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Journal of Applied Pharmaceutical Science Vol. 6 (07), pp. 120-124, July, 2016
Available online at http://www.japsonline.com
Phytochemical Study and Antioxidative Property of Ethanolic
Extract from Termitomyces clypeatus
, Narayan Chandra Mandal
, Anirban Roy
Molecular and Applied Mycology and Plant Pathology Laboratory, Department of Botany, University of Calcutta, Kolkata, West Bengal - 700019, India.
Mycology and Plant Pathology Laboratory, Department of Botany, Visva-Bharati, Santiniketan- 731235, India.
West Bengal Biodiversity Board, Poura
Bhawan, Salt Lake City, Sector III, Kolkata- 700016, India.
Received on: 11/03/2016
Revised on: 22/04/2016
Accepted on: 01/06/2016
Available online: 28/07/2016
The present study documents the phytochemical screening and antioxidant properties of ethanolic extract of
Termitomyces clypeatus. Phytochemical constituents like phenols, flavonoids and ascorbic acid were much
higher than β carotene and lycopene. The analysis of the phenolic compounds performed by HPLC, revealed the
presence of pyrogallol (0.223 µg/mg) and cinnamic acid (0.095 µg/mg). Antioxidant activity was evaluated
through superoxide radical scavenging assay, DPPH radical scavenging assay, chelating ability, reducing power
and total antioxidant capacity determining assays. Their EC
values ranged from 0.21 µg/ml to 3.22 mg/ml.
Result implies that T. clypeatus can not only serve as a food supplement but also be used as treatment for
various oxidative stress related diseases.
Antioxidant, free radicals,
Free radicals and degenerative diseases are associated
with aging and include cancer, cardiovascular disease, immune-
system decline, brain dysfunction and cataracts (Ames et al.,
1993; Khatua et al., 2013). As oxidative damage to DNA,
proteins and other macromolecules accumulates with age; leads
to aging (Fraga et al., 1990; Harman, 1981). Free radicals such as
superoxide, hydrogen peroxide and hydroxyl radicals, which are
mutagens produced by radiation, are also by-products of normal
metabolism (Sies, 1986; Wagner et al., 1992). Our own immune
system at times is unable to combat these reactive oxygen species
(ROS). Hence, the need for antioxidants creeps in. As
carcinogenic properties have been reported for some synthetic
antioxidants, research in the last three decades on the potential
use of natural antioxidants from plants and mushrooms have
received much importance. Extracts from mushroom have
received attention based on their safety and records of health
promotion. It has been also established that they are less
toxic. They may act directly as antioxidant or prevent underlying
* Corresponding Author
oxidative stress related pathological conditions such as cancer
(Chatterjee et al., 2014), heart ailments (Biswas et al., 2011),
diabetes (Biswas and Acharya, 2013), inflammation (Biswas et al.,
2010), gastric ulcer (Chatterjee et al., 2013), hepatic damage
(Acharya et al., 2012; Chatterjee et al., 2012), microbial pathogens
(Rai et al., 2013), parasitic organisms (Mallick et al., 2014,
Mallick et al., 2015) etc. Termitomyces clypeatus R. Heim
commonly known as “bali chatu” by the local people of lateritic
zone of West Bengal has been traditionally used as food for their
flavour, texture and delicacy. Recently a water soluble pure
polysaccharide with average molecular weight of ~1.98 X 10
was isolated from T. clypeatus having strong antioxidative
properties (Pattanayak et al., 2015). As far as our literature survey
says the antioxidant activity of ethanolic rich fraction of this
mushroom from West Bengal has not yet been published. In this
scenario the present investigation was done. We have examined the
antioxidant activity of ethanolic fraction of T. clypeatus employing
various in vitro assay models such as superoxide radical
scavenging activity, DPPH radical scavenging activity, chelating
of ferrous ion, reducing ability and total antioxidant capacity along
with a phytochemical screening for determining the usefulness of
this mushroom as a functional food.
Mitra et al. / Journal of Applied Pharmaceutical Science 6 (07); 2016: 120-124 121
MATERIALS AND METHODS
Collection, Preparation of sample and authentication
The mushroom T. clypeatus was collected from West
Midnapore district of West Bengal. With proper scientific
measures they were brought to the laboratory and cleaned well.
Identification was done following standard literature (Karun and
Sridhar, 2013). The voucher specimen has been deposited in the
Calcutta University Herbarium (CUH) with the accession number
CUH AM 351.
Ethanolic fraction was extracted according to the method
of Dasgupta et al. (2013). Dried and powdered basidiocarps of T.
clypeatus were extracted with ethanol at 25ºC for 2 days and
filtered. After filtration, the residue was re-extracted with ethanol.
After filtration the filtrate was concentrated under reduced
pressure in a rotary evaporator. Now, this concentrated ethanolic
extract of T. clypeatus was stored at 4ºC. The percentage yield
extracts were calculated based on dry weight as:
Yield (%) = (W
× 100)/ W
= weight of extract after solvent evaporation; W
Weight of the minced mushroom.
The content of total phenolic compounds in the ethanolic
extract of T. clypeatus was estimated using Folin-Ciocalteu
reagent (Singleton and Rossi 1965). Gallic acid (10-40 µg) was
used as a standard and expressed as µg/gallic acid equivalents/g of
extract. Aluminium nitrate and potassium acetate were required to
determine total flavonoid content (Park et al., 1997) and quercetin
(5–20 µg) was used as a standard and presented as µg of quercetin
equivalents/ g of extract. Quantification of ascorbic acid was done
by titration against 2, 6-dichlorophenol indophenol dye using
oxalic acid (Rekha et al., 2012). Beta-carotene and lycopene were
estimated by measuring absorbance at 453, 505 and 663 nm
(Nagata and Yamashita, 1992).
Detection of Phenols and flavonoids by HPLC
The phenolic profile of the extract was determined using
eleven standards of Sigma Aldrich (MO, USA) like gallic acid,
chlorogenic acid, vanillic acid, p-coumaric acid, ferulic acid,
myricetin, salicylic acid, quercetin, cinnamic acid, pyrogallol and
kaempferol. For quantitative analysis, a calibration curve (10–50
µg /ml) for each phenolic standard was constructed.
Following methods of Martinez et al. (2001) superoxide
radical scavenging activity of the ethanolic extract of T. clypeatus
(0.2 mg/ml-0.6 mg/ml) was determined. BHA was used as a
positive control. DPPH radical scavenging activity was determined
for concentrations 2.0 mg/ml to 3.0 mg/ml following the protocol
of Shimada et al. (1992). Various concentrations (0.1-0.3 mg/ml)
of the extract were checked if they can chelate ferrous ion, based
on methods stated by Dinis et al. (1994). Reducing power of the
extract was determined spectrophotometrically at 700 nm as per
methods of Oyaizu (1986) using ascorbic acid as standard verses
different concentrations (1.0-2.0 mg/ml). Total antioxidant
capacity assay was also carried out as described by Prieto et al.
(1999) with little modification (Mitra et al., 2014). The activity
was expressed as the number of equivalents of ascorbic acid
value is the half maximal effective value i.e. the
concentration of extract providing 50% of antioxidant activity or
0.5 absorbance. Graphs were plotted based on above data and
values were determined from them.
Data were expressed as mean ± S.D. (Standard
deviation). Means of triplicate analyses were calculated. The
Student’s t test was used for comparison between standard and the
sample. A difference was considered to be statistically significant
when p < 0.05.
RESULTS AND DISCUSSION
The extractive yield of the ethanolic extract of T.
clypeatus was 7%. It was seen to have a good number of
phytochemicals, each of which was responsible to give it a good
antioxidative property. Phenolic compounds are known to be
powerful chain-breaking antioxidants. Their hydroxyl groups
provide them scavenging ability. In this study, the total phenolic
content of the extract was noted to be 5 ± 0.48 μg/mg. Its
estimated flavonoid content was 2.14 ± 0.47 µg/mg. Ascorbic acid
was reported to interact directly with radicals such as O
in plasma, thus preventing damage of cells. Here, ascorbic acid
content was seen to be 1.3 ± 0.11 µg/mg. β-carotene protects
against cancer and cardiovascular diseases. Lycopene, on the other
hand is one of the antioxidants that prevents carcinogenesis and
atherogenesis. β-carotene and lycopene were found in very less
amounts in this extract viz., 0.0014 ± 0.0002 and 0.0011 ± 0.0002
HPLC has been done to predict phenolic composition of
a fraction extracted after thermal processing. As shown in figure
1a and 1b, eleven phenolic substances were analysed and two of
them were detected in the ethanolic extract of T. clypeatus. Our
findings revealed that the dominant phenolic compounds were
pyrogallol (0.223 µg/mg) and cinnamic acid (0.095 µg/mg). The
response of antioxidants to different radical or oxidant sources
may be different. Therefore, no single assay can be capable to
reflect the mechanism of action of all radical sources or
antioxidants in any system. This way, the antioxidant activity of
the samples was assessed through five different methods.
Superoxide anion, which is a reduced form of molecular oxygen,
has been implicated in initiating oxidation reactions associated
with aging (Lavhale and Mishra, 2007). With one unpaired
electron, the superoxide ion is a free radical, and, like di-oxygen, it
is paramagnetic. It plays an important role in formation of other
reactive oxygen species, which induce oxidative damage in lipids,
proteins and DNA. The extract showed superoxide radical
scavenging capacity in a concentration dependant manner (Figure
2 a). The EC
value was 0.33 ± 0.01 mg/ml.
Mitra et al. / Journal of Applied Pharmaceutical Science 6 (07); 2016: 120-124
The method is based on the auto oxidation of riboflavin
in presence of light which in turn reduced NBT to form a blue
colour formazan. EC
value of it was much lower than that of
ethanolic extract of Termitomyces medius (1.40 mg/ml) and
Russula albonigra (0.74 mg/ml) (Mitra et al., 2014; Dasgupta et
The DPPH radical is a stable radical with a maximum
absorbance at 517 nm that can readily undergo reduction by any
antioxidant. The ease and convenience of this reaction has resulted
in the widespread use of it in the free radical-scavenging activity
assessment (Kumar et al., 2008). The DPPH radical scavenging
activity of the ethanolic extract is shown in Figure 2 b. and
is only at a concentration of 3.22 ± 0.4 mg/ml.
Iron generates free radical by the Fenton & Haber-Weiss
reaction. Chelation of metal ions prevents oxyradical generation
and consequent oxidative damage. Chelation is an important
antioxidant mechanism because it reduces concentration of the
catalysing transition metal in LPO (Kumar et al., 2008).The
ethanolic extract has demonstrated reasonable ferrous ion
chelating efficacy (Figure 2 c.) with EC
value at 0.21 ± 0.03
mg/ml, which indicated that it was a far better chelator than
Termitomyces medius (0.68 mg/ml), Russula albonigra (0.81
mg/ml) and Amanita vaginata (0.73 mg/ml) (Mitra et al., 2014,
Dasgupta et al., 2014, Paloi and Acharya, 2014). It is also reported
that chelating agents that form σ-bonds with a metal, are effective
as secondary antioxidants as they reduce the redox potential,
thereby stabilizing the oxidised form of the metal (Srivastava et
al., 2006). The reducing power assay is based on the ability of
sample to reduce yellow ferric form to blue ferrous form by the
action of electron-donating antioxidants (Benzie and Szeto, 1999).
The extract could reduce Fe
with 50 % inhibition capacity
at a concentration of 1.77 ± 0.035 mg/ml (Figure 2 d.). In
comparison to this the EC
value of ethanolic extract of
Termitomyces medius was much higher i.e. 2.05 mg/ml (Mitra et
al., 2014 a). Reductones work by breaking the chain of free
radicals by donating hydrogen atoms (Mitra et al., 2014 b). This
change can be monitored at 700nm, by measuring the intensity of
the Perl’s Prussian blue colour. In the designed experiments the
extract’s reducing power was compared to that of BHA, a
Total antioxidant capacity on the other hand was
measured by the formation of green phosphomolybdenum
complex. The ethanolic extract resulted in the reduction of Mo
(VI) to Mo (V) and form a green phosphate/Mo (V) complex. The
colour intensity was determined with the maximal absorption at
695 nm. Ascorbic acid was used as standard. Total antioxidant
capacity measured had shown that 1mg of extract was as
functional as 1.3 ± 0.39 µg of ascorbic acid (expressed as 100 µg
There were statistically significant differences between
the ethanolic extract of T. clypeatus and the respective standards (p
Fig. 1: HPLC chromatogram. (a) Peaks of standards: 1: gallic acid, 2: chlorogenic acid, 3: vanillic acid, 4: p-coumaric acid, 5: ferulic acid, 6: myricetin, 7:
salicylic acid, 8: quercetin, 9: cinnamic acid, 10: pyrogallol, 11: kaempferol. (b) Peaks of ethanolic extract of Termitomyces clypeatus: (9) cinnamic acid, (10)
Mitra et al. / Journal of Applied Pharmaceutical Science 6 (07); 2016: 120-124 123
Overall, it has been verified that wild ethanolic extract of
edible mushroom T. clypeatus has a variety of phytochemicals
such as phenols, ascorbic acid, flavonoids, β carotene and
lycopene which contribute to the good antioxidant mechanisms
viz., superoxide, DPPH radical scavenging activity, chelation of
ferrous ion activity and reducing ability. Therefore, food
modification through the balanced consumption of this mushroom
is more effective and important than other nutritional supplements
for the primary prevention of acute diseases.
The author PM is grateful and acknowledges Innovation
in Science Pursuit for Inspired Research of Department of Science
& Technology for financial support.
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How to cite this article:
Mitra P, Mandal NC, Roy A,
Acharya K. Phytochemical Study and
Antioxidative Property of Ethanolic Extract from Termitomyces
clypeatus. J App Pharm Sci, 2016; 6 (07): 120-124.