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and Industrial Health

Antioxidant activity via DPPH, gram-positive and gram-negative antimicrobial potential in edible
mushrooms
Nisar Ahmad, Fazal Mahmood, Shahid Akbar Khalil, Roshan Zamir, Hina Fazal and Bilal Haider Abbasi
Toxicol Ind Health 2014 30: 826 originally published online 24 October 2012
DOI: 10.1177/0748233712463775
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Article

Antioxidant activity via DPPH,


gram-positive and gram-negative
antimicrobial potential in edible
mushrooms

Toxicology and Industrial Health


2014, Vol. 30(9) 826834
The Author(s) 2012
Reprints and permissions:
sagepub.co.uk/journalsPermissions.nav
DOI: 10.1177/0748233712463775
tih.sagepub.com

Nisar Ahmad1, Fazal Mahmood2, Shahid Akbar Khalil2,


Roshan Zamir2, Hina Fazal3 and Bilal Haider Abbasi1

Abstract
Edible mushrooms (EMs) are nutritionally rich source of proteins and essential amino acids. In the present
study, the antioxidant activity via 1,1-diphenyl-2-picrylhydrazyl (DPPH) and antimicrobial potential in EMs
(Pleurotus ostreatus, Morchella esculenta, P. ostreatus (Black), P. ostreatus (Yellow) and Pleurotus sajor-caju) were
investigated. The DPPH radical scavenging activity revealed that the significantly higher activity (66.47%) was
observed in Morchella esculenta at a maximum concentration. Similarly, the dose-dependent concentrations
(200, 400, 600, 800 and 1000 mg) were also used for other four EMs. Pleurotus ostreatus exhibited 36.13% activity, P. ostreatus (Black (B)) exhibited 30.64%, P. ostreatus (Yellow (Y)) exhibited 40.75% and Pleurotus sajor-caju
exhibited 47.39% activity at higher concentrations. Furthermore, the antimicrobial potential were investigated
for its toxicity against gram-negative bacterial strains (Escherichia coli, Pseudomonas aeroginosa, Salmonella typhi,
Klebsiella pneumonia, Erwinia carotovora and Agrobacterium tumifaciens), gram-positive bacterial strains (Bacillus
subtilis, Bacillus atrophaeus and Staphylococcus aureus) and a fungal strain (Candida albicans) in comparison with
standard antibiotics. Antimicrobial screening revealed that the ethanol extract of P. ostreatus was active against
all microorganism tested except E. coli. Maximum zone of inhibition (13 mm) was observed against fungus and
A. tumifaciens. P. sajor-caju showed best activities (12.5 mm) against B. subtilis, B. atrophaeus and K. pneumonia.
P. ostreatus (Y) showed best activities against P. aeroginosa (21.83 mm), B. atrophaeus (20 mm) and C. albicans
(21 mm). P. ostreatus (B) exhibited best activities against C. albicans (16 mm) and slightly lower activities against
all other microbes except S. typhi. M. esculenta possess maximum activities in terms of inhibition zone against all
microorganisms tested except S. typhi.
Keywords
Edible mushrooms, antioxidant activity, DPPH, antimicrobial, microbes

Introduction
Mushrooms have long been regarded as the most
scrumptious of foods all over the globe. The majority
of people are unaware of the fact that the mushrooms
available in the markets are only a single representative species of the countless delightful edible types of
mushrooms present in the world. These mushrooms
are found growing everywhere in the fields, yards,
parks, trees and shady floor forests. These ephemeral
plants are often thought to be strange and intolerable
and hence are usually avoided or crushed upon by the
people. These mushrooms that sprout in such reckless
abundance are spicy and mouthwatering and are

eagerly sought by the epicure (Christensen, 1985).


Edible mushrooms (EMs) are the encouraging source

Department of Biotechnology, Faculty of Biological Sciences,


Quaid-i-Azam University, Islamabad, Pakistan
2
Nuclear Institute for Food and Agriculture (NIFA), Peshawar,
Pakistan
3
Pakistan Council of Scientific and Industrial Research (PCSIR)
Laboratories Complex, Peshawar, Pakistan
Corresponding author:
Nisar Ahmad, Department of Biotechnology, Faculty of Biological
Sciences, Quaid-i-Azam University, Islamabad 45320, Pakistan.
Email: nisarbiotech@gmail.com

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Ahmad et al.

827

of proteins, amino acids, vitamins (ascorbic acid,


biotin, cobalamines, niacin, riboflavin and thiamine)
and minerals (phosphorous, potassium and selenium)
(Barros et al., 2007). The EMs are considered as
therapeutic foods to cure diseases such as cancer,
hypercholesterolemia and hypertension (Manzi
et al., 2001). EM produced potent secondary metabolites with antioxidant and antimicrobial properties.
Such EM-derived compounds includes; ergothioneine, phenolics and selenium (Rodriguez-Estrada
et al., 2009). The selenium is an active part of antioxidant enzyme selenoprotein that prevents cellular
damage from toxic free radicals (Vidovic et al.,
2010). EM-derived ergothioneine also belongs to thiol
group, which is a plant-based natural antioxidant. An
antioxidant is a talented molecule that inhibits or
slows down the oxidation of other molecules (Ahmad
et al., 2010b). During stress conditions or incomplete
oxidation, free toxic radicals are produced, which
further start chain reaction and finally damage cells
or tissues (Ahmad et al., 2010a). Medicinal plants and
EM produced active secondary metabolites like phenolics, which is a major group of natural antioxidants
(Ahmad et al., 2011a, 2011b). The antioxidant properties of plants or mushroom-based phenolics are due to
the redox reactions that permitted them to perform as
hydrogen atom donors or reducing agents (Ahmad
et al., 2011c). Therefore, natural antioxidants are the
scavengers of toxic radicals and chain breakers,
complexers of pro-oxidant metal ions and scavengers
of singlet oxygen formation such as reactive oxygen
species, which injured DNA molecule, lipids and
proteins (Ahmad et al., 2010a, 2010b). Mushrooms
are the natural gifts of antimicrobial and antioxidant
compounds. EM must secrete antimicrobial, antifungal and antioxidant metabolites to struggle and stay
alive in natural environment. EM is a solid source
of natural antibiotics due to the presence of cell
wall that has immune-modulatory activities and
secretes extracellular metabolites that compete with
pathogenic microorganisms (Barros et al., 2007).
Moreover, the mycelial secretions of EM are active
against malarial parasite (Plasmodium falciparum)
and protozoa (Isaka et al., 2001; Lovy et al., 1999).
A well known mushroom Lentinus edodes has the
ability to enhance the host immune system against
pathogenic microorganism including Staphylococcus
aureus, Bacillus subtilis and Escherichia coli and also
possess a well-documented antitumor activity (Barros
et al., 2007; Jong and Birmingham, 1993). Not only
the mycelium but also the fruiting body produces

antibacterial and antimicrobial compounds (Akyuz


and Kirbag, 2009). In the literature cited, various
workers all over the world have documented the
antimicrobial potential of various extracts from
mushrooms (Barros et al., 2007; Demirhan et al.,
2007; Gbolagade et al., 2007; Gbolagade and Fasidi,
2005; Gezer et al., 2006; Jonathan and Fasidi, 2003;
Rosa et al., 2003; Solak et al., 2006; Turkoglu et al.,
2006, 2007; Uzun et al., 2004).
EM is a naturally packed gift of antioxidant and
antimicrobial compounds. These natural offerings
should be consumed without any hesitation for health
benefits and spicy savor. The current experiment was
designed to investigate the antioxidant potential via
1,1-diphenyl-2-picrylhydrazyl (DPPH) and antimicrobial efficiency against pathogenic microorganisms.
These different EMs have enough potential to scavenge
noxious free radicals and also inactivate pathogenic
germs. These nutritionally rich mushrooms produce
valuable metabolites that act as natural antioxidants
and antimicrobial agents. Therefore, technologies are
to be developed to extract these natural antioxidants
and antimicrobial compounds from EM. Further
research is required to find minimum inhibitory concentrations in different extracts of these mushrooms.

Material and methods


Preparation of solvent extractions
These EMs were collected from Nuclear Institute for
Food and Agriculture (NIFA), Peshawar, Pakistan. All
five mushrooms were identified and authenticated by
Dr Fazal Mahmood, Deputy Chief Scientist (DCS),
NIFA. Each EM was oven (Heraeus, T-6030, Thermo
Scientific, Langenselbold, Germany) dried. For antimicrobial activity, each EM was powdered, 15 g of milled
materials were individually filled in thimble and
extracted successively with ethanol (150 ml) using a
Soxhelt extraction unit for 72 h. The extracted solution
were concentrated using rotary flash evaporator. Following complete disappearance of ethanol, each extract
was weighed and kept at 4 C in hermetically sealed
vials. Accurately 15 mg of each EM extract were dissolved separately in 1 ml of dimethyl sulfoxide
(DMSO) as a solvent and were used as the test extracts
for antimicrobial assessment.

Gram-positive and gram-negative strains


Gram-positive and gram-negative bacterial strains
including E. coli (ATCC #25922), Pseudomonas

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828

Toxicology and Industrial Health 30(9)

aeroginosa (ATCC #9721) and S. aureus (ATCC


#6538), clinical isolates of Klebsiella pneumoniae,
Salmonella typhi, B. subtilis, B. atrophaeus, Erwinia
carotovora and Agrobacterium tumifaciens and a fungal strain of Candida albicans were all procured from
Pakistan Council of Scientific and Industrial Research
(PCSIR) Laboratories Complex, Peshawar, Pakistan.
These microorganisms were maintained on nutrient
agar medium at 4 C till activity.

Determination of antimicrobial activity


Antimicrobial activity were determined according to
disk diffusion method given by Fazal et al. (2011a,
2012), which is given in terms of diameters of inhibition zone (Figure 6). Antimicrobial activity was determined against six gram-negative strains (E. coli,
P. aeroginosa, S. typhi, K. pneumonia, E. carotovora
and A. tumifaciens), three gram-positive bacterial
strains (B. subtilis, B. atrophaeus and S. aureus) and
a fungal strain (C. albicans). Antimicrobial potential
was recorded for each extract in terms of zones of
inhibition around each disc (measured in millimeter).
Microbial cultures were spread on each nutrient agar
plate. These impregnated plates were then kept for
absorption (15 min) in a refrigerator. Whatman No
1 filter paper discs were placed on these agar media
plates. The stock solutions of the extracts were
applied on these discs in triplicates. Antibiotics
including Tetracycline, Erythromycin, Clotrimazole
and Ciprofloxacin were applied as positive controls
on separate plates against gram-positive bacteria,
gram-negative bacteria and C. albicans, respectively,
while DMSO used for making the stock solution was
applied as negative controls. These plates were then
incubated at 37 C overnight.

DPPH radical scavenging activity


The antioxidant activity (DPPH radical scavenging
activity (DRSA)) was determined according to the
method of Ahmad et al. (2012). Each EM extracts
were calculated in terms of hydrogen donating or radical scavenging ability using the constant radical
(DPPH). The test extracts were prepared in ethanol;
therefore, the DPPH powder was also prepared in similar solvent. Accurately weighed DPPH of 1.25 mg
was dissolved in 20 ml (4X concentration) of ethanol
to obtain stock solution. For activity, 1.0 ml of sample
solution was added to 2.0 ml of DPPH solution in
spectrophotometer cuvette separately. These solution
mixtures were incubation in dark for approximately

30 min at room temperature. After incubation period,


the absorbance of the solution was measured at
517 nm. Lesser absorbance of the reaction mixture
indicated higher DRSA. All tests were carried out in
triplicate. Finally, the radical scavenging activity was
calculated as the percentage of DPPH discoloration
using the following equation
% DRSA 100  1  AE=AD
where AE represents the solution absorbance at
517 nm, when optimum quantity of each mushroom
extract was added to DPPH solution after 30 min of
incubation at room temperature, and AD represents
the absorbance of DPPH solution without tissue
extracts.

Statistical analysis
The experiment was laid out according to CR design
using three replicates for each activity and the experiments were repeated twice. Analysis of variance and
Duncans multiple range test was used for comparison
among treatment means.

Results and discussion


DRSA and antimicrobial activity in
Pleurotus ostreatus
In the present investigation the dose-dependent concentrations (200, 400, 600, 800 and 1000 mg) of
P. ostreatus extract in ethanol revealed that significantly elevated (<37%) antioxidant (%DRSA) activity was recorded in 1000 mg (Figure 1). However,
significantly similar activity was also observed in
800 mg ethanolic portion. Moreover, activity of
>20% was recorded in 400 and 600 mg fractions
of P. ostreatus. Poorer activity (>10%) in terms of
percentage was recorded in 200 mg fraction of P.
ostreatus. % DRSA revealed that as the concentration of the extract increases, the activity also
increases. Similar % DRSA was also reported in
various plant tissues by Ahmad et al. (2010b,
2011b). The current data are an agreement with the
results of Fazal et al. (2011b). Furthermore, for the
antimicrobial activities, the ethanolic extract of P.
ostreatus was best active against C. albicans and
A. tumifaciens forming 13-mm zone in each case,
followed by E. carotovora forming 12.16-mm zone
(Table 1). Moreover 12 mm zones were created
against three types of bacteria including P. aeroginosa, B. subtilis and B. atrophaeus, 11.33 mm

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DPPH radical scavenging activity (%)

Ahmad et al.

829

ab

35
bc

28

21

cd

14
d

was ineffective against S. typhi, while illustrated


best activities against all other tested organisms.
It showed best activity against gram-positive
bacteria B. atrophaeus and against the fungi
C. albicans by showing 20 mm zones in each case.
It also demonstrated paramount activities against
E. coli (15 mm), S. aureus (14 mm), A. tumifaciens
(13 mm), E. carotovora (11.5 mm) and K. pneumoniae
(11.33 mm). The growth of P. aeroginosa and
B. subtilis were equally inhibited by M. esculenta by
forming 11 mm zones.

7
200

600
400
800
Pleurotus ostreatus extracts (g)

1000

Figure 1. Dose-dependent (200, 400, 600, 800 and


1000 mg) antioxidant activity via DPPH in the ethanolic
extract of Pleurotus ostreatus. The activity was determined
using DPPH as a free radical. Values are means of triplicates
with SD. Means with common letters are not significantly
different at p < 0.05. DPPH: 1,1-diphenyl-2-picrylhydrazyl.

against K. pneumoniae followed by 11 mm against


S. aureus. Furthermore, P. ostreatus was least active
against S. typhi (6.75 mm) and was found inactive
against E. coli. Similar antioxidant and antimicrobial
activities against these microorganisms were also
reported by Fazal et al. (2011a) in 11 medicinal
species.

DRSA and antimicrobial activity of Morchella


esculenta
%DRSA in ethanolic extracts of EM was evaluated in
order to compare the potential of each mushroom for
scavenging free radicals. Different doses of M. esculenta (200, 400, 600, 800 and 1000 mg) revealed that
higher activity (<66%) was presented by 1000 mg
fraction. In the overall experiment, of the five Ems,
the M. esculenta is the only species that scavenges
more free radicals (Figure 2). Activity of >60% was
observed in 800 mg ethanolic portion. The 400 and
600 mg fraction exhibited <53% and <57% activity,
respectively. However, <44% activity was recorded
for lower concentration (200 mg) of M. esculenta.
The order of activity for different fractions of
M. esculenta is given as 44.51% (200 mg) < 53.75%
(400 mg) < 57.23% (600 mg) < 61.27% (800 mg) <
66.47% (1000 mg). Furthermore, the antimicrobial
potential against gram-positive and gram-negative
bacteria revealed that the M. esculenta ethanol extract

DRSA and antimicrobial activity of P. ostreatus


(Black)
P. ostreatus (Black (B)) has lower potential than
M. esculenta to scavenge DPPH free radicals. During
the experiment, <24% activity was recorded for
200 mg fraction of extract. While slightly higher
activity of <27% was observed in 400 mg fraction
of extract (Figure 3). Both the 600- and 800-mg
fraction exhibited similar activities (<28%). The
most active fraction was 1000 mg that showed <30%
activity. However, in overall experiment, lower activity was recorded in P. ostreatus (B). P. ostreatus (B)
showed the best anticandidal activity (16 mm).
The best antibacterial activities were recorded against
K. pneumoniae (15 mm) and A. tumifaciens (13 mm).
This was equally effective against B. subtilis and
E. carotovora by forming 11.75 mm inhibitory zone,
followed by 11.5 mm against P. aeroginosa
and 11 mm against B. atrophaeus (Table 1). The
smallest zones of 10 mm were formed both against
S. aureus and E. coli. The activity was absent against
S. typhi.

DRSA and antimicrobial activity of P. ostreatus


(Yellow)
At lower concentration of 200 mg, P. ostreatus (Yellow (Y)) exhibited least activity of <8% in ethanolic
extract. As the concentration increases (400 mg), the
activity also increases (<32%). Significantly, similar
activity (<35%) was also observed for 600 mg. However, <40% activity was recorded for 800 mg extract
of P. ostreatus (Y) (Figure 4). It means that P. ostreatus (Y) has more power than P. ostreatus (B) to scavenge toxic free radicals. Furthermore, maximum
activity of <44% was recorded for higher fraction
(1000 mg) of P. ostreatus (Y) extract. Moreover,
regarding antimicrobial activities, P. ostreatus (Y)

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Pleurotus
ostreatus

11 + 0.2626a
11.53 + 0.56a
11 + 0.076a

12.5 + 0.503a
12.5 + 0.185a
11.67 + 1.15a

NA
10.75 + 0.75a

NA
11.33 + 0.76a

Pleurotus
sajor-caju

NA
10 + 0.34bc

10 + 0.36
11.5 + 0.5bc

bc

P. ostreatus
(Black)
b

NA
14 + 0.36b

15 + 0.17
11 + 1bc

Morchella
esculenta

21 + 0.223a
16 + 0.359a
bc
14 + 0.359 11.75 + 0.31bc
18.5 + 0.27b
13 + 0.238b

NA

NA

15.5 + 0.35
11 + 0.13b

Tetracycline

20 + 0.294a
11.5 + 0.5bc
11.5 + 0.5b
b
13 + 0.4125
15 + 0.174a

15 + 0.183b 11.75 + 0.42bc


11 + 0.452bc
a
bc
20 + 0.169
11 + 0.178
20 + 0.176a
bc
a
11.83 + 1.25
15 + 0.5
11.33 + 0.28bc

NA
NA

10 + 0.19
21.83 + 1.8a

bc

P. ostreatus
(Yellow)

SD along with means with common letters are not significantly different at p < 0.05.

Escherichia coli
NA
Pseudomonas
12 + 0.13a
aeroginosa
Salmonella typhi
6.75 + 0.25b
Staphylococcus
11 + 0.173a
aureus
Bacillus subtilis
12 + 0.257a
Bacillus atrophaeus
12 + 0.218a
Klebsiella
11.33 + 0.76a
pneumoniae
Candida albicans
13 + 0.065a
Erwinia carotovora 12.16 + 0.76a
Agrobacterium
13 + 0.065a
tumifaciens

Test strains

Table 1. Antimicrobial activities in edible mushrooms.a

26 + 0.501a
25 + 0.260a

25 + 0.287

35 + 0.22a

29 + 0.583b

42 + 0.8a

Erythromycin Clotrimazole Ciprofloxacin

Ahmad et al.

831

50

a
DPPH radical scavenging activity (%)

DPPH radical scavenging activity (%)

70
65
b
60

c
cd

55
50
45

40

600
400
800
Morchella esculenta extracts (g)

30

20

10

30.0
b

27.0

24.0

800

1000

31.5

28.5

bc

25.5

600

27.0

25.5

600
400
800
1000
200
Pleurotus ostreatus (Black) extracts (g)

Figure 4. Dose-dependent (200, 400, 600, 800 and


1000 mg) antioxidant activity via DPPH in the ethanolic
extract of Pleurotus ostreatus (Yellow). The activity was
determined using DPPH as a free radical. Values are means
of triplicates with SD. Means with common letters are not
significantly different at p < 0.05. DPPH: 1,1-diphenyl-2picrylhydrazyl.

24.0

DPPH radical scavenging activity (%)

DPPH radical scavenging activity (%)

30.0
b

400

Pleurotus ostreatus (Yellow) extracts (g)

28.5

200

1000

Figure 2. Dose-dependent (200, 400, 600, 800 and


1000 mg) antioxidant activity via DPPH in the ethanolic
extract of Morchella esculenta. The activity was determined
using DPPH as a free radical. Values are means of triplicates
with SD. Means with common letters are not significantly
different at p < 0.05. DPPH: 1,1-diphenyl-2-picrylhydrazyl.
31.5

b
b

40
200

a
ab

48

44
b
40

was most active against P. aeroginosa (21.83 mm)


followed by C. albicans (21 mm), B. atrophaeus
(20 mm), A. tumifaciens (18.5 mm), B. subtilis
(15 mm), E. carotovora (14 mm) and K. pneumoniae
(11.83 mm).This species was least active against
E. coli (10 mm) and was found inactive against
S. typhi and S. aureus.

400

600

36
d
32
200

Figure 3. Dose-dependent (200, 400, 600, 800 and


1000 mg) antioxidant activity via DPPH in the ethanolic
extract of Pleurotus ostreatus (Black). The activity was determined using DPPH as a free radical. Values are means of triplicates with SD. Means with common letters are not
significantly different at p < 0.05. DPPH: 1,1-diphenyl-2picrylhydrazyl.

800

1000

Pleurotus sajor-caju extracts (g)

Figure 5. Dose-dependent (200, 400, 600, 800 and


1000 mg) antioxidant activity via DPPH in the ethanolic
extract of Pleurotus sajor-caju. The activity was determined
using DPPH as free radical. Values are means of triplicates
with SD. Means with common letters are not significantly
different at p < 0.05. DPPH: 1,1-diphenyl-2-picrylhydrazyl.

DRSA and antimicrobial activity of Pleurotus


sajor-caju
Ethanolic extract of P. sajor-caju revealed that
the maximum activity of <47% was present in
elevated concentration (1000 mg). But at lower

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832

Toxicology and Industrial Health 30(9)

Figure 6. Pictorial presentation of some inhibition zones of mushrooms extracts against microorganisms.

concentration of 800 mg, the activity observed was


<41%. In the ethanolic extract of P. sajor-caju,
lower potential of activity was observed in
200 mg (<32%). Significantly, similar activity of
<40% was exhibited by 400 and 600 mg portion
of the extracts (Figure 5). Furthermore, P. sajorcaju showed best activities against both the species
of Bacillus, forming 12.5 mm inhibitory zones
followed by K. pneumoniae (11.67 mm), E. carotovora (11.53 mm) and P. aeroginosa (11.33 mm).
This mushroom was equally effective against
C. albicans and A. tumifaciens showing 11 mm
activity, and least active against S. aureus
(10.75 mm). Both the gram-negative bacteria
E. coli and S. typhi were resistant to the activity
of P. sajor-caju.

Antimicrobial potential of the positive controls


(antibiotics)
Various antibiotics procured from the markets were
also applied as positive controls to check the
resistance of the tested organisms. These include
tetracycline, erythromycin and clotrimazole against
gram-negative and gram-positive bacteria and fungus,
respectively (Table 1). Prepared disc of tetracycline
(30 mg/disc) were applied for E. coli, P. aeroginosa,
E. carotovora and A. tumifaciens that showed
15.5 mm, 11 mm, 11.5 mm and 15 mm zones, respectively. However, S. typhi and K. pneumoniae were
found resistant to tetracycline, so they were also
tested against the most specific antibiotic ciprofloxacin (50 mg/disc) showing 42 mm and 29 mm zones,

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Ahmad et al.

833

respectively. Erythromycin (15 mg/disc) formed largest zone against gram positive B. subtilis (26 mm),
followed by S. aureus and B. atrophaeus (25 mm).
Clotrimazole (50 mg/disc) exhibited 35 mm inhibitory
zone for C. albicans.
Funding
This research received no specific grant from any funding
agency in the public, commercial, or not-for-profit sectors.

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