Structural Characterization of a Novel Glucan from Achatina fulica and

Jan 2, 2014 - A novel glucan designated AFPS-IB was purified from Achatina fulica (China white jade snail) by anion-exchange and gel-permeation chroma...
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Structural Characterization of a Novel Glucan from Achatina f ulica and Its Antioxidant Activity Ningbo Liao,† Shiguo Chen,*,† Xingqian Ye,† Jianjun Zhong,† Xuan Ye,§ Xinzi Yin,† Jenny Tian,∥ and Donghong Liu*,†,‡ †

College of Biosystem Engineering and Food Science, Zhejiang University, 866 Yuhangtang Road, Hangzhou, 310058 Zhejiang, China ‡ Fuli Institute of Food Science, Zhejiang University, Hangzhou, 310058 Zhejiang, China § Department of Materials, Royal School of Mines, The Imperial College of Science, Technology and Medicine, Prince Consort Road, London SW7 2BP, U.K. ∥ Department of Food Science and Agricultural Chemistry, McGill University, Montreal, Quebec H9X 3V9, Canada S Supporting Information *

ABSTRACT: A novel glucan designated AFPS-IB was purified from Achatina f ulica (China white jade snail) by anion-exchange and gel-permeation chromatography. Chemical composition analysis indicated AFPS-IB was composed of glucose, fucose, rhamnose, mannose, and galactose in a molar ratio of 189:2:1:1:2 and with an average molecular weight of 128 kDa. Its structural characteristics were investigated by Fourier transform infrared spectroscopy (FTIR), high performance liquid chromatography (HPLC), gas chromatography mass spectrometry (GC−MS), methylation analysis, nuclear magnetic resonance (NMR) spectroscopy (1H, 13C, H−H COSY, HSQC, TOCSY, and NOESY), and atomic force microscopy (AFM). The glucan mainly consisted of a backbone of repeating (1→4)-α-D-glucose residues with (1→6)-β-D glucosyl branches at random points on the backbone glucose. Antioxidant studies revealed AFPS-IB showed significant DPPH (2,2-diphenyl-1-picrylhydrazyl) radical, superoxide anion (O2−) scavenging activities and high reduction potential. This study suggested that AFPS-IB could be a new source of dietary antioxidants. KEYWORDS: glucan, Achatina f ulica, structure, antioxidant activity



human growth and development.10 Furthermore, it can serve for health care purposes because it boosts the reproductive, immune, and nervous systems.11−13 In China, A. f ulica is currently one of the standard menu items in most restaurants with a yearly output of more than 1 million tonnes. In the last 20 years, an increasing interest in A. f ulica has led to the publication of numerous studies on its nutritional value and components.10−15 Although proteins and lipids have been introduced to contribute to the positive health benefits, they are not the limiting bioactive compounds in A. fulica.14,15 Other specific bioactive compounds are present, which can also play a leading role in the health functions of A. f ulica. In recent years, glucans from mollusks have been known to be good bioactive compounds because of their potential pharmaceutical values.6−9 As an important composition of A. f ulica, the glucans should contribute to the health-beneficial activities. However, the precise chemical structure of A. f ulica glucans is still unknown. Therefore, in this work, a novel water-soluble glucan was extracted and purified from the soft body of A. f ulica for the first time. The molecular weight and chemical structure were investigated by Fourier transform infrared spectroscopy (FTIR), high performance liquid chromatography (HPLC),

INTRODUCTION Polysaccharides widely exist in plants, fungi, bacteria, algae, and animals. Together with proteins and polynucleotides, they are essential biomacromoleules in the life activities and play important roles in intercellular communication, cell adhesion, and molecular recognition in the immune system.1 In recent years, some bioactive polysaccharides isolated from natural sources have attracted much more attention due to their various biological activities affected by different chemical structures.2 Glucans, a type of neutral polysaccharides, also has attracted increasing interest because of their pharmacological activities such as antitumor, immunomodulation, and antioxidation properties.3−5 Liu et al. reported on a water-soluble α-glucan from Strongylocentrotus nudus eggs with significant antitumor activity that was composed of a main chain of (1→4)-linked D-glucopyranosyl with branching points at O-6 of (1→6)-linked 6 D-glucopyranosyl residues. Also, glucans from Bullacta exarata were reported to show antioxidant and antitumor activities.7,8 Several studies have shown that the bioactivities of glucans are highly dependent on their structural characteristics. For example, α-(1→4)-linked backbone usually serves as common food nutrients, whereas β-(1→6)-linked glucose side chains are known as essential for the antioxidant, antitumor, antiviral immunomodulatory, and anticoagulatory activities.9 Achatina f ulica (China white jade snail) is one of the mollusks that has been used as a tonic medicine for the treatment of various diseases. A. fulica contains all nutrients necessary for © 2014 American Chemical Society

Received: Revised: Accepted: Published: 2344

May 2, 2013 January 2, 2014 January 2, 2014 January 2, 2014 dx.doi.org/10.1021/jf403896c | J. Agric. Food Chem. 2014, 62, 2344−2352

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The collected fractions were further chromatographed on a Sephacryl S-300 HR gel filtration column (1.6 cm × 100 cm, Amersham Biosciences, Uppsala, Sweden) eluted with distilled water; fractions were collected and lyophilized to give a white powder consisting of polysaccharides designated AFPS-IA, AFPS-IB, and AFPS-IC. AFPS-IB was subjected to subsequent analyses. The whole procedure is shown in Figure S1. Homogeneity and Molecular Weight Determination. The homogeneity and molecular weight of AFPS-IB were determined by high performance size exclusion chromatography (HPSEC) on a TSKGel G4000 PWXL column (300 mm × 7.5 mm, Tosoh Biosep, Japan), with a column temperature of 40 °C. The polysaccharides were eluted with 0.2 M NaCl at a flow rate of 1.0 mL min−1 and detected by a refractive index detector (Water 2414). Preliminary calibration of the column was performed using dextrin in a range of molecular weights measured in kDa (500, 66.9, 48, 20, 5; Showa-Denko, Japan). Monosaccharide Composition Analysis. The monosaccharide composition was determined using a PMP-HPLC method.20 In brief, polysaccharides (typically 1 mg) were hydrolyzed with 2 M trifluoroacetic acid (TFA) at 110 °C under nitrogen for 8 h with lactose added as an internal standard. The monosaccharide hydrolysate was dried under vacuum and then derivatized with 450 μL of 1-phenyl-3methyl-5-pyrazolone (PMP) solution (0.5 M, in methanol) and 450 μL of 0.3 M NaOH at 70 °C for 30 min. The reaction was stopped by neutralization with 450 μL of 0.3 M HCl before extraction with chloroform (3 × 1 mL). HPLC analyses were performed on an Agilent ZORBAX Eclipse XDB-C18 column (5 μm, 4.6 mm × 150 mm) at 25 °C and UV detection at 250 nm. The mobile phase was 0.05 M KH2PO4 (pH 6.9) with 15% (solvent A) and 40% (solvent B) acetonitrile in water. A gradient of B from 8% to 19% in 25 min was used. Sulfate content was determined by the BaCl2/gelatin method and ionexchange chromatography.21 Methylation Analyses. The polysaccharide fraction (0.5 mg) was dissolved in DMSO (0.5 mL) and O-methylated using two consecutive cycles of NaOH−MeI.22 The per-O-methylated product showed no band in the region of 3600−3300 cm−1. It was then hydrolyzed with 45% (v/v) formic acid at 100 °C for 15 h. The hydrolyzed product was evaporated to dryness, and the residue was then reduced with NaBH4 and acetylated with acetic anhydride to obtain a mixture of partially O-methylated alditol acetates. Qualitative and quantitative analyses were conducted by gas chromatography−mass spectrometry (GC-MS) (Finnigan trace, Thermo Electron Finnigan Co. Ltd., USA) with helium as the carrier gas (2 mL min−1). A capillary column of OV1701 (30 m × 0.25 mm ID, 0.25 μm film thickness; Supelco) was held at 150 °C during the injection and then programmed to increase at 3 °C min−1 to 250 °C (constant temperature). The resulting partially O-methylated alditol acetates were identified by their typical retention times and electron impact spectra. UV, IR, and NMR Analyses. The UV−vis absorption spectra were recorded using a UV-2550 spectrophotometer (Shimadzu, Japan) in the wavelength range of 200−800 nm. The FT-IR spectrum (KBr pellets) of the polysaccharide (2 mg) was recorded on a Nicolet 5700 FTIR (Thermo Electron, Madison, WI, US) in a range of 400−4000 cm−1 at room temperature. For NMR analysis, polysaccharide-containing fractions (about 50 mg) were lyophilized in D2O (99.8%) twice and dissolved in 500 μL of high-quality D2O (99.96%) containing 0.1 μL of acetone. 1 H, 13C NMR experiments were carried out at 600 MHz on a Bruker Avance III 600 spectrometer (Bruker BioSpin Corporation, Fallanden, Switzerland). Spectra were recorded at 60 °C to place the HDO signals with minimal disturbance to carbohydrate protons. The observed 1H chemical shifts were reported relative to the internal acetone (2.23 ppm). Standard homo- and heteronuclear correlated 2D techniques were used for general assignments of the polysaccharide (AFPS-IB): COSY, TOCSY, NOESY, and HSQC.23 Spectra were also carried out at 60 °C. Atomic Force Microscopy (AFM). AFPS-IB was imaged using AFM according to the method described by Sun and Zhang (2006).24 A stock solution (10 mg mL−1) was prepared by adding some AFPS-IB into double-distilled H2O. The solution was diluted to a final

gas chromatography mass spectrometry (GC−MS), methylation analysis, nuclear magnetic resonance (NMR) spectroscopy (1H,13C, H−H COSY, HSQC, TOCSY, and NOESY), and atomic force microscopy (AFM). In addition, in vitro antioxidant activity was investigated by the reducing power, superoxide anion scavenging activity, and the DPPH radical scavenging activity assays.



MATERIALS AND METHODS

Materials and Reagents. The 6-month-old Achatina f ulica (China white jade snails) with an average shell size of 3−5 cm were supplied by the Huzhou Fenren Company (Zhejiang, China). Table 1 shows the composition of A. f ulica soft body in this study.

Table 1. Composition of Achatina fulica

a

composition

wt % in A. f ulica soft part

carbohydrateb proteinb lipidb moisturec ashb

25.75 (0.527)a 71.58 (0.871) 3.76 (0.136) 78.15 (1.673) 5.04 (0.362)

Data were shown as mean (SD), n = 3. bDry weight. cFresh weight.

Monosaccharide standards and disaccharide lactose were purchased from Sigma (St. Louis, MO, USA). Papain (1200 U g−1) and L-cysteine hydrochloride (Cys) were purchased from Fluka (Seelze, Germany). The derivatization reagent 1-phenyl-3-methyl-5-pyrazolone (PMP) was from Sinopharm Chemical Reagents (Shanghai, China). 2,2-Diphenyl-1-picryl-hydrazyl (DPPH), ferrozine, nitroblue tetrazolium (NBT), phenazine methosulfate (PMS), reduced nicotinamide adenine dinucleotide (NADH), and vitamin C were purchased from Sigma-Aldrich Co. All the other reagents used were of analytical grade. Composition Analysis of Achatina f ulica Soft Body. The whole soft body of A. f ulica was oven-dried to constant weight (102 °C for 18−24 h) to determine moisture contents. Lipids were extracted in baths of diethyl ether,16 and protein contents were determined by the Lowry method.17 Ash contents were determined by combusting extracted samples in a muffle furnace at 550 °C for 8 h, and carbohydrates were analyzed using the phenol sulfuric acid method of Dubois et al.18,19 Isolation and Purification of AFPS-IB. Polysaccharides were isolated using a previously described procedure with some modifications.8 The shell of A. f ulica was removed, and fresh muscle was freeze-dried and ground to a powder, which was then defatted using acetone for 24 h. Five grams of defatted powder was suspended in 50 mL of 0.1 M sodium carbonate buffer (pH 6.0). The suspension was shaken for 24 h at 200 rpm and 60 °C after the addition of papain (250 mg), EDTA (73.1 mg), and Cys (39.4 mg). The supernatant was precipitated with four volumes of ethanol at 4 °C overnight and then centrifuged (Beckman Model J-21B centrifuge, Beckman Inst, Inc., Palo Alto, CA, USA) for 30 min at 8000 × g. The precipitate was washed sequentially with alcohol and acetone and then dissolved in 10 mL of ice-cold distilled water. The digestion mixture was cooled to 4 °C, and trichloroacetic acid was added to a final concentration of 5% (w/v). The sample was mixed, allowed to stand for 1 h, and then centrifuged for 20 min at 8000 × g. The supernatant was recovered by decanting before mixing with three volumes of ethanol. The suspension was stored overnight at 4 °C and then centrifuged for 30 min at 8000 × g. The precipitate was washed with absolute ethanol, dissolved in water, and dialyzed against 100 volumes of water. The dialyzate was freeze-dried to obtain crude polysaccharide AFPS. AFPS was dissolved in distilled water, applied to a DEAE-Sephacel column (2.6 cm ×30 cm, Whatman, Brentford, UK), and eluted with distilled water, followed by 0.1 M NaCl and 0.5 M NaCl, respectively. Each fraction of 5 mL was collected at a flow rate of 30 mL h−1 and monitored by the phenol-sulfuric acid method at 490 nm.19 2345

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concentration of 1 μg mL−1. Approximately 5 μL of diluted AFPS-IB solution was dropped on the surface of a mica sample carrier, allowed to dry, and then imaged in air at room temperature. Scanning probe microscopy images were obtained using a Nano Scope III atomic force microscope (Digital Instruments, Santa Barbara, CA, USA) and collected by tapping mode atomic force microscopy (TmAFM). All measurements were performed in air at ambient pressure and 30−40% relative humidity (RH). Reducing Power Assay. The reducing power was determined using the method of Li, Zhou, and Li (2007), with some modifications.25 Samples of 1 mL of different concentrations (2−12 mg mL−1) in phosphate buffer (0.2 M, pH 6.6) were mixed with 1 mL of potassium ferricyanide (1%, w/v) and incubated at 50 °C for 20 min. The reaction was terminated by the addition of 1 mL of TCA (10%, w/v), the solution was then mixed with 0.2 mL of ferric chloride (0.1%, w/v), and the absorbance was measured at 700 nm. Increased absorbance of the reaction mixture indicated greater reducing power. Vitamin C was used as reference material. Reducing power was expressed as a percentage of the activity of a 1 mM solution of vitamin C. Superoxide Radical Scavenging Capacity. The superoxide radical scavenging activity assay was performed using the photoreduction of the NBT (nitroblue tetrazolium) method as described by Li, Zhou, and Han (2006), with some modifications.26 Superoxide radicals were generated in 3 mL of phosphate buffer (0.1 M, pH 7.4) containing 156 μM NADH (reduced form), 52 μM NBT, 20 μM phenazin methosulfate, and varying concentrations of AFPS-IB (2− 12 mg mL−1). The color reaction of superoxide radicals and NBT was detected by monitoring the absorbance at 560 nm. Vitamin C was used as reference material. In the essential control, NADH was substituted with phosphate buffer. The inhibition percentage was calculated using the following formula: Scavenging effect (%) = (AControl 560 − ASample 560)/AControl 560 × 100, where A is the absorbance. DPPH Radical Scavenging Capacity. The DPPH radical scavenging activity was measured using the method reported by Li, Zhou, and Han (2006) with slight modifications.26 Briefly, 0.2 mL of DPPH· solution (400 μM in dehydrated alcohol) was added to 1.0 mL of sample solution, and then 2.0 mL of water was added. The mixture was shaken vigorously for 2−3 min and allowed to stand at room temperature in the dark for 30 min. The absorbance was measured at 517 nm against a blank (water instead of sample and DPPH solution). Vitamin C was used as reference material. The scavenging percentage was calculated by the following equation: Scavenging effect (%) = (AControl 560 − ASample 560)/AControl 560 × 100, where A is the absorbance. Statistical Analysis. Data were reported as the mean ± standard deviation (SD) (n = 3) and evaluated by one-way analysis of variance (ANOVA) followed by the Student’s t-test. Differences were considered to be statistically significant if P < 0.05. All statistical analyses were carried out using SPSS for Windows, Version 16.0 (SPSS Inc., Chicago, IL, USA).

Figure 1. Purification of AFPS-IB. (a) The crude extract was fractionated on a DEAE ion-exchange column and (b) the collected fractions were further purified by gel filtration chromatography on a Sephacryl S-300 HR column.

a single peak on the HPSEC molecular weight profile, corresponding to an average molecular weight of 128 kDa. Monosaccharide composition analysis of AFPS-IB showed that it consisted of Glc, Fuc, Rha, Man, and Gal in a molar ratio of 189:2:1:1:2, respectively. The presence of glucose as the main monosaccharide indicated that AFPS-IB was a glucan. The sulfate content of AFPS-IB was estimated at 2.7% (Table 2). UV and IR Spectrum of AFPS-IB. The polysaccharide AFPS-IB showed negative response to the Lowry test and no absorption at 280 or 260 nm in the UV−vis spectrum, indicating the absence of protein and nucleic acids. Figure 3 shows the IR spectra of the polysaccharide fraction AFPS-IB of A. f ulica. The bands in the region of 3428 cm−1 were assigned to the hydroxyl stretching vibration of the polysaccharide, and those in the region of 2929 cm−1 were due to C−H stretching vibration. A strong band between 950 and 1160 cm−1 in the FT-IR spectrum of AFPS-IB was attributed to the stretching vibrations of the pyranose ring. Absorptions at 917 cm−1 were typical for D-glucose in the pyranose form.27 A characteristic absorption band at 843 cm−1 was also observed, indicating the α-configuration of the sugar units as described previously.28 Structure Data from Methylation Analysis. AFPS-IB was methylated and hydrolyzed to form alditol acetate derivatives, which were prepared and analyzed by GC−MS. As shown in Table 3, the analysis identified 1,5-di-O-acetyl-2,3,4,6tetra-O-methyl-D-glucitol, 1,4,5-tri-O-acetyl-2,3,6-tri-O-methylD-glucitol, and 1,4,5,6-tri-O-acetyl-2,3-di-O-methyl-D-glucitol in a molar ratio of 1:10:1, which indicated the presence of nonreducing-end D-glucopyranosyl, (1→4)-linked D-glucopyranosyl, and (1→4,6)-linked D-glucopyranosyl. The high proportion of (1→4)-linked D-glucopyranosyl indicated AFPS-IB mainly consisted of a (1→4)-linked D-glucopyranosyl backbone. The same proportion of nonreducing-end D-glucopyranosyl and (1→4,6)-linked D-glucopyranosyl indicated the appearance of a



RESULTS AND DISCUSSION Isolation, Purification, and Composition of AFPS-IB. The nutritional ingredients of Achatina f ulica (China white jade snail) are shown in Table 1. Twenty to thirty percent of carbohydrates are included in A. f ulica. The crude polysaccharides (AFPS) were isolated from A. f ulica soft body with a yield of 15.54% (dry weight), which were further separated by ion exchange chromatography on a DEAE-52 column. The polysaccharide fractions (AFPS-I) were pooled and further purified by Sephacryl S-300 HR gel-permeation chromatography (Figure 1a). Three major water-soluble factions, AFPS-IA, AFPS-IB, and AFPS-IC, were collected with yields of 1.12, 5.23, and 2.76% of the dry material, respectively (Figure 2b). Fraction AFPS-IB, which exhibited a larger peak area in the elution profile, was collected for further study. The homogeneity and average molecular weight of AFPS-IB were analyzed by the HPSEC method (Figure 2c). AFPS-IB appeared as 2346

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Figure 2. HPSEC profile of AFPS-IB. Molecular weight determination by HPLC on a TSK-Gel G 4000 SWXL stainless steel column (300 × 7.5 mm) with 0.2 M NaCl at 1 mL/min. Range of molecular weights in kDa, 500; 66.9; 25.

Table 2. Yields, Protein Contents, Sugar Contents, Sulfate Contents, and Mw of Achatina fulica Polysaccharidesa samples yield (%) protein (%) neutral sugar (%) sulfate (%) Mw (kDa) Molar Ratio of Monosaccharides Rha Man Glc Gal Fuc GalN GlcN

AFPS

AFPS-I

AFPS-IA

AFPS-IB

AFPS-IC

15.5(2.3)b 12.3(1.8) 80.6(3.1) 3.2(0.7) --c

8.3(1.3) 9.3(1.9) 87.9(2.8) 3.6(0.2) --c

1.1(0.3) 2.6(0.5) 93.7(3.5) 2.8(0.4) 373(2.6)

5.2(0.8) ND 95.4(5.8) 2.7(0.4) 128(3.8)

2.2(0.5) ND 94.3(1.4) 1.3(0.2) 127(1.7)

6.34(0.3) 1.89(0.2) 13.8(3.5) 28.7(5.7) 16.3(2.7) 8.95(1.7) 25.8(5.6)

5.7(1.2) 2.12(0.4) 12.3(2.7) 25.3(4.3) 16.4(3.4) 7.4(2.3) 36.4(6.3)

0.7(0.1) ND 1.3(0.8) 12.5(2.6) 0.6(0.2) 1.2(0.3) 4.1(1.2)

0.2(0.04) 0.1(0.03) 18.9(4.3) 0.2(0.06) 0.1(0.03) ND ND

ND ND 5.4(1.8) 15.3(2.3) 3.2(1.1) ND 21.7(6.2)

a

AFPS, crude polysaccharide; AFPS-I, separated by ion exchange chromatography (DEAE-52); AFPS-IA, AFPS-IB, AFPS-IC, purified by gelpermeation chromatography (Sephacryl S-300 HR); ND, not detected; Man, mannose; Rha; rhamnose; Glc, glucose; Gal, galactose; Fuc, fucose; GalN, N-acetylgalactosamine; GlcN, N-acetylglucosamine. bData were shown as mean (SD), n = 3. cThe polysaccharide was a mixture.

Figure 3. Infrared spectrum (IR) of the polysaccharide fraction AFPS-IB from Achatina f ulica.

(AFM) is useful to obtain more information about the structure and properties of polysaccharides. The 1H NMR spectrum of AFPS-IB in D2O at 60 °C is shown in Figure 4a. The anomeric region (δH 4.3−5.6 ppm) contained three signals. The three sugar residues in AFPS-IB were arbitrarily labeled as X, Y, and Z, respectively, in a molar ratio 10:1:1. On the basis of their

branched glucopyranosyl at the 6-position of the backbone glucose. Structure Data from NMR and AFM. To make clear the chemical structures and chain conformations of polysaccharides is important to understand their biological activities. Combination of NMR spectroscopy and atomic force microscopy 2347

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observed chemical shifts, X1 at δH 5.35 (JH‑1,H‑2 = 4.3 Hz), Y1 at δH 4.98 ppm (JH‑1,H‑2 = 4.7 Hz), and Z1 at δH 4.48 ppm (JH‑1,H‑2 = 8.0 Hz), the residues X, Y, and Z were designated as α-, α-, and β-hexapyranosyl residues, respectively. The 13C NMR spectra in Figure 4b show three signals in the anomeric region (δC 90− 105 ppm); the signals were assigned to residues Z (δC 102.3 ppm), X (δC 100.8 ppm), and Y (δC 98.8 ppm) from the downfield to the high field, respectively, which were confirmed by crosspeaks in the 1H,13C HSQC spectrum (Figure 5). The detailed proton signals (Table 4) of these three systems from H-1 to H-6 were assigned using the 1H/1H homonuclear

Table 3. GC-MS of Alditol Acetate Derivatives from the Methylated Product of AFPS-IB methylated sugara

Rt molar (min) ratio

2,3,4,6-Me4-Glc 14.82 2,3,6-Me3-Glc

16.96

2,3-Me2-Glc

19.07

a

mass fragment (m/z)

type of linkage

0.97

43, 45, 59, 71, 87, 101, terminal 117, 129, 145, 161, 205 10.23 43, 45, 71, 87, 99, 101, →4)-Glcp-(1→ 113, 117, 129, 131, 143, 161, 173, 233 1.16 43, 45, 85, 87, 101, 117, →4,6)-Glcp-(1→ 142, 159, 201, 261

2,3,4,6-Me4-Glc, 1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl-glucose, etc.

Figure 4. NMR spectrum of the polysaccharide AFPS-IB in D2O. (a) 1H NMR and (b) 13C NMR. 2348

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Table 5. NOE Data for the AFPS-IB Fraction Isolated from Achatina f ulica anomeric proton

NOE contact protons δ

δ

→4)-α-D-Glcp-(1→ X

5.356

→4, 6)-α-D-Glcp-(1→ Y

4.955

β-D-Glcp-(1→ Z

4.482

3.681 3.812 3.615 3.956 3.818 3.772 3.615 3.701 3.681 3.958 3.742 3.973 4.966 3.391 3.523 3.614

glycosyl residue

residue, atom Y X X X X X X Y Y Y Y Y Y Z Z Z

H-4 H-3 H-4 H-5 H-6a H-6b H-4 H-2 H-4 H-5 H-6a H-6b H-6b H-2 H-3 H-4

intensitya,b s s s w s s s w s s s w w s s w

a

The intensities were estimated from visual inspection of the NOESY spectra. bs, strong; w, weak.

Figure 5. 600-MHz H/C-HSQC spectrum of polysaccharide AFPS-IB in D2O at 60 °C. Relevant cross peaks have been labeled.

correlation (1H−1H COSY) (Figure S2) and TOCSY experiments (Figure S3). The carbon signals were assigned based on the assignment of the protons, using 1H,13C HSQC spectrum (Figure 5). For example, the anomeric carbon signal at δ 100.8 ppm for unit X was assigned by the correlation signal of X-H1/C1. The other signals from C2−C6 were similarly assigned as 74.5 (C-2), 75.6 (C-3), 80.1(C-4), 76.1 (C-5), and 63.4 (C-6 ppm). Similarly, the down-shift of the C-4 in unit X was a 4-linked Glc residue, whereras the down shift in the C-4 and C-6 indicated the appearance of the 4,6-linked Glc (Unit Y). The sequence of the glycosyl residues was determined on the basis of a 2D-NOESY NMR experiment (Figure S4, Table 5). The H-1 of residue X has a strong interresidue NOE contact to H-4 of residue Y in addition to intraresidue NOE contacts to H-3, H-4, H-5, H-6a, and H-6b, it is evident that residue X is linked to the 4-position of residue Y. Similarly, residue Y has a strong interresidue NOE contact to H-4 of residue X which indicates that residue Y is linked at the 4-position of residue X. The H-1 of residue Z has a strong interresidue NOE contact to H-6a and medium to H-6b of residue Y in addition to intraresidue NOE contacts to H-2, H-3, and H-4, evidenced that residue Z is linked at the 6-position of residue Y. Based on the above results, we concluded the AFPS-IB was an association of the 4-linked α-glucan chains with (1→6)-β-linked branches. However, the NMR results do not contain information about how the chains are associated, which should be further investigated by AFM studies.

Atomic force microscopy (AFM) is a powerful technique for direct observation of the conformation of individual macromolecules, including polysaccharides.24 The persistence length can be determined from statistical analysis of the change in the tangent direction of polymer chain as a function of segment separation of the chain trajectory.29 The AFM image of AFPSIB is shown in Figure 6. The fiber-like structure and small branches of the AFPS-IB molecule can be observed. The chain length of the backbone was approximately 1140−1540 nm and the height was 20−40 nm. The fiber-like structure of AFPS-IB revealed by AFM was consistent with the results of NMR. NMR spectroscopy can provide useful information about the molecular conformations of polysaccharides. In some cases it is possible to directly confirm the presence of these conformations by AFM imaging of the polysaccharide.30 Thus, the sequence of AFPS-IB was proposed below:

Antioxidant Activity. Figure 7 shows the antioxidant activities of AFPS-IB determined by the DPPH radical, reducing power and superoxide radicals (O2−) assays. Vitamin C was used as reference material. As shown in Figure 7a, AFPS-IB

Table 4. Summary of 1H NMR and 13C NMR Chemical Shifts for AFPS-IB chemical shifts (ppm) glycosidic linkage

H-1/C-1

H-2/C-2

H-3/C-3

H-4/C-4

H-5/C-5

H-6/C-6 3.81a 3.77b 3.74a 3.97b

X

→4)-α-D-Glcp-(1→

5.35/100.8

3.58/75.7

3.82/77.2

3.61/80.3

3.95/72.2

Y

→4, 6)-α-D-Glcp-(1→

4.95/98.8

3.70/74.1

3.73/76.6

3.68/79.5

3.95/73.6

Z

β-D-Glcp-(1→

4.48/102.3

3.39/73.1

3.52/76.2

3.61/69.1

3.69/75.3

63.2 69.9 60.4

a,b

Interchangeable. 2349

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Figure 6. Atomic force microscopy (AFM) image of AFPS-IB. The AFPS-IB concentration was 1 μg mL−1.

showed the ability to scavenge DPPH free radicals. The scavenging effects of the polysaccharide increased with increasing concentrations. The IC50 value of AFPS-IB for DPPH free radicals was 6.34 mg mL−1. The maximum value of AFPS-IB reached 85.1% that of vitamin C at 12.0 mg mL−1. Figure 7b depicts the reducing power of the tested sample. Reducing capacity was expressed as a percentage of the activity of a solution (1 mM) of vitamin C. In the tested sample AFPS-IB, the reducing capacity was positively correlated with sample concentration. The maximum value of AFPS-IB reached 65.6% that of vitamin C. Figure 7c shows the O2− scavenging ability, which indicates that the radical scavenging activity of the samples increased in a concentration dependent manner. The IC50 value of AFPS-IB for O2− radicals was 7.83 mg mL−1. The maximum value of AFPS-IB reached 73.4% that of vitamin C. Glucans, as a source of bioactive macromolecules, have diverse pharmaceutical activities which are dependent on the structural characteristics. The glycosidic linkage and configuration are two main factors determining the bioactivity. AFPSIB, isolated from A. f ulica, is a novel water-soluble glucan composed of (1→4)-α-D-glucose residues in the main chain with (1→6)-β-linked branches at glucose residues. The structure of AFPS-IB is similar to that of starch isolated by Baldwin et al.31 from potato, except for the (1→6)-D-Glcp in AFPS-IB was β-configuration. It is also similar to a glucan characterized by Maity et al.32 from the fruit bodies of an edible hybrid mushroom PCH9FB, except for different molar ratio of (1→4)-α-DGlcp and (1→6)-β-Glcp residues. Antioxidation tests performed in vitro showed that AFPS-IB had higher DPPH radical and superoxide anion (O2−) scavenging activities than that of potato starch, which showed very poor antioxidant activity, and similar to the glucan from the fruit bodies of an edible hybrid

Figure 7. Antioxidant activity of the polysaccharide AFPS-IB and Vc in vitro. (a) The scavenging effect on DPPH radicals; (b) Reducing power was expressed as a percentage of the activity of vitamin C; (c) The scavenging effect on superoxide radicals. Vitamin C was used as reference material. Values are means ± SD of three separate experiments.

mushroom PCH9FB with an IC50 value of 5.00−7.00 mg mL−1.32 The antioxidant mechanism of glucan is not clear, nor is it clear why glucans with different glycosidic linkages and configurations have different free radical scavenging capabilities. The most pronounced hypothesis is that free radicals can abstract hydrogen atoms at all ring C−H bonds of aldoses, uronic acids, and other sites on carbohydrates. The abstraction of hydrogen atom will generate carbon-center radicals. The radicals at carbons which form glycosidic bonds will undergo a β-scission reaction resulting in the breakdown of polysaccharide chains.33 Some free radicals have lone-pair orbitals antiperiplanar to the alkylidene C−H bond. Therefore, glycosidic linkages with different conformations can have different reaction rates with these free radicals, allowing for selectivity in cleaving β-Dlinkages of polysaccharides. 34 A (1→6)-β-D-glucan and (1→4)α-D-glucan complex isolated from mushroom has been found to have strong antioxidant activities.35 The responses to such glucans are likely to be regulated by different conformations of glycosidic linkages. Therefore, it can be hypothesized that the 2350

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conformation of (1→6)-β-D-Glcp residues may be responsible for its antioxidant activity. In summary, this study isolated and characterized a novel water-soluble glucan (AFPS-IB) from the soft body of Achatina f ulica. AFPS-IB was composed of Glc, Fuc, Rha, Man, and Gal in a molar ratio of 189:2:1:1:2, with a molecular weight of 128 kDa. The backbone of AFPS-IB was composed of (1→4)α-D-glucose residues, which occasionally branched at O-6, and terminated with 1→)-β-glucose. Antioxidation tests performed in vitro showed that AFPS-IB had remarkable DPPH radical and superoxide anion (O2−) scavenging activity and high reduction potential. The information of this work and published literature have demonstrated that glucan has a key role for health functions of Achatina fulica. AFPS-IB could be regarded as a potential natural antioxidant. Further studies on other activities of the polysaccharide are currently in progress.



ASSOCIATED CONTENT

S Supporting Information *

Figures S1−S4. This material is available free of charge via the Internet at http://pubs.acs.org.



AUTHOR INFORMATION

Corresponding Authors

*Phone: 86 571 88982169. Fax: 86 571 88982154. E-mail: [email protected] (D.L.). *Phone: 86-571-88982151. E-mail: [email protected] (S.C.). Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS



REFERENCES

This research was financed by the National Natural Science Foundation of China (no. 31371872 and 31301417).

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