Comprehensive Phytochemical Analysis of Various Solvent Extracts of Artemisia judaica and Their Potential Anticancer and Antimicrobial Activities

Solvents play an important role in the extraction process by considerably affecting the amount and nature of secondary metabolites of medicinal plants. Thus, the effect of solvents must be investigated to obtain desired biological properties of plant extracts. In the current study, we extracted aerial parts of Artemisia judaica, native to Saudi Arabia, in three different solvents, including methanol (MeOH), hexane (Hex), and chloroform (Chl). Obtained extracts from the aerial parts of A. judaica were analysed by GC–MS and GC–FID techniques, which resulted in the identification of 46, 18, and 17 phytoconstituents from the Hex, Chl, and MeOH extracts, respectively. All the extracts contain oxygenated terpenes, aliphatic hydrocarbons, and aromatics as major classes of compounds in varying amounts. Among the various phytoconstituents identified, piperitone was the dominant compound and was found in all the extracts in different amounts, specifically, 28.8, 26.1, and 20.1% in the Chl, MeOH, and Hex extracts, respectively. Moreover, all these extracts (Chl, MeOH, and Hex) were tested for the antimicrobial properties on both Gram-positive and negative bacteria as well as for their anticancer properties on four different cell lines including HepG2, DU145, Hela, and A549. Among the different extracts, the Hex and Chl extracts demonstrated identical antimicrobial properties, while the Chl extract showed superior anticancer properties when compare to the other extracts. The higher biological properties of Chl extracts including both antimicrobial and anticancer activities may be attributed to the presence of large amounts of piperitone and/or santonin, which are distinctly present in excess amounts in the Chl extract.


Introduction
Plants are an important source of several pharmaceuticals that are currently used as therapeutics for pain (e.g., morphine); various diseases, including cancer (e.g., vincristine); bacterial and fungal infections (e.g., penicillin); and several heart diseases (e.g., warfarin) [1]. Particularly, in the underdeveloped regions of the world where essential health services are not easily available, plant-based traditional medicines have been proven as life-saving resources [2]. Plants offer extraordinary chemical diversity and excellent capability of producing highly complex novel phytomolecules with varying chemical functionalities [3]. Plants contain a variety of secondary metabolites with diverse properties that are responsible for major organoleptic characteristics of plant-derived foods and beverages, which offer great medical or health benefits. These types of food products and supplements are often referred as "nutraceuticals", which are extensively used in the prevention and treatment of several diseases. In this regard, the functional properties of various plant extracts are being extensively investigated for their use as novel nutraceuticals and functional foods [4,5]. Despite the tremendous potential of plants in modern medicine, among an estimated 350,000 known vascular plant species, a large number of plants still has to be chemically hexane (Hex), chloroform (Chl), and methanol ( was analysed separately to determine their chem logical properties.

Plant Material
Entire aerial parts of A. judaica grown in the part of Saudi Arabia, were procured in April 20 thenticated by Dr. Rajakrishnan Rajagopal from t versity. A specimen sample (AJMED-21) of A. jud

Chemicals
All the chemicals including methanol, chlo grade and purchased from Sigma-Aldrich, Ger riched fractions of volatile constituents such as MA, USA), heptacosane, carvacrol (Sigma Aldric Lancashire, UK), piperitone, caryophyllene oxid were available and used for co-injection/compara

Plant Material
Entire aerial parts of A. judaica grown in the region of Madinah, a city in the Western part of Saudi Arabia, were procured in April 2020. Identifications of A. judaica were authenticated by Dr. Rajakrishnan Rajagopal from the herbarium division of King Saud University. A specimen sample (AJMED-21) of A. judaica is retained with us.

Preparation of A. judaica Extracts
Procured A. judaica plant materials were air-dried at room temperature until constant weight was achieved. The dried plant material was then grounded to a suitable mesh size using a grinder. Obtained plant material (250 g) were first percolated with n-hexane (500 mL) three times at room temperature. After n-hexane extraction, the marc was again subjected to extraction three times with CHCl 3 (500 mL). Finally, the process of extraction was repeated using the residual marc with methanol (500 mL) for three more times at room temperature. Notably, each time, the extraction process was carried out for 3 days for all the solvents used. The resultant n-hexane, chloroform, and methanol extracts were separately dried under vacuum at 40 • C until solvents were completely removed using a Buchi rotary evaporating system (Rotavapor R-215, Buchi, Flawil, Switzerland) equipped with vacuum controller (V-850) and vacuum pump (V-700). These separately dried n-hexane, CHCl 3 , and methanol extracts were used for the screening of anticancer and antimicrobial activities and for GC analysis (Figure 2).

GC and GC-MS Analysis of A. judaica Extracts
In order to identify the chemical constituents of the extracts of A. judaica, dried extracts, i.e., n-hexane and CHCl3 extracts were dissolved in diethylether, whereas methanol extract was dissolved in methanol and subjected to GC-FID and GC-MS analyses. The GC-MS system was equipped with stationary phase columns (HP-5MS) employing the same method as described previously [21]. Detailed methodology is given in Supplementary Materials (Scheme S1). The identified constituents from CHCl3, n-hexane, and methanol extracts of A. judaica and their relative percentages are provided in Table 1 and the constituents are listed according to their elution order on the HP-5MS column.

Calculation of Linear Retention Indices (LRIs)
LRI values of chemical constituents of A. judaica extracts were determined following a previously reported method [21], and they are listed in Table 1. Detailed methodology is provided in Supplementary Materials (Scheme S2).

Identification of Volatile Components
Identification of the chemical constituents of A. judaica extracts were carried out through analysis on a HP-5MS column as described previously [21]. Detailed methodology is provided in Supplementary Materials (Scheme S3) [22][23][24]. GC-MS chromatograms for the identified constituents of n-hexane, chloroform, and methanol extracts of A. judaica on HP-5MS column are given in Figure 3.

GC and GC-MS Analysis of A. judaica Extracts
In order to identify the chemical constituents of the extracts of A. judaica, dried extracts, i.e., n-hexane and CHCl 3 extracts were dissolved in diethylether, whereas methanol extract was dissolved in methanol and subjected to GC-FID and GC-MS analyses. The GC-MS system was equipped with stationary phase columns (HP-5MS) employing the same method as described previously [21]. Detailed methodology is given in Supplementary Materials (Scheme S1). The identified constituents from CHCl 3 , n-hexane, and methanol extracts of A. judaica and their relative percentages are provided in Table 1 and the constituents are listed according to their elution order on the HP-5MS column.

Calculation of Linear Retention Indices (LRIs)
LRI values of chemical constituents of A. judaica extracts were determined following a previously reported method [21], and they are listed in Table 1. Detailed methodology is provided in Supplementary Materials (Scheme S2).

Identification of Volatile Components
Identification of the chemical constituents of A. judaica extracts were carried out through analysis on a HP-5MS column as described previously [21]. Detailed methodology is provided in Supplementary Materials (Scheme S3) [22][23][24]. GC-MS chromatograms for the identified constituents of n-hexane, chloroform, and methanol extracts of A. judaica on HP-5MS column are given in Figure 3.

Anticancer Activity
Cytotoxicity of test extracts was assessed against the human lung adenocarcinoma cell line (A549), human hepatocarcinoma cell line (HepG2), human cervical cancer cell line (HeLa), and human prostate cancer cell line (DU145) using MTT assay [26]. Briefly, 1 × 10 4 exponentially growing cells were seeded into each 96-well plate (counted by Trypan blue exclusion dye method) and allowed to grow until 60-70% confluence, then different concentrations of test extracts were added to the culture medium along with negative (DMSO) and positive controls (Doxorubicin). The plates were incubated for 48 h in a CO 2 incubator at 37 • C with a 90% humidified atmosphere and 5% CO 2 . Then, the media of the wells were replaced with 90 µL of fresh serum-free media and 10 µL of MTT (5 mg/mL of PBS), and the plates were incubated at 37 • C for 2 h. The media was discarded and allowed to dry for 30 min. Later, 100 µL of DMSO was added in each well to dissolve the purple formazan crystals and the absorbance was recorded at 570 nm using Spectra Max plus 384 UV-Visible plate reader (Molecular Devices, Sunnyvale, CA, USA). Each test compound was examined at various concentrations in triplicate and the results are expressed as mean with standard deviation (mean ± SD), (n = 3). One-way ANOVA and Dunnett's post-comparison test were used to analyse the data for significant differences (test vs. control). The statistical significance for the experiment was set at p < 0.05.

Results and Discussion
Herein, our aim was to explore the variability of phytoconstituents of the aerial parts of A. judaica using three different extraction solvents including polar, medium-polar, and non-polar solvents of methanol (MeOH), chloroform (Chl), and hexane (Hex), respectively. In addition, the evaluation of the biological properties including the antibacterial and anticancer activities of these three extracts was also performed. After complete drying and extraction of the samples, the amounts of resultant extracts from different solvents were measured. The extraction process was initiated with 250 g of aerial parts of A. judaica in each solvent, which yielded 4.1 g, 4.4 g, and 4.8 g of plant extract in hexane, chloroform and MeOH, respectively. Notably, different solvents resulted in the variable extract yields, which can be attributed to the nature and quantity of secondary metabolites extracted. In this case, the MeOH extract had the highest yield, which may be due to the higher solubility of polar carbohydrates and glycosides of secondary metabolites in the methanolic solution. The phytochemical analyses of the samples were performed by GC-MS and GC-FID techniques which led to the identification of a total of 46, 18, and 17 chemical constituents from the Hex, Chl, and MeOH extracts, respectively ( Figure 3). All the identified phytoconstituents obtained from the three extracts and their respective proportions are given in the Table 1 according to their elution order on the HP-5MS column. As per the results given in the Table 1, oxygenated monoterpenes were present in significant amounts in all three extracts. In particular, the Hex and MeOH contained 29.0% and 39.0%, respectively, while the Chl extract exhibited the highest percentage of these components, amounting to 42.8% of the total constituents. On the other hand, the oxygenated aliphatic hydrocarbons were present at distant second position in the studied extracts, which were present in the amounts of 14.1%, 6.2%, and 19.6%, in the Hex, Chl, and MeOH extracts, respectively. Apart from these, oxygenated sesquiterpenes, aliphatic hydrocarbons, and aromatics were also present in appreciable amounts. However, there was a large difference between the amount of these components among different extracts. For instance, the Chl extract demonstrated the highest amount of oxygenated sesquiterpenes equivalent to 31.5%, whereas the Hex and MeOH contained 7.9 and 15.1% of these compounds. Similarly, with regards to aliphatic hydrocarbons, the Hex extract contained the highest amount (26.6%), which was followed by the MeOH (10.3%) and Chl (4.8%) extracts. In the case of aromatics, the trend was dominated by Hex (18.3%), which was followed by MeOH (10.9%) and Chl (6.5%) extracts. Apart from these, sesquiterpenes hydrocarbons were also present in lesser amounts, i.e., 3.0, 2.8, and 0.6% in the Chl, MeOH, and Hex extracts, respectively.
Detailed analyses of each extract revealed that the Hex extract demonstrated the presence of highest number of compounds (46), followed by Chl (18) and MeOH (17). Details of all the major components found in the three different extracts are summarized in Figure 4 and their chemical structures are given in Supplementary Materials (Figures S1-S3). Out of 46 components identified in the Hex extract, only a few compounds were present in large amounts while most of the other components existed in negligible concentrations.  From Table 1, it is evident that the Hex extract was mostly dominated by piperitone (20.2%), heptacosane (13.9), 9,19-Cyclo-9β-lanost-24-en-3β-ol, acetate (12.1%), hexacosane (9.5%), trans-ethylcinnamate (9.3%), spathulenol (5.0%), and myrtenyl acetate (4.2%). Among these compounds, most of the components were also present in the other two extracts, Chl and MeOH; however, their amounts vary significantly. Particularly, piperitone was present in large amounts in all three extracts and was the most dominating compound of the Chl (28.8%) and MeOH (26.1%) extracts. Apart from this, myrtenyl acetate, trans-ethylcinnamate, spathulenol, α-santonin, and β-santonin were also found in the From Table 1, it is evident that the Hex extract was mostly dominated by piperitone (20.2%), heptacosane (13.9), 9,19-Cyclo-9β-lanost-24-en-3β-ol, acetate (12.1%), hex-acosane (9.5%), trans-ethylcinnamate (9.3%), spathulenol (5.0%), and myrtenyl acetate (4.2%). Among these compounds, most of the components were also present in the other two extracts, Chl and MeOH; however, their amounts vary significantly. Particularly, piperitone was present in large amounts in all three extracts and was the most dominating compound of the Chl (28.8%) and MeOH (26.1%) extracts. Apart from this, myrtenyl acetate, trans-ethylcinnamate, spathulenol, α-santonin, and β-santonin were also found in the three different extracts in varying quantities. On the other hand, some compounds were specifically found in only one extract, for instance, 9,19-Cyclo-9β-lanost-24-en-3β-ol, acetate (12.1%) and methyl hexadecanoate (13.5%) were specific to the Hex and MeOH extracts, respectively. Literature surveys regarding the phytoconstituents of different contents of the A. judaica population including essential oils, aerial parts, etc. from other countries have mostly indicated the presence of flavonoids, polyphenols, terpenes, etc. [30][31][32]. Notably, similar to the case of A. judaica of Saudi Arabia, piperitone is also present in significant amounts in the A. judaica belonging to the other regions of the world [33][34][35]. Piperitone is an oxygenated monoterpene, which is mainly responsible for the aroma of the plants and is widely used in fragrances, is mostly present in various aromatic plants such as Eucalyptus dives, Micromeria fruticose, Mentha spicata L., etc. [36]. Piperitone exhibits several biological properties such as insecticidal, repellent, and anti-appetent properties [37]. Indeed, in some studies, the high antimicrobial properties of the plant contents are directly attributed to the proportion of piperitone [38]. Apart from this, another compound, santonin, is distinctly present only in Chl in an excessive amount. Both α and β derivatives of santonin were found in the Chl extract in amounts of 17.1 and 13.7%, respectively, and just 7.7 and 5.0% in MeOH and 1.7 and 0.5% in the Hex. Santonin derivatives are sesquiterpene lactones, which are typically isolated from plants and possesses diverse biological properties including antibacterial, anti-inflammation, antimalaria, anticancer, etc. [39,40].
Upon comparing results of the chemical constituents of A. judaica in the present study with those reported from the same species in previous studies [31,33,41,42], it is significant to notice that pipertone was found to be the most versatile compound that was present as a major compound in almost all the volatile oils of A. judaica, except from the oil of A. judaica investigated from Irbid [31], where (E)-ethyl cinnamate was determined as the major constituent. Moreover, ethyl cinnamate was also detected in different proportions in most of the studied oil compositions of A. judaica [33,41] including the present study, as shown in Table 2. However, this compound was not present in the oil of A. judaica studied from Ilizi [42]. These variations in the chemical compositions of A. judaica volatile oils could be attributed to various factors including environmental and climatic conditions and geographic features [42,43]. Ilizi Piperitone (61.9), terpinen-4-ol (4.6) and bornyl acetate (3.0). [42] 3.

Antibacterial Properties
The extracts of A. judaica were tested for their efficiency against Gram-positive and Gram-negative bacterial strains, while Ciprofloxacin, a prescription antibiotic, was employed as a control for the study. It was observed that the methanol extract was effective against S. aureus and K. planticola, which are Gram-positive and Gram-negative bacterial strains, respectively; however, it displayed mild activity against M. luteus and E. coli strains. Furthermore, the hexane extract and chloroform extract showed excellent antibacterial efficiency against the Gram-positive strains S. aureus and M. luteus as well as K. planticola, a Gram-negative strain.
From the results obtained, it is observed that the methanol extract displays significant activity against S. aureus and K. planticola bacterial strains with 3.9 µg/mL and 1.9 µg/mL, respectively, but very mild activity against M. luteus and E. coli (Table 3). Moreover, the extracts obtained from hexane and chloroform are highly active against the tested Grampositive bacterial strains and K. planticola, a Gram-negative bacterial strain. The MIC values obtained against these strains are similar to the control used, i.e., Ciprofloxacin, a prescription antibiotic. While all the extracts, i.e., the hexane, chloroform, and methanol, display mild anti-bacterial activity against the bacterial strain E. coli, it is important to mention here that hexane and chloroform extracts could play a potential role in the development of efficient antibacterial agents in future studies. These two extracts could be recommended for the isolation and identification of an active antibacterial agent from A. judaica.

Anticancer Properties
In addition to the antibacterial studies, the isolated extracts of A. judaica were also tested for their efficiency against various cancer cell lines, such as hepatic cancer cells (HepG2), prostate cancer cells (DU145), cervical cancer cells (Hela), and human lung cancer cells (A549), while Doxorubicin, a prescription anticancer drug, was employed as a control for the study (Table 4). All the extracts showed different levels of activity, and the variations in anticancer activity of the CHCl 3 , methanol, and n-hexane extracts of A. judaica are postulated in Figure 5.  From Table 3, it is evident that all the tested extracts display mild to moderate anticancer activity, with the best IC50 value of 35.41 ± 1.78 μg/mL obtained for the chloroform extract against the DU145 cancer cell line, i.e., the prostate cancer cell line. This activity was comparable to that of the hexane extract as well, for which the IC50 value was 48.49 ± 0.16. On the other hand, the lowest activity was found for the methanol extract of A. judaica against the A549 cell line with an IC50 value of 168.54 ± 5.13 μg/mL. The methanol extract also showed lower activity against the other tested cell lines in comparison to those of the hexane and chloroform extracts. Moreover, careful observation of Table 3 suggests that the hexane extract of A. judaica possessed higher activity against HepG2, Hela, and A549 cancer cell lines compared to those of the chloroform and methanol extracts. Therefore, hexane extract of A. judaica could be considered for further studies to isolate active ingredients for the development of novel anticancer molecules.
It is worth mentioning here that there are no prior reports on the comparative study of anticancer activity of A. judaica extracts obtained from solvents of varying polarities. However, there are some studies which report the anticancer activity of A. judaica extracts using polar solvents such as methanol [17,44,45], unlike the study reported in our manuscript wherein we employed two solvents, i.e., hexane and chloroform, prior to methanol. On comparing anticancer activity results of our methanolic extract with those reported earlier [17,44,45], it was found that the methanolic extract in this study showed mild anticancer activity compared to that reported in previous studies. This might be due to the partition of the active ingredients of A. judaica into hexane and chloroform extracts during the extraction process, as the hexane and chloroform extracts in the present study have also shown significant anticancer activity similar to those reported earlier [17,44,45]. From Table 3, it is evident that all the tested extracts display mild to moderate anticancer activity, with the best IC 50 value of 35.41 ± 1.78 µg/mL obtained for the chloroform extract against the DU145 cancer cell line, i.e., the prostate cancer cell line. This activity was comparable to that of the hexane extract as well, for which the IC 50 value was 48.49 ± 0.16. On the other hand, the lowest activity was found for the methanol extract of A. judaica against the A549 cell line with an IC 50 value of 168.54 ± 5.13 µg/mL. The methanol extract also showed lower activity against the other tested cell lines in comparison to those of the hexane and chloroform extracts. Moreover, careful observation of Table 3 suggests that the hexane extract of A. judaica possessed higher activity against HepG2, Hela, and A549 cancer cell lines compared to those of the chloroform and methanol extracts. Therefore, hexane extract of A. judaica could be considered for further studies to isolate active ingredients for the development of novel anticancer molecules.
It is worth mentioning here that there are no prior reports on the comparative study of anticancer activity of A. judaica extracts obtained from solvents of varying polarities. However, there are some studies which report the anticancer activity of A. judaica extracts using polar solvents such as methanol [17,44,45], unlike the study reported in our manuscript wherein we employed two solvents, i.e., hexane and chloroform, prior to methanol. On comparing anticancer activity results of our methanolic extract with those reported earlier [17,44,45], it was found that the methanolic extract in this study showed mild anticancer activity compared to that reported in previous studies. This might be due to the partition of the active ingredients of A. judaica into hexane and chloroform extracts during the extraction process, as the hexane and chloroform extracts in the present study have also shown significant anticancer activity similar to those reported earlier [17,44,45].

Conclusions
Herein, to determine the effect of extraction solvents on the content of secondary metabolites, antimicrobial and anticancer properties were evaluated for three different extracts (Hex, Chl, and MeOH) of A. judaica grown in Saudi Arabia. All three different extracts of the aerial parts of A. judaica exhibited important disparities in their chemical compositions, and variations in amounts of some lead phytoconstituents were also noticed. In this study, the investigated plant extracts displayed piperitone as the major component, which was present in varied amounts in the different extracts. Among all three different extracts, the Chl extract of A. judaica showed superior antimicrobial and anticancer properties, which could be ascribed to the distinct presence of the large amounts of piperitone (28.8%) and santonin (α =17.1%, β = 13.7%), which are known to demonstrate excellent biological properties. These results offer scientific evidence of the medicinal properties of A. judaica in traditional medicine. A. judaica extracts can prove to be useful resources for the development of plant-based pharmaceuticals, functional foods, and other cosmetic products. However, a detailed biological activity-guided chromatographic analysis is necessary to extract potentially active phytoconstituents from these extracts.