Antioxidant and Cytotoxic Activity of a New Ferruginan A from Olea ferruginea: In Vitro and In Silico Studies

Studies of the ethyl acetate extract bark extract of Olea ferruginea led to the isolation of one new compound Ferruginan A (1) in addition to two known compounds, Ferruginan (2) and cycloolivil (3). Structures of the isolated compounds were confirmed by mass spectrometry (MS) and NMR spectral data. The ethyl acetate fraction and compounds (1–3) were evaluated against breast cancer cell line (MCF-7) and as antioxidants using the free radical scavenging assay. Results revealed that compound 2 exhibits significant antioxidant activity with an IC50 value of 21.74 μg/mL. In addition, the ethyl acetate fraction showed good cytotoxic activity (79.31% inhibition at 250 μg/mL), whereas compounds 1–3 exerted mild cytotoxic activity (IC50 = 8.03–12.01 μg/mL) as compared to the standard (IC50 = 4.41 μg/mL) against MCF-7. Docking studies suggested that antioxidant activity is due to the chelation of compounds with copper present in the active site of tyrosinase. These results suggest that the extract exhibits considerable antioxidant activity, and the isolated compounds exert moderate anticancer activity.


Introduction
Natural products obtained from terrestrial and marine plants have been used as a source of traditional medicines since ancient times. According to the World Health Organi-zation (WHO), people of developing countries rely on natural products for their health care. In the last century, natural products such as Taxol, docetaxel, morphine, cyclosporin A, quinine, simvastain, and lovastatin, among others, have been successfully marketed as safe drugs for the treatment of various diseases. In this respect, around 40% of recent drugs have been developed from natural products [1]. Phenolic compounds, commonly present in fruits and vegetables, are highly bioactive. Currently, these compounds are gaining much attention due to their broad application in the health care systems of several countries. These compounds significantly cure diseases related to free radical-induced stress, cancer, renal, and cardiovascular diseases and act as antiinflammatory and hepatoprotective [2]. Due to their outstanding benefits, there is an increasing demand for these compounds in the pharmaceutical industry. Additionally, the scientific community is paying much attention to natural products [3].
Olea ferruginea is a 10-to 50-feet tall tree belonging to the genus Olea and family Oleaceae. This evergreen plant grows in the mountainous areas of Afghanistan, Kashmir, and Pakistan [4]. In Pakistan, the plant is predominantly found in the hilly regions of Azad Kashmir, Khyber Pakhtunkhwa, and Rawalpindi [5]. Traditionally, the stem bark of Olea ferruginea has been prescribed for the treatment of fever [6], whereas fresh leaf extracts of this plant were used by people suffering from bleeding gums, toothache, skin diseases, skeletal muscular, and dental problems [7,8]. In addition, the fruit of O. ferruginea is a rich source of oil, which can be applied as massage ointment to relieve pain during dislocated bones and rheumatism [9]. Furthermore, research findings indicated that various parts of this plant have shown significant antimalarial, anti-inflammatory, antiperiodic, antileprosy, astringent, antidiabetic, and antiasthmatic properties [10].
The genus Olea is represented by nearly 35 species, and Olea europaea L. is the most cultivated around the globe for its oil production [11]. Within this context, extracts of Olea plant contain numerous phenolic compounds, flavonoids and lignin, and secoiridoid. Due to the significant phenolic content (17-23%), this plant exhibits antioxidant, anticancer, and antibacterial properties [12,13]. Biophenols isolated from other Olea species reduce cancer growth by various mechanisms. These compounds can either cause a cell cycle arrest in a specific phase or inhibit signaling pathways, which promote proliferation of cells [14]. Furthermore, the increasing demand of olive oil encouraged cultivation of Olea europaea in several regions of the world and/or finding alternative sources of olive oil production [15]. For this purpose, Olea ferruginea, also known as wildtype olives, has been investigated as a potent source of ample amounts of olive oil. Olive oil production from wild-type olives will not only help in maintaining balance between demand and supply but also improve the national economy and human health [16]. In this respect, studies are required to authenticate the health-promoting benefits and oil quality and production of Olea ferruginea (wild olive) [17]. Although numerous studies have dealt with the local cultivars, less attention has been given to the phytochemistry and health-promoting benefits of the plant [15,18]. Recently, we have reported on the ethyl acetate extract and the isolation of a new compound, Ferruginan, having antibacterial and antileishmanial activity [19]. Based on the preceding discussion, and in view of the importance of Olea ferruginea and lack of studies pertaining to its health benefits, the current study was designed to evaluate the antioxidant and anticancer activity of ethyl acetate extract of O. ferruginea and to search for new natural compounds present in it having potent antioxidant and chemopreventive properties.

Material and Methods
2.1. General Experimental Procedures. UV-Vis and IR records of isolated compounds were obtained using a spectrophotometer and a Bruker FT-IR spectrophotometer (Japan). Mass spectral data were acquired by employing a FAB-MS spectrometer (Thermo Scientific, United State). Nuclear magnetic resonance spectroscopic data were obtained with the aid of a Bruker Avance (Germany): 1 H (400 MHz) and 13 C NMR (100 MHz). Isolated compounds were purified using polar silica gel purchased from Merck-Germany (mesh size of 70-230), and purity was checked by precoated TLC and visualized under UV light.

Plant
Material. The O. ferruginea plant was collected from the Agriculture Research Institute Tarnab, Peshawar division. Steam, twigs, and leaves were separated and dried in a dark room.

Bioactivity.
The antioxidant activity of ethyl acetate fractions and compounds 1-3 was determined using the 2,2diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay [21]. According to this assay, 7% of DPPH stock solution having a violet color was prepared in methanol. A mixture of 7% DPPH solution (1 mL), extracts and compounds with a concentration of 15-30 μg/mL, and ascorbic acid (positive control) was placed in a labeled test tube for 30 min at room temperature. The optical density (λ max = 517 nm) was measured against a standard by means of a UV/visible spectrophotometer; experiments were carried out in triplicate. The relative radical scavenging activity (%) was calculated as ½1 absorbance of solution with sample and DPPH/absorbance of solution with DPPH × 100.
The cytotoxicity study of ethyl acetate extract and compounds 1-3 against MCF-7 was carried out by using the 2 Oxidative Medicine and Cellular Longevity (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) MTT assay according to a procedure described by Shah et al. [20]. Reduction of the salt to formazan insoluble crystals is the conformation of inhibitory effect of the extract/compounds. Briefly, the MCF-7 cell line at a concentration of 3 × 10 4 was seeded in a 96-well plate and incubated at 37°C overnight under 5% CO 2 . On the next day, the original medium was substituted with fresh medium containing 15-30 μg/mL of test samples and subjected to incubation under similar conditions for 48 h. After 48 h incubation, the MTT and fresh medium was added to achieve 0.5 mg/mL, and the plates were further incubated for 4 h. The formazan crystals were dissolved in DMSO, and absorbance was measured at 550 nm using a spectrophotometer (Thermo Fisher Scientific). The percent MCF-7 cell inhibition was calculated using the following formula: Percent inhibition = OD of the treated well -OD of untreated well ð Þ / ½ OD of the untreated well × 100: Bank. Preparation of downloaded enzymes for binding site determination, 3-D protonation, and energy minimization was conducted as per previously reported methods [22,23]. Structures of the compounds were built using the builder option in the MOE software. The built structures were then energy minimized using the MMFF94X force field in addition to 0.0001 gradient and database. The docking study was carried out using validated parameters including placement/refinement stage and scoring/rescoring functions. Interpretation of docking results was achieved using MOE and discovery studio visualizer.    (Figure 2). The three-spin system was detected in the COSY 45°spectrum ( Figure 2). The correlation of H-5, H-6, and H-11 with each other was noted in the spin system I, while the correlation between H-5 ′ and H-6 ′ was noticed in the spin system II. The connection between H-13 and H-14 was noticed in the spin system III. 1 H-and 13 C-NMR data for 1 are presented in Table 1.

Bioactivity.
Ethyl acetate fraction and isolated compounds 1-3 were tested for free radical scavenging activity     Table 2). The antioxidant potential data of compound 2 matched those reported [24]. The potent antioxidant activity may be due to the presence of phenolic compounds in ethyl acetate (polar) fraction, and five hydroxyl groups (-OH) in compound 2, which support the concept that compounds having higher number of hydroxyl groups (-OH) or other H-donating groups (-NH; -SH) have display antioxidant activity [21]. The anticancer potential of ethyl acetate fraction and compounds 1-3 against breast cancer cell line (MCF-7) was also investigated. Results listed in Table 2 revealed that the percentage inhibition of MCF-7 cell line increases as the concentration (50-250 μg/mL) of ethyl acetate extract and doxorubicin (std) increase. Results also showed that compounds 1-3 exhibit good potential against MCF-7 cell lines (Table 3). These findings support the notion that natural products of Olea species have been shown to exhibit cytotoxic activity toward human cancer cells with little or no effect on normal cells [25].

Docking Studies.
We performed docking studies on tyrosinase, which is a widely dispersed copper containing enzyme. This enzyme has a fundamental role in catalyzing the hydroxylation of mono-and diphenol oxidation reactions [26]. Three-dimensional (3-D) structure of tyrosinase in complex with the antioxidant ligand kojic acid having   Figures 3 and 4. The native compound, kojic acid, is an inhibitor of tyrosinases from various origins such as bacteria and mushrooms [27]. Additionally, 2-D interaction plots indicate that kojic acid chelates with copper ions in a bidentate manner via hydroxyl and carbonyl groups. Moreover, it forms hydrogen bond interactions with Asn205 (active site entrance residue) and His60 (Figure 3(a)). Isolated compound 1 chelates with Cu ions and forms hydrogen bond interactions with Val218 present on a loop adjacent to Cu301 [27]. It also displayed a conventional hydrogen bond interaction with Arg209 positioned near the entrance of the active site (Figure 3(b)).
As for compound 2, the 2-D interaction plot showed that it chelates with copper ions in a bidentate manner via hydroxyl and methoxy oxygen atoms. Conventional hydrogen bond interactions were also observed with Arg209 and Val218 via hydroxyl groups. His60 and His208 displayed π − π interactions with phenyl groups (Figure 4(a)). Compound 3 is oriented toward Cu ion via the methoxy group and forms hydrogen bond interactions with Met215 and Val217. His60 and His208 displayed π − π interactions with phenyl groups, while val218 displayed π-alkyl interactions ( Figure 4(b)).

Conclusions
In addition, findings from this investigation reveal that the isolated compounds and BEPS exhibit significant antiinflammatory effects in both in vitro and in vivo antiinflammatory models, which justifies the use of this plant in Algerian folk medicine. In the study, we have succeeded in isolating a new compound (1) and two known compounds (2 and 3) from O. ferruginea. In addition, findings from this investigation reveal that the ethyl acetate fraction and compound 2 exhibit potent antioxidant activity. Moreover, the ethyl acetate fraction and compounds 1-3 also showed significant anticancer activity against the breast cancer cell line (MCF-7). Docking studies suggest that the isolated compounds show tyrosinase inhibition by chelating with copper present in the active site and thus exhibited antioxidant activity.

Data Availability
The supporting information is available online (see supplementary file).

Conflicts of Interest
The authors declare that they have no conflicts of interest.