Molecular properties and antioxidant activities of polysaccharides isolated from alkaline extract of wild Armillaria ostoyae mushrooms

Article abstract of DOI:10.1016/j.carbpol.2015.05.061

This study aims to discover novel and bioactive polysaccharides (PS) from wild Armillaria ostoyae, a honey mushroom species. Two PS designated AkPS1V-1 (66.6 kDa) and AkPS1V-2 (15.3 kDa) were isolated and fractionated by anion ion exchange (IEC) and size exclusion chromatography (SEC) from the alkaline extract of A. ostoyae mushrooms. AkPS1V-1 was a glucan composed of solely glucose residues and AkPS1V-2 a heteropolysaccharide composed of glucose and galactose at 6:1 molar ratio. AkPS1V-2 exhibited higher antioxidant activities than AkPS1V-1 based on reducing power, radical scavenging and metal chelating assays. The structure of AkPS1V-2 was further analyzed and elucidated as a branched galactoglucan with a backbone composed of (1→6)-β-d-glucopyranosyl, (1→3)-β-d-glucopyranosyl, (1→3)-α-d-galactopyranosyl and (1→3,6)-β-d-glucopyranosyl residues at 3:1:1:1 ratio, and side chain of (1→3)-β-d-glucopyranosyl residue. This is the first report on a pure PS structure and its antioxidant activities from this mushroom species.

Full text of DOI:10.1016/j.carbpol.2015.05.061

Accepted Manuscript
Title: Molecular properties and antioxidant activities of
polysaccharides isolated from alkaline extract of wild
Armillaria ostoyae mushrooms
Author: Ka-Chai Siu Lijian Xu Xia Chen Jian-Yong Wu
PII:
S0144-8617(15)00474-9
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.carbpol.2015.05.061
CARP 9965
To appear in:
Received date:
Revised date:
Accepted date:
1-4-2015
15-5-2015
25-5-2015
Please cite this article as: Siu, K.-C., Xu, L., Chen, X., and Wu, J.-Y.,Molecular
properties and antioxidant activities of polysaccharides isolated from alkaline
extract of wild Armillaria ostoyae mushrooms, Carbohydrate Polymers (2015),
http://dx.doi.org/10.1016/j.carbpol.2015.05.061
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Highlights
 Polysaccharides (PS) were isolated from alkaline extract of Armillaria ostoyae.
 AkPS1V-1 was a 66.6 kDa glucan and AkPS1V-2 a 15.3 kDa galactoglucan.
 AkPS1V-2 was composed of 6:1 Glc/Gal and O-3-linked Glcp branches.
 AkPS1V-2 showed higher antioxidant activities than AkPS1V-1.
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Revised Ms CARBPOL-D-15-00837
Submitted to: Carbohydrate Polymers
(Original Research MS)
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Molecular properties and antioxidant activities of polysaccharides isolated from
alkaline extract of wild Armillaria ostoyae mushrooms
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Ka-Chai Siu a, Lijian Xu b, Xia Chen, Jian-Yong Wu a, 
a
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Department of Applied Biology & Chemical Technology, State Key Laboratory of Chinese
Medicine and Molecular Pharmacology in Shenzhen, The Hong Kong Polytechnic University,
Hung Hom, Kowloon, Hong Kong
b College of Agricultural Resource and Environment, Heilongjiang University, 15000 Harbin,
China
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 Corresponding author:
Tel: +852 3400 8671; E-mail address: jian-yong.wu@polyu.edu.hk (J.Y. Wu).
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Abstract
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This study aims to discover novel and bioactive polysaccharides (PS) from wild Armillaria
ostoyae, a honey mushroom species. Two PS designated AkPS1V-1 (66.6 kDa) and
AkPS1V-2 (15.3 kDa) were isolated and fractionated by anion ion exchange (IEC) and size
exclusion chromatography (SEC) from the alkaline extract of A. ostoyae mushrooms.
AkPS1V-1 was a glucan composed of solely glucose residues and AkPS1V-2 a
heteropolysaccharide composed of glucose and galactose at 6:1 molar ratio. AkPS1V-2
exhibited higher antioxidant activities than AkPS1V-1 based on reducing power, radical
scavenging and metal chelating assays. The structure of AkPS1V-2 was further analyzed and
elucidated as
a
branched galactoglucan with
a
backbone composed of
(16)--D-glucopyranosyl, (13)--D-glucopyranosyl, (13)--D-galactopyranosyl and
(13,6)--D-glucopyranosyl residues at 3:1:1:1 ratio, and side chain of
(13)-β-D-glucopyranosyl residue. This is the first report on a pure PS structure and its
antioxidant activities from this mushroom species.
Keywords: Armillaria ostoyae; Polysaccharides; Extraction; Fractionation; Structure;
Antioxidant activity.
1. Introduction
Edible fungi or mushrooms are nutritious, and healthy foods and many have notable
medicinal properties and bioactivities. Polysaccharides (PS) represent a class of the most
abundant biopolymers in edible fungi which can have a wide range of nutritional and
medicinal functions such as antitumor, immunomodulatory, prebiotic and antioxidant
activities (Stachowiak and Regula, 2012). Extraction is the first important step for
acquirement of the PS from mushrooms. Most of the bioactive (non-starch) PS serve as the
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structural components of fungal cell wall. To make a tough cell wall, these PS form a rigid
and complex network. Both the tough cell wall and the rigid PS property create resistance to
the extraction of PS from mushrooms. While hot water extraction (HWE) can retain the
water-soluble PS, aqueous alkaline extraction is more effective to break the cell wall and to
extract the PS from mushrooms (Latgé, 2007). Several of the well-known antitumor PS were
extracted by alkaline water such as lentinan from Lentinus edodes (Chihara, Hamuro, Maeda,
Arai & Fukuoka, 1970) and MD/D fraction from Grifola frondosa (Mayell, 2001).
The properties and bioactivities of PS depend on the structure characteristics such as
monosaccharide composition, glycosidic bond, degree of branching, and molecular weight
(MW). The most common structure form of antitumor and immunomoactive PS from
mushrooms is -(13) glucan with side chains, such as lentinan from Lentinus edodes and
Schizophyllan from Schizophyllum commune, and some linked with protein/peptide as PSP
complexes (Ooi & Liu, 2000; Zhang, Cui, Cheung & Wang, 2007). In addition to the
well-known antitumor and immunomodulation, antioxidant activities have been widely
evaluated in many recent studies on PS from various edible fungi (Kozarski et al., 2012;
Lindequist, Niedermayer & Jülich, 2005 Vaz et al., 2011). However, most of the fungal PS
tested for antioxidant activities were crude or partially purified PS fractions with complex or
unknown chemical composition, and the actual activity of pure PS is uncertain.
Armillaria fungal species, generally called honey mushrooms for their yellow brown
caps and sweet fragrance, belong to the family Tricholomataceae (Pegler, 2000; Muszynska,
Sulkowska-Ziaja, Wolkowska, & Ekiert, 2011). This species grows symbiotically with
Gastrodia elata (Tin Ma) plant which is a famous Chinese herbal medicine (Cha & Igarashi,
1995). Some Armilliaria species are edible, and consumed around the world, such as A. mella
and A. ostoyae in Europe and China, A. matsutake S. in Japan and A. ponderosa in North
America. Armilliaria mushrooms have significant nutritional and medicinal functions.
Extract of A. mellea and G. elata has been formulated into herbal drug tablets called “Tian Ma
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Mi Huan Jun Pian” in China to treat various diseases such as headache, insomnia,
neurasthenia and infectious diseases (Gao, Li, Zhao & Wang, 2009; Tang & Eisenbrand,
1992). As the major bioactive constituents of Armillaria, PS have shown antitumor,
immunodulating and antidiabetes effects (Kiho, Shiose, Nagai & Ukai, 1992; Lung & Chang,
2013; Vaz et al., 2011; Wu et al., 2012).
A. ostoyae is a popular edible mushroom in northeastern China and is widely
distributed in the forests. However, there is no literature report on the molecular structure and
antioxidant activity of PS from A. ostoyae. In this study, we applied a two-step extraction
protocol, first by HWE to remove proteins and water soluble constituents, and next by
alkaline extraction of wild A. ostoyae mushrooms collected from the Lesser Khingan Range
forest in northeastern China. The crude PS isolated by ethanol precipitation were further
purified and fractionated through a series of steps for further determination of molecular
properties and antioxidant activities.
2. Materials and methods
2.1. Mushroom materials
Wild A. ostoyae mushrooms were collected from the Lesser Khingan Range forest in
the Wuying region in China (Wuying Forestry Bureau) (altitude 320 m, coordinates 48°04' of
Northern latitude and 129°15' of East longitude) in September 2009. The mushroom species
was identified by comparing their morphological traits with reliable reference (Liu, 2004),
and further confirmed by comparing the DNA sequence with the GenBank (National Center
for Biotechnology Information website [http://www.ncbi.nlm.nih.gov]). The mushrooms were
air dried, enclosed within plastic bags, and stored at 25oC before use.
2.2. Extraction and isolation of PS
The mushrooms were dried completely in an oven at 50 oC to constant weight, and
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ground into powder with an electric mill. The mushroom power (100 g) was first defatted
with petroleum ether (PE) and then ethanol (EtOH) for 6 h each. The solid residue was then
subject to hot water extraction (HWE) at 90oC (~2 L distilled water) for the water soluble
constituents. The residue collected after filtration was subject to alkaline extraction with 0.5
M NaOH and 0.05 M NaBH4 in water (~2 L) at 4oC for 6 h for three times. The alkaline
extract was neutralized to pH 7 by acetic acid, and then concentrated by vacuum evaporation
below 45oC. The concentrated extract was subject to sequential ethanol precipitation in three
steps, at 0.3:1, 1:1 and 5:1 EtOH (95%) volume ratio. The liquid mixture was kept at 4 oC for
overnight in each precipitation step. The precipitate was collected by centrifugation and
freeze-dried, yielding the crude PS, AkPS0.3V, AkPS1V and AkPS5V.
2.3. Purification of AkPS1V
The crude PS precipitated in the second step with 1:1 EtOH volume ratio, AkPS1V, was
deproteinized by repeated treatment with Sevag regent (1:4 butanol to chloroform) until no
precipitate formed, and then decolorized with 10% H2O2 at pH 8.5 and room temperature for
5 h. The AkPS1V solution was then dialyzed against distilled water through a 3500 Da MW
cutoff membrane for 5 days and then lyophilized. The partially purified AkPS1V was
fractionated by ion exchange chromatography (IEC) as follows. The AkPS1V was
redissolved in 20 mM Tris-HCl (pH 7.4) and then loaded to 2.6  27 cm DEAE cellulose-52
IEC column (Sigma, St. Louis, MO, USA) which had been equilibrated with Tris-HCl buffer
solution (20 mM, pH 7.4). The column was eluted sequentially with Tris-HCl (20 mM, pH
7.4) containing 0, 0.1, 0.3, 0.5 or 1 M NaCl at a flow rate of 1.5 mL/min. The eluate was
collected with a fractional collector at 4 mL per tube. The carbohydrate content was
monitored by anthrone test and the protein content monitored spectrophotometrically at 280
nm. The PS fraction eluted by the buffer from IEC was collected and further purified by size
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exclusion chromatography (SEC) on a 2.6  60 cm Superdex G-75 column (Sigma). The SEC
column was loaded with 50 mg of PS sample and eluted with 0.3 M (NH3)2CO3 at 0.3
mL/min. The selection of (NH3)2CO3 as the mobile phase for SEC was according to literature
(Siddiqui & Wood, 1971; Powell, Ahmed, Yates & Turnbull, 2010) and the specific
conditions were set based on our preliminary trials. Two fractions abundant in sugar were
collected, dialyzed, and freeze dried, yielding AkPS1V-1 and AkPS1V-2.
2.4. Analysis of AkPS1V-1 and AkPS1V-2
2.4.1. Intrinsic viscosity and molecular weight distribution
The intrinsic viscosity, [η], was determined by Huggins and Kraemer equations,
sp /c = [] + k[]2c
(1)
(2)
(lnr) /c = [] – (1/2-k)[]2c
where sp/c is reduced specific viscosity, (lnr)/c inherent viscosity, and k is a constant
depending on the molecular properties, solvent and experimental conditions. The viscosity of
CPS solution in water was measured at 30°C with an Ubbelohde capillary viscometer (Huang,
Siu, Wang, Cheung & Wu, 2013).
MW distribution of PS fractions was analyzed by high-pressure gel permeation
chromatography (HPGPC) with two columns in series, Ultrahydrogel 250 and Ultrahydrogel
2000 (both 7.8 mm  300 mm dimensions from Waters Co., Milford, MA, USA), with a
Waters 1515 isocratic HPLC pump and a Waters 2414 refractive index detector, as described
by Huang et al. (2013). Distilled water was used as the mobile phase at 0.6 mL/min and 50°C.
PS samples were predissolved in distilled water at 5 mg/mL and centrifuged at 18,000 rpm
for 25 min before injection. Dextran MW standards ranging from 1.0 to 670 kDa (Sigma)
were used for calibration and the peak MW was computed with the Breeze V3.3 software.
2.4.2. FT-IR and NMR spectroscopy
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Infrared (IR) spectrometry of AkPS1V-1 and AkPS1V-2 was performed in the 4000–500
cm-1 region on an Avatar 360 FTIR spectrometer (Thermo Nicolet, Cambridge, UK). Samples
were pressed into tablets with KBr and subjected to 64 scans at 4 cm-1 resolution in reference
to air to obtain an average spectrum in the Spectrum 6.1 software.
1
NMR spectroscopy of AkPS1V-2 including H NMR, 13C NMR and HMBC was
performed on a Bruker AVANCE III 600 spectrometer at 25oC. The PS sample (5 mg) was
lyophilized in deuterium twice and then dissolved in 500 μL 99.8% D2O before analysis.
2.4.3. Monosaccharide composition
Monosaccharide composition of PS fractions was analyzed by HPLC after acid
hydrolysis and 1-phenyl-3-methyl-5-pyrazolone (PMP) reaction as reported previously (Siu,
Chen, & Wu, 2014). In brief, 5 mg of sample was hydrolyzed with 2 M trifluoroacetic acid
(TFA) (2 mL) at 110oC for 4 h. The hydrolysate was then dried under nitrogen in a hot water
bath and re-dissolved in 2 mL water. The hydrolysate solution (450 μL) was mixed with 450
μL of 0.5 M 1-phenyl-3-methyl-5-pyrazolone (PMP) solution in methanol, and 450 μL of 0.3
M NaOH solution and then reacted at 70oC for 30 min. The reaction was stopped by
neutralizing with 450 μL of 0.3 M HCl, and the product was partitioned with chloroform
three times. The aqueous layer was collected and filtered through a 0.45 μM membrane, and
applied to HPLC. The HPLC was performed with an Agilent ZORBAX ECLIPSE XDB-C18
column (150 mm x 4.6 mm) on an Agilent 1100 instrument at 25oC with potassium phosphate
buffered saline (0.05 M, pH 6.9) containing 15% (solvent A) and 40% acetonitrile (solvent B)
as mobile phases, and UV detection at 250 nm. Monosaccharide standards for the
identification and quantification of the corresponding peaks were from Sigma.
2.4.4. Methylation analysis
AkPS1V-2 was subjected to methylation, acid hydrolysis, reduction and acetylation to
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give partially methylated alditol acetates (PMMAs). Methylation was performed according to
documented method (Ciucanu and Kerek, 1984) with minor modifications. In brief, dry
AkPS1V-2 (5 mg) was predissolved in 2.5 mL anhydrous dimethyl sulfoxide (DMSO) with
constant stirring for 3 h. Anhydrous sodium hydroxide (30 mg) was added to the AkPS1V-2
solution in nitrogen atmosphere with stirring for 30 min. Under nitrogen protection, methyl
iodide (0.8 mL) was slowly added to the solution in an ice bath and stirred in dark at room
temperature for 1 h. The reaction was stopped by adding 2.5 mL distilled water and the
excess amount of methyl iodide was removed by vacuum evaporation at 40oC. The partially
methylated sample was extracted with dichloromethane. The dichloromethane solution was
washed with deionized water three times to remove impurities. The partially methylated
AkPS1V-2 was then evaporated to dryness under vacuum in a rotatory evaporator at room
temperature. The methylation process was repeated three times for completion.
The partially methylated AkPS1V-2 was further hydrolyzed with 2 M TFA (1 mL) in a
sealed tube at 110oC for 6 h, and excess TFA was removed with a stream of nitrogen in a
boiling water bath. The dried hydrolysate was redissolved in 1 mL ammonia saturated water
and reduced with excess NaBH4 at room temperature for 12 h. Excess NaBH4 was reacted
with acetic acid (till no bubbles appearing) and the borate acid formed was removed by
co-distillation with methanol. The dried residue was acetylated with 1 mL acetic anhydride in
a sealed tube at 110oC for 2 h to form partially methylated alditol acetate (PMAA). The
PMMA was evaporated to dryness under vacuum, and redissolved in chloroform, and washed
three times with water. The products were analyzed with GC-MS using an Agilent 6890N GC
and 5975 VL MSD through a fused silica capillary column (30 x 0.25mm ID, Agilent
HP-5MS). The column temperature was fixed at 100oC for 3 min, then increased to 250oC at
3oC/min and fixed at 250oC for 10 min. The injector and the detector were fixed at 280oC and
250oC, respectively.
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2.5. Antioxidant activity assays
The antioxidant activity of PS fractions was determined by three chemical assays,
Trolox equivalent antioxidant capacity (TEAC), ferric reducing ability of plasma (FRAP),
and ferrous ion Fe2+ chelating as reported in detail previously (Siu et al., 2014). In all assays,
the PS samples were predissolved in water into a series of dilution. For the TEAC assay in
brief, ABTS+ radicals were generated from 2,2’-azinobis(3-ethylbenzothiazoline-6-sulfonic
acid) (ABTS) oxidation by potassium persulfate (K2S2O8). The ABTS+ solution (500 μl) was
added into various concentrations of PS sample solution in water to attain an initial
absorbance of 0.70 ± 0.02 at 734 nm. The TEAC activity of sample was expressed in μmol
Trolox/g sample. For the FRAP assay, PS sample solution (120 μl) was mixed with 900 μl of
freshly prepared FRAP reagent solution, and incubated at room temperature in dark for 2 h,
followed by measurement of the absorbance at 593 nm using ferrous sulfate (FeSO4) as a
reference. FRAP activity was expressed in μmol FeSO4/g sample. For the Fe2+ chelating assay,
PS sample solution (1 ml) was mixed with 20 μl of 2 mM FeCl2 for 1 min, followed by the
addition of 40 μl of 5 mM ferrozine. The mixture was incubated at room temperature for 10
min, followed by measurement of absorbance at 562 nm. The chelating activity was
expressed in μmol Trolox/g sample.
2.6. Statistical analysis
All experiments were performed in triplicates and the results were expressed as mean ±
standard deviation (SD). Statistical significance of the antioxidant tests was determined by a
one way analysis of variance (ANOVA) followed by Student’s t-test. Difference was
considered to be statistically significant at p<0.05.
3. Results and discussion
3.1. Yields and properties of crude PS from alkaline extract
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Table 1 shows the yields, properties and antioxidant activities of the three crude PS
fractions isolated from the alkaline mushroom extract by sequential EtOH precipitation with
0.3, 1 and 5 volume ratio of EtOH to the extract liquid. The highest yield was achieved at 0.3
EtOH volume ratio, and the yields were much lower in the later steps of precipitation,
suggesting that the concentration of higher MW PS in the water extract was much higher.
EtOH causes the precipitation of PS in an aqueous solution due to its high miscibility with
water to compete for the water molecules bound to the PS surface. With increasing EtOH
concentration in the PS solution, PS become unstable and form aggregate, and then
precipitate. The crude PS with a higher MW is precipitated at a lower EtOH concentration. As
predicted, the intrinsic viscosity (correlated to the average MW) of PS was highest at the
lowest EtOH and vice versa. All crude PS fractions isolated from the alkaline extract were
composed mainly of carbohydrate and protein plus a small portion of phenolic compounds.
The crude PS fraction attained at 5 volume ratio of EtOH also had the highest protein and
phenolic contents as well as the highest antioxidant activities.
Table 1. Yields, chemical contents and antioxidant activities of crude PS fractions isolated
from alkaline extracts of A. ostoyae mushrooms by differential ethanol precipitation with 0.3,
1 and 5 ethanol volume ratios to the extract liquid.
EtOH Yield
Intrinsic
Total content (wt%)
Sugar Protein
Activity *
TEAC
ratio (wt%) viscosity (dL/g)
Phenolics
FRAP
0.3
1
3.30
1.36
0.35
0.25±0.01
0.20±0.01
0.03±0.00
45.8±2.3 34.7±1.9 1.37±0.1
53.3±2.6 10.7±0.1 1.51±0.1
29.1±1.4 43.5±0.7 2.86±0.3
39.9±3.9 14.4±1.3
38.1±2.1 26.7±1.9
115±8.7 53.8±2.7
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249
*TEAC in μM Trolox/g, FRAP in μM Fe2+/g, derived from the calibration curves generated
by Trolox and ferrous sulphate, respectively.
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3.2. MW profiles of purified PS fractions
Since AkPS0.3V was barely soluble in water and AkPS5V had a very low yield,
AkPS1V, which also had the highest total sugar content, was collected for further
fractionation (by IEC and SEC) and structural analysis. On the IEC elution profile of
AkPS1V (Fig. 1a), the first peak eluted out with buffer solution was composed of protein-free
PS, while the two later peaks eluted with 0.1 and 0.3 M NaCl in the buffer solution had
absorbance at 280 nm for protein, and were probably PSPs (supplemental data Fig. 2). The
first and second peaks were major fractions of AkPS1V accounting for 38% and 43% of the
total mass, respectively. The protein-free PS fraction eluted out from SEC column exhibited
two peaks (Fig. 1b), which were collected as AkPS1V-1 (tubes 23-29) and AkPS1V-2 (tubes
33-45), accounting for 37% and 63% of the total mass, respectively. AkPS1V-1 and
AkPS1V-2 both exhibited a sharp, symmetric peak on HPGPC, indicating their MW
homogeneity (supplemental data Fig. 3), and their average MWs were calibrated to 66.5 and
15.3 kDa, respectively. Acid hydrolysis of AkPS1V-1 resulted in glucose residues only while
that of AkPS1V-2 contained glucose and galactose at 6:1 molar ratio according to HPLC
analysis (supplemental data Fig. 4).
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AkPS1V-2
(b)
AkPS1V-1
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Fig. 1. Chromatographic profiles of AkPS1V fractions: (a) AkPS1V on a DEAE-cellulose 52
IEC column (eluted with different concentration of NaCl solution at 1.5 mL/min); (b) Neutral
AkPS1V from (a) eluted out of the Superdex 75 SEC column with 0.3 M (NH3)2CO3.
Absorbance at 620 nm represents total carbohydrate content (relative) determined by
anthrone test.
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3.2. FT- IR spectral characteristics of AkPS1V-1 and AkPS1V-2
The FT-IR spectra of AkPS1V-1 (Fig. 2a) and AkPS1V-2 (Fig. 2b) were characteristic
of PS structure. The broad intense peak at around 3400 cm-1 is attributed to the –OH group
and the peak at around 1380 cm-1 to the –OH bending vibration. The peak at around 2920
cm-1 is assigned to the weak C-H stretching vibration and that at round 1635 cm-1 to the ring
stretching of glucose. The peak at around 1075 cm-1 is attributed to the presence of
pyranoside. The absence of absorption peak at around 1730 cm-1 confirms the absence of
uronic acid.
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893
1384
2914
1635
1073
(a)
3435
895
1638
1381
2925
1076
3399
(b)
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Fig. 2. FT-IR spectra of (a) AkPS1V-1 and (b) AkPS1V-2.
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Table 2 shows the fragments of AkPS1V-2 from methylation analysis including
2,3,4,6-Tetra-O-Me-Glcp,
2,4,6-Tri-O-Me-Glcp/Galp,
2,3,4-Tri-O-Me-Glcp
and
2,4-Di-O-Me-Glcp in a relative molar ratio of 1:2:3:1. This set of results suggests that
AkPS1V-2 was composed of (13,6), (16) and (1) glucopyranosyl and (13) linked
galactopyranosyl/glucopyranosyl residues.
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Table 2. GC-MS result of partially methylated alditol acetates of AkPS1V-2.
Methylated sugar
Mass Fragment (m/z)
Molar
ratio
1
Linkage
type
2,3,4,6-Tetra-O-Me-Glcp 59,71,87,101,117,129,145,161,205
1
2,4,6-Tri-O-Me-Glcp/
2,4,6-Tri-O-Me-Galp
2,3,4-Tri-O-Me-Glcp
2,4-Di-O-Me-Glcp
58,71,87,101,117,129,161,233
2
13
58,71,87,101,117,129,161,189,233
3
16
58,74,87,101,117,129,139,159,173,189,233 1
13,6
14
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292
293
294
295
296
297
298
299
300
301
302
303
304
305
306
307
308
309
310
311
312
The structural characteristics of AkPS1V-2 were further deduced from the NMR spectra
1
(Fig. 3). On the H NMR spectrum (Fig. 3a), the three overlapped peaks in the anomeric
region (δ 4.41 ppm) are attributed to β-anomeric protons, and another peak at δ 4.9 ppm to
α-anomeric proton. The chemical shifts from δ 3.2-4.1 ppm are assigned to protons of H-2 to
H-6. The three β-configuration pyranose units and one α-configuration pyranose unit were
further confirmed by the three anomeric carbon signals around  103 ppm on the 13C NMR
spectrum (Fig. 3b), which are ascribed to (13,6), (16) and (13)-D-glucopyranosyl, and
one anomeric carbon signal in  98 ppm to (13)-D-galactopyranosyl. The relevant sugar
residues were confirmed by labelling the C-1 to C-6 in 13C NMR spectrum according to
reference data (Table 3). The structural characteristics derived from NMR corroborate those
from GC-MS.
3.3. Linkage and structural characteristics of AkPS1V-2
1
Based on the H-13C HMBC spectrum (Fig. 4), the linkage sites and the sequence
among residues were established (Table 3). A cross peak was observed between C-1 (δ 103
ppm) of residue A and H-3 (δ 3.66 ppm) of residue B [A(C1)/B(H3)], suggesting that C-1 of
residue A was linked to H-3 of residue B. Similarly, the cross peaks at δ 97.9/3.66 ppm
[B(C1)/C(H6)], δ 102.8/3.13 ppm [C(C6)/C(H1)], δ 103/3.42 ppm [C(C1)/D(H3)] and δ
102.9/4.02 ppm [D(C1)/A(H6)] suggest that the C-1 of residue B was linked to H-6 of
residue C, C-1 of residue C to H-6 of another residue C, C-1 of residue C to H-1 of residue D,
and C-1 of residue D to H-6 of residue A.
15
Page 15 of 24

[ref]
15
(a)
10
5
[ppm]
3.0
4.5
4.0
3.5
5.5
5.0
[ref]
14
(b)
12
10
8
6
4
2
100
90
70
60
80
[ppm]
313
314
Fig. 3. (a) 1H-NMR spectrum and (b) 13C-NMR spectrum of AkPS1V-2.
315
C(H1)/C(C6)
B(H1)/C(C6)
316
317
318
319
320
D(H3)/C(C1)
C(H6)/B(C1)
321
322
323
B(H3)/A(C1)
A(H6)/D(C1)
A(H3)/T(C1)
324
Fig. 4. HMBC spectrum of AkPS1V-2.
325
326
16
Page 16 of 24

326
327
Table 3. Assignment of 1H NMR and 13C NMR chemical shifts of AkPS1V-2.
Sugar residue
Chemical shifts (ppm)
H1/C1
4.41/
102.8
4.41/
97.9
H2/C2
3.22/
73.4
H3/C3
3.42/
84.2
H4/C4
3.34/
69.7
4.1/
H5/C5
3.51/
74.6
4.1/
H6/C6
3.65, 4.09/
70.1
(A): 3,6)--D-Glcp(1
(B): 3)--D-Galp(1
(C): 6)--D-Glcp(1
(D): 3)--D-Glcp(1
3.94/
70.4
4.03/
80.0
3.6, 3.6/
61.9
71.6
3.36/
69.5
3.21/
69.9
73.5
3.50/
74.9
3.24/
76.0
4.41/
103.0
4.9/
3.20/
73.1
3.38/
75.6
3.72, 4.09/ 68.9
3.25/
73.5
3.17/
85.3
3.33, 3.58/
61.08
102.9
328
329
330
331
Based on all above analytical results, the following structural unit of AkPS1V-2 is
established,
β-D-Glcp
332
333
334
335
336
337
338
339
340
341
342
343
344
345
346
1

3
{6)-β-D-Glcp-(13)--D-Galp-(1[6)--D-Glcp-(1]33)--D-Glcp-(1}n
Various polysaccharides with various molecular structures of have been isolated from
Armillaria species, such as an α-(13) glucan isolated from A. mellea fruiting bodies
(Sánchez , Garcia & Novaes-Ledieu, 1993) and an α-(1→6) glucan from A. tabescens
mycelia (Luo, Xu, Yu, Yang & Zheng, 2008). Some glucans from Armillaria were in the form
of PSP complexes, such as a glucan with β-(13) and β-(16) linkages and a glucan with
α-(13) and α-(14) linkages from A. mellea fruiting bodies (Amar, Delaumèny, & Vilkas,
1976; Sánchez et al., 1993). In addition, various heteropolysaccharides such as xylomannan,
heterogalactan have been isolated from A. mellea (Bouveng et al., 1967; Fraser & Lindberg,
1967; Sun, Liang, Zhang, Tong & Liu, 2009). Up to now, however, there is no literature
report on a PS molecular structure from the A. ostoyae species.
17
Page 17 of 24

347
348
349
350
351
352
353
354
355
356
357
3.4. Antioxidant activities of AkPS1V-1 and AkPS1V-2
Fig. 5 shows the antioxidant activities of AkPS1V-1 and AkPS1V-2 measured by (a)
TEAC, (b) FRAP and (c) ferrous ion chelating assays, respectively, all in a dose-dependent
manner. AkPS1V-2 showed a slightly higher activity than AkPS1V-1 in all three assays (Fig.
5 & supplemental data Table 1). As both PS had the similar chemical composition with
glucose as the major constituent (>86%), the higher antioxidant activity of AkPS1V-2 (15.5
kDa) could be partially attributed to its lower MW than AkPS1V-1 (66.6 kDa). In comparison
of the data in Table 1 and Supplemental data Table 1, the antioxidant activities of AkPS1V-1
and AkPS1V-2 were only significantly different from those of the crude PS, AkPS1V, from
which these two were fractionated. This implies that the antioxidant activity of PS was not
enhanced with the purification process and increase in the chemical purity of PS fractions.
100
358
(a)
80
359
360
361
362
363
364
365
366
367
368
369
370
371
60
AKPS1V-1
40
AKPS1V-2
20
0
0
500
1000
1500
2000
2500
Concentration (ppm)
1000
800
600
400
200
0
(b)
AKPS1V-1
AKPS1V-2
0
200
400
600
800
1000
Concentration (ppm)
372
100
80
60
40
20
0
373
374
375
(c)
AKPS1V-1
AKPS1V-2
18
0
2000
4000
6000
8000 10000
Page 18 of 24
Concentration (ppm)

376
377
378
379
380
381
382
383
384
385
386
387
388
389
390
391
392
393
394
395
396
397
398
399
400
401
Fig. 5. Antioxidant activities of AkPS1V-1 and AkPS1V-2 measured by (a) TEAC, (b) FRAP,
and (c) ferrous ion chelating assays (error bars for SD, n = 3).
Although rhamnose has been shown the most significant correlation with the
antioxidant activity of PS among the monosaccharides (Lo, Chang, Chiu, Tsay & Jen, 2011),
glucans as the major component of crude PS extracted from several medicinal mushrooms
have also shown significant metal chelating and reducing power (Kozarski et al., 2012).
Some purified glucans have also shown antioxidant activities such as the two glucans GLPL1
and GLPL2 from G. lucidum, both containing (13), (14) and (16) glucopyranosyl
residues (Liu, Wang, Pang, Yao & Gao, 2010). The MW of PS is another factor affecting their
bioactivities. In our present study, AkPS1V-2 with a lower MW (15.3kDa) showed higher
radical scavenging, reducing and metal chelating abilities than AkPS1V-1 with a higher MW
(66.6 kDa). The increasing antioxidant activity with MW reduction of PS has also been
demonstrated with lower PS fractions derived from degradation of microbial PS with various
methods such as acid hydrolysis (Yan et al., 2009), microwave radiation (Sun, Wang, Shi &
Ma, 2009), and ultrasonication (Zhou, Yu, Zhang, He & Ma, 2012). Although it is quite
uncertain about the active sites or functional groups responsible for the antioxidant activities
of polysaccharides, the hydroxyl group has been regarded as the most possible group
contributing to the antioxidant action by donating hydrogen atoms to stabilize the radicals
and to terminate the radical chain reaction (Hu, Zhang, & Kitts, 2000). It has been suggested
that, at an equal mass basis, PS with a lower MW may have more reductive hydroxyl
terminals to react with the radical species and tend to have a higher antioxidant activity (Yan
et al., 2009). However, we should not assume that MW and antioxidant activity follow a
simple and proportional relationship. Although the MW difference between AkPS1V-2 and
AkPS1V-1 was nearly four times, the differences in their antioxidant activities were very
small (supplemental data Table 1). As stated early in this report, the bioactivities of PS can be
19
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402
403
404
405
406
407
408
409
410
411
412
413
414
415
416
417
418
419
420
421
422
423
424
425
426
427
affected by several structure characteristics such as monosaccharide composition, glycosidic
bond, degree of branching in addition to MW.
PS including intracellular PS and exopolysaccharides (EPS) from another Armillaria
species, A. mellea, have also shown antioxidant activities such as radical scavenging,
reducing power, metal chelating and protection against lipid peroxidation and DNA damage
(Gao & Wang, 2012; Lung & Yu, 2013; Vaz et al., 2011). However, all these antioxidative PS
were in crude form isolated from hot-water extract by ethanol precipitation without further
purification. Such crude PS often contained significant amounts of proteins and low-MW
pigments and are not able to show the actual activities of pure PS (Kozarski et al., 2012; Siu
et al., 2014).
4. Conclusions
Two pure PS with homogenous MW distributions, AkPS1V-1 (66.6 kDa) and
AkPS1V-2 (15.3 kDa), have been isolated from the alkaline extract of wild A. ostoyae
mushrooms through a series of steps. AkPS1V-1 was composed of solely glucose residues
and recognized as a glucan, and AkPS1V-2 was composed of glucose and galactose at 6:1
molar ratio. AkPS1V-2 exhibited a more significant antioxidant capacity due probably to its
lower MW than AkPS1V-1. The molecule structure of AkPS1V-2 has been determined as a
branched galactoglucan. This is a novel PS from this mushroom species. Further activity tests
in animal models should be performed of the pure PS fractions for more reliable evaluation of
their potential as natural antioxidants for human health.
Acknowledgements
This work was supported by grants from the Hong Kong Government UGC (GRF Projects
PolyU 5036/10P and PolyU 5033/11P) and The Hong Kong Polytechnic University.
20
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