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In Vitro Antibacterial Activity of Citrus limon (L.) Burm against Gentamicin-Resistant Escherichia coli Complemented with in Silico Molecular Docking of Its Major Phytochemicals with Ribosome Recycling Factor

Sakar Emad Ali, Isaac Karimi*, Nasser Karimi, Khosrow Chehri*

Department of Biology,Faculty of Science,  Razi University 67149-67346, Kermanshah, Iran (Islamic Republic of)

CitationCitation COPIED

Ali SE, Chehri K, Karimi N, Karimi I. In Vitro Antibacterial Activity of Citrus limon (L.) Burm against Gentamicin-Resistant Escherichia coli Complemented with in Silico Molecular Docking of Its Major Phytochemicals with Ribosome Recycling Factor. Basic Appl Pharm Pharmacol. 2017 Dec; 1:101

Copyright: © 2017 Ali SE, et al. This is an openaccess article distributed under the terms of the Creative Commons Attribution 4.0 international License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Abstract

Objectives: Due to adverse effects of classic antibiotics and accrual emergence of antibiotic-resistant bacteria, seeking alternative therapeutics would be a necessity. This study was first aimed to investigate in vitro antibacterial activity of hydro-alcoholic extracts derived from peels of Citrus limon (L.) Burm (CL) as anecdotally reported in (Kurdish) ethnomedicine. Then, putative mechanisms which may mediate this antibacterial effect were computationally elucidated. 

Materials and Methods: Antimicrobial activity was determined against strains E. coli ATTCC 25922. Minimum Inhibitory Concentrations (MIC) and Minimum Bactericidal Concentration (MBC) were determined. Molecular docking was performed for major phytocompounds reported in aqueous or hydro-alcoholic extract of peel of CL against Ribosome Recycling Factor (RRF) of E. coli. 

Results: Based on in vitro antibacterial assay, E. coli was resistant to gentamicin while CL showed MIC (32 ± 27 \[\mu g.ml^{-1}\]) and MBC (682 ± 295\[\mu g.ml^{-1}\]) against E. coli. In silico results reported here focused only phytocompounds with binding affinity less than -6 kcal.mol-1 against RRF to screen more antibacterial promising agents. In this regard, the binding affinities of alpha-carotene (-6.7), apigenin (-10.2), beta-carotene (-10.0), betacryptoxanthin (-8.0), d-limonene (-7.6), citral (-7.5), eriocitrin (-12.1), alpha-pinene (-7.5), lycopene (-10.5), and hesperidin (-13.3) with RRF were comparable to gentamicin (-12.6 kcal.\[mol^{-1}\]).

Conclusions: Hesperidin and eriocitrin showed more promising binding affinity with RRF and can be considered as candidate lead molecules for future studies. In sum, our results showed that hydro-alcoholic extract of peel of CL could be a candidate for designing commercial phytobiotics if in-depth antibacterial assays employed in future studies.

Keywords

Citrus limon (L.) Burm; E. coli; In Vitro Antibacterial Assay; In Silico Molecular Docking; Ribosome Recycling Factor

Introduction

Plants have two sets of metabolites, primary and secondary, which are important for their life [1]. Secondary metabolite such as sterols, phenolics, steroids, lignins, and tannins are substances that their presences are not obligatory for growth of plants, but necessary for protecting against (a ) biotic stresses. For instance, secondary metabolites are able to kill or suppress growth of microbes [2].

Medicinal herbs are a group of plants that could be used for drug development due to the presence of secondary metabolites, phytochemicals that are beneficial to prevent diseases and to eliminate pathogenic germs in animals and human [3]. Phytochemicals are able to prohibit peptidoglycan installation, alter bacterial membrane surface hydrophobicity, destruct microbial membrane framework, and as well as modulate bacterial quorum sensing [4]. 

Antibacterial phytochemicals (phytobiotics) and antibiotics inhibit or kill pathogenic bacteria via inhibiting proteins or genes that are essential for the life of bacteria. For example, ribosome recycling is responsible for translating genetic information carried by mRNAs into specific sequences of amino acids and may increase the efficiency of translation by recycling ribosomes from one round of translation to another [5]. In this context, gentamicin is a bactericidal antibiotic that works by irreversibly binding the 30S subunit of the bacterial ribosome, interrupting protein synthesis, this mechanism of action is similar to other aminoglycosides, however we encounter to the emergence of gentamicin resistance bacteria nowadays [6]. 

In this regard, Citrus limon (L.) Burm (CL), commonly known as lemon, is an industrial and medicinal plant belongs to the Rutaceae family [7]. Its specific phytochemical screening revealed presence of alkaloids, tannins, fixed oils, cardiac glycosides, steroids, phenols, flavonoids, carotenoid, pectin, hesperidin, quercetin, lutein, zeaxanthin, β-cryptoxanthin and β-carotene [8]. In vitro antibacterial role of peel of CL is mediated through splitting lipids of bacterial the cell membrane and thus breaching the cell structure and making it more permeable [9]. In this continuum, aqueous extracts of peel of CL showed highest antibacterial activity against gram-positive and gram-negative bacteria including Staphylococcus aureus, Streptococcus pyogenes, Enterococcus faecalis, Streptococcus pneumonia and Pseudomonas aeruginosa [10].

Based on (Kurdish) ethnomedicine, this study was aimed firstly to examine in vitro antibacterial activity of the peel of CL against Escherichia coli which causes human and animal diseases and secondly to decipher in silico docking of its major reported phytochemicals (Figure 1) against ribosome recycling factor as a vital protein of E. coli. 

Figure 1: The phytochemicals that have been reported in the peels of Citrus limon (L.) Burm.

Materials and Methods

Plant preparation

The lemons were collected from different locations in Iran and Iraq since March to October 2016. The collected specie was authenticated by botanist, third author. The peel of CL was air-dried and finely powdered using a blender. To prepare hydro-alcoholic extract, 10 g of powdered peels were extracted twice with 100 ml of 70 % ethanol for 48 h at room temperature. The extracted suspensions were filtered and resulting filtrates were concentrated to complete dryness using a rotary evaporator and then store at −20 °C until further use. For the antibacterial activity assays, the extract was dissolved in Dimethyl Sulfoxide (DMSO) and store at 4 °C as stock solutions. 

In vitro antibacterial assay

We used gentamicin to assay the effects of antibiotics on the strains E. coli (ATTCC® 25922TM) at 5 µg.ml-1 of the antibiotics for each well E. coli was cultured on Mueller Hinton Agar (MHA) and Mueller Hinton Broth (MHB) media. The antibacterial activity of peel of CL on E. coli was measured by Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) methods based on broth microdilution method [11]. Serial dilutions of the extract were prepared in a 96-well micro-titer plate. To each well, 100 μl of indicator solution that prepared by dissolving a 10 mg extract in 1 ml of DMSO and 100 μl of MHB were added. Finally, 50 μl of bacterial suspension (106 colony forming unite (CFU).ml-1) were added to each well to achieve a concentration of 104 CFU.ml-1. The plates were wrapped loosely with cling film to ensure that bacteria did not get dehydrated. The plated were prepared in triplicates, and then were incubated at 37°C for 18-24 hours. 

In silico antibacterial assay

Simulations of the docking between bacterial target protein and reported bioactive compounds of CL were successfully performed using PyRx software (ver. 0.8) and a docking program (VINA WIZARD) using default parameters as described previously [12]. For molecular docking, crystal structures of target proteins were obtained from the Protein Data Bank (PDB) at the Research Collaboratory for Structural Bioinformatics (http://www.RCSB.org). The PDB format of target protein have been edited, optimized and trimmed in Molegro Virtual Docker and Chimera 1.8.1 (http://www. rbvi.ucsf.edu/chimera) before submission to PyRx. The structures of the major phytocompounds of CL were retrieved from ZINC database ver. 12.0 (http://zinc.docking.org/) or DrugBank ver. 5.0 (https:// www.drugbank.ca/; Figure 1). 

After completion of docking procedure, results had shown as binding affinity (kcal.mol-1) values. More negative the binding affinity means better the orientation of the ligand in the binding site. The selected conformer of ligand has been combined with target protein in Molegro Virtual Docker [13] or Chimera 1.8.1 (http://www.rbvi. ucsf.edu/chimera) and their graphical interface has been analyzed with LigPlot+ software [14].

Results and Discussion

 High incidence of nosocomial diseases due to antibiotic resistance, high prices of synthetic antibiotics and hard access to prepare synthetic antibiotics in poor countries lead to overuse of traditional remedies to treat diseases. In this essence, reverse pharmacognosy help scientists to discover natural bioactive compounds which are drug or drug-like candidates. Numerous naturally-occurring components are found in medicinal plant that owing to their antimicrobial properties could be applied as a valuable source for antimicrobial medicines [15].

E. coli causes various diseases of gastrointestinal tract in human and other animals [16]. Strains of E. coli ATTCC 25922 used in this study was resistant to gentamicin. Gentamicin is a bactericidal, massively used antibiotic that cause mRNA decoding mistake, prevent transfer RNA and mRNA translocation, and block ribosome recycling in bacteria [17]. Our results showed that this strain of E. coli ATTCC 25922 was resistant to gentamicin while sensitive to CL. In sum, peel of CL has inhibitory at MIC 32 ± 27 (\[\mu g.ml^{-1}\]) and bactericidal effects at MBC 682 ± 295 (\[\mu g.ml^{-1}\]) on E. coli. In this regard, Fisher and colleagues studied phytochemicals of the CL peel and their antimicrobial activities and reported that the essential oil of peel of CL had good antimicrobial activity against E. coli [18].

In this study, we selected gentamicin as reference antibiotic to compare its in silico affinity with Ribosome Recycling Factor (RRF; PDB code 1WIH) with respect to ligands of peel of CL to decipher which phytocompounds may involve in anti-E coli effects of CL (Table 1 & Figure 2). In this study, we selected RRF as target protein since it is product of the frr gene in E. coli and are responsible for dissociation of ribosomes from mRNA after the termination of translation and recyclizes ribosomes [19].

Based on docking results, gentamicin showed negative binding affinity with RRF at -12.6 (\[kcal.mol^{-1}\]; Table 1) and docked via hydrophobic interactions and hydrogen bonds (Figure 2). In silico findings showed that among phytochemicals found in peel of CL, hesperidin has been docked with lower binding affinity with RRF with respect to gentamicin.

Hesperidin is a flavanone glycoside found in citrus fruits and its aglycone form is called hesperetin. Hesperidin was first isolated from albedo of citrus peels [20]. Hesperidin showed the lowest negative binding affinity against RRF among in silico studied phytochemicals of CL. It docked with RRF using both hydrophobic interactions and hydrogen bonds. Hesperidin showed in vivo and in vitro antimicrobial activities against Aeromonas hydrophila and E. coli, respectively and its bioactive activities are reviewed previously [21-23]. Its pharmacological activities promise its potential to be considered as a drug-like compound.

Eriocitrin, eriodictyol 7-O-rutinoside, is a flavanone-7-Oglycoside which commonly found in lemons [24]. Eriocitrin has been also docked with RRF with very acceptable binding affinity which was in the second rank after hesperidin (Table 1). Similar to its congener, hesperidin, eriocitrin docked with RRF through hydrogen binding and hydrophobic interactions (Figure 2). The antibacterial effects of eriocitrin against gram-positive and gram-negative bacteria have been reported previously [21,25].



Figure 2: The molecular docking of phytochemicals of Citrus limon (L.) Burm (in yellow) against ribosome recycling factor (PDB 1WIH; in cyan)

a) Alpha-carotene Corresponding atoms and non-ligand residues IIe11, Leu8 involved in hydrophobic interactions.

b) Apigenin Corresponding atoms and non-ligand residues His10 involved in hydrophobic interaction. Hydrogen bonds between apigenin and Thr12 and Gly58 shown by dotted red lines.

c) Alpha-pinene Corresponding atoms and non-ligand residues IIe11 and Thr12 involved in hydrophobic interactions.

d) Beta-carotene Corresponding atoms and non-ligand residues Ser5, Ser6, His10, Gly4 and Gly7 and Asp9 involved in hydrophobic interactions.

e) Beta-cryptoxanthin Corresponding atoms and non-ligand residues His10, Gly4 and Gly7 and Asp9 involved in hydrophobic interactions.

f) Citral Corresponding atoms and non-ligand residues IIe11 and Thr12 involved in hydrophobic interactions.

g) D-limonene Corresponding atoms and non-ligand residues IIe11 and Thr12 involved in hydrophobic interactions.

h) Eriocitrin Corresponding atoms and non-ligand residues IIe11and Ser6 involved in hydrophobic interactions. Hydrogen bonds between eriocitrin and Gly7, Gly4, Glu56, Ser3, Ser5 and Thr12 shown by dotted red lines.

i) Hesperidin Corresponding atoms and non-ligand residues Pro75, Asn60, Leu61, Lys76, Val35 and Met59 involved in hydrophobic interactions. Hydrogen bonds between hesperidin and Gln34, Arg71, IIe74, Asn62 and Pro73 shown by dotted red lines.

j) Lycopene Corresponding atoms and non-ligand residues Gly7, Ile11 and Thr12 involved in hydrophobic interactions.

k) Gentamicin Corresponding atoms and non-ligand residues IIe11 involved in hydrophobic interactions. Hydrogen bonds between gentamicin and Ser57, Thr12, His10 shown by dotted red lines.

Lycopene is a symmetrical tetraterpene belongs to the carotenoid family [26]. In a pioneered work, Lee and Lee (2014) reported bactericidal activity of that lycopene against E. coli by inducing reactive oxygen species mediated DNA damage which confirmed by cell division arrest, an indirect marker of the DNA damage repair system, in lycopene-treated E. coli and they proposed that lycopene may be a clinically useful adjuvant for current antimicrobial therapies [27]. In consistent to this experimental study, we also find that lycopene has been docked with RRF through hydrophobic interactions (Figure 2) with suitable binding affinity (Table 1). 

Ligand-Code of ZINC12
Binding affinity \[\left(kcal.mol\right)^{-1}\]
RMSD/upper bound
RMSD/lower bound
Alpha-carotene-8219947
-6.7
8.50
5.00
Apigenin-11726230
-10.2
2.91
2.05
Beta-carotene-85649478
-10.0
17.60
10.95
Beta-cryptoxanthin-70450846
-8.0
17.92
4.55
D-limonene-967513
-7.6
4.23
1.13
Citral-1529208
-7.5
5.51
2.68
Eriocitrin-33963983
-12.1
24.07
20.67
Alpha-pinene-967579
-7.5
3.19
1.091
Lycopene-8214943
-10.5
2. 15
1.15
Hesperidin-8382286
-13.3
26.99
23.78
Gentamicin-DrugBank
-12.6
3.65

1.65


Note: RMSD: root mean-square deviation is the measure of the average distance between the atoms.

Table 1: In silico molecular docking of major phytochemicals of Citrus limon (L.) Burm against ribosome recycling factor (PDB code 1WIH)

Apigenin, 4′, 5, 7-trihydroxyflavone, is a flavonoid found in many plants and citrus species [28,29]. Apigenin also found to be useful as pharmaceutical agents which possess anti-inflammatory and antioxidative, antitumor, and antibacterial activities [30]. In the present study, apigenin has considerable binding affinity to dock with RRF. 

More recently, α-carotene, β-carotene and β-cryptoxanthin have been characterized in peel and pulp extracts of Citrus which inhibited the growth of both gram-positive and gram-negative bacteria [31]. Our results also showed that β-carotene and β-cryptoxanthin and α-carotene have been docked with RRF with considerable binding affinities and may involve in antibacterial effects of CL. The antibacterial and anti-inflammatory effects of these natural pigments are not a new subject, although our in silico findings would be a new insight in this context [32]. In this line, beta-cryptoxanthin belongs to the class of carotenoids and it converted to vitamin A (retinol) in human body and is therefore considered as a pro-vitamin A. it is found mainly in citrus fruit in both free and esterified forms [33]. Betacryptoxanthin has been docked with RFF throughout hydrophobic interactions with considerable binding affinity.

D-limonene is a cyclic terpene found in appreciate amount in citrus species [34]. The antimicrobial activity of d-limonene has been subject of many investigations (e.g.,[35]). In the present study, d-limonene has been docked with RRF via hydrophobic interactions (Figure 2) with moderate binding affinity (Table 1).

Alpha-pinene is a terpenoid compound ubiquitously distributed among plants including citrus species [36]. Alpha-pinene is an anti-inflammatory and broad-spectrum phytobiotic [37]. In this study, α-pinene and citral showed identical binding affinity against RRF (Table 1) and these terpenoid compounds only took part in hydrophobic interactions with RRF (Figure 2a-2k). More specifically, citral or 3, 7-dimethyl-2, 6-octadienal is terpenoid compound found in citrus family and various plants [38]. In this study, citral A or geranial showed acceptable binding affinity with RRF (Table 1). Citral is known as its lemon flavor and has antibacterial effects [39]. 

Conclusions

The emergence of antibiotic-resistant bacteria and hard access to antibiotics in different parts of the world motivated researchers to seek alternative therapeutics. In this continuum, hydro-alcoholic extracts prepared of CL peel, as a waste product, showed significant antibacterial activity against E. coli. In silico findings showed that among phytochemicals found in peel of CL, hesperidin has more negative binding affinity to RRF compared to gentamicin and would be considered as a lead molecule for further attempts to design phytobiotics.

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