Essential oil composition and biomass productivity of Moroccan endemic Thymus satureioides Coss . & Ball . growing in the Agoundis Valley

Thymus satureioides Coss. & Ball. (Th. satureioides) is an endemic thyme of Morocco. Thirty two samples of wild T. satureioides were collected from High Atlas Mountains (Valley of Agoundis). The essential oils (EO) isolated by hydrodistillation from the aerial parts were analysed by gas chromatography-mass spectrometry (GC-MS). The yield of EO ranged from 0.2 to 2.3%. Twenty six components, representing more than 95% of the oil, were fully characterized. Borneol was the main constituent. Its proportion varied between 22.7 and 37.5%. Cluster analysis of the identified components grouped the samples into three main chemotypes, borneol/carvacrol (B/Ca), borneol/camphene/carvacrol (B/C/Ca) and borneol/camphene/α-pinene (B/C/P). Other constituents were identified in significant amounts: α-terpineol (3.1 to 10.6%), 3-carene (1.5 to 10.5%) and p-cymene (2.3 to 8%). The fresh matter productivity average of T. satureioides was around 1.3 t/ha, revealing low potential of exploitation for collectors. This study has shown that the thyme growing in the valley of Agoundis is dominated by borneol chemotypes, showing different level of essential oil yield, according to locations and/or genotyes within general low potential of biomass production.


INTRODUCTION
Thymus belongs to the family of Lamiaceae, subfamily Nepetoideae and tribe Mentheae.The number of species within this genus is estimated between 250 and 350 taxa (species and varieties) of wild growing plants (Morales, 2002;Lawrence and Tucker, 2002;Napoli et al., 2010).According to Jalas (1971), Thymus is divided into eight sections over the world.Thyme species are commonly used as herbal tea, flavouring agents (condiment and spice) and medicinal plants (Morales, 2002).The aromatic and medicinal properties of the genus Thymus have made it one of the most popular medicinal plants (Nickavar et al., 2005;De Lisi et al., 2011).In traditional medicine, leaves and flowering parts of Thymus species are widely used as tonic and herbal tea, antiseptic, antitussive, expectorant and carminative as well as treating colds (Ghasemi, 2009;Rasooli and Mirmostafa, 2002).Thyme oils and in some cases ethnolic extraxt are widely used in pharmaceutical, cosmetic and perfume industry, but also for flavouring and preservation of several food products.Moreover, these species have dermatological benefits against acne and more recently, anti-platelet, antimicrobial, antispasmodic and antioxidant activities have also been demonstrated (Okazaki et al., 2002;Faleiro et al., 2003;Dob et al., 2006;Hazzit et al., 2009;Oh et al., 2009;Dandlen et al., 2010;Meister et al., 1999;Grosso et al., 2010;Giweli et al. 2013).
In Morocco, the Thymus species are widely distributed and found in several areas in Morocco (Jahandiez and Maire, 1934;El Habazi et al., 2006).The rate of endemism in the Thymus genus is 46.6%, representing 7 species (Fennane et al., 2007).Among these endemic species, T. satureioides is the widely natural growing thyme and thus the most exploited, known as the "Moroccan thyme" in worldwide.This is a perennial shrub, locally named "Azukni", in Berber and Zaiitra in Arabic (Bellakhdar, 1997).As for most thyme, this plant possesses culinary, aromatic and especially medicinal uses (Bellakhdar et al., 1991;Bellakhdar, 1997).
The composition of the essential oils (Eos) isolated from aerial parts of T. satureioides harvested from different regions of Morocco has been slightly investigated.Analysis of EOs revealed various compositions.However, in most reported papers, only few samples (one-three samples except one) have been investigated and the reported composition could not be considered as representative of the T. satureioides growing wild in the very vast regions of the country, estimated to thousands of hectares.Furthermore, the few studies undertaken in the past reported biological activity of the EO and the composition of little number of samples.
A study of one essential oil sample, originated from Beni Mellal region, has shown, respectively 52% of borneol, 3% of carvacrol, 27% of thymol and 0.33% of camphene (Jaafari et al., 2007).This study has also shown an antitumor and cytotoxicity activities.Furthermore, antimicrobial and antioxidant activities of EOs of T. satureioides were also investigated by Bouhdid et al. (2006) and Mayaud et al. (2008) in commercial samples from Pronarom (private company) for each, with 26.4 and 27.3% of borneol, respectively.Less antimicrobial effect was also demonstrated on Escherchia coli, Staphylococcus aureus and Bacillus megaterium using an essential oil sample with 31.2% of borneol and 27.4% of camphene content (Tantaoui-Elaraki et al., 1993).In contrast, two compositions from EOs samples collected from Marrakech region were dominated by borneol/carvacrol which represented 30 and 36%, respectively (Jaafari et al., 2007) and 21 and 27%, respectively (El Bouzidi et al., 2013).
Chemical variability of T. satureioides has been partly investigated by Benjilali et al. (1987a, b), who particularly studied different samples (32 samples) from two localities in Morocco: Western and Eastern region of High Atlas (HA) and the Rich region.These authors have reported two types of composition: the first is the most dominant, with borneol which content ranged between 26 to 78%, while the second showed important content of phenols: carvacrol or thymol (35 to 50%).Most studies undertaken on T. satureioides reported the presence at different proportion, of borneol or borneol/carvacrol or phenols (carvacrol and thymol) as shown in Table 1.Except Benjilali et al. (1987a, b), all the studies conducted have been realized on one sample, without any indication on the potential production of biomass.
In general, the exploitation of T. satureioides is realized by local population in the natural growing areas of the species.Collectors, generally women and children, start harvesting plants from May to July.There is no data on the available biomass nor the annual harvested quantities.The Agoundis Valley is considered as one of the richest region where T. satureioides is widely grown and where it is planned to develop with local population in a sustainable way to exploit thyme in the long term.The aim of the present work is to characterize the chemical composition of T. satureioides growing in this valley and the production capacity in term of exploitable biomass.

Plant
Aerial parts 32 samples of T. satureioides Coss.& Ball, were collected at bloom stage on June and July, 2008, from 7 stations in the Agoundis Valley (Figure 1).The geographic coordinates (elevation, latitude and longitude) of each sample were recorded using a global positioning system (GPS) receiver (Table 2).Specimens of plants were taxonomically identified by Pr.Mohamed Ibn Tatou (Scientific Institute of Rabat, Department of Biology).Voucher specimens from each locality were kept at the Herbarium of National Institute of Agricultural Research (INRA, Rabat) and that of the Scientific Institute, under the index number RAB76330.The biomass was estimated using the method of squares.It consists of taking from a unit of space from 2 to 16 m 2 , according to the visual estimation of plant density.Moreso the plant density was high and the surface of squares was small.The vegetative stage was noted and the biomass was determined by direct weighing of harvested plants from each square.A sample from each location was taken for hydrodistillation to determine the quantitative and qualitative extracted essential oil.

Essential oil extraction
Depending on the amount of the plant material available, the airdried parts of the plants were cut into small pieces and submitted to hydro-distillation in a Clevenger-type apparatus from 2 to 3 h.The essential oil was then stored at +4°C in the dark until analysis.The essential oil yields were calculated from dry matter (weight of oil/weight of dry plant).

GC-MS analysis
GC-MS analysis of the essential oil was performed on a TRACE GC ULTRA equipped with non-polar VB5 (5% phenyl; 95% methylpolisyloxane) capillary column (30 m × 0.25 mm × 0.25 µm film thickness), directly coupled to a mass spectrometer (Polaris Q) (EI 70 eV).The temperature of the injector and detector was set at 250 and 300°C, respectively.The oven temperature was programmed from 60 to 200°C at 2°C/min, and then from 200 to 300°C at 20°C/min.Carrier gas was Helium (He) with a flow rate of 1 ml/min.The components of the oil were identified by comparison of their mass spectra with those in the Wiley-NIST 7th edition library of mass spectral data.The percentage composition of the oil sample was calculated from GC-MS peak areas.

Statistical analysis
Samples 30, 31 and 32 are excluded from statistical analy-sis due to the absence of the chemical analysis data.All data were collected and statistically analysed using the XLSTAT software package trial version in order to determine the relationship between the different samples of thyme using the percentage composition of their essential oils.Euclidean distance was selected as a measure of similarity and the Unweighted Pair-Group Method with Arithmetic average (UPGMA) was used for cluster analysis.These analyses will aim to identify groups and possibly recognize chemotypes.

DISCUSSION
The essential oil yields of the aerial parts of T. satureioides varied from 0.20 to 2.29%.This value is similar to that reported previously (Jamali et al., 2012;El Bouzidi et al., 2013).Generally, in the Valley Agoundis, yield of essential oil could be considered as 1%, except areas (Makhzen to Ounain, Table 3) where plants are very old due to the difficult conditions for regeneration (more drought).There is a slight relation between the altitude and the EO yield, as shown in Figure 2B.But according to the observed results in different locations, other factors could explain the variability concerning EO production, among which genetic variation or environment effect (soil fertility, exposition, etc.) have an important role.This result is in contrast with that reported by Vokou et al. (1993), where evidence concerning the increase of EO with altitude was demonstrated.In our case, the valley is already situated in high altitude.
Concerning the biomass productivity, the regions Taghbart, Wijdane and Tanda n'Izm are characterized by favorable vegetative production (deep soils and good exposition to the north side) while in the regions Makhzen, Majjou and Makhzen to Ounanin, soils are generally more shallow and frequently exposed to South side, making the environment conditions much drier.There is however no relation with altitude and biomass production (Figure 2A).Other factors could explain the important variation of biomass.Local population starts cutting thyme from May to July, without any consideration to the survival of the species.In most case we observed that the plants are very old and plant regeneration from seed is compromised, since no plants are kept in the field to set seeds for regenerating new seed stock.

Conclusion
Local population starts cutting thyme from May to July, without any consideration to the survival of the species.
In order to improve this situation, some recommendations have been made to the local population organized under the cooperative of medicinal and aromatic plant exploittation.These recommendations include both some good practices for T. satureioides harvesting and sustainable way for medicinal and aromatic plant in general.
Emphasis is put on the following actions: Avoid cutting thymus from the same place each year.That means exploited fields should be as bi-annual rotation, the rest area has to be respected as it is the case for pasture grazing.This is important to give more chance to the plants to regenerate from seeds or to increase ramifications of old plants.
In the exploited area, collectors should let some plants to set seeds so the seed bank in the soil is constituted.It is also deeply recommended to avoid cutting the very young plants, which are having superficial roots and few ramifications.In the rest area, it is important to install hives of bees, to improve cross pollination and therefore seed production.Finally, it is also recommended to replant thyme in the degraded areas, by sowing seeds collected from some selected plants and grown in the nurseries during the first weeks after autumn.

Figure 2 .
Figure 2. Variation of Th. satureioides biomass production (A) and EO yield (B) with the altitude.

Figure 3 .
Figure 3. Dendrogram obtained by cluster analysis of the percentage composition of EOs.

Table 1 .
Mains compounds (%) of T. satureioides essential oil reported in almost papers.
*Private International society.

Table 2 .
Collection data and productivities (biomass and EO yield) of T. satureioides.
*CADEFA: Cooperative of Agoundis for Development Forestry and agriculture.

Table 3 .
Average of essential oil yield, biomass and borneol content production by region in the Agoundis Valley.

Table 4 .
Chemical composition of EO extracted from 29 samples of Th. satureioides growing in the Agoundis Valley.However, local population should take care on its use, both for medicine use and flavouring foods, due to the presence of borneol and camphene recognized as having harmful effects due to exposure or consumption.