Antioxidant and Antiproliferative Potential of Bioactive Molecules Ursolic Acid and Thujone Isolated from Memecylon edule and Elaeagnus indica and Their Inhibitory Effect on Topoisomerase II by Molecular Docking Approach

The present study aimed to evaluate the antioxidant and antiproliferative potential of ursolic acid and thujone isolated from leaves of Elaeagnus indica and Memecylon edule and their inhibitory effect on topoisomerase II using molecular docking study. The isolated ursolic acid and thujone were examined for different types of free radicals scavenging activity, the antiproliferative potential on U-937 and HT-60 cell lines by adopting standard methods. Further, these compounds were docked with the active site of the ATPase region of topoisomerase II. The findings of the research revealed that ursolic acid harbor strong antioxidant and antiproliferative capacity with low IC50 values than the thujone in all tested methods. Moreover, ursolic acid shows significant inhibition effect on topoisomerase II with a considerable docking score (−8.0312) and GLIDE energy (−51.86 kca/mol). The present outcome concludes that ursolic acid possesses significant antioxidant and antiproliferative potential, which can be used in the development of novel antioxidant and antiproliferative agents in the future.


Introduction
Free radicals are involved in numerous cellular functions including defense mechanisms and cell signaling and are essential for the aerobic metabolism. However, the overproduction of free radicals in cells leads to oxidative stress, consequently causing damage to vital macromolecules, like DNA, lipids, and proteins [1]. Several degenerative related diseases such as cancer, inflammation, atherosclerosis, cataracts, asthma, diabetes mellitus anemia, brain dysfunction, arthritis, liver diseases, and renal problems are predominantly linked to the oxidative stress [2].
Cancer is the malignant illness and the second most leading cause of mortality worldwide. Globally, various types of cancer caused 9.6 million deaths in 2018, approximately 1 in 6 deaths. e people of low-and middle-income countries are highly affected by cancer, due to the change in behavior and diet, and the estimates were around 70% of deaths [3]. In western countries, hormone-dependent cancers such as breast, prostate, and uterine cervix are a common cause of several deaths [4]. Nowadays, different treatments such as chemotherapy, surgery, radiotherapy, and antihormone therapy are used to treat cancer. However, these therapies are expensive and cause an adverse effect on host health. Several plant-derived compounds like vinblastine, vincristine, taxol, and camptothecin were used in the treatment of cancer [5].
us, various research groups around the world have focused on the investigation of plant extract to find a plant-based novel, broad-spectrum, cost-effective, better, and safer anticancer and antioxidant agent from plant materials [6].
Elaeagnus indica (Elaeagnaceae) and Memecylon edule (Melastomataceae) are known to possess various ethnobotanical properties and used to treat various ailments in traditional medicine [2,7,8]. Both plants were reported to have various biological activities, including larvicidal, antibacterial, anti-inflammatory, analgesic, antioxidant, and anticancer activities, and few compounds were identified [1,2,[7][8][9][10][11][12][13][14][15][16][17]. However, there is no data available on the antioxidant and antiproliferative properties of the isolated compound thujone (from Elaeagnus indica). Even though anticancer and antiproliferative activity of ursolic acid (isolated from Memecylon edule) were reported [18][19][20][21], there is no data available on growth inhibitory effect of ursolic acid on the human leukemic monocyte lymphoma (U-937) cells, and antioxidant potential of ursolic acid was the least explored. Moreover, there are no previous studies available on the in silico inhibition interactions of thujone and ursolic acid with topoisomerase II in detail. us, the present investigation aimed to examine the antioxidant and antiproliferative potential of ursolic acid and thujone isolated from leaves of Elaeagnus indica and Memecylon edule and evaluate their inhibitory effect on the topoisomerase II using molecular docking tools. e dried plant materials were pulverized, using an electric grinder, and then sieved through 100-mesh sifter and stored in an airtight container for further use.

Extraction of Plant Materials.
Pulverized plant materials (2 kg) were successively extracted with various organic solvents such as hexane, chloroform, ethyl acetate, acetone, and methanol (1 : 5 solvent ratio) in an increasing polarity manner using a Soxhlet apparatus until the efflux solvents become colorless. e extracts were filtered through Whatman No. 1 filter paper and condensed using a rotary evaporator in vacuum at 40°C which yields greenish crude extracts. ese extracts were stored in an airtight container at 4°C until use.

In Vitro Antioxidant
Studies. Antioxidant potential of isolated compounds were examined on different types of free radicals, that is, DPPH, nitric oxide, hydroxyl, and superoxide radical and ferric reducing antioxidant power assay (FRAP) as per the previous standard methods [22][23][24][25]. Various concentrations (20-100 µg/mL) of the isolated compounds were used in the radical scavenging potential analysis. Similar concentrations of a natural (ascorbic acid) and synthetic compound butylated hydroxyanisole (BHA) were used as reference molecules in all antioxidant investigations.

Antiproliferative Activity.
e human leukemic monocyte lymphoma (U-937) and human acute promyelocytic leukemia (HT-60) cell lines were acquired from the National Institute of Cell Sciences, Pune, India, and maintained in Minimal Essential Medium (MEM) supplemented with 10% (v/v) heat-inactivated Fetal Bovine Serum (FBS), 3% L-glutamine, 100 IU/mL penicillin G, and 100 µg/mL streptomycin in a 5% CO 2 incubator at 37°C. e antiproliferative potential of isolated compounds was detected by methyl thiazolyl diphenyl-tetrazolium bromide (MTT) by adopting the method of Srinivasan et al. [16] on U-937 and HT-60 cell lines.

Preparation of Topoisomerase II.
e crystal structure of the 1QZR complex (ATPase region of topoisomerase II from Saccharomyces cerevisiae) was retrieved from PDB which contains two identical 418 amino acid length polypeptide chains (A and B) with one (s)-4, 4′-(-1-methyl-1,2-ethanediyl) bis-2,6-piperazinedione (CDX), phosphoaminophosphonic acid-adenylate ester (ANP), and magnesium ion in each chain. Two methods were used in the preparation of protein for docking study. In the first method, all the water molecules, one CDX, one magnesium ion, and one ANP were removed and in the second method the the water molecules present in 1QZR were removed.

Statistical Analyses.
All the analyses used in the present research were carried out in triplicate. Data were represented as the mean ± standard deviation of three quotients. e inhibitory concentrations 50 (IC 50 ) were calculated by the curve fitted method using OriginPro 8 software. e significant difference was obtained from the results of Tukey's test (p < 0.05) of ANOVA (SPSS 25.0).

In Vitro Antioxidant Studies.
Both the isolated compounds ursolic acid and thujone expressed good to moderate radical scavenging activity in all tested methods in a concentration-dependent manner (Tables 1-5). Ursolic acid exhibited significant free radical scavenging activity on all tested radicals with the lowest IC 50 /EC 50 values followed by thujone with sustainable IC 50 values. Ursolic acid expressed good Fe 3+ reduction potential in FRAP assay with the least EC 50 value 18.42 ± 0.03 µg/mL ( Table 2) followed by hydroxyl radicals (IC 50 value 29.69 ± 0.44 µg/mL) ( Table 3). NO radical scavenging potential of ursolic acid and thujone is almost similar to high IC 50 value of 70.40 ± 0.88 µg/mL and 77.68 ± 0.58 µg/mL, respectively, which are 2-fold higher than the positive control ascorbic acid (Table 4). Both the ursolic acid (76.92 %) and thujone (72.79 %) show nearly similar percentage of DPPH radical scavenging potential. However, considerable difference was found in the IC 50 values (Table 1). Ursolic acid expressed a significantly high superoxide radical scavenging ability with low IC 50 value of 43.35 ± 0.95 µg/mL, which is lower than both natural and synthetic antioxidant controls, namely, ascorbic acid and BHA, respectively (Table 5).
ujone show the lowest quenching ability on the superoxide radicals with high IC 50 value of 131.78 ± 1.27 µg/mL (Table 6).
e results of the antioxidant potential of thujone revealed that IC 50 values of all radical scavenging activity were two-to threefold higher than the isolated ursolic acid when compared with reference compounds in most of the tested methods. Maximum antiradical activity was found in FRAP method with of EC 50 value of 44.58 ± 0.89 µg/mL. e results of the rest of assays expressed considerable radical scavenging potential of thujone. e present investigation used 5 different assays to assess the antioxidant potential of ursolic acid and thujone. Both ursolic acid and thujone expressed different degree of radical scavenging potential on various tested radicals. e difference in the antioxidant capacity of the tested compounds obtained by the results of FRAP, DPPH radical, hydroxyl radical, nitric oxide radical, and superoxide radical scavenging methods is probably a result of the variation in sensitivity of isolated compounds to the various species of radicals [27]. Moreover, the variation in the reaction media such as lipophilic, hydrophilic, and amphiphilic and nature of radicals and antioxidant molecules have impact on the antiradical potential. For example, DPPH radicals are used to evaluate the antiradical potential of both hydrophilic and hydrophobic antioxidants, whereas nitric oxide assay is used for hydrophilic antioxidants [27]. e difference in the antioxidant potential of ursolic acid and thujone is due to the variation in their structure and functional groups such as free hydroxyl moiety and polarity.
ere are no studies/reports on the antioxidant activity of thujone. However, several reports are present on the antioxidant activity of essential oils from various plants, that is, Salvia officinalis [28], Artemisia herba-alba [29],

BioMed Research International
Artemisia japonica, Artemisia nilagirica [30], and Artemisia absinthium [31], which contains high amount of thujone. Based on the results of the aforementioned studies, thujone exhibited low to moderate antioxidant activities of all tested radicals. Mighri et al. [32] reported that thujone-rich oil from Artemisia herba-alba showed the lower inhibition percentage of antioxidant activity than the positive control (BHA) which strengthens the outcome of the present study. Earlier study on the antioxidant potential of ursolic acid shows similar IC 50 value for the DPPH radicals scavenging activity [33] which supports the findings of the present investigation.

Antiproliferative Activity.
e results of the antiproliferative activity of ursolic acid and thujone expressed notable growth inhibitory effect on both U937 and HL-60 cells in a dose-dependent manner (Table 7). e present study shows ursolic acid harbor higher antiproliferative potential on both U-937 and HL-60 cells than the thujone. Furthermore, the findings of the present investigation revealed that HL-60 cells were more sensitive to ursolic acid than U-937 cells (Figure 1). Ursolic acid possessed a profound inhibitory effect on the proliferation of HL-60 cells (∼77% growth inhibition) with the lowest IC 50 value (26.83 ± 3.07 µmol/mL) followed by U-937 cells (∼80% growth inhibition) with considerable IC 50 value (36.59 ± 0.80 µmol/mL).
ujone shows moderate antiproliferative potential on U-937 cells (∼63% growth inhibition) with a high IC 50 value of 297.42 ± 1.64 µmol/mL (Figure 2). e least cytotoxic activity on HL-60 cell line (∼56% growth inhibition) with a higher IC 50 value of 486.15 ± 2.74 µmol/mL was noticed in thujone. e total number of U937 and HL-60 cells was decreased with increased dose of both ursolic acid and thujone. Distinctive morphological changes in the U937 and HL-60 cells occurred with increasing concentration of ursolic acid and thujone treatment (Figures 1 and 2). Typical apoptotic features such as cell shrinkage and membrane blebbing were found in the U937 and HL-60 cells upon the low-to midand/or high-dose treatment of ursolic acid and thujone [27]. e treatment of ursolic acid and thujone induced the loss of cellular adhesion, echinoid spikes, and blistering. Moreover, the cells are detached from their basal membrane (anoikis) and lost their contact with adjacent cells upon treatment of ursolic acid and thujone at high dose. e increasing concentration of ursolic acid and thujone induced cell death through the necrosis process which, detected with characteristic morphological changes of necrosis in U937 and HL-60 cells such as membrane bubbling, detached from neighbor cells and evaginations [28]. Similarly, morphological changes were reported in the ursolic acid-treated various cancer cells that support the present findings [18][19][20][33][34][35][36].
Ursolic acid is reported to have antiproliferative activity on different types of cancers such as lung, colon, breast, renal, prostate, melanoma, and leukemia [18][19][20][33][34][35][36]. Many reports support the antiproliferative potential of ursolic acid because it might induce the apoptosis process in different mode on various types of cell lines [37,38]. Previously, the antiproliferative activity of ursolic acid on HL-60 cell line was well-reported. It mainly induced apoptosis in HL-60 leukemia cells accompanied by mediating the release of intracellular calcium ions [39], inhibiting DNA synthesis [40] and blocking the cell cycle process [39,41], and inducing Atg5-dependent autophagy [42] and mitochondria dependent apoptosis [19] which supports the findings of the present study. Moreover, many reviews described the mode of action of ursolic acid in the control of cancer cells [18][19][20][21].
Biswas et al. [43] evaluated the antiproliferative and apoptosis-inducing properties of thujone-rich fraction separated from uja occidentalis on melanoma (A375) cell line harbor higher IC 50 (226.18 μg/mL) value. Moreover, the thujone-rich fraction displayed least (∼14%) cytotoxicity on normal peripheral blood mononuclear cell (PBMC) [44] (Biswas et al., 2010). Similarly, Zolotovich et al. [44] reported that thujone exhibits no cytotoxic effect on HeLa cells at high concentrations (100 µg/mL), which supports the outcome of present study. Likewise, Privitera et al. [5] documented that thujone-rich essential oil from Salvia officinalis has no cytotoxic effect on the LNCaP cells in all tested concentrations that strengthen the present findings.

Molecular Docking
Studies. DNA topoisomerases are ubiquitous enzymes that unwind DNA molecule which is  necessary for various DNA dependent biological processes [45]. Generally, two types of topoisomerases are predominantly found in cells, namely, type I and type II. Type I topoisomerases break single strand of duplex DNA that create a gate for the transition of the another DNA strand, whereas type II topoisomerases cut the double strands of DNA and transit another duplex DNA through the gate and then both type I and type II topoisomerases religate the broken DNA strands [45]. Both the type I and type II topoisomerases are involved in the maintenance of the DNA topology. Inhibition of either type I or type II has significant impact on the DNA replication and other biological process. Expression level of topoisomerase II is often elevated in cancer cells [46]. erefore, topoisomerase II is an attractive target for various antitumor and antimicrobial drugs [47]. e clinically successful/approved anticancer drugs which target the topoisomerase II induce topoisomerase II poisoning that leads to the arrest of replication and formation of duplex DNA break as the result cells undergo apoptosis [46]. Previous studies revealed that terpenes and terpenoids are potential inhibitor of topoisomerase II [47,48] and they prefer to bind at the ATPase domain due to the hydrophobic nature of binding site [49]. Ursolic acid and thujone are lipophilic in nature. us, in the present study, topoisomerase II ATPase region was selected as a target.
Ursolic acid and thujone possess a significant binding affinity with topoisomerase II. e H-bond interactions between the ligands and proteins are N-H···O and O-H···O type. Besides the H-bond interactions, van der Waals and hydrophobic interactions with the ATPase region residues of topoisomerase II were noticed. e results of ursolic acid and thujone were compared with the docked pattern of CDX (cocrystallized ligand). e GLIDE energy of ligands, H-bond interactions along with distance, and docking score are given in Table 8. Ursolic acid showed H-bond interactions with the active site r27, Asn142, and Phe362 residues of a single chain of topoisomerase II ( Figure 3) along with other nonbonded interactions with a sustainable docking score (− 4.29) and GLIDE energy (− 37.82 kcal/mol).
Moreover, while ursolic acid docked with both A and B chains displayed H-bond interactions with the Arg77a and r195b residues with notable GLIDE energy (− 51.86 kcal/mol) and docking score (− 8.0312) (Figure 4), thujone expressed H-bond interaction with the Gln365 residue ( Figure 5) when docked with a single chain of topoisomerase II along with hydrophobic interactions with a docking score of − 5.44 and GLIDE energy − 16.96 kcal/mol. While docking, the thujone with two chains of topoisomerase II displayed interactions with Tyr144a and Tyr28b residues ( Figure 6) with a considerable docking score (− 8.18) and GLIDE energy (− 27.38 kcal/mol). e docking results clearly show that ursolic acid is a strong inhibitor of 1QZR and energetically similar to the CDX. Hence, the antiproliferative activity of ursolic acid might be persistent via the findings of molecular docking studies with respect to the inhibition of topoisomerase II.

2.98
GLN-365  BioMed Research International ere are no detailed data available on the docking studies of ursolic acid and thujone with topoisomerase II. Even though earlier reports revealed that ursolic acid has significant inhibition potential on topoisomerase II in in vitro and in vivo assays [50], no detailed data is available on their in silico interactions. Two docking studies report the binding energy of ursolic acid (used as control) with topoisomerase II ATPase site (1QZR) but do not show the interaction between them.
ose studies reported − 21.2 kcal/mol [48] and − 57.7 kJ/mol [51] (equivalent to − 13.79 kcal/mol) as a binding energy of ursolic acid with topoisomerase II which is 2.44-and 3.76-fold higher than the present study, respectively; and the possible reason for the difference in binding energy is variations in the protein preparation and docking platform.
is is the firsthand report of the in silico interactions of ursolic acid and thujone  at the ATPase region of topoisomerase II. e antiproliferative activity of ursolic acid is supported by the docking results which may be achieved due to the inhibition of topoisomerase II.

Conclusion
Overall outcome of this investigation concludes that ursolic acid isolated from M. edule harbors significant antioxidant and antiproliferative potential with low IC 50 values than the thujone isolated from E. indica. HL-60 cells are more sensitive to ursolic acid than the U-936 cells. e outcome of docking results shows that the ursolic acid has a strong affinity with the ATPase active site of topoisomerase II. Ursolic acid might be used as a good molecular template in the discovery of novel and highly potent antioxidant and antiproliferative agents.

Data Availability
No data were used to support this study.

Conflicts of Interest
e authors declare that they do not have any conflicts of interest.