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Radiation Physics and Chemistry 106 (2015) 145150

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Radiation Physics and Chemistry


journal homepage: www.elsevier.com/locate/radphyschem

Considering the antibacterial activity of Zataria multiora Boiss


essential oil treated with gamma-irradiation in vitro and
in vivo systems
Fatemi Faezeh a,n, Dini Salome b, Dadkhah Abolfazl c, Zolfaghari Mohammad Reza d
a
Nuclear Fuel Cycle Research School, Nuclear Science and Technology Research Institute, Tehran, Iran
b
Young Researchers and Elite Club, Karaj Branch, Islamic Azad University, Karaj, Iran
c
Department of Medicine, Faculty of Medicine, Qom Branch, Islamic Azad University, Qom, Iran
d
Department of Microbiology, Faculty of Science, Qom Branch, Islamic Azad University, Qom, Iran

H I G H L I G H T S

 Zataria multiora Boiss essential oil has potential in vitro antimicrobial effect.
 Z. multiora oil has potential antimicrobial effect in vivo system.
 The antibacterial activities of the oil remained after irradiation treatments.

art ic l e i nf o a b s t r a c t

Article history: The aim of the present study was to evaluate the antibacterial activities of essential oils (EOs) obtained
Received 27 January 2014 from the aerial parts of Zataria multiora Boiss against Bacillus cereus, Pseudomonas aeroginosa,
Accepted 13 July 2014 Escherichia coli and Staphylococcus aureus by in vivo and in vitro methods. Also, the effects of gamma-
Available online 19 July 2014
irradiation (0, 10 and 25 kGy) as a new microbial decontamination on the antibacterial activities of
Keywords: Z. multiora were examined. For this purpose, the collected herbs were exposed to radiation at doses of
Zataria multiora 0, 10 and 25 kGy following essential oil (EOs) extraction by steam distillation. Then, the in vitro
Irradiation antibacterial potency of the irradiated and non-irradiated oils was determined by using disc diffusion,
Antibacterial activity agar well diffusion and MIC and MBC determination assays. The in vivo antibacterial activity was also
CLP
studied in sepsis model induced by CLP surgery by Colony forming units (CFUs) determination. The
results showed that the extracted oils were discovered to be effective against all the gram positive and
gram negative pathogens in vitro system. In addition, the oil signicantly diminished the increased CFU
count observed in CLP group. Moreover, the irradiated samples were found to possess the antibacterial
activities as the non-irradiated ones both in vitro and in vivo systems. These data indicated the potential
use of gamma-irradiation as a safe technique for preservation of Z. multiora as a medicinal plant with
effective antibacterial activities.
& 2014 Elsevier Ltd. All rights reserved.

1. Introduction et al., 2007). Also, every year an estimated 18 million people


worldwide develop sepsis with an approximate 30% death rate,
Sepsis is a complex and heterogeneous syndrome dened as a which makes it a serious healthcare and social problem (Slade et
systemic inammatory response to infection (Abraham et al., al., 2003). Studying sepsis in humans is difcult for many factors,
2000). Sepsis is common and carries a high risk of death and so the use of animal models has been proposed as a valuable tool
long-term complications. It remains the primary cause of death in the study of sepsis (Cohen et al., 2001; Fink and Heard, 1990).
from infection despite advances in modern medicine, including There are several experimental animal models (non-surgical and
vaccines, antibiotics, and intensive care (Angus et al., 2001; Engel surgical) to study the laboratory parameters observed in sepsis
(Nemzek et al., 2008). The most widely used sepsis model is
achieved by cecal ligation and puncture (CLP) which is recognized
n
Corresponding author.
as one of the models with the greatest compatibility in clinical
E-mail addresses: ffatemi@aeoi.org.ir, fatemi81@yahoo.com (Fatemi Faezeh). terms (Buras et al., 2005; Rittirsch et al., 2007; Deitch, 2005;

http://dx.doi.org/10.1016/j.radphyschem.2014.07.010
0969-806X/& 2014 Elsevier Ltd. All rights reserved.
146 Fatemi Faezeh et al. / Radiation Physics and Chemistry 106 (2015) 145150

Hubbard et al., 2005). CLP is performed by ligation of the cecum from Z. multiora when exposed to gamma-irradiation at two level
below the ileocecal valve after a midline laparotomy, followed by doses of 10 and 25 kGy both in vivo and in vitro systems.
needle puncture of the cecum. Perforation of the cecum leads to a
polymicrobial peritonitis, followed by translocation of bacteria
into the blood. In CLP, inammation progresses which leads to 2. Material and methods
immune, hemodynamic and biochemical changes, septic shock,
multiple organ failure and death (Wichterman et al., 1980; Deitch, 2.1. Plant material irradiation treatment
2005).
The increasing occurrence of resistance to antibiotics and The Aerial parts of the fresh Z. multiora (Voucher Number:
antimicrobial agents among bacteria is generating a need to nd 41754, Iranian botanical garden (TARI)) were compressed in three
new treatments, and some plant-derived volatile oils and extracts batches containing 50 g with heat-sealed polyethylene pouches
are known to have antibacterial activity (Cowan, 1999). Medicinal and passed by a Co60 source for irradiation at two different doses.
plants could be appropriate alternative treatments which the These doses were selected according to the database on approval
volatile oil and extracts are known to have antibacterial activities for irradiated food described by International Atomic Energy
(Deans et al., 1992; Piccaglia et al., 1993; Kim et al., 2012). Agency which suggests 10 kGy as the maximum approved dose
Zataria multiora Boiss. with the folk name of Avishan-e-Shirazi for decontamination of food supply and 25 kGy as sterile dose in
in Iran is a medicinal plant (herb) belonging to the Lamiaceae which all of the biological contamination were removed
family that grows only in Iran, Pakistan and Afghanistan. It is (International Atomic Energy Agency, 2006). The seeds were
known as avor ingredient in a wide variety of foods and also is subjected to a high dose rate research irradiator (Co60 Gammacell
used in traditional remedies possessed different pharmacological 220 [Atomic Energy of Canada Limited Radiochemical Company],
properties such as antimicrobial and anti-inammatory effects Canada) calibrated with Fricke standard dosimeter which is installed
(Gandomi et al., 2009; Hosseinzadeh et al., 2000; Iranian Herbal in Radiation Application Research School of Atomic Energy Organiza-
Pharmacopoeia, 2002; Sajed et al., 2013). According to our GCMS tion of Iran. The given dose was controlled by the exposure time of
data, the main oil compounds are phenolic compounds like each container to the source. The temperature and dose rate for all the
carvacrol, thymol, p-cymene and gamma-terpinene, which are samples were 2223 1C and 0.37 Gy/s, respectively. The dose range
well-known anti-microbial and anti-fungal agents (Fatemi et al., within the samples was 720% of the nominal dose. The control and
2012; Saleem et al., 2004; Shaee and Javidnia, 1997; Baser, 2008). irradiated samples were stored in plastic containers at room tempera-
The reports indicated the antibacterial effects against different ture (2830 1C) under the same conditions.
bacterial strains (Rahman et al., 2008). The antibacterial activity of
Z. multiora has been shown against a number of Gram-positive 2.2. Essential oil preparation
and Gram-negative bacteria (Mosha et al., 2007; Sadeghzadeh et
al., 2006; Saleem et al., 2004; Abbasgholizadeh et al., 2008). Fazeli Oil extraction of Z. multiora was provided by Clevenger-type
et al. (2007) who reported that Z. multiora hydroalcoholic apparatus (Walton and Brown, 1999), before and after -irradiation.
extracts had considerable antibacterial effects on Staphylococcus The extraction was carried out for 2 h and the oils were stored in
aureus, Bacillus cereus and Salmonella typhimurium (Fazeli et al., dark glass bottles in a freezer ( 20 1C) until further use. The
2007). Further, Sharifar et al. (2007) reported that the EOs of extraction yields for the EOs extracted from non-irradiated and
endemic Z. multiora Bioss strongly inhibited the growth of S. irradiated Z. multiora were approximately 4% (w/w).
aureus, Escherichia coli, Bacillus subtilis and Salmonella typhi espe-
cially the gram-negative ones (Sharifar et al., 2007). Another 2.3. Bacterial strains and growth media
study was shown that various extracts of
Z. multiora have high antibacterial effects on -lactamase produ- E. coli (ATCC 25922), P. aeroginosa (ATCC 27853), B. cereus (ATCC
cing P. aeruginosa strains compared to the standard antibiotics 9634) and S. aureus (ATCC 25923) were cultured in Mueller
(Fallah et al., 2013). Hinton agar (MHA) at 37 1C and maintained on same medium at
On the other hand, one of the major problems associated with 4 1C. All bacterial strains were rst subcultured on agar at 37 1C for
dry commodities is its microbial contamination resulting in quality 1824 h. Bacterial suspension was made in BrainHeart broth
deteriorations (Sharma et al., 1984). These micro organisms are (BHB) to a density of McFarland standards (0.5) and conrmed
present on the surface of the plants and originate from the plant by spectrophotometrical reading at 600 nm (Baron and Finegold,
environment, namely soil, water and air. Many commercial food 1990).
processes are restricted because of adverse effects on the product
(Chmielewski and Migdal, 2005). Nowadays, processing foods 2.4. In vitro determinations of antibacterial activity
including spice and herbs with gamma-radiation is accepted as a
safe and effective method to reduce spoilage and eradicate 2.4.1. Oil dilution solvent
pathogenic microorganisms (Sokhey and Hanna, 1991). Most Bacterial strains were grown on MHA plates using sterile cotton
researches focused on changes in biochemical properties of swabs. 10 L of dimethylsulphoxide (DMSO) loaded on sterile
medicinal plant during irradiation (Khattak et al., 2008; Polovka blank disks were placed on the agar plates and were incubated
and Suhaj, 2010; Machhour et al., 2011). Our previous experiments at 37 1C for 24 h. Bacterial growth inhibition was not observed and
indicated no signicant changes in the antioxidant activity of there was no antibacterial activity on the plates. DMSO was
Z. multiora and Carum carvi oils following treatment with 10 therefore chosen as a safe diluting agent for the oil (Allahghadri
and 25 kGy gamma irradiation (Fatemi et al., 2012, 2011). Despite et al., 2010).
the routine use of gamma-irradiation, our knowledge about the
inuence of radiation on the antimicrobial activity of the medic-
inal plants is not well documented. Therefore, the aim of this study 2.4.2. Agar well diffusion method
was rst to investigate the antibacterial activities of Z. multiora Wells (6 mm in diameter) were made using sterile cork borer in
EOs in vitro systems in compare to in vivo septic rats induced by MHA sterile plates, which had previously been seeded with tested
CLP model. Following this, in this research for the rst time, the bacterial strains. Wells were lled with 25 L of each irradiated
antibacterial activities were considered in essential oils extracted and non-irradiated oils. Plates were incubated at 37 1C. Inhibition
Fatemi Faezeh et al. / Radiation Physics and Chemistry 106 (2015) 145150 147

zones were measured after 24 h of incubation time. All the tests immediately after CLP. 24 h after CLP operation, blood samples
were performed in triplicate. were collected for CFU count.

2.4.3. Disk diffusion method 2.5.3. Colony forming units (CFUs) determination
The agar disk diffusion method (NCCLS M2-A7, 2000) was used Blood was obtained under ether anesthesia from the heart
to evaluate the antibacterial effects of the Z. multiora oils. 0.1 mL puncture and immediately serially diluted 10-fold in sterilized
of bacterial suspension was streaked on the surface of the MHA salts solution. The diluted blood was transferred rapidly to BHI
plate, using a sterile cotton swab. Also, 25 mL of the pure oil sample plates and placed into incubator for 48 h at 37 1C (Hyde et al.,
from each dose and antibiotic (0.3% ciprooxacin, 0.5% chloram- 1990). The number of colonies was counted using a colony counter
phenicol and 0.3% gentamicin) were added on the 6 mm diameter (Sana, Iran).
lter paper disks and then placed on the inoculated agar plates.
The plates were incubated at 37 1C for 2448 h to obtain microbial 2.6. Statistical analysis
growth. The diameters of the inhibition zones were measured in
millimeter. All the tests were performed in triplicate. The experimental results are presented as mean7standard
deviation (SD). Statistical signicance was analyzed by one-way
analysis of variance. P values less than 0.05 was considered
2.4.4. Determination of the minimal inhibitory (MIC) and minimal
signicantly different from the given group within each parameters.
bacterial concentrations (MBC)
The MIC and MBC were assessed according to Baron and
Finegold (1990)s procedure and NCCLS M2-A7 (2000). All tests 3. Results
were performed in BHB. MIC, dened as the lowest concentration
of the oil resulted in a complete inhibition of visible growth in the 3.1. Agar well diffusion method
broth, was determined as follows: standardized suspension of the
test organisms was inoculated in to a series of sterile tubes of BHB The anti-bacterial activity of Z. multiora EOs against four bacterial
containing two-fold dilutions of Z. multiora EOs. A tube without species is summarized in Table 1. Among the bacteria tested, B. cereus
EOs was chosen as control. All tubes were kept at 37 1C for 24 h. was the most sensitive against Z. multiora oil, producing the
Thereafter, the tubes were checked for growth of bacteria con- maximum diameter of inhibition zone (66.570.5 mm) (Po0.05).
sidering turbidity from control tube to higher concentration P. aeroginosa and S. aureus (P40.05) was found more susceptible than
(Mohsenzadeh, 2007; Rasooli, 2007). To determine the MBC of E. coli (Po0.05). Also, E. coli showed the least sensitive compare to B.
oils, all tubes without bacterial growth were cultured on Brain cereus, P. aeroginosa and S. aureus (Po0.05). The antibacterial proper-
Heart Infusion (BHI) agar and incubated at 37 1C for 24 h. The ties of Z. multiora E.O were not affected by irradiation at 10 and
plates were checked for growth of bacterial colonies. MBC was 25 kGy (P40.05).
evaluated as the lowest oil concentration at which no growth
observed on the plates.
3.2. Disk diffusion method

2.5. In vivo evaluation of antibacterial activity The antibacterial effect of the EOs extracted from Z. multiora
against the selected strains by the disk diffusion technique is
2.5.1. Cecal ligation and puncture (CLP) model presented on Table 2. Standard antibiotics were used as positive
Male albino rats of Wistar strain (260280 g) were purchased controls which examined under similar conditions against all
from the Animal laboratory house of Qom Azad University of Iran. bacterial species. The results revealed that B. cereus with maximum
Polymicrobial sepsis in rats was induced by CLP according to the inhibition zone was more susceptible when compared with other
method of Hubbard et al. (2005). Briey, the rats were anesthe- microorganisms (Po0.05). S. aureus and P. aeruginosa (P40.05)
tized by injection (i.p) of ketamine (90 mg/kg b.w) and xylazine displayed more sensitivity to EOs than E. coli (Po0.05). Moreover,
(10 mg/kg b.w) mixture. A small mid abdominal incision (23 cm) E. coli as gram-negative was the most resistant among tested strains
was made and the cecum was exposed. A distended portion of the (Po0.05). The antibacterial action of Z. multiora EOs was found to
cecum just distal to the ileocecal valve was isolated, lled with
fecal content, and tied with a 3-O silk suture in a manner not to
disrupt bowel continuity. The ligated portion of the cecum was Table 1
punctured twice with a 20-ga needle. The cecum was then Diameter of microbial inhibition zone (mm) of Zataria multiora essential oils
replaced in its original position within the abdomen and the determined by Agar well method.
abdomen was then closed with a 3-O suture in two layers. Then,
Microorganisms
the animals were allowed to recover. In the sham-operated rat, the
cecum was exposed, manipulated and returned to the peritoneal Dose (kGy) Escherichia Pseudomonas Bacillus Staphylococcus
cavity without being punctured. After surgery, normal saline coli (ATCC aeroginosa (ATCC cereus aureus (ATCC
(3 ml/100 g b.w) was given subcutaneously to all rats to prevent 25922) 27853) (ATCC 25923)
9634)
dehydration.
0 (control) 18.5 7 0.5c 38.7 7 0.6b 66.57 0.5a 33.27 0.3b
10 197 1c 39.8 7 0.8b 667 1a 32.7 7 0.8b
2.5.2. Animal treatments and blood sampling 25 19.3 7 0.6c 39.3 7 0.8b 66.37 0.6a 32.5 7 0.9b
The animals were divided in to 8 groups (n 5/ group). In
Sham-operated group (SOP), rats undergone laparotomy and nz, No zone.
a
received DMSO as vehicle. In CLP group, animals received vehicle Po 0.05 is considered as the most sensitive as compared to S. aureus and
alone after operation. In treated groups, EOs (100 or 200 mg/kg b. gram-negatives.
b
P o 0.05 is considered as more susceptible than B. cereus and E. coli
w) extracted from irradiated and non-irradiated plants were (P 40.05).
injected intraperitoneally (i.p) immediately after CLP. The EOs c
P o 0.05 is considered as the least sensitive as compared to P. aeroginosa and
prepared from Z. multiora were diluted in DMSO and injected gram-positive.
148 Fatemi Faezeh et al. / Radiation Physics and Chemistry 106 (2015) 145150

be similar to the antibiotics. The antibacterial activity of oils against 3.4. Colony forming units (CFU) determination
S. aureus and B. cereus strains was signicantly comparable to the all
antibiotics (Po0.05). In case of P. aeruginosa, Z. multiora EOs As indicated in Table 4, sepsis induction increased signicantly
exhibited comparable efcacy compared to Chloramphenicol the blood bacterial number in the CLP group as compared to that
(Po0.05). In addition, There are no signicant differences between in control one (P o0.05). In contrast, administration of the rats
irradiated and non-irradiated samples in the antibacterial potency with EOs extracted from fresh Z. mutiora caused to diminish the
(P40.05). bacterial load signicantly (P o0.05). With respect to the non-
irradiated, EOs of the irradiated plant at 10 and 25 kGy did not
show signicant differences in the bacterial load (P o0.05).

3.3. Minimal inhibitory concentration (MIC) and minimal


4. Discussion
bactericidal concentration (MBC)
In this study, the antibacterial activities of the EOs derived from
The MIC and MBC levels of Z. multiora oils against different
irradiated and non-irradiated of Z. mulltiora aerial parts were
kinds of microorganisms are summarized in Table 3. The MIC
performed in vitro systems in compare to in vivo sepsis system
values were found to be in range of 1.04 70.45 to
induced by CLP operation.
4.16 71.08 mg mL  1, while the MBC values were from
The results of in vitro study showed that the EOs exhibited
1.04 70.45 to 6.52 70.00. Among the bacteria tested, P. aeruginosa
strong activity against Gram-positive and Gram-negative bacteria
was the most resiatant organism with the highest rate of MIC and
(Tables 13). Our MIC and MBC ndings are agreement with other
MBC (Po0.05). Similarly, B. cereus, S. aureus and E. coli were
report that among bacterial tested, B. subtilis and S. aureus were
signicantly more sensitive against Z. multiora (P4 0.05). As
the most sensitive organisms followed by E. coli against EOs
shown in Table 3, the equivalent levels of MIC and the MBC were
obtained from Iranian Z. multiora Boiss (Eftekhar et al., 2011).
belonged to B. cereus and S. aureus. The results indicated that there
Also, other investigation of Z. multiora Boiss extract expressed the
were non-signicant differences between irradiated and control
signicant antibacterial activities against both Gram-positive and
samples, so the antibacterial activity of the Z. multiora EOs has
Gram-negative pathogens especially on B. subtilis (Rahman et al.,
remained even after irradiation at 10 and 25 kGy doses (P4 0.05).
2010). Moreover, there was a difference in the size of the zones of
inhibition with previous results (Bokaeian et al., 2013; Mahmoodi
et al., 2012), probably connected to differences in water solubility
Table 2
Diameter of microbial inhibition zone (mm) of Zataria multiora essential oils
and diffusion rates of the compounds, as well as by physiology of
determined by Disk diffusion method. the tested bacteria (Miti-ula et al., 2005). In addition, the
bacteria demonstrating the largest inhibition zones by diffusion
Microorganisms
Table 4
Dose (kGy) Escherichia Pseudomonas Bacillus Staphylococcus
Blood bacterial number (CFU/ml) in sepsis rats treated with Z. multiora ataria
coli (ATCC aeroginosa cereus aureus (ATCC
essential oils before and after - irradiation.
25922) (ATCC 27853) (ATCC 25923)
9634)
Groups Bacterial counts

0 (control) 19.2 7 1.0c 32.8 7 0.8b,e 66.6 70.6a,d 39.3 7 0.8b,d


Laparotomy (DMSO) 0
10 19.8 7 0.3c 32.5 7 0.6b,e 67 71a,d 397 1b,d
CLP (DMSO) 221707 850n
25 19.8 7 0.8c 33.27 0.3b,e 67.7 70.6a,d 39.5 7 0.5b,d
CLP non-irradiated EOs (100 mg/kg b.w) 50007 1100nn
Antibiotics CLP Irridiated Eos (10 kGy) (100 mg/kg b.w) 5040 7 1115nn
Ciprooxacin 23.8 7 0.3 32.7 7 0.6 35.770.6 26.7 7 0.6 CLP Irridiated EOs (25 kGy) (100 mg/kg b.w) 18007 40nn
Chloramphenicol 22 7 0.0 nz 34.8 70.3 20.8 7 0.3 CLP non-irradiated EOs (200 mg/kg b.w) 6380 7 1060nn
Gentamicin 21.7 7 0.6 24.77 0.6 33.370.3 227 0.0 CLP Irridiated EOs (10 kGy) (200 mg/kg b.w) 53007 896nn
CLP Irridiated EOs (25 kGy) (200 mg/kg b.w) 3220 7 720nn
nz, No zone.
a
P o0.05 is considered as the most sensitive as compared to S. aureus and In Sham-operated group(SOP), rats undergone laparotomy and received DMSO as
gram-negatives. vehicle; In CLP group, animals received vehicle alone; CLP Eos groups, Eos
b
Po 0.05 is considered as more susceptible than B. cereus and E. coli. (100 mg/kg or 200 mg/kg) were injected (i.p) immediately after CLP. Values of
c
Po 0.05 is considered as the least sensitive as compared to P. aeroginosa and mean7 S.D obtained from ve rats.
n
gram-positive. P o0.05 is considered signicantly different from laparatomy group within
d
Po 0.05 is considered signicantly different from tests antibiotics for each each group.
nn
bacterium, within each column. P o 0.05 is considered signicantly different from CLP group within
e
P o0.05 is considered signicantly different from Chloramphenicol. each group.

Table 3
MIC and MBC determinations of the Zataria multiora essential oils.

Doses (kGy) 0 10 25

MIC MBC MIC MBC MIC MBC

EO con. EO con. EO con. EO con. EO con. EO con.


Microorganisms Bacillus cereus 1.3 7 0.4 1.3 7 0.4 1.0 7 0.4 1.0 70.4 2.1 70.9 2.17 0.9
Staphylococcus aureus 1.2 7 0.07 1.2 7 0.7 2.17 0.9 2.1 70.9 2.1 70.9 5.2 7 1.8
Escherichia coli 1.3 7 0.4 1.3 7 0.4 2.17 0.9 2.1 70.9 2.1 70.9 2.17 0.9
Pseudomonas aeroginosa 2.6 7 0.9a 4.2 7 1.8a 4.2 7 1.8a 6.5 70.0a 3.1 70.0a 6.5 7 0.0a

EO con., essential oil concentration (mg/mL); MIC, minimal inhibitory concentration; MBC, minimal bactericidal concentration.
a
P o0.05 is considered as the least sensitive as compared to S. aureus, B. cereus and E. coli (Po 0.05).
Fatemi Faezeh et al. / Radiation Physics and Chemistry 106 (2015) 145150 149

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such as antimicrobial and antioxidant activities of tea leaves due to
Gandomi, H., Misaghi, A., Basti, A.A., Bokaei, S., Khosravi, A., Abbasifar, A., Javan, A.J.,
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