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2014 30: 826 originally published online 24 October 2012Toxicol Ind HealthNisar Ahmad, Fazal Mahmood, Shahid Akbar Khalil, Roshan Zamir, Hina Fazal and Bilal Haider Abbasi

mushroomsAntioxidant activity via DPPH, gram-positive and gram-negative antimicrobial potential in edible

  

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Article

Antioxidant activity via DPPH,gram-positive and gram-negativeantimicrobial potential in ediblemushrooms

Nisar Ahmad1, Fazal Mahmood2, Shahid Akbar Khalil2,Roshan Zamir2, Hina Fazal3 and Bilal Haider Abbasi1

AbstractEdible mushrooms (EMs) are nutritionally rich source of proteins and essential amino acids. In the presentstudy, 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) wereinvestigated. The DPPH radical scavenging activity revealed that the significantly higher activity (66.47%) wasobserved 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% activ-ity, P. ostreatus (Black (B)) exhibited 30.64%, P. ostreatus (Yellow (Y)) exhibited 40.75% and Pleurotus sajor-cajuexhibited 47.39% activity at higher concentrations. Furthermore, the antimicrobial potential were investigatedfor its toxicity against gram-negative bacterial strains (Escherichia coli, Pseudomonas aeroginosa, Salmonella typhi,Klebsiella pneumonia, Erwinia carotovora and Agrobacterium tumifaciens), gram-positive bacterial strains (Bacillussubtilis, Bacillus atrophaeus and Staphylococcus aureus) and a fungal strain (Candida albicans) in comparison withstandard antibiotics. Antimicrobial screening revealed that the ethanol extract of P. ostreatus was active againstall microorganism tested except E. coli. Maximum zone of inhibition (13 mm) was observed against fungus andA. 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 againstall other microbes except S. typhi. M. esculenta possess maximum activities in terms of inhibition zone against allmicroorganisms tested except S. typhi.

KeywordsEdible 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 representa-

tive 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

1Department of Biotechnology, Faculty of Biological Sciences,Quaid-i-Azam University, Islamabad, Pakistan2Nuclear Institute for Food and Agriculture (NIFA), Peshawar,Pakistan3Pakistan Council of Scientific and Industrial Research (PCSIR)Laboratories Complex, Peshawar, Pakistan

Corresponding author:Nisar Ahmad, Department of Biotechnology, Faculty of BiologicalSciences, Quaid-i-Azam University, Islamabad 45320, Pakistan.Email: nisarbiotech@gmail.com

Toxicology and Industrial Health2014, Vol. 30(9) 826–834© The Author(s) 2012Reprints and permissions:sagepub.co.uk/journalsPermissions.navDOI: 10.1177/0748233712463775tih.sagepub.com

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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 metabo-

lites with antioxidant and antimicrobial properties.

Such EM-derived compounds includes; ergothio-

neine, phenolics and selenium (Rodriguez-Estrada

et al., 2009). The selenium is an active part of antiox-

idant 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 phe-

nolics, which is a major group of natural antioxidants

(Ahmad et al., 2011a, 2011b). The antioxidant proper-

ties 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, antifun-

gal 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 Staphylococcusaureus, 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 antimicro-

bial 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 con-

centrations 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 antimi-

crobial 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. Fol-

lowing 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 dis-

solved 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

Ahmad et al. 827

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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 fun-

gal 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 inhibi-

tion zone (Figure 6). Antimicrobial activity was deter-

mined 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 rad-

ical scavenging ability using the constant radical

(DPPH). The test extracts were prepared in ethanol;

therefore, the DPPH powder was also prepared in sim-

ilar 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 ¼ 100� 1� AE=AD½ �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 experi-

ments were repeated twice. Analysis of variance and

Duncan’s multiple range test was used for comparison

among treatment means.

Results and discussion

DRSA and antimicrobial activity inPleurotus ostreatus

In the present investigation the dose-dependent con-

centrations (200, 400, 600, 800 and 1000 mg) of

P. ostreatus extract in ethanol revealed that signifi-

cantly elevated (<37%) antioxidant (%DRSA) activ-

ity 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 concentra-

tion 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. aerogi-

nosa, B. subtilis and B. atrophaeus, 11.33 mm

828 Toxicology and Industrial Health 30(9)

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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 Morchellaesculenta

%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. escu-

lenta (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

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.

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 activ-

ity 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 (Yel-

low (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. How-

ever, <40% activity was recorded for 800 mg extract

of P. ostreatus (Y) (Figure 4). It means that P. ostrea-

tus (Y) has more power than P. ostreatus (B) to sca-

venge 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)

7

14

21

28

35

1000800600400

DPP

H r

adic

al s

cave

ngin

g ac

tivity

(%

)

Pleurotus ostreatus extracts (µg)

aab

bc

cd

d

200

Figure 1. Dose-dependent (200, 400, 600, 800 and1000 mg) antioxidant activity via DPPH in the ethanolicextract of Pleurotus ostreatus. The activity was determinedusing DPPH as a free radical. Values are means of triplicateswith SD. Means with common letters are not significantlydifferent at p < 0.05. DPPH: 1,1-diphenyl-2-picrylhydrazyl.

Ahmad et al. 829

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Tab

le1.

Antim

icro

bia

lac

tivi

ties

ined

ible

mush

room

s.a

Tes

tst

rain

sPl

euro

tus

ostr

eatu

sPl

euro

tus

sajo

r-ca

juP.

ostr

eatu

s(Y

ello

w)

P.os

trea

tus

(Bla

ck)

Mor

chel

laes

cule

nta

Tet

racy

clin

eEry

thro

myc

inC

lotr

imaz

ole

Cip

roflo

xac

in

Esc

herich

iaco

liN

AN

A10+

0.1

9bc

10+

0.3

6bc

15+

0.1

7b

15.5

+0.3

5a

Pseu

dom

onas

aero

gino

sa12+

0.1

3a

11.3

3+

0.7

6a

21.8

3+

1.8

a11.5

+0.5

bc

11+

1bc

11+

0.1

3b

Salm

onel

laty

phi

6.7

5+

0.2

5b

NA

NA

NA

NA

NA

42+

0.8

a

Stap

hylo

cocc

usau

reus

11+

0.1

73

a10.7

5+

0.7

5a

NA

10+

0.3

4bc

14+

0.3

6b

25+

0.2

87

a

Bac

illus

subt

ilis

12+

0.2

57

a12.5

+0.5

03

a15+

0.1

83

b11.7

5+

0.4

2bc

11+

0.4

52

bc

26+

0.5

01

a

Bac

illus

atro

phae

us12+

0.2

18

a12.5

+0.1

85

a20+

0.1

69

a11+

0.1

78

bc

20+

0.1

76

a25+

0.2

60

a

Kle

bsie

llapn

eum

onia

e11.3

3+

0.7

6a

11.6

7+

1.1

5a

11.8

3+

1.2

5bc

15+

0.5

a11.3

3+

0.2

8bc

NA

29+

0.5

83

b

Can

dida

albi

cans

13+

0.0

65

a11+

0.2

626

a21+

0.2

23

a16+

0.3

59

a20+

0.2

94

a35+

0.2

2a

Erw

inia

caro

tovo

ra12.1

6+

0.7

6a

11.5

3+

0.5

6a

14+

0.3

59

bc

11.7

5+

0.3

1bc

11.5

+0.5

bc

11.5

+0.5

b

Agr

obac

terium

tum

ifaci

ens

13+

0.0

65

a11+

0.0

76

a18.5

+0.2

7b

13+

0.2

38

b13+

0.4

125

b15+

0.1

74

a

a SDal

ong

with

mea

ns

with

com

mon

lett

ers

are

not

sign

ifica

ntly

diff

eren

tat

p<

0.0

5.

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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.

DRSA and antimicrobial activity of Pleurotussajor-caju

Ethanolic extract of P. sajor-caju revealed that

the maximum activity of <47% was present in

elevated concentration (1000 mg). But at lower

40

45

50

55

60

65

70D

PPH

rad

ical

sca

veng

ing

activ

ity (

%)

Morchella esculenta extracts (µg)

a

b

c

cd

d

1000800600400200

Figure 2. Dose-dependent (200, 400, 600, 800 and1000 mg) antioxidant activity via DPPH in the ethanolicextract of Morchella esculenta. The activity was determinedusing DPPH as a free radical. Values are means of triplicateswith SD. Means with common letters are not significantlydifferent at p < 0.05. DPPH: 1,1-diphenyl-2-picrylhydrazyl.

24.0

25.5

27.0

28.5

30.0

31.5

24.0

25.5

27.0

28.5

30.0

31.5

bc

b

DPP

H r

adic

al s

cave

ngin

g ac

tivity

(%

)

Pleurotus ostreatus (Black) extracts (µg)

a

b

c

1000800600400200

Figure 3. Dose-dependent (200, 400, 600, 800 and1000 mg) antioxidant activity via DPPH in the ethanolicextract of Pleurotus ostreatus (Black). The activity was deter-mined using DPPH as a free radical. Values are means of tri-plicates with SD. Means with common letters are notsignificantly different at p < 0.05. DPPH: 1,1-diphenyl-2-picrylhydrazyl.

10

20

30

40

50

DPP

H r

adic

al s

cave

ngin

g ac

tivity

(%

)

Pleurotus ostreatus (Yellow) extracts (µg)

a

ab

bb

c

1000800600400200

Figure 4. Dose-dependent (200, 400, 600, 800 and1000 mg) antioxidant activity via DPPH in the ethanolicextract of Pleurotus ostreatus (Yellow). The activity wasdetermined using DPPH as a free radical. Values are meansof triplicates with SD. Means with common letters are notsignificantly different at p < 0.05. DPPH: 1,1-diphenyl-2-picrylhydrazyl.

32

36

40

44

48

DPP

H r

adic

al s

cave

ngin

g ac

tivity

(%

)

Pleurotus sajor-caju extracts (µg)

a

b

cc

d

1000800600400200

Figure 5. Dose-dependent (200, 400, 600, 800 and1000 mg) antioxidant activity via DPPH in the ethanolicextract of Pleurotus sajor-caju. The activity was determinedusing DPPH as free radical. Values are means of triplicateswith SD. Means with common letters are not significantlydifferent at p < 0.05. DPPH: 1,1-diphenyl-2-picrylhydrazyl.

Ahmad et al. 831

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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. sajor-

caju showed best activities against both the species

of Bacillus, forming 12.5 mm inhibitory zones

followed by K. pneumoniae (11.67 mm), E. caroto-

vora (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, respec-

tively. However, S. typhi and K. pneumoniae were

found resistant to tetracycline, so they were also

tested against the most specific antibiotic ciprofloxa-

cin (50 mg/disc) showing 42 mm and 29 mm zones,

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

832 Toxicology and Industrial Health 30(9)

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respectively. Erythromycin (15 mg/disc) formed larg-

est 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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