BIOLOGICA NYSSANA 1 (1-2) z December 2010: 83-88

Stojanovi-Radi, Z. et al. z Antimicrobial activity and cytotoxicity of

th

10 SFSES 17-20 June 2010, Vlasina lake

1 (1-2) December 2010: 83-88

Original Article

!
Antimicrobial activity and cytotoxicity of commercial rosemary
essential oil (Rosmarinus officinalis L.)
Zorica Stojanovi-Radi1*, Marija Nei1, Ljiljana omi2, Niko Radulovi3
1

University of Ni, Faculty of Sciences and Mathematics, Department of Biology and

Ecology, Viegradska 33, Ni, Serbia
2
Department of Biology and Ecology, Faculty of Sciences, University of Kragujevac, Radoja
Domanovia 12, Kragujevac, Serbia
3
University of Ni, Faculty of Sciences and Mathematics, Department of Chemistry, irila i
Metodija 2, Ni, Serbia
* E-mail: [email protected]
Abstract:
Stojanovi-Radi, Z., Nei, M., omi, Lj., Radulovi, N.: Antimicrobial activity and cytotoxicity of
commercial rosemary oil (Rosmarinus officinalis L.). Biologica Nyssana, 1 (1-2), December 2010: 83-88.
Rosemary is well known as a spice and widely used plant in ethnomedicine worldwide. In this paper,
commercial essential oil of rosemary was tested for antimicrobial and cytotoxic activity together with its
effect on germination. Antimicrobial activity testing showed moderate effect to both G-positive and Gnegative bacteria. In order to determine its effect to the cell membrane, spectrophotometric analysis was
performed. It was determined that rosemary affects the cell membrane of bacteria. Cytotoxic activity of
Rosmarinus officinalis essential oil had been evaluated. As a plant object, germinative bulbs of Allium cepa
were used. Cytotoxic activity that corresponded to the concentration of essential oil was determined. It had
been noticed that rosemary essential oil affected mitotic phase i.e. it significantly slowed down the mitosis.
Also, investigation of rosemary essential oil's activity to germination was performed. It was determined that
it had high effect to the germination. Concentration of 5 mg/ml completely inhibited the germination of
Triticum vulgare.
Key words: essential oil, cytotoxicity, antimicrobial activity, effect on germination

Introduction

1989). Also, it is used in ethnomedicine as general

stimulant, for improvement of circulation, treatment
of rheumatic pains, hyperglycemia and skin care
(H a m e d o , 2009). Rosemary is known to have
antioxidant and antibacterial properties (S h a h i d i
et al. 1992). P u t n a m et al. (2006) reported that
rosemary essential oil inhibit osteoclast activity and
increase bone density in vitro. Also, cytotoxic
activity of rosemary essential oil has been
demonstrated by several authors (K h a f a g i et al.
2000; E l - M e l e i g y et al. 2010).
The essential oil of rosemary contains several
compounds in high percentage and many others in
traces. These major compounds determine the
biological properties of the essential oil and can act

!
Essential oils are natural, concentrated,
volatile aromatic compounds isolated from plants.
These compounds posses a wide spectrum of
pharmacological activities. The main advantage of
natural agents is that they do not enhance the
antibiotic resistance, a phenomenon encountered
with the long term use of synthetic antibiotics.
Rosemary (Rosmarinus officinalis L.) is plant
belonging to the family Labiateae. This plant is
widely used for many purposes it is well known as
a culinary spice and frequently employed in the
practice of aromatherapy (D a v i d s o n et al.,

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BIOLOGICA NYSSANA 1 (1-2) z December 2010: 83-88

Stojanovi-Radi, Z. et al. z Antimicrobial activity and cytotoxicity of

McFarland
and
confirmed
using
a
spectrophotometer (UV-VIS 1650 Shimatzu, Japan).
Final density of inoculum was 5 x 105. Dilutions of
stock rosemary essential oil solutions were made
with MHB (Mueller Hinton Broth) and inoculums
were added to all wells. One inoculated well was
included to allow control of the broth suitability for
organism growth. One non-inoculated well, free of
antimicrobial agents, was also included to ensure
medium sterility. Bacterial growth was determined
by adding 20 L of 0.5 % triphenyl tetrazolium
chloride (TTC) aqueous solution (S a r t o r a t t o et
al., 2004). Minimal inhibitory concentration (MIC)
was defined as the lowest concentration of the oil
inhibiting visible growth (red colored pellet on the
bottom of the wells after the addition of TTC), while
minimal bactericidal concentration (MBC) was
defined as the lowest oil concentration killing 99.9
% of bacterial cells. To determine MBC, the broth
was taken from each well without visible growth
and inoculated in MHA for 24 h at 37C.
Experiments were done in triplicate and the mean
values are presented.

in synergic manner or regulate one on another

(F a i x o v a & F a i x , 2008). In the papers dealing
with chemical composition of rosemary essential
oil, -pinene is reported as the major component,
followed by 1,8 cineole, camphene, -myrcene,
camphor and borneole (J a m s h i d i et al., 2009,
M o g h t a d e r & A f z a l , 2009). Some studies
reported 1,8 cineole as main component (F u et
al., 2007, D e b e r s a c et al., 2001).
Rosemary is confirmed as antimicrobial agent
against both Gram-positive and Gram-negative
bacteria, as well as against fungi (V a l e r o &
S a l m e r o n , 2003, P r a b u s e e n i v a s a n et al.,
2006, F u et al. 2007a, M o g h t a d e r & A f z a l ,
2009).
In the present study, antimicrobial activity of
commercially available rosemary essential oil was
investigated against a panel of microorganisms for
minimal inhibitory and bactericidal activity. MIC
and MBC were determined using broth
microdilution method. The most sensitive
determined bacterial strain was used to investigate
the mode of action of this oil. In order to evaluate if
the oil exhibits membrane damage effect, testing of
the 260 nm absorbing material leakage was
employed. Also, cytotoxic activity of rosemary
essential oil was determined.

Testing the integrity of the cell membrane

Very important indication of disturbed
membrane integrity is the release of intracellular
components, such as large molecules DNA and
RNA. Since these nucleotides have strong UV
absorption at 260 nm, they are described as 260 nm
absorbing materials (L i u et al., 2009). Leakage of
260 nm absorbing material was determined by
measuring the absorbance of the culture
supernatants according to the modified method of
C a r s o n et al. (2002). Briefly, the cultures of S.
aureus were harvested and twice washed with PBS.
After resuspension in PBS, cells were adjusted to
absorbance of ~ 0.2. At time t = 0 min, essential oil
was added to cell suspensions to give a final
concentrations of MIC and 2 x MIC. Aliquots of the
treated cell suspensions were removed at regular
time intervals, centrifuged at 10 000 x g for 5 min
and absorbance of supernatant was measured at 260
nm and plotted against time. The experiments were
done in triplicate and the mean values are presented.

Material and methods

!
Essential oil, bacterial strains and culture
conditions
Essential oil of rosemary, produced by
Sinefarm, Vrac, Srbija, was purchased from the
local pharmacy.
Bacterial strains used in this study were
Staphylococcus aureus ATCC 25923, S. aureus
(clinical isolate), Escherichia coli (clinical isolate),
Bacillus subtilis ATCC 6633 and Klebsiella
pneumoniae (clinical isolate). Bacterial cultures
were maintained on the nutrient agar (NA) at the
Department
of
Biology
and
Ecology,
Microbiological Laboratory, Faculty of Science and
Mathematics, Ni.
Testing of antimicrobial activity

Allium cepa test

The antimicrobial activity was evaluated

using broth microdilution method (N C C L S ,
2003).
Minimum
inhibitory
concentrations
determination was performed by a serial dilution
method in 96 well microtitre plates. Bacterial strains
were cultivated in Mueller Hinton agar at 37 C for
18 h. Cultures were used for making bacterial
suspensions, whose turbidity was adjusted to 0.5

Cytotoxicity was determined by calculating

the mitotic index of Allium cepa L. root cells after
the 24 h treatment with rosemary essential oil. Small
onion bulbs were placed in 10 ml distilled water in
Petri dishes in an incubator at 251C for 24 h.
After initiating growth, water had been replaced two
more times (after every 24h). After 72 h, water had
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BIOLOGICA NYSSANA 1 (1-2) z December 2010: 83-88

Stojanovi-Radi, Z. et al. z Antimicrobial activity and cytotoxicity of

sensitive strain was S. aureus with both MIC and

MBC values of 1.562 l/ml, while the most resistant
one was Escherichia coli, whose bactericidal
concentration exceeded the tested values.
Determined resistance of E. coli can be attributed to
the outer cell envelope, the feature of all Gramnegative bacteria, which makes them generally more
resistant to all external agents. This is confirmed by
higher MBC values of K. pneumoniae (Gram
negative strain). F u et al. (2007a) investigated
antibacterial activity of rosemary and the results
obtained in our study showed very good agreement
with activity against E. coli, B. subtilis and S.
aureus (1.250 l/ml against all three bacteria)
determined in that paper. M o g h t a d e r &
A f z a l i (2009) reported activity of rosemary
against both Gram positive and negative strains.
Previous investigation of F u et al. (2007) reported
activity of rosemary essential oil against
Propionibacterium acnes in the concentration of
0.56 mg/ml.
In order to determine the mode of action of
rosemary essential oil against the most sensitive of
the tested bacteria, Staphylococcus aureus ATCC
25923, spectrophotometric method was employed.
Since damaged cell membrane increases its
permeability, small molecular weight molecules
such as phosphates and potassium ions first leach
out from the cell, followed by larger molecules,
such as DNA and RNA (C h e n et al, 2002). Since
these nucleotides have strong UV absorption at 260
nm, they are described as 260 nm absorbing
materials. The UV-VIS study on the release of
materials absorbing at 260 nm showed significant
leakage proportional to the oil concentration (Fig.1).
On contrary, control did not showed any releasing
of this material during 120 min period. This
indicates membrane damage related to the addition
of the rosemary essential oil.
For testing of cytotoxicity, onion meristems
(Allium cepa L.) were used as the study material.
They are considered as one of the best biological
models for the study of cytotoxicity, genotoxicity
and environmental pollutants (F i s k e s j o, 1985).
Allium cepa root meristems have been widely used

been replaced with appropriate solution (appropriate

concentration of essential oil of rosemary). Distilled
water and 5 % ethyl alcohol were used as controls.
After the growth, root tips were collected and
fixed. The fixative solution was glacial acetic acid /
absolute alcohol (1/3 v/v). The root tips were kept in
acetic-alcohol solution for 72 h. After fixation, the
root tips were washed with water and then placed
into a 70 % ethanol and stored in a refrigerator until
use.
For examination the root tips were hydrolyzed
in 1N HCl, placed on microscopic slide in 1% acetoorcein and after a few minutes squashed.
For each root tip the number of mitotic and
total meristemic cells was counted in 9 fields using
power (100 x) light microscope. Cells manifesting
different stages of mitosis i.e. interphase and
prophase (P), metaphase (M), anaphase (A) and
telophase (T) were recorded. The mitotic index (MI)
was calculated using following formula:
Mitotic Index = (P+M+A+T) / total cells
Germination test
Three different concentrations of essential oil
were used to determine whether the oil has effect to
germination of Triticum vulgare seeds. Twenty
wheat grains were placed in small Petri dishes and
in an appropriate medium. Distilled water and 5 %
ethanol were used as controls. Concentrations of
rosemary essential oil were 5 mg/ml, 12.5 mg/ml,
25 mg/ml and 50 mg/ml. After 24 h of the treatment,
seeds which germinated were counted and
percentage of germination, compared to positive
control was presented.

Results and disscusion

The results of minimal inhibitory (MIC) and
minimal bactericidal concentrations (MBC) are
presented in Table 1. The testing showed significant
antibacterial activity of rosemary with MIC values
ranging from 1.562 3.125 l/ml, while MBC
ranged from 1.562 to >25.00 l/ml. The most

Table 1. Minimal inhibitory concentrations (MIC) and minimal bactericidal concentrations (MBC) of
rosemary essential oil
Bacterial strain

strain type

S. aureus
S. aureus
E. coli
B. subtilis
K. pneumoniae

ATCC 25923
clinical isolate
clinical isolate
ATCC 6633
clinical isolate

MIC
(l/ml)
1.562
3.125
3.125
1.562
1.562

85

MBC
(l/ml)
1.562
6.250
>25.00
6.250
12.50

Tetracycline
(g/ml)
0.090
0.045
0.045
0.090
0.045

BIOLOGICA NYSSANA 1 (1-2) z December 2010: 83-88

Stojanovi-Radi, Z. et al. z Antimicrobial activity and cytotoxicity of

predictor of antitumor and pesticide activity. Data

showed that rosemary essential oil except high
cytotoxicity, might posses antitumor and pesticide
potential. It is supposed that oxygenated
monoterpenes of essential oil exhibit a variable
degree of cytotoxicity. As a typical lipophilic
substance, it passes through the cell wall and
cytoplasm membranes and disrupts their structure
(F a i x o v a & F a i x , 2008).
K i v a n c & A k g l (1988) reported that the
most active components of the essential oils were
phenols, followed by aldehydes and ketons. One of
the major compound of rosemary essential oil is 1,
8-cineole. A s a n o v a et al. (2003) demonstrated
that this component has very strong cytotoxicity and
pronounced antitumor action. It is possible that this
component has the major role in cytotoxicity of this
essential oil. 1, 8 - cineole exhibits a strong toxic
effect on eukaryotic cells (O b e n g - O f o r i et al.,
1997, S a n t o s et al., 2004).
Another potent compound is -caryophyllene.
It was demonstrated that this substance may not
have direct influence on cytotoxicity of essential oil,
but may impact accumulation of some substances by
increasing permeability of the plasma-membrane. In
that way, it affects and increases cytotoxic effect of
compounds with which it interacts (L e g a u l e &
P i c h e t t e , 2007).
According to all this facts, it is not surprising
that concentration of only 5 mg/ml of rosemary
essential oil totally inhibited germination of wheat.
This was the lowest concentration tested. Other
concentrations also totally inhibited germination of
Triticum vulgare seeds. Limonene, one of the
components of rosemary essential oil, has ability to
moderate proliferation of cells (M a n u e l e et al.,
2008), and in that manner it affects cell mitosis.

for the evaluation of cytotoxicity and anti-mitotic

activity of various compounds (S h e h a b , 1980;
W i l l i a m s & O m o h , 1996; A l - M e s h a l ,
1987). In the present paper, cytotoxicity and antimitotic activity of essential oil of R. officinalis L.
was tested using Allium cepa test. The inhibitory
effect of essential oil of rosemary on the mitotic
activity of root meristems was evaluated and the
effect was compared with distilled water and 5%
ethyl alcohol. The results are presented in Tab. 2.
MIC
2xMIC
CONTROL

1.8

apsorbance at 260 nm

1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
0

20

40

60

80

100

120

time (min)

Figure 1. Release of 260 nm absorbing material

from S. aureus ATCC 25923 cells during the
treatment with MIC and 2 x MIC concentrations of
rosemary essential oil (OD optical density)
Table 2. Mitotic index in Allium cepa (L.) meristem
following incubation with various concentrations of
essential oil of Rosmarinus officinalis (L.)
concentration
d H2O
EtOH
5.00
12.5
25.0
50.0

unit
%
mg/ml
mg/ml
mg/ml
mg/ml

MI
0.424
0.172
0.257
0.235
0.218
0.191

MI in %
42.40
17.20
25.70
23.50
21.80
19.10

Conclusion
Essential oil of rosemary was previously
reported as an important antimicrobial agent with
activity at very low concentrations. Our study
confirmed the previous results regarding MIC and
MBC values of this essential oil. For the first time, it
was determined that rosemary essential oil exhibits
activity by damaging the cell membrane of gram
positive bacteria and further tests are necessary to
explore the exact mechanism of this activity. The
determined cytotoxic and antimicrobial activity of
rosemary essential oil is of great importance in
preservation of food products and also very
important information for its pharmacological
application.

The
results
showed
that
different
concentrations of essential oil have different
percentage of activity, but all of them affect root
growth, length and number. Also, it was
demonstrated that essential oil has inhibitory effect
on proliferation of cells. Higher concentrations of
essential oil exhibit stronger inhibitory effect on
mitosis (Tab. 2). Previous studies revealed that
rosemary essential oil exhibits strong cytotoxicity
against brine shrimp nauplii (K h a f a g i et al.
2000). The test which was taken is not only used for
predicting cytotoxicity, but is also used as a

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Stojanovi-Radi, Z. et al. z Antimicrobial activity and cytotoxicity of

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