Polyphenol Content and Biological Activities of Ruta graveolens L. and Artemisia abrotanum L. in Northern Saudi Arabia

: Natural populations of Ruta graveolens L. and Artemisia abrotanum L. in northern Saudi Arabia may be a rich source of natural polyphenols with potential biological activities. Therefore, tentative high-performance liquid chromatography–diode array detection was used to analyze the polyphenol contents of leaf extracts. R. graveolens mainly contained the phenolic acids chlorogenic acid and p-coumaric acid and the ﬂavonoids rutoside and quercetin, whereas those of A. abrotanum mainly contained the phenolic acids isochlorogenic acid and rosmarinic acid and the ﬂavonoid quercetin. Leaf extracts of both species showed antioxidant activities due to the presence of quercetin, chlorogenic acid, and p-coumaric acid as well as antiproliferative and cytotoxic activities against cancer cells, which may be attributed to necrotic cell accumulation during the early and late apoptotic periods. Both species also exhibited antibacterial activity, although the activity was higher in R. graveolens due to the high contents of quercetin and other polyphenols. Finally, both species exhibited antifungal activities, which were associated with speciﬁc polyphenols. This is the ﬁrst study to conﬁrm the richness of polyphenols and wide spectrum of biological activities in natural populations of R. graveolens and A. abrotanum in northern Saudi Arabia. of


Introduction
Medicinal plants are rich sources of polyphenols, which have important biological activities. For instance, polyphenols may slow aging through their antioxidant activities [1] or control microorganisms growth through their antibacterial and antifungal activities [2,3]. Furthermore, polyphenols have been shown to act as anticancer agents [4][5][6][7] and food preservatives [8].
In the DPPH assay, serial concentrations of leaf extracts were added to 5 mL of 0.004% methanolic DPPH solution freshly prepared. After incubation for 30 mins in the dark at room temperature, the absorbance was measured at 517 nm. The free radical inhibition by DPPH was calculated as follows: The percentage inhibition of antiproliferative activity (IAA) was calculated in triplicates: where (AB 5170nm ) C and (AB 517nm ) s are Abs.517 nm of control and sample, respectively. A standard antioxidant was used (butylated hydroxytoluene, BHT) as a positive control and the inhibition concentration of each sample was compared with that of the BHT and blank. In the β-carotene-bleaching assay the mixture was prepared by dissolving the β-carotene (0.5 mg) in chloroform (1 mL), then adding 25 µL linoleic acid and 200 mg Tween 40. The chloroform was removed by vacuum evaporation, and the distilled water was added (100 mL), followed by vigorous shaking. A 2.5 mL of the mixture were mixed with serial concentrations of leaf extracts, incubated for 48 h at room temperature and the absorbance was measured at 470 nm.
In the FRAP assay, aliquots (100 µL) of leaf extracts/Trolox (Sigma-Aldrich, Berlin, Germany) were added to the FRAP reagent (3 mL), then mixed and incubated for 30 min at 37 • C. The calibration procedure of FRAP was conducted by applying serial dilutions of Trolox (0-0.5 mmol/L), as standard. The absorbance was measured at 593 nm for FRAP. All antioxidant experiments were conducted in triplicates and repeated thrice.
A washing procedure was performed using phosphate-buffered saline (PBS), following which MTT dissolved in PBS (12 mM) was added to the medium and isoprobanol (0.04 N HCl) was mixed. Then, the mixture was left for 40 min, after which absorbance was measured at a wavelength of 570 nm. Positive control (vinblastine sulfate and taxol) and negative control (no treatment) were also prepared. Inhibition activity amount was calculated using the following equation: where (AB 570nm ) c and (AB 570nm ) s are the absorbances of the control and sample, respectively. IC 50 was determined by plotting the percentage of viable cells against the extract concentration in µg/mL.

Flow Cytometry
The IC 50 of MTT was then applied to flow cytometry for investigating the cytotoxic activities of the leaf extracts of R. graveolens and A. abrotanum, following which the apoptotic cell populations were determined (FAC Scan, New York, NY, USA) [4,5]. Briefly, cultured cancer cells in six-well plates were subjected the IC 50 of leaf extracts as well as identified polyphenols (quercetin and isochlorogenic acid) for 48 h. Untreated cells were considered as control. trypsin (0.25%) was used to detach cells in Hank's balanced salt solution (Thermo Fisher Scientific, Berlin, Germany). The cells were stained using the Annexin V apoptosis detection kit (Sigma, St. Louis, MO, USA). By incubation in the dark at 37 • C for 15 min then washing with PBS. The data of flow cytometer is presented in quadrants as percentage: lower left (viable cells), upper left (necrotic cells), lower right (early apoptotic cells), and upper right (late apoptotic cells).

Antibacterial Activities
The antibacterial activities of R. graveolens and A. abrotanum leaf extracts were examined using isolates of Listeria monocytogenes (clinical isolate), Staphylococcus aureus (ATCC 6538), Escherichia coli (ATCC 35210) ria, Bacillus cereus (ATCC 14579), Pseudomonas aeruginosa (ATCC 27853), and Micrococcus flavus (ATCC 10240). A microtiter plate-based protocol (microdilution) was used as described previously [7,[39][40][41][42], wherein serial concentrations of the extracts and pure standards of identified polyphenols that were mixed with bacterial inoculum of 1.0 × 10 4 colony-forming units and 100 µL tryptic soy broth in each well and incubated for 1 day at 37 • C on a rotary shaker. Minimum inhibitory concentration (MIC) was defined as the lowest concentration that caused no visible growth under a binocular microscope, and minimum bactericidal concentration (MBC) was defined as the minimal concentration that eliminated 99.5% of each inoculum as determined using serial subculturing of the leaf extracts (2 µL). Optical density was determined at a wavelength of 655 nm. Streptomycin (0.01-10 mg/mL) and DMSO (1%) were used as positive and negative controls, respectively. Experiments were conducted in triplicate and repeated thrice.

Statistical Analyses
Differences among treatments were expressed as the least significant differences (LSD) in SPSS software. The mean and standard deviation (SD) were calculated from the three replicates (of each assay).

Polyphenol Profiles of the Leaf Extracts
The methanolic leaf extracts of R. graveolens were found to contain four phenolic acids and two flavonoids ( Table 1, Figures 1 and 2A). The major phenolic acids were chlorogenic acid and p-coumaric acid, with protocatechuic acid and caffeic acid present at lower concentrations. In addition, high concentrations of the flavonoids rutoside and quercetin were detected.   The methanolic leaf extracts of A. abrotanum were found to contain seven phenolic acids and one flavonoid ( Table 1, Figures 1 and 2B). The major phenolic acids were isochlorogenic acid and rosmarinic acid, with chlorogenic acid, protocatechuic acid, vanillic acid, caffeic acid, and syringic acid present at lower concentrations. Furthermore, quercetin was identified.

Antioxidant Effects
The methanolic leaf extracts of both R. graveolens and A. abrotanum and the major polyphenols showed strong antioxidant activities (Table 2). However, the methanolic leaf extracts of R. graveolens showed higher antioxidant activity than those of A. abrotanum according to DPPH, β-carotene bleaching, and FRAP assays. Quercetin, chlorogenic acid, and p-coumaric acid, which were identified in R. graveolens, showed strong antioxidant activities, with p-coumaric acid having the lowest IC 50 value. In addition, isochlorogenic acid, rosmarinic acid, and quercitrin, which were identified in A. abrotanum, showed strong antioxidant activities. Indeed, the antioxidant activities of rosmarinic acid and p-coumaric acid were comparable to those of the BHT and Trolox antioxidant standards.

MTT Assay and Flow Cytometry
The antiproliferative activities of the methanolic leaf extracts of R. graveolens and A. abrotanum and the identified polyphenols against different cancer cells were measured using MTT assay. It was found that the leaf extracts of both species and the identified polyphenols had strong antiproliferative activities against all cells, except normal HEK-293 cells (Table 3). In particular, quercetin, chlorogenic acid, isochlorogenic acid, and p-coumaric acid showed strong antiproliferative activities against most cancer cells, with Jurkat being the most resistant cell line. The

Antibacterial Activities of Leaf Extracta and Identified Polyphenols
The methanolic leaf extracts of both R. graveolens and A. abrotanum showed antibacterial activities against a range of bacteria, but R. graveolens showed higher antibacterial activities against all bacteria studied ( Table 4). The most sensitive bacterium (i.e., with the lowest IC50) was S. aureus, whereas the most resistant was M. flavus. Quercitrin, isochlorogenic acid, and p-coumaric acid showed strong antibacterial activities against all bacteria, with the former two having comparable activity to the antibiotic streptomycin. By contrast, rosmarinic acid and quercetin showed moderate-to-low antibacterial activities.

Antifungal Effects
The methanolic leaf extracts of both R. graveolens and A. abrotanum showed moderate antifungal activities against most fungal species studied, but R. graveolens showed higher activity (i.e., lower IC50 values) (Table 5). Quercetin, isochlorogenic acid, p-coumaric acid, quercitrin, and rosmarinic acid also showed antifungal activities, with isochlorogenic acid and quercetin having the highest activities among the studied polyphenols.

Antibacterial Activities of Leaf Extracta and Identified Polyphenols
The methanolic leaf extracts of both R. graveolens and A. abrotanum showed antibacterial activities against a range of bacteria, but R. graveolens showed higher antibacterial activities against all bacteria studied ( Table 4). The most sensitive bacterium (i.e., with the lowest IC 50 ) was S. aureus, whereas the most resistant was M. flavus. Quercitrin, isochlorogenic acid, and p-coumaric acid showed strong antibacterial activities against all bacteria, with the former two having comparable activity to the antibiotic streptomycin. By contrast, rosmarinic acid and quercetin showed moderate-to-low antibacterial activities.

Antifungal Effects
The methanolic leaf extracts of both R. graveolens and A. abrotanum showed moderate antifungal activities against most fungal species studied, but R. graveolens showed higher activity (i.e., lower IC 50 values) (Table 5). Quercetin, isochlorogenic acid, p-coumaric acid, quercitrin, and rosmarinic acid also showed antifungal activities, with isochlorogenic acid and quercetin having the highest activities among the studied polyphenols.

Discussion
Qualitative and quantitative variation in bioactive compound content within populations of the same plant species but with different origins is a fairly well-known phenomenon that has been widely documented [39,43]. In the present study, we investigated the polyphenol contents of leaf extracts of two quite well-known plants that are used as tradtitional medicine and grow naturally at sites that experience harsh weather conditions in northern Saudi Arabia.
We confirmed that methanolic leaf extracts of the Saudi-origin R. graveolens contained four phenolic acids and two flavonoids, among which rutoside (1010.1 mg/100 g DW), quercetin (375.4 mg/100 g DW), chlorogenic acid (356.0 mg/100 g DW), and p-coumaric acid (158.6 mg/100 g DW) were the main metabolites (Table 1). R. graveolens is generally recognized as a plant with a high content of furanocoumarin compounds [44,45], and there is evidence that rutoside and quercetin are the main active flavonoids in this species and may be responsible for its pharmacological functions, which include anti-inflammatory, analgesic, antiandrogenic, antihyperglycemia, antihyperlipidemia, antigout, and anticancer activities [46]. However, phenolic acids may also play an important role in the biological activities of this species. Meinhart et al. [47] previously investigated the availability of chlorogenic acid and its derivatives in various commercial plants in Brazil and confirmed that the leaf extracts of R. graveolens contained chlorogenic acid (103 mg/100 g DW) and 3,5-dicaffeoylquinic acid (132 mg/100 g DW), but not caffeic acid, which is one of the precursors of chlorogenic acid derivatives. By contrast, the Saudi-orgin R. graveolens leaf extracts examined here had 3.5 times higher chlorogenic acid as the Brazilian-origin plants and contained caffeic acid (18.2 mg/100 g DW). In an investigation of leaf extracts of Polish-origin R. graveolens, Ekiert et al. [48] confirmed the presence of protocatechuic acid (114 mg/100 g DW) and p-coumaric acid (5 mg/100 g DW). By contrast, the Saudi-origin leaf extracts contained a 31.5 times higher amount of p-coumaric acid, but a 2.3 times lower amount of protocatechuic acid.
The methanolic leaf extracts of R. graveolens and A. abrotanum showed noticeable antioxidant activities in DPPH, FRAP, and β-carotene bleaching assays, with the former showing the highest antioxidant activity. Diwan and Malpathak [20] suggested that the phenolic compounds in the leaves of R. graveolens originating from central India serve as a good source of antioxidants that could offer potential protective effects against lipid oxidation. Supporting this, we found that the leaf extracts of Saudi-origin R. graveolens contained quercetin, chlorogenic acid, and p-coumaric acid, all of which also showed strong antioxidant activities. Quercetin, which is a flavonoid glycoside that is common in plants, can reduce reactive oxygen species production and activate the acute monocytic leukemia cell line THP-1 in vitro [53] and has strong antioxidant and antiproliferative activities against RAW264.7 cancer cells [54]. Chlorogenic acid, which is considered an ester between caffeic and quinic acids, has the capacity to control oxidative and inflammatory stresses and is strongly recommended for consumption in beverages due to its ability to modulate the number of metabolic pathways, thus reducing oxidative stress [55]. p-Coumaric acid, which is a derivative of cinnamic acid, has strong antioxidant activities that are comparable to standard antioxidants [56,57]. Diwan and Malpathak [20] previously showed that in vitro shoot culture extracts from Indian-origin R. graveolens had DPPH IC 50 values ranging from 33 to 60 µg/mL. However, in the present study, we found that Saudi-origin plants had higher antioxidant activities as revealed by DPPH assay, and similarly Pavić et al. [58] found that choline chloride leaf extracts of R. graveolens originating from Bosnia and Herzegovina had strong antioxidant activities.
The strong antioxidant activity of the A. abrotanum leaf extracts was also attributed to the polyphenols present, which included isochlorogenic acid, rosmarinic acid, chlorogenic acid, and quercitrin. Rosmarinic acid was found to have a strong antioxidant effect, which was comparable to that of the antioxidant standards BHT and Trolox, supporting previous findings [59]. Furthermore, both isochlorogenic acid, which was the major compound in A. abrotanum extracts, and chlorogenic acid showed strong antioxidant activities. Previous studies have also observed strong antioxidant activities of both isochlorogenic acid (3,5-dicaffeoylquinic acid), which has the same structure as chlorogenic acid but with >1 caffeic acid group [60], and chlorogenic acid, which is commonly found in fruits and vegetables [7,61].
MTT assay and flow cytometry revealed that R. graveolens and A. abrotanum leaf extracts had antiproliferative and cytotoxic activities against different cancer cells, similar to quercetin, chlorogenic acid, isochlorogenic acid, and p-coumaric acid. In a previous study, American-origin R. graveolens leaf extracts showed anticancer activities against breast, colon, and prostate cancers [62]. However, the polyphenolic compounds responsible for these effects were not identified. In another study, furanoacridones (alkaloids) were identified in German-origin R. graveolens leaf extracts and were found to be associated with anticancer activities against MCF-7 cells, which included reduced cell proliferation and cell cycle disturbance [63]. In the present study, several polyphenols, including quercetin, isochlorogenic acid, chlorogenic acid, and p-coumaric acid, were identified for the first time in a natural population of R. graveolens, and their activities were associated with the antioxidant, antiproliferative, and cytotoxic activities of this plant. Quercetin is widely recognized as a strong antiproliferative factor against cancer cells and has been shown to suppress the activity of specificity protein 1 and reduces the proliferation of human hepatocellular carcinoma HepG2 cells [64]. It has also been associated with antiprolifrative activities against prostate cancer cells as a synergistic factor to epigallocatechin (polyphenol) in green tea [65] and has recently been described as having cytotoxic activity against lung cancer cells [66]. To the best of our knowledge, the antiproliferative and cytotoxic activities of isochlorogenic acid have not been previously described, making this the first report on the activity of this compound against cancer cells. Chlorogenic acid showed moderate antiproliferative activities against cancer cells in this study, which is in agreement with the findings of previous studies [67,68]. Finally, the antiproliferative activities of p-coumaric acid have rarely been studied, with only two investigations reporting antiproliferative and, to a certain extent, cytotoxic activities of this polyphenol against colon cancer cells [69,70]. Therefore, this is the first report on the activity of this polyphenol against other types of cancer cells.
A. abrotanum leaf extracts also showed antiproliferative activities against cancer cells, which were attributed to the major polyphenols identified, particularly isochlorogenic acid and rosmarinic acid. Tayarani-Najaran et al. [71] found that CH 2 Cl 2 leaf extracts of wild Iranian-origin A. biennis had cytotoxic activities against K562 and HL-60 cancer cells, but they did not identify the responsible polyphenols. Similarly, methanolic flower, leaf, stem, and root extracts of other Artemisia species, including A. absinthium, A. vulgaris, and A. incana, showed cytotoxic activities against MCF-7 cells [72], but no specific polyphenols were associated with these activities.
The leaf extracts of R. graveolens and A. abrotanum had antibacterial activities against a range of bacteria. This antibacterial activity of R. graveolens is attributed to the high contents of specific polyphenols such as chlorogenic acid and p-coumaric acid, but particularly to quercetin. It has been shown that quercetin has strong antibacterial activities against [73], as found in this study, as well as against other bacteria including E. coli and Ps. aeruginosa [74]. Furthermore, chlorogenic acid has strong antibacterial activities [75,76] and p-coumaric acid is a strong antibacterial agent against B. cereus and Salmonella typhimurium, acting synergistically with niacin [77]. However, this is the first comprehensive study to illustrate the wide antibacterial activities of p-coumaric acid. Pavić et al. [58] similarly found that choline chloride leaf extracts of R. graveolens originating from Bosnia and Herzegovina had strong antioxidant and antibacterial activities, with MIC values ranging from 62 to 125 µg/mL against different strains of E. coli, Ps. aeruginosa, B. subtilis, and Staphylococcus aureus. In the present study, we detected relatively lower MIC values, indicating that Saudi-origin R. graveolens have stronger antibacterial activities against microbes.
The leaf extracts of R. graveolens and A. abrotanum showed moderate antifungal activities against most of the fungal species studies. These antifungal activities are strongly associated with the identified polyphenols including quercetin, isochlorogenic acid, p-coumaric acid, quercitrin, and rosmarinic acid. It has previously been shown that quercetin has strong antifungal activities against C. albicans and C. parapsilosis [78]. However, only one study has demonstrated the antifungal activities of p-coumaric acid (against Botrytis cinerea) [79], which contrasts with the wide-spectrum effects observed in the present study, and this is the first report on the antifungal activities of isochlorogenic acid obtained from R. graveolens and A. abrotanum. Oliva et al. [80] reported on the antifungal activities of ethyl acetate leaf extracts of American-origin R. graveolens against Colletotrichum sp. and Botrytis cinerea, which they attributed to the presence of one quinoline alkaloid and four quinolone alkaloids. In the present study, we investigated a larger number of fungal species and found that A. abrotanum showed strong antifungal activities against these fungi, which was attributed to the specific polyphenols it contains. A recent report on A. gmelinii similarly found that ethanolic and chloroform leaf extracts showed antifungal activities against Candida spp. [81], which were attributed to flavonoid, coumarin, and chlorogenic acid contents.

Conclusions
To the best of our knowledge, this is the first study to investigate the polyphenol contents and biological activities of methanolic leaf extracts of natural populations of R. graveolens and A. abrotanum from the Riyadh region of northern Saudi Arabia. Using HPLC-DAD, we identified several polyphenols in the leaf extracts, among which quercetin and isochlorogenic acid were the major components in R. graveolens and A. abrotanum, respectively. More specifically, R. graveolens contained quercetin, chlorogenic acid, p-coumaric acid, protocatechuic acid, caffeic acid, and rutoside, whereas A. abrotanum contained isochlorogenic acid, rosmarinic acid, quercitrin, chlorogenic acid, protocatechuic acid, syringic acid, vanillic acid, and caffeic acid. The detected compounds were identified tentatively by HPLC-DAD method; further analyses, using more modern chromatographic methods, are needed to better understand the composition of studied plants. In addition, the phytochemical analyses conducted in this study was a partial analysis of selected compounds in the extract. For, e.g., fingerprinting purposes, a more sophisticated analysis should be conducted. Most of the polyphenols showed antioxidant activities and consequently were associated with the observed antioxidant activities of the leaf extracts. In addition, the leaf extracts of both species showed antiproliferative and cytotoxic activities against cancer cells, which may be attributed to necrotic cell accumulation during the early and late apoptotic periods. Antibacterial activities were also observed in the leaf extracts of both species and in the identified polyphenols, with the R. graveolens leaf extracts showing higher antibacterial activity, which was attributed to the high content of quercetin and other polyphenols. Similarly, the leaf extracts of both species exhibited antifungal effects, which were attributed to their polyphenol contents and associated with specific polyphenols. This is the first study to confirm the polyphenolic richness and wide spectrum of biological activities associated with natural populations of R. graveolens and A. abrotanum from northern Saudi Arabia.