Dill (Anethum Graveolens L.) Seeds Essential Oil As A Potential Natural Antioxidant And Antimicrobial Agent

Abstract

Synthetic antioxidants and antimicrobial agents can induce many undesired side effects, which attracts interest
of food producers and consumers in finding ingredients of natural origin. The antioxidative and antimicrobial
activity of essential oil from dill (Anethum graveolens L.) seeds was investigated in terms of its possible
application as natural antioxidant and antimicrobial agent. DPPH test and FRAP method have been used for
the investigation of antioxidative activity of essential oil. Disc-diffusion method has been used for investigation
of oil antimicrobial activity on following microorganisms: Staphylococcus aureus, Listeria monocytogenes,
Bacillus subtilis, Escherichia coli, Salmonella enteritidis and Candida albicans. Essential oil, in concentration
of 29 mg/mL, incubated for 60 minutes has shown the highest degree of DPPH radicals’ neutralization
(79.62%). FRAP activity of oil was 40.63 μmol Fe2+/g of essential oil. Essential oil showed the best
antimicrobial activity on Staphylococcus aureus. Furthermore, there was a significant antimicrobial activity on
all investigated microorganisms.

Full text

BIOLOGICA NYSSANA 7 (1)  September 2016: 31-39 Stanojević, Lj.P. et al.  Dill (Anetum graveolens L.) seeds essential oil…
31
Original Article
Received: 18 January 2015
Revised: 19 February 2016
Accepted: 01 Mart 2016
Dill (Anethum graveolens L.) seeds essential oil as a potential
natural antioxidant and antimicrobial agent
Ljiljana P. Stanojević*, Mihajlo Z. Stanković, Dragan J. Cvetković, Bojana R. Danilović,
Jelena S. Stanojević
University of Niš, Faculty of Technology, Bulevar Oslobođenja 124, 16000 Leskovac, Serbia
* E-mail: stanojevic@tf.ni.ac.rs
Abstract:
Stanojević, Lj.P., Stanković, M.Z., Cvetković, D.J., Danilović, B.R., Stanojević, J.S.: Dill (Anethum
graveolens L.) seeds essential oil as a potential natural antioxidant and antimicrobial agent. Biologica
Nyssana, 7 (1), September 2016: 31-39.
Synthetic antioxidants and antimicrobial agents can induce many undesired side effects, which attracts interest
of food producers and consumers in finding ingredients of natural origin. The antioxidative and antimicrobial
activity of essential oil from dill (Anethum graveolens L.) seeds was investigated in terms of its possible
application as natural antioxidant and antimicrobial agent. DPPH test and FRAP method have been used for
the investigation of antioxidative activity of essential oil. Disc-diffusion method has been used for investigation
of oil antimicrobial activity on following microorganisms: Staphylococcus aureus, Listeria monocytogenes,
Bacillus subtilis, Escherichia coli, Salmonella enteritidis and Candida albicans. Essential oil, in concentration
of 29 mg/mL, incubated for 60 minutes has shown the highest degree of DPPH radicals’ neutralization
(79.62%). FRAP activity of oil was 40.63 μmol Fe2+/g of essential oil. Essential oil showed the best
antimicrobial activity on Staphylococcus aureus. Furthermore, there was a significant antimicrobial activity on
all investigated microorganisms.
Key words: Anethum graveolens L., dill seeds, essential oil, antioxidant activity, antimicrobial activity
Apstrakt:
Stanojević, Lj.P., Stanković, M.Z., Cvetković, D.J., Danilović, B.R., Stanojević, J.S.: Etarsko ulje semena
mirođije (Anethum graveolens L.) kao potencijalni prirodni antioksidans i antimikrobni agens. Biologica
Nyssana, 7 (1), Septembar 2016: 31-39.
Sintetski antioksidansi i antimikrobni agensi mogu dovesti do brojnih neželjenih efekata, pa je zato sve veće
interesovanje proizvođača i potrošača hrane za sastojcima prirodnog porekla. Proučavana je antioksidativna i
antimikrobna aktivnost etarskog ulja semena mirođije (Anethum graveolens L.) u cilju moguće primene kao
prirodnog antioksidansa i antimikrobnog agensa. Antioksidativna aktivnost etarskog ulja je određena
primenom DPPH-testa i FRAP metode.Antimikrobna aktivnost ulja je određena disk-difuzionom metodom, na
sledeće mikroorganizme: Staphylococus aureus, Listeria monocytogenes, Bacilus subtilis, Escherichia coli,
Salmonela enteritidis i Candida albicans. Najveći stepen neutralisanja DPPH radikala (79,62%) pokazuje ulje
inkubirano 60 minuta, u koncentraciji 29 mg/mL. FRAP vrednost etarskog ulja iznosi 40,63 μmol Fe2+/g
etarskog ulja. Ulje pokazuje najbolje antimikrobno dejstvo na Staphylococcus aureus. Takođe, postoji značajna
antimikrobna aktivnost na sve ispitivane mikroorganizme.
Key words: Anethum graveolens L., seme mirođije, etarsko ulje, antioksidativna aktivnost, antimikrobna
aktivnost
7 (1) • September 2016: 31-39
DOI: 10.5281/zenodo.159101

BIOLOGICA NYSSANA 7 (1)  September 2016: 31-39 Stanojević, Lj.P. et al.  Dill (Anetum graveolens L.) seeds essential oil…
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Introduction
There are an increasing number of scientific
investigations to find natural products that exhibit
different biological activities, and antioxidant,
antimicrobial and anti-inflammatory activities are the
most commonly studied (T ep e et al., 2004;
B a kka li et al., 2008; M i š i ć et al., 2008).
One of the most important trends in food
industry is discovery of natural antioxidants from
plant material (D el aq ui s, 2002). One of the most
efficient ways of lipid peroxidation inhibition is the
addition of synthetic antioxidants to the oils and
foods, such as ascorbyl palmitate (AP), tert-butyl-4-
hydroxyanisole (BHA), tert-butyl-4-hydroxytoluene
(BHT), propyl gallate (PG), butyl gallate (BG), octyl
gallate (OG), dodecyl gallate (DG). But, synthetic
antioxidants have some undesirable side effects
wherefore natural antioxidants are increasingly used
(M a es t ri et al., 2006).
Plants have long been used for various
infectious diseases treatment and some of these
traditional medicines are still involved in the
treatment of various diseases (M e n d o n ç a -
F i lh o , 2006). Essential oils and herbal extracts, as
sources of natural products, have become interesting
in recent decades. They represent an alternative to
synthetic antioxidants and antimicrobial agents in
food industry (T ep e et al., 2004; H in ne bu rg et
al., 2006) as well as in pharmaceutical industry,
alternative medicine and natural therapy (B u rt ,
2004; T e pe et al., 2004; M i š i ć et al., 2008).
Anethum graveolens L., commonly known as
dill, is an annual and sometimes biennial medicinal
plant from the family Apiaceae (Umbelliferae). Dill
is one of the most significant spices in food industry
(O rh an et al., 2013; L eu ng & F os te r , 2003).
Dill is native plant to Mediterranean region,
southeastern Europe and central southern Asia
(K au r & Ar or a , 2010). Dill herb and dill seeds
have been used as flavoring agent in food industry for
sauces, salads and seafood (P in o et al., 1995;
K a ur & A ro r a , 2010). The food industry often
uses essential oil instead of dill leaves and seeds
(P i no et al., 1995) due to its characteristic aroma
and flavor (Ji ro ve tz et al., 2003). It has been
reported that dill has antimicrobial,
antihyperlipidemic, diuretic, hypotensive,
antispasmodic, antiemetic, laxative effect
(K op pu l a & Ch o i, 2011; Ho ss e in n za de h et
al., 2002; T u c ak o v , 1997) and anticancer activity
(P ee ra k am et al., 2014). Bioactive components of
dill are: essential oil, fatty oil, proteins,
carbohydrates, fiber, mineral elements (potassium,
calcium, magnesium, phosphorous, sodium), vitamin
A and niacin (Ka u r & Ar o r a, 2010). Essential oil
is present in all parts of plant, but its content is the
highest in the seeds (2-5%) (L e un g & F o s t e r ,
2003). The major component in dill seeds essential
oil is carvone (20-60%) (Le un g & F o s te r , 2003;
R a dul es c u et al., 2010, D e la q u is et al., 2002).
Besides carvone, there are also present: limonene,

-
phelandrene,

-pinene,

-terpinene, apiole, dill
apiole, 1,8-cineole, dihydro carvone and p-cymene
(L eu ng & F o st er , 2003; P in o et al., 1995). In
their study, S t a n o j e v i ć et al. (2015) and
coworkers have found a high content of carvone
(about 90%) in dill seeds essential oil from the
territory of Southeast Serbia.
Since dill seeds are one of the most commonly
used spices in Serbian traditional cuisine and food
industry, and, at the same time, dill is a plant with
many medicinal properties, the aim of this study was
to investigate antioxidant activity of essential oil
from dill seeds by two antioxidant assay: DPPH and
FRAP as well as antimicrobial activity against some
intestinal pathogens.
Material and methods
Plant material
The commercial sample of non-disintegrated dill
seeds (Anethi fructus) was purchased („Planta Mell“,
Svrljig, Southeast Serbia) and used for investigations.
Chemicals and reagents
Ethanol, 96% (Centrochem, Zemun, Serbia), 2,4,6-
Tris (2-pyridyl)-1,3,5-triazine (TPTZ reagent), 1,1-
diphenyl-2-picrylhydrazyl (DPPH radical), butylated
hydroxy toluene (BHT), iron (III) chloride
hexahydrate, iron (II) sulfate heptahydrate (Sigma
Chemical Company, St. Louis, USA), dimethyl
sulfoxide (DMSO; BDH, Milan, Italy). All other
chemicals were analytical-grade.
Isolation of essential oil
Essential oil from dill seeds was isolated by classic
Clevenger-type hydrodistillation (cohobation)
according to Ph. Jug. V (2000). Dill seeds (15 g) were
immersed in 300 mL of water in round bottom flask,
and the oil was isolated using a Clevenger-type
apparatus for 3 h. The obtained essential oil was dried
over anhydrous sodium sulfate and used for analysis
(S t a n o j e vi ć et al., 2015).
Antioxidant activity
DPPH assay
Antioxidant activity of essential oil was deterimined
by the use DPPH test (Aq u in o et al., 2002; Ch oi
et al., 2002; S a nc he z-M or en o , 2002).

BIOLOGICA NYSSANA 7 (1)  September 2016: 31-39 Stanojević, Lj.P. et al.  Dill (Anetum graveolens L.) seeds essential oil…
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The essential oil was dissolved in ethanol
(96%) and a series of different concentration solutions
were prepared (0.23 to 29 mg/mL). The ethanol
solution of DPPH radical (1 mL, 3×10-4 mol/L) was
added to 2.5 mL of each essential oil solutions.
Absorbance of one sample was immediately
measured at 517 nm, while the other samples were
incubated at room temperature in the dark, for 20, 30,
45 and 60 minutes, and the absorbance was also
measured at 517 nm (AU). The absorbance at 517 nm
was measured for pure ethanol solution of DPPH
radical prepared as described above – 1 mL of the
DPPH radical (3×10-4 mol/L) diluted with 2.5 mL of
ethanol, Ak), as well as for the essential oil before
treatment with DPPH radical (2.5 mL of essential oil
diluted with 1 mL of ethanol, AB). Free radical
scavenging capacity was calculated by the following
equation (S t a n o j e v i ć et al., 2015a):
DPPH radicals scavenging capacity (%) =
 






K
BU A
AA 100
100
(1)
Essential oil concentration needed for the
neutralization of 50% of the initial DPPH radical
concentration is called EC50 value. This value was
determined by interpolation from the linear
regression analysis in the concentration range
between 0.23 and 29 mg/mL of essential oil added to
the reaction mixture. BHT was used as the reference
compound (EC50 = 0.021 mg/mL).
FRAP assay
The antioxidant activity of essential oil by FRAP
assay is determined using Benzie and Strain method
with some modifications (B e nzi e & S t r a i n ,
1996). FRAP reagent was prepared from acetate
buffer (300 mmol/L, pH = 3.6), TPTZ reagent (10
mmol/L in 40 mmol/L HCl) and FeCl3×6 H2O (20
mmol/L) in 10:1:1 ratio.
Ethanol solution of essential oil (0.1 mL,
concentration 9.25 mg/mL) and 3 mL of FRAP
reagent were added in a test tube. Absorbance was
measured at 593 nm after 30 minutes of incubation at
37 °C against blank control. The calibration curve for
FRAP values determination was obtained by
measuring the absorbance of Fe2+ (0.2 to 1 mmol/L
Fe2SO4×7H2O) standard solution, which was treated
in the same way as the essential oil samples. FRAP
value was expressed as mol Fe2+/g of the essential
oil.
Antimicrobial activity
Microorganisms and substrates. Six microorganisms
were selected to determine the antimicrobial activity:
Staphylococcus aureus (ATCC 25923), Bacillus
subtilis (ATCC 6633), Escherichia coli (ATCC
25922), Listeria monocytogenes (ATCC 19166),
Salmonella enteritidis (ATCC 13076) and Candida
albicans (ATCC 10259). Mediums used for the
growth of the microorganisms: Antibiotic agar no. 1
for microbiology (Merck, Darmstadt, Germany) for
bacteria and Sabouraud dextrose agar (Torlak,
Belgrade) for fungi. Microorganisms are from the
collection of the Microbiological laboratory of the
Faculty of Technology, Leskovac.
Disc-diffusion method. The agar disc-diffusion
method was used for testing antimicrobial activity of
dill seeds essential oil (K ie h l ba u c h et al., 2000).
The mediums were sterilized for 15 minutes in an
autoclave at 121oC. The suspension was prepared
with overnight culture and adjusted to 0.5 McFarland
standard. The inoculum of 0.1 ml of suspension was
added to 10 mL of medium and poured into the Petri
dishes.
For screening, sterilized filter paper disks
(12.7 mm dia., Schleicher & Schuell) were placed on
the surface of inoculated mediums and impregnated
with 60 l of essential oil. Plates were incubated for
24 hours at 37 °C for bacteria, and 48 hours at 25 °C
for yeast. Antimicrobial activity was expressed as the
diameter of inhibition zones (mm) obtained by
investigated sample.
Standardized discs of Amoxicillin (30 μg/disc,
Hemofarm, A.D. Vršac), Cephalexin (30 μg/disc,
Panfarma, Beograd), Amracin (30 μg/disc, Galenika,
A.D. Zemun) and Nystatin (100 U/disc, Bioanalyse)
served as positive controls.
All experiments were carried out in three
replications. Data were expressed as mean ± standard
deviation. The obtained data were analyzed by
Microsoft Excel 2007 and Origin 7 trial.
Results and discussion
Essential oil composition
The moisture content and initial oil content in dill
seeds were 7.33% and 4.0 mL/100 g of dry plant
material, respectively. The yield of essential oil was
2.80 mL/100 g dry plant material (S t a n o j e v i ć et
al., 2015).
In the essential oil twenty nine components
have been identified (99.9% of all components).
These results are presented in our previous studies
where the influence of the technique on the yield,
composition and kinetics of essential oil
hydrodistillation from dill seeds have been
investigated (S t a n o j e vi ć et al., 2015).
It has been found by GC-MS analysis of
essential oil that carvone has the highest content
(85.9%). Carvone content in dill essential oil is
usually 20 to 60% (L e un g & F os t e r, 2003; de

BIOLOGICA NYSSANA 7 (1)  September 2016: 31-39 Stanojević, Lj.P. et al.  Dill (Anetum graveolens L.) seeds essential oil…
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C a rv a lh o et al., 2006). Besides carvone, limonene
(5.1%), cis-dihydrocarvone (3.0%), trans-
dihydrocarvone (2.7%), cis-carveol (1.8%) and
trans-carveol (1.4%), all other components of oil
were identified in much lower concentrations
(S t a n o j e v i ć et al., 2015).
The higher content of carvone in oil from
Serbia compared to its content in oils from other areas
(Bulgaria, Canada, India and Romania) (De la qu is
et al., 2002; J ir ov et z et al., 2003; S in gh et al.,
2005) is probably due to different climatic
conditions, as well as genetic characteristics of seeds
(S t a n o j e vi ć et al., 2015).
Antioxidant activity
Antioxidant activity of essential oil
investigated by DPPH test (ability of oil in different
concentrations to scavenge free DPPH radical) is
shown on Fig. 1 (results for not-incubated and 20, 30,
45 and 60 min incubated samples are represented).
Fig. 1. DPPH radicals scavenging activity by dill
(Anethum graveolens L.) seeds essential oil
It can be noticed that the degree of DPPH
neutralization depends on incubation time, for all
investigated concentrations of oil. The highest degree
of DPPH radicals’ neutralization is for 60 minutes
incubation, in concentration of 29 mg/mL (79.62%).
EC50 values of essential oil are shown in Tab.
1. Non-incubated samples of essential oil have not
achieved EC50 value in the investigated range of
concentrations. The tested synthetic antioxidant has
showed better antioxidant activity compared to the
essential oil. EC50 values of essential oils are lower
than the EC50 values of synthetic antioxidant, BHT.
These synthetic antioxidants are used in food
industry despite their documented undesirable side
effects (T ep e et al., 2005). Based on these results
(Fig. 1 and Tab. 2) it can be concluded that the
incubation time has effect on DPPH radicals’
neutralization. Dill seeds essential oil from Thailand
has shown lower extent of DPPH neutralization (EC50
= 128.49 mg/mL) than oil obtained in our investigation
(N an as o mb at & Wi mu tt i g os ol , 2011).
Table 1. EC50 values of dill (Anethum graveolens
L.) seeds essential oil
Incubation time,
min
EC50, mg/mL*
20
20.27 ± 0.811
30
18.20 ± 1.019
45
13.45 ± 0.807
60
12.20 ± 0.464
*All data represent the mean of tree replications ± standard
deviation (Mean  SD).
Removal of free radicals is very important in
food and food products preservation (H in ne bu rg
et al., 2006; T ep e et al., 2004). Essential oil of dill
seeds can be an alternative to dill as spice in the form
of powdered plant (whole or some parts). Oil is also
a potential source of natural antioxidants, as a
possible alternative to synthetic antioxidants. But, for
this potential application of dill oil it is necessary to
perform in vivo tests, which will be the aim of our
further investigation.
Essential oil of dill seeds has lower DPPH
neutralization activity compared to acetone extracts
of dill seeds (S in gh et al., 2005). Better activity of
extract compared to unstable oil is probably due to
presence of nonvolatile phenol compounds. In
addition, some of the compounds with a different
polarity, which are present in very small amounts in
the extract, are also able to contribute to better
antioxidative activity of extract. Some compounds
can originate in extract during hydrolysis or other
processes of decomposition. Some chemical
reactions initiated by heating can also drive up to
activities changes of complex extract, composed of a
number of compounds with different chemical and
physical properties (Si ng h et al., 2005).
S i nt i m et al. (2015) reported on the
significant effect of the antioxidant capacity of the
dill seed essential oil using the oxygen radical
absorbance capacity (ORAC) method.
In addition, it was found that dill essential oil
from the aerial parts of the plant had antioxidant
activity. Kazemi reported that dill oil exhibited a high
activity in each antioxidant system with a special
attention for β-carotene bleaching test and reducing
power (Kaze mi , 2015).
FRAP value, as a measure of essential oil
antioxidant activity, was 40.63 μmol Fe2+/g of
essential oil. L a do et al. (2004) determined the
antioxidant properties of commercially purchased
essential oils using the FRAP assay (cumin,

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coriander, dill, chamomile, hyssop, lavender, parsley,
rosemary, sage and yarrow). FRAP value of the
investigated dill essential oil was 42.64 μmol/g. The
essential oil, investigated in our work, was incubated
for 30 min at 37 C with FRAP reagent, while La d o
et al. (2004) have been incubated the oil only for 5
minutes. Due to the long incubation time our oil
should express greater FRAP value. However, lower
FRAP value of our essential oil is probably the result
of different chemical composition compared to the
oil used by Lado and coworkers. Different origin of
plant material can be also the reason of such
differences in obtained results. Tab. 2 shows the
FRAP values of dill essential oil as well as literature
FRAP values of some volatile components of oil
(carvone, limonene and linalool).
Table 2. FRAP value of dill (Anethum graveolens L.)
seeds essential oil as well as literature FRAP
values of some volatile oil components
Essential oil or
volatile component
FRAP value,
μmol Fe2+/g essential oil
Dill seeds essential oil
40.63 ± 3.23*
Dill seeds essential oila
42.64a
Carvonea
15.77a
Linaloola
25.73a
Limonenea
37.44a
aL ad o et al. (2004)
*All data represent the mean of tree replications ±
standard deviation (Mean  SD).
The dill essential oil has the highest FRAP
value (Tab. 2), but the individual components
identified in the essential oil also have the reductive
capacities (La do et al., 2004). FRAP value obtained
in our study is most likely the result of reducing
ability of components present in the oil, especially
carvone and limonene, which are present in the
highest content. Such activity of oil is due to the
presence of minor component too, such as
dihydrocarvone, cis- and trans-carveol and linalool.
So, FRAP value of oil is a result of synergistic effect
of all present components since reducing ability of oil
is higher than reducing ability of individual
components (oil shows the highest FRAP activity). A
significant number of natural products investigations
suggest that essential oils have antioxidant activity. It
is believed that antioxidants are directly responsible
for antimutagenic and anticarcinogenic activity due
to their radical scavenging properties. Essential oil
with antioxidant activity could be beneficial for
human health (Ba kk al i et al., 2008).
Antimicrobial activity
Antimicrobial activity of dill essential oil as well as
activity of reference antibiotics are shown in Tab. 3.
Essential oil of dill has effect on all tested
microorganisms. The highest effect was observed on
S. aureus. Tested antibiotics showed much less
activity on this bacterium. It has also a significant
effect on B. subtilis and E. coli.
Staphylococcus aureus is a gram-positive
bacterium that is, among many other harmful effects
to humans, one of the most frequent mastitis agents
in herds of dairy cows (S i ng & P r ak a sh , 2008;
M i la n ov et al., 2010). Milk contaminated with S.
aureus, as well as products from such milk can cause
a variety of infections, by bacteria itself and by their
enterotoxins (S a m a r ž i j a et al., 2007). The
relatively high effect of dill essential oil upon these
bacteria suggests the potential use of oil as a natural
antimicrobial agent in milk products. However, in
order to use the obtained oil in such way, detailed
studies are necessary to establish its minimum
inhibitory concentration as well as its acute toxicity
in particular concentration which would be the goals
of our further studies.
It is significant that investigated essential oil
has a higher effect on B. subtilis compared to widely
used Cephalexin and Amoxicillin antibiotics.
Bacillus species most likely cause alimentary toxic
infections in humans. Toxic infections are
consequence of various food products consumption,
in which starch and proteins are dominating, such as
rice, meat and meat products, desserts, and other
canned food. They are very often present as
contaminants in food of animal and vegetable origin
since they can survive various physical and chemical
conditions because of resistant spores. In addition to
alimentary infection, Bacillus causes a number of
other diseases: septic meningitis, cellulitis, gangrene,
and many eye infections (K o ti r on ta et al., 2000).
Based on this results it can be concluded that dill
seeds essential oil can be used for natural
antimicrobial formulation production.
Essential oil from dill seeds shows higher
antimicrobial activity on E. coli (zone diameter 38
mm) compared to Cephalexin and Amoxicillin
antibiotics. E. coli is considered as a dominant
bacterium species in the digestive tract. The presence
of this bacterium in water and food is a reliable
indicator of fecal contamination. This bacterium
commonly contaminates meat and dairy products, as
well as fruit and vegetables (M ar ko v et al., 2009).
Presence of enteropathogenic E. coli in the food
products can cause vomiting and diarrhea in infants
and young children (S i ng h & P ra ka sh , 2008).

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The good antimicrobial activity of essential oil
against E. coli probably mainly originates from
carvone, which is present in the amount of 86% in the
oil. This result is in accordance with studies of Naigre
and coworkers who determined the effect of carvone
on Enterococcus faecium, Escherichia coli and
Aspergillus niger (Na ig re et al., 1996).
Salmonella are gram-negative bacteria from
Enterobacteriaceae family and they are the most
common causes of food poisoning. Salmonella are
natural inhabitants of animals’ gastrointestinal tract;
they are widespread in soil, water and plants. All
representatives of Salmonella genus are potential
human pathogens. They cause three types of disease
in humans - enteric fever, sepsis and gastroenteritis.
About 95% of Salmonella are ingested through food
and the most common sources of infection are milk
and dairy products, eggs, meat and meat products
(C ox , 2000; M ar ko v et al., 2009). There are
scientific papers on antimicrobial activity of
commercial dill seeds essential oil on Salmonella
typhimurium (D el a qu i s et al., 2002). There is no
data about the effect of dill seeds essential oil from
the Southeast Serbia region on Salmonella species.
The investigated dill seeds oil had a significant effect
against S. enteritidis which is in the range with the
effect of the commercial antibiotics.
L. monocytogenes is a pathogenic bacterium
that leads to listeriosis disease after consumption of
food. This is a particularly dangerous pathogen since
it can survive at low temperatures (foods that are kept
in the fridge). Listeriosis is one of the most common
diseases with a fatal outcome (30%) (M ar ko v et
al., 2009). There are studies of essential oils effects
on L. monocytogenes (De l aq u is et al., 2002). Dill
seeds essential oil has less effect on bacteria
compared to fractions of oils that are rich in carvone
and limonene (De la qu is et al., 2002). Carvone, as
a major component of the oil isolated in our study
exhibited antimicrobial activity against L.
monocytogenes (de C ar v a lh o et al., 2006), so it
is probably the most responsible for the effect of oil
on these bacteria.
Isolated essential oil showed antifungal
activity against C. albicans, which is, again, most
probably due to the high content of carvone in the oil.
Carvone and limonene are the main components of
caraway essential oil which showed strong antifungal
activity against C. albicans. Limonene is a carvone
precursor and it is mainly present in Mentha species
being toxic to most microorganisms. Carvone is
widely used in the manufacture of aromas and
fragrances and it has antifungal activity (Pi na et al.,
2012).
Commercial dill seeds essential oil, with
content of limonene and carvone of 46.3% and 49.5%
respectively, shows antimicrobial activity against
Pseudomonas fragi, Escherichia coli, Salmonella
typhimurium, Listeria monocytogenes,
Staphylococcus aureus and Saccharomyces
cerevisiae. The fractions of essential oils with high
content of limonene (more than 90%), and fractions
with high content of carvone (67-99%) have shown
better activity against gram-positive and gram-
negative bacteria. Thereby, the fractions rich in
carvone have showed weaker activity in some extents
compared to fractions rich in limonene (De l aq u i s
et al., 2002).
Essential oils are complex mixtures and their
biological properties are result of synergistic effects
of all components or major compounds. In most
cases, biological activity of main components only,
like thymol, carvacrol, linalool, terpineol, eugenol,
carvone, geraniol, citronellol, nerol, safrole,
eucalyptol, limonene, cinnamaldehyde, were
analyzed. Generally, the main components of
Table 3. Antimicrobial activity of dill (Anethum graveolens L.) seeds essential oil
Microorganism
Essentail
oil
Antibiotic
Cephalexin
Amracin
Amoxicillin
Nystatin
Inhibition zone diameter, mm
Escherichia coli
38.0 ± 1.52
26.0 ± 1.04
n.t.
28.0 ± 0.84
n.t.
Staphylococcus
aureus
80.0 ± 2.16
26.0 ± 0.78
n.t.
27.0 ± 0.97
n.t.
Salmonela
enteritidis
28.0 ± 0.97
30.0 ± 1.08
25.0
31.0 ± 0.90
n.t.
Listeria
monocytogenes
20.0 ± 0.83
34.0 ± 0.88
n.t.
36.0 ± 0.72
n.t.
Bacillus subtilis
52.0 ± 1.38
48.0 ± 0.96
52.0 ± 0.99
37.0 ± 0.59
n.t.
Candida albicans
18.0 ± 0.57
n.t.
n.t.
n.t.
17.0 ± 0.32
n.t.-not treated; All data represent the mean of tree replications ± standard deviation (Mean  SD).

BIOLOGICA NYSSANA 7 (1)  September 2016: 31-39 Stanojević, Lj.P. et al.  Dill (Anetum graveolens L.) seeds essential oil…
37
essential oils are the ones on which biophysical and
biological properties of oils depend (B ak ka li et
al., 2008).
Antimicrobial activity of dill seeds essential
oil in our study probably originates from carvone,
having in mind literature data about antimicrobial
effect of carvone on a large number of bacteria and
fungi (S in gh et al., 2005). There are also data of
limonene antimicrobial activity, a component that is
represented with about 5% in isolated oil in this study
(D el aq u is et al., 2002; Si ng h et al., 2005). Other
components of the oil probably also contribute to this
activity. The high content of carvone, monotherpenic
ketone, with numerous biological properties,
indicates the possible application of dill seeds
essential oil for medical purposes beside food
industry, as a bioactive product of natural origin.
Antimicrobial activity of dill seeds essential oil is
significant for human and animal pathogens as well
as for food protection (Ba k ka l i et al., 2008).
Conclusion
Essential oils are the source of natural products
with different pharmacological activities. These oils
represent the complex mixture of number
components what is the reason for difficult
explanation of their pharmacological activities. The
presented data on antimicrobial and antioxidant
activities of dill seeds essential oil showed that the
isolated oil (Southeast Serbia, Svrljig), with a high
content of carvone is a potential source of natural
antioxidants and antimicrobial agents. The results
indicate possible application of essential oils in food
and pharmaceutical industry as a safer alternative to
synthetic antioxidants and antimicrobial agents.
Bearing in mind the results obtained in this work, dill
seeds essential oil should be considered for further
investigation with practical applications in different
food and pharmaceutical systems.
Acknowledgements. This research is a part of Project TR-
34012 which is supported by Ministry of Education,
Science and Technological Development of the Republic
of Serbia.
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