Phytochemical Composition and In Vitro Antioxidant, Anti-Inflammatory, Anticancer, and Enzyme-Inhibitory Activities of Artemisia nilagirica (C.B. Clarke) Pamp

Plants have been employed in therapeutic applications against various infectious and chronic diseases from ancient times. Various traditional medicines and folk systems have utilized numerous plants and plant products, which act as sources of drug candidates for modern medicine. Artemisia is a genus of the Asteraceae family with more than 500 species; however, many of these species are less explored for their biological efficacy, and several others are lacking scientific explanations for their uses. Artemisia nilagirica is a plant that is widely found in the Western Ghats, Kerala, India and is a prominent member of the genus. In the current study, the phytochemical composition and the antioxidant, enzyme-inhibitory, anti-inflammatory, and anticancer activities were examined. The results indicated that the ethanol extract of A. nilagirica indicated in vitro DPPH scavenging (23.12 ± 1.28 µg/mL), ABTS scavenging (27.44 ± 1.88 µg/mL), H2O2 scavenging (12.92 ± 1.05 µg/mL), and FRAP (5.42 ± 0.19 µg/mL). The anti-inflammatory effect was also noticed in the Raw 264.7 macrophages, where pretreatment with the extract reduced the LPS-stimulated production of cytokines (p < 0.05). A. nilagirica was also efficient in inhibiting the activities of α-amylase (38.42 ± 2.71 µg/mL), α-glucosidase (55.31 ± 2.16 µg/mL), aldose reductase (17.42 ± 0.87 µg/mL), and sorbitol dehydrogenase (29.57 ± 1.46 µg/mL). It also induced significant inhibition of proliferation in breast (MCF7 IC50 = 41.79 ± 1.07, MDAMB231 IC50 = 55.37 ± 2.11µg/mL) and colon (49.57 ± 1.46 µg/mL) cancer cells. The results of the phytochemical screening indicated a higher level of polyphenols and flavonoids in the extract and the LCMS analysis revealed the presence of various bioactive constituents including artemisinin.


Introduction
Medicinal plants are important sources of various biologically and pharmacologically active compounds [1,2]. Several traditional medicinal plants have been shown to have strong pharmacological properties, such as radical neutralizing, inflammation-preventing, antiproliferative, hypolipidemic, hepatoprotective, neuroprotective, antithrombotic, and immunomodulatory activities [3,4]. Among the various plant families, Asteraceae is one of the most widely utilized ones, and it is also equipped with numerous biological and pharmacological activities. Among the various genera, the Artemesia genus is well-known [5][6][7].
The Artemisia genus and the member species are well-studied for their various biological activities [8][9][10][11][12][13]. Artemisia annua L. has demonstrated significant medicinal benefits because of the presence of artemisinin [14]. Artemisia mongolica is another important member of the genus, which is rich in lactone derivatives of Sesquiterpene and a wide range of pharmacological activities [15]. The different species of the genus were found to have strong antibacterial and antifungal properties against pathogenic organisms in humans, livestock, and plants [16][17][18][19][20]. Antiproliferative and apoptotic effects are attributed to the bioactive compounds and extracts of various species of Artemisia [21][22][23][24].
Artemisia nilagirica is distributed throughout the Western Ghats, India; it has been traditionally applied by various tribal healers in the area for the treatment of infectious diseases and toxicity prevention. The plant has been shown to have significant biological and pharmacological activities based on various in vitro and in vivo studies. The initial studies by Ahameethunisa and Hopper [25] identified the antibacterial potential of the methanol extract of A. nilagirica against 15 bacterial strains. Further, the extract was found to be effective against Mycobacterium smegmatis and M. bovis [26]. The extract was also found to be effective against the malarial parasite Plasmodium falciparum [27].
The anticancer activities of the methanol and ethyl acetate extracts were also elucidated against the human monocytic leukemia cell (THP-1) [28]. Later, studies by Sahu, Meena, Shukla, Chaturvedi, Kumar, Datta, and Arya [24] also supported these results in colorectal cancer cell models. Studies by Raju et al. [29] indicated that the anticancer activity was mediated through the inhibition of TGF-beta signaling. The plant extract was also found to inhibit inflammatory insults in human red blood cell models [30]. The fruit of A. nilagirica was found to have significant antiradical activity via scavenging DPPH and nitric oxide radicals [31]. The essential oil extracted from A. nilagirica was a rich source of monoterpenoid compounds such as thujone, and by virtue of these compounds, the essential oil inhibited the growth of various fungal pathogens [32]. The essential oil was also effective against the phytopathogenic fungal groups of table grapes [33]. Additionally, the essential oil was also effective against various bacterial populations and capable of repelling mosquitoes [34].
Although several studies have reported the preliminary pharmacological activity of the plant, there is no clear-cut information on its quantitative chemical profile and nutritional value. Additionally, the anti-inflammatory properties are yet to be discovered in cell line models, and its mechanism of action is also not specified. Therefore, the present study aimed to analyze the chemical composition of the ethanol extract of Artemisia nilagirica leaves in terms of the bioactive compounds and proximate composition, as well as their antioxidant potential. Further, this study for the first time attempted to analyze the enzymeinhibitory and anti-inflammatory activities of the extract in Raw 264.7 cells stimulated by lipopolysaccharides.

Determination of Proximate Composition of A. nilagirica
The Artemisia species, which includes 200-400 identified plants, are extensively spread in tropical and temperate areas [6]. The importance of the artemisia species in traditional medicine is well established [5]. The plant's antiviral, antifungal, antibiotic, insecticidal, hepatoprotective, and neuroprotective qualities make it useful in both Chinese and Ayurvedic medical systems [35]. The current study examined a specific member Artemisia nilagirica, its phytochemical makeup, and its pharmacological effects.
The physicochemical parameters of the A. nilagirica leaf powder are shown in Table 1. The predominant compounds were carbohydrate, protein, fat, and ash contents. The moisture content was estimated to be 87.4 ± 2.12%.

In Vitro Antioxidant Activities of A. nilagirica Extract
The Artemisia genus members frequently display antioxidant activity [41]; our study also confirmed the antioxidant activity of A. nilagirica for the first time in terms of the radical generation inhibition and reducing potentials. The IC 50 value of the A. nilagirica extract in the anti-DPPH radical assay was estimated to be 23.12 ± 1.28 µg/mL. Likewise, Table 6 shows the other antioxidant activities in terms of the ABTS radical scavenging activity, hydrogen peroxide scavenging potential, and ferric-reducing antioxidant power; the respective IC 50 values were found to be 27.44 ± 1.88, 12.92 ± 1.05, and 5.42 ± 0.19 µg/mL. On the contrary, the level of inhibition of nitric oxide radical generation (IC 50 ) was determined to be 367.09 ± 12.05 µg/mL for the extract. However, in comparison with the standard antioxidant ascorbic acid (Table 6), the activity was much lower in the A. nilagirica extract; further purification of the extract may yield more active antioxidant compounds. The antioxidant properties are attributed to the bioactive compounds identified in the plant via LC-MS. Oxidative stress is the central independent factor that drives many chronic diseases, including cancers [42,43]; hence, the antioxidant properties of the plant may be useful in the management of diseases associated with oxidative stress.

Enzyme-Inhibitory Activities of A. nilagirica Ethanol Extract
The enzyme-inhibitory properties of the extract were analyzed against four enzymes involved in type 2 diabetes mellitus, including α-amylase, α-glucosidase, aldose reductase, and sorbitol dehydrogenase ( Table 7). The IC 50 values for these enzymes were 38.42 ± 2.71, 55.31 ± 2.16, 17.42 ± 0.87, and 29.57 ± 1.46 µg/mL, respectively. Furthermore, α-amylase and α-glucosidase are enzymes involved in carbohydrate metabolism and are common targets of antidiabetic drugs [44]. Similarly, the polyol pathway enzymes, including aldose reductase and sorbitol dehydrogenase, are involved in diabetic complications [45,46]. Hence, the inhibition of these enzymes could result in strong antidiabetic activity for the A. nilagirica extract.

Antiproliferative Activity of the A. nilagirica
Additionally, the results showed the anticancer properties of A. nilagirica in human breast and colon cancer cells. The anticancer activity was analyzed in three cancer cell lines, including MCF-7, MDA-MB-231, and HCT-15. We observed dose-dependent cytotoxicity in these three cell lines (Figure 2). The IC 50 values against the three cells were estimated to be 41.79 ± 1.07, 55.37 ± 2.11, and 49.57 ± 1.46 µg/mL, respectively. In comparison, the standard cyclophosphamide was more toxic to these cells, with respective IC 50 values of 3.12 ± 0.13, 5.74 ± 0.20, and 6.04 ± 0.21 µg/mL. Previous studies have also shown different species of Artemisia in various cancer cells [47][48][49][50]. In addition, the green synthesized nanoparticles from different Artemisia species are also reported to exert antiproliferative effects on cancer cells mediated through apoptotic cell death [23,51,52]. A study by Sahu, Meena, Shukla, Chaturvedi, Kumar, Datta, and Arya [24] indicated that ethyl acetate and hexane fractions of A. nilagirica induced cell death in colon, lung, and breast cancer cells. In addition, the bioactive compounds, including quercetin, apigenin, and eugenol, have also been shown to have significant antiproliferative effects by modulating different signaling pathways [53,54].

Anti-Inflammatory Activity of A. nilagirica
The Artemisia nilagirica extract was shown to inhibit the production of nitric oxide radicals in vitro. Further, the pretreatment of the extract also inhibited cytokine production and inflammatory insults in lipopolysaccharide-stimulated macrophages. The LPS is a microbial component that is known to stimulate inflammatory insults [55,56]. The Artemisia nilagirica leaf ethanol extract (AN) was found to inhibit the lipopolysaccharide-induced activation of macrophages and the subsequent cytokine release. The level of IL-1β was found to be significantly increased after LPS stimulation in macrophages; however, the pretreatment with AN at different doses significantly brought down the IL-1β levels in the macrophages (Table 8). Likewise, the levels of IL-6 and TNF-α also showed a similar increase during LPS exposure, which were successfully brought down by the treatment with different concentrations of A. nilagirica. The level of nitric oxide was determined biochemically and was also significantly elevated in LPS control cells. Pretreatment with 2.5, 5.0, and 7.5 µg/mL of AN successfully brought down the levels to 40.7 ± 1.6 (p < 0.05), 32.2 ± 2.4 (p < 0.05), and 25.7 ± 2.1 (p < 0.01). In addition, it is noted that the high dose of the extract resulted in stronger anti-inflammatory molecules compared to quercetin, which is a well-known anti-inflammatory molecule [57,58]. The LPS is known to stimulate cytokine production in macrophages by upregulating the NF-κB translocation to the nuclear compartment [59,60]. It is, therefore, possible that the A. nilagirica extract may also influence the LPS-induced activation of intracellular NF-KB signaling. Table 8. Effect of the Artemisia nilagirica leaf ethanol extract (AN) against lipopolysaccharide-induced macrophage (Raw 264.7) activation, cytokine release (in pg/mg protein), and nitric oxide production (µM/mg protein).

Nature
Tumor Thus, the study concludes that the Artemisia nilagirica ethanol extract exhibits antioxidant and anti-inflammatory properties in vitro and cultured cells. Further, the extract is also capable of inhibiting the proliferation of various cancer cells. The inhibition of enzymes associated with type 2 diabetes mellitus is also indicative of its anti-diabetic properties. The biological properties of the plant are expected to be due to the bioactive compounds identified in the A. nilagirica extract.

Artemisia Nilagirica (C.B.Clarke) Pamp. Collection and Extraction Using 100% Ethanol
The Artemisia nilagirica plant samples were collected from the Wayanad District, Kerala (11.7917 • N, 76.1716 • E). The mature leaves were carefully cleaned of all kinds of dust via washing. These leaves were dried under shade for 2 weeks and powdered using a mixer grinder; the powder was extracted with 100% ethanol using the Soxhlet method. Briefly, 100 g of the powder was extracted with ethanol at 80 • C for 8 h and the extract was collected, filtered, and concentrated before storage.

Phytochemical Analysis of Artemesia nilagirica
The leaf powder of A. nilagirica was analyzed for the proximate composition according to the methods used by Shukla et al. [61]. The qualitative phytochemical screening was carried out for the detection of alkaloids, flavonoids, glycosides, sterols, and triterpenes by referring to standard protocols [62,63]. The LC-MS analysis (Shimadzu LC-8045, Kyoto, Japan) was used for phytochemical screening [64]; briefly, the C18 column measuring 4.6 × 150 mm and 5 µm in size was used for the study, with methanol (A) and water with 0.1% formic acid (B) as the mobile phase (gradient elution mode). The gradient was set as 95% solution A (0-5 min), 70% solution A (5 to 10 min), 65% solution A (10 to 20 min), 50% solution A (20 to 30 min), and 90% of solution B (until 50 min), with a flow rate of 1.0 mL/min.
The quantitative profiling was estimated in terms of the total polyphenols [65] and total flavonoids [66], and the concentrations of ferulic acid, luteolin, caffeic acid, quercetin, and apigenin were determined using an HPLC analysis according to the same LC-MS conditions mentioned above.

Analysis of the Antioxidant Activity of A. nilagirica Ethanol Extract
The antioxidant activities were determined as the scavenging potentials of different radicals, including diphenyl picryl hydrazyl (DPPH), ABTS [67], and hydrogen peroxide [68]; the reducing potential on ferric ions was also estimated using the procedures described in [69]. The nitric oxide radical removal rate was used as an indicator of the inflammatory process inhibition model [70]. The DPPH was dissolved in methanol (0.1 mM) and varying concentrations of the extract were mixed with it. The solution was incubated for 20 min in the dark at 30 • C and the change in absorbance was used to estimate the percentage inhibition. Likewise, the ABTS radical generated was mixed with different doses of the A. nilagirica extract and the % inhibition was calculated spectrophotometrically. The nitric oxide scavenging was determined using sodium nitroprusside (8 mM) as the radical source; the Griess reagent was used to estimate the nitrite remaining in the treated samples using spectrophotometry at 596 nm.
Ascorbic acid was used as a positive control and standard for the antioxidant assays. The percentage inhibition was determined using the formula

Efficacy of A. nilagirica Ethanol Extract on Activities of Enzymes
The enzyme-inhibitory properties were analyzed against the selected enzymes involved in diabetes and secondary diabetic complications. The inhibitory effect on α-amylase [71], α-glucosidase [72], aldose reductase [73], and sorbitol dehydrogenase [46] was assessed according to the standard methods.

Effect of A. nilagirica Ethanol Extract on Cancer Cell Proliferation
The human breast cancer cell lines MCF7 and MDA-MB-231 and a colon cancer cell line (HCT-15) were collected from NCCS, Pune, India. These cells were maintained in complete MEM, Leibovitz's L-15, and RPMI-1640 media. The cells were selected as they are widely used in the anticancer screening of phytochemicals.
The inhibitory potential of the extract on human cancer cell proliferation (MCF7, MDA-MB-231, and HCT-15) was assessed using the MTT assay [74]. The IC 50 value was determined using probit analysis.

Effect of A. nilagirica Extract on Lipopolysaccharide-Induced Cytokine Production in Macrophages
The murine Raw 264.7 cells were allowed to attach (1 × 10 7 cells/mL) in a 24-well plate in complete growth media. The RPMI-1640 media was used to dilute the different concentrations of A. nilagirica (AN) (2.5, 5.0, and 7.5 µg/mL). Next, the cells were exposed to 1 µg/mL lipopolysaccharide for another 24 h. The protein expression of cytokines such as interleukin-1β and interleukin-6 and the tumor necrosis factor-α release were determined using PeproTech ELISA kits (Rocky Hill, CT, USA), as per the commercially prescribed methods. The nitric oxide release was quantified using the Griess reaction method [64]. Quercetin was used as a standard anti-inflammatory compound in the study.

Presentation of the Data, Software Used, and Statistical Analysis
The accuracy of the results obtained was ensured by conducting three independent assignments, with each having four replicates. Microsoft Excel 2010 was used for data consolidation and verification. The processed data are presented as means ± standard deviations; the IC 50 values were estimated using probit analysis (GraphPad Prism 7.0, San Diego, CA, USA).

Conclusions
Artemisia nilagirica is an ethnomedicinal plant in India. In our study, the ethanol extract of A. nilagirica leaves showed significant antiradical and reducing potentials, which are indicative of its antioxidant potential. The IC 50 values were lower but comparable with those of the standard ascorbic acid. The extract also inhibited enzymes associated with diabetes mellitus, including alpha-amylase and α-glucosidase. Additionally, the extract treatment significantly reduced the proliferative potential of breast and colon cancer cells. In Raw 264.7 macrophages, the pretreatment with the extract inhibited the LPS-stimulated production of cytokines and proved itself to be anti-inflammatory. Most importantly, the higher dose of the extract caused significantly higher activity than the standard quercetin used. Hence, we conclude that the ethanol extract of A. nilagirica leaves has antioxidant, anti-inflammatory, and anticancer properties; further studies on animal models and with bioassay-guided purification are necessary to identify the bioactive components.