Intraspecific chemical variability of the essential oils of Moroccan endemic Origanum elongatum L. (Lamiaceae) from its whole natural habitats

2.1 Plant material

The sampling of O. elongatum plants was performed from wild populations during July 2016 using individual plants sampling method (each sample represents one genotype). 168 individual plants in the flowering stage were collected from 30 populations covering most of the geographical repartition of the O. elongatum, endemic to Morocco. These populations were grouped within 6 regions which all belong to 2 biogeographical areas: the Rif Mountains range including Chefchaouen, Targuist, Al-Hoceima and Taounate regions; and the Middle Atlas range covering Taza and Boulmane regions (see map, Fig. 1). Names of accessions, local climate, elevations and number of samples are reported in the Table 1. M. Ibn Tattou (Scientific Institute of Rabat-Morocco) carried out botanical identification and representative Voucher specimens were deposited in the Herbarium of National Institute of Agronomic Research, Rabat (INRA).

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Fig. 1 Geographical localizations of the 30 studied populations of O. elongatum (noted in Table 1). The maps were generated using ArcGIS Ver. 10.1.

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2.2 Essential oil extraction

Aerial parts from individual plants were dried in the shade and then submitted to hydrodistillation for 3 h using a Clevenger-type apparatus. The amounts of the dry biomass used for distillation ranged from 40 g to 100 g. Essential oil yields were estimated for each sample and expressed by (%); weight of the EOs (g) for 100 g of dry plant material using the following formula reported by Marion et al. (1994): $$\mathit{EO}yield\left(\%\right)=\frac{\mathit{weight}ofEOobtainedbydistillation\left(g\right)}{\mathit{weight}ofdrybiomass\left(g\right)}\ast 100$$

Then, the EOs were stored in a freezer in amber vials until analysis.

2.3 Essential oil analysis

All EO samples were analyzed by GC-FID equipped with two columns of different polarities (to correct for any component coelutions), GC/MS and ¹³C NMR. The GC-FID analysis associated with Retention Indices was used for quantification, whereas GC/MS was used for identification of components in combination with ¹³C NMR following a methodology developed by the University of Corsica (Tomi and Casanova, 2006).

2.4 Statistical analysis

The principal component analysis (PCA) and hierarchical cluster analysis (HCA; Ward‘s method) were performed with PAST (Paleontological Statistics Software Package) 3.14 version (Hammer et al., 2001).

3.1 Chemical variability

To classify the present investigated samples on chemical groups according to their major constituents, the entire data set was analysed by a Cluster Analysis (CA) using the Ward‘s method. Therefore, four different chemical groups have been distinguished. Fig. 3 represents the corresponding dendrogram, while the percentages of the chemical compounds (Mean, Min–Max) of each group are presented in Table 2.

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Fig. 3 Dendrogram obtained by cluster analysis, representing chemical composition similarity relationships among the 168 EO samples of O. elongatum (Group I: carvacrol, Group II: carvacrol/thymol, Group III: carvacrol/p-cymene and Group IV: thymol).

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Group I: this carvacrol-rich group (M 77.45%, 57–88.63%) is the most important one in terms of number of samples, including 100 individual plants (59.52% of samples). Several previous studies reported that the carvacrol constitutes the most dominant compound of the majority of taxa of the genus Origanum (Aboukhalid et al., 2016; Mechergui et al., 2016; Skoula et al., 1999). The second major compound in this group is p-cymene (M 7.63%, 0.20–22.95%) where 4 samples; AH1.3, AH5.2, AH6.3 and Tz5.7 presented high amounts, ranging from 16.78% to 22.95%. Moreover, many atypical compounds were observed in some samples of this group.

Interestingly, two samples exhibited noticeable amounts of terpinene derivatives: α-terpineol (10.76%, Tao4.4) and α-terpinene (7.87%, AH4.5). Regarding α-terpineol, this compound reached higher levels in some other Origanum taxa e.g. 40.3% in O. vulgare subsp. vulgare (Lukas et al., 2013), 55.7% in O. cordifolium (Figuérédo et al., 2005), 68.3% in O. vulgare subsp. virens (Melagari et al., 1995) and up to 73% in O. majorana (Novak et al., 2008). Besides, this compound reached up to 25.8% in the Moroccan O. compactum (Aboukhalid et al., 2016). For α-terpinene (7.87%), the obtained value is the highest one detected in the genus Origanum, the previous studies reported values always lower than 6.6% (Skoula et al., 1999). In addition, several samples exhibited noticeable amounts of phenolic derivatives: around 5% for thymohydroquinone (AH4.6, Tz1.4 and Tz8.3), thymoquinone (6.09%, Tz8.3) and carvacryl methyl oxide (12.66%, Tao4.4). Thymoquinone and thymohydroquinone are, on that subject, very important compounds especially known for their high and strong biological activities such as anticancer, antioxidant, antidiabetic, anti-inflammatory and hypolipidemic activities (Farkhondeh et al., 2017; Taborsky et al., 2012). In fact, both compounds were rarely reported for the genus Origanum, of which thymoquinone is considered typical for O. dictamnus (22.9%) (Skoula et al., 1999), O. syriacum (up to 27.7%) (Toncer et al., 2010; Zgheib et al., 2016) and O. vulgare subsp. hirtum (3.56%) (Skoula et al., 1999), while thymohydroquinone was detected in lower amounts (1–2.5%) in O. dictamnus, O. vulgare subsp. hirtum, O. × minoanum, O. × intercedens (Skoula et al., 1999) and in O. acutidens (Figuérédo et al., 2006c). However, no trace of thymoquinone and thymohydroquinone has been detected in several samples of the Moroccan O. compactum samples, according to recent studies (Aboukhalid et al., 2016; Bakhy et al., 2014). On the other hand, carvacrol methyl oxide was usually found in low percentages in the EOs of some Origanum taxa, whereas, highest amounts were however detected in O. vulgare subsp. glandulosum (22.9%) (Houmani et al., 2002) and O. compactum (36.2%) (Aboukhalid et al., 2016).

Group II: 31 individual plant samples belonging to this group, in which carvacrol (M 55.67%, 40.31–75.57%), thymol (M 22.29%, 8.83–38.16%) and p-cymene (M 8.47%, 2.67–14.39%) represented the most abundant compounds. This group is the most homogeneous since only few differences were noticed particularly in AH2.2, AH2.3, Tz7.5 and Tz8.2, which are characterized by considerable amounts of thymohydroquinone ranging from 2.93 to 4.15%.

Group III: this group is considered as p-cymene rich. It is the least frequent one and consists of 6 individuals (Tz2.4, Tz3.3, Tz6.1, Tz6.4, Tz6.5 and Tz7.6) all sampled from Taza region. The major compounds are carvacrol (M 35.30%, 13.62–51.11%), p-cymene (M 31.53%, 19–43.51%) and thymol (M 14.49%, 0.10–25.82%), while all the other compounds were found in percentage below 3%. Indeed, p-cymene is derived from γ-terpinene and constitutes the precursor to both carvacrol and thymol in their biosynthetic pathway (Crocoll et al., 2010). Previous studies reported p-cymene concentrations higher than those detected in the present study (i) O. vulgare subsp. glandulosum (up to 46%) (Mechergui et al., 2016), (ii) O. vulgare subsp. vulgare 49.7% (Lukas et al., 2013), (iii) O. solymicum 53.1% (Tümen et al., 1994), (iv) O. compactum 58.6% (Aboukhalid et al., 2016), (v) O. syriacum 62.18% (Toncer et al., 2010), (vi) in O. calcaratum (71.61%) (Skoula et al., 1999) and (vii) the highest values (73.6% and 83.7%) were recorded in O. saccatum (Figuérédo et al., 2006c).

Group IV: this group includes 31 individual plants and is considered as thymol-rich (M 62.40%, 42.13–80.79%). Three samples, Ch1.6, Tao3.3 and Bo4.5, presented the highest thymol amount (up to 75%). The thymol chemotype was detected in several Origanum taxa as a major compound (De Mastro et al., 2017; Mechergui et al., 2016) where the highest amounts were recorded in O. compactum (80.7%) (Aboukhalid et al., 2016) and in O. vulgare subsp. hirtum (90.2%) (Vokou et al., 1993). This group is also characterized by carvacrol (M 10.52%, 0.84–35.75%), p-cymene (M 9.79%, 1.02–21.27%), γ-terpinene (M 4.98%, 0.48–20.62%) and linalool (M 2.88%, 0.38–5.87%). The most dissimilar individual among this group is the individual plant Ch1.3 that reached the highest limonene amount (12.58%). This value is the second highest value obtained within all the investigated Origanum taxa after that reported in O. majorana var. majorana (14.3%) (Figuérédo et al., 2006b).

In spite of its limited and specific biogeographical conditions, it seems obvious that O. elongatum share the same major compounds (carvacrol, thymol and p-cymene) with several other Origanum taxa (Skoula et al., 1999), above all with the Moroccan O. compactum species. However, the particularity of the EO profile of this endemic species resides mainly in its minor components, notably thymoquinone and thymohydroquinone which were detected considerably in individual plants from Al-Hoceima and Taza regions.

3.2 Geographical repartition of chemotypes

The principal component analysis (PCA) was realized based on the concentrations of the 10 major compounds of all samples presenting an average percentages higher than 0.5% (α-terpinene, p-cymene, γ-terpinene, trans-sabinene hydrate, linalool, terpinen-4-ol, thymol, carvacrol, (E)-β-caryophyllene and thymohydroquinone). The two first principal components (PC1) and (PC2) highly explained the total data variance (PC1 + PC2 = 98.62%; Fig. 4). In fact, the distribution is highly correlated to the first principal component (PC1 = 94.13%) which displayed a positive correlation with thymol and negatively with carvacrol contents. On the other hand, PC2 was positively related to carvacrol and thymol, and negatively to p-cymene content. Individual plants rich in thymol (right side of Fig. 4) are in majority from samples of the Rif region. Plants rich in p-cymene (lower part of Fig. 4) were mainly represented by samples from Taza region.

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Fig. 4 PCA of 168 oil samples from the 6 regions (Ch: Chefchaouen, Tar: Targuist, Tao: Taounate, AH: Al-Hoceima, Tz: Taza, Bo: Boulmane). The individual plants were represented according to their regions since the circles represent regions from Rif Mountains, while the triangles indicate regions from Middle Atlas range.

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When considering the geographical distribution of the four chemotypes of O. elongatum, carvacrol was the most frequently chemotype, detected in 14 populations from both the Rif Mountains and the Middle Atlas. Besides, the carvacrol/thymol chemotype was detected in 8 populations belonging to the Rif Mountains and in 6 populations from the Middle Atlas. On the other hand, thymol and carvacrol/p-cymene chemotypes exhibited a specific geographical distribution, where thymol was more abundant in the Rif Mountains (detected in 10 of the 15 studied populations) than in the Middle Atlas region (only 4 of the 15 populations) (Figs. 5 and 6). Aboukhalid et al. (2017a) also reported that thymol chemotype is more abundant in O. compactum populations growing in northern Morocco. In contrast, carvacrol/p-cymene chemotype was detected only in 5 populations of the Middle Atlas.

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Fig. 5 Geographical distribution of the chemotypes (the percentage of individuals of the described chemotypes: Group I: carvacrol, Group II: carvacrol/thymol, Group III: carvacrol/p-cymene and Group IV: thymol) in the populations from the Rif Mountains range related to their climatic stages (Ch: Chefchaouen, Tar: Targuist, Tao: Taounate, AH: Al-Hoceima).

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Fig. 6 Geographical distribution of the chemotypes (the percentage of individuals of the described chemotypes: Group I: carvacrol, Group II: carvacrol/thymol, Group III: carvacrol/p-cymene and Group IV: thymol) in the populations from the Middle Atlas range related to their climatic stages (Tz: Taza, Bo: Boulmane).

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Furthermore, 11 homogenous populations growing in different regions have been observed, where Ch1 population presented only thymol rich plants and Bo2 population only carvacrol/thymol rich plants, while, the remaining 9 populations, were completely dominated by the carvacrol chemotype. However, the other populations exhibited remarkable chemical diversity. In addition to the impact of the environmental factors on EOs content, the chemical variability could indicate a high genetic diversity at the (intra)population level (Thompson 2002). In the same way the homogeneous populations at the chemical scale could be a signal of low rate of genetic diversity which could result from danger genetic erosion due to the overharvesting.

Chefchaouen region: it is considered the most important one in terms of Origanum diversity in Morocco, where two others species are growing spontaneously; O. compactum and the endemic O. grosii. The studied population Ch1 was found in the neighbouring area with O. compactum and grows in forest at an elevation of 1216 m. At the chemical level, all the individual plants from this population were found to be rich in thymol (59.53–80.79%).

Targuist region: three populations growing in open areas characterized by elevations around 1500 m represented this region. The majority of individuals harvested in Targuist region were characterized by the dominance of the carvacrol chemotype. In fact, all the analyzed samples belonging to Tar2 population consisted of the carvacrol chemotype. Moreover, almost all individuals from Tar1 population were rich in carvacrol, while the thymol chemotype was recorded only in samples from Tar1 and Tar3 populations. However, the chemotype carvacrol/thymol was exclusive to the population Tar3.

Al-Hoceima region: the six investigated populations were found in open areas, but at different elevations from 681 to 1815 m, among which the first three populations are growing in semi-arid climate, while the other populations were found under humid and sub-humid climates. Populations, AH3, AH4 and AH6, were found entirely rich in carvacrol, whereas populations, AH1, AH2 and AH5, are more heterogeneous and contained carvacrol, thymol and carvacrol/thymol chemotypes with no clear relation with elevation.

Taounate region: the five populations were established in open areas at an elevation below 1000 m. Within this region, we found one plant population (Tao2) with only the carvacrol chemotype. On the other hand, populations Tao1 and Tao3 are represented by plants characterized by the thymol and carvacrol chemotypes, whereas Tar3 and Tao5 populations were distinguished by the presence of three chemotypes, namely carvacrol, thymol and carvacrol/thymol.

Taza region: this region appears to be chemically the more diverse since the eight populations sampled represented all the chemotypes detected in O. elongatum, in addition the original carvacrol/p-cymene chemotype exclusive to this region. At this point, it’s difficult to know if the important amounts of p-cymene detected in the Taza region could be related to some particular pedoclimatic factors and/or genetic peculiarities. Further, studies are needed to address this point. Among the 8 populations, Tz4 is considered homogeneous since all the plants represented the same carvacrol chemotype. However, the other populations are composed by individual plants from two or three chemotypes.

Boulmane region: the seven studied populations grew generally in the forest at elevations higher than 1400 m. The carvacrol chemotype is more dominant in this region, representing all individuals from populations Bo1, Bo5 and Bo6, and more than 75% of the samples from Bo3, Bo4 and Bo7 populations. The carvacrol/thymol chemotype was detected in all the samples from Bo2 population, and only in less than 25% of the samples from Bo3 and Bo7 populations, whereas the thymol chemotype was only found in one population of this region (Bo4).

Until now, few published papers have documented the chemical composition of the EO of O. elongatum. However, these studies are restricted in terms of number of samples and in terms of the explored geographical area. Five populations of O. elongatum originated from the Rif Mountains have been investigated with rich carvacrol (67.34–81.72%) EOs (Ramzi et al., 2017). Another study presented an EO of O. elongatum from the Rif region also rich in carvacrol (79.2%) and noticed that cultivated O. elongatum plants were in the same way dominated by carvacrol (56.1–63.9%) (Figuérédo et al., 2006a). However, Bellakhdar and Il Idrissi (1990) characterized EOs of O. elongatum originated from Middle Atlas rich in thymol 60%, while El Moussaoui et al. (2013) found an EO containing mainly carvacrol, thymol, p-cymene and γ-terpinene.