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Article

Antioxidant Activity of Elderberry Fruits during Maturation

by
Nataša Imenšek
*,
Janja Kristl
,
Tadeja Kraner Šumenjak
and
Anton Ivančič
Faculty of Agriculture and Life Sciences, University of Maribor, Pivola 10, 2311 Hoče, Slovenia
*
Author to whom correspondence should be addressed.
Agriculture 2021, 11(6), 555; https://doi.org/10.3390/agriculture11060555
Submission received: 28 May 2021 / Revised: 11 June 2021 / Accepted: 15 June 2021 / Published: 17 June 2021
(This article belongs to the Special Issue Bioactive Compounds in Fruits)
Browse Figures
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Abstract

:
Antioxidant activity (AOA) in fruits of Sambucus nigra and several new elderberry interspecific hybrids involving S. javanica (JA), S. nigra subsp. nigra (NI), S. nigra subsp. nigra ‘Black Beauty’ (BB), S. nigra subsp. cerulea (CER) and S. racemosa (S. racemosa subsp. racemosa (RAC), S. racemosa subsp. racemosa var. miquelii (MIQ), S. racemosa subsp. tigranii (TIG), S. racemosa subsp. kamtschatica—coreana (KOR) at five maturity stages were investigated by applying DPPH (2,2-diphenyl-1-picrylhydrazyl), ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)), FRAP (ferric reducing antioxidant power) and ORAC (oxygen radical absorption capacity) assays. The aim of the study was to compare different assays for estimating AOA in elderberries, to plan the appropriate harvest time and to determine whether the most promising genotypes could be predicted in advance. All assays used showed that AOA increased significantly during maturation. All assays were significantly correlated with each other and could be reliably used for the AOA determination of elderberries. In fully mature berries, significant correlations were also found between total phenolic (TP) content and AOA by all assays, but no significant correlations were found between total ascorbic acid (TAA) content and AOA by all assays. At the fully mature stage, (the most appropriate stage for harvesting), berries of (JA × NI) × BB and (JA × NI) × MIQ were found to be superior in AOA and TP content. Genotypes with higher/lower AOA and TP content could not be predicted in advance, while TAA showed the opposite.

1. Introduction

Antioxidants are compounds that retard or inhibit the production of free radicals produced by oxidation processes. Free radicals are reactive and unstable molecules or complexes whose chain reactions pass through the enzymatic system and can damage the cells of an organism and cause various diseases [1]. Naturally existing endogenous enzymatic and non-enzymatic antioxidants protect human organism from the reactive oxygen species and their harmful effects. When they are unable to provide complete protection, the need for exogenous antioxidants such as vitamins (E, C, D, K3), ß-carotene, mineral Se and flavonoids increases [2]. Although many synthetic compounds are available in the market, there is an increasing interest in exogenous antioxidants of natural origin that have beneficial nutritional and therapeutic effects. Moreover, many synthetic antioxidants are no longer used in the food industry due to their potential adverse effects on human health and the stringent control that requires expensive testing to evaluate their safety [3]. The search for natural substitutes for synthetic antioxidants has led to the antioxidant evaluation of numerous plant sources. According to Halvorsen et al. [4], among 50 foods analysed that were high in antioxidants, five were berries. Considering typical portion sizes, the authors found that several berries (blackberries, strawberries, cranberries, raspberries and blueberries) and related products (e.g., juice from grape berries) were high on the list of foods high in antioxidants. According to Jakobek et al. [5], Jabłonska-Rys et al. [6] and Tarko et al. [7], elderberry fruits also ranked high in antioxidant activity compared to other commonly used fruits and berries such as blackberries, strawberries, red raspberries, sweet and sour cherries, red and black currants, bilberries, apples, goji berries and cranberries. In addition, juices from some previously mentioned berries had lower antioxidant activity than elderberry fruit juice [8,9,10,11,12].
Elderberries (genus Sambucus) grow as small trees, shrubs or herbs and are widely distributed throughout the world, especially in temperate and subtropical parts of the northern hemisphere [13]. The genus is morphologically complex and characterised by great variability between and within the species. After the last available revision by Bolli [14], the number of recognised species was reduced from 30 to 9. The most widespread species in Europe, European black elderberry (Sambucus nigra subsp. nigra), was recognised as separate species with five subspecies, including the most widespread species in the United States, the American black elderberry (S. nigra subsp. canadensis). According to the available literature, the inflorescences and fruits of these two subspecies have been studied most frequently. In general, the chemical composition of elderberries varies among species and depends on environmental conditions and maturity stage [15,16,17,18,19,20,21]. However, they are known for their high content of some organic compounds (e.g., sugars, organic acids, ascorbic acid, phenolic compounds) [6,22,23,24,25,26,27,28,29,30,31,32,33] that could improve the nutritional value and organoleptic properties of the food. According to data from SPINScan Natural and Information Resources Inc., published by Cavaliere et al. [34], elderberry supplements ranked 8th (over $6.8 million) among the top 20 best-selling botanical dietary supplements in the United States. They increased nearly 50% in 2009 compared to 2008 in the health and natural food channels, including grocery stores, drugstores and mass market retailers, convenience stores, media and network sales and health professionals. Considering only grocery stores, drugstores and mass market retailers, the sales of elderberry supplements also remained relatively stable in 2009 compared to 2008. Increasing consumer awareness of their own health has sparked the interest in elderberry fruits, not only because of their favourable chemical composition but also due to their health benefits, including antibacterial, anti-inflammatory, antidiabetic, protective and antioxidant properties [35].
Various analytical assays are used to evaluate antioxidant activity (AOA). Those based on gas and liquid chromatography, electrochemistry (cyclic voltammetry, amperometry, biamperometry) and spectrometry (DPPH (2,2-diphenyl-1-picrylhydrazyl), ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), FRAP (ferric reducing antioxidant power), PFRAP (potassium ferricyanide reducing power), CUPRAC (cupric reducing antioxidant capacity), ORAC (oxygen radical absorption capacity), HORAC (hydroxyl radical antioxidant capacity), TRAP (total radical trapping antioxidant potential), fluorimetry) are well known [2]. Most spectroscopic techniques are based on colour change, some on measurement of fluorescein fluorescence loos, while others quench chemiluminescence or detect excitation of fluorescence. FRAP assay is based on the ability of antioxidants to reduce Fe3+ to the intensely blue coloured iron and TPTZ complex [Fe2+-(TPTZ)2]2+. On the other hand, ABTS radical scavenging test and DPPH radical scavenging test are characterised by discoloration in the presence of antioxidants. After the reaction of ammonium persulfate, ABTS is converted to its radical cation, ABTS•+, which turns from dark to light green colour when antioxidants are added. On the other hand, the DPPH radical turns from dark purple to yellowish in the presence of antioxidants. ORAC assay is based on the measurement of antioxidant scavenging activity against peroxyl radical induced by 2,2′-azobis(2-methylpropionamidine) dihydrochloride (AAPH) at 37 °C. The spectroscopic techniques mentioned above are generally not directly comparable. They are characterised by different reaction media, sensitivity and working mechanisms (electron transfer or hydrogen atom transfer). In addition, radicals have different antioxidant potentials when reacting with phenolic compounds, they may dissolve differently, and they may have different affinities for other compounds present in the samples. The methods also differ in their endpoint, quantification method and the possibility of measuring both lipophilic and hydrophilic antioxidants [36,37]. However, according to many authors [38,39,40,41], ABTS, FRAP, DPPH and ORAC assays show similar trends and are significantly correlated with each other.
The AOA in fruit extracts of the most commonly used Sambucus species, S. nigra subsp. nigra [22,25,26] and S. nigra subsp. canadensis [29,30] has been studied previously. However, little is known about the antioxidant activity of maturing elderberry fruits [15,16] and even less about the antioxidant activity of fruits of elderberry interspecific hybrids [42]. Therefore, the purpose of the present study was to compare different assays (ABTS, DPPH, FRAP and ORAC) for estimating antioxidant activities in the extracts of maturing fruits of elderberry interspecific hybrids. The aim of this study was also to determine the superior genotypes in AOA and to investigate whether the most promising genetic combinations in terms of AOA, total phenolic and ascorbic content could be predicted in advance.

2. Materials and Methods

2.1. Plant Material

Twenty elderberry bushes from the Plant Gene Bank of the University of Maribor, Faculty of Agriculture and Life Sciences in Hoče near Maribor, Slovenia, were included in the present study. The plant material included the species Sambucus nigra (NI) and fourteen elderberry interspecific hybrids involving S. javanica (JA), S. nigra (NI), S. nigra ‘Black Beauty’ (BB), S. cerulea (CER) and S. racemosa (RAC). Some of them were represented by several bushes. The names S. nigra, S. cerulea and S. racemosa correspond to the names S. nigra subsp. nigra, S. nigra subsp. cerulea and S. racemosa subsp. racemosa respectively, according to the revised classification of Bolli [14]. S. nigra subsp. nigra also includes the cultivated variety named ‘Black Beauty’ (BB) (characterised by purple leaves and inflorescences). S. racemosa is represented by subsp. racemosa in the narrow sense (a local, wildly grown genotype (RAC), S. racemosa subsp. racemosa var. miquelii (MIQ), S. racemosa subsp. tigranii (TIG) and the taxon coreana (KOR) belonging to S. racemosa subsp. kamtschatica. C1 means the first clonal generation (plants developed by cuttings taken on the plant originated from seed).
Elderberries were collected in 2017 (between 7 September and 15 October) from three- to four-year-old bushes containing berries at different stages of maturity. The maturity stages were defined according to the fruit exterior colour and the percentage of fully coloured berries on the infructescence. Infructescences were classified into five groups (Figure 1): UR (containing unripe, green coloured berries that reached their final size); EM (containing berries at early stage of maturity—the exterior of approximately 30% of the berries exhibited the final (in most cases dark purple) colour); AM (containing almost mature berries—the exterior of approximately 70% of berries exhibited the final colour); FM (containing fully mature berries—the exterior of all berries exhibited the final colour (optimal maturity for harvest)); OM (containing overmature berries—the majority of berries were shrivelled due to the loss of moisture).
Immediately after harvest, infructescences were frozen in liquid nitrogen and the fruit stalks were removed. Approximately 100 g of berries were freeze-dried using Christ Alpha 1–2 LD (Vacuumbrand GMBH, Germany) freeze dryer, crushed into a fine powder, vacuum packed and stored at −80 °C until analysis.

2.2. Chemicals and Reagents

The following chemicals (Sigma-Aldrich, St. Louis, Missouri, USA) were used for antioxidant activity assays: 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), potassium persulfate (K2O8S2), 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,4,6-Tris(2-pyridyl)-s-triazine (TPTZ), iron(III) chloride hexahydrate (FeCl3·6H2O), sodium acetate (CH3COONa·3H2O), acetic acid, sodium fluorescein salt, 2,2′-azobis(2-amindinopropane) dihydrochloride (AAPH), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox®), Na2HPO4, NaH2PO4, hydrochloric acid and methanol.

2.3. Extraction

The extraction procedure was performed as described by Ochmian et al. [24], with slight modifications. To 0.40 g of sample, 4 mL of an extraction solution consisting of methanol: water: acetic acid (80:20:0.1, v/v/v) was added. The suspension was sonicated for 15 min and centrifuged at 7500 rpm. The extraction procedure was repeated twice, and the combined supernatants were diluted in a 10 mL volumetric flask.

2.4. Determination of Antioxidant Activity

The ABTS assay was performed according to the method of Mikulic-Petkovsek et al. [42] with slight modifications. First, 1 mL of diluted ABTS•+ solution was added to 50 µL of diluted elderberry extract. The absorbance was measured after 6 min of incubation in the dark at room temperature at 734 nm. The results of the assay were expressed in µmol Trolox equvivalents per g of dry weight (µmol TE/g DW).
DPPH radical scavenging activity was determined as described by Duymus et al. [43] with slight modifications. Diluted elderberry extracts (100 µL) were reacted with 3.9 mL of freshly prepared methanol-DPPH solution. The mixture was incubated in the dark at room temperature for 30 min. After the incubation period, the absorbance at 517 nm was recorded. The inhibition percentage (IP) was calculated using the equation: IP = [(ADPPH − Asample)/(ADPPH)] × 100. According to IP, the antioxidant activity of each sample was calculated and expressed as µmol TE/g DW.
The FRAP assay was performed according to Granato et al. [8] with slight modifications. Three solutions (300 mM acetate buffer, 10 mM TPZT in 40 mM HCl and 20 mM ferric chloride hexahydrate) were prepared and combined (10:1:1, v/v/v) to prepare the FRAP reagent prior to analysis. FRAP reagent was incubated at 37 °C for 10 min and added to 100 µL of each sample extract. After 30 min of incubation at 37 °C in the dark, absorbance was measured at 593 nm. The results were expressed as µmol TE/g DW.
ORAC was performed according to Huang et al. [44] with slight modifications. A stock solution of fluorescein (FLss) (1.33 × 10−3 mM) was prepared in a 75 mM phosphate buffer and stored in the dark at 5 °C. Before analysis, FLss was diluted with phosphate buffer to 8.16 × 10−5 mM fluorescein working solution (FLws). The AAPH solution (153 mM) was prepared fresh daily in 75 mM phosphate buffer and stored in the dark in an ice bath. Before analysis, AAPH was warmed at 37 °C. When using the plates, the outer wells were not used for experimental determinations and were filled with 250 µL of water. The inner (experimental) wells were filled with standards (25 µL of Trolox dilution), samples (25 µL), blanks (25 µL of phosphate buffer), and controls (50 µL of phosphate buffer). To all experimental wells, 150 µL of FLws was added. The plate was then placed in the TECAN INFINITE M1000 PRO microplate reader and incubated for 10 min at 37 °C. After incubation, 25 µL of pre-warmed AAPH was added to all wells to initiate the reaction. Fluorescence was then monitored kinetically, with data recorded every minute for a period of 90 min. ORAC values were calculated according to Cao et al. [45] The results were expressed as µmol TE/g DW.

2.5. Determination of Total Phenolic Content and Total Ascorbic Acid Content

In maturing fruits of elderberry interspecific hybrids, the total phenolic content and total ascorbic content were determined as described in the work of Imenšek et al. in press [46]. In the present study, the obtained results were used only for the calculations of correlations.

2.6. Statistical Analysis

Statistical analysis was conducted using the software package R (Rx64 3.2.5) and IBM SPSS Statistics 25. Group means and standard errors of means were calculated for all numerical variables. To determine differences in AOA determined by different assays among five maturity stages of elderberries, we applied a linear mixed effects model, using the lmer function in the lme4 package [47]. “Maturity stage” was defined as a fixed factor, “plant-ID” as a random factor and time was considered as a covariate. Multiple comparisons were tested using Tukey’s test in the emmeans package [48]. Correlations between observed variables were estimated using Spearman’s rank correlation coefficient. Ward’s method of hierarchical clustering was used to group genotypes into homogeneous subgroups for similarities of the quantitative variables.

3. Results and Discussion

3.1. Antioxidant Activity during Elderberry Maturation Process

To determine the AOA of elderberry fruits at five maturity stages, ABTS, DPPH, FRAP and ORAC assays were applied, and the results are shown in Figure 1. For all assays, AOA increased during maturation of elderberry fruit. The lowest average values at all maturity stages were obtained with the DPPH assay (from 38.8 ± 5.8 in the extracts of unripe berries to 109 ± 14 µmol TE/g DW in overmature berries), followed by ABTS (from 57.4 ± 8.8 to 137 ± 11 µmol TE/g DW) and FRAP values (from 99.1 ± 8.8 to 221 ± 24 µmol TE/g DW). The highest values were obtained with the ORAC assay (from 279 ± 18 to 588 ± 48 µmol TE/g DW). For each assay used, unripe green (UR) elderberries and those at early stage of maturity (EM) were characterised by significantly lower AOA compared to berries at later maturity stages. Almost mature berries (AM) showed significantly lower AOA compared to fully mature (FM) berries for each assay, and overmature berries (OM) were characterised by significantly higher AOA compared to fully mature (FM) berries according to DPPH, FRAP and ORAC assays, while ABTS assay showed no differences. A similar increasing pattern of AOA during maturation was previously reported for cherry fruit [49], mandarin melon berries [50], ginseng berries [51], black and red currants [52] and blackberries [53,54]. The authors explained the increase in AOA during fruit maturation by the concomitant increase in anthocyanins and other phenolic compounds that contribute strongly to AOA [55]. Elderberries are known for their high content of phenolic compounds, especially anthocyanins [56,57]. Since these compounds showed an increasing pattern during the maturation process of elderberries [15,16,17,19,46], a similar pattern could be explained for AOA.
Comparing the AOA of fully mature elderberries with some previous reports on elderberries, our results, when expressed on fresh weight (22.6 µmol TE/g for ABTS, 16.6 µmol TE/g for DPPH, 36.9 µmol TE/g for FRAP and 100 µmol TE/g for ORAC) are in agreement with those reported by Mikulic-Petkovsek et al. [42], Jabłonska-Rys et al. [6], Jakobek et al. [5] for the ABTS assay, Tarko et al. [7] for the DPPH assay, Akbulut et al. [22], Perkins-Veazie et al. [30] for the FRAP assay and Wu et al. [58] for the ORAC assay. The conclusions of the authors were similar, describing elderberries as a raw material with relatively high AOA that could be suitable for use in dietary supplements and the pharmaceutical industry.
The results of the present work showed that the AOA of elderberries was found at each maturity stage in ascending order of DPPH, ABTS, FRAP and ORAC values. The same results have been reported for the AOA of kiwifruit [38] and some Mediterranean edible plants [59]. Our results are also in partial agreement with those of Jimenez et al. [60] who reported higher ORAC values compared to ABTS, DPPH and FRAP values of buriti, but lower FRAP values compared to DPPH and ABTS values. Similar findings were reported by Candido et al. [39] for some medicinal plants. Regarding elderberries, our results are in agreement with those reported by Ozgen et al. [29] who found that FRAP values of fully mature elderberries were proportionally higher than DPPH assay values. The AOA of the samples depends not only on the composition of the extract but also on the conditions of the assay used [61]. DPPH, ABTS and FRAP are based on SET reaction mechanisms (Single Electron Transfer), which could help explain the similarity of their results. However, each method only provides an estimate of AOA based on their reaction reagents and conditions [41]. The redox potential of ABTS•+ is similar to that of Fe (III)-TPTZ, so similar compounds react in the ABTS and FRAP assays. On the other hand, their reaction pH is different (3.6 for FRAP and neutral for ABTS). Since lower pH causes the increase of the redox potential and facilitates electron transfer, FRAP assay values could be higher than ABTS assay values, although ABTS can determine the AOA of hydrophilic and lipophilic antioxidants [62]. Differences between assays may also occur due to different times for radical generation and reaction competition. ABTS•+ radicals require up to 16 h to be generated, whereas DPPH• and Fe (III)-TPTZ can be generated just prior to assay. The reaction of the ABTS assay is complete in 4–6 min, while FRAP and DPPH assay usually require a longer time (30 min) [36]. The DPPH assay might have a lower yield compared to the ABTS assay, probably due to the slower reaction of DPPH• with most antioxidants and lower sensitivity [63]. Although both the ABTS and ORAC assays allow the measurement of AOA of hydrophilic and lipophilic fractions, the ORAC assay values were expected to be the highest because this assay is based on the HAT (Hydrogen Atom Transfer) reaction mechanism, which is pH and solvent independent [36] and is considered to be more sensitive and accurate than other assays studied [39]. Even though DPPH, ABTS, FRAP and ORAC assays have different reaction mechanisms and do not necessarily measure the same activity [36], they clearly showed that the examined fruits of elderberry interspecific hybrids possess variable but considerable antioxidant activities.

3.2. Correlations of AOA Based on Different Assays with Total Phenolic (TP) Content and Total Ascorbic Acid (TAA) Content

Correlations between DPPH, ABTS, FRAP and ORAC assays were calculated for unripe (UR) and fully mature (FM) elderberries to determine if the assays used provide comparable results for AOA in elderberry fruits. Correlations between AOA, the total phenolic (TP) content and total ascorbic acid (TAA) content were also reported for both maturity stages. The results showed that for UR berries the correlations between DPPH, ABTS, FRAP and ORAC were significant and positive (Table 1). On the other hand, only AOA determined by FRAP assay was significantly correlated with TP content in the extracts of UR berries (correlation coefficient 0.734), and AOA determined by ABTS assay was significantly correlated with TAA content. The results were slightly different for FM elderberries (Table 2). We also found significant and positive correlations between all assays, but the correlation coefficients were generally higher than those for UR berries. For FM berries, the results also showed that AOA determined by DPPH, ABTS, FRAP and ORAC assays were significantly correlated with TP content (correlation coefficients 0.544, 0.641, 0.635 and 0.626, respectively), while no significant correlations were found between TAA content and AOA determined by all assays. These results are in agreement with some other reports. Namely, DPPH, ABTS, FRAP and ORAC assays were used for AOA determination in the extracts of buriti fruits [39], sorghum [64], some medicinal plants [60] and other plant extracts [61]. Studies revealed that all the assays used showed a similar pattern and were significantly correlated with each other. In most cases, the authors also agreed that the correlations between assays based on similar mechanisms were stronger than those between assays based on different mechanisms.
For both maturity stages, UR and FM berries, the difference in the results obtained regarding the correlations between AOA, TP and TAA could be partly explained by the difference in chemical composition between these maturity stages. Indeed, the UR berries are characterised by a higher TAA [46] and a lower TP content (mainly due to the absence of anthocyanins) [15,16] than the FM berries. Although phenolic compounds and total ascorbic acid contribute more to antioxidant activity [65], the contribution of phenolic compounds, especially flavonoids, to the total antioxidant activity of fruits is significantly higher than that of total ascorbic acid, which contributes less than 15% [55,65]. Therefore, a high TAA content (such as in UR berries) does not necessarily reflect a high AOA. On the other hand, high/low TP content also does not always result in high/low AOA values, as possible interactions between different types of phenolic compounds and other compounds in fruit extract may cause the incomplete reactions of antioxidants with radicals [66]. However, this was not the case in FM berries, where similar to some other authors [39,64,65,66], significant correlations between AOA assays and TP content were observed. Moreover, our results regarding FM berries are in agreement with those reported by Ma et al. [41], who found significant and positive correlations between TP content and DPPH, ABTS, FRAP and ORAC antioxidant abilities but no significant correlations between AOA and TAA content in mango fruits. Similar results (significant correlation between AOA and TP and no significant correlation between AOA and TAA) were also obtained for some common fruits regarding FRAP assay [67] and some tropical fruits regarding DPPH and ORAC assay [66].

3.3. Clustering of Elderberry Interspecific Hybrids

All analysed genotypes were clustered into four groups (Figure 2). The characteristics of each group (average AOA, TP and TAA content of fully mature berries) are shown in Table 3. The genotypes of groups 4 and 3 were characterised with the lowest AOA and TP contents compared to the other groups. Moreover, JA × (JA × MIQ), (JA × NI) × RAC, (JA × NI) × CER, JA × TIG, JA × KOR and ((JA × NI) × CER) × MIQ from group 4 showed the lowest average AOA according to FRAP and ABTS assays and lowest TP content among the groups. Similar but slightly better results were obtained for the genotypes JA × CER No3 C1, (JA × CER) × (JA × NI) and S. nigra (NI) from group 3, which were found as poorest in AOA according to DPPH and ORAC assays but dominated in TAA content among all groups. More desirable characteristics compared to the previously mentioned genotypes in terms of AOA and TP were obtained for JA × NI, JA × (JA× NI), (JA × NI) × NI and ((JA × NI) × MIQ) × (CER × NI) from group 1. These genotypes had higher AOA according to DPPH, FRAP and ORAC assay and higher average TP content. However, the genotypes (JA × NI) × BB and (JA × NI) × MIQ from group 2 were found to be superior in terms of AOA as per DPPH, FRAP and ORAC assay and TP content but showed a lower average TAA content than the genotypes from group 3 and 4. Nowadays, since there is a great demand for raw materials with high AOA and TP content, the genotypes from groups 1 and 2 could be recommended for further inclusion in breeding processes or use in the food industry. Due to the high TAA content, the genotypes from group 3 may also be useful in breeding programme.
To investigate whether genotypes with favourable chemical composition (AO activity, TP content and TAA content) of fully mature (FM) berries could be predicted in advance based of the known chemical composition of unripe (UR) berries, the correlation coefficients for above mentioned parameters between unripe (UR) and fully mature (FM) were calculated (Table 4). The majority of correlations were not significant, the only exception was TAA (correlation coefficient 0.827). In conclusion, the genotype with higher AO activity and TP content in unripe berries (UR) was, in general, not characterised with higher AO activity and TP content in fully mature (FM) berries, while the opposite results were found for TAA content.

4. Conclusions

DPPH, ABTS, FRAP and ORAC assays showed different AOA levels but a similar increasing pattern of antioxidant activity during elderberry fruit maturation. Moreover, significant and positive correlations were observed between all the methods used for unripe green (UR) and fully mature (FM) stage of elderberry infructescences. The results also showed that total phenolic content strongly contributed to antioxidant activity (positive and significant correlations), while the contribution of total ascorbic acid was lower (no significant correlations). Based on the results, we can conclude that the antioxidant activity of elderberries could be reliably measured using any of the four methods, however, to obtain more comprehensive information on antioxidant properties, the use of more than one method could be recommended.
All the assays used showed significant differences in antioxidant activity between most important stages when planning the harvest time (almost mature (AM), fully mature (FM) and overmature (OM) berries). Considering these results, berries could not be harvested before reaching full maturity (e.g., at the early stage (EM) of maturity). Even though overmature berries showed the highest antioxidant activity, harvesting at this maturity stage could not be recommended due to the overall reduction in yield and quality.
Among the genotypes analysed, berries of (JA × NI) × BB and (JA × NI) × MIQ were found to be superior in antioxidant activity and total phenolic content at full maturity. The results also showed that the most promising genetic combinations (genotypes) regarding antioxidant activity and total phenolic content at full maturity could not be predicted in advance based on the known chemical composition of berries at the beginning of ripening (unripe stage). On the other hand, the results showed that interspecific hybrids with higher total ascorbic acid content in unripe berries were, in general, characterised by a higher total ascorbic acid content in fully mature berries.

Author Contributions

N.I.: performance of the analyses, editing the data, and writing the original manuscript. J.K.: design of the experiment, critical reading and editing of the manuscript. T.K.Š.: statistical processing of the data and writing the part of the manuscript related to the statistical analysis. A.I.: author of the interspecific hybrids, maintenance of the plant material, design of the experiment, critical evaluation and editing of the manuscript. All authors contributed substantially to the final version of the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This research was financially supported by the Ministry of Education, Science and Sport of the Republic of Slovenia, the Programme Research for Improvement of Safe Food and Health P1-0164 (C).

Data Availability Statement

Data available in a publicly accessible repository.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Lobo, V.; Patil, A.; Phatak, A.; Chandra, N. Free radicals, antioxidants and functional foods: Impact on human health. Pharmacogn. Rev. 2010, 4, 118–126. [Google Scholar] [CrossRef] [Green Version]
  2. Pisoschi, A.M.; Negulescu, G.P. Methods for total antioxidant activity determination: A review. Biochem. Anal. Biochem. 2011, 1, 106. [Google Scholar] [CrossRef] [Green Version]
  3. Frankel, E.N. Natural and biological antioxidants in foods and biological systems. Their mechanism of action, applications and implications. Lipid Technol. 1995, 7, 77–80. [Google Scholar]
  4. Halvorsen, B.L.; Carlsen, M.H.; Phillips, K.M.; Bøhn, S.K.; Holte, K.; Jacobs, D.R., Jr.; Blomhoff, R. Content of redox-active compounds (ie, antioxidants) in foods consumed in the United States. Am. J. Clin. Nutr. 2006, 84, 95–135. [Google Scholar] [CrossRef] [Green Version]
  5. Jakobek, L.; Šeruga, M.; Novak, I.; Medvidović-Kosanović, M. Flavonols, phenolic acids and antioxidant activity of some red fruits. Dtsch. Lebensm.-Rundsch. 2007, 103, 369–377. [Google Scholar]
  6. Jabłońska-Ryś, E.; Zalewska-Korona, M.; Kalbarczyk, J. Antioxidant capacity, ascorbic acid and phenolics content in wild edible fruits. J. Fruit Ornamental Plant Res. 2009, 17, 115–120. [Google Scholar]
  7. Tarko, T.; Duda-Chodak, A.; Sroka, P.; Satora, P.; Semik-Szczurak, D.; Wajda, L. Diversity and bioavailability of fruit polyphenols. J. Food Nutr. Res. 2017, 56, 167–178. [Google Scholar]
  8. Granato, D.; Karnopp, A.R.; van Ruth, S.M. Characterization and comparison of phenolic composition, antioxidant capacity and instrumental taste profile of juices from different botanical origins. J. Sci. Food Agric. 2014, 95, 1997–2006. [Google Scholar] [CrossRef]
  9. Bermúdez-Soto, M.J.; Tomás-Barberán, F.A. Evaluation of commercial red fruit juice concentrates as ingredients for antioxidant functional juices. Eur. Food Res. Technol. 2004, 219, 133–141. [Google Scholar] [CrossRef]
  10. Nowak, D.; Goslinski, M.; Szwengiel, A. Multidimensional comparative analysis of phenolic compounds in organic juices with high antioxidant capacity. J. Sci. Food Agric. 2017, 97, 2657–2663. [Google Scholar] [CrossRef] [PubMed]
  11. Jakobek, L.; Šeruga, M.; Medvidović-Kosanović, M.; Novak, I. Anthocyanin content and antioxidant activity of various red fruit juices. Dtsch. Lebensm.-Rundsch. 2007, 103, 58–64. [Google Scholar]
  12. Slatnar, A.; Jakopic, J.; Stampar, F.; Veberic, R.; Jamnik, P. The effect of bioactive compounds on in vitro and in vivo antioxidant activity of different berry juices. PLoS ONE 2012, 7, e47880. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  13. Atkinson, M.D.; Atkinson, E. Sambucus nigra L. J. Ecol. 2002, 90, 895–923. [Google Scholar] [CrossRef]
  14. Bolli, R. Revision of the Genus Sambucus; J. Cramer: Berlin, Germany, 1994. [Google Scholar]
  15. Mathieu, F.; Charlebois, D.; Charles, M.T.; Chevrier, N. Biochemical changes in American elder (Sambucus canadensis) fruits during development. In Proceedings of the Acta Horticulturae, I International Symposium on Elderberry, Columbia, MO, USA, 9–14 June 2013; Thomas, A.L., Ed.; International Society for Horticultural Science: Korbeek-Lo, Belgium, 2015; pp. 61–72. [Google Scholar]
  16. Koss-Mikolajczyk, I.; Lewandowska, A.; Pilipczuk, T.; Kusznierewicz, B.; Bartoszek, A. Composition of bioactive secondary metabolites and mutagenicity of Sambucus nigra L. Fruit at different stages of ripeness. Acta Aliment. 2016, 45, 442–451. [Google Scholar] [CrossRef] [Green Version]
  17. Kaack, K.; Frette, X.C.; Christensen, L.P.; Landbo, A.K.; Meyer, A.S. Selection of elderberry (Sambucus nigra L.) genotypes best suited for the preparation of juice. Eur. Food Res. Technol. 2008, 226, 843–855. [Google Scholar] [CrossRef]
  18. Salvador, A.C.; Rudnitskaya, A.; Silvestre, A.J.D.; Rocha, S.M. Metabolomic-based strategy for fingerprinting of Sambucus nigra L. berry volatile terpenoids and norisoprenoids: Influence of ripening and cultivar. J. Agric. Food Chem. 2016, 64, 5428–5438. [Google Scholar] [CrossRef]
  19. Szaloki-Dorko, L.; Steger-Mate, M.; Abranko, L. Evaluation of colouring ability of main European elderberry (Sambucus nigra L.) varieties as potential resources of natural food colourants. Int. J. Food Sci. Technol. 2015, 50, 1317–1323. [Google Scholar] [CrossRef]
  20. Salvador, A.C.; Rocha, S.M.; Silvestre, A.J.D. Lipophilic phytochemicals from elderberries (Sambucus nigra L.): Influence of ripening, cultivar and season. Ind. Crops Prod. 2015, 71, 15–23. [Google Scholar] [CrossRef]
  21. Zahmanov, G.; Alipieva, K.; Simova, S.; Georgiev, M.I. Metabolic differentiations of dwarf elder by NMR-based metabolomics. Phytochem. Lett. 2015, 11, 404–409. [Google Scholar] [CrossRef]
  22. Akbulut, M.; Ercisli, S.; Tosun, M. Physico-chemical characteristics of some wild grown European elderberry (Sambucus nigra L.) genotypes. Pharmacogn. Mag. 2009, 5, 320–323. [Google Scholar]
  23. Veberic, R.; Jakopic, J.; Stampar, F.; Schmitzer, V. European elderberry (Sambucus nigra L.) rich in sugars, organic acids, anthocyanins and selected polyphenols. Food Chem. 2009, 114, 511–515. [Google Scholar] [CrossRef]
  24. Ochmian, I.; Oszmianski, J.; Skupien, K. Chemical composition, phenolics, and firmness of small black fruits. J. Appl. Bot. Food Qual. 2009, 83, 64–69. [Google Scholar]
  25. Dawidowicz, A.L.; Wianowska, D.; Baraniak, B. The antioxidant properties of alcoholic extracts from Sambucus nigra L. (antioxidant properties of extracts). LWT Food Sci. Technol. 2006, 39, 308–315. [Google Scholar] [CrossRef]
  26. Anton, A.M.; Pintea, A.M.; Rugina, D.O.; Sconta, Z.M.; Hanganu, D.; Vlase, L.; Benedec, D. Preliminary studies on the chemical characterization and antioxidant capacity of polyphenols from Sambucus sp. Dig. J. Nanomater. Biostructures 2013, 8, 973–980. [Google Scholar]
  27. Silva, P.; Ferreira, S.; Nunes, F.M. Elderberry (Sambucus nigra L.) by-products a source of anthocyanins and antioxidant polyphenols. Ind. Crops Prod. 2017, 95, 227–234. [Google Scholar] [CrossRef]
  28. Lee, J.; Finn, C.E. Anthocyanins and other polyphenolics in American elderberry (Sambuclus canadensis) and European elderberry (S. nigra) cultivars. J. Sci. Food Agric. 2007, 87, 2665–2675. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  29. Ozgen, M.; Scheerens, J.C.; Reese, R.N.; Miller, R.A. Total phenolic, anthocyanin contents and antioxidant capacity of selected elderberry (Sambucus canadensis L.) accessions. Pharmacogn. Mag. 2010, 6, 198–203. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  30. Perkins-Veazie, P.; Thomas, A.L.; Byers, P.L.; Finn, C.E. Fruit composition of elderberry (Sambucus spp.) genotypes grown in oregon and missouri, USA. In Proceedings of the Acta Horticulturae, I International Symposium on Elderberry, Columbia, MO, USA, 9–14 June 2015; Thomas, A.L., Ed.; International Society for Horticultural Science: Korbeek-Lo, Belgium, 2015; pp. 219–224. [Google Scholar]
  31. Mudge, E.; Applequist, W.L.; Finley, J.; Lister, P.; Townesmith, A.K.; Walker, K.M.; Brown, P.N. Variation of select flavonols and chlorogenic acid content of elderberry collected throughout the Eastern United States. J. Food Compos. Anal. 2016, 47, 52–59. [Google Scholar] [CrossRef] [Green Version]
  32. Dudonne, S.; Dube, P.; Anhe, F.F.; Pilon, G.; Marette, A.; Lemire, M.; Harris, C.; Dewailly, E.; Desjardins, Y. Comprehensive analysis of phenolic compounds and abscisic acid profiles of twelve native Canadian berries. J. Food Compos. Anal. 2015, 44, 214–224. [Google Scholar] [CrossRef]
  33. Rimpapa, Z.; Toromanovic, J.; Tahirovic, I.; Šapčanin, A.; Sofic, E. Total content of phenols and anthocyanins in edible fruits from Bosnia. Bosnian J. Basic Med. Sci. 2007, 7, 119–122. [Google Scholar] [CrossRef] [Green Version]
  34. Cavaliere, C.; Rea, P.; Lynch, M.E.; Blumenthal, M. Herbal supplement sales rise in all channels in 2009. HerbalGram 2010, 86, 62–65. [Google Scholar]
  35. Milena, V.; Tatjana, M.; Gökhan, Z.; Ivana, B.; Aleksandra, C.; Mohammad, M.F.; Marija, R. Advantages of contemporary extraction techniques for the extraction of bioactive constituents from black elderberry (Sambucus nigra L.) flowers. Ind. Crops Prod. 2019, 136, 93–101. [Google Scholar] [CrossRef]
  36. Prior, R.L.; Wu, X.; Schaich, K. Standardized methods for the determination of antioxidant capacity and phenolics in foods and dietary supplements. J. Agric. Food Chem. 2005, 53, 4290–4302. [Google Scholar] [CrossRef] [PubMed]
  37. Huang, D.; Ou, B.; Prior, R.L. The chemistry behind antioxidant capacity assays. J. Agric. Food Chem. 2005, 53, 1841–1856. [Google Scholar] [CrossRef]
  38. Wang, Y.; Zhao, C.-L.; Li, J.-Y.; Liang, Y.-J.; Yang, R.-Q.; Liu, J.-Y.; Ma, Z.; Wu, L. Evaluation of biochemical components and antioxidant capacity of different kiwifruit (Actinidia spp.) genotypes grown in China. Biotechnol. Biotechnol. Equip. 2018, 32, 558–565. [Google Scholar] [CrossRef] [Green Version]
  39. Cândido, T.L.N.; Silva, M.R.; Agostini-Costa, T.S. Bioactive compounds and antioxidant capacity of buriti (Mauritia flexuosa L.f.) from the Cerrado and Amazon biomes. Food Chem. 2015, 177, 313–319. [Google Scholar] [CrossRef] [PubMed]
  40. Du, G.; Li, M.; Ma, F.; Liang, D. Antioxidant capacity and the relationship with polyphenol and vitamin C in Actinidia fruits. Food Chem. 2009, 113, 557–562. [Google Scholar] [CrossRef]
  41. Ma, X.; Wu, H.; Liu, L.; Yao, Q.; Wang, S.; Zhan, R.; Xing, S.; Zhou, Y. Polyphenolic compounds and antioxidant properties in mango fruits. Sci. Hortic. 2011, 129, 102–107. [Google Scholar] [CrossRef]
  42. Mikulic-Petkovsek, M.; Ivancic, A.; Schmitzer, V.; Veberic, R.; Stampar, F. Comparison of major taste compounds and antioxidative properties of fruits and flowers of different Sambucus species and interspecific hybrids. Food Chem. 2016, 200, 134–140. [Google Scholar] [CrossRef]
  43. Duymus, H.G.; Goger, F.; Baser, K.H.C. In vitro antioxidant properties and anthocyanin compositions of elderberry extracts. Food Chem. 2014, 155, 112–119. [Google Scholar] [CrossRef]
  44. Huang, D.; Ou, B.; Hampsch-Woodill, M.; Flanagan, J.A.; Prior, R.L. High-throughput assay of oxygen radical absorbance capacity (ORAC) using a multichannel liquid handling system coupled with a microplate fluorescence reader in 96-well format. J. Agric. Food Chem. 2002, 50, 4437–4444. [Google Scholar] [CrossRef]
  45. Cao, G.; Alessio, H.M.; Cutler, R.G. Oxygen-radical absorbance capacity assay for antioxidants. Free Radical Biol. Med. 1993, 14, 303–311. [Google Scholar] [CrossRef] [Green Version]
  46. Imenšek, N.; Ivančič, A.; Kraner-Šumenjak, T.; Islamčević-Razboršek, M.; Kristl, J. The effect of maturation on chemical composition and harvest of fruits of diverse elderberry interspecific hybrids. Eur. J. Hortic.Sci. 2021, in press. [Google Scholar]
  47. Bates, D.; Mächler, M.; Bolker, B.; Walker, S. Fitting linear mixed-effects models using lme4. J. Stat. Softw. 2014, 67, 1–48. [Google Scholar] [CrossRef]
  48. Lenth, R. Emmeans: Estimated Marginal Means, Aka Least-Squares Means. R Package Version 1.4.5. 2020. Available online: https://CRAN.R-project.org/package=emmeans (accessed on 30 April 2021).
  49. Mahmood, T.; Anwar, F.; Bhatti, I.A.; Iqbal, T. Effect of maturity on proximate composition, phenolics and antioxidant attributes of cherry fruit. Pak. J. Bot 2013, 45, 909–914. [Google Scholar]
  50. Shin, G.R.; Lee, S.; Lee, S.; Do, S.-G.; Shin, E.; Lee, C.H. Maturity stage-specific metabolite profiling of Cudrania tricuspidata and its correlation with antioxidant activity. Ind. Crops Prod. 2015, 70, 322–331. [Google Scholar] [CrossRef]
  51. Lee, M.Y.; Seo, H.S.; Singh, D.; Lee, S.J.; Lee, C.H. Unraveling dynamic metabolomes underlying different maturation stages of berries harvested from Panax ginseng. J. Ginseng Res. 2020, 44, 413–423. [Google Scholar] [CrossRef] [PubMed]
  52. Mikulic-Petkovsek, M.; Veberič, R.; Stampar, F.; Koron, D. Quality parameters of black and red currants during ripening. In Proceedings of the Acta Horticulture, III Balkan Symposium on Fruit Growing, Belgrade, Serbia, 16–18 September 2015; Milatović, D., Ed.; International Society for Horticultural Science: Korbeek-Lo, Belgium, 2016; pp. 651–656. [Google Scholar]
  53. Acosta-Montoya, Ó.; Vaillant, F.; Cozzano, S.; Mertz, C.; Pérez, A.M.; Castro, M.V. Phenolic content and antioxidant capacity of tropical highland blackberry (Rubus adenotrichus Schltdl.) during three edible maturity stages. Food Chem. 2010, 119, 1497–1501. [Google Scholar] [CrossRef]
  54. Josiane, K.R.; Glenise, B.V.; Rui Carlos, Z. Influence of the degree of maturation on the bioactive compounds in blackberry (Rubus spp.) cv. Tupy. Food Nutr. Sci. 2012, 3, 1453–1460. [Google Scholar]
  55. Wang, H.; Cao, G.; Prior, R.L. Total antioxidant capacity of fruits. J. Agric. Food Chem. 1996, 44, 701–705. [Google Scholar] [CrossRef]
  56. Csorba, V.; Magdolna, T.; Laszlo, A.M.; Kardos, L.; Kovacs, S. Cultivar and year effects on the chemical composition of elderberry (Sambucus nigra L.) fruits. Not. Bot. Horti Agrobot. Cluj-Napoca 2020, 48, 770–782. [Google Scholar] [CrossRef]
  57. Domínguez, R.; Zhang, L.; Rocchetti, G.; Lucini, L.; Pateiro, M.; Munekata, P.E.S.; Lorenzo, J.M. Elderberry (Sambucus nigra L.) as potential source of antioxidants. Characterization, optimization of extraction parameters and bioactive properties. Food Chem. 2020, 330, 127266. [Google Scholar] [CrossRef]
  58. Wu, X.; Gu, L.; Holden, J.; Haytowitz, D.B.; Gebhardt, S.E.; Beecher, G.; Prior, R.L. Development of a database for total antioxidant capacity in foods: A preliminary study. J. Food Compos. Anal. 2004, 17, 407–422. [Google Scholar] [CrossRef]
  59. Gonçalves, S.; Moreira, E.; Andrade, P.B.; Valentão, P.; Romano, A. Effect of in vitro gastrointestinal digestion on the total phenolic contents and antioxidant activity of wild Mediterranean edible plant extracts. Eur. Food Res. Technol. 2019, 245, 753–762. [Google Scholar] [CrossRef]
  60. Jiménez, N.; Carrillo-Hormaza, L.; Pujol, A.; Álzate, F.; Osorio, E.; Lara-Guzman, O. Antioxidant capacity and phenolic content of commonly used anti-inflammatory medicinal plants in Colombia. Ind. Crops Prod. 2015, 70, 272–279. [Google Scholar] [CrossRef]
  61. Dudonne, S.; Vitrac, X.; Coutiere, P.; Woillez, M.; Mérillon, J.-M. Comparative study of antioxidant properties and total phenolic content of 30 plant extracts of industrial interest using DPPH, ABTS, FRAP, SOD, and ORAC assays. J. Agric. Food Chem. 2009, 57, 1768–1774. [Google Scholar] [CrossRef] [PubMed]
  62. Cano, A.; Acosta, M.; Arnao, M.B. A method to measure antioxidant activity in organic media: Application to lipophilic vitamins. Redox Rep. 2000, 5, 365–370. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  63. Martysiak-Żurowska, D.; Wenta, W. A comparison of ABTS and DPPH methods for assessing the total antioxidant capacity of human milk. Acta Sci. Pol. Technol. Aliment. 2012, 11, 83–89. [Google Scholar] [PubMed]
  64. Awika, J.M.; Rooney, L.W.; Wu, X.; Prior, R.L.; Cisneros-Zevallos, L. Screening methods to measure antioxidant activity of sorghum (Sorghum bicolor) and sorghum products. J. Agric. Food Chem. 2003, 51, 6657–6662. [Google Scholar] [CrossRef]
  65. Lee, K.W.; Kim, Y.J.; Kim, D.-O.; Lee, H.J.; Lee, C.Y. Major phenolics in apple and their contribution to the total antioxidant capacity. J. Agric. Food Chem. 2003, 51, 6516–6520. [Google Scholar] [CrossRef] [PubMed]
  66. Mahattanatawee, K.; Manthey, J.A.; Luzio, G.; Talcott, S.T.; Goodner, K.; Baldwin, E.A. Total antioxidant activity and fiber content of select Florida-grown tropical fruits. J. Agric. Food Chem. 2006, 54, 7355–7363. [Google Scholar] [CrossRef] [PubMed]
  67. Guo, C.; Yang, J.; Wei, J.; Li, Y.; Xu, J.; Jiang, Y. Antioxidant activities of peel, pulp and seed fractions of common fruits as determined by FRAP assay. Nutr. Res. 2003, 23, 1719–1726. [Google Scholar] [CrossRef]
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Figure 1. Means and standard errors of means for antioxidant activity of maturing fruits of elderberry interspecific hybrids determined by DPPH (2,2-diphenyl-1-picrylhydrazyl), ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), FRAP (ferric reducing antioxidant power) and ORAC (oxygen radical absorption capacity) assays, respectively. Different letters (a, b, c, or d) above the bars of the same assay indicate significant differences between maturity stages (Tukey, p ≤ 0.05). Maturity stages of infructescences were defined as UR (containing unripe green coloured berries that reached final size); EM (containing berries at early stage of maturity—the exterior of approximately 30% of berries exhibited the final colour); AM (containing almost mature berries—the exterior of approximately 70% of berries exhibited the final colour); FM (containing fully mature berries); OM (containing overmature berries).
Figure 1. Means and standard errors of means for antioxidant activity of maturing fruits of elderberry interspecific hybrids determined by DPPH (2,2-diphenyl-1-picrylhydrazyl), ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), FRAP (ferric reducing antioxidant power) and ORAC (oxygen radical absorption capacity) assays, respectively. Different letters (a, b, c, or d) above the bars of the same assay indicate significant differences between maturity stages (Tukey, p ≤ 0.05). Maturity stages of infructescences were defined as UR (containing unripe green coloured berries that reached final size); EM (containing berries at early stage of maturity—the exterior of approximately 30% of berries exhibited the final colour); AM (containing almost mature berries—the exterior of approximately 70% of berries exhibited the final colour); FM (containing fully mature berries); OM (containing overmature berries).
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Figure 2. Dendrogram of fully mature berries of the 20 elderberry interspecific hybrids studied, obtained by Ward’s method. Abbreviations correspond to S. nigra subsp. nigra (NI), S. nigra subsp. nigra ‘Black Beauty’ (BB), S. nigra subsp. cerulea (CER), S. racemosa subsp. racemosa (RAC), S. racemosa subsp. coreana (KOR), S. racemosa subsp. tigranii (TIG), S. racemosa var. miquelii (MIQ) and S. javanica (JA).
Figure 2. Dendrogram of fully mature berries of the 20 elderberry interspecific hybrids studied, obtained by Ward’s method. Abbreviations correspond to S. nigra subsp. nigra (NI), S. nigra subsp. nigra ‘Black Beauty’ (BB), S. nigra subsp. cerulea (CER), S. racemosa subsp. racemosa (RAC), S. racemosa subsp. coreana (KOR), S. racemosa subsp. tigranii (TIG), S. racemosa var. miquelii (MIQ) and S. javanica (JA).
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Table
Table 1. Spearman correlation coefficients between antioxidant activity (AOA) assays, total phenolic (TP) content and total ascorbic acid (TAA) of unripe green (UR) fruits of elderberry interspecific hybrids.
Table 1. Spearman correlation coefficients between antioxidant activity (AOA) assays, total phenolic (TP) content and total ascorbic acid (TAA) of unripe green (UR) fruits of elderberry interspecific hybrids.
DPPHABTSFRAPORACTP
ABTS0.633 **
FRAP0.529 *0.698 **
ORAC0.524 *0.851 **0.576 **
TP0.0700.2590.734 **0.254
TAA0.4090.594 **0.3520.2860.183
* sig. < 0.05, ** sig. < 0.01.
Table
Table 2. Spearman correlation coefficients between antioxidant activity (AOA) assays, total phenolic (TP) content and total ascorbic acid (TAA) content of fully mature (FM) fruits of elderberry interspecific hybrids.
Table 2. Spearman correlation coefficients between antioxidant activity (AOA) assays, total phenolic (TP) content and total ascorbic acid (TAA) content of fully mature (FM) fruits of elderberry interspecific hybrids.
DPPHABTSFRAPORACTP
ABTS0.764 **
FRAP0.832 **0.885 **
ORAC0.621 **0.720 **0.868 **
TP0.544 *0.641 **0.635 **0.626 **
TAA0.3850.2140.3480.3770.023
* sig. < 0.05, ** sig. < 0.01.
Table
Table 3. Antioxidant activity of four groups of fully mature berries of elderberry interspecific hybrids evaluated by different assays, total phenolic (TP) content and total ascorbic acid (TAA).
Table 3. Antioxidant activity of four groups of fully mature berries of elderberry interspecific hybrids evaluated by different assays, total phenolic (TP) content and total ascorbic acid (TAA).
GroupDPPHFRAPORACABTSTPTAA
196 ± 14208 ± 25527 ± 60130 ± 141130 ± 81158 ± 59
2100 ± 16214 ± 33589 ± 89124 ±132801 ± 246170 ±45
366 ± 21172 ± 26450 ± 24106 ± 251762 ± 76205 ±75
473 ± 19156 ± 36463 ± 5998 ± 171361 ± 170183 ± 125
Values are expressed as mean ± std. deviation of means in µmol TE/g for DPPH, FRAP, ABTS and ORAC, and mg/100 g DW for total ascorbic acid (TAA) content and total phenolic (TP) content. Groups were determined by the Ward’s method and are defined in the dendrogram (Figure 2).
Table
Table 4. Spearman correlation coefficients for antioxidant activity assays, total phenolic (TP) content and total ascorbic acid (TAA) content between unripe (UR) and fully mature (FM) fruits of elderberry interspecific hybrids.
Table 4. Spearman correlation coefficients for antioxidant activity assays, total phenolic (TP) content and total ascorbic acid (TAA) content between unripe (UR) and fully mature (FM) fruits of elderberry interspecific hybrids.
URDPPHABTSFRAPORACTPTAA
FM
DPPH0.384
ABTS 0.300
FRAP 0.232
ORAC −0.155
TP 0.265
TAA 0.827 **
** sig. < 0.01.
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Imenšek, N.; Kristl, J.; Kraner Šumenjak, T.; Ivančič, A. Antioxidant Activity of Elderberry Fruits during Maturation. Agriculture 2021, 11, 555. https://doi.org/10.3390/agriculture11060555

AMA Style

Imenšek N, Kristl J, Kraner Šumenjak T, Ivančič A. Antioxidant Activity of Elderberry Fruits during Maturation. Agriculture. 2021; 11(6):555. https://doi.org/10.3390/agriculture11060555

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Imenšek, Nataša, Janja Kristl, Tadeja Kraner Šumenjak, and Anton Ivančič. 2021. "Antioxidant Activity of Elderberry Fruits during Maturation" Agriculture 11, no. 6: 555. https://doi.org/10.3390/agriculture11060555

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