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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
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Multiple fingerprint analysis for investigating quality control of Flammulina
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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]
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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,
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INTRODUCTION
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Edible mushrooms are important both nutritionally and as a source of drug
39
candidates for use as pharmaceuticals. The golden needle mushroom or enokitake
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(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
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may also affect the bioactivities of the polysaccharides present.
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Assessing the quality of the polysaccharides in F. velutipes fruiting bodies is
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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
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widely available. Fingerprint analysis integrated with chemometrics has proved useful
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for characterization of complex molecular systems and is an identification and
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qualification technique that was approved by the World Health Organization in
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1991.11 The (dis)similarity approaches including principal component analysis (PCA)
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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
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Chemicals. Standards including glucose, ribose, mannose, galactose, xylose and
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6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox) were purchased
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from Sigma-Aldrich (Shanghai, China). 1-phenyl-3-methyl-5-pyrazolone (PMP) was
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purchased from Adamas-beta (Shanghai, China). HPLC grade acetonitrile and water
72
were purchased from Anpel (Shanghai, China). All other chemicals and solvents were
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of the highest commercial grade and were purchased from Sigma-Aldrich (Shanghai,
74
China).
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Source Materials. Ten batches of F. velutipes fruiting bodies as the authentic source
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were provided by Infinitus (China) Company Ltd. and named as ZJ samples after the
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grown location of Zhejiang in China. One each of two foreign sources from Sichuan
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and Fujian named as SC and FJ samples, respectively, were purchased at a local
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market.
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Preparation of Polysaccharide Extracts. Polysaccharides extracts were prepared
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according to the previously reported.14 The fruiting bodies of F. velutipes were
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air-dried at 60 °C for 10 h and ground into fine particles using a DS-Y250 mill
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(Dingshuai Electrics, Shanghai, China) through a 40-mesh screen. 5 g of powder in
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100 mL dd-water was stirred at 80 °C for 4 h, and the extract was cooled to room
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temperature and centrifuged at 4000 rpm for 10 min. The supernatant was collected
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and deproteinized with 60 mL of 5:1 CHCl3–n-BuOH using the Sevag method.15 The
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upper layer was collected and decolorized by adding 30% (v/v) aqueous H2O2. The
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solution was concentrated under vacuum and precipitated with three volumes of 95%
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(v/v) aqueous ethanol, and incubated at 4 °C overnight. Polysaccharide precipitates
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were obtained by centrifugation at 4000 rpm for 10 min and washed sequentially with
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acetone and ether. The polysaccharide extract was dried using nitrogen gas and stored
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in a desiccator for further FT-IR analysis.
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Oxygen Radical Absorbance Capacity (ORAC) Assay. Determination of the
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oxygen radical absorbance capacity of the polysaccharides was performed as
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previously reported 16 using an Infinite F200 PRO microplate reader (Tecan,
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Switzerland). Samples and Trolox standards were prepared in DMSO, and all other
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reagents were prepared in 75 mM phosphate buffer (pH 7.4). Briefly, each well in a
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96-well plate contained 30 µL sample (or DMSO for the blank control), and 225 µL of
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81.63 nM fluorescein. The plate was covered an incubated at 37 °C for 20 min, and 25
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µL of 0.36 M 2,2′-azobis(2-amidinopropane) hydrochloride (AAPH) was added to
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each well to start the reaction, resulting in a final total volume of 280 µL. The
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fluorescence was recorded every 5 min for 1 h (ex/em: 485/538 nm) at 37 °C. Trolox
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equivalents were calculated from the relative area under the curve compared to a
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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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Ferric Reducing Antioxidant Power (FRAP) Assay. The ferric reducing antioxidant
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power assay is based on the reduction of Fe3+-tripyridyltriazine (TPTZ) to a blue
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colored Fe2+-TPTZ.17, 18 The FRAP reagent was prepared by mixing 300 mM acetate
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buffer (pH 3.6), 10 mM TPTZ and 20 mM ferric chloride in a ratio of 10:1:1 (v/v/v).
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Then, 3 mL of FRAP reagent was added to 20 µL of sample or Trolox and incubated
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at 37 °C for 30 min. Absorbance was measured at 590 nm using an Infinite F200 PRO
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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.
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Acid Hydrolysis. Polysaccharides were hydrolyzed using two approaches. For
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complete acid hydrolysis, polysaccharides (10 mg) were hydrolyzed in 2 mL of 2 M
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trifluoroacetic acid (TFA) in an ampoule sealed under a nitrogen atmosphere and
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incubated at 110 °C for 5 h. For partial acid hydrolysis, 2 mL of 0.05 M TFA was
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added and incubated at 110 °C for only 2 h. After cooling to room temperature,
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reactions were centrifuged at 4000 rpm for 5 min, and supernatants were dried at
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reduced pressure. Methanol was twice added and evaporated under vacuum to remove
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residual TFA.
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FT-IR Spectroscopy and Chemometric Analysis. FT-IR spectra of non-hydrolyzed
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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
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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
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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.
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0.2 mL of the standards or hydrolyzed samples was mixed with 0.2 mL of a 0.5 M
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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,
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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
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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.
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Preparation of PMP Derivatives after Partial Acid Hydrolysis. Partially
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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)
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and 0.2 mL 0.3 M NaOH and stirred. Subsequent steps were as described above for
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complete acid hydrolysis.
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HPLC Analysis. Analysis of PMP derivatives was carried out on an Agilent 1260
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HPLC system (Agilent Technologies, USA). Separation was achieved using reverse
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phase elution on a 5 µm Shim-pack VP-ODS column (4.6 mm × 250 mm, 5 µm,
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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:
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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
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wavelength of 245 nm.
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Data Analysis. Antioxidant activity data are reported as means ± standard deviation.
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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
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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
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(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).
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RESULTS AND DISCUSSION
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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
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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
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(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
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were used to visualize data inhomogeneity. A two-dimensional PCA plot of IR
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spectroscopic fingerprints after data pretreatment was constructed in Figure 4.
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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
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Fingerprint of Traditional Chinese Medicine software, and ranged from 0.930 to
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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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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
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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,
373
respectively.
374
polysaccharides. Tests were conducted in triplicate for each batch, with mean values
375
shown and standard deviations depicted by vertical bars. Shown in the same color,
376
columns marked with different letters are significantly different (p
Article
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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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
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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,
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INTRODUCTION
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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
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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.
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Preparation of Polysaccharide Extracts. Polysaccharides extracts were prepared
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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
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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
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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
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added and incubated at 110 °C for only 2 h. After cooling to room temperature,
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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.
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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
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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
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(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
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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
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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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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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(1) Leifa, F.; Pandey, A.; Soccol, C. R., Production of Flammulina velutipes on coffee husk and coffee spent-ground. Braz Arch Biol Techn 2001, 44, 205-212. (2) Song, H. F.; Zhang, Q. B.; Zhang, Z. S.; Wang, J., In vitro antioxidant activity of polysaccharides extracted from Bryopsis plumosa. Carbohyd Polym 2010, 80, 1057-1061. (3) Ma, L.; Chen, H.; Zhang, Y.; Zhang, N.; Fu, L., Chemical modification and antioxidant activities of polysaccharide from mushroom Inonotus obliquus. Carbohyd Polym 2012, 89, 371-378. (4) Thetsrimuang, C.; Khammuang, S.; Sarnthima, R., Antioxidant Activity of Crude Polysaccharides from Edible Fresh and Dry Mushroom Fruiting Bodies of Lentinus sp. Strain RJ-2. International Journal of Pharmacology 2011, 7. 14
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Figure captions
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Figure 1. Flowchart of material pretreatment, extraction, processing, spectroscopic
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and chromatographic fingerprinting, and data handling. *Similarity Evaluation
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System for Chromatographic Fingerprint of Traditional Chinese Medicine.
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Figure 2. ORAC and FRAP values of polysaccharides from F. velutipes fruiting
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bodies. Grey columns indicate ORAC values and white columns represent FRAP
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values. The ZJ-1 to ZJ-10 represent antioxidant values of ten batches of the authentic
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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
