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Novel derivatives of plant monomeric phenolics: act as inhibitors of bacterial cell-to-cell communication
Journal Pre-proof Novel derivatives of plant monomeric phenolics: Act as inhibitors of bacterial cell-tocell communication Nishi Srivastava, Surabhi Tiwari, Kalpna Bhandari, A.K.S. Rawat PII:
S0882-4010(18)31793-5
DOI:
https://doi.org/10.1016/j.micpath.2019.103856
Reference:
YMPAT 103856
To appear in:
Microbial Pathogenesis
Received Date: 18 October 2018 Accepted Date: 5 November 2019
Please cite this article as: Srivastava N, Tiwari S, Bhandari K, Rawat AKS, Novel derivatives of plant monomeric phenolics: Act as inhibitors of bacterial cell-to-cell communication, Microbial Pathogenesis (2019), doi: https://doi.org/10.1016/j.micpath.2019.103856. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.
Novel derivatives of plant monomeric phenolics: act as inhibitors of bacterial cell-to-cell communication Nishi Srivastava1*, Surabhi Tiwari1, Kalpna Bhandari1, A.K.S. Rawat1* 1
Pharmacognosy and Ethnopharmacology Division, CSIR-National Botanical Research Institute, Lucknow-226001, India *E-mail: [email protected], [email protected] Telephone No. 91-522-2297816, 9450601959 Fax: 91-522 2207219
Abstract The aim of the present study was to synthesize novel active Anti-Quorum sensing derivatives from secondary metabolites viz. Gallic acid, Protocatechuic acid and Vanillic acid present in the plant Bergenia ciliata. Efficacy of all synthesize derivatives have been evaluated on the formation of bacterial biofilm and inhibition of cell-to-cell communication. Anti-Quorum Sensing activity and biofilm formation of all synthesize compounds was measured on biomonitor strain Chrobacterium violaceum, ATCC 12472 using standard paper disk-diffusion assay and quantification of violacein pigment. Among all derivatives, five derivatives 3,4,5-Trihydroxybenzoic acid methyl ester (9a), 3,4-Dihydroxy-benzoic acid methyl ester (10a), 3,4,5-Tris-(2,4dichloro-benzyloxy)-benzoic acid methyl ester (12), 3,4,5-Tris-(2,5-dichloro-benzyloxy)-benzoic acid methyl ester (13) and 4-(2,4-Dichloro-benzyloxy)-3-methoxy-benzoic acid methyl ester (15) has shown Anti-Quorum Sensing activity by inhibiting violacein pigment production and biofilm formation without interfering with its growth. The inhibitory effects in violacein pigment production were: positive control (C-30) 72%, (9a), (10a) 47.2%, (12) 27.3%, (13) 40.1% and (15) 22.7% at the concentration of 1 mg/mL and biofilm percent inhibition were found (C-30) 64% (9a) 46.2%, (10a) 40.3%, (12) 18.4%, (13) 35.2%, and (15) 17.3% when compared with the untreated control. Results reveals that synthesize derivatives seems to be good compounds for inhibition and formation of biofilm and AHL-mediated Quorum-sensing mechanism. The present article highlights the importance of derivatives derived from secondary metabolites as potent drug for biofilm formation and inhibition of cell-to-cell communication.
Significance statement In the present manuscript, we have reported synthesis of novel derivatives of Gallic acid and its analogues. These novel derivatives have been found active against Anti-Quorum sensing activity and anti-Biofilm formation. Anti-Quorum sensing and Anti-Biofilm active compounds are of today’s need to combat with microbial infection. Being derived from natural sources ‘secondary metabolites’, they are devoid of development of drug resistant. Development of drug resistant is one of major issue of today’s antimicrobial agents. To overcome drug resistant problem number of newer chemical drugs are introducing in the market and empowering microbes after development of resistant. While natural products such as secondary metabolites are showing promising result to overcome of resistant problems. With this aim we have synthesize novel derivatives and compare their Anti-Quorum and Anti-Biofilm formation with their starting nucleus ‘Gallic acid and its analogue’. On the basis of findings it can be concluded that the synthesize derivatives are active Anti-Quorum sensing and Anti-biofilm formation agents and further can be exploited in assessment of different biological activities.
1. Introduction
Plants can produce a multitude of diverse antimicrobial compounds such as simple phenolics, catechins, quinones, flavanones, polyphenolics, alkaloids, and terpenoids [1-2]. Like microbial antibiotics, these compounds are targeted at killing the pathogen and work via a non-species specific mechanism such as disrupting microbial cell membranes [3]. Recently, it was discovered that plants have another way of dealing with microbes-targeting a cell’s communication system3. One form of intercellular communication in bacteria has been studied since the 1970’s, and is known as Quorum-sensing [4]. Breakdown of this system causes an attenuation of microbial pathogenicity [5]. This process depends on signal molecules called ‘autoinducers’ production, release, and group-wide detection. In gram negative bacteria this autoinducers are typically homoserine lactones (HSLs) [6]. LuxI-type enzymes, and cytoplasmic LuxR-type proteins act as HSL receptors and produces HSLs. Other than LuxR-type proteins Apo–LuxR-type proteins are insoluble in nature. Stability comes by binding of autoinducers to LuxR-type receptors, enabling further processes like dimerization, DNA binding, and transcription of Quorum-sensing target genes. In case of many pathoges LuxI/R-signaling cascades are crucial for virulence, and virulence can be stopped by disabling these circuits with small molecules. The genus Bergenia (family Saxifragraceae) and its species viz. Bergenia ciliata (BC) Bergenia stracheyi (BS) and Bergenia ligulata, is an evergreen perennial herb, generally distributed in Central and East Asia. It is also found in temperate Himalayas from Kashmir to Bhutan at high altitude 7000-10000 feet and in khasia hill at 400 feet [7]. Previous studies on phytochemical analysis of B. ciliata have been shown the isolation of bergenin (C-glycoside of 4-O-methyl Gallic acid), Gallic acid (3,4,5 trihydroxybenzoic acid), (+)catechin, leucocyanidin, (+)-catechin-3-gallate, (+) catechin-7-O-
beta-D-glucopyranoside,
paashaanolactone,
β-sitosterol,
β-sitosterol-D-glucoside,
and
(+)afzelechin [8]. Other than these compounds, the monomeric phenolic compounds viz. Gallic acid, Protocatechuic acid, vanilic acid and syringic acid which possess antioxidant activity are also reported in the plant B. ciliata [9]. Gallic acid and its derivative have been well reported for its antimicrobial activity [10]. Therefore, on the basis of available literature, the present study on evaluation of Anti-Quorum sensing activity in novel semi-synthetic derivatives of monomeric phenolic acids has been proposed. Idea of synthesis of novel derivative has been drawn from reported drug Ketoconazole (1), Itraconazole (2), Econazole (3), Miconazole (4), Fluconazole (5) , Voriconazole (6), Ravuconazole (7) and Fluoxetine (8) which are well established antiCandida drugs (Chart 1). The problem associated with therapeutic use of these drugs accompanied by side effects, resistance and limited bioavailability. To overcome these problems we have targeted plant monomeric phenolic compounds as a lead skeleton for the synthesis of novel antimicrobial agents. On the basis of previous report on introduction of chlorine atom has positive impact on anti-Candida activity, we have introduce 2,4-dichlorobenzyl chloride, 2,5dichlorobenzyl chloride and 3-chlorobenzyl chloride moieties to the Gallic acid and its derivatives [11]. All seven synthesized (9a), (10a), (11a), (12), (13), (14) and (15) derivates were tested on Pseudomonas aeruginosa and Chrobacterium violaceum pathogens. P. aeruginosa is reported as responsible in burn units of hospitals, cystic fibrosis and in implanted medical devices as well as intubation tubes and stents [12]. Chromobacterium violaceum is gramnegative bacillus with single polar flagellum and generally one or two lateral flagella. The disease began with localized skin infection or lymphadenitis on contact with soil or stagnant
water and developed into fulminating septicemia, with multiple abscesses and necrotizing metastatic lesions in the lungs, liver, spleen, skin, brain and lymph nodes, resulting in multiorgan failure [13]. They depend mostly on two major sensing systems that is LuxI/R Quorum-sensing systems and Las and Rhl systems to manage synchronous production of virulence factors and biofilm formation [6-14]. There is different mechanism reported to disrupted the process of QS: (i) reducing the activity of AHL synthase, (ii) inhibiting the formation of autoinducers (iii) AHL degradation, and (iv) mimicking the autoinducers initially using synthetic compounds as analogues of autoinducers In all of possible reported mechanism, AHL degradation has been preferred and applied the most. Plant secondary metabolites have been reported to inhibit QS due to resemblance in their chemical structures to those of signaling molecules and also due to attack on signal receptors (LuxR/LasR). The discovery of Anti-Quorum sensing compounds in plants provides us with yet another type of “antimicrobial” agent. Among all derivatives, five derivatives (9a), (10a), (12), (13) and (15) has shown Anti-Quorum Sensing activity by inhibiting violacein pigment production and biofilm formation without interfering with its growth. 2. Materials and methods 2.1 Derivatization of biomarkers Chemistry All reagents and solvents wee procured from commercial chemical suppliers and used without further purification. IR spectra were recorded on Perkin Elmer 881 and FTIR 8210 PC, Schimadzu spectrophotometers either on KBr discs or in neat. 1H-Nuclear magnetic resonance (NMR) spectra were recorded on Bruker Avance DRX-400 MHz spectrometers in deuterated solvents using TMS as an internal reference. Mass spectra were recorded on JEOL JMS-D-300 spectrometer with the ionization potential of 70 eV and ES mass spectra on Quantro-II, micro
mass. Thin-layer chromatography was performed on Merck precoated silica gel 60 F254 plates. Purity of all tested compounds was ascertained on the basis of their elemental analysis and was carried out on Euro Vector EA 3000 instrument. The melting points were recorded on an electrically heated melting point apparatus and are uncorrected. The
Synthesis
of
novel
derivatives of phenolic acids commenced with the esterification of commercially available Gallic acid (9), Protocatechuic acid (10) and Vanillic acid (11) and is summarized in Scheme-1. Methyl (9a) esters of Gallic acid were prepared in the presence of thionyl chloride and corresponding alcohols. Whilst only methyl ester of Protocatechuic acid (10a) and Vanillic acid (11a) were synthesized for getting the final focused compounds. Thus to a stirred and cooled mixture of 3,4,5-trihydoxybenzoic acid (1)/ 3,4-dihydroxybenzoic acid(2)/ 4-hydroxy,3methoxybenzoic acid (3) (5 mmol), in alcohols, was added thionyl chloride (5 mmol) dropwise. Stirring and cooling continued for 30 min. The reaction mixture was refluxed for 1h, solvent distilled off. The residue obtained was crystallized with methanol to furnish the required esters (9a, 10a & 11a) as off white solid (Scheme-1). Subsequently, condensation of the above phenolic ester (9a, 10a & 11a) with substituted benzyl halides furnished the required benzyl ethers 12-13, 14 & 15. Thus a mixture of methyl ester of 3,4,5-trihydroxybenzoic acid (9a)/ or 3,4-dihydroxybenzoic acid (10a)/ or 4-hydroxy-3-methoxybenzoic acid (11a) (5 mmol) and 2,4dichlorobenzyl chloride/ or 2,5-dichlorobenzyl chloride /or 3-chlorobenzyl chloride (15 mmol for (9a), 10 mmol for (10a), 5 mmol for (11a) with K2CO3 (16 mmol for (9a), 11 mmol for (10a), 6 mmol for (11a) in acetonitrile (10 mL) was heated for 5 h at 900C. After completion of reaction the solid was filtered and solvent was concentrated to give a white solid. The solid was
dissolved in chloroform and washed with distilled water (3 x 5mL). The organic layer was dried over sodium sulphate and concentrated to give the crude product which was purified by column chromatography using methanol:chloroform (1:99) as an eluent to provide the required compounds (12-15). NMR spectra of compounds attached as annexure-1 at the end of the paper. 3,4,5-Trihydroxy-benzoic acid methyl ester (9a) Synthesized from 9; Yield: 84%; M.P: 90°C; MS (ESI) m/z: 185 (100) [M+1]+; 1H NMR (400 MHz, CDCl3): δ 7.06 (s, 2H, ArH), 3.83 (s, 3H); IR (KBr, cm-1): 3855, 3788, 3660, 3372, 3019, 2400, 1438, 1215, 1037, 929, 757, 669; Anal. Calcd. for C8H8O5 : C, 52.38; H, 4.18. Found C, 51.69; H, 3.96. 3,4-Dihydroxy-benzoic acid methyl ester (10a) Synthesized from 10; Yield: 80%; M.P: 129°C; MS (ESI) m/z: 169 (100) [M+1]+; 1H NMR (400 MHz, CDCl3): δ 7.43 (d, 1H, ArH, J = 7.7 Hz), 7.41 (s, 1H, ArH), 6.8 (d, 1H, ArH, J = 8.1 Hz), 3.83 (s, 3H); IR (KBr, cm-1): 3286, 2416, 1692, 1540, 1403, 1317, 1242, 1050, 813, 744; Anal. Calcd. for C8H8O4 : C, 57.24; H, 4.80. Found C, 57.61; H, 4.31. 4-Hydroxy-3-methoxy-bezoic acid methyl ester (11a) Synthesized from 11; Yield: 78%; M.P: 93°C; MS (ESI) m/z: 153 (100) [M+1]+; 1H NMR (400 MHz, CDCl3): δ 7.54 (d, 1H, ArH, J = 2 Hz), 7.52 (s, 1H, ArH), 6.85 (d, 1H, ArH, J = 8 Hz), 3.88 (s, 3H), 3.85 (s, 3H); IR (KBr, cm-1): 3386, 2955, 1699, 1514, 1435, 1373, 1290, 1103, 1029, 983, 874, 778, 766; Anal. Calcd. for C8H8O3 : C, 63.15; H, 5.30. Found C, 63.9; H, 4.92. 3,4,5-Tris-(2,4-dichloro-benzyloxy)-benzoic acid methyl ester (12) Synthesized from 1a; Yield: 74%; M.P: 144°C; MS (ESI) m/z: 662 (100) [M+1]+; 1H NMR (400 MHz, CDCl3): δ 7.53 (s, 3H, ArH), 7.45 (d, 3H, J = 8 Hz), 7.31 (d, 3H, J = 2 Hz), 7.11 (s, 2H, ArH), 5.19 (s, 6H), 3.94 (s, 3H); IR (KBr, cm-1): 3076, 2930, 2359, 1724, 1507, 1451, 1390,
1218, 1096, 1016, 808, 725, 548; Anal. Calcd. for C29H20Cl6O5 : C, 52.68; H, 3.05. Found C, 52.10; H, 3.67. 3,4,5-Tris-(2,5-dichloro-benzyloxy)-benzoic acid methyl ester (13) Synthesized from 1a; Yield: 59%; M.P: 119°C; MS (ESI) m/z: 661 (100) [M+1]+; 1H NMR (400 MHz, CDCl3): δ 7.68 (d, 3H, J = 2.4 Hz), 7.57 (d, 3H, J = 2.4 Hz), 7.27 (s, 3H, ArH), 7.18 (s, 2H, ArH), 5.23 (s, 6H), 3.95 (s, 3H); IR (KBr, cm-1): 3076, 2930, 2359, 1724, 1507, 1432, 1360, 1188, 1096, 1016, 808, 725, 548; Anal. Calcd. for C29H20Cl6O5 : C, 52.68; H, 3.05; Cl, 32.17; O, 31.55; Found C, 53.02; H, 3.45. 3,4-Bis-(3-chloro-benzyloxy)-benzoic acid methyl ester (14) Synthesized from 2a; Yield: 76%; M.P: 148°C; MS (ESI) m/z: 417 (100) [M+1]+; 1H NMR (400 MHz, CDCl3): δ 7.70 (d, 1H, ArH, J = 2 Hz), 7.68 (s, 1H, ArH), 7.48 (m, 2H, ArH), 7.36 (d, 2H, ArH, J = 8 Hz), 7.32 (d, 1H, J = 1.6 Hz), 6.96 (d, 2H, ArH, J = 8.4 Hz), 3.90 (s, 3H); IR (KBr, cm-1): 3847, 3637, 2891, 1721, 1519, 1422, 1377, 1274, 1109, 1038, 992, 774, 679; Anal. Calcd. for C22H18Cl2O4 : C, 63.32; H, 4.35.1 Found C, 63.96; H, 4.65. 4-(2,4-Dichloro-benzyloxy)-3-methoxy-benzoic acid methyl ester (15) Synthesized from 3a; Yield: 68%; M.P: 78°C; MS (ESI) m/z: 342 (100) [M+1]+; 1H NMR (400 MHz, CDCl3): δ 7.48 (d, 1H, ArH, J = 2 Hz), 7.46 (s, 1H, ArH), 7.23 (s, 1H, ArH), 7.12 (d, 1H, ArH, J = 1.4 Hz), 7.09 (d, 1H, J = 2 Hz), 6.71 (d, 1H, ArH, J = 8.4 Hz), 3.79 (s, 3H), 3.74 (s, 3H); IR (KBr, cm-1): 3079, 2967, 1682, 1505, 1434, 1386, 1294, 1099, 821, 764; Anal. Calcd. for C16H14Cl2O4 : C, 56.32; H, 4.14. Found C, 55.98; H, 4.51. 2.2 Determination of Antipathogenic activity: Paper disk-diffusion assay
To detect the Anti-Quorum Sensing activity of the compounds on a biomonitor strain of Chrobacterium violaceum, ATCC 12472, a standard paper disk-diffusion assay was used as described previously6. C. violaceum ATCC 12472 was grown on LB agar and provided with proper antibiotic. LB agar (0.3 %, w/v) volume five milliliters was inoculated with 50 mL of a culture of the bacterium. After preparation the culture solution was poured over the surface of pre-warmed LB agar plates. Compounds to be tested were first dissolved into solvent and after that were pipetted onto sterile paper disks placed on the solidified agar. Later on plates were incubated overnight at 370C and examined for violacein production. Anti-Quorum Sensing activity was analyzed by a colorless, opaque halo zone with viable bacterial cells around the disk. 2.3 Extraction and quantification of violacein Extraction of violacein pigment was carried out as per method of Singh et al., [14]. Bacterial cells were grown in absence and presence of test compounds, lysed by SDS and incubated at room temperature. Quantification of extracted violacein was achieved at A585. The extracted violacein was quantified at A585. 2.4 Anti-biofilm assay In anti-biofilm assay, microtitre dishes of polystyrene were assayed basically while described by Singh et al. [14], with few changes. In short, culture of P. aeruginosa in AB minimal medium incubated overnight, was diluted 1:100 in fresh medium and grown for one more hour. Later on, various concentrations of test compounds solution were pipetted into the wells of the microtitre dishes and incubated for 48 h at 300C. After medium removal, 100 ml of a 1% (w/v) aqueous solution of crystal violet (CV) was added. Subsequent staining at room temperature intended for 20 min, the dye was removed and the wells were washed thoroughly.
After that plates were rinsed to remove planktonic cells, then quantification of surface-attached cells were achieved by solubilizing the dye in ethanol and measuring at A650. 2.5 Statistical analysis Results of Anti-Quorum Sensing activities have been presented as the mean ± standard deviation (SD) of minimum three determinations. Statistical analysis was done using one way ANOVA software and significance of findings was considered at P < 0.05. 3. Results Loss of purple pigment in C. violaceum 12472 is indicative of QS inhibition by the test samples introduced. Out of 7 derivatives screened for Anti-Quorum Sensing activity, 5 derivatives (1 mg/mL each) proved to be effective: (9a), (10a), (12), (13), (15) (Figure -1). The Anti-QS activities of the 5 derivatives were screened using the C. violaceum bioassay. Control discs contained halogenated furanone and ethanol was also used. The positive control furanone (C-30) at 10µg/mL have showed 72% inhibition. As expected, a zone of growth inhibition was observed with gentamycin, a zone of QS inhibition (halo) was seen with the furanone, and no inhibition was apparent with ethanol. Inhibitory effect of these derivatives on violacein pigment production was also measured and violacein production was observed to be inhibited by the derivatives (Scheme-1). The inhibitory effects were: (C-30) 72%, (9a), (10a) 47.2%, (12) 27.3%, (13) 40.1% and (15) 22.7% at the concentration of 1 mg/mL. There was a significant reduction in P. aeruginosa biofilm formation recorded when the bacterium was grown in the presence of 1 mg/mL. Percent inhibitions of biofilm formation were quantified for (C-30) 64% (9a) 46.2%, (10a) 40.3%, (12) 18.4%, (13) 35.2%, and (15) 17.3% when compared with the untreated control (Figure-1 & 2).
4. Discussion In the present work, Anti-Quorum Sensing activities of novel derivatives were examined. Recent research findings have shown that secondary metabolites produce from natural resources viz. plants, fungi, eukaryotic and even animals, interfere with bacterial cell to cell communication system [15-17]. Quorum Quenching could be considered as most effective alternative strategy to combat microbial infection as it reduce the probability of development of multidrug resistant pathogens. Plants are continuously exposed to microbial infection and results into development of sophisticated chemicals to inhibit microbial pathogenesis. Secondary metabolites play an important role as active Anti-Quorum Sensing compounds or lead skeleton in the development of novel Anti-Quorum Sensing drug. Moreover, the identified Quorum Sensing blockers are more reactive and toxic, which raise the concern of using them as medicine in industry and agriculture [16]. Although, targeting cell to cell communication (Quorum Sensing) to control microbial infections in humans, animals and plants seems to produce much promise. Therefore, in the present investigation, we have planned to screen secondary metabolites viz. Gallic acid (9), Protocatechuic acid (10), Vanilic acid (11) and its semi-synthetic derivatives 3,4,5trihydroxybenzoic acid (9a), 3,4-dihydroxybenzoic acid (10a), 4-hydroxy-3-methoxybenzoic acid (11a), 3,4,5-Tris-(2,4-dichloro-benzyloxy)-benzoic acid methyl ester (12), 3,4,5-Tris-(2,5dichloro-benzyloxy)-benzoic acid methyl ester (13), 3,4-Bis-(3-chloro-benzyloxy)-benzoic acid methyl ester (14) and 4-(2,4-Dichloro-benzyloxy)-3-methoxy-benzoic acid methyl ester (15) for their Quorum Sensing activities using disk diffusion assay and anti-biofilm assay.
In the
experiment, C. violaceum 12472 indicator strain was used. Findings of experiments revealed that the Compounds (9a), (10a), (12), (13) and (15) are effectively inhibit Quorum Sensing regulated violacein pigment production in C. violaceum 12472 without interrupting its growth. While, in
zone of growth inhibition, no inhibition was apparent with ethanol. On the basis of findings, it can be mentioned that the negative effect of the secondary metabolites and its novel semisynthetic derivatives on the violacein production is caused by disruption of cell to cell communication (Quorum Sensing signaling system), without inhibition of growth. Inhibition of cell to cell communication without interfering with growth, is highly encoring approach to combat with microbial infection, because there is no probability of development of resistance in bacteria. 5. Conclusion Recent studies have shown that various eukaryotic specimens, including plant, fungi, and even animals produce compounds that interfere with bacterial QS system. Unluckily, most of the QS blockers identified so far are too reactive and toxic, which increases the concern of using these blockers in medicine, agriculture, and industry. However, QS inhibition as a target to control bacterial diseases of human, plant, and animal seems to hold much promise. In this investigation, we therefore decided to screen various derivatives of Gallic acids for their anti-QS property by using different assays including disk diffusion, violacean production, and biofilm formation spectrophotometrically. For disk diffusion assays, C. violaceum 12472, a bioindicator strain was used. Results revealed that the (9a), (10a), (12), (13) and (15) showed Anti-Quorum Sensing activity by inhibiting violacein pigment production and biofilm formation without interfering with its growth. We therefore assumed that the negative effect of the derivatives on violacein production is not caused by inhibition of growth but rather by disruption of QS signaling system. This approach is highly encouraging because when growth is not affected there is no selective pressure for development of resistance in bacteria. Therefore, derivatives of Gallic acid have a
great advantage for human use than toxic anti-QS compounds such as halogenated furanone and convectional antibiotics. Conflict of interest The authors declare that there are no conflicts of interest.
Acknowledgment The authors thank to the Director, CSIR-NBRI Lucknow for his kind support and to make available required resources during this study. First author (NS) is thankful to CSIR (New Delhi) for the award of Senior Research fellowship.
References
1. M.M. Cowan, Plant products as antimicrobial agents, Clin Microbiol Rev, 12 (1999) 564-582.
2. P.M. Dewick, Medicinal Natural Products: A Biosynthetic Approach, Third edn., John Wiley and Sons New York, (2009) .
3. W.D. Bauer, M. Tepletski, Can plants manipulate bacterial quorum sensing, Aust. J. Plant Physiol. 28 (2001) 913-921.
4. S. Schauder, B.L. Bassler, The languages of bacteria, Genes Dev. 15 (2001) 1468-1480.
5. R.S. Smith, B.H. Iglewski, P. aeruginosa quorum-sensing systems and virulence, Curr Opin Microbiol. 6 (2003) 56-60.
6. B.N. Singh, B.R. Singh, R.L. Singh, D. Prakash, R. Dhakarey, G. Upadhyay, H.B. Singh, Oxidative DNA damage protective activity, antioxidant and anti-quorum sensing potentials of Moringa oleifera, Food and Chemical Toxicol. 47 (2009) 1109-1116.
7. Anonymous. The Wealth of India, Raw materials, Council of Scientific and Industrial. Research (CSIR) New Delhi, (1948) 1-179.
8. A.P. Tucci, M.F. Delle, B. Marini, B. Giovanni, Annals 1st super sanita. 5 (1969) 555-556. 9. N. Srivastava, A. Srivastava, S. Srivastava, A.K.S. Rawat, A.R. Khan, HPTLC-densitometric determination and Kinetic studies on antioxidant potential of Monomeric phenolic acids (MPAs) from Bergenia species, RSC Adv. 4 (2014) 52647–52657.
10. A. Borges, C. Ferreira, M.J. Saavedra, M. Simoes, Antibacterial activity and mode of action of ferulic and gallic acids against pathogenic bacteria, Microb Drug Resist. 19 (2013) 256-265. 11. (a) J.C. Song, S. Deresisnski, Curr. Opin. InVest. Drugs 6 (2005) 170–177. (b) W.J. Watkins,
T. E. Renau, In Annual Reports in Medicinal Chemistry; Doherty, A. M., Ed.; Academic Press:
New York 35 (1999) 157-166.
12. G. Slesak, P. Douangdala, S. Inthalad, J. Silisouk, M.Vongsouvath, A. Sengduangphachanh, Catrin E Moore, M. Mayxay, H. Matsuoka, P.N. Newton, Fatal Chromobacterium violaceum septicaemia in northern Laos, a modified oxidase test and post-mortem forensic family G6PD analysis, Ann Clin Microbiol Antimicrob. 8 (2009) 24.
13. B.N. Singh, B.R. Singh, R.L. Singh, D. Prakash, B.K. Sharma, H.B. Singh, Antioxidant and anti-quorum sensing activities of green pod of Acacia nilotica L., Food Chem Toxicol. 47 (2009) 778-786. 14. B.N. Singh, H.B. Singh, A. Singh, B.R. Singh, A. Mishra, C.S. Nautiyal,
Lagerstroemia
speciosa fruit extract modulates quorum sensing-controlled virulence factor production and biofilm formation in Pseudomonas aeruginosa, Microbiology 158(2) (2012) 529-538. 15. A.L. Adonizio, K. Downum, B.C. Bennett, K. Mathee, Anti-quorum sensing activity of medicinal plants in southern Florida, J. Ethnopharmacol. 105 (2006) 427-435.
16. G. Bosgelmez-Tinaz, S. Ulusoy, A. Ugur, O. Ceylan, Inhibition of Quorum Sensing–Regulated Behaviors by Scorzonera sandrasica, Curr. Microbiol. 55 (2007) 114–118.
17. T.B. Rasmussen, T. Bjarnsholt, M.E. Skindersoe, M. Hentzer, P. Kristoffersen, M. Kote, J. Nielsen, L. Eberl, M. Givskov, Screening for quorum-sensing inhibitors (QSI) by use of a novel genetic system, the QSI selector, J. Bactriol. 187 (2005) 1799–1814.
90 80
% Inhibition
70 60 50 40 30 20 10 0 1a 9a
2a 10a
4 12
135
157
C-30 C-30
Derivatives
Figure-1 Inhibitory effect of gallic acid derivatives on production of violacein, (9a), (10a) (12), (13) and (15)
60
% biofilm Inhibition
50 40 30 20 10 0 1a 9a
2a 10a
4 12
5 13
7 15
C-30 C-30
Derivatives
Figure-2 Anti-biofilm formation activity of gallic acid derivaties, (9a), (10a), (12), (13) and (15)
COOH
COOR i
HO
ii
OH HO OH (9)
OHR1O OH (9a) COOR
COOH
OR1 OR1 (12, 13) COOR
i
ii OH
OH OH (10)
OH (10a)
COOH
COOR
OH (11)
COOR
OR1 (14) COOR
i
ii
OCH3
OCH3 OH (11a)
OR1
OCH3 OR1 (15)
(9a, 10a, 11a);
12;
R=CH3
R=CH3; R1=
Cl
H 2C Cl
13;
R=CH3; R1=
Cl
H2 C Cl
14;
R=CH3; R1=
H2C Cl
15;
R=CH3;
R1=
H2 C
Cl Cl
Scheme-1 Reagent and conditions; (i) Thionyl chloride (SOCl2), methanol (CH3OH), 750C, 1h; (ii) K2CO3, 2,4-dichlorobenzyl chloride, 2,5-dichlorobenzyl chloride, 3-chlorobenzyl chloride, acetonitrile (CH3CN)
Annexure-1 (9a) COOCH3
HO
OH OH
(9a)
COOCH3
HO
OH OH
(10a)
COOCH3
OH OH
(10a)
COOCH3
OH OH
(11a) COOCH3
OCH3 OH
(11a)
COOCH3
OCH3 OH
12
COOCH3
Cl O
Cl
Cl
O
O
Cl Cl
Cl
12
COOCH3
Cl O
Cl
Cl
O
O
Cl Cl
Cl
13 COOCH3
Cl Cl
O
O
Cl
O Cl
Cl
Cl
13
COOCH3
Cl Cl
O
O
Cl
O Cl
Cl
Cl
14
COOCH3
O
C
O
Cl
14 COOCH3
O
O
Cl
Cl
15 COOCH3
OCH3
O Cl
Cl
15 COOCH3
OCH3
O Cl
Cl
Annexure-1 1H & 13C-NMR Spectra of derivatives of Gallic acid
S0882-4010(18)31793-5
DOI:
https://doi.org/10.1016/j.micpath.2019.103856
Reference:
YMPAT 103856
To appear in:
Microbial Pathogenesis
Received Date: 18 October 2018 Accepted Date: 5 November 2019
Please cite this article as: Srivastava N, Tiwari S, Bhandari K, Rawat AKS, Novel derivatives of plant monomeric phenolics: Act as inhibitors of bacterial cell-to-cell communication, Microbial Pathogenesis (2019), doi: https://doi.org/10.1016/j.micpath.2019.103856. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.
Novel derivatives of plant monomeric phenolics: act as inhibitors of bacterial cell-to-cell communication Nishi Srivastava1*, Surabhi Tiwari1, Kalpna Bhandari1, A.K.S. Rawat1* 1
Pharmacognosy and Ethnopharmacology Division, CSIR-National Botanical Research Institute, Lucknow-226001, India *E-mail: [email protected], [email protected] Telephone No. 91-522-2297816, 9450601959 Fax: 91-522 2207219
Abstract The aim of the present study was to synthesize novel active Anti-Quorum sensing derivatives from secondary metabolites viz. Gallic acid, Protocatechuic acid and Vanillic acid present in the plant Bergenia ciliata. Efficacy of all synthesize derivatives have been evaluated on the formation of bacterial biofilm and inhibition of cell-to-cell communication. Anti-Quorum Sensing activity and biofilm formation of all synthesize compounds was measured on biomonitor strain Chrobacterium violaceum, ATCC 12472 using standard paper disk-diffusion assay and quantification of violacein pigment. Among all derivatives, five derivatives 3,4,5-Trihydroxybenzoic acid methyl ester (9a), 3,4-Dihydroxy-benzoic acid methyl ester (10a), 3,4,5-Tris-(2,4dichloro-benzyloxy)-benzoic acid methyl ester (12), 3,4,5-Tris-(2,5-dichloro-benzyloxy)-benzoic acid methyl ester (13) and 4-(2,4-Dichloro-benzyloxy)-3-methoxy-benzoic acid methyl ester (15) has shown Anti-Quorum Sensing activity by inhibiting violacein pigment production and biofilm formation without interfering with its growth. The inhibitory effects in violacein pigment production were: positive control (C-30) 72%, (9a), (10a) 47.2%, (12) 27.3%, (13) 40.1% and (15) 22.7% at the concentration of 1 mg/mL and biofilm percent inhibition were found (C-30) 64% (9a) 46.2%, (10a) 40.3%, (12) 18.4%, (13) 35.2%, and (15) 17.3% when compared with the untreated control. Results reveals that synthesize derivatives seems to be good compounds for inhibition and formation of biofilm and AHL-mediated Quorum-sensing mechanism. The present article highlights the importance of derivatives derived from secondary metabolites as potent drug for biofilm formation and inhibition of cell-to-cell communication.
Significance statement In the present manuscript, we have reported synthesis of novel derivatives of Gallic acid and its analogues. These novel derivatives have been found active against Anti-Quorum sensing activity and anti-Biofilm formation. Anti-Quorum sensing and Anti-Biofilm active compounds are of today’s need to combat with microbial infection. Being derived from natural sources ‘secondary metabolites’, they are devoid of development of drug resistant. Development of drug resistant is one of major issue of today’s antimicrobial agents. To overcome drug resistant problem number of newer chemical drugs are introducing in the market and empowering microbes after development of resistant. While natural products such as secondary metabolites are showing promising result to overcome of resistant problems. With this aim we have synthesize novel derivatives and compare their Anti-Quorum and Anti-Biofilm formation with their starting nucleus ‘Gallic acid and its analogue’. On the basis of findings it can be concluded that the synthesize derivatives are active Anti-Quorum sensing and Anti-biofilm formation agents and further can be exploited in assessment of different biological activities.
1. Introduction
Plants can produce a multitude of diverse antimicrobial compounds such as simple phenolics, catechins, quinones, flavanones, polyphenolics, alkaloids, and terpenoids [1-2]. Like microbial antibiotics, these compounds are targeted at killing the pathogen and work via a non-species specific mechanism such as disrupting microbial cell membranes [3]. Recently, it was discovered that plants have another way of dealing with microbes-targeting a cell’s communication system3. One form of intercellular communication in bacteria has been studied since the 1970’s, and is known as Quorum-sensing [4]. Breakdown of this system causes an attenuation of microbial pathogenicity [5]. This process depends on signal molecules called ‘autoinducers’ production, release, and group-wide detection. In gram negative bacteria this autoinducers are typically homoserine lactones (HSLs) [6]. LuxI-type enzymes, and cytoplasmic LuxR-type proteins act as HSL receptors and produces HSLs. Other than LuxR-type proteins Apo–LuxR-type proteins are insoluble in nature. Stability comes by binding of autoinducers to LuxR-type receptors, enabling further processes like dimerization, DNA binding, and transcription of Quorum-sensing target genes. In case of many pathoges LuxI/R-signaling cascades are crucial for virulence, and virulence can be stopped by disabling these circuits with small molecules. The genus Bergenia (family Saxifragraceae) and its species viz. Bergenia ciliata (BC) Bergenia stracheyi (BS) and Bergenia ligulata, is an evergreen perennial herb, generally distributed in Central and East Asia. It is also found in temperate Himalayas from Kashmir to Bhutan at high altitude 7000-10000 feet and in khasia hill at 400 feet [7]. Previous studies on phytochemical analysis of B. ciliata have been shown the isolation of bergenin (C-glycoside of 4-O-methyl Gallic acid), Gallic acid (3,4,5 trihydroxybenzoic acid), (+)catechin, leucocyanidin, (+)-catechin-3-gallate, (+) catechin-7-O-
beta-D-glucopyranoside,
paashaanolactone,
β-sitosterol,
β-sitosterol-D-glucoside,
and
(+)afzelechin [8]. Other than these compounds, the monomeric phenolic compounds viz. Gallic acid, Protocatechuic acid, vanilic acid and syringic acid which possess antioxidant activity are also reported in the plant B. ciliata [9]. Gallic acid and its derivative have been well reported for its antimicrobial activity [10]. Therefore, on the basis of available literature, the present study on evaluation of Anti-Quorum sensing activity in novel semi-synthetic derivatives of monomeric phenolic acids has been proposed. Idea of synthesis of novel derivative has been drawn from reported drug Ketoconazole (1), Itraconazole (2), Econazole (3), Miconazole (4), Fluconazole (5) , Voriconazole (6), Ravuconazole (7) and Fluoxetine (8) which are well established antiCandida drugs (Chart 1). The problem associated with therapeutic use of these drugs accompanied by side effects, resistance and limited bioavailability. To overcome these problems we have targeted plant monomeric phenolic compounds as a lead skeleton for the synthesis of novel antimicrobial agents. On the basis of previous report on introduction of chlorine atom has positive impact on anti-Candida activity, we have introduce 2,4-dichlorobenzyl chloride, 2,5dichlorobenzyl chloride and 3-chlorobenzyl chloride moieties to the Gallic acid and its derivatives [11]. All seven synthesized (9a), (10a), (11a), (12), (13), (14) and (15) derivates were tested on Pseudomonas aeruginosa and Chrobacterium violaceum pathogens. P. aeruginosa is reported as responsible in burn units of hospitals, cystic fibrosis and in implanted medical devices as well as intubation tubes and stents [12]. Chromobacterium violaceum is gramnegative bacillus with single polar flagellum and generally one or two lateral flagella. The disease began with localized skin infection or lymphadenitis on contact with soil or stagnant
water and developed into fulminating septicemia, with multiple abscesses and necrotizing metastatic lesions in the lungs, liver, spleen, skin, brain and lymph nodes, resulting in multiorgan failure [13]. They depend mostly on two major sensing systems that is LuxI/R Quorum-sensing systems and Las and Rhl systems to manage synchronous production of virulence factors and biofilm formation [6-14]. There is different mechanism reported to disrupted the process of QS: (i) reducing the activity of AHL synthase, (ii) inhibiting the formation of autoinducers (iii) AHL degradation, and (iv) mimicking the autoinducers initially using synthetic compounds as analogues of autoinducers In all of possible reported mechanism, AHL degradation has been preferred and applied the most. Plant secondary metabolites have been reported to inhibit QS due to resemblance in their chemical structures to those of signaling molecules and also due to attack on signal receptors (LuxR/LasR). The discovery of Anti-Quorum sensing compounds in plants provides us with yet another type of “antimicrobial” agent. Among all derivatives, five derivatives (9a), (10a), (12), (13) and (15) has shown Anti-Quorum Sensing activity by inhibiting violacein pigment production and biofilm formation without interfering with its growth. 2. Materials and methods 2.1 Derivatization of biomarkers Chemistry All reagents and solvents wee procured from commercial chemical suppliers and used without further purification. IR spectra were recorded on Perkin Elmer 881 and FTIR 8210 PC, Schimadzu spectrophotometers either on KBr discs or in neat. 1H-Nuclear magnetic resonance (NMR) spectra were recorded on Bruker Avance DRX-400 MHz spectrometers in deuterated solvents using TMS as an internal reference. Mass spectra were recorded on JEOL JMS-D-300 spectrometer with the ionization potential of 70 eV and ES mass spectra on Quantro-II, micro
mass. Thin-layer chromatography was performed on Merck precoated silica gel 60 F254 plates. Purity of all tested compounds was ascertained on the basis of their elemental analysis and was carried out on Euro Vector EA 3000 instrument. The melting points were recorded on an electrically heated melting point apparatus and are uncorrected. The
Synthesis
of
novel
derivatives of phenolic acids commenced with the esterification of commercially available Gallic acid (9), Protocatechuic acid (10) and Vanillic acid (11) and is summarized in Scheme-1. Methyl (9a) esters of Gallic acid were prepared in the presence of thionyl chloride and corresponding alcohols. Whilst only methyl ester of Protocatechuic acid (10a) and Vanillic acid (11a) were synthesized for getting the final focused compounds. Thus to a stirred and cooled mixture of 3,4,5-trihydoxybenzoic acid (1)/ 3,4-dihydroxybenzoic acid(2)/ 4-hydroxy,3methoxybenzoic acid (3) (5 mmol), in alcohols, was added thionyl chloride (5 mmol) dropwise. Stirring and cooling continued for 30 min. The reaction mixture was refluxed for 1h, solvent distilled off. The residue obtained was crystallized with methanol to furnish the required esters (9a, 10a & 11a) as off white solid (Scheme-1). Subsequently, condensation of the above phenolic ester (9a, 10a & 11a) with substituted benzyl halides furnished the required benzyl ethers 12-13, 14 & 15. Thus a mixture of methyl ester of 3,4,5-trihydroxybenzoic acid (9a)/ or 3,4-dihydroxybenzoic acid (10a)/ or 4-hydroxy-3-methoxybenzoic acid (11a) (5 mmol) and 2,4dichlorobenzyl chloride/ or 2,5-dichlorobenzyl chloride /or 3-chlorobenzyl chloride (15 mmol for (9a), 10 mmol for (10a), 5 mmol for (11a) with K2CO3 (16 mmol for (9a), 11 mmol for (10a), 6 mmol for (11a) in acetonitrile (10 mL) was heated for 5 h at 900C. After completion of reaction the solid was filtered and solvent was concentrated to give a white solid. The solid was
dissolved in chloroform and washed with distilled water (3 x 5mL). The organic layer was dried over sodium sulphate and concentrated to give the crude product which was purified by column chromatography using methanol:chloroform (1:99) as an eluent to provide the required compounds (12-15). NMR spectra of compounds attached as annexure-1 at the end of the paper. 3,4,5-Trihydroxy-benzoic acid methyl ester (9a) Synthesized from 9; Yield: 84%; M.P: 90°C; MS (ESI) m/z: 185 (100) [M+1]+; 1H NMR (400 MHz, CDCl3): δ 7.06 (s, 2H, ArH), 3.83 (s, 3H); IR (KBr, cm-1): 3855, 3788, 3660, 3372, 3019, 2400, 1438, 1215, 1037, 929, 757, 669; Anal. Calcd. for C8H8O5 : C, 52.38; H, 4.18. Found C, 51.69; H, 3.96. 3,4-Dihydroxy-benzoic acid methyl ester (10a) Synthesized from 10; Yield: 80%; M.P: 129°C; MS (ESI) m/z: 169 (100) [M+1]+; 1H NMR (400 MHz, CDCl3): δ 7.43 (d, 1H, ArH, J = 7.7 Hz), 7.41 (s, 1H, ArH), 6.8 (d, 1H, ArH, J = 8.1 Hz), 3.83 (s, 3H); IR (KBr, cm-1): 3286, 2416, 1692, 1540, 1403, 1317, 1242, 1050, 813, 744; Anal. Calcd. for C8H8O4 : C, 57.24; H, 4.80. Found C, 57.61; H, 4.31. 4-Hydroxy-3-methoxy-bezoic acid methyl ester (11a) Synthesized from 11; Yield: 78%; M.P: 93°C; MS (ESI) m/z: 153 (100) [M+1]+; 1H NMR (400 MHz, CDCl3): δ 7.54 (d, 1H, ArH, J = 2 Hz), 7.52 (s, 1H, ArH), 6.85 (d, 1H, ArH, J = 8 Hz), 3.88 (s, 3H), 3.85 (s, 3H); IR (KBr, cm-1): 3386, 2955, 1699, 1514, 1435, 1373, 1290, 1103, 1029, 983, 874, 778, 766; Anal. Calcd. for C8H8O3 : C, 63.15; H, 5.30. Found C, 63.9; H, 4.92. 3,4,5-Tris-(2,4-dichloro-benzyloxy)-benzoic acid methyl ester (12) Synthesized from 1a; Yield: 74%; M.P: 144°C; MS (ESI) m/z: 662 (100) [M+1]+; 1H NMR (400 MHz, CDCl3): δ 7.53 (s, 3H, ArH), 7.45 (d, 3H, J = 8 Hz), 7.31 (d, 3H, J = 2 Hz), 7.11 (s, 2H, ArH), 5.19 (s, 6H), 3.94 (s, 3H); IR (KBr, cm-1): 3076, 2930, 2359, 1724, 1507, 1451, 1390,
1218, 1096, 1016, 808, 725, 548; Anal. Calcd. for C29H20Cl6O5 : C, 52.68; H, 3.05. Found C, 52.10; H, 3.67. 3,4,5-Tris-(2,5-dichloro-benzyloxy)-benzoic acid methyl ester (13) Synthesized from 1a; Yield: 59%; M.P: 119°C; MS (ESI) m/z: 661 (100) [M+1]+; 1H NMR (400 MHz, CDCl3): δ 7.68 (d, 3H, J = 2.4 Hz), 7.57 (d, 3H, J = 2.4 Hz), 7.27 (s, 3H, ArH), 7.18 (s, 2H, ArH), 5.23 (s, 6H), 3.95 (s, 3H); IR (KBr, cm-1): 3076, 2930, 2359, 1724, 1507, 1432, 1360, 1188, 1096, 1016, 808, 725, 548; Anal. Calcd. for C29H20Cl6O5 : C, 52.68; H, 3.05; Cl, 32.17; O, 31.55; Found C, 53.02; H, 3.45. 3,4-Bis-(3-chloro-benzyloxy)-benzoic acid methyl ester (14) Synthesized from 2a; Yield: 76%; M.P: 148°C; MS (ESI) m/z: 417 (100) [M+1]+; 1H NMR (400 MHz, CDCl3): δ 7.70 (d, 1H, ArH, J = 2 Hz), 7.68 (s, 1H, ArH), 7.48 (m, 2H, ArH), 7.36 (d, 2H, ArH, J = 8 Hz), 7.32 (d, 1H, J = 1.6 Hz), 6.96 (d, 2H, ArH, J = 8.4 Hz), 3.90 (s, 3H); IR (KBr, cm-1): 3847, 3637, 2891, 1721, 1519, 1422, 1377, 1274, 1109, 1038, 992, 774, 679; Anal. Calcd. for C22H18Cl2O4 : C, 63.32; H, 4.35.1 Found C, 63.96; H, 4.65. 4-(2,4-Dichloro-benzyloxy)-3-methoxy-benzoic acid methyl ester (15) Synthesized from 3a; Yield: 68%; M.P: 78°C; MS (ESI) m/z: 342 (100) [M+1]+; 1H NMR (400 MHz, CDCl3): δ 7.48 (d, 1H, ArH, J = 2 Hz), 7.46 (s, 1H, ArH), 7.23 (s, 1H, ArH), 7.12 (d, 1H, ArH, J = 1.4 Hz), 7.09 (d, 1H, J = 2 Hz), 6.71 (d, 1H, ArH, J = 8.4 Hz), 3.79 (s, 3H), 3.74 (s, 3H); IR (KBr, cm-1): 3079, 2967, 1682, 1505, 1434, 1386, 1294, 1099, 821, 764; Anal. Calcd. for C16H14Cl2O4 : C, 56.32; H, 4.14. Found C, 55.98; H, 4.51. 2.2 Determination of Antipathogenic activity: Paper disk-diffusion assay
To detect the Anti-Quorum Sensing activity of the compounds on a biomonitor strain of Chrobacterium violaceum, ATCC 12472, a standard paper disk-diffusion assay was used as described previously6. C. violaceum ATCC 12472 was grown on LB agar and provided with proper antibiotic. LB agar (0.3 %, w/v) volume five milliliters was inoculated with 50 mL of a culture of the bacterium. After preparation the culture solution was poured over the surface of pre-warmed LB agar plates. Compounds to be tested were first dissolved into solvent and after that were pipetted onto sterile paper disks placed on the solidified agar. Later on plates were incubated overnight at 370C and examined for violacein production. Anti-Quorum Sensing activity was analyzed by a colorless, opaque halo zone with viable bacterial cells around the disk. 2.3 Extraction and quantification of violacein Extraction of violacein pigment was carried out as per method of Singh et al., [14]. Bacterial cells were grown in absence and presence of test compounds, lysed by SDS and incubated at room temperature. Quantification of extracted violacein was achieved at A585. The extracted violacein was quantified at A585. 2.4 Anti-biofilm assay In anti-biofilm assay, microtitre dishes of polystyrene were assayed basically while described by Singh et al. [14], with few changes. In short, culture of P. aeruginosa in AB minimal medium incubated overnight, was diluted 1:100 in fresh medium and grown for one more hour. Later on, various concentrations of test compounds solution were pipetted into the wells of the microtitre dishes and incubated for 48 h at 300C. After medium removal, 100 ml of a 1% (w/v) aqueous solution of crystal violet (CV) was added. Subsequent staining at room temperature intended for 20 min, the dye was removed and the wells were washed thoroughly.
After that plates were rinsed to remove planktonic cells, then quantification of surface-attached cells were achieved by solubilizing the dye in ethanol and measuring at A650. 2.5 Statistical analysis Results of Anti-Quorum Sensing activities have been presented as the mean ± standard deviation (SD) of minimum three determinations. Statistical analysis was done using one way ANOVA software and significance of findings was considered at P < 0.05. 3. Results Loss of purple pigment in C. violaceum 12472 is indicative of QS inhibition by the test samples introduced. Out of 7 derivatives screened for Anti-Quorum Sensing activity, 5 derivatives (1 mg/mL each) proved to be effective: (9a), (10a), (12), (13), (15) (Figure -1). The Anti-QS activities of the 5 derivatives were screened using the C. violaceum bioassay. Control discs contained halogenated furanone and ethanol was also used. The positive control furanone (C-30) at 10µg/mL have showed 72% inhibition. As expected, a zone of growth inhibition was observed with gentamycin, a zone of QS inhibition (halo) was seen with the furanone, and no inhibition was apparent with ethanol. Inhibitory effect of these derivatives on violacein pigment production was also measured and violacein production was observed to be inhibited by the derivatives (Scheme-1). The inhibitory effects were: (C-30) 72%, (9a), (10a) 47.2%, (12) 27.3%, (13) 40.1% and (15) 22.7% at the concentration of 1 mg/mL. There was a significant reduction in P. aeruginosa biofilm formation recorded when the bacterium was grown in the presence of 1 mg/mL. Percent inhibitions of biofilm formation were quantified for (C-30) 64% (9a) 46.2%, (10a) 40.3%, (12) 18.4%, (13) 35.2%, and (15) 17.3% when compared with the untreated control (Figure-1 & 2).
4. Discussion In the present work, Anti-Quorum Sensing activities of novel derivatives were examined. Recent research findings have shown that secondary metabolites produce from natural resources viz. plants, fungi, eukaryotic and even animals, interfere with bacterial cell to cell communication system [15-17]. Quorum Quenching could be considered as most effective alternative strategy to combat microbial infection as it reduce the probability of development of multidrug resistant pathogens. Plants are continuously exposed to microbial infection and results into development of sophisticated chemicals to inhibit microbial pathogenesis. Secondary metabolites play an important role as active Anti-Quorum Sensing compounds or lead skeleton in the development of novel Anti-Quorum Sensing drug. Moreover, the identified Quorum Sensing blockers are more reactive and toxic, which raise the concern of using them as medicine in industry and agriculture [16]. Although, targeting cell to cell communication (Quorum Sensing) to control microbial infections in humans, animals and plants seems to produce much promise. Therefore, in the present investigation, we have planned to screen secondary metabolites viz. Gallic acid (9), Protocatechuic acid (10), Vanilic acid (11) and its semi-synthetic derivatives 3,4,5trihydroxybenzoic acid (9a), 3,4-dihydroxybenzoic acid (10a), 4-hydroxy-3-methoxybenzoic acid (11a), 3,4,5-Tris-(2,4-dichloro-benzyloxy)-benzoic acid methyl ester (12), 3,4,5-Tris-(2,5dichloro-benzyloxy)-benzoic acid methyl ester (13), 3,4-Bis-(3-chloro-benzyloxy)-benzoic acid methyl ester (14) and 4-(2,4-Dichloro-benzyloxy)-3-methoxy-benzoic acid methyl ester (15) for their Quorum Sensing activities using disk diffusion assay and anti-biofilm assay.
In the
experiment, C. violaceum 12472 indicator strain was used. Findings of experiments revealed that the Compounds (9a), (10a), (12), (13) and (15) are effectively inhibit Quorum Sensing regulated violacein pigment production in C. violaceum 12472 without interrupting its growth. While, in
zone of growth inhibition, no inhibition was apparent with ethanol. On the basis of findings, it can be mentioned that the negative effect of the secondary metabolites and its novel semisynthetic derivatives on the violacein production is caused by disruption of cell to cell communication (Quorum Sensing signaling system), without inhibition of growth. Inhibition of cell to cell communication without interfering with growth, is highly encoring approach to combat with microbial infection, because there is no probability of development of resistance in bacteria. 5. Conclusion Recent studies have shown that various eukaryotic specimens, including plant, fungi, and even animals produce compounds that interfere with bacterial QS system. Unluckily, most of the QS blockers identified so far are too reactive and toxic, which increases the concern of using these blockers in medicine, agriculture, and industry. However, QS inhibition as a target to control bacterial diseases of human, plant, and animal seems to hold much promise. In this investigation, we therefore decided to screen various derivatives of Gallic acids for their anti-QS property by using different assays including disk diffusion, violacean production, and biofilm formation spectrophotometrically. For disk diffusion assays, C. violaceum 12472, a bioindicator strain was used. Results revealed that the (9a), (10a), (12), (13) and (15) showed Anti-Quorum Sensing activity by inhibiting violacein pigment production and biofilm formation without interfering with its growth. We therefore assumed that the negative effect of the derivatives on violacein production is not caused by inhibition of growth but rather by disruption of QS signaling system. This approach is highly encouraging because when growth is not affected there is no selective pressure for development of resistance in bacteria. Therefore, derivatives of Gallic acid have a
great advantage for human use than toxic anti-QS compounds such as halogenated furanone and convectional antibiotics. Conflict of interest The authors declare that there are no conflicts of interest.
Acknowledgment The authors thank to the Director, CSIR-NBRI Lucknow for his kind support and to make available required resources during this study. First author (NS) is thankful to CSIR (New Delhi) for the award of Senior Research fellowship.
References
1. M.M. Cowan, Plant products as antimicrobial agents, Clin Microbiol Rev, 12 (1999) 564-582.
2. P.M. Dewick, Medicinal Natural Products: A Biosynthetic Approach, Third edn., John Wiley and Sons New York, (2009) .
3. W.D. Bauer, M. Tepletski, Can plants manipulate bacterial quorum sensing, Aust. J. Plant Physiol. 28 (2001) 913-921.
4. S. Schauder, B.L. Bassler, The languages of bacteria, Genes Dev. 15 (2001) 1468-1480.
5. R.S. Smith, B.H. Iglewski, P. aeruginosa quorum-sensing systems and virulence, Curr Opin Microbiol. 6 (2003) 56-60.
6. B.N. Singh, B.R. Singh, R.L. Singh, D. Prakash, R. Dhakarey, G. Upadhyay, H.B. Singh, Oxidative DNA damage protective activity, antioxidant and anti-quorum sensing potentials of Moringa oleifera, Food and Chemical Toxicol. 47 (2009) 1109-1116.
7. Anonymous. The Wealth of India, Raw materials, Council of Scientific and Industrial. Research (CSIR) New Delhi, (1948) 1-179.
8. A.P. Tucci, M.F. Delle, B. Marini, B. Giovanni, Annals 1st super sanita. 5 (1969) 555-556. 9. N. Srivastava, A. Srivastava, S. Srivastava, A.K.S. Rawat, A.R. Khan, HPTLC-densitometric determination and Kinetic studies on antioxidant potential of Monomeric phenolic acids (MPAs) from Bergenia species, RSC Adv. 4 (2014) 52647–52657.
10. A. Borges, C. Ferreira, M.J. Saavedra, M. Simoes, Antibacterial activity and mode of action of ferulic and gallic acids against pathogenic bacteria, Microb Drug Resist. 19 (2013) 256-265. 11. (a) J.C. Song, S. Deresisnski, Curr. Opin. InVest. Drugs 6 (2005) 170–177. (b) W.J. Watkins,
T. E. Renau, In Annual Reports in Medicinal Chemistry; Doherty, A. M., Ed.; Academic Press:
New York 35 (1999) 157-166.
12. G. Slesak, P. Douangdala, S. Inthalad, J. Silisouk, M.Vongsouvath, A. Sengduangphachanh, Catrin E Moore, M. Mayxay, H. Matsuoka, P.N. Newton, Fatal Chromobacterium violaceum septicaemia in northern Laos, a modified oxidase test and post-mortem forensic family G6PD analysis, Ann Clin Microbiol Antimicrob. 8 (2009) 24.
13. B.N. Singh, B.R. Singh, R.L. Singh, D. Prakash, B.K. Sharma, H.B. Singh, Antioxidant and anti-quorum sensing activities of green pod of Acacia nilotica L., Food Chem Toxicol. 47 (2009) 778-786. 14. B.N. Singh, H.B. Singh, A. Singh, B.R. Singh, A. Mishra, C.S. Nautiyal,
Lagerstroemia
speciosa fruit extract modulates quorum sensing-controlled virulence factor production and biofilm formation in Pseudomonas aeruginosa, Microbiology 158(2) (2012) 529-538. 15. A.L. Adonizio, K. Downum, B.C. Bennett, K. Mathee, Anti-quorum sensing activity of medicinal plants in southern Florida, J. Ethnopharmacol. 105 (2006) 427-435.
16. G. Bosgelmez-Tinaz, S. Ulusoy, A. Ugur, O. Ceylan, Inhibition of Quorum Sensing–Regulated Behaviors by Scorzonera sandrasica, Curr. Microbiol. 55 (2007) 114–118.
17. T.B. Rasmussen, T. Bjarnsholt, M.E. Skindersoe, M. Hentzer, P. Kristoffersen, M. Kote, J. Nielsen, L. Eberl, M. Givskov, Screening for quorum-sensing inhibitors (QSI) by use of a novel genetic system, the QSI selector, J. Bactriol. 187 (2005) 1799–1814.
90 80
% Inhibition
70 60 50 40 30 20 10 0 1a 9a
2a 10a
4 12
135
157
C-30 C-30
Derivatives
Figure-1 Inhibitory effect of gallic acid derivatives on production of violacein, (9a), (10a) (12), (13) and (15)
60
% biofilm Inhibition
50 40 30 20 10 0 1a 9a
2a 10a
4 12
5 13
7 15
C-30 C-30
Derivatives
Figure-2 Anti-biofilm formation activity of gallic acid derivaties, (9a), (10a), (12), (13) and (15)
COOH
COOR i
HO
ii
OH HO OH (9)
OHR1O OH (9a) COOR
COOH
OR1 OR1 (12, 13) COOR
i
ii OH
OH OH (10)
OH (10a)
COOH
COOR
OH (11)
COOR
OR1 (14) COOR
i
ii
OCH3
OCH3 OH (11a)
OR1
OCH3 OR1 (15)
(9a, 10a, 11a);
12;
R=CH3
R=CH3; R1=
Cl
H 2C Cl
13;
R=CH3; R1=
Cl
H2 C Cl
14;
R=CH3; R1=
H2C Cl
15;
R=CH3;
R1=
H2 C
Cl Cl
Scheme-1 Reagent and conditions; (i) Thionyl chloride (SOCl2), methanol (CH3OH), 750C, 1h; (ii) K2CO3, 2,4-dichlorobenzyl chloride, 2,5-dichlorobenzyl chloride, 3-chlorobenzyl chloride, acetonitrile (CH3CN)
Annexure-1 (9a) COOCH3
HO
OH OH
(9a)
COOCH3
HO
OH OH
(10a)
COOCH3
OH OH
(10a)
COOCH3
OH OH
(11a) COOCH3
OCH3 OH
(11a)
COOCH3
OCH3 OH
12
COOCH3
Cl O
Cl
Cl
O
O
Cl Cl
Cl
12
COOCH3
Cl O
Cl
Cl
O
O
Cl Cl
Cl
13 COOCH3
Cl Cl
O
O
Cl
O Cl
Cl
Cl
13
COOCH3
Cl Cl
O
O
Cl
O Cl
Cl
Cl
14
COOCH3
O
C
O
Cl
14 COOCH3
O
O
Cl
Cl
15 COOCH3
OCH3
O Cl
Cl
15 COOCH3
OCH3
O Cl
Cl
Annexure-1 1H & 13C-NMR Spectra of derivatives of Gallic acid