Evaluation of Sambucus nigra L. Biopotential as an Unused Natural Resource

Abstract

An unbreakable relationship between plants, nutrition, and health has directed researchers to deeply investigate and characterize the biopotential and medicinal properties of traditional foods. The aim of this study is to analyze and compare the phytochemical composition and biological potential of plant extracts with the idea of defining the most potent extracts as a natural source of bioactive molecules and their application in different industries. We evaluated unused plant species Sambucus nigra L. for investigation of bioactivities as potential natural products. Extracts of fresh elderberry fruits were obtained by modern (microwave-assisted extraction (MAE), ultrasound-assisted extraction (UAE)) and traditional (maceration (MAC)) extraction techniques, using 50% ethanol (50% EtOH) and water (H₂O) of different polarities. In analyzed extracts, rutin and chlorogenic acid were dominant compounds in both 50% EtOH and H₂O extracts, while ursolic acid was identified in 50% EtOH extracts as a terpenic compound with notable concentration. Elderberry extracts were evaluated regarding antioxidant, neuroprotective, antityrosinase, and antidiabetic abilities: MAE extracts had the best overall activity, and in general, 50% EtOH extracts were more potent than water extracts. The correlation of the dominant compound—rutin with all biological activities, indicates the importance of its presence in elderberries. S. nigra fruits showed excellent biopotential and opened possibilities of creating new food products or remedies, which are not present on the market because elderberry extracts are an exceptional source of rutin, chlorogenic acid, and ursolic acid.

1. Introduction

The Sambucus genus (the Adoxaceae family) includes several berry species, and among them is the most interesting and most important black elderberry (Sambucus nigra L.). Black elderberry is a widespread shrub, which grows at higher altitudes on the banks of the rivers, as well as in the brighter forests [1]. For centuries, the species of this genus were wild-growing plants, but nowadays, the extraordinary potential of their fruits and flowers usage have induced plantation cultivating of elderberry across Europe [2]. Montenegrian mountain Ljubišnja, especially canyon of Ćehotina river, is a region rich in wild-grown elderberries. Their exploitation in this area is minor, although it is an unpolluted locality that offers good environmental conditions for the fine growth of elderberries. The elderberry fruits from this area have been used as a traditional remedy for the cold, flu or for the preparation of tea and jams [3], but they have never been applied in industry or subjected to scientific analysis.

Some studies have reported that different types of small fresh berry fruits (raspberry, blackberry, blueberry, and aronia) are extremely rich in biologically active substances [4], which can reduce a risk of several diseases, including cardiovascular, and neurodegenerative ailments, diabetes, and various types of cancers [5,6,7]. Elderberries are receiving much attention due to the high content of bioactive components: anthocyanins, flavonoids, phenolic acids, stilbenes, tannins, and carotenoids [1]. The content and chemical structure of these secondary metabolites are liable for their different biological abilities, such as antioxidant potential to inhibit enzymes involved in the pathogenesis of the different diseases, cytotoxicity, antimicrobial activity, etc. [8]. In order to facilitate their bioavailability and raise qualitative and quantitative composition of extracts, new techniques for isolation are developing extensively [9]. Modern extraction techniques, such as microwave-assisted (MAE) and ultrasound-assisted (UAE) extractions, are considered as an effective method for isolation of various bioactive compounds from plant cells, and they follow the basic principles: maximized extraction yield, customization for industry requirements, avoidance of impurities and toxic compounds, prevented deterioration and loss of functionality during extraction, which all increases the quality of final products [10].

As the previous data showed that some European elderberries exert anti-inflammatory [11], antioxidant [12], antiviral [13], antimicrobial [14], and anticonvulsant [15] activity, the aim of this study was to evaluate biological potential and its correlation with phytochemical composition of fresh elderberry fruits extracts, which were obtained using modern ((MAE), (UAE)) and traditional (maceration (MAC)) extraction techniques. Detailed quantitative and qualitative analysis (LC-MS/MS) of phenolic composition was performed. Neuroprotective, antioxidant, anti-tyrosinase, and anti-diabetic potential of the prepared extracts have been analyzed. Results of the polyphenol content and the analysis of biological activities are closely related to the applied extraction techniques. Therefore, an estimation of the capacities and abilities of the extraction techniques has been made with the goal to obtain highly valuable extracts, which could be used in food, pharmaceutical and cosmetic industry.

2. Materials and Methods

2.1. Plant Material

The fresh elderberry fruits were collected in August 2017 in the mountain of Ljubišnja, Pljevlja (Montenegro). After collection, fresh elderberry fruits were immediately frozen at −20 °C and transported in the laboratory, where different extraction processes were performed for the purpose of obtaining extracts. The specimens voucher (Sambucus nigra L., No. 2-1512) was prepared and identified by Milica Rat, and deposited at the Herbarium of the Department of Biology and Ecology (BUNS Herbarium), University of Novi Sad, Faculty of Sciences, Republic of Serbia.

2.2. Modern and Traditional Extraction Techniques

Ultrasound-assisted extractions (UAE) were performed in a sonication bath (EUP540A, Euinstruments, Paris, France). Briefly, 20.0 g of fresh elderberry fruits was mixed with 300 mL of solvent in 500 mL glass flask. Solvents used for isolation of secondary metabolites were water (H₂O) and 50% ethanol (50% EtOH). The process of sonification lasted for 30 min at a frequency of 40 kHz and with ultrasound power of 70%. Obtained extracts were filtered through filter paper after the extraction process, collected in glass vials [16].

Microwave-assisted extraction (MAE) was performed in an experimental setup described in detail by Zeković et al. (2017) [17]. For each run, 20.0 g of sample and 300 mL of extraction solvents were added in a 500 mL glass flask. Extractions were performed for 30 min at fixed microwave irradiation power (360 W). Water (H₂O) and 50% ethanol (50% EtOH) were separately used as extraction solvents. Extracts were filtered after extraction and collected in glass vials.

Maceration (MAC) as a traditional technique of extraction, was carried out as prescribed by the pharmacopoeia under the laboratory conditions at a temperature of 22 °C in a sheltered, dry place for three days. The same ratio of sample and solvent was used for both modern extraction techniques. After three days, extracts were filtered through filter paper (Whatman, No.1, Cambridge, United Kingdom), and collected in glass vials.

All prepared elderberry extracts were stored at −20 °C to prevent the oxidative processes until analysis.

2.3. Determination of Total Phenolics and Flavonoids Contents

Total phenolics (TPC) and flavonoids (TFC) contents were determined using previously described spectrophotometric methods [18,19]. The results for TPC were expressed as milligrams of gallic acid equivalents per gram of extract (mg GAE/g E), while the result for TFC was expressed as milligrams of rutin equivalents per gram of extract (mg RE/g E).

2.4. Determination of Total Anthocyanins Content

The content of the total monomeric anthocyanins (TAC) was determined using a pH differential method adapted to 96-well plates according to the previously published procedure [20]. Extraction techniques were applied and two solvents were acidified with 1% of CH₃COOH. Total anthocyanins content was expressed as milligrams of cyanidin-3-glucoside equivalents per gram of extract (mg Cy-3-G/g E).

2.5. Determination of Total Tannins Content

The tannin content (TTC) was determined by using insoluble polyvinylpyrrolidone, which binds the tannins. 500 μL of each extract, was mixed with 500 μL of citrate buffer and 50 mg polyvinylpyrrolidone, vortexed, incubated for 15 min at 4 °C and centrifuged (15 min at 3500 rpm). In supernatant, non-tannin phenolics were determined in the same manner as the total phenols. The total tannin content was obtained as the difference between the total content of the phenolics and the non-tannin phenolics [21]. The total tannin content (TTC) was expressed as milligrams of galic acid equivalents per gram of extract (mg GAE/g E).

2.6. LC-MS/MS Analysis of Phenolic Compounds

The quantification of the 48 phenolic compounds in fresh fruits of elderberry extracts was carried out using the LC-MS/MS method published by Orčić et al. [22] and Šibul et al. [23]. Samples and standards were analyzed using Agilent Technologies 1200 Series high-performance liquid chromatography coupled with Agilent Technologies 6410A Triple Quad tandem mass spectrometer with an electrospray ion source and controlled by Agilent Technologies MassHunter Workstation Software-Data Acquisition (ver. B.03.01). For all the compounds, peak areas were determined using Agilent MassHunter Workstation Software-Qualitative Analysis (ver. B.03.01). Calibration curves were plotted and concentrations of samples were calculated using the OriginLabs Origin Pro (ver. 8.0) software.

2.7. Determination of Biological Activity

2.7.1. Antioxidant Properties

Radical Scavenging Activity

The radical scavenging properties of the extracts were estimated by described method [24] against 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) and 2,2′-azinobis(3-ethylbenzothiazoline)-6-sulfonic acid radical cation (ABTS). These UV–vis-based methods are based on the decolorization of free radicals by plant sample. In brief, for DPPH assay, 20 μL of extract was dissolved in methanol in several concentrations, was mixed with 180 μL of 0.1 mM DPPH solution. Discoloration of mixtures was measured at 517 nm after 30 min. For the ABTS assay, ABTS radicals are produced by the reaction of 7 mM ABTS solution and 2.45 mM potassium persulfate at room temperature. Before the reaction started, the ABTS solution was diluted with methanol to an absorbance of 0.700 ± 0.02 at 734 nm. Afterward, extract (500 μL) was added to the ABTS (1 mL) and mixed. The sample absorbances were read at 734 nm after 30 min incubation at room temperature. Results were expressed as milligrams Trolox equivalents per gram of extract (mg TE/g E). On the other hand, a test of nitric oxide radical scavenging capacity was determined according to previously described procedures [25].

Reducing Power Activity

The cupric ion reducing capacity (CUPRAC) and the ferric reducing antioxidant power (FRAP) assays were performed to evaluate the reducing power of the extracts. These assays are based on reducing the cupric and ferrous ions. For CUPRAC experiments, prepared extracts (250 μL) was added to the reaction mixture containing CuCl₂ (500 μL, 10 mM), neocuproine (500 μL, 7.5 mM), and NH₄Ac buffer (500 μL, 1 M, pH 7.0). Then, preparation of the blank was carried out by adding extracts (250 μL) to the reaction mixture (1.5 mL) without CuCl₂. Finally, the sample and blank absorbances read at 450 nm after 30 min incubation at room temperature. The absorbance of the blank was subtracted from the absorbance of samples. In the FRAP test, the extract (50 μL) was added to FRAP reagent (1 mL) containing acetate buffer (0.3 M, pH 3.6), 2,4,6-tris(2-pyridyl)-s-triazine (TPTZ) (10 mM) in 40 mM HCl and ferric chloride (20 mM) in a ratio of 10:1:1 (v/v/v). Then, the sample absorbances were read at 593 nm after a 30 min incubation at room temperature. Results were expressed as milligrams Trolox equivalents per gram of extract (mg TE/g E) [24].

Total Antioxidant Activity

The total antioxidant activity of the extracts was examined by phosphomolybdenum method. Using this method, an aliquot of 20 μL of the extracts was combined with 200 μL of reagent solution in an Eppendorf tube including 0.6 M sulfuric acid, 28 mM sodium phosphate, and 4 mM ammonium molybdate. The samples absorbances were read at 695 nm after 90 min incubation at 95 °C. Total antioxidant capacity was expressed as equivalents of Trolox as determined by the equation of the standard Trolox curve [24].

Metal Chelating Activity

The metal chelating activity of the extracts was estimated using ferrous ions method as antioxidant activity. In this method, the chelation ability of the natural compounds is a sign of their antioxidant potential and can therefore be determined by this test. In brief, 1 mL of extract was added to FeCl₂ solution (25 μL, 2 mM). The reaction was initiated by the addition of 5 mM ferrozine (0.2 mL). Similarly, a blank was prepared by adding sample solution (1 mL) to FeCl₂ solution (25 μL, 2 mM) and water (100 μL) without ferrozine. Then, the sample and blank absorbances were read at 562 nm after 10 min incubation at room temperature. The absorbance of the blank was subtracted from the absorbance of samples. Results were expressed as milligrams EDTA equivalents per gram of extract (mg EDTA/g E) [24].

2.7.2. Enzyme Inhibitory Assessment

Neuroprotective Activity (Inhibition Acetylcholinesterase AChE and Butyrylcholinesterase BChE)

Cholinesterase (ChE) inhibitory activity was measured using method [24]. Sample solution (50 μL) was mixed with DTNB (125 μL) and AChE (or BChE) solution (25 μL) in Tris-HCl buffer (pH 8.0) in a 96-well microplate and incubated for 15 min at 25 °C (±1 °C). The reaction was initiated with the addition of acetylthiocholine iodide (ATCI) or butyrylthiocholine chloride (BTCl) (25 μL). Similarly, a blank was prepared by adding sample solution to all reaction reagents without enzyme (AChE or BChE) solution. The sample and blank absorbances were read at 405 nm after incubation at 25 °C (±1 °C) for 10 min. The absorbance of the blank was subtracted from the absorbance of samples and the cholinesterase inhibitory activity was expressed as milligrams equivalents of galantamine per gram of extract (mg GALAE/g E).

Antityrosinase Activity (Inhibition Tyrosinase)

Tyrosinase inhibitory activity will be measure using the modified dopachrome method with L-DOPA as substrate. Sample solution (25 μL) was mixed with tyrosinase solution (40 μL) and phosphate buffer (100 μL, pH 6.8) in a 96-well microplate and incubated for 15 min at 25 °C (±1 °C). The reaction was initiated with the addition of L-DOPA (40 μL). Similarly, a blank was prepared by adding sample solution to all reaction reagents without enzyme (tyrosinase) solution. The sample and blank absorbances were read at 492 nm after incubation at 25 °C (±1 °C) for 10 min. The absorbance of the blank was subtracted from the absorbance of samples and the tyrosinase inhibitory activity was expressed as milligrams equivalents of kojic acid per gram of extract (mg KAE/g E) [24].

Antidiabetic Activity (Inhibition of α-Amylase and α-Glucosidase)

α-amylase inhibitory activity will be perform using before described method [24]. Sample solution (25 μL) was mixed with α-amylase solution (50 μL) in phosphate buffer (pH 6.9 with 6 mM sodium chloride) in a 96-well microplate and incubated for 10 min at 37 °C (±1 °C). After pre-incubation, the reaction was initiated with the addition of starch solution (50 μL, 0.05%). Similarly, a blank sample was prepared by adding sample solution to all reaction reagents without enzyme (α-amylase) solution. The reaction mixture was incubated for 10 min at 37 °C (±1 °C). The reaction was stopped with the addition of HCl 25 μL, 1 M). This was followed by addition of the iodine-potassium iodide solution (100 μL). The sample and blank absorbances were read at 630 nm. The absorbance of the blank was subtracted from the absorbance of samples and the α-amylase inhibitory activity was expressed as milligrams equivalents of acarbose per gram of extract (mg ACAE/g E).

Inhibition of α-glucosidase—sample solution (50 μL) was mixed with glutathione (50 μL), α-glucosidase solution (50 μL) in phosphate buffer (pH 6.8) and p-Nitrophenyl-β-D-Galactopyranoside (PNPG, 50 μL) in a 96-well microplate and incubated for 15 min at 37 °C (±1 °C). Similarly, a blank sample was prepared by adding sample solution to all reaction reagents without enzyme (α-glucosidase) solution. The reaction was stopped with the addition of sodium carbonate (50 μL, 0.2 M). The sample and blank absorbances were read at 400 nm. The absorbance of the blank was subtracted from the absorbance of samples and the α-glucosidase inhibitory activity was expressed as milligrams equivalents of acarbose per gram of extract (mg ACAE/g E) [24].

2.8. Statistical Analysis

The results of the investigation were expressed as mean values ± SD of three different examinations. The differences between the samples were evaluated using a one-way analysis of variance (ANOVA) followed by a comparison of the means by Tukey HSD test (p ≤ 0.05). Correlations between the antioxidant/enzyme inhibitor activity and TPC, TFC, TAC, TTC, rutin, chlorogenic acid, and ursolic acid were determined using the Pearson correlation test. Correlation coefficients were considered highly significant at p < 0.05. The variability of the phenolic composition and antioxidant/enzyme inhibitor activity of fresh elderberry extracts and the standards investigated were assessed separately by using the principal component analysis (PCA). The data analyzed were standardized to account for the different magnitude, hence the responses and parameters contribute equally to the data set variance and to the principal component calculation. All statistical analyses were performed using STATISTICA version 13.2. (StatSoft, Dell).

3. Results and Discussion

3.1. Total Bioactive Compounds and Phenolic Components

3.1.1. Content of Total Bioactive Compounds

The results of analysis of the total content of secondary metabolites in elderberry extracts are summarized in Figure 1.

All examined extracts contained significant levels of bioactive components; 50% EtOH was generally more efficient solvent than water, while MAC was the most suitable technique for extraction biologically active compounds. Namely, 50% EtOH extracts produced by MAC are characterized by the highest total content of phenols, flavonoids, anthocyanins, and tannins. The amount of total phenols and anthocyanins in the 50% EtOH MAC extract was significantly different (p < 0.05) from the content in MAE and UAE extracts, while the presence of total tannins did not differ significantly between MAC, MAE, and UAE extracts. Moreover, there was no statistically significant difference in the content of total phenols, anthocyanins, and tannins between MAE and UAE extracts. Regarding the content of total flavonoids, the MAE extract was the best source of total flavonoids, while somewhat lower content was observed in the MAC extract. The UAE extract was distinguished as the poorest source of total flavonoids. Regarding water extracts, the results showed that MAE was the most effective technique for the isolation of biologically valuable compounds. These outcomes, based on the usage of solvents with different polarity are in the line with previous results: 50% EtOH was demonstrated as more efficient than H₂O [26].

The presented results indicated that MAC was an extraction technique whose application resulted in the most effective isolation of biologically active molecules. The highlighting of MAC as the dominant extraction technique for the extraction of secondary metabolites could be related to the duration of the isolation process and the conditions under which the isolation of biologically active compounds was carried out. Namely, MAC lasted for 72 h, without the influence of temperature, so it can be assumed that the degradation of thermolabile phenolic compounds was avoided during this process, and, on the other hand, the length of the process of isolation resulted in the maximal release of non-phenolic structures from the plant matrix. Accordingly, the influence of temperature in MAE and the strength of ultrasound in the UAE could cause degradation of thermolabile compounds [27]. Comparing obtained results with previously reported content of phenolic compounds in the other berry fruits, it could be noticed that examined extracts have higher content than raspberry and blueberry [28], but still inferior to the phenolics amount in blackberry [29]. The diverse content of bioactive compounds in berries could be caused by different methods of sample preparation, climatic and geographic factors, as well as the composition of the soil on which these plants grew [30].

The spectrophotometric methods, which have been used to estimate the total content of phenolic compounds, are not selective, so further analysis was performed by using the accurate and sensitive LC-MS/MS technique.

3.1.2. Phenolic Profile

Quantitative analysis of 48 selected phenolics in obtained extracts was performed by LC-MS/MS technique and it resulted in the quantitative determination of 18 compounds (

Table 1. Concentration * of detected ** phenolic acids in elderberry extracts (µg/mg extract).

Extraction Technique	UAE	MAE	MAC	UAE	MAE	MAC
Solvent	H2O			50% EtOH
Phenolic Acids and Ursolic Acid	μg/mg
p-OH benzoic acid	0.093 ± 0.006 d	0.112 ± 0.002 c	0.126 ± 0.008 a	0.113 ± 0.007 c	0.110 ± 0.007 c	0.119 ± 0.007 b
Protocatechuic acid	0.316 ± 0.025 d	1.188 ± 0.070 a	0.285 ± 0.021 e,d	0.244 ± 0.020 e	0.513 ± 0.025 b	0.406 ± 0.032 c
p-Coumaric acid	0.040 ± 0.004 d	0.056 ± 0.005 b	0.050 ± 0.004 c	0.034 ± 0.003 e	0.061 ± 0.006 a	0.041 ± 0.004 d
Gallic acid	0.011 ± 0.001 d	0.029 ± 0.003 a	0.017 ± 0.002 c	0.009 ± 0.001 e	0.023 ± 0.002 b	0.011 ± 0.001 d
Caffeic acid	0.021 ± 0.001 c	0.026 ± 0.002 b	0.079 ± 0.006 a	0.027 ± 0.002 b	0.024 ± 0.002 b,c	0.024 ± 0.002 b,c
Quinic acid	21.58 ± 2.148 b,c	21.15 ± 2.005 c	20.56 ± 1.956 d	17.16 ± 1.716 e	22.37 ± 2.227 a	21.71 ± 2.111 b
Chlorogenic acid	0.881 ± 0.093 a,b	0.935 ± 0.279 a	0.643 ± 0.001 d	0.714 ± 0.131 d	0.735 ± 0.015 c,d	0.824 ± 0.015 b,c
Ursolic acid	0.002 ± 0.000 d	<LoD *	<LoD	2.486 ± 0.075 c	6.623 ± 0.255 a	4.978 ± 0.149 b

±3SD. Means within each row with different superscripts ^(a–e) differ significantly (p ≤ 0.05) * <LoD limit of detection ** Analyzed, but not detected: Apiin, Apigenin, Apigenin 7-O-glucoside, Baicalin, Chrysoeriol, Daidzein, Genistein, Epigallocatechin gallate, Luteolin, Luteolin 7-O-glucoside, Matairesinol, Myricetin, Sinapic acid, Syringic acid, Secoisolariciresinol, Vitexin.

and

Table 2. Concentration * of detected ** flavonoids in elderberry extracts (µg/mg extract).

Extraction Technique	UAE	MAE	MAC	UAE	MAE	MAC
Solvent	H2O			50% EtOH
Flavonoids	μg/mg
Naringenin	0.003 ± 0.001 d	0.006 ± 0.000 c	0.003 ± 0.000 d	0.007 ± 0.001 b	0.007 ± 0.001 c	0.010 ± 0.001 a
Epicatechin	<LoD	<LoD	<LoD	0.089 ± 0.009 a	0.070 ± 0.007 b	0.081 ± 0.008 a,b
Quercetin	0.145 ± 0.012 e	0.189 ± 0.015 d	0.168 ± 0.013 d,e	0.334 ± 0.027 c	0.395 ± 0.018 b	0.466 ± 0.038 a
Isorhamentin	<LoD	0.007 ± 0.001 b	<LoD	0.004 ± 0.000 d	0.010 ± 0.001 a	0.005 ± 0.001 c
Baicalein	<LoD	0.036 ± 0.004 c	<LoD	0.029 ± 0.003 d	0.042 ± 0.004 a	0.039 ± 0.004 b
Kaempferol	0.005 ± 0.000 c	0.005 ± 0.000 c	0.003 ± 0.000 d	0.012 ± 0.001 b	0.013 ± 0.001 b	0.022 ± 0.002 a
Kaempferol 3-O-glucoside	0.023 ± 0.001 d	0.017 ± 0.001 e	0.024 ± 0.001 c	0.024 ± 0.001 b	0.028 ± 0.001 a	0.024 ± 0.001 c
Quercetin 3-O-hexoside	0.273 ± 0.003 b	0.290 ± 0.008 b	0.273 ± 0.025 b	0.292 ± 0.030 b	0.340 ± 0.017 a	0.372 ± 0.041 a
Rutin	7.260 ± 0.098 b,c	6.302 ± 0.353 c,d	5.092 ± 0.145 d	7.388 ± 0.955 b,c	8.086 ± 0.861 a,b	8.807 ± 1.071 a
Diosmetin	0.001 ± 0.000 d	0.002 ± 0.000 b	<LoD	0.001 ± 0.000 c	0.003 ± 0.000 a	0.002 ± 0.000 b

±3SD. Means within each row with different superscripts ^(a–e) differ significantly (p ≤ 0.05). * <LoD limit of detection ** Analyzed, but not detected: Apiin, Apigenin, Apigenin 7-O-glucoside, Baicalin, Chrysoeriol, Daidzein, Genistein, Epigallocatechin gallate, Luteolin, Luteolin 7-O-glucoside, Matairesinol, Myricetin, Sinapic acid, Syringic acid, Secoisolariciresinol, Vitexin.

).

The analysis revealed chlorogenic acid as the most abundant phenolic acid in extracts of fresh elderberry fruits, particularly in H₂O extracts. The highest content of chlorogenic acid was observed in H₂O MAE (0.935 µg/mg), while the lowest was found in UAE and MAC extracts (0.881 and 0.642 µg/mg, respectively). The presence of chlorogenic acid in 50% EtOH extracts of fresh elderberry fruits ranged from 0.714–0.824 µg/mg, with no statistically significant difference between extracts obtained by modern and traditional extraction techniques. Furthermore, H₂O extracts obtained by modern extraction techniques were better sources of protocatechuic acid than corresponding 50% EtOH extracts. Regarding extracts obtained by the traditional extraction technique, it was observed that 50% EtOH MAC extract was richer in protocatechuic acid than H₂O MAC extract.

p-Hydroxy benzoic acid (p-OH) has been identified in the fresh elderberry fruit extracts, with the highest concentration in water and ethanol extracts (0.126 and 0.119 μg/mg, respectively) obtained by MAC. The lower content of p-OH benzoic acid was observed in the extracts obtained by modern extraction techniques.

Due to the non-polar characteristics of the terpene structure, the presence of ursolic acid was expected in ethanol extracts. MAE extract was the richest source of ursolic acid (6.623 μg/mg), followed by MAC (4.978 μg/mg), while the lowest ursolic acid content was recorded in UAE extract (2.486 μg /mg). The presence of ursolic acid in fresh elderberry fruit extracts in high concentrations is of great importance for biological potential. Namely, ursolic acid is a potent natural agent that has the ability to prevent the proliferation of tumor cells, protect mental health and also has anti-oxidative, anti-inflammatory, and antimicrobial activity. Due to its high content, use of fresh elderberry fruits extracts as a potential antitumor agent can be implicated [31].

In terms of flavonoids, elderberry extracts are characterized by the dominant presence of rutin, quercetin, and quercetin-3-O-hexoside. Rutin was more prevalent in ethanol than in water extracts, and its highest concentration was identified in the extracts obtained by MAC, MAE, and UAE extraction (8.807, 8.086, and 7.388 μg/mg, respectively). The presence of rutin in UAE and MAE water extracts did not differ significantly (7.260 and 6.302 μg/mg, respectively). The higher concentration of rutin in ethanol extracts is caused by its poor solubility in water solutions; therefore, the selection of a suitable extraction technique will largely contribute to the more efficient isolation of the rutin from the plant material. A large number of food products that are present in the US market contain rutin in their composition, which is of utmost importance for the promotion of health and the enrichment of products with natural products [32].

Another very potent, biologically active compound, quercetin, was, next to rutin, the most prevalent flavonoid in fresh elderberry fruit extracts. The content of quercetin in the analyzed extracts follows the trend of rutin content, thus quercetin was mostly present in ethanol, while lower concentrations were detected in water elderberry extracts. The highest presence of quercetin was detected in MAC extract (0.466 μg/mg), while extracts obtained by modern extraction techniques were a weaker source of quercetin. Regarding water extracts, MAE extract was a better source of quercetin than MAC and UAE extracts.

An important carrier of the biological potential of elderberry extracts was quercetin-3-O-hexoside, which was most prevalent in ethanol MAC and MAE extracts (0.372 and 0.340 μg/mg, respectively). The use of two different solvents for isolation of quercetin-3-O-hexoside in the case of UAE ethanol and water extracts did not cause significantly different results.

Quinic acid is a plant product, a precursor in the synthesis of phenolic compounds, stored mainly in the bark of the plants. In this study, quinic acid was identified in significant concentration, which indicates the presence of different phenolic compounds, even those not included in this analysis.

Comparison of the results acquired by phytochemical analysis of elderflower [26], and elderberry extracts, obtained in the same way (regarding extraction techniques, solvents, and experimental conditions) points out that the same classes of natural compounds are dominant in both plant parts. The main flavonoid in the examined extracts was rutin, while chlorogenic acid was the dominant phenolic acid, and the 50% EtOH extracts of elderflowers and fruits can be highlighted as very valuable sources of ursolic acid.

The lower content of phenolic compounds in elderberry extracts, in regard to elderflower extracts, could be explained by the fact that phenolic compounds are significantly accumulated on the surface of the fruits, as their biosynthesis depends on the light. Moreover, flavonoids are concentrated in the skin of the fruits, and even the preparation for extraction and the extraction process can lead to the disintegration of the fruits, and consequently decrease in the flavonoids content. Furthermore, the content of phenolic compounds depends on the physiological state of the plant and may vary as a result of the equilibrium between biosynthesis and metabolism [33]. The chemical composition of the elderberry fruits investigated by Natić et al. [34] confirmed that the dominant compounds were rutin, quercetin-3-O-hexoside, chlorogenic acid, and protocatechuic acid. In the framework of their study, the mentioned group of researchers also determined the presence of arbutin, which was not detected in the conducted research, which was a clear indicator of the difference between the same plant species growing in another geographical area.

In order to determine which phenolic compound stands out, and to emphasize the difference between the extracts, a PCA analysis was performed (Figure 2). The first two components (F1 and F2) accounted for 71.30% of the interpreted variance if phenolic acids and flavonoids are regarded. 50% EtOH extracts were positioned on the left side of the diagram, in which the MAE and MAC occupy the central part, mainly due to the prominent content of rutin, ursolic acid, quercetin-3-O-hexoside, and kaempferol, which could be considered as the differentiation factors. H₂O extracts are located on the right side of the diagram, where UAE and MAC occupy positions on the upper right side, mainly caused by the outstanding content of chlorogenic acid. The extracts obtained by the MAE are placed in the lower part of the right side of the diagram (protocatechuic, gallic and p-coumaric acids influence on variance). This way of grouping and positioning of the examined extracts could be explained by the use of two extraction solvents, which differ in the nature of polarity: the position of the H₂O extract on the right side of the diagram can be related to the higher content of polar compounds phenolic acids.

3.2. Biological Activity

3.2.1. Antioxidant Capability

The antioxidant ability of extracts of fresh elderberry fruits was evaluated by using a variety of different in vitro assays: radical scavenging (ABTS^•+, DPPH^• and ^•NO), reducing power (FRAP and CUPRAC), total antioxidant activity (PM), and metal chelating (MC) assays. The results are presented in

Table 3. Antioxidant properties of fresh fruit elderberry extracts.

Extraction Technique	Solvent	ABTS (mg TE/g E) *	DPPH (mg TE/g E) *	NO (mg TE/g E) *	FRAP (mg TE/g E) *	CUPRAC (mg TE/g E) *	PM (mmol TE/g E) **	MC mg (EDTA/g E) ***
MAE	50% EtOH	157.99 ± 0.03 a	76.28 ± 0.01 a	0.13 ± 0.33 b,c	175.86 ± 0.01 a	217.00 ± 0.01 a	1.80 ± 0.005 b	56.41 ± 0.01 b
UAE		135.24 ± 0.01 b	53.32 ± 0.03 e	0.15 ± 0.45 b	159.79 ± 0.01 c	192.81 ± 0.02 c	1.44 ± 0.01 f	58.88 ± 0.01 a
MAC		156.14 ± 2.31 a	73.31 ± 0.01 b	0.21 ± 0.48 a	169.32 ± 0.01 b	208.49 ± 0.01 b	1.91 ± 0.01 a	52.11 ± 0.01 c
MAE	H2O	134.47 ± 0.02 b	60.50 ± 0.02 c	0.18 ± 0.66 a	147.59 ± 0.01 d	170.31 ± 0.005 d	1.51 ± 0.00 d	13.22 ± 0.02 f
UAE		115.08 ± 0.07 c	53.20 ± 0.10 e	0.13 ± 0.19 b,c	140.02 ± 0.02 e	153.08 ± 0.01 f	1.53 ± 0.005 d	25.27 ± 0.01 e
MAC		113.00 ± 0.01 c	54.30 ± 0.04 d	0.13 ± 0.37 c	133.39 ± 5.20 f	158.42 ± 0.01 e	1.70 ± 0.01 c	27.64 ± 0.01 d

* mg Trolox equivalent per g of extract, ** mmol Trolox equivalent per g of extract, *** mg EDTA equivalent per g of extract ± 3SD. Means followed by different letters ^(a–f) differ significantly at p < 0.05.

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The examined extracts have achieved excellent results in terms of antioxidant ability. The investigated radical scavenger activity ranged from 113.00 to 157.99 mg TE/g E for scavenging ABTS^•+, while the values for the DPPH^• ranged from 53.20 to 76.28 mg TE/g E.

The extracts obtained by MAE and 50% EtOH as solvent exhibited the highest scavenger activity, while UAE showed the lowest potential. Additionally, the activity of MAC was in the vicinity of MAE ability, which highlights that MAC is a competitive approach to MAE extraction. Considering water extracts, the dominant activity of MAE extracts was observed, while UAE and MAC have attained approximately similar activity. Neutralization of ^•NO radicals may contribute to a better forecast of the in vivo biological potential of fresh elderberry fruit extracts [35]. The elderberry extracts have shown good potential to inhibit the ^•NO radical, where 50% EtOH MAC and H₂O MAE extracts were particularly active. Regarding the reduction power capacity, examined by FRAP and CUPRAC tests, the extracts achieved great activity and the same trend as a radical scavenger activity.

The 50% EtOH UAE extract and H₂O MAC extract exhibited the best chelating effect in the MC assay (58.88 and 27.64 mg EDTAE/g E, respectively). In the PM test, 50% EtOH and H₂O MAC extracts (1.91 and 1.70 mmol TE/g E, respectively) have shown the best effect and expressed their domination to the extracts obtained by modern extraction techniques.

LC-MS/MS analysis showed that elderberry fruits contain a significant amount of both phenolic and terpene compounds, and it could be assumed that the expressed antioxidant activity was the result of their activity. Regarding phytochemical composition and antioxidant activity relationship, the content of rutin was strongly correlated with potency of ABTS^•+ scavenging, FRAP, and CUPRAC tests, and moderately in other antioxidant assays. Further, due to the high correlation between the amount of chlorogenic acid and antioxidant activity, it can be concluded that this compound, besides rutin, contributes to the overall notable antioxidant activity of extracts. Ursolic acid was in excellent correlation with the ability of the extracts to neutralize ABTS^•+ and DPPH^• free radicals, as well as to chelating metal ions. A very good correlation of ursolic acid was achieved with reduction capacity and overall antioxidant potential of analyzed elderberry fruit extracts (Table S1, Supplementary Material).

The antioxidant potential of fresh fruits of elderberry extracts in this investigation was compared to the antioxidant potential of elderflower extracts of the same plant species, which was collected at the same locality and obtained according to the same procedure [26]. 50% EtOH and H₂O elderflowers extract realized the stronger antioxidant potential in radical scavenger, reducing power and MC assays, while elderberry extracts achieved slightly better activity than elderflowers using PM assay, but with small differences in the obtained values. Comparing the antioxidant potential of the investigated elderberry fruit extracts with the antioxidant capacity of the elderberry fruit extracts tested in the study Natić et al. [34], it was observed that the tested extracts in this study achieved a better antioxidant potential when it came to the scavenger activity of DPPH^• radical. It is important to emphasize that the extracts examined by Natić et al. [34] did not have the ability to neutralize the ^•NO radical.

The difference in the obtained results could be explained by the application of different techniques of isolation of bioactive molecules, the use of different solvents, their efficiency, as well as the collection of plant material growing at different localities, which greatly contributes to the biopotential and future use of the plant species S. nigra collected in the territory of Montenegro.

3.2.2. Enzyme Inhibitor Activity of Fresh Fruits of Elderberry Extracts

Natural products and their biological potential are very interesting subjects, not only for the scientific community but also for different groups of consumers, as well as a substituent of already existing synthetic medicines in the prevention of various diseases of modern society [36]. The special importance of this paper is reflected in the assessment of fresh elderberry fruits and a small number of literary data on their potency to prevent the development of some pathological states caused by overactivity of the enzymes [37]. The results of the enzyme inhibition activities are shown in

Table 4. Enzyme inhibitor capability of fresh fruits of elderberry extracts.

Extraction Technique	Solvent	AChE Inhibition (mg GALAE/g E) *	BChE Inhibition (mg GALAE/g E) *	Tyrosinase Inhibition (mg KAE/g E) **	α-Amylaseinhibition (mmol ACAE/g E) ***	α-Glucosidase Inhibition (mmol ACAE/g E) ***
MAE	50% EtOH	4.93 ± 0.02 c	3.74 ± 0.01 b	243.70 ± 0.05 a	0.33 ± 0.00 a	0.056 ± 0.05 a
UAE		5.05 ± 0.001 a	4.00 ± 0.05 a	240.41 ± 0.01 b	0.31 ± 0.01 a	0.083 ± 0.05 a
MAC		5.09 ± 0.005 a	1.29 ± 0.01 c	102.11 ± 0.01 c	0.08 ± 0.01 b	0.086 ± 0.005 a
MAE	H2O	4.50 ± 0.01 e	0.51 ± 0.01 e	96.63 ± 0.01 d	0.07 ± 0.00 b,c	0.080 ± 0.06 a
UAE		4.78 ± 0.005 d	1.26 ± 0.01 c	82.95 ± 0.001 e	0.06 ± 0.00 c	0.06 ± 0.00 a
MAC		5.00 ± 0.05 b	1.09 ± 0.01 d	80.54 ± 0.001 f	0.06 ± 0.00 c	0.043 ± 0.00 a

* mg galantamine equivalent per g of extract, ** mg kojic acid equivalent per g of extract, *** mmol acarbose equivalent per g of extract ± 3SD. Means followed by different letters ^(a–f) differ significantly at p < 0.05.

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Examining the ability of extracts of fresh elderberry fruits to inhibit AChE and BChE enzymes is of particular importance in the prevention of the development of Alzheimer’s disease, some types of leukemia, and tumorigenesis. According to scientific estimates, in elderly populations, Alzheimer’s disease is the third disorder in frequency, placed immediately after heart disease and cancer [38].

The investigated extracts achieved excellent inhibitory activity against both cholinesterases (AChE and BChE). 50% EtOH extracts achieved better inhibitory potential against cholinesterases than H₂O extracts.

Although obtained by the traditional extraction technique, ethanol MAC extract achieved an excellent result in inhibition of AChE overactivity, with no statistically significant difference to ethanol UAE extract, obtained by a modern extraction technique (5.09 for MAC and 5.05 for UAE mg GALAE/g E, respectively).

The results of the regression analysis showed that rutin and chlorogenic acid contents are highly correlated with AChE and BChE inhibition potential, indicating that they, as the most dominant representatives of flavonoids and phenolic acids, significantly contributed to overall cholinesterases inhibition. Ursolic acid correlates moderately with the capacity of the elderberry extract to provide neuroprotective protection (Table S1, Supplementary Material).

Changes that may occur in the activity of enzyme tyrosinase could lead to the skin exposed, hyperpigmentation, and could cause melanoma [39]. In order to find novel natural tyrosinase inhibitors, the analysis of fresh elderberry extracts has resulted in excellent inhibitory activity and is summarized in Table 4. Achieving the best inhibitory ability was 50% EtOH MAE, 243.70 mg KAE/g E, while the inhibitory capacity of 50% EtOH of the UAE extract was 240.41 mg KAE/g E. MAC extract accomplished the lowest tyrosinase inhibition value: 102.11 mg KAE/g E. H₂O extracts realized good results in terms of anti-tyrosinase activity, but the obtained values were lower in relation to 50% EtOH extracts and ranged from 80.54 to 96.63 mg KAE/g E. The presence of secondary metabolites in elderberry extracts, especially rutin, chlorogenic acids, and ursolic acid, which correlate well with tyrosinase inhibition, enabled the analyzed extracts to achieve very good inhibition of tyrosinase (Table S1, Supplementary Material). The results achieved by fresh elderberry extracts in the process of inhibition of excessive tyrosinase enzyme activity, especially EtOH MAE extract could be considered as a starting point and a potential guideline for the formulation of potentially new products based on the protective role of the skin.

Preventing the development of diabetes as the leading chronic disease of modern society is based on the inhibition of excessive activity of the enzymes α-amylase and α-glucosidase. In this regard, fresh elderberry extracts were examined as potential natural inhibitors of these enzymes. Investigated elderberry extracts exerted strong inhibitory power against α-amylase than α-glucosidase, where all the extraction techniques leaded to an approximately similar result. In the ability to inhibit α-amylase and α-glucosidase, there was no statistically significant difference between the 50% EtOH extracts of all three extraction techniques. In terms of correlation, the best correlation was observed between the content of rutin and α-glucosidase inhibition, so rutin probably had the strongest effect on this enzyme inhibition (Table S1, Supplementary Material). To achieve the general conclusion on antioxidant and enzyme inhibitor activities and possible classification of the samples, PCA was carried out using the datasets containing variables related to antioxidant and enzyme inhibitor activities (Figure 3). PCA revealed 50% EtOH MAC as the most potent overall antioxidant agent and pointed out 50% EtOH MAE as the most potent enzyme inhibitor factor. The first and the second principal components (Factor 1 and 2) explained 61.69 and 26.68% of the total variance, respectively. All antioxidant activities are positively correlated with Factor 2, while H₂O extracts appeared in a compact group in the right area of the plot, demonstrating moderate activity in all of the tests applied. This kind of separation implicates that the biological activity of extracts, due to possibilities of different compound extraction, could be linked with used solvent.

4. Conclusions

Modern and traditional extraction techniques were applied to obtain elderberry extracts and characterized by their polyphenolic profile, antioxidant potential, and enzyme-inhibitory activity. The highest content of dominant phenolic acids (chlorogenic acid and protocatechuic acid) was observed in water extract obtained by microwave-assisted extraction, while ursolic acid was the most abundant in ethanol extract obtain by same extraction process. The most common flavonoids (rutin and quercetin-3-O-hexoside) were identified in the greatest concentration in ethanol extract obtained by MAE extraction. The most potent antioxidant activity towards ABTS^•+ and DPPH^• radicals, reducing the power of ferric and cupric ions antioxidant activity were observed in EtOH MAE extract, which was explained by a high concentration of phenolic compounds in this sample. Ethanol extract obtained by modern UAE extraction showed the strongest influence towards inhibition of AChE and BChE enzymes. On the other hand, ethanol MAE extract expressed the greatest inhibition of tyrosinase, α-amylase, and α-glucosidase. Generally, EtOH extracts achieved better bioactivity than water extracts. Hence, it could be concluded that MAE represents an excellent alternative for the traditional extraction process since certain samples exhibited advantages in terms of phenolic acids and flavonoids. MAE extraction technique could be particularly useful for the environmentally-friendly production of elderberry extracts rich in secondary metabolites. According to the present results, elderberry represents an excellent natural resource for the isolation of valuable bioactive compounds, and the application of new isolation techniques provides an additional advantage to their processing and properties.