Vol.4, No.9, 449-454 (2013) Agricultural Sciences
http://dx.doi.org/10.4236/as.2013.49060
Genetic variation of oregano (Origanum vulgare L.)
for etheric oil in Albania
Fetah Elezi1, Fatbardh Plaku1, Alban Ibraliu1, Gose Stefkov2, Marija Karapandzova2,
Svetlana Kulevanova2, Sali Aliu3*
1Department of Plant Production, Agricultural University in Tirana, Tirana, Albania
2Faculty of Pharmacy, University Ss.Cyril and Methodius, Skopje, Macedonia
3Faculty of Agriculture, Department of Crop Science, University of Prishtina, Prishtina, Kosovo;
*Corresponding Author:
Received 11 January 2013; revised 11 February 2013; accepted 15 March 2013
Copyright © 2013 Fetah Elezi et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
In this study are presented the results of studies
for oregano in 16 locations (sites) of Albania for
the content of the quantity of oils and their frac-
tions. Albania is the one of the important diver-
sity centers for Oregano because of their adap-
tation to diverse agro ecological conditions as a
result of natural selection. The design of the
experiment was based on split plot methods.
From the total samples collected, were selected
16 mostly widespread samples of natural popu-
lations of origano (Origanum vulgare L. sp. vul-
gare and sp. hirtum). Essential oil isolation from
oregano was performed by hydro distillation.
Identification of the components was made by
comparing mass spectra of components in es-
sential oils with those from Nist, Wiley and Ad-
ams mass spectra libraries. The results show
that the total oil content varies from 3.45% to
0.1%. The populations of Oregano in the part of
southern Albania have a higher amount of ethe-
ric oil compared with those of the North part.
The higher content of car vacrol in 55.63%, of the
samples was determined in sites of the south-
ern part of Albania, while in the other two inves-
tigated area central and north part it was 55.63%
and 48.49% respectively. The same indicators
were realised also for content of linalool and
thymol, while North area has sites with high
content of caryophyllen-oxide and β-pinen 3.
But, in all sites, the oils of thymol and carv acrolit
were dominant.
Keywords: Origanum; Essential Oil; Carvacrol;
Thymol
1. INTRODUCTION
The genus Origanum is a member of the Lamiaceae
family and has a complex taxonomy [1]. O. vulgare plays
a primary role among culinary herbs in world trade [2]. It
is distributed all over Europe, West and Central Asia up
to Taiwan [3]. The use of O. vulgare as medicinal plant is
believed to be due to biological properties of p-cymene
and carvacrol. Bernáth [4] has noted that there are intras-
pecific taxa of oregano having no “oregano” character
that is based on the presence of carvacrol. Oregano is the
common name for a general aroma and flavour primarily
derived from more than 60 plant species used all over the
world as a spice [5]. Four main groups of plants com-
monly used for culinary purposes can be distinguished,
i.e., Greek oregano (Origanum vulgare ssp. hirtum (Link)
Ietswaart); Spanish origano (Coridohymus capitatus (L.)
[2]; Turkish oregano (Origanum onites L.); and Mexican
oregano (Lippia graveolens HBK [6]. Origano is the
commercial name of those Origa num species that are
rich in the phenolic monoterpenoids, mainly carvacrol
and occasionally thymol [7]. A number of chemically
related compounds i.e. p-cymene; γ-terpinene, carvacrol
methyl ethers, thymol methyl ethers, carvacrol acetates
and thymol acetates; as well as p-cymenene, p-cy-
men-8-ol, p-cymen-7-ol, thymoquinone, and thymohy-
droquinone are present in the oil of Origanum vulgare
which is extremely rich in essential oils (up to 7%) with
carvacrol as a major constituent present in very high
quantity (75% - 95%), followed by p-cymene (4% - 14%)
and γ-terpinene (1% - 10%). It seems possible that the
uses of the plant in traditional medicine can be attributed
to the known biological properties of p-cymene and car-
vacrol [8]. Many of the studies confirmed the medicinal
effects of oregano for human health. The Origanum spe-
cies, which are rich in essential oils, have been used for
thousands of years as spices and as local medicines in

F. Elezi et al. / Agricultural Sciences 4 (2013) 449-454
450
traditional medicine [9]. About 20 European public in-
stitutions hold genetic resources of different species of
oregano [10]. Marjoram (Origanum vulgare L.) is one of
medicinal aromatic plants found wholesale almost in all
areas of Albania, which is a perennial plant usually grows
in dry area in smaller groups. Oregano plants are collect-
ed from natural habitats and used as raw materials in the
pharmaceutical, cosmetic and food industry [11]. But
many countries start to cultivate it in different areas. In
botanical aspects, the oregano populations differ from
one to another, that is they vary depending on the content
and composition of essential oils [12]. From the quantity
and quality of essential oils the values of this plant were
determined [11,12]. The different results showed that the
effects of oregano antioxidante are associated with high
content of essential oils, thymol and carvacrols, and
these are the main ingredients in oregano oil. The con-
tents of Thymol and Carvacrol in oregano give it differ-
ent properties [13]. According to the studies, carvacrol is
a powerful bactericidal agent, and provides protection
against mold and other common bacteria. The main ob-
jective in our study was to investigate the different re-
gions and to find the variation for oil content in oregano
plant populations.
2. MATERIAL AND METHODS
2.1. Collection Sites
The research expedition was organized in 2012 in the
whole territory of Albania. During this expedition are
identified and collected 62 accessions. At each location
were taken of the 50 samples which derived a main rep-
resentativ sample. From the total samples collected, were
selected 16 samples mostly widespread of natural popu-
lations of origano (Origanum vulgare L. sp. vulgare and
sp. hirtum). Those samples you perform analyzes for
content of oils and their components. The confirmation
of 53 essential oil was made to analyses: β-Pinene, p-
Cymene, γ-Terpinene, Linalool, Terpinene-4-ol, Thymol,
Carvacrol dhe Caryophyllene oxide. The overground parts
of the flowering plants (20 - 25 cm from the top) were
collected during the summer of 2012. The plant material
was air dried, packed in paper bags and kept in a dark
and cool place until analysis. Plant identity was verified
and voucher specimens were deposited at the Institute of
Pharmacognosy, Faculty of Pharmacy, Skopje.
2.2. Essential Oil Isolation
Essential oil isolation from oregano was performed by
hydro distillation in all-glass Clevenger apparatus fol-
lowing this procedure: 20 g of the plant material was
stored in 500 mL flask where 250 mL of water R was
used as distillation liquid and 0.5 mL of xylene R was
added in the graduate tube. The Distillation was per-
formed for 2.5 h with a rate of 2 - 3 mL/min.
GC and GC-MS analyses: Agilent 7890А Gas Chro-
matography system equipped with flame ionization de-
tector (FID) and Agilent 5975C Mass Quadrupole detec-
tor as well as capillary flow technology which enable
simultaneous analysis of the sample on both detectors.
HP-5 ms (30 m × 0.25 mm, film thickness 0.25 m) cap-
illary column was used. Operating conditions were as
follows: GC Method for essential oils: oven temperature
60˚C (0 min), 3˚C/min to 240˚C (held for 1 min) and
10˚C/min to 280˚C (held for 1 min); helium as carrier
gas at a flow rate of 1 mL/min; injector T = 220˚C and
FID T = 270˚C. 1 L of injection volume was injected at
split ratio 1:1. The mass spectrometry conditions were:
ionization voltage 70 eV, ion source temperature 230˚C,
transfer line temperature 280˚C and mass range from 50 -
500 Da. The MS was operated in scan mode. GC Method
for Head Space: oven temperature 60˚C, 20˚C/min to
280˚C; helium as carrier gas at a flow rate of 1 mL/min;
injector T = 260˚C and FID T = 270˚C. 1000 L of injec-
tion volume was injected at split ratio 1:1. The mass
spectrometry conditions were: ionization voltage 70 eV,
ion source temperature 230˚C, transfer line temperature
280˚C and mass range from 50 - 500 Da. The MS was
operated in scan mode. Head Space method: Incubation
Temperature 80˚C, Incubation Time 5.00 m:ss, Syringe
Temperature 85˚C, Agitator Speed 500 rpm, Fill Speed
500 µl/s, Pullup Delay 500 ms, Inject to GC, Injection
speed 500 µl/s, Pre Inject Delay 500 ms, Post Inject De-
lay 500 ms, Flush Time (m:ss) 0:10, GC Run time (m:ss)
10:00. Identification of the components: Identification of
the components was made by comparing mass spectra of
components in essential oils with those from Nist, Wiley
and Adams mass spectra libraries, by AMDIS (Auto-
mated Mass Spectral Deconvolution and Identification
System) and by comparing literature and estimated Ko-
vat’s (retention) indices that were determined using mix-
ture of homologous series of normal alkanes from C9 to
C25 in hexane, under the same above mentioned condi-
tions. The percentage ratio of the components was com-
puted by the normalization method of the GC/FID peak
areas and average values were taken into further consid-
eration (n = 3).
2.3. Statistical Analyses
All statistical analyses were performed with the SPSS
software (version 15.0, SPSS) [14]. Means values and
variation coefficients were used in the statistical analyses.
Effects of the studied traits were evaluated by ANOVA.
In order to assess the differentiation of plants of oregano
based on all variables that were measured, the Canonical
Discriminate Analyses (CDA) was applied.

F. Elezi et al. / Agricultural Sciences 4 (201 3) 449-454 451
3. RESULTS
The our results for the oil content in the samples of 16
locality in Albania are presented in Table 1. The present
collection of oregano showed appreciable genotypic
variation in content of etheric oil compounds. In our re-
sults, the genetic variation varied from +167.53 to
−93.42%.
The data which are showed in Ta bl e 1 indicates that
the total oil content varies from 3.45% (Vagalat-Delvin)
to 0.1% (Baz-Burrel), which is one a wide variation
between plants in different locality. Figure 1 is presented
the genetic variation compare with experimental average
values µ at all locality collected in Albania. From the
total locality (16), 50% or 8% locality showed postive
values for oil content and 50% of locality are determined
under experimental average value µ.
Table 2 showed the data of variation on the site of es-
sential oils content in South part in Albania. Referring to
the two main ingredients of etheric oil of oregano, thy-
mol and carvacrol contents are higher in the South part
(carvacrol = 68.4, thymol = 6.87) while in the central
area the obtained results for content of carvacrol were
55.63% and thymol 5.30%. In the Northern part, the con-
tent with these two components were lower (carvacrol =
48.49%, and thymol = 4.19%) compare with south and
central part of Albania. Referring to the average content
of essential oil fractions, the sites of central area regions
Tab le 1. The oil content in different regions-locality in Alba-
nia.
Number Regions-Locality Content of etheric
oil (EO) % GV* %
1 Vagalat Delvinë 3.45 167.53
2 Mal i gjerë
Girokastër 1.9 25
3 Llogara Vlorë 3.4 123.68
4 Qafë e
Koshovicës Fier 2 31.57
5 Tozhar Berat 2.5 64.47
6 Cukalat Berat 3.2 110.52
7 Roshnik Beart 0.6 −60.62
8 Tregan Elbasan 0.6 −60.52
9 Kashar Tiranë 1.8 18.42
10 Shëngjergj Tiranë 0.4 −73.68
11 Shesh Tiranë 2.9 90.78
12 Baz Burrel 0.1 −93.42
13 Maqellarë Dibër 0.3 −80.26
14 Kala e Dodës
Dibër 0.4 −73.68
15 Dragobi Tropojë 0.5 −67.1
16 Lepush Vermosh 0.4 −73.68
Average 1.53
GV*: genetic variation.
Figure 1. The genetic variation of etheric oil content.
compare to the south part distinct with higher of p-cy-
mene (6.19%) or more +2.38% compare to the south part
(3.81%), and +5.28% higher than north part regions
(0.91%). Compared the average values for p-cymene
6.19% with experimental values µ (2.80%) the differ-
ences were +3.39% or expressed in relative values 121.07%,
significantly higher. The differences between three re-
gions for Caryophyllen oxide content are with higher
significance. The total average values for this component
are 2.11%, while the higher values are determined in
north part 2.18%. Compare to average values the differ-
ences are +0.07% which are none significantly for level
of probability. The central and south parts are character-
ized with lower quantity of Caryophyllen oxide content
2.18% and 1.96% respectively. The differences between
these two regions are +0.22% or 10.42% higher for cen-
tral part of Albania. With higher percentage of β-pinene
is characterized the area in site of Northern Albania with
average values of 0.34%. Also, the regions in the south
par for these compounds are showed the lower content of
β-pinene (0.15%). So, the differences among the two
regions are +0.19% or expressed in relative values are
172.72%, significantly higher differences.
The content of γ-Terpinen in South part varies from
7.87% in the Qafa e Koshovices (Fier) to 0.19% in Berat
Roshnik. In the north part, the content of γ-Terpinene
ranges from 9.12% to 0.48%. The differences within
locality inside one regions are +8.64%. The amount of
the carvacrol in South part ranges from 82.76%. Amount
of the carvacrol in south part was characterized with
huge genetic variation which ranges from 82.76% to
0.66% (Tozhar-Berat). In Central part, the values also are
with higher differences from 72.53 (Kashar-Tirane) to
33.875 (Shengjergj-Tirane). Results are presented in Ta-
ble 2. Table 3 describes the canonical discriminate func-
tions, the eigenvalue, percentages of variation of each
function and the cumulative variance of the three dis-
criminate functions. This table also shows the stan- dar-
dized elements of structure matrix. The three canoni- cal
discriminate functions were significant (p < 0.000). It is
important to point out the great relevance of the first

F. Elezi et al. / Agricultural Sciences 4 (201 3) 449-454
452
Table 2. Average values for essential oil content in Oregano at different part of Albania.
OPEN A CCESS

F. Elezi et al. / Agricultural Sciences 4 (201 3) 449-454 453
Table 3. Summary statistics for canonical discriminant stan-
dardized functions.
Functions 1 2 3
Eigenvalue 17170.58 1130.05 11.21
Percentage of variation 99.9 0.1 0
Cumulative percentage 99.9 100 100
Canonical correlation 1 1 0.95
Functions at group centroides
β-Pinene −512.46 3.78 −1.397
p-Cymene −228.43 −64.288 3.015
γ-Terpinene −421.181 −6.484 −3.542
Linalool −519.61 36.626 2.696
Terpinen-4-ol −500.65 4.354 −0.932
Thymol −179.23 −0.546 −3.709
Carvacrol 2812.387 5.395 0.152
Caryophyllen oxide −450.798 21.162 3.716
df 7 7 7
Significance <0.000 <0.000 <0.000
Elements of Structure Matrix
Locality 1* 0.55* 0.83** 0.24
locality 2 0.078 −0.027 0.997**
Locality 3 0.52* 0.249 0.967**
*Largest absolute correlation between each variable and any discriminant
function; *Locality 1 (South part of Albania); Locality 2 (Central part of
Albania); Locality 3 (North part of Albania).
two discriminate functions justifying 99.9% and 0.1% of
the variability. The first discriminate function showed a
significant positive correlation with the locality 1 (0.55)
and locality 3 (0.52) following by second and third dis-
criminate function (0.83) and 0.99. But, the negative
correlation (−0.027) was determined between second
discriminate function and locality 2. Results are pre-
sented in Table 3.
4. DISCCUSION
The essential oil of oregano is composed of carvacrol
and thymol as dominant components, followed by; ter-
pinene, p-cymene, linalool, terpinen-4-ol and sabinene
hydrate [5]. Results of various studies indicated that the
antioxidant effects of oregano might be related to the
dominant components, carvacrol and thymol, of the es-
sential oil [15,16]. Origanum vulgare L. is the species
with the highest variability in the genus Origanum. Nev-
ertheless, diversity, genetic resources and potential for
utilization of O. vulgare have not yet been fully explored
so that extended research on oregano germplasm is nec-
essary [17]. The oil content is an important indicator
which associated with value of oregano. Also, Ayala [18]
they reported different values in Origanum spp for oil
content which varied to 8.8%. The samples from the
southern part of Albania has a high amount of etheric oil
compared with those of the North zone. The variation for
the oil content was found also within an area. The
different variation of essential oils in oregano were re-
ported by numerous researches [10,19]. Obtained results
by our study for analysis of the whole content of etheric
oils showed that sites of southern part of Albania
distinguished for higher content of γ-carvacrol on
average values for with 68.4%, γ-Terpinene and 1.72%,
Thymol and 6.87%. Gounaris et al. [20] in their research
reported results for γ-Terpinene (10%), p-Cymene
(5.98%), thymol (0.40%), Carvacrol (58.73%). A similar
effect was also reported by Azizi [5] which showed re-
sults for β-pinene (0.1%), p-cymene (5.3%), γ-Ter-
pinene (8.1%), thymol (0.3%), Carvacrol (77.4%). From
these obtained data the content of the different fractions
of essential oils is closely linked to the area where ore-
gano is so widespread which have an ecological impact
on their selection. According to Putievsky et al. [21] es-
sential oil content of oregano was higher in full bloom
stage than in the stage of start flowering. This hypothesis
gives us some preliminary information that local popula-
tions that are selected and adapted to the agro ecological
conditions for centuries are more tolerant to environ-
mental stress [22]. An early study by Azizi et al. [23] for
interactions factors showed that dry matter production
and essential oil content of Origanum vulgare L. can be
significantly affected by environmental and agronomical
conditions including nitrogen fertilization and soil mois-
ture regime, whereas percentage of main compounds of
essential oil such as carvacrol, terpinene and p-cymene
remained unaffected.
5. CONCLUSION
The content of the total amount of etheric oil is char-
acterized with different variation in different regions.
From our results, etheric oil with higher content was
characterized in the south part in Albania. The quantity
and content of oils fractions depend on the area where
the samples were taken. So, carvacrol with higher con-
tent in 55.63% of the samples is coming from sites of
Southern Albania, while in the other two areas, in Central
and Northern area is 55.63% to 48.49% respectively. The
same results were found for content of thymol, linalool’s
compounds, while North area had high content of caryo-
phyllen-oxide and β-pinen. In all sites, the dominant oils
were thymolit and carvacrol. In many cases, the essential
components of oregano oil are determined by the geno-
type structure, but the environmental effects have a small
effect. From the results of chromatographic analysis, it
can be concluded that the populations have significantly
changed in the composition of essential oils. The results
of this study can be a good base for selection criteria to

F. Elezi et al. / Agricultural Sciences 4 (201 3) 449-454
454
establish the programs to improve the oregano plants.
6. ACKNOWLEDGEMENTS
The authors are thankful to institute of Pharmacognosy, Faculty of
Pharmacy in Skopje for providing facilities to conduct this research.
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