Phenolics and anthocyanins in berries of European cranberry and their antimicrobial activity

European cranberry is a berry plant rich in biologically active substances, making it valued by both the phyto-pharmaceutical and food industries. The aim of this study was to examine the accumulation of phenolic compounds and anthocyanins in berries of European cranberry and to assess their antibacterial activity. Material and methods. Different wild clones of European cranberry were investigated according to berry weight and the amounts of total phenolics and anthocyanins. Anthocyanin profiles of extracts were evaluated by HPLC, whereas the antimicrobial properties were determined by the agar well diffusion method. A strong negative correlation between berry weight and the amount of anthocyanins was found. The amount of total phenolics among different cranberry clones in the field collection ranged from 224.0 mg/100 g to 498.0 mg/100 g, and the amount of total anthocyanins ranged from 40.7 mg/100 g to 207.3 mg/100 g. Quantitative HPLC-UV analysis revealed six anthocyanins in the berries of European cranberry, among which the anthocyanin peonidin-3-galactoside was most prevalent. Conclusions. Investigation of the antimicrobial properties showed that European cranberry extracts inhibited the growth of wide range of human pathogenic bacteria, both gram-negative (Escherichia coli and Salmonella typhimurium) and gram-positive (Enterococcus faecalis, Listeria monocytogenes, Staphylococcus aureus, and Bacillus subtilis).


Introduction
The European cranberry Vaccinium oxycoccos L. is a common species in the oligotrophic and mesotrophic bogs.The largest V. oxycoccos fields of Lithuania are located in the strictly protected areas Žuvintas, Čepkeliai, and Kamanos.Sweet-and-sour berries ripen under Lithuanian climate conditions from the middle of August until the end of September (1).The unique collection of genetic resources of this species was established at Kaunas Botanical Garden of Vytautas Magnus University with the purpose to preserve valuable clones selected in the strictly protected areas Čepkeliai, Žuvintas, Kamanos, and in other bogs as well.
The consumption of these valuable berries is a long-lasting tradition in Lithuania, and V. oxycoccos was much appreciated in folk medicine as well.An increased interest in fruit with large amounts of biologically active substances has led to biochemical evaluations of cranberry.For example, the latest evaluations of the closely related American cranberry species Vaccinium macrocarpon Aiton have revealed a very valuable biochemical composition of these berries.Phenolic compounds in cranberries are a diverse group that includes anthocyanins, flavonoids, proanthocyanidins, and phenolic acids.These compounds have been identified as strong antioxidants, with the potential to prevent oxidative damage and protect against cardiovascular diseases and some cancers (2)(3)(4)(5).Phenolic phytochemicals are secondary metabolites distinguished for their ability to protect plants against biological and environmental stresses, such as fungal or bacterial infections (6).The red color of cranberry fruit is also due to the presence of anthocyanins.The anti-inflammatory and antiulcer properties of these compounds are particularly important (7,8).
In recent years, investigations of bioactive berry compounds have increased considerably.Different studies revealed that phenolic compounds of raspberry, blueberry, cloudberry, and American cranberry inhibit the growth of human pathogenic bacteria, such as Salmonella, Staphylococcus, and Helicobacter (9)(10)(11).An interest in the composition of European cranberry berries has been intensified because of the increased awareness of their possible positive health effects.
The aim of the present study was to examine the accumulation of phenolic compounds and anthocyanins in berries of European cranberry and to assess their antibacterial activity.

Material and methods
Plant material.The material for the evaluations was collected in the strictly protected reserve Žuvintas from 1996 to 1999 and other mesotrophic bogs in eastern Lithuania in 1995, where a great morphological variation had been noticed.The collected runners of 21 clones, conspicuous in different shape and berry size, were planted in the experimental plot of the Kaunas Botanical Garden of Vytautas Magnus University, were raised under the same growth conditions, and then were evaluated.Clones were transplanted to the field to avoid the influence of various ecological factors in natural habitats.
Morphological characterization.Berry weight and leaf area were quantified.The average weight of a berry was calculated by weighing 50 berries in three replications.The mean area of a leaf was determined by scanning 30 leaves with a scanner (HP Scan Jet 3600) in three replications and applying two noise removal filters.The image threshold was set with userchosen values, and the amount of dark pixels representing the actual area of leaves was counted.
Reagents.Reagents used to estimate the amounts of total anthocyanins and phenolics were of analytical grade.Purified cyanidin-3-rutinoside was donated by the Danish Institute of Agricultural Science (Department of Fruit, Vegetable and Food Science, Tjele, Denmark).A gallic acid standard was purchased from Sigma-Aldrich Chemie GmbH (Steinheim, Germany).
Biochemical evaluation.Berry samples used to evaluate the amounts of total phenolics and anthocyanins were gathered during the stage of mass ripening.
Analysis of total phenolic compounds.The amount of total phenolics in the cranberry extracts was determined with the Folin-Ciocalteu reagent according to the method of Slinkard and Singleton, using gallic acid as a standard (12).The reagent was prepared by diluting a stock solution (Sigma-Aldrich Chemie GmbH, Steinheim, Germany) with distilled water (1:10, v/v).Samples (1.0 mL, two replicates) were introduced into test cuvettes, and then 5.0 mL of Folin-Ciocalteu's phenol reagent and 4.0 mL of Na 2 CO 3 (7.5%)were added.The absorbance of all samples was measured at 765 nm using the Genesys10 UV/ Vis spectrophotometer (Thermo Spectronic, Rochester, USA) after incubation at 20°C for 1 h.Results were expressed as milligrams of gallic acid equivalent (GAE) per 100 grams of fresh weight.
Analysis of anthocyanins.Pigments were extracted from 5 g of frozen berries with acidified (0.1 N HCl, v/v), 95% (v/v) food-grade ethanol (13).The berries were ground with quartz sand, and the extraction was continued with 20-mL portions of solvent until the sample became colorless.The extract was diluted with acidified ethanol.The absorption was measured on a spectrophotometer Genesys-5 (Thermo Spectronic, Rochester, USA) at 544 nm.The concentration of anthocyanins was determined from the calibration curve, which was constructed by measuring the absorption of cyanidin-3-rutinoside (MW 595.2, ε=28.800)reference solutions.The concentration of anthocyanins (C) was calculated using the following formula and expressed in milligrams of cyanidin-3-rutinoside in 100 g of berries: , where c is the concentration of anthocyanins in mg/L obtained from the calibration curve, V is the volume of the extract in mL, k is the dilution factor, and m is the amount of berries used for the extraction in g.
The elution sequence of anthocyanins was confirmed by the HPLC-MS method.Mass spectra were registered by a Hewlett Packard 1100 MS (Agilent Medicina (Kaunas) 2009; 45 (12) Phenolics and anthocyanins in berries of European cranberry and their antimicrobial activity Technologies), operating in nitrogen flow at atmospheric pressure, and by applying electrical ionization.The voltage in the capillary was 4500 V; the voltage of fragmentation was 100 V.The temperature was 250 o C. The scanning range was 100-1000 m/z with an interval of 0.1 m/z.
Assessment of the antimicrobial activity.Antimicrobial activity of the extracts was performed on the following gram-positive bacterial test cultures: Listeria monocytogenes (ATCC 19117), Bacillus subtilis (ATCC 6633), Enterococcus faecalis (ATCC 29212), Staphylococcus aureus (ATCC 25923), and also on the gram-negative bacterial test cultures Escherichia coli (ATCC25922), Salmonella typhimurium (ATCC 14028).The antimicrobial properties were evaluated by the agar well diffusion method.Bacteria were grown in peptone-soy bouillon (LAB 04, LAB M) for 24 hours at 37°C.After cultivation, test culture cells were mixed using a minishaker MS 1 (Wilmington, USA), and the cell suspensions were adjusted according to McFarland No. 0.5 standard (14).The suspension of bacteria cells was introduced into the dissolved media and cooled to 47°C; 20 mL was pipetted into a 90-mm-diameter Petri plate.Wells 8 mm in diameter were pushed in the agar and filled with 50 µL of the ethanol extracts of berries.The plates were incubated overnight at 37°C.Data analysis.The data were subjected to multivariate statistical analysis.Differences among the cranberry clones were tested by one-way analysis of variance (ANOVA).

Results and discussion
Morphological variability.Significant differences among clones of V. oxycoccos were ascertained in calculating the mean values of berry weight and leaf area (Table 1).
A high positive correlation between leaf area and berry weight was found with a coefficient of correlation of r=0.812.A regression analysis revealed that the interrelationship between leaf area and berry weight could be best expressed by the exponential equation y(x)=0.26708•e^3.18506x, with the coefficient of regression R 2 x/y =0.702.Variability of phenolic and anthocyanin amounts.The differences in total phenolic and anthocyanin amounts among clones of European cranberry were ascertained.The clones accumulated from 224.1 mg/ 100 g to 498.2 mg/100 g of phenolic compounds.The most valuable clones V416, V411, V425, V407, and V413, which accumulated 498.2, 481.8, 485.9, 431.5, and 428.6 mg/100 g of phenolic compounds, respectively, were selected.Clone V425 was notable for its exceptionally large amount of anthocyanins, 207.3 mg/ 100 g.According to the results of the present study, anthocyanins comprised 18.3% to 42.7% of total phenolic content.As several authors have reported (15,16), in bilberries, red raspberries, and lingonberries, anthocyanins accounted for 90%, 30%, and 22% of their phenolic profiles, respectively.In the high bush blueberry, anthocyanins make up on average 20-30% of total phenolic content (17).Berries of a related species V. macrocarpon accumulate on average 192.3-520.8mg/100 g of phenolic compounds (18).V. oxycoccos berries have one of the richest sources of phenolic phytochemicals because they contain large amounts of total phenolic compounds including anthocyanins (Table 2).
Separation of cranberry anthocyanins by HPLC-UV revealed six anthocyanins.Mass spectra showed the elution sequence to be as follows: cyanidin-3-galactoside and cyanidin-3-glucoside (449 m/z), cyanidin-3-arabinoside (419 m/z), peonidin-3-galactoside and peonidin-3-glucoside (463 m/z), and peonidin-3arabinoside (433 m/z).Masses for the aglycones are the following: cyanidin 287 m/z and peonidin 301 m/z.The order of elution of the glycosides from the C 18 column is galactoside before glucoside, which is before arabinoside.The proportion of individual anthocyanins in berries depends on the clone of the cran- berry (Table 3).In some clones, cyanidin-3-glucoside was not detected (V409, V475, V407, V423, and V488), and in other clone, peonidin-3-glucoside was not detected (V407).The dominant anthocyanin was peonidin-3-galactoside; on average, it amounted to 30% of total anthocyanin content.The average composition of other anthocyanins was the following: cyanidin-3-galactoside, 19.8%; cyanidin-3-glucoside, Medicina (Kaunas) 2009; 45 (12) Phenolics and anthocyanins in berries of European cranberry and their antimicrobial activity 3.4%; cyanidin-3-arabinoside, 21.7%; peonidin-3glucoside, 7.4%; and peonidin-3-arabinoside, 17.4%.Berry weight was found to be negatively correlated with anthocyanin content (r=-0.744).A regression analysis revealed the interrelationship between berry weight and amount of anthocyanins is expressed by the linear equation (Fig. 1).It shows that berry size was an important factor for determining the amount of total anthocyanins, with a regression coefficient of R 2 x/y =0.554.An analogous trend was also detected for the amount of total phenolics, the interrelationship of which with berry weight could be best expressed by the hyperbolic equation (Fig. 2) with a regression coefficient of R 2 x/y =0.22.The same trend was established for V. macrocarpon; i.e., the levels of phenolic compounds were negatively correlated with hor- Berry weight, g Total anthocyanins, mg/100 g ticultural traits such as yield and fruit size (4).Meanwhile, the results obtained by Moyer et al. showed that there was a strong positive correlation between fruit size and amount of total anthocyanins in V. corymbosum, but no correlation was found across other Vaccinium species investigated (26,27).
Antibacterial activity.Investigation of the antimicrobial properties showed that European cranberry extracts inhibited the growth of wide range of human pathogenic bacteria, both gram-negative and grampositive.Listeria monocytogenes and Enterococcus faecalis were the most sensitive (average zones of inhibition were 20.35 and 19.71 mm, respectively), Salmonella typhimurium and Staphylococcus aureus were found to be of moderate resistance, and Escherichia coli was the least sensitive (Table 4).A comparison of results from the different clones showed that the largest zones of inhibition were made by the extract of the clones V409 (16.00-23.66mm), V481 (15.00-26 mm), and V488 (16.00-22.00mm).Extracts of less active clones V487 and V478 showed zones of 14.00-17.00mm and 13.00-19.00mm in diameter, respectively.
Several authors have reported that phenolic berry extracts inhibited the growth of Salmonella, Escherichia, and Staphylococcus species (10,11,28).Berry phenolics affect the growth of bacterial species in different mechanisms.There seem to be complex interactions between pH of the growth media and antimicrobial effects of the berry phenolics varying in different bacterial species (10).It is suggested that the antimicrobial activity of berries may enhance shelf life of food products and act as new type antimicrobials, which may control a wide range of pathogens.Natural food preservatives targeted to foods that are easily contaminated by Salmonella and Staphylococcus are highly desired (28,29).Cranberry-derived antimicrobials could be included as effective supplements to traditional antimicrobial preparations and treatment measures.

Fig. 1 .Fig. 2 .
Fig. 1.Relation between the total amount of anthocyanins and berry weight of Vaccinium oxycoccos

Table 4 . The antibacterial influence of Vaccinium oxycoccos extracts on test cultures
Phenolics and anthocyanins in berries of European cranberry and their antimicrobial activitythe selection of clones with putative large amounts of phenolics and anthocyanins.Selected clones of European cranberry with large amounts of phenolic compounds could be used for breeding new cultivars with desired biochemical characteristics.Our study indicates that European cranberry may act as antimicrobials, which control a wide range of pathogens.Antimicrobial berry compounds could have important app-lications in the future as natural antimicrobial agents for the food industry as well as for the field of medicine.