Biochemical Profile and Antioxidant Activity of Fresh Fruits from Apple Genotypes

Abstract

This study investigates the biochemical profile and antioxidant activity of different apple genotypes developed through breeding as compared to three established cultivars, providing valuable insights for improving the nutritional quality of apples. The research analyzes the content of bioactive compounds such as polyphenols (TPC), flavonoids (TFC), tannins (TTC), and anthocyanins (TAC) as well as sugars content to determine nutritional variability between genotypes. Antioxidant activity was assessed by standardized methods, correlating the results with biochemical profiles. The content of bioactive compounds including polyphenols, tannins, flavonoids, and anthocyanins varied significantly between the studied apple genotypes, with the H18/6 genotype showing the highest values for TPC, TFC, and TAC (839.08 mg GAE/100 g; 130.39 mg CE/100 g, and 19.04 mg C3GE/100 g, respectively), highlighting the importance of varietal characterization for nutraceutical potential. Lycopene and β-carotene, carotenoid compounds with antioxidant properties, were present in apples only in low concentrations, ranging between 0.25 and 0.95 mg/100 g for lycopene and 0.03 and 0.50 mg/100 g for β-carotene, with higher levels observed in genotypes with more intense colors. This study contributes to the identification of genotypes with added value that are useful in improving human health and developing functional food products.

1. Introduction

Apples (Malus domestica) are among the most consumed and appreciated fruits globally due to their nutritional value and benefits for health. Widespread increasing consumption of apples, their juices, and derived products along with their rich phytochemical profile highlight their significant potential to positively influence human health [1]. The diversity of apple genotypes offers a wide range of biochemical characteristics, such as the content of polyphenols, flavonoids, vitamins, organic acids, and natural sugars, all of which contribute to the nutritional profile of the fruit [2,3]. Apples contain phenolic compounds like flavonoids, phenolic acids, and tannins, which contribute to the fruit’s antioxidant activity, neutralizing free radicals, protecting cells from oxidative stress, and reducing the risk of chronic conditions while enhancing its nutritional quality and providing health benefits [3,4]. More than 60 phenolic compounds have been identified in apples. They are part of the plant’s secondary metabolism and perform essential functions such as growth, defense against pathogens, color, and aroma. They are also vital for growth and reproduction and are mainly synthesized when the plant is submitted to stressful conditions such as infections, wounds, and ultraviolet radiation [5]. Phenolic compounds are organic compounds with at least one phenolic unit and two or more hydroxyl groups in the structure [5] and fall into two groups: flavonoids and non-flavonoids, the latter including phenolic acids, stilbenes, lignans, and tannins [5]. Apples contain six subclasses of flavonoids, namely flavanols, flavones, flavanones, monomeric and oligomeric flavanols, dihydrochalcones, and anthocyanins [5,6,7,8]. Tannins are water-soluble polyphenolic substances capable of forming complexes with proteins, polysaccharides, nucleic acids, steroids, alkaloids, and saponins [9]. Depending on their chemical structure and properties, apples contain both hydrolysable and condensed tannins [10,11]. Their complexity depends on the flavanol or flavandiol units, which vary among constituents and within sites for interflavan bond formation [12,13].

The biochemical profile of apples varies significantly depending on the geographical area of cultivation due to the influence of multiple environmental factors, such as climate, soil type, altitude, and specific agricultural practices. Environmental conditions are essential factors in determining the content of bioactive compounds in fruits, such as polyphenols, flavonoids, vitamins, and minerals [4,14]. The content of soluble sugars and organic acids in apples differs depending on the cultivar and can be influenced by factors such as light intensity, sun exposure, and other environmental conditions [15,16]. At the same time, Mignard et al. [17] considered that the geographical area of cultivation plays an essential role in determining the resulting metabolite profiles. The antioxidant activity of apples is one of their main benefits, as it is associated with the prevention of chronic illnesses such as cardiovascular diseases, diabetes, and certain types of cancer. Among antioxidants, it has been estimated that the total content of phenolic compounds, flavonoids, anthocyanins, and ascorbic acid has a major contribution to the overall antioxidant activity of the fruit [18]. Furthermore, the variations between genotypes in terms of the concentration and composition of these compounds provide a valuable opportunity for the selection and improvement of apple varieties. Previous research has highlighted the differences between genotypes, emphasizing their influence and that of climate as the main factors determining metabolite profiles and fruit characteristics [19,20]. In this context, this study aims to evaluate the biochemical profile and antioxidant activity of different apple genotypes to characterize genetic diversity and identify those with superior functional and nutritional potential. The research hypothesis is that genotypes developed through breeding programs exhibit enhanced biochemical and antioxidant properties compared to established cultivars. Over the years, parental varieties with valuable genetic traits have been used to develop disease-resistant apple varieties [21]. Through a comparative analysis of fresh fruit from newly bred genotypes and three commercial cultivars, this study provides valuable insights for genetic improvement and the enhancement of apple nutritional quality.

2. Materials and Methods

2.1. Materials and Location

Fourteen genotypes of apple (Malus domestica) were selected for this study: three cultivars (‘Valery’, ‘Florina’, and ‘George’) and eleven elites found in the selection field [21]. All fruit materials were taken from 10 trees of each genotype from the experimental orchard of the Research and Development Station for Fruit Cultivation Voinești, located in Voinești, Romania (45°4′12″ N 25°14′58″ E), at an altitude of 400–600 m. The climate is temperate, with summers without excessive heat and milder winters, when the temperature rarely drops below −20 °C. The average multiannual temperature (normal for the area) is 8.8 °C, and the sum of annual rainfall is 782 mm. The area is protected from strong winds due to the existence of slopes on both sides of Dâmboviţa River Valley.

2.2. Preparation of Extracts

For the biochemical analyses, the fresh fruits (approximately 1 kg for each cultivar) were washed with drinking water and dried with a paper towel. After removing the seeds, the apples (pulp and skin) were cut into pieces, homogenized using a vertical mixer, and stored in hermetically sealed glass jars at 3–4 °C. Three samples were taken from each well-homogenized puree for each analysis.

Next, 1 g of each fruit puree was mixed with 10 mL of either distilled water or an ethanol–water solution (8:2, v/v). The mixtures were vortexed at 3000 rpm for 2 min using a Corning-Labnet Vortex Mixer VX-200 system (Corning Life Sciences, Tewksbury, MA, USA) and subjected to ultrasonic treatment (40 kHz; ULTR-2L0-001; Labbox Labware, Migjorn, Spain) at 99 °C for 30 min for aqueous extracts or at room temperature for 60 min for ethanolic extracts. Aqueous extracts were filtered, while ethanolic extracts were centrifuged at 6500 rpm (Spectrafuge 6C Research Centrifuge; Labnet International Inc., Edison, NJ, USA) for 30 min. The supernatants were collected for the analysis of tannins, sugars, polyphenols, flavonoids, anthocyanins, and antioxidant activity [13].

2.3. Determination of Moisture and Ash Content

Moisture (water content) and ash (mineral content) were determined gravimetrically. Moisture content was determined by oven drying at 105–110 °C and ash content by calcining at 550–600 °C to constant mass. The results are presented in percentage (%) [22].

2.4. Determination of Sugar Content

Total sugar content (TSC) was measured using a colorimetric method [22]. A mixture of 0.1 mL aqueous extract, 0.9 mL distilled water, 5 mL concentrated sulfuric acid, and 1 mL of 5% phenol was heated at 100 °C for 15 min. Absorbance was read at 490 nm, and TSC was calculated using a glucose calibration curve and is expressed as g glucose equivalents (GluE)/100 g fresh weight (FW).

2.5. Determination of Antioxidant Activity (DPPH)

The antioxidant activity was evaluated using the DPPH (2,2-Diphenyl-1-picrylhydrazyl) assay, a method based on the reduction of the DPPH radical. A mixture of 2.97 mL DPPH solution and 0.03 mL extract was shaken and incubated in the dark for 20 min. Absorbance was measured at 517 nm [22].

2.6. Determination of Total Polyphenol Content

Total polyphenol content (TPC), expressed as mg GAE/100 g FW, was determined using a colorimetric method adapted from Stamin et al. [22]. The reaction involved mixing 1 mL ethanolic extract, 6.5 mL distilled water, 0.5 mL Folin–Ciocalteu reagent, and 2 mL of 10% sodium carbonate. After a 2 h dark incubation, absorbance was measured at 760 nm.

2.7. Determination of Total Tannin Content

Total tannin content (TTC), expressed as mg GAE/100 g FW, was determined using a colorimetric method adapted from Stamin et al. [22]. A mixture of 1 mL aqueous extract, 6.5 mL distilled water, 0.5 mL Folin–Ciocalteu reagent, and 2 mL of 10% sodium carbonate was incubated in the dark for 60 min. Absorbance was measured at 760 nm.

2.8. Determination of Total Flavonoid Content

Total flavonoid content (TFC), expressed as mg catechin equivalents (EC)/100 g FW, was determined spectrophotometrically [22]. A mixture of 5 mL distilled water, 2 mL ethanolic extract, 0.5 mL 5% sodium nitrite, 0.5 mL 10% aluminum chloride, and 2 mL 1 M sodium hydroxide was prepared. Absorbance was measured at 510 nm using a catechin calibration curve.

2.9. Determination of Total Anthocyanin Content

Total anthocyanin content (TAC), expressed as mg C3GE/100 g FW, was determined using a modified differential pH method from Angraini et al. [23]. Ethanolic extracts were diluted with pH 1.0 (potassium chloride) and pH 4.5 (sodium acetate) buffers. Absorbance was measured at 520 and 700 nm after 15 min.

2.10. Determination of Carotenoid Content (Lycopene and β-Carotene)

Lycopene and β-carotene concentrations (mg/100 g FW) were determined using a colorimetric method [22]. Supernatants from 3 g of fruit puree extracted with hexane, ethanol, and acetone (2:1:1) were analyzed at 470 nm and 503 nm using extinction coefficients in hexane.

2.11. Statistical Analysis

All assays were performed in triplicate, and results are presented as mean ± SD. Data were analyzed using Excel and SPSS Trial Version 26.0. Duncan’s multiple range test was used to assess genotype differences (p < 0.05). The Pearson correlation coefficient was used to evaluate the linear correlation between parameters.

3. Results and Discussion

3.1. Moisture, Ash, Sugars, and DPPH• Content

Table 1. Moisture content, ash, total sugar content (TSC), and antioxidant activity (DPPH• inhibition%) in fruits from apple genotypes *.

Genotype	Moisture Content (%)	Ash (%)	TSC (gGluE/100 g)	DPPH• (%)
‘Valery’	76.75 ± 0.19 l	0.38 ± 0.01 e	9.91 ± 0.06 a	12.07 ± 0.46 i
‘Florina’	83.26 ± 0.03 c	0.33 ± 0.01 g	8.94 ± 0.15 d	15.50 ± 0.06 e
‘George’	83.99 ± 0.19 b	0.29 ± 0.01 i	7.78 ± 0.14 f	16.13 ± 0.04 d
H1/20–10	80.70 ± 0.04 f	0.33 ± 0.01 g	9.27 ± 0.13 bc	15.44 ± 0.12 e
H14/1	77.30 ± 0.18 k	0.67 ± 0.01 a	9.48 ± 0.16 b	17.55 ± 0.06 c
H8/6	83.47 ± 0.18 c	0.26 ± 0.01 j	6.98 ± 0.23 g	15.45 ± 0.06 e
H2/3	83.42 ± 0.11 c	0.39 ± 0.01 d	6.86 ± 0.13 g	13.01 ± 0.06 h
H1/55	84.70 ± 0.05 a	0.17 ± 0.01 k	7.01 ± 0.17 g	14.75 ± 0.02 f
H4/42	78.95 ± 0.10 i	0.59 ± 0.01 b	9.82 ± 0.39 a	11.39 ± 0.16 k
H1/28	82.94 ± 0.11 d	0.32 ± 0.01 h	7.16 ± 0.13 g	11.46 ± 0.05 k
H8/1	80.41 ± 0.11 g	0.31 ± 0.01 h	7.70 ± 0.14 f	18.47 ± 0.04 b
H19/6	79.69 ± 0.04 h	0.37 ± 0.01 f	8.18 ± 0.18 e	13.41 ± 0.12 g
H18/6	81.50 ± 0.14 e	0.33 ± 0.01 g	7.60 ± 0.15 f	19.90 ± 0.04 a
H4/44	77.60 ± 0.03 j	0.44 ± 0.01 c	9.01 ± 0.20 cd	10.71 ± 0.07 l
Mean	81.05 ± 2.61	0.37 ± 0.12	8.26 ± 1.09	14.66 ± 2.73

* Note: Values are expressed as mean ± standard deviation; Different letters indicate statistically significant differences between genotypes (Duncan multiple range test, p < 0.05).

shows significant differences (p < 0.05) between the studied genotypes for all evaluated parameters. These variations in moisture content, ash content, sugar levels, and antioxidant activity (DPPH•) are probably influenced by genetic diversity (genotype) [17,24,25]. In our study, the moisture content of fresh apple fruits showed significant differences between cultivars. Understanding these differences is essential for choosing suitable genotypes for both commercial purposes and for further research aimed at optimizing apple fruit quality. The average moisture content for all apple genotypes was 81.05%, ranging from 76.75% (‘Valery’) to 84.70% (H1–55). Kalkisim et al. [26] reported a moisture content ranging from 80% to 90.2% for local apple varieties grown in Turkey. Similarly, Campeanu et al. [27] reported moisture values ranging from 76.69% to 88.37% for seven different apple varieties. These ranges reflect the specific variations of both environmental conditions and genetic characteristics of the analyzed varieties. For ash content, the highest value (0.67%) was found in the H14/1 genotype, while the lowest (0.17%) was in the H1–55 genotype, with an average of 0.37%. Analysis of the ash content of apple fruits also revealed significant variations between genotypes (p < 0.05), with these differences being influenced by genetic variability given that the specific environmental conditions in which the fruits were grown were identical. In the study conducted by Kalkisim et al. [26], the ash content was determined in the range of 0.6–1.57%. Ash content is an indicator of the mineral composition of fruits and can influence their nutritional and technological properties. Genotypes with high ash content (such as H14/1) may be beneficial for mineral rich diets, as they indicate a higher presence of essential elements such as potassium, calcium, or magnesium. Genotypes with lower ash content (such as H1/55) may have a more specific use in processed products where excess minerals could affect the stability or taste of the final product. The findings of this study offer valuable insights for apple genotype breeding, focusing on improving the nutritional profile of the fruit and supporting decisions on their use according to market and consumer requirements. The average sugar content (TSC) was 8.26 g GluE/100 g, with significant genotype differences (p < 0.05) ranging from 6.86 g GluE/100 g (H2/3) to 9.91 g GluE/100 g (’Valery’).

These values are similar to the ones reported by Mignard et al. [17], where the average value identified in five apple varieties varied between 61.85 and 121.61 g/kg. Based on Duncan test and the significance of the differences between varieties, a grouping of varieties according to sugar content can be observed. A first category is represented by genotypes with a high sugar content (9.82–9.91; H4/42 and ‘Valery’), and at the opposite pole are four genotypes with a sugar content between 6.86 and 7.16 gGluE/100 g (H8/6, H2/3, H1/55, and H1/28). From the breeder’s point of view, the high sugar content should be balanced with other factors, such as aroma, acidity, adaptability, and market preferences, aiming to develop varieties that are both pleasing to consumers and efficient for producers. The correct choice of parents in crossbreeding programs is crucial from the point of view of sugar content given that parental cultivars with high sugar content will transmit this characteristic to the offspring, and soluble sugars along with organic acids are essential in shaping the taste of fruits [28]. The antioxidant activity of apples is an important nutritional and medicinal feature due to the presence of bioactive compounds that neutralize free radicals and protect cells from oxidative stress [29]. The antioxidant activity of apples is mainly attributed to the phenolic compounds present in apples. Antioxidant activity showed an average free radical inhibition of 14.66%, with a 5.24% difference from the highest value of 19.90% observed in the H18/6 genotype.

3.2. The Content of Phenolic Compounds

Table 2. The phenolic compound content in fresh apple fruits *.

Genotype	TPC (mg GAE/100 g)	TTC (mg GAE/100 g)	TFC (mg CE/100 g)	TAC (mg C3GE/100 g)
‘Valery’	520.07 ± 6.03 g	316.06 ± 1.03 e	99.76 ± 4.19 b	17.39 ± 0.15 e
‘Florina’	499.30 ± 3.97 h	306.60 ± 2.94 d	82.74 ± 3.71 cd	17.99 ± 0.15 bc
‘George’	693.28 ± 4.75 e	361.94 ± 1.59 a	90.83 ± 5.20 c	17.99 ± 0.15 bc
H1/20–10	648.77 ± 4.26 f	257.16 ± 1.92 f	100.32 ± 4.52 b	17.76 ± 0.16 cd
H14/1	793.22 ± 8.19 b	299.27 ± 2.10 e	102.24 ± 4.79 b	19.00 ± 0.14 a
H8/6	759.46 ± 9.62 c	324.97 ± 2.03 b	105.53 ± 5.41 b	17.51 ± 0.15 de
H2/3	382.33 ± 4.17 k	174.52 ± 1.33 i	72.13 ± 2.92 ef	18.14 ± 0.14 b
H1/55	449.95 ± 4.20 j	256.75 ± 2.18 f	84.06 ± 3.80 cd	17.59 ± 0.15 de
H4/42	729.40 ± 2.37 d	118.06 ± 1.73 l	63.67 ± 2.14 g	17.54 ± 0.15 de
H1/28	377.63 ± 2.63 k	138.95 ± 1.28 j	77.52 ± 2.91 de	17.53 ± 0.16 de
H8/1	514.81 ± 3.97 g	186.02 ± 4.69 h	128.19 ± 6.82 a	18.16 ± 0.14 b
H19/6	483.16 ± 3.03 i	127.65 ± 4.04 k	83.61 ± 3.50 cd	17.67 ± 0.15 de
H18/6	839.08 ± 3.52 a	252.05 ± 0.85 g	130.39 ± 6.87 a	19.04 ± 0.14 a
H4/44	379.53 ± 2.56 l	108.41 ± 1.48 m	67.96 ± 2.25 fg	18.26 ± 0.14 b
Mean	576.43 ± 159.01	230.60 ± 84.49	92.07 ± 20.22	17.97 ± 0.52

* Note: Values are expressed as mean ± standard deviation; Different letters indicate statistically significant differences between genotypes (Duncan multiple range test, p < 0.05); TPC = total polyphenol content, TTC = total tannin content, TFC = concentration of flavonoids, and TAC = total anthocyanin content.

shows the phenolic compound content in fresh apple fruits, with significant differences observed between the genotypes for all identified parameters (p < 0.05). These variations may result from the complex interactions between cultivar genetics, growing conditions, and post-harvest factors [30]. Regarding the content of bioactive compounds, the concentration of TPC varied among the apple genotypes. TPC, known for its antioxidant properties, varied greatly among the genotypes, with the limits of variation being between 377.63 (H1/28) and 839.08 mg GAE/100 g (H18/6). The apple cultivars ‘Valery’, ‘Florina’, and ‘George’ showed a lower content of phenolic compounds, with values 1.21 to 1.68 times lower compared to the H18–6 genotype.

Although the cultivars ‘Valery’, ‘Florina’, and ‘George’ showed a high content of phenolic compounds, some genotypes in the testing period (H18/6, H14/1, H8/6, and H4/42) demonstrated an even higher content, highlighting a significant beneficial potential for health. The concentrations determined for the analyzed genotypes were, however, higher than those reported by Piagentini and Pirovani [31] for the combination of peel and pulp in five apple varieties. Also, significant differences between apple cultivars in terms of the content of phenolic compounds were also observed by Mohammed et al. [32]. In their study, the maximum concentration was 1154.65 μg EAG/g extract, while the minimum value was 82.63 μg EAG/g extract. Such genotype differences are commonly reported in the literature, which highlights that polyphenol levels are influenced by factors including geographical origin, genotype, varietal profile, and various abiotic and biotic conditions [32,33]. Among all genotypes studied, total tannins (TTC) stand out by with the highest percentage of total phenolic compounds (TPC), followed, in descending order, by total flavonoids (TFC) and total anthocyanins (TAC). The differences between these classes of phenolic compounds are significant in terms of order of magnitude. The highest content of tannins (TTC) was identified in the cultivar ‘George’, with a value of 361.94 mg GAE/100 g, representing 62.8% of the average value of TPC. Regarding flavonoids (TFC), the genotype H18/6 reached the maximum value, with 130.39 mg CE/100 g, contributing 22.6% to the average TPC. On the other hand, anthocyanins (TAC) represent the least-abundant class of polyphenolic compounds in apples, with the H18/6 genotype recording the highest anthocyanin content, 19.04 C3GE/100 g, which corresponds to only 3.3% of the average TPC. These results highlight the significant variations between genotypes (p < 0.05) within the same group of compounds in terms of composition, underlining their importance in the nutraceutical characterization of apple fruits. According to the results obtained by Bahukhandi et al. [33] regarding the content of phenolic compounds and tannins in various apple genotypes, the levels of phenolic compounds and tannins varied considerably between different cultivars; the highest concentrations of total tannins were observed in the peel of the studied genotypes (1.37–21.74 mgTAE/g) compared to the whole fruit (0.86–16.47 mgTAE/g). Compared to the literature data, our results are considerably lower. For example, according to Vrhovsek et al. [34], the average total polyphenol content in apples ranged between 66.2 and 211.9 mg/100 g in fresh fruit.

3.3. The Content of Carotenes (Lycopene and β-Carotene)

Lycopene and β-carotene are two carotenoid pigments that contribute to the color of fruits and vegetables and play an important role in health due to their antioxidant properties. However, in case of apples, these compounds are not present in significant concentrations, unlike in other fruits [35]. Apples contain extremely low concentrations of lycopene or may even not contain this compound at all [36]. The concentrations determined in the analyzed genotypes ranged between 0.25 and 0.95 mg/100 g (

Table 3. Carotene content (lycopene and β-carotene) in fresh apple fruits *.

Genotype	Lycopene (mg/100 g)	β–Carotene (mg/100 g)
‘Valery’	0.51 ± 0.01 f	0.09 ± 0.02 f
‘Florina’	0.25 ± 0.01 l	0.09 ± 0.04 fg
‘George’	0.29 ± 0.01 k	0.13 ± 0.01 e
H1/20–10	0.87 ± 0.01 c	0.06 ± 0.01 gh
H14/1	0.49 ± 0.01 g	0.16 ± 0.01 d
H8/6	0.34 ± 0.01 i	0.04 ± 0.01 hi
H2/3	0.25 ± 0.01 l	0.05 ± 0.01 hi
H1/55	0.88 ± 0.01 b	0.03 ± 0.01 i
H4/42	0.48 ± 0.01 e	0.24 ± 0.01 h
H1/28	0.31 ± 0.01 j	0.16 ± 0.01 d
H8/1	0.95 ± 0.01 a	0.24 ± 0.01 c
H19/6	0.85 ± 0.01 d	0.50 ± 0.03 a
H18/6	0.48 ± 0.01 h	0.44 ± 0.03 b
H4/44	0.55 ± 0.01 ef	0.12 ± 0.01 k
Mean	0.54 ± 0.24	0.17 ± 0.14

* Note: Values are expressed as mean ± standard deviation. Different letters indicate statistically significant differences between genotypes (Duncan multiple range test, p < 0.05).

). β-carotene, a precursor of vitamin A, is found in small quantities in apples, with the concentrations identified being between 0.03 (H1/55) and 0.50 mg/100 g (H19/6). The concentration varied depending on the genotype: those with more intense colors (yellow-orange or reddish) generally had higher levels of beta-carotene than those with a green color. Although the concentrations of lycopene and β-carotene are low, their variability depending on the fruit color suggests the nutraceutical potential of genotypes with more intense colors.

Table 4. Correlations between bioactive compounds found in apple genotypes.

	TPC	TTC	TFC	TAC	Lycopene	Β-Carotene	DPPH•
TPC	1	0.487 **	0.501 **	0.378 *	−0.094	0.232	0.599 **
TTC	0.487 **	1	0.446 **	0.090	−0.246	−0.383 *	0.519 **
TFC	0.501 **	0.446 **	1	0.414 **	0.294	0.281	0.841 **
TAC	0.378 *	0.090	0.414 **	1	−0.084	0.312 *	0.613 **
Lycopene	−0.094	−0.246	0.294	−0.084	1	0.254	0.188
Β-carotene	0.232	−0.383 *	0.281	0.312 *	0.254	1	0.253
DPPH•	0.599 **	0.519 **	0.841 **	0.613 **	0.188	0.253	1

* Correlation is significant at the 0.05 level (2-tailed). ** Correlation is significant at the 0.01 level (2-tailed). TPC = total polyphenol content, TTC = total tannin content, TFC = concentration of flavonoids, TAC = total anthocyanin content, and DPPH• = antioxidant activity.

shows the correlation matrix and highlights the statistical relationships between the analyzed variables, highlighting the link between antioxidant activity and bioactive compounds. In the analysis, antioxidant activity recorded significant positive correlations with several parameters, suggesting that these compounds directly contribute to the free radical neutralization capacity. Antioxidant activity exhibited significant positive correlations with TPC (r = 0.599, p < 0.01), TTC (r = 0.519, p < 0.01), TFC (r = 0.841, p < 0.01), and TAC (r = 0.613, p < 0.01). The strong positive correlation between antioxidant activity and TPC suggests that polyphenols play a key role in antioxidant activity by inhibiting oxidative processes.

This relationship emphasizes that polyphenols are among the most important contributors to the antioxidant capacity of the samples [37]. Antioxidant activity was also positively correlated with TTC, suggesting the involvement of tannins in protecting against oxidative stress. This property is strongly associated with their chemical structure, characterized by phenolic rings capable of interacting with various molecules and acting as electron scavengers to neutralize ions and free radicals [38,39].

The strong correlation between antioxidant activity and TTC suggests that flavonoids are the key bioactive compounds driving antioxidant activity. Known for their powerful antioxidant properties, flavonoids prevent free radical damage by scavenging reactive oxygen species, activating antioxidant enzymes, inhibiting oxidases, and reducing α-tocopheryl radicals [40]. Antioxidant activity was closely associated with TAC, suggesting a significant contribution of anthocyanins to antioxidant capacity. Anthocyanins, natural pigments from the flavonoid class, are recognized for their antioxidant power and their role in preventing oxidative stress [41].

4. Conclusions

In conclusion, this study emphasizes the considerable variability in bioactive compounds (polyphenols, flavonoids, tannins, and anthocyanins) and antioxidant activity across apple genotypes, underscoring the significance of these differences for breeding. From a breeding perspective, the results indicate that the careful selection of parents and development of genotypes with high content of polyphenols and flavonoids could improve the nutraceutical value of the fruit, offering a significant potential to achieve attractiveness. This would allow for more attractive varieties for consumers, with increased benefits for human health. The contributions of this study are essential for the identification and promotion of genotypes with added value, which is relevant for the development of functional products as well as for increasing the market competitiveness of these fruits.