Phytochemical Analysis, Antioxidant Potential, and Cytotoxicity Evaluation of Traditionally Used Artemisia absinthium L. (Wormwood) Growing in the Central Region of Saudi Arabia

Artemisia absinthium, a plant distributed worldwide, has been reported for its numerous traditional uses, and its phytoconstituents have been investigated in several previous publications. The current study was designed to investigate the chemistry and quality; i.e., the antioxidant and cytotoxic activities, of A. absinthium volatile oil from plant species growing in the central area of Saudi Arabia compared to reported data for the plant growing in other parts of the world. Gas chromatography–mass spectrometry (GC-MS) and gas chromatography with flame ionization detector (GC-FID) spectroscopic analyses, in addition to in vitro antioxidant and cytotoxic assays, were conducted to fulfill the aims, and integrated the study’s conclusion. A total of 34 compounds representing 99.98% of the essential oil of the plant were identified; among them, cis-davanone was found at the highest concentration (52.51%) compared to the other constituents. In addition, α-gurjunene (7.15%), chamazulene (3.38%), camphene (3.27), γ-eudesmol (2.49%), pinocarvone (2.18%), and ocimenone (2.03%) were also identified as major constituents of the plant’s essential oil. The total percentage of davanones (53%) was the highest percentage found in the plant species growing elsewhere in the world. The antioxidant assays; i.e., the total antioxidant capacity (TAC), ferric-reducing antioxidant power (FRAP), and 2,2-diphenyl-1-picrylhydrazyl-scavenging activity (DPPH-SA), evidenced the potential in vitro antioxidant activity of the A. absinthium essential oil, with 35.59, 10.54, and 24.00 mg Trolox equivalent per gram of the essential oil. In addition, the metal-cheating activity (MCA) of the essential oil was measured at 29.87 mg ethylenediaminetetraacetic acid (EDTA) equivalent per gram of the essential oil. Moreover, a limited cytotoxic effect of the essential oil against all tested cell lines was observed, which might be considered as an indicator of the safety of A. absinthium as a worldwide edible plant. In conclusion, the study confirmed the variations in the A. absinthium essential oil constituents in response to the environmental conditions. The study also highlighted the potential health benefits of the plant’s essential oil as an antioxidant agent.


Introduction
Aromatic plants have been utilized for human use as food, flavoring agents, and medicaments for the treatment of various diseases. They are also considered as chief materials in many industries; e.g., in pharmaceutical preparation and perfume preparation. Aromatic-therapy with plants and their derived volatile oils has been given a high economic value around the world [1]. Several factors affecting the quality and diversity of volatile oils in particular aromatic plants, have been reported [2,3]. Among them, the major factor is the variation in environmental conditions in the area where the plant grows [4][5][6]. Therefore, the production, chemistry, and quality of the volatile oil of a particular plant could Table 1. Major volatile constituents of A. absinthium growing in different locations.

Location
Major Volatile Constituents Oil Production Ref.
The current study investigated a phytochemical analysis of A. absinthium volatile oils for the plant species growing in the central area of Saudi Arabia. In addition, the effects of environmental conditions on the volatile constituents of the plant were also discussed by comparing the results of the current analysis with the reported volatile constituents of the plant. The study also provided data on the antioxidant and cytotoxic effects of the volatile oil of A. absinthium.

Essential Oil Production and Constituents of A. absinthium
A. absinthium is one of the most widely distributed and commonly used aromatic plants in the world. Its volatile constituents have been investigated and described for the plant's species growing in different locations and countries; e.g., Egypt, Tunisia, Algeria, Spain, Germany, Siberia, Lithuania, and Turkey. However, these different countries varied in their environmental conditions, which affected the plant's volatile oil constituents and production (Table 1), and subsequently the quality and biological activities of the oil. In the current study, the volatile oil of the A. absinthium species growing in the central region of Saudi Arabia was distilled and analyzed using GC-MS and GC-FID spectrometry (GC-chromatogram is available in the supplementary file). The result obtained for the plant's volatile oil production (0.94 ± 0.03% w/w) was slightly higher than the reported percentage of volatile oil production obtained from same species growing in the southwest region of Saudi Arabia (0.83% v/w), at a distance of more than 1000 km [24,33] away from the Qassim region. This large distance between Qassim on one side and Al-Baha and Jazan on the other side was accompanied by distinct variations in the environmental conditions of these locations. In March, for the plants collected in the current study and in the reported study [24], temperatures of 7-18 • C and 17-27 • C and humidity levels of 83% and 25% were recorded for the Jazan and Qassim regions, respectively. In addition, Qassim is characterized by flat land, while Jazan and Al-Baha are mostly mountainous [55]. Moreover, the soil contents also vary, as Qassim is mostly characterized by the presence of a high salt content in the soil. The environmental conditions' effects on the plant constituents were clearly demonstrated by comparing the results obtained using the GC analysis of the volatile oil sample of A. absinthium growing in central Saudi Arabia (Table 2) with the reported results of the plant's volatile constituents from other areas of Saudi Arabia and the world.
The chemotypic variations among the A. absinthium plants growing in different locations might be clearly determined by comparing the major constituent of the plant in the current study; i.e., cis-davanone, which was also reported to be the major constituent of the plant samples collected from other regions of Saudi Arabia [24,33] compared to the major constituents; e.g., chamazulene, cineole, and trans-sabinyl acetate, reported in the plant samples growing in other regions of the world [38][39][40]42]. In addition, the colors of the A. absinthium essential oils from different regions were varied, and seemed be associated with the major constituents of the plant's essential oils [23]. For instance, the essential oils obtained from A. absinthium growing in Tunisia, Algeria, China, and southern Saudi Arabia were reported to be dark blue in color, which was attributed to the presence of chamazulene (approximately ≥8%) as one of the major constituents of the plants' essential oils [24,34,35,[61][62][63]. In addition, essential oils obtained from Lithuanian A. absinthium were described in the literature as dark brown in color, with major constituents including transsabinyl acetate, β-pinene, trans-thujone, cis-thujone, and myrcene; however, chamazulene did not exceed a concentration of 1.5% [37,38]. Also, a yellow-colored essential oil was obtained from A. absinthium growing in Tajikistan, in which chamazulene was detected as one of the minor constituents of the essential oil [40]. The essential oil of A. absinthium obtained in this study was yellow in color, which might be attributed to the lower concentration of chamazulene (3.38%) compared to the reported concentration of the compound in the blue A. absinthium essential oil. The yellow color of the essential oil in the current study might also be attributed to the presence of other major constituents; e.g., cis-davanone (52.51%), α-gurjunene (7.15%), and camphene (3.27%). When compared to the current analysis of the plant's essential oil, the blue-colored chamazulene was also identified at a higher concentration in the volatile oil of A. absinthium growing in the southern area of Saudi Arabia, at a concentration of 8.2% versus 5.04% for the species of the plant growing in Jazan and AL-Baha, respectively [24,33]. This significant variation in the chamazulene concentration among the Saudi Arabian A. absinthium species seems to be a direct reason for the color variation (blue to yellow), and also might be associated with a variation in the biological activities of the plant's essential oil. However, identification of semisaturated azulenes; i.e., α-gurjunene (7.15%), γ-gurjunene (0.86%), and guaiol (0.49%), in addition to 3.38% of the chamazulene, in the current GC-MS and GC-FID analyses was an indication of the liability of the plant to biosynthesize azulenes as its major sesquiterpene constituents ( Table 2). The representation of these azulene derivatives could be also indicative of part of the possible chemical bioconversion in the plant's essential oil constituents ( Figure 1). also might be associated with a variation in the biological activities of the plant's essential oil. However, identification of semisaturated azulenes; i.e., α-gurjunene (7.15%), γ-gurjunene (0.86%), and guaiol (0.49%), in addition to 3.38% of the chamazulene, in the current GC-MS and GC-FID analyses was an indication of the liability of the plant to biosynthesize azulenes as its major sesquiterpene constituents ( Table 2). The representation of these azulene derivatives could be also indicative of part of the possible chemical bioconversion in the plant's essential oil constituents ( Figure 1).

Antioxidant Activity of A. absinthium Essential Oil
Four different in vitro assays were conducted to evaluate the antioxidant potency of the A. absinthium essential oil. These assays measured the reducing effect (i.e., TAC and FRAP), scavenging ability (i.e., DPPH), and metal-chelating activity (i.e., MCA) of the

Antioxidant Activity of A. absinthium Essential Oil
Four different in vitro assays were conducted to evaluate the antioxidant potency of the A. absinthium essential oil. These assays measured the reducing effect (i.e., TAC and FRAP), scavenging ability (i.e., DPPH), and metal-chelating activity (i.e., MCA) of the essential oil of the plant. The measurements were conducted in a comparable manner to the standard antioxidant compound, Trolox, and standard metal-chelating agent, EDTA. Therefore, the results were expressed as Trolox equivalents in the TAC, DPPH, and FRAP; however, the result of the MCA was expressed as the EDTA equivalent (Table 3). The results shown in Table 3 represent data obtained from three independent measurements; means ± SDs were calculated. TAC, total antioxidant capacity in mg Trolox equivalent per gram of the essential oil; FRAP, ferric-reducing antioxidant power in mg Trolox equivalent per gram of the essential oil; DPPH-SA, 2,2-diphenyl-1picrylhydrazyl-scavenging activity in mg Trolox equivalent per gram of the essential oil; MCA, metal-chelating activity in mg EDTA equivalent per gram of the essential oil.
The antioxidant activities of A. absinthium essential oils have been recorded several times in the literature [35,43,44,[64][65][66][67]. The methods that were used in most of the reports were different from the methods used in this study; therefore, comparing current antioxidant results to the reported data might be conflicting. However, few studies reported similar antioxidant methods. For instance, a phosphomolybdenum assay (TAC) was conducted using standard calibration curves obtained for α-tocopherol compared to the essential oil obtained from A. absinthium growing in Turkey [68]. The result was measured as 2.89 ± 0.16 mM α-tocopherol equivalent, compared to 35.59 ± 1.86 for the total antioxidant capacity of the A. absinthium essential oil in the current work. The ferricreducing antioxidant capacity (FRAP) of the essential oil of A. absinthium growing in Iran was calculated as 10.67 ± 0.45 mg/g gallic acid equivalent, which was significantly lower than the current result for FRAP, at 24.00 ± 0.13 mg/g of the essential oil as the Trolox equivalent [44].
The overall results represented in Table 3 revealed the potential antioxidant power of the volatile oil of A. absinthium growing in the central area of Saudi Arabia, and also indicated that the environmental conditions of the area where the aromatic plants grew represented a primary factor affecting the chemistry and quality of the essential oils obtained from these plants. The current higher antioxidant activity of the essential oil of A. absinthium could be attributed to the presence of a high percentage of davanones, which were found collectively to be more than 53%. Plants rich in davanones have been reported for their interesting antioxidant activities; specifically, scavenging free radicals [58,59]. The higher percentages of oxygenated sesquiterpenes in the essential oil of the plant could be also a reason for the higher antioxidant activity in the current results. The oxygenated sesquiterpenes, guaiol (0.49%) and globulol (1.38%), have shown antioxidant power against thiobarbituric-acid-reactive species [69]. In addition, the presence of ocimene (1.65%); Plants 2022, 11, 1028 7 of 14 1, 8-cineole (0.87%); α-terpinene (2.68%); cis-linalool oxide (1.55%); camphene (3.27%); and α-thujene (0.35%), which have been reported for their antioxidant effects as scavengers of the thiobarbituric-acid-reactive species and linoleic acid peroxidation [69], could also affect the antioxidant activity of A. absinthium.

Cytotoxic Activity of A. absinthium Essential Oil
The cytotoxic effects of the A. absinthium essential oil were evaluated at five different concentrations (in a range of 0.01 to 100 µg/mL) against three cancer cell lines; i.e., A-431, MCF7, and PANC-1, as well as one normal cell line; i.e., HSF, using the standard SRB (sulforhodamine B) assay (Table 4, Figure 2). An SRB assay is used to measure cell masses and proliferation in an acidic medium. The SRB binds quantitatively to cell proteins, and can be extracted using a basic solvent [70]. The blank absorbances were measured (0.02 to 0.04 A.U.) and were subtracted from the absorbances of the tests. The negativecontrol absorbances were also measured as 1.6373, 1.344, 2.287, and 2.816 for the MCF-7, Panc-1, A-341, and HSF cell lines, respectively (the raw data are available in the supplementary file, Table S1-S4). The results presented in Table 4 revealed that the A. absinthium essential oil showed negligible cytotoxicity against all the tested cancer cell lines, and did not exceed 8.27%, 12.77%, and 11.10% of the activity against the three cancer cell lines MCF7, PANC-1, and A-431, respectively. The weak cytotoxic effect of the A. absinthium essential oil was clearly demonstrated when it was compared with the results obtained from the positive control, doxorubicin (DOX). At all concentrations;, i.e., 0.01-100 µg/mL, DOX's antiproliferative effects against all cell lines were much stronger compared to the A. absinthium essential oil. In addition, doxorubicin at the highest concentration (100 µg/mL) showed increased cytotoxic activity by 8.8×, 6.3×, and 5.9× times compared to the A. absinthium essential oil against the MCF-7, Panc-1, and A-431 cancer cell lines, respectively (Table 4).

Plant Collection and Volatile Oil Preparation
The plant materials as aerial parts (obtained from   It was noteworthy that the susceptibility of the HSF normal cell line to the essential oil of A. absinthium was higher than that of the cancer cell lines at all concentrations ( Figure 2). The higher sensitivity of the HSF cell line to the A. absinthium essential oil compared to the other cancer cell lines was also clearly demonstrated as shown in Figure 2 and Table 4, which show a significantly higher cytotoxic effect of the plant's essential oil against the HSF cells (14.56%) compared to the MCF-7, Panc-1, and A-431 cell lines. The overall lower cytotoxic effect of the A. absinthium essential oil might be considered to signal a higher safety of this plant and its essential oil, primarily based on this plant being used as food without any record of toxicity or side effects [71,72]. A cytotoxic effect of A. absinthium essential oil from different regions of the world has been reported. The essential oil of the plant growing in Lithuania [73] showed a cytotoxic effect on brine shrimp, with IC 50 values in the range of 15-31 µg/mL; however, this toxicity could be attributed to the presence of a higher concentration of thujones, which are well-known cytotoxic monoterpenes [72], at a concentration of 12.3% in the Lithuanian plant [73]. However, the total percentage of trans-thujone, the only thujone found in the plant's essential oil, was calculated in current study as 1.14% of the plant's total essential oil constituents.

Plant Collection and Volatile Oil Preparation
The plant materials as aerial parts (obtained from ≈100 plants) were collected in March 2020 from private gardens in Qassim, Saudi Arabia. The plant materials were identified as Artemisia absinthium L. by the botanist at the Department of Plant Protection and Production, College of Agriculture, Qassim University. The plant samples were kept at the Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, Qassim University. The plant materials were dried under shade in the conditions of the room for 10 days and ground to powdered form. The volatile oil was distilled from the ground plant's aerial parts using a Clevenger apparatus in which 450 g of the plant materials were divided into three equal parts (150 g each). Each part was suspended separately in 1 L of distilled water in a conical flask and allowed to boil for 3 h over a heating mantle. The distilled oils were separated from the aqueous layer using a separating funnel, and then dried over anhydrous sodium sulfate powder. The total yield of the oil was calculated as the mean of three distillation processes (0.94 ± 0.03% w/w) of the dried aerial-part materials of the plant.

Gas Chromatography-Mass Spectroscopy Analysis of the Volatile Oil
An Agilent 8890 GC system attached to a PAL RTC 120 autosampler equipped with a HP-5 capillary column (30 m, 250 µm i.d., 0.25 µm film thickness) was used to separate and analyze the essential-oil components of the A. absinthium. The system was also equipped with an Agilent 9977B GC/MSD mass spectrometer (Agilent technology, Santa Clara, CA, USA). The initial isothermal column temperature was kept at 50 • C for 2 min. The column temperature was automatically adjusted upward until reaching 220 • C in intervals of 5 • C/min. Finally, the column temperature was elevated to 280 • C (at rate of 10 • C/min) and remained at 280 • C for 10 min (isothermal). In addition, the temperature of the injector was kept constant at 230 • C. The transfer-line, ionization-source, and mass-analyzer temperatures were 280 • C, 230 • C, and 150 • C, respectively. The diluted samples (1% v/v) Plants 2022, 11, 1028 9 of 14 were injected in split mode (at a split ratio of 1:15). The analysis running time was ≈ 65 min, and the carrier gas (helium) flow rate was kept constant (1 mL/min).

Gas Chromatography-Flame-Ionization Detector (GC-FID) Analysis of the Volatile Oil
The GC-FID analysis of the A. absinthium volatile oil was conducted using a Perkin Elmer Auto System XL instrument that was equipped with a flame-ionization detector (FID). The column used in the oil separation was a ZB-Wax fused silica capillary column (60m × 0.25mm i.d.). The initial temperature of the oven was maintained at 50 • C, and programmed to rise from 50 • C to 220 • C at a rate of 3 • C/min. However, the injector and detector temperatures were 240 • C and 250 • C, respectively. The carrier gas used in the separation was helium, which was passed through the column at a flow rate of 1 mL/min.

Identification of the Volatile Constituents
The volatile oil constituents of the A. absinthium were identified by comparing the experimental retention index (RI) of each volatile compound with the retention index obtained for a series of n-alkenes (C 8 to C 40 ) analyzed under similar conditions of experimentation. The reported RI recorded at comparable GC conditions was also used in the compounds identification. In addition, retention time, mass-fragmentation patterns of the peaks, and mass-spectral libraries (NIST-11 and Wiley database) were all used in the identification of the volatile oil constituents of the plant. The areas under the peaks were used to calculate the relative percentages of the constituents.

Antioxidant Activity of A. absinthium Volatile Oil
The antioxidant activity of the A. absinthium volatile oil was measured using four in vitro assays as described in the following.

Total Antioxidant Capacity (TAC)
The TAC of the A. absinthium volatile oil was evaluated according to the method of Aroua et al. [74] using a molybdate reagent assay. The reagent was formed by dissolving ammonium molybdate ((NH 4 ) 2 MoO 4 ) in a sodium phosphate buffer (28 mM) to a final concentration of 4 mM, and finally adding H 2 SO 4 (0.6 M). The working solution was prepared as follows: the diluted A. absinthium volatile oil (0.4 mL of methanol containing 200 µg of the volatile oil) was mixed with the molybdate reagent (3.6 mL) in a 20 mL stopped test tube. The tube contents were thoroughly mixed and kept in a 90 • C heated water bath for 30 min. A blank test was also prepared similarly to the previous method, but without involving the oil sample (the oil sample was replaced by 0.4 mL of distilled water). After cooling the mixture to room temperature, the intensity of resulting color was measured at 695 nm. The TAC of the A. absinthium volatile oil was calculated as the mean of three replicates equivalent to the well-known antioxidant standard, Trolox.

DPPH Scavenging Activity
The scavenging capability of A. absinthium volatile to the stable-free radical, DPPH, was measured and calculated as Trolox equivalents [75]. The DPPH working solution was prepared by dissolving 12 mg of the DPPH in 0.1 L of methanol. Then, 1 mL of the A. absinthium volatile oil (diluted in a methanol solution and containing 200 µg of the volatile oil) and 1 mL of DPPH working solution was thoroughly mixed and kept at room temperature in a dark place for 30 min before measuring the reduction in the color intensity of the DPPH using a spectrophotometer at 517 nm. The method was repeated three times; methanol was used as a blank, and the scavenging activity of the A. absinthium volatile oil was calculated using an equation of the Trolox calibration curve against the DPPH.

Ferric-Reducing Antioxidant Power (FRAP) Assay
The method of Benzie and Strain [76] was used to measure the FRAP activity of the A. absinthium volatile oil. The FRAP reagent was freshly prepared using the described method [77]. In test tubes, 0.1 mL of the diluted A. absinthium in methanol (a methanol solution containing 200 µg of the oil) was thoroughly mixed with the FRAP reagent (2 mL). The tubes were kept at room temperature for 30 min to develop the reaction color, which was measured at 593 nm. The FRAP activity of the A. absinthium volatile oil was calculated as mg Trolox equivalent/g of the volatile oil from three independent experiments using an equation of the FRAP-Trolox calibration curve.

Metal-Chelating-Activity Assay
The iron-chelating activity of the A. absinthium volatile oil was measured using the method of Zengin et al. [78]. First, 2 mL of the A. absinthium volatile oil diluted in methanol (a methanol solution with 200 µg of the plant's volatile oil) and 25 µL of FeCl 2 (2 mM) were thoroughly mixed with ferrozine (100 µL). A blank mixture consisting of all the previous ingredients, but without ferrozine, was prepared in a similar manner. The blank and test mixtures were measured at 562 nm. The chelating activity of the A. absinthium volatile oil was calculated using an EDTA calibration curve and expressed as mean of three independent experiments using equivalents to EDTA.

Cytotoxic Assay
The antiproliferative effect of the A. absinthium volatile oil was tested against HSF (normal human skin fibroblast cells), A-431 (human epidermoid skin carcinoma), MCF7 (breast adenocarcinoma), and PANC-1 (pancreatic cancer) cell lines, using a standard SRB (sulforhodamine B) assay [70]. Exactly 100 µL of each cell type (at a density of 5 × 10 3 cells/mL) was seeded in a 96-well microtiter plate in RPMI 1640 medium and incubated for 24 h. Cells were treated with another 100 µL aliquot of the medium containing the A. absinthium volatile oil at various concentrations; i.e., 0.01, 0.1, 1, 10, and 100 µg/mL. After 72 h of drug exposure, cells were fixed by replacing the medium with 150 µL of 10% trichloroacetic acid (TCA) and incubated at 4 • C for 1 h. The TCA solution was removed, and the cells were washed 5 times with distilled water. Aliquots of 70 µL SRB solution (0.4% w/v) were added and incubated in a dark place at room temperature for 10 min. The plates were washed 3 times with 1% acetic acid and allowed to air-dry overnight. Then, 150 µL of TRIS (10mM) was added to dissolve the protein-bound SRB stain; the absorbance was measured at 540 nm using a BMGLA BTECH ® -FLUO star Omega microplate reader (Ortenberg, Germany). A blank composed of the medium and the A. absinthium volatile oil without cells was prepared and treated with the SRB in the same manner, and was used to subtract its value from the absorbance values of the tests. In addition, control (untreated cells) and standard tests (doxorubicin) were prepared for each cell line and similarly treated with the SRB solution. The viability percentages were calculated as follows:

Statistical Analysis
The results are stated as the mean ± standard error of the mean/standard deviation. A post hoc test was performed using Tukey's multigroup comparison in GraphPad Prism 8.0.2. The significance value was set at p < 0.05.

Conclusions
In the present study, the volatile oil constituents of A. absinthium, the plant species growing in the central area of Saudi Arabia, were isolated and analyzed using GC-MS and GC-FID spectrometric methods. The results of the study revealed distinct variations in the essential-oil constituents of the plant growing in central region of Saudi Arabia and those species of the plant growing in other regions. The higher percentages of davanones (collectively 53%) recorded in the current study represented a clear sign of the environmental effects on the essential-oil constituents of the aromatic plants. The antioxidant and cytotoxic activities of the plant's essential oil were also evaluated in the current study, and revealed potential health benefits associated with higher antioxidant and lower cytotoxic activity levels obtained for the essential oil of A. absinthium. An in vivo antioxidant evaluation of the A. absinthium essential oil using animal experimentations, as well as preclinical and clinical trials, are highly recommended for such an important plant.