INFLUENCE OF GRAPE (VITIS VINIFERA) POLYPHENOLS ON DENTAL BIOFILM RELATED ORAL DISEASES

• Sponsoring Institution: State Agricultural Experiment Station
• Project Status: COMPLETE
• Funding Source: STATE
• Reporting Frequency: Annual
• Accession No.: 0206283
• Project Start Date: Dec 1, 2005
• Project End Date: Nov 30, 2008
• Program Code: [(N/A)]- (N/A)

Recipient Organization
N Y AGRICULTURAL EXPT STATION
(N/A)
GENEVA,NY 14456

Performing Department
GENEVA - FOOD SCIENCE & TECH

Non Technical Summary
Dental caries, despite significant advances, afflicts more than 95 percent of the adult population in the United States and continues to be a major reason for attendance at emergency rooms and a leading cause for absence from work. Dental caries results from the interaction of specific bacteria with constituents of the diet on a susceptible tooth surface. Grapes and its main product, wine, contain a variety of biological compounds such as polyphenols that can affect the formation of plaque and cavities. This research focus on the influence of polyphenols from grape on many of the biological aspects involved in the pathogenesis of dental plaque (biofilm) formation and caries, and their mechanisms of action in vitro and in vivo. The grape compounds will be extracted and separated to determine which fractions are effective to promote oral health and the concentration required to obtain the benefits. We will also study the variation in the type and levels of grape polyphenols due to grape cultivar and processing conditions.

Animal Health Component

30%

Research Effort Categories

Basic

70%

Applied

30%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
702	1131	1010	100%

Knowledge Area
702 - Requirements and Function of Nutrients and Other Food Components;

Subject Of Investigation
1131 - Wine grapes;

Field Of Science
1010 - Nutrition and metabolism;

Keywords

grape polyphenols

oral health

Goals / Objectives
Our main objective is to study the influence of polyphenols from grape on many of the biological aspects involved in the pathogenesis of dental plaque (biofilm) formation and caries, and their putative mechanisms of action in vitro and in vivo. We will study the effectiveness of the different grape polyphenols fractions on oral health, the effect of grape cultivar on polyphenols levels and composition, and the role of grape processing in the retention, composition and yield of active fractions prepared from grapes and grape products.

Project Methods
Grape varieties typically used for wine production will be use as the source of polyphenols, as previous studies have shown that wine varieties have higher phenolic content than table grape varieties, such as Cabernet Franc, Pinot Noir, Baco Noir and/or other wine/juice varieties. We will use polyphenols fractions of wine grapes at concentration normally found in the whole food product (ranging from 50 to 5,000 mg/kg, depending on polyphenol fraction and grape varietal). The preparation and analysis of grape polyphenols fractions constitute the start of the trials to determine which fractions are effective to promote oral health. Several grape polyphenols fractions from 3 grape varieties will be tested: (1) Main extract that contains all the polyphenols; (2) Anthocyanin fraction; (3) Polyphenols other than anthocyanins; (4) Phenolic acids; (5) Neutral polyphenols (catechins, proanthocyanidins and flavonols); (6) Proanthocyanidins; (7) Flavonols. Initially, we will test the anthocyanin, phenolic acids, proanthocyanidins and flavonols fractions because these classes of compounds are often associated with the beneficial effects of grape and wine; flavonols, phenolic acids and proanthocyanidins have shown biological effects against mutans streptococci. In addition, we will evaluate the use of pomace (grape skins and seeds) as possible raw materials for grape polyphenols. The three grape varieties selected will be analyzed for total phenols by the colorimetric method developed by Singleton and Rossi using the Folin-Ciocalteau reagent, total monomeric anthocyanin by the pH-differential method and total proanthocyanidins by transformation into cyaniding. This information will be used to estimate the yield of different fractions from the selected grapes. HPLC analysis of grape fractions will provide specific information on the concentration of individual compounds. The samples will be analyzed using a reversed-phase HPLC system with a C18 reversed-phase analytical column and a diode array detector. The detector will be set at 280, 320, 370 nm and 520 nm for tentative identification and quantification of known compounds against available standards. Acid hydrolysis will be performed in some phenolic fractions to simplify the HPLC profiles. Unidentified compounds in phenolic fractions that show activity will be isolated from HPLC runs to pursue further identification. The extraction of polyphenolic compounds present in grapes will be performed with the ultrasound-assisted aqueous methanol/ethanol extraction of freeze-dried powdered grapes. The main extract, which contains anthocyanins, phenolic acids, proanthocyanidins and flavonols, will be separated into major fractions to determine which fractions possess activity. The separation will be performed by solid-phase extractions, solvent extraction with pH control and by HPLC. The solvent will be evaporated from extracted fractions and the compounds will be resolubilized with deionized distilled water for analysis and activity testing.

Progress 12/01/05 to 11/30/08

Outputs
OUTPUTS: Grapes are rich in polyphenols, which are known to possess health benefits and antimicrobial activity against Streptococcus mutans, the primary microorganism responsible for dental caries. However, the phenolic content is variable depending on grape variety, and may be modified during vinification. Based on our research activites, we have determned that red wine grape pomaces are promising sources of active polyphenolic extracts for oral disease prevention. By utilizing chemical, physical and biological methods, we were able to fractionate the main phenolic extracts into powders with different antimicrobial effectiveness, and the flavan-3-ol content appears to be the best indicator for activity. Further studies related to inhibition of biofilm formation and with animal models indicated that phenolic grape extracts composed mainly of flavan-3-ols (proanthocyanidins) and flavonols are promising natural antimicrobial agents for prevention of oral disease. The potential application is the production of a natural product from a current wine byproduct (waste) that can reduce the incidence of plaque formation and caries. These findings have been disseminated through technical presentations at professional meetings and through publications. In addition, two graduate students participated in the project and received their doctorate degrees. PARTICIPANTS: Partner organizations: University of Rochester, Medical Center for Oral Biology. Cornell University, Department of Food Science and Technology. Collaborators: Dr. Hyun Koo, Project Director, University of Rochester, [e-mail]. Dr. Olga Padilla-Zakour, CoPD, Cornell University, [e-mail]. Professional Development: PhD Graduate students Illeme Bonsi and Joanne Thimothe, Cornell University. These students worked on this project and obtained their PhD degrees in 2008. TARGET AUDIENCES: Target audiences are food scientists, dentists, and enologists. In addition, the wine/juice industry and dental health industry. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
We examined the chemical composition and biological activity of phenolic extracts prepared from several wine grape varieties and their fermented by-product of winemaking (pomace) on many of the biological aspects involved in the pathogenesis of dental biofilm formation and caries development using in vitro and in vivo models. Grape phenolic extracts were obtained from Vitis vinifera varieties Cabernet Franc and Pinot Noir, and Vitis interspecific hybrid varieties Baco Noir and NoiretTM. Polyphenols were extracted with methanol/ethanol/water and separated by solid phase extraction using C18 cartridges to produce powdered main (total) phenolic and whole seed extracts, which were fractionated into anthocyanin, neutral, polymeric, oligomeric and monomeric fractions. Extracts were analyzed for total phenolic, anthocyanin and flavan-3-ol content, and for individual phenolic composition by HPLC. The anthocyanin and flavan-3-ols content were highly variable depending on grape variety and type of extract. The flavan-3-ol content ranged from 48 to 76 g CE/100g. The polymeric fraction had the most yield (4.8 g extract/100g fresh weight). Nevertheless, all grape phenolic extracts, except anthocyanin fraction, remarkably inhibited glucosyltransferases (Gtfs) B and C (70-85% inhibition) at concentrations as low as 62.5 ug/ml. Furthermore, the glycolytic pH-drop by S. mutans cells was inhibited by the grape extracts without affecting the bacterial viability; an effect that can be attributed to partial inhibition of F-ATPase activity (30-65% inhibition at 125 ug/ml). The biological activity of fermented pomace was either as effective as or significantly better than whole fruit grape extracts. Topical applications of pomace extracts significantly reduced the formation and accumulation of S. mutans biofilms on the surface of saliva-coated HA discs. The reduction of the biomass of the treated-biofilms was proportional to that of extracellular insoluble polysaccharides content in the biofilm matrix (up to 50% reduction). Bioassay-guided fractionation of the pomace extract yielded a highly bioactive extract containing a mixture of proanthocyanidins (PACs) and flavonols based on its abilities to inhibit (i) glucan synthesis by surface adsorbed Gtfs, (ii) acid production and acid tolerance of S. mutans, and (iii) formation and accumulation of biofilms. Furthermore, the influence of PACs+flavonols-enriched fraction, alone or in combination with fluoride, on development of dental caries was examined using Koo's rodent model. Results have shown that the bioactive grape fraction may exhibit cariostatic effect, and could enhance the effectiveness of fluoride in vivo; it also appears that PACs and flavonols are active constituents. Red wine grapes and pomace are promising sources of active polyphenolic extracts for oral disease prevention.

Publications

• Bonsi, I.A., Thimothe, J., Koo. H., Worobo, R.W., and Padilla-Zakour, O.I. 2008. Pinot noir grape polyphenolic extracts: Chemical composition and antimicrobial effects against Streptococcus mutans and Alicyclobacillus acidoterrestris. Institute of Food Technologists Annual Meeting & Food Expo, New Orleans, La. Abstract 056-01.
• Thimothe, J. 2009. Influence of red wine grape phenolic extracts on glucosyltransferase activity, acidogenicity and biofilm formation of Streptococcus mutans. Ph.D. Dissertation, Cornell University.
• Bonsi. I. A. 2009. Effect of processing on phenolic composition of wine grapes and their products, and production and characterization of grape phenolic extracts with biological activity against Streptococcus mutans and Alicyclobacillus acidoterrestris. Ph.D. Dissertation, Cornell University.

Progress 10/01/06 to 09/30/07

Outputs
activity against virulence traits of S. mutans, and selected four varieties to continue the studies: Cabernet Franc, Pinot noir, Baco Noir and Noiret. Polyphenols from grapes and pomace were extracted with solvents and the main extracts were prepared by solid phase separation using C18 SepPak cartridges, and freeze-dried to obtain a powdered extract. Total phenolic, total anthocyanin and flavan-3-ol contents were measured in the powders and expressed as grams gallic acid equivalents (g GAE/100 g), malvidin equivalents (g ME/100 g), and catechin equivalents (g CE/100 g). Phenolic profiles were analyzed by HPLC. Results showed that the pomace extracts were as effective as the grape extracts and were selected for further fractionation to determine what kind of compounds presented the highest antimicrobial activity. Cabernet Franc and Pinot noir pomace were used to prepare different phenolic fractions: acid, neutral and anthocyanin. Neutral fractions were the most active and had the highest flavan-3-ol content at 50 and 38 g CE/100g for Pinot noir and Cabernet Franc respectively. Phenolic extracts from separated seeds from pomace were also prepared although they were not as effective. Further studies with additional fractionation are being conducted.

Impacts
Red wine grape pomaces are promising sources of active polyphenolic extracts for oral disease prevention. Fractionation of the main phenolic extracts resulted in different antimicrobial effectiveness, and therefore, the flavan-3-ol content appears to be the best indicator for activity. Further studies related to inhibition of biofilm formation and with animal models will determine what fractions and classes of phenolic compounds are the most effective. The potential application is the production of a natural product from a current wine byproduct that can inhibit pathogenic bacteria. These findings are being disseminated through technical presentations at professional meetings and through publications.

Publications

• Bonsi, I., Thimothe, J., Koo, H. and Padilla-Zakour, O.I. 2007. Chemical composition of phenolic extracts from wine grapes and pomace in relation to oral health. Institute of Food Technologists Annual Meeting & Food Expo, Orlando, FL. Abstract 146-05.
• Thimothe, J. Bonsi, I., Padilla-Zakour, O.I. and Koo, H. 2007. Chemical characterization of red wine grape (Vitis vinifera and Vitis interspecific hybrids) and pomace phenolic extracts and their biological activity against Streptococcus mutans. J. Agric. Food Chem., ASAP Article, 10.1021/jf0722405. Web Release Date: November 14, 2007.

Progress 01/01/06 to 12/31/06

Outputs
Grapes are rich in polyphenols, which are known to possess health benefits and antimicrobial activity against Streptococcus mutans, the primary microorganism responsible for dental caries. Wine grapes are richer in polyphenols than table grapes but demand higher prices. Pomace, a waste product from the winemaking process, could be used as a source of polyphenols if antimicrobial activity persists after processing. Our objective was to evaluate the phenolic composition of selected New York wine grape varieties and their pomace in relation to their activity against oral biofilm related diseases. Nine wine grapes and their pomace, obtained after grapes were crushed, fermented on skins and pressed, were freeze-dried and ground. Polyphenols were extracted by solvent extraction and analyzed for total phenolic and anthocyanin contents. Four red varieties were selected for extract preparation based on composition: Cabernet Franc, Pinot Noir, Baco Noir and Noiret. Polyphenolic extracts were prepared by solid phase extraction using C18 SepPak cartridges, and freeze-dried to obtain a powdered extract. Total phenolic, total anthocyanin and flavan-3-ol contents were measured in the powders and expressed as grams gallic acid equivalents (g GAE/100 g), malvidin equivalents (g ME/100 g), and catechin equivalents (g CE/100 g). Phenolic profiles were analyzed by HPLC. Pinot Noir, Cabernet Franc and Lemberger pomace had the highest concentration of total phenols while Noiret grapes had the highest content of anthocyanins. Chemical composition of powdered extracts showed significant differences due to variety and processing. Pinot Noir pomace and grapes had the highest polyphenolic (61.8 g GAE) and flavan-3-ol (56.7 g CE) content per 100 gram extract respectively which corresponded to high activity against virulence traits of S. mutans. The highest anthocyanin content (22.7 g ME/100 g) was measured in Noiret grape extract.

Impacts
Red wine grapes and pomace are promising sources of active polyphenolic extracts for oral disease prevention. Further research will determine the specific fractions or compounds responsible for the activity and the feasibility of using these extracts for commercial application.

Publications

• No publications reported this period