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Research Journal of Biotechnology Vol.

7 (2) May (2012)


Res. J. Biotech

Effect of Medicinal Mushroom, Auricularia auricula-


judae, polysaccharides against EAC cell lines Ramasamy
Gurusamy* and Rajarajan Arthe
Department of Biotechnology, Vivekanandha College of Engineering for Women, Tiruchengode, Tamilnadu, INDIA
*guruibt@gmail.com
Abstract shown anticancer activity and it is found to be Beta-glucan
One of the major causes of mortality which is in the cell walls of mushrooms where it is
worldwide is cancer. Increasing research on herbal firmly bound to other molecules responsible for the
medicine has revealed its importance in treating many strength and shape stability of the cells. Besides beta-
glucan, the cell wall also contains other saccharides and
diseases including cancer. The present study was
proteins that are linked and bound together to form a solid
carried out to evaluate the antitumor activity of crude structure which makes it responsible for anticancer activity.
polysaccharide extract of Auricularia auricula-judae However, in spite of traditional use, pharmacology of its
on Ehrlich Ascites Carcinoma (EAC) model in mice. parts has not yet been explored scientifically. The present
After inoculation of EAC cells into mice, treatment investigation was carried out to evaluate the anticancer
with Auricularia auricula-judae (AAE) (200 mg/kg) property of Auricularia auricula-judae against Ehrlich
was continued for 9 days. The effect of drug response Ascites Carcinoma (EAC) tumor model.
was evaluated by the study of tumor growth response
including study of hematological parameters, Material and Methods
biochemical analysis, chromosomal disintegration Biochemical screening: The preliminary biochemical
assay and in vitro cytotoxicity. Experimental results screening was carried out by estimating the moisture

revealed that the polysaccharide extract of content 1, carbohydrate 9, nitrogen 20, crude protein 6, amino
16 3 12 8 13
nitrogen , fat , magnesium , phosphorus , crude fibres
Auricularia auricula-judae possesses significant
crude mushroom extract as well as various purified
anticancer activity due to the presence of
fractions, including proteins and polysaccharides, have
polysaccharides like Beta-Glucans which may be in
response to its cytotoxicity.

Keywords: Auricularia auricula-judae, AAE, EAC cells,


Beta-Glucans, Cytotoxicity.

Introduction
Over the past few decades, cancer has remained as
the largest cause of mortality worldwide and the number of
individuals living with cancer is steadily expanding11.
Hence, a major portion of the current pharmacological
research is involved with the anticancer drug design
customized to fit new molecular targets21. Due to the
enormous propensity of plants, which synthesize a variety
of structurally diverse bioactive compounds, the plant
kingdom is a potential source of chemical constituents with
10
antitumor and cytotoxic activities . Traditionally various
4
plants have long been used in the treatment of cancer .

Auricularia auricula-judae is a common jelly


fungus widely distributed throughout North America and
Europe. It is brown, irregular, wavy, rubbery and
gelatinous; it grows on wood; it is edible but with no
distinctive taste and is widely cultivated throughout the
world for use as mushroom as well as medicine. The
mushroom has been used traditionally as medicine in
many countries as anti-diabetic, antitumor, antihyperten-
sive, anti-inflammatory, immunomodulatory and anti-
bacterial agents. Several in vitro and in vivo studies with

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Research Journal of Biotechnology Vol. 7 (2) May (2012)
Res. J. Biotech
and ash content of the dried powder of Auricularia
auricula-judae and the percentage of each molecule are
tabulated in table 1.

Extraction of polysaccharides from Auricularia


auricula-judae: The anti-cancer compounds are extracted
from mushroom fruit-bodies. In the initial step dried
mushroom powder was repeatedly heated in 80% ethanol to
extract and eliminate low molecular weight substances.
Crude fractions 1, 11 and 111 are obtained from the
remaining ethanol extract residue by extraction with water
(100C, 3h) , 1% ammonium oxalate (100C, 6h) and 5%
sodium hydroxide (80C, 6h)15. The obtained fractions
were evaporated, dried and purified. The prepared extracts
were used for the in vitro and in vivo studies of anticancer
activity.

Purification of polysaccharides: Column chromatography


was performed with DEAE-cellulose. Volumes of 10 ml of
the extracts (different polysaccharides obtained) at 400
mg/L were applied. The column was eluted with deionized
water, NaCl at different concentrations (0.05, 0.10 and 0.50
M), NaHCO3 (0.3 M) and NaOH (0.1 M) successively at
the flow rate of 30 ml/h. The isolated fractions were
measured by the phenol-sulphuric acid method. Finally,
different fractions of isolated polysaccharides were
obtained and lyophilized for 48 h for other assays. Since
the yields of the purified fractions were very low, crude
polysaccharides extract were prepared for in vivo studies.

Anti-Cancer Screening
Cell line: Cancer cell lines, Ehrlich ascites carcinoma
(EAC), to induce cancer in animal model (mice) were

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Research Journal of Biotechnology Vol. 7 (2) May (2012)
Res. J. Biotech
obtained from Amala Cancer Research Center, Amala protein and Packed Cell Volume (PCV) were determined.
Nagar, Kerala, India. The cells were maintained as ascites
tumor in Swiss Albino mice by intraperitoneal injunction of Disintegration assay of Chromatin by Toluidene blue:
1106 viable cells. The smear of culture was air dried and it was fixed in the
mixture of ethanol: acetic acid (3:1) for half an hour. The
Animals: Swiss albino mice (25-30 g) were procured from slide was stained using 0.2mg/ml toluidene blue for 30
KMCH, Coimbatore, Tamilnadu, India and used throughout seconds and washed using distilled water and the
the study. They were housed in microlon boxes in a disintegrated chromatin observed under light microscope
controlled environment (temperature 252C and 12 h (40X).
dark/light cycle) with standard laboratory diet, water at
libitum approved by the Institutional Animal Ethical Results and Discussion
Committee (IAEC). In the current study of tumor growth response,
AAE treatment significantly reduced packed cell volume
Experimental Protocol : Healthy Swiss albino male mice and viable cell count compared to those of EAC control
were weighed and divided into three groups (n=6). EAC mice, along with increase in nonviable cell count in the
cells (1 106 cells/ mouse) where injected i.p. to each treated groups.
mouse of each group except normal saline group. This was
taken as day 0. Extract treatment was continued for Haemotological parameters of tumor bearing mice
subsequent 9 days starting from day 1. On 10th day, 24h on the day 14 showed significant changes when compared
after the last dose, mice were sacrificed from each group. to normal mice. The total WBC count, protein and PCV
Before that the animal blood was collected to evaluate the were found to increase with a reduction in the haemoglobin
hematological and biochemical parameters. The groups and content of RBC. The differential count of WBC showed
the design of the experiment were as follows:14 that the percentage of neutrophils increased while that of
Group I : Normal Saline Group lymphocytes decreased. Treatment with AAE brought back
Group II: Tumor control - EAC (1X106 cells / mouse, i.p.) the haemoglobin content, RBC and WBC counts to the near
Group III: Treated -EAC (1x106 cells / mouse, i.p.) + AAE normal.
(200mg/kg b.wt, p.o.)
Nuclear staining studies of the Cancer cells
Antitumor activity of AAE was assessed by
revealed the damages made by the extract on cancer cells.
observation of changes with respect to the following
Cytotoxicity assay and the disintegrated chromatin were
parameters:
observed in the ascetic fluid and the percentage of
disintegration was nearly same in all the three categories.
Tumor cell count: The ascetic fluid was taken in a WBC
The percentage of cell impact was found to be 33% through
pipette and diluted 100 times. Then a drop of the diluted
cell counting. The anticancer activity was also tested by
cell suspension was placed on the Neubauer counting
culturing the treated and tumor control samples in MEME
chamber and the numbers of cells in the 64 small squares
medium from the doubling time of cells.
were counted [Table 2].
Khan et al2 studied the anticancer activity against
Cytotoxicity Assay: In vitro short term cytotoxic activity18
EAC cells by examining the survival time of mice and the
of Auricularia auricula-judae was determined using EAC
cytotoxicity test. In the present study performed
cells directly from the control and test mice
cytotoxicity test and chromatin disintegration methods
intraperitoneally.100l of 0.4% Trypan blue dye solution
before and after the treatment in mice model was employed
was added to both sample and mixed well. After mixing,
to identify the live and dead cells.
the live and dead cells were counted in haemocytometer
and the percentage of cytotoxicity was calculated. Tetsuro Ikekawa et al19 reported that the aqueous
extract of seven edible mushrooms including Auricularis
% of Cytotoxicity = No. of dead cells / No. of live cells + showed inhibition towards tumour development. Daba and
No. of dead cells X 100. Ezeronye7 showed the anticancer property of poly-
saccharide isolated from higher basidiocytes. In the present
Haematological Study: In order to detect the influence of study, the crude polysaccharide extract of Auricularia
mushroom on hematological status of EAC bearing mice, a auricula-judae exhibited 33% inhibition against the EAC
comparison was made among three groups (n=6) of mice cell lines.
on the 14th day after inoculation. The groups comprised of
(1) Tumor bearing mice (2) Tumor bearing mice treated
with AE (200 mg/Kg/day, p.o. for 9 days) (3) Control mice Conclusion
(Normal Saline group). Blood was drawn from each mice EAC has a resemblance with human tumors which
by the retro orbital plexus method and the White Blood are most sensitive to chemotherapy due to the fact that it is
Cell count (WBC), Red Blood Cells (RBC) hemoglobin, undifferentiated and it has a rapid growth rate. Due to this
resemblance, EAC cell line can be used as a model for

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Research Journal of Biotechnology Vol. 7 (2) May (2012)
Res. J. Biotech
human cancer17. Tetsuro Ikekawa et al19 reported that 200
mg/Kg body weight of crude Auricularis extract showed
anticancer activity against Sarcoma 180. The present study
revealed that the polysaccharide extract of Auricularia
auricula-judae is effective against EAC induced Swiss
albino model with the dose of 200 mg / Kg showing significant
anticancer activity and we also conclude that the
polysaccharides play a major role in producing the
anticancer activity.
a. Live Cells without stain b. Dead cells with stain
Acknowledgement Figure 2: Cytotoxicity Assay
We gratefully thank Dr. M. Karunanithi, Chairman
and Secretary for providing facilities to carry out this
investigation. Disintegrated
Chromatin
Table 1
Biochemical screening

S.N. Components % present in


Auricularia
auricula-judae
Normal
1 Moisture 12.8
Chromatin
2 Carbohydrate 31.3
Structure
3 Nitrogen 1.5
4 Crude protein 6.5
5 Amino nitrogen 3.4 Figure 3: Chromatin Disintegration by Toluidene Blue
6 Fat 1.2
7 Magnesium 0.36 References
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Figure 1: Anticancer screening phosphorous and phosphatase with N-phenyl-p-phenylane-

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15. Mizuno T., The extraction and development of antitumour- (Received 25th January 2012, accepted 20th April 2012)
active polysaccharides from medicinal mushrooms in Japan,

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