Essential oil composition and variability of Artemisia herba-alba Asso. growing in Tunisia: comparison and chemometric investigation of different plant organs

a University Manouba, ISBST, BVBGR-LR11ES31, Biotechpole Sidi Thabet, 2020, Ariana, Tunisia. b Faculty of Sciences of Bizerte, Jarzouna Bizerte 7021, University of Carthage, Tunisia. c UR Ecophysiologie Environnementale et Procédés Agroalimentaires, Biotech Pole de Sidi Thabet. Université de la Manouba, Tunisie. d Faculty of Sciences and Arts in Balgarn PO BOX 60 BalgarnSabt Al Olaya 61985, University of Bisha, Saudi Arabia. e Department of Pharmaceutical Sciences, Bioorganic Chemistry and Biopharmaceutics, University of Pisa, Via Bonanno 33, 56126, Pisa, Italy.


INTRODUCTION
The genus Artemisia is one of the largest and most widely distributed genera of Asteraceae family includes 400 species (Judd et al., 2002).Artemisia species are of great socio-Further, the plant is widely used in traditional medicine for the treatment of diabetes, bronchitis, diarrhea, hypertension, and neuralgias (Tahraoui et al., 2007;Mahomoodally et al., 2013).EOs of this species are known for its therapeutic disinfectant, anthelminthic and antispasmodic virtues (Hatimi et al., 2001).The aim of this paper is to provide more information on the chemical composition of the EOs extracted from different parts of A. herbaalba collected in the Center of Tunisia.

Plant material
Artemisia herba-alba aerial (leaves and stems) and roots parts were collected from Chrarda locality in the Center of Tunisia (Fig. 1).Plant identification was carried out by Pr.MA Nabli, botanist at the Faculty of Sciences of Tunis-Tunisia.All samples were shade dried with ventilation for 15 days at room temperature.The plant used parts was cut into small pieces and subjected to hydrodistillation using a Clevenger-type apparatus (Clevenger, 1928) for 4 h.The oil was collected and stored at 4°C in amber vials before analysis.

Gas Chromatographic-Mass Spectral Analysis
The GC analyses were accomplished with a HP-5890 Series II instrument equipped with HP-WAX and HP-5 capillary columns (30 m × 0.25 mm, 0.25 µm film thickness).The temperature program was as follows: 60°C for 10 min, ramp of 5°C/min up to 220 °C; injector and detector temperatures 250°C; carrier gas nitrogen (2 mL/min); detector dual FID; split ratio 1:30; injection of 0.5 µL.The identification of the constituents was performed, for both columns, by comparison of their retention times with those of pure authentic samples and by mean of their linear retention indices (L.R.I) relative to the series of nhydrocarbons.The relative proportions of the essential oils constituents were percentages obtained by FID peak-area normalization.GC-EIMS analyses were performed with a Varian CP-3800 gas chromatograph equipped with a DB-5 capillary column (30 m×0.25 mm, coating thickness 0.25 µm) and a Varian Saturn 2000 ion trap mass detector.Analytical conditions were as follows: injector and transfer line temperature 220 and 240 °C, respectively; oven temperature was programmed from 60 to 240 °C at 3 °C/min; carrier gas helium at 1 mL/min; injection of 0.2 µL (10% hexane solution); split ratio 1:30.Identification of the constituents was based on comparison of the retention times with those of the authentic samples, comparing their L.R.I. relative to the series of n-hydrocarbons and on computer matching against commercial (NIST 98 and ADAMS) and home-made library mass spectra, built up from pure substances and components of known oils and MS literature data (Adams, 2009).

RESULTS AND DISCUSSION
The EO yields were respectively of 1.86%, 0.42%, 0.25% and 0.1% for the leaves, the leaves/stems, the stems and the roots, respectively.According to Haouari and Ferchichi (2009), the oil yield varied between 0.68% and 1.93% in EOs of Artemisia herba alba growing in the South of Tunisia.The chemical composition of the analyzed oils is reported in Table 1.Altogether, 152 compounds were identified in these four EOs, accounting for 99.7%, 99.7%, 98.7% and 91.3% of the whole oils, respectively.All the EOs obtained from the different parts were characterized by a high content of α-Thujone.It is important to announce that in the EOs of roots, α-Thujone was less represented (18.2%), followed by camphor (14.6%) and curcumen-15-al (14.3%).Curcumen-15-al has been reported for the first time in Artemisia herba-alba oil and the correspondent oil should be considered as a new chemotype.α-Thujone has been reported as the major constituent of the Artemisia herba-alba essential oil originating from Tunisian semi-arid and south region (Akrout, 2004;Kadri et al., 2011), Jordan (Hudaib and Aburjai, 2006), Algeria (Belhatta et al., 2014), Morocco (Paolini et al., 2010) and Israel (Fleisher et al., 2002).This monoterpenic cetone confers to this plant its characteristic smell of Mentha and its bitter taste.Furthermore, leaves EO was characterized by the highest α-Thujone amount (45.5%).β-Thujone (11.4%) was the second component followed by trans-sabinyl acetate (10.1%), 1,8-cineole (7.4%), camphor (6.8%) and isoborneol (3.4%).The main components of this oil differed from those reported by Akrout et al. (2010), and according to them, β-Thujone was the main component of the oil extracted from the leaves of Artemisia herba-alba growing in the South of Tunisia.Furthermore, in this oil, oxygenated terpenes were the most represented compounds (93.8%), but oxygenated monoterpenes prevailed over oxygenated sesquiterpenes (93.3% vs. 0.5%, respectively).Terpene hydrocarbons were present in lower amount (5.7%) divided between monoterpene (4.6%) and sesquiterpene hydrocarbons (1.1%).Haouari and Ferchichi (2009) reported that the main components were cineole, thujones, chrysanthenone, camphor, borneol, chrysanthenyl acetate, sabinyl acetate, davana ethers and davanone in Eos of Artemisia herba alba growing in the South of Tunisia.They also reported Twelve samples characterized by monoterpenes as major components amounting to more than 57% of the total oil, three had last three samples had approximately the same percentage of monoterpenes and sesquiterpenes.
The EO of roots was dominated by α-Thujone whose amount was lower than in the other oils (18.2%).Besides, this oil was characterized by high camphor and curcumen-15-al percentages (14.6% and 14.3%, respectively).It seems that curcumen-15-al was exclusive of EO; however it was detected only in very small amount in the stems (0.3%).It should be noted that this oil type was not reported in literature because.It is reported at the first time that such codominance of 3 main components of α-thujone, camphor and curcumen-15-al has been reported in Artemisia herba-alba oils.Curcumen-15-al should be considered as a new chemotype of Artemisia herba-alba.Like αthujone, βthujone was found with a lower percentage than that found in the others examined oils (5.7% vs. 11.4% and 8.2%).

CONCLUSION
Artemisia herba-alba EOs were characterized by qualitative and quantitative differences depending on the part of the plant.The variability was especially related to the proportions of constituents and relatively to the presence of new compounds or the absence of particular ones.It has been suggested that the variation in EO yield and the composition could be due to the activity of enzymes responsible for the biosynthesis of volatile oils (Hendawy and Khaled, 2005).
According to our results, it seems that chemical composition of Artemisia herba-alba essential oil varied significantly with the part of the plant.This characteristic should contribute to the understanding of the pharmacological activities of the herb.Furthermore, it must be taken into account when the plant could be used as aroma source and also in its valorization in many industrial sectors in relation to the type of volatiles accumulated.
Faculty of Sciences and Arts in Balgarn, University of Bisha, Saudi Arabia for his contribution in the correction of English language.

Table 1 :
Composition (in% of the total identified EO) a of the essential oils of leaves, leaves/stems, stems and roots of Artemisia herba-alba from center Tunisia.