International Journal of Biological Macromolecules 115 (2018) 221–226

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International Journal of Biological Macromolecules

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Purification and structural elucidation of a water-soluble polysaccharidefrom the fruiting bodies of the Grifola frondosa

Anqiang Zhang a,⁎, Jiaying Deng a, Shuying Yu a, Fuming Zhang b,c,d, Robert J. Linhardt b,c,d, Peilong Sun a

a Department of Food Science and Technology, Zhejiang University of Technology, Hangzhou 310014, Chinab Department of Chemistry and Chemical Biology, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY 12180, USAc Department of Chemical and Biological Engineering, Biological Science and Biomedical Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy,NY 12180, USAd Department of Biological Science and Biomedical Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY 12180, USA

⁎ Corresponding author.E-mail address: zhanganqiang@zjut.edu.cn (A. Zhang)

https://doi.org/10.1016/j.ijbiomac.2018.04.0610141-8130/© 2018 Elsevier B.V. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 2 October 2017Received in revised form 9 April 2018Accepted 11 April 2018Available online 13 April 2018

Grifola frondosa is a polypore mushroom, which has been used for many centuries by traditional Chinese andJapanese herbalists as a medicinal mushroom. A water-soluble polysaccharide (code as GFP30-2-a, molecularweight: 2.04 × 106 Da) was isolated from the fruiting bodies of G. frondosa by hot water extraction, ethanol pre-cipitation and chromatography. Its structure was elucidated from its monosaccharide composition, methylationanalysis, together with 1D NMR (1H NMR and 13C NMR) and 2D NMR (COSY, TOCSY, HSQC, HMBC and NOESYspectra). GFP30-2-a consists of D-Glc and D-Gal in the molar ratio of 1:0.098 and the structure of the repeatingunits was identified to be β D Glcp (1→ [4) α D Glcp (1→ 4)α D Glcp (1]m → 4)α D Glcp.

© 2018 Elsevier B.V. All rights reserved.

Keywords:Grifola frondosaPolysaccharidePurificationStructural elucidation

1. Introduction

Grifola frondosa has been used as an important medicinal mushroom,a food delicacy, and a food supplement in ancient and modern Asia [1].Many reports described that G. frondosa as a medicinal and edible mush-room boosts the immunological system [2]. Over the past several de-cades, many researchers have ascribed a variety of biological activitiesto the polysaccharides isolated and purified from G. frondosa, includingantitumor and immunomodulatory activities [3], anti-oxidative activity[4], and hypoglycemic activity [5], and effects on hematopoietic stemcells [6] and skin [7]. These studies have motivated scientists to pursuethe isolation and structural identification of G. frondosa polysaccharides.Ohno and colleagues reported G. frondosa polysaccharides obtainedfrom the hotwater contained a large amount of (1→ 4)α glucans [8]. Re-search, on the structure-function relationship of highly purified andwell-characterized polysaccharides fromG. frondosa, is needed. The aim of thispaper is to fully elucidate the structure of GFP30-2-a using multiplemethods includingmonosaccharide composition andmethylation analy-sis in conjunction with 1D NMR and 2D NMR. These structural datashould further contribute to understanding the relationship betweenthe structural properties and biological activities.

.

2. Materials and methods

2.1. Materials

Dried fruiting bodies were provided by Hangzhou Baishanzu Biologi-cal Technology co., Ltd. (Hangzhou, China) and identified by Prof.Weiming Cai (Zhejiang Academy of Agricultural Sciences, Hangzhou,China). DEAE-Sepharose Fast Flow and Sephacryl S-300 High-Resolutionwere purchased fromGEHealthcare. Dextrans andmonosaccharide stan-dards (D-Gal, L-Fuc, L-Rha, D-Man, D-Xyl, D-Glc) were from Sigma. HPLCwas carried out on a waters 1525 HPLC system (1525 HPLC Pump, 2414Refractive Index Detector). GC analysis was carried out using an Agilent7890A instrument. GC–MS analysis was carried out using a Aglient7890A /5975C and NMR spectra were determined with a Varian INOVA500. All the other reagents were of analytical reagent grade and made inChina.

2.2. Isolation and purification of polysaccharide

After pretreatment by immersion in 95% alcohol overnight (12 h)three-times, the dried fruiting bodies of G. frondosa (1000 × g) were ex-tracted three-times (2 h each time) with boiling water. The aqueous ex-tracts were combined and concentrated into one-tenth of the originalvolume, and 95% (v/v) alcohol was added slowly until the final alcoholconcentration reached 30% (v/v). The mixture was kept overnight in an

222 A. Zhang et al. / International Journal of Biological Macromolecules 115 (2018) 221–226

explosion-proof refrigerator at 4 °C to precipitate the polysaccharides.The precipitate was recovered by centrifugation and defined as GFP30.This material was dissolved in water and the insoluble residue wasremoved by centrifugation. The supernatant containing solublepolysaccharide, was applied to a DEAE-Sepharose Fast Flow column (XK26 mm × 100 cm),and eluted with water, followed stepwise elutionwith 0.1, 0.2, 0.4, 0.6 and 2 mol/L aq. NaCl. Fractions were collectedusing a fraction collector and compounds were detected by thephenol sulfuric acid method, and the GFP30-2, obtained by elutionwith 0.1 mol/L NaCl, was further purified using Sephacryl S-300 (XK26 mm × 100 cm) High-Resolution chromatography. The main fractionwas collected, concentrated, and lyophilized to obtain a white solidconsisting of purified G. frondosa polysaccharide (GFP30-2-a).

2.3. Homogeneity and molecular weight

The homogeneity and molecular weight of GFP30-2-a was deter-mined by HPLC on a Waters 1525 system equipped with a TSK-gelPWXL G5000 column and a refractive index detector (RID), eluted withdistilled water as themobile phase at a flow rate of 0.8mL/min. The col-umnwas kept at 30.0±0.1 °C. The linear regressionwas calibratedwithT-series Dextran standards (Mw 1 k, 5 k, 12 k, 80 k, 150 k, 270 k, 670 k,1400 k and 2000 kDa). The samplewas prepared as 2.0mg/mL solution,and 20 μL of solution was analyzed.

2.4. Spectroscopic methods

Fourier-transform infrared (FT-IR) spectra of the polysaccharideswere recorded with a Nicolet 6700 FT-IR spectrometer [9] using theKBr disk method.

2.5. Monosaccharide composition analysis

GFP30-2-a (2 mg) was hydrolyzed with 2 mol/L trifluoroacetic acid(TFA, 4 mL) at 110 °C for 2 h. The excess acid was removed by vacuumevaporationwithmethyl alcohol (MeOH) after the hydrolysis. The hydro-lysate was converted into its alditol acetates [10]. Then the hydrolyzedproducts were reduced with NaBH4 (30 mg) and acetylated with aceticanhydride, and the standard monosaccharide was derived with same

Fig. 1. The high-performance size exclusion chromatography profi

method for 6 repeat and parallel experiments. The acetylated derivativesof GFP30-2-a were analyzed by gas chromatography (GC) using anAgilent 7890A instrument equipped with an HP-5 capillary column(30 m × 0.25 mm × 0.25 μm) a flame-ionization detector (FID). Thetemperature program was as follows: increasing from 120 °C (1 min) to240 °C at 10 °C/min and holding at 240 °C for 5 min. The temperaturesof both injector and detectorwere set at 250 °C. Nitrogenwas used as car-rier gas.

2.6. Methylation analysis

GFP30-2-a (2 mg) was dissolved in 0.2 mL of dimethyl sulfoxide(DMSO) and methylated by treatment with NaOH/DMSO (0.2 mL) sus-pension and iodomethane (0.2 mL) by the method [11]. Complete meth-ylation was confirmed by the disappearance of the OH band(3200–3700 cm−1) in the IR spectrum. The reaction mixture was ex-tracted with CHCl3, and the solvent was then removed by evaporation.The permethylated polysaccharide was hydrolyzed by treatment withHCO2H (88%, 3 mL) at 100 °C for 3 h, evaporated to dryness and furtherhydrolyzed with 2 M TFA (4 mL) at 100 °C for 6 h. The partially methyl-ated sugar in the hydrolysate were reacted with NaBH4 and acetylatedwith AC2O, and the methylated alditol acetates were analyzed by GC–MS. The applied temperature programwas as follows: oven temperaturewas initially set at 120 °C, increasing to 240 °C at a rate of 10 °C/min andthen held at 240 °C for 6.5 min. The injector and detector heater temper-atures were kept at 250 °C. Helium was used as the carrier gas.

2.7. NMR analysis

GFP30-2-a (30 mg) was exchanged with deuterium by lyophiliz-ing three-times from D2O (0.5 mL). GFP30-2-a was finally dissolvedin 0.5 mL D2O for NMR analysis. The 1H NMR and 13C NMR (25 °C)spectra were determined in 5-mm tubes using a Varian INOVA 500NMR spectrometer. 1H chemical shifts were referenced to residualHOD at δ 4.78 ppm (25 °C) as the internal standard. 13C chemicalshifts were determined to DSS (δ 0.00 ppm) as external standard.COSY, TOCSY and HMQC spectra were used to assign the signals.HMBC and NOESY spectra were used to assign inter-residue linkagesand sequences.

le of the polysaccharide GFP30-2-a isolated from G. frondosa.

4000 3500 3000 2500 2000 1500 1000 500

70

75

80

85

90

95

100

T%

Wavenumbers(cm-1)

3412.8

2929.7

1637.0

1420.81367.0

1153.9

1081.1

1023.5

927.6

896.7512

Fig. 2. The FT-IR spectrum of the polysaccharide GFP30-2-a isolated from G. frondosa.

223A. Zhang et al. / International Journal of Biological Macromolecules 115 (2018) 221–226

3. Results and discussion

3.1. Purification and physicochemical properties of GFP30-2-a

A water-soluble polysaccharide named as GFP30-2-a was ob-tained from the fruiting bodies of G. frondosa by anion exchangeand gel-permeation chromatography, which was shown to be a ho-mogeneous polysaccharide by the observation of a single symmetri-cal peak by high-performance liquid chromatography on a TSK GelG5000 PWXL column (Fig. 1). Its molecular weight was determinedas about 2.04 × 106 Da based on a calibration curve of dextran stan-dards. The absence of absorption at 280 nm and 260 nm in a UV scan-ning spectrum indicated GFP30-2-a contained no protein or nucleicacids.

The FT-IR spectrum (Fig. 2) of GFP30-2-a showed the characteris-tic absorptions for the polysaccharide. A broad and intense IR band at

Fig. 3. The GC profile of the mixe

3412.8 cm−1 was assigned to hydroxyl stretching vibration and theband at 2929.7 cm−1. The C\\H stretching vibration band occurredat 2929.7 cm−1 and bending vibration peaks around 1367.0 cm−1.The stretching vibrations of C\\O\\C and C\\O\\H contributed theabsorption peaks in the range from 1000.0 to 1200.0 cm−1. Further-more, the peak at 1637.0 cm−1 was ascribed to a small amount of as-sociated water. The absorption bands at 896.7 cm−1 and 858.5 cm−1

suggested that GFP30-2-a contained the sugar moieties with α- andβ-anomeric configurations. Absorption at about 1730.0 cm−1 wasnot observed, demonstrating GFP30-2-a was free of uronic acids.

3.2. Structural analysis of GFP30-2-a

The results of acetylation analysis of standard monosaccharide mix-ture are as shown in Fig. 3, and the corresponding monosaccharide andpeak time and response factor are in Table 1. RSD1 is the relative

d standard monosaccharide.

Table 1Monosaccharide analysis of mixed standard monosaccharide by GC.

Monosaccharide Retent times (min) RSD1(%) RSD2(%) Response factor

L Rhamnose 14.202 0.71 0.98 1.00

L Fucose 14.413 0.82 1.21 0.95

L Arabinose 14.511 1.02 1.41 0.79

D Xylose 14.813 1.31 1.92 0.91

D Mannose 20.120 1.23 1.21 0.79

D Glucose 20.243 0.99 0.65 0.90

D Galactose 20.544 1.27 1.01 0.62

Fig. 4. The GC profile of monosaccha

Fig. 5. The 500-MHz 1H NMR spectrum of the GFP3

224 A. Zhang et al. / International Journal of Biological Macromolecules 115 (2018) 221–226

standard deviation of six repeated tests (intra-group deviation) andRSD2 is the relative standard deviation of six parallel trials (inter-group deviation). The results showed that both RSD1 and RSD2 are lessthan 5%, indicating that acetylation analysis is feasible. The instrumentis stable and reliable, and the response factor of each monosaccharidecomponent relative to the instrument can be calculated from the per-cent content of average peak area.

The composition of GFP30-2-a determined by GC (Fig.4) as alditolacetate indicated that it consists of D-Glc and D-Gal, in the molar ratioof 1:0.098. Methylation analysis was used for identifying thesubstitution of the monosaccharide units constituting a polysaccharide.

ride composition of GFP30-2-a

0-2-a isolated from G. frondosa in D2O at 25 °C.

Fig. 6. The 500-MHz HMQC spectrum of the GFP30-2-a isolated from G. frondosa in D2O at 25 °C.

225A. Zhang et al. / International Journal of Biological Macromolecules 115 (2018) 221–226

GFP30-2-a was methylated, acetylated, and then analyzed by GC–MS.The GC–MS results showed that the glucose residue with mass mainlyfragments 43,71,87,101,117,129,143,161,173,233 showed that it ismainly 1,4-linked-Glcp, the other mass mainly fragments43,71,83,101,117,129,145,161,205 showed that it is terminal-Glcp orterminal-Galp. From the integrated areas of corresponding peaks, theratio of these linkages was calculated approximately as 1.0:0.12.

NMR analysis was implemented to further determine the structuralcharacteristics of the polysaccharide. The 1H NMR spectrum (Fig.5) ofthe polysaccharide mainly contained signals for four anomeric protonsat δ 4.56–5.31 along with the HSQC spectrum (Fig.6), which wereassigned as Residue-a (δ 5.31), Residue-b (δ 5.13), Residue-c (δ 4.87)and Residue-d (δ 4.56), respectively. Other sugar protonswere seriouslyoverlapped in the region of δ 3.18–3.91. The 13C NMR spectrum of thepolysaccharide mainly contained signals for four anomeric carbons atδ 91.88–99.77. Sugar ring.

For Residue a and b all the 1H resonances for residue awere assignedfrom the 1H\\1H COSY spectrum and confirmed from the TOCSY spec-trum. Magnetization relayed well through the spin system, as expected

Table 2Chemical shifts data for GFP30-2-a isolated from G. frondosa.a

Residue proton or carbon

1 2

→1) α D Glcp (4→ (a) HC

5.3199.77

3.4871.38

→4) α D Glcp (b) HC

5.1391.88

3.4668.47

α D Galp (1→ (c) HC

4.8798.63

3.4968.91

β D Glcp (1→ (d) HC

4.5695.66

3.1874.00

a Underlined bold numbers represent glycosylation sites.

for the gluco-configuration and all crosspeaks were clearly visible [12].On the basis of the proton assignments, the chemical shifts of C-1to C-6 were readily obtained from the HSQC spectrum. H-1 appearedas a singlet (JH-1, H-2 b 4 Hz) in the 1H NMR spectrum, and its chemicalshift was greater than 4.8 ppm, demonstrating that residue a and bhad α-configurations at their anomeric centers. The combination ofthese data identified residue a as →1) α D Glcp (4→ and residue bas →4) α D Glcp.

As to Residue c the 1H resonances for H-1,-2,-3 and H-4 wereassigned from the crosspeaks in the 1H\\1H COSY and TOCSY spectra.The H-5 resonance was assigned from the H-3/H-4 and H-4/H-5crosspeaks in the NOESY spectrum (Reddy et al., 1998). The H-5, H-6aand H-6b resonances were then obtained from the TOCSY spectrum.13C resonances were assigned from the HMQC spectrum. H-4 displaysstrong NOEs to both H-3 and H-5, which indicated that residue c wasa Gal-type residue. The H-1 appeared as a singlet (JH-1,H-2 b 3 Hz) inthe NOESY spectrum and H-1/H-2 intra-residue correlations in theNOESY spectrum indicated that residue c had an α-configuration.Thus, residue c was identified as α D Galp (1→.

3 4 5 6a 6b

3.8773.18

3.5576.74

3.6272.59

3.6860.44

3.74

3.8570.03

3.5376.82

3.6069.95

3.4260.49

3.73

3.9171.18

3.6371.32

3.5169.03

3.5760.53

3.51

3.6776.26

3.3169.59

3.3875.94

3.5060.32

3.56

Table 3Interglycosidic correlations from HMBC spectrum of GFP30-2-a isolated from G. frondosa.

Residue Proton Proton correlation a

→1) α D Glcp (4→ (a) H-1 (a; 5.31)H-4 (a; 3.55)

71.38 (a; C-2), 76.74 (a; C-4),72.59 (a; C-5)99.77 (a; C-1)

→4) α D Glcp (b) H-3 (b; 3.85)H-4 (b; 3.53)H-5 (b; 3.60)

76.82 (b; C-4)99.77 (a; C-1), 69.95 (b;C-5)60.49 (b; C-6)

α D Galp (1→ (c) H-3 (c; 3.91) 71.32 (c;C-4)β D Glcp (1→ (d) H-1(d; 4.56)

H-2 (d; 3.18)H-4 (d; 3.31)

76.74 (a; C-4)72.26 (d; C-3)75.94 (d;C-5)

a Inter-residue correlations are shown in bold font.

226 A. Zhang et al. / International Journal of Biological Macromolecules 115 (2018) 221–226

1H resonances for residue d were readily assigned from the 1H\\1HCOSY and TOCSY spectra. 13C resonances were assigned from theHSQC spectrum. The H-1 appeared as a doublet (JH-1, H-2 = 7.5 Hz) inthe 1H NMR spectrum and its chemical shift was less than 4.8 ppm,demonstrating that residue d was β-D-Glcp-(1→.

Comparison of the chemical shifts data (Table 2) for residues a–dwith those reported for glycosides [13] permitted identification of resi-due a as 1,4-linked α Glcp, residue b as terminal end 4-linked α D Glcp,residue c as reducing end α D Galp and residue d as reducing endα D Glcp.

The sequence of glycosyl residues were determined from HMBCspectral studies and confirmed by NOESY experiments. HMBC experi-ments (Table 3) showed clear correlations between H-1of residue dand C-4 of residue a, between H-1 of residue a and C-4 of residue a, be-tween H-4 of residue b and C-1 of residue a. between the H-1 to H-6 ofresidue c did not correlate to residues a, b andd. Inter-residueNOEs cor-relations (Table 4) were observed H-1 of d and H-4 of residue a, be-tween H-1 of residue a and C-4 of residue a, between H-4 of residue band H-1 residue a. the H-1 to H-6 of residue c also did not correlate toresidues a, b and d.

The combined chemical and NMR data permitted the determinationof the structure of the repeating units of the GFP30-2-a, which also con-tains a minor of terminal residue α-D-Galp.

β D Glcp (1→ [4)α D Glcp (1→4)α D Glcp (1] m → 4)α D Glcp

4. Conclusions

In this study, a water-soluble polysaccharide had been successfullyisolated from G. frondosa fruiting body by boiling-water combinedwith DEAE ion exchange and size exclusion chromatographic tech-niques. Its molecular weight was measured to be 2.04 × 106 Da byusing HPLC. Sugar composition showed that GFP30-2-a is composed ofD-Glc and D-Gal in a molar ratio of 1:0.098. Methylation analysis to-gether with 1H and 13C NMR spectroscopy and 2D NMR establishedthat GFP30-2-a is consisted of (1 → 4)α D Glcp backbone and containsa minor of terminal residue β D Galp. Future research on structuresand functions relationship of G. frondosa polysaccharides is required tostudy, whichwill help scientists to designmore functional foods for po-tential health promoting.

Table 4NOEs data for the GFP30-2-a isolated from G. frondosa.

Residue Proton Intra-correlation a

→1)α D Glcp (4→ (a) H-1 3.55 (a; H-4), 3.87 (a; H-3)→4) α D Glcp (b) H-1

H-43.46 (b; H-2), 3.85 (b, H-3)5.31 (a; H-1)

α D Galp (1→ (c) H-1H-4

3.49 (c; H-2)3.91 (c; H-3), 3.51 (c; H-5)

β D Glcp (1→ (d) H-1 3.18 (d; H-2), 3.38 (d; H-5),3.55 (a; H-4), 3.67 (d; H-3)

a Inter-residue NOEs are in bold.

Acknowledgements

The study was supported by Natural Science Foundation of ZhejiangProvince No. LY17C200017.

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