H

O O

H H3CO H H H H O OH

Fig. 2. HMBC correlations observed for 1 and 2. Y. Li et al. / Food Chemistry 141 (2013) 1614–1618 1617

HRESI-MS. Compound 2 displayed moderate DPPH scavenging activity. Although needing further investigation, the current research on N. lepideus demonstrates that the solid culture of this mushroom on cooked rice could be used as good functional food.

Acknowledgements

This work was supported by National Key Basic Research Pro- ject of China (2009CB522300), and Grants from NSFC (31000036 and 21072219).

References

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