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FULL COMMUNICATION
Composition and Antiproliferative Effect of Essential Oil of Origanum vulgare
Against Tumor Cell Lines
Karine Rech Begnini,
1
Fernanda Nedel,
1,2
Rafael Guerra Lund,
1–3
Pedro Henrique de Azambuja Carvalho,
3
MariaReginaAlvesRodrigues,
4
Fa
´tima Tereza Alves Beira,
2
and Francisco Augusto Burkert Del-Pino
2
1
Department of Physiology and Pharmacology, Institute of Biology, Federal University of Pelotas, Pelotas,
Rio Grande do Sul, Brazil.
2
Post-Graduate Program in Dentistry,
3
Pelotas Dental School, and
4
Institute of Chemistry and Geosciences,
Federal University of Pelotas, Pelotas, Rio Grande do Sul, Brazil.
ABSTRACT Cancer is a leading cause of death and is responsible for one in eight deaths worldwide. The use of herbs as
complementary medicine for cancer, especially advanced cancer, has recently increased. The aim of this study was to evaluate
in vitro, the antiproliferative effect of Origanum vulgare against human breast adenocarcinoma ( MCF-7), and human colon
adenocarcinoma (HT-29). The essential oil (EO) was extracted from a bought amount of O. vulgare dried leaves and analyzed
in a gas chromatograph interfaced with a mass selective detector. The cytotoxicity test was performed by sulforhodamine B
assay. The results show that the EO is composed mostly of 4-terpineol and induces a high cytotoxicity effect in HT-29. In the
MCF-7 cell line the EO was less effective. In conclusion, this study showed that O. vulgare main component is 4-terpineol and
was effective in inducing cancer cell growth inhibition.
KEY WORDS: antiproliferative assay cancer cell lines Origanum vulgare plant extracts
INTRODUCTION
According to the World Health Organization,
cancer is a leading cause of death and is responsible for
one in eight deaths worldwide.
1
Cancer is a generic term for
a large group of diseases that can affect any part of the body
and has diverse risk factors and epidemiology. It can origi-
nate from most cell types and organs of the human body and
is characterized by relatively unrestrained proliferation of
cells that can invade beyond normal tissue boundaries and
metastases to distant organs.
2
The most frequent types of
cancer are breast, prostate, lung, colorectal, stomach, and
liver,
3
and the numbers of deaths from cancer worldwide are
projected to continue to rise to over 11 million in 2030.
4
The use of herbs as complementary medicine for cancer,
especially advanced cancer, has recently increased.
5,6
Questions concerning the safety of synthetic agents have
increased the interest in the use of natural compounds and
have encouraged more detailed studies of plant resources,
which are a rich source of bionutrients or bioactive phyto-
chemicals.
7
One new approach to cancer therapy focuses on
anticancer and antimetastatic agents with little or no cyto-
toxic activity, such as the use of plant foods and their es-
sential oils (EOs), which have a potential antioxidant effect.
8
EOs are volatile, natural complex compounds character-
ized by a strong odor and are formed by aromatic plants as
secondary metabolites.
9
Origanum vulgare (oregano) is an
annual, perennial, and shrubby herb native to the Mediter-
ranean and has been used for many years as a medicinal
plant with health-aiding properties such as its powerful an-
tibacterial and antifungal properties.
8
Oregano belongs to
the Lamiaceae family and it is well-known that Lamiaceae
spices have potent antioxidant properties, mostly due to the
polyphenolic compounds.
10,11
Phenolic phytochemicals are
a large group of substances, which are found in significant
quantities in vegetables, fruits, spices, and seeds, and are
thought to promote optimal health partly through their scav-
enging effects in protecting cellular components against free
radicals.
7
Recent findings revealed the antimicrobial, fungicidal,
insecticidal, and antioxidant potential of the EO and extract
of Origanum, which raised great pharmaceutical and in-
dustrial interest in oregano.
10
Natural antioxidants can
protect the human body from free radicals and could retard
the progress of many chronic diseases.
11
Oxidation of lipids
is associated with cell membrane damage, aging, heart
disease, and cancer in living organisms.
11
Studies have
shown that components of oregano EOs, that is, carvacrol,
Manuscript received 2 March 2013. Revision accepted 18 June 2014.
Address correspondence to: Rafael Guerra Lund, DDS, MD, PhD, Post-Graduate Program
in Dentistry, Pelotas Dental School, Federal University of Pelotas (UFPel), Gonc¸alves
Chaves St., 457/503, Pelotas 96015–560, RS, Brazil, E-mail: rafael.lund@gmail.com
JOURNAL OF MEDICINAL FOOD
J Med Food 17 (10) 2014, 1–5
#Mary Ann Liebert, Inc., and Korean Society of Food Science and Nutrition
DOI: 10.1089/jmf.2013.0063
1
thymol and protocatechuic acid, possesses strong antioxi-
dant properties and can act like potent anticancer and anti-
melanogenic compounds.
12–14
Modulation of carcinogenic
and mutagenic effects by inhibitors from plant origin has
been of crucial importance for the final outcome of some
biological effects, particularly for cancer.
7
Therefore, the present study aimed to evaluate in vitro the
antiproliferative effect of O. vulgare against human breast
adenocarcinoma (MCF-7) and human colon adenocarci-
noma (HT-29).
MATERIALS AND METHODS
Material
Samples of oregano (dried leaves from Chile) were pur-
chased from TecPharma Importac¸a
˜o de Produtos Quı
´micos
e Farmace
ˆuticos (TECPHARMA), a major manufacturer of
chemical and pharmaceutical products. These samples were
manually crushed and stored under nitrogen atmosphere.
The analytical standards thymol, a-terpinene, and a-terpinene
were purchased from Fluka
and the analytical standards
a-terpineol, 4-terpineol, camphene, carvacrol, limonene, a-
pinene, a-pinene, myrcene, p-cymene, 1,8-cineole, terpino-
lene, and linalool were purchased from Sigma-Aldrich
.
Solutions of each standard were prepared (1000 mg/L) using
dichloromethane (Merck; pa grade, bidistilled) and stored
under refrigeration.
Extraction procedure
The EO was extracted by hydrodistillation over a period
of 4 h using a Clevenger apparatus and the yield of oil was
recorded at every 5 min. The density of the EO measured by
the gravimetric method was 0.92 g/mL at room temperature
(20C). After hydrodistillation, water was removed by de-
cantation and the EOs obtained were stored at 4C in a dark-
colored container to prevent light-sensitive decomposition.
Analytical methods
The EO was analyzed in a gas chromatograph interfaced
with a mass selective detector—GC/MS (Shimadzu 5050A),
using a capillary column DB-5 (30 m ·0.25 mm ·0.25 mm)
and a flow rate of 1 mL/min, in electronic impact mode of
70 eV and in split mode (split ratio 1:50). The following
temperature gradient was used: 40C (0 min)—2C/min—
145C—10C/min—280C (10 min). The interface temper-
ature was maintained at 280C. The identification of major
compounds was accomplished by comparing their retention
times with those of authentic standards, and by comparison of
their mass spectra with those from the equipment library.
Compositions were then expressed as percentages of nor-
malized peak areas.
Cell lines
The cell lines under investigation were human breast
adenocarcinoma (MCF-7) and human colon adenocarci-
noma (HT-29). They were purchased from RJCB Collection
(Rio de Janeiro Cell Bank). The cells were cultured in
Dulbecco’s modified Eagle’s medium (Gibco
), supple-
mented with 10% fetal bovine serum (Gibco) and incubated
at 37C in a humidified atmosphere containing 5% CO
2
,as
described previously.
15–17
Cytotoxicity assay
According to the cells growth profile, cells were seeded
with a density of 2 ·10
4
cell/well and incubated at 37Cina
humidified atmosphere containing 5% CO
2
. After 24 h, the
cells were treated with the EO, which was diluted in di-
methylsulfoxide to produce nine concentrations, ranging
from 10 to 500 mg/mL. Of each concentration, 100 lL/well
was added to the plates in triplicates. The final dilution used
for treating the cells contained not more than 1% of the
initial solvent, this concentration being used in the solvent
control wells. This concentration of dimethylsulfoxide did
not significantly influence the proliferation rate as compared
to media alone. The plates were incubated for 72 h. At the
end of the exposure time, cell growth was analyzed using the
sulforhodamine B (SRB) assay.
SRB assay
After incubation for 72 h, adherent cell cultures were fixed
in situ by adding 50 lL of cold 40% (w/v) trichloroacetic acid
and incubated for 60min at 4C. The supernatant was then
discarded, and the plates were washed five times with deio-
nized water and then dried. Of the SRB solution (0.4% w/v in
1% acetic acid), 50 lL was added to each well and incubated
for 30 min at room temperature. Unbound SRB was removed
by washing five times with 1% acetic acid. Then, the plates
were air-dried and 100 lLof10mMTrisbasepH10.5
(Sigma
) was added to each well to solubilize the dye. The
plates were shaken gently for 20 min on a plate shaker, and
the optical density was determined in an ELISA multiplate
reader (Thermo Plate TP-Reader; Thermo Fisher Scientific)
using a wavelength filter of 492 nm. The cell growth in-
hibition was calculated as the percentage inhibition of cell
growth and was determined as follows: inhibitory rate =
(1 – Abs
492treated cells
/Abs
492control cells
)·100. All observations
were validated by at least two independent experiments, and
for each experiment, the analyses were performed in triplicate
Statistical analyses
Data sets from the SRB assay were analyzed using a
two-way ANOVA followed by a Tukey test for multiple
comparisons. Two factors were considered: the cell type
and the concentration of the compound. Significance was
considered at P<.05 in all analyses. The data are expressed
as the mean –SEM.
RESULTS
EO composition
The EO was obtained from O. vulgare and its composi-
tion is listed in Table 1. The table shows the retention
2BEGNINI ET AL.
indices calculated on the DB-5 column and the percentages
of the detected compounds. Retention times of the sample
components were calculated on the basis of homologous
compounds under the same conditions, and the compounds
were identified by injection of standards and/or by com-
paring the mass spectra with the equipment library.
The average extraction yield of oregano EO was deter-
mined to be 1.33 –0.16 wt%, achieved after about 60-min
extraction; this value is similar to that found by Rodrigues
et al.
18
and Busatta et al.
20
The chromatogram of O. vulgare
EO (Fig. 1) shows that 4-terpineol (peak 20 and percent
area of 41.17) is the major component, followed by thymol
(peak 28, percent area of 21.95), c-terpinene (peak 12,
percent area of 5.91), and carvacrol (peak 29, percent area
of 4.71).
Antiproliferative activity
The cell growth inhibition of O. vulgare was determined
by the cell proliferation assay using the MCF-7 and HT-29
cell lines. As shown in Figure 2, the EO was most effective
in the HT-29 cell line when compared with MCF-7. In both
cell types, the concentration of 10 mg/mL was not effective;
in HT-29 cells, the percent of cell growth inhibition was
8.18% and in MCF-7 cells, this concentration induced cell
proliferation. The most effective concentration for HT-29
and MCF-7 was 50 mg/mL: 60.8% and 48.9%, respectively;
however, an increase in the EO concentration did not en-
hance the cell growth inhibition (Fig. 2).
DISCUSSION
O. vulgare L. is the most variable species of the genus
Origanum, and variations in the composition of the EO have
been the topic in the reports of several researchers.
12,10
In
the composition of the EO of oregano, great variations in the
major and minor constituents were recorded. Several oreg-
ano species are characterized by the presence of two major
components, thymol and carvacrol. Another intermediate
type would contain a high content of two monoterpene hy-
drocarbons, c-terpinene or p-cymene, which are biogenetic
precursors of thymol and carvacrol. However, some species
were found with high values of linalool and others with
monoterpenes and sesquiterpenes.
12,18
The EO composition and yield of Origanum species vary
with the origin of these plants and other factors, mainly the
geographical and time of harvest.
19
Table 1. Chemical Composition of the Essential Oil
of Origanum vulgare, Based on Retention Time
and Concentration (%)
Peak Compounds Rt Peak area C (%)
1a-thujene 10.07 52 0.12
2a-pinene
a
——ND
3 Sabinene 12.58 72 0.18
4b-pinene
a
——ND
5 Myrcene
a
13.76 80 0.2
6a-phellandrene 14.41 651 1.73
7a-terpinene
a
15.19 913 2.43
8p-cymene
a
15.67 423 1.13
9 Limonene
a
15.94 908 2.59
10 1,8-cineole
a
——ND
11 Cis/trans b-ocimene 16.76 55 0.13
12 a-terpinene
a
17.99 2.143 5.91
13 Terpinolene
a
19.96 544 1.63
14 Linalool
a
20.88 709 2.07
15 Trans-p-menthenol 22.19 31 0.10
16 Borneol 25.21 108 0.36
17 4-terpineol
a
26.13 11.286 41.17
18 a-terpineol
a
27.05 1.548 4.98
19 Trans-piperitol 27.44 49 0.17
20 Methyl thymol eter 30.28 176 0.58
21 Methyl carvacrol Eter 30.90 942 3.04
22 Geraniol/nerol 31.89 147 0.51
23 Thymol
a
34.45 6.646 21.95
24 Carvacrol
a
35.00 1.415 4.71
25 Geranil/neril Acetate 40.56 70 0.21
26 b-caryophyllene 42.55 909 3.22
27 Spathulenol 52.38 216 0.81
28 Caryophyllene 52.75 20 0.07
a
Compounds identified by comparison with standards compounds; other
compounds identified by literature data and Willey Library.
C, normalized peak areas without using the correction factors; ND, not
detected (based in Rt of standards compounds); Rt, retention time.
FIG. 1. Chromatogram of oregano essential oil (EO) obtained by hydrodistillation. Peaks identification according to Table 1.
OREGANO’S COMPOSITION AND ANTIPROLIFERATIVE EFFECT 3
Additionally, it is important to know the botanical origin
of oregano as well as to do the investigation of the chemical
constituents of this species. With regard to the taxonomic
viewpoint, it is difficult to establish a correspondence be-
tween subspecies of O. vulgare and composition of its EO.
In this study, 4-terpineol was the main component of Or-
iganum EO, followed by thymol, c-terpinene, and carvacrol.
This chemical profile is very similar to that found by Busatta
et al.
20
where 4-terpineol, c-terpinene, carvacrol, and thymol
were the major components. Comparison of the data pro-
duced herein with previous reports showed that the che-
motype variability has a straight relationship with the
geographical region where the species is found.
8,10,11
Re-
ports
2,21,22
have described that oregano grown in a Medi-
terranean climate contains higher amount of phenols,
whereas oregano from the inland contains a higher amount
of terpene alcohols. In a study of Russo et al.,
23
the authors
evaluated the chemical composition of the EO of 24 samples
of O. vulgare, hirtum variety that grows in Southern Italy by
GC/MS. A total of 56 compounds were identified, the major
compounds were carvacrol (0.12–56.63%) and thymol
(7.91–53.62%). This same species, hirtum variety, grown in
Northern Italy, was analyzed by Bocchini and coworkers
24
and they found a group of oregano with high content of
thymol, carvacrol, and linalool, other with a large variation
among the sesquiterpenes, and a third group with loads of
sesquiterpenes.
Furthermore, the technique of obtaining the EO may in-
fluence the quality of the extract obtained and the amount of
extracted aromatics. Parameters that affect the distribution
coefficients of the main components present in the EO of
O. vulgare, as the fraction of monoterpene hydrocarbons,
depend on the equilibrium time, temperature, and density of
the solvent used in the extraction process.
19
Comparing the
extraction process with those obtained from the supercritical
CO
2
extract indicated that the extraction method plays an
important role in the final extract’s composition,
18
espe-
cially with regard to the hydrocarbon terpene fraction.
MCF-7 is an adenocarcinoma cell line recommended as
one of the models for breast cancer tissue by the National
Cancer Institute (NCI). The cell line expresses estrogen
receptors and has been well studied and documented.
22
In
the present study, the Origanum EO was less effective in the
MCF-7 cell type and was able to induce cytotoxicity in a
concentration of 50 mg/mL (cell growth inhibition of
48.9%). The increase in the EO concentration did not en-
hance the cell growth inhibition. In the HT-29 cell line, the
EO was significantly more effective (cell growth inhibition
of 60.8%) and also presented the same characteristic of in-
ducing cytotoxicity at 50 mg/mL and the nonenhancement
of cell growth inhibition values with the increase of EO
concentration. These results suggest that the EO could have
a selective activity and therefore offer an opportunity to
investigate its use as a therapeutic agent.
The cytotoxic activity of oregano oil can be attributed to
the action of its principal phenolic components, carvacrol,
and thymol, which exhibit significant anticancer and anti-
mutagenic activity when tested separately.
7,13
Arunasree
13
demonstrated the anticancer effects of carvacrol in MDA-MB
231, a human metastatic breast cancer cell line. These authors
showed that carvacrol-treated cells exhibited prominent
morphological changes like cell shrinkage with rounding of
cells and formation of membrane blebs characteristic of ap-
optosis. Other studies have suggested that carvacrol might be
potentially useful in counteracting free radical-mediated in-
juries and in DNA damage by the ability to enhance the
levels of antioxidants along with its antilipid peroxidative
activity,
25,26
what can be a beneficial action of carvacrol
against pathological alterations like melanogenesis and cancer.
In conclusion, it was found that the O. vulgare EO main
component is 4-terpineol and that the oil presents a signif-
icant effect on the cancer cell line tested, which could be
associated with the major components of the extracted oil.
ACKNOWLEDGMENT
This study was made possible by an undergraduate fel-
lowship provided by the National Council of Scientific and
Technology Development of Brazil (CNPq).
FIG. 2. Cell growth inhibition of MCF-7 and HT-29 cell lines by
Origanum vulgare EO. Uppercase letters indicate significant differ-
ences between tumor cell type and lowercase letters indicate signif-
icant differences in the concentration used. A P-value <.05 was
considered significant (Tukey test).
4BEGNINI ET AL.
AUTHOR DISCLOSURE STATEMENT
The authors declare that they have no conflicts of interest
concerning this article.
REFERENCES
1. American Cancer Society: Global Cancer Facts & Figures 2nd
Edition. Atlanta: American Cancer Society; 2011. www.cancer.org/
research/cancerfactsfigures/globalcancerfactsfigures/global-facts-
figures-2nd-ed (accessed July 15, 2013).
2. Straton MR, Campbell PJ, Futreal A: The cancer genome. Nature
2009;458:719–724.
3. Ferlay J, Shin HR, Bray F, Forman D, Mathers C, Parkin DM:
GLOBOCAN 2008, Cancer Incidence and Mortality Worldwide:
IARC CancerBase No. 10 (Online), 2010. International Agency
for Research on Cancer, Lyon, France. http://globocan.iarc.fr
(accessed December 5, 2012).
4. World Health Organization: Mortality Database WHO Statistical
Information System. 2011. www.who.int/whosis/mort/download/
en/index.html (accessed November 28, 2012).
5. Powell CB, Fung P, Jackson J, Dall’Era J, Lewkowicz D, Cohen
I, Smith-McCune K: Aqueous extract of herba Scutellaria bar-
batae, a Chinese herb used for ovarian cancer, induces apoptosis
of ovarian cancer cell lines. Gynecol Oncol 2003;91:332–340.
6. Nedel F, Begnini K, Carvalho PH, Lund RG, Beira FT, Del Pino
FA: Antiproliferative activity of flower hexane extract obtained
from mentha spicata associated with mentha rotundifolia against the
MCF7, KB, and NIH/3T3 cell lines. JMedFood2012;15:955–958.
7. Aydin S, Seker E: Effect of an aqueous distillate of Origanum
onites L. on isolated rat fundus,duodenum and ileum: evidence for
the role of oxygenated monoterpenes. J Pharm 2005;60:147–150.
8. Karakaya S, El SN, Karago
¨zulu
¨N, Sahin S: Antioxidant and
antimicrobial activities of essential oils obtained from Oregano
(Origanum vulgare ssp. hirtum) by using different extraction
methods. J Med Food 2011;14:645–652.
9. Bakkali F, Averbeck S, Averbeck D, Idaomar M: Biological
effects of essential oils—a review. Food Chem Toxicol 2008;46:
446–475.
10. Verma RS, Padalia RC, Chauhan A, Verma RK, Yudav AK,
Singh HP: Chemical diversity in Indian ore
´gano (Organum vul-
gare L.). Chem Biodivers 2010;7:2054–2064.
11. Hossain MB, Brunton NP, Barry-Ryan C, Martin-Diana AB,
Wilkinson M: Characterization of phenolics composition in La-
miaceae spices by LC-ESI-MS/MS. J Agric Food Chem 2010;58:
10576–10581.
12. Al-Kalaldeh JZ, Abu-Dahab R, Afifi FU: Volatile oil composi-
tion and antiproliferative activity of Laurus nobilis, Origanum
syriacum, Origanum vulgare, and Salvia triloba against human
breast adenocarcinoma cells. Nutr Res 2010;30:271–278.
13. Arunasree KM: Anti-proliferative effects of carvacrol on a hu-
man metastatic breast cancer cell line, MDA-MB 231. Phyto-
medicine 2012;17:581–588.
14. Chou TH, Ding HY, Hung WJ, Liang CH: Antioxidative char-
acteristics and inhibition of alpha-melanocyte-stimulating hor-
mone-stimulated melanogenesis of vanillin and vanillic acid
from Origanum vulgare.Exp Dermatol 2010;19:742–750.
15. Henn S, Nedel F, de Carvalho RV, Lund RG, Cenci MS, Pereira-
Cenci T, Demarco FF, Piva E: Characterization of an antimicro-
bial dental resin adhesive containing zinc methacrylate. JMater
Sci Mater Med 2011;22:1797–1802.
16. de Vasconcelos A, Campos VF, Nedel F, Seixas FK, Dellagostin
OA, Smith KR, de Pereira CM, Stefanello FM, Collares T,
Barschak AG: Cytotoxic and apoptotic effects of chalcone de-
rivatives of 2-acetyl thiophene on human colon adenocarcinoma
cells. Cell Biochem Funct 2013;31:289–297.
17. Nedel F, Campos VF, Alves D, McBride AJ, Dellagostin OA,
Collares T, Savegnago L, Seixas FK: Substituted diaryl dis-
elenides: cytotoxic and apoptotic effect in human colon adeno-
carcinoma cells. Life Sci 2012;91:345–352.
18. Rodrigues MR, Krause LC, Caramao EB, dos Santos JG,
Dariva C, Vladimir de Oliveira J: Chemical composition and
extraction yield of the extract of Origanum vulgare obtained
from sub- and supercritical CO2. J Agric Food Chem 2004;52:
3042–3047.
19. Rodrigues MRA: Studies of Essential Oils Present in Marjoram
and Oregano. 2002. [Ph.D. thesis in Chemistry, 148f.]. Chemistry
Institute, Federal University of Rio Grande do Sul, Porto Alegre,
RS, Brazil.
20. Busatta C, Mossi AJ, Rodrigues MRA, Cansian RL, De Oliveira
JV: Evaluation of Origanum vulgare essential oil as antimicro-
bial agent in sausage. Braz J Microbiol 2007;38:610–616.
21. Hristova R, Ristc M, Brkic D, Stefkov G, Kulevanova S: Com-
parative analysis of essential oil composition of Origanum vul-
gare from Macedonia and commercially available Origani herba.
Acta Pharm 1999;49:299–305.
22. Shoemaker RH: The NCI60 human tumour cell line anticancer
drug screen. Nat Rev Cancer 2006;6:813–823.
23. Russo M, Galletti GC, Bocchini P, Carnacini A: Essential oil
chemical composition of wild populations of Italian oregano
spice (Origanum vulgare ssp. hirtum (link) ietswaart): a pre-
liminary evaluation of their use in chemotaxonomy by cluster
analysis. 1. Inflorescences. J Agric Food Chem 1998;46:3741–
3746.
24. D’Antuono LF, Galletti GC, Bocchini P: Variability of essential
oil content and composition of Origanum vulgare L. populations
from a North Mediterranean area (Liguria region, Northern Ita-
ly). Ann Bot 2000;86:471–478.
25. Yanishlieva NV, Marinova EM, Gordon MH, Reneva VG: Ac-
tivity and mechanism of action of thymol and carvacrol in two
lipid systems. Food Chem 1999;64:59–66.
26. Aristatile B, Al-Numair KS, Veeramani C: Protective effect of
carvacrol on oxidative stress and cellular DNA damage induced
by UVB irradiation in human peripheral lymphocytes. J Biochem
Mol Toxicol 2010 [Epub ahead print]; DOI: 10.1002/jbt.20355.
OREGANO’S COMPOSITION AND ANTIPROLIFERATIVE EFFECT 5