plants

Article
Phenolic Composition and Antioxidant Activity of Peel, Pulp
and Seed Extracts of Different Clones of the Turkish Grape
Cultivar ‘Karaerik’
Muhammed Kupe 1 , Neva Karatas 2 , Mehmet Settar Unal 3 , Sezai Ercisli 1, * , Mojmir Baron 4
and Jiri Sochor 4

1 Department of Horticulture, Faculty of Agriculture, Atatürk University, Erzurum 25240, Turkey;

[email protected]
2 Department of Nutrition and Dietetics, Faculty of Health Sciences, Ataturk University,
Erzurum 25240, Turkey; [email protected]
3 Department of Horticulture, Faculty of Agriculture, Sirnak University, Sirnak 73000, Turkey;
[email protected]
4 Department of Viticulture and Enology, Faculty of Horticulture, Mendel University in Brno, Valticka 337,
691 44 Lednice, Czech Republic; [email protected] (M.B.); [email protected] (J.S.)
* Correspondence: [email protected]; Tel.: +90-535-639-56-07

Abstract: The Erzincan plain is one of the richest regions in Turkey in terms of plant biodiversity. In
this region, the famous grape cultivar ‘Karaerik’ has always dominated grape production due to its



berry characteristics. The cultivar shows great morphological variation at clonal level. In this study,


the total phenolic content and antioxidant activity of peel, pulp and seed extracts of nine ‘Karaerik’
Citation: Kupe, M.; Karatas, N.; clones sampled from same location were investigated. The Folin–Ciocalteu method was used to
Unal, M.S.; Ercisli, S.; Baron, M.;
determine the total phenolic content of peel, pulp and seed extracts of nine clones. To determine
Sochor, J. Phenolic Composition and
antioxidant activity, three well known assays such as DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate),
Antioxidant Activity of Peel, Pulp
FRAP (Ferric Reducing Antioxidant Power) and TEAC (Trolox Equivalent Antioxidant Capacity)
and Seed Extracts of Different Clones
of the Turkish Grape Cultivar
were used. In addition, the correlation between total phenol content and DPPH, FRAP and TEAC
‘Karaerik’. Plants 2021, 10, 2154. was determined. Results showed that among the tissues, seed samples in berries of all clones had
https://doi.org/10.3390/ the highest total phenol content and antioxidant activity determined by three assays. Seed samples
plants10102154 were followed by peel and pulp for total phenolic content and antioxidant activity. Among the nine
‘Karaerik’ clones, Clone 8 had the highest total phenolic content (149 mg GAE/100 g FW) while
Academic Editors: Nijole Savickienė Clone 3 had the lowest (111 mg GAE/100 g FW). Peel, pulp and seed samples of nine ‘Karaerik’
and Lina Raudone clones showed strong antioxidant activity in DPPH, FRAP and TEAC assays. In particular, grape
seeds were found rich for better in phenolic compounds including gallic acid, quercetin, catechin,
Received: 14 September 2021
chlorogenic acid, caffeic acid and p-coumaric acid. Clones such as 7, 8 and 9 higher antioxidant
Accepted: 4 October 2021
activity may present great potential for grape breeders and the food industry as well as health-
Published: 11 October 2021
conscious consumers.

Publisher’s Note: MDPI stays neutral

Keywords: grape; antioxidant activity; peel; pulp; seed
with regard to jurisdictional claims in
published maps and institutional affil-
iations.

1. Introduction
Turkey has a superior geographical and ecological advantage in terms of the culti-
Copyright: © 2021 by the authors.
vation of horticultural plants including fruits, vegetables and grapes. Due to its different
Licensee MDPI, Basel, Switzerland.
climatic conditions, many fruit and vegetable species and two main Vitis species (Vitis
This article is an open access article
vinifera and Vitis labrusca) have been grown in different regions of the country since ancient
distributed under the terms and times. Located in both the Near East and the Mediterranean basins, Turkey is the place
conditions of the Creative Commons of the genetic origin of many horticultural species. This situation becomes even more
Attribution (CC BY) license (https:// advantageous when combined with the advantage of ecology. As a matter of fact, out of
creativecommons.org/licenses/by/ 138 fruit and 100 vegetable species cultivated in the world today, more than 80 fruit species
4.0/). and 50 vegetable species can be grown in Turkey [1–5].

Plants 2021, 10, 2154. https://doi.org/10.3390/plants10102154 https://www.mdpi.com/journal/plants

Plants 2021, 10, 2154 2 of 15

It is strongly recommended to consume horticultural crops including grape berries

daily due to their high phenolic compounds, which have beneficial effects on human
health. Phenolic compounds, widely found in particular horticultural crops, are secondary
metabolites produced in the shikimic acid of plants and pentose phosphate through phenyl-
propanoid metabolization [6]. They include benzene rings and form simple form phenolic
to highly polymerized compounds [7]. Phenolic substances or polyphenols are distributed
throughout the entire metabolic process in plants and contain numerous compounds such
as flavonoids, phenolic acids, anthocyanins, etc. [8]. Horticultural crops are the main
sources of phenolic compounds in the human diet compared to other crops. Plant polyphe-
nols as natural and dietary antioxidants in human health and disease might offer some
protection against oxidative damage [9]. The majority of horticultural crops especially,
grapes, small fruits and berries are rich in phenolic compounds, for example phenolic acids
and flavonoids, and promote health benefits by reducing the risk of metabolic syndrome
and related complications such as type 2 diabetes [10]. Moreover, the antioxidant com-
pounds found in horticultural crops are mainly phenolic and include compounds such as
tocopherols, carotenoids, phenolic acids (benzoic acid derivatives and cinnamon acids),
flavonoids, and dipropenes. Secondary plant-derived metabolites, including phenolic com-
pounds, have a potent potential to clear free radicals that exist in all parts of the plant, such
as the leaves, fruits, seeds, roots, and skin [11]. Studies have shown that many factors, such
as climatic, geographic, genetic, extraction methods, etc., affect the amount of secondary
metabolites in plants [12,13].
Grape (Vitis vinifera) berries are rich in phytochemicals and these substances may be
important for reducing chronic illnesses, such as cancer, cardiovascular diseases, ischemic
stroke, neurodegenerative disorders and aging [14–17]. Grapes are one of the richest
sources of phenolic substances and antioxidant compounds among fruits. Therefore, grape
berries have been broadly studied due to their composition in phenolic substances and
antioxidant compounds and their potential beneficial effects on human health. Studies
have shown that grape cultivars have differences in terms of phenolic substances and
antioxidant capacity. Moreover, different plant parts such as leaves, seeds, berry peel, and
berry pulp show differences in terms of antioxidant activity. Among grapes, black varieties
and their products contain rich nutritional and phenolic content [18–23].
Along with cultivars and plant parts, many other factors such as environment, viti-
cultural practice, rootstocks, etc., affect phenolic compounds and the antioxidant activity
of grape berries [24–27]. In addition, during the long cultivation periods of grapevines,
mutations occur in vineyards and grape varieties generate populations of individuals that
may present certain differences. This intra-varietal diversity is expressed in many ways:
growth habit, vigour, fertility, phenological cycle, accumulation of sugars, aromatic and/or
polyphenolic potential, size and shape of bunches and berries, compactness of bunches,
ampelographic characteristics (color of organs and indentation of the leaves), etc. Thus,
clonal variation is common in grape cultivars and different clones of same cultivar affect
their antioxidant activity and have an influence on the antioxidant activity [28].
The Erzincan plain, located in eastern Anatolia, has a long tradition of viticulture.
‘Karaerik’ cultivar is the most important black grape variety grown in Erzincan and is
used for the production of high-quality table grapes. The cultivar has large, attractive
black berries with a perfect sugar–acid balance. ‘Karaerik’ means black plum in English.
The origin of this cultivar is unknown and shows low adaptation capacity; thus, they are
not successfully introduced in the other grape-growing regions with different climatic
conditions in Turkey. The plantations of this cultivar in Erzincan have a heterogeneous
population [29,30]. Using the complex selection criteria, it is possible to make a better
clonal selection from this cultivar [31]. In the Erzincan plain, the ‘Karaerik’ cultivar exhibits
different clones that vary in their berry biochemical properties [32]. Clonal selection is
considered a very important tool for grapevine genetic improvement. In most of the
grapevine-growing regions of the world, selection has always been a successful breeding
method. Clonal selection is a means of intensive improvement successfully adapted in
Plants 2021, 10, 2154 3 of 15

several varieties such as White Riesling, Muscat d’Adda, Pinot sp., Steinschiller, Hárslevelû,
Welsch/Italian Riesling, Müller Thurgau, etc.
The aim of this study was to evaluate the phenolic compounds and antioxidant
activity in peel, pulp and seeds of nine ‘Karaerik’ clones in order to find differences and/or
similarities between ‘Karaerik’ clones.

2. Materials and Methods

2.1. Plant Materials, Sampled Vineyards and Location
Nine ‘Karaerik’ clones were harvested from different vineyards in the Uzumlu district
of Erzincan in the 2020 growing season. The sampled vineyards are around 15 years old
with a specific ‘Baran’ training system. The berries were picked homogenously and their
laboratory analyses were conducted after the morphological measurements.

2.2. Morphological Traits

Harvest period, number of seed, cluster weight, cluster form, berry weight and
berry color were performed in the full ripening period, in a sample of 3 kg of clusters.
Characterization of the berries (weight and peel color) was performed in a representative
sample of 40 berries taken randomly form the middle part of clusters [33]. Analysis was
performed in duplicate.

2.3. Extraction
The fresh grape pulp, peel and seed extracts were obtained by [34]. The extraction
sample (2 g) was put into centrifuge tube and then 8 ml acidic methanol–water (60:40, v/v
pH 2) was added. The centrifuge tube was vortexed for 1 min and shaken for 1 h. After
that, the sample was centrifuged at 9000 rpm/10 min at 4 ◦ C to recover the supernatant. A
total of 8 mL of acetone–water (70:30) was added to the residue before it was stirred and
sonicated for 1 and 15 min, respectively, and centrifuged again at 4 ◦ C and 9000 rpm/10 min.
The last extraction was repeated without sonication. The obtained supernatants transferred
to a 25 mL volumetric flask, and acetone–water (70:30) was added to reach a final volume
of 25 mL. Finally, the extract was stored at −80 ◦ C until analysis.

2.4. Total Phenol Content

The Folin–Ciocalteau method [35] was used to determine the total phenolic content in
peel, pulp and seed samples of cv. ‘Karaerik’ grape clones. To achieve this, 9 mL of 80%
methanol was added to 1 mL of the sample and the mixture was centrifuged at 5500 rpm for
10 min. After that, 50 µL of supernatant was added to 250 µL of the Folin–Ciocalteu reagent.
Then, 750 µL 20% (w/v) Na2 CO3 was supplemented and incubated for 2 h. Afterward, the
absorbance was measured at 760 nm against a blank surface. The results are expressed as
milligrams of GAE (gallic acid equivalent) per 100 g of fresh weight (FW). Analysis was
performed in duplicate.

2.5. Total Antioxidant Capacity Measurement

2.5.1. 1,1-. Diphenyl-2-picryl-hydrazyl Assay
Brand-Williams et al.’s [36] method was used to determine the 1,1-Diphenyl-2-picryl-
hydrazyl (DPPH) scavenging capacity of peel, pulp and seed parts in berries of cv. ‘Karaerik’
clones. First, DPPH powder was dissolved in methanol to obtain an absorbance of
0.7 ± 0.02 at 517 nm. An amount of 20 µL test sample was diluted with water, Trolox
standard or blank (distilled water) and was placed in a 96-well microplate, after which
280 µL of working DPPH solution was added. A total of 30 min later at 30 ◦ C, the ab-
sorbance was measured at 517 nm using a microplate reader. Aqueous solutions of Trolox
(50–500 µM) were used for calibration. Finally, the obtained results were expressed micro-
moles of Trolox equivalent (TE) per 100 g of fresh weight (µmol of TE/100 g of FW).
Plants 2021, 10, 2154 4 of 15

2.5.2. Ferric Reducing Antioxidant Power

Benzie and Strain’s [37] method was used for ferric reducing antioxidant power
(FRAP) determination. The amount of 20 µL of the test sample, diluted with water, or
Trolox standard, or ferrous sulphate standard or blank (distilled water) was placed in
a 96-well microplate, after which 280 µL of the FRAP reagent (containing TPTZ, FeCl3
and acetate buffer), freshly prepared and warmed at 37 ◦ C, was added. After 30 min,
the absorbance at 595 nm was measured. The standard curves were constructed using
FeSO4 (115–1150 µM) and Trolox solutions (20–400 µM) and the results were expressed as
micromoles of Trolox equivalent (TE) per 100 g of fresh weight (µmol of TE/100 g of FW).

2.5.3. Trolox Equivalent Antioxidant Capacity

Re et al.’s [38] method was used for Trolox equivalent antioxidant capacity (TEAC)
determination. To obtain ABTS stock solution, 7 mM ABTS and 2.45 mM potassium
persulphate in a volume ratio 1:1 was prepared and, after that, incubated in the dark for
16 h. ABTS stock solution was diluted with phosphate buffer 5 mM at pH = 7.4 to obtain
an absorbance of 0.7 ± 0.02 at 730 nm. An amount of 30 µL of the test sample, diluted
with water, or Trolox standard or blank (distilled water) was placed in a 96-well microplate,
after which 270 µL of radical ABTS was added. A total of 30 min later, the absorbance was
measured at 730 nm. Aqueous solutions of Trolox concentrations (20–200 µM) were used
for calibration and the results were expressed as micromoles of Trolox equivalent (TE) per
100 g of fresh weight (µmol of TE/100 g of FW).

2.6. Phenolic Compounds

The phenolic compounds in grape samples were determined following the procedure
described by Rodriguez-Delgado et al. [39]. About 50 g sample for each sample was
transferred to a centrifuge tube, mixed homogeneously, then diluted 1:1 with distilled
water and centrifuged at 15,000× g for 15 min. The supernatant was passed through
a 0.45 µm Millex-HV Hydrophilic PVDF membrane filter, then injected into the HPLC
system (gradient). The chromatographic separation in Agilent 1100 series HPLC took
place in a DAD (photodiode array detector) detector (Agilent, Waldbronn, Germany) with
250 mm × 4.6 mm, 4m ODS column (HiChrom, New Jersey, USA). The following solvents
in water with a flow rate of 1 mL/min and 20 µl injection volume were used for spectral
measurements taken at both 254 nm and 280 nm: as mobile phase solvent A, methanol–
acetic acid–water (10:2:88) and Solvent B, methanol–acetic acid–water (90:2:8). The results
were expressed as mg/100 g FW.

2.7. Statistical Analysis

The study was planned as four replication including 10 samples per replicate. In the
statistical evaluations, Windows SPSS 20 (IBM Corp. Armonk, NY, USA) was used and the
differences between the means was evaluated by subjecting to ANOVA variance analysis
and determined with Duncan multiple comparison test (p < 0.05). The Pearson correlation
analysis between antioxidant activity and total phenolic content was performed.

3. Results and Discussion

3.1. Morphological Traits
Table 1 presents some important morphological traits of the nine clones of ‘Karaerik’
grape cultivar. The clones exhibited diversity for most of the searched morphological
parameters.
In terms of harvest season, Clones 1, 7 and 9 were found earlier than the rest of the
Clones. (Table 1). Clones 9 had the lowest number seeds per berry, Clones 1, 4, 6, 7 and 8
had 2-3 seeds per berry and Clones 2 and 3 had the highest average of the seeds per berry
(3-4) which reflects to the larger size of the berries obtained (6.13 g for Clone 2 and 6.90 g
for Clone 3).
Plants 2021, 10, 2154 5 of 15

Table 1. Some morphological traits of nine ‘Karaerik’ clones.

Harvest Number of Cluster Berry Berry Peel

Locations Clones Cluster Form Usage
Period Seed Weight (g) Weight (g) Color
Winged
Üzümlü Clone 1 Mid-season 2–3 460 5.86 Black Table
cylindrical
Winged
Üzümlü Clone 2 Late season 3–4 505 6.13 Black Table
cylindrical
Winged
Üzümlü Clone 3 Late season 3–4 587 6.90 Black Table
cylindrical
Üzümlü Clone 4 Late season 2–3 Winged conical 433 5.21 Black Table
Irregular
Üzümlü Clone 5 Mid-late 2–3 456 5.60 Black Table
winged conical
Irregular
Üzümlü Clone 6 Mid-late 2–3 412 5.42 Black Table
winged conical
Üzümlü Clone 7 Mid-season 2–3 Conical 390 5.03 Black Table
Üzümlü Clone 8 Mid-late 2–3 Conical 383 4.67 Black Table
Üzümlü Clone 9 Mid-season 1–2 Conical 346 4.59 Black Table
Mid-season: 1 September–1 October; Mid-late: 10 September–10 October; Late season:15 September–15 October.

Clone 9 gave the lowest berry weight (4.59 g, which had the lowest number of seeds)
(Table 1). All clones had a black berry color and all of them were to be used only for table
consumption due to their perfect fresh berry characteristics (larger size, attractive color,
unique sugar-acid balance, thick peel).
Cluster form was found to be different among Clones (Figure 1), with Clones 1, 2 and
3 having a winged cylindrical form, Clone 4 having a winged conical form, Clones 5 and 6
having an irregular winged conical form and the rest of the Clones having a conical cluster
form (Table 1). The average cluster weight of the Clones was found to be between 346 and
587g, with the highest cluster weight obtained from Clone 3.

Figure 1. ‘Karaerik’ grape cultivars (in ‘Baran’ training system).

According to OIV [33], cluster weight of all Clones was found to be over 300 g and
classified as large. In grapes, cluster and berry weight are strongly affected by cultivar,
altitude, cluster thinning, etc. Kok et al. [22] reported cluster weight in grape cultivars be-
tween 232 and 560 g, which indicated good agreement with our result. Dilli and Kader [40]
studied table grape cultivars widely grown in Turkey and reported that the cultivars were
harvested early, mid and late period. They also found that common table cultivars had
Plants 2021, 10, 2154 6 of 15

a large cluster weight and 1–4 seeds per berry. Good quality in table grapes represents a
combination of medium-sized clusters of uniformly large, perfect berries with the charac-
teristic color, pleasing flavor, and the texture of the cultivar. Uniform color formation and
suitability for transportation are also desirable traits for table grapes [41].
Our results are in agreement with the above studies [22,39]. The differences could
be effects of the different cultivars, altitude, ecology, etc. In the literature, there were
studies that determined the morphological characteristics of grape cultivars, indicating a
wide variability in cluster weight, cluster form, seed number per berry, berry color and
berry weight, according to cultivars and treatments [42–44]. We found great diversity in
particular cluster forms even in the same vineyards.

3.2. Total Phenolic Content

The results of the total phenolic content of peel, pulp and seed samples of nine
‘Karaerik’ grape clones are presented in Table 2. The Clones showed statistically significant
differences each other (p < 0.05) in terms of peel, pulp and seed total phenolic content
(Table 2).
Among the nine ‘Karaerik’ clones, Clone 8 had the highest total phenolic content in
peel as 149 mg GAE/100 g FW, and followed by in descending order Clone 9 (143 mg
GAE/100 g FW) > Clone 5 (138 mg GAE/100 g FW) > Clone 7 (134 mg GAE/100 g FW) >
Clone 2 (129 mg GAE/100 g FW) > Clone 6 (126 mg GAE/100 g FW) > Clone 4 (121 mg
GAE/100 g FW) > Clone 1 (117 mg GAE/100 g FW) > Clone 3 (111 mg GAE/100 g FW),
respectively (Table 2).
For pulp, the Clones exhibited the lowest total phenolic content, which varied from
2.775 to 3.715 mg GAE/100 g FW (Table 2).
The seed of all Clones displayed the highest total phenolic content. The differences
total phenolic content in seeds of nine Clones was found to be significant at p < 0.05 level.
Clone 6 had the highest total phenolic content at 245 mg GAE/100 g, followed by Clone
9 at 238 mg GAE/100 g FW, whereas the lowest total phenolic content was observed in
Clone 1 and Clone 4 at 201 and 207 mg GAE/100 g FW, respectively (Table 2).

Table 2. Total phenolic content in berries of nine ‘Karaerik’ grape clones.

Total phenolic content (mg GAE/100 g FW)

Clones
Peel Pulp Seed
Clone 1 117cd 2.816ef 204e
Clone 2 129c 2.775f 218d
Clone 3 111d 2.904e 211de
Clone 4 121cd 3.112d 207e
Clone 5 138b 3.178d 222cd
Clone 6 126c 3.386c 245a
Clone 7 134b 3.514b 233bc
Clone 8 149a 3.490bc 229c
Clone 9 143ab 3.715a 238b
Different letters in the same column indicate significant differences (p < 0.05).

The results clearly indicated that grape seeds are richer sources of total phenolic
content than peel and pulp and also peel was found to be richer than pulp for all nine
Karaerik grape clones. It is also evident that there are clonal variations among grape clones
in terms of total phenolic content (Table 2). In fact, there were a few studies comparing
the total phenolic content of peel, pulp and seed samples of different grape cultivars and
even a very limited number of studies has been carried out on different clones of a single
cultivar. Yi et al. [45] observed great variability in juices among grape cultivars in terms
of total phenolic content in the range of 44–184 mg GAE/100 g fresh samples, which is
Plants 2021, 10, 2154 7 of 15

in accordance with our results. In Spain, Ruiz-Torralba et al. [46] used berries of two
white and red grape cultivars and reported total phenolic content values between 124
and 151 mg GAE/g FW. A large number of grape cultivars were used in total phenol
content analysis in Italy and great variation has been observed among cultivars in terms
of total phenolic content (92–468 mg GAE/100 g FW [47]. In middle Anatolia, Gundesli
et al. [48] determined the average total phenol content of 222 mg GAE/g FW in berries of
the Kabarcik grape cultivar. In China, Liu et al. [49] used a large number of diverse grape
cultivars including white, red and black colored cultivars and found total phenol content
varied from 29 to 140 mg GAE/100 g FW. In China, a total six red peel colored grape
cultivars was used in an experiment and the total phenolic content was found to be the
highest in seeds, followed by peels and pulps, indicating similarities with our study [50].
Yilmaz et al. [51] produced a comprehensive study to determine the total phenolic content
and antioxidant activity of 22 grape cultivars including seven white and 15 red grapes
grown in the Marmara region of Turkey. They found that total phenolic contents of grape
pulp, seed and peel parts ranged from 9.26 to 62.29, from 162.29 to 326.18 and from 96.61
to 167.42 mg gallic acid equivalents/100 g fresh weight among cultivars, respectively.
Our finding is coincided with the results of Yilmaz et al. [51]. Previous studies indicated
that total phenolic content vary among plant organs of grape, cultivars, clones, growing
location, climate, soil, temperature, cultural practices, ripening stage, training system,
etc. [51–55]. Phenolic compounds contribute the color and taste characteristics of grapes
and they also significantly contribute to the antiradical and antioxidant properties of grape
berries [56]. The contributions of grape juice, pulp, peel and seeds to the total phenolic
contents of grape berries were reported to be 5, 1, 30 and 64%, respectively [57]. Karaman
et al. [58] reported that total phenolic content cultivar dependent and seeds were found to
be richer than peels of all six grape cultivars used.

3.3. Total Antioxidant Capacity

3.3.1. DPPH Assay
DPPH (1,1-diphenyl-2-picrylhydrazyl) analysis is one of the best-known, accurate, and
frequently employed methods for evaluating antioxidant activity in plant materials [59].
The DPPH scavenging against ‘Karaerik’ grape peel, pulp and seed extracts was found
in the descending order of seed>peel>pulp for all nine Clones (Table 3). There were
statistically significant differences among Clones for DPPH scavenging activity (p < 0.05).
The highest DPPH radical scavenging were observed in Clone 9 as 1918 µmol Trolox/100 g
FW while the lowest values were obtained from Clone 1 as 1510 µmol Trolox/100 g FW. The
peel samples of nine ‘Karaerik’ grape clones exhibited DPPH radical scavenging between
1080–1340 µmol Trolox/100 g FW. The lowest DPPH scavenging has been observed in
pulp samples were in range of 276–346 µmol Trolox/100 g FW, respectively (Table 3). It is
clear that ‘Karaerik’ grape seeds and peels extracts belongs to different clones significantly
(p < 0.05) inhibited DPPH free radicals’ generation. The studies conducted in different parts
of the world on grape peel and pulp. For example, Choi et al. [60] reported higher DPPH
radical scavenging activity for Campbell Early grape seed extracts than pulp in Korea.
Fahmi et al. [61] used DPPH assay to estimate antioxidant activity in grapes and found
that different grape cultivar extracts exhibited variable DPPH radical scavenging effect.
Farhadi et al. [62] used six grape seeds and pulps for antioxidant capacity analysis and
found DPPH inhibition was the highest in the seed extracts. Mandić et al. [63] reported that
grape species, individual cultivars, cultivation condition, maturation stage, and seasonal
variations may affect phenolic biosynthesis and antioxidant capacity in grape seed, peel
and flesh. Yilmaz et al. [51] indicated that the seeds of grape cultivars are the best source of
antioxidants, followed by peels and then pulps by using DPPH, FRAP and TEAC assays.
In a study, Anastasiadi et al. [64] reported that regardless of the assay method, grape seeds
have the best antioxidant activity compared to peel and pulp. Shen et al. [50] observed that
DPPH scavenging effects of the pulp and peel of grape cultivars were remarkably lower
than those of the seeds.
Plants 2021, 10, 2154 8 of 15

Table 3. DPPH assay results of ‘Karaerik’ grape clones.

DPPH (µmol Trolox/100 g FW)

Clones
Peel Pulp Seed
Clone 1 1152c 289bc 1510e
Clone 2 1186bc 303bc 1778c
Clone 3 1080d 276c 1687d
Clone 4 1125cd 282bc 1542e
Clone 5 1260ab 322ab 1744c
Clone 6 1242b 310b 1865b
Clone 7 1286ab 330ab 1851b
Clone 8 1340a 347a 1846b
Clone 9 1316ab 341ab 1918a
Different letters in the same column indicate significant differences (p < 0.05).

3.3.2. FRAP Assay

The antioxidant activities determined by FRAP assay in peel, pulp and seeds of nine
Clones were analyzed and expressed µmol Trolox/100 g FW. The Clones differed each
other significantly (p < 0.05) in terms of FRAP values (Table 4).

Table 4. FRAP assay results of ‘Karaerik’ grape clones.

FRAP (µmol Trolox/100 g FW)

Clones
Peel Pulp Seed
Clone 1 3620cd 82e 42600cd
Clone 2 3940bc 97cd 45260bc
Clone 3 3544d 77e 39880d
Clone 4 3870c 90cd 43700c
Clone 5 3986bc 104c 48670b
Clone 6 4235b 86d 48020b
Clone 7 4436ab 112b 49300ab
Clone 8 4127bc 128a 52460a
Clone 9 4610a 120ab 50640ab
Different letters in the same column indicate significant differences (p < 0.05).

As shown in Table 4, the highest FRAP values were expressed from seeds and followed
by peel and pulp. The seed extract from Clone 8 had the highest FRAP value (52460 µmol
Trolox/100 g FW), whilst that of Clone 9 and Clone 7 seeds showed the second and third
highest activity (50640 and 49300 µmol Trolox/100 g FW, respectively). In the FRAP
assay, among the seed samples, the lowest activity was observed in Clone 3 as 39880 µmol
Trolox/100 g FW. The FRAP values of peel and pulp samples of nine ‘Karaerik’ grape clones
varied from 3544 (Clone 3) to 4610 (Clone 9) µmol Trolox/100 g FW and 77 (Clone 3) to 128
(Clone 8) µmol Trolox/100 g FW. Overall, the peel, pulp and seeds of Clone 3 exhibited
the lowest FRAP value (Table 4). Liu et al. [49] used pulp samples of 30 common grape
cultivars with white, red and black peel color in China and found FRAP values in the
range of 59–612 µmol Trolox/100 g FW. Our FRAP results showed some similarities with
this study.
In another study, Fu et al. [65] used whole berry samples of four grape cultivars and
found FRAP values between 173 and 1012 µmol Fe (II)/100 g FW. In another study [66]
showed that FRAP values of 56 wild edible fruits quite variable and ranged from 67 to
14300 µmol Fe (II)/100 g FW. Sochorova et al. [67] showed that grape seeds had high content
Plants 2021, 10, 2154 9 of 15

of antioxidant components and the differences in content mainly belongs to individual

cultivars. Shen et al. [50] used six grape cultivars in FRAP analysis and found that the seeds
had higher FRAP values than pulp. Ruiz-Torralba et al. [46] used whole berries of white
and red grape cultivars and reported FRAP values between 738 and 786 µmol Trolox/100 g
FW, which is in agreement with our present results. Yilmaz et al. [51] used a large number
of grape seed, pulp and peel extracts and found that FRAP values were the highest in seed
samples. They reported that the pulps of red cultivars had higher FRAP values than white
cultivars. Among the red cultivars, the pulps of Hamburg Misketi had an FRAP value of
297 µmol TE/100 g FW. Our results were in good agreement with this report. Most of the
studies showed that grape seed contains higher FRAP values than peels and also that peel
has higher FRAP values than pulps [50,51,58,68]. Gokturk Baydar et al. [69] demonstrated
that seeds of Narince grape cultivar had the highest antioxidant activity than pulp.

3.3.3. TEAC Assay

The antioxidant activity of peel, pulp and seeds of nine ‘Karaerik’ grape clones was
also performed using the Trolox equivalent antioxidant capacity (TEAC) assay. Results of
TEAC assay in peel, pulp and seed extracts of nine ‘Karaerik’ grape clones are presented in
Table 5. Statistically significant differences among cultivars for peel, pulp and seed extracts
are found (p < 0.05). For each studied clone, the highest TEAC values was observed in
seeds and followed by peel, and the lowest TEAC values are evident in pulp samples
(Table 5). For seed extracts, The TEAC values were found between 1464 (Clone 3) and 1730
(Clone 9) µmol Trolox/100 g FW. For peel and pulp extracts, those values were within
in range of 310 (Clone 3) to 414 (Clone 9) µmol Trolox/100 g FW and 50 (Clone 3) to 98
(Clone 7) µmol Trolox/100 g FW, respectively (Table 5). Overall, the lowest TEAC values of
peel, pulp and seed extracts was observed from ‘Karaerik’ Clone 3. TEAC results clearly
indicated that there was a great variability among ‘Karaerik’ clones in terms of TEAC
values and results also showed that grape seeds are better sources of antioxidants among
grape plant parts even among horticultural crops. Costa et al. [70] also found that grape
berries are rich for antioxidants by using the TEAC method. Ruiz-Torralba et al. [46]
used whole berries of white and red grape cultivars and reported FRAP values 971 µmol
TE/100 g FW for red and 1097 µmol TE/100 g FW for white cultivars. Liu et al. [49] used
30 grape cultivars grown in China and determined the TEAC values of whole berries in
the range of 339–4839 µmol TE/100 g FW, respectively indicating similarities with our
study. Sochorova et al. [67] indicated that the grape seed extract was rich both in oligomeric
and polymeric flavanols; therefore, extracts from grape seeds and peels contained great
amounts of antioxidants. Weidner et al. [71] studied phenolic compounds isolated from
seeds of European and Japanese species of grapevine (Vitis vinifera and Vitis coignetiae) and
found that seeds contained great amounts of tannins and detectable levels of catechins and
p-coumaric, ferulic and caffeic acids that have antioxidant effect. Yilmaz et al. [51] used a
large number of grape cultivars with different berry peel color and found great variability
both among cultivars and plant parts (peel, pulp and seeds) in terms of antioxidant activity
by using TEAC assay. They also indicated that DPPH, FRAP and TEAC assays were useful
to determine antioxidant activity of different plant parts of grapes.
Plants 2021, 10, 2154 10 of 15

Table 5. TEAC assay results of ‘Karaerik’ grape clones.

TEAC (µmol Trolox/100 g FW)

Clones
Peel Pulp Seed
Clone 1 322bc 56e 1490bc
Clone 2 348bc 66d 1530bc
Clone 3 310c 50f 1464c
Clone 4 340bc 60def 1510bc
Clone 5 382ab 70cd 1610b
Clone 6 360b 76c 1580bc
Clone 7 374ab 98a 1682ab
Clone 8 394ab 80bc 1702ab
Clone 9 414a 87b 1730a
Different letters in the same column indicate significant differences (p < 0.05).

3.3.4. Correlations between TPC and Antioxidant Activity

The correlations between the total phenol content (TPC) with antioxidant activity
(DPPH, FRAP and TEAC) in peel, pulp and seeds of nine clones of ‘Karaerik’ grape cultivar
are shown in Table 6.

Table 6. Correlation between the TPC and antioxidant capacities by DPPH, FRAP and TEAC.

TPC DPPH FRAP TEAC

Heading
Peel Pulp Seed Peel Pulp Seed Peel Pulp Seed Peel Pulp Seed
TPC 1.00 1.00 1.00
DPPH 0.84 ** 0.82 * 0.87 ** 1.00 1.00 1.00
FRAP 0.81 ** 0.75 * 0.80 ** 0.72 * 0.68 * 0.70 * 1.00 1.00 1.00
TEAC 0.85 ** 0.82 ** 0.80 ** 0.55 * 0.50 * 0.52 * 0.73 * 0.65 * 0.67 * 1.00 1.00 1.00
TPC: Total Phenol Content; *:(p < 0.05); **:(p < 0.01).

Considering the correlation coefficient, the TPC of grape peel, pulp and seeds showed
a high correlation coefficient (0.8 < r < 1) with DPPH (0.84, 0.82 and 0.87), FRAP (0.81, 0.75
and 0.80) and TEAC (0.85, 0.82 and 0.80). DPPH showed a moderate positive correlation
(0.5 < r < 0.8) with FRAP (0.72, 0.68 and 0.70) and TEAC (0.55, 0.50 and 0.52) based on peel,
pulp and seed. In addition, FRAP also showed a moderate positive correlation (0.5 < r < 0.8)
with TEAC (0.73, 0.65 and 0.67) (Table 6). This correlation helps us to understand the
contribution of the total phenol content to the antioxidant capacity of grape pulp, peel and
seeds in findings reported previously [72–74]. Clarke et al. [75] found that high correlation
of DPPH, FRAP, TEAC and TPC indicates redundancy in the use of all three assays to
screen for the antioxidant activity in extracts of plants.

3.4. Phenolic Compounds

It has been observed that phenolic compounds show a wide variation both in berry
parts and clones of ‘Karaerik’ grape cultivar (Tables 7–9). Phenolic compounds, except
ferulic acid, chlorogenic acid, quercetin, myricetin and vanillic acid in the peel of ‘Karaerik’
clones, showed significant differences at p ≤ 0.05 level (Table 7). All ‘Karaerik’ clones had
the highest amount of syringic acid (51.1–73.6 mg/100 g FW) in its peel, and followed by
caffeic acid (22.0–35.7mg/100 g FW) and gallic acid (10.4–19.1 mg/100 g FW), respectively.
Plants 2021, 10, 2154 11 of 15

Table 7. Phenolic compounds in peel of ‘Karaerik’ clones (mg/100 g FW).

p- Caffeic Syringic Gallic Ferulic Chlorogenic Vanillic

Clones Quercetin Myricetin
coumaric Acid Acid Acid Acid Acid Acid
Clone 1 5.2ab 25.0bc 57.6de 13.2bc 3.8NS 4.2NS 0.8NS 0.6NS 0.2NS
Clone 2 5.5ab 26.2bc 58.2de 14.7bc 1.9 2.4 1.2 0.4 0.3
Clone 3 4.2bc 23.8bc 59.6d 16.0b 2.0 2.6 0.9 0.5 0.3
Clone 4 4.9b 22.0c 51.1f 11.2bc 1.7 3.0 1.1 0.5 0.2
Clone 5 6.2ab 28.4ab 56.0e 10.4c 3.2 6.0 2.4 1.7 0.5
Clone 6 5.9ab 27.7b 54.3cd 16.8b 3.0 3.6 1.4 0.8 0.2
Clone 7 6.8ab 31.0ab 70.3b 14.3bc 3.8 4.0 1.7 0.9 0.5
Clone 8 7.9a 35.7a 73.6a 17.8ab 2.4 2.9 1.0 0.8 0.6
Clone 9 7.6ab 32.6ab 68.2c 19.1a 3.6 2.5 1.9 0.6 0.5
Different letters in the same column indicate significant differences (p < 0.05); NS: Non significant.

It was determined that all ‘Karaerik’ clones had negligible amounts of phenolic com-
pounds in pulp. Moreover, all phenolic compounds determined in pulp of nine ‘Karaerik’
grape clones were found insignificant (Table 8).
In the experiment, the seeds of all clones exhibited higher phenolic compound than
peel and pulp samples. Gallic acid content in seeds of the examined nine clones generally
had higher than the other phenolics. The highest gallic acid content was obtained from
Clone 5 as 110.1 mg/100 g FW, while the lowest gallic acid content was determined to be in
the Clones 8 and 4 as 95.0 and 95.6 mg/100 g FW, respectively. Next to gallic acid, quercetin
was found to be the second most important phenolic compound in ‘Karaerik’ grape seeds,
and its concentration varied from 57.5 to 72.1 mg/100 g FW, respectively (Table 9). Pantelic
et al. [76] reported that grape seeds rich for phenolic compounds including gallic acid,
syringic acid, quercetin, caffeic acid, chlorogenic acid and seeds followed by peel and
pulp in terms of phenolic compounds which is good agreement with our results. Rusjan
et al. [77] also indicated that grape berries are rich inphenolic compounds. Gokcen et al. [78]
reported that the phenolic compounds widely found in grape berries are syringic acid,
vanillic acid, gallic acid, p-coumaric acid, caffeic acid and ferulic acid. Gokturk Baydar
et al. [53] reported that grape seeds richer than peels in terms of phenolic compounds
including syringic acid, vanillic acid, gallic acid, p-coumaric acid, caffeic acid and ferulic
acid. Phenolic compounds play a role in many physiological events such as color and taste
formation in plants.

Table 8. Phenolic compounds in pulp of ‘Karaerik’ clones (mg/100 g FW).

p-
Caffeic Syringic Gallic Ferulic Chlorogenic Vanillic
Clones coumaric Quercetin Myricetin
Acid Acid Acid Acid Acid Acid
acid
Clone 1 0.2NS 0.9NS 1.1NS 0.5NS 0.2NS 0.3NS 0.1NS 0.2NS 0.3NS
Clone 2 0.1 0.4 0.6 0.3 0.4 0.3 ND ND ND
Clone 3 0.3 0.6 0.8 0.3 ND 0.1 0.1 0.1 0.1
Clone 4 0.1 0.5 0.8 0.5 0.1 0.2 0.2 ND ND
Clone 5 0.2 0.3 0.6 0.4 ND ND ND 0.1 0.1
Clone 6 0.3 0.6 1.4 0.5 0.1 0.1 0.1 0.2 ND
Clone 7 0.1 1.1 1.0 0.4 ND 0.5 ND ND 0.1
Clone 8 0.4 0.7 1.2 0.4 0.2 ND 0.2 0.1 ND
Clone 9 0.4 1.0 1.0 0.6 0.3 ND 0.1 0.1 0.2
Different letters in the same column indicate significant differences (p < 0.05): NS: Non Significant; ND: Non Determined.
Plants 2021, 10, 2154 12 of 15

Table 9. Phenolic compounds in seed of ‘Karaerik’ clones (mg/100 g).

Gallic Chlorogenic p-coumaric Caffeic Syringic

Clones Catechin Quercetin Myricetin
Acid Acid Acid acid Acid
Clone 1 23.2d 60.4bc 97.3bc 8.6ab 1.4NS 2.3NS 1.7NS 1.9NS
Clone 2 26.6c 63.5b 102.2b 11.9a 1.0 2.8 1.1 1.4
Clone 3 20.6de 57.5c 104.9abc 10.2ab 2.1 1.9 2.2 2.0
Clone 4 19.5e 66.4ab 95.6c 4.1b 3.1 3.0 2.7 3.0
Clone 5 25.0cd 60.9bc 110.1a 7.8ab 4.2 1.9 2.0 1.5
Clone 6 31.1ab 70.4ab 104.0 9.8ab 4.0 3.7 4.4 2.9
Clone 7 29.4bc 65.0ab 99.2bc 6.8ab 2.7 4.5 4.5 ND
Clone 8 33.2a 72.1a 95.0c 4.0b 3.2 3.8 2.9 3.1
Clone 9 30.1b 63.3b 106.8 7.3ab 3.6 3.0 4.2 3.3
Different letters in the same column indicate significant differences (p < 0.05): NS: Non Significant; ND: Non Determined.

4. Conclusions
The results obtained showed that the seeds of nine ‘Karaerik’ grape clones had higher
total phenolic content and antioxidant activity values than peel and pulp. In addition,
the total phenolic content and antioxidant activity was found clone dependent. Is is very
important in grape breeding to use better clones in breeding activities in order to obtain
nutraceutical rich ‘Karaerik’ plants. Moreover, the results indicated that the ‘Karaerik’
grape clones studied in this research may have great potential to be exploited by the grape
processing industry. Further studies will be focused on the more detailed analysis in the
different parts of these clones to clarify the beneficial effects on human health. In this
context, the most promising clones—such as 7, 8 and 9— that have higher antioxidant
activity may have present great potential for grape breeders, the food industry and health-
conscious consumers. Clones obtained from the study could be used to establishing a base
of vineyards where propagation can start to supply growers and propagators.

Author Contributions: Conceptualization, M.S.U.; N.K.; M.K. and S.E; data curation, M.S.U.; N.K.;
S.E. and M.K.; formal analysis, S.E.; M.S.U. and N.K.; methodology, S.E. and M.K.; project adminis-
tration, M.S.U. and S.E.; visualization, M.K.; M.B. and J.S; writing—original draft, S.E.; M.B. and J.S;
writing—review and editing, S.E.; M.B. and J.S. All authors have read and agreed to the published
version of the manuscript.
Funding: This study supported by the project CZ.02.1.01/0.0/0.0/16_017/0002334 Research Infras-
tructure for Young Scientists; this is co-financed by Operational Programme Research, Development
and Education.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: All-new research data were presented in this contribution.
Conflicts of Interest: The authors declare that they have no conflicts of interest.

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