Chemical Variability and Anti‐Inflammatory Activity of Rosmarinus officinalis L. Leaf Essential Oil from Algerian Sahara

The chemical composition of 71 oil samples from the leaves of Rosmarinus officinalis L., harvested in three provinces: Naâma (Western Algeria), Béchar and Adrar (Algerian Sahara), was investigated by GC‐FID, GC/MS and 13CNMR. In total, 52 compounds were identified accounting for 88.8 % to 99.9 % of the total composition. The chemical composition of the oils was largely dominated by monoterpenes, with 1,8‐cineole (9.7–70.2 %), camphor (0.3–31.0 %) being the major compounds followed by borneol (0.3–21.0 %), α‐pinene (4.5–14.5 %), β‐pinene (0.1–12.0 %), linalool (0.7–9.9 %) and verbenone (up to 11.1 %) which was present only in the samples harvested in Adrar. All compositions (71 samples) were submitted to statistical analysis. Combination of hierarchical clustering dendrogram and principal component analysis suggested the existence of three groups (one of these being subdivided into two sub‐groups) which were distinguished on the basis of 1,8‐cineole, camphor and verbenone contents. Four essential oil samples, containing 1,8‐cineole and/or camphor as main components, exhibited anti‐inflammatory activity against lipoxygenase, with IC50 values in the range 93 to 155 μg/mL.


Introduction
The genus Rosmarinus belonging to the Lamiaceae family, is very fragrant perennial woody shrubs or sub-shrubs.In Algeria, two species are mentioned: Rosmarinus officinalis L. and an endemic species namely R. tournefortii de Noé (synonym, R. eriocalyx).Rosmarinus officinalis, commonly known in Algeria as "Klil" or "Lazir" has inflorescences and calyx with very short pruinose hairs, constituted by tightly applied hairs.Inflorescences are in very short spikes, with squamiform bracts 1-2 mm, quickly caduceus. [1]R. officinalis, an evergreen shrub usually growing in the garrigue and thin forest, is widespread throughout the Mediterranean basin and widely cultivated; it extends from Portugal to Turkey in the Northern side and from Eastern Morocco to Cyrenaica (Libya) in the Southern side.It is found in various bioclimatic stages, from sub-humid to the upper arid. [2]As an aromatic species, rosemary is widely used in the Mediterranean culinary as spice and as food flavoring.In Algeria (Naâma, Béchar and Adrar provinces), R. officinalis has been used in folk medicine for many purposes such as stomachache, menstrual problems, antihypertensive, cholagogue..4] Widely used in aromatherapy, rosemary essential oil is the first "chemotyped" essential oil.According to the literature, it is classified in three chemotypes, namely, cineoliferum (high content in 1,8-cineole), camphoriferum (high content of camphor) [5] and verbenoniferum (fair content of verbenone) from Corsica and Sardinia. [6]R. officinalis chemotypes display specific biological activities: ct.cineole is used against pulmonary affections, bronchitis, sinusitis, and asthma, ct.camphor is used as antalgic, muscular relaxant, and against rheumatism pains, and lastly ct.verbenone purify blood, drains toxins, and it is appreciated as lypolitic.
In continuation of our on-going work devoted to the valorization of aromatic and medicinal plants from Algeria, [37] the aim of the present study was to investigate the chemical composition of essential oils isolated from R. officinalis growing wild or cultivated in the Algerian Sahara, namely in Naâma, Béchar and Adrar provinces and to evidence an homogeneity or a chemical variability of the compositions.In addition, the antiinflammatory activity of four samples, chosen according to their chemical profiles, was assessed by in vitro lipoxygenase inhibition assay.

Essential oil yield
The essential oil yields (w/w) calculated from dry material varied drastically from sample to sample ranging from 0.66 to 2.76 % (Table 1).The highest yields were obtained for Béchar and  Naâma provinces (0.77-2.76 %) and the lowest were observed for cultivated plants harvested in Adrar (0.66-0.95 %).However, an appreciable variation was observed between the two harvest periods.Indeed, the yield values obtained in spring were more or less 15-20 % higher than those observed in autumn.

Chemical analysis of four selected essential oil samples
[40] The detailed compositions of four oil samples (two cineole-rich oil samples and two camphor-rich oil samples) are reported in Table 2, including also the mode of identification of every component.In total, 52 components have been identified.They accounted for 91.4-98.7 % of the whole composition.As expected, all the samples contained mainly oxygenated monoterpenes as well as monoterpene hydrocarbons.Sesquiterpene hydrocarbons and oxygenated sesquiterpenes were scarce and always at low content.

Chemical variability of R. officinalis from Algerian Sahara
Data obtained from the chemical composition of the 71 samples have been submitted to statistical analysis, Principal Component Analysis (PCA) and Hierarchical Cluster Analysis (HCA).Three groups were differentiated, they contained 10, 30 and 31 oil samples, respectively (Figure 2).Group III has been divided into two sub-groups, IIIA, (23 oil samples) and IIIB (8 oil samples).Mean value (M), standard deviation (SD) as well as minimum and maximum content have been reported in Table 3 for every component present at appreciable content.It could be highlighted that the index of dissimilarity was high and therefore, qualitative and quantitative differences between groups may be discussed.
Concerning the repartition of essential oil samples with respect to their geographical area, it could be highlighted, going to north to south, that: Going from Naâma, Béchar, and Adrar, it could be highlighted that oil samples from Naâma and Béchar belonged mainly to the cineole chemotype, while oil samples from the locality of harvest located most southerly, Adrar, all belonged to the camphor chemotype, although they contained appreciable amounts of verbenone, compound that characterize the third chemotype of R. officinalis essential oil.It should be kept in mind that the Adrar oil samples were isolated from cultivated plants of undetermined origin.Therefore, genetic factors may be responsible of the specific composition of the essential oil samples.

In vitro Anti-Inflammatory Activity
Inflammation is a physiological response to a variety of agents including infectious microorganisms, toxic chemical compounds and physical injury.There are many diseases that are associated with the inflammation process.So, chronic inflammation increases the risk for skin inflammation, autoimmune diseases such as arthritis and diabetes, Alzheimer's disease and various cancers.[46] Therefore, the elimination of inflammation may represent a valid strategy for cancer prevention and therapy.For that purpose, we evaluated the in vitro anti-inflammatory potential of four samples of R. officinalis leaf essential oil (B5, B34, N14 and A1) by determining their ability to inhibit lipoxygenase (LOX).The inhibition percentages of LOX are presented in Table 4.
All essential oil samples exhibited an inhibition of LOX activity and have dose dependent, with good percentages of inhibition ranging between 25 % and 96 % (Table 4).Indeed, at 0.3 and 0.4 μL/mL, the four oil samples showed an appreciable percentage of inhibition in the range 68.1 to 84.2 %, while this inhibition was very expressed for sample A1 (96.0 % at 1.6 μl/ mL).The IC 50 values (concentration at which 50 % of the lipoxygenase was inhibited) were determined for the R. officinalis leaf essential oil and for the nordihydroguaiaretic acid (NDGA), which used as a reference in anti-inflammatory activity.The IC 50 values, presented in Table 4, allow us to evaluate and compare the effectiveness of essential oils.So, the strongest anti-inflammatory ability was noted for sample A1 (IC 50 = 93 μg/ mL), which contained camphor as major compound (23.2 %) accompanied by cineole (13.2 %) and α-pinene (10.0 %).This sample was also characterized by an appreciable amount of pcymene (2.3 % vs. 0.5-0.6 %), limonene (4.0 % vs. 0.8-1.9%) and iso pinocamphone (1.3 % vs. 0.0 %) which probably participated to this good anti-inflammatory activity of the sample A1.However, in comparison with standard anti-inflammatory (NDGA: IC 50 = 11 μg/mL), the essential oils of the samples tested were found to be less active.
In parallel, the anti-inflammatory activity of various cineolerich essential oils has been investigated.Indeed, Viljoen et al, [48] evaluated the anti-inflammatory activity of essential oil of 17 indigenous Agathosma species.All the essential oils exhibited in vitro anti-inflammatory activity with that of A. namaquensis (cineole, 23.5 %) being the most potent (IC 50 = 31.54μg/mL) although it displayed a low activity by compared to the standard (NDGA: IC 50 value of 2.39 μg/mL).Similarly, Albano et al. [49] reported a poor activity for the cineole-rich (49 and 59 %, respectively) essential oils of T. mastichina (IC 50 = 1084 μg/ mL) and Salvia officinalis (IC 50 = 827.9μg/mL), the IC 50 value of positive control (NDGA) was 63.7 μg/mL.

Conclusions
Seventy-one essential oil samples have been isolated by hydrodistillation from R. officinalis plants growing wild or cultivated in the Algerian Sahara, the localities of harvest (particularly Béchar and Adrar) being located most southerly than any other reported in the literature.The compositions of 71 samples displayed a fair chemical variability, three groups (one group subdivided into two sub-groups) being differentiated on the basis of 1,8-cineole, camphor and verbenone contents.Four oil samples have been tested to investigate their anti-inflammatory

Experimental Part Plant Material
In total, leaves from seventy-one individual plants of R. officinalis were collected in three provinces of Algeria: Naâma, Béchar and Adrar (Figure 1).

Oil Distillation
Dry leaves (30-300 g) were submitted to hydrodisillation for 2 h using a Clevenger-type apparatus.Yields have been calculated from dry material (w/w).

Analytical GC
GC analyses were performed on a Perkin-Elmer Clarus 500 gas chromatograph (FIame Ionization Detector) equipped two fused silica capillary columns (50 m×0.22 mm, 0.25 μm film thickness), BP-1 (polydimethyl siloxane) and BP-20 (polyethylene glycol).The oven temperature was programmed from 60 °C to 220 °C at 2 °C/min and then held isothermal at 220 °C for 20 min, injector temperature: 250 °C; detector temperature: 250 °C; carrier gas: hydrogen (1.0 mL/ min); split: 1/60.The relative proportions of the oil constituents were expressed as percentages obtained by peak area normalization, without using correcting factors.Retention indices (RI) were determined relative to the retention times of a series of n-alkanes with linear interpolation ("Target Compounds" software from Perkin-Elmer).C NMR analysis was performed on a Bruker AVANCE 400 Fourier Transform spectrometer operating at 100.623 MHz for 13 C, equipped with a 5 mm probe, in deuterated chloroform (CDCl 3 ), with all shifts referred to internal tetramethylsilane (TMS). 13C NMR spectra were recorded with the following parameters: pulse width (PW), 4 μs (flip angle 45°); acquisition time, 2.73 s for 128 K data table with a spectral width (SW) of 220 000 Hz (220 ppm); CPD mode decoupling; digital resolution 0.183 Hz/pt.The number of accumulated scans ranged 2 000-3 000 for each sample (around 40 mg of oil in 0.5 mL of CDCl 3 ).Exponential line broadening multiplication (1.0 Hz) of the free induction decay was applied before Fourier transformation.

Identification of Components
[40] In the investigated samples individual components were identified by NMR at contents as low as 0.3-0.4%.

Anti-inflammatory Activity
The anti-inflammatory capacity of four samples (One sample from each PCA-HCA group: B5, B34, N14 and A1) of R. officinalis essential oil was evaluated by in vitro lipoxygenase inhibition assay.Lipoxygenase from Glycine max (soybean) (EC 1.13.11.12) type IÀ B inhibiting activity was essayed spectrophotometrically as described by Bayala et al. [46] with slight modifications.It was performed by recording the rate of change in absorbance at 234 nm.Indeed, the increase of absorbance at 234 nm due to formation of 13-hydroperoxides of linoleic acid (substrate used for LOX inhibition activity assay). [46]iefly, 20 μL of the enzyme solution (at the final concentration of 200 U/mL) was prepared in boric acid buffer (0.2 M; pH 9.0), mixed with 20 μL of essential oil solution in DMSO (0.05 to 1.6 μL/mL, final concentration), and dissolved in 1940 μL of boric acid buffer and then incubated at 25 °C for 5 min.The reaction started by addition of 20 μL of substrate solution (linoleic acid, 25 mM) and the mixture was incubated for 4 min.Then placed in a boiling water bath and boiled for 3 min.The absorbance was measured at 234 nm.Nordihydroguaiaretic acid (NDGA) was used as reference standard.All tests were performed on triplicate.The percentage of lipoxygenase inhibition was calculated according to the following equation: Inhibition % ¼ Absorbance control Absorbance sample Absorbance control x 100 The IC 50 value was calculated by the concentration of R. officinalis EO inhibiting 50 % of LOX activity.

Figure 1 .
Figure 1.Sampling provinces of R. officinalis from Algeria.

Figure 2 .
Figure 2. Hierarchical Cluster Analysis of the compositions of the 71 essential oil samples isolated from leaves of R. officinalis from Algerian Sahara.

Table 1 .
Yields (%) of essential oils isolated from leaves of R. officinalis harvested in three provinces of Algerian Sahara.

Table 2 .
Chemical composition of four essential oil samples isolated from leaves of Saharan R. officinalis.

Table 3 .
Chemical variability of R. officinalis leaf oil from Algerian Sahara.

Table 4 .
In vitro anti-inflammatory activity of R. officinalis leaf essential oil (Percentage Inhibition of LOX and IC 50 values).against lipoxygenase.IC 50 values ranged between 93 and 155 μg/mL compared with the standard NDGA, IC 50 value, 11 μg/mL.It could be highlighted that R. officinalis growing wild or cultivated in arid provinces produce secondary metabolites similar to those found in temperate countries.In the domain of EOs, this point looks encouraging in view of the climatic change. activity