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Determination of antioxidant and antimicrobial properties of Agaricus

bisporus from Romanian markets

Article  in  Analele Universitatii "Ovidius" Constanta - Seria Chimie · January 2012

DOI: 10.2478/v10310-012-0007-4

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 10.2478/v10310-012-0007-4
Ovidius University Annals of Chemistry Volume 23, Number 1, pp.47-52, 2012
Determination of antioxidant and antimicrobial properties of Agaricus bisporus
from Romanian markets
Emanuel VAMANU

University of Agronomical Sciences and Veterinary Medicine Bucharest, Faculty of Biotechnology 59 Marasti
Blvd, 011464, Bucharest, Romania
___________________________________________________________________________________________

Abstract The antioxidant action of ethanolic extract from Agaricus bisporus which is available in the
supermarkets of Bucharest, Romania, was assessed by determining its reducing power and its radical scavenging
activity. The determinations were made by analyzing the freeze-dried ethanolic extract. The radical scavenging
activity was determined using 2, 2-diphenyl-1-picrylhydrazyl (DPPH), superoxide radical, nitric oxide radical
and hydrogen peroxide scavenging assays. Total phenols, flavonoids, ascorbic acid, β-carotene and lycopene
were also determined. The ethanolic extract from A. bisporus could be a natural antioxidant and antimicrobial
source against the tested organisms, as demonstrated by the minimum inhibitory concentration values.

Keywords: mushroom, freeze dried, scavenging activity, MIC.

___________________________________________________________________________________________

1. Introduction objective of the study was to determine the

Agaricus bisporus is well known by several antioxidant properties of ethanolic extract from dried
names, such as the common mushroom, button mushrooms sold in the supermarkets of Bucharest.
mushroom, white mushroom and champignon The antioxidant properties were assayed in terms of
mushroom. This mushroom may be cultivated with antioxidant activity by their reducing power and
minimum effort and, therefore, it is most frequently their scavenging abilities on radicals. The amounts
found sold along with other market produce. It is of potential antioxidant components in the ethanolic
traded all year round, registering the highest sales extract were also determined. The second objective
volumes among the edible mushrooms. The of the study was to evaluate the antibacterial activity
marketed mushrooms are known for their rich source against Gram-negative and Gram-positive bacteria,
of biologically active compounds (phenolic and two Candida strains.
compounds, carotenoid compounds and ascorbic
acid), which offer protection to the human body in a 2. Experimental
natural way [1]. Moreover, they contain proteins and Preparation of samples. Mushrooms A.
nutritive fibers, as well as other vitamins necessary bisporus (trays weighing 500 g, collected over one
for the normal functioning of the human body. day) were purchased from the Bucharest
By way of previous researches, it was noticed supermarkets. Mushrooms without any damage were
that A. bisporus has a significant antioxidant activity chosen and dried in a stream of dry air. The drying
as compared with Pleurotus sajor-caju, Volvariella process took place at a constant temperature of 25
volvaceae or Pleurotus ostreatus. The most °C in the oven for 15 days, until a constant weight
significant antimicrobial activity was observed in was reached.
Staphylococcus aureus for ethyl acetate extracts [2]. Obtaining of extracts. The dried samples were
Such studies demonstrate that, besides its use in the subjected to 70% ethanol extraction; 10 g of the
food industry, these mushrooms also have a dried mushroom sample was extracted using 100 mL
therapeutic role, by integration thereof, in nutritional solvent (ethanol), over a period of 24 h, at 25 °C and
supplements. In the case of A. bisporus, at the at 150 rpm. The extract was filtered using a
phenols composition which prevails in most Whatman No. 4 filter paper [4,5]. The solvents used
mushrooms, ergothioneine is added [3]. The first for extraction were removed using a rotary vacuum

ISSN-1223-7221 © 2012 Ovidius University Press

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48 Development of antioxidant and antimicrobial.... / Ovidius University Annals of Chemistry 23 (1), 47-52 (2012)

evaporator Buchi R215, with a vacuum controller V- with molar absorbtivity of 81 M-1 cm-1. Each extract
850, and a heating bath for parallel evaporation (100 µg mL-1) was added to the hydrogen peroxide
Multivapor P-6, at 50 °C under vacuum. The solution (0.6 mL). The absorbance of hydrogen
resulting extracts were lyophilized in a freeze-dryer peroxide at 230 nm was determined after 10 min
ALPHA 1-2/LD plus (Martin Christ GmbH), at –55 against a blank containing phosphate buffer without
°C, for 48 h. Extract yield (g/g) was expressed as the hydrogen peroxide [9].
weight of extract (g) per gram of dry mushroom of Superoxide radical scavenging (SO) assay. The
A. bisporus [6]. reaction mixture contained Nitroblue tetrazolium
Antioxidant activity (DPPH free radical (0.1 mM) and Nicotinamide adenine dinucleotide
scavenging activity) of ethanolic mushroom extract. (0.1 mM) with or without sample to be assayed in a
The antioxidant activity of the plant extracts and the total volume of 1 mL of Tris-HCl buffer (0.02 M,
standard (ascorbic acid) was assessed on the basis of pH 8.3). The reaction was started by adding
the radical scavenging effect of the stable 1, 1- Phenazine methosulphate (10 µM) to the mixture
diphenyl-2-picrylhydrazyl (DPPH)-free radical and change in the absorbance was recorded at 560
activity. The different concentration of the extracts nm. The percent inhibition was calculated against a
(2-10 mg/mL) was prepared in ethanol. 0.002% of control without test sample [10].
DPPH was prepared in ethanol and 1 mL of this The total phenolic and flavonoid content of
solution was mixed with 1 mL of sample solution ethanolic extract, and several organic fractions, were
and standard solution separately. These solution determined using Folin-Ciocalteu reagent and
mixtures were kept in dark for 30 min and optical aluminium chloride colorimetric method,
density was measured at 517 nm using Helios λ respectively [10].
spectrophotometer. % inhibition was calculated β-carotene and lycopene determination. The
using the formula given below (14): (A – B)/A × dried ethanolic extract (100 mg) was vigorously
100, where A - optical density of the blank and B - shaken with 10 mL of acetone–hexane mixture (4:6)
optical density of the sample [5]. for 1 min and filtered through Whatman No. 4 filter
Reducing power. Different extract concentration paper. The absorbance of the filtrate was measured
(2 to 10 mg/mL) in ethanol (2.5 mL) was mixed with at 453, 645 and 663 nm. Contents of β-carotene and
2.5 mL of 200 mM sodium phosphate buffer (pH lycopene were calculated according to the following
6.6) and 2.5 mL of 1% potassium ferricyanide, and equations: lycopene (mg/100 mL) = -0.0458 A663 +
the mixture was incubated at 50°C for 20 min. Then, 0.372 A505 - 0.0806 A453; β-carotene (mg/100 mL) =
2.5 mL of 10% trichloroacetic acid was added, and 0.216 A663 - 0.304 A505 + 0.452 A453 [11].
the mixture was centrifuged at 200 ×g for 10 min. Ascorbic acid determination. Ascorbic acid
The upper layer (2.5 mL) was mixed with 2.5 mL of contents of the extract were determined
deionized water and 0.5 mL of 0.1% ferric chloride. spectrophotometrically by metaphosphoric acid
The absorbance was measured at 700 nm against a extraction of 2,6-dichlorophenol indophenol dye
blank. Ascorbic acid was used as standard [7]. using a Helios spectrophotometer in a wavelength of
Nitric oxide radical scavenging assay. The 500 nm with a 1 cm quartz cell. The ascorbic acid
procedure is based on the method [8] where sodium content was reported as mg/100 mL [12].
nitroprusside in aqueous solution at physiological Determining the antimicrobial capacity
pH spontaneously generates nitric oxide, which (Minimum Inhibitory Concentration - MIC). For
interacts with oxygen to produce nitrite ions that can tests, Escherichia coli CBAB 2, Bacillus cereus
be estimated using Greiss reagent. Scavengers of CMGB 215, Listeria innocua CMGB 218,
nitric oxide compete with oxygen leading to reduced Staphylococcus aureus ATCC 6588, Pseudomonas
production of nitrite ions. aeruginosa ATCC 15442, Candida albicans ATCC
Hydrogen peroxide radical scavenging (SO) 20231, Candida sp. ICCF 15 strains were used. Each
assay. A solution of hydrogen peroxide (2 mmol L-1) strain was inoculated separately (106 UFC/mL) in a
was prepared in a phosphate buffer (pH 7.4). The Petri dish on which LB/YPG agarised medium was
hydrogen peroxide concentration was determined poured. The extract was incorporated into nutrient
spectrophotometrically from absorption at 230 nm agar at concentrations from 1 mg/mL to 25 mg/mL.

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 E.Vamanu / Ovidius University Annals of Chemistry 23 (1), 47-52 (2012) 49

20 µL of extract were added and the dish was kept justifies the researches on new sources of bioactive
30 minutes to absorb the extract. Then it was compounds.
inserted in the thermostat, at 28 – 300C, for 24 hours.
The minimum inhibitory concentrations (MICs) of 2,2-diphenyl-1-picrylhydrazyl scavenging activity
the extract for each test microorganism were 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical is
regarded as the agar plate with the lowest a very stable one when it accepts an electron. The
concentrations without growth. [13]. action of antioxidants determines the reduction of
Statistical analysis. All the essays were assessed absorbance to 517 nm. The reduction of absorbance
in triplicate, and the results were expressed as mean caused by the antioxidants determines a change in
color from violet to yellow [14].
±SD values of three observations (p <0.05). The
Thus, DPPH is normally used as a sublayer to
mean values and standard deviation were calculated assess the antioxidant activity of an extract [15].
with the EXCEL program from Microsoft Office Figure 1 presents (p <0.01) the reduction of the
2010 package. DPPH radicals’ concentration due to the ethanolic
extract in A. bisporus and the standard (ascorbic
3. Results and Discussions acid). At a concentration of 10 mg/mL, the
scavenging ability of the DPPH radical was
Ethanol is a bipolar solvent with high approximately 43% lower than that of the standard,
penetration power, therefore, being mainly used in and by 31% as compared with the ethanolic extract
extractions of biologically active compounds from from A. blazei. The study confirms the moderate
natural products. Using a mixture of ethanol:distilled antioxidant effect of the ethanolic extract from A.
water of 70:30 (v/v), an extraction yield of 4.73 ± blazei. Similar values were obtained for strains of
0.32% was obtained. The extraction yield of Pleurotus. They were lower than those of the extract
phenolic compounds in A. bisporus was 0.19 ± from Coprinus comatus, approximately 65%, for
0.12%. similar concentrations of extract [16]. This result is
compliant with prior researches.
In vitro antimicrobial activity
The results of the in vitro antimicrobial activity Reducing power
of ethanolic extract are detailed in Table 1. All Figure 2 presents the reducing power of the
extracts from A. bisporus have antimicrobial activity ethanolic extract as compared with the ascorbic acid.
against the potentially pathogenic strains tested. This It is well known that the reducing power is
is due to the fact that the ethanolic extract contains associated with the antioxidant activity and the total
various components with antimicrobial activity. The quantity of phenols. The yellow color of the testing
freeze-dried extract displayed maximum inhibition solution changes to various tones of green-blue,
(MIC 5 mg/mL) towards Escherichia coli CBAB 2, depending on the reducing power of each extract or
and also against S. aureus ATCC 6588 (Gram- on the concentration thereof. The presence of
negative). In exchange, Pseudomonas aeruginosa antioxidants in an extract determines the reduction
ATCC 15442 proved to be the most resistant strain, of Fe3+ to Fe2+ [17, 18, 19]. The reducing power of
with a minimum inhibitory concentration value of 15 the ethanolic extract increased with its
mg/mL. The antimicrobial activity on the tested concentration.
strains confirms the use of such raw extracts, and
Table 1. Minimum inhibitory concentration (MIC) of Agaricus bisporus ethanolic extract
Antimicrobial activity Ampicillin
Tested strains
(mg/mL) (µg/mL)
Escherichia coli CBAB 2 5 22
Bacillus cereus CMGB 215 12.5 22
Listeria innocua CMGB 218 7.5 10
Staphylococcus aureus ATCC 6588 5 23
Pseudomonas aeruginosa ATCC 15442 15 28
Candida albicans ATCC 20231 7.5 25
Candida sp. ICCF 15 7.5 25

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50 Development of antioxidant and antimicrobial.... / Ovidius University Annals of Chemistry 23 (1), 47-52 (2012)

Table 2. EC50 values (mg/mL) obtained in the antioxidant activity of ethanolic extract from Agaricus bisporus
EC50 values
DPPH scavenging activity 3.59±0.08
Reducing power 4.71±0.15
Nitric oxide scavenging activity 4.22±0.29
Superoxide anion scavenging activity 10.00±1.37
Hydrogen peroxide scavenging activity 2.75±0.2

The results obtained (for 10 mg/mL) are similar 1,6

to those for A. arvensis and Sarcodon imbricatus, at 1,4

Agaricus bisporus Ascorbic Acid

a concentration of 5 mg/mL [11]. Also, the values of

Reducing power (700 nm)

1,2
the reducing power are similar to those obtained 1
from the fructification bodies of the mushrooms P.
0,8
ferula (0.57), P. ostreatus (0.56), and even higher
for the extract from Boletus edulis (0.44) at 13.72% 0,6

[17]. In exchange, as compared with the standard 0,4

(ascorbic acid), the results were, on average, 62.5% 0,2

lower, which is a moderate reducing power. These 0

results are compliant with the scavenging ability of 2 4 6 8 10

the DPPH radical. Sample concentration (mg/mL)

120
Fig. 2. Reducing power of the ethanolic extract from
DPPH scavenging activity (%)

Agaricus bisporus Ascorbic acid

100 Agaricus bisporus
80
100
Agaricus bisporus Ascorbic acid
Nitric oxide scavenging activity (%)

60 90

80
40 70

60
20
50

0 40
2 4 6 8 10
Sample concentration (mg/mL) 30

20

10
Fig. 1. DPPH radical scavenging activity of the 0
ethanolic extract from Agaricus bisporus 2 4 6
Sample concentration (mg/mL)
8 10

Nitric oxide radical scavenging activity

The nitric oxide may spontaneously occur by Fig. 3. Nitric oxide scavenging activity of the
oxidation. It is very unstable and reacts with oxygen. ethanolic extract from Agaricus bisporus
It is a strong mediator for certain physiological
processes, such as relaxation of the soft muscles and Superoxide anion Radical Scavenging Activity
neuronal signaling, while the deficit of nitric oxide is This radical is involved in the acceleration of
associated with hypertension [20,21]. The ethanolic aging processes. Superoxide anions play an
extract inhibits the nitric oxide depending on the important role in the formation of other reactive
concentration (Fig. 3). At a concentration of 10 oxygen species, such as singlet oxygen, hydrogen
mg/mL, the binding ability was 27.74% lower than peroxide and hydroxyl radical, which induce
that of the standard 67.2%. Thus, the EC50 value was oxidative damage in DNA, lipids and proteins [22].
4.22 ± 0.29 mg/mL, and the standard 0.08 ± 0.01 Figure 4 indicates a reduced inhibiting ability of the
mg/mL (Table 2). superoxide radical. An ability of 50% was

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 E.Vamanu / Ovidius University Annals of Chemistry 23 (1), 47-52 (2012) 51

determined for 10 mg/mL, and the ascorbic acid The validation of the results obtained is
used as standard displayed a value of 97% for the supported by the values which correspond to recent
same concentration of the sample. The researches in which both the antioxidant activity and
determinations are confirmed by the EC50 value of the reducing power are concerned (Table 2). In this
the standard, i.e. 0.6 ± 0.02 mg/mL. case, the values for EC50 were less than 10 mg/mL,
with the exception of the superoxide anion
100 scavenging activity. A very good correlation
coefficient (r2) was calculated between the quantity
Agaricus bisporus Ascorbic acid
Superoxide anion scavenging activity (%)

90

80 of phenols and the DPPH scavenging activity

70
(0.7864), reducing power (0.9843), nitric oxide
60
scavenging activity (0.9346), superoxide anion
50
scavenging activity (0.935) and hydrogen peroxide
40

30
scavenging activity (0.8956). In exchange, the
20
correlation coefficient between the content of
10
flavonoids and DPPH scavenging activity was
0 0.0776, reducing power (0.0027), nitric oxide
2 4 6 8 10 scavenging activity (0.0928), superoxide anion
Sample concentration (%)
scavenging activity (0.0087) and hydrogen peroxide
Fig. 4. Superoxide radical scavenging activity of the scavenging activity (0.0076). These low values of
ethanolic extract from Agaricus bisporus the correlation coefficient (r2) prove the different
roles exercised by the flavonoids as compared with
phenols on various species of free radicals [24].
In the ethanolic extract from A. bisporus,
Hydrogen peroxid scavenging activity (%)

100
Agaricus bisporus Ascorbic acid
compounds with antioxidant effect were identified
80
(Table 3). A very small quantity of carotenoid
60 compounds, especially lycopene, was identified, as
compared with other extracts from A. bisporus [25]
40
and P. ostreatus [26].
20
Table 3. Antioxidant contents of ethanolic extract
0 from Agaricus bisporus
2 4 6 8 10
Sample concentration (mg/mL)
Antioxidant components Ethanolic extract
Fig. 5. Hydrogen peroxide radical scavenging Total phenols
19.74±0.3
activity of the ethanolic extract from (mg gallic acid/g extract)
Agaricus bisporus Flavonoids
123±0.62
(mg quercetina/g extract)
Scavenging of hydrogen peroxide Ascorbic acid (mg/g extract) 0.01±0.01
Hydrogen peroxide is formed in vivo by enzymatic β-carotene (µg/100 g extract) 9±0.5
oxidation processes. It may go through the cellular Lycopene (µg/100 g extract) 0.1±0. 005
membrane, having significant oxidation abilities. On the other hand, the total determined quantity
Although not toxic in itself, its negative role results of phenols and flavonoids was higher than in the
from the ability to generate hydroxyl radicals at case of certain species related to or belonging to the
cellular level [23]. The biological value of the same species in the case of methanolic extract [27].
ethanolic extract from A. bisporus comes from the
high inhibiting ability of the hydrogen peroxide. 4. Conclusions
This conclusion is strengthened by the low value of A. bisporus has significant antioxidant
EC50, 2.75 ± 0.2 mg/mL (Table 2), as compared with activities and free radicals binding abilities. The
that of the ascorbic acid, 1.08 ± 0.13 mg/mL. extract represents a source of compounds useful in

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 52 Development of antioxidant and antimicrobial.... / Ovidius University Annals of Chemistry 23 (1), 47-52 (2012)

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