Antimicrobial activity of phenolic compounds isolated from Caesalpinia pulcherrima


 IntroductionMultidrug resistance is an alarming issue worldwide. Herbal plants are being used as therapeutic agents since ages. One such plant is Caesalpinia pulcherrima, known for its beneficial effects. Therefore, this study was planned to isolate pure compounds and check its antimicrobial activity along with synergism.MethodologyAntimicrobial susceptibility pattern of MDR clinical bacterial strains against phenolic compounds was performed by Agar well diffusion method. Minimum inhibitory concentration (MIC) was determined by tube dilution and micro broth dilution methods. Synergistic combinations of Methyl gallate with antibiotics were assessed by checkerboard titration method. Scanning Electron Microscopy was conducted to analyse the changes in cell morphology of MDR E. coli and Methicillin-Resistant Staphylococcus aureus after treatment with Methyl gallateResultsMethyl gallate showed promising antimicrobial activity (250–500 µg/ml), whereas, Ethyl gallate, Syringic acid and Gallic acid trimethyl ether showed MIC (500–2500 µg/ml). Time kill kinetics assays of Methyl gallate showed bactericidal activity. Methyl gallate showed good synergistic combinations with Amoxycillin and Ciprofloxocin. Scanning electron microscopy of Methyl gallate depicted profound disruption in the cell wall, incomplete cell division and distorted structure of MDR bacteria.ConclusionsPhenolic compounds were found to be a potential source of therapeutic substances as compared to the conventional antibiotics. Further studies are required to explore their mechanism of action.


Introduction
The discovery of antibiotics is one of the greatest achievements in the history of medical sciences. Antibiotics are not only involved in the treatment of infections but also for the assurance of successful surgical interventions [1]. Despite all the facts regarding their effectiveness, the evolution of resistance against antibiotics is a distressing issue. Natural resistance was found in some bacteria even before the invention of antibiotics. There are many factors associated with the emergence of resistance such as mutations in microbes, inappropriate use and over the counter sale of antibiotics. Herbal medicine has been in use in the treatment of various diseases since ages [2]. It has been reported that more than 80% of world's population depends on herbal medicine for various illnesses [3]. There is a rich history of utilization of herbal medication and transfer of knowledge from generation to generation. The WHO has been involved in collecting and documenting the bene ts of herbal medicine from the natives of different cultural backgrounds [4]. Many studies have been and are conducted on plants to authenticate the claim of their use in herbal medicine. Health bene ts of medicinal plants are due to the presence of secondary metabolites called phenolic compounds [5]. The bene ts of these compounds are related to their consumption as food and their bioavailability [6]. Caesalpinia pulcherrima is an ornamental plant, inhabitant to Central America but also found in Africa, Asia and Australia [7]. Extracts from different parts of the plant have been used as anti-convulsant, anti-in ammatory and immuno-stimulating agents [8]. The stem was found to be useful as an abortifacient and antiulcer agent [9]. The idea of synergy between herbal drugs and antibiotics is a new approach to treating multidrug-resistant bacteria. Antimicrobial activity of extracts of different parts of this plant has been explored in our previous study [10]. After obtaining promising results, we planned this study and isolated pure compounds from fresh pods and performed antimicrobial activity against MDR (multi-drug resistant) bacteria alone and in combination with antimicrobials and also checked its effect on bacterial cell morphology.

Preparation of plant extracts and isolation of Pure Compounds
The methanol extract of pods (CP-Pods-M 292gms) was subjected to solvent separation which gave hexane (P.E), ethyl acetate (EA), butanol (But) and aqueous (Aq) phases separately. A small quantity (1.6 g) of EA phase (35.6g) was fractionated using different ratios of PE:EA mixture, which led to the isolation of a pure compound in one of the above fractions (PE:EA 1:1), identi ed as methyl gallate through spectral studies. Major quantity of EA phase (34.0 g) was subjected to VLC, which afforded 23 fractions. The fraction VLC-5 afforded Methyl-4-hydroxy benzoic acid, Fraction VLC-6 afforded Ethyl gallate, Fraction VLC-7 and VLC-8 afforded methyl gallate in large quantity (1.4 % wet weight base), while fraction 9 was found to be a mixture of methyl gallate and gallic acid. Based on this work, phenolic compounds (Dodecyl gallate, Propylgallate, Vanillic acid, Syringic acid, Ethyl-4 hydroxy benzoic acid, Gallic acid trimethyl ether) obtained commercially were used in the present study.

Antimicrobial Activity of Plants and Phenolic compounds
Antimicrobial activity of phenolic compounds (Gallic acid, Methyl gallate, Ethyl gallate, Propyl gallate, Dodecyl gallate, Syringic acid, Vanillic acid, Para hydroxybenzoic acid, Methyl 4 hydroxy benzoic acid, Ethyl 4 hydroxy benzoic acid, Gallic acid trimethyl ether) was checked against MDR clinical bacterial isolates and ATCC reference strains. Antimicrobial susceptibility pattern of MDR clinical bacterial strains against phenolic compounds was assessed by Agar well diffusion method [11]. Minimum inhibitory concentration (MIC) was determined by tube dilution and micro broth dilution methods [12].

Agar Well Diffusion Method
Antimicrobial activity of phenolic compounds was tested against MDR clinical pathogen by agar well diffusion method. The stock solution of all plant extracts was prepared in sterile DMSO. The overnight grown culture was inoculated in Mueller Hinton Broth, incubated for 3-4 hours in aerobic conditions at 37ºC to get a log phase culture. The turbidity of culture was matched with 0.5 Mac Farland index. Mueller Hinton Agar plates were seeded with bacterial culture. 20µl of extracts were added in the wells (diameter = 6mm) made by sterile borer and incubated for 24 hours at 37ºC in aerobic conditions. Results were recorded by measuring zones of inhibition around wells. The experiment was run in triplicate on three different occasions

Minimum Inhibitory Concentration By Micro broth Dilution Method
Phenolic compounds as well as conventional antibiotics that lost e cacy were checked to determine their MIC. Two-fold serial dilutions of extracts, fractions, phenolic compounds and conventional antibiotics were prepared in Mueller Hinton broth in 96-well at bottomed plates. 20 µl of inoculums 10 6 CFU/ml were added in each well to keep a total volume at 200 µl. Micro titer plates were incubated at 37ºC for 24 hours. Dilution with no turbidity was considered as MIC.
The experiment was run in triplicate on three different occasions.

Minimum Inhibitory Concentration By Tube Dilution Method
MIC of phenolic compounds as well as conventional antibiotics was recon rmed by tube dilution method. 1ml MH broth was added in sterile test tubes with inoculums (10 6 CFU/ml). Tubes without extracts but containing MH broth and inoculum were designated as positive control, whereas negative control had extract and MH broth.
Minimum Bactericidal Activity: MBC was determined by taking 100 µl of MHB from the wells and tubes containing plant extracts showing no apparent turbidity and subcultured on MHA plates. The lowest concentration without any growth was considered as MBC value [13].

Time Kill Kinetics Assay:
Time Kill Kinetics was performed to study the effect of pure Methyl gallate on the growth of MDR bacterial isolates [14]. MRSA (n = 10), MDR E. coli (n = 10) and control ATCC cultures of S. aureus and E. coli were selected for this study. 20 ml cation adjusted MHB with the absolute concentration of 0.5, 1and 2× MIC of Methyl gallate. The log phase culture of bacteria with the inoculums 10 5 CFU/ml was transferred to the ask. The ask was incubated for 24 hours at 37ºC. 100 µl was taken from the ask and plated on MHA at 2,4,6,8 and 24 hours time interval. The plates were further incubated for 24 hours at 37ºC to check CFU/ml. The experiment was run in replicate on three different occasions.
Antimicrobial activity of Methyl gallate with Cipro oxocin and Amoxycillin: The emergence of multidrug resistance is on the rise; therefore, there is an urgent need for exploring alternative therapeutic regime, which may include a combination of plant-derived substances with antibiotics that have lost e cacy. In the present study, different combinations of Amoxycillin and Cipro oxocin with Methyl gallate were studied by checkerboard titration method as demonstrated in earlier study [15]. The assay was performed in at bottomed sterile 96 well-bottomed plate. Methyl gallate with its two-fold serial dilutions was performed in all rows. Dilutions of antibiotics were made in separate test tubes. Antibiotics with their dilutions were added to all columns with equal quantity. Each well of 96 well plate had same combinations of compound and antibiotics. Inoculum size was adjusted to 10 6 CFU/ml in each well. The highest concentration of Cipro oxocin and Amoxycillin was 650µg/ml and 325µg/ml, respectively. Moreover, the highest concentration of Methyl gallate was 500µg/ml. Fractional Inhibitory concentration index (FICI) was measured as the sum of MIC of agent with combination divided by MIC of agent alone. Results of FICI were evaluated as synergy = ≤ 0.5, additive effect = > 1to 2, no effect or antagonism = > 2.
Effect of Methyl gallate isolated from C. pulcherrima on Morphology of Bacterial Cells by Scanning Electron Microscopy (SEM): In order to study the effect of subinhibitory concentrations of Methyl gallate on bacterial cell morphology by SEM, MRSA and MDR, E. coli was grown in MH broth with 0.5 MIC of compound for 24 hours at 37 ºC. After incubation, the cells were washed thrice at 10000 rpm with sterile deionised water and resuspended in PBS. A drop of culture was xed with ethanol on 4mm glass slides. Negative staining was performed with 0.2% uranyl acetate for 30  Effects of Methyl gallate on Growth Kinetics of MDR Bacteria: Time kill kinetics of gram-positive bacteria MRSA and S. aureus ATCC 29213 and gram-negative bacteria MDR E. coli and E. coli ATCC 25922 was carried out to study the effect of Methyl gallate on their growth. Ten strains (n = 10) of each species were included in this study. MRSA and S. aureus ATCC 29213 showed a reduction of CFU within 2 hours and complete bactericidal activity was observed at 24 hours and 6 hours, respectively at MIC and 2×MIC. Moreover, at 0.5×MIC, bacterial count was reduced at 5 hours in MRSA. In case of S. aureus ATCC 29213, a steady decline in CFU till 6 hours and then an increase in bacterial count were noticed (Fig. 1,2). Figure 3 shows concentration-dependent fall in CFU of E. coli ATCC 25922, less than the MIC showed lag phase for 4 hours and then started to fall between 4 to 6 hrs, and again multiplied. At MIC and 2×MIC, a drastic reduction in growth was recorded after 2 hours and complete bactericidal activity after 6 hours of post-incubation. Figure 4 shows a slight reduction in CFU of MDR. E. coli was noticed in culture treated with 0.5 MIC to 5 hours. Culture grown in 2×MIC showed complete inhibition of growth after 8 hours. At MIC inhibition, bacterial growth was observed till 8 hours and cidal activity was noticed at 24 hours. Figure #5 shows prominent structural changes in the cell wall of MDR E. coli. The surface of the cell was rough, disrupted with expulsion of cell contents. Arrow indicated the appearance of depression on the surface of the cell (C). Incomplete cell division and septum formation were also noticed as shown by arrows (D). There was a signi cant malformation, typical long rod shape converted into small rods and cocci. Morphological changes in MRSA after treatment with Methyl gallate are shown in Fig. 6. Overall, the size of cells was bigger with a distorted surface (C and D) as compared to untreated cells (A and B). Septum formation was as indicated by arrow leading to incomplete separation of daughter cells (D and E).

Antimicrobial Activity of New Synergistic Combinations of Methyl gallate:
In order to develop e cient therapeutic combinations against MDR bacterial isolates, Methyl gallate, a phenolic compound isolated from C. pulcherrima, in combination with commonly used conventional antibiotics was evaluated for its antimicrobial activity.

Synergistic Combinations of Cipro oxocin with Methyl gallate:
When synergistic combinations of Methyl gallate and Cipro oxacin were tested against MDR E.coli, there was a signi cant reduction of MICs of Cipro oxocin and Methyl gallate from512 µg/ml to 0.125 µg/ml and 500 µg/ml to 15.6 µg/ml, respectively. However, MICs of Cipro oxacin were still higher than the breakpoint values. Average FICI (fractional inhibitory concentration index) was (0.137 ± 0.11) suggesting good synergism between the two combinations. MIC of Cipro oxacin failed to reach its breakpoint, therefore, no zone of inhibition around Cipro oxacin disc placed in MHA incorporated with (0.2 ×MIC) of Methyl gallate was observed ( Table 2).

Synergistic Combinations of Amoxycillin with Methyl gallate:
A synergistic combination of Amoxicillin and Methyl gallate was observed against MRSA, with a remarkable fall in MICs of Amoxycillin and Methyl gallate from 256µg/ml to 0.0625µg/ml and 500 µg/ml to 31.25µg/ml, respectively. Sub-inhibitory concentration (0.2 ×MIC) of Methyl gallate was incorporated in MHA plate. 10mm zone of inhibition of Amoxicillin was noted and compared with control MHA plates. Average FICI (0.225 ± 0.162) exhibited potent synergistic antimicrobial combination (Table 3).

Discussion
Multi drug-resistant bacterial infections are responsible for greater portion of infectious disease burden worldwide. For the last twenty years, the emergence of antimicrobial resistance along with the adverse effects of antibiotics has directed the researchers for the exploration of new antimicrobial substances speci cally from herbal extracts, which can be able to solve the above problems [1]. Moreover, the therapeutic potential of herbal plants has been studied and identi ed to use them as a source of new drugs. According to the guidelines of WHO in 1997, bene cial plants can be used as an alternative of drugs [2].
During the course of the study, different clinical samples were collected from patients suffering from a variety of infections. These samples were processed for isolation of MDR human pathogens, which were screened against different antibiotics in order to determine their antibiotic susceptibility pattern. Methyl gallate exhibited remarkable antimicrobial activity against both MDR gram-positive (MIC = 250 µg/ml) and gram-negative bacteria (MICs = 125-250µg/ml). According to one study, phytochemicals with MIC between 100-1000µg/ml are considered as potential antimicrobial agents [3]. Earlier reports stated the antimicrobial activity of Methyl gallate against MRSA with (MIC = 1.25mg/ml) [4,5]. MRSA is one of the most important pathogens that accounts for respiratory tract, skin, and surgical site infections with high mortality and morbidity [6]. Previous studies indicated the antimicrobial activity of Methyl gallate against non-MDR E. coli, S. typhi and Vibrio Cholerae [7,8]. Time kill kinetics study of Methyl gallate showed dose-dependent bactericidal activity in both gram-negative and gram-positive bacteria. Profound effects of Methyl gallate on cell morphology of MRSA and MDR E. coli were seen by Scanning Electron Microscopy. Disruption in cell wall, incomplete cell division and distorted structure suggested that the site of action of Methyl gallate was cell wall.
It was reported that the antimicrobial activity of alkyl esters of GA depends on speci c lipophilic characteristics, assuming that cytoplasmic membrane could be a possible site of action. There are various modes of actions regarding the antimicrobial activities of phenolic compounds including interaction with including sulfhydryl group, change in permeability of cytoplasmic membrane, inactivation of enzymes, insoluble complexes with amino acids and proteins.
One more study explained the antibacterial activity of Ethyl gallate against P. aeruginosa ATCC 27853 [10]. However, Dodecyl gallate and Propyl gallate did not show antimicrobial activity against MDR bacteria in the present study but another study reported good antimicrobial activity of both compounds against MRSA [5]. Propyl gallate performed antibacterial activity against non-MDR E. coli, P. aeruginosa and S. typhi [11]. Gram-negative bacteria have specialized cell wall structure, therefore, they are di cult to kill as compared to gram-positive bacteria. The diffusion of antibiotic and active compounds is obstructed due to the presence of periplasmic space and murein layer. Mutation in active e ux pumps out anti-bacterial agent through the e ux pumps, which further promotes the development of intrinsic resistance for gram-negative bacteria [12]. Chemical structure including saturated chain length position and number in the benzene ring of phenolic compounds plays a key role in determining their anti-microbial activity. Phenolic acids had lower antimicrobial activity compared with their butyl and methyl ester [13]. The antimicrobial effect increased with increasing length of the alkyl chain [14].
Hydroxybenzoic and hydroxycinnamic acids occurring in plants exhibit diversity with respect to the number of hydroxyl or methoxy groups. Alkyl gallates disrupt the respiratory mechanism of bacteria by acting as pro-oxidants, which produce free radicles. These ROS cause oxidation of unsaturated fatty acids in the cell membrane, leading to disruption of its structure and function [15]. However, current knowledge on structure-function relationships of the antimicrobial activity of phenolic acids does not account for this diversity of compounds. Para hydroxy benzoic acid was found to be a potent antimicrobial agent against MRSA but the present study exhibited no antimicrobial activity of Parahydroxy benzoic acid and its ethyl and methyl esters against MDR grampositive and gram-negative bacteria [5]. Methyl 4 hydroxy benzoic acid showed weak antimicrobial activity against non-MDR P. aeruginosa, E. coli and S. aureus [16]. Similarly, Vanillic acid exhibited no antimicrobial activity in the present study. Earlier reports displayed antimicrobial activity against MRSA, MDR E. coli and P. mirabilus. Antimicrobial activity of Trimethyl gallic acid against non-MDR gram-positive and gram-negative bacteria was reported (350). The previous study explained antimicrobial activity of Syringic acid against MRSA, MDR E. coli and P. mirabilus, which supports the results of the present study [17].
The absence of antibacterial activity does not mean that there is no potential of inhibiting microbes. It may be possible that the active compound was not in su cient quantity or diluted with other non-antimicrobial compounds. Sometimes seasonal variations, geographical area, age of plant and method of extraction also in uence the activity of the compound [18]. Extraction techniques are usually based on the chemical nature of compounds and their solubility in polar and non-polar solvents [19].
The approach of using antimicrobial substances that suppress the mechanism of antimicrobial resistance, hence increasing the e ciency of antimicrobial product, is known as synergism [20]. Synergistic combinations of different drugs or with herbal formulations play an important role in treating antibiotic resistance. Alkyl gallates are documented to possess resistance modifying action on various antibiotics against MRSA [21]. Therefore, we used different combinations of antibiotics with Methyl gallate. Our study exhibited strong synergistic combinations of Cipro oxacin and Amoxycillin with Methyl gallate against MDR E. coli and MRSA, respectively. An earlier study explained synergistic combination of Cipro oxacin and Methyl gallate against non-MDR S. typhi [22]. There are different mechanisms involved in the augmentation of the antibacterial activity of antibiotics by natural compounds such as interaction on multiple target sites in bacteria; bioavailability and solubility of antibiotics or aimed for a speci c bacterial resistance mechanism [23]. These interactions prevent the activity of drugs from enzyme degradation and alterations in transport proteins, thus bypassing the mechanism of antimicrobial resistance [24]. Amoxycillin is a β-lactam antibiotic that inhibits bacterial cell wall synthesis by binding one or more of the penicillin-binding proteins (PBPs). Stimulation of mecA gene and gene variants resulted in the formation of PBP2a, which attaches to lower a nity with antibiotics and leads to drug resistance [25]. We assume that methyl gallate interferes with the synthesis of PBP2a in cell wall of bacteria. This could be the explanation of synergistic combinations in the present study.

Conclusions
Our study suggested that phenolic compounds are potential sources of therapeutic substances as compared with the conventional antibiotics that have failed to treat multi drug-resistant infections. Synergistic combinations of Methyl gallate with Cipro oxocin and Amoxycillin against MDR E. coli and MRSA have been found to be effective against MDR bacteria but detailed mode of action and toxicity pro les are needed to be explored.

Consent for publication:
Not applicable Availability of data and material: The datasets generated and/or analysed during the current study are not publicly available but are available from the corresponding author on reasonable request.

Declation of Interests:
All authors have no con ict of interest.