Comparison of phenolics, antioxidant capacity and total phenol bioaccessibility of Ribes spp. grown in Turkey

On this study, anthocyanins (13), flavanols (6), phenolic acids (10), flavonol glycosides (17), antioxidant capacity, and bioaccessible phenolic content of Ribes spp., grown in Turkey were investigated. Ondividual phenolic compounds were identified and quantified with LC-QTDF/MS in red and black currants and hybrid Jostaberry. Significant variations in the individual phenolic compounds could be observed between the different cultivars. On all black currant cultivars, cyanidin 3,5-di-D-glucoside was the predominant anthocyanin compounds ( p ≤ 0.01). Cyanidin 3-D-sambubioside and cyanidin 3-D-rutinoside were detected in red currants. On all Ribes cultivars quercetin 3-D-rutinoside was the major flavonol glycoside and epigallocatechin found as the dominant flavonol compound. Rosenthal had the highest amount of total phenols, antioxidant capacity levels by DPPH and CUPRAC. Boskoop Giant characterized with the highest amount of total anthocyanin and bioaccessibility of phenolic compounds (84.27%).


Introduction
Berries have remarked for their nutritional and bioactive properties based on good source of polyphenolic compounds (Anttonen & Karjalainen, 2006). Many research announced that polyphenolic compounds, mostly flavonoids, and anthocyanins have amazing health-promoting properties (Kim et al., 2005). Also, epidemiological studies indicate that the consumption of anthocyanins prevents diabetes, oxidative stress, and the risk of cardiovascular disease (Zafra-Stone et al., 2007). Total phenols and total anthocyanins have been reported as the main contributors to the antioxidant effect of many berries (Tabart et al., 2006;Djordjević et al., 2010). On literature, berry flavonols such as myricetin, quercetin, kaempferol, and their derivatives concentrations show an alteration which could be explained by cultivars, their growth conditions, maturation, environmental condition and the methodological procedures applied (Mikulic-Petkovsek et al., 2012).
Despite the enormous research on the total phenolic content of Ribes spp., studies investigating their bioaccessibility are scarce (Bordonaba & Terry, 2008;Contessa et al., 2013;Nour et al., 2013). Determination of total phenolic content bioavailability is important because only gastrointestinal sustainable phenolic can be bioaccessible for absorption (Chiang et al., 2013). Thus, the health effects of polyphenols depend on the amount consumed and on their bioavailability which appears to differ greatly. For this reason, knowledge of bioavailability is necessary for exploring the health effects of polyphenols in the fruit. Most polyphenols exist in the form of glycosides, esters, or polymers which cannot be absorbed in their native form in foods. Therefore, some polyphenols can be absorbed less efficiently and bioavailability than others, even though they are present large amounts. However, bioavailability research has practical, technical and ethical difficulties. Therefore, there is a need to develop and use in vitro models that mimic the physiological processes that occur in human digestion. (Minekus et al., 2014). Because of this, the impact of bioaccessibility on the stability of phenolic compounds has been one of the more widely investigated topics during the last decade.
This research aimed to investigate certain cultivars of Ribes species grown in Turkey for their content and composition of individual phenolic compounds including anthocyanins, flavonols, phenolic acids and flavonol glycosides, antioxidant capacities, total anthocyanin content, total polyphenolic content, and their bioaccessibility. For the detection of bioaccessibility, samples were processed by an in vitro digestive enzymatic extraction that mimics the conditions in the gastrointestinal tract. The polyphenol composition of the berry fruits has been characterized utilizing quadrupole time of flight mass spectrometry (LC QTDF/MS) and for other bioactive characteristics, spectrophotometric techniques were used.

Plant material
Black currant (R. Nigrum L. cv. Boskoop Giant, Rosenthal, Goliath); red currant (R. Rubrum L. cv. Red Lake) and a cross between black currant and gooseberry (R. Nidigrolaria L. cv. Jostaberry) fruits were harvested from Bursa, Turkey. For this study, we selected 5 fields in the same region and nearby. 25 kg healty fruit were harvested at commercial ripening from different parts of the field for each cultivar. The fruits were transferred to the laboratory and the moisture content of all cultivars were immediately analyzed. The samples were frozen (-80 °C) and stored for analyses.

Extraction of extractable, hydrolyzable and bioaccessible fractions of phenols
Extractable, hydrolysable, and bioaccessible fraction were extracted according to Vitali et al. (2009) with slight modifications. The extractable and hydrolyzable fraction was used for total phenolic content, antioxidant capacity, total anthocyanins, and individual phenolic compounds analysis (Vitali et al., 2009). For the determination of bioaccessible fractions, investigated Ribes varieties were processed by an in vitro digestive enzymatic extraction that mimics the conditions in the gastrointestinal tract.

Determination of total phenolic content and antioxidant capacity
The extractable, hydrolyzable and bioaccessible fraction of total phenolic content of fruits was determined using Folin-Ciocalteu method (Singleton et al., 1999). Gallic acid was used as a standard and the results were expressed as mg GAE/g dw.
A calibration curve was prepared, Trolox (6-hydroxy-2,5,7,8tetramethylchromane-2-carboxylic acid) and the results were expressed as µmol TE/g dw for each method. TPC, CUPRAC and DPPH results were calculated as the sum of extractable and hydrolyzable fraction extracts.

Total anthocyanin
Anthocyanin quantification was performed by the pH-differential method (Giusti & Wrolstad, 2001). Calculation of the anthocyanins concentration was based on a cyanidin-3-glucoside molar extinction coefficient 26,900 and a molecular mass of 449.2 g/mol. Results were expressed as milligrams of cyanidin-3-glucoside equivalents per 100 g of dry weight basis.

Individual phenolic compounds
Odentification and quantification of anthocyanins and flavonols, phenolic acids and flavonol glycosides in Ribes fruits were carried out with an Agilent 6550 LC-QTDF/MS-ESO (Agilent Technologies, USA) system equipped with a Poroshell 120 EC-C 18 column (4.6 × 100 mm, 2.7 µm film thickness). For elution of these compounds, the mobile phase consisted of two solventsI: formic acid (1%, v/v) in water (A) and formic acid (1%, v/v) in acetonitrile (B). All phenolic compounds were identified using an LC-QTDF mass spectrometer with electrospray ionization (ESO) operating in the negative (non-anthocyanin phenolic compounds) and positive (for anthocyanins) ion mode. Odentification of the components was carried out by comparing the retention times and MS2 fragmentation in currant samples. For all compound, MS identification was confirmed with authentic standards. The MS data are presented in Table 1. Concentrations were expressed in mg/kg dw.
For non-anthocyanin and anthocyanin compounds of samples, the total run time was 20, 40 min, with 8, 5 min of equilibration treatment performed before each analysis, the analysis was carried out using full-scan data-dependent MS2 scanning from m/z 50 to 3000, 100 to 1600, flow rate was 0.6, 0.3 mL/min, column temperature was set at 40 °C, 25 °C, nitrogen was used as nebulizing and drying gas at a pressure of 35, 45 psi and the flow rate was adjusted to 14, 12 L/min. respectively.

Statistical analysis
All results were expressed as mean values ± standard deviation (SD) for six replicates. Statistical analyses were performed with JMP software, v.9.0.2 (SAS, USA). Fisher's LSD test was used to compare the means among treatments with significant differences (p ≤ 0.01).

Total phenolic content
The content of extractable, hydrolyzable and bioaccessible phenolics of Ribes spp. cultivars are presented in Figure 1. Significant differences in extractable fraction, hydrolyzable fraction and total phenolic content among the different species were recorded (p < 0.01). Generally, total phenolic content was in fruits vary according to numerous genetic, environmental, and technologic factors (Manach et al., 2004).
The highest total phenolic content (123.22 mg GAE/g dw) detected in Rosenthal cultivar, followed by Boskoop giant (112.01 mg GAE/g dw). Mikulic-Petkovsek et al. (2013) recorded that total phenolic concentrations 3519.2 and 3774.1 mg GAE/kg fw for Goliath and Rosenthal, respectively. Dur finding higher than these results for both varieties, but Mikulic-Petkovsek et al.
(2015) 4525.26-6803.41 mg GAE/100 g fw of total phenolic content detected in Rosenthal varieties, which is higher than our findings.
Red Lake cultivar showed the highest content of hydrolyzable phenols, while the lowest was recorded in hybrid Jostaberry cultivar (Table 2). On terms of total phenolic content, Red lake cultivar contained the lowest amount among all species. On literatüre, total phenolic content was found that 0.418 g GAE 100 g -1 fw (Plessi et al., 2007) and 1115-1193 mg GAE 100/g (Pantelidis et al., 2007) in red lake cultivar; and 18.94-35.85 mg GAE/g dw (Woznicki et al., 2015) and 251.9 mg GAE/100 g fw (Djordjević et al., 2014) in black currant cultivar. On comparison with those researches, the results obtained by our study of total phenolic content in both red and black currants are much higher than other results.
On Jostaberry relatively high amount of total phenolic content was detected (1809.24 mg GAE/kg fw), the amount of this cultivar was following by the report of Mikulic-Petkovsek et al. (2015) results. Bioaccessibility of total phenolic content in investigated Ribes spp. cultivars ranged from 69.91% to 89.07%. Data on bioaccessibility of total phenolic content from Ribes spp. are quite limited.

Antioxidant capacity
The antioxidant capacities of the samples were determined by DPPH and CUPRAC methods. On the comparison of the levels of DPPH and CUPRAC antioxidant capacities among Ribes varieties, differences were observed (p < 0.01) ( Table 2). According to our results, Ribes nigrum (black currant) varieties had the highest antioxidant capacity among all Ribes species.
High antioxidant potential of black currant has been reported previously (Ehala et al., 2005) and this property correlated with its high content of phenolic compounds. DPPH values of Ribes species ranged from 68.61 to 181.38 µmol TE/g dw. The highest DPPH antioxidant capacity was detected in Boskoop giant followed by Rosenthal cultivar, whereas the lowest value was detected in Red lake cultivar.
The values of CUPRAC were in the range of 299.64 µmol TE/g dw (Red lake) to 914.31 µmol TE/g dw (Rosenthal) in Ribes species. Following the Rosenthal, the highest level of CUPRAC was detected in Boskoop giant and Goliath cultivar, respectively.

Phenolic profile
Phenolic compounds of Ribes species include anthocyanins, phenolic acids and flavonols being the dominant group (Anttonen & Karjalainen, 2006). Significant variations (p ≤ 0.01) in the individual phenolic compounds could be observed among the different Ribes species cultivars (Table 3 and 4). These differences among fruits for the quantity and the composition of phenolic compounds might be explained by several factors such as the genotype, phenotypic differences, species, cultivar properties, and growing condition (Strack, 1997).
Although Jostaberry had the same anthocyanin compounds as black currant, the concentrations of these anthocyanins were low. On Jostaberry the prevailing anthocyanins had cyanidin glycosides yielding dark blue or almost black fruit color. Cyanidin 3-D-rutinoside was found as the major anthocyanin compound, followed by cyanidin 3,5-di-D-glucoside and delphinidin 3-D-rutinoside.

Phenolic acid compounds
Ondividual phenolic acid compounds levels changed significantly in tested samples (Table 3). On genaral, vanillic acid, 2-hydroxybenzoic acid and protocatechuic acid were abundant in all Ribes species. The highest concentration of 2-hydroxybenzoic acid and protocatechuic acid were detected in Goliath cultivar (42.94 mg/kg dw and 21.65 mg/kg dw, respectively) and vanillic acid in Rosenthal cultivar (52.28 mg/kg dw). On total, the highest total individual phenolic acid was found in Goliath cultivar, while the lowest content was determined in Boskoop giant cultivar. Ellagic acid, 4-hydroxybenzoic acid and gallic acid were determined as the lowest phenolic acid compounds in all cultivar.
On literature, it was found that the differences for concentration of phenolic acid compounds among the Ribes species (Gavrilova et al., 2011;Mikulic-Petkovsek et al., 2015). Ot might be that cultivar, origin of the fruit, maturation period, agricultural teratment and growing condition caused these differences.

Flavanol compounds
The mean (± standard deviation) and range of the concentrations of individual flavanol compounds in Ribes species are given in Table 4. On statistical evaluation, it was found that the differences were important (p ≤ 0.01) among cultivars. Red lake cultivar was the richest species in terms of the total flavonol content (320.65 mg/kg dw), followed by Jostaberry cultivar (225.16 mg/kg dw). Red lake, Jostaberry, Boskoop giant and Goliath cultivars contained a high amount of epigallocathechin. After epigallocatechin, catechin, and epicatechin were the abundant flavonol compounds in all cultivars. The common flavonols present in different parts of black currant plants are epigallocatechin, gallocatechin, catechin, epicatechin and epigallocatechin gallate (Tabart et al., 2011) and epigallocatechin concentration in black currant were in the range of 5.86-5.95 mg/100 g fw, 27.9 mg/kg fw in Goliath and 36.3 mg/kg fw in Rosenthal cultivar (Gavrilova et al., 2011;Mikulic-Petkovsek et al., 2015;Mikulic-Petkovsek et al., 2016). When these flavanol levels were compared with the results obtained in this study, epigallocatechin concentration in black currant was different; however different growing condition, maturation, and cultivar were examined.

Flavonol glycoside compounds
Six (6) glycosides from the group of quercetin derivatives, three (3) glycosides from the group of myricetin, three (3) glycosides from the group of isorhamnetin, four (4) glycosides from the group of kaempferol and one (1) glycoside from the group of syringetin have been investigated in our study. Significant variations in flavonol glycoside compounds could be observed between the different Ribes species (Table 4). Myricetin 3-D-galactoside, quercetin 3-glucoside, quercetin 3-D-galactoside, quercetin, kaempferol 3-B-D glucoside compounds were not detected any Ribes species. The amount of isorhamnetin, isorhamnetin 3-D-glucoside, and syringetin-3-glucoside were quite low in all species.
Quercetin 3-D-rutinoside concentration ranged from 105.64 to 257.42 mg/kg dw was the major flavonol glucoside compound in all Ribes species, except Goliath cultivar. Quercetin  Mattila et al. (2016) reported myricetin glycosides as the main flavonol. These difference may be due to the difficulty in quantifying myricetin, as this flavonol is unstable and sensitive to interference from other compounds (Justesen et al., 1998). Gavrilova et al. (2011) reported that quercetin 3-D-rutinoside level found between 0.47-1.89 mg/100 g fw in red currants and 4.24-4.58 mg/100 g fw in black currants, these findings were in line with our results. Myricetin has been quantified in all Ribes cultivars analyzed; however, levels of these compound in red currant were significantly higher than black currant cultivars. The third prevailing flavonol compounds belonged to the group of kaempferol derivatives (6.46-31.47 mg/kg dw) in all species. On our research, the highest levels of total kaempferol compounds have been measured in the Jostaberry (31.47 mg/kg dw), followed by Red lake (27.25 mg/kg dw), on the contrary, Paunović et al. (2017) reported the amount of kaempferol was very low in all cultivars.
Red lake cultivar had the highest amount of total individual flavonol glycoside and especially quercetin (258.06 mg/kg dw) and myricetin (184.77 mg/kg dw) derivatives were present in high amount and followed by kaempferol derivatives (27.25 mg/kg dw) in this cultivar. For Jostaberry, total individual flavonol glycoside concentration was found 233.13 mg/kg dw. Quercetin 3-D-rutinoside level was the most abundant among the flavonol glycoside quantified, while syringetin 3-glucoside was not determined. Mikulic-Petkovsek et al. (2015) reported that flavonols were only present in small amounts in Ribes species ranging from 5% to 11% of total analyzed phenolic compounds and flavonols ranged from 36.12 to 53.94 mg/kg fw in Jostaberry.
Ot is difficult to make a direct comparison of bioactive compounds found in our research and those reported by other authors in Ribes species, since growing conditions, genotype, species, cultivar, fruit maturity, agro techniques, climatic factors, geographic region, and different extraction methods may affect the composition and concentration of phenolic compounds in these fruits (Strack, 1997;Tabart et al., 2006;Rubinskiene et al., 2005;Kellogg et al., 2010;Vagiri et al., 2013;Mikulic-Petkovsek et al., 2013). The data obtained from fruits, all in the same field and therefore cultivated in the same conditions, have confirmed that the genome and cultivar is the principal factor that determines the contents and compositions of phenolic compounds (Plessi et al., 2007).
The results of our study indicate that Ribes species, especially black currants are an exceptionally rich source of phenolic compounds and presents a nutritionally rich and healthy fruit. On all black currant cultivars cyanidin 3,5-di-D-glucoside, delphinidin 3-D-rutinoside, and cyanidin 3-D-rutinoside were the predominant anthocyanin compounds, respectively and in red currants only cyanidin 3-D-sambubioside and cyanidin 3-D-rutinoside were detected. On almost all Ribes species fruits quercetin 3-D-rutinoside was the major flavonol glycoside and epigallocatechin found as the dominant flavanol compound. Ot was found that black currants had the highest bioactive properties. Among the black currants, Rosenthal cultivar had the highest amounts of total phenols and antioxidant capacity levels, whereas Boskoop Giant cultivar characterized with the best content of anthocyanins and bioaccessible total phenolic content.
The wide cultivar of the phenolic compounds and values of antioxidant capacities found in the studied Ribes species fruit and implies their potential beneficial effects for human health. The obtained data can be used to encourage people to consume more these healthy fruits and can be also used further studies aimed at introducing promising varieties for cultivation.