Structural Characterization and Biological Activities of a Novel

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Structural Characterization and Biological Activities of a Novel Polysaccharide from Cultured Cordyceps militaris and Its Sulfated Derivative Yongshuai Jing, Jianhua Zhu, Ting Liu, Sixue Bi, Xianjing Hu, Zhiyan Chen, Liyan Song, Wenjie Lv, and Rongmin Yu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf505915t • Publication Date (Web): 18 Mar 2015 Downloaded from http://pubs.acs.org on March 22, 2015

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Journal of Agricultural and Food Chemistry

1

Structural Characterization and Biological Activities of a Novel

2

Polysaccharide from Cultured Cordyceps militaris and Its

3

Sulfated Derivative

4 5

Yongshuai Jing†,‡, Jianhua Zhu*,§, Ting Liu⊥, Sixue Bi†, Xianjing Hu†, Zhiyan Chen⊥, Liyan

6

Song*, , Wenjie Lv†, Rongmin Yu*,†,§

7

†

⊥

Biotechnological Institute of Chinese Materia Medica, Jinan University, 601 Huangpu

8

Avenue West, Guangzhou 510632, China. ‡

9 10 11

Technology, 26 Yuxiang Street, Shijiazhuang 050018, China. §

College of Pharmacy, Department of Natural Medicinal Chemistry, Jinan University, 601

12 13 14

College of Chemistry and Pharmaceutical Engineering, Hebei University of Science and

Huangpu Avenue West, Guangzhou 510632, China. ⊥

College of Pharmacy, Department of Pharmacology, Jinan University, 601 Huangpu Avenue West, Guangzhou 510632, China.

15 16

*Corresponding

17

[email protected] (R. M. Yu). Tel: +86-20-85222069; E-mail Address: [email protected] (J. H. Zhu).

18

Tel: +86-20-85228205; E-mail Address: [email protected] (L. Y. Song).

authors:

Tel:

+86-20-85220386;

fax

+86-20-85224766.

19 20 21 22 23 24

1

ACS Paragon Plus Environment

E-mail

Address:


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25 26

ABSTRACT: A novel polysaccharide (CMPA90-1, 1) was isolated from the cultured

27

fruiting bodies of Cordyceps militaris. The chemical structure of 1 was elucidated by

28

acid hydrolysis, periodate oxidation, Smith degradation, methylation analysis, along

29

with FT-IR, HPAEC-PAD, GC-MS, 1D (1H and

30

HMBC). Sulfation of 1 by chlorosulfonic acid-pyridine (CSA-Pyr) method led to

31

synthesis of its sulfated analog (CMPA90-M1, 2). The ultrastructures of both 1 and 2

32

were further characterized by SEM and AFM. The results of antioxidant assays

33

showed that 1 and 2 exhibited free radical-scavenging effects, ferrous ion-chelating

34

ability and reducing power. Also, in cytotoxicity assay, 1 and 2 showed inhibitory

35

activity against A549 cells, with the IC50 values of 39.08 and 17.33 μg/mL,

36

respectively.

37

KEYWORDS: Cordyceps militaris, polysaccharide, sulfated modification, structural

38

characterization, antioxidant activity, antitumor activity

13

C NMR) and 2D NMR (HSQC and

39 40 41

INTRODUCTION

42

Cordyceps militaris (Fr.) Link was found to have effects on replenishing the kidney and

43

soothing the lung for the therapy of hyposexualities, hyperglycemia, hyperlipidemia, renal

44

dysfunction, and liver disease,1 which are similar with those of the precious and costful

45

Chinese traditional medicine Ophiocordyceps sinensis (Cordyceps sinensis). As such, C.

46

militaris (Fr.) Link, instead of O. sinensis, has been used in the formulation of nutraceuticals

47

and functional foods in China and South East Asia, and even might more widely replace O.

48

sinensis in the application of health enhancement, disease prevention, and disease treatment.2,3

49

Recent investigations have shown that the polysaccharides from C. militaris possess various

50

biological

activities

including

antioxidation,

immunomodulation,

2


antitumor

and

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51


anti-inflammation.4

52

Structure features of polysaccharides, i.e. monosaccharide composition, types of

53

glycosidic bonds, extent of polymerization, molecular weight, degrees of branching, and 3-D

54

conformation lead to their various biological activities.5 Changes in functionality and

55

conformation of polysaccharides via synthetic modification (e.g. sulfation, phosphorylation,

56

methylation and carboxymethylation method) could alter or improve their biological

57

properties.6,7 Among these modification strategies, sulfation is one of the most effective

58

approaches and has been used to improve a variety of bioactivities of many well-known

59

polysaccharides, such as anticoagulant, immunostimulant, hypoglycemic, antioxidant,

60

antitumor, and bile acid-binding properties8-10.

61

Although an increasing number of efforts on the molecular modification and structural

62

improvement of polysaccharides to pursuit for strong or new biological activities, have been

63

taken, the structural modification of polysaccharides from C. militaris by sulfation and the

64

biological activities of their sulfated derivatives have not been reported yet.

65

In the current study, we report the extraction, purification and structural characterization

66

of a novel polysaccharide (CMPA90-1, 1) from the cultured C. militaris, as well as its sulfated

67

derivative (CMPA90-M1, 2) for the first time. Scanning electron microscopy (SEM), atomic

68

force microscope (AFM) and Fourier transform infrared spectroscopy (FT-IR) were

69

conducted to analyze and compare the morphological and structural characteristics of 1 and 2.

70

In addition, their antioxidant and antitumor activities were evaluated in vitro.

71 72

MATERIALS AND METHODS

3



73

Materials. The cultured fruiting bodies of C. militaris (No. 201301028) were purchased

74

from Jiangmen Honghao Bioscience and Technology Corporation, Jiangmen, China. The

75

material was identified by Professor R. M. Yu, College of Pharmacy, Jinan University, China.

76

Experimental Reagents and Materials. Sephadex G-25 and DEAE cellulose-52 were

77

obtained from Whatman Ltd. Sephacryl S-300 HR was obtained from Amersham Biosciences.

78

XAD-7 macroporous adsorption resin, dimethyl sulfoxide (DMSO), 3-(4,5-dimethylthiazol-

79

2-y1)-2,5-diphenyltetrazolium bromide (MTT), penicillin G and streptomycin were obtained

80

from Sigma Chemical Co. (St. Louis, MO, USA). Ascorbic acid (Vitamin C, Vc),

81

chlorosulfonic acid (CSA), pyridine (Pyr), hydrogen peroxide (H2O2), potassium ferricyanide

82

[K3Fe(CN)6] and ferrous sulfate (FeSO4) were obtained from Guangzhou Chemical Reagent

83

Company, China. Human lung adenocarcinoma cell line A549 was obtained from Shanghai

84

Institutes for Biological Sciences, Chinese Academy of Sciences. RPMI-1640 medium and

85

fetal bovine serum (FBS) were purchased from Hyclone (Logan, UT, USA). All reagents were

86

of analytical grade.

87

Extraction, Isolation and Purification of 1. The dried powder of cultured C. militaris

88

(500 g) was extracted in 0.5 L of distilled water using a domestic blender for 5 min. The

89

mixture was added to a solution of simulated gastric juice (9.5 L, pH 1.5) which was used to

90

mimic the conditions of the stomach according to the method of Golovchenko 11 with some

91

modifications. Simulated gastric juice contained HCl (1.34 g/L), NaCl (2.16 g/L), KH 2PO4

92

(0.63 g/L), CaCl2 (0.12 g/L), KCl (0.39 g/L), and pepsin (0.53 g/L). Extraction was conducted

93

for 4 h at 37 °C. After the filtered extract was centrifuged at 5000 g for 15 min, the

94

supernatant was concentrated with a rotary evaporator at 60 °C under vacuum. The mixture

4


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95

was obtained by ethanol precipitation at the final concentration of 80% (v/v) of ethanol at

96

4 °C overnight and then centrifuged at 5000 g for 15 min to precipitate proteins and

97

high-molecular-weight polysaccharides.12 The supernatant (CMPA) was collected and

98

lyophilized.

99

CMPA was fractionated on XAD-7 macroporous adsorption resin column (2.6 × 95 cm),

100

eluted with distilled water at a flow rate of 2 mL/min.13 The elute (20 mL) was collected and

101

then determined by the phenol–sulfuric acid method. Fractions 10-30 corresponding to the

102

major peak were combined, concentrated, dialyzed (Mw cut off: 500 Da), and lyophilized to

103

obtain CMPA90.

104

CMPA90 was dissolved in distilled water, filtered through a filter (0.45μm) and purified

105

on DEAE cellulose-52 anion exchange column (2.6 × 40 cm), eluted with distilled water and

106

a linear gradient from 0 to 1.0 M NaCl at a flow rate of 0.5 mL/min. The eluate (5 mL/tube)

107

was collected automatically and carbohydrates were assayed by the phenol-sulfuric acid

108

method. A sharp peak was collected, dialyzed and further purified by a Sephadex G-25

109

column (1.6 × 70 cm) eluting with distilled water. The flow rate was maintained at 0.3

110

mL/min. Consequently, a polysaccharide was obtained and coded CMPA90-1 (1), with

111

(c 1.0, H2O).

112

Analytical Methods. The content of total sugar and uronic acid was determined by the

113

phenol-sulfuric acid colorimetric method and the carbazole-sulfuric acid method,

114

respectively.14,15 Optical rotation was assayed by a Jasco P-1020 polarimeter. The sulfur

115

content was determined by Wang’s method.9 The calibration curve was prepared with sodium

116

sulfate as standard. The degree of substitution (DS) was calculated based on the following

5



117

equation:

118 119

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DS 

Determination

of

1.62  S% 32  1.02  S%

Homogeneity

and

Molecular

Weight.

Gel

permeation

120

chromatography (GPC) was used to determine the homogeneity and molecular weights of

121

samples. Standard dextrans (blue dextran, T-200, T-70, T-40, T-10, T-7 and T-4) and glucose

122

were passed through a Sephacryl S-300 HR column (1.6 × 70 cm), and elution volumes were

123

plotted against the logarithms of their respective molecular weights. Elution volumes of

124

samples were then plotted in the same graph, and molecular weights of the polysaccharides

125

were measured.16

126

Monosaccharide Composition Analysis. 1 (5 mg) was dissolved in 2mL of 2 M

127

trifluoroacetic acid (TFA) in a sealed tube and hydrolyzed at 110 °C for 6 h. The excess acid

128

was evaporated with a rotary evaporator after the hydrolysis was completed. The

129

monosaccharides

130

chromatography coupled with pulsed amperometric detection (HPAEC-PAD). The

131

hydrolysate (1 mg/mL) was used for the ionic chromatography analysis by HPAEC-PAD on

132

the Dionex ICS-2500 system, followed by the elution with a mixture of water and NaOH (200

133

mM) in the volume ratio of 92:8.17

contents

were

measured

by

high-performance

anion

exchange

134

Partial Hydrolysis. 1 (30 mg) was partially hydrolyzed with 0.05 M TFA at 100 °C for 6

135

h. The hydrolysate was dialyzed in a dialysis bag (Mw cut off: 500 Da) against distilled water

136

for 48 h. The fraction out of the dialysis bag was collected. The excess TFA in the fraction

137

was removed by co-distillation with MeOH, and the fraction was evaporated with a rotary

138

evaporator, named fraction 1. The fraction in the dialysis bag was evaporated to dryness, and

6


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139

then hydrolyzed with 0.5 M TFA. The hydrolysate was dialyzed, and the fraction out of the

140

dialysis bag (namely fraction 2) and the fraction in the dialysis bag (namely fraction 3) were

141

concentrated, respectively. Fractions 1, 2 and 3 were hydrolyzed with 2 M TFA and

142

determined with HAPEC-PAD.16

143

Periodate Oxidation-Smith Degradation. 1 (15 mg) was dissolved in 5 mL of distilled

144

water, and 25 mL of NaIO4 (15 mM) was added. The solution was kept in the dark at 4 °C. 0.1

145

mL aliquots were taken out at 6 h intervals, diluted to 5 mL with distilled water, and measured

146

in a spectrophotometer at 223 nm, until the value became stable. The consumption of HIO4

147

was analyzed by a spectrophotometric method and the production of formic acid was

148

determined by titration with NaOH (0.060 M). The residue was collected and reduced with

149

NaBH4 (30 mg) for overnight.16 After neutralization and dialysis, the retentate was lyophilized

150

and hydrolyzed with TFA (2 M) at 100 °C for 6 h. The products were acetylated and then

151

determined by gas chromatography (GC).18

152

Methylation Analysis. In order to analyze the glycosyl linkages, 1 (20 mg) was

153

methylated six times according to the method described in the literature.19 Disappearance of

154

the OH band (3200−3700 cm-1) in the IR spectrum indicated the implementation of complete

155

methylation. The methylated products were hydrolyzed, reduced, and acetylated to produce

156

alditol acetates which were analyzed by GC-MS.20 Peaks of methylated products were

157

identified by their mass spectra. The relative molar ratios of products were estimated from the

158

peak areas and corresponding response factors in GC.

159

NMR Spectroscopy. 1 (60 mg) was dried in a vacuum over P2O5 for 6 h, and then

160

deuterium-exchanged by lyophilization with D2O three times to completely replace H with D.

7



161

After that, the sample was dissolved in D2O at room temperature for 3 h before NMR analysis.

162

1D and 2D NMR spectra were obtained on an AMX 500 MHz FT NMR spectrometer (Bruker,

163

Germany).

164

Sulfated Modification of 1. CSA (6 mL) was added dropwise into anhydrous pyridine

165

(12 mL) filled in three-necked flask, under continuous stirring and cooling in ice water bath.9

166

All chemical reactions were completed in 40 min and the sulfating reagent was obtained. 1

167

(500 mg) was suspended in 20 mL of anhydrous formamide at room temperature, and then the

168

mixture was stirred for 30 min. After the sulfating reagent was added, the mixture was kept

169

stirred for 2 h at 70 °C. The product was neutralized with 2.0 M NaOH solution at room

170

temperature, and then concentrated and precipitated with 95% ethanol (3 times of volume).

171

The sediment was then re-dissolved in water and intensively dialyzed against tap water (24 h)

172

and then distilled water (48 h) to remove pyridine, salt and potential degradation products. 6

173

Consequently, a sulfated polysaccharide, namely CMPA90-M1 (2) was collected after

174

lyophilization and saved in dryness box.

175

Fourier Transformed Infrared (FT-IR) Analysis. The infrared spectra of 1 and 2 were

176

determined using an FT-IR spectrometer (Perkin-Elmer, USA). The dried powder of each

177

sample was ground with KBr powder and then pressed into 1 mm pellets for FT-IR

178

measurement in a frequency range of 4000-500 cm-1.

179

Ultrastructures of 1 and 2. Scanning electron microscope (SEM) images of 1 and 2

180

were obtained by an environmental scanning electron microscope (ESEM) (Philips XL-30,

181

The Netherlands). The dried powder of each sample was directly placed on separate specimen

182

holders by double-sided adhesive tapes and then sputtered with gold powder using sputter

8


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183

coater.21 Finally, the samples were observed with 2000- and 10000-fold magnification at 5.0

184

kV under a high vacuum condition, respectively.

185

The ultrastructures of 1 and 2 were also observed using an atomic force microscope

186

(AFM) (Bioscope Catylyst Nanoscope-V, USA). Each sample was dissolved in doubly

187

distilled water (1 μg/mL) and stirred by a magnetic stirrer apparatus for 4 h. After that, the

188

solution (5 μL) was dropped onto freshly cleaved mica substrate and allowed to air-dry at

189

room temperature. The AFM was determined in the contact-mode.22 A tube-type piezoelectric

190

scanner ( 4 × 4 μm) and a Si3N4 probe (Olympus, Japan) were employed, and images were

191

obtained simultaneously with 256 × 256 pixels at a scanning rate of 1.0 Hz per line.

192

Measurement of DPPH Radical-scavenging Activity. The free-radical scavenging

193

capacity was measured using the DPPH test according to the method of Braca.23 Briefly, each

194

of 1 and 2 solution (10 μL) at varying concentrations (0, 25, 50, 100, 200, 400, 800 and 1600

195

μg/mL) was added to DPPH ethanol solution (190 μL, 0.2 mM). For the positive control,

196

sample was substituted by Vc. The solution was kept at room temperature for 30 min, and the

197

absorbance was measured at 517 nm. The percentage of DPPH radical-scavenging capability

198

was calculated according to the following equation:

199

scavenging ability (%)  [(A 0 - A1 )]/A 0  100%

200

where A0 was the absorbance in the absence of sample and A1 was the absorbance in the

201

presence of sample.

202

Measurement of Hydroxyl Radical-scavenging Activity. The hydroxyl radical-

203

scavenging activity was examined using the method described previously.17 Vc was used as

204

the positive control. All values were determined in there replicates. The percentage of

9



205 206

hydroxyl radical-scavenging activity was calculated from the following equation: scavenging ability (%)  [(A 2 - A1 )]/[(A 0 - A1 )]  100%

207

where A0 was the absorbance of the blank control (without of H2O2), A1 was the absorbance in

208

the absence of sample, and A2 was the absorbance in the presence of sample.

209

Measurement of Reducing Power. The reducing power was tested referring to the

210

ferric-reducing antioxidant power (FRAP) assay.24 Samples (0-1600 μg/mL, 2.5 mL) were

211

mixed with 2.5 mL of 0.2 M phosphate buffer (pH 6.6) and 5 mL of 1% (w/v) potassium

212

ferricyanide [K3Fe(CN)6]. After incubated at 50 °C for 20 min, the reaction mixture was

213

mixed with 2.5 mL of 10% (w/v) trichloroacetic acid. The mixture was then centrifuged at

214

5000 g for 10 min. 2.5 mL of the supernatant was mixed with the same volume of distilled

215

water and 0.5 mL of 0.1% (w/v) ferric chloride. The absorbance of the resulting solution was

216

measured at 700 nm after 10 min. Vc was used as the positive control.

217

Measurement of Ferrous Ion-chelating Activity. The ferrous ion-chelating activity was

218

estimated using the formation of ferrous iron–ferrozine complex.25 Samples at different

219

concentrations (0-1600 μg/mL) were mixed with deionized water (3.7 mL), and then reacted

220

with 0.1 mL of 2.0 mM FeSO4 and 0.2 mL of 5.0 mM ferrozine. After the reaction for 10 min,

221

the absorption was measured at 562 nm. For the positive control, sample was substituted with

222

EDTA. The chelating activity on ferrous ions was calculated as the following equation:

223

chelating ability (%)  [ ( A0 - A1 ) ] / A 0 ] 1 0 0 %

224

where A0 was the absorbance of the control (deionized water, instead of sample), and A 1 was

225

the absorbance of the sample mixed with reaction solution.

226

Cell Culture and Cytotoxicity Assay. A549 cells were cultured in RPMI-1640 medium

10


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227

containing 10% FBS, 100 units/mL penicillin G and 100 μg/mL streptomycin, in a cell

228

incubator with 5% CO2 at 37 °C. Experiments were performed when cell growth was

229

approximately 80% confluent. Three independent experiments were performed. The

230

cytotoxicity of 1 against A549 cell line was determined using the MTT assay.26 Briefly, A549

231

cells in logarithmic phase were seeded in 96-well culture dishes at a density of 3000 cells/well.

232

24 hours later, the cells were treated with 1 and 2 (final concentrations: 5.6, 16.7, 50, 150 and

233

450 µg/ml), respectively. Cisplatin was used as the positive control. After treatment of 48 h,

234

20 μL of MTT (5 mg/mL) was added to each well and the cells were incubated for another 4 h

235

at 37 °C. After the media were removed, 200 μL of dimethyl sulphoxide (DMSO) was added

236

to each well to dissolve the cellular crystalline deposits and the absorbance was determined

237

spectrophotometrically at 570 nm. The inhibition rate (I %) was calculated according to the

238

formula below:

Inhibition rate ( I %) 

239

A570nm,control  A570nm,sample A570nm,control  A570nm,blank

100%

240

Statistical Analysis. Data were analyzed using statistical analysis software SPSS 11.5

241

and expressed as mean value ± standard deviation (SD) for three independent experiments.

242

Statistical significance was defined as P < 0.05 by analysis of variance (ANOVA) followed by

243

Dunnett’s tests.

244 245

RESULTS AND DISCUSSION

246

Isolation, Purification and Chemical Composition of 1. Crude polysaccharide (20.18

247

g) was obtained from the cultured fruiting bodies of C. militaris. The subsequent purification

248

led to a single and symmetrical sharp peak of 1 by analysis of a gel-filtration chromatography 11



249

on a Sephacryl S-300 HR column with the phenol-sulfuric acid assay. The resulting 1 also

250

showed the same optical rotation in varying low-content aqueous ethanol. The uronic acid

251

content of 1 was below the detection limit. The average molecular weight of 1 was

252

determined as 9.3 kDa by GPC method on a Sephacryl S-300 HR column. The total sugar

253

content of 1 was 98.4% (w/w), and the result of m-hydroxybiphenyl colorimetric test was

254

negative.

255

Structural Characterization of 1. 1 was acidically hydrolyzed and three

256

monosaccharides, arabinose, mannose and galactose, were identified in the hydrolysate of 1

257

by HAPEC-PAD analysis, with relative ratios of 1.00:2.89:2.03. The partial acid hydrolysis of

258

1 afforded three fractions (1, 2 and 3) which were subsequently analyzed by HAPEC-PAD

259

(Table 1). The component of fraction 3 (the precipitate in the sack) indicated that galactose

260

might be the structural backbone of 1. The results of fractions 1 and 2 suggested that 1 had the

261

branched structure consisting of arabinose and mannose, with mannose as terminal unit.

262

The results of periodate oxidized-Smith degradations were analyzed by GC and shown in

263

Table 1. No mannose was found in the resulting oxidation products, thus the linkage manner

264

of mannose could be (1→), (1→2), (1→6), (1→2,6), (1→4) and (1→4,6), which might lead

265

to oxidization of mannose into glycerol and erythritol. The presence of arabinose and

266

galactose revealed that at least partial residues of arabinose and galactose were linked by the

267

manners of (1→3), (1→2,3), (1→2,4), (1→3,4) or (1→2,3,4), which are stable under the

268

condition of the oxidation.27 The results of methylation analysis (Table 2) showed the

269

presence of five components, namely, 2,5-Me2-Ara, 2,3-Me2-Ara, 2,3,4,6-Me4-Man,

270

3,4,6-Me3-Man, 2,4-Me2-Gal with a molar ratio of 1.00:1.08:3.22:0.89:6.19. Based on the

12


Page 12 of 33

Page 13 of 33


271

standard data in the CCRC Spectral Database for PMAA’s, the linkage manners of arabinose

272

were deduced as (1→3) and (1→5) , while those of mannose were (1→) and (1→2), and the

273

those of galactose were (1→3,6). This finding was in consistent with the linkage of terminal

274

and branched residues. Moreover, the molar ratios also showed a good correlation with the

275

monosaccharide composition analysis of 1 described above.

276

As shown in Figure 1, the characteristic absorption peaks of polysaccharide showed in

277

the infrared spectrum of 1 at 3436, 2961, 1641, 1413 and 1077 cm-1. Absence of absorbance

278

band near 1243 and 810 cm−1 indicated lack of sulfate ester in 1. The absorption peaks at 866

279

and 896 cm-1 confirmed the co-existence of α- and β-glycosidic bonds. No band around 1740

280

cm−1 was observed, confirming that 1 did not contain uronic acid.17,28

281

NMR Spectroscopy Analysis. The planar structure of 1 was established by analysis of

282

its 1D and 2D NMR spectra, together with comparison with the reported NMR data.29-32 Five

283

pairs of 1H and 13C signals of anomeric CH attributed to 5 different types of glycosidic bonds

284

were assigned at δ 5.04/105.89, 5.12/104.15, 5.07/100.60, 5.10/99.33 and 4.98/98.08 ppm

285

based on their chemical shifts and the corresponding HSQC correlations (Figure 2A). These

286

five types of glycosidic bonds indicated the existence of five structural residues in 1, i.e.

287

residues A, B, C, D and E, respectively. According to 1H and 13C NMR spectra of the standard

288

arabinose (data not shown), 30 as well as the HSQC correlations, the residue A was determined

289

as an arabinose residue, and the remaining signals of the residue A were assigned as δ 81.53/δ

290

4.01(CH-2), 78.35/4.05 (CH-3), 86.86/4.12 (CH-4) and 68.40/3.85 (CH2-5), respectively.

291

Similarly, the NMR signals of the other residues were assigned (Table 3).

292

Five residues were connected together by the analysis of the HMBC spectrum (Figure

13



Page 14 of 33

293

2B) of 1. In the HMBE spectrum, the following correlations were observed: E H-6 (δ 3.75)

294

and B C-1 (δ 104.15), D H-2 (δ 4.10) and C C-1 (δ 100.60), B H-3 (δ 3.74) and C C-1 (δ

295

100.60), A H-1 (δ 5.04) and A C-4 (δ 86.86), A H-1 (δ 5.04) and A C-2 (δ 81.53), C H-1 (δ

296

5.07) and B C-3 (δ 81.11), C H-1 (δ 5.07) and D C-2 (δ 75.50), E H-1 (δ 4.98) and E C-2 (δ

297

70.62), B H-1 (δ 5.12) and E C-6 (δ 67.20), A H-1 (δ 5.04) and E C-6 (δ 67.20). The

298

cross-peaks at δ 3.75/104.15 and δ 5.12/67.20 were observed in HMBC. Since the signal at δ

299

3.75 corresponded to H-6 in residue E, δ 104.15 to C-1 in residue B, δ 5.12 to H-1 in residue

300

B, and δ 67.20 to C-6 in residue E, it could be concluded that the residues E and B were

301

connected to each other as E-(6→1)-B. Moreover, the cross-peaks at δ 5.07/75.50 and δ

302

4.10/100.60 were assigned to the inter-residue C H-1, D C-2 and the inter-residue D H-2, C

303

C-1, respectively, indicating that the residues C and D were connected to each other as

304

C-(1→2)-D. Similarly, the following sequences were established: E-(6→1)-A and C-(1→3)-B.

305

Therefore, the structure of 1 was established, with a backbone composed of (1→3)-linked

306

α-D-galactose, and 3 branches, all of which substitute at O-6 of α-D-galactose, consisted of

307

(1→2)-linked α-L-mannose, (1→5)-linked α-L-arabinose and (1→3)-linked β-L-arabinose,

308

respectively, with α-L-mannose as terminal unit (Figure 3).

309

Structure analysis of 2. 2 was prepared from 1 by CSA-Pyr method.6 The GPC analysis

310

of 2 showed a single and symmetric sharp peak and the average molecular weight of 2 was

311

determined as 8.9 kDa. The specific rotation of 2 was determined as

312

H2O). The sulfur content was 3.9 % (w/w), and the DS value was 0.23 in 2. Comparing to 1,

313

two characteristic absorption bands attributed to sulfate groups occurred in the FT-IR

314

spectrum of 2, one at 1243 cm-1 corresponding to an asymmetrical S=O stretching vibration,

14


(c 1.0,

Page 15 of 33


315

and the other at 810 cm-1 corresponding to a symmetrical C-O-S vibration associated with a

316

C-O-SO3 group, which indicated the sulfate groups were successfully introduced into 233.

317

Ultrastructures of 1 and 2. Chemical modification could change the spatial structure of

318

polysaccharides and thus affect the structure-activity relationship.34 Scanning electron

319

micrographs (SEM) of 1 and 2 at magnifications of 2000 and 10000 showed that 1 had a

320

rough surface with a sheet-like appearance composed of many small lumpish particles and

321

irregular pores, while 2 appeared to be a smooth surface with pore openings, which consisted

322

mainly of randomly distributed individual spherical particles, with diameters of

323

approximately 2 μm (Figure 4A and 4B). The different surface topography of the

324

polysaccharides was probably due to the sulfated modiﬁcation and changes of

325

physicochemical properties, so SEM could be set as a characteristic to qualitatively

326

identify sulfated polysaccharides.

327

AFM has been applied as a valuable metrological tool to characterize surface topology

328

on the nanometer scale of particles, macromolecules absorbed to surfaces, biopolymers, and

329

the linear and circular triple helix structures of polysaccharides.34

330

Herein, AFM images showed that 1 aggregated to form homogeneous, smooth, and

331

elastic dome-shaped particles (diameter ranged from 150 to 300 nm and height was

332

approximately 15 nm), while 2 particles aggregated to form a coral-like structure, with

333

dimensions ranged from 500 to 1000 nm in length, 100 to 300 nm in width, and 2 to 3 nm in

334

height (Figure 5A and 5B). The size of polysaccharide chains of both 1 and 2 were over 1.0

335

nm which was larger than the usual size (approximately 0.1–1.0 nm).34 Aggregation of

336

polysaccharides occurs from their side branches entangling each other, and the substitution of

15



337

the hydroxyl group of polysaccharides by sulfate group can cause twisting and converting of

338

the sugar ring conformation which leads to decrease of the size of the resulting sulfated

339

polysaccharides.

340

Scavenging Activity of 1 and 2 against DPPH Radicals. As a stable free-radical

341

compound with maximum absorbance at 517 nm, DPPH has been widely used to determine

342

the free radical-scavenging capacity of various antioxidative samples.35 As shown in Figure 6A,

343

1, 2 and Vc scavenged DPPH radicals to different degree in the given concentration range.

344

The IC50 values of 2 and Vc were 285.33 and 104.17 μg/mL, respectively.

345

Scavenging Activity of 1 and 2 against Hydroxyl Radicals. The hydroxyl radical

346

generated by the Fenton reaction in this system was scavenged by samples with antioxidant

347

activity. The scavenging activity of 1, 2 and Vc against hydroxyl radicals was shown in Figure

348

6B. The IC50 values of CMPA90-M1 and Vc were 405.62 and 136.54 μg/mL, respectively.

349

Reducing Power of CMPA90-1 and CMPA90-M1. Figure 6C depicted the increase in

350

reducing power of 1, 2 and Vc at the gradient concentrations. Higher absorbance of the

351

reaction mixture indicated stronger reducing power of samples. The reducing abilities of 1, 2

352

and Vc at 400 μg/mL were 0.49, 0.82 and 1.21, respectively.

353

Ferrous Ion-Chelating Effect of 1 and 2. The chelating abilities of 1, 2 and EDTA on

354

Fe2+ were shown in Figure 6D. In the range of concentrations from 25 to 1600 μg/mL, they

355

were found to possess more potent chelating effect on Fe2+ in a concentration-dependent

356

manner. The EC50 values of CMPA90-M1 and EDTA were 385.68 and 107.14 μg/mL,

357

respectively.

358

Cytotoxicity of 1 and 2 against A549 cells. The cytotoxicity induced by 1 and 2 was

16


Page 16 of 33

Page 17 of 33


359

investigated in human cancer cell line A549 using MTT assay. As shown in Figure 7, 1 and 2

360

exhibited the capability of anti-proliferation in A549 cells, with the IC50 values of 39.08 and

361

17.33 μg/mL, respectively. Cisplatin exhibited the capability of anti-proliferation against

362

A549 cells, with the IC50 value of 0.70 μg/mL.

363

In conclusion, a novel polysaccharide (1) was isolated from the cultured fruiting bodies

364

of C. militaris contains predominantly D-galactose, L-mannose and L-arabinose. The result

365

showed that the structural characteristics of 1 is different from that of the polysaccharides

366

previously found from Cordyceps sp. in monosaccharide ratios, molecular weights,

367

etc.1,2,4,36,37 Sulfation of 1 by CSA-Pyr method led to its sulfate derivative (2). SEM and AFM

368

exhibited that the ultrastructure of 2 much differed from that of 1, which could result in an

369

improvement of antioxidant and cytotoxic activities. Relative to 1, 2 showed more noticeable

370

free radical-scavenging activity, ferrous ion-chelating ability and reducing power, and

371

stronger anti-proliferative effect on A549 cells. The results of this study indicate that some

372

specific substitutions occurring on original groups of polysaccharides might alter or improve

373

their physicochemical and biological properties. This research may provide new insights into

374

the development and application of polysaccharides from C. militaris.

375 376

AUTHOR INFORMATION

377

*Corresponding authors: Tel: +86-20-85220386; fax +86-20-85224766. E-mail Address:

378

[email protected] (R. M. Yu). Tel: +86-20-85222069; E-mail Address: [email protected] (J.

379

H. Zhu). Tel: +86-20-85228205; E-mail Address: [email protected] (L. Y. Song).

380

17



381

ACKNOWLEAGMENTS

382

This research work was financially supported by Major National Science and Technology

383

Projects / Significant New Drugs Creation (No. 2011ZX09102-001-33). The authors thank Dr.

384

Dongbo Yu from The University of Chicago Medical Center, USA, for proof-reading our

385

manuscript.

386 387

REFERENCES

388

(1) Won, S. Y.; Park, E. H. Anti-inflammatory and related pharmacological activities of cultured

389

mycelia and fruiting bodies of Cordyceps militaris. J. Ethnopharmacol. 2005, 96, 555−561.

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(2) Yu, R. M.; Song, L. Y.; Zhao, Y.; Bin, W.; Wang, L.; Zhang, H.; Wu, Y. H.; Ye, W. C.; Yao, X. S.

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Isolation and biological properties of polysaccharide CPS-1 from cultured Cordyceps militaris.

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Fitoterapia, 2004, 75, 465−472.

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Structural elucidation and biological activity of a novel polysaccharide by alkaline extraction from

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cultured Cordyceps militaris. Carbohydr. Polym. 2009, 75, 166−171.

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characterization of polysaccharides from Tricholoma matsutake and their antioxidant and antitumor

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determination of sugars and related substances. Anal. Chem. 1956, 28, 350−356.

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and antioxidant activity of a novel polysaccharide isolated from pulp tissues of Litchi chinensis. J.

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characterization and antioxidant activity of a novel polysaccharide isolated from Ginkgo biloba. Int. J.

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polysaccharides from pericarp tissues of litchi (Litchi chinensis Sonn.) fruit in relation to their

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(19) Hakomori, S. A rapid premethylation of glycolipid, and polysaccharide catalyzed by 19



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methylsulfinyl carbanion in dimethyl sulfoxide. J. Biochem. 1964, 55, 205−208.

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antioxidant and α-amylase inhibitory activities of corn silk polysaccharides. Carbohydr. Polym. 2013,

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polysaccharide POJ-U1a extracted by ultrasound from Ophiopogon japonicus. Fitoterapia, 2012, 83,

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antioxidants and inhibitors of cell proliferation in vitro. Food and Chem. Toxicol. 2005, 43, 1073−1081.

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(25) Dinis, T. C.; Madeira, V. M.; Almeida, L. M. Action of phenolic derivatives (acetaminophen,

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salicylate, and 5-aminosalicylate) as inhibitors of membrane lipid peroxidation and as peroxyl radical

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scavengers. Arch. Biochem. Biophy. 1994, 315, 161−169.

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(26) Hu, X. J.; Song, L. Y.; Huang, L. J.; Zhen, Q.; Yu, R, M. Antitumor effect of a polypeptide fraction

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from Arca subcrenata in vitro and in vivo. Mar. Drugs, 2012, 10, 2782−2794.

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(27) Abdel-Akher, M.; Hamilton, J. K.; Montgomeny, R.; Smith, F. A new procedure for the

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(28) Sun, Y. X.; Liang, H. T.; Zhang, X. T.; Tong, H. B.; Liu, J. C. Structural elucidation and

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immunological activity of a polysaccharide from the fruiting body of Armillaria mellea. Bioresour.

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Technol. 2009, 100, 1860−1863.

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(29) Bi, H. T.; Gao, T. T.; Li, Z. H.; Ji, L.; Yang, W.; Iteku, B. J.; Liu, E. X.; Zhou, Y. F. Structural

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elucidation and antioxidant activity of a water-soluble polysaccharide from the fruit bodies of Bulgaria

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inquinans (Fries). Food Chem. 2013, 138, 1470−1475.

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(Anogeissus latifolia) Part III: Structure characterization of a globular polysaccharide fraction by 1D,

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(32) Mandal, E. K.; Maity, K.; Maity, S.; Gantait, S. K.; Behera, B.; Maiti, T. K.; Sikdar, S. R.; Islama,

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S. S. Chemical analysis of an immunostimulating (1-4)-, (1-6)-branched glucan from an edible

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mushroom, Calocybe indica. Carbohydr. Res. 2012, 347, 172−177.

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(33) Maciel, J. S.; Chaves, L S.; Souza, B. Structural characterization of cold extracted fraction of

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soluble sulfated polysaccharide from red seaweed Cracilaria birdiae. Carbohydr. Polym. 2008, 77,

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(34) Wang, Y. F.; Peng, Y. H.; Wei, X. L.; Yang, Z. W.; Xiao, J. B.; Jin, Z. Y. Sulfation of tea

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polysaccharides: Synthesis, characterization and hypoglycemic activity. Int. J. Biol. Macromol. 2010,

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(35) Benvenuti, S.; Pellati, F.; Melegari, M.; Bertelli, D. Polyphenols, anthocyanins, ascorbic acid, and

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radical scavenging activity of Rubus, Ribes, and Aronia. J. Food Sci. 2004, 69, 164−169.

483

(36) Lee, J. S.; Kwon, J. S.; Yun, J. S.; Pahk, J. W.; Shin, W. C.; Lee, S. Y. Structural characterization of

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immunostimulating polysaccharide from cultured mycelia of Cordyceps militaris. Carbohydr. Polym.

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486

(37) Jing, Y. S.; Cui, X. L.; Chen, Z. Y.; Huang, L. J.; Song, L. Y.; Yu, R. M. Elucidation and biological

487

activities of a new polysaccharide from cultured Cordyceps militaris. Carbohydr. Polym. 2014, 102,

488

288–296.

489 490 491 492 493 494 495 496 497 498 21



499

Page 22 of 33

Table 1. Results of HAPEC-PAD Analysis for the Degradation Products of 1 molar ratios

500

arabinose

mannose

galactose

CMPA90-1

1.00

2.89

2.03

fraction 1

n.d.

1.00

n.d.

fraction 2

2.33

1.00

n.d.

fraction 3

n.d.

n.d.

1.00

Smith degradation

1.00

n.d.

3.15

n.d., not detected.

501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 22


Page 23 of 33

521


Table 2. GC-MS Results of Methylated Products of 1 methylation

retention

molar

mass fragments

linkage

product

time (min)

ratio

(m/z)

type

2,5-Me2-Ara

15.22

1.00

87, 101, 117, 129, 189

1→3

2,3-Me2-Ara

16.28

1.08

87, 113, 117, 129, 233

1→5

2,3,4,6-Me4-Man

17.06

3.22

71, 87, 101, 117, 129, 161, 205

T→

3,4,6-Me3-Man

17.84

0.89

71, 87, 101, 129, 161, 189

1→2

2,4-Me2-Gal

18.94

6.19

43, 87, 117, 129, 189

1→3,6

522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542

23



543

Page 24 of 33

Table 3. Assignment of 13C NMR and 1H NMR Chemical Shifts of 1 sugar residue

A →5)-α-L-Ara(1→

B

C

D

→3)-β-L-Ara(1→

α-L-Man(1→

→2)-α-L-Man(1→

E →3,6)-α-D-Gal(1→

chemical shifts (ppm) C1/H1

C2/H2

C3/H3

C4/H4

C5/H5

105.89/

81.53/

78.35/

86.86/

68.40/

5.04

4.01

4.05

4.12

3.85

104.15/

73.92/

81.11/

73.04/

61.10/

5.12

4.10

3.74

3.68

3.80

100.60/

69.40/

69.89/

66.20/

72.50/

60.86/

5.07

4.00

3.87

3.95

3.72

3.68

99.33/

75.50/

66.03/

65.99/

72.80/

60.86/

5.10

4.10

3.85

3.59

3.61

3.85

98.08/

70.62/

76.00/

70.01/

74.78/

67.20/

4.98

3.48

4.05

3.95

4.17

3.75

544 545 546 547 548 549 550 551 552 553

24


C6/H6

Page 25 of 33


554

Figure captions

555

Figure 1. FT-IR spectra of 1 and 2.

556

Figure 2. (A) 1H-13C HSQC spectrum of CMPA90-1. (B) 1H-13C HMBC spectrum of 1.

557

Figure 3. Predicted structure of 1.

558

Figure 4. SEM images of 1(A) and 2(B).

559

Figure 5. AFM images of 1(A) and 2(B).

560

Figure 6. Antioxidant activities of 1 and 2. (A) DPPH radical-scavenging activity, (B)

561

Hydroxyl radical-scavenging activity, (C) Reducing power, (D) Ferrous ion-chelating effect.

562

Values are means ± SD of three separate experiments.

563

Figure 7. Inhibitory effects of 1 and 2 on human tumor cell growth. A549 cells were treated

564

by 1 and 2 at different concentrations for 48 h in 96-well plates, respectively. The absorbance

565

of the culture was measured at 570 nm. Data are presented as mean ± SD of three separated

566

experiments.

567 568 569 570 571 572 573 574 575

25



576 577 578 579

Figure 1

580

Jing, et al.

581 582 583 584 585 586 587 588 589 590

26


Page 26 of 33

Page 27 of 33


591 592 593

Figure 2

594

Jing, et al.

595

27



C

D (6

3)-α-D-Gal-(1

596

C

5)-Ara-L-α-(1

3)-α-D-Gal-(1 n (6

6)

598 Figure 3

600

Jing, et al.

1)-β-L-Ara-(3

B

A

597

599

1)-α-L-Man

1)-α-L-Man-(2

E

E

E Man-L-α-(1

3)-α-D-Gal-(1

Page 28 of 33

601 602 603 604 605 606 607 608 609 610 611 612 613 614

28


1)-α-L-Man

C

Page 29 of 33


615 616 617 618 619

Figure 4

620

Jing, et al.

621 622 623 624 625 626 627

29



628 629 630 631

Figure 5

632

Jing, et al.

633 634 635 636 637

30


Page 30 of 33

Page 31 of 33


638 639 640 641

Figure 6

642

Jing, et al.

643 644 645 646 647 648 649 650 651

31



652 653 654 655 656 657

Figure 7

658

Jing, et al.

659 660 661 662 663 664 665 666 667

32


Page 32 of 33

Page 33 of 33


TOC Graphic

668 669

(6

3)-¦Á-D-Gal-(1

3)-¦Á-D-Gal-(1

1)-¦Á-L-Man

1)-¦Á-L-Man-(2

SO4

3)-¦Á-D-Gal-(1 n

Man-L-¦Á-(1

5)-Ara-L-¦Á-(1

(6

6)

1)-¦Â-L-Ara-(3

1)-¦Á-L-Man

670 671

33


2

Structural Characterization and Biological Activities of a Novel

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