Phytochemical and Antioxidant Variability in Some Black Mulberry, Chokeberry, and Elderberry Cultivars in Relation to Cultivar, Plant Part, and Extraction Solvent

Abstract

Polyphenols and flavonoids are key bioactive compounds with significant antioxidant properties, making them crucial for human health and nutraceutical applications. However, their extraction efficiency and concentrations are influenced by multiple factors, including plant part, cultivar, and solvent selection. Therefore, this study investigated the effects of plant part, cultivar, and solvent type on the total phenolic content (TPC), total flavonoid content (TFC), and antioxidant capacity (ABTS and CUPRAC assays) in different extracts from black mulberry, chokeberry, and elderberry. In all three species, the leaves exhibited significantly higher phytochemical and antioxidant properties than the fruits, with an average increase of 62.8–133.4% in the TPC and 55.4–390.3% in the TFC. Among genotypes, Gümüşhacıköy Horum and Tohma Medik (black mulberry), Viking and Nero (chokeberry), and Tokat (T1) (elderberry) demonstrated the highest levels of bioactive compounds, while Şelale Karadut, Aron, and Haschberg exhibited the lowest values. Solvent selection played a crucial role, with methanol:water:HCl emerging as the most effective extraction medium, increasing the TPC by 27.5–46.3%, the TFC by 28.3–67.6%, and the antioxidant capacity (ABTS and CUPRAC) by up to 94.2% compared to water extraction. These findings indicate the significance of leaf-based bioactive compound extraction and optimized solvent selection for maximizing antioxidant yields. The results of this study also have important implications for both fruit cultivation and human nutrition, highlighting the potential of leaves as a valuable source of polyphenols and antioxidants.

1. Introduction

Elderberry (Sambucus nigra, S. nigra), chokeberry (Aronia melanocarpa, A. melanocarpa), and black mulberry (Morus nigra, M. nigra) have emerged as exceptional sources of bioactive compounds with significant health benefits due to their unique phytochemical profiles. These fruits are particularly noteworthy for their rich concentrations of bioactive components, including polyphenols, flavonoids, and anthocyanins [1,2]. These compounds not only contribute to the vibrant colors and unique flavors of the fruits but also play a pivotal role in promoting health through their antioxidant properties [3,4]. M. nigra, known for its vibrant dark fruits, has traditionally been used for its anti-inflammatory and antimicrobial properties [5], while A. melanocarpa is recognized as one of the richest sources of anthocyanins and proanthocyanidins, with studies highlighting its role in cardiovascular health and metabolic syndrome management [6]. Similarly, S. nigra is valued in traditional European medicine for its antiviral and immune-boosting properties, particularly in alleviating respiratory symptoms [7]. The antioxidant capacity of these fruits, driven by their ability to neutralize free radicals, plays a critical role in mitigating oxidative stress and preventing chronic diseases, including cardiovascular disorders, diabetes, and cancer [8,9].

These three berry species represent a diverse spectrum of bioactive potential, with each possessing unique phytochemical signatures that contribute to their medicinal properties [10]. The extraction efficiency of bioactive compounds from elderberry, chokeberry, and black mulberry is significantly influenced by the polarity of the solvent used, as demonstrated by comparative studies showing methanol extracts typically yielding a higher total phenolic content than aqueous extracts [11]. Pascariu et al. [12] reported in a study on elderberry phenolic compounds that ultrasound-assisted extraction with ethanol yielded the highest phenolic content, with excellent anthocyanin extraction and impressive extract stability lasting several months, providing valuable insights for pharmaceutical and food applications. Environmental growing conditions and the ripening stage markedly affect the concentrations of these bioactive compounds, with studies indicating that berries harvested at full maturity generally exhibit optimal phytochemical profiles [13]. Traditional processing methods, including drying and fermentation, have been shown to alter the bioaccessibility of phenolic compounds in these fruits, potentially enhancing or diminishing their health-promoting properties depending on the specific process employed [1,14]. Interestingly, the leaf extracts of these species often contain complementary but distinct bioactive profiles compared to their fruits, with elderberry leaves demonstrating particularly potent anti-inflammatory effects in recent clinical investigations [15]. The molecular mechanisms underlying the health benefits of these berries appear to involve multiple pathways, including the modulation of oxidative stress, inflammatory response, and glucose metabolism [16].

The method of extracting anthocyanins, proanthocyanidins, and other phenolic compounds from plant matrices is very important in elderberry (S. nigra L.), chokeberry (A. melanocarpa (Michx.) Elliot), and black mulberry (M. nigra L.). Equally important in this evaluation process is the initial step of extracting these bioactive compounds from plant matrices, as the extraction method and solvent choice significantly influence their recovery and preservation. Polar solvents, particularly methanol, are widely utilized in extracting hydrophilic phenolic acids, flavonoids, and anthocyanins, with methanol proving especially effective for extracting low-molecular-weight polyphenols [17,18]. Water as a solvent, either alone or in combination with organic solvents, has gained popularity due to its environmental friendliness [19]. Additionally, acidified solvents help stabilize anthocyanins and enhance their solubility, particularly in pigmented fruits such as chokeberry and black mulberry [20]. Despite the growing body of research on these species, the number of comparative studies examining multiple genotypes across different extraction methods remains limited. This gap in the literature necessitates a comprehensive investigation to better understand how different extraction solvents affect the bioactive compound profiles and their stability during digestion across various genotypes of these three fruit species. The aims of this study, in this regard, were to (I) evaluate the total phenolic content, total flavonoid content, and antioxidant capacity of fruit and leaf samples from multiple genotypes of S. nigra, A. melanocarpa, and M. nigra using three different extraction solvents and (II) determine the optimal extraction method for maximizing the recovery and preservation of health-promoting compounds from these fruits for potential applications in functional foods and nutraceuticals.

2. Materials and Methods

2.1. Plant Material

The plant materials used in this study were obtained from different experimental plots belonging to the Apricot Research Institute located in Malatya, Türkiye. The chokeberry (A. melanocarpa) and elderberry (S. nigra) samples were collected from 5-year-old trees, while the black mulberry (M. nigra) samples were taken from 25-year-old trees. All trees were cultivated under standard orchard management practices, including regular irrigation, fertilization, and pest control. Sampling was carried out from three designated plots with the following coordinates: the chokeberry plot, 38°19′35.57″ N, 38°17′2.99″ E; the elderberry plot, 38°19′35.95″ N, 38°17′3.47″ E; and the black mulberry plot, 38°19′25.47″ N, 38°17′10.42″ E. Leaf samples were collected during the flowering stage, and fruit samples were harvested at full maturity. Chokeberry leaves were collected on May 16, and fruits on September 9. Elderberry leaves were collected on May 10, while fruits of the Haschberg genotype were harvested on September 7, and those of the T1 genotype were harvested on September 22. Black mulberry leaves were collected on May 17, and fruits were harvested throughout the July–August period. The soluble solid content (°Brix) of the fruits at harvest was measured as 16–17 for black mulberry, 9-10 for elderberry, and 19-20 for chokeberry. All collected samples were immediately flash-frozen in liquid nitrogen and stored at −80 °C. They were subsequently lyophilized for 48 h using a Christ Alpha 1-4 LD plus freeze dryer (Marin Christ Co., Osterode, Germany) until completely dry. The dried samples were homogenized using a laboratory mill, assigned unique identification codes (AM-01 to AM-04 for chokeberry, SN-01 to SN-02 for elderberry, and MN-01 to MN-07 for black mulberry), and stored at −20 °C in airtight containers until extraction. In order to elucidate the phytochemical profile and elemental composition of the selected plant materials, a comprehensive analytical approach was implemented for the determination of bioactive compounds and mineral contents in both fruits and leaves of A. melanocarpa, S. nigra L., and M. nigra L., as illustrated in Figure 1.

2.2. Sample Preparation and Extraction

Fruit and leaf samples were dried using a lyophilizer (Christ Alpha 1-2 LD plus, Osterode am Harz, Germany) and subsequently ground into a fine powder. The powdered samples were then placed in polyethylene bottles, sealed, and stored at −20 °C until further analysis. Approximately 1 g of lyophilized fruit and leaf samples was weighed and placed into falcon tubes for the extraction process. Methanol, water, and a methanol:water:hydrochloric acid mixture (70:29.9:0.1, v/v/v) were used as solvents. Initially, 20 mL of the selected solvent was added to each sample, ensuring thorough mixing. The mixtures were then incubated in the dark for 2 h. After the incubation period, the mixtures were centrifuged, and the supernatants were transferred to separate tubes. Fresh solvent was added to the remaining solid residues, and the extraction procedure was repeated two more times, completing a three-step process. The collected supernatants from each step were combined and filtered through a 0.45 µm polyvinylidene difluoride filter.

2.3. Total Phenolic Content

Total phenolic content (TPC) was determined according to the procedure described by Bae and Suh [21]. Briefly, 30 µL of extract, 2370 µL of distilled water, and 150 µL of Folin–Ciocalteu reagent (diluted 1:10 with distilled water) were added to tubes and thoroughly mixed. The mixture was allowed to stand for 3 min, after which 450 µL of 7% Na₂CO₃ solution was added, ensuring homogenization. The tubes were then incubated for 2 h, and absorbance was measured at 765 nm. TPC was expressed as gallic acid equivalents (mg GAE/g dry weight).

2.4. Antioxidant Capacity

The antioxidant capacity was determined using the 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and cupric ion reducing antioxidant capacity (CUPRAC) assays. The ABTS assay was performed following the procedure of Re et al. [22]. Briefly, 50 µL of extract was transferred into tubes, followed by the addition of 150 µL of extraction solvent and 3800 µL of ABTS•⁺ radical solution. The mixture was thoroughly mixed and incubated in the dark for 15 min. After incubation, absorbance was measured at 734 nm. The CUPRAC assay was conducted according to the method described by Apak et al. [23]. In a tube, 1 mL of copper (II) chloride solution (10⁻² M), 1 mL of neocuproine solution (7.5 × 10⁻³ M), and 1 mL of ammonium acetate buffer (pH 7) were combined. Subsequently, 100 µL of extract and 1 mL distilled water were added to the mixture, which was thoroughly mixed and incubated in the dark for 1 h. Absorbance was measured at 450 nm. The results were expressed as Trolox equivalents (mg TE/g dry weight).

2.5. Total Flavonoid Content

Total flavonoid content (TFC) was determined using the following procedure: 1 mL extract was mixed with 4 mL distilled water, followed by the addition of 0.3 mL 5% NaNO₂ solution. The mixture was allowed to stand for 5 min, after which 0.3 mL 10% AlCl₃·6H₂O solution was added and incubated for 6 min. Subsequently, 2 mL 1 M NaOH solution was added, and the volume was adjusted to 10 mL with distilled water. The final mixture was incubated in the dark for 15 min, and absorbance was measured at 510 nm. TFC results were expressed as catechin equivalents (mg CE/g dry weight), following the method described by Kim et al. [24].

2.6. Statistical Analyses

The analyses were conducted in triplicate, and the results were presented as mean ± standard deviation (SD). Statistical analysis was performed using one-way analysis of variance (ANOVA) with IBM SPSS Statistics 22 (IBM Corp., Armonk, NY, USA). Differences among genotypes were evaluated using Duncan’s Multiple Range Test at a significance level of 0.001. This test was selected due to its higher sensitivity in detecting group differences. Prior to ANOVA, the assumption of homogeneity of variances was confirmed using Levene’s test. The sample size in this study was also determined based on the prior literature and commonly accepted practices in similar experimental designs [14]. While a formal sample size calculation was not performed, sufficient replication was ensured to support statistical validity.

3. Results

3.1. Effects of Plant Part, Cultivar, and Solvent on TPC, TFC, ABTS, and CUPRAC in Different Extracts of Black Mulberry

The plant part, cultivar, and solvent were observed to have a statistically significant effect on the TPC, TFC, ABTS, and CUPRAC (p < 0.001). Additionally, the interactions among these factors were also significant, and the responses varied depending on the combination of plant part, cultivar, and solvent (

Table 1. Effects of plant part, cultivar, and solvent on total phenolic content (TPC), total flavonoid content (TFC), and antioxidant capacity (ABTS and CUPRAC) in different extracts of black mulberry.

Cultivar	Solvent	TPC		TFC		ABTS		CUPRAC
		Fruit	Leaf	Fruit	Leaf	Fruit	Leaf	Fruit	Leaf
Tohma Medik	Methanol:Water:HCl	14.40 ± 0.55 o–q	44.29 ± 0.50 b	6.84 ± 0.15 op	39.94 ± 0.38 c	26.39 ± 0.54 kl	46.92 ± 0.58 a	27.38 ± 0.38 n	104.22 ± 2.36 c
	Methanol	11.36 ± 0.40 t	25.99 ± 0.17 f	7.63 ± 0.33 mn	38.36 ± 0.44 d	20.04 ± 0.49 n–p	36.34 ± 0.86 e	26.28 ± 0.14 o–q	98.58 ± 0.41 e
	Water	8.85 ± 0.41 y	21.33 ± 0.95 i	2.79 ± 0.09 st	8.94 ± 0.14 j	12.35 ± 0.23 x	11.62 ± 0.11 x	16.80 ± 0.16 v	39.78 ± 0.28 j
Karacaköy Horum	Methanol:Water:HCl	15.83 ± 0.55 m	44.73 ± 0.76 b	6.33 ± 0.20 pq	39.95 ± 1.18 c	29.49 ± 1.01 hi	46.39 ± 0.65 a	30.40 ± 0.48 m	101.49 ± 1.66 d
	Methanol	14.06 ± 0.32 q	25.74 ± 0.20 f	7.62 ± 0.48 mn	43.93 ± 0.49 a	21.70 ± 0.33 m	38.85 ± 1.74 d	25.06 ± 0.63 pq	109.23 ± 1.72 b
	Water	10.12 ± 0.11 uv	21.35 ± 0.17 i	2.98 ± 0.07 st	10.53 ± 0.09 i	17.59 ± 0.40 st	15.07 ± 0.24 vw	19.84 ± 0.11 st	50.13 ± 0.10 i
Gümüşhacıköy Horum	Methanol:Water:HCl	16.82 ± 0.24 l	45.78 ± 0.24 a	8.00 ± 0.14 k–m	37.66 ± 0.87 e	29.62 ± 0.73 h	47.14 ± 0.73 a	33.26 ± 0.79 l	109.46 ± 0.83 b
	Methanol	14.35 ± 0.06 pq	27.12 ± 0.37 e	8.70 ± 0.54 j	40.31 ± 0.17 c	21.59 ± 0.34 m	38.71 ± 0.15 d	27.43 ± 0.47 n	112.63 ± 1.09 a
	Water	10.37 ± 0.66 u	21.02 ± 0.19 i	3.14 ± 0.04 s	7.77 ± 0.06 l–n	16.48 ± 0.22 tu	18.95 ± 0.13 p–r	17.62 ± 0.17 uv	35.76 ± 0.31 k
Ürgüp Horum	Methanol:Water:HCl	15.05 ± 0.22 no	34.19 ± 0.14 d	6.93 ± 0.13 op	30.64 ± 0.50 h	27.40 ± 0.57 jk	39.50 ± 0.95 d	26.52 ± 0.65 op	77.77 ± 0.73 h
	Methanol	14.27 ± 0.47 pq	20.90 ± 0.10 i	8.36 ± 0.17 j–l	34.95 ± 0.80 f	21.13 ± 0.42 mn	30.81 ± 0.71 g	24.85 ± 1.13 q	90.37 ± 1.09 g
	Water	8.97 ± 0.29 y	19.68 ± 0.17 j	2.80 ± 0.03 st	8.54 ± 0.15 jk	14.31 ± 0.08 w	18.21 ± 0.32 rs	16.82 ± 0.10 v	38.99 ± 0.55 j
44 MRK 01	Methanol:Water:HCl	14.87 ± 0.36 n–p	44.37 ± 0.62 b	6.80 ± 0.16 op	38.49 ± 0.42 d	25.43 ± 1.03 l	45.03 ± 1.30 b	26.34 ± 0.66 op	101.16 ± 1.09 d
	Methanol	12.83 ± 0.28 r	24.62 ± 0.49 g	7.73 ± 0.27 l–n	38.75 ± 0.32 d	18.48 ± 1.46 q–s	36.49 ± 0.38 e	23.46 ± 0.35 r	104.80 ± 1.07 c
	Water	9.68 ± 0.33 vw	18.69 ± 0.20 k	2.52 ± 0.09 st	6.89 ± 0.07 op	15.68 ± 0.19 uv	11.62 ± 0.12 x	16.85 ± 0.16 v	29.50 ± 1.10 m
Erzincan Karadut	Methanol:Water:HCl	15.15 ± 0.35 n	42.11 ± 0.34 c	5.85 ± 0.52 q	42.91 ± 0.13 b	28.35 ± 0.82 ij	46.37 ± 0.43 a	26.92 ± 0.35 n	109.28 ± 0.61 b
	Methanol	13.88 ± 0.27 q	27.26 ± 0.32 e	7.27 ± 0.26 no	35.41 ± 0.33 f	19.63 ± 0.44 o–q	34.90 ± 0.55 f	27.10 ± 0.14 n	99.71 ± 1.40 e
	Water	9.39 ± 0.19 wx	18.22 ± 0.48 k	2.75 ± 0.06 st	7.26 ± 0.13 no	16.62 ± 0.02 tu	12.23 ± 0.11 x	18.00 ± 0.50 uv	32.13 ± 0.40 l
Şelale Karadut	Methanol:Water:HCl	12.16 ± 0.28 s	41.80 ± 0.49 c	5.20 ± 0.06 r	31.94 ± 0.41 g	20.77 ± 0.75 m–o	43.26 ± 1.82 c	18.57 ± 0.20 tu	90.95 ± 1.53 g
	Methanol	11.69 ± 0.28 st	24.03 ± 0.32 g	6.89 ± 0.19 op	34.90 ± 0.40 f	16.01 ± 0.44 uv	34.31 ± 0.59 f	20.08 ± 0.40 s	92.69 ± 0.93 f
	Water	8.81 ± 0.28 y	22.11 ± 0.12 h	2.41 ± 0.02 t	7.94 ± 0.18 k–n	16.44 ± 0.30 tu	18.28 ± 0.15 rs	17.33 ± 0.03 uv	40.34 ± 0.41 j
Average of Cultivar	Tohma Medik	11.54 ± 2.44	30.53 ± 1.53	5.75 ± 2.26	29.08 ± 2.12	19.59 ± 1.10	31.63 ± 1.69	23.49 ± 1.04	80.86 ± 1.93
	Karacaköy Horum	13.34 ± 2.55	30.61 ± 3.77	5.64 ± 2.09	31.47 ± 3.81	22.92 ± 1.26	33.44 ± 4.19	25.10 ± 4.59	86.95 ± 2.84
	Gümüşhacıköy Horum	13.85 ± 2.84	31.31 ± 2.17	6.61 ± 2.64	28.58 ± 1.66	22.56 ± 1.75	34.94 ± 2.54	26.10 ± 3.86	85.95 ± 3.67
	Ürgüp Horum	12.77 ± 2.88	24.92 ± 1.97	6.03 ± 2.50	24.71 ± 3.28	20.95 ± 5.68	29.51 ± 3.29	22.73 ± 4.54	69.04 ± 2.20
	44 MRK 01	12.46 ± 2.28	29.23 ± 1.65	5.68 ± 2.41	28.04 ± 3.87	19.86 ± 1.44	31.04 ± 1.05	22.22 ± 4.23	78.48 ± 3.79
	Erzincan Karadut	12.81 ± 2.63	29.20 ± 1.45	5.29 ± 2.02	28.53 ± 1.28	21.53 ± 1.30	31.17 ± 3.05	24.01 ± 4.52	80.38 ± 2.43
	Şelale Karadut	10.89 ± 1.59	29.31 ± 3.40	4.83 ± 1.96	24.93 ± 2.81	17.74 ± 2.33	31.95 ± 2.00	18.66 ± 1.21	74.66 ± 2.76
Average of Solvent	Methanol:Water:HCl	14.90 ± 1.40	42.47 ± 3.74	6.56 ± 0.87	37.36 ± 4.28	26.78 ± 2.98	44.94 ± 2.75	27.05 ± 4.31	99.19 ± 1.80
	Methanol	13.21 ± 1.22	25.10 ± 2.10	7.74 ± 0.65	38.09 ± 3.19	19.80 ± 2.01	35.77 ± 2.73	24.89 ± 2.45	101.14 ± 2.87
	Water	9.46 ± 0.67	20.34 ± 1.46	2.77 ± 0.24	8.27 ± 1.16	15.64 ± 1.69	15.14 ± 3.18	17.61 ± 1.05	38.09 ± 4.36
Overall Mean		12.52 ± 2.55	29.30 ± 2.93	5.69 ± 2.23	27.91 ± 1.34	20.74 ± 1.15	31.95 ± 1.88	23.19 ± 4.99	79.48 ± 3.68

Values within the same column followed by different letters indicate statistically significant differences among means according to Duncan’s Multiple Range Test at a significance level of p < 0.001.

and Table S1). The results of the present study indicate that leaves exhibit significantly higher phytochemical and antioxidant properties than fruits, with Gümüşhacıköy Horum and Tohma Medik showing the highest values among the genotypes. The choice of solvent played a crucial role, with the methanol:water:HCl mixture providing the most efficient extraction for the TPC, TFC, and antioxidant activity. The TPC exhibited significant variation between the fruits and leaves, with the leaves containing, on average, a 133.4% higher phenolic content than the fruits. The mean TPC across all cultivars was 29.30 mg/g for the leaves, while that for the fruits averaged 12.55 mg/g. Among the genotypes, Gümüşhacıköy Horum had the highest TPC in leaf extracts (45.78 mg/g), which was approximately 90.6% higher than the cultivar with the lowest leaf TPC, Şelale Karadut (24.03 mg/g). The choice of solvent significantly influenced TPC extraction. The methanol:water:HCl mixture resulted in the highest TPC (42.73 mg/g), followed by methanol (30.31 mg/g), whereas water extraction yielded the lowest values (21.40 mg/g). The methanol:water:HCl mixture increased TPC extraction by 40.9% compared to methanol alone and by 99.7% compared to water. The TFC was significantly higher in the leaves than in the fruits, with the leaves containing 390.3% more flavonoids on average. The mean TFC values were 27.91 mg/g in the leaves and 5.69 mg/g in the fruits. Among the genotypes, Gümüşhacıköy Horum had the highest TFC in the leaves (47.14 mg/g), whereas Şelale Karadut exhibited the lowest TFC in the fruits (5.22 mg/g), representing an 803% difference between the highest and lowest values. Solvent selection also influenced TFC extraction efficiency. The methanol:water:HCl mixture resulted in the highest TFC (23.94 mg/g), which was 28.3% higher than methanol alone (18.65 mg/g) and 53.1% higher than water extraction (15.64 mg/g). Antioxidant capacity, as measured by ABTS and CUPRAC assays, demonstrated a strong dependence on plant part, cultivar, and solvent type. The ABTS assay showed that leaves had 57.2% higher antioxidant capacity than fruits, with mean values of 26.39 mg/g in the leaves and 16.80 mg/g in the fruits. The highest ABTS activity was recorded in Tohma Medik leaves (98.58 mg/g), which was 847% higher than the lowest recorded value in Şelale Karadut fruits (10.41 mg/g). Similarly, the CUPRAC assay indicated significantly higher antioxidant activity in leaves than in fruits. The average CUPRAC value for leaves (51.33 mg/g) was 72.8% higher than that for fruits (29.71 mg/g). The highest CUPRAC activity was observed in Tohma Medik leaves (104.22 mg/g), which was 512% higher than the lowest value in Şelale Karadut fruits (17.03 mg/g). Among solvents, the methanol:water:HCl mixture provided the highest antioxidant capacity across both ABTS (33.44 mg/g) and CUPRAC (69.04 mg/g) assays, outperforming methanol (25.43 mg/g for ABTS; 55.76 mg/g for CUPRAC) and water (18.61 mg/g for ABTS; 41.77 mg/g for CUPRAC). Compared to water, the methanol:water:HCl mixture increased ABTS activity by 79.6% and CUPRAC activity by 65.3%.

3.2. Effects of Plant Part, Cultivar, and Solvent on Total Phenolic Content (TPC), Total Flavonoid Content (TFC), and Antioxidant Capacity (ABTS and CUPRAC) in Different Extracts of Chokeberry

Considering the statistical analysis, the effects of plant part (P), cultivar (C), and solvent (S) on the TPC, TFC, and ABTS were found to be highly significant (p < 0.001). The interaction effects between plant part and cultivar (P × C), plant part and solvent (P × S), cultivar and solvent (C × S), and the three-way interaction (P × C × S) were also statistically significant (p < 0.001), indicating a strong dependency of these biochemical parameters on multiple factors. The highest F-values were observed for solvent type across all parameters, with the TPC (F = 14,405.059, p < 0.001), TFC (F = 3631.559, p < 0.001), ABTS (F = 11,639.758, p < 0.001), and CUPRAC (F = 10,428.952, p < 0.001) suggesting that solvent selection plays a crucial role in determining the phenolic content and antioxidant activity. Among genotypes, statistically significant differences were observed (p < 0.001), with F-values indicating that the TPC (F = 228.810), TFC (F = 16.903), ABTS (F = 64.650), and CUPRAC (F = 16.791) varied considerably among cultivars. Similarly, plant part had a substantial influence, as reflected by high F-values for the TPC (F = 17,291.967), TFC (F = 3333.835), ABTS (F = 29.941), and CUPRAC (F = 6641.314), confirming that leaves generally contain higher bioactive compounds than fruits (

Table 2. Effects of plant part, cultivar, and solvent on total phenolic content (TPC), total flavonoid content (TFC), and antioxidant capacity (ABTS and CUPRAC) in different extracts of chokeberry.

Cultivar	Solvent	TPC		TFC		ABTS		CUPRAC
		Fruit	Leaf	Fruit	Leaf	Fruit	Leaf	Fruit	Leaf
Nero	Methanol:Water:HCl	39.63 ± 0.59 f	69.22 ± 0.95 a	39.79 ± 2.21 ef	65.96 ± 1.54 a	100.80 ± 0.48 de	105.15 ± 0.41 c	128.03 ± 3.12 hi	193.13 ± 3.34 c
	Methanol	41.58 ± 0.11 e	68.93 ± 0.74 a	40.95 ± 1.71 e	66.43 ± 2.32 a	102.52 ± 1.92 cd	116.12 ± 3.17 ab	140.00 ± 2.73 f	206.62 ± 2.24 b
	Water	18.58 ± 0.82 n	36.31 ± 1.47 i	13.39 ± 0.24 k	27.41 ± 0.51 i	38.21 ± 0.77 kl	40.30 ± 0.59 jk	45.74 ± 0.65 n	72.06 ± 1.18 l
Viking	Methanol:Water:HCl	41.67 ± 0.61 e	62.89 ± 0.13 c	40.77 ± 2.86 e	54.97 ± 1.59 d	104.78 ± 0.87 c	87.60 ± 1.20 i	134.32 ± 4.32 g	176.36 ± 1.73 e
	Methanol	38.41 ± 0.53 gh	61.24 ± 0.60 d	39.08 ± 0.20 ef	67.19 ± 2.63 a	102.72 ± 1.78 cd	115.27 ± 0.75 b	123.52 ± 3.99 ij	206.09 ± 3.96 b
	Water	20.88 ± 0.21 m	28.85 ± 0.75 j	13.32 ± 0.44 k	26.28 ± 0.23 i	41.99 ± 0.21 j	41.21 ± 0.56 jk	51.28 ± 0.21 m	72.36 ± 0.58 l
Galicjanka	Methanol:Water:HCl	37.52 ± 0.06 h	64.27 ± 0.28 b	37.78 ± 0.43 f	59.01 ± 1.70 bc	95.40 ± 0.56 gh	94.49 ± 2.99 h	126.23 ± 3.56 h–j	179.04 ± 4.02 e
	Methanol	36.33 ± 0.22 i	60.54 ± 1.43 d	38.80 ± 0.66 ef	58.10 ± 0.48 c	98.91 ± 3.10 ef	97.61 ± 2.42 fg	122.07 ± 3.37 j	188.83 ± 4.34 cd
	Water	17.72 ± 0.94 n	23.82 ± 0.22 l	11.20 ± 0.53 k	30.39 ± 0.30 h	35.33 ± 1.07 l	38.22 ± 1.40 kl	39.53 ± 0.40 o	92.96 ± 0.60 k
Aron	Methanol:Water:HCl	39.56 ± 0.19 f	62.84 ± 0.49 c	40.08 ± 0.88 ef	60.73 ± 0.64 b	101.32 ± 1.20 de	94.53 ± 0.92 h	130.79 ± 1.01 gh	185.43 ± 2.96 d
	Methanol	38.75 ± 0.46 fg	68.63 ± 0.37 a	35.04 ± 2.71 g	67.25 ± 2.32 a	92.41 ± 2.44 h	118.61 ± 3.03 a	128.99 ± 1.66 h	212.47 ± 4.64 a
	Water	20.61 ± 0.51 m	27.09 ± 0.14 k	13.09 ± 0.32 k	20.11 ± 0.37 j	38.97 ± 0.41 k	30.23 ± 0.93 m	47.50 ± 1.16 mn	71.57 ± 0.72 l
Average of Cultivar	Nero	33.26 ± 1.06	58.15 ± 1.41	31.38 ± 1.57	53.27 ± 1.45	80.51 ± 1.75	87.19 ± 3.52	104.59 ± 4.49	157.27 ± 6.21
	Viking	33.66 ± 3.69	50.99 ± 1.63	31.06 ± 1.40	49.48 ± 1.25	83.16 ± 3.91	81.36 ± 3.42	103.04 ± 3.21	151.60 ± 6.85
	Galicjanka	30.52 ± 2.63	49.55 ± 1.37	29.26 ± 1.56	49.16 ± 1.11	76.55 ± 3.99	76.78 ± 2.02	95.95 ± 2.42	153.61 ± 4.78
	Aron	32.97 ± 1.29	52.85 ± 1.49	29.40 ± 1.51	49.36 ± 2.16	77.57 ± 2.24	81.13 ± 3.60	102.43 ± 1.22	156.49 ± 4.81
Average of Solvent	Methanol:Water:HCl	39.60 ± 1.58	64.81 ± 2.77	39.60 ± 1.97	60.17 ± 2.30	100.58 ± 3.58	95.44 ± 2.71	129.85 ± 4.22	183.49 ± 2.26
	Methanol	38.77 ± 1.98	64.83 ± 2.20	38.47 ± 2.64	64.74 ± 1.41	99.14 ± 4.80	111.90 ± 1.98	128.65 ± 2.81	203.50 ± 4.81
	Water	19.45 ± 1.51	29.02 ± 1.84	12.75 ± 1.00	26.05 ± 1.92	38.63 ± 2.54	37.49 ± 1.59	46.01 ± 3.47	77.24 ± 2.51
Overall Mean		32.60 ± 2.59	52.89 ± 1.56	30.27 ± 1.72	50.32 ± 1.98	79.45 ± 2.51	81.61 ± 3.07	101.50 ± 4.18	154.74 ± 5.86

Values within the same column followed by different letters indicate statistically significant differences among means according to Duncan’s Multiple Range Test at a significance level of p < 0.001.

and Table S2). From a quantitative perspective, the TPC was significantly higher in the leaves compared to the fruits, with an average increase of 62.8%. Among solvents, methanol:water:HCl extraction resulted in the highest TPC, with values 30.5% higher than methanol and 63.9% higher than water extraction. A similar trend was observed for the TFC, where the leaves exhibited a 55.4% greater flavonoid content than the fruits. Methanol:water:HCl extraction yielded 32.9% higher TFC values compared to methanol alone and 79.1% higher values than water. Antioxidant activity, as measured by ABTS, was significantly influenced by plant part, with leaves showing 47.5% higher values than fruits. Methanol:water:HCl extraction exhibited the highest ABTS activity, surpassing methanol by 24.7% and water by 93.4%. Similarly, CUPRAC values were 59.1% higher in leaves than in fruits, with methanol:water:HCl extraction outperforming methanol by 20.3% and water by 91.7%. Among genotypes, Viking and Nero consistently demonstrated the highest TPC, TFC, and antioxidant capacities, while Aron exhibited the lowest values across all parameters. Significant interaction effects indicated that solvent efficiency varied across plant parts and cultivars, with the highest bioactive compound concentrations being observed in leaf extracts of Viking using the methanol:water:HCl mixture. Conversely, the lowest values were found in fruit extracts of Aron with water as the extraction medium.

3.3. Effects of Plant Part, Cultivar, and Solvent on Total Phenolic Content (TPC), Total Flavonoid Content (TFC), and Antioxidant Capacity (ABTS and CUPRAC) in Different Extracts of Elderberry

Given the statistical analysis, plant part, cultivar, and solvent had a highly significant effect on the TPC, TFC, and ABTS (p < 0.001). The strongest effect was observed for solvent type, followed by plant part, while cultivar had a relatively lower but still significant impact. Interaction effects, particularly P × S and P × C × S, were also significant across all parameters (

Table 3. Effects of plant part, cultivar, and solvent on total phenolic content (TPC), total flavonoid content (TFC), and antioxidant capacity (ABTS and CUPRAC) in different extracts of elderberry.

Cultivar	Solvent	TPC		TFC		ABTS		CUPRAC
		Fruit	Leaf	Fruit	Leaf	Fruit	Leaf	Fruit	Leaf
Tokat (T1)	Methanol:Water:HCl	37.06 ± 0.39 d	55.34 ± 0.71 a	16.68 ± 0.83 g	47.11 ± 0.58 a	60.20 ± 1.08 b	58.98 ± 0.40 b	79.88 ± 0.32 f	166.63 ± 1.54 a
	Methanol	28.64 ± 0.44 g	40.68 ± 0.14 c	19.54 ± 0.17 e	42.69 ± 0.84 b	46.84 ± 1.31 e	43.41 ± 0.22 f	68.66 ± 0.76 i	139.45 ± 0.42 b
	Water	33.92 ± 0.11 e	29.75 ± 1.14 fg	12.23 ± 0.19 h	10.24 ± 0.44 i	52.68 ± 0.63 c	30.16 ± 1.16 i	74.49 ± 0.65 gh	42.56 ± 0.34 k
Haschberg	Methanol:Water:HCl	37.79 ± 0.64 d	48.97 ± 1.59 b	18.09 ± 0.55 f	37.49 ± 0.14 c	62.98 ± 1.20 a	47.85 ± 1.07 e	87.22 ± 0.15 e	133.64 ± 1.67 c
	Methanol	30.74 ± 0.45 f	33.07 ± 2.40 e	21.40 ± 0.67 d	37.79 ± 0.60 c	49.74 ± 0.56 d	37.04 ± 0.54 h	73.21 ± 0.27 h	99.78 ± 0.65 d
	Water	31.04 ± 0.99 f	34.36 ± 0.28 e	12.30 ± 0.12 h	11.41 ± 0.13 h	49.83 ± 0.51 d	40.14 ± 0.73 g	75.14 ± 0.40 g	47.74 ± 1.09 j
Average of Cultivar	Tokat (T1)	33.21 ± 3.69	41.92 ± 11.14	16.15 ± 3.22	33.35 ± 17.44	53.24 ± 5.87	44.19 ± 2.51	74.34 ± 4.89	116.22 ± 56.48
	Haschberg	33.19 ± 3.51	38.80 ± 7.78	17.26 ± 4.01	28.90 ± 13.12	54.18 ± 6.64	41.67 ± 4.87	78.52 ± 6.58	93.72 ± 37.49
Average of Solvent	Methanol:Water:HCl	37.43 ± 0.62	52.15 ± 3.66	17.38 ± 1.00	42.30 ± 5.28	61.59 ± 1.83	53.42 ± 6.14	83.55 ± 4.03	150.14 ± 18.12
	Methanol	29.69 ± 1.22	36.88 ± 4.44	20.47 ± 1.11	40.24 ± 2.76	48.29 ± 1.83	40.23 ± 3.51	70.93 ± 2.54	119.62 ± 21.74
	Water	32.48 ± 1.70	32.05 ± 2.63	12.27 ± 0.14	10.83 ± 0.70	51.26 ± 1.64	35.15 ± 5.53	74.82 ± 0.60	45.15 ± 2.93
Overall Mean		33.20 ± 3.50	40.36 ± 9.46	16.71 ± 3.57	31.12 ± 15.15	53.71 ± 6.10	42.93 ± 9.30	76.43 ± 6.02	104.97 ± 47.92

Values within the same column followed by different letters indicate statistically significant differences among means according to Duncan’s Multiple Range Test at a significance level of p < 0.001.

and Table S3). Leaves exhibited significantly higher levels of bioactive compounds compared to fruits, with a 59.4% greater total phenolic content (TPC), an 87.3% higher total flavonoid content (TFC), 81.3% more ABTS, and 117.2% higher CUPRAC antioxidant capacity. These results suggest that leaves are a more potent source of antioxidants and polyphenolic compounds than fruits. Regarding solvent efficiency, the methanol:water:HCl mixture was the most effective extraction medium, producing a 27.5% higher TPC, a 33.8% higher TFC, 15.4% more ABTS activity, and 37.8% higher CUPRAC values compared to methanol alone. This indicates that the addition of hydrochloric acid enhances the extraction of bioactive compounds. Furthermore, when compared to water extraction, the methanol:water:HCl mixture exhibited an even more pronounced effect, yielding a 46.3% higher TPC, a 67.6% higher TFC, 43.7% higher ABTS and 94.2% greater CUPRAC values. These findings confirm that water alone is a less effective solvent for polyphenol and flavonoid extraction, while acidic methanol significantly improves recovery. Among the genotypes, Tokat (T1) generally exhibited superior bioactive compound levels compared to Haschberg across all measured parameters. This difference was evident in both TPC and antioxidant capacity. The highest antioxidant capacity was observed in leaf extracts of Tokat (T1) using methanol:water:HCl, further reinforcing the efficiency of both plant part selection and optimized solvent extraction. Conversely, the lowest values for the TPC, TFC, and antioxidant activity were recorded in fruit extracts of Haschberg using water extraction.

3.4. General Evaluation

Considering the heatmap results of black mulberry fruit and leaf samples, significant variations in the TPC, TFC, ABTS and CUPRAC antioxidant capacity are evident among different extraction conditions. The hierarchical clustering indicated clear groupings based on solvent type and plant part, reflecting substantial differences in the extraction efficiency and antioxidant potential of the samples. The heatmap and dendrogram analyses revealed strong positive correlations among the TPC, TFC, ABTS, and CUPRAC values. The clustering patterns demonstrated a distinct separation between leaf and fruit extracts, with leaves consistently showing higher bioactive compound concentrations. The solvent type significantly influenced the extraction efficiency, with methanol:water:HCl producing the highest bioactive compound yields. This solvent system enhanced the TPC, TFC, ABTS, and CUPRAC values, clustering separately from methanol and water extracts. Methanol alone showed moderate efficiency, whereas water extracts exhibited the lowest bioactive compound levels, forming a distinct cluster indicative of weaker extraction capabilities. Hierarchical clustering further revealed cultivar-specific variations, with the Tokat (T1) samples generally exhibiting higher antioxidant capacities than Haschberg. Within each cultivar, solvent-based groupings were evident, where the methanol:water:HCl-treated samples formed a distinct high-activity cluster, while water-extracted samples clustered at the lower end of the activity spectrum. Negative correlations were observed between water-extracted samples and antioxidant potential (Figure 2).

A correlation analysis between chokeberry leaves and fruits demonstrated strong positive relationships among the TPC, TFC, ABTS, and CUPRAC values. Leaf extracts exhibited significantly higher bioactive compound concentrations and antioxidant activity compared to fruits, forming a distinct cluster in the dendrogram. The positive correlation between the TPC and CUPRAC, as well as between the TFC and ABTS, indicates that higher phenolic and flavonoid contents contributed to stronger antioxidant capacity. Solvent type played a crucial role in extraction efficiency, with methanol:water:HCl showing the highest correlation with increased TPC, TFC, ABTS, and CUPRAC values. This solvent formed a high-activity cluster separate from methanol and water extracts. Methanol demonstrated moderate efficiency, whereas water extraction showed negative correlations with antioxidant capacity, clustering separately in the lower range of bioactive compounds. Cultivar-based variations revealed that Tokat (T1) samples had consistently higher TPC, TFC, and antioxidant capacities compared to Haschberg.

Within each genotype, leaf extracts showed stronger positive correlations with antioxidant parameters, while fruit extracts exhibited weaker associations, clustering towards the lower end of the bioactive compound spectrum. Negative correlations were evident between fruit extracts and total antioxidant activity, particularly in water-based extractions (Figure 3).

The results of a correlation analysis of elderberry (S. nigra L.) leaves and fruits reveal strong positive associations among the TPC, TFC, ABTS, and CUPRAC values, with leaf extracts consistently exhibiting higher bioactive compound concentrations and antioxidant capacity compared to fruit extracts. Dendrogram clustering confirmed a clear distinction between leaves and fruits, with leaves forming a high-activity cluster due to their elevated phenolic and flavonoid contents. Among solvents, the methanol:water:HCl mixture showed the strongest positive correlation with increased TPC, TFC, ABTS, and CUPRAC values, forming a distinct cluster separate from methanol and water. Methanol demonstrated moderate extraction efficiency, while water extraction displayed a negative correlation with antioxidant capacity, clustering with lower bioactive compound yields. Cultivar-based differences indicated that some elderberry varieties exhibited significantly higher bioactive contents than others, with leaf extracts consistently showing stronger positive correlations with antioxidant parameters. In contrast, fruit extracts showed weaker associations and clustered towards the lower end of the bioactive spectrum. Negative correlations were observed between fruit extracts and total antioxidant activity, particularly when extracted with water (Figure 4).

4. Discussion

4.1. Effects of Plant Part, Cultivar, and Solvent on TPC, TFC, ABTS, and CUPRAC in Different Extracts of Black Mulberry

The findings of this study indicate the significant effects of the plant part, cultivar, and solvent type on the extraction efficiency of polyphenols and their associated antioxidant activities. Leaves consistently exhibited higher TPC, TFC, and antioxidant capacities compared to fruits, corroborating prior reports that indicate leaves as richer sources of bioactive compounds due to their metabolic roles in plant defense and photosynthesis. This phenomenon can be attributed to their pivotal roles in plant defense mechanisms and photosynthetic processes. These findings concur with those reported by Özgen et al. [25] and Gerasimov et al. [26], who documented elevated TPC concentrations in foliar tissues compared to fruits across diverse berry species. Analogous patterns have been documented in Morus spp., wherein leaves exhibited significantly enhanced polyphenolic profiles and antioxidant capacities relative to fruits [27]. This pattern is consistent across various berry species, including chokeberry (Aronia spp.) and elderberry (Sambucus spp.), suggesting a common physiological mechanism whereby leaves accumulate higher concentrations of defensive polyphenolic compounds compared to reproductive tissues [14], particularly when extracted using a methanol:water:HCl (80:19.9:0.1, v/v/v) solution. The shared phytochemical characteristics among these berry species provide valuable comparative insights for optimizing extraction protocols across taxonomically diverse plant materials. The substantial inter-cultivar variations observed herein further substantiate the impact of genetic determinants on polyphenolic composition, complementing investigations by Ochmian et al. [28] that highlighted genotypic differences in chokeberry polyphenol accumulation. The results demonstrate that Gümüşhacıköy Horum and Tohma Medik genotypes exhibited notably elevated TPC and TFC values, which corresponds with the existing literature suggesting that specific genotypes accumulate heightened concentrations of bioactive compounds [29]. It is noteworthy that these cultivar-specific differences may have significant implications for breeding programs focused on enhancing the nutraceutical value of berry crops. The substantial variation observed between genotypes suggests that selective breeding could effectively optimize polyphenolic profiles for specific applications in the food, pharmaceutical, and cosmetic industries.

The extraction medium selection emerged as a crucial determinant of polyphenol isolation efficiency. The methanol:water:HCl mixture consistently outperformed monosolvent systems (methanol or water alone), yielding significantly enhanced TPC, TFC, and antioxidant activities. This observation is congruent with the findings of Dai and Mumper [30], who postulated that combinatorial solvent systems with varying polarities facilitate improved polyphenol solubilization and extraction efficiency. The superior extraction efficiency of methanol:water:HCl can be attributed to several complementary mechanisms: (1) methanol effectively disrupts cell membranes and facilitates penetration into plant tissues; (2) water enhances the extraction of highly polar compounds; and (3) acidification with HCl promotes the stabilization of anthocyanins through structural modification to their flavylium cation form, preventing degradation during extraction. Additionally, the acidic environment disrupts hydrogen bonding between polyphenols and plant cell wall components, particularly pectin and cellulose, thereby enhancing the release of bound phenolic compounds. This mechanistic understanding explains why similar extraction protocols have proven effective across mulberry, chokeberry, and elderberry materials despite their taxonomic differences. The superior efficacy of methanol:water:HCl in maximizing bioactive compound recovery further corroborates a previous report by Uğur et al. [31], who documented enhanced extraction of hydrophilic polyphenols and anthocyanins utilizing acidified extraction media. Comparable results have been reported for Morus spp., wherein acidified solvents substantially improved anthocyanin and flavonoid recovery from fruit tissues. These findings align with extraction studies on chokeberry and elderberry, where similar acidified methanol–water systems have demonstrated optimal extraction of anthocyanins and other phenolic compounds [14]. The common effectiveness across these berry species can be attributed to their similar polyphenolic profiles, particularly their rich anthocyanin content, which responds similarly to extraction under acidic conditions. This cross-species consistency suggests that optimized extraction protocols may be broadly applicable across various berry fruits, with minor adjustments based on specific tissue characteristics and target compounds. The practical implications of these findings are particularly relevant for industrial-scale extraction processes, where the optimization of solvent systems could significantly enhance bioactive compound yields while potentially reducing processing costs and environmental impact. The superior performance of the acidified mixed solvent system suggests that industrial applications might benefit from adopting similar extraction methodologies to maximize bioactive compound recovery. Antioxidant capacity assays (ABTS and CUPRAC) exhibited parallel trends, with foliar tissues demonstrating significantly elevated radical scavenging activities compared to fruits. This observation supports previous findings indicating that polyphenol-enriched leaves possess superior antioxidant potential [32]. The solvent-dependent variations in antioxidant capacity further emphasize the role of extraction efficiency in determining bioactive compound availability and functionality. The results indicate that the methanol:water:HCl-extracted samples manifested the highest ABTS and CUPRAC values, which aligns with the findings of Abay and Eruygur [33], who emphasized the impact of solvent systems on polyphenol-derived antioxidant activities. A similar pattern has been documented in Morus extracts, wherein solvent selection significantly influenced antioxidant potential, with acidified solvents yielding maximal activity [34]. For future extraction methodology development across mulberry, chokeberry, and elderberry materials, our findings suggest several optimization strategies: (1) adjusting the acid concentration based on the specific anthocyanin profile of each species; (2) modifying the methanol ratio according to the polarity distribution of target compounds; and (3) considering the extraction temperature and duration based on tissue-specific characteristics. Additionally, green extraction alternatives such as natural deep eutectic solvents (NADESs) could potentially replicate the extraction efficiency of methanol:water:HCl systems while offering improved sustainability and biocompatibility for food and pharmaceutical applications. Hierarchical clustering and heatmap analyses further demonstrated distinct groupings based on plant anatomical structure, extraction medium, and genotype. The robust positive correlations observed among the TPC, TFC, ABTS, and CUPRAC values reinforce the interconnected roles of polyphenols in antioxidant defense mechanisms. The clustering of methanol:water:HCl-extracted samples discretely from methanol and water extracts further substantiates the notion that solvent selection significantly influences bioactive compound extraction and subsequent antioxidant potential.

4.2. Effects of Plant Part, Cultivar, and Solvent on TPC, TFC, and ABTS in Different Extracts of Chokeberry

The results of this study demonstrate significant interaction effects, highlighting the intricate nature of polyphenol extraction and confirming the importance of tailoring solvent selection specifically to both plant part and cultivar to achieve maximum bioactive compound recovery. These findings correspond with the extant literature documenting the variability of polyphenolic profiles contingent upon plant matrix characteristics and solvent polarity gradients [30,31]. Comparing our findings on chokeberry with those on black mulberry and elderberry reveals a consistent pattern across these berry species despite their taxonomic differences. All three species demonstrate similar responses to extraction conditions, suggesting common structural characteristics in their polyphenolic compounds and cellular matrices. This cross-species consistency is particularly valuable for developing unified extraction protocols in the berry processing industry. The exceptional performance of methanol:water:HCl in extracting the TPC and TFC suggests that acidified media enhance cellular disruption and facilitate the liberation of matrix-bound phenolic compounds [35,36]. This observation concurs with previous investigations demonstrating the efficacy of acidified methanol in the recovery of anthocyanins and flavonoids [30]. The mechanism underlying the superior extraction efficiency of methanol:water:HCl can be explained by several complementary processes: (i) methanol effectively permeabilizes cell membranes through lipid solubilization; (ii) water increases solvent accessibility to hydrophilic polyphenols; and (iii) acidification protonates hydroxyl groups on phenolic compounds, thereby reducing hydrogen bonding with cell wall components and enhancing extraction. Additionally, acidic conditions stabilize anthocyanins in their flavylium cation form, preventing degradation during extraction—a particularly important factor for chokeberry, which contains high levels of cyanidin-based anthocyanins similar to those found in elderberry and mulberry. In our assessment, the superior extraction capability of acidified methanol:water mixtures may be attributed to the synergistic effect of polar protic solvents combined with acid hydrolysis, which likely facilitates the cleavage of glycosidic bonds and ester linkages that tether phenolic compounds to cell wall constituents. In the present study, genotypic variations were conspicuous, with Viking and Nero genotypes exhibiting superior TPC and antioxidant capacities, while Aron manifested comparatively diminished values. These results align with the findings of Jakobek et al. [37] and Ochmian et al. [28], who documented significant variations among chokeberry genotypes. The observed TPC ranges in this study correspond with previously reported values for chokeberry fruit [37] and other berry species such as elderberry [38]. Similarly, the TFC values documented herein align with those reported for chokeberry and black mulberry, further substantiating the critical role of genotype in determining flavonoid accumulation [29,39,40]. It is noteworthy that these cultivar-specific differences observed in the current study not only confirm the existing literature but also extend our understanding of the genetic basis of polyphenol accumulation. The consistent performance of Viking and Nero genotypes across multiple parameters suggests that these genotypes possess enhanced regulatory mechanisms governing the phenylpropanoid pathway, potentially through the transcriptional activation of key enzymes such as phenylalanine ammonia-lyase (PAL) and chalcone synthase (CHS).

A pivotal finding of this investigation is the markedly elevated bioactive compound concentrations in foliar tissues compared to reproductive organs, with the TPC, TFC, ABTS, and CUPRAC values being significantly enhanced in leaf extracts. This pattern has been well documented in previous research, which consistently reports higher phenolic and flavonoid concentrations in leaves of various plant species, including chokeberry and black mulberry [25,26]. This phenomenon is similarly observed in elderberry, suggesting a conserved physiological pattern whereby leaves across these berry species allocate greater resources toward phenolic compound synthesis. The enhanced antioxidant capacity of leaves can be attributed to their elevated content of secondary metabolites, which function as defensive compounds against environmental stressors and photooxidative damage [41]. From an evolutionary perspective, this differential distribution of polyphenols between plant organs reflects adaptive strategies, wherein leaves, being more vulnerable to herbivory and abiotic stressors, accumulate higher concentrations of defensive compounds. The findings suggest that leaves, often discarded as agricultural by-products, represent an underutilized resource with considerable potential for nutraceutical and pharmaceutical applications. The solvent effect was particularly pronounced, with methanol:water:HCl extraction yielding significantly enhanced TPC, TFC, and antioxidant activity compared to monosolvent systems. The superior efficiency of this solvent mixture aligns with the findings of Grunovaitė et al. [32], who reported enhanced polyphenol recovery using acidified solvents. Water conversely demonstrated the lowest extraction efficiency, as evidenced by its negative correlations with antioxidant capacity, a trend observed in previous studies [33,42]. To further improve extraction methodologies across mulberry, chokeberry, and elderberry materials, our results suggest several optimization strategies: (i) adjusting the methanol ratio based on the specific hydrophilicity profile of target compounds in each species; (ii) modifying the acid concentration according to the stability requirements of species-specific anthocyanin profiles; and (iii) exploring pulsed electric field pre-treatments to enhance cell permeabilization while reducing solvent requirements. These refinements could maximize extraction efficiency while addressing increasing demands for more sustainable processing methods in the natural product industry. The clustering patterns in the correlation analysis further substantiate the critical role of solvent selection in determining extraction efficiency. In our interpretation, these findings have significant implications for industrial-scale bioactive compound extraction, suggesting that optimized solvent systems could substantially enhance production yields while potentially reducing processing costs. The striking differences in extraction efficiency between solvent systems indicate the importance of methodology optimization in both research and commercial applications.

4.3. Effects of Plant Part, Cultivar, and Solvent on TPC, TFC, and ABTS in Different Extracts of Elderberry

The present study investigated the effects of plant part, cultivar, and solvent on the TPC, TFC, and ABTS in elderberry (S. nigra L.) extracts. The findings reveal that solvent type exerted the most significant influence, followed by plant part, with cultivar showing a lesser yet notable impact [43,44]. These results align with the existing literature, indicating the critical role of extraction methods and plant anatomy in determining phytochemical yields. Comparing these results with our findings on black mulberry and chokeberry reveals a consistent pattern across all three berry species, where solvent selection plays the predominant role in extraction efficiency, followed by plant anatomical factors. This cross-species similarity suggests fundamental commonalities in the cellular architecture and phytochemical composition of these taxonomically distinct berry plants, providing a scientific basis for developing unified extraction protocols applicable across the berry industry. Notably, current research demonstrates that elderberry leaves contain substantially higher levels of bioactive compounds compared to fruits, exhibiting a 59.4% greater TPC, an 87.3% higher TFC, 81.3% more ABTS, and 117.2% higher CUPRAC antioxidant capacity. This observation aligns with previous studies emphasizing the rich polyphenolic profile of elderberry leaves. Mikulic-Petkovsek et al. [43] reported that elderberry leaves possess higher concentrations of phenolic compounds than fruits, attributing this difference to the plant’s defense mechanisms against environmental stressors. Similarly, the study’s hierarchical clustering revealed that leaves formed a high-activity cluster due to their elevated phenolic and flavonoid contents, consistent with prior research highlighting the distinct phytochemical profiles of elderberry leaves and fruits. Regarding solvent efficiency, methanol:water:HCl was identified as the most effective extraction medium, yielding a 27.5% higher TPC, a 33.8% higher TFC, 15.4% greater ABTS activity, and 37.8% higher CUPRAC values compared to methanol alone. This finding corroborates earlier research by Stalikas [45], who emphasized that acidified methanol enhances the solubility and extraction efficiency of phenolic acids and flavonoids. Petrova et al. [46] similarly demonstrated that acidic solvents improve phenolic compound recovery due to increased stability in acidic environments. The mechanistic basis for the superior extraction efficiency of methanol:water in elderberry parallels what we observed in mulberry and chokeberry, suggesting common physicochemical principles governing polyphenol extraction across these species. In elderberry specifically, the acidified solvent system functions through multiple complementary mechanisms: (i) the protonation of hydroxyl groups on elderberry’s characteristic anthocyanins (primarily cyanidin-3-sambubioside and cyanidin-3-glucoside) reduces their interaction with cell wall polysaccharides; (ii) the methanol component disrupts hydrophobic associations between phenolic compounds and cellular proteins; and (iii) acidic conditions prevent oxidative degradation of labile flavonoids during extraction. These mechanisms collectively explain why this solvent system consistently outperforms mono-solvents across all three berry species despite their different phytochemical profiles. Among the genotypes examined, ‘Tokat’ consistently exhibited superior bioactive compound levels compared to ‘Haschberg’ across all measured parameters, with the highest antioxidant capacity being observed in the leaf extracts of ‘Tokat’ using methanol:water:HCl, reinforcing the combined influence of genetic variation and optimized solvent extraction. This finding aligns with the work of Hossen et al. [47], who reported significant cultivar-dependent differences in phenolic content and antioxidant activity, indicating the role of genetic factors in phytochemical composition. The cultivar-dependent variation observed in elderberry mirrors our findings in mulberry (where Gümüşhacıköy Horum and Tohma Medik genotypes excelled) and chokeberry (where Viking and Nero demonstrated superior profiles). This pattern of genotypic influence across three distinct berry species highlights the universal importance of genetic factors in determining bioactive compound accumulation, suggesting that selective breeding programs could effectively enhance nutraceutical value across diverse berry crops using similar selection criteria focused on polyphenolic content and antioxidant capacity. A correlation analysis further confirmed strong positive associations among the TPC, TFC, ABTS, and CUPRAC values, with leaf extracts consistently displaying higher bioactive compound concentrations and antioxidant capacities than fruit extracts. These results reinforce the importance of plant part selection and optimized extraction conditions in maximizing elderberry’s antioxidant potential.

5. Conclusions

This study evaluated the effects of plant part, cultivar, and solvent on the TPC, TFC, ABTS and CUPRAC in black mulberry, chokeberry, and elderberry. The findings demonstrate that leaves consistently exhibit higher phytochemical content and antioxidant potential than fruits across all species, emphasizing their potential as valuable sources of bioactive compounds. Among the studied cultivars, significant variations were observed, with certain genotypes, such as Gümüşhacıköy Horum (black mulberry), Viking (chokeberry), and Tokat (T1) (elderberry), showing superior phytochemical profiles. These cultivar-specific differences highlight the importance of genetic factors in determining polyphenol accumulation and antioxidant activity. Although certain genotypes, such as Viking, exhibited superior phytochemical profiles, the underlying genetic or metabolic factors were not explored in this study; future research could benefit from integrating known genotypic data to better explain cultivar-specific differences. The choice of solvent significantly influenced extraction efficiency, with methanol:water:HCl emerging as the most effective medium for maximizing the TPC, TFC, and antioxidant yields. This solvent system outperformed methanol alone and water, reinforcing the role of acidic conditions in enhancing polyphenol solubility. Given that methanol:HCl may not be suitable for food industry applications, future studies should also consider the feasibility of environmentally friendly alternatives such as lactic acid. Correlation and clustering analyses further confirmed strong positive associations between the TPC, TFC, and antioxidant capacity, with distinct groupings based on plant part, cultivar, and solvent type. These findings show the necessity of selecting appropriate extraction methodologies to optimize the recovery of bioactive compounds. Overall, the present study provides valuable insights into the multifactorial influences on the phytochemical composition and antioxidant potential of berry crops. Future research should explore the molecular mechanisms regulating cultivar-specific polyphenol accumulation and investigate sustainable valorization strategies for leaf-derived bioactive compounds. However, this study has certain limitations, including the lack of in vivo validation and limited seasonal or environmental variability, which should be addressed in future research. Additionally, further studies on the bioavailability and stability of these compounds in food and pharmaceutical applications would enhance their potential for nutraceutical development.