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Anti-inflammatory diterpenes and steroids from peels of the cultivated edible mushroom Wolfiporia cocos
Phytochemistry Letters 36 (2020) 11–16
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Anti-inflammatory diterpenes and steroids from peels of the cultivated edible mushroom Wolfiporia cocos
T
Baosong Chena,b, Sixian Wangc, Gaoqiang Liuc, Li Baoa,*, Ying Huangd, Ruilin Zhaoa, Hongwei Liua,b,* a
State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences. No.1 Beichenxi Road, Chaoyang District, Beijing, PR China Savaid Medicine School, University of Chinese Academy of Sciences, Beijing, PR China c Hunan Provincial Key Laboratory of Forestry Biotechnology & International Cooperation Base of Science and Technology Innovation on Forest Resource Biotechnology, Central South University of Forestry & Technology, Changsha, PR China d State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences. No.1 Beichenxi Road, Chaoyang District, Beijing, PR China b
ARTICLE INFO
ABSTRACT
Keywords: Wolfiporia cocosditerpene Steroid Anti-Inflammatory Antimicrobial activity
Chemical investigation on the peels of the cultivated edible mushroom Wolfiporia cocos led to the identification of 13 diterpenes and 4 steroids, including two new abietane diterpenes (1 and 2) and one new pregnane steroid (14). Structures of new compounds were determined by analysis of NMR, MS, and electronic circular dichroism (ECD) data. All compounds were evaluated for cytotoxicity (K562 and HepG2), antimicrobial and anti-inflammatory activity. Compounds 15, 17 showed strong cytotoxicity on K562 cells with the IC50 values of 5.4 and 7.5 μM, respectively. Compounds 4, 9 and 17 displayed medium antibacterial activity against Staphylococcus aureus with MICs of 31.3, 48.5 and 12.5 μM, respectively. Compounds 1-10, 14 and 16 showed inhibitory activity on the NO (nitric oxide) release in LPS-induced RAW 264.7 cells with IC50 values at the range of 16.8–75.8 μM. This work confirms the potential of peels of W. cocos in the treatment of infection diseases.
1. Introduction Wolfiporia cocos (F.A. Wolf) Ryvarden & Gilb (syn. Poria cocos) is a subterranean edible and medical fungus growing on the roots of pine trees (Dong et al., 2017). The decorticated sclerotia of W. cocos, commercially called Fuling (Hoelen in Japan), is widely used as functional food and traditional Chinese medicine in Asian countries due to its effects on diuresis, invigorating the spleen, and tonifying, tranquilizing the heart, and soothing the spirit (Wang et al., 2015; Li et al., 2014; T.S.P.C.o.t.P.s.R.o. China, 2015; Zeng et al., 2019). W. cocos has a high content in polysaccharides and triterpenoids (Lai et al., 2016). Triterpenes with diverse pharmacological activities have been reported, such as poricoic acid C with anti-inflammatory property (Lee et al., 2017), poricoic acid G with cytotoxicity activity against HL60 (Ukiya et al., 2002), and dehydrotrametenolic acid with inhibition against 5hydroxytryptamine 3A (5-HT3A) receptor (Lee et al., 2009). In our early work, we isolated and identified forty-seven lanostane triterpenoids including sixteen new compounds from the peels of W. cocos and reported their cytotoxicity, hypoglycemic and hypolipidemic activities (Chen et al., 2019). In this study, we conducted a further
detailed investigation on the peel extract of W. cocos for non-triterpene components. As a result, two new abietane diterpenes (1 and 2) and one new pregnane steroid (14) were obtained along with fourteen known compounds (Fig. 1). All compounds were evaluated for their cytotoxic, antimicrobial and anti-inflammatory activities in lipopolysaccharide (LPS)-stimulated RAW 264.7 cell. 2. Material and method 2.1. General experimental procedures UV and IR spectra were obtained on a Thermo Genesys-10S UV–vis spectrophotometer and on a Nicolet IS5 FT-IR spectrophotometer, respectively. Optical rotations were recorded on an Anton Paar MCP 200 Automatic Polarimeter. CD spectra were acquired using an Applied Photophysics Chirascan spectropolarimeter. NMR spectral data were recorded with a Bruker Avance-500 spectrometer in CDCl3, (δH 7.26/δC 77.16) or C5D5N (δH 8.74, 7.58 and 7.22/δC 150.4, 135.9 and 123.9). HSQC and HMBC experiments were optimized for 145.0 and 8.0 Hz, respectively. HRESIMS data were obtained on an Agilent Accurate-
⁎ Corresponding Authors at: State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, No.1 Beichenxi Road, Beijing 100101, PR China. E-mail addresses: [email protected] (B. Li), [email protected] (L. Hongwei).
https://doi.org/10.1016/j.phytol.2020.01.005 Received 16 October 2019; Received in revised form 24 December 2019; Accepted 15 January 2020 1874-3900/ © 2020 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.
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Fig. 1. Structures of compounds 1-17.
Mass-Q-TOF LC/MS 6520 instrument. Optical density (OD) absorbance data were detected through Spectra Max 190 microplate reader. Solvents including methanol, dichloromethane, petroleum ether 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 spraying with 10 % H2SO4 followed by heating. Silica gel (150 − 250 μm, Qingdao Haiyang Chemical Co., Ltd.), octadecylsilyl (ODS) (50 μm, YMC CO., LTD) and Sephadex LH-20 (Amersham Biosciences) were used for column chromatography (CC). HPLC separation was performed on Shimadzu LC6AD with SPD-20A detector using an ODS column (C18, 250 × 9.4 mm, YMC Pak, 5 μm) at a flow rate of 2.0 mL/min.
Table 1 1 H and 13C NMR Data for compounds 1 and 2. Position
2.2. Fungal material The peels of W. cocos were collected from Baoshan City, Yunnan Province, China, and identified by Prof. Ruilin Zhao from State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences. A voucher specimen (HMAS 255,464) had been deposited in the Herbarium of the Institute of Microbiology, Chinese Academy of Sciences, Beijing (HMAS). 2.3. Extraction and isolation The dried and powdered peels of W. cocos (2.0 kg) were extracted as previously reported (Chen et al., 2019). Twenty fractions (Fr. 1−Fr. 20) were obtained by silica gel CC eluted with a gradient of n-hexane/ethyl acetate (100:0 to 0:100, v/v). Fr.10 (n-hexane-EtOAc, 10:1, 10.2 g) containing compounds with the characteristic UV spectrum of abietane diterpenes was further subjected to ODS CC eluted with MeOH in water (from 20 to 100 %, v/ v) to give seven subfractions (Fr. 10.1-Fr.10.7). Fr.10.2 (800 mg) was separated by semipreparative HPLC (98 % MeOH-H2O, 2 mL/min) to yield compounds 14 (2.8 mg, tR = 15.4 min), 15 (14.6 mg, tR = 28.0 min), and 16 (11.3 mg, tR = 34.7 min). Compounds 8 (7.8 mg, tR = 25.7 min), 7 (3.5 mg, tR = 32.5 min), 5 (4.2 mg, tR = 37.8 min), 6 (6.5 mg, tR =43.2 min), and 9 (7.5 mg, tR = 43.2 min) were purified from Fr.10.3 by semi-preparative HPLC (MeCN-H2O, 50:50, 2 mL/min). Fr.10.4 was separated on a Sephadex LH-20 column (CH2Cl2-MeOH, 1:1, v/v) to give six subfractions (Fr.10.4.1-Fr.10.4.6). Subfraction Fr.10.4.4 was purified by semi-preparative HPLC using 78 % MeOH in water to yield compounds 3 (21.6 mg, tR = 25.1 min), 13 (8.5 mg, tR = 32.6 min), 2 (3.3 mg, tR = 35.4 min), 1 (2.2 mg, tR = 39.4 min), 10 (3.5 mg, tR = 44.6 min), and 17 (6.7 mg, tR = 51.9 min). Compounds 4 (3.8 mg, tR =27.4 min), 11 (8.8 mg, tR = 35.4 min) and 12 (4.6 mg, tR = 40.0 min) were obtained from Fr.10.5 by semi-preparative HPLC (MeCN-H2O, 80:20, 2 mL/min). 7α-Ethoxycallitirisic acid (1). White gum, [α]25 D = +46.8 (c 0.05, CH2Cl2); UV (MeOH) λmax (log ε) = 212 (3.43) nm; CD (c 0.7 × 10−3 M, MeOH) λmax (Δε) = 224 (14.61) nm; IR (neat) νmax = 2956, 2927, 2852, 1697, 1606, 1456, 1277, 1077, 823, 731 cm-1; 1H and 13C NMR data, see Table 1. Positive HRESIMS m/z 345.2436 [M+H]+ (calcd for
1
2
δC[a]
δH[b], mult (J in Hz)
δC[a]
δH[b], mult (J in Hz)
1
37.6
37.8
2
18.5
2.29, dt (13.0, 2.1) 1.54, td (13.0, 4.1) 1.77, m
3
36.1
4 5 6
47.1 40.0 25.9
2.29, 1.46, 1.79, 1.72, 1.78, 1.75,
m m m m m m
7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
75.1 134.3 146.7 37.4 124.1 126.4 146.1 128.6 33.6 24.0 23.8 183.1 16.3 24.2 63.9
2.26, 1.91, 1.87, 4.65,
m m m dd (9.8, 7.4)
22
15.6
[a] [b]
1.93, m 1.70, m 2.58, dd (11.7, 3.0) 1.88, m 4.39, dd (4.1, 3.0)
7.19, d (8.2) 7.12, d (8.2) 7.13, 2.89, 1.25, 1.27,
s p (6.9) d (6.9) d (6.9)
1.31, 1.21, 3.67, 3.53, 1.26,
s s m m t (7.0)
18.4 36.6 47.1 42.9 28.3 77.4 135.5 146.9 37.3 123.8 125.6 146.2 125.8 33.6 24.1 23.8 182.4 16.3 25.5 63.1 15.7
7.14, d (8.2) 7.08, dd (8.2, 2.0) 7.31, 2.87, 1.22, 1.22,
d (2.0) m d (6.9) d (6.9)
1.31, 1.25, 3.70, 3.58, 1.28,
s s dq (9.1, 7.0) dq (9.1, 7.0) t (7.0)
Recorded at 125 MHz. Recorded at 500 MHz.
C22H33O3, 345.2430). 7β-Ethoxycallitirisic acid (2). White gum, [α]25 D = +39.8 (c 0.05, CH2Cl2); UV (MeOH) λmax (log ε) = 212 (3.43) nm; CD (c 0.8 × 10−3 M, MeOH) λmax (Δε) =209 (6.21), 251 (-3.15), 293 (-3.28), 327 (305) nm; IR (neat) νmax = 2956, 2927, 2867, 1694, 1456, 1247, 1077, 823, 731 cm-1; 1H and 13C NMR data, see Table 1. Positive HRESIMS m/z 345.2434 [M+H]+ (calcd. for C22H33O3, 345.2430). Poriaprogesterol A (14). White gum, [α]25 D = +16.0 (c 0.05, CH2Cl2); UV (MeOH) λmax (log ε) = 200 (3.48), 242 (1.23) nm; CD (c 1.1 × 10−3 M, MeOH) λmax (Δε) = 206 (2.34), nm; IR (neat) νmax = 3283, 2933, 2857, 2827, 1726, 1444, 1216, 1073, 998 cm-1; 1H and 13C NMR data, see Table 2. Positive HRESIMS m/z 463.3055 [M+H]+ (calcd. for C27H43O6, 463.3060). 2.4. ECD calculation Systematic conformation analysis of 1a was conducted with CONFLEX (version 7 Rev. A; CONFLEX Corporation) using the MMFF94 molecular mechanics force field. Optimization with DFT calculation at the B3LYP/6−31 G(d) level in MeOH by the Gaussian09 program 12
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2.6. Cytotoxicity
Table 2 1 H and 13C NMR Data for compound 14 in pyridine–d5. Position
δC[a]
δH[b],mult (J in Hz)
1
32.8
2
25.9
3 4
69.7 32.4
5 6 7 8 9 10 11
35.6 29.4 118.6 136.8 49.5 34.5 21.0
1.41, 1.48, 1.54, 1.81, 5.20, 1.38, 1.66, 1.82, 1.68, 5.88,
12
39.2
13 14 15 16
43.0 62.5 69.7 38.4
17 18 19 20 21 1′ 2′
56.8 13.9 12.2 68.5 24.6 170.8 42.0
3′ 4′
75.7 41.3
5′ 6′
71.6 76.0
[a] [b] [c]
Cell lines K562 (human bone marrow chronic myelogenous leukemia), and HepG2 (liver hepatocellular cells) were purchased from National Infrastructure of Cell Line Resource. Cytotoxicity assay was performed according to the method previously reported (Tao et al., 2016). After treating cells with the tested compounds at different doses (the maximum final concentration of DMSO is 0.5 %) for 48 h, 5 μL of cell counting kit-8(CCK8) was added to each well, and incubated for another 4 h. Cisplatin (J&K Scientific Ltd., purity ≥ 98 %) was used as positive control. The assay plate was read at 450 nm using a microplate reader. The inhibition rate was calculated with the following formula: Inhibitory rate (%) = [1-(OD treated/ OD control)] × 100 % and IC50 was calculated by plotting inhibitory rate vs. test concentrations.
14
m m m m t (2.9) o [c] m m m dd (5.3, 2.5)
1.76, m
2.7. Anti-inflammatory activity
1.55, 1.43, 1.93, 1.31,
m m d (12.1) td (12.1, 4.2)
2.28, 4.63, 2.72, 2.51, 2.03, 0.77, 0.78, 3.95, 1.42,
d (9.3) m dt (2.7) m m s s m d (6.2)
3.18, 2.98, 4.65, 2.46, 2.03, 4.64, 4.15, 3.88,
dd dd m m m m dd dd
The anti-inflammatory activity of compounds was evaluated using LPS-induced RAW 264.7 cells as previously reported (Meng et al., 2019). RAW 264.7 macrophages cells (105 cells/well) were suspended in 100 μL of DMEM supplemented with 10 % fetal bovine serum, precultured in 96-well microplates at 37 °C and 5 % CO2 in air for 24 h, then cultured for additional 24 h after administering the test compounds (100, 50, 25, 12.5, 6.3, 3.1 μM) and the positive control of hydrocortisone with or without 3 μg/mL LPS. Nitric Oxide (NO) production in each well was assessed by measuring the accumulation of nitrite in the culture medium using Griess reagent. Cytotoxicity was determined by CCK8 colorimetric assay.
(15.2, 7.6) (15.2, 5.9)
3. Results and discussion The ethanol extracts (150 g) of W. cocos were separated by a comprehensive chromatographic process to afford thirteen abietane diterpenes (1-13) and four steroids (14-17) (Fig. 1). By comparing NMR and MS data with the corresponding compounds in the literatures, known compounds were identified as dehydroabietic acid (3) (Surendra et al., 2014), 7β-hydroxyabieta-8,11,13-trien-18-oic acid (4) (Ozsen et al., 2017), 15-hydroxydehydroabietic acid (5)(Li et al., 2016), 15methoxy-8,11,13-abietatriene-18-oic acid (6) (Wang et al., 2010), 15hydroxy-7-oxo-8,11,13-abietatrien-18-oic acid (7) (Matsumoto et al., 1988), 18-oxoferruginol (8) (Kinouchi et al., 2000), 7-oxy-8,11,13abietatrien-18-oic acid (9) (Zhou and Zhou, 2018), abieta-7,17-diene12α-methoxy-18-oic acid (10) (Wu et al., 2010), abieta-8,11,13,15tetraen-18-oic acid (11) (Tanaka et al., 1997), 6,8,11,13-abietatrien-18oic acid (12) (Lu Yi, 2011), 15-oxo-17-norabieta-8,11,13-trien-18-oic acid (13) (Yang et al., 2008), ergosta-4, 6, 8(14),22-tetraen-3-one (15) (Wang et al., 2019), β-sitostenone (16) (Luo et al., 2009), demethylincisterol A3 (17) (Amagata et al., 2013), respectively. The structures of the new compounds were determined by extensive spectroscopic experiments. 7α-Ethoxycallitirisic acid (1) gave a protonated molecule peak at m/ z 345.2436 [M+H]+ (calcd. for C22H33O3, 345.2430) in the HREIMS spectrum, suggesting a molecular formula of C22H32O3. The 1H and 13C NMR (Table 1) spectra of 1 exhibited two singlet methyls at δH 1.31, and 1.21, two doublet methyl signals at δH 1.25 (d, J =6.9 Hz) and 1.27 (d, J = 6.9 Hz), one triplet methyl at δH 1.26 (t, J = 7.0 Hz) together with one oxygenated methylene at δH 3.67 (m) and 3.53 (m), one oxygenated methine at δH 4.39 (dd, J= 4.1, 3.0 Hz), three aromatic protons at δH 7.12 (dd, J= 8.2 Hz), 7.13 (s) and 7.19 (d, J = 8.2 Hz) for a 1,2,4-trisubstituted benzene moiety, as well as twenty-two carbon signals including a carboxylic carbon (δC 183.1), six aromatic carbons (δC 146.7, 146.1, 134.3, 128.6, 126.4 and 124.1), two oxygenated carbons (δC 75.1 and 63.9), and thirteen sp3 carbons. The 1H-1H COSY correlations (Fig. 2) gave the spin systems of H2-1/H2-2/H2-3, H-5/H26/H-7, H-11/H-12, H3-16/H-15/H3-17, and H2-21/H3-22, which together with HMBC correlations (Fig. 2) of H-5 with C-3, C-4, C-6, C-7,
(9.0, 2.3) (9.0, 4.7)
Recorded at 125 MHz. recorded at 500 MHz. o means overlap.
(Revision C.01, Gaussian Inc.) afforded the MMFF minima (Frisch et al., 2010). At the B3LYP/6−31 G(d) level, the exciting states were calculated using time-dependent density-functional theory (TDDFT) methodology for 1a. The overall ECD spectra were then produced based on Boltzmann weighting of each conformer as described in literature (Xue et al., 2019). 2.5. Antimicrobial assay Antimicrobial activities of compounds 1-17 were evaluated in triplicate according to the National Center for Clinical Laboratory Standards (NCCLS) recommendations using broth micro dilution method (Basnet et al., 2019). Bacteria, S. aureus (CGMCC 1.2465), B. subtilis (ATCC 6633) and E. coli (CGMCC 1.2340), and the fungus C. albicans (ATCC 18,804) were used as test strains. The resulting values were compared with that of the positive controls of vancomycin hydrochloride (Sigma, purity > 900 μg/mg) for S. aureus and B. subtilis, ampicillin (Sigma, purity ≥ 99 %) for E. coli, and amphotericin B (Sigma, approximately 80 %) for C. albicans. All samples for the antibacterial test were dissolved in DMSO. After 24 h incubation, the absorbance was determined at 600 nm by a microplate reader. The MIC value was determined as the lowest concentration inhibiting microbial growth. 13
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Fig. 2. Key 1H-1H COSY and HMBC correlations of compounds 1, 2 and 14.
Fig. 3. Key NOE correlations of c ompounds 1, 2 and 14.
C-10, C-19, and C-20, H3-19 with C-3, C-4, C-5 and C-18, H3-20 with C1, C-9 and C-10, H2-6 and H-7 with C-8, H3-16, H3-17 and H-15 with C13 established the skeleton structure of 1. The HMBC correlations from the H-7 (δH 4.39) to C-21 (δC 63.9) verified the ethyl group attaching the 7−OH. Compound 1 was assigned to be an ethyl ether derivative of 7-hydroxyabieta-8, 11, 13-trien-18-oic acid (4) (Ozsen et al., 2017). The
α configuration of H-5 and β configuration for H-7, CH3-19 and CH3-20 were established by NOE correlations (Fig. 3) of H-1α with H-5, H-1β with H3-20 and H3-19, H-7 with H3-20 indicated. To determine the absolute configurations by ECD calculation method, the structure of 1 were simplified to two stereoisomers 1a (4R/5R/7R/10S) and 1b (4S/ 5S/7S/10R). As shown in Fig. 4, the calculated ECD curve of 1a was in 14
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Table 3 Inhibitory activity of NO production. Compounds
IC50 (95%CI), μM
1 2 3a 4 5 6 7 8 9 10 11 a 12 13 a 14 16 a Hydrocortisone
52.1 75.8 33.9 60.8 50.9 33.9 31.7 14.7 17.1 43.2 50.4 31.6 44.1 36.0 16.8 22.4
(34.1–81.4) (53.2–97.1) (26.1–44.5) (40.3–94.5) (35.3–74.6) (21.5–54.7) (24.7–40.8) (11.8–18.1) (11.9–24.2) (30.6–61.6) (34.5–76.9) (20.8–48.4) (29.4–67.0) (26.2–49.6) (10.5–26.6) (18.3–27.4)
All data are presented as the mean of IC50 values with lower and upper 95 % confidence interval (CI) from triplicate measurement (n = 3); a, the tested concentration start from 50 μM.
Fig. 4. Experimental ECD (1) and calculated ECD (1a and 1b) spectra.
(liver hepatocellular cells). Compounds 15 and 17 showed strong cytotoxicity against K562 cells with the IC50 values of 5.4 (3.3–11.7) and 7.5 (4.5–12.7) μM, respectively, while the positive drug cisplatin had an IC50 value of 3.8 (1.2–5.2) μM. All other compounds were inactive against the growth of K562 cells at the concentration of 100 μM. In assays with HepG2 cells, none of tested compounds showed cytotoxicity at the dose of 100 μM. In antimicrobial assay, compounds 4, 9 and 17 displayed moderate antibacterial activity against S. aureus with MICs of 31.3, 48.5 and 12.5 μM, respectively. Vancomycin hydrochloride was used as the positive control with a MIC value of 2.1 μM. None of the tested compounds showed inhibition against B. subtilis, E. coli and C. albicans at the dose of 100 μM. Compounds 1-10, 14 and 16 that did not influence the growth of RAW 264.7 cells at the dose of 100 μM were assayed for their antiinflammatory activity by using LPS-induced RAW 264.7 cells. They showed inhibitory activity against the NO (nitric oxide) release in LPSinduced RAW 264.7 cells with IC50 values in the range of 16.8–75.8 μM (Table 3). Compounds 15 and 17 showing strong cytotoxicity on RAW 264.7 in the range of 20−100 μM were not tested. Compounds 8, 9 and 16 exhibited stronger inhibitory activity than the positive control of hydrocortisone. Analysis of the structure-activity relationship between abietane diterpenes and anti-inflammatory bioactivity suggests that the carbonylation at C-7 benefits for the nitric oxide inhibition activity. In conclusion, thirteen diterpenes including two new abietane diterpenes (1 and 2) and four steroids including one new pregnane-type steroid (14) were isolated and identified from the peels of the cultivated edible mushroom W. cocos. Diterpenes showing antimicrobial and NO inhibitory activities support the therapeutic effect of peels of W. cocos on infectious diseases.
good agreement with the experimental CD spectrum of 1. Thus, the absolute configuration of 1 was established as 4R, 5R, 7R and 10S. 7β-Ethoxycallitirisic acid (2) had the same molecular formula and same planar structure as that of 1, as supported by analysis of HRESIMS, 1D- and 2D-NMR data (Table 1 and Fig. 2). A detailed comparison of the 1H and 13C NMR data between 1 and 2 showed the biggest difference at C-7. The ROESY experiment showed NOE correlations (Fig. 3) of H-5 with H-7 and H-1α, H-1β with H3-20 and H3-19, which indicated the α-orientation of H-5 and H-7 and the β-orientation for CH3-19 and CH3-20. Considering the same biogenetic origin between 1 and 2, the absolute configuration of 2 was deduced to be 4R, 5R, 7S and 10S. Poriaprogesterol A (14) was obtained as a white gum. On the basis of the HRESIMS data at m/z 463.3055 [M+H]+ (calcd. for C27H43O6, 463.3060), the molecular formula of 14 was determined to be C27H42O6. Two singlet methyls at δC/H 13.9/0.77 and 12.2/0.78, one doublet methyl at δC/H 24.6/1.42 (d, J = 6.2 Hz), one oxygenated methylene δC/H 76.0/4.15 (dd, J = 9.0, 2.3 Hz) and 3.88 (dd, J = 9.0, 4.7 Hz), five oxygenated methine δC/H 69.7/5.20 (t, J =2.9 Hz), 69.7/ 4.63 (m), 68.5/3.95 (m), 75.7/4.65 (m), 71.6/4.64 (m), one double bond at δC/H 118.6/5.88 (dd, J = 5.3, 2.5 Hz) and δC 136.8, one ester carbonyl carbon at δC 170.8 were observed in the 1H and 13C-NMR spectra of 14 (Table 2). The 1H-1H COSY correlations assigned to four spin coupling systems in combination with the HMBC correlations from H3-18 to C-12, C-13, C-14 and C-17, from H3-19 to C-1, C-5, C-9 and C10, from H2-4, H-5 and H2-6 to C-10, from H-9 to C-10, C-7, C-8 and C14, from H-14 to C-7, C-8, C-12, C-13 and C-17, and from H-3′ to C-5′ and C-6′ (Fig. 2) elucidated two substructures of pregn-7-en-3,15,20triol and 2-(4-hydroxytetrahydrofuran-2-yl)acetic acid. The HMBC correlation from H-3 to C-1′ determined the linkage between pregn-7en-3, 15, 20-triol and 2-(4-hydroxytetrahydrofuran-2-yl)acetic acid. The α configuration for H-3, H-5, H-9, H-14 and H-17 and β configuration for H-15, H3-18 and H3-19 in the pregnane skeleton were assigned by the NOE correlations H-5 with H-9 and H-3, H-14 with H-9, H-17 and H-12α (δH 1.31), H-15 with H3-18, H3-19 and H3-18 with H12β (δH 1.93) (Fig. 3). The NOE correlation of H-2′ with H-5′ indicated that H2-2′ and H-5′ were on the same side of furan ring. With little quantity in hand, the absolute stereochemistry was left unsolved in this study. Two pregnane steroids have been previously reported from W. cocos (Chen et al., 2018; Tong et al., 2010). All compounds were tested for cytotoxicity on cell lines K562 (human bone marrow chronic myelogenous leukemia) and HepG2
Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements This work was supported by the National Key R&D program of China (No. 2018YFD0400203 and 2017YFE0108200) and National Natural Science Foundation of China (Grant 81673334). Dr. Jinwei Ren and Dr. Wenzhao Wang (State Key Laboratory of Mycology, Institute of 15
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Microbiology, Chinese Academy of Sciences) are appreciated for their help in measuring the NMR and MS data.
Methods Chem. (2014), 1–9. Li, H.L., Song, H.C., Zhang, Y., Chen, Y.G., 2016. Chemical constituents of the barks of Podocarpus macrophyllus. Chem. Nat. Compd. 52, 539–541. Lu Yi, H.Y., 2011. Abietane diterpenes from Callicarpa pedunculata. Nat. Prod. Res. Dev. 23, 66. Luo, J.R., Ma, Q.Y., Zhao, Y.X., Yi, T.M., Li, C.S., Zhou, J., 2009. Palaeophytochemical components from the miocene-fossil wood of Pinus griffithii. J. Chin. Chem. Soc. 56, 600–605. Matsumoto, T., Imai, S., Sunaoka, Y., Yoshinari, T., 1988. The conversion of (+)-dehydroabietic acid into steroidal hormones. B. Chem. Soc. Jpn. 61, 723–727. Meng, X., Che, C.C., Zhang, J.M., Gong, Z.J., Si, M.R., Yang, G., Cao, L., Liu, J.F., 2019. Structural characterization and immunomodulating activities of polysaccharides from a newly collected wild Morchella sextelata. Int. J. Biol. Macromol. 129, 608–614. Ozsen, O., Kiran, I., Dag, I., Atli, O., Ciftci, G.A., Demirci, F., 2017. Biotransformation of abietic acid by fungi and biological evaluation of its metabolites. Process Biochem. 52, 130–140. Surendra, K., Rajendar, G., Corey, E.J., 2014. Useful catalytic enantioselective cationic double annulation reactions initiated at an internal pi-bond: method and applications. J. Am. Chem. Soc. 136, 642–645. T.S.P.C.o.t.P.s.R.o. China, 2015. Pharmacopoeia of the People’s Republic of China 2015. China Medical Science and Technology Press. Tanaka, R., Ohtsu, H., Matsunaga, S., 1997. Abietane diterpene acids and other constituents from the leaves of Larix kaempferi. Phytochemistry 46, 1051–1057. Tao, Q.Q., Ma, K., Bao, L., Wang, K., Han, J.J., Wang, W.Z., Zhang, J.X., Huang, C.Y., Liu, H.W., 2016. Sesquiterpenoids with PTP1B inhibitory activity and cytotoxicity from the edible mushroom Pleurotus citrinopileatus. Planta Med. 82, 639–644. Tong, X.G., Liu, J.L., Cheng, Y.X., 2010. A new pregnane steroid from Poria cum Radix pini. J. Asian Nat. Prod. Res. 12, 419–423. Ukiya, M., Akihisa, T., Tokuda, H.M., Oshikubo, M., Nobukuni, Y.J.J.P., 2002. Inhibition of tumor-promoting effects by poricoic acids G and H and other lanostane-type triterpenes and cytotoxic activity of poricoic acids A and G from Poria cocos. J. Nat. Prod. 65, 462–465. Wang, W.H., Dong, H.J., Yan, R.Y., Li, H., Li, P.Y., Chen, P., Yang, B., Wang, Z.M., 2015. Comparative study of lanostane-type triterpene acids in different parts of Poria cocos (Schw.) Wolf by UHPLC-Fourier transform MS and UHPLC-triple quadruple MS, J Pharmaceut. Biomed. 102, 203–214. Wang, B., Ju, J., He, X.F., Yuan, T., Yue, J.M., 2010. Three new terpenoids from Pinus yunnanensis. Helv. Chim. Acta 93, 490–496. Wang, Z.R., Li, G., Ji, L.X., Wang, H.H., Gao, H., Peng, X.P., Lou, H.X., 2019. Induced production of steroids by co-cultivation of two endophytes from Mahonia fortunei. Steroids 145, 1–4. Wu, L.A., Li, Y.L., Li, S.M., Yang, X.W., Xia, J.H., Zhou, L., Zhang, W.D., 2010. Systematic phytochemical investigation of Abies spectabilis. Chem. Pharm. Bull. 58, 1646–1649. Xue, G.M., Zhu, D.R., Han, C., Wang, X.B., Luo, J.G., Kong, L.Y., 2019. Artemisianins A-D, new stereoisomers of seco-guaianolide involved heterodimeric [4+2] adducts from Artemisia argyi induce apoptosis via enhancement of endoplasmic reticulum stress. Bioorg. Chem. 84, 295–301. Yang, X.W., Li, S.M., Feng, L., Shen, Y.H., Tian, J.M., Liu, X.H., Zeng, H.W., Zhang, C., Zhang, W.D., 2008. Abiesanordines A-N: fourteen new norditerpenes from Abies georgei. Tetrahedron 64, 4354–4362. Zeng, G.P., Li, Z., Zhao, Z., 2019. Comparative analysis of the characteristics of triterpenoid transcriptome from different strains of Wolfiporia cocos. Int. J. Mol. Sci. 20, 3703. Zhou, Z., Zhou, T.T., 2018. Synthesis and antibacterial activity of C-7 acylhydrazone derivatives of dehydroabietic acid. J. Chem. Res. 8, 405–407.
Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.phytol.2020.01.005. References Amagata, T., Tanaka, M., Yamada, T., Chen, Y.P., Minoura, K., Numata, A., 2013. Additional cytotoxic substances isolated from the sponge-derived Gymnascella dankaliensis. Tetrahedron Lett. 54, 5960–5962. Basnet, B.B., Chen, B.S., Suleimen, Y.M., Ma, K., Guo, S.Y., Bao, L., Huang, Y., Liu, H.W., 2019. Cytotoxic secondary metabolites from the endolichenic fungus Hypoxylon fuscum. Planta Med. 85, 1088–1097. Chen, B.S., Zhang, J.J., Han, J.J., Zhao, R.L., Bao, L., Huang, Y., Liu, H.W., 2019. Lanostane triterpenoids with glucose-uptake-stimulatory activity from peels of the cultivated edible mushroom Wolfiporia cocos. J. Agr. Food Chem. 67, 7348–7364. Chen, T., Kan, Y.J., Chou, G.X., Zhang, C.G., 2018. A new highly oxygenated pregnane and two new 5-hydroxymethylfurfural derivatives from the water decoction of Poria cocos. J. Asian Nat. Prod. Res. 20, 1101–1107. Dong, H.J., Xue, Z.Z., Geng, Y.L., Wang, X., Yang, B., 2017. Lanostane triterpenes isolated from epidermis of Poria cocos. Phytochem. Lett. 22, 102–106. Frisch, T.G., MJ, S.H.B., Scuseria, G.E., Robb, M.A., Cheeseman, J.R., Scalmani, G., Barone, V., Mennucci, B., Petersson, G.A., Nakatsuji, H., Caricato, M., Li, X., Hratchian, H.P., Izmaylov, A.F., Bloino, J., Zheng, G., Sonnenberg, J.L., Hada, M., Ehara, M., Toyota, K., Fukuda, R., Hasegawa, J., Ishida, M., Nakajima, T., Honda, Y., Kitao, O., Nakai, H., Vreven, T., Montgomery Jr, J.A., Peralta, J.E., Ogliaro, F., Bearpark, M., Heyd, J.J., Brothers, E., Kudin, K.N., Staroverov, V.N., Keith, T., Kobayashi, R., Normand, J., Raghavachari, K., Rendell, A., Burant, J.C., Iyengar, S.S., Tomasi, J., Cossi, M., Rega, N., Millam, J.M., Klene, M., Knox, J.E., Cross, J.B., Bakken, V., Adamo, C., Jaramillo, J., Gomperts, R., Stratmann, R.E., Yazyev, O., Austin, A.J., Cammi, R., Pomelli, C., Ochterski, J.W., Martin, R.L., Morokuma, K., Zakrzewski, V.G., Voth, G.A., Salvador, P., Dannenberg, J.J., Dapprich, S., Daniels, A.D., Farkas, O., Foresman, J.B., Ortiz, J.V., Cioslowski, J., Fox, D.J., 2010. Gaussian 09, Revision C.01. Gaussian, Inc., Wallingford CT. Kinouchi, Y., Ohtsu, H., Tokuda, H., Nishino, H., Matsunaga, S., Tanaka, R., 2000. Potential antitumor-promoting diterpenoids from the stem bark of Picea glehni. J. Nat. Prod. 63, 817–820. Lai, K.H., Lu, M.C., Du, Y.C., El-Shazly, M., Wu, T.Y., Hsu, Y.M., Henz, A., Yang, J.C., Backlund, A., Chang, F.R., Wu, Y.C., 2016. Cytotoxic lanostanoids from Poria cocos. J. Nat. Prod. 79, 2805–2813. Lee, S., Lee, D., Lee, S.O., Ryu, J.Y., Choi, S.Z., Kang, K.S., Kim, K.H., 2017. Anti-inflammatory activity of the sclerotia of edible fungus, Poria cocos Wolf and their active lanostane triterpenoids. J. Funct. Foods 32, 27–36. Lee, J.H., Lee, Y.J., Shin, J.K., Nam, J.W., Nah, S.Y., Kim, S.H., Jeong, J.H., Kim, Y., Shin, M., Hong, M., Seo, E.K., Bae, H., 2009. Effects of triterpenoids from Poria cocos Wolf on the serotonin type 3A receptor-mediated ion current in Xenopus oocytes. Eur. J. Pharmacol. 615, 27–32. Li, Y., Zhang, J., Zhao, Y.L., Li, Z.M., Li, T., Wang, Y.Z., 2014. Characteristic fingerprint based on low polar constituents for discrimination of Wolfiporia extensa according to geographical origin using uv spectroscopy and chemometrics methods. J. Anal.
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Anti-inflammatory diterpenes and steroids from peels of the cultivated edible mushroom Wolfiporia cocos
T
Baosong Chena,b, Sixian Wangc, Gaoqiang Liuc, Li Baoa,*, Ying Huangd, Ruilin Zhaoa, Hongwei Liua,b,* a
State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences. No.1 Beichenxi Road, Chaoyang District, Beijing, PR China Savaid Medicine School, University of Chinese Academy of Sciences, Beijing, PR China c Hunan Provincial Key Laboratory of Forestry Biotechnology & International Cooperation Base of Science and Technology Innovation on Forest Resource Biotechnology, Central South University of Forestry & Technology, Changsha, PR China d State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences. No.1 Beichenxi Road, Chaoyang District, Beijing, PR China b
ARTICLE INFO
ABSTRACT
Keywords: Wolfiporia cocosditerpene Steroid Anti-Inflammatory Antimicrobial activity
Chemical investigation on the peels of the cultivated edible mushroom Wolfiporia cocos led to the identification of 13 diterpenes and 4 steroids, including two new abietane diterpenes (1 and 2) and one new pregnane steroid (14). Structures of new compounds were determined by analysis of NMR, MS, and electronic circular dichroism (ECD) data. All compounds were evaluated for cytotoxicity (K562 and HepG2), antimicrobial and anti-inflammatory activity. Compounds 15, 17 showed strong cytotoxicity on K562 cells with the IC50 values of 5.4 and 7.5 μM, respectively. Compounds 4, 9 and 17 displayed medium antibacterial activity against Staphylococcus aureus with MICs of 31.3, 48.5 and 12.5 μM, respectively. Compounds 1-10, 14 and 16 showed inhibitory activity on the NO (nitric oxide) release in LPS-induced RAW 264.7 cells with IC50 values at the range of 16.8–75.8 μM. This work confirms the potential of peels of W. cocos in the treatment of infection diseases.
1. Introduction Wolfiporia cocos (F.A. Wolf) Ryvarden & Gilb (syn. Poria cocos) is a subterranean edible and medical fungus growing on the roots of pine trees (Dong et al., 2017). The decorticated sclerotia of W. cocos, commercially called Fuling (Hoelen in Japan), is widely used as functional food and traditional Chinese medicine in Asian countries due to its effects on diuresis, invigorating the spleen, and tonifying, tranquilizing the heart, and soothing the spirit (Wang et al., 2015; Li et al., 2014; T.S.P.C.o.t.P.s.R.o. China, 2015; Zeng et al., 2019). W. cocos has a high content in polysaccharides and triterpenoids (Lai et al., 2016). Triterpenes with diverse pharmacological activities have been reported, such as poricoic acid C with anti-inflammatory property (Lee et al., 2017), poricoic acid G with cytotoxicity activity against HL60 (Ukiya et al., 2002), and dehydrotrametenolic acid with inhibition against 5hydroxytryptamine 3A (5-HT3A) receptor (Lee et al., 2009). In our early work, we isolated and identified forty-seven lanostane triterpenoids including sixteen new compounds from the peels of W. cocos and reported their cytotoxicity, hypoglycemic and hypolipidemic activities (Chen et al., 2019). In this study, we conducted a further
detailed investigation on the peel extract of W. cocos for non-triterpene components. As a result, two new abietane diterpenes (1 and 2) and one new pregnane steroid (14) were obtained along with fourteen known compounds (Fig. 1). All compounds were evaluated for their cytotoxic, antimicrobial and anti-inflammatory activities in lipopolysaccharide (LPS)-stimulated RAW 264.7 cell. 2. Material and method 2.1. General experimental procedures UV and IR spectra were obtained on a Thermo Genesys-10S UV–vis spectrophotometer and on a Nicolet IS5 FT-IR spectrophotometer, respectively. Optical rotations were recorded on an Anton Paar MCP 200 Automatic Polarimeter. CD spectra were acquired using an Applied Photophysics Chirascan spectropolarimeter. NMR spectral data were recorded with a Bruker Avance-500 spectrometer in CDCl3, (δH 7.26/δC 77.16) or C5D5N (δH 8.74, 7.58 and 7.22/δC 150.4, 135.9 and 123.9). HSQC and HMBC experiments were optimized for 145.0 and 8.0 Hz, respectively. HRESIMS data were obtained on an Agilent Accurate-
⁎ Corresponding Authors at: State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, No.1 Beichenxi Road, Beijing 100101, PR China. E-mail addresses: [email protected] (B. Li), [email protected] (L. Hongwei).
https://doi.org/10.1016/j.phytol.2020.01.005 Received 16 October 2019; Received in revised form 24 December 2019; Accepted 15 January 2020 1874-3900/ © 2020 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.
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Fig. 1. Structures of compounds 1-17.
Mass-Q-TOF LC/MS 6520 instrument. Optical density (OD) absorbance data were detected through Spectra Max 190 microplate reader. Solvents including methanol, dichloromethane, petroleum ether 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 spraying with 10 % H2SO4 followed by heating. Silica gel (150 − 250 μm, Qingdao Haiyang Chemical Co., Ltd.), octadecylsilyl (ODS) (50 μm, YMC CO., LTD) and Sephadex LH-20 (Amersham Biosciences) were used for column chromatography (CC). HPLC separation was performed on Shimadzu LC6AD with SPD-20A detector using an ODS column (C18, 250 × 9.4 mm, YMC Pak, 5 μm) at a flow rate of 2.0 mL/min.
Table 1 1 H and 13C NMR Data for compounds 1 and 2. Position
2.2. Fungal material The peels of W. cocos were collected from Baoshan City, Yunnan Province, China, and identified by Prof. Ruilin Zhao from State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences. A voucher specimen (HMAS 255,464) had been deposited in the Herbarium of the Institute of Microbiology, Chinese Academy of Sciences, Beijing (HMAS). 2.3. Extraction and isolation The dried and powdered peels of W. cocos (2.0 kg) were extracted as previously reported (Chen et al., 2019). Twenty fractions (Fr. 1−Fr. 20) were obtained by silica gel CC eluted with a gradient of n-hexane/ethyl acetate (100:0 to 0:100, v/v). Fr.10 (n-hexane-EtOAc, 10:1, 10.2 g) containing compounds with the characteristic UV spectrum of abietane diterpenes was further subjected to ODS CC eluted with MeOH in water (from 20 to 100 %, v/ v) to give seven subfractions (Fr. 10.1-Fr.10.7). Fr.10.2 (800 mg) was separated by semipreparative HPLC (98 % MeOH-H2O, 2 mL/min) to yield compounds 14 (2.8 mg, tR = 15.4 min), 15 (14.6 mg, tR = 28.0 min), and 16 (11.3 mg, tR = 34.7 min). Compounds 8 (7.8 mg, tR = 25.7 min), 7 (3.5 mg, tR = 32.5 min), 5 (4.2 mg, tR = 37.8 min), 6 (6.5 mg, tR =43.2 min), and 9 (7.5 mg, tR = 43.2 min) were purified from Fr.10.3 by semi-preparative HPLC (MeCN-H2O, 50:50, 2 mL/min). Fr.10.4 was separated on a Sephadex LH-20 column (CH2Cl2-MeOH, 1:1, v/v) to give six subfractions (Fr.10.4.1-Fr.10.4.6). Subfraction Fr.10.4.4 was purified by semi-preparative HPLC using 78 % MeOH in water to yield compounds 3 (21.6 mg, tR = 25.1 min), 13 (8.5 mg, tR = 32.6 min), 2 (3.3 mg, tR = 35.4 min), 1 (2.2 mg, tR = 39.4 min), 10 (3.5 mg, tR = 44.6 min), and 17 (6.7 mg, tR = 51.9 min). Compounds 4 (3.8 mg, tR =27.4 min), 11 (8.8 mg, tR = 35.4 min) and 12 (4.6 mg, tR = 40.0 min) were obtained from Fr.10.5 by semi-preparative HPLC (MeCN-H2O, 80:20, 2 mL/min). 7α-Ethoxycallitirisic acid (1). White gum, [α]25 D = +46.8 (c 0.05, CH2Cl2); UV (MeOH) λmax (log ε) = 212 (3.43) nm; CD (c 0.7 × 10−3 M, MeOH) λmax (Δε) = 224 (14.61) nm; IR (neat) νmax = 2956, 2927, 2852, 1697, 1606, 1456, 1277, 1077, 823, 731 cm-1; 1H and 13C NMR data, see Table 1. Positive HRESIMS m/z 345.2436 [M+H]+ (calcd for
1
2
δC[a]
δH[b], mult (J in Hz)
δC[a]
δH[b], mult (J in Hz)
1
37.6
37.8
2
18.5
2.29, dt (13.0, 2.1) 1.54, td (13.0, 4.1) 1.77, m
3
36.1
4 5 6
47.1 40.0 25.9
2.29, 1.46, 1.79, 1.72, 1.78, 1.75,
m m m m m m
7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
75.1 134.3 146.7 37.4 124.1 126.4 146.1 128.6 33.6 24.0 23.8 183.1 16.3 24.2 63.9
2.26, 1.91, 1.87, 4.65,
m m m dd (9.8, 7.4)
22
15.6
[a] [b]
1.93, m 1.70, m 2.58, dd (11.7, 3.0) 1.88, m 4.39, dd (4.1, 3.0)
7.19, d (8.2) 7.12, d (8.2) 7.13, 2.89, 1.25, 1.27,
s p (6.9) d (6.9) d (6.9)
1.31, 1.21, 3.67, 3.53, 1.26,
s s m m t (7.0)
18.4 36.6 47.1 42.9 28.3 77.4 135.5 146.9 37.3 123.8 125.6 146.2 125.8 33.6 24.1 23.8 182.4 16.3 25.5 63.1 15.7
7.14, d (8.2) 7.08, dd (8.2, 2.0) 7.31, 2.87, 1.22, 1.22,
d (2.0) m d (6.9) d (6.9)
1.31, 1.25, 3.70, 3.58, 1.28,
s s dq (9.1, 7.0) dq (9.1, 7.0) t (7.0)
Recorded at 125 MHz. Recorded at 500 MHz.
C22H33O3, 345.2430). 7β-Ethoxycallitirisic acid (2). White gum, [α]25 D = +39.8 (c 0.05, CH2Cl2); UV (MeOH) λmax (log ε) = 212 (3.43) nm; CD (c 0.8 × 10−3 M, MeOH) λmax (Δε) =209 (6.21), 251 (-3.15), 293 (-3.28), 327 (305) nm; IR (neat) νmax = 2956, 2927, 2867, 1694, 1456, 1247, 1077, 823, 731 cm-1; 1H and 13C NMR data, see Table 1. Positive HRESIMS m/z 345.2434 [M+H]+ (calcd. for C22H33O3, 345.2430). Poriaprogesterol A (14). White gum, [α]25 D = +16.0 (c 0.05, CH2Cl2); UV (MeOH) λmax (log ε) = 200 (3.48), 242 (1.23) nm; CD (c 1.1 × 10−3 M, MeOH) λmax (Δε) = 206 (2.34), nm; IR (neat) νmax = 3283, 2933, 2857, 2827, 1726, 1444, 1216, 1073, 998 cm-1; 1H and 13C NMR data, see Table 2. Positive HRESIMS m/z 463.3055 [M+H]+ (calcd. for C27H43O6, 463.3060). 2.4. ECD calculation Systematic conformation analysis of 1a was conducted with CONFLEX (version 7 Rev. A; CONFLEX Corporation) using the MMFF94 molecular mechanics force field. Optimization with DFT calculation at the B3LYP/6−31 G(d) level in MeOH by the Gaussian09 program 12
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2.6. Cytotoxicity
Table 2 1 H and 13C NMR Data for compound 14 in pyridine–d5. Position
δC[a]
δH[b],mult (J in Hz)
1
32.8
2
25.9
3 4
69.7 32.4
5 6 7 8 9 10 11
35.6 29.4 118.6 136.8 49.5 34.5 21.0
1.41, 1.48, 1.54, 1.81, 5.20, 1.38, 1.66, 1.82, 1.68, 5.88,
12
39.2
13 14 15 16
43.0 62.5 69.7 38.4
17 18 19 20 21 1′ 2′
56.8 13.9 12.2 68.5 24.6 170.8 42.0
3′ 4′
75.7 41.3
5′ 6′
71.6 76.0
[a] [b] [c]
Cell lines K562 (human bone marrow chronic myelogenous leukemia), and HepG2 (liver hepatocellular cells) were purchased from National Infrastructure of Cell Line Resource. Cytotoxicity assay was performed according to the method previously reported (Tao et al., 2016). After treating cells with the tested compounds at different doses (the maximum final concentration of DMSO is 0.5 %) for 48 h, 5 μL of cell counting kit-8(CCK8) was added to each well, and incubated for another 4 h. Cisplatin (J&K Scientific Ltd., purity ≥ 98 %) was used as positive control. The assay plate was read at 450 nm using a microplate reader. The inhibition rate was calculated with the following formula: Inhibitory rate (%) = [1-(OD treated/ OD control)] × 100 % and IC50 was calculated by plotting inhibitory rate vs. test concentrations.
14
m m m m t (2.9) o [c] m m m dd (5.3, 2.5)
1.76, m
2.7. Anti-inflammatory activity
1.55, 1.43, 1.93, 1.31,
m m d (12.1) td (12.1, 4.2)
2.28, 4.63, 2.72, 2.51, 2.03, 0.77, 0.78, 3.95, 1.42,
d (9.3) m dt (2.7) m m s s m d (6.2)
3.18, 2.98, 4.65, 2.46, 2.03, 4.64, 4.15, 3.88,
dd dd m m m m dd dd
The anti-inflammatory activity of compounds was evaluated using LPS-induced RAW 264.7 cells as previously reported (Meng et al., 2019). RAW 264.7 macrophages cells (105 cells/well) were suspended in 100 μL of DMEM supplemented with 10 % fetal bovine serum, precultured in 96-well microplates at 37 °C and 5 % CO2 in air for 24 h, then cultured for additional 24 h after administering the test compounds (100, 50, 25, 12.5, 6.3, 3.1 μM) and the positive control of hydrocortisone with or without 3 μg/mL LPS. Nitric Oxide (NO) production in each well was assessed by measuring the accumulation of nitrite in the culture medium using Griess reagent. Cytotoxicity was determined by CCK8 colorimetric assay.
(15.2, 7.6) (15.2, 5.9)
3. Results and discussion The ethanol extracts (150 g) of W. cocos were separated by a comprehensive chromatographic process to afford thirteen abietane diterpenes (1-13) and four steroids (14-17) (Fig. 1). By comparing NMR and MS data with the corresponding compounds in the literatures, known compounds were identified as dehydroabietic acid (3) (Surendra et al., 2014), 7β-hydroxyabieta-8,11,13-trien-18-oic acid (4) (Ozsen et al., 2017), 15-hydroxydehydroabietic acid (5)(Li et al., 2016), 15methoxy-8,11,13-abietatriene-18-oic acid (6) (Wang et al., 2010), 15hydroxy-7-oxo-8,11,13-abietatrien-18-oic acid (7) (Matsumoto et al., 1988), 18-oxoferruginol (8) (Kinouchi et al., 2000), 7-oxy-8,11,13abietatrien-18-oic acid (9) (Zhou and Zhou, 2018), abieta-7,17-diene12α-methoxy-18-oic acid (10) (Wu et al., 2010), abieta-8,11,13,15tetraen-18-oic acid (11) (Tanaka et al., 1997), 6,8,11,13-abietatrien-18oic acid (12) (Lu Yi, 2011), 15-oxo-17-norabieta-8,11,13-trien-18-oic acid (13) (Yang et al., 2008), ergosta-4, 6, 8(14),22-tetraen-3-one (15) (Wang et al., 2019), β-sitostenone (16) (Luo et al., 2009), demethylincisterol A3 (17) (Amagata et al., 2013), respectively. The structures of the new compounds were determined by extensive spectroscopic experiments. 7α-Ethoxycallitirisic acid (1) gave a protonated molecule peak at m/ z 345.2436 [M+H]+ (calcd. for C22H33O3, 345.2430) in the HREIMS spectrum, suggesting a molecular formula of C22H32O3. The 1H and 13C NMR (Table 1) spectra of 1 exhibited two singlet methyls at δH 1.31, and 1.21, two doublet methyl signals at δH 1.25 (d, J =6.9 Hz) and 1.27 (d, J = 6.9 Hz), one triplet methyl at δH 1.26 (t, J = 7.0 Hz) together with one oxygenated methylene at δH 3.67 (m) and 3.53 (m), one oxygenated methine at δH 4.39 (dd, J= 4.1, 3.0 Hz), three aromatic protons at δH 7.12 (dd, J= 8.2 Hz), 7.13 (s) and 7.19 (d, J = 8.2 Hz) for a 1,2,4-trisubstituted benzene moiety, as well as twenty-two carbon signals including a carboxylic carbon (δC 183.1), six aromatic carbons (δC 146.7, 146.1, 134.3, 128.6, 126.4 and 124.1), two oxygenated carbons (δC 75.1 and 63.9), and thirteen sp3 carbons. The 1H-1H COSY correlations (Fig. 2) gave the spin systems of H2-1/H2-2/H2-3, H-5/H26/H-7, H-11/H-12, H3-16/H-15/H3-17, and H2-21/H3-22, which together with HMBC correlations (Fig. 2) of H-5 with C-3, C-4, C-6, C-7,
(9.0, 2.3) (9.0, 4.7)
Recorded at 125 MHz. recorded at 500 MHz. o means overlap.
(Revision C.01, Gaussian Inc.) afforded the MMFF minima (Frisch et al., 2010). At the B3LYP/6−31 G(d) level, the exciting states were calculated using time-dependent density-functional theory (TDDFT) methodology for 1a. The overall ECD spectra were then produced based on Boltzmann weighting of each conformer as described in literature (Xue et al., 2019). 2.5. Antimicrobial assay Antimicrobial activities of compounds 1-17 were evaluated in triplicate according to the National Center for Clinical Laboratory Standards (NCCLS) recommendations using broth micro dilution method (Basnet et al., 2019). Bacteria, S. aureus (CGMCC 1.2465), B. subtilis (ATCC 6633) and E. coli (CGMCC 1.2340), and the fungus C. albicans (ATCC 18,804) were used as test strains. The resulting values were compared with that of the positive controls of vancomycin hydrochloride (Sigma, purity > 900 μg/mg) for S. aureus and B. subtilis, ampicillin (Sigma, purity ≥ 99 %) for E. coli, and amphotericin B (Sigma, approximately 80 %) for C. albicans. All samples for the antibacterial test were dissolved in DMSO. After 24 h incubation, the absorbance was determined at 600 nm by a microplate reader. The MIC value was determined as the lowest concentration inhibiting microbial growth. 13
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Fig. 2. Key 1H-1H COSY and HMBC correlations of compounds 1, 2 and 14.
Fig. 3. Key NOE correlations of c ompounds 1, 2 and 14.
C-10, C-19, and C-20, H3-19 with C-3, C-4, C-5 and C-18, H3-20 with C1, C-9 and C-10, H2-6 and H-7 with C-8, H3-16, H3-17 and H-15 with C13 established the skeleton structure of 1. The HMBC correlations from the H-7 (δH 4.39) to C-21 (δC 63.9) verified the ethyl group attaching the 7−OH. Compound 1 was assigned to be an ethyl ether derivative of 7-hydroxyabieta-8, 11, 13-trien-18-oic acid (4) (Ozsen et al., 2017). The
α configuration of H-5 and β configuration for H-7, CH3-19 and CH3-20 were established by NOE correlations (Fig. 3) of H-1α with H-5, H-1β with H3-20 and H3-19, H-7 with H3-20 indicated. To determine the absolute configurations by ECD calculation method, the structure of 1 were simplified to two stereoisomers 1a (4R/5R/7R/10S) and 1b (4S/ 5S/7S/10R). As shown in Fig. 4, the calculated ECD curve of 1a was in 14
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Table 3 Inhibitory activity of NO production. Compounds
IC50 (95%CI), μM
1 2 3a 4 5 6 7 8 9 10 11 a 12 13 a 14 16 a Hydrocortisone
52.1 75.8 33.9 60.8 50.9 33.9 31.7 14.7 17.1 43.2 50.4 31.6 44.1 36.0 16.8 22.4
(34.1–81.4) (53.2–97.1) (26.1–44.5) (40.3–94.5) (35.3–74.6) (21.5–54.7) (24.7–40.8) (11.8–18.1) (11.9–24.2) (30.6–61.6) (34.5–76.9) (20.8–48.4) (29.4–67.0) (26.2–49.6) (10.5–26.6) (18.3–27.4)
All data are presented as the mean of IC50 values with lower and upper 95 % confidence interval (CI) from triplicate measurement (n = 3); a, the tested concentration start from 50 μM.
Fig. 4. Experimental ECD (1) and calculated ECD (1a and 1b) spectra.
(liver hepatocellular cells). Compounds 15 and 17 showed strong cytotoxicity against K562 cells with the IC50 values of 5.4 (3.3–11.7) and 7.5 (4.5–12.7) μM, respectively, while the positive drug cisplatin had an IC50 value of 3.8 (1.2–5.2) μM. All other compounds were inactive against the growth of K562 cells at the concentration of 100 μM. In assays with HepG2 cells, none of tested compounds showed cytotoxicity at the dose of 100 μM. In antimicrobial assay, compounds 4, 9 and 17 displayed moderate antibacterial activity against S. aureus with MICs of 31.3, 48.5 and 12.5 μM, respectively. Vancomycin hydrochloride was used as the positive control with a MIC value of 2.1 μM. None of the tested compounds showed inhibition against B. subtilis, E. coli and C. albicans at the dose of 100 μM. Compounds 1-10, 14 and 16 that did not influence the growth of RAW 264.7 cells at the dose of 100 μM were assayed for their antiinflammatory activity by using LPS-induced RAW 264.7 cells. They showed inhibitory activity against the NO (nitric oxide) release in LPSinduced RAW 264.7 cells with IC50 values in the range of 16.8–75.8 μM (Table 3). Compounds 15 and 17 showing strong cytotoxicity on RAW 264.7 in the range of 20−100 μM were not tested. Compounds 8, 9 and 16 exhibited stronger inhibitory activity than the positive control of hydrocortisone. Analysis of the structure-activity relationship between abietane diterpenes and anti-inflammatory bioactivity suggests that the carbonylation at C-7 benefits for the nitric oxide inhibition activity. In conclusion, thirteen diterpenes including two new abietane diterpenes (1 and 2) and four steroids including one new pregnane-type steroid (14) were isolated and identified from the peels of the cultivated edible mushroom W. cocos. Diterpenes showing antimicrobial and NO inhibitory activities support the therapeutic effect of peels of W. cocos on infectious diseases.
good agreement with the experimental CD spectrum of 1. Thus, the absolute configuration of 1 was established as 4R, 5R, 7R and 10S. 7β-Ethoxycallitirisic acid (2) had the same molecular formula and same planar structure as that of 1, as supported by analysis of HRESIMS, 1D- and 2D-NMR data (Table 1 and Fig. 2). A detailed comparison of the 1H and 13C NMR data between 1 and 2 showed the biggest difference at C-7. The ROESY experiment showed NOE correlations (Fig. 3) of H-5 with H-7 and H-1α, H-1β with H3-20 and H3-19, which indicated the α-orientation of H-5 and H-7 and the β-orientation for CH3-19 and CH3-20. Considering the same biogenetic origin between 1 and 2, the absolute configuration of 2 was deduced to be 4R, 5R, 7S and 10S. Poriaprogesterol A (14) was obtained as a white gum. On the basis of the HRESIMS data at m/z 463.3055 [M+H]+ (calcd. for C27H43O6, 463.3060), the molecular formula of 14 was determined to be C27H42O6. Two singlet methyls at δC/H 13.9/0.77 and 12.2/0.78, one doublet methyl at δC/H 24.6/1.42 (d, J = 6.2 Hz), one oxygenated methylene δC/H 76.0/4.15 (dd, J = 9.0, 2.3 Hz) and 3.88 (dd, J = 9.0, 4.7 Hz), five oxygenated methine δC/H 69.7/5.20 (t, J =2.9 Hz), 69.7/ 4.63 (m), 68.5/3.95 (m), 75.7/4.65 (m), 71.6/4.64 (m), one double bond at δC/H 118.6/5.88 (dd, J = 5.3, 2.5 Hz) and δC 136.8, one ester carbonyl carbon at δC 170.8 were observed in the 1H and 13C-NMR spectra of 14 (Table 2). The 1H-1H COSY correlations assigned to four spin coupling systems in combination with the HMBC correlations from H3-18 to C-12, C-13, C-14 and C-17, from H3-19 to C-1, C-5, C-9 and C10, from H2-4, H-5 and H2-6 to C-10, from H-9 to C-10, C-7, C-8 and C14, from H-14 to C-7, C-8, C-12, C-13 and C-17, and from H-3′ to C-5′ and C-6′ (Fig. 2) elucidated two substructures of pregn-7-en-3,15,20triol and 2-(4-hydroxytetrahydrofuran-2-yl)acetic acid. The HMBC correlation from H-3 to C-1′ determined the linkage between pregn-7en-3, 15, 20-triol and 2-(4-hydroxytetrahydrofuran-2-yl)acetic acid. The α configuration for H-3, H-5, H-9, H-14 and H-17 and β configuration for H-15, H3-18 and H3-19 in the pregnane skeleton were assigned by the NOE correlations H-5 with H-9 and H-3, H-14 with H-9, H-17 and H-12α (δH 1.31), H-15 with H3-18, H3-19 and H3-18 with H12β (δH 1.93) (Fig. 3). The NOE correlation of H-2′ with H-5′ indicated that H2-2′ and H-5′ were on the same side of furan ring. With little quantity in hand, the absolute stereochemistry was left unsolved in this study. Two pregnane steroids have been previously reported from W. cocos (Chen et al., 2018; Tong et al., 2010). All compounds were tested for cytotoxicity on cell lines K562 (human bone marrow chronic myelogenous leukemia) and HepG2
Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements This work was supported by the National Key R&D program of China (No. 2018YFD0400203 and 2017YFE0108200) and National Natural Science Foundation of China (Grant 81673334). Dr. Jinwei Ren and Dr. Wenzhao Wang (State Key Laboratory of Mycology, Institute of 15
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Microbiology, Chinese Academy of Sciences) are appreciated for their help in measuring the NMR and MS data.
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