Phytochemical profiles of wild blackberries, black and white mulberries from southern Bulgaria

ABSTRACT

Sugars (glucose and fructose), organic acids (citric acid, tartaric acid, oxalic acid, malic acid, succinic acid and fumaric acid), vitamin C, phenolic compounds (catechin, rutin, quercetin, chlorogenic acid, ferulic acid, o-coumaric acid, p-coumaric acid, caffeic acid, syringic acid, vanillic acid and gallic acid) and antioxidant capacity (Trolox equivalent antioxidant capacity, TEAC assay) were determined in fruits of wild blackberry, black and white mulberries from southern Bulgaria. Malic acid was the predominant organic acid for the black and white mulberries. Citric and malic acids were represented with the highest content in blackberries. The highest fructose concentration was observed in blackberries (16.187 g·100 g⁻¹). Black mulberries showed the highest antioxidant activity (12.230 µmol TE g⁻¹), followed by white mulberries (8.6133 µmol TE g⁻¹) and blackberries (4.490 µmol TE g⁻¹). Chlorogenic acid and rutin were the main phenolics in the analysed fruits. The results illustrated significant phytochemical profiles of the studied berries, which could contribute to the medicinal industry and provide valuable genetic resources for breeding programmes.

KEYWORDS:

• Antioxidants
• blackberry
• mulberry
• organic acids
• phenolic compounds

Introduction

Horticultural plants show wide variability in terms of morphological, biochemical and molecular characteristics.[1–5] The Balkan Peninsula is one of the main genetic diversity centres for numerous fruit species with diverse phytochemical profiles and potential health benefits important in pharmaceutical and functional food industries.[5] There are many fruit species with undefined phytochemical composition with potency for the food industry and breeding programmes.[6,7]

Berries are very popular in the human diet due to their high anthocyanin content and antioxidant activities with potential health benefits on retarding aging, decreasing the risk of cardiovascular diseases and cancer.[8–10] Therefore, many studies have been conducted on the chemical composition of berries. Mikulic-Petkovsek et al. [11] reported the sugars, organic acids and total phenolics of wild and cultivated berry species grown in central Slovenia. Organic acids, phenolic compounds, sugar composition, vitamin C (ascorbic acid) content and total antioxidant capacity of white (Morus alba L.), black (Morus nigra L.) and red (Morus rubra L.) mulberries from the eastern Anatolia region of Turkey [8,12] and blackberries grown in Adana province of the Mediterranean region, Turkey [13] have been investigated. Huang et al. [14] analysed the phenolic composition and antioxidant activities of blueberry, blackberry and strawberry species grown in China. Sánchez-Salcedo et al. [10] investigated the anthocyanins and their antioxidant capacities in some promising black and white mulberries from Spain. Connor et al. [15] highlighted the hereditary and environmental variability in anthocyanins and their antioxidant activities in blackberries. Sellappan et al. [16] reported the phenolic composition of wild blackberries from Georgia. Likewise, there are many investigations on the phytochemical content of blackberries under diverse ecological conditions.[17–19]

The phytochemical compositions of various berries grown in different regions of Bulgaria were investigated.[17,20,21] However, there is no information on the chemical composition of wild blackberries and mulberries from southern Bulgaria. Therefore, the objective of this research was to identify and quantify sugars (glucose and fructose), organic acids (citric acid, tartaric acid, malic acid, succinic acid and fumaric acid), vitamin C, phenolic composition (catechin, rutin, quercetin, oxalic acid, chlorogenic acid, ferulic acid, o-coumaric acid, p-coumaric acid, caffeic acid, syringic acid, vanillic acid and gallic acid) and antioxidant activities (Trolox equivalent antioxidant capacity, TEAC assay) of wild blackberries, black and white mulberries from southern Bulgaria.

Materials and methods

Plant material

A total of 1 kg of ripe fruits of wild blackberry (Rubus fruticosus L.), white mulberry (Morus alba L.) and black mulberry (Morus nigra L.) were collected during the summer of 2013 from the region of the Municipality of Haskovo, southern Bulgaria.

Detection of organic acids

The modified method of Bevilacqua and Califano [22] was used for detection and quantification of organic acids. Berries were mashed within cheesecloth. Juice samples were stored at −20 °C and thawed overnight at room temperature prior to analysis. Twenty millilitres of 0.009 N H₂SO₄ was added to 5 mL of sample and mixed with a shaker (Heidolph Unimax 1010, Schwabach, Germany) for 1 h, then centrifuged for 15 min at 150,00 × g. The supernatants were filtered twice through an 0.45 μm membrane filter (Millipore Millex-HV Hydrophilic PVDF, Millipore, Temecula, CA, USA) and run on SEP-PAK C18 cartridge using Aminex column (HPX – 87 H, 300 mm × 7.8 mm, Bio-Rad Laboratories, Richmond, CA, USA). The absorbance range was set as 214 and 280 nm. An Agilent package with diode array detector (DAD) was adopted.

Detection of phenolic compounds

The modified method of Rodriguez-Delgado et al. [23] was used to define gallic acid, catechin, chlorogenic acid, caffeic acid, p-coumaric acid, o-coumaric acid, ferulic acid, vanillic acid, rutin, syringic acid and quercetin content. Juice samples were diluted with distilled water in a ratio of 1:1 and centrifuged for 15 min at 15,000 × g. Supernatants were filtered twice through an 0.45 μm membrane filter (Millipore Millex-HV Hydrophilic PVDF, Millipore, Temecula, CA, USA), then loaded onto an 1100 (Agilent, Santa Clara, CA, USA) high-performance liquid chromatograph (HPLC) using a DAD detector (Agilent) and 250 × 4.6 mm, 4 μm octadecylsilane (ODS) column (HiChrom, Chadds Fors, PA, USA). Solvent A methanol:acetic acid:water (10:2:28) and solvent B methanol:acetic acid:water (90:2:8) were used as a mobile phase. Spectral measurements were performed at 254 and 280 nm, 1 mL min⁻¹ flow rate and 20 μL injection volume.

Sugar analysis

The modified method of Melgarejo et al. [24] was utilized in sugar analysis. After homogenization in a shaker, juice samples were centrifuged for 2 min at 12,000 × g. The supernatants were filtered and run on an SEP-PAK C18 column (pore size 125 Å). Then, 85% solvent B (acetonitrile liquid phase) was loaded onto HPLC with μBondapak-NH2 column (Waters Inc., New Castle, DE, USA). A refractive index detector was used for elution. The sugar composition in the berries was identified based on fruit juice standards.

Analysis of ascorbic acid

Five millilitres of each sample juice was blended with meta-phosphoric acid solution and then, centrifuged for 10 min at 6500 × g at 4 °C. An 0.5 mL aliquot of supernatant was complemented to 10 mL with % 2.5 meta-phosphoric solution, after which it was filtered through an 0.45 μm Teflon filter. Samples were loaded onto HPLC with a C18 column (Phenomenex Luna C18, 250 × 4.60 mm, 5 μm) at 25 °C. Ultra distilled water was set as a mobile phase at 2.2 pH. A DAD detector was used at 254 nm wavelength. Readings were performed at different L-ascorbic acid levels (Sigma A5960, St. Louis, MO, USA) (50, 100, 500, 1000 and 2000 μL L⁻¹).[25]

Determination of Trolox equivalent antioxidant capacity

For TEAC analysis, 2, 2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) was dissolved with potassium persulphate in a buffer.[26] ABTS(+) was diluted with 20 mmol L⁻¹ sodium acetate buffer at 4.5 pH and 734 nm wavelength, 0.700 ± 0.01 for long storage. Three millilitres of ABTS(+) solution and 20 μL of sample fruit extract mixture were incubated for 10 min and used for spectrophotometric readings.

Statistical analysis

Descriptive statistics of all phytochemical parameters for each sample of blackberry and mulberries are given as mean values from six independent experiments with standard error (±SE). One-way analysis of variance (ANOVA) was used for assessment of the differences in the phytochemical parameters between white mulberry, black mulberry and blackberry. Mean separation was performed by Duncan's test. Statistical evaluations were made via IBM SPSS Statistics for Windows (Version 22.0, Armonk, NY, USA) at 5% significance level.

Results and discussion

According to the ANOVA results, significant differences between blackberry, black and white mulberries were observed for the analysed phytochemical parameters (p < 0.05) (Tables 1–3).

Table 1. Organic acid profiles of wild blackberry, black and white mulberry accessions from southern Bulgaria.

	Blackberry	Black mulberry	White mulberry
Citric acid (g·100 g^−1)	0.953 ± 0.014^b	1.163 ± 0.026^a	0.840 ± 0.0153^c
Tartaric acid (g·100 g^−1)	ND	0.233 ± 0.018^b	0.316 ± 0.008^a
Malic acid (g·100 g^−1)	0.910 ± 0.020^c	1.947 ± 0.009^b	2.106 ± 0.037^a
Succinic acid (g·100 g^−1)	0.297 ± 0.0145^b	0.140 ± 0.005^c	0.360 ± 0.011^a
Fumaric acid (g·100 g^−1)	0.020 ± 0.000^b	0.023 ± 0.003^b	0.033 ± 0.003^a

Note: ND, not detected.

^a,b,cSame letters within each row indicate significant differences between means (p < 0.05).

Table 2. Sugars, vitamin C and antioxidant activity of wild blackberry, black and white mulberries from southern Bulgaria.

	Blackberry	Black mulberry	White mulberry
Fructose (g·100 g^−1)	16.187 ± 0.272^a	8.610 ± 0.046^b	5.270 ± 0.035^c
Glucose (g·100 g^−1)	8.507 ± 0.133^b	9.180 ± 0.044^a	6.143 ± 0.055^c
Vitamin C (mg·100 g^−1)	13.330 ± 0.049^b	13.160 ± 0.032^c	16.687 ± 0.023^a
TEAC (µmol TE g^−1)	4.490 ± 0.120^c	12.230 ± 0.064^a	8.613 ± 0.027^b

^a,b,cSame letters within each row indicate significant differences between means (p < 0.05).

Table 3. Phenolic compounds of the wild blackberry, white and mulberries from southern Bulgaria.

	Blackberry	Black mulberry	White mulberry
Catechin (mg g^−1)	0.082 ± 0.004^a	0.063 ± 0.003^b	0.035 ± 0.001^c
Rutin (mg g^−1)	0.842 ± 0.061^c	1.334 ± 0.005^a	1.114 ± 0.005^b
Quercetin (mg g^−1)	0.141 ± 0.003^a	0.065 ± 0.001^c	0.082 ± 0.002^b
Chlorogenic acid (mg g^−1)	2.324 ± 0.008^a	2.126 ± 0.008^b	1.663 ± 0.003^c
Ferulic acid (mg g^−1)	0.100 ± 0.004^a	0.024 ± 0.001^c	0.065 ± 0.002^b
o-Coumaric acid (mg g^−1)	0.098 ± 0.003^b	0.166 ± 0.003^a	0.051 ± 0.002^c
p-Coumaric acid (mg g^−1)	0.098 ± 0.003^a	0.061 ± 0.001^b	0.045 ± 0.001^c
Caffeic acid (mg g^−1)	0.178 ± 0.002^a	0.108 ± 0.001^c	0.122 ± 0.001^b
Syringic acid (mg g^−1)	0.159 ± 0.004^a	0.037 ± 0.001^c	0.049 ± 0.001^b
Vanillic acid (mg g^−1)	0.052 ± 0.002^a	0.036 ± 0.001^b	0.039 ± 0.002^b
Gallic acid (mg g^−1)	0.312 ± 0.003^b	0.429 ± 0.004^a	0.141 ± 0.003^c
Oxalic acid (g·100 g^−1)	0.087 ± 0.003	ND	ND

Note: ND, not detected.

^a,b,cSame letters within each row indicate significant differences between means (p < 0.05).

Organic acids

The descriptive statistics obtained for the organic acid composition of blackberry, black and white mulberries growing wildly in southern Bulgaria are given in Table 1. The content of tartaric acid in blackberries was non-detectable (ND). The concentration ranges were as follows: white mulberry > black mulberry > blackberry (ND) for tartaric acid; black mulberry > blackberry > white mulberry for citric acid; white mulberry > black mulberry > blackberry for malic acid; white mulberry > blackberry > black mulberry for succinic acid and white mulberry > black mulberry > blackberry for fumaric acid.

Blackberry

Mikulic-Petkovsek et al. [11] reported that tartaric acid was not present in the wild blackberries from Slovenia. In their study, the ranges of the other organic acids were as follows: citric acid (4.1–5.6 g kg⁻¹), malic acid (1.11–2.05 g kg⁻¹), fumaric acid (30.7 and 34.4 mg kg⁻¹) and shikimic acid (89.3 and 28.2 mg kg⁻¹). Our results (Table 1) are in agreement with theirs. Sensoy et al. [27] also reported organic acid concentrations for wild blackberry accessions from Van province, eastern Turkey, which were as follows: citric acid (5.69–12.01 g kg⁻¹), malic acid (1.41–6.34 g kg⁻¹), oxalic acid (0.08–0.19 g kg⁻¹), succinic acid (1.23–3.28 g kg⁻¹) and fumaric acid (0.01–0.07 g kg⁻¹). The differences between the data reported by Sensoy et al. [27] and our results could be a result of regional, geographical, climate factors or seasonal variations, etc.

Black mulberry

Organic acids are considered as a ripening index, and the balance between organic acids and sugars affects the taste of the berries. Drug industries utilize organic acids as antioxidants and preservative substances.[11] Koyuncu [28] demonstrated some range values for citric acid (8.80–823.40 mg g⁻¹), tartaric acid (3.05–5.95 mg g⁻¹), malic acid (35.55–198.50 mg g⁻¹), oxalic acid (0.348–1.176 mg g⁻¹) and fumaric acid (0.015–0.033 mg g⁻¹) in wild black mulberries from Isparta province, western Turkey. Eyduran et al. [8] detected and quantified higher values in citric acid (0.733–1.033 g·100 g⁻¹), tartaric acid (0.147–0.297 g·100 g⁻¹) of wild black mulberries compared to our results. The concentrations of malic acid (1.210–3.040 g·100 g⁻¹), succinic acid (0.117–0.360 g·100 g⁻¹) and fumaric acid (0.010–0.107 g·100 g⁻¹) were within the ranges reported by Eyduran et al.[8] The citric acid (0.820 g·100 g⁻¹), tartaric acid (0.290 g·100 g⁻¹), malic acid (3.073 g·100 g⁻¹), succinic acid (0.1133 g·100 g⁻¹) and fumaric acid (0.120 g·100 g⁻¹) contents of wild black mulberry native to the central district of Igdir, Turkey were examined by Gecer et al.[29] In our study, the contents of citric and succinic acids were found to be higher, but the contents of other organic acids were lower in comparison to those reported by Gecer et al.[29] This could be attributed to the difference in the genetic structure and environmental conditions. Spanish black mulberries [2] showed lower citric acid (0.14–0.66 g·100 g⁻¹), malic acid (0.41–0.79 g·100 g⁻¹) and tartaric acid (0.03–0.04 g·100 g⁻¹) concentrations when compared with our results (Table 1). Jiang and Nie [30] reported that black mulberries have the richest organic acid contents (succinic acid 6.48 mg g⁻¹, acetic acid 3.55 mg g⁻¹, malic acid 2.62 mg g⁻¹ and citric acid 0.71 mg g⁻¹) in comparison to white (M. alba L.), Russian (M. alba var. tatarica L.) and black (M. nigra L.) mulberries grown in Xinjiang province, north-west China. Mikulic-Petkovsek et al. [11] did not detect tartaric acid in wild black mulberries native to central Slovenia, which was confirmed in our experiments (Table 1). Malic acid was predominant among the organic acids studied by us, which was consistent with previous reports.[3,24] However, trace levels of fumaric acid were detected in the wild blackberries, in agreement with Mikulic-Petkovsek et al.[11]

White mulberry

Malic acid was determined to be the predominant organic acid for white mulberry, which is consistent with the results of Eyduran et al. [8] and Gecer et al. [29] in Turkish wild white mulberries and Sanchez et al. [7] in Spanish white mulberries. The concentrations of malic acid, citric and tartaric acid of our samples in white mulberries were higher than those in the white mulberries investigated by Sanchez et al.[7] Eyduran et al. [8] found a statistically significant difference in citric acid (0.687 vs. 0.480 g·100 g⁻¹), tartaric acid (0.153 vs. 0.430 g·100 g⁻¹), malic acid (2.103 vs. 1.130 g·100 g⁻¹), succinic acid (0.267 vs. 0.437 g·100 g⁻¹) and fumaric acid (0.100 vs. 0.123 g·100 g⁻¹) between wild white mulberries (76-IGD-4 and 76-IGD-5) native to Igdir province (Melekli district), Turkey (p < 0.05). The investigated white mulberry accessions in our study were higher in citric and fumaric acids contents in comparison to the white mulberries (76-IGD-4 and 76-IGD-5) reported by Eyduran et al.,[8] whereas their malic acid content was similar to that in 76-IGD-4 and almost twofold higher than in the 76-IGD-5 white mulberry accession. Gecer et al. [29] identified and quantified the citric acid (0.6367 g·100 g⁻¹), tartaric acid (0.1500 g·100 g⁻¹), malic acid (2.1333 g·100 g⁻¹), succinic acid (0.2500 g·100 g⁻¹) and fumaric acid (0.1067 g·100 g⁻¹) concentrations in wild white mulberries of Igdir province, Turkey. Compared to those in the Bulgarian white mulberries analysed in the present research, the concentrations of citric, succinic and tartaric acids reported in [29] were lower, but the values of fumaric acid were higher. The malic acid content measured in our experiments was similar to that previously reported by Gecer et al. [29] and in agreement with Eyduran et al.[8] The accessions analysed by us had a much higher content of citric acid (0.393 g·100 g⁻¹), tartaric acid (0.223 g·100 g⁻¹), succinic acid (0.168 g·100 g⁻¹) and fumaric acid (0.024 g·100 g⁻¹) compared to those reported by Gundogdu et al. [12] in white (M. alba) mulberries from the eastern part of Turkey.

Sugars, vitamin C and antioxidant activity

The descriptive statistics for the sugars and vitamin C content and antioxidant activity (TEAC assay) of wild blackberry, black and white mulberries from southern Bulgaria are presented in Table 2. The concentration ranges were as follows: fructose (blackberry > black mulberry > white mulberry), glucose (black mulberry >blackberry > white mulberry), TEAC (black mulberry >white mulberry > blackberry) and vitamin C (white mulberry> blackberry > black mulberry). Significant differences between black and white mulberries have been reported.[3,7,24]

Blackberry

The data about the phytochemical parameters in blackberry available so far are still not understood well; therefore, more studies should be performed. In our experiments, the glucose and fructose sugar contents were shown to be much higher when compared to those reported by Hassimotto et al. [31] in other blackberry cultivars grown in Brazil: Caingangue (2.44 and 1.91 g·100 g⁻¹), Brazos (1.69 and 1.15 g·100 g⁻¹), Tupy (2.53 and 2.02 g·100 g⁻¹), Guarani (2.08 and 1.70 g·100 g⁻¹) and Seleção 97 (2.60 and 2.11 g·100 g⁻¹). Mikulic-Petkovsek et al. [11] reported glucose (35.30 g kg⁻¹), fructose (35.40 g kg⁻¹) and sucrose content (1.26 g kg⁻¹) in wild blackberries, and highlighted that the wild berries are richer in phytochemical content than the cultivated ones. Kafkas et al. [13] described the fructose (33.8, 25.1, 25.5, 21.1 and 30.2 mg g⁻¹), glucose (26.1, 16.9, 16.6, 15.8 and 20.3 mg g⁻¹) and sucrose content (2.6, 1.5, 1.4, 1.2 and 2.0 mg g⁻¹) in Navaho, Chester Thornless, Jumbo, Bursa 2 and Loch Ness blackberry cultivars, respectively, grown in the Mediterranean region of Turkey. The glucose and fructose contents of the wild blackberry accession studied in this research were markedly richer compared to the amounts described by Zielinski et al. [19] in Brazos and Tupy blackberries grown in southern Brazil: glucose (3.11 and 3.30 g·100 g⁻¹) and fructose (2.86 and 2.49 g·100 g⁻¹). Sensoy et al. [27] quantified the fructose (8.84–16.31 g kg⁻¹) and glucose (1.48–3.89 g kg⁻¹) contents in wild blackberries from Van, Turkey. In our study, the vitamin C content in blackberries (13.330 mg·100 g⁻¹) was higher than that in the findings of Ochmian et al. [32] in wild blackberries from Poland (11 mg·100 g⁻¹). However, higher levels of vitamin C (14.05–17.15 mg·100 g⁻¹) are reported in wild blackberries from Coruh valley of Turkey.[33] Comparative analysis of our results (Table 2) with those of other authors showed that Donno et al. [34] reported very high vitamin C content (45.07 mg·100 g⁻¹) in blackberries from northern Italy. Souza et al. [35] also measured very high vitamin C concentration (52.41 mg·100 g⁻¹) in Brazilian blackberries. Hassimotto et al. [31] quantified the vitamin C content in Caingangue (21.0 mg·100 g⁻¹), Brazos (9.9 mg·100 g⁻¹), Tupy (14.4 mg·100 g⁻¹), Guarani (11.9 mg·100 g⁻¹) and Seleção 97 (15.6 mg·100 g⁻¹) blackberry cultivars grown in Brazil. Vasco et al. [36] showed vitamin C content of 10–11 mg·100 g⁻¹ in Andean blackberry of Ecuador, which was lower than the amount measured in our study. Paulovicsová et al. [37] reported high vitamin C content (44.01 mg·100 g⁻¹) in thorn-free blackberry genotype grown in Slovakia. Regarding the antioxidant activity, Bernal et al. [38] demonstrated 30.22 µmol Trolox g⁻¹ in blackberries. Vasco et al. [36] found 2167 mg gallic acid equivalent (GAE) 100 g⁻¹ total soluble phenolic compound content in Andean blackberry. Siriwoharn and Wrolstad [39] obtained that the total phenolic amounts in Evergreen and Marion blackberries from Woodburn (the United States) were 822 and 844 mg GAE 100 g⁻¹, respectively. The differences in the data in the literature might be ascribed to the various agro-climatic conditions (light, soil, temperature and humidity), heritable variability (cultivar and species) and cultural practices, etc.

Black mulberry

Regarding the vitamin C content, our results (Table 2) showed considerably lower levels than the 32.25 mg·100 g⁻¹ previously reported by Iqbal et al. [40] in black mulberries from Pakistan. Our data for the vitamin C concentration in black mulberry were in line with the findings of Eyduran et al. [8] (10.123–16.293 mg·100 g⁻¹ for wild grown black mulberries). In the samples analysed in our study, there was higher content of glucose and fructose than reported by Gundogdu et al. [12] (7.748 and 5.634 g·100 g⁻¹, respectively). The contents of the studied parameters in our study were also higher than the results of Ozgen et al. [26] for glucose (5.50–7.12 g·100 mL⁻¹), fructose (4.86–6.41 g·100 mL⁻¹) and vitamin C content (0.003–100 mL⁻¹). These sugars, however, were almost half the concentrations reported by Mikulic-Petkovsek et al. [11] for wild black mulberries from Slovenia.

Lale and Ozcagiran [41] and Ercisli et al. [42] determined vitamin C content of 16.6 and 20.79 mg·100 mL⁻¹ in black mulberries, respectively. Paulovicsová et al. [37] showed significantly higher vitamin C content (70.53 mg·100 g⁻¹) in black mulberries from Slovakia compared to our results.

The TEAC of wild black mulberries (76-IGD-1, 76-IGD-2 and 76-IGD-3) has been determined to be 11.297, 14.400 and 10.167 µmol TE g⁻¹ by Eyduran et al.[8] Gecer et al. [29] reported a lower 9.1667 µmol TE g⁻¹ TEAC in comparison to our results, which were within the values of both Ozgen et al. [26] and Eyduran et al.[8] However, the present TEAC value for black mulberries was lower than those of Gundogdu et al.[12]

White mulberry

The glucose and fructose contents were quantified (6.1413 and 5.2700 g·100 g⁻¹) for Bulgarian white mulberries. These values were lower compared to those reported by Gecer et al. [29] (8.3100 and 7.693 g·100 g⁻¹) and Eyduran et al. [8] for 76-IGD-4 wild mulberry, but higher than the relevant content in 76-IGD-5.[8]

The vitamin C content in the white mulberries analysed by us was 16.687 mg·100 g⁻¹, slightly higher than the amount reported by Gecer et al. [29] (16.420 mg·100 g⁻¹) in wild white mulberry from eastern Turkey and by Imran et al. [43] (15.20 mg·100 g⁻¹) in white mulberries from Pakistan. Our findings were between the values in 76-IGD-4 (18.223 mg·100 g⁻¹) and 76-IGD-5 (13.400 mg·100 g⁻¹) white mulberries from eastern Turkey studied by Eyduran et al. [8] A vitamin C content of 22.4 mg·100 mL⁻¹ has been shown in white mulberries from north-east Anatolia, Turkey.[39] When compared with our results, Gundogdu et al. [12] reported an amount of 24.422 (mg·100 g⁻¹) vitamin in white mulberries from Van, Turkey. Eyduran et al. [8] found 9.273 (TEAC µmol TE g⁻¹) antioxidant activity in 76-IGD-5 white mulberry, slightly higher than the 8.613 (µmol TE g⁻¹) TEAC average in Bulgarian wild white mulberries. Although the antioxidant activity in Bulgarian white mulberries was higher than the TEAC values identified by Eyduran et al. [8] and Gecer et al. [29] (6.1667 and 6.170 µmol TE g⁻¹ for 76-IGD-5), other authors, for example Gundogdu et al.,[12] have found a significantly lower TEAC value for white mulberries (4.494 (µmol TE g⁻¹) from Van, Turkey. All these variations could be attributed to genotype, various environmental conditions and extraction methods.

Phenolic compounds

The descriptive statistics for 12 phenolic compounds extracted from the wild grown blackberry, black and white mulberries accessions from southern Bulgaria are shown in Table 3.

Blackberry

Hassimotto et al. [31] identified some bioactive compounds of blackberries (Caingangue, Brazos, Tupy, Guarani and Seleção 97) grown in Brazil and found, respectively, quercetin contents of 19.0, 8.0, 7.8, 13.3 and 9.0 mg·100 g⁻¹. Ochmian et al. [32] determined 0.9 mg·100 g⁻¹ p-coumaric acid in blackberries from Poland. Rutz et al. [44] phytochemically evaluated the influence of maturity stage on phenolic compounds of blackberry cv. Tupy cultivated in Brazil and detected gallic acid (144.30 mg·100 g⁻¹), catechin (19.08 mg·100 g⁻¹), In their study, caffeic acid and quercetin were below the limits of detection. They found epicatechin (466.22 mg·100 g⁻¹), ferulic acid (0.197 mg·100 g⁻¹) and ellagic acid (3.811 mg·100 g⁻¹) at its maturity stage. Sellappan et al. [16] investigated several pyhtochemical properties of Choctaw and Kiowa blackberry cultivars from Georgia (United States) and suggested that the total anthocyanins and polyphenols could be correlated with TEAC values. They also reported caffeic acid (1.38–3.64 mg·100 g⁻¹), p-coumaric acid (2.08–0.40 mg·100 g⁻¹), ferulic acid (2.51–2.99 mg·100 g⁻¹), phenolic acids and catechin (312.86–265.75 mg·100 g⁻¹). Mikulic-Petkovsek et al. [11] showed that wild raspberry, strawberry and blackberries from Slovenia contain twofold to fivefold more phenolic compounds in comparison to cultivated ones, which would be a valuable gene source for breeding programmes.

Black mulberry

Recent studies defined antioxidant activity with potential benefits on human health and closely related with the phytochemical profiles of the berries.[45] Our findings (Table 3) are in agreement with those of Eyduran et al.,[8] who detected a number of phenolic compounds neutralizing free radicals and quantified catechin (0.046–0.085 mg g⁻¹), rutin (0.820–1.365 mg g⁻¹), quercetin (0.067–0.137 mg g⁻¹), chlorogenic acid (0.759–2.339 mg g⁻¹), ferulic acid (0.023–0.063 mg g⁻¹), o-coumaric acid (0.034–0.129 mg g⁻¹), p-coumaric acid (0.035–0.111 mg g⁻¹), caffeic acid (0.094–0.158 mg g⁻¹), syringic acid (0.053–0.119 mg g⁻¹), vanillic acid (0.011–0.062 mg g⁻¹) and gallic acid (0.105–0.410 mg g⁻¹) in wild black mulberries from eastern Anatolia. Compared to our results, Gundogdu et al. [12] have reported higher levels of catechin (0.075 mg g⁻¹), rutin (1.423 mg g⁻¹), quercetin (0.113 mg g⁻¹), chlorogenic acid (3.106 mg g⁻¹), ferulic acid (0.064 mg g⁻¹), o-coumaric acid (0.134 mg g⁻¹), p-coumaric acid (0.129 mg g⁻¹), caffeic acid (0.131 mg g⁻¹) and syringic acid (0.103 mg g⁻¹), but lower levels of gallic acid (0.150 mg g⁻¹) and similar ones for vanillic acid (0.036 mg g⁻¹) in black mulberries from eastern Anatolia of Turkey. Sánchez-Salcedo et al. [10] determined lower concentrations of caffeic acid (0.01–0.03 mg g⁻¹) and p-coumaric acid (0.02–0.04 mg g⁻¹), but higher values for vanillic acid (0.06–0.14 mg g⁻¹) in mulberry cultivars in Spain compared to those measured in our study.

White mulberry

Gundogdu et al. [12] quantified gallic acid (0.215 mg g⁻¹), catechin (0.037 mg g⁻¹), chlorogenic acid (0.119 mg g⁻¹), caffeic acid (0.133 mg g⁻¹), syringic acid (0.049 mg g⁻¹), p-coumaric acid (0.047 mg g⁻¹), ferulic acid (0.033 mg g⁻¹), o-coumaric acid (0.015 mg g⁻¹), vanillic acid (0.008 mg g⁻¹), rutin (1.111 mg g⁻¹) and quercetin (0.015 mg g⁻¹) in fruits of white mulberry grown in eastern Turkey. When compared to the results reported by Gundogdu et al.,[12] the white mulberries in our study showed higher content of chlorogenic acid, ferulic acid, o-coumaric acid and vanillic acid, but lower amounts of quercetin and gallic acid, and were in agreement with the catechin, caffeic acid, syringic acid, p-coumaric acid and rutin content. Our results, with the exception of vanillic acid, showed much higher values than those determined in the study of Sánchez-Salcedo et al.,[10] who found vanillic acid (0.07–0.11 mg g⁻¹), caffeic acid (0.01–0.03 mg g⁻¹) and ferulic acid (0.002–0.03 mg g⁻¹) in four Spanish white mulberry clones, MA1, MA2, MA3 and MA4. In another study, Eyduran et al. [8] detected catechin (0.032–0.070 mg g⁻¹), rutin (0.750–0.925 mg g⁻¹), quercetin (0.101–0.045 mg g⁻¹), chlorogenic acid (2.667–0.980 mg g⁻¹), o-coumaric acid (0.062–0.026 mg g⁻¹), caffeic acid (0.094–0.134 mg g⁻¹) and vanillic acid (0.074–0.017 mg g⁻¹) in white mulberries from eastern Turkey. However, except for rutin, the concentrations were lower than those reported by Eyduran et al.,[8] who found syringic acid (0.115–0.060 mg g⁻¹), p-coumaric acid (0.065–0.127 mg g⁻¹), ferulic acid (0.110–0.141 mg g⁻¹) and gallic acid (0.206–0.214 mg g⁻¹).

Natic et al. [6] pyhtochemically characterized 11 white mulberries from North Serbia and identified caffeic acid (0.11–0.92 mg kg⁻¹), chlorogenic acid (0.43–22.12 mg kg⁻¹), rutin (0.65–77.28 mg kg⁻¹), p-coumaric acid (0.24–1.42 mg kg⁻¹), ferulic acid (0.57–29.49 mg kg⁻¹) and gallic acid (0.38–0.86 mg kg⁻¹). Radojkovic et al. [46] identified rutin (43.50 mg·100 g⁻¹), chlorogenic acid (33.00 mg·100 g⁻¹), quercetin (3.70 mg·100 g⁻¹) and gallic acid (14.50 mg·100 g⁻¹) in white mulberries from Serbia. Arfan et al. [47] have found different values for chlorogenic acid (15.0 and 17.7 mg g⁻¹) and rutin (26.2 and 32.3 mg g⁻¹) in white mulberries from Pakistan using methanol and acetone extraction solvents, with respect to our results. This difference could be attributed to the different solvent types used, as suggested from the study of Memon et al.,[48] who used three extraction techniques (sonication, magnetic stirring and homogenization) to identify chlorogenic acid (20.47, 17.03 and 24.45 mg·100 g⁻¹, respectively), syringic acid (9.19, 6.31 and 8.48 mg·100 g⁻¹), vanillic acid (4.57, 3.95 and 3.70 mg·100 g⁻¹) and gallic acid (5.81, 4.32 and 3.57 mg·100 g⁻¹) in mulberries from Pakistan. The results from our study were consistent with those of Memon et al.,[48] who identified chlorogenic acid as the main phenolic compound in white mulberries.

There is increasing interest in berries phytochemical profiles showing antioxidant properties that are important in human diet. Wild berries are in general a rich source of phenolics, and the range of phytochemicals could vary according to the genotype and environmental conditions. Another factor is the extraction method used and the discrepancies in the phytochemical profiles of berries in different reports could be circumvented, at least in part, by using the same extraction methods. Taking this into consideration, overall our results suggest that the wild berries in the region studied by us could be considered promising resources in breeding programmes and the pharmaceutical industry.

Conclusions

The present results showed that malic acid was the major organic acid in wild black and white mulberries collected in southern Bulgaria, whereas citric and malic acids were the predominant organic acids in the wild blackberries. Blackberry fructose content (16.187 g·100 g⁻¹) was higher than that in wild black and white mulberries. The highest antioxidant activity (TEAC, assay) was observed in wild black mulberry (12.230 µmol TE g⁻¹), followed by white mulberry (8.613 µmol TE g⁻¹) and blackberry (4.490 µmol TE g⁻¹). Chlorogenic acid and rutin were the main phenolics in wild blackberry, black and white mulberries. Oxalic acid was present only in wild blackberry. The results from this investigation revealed valuable information about the phytochemical characteristics of wild blackberry, black and white mulberries native to the flora of southern Bulgaria, which could be a valuable source in breeding programmes and the pharmaceutical industry.

Disclosure statement

No potential conflict of interest was reported by the authors.