Morshedloo2018 Essential Oil Profile of Oregano
Morshedloo2018 Essential Oil Profile of Oregano
Morshedloo2018 Essential Oil Profile of Oregano
A R T I C LE I N FO A B S T R A C T
Keywords: Oregano (Origanum vulgare L.) is one of the most commercially important herbs in the Lamiaceae family. It is a
Origanum vulgare L. rich natural source of bioactive components including phenolic glucosides, flavonoids, tannins, sterols, tri-
Chemotypes terpenes, resins and essential oil. In the present study, the variation of the essential oil compositions among
Carvacrol seven populations of Iranian oregano, originating from different bioclimate and geographical zones was in-
Germplasm
vestigated over two harvest years (2014 and 2015), and under controlled soil and climate conditions. The es-
Chemical diversity
sential oil content showed a wide variability, ranging from 0.12% to 1.76% (v/w), correlated to the chemical
profile. GC-FID and GC–MS analyses of the essential oils characterized a total of forty-two constituents in or-
egano populations. Carvacrol (0.3–46.8%), linalyl acetate (0.2–44.3%), (Z)-α-bisabolene (0.0–40.3%), (E)-β-
caryophyllene (0.0–24.0%), and caryophyllene oxide (0.1–21.3%) were identified as the main components of the
essential oils, depending on the population and harvest year. The highest amounts of these components were
recognized in the essential oil of Baneh, Rasht, Gilan, Kaleybar and Ardabil populations, respectively. According
to cluster and principal component analyses (PCA), the studied populations were grouped into four main che-
motypes: i.e., chemotype I (carvacrol), chemotype II ((Z)-α-bisabolene), chemotype III (linalyl acetate), che-
motype IV (caryophyllene oxide/germacrene D/(E)-β-caryophyllene). Variability of essential oil constituents in
oregano populations studied can primarily be explained by differences in efficiency and/or activity of the me-
thylerythritol 4-phosphate (MEP) and mevalonate pathways. Intraspecific chemical variability in Iranian or-
egano provides possibility of selection of those batches with specific aromas and chemical profiles for industrial
intentions. The results also provided new insight for development of effective conservation strategies, domes-
tication and breeding programs in Iranian O. vulgare germplasm.
⁎
Corresponding author.
E-mail address: [email protected] (S.A. Salami).
https://doi.org/10.1016/j.indcrop.2018.03.049
Received 18 December 2017; Received in revised form 17 February 2018; Accepted 23 March 2018
0926-6690/ © 2018 Elsevier B.V. All rights reserved.
M.R. Morshedloo et al. Industrial Crops & Products 119 (2018) 183–190
Table 1
Origin, geographical characteristics and voucher numbers of studied Origanum vulgare populations.
Voucher number Habitat Altitude (m) Latitude (N) Longitude (E) Subspecies Area of sampling collection Populations
and subsp. viridulum (Morshedloo et al., 2017a; Kokkini, 1996; controlled glass greenhouse at the College of Agriculture and Natural
Ietswaart and Ietswaart, 1980). The first three subspecies are rich in Resources, University of Tehran, Iran, over 2014 to 2015. The seeds
essential oil, whereas the remaining ones are essential oil poor plants were obtained from the gene bank of the Forest and Rangeland
(Padulosi, 1997). In Iran, the Origanum genus is represented by three Research Institute, Tehran, Iran, and germinated in a coco peat: perlite
species including O. vulgare, O. strobilaceum and O. laevigatum (Jamzad, mixture, (70:30, w: w), in a plastic germination tray.
2012). In this country O. vulgare occurs with three subspecies (subsp. In this study, twenty uniformly sized seedlings from each popula-
viridulum, subsp. viride and subsp. gracile) (Jamzad, 2012; Rechinger, tion, were transplanted from seedling bed into seven L pots (two plant
1982). per pot) thirty-two days after seeding. The soil mixture used in this
According to the extensive phytochemical studies on the essential experiment was a clay soil. The soil material was taken from the layer of
oil composition of O. vulgare and other related species, a wide chemical 0–20 cm, sieved, homogenized and mixed with sand and leaf mold (soil:
diversity with a considerable intraspecific qualitative and quantitative sand: leaf mold = 2:1:1w/w). Each pot was supplemented with 6.9 mg
variation in constituents is found (Mechergui et al., 2016; Lukas et al., P, 1.4 mg N, and 12.8 mg K to warrant the optimal nutrient supply for
2015; Moradi et al., 2014; Béjaoui et al., 2013; Lukas et al., 2013; plant growth. The mixed soil was adjusted to a pH 7.2 and electrical
Crocoll et al., 2010; Verma et al., 2010; Azizi et al., 2009; de Barros conductivity (EC) of 1.2 dS m−1. The pots containing the seedlings were
et al., 2009; Mockute et al., 2001; D’Antuono et al., 2000; Vokou et al., placed in a glass greenhouse (longitude 50°59′51” E, latitude 35°48°20”
1993). This variability could be related to the effect of variables such as N, altitude 1343 m a.s.l.). During the experiment, the average minimum
genetic factors, geographical distribution, plant part studied, collection and maximum temperatures inside the greenhouse were 16.5 °C and
time, methods of extraction, environmental conditions, etc. 32.5 °C, respectively, under natural light conditions. Two harvests were
(Morshedloo et al., 2015a, 2015b; Padulosi, 1997). According to pre- performed at the full flowering stage in 2014 and 2015 (the whole
vious studies, individuals rich in essential oil usually accumulate large plants were used for extraction of essential oil). To warrant a good
amounts of phenolic monoterpenes such as carvacrol, thymol and their comparison, the stems of the experimental oregano plants were uni-
biosynthetic precursors γ-terpinene and p-cymene, whereas the plants formly cut at five cm above the soil just before the beginning of the
with poor essential oil content are often characterized by high amounts second growing season. Voucher specimens were collected at flowering
of sesquiterpenes (such as germacrene D, (E)-β-caryophyllene, γ- stage, and deposited in the herbarium of the College of Agriculture and
muurolene and caryophyllene oxide), acyclic monoterpenoids (such as Natural Resources, University of Tehran, Karaj, Iran. The origin, geo-
linalool and/or linalyl acetate, β-ocimene or myrcene) and/or bicyclic graphical characteristics and voucher numbers of studied populations
‘sabinyl’-type monoterpenoids (mainly sabinene and cis-/trans-sabinene are presented in Table 1.
hydrate) (Lukas et al., 2015; Azizi et al., 2009; Skoula and Harborne,
2002; Padulosi, 1997). 2.2. Essential oil isolation
Although there are some reports on the volatile constituents of O.
vulgare growing wild in Iran (Moradi et al., 2014; Andi et al., 2012), to Plant materials were air dried in the shade for one week after har-
the best of our knowledge there are no systematic and comprehensive vesting at flowering stage. To isolate the essential oil, 20 g of dried
studies undertaken to explore the essential oil variability in Iranian leaves and inflorescences were hydro-distilled for 3 h using a Clevenger-
oregano populations. Therefore, the present study was performed to type apparatus according to the method reported in the European
characterize the chemical compositions of seven Iranian O. vulgare Pharmacopoeia (Council of Europe (COE), 2007). Isolated essential oils
populations, originating from different bioclimate and geographical were dried over anhydrous sodium sulphate and kept at −20 °C until
zones. The plants were grown in similar climatic conditions in order to analysis. Essential oil content was measured as relative percentage units
avoid the effects of environmental factors and growing conditions on (volume in ml of oil for 100 g of dry weight of plant). For GC-FID and
the production of secondary metabolites. Cluster analysis (CA) and GC–MS analysis, the essential oils were transferred to the Mass Spec-
principal component analyses (PCA) was performed to characterize the trometry Center, University of Massachusetts, Amherst, USA.
chemotypes based on their main volatile components. As most of or-
egano from Iran was collected from wild sources without focusing on
2.3. GC-FID and GC/MS analyses
the specific subspecies and chemotype, the presented study, aimed at
detecting interesting chemotypes (with genetically differences from
Gas chromatography analysis was performed using a Shimadzu GC-
others) for industrial use, breeding programs and development of ef-
FID Model 2014 (Japan), equipped with a Supelco fused-silica gel ca-
fective conservation strategies.
pillary RTX-5 column (30 m length, 0.25 mm diameter, and 0.25 μm
film thickness). The oven temperature was programmed at 60 °C for
2. Materials and methods 5 min, then from 60 to 210 °C at 3 °C/min, and held at 210 °C for
10 min. Both injector and detector temperatures were 230 °C. Helium
2.1. Plant and soil materials was used as carrier gas with a flow rate of 1 mL/min. The samples were
injected using the split sampling technique, with a ratio of 1:30. The
To diminish the effects of seasons, climate, and growing conditions percentage composition of the essential oils was computed by the
on essential oil yield and composition, seven populations of O. vulgare normalisation method from the GC peak areas, without using correction
were gathered from different bioclimatic zones of Iran, and seeded in a factors (Venditti et al., 2015). The GC–MS unit consisted of an Agilent
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M.R. Morshedloo et al. Industrial Crops & Products 119 (2018) 183–190
6890N-5973A (Agilent Technologies, USA) gas chromatograph that the essential oil yield varies over different harvest years
equipped with a DB-5MS capillary column (30 m length, 0.25 mm (Mechergui et al., 2016). Our results showed that there were no sig-
diameter, and 0.25 μm film thickness). The injector temperature was nificant differences among the essential oil contents of populations
250 °C and the initial GC oven temperature was 50 °C, held for 5 min, from Ardabil, Namin, Arasbaran, Gilan, Kaleybar and Chalus in 2014.
then raised to 240 °C at 3 °C/min and held for 10 min. Helium was used In 2015, however, the populations of Baneh (1.91%) and Namin
as the carrier gas with a flow rate of 1 mL/min. Furthermore, the (0.56%) exhibited significantly higher essential oil content than the
electron energy (EI) was 70 eV, the transfer line temperature was others. Previous studies confirmed a wide range of variability occurring
270 °C, and the acquisition scan was from 50 to 240 m/z. The essential in different populations and subspecies of O. vulgare (Lukas et al., 2015;
oil constituents were identified by co-injection with available authentic Verma et al., 2010). As a matter of fact, O. vulgare subsp. viridulum and
standards (Sigma–Aldrich, USA) and by computer matching with the O. vulgare subsp. virens were classified as essential oil-poor plants (oil
following MS libraries: WILEY275, NIST 05, ADAMS, and a “home- yield < 1%, w/w), whereas O. vulgare subsp. gracile (Baneh population)
made” library. The coherence of linear retention indices determined by was defined as an essential oil-rich subspecies (about 2% or more)
reference to a homologous series of n-alkanes, C8-C40; (Sigma, USA) (Kokkini, 1996). Verma et al. (2010) reported that the essential oil
with respect to those reported in literature (Adams, 2007; NIST 05, content in different populations of Indian oregano varied from 0.07 to
2005) was used as an additional identification method (Morshedloo 0.80%. In the present study, the population from Baneh, with individual
et al., 2017a). plants exhibiting the highest values of oil content, showed the highest
potential to be selected for maximizing the oregano essential oil yield.
2.4. Statistical analysis
3.2. Chemical profile of essential oils
A completely randomized experimental design was conducted with
The results of GC-FID and GC–MS analysis are shown in Tables 2
three replications. Dataset from chemical constituents of studied po-
and 3. A total of forty-two components in the first year (accounting for
pulations were subjected to multivariate analyses using the IBM SPSS
84.57–98.09% of the total compositions) and forty-one components in
Statistics v. 23.0 (SPSS, Chicago, IL, USA). All data were subjected to
the second year (accounting for 79.89–96.82% of the total composi-
analysis of variance (ANOVA) followed by the LSD test at P < 0.05
tions) were identified. The essential oil chemical profile were extremely
level. Hierarchical cluster analysis was performed based on the squared
variable. In addition, there were qualitative and quantitative differ-
Euclidean distance coefficients according to the Euclidean distances
ences betweeen two harvest years. Regardless of the year, the mono-
method. In addition, to determine the correlation among the seven
terpenoids were mainly consist of carvacrol, linalyl acetate, p-cymene,
Iranian oregano populations based on essential oil compositions (for
γ-terpinene, trans-sabinene hydrate, (Z)-β-ocimene and sabinene.
each population the average of six replicates in 2014 and 2015 years
Among the sesquiterpenoids (Z)-α-bisabolene, (E)-β-caryophyllene,
was considered), and to identify the main components influencing the
caryophyllene oxide, germacrene D, elemol and β-bisabolene were the
variability, the data matrix composed of 287 data (41 variables × 7
most abundant compounds. The shared components, however, ex-
populations) was subjected to principal component analysis (PCA) by
hibited a relatively high variation in levels.
STATISTICA 7.1 (Stat Soft Italia srl, 2005, www.statsoft.it). Eigenvalues
Carvacrol (0.3–46.8%), linalyl acetate (0.2–44.3%), (Z)-α-bisabo-
were calculated using a covariance matrix among 41 chemical com-
lene (0.0–40.3%), (E)-β-caryophyllene (0.0–24.0%) and caryophyllene
pounds as input, and the two-dimensional PCA biplot, including both
oxide (0.1–21.3%) were identified as the main compounds in the es-
oregano populations and volatile constituents, was built.
sential oils, depending on the populations and harvest year. The highest
amounts of these components were recognized in the essential oil from
3. Results and discussions Baneh, Rasht, Gilan, Kaleybar and Ardabil populations, respectively.
Previous studies have reported a large chemical diversity in oregano
3.1. Essential oil content essential oils from different parts of the world (Mechergui et al., 2016;
Lukas et al., 2015; Vazirian et al., 2015; Raina and Negi, 2014; Crocoll
There was a significant difference in essential oil content among the et al., 2010; Verma et al., 2010, Azizi et al., 2009; Sezik et al., 1993;
studied populations. According to the results, the essential oil content Giuliani et al., 2013) strengthening the fact that O. vulgare is a very
varied from 0.12 (for Gilan and Kaleybar populations) to 1.76%, (for complex species of the Lamiaceae family (Ietswaart and Ietswaart,
Baneh population) (Fig. 1). In both harvest seasons, population of 1980). Carvacrol, the major compound of the Baneh population, has
Baneh showed a significantly higher essential oil content than the other been previously reported as the main component of oregano essential
six investigated populations. In addition, the essential oil content of oil in many studies (Shiwakoti et al., 2016; Lukas et al., 2015;
oregano populations harvested in 2015, was higher than those har- Mechergui et al., 2010; Azizi et al., 2009; Baser et al., 1994). In
vested in 2014. Previous studies on oregano populations demonstrated agreement to our results, linalyl acetate (Andi et al., 2012; De Martino
et al., 2009; Afsharypour et al., 1997), β-caryophyllene, germacrene D
(Lukas et al., 2015; Shafaghat, 2011; Mockutë et al., 2004; Mockute
et al., 2001) and caryophyllene oxide (Lukas et al., 2015; Mockutë
et al., 2004) were often reported as the major components of oregano
essential oils. Previous studies have demonstrated that several factors
affecting the plant secondary metabolites such as environmental con-
ditions, season of collection, age of plants, and/or genetic factors
(Morshedloo et al., 2017b; Mechergui et al., 2016; Morshedloo et al.,
2015a, 2015b; Aprotosoaie et al., 2010). However, the obtained results
from our study showed that the essential oil chemical compositions of
oregano are strongly influenced by genetic factors as the plant were
grown in similar climatic conditions.
It is worth to note that the amount of carvacrol in Baneh population,
increased from one year to another (39.0% and 46. 9%, over 2014 and
Fig. 1. Essential oil content of Origanum vulgare populations (average of 2014 2015, respectively). On the other hand, the amount of p-cymene and
and 2015 harvest years). carvacrol methyl ether decreased (Tables 2 and 3). The amount of
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Table 2
The chemical composition of essential oils of Origanum vulgare populations in 2014 harvest year.
N Components Populations
a b
RI LIT RI Baneh Ardabil Naim Arasbaran Gilan Kaleybar Chalus LSDc IDd
1 α-Thujene 923 924 1.40 ± 0.08 0.25 ± 0.03 0.13 ± 0.04 0.40 ± 0.01 0.35 ± 0.03 0.39 ± 0.01 0.28 ± 0.14 0.11 Std
2 α-Pinene 930 932 0.72 ± 0.04 0.13 ± 0.00 0.25 ± 0.03 0.17 ± 0.01 0.53 ± 0.03 0.19 ± 0.00 0.16 ± 0.10 0.09 Std
3 Camphene 946 946 0.08 ± 0.01 – 0.37 ± 0.19 tr. – 0.05 ± 0.00 0.22 ± 0.18 0.25 RI, MS
4 Sabinene 970 969 0.10 ± 0.05 1.24 ± 0.03 4.11 ± 1.60 1.49 ± 0.29 11.52 ± 2.02 2.14 ± 0.11 0.62 ± 0.27 2.98 Std
5 β-Pinene 975 974 0.19 ± 0.01 – – – – – – 0.01 Std
6 3-Octanone 984 979 0.79 ± 0.31 0.87 ± 0.25 1.71 ± 0.42 0.89 ± 0.01 0.74 ± 0.41 1.19 ± 0.27 0.39 ± 0.03 0.87 RI, MS
7 Myrcene 987 988 2.24 ± 0.05 0.95 ± 0.07 1.98 ± 0.03 1.45 ± 0.08 1.12 ± 0.02 1.18 ± 0.01 1.38 ± 0.33 0.25 Std
8 α-Phellandrene 1005 1004 0.19 ± 0.01 – – – tr. tr. tr. 0.02 RI, MS
9 δ-3-Carene 1007 1008 0.07 ± 0.00 – tr. tr. tr. tr. 0.01 RI, MS
10 α-Terpinene 1015 1014 1.90 ± 0.25 0.11 ± 0.04 0.07 ± 0.00 0.14 ± 0.06 0.20 ± 0.05 0.33 ± 0.07 0.20 ± 0.03 0.09 Std
11 p-Cymene 1023 1020 11.63 ± 1.70 4.55 ± 0.27 1.14 ± 0.44 5.73 ± 0.15 4.10 ± 0.27 5.95 ± 0.07 2.02 ± 0.47 2.09 Std
12 Limonene 1027 1024 – 0.04 ± 0.00 0.50 ± 0.06 0.07 ± 0.01 – tr. 0.26 ± 0.04 0.07 Std
13 β-Phellandrene 1028 1025 – 0.07 ± 0.01 – tr. – tr. tr. 0.02 RI, MS
14 (Z)-β-Ocimene 1034 1032 0.80 ± 0.09 2.70 ± 0.65 5.77 ± 0.94 8.04 ± 1.92 2.39 ± 0.44 5.31 ± 0.42 2.63 ± 0.47 2.67 RI, MS
15 (E)-β-Ocimene 1045 1044 0.39 ± 0.19 1.87 ± 0.94 5.17 ± 0.01 3.35 ± 0.74 1.02 ± 0.25 1.36 ± 0.09 1.24 ± 0.14 1.42 RI, MS
16 γ-Terpinene 1056 1054 14.16 ± 2.77 0.54 ± 0.20 0.54 ± 0.15 1.27 ± 0.33 0.73 ± 0.27 1.40 ± 0.39 1.07 ± 0.20 3.27 Std
17 Terpinolene 1083 1086 tr. – – tr. 0.19 ± 0.13 tr. 0.18 ± 0.02 0.15 RI, MS
18 trans-Sabinene hydrate 1099 1098 0.08 ± 0.03 tr. 12.10 ± 0.26 0.44 ± 0.09 0.24 ± 0.04 2.69 ± 0.79 12.20 ± 2.36 1.87 Std
19 Borneol 1170 1165 0.23 ± 0.12 0.11 ± 0.01 0.14 ± 0.04 0.11 ± 0.03 0.80 ± 0.30 0.10 ± 0.00 0.14 ± 0.11 0.38 RI, MS
20 Terpinen-4-ol 1178 1174 tr. 0.18 ± 0.06 3.62 ± 0.24 0.06 ± 0.01 0.14 ± 0.01 0.08 ± 0.00 2.55 ± 1.37 0.94 RI, MS
21 α-Terpineol 1193 1186 0.06 ± 0.01 – – – – 0.10 ± 0.01 – 0.01 RI, MS
22 cis-Dihydro carvone 1196 1191 tr. – – – 0.10 ± 0.04 tr. – 0.04 Std
23 Thymol methyl ether 1228 1232 0.07 ± 0.03 – – – 1.26 ± 0.95 – – 1.09 RI, MS
24 Carvacrol methyl ether 1237 1241 5.97 ± 2.03 0.40 ± 0.10 – 0.41 ± 0.06 1.19 ± 0.77 0.53 ± 0.10 0.20 ± 0.06 2.49 RI, MS
25 Linalyl acetate 1252 1257 tr. 1.72 ± 0.74 37.36 ± 2.96 0.33 ± 0.04 0.24 ± 0.02 0.23 ± 0.03 44.35 ± 0.23 3.50 RI, MS
26 Thymol 1289 1289 3.69 ± 0.53 0.52 ± 0.16 tr. 0.74 ± 0.25 1.51 ± 0.39 1.75 ± 0.26 0.22 ± 0.19 0.88 Std
27 Carvacrol 1298 1298 46.86 ± 2.47 3.18 ± 0.80 0.36 ± 0.16 1.27 ± 0.77 5.23 ± 1.35 3.05 ± 0.20 1.25 ± 0.85 3.51 Std
28 Carvacrol acetate 1362 1370 – 0.05 ± 0.00 1.30 ± 0.07 0.11 ± 0.01 – 0.10 ± 0.01 1.65 ± 0.16 0.13 RI, MS
29 (E)-β-Caryophyllene 1414 1417 1.01 ± 0.30 13.84 ± 0.76 3.70 ± 0.47 22.76 ± 1.79 0.22 ± 0.09 23.97 ± 0.65 3.08 ± 0.64 2.63 Std
30 α-trans-Bergamotene 1429 1432 0.06 ± 0.01 0.12 ± 0.00 tr. 0.15 ± 0.01 0.53 ± 0.27 0.20 ± 0.06 0.14 ± 0.02 0.31 RI, MS
31 (E)-β-Farnesene 1451 1454 0.14 ± 0.04 2.42 ± 0.05 0.55 ± 0.03 2.45 ± 0.51 0.50 ± 0.23 3.38 ± 0.09 0.24 ± 0.02 0.65 RI, MS
32 α-Humulene 1450 1452 0.06 ± 0.03 0.24 ± 0.01 0.15 ± 0.05 0.46 ± 0.21 1.53 ± 0.52 0.61 ± 0.06 0.15 ± 0.01 0.65 Std
33 Germacrene D 1482 1484 tr. 7.40 ± 7.40 4.70 ± 0.49 19.50 ± 2.75 0.11 ± 0.06 15.93 ± 0.41 0.98 ± 0.51 3.21 RI, MS
34 β-Bisabolene 1504 1505 0.39 ± 0.04 5.20 ± 0.12 0.96 ± 0.36 7.44 ± 0.52 4.24 ± 0.27 11.78 ± 1.68 0.56 ± 0.12 2.08 RI, MS
35 δ-Amorphene 1513 1511 tr. 0.35 ± 0.14 tr. 0.10 ± 0.01 0.35 ± 0.28 0.12 ± 0.00 tr. 0.36 RI, MS
36 β-Sesquiphellandrene 1520 1521 – 0.06 ± 0.01 0.16 ± 0.08 0.08 ± 0.01 0.07 ± 0.03 0.43 ± 0.01 – 0.10 RI, MS
37 (Z)-α-Bisabolene 1538 1521 3.23 ± 0.36 0.35 ± 0.03 0.05 ± 0.01 0.27 ± 0.03 38.17 ± 3.37 0.42 ± 0.00 7.33 ± 1.66 4.04 RI, MS
38 Elemol 1545 1548 – 14.09 ± 1.04 1.49 ± 0.41 0.56 ± 0.03 – 0.40 ± 0.00 – 0.97 RI, MS
39 Spathulenol 1571 1577 – – – 0.51 ± 0.09 – – – 0.1 RI, MS
40 Caryophyllene oxide 1576 1582 0.11 ± 0.03 13.33 ± 1.38 1.59 ± 0.11 6.40 ± 1.43 3.11 ± 0.98 5.45 ± 0.28 1.01 ± 0.31 2.57 Std
41 Shyobunol 1687 1688 0.06 ± 0.03 3.06 ± 0.41 0.27 ± 0.08 2.25 ± 0.22 1.18 ± 0.04 1.17 ± 0.16 0.07 ± 0.01 0.57 RI, MS
Grouped compounds
Monoterpene 33.90 12.43 20.02 22.20 22.17 18.41 10.29
hydrocarbons
Oxygenated 57.02 6.16 54.90 3.47 10.70 8.63 62.55
monoterpens
Sesquiterpene 4.96 29.96 10.31 53.21 45.72 56.83 12.49
hydrocarbons
Oxygenated 0.15 30.47 3.34 9.71 4.29 7.02 1.08
sesquiterpens
Other 0.79 0.87 1.71 0.89 0.74 1.19 0.39
Total identified (%) 96.82 79.89 90.27 89.47 83.63 92.07 86.79
linalyl acetate in population from Namin, however, slightly increased Furthermore, the harvest years affected the main compositions of the
from 33.8% to 37.4% in 2014 and 2015 respectivly. The amount of p- essential oils of Echinophora tenuifolia subsp. sibthorpiana leaves,
cymene in the essential oil of the populations from Ardabil, Arasbaran, growing wild in Middle Anatolia. Methyl-eugenol, δ-3-carene and p-
Gilan and Chalus was decreased over 2014 to 2015. Previous studies cymene were found as the main constituents of the essential oils were
have demonstrated a broad variation in the essential oil composition depending on the harvest years (Chalchat et al., 2011). On the contrary,
from different species, depending on the harvest year. The work of chemical compositions of the essential oils of Rosmarinus officinalis L.
Mechergui et al. (2016) on O. vulgare subsp. glandulosum populations were not significantly affected by different harvest years (Atti-Santos
showed qualitative and quantitative differences in the chemical com- et al., 2005).
position of the essential oils from one harvest year to another. There was a significant variability in the amounts of the main
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Table 3
The chemical composition of essential oils of Origanum vulgare populations in 2015 harvest year.
N. Components Populations
a b
RI LIT RI Baneh Ardabil Namin Arasbaran Gilan Kaleybar Chalus LSDc IDd
1 α-Thujene 923 924 0.88 ± 0.11 0.32 ± 0.05 0.25 ± 0.02 0.36 ± 0.06 0.30 ± 0.00 0.29 ± 0.03 0.39 ± 0.07 0.16 Std
2 α-Pinene 930 932 0.79 ± 0.10 0.47 ± 0.08 0.24 ± 0.01 0.25 ± 0.02 0.35 ± 0.02 0.19 ± 0.01 0.28 ± 0.05 0.14 Std
3 Camphene 946 946 – – 0.19 ± 0.05 0.05 ± 0.00 – 0.05 ± 0.00 0.33 ± 0.11 0.09 RI, MS
4 Sabinene 970 969 0.89 ± 0.2 2.69 ± 0.29 3.32 ± 0.83 2.23 ± 0.18 6.45 ± 0.29 1.68 ± 0.31 1.85 ± 0.12 1.51 Std
5 β-Pinene 975 974 0.20 ± 0.06 – – – – – – 0.06 Std
6 3-Octanone 984 979 5.46 ± 1.47 3.77 ± 0.38 3.01 ± 0.49 4.26 ± 1.20 0.90 ± 0.01 2.19 ± 0.31 1.47 ± 0.01 2.31 RI, MS
7 Myrcene 987 988 0.89 ± 0.23 2.07 ± 0.14 1.44 ± 0.06 1.39 ± 0.23 1.50 ± 0.06 1.14 ± 0.12 1.25 ± 0.12 0.56 Std
8 3-Octanol 996 988 – 2.91 ± 0.81 1.24 ± 0.15 1.51 ± 0.22 – – 1.69 ± 0.37 1.51 RI, MS
9 α-Phellandrene 1005 1004 0.23 ± 0.07 – – 0.20 ± 0.05 tr. tr. 0.03 ± 0.01 0.09 RI, MS
10 δ-3-Carene 1007 1008 0.02 ± 0.00 – 0.56 ± 0.01 0.23 ± 0.06 – 0.43 ± 0.01 0.02 ± 0.01 0.07 RI, MS
11 α-Terpinene 1015 1014 1.55 ± 0.10 tr. tr. 0.39 ± 0.05 0.12 ± 0.01 0.34 ± 0.01 0.26 ± 0.01 0.98 Std
12 p-Cymene 1023 1020 14.80 ± 0.40 13.89 ± 1.05 5.00 ± 0.19 9.89 ± 1.51 11.11 ± 0.58 7.49 ± 0.80 10.83 ± 1.23 2.99 Std
13 Limonene 1027 1024 – 0.07 ± 0.02 0.37 ± 0.02 – – 0.06 ± 0.00 0.06 ± 0.00 0.03 Std
14 β-Phellandrene 1028 1025 tr. 0.09 ± 0.00 – 1.04 ± 0.09 – 0.06 ± 0.00 0.06 ± 0.03 0.10 RI, MS
15 (Z)-β-Ocimene 1034 1032 3.84 ± 0.15 5.51 ± 1.11 5.59 ± 0.58 6.12 ± 2.05 1.15 ± 0.01 5.88 ± 1.63 2.98 ± 1.80 3.52 RI, MS
16 (E)-β-Ocimene 1045 1044 0.59 ± 0.17 0.23 ± 0.00 1.45 ± 0.34 1.38 ± 0.31 0.30 ± 0.01 0.48 ± 0.04 0.59 ± 0.36 0.61 RI, MS
17 γ-Terpinene 1056 1054 11.10 ± 0.61 0.07 ± 0.00 0.69 ± 0.12 2.04 ± 0.42 0.23 ± 0.00 0.73 ± 0.14 0.99 ± 0.71 0.98 Std
18 Terpinolene 1083 1086 0.22 ± 0.01 – – 0.13 ± 0.05 tr. 0.07 ± 0.01 0.12 ± 0.01 0.05 RI, MS
19 trans-Sabinene hydrate 1099 1098 tr. tr. 9.13 ± 0.68 0.24 ± 0.02 0.18 ± 0.01 1.38 ± 0.33 5.45 ± 0.35 0.89 Std
20 Borneol 1170 1165 0.99 ± 0.08 0.07 ± 0.01 0.37 ± 0.07 0.17 ± 0.03 0.27 ± 0.03 0.15 ± 0.03 0.27 ± 0.07 0.01 RI, MS
21 Terpinen-4-ol 1178 1174 tr. 0.06 ± 0.01 3.47 ± 0.38 0.40 ± 0.06 0.21 ± 0.01 0.08 ± 0.01 0.84 ± 0.52 0.56 RI, MS
22 α-Terpineol 1193 1186 0.05 ± 0.00 – – – – 0.20 ± 0.06 – 0.06 RI, MS
23 cis-Dihydro carvone 1196 1191 – – – tr. tr. 0.07 ± 0.00 – 0.02 Std
24 Thymol methyl ether 1228 1232 0.15 ± 0.06 – – 0.15 ± 0.07 0.52 ± 0.00 – – 0.10 RI, MS
25 Carvacrol methyl ether 1237 1241 9.69 ± 1.69 0.49 ± 0.05 – 1.25 ± 0.54 0.30 ± 0.03 0.66 ± 0.01 0.33 ± 0.01 2.04 RI, MS
26 Linalyl acetate 1252 1257 tr. 0.39 ± 0.03 33.83 ± 3.30 0.31 ± 0.06 0.70 ± 0.01 0.29 ± 0.03 39.74 ± 6.27 6.65 RI, MS
27 Thymol 1289 1289 2.42 ± 0.65 0.17 ± 0.04 tr. 0.12 ± 0.01 0.20 ± 0.00 0.85 ± 0.20 – 0.78 Std
28 Carvacrol 1298 1298 39.0 ± 2.87 0.50 ± 0.04 0.65 ± 0.14 0.87 ± 0.24 0.80 ± 0.03 1.60 ± 0.52 0.95 ± 0.35 2.4 Std
29 Carvacrol acetate 1362 1370 – tr. 1.63 ± 0.09 – – 0.09 ± 0.04 1.88 ± 0.93 0.62 RI, MS
30 (E)-β-Caryophyllene 1414 1417 0.49 ± 0.17 13.88 ± 1.36 3.23 ± 0.46 18.83 ± 0.31 0.07 ± 0.00 20.14 ± 0.94 6.57 ± 2.29 2.51 Std
31 α-trans-Bergamotene 1429 1432 tr. 0.31 ± 0.11 – tr. 0.60 ± 0.04 0.11 ± 0.00 0.11 ± 0.01 0.12 RI, MS
32 (E)-β-Farnesene 1451 1454 tr. 1.77 ± 0.33 0.91 ± 0.12 2.30 ± 0.31 0.33 ± 0.06 3.33 ± 0.12 0.24 ± 0.09 0.55 RI, MS
33 α-Humulene 1450 1452 tr. 0.29 ± 0.02 0.15 ± 0.07 0.30 ± 0.06 4.59 ± 0.58 0.47 ± 0.24 0.14 ± 0.02 0.72 Std
34 Germacrene D 1482 1484 0.06 ± 0.01 5.06 ± 0.21 4.05 ± 0.77 13.02 ± 1.07 0.90 ± 0.00 13.79 ± 0.57 0.31 ± 0.19 3.32 RI, MS
35 β-Bisabolene 1504 1505 0.63 ± 0.02 2.87 ± 0.31 1.29 ± 0.42 1.98 ± 0.52 3.78 ± 0.29 1.59 ± 0.40 0.82 ± 0.14 1.01 RI, MS
36 δ-Amorphene 1513 1511 tr. 0.10 ± 0.00 – – 0.13 ± 0.00 0.11 ± 0.00 tr. 0.01 RI, MS
37 β-Sesquiphellandrene 1520 1521 – 0.05 ± 0.00 0.05 ± 0.01 – 0.11 ± 0.03 0.23 ± 0.05 – 0.06 RI, MS
38 (Z)-α-Bisabolene 1538 1521 2.67 ± 0.76 0.25 ± 0.03 1.75 ± 0.49 0.05 ± 0.02 40.29 ± 2.44 0.37 ± 0.03 3.62 ± 1.81 2.28 RI, MS
39 Elemol 1545 1548 – 12.94 ± 1.70 1.67 ± 0.44 0.32 ± 0.05 – – – 2.00 RI, MS
40 Spathulenol 1571 1577 – – 1.10 ± 0.32 – – – – 0.00 RI, MS
41 Caryophyllene oxide 1576 1582 0.24 ± 0.09 21.34 ± 1.95 4.56 ± 0.50 10.89 ± 1.29 5.74 ± 0.87 18.40 ± 2.68 4.85 ± 0.96 4.27 Std
42 Shyobunol 1687 1688 tr. 1.75 ± 0.20 0.42 ± 0.05 1.82 ± 0.70 2.80 ± 0.29 1.80 ± 0.19 0.17 ± 0.13 0.93 RI, MS
Grouped compounds
(%)
Monoterpene 36.03 25.42 19.1 25.70 21.55 18.90 20.01
hydrocarbons
Oxygenated 52.37 1.72 49.09 3.54 3.20 5.35 49.45
monoterpens
Sesquiterpene 3.99 24.57 11.42 36.51 50.8 40.11 11.81
hydrocarbons
Oxygenated 0.27 36.03 7.73 13.03 8.54 20.19 5.02
sesquiterpens
Other 5.45 6.68 4.25 5.77 0.9 2.19 3.16
Total identified (%) 98.09 94.42 91.59 84.57 85.00 86.75 89.44
chemical classes in the essential oils (Tables 2 and 3). Oxygenated (Mechergui et al., 2016; Verma et al., 2010). In accordance with
monoterpenes represented the major fraction in the essential oils from Aprotosoaie et al. (2010), a significant increase in the monoterpenes
Baneh (52.37–57.02%), Namin (49.09–54.90%), and Chalus level from 12.55% (2002) to 19.29% (2003) was noted in fennel fruits
(49.45–62.55%) populations in both 2014 and 2015. Interestingly, the (Foeniculum vulgare Mill.). According to our results, in both studied
amount of these components increased in all three mentioned popula- years sesquiterpene hydrocarbons were the major fraction in the es-
tions during the second harvest year. Similar results were reported for sential oils from Arasbaran (36.51 and 53.21%), Gilan (50.8 and
some populations of O. vulgare growing in North Africa and India 45.72%) and Kaleybar (40.11and 56.83%), while oxygenated
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M.R. Morshedloo et al. Industrial Crops & Products 119 (2018) 183–190
sesquiterpenes constituted the major fraction in the sample from Ar- Overall, the seven populations were clustered in 4 groups being char-
dabil (36.03% and 30.47%). These classes were revealed as the pre- acterized by different major compounds (Fig. 3A). Population from
dominant components in some populations growing in India (Verma Baneh was in the left upper side of the score plot and was correlated
et al., 2010) and Europe (Lukas et al., 2015; Mockutë et al., 2004). Our with high amounts of carvacrol. Populations from Namin and Chalus
results demonstrated that chemical compositions of the Iranian oregano were those in the right side of the score plot and were characterized by
may be strongly influenced by genetic factors, as we removed the effect high levels of linalyl acetate. Population from Gilan was in the middle
of environmental and climate conditions by planting the oregano po- part of the score plot being correlated with (Z)-α-bisabolene and, to a
pulations in the same soil, temperature and humidity inside the glass minor extent, γ-terpinene. Finally, populations from Ardabil, Kaleybar
greenhouse. The documented chemical differences may also depend on and Arabsbaran were those in the lower side of the score plot and were
the variability in the structure and distribution of glandular trichomes rich in sesquiterpenes such as (E)-β-caryophyllene, germacrene D and
during the growing seasons (Giuliani et al., 2013). Further studies are cayophyllene oxide. Therefore, PCA permitted to classify the Iranian
needed in order to elucidate the intraspecific genetic variability of Ir- oregano populations into four chemotypes, namely I carvacrol, II linalyl
anian oregano. acetate, III (Z)-α-bisabolene and IV sesquiterpenes ((E)-β-car-
yophyllene,/germacrene D/cayophyllene oxide).
According to the present and previous studies, O. vulgare is an ex-
3.3. Characterization of chemotypes tremely variable species and many different chemotypes have been
previously reported from this valuable herb (Lukas et al., 2015; Verma
A cluster analysis (CA) and principal component analysis (PCA) et al., 2010; De Martino et al., 2009; Russo et al., 1998). Such differ-
were performed to determine the chemotypes in oregano populations, ences in chemical components of oregano populations offer a wide
based on their essential oil constituents. Similar patterns were observed range for selection of desired chemotypes, as a part of domestication
among constructed dendrograms in both harvest years. According to and breeding programs of this valuable species for industrial purposes
the constructed dendrogram, the investigated populations were divided (Aboukhalid et al., 2016).
into four main groups, each representing a separate chemotype (Fig. 2). This study has contributed to the primary knowledge of Iranian O.
Cluster analysis exhibited a distinct separation of the population from vulgare wild populations cultivated in the same agricultural conditions.
Baneh (O. vulgare subsp. gracile) and classified this population into the The chemical variation observed among oregano populations according
carvacrol chemotype (chemotype I). γ-Terpinene and p-cymene, how- to their genetics and geographic origin (bioclimatic distribution) im-
ever, were the other main components of this population. Previous poses that conservation strategies should be made appropriately, taking
studies on oregano populations demonstrated that O. vulgare subsp. into account genetic and environmental factors. Different chemotypes
gracile is richer in essential oil and usually accumulate large amounts of of the plant in Iran and other countries can be utilized for the genetic
phenolic monoterpenes deriving from the ‘cymyl’-pathway (mainly improvement in order to produce high quality oregano.
carvacrol and its biosynthetic precursor's γ-terpinene and p-cymene)
(Morshedloo et al., 2017a; Lukas et al., 2015; Moradi et al., 2014;
Kokkini, 1996). Chemotype II was composed of two populations 4. Conclusion
(Namin and Chalus) characterized by high amount of linalyl acetate
(33.83–44.35%). A similar chemotype was previously recorded in Al- The present study was performed to characterize the chemical di-
bania (Lukas et al., 2015), Italy (De Martino et al., 2009) and Iran versity of oregano populations were from collected different regions of
(Afsharypour et al., 1997). Chemotype III was composed of one popu- Iran. A considerable qualitative and quantitative variation was ob-
lation (Gilan) with (Z)-α-bisabolene (38.17–40.29%) as the major served in the chemical profile of the investigated essential oils.
compound. The high content of caryophyllene oxide, followed by ger- Carvacrol, linalyl acetate, (Z)-α-bisabolene, (E)-β-caryophyllene, car-
macrene D and (E)-β-caryophyllene differentiated the populations from yophyllene oxide, germacrene D and elemol were the major compo-
Ardabil, Arasbaran and Kaleybar as distinct chemotype (chemotype IV: nents of the essential oils depending on the population and/or sub-
(E)-β-caryophyllene/germacrene D/cayophyllene oxide). species. In some cases, the amount of components was affected over two
A similar grouping was obtained by PCA. The score and loading different harvest years. Cluster analysis grouped the studied popula-
plots obtained by analyzing the average values of essential oil compo- tions into four distinct groups which were not mostly in accordance
sitions of seven oregano populations over 2014 and 2015 harvest years, with their geographical origins, indicating an important role of genetic
are reported in Fig. 3 where the 2D graphical representation of PCA factors than geographical origin. Present of such variability in the
represents 71.76% of the total variance in the data set. The components chemical profile of studied populations enabled selection of those with
affording the highest contribution to the variability of data were linalyl specific aroma or bioactive constituents for use in relevant industries,
acetate (values of eigenvectors: 18.16; 4.31) in the first PC and carva- and suggested that the in situ and ex situ conservation strategies should
crol (values of eigenvectors: −8.43; 11.40) and (E)-β-caryophyllene concern all populations, representing the different chemotypes.
(values of eigenvectors: −2.06; −8.42) in the second PC (Fig. 3B).
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M.R. Morshedloo et al. Industrial Crops & Products 119 (2018) 183–190
Fig. 3. Score plot (PCA) obtained from the main variation of volatile compositions among Iranian oregano populations over 2014 and 2015 harvest years. A) The PCA
loading plot for volatile constituents which explains 41.57% of the variation on horizontal axis (PC 1) and 30.19% on the vertical axis (PC 2).
Acknowledgements growth and some physiological characteristics of Staphylococcus aureus strains iso-
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Center, University of Massachusetts, Amherst, USA) for his kind assis- (Liguria Region, Northern Italy). Ann. Bot. 86, 471–478.
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by research grant from University of Massachusetts, Amherst, USA, Origanum vulgare L. ssp hirtum (Link) Ietswaart growing wild in Campania (Southern
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