Compactum Grown in All Natural Habitats in Morocco

1126 Chem. Biodiversity 2016, 13, 1126 – 1139

FULL PAPER

Chemical Polymorphism of Origanum compactum Grown in All Natural Habitats in Morocco

by Kaoutar Aboukhalid*a)b), Abdeslam Lamirib), Monika Agacka-Mołdochc), Teresa Doroszewskac), Ahmed Douaikd), Mohamed Bakhaa)e), Joseph Casanovaf), Felix Tomif), Nathalie Machong), and Chaouki Al Faiza) a) Institut National de la Recherche Agronomique, UR Plantes Aromatiques et Medicinales, INRA, CRRA-Rabat, PB 6570, 10101 Rabat, Morocco (phone: +212661265485, e-mail: [email protected]) b) Laboratoire de Chimie Appliquee et Environnement, Faculte des Sciences et Techniques, Universite Hassan I, BP 577, 26000 Settat, Morocco c) 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, Morocco e) Laboratoire de Biologie et Sante, Faculte des sciences, Universite Abdelmalek Essaadi,^ BP 2121, 93002 Tetouan, Morocco f) UMR 6134 SPE, Equipe Chimie et Biomasse, Universite de Corse-CNRS, Route des Sanguinaires, FR-20000 Ajaccio g) UMR 7204 CESCO, Departement d’Ecologie et gestion de la Biodiversite, Museum 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.

endemic to Morocco and southern Spain [2]. O. com- Introduction pactum BENTH. is the most widespread species in Mor- The genus Origanum is a taxonomically complex group occo, extending from the Middle Atlas region delimited of aromatic plants that are used all over the world for by Beni mellal, Azrou, Khenifra, and Oulmes up to the their aromatic and medicinal properties and as a culinary occidental and central Rif region, including the provinces herb [1]. According to Ietswaart’s classification [2], the of Tangier-Tetouan, Chefchaouen, Taounate, and Ouaz- genus Origanum has been divided into 38 species, 6 sub- zane [4][5]. O. compactum BENTH., known locally as species, and 17 hybrids, arranged in three groups ‘Zaatar’,^ constitutes one of the most appreciated aromatic and 10 sections. The genus Origanum has a local distri- herbs, widely used in Moroccan folk medicine in the form bution mostly around the Mediterranean basin, and it is of infusions and decoctions to threat broncopulmonary, characterized by a large morphological and chemical gastric acidity, gastrointestinal diseases, and numerous diversity [3]. infections [6]. Due to its pleasant flavor and spicy fra- In Morocco, the genus Origanum is represented by grance, O. compactum is the aromatic ingredient of five taxa, three of which, O. elongatum (BONNET)EMB & choice for flavoring some traditional dishes (barley soup, MAIRE, O. grosii PAU &FONT QUER, and O. frontqueri couscous, etc.). PAU are endemic to the central Rif region. O. vulgare Steam distillation of aerial parts of O. compactum subsp. virens (HOFFM.etLINK)IETSWAART is also common BENTH. produces an essential oil (EO), which is appre- to the Iberian Peninsula, while O. compactum BENTH.is ciated for its aromatic and medicinal properties:

© 2016 Wiley-VHCA AG, Zurich€ DOI: 10.1002/cbdv.201500511 Chem. Biodiversity 2016, 13, 1126 – 1139 1127 antifungal [7 – 9], antibacterial [10][11], and antioxidant plant. Nevertheless, the domestication of wild medicinal effects [12]. Previous studies on Moroccan O. com- species requires a good understanding of the chemical pactum BENTH. EOs revealed a wide chemical diversity. and genetic diversity within the species. Although vari- Compositions were dominated either by carvacrol or by ous studies have been carried out in order to charac- thymol. Mixed types, combining both thymol and terize Moroccan O. compactum BENTH. EOs, these carvacrol, and types containing a high level of precur- studies were restricted to a limited number of samples. sors, c-terpinene and p-cymene, have also been re- Moreover, these studies did not cover the entire area ported [13 – 15]. The various compositions have about wild-growing O. compactum BENTH. where this been summarized in a previous paper that described plant still subsists, and only few studies specified the also the chemical variability observed on 36 oil samples geographical origin of samples. Furthermore, only the isolated from plants harvested in three Moroccan EO composition of mixed plant samples was reported provinces: Chefchaouen, Larache, and Tetouan [4]. and no study to date has been undertaken at intrapop- Two-thirds of the samples exhibited carvacrol as major ulation level. For instance, most Origanum species have component. cross-pollinated reproductive system [15], which can Nowadays, O. compactum BENTH. is considered as a lead to a high level of genetic polymorphism within threatened species due to a dramatic population decline populations. This variation may eventually influence the caused by various factors: overexploitation, drought, genetic control of accumulation of specific compounds overgrazing, combined with unsustainable and destruc- among the secondary metabolites [16]. In this paper, tive methods of harvesting, through up-rooting in the we report an analysis of the EO composition of wild, and collecting essentially during the flowering per- O. compactum BENTH. individual plants distributed all iod, before seed set. Since the decline of natural popu- over the Moroccan territory. This is the first report of lations, an urgent attempt to set up a domestication a deep study on native populations of O. compactum program should be initiated to ensure the conservation BENTH. Such information would be fundamental to pro- and a sustainable utilization of this valuable medicinal 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.

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 A40 Brikcha 194 – 197 307 Subhumid 1.42 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 A52 Brikcha 263 – 267 188 Subhumid 1.63 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 A59 Sidi Redouane 303 – 307 200 Subhumid 1.56 A60 Sidi Redouane 308 – 314 181 Subhumid 1.61 A61 Mzefroun 315 – 319 242 Subhumid 1.89

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 A66 Brikcha 336 – 341 162 Subhumid 2.42 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 Oulmes 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

national scale, by the selection of the most valuable dry matter (Table 1), depending on the accession ori- and performant chemotypes or/and establish in situ gin. Our results revealed noticeable spatial variation in conservation program. EO yield of O. compactum. The bioclimatic differences among the 12 investigated regions seem to have a significant effect on the EO content. Populations dis- Results and Discussion tributed under a semiarid climate showed the highest Individual plants of O. compactum have been collected levels of EO yield (average: 2.15%), while accessions from all the Moroccan sites were they grow sponta- located under a subhumid climate, displayed a rela- neously. In detail, 527 plants have been collected in 88 tively lower EO content (average: 1.71%). The lowest locations covering most of the natural habitats of the EO yield (average: 1.46%) was recorded in the north- species in Morocco (Fig. 1). The prospected areas were: ern part of the country, Tangier-Tetouan and i) the northern region (provinces of Tangier-Tetouan, Chefchaouen provinces, exposed to humid climate. One Chefchaouen, Ouazzane, and Taounate) that provided population from Chefchaouen (A34) was located in a 57% of the samples; ii) the central Morocco (Bensli- perhumid climate. This sample was characterized by a mane, Rommani, Oulmes, Moulay Driss Zerhoun, relatively low EO content (0.91%). Thus, the observed and Sidi Kacem, 23% of the samples); iii) the Middle yields of EOs increase significantly from humid to arid Atlas (Azrou, Khenifra, and Beni Mellal, 20% of the zones (Fig. 2). In general, it is recognized that plants samples). growing in arid areas tend to produce high levels of Various regions have been explored for the first time: EO as an adaptive mechanism in response to Benslimane, Azrou, Khenifra, Beni Mellal, Tangier, Sidi water stress [17]. For instance, Azizi et al. [18] demon- Kacem, and Moulay Driss Zerhoun. strated that water deficiency increases EO content of O. vulgare. Otherwise, no significant correlation in the EO yields Essential-Oil Yield related to the altitude was observed. These findings agree Yield of EOs isolated from the aerial parts of 88 with Bakhy et al. [4] for O. compactum, but they differ O. compactum populations varied drastically from sam- with Vokou et al. [17] and Kokkini and Vokou [19] who ple to sample. Yields ranged from 0.67 to 2.88% of the emphasized that altitude is the most important

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 Moroccan Origanum compactum. environmental factor inﬂuencing the oil content of O. vul- Before doing statistical analysis, four oil samples iso- gare subsp. hirtum. lated from aerial parts of O. compactum were subjected to quantitative determination using nonane as internal standard and correction factors according to Costa et al. Essential Oils Chemical Variability [20] and Bicchi et al. [21]: a carvacrol-rich oil sample, a The 527 individual plants belonging to 88 populations of thymol-rich oil sample and two mixed types, carvacrol/ O. compactum were analyzed by GC/MS. The chemical p-cymene and thymol/p-cymene. Results are reported in composition of the EOs can be summarized by a mixture Table 2. of 34 predominant mono- and sesquiterpenes. The The 527 compositions were subjected to principal monoterpene fraction (69 – 99.9%) was dominant and component analysis (PCA). Twelve major compounds consisted mainly of oxygenated monoterpenes detected at an average concentration higher than 0.5% (21.4 – 99.1%), followed by monoterpene hydrocarbons have been considered for the statistical analysis (a-thu- (0.3 – 76.8%). Carvacrol (up to 96.3%), thymol (up to jene, myrcene, a-terpinene, p-cymene, c-terpinene, cis- 80.7%), a-terpineol (up to 25.8%), and carvacryl methyl sabinene hydrate, linalool, a-terpineol, carvacryl methyl ether (up to 36.2%) were the major oxygenated mono- ether, thymol, carvacrol, and (E)-b-caryophyllene). terpenes, while c-terpinene (up to 35.2%) and p-cymene These components constituted 86.2 – 99.6% of the total (up to 58.6%) were the most highly represented oils. PCA reduced the 12 variables to four principal compounds of the monoterpene hydrocarbons class components with eigenvalues higher than 1. The ﬁrst (Fig. 3). The sesquiterpene fraction occurred only in smal- principal component (PC1) underlines the positive cor- ler proportions (up to 12.4%), (E)-b-caryophyllene (up to relation between a-thujene, myrcene, a-terpinene, and 11.5%) being its main component (average: 0.7%). c-terpinene. In contrast, PC1 is negatively related to the www.cb.wiley.com © 2016 Wiley-VHCA AG, Zurich€ Chem. Biodiversity 2016, 13, 1126 – 1139 1131

Table 2. Composition of four oil samples representative of each characterized by high amount of a-thujene, myrcene, defined group of the Moroccan Origanum compactum (main compo- a-terpinene, and c-terpinene. With respect to PC2, most nents, Contents (g/100 g) calculated using correction factors) of the oil samples from the 12 regions showed interme- Compounda) Group 1 Group 2 Group 3 SG2 Group 4 diate to high negative scores, implying their high carvacrol content, however, some samples have particularly a -Thujene 1.0 0.0 0.4 0.4 high positive scores. These samples exclusive to Ouaz- a-Pinene 0.4 0.1 0.3 0.3 Camphene 0.1 trb) 0.1 0.1 zane province (samples 214, 246, 247, and 248) are Sabinene 0.1 0.0 0.1 0.1 characterized by their very high thymol content. b-Pinene 0.2 0.0 0.0 0.0 Regarding PC3, oil samples 198, 226, 227, 259, and 343 Oct-1-en-3-ol 1.3 0.4 0.5 0.2 from Ouazzane province showed the highest positive Octan-3-one 1.9 0.2 0.4 1.3 scores and, consequently, they have the highest p-cym- Myrcene 0.1 0.0 1.2 1.2 ene and (E)-b-caryophyllene content. Concerning PC4, a-Phellandrene 0 0.1 1.2 0.1 3-Carene 0.1 0.0 0.0 0.1 samples 14, 8, 10, 4, 200, 12, 16, 2, 23, 202, and 22 a-Terpinene 1.4 0.0 1.2 1.5 from Tangier-Tetouan and Ouazzane provinces showed p-Cymene 48.1 3.9 37.5 24.3 the highest positive scores. These samples are character- Limonene 0.3 0.1 0.2 0.3 ized by their very high a-terpineol and carvacryl methyl b -Phellandrene 0.2 0.0 0.1 0.2 ether content (Fig. 7). Cluster Analysis (CA) was per- b-Ocimene 0.0 0.0 0.0 0.0 formed to classify and differentiate the analyzed c-Terpinene 0.1 0.1 3.4 6.2 cis-Sabinene hydrate 0.1 1.0 0.1 0.1 O. compactum samples according to their major con- Terpinolene 0.0 0.0 0.0 0.1 stituents. Fig. 8 presents the corresponding dendrogram Linalool 2.0 1.0 1.9 1.5 using Ward’s method. Considering the 12 major con- Borneol 0.2 0.2 tr 0.1 stituents of the 527 samples, four major groups were Terpinen-4-ol 1.0 0.7 0.4 0.7 defined, whose composition is summarized in Table 4. a-Terpineol 9.3 1.4 0.2 3.3 Thymol methyl ether 2.1 0.0 0.0 0.0 The resulting dendrogram reported in Fig. 8 reflects the Thymol 0.1 4.1 35.2 56.1 qualitative heterogeneity of wild Moroccan O. com- Carvacrol 16.6 79.3 2.4 3.0 pactum EOs and showed the existence of high intrapop- (E)-b-caryophyllene 0.9 0.2 2.2 0.8 ulation variability within the EOs. The following groups a-Humulene tr 0.0 0.1 tr have been defined: Caryophyllene ether 0.4 2.1 0.7 0.4 Group I: This group was represented by 107 samples a) Compounds listed in order of elution on the nonpolar HP-5MS col- widespread along the distribution range of the species. umn. Percentages calculated using correction factors according to Carvacrol (34.8 – 65.6%, M = 54.9%), p-cymene Costa et al. [20] and Bicchi et al. [21]. The highest values are in bold. b) (5.9 – 36.4%, M = 15.7%), and c-terpinene (2.6 – 35.2%, < tr, Traces amounts ( 0.1%). M = 18.4%) were the major components. One sample from Ouazzane (A66) was the most dissimilar within the group for its considerable amount of (E)-b-caryophyllene content of cis-sabinene hydrate and linalool (Fig. 4). (11.5%), found at insignificant amount in all the remain- The second component (PC2) is the expression of the ing samples belonging to this group. negative correlation between thymol and carvacrol Group II: This group is the largest in terms of num- (Fig. 4). The data presented in Fig. 5 shows the distri- ber of samples including 274 individuals (52% of sam- bution of the individuals in the space of the first two ples) and representing the most typical EO profile of principal components (PC1 + PC2). These latter Moroccan wild O. compactum. Carvacrol is the main explained cumulatively 57.6% of the total variance component (44 – 96.6%, M = 76.2%). Interestingly, (Table 3). PC2 clearly separated O. compactum plants among the carvacrol-rich oils, the highest content rich in thymol, observed mainly on the left side of (90.2 – 96.7%) was observed in 24 individuals from Fig. 5, from those that contain high amounts of car- Benslimane (A13 and A14), Ouazzane (A53, 55, 56, 57, vacrol, observed mainly on the right side. Along the 58, 59, 60, 62, 65, and 66), Oulmes (A87), Taounate second axis, it is possible to identify a zone of disconti- (A18), and Moulay Driss Zerhoun (A81). This is the nuity near the zero point. This is because no sample highest percentage of this compound detected up today contained simultaneously low concentrations of car- in O. compactum EOs. Very few papers have reported vacrol and thymol. The third component, describing an oregano chemotype characterized by such excep- 10.3% of the total variance, is positively correlated with tional amount of carvacrol. Indeed, Koc et al. [22] p-cymene and (E)-b-caryophyllene, while the last factor, revealed that carvacrol (up to 93%) was the dominant explaining 8.9% of the data variability, is positively volatile component of Turkish O. bilgeri. For the Greek related to the content of a-terpineol and carvacryl oregano (O. vulgare subsp. hirtum), carvacrol was also methyl ether (Fig. 6). With regard to PC1, six of the detected in a substantial amount (93.8 – 95%) [19][23]. 527 oil samples (13, 92, 354, 435, 446, and 496) have This exceptional carvacrol content in O. compactum the strongest positive scores, indicating that they are plants reflects the particular importance of this species

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.

and the high potential to produce improved rich car- Among this subgroup, 10 of the surveyed plants, local- vacrol varieties. In fact, carvacrol is regarded as the ized in Tangier-Tetouan (A1, A3, and A4), and Ouazzane most required component in the oregano EOs. How- regions (A41) were characterized by the preeminence of ever, the role of the other minor components should carvacryl methyl ether, dominating for some samples, the not be neglected as they have been reported to act as four monoterpenes involved in the phenolic biosynthetic synergists [24]. Exploring the results of statistical analy- pathway. The exceptionally high content of carvacryl sis, some samples stand out from the others for some methyl ether (20.1 – 33.8%) in these samples is remarkable particular composition. In fact, 17 samples originated as this compound is usually detected in the whole genus from Tangier-Tetouan (A1, 2, 3, and 4) and Che- Origanum in either negligible amounts or traces only. A fchaouen (A30, 34, 35, and 36) have shown a relatively few papers have reported an oregano chemotype character- high content of carvacryl methyl ether (4.6 – 19.9%) ized by the presence of carvacryl methyl ether at such and a-terpineol (0.6 – 20.1%) in comparison with the appreciable amount but never in O. compactum. Hazzit average content, lower than 1% in all other samples of et al. [25] referred to O. ﬂoribundum oil sample with this group. noticeable amount (6.9%) of carvacryl methyl ether. High Group III: This group appeared less homogenous level of carvacryl methyl ether (11.4%) was also recorded and shows high content of p-cymene, c-terpinene, carva- in O. vulgare subsp. glandulosum from Algeria [26]. Fur- crol, and carvacryl methyl ether. Based on the relative thermore, eight samples (A3, 21, 33, 34, 36, 43, 47, and 82) abundance of these compounds, this group could be originated from Tangier-Tetouan, Taounate, Chefchaouen, subdivided into two subgroups with distinct characteris- Ouazzane, and Moulay Driss Zerhoun, were distinguished tics: by the highest amount of p-cymene (37.5 – 58.6%). High Subgroup 1: This subgroup contains 34 samples; p-cym- level of p-cymene was also identiﬁed in O. glandulosum ene (4.7 – 58.6%, M = 28.8%), c-terpinene (4.3 – 34.2%, from Tunisia (36 – 46%) [27]. M = 18.8%), carvacrol (0.5 – 42.2%, M = 26.3%), and car- Subgroup 2: This subgroup composed of 29 samples vacryl methyl ether (0 – 36.2%, M = 9.9%) were the most originated from Chefchaouen, Ouazzane, and Taounate. relevant components. a-Terpineol reached considerable Thymol, (16 – 52.2%, M = 31.3%), carvacrol contents in samples 8, 10 (A3), and 16 (A4) (up to 12.5, (0.2 – 50.6%, M = 14.5%), p-cymene (4.1 – 44.2%, M = 15.4, and 16.6%, respectively). 24%), and c-terpinene (1 – 27.8%, M = 16.9%) were the www.cb.wiley.com © 2016 Wiley-VHCA AG, Zurich€ Chem. Biodiversity 2016, 13, 1126 – 1139 1133

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], Oulmes [OM], Rommani [RM], Sidi Kacem [SK], and Moulay Driss Zerhoun [ZR]) in the PC1/PC2 plan.

most abundant compounds. Four O. compactum plants Table 3. Loadings of 12 compounds on the ﬁrst four PCs grown in Ouazzane and Chefchaouen (A24, 25, 27, and Compound Factor 1 Factor 2 Factor 3 Factor 4 31), showed the codominance of the phenolic compounds – – a À À thymol (33.6 52.2%) and carvacrol (31 50.6%). -Thujene 0.82 0.04 0.34 0.20 – c – Myrcene 0.85 0.03 0.31 À0.18 p-Cymene (4.1 8.4%) and -terpinene (1 10.1%) were a-Terpinene 0.87 0.10 0.38 À0.13 less represented. p-Cymene 0.36 0.11 0.68 0.05 Among this subgroup, two samples, 153 and 449, c-Terpinene 0.85 0.20 0.26 À0.05 from Chefchaouen (A30) and Khenifra (A74), respec- cis-Sabinene hydrate À0.71 0.05 0.17 À0.24 tively, were clearly outstanding and may not be repre- À À Linalool 0.74 0.09 0.30 0.14 sentative as a new chemotype since only one sample a-Terpineol 0.23 0.33 À0.08 0.71 Carvacryl methyl ether À0.01 À0.09 0.13 0.88 characterize these chemotypes. Sample 153 (A30) Thymol À0.10 0.97 À0.06 À0.02 showed a dominance of thymol (40.8%) and a-terpineol Carvacrol À0.26 À0.88 À0.33 À0.20 (25.8%). Compositions with high percentages of a-terpi- (E)-b-Caryophyllene À0.01 0.04 0.67 0.02 neol (41.5%), as described in O. ramonense [28], O. ma- Eigenvalue 4.72 2.18 1.23 1.06 jorana (up to 73%) [29], and O. vulgare subsp. vulgare % of variance 39.4 18.2 10.3 8.9 (up to 40.4%) [30] is rather rare in the genus Orig- Cumulative % of 39.4 57.6 67.9 76.8 the variance anum. Sample 449 (A74) displayed an important quantity of thymyl methyl ether (19.6%), together with a The highest ones (> 0.5 threshold) are in bold. relatively high amount of thymol (23.5%) and p-cymene

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.

(11.3%) while carvacrol was detectable in negligible wild O. compactum grown in different areas of Morocco amount (1.3%). High level of thymyl methyl ether confirms the high chemical polymorphism reported for (36.2%) was recorded in O. vulgare subsp. hirtum culti- the genus by many authors [35 – 37]. Four compounds, vated in Italy [31] and O. vulgare subsp. glandulosum c-terpinene, p-cymene, thymol, and carvacrol, are partic- from Algeria (16.3%) [26]. Furthermore, this individual ularly involved in the partitioning between groups and accumulated the highest amounts of germacrene D subgroups. Chemotypes in plant species have genetically (12.0%) and caryophyllene ether (6.1%). Considerable codified enzymatic equipment which directs biosynthesis amounts of germacrene D was reported in O. vulgare to the preferential formation of a definite compound. In from Lithuania (10.0 – 16.2%) [32] and from India (up these phenolic compounds, c-terpinene is the component to 13.3%) [33], and in O. vulgare subsp. gracile and involved in the aromatization process which results in O. vulgare subsp. vulgare from Turkey, with 15.8 and the formation of p-cymene, that is the precursor of oxy- 17.8%, respectively [34]. genated derivatives, thymol or carvacrol [38]. Group IV: This group consists of 83 individuals, having The chemical diversity of EOs was particularly evi- a particular occurrence in Chefchaouen, Taounate, dent within populations belonging to the Occidental Ouazzane, and Tangier-Tetouan regions. Thymol and Central Rif regions. In Tangier-Tetouan popula- (45 – 80.7%, M = 60.0%) was identified as the major tions, the occurrence of carvacryl methyl ether, a minor monoterpene of this chemotype while carvacrol compound of O. compactum, in so high amounts (up to (0.4 – 15.2%) presented the lowest proportions with an 36.2%), could be considered as a specific regional char- average of 4.1%. In this group, p-cymene, c-terpinene, and acteristic. The rare a-terpineol was also well represented a-terpineol varied to a great extent (0.4 – 2.9%, 0 – 31.3%, in the northern part of the country with a particular and 0.1 – 18.6%, respectively). concentration in Tangier-Tetouan region (up to 25.8%). The chemical variability found for the composition Furthermore and as previously mentioned, carvacrol of the EOs from such large number of accessions of type was the most common in almost all populations www.cb.wiley.com © 2016 Wiley-VHCA AG, Zurich€ Chem. Biodiversity 2016, 13, 1126 – 1139 1135

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], Oulmes [OM], Rommani [RM], Sidi Kacem [SK], and Moulay Driss Zerhoun [ZR]) in the PC3/PC4 plan.

originating from the Middle Atlas region and the Cen- Regional speciﬁcity in terms of some emerging tral Morocco. Among these samples, a pure carvacrol chemotypes could be also considered to choose the best chemotype and chemotypes exhibiting high content of genetic material to be involved in the breeding program. the precursors, p-cymene and c-terpinene, were also Thus and based on the results of EO composition and recorded. Moreover, different chemotypes were found comparing, the EO yield in O. compactum accessions, within the same population, therefore, the common har- Benslimane, Rommani, Oulmes, Moulay Driss Zerhoun, vesting techniques, which includes mixed plants and Sidi Kacem populations could be recommended as collected from different individuals, may explain why parental material for direct domestication or breeding previously investigated O. compactum EOs allowed the program, exploiting the highest oil yield (up to 2.88%) detection of one chemotype in a given geographical and exhibiting the exceptional carvacrol content (up to area, it was in fact the dominant chemotype. 96%). Unfortunately, these populations are under a seri- ous overharvesting pressure in the wild, engendering gradual degradation of wild populations. Obviously, with Conclusions the decrease in wild populations, this variability will The EO of O. compactum showed a high chemical shrink more and more, until the extinction of some polymorphism. A high presumably genetic effect important chemotypes. Faced with this situation, the explains the variation of EO components observed, in situ as well as ex situ germplasm conservations are of although environmental factors may possibly account for particular importance and constitute an efﬁcient alterna- some parts of this variation. This could be of great tive to overcome the overexploitation from the wild and interest for breeding program, aiming to select given resulting genetic erosion. Furthermore, the results desired chemotypes. obtained in this exhaustive study give further contribution

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

to the understanding of the genetic background of the depicted on a geographic information system map using species. ArcGis 10.1 software (Fig. 1). The prospected areas were as follow: We thank the French and Moroccan collaborative pro- Middle Atlas: Azrou, Khenifra, and Beni Mellal. gram (PRAD) for ﬁnancial support. Northern region: Tangier-Tetouan, Chefchaouen, Ouaz- zane, and Taounate. Central Morocco: Benslimane, Rommani, Oulmes, Moulay Experimental Part Driss Zerhoun, and Sidi Kacem. It is worth mentioning that the following regions have Surveyed Populations and Sampling not been previously explored: Benslimane, Azrou, Kheni- Collection trips were organized to the whole territory fra, Beni Mellal, Tangier, Sidi Kacem, and Moulay Driss of Morocco. During these expeditions, 88 accessions of Zerhoun. Voucher specimens of representative individuals O. compactum plants, i.e., 527 individual plants, were from each locality were deposited with the Herbarium of collected from their natural habitats. The 88 O. com- National Institute of Agronomic Research, Rabat pactum populations were sampled over 2013/2014 (be- (INRA). tween March and June). The sampling strategy was designed to cover most of the remaining natural area Essential Oil Isolation of the species in Morocco. The distance between individuals exceeded 15 – 20 m, to avoid collection from The aerial parts of the collected samples were submit- close parents. Each sample was labeled and the location ted to hydrodistillation for 2 h 30 min using a Cle- was recorded using a global positioning system receiver. venger-type apparatus. Extracted EOs were stored and ° The spatial distribution of investigated populations was kept under refrigeration at 4 C until their analysis by

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 Æ 0.3 0.3 Æ 0.3 0.9 Æ 0.7 0.7 Æ 0.4 0.4 Æ 0.3 RI,MS a-Pinene 931 0.4 Æ 0.1 0.1 Æ 0.1 0.4 Æ 0.3 0.3 Æ 0.2 0.2 Æ 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 Æ 0trtrtrtrRI,MS Oct-1-en-3-ol 977 0.3 Æ 0.2 0.4 Æ 0.3 0.3 Æ 0.3 0.4 Æ 0.3 0.4 Æ 0.3 RI,MS Octan-3-one 985 0.3 Æ 0.2 0.4 Æ 0.3 0.6 Æ 0.5 0.5 Æ 0.4 0.6 Æ 0.4 RI,MS Myrcene 990 1.1 Æ 0.3 0.4 Æ 0.4 0.8 Æ 0.5 1.0 Æ 0.5 0.6 Æ 0.4 RI,MS a-phellandrene 1004 0.2 Æ 0.1 tr 0.1 Æ 0.1 0.1 Æ 0.1 tr RI,MS 3-Carene 1009 0.1 Æ 0trtrtrtrRI,MS a-Terpinene 1015 2.2 Æ 0.5 0.7 Æ 0.6 2.1 Æ 1.0 2.0 Æ 1.0 1.2 Æ 0.9 RI,MS p-Cymene 1023 15.7 Æ 6.7 7.4 Æ 5.1 28.8 Æ 14.1 24.0 Æ 13.1 9.7 Æ 5.5 RI, MS, CoI Limonene 1027 0.4 Æ 0.1 0.1 Æ 0.1 0.4 Æ 0.2 0.4 Æ 0.2 0.2 Æ 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 Æ 6.2 5.8 Æ 4.6 18.8 Æ 9.1 16.9 Æ 7.7 12.7 Æ 8.2 RI, MS, CoI cis-Sabinene hydrate 1065 0.6 Æ 0.2 0.9 Æ 0.4 0.5 Æ 0.3 0.7 Æ 0.3 0.8 Æ 0.3 RI,MS Non-1-en-3-ol 1079 tr tr 0.1 Æ 0.2 tr tr RI,MS Terpinolene 1087 tr tr tr tr tr RI,MS Linalool 1100 0.8 Æ 0.4 1.4 Æ 0.7 1.0 Æ 0.6 1.3 Æ 0.5 1.4 Æ 0.8 RI, MS, CoI Camphor 1142 tr tr tr tr tr RI,MS Borneol 1164 0.2 Æ 0.1 0.2 Æ 0.2 0.1 Æ 0.1 0.1 Æ 0.1 0.2 Æ 0.3 RI,MS Terpinen-4-ol 1176 0.3 Æ 0.1 0.4 Æ 0.2 0.2 Æ 0.1 0.3 Æ 0.1 0.4 Æ 0.2 RI,MS a-Terpineol 1189 0.7 Æ 1.0 1.6 Æ 2.8 3.6 Æ 4.6 1.9 Æ 2.6 3.4 Æ 4.0 RI,MS Carvone 1196 tr tr tr tr tr RI,MS Thymyl methyl ether 1235 tr tr tr 0.3 Æ 1.0 tr RI,MS Carvacryl methyl ether 1244 0.1 Æ 0.4 0.7 Æ 2.7 9.9 Æ 12.6 0.7 Æ 1.5 0.6 Æ 1.2 RI,MS Thymol 1293 0.3 Æ 1.0 0.4 Æ 1.8 1.8 Æ 4.1 31.3 Æ 8.1 60.0 Æ 10.1 RI, MS, CoI Carvacrol 1311 54.9 Æ 6.5 76.2 Æ 10.1 26.3 Æ 9.2 14.5 Æ 17.5 4.1 Æ 2.8 RI, MS, CoI (E)-b-Caryophyllene 1417 0.7 Æ 1.1 0.5 Æ 0.5 1.1 Æ 1.4 1.0 Æ 0.8 0.8 Æ 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 Æ 0.4 tr ––RI,MS Caryophyllene ether 1580 0.1 Æ 0.3 0.3 Æ 0.6 0.2 Æ 0.2 0.2 Æ 0.2 0.4 Æ 1.0 RI,MS Monoterpene 30.0 – 57.8 0.3 – 33 12.4 – 76.8 5.4 – 73.4 0.7 – 49.0 hydrocarbons (MH) Oxygenated 40.6 – 68.3 64 – 99.1 21.4 – 85.0 22.5 – 91.3 49.4 – 97.5 monoterpenes (OM) Sesquiterpene 0.2 – 11.98 0 – 4.2 0 – 6.6 0 – 2.9 0 – 6.3 hydrocarbons (SH) Oxygenated 0 – 2.8 0 – 6.0 0 – 1.0 0 – 1.0 0 – 5.7 sesquiterpenes (OS) Other oxygenated 0 – 1.0 0 – 2.7 0 – 1.3 0 – 1.0 0 – 1.5 compounds (OC) Total identified [%] 95.2 – 99.9 94.7 – 99.8 95.3 – 99.9 97.9 – 99.9 95 – 99.9 a b ) Compounds listed in order of elution on the nonpolar HP-5MS column. ) RI, Retention index determined relative to n-alkanes (C8 – C24) on the nonpolar HP-5MS column. c) Contents are given as mean Æ 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%).

GC/MS. The determined EO content was based on air- 0.25 lm ﬁlm thickness). He was used as the carrier gas dry matter. at a ﬂow rate of 1 ml/min with a constant linear velocity of 36.4 cm/s. The temp. was of 220 °C in the injector and used in split mode. Oven temp. was GC/MS Analysis programmed from 50 to 150 °C at rate of 3 °C/min, GC/MS Analyses were performed using an Agilent GC/ holding at 150 °C for 10 min then to 250 °C with MSD system (Agilent Technologies 7890/5975) equipped 10 °C/min. For GC/MS detection, an electron ionization with HP-5MS (apolar, 5% phenyl methyl siloxane) system was used with ionization energy of 70 eV. MSD fused SiO2 capillary column (30 m 9 0.25 mm i.d., transfer line temp. was 250 °C and MSD quadrupole

© 2016 Wiley-VHCA AG, Zurich€ www.cb.wiley.com 1138 Chem. Biodiversity 2016, 13, 1126 – 1139 temp. 150 °C. A quantity of 1 ll of each sample was [4] K. Bakhy, O. Benlhabib, A. Bighelli, J. Casanova, F. Tomi, C. injected and the split ratio was 1:30. The ion source Al Faiz, Am. J. Essent. Oils Nat. Prod. 2014, 1,9. temp. was set at 230 °C. [5] A. Benabid, ‘Flore et ecosystemes du Maroc. Evaluation et preservation de la biodiversite’, Edition Ibis Press, Paris, 2000, p. 360. [6] J. Bellakhdar, ‘La pharmacopee marocaine traditionnelle. GC (Flame-Ionization Detector) analysis Medecine arabe ancienne et savoir populaire’, Ibis Press, Mor- GC Analyses were carried out with a Clarus 500 occo, 1997. [7] C. Bouchra, M. Achouri, L. M. I. Hassani, M. Hmamouchi, PerkinElmer Autosystem apparatus equipped with two J. Ethnopharmacol. 2003, 89, 165. flame-ionization detectors and fused capillary columns [8] M. Zyani, D. Mortabit, S. El Abed, A. Remmal, S. I. Koraichi, (50 m 9 0.22 mm i.d., film thickness 0.25 lm), BP-1 Int. Res. J. Microbiol. 2011, 2, 104. (dimethylpolysiloxane), and BP-20 (polyethylene glycol). [9] F. Fadel, D. Ben Hmamou, R. Salghi, B. Chebli, O. Benali, A. The carrier gas was He with a linear velocity of 1.0 ml/ Zarrouk, E. E. Ebenso, A. Chakir, B. Hammouti, Int. J. Elec- min. The oven temp. was programmed from 60 to trochem. Sci. 2013, 8, 11019. ° ° [10] F. Ben Hammou, S. N. Skali, M. Idaomar, J. Abrini, Afr. J. 220 Cat2 C/min and then held isothermal (20 min). Biotechnol. 2011, 10, 15998. ° The injector temp. was 250 C (injection mode: split 1/60). [11] H. Sbayou, N. Oubrim, B. Bouchrif, B. Ababou, K. Boukach- The detector temp. was 250 °C. 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. Identification and Quantification of Components [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- The characterization of components was achieved on nol. 1986, 19, 22. the basis of: i) comparison of their mass spectra with [15] A. Kheyr-Pour, J. Hered. 1981, 72, 45. those of authentic reference compounds when possible. [16] J. Gershenzon, R. Croteau, in ‘Biochemistry of the Mevalonic Further identification was confirmed by comparing the Acid Pathway to Terpenoids’, Eds. G. H. N. Towers, H. A. Stafford, Plenum Press, New York, 1990, p. 99. mass spectra with those recorded in NIST mass spectral [17] D. Vokou, S. Kokkini, J. M. Bessiere, Biochem. Syst. Ecol. library and Adams terpene library [39], ii) comparison 1993, 21, 287. of their retention indices (RI)onHP-5MS determined [18] A. Azizi, F. Yan, B. Honermeier, Ind. Crops Prod. 2009, 29, 554. with reference to a homologous series of n-alkanes [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, (C8 – C24) under the same operating conditions, with those of authentic compounds or literature data. For M. R. Valentino, P. Dugo, G. Dugo, L. Mondello, Flavour Fragrance J. 2008, 23, 40. semiquantification purposes, the normalized peak area [21] C. Bicchi, E. Liberto, M. Matteodo, B. Sgorbini, L. Mondello, of each component was used without any correction B. d’Acampora Zellner, R. Costa, P. Rubiolo, Flavour Fra- factors to establish abundances. Quantitative determina- grance J. 2008, 23, 382. tion of individual components, using nonane as internal [22] S. Koc, E. Oz, I. Cinbilgel, L. Aydin, H. Cetin, Parasitology standard and correction factors according to Costa et al. 2013, 193, 316. [20] and Bicchi et al. [21] was applied to four oil [23] G. Economou, G. Panagopoulos, P. Tarantilis, D. Kalivas, V. Kotoulas, I. S. Travlos, M. Polysiou, A. Karamanos, Ind. Crops samples. Prod. 2011, 33, 236. [24] D. Stojkovic, J. Glamoclija, A. Ciri c, M. Nikolic, M. Ristic, J. Siljegovic, M. Sokovic, Arch. Biol. Sci. 2013, 65, 639. Statistical Analysis [25] M. Hazzit, A. Baaliouamer, M. L. Faleiro, G. Miguel, J. Agric. Food Chem. 2006, 54, 6314. The data were subjected to multivariate statistical [26] Z. Houmani, S. Azzoudj, G. Naxakis, M. Skoula, J. Herbs analyses using the Statistical Analysis System Software. Spices Med. Plants 2002, 9, 275. PCA was performed to identify possible relationships [27] K. Mechergui, J. A. Coelho, M. C. Serra, S. B. Lamine, S. between the components. CA was carried out to Boukhchina, M. L. Khouja, J. Sci. Food Agric. 2010, 90, 1745. determine the various groups to which the different [28] A. Danin, U. Ravid, K. Umano, T. Shibamoto, J. Essent. Oil samples refer. Hierarchical clustering was performed Res. 1997, 9, 411. [29] J. Novak, B. Lukas, C. Franz, J. Essent. Oil Res. 2008, 20, 339. according to the Ward’s variance minimization method. [30] B. Lukas, C. Schmiderer, J. Novak, Biochem. Syst. Ecol. 2013, 50, 106. REFERENCES [31] D. Mockute, B. Genovaite, A. Judzentiene, Biologija 2004, 4, 44. [32] D. Mockute, G. Bernotiene, A. Judzentiene, Phytochemistry [1] S. E. Kintzios, ‘Oregano: the Genera Origanum and Lippia’, 2001, 57, 65. CRC Press, Boca Raton, FL, 2002. [33] D. Bisht, C. S. Chanotiya, M. Rana, M. Semwal, Ind. Crops [2] J. H. Ietswaart, ‘A Taxonomic Revision of the Genus Origanum Prod. 2009, 30, 422. € (Labiatae)’, Leiden Botanical Series Vol. 4, Leiden University [34] E. Sezik, G. Tumen,€ N. Kirimer, T. Ozek, K. H. C. Baser, Press, The Haag, 1980. J. Essent. Oil Res. 1993, 5, 425. [3] S. Kokkini, in ‘Oregano, Proceedings of the IPGRI International [35] L. F. D’Antuono, G. C. Galleti, P. Bocchini, Ann. Bot. 2000, Workshop on Oregano’, Ed. S. Padulosi, IPGRI, Rome, Italy, 86, 471. 1997, p. 2.

[36] G. Figuerdo, P. Cabassu, J. C. Chalchat, B. Pasquier, J. Essent. [39] R. P. Adams, ‘Identiﬁcation of Essential Oil Components by Oil Res. 2006, 21, 134. Gas Chromatography/Mass Spectrometry’, 4th edn., Allured [37] R. S. Verma, R. C. Padalia, A. Chauhan, R. K. Verma, A. K. Publishing Corporation, Carol Stream, 2007. Yadav, H. P. Singh, Chem. Biodiversity 2010, 7, 2054. [38] A. J. Poulose, R. Croteau, Arch. Biochem. Biophys. 1978, 187, Received December 25, 2015 307. Accepted June 3, 2016