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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:

© 2016 Wiley-VHCA AG, Z€urich DOI: 10.1002/cbdv.201500511

1126 Chem. Biodiversity 2016, 13, 1126 – 1139

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

generated using ArcGIS Ver. 10.1.

Chem. Biodiversity 2016, 13, 1126 – 1139 1127

© 2016 Wiley-VHCA AG, Z€urich www.cb.wiley.com

Table 1. Origin, accession number, sampling locations, altitude, climate (the extraction of bioclimatic parameters was conducted using

ArcGIS 10.1 software), and essential-oil yield of each Origanum compactum population studied

Region Accession no. Collection site Samples No. Altitude [m] Climate EO Yield [%]

Tangier-Tetouan A1 Ain Lahcen 1 – 5 200 Humid 0.82

A2 Khmiss Anjra 6 – 7 197 Humid 0.94

A3 Khmiss Anjra 8 – 15 224 Humid 1.10

A4 Melloussa 16 – 23 391 Humid 0.88

Sidi Kacem A5 Col Zeggota 24 – 29 560 Semiarid 2.67

A6 Tnine Srafeh 30 – 35 206 Semiarid 2.28

Benslimane A7 Bouznika toward Benslimane 36 208 Semiarid 2.12

A8 Krama forest 37 – 41 233 Semiarid 1.81

A9 Benslimane forest 42 – 44 224 Subhumid 2.34

A10 Oued Cherrat 45 – 47 252 Semiarid 1.54

A11 Ain dakhla 48 – 54 223 Subhumid 2.08

A12 Khatouat 55 248 Semiarid 2.61

A13 Benslimane toward Sidi Yahya Zaer 56 – 59 382 Semiarid 1.88

A14 Benslimane toward Rommani 60 – 65 223 Semiarid 2.16

A15 Benslimane toward Rommani 66 – 73 371 Semiarid 2.09

Taounate A16 Ouartzagh 74 – 75 303 Subhumid 2.22

A17 Oudka 76 – 80 1076 Humid 1.63

A18 Tabouda 81 – 88 438 Humid 1.42

A19 Kissane 89 – 95 605 Subhumid 1.92

A20 Sidi Mokhfi 96 – 106 432 Humid 1.61

A21 Zrizer 107 352 Humid 1.86

A22 Timezgana 108 – 112 297 Humid 1.94

A23 Galaz 113 – 116 369 Subhumid 1.79

A24 Ghafsai 117 – 119 397 Humid 1.43

A25 Beni zeroual 120 – 121 667 Subhumid 1.64

A26 Beni zeroual 122 – 125 220 Humid 1.89

A27 Bibane 126 – 130 524 Humid 1.58

A28 Tafrant 131 – 135 373 Subhumid 1.91

Chefchaouen A29 Talassemtane 136 – 142 881 Humid 0.89

A30 Akchour 143 – 150 307 Humid 0.95

A31 Talambote 151 – 153 399 Humid 1.34

A32 Akchour 154 – 160 957 Humid 1.23

A33 Jbel Meggou 161 – 165 845 Humid 0.84

A34 Jbel Tissouka 166 – 171 831 Perhumid 0.91

A35 Beni bouhlou 172 – 173 891 Humid 1.16

A36 Assifane 174 – 180 601 Humid 0.96

A37 Beni Ahmed 181 – 185 426 Humid 1.68

Ouazzane A38 Ain beida 186 – 190 344 Humid 2.12

A39 Brikcha 191 – 193 235 Subhumid 1.96


A41 Oued loukouss 198 – 203 126 Humid 1.78

A42 Mokrisset 204 – 210 498 Subhumid 1.26

A43 Mokrisset 211 – 217 340 Humid 1.64

A44 Zoumi 218 – 223 244 Subhumid 0.93

A45 Zoumi 224 – 230 330 Subhumid 1.79

A46 Kalaat Bouqorra 231 – 234 150 Humid 2.43

A47 Souk el had 235 – 241 173 Humid 1.45

A48 Ouazzane 242 – 247 593 Humid 1.89

A49 Mokrisset 248 – 253 589 Humid 1.58

A50 Zoumi 254 – 258 256 Subhumid 1.99

A51 Jabriyine 259 – 262 252 Subhumid 1.22


A53 Asjen 268 – 272 140 Subhumid 1.28

A54 Asjen 273 – 278 273 Subhumid 1.92

A55 Teroual 279 – 284 322 Subhumid 0.67

A56 Zghira 285 – 290 341 Subhumid 0.86

A57 Ain dorij 291 – 296 268 Subhumid 1.94

A58 Sidi Redouane 297 – 302 220 Subhumid 1.67



A61 Mzefroun 315 – 319 242 Subhumid 1.89


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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 [%]

A62 Bni Qolla 320 – 322 302 Subhumid 1.85

A63 Mesmouda 323 – 324 243 Subhumid 1.99

A64 Mesmouda 325 – 330 172 Subhumid 2.2

A65 Bni Qolla 331 – 335 226 Subhumid 1.63


A67 Bab Joughmar 342 – 350 194 Humid 1.92

Azrou A68 Ranch Adarouch 351 – 367 1071 Subhumid 1.66

A69 Azrou toward Elhajeb 368 – 389 1332 Subhumid 1.12

A70 Elhajeb 390 – 392 1385 Subhumid 1.08

Beni Mellal A71 Tassemit 393 – 417 1385 Semiarid 1.62

A72 Ain Aserdoun 418 – 431 1248 Semiarid 1.1

A73 Beni Mellal toward Ksiba 432 – 439 1210 Semiarid 1.86

Khenifra A74 Khenifra toward Oum R’bia 440 – 449 1239 Subhumid 2.18

A75 Arougou 450 – 455 1032 Subhumid 1.56

Rommani A76 Had Ghoualem 456 – 460 610 Semiarid 2.33

A77 Merchouch 461 – 464 640 Semiarid 1.80

A78 Merchouch 465 – 469 667 Semiarid 2.48

A79 Ain Aouda 470 – 476 443 Semiarid 1.79

A80 Gara 477 – 487 360 Semiarid 2.28

Moulay Driss Zerhoun A81 Moussaoua 488 – 491 879 Semiarid 2.58

A82 Moussaoua 492 – 493 722 Semiarid 2.77

A83 Sidi Ali 494 – 503 904 Semiarid 2.18

Oulm�es A84 Boukachmir 504 – 505 1002 Subhumid 2.02

A85 Tiddas 506 – 514 880 Subhumid 2.39

A86 Sidi Moussa 515 – 522 1071 Subhumid 1.78

A87 Harcha 523 – 524 957 Semiarid 2.45

A88 Ait Ikkou 525 – 527 1058 Semiarid 2.88



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)

Compounda) Group 1 Group 2 Group 3 SG2 Group 4

a-Thujene 1.0 0.0 0.4 0.4

a-Pinene 0.4 0.1 0.3 0.3

Camphene 0.1 trb) 0.1 0.1

Sabinene 0.1 0.0 0.1 0.1

b-Pinene 0.2 0.0 0.0 0.0

Oct-1-en-3-ol 1.3 0.4 0.5 0.2

Octan-3-one 1.9 0.2 0.4 1.3

Myrcene 0.1 0.0 1.2 1.2

a-Phellandrene 0 0.1 1.2 0.1

3-Carene 0.1 0.0 0.0 0.1

a-Terpinene 1.4 0.0 1.2 1.5

p-Cymene 48.1 3.9 37.5 24.3Limonene 0.3 0.1 0.2 0.3

b-Phellandrene 0.2 0.0 0.1 0.2

b-Ocimene 0.0 0.0 0.0 0.0

c-Terpinene 0.1 0.1 3.4 6.2

cis-Sabinene hydrate 0.1 1.0 0.1 0.1

Terpinolene 0.0 0.0 0.0 0.1

Linalool 2.0 1.0 1.9 1.5

Borneol 0.2 0.2 tr 0.1

Terpinen-4-ol 1.0 0.7 0.4 0.7

a-Terpineol 9.3 1.4 0.2 3.3

Thymol methyl ether 2.1 0.0 0.0 0.0

Thymol 0.1 4.1 35.2 56.1

Carvacrol 16.6 79.3 2.4 3.0

(E)-b-caryophyllene 0.9 0.2 2.2 0.8

a-Humulene tr 0.0 0.1 tr

Caryophyllene ether 0.4 2.1 0.7 0.4

a) Compounds listed in order of elution on the nonpolar HP-5MS col-

umn. Percentages calculated using correction factors according to

Costa et al. [20] and Bicchi et al. [21]. The highest values are in bold. b)

tr, Traces amounts (< 0.1%).



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],

Tangier-Tetouan [TT], Azrou [AZ], Beni Mellal [BM], Khenifra [KN], Benslimane [BS], Oulm�es [OM], Rommani [RM], Sidi Kacem [SK], and

Moulay Driss Zerhoun [ZR]) in the PC1/PC2 plan.

Table 3. Loadings of 12 compounds on the first four PCs

Compound Factor 1 Factor 2 Factor 3 Factor 4

a-Thujene 0.82 �0.04 0.34 �0.20

Myrcene 0.85 0.03 0.31 �0.18

a-Terpinene 0.87 0.10 0.38 �0.13

p-Cymene 0.36 0.11 0.68 0.05

c-Terpinene 0.85 0.20 0.26 �0.05

cis-Sabinene hydrate �0.71 0.05 0.17 �0.24

Linalool �0.74 �0.09 0.30 0.14

a-Terpineol 0.23 0.33 �0.08 0.71Carvacryl methyl ether �0.01 �0.09 0.13 0.88

Thymol �0.10 0.97 �0.06 �0.02

Carvacrol �0.26 �0.88 �0.33 �0.20

(E)-b-Caryophyllene �0.01 0.04 0.67 0.02

Eigenvalue 4.72 2.18 1.23 1.06

% of variance 39.4 18.2 10.3 8.9

Cumulative % of

the variance

39.4 57.6 67.9 76.8

The highest ones (> 0.5 threshold) are in bold.



(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],

Tangier-Tetouan [TT], Azrou [AZ], Beni Mellal [BM], Khenifra [KN], Benslimane [BS], Oulm�es [OM], Rommani [RM], Sidi Kacem [SK], and

Moulay Driss Zerhoun [ZR]) in the PC3/PC4 plan.



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:

Middle Atlas: Azrou, Khenifra, and Beni Mellal.Northern region: Tangier-Tetouan, Chefchaouen, Ouaz-

zane, and Taounate.Central Morocco: Benslimane, Rommani, Oulm�es, Moulay

Driss Zerhoun, and Sidi Kacem.

It is worth mentioning that the following regions have

not been previously explored: Benslimane, Azrou, Kheni-fra, Beni Mellal, Tangier, Sidi Kacem, and Moulay Driss

Zerhoun. Voucher specimens of representative individualsfrom each locality were deposited with the Herbarium of

National Institute of Agronomic Research, Rabat(INRA).

Essential Oil Isolation

The aerial parts of the collected samples were submit-ted to hydrodistillation for 2 h 30 min using a Cle-

venger-type apparatus. Extracted EOs were stored andkept under refrigeration at 4 °C until their analysis by

Fig. 8. Two-dimentional dendrogram obtained by cluster analysis, representing chemical composition similarity relationships among the 527Ori-

ganum compactum oil samples.



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

Compounda) RIb) Content [%]c) Identificationd)

Group 1 Group 2 Group 3 SG1 Group 3 SG2 Group 4

a-Thujene 925 1.0 � 0.3 0.3 � 0.3 0.9 � 0.7 0.7 � 0.4 0.4 � 0.3 RI, MS

a-Pinene 931 0.4 � 0.1 0.1 � 0.1 0.4 � 0.3 0.3 � 0.2 0.2 � 0.3 RI, MS

Camphene 946 tr tr tr tr tr RI, MS

Sabinene 971 tr tr tr tr tr RI, MS

b-Pinene 974 0.1 � 0 tr tr tr tr RI, MS

Oct-1-en-3-ol 977 0.3 � 0.2 0.4 � 0.3 0.3 � 0.3 0.4 � 0.3 0.4 � 0.3 RI, MS

Octan-3-one 985 0.3 � 0.2 0.4 � 0.3 0.6 � 0.5 0.5 � 0.4 0.6 � 0.4 RI, MS

Myrcene 990 1.1 � 0.3 0.4 � 0.4 0.8 � 0.5 1.0 � 0.5 0.6 � 0.4 RI, MS

a-phellandrene 1004 0.2 � 0.1 tr 0.1 � 0.1 0.1 � 0.1 tr RI, MS

3-Carene 1009 0.1 � 0 tr tr tr tr RI, MS

a-Terpinene 1015 2.2 � 0.5 0.7 � 0.6 2.1 � 1.0 2.0 � 1.0 1.2 � 0.9 RI, MS

p-Cymene 1023 15.7 � 6.7 7.4 � 5.1 28.8 � 14.1 24.0 � 13.1 9.7 � 5.5 RI, MS, CoI

Limonene 1027 0.4 � 0.1 0.1 � 0.1 0.4 � 0.2 0.4 � 0.2 0.2 � 0.2 RI, MS

b-Phellandrene 1029 tr tr tr tr tr RI, MS

b-Ocimene 1037 tr tr tr tr tr RI, MS

c-Terpinene 1057 18.4 � 6.2 5.8 � 4.6 18.8 � 9.1 16.9 � 7.7 12.7 � 8.2 RI, MS, CoI

cis-Sabinene hydrate 1065 0.6 � 0.2 0.9 � 0.4 0.5 � 0.3 0.7 � 0.3 0.8 � 0.3 RI, MS

Non-1-en-3-ol 1079 tr tr 0.1 � 0.2 tr tr RI, MS

Terpinolene 1087 tr tr tr tr tr RI, MS

Linalool 1100 0.8 � 0.4 1.4 � 0.7 1.0 � 0.6 1.3 � 0.5 1.4 � 0.8 RI, MS, CoI

Camphor 1142 tr tr tr tr tr RI, MS

Borneol 1164 0.2 � 0.1 0.2 � 0.2 0.1 � 0.1 0.1 � 0.1 0.2 � 0.3 RI, MS

Terpinen-4-ol 1176 0.3 � 0.1 0.4 � 0.2 0.2 � 0.1 0.3 � 0.1 0.4 � 0.2 RI, MS

a-Terpineol 1189 0.7 � 1.0 1.6 � 2.8 3.6 � 4.6 1.9 � 2.6 3.4 � 4.0 RI, MS

Carvone 1196 tr tr tr tr tr RI, MS

Thymyl methyl ether 1235 tr tr tr 0.3 � 1.0 tr RI, MS

Carvacryl methyl ether 1244 0.1 � 0.4 0.7 � 2.7 9.9 � 12.6 0.7 � 1.5 0.6 � 1.2 RI, MS

Thymol 1293 0.3 � 1.0 0.4 � 1.8 1.8 � 4.1 31.3 � 8.1 60.0 � 10.1 RI, MS, CoI

Carvacrol 1311 54.9 � 6.5 76.2 � 10.1 26.3 � 9.2 14.5 � 17.5 4.1 � 2.8 RI, MS, CoI

(E)-b-Caryophyllene 1417 0.7 � 1.1 0.5 � 0.5 1.1 � 1.4 1.0 � 0.8 0.8 � 0.8 RI, MS, CoI

a-Humulene 1452 tr tr tr tr tr RI, MS

b-Bisabolene 1502 tr tr tr tr tr RI, MS

Germacrene D 1532 tr 0.1 � 0.4 tr – – RI, MS

Caryophyllene ether 1580 0.1 � 0.3 0.3 � 0.6 0.2 � 0.2 0.2 � 0.2 0.4 � 1.0 RI, MS

Monoterpene

hydrocarbons (MH)

30.0 – 57.8 0.3 – 33 12.4 – 76.8 5.4 – 73.4 0.7 – 49.0

Oxygenated

monoterpenes (OM)

40.6 – 68.3 64 – 99.1 21.4 – 85.0 22.5 – 91.3 49.4 – 97.5

Sesquiterpene

hydrocarbons (SH)

0.2 – 11.98 0 – 4.2 0 – 6.6 0 – 2.9 0 – 6.3

Oxygenated

sesquiterpenes (OS)

0 – 2.8 0 – 6.0 0 – 1.0 0 – 1.0 0 – 5.7

Other oxygenated

compounds (OC)

0 – 1.0 0 – 2.7 0 – 1.3 0 – 1.0 0 – 1.5

Total identified [%] 95.2 – 99.9 94.7 – 99.8 95.3 – 99.9 97.9 – 99.9 95 – 99.9

a) Compounds listed in order of elution on the nonpolar HP-5MS column. b) RI, Retention index determined relative to n-alkanes (C8 – C24)

on the nonpolar HP-5MS column. c) Contents are given as mean � standard deviation. d) Identification method: RI, identification based on

RI; MS, identification based on mass spectra; CoI, coinjection with commercial standard. e) tr, Traces amounts (< 0.1%).



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.

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Received December 25, 2015

Accepted June 3, 2016



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