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EDITOR-IN-CHIEF
DR. PAWAN K AGRAWAL
Natural Product Inc.
7963, Anderson Park Lane,
Westerville, Ohio 43081, USA
agrawal@naturalproduct.us
EDITORS
PROFESSOR ALEJANDRO F. BARRERO
Department of Organic Chemistry,
University of Granada,
Campus de Fuente Nueva, s/n, 18071, Granada, Spain
afbarre@ugr.es
PROFESSOR ALESSANDRA BRACA
Dipartimento di Chimica Bioorganicae Biofarmacia,
Universita di Pisa,
via Bonanno 33, 56126 Pisa, Italy
braca@farm.unipi.it
PROFESSOR DEAN GUO
State Key Laboratory of Natural and Biomimetic Drugs,
School of Pharmaceutical Sciences,
Peking University,
Beijing 100083, China
gda5958@163.com
PROFESSOR YOSHIHIRO MIMAKI
School of Pharmacy,
Tokyo University of Pharmacy and Life Sciences,
Horinouchi 1432-1, Hachioji, Tokyo 192-0392, Japan
mimakiy@ps.toyaku.ac.jp
PROFESSOR STEPHEN G. PYNE
Department of Chemistry
University of Wollongong
Wollongong, New South Wales, 2522, Australia
spyne@uow.edu.au
PROFESSOR MANFRED G. REINECKE
Department of Chemistry,
Texas Christian University,
Forts Worth, TX 76129, USA
m.reinecke@tcu.edu
PROFESSOR WILLIAM N. SETZER
Department of Chemistry
The University of Alabama in Huntsville
Huntsville, AL 35809, USA
wsetzer@chemistry.uah.edu
PROFESSOR YASUHIRO TEZUKA
Faculty of Pharmaceutical Sciences
Hokuriku University
Ho-3 Kanagawa-machi, Kanazawa 920-1181, Japan
y-tezuka@hokuriku-u.ac.jp
PROFESSOR DAVID E. THURSTON
Department of Pharmaceutical and Biological Chemistry,
The School of Pharmacy,
University of London, 29-39 Brunswick Square,
London WC1N 1AX, UK
david.thurston@pharmacy.ac.uk
HONORARY EDITOR
PROFESSOR GERALD BLUNDEN
The School of Pharmacy & Biomedical Sciences,
University of Portsmouth,
Portsmouth, PO1 2DT U.K.
axuf64@dsl.pipex.com
ADVISORY BOARD
Prof. Viqar Uddin Ahmad
Karachi, Pakistan
Prof. Giovanni Appendino
Novara, Italy
Prof. Yoshinori Asakawa
Tokushima, Japan
Prof. Roberto G. S. Berlinck
So Carlos, Brazil
Prof. Anna R. Bilia
Florence, Italy
Prof. Maurizio Bruno
Palermo, Italy
Prof. Csar A. N. Cataln
Tucumn, Argentina
Prof. Josep Coll
Barcelona, Spain
Prof. Geoffrey Cordell
Chicago, IL, USA
Prof. Fatih Demirci
Eskiehir, Turkey
Prof. Dominique Guillaume
Reims, France
Prof. Ana Cristina Figueiredo
Lisbon, Portugal
Prof. Cristina Gracia-Viguera
Murcia, Spain
Prof. Duvvuru Gunasekar
Tirupati, India
Prof. Hisahiro Hagiwara
Niigata, Japan
Prof. Kurt Hostettmann
Lausanne, Switzerland
Prof. Martin A. Iglesias Arteaga
Mexico, D. F, Mexico
Prof. Leopold Jirovetz
Vienna, Austria
Prof. Vladimir I Kalinin
Vladivostok, Russia
Prof. Niel A. Koorbanally
Durban, South Africa
NPC
2014
Vol. 9
No. 11
1633 - 1636
Zabol Medicinal Plants Research Center, Zabol University of Medical Sciences, Zabol, Iran
Department of Pharmacognosy, Faculty of Pharmacy, Zabol University of Medical Sciences, Zabol, Iran
c
Department of Bacteriology and Virology, Shiraz Medical School, Shiraz University of Medical Sciences, Shiraz, Iran
d
Department of Range and Watershed Management, Faculty of Natural Resources, University of Zabol, Iran
e
Department of Chemistry, University of Alabama in Huntsville, Huntsville, Alabama 35899, USA
f
Department of Pharmacognosy, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran
g
Medicinal Plants Research Center, Tehran University of Medical Sciences, Tehran, Iran
b
m.hoseinialfatemi@gmail.com
Received: August 29th, 2014; Accepted: September 16th, 2014
The present study investigated the chemical composition of the essential oil (EO) from aerial parts (flowering stage) of Pulicaria vulgaris Gaertn. by GCMS.
Also, the antimicrobial activity of the EO against Gram-positive bacteria (Bacillus cereus and Staphylococcus aureus), Gram-negative bacteria (Escherichia
coli and Pseudomonas aeruginosa) and fungi (Aspergillus niger and Candida albicans) was tested. In total, 23 compounds were recognized, accounting for
98.08 % of the EO. The main compounds in the EO were thymol (50.22%), p-menth-6-en-2-one (carvotanacetone, 20.2%), thymol isobutyrate (16.88%),
menthan-2-one (4.31%), 1-methyl-1,2-propanedione (4.13%), 2,5-dimethoxy-p-cymene (4.01%), myrtenol (1.22%), linalool (1.1%), and -myrcene (1.9%).
Results of antibacterial test of P. vulgaris essential oil showed that all assayed concentrations significantly inhibited the growth of B. cereus, S. aureus, E. coli,
and P. aeruginosa at P < 0.05. MIC for B. cereus, S. aureus, E. coli, P. aeruginosa was 17.5, 25.2, 19.4 and 33.2 g/mL respectively; antifungal screening of
the essential oil of P. vulgaris showed that the oil significantly inhibited the growth of A. niger and C. albicans (MIC = 15.5 and 9.9 g/mL, respectively).
Results of cytotoxicity assay showed that the essential oil exhibited a significant cytotoxic activity against both cell lines. In case of MCF-7 and Hep-G2 cell
lines, IC50 of the essential oil were 5.36 and 7.16 g/ml, respectively. The potent antimicrobial and cytotoxic activities of the EO may be attributed to its high
contents of thymol, carvotanacetone and thymol isobutyrate. Antimicrobial and antitumor chemotherapies are showing diminishing effectiveness because of
emergence of drug-resistance. Hence, using efficient natural chemotherapeutic agents such as Pulicaria vulgaris essential oil with fewer side effects is an
encouraging approach to fight cancer and infectious diseases in medicine, agriculture, food science and related fields.
Keywords: Antibacterial, Antifungal, Anticancer, Cytotoxicity assay, Carvotanacetone, Thymol.
Sharifi-Rad et al.
RI
908
941
989
1020
1098
1145
1150
1152
1183
1188
1199
1222
1235
1246
1293
1299
1306
1403
1406
1425
1470
1498
1546
Relative %
0.03
0.01
1.9
0.4
1.1
4.13
0.04
0.2
0.81
1.22
4.31
0.9
0.01
20.2
40.22
0.3
0.1
0.01
0.4
4.01
0.4
16.88
0.5
B. cereus
S. aureus
E. coli
P. aeruginosa
A. niger
C. albicans
30.2 0.0 i
33.3 0.1 h
38.7 0.3 g
42.90.5 f
51.20.0 e
57.6 0.3 d
62.3 0.2 c
76.5 0.2 b
81.0 0.0 a
1.50.0 k
29.5 0.2 g
17.5
25.5 0.0 h
25.8 0.0 h
26.0 0.1g
28.12 0.0 f
34.2 0.1 e
39.1 0.0 d
42.4 0.2 c
52.8 0.3 b
68.7 0.3 a
1.10.0 j
23.9 0.1 i
25.2
31.2 0.0 g
34.5 0.1 f
35.0 0.0 e
43.7 0.3 d
48.5 0.1 c
48.8 0.3 c
49.7 0.2 c
51.2 0.1b
72.1 0.0 a
1.70.1 h
35.9 0.4 e
19.4
12 0.0 i
12.5 0.0 i
14.4 0.0 h
15.9 0.1 g
16.30.0 f
18.5 0.5 d
25.6 0.1 c
32.4 0.0 b
39.5 0.0 a
2.00.0 j
17.5 0.0 e
37.2
8.5 0.3 j
10.5 0.2 i
14.4 0.1 g
18.5 0.2 f
28.3 0.0 e
36.8 0.5 d
39.5 0.1c
77.9 0.4 b
81 0.5 a
4.1 0.0 j
13.5 0.1 h
15.5
13 0.1 h
15.1 0.0 g
19.5 0.4 f
21 0.9 e
36.5 0.2 d
36.7 0.2 d
41.1 0.0 c
87.7 0.3 b
98.2 0.0 a
4.7 0.3 j
12.1 0.0 i
9.9
Data are expressed as means SD of inhibition zone diameter (mm) for different concentration of essential oil controls and minimum inhibitory concentration (MIC) (g/mL). The
values with different letters within a column are significantly different (P < 0.05; LSD).
Table 3: In vitro cytotoxic activity of Pulicaria vulgaris essential oil on MCF-7 and Hep-G2 tumor cell lines.
Concentrations
(g/mL)
0
1.56
3.125
6.25
12.5
25
50
*IC50
Hep-G2
100
78.56 2.45 a
69.33 1.22 b
52.14 0.55 c
41.23 1.12 d
34.11 0.25 e
27.77 2.88 f
7.16 g/ml
Hep-G2
100
72.33 1.55 a
58.11 0.77 b
49.73 0.95 c
22.54 1.78 d
18.25 1.22 d
12.12 0.53 e
6.11 g/ml
IC50: sample concentration required to inhibit tumor cell line proliferation by 50%. Data are expressed as means SD of %viability for different concentration of essential oil and
controls. Different letter showed the significant different between Pulicaria vulgaris essential oil concentrations at P < 0.05.
be the major group in the leaf oil (99.47%), which consisted almost
entirely of p-menth-6-en-2-one (carvotanacetone, 98.59%). The
essential oil showed moderate antimicrobial activity against Grampositive bacteria and C. albicans. However, no activity was shown
against Gram-negative bacteria. The oils showed a significant
cytotoxic activity against both MCF-7 and Hep-G2 (IC50 = 3.8 and
5.1 g/mL, respectively). They suggested that the potent cytotoxic
and moderate antimicrobial activities of this oil may be attributed to
its high content of p-menth-6-en-2-one (carvotanacetone). In
addition, El-Kamali et al. [13] reported that carvotanacetone, with
55.87%, was the major component in essential oil of Pulicaria
undulata aerial parts from Sudan. In our study, we observed this
compound in P. vulgaris leaf oil (Table 1).
To the best of our knowledge, this is the first study on the chemical
composition and bioactivity of the leaf essential oil of Pulicaria
vulgaris. However, in-vivo studies on this plant are necessary to
determine toxicity of the active ingredients, their side effects,
pharmacokinetic characteristics, serum-attainable levels and
diffusion in various body sites. The antimicrobial, antifungal and
cytotoxicity activities may be enhanced if the active constituents are
purified and sufficient dosages are determined for suitable
administration. This may go a long way in preventing the
administration of inappropriate concentrations, a usual practice
between many traditional physicians.
Experimental
Plant Material: Pulicaria vulgaris Gaertn. was collected during the
flowering period, March 2013, from the area surrounding Hamun
Lake, Zabol (Coordinates: 31 1 43 N, 61 30 4 E), in Sistan and
Baluchestan Province of Iran. The taxonomic identification of plant
materials was confirmed by a botanist at the herbarium affiliated to
Shahid Beheshti University of Iran. The collected plant materials
were dried in the shade. A voucher specimen has been deposited in
the Zabol Medicinal Plants Research Center, Zabol University of
Medical Sciences of Iran.
Essential Oil Extraction: The dried aerial parts (leaves, stems and
flowers) (200 g) of P. vulgaris were subjected to hydrodistillation
for 3 hours using a Clevenger-type apparatus in accordance with
the method outlined by the British Pharmacopeia [14]. The obtained
essential oil was dried over anhydrous sodium sulfate (SigmaAldrich, USA) and kept at 4C until analysis and further assays.
GC-FID and GC-MS Analysis: The P. vulgaris essential oil was
analyzed on an Agilent gas chromatograph (GC-FID) Model 6890,
equipped with a HP-5 MS fused silica capillary column having
(5%-phenyl)-methylpolysiloxane stationary phase (30 m length
0.25 mm internal diameter and 0.25 m film thickness),
programmed from 50C (5 min) to 250C at 5C/min and held for 5
min. Injector and flame ionization detector temperatures were 280
and 300C, respectively. The essential oil was diluted in acetone in
3.5% (v/v), and 1 L was injected in split mode (1/60). Hydrogen
was used as a carrier gas (1.0 mL/min). The gas chromatography
mass spectrometry (GCMS) analysis was carried out using
Hewlett-Packard 6890 mass selective detector coupled with a
Hewlett-Packard 6890 gas chromatograph operating at 70 eV
ionization energy, equipped with a HP-5MS capillary column (30 m
0.25 mm i.d. 25 m film thickness) with He as the carrier gas
and split ratio 1:50. Retention indices (RI) were determined using
retention times of n-alkanes that were injected after the essential oil
according to the same chromatographic conditions. Components
were recognized by comparison of mass spectral fragmentation
patterns and retention indices (HP-5) with Mass Finder 2.1 Library
data published mass spectra data, Wiley 7n.L Mass Spectral Library
(Wiley, New York, NY, USA) and additional literature sources
[15]. The relative percentages of the compounds of the essential oil
were attained according to the peak area in the chromatogram [16].
Antimicrobial Assays: The essential oils were tested against four
bacterial and two fungal strains purchased from Persian Type
Culture Collection, Tehran, Iran. The microbes used in this study
included two Gram-positive bacteria (Bacillus cereus and
Staphylococcus aureus), two Gram-negative bacteria (Escherichia
coli and Pseudomonas aeruginosa), and two fungi (Aspergillus
niger and Candida albicans). The bacteria and fungi were cultured
at 37C for 1524 h and the densities were adjusted to 0.5
McFarland standards (108 CFU/mL) at A530 nm. Antimicrobial tests
were carried out by the disc diffusion method [17]. Microbial
suspensions (200 L) of each organism were spread on nutrient
agar (Merck, Germany) plates (100 mm 15 mm). Discs (6 mm
diameter) were impregnated with 20 L of different concentrations
5, 10, 15, 20, 30, 40, 50, 90 and 120 g/mL of essential oil in
DMSO and placed on the inoculated agar. All the plates were
incubated at 37C for 24 h. Ketoconazole, ampicillin and
gentamicin (10 mg/disc) were positive control for fungi, grampositive and gram-negative bacteria, respectively. Also dimethyl
sulfoxide (DMSO) (Merck, Germany) was used as the negative
control. Antimicrobial activity was appraised by measuring the zone
of inhibition. Minimum inhibitory concentration (MIC) was
determined using serial dilutions of the essential oils (0200
g/mL) using microdilution test confirmed by Clinical and
Laboratory Standards Institute (CLSI) [18]. In LuriaBertani media
the bacteria and fungi strains were suspended and the densities were
regulated to 0.5 McFarland standards at 530 nm (108 CFU/mL). The
essential oil (100 L) and the bacteria and fungi suspensions (100
L) were added to microtiter plates and incubated at 37C for 24 h.
In this study, growth control was medium with bacteria and fungi
but without essential oil. Sterility control was medium without
bacteria and fungi. The growth in each well with that of the growth
in the control well was compared. The MIC were visually discerned
in comparison with the growth in the control well and determined as
the lowest concentration of the components with >95% growth
inhibition.
Cytotoxicity Assay: The mammalian cell lines MCF-7 cells (human
breast cancer cell line) and Hep-G2 (human liver cancer cell line)
were used for cytotoxicity assay. The cells were reproduced in
Dulbeccos modified Eagles Medium supplemented with 10%
heat-inactivated fetal bovine serum (Sigma Chemical Co., St. Louis,
Mo, USA), 1% L-glutamine, HEPES (N-2-hydroxyethylpiperazineN-2-ethane sulfonic acid) buffer and 50 g/mL gentamicin (Sigma
Chemical Co., St. Louis, Mo, USA). All cells were well kept at 37
C in a humidified atmosphere with 5% CO2 and were sub-cultured
three times a week. A modification of the crystal violet staining
(CVS) method described by Saotome et al. [19] was used to assess
cytotoxic activity. Briefly, in a 96-well tissue culture microplate, the
cells were incubated a cell concentration of 1 104 cells per well in
100 L of growth medium. Fresh medium containing various
concentrations of the essential oil was added after 24 h of seeding at
37C. Serial two-fold dilutions of the assayed essential oil was
added to confluent cell monolayers distribute into 96-well, flatbottomed microtiter plates applying a multichannel pipette. For a
period of 48 h, the microtiter plates were incubated at 37C in a
humidified incubator with 5% CO2. Then by a colorimetric method
the viable cells yield was determined. Briefly, media were aspirated
and a 1% (v/v) crystal violet solution in methanol was added to each
well. After 45 min, the stains were removed and the plates were
carefully rinsed with distilled water. Then, 0.2 mL of glacial acetic
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Dragoljub L. Miladinovi, Budimir S. Ili, Branislava D. Koci and Marija D. Miladinovi
Chemical Composition and Biological Activity of Pulicaria vulgaris Essential Oil from Iran
Javad Sharifi-Rad, Abdolhossein Miri, Seyedeh Mahsan Hoseini-Alfatemi, Majid Sharifi-Rad, William N. Setzer and
Abbas Hadjiakhoondi
Activity against Microorganisms Affecting Cellulosic Objects of the Volatile Constituents of Leonotis nepetaefolia
from Nicaragua
Simona Casiglia, Maurizio Bruno and Felice Senatore
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Accounts/Reviews
Nutrigenomics of Essential Oils and their Potential Domestic Use for Improving Health
Jos Antonio Cayuela Snchez and Abdelaziz Elamrani
Biotechnological Valorization of Pectinolytics and Their Industrial Applications: A Review
Muhammad Irshad, Muhammad Asgher, Zahid Anwar and Aftab Ahmad
Anticancer Agents from Diverse Natural Sources
Jabeena Khazir, Darren L. Riley, Lynne A. Pilcher, Pieter De-Maayer and Bilal Ahmad Mir
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