Rosmarinus officinalis essential oils from Morocco: new advances on extraction, GC/MS analysis, and antioxidant activity

Abstract The essential oils (EOs) of Rosmarinus officinalis (Ro) are from two cities located in the eastern region of Morocco (Taourirt and Jerrada) were extracted using the steam distillation method in two cooperatives, namely "Belahssan" and "Beni Yaala Zkara", respectively. The chemical composition was determined by gas chromatography coupled with mass spectrometry (GC/MS), in which 1,8-cineole (53.6%), α-pinene (12.3%), and camphor (9.6%) represented the major compounds for essential oil of Rosmarinus officinalis from Taourirt (RoEOT). While, 1,8-cineole (42.3%), α-pinene (11.6%), and camphor (10.5%) were predominant in that of Jerada Rosmarinus officinalis (RoEOJ). The antioxidant activity of the two essential oils was assessed using, the free radical scavenging activity against the DPPH•, the ferric reducing power assay (FRAP), and the β-carotene bleaching technique. Graphical Abstract


Introduction
Antioxidants are compounds that can delay or inhibit the oxidation of lipids or other molecules by inhibiting the initiation or the propagation of oxidizing chain reactions (Velioglu et al. 1998). There are two basic categories of antioxidants: natural and synthetic antioxidants among which the second class has been found to cause long-term toxicological effects, e.g. carcinogenicity (Tahri et al. 2015). The application of antioxidants is the best strategy to prevent oxidation reactions. Synthetic antioxidants such as BHT, TBHQ, and BHA have various human health effects, including allergy, headache, asthma, and dermatitis. Therefore, there is a general desire to replace synthetic antioxidants with natural ones (Kivrak et al. 2009;Shahidi. 2000). A large number of medicinal plants and their purified constituents have shown beneficial therapeutic potential. Various herbs and spices have been reported to exhibit antioxidant activity. Ro is a good natural source of antioxidant compounds. It is widely used in the food industry to prevent possible oxidative and microbial degradation of foods (Georgantelis et al. 2007). Ro is an aromatic plant of the Lamiaceae family that comprises a large number of flowering plants of widespread distribution, which have been classified into some 236 genera and 6900-7200 species (Mohammadhosseini 2016). Ro is an evergreen shrub reaching a height of up to 1.5 m, that grows spontaneously in many Mediterranean countries. It has been widely used since antiquity in culinary, cosmetics, and medicinal products. Many studies have demonstrated that it is a very efficient plant as a natural antioxidant (Oualdi et al. 2021). This research studies the chemical composition of EO isolated from the Ro plant in Morocco (Jerada and Taourirt region) and their antioxidant activities.

Results and discussion
2.1. Analysis of essential oil by gas chromatography Table S1 represents the chemical composition identified in the Rosmarinus officinalis essential oil (RoEO), collected from two locations in eastern Morocco (Jerada and Taourirt), and extracted by steam distillation in two cooperatives, namely "Belahssan" and "Beni Yaala Zkara".
The 1,8-cineole represented with almost 40% was the main component of RoEO originating from Tunisia, Turkey, Morocco, and Italy. While the RoEOs from Spain, France, and Greece were characterized by the presence of 1,8 cineole, a-pinene, and camphor as major components with approximately equal ratios (20-30%). Finally, EO rich with myrcene chemotype has been reported in the rosemary of South America (Ra skovi c et al. 2014).

Antioxidant activities of RoEO
The antioxidant activity of the RoEOs was assessed by triplicate assays with different in vitro tests e.g. the DPPH, FRAP, and the b-carotene bleaching technique, and the results are drafted in Table S2.
DPPH method is based on the reduction of alcoholic DPPH solution in the presence of hydrogen donating antioxidant (AH) due to the formation of non-radical form DPPH-H by the reaction DPPHþ AH ! DPPH-H þ A. The remaining DPPH measured at 517 nm after 30 min, corresponds inversely to the radical scavenging activity of the antioxidant, the sensitivity of the method is determined by the strong absorption of DPPH (Koleva et al. 2002). Antioxidant activity increases with increasing concentration of EO, and the half inhibitory concentrations (IC 50 ) of the free radical scavenger activity of RoEOT and RoEOJ are respectively, 13.9 mg/ml and 5.6 mg/ml. While the ascorbic acid gave an IC 50 of 21.6 10 À3 mg/ml. FRAP which is the reducing capability of the RoEOs was measured at 700 nm by the transformation of the ferric iron (Fe 3þ ) to ferrous iron (Fe 2þ ) in the presence of the RoEO. Increased absorbance of the reaction mixture indicates increased reducing power (Anandjiwala et al. 2008). The results show that the reducing power increased with the increase of concentration of the EO; it is reported that the extract reaches the optical density of 0.5 at a concentration of 488.4 mg/ml for RoEOT and 212.8 mg/ ml for RoEOJ, while ascorbic acid reaches this value at a concentration of 0.06 mg/ml. b-Carotene bleaching consists in measuring at 470 nm the discoloration of b-carotene resulting from its oxidation by the decomposition products of linoleic acid. The addition of antioxidants in plant form induces a delay in the discoloration kinetics of b-carotene (Bourkhiss et al. 2010). The result shows the evolution of the antioxidant activity of RoEO and BHA as reference according to different concentrations, and it shows that the percentage of inhibition increased with the increase of concentration of the EO. IC 50 of the anti-bleaching of b-carotene for RoEOT and RoEOJ are respectively 27.4 and 12.9 mg/ml. While the BHA used as positive control had an IC 50 of 2.2 10 À3 mg/ml. Antioxidant activities of EOs from aromatic plants are mainly attributed to the active compounds identified. This can not only be due to the high percentage of main constituents but also to the presence of other constituents in small quantities or synergy among them (Tahri et al. 2015). The activities of EOs such as antioxidants depend not only on their structural features but also on many other factors, such as concentration, temperature, light, type of substrate, and physical state of the system, as well as on microcomponents acting as pro-oxidants (Viuda-Martos et al. 2010). RoEO contains a broad set of natural compounds, especially oxygenated monoterpenes, monoterpene hydrocarbons, and sesquiterpene hydrocarbons. RoEO exhibits a lower activity compared with that ascorbic acid and BHA. The low antioxidant activity of RoEO can be attributed to the absence of different bioactive compounds that usually belong to flavonoids, phenolic compounds, etc. which are mainly responsible for the relevant prominent biological activities, particularly antioxidant properties (Mohammadhosseini et al. 2021).

Plant material and essential oil
The detailed description is provided in the supplementary material ( Figure S1).

Gas chromatography-mass spectrometry (GC-MS)
The detailed description is provided in the supplementary material.

Dpph radical-scavenging activity
The detailed description is provided in the supplementary material.

Reducing power determination FRAP
The detailed description is provided in the supplementary material.

b-Carotene bleaching test
The detailed description is provided in the supplementary material.

Conclusion
The EOs were extracted by the steam distillation method in some cooperatives of eastern Morocco. GC/MS analysis of EOs showed a great richness with different bioactive compounds mainly 1,8-cineole, a-pinene, and camphor. The results obtained when evaluating the antioxidant activity against the DPPH, b-carotene bleaching, and FRAP are in good correlation. RoEOJ was found to be more effective than RoEOT. The oxygenated monoterpenes (such as 1,8-cineole and camphor) could be responsible for the antioxidant activity of RoEO. From a general point of view, phenols were confirmed to possess the highest antioxidant activity. In particular, some monoterpene hydrocarbons, namely, terpinolene, and c-terpinene showed a significant protective action, whereas among the oxygenated components, besides the aforesaid phenols, allylic alcohols manifested an appreciable activity. Sesquiterpene hydrocarbons and non-isoprenoid components showed a low antioxidant effect. Many perspectives are desirable, following this phytochemical study of Ro such as antimicrobial study, biopesticide activity of EO, and its toxicity.