Cytotoxic, antimicrobial and antioxidant activities and phytochemical analysis of Artemisia judaica and A. sieberi in Saudi Arabia

Cancer and microbial infectious diseases are considered a global threat. Plants have been investigated across the world to exploit their potential anticancer and antimicrobial effective agents. In this study, two medicinal plant species native to Saudi Arabia, namely; Artemisia judaica and Artemisia sieberi were screened to assess their antioxidant, anticancer and antimicrobial potential activities as well as phytochemical compositions. The collected aerial parts were extracted by maceration with methanol. Cytotoxic and antimicrobial activities were investigated using the MTT and MIC assays, respectively. Free radical scavenging and antioxidant potential were assessed respectively by 2, 2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) methods. The total flavonoid and phenolic contents of the crude methanolic extract were quantified using standard methods. Artemisia judaica displayed a strong cytotoxicity compared to A. sieberi while both species showed approximately similar bacterial and fungal growth inhibition. In contrast, A. sieberi displayed the highest phenol and flavonoid contents between the two species which was consistent with the higher antioxidant activity found in A. sieberi . It is concluded that both Artemisia species could be a promising source of antioxidant, anticancer and antimicrobial agents.


INTRODUCTION
For millennia, plants have played a crucial role in preserving and enhancing humans' quality of life. Moreover, plants were and still represent a rich source of new therapeutic agents for the treatment of several primary health care ailments. In addition, there are several reports available where natural products have shown their promising potential to cure complex diseases (Newman and Cragg, 2016;Mushtaq et al., 2018;Thomford et al., 2018;Che and Zhang, 2019). In the area of cancer treatment, which now represents the most common and second most lethal disease in the world (Siegel et al., 2020), medicinal plants contribute to anticancer drugs that inhibit tumor progression without many side effects (Dall'Acqua, 2014;Khazir et al., 2014;Iqbal et al., 2017). Therefore, plants are still being investigated across the world to exploit their potential of anticancer agents. Moreover, microbial infectious diseases are also considered a global threat. Although a large number of new antibiotics have been introduced by pharmacological industries, the microbial resistance to them has increased (Darsanaki and Lisar, 2014). Therefore, increasing attention has turned to medicinal plant as a potential source of new antimicrobials (Ali-Shtayeh et al., 1998).
Artemisia (Asteraceae), popularly known as "Sage Brush" or "Wormwood," is a genus of small herbs and shrubs of about 500 species, which are mainly found in Asia, Europe and North America (Bora and Sharma, 2011) but occurs to a lesser extent in the southern hemisphere. It is widely used in folk medicine for several ailments, including coughing (Tan et al., 1998), lowering pain (Morshedi et al., 2011), reducing phlegm (Martínez et al., 2012) and hypertension (Ben-Nasr et al., 2013). Several studies conducted in this genus have revealed their potential in antimalarial, antinociception and antimicrobial activities (Zhang et al., 2017). Artemisia judaica (Arabic name, Shih Balady) is a fragranced shrub that grows widely in the Arabian area (Acheuk et al., 2017). This species is used as a tea in Egypt Sinai and Saudi Arabia. A. judaica has antioxidant (El-Massry et al., 2002), antibacterial (Liu et al., 2003), anti-malarial (Liu et al., 2004), and anti-inflammatory properties (El-Sayed et al., 2013). Artemisia sieberi on the other hand, is a perennial shrub that grows abundantly in arid areas of Middle Eastern countries (Abouhosseini et al., 2016) and other parts of the world (Shadi and Saharkhiz, 2016). It is used as anthelminthic in Middle East traditional medicine (Mahboubi, 2017) and to treat various diseases, including diabetes mellitus in Jordan (Irshaid et al., 2012). A. sieberi is also reported to be used as an herbal medicine for treating high blood pressure and gastrointestinal ailments (Bidgoli et al., 2013). Saudi Arabia has a diversity of plant species that grow in harsh conditions that make them medicinally promising (Osman et al., 2014). However, investigation into the biological properties of Artemisia genera in Saudi Arabia remains insufficient. Therefore, this investigation aims to report on the phytochemicals composition, antioxidant, cytotoxic as well as antimicrobial activities of A. judaica and A. sieberi. The study will be of value in highlighting the importance of Artemisia species as a natural resources for a novel compounds in addition to assert its beneficial effects in folk medicine.

Plant collection and authentication
The aerial parts (leaves and stems) of A. judaica and A. sieberi were collected during winter season (February, 2015) from Al-Ghat,

Extracts preparation
The aerial parts of A. judaica and A. sieberi were dried at 45°C and grounded. 100 g of each plant powder was extracted with methanol (500 ml) using the maceration method. The liquid extract was concentrated under vacuum to get 11 g crude methanol extract for A. sieberi and 15 g crude methanol extract for A. judaica, both extracts were kept in a fridge until use.

Estimation of total phenol and flavonoid contents
Total phenolic content of A. judaica and A. sieberi extracts was determined by the standard Folin Ciocalteau spectrophotometric method (Slinkard and Singleton, 1977;Sulaiman et al., 2013). Briefly, 0.5 ml of each extract was added to 0.1 ml of Folin-Ciocalteau reagent (0.5 N) and the contents of the flask were mixed thoroughly. Later 2.5 ml of Sodium carbonate (Na 2 CO 3 ) was added, mixed and incubated for 0.5 h. The optical density was measured at 760 nm utilizing UV-Visible spectrophotometer. The total phenolic contents were expressed as mg gallic acid equivalents (GAE)/g of the extract. For total flavonoids content, standard aluminium chloride spectrophotometric method according to Mervat et al. (2009) was used. In brief, 1 ml of each extract at a concentration of 1 mg/ml was taken. Then 1 ml of AlCl 3 (10%) was added sequentially. The test solution was vigorously shaken. Absorbance was recorded at 415 nm after 30 min of incubation. A standard calibration plot was generated at 415 nm using known concentrations of quercetin. From calibration plot, flavonoid concentrations in each sample were calculated and expressed as mg quercetin equivalent (QE) /g of the extract.

DPPH radical-scavenging activity
A. judaica and A. sieberi antioxidative activity was estimated by DPPH (2, 2-diphenyl-1-picrylhydrazyl) method as reported by Brand-Williams et al. (1995) and Hussein et al. (2019). Various concentrations of A. judaica and A. sieberi extracts (10, 50, 100, 500 and 1000 μg/mL) were prepared. Thereafter, 0.5 mL of each concentration was mixed with 0.125 mL DPPH and 0.375 mL methanol and incubated for 0.5 h. The optical density was measured at λmax = 517 nm. Ascorbic acid was used as a positive control. Radical scavenging activity was calculated as the following formula: % of radical scavenging activity = (Abs control -Abs extract / Abs control) × 100

ABTS radical cation scavenging activity
The antioxidative activity of A. judaica and A. sieberi was estimated utilizing ABTS method as reported by Li et al. (2009), with minor modification. Aqueous solutions of ABTS (7 mmol/L) and potassium persulfate (2.45 mmol/L) were prepared. After 12 h standing in the dark, the two solutions were mixed and incubated for 0.5 h, followed by standing in refrigerator for 24 h, then diluted in ethanol.
To ABTS solution 50 µg/ml (1:1) different prepared concentration of each extract (10, 50, 100, 500 and 1000 μg/mL) were pipetted to initiate the reaction for achieving a calibration curve. The absorbance was read at wavelength λ734 nm using UV-vis spectrophotometer. Ethanol (95%) was used as a blank. ABTS (50 µg/ml) solution was used as control. Ascorbic acid was used as standard. Three replicates for standard and each extract were used for analysis. The percentage of antioxidant capacity for each extract was determined based on the reduction of ABTS absorbance by calculation using the following formula (Li et al., 2011).

Cytotoxic activity (MTT assay)
To assess the effect of the A. judaica and A. sieberi extracts on the growth of different malignant cell lines (MCF-7 breast, LoVo colon and HepG2 liver) the MTT assay was performed according to Mothana et al. (2019). In brief, 50,000 cells/well were added into cell culture plate (24-well). After 24 h of incubation at 37°C with 5% CO 2 , the cells were treated with methanolic extracts of A. judaica and A. sieberi at (0, 25, 50,100 and 200 µg/ mL) concentrations. The viability of the cells was determined after 48h. 100 μL of MTT (5 mg/mL) was added to each well and incubated for 4 h. The media was removed and 1000 μL of acidified isopropanol was pipetted into each well to solubilize the formazan crystals. The absorbance was then measured at 540 nm using a (Bio-Tek, USA) plate reader.

Minimum inhibitory concentrations
The minimum inhibitory concentrations (MICs) for methanolic extracts of A. judaica and A. sieberi was assessed using micro-well dilution method as previously method stated by Mann and Markham (1998)

Statistical analysis
Data represent the mean ± standard deviation of three determinations. The IC 50 values of MTT assay were determined using OriginPro8.5 Software (Massachusetts, USA).

Total phenol and total flavonoid contents
Both A. sieberi and A. judaica showed different total phenolic and flavonoid content. As shown in Figure 1, A. sieberi exhibited higher total phenolic and flavonoid content (122.1 mg of GAE/g) powder weight than A. judaica which exhibited a lower total phenolic content (117.5 of GAE/g). Furthermore, A. sieberi showed higher flavonoid content (36.5 mg of QE/g) than that of A. judaica which showed a lower flavonoid content (28.7 of QE/g) equivalent.

Antioxidant activity
Both species showed dose dependent scavenging activities ( Table 1). The results of this study reported that A. sieberi showed higher antioxidant activity than A. judaica.

Cytotoxic activity
Extracts of both species exhibited anti-proliferation activity in a dose dependent manner as illustrated by Figure 2. The calculated IC 50 values ranged between 42 and 93 µg/ml (Table 2). However, A. judaica showed a higher effect against all cell lines than A. sieberi, and the activity was stronger against HepG2, followed by MCF-7 and finally LoVo.

DISCUSSION
Medicinal plants have proven their pharmacological activities historically and nowadays still represent a valuable source of new drugs reaching the market (Atanasov et al., 2015;Che and Zhang, 2019). Due to their advantages for human health, researchers have long shown interest in phenolic and flavonoids agents in  In the columns, means ± SD with different letters notification are significant at (P < 0.05) (n = 3).  medicinal plants (Tungmunnithum et al., 2018). In this study, we reported that A. judaica and A. sieberi are considered as a promising source of phenolic and flavonoids compounds. Our findings are consistent with several studies that documented the presence of these constituents in same species grown in different regions of the world (Azimian and Roshandel, 2015;Bakr, 2015;Allam et al., 2019;Ranjbar et al., 2020). The antioxidant capacity of phenolic and flavonoids compounds is well documented in several in vitro studies and they have been shown strong capacity of scavenging of several non-physiological radicals such as DPPH and ABTS ABTS (Pietta, 2000;Kosar et al., 2003;Payet et al., 2005;Cai et al., 2006;Kumar and Pandey, 2013). In this study, total phenols and flavonoids contents have been quantified from the aerial part of A. judaica and A. sieberi, which justifies the high antioxidant activity reported for the methanol extracts. It was noticed that extracts from both species exhibited a notable antioxidant activity in both DPPH and ABTS methods with A. sieberi demonstrating the highest activity than A. judaica. In a previous study, Al-Mustafa and and Al-Thunibat (2008) demonstrated that Jordanian A. judaica exhibited an antioxidant activity (DPPH-TEAC = 18.3 ± 0.6 mg g -1 and ABTS-IC 50 = 104.6 ± 0.9) which is close to our findings. In addition, various species of Artemsia showed variation in their antioxidant capacity (Lopes-Lutz et al., 2008), which can be attributed to the quantity of phenolic compounds, including flavonoid, and climate and edaphic characteristics of the geographical areas.
Different forms of cancer are constantly gaining resistance to current drugs, creating a need for the discovery of new drugs. In this work, the methanol extracts of A. sieberi and A. judaica showed remarkable cytotoxic activity against all tested cancer cell lines. This could be due to the presence of artemisinin and other sesquiterpene lactones, which have been reported before for both species (Arab et al., 2006;Abbas et al., 2017). Artemisinin which is a sesquiterpene lactone demonstrated anticancer activity when tested in vitro and in vivo (Ferreira et al., 2010). Artemisinin was also found to be a good antibacterial, antifungal, antileishmanial, and antitumor agent (Appalasamy et al., 2014).
Due to pathogens developing resistance to existing drugs, millions of lives are lost every year owing to infectious diseases (Nicolaou et al., 2009;Yu et al., 2010). The ability of extracts to combat different species of microorganisms was explored. Assays involving in vitro MIC methodologies are commonly used and provide data on the lowest concentration of an antimicrobial that inhibits the visible growth of a microorganism. The value of MIC is an accepted standard for measuring the sensitivity of microorganisms (e.g. bacteria in their planktonic phase) to inhibitors (Kalemba and Kunicka, 2003). Thus, the minimum inhibitory concentrations (MIC) of the A. judaica and A. sieberi extracts were tested to determine the antimicrobial effectiveness (Table 3). Artemisia sieberi was found to have similar activities of A. judaica against all microbes, except Gram-negative bacteria (E. coli and P. vulgaris), which were resistant against both plant species. The antibacterial activity of these species could be attributed to artemisinin, which had been tested on a wide range of bacteria (Appalasamy et al., 2014). Our results are in conformity with the study of Guetat et al. (2017) who reported that A. sieberi as well as A. scoparia and A. judaica possess high antibacterial activity.

Conclusion
Our results confirm the potential use of A. judaica and A. sieberi species in folk medicine and in primary health care. Our data support the antioxidant properties of these species which can interfere with oxidative stress and hence minimizing the risk of developing complex diseases. In addition, our results documented the anticancer properties of A. judaica and A. sieberi species suggesting the potential use of these species as a source of anticancer agents. The obtained results also provide a basis for a further investigation to characterize new compounds from these species that could be used as anticancer and antimicrobial agents. Further in vitro and in vivo investigation is still necessary to examine the reported bioactivity to confirm the beneficial uses of Artemisia species against cancer and infectious diseases.

CONFLICT OF INTERESTS
The authors have not declared any conflict of interests.