Chemical Composition and Antioxidant Activity of Essential Oil from Daucus reboudii Coss., an Endemic Plant of Algeria

: Plants of the Daucus genus (Apiaceae) are used worldwide as traditional medicines or culinary ingredients. In this work, in order to explore a new chemotype, the essential oil obtained from the aerial parts of Daucus reboudii Coss., collected from the National Park of Gouraya (Bejaïa, Algeria), was analyzed by GC-MS. Twenty-eight compounds were identiﬁed, accounting for 96.6% of the total oil. (E)-anethol was the main constituent (59.4%), followed by estragol (21.2%) and dodecanal (4.4%). (E)-anethol is an uncommon constituent of Daucus genus, hence it could be considered as a marker that contributes to differentiating D. reboudii from other species. Metal chelating, ABTS • + and DPPH • assays were performed to determine the antioxidant activity. The highest activity was revealed by the DPPH • method, where D. reboudii essential oil showed a signiﬁcantly higher activity compared to the reference standard BHT at doses of 50 and 100 µ g/mL. Results suggest that the essential oil from D. reboudii could have a potential use in the food industry as food preservative. Nevertheless, further studies are needed to assess its applicability, and to elucidate also the composition of non-volatile compounds of this plant.


Introduction
Because of their essential oils in particular, plants of the Apiaceae family have been used as spices or raw medicines since antiquity. Most of the species of the genus Daucus are encountered in Africa, Europe, West Asia and North America, and several species of this family (the carrot family) provide us with economically important food products. Among the others, Daucus genus is widely diffused, and Daucus carota is one of the most commonly used as a vegetable for human nutrition [1].
Many Daucus species are rich sources of bioactive compounds, such as essential oils, polyacetylenes and flavonoids [2][3][4], that are considered as the main contributors of their medicinal properties. These latter comprise stomachic, carminative and diuretic properties [5].
In Algeria there are about 25 Daucus species, nine of which are endemic: D. alatus, D. gracilis, D. grandiflorus, D. jolensis, D. micranthus, D. paralias, D. serotinus, D. stenopterus and D. reboudii [6]. D. reboudii Coss. (Figure 1), a species close to the world-diffused D. carota, is an endemic plant of Algeria and Tunisia. In Algeria, it grows exclusively in The essential oil composition of many Apiaceae species has been already reported. Among Daucus, the essential oil from the species carota has been extensively studied, especially from the aerial parts [8][9][10] and seeds [11]. Nevertheless, to the best of our knowledge, only one study regarding the composition of essential oil from endemic D. reboudii is present in the literature, reporting a high content of monoterpenes hydrocarbons (69.3%) and oxygenated monoterpenes (14.9%) [7].

Plant Material and Extraction of Essential Oil
Aerial parts of D. reboudii were collected during the ripening stage near the National Park of Gouraya, Bejaïa, Algeria. Plants were identified by Dr. Khellaf Rebbas and a voucher sample was deposited at the Department of Natural and Life Sciences, Faculty of Sciences, University of M'sila, Algeria (D. reboudii voucher number ST/RK N • 05). The dried plant material (200 g) was coarsely cut and distilled in a Clevenger-like hydrodistillator for 2 h.

GC-MS Analysis
Gas chromatography-mass spectrometry (GC-MS) analyses were performed using a Varian CP-3800 GC equipped with a DB-5 capillary column (30 m × 0.25 mm; coating thickness 0.25 µm) coupled with a Varian Saturn 2000 mass detector. Analytical conditions were as follows: injector and transfer line temperatures were set at 220 and 240 • C, respectively; the oven temperature was programmed from 60 • C to 240 • C at 3 • C/min, using helium at 1 mL/min as carrier gas; injection of 0.2 µL; split ratio 1:30.

Identification of Volatile Components
The volatile constituents were identified by comparing their retention times with those of pure samples, by comparison of their linear retention indices relative to the series of n-hydrocarbons, and by computer matching against commercial (NIST 2000 andADAMS 2007) and homemade libraries of mass spectra built up from pure substances and MS literature data [12,13].

Determination of Antioxidant Activity
The free radical-scavenging activity of D. reboudii was determined spectrophotometrically by three different methods. The DPPH • assay was performed in accordance with a previously published protocol [14]. Briefly, 100 µL of essential oil dissolved in methanol was added to 1900 µL of a methanol DPPH solution. After vortexing, the solution was kept at room temperature for 30 min in the dark. The absorbance was measured at 517 nm, using a solution of DPPH in methanol as control. The scavenging activity on DPPH • was expressed as inhibition percentage using the equation reported in [14].
The ABTS •+ scavenging activity was tested according to Re et al. [15], and the results were compared with the absorbance values of the reference standards butylated hydroxytoluene (BHT) and α-tocopherol. Briefly, before use, the aqueous 7 mM ABTS •+ solution was incubated for 15 h in the dark at room temperature. Then, 100 µL of essential oil dissolved in methanol was added to 1900 µL of the ABTS •+ solution. After vortexing, the mixture was kept at room temperature for 10 min in the dark, after which the absorbance was measured at 734 nm, using an aqueous solution of ABTS •+ as control. The scavenging activity on ABTS •+ was expressed as inhibition percentage using the equation reported in [15].
The metal chelating activity by the ferrene-Fe 2+ complexation assay was carried on following the method of Decker [16], using ethylenediaminetetracetic acid (EDTA) as reference standard. Briefly, 40 µL of essential oil was added to 40 µL of a 0.2 mM FeCl 2 solution. The reaction was initiated after the addition of 80 µL of ferene solution (0.5 mM). The mixture was vortexed and kept at room temperature for 10 min. The absorbance was recorded at 593 nm, and the inhibition percentage of ferrous ion chelating was calculated using the equation reported in [16].

Statistical Analysis
The antioxidant activities tests were performed in triplicate, and the results are expressed as mean ± standard deviation (SD). Student's t-test procedures were used for determination of significant differences between means, and p-values <0.05 were considered as statistically significant.

Chemical Composition of the Essential Oil
The yield of the essential oil obtained from the aerial parts of D. reboudii was 0.9% (w/w). The chemical composition obtained by GC-MS is summarized in Table 1. Characteristic MS spectra of the identified compounds are reported in the Supplementary Materials.
Twenty-eight compounds were identified, representing 96.6% of the whole oil. A high content of oxygenated monoterpenes (82.1%) was observed, mainly because of the high percentage of (E)-anethol (59.4%) and estragol (21.2%). The second most representative chemical class of constituents was that of non-terpene derivatives (6.6%), mostly characterized by dodecanal (4.4%) and tetradecanal (0.7%), followed by monoterpene hydrocarbons (4.9%), largely represented by limonene (3.6%), myrcene (0.6%) and α-pinene (0.3%). Sesquiterpene hydrocarbons (1.7%) were another important class of volatiles in D. reboudii essential oil, represented mainly by β-caryophyllene (0.4%) and germacrene D (0.4%). Oxygenated sesquiterpenes were the less represented (1.3%). In previous studies, the chemical composition of essential oils from different Daucus species has been largely investigated. These studies revealed the predominance of monoterpenes and/or sesquiterpenes, and sometimes phenylpropanoids, as major fractions. Indeed, the essential oils of D. gingidium ssp. gingidium and D. carota ssp. carota (from Italy, Lithuania and Poland) were dominated by the monoterpenes α-pinene and sabinene [5,17,18], whereas the main constituents of the oil of D. carota (from China) and D. carota var. sativa (from Egypt) were the sesquiterpenes carotol and daucol [19,20]. In the case of D. sahariensis and D. carota ssp. hispanicus, the major compound was the phenylpropanoid myristicin [21,22]. Moreover, in the oil of D. carota ssp. maximus, trans-methyl isoeugenol and β-bisabolene were identified as the main volatiles [8]. Nevertheless, in the aerial parts of D. reboudii growing in El Tarf, Algeria, Djarri et al. [7] found the monoterpenes α-pinene (39.7%) and sabinene (21.2%) as the main constituents. Our results showed that the chemical composition of the D. reboudii essential oil growing in Bejaïa, Algeria, was significantly different, mainly due to the presence of the oxygenated monoterpenes (E)anethol and estragole. This result suggests that the Daucus species considered in this work could represent a new chemotype of D. reboudii, although further genetic and epigenetic investigations will be required. Table 2 shows the inhibition (%) of the free radical scavenging activities by DPPH • , ABTS •+ and metal chelating assays. According to the results, in the DPPH • test the essential oil from D. reboudii showed an inhibition percentage comparable to those of the reference compounds BHT and α-tocopherol, and at the doses of 50 and 100 µg/mL it was significantly (p < 0.05) higher than that of BHT. On the other hand, in the ABTS •+ assay, the essential oil demonstrated mild antioxidant activity compared to the same reference compounds, while in the metal chelating assay, the essential oil showed a very small inhibition percentage compared to reference EDTA at all the concentrations tested. Although a correlation analysis was not performed, considering literature data we could propose that the activity of D. reboudii essential oil in the ABTS •+ assay could be related mainly to (E)-anethol, carvacrol and γ-terpinene. In fact, these compounds have been already reported to exert significant antioxidant activity in the ABTS •+ assay [23]. On the other hand, considering that both (E)-anethol and estragole, the main volatile constituents identified in D. reboudii essential oil, have been reported as scarcely active in the DPPH • assay [24], we can exclude their significant contribution to the activity observed in our study, which could be better associated to a synergism between the main compounds and the other phytoconstituents [24]. Nevertheless, further experimental studies on D. reboudii essential oil are needed.

Conclusions
This study reports the chemical composition and the antioxidant activity of the essential oil from the aerial parts of D. reboudii from Algeria. The results show significant differences between the chemical composition of the essential oil from D. reboudii compared to those from other Daucus species. The compounds that mostly characterized D. reboudii were the oxygenated monoterpenes (E)-anethol and estragole. Our study highlighted a possible new chemotype [(E)-anethol chemotype] in D. reboudii species, confirming the chemical polymorphism occurring either in the species or in the Daucus genus. The antioxidant capacity of the oil was evaluated using three complementary assays, namely metal chelating, ABTS •+ and DPPH • assays. The highest antioxidant activity was observed with DPPH • method, where D. reboudii showed a significantly higher activity compared to BHT at 50 and 100 µg/mL. This result suggests that the essential oil from D. reboudii could have a potential use in the food industry as food preservative. Nevertheless, further studies are needed to explore other bioactivities of D. reboudii essential oil and to elucidate also the composition of non-volatile compounds.