RELATION BETWEEN POLYPHENOLS CONTENT AND SKIN COLOUR IN SOUR CHERRY FRUITS

Fruit skin colour plays a major role in quality assessment of food, significantly determining consumer`s choice. Colour of sour cherries depends on anthocyanins which are phenolic compounds (flavonoids) present in high amounts in fruits. The aim of this study was to determine a possible relation between polyphenols (total phenolics and anthocyanins) and colour parameters of fruit skin of sour cherries. The plant material used in this study was twenty two sour cherry genotypes from an orchard of Agricultural Institute Osijek. Total phenolics and anthocyanins contents as well as colour parameters (L, a, b, h and C) of fruit skin were determined. Variability between sour cherry genotypes in total phenolics and anthocyanins as well as in colour parameters was revealed. Total polyphenols content varied from 462.7 to 1049.0 mg GAE/100 g of fresh weight, while total anthocyanins ranged from 160.1 to 495.6 mg CGE/100 g of fresh weight. A significant positive correlation was found between polyphenols and anthocyanins content. Total phenolics content had a significant negative correlation with colour parameters b* and h, while anthocyanins content negatively correlated with colour parameters L*, b* and h. According to the obtained results, genotypes Maraska, Heimanns Konservenweichsel and Rexelle are the richest genotypes in polyphenols and anthocyanins content.


Introduction
Sour cherry (Prunus cerasus L.) is a species with the smallest fruit size among stone fruits belonging to the Rosaceae family.The main characteristics of sour cherry fruit quality are colour, sweetness, sourness and firmness.In sour cherries, the ripening process is related with accumulation of polyphenolic compounds, especially anthocyanins and products of chlorophyll degradation.Similarly, polyphenolics, mainly concentrated in fruit skin, contribute to the sensory and organoleptic qualities of fruits, in particular taste and astringency (Ferretti et al., 2010).
Polyphenols are secondary metabolites of plants involved in antioxidative defence against biotic and abiotic stresses.Current epidemiological studies strongly support a contribution of polyphenols in the prevention of cardiovascular diseases, cancers, and osteoporosis as well as neurodegenerative diseases and diabetes mellitus (Scalbert et al., 2005).Most of these beneficial effects of polyphenols on human health involve their interaction with cellular signalling pathways and related machinery that mediate cell function under both normal and pathological conditions (Vauzour et al., 2010).
Small coloured fruits, such as cherries and berries are important source of polyphenols.In fruits, polyphenols may contribute to the bitterness, astringency, colour, flavour, odour and oxidative stability (Pandey and Rizvi, 2009).Colour is the most important indicator of maturity and quality in many fruit species.Fruit colouration is mainly influenced by the concentration and distribution of various anthocyanins in the skin.Although all cherry cultivars have some anthocyanin pigmentation in the skins, the amounts vary tremendously (Chaovanalikit and Wrolstad, 2004).Total anthocyanin content and the anthocyanin fractions differ according to the sour cherry variety (Šimunić et al., 2005).The main anthocyanins found in sour cherries are cyanidin-3-glucoside, cyanidin-3-glucosylrutinoside, cyanidin-3-sophoroside, and cyanidin-3-rutinoside.According to Kirakosyan et al. (2009) cyanidin derivatives represent about 93% of total anthocyanins present in cultivars Montmorency and Balaton.The glycosides release the anthocyanidin aglycone by hydrolysis, but aglycones are rarely found in fresh plant material.As compared to other cherries, sour cherries contain the highest amount of anthocyanins, which are major contributors to the cherries antioxidant capacity (Jakobek et al., 2007).
Visual colour of fruits is subjective, particularly at hue junctions, and in an attempt to avoid this, instrumental measurements and several colour systems are used.The colorimetric CIELab system is widely used for fruit colour assessment.According to CIELab scale, any given colour is located as a point in a threedimensional space located by lightness coefficient (L * ) and two coordinates (a * and b * ).Parameters hue angle (h) and chroma (C) are mathematically derived from the L * , a * and b * values.Numerous studies have been done on anthocyanins and colour change in sweet cherries (Mozetič et al., 2004;Gonçalves et al., 2007), while there is no enough information on relation between anthocyanin content and skin colour in sour cherries (Pedisić et al., 2009).The chromatic parameters L * , a * , b * and hue angle correlated negatively with total anthocyanin levels in sweet cherry (Gonçalves et al., 2007) and weakly correlated in plums (Usenik et al., 2009).
The aim of this study was to determine total phenolics and anthocyanins content as well as possible relation between phenolics (total phenols and anthocyanins) and colour parameters of fruit skin in twenty two sour cherry genotypes.

Material and Methods
The plant material used in this study was twenty two sour cherry genotypes from the orchard of Agricultural Institute Osijek in 2011 season.The harvesting was made at the commercial maturity stage, when the fruits have developed the characteristic colour, ranging from 15 th June to 5 th July.Immediately after harvesting, fruits were frozen at -20ºC.Frozen sour cherries were homogenized in blender, weighed (~1 g), and total phenolics, including anthocyanins, were extracted with 10 ml of acidified methanol (1% hydrochloric acid) for 60 min in an ultrasonic bath.After centrifugation at 4,000 rpm for 15 min, supernatant was used for spectrophotometrical determination of total phenolics and antocyanins.
Total phenolics were determined by Folin-Ciocalteu method (Slinkard and Singleton, 1977) modified in the micro method by Waterhouse (2009).An aliquot (100 µl) of diluted sour cherry extract (dilution factor 3) was mixed with 2,000 µl of 2% (w/v) sodium carbonate solution and 100 µl of Folin-Ciocalteu reagent.After incubation at room temperature for 30 min in the dark, the absorbance was read against the blank at 765 nm (Specord 200, Analytic Jena, Germany).Total polyphenols were expressed as mg of gallic acid equivalents (GAE) per 100 g of fruit fresh weight (FW).
Total anthocyanins were estimated by a pH-differential method (Giusti and Wrolstad, 2001).Two dilutions of extract (dilution factor 10) were prepared, one with potassium chloride buffer (0.025 M; pH 1.0) and the other with sodium acetate buffer (0.4 M; pH 4.5).The absorbance was measured simultaneously at 510 and 700 nm after l5 min of incubation at room temperature in the dark.The content of total anthocyanins was expressed in mg of cyanidin-3-O-glucoside equivalents (CGE) per 100 g of fruit fresh weight using a molar extinction coefficient (ε) of cyanidin-3-O-glucoside of 26,900 dm 3 mol -1 cm -1 and molar weight (MW) (449.2 g mol -1 ).
Colour parameters (L * , a * , b * , h and C) of fruit skin were determined according to CIELab method with colorimeter CR-400 (Konica Minolta) on thirty fruits in four replicates per genotype.
Data on total phenolics and anthocyanins content as well as colour parameters were subjected to one-way ANOVA and LSD test for post hoc analysis.In addition, correlation coefficients between measured parameters were calculated.Differences were considered significant at p ≤ 0.05.The similarity of 22 investigated genotypes according to total phenolics and anthocyanins content as well as colour parameters was tested by cluster analysis using Unweighted pair group method with arithmetic mean (UPGMA) and Euclidian distances.All statistical analyses were performed by the programme Statistica 7.1.(StatSoft, Inc., 2005, Tulsa, OK, USA).

Results and Discussion
Quantitative differences in fruit polyphenols content were observed among investigated genotypes (Table 1).Total phenolics content ranged from 462.7 to 1049.0 mg GAE/100 g fresh weight in genotypes Kereška and Heimanns Konservenweichsel, respectively.Genotypes Maraska, Maraska type ST, Rexelle and Heimanns Konservenweichsel revealed similar content of polyphenols around 1,000 mg GAE/100 g of fresh weight.All Oblačinska types had total polyphenols content in range from 595 to 773 mg GAE/100 g of fresh weight.Our results are in accordance with findings of Khoo et al. (2011) who reported that the total phenolic content in sour cherry ranged from 74 to 754 mg GAE/100 g of fresh weight while Kirakosyan et al. (2009) found some higher values of total phenolics in sour cherry in range from 674 to 1266 mg GAE/100 g of dry weight.However, some other authors (Dragović-Uzelac et al., 2007;Jakobek et al., 2007) obtained lower amounts of total polyphenols in sour cherry fruits.This variation in phenolics content may be influenced by genotype as well as growing season (Bolling et al., 2010).
In our investigation, total anthocyanins content ranged from 160.1 to 495.6 mg CGE/100 g of fresh weight.The lowest amount of total anthocyanins was found in genotype Kereška (160.1).Genotypes Maraska and Maraska type ST had the highest anthocyanins content among all investigated genotypes with values of 495.6 and 480.8 mg CGE/100 g, respectively.Pedisić et al. (2010) investigated total anthocyanins in fruits of three Maraska ecotypes and found anthocyanins content in range from 318 to 1,975 mg CGE/100 g of dry weight depending on maturity stage.Also, Pedisić et al. (2009), Jakobek et al. (2007), Kirakosyan et al. (2009) found similar or lower amounts of total anthocyanins than obtained in our investigation.
One of the objectives of this investigation was to consider the correlation between polyphenols and anthocyanins.Total phenolic content and total anthocyanin content were strongly correlated with correlation coefficient of 0.76 (p < 0.001) which is shown in Table 3. Significant correlations between total phenolics content and anthocyanin fractions were found in sour cherry juice (Damar and Ekşi, 2012) and in acidified methanolic extracts (Khoo et al., 2011), while Filimon et al. (2011) did not find a significant correlation between these parameters in acidified ethanolic extracts from sour cherry fruits.The chromatic characteristics of the fruits, expressed as parameters L * , a * , b * and h are shown in Table 2.There were significant differences (p ≤ 0.05) in all colour parameters among the investigated sour cherry genotypes.Parameter L * , which represents lightness of sample, varied among investigated genotypes from 25.6 in Cigany type VS10-13 to 27.8 in genotype Heimanns Konservenweichsel.Redness of samples, estimated by parameter a * , ranged from 8.6 in genotype Kelleris 16 to 15.2 in Oblačinska type 15, while yellowness, estimated by parameter b * , varied from 1.5 in genotype Kelleris 16 to 3.8 in Oblačinska type 15.
As instrumental colour L * , a * and b * parameters give an estimate of lightness, redness or yellowness, computation of hue angle (h) and chroma (C), which is computed from a * and b * coordinates data, gives a more reliable comparison and interpretation of colour data.Chroma represents 'richness of colour' or colour intensity, while hue angle depicts how an average person will perceive that colour (Siddiq et al., 2011).Values of hue angle in investigated genotypes varied from 9.4 to 13.9, whereas chroma values varied from 8.7 to 15.7 in genotypes Kelleris 16 and Oblačinska type 15, respectively.Variation in hue angle of investigated genotypes could be connected with differences in polyphenols and anthocyanins composition, as well as with anthocyanins interactions with other compounds at relatively low pH values of fruits so-called co-pigmentation (Gonçalves et al., 2007).Genotypes Kelleris 16 and Oblačinska type 15 were proven to have minimal or maximal values of almost all colour parameters obtained in this investigation.In fruits of genotypes that have higher anthocyanin content (both types of Maraska), hue angle and chroma were lower than those in genotypes that have lower anthocyanin level (for example genotype Kereška).Our results are in accordance with previously reported data of Gonçalves et al. (2007).All results on colour parameters obtained in our investigation are in accordance with available literature data (Mozetič et al., 2004;Gonçalves et al., 2007;Pedisić et al., 2010;Kasim et al., 2011;Siddiq et al., 2011).
The influence of ripeness on accumulation of polyphenols and anthocyanins and consequently development of colour has been already proved (Gonçalves et al., 2007;Usenik et al., 2009).Therefore, it is expected to find the correlation between phenolic compounds and colour parameters.Data presented in Table 3 indicates that total phenolics were in a weak, but significant negative correlation (p≤0.05) with colour parameters b * and h (correlation coefficients were -0.26 and -0.42, respectively), whereas total anthocyanins were also in a weak, but significant negative correlation (p≤0.05) with L * , b * and h (correlation coefficients were -0.35, -0.28 and -0.46, respectively).Lancaster et al. (1997) found linear correlation between anthocyanins content and hue angle in several fruit and vegetable species.Moderate to high negative correlations were found between total anthocyanin content and colour parameters (L * , b * , h and chroma) in sweet cherry fruits (Mozetič et al., 2004).Furthermore, total anthocyanins level was negatively correlated with colour parameters (a * , b * , h and C) in fruits of cherry laurel (Prunus laurocerasus L.) (Kasim et al., 2011).Table 3. Correlation coefficients (r) and the probability levels for total phenolic and anthocyanin content as well as colour parameters of 22 sour cherry genotypes.Cluster analysis revealed the presence of three groups within the analysed sour cherry genotypes according to their content of total phenolics and anthocyanins, as well as colour parameters (Figure 1).The dendrogram shows that the genotypes belonging to the first group were Maraska, Heimans Konservenweichsel and Rexelle, whereas the second group consisted of only one genotype (Kereška).All other investigated genotypes belonged to the third group.Generally, genotypes were mostly grouped based on total phenolics and anthocyanins content rather than on colour parameters.
Figure 1.Cluster analysis of total polyphenols and anthocyanins as well as colour parameters in 22 sour cherry genotypes.

Conclusion
In conclusion, there is variability between sour cherry genotypes in polyphenols and anthocyanins content as well as in colour parameters.Within investigated sour cherry genotypes, total polyphenols and anthocyanins were significantly negatively correlated with colour parameters, especially b * and h, while L * value correlated only with total anthocyanins level.The present study revealed that sour cherry fruits are a significant source of anthocyanins, especially genotypes Maraska, Rexelle and Heimanns Konservenweichsel, but fruit colour depends also on other pigments present in fruits.

Table 1 .
Mean values ± standard deviation of total phenolics and anthocyanins content in 22 sour cherry genotypes.Significant differences according to Fisher LSD test (p ≤ 0.05) are designated by a lower-case letters.

Table 2 .
Mean values ± standard deviation of colour parameters in 22 sour cherry genotypes.Significant differences according to Fisher LSD test (p ≤ 0.05) are designated by a lower-case letters.