Antioxidant activity of oregano essential oil (Origanum vulgare L.)


BIOLOGICA NYSSANA 7 (2)  December 2016: 131-139  Stanojević, Lj.P. et al.  Antioxidant activity of oregano essential oil… 

  

 131 

 

 

 

Original Article 

Received: 30 June 2016 

Revised: 31 October 2016 

Accepted: 24 November 2016  

 

Antioxidant activity of oregano essential oil (Origanum vulgare L.) 

Ljiljana P. Stanojević*, Jelena S. Stanojević, Dragan J. Cvetković, Dušica P. Ilić 

Faculty of Technology, University of Niš, Bulevar Oslobodenja 124, 16000 Leskovac, Serbia  

* E-mail: stanojevic@tf.ni.ac.rs 
 

 

Abstract:  

Stanojević, Lj.P., Stanojević, J.S., Cvetković, D.J., Ilić, D.P.: Antioxidant activity of oregano essential oil 

(Origanum vulgare L.). Biologica Nyssana, 7 (2), December 2016: 131-139. 

Essential oil obtained from oregano (Origanum vulgare L.) by Clevenger-type hydrodistillation and 

hydromodulus 1:10 m/v during 180 minutes, has been investigated in this work. Qualitative and quantitative 

composition of the oil was determined by GC-MS and GC-FID spectrometry. Antioxidant activity of the 

obtained oil was examined spectrophotometrically by DPPH test (after 20, 30, 45 and 60 minutes of incubation) 

and TBA-MDA assay. The yield of essential oil was 4.1 mL/100 g of plant material. Seven components were 

identified: α-thujene, myrcene, α-terpinene, o-cymene, γ-terpinene, thymol and carvacrol. The major 

components were thymol (45%) and carvacrol (37.4%). Oil incubated for 60 minutes has shown the best 

antioxidant activity according to DPPH test. The concentrations of essential oil, required for neutralization of 

50% of initial DPPH radical concentration (EC50), were 0.761, 0.590, 0.360 and 0.326 mg/mL, after 20, 30, 45 

and 60 minutes of incubation, respectively. Lipid peroxidation inhibition of 92.3% was achieved by 1.35 

mg/mL essential oil concentration. The results obtained indicate that oregano essential oil is a good source of 

natural antioxidants with potential application in food and pharmaceutical industries, as a safer alternative to 

the synthetic antioxidants. 

Key words: Origanum vulgare L., essential oil, antioxidant activity, GC-MS analysis 

 

Apstrakt: 

Stanojević, Lj.P., Stanojević, J.S., Cvetković, D.J., Ilić, D.P.: Antioksidativna aktivnost etarskog ulja 

vranilove trave (Origanum vulgare L.). Biologica Nyssana, 7 (2), December 2016: 131-139. 

U ovom radu je ispitivano etarsko ulje vranilove trave (Origanum vulgare L.) dobijeno Clevenger 

hidrodestilacijom pri hidromodulu 1:10 m/v, u toku 180 minuta. Kvalitativni i kvantitativni sastav dobijenog 

etarskog ulja određen je GC-MS i GC-FID spektrometrijom. Antioksidativna aktivnost ulja je određena 

spektrofotometrijski, DPPH testom (posle 20,30, 45 i 60 minuta inkubacije) i TBA-MDA testom. Prinos 

etarskog ulja bio je 4.1 mL/100 g biljnog materijala. Identifikovano je sedam komponenti: α-tujen, mircen, α-

terpinen, o-cimen, γ-terpinen, timol i karvakrol. Kao glavne komponente identifikovane su timol (45%) i 

karvakrol (37,4%). Najbolju DPPH-antioksidativnu aktivnost pokazalo je ulje nakon 60 min inkubacije. 

Koncentracije etarskog ulja neophodne za neutralizaciju 50% od početne koncentracije DPPH radikala (EC50 

vrednosti) bile su 0,761; 0,590; 0,360 i 0,326 mg/mL, nakon 20, 30, 45 i 60 min inkubacije, respektivno. 

Inhibicija lipidne peroksidacije od 92,3% je postignuta sa 1,35 mg/mL eterskog ulja. Dobijeni rezultati sugerišu 

da dobijeno etarsko ulje vranilove trave predstavlja dobar izvor prirodnih antioksidanasa sa potencijalnom 

primenom u prehrambenoj i farmaceutskoj industriji kao bezbednija alternartiva sintetskim antioksidansima. 

Ključne reči: Origanum vulgare L., etarsko ulje, antioksidativna aktivnost, GC-MS analiza 
  

7 (2) • December 2016: 131-139 

 

12th SFSES • 16-19 June 2016, Kopaonik Mt 

 

DOI: 10.5281/zenodo.200410 



BIOLOGICA NYSSANA 7 (2)  December 2016: 131-139  Stanojević, Lj.P. et al.  Antioxidant activity of oregano essential oil… 

 

 132 

Introduction 

The oxidation process is one of the major causes of 

food spoilage, which results in rancidity and 

deterioration of the nutritional quality, color, flavor, 

texture, and safety of foods (B e t a i e b  et al., 2010). 

A significant number of herbs is used as natural 

preservatives in food industry. Besides being used to 

achieve the proper flavor and to intensify the flavors 

some spices and herbs exhibit antioxidant effects 

which is of great importance for food industry 

(S t a n k o v i ć  & S t a n o j e v i ć , 2014; 

T o n g n u a n c h a n  et al., 2014). 

Plants rich in antioxidant compounds are 

present in food industry (B e t a i e b  et al., 2010). 

There are evident requirements for increasing 

application of natural antioxidants obtained from 

plant material. Undesirable side effects of synthetic 

antioxidants, such as butyl hydroxy anisole (BHA) 

and butylated hydroxytoluene (BHT), which are 

associated with their toxic and carcinogenic effects 

(M a e s t r i  et al., 2006), are the reason of such 

requirements.  

Essential oils are very heterogeneous group of 

complex mixtures of secondary plant metabolites. 

Composition of essential oil may be different 

between different species or varieties, related to 

different cultivation, origin, vegetative stage and 

growing seasons of the plant (V a z i r i a n  et al., 

2015). Beside aroma, odor and fragrance of many of 

the oils, some of them have also been confirmed to 

possess antioxidant activities (B e t a i e b  et al., 2010; 

V a z i r i a n  et al., 2015). Essential oils are also 

widely used in perfumes, cosmetics, aromatherapy 

and nutrition (B u r t , 2004; B a k k a l i  et al., 2008).  

Earlier studies have been shown that, among 

the herbs and spices which are extensively studied, 

the plants from the Lamiaceae (Labiatae) family 

possess a significant antioxidant activity 

(T s i m i d o u  & B o s k o u , 1994; L a g o u r i  et al., 

1993). Within this family oregano (Origanum 

vulgare L.) is probably one of most widely used 

aromatic plant which essential oils are particularly 

rich in mono- and sesquiterpenes (D e  F a l c o  et al., 

2013).  

The genus Origanum includes around 38 

species, most of which are indigenous to the 

Mediterranean, Euro-Siberian and Irano-Siberian 

regions (S a h i n  et al., 2004). Origanum vulgare L. 

is one of the most widely among all the species within 

the genus which distributed all over the Europe, West 

and Central Asia up to Taiwan (S a h i n  et al., 2004; 

R a d u š i e n e  et al, 2008). It is one of the most 

important culinary herbs in the world. Leaves and 

flowers of oregano are traditionally used to cure 

cough and sore throats and for relieve of 

gastrointestinal disorders. Origanum vulgare is a 

source of essential oils and phenolic metabolites 

(R a d u š i e n e  et al, 2008).  

The main bioactive components of oregano are 

phenolic components (S e g e i t - K u j a w a  et al., 

1990; K u l e v a n o v a  et al., 2001; L e u n g  & 

F o s t e r , 2003; R a d u š i e n ė  et al., 2008) and 

essential oil (L e u n g  & F o s t e r , 2003). Origanum 

vulgare contains 0.1-1.0% of essential oil composed 

of thymol, carvacrol, -bisabolene, caryophyllene, p-
cimene, borneol, linalool, linalyl acetate, geranyl 

acetate, -pinene, -pinene, -terpinene, with highly 
variable relative proportion, depending on source 

(L e u n g  & F o s t e r , 2003).  

Oregano essential oils have been shown to 

possess antioxidant, antibacterial, antifungal, 

diaphoretic, carminative, antispasmodic and 

analgesic activities (D e  F a l c o  et al., 2013). 

Thymol and carvacrol, usually the major phenols 

present in oregano, have strong fungicidal, 

anthelmintic, irritant, and other properties (L e u n g  

& F o s t e r , 2003). 

Based on numerous studies it was established 

that oregano essential oil, rich in thymol and 

carvacrol, has a significant antioxidant activity in the 

process of the lard oxidation (L a g o u r i  et al., 1993; 

T s i m i d o u  & B o s k o u ,1994). Y a n i s h l i e v a  

and M a r i n o v a  (1995) examined the antioxidant 

activity of hexane extracts from oregano grown in 

Bulgaria, and the mechanism of action of pure 

thymol and carvacrol (Y a n i s h l i e v a  et al.,1999), 

while Kulišić and coworker presented different 

methods for antioxidant activity of oregano essential 

oil testing (K u l i š i ć  et al., 2004). 

The aim of the present study was to examine 

the antioxidant properties of oregano essential oil, 

originated from Serbia, by using two different 

methods, namely, DPPH test, and TBA-MDA assay.  

 

Material and methods 

Plant material 

The commercial sample of aerial parts of Origanum 

vulgare L. (Origani herba) was purchased from the 

local health food store in Leskovac, Serbia. 

According to the declaration, the material used for 

investigations originates from Serbia (packed by: 

MALINA-IMPEX d.o.o. Popučke b.b., Valjevo) 

Chemicals and reagent 

Ethanol, 96% (Centrochem, Zemun, Serbia), 1,1-

diphenyl-2-picrylhydrazyl (DPPH radical), butylated 

hydroxy toluene (BHT), thiobarbituric acid (TBA), 

2,2'-azobis (2-methylpropionamidine) dihydro-

chloride (AAPH) (Sigma Chemical Company, St. 

Louis, USA), trichloroacetic acid (TCA) (J.T. Baker, 



BIOLOGICA NYSSANA 7 (2)  December 2016: 131-139  Stanojević, Lj.P. et al.  Antioxidant activity of oregano essential oil… 

  

 133 

VA Deventer, Netherlands). Phospholipids 

(Phospholipon® 90; PL90) were a gift from 

Phospholipid GmbH (Cologne, Germany). 

According to the declaration PL90 mixture consisting 

of phosphatidylcholine 94.6%, lyso-phosphatidyl-

choline 1.3%; fatty acid: palmitic acid 12 ± 2%, 

stearic acid 3 ± 1%, oleic acid 10 ± 3%, linoleic acid 

66 ± 5%, linoleic acid 5% ± 2; peroxide number 1.4. 

Isolation of essential oil 

Essential oil from aerial parts of O. vulgare was 

isolated by classic Clevenger-type hydrodistillation 

according to Ph. Jug. V (2000). Plant material (50 g) 

was immersed in 500 mL of water in round bottom 

flask, and the oil was isolated using a Clevenger-type 

apparatus for 180 min. The resulting essential oil was 

dried over anhydrous sodium sulfate, filtered and 

stored at +4 °C in a well-filled, airtight container, 

protected from light, until the analysis. 

GC-MS and GC-FID analysis 

GC-MS analysis of the oregano essential oil was 

performed on Agilent Technologies 7890B gas 

chromatograph, equipped with weakly polar, silica 

capillary column, HP-5MS (5% diphenyl- and 95% 

dimethyl-polysiloxane, 30 m x 0.25 mm, 0.25 μm 

film thickness; Agilent Technologies, USA) and 

coupled with inert, selective 5977A mass detector of 

the same company. One μl of the sample dissolved in 

diethyl ether in the concentration of 1000 ppm was 

injected in 20:1 split mode. Helium was used as the 

carrier gas, at a constant flow rate of 1 mL/min. The 

oven temperature was programmed from 50 °C for 

2.25 minutes and then increased to 290 °C at the rate 

of 4 °C/min. Temperatures of the MSD transfer line, 

ion source and quadruple mass analyzer were set at 

300 °C, 230 °C and 150 °C, respectively. The 

ionization voltage was 70 eV and mass range m/z 35-

650.  

GC-FID analysis was carried out under 

identical experimental conditions as GC-MS. The 

temperature of the flame-ionization detector (FID) 

was set at 300°C. Data processing was performed 

using MSD ChemStation, MassHunter Qualitative 

Analysis and AMDIS 32 softwares (Agilent 

Technologies, USA). Retention indices of the 

components from the analyzed samples were 

experimentally determined using a homologous 

series of n-alkanes from C8-C20 as standards. 

Compounds identification was based on the 

comparison of their retention indices (RIexp – Tab. 1) 

with those available in literature (Adams, 2007) (RIlit 

– Tab. 1), as well as their mass spectra with those 

from Willey, NIST and RTLPEST libraries. The 

percentage composition of particular components in 

the essential oil was determined on the basis of 

automatically integrated peak areas of the GC-FID 

signal. 

Antioxidant activity  

DPPH assay 

Antioxidant activity of oregano essential oil was 

determined by DPPH test (A q u i n o  et al., 2002; 

C h o i  et al., 2002; S a n c h e z - M o r e n o , 2002). 

The essential oil was dissolved in ethanol (96%) and 

a series of different concentration solutions were 

prepared (0.098 to 12.5 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 (AS). 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, AC), 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 eq. 1 

(S t a n o j e v i ć  et al., 2015): 

DPPH rsc (%) =   









C

BS
A

AA
100

100  ......... (1) 

rsc- radicals scavenging capacity 

EC50 value was defined as essential oil concentration 

needed for the neutralization of 50% of the initial 

DPPH radical concentration. This value was 

determined by interpolation from the linear 

regression analysis in the concentration range 

between 0.098 and 0.391 mg/mL of essential oil 

added to the reaction mixture. BHT was used as the 

reference compound. 

TBA-MDA assay  

Thiobarbituric acid – malondialdehyde (TBA-MDA) 

test is one of the most commonly used tests for lipid 

peroxidation process monitoring in vitro. It’s based 

on heating of sample with TBA in acidic 

environment, i.e. on TBA and MDA reaction. MDA 

is end-product of lipid peroxidation formed during 

hydroperoxide degradation which build pink 

chromogen ([TBA]2-malondialdehyde adduct) with 

absorption maximum at 532 nm. Colored complex is 

formed by condensation of 2 moles of TBA and 1 

mole of MDA only from the fatty acid chains that 

contain at least three double bonds (H a l l i w e l l  & 

C h i r i c o , 1993; L a g u e r r e  et al., 2007). The 

antioxidant activity of oregano essential oil was 

determined by the method developed for caroteinoid, 



BIOLOGICA NYSSANA 7 (2)  December 2016: 131-139  Stanojević, Lj.P. et al.  Antioxidant activity of oregano essential oil… 

 

 134 

flavonoids, extracts and some potential antioxidants 

(C v e t k o v i c  & M a r k o v i c , 2008; 

C v e t k o v i c  et al., 2011; Z v e z d a n o v i ć  et al., 

2014; S t a n o j e v i ć  et al., 2015a), with some 

modifications. "Sample" contains 0.3 cm3 of PL90 

methanolic solution (1·10-2 mol/L), and essential oil 

ethanolic solution (0.042-5.375 mg/mL) in 2:1 (v/v) 

ratio. Lipid peroxidation was initiated by addition of 

0.2 cm3 of aqueous solution of thermal azo-initiator 

AAPH (2.2·10-2 mol/L) during 3 h at a temperature 

of 40 °C, protected from light. After incubation, 1 mL 

of aqueous solution of TCA (5.5%), 0.5 mL of 

methanolic solution of BHT (1∙10-3 mol/L) and 0.5 

mL of TBA (4.2∙10-2 mol/L in 5∙10-2 mol/L NaOH) 

were added to the reaction mixture. The mixture was 

incubated for 10 min at 65 °C and then centrifuged 

for 5 min at 13800 rpm. The increase in absorbance 

of the supernatant at 532 nm represents an absorption 

maximum of generated TBA-MDA complex. 

The absorbance of PL90 solution, where lipid 

peroxidation is initiated with AAPH, treated with 

TBA (”control“), as well as of PL90 solution, without 

lipid peroxidation initiation, but also treated with 

TBA (”blank“). Lipid peroxidation inhibition was 

calculated by the eq. 2 (Stanojević et al., 2015a): 

 
100

BC

SC

 - AA

) - A(A
 LPI (%)  ............................. (2) 

LPI- inhibitionxidationLipid pero  

where AC – represents the absorbance of ”control“; AS 

– absorbance of ”sample“; AB – absorbance of 

”blank“. BHT (0.0125-0.05 mg/mL) was used as the 

reference compound. 

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 

Hydrodistillation kinetics and oregano essential 

oil composition 

Figure 1 shows the influence of hydrodistillation 

time on the essential oil yield. Maximal essential oil 

yield of 4.1 mL/100g of plant material was achieved 

after 180 minutes. The hydrodistillation curve shows 

that there are two different periods of 

hydrodistillation (Fig. 1). The essential oil was 

evaporated out from the surface from the cells of 

plant material in the first period (the fast oil 

hydrodistillation). In the second, slow oil 

hydrodistillation period, a slow molecular diffusion 

of the essential oil from internal part from cells of 

plants material occurred.  

 

Fig. 1. Hydrodistillation kinetics of oregano 

essential oil 

In the Tab. 1 and on Fig. 2 the results of GC–

MS analysis of oregano essential oil are presented. 

Seven components were identified in essential oil: α-

thujene, myrcene, α-terpinene, o-cymene, γ-

terpinene, thymol and carvacrol. The major 

components were thymol (45%) and carvacrol 

(37.4%).  

The content of thymol and carvacrol in the 

oregano essential oil from various regions was 

different: 35% and 32% of thymol and carvacrol, 

respectively, from Dalmacia (K u l i š i ć  et al., 2004), 

0.8 and 0.6% respectively, from Turkey (S a h i n  et 

al., 2004), 1.1 and 14.3% respectively, from Southern 

Italy (D e  F a l c o  et al., 2013), 37.1 and 9.6 

respectively, from Iran (V a z i r i a n  et al., 2015), 

31.8 and 0.2%, respectively from Portugal (G a l e g o  

et al, 2008). Other investigators reported a lower 

content of carvacrol in oregano essential oil (less than 

35%) compared to the oregano from Serbia.  

These changes of thymol and carvacrol content 

in the oregano essential oil, and changes in the 

compositions of the oil might arise from several 

environmental reasons (climatic, seasonal, 

geographical), used drying method of plant material 

and genetic differences of the plant material (F a b e r  

et al., 1997; C a l l a n  et al., 2007; G h a s s e m i -

G o l e z a n i  et al., 2008; F i g i e l  et al, 2010; 

S t a n o j e v i ć  et al., 2011; D e  F a l c o  et al, 2013). 

Significantly higher content of this two 

bioactive components with an array of different 

biological activities is especially important (U l t e e  

et al., 2002; N o s t r o  et al., 2007; W a n g  et al., 

2009; M a t h e l a  et al., 2010; M e h d i  et al., 2011). 

The major components are believed to be mainly 

responsible for the biological activity of this 

B a k k a l i , 2008). Based on the results obtained in 



BIOLOGICA NYSSANA 7 (2)  December 2016: 131-139  Stanojević, Lj.P. et al.  Antioxidant activity of oregano essential oil… 

  

 135 

particular essential oil (B a i l e r  et al., 2001; our 

investigation, the oregano essential oil from Serbia 

could be a potential natural source of thymol and 

carvacrol. 

Antioxidant activity 

DPPH test is most commonly in vitro method for 

antioxidant activities determination of plant extracts 

and essential oils. It is very suitable and useful for the 

antioxidant activity determination since it’s fast and 

sufficiently sensitive (M o l y n e u x , 2004). This 

method is based on hydrogen atoms or electrons 

exchange between antioxidant molecules and DPPH 

radicals in the solution (S a n c h e z - M o r e n o  et al., 

2002).  

DPPH radical scavenging activity of isolated 

oregano essential oil was shown on Fig. 3, while the 

EC50 values are listed in Tab. 2. Degree of DPPH 

radical neutralization depends on oil concentration as 

well as the incubation time - it increases with the 

concentration increase which is the lowest for non 

incubated samples (Fig. 3). The highest antioxidant 

activity (about 95%) has been measured after 60 

minutes of incubation. The EC50 (DPPH) value of 

 

Fig. 2. GC-FID chromatogram of oregano essential oil 

 

Table 1. Chemical composition of Origanum vulgare essential oil 

 

No. tret., min Compound RI
exp RIlit 

Method of 

identification 

Content, 

% 

  
Monoterpene 

hydrocarbons (14.4%) 
    

1 9.78 α-Thujene 925 924 RI, MS 0.4 

2 11.99 Myrcene 987 988 RI, MS 1.0 

3 12.93 α-Terpinene 1014 1014 RI, MS 1.1 

4 13.22 o-Cymene 1022 1022 RI, MS 4.4 

5 14.46 γ-Terpinene 1057 1054 RI, MS 7.5 

 Phenols (82.4%)  

6 22.59 Thymol 1292 1289 RI, MS 45.0 

7 22.92 Carvacrol 1300 1298 RI, MS 37.4 

 Total 96.8% 

tret.: Retention time; RI
lit a,b-Retention indices from literature (Adams, 2007), respectively; RIexp: Experimentally 

determined retention indices using a homologous series of n-alkanes (C8-C20) on the HP-5MS column. MS: constituent 

identified by mass-spectra comparison; RI: constituent identified by retention index matching. 

 



BIOLOGICA NYSSANA 7 (2)  December 2016: 131-139  Stanojević, Lj.P. et al.  Antioxidant activity of oregano essential oil… 

 

 136 

synthetic antioxidant BHT was 0.021 mg/mL, 

indicating better antioxidant activity compared to the 

tested essential oil. Mentioned antioxidant is the most 
commonly used synthetic antioxidant, but with 

harmful effects in human body (I t o  et al., 1986). So, 

presented results suggest that oregano essential oil 

can be potentially used as a safer alternative to 

synthetic antioxidants in pharmaceutical and food 

industries. 

 

Fig. 3. DPPH antioxidant activity of oregano 

essential oil 

K u l i š i ć  et al. (2004) and S a h i n  et al. 
(S a h i n  et al., 2004) have come to similar results in 

their investigations. In earlier reports, thymol and 

carvacrol, in particular, were found to be main 

antioxidant components of essential oil from 

different Origanum species (B a r r a t a  et al., 1998; 

R u b e r t o  et al., 2002; P u e r t e s -M e j i a  et al., 

2002). Because the main components of the oregano 

essential oil in our investigations are phenolics 

thymol and carvacrol, they are probably mostly 

responsible for the high degree of DPPH radical 

neutralization (Y a n i s h l i e v a  & M a r i n o v a , 

1995; K u l i š i ć  et al., 2004; S a h i n  et al., 2004). 

The TBA method is sensitive and achieves 

reproducible results. This method is preferable in 

order to obtain useful data in an environment similar 

to the real-life situation (K u l i š i ć  et al., 2004). Fig. 

4 shows degree of lipid peroxidation inhibition by 

investigated oregano essential oil, while Tab. 2 

shows the EC50 value (TBA-MDA) of the oil.  

 
Fig. 4. Inhibition of lipid peroxidation by oregano 

essential oil (TBA-MDA test) 

Based on the presented results it can be 

concluded that isolated essential oil from oregano 

shows good concentration dependent antioxidant 

activity. The highest degree of lipid peroxidation 

inhibition was achieved by essential oil in 

concentration of 3.0 mg/mL. The EC50 (TBA-MDA) 

value of synthetic antioxidant BHT was 0.0185 

mg/mL. 

K u l i š i ć  et al. (2004) reported antioxidant 

activity of oregano essential oil from Dalmacia and 

its fractions, measured by TBA method. In addition, 

C a p e c k a  et al. (2005) found that O. vulgare 

showed the strongest inhibition of linolenic acid 

peroxidation. 

Our results of antioxidant activity are in 

accordance with S a h i n  et al. (2004) who reported 

that essential oil of Origanum vulgare ssp. vulgare 

possesses antioxidant compounds, and therefore can 

be used as a natural preservative in food and/or 

pharmaceutical industry.  

Presented results confirm that oregano 

essential oil possesses remarkable antioxidant 

activities as assessed by two different methods. This 

biological effect is probably due to the presence of 

thymol and carvacrol, but possible synergistic effect 

Table 2. EC50 values of oregano essential oil 

EC50 (DPPH), mg/mL
 

20 min 30 min 45 min 60 min 

0.761  0.003 0.590  0.005 0.360  0.004 0.326  0.002 

EC50 (TBA-MDA), mg/mL 

0.455  0.004 

 



BIOLOGICA NYSSANA 7 (2)  December 2016: 131-139  Stanojević, Lj.P. et al.  Antioxidant activity of oregano essential oil… 

  

 137 

among other compounds in the oil can be also 

suggested.  

 

Conclusion 

The presented results indicate that the oregano 

essential oil could be used as potential source of 

natural antioxidants for the food, pharmaceutical and 

chemical industry. Therefore, oregano essential oil 

represents the alternative to synthetic additives that 

exhibit toxic and carcinogenic effects. So, it is 

interesting to investigate its application as natural 

antioxidant additive in some final food and 

pharmaceutical products, for preservation and/or 

extension the shelf-life of raw and processed foods as 

well as pharmaceuticals. In addition, the results of 

antioxidant activity in the present study suggested 

that use of oregano is not just reasonable but it should 

be even favored in the traditional Serbian cuisine.  

Acknowledgements. This work is part of the research 

project "Plant and synthetic bioactive products of new 

generation", no. TR 34012, financed by the Ministry of 

Education, Science and Technological Development of 

Republic of Serbia. 

  

References 

Aquino, R., Morelli, S., Tomaino, A., Pellegrino, M., 

Saija, A., Grumetto, L., Puglia, C., Ventura D., 

Bonina, F., Grumetto, L. 2002: Antioxidant and 

photoprotective activity of a crude extract of 

Culcitium reflexum H.B.K. Leaves and their 

major flavonoids. Journal of Ethnopharma-

cology, 79: 183–191. 

Bailer, J., Aichinger, T., Hackl, G., Hueber, K., 

Dachler, M. 2001: Essential oil content and 

composition in commercially available dill 

cultivars in comparison to caraway. Industrial 

Crops and Products,14: 229–239. 

Bakkali, F., Averbeck, S., Averbeck, D., Idaomar, M. 

2008: Biological effects of essential oils – a 

review. Food and Chemical Toxicology, 46: 446–

475. 

Barrata, M.D.S., Dorman, H.J.D., Deans,S.G., 

Figueiredo, A.C., Barroso, J.G., Ruberto,G. 1998: 

Chemical composition, antimicrobial and 

antioxidative activity of laurel, sage, rosemary, 

oregano and coriander essential oils. Journal of 

Essential Oil Research, 10: 618–627. 

Bettaieb, I., Bourgou, O., Wannes, W. A., Hamrouni, 

I., Limam, F. , Marzouk, B. 2010: Essential oils, 

phenolics, and antioxidant activities of different 

parts of Cumin (Cuminum cyminum L.). Journal 

of Agricultural and Food Chemistry, 58: 10410–

10418. 

Burt, S. 2004: Essential oils: their antibacterial 

properties and potential applications in foods – a 

review. International Journal of Food 

Microbiology, 94: 223–253. 

Callan, N.W.,, Johnson, D. L., Westcott, M.P., 

Welty, L.E. 2007: Herb and oil composition of dill 

(Anethum graveolens L.): Effects of crop maturity 

and plant density. Industrial Crops and Products, 

25: 282–287. 

Capecka, E., Mareczek, A., Leja, M. 2005: 

Antioxidant activity of fresh and dry herbs of 

Lamiaceae species. Food Chemistry, 93(2): 223–

226. 

Choi, W.C., Kim, C.S., Hwang, S.S., Choi, K.B., 

Ahn, J.H., Lee, Y.M., Park. H.S., Kim, K.S., Lee, 

Y.M. 2002: Antioxidant activity and free radical 

scavenging capacity between Korean medicinal 

plants and flavonoids by assay-guided 

comparison. Plant Science, 163: 1161–1168. 

Cvetković, D., Marković, D. 2008: UV-induced 

changes in anti-oxidant capacities of selected 

carotenoids toward lecithin in aqueous solution. 

Radiation Physics and Chemistry, 77: 34–41.  

Cvetković, D., Marković, D., Cvetković, D., 

Radovanović, B. 2011: Effects of continuous UV-

irradiation on the antioxidant activities of 

quercetin and rutin in solution in the presence of 

lecithin as the protective target. Journal of the 

Serbian Chemical Society, 76: 973–985. 

De Falco, E., Mancini, E., Roscigno, G., Mignola, E., 

Taglialatela-Scafati, O., Senatore, F. 2013: 

Chemical composition and biological activity of 

essential oils of Origanum vulgare L. subsp. 

vulgare L. under different growth conditions. 

Molecules, 18: 14948–14960. 

Faber, B., Bangert, K., Mosandl, A., 1997: GC-IRMS 

and enantioselective analysis in biochemical 

studies in dill (Anethum graveolens L.). Flavour 

and Fragrance Journal, 12: 305–314. 

Figiel, A., Szumny, A., Gutiérrez-Ortíz, A., 

Carbonell-Barrachina, A.A. 2010: Composition 

of oregano essential oil (Origanum vulgare) as 

affected by drying method. Journal of Food 

Engineering, 98: 240–247. 

Galego, L., Almeida, V., Gonçalves, V., Costa, M., 

Monteiro, I., Matos, F., Miguel, G. 2008: 

Antioxidant activity of the essential oils of 

Thymbra capitata, Origanum vulgare, Thymus 

mastichina and Calamintha baetica. Acta 

Horticulture, 765:325-34. 

Ghassemi-Golezani, K., Andalibi, B., Zehtab-

Salmasi, S., Saba, J. 2008: Effects of water stress 

during vegetative and reproductive stages on seed 

yield and essential oil content of dill (Anethum 

graveolens L.). Journal of Food, Agriculture and 

Environment, 6: 282–284.  

https://www.google.rs/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&cad=rja&uact=8&ved=0ahUKEwifsfufic_NAhVHORQKHclHDJ0QFggaMAA&url=http%3A%2F%2Fpubs.acs.org%2Fjournal%2Fjafcau&usg=AFQjCNH5PAfTp8G3y2WT0pD14ztU8PQzEg&sig2=2r5ovV3iL1OeynBO5m-Cbg&bvm=bv.125801520,d.bGs
https://www.google.rs/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&cad=rja&uact=8&ved=0ahUKEwifsfufic_NAhVHORQKHclHDJ0QFggaMAA&url=http%3A%2F%2Fpubs.acs.org%2Fjournal%2Fjafcau&usg=AFQjCNH5PAfTp8G3y2WT0pD14ztU8PQzEg&sig2=2r5ovV3iL1OeynBO5m-Cbg&bvm=bv.125801520,d.bGs


BIOLOGICA NYSSANA 7 (2)  December 2016: 131-139  Stanojević, Lj.P. et al.  Antioxidant activity of oregano essential oil… 

 

 138 

Halliwell, B., Chirico, S. 1993: Lipid peroxidation: 

its mechanism measurement, and significance. 

American Journal of Clinical Nutrition, 57: 715S-

725S.  

Ito, N., Hirose, M., Fukushima, H., Tsuda, T., Shirai, 

T., Tatenatsu, M. 1986: Studies on antioxidants: 

their carcinogenic and modifying effects on 

chemical carcinogens. Food and Chemical 

Toxicology, 24:1071–1092. 

Kulevanova, S., Stefova, M., Stefkov, G., Stafilov. T. 

2001: Identification, isolation, and determination 

of flavones in Origanum vulgare from 

Macedonian flora. Journal of Liquid 

Chromatography & Related Technologies, 24(4): 

589–600. 

Kulišić, T., Radonić, A., Katalinić, V., Miloš, M. 

2004: Use of different methods for testing 

antioxidative activity of oregano essential oil. 

Food Chemistry, 85: 633–640. 

Lagouri, V., Blekas, G., Tsimidou, M., Kokkini, S., 

Boskou, D. 1993: Composition and antioxidant 

activity of essential oils from Oregano plants 

grown wild in Greece. Zeitschrift für 

Lebensmittel-Untersuchung und -Forschung, 

197: 20–23. 

Laguerre, M., Lecomte, J., Villeneuve, P. 2007: 

Evaluation of the ability of antioxidants to 

counteract lipid oxidation: existing methods, new 

trends and challenges. Progress in Lipid 

Research, 46: 244–282.  

Leung, A.Y., Foster, S. 2003: Encyclopedia of 

common natural ingredients (used in food, drugs, 

and cosmetics), Second Edition, A John Wiley & 

Sons, Inc., Hoboken, New Jersey. 398–399. 

Maestri, D.M., Nepote, V., Lamarque, A.L., Zygadlo, 

J.A. 2006. Natural products as antioxidants. In: 

Imperato F. (ed.), Phytochemistry: advances in 

research, Research Signopost: 105–135, Kerala, 

India.  

Mathela, C. S., Singh, K. K., Gupta, V. K. 2010: 

Syntesis and in vitro antibacterijal activity of 

thymol and carvacrol derivates. Acta Poloniae 

Pharmaceutican Drug Research, 67(4): 375–380.  

Mehdi, S.J., Ahmad, A., Irshad, M., Manzoor, N., 

Rizvi, M.M.A. 2011: Cytotoxic effect of 

Carvacrol on human cervical cancer cells. Biology 

and Medicine, 3(2): 307–312. 

Molyneux, P., 2004: The use of the stable free radical 

diphenylpicrylhydrazyl (DPPH) for estimating 

antioxidant activity. Songklanakarin Journal of 

Science and Technology, 26(2): 211–219. 

Nostro, A., Sudano Roccaro,A., Bisignano, G., 

Marino, A., Cannatelli, M.A., Pizzimenti, F.C., 

Cioni, P.L., Procopio. F. and Blanco, A.R. 2007: 

Effects of oregano, carvacrol and thymol on 

Staphylococcus aureus and Staphylococcus 

epidermidis biofilms. Journal of Medical 

Microbiology, 56: 519–523.  

Pharmacopoeia Jugoslavica V, 2000: Savremena 

administracija, Beograd (in Serbian), Vol. 1: 118. 

Puertes-Mejia, M., Hillebrand,S., Stashenko,E., 

Winterhalter, P. 2002: In vitro radical scavenging 

activity of essential oils of Columbian plants and 

fractions from oregano (Origanum vulgare L.) 

essential oil. Flavour and Fragrance Journal, 17: 

380–384. 

Radušienė, J., Ivanauskas, L., Janulis, V., Jakštas, V. 

2008: Composition and variability of phenolic 

compounds in Origanum vulgare from Lithuania. 

Biologia, 54(1): 45–49. 

Ruberto, G., Barrata, M.T., Sari, M., Kaabexhe, M. 

2002: Chemical composition and antioxidant 

activity of essential oils from Algerian Origanum 

glandulosum Desf. Flavour and Fragrance 

Journal, 17: 251–254. 

Sahin, F., Gulluce, M., Daferera, D., Sokmen, A., 

Sokmen, M., Polissiou, M., Agar, G., Ozer, H. 

2004: Biological activities of the essential oils and 

methanol extract of Origanum vulgare ssp. 

vulgare in the Eastern Anatolia region of Turkey. 

Food Control, 15: 549–557. 

Sanchez-Moreno, C. 2002: Methods used to evaluate 

the free radical scavenging activity in foods and 

biological systems. Food Science and Technology 

International, 8(3), 121–137.  

Segeit–Kujawa, E., Michalowska, A. 1990: 

Determination of flavonoids in Hb. Origani. 

Herba Polonica, 36(3): 79–82. 

Stanković, M.Z., Stanojević, LJ.P., Tehnologija 

lekovitog i začinskog bilja, Tehnološki fakultet, 

Leskovac, 2014. 113 p. 

Stanojević, J.S., Stanojević, Lj.P., Cvetković, D.J., 

Danilović B.R., 2015: Chemical composition, 

antioxidant and antimicrobial activity of the 

turmeric essential oil (Curcuma longa L.). 

Advanced technologies, 4(2): 19–25. 

Stanojević, Lj.P., Stanojević, J.S., Cvetković, D. J., 

Cakić, M.D., Ilić, D.P. 2015a: Antioksidativna 

aktivnost etanolnog ekstrakta lista gajene jagode 

(Fragariae folium). Hemijska Industrija, 69(5): 

567–576. 

Stanojević, Lj., Stanković, M., Cakić, M., Nikolić, 

V., Nikolić, Lj., Ilić, D., Radulović, N. 2011: The 

effect of hydrodistillation techniques on yield, 

kinetics, composition and antimicrobial activity 

of essential oils from flowers of Lavandula 

officinalis L. Hemijska Industrija, 65: 455–463. 

Tongnuanchan, P., Benjakul, S. 2014: Essential oils: 

extraction, bioactivities, and their uses for food 

preservation. Journal of Food Science, 79(7): 

R1231–R1249.  



BIOLOGICA NYSSANA 7 (2)  December 2016: 131-139  Stanojević, Lj.P. et al.  Antioxidant activity of oregano essential oil… 

  

 139 

Tsimidou, M., Boskou, D. 1994: Antioxidant activity 

of essential oils from the plants of the Lamiaceae 

family. In: G. Charalambous, Spices, herbs and 

edible fungi, 273–284, Amsterdam: Elsevier.  

Ultee, A., Bennik, M. H. J., Moezelaar, R. 2002: The 

phenolic hydroxyl group of carvacrol is essential 

for action against the food-borne pathogen 

Bacillus cereus. Applied and Environmental 

Microbiology, 68(4):1561–1568.  

Vazirian, M., Mohammadi, M., Farzaei, M.H., Amin, 

G., Amanzadeh, Y. 2015: Chemical composition 

and antioxidant activity of Origanum vulgare 

subsp. vulgare essential oil from Iran. Research 

Journal of Pharmacognosy, 2(1): 41–46. 

Wang, Q., Gong, J., Huang, X., Yu, H., Xue. F. 2009: 

In vitro evaluation of the activity of 

microencapsulated carvacrol against Escherichia 

coli with K88 pil. Journal of Applied 

Microbiology 1, 107(6): 1781–178. 

Yanishlieva, N.V., Marinova, E.M., Gordon, M.H., 

Raneva, V.G. 1999): Antioxidant activity and 

mechanism of action of thymol and carvacrol in 

two lipid systems. Food Chemistry, 64: 59–66. 

Yanishlieva, N.V., Marinova, E.M. 1995: 

Antioxidant activity of selected species of the 

family Lamiaceae grown in Bulgaria. Die 

Nahrung, 39: 458–463. 

Zvezdanović, J., Daskalova, L., Yancheva, D., 

Cvetković, D., Marković, D., Anderluh, M., 

Šmelcerović, A. 2014: 2-Amino-5-

alkylidenethiazol-4-ones as promising lipid 

peroxidation inhibitors. Monatshefte Fur 

Chemie,145: 945–952.