Anti-MRSA Constituents from Ruta chalepensis (Rutaceae) Grown in Iraq, and In Silico Studies on Two of Most Active Compounds, Chalepensin and 6-Hydroxy-rutin 3′,7-Dimethyl ether

Ruta chalepensis L. (Rutaceae), a perennial herb with wild and cultivated habitats, is well known for its traditional uses as an anti-inflammatory, analgesic, antipyretic agent, and in the treatment of rheumatism, nerve diseases, neuralgia, dropsy, convulsions and mental disorders. The antimicrobial activities of the crude extracts from the fruits, leaves, stem and roots of R. chalepensis were initially evaluated against two Gram-positive and two Gram-negative bacterial strains and a strain of the fungus Candida albicans. Phytochemical investigation afforded 19 compounds, including alkaloids, coumarins, flavonoid glycosides, a cinnamic acid derivative and a long-chain alkane. These compounds were tested against a panel of methicillin-resistant Staphylococcus aureus (MRSA) strains, i.e., ATCC 25923, SA-1199B, XU212, MRSA-274819 and EMRSA-15. The MIC values of the active compounds, chalepin (9), chalepensin (10), rutamarin (11), rutin 3′-methyl ether (14), rutin 7,4′-dimethyl ether (15), 6-hydroxy-rutin 3′,7-dimethyl ether (16) and arborinine (18) were in the range of 32–128 µg/mL against the tested MRSA strains. Compounds 10 and 16 were the most active compounds from R. chalepensis, and were active against four out of six tested MRSA strains, and in silico studies were performed on these compounds. The anti-MRSA activity of compound 16 was comparable to that of the positive control norfloxacin (MICs 32 vs 16 μg/mL, respectively) against the MRSA strain XU212, which is a Kuwaiti hospital isolate that possesses the TetK tetracycline efflux pump. This is the first report on the anti-MRSA property of compounds isolated from R. chalepensis and relevant in silico studies on the most active compounds.


Introduction
Antibiotic resistance is a global public health problem and is most common in developing countries [1]. According to the World Health Organization (WHO), every year, microbial resistance to antibiotics causes more than 60,000 deaths worldwide, out of which 77% are children [2]. Microorganisms, particularly bacteria, develop resistance to antimicrobial drugs, mainly because of clinical, cellular and molecular factors. Instances of misuse and over-prescription of antibiotics have become common practices in developing

Results and Discussion
Leaves, stem, fruits and roots of R. chalepensis, collected from Iraq, were Soxhlet extracted separately, but sequentially with n-hexane, dichloromethane (DCM) and methanol (MeOH), followed by screening for antimicrobial activity against two Gram-positive (Staphylococcus aureus and Micrococcus luteus), two Gram-positive bacterial strains (Escherichia coli and Pseudomonas aeruginosa) and one strain of the fungus Candida albicans. All three extracts from leaves, stems and fruits of R. chalepensis revealed low to moderate levels of antimicrobial activities against all the tested organisms with varied minimum inhibitory concentration (MIC) values (Table 1). However, in the case of the roots, only the MeOH extract showed activity (MIC 1.25-5 mg/mL) against the test bacteria and fungus (Table 1). Following the initial antimicrobial screening of the crude extracts of R. chalepensis, the MICs of 6.25 × 10 −1 mg/mL was chosen as the minimum threshold of activity for any extract for further analysis, leading to the isolation of compounds responsible for their antimicrobial activity. Many previous studies on R. chalepensis documented its antimicrobial activity using different plant parts and methods [9][10][11][12]. However, there is no report on the antimicrobial activity studies of R. chalepensis using the modified microtitre assay, or against MRSA strains. Additionally, no in silico studies have ever been conducted on anti-MRSA compounds present in this plant.
Thirteen of the 19 isolated compounds from R. chalepensis were tested for activity against six MRSA strains ( Table 3). The results revealed significant anti-MRSA activity of most of these compounds against the tested strains with different MIC values (64-256 µg/mL). However, γ-fagarine (1), bergapten (6), isopimpineline (7) and graveoline (12) did not show any activity against any of the tested MRSA strains (Table 3). Kokusaginine (3) and rutin (13) were found to be active, albeit at a high concentration (256 µg/mL), only against the MRSA strain MRSA274819, but were inactive against the other five MRSA strains. While chalepin (9), chalepensin (10) and rutamarin (11) are all prenylated furanocoumarin derivatives, they caused different levels of inhibitions because of subtle structural differences. The order of anti-MRSA, the potency of these compounds was 10 > 11 > 9. Functional groups were the main differences among these three compounds contributing to their differences in lipophilicity. All three compounds are 3-substituted furanocoumarins, among which, except for 10, the other two compounds are dihydrofuranocoumarins. Rutamarin (11), which is simply an acetylated product of chalepin (9) was more active than 9, presumably because of more lipophilicity caused by acetylation. As two other furanocoumarins, bergapten (6) and isopimpineline (7) were inactive, and none of them has any prenylation at C-3 of the coumarin nucleus like in compounds 9-11, it appears that 3-prenylation is another key determinant of anti-MRSA activity.  6-Hydroxy-rutin 3 -7-dimethyl ether (16), rutin (13) and rutin 3 -methyl ether (14) are flavonoid glycosides that only have differences in the presence/absence and in the number of methyl ether groups in them, offering varying degrees of lipophilicity. Rutin (13) does not contain an OMe group, while compounds 14 has an OMe group on 3 position, and 16 has two OMe groups at positions 3 and 7. In addition, in 14, a hydroxyl group occupies position 6. The highest anti-MRSA potency of compound 16 may be because of the different functional groups and their unique positions that make this compound the most lipophilic among these three compounds. The order of anti-MRSA potency in these compounds was 16 > 14 > 13. This order was also observed in their antimicrobial activity against other test organisms [5]. It is noteworthy that the anti-MRSA activity of compound 16 was quite comparable to that of the positive control norfloxacin (MICs 32 vs. 16 g/mL, respectively). Compound 18 is an acridone alkaloid containing three methyl groups, two of which are oxygenated. This compound has been reported to have many pharmaceutical applications, such as antimicrobial, antiviral, antiplasmodial, antimalarial and anticancer agents, and not surprisingly, as an anti-MRSA agent [33][34][35]. This is the first report on the evaluation of the anti-MRSA effect of isolated compounds, from R. chalepensis, against several MRSA strains.
Chalepensin (10) and 6-hydroxy-rutin 3 ,7-dimethyl ether (16), being the most active anti-MRSA compounds found in R. chalepensis in the present study, were subjected to in silico studies to have an understanding of to what extent these compounds (10 and 16) are able to bind to MRSA proteins, and also their drug−like physicochemical characters. The structures of these compounds were optimized ( Figure 2) using the Schrodinger suite platform 7.0. [36,37] as the optimization is essential for better understanding interaction patterns and their degree of bonding during the associations. Figures 3-6 show the bonded ligands (10 and 16) resulting from hydrogen bonding interaction with the target molecules of MRSA, and potential target protein structures of MRSA. To study various bioactivity predictions of the compounds (10 and 16), the Circos modelling study was performed with all the functional targeted proteins taken into consideration and the therapeutic potential of the compounds were predicted by the Circos associated prediction model 3.0.1. (Figures 6 and 7) [38]. It can be mentioned here that Circos is a software package for visualizing data and information, and it visualizes data in a circular layout, which makes Circos ideal for exploring relationships between objects or positions. Besides, a circular layout is advantageous, not least being the fact that it is attractive. Associated programming was performed by the Hex server [39]. The results were built up and visualized using the Python server 7.0.1 and the Python with the modeling package. The results of both were screened for the MRSA strains and target proteins like the integrase, penicillin binding proteins (PBPs) [40], pyruvate kinase, and tail−anchored proteins (TaPs) with their specified active site.
Chalepensin (10) and 6-hydroxy-rutin 3′,7-dimethyl ether (16), being the mos anti−MRSA compounds found in R. chalepensis in the present study, were subjecte silico studies to have an understanding of to what extent these compounds (10 and able to bind to MRSA proteins, and also their drug−like physicochemical characte structures of these compounds were optimized (Figure 2) using the Schrodinger sui form 7.0. [36,37] as the optimization is essential for better understanding interacti terns and their degree of bonding during the associations. Figures 3-6 show the b ligands (10 and 16) resulting from hydrogen bonding interaction with the target mo of MRSA, and potential target protein structures of MRSA. To study various bioa predictions of the compounds (10 and 16), the Circos modelling study was perf with all the functional targeted proteins taken into consideration and the therapeu tential of the compounds were predicted by the Circos associated prediction mode ( Figures 6 and 7) [38]. It can be mentioned here that Circos is a software package fo alizing data and information, and it visualizes data in a circular layout, which mak cos ideal for exploring relationships between objects or positions. Besides, a circu out is advantageous, not least being the fact that it is attractive. Associated progra was performed by the Hex server [39]. The results were built up and visualized us Python server 7.0.1 and the Python with the modeling package. The results of bot screened for the MRSA strains and target proteins like the integrase, penicillin b proteins (PBPs) [40], pyruvate kinase, and tail−anchored proteins (TaPs) with thei fied active site.           In Circos modelling, chalepensin (10) demonstrated its predicted bioactivity implicating translational regulation, transportation of the small molecules, membrane proteins and energy metabolism. The important association and activity could deduce its potency as an inflammatory agent ( Figure 6). However, the Circos modelling with 6-hydroxy-rutin 3′,7-dimethyl ether (16) predicted bioactivity, implicating its chemotaxis role, associated with the DNA replication, modulators of the putative enzymes, membrane proteins and energy metabolism. The important association and activity could deduce its potency as an antireplicative agent and its possible role in the RNA processing and amino−acid biosynthesis. The PASS (Prediction of Activity Apectra for Substances) prediction analysis [41] of compounds 10 and 16 revealed their potency in interacting with various enzymes, associated with various bioactivities as well as potential adverse effects and toxicities (Table 4). It was found that compound 10 could potentially generate itchiness and eye irritation, while compound 16 could trigger metabolic acidosis.  (16) with its predicted bioactivity implicating chemotaxis role, associated with the DNA replication, modulators of the putative enzymes, membrane proteins and energy metabolism. The important association and the activity deduce its potency as antireplicative property and its role in the RNA processing and amino-acid biosynthesis. Different colors indicate various pivotal cellular processes shown by different letters and different gradients explain the degree of the interrelated network.
In Circos modelling, chalepensin (10) demonstrated its predicted bioactivity implicating translational regulation, transportation of the small molecules, membrane proteins and energy metabolism. The important association and activity could deduce its potency as an inflammatory agent ( Figure 6). However, the Circos modelling with 6-hydroxy-rutin 3 ,7-dimethyl ether (16) predicted bioactivity, implicating its chemotaxis role, associated with the DNA replication, modulators of the putative enzymes, membrane proteins and energy metabolism. The important association and activity could deduce its potency as an antireplicative agent and its possible role in the RNA processing and amino-acid biosynthesis. The PASS (Prediction of Activity Apectra for Substances) prediction analysis [41] of compounds 10 and 16 revealed their potency in interacting with various enzymes, associated with various bioactivities as well as potential adverse effects and toxicities (Table 4).

Bioactivities
Pa (Probability to be Active) Pi (Probability to be Inactive) 10  Molecular docking interaction studies on two anti-MRSA compounds, chalepensin (10) and 6-hydroxy-rutin 3 ,7-dimethyl ether (16), against MRSA target proteins revealed their interactions at various levels with integrase, tail−anchored proteins (TaPs), penicillin binding proteins (PBPs) and pyruvate kinase (Table 5), and their hydrogen bonding abilities with those proteins (Table 6). Chalepensin (10) was found to possess significant docking ability with PBPs with an e-score of −21.6229 (Figure 8), while that of compound 16 was −9.3219. However, 6-hydroxy-rutin 3 ,7-dimethyl ether (16) docked better with integrase (−17.331) than compound 10 (−8.933). Both compounds (10 and 16) could potentially bind with tail−anchored proteins (TaPs) and pyruvate kinase to similar extents (Table 5).  The active interactive residues include GLN-216 having hydrogen bonding with LYS-218. These amino acid residues mark the integrity of the PBPs and masking of these key residues can offer inactivation of PBPs that are responsible for the ineffectiveness of the strain. It can be noted that the integrase protein is responsible for the efflux of the drug and inactivation of this protein results in inhibition by targeting the residues ASP-111 and ARG-168.
While compound 10 was predicted to be a good blood-brain-barrier (BBB) permeant and CYP2D6 inhibitor, compound 16 was not (Table 7). It can be noted that BBB is one of the parameters that are assessed in in silico studies on potential drug molecules to have better understanding of their pharmacology as well as probable toxicity to the brain. However, in the context of anti-MRSA activity, BBB may not be that important, but could be relevant to any probable toxicity of these compounds towards the brain. While compound 10 was predicted to be a good blood-brain-barrier (BBB) permeant and CYP2D6 inhibitor, compound 16 was not (Table 7). It can be noted that BBB is one of the parameters that are assessed in in silico studies on potential drug molecules to have better understanding of their pharmacology as well as probable toxicity to the brain. However, in the context of anti-MRSA activity, BBB may not be that important, but could be relevant to any probable toxicity of these compounds towards the brain.
Both compounds should have high gastrointestinal (GI) absorption and no violation of the Lipinski rule, which states that an orally active drug has no more than one violation of the following criteria: no more than five hydrogen bond donors (the total number of nitrogen-hydrogen and oxygen-hydrogen bonds), no more than 10 hydrogen bond acceptors (all nitrogen or oxygen atoms), a molecular mass <500 Daltons and an octanolwater partition coefficient that does not exceed five. From the in silico studies with anti-MRSA compounds 10 and 16, it was apparent that these compounds could bind with certain MRSA protein targets, predominantly through hydrogen bonding, as well as van de  Table 7. Interaction of chalepensin (10) and 6-hydroxy-rutin 3 ,7-dimethyl ether (16) with CYPs, solubility and gastrointestinal (GI) absorption, as determined by the Silicos-IT chemoinformatic software (silicos-it.be.s3-website-eu-west-1. amazonaws.com, accessed on 7 February 2021). Both compounds should have high gastrointestinal (GI) absorption and no violation of the Lipinski rule, which states that an orally active drug has no more than one violation of the following criteria: no more than five hydrogen bond donors (the total number of nitrogen-hydrogen and oxygen-hydrogen bonds), no more than 10 hydrogen bond acceptors (all nitrogen or oxygen atoms), a molecular mass <500 Daltons and an octanolwater partition coefficient that does not exceed five. From the in silico studies with anti-MRSA compounds 10 and 16, it was apparent that these compounds could bind with certain MRSA protein targets, predominantly through hydrogen bonding, as well as van de Waals forces. It was also apparent that these compounds could possess various other bioactivities, as listed in Table 4, with minimum side effects or adverse reactions.

Plant Materials
Leaves, stem bark, fruits and roots of R. chalepensis L. were collected from Diyala, Central Iraq (N 33.79684 E 44.623337) during September 2015, air-dried at room temperature, and ground to a fine powder using a coffee grinder. A voucher specimen (No. 33396) for this collection was deposited at the National Herbarium in Iraq.

Extraction
The air-dried ground fruits (103 g), leaves (98 g), stems (81 g) and roots (110 g) of R. chalepensis were extracted separately and sequentially with n-hexane, dichloromethane (DCM) and methanol (MeOH) (Loughborough, UK) using a Soxhlet apparatus (900 mL, ten cycles each). The crude extracts were concentrated to dryness using a rotary evaporator and stored at 4 • C for further work. Only the MeOH extract showed antimicrobial activity in the initial in vitro antimicrobial screening using resazurin as an indicator of cell growth [5,32], and was subjected to further fractionation, leading to the isolation of antimicrobial compounds.
All bacterial and fungal strains were cultured on nutrient agar (Oxoid), followed by incubation at 37 • C for 24 h prior to MIC determination using the resazurin assay. Ciprofloxacin was used as a positive control for bacterial strains, and nystatin for C. albicans. Resazurin solution, prepared by dissolving 4 mg of resazurin in 20 mL of sterile distilled water, was used in this assay as an indicator of cell growth. The antimicrobial method used during the study was as described by Reference [32].
Briefly, plates were prepared under aseptic conditions. A sterile 96-well plate was labelled. A volume of 100 µL of test material in 10% (v/v) DMSO (10 mg/mL for crude extracts) was pipetted into the first row of the plate. To all other wells, 50 µL of normal saline was added. Serial dilutions were performed using a multichannel pipette. Tips were discarded after use, such that each well had 50 µL of the test material in serially descending concentrations. Nutrient broth and 10 µL of resazurin indicator solution was added to each 30 µL well. Finally, 10 µL of bacterial suspension (5 × 10 5 cfu/mL) was added to each well. Each plate was wrapped loosely with cling film to ensure that bacteria did not become dehydrated. Each plate had a set of controls: a column with a broad-spectrum antibiotic as a positive control (usually ciprofloxacin in serial dilution), a column with all solutions with the exception of the test compound, and a column with all solutions with the exception of the bacterial solution adding 10 µL of nutrient broth instead. The plates were prepared in triplicates and placed in an incubator set at 37 • C for 18-24 h. The color change was then assessed visually. Any color changes from purple to pink was recorded as positive. The lowest concentration at which color change occurred was taken as the MIC value. The average of three values was calculated and that was the MIC for the test material and bacterial strain.

Solid-Phase Extraction (SPE)
A portion (2 g) of the active MeOH extract of each plant part was subjected to SPE on a Strata C 18 reversed-phase cartridge (20 g, Phenomenex, Macclesfield, UK), eluted with a step-gradient using water-MeOH mixture of decreasing polarity, water:MeOH 80:20, 50:50, 20:80 and 0:100 (200 mL each), to obtain four SPE fractions I-IV, respectively. All SPE fractions were dried using a rotary evaporator followed by freeze-drying and stored in sealed vials in a fridge at 4 • C for further work.

Assessment of Anti-MRSA Activity
All chemicals for the anti-MRSA assay was purchased from Sigma-Aldrich (Gillingham, UK), Cation-adjusted Mueller-Hinton broth was sourced from Oxoid Microbiology Products, UK, and was adjusted to have 20 and 10 mg/L of Ca 2+ and Mg 2+ ions, respectively. The Staphylococcus aureus strains used in this study were ATCC25923 (a standard laboratory strain sensitive to antibiotics like tetracycline), SA1199B, XU212, MRSA-274819, MRSA340702 and EMRSA15 [9]. SA1199B overexpresses the NorA MDR efflux pump [42] and XU212 is a Kuwaiti hospital isolate that is a MRSA strain possessing the TetK tetracycline efflux pump [9], whereas the EMRSA 15 strain [43] was epidemic in the UK. All these were obtained from the National Collection of Type Cultures (NCTC). The assay protocol was exactly as described by Nurunnabi et al. [44]. Norfloxacin, a well-known antibiotic, was used as the positive control.
Briefly, an inoculum density of 5 × 10 5 colony-forming units of each bacterial strain was prepared in normal saline (9 g/L) by comparison to a 0.5 MacFarland turbidity standard. The inoculum (125 µL) was added to all wells, and the microtitre plate was incubated at 37 • C for the corresponding incubation time. For MIC determination, 20 µL of a 5 mg/mL methanolic solution of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) was added to each of the wells and incubated for 20 min. Bacterial growth was indicated by a color change. The minimum inhibitory concentrations (MICs) were determined using the broth microdilution method according to National Committee for Clinical Laboratory Standards with modification using nutrient broth as the medium.
3.9. In Silico Studies with Two Most Active Anti-MRSA Compounds from This Plant, Chalepensin (10) and 6-Hydroxy-rutin 3 ,7-dimethyl Ether (16) In silico studies with two most active anti-MRSA compounds from this plant, chalepensin (10) and 6-hydroxy-rutin 3 ,7-dimethyl ether (16), were conducted using a variety of methods and protocols, as described in the Results and Discussion Sections earlier. Briefly, the structures of these compounds were optimized using the Schrodinger suite platform 7.0. (Schrodinger, Cambridge, UK) [36,37]. To study the various bioactivity prediction of the compounds (10 and 16), the Circos modelling study was performed with all the targeted proteins taken into consideration and the therapeutic potential of the compound were predicted by the Circos associated prediction modelling 3.0.1. (Echelon Innovation Centre, Vancouver, Canada) [38]. Associated programming was carried out with the Hex server [39]. The results were built up and visualized using the Python server 7.0.1 and the Python with the modeling package. The PASS prediction [41] analysis was used to predict the potency of these compounds in interacting with various enzymes, associated with various bioactivities as well as potential adverse effects and toxicities.

Conclusions
The present work generated the first comprehensive phytochemical report on the analysis of Iraqi R. chalepensis species, along with their antimicrobial activity using the modified microtitre assay. This is also the first report on the antibacterial activity of the compounds isolated from R. chalepensis against a panel of methicillin-resistant Staphylococcus aureus (MRSA). The outcome of this study demonstrated that at least seven of the isolated compounds from various parts of R. chalepensis possess reasonable anti-MRSA property. Among the active compounds, chalepensin (10), and 6-hydroxy-rutin 3 ,7-dimethyl ether (16) appear to be the most active compounds from R. chalepensis and are active against four out of the six tested MRSA strains. In silico studies on compounds 10 and 16 revealed that both compounds should have high GI absorption and no violation of the Lipinski rules, meaning 'drug-like' characters in these compounds, and it was also apparent that these compounds could bind with certain MRSA protein targets, predominantly through hydrogen bonding as well as van de Waals forces. Based on the current findings, it can be assumed that these two compounds might be utilized as structural templates for generating structural analogues and developing potential anti-MRSA therapeutic agents.