Anti-MRSA Constituents from Ruta Chalepensis (Rutaceae)

molecules Article 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 30,7-Dimethyl ether Shaymaa Al-Majmaie 1, Lutfun Nahar 2,* , M. Mukhlesur Rahman 3 , Sushmita Nath 1, Priyanka Saha 4, Anupam Das Talukdar 5, George P. Sharples 1 and Satyajit D. Sarker 1,* 1 Centre for Natural Products Discovery, School of Pharmacy and Biomolecular Sciences, Liverpool John Moores University, James Parsons Building, Byrom Street, Liverpool L3 3AF, UK; [email protected] (S.A.-M.); [email protected] (S.N.); [email protected] (G.P.S.) 2 Laboratory of Growth Regulators, Institute of Experimental Botany ASCR & Palacký University, Šlechtitel ˚u27, 78371 Olomouc, Czech Republic 3 Medicines Research Group, School of Health, Sport and Bioscience, University of East London, Water Lane, London E15 4LZ, UK; [email protected] 4 Cancer Biology and Inflammatory Disease Division, CSIR-Indian Institute of Chemical Biology, Kolkata, West Bengal 700032, India; [email protected] 5 Department of Life Science and Bioinformatics, Assam University, Silchar, Assam 788011, India; Citation: Al-Majmaie, S.; Nahar, L.; [email protected] or [email protected] Rahman, M.M.; Nath, S.; Saha, P.; * Correspondence: [email protected] (L.N.); [email protected] (S.D.S.); Tel.: +44-(0)-15-1231-2096 (S.D.S.) Talukdar, A.D.; Sharples, G.P.; Sarker, S.D. Anti-MRSA Constituents from Abstract: Ruta chalepensis L. (Rutaceae), a perennial herb with wild and cultivated habitats, is well Ruta chalepensis (Rutaceae) Grown in known for its traditional uses as an anti-inflammatory, analgesic, antipyretic agent, and in the Iraq, and In Silico Studies on Two of treatment of rheumatism, nerve diseases, neuralgia, dropsy, convulsions and mental disorders. The Most Active Compounds, antimicrobial activities of the crude extracts from the fruits, leaves, stem and roots of R. chalepensis Chalepensin and 6-Hydroxy-rutin were initially evaluated against two Gram-positive and two Gram-negative bacterial strains and a 30,7-Dimethyl ether. Molecules 2021, strain of the fungus Candida albicans. Phytochemical investigation afforded 19 compounds, including 26, 1114. https://doi.org/10.3390/ molecules26041114 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) Academic Editors: strains, i.e., ATCC 25923, SA-1199B, XU212, MRSA-274819 and EMRSA-15. The MIC values of the 0 Agnieszka Ludwiczuk and active compounds, chalepin (9), chalepensin (10), rutamarin (11), rutin 3 -methyl ether (14), rutin Yoshinori Asakawa 7,40-dimethyl ether (15), 6-hydroxy-rutin 30,7-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 Received: 27 January 2021 compounds from R. chalepensis, and were active against four out of six tested MRSA strains, and in Accepted: 18 February 2021 silico studies were performed on these compounds. The anti-MRSA activity of compound 16 was Published: 19 February 2021 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 Publisher’s Note: MDPI stays neutral pump. This is the first report on the anti-MRSA property of compounds isolated from R. chalepensis with regard to jurisdictional claims in and relevant in silico studies on the most active compounds. published maps and institutional affil- iations. Keywords: Ruta chalepensis; Rutaceae; chalepensin; rutin; anti-MRSA; in silico Copyright: © 2021 by the authors. 1. Introduction Licensee MDPI, Basel, Switzerland. This article is an open access article Antibiotic resistance is a global public health problem and is most common in de- distributed under the terms and veloping countries [1]. According to the World Health Organization (WHO), every year, conditions of the Creative Commons microbial resistance to antibiotics causes more than 60,000 deaths worldwide, out of which Attribution (CC BY) license (https:// 77% are children [2]. Microorganisms, particularly bacteria, develop resistance to antimi- creativecommons.org/licenses/by/ crobial drugs, mainly because of clinical, cellular and molecular factors. Instances of 4.0/). misuse and over-prescription of antibiotics have become common practices in developing Molecules 2021, 26, 1114. https://doi.org/10.3390/molecules26041114 https://www.mdpi.com/journal/molecules Molecules 2021, 26, 1114 2 of 17 countries [3]. The unlicensed medicine suppliers, uncontrolled antibiotic sales, and avail- ability of over-the-counter antibiotics without a prescription in developing countries, have led to an exponential increase in drug resistance [4]. This dire situation of antimicrobial drug resistance has prompted the search for novel compounds, particularly those from natural sources, with potential antimicrobial properties against various drug-resistant microbial strains. Ruta chalepensis L. (Fam. Rutaceae), commonly known as ‘Fringed rue’, is an Iraqi medicinal plant, endemic to Eurasia and North Africa, and is cultivated elsewhere [5]. This plant is known for its traditional medicinal applications in the treatment of convulsions, dropsy, fever, mental disorders, menstrual problems, microbial infections, neuralgia, rheumatism, and other bleeding and nervous disorders [5–8]. Previous phytochemical studies on this plant demonstrated the presence of alkaloids, anthraquinones, cardiac glycosides, coumarins, flavonoids, saponins, tannins and terpenoids, whereas pharmacological evaluations established its analgesic, anthelmintic, anti-acetylcholinesterase, anticancer, anti-inflammatory, antimicrobial, antioxidant and antiparasitic properties [5]. We have recently reported the isolation, characterization and antimicrobial activity of three flavonol glycosides, including a new one, 6-hydroxy-rutin 30,7-dimethyl ether (16), from the fruits of this plant [5]. However, to the best of our knowledge, no anti-MRSA (methicillin-resistant Staphylococcus aureus) compound was reported from R. chalepensis before, and no in silico study has been conducted on the antimicrobial compounds from this plant. We now report on the isolation, identification, anti-MRSA activity of several compounds from R. chalepensis, collected in Iraq, against a panel of MRSA strains including, ATCC25923 (a standard laboratory strain sensitive to antibiotics like tetracycline), SA1199B, XU212, MRSA-274819 and EMRSA15, and also in silico studies on the two most active anti-MRSA compounds from this plant, chalepensin (10) and 6-hydroxy-rutin 30,7-dimethyl ether (16). 2. Results and Discussion Leaves, stem, fruits and roots of R. chalepensis, collected from Iraq, were Soxhlet ex- tracted 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 (Es- cherichia 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 (Table1). 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 (Table1 ). 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 [9–12]. 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. Molecules 2021, 26, 1114 3 of 17 Table 1. Antimicrobial activity of the n-hexane, DCM and MeOH extracts of various plant parts (leaves, stem, fruits and roots) of Ruta chalepensis *. Plant Parts (MICs in mg/mL) Bacteria and Fungi Extract Leaves Stem Fruits Roots n−Hexane 6.25 × 10−1 6.25 × 10−1 3.12 × 10−1 N/A E. coli DCM 3.12 × 10−1 6.25 × 10−1 3.12 × 10−1 N/A Gram-Negative Methanol 6.25 × 10−1 3.12 × 10−1 6.25 × 10−1 5 Bacteria n−Hexane 6.25 × 10−1 6.25 × 10−1 3.12 × 10−1 N/A P. aeruginosa DCM 3.12 × 10−1 6.25 × 10−1 6.25 × 10−1 N/A Methanol 6.25 × 10−1 3.12 × 10−1 1.56 × 10−1 2.5 n−Hexane 1.56 × 10−1 6.25 × 10−1 3.12 × 10−1 N/A M. luteus DCM 1.56 × 10−1 6.25 × 10−1 7.81 × 10−2 N/A Gram-Positive Methanol 1.95 × 10−2 7.81 × 10−2 3.90 × 10−2 1.25 Bacteria n−Hexane 6.25 × 10−1 6.25 × 10−1 6.25 × 10−1 N/A S. aureus DCM 6.25 × 10−1 6.25 × 10−1 6.25 × 10−1 N/A Methanol 3.12 × 10−1 3.12 × 10−1 3.12 × 10−1 5 n−Hexane 6.25 × 10−1 1.95 × 10−1 1.56 × 10−2 N/A Pathogenic Fungi C. albicans DCM 3.12 × 10−1 3.12 × 10−1 3.90 × 10−2 N/A Methanol 1.95 × 10−2 7.81 × 10−2 3.90 × 10−2 2.5 * Positive controls used were ciprofloxacin against bacterial strains and nystatin against the fungal strain, and the MIC values are shown in Table2. Table

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