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Article Cite This: J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Synthesis and Evaluation of Antimicrobial and Antibiofilm Properties of A‑Type Procyanidin Analogues against Resistant Bacteria in Food Alfonso Alejo-Armijo,† Nicolás Glibota,‡ María P. Frías,§ Joaquín Altarejos,† Antonio Gálvez,‡ Sofía Salido,*,† and Elena Ortega-Morente*,‡ †

Departamento de Química Inorgánica y Orgánica, ‡Departamento de Ciencias de la Salud, and §Departamento de Estadística e Investigación Operativa, Facultad de Ciencias Experimentales, Universidad de Jaén, Campus de Excelencia Internacional Agroalimentario, ceiA3, 23071 Jaén, Spain S Supporting Information *

ABSTRACT: Natural A-type procyanidins have shown very interesting biological activities, such as their proven antiadherence properties against pathogenic bacteria. In order to find the structural features responsible for their activities, we describe herein the design and synthesis of six A-type procyanidin analogues and the evaluation of their antimicrobial and antibiofilm properties against 12 resistant bacteria, both Gram positive and Gram negative, isolated from organic foods. The natural A-type procyanidin A-2, which had known antiadherence activity, was also tested as a reference compound for the comparative studies. Within the series, analogue 4, which had a NO2 group on ring A, showed the highest antimicrobial activity (MIC of 10 μg/mL) and was one of the best molecules at preventing biofilm formation (up to 40% decreases at 100 μg/mL) and disrupting preformed biofilms (up to 40% reductions at 0.1 μg/mL). Structure−activity relationships are also analyzed. KEYWORDS: A-type procyanidin analogues, procyanidin A-2, flavylium salts, antimicrobial activity, antibiofilm activity, resistant bacteria

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INTRODUCTION There is increasing concern about the growing number of foodborne-illness outbreaks caused by some pathogens coupled with the high rate of antibiotic resistance associated with foodborne infections.1 There has therefore been interest in developing new types of effective antimicrobial compounds derived from natural sources.2 Many of the phytochemicals and plant extracts evaluated as to their bactericidal activities against foodborne pathogens have been active against both nonresistant (susceptible) and antibiotic-resistant (resistant) bacteria in buffers and in liquid and solid foods.3,4 However, there is still a need to develop new, preferably inexpensive, alternatives to standard antibiotics for use against antibioticresistant bacteria, thereby providing more options for the treatment of livestock and poultry and reducing the exposure of humans to resistant bacteria. A challenging objective is to develop candidates that could be incorporated into formulations to reduce both susceptible and resistant pathogens in animal feeds, food-animal environments, and human foods. Foodborne bacteria are recurringly found on wet surfaces in the food industry and in refrigerated food factories, despite routine cleaning and disinfection.5 A bacterium’s ability to adhere to and grow on a variety of surfaces found in food-processing plants, including stainless steel, rubber, glass, and polypropylene, and its capacity to form biofilms on biotic or abiotic surfaces6,7 are the main factors resulting in bacteria being continuously introduced into processing environments and cross contaminating food-contact surfaces, equipment, floors, drains, and other locations. Thus, the antibiofilm properties of candidates are of prime importance when studying their possible use in the food-industry environment. © XXXX American Chemical Society

Being aware of the current interest in proanthocyanidins, natural polyphenols structurally based on flavan-3-ols,8 we have recently investigated the antimicrobial and antibiofilm activities of two proanthocyanidins, belonging to a subgroup of procyanidins, isolated from laurel (Laurus nobilis L.)-wood extracts against several foodborne microorganisms.9 One of them was an A-type procyanidin (cinnamtannin B-1), and the other one was a B-type procyanidin (procyanidin B-2, Figure 1). A-type procyanidins have at least a double interflavanyl linkage between two (epi)catechin units, whereas B-type procyanidins have single interflavan-bond linkages among monomers. In that work, cinnamtannin B-1 was found to have higher antimicrobial activity than procyanidin B-2,9 which is consistent with the higher activity of A-type procyanidins versus B-type procyanidins previously reported in relation to, for example, their antiadherence properties against uropathogenic Escherichia coli.10 It has been suggested that the conformational rigidity that the double interflavanyl linkages give to the A-type procyanidins provide these molecules with favorable antiadhesion activities compared with those of the Btype procyanidins.10 In order to broaden the knowledge about structural features that could influence the antimicrobial and antibiofilm activities of A-type procyanidins against foodborne pathogens, we have now designed a set of analogues to the Atype procyanidin A-2, the structurally simplified version of cinnamtannin B-1 (Figures 1 and 2), and we have tested their antimicrobial and antibiofilm activities against a selection of Received: January 29, 2018 Revised: February 9, 2018 Accepted: February 10, 2018

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DOI: 10.1021/acs.jafc.8b00535 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Figure 1. Structures of cinnamtannin B-1 (an A-type procyanidin) and procyanidin B-2 (a B-type procyanidin), both isolated from laurel (Laurus nobilis L.) wood and evaluated against foodborne pathogens,9 and procyanidin A-2 (an A-type procyanidin), the reference compound used in this work.

Figure 2. Synthetic route for the preparation of A-type procyanidin analogues 1−6 from flavylium salts 13−17 and catechin or phloroglucinol. Reagents and conditions: (i) H2SO4, HOAc; (ii) MeOH or EtOH, HCl(g); (iii) MeOH (23−60% from the starting aldehyde); (iv) MeOH/H2O, MW (12% from the starting aldehyde); (v) MeOH, MW (27% from the starting aldehyde).

analogues following a procedure based on flavylium chemistry,11 their antimicrobial and antibiofilm activities against 12

resistant bacteria previously isolated from organic foods. We therefore describe here the synthesis of the procyanidin B


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and a red solid precipitated. The solid was filtered off, carefully washed with Et2O, and dried. 3′,4′-Dihydroxyflavylium hydrogen sulfate (13) was obtained by procedure A with 2-hydroxybenzaldehyde (7, 0.1 mL, 1 mmol) and 11 (0.152 g, 1 mmol) as a red solid (0.301 g). 3′,4′,7-Trihydroxyflavylium hydrogen sulfate (15) was obtained by procedure A with 2,4-dihydroxybenzaldehyde (9, 0.138 g, 1 mmol) and 11 (0.152 g, 1 mmol) as a red solid (0.277 g). 6-Nitro-3′,4′-dihydroxyflavylium hydrogen sulfate (16) was obtained by procedure A with 2-hydroxy-5-nitrobenzaldehyde (10, 0.167 g, 1 mmol) and 11 (0.152 g, 1 mmol) as a red solid (0.293 g). General Procedure B for the Synthesis of Flavylium Salts 14 and 17. As shown in Figure 2, a mixture of a benzaldehyde derivative (7 or 8) and an acetophenone derivative (11 or 12) in absolute ethanol or methanol was saturated with dry HCl gas for 1 h. The reaction mixture was stirred overnight following a procedure similar to that described by Kraus et al.14 Then, the solvent was removed, Et2O was added, and a solid precipitated. The solid was filtered off, carefully washed with Et2O, and dried. 3′,4′,5,7-Tetrahydroxyflavylium chloride (14) was obtained by procedure B with 2,4,6-triacetoxybenzaldehyde (8, 0.280 g, 1 mmol), prepared from 2,4,6-trihydroxybenzaldehyde according to the literature,15 and 3′,4′-dihydroxyacetophenone (11, 1.064 g, 7 mmol) as a brown solid (0.122 g). In this case, a solution of compound 8 in MeOH (30 mL) was added to a solution of 11 in MeOH (40 mL, previously saturated with dry HCl(g)). The resulting solid was recrystallized in MeOH/EtOAc (8:2). 3-Chloro-3′,4′-dihydroxyflavylium chloride (17) was obtained by procedure B with 2-hydroxybenzaldehyde (7, 0.4 mL, 4 mmol), 2chloro-3′,4′-dihydroxyacetophenone (12, 0.745 g, 4 mmol), and EtOH (40 mL) as a purple solid (1.181 g). General Procedure C for the Synthesis of A-type Procyanidin Analogues 1 and 4−6. As shown in Figure 2, a mixture of a flavylium salt (13, 16, or 17) and (+)-catechin hydrate or phloroglucinol (0.5 mmol) in absolute methanol (8 mL) was stirred overnight at 50 °C following a similar procedure to that described by Kraus et al.16 Then, the solvent was removed, and the crude was purified by semipreparative HPLC or SEC. 2-(3′,4′-Dihydroxyphenyl)chromane-(4→8,2→O-7)-catechin (analogue 1) was synthesized by procedure C with flavylium salt 13 (0.168 g) and (+)-catechin hydrate (0.154 g, 0.5 mmol). The crude was purified by semipreparative HPLC. Separation was carried out with H2O/CH3COOH (99.8:0.2, v/v, solvent A) and MeOH/CH3COOH (99.8:0.2, v/v, solvent B) at a flow rate of 5 mL/min, a linear gradient from 45−70% B for 15 min, 70% B for 3 min, a linear gradient from 70−100% B for 2 min, and 5 min to return to the initial conditions. Pure analogue 1 (a white foam) was obtained as a 1.3:1 mixture of diastereomers (0.084 g, 23% from aldehyde 7). 2-(3′,4′-Dihydroxyphenyl)-6-nitrochromane-(4→4′′,2→O-5′′)phloroglucinol (analogue 4) was synthesized by procedure C with flavylium salt 16 (0.191 g) and phloroglucinol (0.063 g, 0.5 mmol). The crude was purified by SEC. Purification on a Sephadex LH-20 and elution with MeOH/H2O (90:10) yielded pure analogue 4 as a brownish solid (0.160 g, 60% from aldehyde 10). 3-Chloro-2-(3′,4′-dihydroxyphenyl)chromane-(4→4′′,2→O-5′′)phloroglucinol (analogue 5) was synthesized by procedure C with flavylium salt 17 (0.154 g) and phloroglucinol (0.063 g, 0.5 mmol). The crude was purified by semipreparative HPLC. Separation was carried out with H2O/CH3COOH (99.8:0.2, v/v, solvent A) and MeOH/CH3COOH (99.8:0.2, v/v, solvent B) at a flow rate of 5 mL/ min, a linear gradient from 35−50% B for 10 min, 50% B for 5 min, a linear gradient from 50−100% B for 5 min, and 5 min to return to the initial conditions. Pure analogue 5 was obtained as a white foam (0.123 g, 60% from aldehyde 7). 3-Chloro-2-(3′,4′-diacetoxyphenyl)chromane-(4→4′′,2→O-5′′)1,3-diacetoxyphloroglucinol (analogue 6) was obtained by procedure C with flavylium salt 17 (0.154 g) and phloroglucinol (0.063 g, 0.5 mmol). Then, the solvent was removed, the crude was dissolved in Ac2O (3.6 mL, 19.6 mmol), and KOAc was added (0.127 g, 0.66 mmol). The mixture was stirred at 85 °C for 48 h and poured into a

Gram-positive and Gram-negative resistant bacteria previously isolated from organic foods, and the resulting structure−activity relationships.

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MATERIALS AND METHODS

Chemicals and Instruments. The following commercially available reagents and standard were used without further purification: phloroglucinol, (+)-catechin, aldehydes 8 and 9, and ketone 12 (Sigma-Aldrich Chemie, Steinheim, Germany); aldehyde 7 (Acros Organics, Geel, Belgium); aldehyde 10 and ketone 11 (Alfa Aesar, Karlsruhe, Germany); and procyanidin A-2 (99%, Extrasynthese, Genay Cedex, France). All the solvents used in the chemical syntheses and preparative chromatographies were used as received (Panreac, Barcelona, Spain). The methanol and ethanol used for the synthesis of the A-type analogues were dried according to standard methods.12 The methanol used for high-performance liquid chromatography (HPLC) was of HPLC grade (VWR Chemicals, Prolabo, Fontenay-sous-Bois, France). Deuterated methanol (CD3OD), acetonitrile (CD3CN), and chloroform (CDCl3) were used to prepare solutions of purified compounds for nuclear magnetic resonance (NMR). In some cases, some drops of D2O were added to exchange with the hydroxyl hydrogens in order to simplify the signals observed in the spectrum. For the flavylium salts, DCl was added to acidify the solutions. Analytical thin-layer chromatography (TLC) was performed on silica gel 60 F254 precoated aluminum sheets (0.25 mm, Merck Chemicals, Darmsdadt, Germany). Silica gel 60, 200−400 mesh (Merck Chemicals), was used for the silica-gel column chromatography (CC), and Sephadex LH-20 (Sigma-Aldrich Chemie) was used for the size-exclusion chromatography (SEC). Analytical-HPLC analyses were performed on a C18 reversed-phase Spherisorb ODS-2 column, 250 × 3 mm i.d., 5 μm (Waters Chromatography Division, Milford, MA). The best separation was obtained with H2O/CH3COOH (99.8:0.2, v/v, solvent A) and methanol/CH3COOH (99.8:0.2, v/v, solvent B) at a flow rate of 0.7 mL/min with a linear gradient from 30−100% B for 25 min followed by 100% B for 15 min and a return to the initial conditions in 5 min. The total run time excluding equilibration was 45 min. Semipreparative-HPLC separations were performed on a C18 reversed-phase Spherisorb ODS-2 column, 250 × 10 mm i.d., 5 mm (Waters), on the instrument described above at flow rate of 5 mL/min. 1 H NMR and 13C NMR spectra were recorded on an Avance 400 spectrometer (Bruker Daltonik GmbH, Bremen, Germany) operating at 400.13 and 100.03 MHz for 1H and 13C, respectively. The chemical shifts (in ppm) were expressed with respect to tetramethylsilane (TMS), the internal reference. The coupling constants (J) are quoted in hertz (Hz). The following abbreviations are used: s, singlet; d, doublet; t, triplet; m, multiplet; br s, broad singlet; br d, broad doublet; dd, doublet of doublets; ddd, doublet of doublets of doublets; dt, doublet of triplets. The complete assignments of the 1H and 13C signals were performed by analyses of the correlated homonuclear (COSY) and heteronuclear (HMBC, HSQC) spectra. Ultraviolet (UV) spectra were obtained in methanol on a Cary 4000 UV−vis spectrophotometer (Varian Inc., Palo Alto, CA). For the flavylium salts, HCl was added to acidify the solution. Infrared (IR) spectra were recorded on a Tensor 27 Fourier-transform infrared (FTIR) spectrometer (Bruker Optik GmbH, Ettlingen, Germany) fitted with a single-reflection attenuated-total-reflection (ATR) accessory. High-resolution mass spectra (HRMS) were recorded on a 6520B quadrupole time-of-flight (QTOF) mass spectrometer (Agilent Technologies, Santa Clara, CA). The microwave-assisted reaction was carried out with a CEM microwave synthesizer (CEM Discover, Matthews, NC). General Procedure A for the Synthesis of Flavylium Salts 13, 15, and 16. As shown in Figure 2, a mixture of a benzaldehyde derivative (7, 9, or 10; 1 mmol), 3′,4′-dihydroxyacetophenone (11, 1 mmol), 98% H2SO4 (0.3 mL, 5.4 mmol), and HOAc (1.3 mL) was stirred overnight at room temperature following a similar procedure to that described by Calogero et al.13 Then, Et2O (30 mL) was added, C


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Journal of Agricultural and Food Chemistry flask containing 50 g of crushed ice. The solution was then extracted with EtOAc (20 mL), and the organic phase was washed with brine and dried over anhydrous Na2SO4. The solvent was removed, and the crude was purified by silica-gel chromatography with EtOAc and hexane as the eluents, yielding pure analogue 6 as a white foam (0.133 g, 46% from aldehyde 7). General Procedure D for the Synthesis of A-type Procyanidin Analogues 2 and 3. As shown in Figure 2, a mixture of a flavylium salt (14 or 15) and phloroglucinol (0.2 mmol) in a mixture of MeOH (3 mL) and an aqueous buffer (pH 5.8, 3 mL) or in absolute MeOH (6 mL) was heated in a microwave reactor at 80 °C until the phloroglucinol was consumed, a similar procedure to that described by Selenski and Pettus.15 Then, the solvent was removed and the crude was purified by semipreparative HPLC. Separation was carried out with H2O/CH3COOH (99.8:0.2, v/v, solvent A) and MeOH/CH3COOH (99.8:0.2, v/v, solvent B) at a flow rate of 5 mL/ min, a linear gradient from 35−50% B for 10 min, a linear gradient from 50−100% B for 5 min, and 5 min to return to the initial conditions. 2-(3′,4′-Dihydroxyphenyl)-5,7-dihydroxychromane-(4→4′′,2→O5′′)-phloroglucinol (analogue 2) was obtained by procedure D with flavylium salt 14 (0.062 g) and phloroglucinol (0.025 g, 0.2 mmol) in a mixture of MeOH (3 mL) and an aqueous buffer (pH 5.8, 3 mL) as a white foam (0.023 g, 12% from aldehyde 8). 2-(3′,4′-Dihydroxyphenyl)-7-hydroxychromane-(4→4′′,2→O-5′′)phloroglucinol (analogue 3) was obtained by procedure D with flavylium salt 15 (0.070 g) and phloroglucinol (0.025 g, 0.2 mmol) in absolute MeOH (6 mL) as a white foam (0.026 g, 27% from aldehyde 9). Antimicrobial Activity. The antimicrobial activities of analogues 1−6 were tested on bacterial strains previously isolated in our laboratory from organic foods, identified, and classified as resistant to biocides and antibiotics (Table 1).17 The compounds (with purities higher than 95% according to 1H NMR) were dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich Chemie) to concentrations of 10

mg/mL. These solutions were stored at −20 °C and serially diluted in tryptic soy broth (TSB, Scharlab, Barcelona, Spain) for the antimicrobial and antibiofilm assays. Procyanidin A-2 was used as a standard within this study in order to compare its effect with those of the analogues. The antimicrobial activities of procyanidin A-2 and analogues 1−6 were initially screened by the standard agar-diffusion method, and then they were subjected to minimal-inhibitoryconcentration (MIC) tests. Standard Agar-Diffusion Method. The indicator strains were grown in Mueller-Hinton broth (MHB, Becton Dickinson, Sparks, MD) at 37 °C. For the antimicrobial-activity analysis, 100 μL of each microbial suspension adjusted to 1.0 × 108 colony forming units (CFU)/mL was poured onto plates with 15 mL of Mueller-Hinton agar medium (MHA) and dispersed using a microbiological rake. The plates were allowed to dry, and 5 μL of one of the 10-fold serially diluted purified compounds (1 mg/mL starting concentration) was placed on the surface of a test MHA plate and incubated at 37 °C for 24 h. The vehicle (DMSO at the same concentration reached in each serial dilution of the compounds tested) was used as the negative control, and all the samples were analyzed in triplicate. The antimicrobial activity was expressed in terms of the average diameter of the zone of inhibition in millimeters. A zone of inhibition ≤5 mm was interpreted as the absence of antimicrobial activity. Minimal-Inhibitory-Concentration Test. MIC values were determined by the broth microdilution method in 96-well microtiter plates (Becton Dickinson Labware, Franklin Lakes, NJ) as recommended by the Clinical and Laboratory Standards Institute.18 Briefly, dilutions of each substance (ranging from 1 to 500 μg/mL, according to the results obtained in the preliminary screenings of the antimicrobial activities in the agar-diffusion tests) were incubated with microbial suspensions adjusted to 5 × 105 CFU/mL in TSB.19 Growth and sterility controls were included for each isolate, and the vehicle was included as a negative control. The microtiter plates were incubated at 37 °C, and readings were performed after 20 h of incubation by visual reading and optical-density (OD 595 nm) determination in an iMark microplate reader (BioRad, Madrid, Spain). The MIC value was defined as the lowest compound concentration that prevented cell growth after a 20 h incubation. The reported MIC data were from three independent assays. Biofilm-Formation-Inhibition Assay. The inhibition of biofilm formation by procyanidin A-2 and analogues 1−6 was measured by the crystal-violet-stain method described by Djordjevic et al.20 with some modifications. Bacterial suspensions (105 CFU in TSB) were incubated with increasing concentrations of each compound (from 0.1 to 100 μg/mL) or without a treatment in a 96-well plate (24 h, 30 °C). The wells were washed with tap water, and the biofilms were fixed with methanol. The plate was stained with 0.3% crystal violet for 5 min, washed, and reconstituted with absolute ethanol. The absorbance was read at 595 nm on an iMark microplate absorbance reader (BioRad). Disruption of Preformed Biofilm. The disruption of preformed biofilms was assayed as previously described.21,22 After biofilms formed (24 h, 30 °C) in a 96-well plate, the contents of all of the experimental and control wells were aspirated, the wells were gently washed twice with sterile physiological saline, and 100 μL of the appropriate diluted compound was added. Wells without any treatment served as the positive controls. After an additional incubation (24 h, 30 °C), the crystal-violet stain was performed as described for the biofilmformation-inhibition assay. Statistical Analysis. All the experiments were carried out in triplicate. The average data ± the standard deviations of the absorbances at OD595 were determined with Excel (Microsoft Corporation, Redmond, WA). A t test was performed at the 95% confidence level with Statgraphics Plus version 5.1 (Statistical Graphics Corporation, Rockville, MD), in order to determine the statistical significance of the data corresponding to the biofilm-formation inhibition and disruption of preformed biofilms.

Table 1. Identification of Bacterial Isolates from Organic Foods with High Levels of Biocides or Antibiotic Tolerancesa isolate

species

sample of origin

UJA27q UJA11e

Bacillus cereus Enterococcus casselif lavus Enterococcus faecium

eggplant spelt wholemeal flour spelt wholemeal flour potato

UJA11c

UJA35h

Staphylococcus aureus Staphylococcus saprophyticus Lactobacillus casei

UJA37p UJA7m UJA29o

Enterobacter sp. Pantoea agglomerans Pantoea agglomerans

UJA32j UJA40k

Klebsiella terrigena Salmonella sp.

UJA40l

Salmonella sp.

UJA34f UJA27g

eggplant cheese from raw milk tomato guacamole lettuce carrot Moroccan red pepper Moroccan red pepper

antibiotic and biocide resistanceb AM, BC, CE, CH, HDP AM, EM, AB, CE, CF, HDP AM, EM, AB, CE, CF, CH, HDP CX, EM, CE, CH, HDP, TC EM, CF, CH, HDP AM, CP, CX, EM, NF, CE, CH CE, CF, CH, HDP CP, CX, CE, CH AM, CP, CX, EM, CE, CH, TC AM, CX, CE, CF, CH AM, CX, EM, CF, CH CF, CH, HDP

a

These strains were previously isolated in our laboratory from organic foods, identified, and characterized as resistant to biocides and antibiotics.17 bAM, amoxicillin; CP, ciprofloxacin; CX, cefuroxime; EM, erythromycin; NF, nitrofurantoin; AB, didecyldimethylammonium bromide; BC, benzalkonium chloride; CE, cetrimide; CF, hexachlorophene [2,20-methylenebis(3,4,6-trichlorophenol)]; CH, chlorhexidine; HDP, hexadecylpyridinium chloride; TC, triclosan. D


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RESULTS AND DISCUSSION Procyanidin A-2, a natural product that has shown adherence inhibition (Figure 1),23 was proposed in this work as reference compound for comparative purposes. Its antimicrobial and antibiofilm activities against resistant bacteria have been compared with those of analogues 1−6 (Figure 2), which are closely related to procyanidin A-2. These analogues keep its Alinkage and catechol unit (ring B) but differ in the substitution patterns on rings A and C and in the nature of the lower monomer, which is catechin for analogue 1 and phloroglucinol for analogues 2−6. To our knowledge, the activities of analogues 1−6 are reported here for the first time. Chemistry. The synthetic route followed to prepare analogues 1−6 is outlined in Figure 2. These compounds were synthesized by nucleophilic attacks of (+)-catechin or phloroglucinol on flavylium salts 13−17, which were prepared through the acid-catalyzed condensation of o-hydroxybenzaldehyde and the derivatives (7−10) with 3′,4′-dihydroxyacetophenone and a chloro derivative (11 or 12). The flavylium salts were prepared following two different procedures. The classic method, which uses a solution of sulfuric acid in acetic acid,13 could be successfully applied for the synthesis of salts 13, 15, and 16. However, this conventional method did not allow us to prepare salts 14 and 17. Instead, an unidentified polymer was formed. In order to get salts 14 and 17, several modifications of the above method had to be assayed. Finally, the use of a dry hydrogen chloride stream over a methanol/ethanol solution of the acetophenone derivative (and the subsequent addition of the benzaldehyde derivative) or over a mixture of both the acetophenone and benzaldehyde derivatives allowed, respectively, the synthesis of salts 14 and 17 following similar protocols to those described in the literature.14,15 These colored solids (13−17) were used without further purification to perform the synthesis of analogues 1−6, for which it was also necessary to follow two different procedures. The use of one or the other method depended exclusively on the electronic density of the flavylium salt. Thus, flavylium salts with relatively lower electronic densities, such as 13, 16, and 17, were able to react with (+)-catechin and phloroglucinol in methanol at 50 °C to give analogues 1, 4, and 5 in moderate yields (23−60% from the starting aldehyde),16 whereas the flavylium salts with relatively higher electronic densities, such as 14 and 15, only reacted with phloroglucinol to give analogues 2 and 3 (12−27% from the starting aldehyde) when microwave irradiation was applied.15 Analogue 6 was prepared from analogue 5 by simple acetylation. It seemed that the microwave radiation enhanced the reactivity of the flavylium salts with electron-donating substituents on ring A over those with nucleophiles, although it reduced the stability of the resulting bicyclic structure, causing a diminution of the overall outcome. The structures of the synthesized compounds were characterized by IR, UV−vis, 1H NMR, 13C NMR, and 2D NMR spectroscopy as well as by HRMS spectrometry. The spectroscopic data of the known compounds, 13,13 14,15 15,13 16,16 analogue 1,16 analogue 2,15 and analogue 4,16 were consistent with the literature. Out of the 11 synthetic compounds, 4 of them (salt 17 and analogues 3, 5, and 6) were new. Antimicrobial Activity. The target strains used in this study had been previously isolated from organic foods, identified, and classified as highly tolerant to biocides and/or antibiotics (Table 1).17 The standard agar-diffusion method was

used to procure preliminary screenings of the antimicrobial activities of procyanidin A-2 and the complete set of analogues. The zones of inhibition of the most active compounds at concentrations of 1 mg/mL on the target strains are shown in Table 2. The best antimicrobial-activity results were found with Table 2. Zones of Inhibition of the Most Active Procyanidin Analogues at Concentrations of 1 mg/mLa zone of inhibition (mm) isolate

analogue 1

analogue 4

analogue 5

B. cereus UJA27q E. casselif lavus UJA11e E. faecium UJA11c S. aureus UJA34f S. saprophyticus UJA27g L. casei UJA35h Enterobacter sp. UJA37p P. agglomerans UJA7m P. agglomerans UJA29o K. terrigena UJA32j Salmonella sp. UJA40k Salmonella sp. UJA40l

10 7 1000


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Table 4. Significant Effects of Procyanidin A-2 and A-Type Procyanidin Analogues 1−6 on Biofilm Formation and on the Disruption of Preformed Biofilms of Resistant Strains from Organic Foodsa isolate (active concentration) compound procyanidin A-2 analogue 1

analogue 2 analogue 3

analogue 4

analogue 5

inhibition of biofilm formation

disruption of preformed biofilms

UJA27q (1 μg/mL*) UJA11c (100 μg/mL*) UJA34f (100 μg/mL*) UJA35h (1 μg/mL*)

UJA40k (1 μg/mL**) UJA27g (100 μg/mL*, 1 μg/mL**) UJA11c (10 μg/mL*)

UJA27q (1 μg/mL*, 0.1 μg/mL*) UJA35h (1 μg/mL*, 0.1 μg/mL*) UJA40k (0.1 μg/mL*) UJA11c (100 μg/mL*, 10 μg/mL*) UJA32j (100 μg/mL*) UJA40k (100 μg/mL*)

UJA32j (0.1 μg/mL*) UJA37p (1 μg/mL*) UJA40k (1 μg/mL*) UJA40l (1 μg/mL*)

analogue 6

a

UJA11c (10 μg/mL*, 1 μg/mL*, 0.1 μg/mL*) UJA34f (10 μg/mL*, 0.1 μg/mL*) UJA35h (10 μg/mL*, 1 μg/mL**, 0.1 μg/mL*) UJA37p (10 μm/mL**, 1 μg/mL*, 0.1 μg/mL**) UJA40k (10 μg/mL*, 1 μg/mL*, 0.1 μg/mL*) UJA11c (10 μg/mL*, 1 μg/mL*, 0.1 μg/mL*) UJA34f (10 μg/mL*, 0.1 μg/mL*) UJA35h (10 μg/mL*, 0.1 μg/mL*) UJA37p (10 μg/mL**, 1 μg/mL*, 0.1 μg/mL**) UJA40k (1 μg/mL*, 0.1 μg/mL*) UJA35h (10 μg/mL*, 1 μg/mL**, 0.1 μg/mL*) UJA37p (10 μg/mL*, 0.1 μg/mL*) UJA40k (10 μg/mL*, 1 μg/mL*)

*p < 0.05, **p < 0.01.

the strains except B. cereus UJA27q (MIC of 50 μg/mL) and L. casei UJA35h (MIC of 100 μg/mL). The best antimicrobial effects against the resistant strains (mainly the Gram positives) were found for analogue 4, which showed MIC values of 10 μg/ mL against B. cereus UJA27q and S. saprophyticus UJA27g and 50 μg/mL against all the remaining strains analyzed except for K. terrigena UJA32j (MIC value of 100 μg/mL) and Salmonella sp. UJA40l (MIC of 1 mg/mL). The bioassay results suggest that the absence of electrondonating groups attached to ring A enhances the antimicrobial activities of the analogues. Moreover, the presence of an electron-withdrawing group such as −NO2 on ring A markedly increases the activity, so analogue 4 stands out above the rest of studied compounds. Effect of Procyanidin Analogues on Biofilm Formation. Subinhibitory concentrations of procyanidin A-2 and analogues 1−6 were tested in order to ascertain their effects on biofilm formation by the selected resistant strains. The results are shown in Table 4. Biofilm formation by the target strains was not significantly inhibited in the presence of procyanidin A2 or analogue 2 or 6 at any of the concentrations tested, showing minor increases or decreases in the capacity of biofilm formation depending upon the concentration tested and the target strain. Analogue 1 showed an inhibitory effect on the biofilm formation of Gram-positive strains. It induced 40% inhibitions (p < 0.05) on the formation of biofilms by E. faecium UJA11c and S. aureus UJA34f at the highest concentration tested (100 μg/mL), as well a 22% reduction on the formation of biofilms by L. casei UJA35h at a concentration of 1 μg/mL (p < 0.05). The remaining resistant strains showed no significant alterations in their biofilm formation in the presence of this analogue. Analogue 3 induced significant inhibitions, about 30%, of biofilm formation by B. cereus UJA27q and L. casei UJA35h at concentrations as low as 1 and 0.1 μg/mL. Salmonella sp. UJA40K also showed a 30%

inhibition of its biofilm formation in the presence of 0.1 μg/mL analogue 3 (p < 0.05). However, as the concentration of this compound increased to 100 μg/mL, the inhibitory effect decreased. Analogue 4 induced a significant decrease (30−40%) in biofilm formation by E. faecium UJA11c, K. terrigena UJA32j, and Salmonella sp. UJA40k at 100 μg/mL (p < 0.05). This inhibitory effect was only maintained on E. faecium UJA11c when the dose was lowered to 10 μg/mL. The remaining concentrations tested had no significant effect on any of the strains analyzed. Analogue 5 showed a potent inhibitory effect on biofilm formation by Gram-negative strains at low concentrations. This compound induced a 30% inhibition (p < 0.05) of the formation of biofilms by Enterobacter sp. UJA37p and by strains UJA40k and UJA40l, both identified as Salmonella, at concentrations as low as 1 μg/mL. This compound was also able to inhibit the biofilm formation of K. terrigena UJA32j at 0.1 μg/mL. However, as the concentration of this compound increased to 100 μg/mL, the inhibitory effect decreased, so the highest concentrations tested induced minor decreases in biofilm formation, which were not statistically significant. We have previously described a similar paradoxical effect by cinnamtannin B-1 on the formation of biofilms by E. coli,9 and it had also been previously described when studying the effect of cranberry proanthocyanidins24 and of echinocandins25 on Candida albicans biofilm formation. This effect has been linked to the known tendency of some polyphenols to self-associate in aqueous solutions when the concentration increases26 or to form aggregates with peptides and proteins.27 Both binding processes should necessarily lead to lower effective concentrations of the polyphenols in the media. Regarding the structure−activity relationship on the inhibition of biofilm formation, a trend is found for the seven tested compounds that is similar to their corresponding antimicrobial activities reported above. Analogues 1, 4, and 5 are again among the F


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supposed to be similar for all of the compounds tested on the basis of the cathecol moiety present in all of them, whereas great differences in the antimicrobial and antibiofilm activities have been observed that depend on the substitution patterns on rings A and C. Thus, our results suggest that the iron-chelating ability of procyanidins could not play an essential role in the antibiofilm activities of these compounds, as had been previously suggested by Rane et al.24 In conclusion, we have reported herein the synthesis of six Atype procyanidin analogues using flavylium chemistry, their abilities in inhibiting the growth of 12 resistant bacteria isolated from organic foods, their inhibitory effects on biofilm formation, and their abilities in the disruption of preformed biofilms. These activities have been compared with those of the natural product procyanidin A-2, an active A-type procyanidin used as a reference compound. In all cases, the synthetic compounds were found to show higher antimicrobial activities than procyanidin A-2. Taking into consideration both the antimicrobial and antibiofilm activities, some structure−activity relationships for the tested compounds may be deduced as follows: (a) the absence of OH groups on rings A and C improves the activity (analogue 1 vs procyanidin A-2); (b) a decrease in the size of the bottom monomer also enhances the effectiveness of the derivative (analogues 3−6 vs analogue 1), with the exception of analogue 2; (c) the absence of electrondonating groups on ring A increases the activity (analogues 1 and 4−6 vs analogues 2 and 3); and (d) a relative higher polarity improves the compound’s activity (analogue 5 vs analogue 6). Thus, compound 4, with an electron-withdrawing group on ring A, seems to emerge as a new leading structure for further structure−activity studies and the potential development of new food preservatives and biocides. A-type procyanidin analogues, with their ability to inhibit microbial growth and biofilm formation, may stimulate the market as food preservatives or sanitizers for processing equipment where foodborne pathogens reside. These compounds, with proven antimicrobial and antibiofilm properties, could also be useful in obtaining activated plastics or films for food packaging.

most active compounds against biofilm formation, although analogue 3 also shows activity this time despite the presence of an electron-donating group (−OH) at C-7. However, the occurrence of two −OH groups at the same time in ring A continued to be unfavorable for the inhibitory effects of procyanidin A-2 and analogue 2. Effect of Procyanidin Analogues on the Disruption of Preformed Biofilms. When we allowed the bacteria to attach and form biofilms for 24 h before treatment, exposure to analogues 4, 5, and 6 for an additional 24 h provided the best results on the reduction of preformed biofilms compared with the untreated control for both the Gram-positive and Gramnegative resistant strains (Table 4). Analogues 4 and 5 showed similar effects, with reductions of 30−40% of the biofilms preformed by E. faecium UJA11c, S. aureus UJA34f, L. casei UJA35h, Enterobacter sp. UJA37p, and Samonella sp. UJA40k at 10, 1, and 0.1 μg/mL. The differences were especially significant (p < 0.01) for analogue 4 on L. casei UJA35h at 1 μg/mL and on Enterobacter sp. UJA37p at 10 and 0.1 μg/mL. Analogue 5 also showed the highest effect (p < 0.01) on Enterobacter sp. UJA37p preformed biofilms at 10 and 0.1 μg/ mL. Analogue 6 induced a significant reduction of biofilms preformed by L. casei UJA35h, Enterobacter sp. UJA37p, and Samonella sp. UJA40k at 10 μg/mL; by L. casei UJA35h and Samonella UJA40k at 1 μg/mL; and by L. casei UJA35h and Enterobacter sp. UJA37p at concentrations as low as 0.1 μg/mL. In the final case, the effects were especially significant (p < 0.01). For the remaining tested compounds, the results on the disruption of preformed biofilms were less significant. Procyanidin A-2 only induced a significant reduction (p < 0.05) of biofilms preformed by B. cereus UJA27q at 1 μg/mL. Analogue 1 induced reductions of about 50% (p < 0.01) in S. saprophyticus UJA27g and Salmonella sp. UJA40k preformed biofilms at 1 μg/mL and 35% reductions (p < 0.05) of S. saprophyticus UJA27g preformed biofilms at 100 μg/mL, and analogue 2 only induced a significant disruption of biofilms preformed by E. faecium UJA11c at 10 μg/mL (p < 0.05). We previously reported that the concentration of procyanidin B-2 and disruption of preformed biofilms varies inversely in S. aureus and resistant E. faecalis UJA27t.9 This study again showed that the best results on the disruption of preformed biofilms occurred when low concentrations were used, although strictly inverse proportions were not found. Once more, analogue 4 appeared as one of the compounds in the series with better behavior on the disruption of preformed biofilms along with derivatives 5 and 6. In terms of structure−activity relationships, it seemed to indicate again that the absence of electron-donating groups on ring A might increase a compound’s effects on biofilm disruption. The ability of procyanidins to prevent biofilm formation has been attributed to their ability to inhibit the production of exopolysaccharides,28 prevent bacterial coaggregation,29 reduce bacterial hydrophobicity, and alter cell-surface molecules.30 Previous studies by Dean et al.21 suggest that the antimicrobial activity and antibiofilm activity of a compound may be due to different mechanisms; the antimicrobial activity could be directed by a physical interaction between the substance and the bacterial membrane, whereas the antibiofilm activity could be mediated by alterations of bacterial gene expression. Moreover, procyanidins can also prevent the P-fimbriae adhesion of bacteria in vitro and in vivo,31 so multiple mechanisms may be responsible for their effects on microbial viability and biofilm formation. The iron-starvation abilities are

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.8b00535. Physical, spectroscopic, and spectrometric data of the synthesized compounds; 1H NMR and 13C NMR spectra of the new compounds and those biologically evaluated in this work; effects of procyanidin A-2 and the A-type procyanidin analogues 1−6 on biofilm formation and on the disruption of preformed biofilms by resistant strains from organic foods (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*Tel.: +34 953 212746, Fax: +34 953 211876, E-mail: ssalido@ ujaen.es (S.S.). *Tel.: +34 953 212004, Fax: +34 953 212943, E-mail: [email protected] (E.O.-M.). ORCID

Antonio Gálvez: 0000-0002-5894-5029 Sofía Salido: 0000-0003-2319-7873 G


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Journal of Agricultural and Food Chemistry Funding

from organic foods: Sensitivity to biocides and antibiotics. Food Control 2012, 26, 73−78. (18) Clinical and Laboratory Standards Institute (CLSI). Performance standards for antimicrobial susceptibility testing; Twenty-fourth informational supplement, Document M100eS24, No. 3; CLSI: Wayne, PA, 2015; Vol. 34. (19) Gadea, R.; Fernández-Fuentes, M. A.; Pérez-Pulido, R.; Gálvez, A.; Ortega, E. Adaptive tolerance to phenolic biocides in bacteria from organic foods: Effects on antimicrobial susceptibility and tolerance to physical stresses. Food Res. Int. 2016, 85, 131−143. (20) Djordjevic, D.; Wiedmann, M.; McLandsborough, L. A. Microtiter plate assay for assessment of Listeria monocytogenes biofilm formation. Appl. Environ. Microbiol. 2002, 68, 2950−2958. (21) Dean, S. N.; Bishop, B. M.; van Hoek, M. L. Natural and synthetic cathelicidinpeptides with anti-microbial and anti-biofilm activity against Staphylococcus aureus. BMC Microbiol. 2011, 11, 114− 126. (22) Ulrey, R. K.; Barksdale, S. M.; Zhou, W.; van Hoek, M. L. Cranberry proanthocyanidins have anti-biofilm properties against Pseudomonas aeruginosa. BMC Complementary Altern. Med. 2014, 14, 499−511. (23) Foo, L. Y.; Lu, Y.; Howell, A. B.; Vorsa, N. A-Type proanthocyanidin trimers from cranberry that inhibit adherence of uropathogenic P-fimbriated Escherichia coli. J. Nat. Prod. 2000, 63, 1225−1228. (24) Rane, H. S.; Bernardo, S. M.; Howell, A. B.; Lee, S. A. Cranberry-derived proanthocyanidins prevent formation of Candida albicans biofilms in artificial urine through biofilm- and adherencespecific mechanisms. J. Antimicrob. Chemother. 2014, 69, 428−436. (25) Melo, A. S.; Colombo, A. L.; Arthington-Skaggs, B. A. Paradoxical growth effect of caspofungin observed on biofilms and planktonic cells of five different Candida species. Antimicrob. Antimicrob. Agents Chemother. 2007, 51, 3081−3088. (26) Goto, T.; Yoshida, K.; Yoshikane, M.; Kondo, T. Chiral stacking of a natural flavone, flavocommelin, in aqueous solutions. Tetrahedron Lett. 1990, 31, 713−716. (27) Charlton, A. J.; Baxter, N. J.; Khan, M. L.; Moir, A. J. G.; Haslam, E.; Davies, A. P.; Williamson, M. P. Polyphenol/peptide binding and precipitation. J. Agric. Food Chem. 2002, 50, 1593−1601. (28) Steinberg, D.; Feldman, M.; Ofek, I.; Weiss, E. I. Effect of a high-molecular-weight component of cranberry on constituents of dental biofilm. J. Antimicrob. Chemother. 2004, 54, 86−89. (29) Weiss, E. I.; Lev-Dor, R.; Kashamn, Y.; Goldhar, J.; Sharon, N.; Ofek, I. Inhibiting interspecies coaggregation of plaque bacteria with a cranberry juice constituent. J. Am. Dent. Assoc., JADA 1998, 129, 1719−1723. (30) Yamanaka, A.; Kimizuka, R.; Kato, T.; Okuda, K. Inhibitory effects of cranberry juice on attachment of oral streptococci and biofilm formation. Oral Microbiol. Immunol. 2004, 19, 150−154. (31) Amer, L. S.; Bishop, B. M.; van Hoek, M. L. Antimicrobial and antibiofilm activity of cathelicidins and short, synthetic peptides against. Biochem. Biophys. Res. Commun. 2010, 396, 246−251.

This study was financially supported by the University of Jaén ́ A.A.-A. and the Andalusian Government (Junta de Andalucia). was the recipient of a predoctoral fellowship granted by the University of Jaén. Part of the work was supported by the ́ Centro de Instrumentación Cientifico-Té cnica of the University of Jaén. Notes

The authors declare no competing financial interest.

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REFERENCES

(1) Pisoschi, A. M.; Pop, A.; Georgescu, C.; Turcus, V.; Olah, N. K.; Mathe, E. An overview of natural antimicrobials role in food. Eur. J. Med. Chem. 2018, 143, 922−935. (2) Friedman, M. Antibiotic-resistant bacteria: prevalence in food and inactivation by food-compatible compounds and plant extracts. J. Agric. Food Chem. 2015, 63, 3805−3822. (3) Friedman, M.; Henika, P. R.; Levin, C. E.; Mandrell, R. E. Antibacterial activities of plant essential oils and their components against Escherichia coli O157:H7 and Salmonella enterica in apple juice. J. Agric. Food Chem. 2004, 52, 6042−6048. (4) Friedman, M.; Buick, R.; Elliott, C. T. Antibacterial activities of naturally occurring compounds against antibiotic-resistant Bacillus cereus vegetative cells and spores, Escherichia coli, and Staphylococcus aureus. J. Food Prot. 2004, 67, 1774−1778. (5) Carpentier, B.; Cerf, O. Review persistence of Listeria monocytogenes in food industry equipment and premises. Int. J. Food Microbiol. 2011, 145, 1−8. (6) Lundén, J. M.; Miettinen, M. K.; Autio, T. J.; Korkeala, H. J. Persistent Listeria monocytogenes strains show enhanced adherence to food contact surface after short contact times. J. Food Prot. 2000, 63, 1204−1207. (7) Latorre, A.; van Kessel, A. J. S.; Karns, J. S.; Zurakowski, M. J.; Pradhan, A. K.; Boor, K. J.; Jayarao, B. M.; Houser, B. A.; Daugherty, C. S.; Schukken, Y. H. Biofilm in milking equipment on a dairy farm as a potential source of bulk tank milk contamination with Listeria monocytogenes. J. Dairy Sci. 2010, 93, 2792−2802. (8) Sullivan, I. Proanthocyanidins. Food sources, antioxidant properties and health benefits; Nova Science Publishers: New York, NY, 2015. (9) Alejo-Armijo, A.; Glibota, N.; Frías, M. P.; Altarejos, J.; Gálvez, A.; Ortega-Morente, E.; Salido, S. Antimicrobial and antibiofilm activities of procyanidins extracted from laurel wood against a selection of foodborne microorganisms. Int. J. Food Sci. Technol. 2017, 52, 679− 686. (10) Howell, A. B.; Reed, J. D.; Krueger, C. G.; Winterbottom, R.; Cunningham, D. G.; Leahy, M. A-Type cranberry proanthocyanidins and uropathogenic bacterial anti-adhesion activity. Phytochemistry 2005, 66, 2281−2291. (11) Alejo-Armijo, A.; Salido, S.; Altarejos, J.; Parola, A. J.; Gago, S.; Basilio, N.; Cabrita, L.; Pina, F. Effect of methyl, hydroxyl, and chloro substituents in position 3 of 3′,4′,7-trihydroxyflavylium: stability, kinetics, and thermodynamics. Chem. - Eur. J. 2016, 22, 12495−12505. (12) Armarego, W. L. F.; Chai, C. L. L. Purification of laboratory chemicals; Butterworth Heinemann: Burlington, 2003. (13) Calogero, G.; Sinopoli, A.; Citro, I.; Di Marco, G.; Petrov, V.; Diniz, A. M.; Parola, A. J.; Pina, F. Synthetic analogues of anthocyanins as sensitizers for dye-sensitized solar cells. Photochem. Photobiol. Sci. 2013, 12, 883−894. (14) Kraus, M.; Biskup, E.; Richling, E.; Schreier, P. Synthesis of [4-14C]-pelargonidin chloride and [4-14C]-delphinidin chloride. J. Labelled Compd. Radiopharm. 2006, 49, 1151−1162. (15) Selenski, C.; Pettus, T. R. R. (±)-Diinsininone: made nature’s way. Tetrahedron 2006, 62, 5298−5307. (16) Kraus, G. A.; Yuan, Y.; Kempema, A. A convenient synthesis of type A procyanidins. Molecules 2009, 14, 807−815. (17) Fernández-Fuentes, M. A.; Ortega-Morente, E.; Abriouel, H.; Pérez-Pulido, R.; Gálvez, A. Isolation and identification of bacteria H


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