Deep Profiling of Immunosuppressive Glycosphingolipids and

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Bioactive Constituents, Metabolites, and Functions

Deep Profiling of Immunosuppressive Glycosphingolipids and Sphingomyelins in Wild Cordyceps Jianing Mi, Yuwei Han, Yingqiong Xu, Junping Kou, Wen-Jia Li, Jing-Rong Wang, and Zhi-Hong Jiang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b02706 • Publication Date (Web): 30 Jul 2018 Downloaded from http://pubs.acs.org on August 3, 2018

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Journal of Agricultural and Food Chemistry

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Deep Profiling of Immunosuppressive Glycosphingolipids and

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Sphingomyelins in Wild Cordyceps

3

Jianing Mia, Yuwei Hanb, Yingqiong Xub, Junping Koub, Wen-Jia Lid, Jing-Rong Wanga* and

4

Zhi-Hong Jianga,c*

5

a

6

Research in Medicine and Health, Macau University of Science and Technology, Macau,

7

China.

8

b

9

Complex Prescription of TCM, China Pharmaceutical University, 639 Longmian Road,

State Key Laboratory of Quality Research in Chinese Medicine, Macau Institute for Applied

Jiangsu Key Laboratory of TCM Evaluation and Translational Research, Department of

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Nanjing 211198, China.

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c

12

Medicine, Guangzhou, China.

13

d

14

Pharm Co. Ltd, Guangdong 523850, China.

15

* Corresponding authors.

16

E-mail address: [email protected] (Z.-H. Jiang) and [email protected] (J.-R. Wang).

International Institute for Translational Chinese Medicine, Guangzhou University of Chinese

Key Laboratory of State Administration of Traditional Chinese Medicine, China HEC

1

ACS Paragon Plus Environment


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Abstract

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Deep profiling of glycosphingolipids and sphingomyelins in wild Cordyceps was

19

carried out by using offline chromatographic enrichment followed by ultrahigh

20

performance liquid chromatography-ultrahigh definition-quadrupole time of flight

21

mass spectrometry (UHPLC-UHD-Q-TOF-MS). A total of 119 glycosphingolipids

22

(72 new ones) and 87 sphingomyelins (43 new ones) were identified from wild

23

Cordyceps on the basis of the accurate mass and MS/MS fragmentations, isotope

24

patterns, sphingolipid (SPL) database matching, confirmation by SPL standards, and

25

reversed-phase liquid chromatographic retention rule. This study is the most

26

comprehensive report on the identification of glycosphingolipids and sphingomyelins

27

from fungus. Subsequent lipopolysaccharide-induced mouse splenic lymphocyte

28

proliferation assay showed that Cordyceps glycosphingolipid fraction exhibits higher

29

immunosuppressive activity compared to Cordyceps sphingomyelins. Our findings

30

provided an insight into chemical diversity of sphingolipids in Cordyceps and

31

chemical evidence for the therapeutic application of wild Cordyceps.

32

Keywords:

33

UHPLC-Q-TOF-MS; immunosuppressive activity

wild

Cordyceps;

glycosphingolipids;

sphingomyelins;

2


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1. Introduction

35

Cordyceps (Chinese caterpillar fungus), a precious herb with a long and

36

illustrious history, consists of the stroma of the fungus Cordyceps sinensis (Berk.)

37

Sacc. in the family Hypocreaceae and the dead caterpillar of Hepialus armoricanus

38

belonging to family Hepialidae. A considerable number of studies have demonstrated

39

that Cordyceps exists immunomodulatory [1, 2], anti-inflammatory [3, 4], anticancer

40

[5, 6], and kidney-protective bioactivities [7] and so on. It is used for the treatment of

41

deficiency of kidney essence, impotence and seminal emission, limp aching in the

42

lower back and knees, chronic cough and dyspnea of deficiency type and so on [8].

43

The clinical efficacy has led to an ever-increasing demand on this high-priced

44

biological commodity [9], driving studies on active ingredients for the rational use

45

and development of this precious herb. Many chemical components, e.g.,

46

polysaccharides, nucleotides,

47

sphingolipids (SPL), were isolated from Cordyceps [10-12].

D-mannitol,

ergosterol, aminophenol, fatty acids and

48

As two predominant subclasses of SPLs, glycosphingolipids and sphingomyelins

49

are derived from ceramides by addition of various headgroups at C1 hydroxyl group

50

via glycosidic bonds and phosphodiester linkages, respectively [13]. In addition to

51

crucial role of endogenous glycosphingolipids and sphingomyelins in various

52

biological procedures, these complex SPLs have also been demonstrated to have

53

important pharmacological effects. For instance, α-galactosylceramide isolated from

54

the marine sponge Agelas mauritianus, a potent activator of iNKT cells, could

55

promote immunotolerance [14-16]. Interestingly, its altered analogues, such as 3



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(2S,3S,4R)-1-O-(α-D-galactosyl)-2-tetracosanoylamino-1,3,4-nonanetriol

and

57

hydroxylated KRN7000 have been reported as immunosuppressant [17, 18]. Recent

58

study also suggested that two prenylated glycosphingolipids (Plakoside A and B)

59

exhibit strong immunosuppressive activity [19]. Additionally, evidence showed that

60

sphingomyelins have effects on atherosclerosis and colon carcinogenesis [20-23].

61

These strongly suggested that glycosphingolipids and sphingomyelins are

62

pharmacologically active constituents of natural medicines. Therefore, the

63

comprehensive profiling of the glycosphingolipids and sphingomyelins in Cordyceps

64

is desperately needed for their pharmacological study.

65

LC-MS is a very useful technique for the detection and structure elucidation of

66

SPLs. To date, the in-depth profiling of SPLs in the upper layer of human skin [24],

67

human lung sputum [25], Arabidopsis thaliana and Camelina sativa [26] has been

68

carried out by using LC-electrospray ionization (ESI)-MS. However, comprehensive

69

sphingolipidome study of wild Cordyceps by using LC-MS is challenging, especially,

70

the analysis of low-abundance SPLs by LC-MS is aggravated by serious ionization

71

suppression of matrix. Therefore, exploring highly sensitive analysis strategies for the

72

comprehensive

73

sphingomyelins is of great interest.

identification

of

wild

Cordyceps

glycosphingolipids

and

74

We herein carried out a deep profiling of glycosphingolipids and sphingomyelins

75

in Cordyceps by using column chromatography including silica gel and amino silica

76

gel followed by UHPLC-MS approach [27-29]. Then, we investigated the

77

immunosuppressive activity of Cordyceps glycosphingolipid and sphingomyelin 4


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78

fractions using lipopolysaccharide (LPS) and concanavalin A (Con A)-induced mouse

79

splenic lymphocyte proliferation models [30].

80

2. Materials and Methods

81

2.1. Chemicals and animals

82

Formic acid (LC-MS grade), acetic acid (LC-MS grade), and dimethyl sulfoxide

83

(DMSO, ≥ 99%) were acquired from Sigma-Aldrich (MO, USA). Methanol (MeOH,

84

LC-MS grade), isopropanol (LC-MS grade), acetone (HPLC grade), ethyl acetate

85

(HPLC grade), chloroform (CHCl3, HPLC grade) and n-hexane (HPLC grade) were

86

obtained from Avantor Performance Materials, Inc. (PA, USA). LPS, Con A,

87

potassium hydroxide (≥ 85%), and ammonium acetate (≥ 98%) were acquired from

88

Sigma-Aldrich (MO, USA). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

89

bromide (MTT) and RPMI 1640 were obtained from Amersco LLC. (OH, USA) and

90

Gibco (NM, USA), respectively. Fetal Bovine Serum (FBS) was acquired from

91

Zhejiang Tian Hang Biological Technology Stock Co., Ltd. (Zhejiang, China).

92

Sphingomyelin standards SM (d18:1/16:0), SM (d18:1/17:0), SM (d18:1/18:0) and

93

SM (d18:1/24:0) were acquired from Avanti Polar Lipids (AL, USA). SM

94

(d18:1/20:0), SM (d18:1/22:0) and Hex-Hex-Hex-Cer (d18:1/24:0) standards were

95

acquired from Matreya LLC (PA, USA). Davisil® chromatographic silica gel

96

(Particle size 10-14 µm) and amino silica media (Particle size 35-70 µm) were

97

acquired from W. R. Grace & Co.-Conn. (MD, USA). Water was prepared using a

98

Milli-Q system (Millipore, Billerica, MA). SM (d18:0/16:0), SM (d18:0/17:0), SM

5



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99

(18:0/18:0), SM (d18:0/20:0), SM (d18:0/22:0) and SM (d18:0/24:0) were

100

synthesized by using hydrogen gas and 10% Pd on charcoal (Aldrich-Sigma, MO,

101

USA), and the products were verified by UHPLC-MS/MS. 20 to 22 g ICR mice were

102

acquired from the Comparative Medicine Center of Yangzhou University, China.

103

2.2. Glycosphingolipid and sphingomyelin fractions preparation

104

SPL fractions were prepared according to the protocol [27-29]. In brief, the crude

105

SPLs extract was obtained from the dried wild Cordyceps with MeOH/CHCl3 (2:1,

106

v/v and 1:2, v/v) by incubation and ultrasound-assisted extraction. Then, the

107

enrichment of glycosphingolipids or sphingomyelins was carried out using silica gel

108

column chromatography (4.2×29 cm) with precondition of CHCl3 (6 bed volume

109

(BV)), washing of CHCl3 (6 BV) and MeOH/acetone (1:9, v/v) (3.5 BV), and elution

110

of MeOH (7.5 BV). Elution fractions were collected by 0.1 BV each fraction and

111

were analyzed by UHPLC-MS. Enriched glycosphingolipid or sphingomyelin

112

fractions were combined together and dried under vacuum for the further purification.

113

Then, the purification of glycosphingolipid or sphingomyelin fractions were carried

114

out respectively using NH2 silica gel column chromatography (4.2×25 cm) with

115

precondition of n-hexane (6 BV), washing with mobile phases including n-hexane (2

116

BV), ethyl acetate/n-hexane (15:85, v/v) (6 BV), and MeOH/CHCl3 (1:23, v/v) (5

117

BV), and elution of MeOH/acetone (1.35:9, v/v) (5 BV) and MeOH/CHCl3 (1:2, v/v)

118

(5 BV) by 0.1 BV each fraction. 50 of MeOH/acetone eluting fractions collected were

119

analyzed by UHPLC-MS. Enriched glycosphingolipid fractions were combined

120

together. CHCl3/MeOH eluting fractions were selectively combined together based on 6


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121

their UHPLC-MS analysis to give sphingomyelin fraction. The blank sample without

122

Cordyceps material was prepared and purified by the same procedure with SPLs

123

fraction to give the blank sample. The glycosphingolipid and sphingomyelin fractions

124

were dried and dissolved in methanol, and filtered using a 0.22 µm filter for

125

UHPLC-MS analysis.

126

2.3. UHPLC-UHD-Q-TOF-MS conditions

127

The detection of glycosphingolipids and sphingomyelins was performed on an

128

UHPLC system (Agilent 1290, CA, USA) with an Eclipse Plus C18 column (Agilent,

129

100×2.1 mm, 1.8 µm) coupled with a ESI-iFunnel-Q-TOF mass spectrometer (Agilent

130

UHD 6550, CA, USA) in the positive ion mode, following the optimized UHPLC-MS

131

approach [27-29].

132

2.4. Bioassay of immunosuppress activity

133

The immunosuppressive bioassay of fractions of Cordyceps glycosphingolipid

134

and sphingomyelin was carried out according to the protocol [29]. In brief, splenic

135

lymphocytes were obtained from ICR mice and were cultured in 96-well plate.

136

Compared to the control group and model group (LPS or Con A model), 50 µL of

137

glycosphingolipid and sphingomyelin fractions with nine final concentrations (0.1, 0.3,

138

1, 3, 10, 30, 100, 300 and 600 µg/mL) were added into each well in the test group (n =

139

5). After the culture of 48 h, the MTT assay was used to evaluate the

140

immunosuppressive bioassay of these fractions. The half maximal inhibitory

141

concentration (IC50) values were determined using the GraphPad Prism 5 software.

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Student’s t-test or Dunnett’s test was employed in data analysis. This study were

143

carried out according to the guide of the Animal Ethics Committee of China

144

Pharmaceutical University.

145

2.5. SPL nomenclature

146

According to the nomenclature system of LIPID MAPS (Lipidomics Gateway),

147

annotation of ceramide moiety of glycosphingolipids denotes hydroxyl-group number

148

(d or t means two or three hydroxyl groups) in sphingoid backbone, carbon number of

149

sphingoid backbone, double bond number in sphingoid backbone, carbon number of

150

N-acyl chain, double bond number in N-acyl chain, and hydroxyl-group number in

151

N-acyl chain (e.g., in d18:1/24:0(OH), OH means one hydroxyl group); and

152

annotation of headgroup denotes sugar residues composition and sequence (e.g., in

153

Fuc-Hex-GalNAc-Cer,

154

N-acetyl-D-galactosamine, respectively; Cer means ceramide). For sphingomyelins,

155

annotation of sphingoid backbone and N-acyl chain is same with glycosphingolipids,

156

SM means sphingomyelin with phosphocholine group. In some sphingomyelins,

157

annotation of total carbon number and total double bond number denotes the sum of

158

those in ceramide moiety.

159

3. Results and Discussions

160

3.1. Enrichment of glycosphingolipids and sphingomyelins

Fuc,

Hex

and

GalNAc

mean fucose,

hexose and

161

By using the column chromatographic enrichment strategies and UHPLC-MS,

162

blank sample, glycosphingolipid-enriched fraction, and sphingomyelin-enriched 8


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fraction were prepared and determined (Fig. 1A, 1B and 1C). A significant increase of

164

the MS signal response was achieved in the LC-MS analysis of low-level SPL species

165

benefiting from the enrichment. This can be exemplified by the discovery of some

166

low-content SPLs such as polyunsaturated glycosphingolipids [e.g., HexCer

167

(d14:2/26:3(OH)) (36)] and polyhydroxyl and/or polyunsaturated sphingomyelins

168

[e.g., SM (t14:1/27:3(OH)) (185)], whose MS signal response were enhanced after the

169

purification (Fig. 1D and 1E). Additionally, in SPL-enriched fractions, the ionization

170

suppression of non-targeted species for glycosphingolipids or sphingomyelins was

171

dramatically reduced and the signal-to-noise ratio of the MS signal response of

172

low-content SPLs was significantly increased to avoid being masked. For example,

173

the relative abundance of precursor ion at m/z 724 of glycosphingolipid 7 was

174

enhanced by approximately 30-fold in the MS spectra of scanning at retention time

175

(RT) of 12.60 min (Fig. 1F and 1G), and the signal to nose ratio of the MS signal

176

response of SM (t16:0/18:0) (180) was improved by 3-fold after the enrichment

177

procedure (Fig. 1H and 1I). All evidence supported that the enrichment strategy was

178

important

179

sphingomyelins in Cordyceps. Based on the amount of SPL fractions enriched, it

180

could be stated that the calculated average content of individual SPLs in raw wild

181

Cordyceps is low (about 0.03 mg/g for each glycosphingolipid; about 0.004 mg/g for

182

each sphingomyelin).

183

3.2. Identification and characterization of glycosphingolipids in wild Cordyceps

for

the

comprehensive

identification

of

glycosphingolipids

and

9



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184

UHPLC-Q-TOF-MS experiments were performed to determine the exact

185

molecular masses of the ions observed in Cordyceps SPL fractions, which reveals the

186

molecular formula and the degree of unsaturation in the ions. Based on which, SPL

187

candidates were selected for the further MS/MS experiments by matching the

188

comprehensive theoretical SPL database constructed in our lab with the

189

building-block approach [31].

190

3.2.1. Structural elucidation of glycosphingolipids using UHPLC-Q-TOF-MS

191

Based

on

the

high-resolution

MS

and

MS/MS

data,

structures

of

192

glycosphingolipids were elucidated according to the characterized fragments. This can

193

be exemplified by the structural elucidation of novel glycosphingolipid 68 with

194

[M+H]+ ion at m/z 744.5663 (Supporting Information Table S1). Based on its accurate

195

mass, compound 68 was matched with a glycosphingolipid candidate with the formula

196

of C41H77NO10 and two double bonds. MS/MS experiments on the [M+H]+ ion at m/z

197

744 provided characteristic fragments which revealed some aspects of the structure of

198

glycosphingolipid 68 (Fig. 2). Firstly, one 162 gap in the pattern of these

199

fragmentations of m/z 744 to m/z 582, was observed as a result of loss of one hexosyl

200

group. This information indicated that glycosphingolipid 68 belongs to hexosyl

201

ceramides. Secondly, the neutral loss of three H2O from these fragments at m/z 744,

202

m/z 726, m/z 708 and m/z 690, suggested that there are three hydroxyl groups in 68. In

203

addition, the information of ions at m/z 564 ([M-Hex-H2O+H]+), m/z 546

204

([M-Hex-2H2O+H]+),

205

([M-Hex-3H2O+H]+),

m/z m/z

534 516

([M-Hex-H2O-HCHO+H]+), ([M-Hex-2H2O-HCHO+H]+)

and

m/z m/z

528 510 10


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206

([M-Hex-4H2O+H]+) supported that three hydroxyl groups are in 68. Thirdly, the ion

207

at m/z 412 was yielded by the cleavage of C6-C7 bond in sphingoid backbone with

208

loss of a H2O and C1″-C2″ and C5″-O bonds in sugar residue [32]. Additionally, the

209

ion at m/z 384 was produced by the cleavage of C6-C7 bond in sphingoid backbone

210

with the dehydration happened at position C4 and C1″-O bond linked ceramide

211

moiety and sugar residue. Based on these fragment clues, a double bond was inferred

212

to be located at position C8 in sphingoid backbone [33, 34]. The ion at m/z 384 further

213

produced ions at m/z 366, m/z 348 and m/z 330 by loss of three H2O, indicating that

214

one hydroxyl group exists in N-acyl chain. Fourthly, a cleavage of N-C1′ bond in

215

ceramide moiety gave rise to the ion at m/z 312 ([M-Hex-H2O-C16 FA+H]+) which

216

yielded ions at m/z 312, m/z 294, m/z 276 and m/z 264, reflecting a C19

217

dehydrophytosphingosine backbone. Finally, the ions at m/z 312.2579 and m/z

218

270.2415 were yielded from the ion at m/z 582 by the cleavage of C2-C3 bond and the

219

cleavage of N-C2 bond, respectively, revealing a C16 fatty acid chain with a double

220

bone and a hydroxyl group. This conclusion of hydroxylated N-acyl chain was

221

confirmed by the neutral loss of H2O from ion at m/z 270 to ion at m/z 252. Based on

222

fragment clues mentioned above, the structure of HexCer (t19:1/16:1(OH)) (68) was

223

elucidated.

224

For glycosphingolipids with two or three sugar residues, the neutral loss

225

corresponding to various types of sugar residues, are decisive for the identification of

226

sugar composition and sequence. For example, in the MS/MS spectrum of

227

glycosphingolipid 112 (Fig. 3A), a 146 gap from the ions m/z 1048 to m/z 902 was 11



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228

observed, suggesting a fucosyl unit located in the end of the sugar chain. Then two

229

162 gaps in the pattern of these fragmentations of m/z 902 to m/z 746 and m/z 746 to

230

m/z 578, respectively, were observed as a result of sequential loss of two hexosyl

231

groups. Complementary support was given by the ions at m/z 578, m/z 560 and m/z

232

548 corresponding to the ceramide moiety, ion at m/z 356 reflecting a C22 fatty acid

233

chain with a hydroxyl group, and the ions at m/z 240, m/z 222 and m/z 210

234

representative of the sphingoid backbone. Hence this glycosphingolipid was identified

235

as Fuc-Hex-Hex-Cer (d15:1/22:0(OH)) (112). To validate the structural elucidation of

236

the headgroup with three sugar units, Hex-Hex-Hex-Cer (d18:1/24:0) standard was

237

used to provide the MS/MS pattern reflecting sugar residues composition and

238

sequence (Fig. 3B). The very similar MS/MS patterns were observed in both MS/MS

239

spectra of Fuc-Hex-Hex-Cer (d15:1/22:0(OH)) (112) and Hex-Hex-Hex-Cer

240

(d18:1/24:0) standard, which supported the identification of glycosphingolipid 112.

241

Based on the structural elucidation stratagem of glycosphingolipids, the feature

242

ions representative of sphingoid backbone and N-acyl chain of SPLs, and the neutral

243

loss are used for the identification of other glycosphingolipids. The feature ions

244

representative of sphingoid backbone is critical for the identification of various

245

subclasses of glycosphingolipids. For example, in the MS/MS spectra, the sphingoid

246

backbones of d14:0, d14:2, d16:0, d18:1, d19:2, and t18:1 were elucidated by these

247

producted ions at m/z 210, m/z 206, m/z 238, m/z 264, m/z 276 and m/z 262 by loss of

248

H2O and sugar residues, as well as the elimination of the FA chain from ceramide

249

moiety. The feature ions representative of N-acyl chain is very important assistance 12


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250

for the identification of N-acyl chain in glycosphingolipids. For example, the N-acyl

251

chains of 16:0, 20:0, 24:1, and 16:1(OH) were identified by these producted ions at

252

m/z 256, m/z 312, m/z 366, and m/z 252 which yield via the cleavage of C2-N bond in

253

ceramide moiety. Neutral loss of glycan unit (e.g., 162 u for Hex group and 146 u for

254

Fuc group) can provide

255

glycosphingolipids.

256

3.2.2. Applications of the reversed-phase liquid chromatographic retention rule in

257

identification of glycosphingolipids

important information for the identification of

258

The reversed-phase liquid chromatographic retention time (RT) rule [29, 35] was

259

used to identify glycosphingolipids without enough information of the diagnostic ion.

260

For

261

(d19:2/24:1(OH)), Fuc-Hex-Hex-Cer (d14:1/23:0), Fuc-Hex-Hex-Cer (d14:1/24:0)

262

and Fuc-Hex-Hex-Cer (d14:1/22:0(OH)) (Table S1 and Fig. S1, glycosphingolipids

263

16, 59, 61, 99, 100 and 109).

264

3.3. Identification and characterization of sphingomyelins in wild Cordyceps

265

3.3.1. Structural elucidation of sphingomyelins by using UHPLC-Q-TOF-MS

example,

HexCer

(d16:2/21:1),

HexCer

(d19:2/22:0(OH)),

HexCer

266

The structures of sphingomyelins were elucidated according to the accurate mass

267

of the parent ion and the feature ion at m/z 184 (protonated phosphocholine fragment).

268

In MS/MS spectrum of compound 121 (Fig. 4), a major protonated phosphocholine

269

fragment ion at m/z 184 and a few of low-abundance product ions, e.g., m/z 166, m/z

270

124, m/z 104, m/z 86 and m/z 60, were obviously observed. Combined with its 13



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271

accuracy mass of [M+H]+ ion at m/z 675.5433, compound 121 was assigned as a

272

sphingomyelin, differentiating other subclasses of phospholipids with an ion at m/z

273

184, e.g., glycerophosphocholines. Only a weak peak of m/z 208 ion reflecting the

274

d14:1 sphingoid backbone [36] but no ion from the fatty acid chain was observed in

275

its MS/MS spectrum. According to the fragment clues discussed above, compound

276

121 was inferred to be SM (d14:1/18:0). The fragment ion at m/z 184 and N-acyl

277

chain feature ions of SPLs (e.g., ions at m/z 210, m/z 206, m/z 222, m/z 236, m/z 264,

278

and m/z 276 reflecting d14:0, d14:2, d15:1, d16:1, d18:1, and d19:2 sphingoid

279

backbone) are used for the identification of other sphingomyelins. Regarding lack of

280

N-acyl chain feature ion, sphingomyelins were identified as sphingomyelins analogs

281

expressed as total carbon number and number of total unsaturation degree of

282

sphingomyelins.

283

3.3.2. Applications of commercial and synthesized standards in the confirmation of

284

sphingomyelins

285

To confirm sphingomyelins identified from wild Cordyceps, 6 commercial

286

sphingomyelin standards (Fig. S2, sphingomyelins 151, 153, 155, 158, 160 and 165)

287

and 6 authentic sphingomyelins synthesized by us (Fig. S2, sphingomyelins 152, 154,

288

156, 159, 161 and 166) were employed in UHPLC-MS experiment. It can be seen in

289

Fig. S3 that 12 sphingomyelins identified from wild Cordyceps were co-eluted with

290

corresponding sphingomyelin standards. Furthermore, 12 sphingomyelins identified

291

from wild Cordyceps were confirmed by corresponding sphingomyelin standards via

292

comparing their high-resolution MS and MS/MS spectra (Fig. S4). 14


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293

3.3.3. Applications of the reversed-phase liquid chromatographic retention rule in

294

identification of sphingomyelins

295

Another application of the chromatographic RT rule is helpful for assignations of

296

dihydrosphingomyelins 150 and 157, whose MS/MS data without characteristic ions

297

reflecting sphingoid backbone are not enough for their identification. As important

298

evidence for their structural elucidation, the RT at 12.07 min of SM (d18:0/15:0) (150)

299

and RT at 14.52 min of SM (d18:0/19:0) (157) are suitable to SM (d18:0/x:0) linear

300

regression model with R2 = 0.9995 (Fig. S5) [29, 35].

301

3.4. Chemical characteristics of glycosphingolipids and sphingomyelins identified

302

from wild Cordyceps

303

Via the rigorous identification of glycosphingolipids and sphingomyelins, a total

304

of 119 glycosphingolipids and 87 sphingomyelins were found in wild Cordyceps.

305

Among which, newly characterized glycosphingolipids and sphingomyelins

306

significantly enlarged the diversification of natural SPLs. Novel glycosphingolipids in

307

Cordyceps can be reflected in: 1) monohexosylceramides with t19:1 and t19:2

308

back-bones (68, 69); 2) dihexosylceramides with new sphingoid backbones, such as

309

d14:2, d16:2 and d19:2 (72, 84 and 76); 3) glycosphingolipids with three sugar

310

moieties with new sphingoid backbones, e.g., C14, C15, C16 and C17 sphingoid

311

backbones (93, 95, 102, 104, 107, 111, 113 and 117); t18:1 backbone (119); 4)

312

glycosphingolipids with hydroxyl fatty acid chain, e.g., HexCer (d15:1/20:0(OH)) (37)

313

and Hex-Hex-Cer (d14:1/20:0(OH)) (78) (Fig. 5). On the other hand, the features of

314

novel sphingomyelins are predominantly those: 1) with new short sphingoid 15



Page 16 of 31

315

backbones; 2) with new long odd-numbered carbon sphingoid backbones including

316

d19:2 (177) and t19:1 (188) backbones; 3) polyhydroxylated. The results illustrated

317

that Cordyceps contains lots of novel sphingomyelins with very short sphingoid

318

backbones, e.g., d14:0, d14:1, d14:2, d15:1 and d15:2 sphingomyelins (120, 121, 123,

319

139 and 140); t14:0, t14:1, t16:0 and t16:1 sphingomyelins (183, 179-181).

320

Additionally, the identification of polyhydroxylated sphingomyelins, e.g., SM

321

(t14:0/25:3(OH)) (183), SM (t18:0/24:1(OH)) (187) and SM (t19:1/16:0(OH)) (188),

322

indicated that the number of hydroxyls of sphingomyelins is increased from 2 to 3

323

(Fig. 6).

324

3.5.

325

sphingomyelins fractions

Immunosuppressive

activities

of

Cordyceps

glycosphingolipids

and

326

Bioassays showed that Cordyceps glycosphingolipids and sphingomyelins

327

fractions could inhibit the proliferation of mouse splenic lymphocyte induced by LPS

328

and Con A in a dose-dependent manner (Fig. S6). Although these two fractions

329

appeared similar immunosuppressive activity (their IC50 values were determined to be

330

8.24 µg/mL and 4.18 µg/mL, respectively) for the proliferation of mouse splenic

331

lymphocyte induced by Con A, the IC50 values of glycosphingolipid and

332

sphingomyelin fractions were determined to be 11.82 µg/mL and 278.90 µg/mL in

333

LPS-induced mouse splenic lymphocyte proliferation model, respectively. Which

334

showed that Cordyceps glycosphingolipids exhibit higher immunosuppressive activity

335

compare to Cordyceps sphingomyelins.

16


Page 17 of 31


336

Abbreviations:

337

UHPLC-UHD-Q-TOF-MS, ultrahigh performance liquid chromatography-ultrahigh

338

definition-quadrupole time of flight mass spectrometry; SPL, sphingolipids; ESI,

339

electrospray ionization; LPS, lipopolysaccharide; Con A, concanavalin A; MeOH,

340

methanol;

341

5-diphenyltetrazolium bromide; FBS, Fetal Bovine Serum; BV, bed volume; IC50,

342

The half maximal inhibitory concentration; FA, fatty acid; RT, retention time;

343

Acknowledgments

344

This research was supported by the Macau Science and Technology Development

345

Fund (Project No.: 015/2017/AFJ).

346

Conflict of interest

347

Authors declare no conflict of interest

348

Supplementary data

349

Supplementary data related to this article can be found on line. Table S1. SPLs

350

identified

351

glycosphingolipids. Figure S2. The synthetic reactions of 6 dihydrosphingomyelins in

352

this work. Figure S3, S4, and S5. Identification of sphingomyelins from wild

353

Cordyceps. Figure S6. Effects of glycosphingolipid and sphingomyelin fractions on

354

the proliferation inhibition ratio in LPS-induced mouse splenic lymphocytes and Con

355

A-induced mouse splenic lymphocytes.

CHCl3,

from

wild

chloroform;

Cordyceps.

MTT,

Figure

S1.

3-(4,5-dimethylthiazol-2-yl)-2,

The

UHPLC

RT

rule

of

17



Page 18 of 31

356

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Page 24 of 31

467

Figure Captions

468

Figure 1. Enrichment of glycosphingolipids and sphingomyelins by using silica

469

gel and amino silica gel column chromatographies. Base peak chromatograms

470

(BPCs) of blank sample (A), glycosphingolipid fraction (B) and sphingomyelin

471

fraction (C); Comparison of extracted ion chromatograms (EICs) of HexCer

472

(d14:2/26:3(OH)) (D) and SM (t14:1/27:3(OH)) (E), comparison of MS spectra of

473

HexCer (d14:2/22:1) (F and G), comparison of EICs and corresponding MS spectra of

474

SM (t16:0/18:0) (H and I) between samples before and after the enrichment

475

procedure.

476

Figure 2. The MS/MS spectrum of HexCer (t19:1/16:1) (68).

477

Figure 3. The MS/MS spectra of Fuc-Hex-Hex-Cer (d15:1/22:0(OH)) (112) (A)

478

and Hex-Hex-Hex-Cer (d18:1/24:0) standard (B).

479

Figure 4. The MS/MS spectrum of SM (d14:1/18:0) (121) and the proposed

480

fragmentation pathways.

481

Figure 5. Structures of representative new glycosphingolipid analogues.

482

Figure 6. Structures of representative new sphingomyelin analogues.

24


Page 25 of 31


483

25



Figure 1. Enrichment of glycosphingolipids and sphingomyelins by using silica gel and amino silica gel column chromatographies. Base peak chromatograms (BPCs) of blank sample (A), glycosphingolipid fraction (B) and sphingomyelin fraction (C); Comparison of extracted ion chromatograms (EICs) of HexCer (d14:2/26:3(OH)) (D) and SM (t14:1/27:3(OH)) (E), comparison of MS spectra of HexCer (d14:2/22:1) (F and G), comparison of EICs and corresponding MS spectra of SM (t16:0/18:0) (H and I) between samples before and after the enrichment procedure. 196x269mm (300 x 300 DPI)


Page 26 of 31

Page 27 of 31


Figure 2. The MS/MS spectrum of HexCer (t19:1/16:1) (68). 181x201mm (300 x 300 DPI)



Figure 4. The MS/MS spectrum of SM (d14:1/18:0) (121) and the proposed fragmentation pathways. 128x132mm (300 x 300 DPI)


Page 28 of 31

Page 29 of 31


Figure 5. Structures of representative new glycosphingolipid analogues. 172x206mm (300 x 300 DPI)



Figure 6. Structures of representative new sphingomyelin analogues. 126x123mm (300 x 300 DPI)


Page 30 of 31

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