Chemical composition and antioxidant activity of Jordanian Artemisia judaica L. as affected by different drying methods

ABSTRACT Aerial parts of Artemisia judaica from Jordan were subjected to different drying methods, including shade (ShD), sun (SD), oven (OD) drying at different temperatures in addition to microwave drying (MWD). Essential oils extracted from these samples were assayed for their chemical composition and antioxidant activity. GC/MS analysis of the different oil samples revealed qualitative and quantitative differences. All samples contained high concentration levels of oxygenated monoterpenes (41.69%-68.56%) followed by esters (8.32–36.65%). The essential oil extracted from ShD plant material exhibited the strongest DPPH and ABTS radical scavenging activities while oil samples obtained from the SD method showed the strongest ferrous ion chelating activity (FIC). Cluster analysis (CA) and principal component analysis (PCA) were used to identify the effect of drying methods on the essential oil composition obtained from A. judaica. The different essential oils were classified into two clusters corresponding to natural and artificial drying methods.


Introduction
Artemisia L. belonging to the Asteraceae family (formerly known as Compositae), is a large and diverse genus of containing about 500 temperate annual, biennial, and shrubby species. [1] Members of this genus are strongly aromatic, well recognized for their bitter taste that is attributed mainly to the presence of terpenoids and sesquiterpene lactones, and most importantly are known for their nutritional and ethnomedicinal values. [2,3] Previous phytochemical studies on different Artemisia species revealed the presence of terpenoids, flavonoids, phenolics, and alkaloids. [2] Previous investigations designed to identify essential oil constituents revealed the presence of different classes of terpenoids in addition to phenylpropanoids. [4,5] In Jordan, different Artemisia species are known to grow wild in hot arid and semiarid regions, mainly reported in extreme desert habitats of the Irano-Turanian and east Saharo-Arabian biogeographical zones. Five Artemisia species were reported in the Flora of Jordan, including A. arborescens L., A. jordanica Danin, A. judaica L., A. monosperma Delile, and A. sieberi Besser. [6] Thorough literature survey clearly showed that Artemisia species of Jordanian origin were seldom investigated for their essential oil composition and biological activity. The essential oil obtained from fresh aerial parts of A. sieberi contained high concentration levels of oxygenated monoterpenes (39.3%) and have antibacterial, antifungal, antipyretic, anti-inflammatory, antioxidant, and antidiabetic activities. [7] Artemisia judaica L., known also in Jordan as Beithran, is considered as one of the most reputable medicinal plants that is prescribed by local traditional healers for the treatment of many ailments including hepatitis, cancer, malaria, gastrointestinal disorders, skin disorders and inflammations caused by fungal, bacterial or viral infections. [8] The plant is used in its fresh form during the spring season and in its dried form during other seasons of the year. Previous investigation showed that the essential oil obtained from the fresh aerial parts of A. judaica contained high concentration levels of oxygenated monoterpenes (68.7%) and exerted effective antibacterial and antifungal activities. [9] It is well known that different drying methods affect both essential oils' yield and chemical composition. [10][11][12][13][14][15][16][17][18][19] Therefore, the objective of this work was to investigate the effects of different drying methods on the yield, the chemical composition and antioxidant activity of the oil obtained from the aerial parts of A. Judaica from Jordan.

Plant material
The aerial parts of A. Judaica were collected (2.0 kg) during the full flowering stage from its natural habitats located near Saudi-Jordan borders (29°16ʹ42.6" N 35°59ʹ20.6" E; March 2017). The identity of the plant was confirmed using characteristics related to growth habits and morphological attributes in regional floras [20] and was further confirmed by Prof. Dr Jamil Al-Lahham, Department of Biological Sciences -Faculty of Science, Yarmouk University, Irbid, Jordan. A voucher specimen was deposited in the herbarium of the Department of Biological Sciences-Yarmouk University, Irbid, Jordan (YU/09/ CA/1003),

Drying conditions
Fresh aerial parts of A. judaica were subjected to different drying methods, immediately after collection. The different drying methods included shade drying (ShD), sun drying (SD), oven drying (OD) and microwave drying (MWD). In the ShD method, the aerial parts of the plant (250 g) were dried in a dark and dry room with appropriate ventilation for 4 weeks (until constant weight was achieved). The temperature of the room was 25 ± 2°C.
In SD, the aerial parts (250 g) were dried on paper trays under direct sunlight at temperatures between 19°C and 36°C for 7 days. OD was performed in a ventilated oven at two temperatures, 40°C and 60°C for 3 days. MWD (100 W) was performed for 3 min.

Extraction of essential oils
Fresh aerial parts (250 g) were chopped and each dried sample was milled to a fine powder and then suspended in 250 mL distilled water. The oils were extracted by hydro-distillation for 3 h using a Clevenger-type apparatus. The different oils obtained were pooled using hexane, dried and then were stored in sealed amber vials at 4°C until analysis. [21][22][23][24]

GC and GC-MS analyses
A sample of 1 μL of each oil was diluted to 10.0 μL with GC grade n-hexane, and then analyzed by GC-MS (Varian Chrompack CP-3800 GC/MS, Saturn, Netherlands) equipped with a DB-5 GC-column (5% diphenyl, 95% dimethyl polysiloxane, 30 m × 0.25 mm i.d., 0.25-μm film thicknesses). In the MS detector, an electron ionization mode of 70 eV was used. The temperature in the MS source was set at 180°C. The temperature column was also programmed from 60°C for 1 min (isothermal) to 246°C at a constant rate of 3°C/min, with the lower and upper temperatures being held for 3 min. Helium was used as a carrier gas (0.9 mL/min).
Quantitative analysis was performed using GC/FID instrument (Hewlett -Packard HP-8590, USA) equipped with optima-5 column (5% diphenyl, 95% dimethyl polysiloxane; 30 m × 0.25 mm, 0.25 μm film thickness) and a split-splitless injector (split ratio 1:50). The temperature of the oven was increased from 60°C to 250°C at a rate of 3°C/min, then held constant at 250°C for 5 min. The temperatures of the injector and detector were maintained at 250°C and 300°C, respectively. The relative peak areas were used to calculate the relative percent concentrations of the detected compounds. A standard solution of C 8 -C 20 n-alkanes mixture was analyzed under the same chromatographic conditions.

Identification of oil constituents
Chemical constituents of the different essential oils were identified by comparison of their mass spectra with those found in the database library (Wiley 275 library, New York, USA) or with authentic compounds and then confirmed by comparison of their Kovats retention indices with those of authentic compounds and/or literature. [25,26]

Antioxidant activity
The antioxidant activity of the essential oils was determined using DPPH˙ (1,1-diphenyl-2-picrylhydrazyl) radical), ABTS˙+ (2,2ʹ-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) and Ferrous Ion Chelating effect (FIC) assay methods and according to the procedure described in the literature. [27][28][29][30] The percentage of scavenging activity was calculated using the equation: where A c is the absorbance of the control and A s is the absorbance in the presence of either the oil or the positive controls. Nonlinear regression analysis of GraphPad Prism 6 (GraphPad Software, San Diego, California, USA) was applied for the determination of IC 50 in all of the antioxidant assays. Each concentration was tested three times in three independent experiments.

Statistical analysis
R 3.6.1 (The R Foundation of Statistical Computing) with devtools, ggbi plot, psych, and GPArotation packages were used for the data processing and chemometric analysis.

Essential oil yield
The yields of the essential oils (EO, yellow color) obtained in this current investigation are listed in Table 1. It was noticed that dried samples, regardless of the drying method, had higher yields when compared to the yield obtained from fresh samples and this was in total agreement with previous reports. [18] ShD and OD-40 samples showed the highest EO content (0.91 ± 0.05, 0.85 ± 0.03, respectively), while SD was recognized with lowest content (0.64 ± 0.01%). Our results showed that increasing temperature (from 40°C to 60°C) caused a significant decrease in the EO content.

Essential oils' composition
The results obtained from GC/MS analysis of the different essential oils (EO), obtained from fresh aerial parts of A. judaica and those obtained from plant after subjected to different drying methods) are summarized in (Table 2). Figure 1 shows GC chromatogram of a representative sample analysis. A total of 55 different components were identified in the different hydro-distilled oils. The essential oil obtained from fresh samples was dominated by oxygenated monoterpenes (OM) that amounted to 41.69% of the total composition. The main components detected in this sample included E-ethyl cinnamate (21.46%), artemisia ketone (20.76%), davanone (16.78%), Z-ethyl cinnamate (12.13%), yomogi alcohol (5.15%), artemisyl acetate (4.70%), and chrysanthenone (4.60%). GC/MS analysis of the essential oils extracted from the plant after being subjected to different drying methods showed qualitative and quantitative differences among the main classes and their components ( Table 2). Despite these differences, all essential oils were dominated by OM and the highest percent was recorded in SD and ShD EO (68.56, 67.56%, respectively), while OD-40 had relatively the lowest content (45.72%) of this class. In ShD and SD oil samples, this class was mainly represented by chrysanthenone (31.24%, 21.42%, respectively). Other components included camphor (16.00, 8.61%); piperitone (14.70, 10.27%) and E-ethyl cinnamate (3.92%, 6.03%).
Careful inspection of the chemical composition of the MWD-EO revealed relatively high concentration levels of OM (54.03%), followed by esters (23.38%) and the highest OS content (9.40%) when compared to its content in other oils obtained by the other drying methods. Again, chrysanthenone (15.63%), E-ethyl cinnamate (15.34%), piperitone (10.64%), and artemisia ketone (9.96%) were the main components detected in this oil ( Table 2). Our results indicated that the concentration of the different classes of compounds detected in the different oil samples varied with the drying method, which was in total agreement with the previous studies performed on other aromatic plants. [10][11][12][13][14][15][16][17][18][19] It was also noticed increasing oven temperature without pre-drying treatment reduced significantly the content of OM, increased OS amounts but did not affect the content of both monoterpene and sesquiterpene hydrocarbons (MH & SH, respectively). The content of MH, OM and SH in ShD and SD oils were generally higher when compared to their contents in the oils obtained from fresh sample. On the contrary, low concentration levels of both OS and ester components were detected in ShD and SD oil samples as compared to their content in oil obtained from the fresh sample (Table 2).

Statistical analysis
In the current work, the data listed in Table 2 have been subjected to principal component analysis (PCA) to explore the differences in the chemical composition of the essential oils obtained by the different drying methods used in this investigation. PCA has been used to investigate similarities or differences in the chemical composition of the essential oils obtained from A. judaica samples subjected to natural and artificial drying methods. Therefore, only the first few PCs were studied, the best result was obtained upon using a two-dimensional PCA score model that accounted for about 71% of the total variation in the dataset using the first two PCs. Figure 2 represents the resulted score PCA plot. Two crossed clusters were obtained, one cluster referred to the essential oil data subjected to natural drying methods (ShD and SD) and the other one was related to the data belonging to artificial drying methods (OD-40, OD-60 and MWD). In this figure, every point in the two clusters represents the data obtained from the specific drying method. In general, crossed clusters indicated some similarities in the chemical compositions among the different drying methods, especially in the composition of oils obtained from SD (2) and MWD (6) samples. Further investigation of the data using the PCA has been carried out in an attempt to identify the major chemical constituents responsible for the above behavior. Hence, a bi-plot (scores and loadings) PCA was created and displayed in Figure 3. As could be deduced from this figure, it is clear that cineol, Figure 1. Representative gas chromatograms for oils extracted from A. judaica obtained by hydro-distillation at different drying methods in comparison to the fresh sample. The numbers above the peaks indicate the major chemicals that were identified and listed by the same numbers in Table 2.
located at the center of the crossed clusters, had the greatest impact on the similarity in the first PC, and γ-muurolene, germacrene D, yomogi alcohol, artemisyl acetate, contributed to a less extent to the similarity of the same PC.
For more analysis, CA was applied to the same data matrix used for the PCA. The resulted dendrogram of this test is shown in Figure 4 using Complete Linkage distances between the sample drying methods. The obtained results were very consistent with those obtained from the PCA application. SD and MWD obtained oils were strongly related to each other, while the composition of OD samples was strongly related to the composition obtained from fresh plant material.

Antioxidant activity
The essential oils obtained from the fresh, ShD, SD, OD's, and MWD of A. judaica were evaluated for their antioxidant activities according to the procedure described in the literature using the DPPH, ABTS and FIC assay methods, results are summarized in Table 3. [27][28][29][30] All essential oils showed good antioxidant activity. The oils obtained from natural drying methods (ShD and SD) exhibited higher scavenging effect than the ones obtained from the artificial drying methods (OD-40, OD-60 and MWD). The good DPPH and ABTS radical scavenging activity of ShD and SD EO samples may be attributed to their high content of OM in their essential oils. In general, the antioxidant activity power of OM could be attributed mainly to their ability to undergo antioxidation, characterized by a very fast termination process thereby reducing the overall rate of oxidation. [34] The observed antioxidant activity of the investigated samples indicates that A. judaica essential oil, in general, is a good antioxidant candidate in pharmaceutical and food industries. [35]

Conclusion
GC-MS analysis of the different essential oils obtained from the aerial parts of A. judaica after being subjected to different natural and artificial drying methods revealed qualitative and quantitative differences in the chemical composition of these oils. The oil extracted from the plant left to dry in shade contained the highest value of terpenes when compared to the content in the essential oils obtained by the other drying methods. Additionally, this essential oil had the highest antioxidant activity indicating its importance as a source for natural antioxidants. Values expressed are mean ± SD of three parallel measurements. Mean values are significantly different (p < 0.05).