Analyses of active antioxidant polysaccharides from four edible mushrooms

Accepted Manuscript Analyses of active antioxidant polysaccharides from four edible mushrooms

Jingmin Yan, Lei Zhu, Yunhe Qu, Xian Qu, Meixia Mu, Mengshan Zhang, Gul Muneer, Yifa Zhou, Lin Sun PII: DOI: Reference:

S0141-8130(18)33596-7 https://doi.org/10.1016/j.ijbiomac.2018.11.079 BIOMAC 10956

To appear in:

International Journal of Biological Macromolecules

Received date: Revised date: Accepted date:

15 July 2018 16 September 2018 12 November 2018

Please cite this article as: Jingmin Yan, Lei Zhu, Yunhe Qu, Xian Qu, Meixia Mu, Mengshan Zhang, Gul Muneer, Yifa Zhou, Lin Sun , Analyses of active antioxidant polysaccharides from four edible mushrooms. Biomac (2018), https://doi.org/10.1016/ j.ijbiomac.2018.11.079

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ACCEPTED MANUSCRIPT Analyses of Active Antioxidant Polysaccharides from Four Edible Mushrooms

Jingmin Yan, Lei Zhu, Yunhe Qu, Xian Qu, Meixia Mu, Mengshan Zhang, Gul Muneer, Yifa Zhou, Lin Sun*

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Jilin Province Key Laboratory on Chemistry and Biology of Changbai Mountain

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Natural Drugs, School of Life Sciences, Northeast Normal University, Changchun

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130024, PR China

E-mail address: [email protected]

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Tel./fax: +86 431 85099350

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Corresponding author: Lin Sun

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Jingmin Yan, e-mail address: [email protected]

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Lei Zhu, e-mail address: [email protected] Yunhe Qu, e-mail address: [email protected]

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Xian Qu, e-mai address: [email protected] Meixia Mu, e-mail address: [email protected]

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Mengshan Zhang, e-mail address: [email protected] Gul Muneer, e-mail address: [email protected] Yifa Zhou, e-mail address: [email protected]

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ACCEPTED MANUSCRIPT Abstract Four water-soluble polysaccharides were extracted from Pleurotus eryngii, Flammulina velutipes, Pleurotus ostreatus and white Hypsizygus marmoreus. Using anion exchange and gel permeation chromatography, a neutral and an acidic fraction were purified from each water-soluble polysaccharide. Their molecular weights were

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all around 20 kDa except that the acidic polysaccharide from Pleurotus ostreatus

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(named WPOPA) had a lower molecular weight of 5 kDa. Four neutral

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polysaccharides were mainly composed of galactose (42.7% ~ 69.1%), followed by Man (19.4% ~ 39.3%) and Glc (1.1% ~ 15.9%). Four acidic polysaccharides

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contained glucose (59.0% ~ 81.8%) as major sugar and minor glucuronic acid (4.5% ~ 9.5%). Acidic polysaccharides exhibited stronger antioxidant activities than neutral

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fractions, and WPOPA showed the best antioxidant effects. Structural analysis indicated WPOPA had β-(1→6)-glucan backbone branched at O-3 by β-1,3-D-Glcp,

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t-β-D-Glcp and t-β-D-GlcpA. This investigation would be useful for screening natural

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antioxidants and significant in developing mushroom polysaccharides as functional

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foods.

Keywords: mushroom polysaccharides; structural characterization; antioxidant

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activities

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ACCEPTED MANUSCRIPT 1. Introduction Mushrooms are very nutritional edible fungi, and possess many medicinal values and bioactivities. Polysaccharides are major active constituents in edible mushrooms. There have been a lot of studies about polysaccharides extracted from fruiting bodies, mycelium,

sclerotia

and

fermentation

broth

of

edible

mushrooms

[1-4].

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Polysaccharides from mushrooms not only play an important role in the growth and

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development of fungi organisms but also have wide-ranging bioactivities, such as

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antitumor [5], antibacterial [6], immunomodulatory [7] and hypoglycemic activity [8]. Recently, the antioxidant activities of mushrooms and their polysaccharides have

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garnered considerable attention [9]. For example, various polysaccharides isolated from Pleurotus eryngii [10], Coprinus comatus [11], Armillaria mellea [12], Grifola

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frondosa [13], Russula albonigra [14] and bachu mushroom [15] have shown to exhibit potent antioxidant effects on oxygen radicals. Although these polysaccharides

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have been the subject of intense research [16], the relationship between antioxidant

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activity and the chemical properties of mushroom polysaccharides, such as sugar composition, molecular weight and chemical structure, remains unclear. In order to

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develop the mushroom-derived polysaccharides as natural antioxidant, it is necessary to identify the structural features of those polysaccharides which promote the

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antioxidant activity. In this paper, we extracted and purified polysaccharides from four species of edible mushrooms (Pleurotus eryngii, Flammulina velutipes, Pleurotus ostreatus and white Hypsizygus marmoreus), compared their antioxidant activities together with sugar composition and molecular weight properties, and elucidated the structural features of the most potent polysaccharides with antioxidant activities. These data will be helpful for investigating the relationship between

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ACCEPTED MANUSCRIPT mushroom polysaccharide structure and antioxidant activity and development of mushroom polysaccharide in functional foods or medicine.

2. Materials and methods 2.1 Materials

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Fresh fruiting bodies from Pleurotus eryngii, Flammulina velutipes, Pleurotus

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ostreatus and white Hypsizygus marmoreus were purchased from Changchun, Jilin

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Province, China, and identified by using species-specific ITS rDNA PCR. DEAEcellulose was purchased from Shanghai Chemical Reagent Research Institute

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(Shanghai, China). Sepharose CL-6B was purchased from GE healthcare (Pittsburgh, USA). Bio-Gel P-2 was purchased from Bio-Rad (California, USA). β-1,3-glucanase

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and β-glucuronidase were purchased from Megazyme (Ireland). All other chemicals

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were of analytical grade and commercially available or produced in China.

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2.2 Polysaccharide extraction and purification Fruiting bodies were defatted with 95% ethanol (material/ethanol, w/v, 1:10),

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and the residues were extracted with distilled water (material/dH2O, w/v, 1:20) three times at 100°C for 4 h each time. The extracts were concentrated under vacuum at

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60°C and precipitated using 4 volumes of 95% ethanol at 4°C for 12 h. After centrifugation (4000 rpm, 15 min), the precipitate was collected and re-dissolved in water, then dialyzed and lyophilized. Four water-soluble polysaccharide-enriched fractions were obtained from Pleurotus eryngii (named WPEP), Flammulina velutipes (named WFVP), Pleurotus ostreatus (named WPOP) and White Hypsizygus marmoreus (named WHMP), respectively.

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ACCEPTED MANUSCRIPT Each water-soluble polysaccharide-enriched fraction was dissolved in distilled water and applied to DEAE-cellulose column (8.0 × 20 cm, Cl-). The column was eluted with distilled water and 0.3 M NaCl, giving one neutral polysaccharide and one acidic polysaccharide fraction, respectively. Both fractions were further purified by Sepharose CL-6B column, giving purified neutral and acidic polysaccharide fractions

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(Figure S2~Figure S4).

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2.3 General methods

Total carbohydrate content was determined by the phenol-sulfuric acid protocol

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with glucose as standard [17]. Uronic acid content was determined by using the colorimetric method of Filisetti-Cosi & Carpita [18] with glucuronic acid as the

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standard. Protein content was determined by using the Bradford assay with bovine serum albumin (BSA) as standard [19]. Glycogen-like polysaccharide was detected by

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the I2-KI assay [20]. Molecular weights were determined by using high performance

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gel-permeation chromatography (HPGPC) with a TSK-gel G-3000 PWXL column (7.8 × 300 mm, TOSOH, Japan) coupled to a Shimadzu high performance liquid

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chromatography (HPLC) system.

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2.4 Monosaccharide composition analysis Polysaccharide sample (2 mg) was hydrolyzed first with anhydrous methanol containing 1 M HCl at 80°C for 16 h and then with 2 M TFA at 120°C for 1 h. The released monosaccharides were derived by 1-phenyl-3-methyl-5-pyrazolone (PMP) and analyzed by HPLC as previously described [21]. Methyl-galactose (Me-Gal) was determined by UPLC-MS. Briefly, PMP- derived monosaccharides were analyzed by Waters UPLC system equipped with an Acquity UPLC BEH C18 column (2.1 mm × 5

ACCEPTED MANUSCRIPT 50 mm), coupled to Amazon Speed ETD mass spectrometer using positive electrospray as the ionization process, and the mass scan range was m/z 0~1200. Monosaccharide composition of acidic polysaccharide was further determined by using high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD, Dionex ICS-5000+ DC) and a CarboPac PA-20 column (150

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× 3 mm) [22]. The gradient elution program was as follows: 0~20 min, 2 mM NaOH;

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20~35 min, 2 mM NaOH and 0-200 mM CH3COONa; 35~45 min, 200 mM NaOH.

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The elution was run at 35°C with a flow rate of 0.4 ml/min.

To confirm the type of uronic acid in acidic polysaccharide, carboxyl-reduction

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of these fractions were carried out by using the reported method [23]. Carboxyl groups were first activated with carbodiimide then reduced by NaBD4 to give the

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carboxyl-reduced polysaccharides, followed by hydrolysis and derivatization into alditol acetates. The resulting alditol acetates were analyzed by GC (7890B, Agilent,

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USA) with an HP-35ms capillary column (30 m × 0.32 mm × 0.25 mm). The oven

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temperature was programed from 140°C (hold for 2 min) to 210°C (hold for 3 min) at 5°C/min, then up to 260°C (hold for 5 min) at 2°C/min, finally up to 300°C (hold for

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1 min) at 20°C/min. Both temperature of inlet and detector were 300°C. Helium was

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used as carrier gas.

2.5 Fourier transform infrared spectroscopy Polysaccharides were ground with KBr powder and then pressed into a 1 mm pellet for Fourier transform infrared (FT-IR) measurements. FT-IR spectra were obtained using a Spectrum Two FT-IR spectrometer in the range of 4000-400 cm−1 (Perkin Elmer, USA).

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ACCEPTED MANUSCRIPT 2.6 Methylation analysis Methylation analysis was carried out according to the method of Needs and Selvendran [24]. In brief, polysaccharide (10 mg) was dissolved in DMSO (1.5 ml) and methylated with a suspension of NaOH/DMSO (1.5 ml) and iodomethane (2.0 ml). The reaction mixture was extracted with dichloromethane (CH2Cl2), and then the

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solvent was removed by vacuum evaporation. Complete methylation was confirmed

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by the disappearance of the -OH band (3200-3400 cm-1) in the FT-IR spectrum. The

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per-O-methylated polysaccharide was hydrolyzed subsequently by using HCOOH (85%, 1 ml) for 4 h at 100°C and then CF3COOH (2 M, 1 ml) for 6 h at 100°C. The

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partially methylated sugars in the hydrolysate were reduced by using NaBH4 and acetylated. The resulting alditol acetates were analyzed by GC-MS (7890B-5977B,

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Agilent, USA) with an HP-5ms capillary column (30 m × 0.32 mm × 0.25 mm). The oven temperature was programed from 120°C (hold for 1 min) to 210°C (hold for 2

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min) at 3°C/min, then up to 260°C (hold for 4 min) at 10°C/min. Both temperature of

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was 50-500 m/z.

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inlet and detector were 300°C. Helium was used as carrier gas. The mass scan range

2.7 Periodate oxidation and Smith degradation

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WPOPA was first dissolved in dH2O (20 mg in 12.5 ml) and 30 mM NaIO4 (12.5 ml) was added. The solution was maintained for 48 h in the dark at 4°C. Then, ethylene glycol (0.5 ml) was added to stop the reaction. The solution was dialyzed (MWCO 1 kDa for 24 h) against tap water and dH2O respectively, then reduced by using NaBH4 (70 mg/ml) for 16 h. After neutralization with HOAc and dialysis, the solution was concentrated to small volume and submitted to mild acid hydrolysis with 0.5 M TFA at 25°C for 15 h. Then, the reaction solution was dialyzed against tap 7

ACCEPTED MANUSCRIPT water and dH2O for 24 h, respectively. The dialyzed product was collected and lyophilized to give smith-degraded WPOPA [25].

2.8 NMR spectroscopy 1

H,

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C, HSQC and HMBC NMR spectra were recorded at 20oC on a Bruker

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C NMR. Sample (20.0 mg)

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operating at 600 MHz for 1H NMR and 150 MHz for

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Avance 600 MHz spectrometer (Germany) with a Bruker 5 mm broadband probe,

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was dissolved in D2O (0.5 ml) and centrifuged to remove any undissolved

2.9

Enzymatic

hydrolysis

high

performance

anion

exchange

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chromatography analyses

and

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polysaccharide. Data were analyzed using standard Bruker software.

WPOPA was dissolved in 10 mM CH3COONH4 buffer (containing 50 mM KCl)

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to 4 mg/ml. β-Glucuronidase was added (1.1 U/mg) and the reaction was carried out

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at 37oC with continuous agitation for 24 h. After inactivation of the enzyme and centrifugation, diluted 100 times into 200 μl with ddH2O, and analyzed by high

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performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD, Dionex ICS-5000+ DC) by using a CarboPac PA-200 column (250 ×

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3 mm) [22]. The gradient elution program was as follows: 0~2 min, 10% 1 M NaOH and 10% 1 M CH3COONa; 2~5 min, 10% 1 M NaOH and 40% 1 M CH3COONa; 5~25 min, 10% 1 M NaOH and 70% 1 M CH3COONa; 26~31 min, 20% 1 M NaOH and 70% 1 M CH3COONa; 31~35 min, 10% 1 M NaOH and 10% 1 M CH3COONa. All samples were run at 35°C with a flow rate of 0.5 ml/min. Glucuronic acid (GlcA) was used as the standard.

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ACCEPTED MANUSCRIPT WPOPA was dissolved in 50 mM acetate buffer (pH 5.0) to 5 mg/ml and β-1,3glucanase (0.15 U/mg) was added. Digestion was carried out at 40 oC with continuous agitation for 12 h. After enzyme inactivation by boiling for 10 min and centrifugation at 10000 rpm for 5 min, the degradation product was separated by Bio Gel P-2 column. An oligosaccharide fraction (WPOPA-Oligo) was obtained and its

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monosaccharide composition was detected by HPLC.

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2.10 Determination of antioxidant activities 2.10.1 Measurement of reducing power

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The ability of polysaccharides to reduce ferrous ions was determined according to the reported method with some modifications [26]. Briefly, 2.5 ml of phosphate

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buffer (pH 6.6) and 2.5 ml of 1% K3Fe(CN)6 were added to a 1 ml sample solution at different concentrations (0.5 mg/ml to 10 mg/ml), and the mixture was incubated at

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50°C for 20 min. After cooling down, 1 ml of 10% TCA was added and then

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centrifuged at 4000 rpm for 15 min. After that, 2.5 ml of the supernatant was mixed with 2.5 ml distilled water and 2.5 ml 0.1% FeCl3 for 10 min. The absorbance at 700

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nm was then determined. Ascorbic acid was used as positive control, and distilled water was the negative control. The ability of different polysaccharide to reduce Fe3+

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was expressed as:

Reducing power=Asample- Acontrol

2.10.2 Hydroxyl radical scavenging activity The hydroxyl radical scavenging activity was evaluated as reported by previous literature with minor modifications [27]. Briefly, 1.0 ml of 9 mM FeSO4 and 1.0 ml of 9 mM salicylic acid (dissolved in alcohol) were added to 0.5 ml of sample solution at 9

ACCEPTED MANUSCRIPT different concentrations (0.5 mg/ml to 10 mg/ml). After that, 8.8 mM H2O2 was added to reaction mixture incubated for 30 min at 25°C. The absorbance at 510 nm was then measured. Ascorbic acid and distilled water were used as the positive and negative controls, respectively. The hydroxyl radical scavenging activity was calculated as:

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Hydroxyl radical scavenging activity (%) = (Acontrol - Asample)/Acontrol × 100%.

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2.10.3 Superoxide anion radical scavenging activity

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The ability of polysaccharides to scavenge superoxide anion radical was measured according to the reported method with some modifications [28]. Briefly, 1

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ml sample solution at various concentrations (0.5 mg/ml to 10 mg/ml) was mixed with 1 ml 300 μM nitroblue tetrazolium (NBT) and 1 ml 936 μM reduced form of

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nicotinamide-adenine dinucleotide (NADH), then 1 ml 120 μM phenazin methosulfate (PMS) was added to the reaction mixture and incubated for 5 min at

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25°C. The absorbance at 560 nm was determined. Ascorbic acid was used as positive

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control, and distilled water was used as negative control. The ability of polysaccharides to scavenge superoxide anion radical was calculated as:

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Superoxide anion scavenging activity (%)= (Acontrol - Asample)/Acontrol × 100%

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2.10.4 DPPH radical scavenging activity The ability of polysaccharides to scavenge DPPH radical was assessed by using the reported method [29]. Briefly, 1 ml sample solution at various concentrations (0.5 mg/ml to 10 mg/ml) was mixed with 4 ml 0.004% DPPH solution (dissolved with methanol) and shaken vigorously. The mixture was placed in the dark for 30 min, and the absorbance at 517 nm was immediately measured. Ascorbic acid was used as

10

ACCEPTED MANUSCRIPT positive control, and distilled water was used as negative control. The ability of polysaccharides to scavenge DPPH radical was calculated as: DPPH radical scavenging activity (%) = (Acontrol - Asample)/Acontrol × 100%

2.11 Statistical analysis

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Antioxidant activity results were expressed as mean ± standard deviation (SD)

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from three independent experiments. The data were analyzed for significance using

SC

the Student’s t test, and P value less than 0.05 was considered statistically significant.

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IC50 values were calculated using SPSS 19.0 software.

3. Results and discussion

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3.1 Extraction of polysaccharides from four edible mushrooms Four water-soluble polysaccharide-enriched fractions were extracted from the

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fruiting bodies of Pleurotus eryngii (WPEP), Flammulina velutipes (WFVP),

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Pleurotus ostreatus (WPOP) and White Hypsizygus marmoreus (WHMP) by using hot water extraction and ethanol precipitation. Their yields were 0.44%, 0.47%,

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0.88% and 0.53%, respectively, relative to the wet weights of mushroom materials. Monosaccharide composition analysis indicated that all polysaccharides were mainly

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composed of glucose (Glc), galactose (Gal) and mannose (Man) (Table 1), with the content of Glc being highest in WHMP. In addition, all polysaccharides contained minor glucuronic acid (GlcA). WPEP and WPOP also contained small amounts of methyl-galactose (Me-Gal). Although these polysaccharides contained large amounts of Glc, the I2-KI assay indicated that they did not contain glycogen-like glucans.

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ACCEPTED MANUSCRIPT 3.2 Antioxidant activities of four mushroom polysaccharides The antioxidant activities of four mushroom polysaccharides (WPEP, WFVP, WPOP and WHMP) were evaluated by ferric-reducing power assay and scavenging activities for three kinds of radicals including hydroxyl, superoxide anion and DPPH radicals (Figure 1). The results indicated that WPEP, WFVP, WPOP and WHMP all

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exhibited antioxidant activities in a dose dependent manner over the range of 0.5

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mg/ml to 10 mg/ml, but antioxidant effects of the four mushroom polysaccharides

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toward different free-radicals were different. In general, WPOP displayed the best antioxidant activities among the four mushroom polysaccharides with regard to

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different assays, although its antioxidant activity was weaker than that of ascorbic acid. As these polysaccharides were all mixtures, to discuss the structure-antioxidant

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activity relationship, they were further purified into homogeneous fractions.

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3.3 Fractionation of mushroom polysaccharides

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WPEP, WFVP, WPOP and WHMP were all first fractionated by anion-exchange chromatography. From the chromatography procedure, one neutral fraction was eluted

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with distilled water, and one acidic fraction was eluted with 0.3 M NaCl from each polysaccharide. These fractions were then further separated by using gel-permeation

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chromatography

(Figure

2a).

Following

separation,

four

purified

neutral

polysaccharide fractions (WPEPN, WFVPN, WPOPN and WHMPN) and four purified acid polysaccharide fractions (WPEPA, WFVPA, WPOPA and WHMPA) were obtained from different mushroom polysaccharides (Figure 2a). Their molecular weights were all around 20 kDa determined by HPGPC (Figure 2b, 2c and Table 2), except for WPOPA which had a much lower molecular weight (5.1 kDa) than other fractions. Monosaccharide compositions of these polysaccharide fractions were 12

ACCEPTED MANUSCRIPT determined by HPLC and listed in Table 2. All of the neutral polysaccharides (WPEPN, WFVPN, WPOPN and WHMPN) contained Gal as the major monosaccharide, followed by Man and Glc. WPEPN and WPOPN also contained some Me-Gal residues. Therefore, these neutral fractions might be heterogalactans. Acid polysaccharide fractions (WPEPA, WFVPA, WPOPA and WHMPA) were all

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mainly composed of Glc, together with small amounts of Man, Gal and GlcA,

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indicating they were heteroglucans. Their monosaccharide compositions were further

SC

identified by HPAEC as shown in Table 2 and Figure S5, which were similar to HPLC results. To prove the existence of GlcA, carboxyl-reduction of these acid

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polysaccharides together with GC analysis were also performed. The content of Glc increased obviously after reduction compared with non-reduced acidic polysaccharide

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fractions (Table S1), suggesting that GlcA indeed existed in these acidic

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3.4 FT-IR spectra analysis

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polysaccharides.

The FT-IR spectra of all the neutral and acid polysaccharide fractions were

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presented in Figure 3. Their spectra were similar to each other. The intense absorption band near 3400 cm-1 (3394 cm-1 or 3374 cm-1) was associated with the stretching

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vibration of O-H. The weak band around 2930 cm-1 (2929 cm-1 or 2927 cm-1) was attributed to C-H stretching vibration [30]. Bands observed at 1143 cm-1 were typical for C-O-C stretching vibration [31]. The asymmetrical stretching bands around 1650 cm-1 (1648 cm-1 or 1645 cm-1) and the weaker symmetric stretching bands near 1409 cm-1 were attributed to asymmetric and symmetric stretching of C=O. The band near 1080 cm-1 (1082 cm-1 or 1075 cm-1) indicated the presence of pyranose ring and the

13

ACCEPTED MANUSCRIPT weak bands around 858 cm-1 and 896 cm-1 indicated the presence of α-linked and βlinked glycosyl residues, respectively [32].

3.5 Antioxidant activities of purified mushroom polysaccharide fractions The antioxidant activities of four neutral polysaccharide fractions (WPEPN,

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WFVPN, WPOPN and WHMPN) and four acid polysaccharide fractions (WPEPA,

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WFVPA, WPOPA and WHMPA) were determined by using ferric-reducing power

SC

assay and three radical scavenging assays. As shown in Figure 4, WPEPA, WFVPA, WPOPA and WHMPA all displayed antioxidant activities in these four different

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assays. Their antioxidant abilities increased with the increase of concentration from 0.5 to 10 mg/ml. Among these acid polysaccharides, WPOPA exhibited the highest

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antioxidant activities with regard to four different assays, but still lower than that of positive ascorbic acid. At the concentration of 10 mg/ml, the reducing power of

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WPOPA reached to 0.428, the hydroxyl radicals scavenging ratio of WPOPA reached

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to 50.0%, the superoxide anion radicals scavenging ratio of WPOPA reached to 90.1%, and the DPPH radicals scavenging ratio of WPOPA reached to 97.4%. Within

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the test dosage range, the IC50 values of WPOPA towards these four assays were 9.286 ± 0.037 mg/ml, 10.284 ± 0.018 mg/ml, 0.572 ± 0.013 mg/ml and 2.138 ± 0.037

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mg/ml, respectively (Table 3). However, four neutral polysaccharides WPEPN, WFVPN, WPOPN and WHMPN almost had no antioxidant activities except for WPOPN and WFVPN exhibiting some superoxide anion radicals scavenging abilities. Therefore, acid polysaccharides are the main antioxidant functional components of four mushroom polysaccharides extracted from Pleurotus eryngii, Flammulina velutipes, Pleurotus ostreatus and White Hypsizygus marmoreus. WPOPA showed the highest antioxidant abilities, which was consistent with the result that water14

ACCEPTED MANUSCRIPT soluble polysaccharides from Pleurotus ostreatus (WPOP) had the best antioxidant effects compared with other mushroom polysaccharides. According to above analysis, the antioxidant activities of neutral and acid mushroom polysaccharides in various reactive systems were different, which might be due to the structural difference of these polysaccharides. Based on monosaccharide

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composition and NMR spectrum results, heteroglucans might have better antioxidant

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activities than heterogalactans. This result was similar to antioxidant activities of

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glucans from Hericium erinaceus [33]. The possible antioxidant mechanism might be that hereroglucans are the hydrogen carrier, and hydrogen could combine with

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radicals and form a stable radical to terminate the radical chain reaction [34]. Compared with other heteroglucans (WPEPA, WFVPA and WHMPA), WPOPA has

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the highest antioxidant abilities. The possible reason is that WPOPA has lower molecular weight (5.1 kDa) and higher GlcA content (9.5%) than other fractions.

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Similar results have been found in previous report that polysaccharide fraction TPC-3

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from Camellia Sinensis had the lowest molecular weight and showed the highest antioxidant activities [35]. Uronic acid group in hereroglucans might combine with

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the radical ions and end the damaging chain reaction. Therefore, higher content of GlcA in WPOPA resulted in higher antioxidant activity [34-36]. To better understand

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the structure-antioxidant activity relationship, the structures of acidic polysaccharides were further characterized.

3.6 Structural analysis of acidic polysaccharides 3.6.1 13C-NMR spectra analysis Structural features of acidic polysaccharide fractions were characterized by 13CNMR spectra. As shown in Figure 5, four acidic polysaccharide fractions exhibited 15

ACCEPTED MANUSCRIPT similar spectra. Six typical signals at 104.78, 74.86, 77.38, 71.26, 76.61 and 70.60 ppm were assigned to C-1, C-2, C-3, C-4, C-5 and C-6 of β-1,6-D-Glcp residues, respectively[37]. Signals at 104.63 ppm and 104.80 ppm were assigned to the anomeric carbons of β-1,3-D-Glcp and t-β-D-Glcp, respectively [38]. Signals at 85.38 ppm and 86.13 ppm were assigned to C-3 of β-1,3,6-D-Glcp and β-1,3-D-Glcp

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residues, respectively. Other signals at 104.30 ppm and 99.64 ppm were assigned to

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the anomeric carbons of β-D-Manp and α-D-Galp, respectively [39]. Weak signals at

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175.5 ppm were assigned to carboxyl group of GlcpA. These results combined with monosaccharide composition analysis indicated that these acid polysaccharide

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fractions were heteroglucans, which might have an β-1,6-D-Glcp backbone and was substituted at O-3 of Glc by β-D-Manp, α-D-Galp or t-β-D-GlcpA residues. These

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results indicated that acidic polysaccharide fractions had similar structural features. Among them, the fraction (WPOPA) from Pleurotus ostreatus showed the best

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antioxidant effects. Thus, the fine structure of WPOPA was further analyzed by

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methylation and GC-MS, periodate oxidation and smith degradation, 2D-NMR

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spectra, enzymatic hydrolysis and HPAEC-PAD.

3.6.2 Methylation analysis of WPOPA

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To determine the glycosidic linkage types of WPOPA, methylation and GC-MS analysis was performed. The result of the linkage composition was shown in Table 4. The major methylated product of WPOPA was 1,6-linked Glcp (56.3%), along with 1,3,6-Glcp (13.2%), 1,3-Glcp (10.9%) and non-reducing terminal Glcp (11.9%). It was deduced that 1,6-linked Glcp residues formed the backbone in WPOPA, and of which 18.9% were branched at O-3 position. Other linkages such as 1,3-Glcp and non-reducing terminal Glcp might exist as side chains. In addition, a few 1,4-linked 16

ACCEPTED MANUSCRIPT Manp (4.5%) and 1,6-linked Galp (3.2%) were detected, which might be also as side chains.

3.6.3 Periodate oxidation and Smith degradation Periodate oxidation and smith degradation was carried out to determine the most

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probable main chain of WPOPA. Periodate oxidation result showed that WPOPA

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consumed 119.7 mmol periodate and produced 57.57 mmol formic acid, suggesting

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that 1,6-linked and 1-linked glucosyl residues in WPOPA were destroyed. The retained fraction obtained after smith degradation was only 25.0% of WPOPA, which

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might be in the form of 1,3- and 1,3,6-linkage. This was consistent with methylation analysis result that total content of 1,3,6-Glc and 1,3-Glc was 24.1%. Besides, the

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molecular weight of degradation product decreased significantly (Figure S6) compared with WPOPA. These results suggested that the backbone of WPOPA

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should be 1,6-linked Glcp which were severely broken by periodate while small

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amounts of 1,3- linked Glcp side chains were left.

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3.6.4 NMR analysis of WPOPA

The structure of WPOPA was further analyzed by 1H NMR and 2D NMR

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spectroscopy (13C NMR has been shown in Figure 5). In the 1H NMR spectrum (Figure 6a), the signal at 4.46 ppm was assigned to anomeric protons of β-1,6-D-Glcp and β-1,3,6-D-Glcp, and signals at 4.68 and 4.65 ppm which might be over-lapped with signal of HDO were assigned to anomeric protons of β-1,3-D-Glcp and t-β-DGlcp, respectively [38]. The signal at 4.94 ppm was attributed to anomeric proton of α-D-1,6-linked Galp residue [39]. Other signals were assigned according to HSQC spectrum (Figure 6b, Table 5). The signals at 4.46/104.78 ppm (H-1/C-1), 3.75/85.38 17

ACCEPTED MANUSCRIPT ppm (H-3/C-3) and 4.15,3.78/70.60 ppm (H-6/C-6) were assigned to β-1,3,6-D-Glcp. The signals appeared at 3.68/86.13 ppm and 3.84;3.66/62.50 ppm were assigned to H3/C-3 and H-6/C-6 of β-1,3-D-Glcp residues [38]. Some weak signals at 4.94/99.64 ppm (H-1/C-1) and 3.85;3.59/68.33 ppm (H-6/C-6) indicated the presence of 1,6linked α-D-Galp [39], and the signal at 3.64/82.67 ppm was attributed to H-4/C-4 of

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β-1,4-D-Manp. In HMBC spectrum, the cross peaks of both anomeric protons and

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carbons of the glycosyl residues AH-1/AC-6, AH-2/AC-1, AH-6a/AC-1, AH-6a/BC-1, BC-3/CH-1, CC-3/DH-1, CC-1/CH-3, CC-4/CH-3, and DC-1/DH-2 were observed

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(Figure 6c). Hence, WPOPA was possibly composed of (1→6)-linked β-D-glucan

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backbone and branched at O-3 position by β-1,3-D-Glcp and single-unit β-Glcp or minor α-1,6-D-Galp and β-1,4-D-Manp as side chains. These results were consistent

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with the analysis by methylation and GC-MS.

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3.6.5 Enzymatic hydrolysis of WPOPA and HPAEC-PAD analyses

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WPOPA contained 9.5% of GlcA residues, while methylation and NMR analysis did not give more structural information about this sugar. To detect the glycosidic

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linkage of GlcpA in WPOPA, enzymatic hydrolysis and HPAEC-PAD analyses were performed. WPOPA was first hydrolyzed by β-glucuronidase, and the degraded

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products were determined by HPAEC-PAD. As shown in Figure 7a, single unit of GlcA residue was produced after hydrolysis by β-glucuronidase, which indicated that t-β-D-GlcpA residue was present in WPOPA. The terminal β-D-GlcpA might be directly attached to β-(1→6)-glucan backbone through O-3 or linked to the backbone by β-1,3-D-Glcp. To determine if t-β-D-GlcpA was linked to β-1,3-D-Glcp, β-1,3glucanase was used to hydrolyze WPOPA. The degradation product was fractionated by Bio-Gel P-2 and one oligosaccharide fraction (WPOPA-Oligo) was obtained 18

ACCEPTED MANUSCRIPT (Figure 7b). Monosaccharide composition result showed that WPOPA-Oligo was only composed of Glc (Figure 7c), suggesting GlcpA might be not linked to β-1,3-D-Glcp. Therefore, we speculated that most of GlcpA residues in WPOPA were in the form of t-β-D-GlcpA and might exist as side chains, which were connect to the (1→6)-linked β-D-glucan backbone through O-3. Our result was similar with GlcpA residues which

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have been found in acidic heteropolysaccharides from Auricularia auricular [40],

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Tremella aurantialba [41] and Tremella fuciformis [42], in which t-β-D-GlcpA units

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were observed as the side chains attached to O-2 of α-D-1,3-linked Manp backbone.

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According to above results, the possible structural model was established as following:

WPOPA showed better antioxidant activities in our study. Similar observations have been found in other reports that β-D-glucans from mushrooms were effective

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antioxidants. One glucan fraction, LB-1b, isolated from pleurotus abalonu exhibited

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high antioxidant ability. LB-1b also contained minor GlcpA residues. However, the main chain of LB-1b was β-1,4-D-Glcp which was different from WPOPA, and the linkage type of GlcpA was not identified in it [43]. Higher molecular weight βglucans (~200 kDa) from Russula albonigra [44] and Entoloma lividoalbum [45] also exhibited potent antioxidant activities. Their backbone structures were similar to WPOPA but contained no GlcpA residues. Thus, the β-glucan isolated from Pleurotus ostreatus in this study might be a new potent natural antioxidant.

19

ACCEPTED MANUSCRIPT 4. Conclusion In the present work, four water-soluble enriched polysaccharides were extracted from the fruiting bodies of four different species of mushrooms and further purified by anion-exchange and gel-permeation chromatography. Antioxidant activity analyses indicated that acidic polysaccharides which contained large amounts of Glc along

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with minor GlcA exhibited stronger antioxidant activities than neutral polysaccharides

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(hererogalactans). Among acidic polysaccharides, the fraction from Pleurotus ostreatus (named WPOPA) showed the best antioxidant effects. Structure analysis

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showed that WPOPA was composed of β-1,6-D-Glcp backbone, branched at O-3 of

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Glcp by β-1,3-D-Glcp, single-unit β-Glcp and t-β-D-GlcpA. In this regard, it was

antioxidant in functional foods.

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Acknowledgments

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supposed that β-glucans containing GlcA from mushrooms could be candidates as

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This work was supported by the National Natural Science Foundation of China (No: 31500274, 31770852), the University S & T Innovation Platform of Jilin

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Province for Economic Fungi (#2014B-1) and Natural Science Foundation of

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Changchun City of China (17DY026).

Author Contributions LS and YFZ designed and conceived the study, and revised the manuscript. JMY performed the chemical analysis of polysaccharides and drafted the manuscript. LZ carried out the antioxidant assay. YHQ, XQ, MXM, MSZ and MG performed the polysaccharides extraction, isolation and purification. All authors read and approved the final manuscript. The authors declare that they have no conflict of interest. 20

ACCEPTED MANUSCRIPT Conflicts of Interest

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The authors declare no conflict of interest.

21

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Structural, immunological, and antioxidant studies of β-glucan from edible mushroom entoloma

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CE

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lividoalbum, Carbohydr. Polym. 123 (2015) 350-358.

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ACCEPTED MANUSCRIPT Table 1. Chemical characterization of the polysaccharides extracted from four species of edible mushrooms.

Mushrooms

Pleurotus

Flammulina

Pleurotus

White

eryngii

velutipes

ostreatus

Hypsizygus

polysaccharides

marmoreus

0.44

0.47

Total sugar (%)

86.7

87.4

Protein (%)

5.3

4.3

Uronic acid (%)

2.3

Ash (%)

2.3

WHMP 0.53

85.4

87.7

2.9

1.2

1.7

3.1

2.2

2.7

3.3

1.8

59.0

59.1

80.6

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0.88

36.3

Galactose (Gal)

23.4

18.5

22.0

10.2

Mannose (Man)

25.3

14.6

15.8

4.9

Rhamnose (Rha)

3.2

1.8

0.6

--

2.2

1.5

3.7

2.4

Galacturonic acid (GalA)

0.3

--

1.1

--

Methyl-Galactose (Me-Gal)

8.7

--

2.2

--

0.5

1.3

--

1.6

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Glucuronic acid (GlcA)

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Glucose (Glc)

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Sugar composition (%)

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Yield (%)

WPOP

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WFVP

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WPEP

Fucose (Fuc)

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Yield in relation to the wet weights of mushroom materials. Pleurotus eryngii polysaccharide (WPEP); Flammulina velutipes polysaccharide (WFVP); Pleurotus ostreatus polysaccharide (WPOP); White Hypsizygus marmoreus polysaccharide (WHMP).

27

ACCEPTED MANUSCRIPT Table 2. Molecular weight and monosaccharide composition of eight sub-fractions isolated from four species of mushroom polysaccharides.

Mushrooms

Yield

Mw

Monosaccharide composition (mol%)

(%)

kDa

Glc

Gal

Man

GlcA

Fuc

Me-Gal

Fractions

WPEPN

52.7

21.4

9.2a

42.7a

39.3a

--

--

11.7a

eryngii

WPEPA

20.1

18.6

81.5a;80.2b

4.6a;3.1b

9.5a;9.2b

4.5a;6.6b

--

--

Flammulin

WFVPN

42.3

19.8

15.9a

58.2a

19.4a

--

6.6a

--

avelutipes

WFVPA

17.8

23.4

59.0a;61.1b

8.5a;6.9b

13.7a;14.2b

4.1a;5.0b

5.0a;5.8b

--

Pleurotus

WPOPN

60.7

20.0

10.9a

48.8a

--

1.4a

7.4a

ostreatus

WPOPA

19.3

5.1

81.8a;83.0b

5.1a;2.2b

3.6a;2.8b

9.5a;10.6b

--

--

Hypsizygus

WHMPN

54.7

19.6

1.1a

69.1a

22.2a

--

7.6a

--

marmoreus

WHMPA

13.3

18.6

79.5a;78.9b

4.4a;4.9b

4.8a;5.3b

--

--

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Pleurotus

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22.5a

11.3a;9.9b

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Yield in relation to the crude polysaccharides. aThe monosaccharide composition of polysaccharide fractions were determined by HPLC. Polysaccharide fractions were first hydrolyzed with methanol containing 1 M HCl and then with 2 M TFA, after that, the hydrolysates were derived by 1-phenyl-3-methyl-5-pyrazolone (PMP) and analyzed

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by HPLC with UV detector at 245 nm; bThe monoshaccharide compositions of acidic polysaccharide fractions

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were determined by HPAEC-PAD. Acidic polysaccharide fractions were hydrolyzed, and then determined by

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using HPAEC-PAD on a CarboPac PA-20 column (150 × 3 mm).

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ACCEPTED MANUSCRIPT Table 3. Antioxidant activities of polysaccharides from four edible mushrooms. polysaccharide

IC50 values (mg/ml) Reducing power

•OH-

•O2-

DPPH

WPEPA

19.256 ± 0.028b

32.702 ± 0.071c

--

31.075 ± 0.038c

WPOPA

9.286 ± 0.037a

10.284 ± 0.018a

0.572 ± 0.013a

2.138 ± 0.037a

WHMPA

20.358 ± 0.022c

18.130 ± 0.021b

3.904 ± 0.027c

42.030 ± 0.071d

WFVPA

18.429 ± 0.073b

19.315 ± 0.037b

2.297 ± 0.014b

17.280 ± 0.046b

WPEPN

--

--

--

--

WPOPN

--

--

4.469 ± 0.071d

WHMPN

--

--

WFVPN

--

--

--

--

--

3.375 ± 0.044c

--

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RI

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fractions

Values are presented as the means ± SD (n=3), and labeled by different superscript letters imply significantly

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differences (p < 0.05). The sequence of the letters in the alphabet means the order of the antioxidant ability for

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these purified polysaccharide fractions.

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ACCEPTED MANUSCRIPT Table 4. Linkage type analysis of WPOPA by GC-MS Linkages

Molar percent (%)

Mass fragments(m/z)

2,3,4-Me3-Glcp

1,6-

56.3

101,117,129,161,173,189,233

2,4-Me2-Glcp

1,3,6-

13.2

117,129,159,189,233,261,305

2,4,6-Me3-Glcp

1,3-

10.9

101,117,129,161,189,233,277

2,3,4,6-Me4-Glcp

1-

11.9

101,117,129,145,161,205

2,3,6-Me3-Manp

1,4-

4.5

101,117,129,161,233

2,3,4-Me3-Galp

1,6-

3.2

101,117,129,161,189,233

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Methylated sugars

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ACCEPTED MANUSCRIPT Table 5. HSQC spectral assignments of WPOPA.

(D)t-β-D-Glcp

(E)β-1,4-D-Manp

6

H

4.46

3.26

3.42

3.40

3.57

4.15;3.78

C

104.78

74.86

77.38

71.26

76.61

70.60

H

4.46

3.37

3.75

3.46

3.60

4.15;3.78

C

104.78

74.76

85.38

71.39

76.37

70.60

H

4.67

3.31

3.68

3.33

3.60

3.84;3.66

C

104.63

75.19

86.13

71.40

76.37

62.50

H

4.65

3.45

3.71

C

104.80

74.64

77.52

H

4.72

3.62

3.72

C

104.30

72.00

H

4.94

3.84

C

99.64

71.68

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31

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5

D

(F)α-1,6-D-Galp

4

RI

(C)β-1,3-D-Glcp

3

3.56

3.60

3.86;3.66

72.02

76.37

62.50

3.64

3.82

--

77.64

82.67

77.06

--

3.97

4.00

4.04

3.85;3.59

73.33

71.71

72.11

68.33

SC

(B)β-1,3,6-D-Glcp

2

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(A)β-1,6-D-Glcp

1

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Linkage type

ACCEPTED MANUSCRIPT Figure captions Figure 1. Antioxidant activities of four polysaccharides. (a) Reducing power of polysaccharides on Fe3+. (b) Scavenging ability of polysaccharides on hydroxyl radical (•OH-). (c) Scavenging ability of polysaccharides on superoxide anion radical (•O2-). (d) Scavenging ability of polysaccharide on DPPH radical. Vc as the positive control. Data are shown as the mean ± SD,

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(n=3, p < 0.05). All experiments were performed in triplicate. Figure 2. Purification and molecular weight determination of polysaccharides. (a) Fractionation

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scheme for polysaccharide fractions by anion-exchange and gel-permeation chromatography; (b)

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Molecular weight of purified neutral polysaccharide fractions; (c) Molecular weight of purified acidic polysaccharide fractions.

WPEPA, WPOPA, WHMA and WFVPA).

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Figure 3. FT-IR spectra of polysaccharide fractions (WPEPN, WPOPN, WHMPN, WFVPN,

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Figure 4. Antioxidant activities of purified polysaccharide fractions on their (a) reducing power; (b) ability to scavenge hydroxyl radical; (c) ability to scavenge superoxide anion radical; and (d) ability to scavenge DPPH radicals. Vc as the positive control. Data are shown as the mean ± SD,

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(n=3, p < 0.05). All the experiments were performed in triplicate.

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Figure 5. 13C-NMR spectra of WPEPA, WPOPA, WHMPA and WFVPA. Figure 6. 1D and 2D NMR spectra of WPOPA (a) 1H-NMR spectrum; (b) HSQC spectrum (c)

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HMBC spectrum.

Figure 7. Analysis for linkage type of glucuronic acid (GlcpA). (a)Enzymatic hydrolysis of

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WPOPA and HPAEC-PAD analyses; (b) Enzymatic hydrolysis product of WPOPA was purified by using Bio-Gel P-2 column; (c) Monosaccharide composition of WPOPA-Oligo.

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International Journal of Biological Macromolecules-Jingmin Yan-Figure 5.

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International Journal of Biological Macromolecules-Jingmin Yan-Figure 6.

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International Journal of Biological Macromolecules-Jingmin Yan-Figure 7.

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ACCEPTED MANUSCRIPT Highlights 1. Water-soluble polysaccharides were obtained from four species of edible mushrooms. 2. Four neutral fractions and four acidic fractions were isolated and purified.

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3. Acidic fractions exhibited stronger antioxidant effects than neutral fractions. 4. β-(1→6)-glucan branched at O-3 by β-D-Glcp and β-D-GlcpA might be

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responsible for antioxidant activities.

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