Article pubs.acs.org/JAFC
Chemical Constituents and Their Bioactivities of Mushroom Phellinus rhabarbarinus Tao Feng,† Jin-Long Cai,‡ Xue-Mei Li,† Zhong-Yu Zhou,§ Zheng-Hui Li,*,† and Ji-Kai Liu*,† †
College of Pharmacy, South-Central University for Nationalities, Wuhan 430074, China School of Agriculture and Biological Technic, Yunnan Agricultural University, Kunming 650201, China § Key Laboratory of Plant Resources Conservation and Sustainable Utilization, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, China J. Agric. Food Chem. 2016.64:1945-1949. Downloaded from pubs.acs.org by UNIV OF SOUTH DAKOTA on 09/12/18. For personal use only.
‡
S Supporting Information *
ABSTRACT: Phellinus rhabarbarinus soaked in wine has folk usages by local residents of Ailao mountain of Yunnan province, China, which were to daub the wound to prevent infection and to drink to enhance immunity and treat other diseases such as cough, gastritis, and cancer. Systemic investigation on the chemical constituents of fruiting bodies of P. rhabarbarinus resulted in the isolation of 11 lanostane triterpenoids (1−10) including three new ones, namely, phellibarins A−C (1−3), together with five ergosterols (11−15). This is the first time reporting secondary metabolites of P. rhabarbarinus. Compounds 2, 3, 7, and 8 showed inhibitory activities against nitric oxide (NO) production in LPS-activated RAW264.7 macrophages, whereas compounds 2−4, 6, 7, and 10 exhibited cytotoxicities against human cancer cell lines. The results of this assessment suggested that the lanostane triterpenoids in fruiting bodies of P. rhabarbarinus played key roles in its folk usages. KEYWORDS: Phellinus rhabarbarinus, lanostane triterpenoid, NO production inhibition, cytotoxicity
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INTRODUCTION Fungi are widely known as high-performance creators of structurally and biologically diverse natural products.1−7 Mushrooms, which are a group of fungi called macromycetes, have traditionally played important roles in agriculture, food, and medicine.1−7 Species of the genus Phellinus are widely known as harmful fungi that cause white pocket rot and severe plant diseases such as canker or heart rot in living trees.8 However, certain Phellinus species, including Phellinus igniarius, have been used as traditional medicines.9,10 Chemical investigations of these species have described numerous natural products, particularly polysaccharides and polyphenols.11−19 The aqueous extract of Phellinus rhabarbarinus was reported to exhibit strong anti-HIV-1 activity. 20 In China, P. rhabarbarinus widely grows on the Ailao mountain of Yunnan province, where the residents soaked it with wine for folk usages, such as daubing the wound to prevent infection and drinking to enhance immunity and treat other diseases such as cough, gastritis, and cancer. Our biological activity experiments indicated that the EtOAc layer of crude EtOH exact of P. rhabarbarinus exhibited certain inhibitory activity against nitric oxide production in LPS-activated RAW264.7 macrophages and cytotoxicity against human cancer cell lines, in good agreement with the folk usage of P. rhabarbarinus. These results prompted us to make an investigation of its chemical constituents. Our systematic isolation of EtOH extract of P. rhabarbarinus gave a number of lanostane triterpenoids (1−10) including three new ones, namely, phellibarins A−C (1−3), together with five known ergosterols (11−15) (Figure 1). All these compounds were evaluated for their inhibitory activities against NO production in LPS-activated RAW264.7 macrophages and their cytotoxicity against five human cancer cell lines. © 2016 American Chemical Society
MATERIALS AND METHODS
Instrumentation. Melting points were measured using a WRX-4 micro melting point apparatus. OR were measured with an Horiba SEPA-300 polarimeter. IR spectra were obtained using a Bio-Rad FtS135 spectrometer. NMR spectra were measured on a Bruker AV600 MHz spectrometer with tetramethylsilane as the internal standard. ESI-MS and HR-ESI-MS were recorded with an APIQSTARPulsar 1 spectrometer. Column chromatography (CC) was performed with normal-phase silica gel, Sephadex LH-20, and reversed-phase C18 silica gel. A BUCHI C-615 instrument was used for MPLC preparation equipped with C18 silica gel columns. An Agilent 1100 series instrument equipped with Agilent ZORBAX SB-C18 column (5 μm, 4.6 mm × 150 mm) was used for HPLC analysis, and a semipreparative Agilent ZORBAX SB-C18 column (5 μm, 9.4 mm × 150 mm) was used for the sample preparation. Fractions were monitored by TLC, and spots were visualized by heating TLC plates sprayed with 10% H2SO4 in ethanol. Fungus Material. Fruiting bodies of P. rhabarbarinus were collected from the Ailao mountain of Yunan province, China, in September 2014, and identified by Dr. Yu-Cheng Dai (Beijing Forestry University). The voucher specimen (20140908004) was deposited at College of Pharmacy, South-Central University for Nationalities, China. Extraction and Isolation. The air-dried (80% water removed) mushroom materials (220 g) were extracted three times with EtOH (1 L) to obtain a crude extract (32.5 g), which was partitioned by H2O and EtOAc (1:1) to give an EtOAc extract (7.6 g). The latter was subjected to silica-gel CC and eluted with a gradient system of petroleum ether/Me2CO (from 20:1 to 1:1) to give six fractions (A− F). Fraction B (0.8 g) was separated by silica gel (petroleum ether/ Received: Revised: Accepted: Published: 1945
January 13, 2016 February 22, 2016 February 23, 2016 February 23, 2016 DOI: 10.1021/acs.jafc.6b00176 J. Agric. Food Chem. 2016, 64, 1945−1949
Article
Journal of Agricultural and Food Chemistry
Figure 1. Structures of compounds 1−15 from fruiting bodies of Phellinus rhabarbarinus. 10% FBS under a humidified atmosphere with 5% CO2 at 37 °C. After 24 h of preincubation, cells were treated with serial dilutions of the test compound, up to a maximum concentration of 25 μM, in the presence of 1 μg/mL LPS (Sigma) for 18 h. The compound was dissolved in DMSO and further diluted in medium to produce different concentrations. NO production in each well was assessed by adding 100 μL of Griess reagent (reagents A and B, Sigma) to 100 μL of each supernatant from the LPS-treated or LPS- and compound-treated cells in triplicate. After a 5 min of incubation, the absorbance of samples was measured at 570 nm with a 2104 Envision multilabel plate reader (PerkinElmer Life Sciences, Inc., Boston, MA, USA). MG-132 was used as a positive control (IC50 = 0.2 μM). Cytotoxic Assay. Compounds 1−15 were evaluated for their cytotoxicity against five human cancer cell lines: breast cancer SK-BR3, hepatocellular carcinoma SMMC-7721, human myeloid leukemia HL-60, pancreatic cancer PANC-1, and lung cancer A-549 cells. Cells were cultured in RPMI-1640 or in DMEM medium (Hyclone, USA) supplemented with 10% fetal bovine serum (Hyclone, USA) in 5% CO2 at 37 °C. The cytotoxicity assay was performed according to 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) method in 96-well microplates.21 Briefly, 100 μL of adherent cells were seeded into each well of 96-well cell culture plates and allowed to adhere for 12 h before addition of test compounds, whereas suspended cells were seeded just before drug addition with initial density of 1 × 105 cells/mL. Each tumor cell line was exposed to the test compound at concentrations of 0.0625, 0.32, 1.6, 8, and 40 μM in triplicate for 48 h, and all tests were done in twice with cisplatin (Sigma, USA) as a positive control. After compound treatment, cell viability was detected, and a cell growth curve was graphed. IC50 values were calculated by Reed and Muench’s method.22
Me2CO from 15:1 to 8:1) to afford 12 (4.8 mg) and a crystal from a subfraction. The crystal was washed using the mixture of petroleum ether and Me2CO to obtain 11 (123 mg). Fraction C (2.3 g) was first separated by silica gel (petroleum ether/Me2CO from 10:1 to 5:1), then purified by HPLC (MeCN/H2O from 5:5 to 8:2) to afford 1 (1.8 mg), 2 (3.2 mg), 3 (2.7 mg), 4 (7.7 mg), 6 (12 mg), and 7 (5.3 mg). Fraction D (0.9 g) was separated by CC using RP-C18 (MeOH/H2O from 6:4 to 9:1) to give four subfractions (D1−D4). Fraction D2 (230 mg) was separated by reverse-phase C18 (MeOH/H2O from 5:5 to 8:2) to afford 8 (4.3 mg) and 9 (7.9 mg). Fraction D3 (27 mg) was purified by Sephadex LH-20 (MeOH) to afford 5 (34 mg). Fraction E (1.5 g) was separated by reserve-phase C18 (MeOH/H2O from 4:6 to 6:4), then purified by HPLC (a gradient of MeCN/H2O from 6:4 to 7:3) to afford 10 (12 mg), 13 (5.3 mg), 14 (2.1 mg), and 15 (3.3 mg). Physical Data for Phellibarin A (1). Colorless needles (MeOH); mp 132−133 °C; [α]25 D 13.4 (c 0.20, CHCl3); UV (MeOH) λmax (log ε) 203 (3.49), 241 (3.52) nm; IR (KBr) υmax 3447, 2933, 2869, 1698, 1644, 1379, 1217, 1021, 754 cm−1; 1H and 13C NMR data in Table 1; positive-ion HRESIMS m/z 479.3489 (calcd for C30H48O3Na [M + Na]+, 479.3496). Physical Data for Phellibarin B (2). White powder; [α]25 D 22.3 (c 0.18, CHCl3); IR (KBr) υmax 3544, 3073, 1718, 1694, 1645, 1456, 1386, 1375 cm−1; 1H and 13C NMR (150 MHz) data in Table 1; negetive-ion HRESIMS m/z 477.3411 (calcd for C30H46O3Na [M + Na]+, 477.3339). Physical Data for Phellibarin C (3). Colorless oil; [α]25 D 37.6 (c 0.10, CHCl3); IR (KBr) υmax 3544, 3073, 1712, 1645, 1456, 1386, 1375 cm−1; 1H and 13C NMR data in Table 1; negetive-ion HRESIMS m/z 501.3564 (calcd for C30H48O3·COOH [M + COOH]−, 501.3585). Nitric Oxide Production in RAW 264.7 Macrophages. Murine monocytic RAW264.7 macrophages were dispensed into 96-well plates (2 × 105 cells/well) containing RPMI 1640 medium (HyClone) with 1946
DOI: 10.1021/acs.jafc.6b00176 J. Agric. Food Chem. 2016, 64, 1945−1949
Article
Journal of Agricultural and Food Chemistry Table 1. 1H and 13C NMR Spectroscopic Data of 1−3 in CDCl3a 2
a
3
4
no.
δH
δC
δH
δC
δH
δC
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
1.52 (m); 1.42 (m) 1.90 (m); 1.58 (m) 3.40 (br. s)
30.2 25.8 76.2 37.7 44.3 18.3 26.2 135.0 133.9 37.0 21.1 31.3 45.1 49.8 31.3 28.1 46.1 15.7 19.1 42.8 16.5 80.9 201.6 121.0 158.2 28.4 21.6 28.4 22.3 24.3
1.95 (m); 1.60 (m) 2.56 (m); 2.38 (m)
36.2 34.8 218.1 47.6 51.3 19.6 26.5 135.1 133.5 37.0 21.2 30.9 45.1 49.8 31.4 28.1 46.0 15.9 18.9 42.7 16.9 81.1 201.5 120.9 158.3 28.3 21.6 26.3 21.5 24.4
1.95 (m); 1.60 (m) 2.56 (m); 2.38 (m)
36.2 34.8 218.2 47.6 51.3 19.6 26.5 135.2 133.5 37.1 21.2 31.0 44.9 49.7 31.2 27.5 47.5 16.1 18.9 42.8 12.7 74.6 125.2 140.7 71.0 30.0b 30.2b 26.3 21.5 24.4
1.48 (m) 1.56 (m); 1.48 (m) 2.01 (m)
2.02 (m); 1.97 (m) 1.68 (m); 1.60 (m)
1.60 1.86 1.85 0.69 0.96 1.98 0.91 4.10
(m); 1.18 (m) (m); 1.30 (m) (m) (s) (s) (m) (d, 6.8) (br. s)
6.08 (s) 1.94 2.17 0.94 0.85 0.84
(s) (s) (s) (s) (s)
1.58 (m) 1.58 (m) 2.05 (m)
2.01 (m) 1.71 (m); 1.64 (m)
1.62 1.86 1.87 0.71 1.09 2.00 0.94 4.08
(m); 1.21 (m) (m); 1.29 (m) (m) (s) (s) (m) (d, 6.8) (br. s)
6.09 (s) 1.94 2.17 1.06 1.04 0.85
(s) (s) (s) (s) (s)
1.57 (m) 1.59 (m) 2.06 (m)
2.01 (m) 1.68 (m); 1.63 (m)
1.62 1.84 1.40 0.73 1.09 1.76 0.91 4.18 5.64 5.80
(m); 1.20 (m) (m); 1.41 (m) (m) (s) (s) (m) (d, 6.8) (d, 7.0) (dd, 15.6, 7.0) (d, 15.6)
1.31 1.31 1.06 1.04 0.81
(s) (s) (s) (s) (s)
δ in ppm. binterchangeable.
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RESULTS AND DISCUSSION
Structure Elucidation. Compound 1, colorless needles, possessed a molecular formula C30H48O3 with seven degrees of unsaturation as established by the HRESIMS at m/z 479.3489 [M + Na]+ (calcd for C30H48O3Na, 479.3496). In the 1H NMR spectrum (Table 1), signals for eight CH3 (including seven singlets and one doublet) were readily observed. Of them, signals at δH 1.94 and 2.17 (each 3H, s, H-26 and H-27, respectively) suggested the connection of these CH3 to olefinic carbon(s). In addition, signals at δH 3.40 (1H, dd, J = 2.6, 2.6 Hz, H-3) and 4.10 (1H, d, J = 2.2 Hz, H-22) indicated the existence of two oxygenated CH2 in 1, whereas an olefinic proton at 6.08 (1H, s, H-24) was also observed. The 13C NMR spectrum displayed 18 carbon resonances ascribable to 8 CH3, 8 CH2, 6 CH, and 7 quaternary carbons on the basis of HSQC spectrum (Table 1). These data suggested that compound 1 possessed a lanostane triterpenoid backbone similar to that of igniaren D, which was also isolated as known compound 4,23 especially the data from C-1 to C-21. In the 1H−1H COSY spectrum, the correlation between δH 4.10 (1H, d, J = 2.2 Hz, H-22) and δH 1.98 (1H, m, H-20) indicated that C-22 was substituted by a hydroxy group (Figure 2). In addition, the HMBC correlations from H-24 and H-22 to δC 201.6 (s, C-23), as well as HMBC correlations from δH 1.94 (3H, s, H-26) and 2.17 (3H, s, H-27) to δC 158.2 (s, C-25), revealed a 24,25unsaturated-23-keto moiety (Figure 2). The coupling constant
Figure 2. Key 2D NMR correlations of 1 and 3.
of H-3 (br. s) indicated α form of 3-OH. In addition, the data of the side chain were in good agreement with those of perviridisinol A,24 which indicated that C-22 should be S form. Detailed analyses of 2D NMR data identified the structure of 1 which was given the name phellibarin A. Compound 2 was isolated as white powder. Its molecular formula C30H46O3 was determined by HRESIMS at m/z 477.3411 [M + Na]+ (calcd for C30H46O3Na, 477.3339). The NMR data of 2 were in good agreement with those of 1 except that 3-OH was replaced by a carbonyl group, which was supported by HMBC correlations from H-2 to δH 218.1 (s, C3). Detailed analyses of other NMR data suggested that the other parts of 2 were the same as those of 1. Therefore, compound 2 was identified and named phellibarin B. Compound 3 was isolated as a colorless oil. Its MS and NMR data (Table 1) suggested that compound 3 was also a lanostane triterpenoid, which displayed the patterns similar to those of 2. 1947
DOI: 10.1021/acs.jafc.6b00176 J. Agric. Food Chem. 2016, 64, 1945−1949
Article
Journal of Agricultural and Food Chemistry However, two olefinic protons at δH 5.64 (1H, dd, J = 15.6, 7.0 Hz, H-23) and 5.80 (1H, d, J = 15.6 Hz, H-24) and one quaternary carbon at δC 71.0 indicated variety of the side chain. The 1H−1H COSY correlation between δH 4.18 (1H, d, J = 7.0 Hz, H-22) and H-23 (Figure 2) as well as coupling constants of J = 15.6 Hz for H-23 and H-24 suggested a double bond placed between C-23 and C-24 with E form. In addition, the HMBC correlations from δH 1.31 (s, each 3H for H-26 and H-27) and H-24 to δC 71.0 (s, C-25) indicated that C-25 was oxidized into a quaternary carbon (Figure 2) which allowed Me-26 and Me27 to display as singlets in the 1H NMR spectrum. Detailed analyses of other NMR data suggested that the other parts of 3 were the same as those of 2. The NMR data of the side chain of 3 were in good agreement with those of the similarly structured (22R)-cycloart-23-ene-3β,22α,25-triol.25 Systemic study of the chemical constituents of P. rhabarbarinus led to the isolation of other seven known compounds including igniaren D (4),23 24-methylenelanost-8ene-3b,22-diol (5),26 igniaren C (6),23 gilvsin A (7),26 gilvsin C (8),26 igniaren B (9),23 gilvsin D (10),26 (22E,24R)-ergosta5,7,22-triene-3β-ol (11), (22E,24R)-5α,6α-epoxyergosta-8(14),22-dien-3β,7α-diol (12),27 (22E,24R)-5α,6α-epoxyergosta-8,22-dien-3β,7α-diol (13),27 (22E,24R)-ergosta-7,22-dien3β,5α,6β-triol (14),28 and (22E,24R)-3β,5α,9α-trihydroxyergosta-7,22-dien-6-one (15)29 (Figure 1). Biological Activity Assays. Compounds 1−15 were evaluated for their inhibitory activities against NO production in LPS-activated RAW264.7 macrophages and their cytotoxicities against five human cancer cell lines. This assessment revealed that 2, 3, 7, and 8 exhibited inhibitory activities against nitric oxide production in LPS-activated RAW264.7 macrophages, with IC50 values of 22.3, 20.2, 19.3, and 22.3 μM, respectively, whereas the other compounds were inactive (IC50 > 30 μM). In addition, compounds 2−4, 6, 7, and 10 also demonstrated comparable inhibitory activities across the tested cell lines, with IC50 values equal to those of cisplatin (Table 2); the other compounds were inactive (IC50 > 40 μM).
major components in P. rhabarbarinus, whereas the lanostane triterpenoids exhibited NO production inhibitory and cytotoxic activities, suggesting that lanostane triterpenoids in fruiting bodies of P. rhabarbarinus played key roles in its folk usages.
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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.6b00176. NMR and MS of compounds 1−3. (PDF)
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a
HL-60
SMMC-7721
A-549
MCF-7
SW480
2 3 4 6 7 10 cisplatin
13.9 33.1 >40 15.5 14.6 22.9 1.8
28.8 10.7 19.1 >40 17.7 23.7 14.3
15.0 13.7 20.0 22.4 18.5 11.4 7.3
10.2 18.6 14.3 >40 8.8 12.5 14.7
14.5 22.7 39.1 >40 17.8 35.0 11.0
AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected]. *E-mail:
[email protected]. Funding
We are grateful to the National Key Technology Support Program, China (2013BAI11B02), National Natural Science Foundation of China (81373289), West Light Foundation of The Chinese Academy of Sciences (2013312D11016), and Guangdong Provincial Key Laboratory of Applied Botany, South China Botanical Garden, CAS (AB2015001). Notes
The authors declare no competing financial interest.
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ABBREVIATIONS USED MP, melting point; OR, optical rotations; IR, infrared spectroscopy; NMR, nuclear magnetic resonance; ESI-MS, electrospray ionization-mass spectrometry; HR-ESI-MS, highresolution electrospray ionization-mass; CC, column chromatography; MPLC, middle-performance liquid chromatography; HPLC, high-performance liquid chromatography; TLC, thinlayer chromatography; HSQC, 1H-detected heteronuclear single-quantum coherence spectroscopy; HMBC, 1H-detected heteronuclear multiple bond coherence spectroscopy; COSY, homonuclear correlation spectroscopy; ROESY, rotating-frame Overhauser effect spectroscopy
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Table 2. Cytotoxicities of Compounds 2−4, 6, 7, and 10a entry
ASSOCIATED CONTENT
S Supporting Information *
REFERENCES
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IC50, repored in μM.
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DOI: 10.1021/acs.jafc.6b00176 J. Agric. Food Chem. 2016, 64, 1945−1949
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Journal of Agricultural and Food Chemistry
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DOI: 10.1021/acs.jafc.6b00176 J. Agric. Food Chem. 2016, 64, 1945−1949
