Antioxidant Capacity , Total Phenols , Total Flavonoids and Colour Component of Cornelian Cherry ( Cornus mas L . ) Wild Genotypes

A number of selected cornelian cherry wild genotypes were investigated regarding antioxidant capacity, total phenols, total flavonoids and colour component. The total phenolic, total flavonoids and antioxidant activity of methanol extract of cornelian cherry fruits were determined using Folin-Ciocalteu, colorimetric and DPPH methods, respectively. Total phenolic content ranged from 163.69 (S1) to 359.28 (H2) mg GAE 100 g FW. Genotypes H2 and H3 had the highest total phenolic content (359.28 and 343.50 mg GAE 100 g FW, respectively), total flavonoids (54.26 and 64.48 mg QE 100 g FW, respectively) and antioxidant capacity (2.39 and 2.71 mmolTrolox100g FW, respectively). Significant differences of yellow/blue color component (b*), red/ green (a*) color component, values of lightness (L*) and chroma (C) were obtained for extracts from fruits. The present study shows the potential of certain cornelian cherry genotypes as valuable source of natural antioxidants and gene stock for breeding programs.


Introduction
Cornus mas L. (cornelian cherry) is a species from Cornaceae family, native to southern Europe and south western Asia.In Romania it is part of the spontaneous flora and is found in the forests spread on the plains and hills (Cornescu and Cosmulescu, 2017).Commonly, the most frequent use of cornelian cherry fruits is to produce different drinks, syrups, gels, jams and other traditional products.C. mas fruits have been used for centuries as traditional cuisine and folk medicine in various countries of Europe and Asia (Dinda et al., 2016).Plants are valuable sources of bioactive compounds with an important role in nutrition and human health.Among the natural compounds, phenols and flavonoids have been found to be an important part of human diet and are considered as active principles in many herbs.There are investigations regarding the chemical properties of cornelian cherry fruits.C. mas fruits collected in wild flora are a valuable source of natural antioxidants and deserve attention in preservation of genetic and biological diversity (Bajic-Ljubicic et al., 2018).A high number of bioactive compounds have been identified in C. mas fruits, among which flavonoids exert favorable health effects, especially by acting as potent antioxidants (Moldovan and David, 2017).Cornelian cherry cultivars have high biological efficiency-antioxidant activity, total phenolic content and content of ascorbic acid (Rop et al., 2010;Hassanpour et al., 2012).According to Pawlowska et al. (2010), it looks like the fruits of cornelian cherry revealed the presence of considerable amounts of flavonoids.Cyanidin-3-rhamnosylgalactoside, cyanidin-3galactoside and delphinidin-3-galactoside, were isolated and identified from the berries of C. mas by Du and Francis (1973).There is a variety of cornelian cherry genotypes with different physical-chemical characteristics.The investigation made by Popovic et al. (2012) show that the fruits are a significant source of phenolic compounds and anthocyanins with high antioxidant activity, which varied greatly among the genotypes.Significant variability has also been found in other constituents as well, such as vitamin C, total sugars, organic acid, flesh lipids, linoleic acid in the seed (Brindza et al., 2007).In most cornelian cherry fruits, studied by Kucharska et al. (2015) differences of origin do affect the quantitative but not the qualitative composition and pelargonidin 3-galactopyranoside was dominant.

Phenolic compounds
HPLC analyses were performed on a Finningan Surveyor Plus system (Thermo Electron Corporation, San Jose, CA, USA) including a vacuum degasser, a Surveyor Plus LCPMPP pump, a Surveyor Plus ASP autosampler, a PDA5P diode array detector with 5 cm flow cell and a Chrom Quest 4.2 system manager as data processor.Separation was performed using a reversed-phase Hypersil Gold C18 column provided by Thermo Electron Corporation (USA).Chromatographic conditions were similar to those described by Nour et al. (2013), Cosmulescu et al. (2017).Phenolic compounds were expressed in mg/100g fresh weight (mg 100 g -1 FW).

Measurement of color by the CIEL*a*b* system
Absorbance of undiluted samples was measured in quartz cuvettes with a path length of 0.1 cm from 350 to 850 nm using an Evolution 600 spectrophotometer (Cosmulescu et al., 2014).L* (lightness), a* (red/green colour component), b* (yellow/blue colour component) were determined using the color analysis program VL-ColorCalc.Chroma (C) or saturation and hue angle (H) or tone was determined considering McGuire (1992): C = (a* 2 +b* 2 ) 1/2 ; H = arctangent of b*/a*.

Statistical analysis
Results were expressed as mean ± standard error of three samples analyzed separately.Statistical comparisons between samples were performed with Duncan t-test for independent observations (using Statgraphics Centurion XVI software).Differences were considered significant at p < 0.05.

Total phenolics, total flavonoid, individual phenolic compounds and antioxidant capacity
The results obtained have indicated significant differences (p < 0.05) in antioxidant capacity, total phenols and total flavonoids content between the genotypes analyzed (Table 1).Total phenolic content ranged from 163.69 (S1) to 359.28 (H2) mg GAE 100 g -1 FW.A content of 315.3 mg 100g -1 FW phenolics was found by Hashempour et al. (2010) in cornelian cherry genotypes from Iranian native populations.A variation of the content in total phenolic content was also found by Rop et al. (2010) in 12 cultivars of cornelian cherry (2.61 to 8.11 g GAE kg -1 FM).These differences may be due to genetic factors and different ability to synthesize secondary metabolites of genotypes.In this study, the content of polyphenols is higher than the content found by Cosmulescu et al. (2017) (192.602).A content of 2.81-5.79mg g -1 total phenolics was found by Tural and Koca Romania is rich in cornelian cherry populations, and as a result it is important to select spontaneous flora of genotypes with important characteristics, whose commercial cultivation could be successfully practiced.The objective of this research was to examine the antioxidant activity, total phenols and total flavonoids of six cornelian cherry that originate from the spontaneous flora of Romania, in order to identify valuable genotypes as a source of natural antioxidants and gene stock for breeding programs.

Materials
Materials were obtained from fruits of six genotypes of cornelian cherry selected from spontaneous flora in the following areas in Romania: Strimba-Jiu (S1) and Calaparu (C1 and C2) in the town of Turceni, Gorj county (44°40'0"N 23°23'0"E) and from the village Hartagani (H1, H2 and H3), Baita village in Hunedoara county (46°01'52"N 22°53'34"E).During 2017, fruits of the above genotypes were picked at the stage of full maturity (consumption ripeness).Each sample consisted of 500 g of fruits per genotype.All samples were frozen at -20 °C until use.

Extraction procedure
Pulp (50 g) from each cultivar have been finely chopped and then homogenized (using an IKA T-25 ULTRA-TURRAX mixer-homogenizer); 2 g of product have been combined with 20 mL methanol, with 2% hydrochloric acid and put in sealed polyethylene flasks; and then were kept for 60 minutes at a temperature of 30 °C under the ultrasounds action.

Determination of total phenol content
The method described by Cosmulescu et al. (2015) was used to determine total phenolics content in the extracts.The absorbance of mixture was measured at 765 nm using an Evolution 600 UV/VIS spectrophotometer (Thermo Scientific, USA).Gallic acid was used to prepare the standard curve (0-250 mg/L).Gallic acid was used as reference standard and results were expressed as gallic acid equivalents in milligrams per 100g fresh weight (mg GAE 100g -1 FW).

Total flavonoids content
Determination of flavonoids was done by aluminum nitrate colorimetric method described by Cosmulescu et al. (2015).Quercetin was used for preparing the standard curve (0-100 mg/L).The results were expressed as milligrams of quercetin equivalents per 100g fresh weight (mg QE 100g -1 FW).

Antioxidant activity
The scavenging capacity of the 2,2-diphenyl-1picrylhydrazyl (DPPH) free radical was monitored according to the method reported by Cosmulescu et al. (2015).Antioxidant capacity was expressed in mmolTrolox100g -1 .
(2008) in the cornelian cherry fruits.In a study by Pawlowska et al. (2010), the fruits of cornelian cherry revealed the presence of considerable amounts of flavonoids, which supports the antioxidant and nutritional potential of this plant species.In our study, the content of flavonoids differs from one genotype to another and the highest content of total flavonoid was determined in H3 genotype (64.48 mg QE 100g -1 ) followed by H2 genotype (54.26 mg QE 100g -1 ).A high content of total flavonoids (669 mg catechin 100 g -1 FW) was found by Hassanpour et al. (2011) in the cornelian cherry genotypes of Iran's spontaneous flora.The DPPH radical scavenging effects of cornelian cherry fruit extracts are given in Table 1.Among six cornelian cherry types that have different results, the results have a variation between 1.24 and 2.71 mmolTrolox100g -1 FW.The variability of total antioxidant activity was equally high (from 3.30 to 9.54 g AAE kg -1 FM) to 12 cornelian cherry genotypes cultivated in Czech Republic (Rop et al., 2010).According to Table 1, the highest antiradical activity was found H3 genotype (2.71) followed by H2 (2.39).Popovic et al. (2012) believe that cornelian cherry fruits possess high antioxidant activity and that there is a huge variation among C. mas genotypes in terms of antioxidant activity.Also, some authors have reported potential antioxidant activity of cornelian cherry leaves and flowers (Stankovic et al., 2014;Hassanpour et al., 2011;Islamovic et al., 2014).Comparing the antioxidant activity of Cornus vs Crataegus extract, antioxidant activity was found to be significantly higher (1.5-1.7 times) for Cornus (Babalica-Petrescu et al., 2014).Among the individual phenolic compounds (Table 2), gallic acids were determined in higher amounts (14.49mg/100g), followed by coumaric acid (13.79 mg/100g), ellagic acid (5.71mg/100g), salicylic acid (1.43 mg/100g), ferulic acid (1.25 mg/100g) and synaptic acid (0.19 mg/100g).Milenkovic-Andjelkovic et al. (2015) reported that ellagic acid was predominant phenol acid in cornelian cherry fruit and leaf extracts, followed by chlorogenic and gallic acids.

Measurement of color by the CIEL*a*b* system
To investigate color of the extracts obtained from the fruits of cornelian cherry, lightness and color components were measured.Table 3 presents the results of color evaluation of the fruits extracts using the CIEL*a*b* color system.Significant differences of yellow/blue color component (b*), red/ green (a*) color component, values of lightness (L*) and chroma (C) were obtained for extracts from fruits.The maximum for L (lightness) is 100, representing a perfectly reflecting diffuser and the minimum is zero which represents black.The a* and b* axes have no specific numerical limits, where the positive a* is red and the negative a* is green.In the present study, the a* values were in range of 12.05 to 21.8, which is higher than those reported by Tural and Koca (2008) (6.25 -15.59).Positive and negative b* are yellow and blue, respectively (Mohebbi et al., 2009).Values b* ranged from 10.95, for samples H2, to 4.15, for samples C2.In other studies b* values are in range from 6.64 to 8.12 (Islamovic et al., 2014), 1.22 to 11.35 (Drkenda et al., 2014) and from 3.46 and 6.64 (Tural and Koca, 2008).Table 2 shows that lightness (L*) has values between 44.45 and 48.8.L* is the indicator of lightness-darkness and the higher it is, the lighter the extracts.Indicators a* and b* varies from one genotype to another, the values are positive, thus pointing out a greater contribution of red and yellow components.Together the three parameters lightness (L*), saturation (C*), hue angle or tone (H o ) all contribute tothe color observedin each sample.Intensity or saturation (C) is higher in the H2 genotype (24.39) and hue angle (H°) at the same genotype (26.66).Hue angle presented values between 19.00-26.66°from red purple (0°) to yellow (90°).A value of 39.7 and 20.7, respectively, was reported by Gunduz et al. (2013) to cornelian cherries fruit for chroma (C) and tone (H o ) in the stage of dark red.

Conclusions
C. mas fruits collected in wild flora are a valuable source of natural antioxidants and gene stock for breeding programs.The present study shows the potential value of the cornelian cherry germplasm in Romania.
in genotypes previously analyzed.According to the results obtained byCosmulescu et al.

Table 1 .
Antioxidant activity, total phenolics content and total flavonoids content of wild cornelian cherry fruits Data expressed as means ± SE of three samples analyzed separately.* Different superscript letters within the same column indicate significant differences (P < 0.05) *

Table 2 .
Descriptive statistics for individual phenolic compounds in wild cornelian cherry fruits genotypes (mg/100 g FW)

Table 3 .
CIEL*a*b* parameters for three differently extracts in wild cornelian cherry fruits All data are expressed as average value±standard deviation.Different superscript letters within the same column indicate significant differences (P < 0.05) among genotypes.