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Natural Product Communications
Chemical Composition and Biological Activity of Pulicaria vulgaris
Essential Oil from Iran
2014
Vol. 9
No. 11
1633 - 1636
Javad Sharifi-Rada,b, Abdolhossein Miria,b, Seyedeh Mahsan Hoseini-Alfatemic,*, Majid Sharifi-Radd,
William N. Setzere and Abbas Hadjiakhoondif,g
a
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
[email protected]
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 GC–MS.
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 β-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 µg/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 µg/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 μg/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.
Many microorganisms are responsible for drug-resistant infections,
and therefore, alternative chemotherapeutic options are necessary
and have been the focus of many researchers worldwide [1]. In
recent years, medicinal plant therapy has been shown to be useful
for treatment of many human and animal diseases. However,
although plants have been immensely utilized in traditional healing
systems, only in a few cases have their curative potentials in human
diseases been proven [2].
The Asteraceae is known for its wide range of floral variation and
reproductive attributes and it is the biggest dicotyledonous family
with 1,535 genera and 23,000 species (except single genus and
species). The genus Pulicaria Gaertn., according to Iranica flora,
includes five species that exist in Iran: P. dysenterica (L.) Bernh., P.
arabica (L.) Cass., P. gnaphalodes (Vent.) Boiss., P. salvifolia
Bunge and P. vulgaris Gaertn. [3]. P. vulgaris is an annual plant
that has many branched reddish stems and small (6-12 mm) yellow
flower heads. To our knowledge, there have been no previous
studies on the chemical composition and antimicrobial activities of
P. vulgaris essential oil.
The chemical composition of P. vulgaris is shown in Table 1.
Examination of the essential oil showed the main components 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 β-myrcene (1.9%).
The antibacterial and antifungal results of disc diffusion test are
summarized in Table 2. P. vulgaris essential oil significantly
inhibited the growth of Gram-positive bacteria, S. aureus and
B. cereus, as well as the Gram-negative bacterium, E. coli in a dosedependent manner. At concentration of 120 μg/mL (the maximum
concentration that used in this test) of the plant essential oil,
inhibition zones were for B. cereus S. aureus, E. coli, and
P. aeruginosa 81, 68.7, 72.1, and 39.5 mm, respectively. In
addition, the essential oil showed significant inhibition at all tested
concentrations of the growth of C. albicans and A. niger at P < 0.05,
except at concentrations of 30 and 40 μg/mL of essential oil on
C. albicans that showed no significant different between these
concentrations (P < 0.05). MIC for B. cereus, S. aureus, E. coli,
P. aeruginosa, A. niger and C. albicans were 17.5, 25.2, 19.4, 37.2,
15.5 and 9.9 µg/mL of essential oil, respectively (Table 2).
The cytotoxic activity of P. vulgaris essential oil on MCF-7 and
Hep-G2 tumor cell lines was determined using CVS method and
vinblastine as a reference drug. The response parameter (IC50) was
calculated for each of the cell lines (Table 3). P. vulgaris essential
oil showed concentration-dependent decrease in surviving fractions
of MCF-7 and Hep-G2 cell lines. The essential oil exhibited a
1634 Natural Product Communications Vol. 9 (11) 2014
Sharifi-Rad et al.
significant cytotoxic activity against both cell lines. In case of
MCF-7, IC50 of the essential oil (IC50 = 5.36 μg/mL) was less than
that of the reference drug used (IC50 = 7.47 μg/mL) showing its
higher cytotoxic potentiality. The Hep-G2 cell lines IC50 of essential
oil (IC50 = 7.16 μg/mL) was close to that of the reference drug (IC50
= 6.11μg/mL).
Table 1: Composition of the essential oil (EO) of Pulicaria vulgaris.
Compounds
Pyridine
α-Pinene
β-Myrcene
α-Terpinene
Linalool
1-Methyl-1,2-propanedione
Camphor
neo-3-Thujanol
α-Terpineol
Myrtenol
Menthan-2-one
trans-Carveol
Thymol methyl ether
p-Menth-6-en-2-one
Thymol
Carvacrol
Modhephene
Italicene
β-Cubebene
2,5-Dimethoxy-p-cymene
Neryl isobutyrate
Thymol isobutyrate
α-Cadinol
a
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
RI = Retention index with respect to a homologous series of n-alkanes.
Use of herbs as a source of medicine continues to be a significant
component of the health care system [4]. Understanding of the
chemical composition of plants is profitable, not only for the
discovery of curative factors, but also since such knowledge may be
of value in revealing new source of such economic materials as oils,
saponins, flavonoids, tannins, and essential oils as precursors for
modification and optimization of activity [5].
Al-Hajj et al. [6] studied chemical composition of essential of
Pulicaria inuloides aerial part in flowering stage. They found the
main components identified in the oil to be carvotanacetone
(47.34%) and hexadecanoic acid (12.82%). Mumivand et al. [7]
studied the chemical composition of the essential oil from flowering
aerial parts of Pulicaria dysenterica (L.) Bernh, growing wild in
Iran. Nineteen compounds, representing about 96.0% of the oil,
were identified. Oxygenated sesquiterpenes (43.5%) and
sesquiterpene hydrocarbons (41.5%) made up the major fraction of
the oil. Also, ar-curcumene (28.3%), epi-α-cadinol (16.4%) and
(E)-coniferyl alcohol (11.0 %) were the main ingredients of the oil.
Algabr et al. [8] investigated the essential oil of aerial parts of
Pulicaria jaubertii collected from Yemen. The major components
identified in this oil were carvotanacetone (63.96%), 1-methyl-1,2propanedione (5.89%), hexadecanoic acid (3.99%), 2,5-dimethoxyp-cymene (3.31%), ar-curcumene (3.28%) and absence of nonoxygenated terpenes. Ali et al. [9] investigated the chemical
composition of the essential oil obtained from the leaves of
Pulicaria undulata Gamal Ed Din. The main components of P.
undulata oil were the oxygenated monoterpenenes, carvotanacetone
(91.4%) and 2,5-dimethoxy-p-cymene (2.6%). The oil showed the
strongest bactericidal activity against S. aureus and methicillinresistant S. aureus, as well as C. albicans. Hanbali et al. [10]
analyzed essential oils of aerial parts of the Pulicaria odora (L.)
from Morocco. They collected plants at full flowering stage, from
Mrissat, province of Shoul (70 km east of Rabat) of Morocco.
Twenty-seven ingredients were identified, including thymol
(47.83%) and its isobutyrate ester (30.05%) as the main components
in the oil. In addition, the oil was assayed against seven bacteria at
various concentrations (5 and 10 µg/disc). Results showed that the
oil exhibited a significant antibacterial activity against C
Streptococcus (IPT 2-035), Bacillus cereus (IPL 58605),
Enterococcus faecalis (CIP 103214) and Pseudomonas aeruginosa
(CIP A 22). The bacteriostatic characteristic of the oil are suspected
to be associated with the high thymol content, which has been
assayed previously and was discovered to have a significant
antibiotic activity [11]. Fawzy et al. [12] evaluated chemical
composition and biological evaluation of essential oils of Pulicaria
jaubertii leaf. This investigation led to the identification of 16
constituents in the plant. Oxygenated monoterpenes were found to
Table 2: Antimicrobial activity of essential oil of Pulicaria vulgaris against microorganisms.
Essential oil
(µg/mL)
5
10
15
20
30
40
50
90
120
Dimethyl sulfoxide
Ampicillin
Gentamicin
Ketoconazole (µg/mL)
MIC
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
42.9±0.5 f
51.2±0.0 e
57.6± 0.3 d
62.3± 0.2 c
76.5± 0.2 b
81.0 ± 0.0 a
1.5±0.0 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
1.1±0.0 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
1.7±0.1 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
16.3±0.0 f
18.5± 0.5 d
25.6± 0.1 c
32.4± 0.0 b
39.5± 0.0 a
2.0±0.0 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
a
Data are expressed as means ± SD of inhibition zone diameter (mm) for different concentration of essential oil controls and minimum inhibitory concentration (MIC) (μg/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
(µg/mL)
0
1.56
3.125
6.25
12.5
25
50
*IC50
% Viability against essential oil
MCF-7
100
68.33 ± 1.92 a
54.77 ± 3.21 b
48.64 ± 2.55 c
39.61 ± 1.22 d
26.75 ± 2.95 e
19.77 ± 3.22 f
5.36 µg/ml
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 µg/ml
% Viability against vinblastine sulfate
MCF-7
100
62.33 ± 1.11 a
59.64 ± 2.51 b
52.71 ± 1.44 c
42.14 ± 1.89 d
17.54 ± 0.95 e
10.69 ± 1.52 f
7.47 µg/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 µg/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.
Composition and bioactivity of Pulicaria vulgaris essential oil
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 μg/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 4°C 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 μm film thickness),
programmed from 50°C (5 min) to 250°C at 5°C/min and held for 5
min. Injector and flame ionization detector temperatures were 280
and 300°C, respectively. The essential oil was diluted in acetone in
3.5% (v/v), and 1 μL was injected in split mode (1/60). Hydrogen
was used as a carrier gas (1.0 mL/min). The gas chromatography–
mass spectrometry (GC–MS) 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 µm 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
Natural Product Communications Vol. 9 (11) 2014 1635
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 37ºC for 15–24 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 µL) of each organism were spread on nutrient
agar (Merck, Germany) plates (100 mm × 15 mm). Discs (6 mm
diameter) were impregnated with 20 µL of different concentrations
5, 10, 15, 20, 30, 40, 50, 90 and 120 µg/mL of essential oil in
DMSO and placed on the inoculated agar. All the plates were
incubated at 37ºC 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 (0–200
µg/mL) using microdilution test confirmed by Clinical and
Laboratory Standards Institute (CLSI) [18]. In Luria–Bertani 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 µL) and the bacteria and fungi suspensions (100
µL) were added to microtiter plates and incubated at 37°C 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
Dulbecco’s modified Eagle’s 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 μg/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 μL of growth medium. Fresh medium containing various
concentrations of the essential oil was added after 24 h of seeding at
37°C. 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 37°C 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
1636 Natural Product Communications Vol. 9 (11) 2014
Sharifi-Rad et al.
acid-ethanol mixture (1.0 mL glacial acetic acid in 100 mL 50%
ethanol) to all wells and mixed thoroughly. The colorimetric
appraisal of fixed cells was carried out by determining the
absorbance in an automatic Microplate reader at 595 nm. The
concentration at which the growth of cells was inhibited to 50% of
the control (IC50) was calculated by using the formula:
Statistical Analysis: Data were subjected to analysis of variance
following a completely random design to determine the least
significant difference (LSD) at P < 0.05 using SPSS v. 11.5 (IBM
SPSS, New York, USA). The essential oil was prepared in triplicate
for chemical composition, antifungal, antibacterial and cytotoxicity
assays.
EXP (LN (conc >50%) ‐ ((signal >50%‐50)/ (signal >50%‐signal
<50%)*LN (conc >50%/conc <50%)))
Acknowledgments – The authors would like to express their
sincere appreciation to Professor Louis Maes, Faculty of
Pharmaceutical, Biomedical and Veterinary Sciences, University of
Antwerp, Belgium, for supplying the excel worksheet for
calculating the IC50 for the essential oil. The authors are very
grateful to Department of Bacteriology and Virology, Shiraz
Medical School, Shiraz University of Medical Sciences, Shiraz, Iran
and Department of Range and Watershed Management, Faculty of
Natural Resources, University of Zabol for supports this study.
using an automated excel worksheet developed by Professors Maes
and Cos of Antwerp University. In this test, the control cells were
incubated without test sample and with or without
dimethylsulfoxide (DMSO). Vinblastine sulfate was used as
standard antitumor drug.
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Natural Product Communications Vol. 9 (11) 2014
Published online (www.naturalproduct.us)
An in vitro Antibacterial Study of Savory Essential Oil and Geraniol in Combination with Standard Antimicrobials
Dragoljub L. Miladinović, Budimir S. Ilić, Branislava D. Kocić and Marija D. Miladinović
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
José Antonio Cayuela Sánchez 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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Natural Product Communications
2014
Volume 9, Number 11
Contents
Original Paper
Page
Sesinoside, a New Iridoid Glucoside from Sesame (Sesamum indicum) Seedlings
Ryo Takase, Tsuyoshi Hasegawa, Kosumi Yamada, Koji Hasegawa and Hideyuki Shigemori
A New Sesquiterpene Lactone and Other Constituents of Moquiniastrum polymorphum subsp. floccosum (Asteraceae)
Regiane L. B. Strapasson, André Luis Rüdiger, Robert A. Burrow, Andersson Barison and Maria Élida A. Stefanello
Further Degraded Diterpenoids from the Stems of Trigonostemon lii
Cheng-Jian Tan, Ying-Tong Di and Xiao-Jiang Hao
Antitumor Screening of Pterodon pubescens Terpenic Fraction Indicates High Sensitivity for Lymphocytic Leukemia Cells
Thiago Martino, Monica F. Pereira, Carlos R.M. Gayer, Sergio R. Dalmau, Marsen G.P. Coelho and Kátia C.C. Sabino
Dammarane Terpenoids from the Fruits of Dysoxylum mollissimum
Cholpisut Tantapakul, Wisanu Maneerat, Tawanun Sripisut, Thunwadee Ritthiwigrom and Surat Laphookhieo
Anti-tumor Activities of Triterpenes from Syzygium kusukusense
Li-Yuan Bai, Wei-Yu Lin, Chang-Fang Chiu and Jing-Ru Weng
Non-Competitive Inhibition of Acetylcholinesterase by Bromotyrosine Alkaloids
Opeyemi J. Olatunji, Akintayo L. Ogundajo, Ibrahim A. Oladosu, Kanokwan Changwichit, Kornkanok Ingkaninan,
Supreeya Yuenyongsawad and Anuchit Plubrukarn
Facile, Protection-Free, One-Pot Synthesis of Aureusidin
Quoc Vuong Nguyen, Phuong Diep Thi Lan, Hang Pham Thi, Van Chien Vu, Tuan Nguyen Le, Van Minh Chau and Van Cuong Pham
Profiling of Flavonols in Seeds and Sprouts of Luffa cylindrica
Soon-Mi Shim and Tae-Sik Park
Direct Identification and Characterization of Phenolic Compounds from Crude Extracts of Buds and Internodes of
Grapevine (Vitis vinifera cv Merlot)
Said Qsaib, Nuno Mateus, Fatima Ez-zohra Ikbal, Lala Aicha Rifai, Victor de Freitas and Tayeb Koussa
Phytochemical Analysis, Antioxidant and Anti-Inflammatory Activity of Calyces from Physalis peruviana
Reina M. Toro, Diana M. Aragón, Luis F. Ospina, Freddy A. Ramos and Leonardo Castellanos
Phenolic Derivatives from Radix Astragali and their Anti-inflammatory Activities
Wei Chen, Ying-Ying Zhang, Zhuo Wang, Xiao-Hua Luo, Wan-Chun Sun and Hong-Bing Wang
Isolation of Hydroxyoctaprenyl-1’,4’-hydroquinone, a new Octaprenylhydroquinone from the Marine Sponge
Sarcotragus spinosulus and Evaluation of its Pharmacological Activity on Acetylcholine and Glutamate Release in the
Rat Central Nervous System
Angela Bisio, Ernesto Fedele, Anna Pittaluga, Guendalina Olivero, Massimo Grilli, Jiayang Chen, Giacomo Mele,
Nicola Malafronte, Nunziatina De Tommasi, Fabio Ledda, Renata Manconi, Roberto Pronzato and Mario Marchi
PTP1B Inhibitory Effect of Alkyl p-Coumarates from Calystegia soldanella
Jung Im Lee, In-Hye Kim, Youn Hee Choi, Eun-Young Kim and Taek-Jeong Nam
Anti-inflammatory and Anti-oxidative Activities of Polyacetylene from Dendropanax dentiger
Shih-Chang Chien, Yen-Hsueh Tseng, Wei-Ning Hsu, Fang-Hua Chu, Shang-Tzen Chang, Yueh-Hsiung Kuo and Sheng-Yang Wang
Identification of the Country of Growth of Sophora flavescens using Direct Analysis in Real Time Mass Spectrometry (DART-MS)
Eriko Fukuda, Yoshihiro Uesawa, Masaki Baba, Ryuichiro Suzuki, Tatsuo Fukuda, Yoshiaki Shirataki and Yoshihito Okada
Physicochemical Properties of Honey from Marche, Central Italy: Classification of Unifloral and Multifloral Honeys by
Multivariate Analysis
Cristina Truzzi, Silvia Illuminati, Anna Annibaldi, Carolina Finale, Monica Rossetti and Giuseppe Scarponi
Antifungal Activity of Plant Extracts against Aspergillus niger and Rhizopus stolonifer
Venkatasaichaitanya Surapuram, William N. Setzer, Robert L. McFeeters and Hana McFeeters
GC/MS Analysis of the Essential Oil of Leucas indica from India
Rajesh K. Joshi
Composition of Headspace Volatiles and Essential Oils of Three Thymus Species
Gordana Stojanović, Olga Jovanović, Goran Petrović, Violeta Mitić, Vesna Stankov Jovanović and Snežana Jovanović
Constituents of Essential Oils from the Leaf and Flower of Plumeria alba Grown in Nigeria
Oladipupo A. Lawal, Isiaka A. Ogunwande and Andy R. Opoku
Anticonvulsant Activity of Citrus aurantium Blossom Essential Oil (Neroli): Involvment of the GABAergic System
Taravat Azanchi, Hamed Shafaroodi and Jinous Asgarpanah
Skin Regeneration Effect and Chemical Composition of Essential Oil from Artemisia montana
Mi-So Yoon, Kyung-Jong Won, Do Yoon Kim, Dae il Hwang, Seok Won Yoon, Bokyung Kim and Hwan Myung Lee
Chemical Composition and Antimicrobial Activity of the Essential Oils of Pinus peuce (Pinaceae) Growing Wild
in R. Macedonia
Marija Karapandzova, Gjose Stefkov, Ivana Cvetkovikj, Elena Trajkovska-Dokik, Ana Kaftandzieva and Svetlana Kulevanova
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Natural Product Communications
Chemical Composition and Biological Activity of Pulicaria vulgaris
Essential Oil from Iran
2014
Vol. 9
No. 11
1633 - 1636
Javad Sharifi-Rada,b, Abdolhossein Miria,b, Seyedeh Mahsan Hoseini-Alfatemic,*, Majid Sharifi-Radd,
William N. Setzere and Abbas Hadjiakhoondif,g
a
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
[email protected]
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 GC–MS.
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 β-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 µg/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 µg/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 μg/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.
Many microorganisms are responsible for drug-resistant infections,
and therefore, alternative chemotherapeutic options are necessary
and have been the focus of many researchers worldwide [1]. In
recent years, medicinal plant therapy has been shown to be useful
for treatment of many human and animal diseases. However,
although plants have been immensely utilized in traditional healing
systems, only in a few cases have their curative potentials in human
diseases been proven [2].
The Asteraceae is known for its wide range of floral variation and
reproductive attributes and it is the biggest dicotyledonous family
with 1,535 genera and 23,000 species (except single genus and
species). The genus Pulicaria Gaertn., according to Iranica flora,
includes five species that exist in Iran: P. dysenterica (L.) Bernh., P.
arabica (L.) Cass., P. gnaphalodes (Vent.) Boiss., P. salvifolia
Bunge and P. vulgaris Gaertn. [3]. P. vulgaris is an annual plant
that has many branched reddish stems and small (6-12 mm) yellow
flower heads. To our knowledge, there have been no previous
studies on the chemical composition and antimicrobial activities of
P. vulgaris essential oil.
The chemical composition of P. vulgaris is shown in Table 1.
Examination of the essential oil showed the main components 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 β-myrcene (1.9%).
The antibacterial and antifungal results of disc diffusion test are
summarized in Table 2. P. vulgaris essential oil significantly
inhibited the growth of Gram-positive bacteria, S. aureus and
B. cereus, as well as the Gram-negative bacterium, E. coli in a dosedependent manner. At concentration of 120 μg/mL (the maximum
concentration that used in this test) of the plant essential oil,
inhibition zones were for B. cereus S. aureus, E. coli, and
P. aeruginosa 81, 68.7, 72.1, and 39.5 mm, respectively. In
addition, the essential oil showed significant inhibition at all tested
concentrations of the growth of C. albicans and A. niger at P < 0.05,
except at concentrations of 30 and 40 μg/mL of essential oil on
C. albicans that showed no significant different between these
concentrations (P < 0.05). MIC for B. cereus, S. aureus, E. coli,
P. aeruginosa, A. niger and C. albicans were 17.5, 25.2, 19.4, 37.2,
15.5 and 9.9 µg/mL of essential oil, respectively (Table 2).
The cytotoxic activity of P. vulgaris essential oil on MCF-7 and
Hep-G2 tumor cell lines was determined using CVS method and
vinblastine as a reference drug. The response parameter (IC50) was
calculated for each of the cell lines (Table 3). P. vulgaris essential
oil showed concentration-dependent decrease in surviving fractions
of MCF-7 and Hep-G2 cell lines. The essential oil exhibited a
1634 Natural Product Communications Vol. 9 (11) 2014
Sharifi-Rad et al.
significant cytotoxic activity against both cell lines. In case of
MCF-7, IC50 of the essential oil (IC50 = 5.36 μg/mL) was less than
that of the reference drug used (IC50 = 7.47 μg/mL) showing its
higher cytotoxic potentiality. The Hep-G2 cell lines IC50 of essential
oil (IC50 = 7.16 μg/mL) was close to that of the reference drug (IC50
= 6.11μg/mL).
Table 1: Composition of the essential oil (EO) of Pulicaria vulgaris.
Compounds
Pyridine
α-Pinene
β-Myrcene
α-Terpinene
Linalool
1-Methyl-1,2-propanedione
Camphor
neo-3-Thujanol
α-Terpineol
Myrtenol
Menthan-2-one
trans-Carveol
Thymol methyl ether
p-Menth-6-en-2-one
Thymol
Carvacrol
Modhephene
Italicene
β-Cubebene
2,5-Dimethoxy-p-cymene
Neryl isobutyrate
Thymol isobutyrate
α-Cadinol
a
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
RI = Retention index with respect to a homologous series of n-alkanes.
Use of herbs as a source of medicine continues to be a significant
component of the health care system [4]. Understanding of the
chemical composition of plants is profitable, not only for the
discovery of curative factors, but also since such knowledge may be
of value in revealing new source of such economic materials as oils,
saponins, flavonoids, tannins, and essential oils as precursors for
modification and optimization of activity [5].
Al-Hajj et al. [6] studied chemical composition of essential of
Pulicaria inuloides aerial part in flowering stage. They found the
main components identified in the oil to be carvotanacetone
(47.34%) and hexadecanoic acid (12.82%). Mumivand et al. [7]
studied the chemical composition of the essential oil from flowering
aerial parts of Pulicaria dysenterica (L.) Bernh, growing wild in
Iran. Nineteen compounds, representing about 96.0% of the oil,
were identified. Oxygenated sesquiterpenes (43.5%) and
sesquiterpene hydrocarbons (41.5%) made up the major fraction of
the oil. Also, ar-curcumene (28.3%), epi-α-cadinol (16.4%) and
(E)-coniferyl alcohol (11.0 %) were the main ingredients of the oil.
Algabr et al. [8] investigated the essential oil of aerial parts of
Pulicaria jaubertii collected from Yemen. The major components
identified in this oil were carvotanacetone (63.96%), 1-methyl-1,2propanedione (5.89%), hexadecanoic acid (3.99%), 2,5-dimethoxyp-cymene (3.31%), ar-curcumene (3.28%) and absence of nonoxygenated terpenes. Ali et al. [9] investigated the chemical
composition of the essential oil obtained from the leaves of
Pulicaria undulata Gamal Ed Din. The main components of P.
undulata oil were the oxygenated monoterpenenes, carvotanacetone
(91.4%) and 2,5-dimethoxy-p-cymene (2.6%). The oil showed the
strongest bactericidal activity against S. aureus and methicillinresistant S. aureus, as well as C. albicans. Hanbali et al. [10]
analyzed essential oils of aerial parts of the Pulicaria odora (L.)
from Morocco. They collected plants at full flowering stage, from
Mrissat, province of Shoul (70 km east of Rabat) of Morocco.
Twenty-seven ingredients were identified, including thymol
(47.83%) and its isobutyrate ester (30.05%) as the main components
in the oil. In addition, the oil was assayed against seven bacteria at
various concentrations (5 and 10 µg/disc). Results showed that the
oil exhibited a significant antibacterial activity against C
Streptococcus (IPT 2-035), Bacillus cereus (IPL 58605),
Enterococcus faecalis (CIP 103214) and Pseudomonas aeruginosa
(CIP A 22). The bacteriostatic characteristic of the oil are suspected
to be associated with the high thymol content, which has been
assayed previously and was discovered to have a significant
antibiotic activity [11]. Fawzy et al. [12] evaluated chemical
composition and biological evaluation of essential oils of Pulicaria
jaubertii leaf. This investigation led to the identification of 16
constituents in the plant. Oxygenated monoterpenes were found to
Table 2: Antimicrobial activity of essential oil of Pulicaria vulgaris against microorganisms.
Essential oil
(µg/mL)
5
10
15
20
30
40
50
90
120
Dimethyl sulfoxide
Ampicillin
Gentamicin
Ketoconazole (µg/mL)
MIC
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
42.9±0.5 f
51.2±0.0 e
57.6± 0.3 d
62.3± 0.2 c
76.5± 0.2 b
81.0 ± 0.0 a
1.5±0.0 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
1.1±0.0 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
1.7±0.1 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
16.3±0.0 f
18.5± 0.5 d
25.6± 0.1 c
32.4± 0.0 b
39.5± 0.0 a
2.0±0.0 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
a
Data are expressed as means ± SD of inhibition zone diameter (mm) for different concentration of essential oil controls and minimum inhibitory concentration (MIC) (μg/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
(µg/mL)
0
1.56
3.125
6.25
12.5
25
50
*IC50
% Viability against essential oil
MCF-7
100
68.33 ± 1.92 a
54.77 ± 3.21 b
48.64 ± 2.55 c
39.61 ± 1.22 d
26.75 ± 2.95 e
19.77 ± 3.22 f
5.36 µg/ml
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 µg/ml
% Viability against vinblastine sulfate
MCF-7
100
62.33 ± 1.11 a
59.64 ± 2.51 b
52.71 ± 1.44 c
42.14 ± 1.89 d
17.54 ± 0.95 e
10.69 ± 1.52 f
7.47 µg/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 µg/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.
Composition and bioactivity of Pulicaria vulgaris essential oil
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 μg/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 4°C 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 μm film thickness),
programmed from 50°C (5 min) to 250°C at 5°C/min and held for 5
min. Injector and flame ionization detector temperatures were 280
and 300°C, respectively. The essential oil was diluted in acetone in
3.5% (v/v), and 1 μL was injected in split mode (1/60). Hydrogen
was used as a carrier gas (1.0 mL/min). The gas chromatography–
mass spectrometry (GC–MS) 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 µm 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
Natural Product Communications Vol. 9 (11) 2014 1635
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 37ºC for 15–24 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 µL) of each organism were spread on nutrient
agar (Merck, Germany) plates (100 mm × 15 mm). Discs (6 mm
diameter) were impregnated with 20 µL of different concentrations
5, 10, 15, 20, 30, 40, 50, 90 and 120 µg/mL of essential oil in
DMSO and placed on the inoculated agar. All the plates were
incubated at 37ºC 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 (0–200
µg/mL) using microdilution test confirmed by Clinical and
Laboratory Standards Institute (CLSI) [18]. In Luria–Bertani 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 µL) and the bacteria and fungi suspensions (100
µL) were added to microtiter plates and incubated at 37°C 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
Dulbecco’s modified Eagle’s 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 μg/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 μL of growth medium. Fresh medium containing various
concentrations of the essential oil was added after 24 h of seeding at
37°C. 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 37°C 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
1636 Natural Product Communications Vol. 9 (11) 2014
Sharifi-Rad et al.
acid-ethanol mixture (1.0 mL glacial acetic acid in 100 mL 50%
ethanol) to all wells and mixed thoroughly. The colorimetric
appraisal of fixed cells was carried out by determining the
absorbance in an automatic Microplate reader at 595 nm. The
concentration at which the growth of cells was inhibited to 50% of
the control (IC50) was calculated by using the formula:
Statistical Analysis: Data were subjected to analysis of variance
following a completely random design to determine the least
significant difference (LSD) at P < 0.05 using SPSS v. 11.5 (IBM
SPSS, New York, USA). The essential oil was prepared in triplicate
for chemical composition, antifungal, antibacterial and cytotoxicity
assays.
EXP (LN (conc >50%) ‐ ((signal >50%‐50)/ (signal >50%‐signal
<50%)*LN (conc >50%/conc <50%)))
Acknowledgments – The authors would like to express their
sincere appreciation to Professor Louis Maes, Faculty of
Pharmaceutical, Biomedical and Veterinary Sciences, University of
Antwerp, Belgium, for supplying the excel worksheet for
calculating the IC50 for the essential oil. The authors are very
grateful to Department of Bacteriology and Virology, Shiraz
Medical School, Shiraz University of Medical Sciences, Shiraz, Iran
and Department of Range and Watershed Management, Faculty of
Natural Resources, University of Zabol for supports this study.
using an automated excel worksheet developed by Professors Maes
and Cos of Antwerp University. In this test, the control cells were
incubated without test sample and with or without
dimethylsulfoxide (DMSO). Vinblastine sulfate was used as
standard antitumor drug.
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Natural Product Communications Vol. 9 (11) 2014
Published online (www.naturalproduct.us)
An in vitro Antibacterial Study of Savory Essential Oil and Geraniol in Combination with Standard Antimicrobials
Dragoljub L. Miladinović, Budimir S. Ilić, Branislava D. Kocić and Marija D. Miladinović
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
José Antonio Cayuela Sánchez 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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Natural Product Communications
2014
Volume 9, Number 11
Contents
Original Paper
Page
Sesinoside, a New Iridoid Glucoside from Sesame (Sesamum indicum) Seedlings
Ryo Takase, Tsuyoshi Hasegawa, Kosumi Yamada, Koji Hasegawa and Hideyuki Shigemori
A New Sesquiterpene Lactone and Other Constituents of Moquiniastrum polymorphum subsp. floccosum (Asteraceae)
Regiane L. B. Strapasson, André Luis Rüdiger, Robert A. Burrow, Andersson Barison and Maria Élida A. Stefanello
Further Degraded Diterpenoids from the Stems of Trigonostemon lii
Cheng-Jian Tan, Ying-Tong Di and Xiao-Jiang Hao
Antitumor Screening of Pterodon pubescens Terpenic Fraction Indicates High Sensitivity for Lymphocytic Leukemia Cells
Thiago Martino, Monica F. Pereira, Carlos R.M. Gayer, Sergio R. Dalmau, Marsen G.P. Coelho and Kátia C.C. Sabino
Dammarane Terpenoids from the Fruits of Dysoxylum mollissimum
Cholpisut Tantapakul, Wisanu Maneerat, Tawanun Sripisut, Thunwadee Ritthiwigrom and Surat Laphookhieo
Anti-tumor Activities of Triterpenes from Syzygium kusukusense
Li-Yuan Bai, Wei-Yu Lin, Chang-Fang Chiu and Jing-Ru Weng
Non-Competitive Inhibition of Acetylcholinesterase by Bromotyrosine Alkaloids
Opeyemi J. Olatunji, Akintayo L. Ogundajo, Ibrahim A. Oladosu, Kanokwan Changwichit, Kornkanok Ingkaninan,
Supreeya Yuenyongsawad and Anuchit Plubrukarn
Facile, Protection-Free, One-Pot Synthesis of Aureusidin
Quoc Vuong Nguyen, Phuong Diep Thi Lan, Hang Pham Thi, Van Chien Vu, Tuan Nguyen Le, Van Minh Chau and Van Cuong Pham
Profiling of Flavonols in Seeds and Sprouts of Luffa cylindrica
Soon-Mi Shim and Tae-Sik Park
Direct Identification and Characterization of Phenolic Compounds from Crude Extracts of Buds and Internodes of
Grapevine (Vitis vinifera cv Merlot)
Said Qsaib, Nuno Mateus, Fatima Ez-zohra Ikbal, Lala Aicha Rifai, Victor de Freitas and Tayeb Koussa
Phytochemical Analysis, Antioxidant and Anti-Inflammatory Activity of Calyces from Physalis peruviana
Reina M. Toro, Diana M. Aragón, Luis F. Ospina, Freddy A. Ramos and Leonardo Castellanos
Phenolic Derivatives from Radix Astragali and their Anti-inflammatory Activities
Wei Chen, Ying-Ying Zhang, Zhuo Wang, Xiao-Hua Luo, Wan-Chun Sun and Hong-Bing Wang
Isolation of Hydroxyoctaprenyl-1’,4’-hydroquinone, a new Octaprenylhydroquinone from the Marine Sponge
Sarcotragus spinosulus and Evaluation of its Pharmacological Activity on Acetylcholine and Glutamate Release in the
Rat Central Nervous System
Angela Bisio, Ernesto Fedele, Anna Pittaluga, Guendalina Olivero, Massimo Grilli, Jiayang Chen, Giacomo Mele,
Nicola Malafronte, Nunziatina De Tommasi, Fabio Ledda, Renata Manconi, Roberto Pronzato and Mario Marchi
PTP1B Inhibitory Effect of Alkyl p-Coumarates from Calystegia soldanella
Jung Im Lee, In-Hye Kim, Youn Hee Choi, Eun-Young Kim and Taek-Jeong Nam
Anti-inflammatory and Anti-oxidative Activities of Polyacetylene from Dendropanax dentiger
Shih-Chang Chien, Yen-Hsueh Tseng, Wei-Ning Hsu, Fang-Hua Chu, Shang-Tzen Chang, Yueh-Hsiung Kuo and Sheng-Yang Wang
Identification of the Country of Growth of Sophora flavescens using Direct Analysis in Real Time Mass Spectrometry (DART-MS)
Eriko Fukuda, Yoshihiro Uesawa, Masaki Baba, Ryuichiro Suzuki, Tatsuo Fukuda, Yoshiaki Shirataki and Yoshihito Okada
Physicochemical Properties of Honey from Marche, Central Italy: Classification of Unifloral and Multifloral Honeys by
Multivariate Analysis
Cristina Truzzi, Silvia Illuminati, Anna Annibaldi, Carolina Finale, Monica Rossetti and Giuseppe Scarponi
Antifungal Activity of Plant Extracts against Aspergillus niger and Rhizopus stolonifer
Venkatasaichaitanya Surapuram, William N. Setzer, Robert L. McFeeters and Hana McFeeters
GC/MS Analysis of the Essential Oil of Leucas indica from India
Rajesh K. Joshi
Composition of Headspace Volatiles and Essential Oils of Three Thymus Species
Gordana Stojanović, Olga Jovanović, Goran Petrović, Violeta Mitić, Vesna Stankov Jovanović and Snežana Jovanović
Constituents of Essential Oils from the Leaf and Flower of Plumeria alba Grown in Nigeria
Oladipupo A. Lawal, Isiaka A. Ogunwande and Andy R. Opoku
Anticonvulsant Activity of Citrus aurantium Blossom Essential Oil (Neroli): Involvment of the GABAergic System
Taravat Azanchi, Hamed Shafaroodi and Jinous Asgarpanah
Skin Regeneration Effect and Chemical Composition of Essential Oil from Artemisia montana
Mi-So Yoon, Kyung-Jong Won, Do Yoon Kim, Dae il Hwang, Seok Won Yoon, Bokyung Kim and Hwan Myung Lee
Chemical Composition and Antimicrobial Activity of the Essential Oils of Pinus peuce (Pinaceae) Growing Wild
in R. Macedonia
Marija Karapandzova, Gjose Stefkov, Ivana Cvetkovikj, Elena Trajkovska-Dokik, Ana Kaftandzieva and Svetlana Kulevanova
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