Triterpenoids from Ganoderma lucidum and their Potential Anti

Article Cite This: J. Agric. Food Chem. 2019, 67, 5147−5158

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Triterpenoids from Ganoderma lucidum and Their Potential Antiinflammatory Effects Yan-Li Wu,†,⊥ Fei Han,†,⊥ Shan-Shan Luan,‡,⊥ Rui Ai,† Peng Zhang,† Hua Li,*,†,‡ and Li-Xia Chen*,† †

Wuya College of Innovation, School of Pharmacy, Key Laboratory of Structure-Based Drug Design & Discovery, Ministry of Education, Shenyang Pharmaceutical University, Shenyang 110016, China ‡ School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China

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ABSTRACT: Ganoderma lucidum, as food, tea, dietary supplement, and medicine, is widely used in China and Eastern Asian countries. In order to discover its anti-inflammatory constituents and provide some references for the usage of G. lucidum and G. sinense, two official species in China, the fruiting bodies of G. lucidum were studied, leading to the isolation of six new triterpenoids (1−6) and 27 known analogues (7−33). Compound 4 exhibited the most potent inhibition on nitric oxide (NO) production induced by lipopolysaccharide (LPS) in RAW264.7 macrophage cells. The production of IL-6 and IL-1β, as well as the expression of iNOS, COX-2, and NF-κB were dose-dependently reduced by 4. The phosphorylations of IκBα and IKKβ in LPS-induced macrophage cells were blocked by 4. Therefore, 4 could be used as a potential anti-inflammatory candidate and the total triterpenoids might be developed as value-added functional food for the prevention of inflammation. In combination of previous studies, it should be cautious for the interchangeable usage of G. lucidum and G. sinense. KEYWORDS: Ganoderma lucidum, triterpenoids, anti-inflammation

1. INTRODUCTION Mushrooms have always been a popular food for their delicious taste and being rich in nutrient elements for thousands of years. Some of them are also used as potent food supplements and medicinal sources for human beings.1 Ganoderma lucidum (Lingzhi in Chinese, Reishi in Japanese, and Youngzhi in Korean), a kind of famous edible and medicinal mushroom, has been commonly used as functional food and traditional medicine for regulating immunity and promoting health in China and other Eastern Asian countries.2,3 The development and utilization of G. lucidum have made great progress in China, and a large number of artificially cultivated G. lucidum is exported to many countries as food, tea, dietary supplements, and raw materials for further processing, which has brought great benefits to the planting industry of edible and medicinal mushrooms.4 The mushrooms of G. lucidum could prevent and treat various diseases, such as bronchitis, asthma, hypercholesterolaemia, hepatitis, hypertension, neurasthenia, leucopenia, and cancer,5 with a wide range of biological activities such as anti-inflammation,6,7 immune regulation,8,9 hepatoprotection,10 and antitumor.11,12 G. lucidum and its closely related species G. sinense are suggested be used interchangeably as Lingzhi according to Chinese Pharmacopoeia.2,3,13,14 Chemical investigations on the two species of Ganoderma based on multiple analytic technologies or chemical separation have been carried out, and triterpenoids and polysaccharides were determined as the major constituents13,15−22 of G. lucidum, while studies on G. sinense are relatively few, showing the presence of meroterpenoids, steroids, alkaloids, triterpenoids, and polysaccharides.13,23−28 Comparing the chemical constituents from two official species of Ganoderma genus demonstrated the significant difference on type and content of triterpenoids by © 2019 American Chemical Society

high performance liquid chromatography coupled with photodiode array (HPLC-PDA) and high performance liquid chromatography−mass spectrometry (LC−MS) analyses, with about 10 times higher in G. lucidum than G. sinense, and lack of common triterpenes in G. sinense.13,15 While the similar chemical features of polysaccharides between both species were observed,13,29 they also showed similar antitumor and immunomodulating activities.29 Although polysaccharides as the active principles could explain to some extent the official use of both species as Lingzhi, the influence of numerous small-molecular constituents and their different bioactivities in both species remains to be further investigated and provides a deeper explanation for their exchangeable usage in China. Nuclear factor-κB (NF-κB) signaling pathway has been proved to play an important role in inflammation and immune responses, and NF-κB can be activated by the phosphorylation of IκB by IκB kinase (IKK) complex, mainly by IKKβ.30,31 NFκB activation is usually induced by the translocation to nucleus of its dimers, which can bind with DNA to trigger the expressions of a series of inflammatory cytokines such as interleukin (IL)-6 and IL-1β, and stress response proteins including cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS).32 Nitric oxide (NO) is produced by iNOS, and excessive amounts of NO can lead to multiple inflammation-related diseases, such as allergic rhinitis, arthritis, and bowel diseases.33−35 In order to discover the anti-inflammatory constituents from G. lucidum and further provide some references for the usage of Received: Revised: Accepted: Published: 5147

February April 15, April 17, April 17,

20, 2019 2019 2019 2019 DOI: 10.1021/acs.jafc.9b01195 J. Agric. Food Chem. 2019, 67, 5147−5158

Article

Journal of Agricultural and Food Chemistry Table 1. 1H and

13

C NMR Spectroscopic Data for Compounds 1−3 1a

a

position

δC

1a 1b 2a 2b 3 4 5 6a 6b 7 8 9 10 11 12a 12b 13 14 15 16a 16b 17 18 19 20 21 22a 22b 23a 23b 24a 24b 25a 25b 26 27 28a 28b 29 30 CH3CO CH3CO OCH3

37.2, CH2

δH (J in Hz) 2.67, 2.46, 1.73, 1.73, 3.65,

27.7, CH2 71.7, 43.9, 45.2, 34.2,

CH C CH CH2

δC

m d (12.8) m m dd (11.6, 5.2)

27.7, CH2 71.7, 44.0, 45.2, 34.2,

49.3, C 59.9, C 208.8, C 38.7, CH2

2.84, 1.96, 2.64, 0.81, 1.36, 2.32, 0.97, 2.56, 2.35,

CH CH3 CH3 CH CH3 CH2

CH C CH CH2

200.9, C 153.6, C 147.6, C 41.6, C 195.8, C 81.0, CH

5.70, s

49.7, C 59.9, C 209.2, C 38.4, CH2

m dd (18.0, 8.0) m s s m d (4.4) m m

46.7, 12.9, 18.8, 34.0, 20.8, 31.3,

211.0, C


32.8, CH2

47.9, CH2

2.82, m 2.51, m 2.84, m

36.9, CH 179.9, C 17.9, CH3 65.5, CH2

1.17, 3.53, 3.24, 0.77, 1.72, 2.21,

12.8, CH3 21.8, CH3 21.0, CH3 171.8, C 1

3b δH (J in Hz)

37.2, CH2

2.07, d (12.4) 2.64, m 1.16, m

200.8, C 153.6, C 147.5, C 41.6, C 195.7, C 80.9, CH

46.2, 12.8, 18.9, 30.4, 22.4, 49.3,

2a 2.68, 2.47, 1.74, 1.74, 3.66,

m dd (14.8, 2.0) m m m

2.07, m 2.69, m 2.61, m

5.70, s

2.86, 2.09, 2.60, 0.83, 1.36, 1.76, 1.02, 1.86, 1.30, 2.40, 2.29,

dd (18.3, 9.6) m m s s m d (6.4) m m m m

177.8, C

12.8, CH3 21.9, CH3

21.0, CH3 171.9, C 13

36.3, CH2 34.2, CH2 214.4, C 47.9, C 52.9, CH 40.0, CH2 213.0, C 54.1, CH 59.6, CH 36.6, C 208.0, C 52.8, CH2 49.9, 50.0, 74.3, 38.3,

C C CH CH2

47.7, 16.6, 13.3, 35.2, 18.0, 30.9,


31.0, CH2

δH (J in Hz) 3.03, 1.89, 2.72, 2.35,

m m m m

1.67, m 2.54, m 2.40, m 2.85, d (13.2) 2.35, d (13.2)

2.67, d (14.0) 2.38, d (14.0)

4.05, 1.99, 1.76, 1.99, 0.78, 1.44, 1.77, 0.82, 1.78, 1.30, 2.36, 2.23,

t (6.4) m m m s s m d (6.7) m m m m

174.3, C

65.5, CH2

d (4.4) d (11.8) d (11.8) s s s

δC

b

3.53, 3.24, 0.77, 1.73,

d (11.2) d (11.2) s s

21.4, CH3

1.07, s

25.4, CH3 12.6, CH3

1.05, s 1.20, s

51.7, CH3

3.65, s

2.19, s

1

Measured in CD3OD; 400 MHz for H NMR; 100 MHz for C NMR. Measured in CDCl3; 400 MHz for H NMR; 100 MHz for 13C NMR. Shimadzu UV 2201 UV−vis spectrophotometer (Shimadzu Corporation, Kyoto, Japan). Infrared (IR) absorption spectra [4000−400 cm−1; potassium bromide (KBr) disks] were performed on a Bruker IFS 55 spectrometer (Bruker Optics, Ettlingen, Germany). Nuclear magnetic resonance (NMR) experiments including proton nuclear magnetic resonance (1H NMR), carbon nuclear magnetic resonance (13C NMR), nuclear Overhauser enhancement spectroscopy (NOESY), heteronuclear single quantum correlation (HSQC), and heteronuclear multiple-bond correlation (HMBC) were measured on Bruker AV-600 or ARX-400 spectrometers (Bruker Biospin, Fallanden, Switzerland). Chemical values are expressed in δ (ppm) relative to tetramethylsilane (TMS), and the coupling constants are depicted as J (Hz). High resolution electron spray ionization mass (HRESIMS) data were collected on an Agilent 6210 TOF mass spectrometer (Palo Alto, USA). Silica gel (200−300 mesh, Qingdao

both official species of Lingzhi in China, the fruiting bodies of G. lucidum were sequentially investigated herein based on our previous study on G. sinense.25−28 As a result, six new triterpenoids (1−6) and 27 known analogues (7−33) were isolated and characterized from G. lucidum. Their inhibitory effects on NO production in LPS-stimulated RAW 264.7 macrophages and anti-inflammation mechanism were preliminarily investigated herein.

2. MATERIALS AND METHODS 2.1. General. Melting point was tested with an X-4 digital display micromelting point apparatus (uncorrected). Optical rotation data were recorded on a PerkinElmer 241 polarimeter (PerkinElmer, Waltham, MA, USA). Ultraviolet (UV) spectra were measured on a 5148

DOI: 10.1021/acs.jafc.9b01195 J. Agric. Food Chem. 2019, 67, 5147−5158

Article

Journal of Agricultural and Food Chemistry Table 2. 1H and

13

C NMR Spectroscopic Data for Compounds 4−6 4a

position

δC

1a 1b 2a 2b 3 4 5 6a 6b 7 8 9 10 11 12a 12b 13 14 15 16a 16b 17 18 19 20 21 22a 22b 23a 23b 24a 24b 25 26 27 28 29 30 CH3CO CH3CO OCH3

35.3, CH2 34.2, CH2 215.3, C 46.8, C 49.3, CH 37.2, CH2 204.7, C 151.2, C 152.7, C 39.3, C 201.4, C 52.1, CH2 47.8, 52.9, 72.3, 36.4,

C C CH CH2

48.4, 17.6, 17.9, 35.7, 18.1, 30.9,


31.2, CH2

5b δH (J in Hz) 2.96, 1.80, 2.63, 2.51,

m m m m

2.28, m 2.62, m 2.47, m

2.82, d (17.4) 2.58, d (17.4)

4.29, 1.97, 1.97, 1.82, 0.87, 1.27, 1.41, 0.87, 1.81, 1.34, 2.38, 2.26,

t (7.4) m m m s s m d (6.8) m m m m

174.4, C

δC

δH (J in Hz)

36.9, CH2

2.77, 1.55, 2.58, 2.40,

35.3, CH2 220.0, C 47.9, C 49.9, CH 30.0, CH2

1.80, 2.05, 1.68, 4.61,

69.7, CH 162.3, C 141.1, C 39.2, C 202.3, C 53.5, CH2 48.5, 55.8, 73.3, 31.5,

C C CH CH2

51.7, 19.6, 20.0, 74.9, 27.0, 55.5,

CH CH3 CH3 C CH3 CH2

1.12, s 1.15, s 1.19, s

51.8, CH3

3.67, s

m m m m

br d (13.2) dd (11.6, 6.8) m dd (10.0, 6.8)

2.87, d (16.0) 2.51, d (16.0)

4.82, 2.40, 1.68, 2.30, 1.12, 1.24,

dd (10.0, 6.8) m m br t (10.4) s s

1.31, s 2.89, d (14.6) 2.53, d (14.6)

211.2, C 49.2, CH2

20.5, CH3 27.6, CH3 20.7, CH3

6b

2.87, m 2.58, m 2.85, m

36.2, CH 180.0, C 17.6, CH3 27.9, CH3 21.2, CH3 20.6, CH3

1.17, 1.12, 1.09, 1.27,

d (6.8) s s s

δC

δH (J in Hz)

37.7, CH2 28.2, CH2 78.2, 40.3, 52.8, 34.5,

CH C CH CH2

201.3, C 153.4, C 147.2, C 41.9, C 195.6, C 81.2, CH

2.72, 2.49, 1.69, 1.69, 3.21,

t (14.4) d (13.2) m m dd (12.0, 4.6)

1.61, m 2.66, m 1.20, m

5.67, s

49.4, C 59.8, C 208.9, C 37.4, CH2

67.8, CH

2.85, 2.02, 2.64, 0.79, 1.34, 1.71, 1.07, 1.64, 1.48, 4.54,

143.9, CH

6.57, d (9.0)

46.5, 13.0, 18.4, 30.9, 22.5, 43.1,


130.7, C 171.8, C 13.4, CH3 28.4, CH3 16.3, CH3 21.7, CH3 21.0, CH3 171.8, C

1.90, 1.00, 0.88, 1.73, 2.14,

dd (18.0, 9.8) dd (18.0, 8.4) m s s m d (6.6) m m dd (7.4,4.8)

s s s s s

a

Measured in CDCl3; 600 MHz for 1H NMR; 150 MHz for 13C NMR. bMeasured in CD3OD; 600 MHz for 1H NMR; 150 MHz for 13C NMR. 2.3. Extraction and Isolation. The fruiting bodies of G. lucidum (9.0 kg) were cut into small pieces (about 2 cm) and extracted with 80% ethanol (EtOH, 90 L × 2 h × 2) to afford a total extract (240.7 g) after concentrating in vacuo. Then the extract was suspended in water (H2O, 5 L) and partitioned successively with the same volume of cyclohexane, ethyl acetate (EtOAc), and n-butanol (n-BuOH) for three times. The EtOAc extract (131.1 g) was subjected to silica gel CC (10 × 80 cm) and eluted with dichloromethane/methanol (CH2Cl2/MeOH, 100:1, 50:1, 20:1, 10:1, 5:1, 2:1, 1:1, and 0:1, v/v) to afford six fractions (CE1−CE6) based on silica gel TLC analyses. Separation of CE2 (8.2 g) on an ODS column (2.5 × 30 cm) with MeOH/H2O (30:70, 50:50, 70:30, and 100:0, v/v) as eluent yielded fractions CE21−CE25. CE23 (2.8 g) was separated on HPLC (58% MeOH/H2O) to obtain four fractions (CE231−CE234). Compounds 26 (25.7 mg) and 27 (27.6 mg) were further purified from fraction CE232 (100.5 mg) by preparative HPLC [50% acetonitrile/H2O (MeCN/H2O)]. CE233 (500.2 mg) was separated by preparative HPLC (45% MeCN/H2O) to give 11 (22.4 mg) and two subfractions

Marine Chemical Factory, Qingdao, China), Sephadex LH-20 (Pharmacia, Amersharm, Sweden), and octadecyl silica gel (Merck Chemical Company Ltd., Darmstadt, Germany) were applied for column chromatography (CC). Silica gel GF254 for thin layer chromatography (TLC) was bought from Qingdao Marine Chemical Factory (Qingdao, China). Prepared HPLC was performed on an LC6AD liquid chromatography equipped with a SPD-10AVP UV/vis detector (Shimadzu, Kyoto, Japan) and an ODS-A column (250 × 20 mm, 5 μm, 120 Å, YMC Co. Ltd.). All reagents (Tianjin Damao Chemical Company, Tianjin, China) were HPLC or analytical grade. Spots on TLC plates were visualized under UV light and by spraying with anisaldehyde-H2SO4 reagent. 2.2. Fungal Material. The fruiting bodies of G. lucidum (Leyss ex Fr) Karst were provided by Jiangsu Xinxian Pharmaceutical Co. Ltd. (Jiangsu, China), and authenticated by Professor Jincai Lu, School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University. A voucher specimen (LZ-15-058) was preserved in our laboratory. 5149


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Journal of Agricultural and Food Chemistry

12β-Acetoxyganoderic Acid θ (6). White powder (MeOH); [α]25 D +93.3 (c 0.045, MeOH); UV (MeOH) λmax (log ε) 210 (4.6) nm; 1H 13 NMR (600 MHz, CDCl3) and C NMR (150 MHz, CDCl3) data, see Table 2; HRESIMS (negative) m/z 571.2919 [M − H]− (calcd for C32H43O9−, 571.2907). 2.4. Preparation of (R)- and (S)-MTPA Esters of 6. Compound 6 (1.0 mg) was divided equally into two NMR tubes and dried under vacuum for 12 h. (R)-Methoxy-α-(trifluoromethyl)phenylacetyl chloride [(R)-MTPA-Cl, 10 μL) and deuterated pyridine (0.5 mL) were added into one NMR tube under the protection of nitrogen (N2). The reaction was performed at room temperature for hours and monitored by 600 MHz NMR. The 1H NMR spectrum was measured on 600 MHz NMR in pyridine-d5 (Figure S51). Similarly, (S)-MTPACl (10 μL) and deuterated pyridine (0.5 mL) were added into another NMR tube and reacted at room temperature to yield (S)MTPA ester derivative, and the 1H NMR spectrum was recorded on 600 MHz NMR in pyridine-d5 (Figure S52). 2.5. Cell Cultures. Mouse RAW264.7 macrophage cells were obtained from ATCC and cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS, Sigma), 100 U/mL penicillin (Hyclone), and 100 μg/mL streptomycin (Hyclone) with 5% CO2/95% air (v/v) at 37 °C. The cells were divided into three groups: dimethyl sulfoxide (DMSO) control group, LPS (1 μg/mL) group, and LPS (1 μg/mL) plus compounds group. 2.6. CCK-8 Assay. The viability of mouse RAW264.7 macrophage cells was determined by CCK-8 (Cell counting Kit-8, Beyotime, Shanghai, China). The RAW264.7 cells were seeded into 96-well plate (Nest, Biotech, China) with the density of 5 × 104 cells/mL. After treatment with series concentrations of compound 4 (0−200 μM) for 24 h, the absorbance (540 nm) was recorded on a microplate reader following the manufacturer’s instructions. 2.7. Bioassay for NO Production. Nitrite, as an indicator of NO production, was measured for its concentration in medium according to Griess method.36 RAW 264.7 cells were inoculated into 96-well plates with 2 × 104 cells/well and were cultured overnight. After replacement with new medium, cells were stimulated with 1 μg/mL of LPS with or without tested compounds and incubated for 24 h at 37 °C. The cell-free supernatant and Griess reagent were completely mixed with the same amount of 100 μL. Absorbance of the final product was measured at 540 nm on a microplate reader. The nitrite concentration and inhibitory rate were calculated according to the standard calibration curve. The inhibitory effect of the tested compounds on LPS-induced NO production was described as IC50 values. 2.8. ELISA Assay. The secretion of the inflammatory cytokines in RAW264.7 cell supernatants was detected after treated with compound 4. Cell supernatants were collected , and ELISA kits (Boster, China) were applied for determination of the production of IL-1β and IL-6 following the manufacturer’s instructions. 2.9. Western Blot Assay. Western blot analysis was performed to evaluate the expression of inflammation-associated proteins, such as iNOS, COX-2, and NF-κB pathway proteins. Cells were collected, then resuspended with radio immunoprecipitation assay (RIPA) buffer supplemented with 0.1 mM PMSF protease inhibitor. The cell suspension was lysed by vortex on ice and then centrifuged at a high speed of 20,000 × g to get supernatant. The total protein concentrations were determined by BCA protein assay kit. The same amount of proteins for each sample was loaded to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the proteins separated on the gel were transferred onto a polyvinylidene difluoride (PVDF) membrane. After blocked with 5% skim milk, the membranes were then incubated with specific primary antibodies at 4 °C overnight, followed by incubation with corresponding secondary antibodies. The protein blots were finally detected by enhanced chemiluminescence (ECL) system. 2.10. Immunofluorescence Assay. To observe the nuclear translocation of NF-κB, macrophage cells were cultured in a glass chamber to 50−60% confluence, and then treated with DMSO and 20 or 40 μM compound 4. Two hours later, cells were stimulated with 1 μg/mL LPS for 12 h. After washing with phosphate buffer saline

CE2332 and CE2334. CE2332 (121.1 mg) was further purified by preparative HPLC (60% MeOH/H2O) to give 7 (32.4 mg) and 24 (16.8 mg). Subfraction CE2334 (50.9 mg) yielded 20 (11 mg) through purification on a preparative HPLC (70% MeOH/H2O). Separation of CE234 (918.9 mg) by preparative HPLC (45% MeCN/ H2O) afforded 3 (29.3 mg), 4 (8.0 mg), 6 (14.9 mg), 17 (227.9 mg), 21 (4.2 mg), and CE2344 (512.4 mg). Compounds 9 (219.0 mg) and 16 (294.3 mg) were obtained from fraction CE2344 by preparative HPLC (60% MeOH/H2O). Fraction CE3 (3.3 g) was separated by ODS CC (2.5 × 30 cm) with MeOH/H2O (10:90, 30:70, 50:50, 70:30, and 100:0, v/v) as eluent to give five subfractions (CE31− CE35). Fr. CE33 (1.5 g) was further separated by preparative HPLC (65% MeOH/H2O) to give three subfractions (CE331−CE333). Purification of CE331 (507.9 mg) by preparative HPLC (65% MeOH/H2O) afforded 8 (10.7 mg), 10 (21.9 mg), 12 (174.4 mg), 22 (13.0 mg), 23 (27.6 mg), and 28 (20.2 mg). Compound 25 (28.3 mg) was obtained from fraction CE332 (86.1 mg) by preparative HPLC (40% MeCN/H2O). Separation of CE333 (309.7 mg) on preparative HPLC eluted with 40% MeCN/H2O afforded 15 (141.1 mg). CE4 (2.2 g) was separated on an ODS column (2.5 × 30 cm) eluted with MeOH/H2O (30:70, 50:50, 70:30, and 100:0, v/v) to afford fractions CE41−CE45. Purification of CE43 (307.9 mg) by preparative HPLC (65% MeOH/H2O) yielded 1 (8.2 mg), 2 (10.4 mg), 5 (20.1 mg), 14 (4.2 mg), and 32 (2.2 mg). Separation of CE5 (3.3 g) on an ODS column (2.5 × 30 cm) with MeOH/H2O (30:70, 50:50, 70:30, and 100:0 v/v) as eluent yielded fractions CE51−CE55. CE53 (1.0 g) was separated by using silica gel CC (2 × 30 cm) and eluted with CH2Cl2/MeOH (100:1, 50:1, 20:1, 10:1, 5:1, 2:1, 1:1, and 0:1, v/v) to give four fractions (CE531−CE534). CE531 (31.0 mg) yielded 19 (6.3 mg) through purification on preparative HPLC (60% MeOH/H2O). CE532 (200.9 mg) was further purified by preparative HPLC (35% MeCN/H2O) to yield 18 (10.9 mg), 30 (2.7 mg), and 31 (40.5 mg). Purification of CE533 (309.8 mg) by preparative HPLC (50% MeOH/H2O) afforded 13 (120.0 mg). Fraction CE6 (1.2 g) was separated further by ODS CC (2.5 × 30 cm) eluted with MeOH/H2O (10:90, 30:70, 50:50, 70:30, and 100:0, v/v) to give five subfractions (CE61−CE65). CE63 (156.3 mg) was further separated by preparative HPLC (40% MeCN/H2O) to give 29 (36.0 mg) and 33 (5.8 mg). 12β-Acetoxy-3β,28-dihydroxy-7,11,15,23-tetraoxo-5α-lanosta-8en-26-oic acid (1). White powder (MeOH); [α]25 D +67 (c 0.125, MeOH); UV (MeOH) λmax (log ε) 256 (4.03) nm; IR (KBr) νmax 3427, 2920, 2360, 1641, 1464, 1384, 1123, 619 cm−1; 1H NMR (400 MHz, CD3OD) and 13C NMR (100 MHz, CD3OD) data, see Table 1; HRESIMS (negative) m/z 587.2866 [M − H]− (calcd for C32H43O10−, 587.2856). Lucidenic Acid R (2). White powder (MeOH); [α]25 D +61 (c 0.095, MeOH); UV (MeOH) λmax (log ε) 256 (3.99) nm; IR (KBr) νmax 3433, 2920, 1745, 1463, 1384, 1118, 619 cm−1; 1H NMR (400 MHz, CD3OD) and 13C NMR (100 MHz, CD3OD) data, see Table 1; HRESIMS (negative) m/z 531.2595 [M − H]− (calcd for C29H39O9−, 531.2594). Methyl Lucidenate K (3). Colorless needles (MeOH); mp 179− 182 °C; [α]25 D +113 (c 0.205, MeOH); UV (MeOH) λmax (log ε) 257 (4.04) nm; IR (KBr) νmax 3435, 2921, 1737, 1462, 1384, 1170, 617 cm−1; 1H NMR (400 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3) data, see Table 1; HRESIMS (negative) m/z 473.2903 [M − H]− (calcd for C28H41O6−, 473.2903). Methyl Lucidenate L (4). White powder (MeOH); [α]25 D +118 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 265 (5.95) nm; IR (KBr) νmax 3435, 2922, 1638, 1384, 1115, 1017, 619 cm−1; 1H NMR (600 MHz, CDCl3) and 13C NMR (150 MHz, CDCl3) data, see Table 2; HRESIMS (negative) m/z 471.2754 [M − H]− (calcd for C28H39O6−, 471.2747). 7β,15α,20-Trihydroxy-3,11,23-trioxo-5α-lanosta-8-en-26-oic Acid (5). White powder (MeOH); [α]25 D +121.1 (c 0.185, MeOH); UV (MeOH) λmax (log ε) 252 (4.3) nm; 1H NMR (600 MHz, CDCl3) and 13C NMR (150 MHz, CDCl3) data, see Table 2; HRESIMS (negative) m/z 531.2967 [M − H]− (calcd for C30H43O8−, 531.2958). 5150


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Figure 1. Structures of compounds 1−33. (PBS), cells were fixed in paraformaldehyde and then blocked with 5% BSA for 1 h. The glass chambers were incubated with NF-κB antibody at 1:1000 dilution at 4 °C overnight, followed by a secondary AlexaFluor488 antibody at 1:500 dilution for 30 min in the dark. The cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) for 30 min at 37 °C. Finally, the nuclear translocation was observed using a fluorescence microscope (Nikon). 2.11. Statistical Analysis. At least triplicate experiments were conducted to obtain all results, and the data are displayed as mean ± SD. GraphPad Prism, version 4.00, and SPSS software, version 22.0,

with one-way ANOVA and Student’s t test were used for the determination of statistical significance (p < 0.05).

3. RESULTS AND DISCUSSION Study on constituents of the fruiting bodies of G. lucidum yielded six new triterpenoids (1−6) and 27 known analogues (7−33) including methyl ganoderate C1 (7),37 12-acetoxyganoderic acid D (8),38 methyl ganoderate F (9),39 ganoderic acid E (10),40 methyl ganoderate E (11),37 ganoderic acid F 5151


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Figure 2. Key HMBC correlations of compounds 1−6.

Figure 3. Selected NOESY correlations of compounds 1−6.

(12),40 ganoderic acid C (13),41 methyl ganoderate C (14),41 ganoderic acid J (15),42 methyl lucidenate D2 (16),39 methyl lucidenate A (17),37 ganoderenic acid C (18),40 ganoderenic acid A (19),40 methyl lucidenate H (20),43 ganoderenic acid K (21),38 12β-acetoxy-7β-hydroxy-3,11,15,23-tetraoxo-5α-lanosta-8,20-dien-26-oic acid (22),44 12β-acetoxy-3,7,11,15,23-

pentaoxo-lanosta-8,20-dien-26-oic acid (23),45 ganoderenic acid B (24),40 ganoderenic acid G (25),46 methyl ganoderenate D (26),47 methyl ganoderate P (27),48 ganoderenic acid F (28),46 ganoderenic acid D (29),40 ganoderic acid η (30),49 ganoderic acid ζ (31),49 lucidone F (32),50 and ganoderic acid I (33)42 (Figure 1). 5152


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The HRESIMS of compound 2 gave a pseudo molecular ion peak at m/z 531.2595 [M − H]− (calcd for C29H39O9−, 531.2594), suggesting its formula as C29H40O9. Its 1H and 13C NMR data (Table 1) resembled those of lucidenic acid E,52 except for the missing of a methyl signal at C-25 (δC 27.9/δH 1.03), and the appearance of one oxygenated methene (δC 65.5/δH 3.53, 3.24) in 2, revealing the hydroxylation of C-25. This supposition was further supported by the HMBC correlations (Figure 2) of H2-25 with C-3/C-4/C-5/C-26. In the NOESY spectrum, H-3 correlated to H-5/H-25, and H-12 to CH3-27, demonstrating a β-orientation for 3-OH and CH3COO-12 (Figure 3). As a result, the structure of 2 was determined as lucidenic acid R. Compound 3 possessed a molecular formula of C28H42O6 as determined via HRESIMS peak at m/z 473.2903 [M − H]− (calcd for C28H41O6−, 473.2903), implying eight indices of hydrogen deficiency. Its 13C NMR spectroscopic data showed close similarity with methyl 8β,9α-dihydroganoderate J,53 especially for the carbon signals of rings A−D. The HMBC spectrum revealed obvious correlations of CH3-21 (δH 0.82) with C-17/C-20/C-22, H-23b (δH 2.23) with C-22/C-24, and H3-OCH3 (δH 3.65) with C-24, confirming the linkage of the five-carbon side chain (from C-20 to C-24) (Figure 2). In its NOESY spectrum, H-15 (δH 4.05) correlated to CH3-18 (δH 0.78), demonstrating an α-orientation for 15-OH (Figure 3). The structure of compound 3 was thus established as methyl lucidenate K. The molecular formula of compound 4 was deduced as C28H40O6 by its 13C NMR data and [M − H]− at m/z 471.2754 (calcd for C28H39O6−, 471.2747) in the negative HRESIMS. Its 1H and 13C NMR data (Table 2) were almost consistent with those of 3, except that the resonances at δC

Figure 4. Selected values of Δδ (S-R) of the MTPA esters of 6.

The molecular formula of compound 1 was established as C32H44O10 via HRESIMS (m/z 587.2866 [M − H]−, calcd for C32H43O10−, 587.2856) and 13C NMR spectroscopic data, implying 11 indices of hydrogen deficiency. The 1H and 13C NMR data (Table 1) suggested the presence of seven methyls, two oxygenated methines, one olefinic bond, four ketone carbonyls, and two ester or carboxyl groups. Its 1H and 13C NMR data (Table 1) were similar to those of ganoderic acid H previously isolated from G. lucidum,51 except for a major difference at C-28. The methyl group at C-28 (δC 27.9/δH 1.03) in ganoderic acid H was replaced by an oxygenated methene (δC 65.5/δH 3.53, 3.24) in 1, demonstrating the linkage of a hydroxyl group to C-28. The key HMBC (Figure 2) from H2-28 to C-3/C-4/C-5/C-29 confirmed the above supposition. In its NOESY spectrum, H-3 correlated to H-28/ H-5, and H-30 to H-5/H-12, demonstrating a β-orientation for both 3-OH and CH3COO-12 (Figure 3). Thus, the structure of compound 1 was identified as 12β-acetoxy-3β,28-dihydroxy7,11,15,23-tetraoxo-5α-lanosta-8-en-26-oic acid.

Figure 5. Inhibitory effect of compound 4 on NO production and cell viability of RAW264.7 cells. (A) NO inhibition of all the isolated compounds at 50 μM was examined. (B) IC50 value of dexamethasone (Dex) was 7.9 ± 1.3 μM. (C) IC50 value of compound 4 was 38.6 ± 1.0 μM. (D) RAW264.7 cell viability after treatment with compound 4 was detected by CCK-8 assays. *, p < 0.5, **, p < 0.01, compared with control (treated with 0 μM compound 4). 5153


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Figure 6. Inhibitory effect of compound 4 on the expression of pro-inflammatory cytokines in RAW264.7 cells. (A,B) Cytokine levels of IL-6 and IL-1β in the RAW264.7 cell medium after exposure to compound 4 were examined by ELISA. ###, p < 0.001, compared with the control that was neither administrated with compound 4 nor LPS. *, p < 0.05, **, p < 0.01, and ***, p < 0.001, compared with that only treated with LPS.

Figure 7. Effects of compound 4 on the protein expressions of iNOS and COX-2 in RAW264.7 cells. (A) Protein expressions of iNOS and COX-2 were down-regulated by compound 4 using Western blot. (B,C) Quantitative analysis of the Western blot. ###, p < 0.01, compared with the control that was treated without compound 4; **, p < 0.01 and ***, p < 0.001, compared that only stimulated with LPS.

59.6 and 54.1 in 3 were replaced by δC 152.7 and 151.2 in 4, suggesting the presence of a double bond between C-8 and C9 in 4. Its HMBC spectrum showed correlations of H-6/H-15/ H-27 with C-8, and H-1/H-12/H-19 with C-9, confirming the above speculation (Figure 2). The NOE correlation of H-15 (δH 4.29) with CH3-18 (δH 0.87) demonstrated an αorientation for 15-OH (Figure 3). As a result, the structure of 4 was determined as methyl lucidenate L. Compound 5, white powder, has a molecular formula of C30H44O8 based on negative HRESIMS data at m/z 531.2967 [M − H]− (calcd for C30H43O8−, 531.2958). Comparison of its 1 H and 13C NMR data (Table 2) with those of methyl ganoderate I54 showed an obvious difference at C-3 and C-15 in rings A−D. The absence of an oxygenated methine at C-3 (δC 78.4/δH 3.22) and a carbonyl at C-15 (δC 217.7) in methyl ganoderate I, and the existence of an oxygenated methine at δC 73.3/δH 4.82 and a carbonyl at δC 220.0 in 5, suggested that C3 might be a carbonyl and a hydroxyl group attached to C-15

in 5. The HMBC correlations of H-15 (δH 4.82) with C-16/C30, and H2-2 (δH 2.58/2.40) with C-1/C-3/C-10 confirmed the above supposition (Figure 2). The 7-OH and 15-OH in 5 were determined to be β- and α-oriented, respectively, according to the NOESY correlations of H-7 with H-5/CH330, and H-15 with CH3-18 (Figure 3). Therefore, the structure of compound 5 was assigned as 7β,15α,20-trihydroxy-3,11,23trioxo-5α-lanosta-8-en-26-oic acid. Compound 6 was determined to possess the molecular formula of C32H44O9 based on HRESIMS data at m/z 571.2919 [M − H]− (calcd for C32H43O9−, 571.2907) and its 13C NMR data. The 1H and 13C NMR data (Table 2) were almost identical to those of the known compound ganoderic acid θ,49 except for the presence of acetoxyl signals [δH 2.14 (3H, s); δC 171.8, 21.0] in 6. The HMBC spectrum revealed obvious correlations CH3 (δH 2.14) in the acetoxyl group with C-12/carbonyl carbon (δC 171.8), and H-12 (δH 5.67) with C11/C-13/carbonyl carbon (δC 171.8), confirming that the 5154


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Figure 8. Inhibitory effect of compound 4 on the activation of NF-κB signaling pathway in LPS-induced RAW 264.7 cells. (A) Western blots of NF-κB p65, IκBα, p-IκBα, IKKβ, and p-IKKβ. (B) Compound 4 inhibited LPS-induced nuclear translocation of NF-κB p65. RAW264.7 cells were stained for nuclei (DAPI, blue) and NF-κB (green).

To evaluate whether compound 4 inhibited the inflammatory response in LPS-induced macrophage RAW264.7, the levels of pro-inflammatory cytokines IL-1β and IL-6 were measured by ELISA. As expected, LPS stimulation significantly elevated the release of IL-1β and IL-6. Meanwhile, compound 4 treatment dose-dependently reduced the LPS-induced cytokine increase (Figure 6A,B). The inhibition of NO in macrophage cells was closely related to the down-regulated level of iNOS expression.55 COX-2, as an important pro-inflammatory protein, is also connected with many inflammatory-associated diseases.56 Therefore, the expression levels of iNOS and COX-2 were measured in RAW264.7 cells after LPS stimuli with or without compound 4. As a result, LPS treatment dramatically enhanced the expressions of iNOS and COX-2, which were reduced by compound 4 dose-dependently (Figure 7). These potent anti-inflammatory effects of compound 4 prompted us to study its probable mechanism. NF-κB was proved to be a nuclear transcription factor and play a key role in inflammatory response.57 The stimulation of pro-inflammatory factors induces phosphorylation of IKKβ and IκBα in inflammatory state. Then, unbounded p65 separates from IκBα and subsequently translocates into nucleus to activate transcriptions of its target genes.58,59 Our results demonstrated that compound 4 inhibited LPS-triggered activation of NF-κB signaling pathway. As shown in Figure 8A, the expression of NF-κB in RAW264.7 cells was up-regulated by LPS and then was reduced by compound 4 at 10 μΜ. Meanwhile,

acetoxyl group was attached to C-12 (Figure 2). The NOESY cross-peaks of H-3 correlating to H-28/H-5, and H-12 correlating to CH3-30 suggested that 3-OH and CH3COO12 were both β-orientated (Figure 3). The (S)- and (R)MTPA ester derivatives of 6 at C-23 were synthesized from (R)-(+)-MTPA-Cl and (S)-(−)-MTPA-Cl, respectively. The Δδ-values demonstrated a 23S configuration (Figure 4). As a result, the structure of 6 was identified as 12β-acetoxyganoderic acid θ. All the isolated compounds (1−33) were assessed for their inhibitory activities at 50 μM against NO production triggered by LPS in mouse macrophage cell RAW264.7. Among all these compounds, 4 and 9 showed stronger inhibition against NO production (inhibition rate >50%) than other compounds (Figure 5A, Supporting Information Table S1). Since inhibition rate of 4 is higher than 9 at 50 μM, the IC50 value of 4 was further assayed, and the result was 38.6 ± 1.0 μM (Figure 5C), which was chosen for further anti-inflammatory research. Dexamethasone (Dex, IC50 = 7.9 ± 1.3 μM) was employed as a positive control (Figure 5B). The cytotoxicity of 4 on RAW264.7 cells was measured to exclude the possibility that the inhibition of NO production was owing to its cytotoxic effects. This result demonstrated no significant inhibitory effects of 4 on cell viability at the concentration up to 50 μM and its IC50 was greater than 200 μM (Figure 5D). Therefore, in the subsequent experiments, the concentration of this compound was controlled to be less than 50 μM. 5155


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201602689], and Shenyang Planning Project of Science and Technology [grant number 18-013-0-46].

phosphorylated IκBα (p-IκBα) and phosphorylated IKKβ (pIKKβ) were increased after pretreated with LPS, and administration with compound 4 obviously inhibited the phosphorylation of both IκBα and IKKβ (Figure 8A). Moreover, treatment of compound 4 blocked the nuclear translocation of p65 induced by LPS (Figure 8B). Taken together, the anti-inflammatory mechanism of compound 4 is related to the suppression of NF-κB activation in macrophage RAW264.7 cells. In summary, six new triterpenoids (1−6) and 27 known analogues (7−33) were separated from the fruiting bodies of G. lucidum. Their structures were established using analyses of spectroscopic data, Mosher method, and comparison with literature data. Among them, compound 4 displayed the strongest inhibitory effect against LPS-induced NO production in RAW264.7 macrophage cells. Furthermore, compound 4 attenuated the production of pro-inflammatory cytokines including IL-6 and IL-1β in LPS-induced RAW264.7 cells. The expression levels of iNOS, COX-2, and NF-κB were effectively decreased, and the phosphorylation levels of IκBα and IKKβ were inhibited by compound 4 in LPS-induced macrophage cells. These findings provided a preliminary molecular and bioactivity basis for the anti-inflammatory efficacy of G. lucidum and revealed that triterpenoid 4 might be used as a potential anti-inflammatory candidate and that the total triterpenoids might be developed as value-added functional food for the prevention of inflammation. In combination with our previous study25−28 and the published literatures,13,15−24,29 the differences on small-molecular constituents and their bioactivities between G. sinense and G. lucidum suggest that it should be cautious for their interchangeable usage. Our research can expand the potential applications of G. lucidum and further provide references for the development and utilization of G. lucidum as functional foods.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.9b01195.

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REFERENCES

Spectra and inhibition rate against NO production of all compounds (PDF)

AUTHOR INFORMATION

Corresponding Authors

*Tel: +86-24-23986463. E-mail: [email protected] (L.-X.C.). *Tel: +86-24-23986463. E-mail: [email protected] (H.L.). ORCID

Hua Li: 0000-0003-1903-836X Li-Xia Chen: 0000-0003-2196-1428 Author Contributions ⊥

These authors contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by National Natural Science Foundation of China (NSFC) [grant numbers 81773594, U1803122, U1703111, 81473254, and 81773637], Liaoning Province Natural Science Foundation [grant number 5156


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