The full range of biological properties of the essential oils is linked to concrete chemical compositions which have shown efficacy in the experiments. These properties are usually attributed to the major components of the essential oils. However, in such a complex mixture, the possible effects or synergistic and antagonistic interactions between compounds are not negligible (Bakkali et al., 2008; Dhami and Mishra 2015). Phytochemical variation is a common phenomenon in aromatic and medicinal species. As a consequence of this fact, and unlike synthetic medicines, food preservatives or insecticides which are chemically consistent and stable, essential oils and other herbal products are rich and complex mixtures linked to inherent biological variation. In this regard it should be noted that in most cases, promising
Jaime Usano-Alemany, Jesús Palá-Paúl and David Herráiz-Peñalver 30
beneficial effects of essential oils are debased by spot samples or not well defined raw materials with lack of detail traceability regarding cultivation managements, collection times or edaphoclimatic conditions. To this extent, there is a definite necessity for chemical industries to manage high-quality botanicals, dealing with standardised or standardisable active compounds and matching the triple constraint ŘqualityŔsafetyŔefficacyř (Carrubba and Scalenghe 2012).
The first move towards this goal is to carry out a comprehensive and in-depth study of natural wild populations of potentially attractive market-based essential oil plants. These efforts have been notably intensive in the Mediterranean region where a vast number of species are well known by their essential oils. The content of the corresponding relevant secondary plant products in general is lower in plants grown in a moderate Atlantic climate than in those grown in semi-arid regions (Selmar and Kleinwächter, 2013b) and this is even more pronounced for the essential oils. The Mediterranean climate, with its typical warm dry summers and cool winters encompasses a variety of sub-climates mostly with drought periods which may last up to 5 or 6 months, during summer. Accordingly, natural epigenetic variations in natural systems allow the study of correlations between DNA methylation levels, environmental conditions and phenotypic expression (Lira-Medeiros et al., 2010). DNA methylation may thus affect phenotypic plasticity in response to adverse environmental conditions (Han and Wagner 2013), upregulating the production of diverse secondary metabolites. In this context, natural populations of most species are usually heterogenous and different essential oil profiles and volatile compositions can be discerned (Muñoz-Bertomeu et al., 2007; Ormeño et al., 2007; Rzepa et al.,2009; Herráiz-Peñalver et al., 2010, 2013;
Rajabi et al., 2014; Usano-Alemany et al., 2014a). In the last decades, the description of variations in the chemical composition of essential oils in wild populations has been the subject of intensive research. The major monoterpenes, sesquiterpenes and even diterpenes show pronounced dynamics during vegetative cycle that have been confirmed in numerous species and under different geographical conditions (Table 4). As an example, the phenological investigation of wild populations of Hypericum triquetrifolium showed that the main essential oil compounds varied with the phenological stage (Hosni et al., 2011). The oils collected at the vegetative stage were found to be rich in both aliphatic and branched hydrocarbons with oxygenated compounds with mono and sesquiterpenes less represented. At the flowering stage, the oils consisted mainly of sesquiterpene hydrocarbons whilst at the fruiting stage the essential oils were distinguished by their higher content of oxygenated compounds (mono and sesquiterpenes). Sesquiterpenes were also higher in wild collected flowering plants of Thymus algeriensis (Zouari et al., 2012). Similar variations were found in Hypericum perforatum when wild populations were subjected to different collection times (Schwob et al., 2004). Higher content of oxygenated compounds during fruit maturation-vegetative has also been reported for Salvia lavandulifolia (Usano-Alemany et al., 2014b).
Nonetheless, in Thymus caramanicus oxygenated compounds outstanding as the major portion throughout the whole phenological cycle (Nejad Ebrahimi et al., 2008).
Table 4. Essential oil main compounds reported from different phenological stages in representative wild
Vegetative Floral budding Flowering Fruiting
1. β-Caryophyllene
5. α-Pinene/1,8-Cineole/Camphor 10.2/15.0/14.3 - 8.5/13.7/12.0 - Strong No
6. p-Cymene /γ-Terpinene/Thymol 33.0/12.6/16.1 - 30.4/14.3/26.2 40.8/9.7/23.3 Strong No
7.1.
13. 1,8-Cineole/Linalool /Camphor - - 32.6/30.4/13.3 29.1/38.2/15.3 Weak 3526:2015
14. 1,8-Cineole/Borneol /Camphor 31.8/20.0/13.8 - 13.6/10.3/4.3 17.3/14.0/15.3 Strong 3515:2012
Table 4. (Continued)
Vegetative Floral budding Flowering Fruiting
15. Geraniol/Geranyl acetate - 57.5/28.6 68.0/14.2 75.0/5.56 Strong 4727:2014
16.1
19. β-Pinene/Pinocamphone 20.0/39.2 25.7/29.6 8.5/54.0 7.1/57.5 Strong 9841:2013
20.1
(a) Species correspond to those reported in Table 2.
*All data presented are referred to mean values published as %, † mg kg-1.
Table 5. Phenological variation in the content (%) of 1,8-cineole in the essential oil of cultivated clonal lines of Salvia lavandulifolia
COMPOUND Harvesting events in one year
I II III IV V VI
1,8-cineole
Clones HSD A C B B AB AB
A2 de 37.9 24.5 13.3 24.5 25.2 18.3
A3 e 25.5 17.7 18.4 11.6 16.1 5.7
B1 bc 53.1 16.8 38.2 42.7 46.7 43.7
B2 bc 47.9 17.5 41.0 43.2 44.3 42.7
B3 ab 51.8 27.8 43.3 49.4 48.8 57.0
C1 de 29.6 13.8 17.3 24.9 38.6 31.6
C2 e 24.2 18.2 11.3 8.2 15.6 15.0
C3 ab 54.1 31.3 40.6 42.1 56.2 52.1
D1 a 62.7 48.8 52.5 49.1 56.8 54.3
D2 cd 46.0 22.0 24.3 34.4 37.2 31.2
D3 abc 49.5 10.8 49.2 50.6 47.5 43.0
Average per harvesting event 43.8 22.7 31.8 34.6 39.4 35.9 Cells in bold indicate samples from group 1 with low content of 1,8-cineole (μ = 19.74%). The rest of
cells belong to group 2 with high content of 1,8-cineol (μ = 47.13%). The methodologies used were the k-mean and Mahalanobis distance for the group separation and for the setting of groups differences, respectively, by using the major 15 compounds of the essential oil in cultivated plants.
Different uppers and lower cases in the row and in the column, respectively, indicate significant differences in relative percentage (p < 0.05). The phenological stages were the following: I (vegetative), II (new leaves formation), III (flowering), IV (late flowering) V (fruit maturation), VI (vegetative II). Table extracted from Usano-Alemany et al. (2014b) with modifications.
The evolution of main terpenoids according to the phenology of the plants is highly complex and difficult to clarify due to the proved heterogeneity. Chemical profiles may be different between species and also within species regarding different locations and growing conditions (Tounsi et al., 2011). As previously mentioned, several factors can influence the qualitative and quantitative changes in the chemical profile and they must be handled accordingly in each particular case. In this regard, cultivation of medicinal and aromatic plants is presented as the best way to reduce these sources of variation generating material grown under common conditions in contrast to wild collection. This fact would help to solve the lack of reproducibility of plant-based products and to achieve assurance in quality and traceability. However, to minimize the above mentioned uncertainties, an appropriate characterization of propagating material is needed in order to promote a better cultivation management.
Genetic variation between plants can be a major source of variation (Gorelick and Nirit 2014) in plant secondary metabolites does so for essential oils, and it can considerably affect the amounts and type of metabolites produced. Accordingly, studies carried out with particular genotypes have shown great stability on their chemical profiles hence the investigation on cultivated clones has been proved to be the best method for monitoring real phenological changes in the composition of the essential oils (Jordán et al., 2006; Shojaiefar et al., 2015; Smitha and Virendra 2015; Usano-Alemany., in press). Monitoring provides
Jaime Usano-Alemany, Jesús Palá-Paúl and David Herráiz-Peñalver 34
valuable information on optimum harvesting period and real phenological influences on essential oil chemical composition which is a key reporting for commercial growers (Table 5). Accordingly, 1,8-cineole showed its lower content in a clear manner in Salvia lavandulifolia when new leaves begin to grow in Spring even though the relative percentage is remarkable linked to concrete clonal lines (Usano-Alemany et al., 2014b). Some clonal lines had low values throughout the phenology. Therefore, chemical variability and lack of reproducibility are the primary reasons why essential oils and other plant extracts are not yet utilized on a large scale even though a plethora of new bioactivities and uses are described each year (Christaki et al., 2012; Hyldgaard, 2012; Amorati et al., 2013; Ellse and Wall 2014).