Structural features and antioxidant activity of a new galactoglucan from edible mushroom Pleurotus djamor

Article abstract of DOI:10.1016/j.ijbiomac.2020.11.131

A new water soluble galactoglucan with apparent molecular weight ~1.61 × 10⁵ Da, was isolated from the edible mushroom Pleurotus djamor by hot water extraction followed by purification through dialysis tubing cellulose membrane and sepharose 6B column chromatography. The sugar analysis showed the presence of glucose and galactose in a molar ratio of nearly 3:1 respectively. The structure of the repeating unit in the polysaccharide was determined through chemical and NMR experiments as:[Formula presented] In vitro antioxidant studies showed that the PDPS exhibited hydroxyl radical scavenging activity (EC₅₀ = 1.681 ± 0.034 mg/mL), DPPH radical scavenging activity (EC₅₀ = 3.83 ± 0.427 mg/mL), reducing power (EC₅₀ = 4.258 ± 0.095 mg/mL), and ABTS radical quenching activity (EC₅₀ = 0.816 ± 0.077 mg/mL). So, PDPS should be explored as a natural antioxidant.

Full text of DOI:10.1016/j.ijbiomac.2020.11.131

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Structural features and antioxidant activity of a new galactoglucan
from edible mushroom Pleurotus djamor
Gajendra Nath Maity, Prasenjit Maity, Somanjana Khatua,
Krishnendu Acharya, Sudipta Dalai, Soumitra Mondal
PII:
S0141-8130(20)35056-X
DOI:
https://doi.org/10.1016/j.ijbiomac.2020.11.131
BIOMAC 17294
Reference:
To appear in:
International Journal of Biological Macromolecules
Received date:
Revised date:
Accepted date:
17 July 2020
16 November 2020
18 November 2020
Please cite this article as: G.N. Maity, P. Maity, S. Khatua, et al., Structural features
and antioxidant activity of a new galactoglucan from edible mushroom Pleurotus djamor,
International Journal of Biological Macromolecules (2018), https://doi.org/10.1016/
j.ijbiomac.2020.11.131
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Structural features and antioxidant activity of a new galactoglucan from
edible mushroom Pleurotus djamor
a,c
Gajendra Nath Maity , Prasenjit Maity b,d, Somanjana Khatuae, Krishnendu Acharyae, Sudipta
Dalaif, Soumitra Mondal b*
a
Department of Microbiology, Panskura Banamali College, Panskura, Purba Midnapore, Pin-
721152,West Bengal, India.
b
Department of Chemistry, Panskura Banamali College, Panskura, Purba Midnapore, Pin-
721152,West Bengal, India.
c
Department of Microbiology, Asutosh College, 92, S.P. Mukherjee Road, Kolkata- 700026,
West Bengal, India.
d Department of Chemistry, Sabang Sajanikanta Mahavidyalaya, Lutunia, Paschim Midnapore,
Pin-721166, West Bengal, India.
e
Molecular and Applied Mycology and Plant Pathology Laboratory, Department of Botany,
University of Calcutta, 35, Ballygunge Circular Road, Kolkata 700019, West Bengal, India
f Department of Chemistry, Vidyasagar University, Midnapore, 721102, West Bengal, India
* Corresponding author: Soumitra Mondal
E. mail: mondalsoumitra78@yahoo.com
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Abstract
A new water soluble galactoglucan with apparent molecular weight 1.61 × 105 Da, was
isolated from the edible mushroom Pleurotus djamor by hot water extraction followed by
purification through dialysis tubing cellulose membrane and sepharose 6B column
chromatography. The sugar analysis showed the presence of glucose and galactose in a molar
ratio of nearly 3:1 respectively. The structure of the repeating unit in the polysaccharide was
determined through chemical and NMR experiments as:
6)--D-Glcp-(1 4)--D-Glcp-(1 6)--D-Glcp-(1
3

1
-D-Galp
In vitro antioxidant studies showed that the PDPS exhibited hydroxyl radical scavenging
activity (EC50= 1.681 ± 0.034 mg/ml), DPPH radical scavenging activity (EC50= 3.83 ± 0.427
mg/ml), reducing power (EC50= 4.258 ± 0.095 mg/ml), and ABTS radical quenching activity
(EC50= 0.816 ± 0.077 mg/ml). So, PDPS should be explored as a natural antioxidant.
Keywords: Pleurotus djamor, Galactoglucan, Antioxidant activity
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Abbreviations: AcOH= Acetic acid; ABTS=2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonate);
BHA= Butylated hydroxyl anisole; CF3COOH= Trifluoro acetic acid; DEPT= Distortionless
enhancement by polarization transfer; DPPH=‎ Diphenyl–picrylhydrazine; DMSO= Dimethyl
sulfoxide; DQF-COSY= Double-quantum filtered correlation spectroscopy; GLC= Gas-liquid
chromatography; GLC-MS= Gas-liquid chromatography mass spectrometry; HSQC=
Heteronuclear single quantum coherence; NMR= Nuclear magnetic resonance; NOESY=
Nuclear overhauser enhancement spectroscopy; PDPS= Pleurotus djamor polysaccharide
(heteroglycan); P2O5= Phosphorus pentoxide; TOCSY= Total correlation spectroscopy.
1. Introduction
Edible mushrooms are very important foodstuff and folk medicine all over the world [1,
2]. From edible mushrooms prepared hot aqueous solutions, containing mostly polysaccharides,
were considered as a medicine since ancient times [3]. Polysaccharides from edible mushrooms
are considered as an antitumor agent [4-8]. Specifically some edible mushrooms, Lentinus,
Hericium, Grifola, Flammulina, Pleurotus, and Tremella have highly medicinal and functional
properties [9]. Pleurotus genus (Class Basidiomycetes) is among the most popular and edible
macrofungi which are commonly regarded as oyster mushrooms. This species is widely
cultivated in several countries especially due to its flavour, texture and health benefits [10].
Generally, Pleurotus spp. is grown in the woody forests of the earth [11, 12]. These mushrooms
contain mainly carbohydrates, proteins, dietary fiber, minerals, some essential amino acids, and
vitamins [13]. Pleurotus spp. has been demonstrated in many previous studies to possess
valuable medicinal attributes such as antioxidant, immunomodulatory, hypocholesterolemic,
anti-inflammatory, anti-hyperglycaemic, anti-genotoxic, anti-tumour, anti-viral, anti-Human
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Immunodeficiency Virus (HIV), hepatoprotective, anti-mutagenic, anti-allergic and anti-ageing
effects [14]. The genus encompasses about 50 species of which nearly 26 taxa have been
included from India. One such common oyster mushroom is Pleurotus djamor that is appreciated
worldwide as food encouraging artificial cultivation and economic benefit. In earlier
experimental studies, P. djamor has been reported to possess strong anti-inflammatory,
analgesic, antioxidant, antimicrobial, antiplatelet and antipyretic activity [15]. They have been
reported to modulate the immune system, and to inhibit tumor growth [11, 16]. The cultivation of
the genus Pleurotus such as Pleurotus sajorcaju, Pleurotus citrinopileatus, Pleurotus ostreatus,
Pleurotus florida and Pleurotus djamor has been increasing at a rapid rate [17]. Previously a
non-toxic water insoluble β-glucan was isolated from the alkaline extract of the fruit body of
Pleurotus djamor, and the detailed structural investigation and study of cytotoxic effect were
carried out and reported [18]. A new water soluble polysaccharide (PDPS) has been isolated
from the aqueous extract of the edible mushroom Pleurotus djamor through gel permeation
chromatography. The present study was carried out to evaluate the detailed structural
investigation and antioxidant activities of PDPS.
2. Materials and methods
2.1. Isolation and purification of the polysaccharide
Dry fruiting body of edible mushroom Pleurotus djamor (500 g) were extracted by
distilled water at 100 ◦C for 7 h. The mixture was kept overnight and filtered through fresh linen
cloth. The supernatant was collected after centrifugation at 8000 rpm at 4◦C for 1 h and then
precipitated by adding 5 times volumes of ethanol. The precipitate was dialyzed through tubing
cellulose membrane against distilled water for 2 days. The dialyzed material was freeze-dried to
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obtain crude polysaccharide (800 mg). During purification process, a single homogeneous
fraction (test tube 26-37) was collected and freeze-dried [19, 20]. The same route was repeated
several lots to get 120 mg of PDPS for further analysis.
2.2. Determination of molecular weight
The apparent molecular weight of the PDPS was determined by gel-permeation
chromatography [19]. Standard dextrans T-200, T-70, and T-40 were passed through a Sepharose
6B column, and then the elution volumes were plotted against the logarithms of their respective
molecular weights. The elution volume of PDPS was plotted in the same graph and the average
molecular weight of PDPS was measured.
2.3. Absolute configuration of monosaccharide
According to the method Gerwig, Kamerling, and Vliegenthart, the D and L notation of a
sugar was evaluated [19, 21]. PDPS (1.0 mg) was hydrolyzed with CF3COOH, and then the acid
was removed by co-distillation with water. 250 l of 0.625 M HCl solution treated with R- ()-2-
butanol was added to the hydrolyzed product and heated at 80 C for 16 h. Then the reactants
were evaporated and TMS-derivatives were prepared with N,O-bis (trimethylsilyl)
trifluroacetamide (BSTFA). The products were analyzed by GLC using a capillary column SPB-
1 (30 m  0.26 mm) with a temperature program (3 C/min) from 150 to 210 C. The resulting
2,3,4,6-tetra-O-TMS-(+)-2-butylglycosides were identified by comparison with those prepared
from the D and L enantiomers of different monosaccharides.
2.4. Optical rotation
Optical rotation was measured through a Jasco Polarimeter model P-1020 at 31 °C.
2.5. Monosaccharide analysis
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The neutral monosaccharide composition of the PDPS was determined by gas liquid
chromatography (GLC) analysis. PDPS (3.0 mg) was hydrolyzed with 2 M CF3COOH (2 mL) at
100 °C in a round-bottom flask for 18 h in a boiling water bath. The excess acid was completely
removed by co-distillation with water. Then, the hydrolyzed product was reduced with NaBH4 (8
mg), followed by acidification with dilute CH3COOH, and then co-distilled with pure CH3OH to
remove excess boric acid. The reduced sugars were acetylated with 1:1 pyridine–acetic
anhydride in a boiling water bath for 1 h to give the alditol acetates, which were analyzed by
GLC [18, 19].
2.6. Methylation analysis
The PDPS (4.0 mg) was methylated according to the method of Ciucanu and Kerek
method where distilled DMSO and finely grounded NaOH were used. The methylated product
was then hydrolyzed with 90 % formic acid (1 ml) at 100 C for 1 h and excess formic acid was
evaporated by co-distillation with distilled water. The hydrolyzed product was then reduced with
sodium borohydride and acetylated with pyridine-acetic anhydride (1:1). The alditol acetates of
methylated sugars were analyzed by GLC-MS [18, 19].
3.7. Periodate oxidation
PDPS (10 mg) was added to 2 mL 0.1 M sodium metaperiodate solution and the mixture
was kept in the dark for 72 h at room temperature. The excess periodate was destroyed by adding
1,2-ethanediol and the solution was dialyzed against distilled water for 2 h. The volume of the
dialyzed material was concentrated to 2–3 ml. This material was reduced with NaBH4, 12 h,
neutralized with 50% AcOH, and dialyzed with distilled water and finally freeze dried. The
periodate-oxidized material was divided into two parts. One portion was hydrolyzed with 2 M
CF3COOH for 18 h and this hydrolyzed material was used for alditol acetates preparation as
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usual for GLC analysis. Another portion was methylated, followed by preparation of alditol
acetates which were analyzed by GLC-MS [18, 19].
2.8. NMR analysis
PDPS was dried over P2O5 in vacuum for several days and then exchanged with
deuterium followed by lyophilizing with D2O (99.96 % atom 2H, Aldrich) for four times. The 1H
13
and C NMR experiments were carried out at 500 MHz and 125 MHz, respectively with a
Bruker Avance DPX-500 spectrometer. The 1H, 13C, TOCSY, DQF-COSY, NOESY, and HSQC
1
NMR spectra were recorded in D2O at 30 ºC. The H NMR spectrum was recorded by
suppressing the HOD signal (fixed at δ 4.70) using the WEFT pulse sequence using acetone as
internal standard fixing methyl proton signal at δ 2.19. Acetone was used as an internal standard
(δ 31.05 ppm) for 13C spectrum. The 2D-DQF-COSY experiment was performed using standard
BRUKER software. The TOCSY experiment was recorded at mixing time of 150 ms, and
complete assignment required several TOCSY experiments having mixing times ranging from 60
to 300 ms. The NOESY mixing delay was 300 ms.
2.9. Antioxidant properties
2.9.1. Hydroxyl radical scavenging assay
For the assay of hydroxyl radical scavenging activity, a 1 ml reaction mixture was
prepared consisting of KH2PO4–KOH buffer (20 mM, pH 7.4), 2-deoxy-D-ribose (2.8 mM),
variable concentration (0.5–2.5 mg/ml) of PDPS, FeCl3(100 mM), EDTA (100 mM), ascorbate
(100 mM) and H2O2 (1 mM) [22]. It was incubated at 37 ℃ for 1 h. Then the reaction mixture
was added to 2 ml thiobarbituric acid (TBA) and trichloroacetic acid (TCA) solution (3.75%
(w/v) TBA, 0.15% (w/v) TCA and 0.25 N HCl) and the whole mixture were incubated in a
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boiling water bath for 15 min. A pink colour was developed. After cooling, absorbance was
measured at 535 nm. BHA was used as standard. The scavenging activity was calculated using
the following equations:
Hydroxyl radical Scavenging rate (%) = {(A0-A1)/ A0} × 100
Where A0 was the absorbance of the control and A1 was the absorbance in the presence of
sample.
2.9.2. DPPH radical scavenging capacity
A methanol DPPH-solution (0.2 mL) would be added to 1.0 mL of sample solution, and
2.0 mL of water was added. After 30 min the absorbance will be measured at 517 nm using a
spectrophotometer [23, 24]. BHA was used as positive control. The DPPH radical scavenging
activity was calculated by the following formula:
Scavenging activity (%) = {(A0-A1)/ A0} × 100
Where A0 was the absorbance of the control and A1 was the absorbance in the presence of
sample.
2.9.3. ABTS radical scavenging activity
ABTS radicals were generated freshly by adding 2.45 mM of potassium persulfate in 7
mM ABTS solution and the mixture was incubated overnight. After 12–16 h, ABTS•+ solution
was diluted to an absorbance of 0.7 ± 0.02 at 750 nm. Further, the radicals were allowed to react
with extracts at variable doses in 200 μl reaction mixture in 96 well plates. The plate was shaken
at medium speed for 10 seconds and absorbance was perceived at 750 nm [23, 24]. BHA was
used as a positive control. Scavenging degree was deliberated by adopting the equation as
mentioned below:
Quenching effect (%) = {(A0–A1)/ A0} × 100
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Where A0 and A1 were the absorbance of negative control and experimental sets respectively.
2.9.4. Determination of reducing power
The reducing power of was determined according to the method described by Oyaizu
[31]. Various concentrations of PDPS (1-6 mg/mL) were mixed with 2.5 mL sodium phosphate
buffer (0.2 M, pH 6.6) and 2.5 mL of potassium ferricyanide (1 %). The mixture was incubated
for 20 min and then 2.5 mL of trichloroacetic acid (10 %) was added. 2.5 mL of solution mixture
was mixed with 2.5 mL distilled water and 0.5 mL FeCl3 (0.1 %) and incubated for 15 min. The
absorbance was measured at 700 nm against buffer [23, 24]. BHA was used as standard. EC50
value is the effective concentration at which the absorbance was 0.5 for reducing power.
Fig. 1
Table 1a
3. Results and discussion
3.1. Isolation, purification and chemical analysis of PDPS
800 mg of crude polysaccharide was isolated from hot water extraction of fruit bodies of
Pleurotus djamor. 18 mg of pure PDPS was found from 25 mg crude polysaccharide through
sepharose 6B column (Fig. 1). The same method was repeated several times to get 120 mg of
31
PDPS. The PDPS showed specific rotation [α]D - 3.47 (c 0.10, H2O). The average molecular
weight of PDPS was estimated as ~1.61 × 105 Da. Chemical composition analysis by the phenol-
sulphuric acid method [20], shows the total carbohydrate content of PDPS was up to 98.8 ±
1.2%. GLC analyses of the alditol acetates of this polysaccharide revealed the presence of
glucose and galactose in a molar ratio nearly 3:1. The absolute configuration of monosaccharides
were determined by the method of Gerwig et al. (1978) and found that all the monomeric sugar
units had the D configuration. The molecular weight, sugar composition, absolute configuration,
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and specific rotation of the PDPS were shown in Table 1a. GLC–MS study of the methylated
product showed the presence of four components as presented in Table 1b. This result indicated
that (1→3,6)-linked glucopyranosyl, (1→4) linked glucopyranosyl, (1→6)-linked
glucopyranosyl and terminal galactopyranosyl moieties were present in the PDPS. GLC study
showed that only glucose was present in periodate oxidized reduced material of PDPS. GLC–MS
study of alditol acetates of periodate oxidized reduced methylated product showed that (1 →
3,6)-Glcp was unaffected during oxidation while other residues were destroyed during oxidation.
Table 1b
Fig. 2a
Fig. 2b
Table 2a
3.2. NMR and structural analysis of PDPS
1
The H NMR spectrum (500 MHz, 30 °C, Fig. 2a) of the PDPS showed four anomeric
signals at 4.65, 4.64, 4.44 and 4.43 ppm. They were chosen as A, B, C, D according to their
falling anomeric proton chemical shifts (Table 2a). In 13C NMR spectrum (125 MHz, Fig. 2b) at
30 °C, four signals were found in the anomeric region at 103.0, 102.7, 102.5 and 102.3 ppm.
All the 1H and 13C signals (Table 2a) were assigned using DQF-COSY, HSQC (Fig. 2c),
and TOCSY (Fig. 2d) spectrum. Continue with the attribution of signals but correlate H and C
chemical shifts (Fig. 2c, Table 2a) to a given unit: δ 4.65/103.0 (A), 4.64/102.7 (B), 4.44/102.5
(C) and 4.43/102.3 (D).
Fig. 2c
Fig. 2d
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The β-configuration of the residues (A-D) was confirmed by low resonance frequency of
H-1 (4.43-4.65 ppm) and high resonance frequency of C-1 (102.3-103.0 ppm) signals [22, 24-
26]. The downfield shifts of C-4 (δ 78.7) with respect to standard value of methyl glycoside
indicated that residue A was (1 → 4)-linked β-D-Glcp. In residue B, all the proton and carbon
chemical shifts values were found nearly to the standard value of methylgalactosides. Thus,
residue B was terminal β-D-Galp. The downfield shifts at C-3 (δ 84.4) and C-6 (δ 68.6) with
respect to standard value of methyl glycoside indicated that residue C was (1 → 3,6)-β-D-Glcp.
Since the downfield shifts of C-6 (δ 68.7) with respect to standard value of methyl glycoside
indicated that residue D was (1 → 6)-linked β-D-Glcp [27, 28]. The O-6 substitution of residues
C and D were confirmed from the respective reverse peak in the DEPT-135 spectrum (Fig. 2b).
Table 2b
Fig. 3
The sequence of glycosyl residues (A to D) was determined from the NOESY experiment
(Table 2b, Fig. 3). The NOESY experiment showed the inter-residual contacts: A-H1/D-H6a, D-
H6b; B-H1/C-H3; C-H1/A-H4, D-H1/C-H6a, C-H6b along with other intra-residual contacts
(Fig. 3). The above NOESY connectivities established the following sequences: A (1 → 6) D; B
(1 → 3) C; C (1 → 4) A; D (1 → 6) C.
Based on the chemical and NMR spectral evidences the probable structure of repeating unit in
the PDPS was proposed as:
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C
A
D
6)--D-Glcp-(1 4)--D-Glcp-(1 6)--D-Glcp-(1
3

1
-D-Galp
B
3.3 Evaluation of antioxidant activities
3.3.1 Hydroxyl radical scavenging activity
Firstly, hydroxyl radical (ȮH) scavenging potential was determined as the radical can
damage biomolecules immediately after generation. This most reactive radical can be formed
inside human body and can damage DNA by attacking purines, pyrimidines and deoxyribose
resulting pathogenesis of several kinds of diseases [29]. Thus, direct scavenging of ȮH by
antioxidants in a biological system is highly required to combat against oxidative stress. In this
context, classic Fenton’s reaction is generally followed to determine ȮH quenching effect of
natural or synthetic product. The synthesized radical then can react with deoxyribose forming
MDA that can be measured as a pink MDA–TBA chromogen [30]. When test sample is added to
the reaction mixture, the reaction progression is inhibited resulting decolourization of solution.
Following the method, effect of PDPS on ȮH was evaluated where extremely potent activity was
found that increased gradually with rise of concentration (Fig 4a). Results showed that the
polysaccharides inhibited 8.57%, 37.52% and 55.44% radicals at the level of 0.1, 1 and 2 mg/ml
respectively. EC50 value of PDPS was found 1.681 ± 0.034 mg/ml. EC50 value is the effective
concentration at which 50% of the ȮH were scavenged.
Fig 4a
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3.3.2 DPPH radical scavenging activity
The DPPH radical scavenging method is an easy, sensitive, valid, accurate and economic
mean to evaluate antioxidant potential of investigating drug(s). It is characterized by virtue of
delocalisation of spare electron over the molecule giving rise to deep violet colour. After mixing
the DPPH• solution with a substance that can donate hydrogen atom or electron, the radical is
transformed to yellow coloured reduced form i.e. diphenyl–picrylhydrazine [31]. Thus,
magnitude of lowering in signal intensity of reactant solution is directly proportional to increased
antioxidant activity. As presented in Fig 4b, PDPS possessed moderate DPPH• scavenging
abilities that increased in dose dependent manner. At the level of 2 and 4 mg/ml, the
polysaccharides quenched radicals at the rate of 27.41% and 52.31% respectively. EC50 value of
PDPS was found 3.83 ± 0.427 mg/ml.
Fig 4b
Fig 4c
3.3.3 ABTS radical quenching activity
ABTS salt can generate blue–green ABTS•+ by reacting with a strong oxidizing agent like
potassium persulfate. However, the chromophore can quickly be converted back to its colourless
neutral form in presence of hydrogen–donating antioxidant compounds [30]. Thus, the radical
scavenging performance of antioxidative molecule can be determined by monitoring decrease in
absorbance. According to the result, PDPS possessed strong radical quenching potential that
amplified steadily with increase of concentration (Fig 4c). The polysaccharides quenched 17.4%
and 35.08% ABTS•+ respectively at the level of 0.1 and 0.5 mg/ml and the effect was further
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amplified to 59.64% at the concentration of 1 mg/ml. EC50 value of PDPS was found 0.816 ±
0.077 mg/ml.
3.3.4 Reducing power
Ferric reducing antioxidant power assay is a widely used, simple, sensitive, precise and
inexpensive method that uses antioxidants as reductants in a redox-linked colorimetric reaction.
The method is based on reduction of ferric iron (Fe3+) by electron-donating antioxidants under
investigation to its ferrous form (Fe2+). Consequently, the iron colorimetric probe complex
develops a dark blue colour product which can be measured at 540-600 nm [32]. Thus, increase
in absorbance denotes an increment in reducing power as well as antioxidant effects. At
concentration of 1.0 mg/ml, the absorbance value of PDPS was 0.142 but in case of BHA, the
value was 1.2. The PDPS showed the absorbance value 0.587 at 5.0 mg/ml, whereas the BHA
showed the value of 1.2 at the same concentration. Reducing power of the PDPS increases with
the increase in concentration. At concentrations of 2.0, 3.0, and 4.0 mg/mL the absorbance value
of BHA was fixed at 1.2 whereas the value of PDPS increases to 0.25, 0.354 and 0.471
respectively. EC50 value of PDPS was found 4.258 ± 0.095 mg/ml (Fig. 4d). This result suggests
that PDPS has potency to donate electron to reactive free radicals, converting them into more
stable non reactive species and terminating the free radical chain reaction.
Fig 4d
4. Conclusion
A water soluble galactoglucan (PDPS) was isolated from the aqueous extract of an edible
mushroom Pleurotus djamor and characterized using chemical and 1D/2D NMR experiments.
PDPS had an average molecular weight of 1.61 × 105 Da and was composed of glucose and
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galactose. The repeating unit was composed of a backbone containing two (1→6)-β-D-
glucopyranosyl, and one (1→4)-β-D-glucopyranosyl residues. One of (1→6)-linked glucose
units was branched at O-3 position with terminal β-D-galactopyranosyl residue. The PDPS
showed hydroxide radical scavenging activity (EC50=1.681 ± 0.034 mg/ml), DPPH radical
scavenging activity (EC50=3.83 ± 0.427 mg/ml), ABTS radical scavenging activity (EC50=0.816
± 0.077 mg/ml), and reducing power (EC50= 4.258 ± 0.095 mg/ml). So PDPS could be used as a
natural antioxidant and also employed as an ingredient in dietary health or functional products
due to its antioxidant advantages.
Acknowledgements
The authors are thankful to Mr. Barun Majumder, Bose Institute, Kolkata for preparing NMR
spectra. The DST, Govt. of India (Ref. No. SR/FST/College-295 dt 18th Nov. 2015 (DST-FIST)
is acknowledged for financial support.
Conflict of Interest
The authors declare no Conflict of Interest.
Author Contribution Statement
Gajendra Nath Maity: Methodology, analysis and Writing
Prasenjit Maity: Conceptualization, Writing and Editing
Somanjana Khatua: Analysis and Writing
Krishnendu Acharya: Supervision
Sudipta Dalai: Supervision
Soumitra Mondal: Editing and Supervision
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Figure caption
Fig. 1. Gel permeation chromatogram of crude polysaccharide isolated from an edible mushroom
Pleurotus djamor using Sepharose 6B column.
Fig. 2 a) 1H NMR spectrum (500 MHz, D2O, 30 °C) of the PDPS isolated from the fruit body of
Pleurotus djamor. b) 13C NMR spectrum (125 MHz, D2O, 30 ºC) of a PDPS isolated from an
edible mushroom Pleurotus djamor. Part of DEPT-135 spectrum (D2O, 30 ºC) of a PDPS
isolated from an edible mushroom Pleurotus djamor (inset).
Fig. 2 c) The HSQC spectrum (D2O, 30 °C) of the PDPS isolated from the fruit body of
Pleurotus djamor. d) TOCSY spectrum of the PDPS, isolated from an edible mushroom
Pleurotus djamor.
Fig. 3. Part of NOESY spectrum of a PDPS isolated from an edible mushroom Pleurotus djamor.
The NOESY mixing time was 300 ms.
Fig. 4. (a) Hydroxyl radical scavenging activity of the PDPS. Results are the mean ± SD of five
separate experiments, each in triplicate. (b) DPPH radical scavenging activity of the PDPS.
Results are the mean ± SD of four separate experiments, each in triplicate. (c) ABTS radical
scavenging activity of the PDPS. Results are the mean ± SD of five separate experiments, each in
triplicate. (d) Determination of reducing power of the PDPS. Results are the mean ± SD of five
separate experiments, each in triplicate.
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Table 1a. Components of monosaccharide and properties of PDPS from Pleurotus djamor
Properties
31
Data
- 3.47 (c 0.10, H2O)
~1.61 × 105 Da
[α]D
Molecular weight
Monosaccharide component
Molar ratio of sugar unit
absolute configuration
Glucose, Galactose
Glucose (3) : Galactose (1)
D
Table 1b
GLC-MS analysis of methylated polysaccharide (PDPS) isolated from mushroom Pleurotus
djamor.
Methylated sugars
Molar
ratio
Linkage type
Major Mass Fragments (m/z)
2,3,6-Me3-Glc
2,4-Me2-Glc
1
1
1
1
→4)-Glcp-(1→
→3,6)-Glcp-(1→
→6)-Glcp-(1→
Galp-(1→
43,45,71,87,99,101,113,117,129,131,161,173,233
43,58, 87,101,117,129,139,159,189,201,233
43, 45, 58, 71, 87, 99, 101, 117, 129, 161, 173, 189, 233
43, 45, 59, 71, 87, 101, 117, 129, 161, 205
2,3,4-Me3-Glc
2,3,4,6-Me4-Gal
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Table 2a
1
13
The Ha and Cb NMR chemical shifts for the polysaccharide (PDPS) isolated from mushroom
Pleurotus djamor D2O at 30 C.
Glycosyl residue
H1/C1
4.65
H2/C2
3.30
73.3
3.31
69.5
3.44
73.1
3.27
73.1
H3/C3
3.52
74.8
3.56
74.8
3.70
84.4
3.56
75.5
H4/C4
3.56
78.7
3.43
68.1
3.56
70.0
3.41
69.5
H5/C5
3.43
75.9
3.41
75.9
3.67
75.8
3.66
74.1
H6b,H6a /C6
3.87, 3.72
60.0
→4)-β-D-Glcp-(1→
103.0
4.64
A
β-D-Galp-(1→
3.83, 3.65
60.7
102.7
4.44
B
→3,6)-β-D-Glcp-(1→
4.12, 3.51
68.6
102.5
4.43
C
→6)-β-D-Glcp-(1→
3.80, 3.41
68.7
102.3
D
a
Values of the 1H chemical shifts were recorded with respect to the HOD signal fixed at  4.70
at 30 C.
b
13
Values of the C chemical shifts were recorded with reference to acetone as the internal
standard and fixed at  31.05 at 30 C.
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Table 2b
NOE effects of PDPS, observed in the NOESY spectrum recorded in D2O at 30 C.
Unit
NOE signals
Intraunit
From
Interunit
A-H1
A-H2, A-H3, A-H5
B-H2, B-H3, B-H5
C-H2, C-H3, C-H5
D-H2, D-H3, D-H5
D-H6a, D-H6b
A
B
C
D
B-H1
C-H1
D-H1
C-H3
A-H4
C-H6a, C-H6b
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Author Statement
The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.
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Highlights
 A galactoglucan (PDPS) was isolated from edible mushroom Pleurotus djamor.
 Its structure was established by chemical and 1D/2D NMR analyses.
 It showed antioxidant activities.
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Figure 1

Figure 2

Figure 3

Figure 4