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Antitumor Activity of β-glucan Extracted from Pleurotus eryngii

Article in Indian Journal of Forensic Medicine and Toxicology · November 2020

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2492 Indian Journal of Forensic Medicine & Toxicology, July-September 2020, Vol. 14, No. 3

Antitumor Activity of β-glucan Extracted from


Pleurotus eryngii

Ali Z. Al-Saffar1, Noora A. Hadi2, Hadeel Mohamed Khalaf3


1Assist. Prof., 2Lecturer, 3Lecturer, Al-Nahrain University, College of Biotechnology,
Dept. of Molecular and Medical Biotechnology, Baghdad, Iraq

Abstract
Pleurotus eryngii, a type of edible mushroom that exhibit various pharmacological properties, including
antioxidant and anticancer effects. In the present study, extracted β-glucan from the P. eryngii was tested as
an antioxidant and anti-tumor factor. β-glucan was extracted and analyzed by HPLC and FT-IR. Analytical
results showed more than 90% similarity in chemical structure and purity. Potential antioxidant activity of
β-glucan was examined using 2, 2-diphenyl-1-picrylhydrazyl (DPPH) and compared with ascorbic acid.β-
glucan confirmed a potential scavenging activity. The anticancer activity of theβ-glucan was assessed using
different concentrations (6.25 to 400 µgmL-1) on MCF-7 and HepG2 cell lines. P. eryngiiβ-glucan exerted
a dose-dependent reduction in MCF-7 and HepG2 cell viability with an IC50 of 280.00 and 539.5 µg mL-1,
respectively. At the same time, no significant effect was recorded on normal cell line WRL 68. The obtained
results are expected that could be used to develop P. eryngiiβ-glucanas an antitumor drug.

Keywords: MTT, pleurotus eryngii, Cytotoxicity, β-glucan, Antioxidant, MCF-7, HepG2.

Introduction glucuronic acid, xylose, galactose, mannose, arabinose


or ribose(2).
Mushrooms have attracted a great deal of interest in
many areas of food and bio-pharmaceutical research and Pleurotus eryngii is an edible mushroom,considered
are well known for their nutritional and medicinal values to be a health food not only for low fat and calories but
(1). Several major components with immunomodulatory
also being rich in amino acids, vitamins, and dietary
and/or antitumor activity have been isolated from fiber(3). In addition, β-glucans of P. eryngii received
mushrooms. These include mainly polysaccharides, such an increasing interest for its bioactive properties
as β-glucans, polysaccharo-peptides, polysaccharide- including antitumor, immunomodulator, antioxidant and
protein conjugates, and proteins. β-glucans have a wide antiallergic activities. Studies have indicated that the
range of biological activities. Mushroom β-glucan polysaccharides isolated from P. eryngii were mostly
polysaccharides are fibers that mostly present as β-glucans, which exhibited potential activities(4).
linear and branched chains with different types of
glycosidic linkages, such as (1-3), (1-6)- β-glucans and Chemically, β-glucans are heterogeneous, non-
(1-3)-α-glucans, others are heteroglycans containing starch polysaccharides, which form the structural
compounds of the cell wall of certain microorganisms,
including yeast and algae, mushrooms, and grains, such
as oats and wheat. β-glucans may be insoluble or soluble.
Insoluble β-glucans fibers consist of β-(1-3/1-4)-D-
Corresponding Author:
linked glucose units, whereas soluble viscous β-glucans
Ali Z. Al-Saffar fibers consist of β-(1-3)/1-6)-D-linked glucose (5).
Assist. Prof., Al-Nahrain University, College of
Biotechnology, Dept. of Molecular and Medical Many chemical compounds identified as specific
Biotechnology, Baghdad, Iraq agents for inhibiting cancer cell proliferation were also
e-mail: ali.btc80@gmail.com showed significant toxicity toward normal cells, as well
Indian Journal of Forensic Medicine & Toxicology, July-September 2020, Vol. 14, No. 3 2493
as their side effects. Many potential anticancer drugs into HPLC column for analysis. The separation occurred
have considerable side effects(6). Therefore, discovery on liquid chromatography and, the eluted peaks were
of new safer drugs with potential activity against tumor monitored by UV-Vis 10 A-SPD spectrophotometer.
has become an important goal of research in biomedical
sciences. Polysaccharides from mushroom sources FT-IR (Fourier Transformed Infrared) Analysis:
can stimulate immune cells, including macrophages, The chemical structure of β-glucan was analyzed using
granulocytes, nature killer cells and monocytes to trigger FT-IR spectrometry (Shimadzu IR Prestige-21– Japan).
cytokine production and thus stimulating the immune The FTIR spectrum was utilized to detect the functional
system(7). groups of glucan structure compared with the standard.
This was done under FT-IR spectrometry in wavelength
In same point, this study was conducted first to range 4000-400 cm-1 and at a resolution of 8 cm-1. This
extract β-glucans from P. eryngii, and evaluate the test involved mixing an equal volume of glucan sample
cytotoxic activity of the extracted β-glucanagainst tumor and standard (2 mg) with potassium bromide (KBr) (100
cell lines HepG2 and MCf-7, as approach in developing mg), then grinding the mixture by special grinder until
a mushroom polysaccharides to use either individually soft and fine powder obtained. The sample was loaded in
or in combination with medicinal drugs combination in target mold and analyzed (9).
cancer treatment.
Determination of Carbohydrate Content:
Materials and Method Carbohydrate content was calculated by multiplying the
reducing sugar content which was determined depending
Mushroom Strain: Pleurotus eryngii strain was on Fehling’s reducing method(10). Briefly, 10 g sample
kindly provided and authenticated by Dr. Ahmed A. was mixed with 20 mL sulphuric acid (0.5 M). Reflux
Kareem, Department of Organic Farming, Ministry of was then performed in a sand bath for 2.5 hours. The
Agriculture, Baghdad, Iraq. residue was washed after filtration (Whatman filter No.
β-Glucan Extraction from P. eryngii: β-glucan 1) with warm dH2O. The solution was then neutralized
was extracted using a water extraction method(8). In with Na2CO3 powder and the mixture’s volume was
brief: dried fruit bodies of P. eryngiiwere powdered. The completed to 100 mL with dH2O. Titrations were
powder was mixed with ddH2O in ratio of 1:20 (wt/v). performed using 5 mL Fehling’s solution (equal volumes
The pH of the mixture was adjusted to 7.0 using 20% of solution A and B) pipetted into a conical flask and
Na2Co3. Mixture heated to 90°C for 6 h with shaking (100 aliquot of 5 mL dH2O was added. The solution was then
rpm). After heating process, the mixture was centrifuged boiled for 15 seconds. Methylene blue indicator (a few
at 8000 rpm for 10 min at 4°C. The pellet was discarded, drops) was then titrated with the solution until the color
and the supernatant was transferred to new container changed from blue to green. The carbohydrate content
and the pH was further adjusted to 4.5 using 2M HCl. was then calculated according to following equation:
The solution was centrifuged at 8000 rpm for 30 min at Carbohydrate Content (%) =
4°C. Pellet which contained proteins was discarded and
5 × 0.005 × 100 × 100
supernatant was mixed with absolute ethanol in a ratio of × 0.9%
1:1 and left for 12 h at 4°C to precipitate the beta-glucan. V × 10 × W
The mixture was centrifuged at 3000 rpm for 10 min at
Where V = volume of sample solution (titration volume) and W =
4°C. Finally, the pellet was homogenized with absolute weight of powdered sample.
ethanol and then oven-dried at 60°C.
Antioxidant Activity
β-Glucan Analysis by High Performance Liquid
Chromatography (HPLC): The samples and standard Antioxidant activity of extracted β-glucanwas
were analyzed by HPLC (SYKAM, Germany) supplied detected using DPPH (Sigma Aldrich, USA) for free
with S2100 quaternary gradient pump andfluorescence radical scavenging assay(11). Scavenging potential
detector RF-20A (UV280). The condition analysis of of β-glucan against DPPH radicals was determined
β-glucan; mobile phase: dH2O and orthophosphoric acid spectrophotometrically (Aquarius, Cecil, Italy). Colour
(90:10 v/v); column: C18–ODS (25cm x 4.6 mm);Flow change (from deep- violet to light- yellow) when DPPH
rate = 0.7 mL min-1. Preparation of sample: 1 mg reduced was measured at 517 nm. In our experiment, set
dissolved in 25 mL dH2O and then 20 µLwas injected of concentrations (12.5, 25, 50, 100 and 200 µg mL-1)
2494 Indian Journal of Forensic Medicine & Toxicology, July-September 2020, Vol. 14, No. 3
were used. Ascorbic acid was used as positive control. The inhibition (%) of radicals by β-glucan was calculated
according to the formula:

Cytotoxicity Assay Statistical Analysis: Data were expressed as


means±standard deviation (SD) and analyzed by a
Cell Culture and Maintenance: Human breast one-way analysis of variance (ANOVA) followed with
carcinoma cells (HepG2), human breast adenocarcinoma Dunn’s test using GraphPad Prism(Graph Pad Software
cells (MCF-7) and one non-carcinogenic liver cell line Inc.). A p ≤0.05 was considered to indicate a statistically
(WRL68) were kindly provided from Biotechnology significant difference between groups.
Research Center, Al-Nahrain University. Cells were
maintained and cultured in RPMI-1640 (Sigma Aldrich, Results and Discussions
USA) supplemented with 10% fetal bovine serum, 100
U mL-1 penicillin G and 100 µg mL-1 streptomycin. Dried fruiting bodies of P. eryngii was subjected
Cells (3 x 104 cell mL-1) were seeded into tissue culture to β-glucan extraction which depended on heating-
flasks and allowed to grow at approximately 80 to acid extraction steps. This method is characterized
90% confluence monolayer (24 to 48 h). Cultures were by its ability to extract glucan from mushrooms with
maintained at 37ºC in CO2incubatorwith humidified significant quantities, limited use of organic solvents
atmosphere. Gentle trypsinization (50 mg mL-1 of and time saving. The total yield of extracted glucan was
trypsin) was used for harvesting the cells(12). 7.9%. Previously reported that the total yield of glucan
extracted from P. eryngiiwas 6% (14). The advantages
MTT: The cytotoxic effect of β-glucan against of this procedure were heating, and extensive acid
HepG2, MCF-7 and WRL68 was estimated using treatment followed byethanol application which leads
3-(4,5-Dimethylthyiazol-2-yl)-2,5-diphenyltetrazolium to β-glucan precipitation and dissolve or remove most
bromide (MTT) assay (13). Briefly, cells were cultured the proteins, mannan, nucleic acids and others. The
in 96-well plates and incubated until cells reach to impurities affect the physical and chemical properties
80% confluence. Medium was removed and 200 µL of β-glucanand may cause reducing in its ability to be
of different β-glucanconcentrations (25 to 400 µgmL- soluble in water (15). Furthermore, the carbohydrate
1
RPMI-1640 serum free medium)were added to content for the extracted β-glucanwas 54%, indicating
the respective wells containing the cells. Wells with purity and method of choice for β-glucan extraction.
untreated cells were used as the negative control. After
24 h, 10 µL of MTT (Sigma Aldrich, USA) was added to Regarding HPLC analysis, results in Fig. (1) revealed
each well.Plates were further incubated at 37ºC, 5% CO2 one major peak in sample of the extracted β-glucan at
for 4 h. Themedium was then carefully removed and retention time 3.16 min (487.633 mAU) with an overall
100 µL of dimethyl sulfoxide was added per well and area percentage of 90%, which indicated the purity of
incubated for5 min.Absorbance at 540 nm was measured the extracted β-glucan by comparing with the standard
using an ELISA microplate reader. The percentage of which exhibited almost the same retention time at 2.94
viability (%) was calculated according to the following min (377.215 mAU). Purity of 90% gave an indication
formula: for the successful β-glucan extraction method. HPLC
was used for detecting the purity of polysaccharides
Viability (%) = OD control− OD sample/OD Control including β-glucan extracted from mushrooms and
×100 yeasts (16).

Cytotoxicity of each sample was expressed as IC50


value.
Indian Journal of Forensic Medicine & Toxicology, July-September 2020, Vol. 14, No. 3 2495

Fig. 1: HPLC chromatogram for (Top) β-glucan standard and (Down) the test sample.

FT-IR analysis of P. eryngiiβ-glucan with represent the existence of C-O-C group(18). In addition,
absorption range of 4000-400 cm-1 to was compared hydroxyl groups and carboxyl groups were detected
with the resulted functional groups of the standard. Fig. at band 2923.88 cm-1, these groups are features of
(2A), shows that the band range of ~1027.99 cm-1 is a carbohydrate structure(19). Moreover, both sample and
characteristic feature of polysaccharides and assigned standard showed high degree of similarity with respect
for β-1,4 glucans(17), the absorbance peak at this band to overall spectra abortion.
2496 Indian Journal of Forensic Medicine & Toxicology, July-September 2020, Vol. 14, No. 3

Fig. 2: FT-IR Spectra of (A) extracted P. eryngii β-glucan sample (B) standard.

The scavenging activity of P. eryngiiβ-glucan was which display many biological activities including
estimated using increasing concentrations of β-glucan. antioxidant(20). Antioxidant activity of β-glucan is
Results in Fig. (3) demonstrate a potential free radical highly dependent on mushroom source and method of
scavenging capability of P. eryngiiβ-glucan with extraction. Our results are in agreement with Roncero-
calculated IC50 value of 39.3 µg mL-1. By comparing with Ramoset al.,(21) which described the antioxidant activity
ascorbic acid (IC50 27.47 µg mL-1), P. eryngiiβ-glucan of β-glucan from different mushroom sources including
showed no significant differences in the pattern of free P. eryngii. Another study involving edible mushrooms
radicles reduction among all the tested concentrations. revealed that β-glucan exhibited antioxidant activity 64
β-glucanare the most abundant forms of polysaccharides to 93% reduction of DPPH (22).
Indian Journal of Forensic Medicine & Toxicology, July-September 2020, Vol. 14, No. 3 2497

Fig. 3: Mean (%) DPPH scavenging activity of P. eryngii β-glucan with respect to ascorbic acid. NS: Non-
Significant.

Various concentrations of P. eryngiiβ-glucan MCF-7 and HepG2 viability. The effect of MCF-7 and
were tested againstMCF-7 and HepG2 tumor cells. HepG2 viability by P. eryngiiβ-glucanexhibited a dose
In the present study we used the MTT assay, which dependent pattern of reduction with a calculated IC50 of
is universally used to evaluate the cytotoxic potency 280.00 and 539.5 µg mL-1, respectively. On the other
of drugs in vitro(12). Results in Fig. (4) shows that the hand, P. eryngiiβ-glucan hadslight toxic effect on the
more increasing in β-glucan dose the more reduction in cell viability of normal cells WRL68.

Fig. 4: MTT assay for P. eryngii β-glucan against (A) MCF-7 (B) HepG2 with corresponding normal cell line
WRL68. Mean (±SD) of viability was detected after 24 h treatment. Differences considered significant:
**p ≤ 0.01. NS: Non-Significant, SD: Standard Deviation.

The biological, immunological and pharmacological cells indicated a significant dose-dependent inhibition
activities of glucans extracted from edible mushroom are of cell proliferation and exerted direct cytotoxicity
mainly attributed to β-glucan(23). The cytotoxic activity after 24 h. Many reports indicated the anti-proliferative
of P. eryngiiβ-glucan against different types of tumor effect of β-glucan extracted from different mushroom
2498 Indian Journal of Forensic Medicine & Toxicology, July-September 2020, Vol. 14, No. 3
sources. The effect of β-glucanin vitro against different and quality of life in ovarian and breast cancer
tumor cell lines was well demonstrated (23). In addition, patients. Current opinion in obstetrics &
pervious finding indicated that the toxicity of β-glucan gynecology. 2006;18(1):24-8.
on human pigmented malignant melanoma (Me45) cell 7. Cha YJ, Alam N, Lee JS, Lee KR, Shim MJ, Lee
line increased by increasing β-glucan concentration MW, et al. Anticancer and Immunopotentiating
with viability reduction reached up to 19%. Moreover, Activities of Crude Polysaccharides from Pleurotus
it was proved that glucans in nature have low toxicity on nebrodensis on Mouse Sarcoma 180. Mycobiology.
normal cells and well tolerated by patients treated with 2012;40(4):236-43.
glucan combination(24).
8. Wood PJ. Physicochemical properties and
physiological effects of the (1----3)(1----4)-beta-
Conclusions
D-glucan from oats. Advances in experimental
We can conclude that the extracted P. eryngiiβ- medicine and biology. 1990;270:119-27.
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promising anti-proliferative potential on tumor cells resolution titrator: a new approach to studying
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10. Katoch R. Carbohydrate Estimations. In: Analytical
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ESTIMATION OF THE WHOLE FLAVONOID,
Conflict of Interest: Non
ANTIOXIDANT, ANTI BACTERIAL
Funding: Self-funding. CHALLENGE CONCERNING VIOLA
ODORATA (BANAFSHA) METHANOLIC
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