Triterpenoids from Ganoderma lucidum and Their Potential Anti

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Bioactive Constituents, Metabolites, and Functions

Triterpenoids from Ganoderma lucidum and their Potential Anti-inflammatory Effects Yanli Wu, Fei Han, Shanshan Luan, Rui Ai, Peng Zhang, Hua Li, and Lixia Chen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b01195 • Publication Date (Web): 17 Apr 2019 Downloaded from http://pubs.acs.org on April 18, 2019

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Journal of Agricultural and Food Chemistry

1

Triterpenoids from Ganoderma lucidum and their Potential

2

Anti-inflammatory Effects

3 4

Yan-Li Wu,†,⊥ Fei Han,†,⊥Shan-Shan Luan,‡,⊥Rui Ai,† Peng Zhang,† Hua Li,*,†,‡ and

5

Li-Xia Chen*,†

6 7

†

8

Structure-Based Drug Design & Discovery, Ministry of Education, Shenyang

9

Pharmaceutical University, Shenyang 110016, China.

Wuya College of Innovation, School of Pharmacy, Key Laboratory of

10

‡

11

Technology, Wuhan, 430030, China.

School of Pharmacy, Tongji Medical College, Huazhong University of Science and

12 13 14 15 16 17 18

1

ACS Paragon Plus Environment


19

ABSTRACT

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Ganoderma lucidum, as food, tea, dietary supplement, and medicine, is widely used in

21

China and Eastern Asian countries. In order to discover its anti-inflammatory

22

constituents and provide some references for the usage of G. lucidum and G. sinense,

23

two official species in China, the fruiting bodies of G. lucidum were studied, leading

24

to the isolation of six new triterpenoids (1-6) and 27 known analogues (7-33).

25

Compound 4 exhibited the most potent inhibition on nitric oxide (NO) production

26

induced by lipopolysaccharide (LPS) in RAW264.7 macrophage cells. The production

27

of IL-6 and IL-1β, as well as the expression of iNOS, COX-2 and NF-κB were

28

dose-dependently reduced by 4. The phosphorylations of IκBα and IKKβ in

29

LPS-induced macrophage cells were blocked by 4. Therefore, 4 could be used as a

30

potential anti-inflammatory candidate and the total triterpenoids might be developed

31

as value-added functional food for the prevention of inflammation. In combination of

32

previous studies, it should be cautious for the interchangeable usage of G. lucidum

33

and G. sinense.

34 35

Keywords: Ganoderma lucidum; Triterpenoids; Anti-inflammation

2


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1. INTRODUCTION

37

Mushrooms have always been a popular food for their delicious taste and being

38

rich in nutrient elements for thousands of years. Some of them are also used as potent

39

food supplements and medicinal sources for human beings [1]. Ganoderma lucidum

40

(Lingzhi in Chinese, Reishi in Japanese, and Youngzhi in Korean), a kind of famous

41

edible and medicinal mushroom, has been commonly used as functional food and

42

traditional medicine for regulating immunity and promoting health in China and other

43

Eastern Asian countries [2-3]. The development and utilization of G. lucidum have

44

made great progress in China, and a large number of artificially cultivated G. lucidum

45

is exported to many countries as food, tea, dietary supplements, and raw materials for

46

further processing, which has brought great benefits to the planting industry of edible

47

and medicinal mushrooms [4]. The mushrooms of G. lucidum could prevent and treat

48

various diseases, such as bronchitis, asthma, hypercholesterolaemia, hepatitis,

49

hypertension, neurasthenia, leucopenia and cancer [5], with a wide range of biological

50

activities such as anti-inflammation [6-7], immune regulation [8-9], hepatoprotection

51

[10], and antitumor [11-12].

52

G. lucidum and its closely related species G. sinense are suggested be used

53

interchangeably as Lingzhi according to Chinese Pharmacopoeia [2-3,13-14].

54

Chemical investigations on the two species of Ganoderma based on multiple analytic

55

technologies or chemical separation have been carried out, and triterpenoids and

56

polysaccharides were determined as the major constituents [13,15-22] of G. lucidum.

57

While, studies on G. sinense are relatively few, showing the presence of 3



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meroterpenoids, steroids, alkaloids, triterpenoids, and polysaccharides [13,23-28].

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Comparing the chemical constituents from two official species of Ganoderma genus

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demonstrated the significant difference on type and content of triterpenoids by high

61

performance liquid chromatography coupled with photodiode array (HPLC-PDA) and

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high performance liquid chromatography-mass spectrometry (LC-MS) analyses, with

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about 10 times higher in G. lucidum than G. sinense, and lack of common triterpenes

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in G. sinense [13,15]. While, the similar chemical features of polysaccharides

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between both species were observed [13,29], and they showed similar antitumor and

66

immunomodulating activities [29]. Although polysaccharides as the active principles

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could explain to some extent the official use of both species as Lingzhi, the influence

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of numerous small-molecular constituents and their different bioactivities in both

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species remains to be further investigated and provides a deeper explanation for their

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exchangeable usage in China.

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Nuclear factor-κB (NF-κB) signaling pathway has been proved to play an

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important role in inflammation and immune responses, and NF-κB can be activated

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by the phosphorylation of IκB by IκB kinase (IKK) complex, mainly by IKKβ [30-31].

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NF-κB activation is usually induced by the translocation to nucleus of its dimmers

75

which can bind with DNA to trigger the expressions of a series of inflammatory

76

cytokines such as interleukin (IL)-6 and IL-1β, and stress response proteins including

77

cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) [32]. Nitric

78

oxide (NO) is produced by iNOS and excessive amounts of NO can lead to multiple

79

inflammation-related diseases, such as allergic rhinitis, arthritis, and bowel diseases 4


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[33-35].

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In order to discover the anti-inflammatory constituents from G. lucidum and

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further provide some references for the usage of both official species of Lingzhi in

83

China, the fruiting bodies of G. lucidum were sequentially investigated herein based

84

on our previous study on G. sinense [25-28]. As a result, 6 new triterpenoids (1-6) and

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27 known analogues (7-33) were isolated and characterized from G. lucidum. Their

86

inhibitory effects on NO production in LPS-stimulated RAW 264.7 macrophages and

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anti-inflammation mechanism were preliminarily investigated herein.

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2. MATERIALS AND METHODS

89

2.1 General

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Melting point was tested with an X-4 digital display micromelting point apparatus

91

(uncorrected). Optical rotation data were recorded on a PerkinElmer 241 polarimeter

92

(Perkin-Elmer, Waltham, MA, USA). Ultraviolet (UV) spectra were measured on a

93

Shimadzu UV 2201 UV-VIS spectrophotometer (Shimadzu Corporation, Kyoto,

94

Japan). Infrared (IR) absorption spectra [4000−400 cm−1; potassium bromide (KBr)

95

disks] were performed on a Bruker IFS 55 spectrometer (Bruker Optics, Ettlingen,

96

Germany). Nuclear magnetic resonance (NMR) experiments including proton nuclear

97

magnetic resonance (1H-NMR), carbon nuclear magnetic resonance (13C-NMR),

98

nuclear overhauser enhancement spectroscopy (NOESY), heteronuclear single

99

quantum correlation (HSQC), and heteronuclear multiple-bond correlation (HMBC)

100

were measured on Bruker AV-600 or ARX-400 spectrometers (Bruker Biospin,

101

Fallanden, Switzerland). Chemical values are expressed in δ (ppm) relative to 5



102

tetramethylsilane (TMS), and the coupling constants are depicted as J (Hz). High

103

resolution electron spray ionization mass (HRESIMS) data were collected on an

104

Agilent 6210 TOF mass spectrometer (Palo Alto, USA). Silica gel (200−300 mesh,

105

Qingdao Marine Chemical Factory, Qingdao, China), Sephadex LH-20 (Pharmacia,

106

Amersharm, Sweden), and octadecyl silica gel (Merck Chemical Company Ltd.,

107

Darmstadt, Germany) were applied for column chromatography (CC). Silica gel

108

GF254 for thin layer chromatography (TLC) was bought from Qingdao Marine

109

Chemical Factory (Qingdao, China). Prepared HPLC was performed on an LC-6AD

110

liquid chromatography equipped with a SPD-10AVP UV/vis detector (Shimadzu,

111

Kyoto, Japan) and an ODS-A column (250 × 20 mm, 5μm, 120 Å, YMC Co. Ltd.).

112

All reagents (Tianjin Damao Chemical Company, Tianjin, China) were HPLC or

113

analytical grade. Spots on TLC plates were visualized under UV light and by spraying

114

with anisaldehyde-H2SO4 reagent.

115

2.2 Fungal material

116

The fruiting bodies of G. lucidum (Leyss ex Fr) Karst were provided by Jiangsu

117

Xinxian Pharmaceutical Co. Ltd. (Jiangsu, China), and authenticated by Professor

118

Jincai Lu, School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical

119

University. A voucher specimen (LZ-15-058) was preserved in our laboratory.

120

2.3 Extraction and isolation

121

The fruiting bodies of G. lucidum (9.0 kg) were cut into small pieces (about 2 cm)

122

and extracted with 80% ethanol (EtOH, 90 L × 2 h × 2) to afford a total extract (240.7

123

g) after concentrating in vacuo. Then the extract was suspended in water (H2O, 5 L), 6


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and partitioned successively with the same volume of cyclohexane, ethyl acetate

125

(EtOAc), and n-butanol (n-BuOH) for three times. The EtOAc extract (131.1 g) was

126

subjected to silica gel CC (10 × 80 cm) eluted with dichloromethane/methanol

127

(CH2Cl2/MeOH, 100:1, 50:1, 20:1, 10:1, 5:1, 2:1, 1:1, and 0:1, v/v) to afford six

128

fractions (CE1−CE6) based on silica gel TLC analyses. Separation of CE2 (8.2 g) on

129

an ODS column (2.5 × 30 cm) with MeOH/H2O (30:70, 50:50, 70:30, and 100:0, v/v)

130

as eluent yielded fractions CE21−CE25. CE23 (2.8 g) was separated on HPLC (58%

131

MeOH/H2O) to obtain four fractions (CE231−CE234). Compounds 26 (25.7 mg) and

132

27 (27.6 mg) were further purified from fraction CE232 (100.5 mg) by preparative

133

HPLC [50% acetonitrile/H2O (MeCN/H2O)]. CE233 (500.2 mg) was separated by

134

preparative HPLC (45% MeCN/H2O) to give 11 (22.4 mg), and two subfractions

135

CE2332 and CE2334. CE2332 (121.1 mg) was further purified by preparative HPLC

136

(60% MeOH/H2O) to give 7 (32.4 mg) and 24 (16.8 mg). Subfraction CE2334 (50.9

137

mg) yielded 20 (11 mg) through purification on a preparative HPLC (70%

138

MeOH/H2O). Separation of CE234 (918.9 mg) by preparative HPLC (45%

139

MeCN/H2O) afforded 3 (29.3 mg), 4 (8.0 mg), 6 (14.9 mg), 17 (227.9 mg), 21 (4.2

140

mg), and CE2344 (512.4 mg). Compounds 9 (219.0 mg) and 16 (294.3 mg) were

141

obtained from fraction CE2344 by preparative HPLC (60% MeOH/H2O). Fraction

142

CE3 (3.3 g) was separated by ODS CC (2.5 × 30 cm) with MeOH/H2O (10:90, 30:70,

143

50:50, 70:30, and 100:0, v/v) as eluent to give five subfractions (CE31−CE35). Fr.

144

CE33 (1.5 g) was further separated by preparative HPLC (65% MeOH/H2O) to give

145

three subfractions (CE331−CE333). Purification of CE331 (507.9 mg) by preparative 7



146

HPLC (65% MeOH/H2O) afforded 8 (10.7 mg), 10 (21.9 mg), 12 (174.4 mg), 22

147

(13.0 mg), 23 (27.6 mg), and 28 (20.2 mg). Compound 25 (28.3 mg) was obtained

148

from fraction CE332 (86.1 mg) by preparative HPLC (40% MeCN/H2O). Separation

149

of CE333 (309.7 mg) on preparative HPLC eluted with 40% MeCN/H2O afforded 15

150

(141.1 mg). CE4 (2.2 g) was separated on an ODS column (2.5 × 30 cm) eluted with

151

MeOH/H2O (30:70, 50:50, 70:30, and 100:0, v/v) to afford fractions CE41-CE45.

152

Purification of CE43 (307.9 mg) by preparative HPLC (65% MeOH/H2O) yielded 1

153

(8.2 mg), 2 (10.4 mg), 5 (20.1 mg), 14 (4.2 mg), and 32 (2.2 mg). Separation of CE5

154

(3.3 g) on an ODS column (2.5 × 30 cm) with MeOH/H2O (30:70, 50:50, 70:30, and

155

100:0 v/v) as eluent yielded fractions CE51−CE55. CE53 (1.0 g) was separated by

156

using silica gel CC (2 × 30 cm) and eluted with CH2Cl2/MeOH (100:1, 50:1, 20:1,

157

10:1, 5:1, 2:1, 1:1, and 0:1, v/v) to give four fractions (CE531−CE534). CE531 (31.0

158

mg) yielded 19 (6.3 mg) through purification on preparative HPLC (60%

159

MeOH/H2O). CE532 (200.9 mg) was further purified by preparative HPLC (35%

160

MeCN/H2O) to yield 18 (10.9 mg), 30 (2.7 mg), and 31 (40.5 mg). Purification of

161

CE533 (309.8 mg) by preparative HPLC (50% MeOH/H2O) afforded 13 (120.0 mg).

162

Fraction CE6 (1.2 g) was separated further by ODS CC (2.5 × 30 cm) eluted with

163

MeOH/H2O (10:90, 30:70, 50:50, 70:30, and 100:0, v/v) to give five subfractions

164

(CE61−CE65). CE63 (156.3 mg) was further separated by preparative HPLC (40%

165

MeCN/H2O) to give 29 (36.0 mg) and 33 (5.8 mg).

166

12β-Acetoxy-3β,28-dihydroxy-7,11,15,23-tetraoxo-5α-lanosta-8-en-26-oic acid

167

(1): White powder (MeOH); [α] D + 67 (c 0.125, MeOH); UV (MeOH) λmax (log ε) 256

25

8


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(4.03) nm; IR (KBr) νmax 3427, 2920, 2360, 1641, 1464, 1384, 1123, 619 cm−1; 1H

169

NMR (400 MHz, CD3OD) and

170

HRESIMS (negative) m/z 587.2866 [M - H]- (calcd for C32H43O10-, 587.2856).

171

13C

NMR (100 MHz, CD3OD) data, see Table 1;

25

Lucidenic acid R (2): White powder (MeOH); [α] D + 61 (c 0.095, MeOH); UV

172

(MeOH) λmax (log ε) 256 (3.99) nm; IR (KBr) νmax 3433, 2920, 1745, 1463, 1384,

173

1118, 619 cm−1; 1H NMR (400 MHz, CD3OD) and 13C NMR (100 MHz, CD3OD)

174

data, see Table 1; HRESIMS (negative) m/z 531.2595 [M - H]- (calcd for C29H39O9-,

175

531.2594). 25

176

Methyl lucidenate K (3): Colorless needles (MeOH); mp 179-182˚C; [α] D + 113

177

(c 0.205, MeOH); UV (MeOH) λmax (log ε) 257 (4.04) nm; IR (KBr) νmax 3435, 2921,

178

1737, 1462, 1384, 1170, 617 cm−1; 1H NMR (400 MHz, CDCl3) and 13C NMR (100

179

MHz, CDCl3) data, see Table 1; HRESIMS (negative) m/z 473.2903 [M - H]- (calcd

180

for C28H41O6-, 473.2903). 25

181

Methyl lucidenate L (4): White powder (MeOH); [α] D + 118 (c 0.1, MeOH); UV

182

(MeOH) λmax (log ε) 265 (5.95) nm; IR (KBr) νmax 3435, 2922, 1638, 1384, 1115,

183

1017, 619 cm−1; 1H NMR (600 MHz, CDCl3) and 13C NMR (150 MHz, CDCl3) data,

184

see Table 2; HRESIMS (negative) m/z 471.2754 [M - H]- (calcd for C28H39O6-,

185

471.2747).

186

7β,15α,20-Trihydroxy-3,11,23-trioxo-5α-lanosta-8-en-26-oic acid (5): white

187

powder (MeOH); [α] D + 121.1 (c 0.185, MeOH); UV (MeOH) λmax (log ε) 252 (4.3)

188

nm; 1H NMR (600 MHz, CDCl3) and 13C NMR (150 MHz, CDCl3) data, see Table 2;

189

HRESIMS (negative) m/z 531.2967 [M - H]- (calcd for C30H43O8-, 531.2958).

25

9



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25

190

12β-Acetoxyganoderic acid θ (6): white powder (MeOH); [α] D + 93.3 (c 0.045,

191

MeOH); UV (MeOH) λmax (log ε) 210 (4.6) nm; 1H NMR (600 MHz, CDCl3) and 13C

192

NMR (150 MHz, CDCl3) data, see Table 2; HRESIMS (negative) m/z 571.2919 [M -

193

H]- (calcd for C32H43O9-, 571.2907).

194

2.4 Preparation of (R)- and (S)-MTPA esters of 6

195

Compound 6 (1.0 mg) was divided equally into two NMR tubes and dried under

196

vacuum

for

12

hours.

(R)-Methoxy-α-(trifluoromethyl)phenylacetyl

chloride

197

[(R)-MTPA-Cl, 10 μL) and deuterated pyridine (0.5 mL) were added into one NMR

198

tube under the protection of nitrogen (N2). The reaction was performed at room

199

temperature for hours and monitored by 600 MHz NMR. The 1H NMR spectrum was

200

measured on 600 MHz NMR in pyridine-d5 (Figure S51). Similarly, (S)-MTPA-Cl

201

(10 μL) and deuterated pyridine (0.5 mL) were added into another NMR tube and

202

reacted at room temperature to yield (S)-MTPA ester derivative, and the 1H NMR

203

spectrum was recorded on 600 MHz NMR in pyridine-d5 (Figure S52).

204

2.5 Cell cultures

205

Mouse RAW264.7 macrophage cells were obtained from ATCC and cultured in

206

Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum

207

(FBS, Sigma), 100 U/mL penicillin (Hyclone), and 100 μg/mL streptomycin (Hyclone)

208

with 5% CO2/95% air (v/v) at 37 °C. The cells were divided into three groups:

209

dimethyl sulfoxide (DMSO) control group, LPS (1 μg/mL) group, LPS (1 μg/mL)

210

plus compounds group.

211

2.6 CCK-8 assay 10


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The viability of mouse RAW264.7 macrophage cells was determined by CCK-8

213

(Cell counting Kit-8, Beyotime, Shanghai, China). The RAW264.7 cells were seeded

214

into 96-well plate (Nest, Biotech, China) with the density of 5×104 cells/mL. After

215

treatment with series concentrations of compound 4 (0–200 μM) for 24 h, the

216

absorbance (540 nm) was recorded on a microplate reader following the

217

manufacturer’s instructions.

218

2.7 Bioassay for NO production

219

Nitrite, as an indicator of NO production, was measured for its concentration in

220

medium according to Griess method [36]. RAW 264.7 cells were inoculated into

221

96-well plates with 2 × 104 cells/well and were cultured overnight. After replacement

222

with new medium, cells were stimulated with 1 μg/mL of LPS with or without tested

223

compounds and incubated for 24 h at 37 °C. The cell-free supernatant and Griess

224

reagent were completely mixed with the same amount of 100 μL. Absorbance of the

225

final product was measured at 540 nm on a microplate reader. The nitrite

226

concentration and inhibitory rate were calculated according to the standard calibration

227

curve. The inhibitory effect of the tested compounds on LPS-induced NO production

228

was described as IC50 values.

229

2.8 ELISA assay

230

The secretion of the inflammatory cytokines in RAW264.7 cell supernatants was

231

detected after treated with compound 4. Cell supernatants were collected and ELISA

232

kits (Boster, China) were applied for determination of the production of IL-1β and

233

IL-6 following the manufacturer’s instructions. 11



234

2.9 Western blot assay

235

Western blot analysis was performed to evaluate the expression of

236

inflammation-associated proteins, such as iNOS, COX-2 and NF-κB pathway proteins.

237

Cells were collected, then resuspended with radio immunoprecipitation assay (RIPA)

238

buffer supplemented with 0.1 mM PMSF protease inhibitor. The cell suspension was

239

lysed by vortex on ice, and then centrifuged at a high speed of 20,000 × g to get

240

supernatant. The total protein concentrations were determined by BCA protein assay

241

kit. The same amount of proteins for each sample was loaded to 10% sodium dodecyl

242

sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and the proteins separated on

243

the gel were transferred onto a polyvinylidene difluoride (PVDF) membrane. After

244

blocked with 5% skim milk, the membranes were then incubated with specific

245

primary antibodies at 4 °C overnight, followed by incubated with corresponding

246

secondary antibodies. The protein blots were finally detected by enhanced

247

chemiluminescence (ECL) system.

248

2.10 Immunofluorescence assay

249

To observe the nuclear translocation of NF-κB, macrophage cells were cultured in

250

a glass chamber to 50–60% confluence, and then treated with DMSO, 20 μM or 40

251

μM compound 4. Two hours later, cells were stimulated with 1 μg/mL LPS for 12 h.

252

After washing with phosphate buffer saline (PBS), cells were fixed in

253

paraformaldehyde and then blocked with 5% BSA for 1 h. The glass chambers were

254

incubated with NF-κB antibody at 1:1000 dilution at 4 °C overnight, followed by a

255

secondary AlexaFluor488 antibody at 1:500 dilution for 30 min in the dark. The cell 12


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nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI) for 30 min at 37 °C.

257

Finally, the nuclear translocation was observed using a fluorescence microscope

258

(Nikon).

259

2.11 Statistical Analysis

260

At least triplicate experiments were conducted to obtain all results and the data are

261

displayed as mean ± SD. GraphPad Prism, version 4.00 and SPSS software, version

262

22.0 with one-way ANOVA and Student’s t-test were used for the determination of

263

statistical significance (p < 0.05).

264

3. RESULTS AND DISCUSSION

265

Study on constituents of the fruiting bodies of G. lucidum yielded six new

266

triterpenoids (1-6), and 27 known analogues (7-33) including methyl ganoderate C1

267

(7) [37], 12-acetoxyganoderic acid D (8) [38], methyl ganoderate F (9) [39],

268

ganoderic acid E (10) [40], methyl ganoderate E (11) [37], ganoderic acid F (12) [40],

269

ganoderic acid C (13) [41], methyl ganoderate C (14) [41], ganoderic acid J (15) [42],

270

methyl lucidenate D2 (16) [39], methyl lucidenate A (17) [37], ganoderenic acid C

271

(18) [40], ganoderenic acid A (19) [40], methyl lucidenate H (20) [43], ganoderenic

272

acid

273

12β-acetoxy-7β-hydroxy-3,11,15,23-tetraoxo-5α-lanosta-8,20-dien-26-oic acid

274

[44], 12β-acetoxy-3,7,11,15,23-pentaoxo-lanosta-8,20-dien-26-oic acid (23) [45],

275

ganoderenic acid B (24) [40], ganoderenic acid G (25) [46], methyl ganoderenate D

276

(26) [47], methyl ganoderate P (27) [48], ganoderenic acid F (28) [46], ganoderenic

277

acid D (29) [40], ganoderic acid η (30) [49], ganoderic acid ζ (31) [49], lucidone F (32)

K

(21)

13


[38], (22)


278

Page 14 of 50

[50], ganoderic acid I (33) [42] (Figure 1).

279 21

R4 18 O

17

19

1

R1

26

R4

COOR6

O

O

30

R2

R3

R2 O -H, -OH -H, -OH O O O O -H, -OH -H, -OH O

25

26

R3 O O O O O O O -OH, -H -OH, -H -OH, -H

R4 AcO H AcO AcO H H AcO H H H

R5 OH H H H H H H H H H

R6 H Me H Me H Me H H Me H

R2

COOCH3

O

OH

O

R4 18

O

COOH

O

HO 6 21

R4 18

26

19

26

O

R1 18: -H, -OH 19: O 24: -H, -OH 25: O 26: O 28: O 29: O

27

R3 30

R1

28

R2 R3 -H,-OH -OH, -H -H, -OH -OH, -H -H, -OH O -OH,-H O -H, -OH O O O -H, -OH O

R4 H H H H H H H

R5 H H H H Me H H

HO

280 281

28

R2

R2 -H, -OH -H, -OH O -H, -OH

R3

R3  O O O

R4 AcO AcO AcO AcO

COOH

O

O

O

R1 30: -H, -OH 31: O

O R2 OH H

OH

R5 H H H Me

OH

O HO

R1

29

R1 21: -H, -OH 22: O 23: O 27: -H, -OH

R2 O

COOR5

O

19 30

O

27

COOR5

R2

R6 H Me Me Me Me

HO

OH OH

O

R1

R5 OH H H H OH

O O

5 O

R4 AcO H AcO H H

O

COOH

O

21

R3

R1 R2 R3 2: -H, -OH O O 4: -OH, -H O O 16: O O O 17: -H, -OH O O 20: -H, -OH -H, -OH O

O

3

29

COOR6

R5

OH

O

24

23

27

R1

R5 R1 1: -H, -OH 7: O 8:  9: O 10: O 11: O 12: O 13: -H, -OH 14: -H, -OH 15: O

20 22

27 10 5 28

29

21

20

O

OH HO

32

OH

COOH

O

33

Figure 1. Structures of compounds 1-33.

282 283 284

The molecular formula of compound 1 was established as C32H44O10 via HRESIMS (m/z 587.2866 [M - H]-, calcd for C32H43O10-, 587.2856) and 14


13C

NMR

Page 15 of 50


285

spectroscopic data, implying 11 indices of hydrogen deficiency. The 1H and 13C NMR

286

data (Table 1) suggested the presence of seven methyls, two oxygenated methines,

287

one olefinic bond, four ketone carbonyls, and two ester or carboxyl groups. Its 1H and

288

13C

289

from G. lucidum [51], except for a major difference at C-28. The methyl group at

290

C-28 (δC 27.9/δH 1.03) in ganoderic acid H was replaced by an oxygenated methene

291

(δC 65.5/δH 3.53, 3.24) in 1, demonstrating the linkage of a hydroxyl group to C-28.

292

The key HMBC (Figure 2) from H2-28 to C-3/C-4/C-5/C-29 confirmed the above

293

supposition. In its NOESY spectrum, H-3 correlated to H-28/H-5, and H-30 to

294

H-5/H-12, demonstrating a β-orientation for both 3-OH and CH3COO-12 (Figure 3).

295

Thus,

296

12β-acetoxy-3β,28-dihydroxy-7,11,15,23-tetraoxo-5α-lanosta-8-en-26-oic acid.

NMR data (Table 1) were similar to those of ganoderic acid H previously isolated

the

structure

of

compound

1

15


was

identified

as


O

O O

O COOH

O

HO

O

OH

HO

O OH

1

COOCH3

O

OH

O

COOCH3

O

O

4

O OH

297 298

O O

COOH

OH

OH

O

OH

O

O

2

3

O

COOH

O

O

O

O

Page 16 of 50

HO

O COOH HO O

O 6

5

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

16


Page 17 of 50


1

2

3

4

6 5

299 300

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

301 302

The HRESIMS of compound 2 gave a pseudo molecular ion peak at m/z 531.2595

303

[M - H]- (calcd for C29H39O9-, 531.2594), suggesting its formula as C29H40O9. Its 1H

304

and 13C NMR data (Table 1) resembled to those of lucidenic acid E [52], except for

305

the missing of a methyl signal at C-25 (δC 27.9/δH 1.03), and the appearance of one

306

oxygenated methene (δC 65.5/δH 3.53, 3.24) in 2, revealing the hydroxylation of C-25.

307

This supposition was further supported by the HMBC correlations (Figure 2) of H2-25

308

with C-3/C-4/C-5/C-26. In the NOESY spectrum, H-3 correlated to H-5/H-25, and

309

H-12 to CH3-27, demonstrating a β-orientation for 3-OH and CH3COO-12 (Figure 3).

310

As a result, the structure of 2 was determined as lucidenic acid R. 17



Page 18 of 50

311

Compound 3 possessed a molecular formula of C28H42O6 as determined via

312

HRESIMS peak at m/z 473.2903 [M - H]- (calcd for C28H41O6-, 473.2903), implying 8

313

indices of hydrogen deficiency. Its

314

similarity with methyl 8β, 9α-dihydroganoderate J [53], especially for the carbon

315

signals of rings A–D. The HMBC spectrum revealed obvious correlations of CH3-21

316

(δH 0.82) with C-17/C-20/C-22, H-23b (δH 2.23) with C-22/C-24, and H3-OCH3 (δH

317

3.65) with C-24, confirming the linkage of the five-carbon side chain (from C-20 to

318

C-24) (Figure 2). In its NOESY spectrum, H-15 (δH 4.05) correlated to CH3-18 (δH

319

0.78), demonstrating an α-orientation for 15-OH (Figure 3). The structure of

320

compound 3 was thus established as methyl lucidenate K.

13C

NMR spectroscopic data showed close

321

The molecular formula of compound 4 was deduced as C28H40O6 by its 13C NMR

322

data and [M - H]- at m/z 471.2754 (calcd for C28H39O6-, 471.2747) in the negative

323

HRESIMS. Its 1H and 13C NMR data (Table 2) were almost consistent with those of 3,

324

except that the resonances at δC 59.6 and 54.1 in 3 were replaced by δC 152.7 and

325

151.2 in 4, suggesting the presence of a double bond between C-8 and C-9 in 4. Its

326

HMBC

327

H-1/H-12/H-19 with C-9, confirming the above speculation (Figure 2). The NOE

328

correlation of H-15 (δH 4.29) with CH3-18 (δH 0.87) demonstrated an α-orientation for

329

15-OH (Figure 3). As a result, the structure of 4 was determined as methyl lucidenate

330

L.

spectrum

showed

correlations

of

H-6/H-15/H-27

331 332 18


with

C-8,

and

Page 19 of 50


333

Compound 5, white powder, has a molecular formula of C30H44O8 based on

334

negative HRESIMS data at m/z 531.2967 [M - H]- (calcd for C30H43O8-, 531.2958).

335

Comparison of its 1H and 13C NMR data (Table 2) with those of methyl ganoderate I

336

[54], showed an obvious difference at C-3 and C-15 in rings A–D. The absence of an

337

oxygenated methine at C-3 (δC 78.4/δH 3.22) and a carbonyl at C-15 (δC 217.7) in

338

methyl ganoderate I, and the existence of an oxygenated methine at δC 73.3/δH 4.82

339

and a carbonyl at δC 220.0 in 5, suggested that C-3 might be a carbonyl and a

340

hydroxyl group was attached to C-15 in 5. The HMBC correlations of H-15 (δH 4.82)

341

with C-16/C-30, and H2-2 (δH 2.58/2.40) with C-1/C-3/C-10 confirmed the above

342

supposition (Figure 2). The 7-OH and 15-OH in 5 were determined to be β- and

343

α-oriented, respectively, according to the NOESY correlations of H-7 with

344

H-5/CH3-30, and H-15 with CH3-18 (Figure 3). Therefore, the structure of compound

345

5 was assigned as 7β,15α,20-trihydroxy-3,11,23-trioxo-5α-lanosta-8-en-26-oic acid.

346

Compound 6 was determined to possess the molecular formula of C32H44O9 based

347

on HRESIMS data at m/z 571.2919 [M - H]- (calcd for C32H43O9-, 571.2907) in the

348

and its 13C NMR data. The 1H and 13C NMR data (Table 2) were almost identical to

349

those of the known compound ganoderic acid θ [49], except for the presence of

350

acetoxyl signals [δH 2.14 (3H, s); δC 171.8, 21.0] in 6. The HMBC spectrum revealed

351

obvious correlations CH3 (δH 2.14) in the acetoxyl group with C-12/carbonyl carbon

352

(δC 171.8), and H-12 (δH 5.67) with C-11/C-13/carbonyl carbon (δC 171.8),

353

confirming that the acetoxyl group was attached to C-12 (Figure 2). The NOESY

354

cross-peaks of H-3 correlating to H-28/H-5, and H-12 correlating to CH3-30 19



355

suggested that 3-OH and CH3COO-12 were both β-orientated (Figure 3). The (S)- and

356

(R)-MTPA ester derivatives of 6 at C-23 were synthesized from (R)-(+)-MTPA-Cl

357

and (S)-(–)-MTPA-Cl, respectively. The Δδ-values demonstrated a 23S configuration

358

(Figure 4). As a result, the structure of 6 was identified as 12β-acetoxyganoderic acid

359

θ.

O 0.00

-0.02

O

-0.04

O

-0.02 -0.0 -0.0 4 3

+0.06

RO

COOH

-0.01

HO

360 361 362

-0.03

O

O

+0.03

6: R=H 6a: R=(S)-MTPA-ester 6b: R=(R)-MTPA-ester Figure 4. Selected values of Δδ (S-R) of the MTPA esters of 6.

363

20


Page 20 of 50

Page 21 of 50


Table 1. 1H and 13C NMR Spectroscopic Data for Compounds 1-3. 1a position

δC

1a

37.2, CH2

1b 2a

2a δH (J in Hz) 2.67, m

3b

δC

δH (J in Hz)

δC

δH (J in Hz)

37.2, CH2

2.68, m

36.3, CH2

3.03, m

2.46, d (12.8) 27.7, CH2

2b

1.73, m

2.47, dd (14.8, 2.0) 27.7, CH2

1.73, m

34.2, CH2

1.74, m 71.7, CH

3.66, m

2.72, m 2.35, m

3

71.7, CH

4

43.9, C

5

45.2, CH

2.07, d (12.4)

45.2, CH

2.07, m

52.9, CH

1.67, m

6a

34.2, CH2

2.64, m

34.2, CH2

2.69, m

40.0, CH2

2.54, m

6b

3.65, dd (11.6, 5.2)

1.74, m

1.89, m

44.0, C

1.16, m

214.4, C 47.9, C

2.61, m

2.40, m

7

200.8, C

200.9, C

213.0, C

8

153.6, C

153.6, C

54.1, CH

2.85, d (13.2)

9

147.5, C

147.6, C

59.6, CH

2.35, d (13.2)

10

41.6, C

41.6, C

36.6, C

11

195.7, C

195.8, C

208.0, C

12a

80.9, CH

5.70, s

81.0, CH

5.70, s

52.8, CH2

12b

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

13

49.3, C

49.7, C

49.9, C

14

59.9, C

59.9, C

50.0, C

15

208.8, C

209.2, C

74.3, CH

4.05, t (6.4)

16a

38.7, CH2

38.3, CH2

1.99, m

16b 17

2.84, m

38.4, CH2

1.96, dd (18.0, 8.0) 46.2, CH

2.64, m

2.86, dd (18.3, 9.6) 2.09, m

46.7, CH

2.60, m

21


1.76, m 47.7, CH

1.99, m


18

12.8, CH3

0.81, s

12.9, CH3

0.83, s

16.6, CH3

0.78, s

19

18.9, CH3

1.36, s

18.8, CH3

1.36, s

13.3, CH3

1.44, s

20

30.4, CH

2.32, m

34.0, CH

1.76, m

35.2, CH

1.77, m

21

22.4, CH3

0.97, d (4.4)

20.8, CH3

1.02, d (6.4)

18.0, CH3

0.82, d (6.7)

22a

49.3, CH2

2.56, m

31.3, CH2

1.86, m

30.9, CH2

1.78, m

22b 23a

2.35, m

1.30, m

211.0, C

32.8, CH2

23b 24a 25a

2.40, m

1.30, m 31.0, CH2

2.29, m 47.9, CH2

24b

2.82, m

177.8, C

2.36, m 2.23, m

174.3, C

2.51, m 36.9, CH

2.84, m

65.5, CH2

25b

3.53, d (11.2)

21.4, CH3

1.07, s

3.24, d (11.2)

26

179.9, C

27

17.9, CH3

1.17, d (4.4)

28a

65.5, CH2

3.53, d (11.8)

28b

12.8, CH3

0.77, s

25.4, CH3

1.05, s

21.9, CH3

1.73, s

12.6, CH3

1.20, s

21.0, CH3

2.19, s 51.7, CH3

3.65, s

3.24, d (11.8)

29

12.8, CH3

0.77, s

30

21.8, CH3

1.72, s

CH3CO

21.0, CH3

2.21, s

CH3CO

171.8, C

171.9, C

OCH3

364 365

Page 22 of 50

a Measured

for 1H

NMR; 100 MHz

for 13C

in CD3OD; 400 MHz

b Measured

in CDCl3; 400 MHz for 1H NMR; 100 MHz for 13C NMR.

NMR.

366

22


Page 23 of 50


Table 2. 1H and 13C NMR Spectroscopic Data for Compounds 4-6. 4a position

δC

1a

35.3, CH2

1b 2a

5b δH (J in Hz) 2.96, m

6b

δC

δH (J in Hz)

δC

δH (J in Hz)

36.9, CH2

2.77, m

37.7, CH2

2.72, t (14.4)

1.80, m 34.2, CH2

2b

2.63, m

1.55, m 35.3, CH2

2.51, m

2.58, m

2.49, d (13.2) 28.2, CH2

2.40, m

1.69, m

3

215.3, C

4

46.8, C

5

49.3, CH

2.28, m

49.9, CH

1.80, br d (13.2)

52.8, CH

1.61, m

6a

37.2, CH2

2.62, m

30.0, CH2

2.05, dd (11.6, 6.8)

34.5, CH2

2.66, m

6b

220.0, C

1.69, m

78.2, CH

47.9, C

40.3, C

2.47, m

1.68, m

204.7, C

69.7, CH

8

151.2, C

162.3, C

153.4, C

9

152.7, C

141.1, C

147.2, C

10

39.3, C

39.2, C

41.9, C

11

201.4, C

12a

52.1, CH2

12b

4.61, dd (10.0, 6.8)

1.20, m

7

202.3, C 2.82, d (17.4)

201.3, C

195.6, C

53.5, CH2

2.58, d (17.4)

2.87, d (16.0)

81.2, CH

47.8, C

48.5, C

49.4, C

14

52.9, C

55.8, C

59.8, C

15

72.3, CH

4.29, t (7.4)

73.3, CH

4.82, dd (10.0, 6.8)

208.9, C

16a

36.4, CH2

1.97, m

31.5, CH2

2.40, m

37.4, CH2

17

1.97, m 48.4, CH

1.82, m

5.67, s

2.51, d (16.0)

13

16b

3.21, dd (12.0, 4.6)

1.68, m 51.7, CH

2.30, br t (10.4) 23


2.85, dd (18.0, 9.8) 2.02, dd (18.0, 8.4)

46.5, CH

2.64, m


18

17.6, CH3

0.87, s

19.6, CH3

1.12, s

13.0, CH3

0.79, s

19

17.9, CH3

1.27, s

20.0, CH3

1.24, s

18.4, CH3

1.34, s

20

35.7, CH

1.41, m

74.9, C

30.9, CH

1.71, m

21

18.1, CH3

0.87, d (6.8)

27.0, CH3

1.31, s

22.5, CH3

1.07, d (6.6)

22a

30.9, CH2

1.81, m

55.5, CH2

2.89, d (14.6)

43.1, CH2

1.64, m

22b 23a

1.34, m 31.2, CH2

23b 24a

2.38, m

2.53, d (14.6) 211.2, C

1.48, m 67.8, CH

4.54, dd (7.4,4.8)

143.9, CH

6.57, d (9.0)

2.26, m 174.4, C

49.2, CH2

24b

2.87, m 2.58, m

25

20.5, CH3

1.12, s

36.2, CH

26

27.6, CH3

1.15, s

180.0, C

27

20.7, CH3

1.19, s

17.6, CH3

1.17, d (6.8)

13.4, CH3

1.90, s

28

27.9, CH3

1.12, s

28.4, CH3

1.00, s

29

21.2, CH3

1.09, s

16.3, CH3

0.88, s

30

20.6, CH3

1.27, s

21.7, CH3

1.73, s

CH3CO

21.0, CH3

2.14, s

CH3CO

171.8, C

OCH3

367 368

Page 24 of 50

51.8, CH3

2.85, m

171.8, C

3.67, s

a Measured

in CDCl3; 600 MHz for 1H NMR; 150 MHz for 13C NMR.

b Measured

in CD3OD; 600 MHz for 1H NMR; 150 MHz for 13C NMR.

130.7, C

24


Page 25 of 50


369

All the isolated compounds (1-33) were assessed for their inhibitory activities at

370

50 μM against NO production triggered by LPS in mouse macrophage cell

371

RAW264.7. Among all these compounds, 4 and 9 showed stronger inhibition against

372

NO production (inhibition rate >50%) than other compounds (Figure 5A, Supporting

373

information Table S1). Since inhibition rate of 4 is higher than 9 at 50 μM, the IC50

374

value of 4 was further assayed and the result was 38.6 ± 1.0 μM (Figure 5C), which

375

was chosen for further anti-inflammatory research. Dexamethasone (Dex, IC50 = 7.9 ±

376

1.3 μM) was employed as a positive control (Figure 5B).

377

The cytotoxicity of 4 on RAW264.7 cells was measured to exclude the possibility

378

that the inhibition of NO production was owing to its cytotoxic effects. This result

379

demonstrated no significant inhibitory effects of 4 on cell viability at the

380

concentration up to 50 μM and its IC50 was greater than 200 μM (Figure 5D).

381

Therefore, in the subsequent experiments, the concentration of this compound was

382

controlled to be less than 50 μM.

25



383 384 385 386 387 388

Figure 5. The 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) The IC50 value of dexamethasone (Dex) was 7.9 ± 1.3 μM. (C) The IC50 value of compound 4 was 38.6 ± 1.0 μM. (D) The RAW264.7 cell viability after treatment with compound 4 was detected by CCK-8 assays. *, p

Triterpenoids from Ganoderma lucidum and Their Potential Anti

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