Industrial Crops and Products 84 (2016) 224–229

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Industrial Crops and Products

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Volatile composition, antimicrobial, cytotoxic and antioxidant

evaluation of the essential oil from Nepeta sintenisii Bornm.
Abolfazl Shakeri a , Fatemeh Khakdan b , Vahid Soheili c , Amirhossein Sahebkar d,e ,
Rezvan Shaddel f , Javad Asili g,∗
a
Student Research Committee, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran
b
Department of Biotechnology, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran
c
Department of Drug control, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran
d
Biotechnology Research Center, Mashhad University of Medical Sciences, Mashhad, Iran
e
Metabolic Research Centre, Royal Perth Hospital, School of Medicine and Pharmacology, University of Western Australia, Perth, Australia
f
Department of Food Science and Technology, Faculty of agriculture, University of Tabriz, Tabriz, Iran
g
Department of Pharmacognosy, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran

a r t i c l e i n f o a b s t r a c t

Article history: The aim of this study was to investigate volatile composition of the essential oil (EO) of Nepeta sintenisii
Received 8 July 2015 Bornm. (N. sintenisiig), and its antioxidant, antimicrobial and cytotoxic activities. The EO was obtained by
Received in revised form 19 October 2015 means of hydrodistillation and its components were analyzed using gas chromatography and mass spec-
Accepted 13 December 2015
trometry (GC/MS). The main constituents of the EO were found to be 4a␣,7␣,7a␤-Nepetalactone (51.74%),
Available online 13 February 2016
␤-Farnesene (12.26%), 4a␣,7␣,7a␣-Nepetalactone (8.01%), Germacrene-D (5.01%), and 4a␣,7␤,7a␣-
Nepetalactone (3.71%); these together constituted 95.81% of the extracted oil. The antimicrobial activity
Keywords:
of the EO against eleven bacterial strains and one fungus was studied. The EO showed potent and
Nepeta sintenisii
Lamiaceae
broad-spectrum antibacterial effects against both Gram-positive and Gram-negative bacteria, and also
Volatile oil possessed antifungal activity against Candida albicans. In vitro cytotoxicity evaluation against four cell
Nepetalactone lines of human ovarian carcinoma (A2780), cervical cancer (Hela), human colon adenocarcinoma (LS180),
Biological activity and human breast adenocarcinoma (MCF-7), and Human Umbilical Vein Endothelial Cells (HUVEC (as
a normal cell line), showed a potent anti-proliferative activity of the oil with the lowest IC50 value
(20.37 ␮g/mL) belonging to the Hela cells. The IC50 value for normal human cell line (HUVEC) was
219 ␮g/mL. Also, antioxidant activity of the EO was evaluated using 1,1-diphenyl-2-picrilhydrazyl (DPPH)
and ferric reducing antioxidant power (FRAP) methods. The oil exhibited weak antioxidant activity in
DPPH test (IC50: 7.16 mg/mL) and FRAP (IC50: 0.82 mM Fe2+ /mg EO) assay. The present results suggested
promising cytotoxic, antibacterial, and antifungal properties for the N. sintenisii EO.
© 2016 Elsevier B.V. All rights reserved.

1. Introduction

EOs and secondary metabolites of plants have many applica-

Abbreviations: N. sintenisii, Nepeta sintenisii Bornm; EO, essential oil; GC/MS,
gas chromatography and mass spectrometry; A2780, human ovarian carcinoma;
Hela, cervical cancer cells; LS180, human colon adenocarcinoma cell line; MCF-7,
human breast adenocarcinoma; HUVEC, human umbilical vein endothelial cells;
DPPH, 1,1-diphenyl-2-picryl-hydrazil; FBS, fetal bovine serum; SCDA, soybean
casein digest agar; MHB, Mueller-Hinton broth; MTT, 3-(4,5-dimethylthiazole-2-
yl)-2,5-biphenyl tetrazolium bromide; TTC, 2,3,5-triphenyltetrazolium chloride;
SDB, dextrose broth; BHT, butylhydroxytoluene; SDS, sodium dodecyl sulfate; RI,
retention indices; PTCC, Persian type culture collection; ATCC, American type culture
collection; MIC, minimum inhibitory concentration; MBC, minimum bactericidal
concentration; MFC, minimum fungicidal concentration; DOX, doxorubicin.
∗ Corresponding author. Fax: +98 511 8823251.
E-mail addresses: asilij@mums.ac.ir, shakeria912@mums.ac.ir (J. Asili).
tions in medicine as well as food and cosmetics industry (Sun et al.,
2015). EOs have several health benefits including antioxidant and
antimicrobial properties (Yu et al., 2004). These properties make
EOs possible substitutes for synthetic antioxidant and antimicrobial
agents (Dashipour et al., 2015). However, since EOs are concen-
trated mixtures and may exhibit higher toxicity compared with the
original plant or crude extracts, investigation of cytotoxic effects of
EOs is necessary prior to any therapeutic application. Nepeta is a
large genus belonging to the Lamiaceae family. This genus com-
prises about 280 species distributed mainly in Europe, Asia, and
some parts of Africa (Rechinger, 1982). Iran is one of the habitats
of this genus with 75 species and approximately 53% endemics

http://dx.doi.org/10.1016/j.indcrop.2015.12.030
0926-6690/© 2016 Elsevier B.V. All rights reserved.
 A. Shakeri et al. / Industrial Crops and Products 84 (2016) 224–229
(Sefidkon et al., 2006). Several species of this genus are widely
used in the Iranian folk medicine for their antispasmodic, expec-
torant, diuretic, antiseptic, antitussive, antiasthmatic, carminative,
and febrifuge activities (Formisano et al., 2011; Hussain et al., 2010).
Phytochemical analyses of Nepeta spp. have revealed the presence
of several bioactive phytochemicals such as phenolics, flavonoids,
and terpenoids (Formisano et al., 2011). Part of the health benefits
and biological activities of this genus maybe attributed to its EO.
Due to the multiple biological activities of Nepeta species and their
use in folk medicine, we aimed to study the EO composition and
biological activities of one of the species of this genus i.e., Nepeta
sintenisii Bornm. N. sintenisii is an herbaceous wild plant endemic to
Iran that grows to a height of 40–60 cm. The plant has white flower
characterized by hairs that cover all parts of the plant (Jamzad,
2012; Rechinger, 1982). To the best of our knowledge, there has no
previous study on the biological activity of the EO of N. sintenisii.
We report here the volatile composition of the EO obtained from
the aerial parts of N. sintenisii and its antimicrobial, cytotoxic and
antioxidant activities.
2. Materials and methods
2.1. Plant material
The whole aerial parts of N. sintenisii were collected during
the flowering stage from Neyshabur, Khorasan-Razavi province,
Iran, in July 2014. The plant was identified by Mrs. Souzani and
a voucher specimen (no. 11258) was deposited at the Herbarium of
the Department of Pharmacognosy, School of Pharmacy, Mashhad
University of Medical Sciences, Mashhad, Iran.
2.2. Isolation of EO
EO was isolated by hydro-distillation of air-dried materials using
a Clevenger-type apparatus. The colorless oil was obtained with a
yield of 0.5% v/w. The oil was dried over anhydrous sodium sulphate
and stored at 4◦ C in the dark until further testing.
2.3. GC–MS analysis
GC–MS analyses were performed using an Agilent 5975 appa-
ratus with a HP-5 MS column (30 m × 0.25 mm i.d., 0.25 ␮m film
thicknesses) interfaced with a quadruple mass detector and a
computer equipped with Wiley 7n.L library. Other analytical set-
tings were: oven temperature: 50 ◦ C (5 min), 50–250 ◦ C (3 ◦ C/min),
250 ◦ C (10 min); injector temperature 250 ◦ C; injection volume:
0.1 ␮L; split ratio: 1:50; carrier gas: helium at 1.1 mL/min; ioniza-
tion potential: 70 eV; ionization current: 150 ␮A; and mass range:
35–465. The linear retention indices (RI) were calculated for all
components using retention times (RT) of a homologous series of
n-alkanes (C6-C21) that were injected in conditions equal to the
sample one. Identification of the components of the EO was based
on retention indices relative to n-alkanes and computer matching
with the Wiley 7n.L library as well as comparisons of the fragmen-
tation pattern of the mass spectra with the data published in the
literature (Adams, 2007). Quantification of the relative amount of
the individual components was performed according to the area
under the curve method without consideration of calibration factor.
2.4. Determination of antimicrobial activity
2.4.1. Bacterial strains and culture media
Ten microorganisms including Bacillus cereus (Persian type
culture collection [PTCC] 1247), Bacillus subtilis (American type cul-
ture collection [ATCC] 6633), Listeria monocytogenes (PTCC 1165),
Staphylococcus aureus (ATCC 43300), Staphylococcus epidermidis
225
(DSMZ 3270), Lactobacillus plantarum (PTCC 1058), Lactobacillus
sakei (PTCC 1712) and Micrococcus luteus (ATCC 9341) were selected
as Gram-positive strains, while Salmonella typhi (PTCC 1609) and
Escherichia coli (PTCC 1330) were selected as Gram-negative bac-
teria. The over-night growth culture of each micro-organism on
◦
soybean-casein digest agar (SCDA) medium at 37 C was used
for anti-microbial screening tests, and the inoculation was set
to 106 CFU/mL sterile normal saline (0.9%). EO was added to
the co-solvent, dimethyl sulfoxide (DMSO), and then dissolved in
Mueller-Hinton broth (MHB) medium. To prepare the first concen-
tration (10% v/v, 90 ␮g/mL), 864 ␮L of EO was dispersed in 432 ␮L
DMSO, and MHB medium was added gradually to the final vol-
ume of 8.420 mL. Other concentrations were prepared serially using
two-fold dilution method. Then, 180 ␮L of each concentration as
well as 20 ␮L of bacterial suspensions (106 CFU/mL) was inocu-
lated in a 96-well culture plate. The test was performed for each
bacterium in duplicate. To test the sterility of media, MHB was
inoculated in a separate well as a negative control. The cultured
bacteria in MHB were also used as positive controls. The plates were
◦
incubated at 37 C for an overnight period and the bacterial growth
in the wells were evaluated by adding of a colorimetric indicator,
2,3,5-triphenyltetrazolium chloride (TTC) (20 ␮L of the 5 mg/mL
stock solution). The plates were then incubated at 37 ◦ C for 1 h.
The minimum inhibitory concentration (MIC) for each microorgan-
ism was the first concentration with no color alteration. Minimum
bactericidal concentration (MBC) was determined by culturing the
wells containing EO with concentrations equal or greater than MIC,
on the surface of plates containing Mueller-Hinton agar medium
◦
(overnight, 37 C) (Andrews, 2001; Eloff, 1999).
2.4.2. Anti-fungal activity
To evaluate the anti-fungal activity of the EO, Candida albicans
(PTCC 5027) was used as the test strain. First, the growth culture
of the microorganism on SCDA (48 h, 25 ◦ C) was employed and set
to 106 CFU/mL by sterile normal saline (0.9%). Thereafter, the first
concentration of EO (10% v/v, 90 ␮g/mL) was prepared and seri-
ally diluted with the above-mentioned method. All other levels of
the experiment were performed as described for the anti-bacterial
test. Positive and negative controls were used and the plate was
◦
incubated at 25 C for 48 h. The MIC was evaluated following the
addition of TTC (20 ␮L of a 5 mg/mL stock solution) and a 24 h
incubation period. The minimum fungicidal concentration (MFC)
was determined with the same method as MBC (Andrews, 2001;
Oliveira et al., 2011).
2.5. Cytotoxic activity
2.5.1. Cell culture
Human ovarian carcinoma (A 2780), cervical cancer cells (Hela),
human colon adenocarcinoma cell line (LS180), human breast
adenocarcinoma (MCF-7) and Human Umbilical Vein Endothelial
Cells (HUVEC; used as a normal cell line) were provided by the
National Cell Bank of Iran, Pasteur Institute, Tehran, Iran. Cells
were maintained in RPMI 1640 supplemented with 10% fetal bovine
serum 1% (w/v), glutamine, penicillin (100 U/mL) and streptomycin
(100 ␮g/mL). Cultures were incubated at 37 ◦ C under 5% CO2 in a
humidified atmosphere.
2.5.2. Cell viability assay
The EO was screened for its cytotoxic activity using the MTT
[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bro-
mide] assay against the aforementioned cancer cell lines Cell
viability following exposure to EOs was estimated using the MTT
reduction assay (Firuzi et al., 2010; Mosmann, 1983). A2780,
Hela, LS180, MCF7 and HUVEC cells were seeded at a density of
105 , 25 × 103 , 105 , 104 , and 3 × 103 cells/well (100 ␮L per well)
226 A. Shakeri et al. / Industrial Crops and Products 84 (2016) 224–229
into 96-well plates in a humidified atmosphere at 37 ◦ C in 5%
CO2 , respectively. Control wells contained no EO and blank wells
contained only the growth medium for background correction.
Doxorubicin (DOX) was used as a positive control. After an
overnight incubation at 37 ◦ C to allow cell attachment, EO was
solubilized in DMSO, and then diluted in culture medium for use.
The maximum concentration of DMSO in the wells was kept below
0.5%. The EO dilutions (5–400 ␮g mL−1 ) were added to triplicate
wells and cells were incubated for a further 24 h. At the end of
the incubation, the medium was removed and MTT was added
to each well at a final concentration of 0.5 mg/mL, and plates
were incubated for another 4 h at 37 ◦ C. The formazan crystals,
which are formed by the action of dehydrogenase enzyme in the
mitochondria of viable cells, were solubilized in 200 ␮L DMSO.
The optical density was measured at 570 nm with a background
correction at 655 nm using a Bio-Rad microplate reader (Model
680). The percentage of inhibition of viability compared with
control wells was calculated and IC50 values were determined
using GraphPad Prism 5.0 program (Graph Pad Software, San
Diego, CA, USA). The values of the blank wells were subtracted
from each well of treated and control cells, and the percentage
viability was determined as formulated below:
[(At − Ab) × 100]
Viability (%) =
Ac − Ab
Where At, Ab and Ac are absorption of treated, blank and control
wells.
2.6. Determination of antioxidant capacity
2.6.1. DPPH radical scavenging assay
The in vitro antioxidant potential of EO in terms of 1,1-diphenyl-
2-picrylhydrazyl (DPPH) free radical scavenging was determined
according to the standard procedure (Braca et al., 2001). A volume
of 50 ␮L of different concentrations of EO (0.125–12.5 mg/mL) was
added to 50 ␮L of 0.1 mM DPPH solution (in methanol) in a 1:1
ratio. The reaction mixture was incubated at 37 ◦ C for 30 min in
darkness. The absorbance of the sample at 517 nm was measured
using a 96-well microplate reader and was then compared with that
of a control solution containing the reaction mixture amended with
50 ␮L of methanol instead of EO. Ascorbic acid (1.5–12.5 ␮g/mL)
was used as the standard reference compound, and the percentage
of DPPH free radical scavenging activity was calculated using the
following equation:
A − A 
0 t
Scavenging (%) = × 100
A0
where, A0 is the absorbance of the control solution and At is the
absorbance of the test solution.
2.6.2. FRAP assay
FRAP assay was conducted according to the method described
by Benzie and Strain (Benzie and Strain, 1996), with some modifi-
cations. TPTZ (2,4,6-tripyridyl-S-triazine) solution (10 mM) in HCl
(40 mM), FeCl3 (20 mM) and acetate buffer (0.3 M, pH 3.6) were the
solutions needed for this method. Acetate buffer, FeCl3 and TPTZ
were mixed before use. This mixture was heated to 37 ◦ C, and then
mixed with 20 ␮L of EO (1 mg/mL) and 180 ␮L of FRAP reagent in
a 96-well microplate, followed by incubationat 37 ◦ C for 10 min.
The absorbance of the mixture was measured at 593 nm. A cali-
bration curve of ferrous sulphate (0.125–2.5 mM) was constructed,
and results were expressed in mM Fe2+ /mg dry weight extract. The
relative activity of the EO was compared to that of l-ascorbic acid
(ranging from 1.25 to 100 ␮g/mL).
3. Results and discussion
3.1. Chemical composition of the essential oil
The use of EOs for medical purposes has been the subject of
increasing research in recent decades. This surge of interest in EO
research is due to the diverse biological activities, simple oil extrac-
tion techniques, and natural origin of EOs. This study evaluated the
volatile composition and biological activities of the EO from N. sin-
tenisii. The yield of the EO was 0.5% (v/w) on a dry weight basis.
The identified compounds, their percentages as well as the Kovats
Indices are listed in Table 1. In the case of N. sintenisii, fifty-one con-
stituents were identified in the EO and accounted for 95.81% of the
total oil composition. The oil composition is dominated by the pres-
ence of oxygenated monoterpenes comprising 64.69% from total,
followed by sesquiterpene hydrocarbons (27.81%). The principal
chemical constituents were found to be 4a␣,7␣,7a␤-nepetalactone
(51.74%), ␤-farnesene (12.26%), 4a␣,7␣,7a␣-nepetalactone (8.01%),
germacrene-D (5.01%), and 4a␣,7␤,7a␣-nepetalactone (3.71%).
Nepetalactones have been previously reported as common compo-
nents of the EOs of many Nepeta species, and represented 63.46%
of the total composition of N. sintenisii oil. Similarly, Sajjadi (2005)
reported 4a␤,7␣,7a␤-nepetalactone (23.4%) as a major compound
in N. sintenisii EO collected from the Charat region (Savadkooh,
Mazandaran Province, Iran). On the contrary, our results are dif-
ferent from those reported by Abad et al. (2011) on the volatile
composition of the same plant collected from the Darkesh Protected
Area, Bojnourd (North Khorassan Province, Iran), in which alpha-
terpinolene (47.86%) was the major compound. Such differences
might have been derived from inter-species biodiversity as well as
variations in climatic, seasonal and geographic conditions, the stage
of plant development, harvest period, and distillation technique
used.
3.2. Antimicrobial activity
The results of antimicrobial activity testing of the EO are summa-
rized in Table 2. N. sintenisii EO had a potent effect on C. albicans and
both Gram-positive and Gram-negative bacteria. Both MIC and MBC
values against tested strains were between 1.4–22.5 ␮g/mL. The
results also presented that both MIC and MFC values against C. albi-
cans are <0.35 ␮g/mL. The antimicrobial effects of the N. sintenisii
EO against susceptible microorganisms were higher than those
previously reported for other species of Nepeta such as N. binalu-
densis Jamzad (Mohammadpour et al., 2013), N. glomerata (Rigano
et al., 2011), N. cataria (Adiguzel et al., 2009), and N. deflersiana
(Mothana, 2012). The mechanism(s) of the antimicrobial action of
EOs are still not completely understood. However, the amphiphilic
nature of terpenoids due to the presence of hydrophobic skeleton
and hydrophilic functional groups enables easy transport of these
phytochemicals across biological membranes of microbial and
mammalian cells, thereby allowing them to reach cytosol and inter-
act with key intracellular biomolecules (Derouiche et al., 2013). The
antimicrobial activity of N. sintenisii EO may be attributed to the
diversity of bioactive compounds, such as nepetalactones (com-
prising 63.46% of the oil), in the EO. It has been previously reported
that antibacterial and fungicidal activities of N. cataria, N. sibirica
and N. rtanjensis oils are exerted, at least in part, by nepetalactones
(Nestorović et al., 2010; Safaei-Ghomi et al., 2009). This effect of
nepetalactones might be attributed to their higher water solubility
and diffusion coefficient through the medium and also their high
hydrogen bonding potential (Kumar et al., 2014). However, some
studies have shown that the whole EO has a greater antibacterial
activity compared with the isolated major components (Mahboubi
and Kazempour, 2011). The antimicrobial results observed in this
study are particularly important for B. cereus, L. monocytogenes, S.
 A. Shakeri et al. / Industrial Crops and Products 84 (2016) 224–229 227

Table 1 3.3. Cytotoxic activity

Volatile composition of the essential oil obtained from the aerial parts of N. sintenisii.

No. Compounds % KIa RTb In the present study, cytotoxicity of the N. sintenisii EO was mea-
1 2E-Hepten-1-ol 0.01 965 9.4691 sured (using MTT assay) on four cancer cell lines, namely human
2 3Z-Octen-2-ol 0.02 978 10.075 ovarian carcinoma (A 2780), cervical cancer cells (Hela), human
3 Myrcene 0.02 990 10.594 colon adenocarcinoma (LS180), and human breast adenocarcinoma
4 ␣-Terpinene 0.01 1017 11.683 (MCF-7), and also on Human Umbilical Vein Endothelial Cells
5 2-Acetyl-5-methyl-Furan 0.07 1037 12.354
(HUVEC) as a normal cell line. The cells were subjected to increas-
6 Z-␤-Ocimene 0.05 1037 12.884
7 E-␤-Ocimene 0.21 1050 13.374 ing doses of the EO ranging from 5 to 400 ␮g/mL. The growth of cell
8 ␥-Terpinene 0.05 1059 13.782 lines was inhibited by N. sintenisii EO in a concentration-dependent
9 n-Octanol 0.01 1068 14.551 manner after exposure to the EO for 24 h (Table 3). Cytotoxic activ-
10 2,2-Dimethyl-3,4-Octadienal 0.01 1103 14.674
ity of the EO against Hela cells (IC50 : 20.37 ␮g/mL) was significantly
11 Linalol 0.03 1096 15.903
12 Camphor 0.01 1146 17.833 stronger compared with other cell lines with. Extracts with IC50
13 Borneol 0.04 1169 18.905 values lower than 30 ␮g/mL against experimental tumor cell lines
14 ␣-Terpineol 0.02 1188 20.164 could be considered as promising candidates for the development
15 Methyl salicylate 0.04 1191 20.292 of anticancer drugs (de Oliveira et al., 2015). Another important
16 Methyl ether thymol 0.01 1235 22.327
finding was the selective toxicity of N. sintenisii EO for cancer cell
17. Pulegone 0.05 1237 22.408
18 Thymol 0.20 1290 25.142 lines and the weak cytotoxicity (IC50 : 219 ␮g/mL) on the HUVEC
19 Carvacrol 0.86 1299 25.270 cell line. However, the cytotoxic effects of the tested EO on A2780
20 4a␣,7␣,7a␣-Nepetalactone 8.01 1360 27.899 (0.038 ␮g/mL), Hela (0.035 ␮g/mL), LS180 (0.030 ␮g/mL), and MCF-
21 ␣-Copaene 0.20 1376 28.464
7 (0.029 ␮g/mL) cells were weaker than those of DOX as a positive
22 Daucene 0.22 1381 28.826
23 4a␣,7␣,7a␤-Nepetalactone 51.74 1387 29.438
control. Cytotoxicity of EOs may be exerted through several mech-
24 4a␣,7␤,7a␣-Nepetalactone 3.71 1392 29.525 anisms including depolarization and disruption of cell membrane
25 ␣-Funebrene 0.03 1402 29.933 integrity, increasing membrane permeability, reducing the activity
26 Trancs-caryophyllene 2.38 1408 30.318 of membrane-bound enzymes (Carnesecchi et al., 2001); alter-
27 ␤-Ylangene 0.06 1420 30.708
ation of the mevalonate pathway of metabolism (Elson, 1995);
28 ␣-Trans-bergamotene 0.05 1434 31.064
29 ␤-Funebrene 0.11 1414 31.425 or induction of apoptosis (Kumar et al., 2008). EOs isolated from
30 ␤-Humulene 0.50 1438 31.687 other Nepeta species such as N. ucrainica L. spp. kopetdaghensis
31 ␤-Farnesene 12.26 1456 32.125 (Shakeri et al., 2014), N. cataria (Reichling et al., 2009) and N. men-
32 cis-Cadina-1(6), 4-diene 0.02 1463 32.568
thoides Boiss & Buhse (Kahkeshani et al., 2014) have been reported
33 Germacrene-D 5.01 1485 32.876
34 ar-Curcumene 0.13 1480 33.028
to exhibit cytotoxic effects against different types of human can-
35 Amorpha-4,7(11)-diene 0.02 1481 33.261 cer celllines. The cytotoxic activity of N. sintenisii EO has not been
36 Bicyclogermacrene 0.64 1500 33.471 studied sofar. For example, nepetalactone, as the main volatile com-
37 Epizonarene 0.03 1501 33.663 ponents of the N. sintenisii oil, have been reported to inhibit the
38 Germacrene A 0.15 1509 33.786
growth of HL60 myeloma and Kato III stomach carcinoma cell lines
39 Eugenol acetate 0.58 1522 33.978
40 ␤-Bisabolene 2.70 1505 34.071 (Tsuruoka et al., 2012). However, minor components could also
41 ␦-Cadinene 0.42 1513 34.182 contribute to the cytotoxic activity of the oil. It is also possible
42 ␤-Sesquiphellandrene 2.81 1522 34.648 that the minor components maybe involved in some type of syner-
43 ␥-Z-Bisabolene 0.06 1515 34.940
gism with the other active compounds, which deserves attention
44 ␣-Calacorene 0.01 1545 35.301
45 Spathulenol 0.27 1578 36.595
in future studies.
46 Caryophyllene oxide 0.35 1583 36.782
47 Salvial-4(14)-en-1-one 0.06 1594 37.195 3.4. Antioxidant activity
48 Guaiol 0.22 1600 38.041
49 epi-␣-Cadinol 1.15 1640 39.020
Some reports have supported the relationship between cyto-
50 epi-␣-Muurolol 0.09 1641 39.498
51 8-Cedren-13-ol 0.10 1689 40.629 toxic and antioxidant activities of natural products (Hou et al.,
2007). Antioxidant activity of the EO from N. sintenisii was exam-
Major groups %
Monoterpene hydrocarbons 0.34
ined using DPPH and FRAP assay (Table 4). Antioxidant activity in
Oxygenated monoterpenes 64.69 the DPPH method is thought to be either due to the hydrogen-
Sesquiterpene hydrocarbons 27.81 donating capacity or radical scavenging activity of the compound.
Oxygenated sesquiterpenes 2.24 The scavenging activity of N. sintenisii EO was tested at concentra-
Others 0.73
tions ranging from 0.125 to 12.5 mg/mL. The EO exhibited weak
Total identified 95.81
activity to reduce the stable radical DPPH into yellow-colored
a
Kovats index on the HP-5 column. diphenylpicrylhydrazine, with an IC50 value of 7.16 mg/mL. The
b
Retention time.
radical scavenging activity of EO was weaker than that of the posi-
tive control ascorbic acid (IC50 : 0.006 mg/mL). Also, the FRAP value
aureus, S. epidemidis, S. typhi, E. coli and C. albicans because these for the EO (1 mg/mL) was 0.82 mM Fe2+ /mg, which is significantly
strains are classified in the second hazard group of biological agents lower than that of ascorbic acid as a standard (16.4 mM Fe2+ /mg).
according to the Advisory Committee on Dangerous Pathogens Strong antioxidant activity of EOs has been attributed to their
(ACDP). ACDP defines these pathogens as agents that can result in phenolic constituents such as thymol, carvacrol and eugenol
human disease and possibly a risk to staffs, but generally efficient (Miguel, 2010). Therefore, the moderate antioxidant activity may
prophylactic or treatment measures are available. Inhibition of the be attributed, at least in part, to the low occurrence of such com-
growth of the above-mentioned microorganisms is also important pounds (<1% of total oil composition) in the N. sintenisii oil. There
owing to their role in cereal, water, meat, milk, and egg contamina- has been scarce data for the antioxidant effects of nepeta lactones
tion (Atlas, 2006). Hence, N. sintenisii oil is a potential antibacterial or nepeta lactone-rich EOs. The present findings are in accor-
and antifungal agent that may find wider applications in medicine dance with previous results on the weak antioxidant activity of
or food industry. EOs obtained from other Nepeta species (Adiguzel et al., 2009; Alim
228 A. Shakeri et al. / Industrial Crops and Products 84 (2016) 224–229

Table 2
Antimicrobial activity of the N. sintenisii essential oil expressed as minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and minimum
fungicidal concentration (MFC).

Strain Strain Gram MIC value (␮g/mL) MBC value MFC value
(␮g/mL) (␮g/mL)

Bacillus cereus PTCC 1247 Positive 5.62 5.62 –

Bacillus subtilis. ATCC 6633 Positive 1.40 2.80 –
Listeria monocytogenes PTCC 1165 Positive 5.62 5.62 –
Staphylococcus aureus ATCC 43300 Positive 1.40 1.40 –
Staphylococcus epidermidis DSMZ 3270 Positive 11.25 11.25 –
Lactobacillus plantarum PTCC 1058 Positive 1.40 2.80 –
Lactobacillus sakei PTCC 1712 Positive 1.40 2.80 –
Micrococcus luteus ATCC 9341 Positive 1.40 5.62 –
Salmonella typhi PTCC 1609 Negative 1.40 1.40 –
E. coli PTCC 1330 Negative 22.50 22.50 –
Candida albicans PTCC 5027 – <0.35 – <0.35

Table 3 Alim, A., Goze, I., Cetin, A., Atas, A.D., Cetinus, S.A., Vural, N., 2009. Chemical
Cytotoxic activity of the N. sintenisii essential oil. composition and in vitro antimicrobial and antioxidant activities of the
essentialoil of Nepeta nuda L. subsp Albiflora (Boiss.) gams. Afr. J. Microbiol.
IC50 (␮g/mL) Res. 3, 463–467.
Andrews, J.M., 2001. Determination of minimum inhibitory concentrations. J.
MCF7 LS180 Hela A2780 HUVEC
Antimicrob. Chemother. 48, 5–16.
Cells 43.75 ± 1.07 42.64 ± 1.07 20.37 ± 3.13 51.98 ± 2.23 219 ± 1.28 Atlas, R.M., 2006. Microbiological Media for the Examination of Food. CRC Press,
DOX 0.029 ± 1.50 0.030 ± 1.94 0.035 ± 3.82 0.038 ± 7.75 0.021 ± 2.90 Boca Raton.
Benzie, I.F., Strain, J., 1996. The ferric reducing ability of plasma (FRAP) as a
measure of antioxidant power: the FRAP assay. Anal. Biochem. 239, 70–76.
Table 4 Braca, A., Tommasi, N.D., Bari, L.D., Pizza, C., Politi, M., Morelli, I., 2001. Antioxidant
principles from Bauhinia terapotensis. J. Nat. Prod. 64, 892–895.
Total antioxidant capacity of the N. sintenisii essential oil according to the DPPH and
Carnesecchi, S., Schneider, Y., Ceraline, J., Duranton, B., Gosse, F., Seiler, N., Raul, F.,
FRAP assays.
2001. Geraniol, a component of plant essential oils, inhibits growth and
Sample DPPH (IC50 , mg/mL) FRAP (mM Fe2+ /mg EO) polyamine biosynthesis in human colon cancer cells. J. Pharmacol. Exp. Ther.
298, 197–200.
EO 7.16 0.82 Dashipour, A., Razavilar, V., Hosseini, H., Shojaee-Aliabadi, S., German, J.B., Ghanati,
Ascorbic acid 0.006 16.40 K., Khakpour, M., Khaksar, R., 2015. Antioxidant and antimicrobial
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Chemical composition, antimicrobial and antioxidant activities of the essential
between antioxidant and cytotoxic activities of the EO. oils of Santolina africana flowers, endemic in Algeria. J. BioSci. Biotechnol. 2.
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product which deserves to be further studied for its potential ther- Hou, J., Sun, T., Hu, J., Chen, S., Cai, X., Zou, G., 2007. Chemical composition,
cytotoxic and antioxidant activity of the leaf essential oil of Photinia serrulata.
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Future research is encouraged to find the major volatile compo- Hussain, J., Rehman, N.U., Hussain, H., 2010. Chemical constituents from Nepeta
nents that are responsible for the observed biological effects of clarkei. Biochem. Syst. Ecol. 38, 823–826.
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the EO, and also determine the synergistic effects of this EO with Kahkeshani, N., Razzaghirad, Y., Ostad, S.N., Hadjiakhoondi, A., Shams Ardekani,
standard cytotoxic agents used in cancer chemotherapy. M.R., Hajimehdipoor, H., Attar, H., Samadi, M., Jovel, E., Khanavi, M., 2014.
Cytotoxic, acetylcholinesterase inhibitor and antioxidant activity of Nepeta
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Acknowledgment Kumar, A., Malik, F., Bhushan, S., Sethi, V.K., Shahi, A.K., Taneja, S.C., Qazi, G.N.,
Singh, J., 2008. An essential oil and its major constituent isointermedeol induce
The authors are indebted to the Student Research Committee apoptosis by increased expression of mitochondrial cytochrome c and apical
death receptors in human leukaemia HL-60 cells. Chem. Biol. Interact. 171,
at the School of Pharmacy of the Mashhad University of Medical 332–347.
Sciences (Mashhad, Iran) for financial support of this project. Kumar, V., Mathela, C., Tewari, G., Singh, D., 2014. Antifungal activity of Nepeta
elliptica Royle ex Benth. oil and its major constituent (7R)-trans,
trans-nepetalactone: a comparative study. Ind. Crops Prod. 55, 70–74.
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