(615b) Relationship Between Antimicrobial and Antioxidant Activities of Polyphenols against Streptococcus Mutans

1. Introduction Phenolic compounds found in several common foods hold many antioxidant and antimicrobial qualities. Polyphenols appear to serve a great purpose in aiding in human health to scavenge free-radicals and microorganisms within the body. The main source of natural phenolic compounds in the human diet is from vegetables, fruits, and plants. The antimicrobial benefits of polyphenolic compounds span from medical use to cosmetics and food preservations. Like for medical uses, safety is a great concern since cosmetics are applied to the hair, skin, and teeth. One such benefit of using polyphenolic compounds in cosmetics is preservation and protection against antimicrobial contaminants, while simultaneously protecting the body from carcinogenic free radicals. We examined both antimicrobial and antioxidant effects of several natural polyphenols to investigate the relationship between the two effects. Low molecular weight polyphenols examined include: ascorbic acid, gallic acid, quercetin, and salicylic acid. Tannic acid is the only high molecular weight polyphenol. Polyphenols: 1. ascorbic acid, 2. gallic acid, 3. salicylic acid, 4. quercetin, 5. tannic acid Antioxidant properties were determined by following the DMPD (N,N-dimethyl-p-phenylenediamine dihydrochloride) spectrophotometric method, cyclic voltammetric analysis, and amperometric reduction of DMPD. The list of polyphenols chosen includes some water soluble antioxidants present in blood, as well as methanol soluble antioxidants present in certain foods and drinks. Antimicrobial activities of the polyphenols were measured using Streptococcus mutans, a Gram - positive bacteria. S.mutans is well known for causing dental caries. Dental caries are initiated by the potent, cariogenic streptococcus mutans that forms plaque on teeth. Polyphenolic extracts have been shown to prevent dental plaque by inhibiting the activity of glucosyltransferase enzyme produced by S. mutans in vitro. The antimicrobial activities of polyphenols were assessed against Streptococcus mutans by minimum inhibitory concentration assays using the plate dilution technique. Minimum inhibitory concentrations are important in diagnostic laboratories to confirm the resistance of microorganisms to an antimicrobial agent, and also monitor the activity of new antimicrobial agents. The contents of this paper include comparisons of the antioxidant and free radical scavenging ability of polyphenols, along with comparisons of the antimicrobial activities of polyphenols against Streptococcus mutans. A relationship between the antimicrobial and antioxidant activities was established based on the results. 2. Materials 2.1 Reagents DMPD (N,N-dimethyl-p-phenylenediamine), DMF (N,N-Dimethylformamide), ascorbic acid, gallic acid, tannic acid, salicylic acid, and trolox were obtained from Sigma-Aldrich (St. Louis USA). Quercetin was obtained from Fischer Scientific (USA). Streptococcus mutans ATCC 25175 was obtained from American Type Culture Collection (Rockville, MD). 2.2 Equipment A Cylic voltameter (Gamry Instruments) with three-electrode chemical cell and a gold electrode as the working electrode, a platinum electrode for the counter electrode, and an Ag/AgCl electrode as the reference electrode was used for antioxidant effects of polyphenols. The electrochemical cell was connected to the computer for data collection. DMPD absorbance was measured at 550 nm with a Schimadzu UV-2401 PC UV-VIS recording spectrophotometer connected to the computer for data collection. Branson sonicator was used for cleaning the cyclic voltameter gold electrode. 3. Methods 3.1 DMPD Spectrophotometric Method A 100 mM DMPD∙+ solution was prepared by dissolving 0.209 g of DMPD in 10 mL of deionized water; 1 mL of this mixture was added to 100 mL of 0.1 M acetate buffer, at pH 5.25; the purple colored radical cation was then created by the addition of 0.2 mL of a 0.05 M ferric chloride solution to the acetate buffer. One mL of the final DMPD∙+ solution was placed in a quartz cuvette, and the absorbance was taken at 550 nm. This was the maximum absorbance reading recorded in the visible light region between 850-250 nm in the acetate buffer. The solution was allowed to stabilize until the optical density reading fell within 0.900 ± 0.100 units of absorbance. When the stable DMPD∙+ solution was achieved, with optical density in the proper range, 150 µL of 1 mM antioxidant solution, of various antioxidant compounds, was added to the quartz cuvette. After 10 minutes the absorbance was taken again, to measure the color change observed after the addition of the antioxidant compound. The resultant change in absorption was recorded as the percentage inhibition of the radical cation solution. In the presence of an antioxidant that can donate a hydrogen atom to the DMPD∙+ molecule, the DMPD∙+ solution changes from purple to a clear solution. 3.2 Electrochemical Experimentation by Cyclic Voltammetry The gold electrode was polished with gamma micropolish II deacclomerated alumina 0.3 for 3 minutes, followed by 0.05 for 3 minutes. After polishing, the gold electrode was sonicated in an acetone bath for 10 minutes, followed by sonication in a water bath for 10 minutes before each use. 30 mL samples of 1 mM polyphenol solutions were studied experimentally by way of cyclic voltammetry. Individual polyphenol samples were dissolved in 40-60% methanol-water, 0.033 M potassium phosphate buffer solutions, at pH 7.4, with 0.033 M KCl added as an auxiliary electrolyte. 3.3 Electrochemical Experimentation by Amperometry Amperometric determination of the antioxidant activity of polyphenols follows the same reaction as cyclic voltammetry. Antioxidant activity is determined by the electrochemical reduction of the DMPD free radical by the antioxidant compound. From the cyclic voltammetric curves the region for a proper working potential was determined from the range of reduction potentials recorded for DMPD and each polyphenol. Current-time diagrams showing the response of the gold electrode were obtained for the addition of the DMPD radical cation, and reduction of the DMPD radical cation by the polyphenol solutions. Time and quantitative results were recorded for the antioxidant capacity for each individual polyphenol. 3.4 Antimicrobial Media A broth medium designated as GY (Glucose Yeast extract) broth was prepared as a medium essentially free of sucrose. The broth contained (per liter): NaCl, 2 g; KH2PO4, 4.02 g; Na2HPO4, 2 g; K2CO3, 1 g; MgSO4, 0.120 g; MnSO4.H2O, 15 mg; Glucose, 2.5 g; Yeast extract, 10 g. Glucose was autoclaved separately and combined with the broth aseptically. 3.5 Antibacterial activity of polyphenols against Streptococcus mutans The growth of the bacteria in the presence of polyphenols was monitored by measuring the live cell counts in colony forming units (CFU). The polyphenols were first dissolved in DMF (N,N-Dimethylformamide), and then added to 10 mL of sterile media in the petriplates, resulting in 1% DMF concentration. Streptococcus mutans was grown overnight in the broth at 37°C with shaking until the growth reached the stationary phase. Next 0.1 mL of the solution was added to each of the petriplates containing the polyphenols. The growth was monitored by transferring 0.1 mL of each polyphenol from the petriplates to a micro centrifuge tube containing 0.9 mL of 0.1 M phosphate buffered saline (pH ? 7.0) and performing serial dilutions. 0.1 mL of the solution from each micro centrifuge tube was plated and the CFU was found by counting the number of colonies formed on the agar plates after incubation for 24 - 48 hrs at 37°C. The lowest concentration of a sample that inhibits the visible growth of a microorganism after overnight incubation was defined as the minimum inhibitory concentration (MIC). MIC was estimated at least 3 times in each experiment. 4. Results and Discussion The results of the spectrophotometric examination at 550 nm, and the amperometric examination of antioxidant activity are in good correlation. A relationship was identified between the antioxidant ability of polyphenols and the antimicrobial properties of polyphenols studied. 5. References Fogliano, V., Verde, V., Randazzo, G. and Ritieni, A., 1999. Method for measuring antioxidant activity and its application to monitoring the antioxidant capacity of wines. J. Agric. Food Chem. 47, pp. 1035?1040. Yakovleva, K. E., Kurzeev, S. A., Stepanova, E. V., Fedorova, T. V., Kuznetsov, B. A., and Koroleva, O. V., 2007. Characterization of Plant Phenolic Compounds by Cyclic Voltammetry. Applied Biochemistry and Microbiology 43, pp. 661-668 Milardovic, S., Ivekovic, D., and Grabaric, B. S., 2005. A novel amperometric method for antioxidant activity determination using DPPH free radical. Bioelectrochemistry 68, pp. 175-180