Multiple-Fingerprint Analysis for Investigating Quality Control of

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Multiple fingerprint analysis for investigating quality control of Flammulina velutipes fruiting body polysaccharides Pu Jing, Shu-Juan Zhao, Manman Lu, Zan Cai, Jie Pang, and Lihua Song J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf504349r • Publication Date (Web): 05 Nov 2014 Downloaded from http://pubs.acs.org on November 8, 2014

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Journal of Agricultural and Food Chemistry

1

Multiple fingerprint analysis for investigating quality control of Flammulina

2

velutipes fruiting body polysaccharides Pu Jing†, Shu-Juan Zhao†, Man-Man Lu†, Zan Cai†, Jie Pang‡, Li-Hua, Song†,*

3 4 5

†

6

(South), Bor S. Luh Food Safety Research Center, School of Agriculture & Biology,

7

Shanghai Jiao Tong University, Shanghai 200240, China

8

‡

9 10 11

Research Center for Food Safety and Nutrition, Key Lab of Urban Agriculture

College of Food Science, Fujian Agriculture and Forestry University, Fujian 350002, China

* Author to whom correspondence should be addressed: Tel: +86-2134205717; E-mail: [email protected]; [email protected]

12 13 14 15 16 17 18

1

ACS Paragon Plus Environment


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ABSTRACT: Quality control issues overshadow potential health benefits of the

20

edible mushroom Flammulina velutipes, with the detection and isolation of

21

polysaccharides posing particular problems. In this study, multiple fingerprint analysis

22

was performed using chemometrics to assess polysaccharide quality and antioxidant

23

activity of F. velutipes fruiting bodies from different sources. The authentic source

24

exhibited differences in both oxygen radical absorbance capacity and ferric reducing

25

antioxidant power from foreign sources. IR spectroscopic/HPLC chromatographic

26

fingerprints of polysaccharide extracts from the authentic source were established and

27

applied to assess polysaccharide quality of foreign sources. Analysis of IR fingerprints

28

using Pearson correlation coefficient gave correlation coefficient r values of 0.788 and

29

0.828 for two foreign sources, respectively, indicating distinctness from the authentic

30

source. Analysis of HPLC fingerprints using the supervised method by Traditional

31

Chinese Medicine could not discriminate between sources (r>0.9), but principal

32

component analysis of IR and HPLC fingerprints distinguished the foreign sources.

33 34

Flammulina

velutipes,

polysaccharide,

35

KEYWORDS:

36

fingerprint analysis, similarity, principal component analysis

antioxidant

activity,

2


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37

INTRODUCTION

38

Edible mushrooms are important both nutritionally and as a source of drug

39

candidates for use as pharmaceuticals. The golden needle mushroom or enokitake

40

(Flammulina velutipes) is the fourth most popular edible mushroom worldwide due to

41

its delicious taste and high nutritional properties containing a low calorie and fat

42

content, and high proportion of essential amino acids, fibre and vitamins.1 Numerous

43

reports

44

polysaccharides,2-4 and F. velutipes serves as an excellent source of both fibre and

45

polysaccharides that exhibit antioxidant,2 cholesterol-lowering,5 anti-inflammatory,6

46

immunomodulatory,7 and anti-tumor activities.8

have

highlighted

the

strong

antioxidant

activity

of

mushroom

47

The antioxidant and other biological activities of polysaccharides is dependent on

48

various structural parameters including monosaccharide composition, main chain

49

glycosidic bond type, the nature and degree of polymerization and branching, and the

50

flexibility and spatial configuration of the glycan chains.9, 10 The source of F. velutipes

51

may also affect the bioactivities of the polysaccharides present.

52

Assessing the quality of the polysaccharides in F. velutipes fruiting bodies is

53

currently hindering the potential health benefits, because the detection and separation

54

of carbohydrates, especially long-chain polysaccharides, is highly challenging. Simple

55

and reliable analytical techniques to assess the complex polysaccharides are not yet

56

widely available. Fingerprint analysis integrated with chemometrics has proved useful

57

for characterization of complex molecular systems and is an identification and

58

qualification technique that was approved by the World Health Organization in

3



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1991.11 The (dis)similarity approaches including principal component analysis (PCA)

60

have been applied to assess authenticity or detect adulteration in foods and herbs.12, 13

61

In

62

chromatographic

63

polysaccharides and their antioxidant activity of F. velutipes fruiting bodies from

64

authentic and foreign sources. The multiple fingerprints of polysaccharides from the

65

qualified or authentic source were established and applied to detect foreign sources.

this

study,

multiple

fingerprint

approaches

and

analysis

chemometrics

involving was

spectroscopic

performed

to

and assess

66 67

MATERIALS AND METHODS

68

Chemicals. Standards including glucose, ribose, mannose, galactose, xylose and

69

6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox) were purchased

70

from Sigma-Aldrich (Shanghai, China). 1-phenyl-3-methyl-5-pyrazolone (PMP) was

71

purchased from Adamas-beta (Shanghai, China). HPLC grade acetonitrile and water

72

were purchased from Anpel (Shanghai, China). All other chemicals and solvents were

73

of the highest commercial grade and were purchased from Sigma-Aldrich (Shanghai,

74

China).

75 76

Source Materials. Ten batches of F. velutipes fruiting bodies as the authentic source

77

were provided by Infinitus (China) Company Ltd. and named as ZJ samples after the

78

grown location of Zhejiang in China. One each of two foreign sources from Sichuan

79

and Fujian named as SC and FJ samples, respectively, were purchased at a local

80

market.

4


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Preparation of Polysaccharide Extracts. Polysaccharides extracts were prepared

83

according to the previously reported.14 The fruiting bodies of F. velutipes were

84

air-dried at 60 °C for 10 h and ground into fine particles using a DS-Y250 mill

85

(Dingshuai Electrics, Shanghai, China) through a 40-mesh screen. 5 g of powder in

86

100 mL dd-water was stirred at 80 °C for 4 h, and the extract was cooled to room

87

temperature and centrifuged at 4000 rpm for 10 min. The supernatant was collected

88

and deproteinized with 60 mL of 5:1 CHCl3–n-BuOH using the Sevag method.15 The

89

upper layer was collected and decolorized by adding 30% (v/v) aqueous H2O2. The

90

solution was concentrated under vacuum and precipitated with three volumes of 95%

91

(v/v) aqueous ethanol, and incubated at 4 °C overnight. Polysaccharide precipitates

92

were obtained by centrifugation at 4000 rpm for 10 min and washed sequentially with

93

acetone and ether. The polysaccharide extract was dried using nitrogen gas and stored

94

in a desiccator for further FT-IR analysis.

95 96

Oxygen Radical Absorbance Capacity (ORAC) Assay. Determination of the

97

oxygen radical absorbance capacity of the polysaccharides was performed as

98

previously reported 16 using an Infinite F200 PRO microplate reader (Tecan,

99

Switzerland). Samples and Trolox standards were prepared in DMSO, and all other

100

reagents were prepared in 75 mM phosphate buffer (pH 7.4). Briefly, each well in a

101

96-well plate contained 30 µL sample (or DMSO for the blank control), and 225 µL of

102

81.63 nM fluorescein. The plate was covered an incubated at 37 °C for 20 min, and 25

5



103

µL of 0.36 M 2,2′-azobis(2-amidinopropane) hydrochloride (AAPH) was added to

104

each well to start the reaction, resulting in a final total volume of 280 µL. The

105

fluorescence was recorded every 5 min for 1 h (ex/em: 485/538 nm) at 37 °C. Trolox

106

equivalents were calculated from the relative area under the curve compared to a

107

Trolox standard curve prepared under the same experimental conditions. Reactions

108

were conducted in triplicate and results are expressed as micromoles of Trolox

109

equivalents per gram of polysaccharides.

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110 111

Ferric Reducing Antioxidant Power (FRAP) Assay. The ferric reducing antioxidant

112

power assay is based on the reduction of Fe3+-tripyridyltriazine (TPTZ) to a blue

113

colored Fe2+-TPTZ.17, 18 The FRAP reagent was prepared by mixing 300 mM acetate

114

buffer (pH 3.6), 10 mM TPTZ and 20 mM ferric chloride in a ratio of 10:1:1 (v/v/v).

115

Then, 3 mL of FRAP reagent was added to 20 µL of sample or Trolox and incubated

116

at 37 °C for 30 min. Absorbance was measured at 590 nm using an Infinite F200 PRO

117

microplate reader (Tecan, Switzerland). Trolox was used as a standard and the

118

appropriate sample dilutions were determined. Reactions were conducted in triplicate

119

and results are reported as Trolox equivalents (TE) per gram of polysaccharides.

120 121

Acid Hydrolysis. Polysaccharides were hydrolyzed using two approaches. For

122

complete acid hydrolysis, polysaccharides (10 mg) were hydrolyzed in 2 mL of 2 M

123

trifluoroacetic acid (TFA) in an ampoule sealed under a nitrogen atmosphere and

124

incubated at 110 °C for 5 h. For partial acid hydrolysis, 2 mL of 0.05 M TFA was

6


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added and incubated at 110 °C for only 2 h. After cooling to room temperature,

126

reactions were centrifuged at 4000 rpm for 5 min, and supernatants were dried at

127

reduced pressure. Methanol was twice added and evaporated under vacuum to remove

128

residual TFA.

129 130

FT-IR Spectroscopy and Chemometric Analysis. FT-IR spectra of non-hydrolyzed

131

polysaccharides were obtained using a PerkinElmer Spectrum 100 FT-IR

132

Spectrometer. Spectra were recorded in absorbance mode from 4000 to 400 cm−1 at a

133

resolution of 4 cm−1. Four replicate spectra were collected for each sample. Spectra

134

from each batch of ZJ samples were exported to EXCEL 2010 to calculate the mean

135

chromatograph. The Pearson correlation coefficient r (-1 ≤ r ≥ 1) was calculated to

136

evaluate the similarity of fingerprints using Eq. 1.13 r=

137

− − ∑ =1( − )( − ) 2

2

− ∑ =1( − ) ∑=1(−− )

Eq.1

138

where xi and yi are the ith elements of x and y (from two fingerprints), n is the number

139

of variables in the fingerprints, x and

140

set and SC or FJ samples. The calculation was performed using the PEARSON

141

function in EXCEL 2010 (Microsoft).

y

are means or averages of the ZJ sample

142 143

Preparation of PMP Derivatives after Complete Acid Hydrolysis. Monosaccharide

144

standards or hydrolyzed polysaccharide samples were dissolved in 10 mL dd-water.

145

0.2 mL of the standards or hydrolyzed samples was mixed with 0.2 mL of a 0.5 M

146

methanol solution of 1-phenyl-3-methyl-5-pyrazolone (PMP) and 0.2 mL of 0.3 M 7



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NaOH, and stirred at 70 °C for 60 min. Following cooling to room temperature,

148

reactions were neutralized with 0.2 mL of 0.3 M HCl and 1 mL chloroform was added

149

and stirred for 30 s before centrifugation at 2000 rpm for 5 min. The chloroform phase

150

was discarded and the extraction process was repeated a further two times to remove

151

the excess PMP reagent. The aqueous layer was collected, made up to 5 mL with

152

dd-water, and filtered through a 0.45 µm membrane.

153 154

Preparation of PMP Derivatives after Partial Acid Hydrolysis. Partially

155

hydrolyzed polysaccharides were dissolved in 2 mL dd-water and 0.5 mL was mixed

156

with 0.2 mL of a 0.5 M methanol solution of 1-phenyl-3-methyl-5-pyrazolone (PMP)

157

and 0.2 mL 0.3 M NaOH and stirred. Subsequent steps were as described above for

158

complete acid hydrolysis.

159 160

HPLC Analysis. Analysis of PMP derivatives was carried out on an Agilent 1260

161

HPLC system (Agilent Technologies, USA). Separation was achieved using reverse

162

phase elution on a 5 µm Shim-pack VP-ODS column (4.6 mm × 250 mm, 5 µm,

163

Shimadzu, Kyoto, Japan) fitted with a 4.6 mm x 10 mm Shim-pack GVP-ODS guard

164

column (Shimadzu, Kyoto, Japan). The chromatographic conditions were as follows:

165

flow rate = 0.8 mL/min; sample injection volume = 20 µL; column temperature =

166

30°C; the mobile phase consisted of 82% (v/v) 0.05 M sodium phosphate (pH 6.8)

167

containing 18% (v/v) acetonitrile for isocratic elution. Spectra were collected at a

168

wavelength of 245 nm.

8


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169 170

Data Analysis. Antioxidant activity data are reported as means ± standard deviation.

171

One-way ANOVA and the LSD test at the level of 0.05 were used to identify

172

differences in means using SPSS for Windows (version rel. 10.05, 1999, SPSS Inc.,

173

Chicago, IL). All material pretreatment, extraction, processing, and spectroscopic/

174

chromatographic fingerprinting and data handling were performed as presented on the

175

flowchart (Figure 1). The similarity of FT-IR fingerprints was determined using the

176

Pearson correlation coefficient r calculated using EXCEL 2010. The similarity of

177

HPLC fingerprints was determined using the Similarity Evaluation System for

178

Chromatographic Fingerprint of Traditional Chinese Medicine Version 2004A

179

(Chinese Pharmacopoeia Committee), which was designed specifically for similarity

180

analyses of LC and GC fingerprints and has been recommended by the State Food and

181

Drug Administration of China.19,

182

chromatographic profiles of samples were calculated. The chemical fingerprints of

183

polysaccharides from 12 batches of F. velutipes fruiting bodies were analyzed by

184

similarity analysis and principal component analysis (PCA) performed using

185

SIMCA-P 11.5 (Umetrics AB, Sweden).

186

RESULTS AND DISCUSSION

187

Antioxidant Activity of Polysaccharides from F. velutipes Fruiting Bodies.

188

Oxygen radical absorbance capacity (ORAC) and ferric reducing antioxidant power

189

(FRAP) assays were performed to investigate the antioxidant activity of

190

polysaccharide samples and to determine whether different radical systems influenced

20

The correlation coefficients of complete

9



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191

the results. Polysaccharides from the authentic source (ZJ-1 to ZJ-10 samples) and the

192

foreign sources (SC and FJ samples) contributed toward the observed ORAC and

193

reducing power in Figure 2. The influence of F. velutipes polysaccharides on reactive

194

oxygen species (Figure 2) showed that the antioxidant capacity was highest in

195

fruiting bodies from FJ, and similar in SC and most of ZJ samples (p >0.05), with

196

ORAC values between 32-52 µmol TE/g polysaccharides. The reducing power serves

197

as a significant indicator of potential antioxidant activity, and this was found to be

198

similar in fruiting bodies from most of ZJ samples and SC, and to be the lowest in FJ

199

(p >0.05), in the range of 5-11 µmol TE/g polysaccharides. Variation in antioxidant

200

activities of extracts from different sources of F. velutipes fruiting bodies indicated a

201

structural dissimilarity of polysaccharides, which might impact other biological

202

activities of F. velutipes fruiting bodies .9, 10

203 204

The quality of polysaccharides in fruiting bodies can not be determined from

205

mushroom appearance alone, and existing high-throughput methods are unable to

206

differentiate between authentic and foreign sources because distinguishing between

207

complex polysaccharide structures is highly challenging. Therefore, alternative

208

approaches are needed for quality control of F. velutipes fruiting bodies.

209 210

FT-IR Fingerprinting: Similarity Analysis and PCA. The spectroscopic

211

fingerprints of non-hydrolyzed polysaccharide extracts from 10 batches of qualified

212

ZJ samples were analyzed by FT-IR, and the ZJ-7 fingerprint is shown as an example

10


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213

(Figure 3a). The absorption peaks at 3400, 2944, and 1420 cm−1 correspond to O–H,

214

C–H and carboxyl C–O group stretching, whereas the band in the region of 1246 cm−1

215

results from a O–H deformation vibration. Stretching peaks at 934, 885, and 882 cm−1

216

may derive from β-glycosidic linkages between the sugars, and water molecules

217

bound to the polysaccharides contributed to the absorption band at 1643 cm−1. FT-IR

218

chromatographs of SC and FJ samples (Figures 3b and 3c) were similar to that of ZJ,

219

but key differences in the transmission % of some peaks were apparent.

220

The similarity of 12 separate batches of F. velutipes fruiting bodies from various

221

sources was evaluated by calculating the correlation coefficient with the original data.

222

All spectra were exported to Excel 2010 and the correlation coefficient r was

223

calculated. The fingerprints of SC and FJ were compared with the standard profiles

224

derived from ZJ samples. A value of 1 indicates a perfect correlation with the standard

225

profile.13 For 10 batches of ZJ samples, r values varied between 0.973 to 0.996,

226

indicating a high degree of similarity (r >0.9). The r values for SC and FJ samples

227

were 0.788 and 0.828, respectively, indicating significant differences from the ZJ

228

samples.

229

Principal component analysis was applied to confirm the discrimination of sources

230

of F. velutipes fruiting bodies based on FT-IR fingerprint data of raw polysaccharide

231

extracts. On the basis of Kaiser’s stopping rule, only factors with eigenvalues over 1

232

were considered in the analysis.21 The 10 characteristic chromatographic peaks

233

(Figure 3) were processed using PCA, and factors 1 and 2 had eigenvalues of 8.60

234

and 1.07, representing 96.7% of the cumulative variance. Therefore, factors 1 and 2

11



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235

were used to visualize data inhomogeneity. A two-dimensional PCA plot of IR

236

spectroscopic fingerprints after data pretreatment was constructed in Figure 4.

237

Scattered small triangles representing PCA scores showed that the authentic samples

238

(ZJ-1 to ZJ-10) were clustered closely, whereas the SC and FJ samples were much

239

diffuse. This suggests that PCA of IR fingerprint data could be used to discriminate

240

authentic and foreign sources of F. velutipes fruiting bodies.

241 242

HPLC Fingerprinting: Similarity Analysis and PCA. The monosaccharide

243

composition of polysaccharides of F. velutipes fruiting bodies was investigated using

244

HPLC following a complete acid hydrolysis, and revealed the presence of glucose,

245

mannose, galactose, ribose, and xylose (Figure 5). This profile differed significantly

246

from mycelial tissue, in which glucose was the only monosaccharide identified.14

247

Glucose, with a molar ratio of 85.04%, was the predominant monosaccharide,

248

followed by mannose (6.81%), galactose (6.09%), xylose (1.65%), and ribose

249

(0.39%). The fruiting bodies of F. velutipes were also previously reported to contain

250

arabinose22 and fructose.23

251

Similarity analyses were applied to HPLC fingerprints following partial acid

252

hydrolysis since there were too few peaks in the HPLC fingerprints after complete

253

acid hydrolysis. The HPLC profiles of 12 batches of F. velutipes fruiting bodies and

254

the mean chromatogram from the 10 ZJ batches (Figure 6a) revealed 17 characteristic

255

peaks suitable for similarity analysis. The correlation coefficients of the 10 ZJ batches

256

were calculated using the Similarity Evaluation System for Chromatographic

12


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Fingerprint of Traditional Chinese Medicine software, and ranged from 0.930 to

258

0.998. The r values for SC and FJ samples were 0.955 and 0.982, respectively

259

(Figures 6b and 6c), suggesting the similarity software was not able to discriminate

260

between the sources.

261

PCA was applied to the HPLC profiles from pre-column PMP derivatization after

262

partial acid hydrolysis. The relative peak area of 17 characteristic chromatographic

263

peaks (Figure 6) was used for PCA, and three factors had eigenvalues of 7.18, 2.22,

264

and 1.49,

265

three-dimensional PCA plot of HPLC chromatographic profiles was constructed in

266

Figure 7. Scattered dots representing the PCA scores showed that the qualified

267

samples (ZJ-1 to ZJ-10) were clustered closely in geometric space, whereas the SC

268

and FJ samples were much diffuse. This suggests that PCA of partially hydrolyzed

269

PMP-derivatives could be used to discriminate between authentic and foreign sources

270

of F. velutipes fruiting bodies.

representing 90.7% of the cumulative

variance.

Therefore, a

271 272

Spectroscopic and chromatographic fingerprints using supervised/ unsupervised

273

data analysis techniques including similarity parameters (i.e. correlation coefficient)

274

and principal component analysis have been applied to detect foreign sources of F.

275

velutipes fruiting bodies. IR spectroscopic fingerprints using correlation coefficient

276

and principal component analysis proved capable of discriminating sources of F.

277

velutipes

278

TCM-supervised method could not discriminate between sources, but principal

fruiting

bodies.

HPLC

chromatographic

fingerprints

using

the

13



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279

component analysis of HPLC fingerprints could detect the foreign sources. Generally

280

IR fingerprinting with unsupervised chemometrics is potent to be a reliable

281

identification and qualification method suitable for quality control of F. velutipes

282

fruiting bodies. PCA is a widely used mathematical approach for reducing the

283

dimensionality of original data by introducing a small number of underlying factors

284

without losing too much information. In this study, PCA is practicable for analyzing

285

both IR spectroscopic and HPLC chromatographic fingerprint data to determine the

286

quality of polysaccharides between different sources of F. velutipes fruiting bodies.

287

These findings should be taken into account for quality control of F. velutipes fruiting

288

bodies or foods rich in bioactive polysaccharides.

289 290 291 292

FUNDING This study was founded by the National Nature Science Foundation of China (Grant No. 31371756).

293 294

REFERENCES

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(20) Liang, X. R.; Wu, H.; Su, W. K., A Rapid UPLC-PAD Fingerprint Analysis of Chrysanthemum morifolium Ramat Combined with Chemometrics Methods. Food Anal Method 2014, 7, 197-204. (21) Kaiser, H. F., The application of electronic computers to factor analysis. Educational and Psychological Measurement 1960, 20, 141-151. (22) Otagiri, K.; Ohkuma, T.; Ikekawa, T.; Tanaka, S., Intensification of antitumor-immunity by protein-bound polysaccharide EA6, derived from Flammulina velutipes (Curt. ex Fr) Sing. combined with murine leukemia L1210 vaccine in animal experiments. J. Pharm. Dyn. 1983, 6, 96-104. (23) Mukumoto, T.; Yamaguchi, H., The chemical structure of a mannofucogalactan from the fruit bodies of Flammulina velutipes (Fr.) Sing. Carbohydr. Res. 1997, 59, 614-621.

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Figure captions

366

Figure 1. Flowchart of material pretreatment, extraction, processing, spectroscopic

367

and chromatographic fingerprinting, and data handling. *Similarity Evaluation

368

System for Chromatographic Fingerprint of Traditional Chinese Medicine.

369

Figure 2. ORAC and FRAP values of polysaccharides from F. velutipes fruiting

370

bodies. Grey columns indicate ORAC values and white columns represent FRAP

371

values. The ZJ-1 to ZJ-10 represent antioxidant values of ten batches of the authentic

372

source; SC and FJ are the antioxidant values of one each of two foreign sources,

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respectively.

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polysaccharides. Tests were conducted in triplicate for each batch, with mean values

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shown and standard deviations depicted by vertical bars. Shown in the same color,

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columns marked with different letters are significantly different (p

Multiple-Fingerprint Analysis for Investigating Quality Control of

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