Genetic variation of oregano (<i>Origanum vulgare</i> L.) for etheric oil in Albania

doi:10.4236/as.2013.49060

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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: [email protected]

Received 11 January 2013; revised 11 February 2013; accepted 15 March 2013

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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