Chemical Polymorphism of Origanum compactum Grown …webagris.inra.org.ma/doc/douaik016.pdf · Chemical Polymorphism of Origanum compactum Grown in All ... UR Plantes Aromatiques
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
FULL PAPER
Chemical Polymorphism of Origanum compactum Grown in All Natural Habitats inMorocco
by Kaoutar Aboukhalid*a)b), Abdeslam Lamirib), Monika Agacka-Mołdochc), Teresa Doroszewskac), Ahmed Douaikd),
Mohamed Bakhaa)e), Joseph Casanovaf), F�elix Tomif), Nathalie Machong), and Chaouki Al Faiza)
a) Institut National de la Recherche Agronomique, UR Plantes Aromatiques et M�edicinales, INRA, CRRA-Rabat, PB 6570,
10101 Rabat, Morocco (phone: +212661265485, e-mail: [email protected])b) Laboratoire de Chimie Appliqu�ee et Environnement, Facult�e des Sciences et Techniques, Universit�e Hassan I, BP 577,
26000 Settat, Moroccoc) Institute of Soil Science and Plant Cultivation, State Research Institute, ul. Czartoryskich 8, PL-24-100 Puławy
d) Institut national de la Recherche Agronomique, UR Environnement et Conservation des Ressources Naturelles, INRA,
CRRA-Rabat, PB 6570, 10101 Rabat, Moroccoe) Laboratoire de Biologie et Sant�e, Facult�e des sciences, Universit�e Abdelmalek Essaadi, BP 2121, 93002 T�etouan, Morocco
f) UMR 6134 SPE, Equipe Chimie et Biomasse, Universit�e de Corse-CNRS, Route des Sanguinaires, FR-20000 Ajacciog) UMR 7204 CESCO, D�epartement d’Ecologie et gestion de la Biodiversit�e, Mus�eum National d’Histoire Naturelle, 55 rue
Buffon, FR-75005 Paris
Origanum compactum L. (Lamiaceae) is one of the most important medicinal species in term of ethnobotany in Morocco.
It is considered as a very threatened species as it is heavily exploited. Its domestication remains the most efficient way to
safeguard it for future generations. For this purpose, wide evaluation of the existing variability in all over the Moroccan
territory is required. The essential oils of 527 individual plants belonging to 88 populations collected from the whole
distribution area of the species in Morocco were analyzed by GC/MS. The dominant constituents were carvacrol (0 – 96.3%),
thymol (0 – 80.7%), p-cymene (0.2 – 58.6%), c-terpinene (0 – 35.2%), carvacryl methyl ether (0 – 36.2%), and a-terpineol(0 – 25.8%). While in the Middle Atlas region and the Central Morocco mainly carvacrol type samples were found, much
higher chemotypic diversity was encountered within samples from the north part of Morocco (occidental and central Rif
regions). The high chemical polymorphism of plants offers a wide range for selection of valuable chemotypes, as a part of
breeding and domestication programs of this threatened species.
Keywords: Origanum compactum, Essential oils, Chemical variability, Morocco.
Introduction
The genus Origanum is a taxonomically complex group
of aromatic plants that are used all over the world fortheir aromatic and medicinal properties and as a culinary
herb [1]. According to Ietswaart’s classification [2], thegenus Origanum has been divided into 38 species, 6 sub-
species, and 17 hybrids, arranged in three groupsand 10 sections. The genus Origanum has a local distri-
bution mostly around the Mediterranean basin, and it ischaracterized by a large morphological and chemical
diversity [3].In Morocco, the genus Origanum is represented by
five taxa, three of which, O. elongatum (BONNET) EMB &MAIRE, O. grosii PAU & FONT QUER, and O. frontqueri
PAU are endemic to the central Rif region. O. vulgare
subsp. virens (HOFFM. et LINK) IETSWAART is also commonto the Iberian Peninsula, while O. compactum BENTH. is
endemic to Morocco and southern Spain [2]. O. com-
pactum BENTH. is the most widespread species in Mor-occo, extending from the Middle Atlas region delimited
by Beni mellal, Azrou, Khenifra, and Oulm�es up to theoccidental and central Rif region, including the provinces
of Tangier-Tetouan, Chefchaouen, Taounate, and Ouaz-zane [4][5]. O. compactum BENTH., known locally as‘Zaatar’, constitutes one of the most appreciated aromatic
herbs, widely used in Moroccan folk medicine in the formof infusions and decoctions to threat broncopulmonary,
gastric acidity, gastrointestinal diseases, and numerousinfections [6]. Due to its pleasant flavor and spicy fra-
grance, O. compactum is the aromatic ingredient ofchoice for flavoring some traditional dishes (barley soup,
couscous, etc.).Steam distillation of aerial parts of O. compactum
BENTH. produces an essential oil (EO), which is appre-ciated for its aromatic and medicinal properties:
antifungal [7 – 9], antibacterial [10][11], and antioxidant
effects [12]. Previous studies on Moroccan O. com-
pactum BENTH. EOs revealed a wide chemical diversity.Compositions were dominated either by carvacrol or by
thymol. Mixed types, combining both thymol andcarvacrol, and types containing a high level of precur-
sors, c-terpinene and p-cymene, have also been re-ported [13 – 15]. The various compositions have
been summarized in a previous paper that describedalso the chemical variability observed on 36 oil samples
isolated from plants harvested in three Moroccanprovinces: Chefchaouen, Larache, and Tetouan [4].
Two-thirds of the samples exhibited carvacrol as majorcomponent.
Nowadays, O. compactum BENTH. is considered as athreatened species due to a dramatic population decline
caused by various factors: overexploitation, drought,overgrazing, combined with unsustainable and destruc-
tive methods of harvesting, through up-rooting in thewild, and collecting essentially during the flowering per-
iod, before seed set. Since the decline of natural popu-lations, an urgent attempt to set up a domestication
program should be initiated to ensure the conservationand a sustainable utilization of this valuable medicinal
plant. Nevertheless, the domestication of wild medicinal
species requires a good understanding of the chemicaland genetic diversity within the species. Although vari-ous studies have been carried out in order to charac-
terize Moroccan O. compactum BENTH. EOs, thesestudies were restricted to a limited number of samples.
Moreover, these studies did not cover the entire areaabout wild-growing O. compactum BENTH. where this
plant still subsists, and only few studies specified thegeographical origin of samples. Furthermore, only the
EO composition of mixed plant samples was reportedand no study to date has been undertaken at intrapop-
ulation level. For instance, most Origanum species havecross-pollinated reproductive system [15], which can
lead to a high level of genetic polymorphism withinpopulations. This variation may eventually influence the
genetic control of accumulation of specific compoundsamong the secondary metabolites [16]. In this paper,
we report an analysis of the EO composition ofO. compactum BENTH. individual plants distributed all
over the Moroccan territory. This is the first report ofa deep study on native populations of O. compactum
BENTH. Such information would be fundamental to pro-mote a domestication program of the species at
Fig. 1. Geographical distribution of the 88 Origanum compactum accessions (noted A in Table 1) sampled from the 12 regions. The map was
national scale, by the selection of the most valuableand performant chemotypes or/and establish in situ
conservation program.
Results and Discussion
Individual plants of O. compactum have been collected
from all the Moroccan sites were they grow sponta-neously. In detail, 527 plants have been collected in 88
locations covering most of the natural habitats of thespecies in Morocco (Fig. 1). The prospected areas were:
i) the northern region (provinces of Tangier-Tetouan,Chefchaouen, Ouazzane, and Taounate) that provided
57% of the samples; ii) the central Morocco (Bensli-mane, Rommani, Oulm�es, Moulay Driss Zerhoun,
and Sidi Kacem, 23% of the samples); iii) the MiddleAtlas (Azrou, Khenifra, and Beni Mellal, 20% of thesamples).
Various regions have been explored for the first time:Benslimane, Azrou, Khenifra, Beni Mellal, Tangier, Sidi
Kacem, and Moulay Driss Zerhoun.
Essential-Oil Yield
Yield of EOs isolated from the aerial parts of 88O. compactum populations varied drastically from sam-
ple to sample. Yields ranged from 0.67 to 2.88% of the
dry matter (Table 1), depending on the accession ori-gin. Our results revealed noticeable spatial variation inEO yield of O. compactum. The bioclimatic differences
among the 12 investigated regions seem to have a sig-nificant effect on the EO content. Populations dis-
tributed under a semiarid climate showed the highestlevels of EO yield (average: 2.15%), while accessions
located under a subhumid climate, displayed a rela-tively lower EO content (average: 1.71%). The lowest
EO yield (average: 1.46%) was recorded in the north-ern part of the country, Tangier-Tetouan and
Chefchaouen provinces, exposed to humid climate. Onepopulation from Chefchaouen (A34) was located in a
perhumid climate. This sample was characterized by arelatively low EO content (0.91%). Thus, the observed
yields of EOs increase significantly from humid to aridzones (Fig. 2). In general, it is recognized that plants
growing in arid areas tend to produce high levels ofEO as an adaptive mechanism in response to
water stress [17]. For instance, Azizi et al. [18] demon-strated that water deficiency increases EO content of
O. vulgare.Otherwise, no significant correlation in the EO yields
related to the altitude was observed. These findings agreewith Bakhy et al. [4] for O. compactum, but they differ
with Vokou et al. [17] and Kokkini and Vokou [19] whoemphasized that altitude is the most important
Table 1. (cont.)
Region Accession no. Collection site Samples No. Altitude [m] Climate EO Yield [%]
environmental factor influencing the oil content of O. vul-
gare subsp. hirtum.
Essential Oils Chemical Variability
The 527 individual plants belonging to 88 populations ofO. compactum were analyzed by GC/MS. The chemical
composition of the EOs can be summarized by a mixtureof 34 predominant mono- and sesquiterpenes. The
monoterpene fraction (69 – 99.9%) was dominant andconsisted mainly of oxygenated monoterpenes
(21.4 – 99.1%), followed by monoterpene hydrocarbons(0.3 – 76.8%). Carvacrol (up to 96.3%), thymol (up to
80.7%), a-terpineol (up to 25.8%), and carvacryl methylether (up to 36.2%) were the major oxygenated mono-
terpenes, while c-terpinene (up to 35.2%) and p-cymene(up to 58.6%) were the most highly representedcompounds of the monoterpene hydrocarbons class
(Fig. 3). The sesquiterpene fraction occurred only in smal-ler proportions (up to 12.4%), (E)-b-caryophyllene (up to
11.5%) being its main component (average: 0.7%).
Before doing statistical analysis, four oil samples iso-lated from aerial parts of O. compactum were subjectedto quantitative determination using nonane as internal
standard and correction factors according to Costa et al.[20] and Bicchi et al. [21]: a carvacrol-rich oil sample, a
thymol-rich oil sample and two mixed types, carvacrol/p-cymene and thymol/p-cymene. Results are reported in
Table 2.The 527 compositions were subjected to principal
component analysis (PCA). Twelve major compoundsdetected at an average concentration higher than 0.5%
have been considered for the statistical analysis (a-thu-jene, myrcene, a-terpinene, p-cymene, c-terpinene, cis-
sabinene hydrate, linalool, a-terpineol, carvacryl methylether, thymol, carvacrol, and (E)-b-caryophyllene).These components constituted 86.2 – 99.6% of the totaloils. PCA reduced the 12 variables to four principal
components with eigenvalues higher than 1. The firstprincipal component (PC1) underlines the positive cor-
relation between a-thujene, myrcene, a-terpinene, andc-terpinene. In contrast, PC1 is negatively related to the
Fig. 2. Mean essential-oil yield (%) of the 88 Origanum compactum accessions studied according to the four bioclimatic stages (semiarid,
subhumid, humid, and perhumid).
Fig. 3. Major monoterpenes in the essential oils of MoroccanOriganum compactum.
content of cis-sabinene hydrate and linalool (Fig. 4).The second component (PC2) is the expression of thenegative correlation between thymol and carvacrol
(Fig. 4). The data presented in Fig. 5 shows the distri-bution of the individuals in the space of the first two
principal components (PC1 + PC2). These latterexplained cumulatively 57.6% of the total variance
(Table 3). PC2 clearly separated O. compactum plantsrich in thymol, observed mainly on the left side of
Fig. 5, from those that contain high amounts of car-vacrol, observed mainly on the right side. Along the
second axis, it is possible to identify a zone of disconti-nuity near the zero point. This is because no sample
contained simultaneously low concentrations of car-vacrol and thymol. The third component, describing
10.3% of the total variance, is positively correlated withp-cymene and (E)-b-caryophyllene, while the last factor,
explaining 8.9% of the data variability, is positivelyrelated to the content of a-terpineol and carvacryl
methyl ether (Fig. 6). With regard to PC1, six of the527 oil samples (13, 92, 354, 435, 446, and 496) have
the strongest positive scores, indicating that they are
characterized by high amount of a-thujene, myrcene,
a-terpinene, and c-terpinene. With respect to PC2, mostof the oil samples from the 12 regions showed interme-diate to high negative scores, implying their high car-
vacrol content, however, some samples have particularlyhigh positive scores. These samples exclusive to Ouaz-
zane province (samples 214, 246, 247, and 248) arecharacterized by their very high thymol content.
Regarding PC3, oil samples 198, 226, 227, 259, and 343from Ouazzane province showed the highest positive
scores and, consequently, they have the highest p-cym-ene and (E)-b-caryophyllene content. Concerning PC4,
samples 14, 8, 10, 4, 200, 12, 16, 2, 23, 202, and 22from Tangier-Tetouan and Ouazzane provinces showed
the highest positive scores. These samples are character-ized by their very high a-terpineol and carvacryl methyl
ether content (Fig. 7). Cluster Analysis (CA) was per-formed to classify and differentiate the analyzed
O. compactum samples according to their major con-stituents. Fig. 8 presents the corresponding dendrogram
using Ward’s method. Considering the 12 major con-stituents of the 527 samples, four major groups were
defined, whose composition is summarized in Table 4.The resulting dendrogram reported in Fig. 8 reflects the
qualitative heterogeneity of wild Moroccan O. com-
pactum EOs and showed the existence of high intrapop-
ulation variability within the EOs. The following groupshave been defined:
Group I: This group was represented by 107 sampleswidespread along the distribution range of the species.
Carvacrol (34.8 – 65.6%, M = 54.9%), p-cymene(5.9 – 36.4%, M = 15.7%), and c-terpinene (2.6 – 35.2%,
M = 18.4%) were the major components. One samplefrom Ouazzane (A66) was the most dissimilar within the
group for its considerable amount of (E)-b-caryophyllene(11.5%), found at insignificant amount in all the remain-ing samples belonging to this group.
Group II: This group is the largest in terms of num-ber of samples including 274 individuals (52% of sam-
ples) and representing the most typical EO profile ofMoroccan wild O. compactum. Carvacrol is the main
component (44 – 96.6%, M = 76.2%). Interestingly,among the carvacrol-rich oils, the highest content
(90.2 – 96.7%) was observed in 24 individuals fromBenslimane (A13 and A14), Ouazzane (A53, 55, 56, 57,
58, 59, 60, 62, 65, and 66), Oulm�es (A87), Taounate(A18), and Moulay Driss Zerhoun (A81). This is the
highest percentage of this compound detected up todayin O. compactum EOs. Very few papers have reported
an oregano chemotype characterized by such excep-tional amount of carvacrol. Indeed, Koc et al. [22]
revealed that carvacrol (up to 93%) was the dominantvolatile component of Turkish O. bilgeri. For the Greek
oregano (O. vulgare subsp. hirtum), carvacrol was alsodetected in a substantial amount (93.8 – 95%) [19][23].
This exceptional carvacrol content in O. compactum
plants reflects the particular importance of this species
Table 2. Composition of four oil samples representative of each
defined group of the Moroccan Origanum compactum (main compo-
nents, Contents (g/100 g) calculated using correction factors)
and the high potential to produce improved rich car-vacrol varieties. In fact, carvacrol is regarded as themost required component in the oregano EOs. How-
ever, the role of the other minor components shouldnot be neglected as they have been reported to act as
synergists [24]. Exploring the results of statistical analy-sis, some samples stand out from the others for some
particular composition. In fact, 17 samples originatedfrom Tangier-Tetouan (A1, 2, 3, and 4) and Che-
fchaouen (A30, 34, 35, and 36) have shown a relativelyhigh content of carvacryl methyl ether (4.6 – 19.9%)
and a-terpineol (0.6 – 20.1%) in comparison with theaverage content, lower than 1% in all other samples of
this group.Group III: This group appeared less homogenous
and shows high content of p-cymene, c-terpinene, carva-crol, and carvacryl methyl ether. Based on the relative
abundance of these compounds, this group could besubdivided into two subgroups with distinct characteris-
tics:Subgroup 1: This subgroup contains 34 samples; p-cym-
ene (4.7 – 58.6%, M = 28.8%), c-terpinene (4.3 – 34.2%,M = 18.8%), carvacrol (0.5 – 42.2%, M = 26.3%), and car-
vacryl methyl ether (0 – 36.2%, M = 9.9%) were the mostrelevant components. a-Terpineol reached considerable
contents in samples 8, 10 (A3), and 16 (A4) (up to 12.5,15.4, and 16.6%, respectively).
Among this subgroup, 10 of the surveyed plants, local-ized in Tangier-Tetouan (A1, A3, and A4), and Ouazzaneregions (A41) were characterized by the preeminence of
carvacryl methyl ether, dominating for some samples, thefour monoterpenes involved in the phenolic biosynthetic
pathway. The exceptionally high content of carvacrylmethyl ether (20.1 – 33.8%) in these samples is remarkable
as this compound is usually detected in the whole genusOriganum in either negligible amounts or traces only. A
few papers have reported an oregano chemotype character-ized by the presence of carvacryl methyl ether at such
appreciable amount but never in O. compactum. Hazzit
et al. [25] referred to O. floribundum oil sample with
noticeable amount (6.9%) of carvacryl methyl ether. Highlevel of carvacryl methyl ether (11.4%) was also recorded
in O. vulgare subsp. glandulosum from Algeria [26]. Fur-thermore, eight samples (A3, 21, 33, 34, 36, 43, 47, and 82)
originated from Tangier-Tetouan, Taounate, Chefchaouen,Ouazzane, and Moulay Driss Zerhoun, were distinguished
by the highest amount of p-cymene (37.5 – 58.6%). Highlevel of p-cymene was also identified in O. glandulosum
from Tunisia (36 – 46%) [27].Subgroup 2: This subgroup composed of 29 samples
originated from Chefchaouen, Ouazzane, and Taounate.Thymol, (16 – 52.2%, M = 31.3%), carvacrol
(0.2 – 50.6%, M = 14.5%), p-cymene (4.1 – 44.2%, M =24%), and c-terpinene (1 – 27.8%, M = 16.9%) were the
Fig. 4. Loading plot for the principal component analysis: oil components in the PC1/PC2 plan, including a-thujene, myrcene, a-terpinene,c-terpinene, cis-sabinene hydrate, linalool, thymol, and carvacrol.
most abundant compounds. Four O. compactum plantsgrown in Ouazzane and Chefchaouen (A24, 25, 27, and31), showed the codominance of the phenolic compounds
thymol (33.6 – 52.2%) and carvacrol (31 – 50.6%).p-Cymene (4.1 – 8.4%) and c-terpinene (1 – 10.1%) were
less represented.Among this subgroup, two samples, 153 and 449,
from Chefchaouen (A30) and Khenifra (A74), respec-tively, were clearly outstanding and may not be repre-
sentative as a new chemotype since only one samplecharacterize these chemotypes. Sample 153 (A30)
showed a dominance of thymol (40.8%) and a-terpineol(25.8%). Compositions with high percentages of a-terpi-neol (41.5%), as described in O. ramonense [28], O. ma-
jorana (up to 73%) [29], and O. vulgare subsp. vulgare
(up to 40.4%) [30] is rather rare in the genus Orig-
anum. Sample 449 (A74) displayed an important quan-
tity of thymyl methyl ether (19.6%), together with arelatively high amount of thymol (23.5%) and p-cymene
Fig. 5. Score plot for the principal component analysis: oil samples from the 12 regions (Chefchaouen [CC], Ouazzane [OZ], Taounate [TN],
(11.3%) while carvacrol was detectable in negligibleamount (1.3%). High level of thymyl methyl ether(36.2%) was recorded in O. vulgare subsp. hirtum culti-
vated in Italy [31] and O. vulgare subsp. glandulosum
from Algeria (16.3%) [26]. Furthermore, this individual
accumulated the highest amounts of germacrene D(12.0%) and caryophyllene ether (6.1%). Considerable
amounts of germacrene D was reported in O. vulgare
from Lithuania (10.0 – 16.2%) [32] and from India (up
to 13.3%) [33], and in O. vulgare subsp. gracile andO. vulgare subsp. vulgare from Turkey, with 15.8 and
17.8%, respectively [34].Group IV: This group consists of 83 individuals, having
a particular occurrence in Chefchaouen, Taounate,Ouazzane, and Tangier-Tetouan regions. Thymol
(45 – 80.7%, M = 60.0%) was identified as the majormonoterpene of this chemotype while carvacrol
(0.4 – 15.2%) presented the lowest proportions with anaverage of 4.1%. In this group, p-cymene, c-terpinene, anda-terpineol varied to a great extent (0.4 – 2.9%, 0 – 31.3%,and 0.1 – 18.6%, respectively).
The chemical variability found for the compositionof the EOs from such large number of accessions of
wild O. compactum grown in different areas of Moroccoconfirms the high chemical polymorphism reported forthe genus by many authors [35 – 37]. Four compounds,
c-terpinene, p-cymene, thymol, and carvacrol, are partic-ularly involved in the partitioning between groups and
subgroups. Chemotypes in plant species have geneticallycodified enzymatic equipment which directs biosynthesis
to the preferential formation of a definite compound. Inthese phenolic compounds, c-terpinene is the component
involved in the aromatization process which results inthe formation of p-cymene, that is the precursor of oxy-
genated derivatives, thymol or carvacrol [38].The chemical diversity of EOs was particularly evi-
dent within populations belonging to the Occidentaland Central Rif regions. In Tangier-Tetouan popula-
tions, the occurrence of carvacryl methyl ether, a minorcompound of O. compactum, in so high amounts (up to
36.2%), could be considered as a specific regional char-acteristic. The rare a-terpineol was also well represented
in the northern part of the country with a particularconcentration in Tangier-Tetouan region (up to 25.8%).
Furthermore and as previously mentioned, carvacroltype was the most common in almost all populations
Fig. 6. Loading plot for the principal component analysis: oil components in the PC3/PC4 plan, including p-cymene, (E)-b-caryophyllene,a-terpineol, and carvacryl methyl ether.
originating from the Middle Atlas region and the Cen-tral Morocco. Among these samples, a pure carvacrolchemotype and chemotypes exhibiting high content of
the precursors, p-cymene and c-terpinene, were alsorecorded. Moreover, different chemotypes were found
within the same population, therefore, the common har-vesting techniques, which includes mixed plants
collected from different individuals, may explain whypreviously investigated O. compactum EOs allowed the
detection of one chemotype in a given geographicalarea, it was in fact the dominant chemotype.
Conclusions
The EO of O. compactum showed a high chemical
polymorphism. A high presumably genetic effectexplains the variation of EO components observed,although environmental factors may possibly account for
some parts of this variation. This could be of greatinterest for breeding program, aiming to select given
desired chemotypes.
Regional specificity in terms of some emergingchemotypes could be also considered to choose the bestgenetic material to be involved in the breeding program.
Thus and based on the results of EO composition andcomparing, the EO yield in O. compactum accessions,
Benslimane, Rommani, Oulm�es, Moulay Driss Zerhoun,and Sidi Kacem populations could be recommended as
parental material for direct domestication or breedingprogram, exploiting the highest oil yield (up to 2.88%)
and exhibiting the exceptional carvacrol content (up to96%). Unfortunately, these populations are under a seri-
ous overharvesting pressure in the wild, engenderinggradual degradation of wild populations. Obviously, with
the decrease in wild populations, this variability willshrink more and more, until the extinction of some
important chemotypes. Faced with this situation, thein situ as well as ex situ germplasm conservations are of
particular importance and constitute an efficient alterna-tive to overcome the overexploitation from the wild and
resulting genetic erosion. Furthermore, the resultsobtained in this exhaustive study give further contribution
Fig. 7. Score plot for the principal component analysis: oil samples from the 12 regions (Chefchaouen [CC], Ouazzane [OZ], Taounate [TN],
to the understanding of the genetic background of thespecies.
We thank the French and Moroccan collaborative pro-
gram (PRAD) for financial support.
Experimental Part
Surveyed Populations and Sampling
Collection trips were organized to the whole territoryof Morocco. During these expeditions, 88 accessions ofO. compactum plants, i.e., 527 individual plants, were
collected from their natural habitats. The 88 O. com-
pactum populations were sampled over 2013/2014 (be-
tween March and June). The sampling strategy wasdesigned to cover most of the remaining natural area
of the species in Morocco. The distance between indi-viduals exceeded 15 – 20 m, to avoid collection from
close parents. Each sample was labeled and the locationwas recorded using a global positioning system receiver.
The spatial distribution of investigated populations was
depicted on a geographic information system map usingArcGis 10.1 software (Fig. 1).The prospected areas were as follow:
GC/MS. The determined EO content was based on air-dry matter.
GC/MS Analysis
GC/MS Analyses were performed using an Agilent GC/MSD system (Agilent Technologies 7890/5975) equipped
with HP-5MS (apolar, 5% phenyl methyl siloxane)fused SiO2 capillary column (30 m 9 0.25 mm i.d.,
0.25 lm film thickness). He was used as the carrier gasat a flow rate of 1 ml/min with a constant linearvelocity of 36.4 cm/s. The temp. was of 220 °C in the
injector and used in split mode. Oven temp. wasprogrammed from 50 to 150 °C at rate of 3 °C/min,
holding at 150 °C for 10 min then to 250 °C with10 °C/min. For GC/MS detection, an electron ionization
system was used with ionization energy of 70 eV. MSDtransfer line temp. was 250 °C and MSD quadrupole
Table 4. Mean percentages and standard deviations of Moroccan Origanum compactum essential oils
temp. 150 °C. A quantity of 1 ll of each sample was
injected and the split ratio was 1:30. The ion sourcetemp. was set at 230 °C.
GC (Flame-Ionization Detector) analysis
GC Analyses were carried out with a Clarus 500
PerkinElmer Autosystem apparatus equipped with two
flame-ionization detectors and fused capillary columns(50 m 9 0.22 mm i.d., film thickness 0.25 lm), BP-1
(dimethylpolysiloxane), and BP-20 (polyethylene glycol).The carrier gas was He with a linear velocity of 1.0 ml/
min. The oven temp. was programmed from 60 to220 °C at 2 °C/min and then held isothermal (20 min).
The injector temp. was 250 °C (injection mode: split 1/60).The detector temp. was 250 °C.
Identification and Quantification of Components
The characterization of components was achieved onthe basis of: i) comparison of their mass spectra with
those of authentic reference compounds when possible.Further identification was confirmed by comparing the
mass spectra with those recorded in NIST mass spectrallibrary and Adams terpene library [39], ii) comparison
of their retention indices (RI) on HP-5MS determinedwith reference to a homologous series of n-alkanes
(C8 – C24) under the same operating conditions, withthose of authentic compounds or literature data. For
semiquantification purposes, the normalized peak areaof each component was used without any correctionfactors to establish abundances. Quantitative determina-
tion of individual components, using nonane as internalstandard and correction factors according to Costa et al.
[20] and Bicchi et al. [21] was applied to four oilsamples.
Statistical Analysis
The data were subjected to multivariate statistical
analyses using the Statistical Analysis System Software.PCA was performed to identify possible relationships
between the components. CA was carried out todetermine the various groups to which the differentsamples refer. Hierarchical clustering was performed
according to the Ward’s variance minimization method.
REFERENCES
[1] S. E. Kintzios, ‘Oregano: the Genera Origanum and Lippia’,
CRC Press, Boca Raton, FL, 2002.
[2] J. H. Ietswaart, ‘A Taxonomic Revision of the Genus Origanum
(Labiatae)’, Leiden Botanical Series Vol. 4, Leiden University
Press, The Haag, 1980.
[3] S. Kokkini, in ‘Oregano, Proceedings of the IPGRI International
Workshop on Oregano’, Ed. S. Padulosi, IPGRI, Rome, Italy,
1997, p. 2.
[4] K. Bakhy, O. Benlhabib, A. Bighelli, J. Casanova, F. Tomi, C.
Al Faiz, Am. J. Essent. Oils Nat. Prod. 2014, 1, 9.
[5] A. Benabid, ‘Flore et �ecosyst�emes du Maroc. �Evaluation et
pr�eservation de la biodiversit�e’, Edition Ibis Press, Paris, 2000,
p. 360.
[6] J. Bellakhdar, ‘La pharmacop�ee marocaine traditionnelle.
M�edecine arabe ancienne et savoir populaire’, Ibis Press, Mor-
occo, 1997.
[7] C. Bouchra, M. Achouri, L. M. I. Hassani, M. Hmamouchi,
J. Ethnopharmacol. 2003, 89, 165.[8] M. Zyani, D. Mortabit, S. El Abed, A. Remmal, S. I. Koraichi,
Int. Res. J. Microbiol. 2011, 2, 104.
[9] F. Fadel, D. Ben Hmamou, R. Salghi, B. Chebli, O. Benali, A.
Zarrouk, E. E. Ebenso, A. Chakir, B. Hammouti, Int. J. Elec-
trochem. Sci. 2013, 8, 11019.
[10] F. Ben Hammou, S. N. Skali, M. Idaomar, J. Abrini, Afr. J.
Biotechnol. 2011, 10, 15998.
[11] H. Sbayou, N. Oubrim, B. Bouchrif, B. Ababou, K. Boukach-
abine, S. Amghar, Int. J. Engineer. Res. Technol. 2014, 3, 3562.
[12] S. Bouhdid, S. N. Skali, M. Idaomar, A. Zhiri, D. Baudoux, M.
Amensour, J. Abrini, Afr. J. Biotechnol. 2008, 7, 1563.[13] C. O. Van Den Broucke, J. A. Lemli, Planta Med. 1980, 41, 264.
[14] B. Benjilali, H. Richard, O. Britaux, Lebensm. Wiss. U. Tech-
nol. 1986, 19, 22.
[15] A. Kheyr-Pour, J. Hered. 1981, 72, 45.[16] J. Gershenzon, R. Croteau, in ‘Biochemistry of the Mevalonic
Acid Pathway to Terpenoids’, Eds. G. H. N. Towers, H. A.
Stafford, Plenum Press, New York, 1990, p. 99.
[17] D. Vokou, S. Kokkini, J. M. Bessi�ere, Biochem. Syst. Ecol.
1993, 21, 287.
[18] A. Azizi, F. Yan, B. Honermeier, Ind. Crops Prod. 2009, 29, 554.
[19] S. Kokkini, D. Vokou, Flavour Fragrance J. 1989, 4, 1.[20] R. Costa, B. d’Acampora Zellner, M. L. Crupi, M. R. De Fina,
M. R. Valentino, P. Dugo, G. Dugo, L. Mondello, Flavour
Fragrance J. 2008, 23, 40.
[21] C. Bicchi, E. Liberto, M. Matteodo, B. Sgorbini, L. Mondello,
B. d’Acampora Zellner, R. Costa, P. Rubiolo, Flavour Fra-
grance J. 2008, 23, 382.
[22] S. Koc, E. Oz, I. Cinbilgel, L. Aydin, H. Cetin, Parasitology
2013, 193, 316.[23] G. Economou, G. Panagopoulos, P. Tarantilis, D. Kalivas, V.
Kotoulas, I. S. Travlos, M. Polysiou, A. Karamanos, Ind. Crops
Prod. 2011, 33, 236.[24] D. Stojkovi�c, J. Glamo�clija, A. �Ciri�c, M. Nikoli�c, M. Risti�c, J.
�Siljegovi�c, M. Sokovi�c, Arch. Biol. Sci. 2013, 65, 639.
[25] M. Hazzit, A. Baaliouamer, M. L. Faleiro, G. Miguel, J. Agric.
Food Chem. 2006, 54, 6314.[26] Z. Houmani, S. Azzoudj, G. Naxakis, M. Skoula, J. Herbs
Spices Med. Plants 2002, 9, 275.
[27] K. Mechergui, J. A. Coelho, M. C. Serra, S. B. Lamine, S.
Boukhchina, M. L. Khouja, J. Sci. Food Agric. 2010, 90, 1745.[28] A. Danin, U. Ravid, K. Umano, T. Shibamoto, J. Essent. Oil
Res. 1997, 9, 411.
[29] J. Novak, B. Lukas, C. Franz, J. Essent. Oil Res. 2008, 20, 339.
[30] B. Lukas, C. Schmiderer, J. Novak, Biochem. Syst. Ecol. 2013,50, 106.
[31] D. Mockute, B. Genovaite, A. Judzentiene, Biologija 2004, 4, 44.
[32] D. Mockute, G. Bernotiene, A. Judzentiene, Phytochemistry
2001, 57, 65.
[33] D. Bisht, C. S. Chanotiya, M. Rana, M. Semwal, Ind. Crops
Prod. 2009, 30, 422.
[34] E. Sezik, G. T€umen, N. Kirimer, T. €Ozek, K. H. C. Baser,
J. Essent. Oil Res. 1993, 5, 425.
[35] L. F. D’Antuono, G. C. Galleti, P. Bocchini, Ann. Bot. 2000,