Bioactive Compounds from Elderberry: Extraction, Health Benefits, and Food Applications

Abstract

Elderberries are appreciated for their antioxidant properties. Sambucus nigra L. is an extremely abundant plant in the wild flora of Romania, but it is underutilized. Elderberry is used in modern and traditional medicine due to the complex chemical composition of the fruit. The content of phenolic compounds is high (516–8974 mg/100 g DW), of which the most abundant are anthocyanins. Phenolic compounds are known for their beneficial effects on the body. Numerous studies have demonstrated the antioxidant capacity, antibacterial, antiviral, antidiabetic, and anticancer properties of the fruit. It is considered that most of the therapeutic properties of elderberries can be correlated with the antioxidant activity they have. S. nigra fruits are also used in the food industry. Some studies have shown that the therapeutic properties of elderberries can also be found in the products obtained from them. Therefore, this review aimed to describe the chemical composition of elderberries and products obtained from them, the positive effects on the body, and the methods by which the bioactive compounds can be extracted from the fruits and analyzed. This manuscript is useful for extraction optimization and characterization in order to valorize new functional foods, food supplements, and also in new pharmaceutical products.

1. Introduction

S. nigra is one of the 20 species that are part of the Adoxaceae (Caprifoliaceae) family. It is found in both subtropical and temperate areas [1]. The fruits are globose, small in size (0.4–0.6 cm in diameter), and dark purple (almost black) in the case of S. nigra. The color of the fruits can vary depending on the variety, from bright red to dark purple. The fruits are grouped in large clusters, between 15 and 22 cm [2,3]. S. nigra L. is known as ‘European elderberry’, ‘European black elderberry’, ‘European elder’, ‘black elderberry’, ‘elderberry’, ‘black elder’, or ‘elder’. This plant has low requirements in terms of growing conditions, tolerating even eutrophic soils (disturbed soils) [4]. S. nigra can be cultivated, but the fruits are harvested in large quantities from the wild flora. It often grows at the edge of forests, in sunny locations, in temperate and subtropical regions of the Northern Hemisphere, as well as in Asia, North Africa, and North America. S. nigra has a high content of phenolic compounds with pharmacological properties, which is why it was studied for use in the ecological pharmaceutical industry [5,6]. Due to the complex composition of the fruits, the high availability of the plants in the wild flora and the simple growing conditions, interest in the use of elderberries has increased in recent decades [7,8]. At an industrial level, elderberries (EDBs) are mainly used as dyes due to the large amounts of pigments they contain (especially anthocyanins). They are also used as preventive or curative treatments because their health benefits have been proven by numerous studies [9]. The fruits and the vegetables that are consumed on a daily basis contain different levels of phenolic compounds. However, since a regular diet cannot provide the necessary antioxidants, the production of functional foods, which contain greater amounts of added antioxidants, is justified [10].

The extraction of phenolic compounds from matrices that are complex from a chemical point of view, containing a series of secondary metabolites with different chemical structures and biological properties, involves the control of many factors of the extraction process (for example, the extraction method, the number of materials, the type of solvent, the pH, the temperature, and the extraction time) [11]. Choosing the most efficient extraction method and optimizing the extraction parameters lead to an increase in the level of extracted bioactive molecules.

The aim of this review was to highlight the chemical composition of EDB and to present the products from EDB or those in which EDB was used, the compositional changes that occur after processing, the benefits that fruits can have for human health, the extraction methods, and analysis of bioactive compounds.

2. Chemical Composition

The chemical composition of S. nigra fruits can vary greatly. The factors that contribute to the compositional variations are the variety, the climatic conditions, the growing location, and the ripening stage [12].

2.1. Macronutrients

The main macronutrient in fresh EDB is water (71–78%). The content of total soluble solids (with glucose and fructose as main components) can vary quite a lot (6.8–14.14 g/100 g fresh weight (FW), and the ash content varies less (0.92–1.02 g/100 g FW). The content of total sugar in elderberries ranged from 6.85 to 10.42 g/100 g FW. The fruits can be considered a good source of fiber, containing 1.65% cellulose, 0.16% pectin, 0.23% pectic acid, and 0.04% protopectin [12,13,14,15,16]. Peptic polysaccharides contained arabinose, rhamnose, xylose, mannose, galactose, glucose, glucuronic acid, galacturonic acid, and 4-O-methylation of glucuronic acid [17]. An analysis of individual sugar compounds revealed the presence of glucose (29.03–50.23 g/kg FW), fructose (26.81–52.25 g/kg FW), and sucrose in small quantities (0.47–10.46 g/kg FW) [15,16,18]. According to Thomas et al. [19], the genotype, place of growth, and climatic conditions of that year affected the levels of glucose and fructose in fruits.

Protein content varies less (2.7–2.97 g/100 g FW), including nine essential amino acids out of a total of sixteen identified amino acids [12]. Vulić et al. [16] identified 2.84 g proteins/mL, of which the most important amino acids were glutamic acid (0.311%), aspartic acid (0.303%), alanine (0.238%), leucine (0.205%), and tyrosine (0.198%).

Elderberries have a low fat content (0.35 g/100 g FW) [14]; fruits contain 0.01 g/100 g essential oil, which is composed of over 30 different compounds, including phenyl aldehydes (3–25.8% of the oil composition) and furfural (18%) [20,21]. The diversity of lipids is very high: 12.96–14.21 g/100 g monounsaturated fatty acids (MUFA), 75.15–77.69 g/100 g polyunsaturated fatty acids (PUFA), and the amount of saturated fatty acids (SFA) reached 9.35–10.64 g/100 g. The most abundant individual fatty acids were linoleic acid (C18:2n-6; ω-6; 39.47–42.4%), α–linolenic acid (C18:3n-3; ω-3; 32–41%), oleic acid (C18:1n-9; ω-9; 12.6–13.2%), palmitic acid (C16:0; 6.59–9.3%), and stearic acid (∼1.7% in all samples). Fats are accumulated mostly in elderberry seeds (22.4 g/100 g) and seed flour (15.9 g/100 g) [12,14,22,23,24]. Fazio et al. [24] reported a quantity of 4.21 g/100 mg seeds oil MUFA, 21.54 g/100 mg seeds oil PUFA, and 4.81 mg/100 mg seeds oil SFA.

2.2. Vitamins

The vitamins that have been identified are part of the B complex (B2—65 mg/100 g; B7—17 mg/100 g; B9—1.8 mg/100 g), vitamin C was also noted in smaller quantities in wild fruits compared to cultivated fruits (34.10–116.70 mg/100 g FW), and vitamin A (600 IU/100 g) was also present [12,16,20,21,25,26,27,28].

The oil obtained from EDB seeds contains 0.49 µg α-Tocopherol/g_oil and 2.63 µg γ-Tocopherol/g_oil. Tocopherols in vegetable oils protect polyunsaturated fatty acids from oxidation. Overall, α-Tocopherol has the highest vitamin E bioactivity, while γ-Tocopherol shows a better antioxidant activity. These data indicate that EDB seeds may serve as natural sources of beneficial compounds for the body [24].

2.3. Phenolic Compounds

The content of phenolic compounds in fruits varies widely, e.g., 4917–8974 mg/100 g DW [12], 1136 mg/100 g DW, 827 mg/100 g DW, or 516 mg/100 g DW [25]. The content of phenolic compounds varies depending on the growing seasons [29], the ripening stage of the fruits, and the variety [30]. Each polyphenolic compound undergoes a series of specific transformations during the ripening process [31]. The most common flavor perceptions of phenolic compounds are astringency and bitterness, elicited primarily by flavonol polymers (proanthocyanidins or condensed tannins) [32].

The flavonoids (Figure 1) are a class of phenolic compounds that exhibit antioxidant properties. The anthocyanins and the quercetins are flavonoids [33]. Anthocyanins are the major phenolic compounds in the mature fruits of elderberry (560–1347 mg/100 g FW), present mostly as cyanidin derivatives [34,35,36,37,38]. ‘Anthocyanins are water-soluble glycosides or acylglycosides of anthocyanidins being oxygenated derivatives of flavylium salts’ [12]. They are considered chemopreventive agents because they have the ability to reduce oxidative stress by scavenging free radicals. They are used in the food industry to color food bright red, blue or purple, being an alternative to synthetic dyes. Their use as coloring agents has as advantages the potential therapeutic effect, but also safety for the body and the environment [12].

Following the analysis of elderberries that came from plants of several varieties, it was found that the content of anthocyanins in 100 g FW was 529 and 664 mg in the ‘Haschberg’ variety, 877 and 1815 mg in the ‘Sampo’ variety, 846 and 1634 mg in the cultivar ‘Samyl’ [25]. Brønnum-Hansen and Hansen [39] reported that 65.7% of the total anthocyanins were represented by cyanidin 3-glucoside and 32.4% of the total anthocyanins by cyanidin 3-sambubioside. Cyanidin-3-sambubioside accounted for 32.4–73.2% of anthocyanins [6,40,41], 269–656 mg/100 g FW [15,29,38,42,43]. Dawidowicz [44] identified higher amounts of cyanidin 3-sambubioside (270.5–277 mg/100 g) than cyanidin 3-glucoside (214.1–251.8 mg/100 g) in the alcoholic extracts of EDB. Chrysanthemin was quantified in proportions of 65.7% of the total anthocyanins, 221.4–739.8 mg/100 g FW. Other anthocyanins were also identified, but in smaller quantities: cyanidin-3-sambubioside-5-glucoside (16.0–82.6 mg/100 g FW), cyanidin-3,5-diglucoside (7.41–23.29 mg/100 g FW), callistephin (0.3–1.80 mg/100 g FW), and pelargonidin-3-sambubioside (0.025 mg/100 g FW) [41,43]. Besides these compounds, the presence of chrysanthemin monoglucuronide, peonidin monoglucuronide, peonidin-3-sambubioside, and peonidin-3-glucoside was also reported [34,45]. The degree of ripeness of the fruit influences the content of anthocyanins (ripe fruits have a higher content), especially the amount of cyanidin-3-sambubioside-5-glucoside, cyanidin-3-sambubioside, and cyanidin-3-glucoside [31]. Since anthocyanins are unstable compounds, the processing conditions of the products containing elderberries are important, especially the temperature used [25]. Proanthocyanidins are monomers (epicatechin 63.71 mg/kg FW), dimers (10.62 mg/kg FW), trimers (5.63 mg/kg FW),tetramers, pentamers, and hexamers (10.80 mg/kg FW) [38,46].

Quercetin-3-O-rutinoside (rutin) was the main flavanol glucoside in elderberries (13.8–31.33 mg/100 g FW; 1.09–1.54 mg/g extract), followed by isoquercetin (4.35 mg/100 g FW; 0.8–0.3 mg/g extract), astragalin (0.11–0.18 mg/g extract), quercetin acetyl hexosides (0.466 mg/100 g FW), quercetin hexoside, and pentoside (0.336 mg/100 g FW). The genotype of the plants and the growing conditions had a major influence on the biochemical profile of the fruits, including the content of anthocyanins and flavonols [15,19,44,46].

The content of organic acids can vary quite a lot: from the range of 0.4–0.6% [15] to the range of 1.0–1.3% [12]. Among the identified phenolic acids, the largest amount was represented by the 5-O-trans-caffeoylquinic acid (15.38 mg/100 g FW), followed by 3-O-caffeoylquinic acid (8.8 mg/100 g FW), 4-O-caffeoylquinic acid (2.86 mg/100 g FW), 5-O-cis-caffeoylquinic acid (4.01 mg/100 g FW), 3-O-feruloylquinic acid (1.87 mg/100 g FW), 3-O-p-coumaroylquinic acid (1.19 mg/100 g FW), 3,5-di-O-caffeoylquinic acid (0.34 mg/100 g FW), and 4,5-di-O-caffeoylquinic acid (0.22 mg/100 g FW) [46]. Fazio et al. [24] reported 0.04 mg ellagic acid/g_seeds.

Przybylska-Balcerek et al. [25] reported that the predominant phenolic acids were chlorogenic acid (139.09 mg/g extract), sinapic acid (72.84 mg/g extract), and t-cinnamic acid (51.29 mg/g extract). The major organic acid in ripe elderberry fruits was citric acid (308–966 mg/100 g FW), followed by malic acid (97–131 mg/100 g FW), shikimic acid (14–93 mg/100 g FW), and fumaric acid (10–29 mg/100 g FW) [13,15,18]. The analysis of wild and cultivated elderberries showed that there is a compositional difference between them. Unripe wild elderberries had a higher content of organic acids and sugars. Unripe cultivated elderberries (‘Ljubostinja’ variety) had a higher chlorogenic acid content. Both ripe and unripe cultivated fruits showed a large amount of quercetin and cyanidin derivatives [13].

2.4. Cyanogenic Glycosides

EDB contains cyanogenic glycosides, which are harmful bioactive compounds from plants that are derived from amino acids. The most abundant of them are sambunigrin (0.08–0.77 µg/g FW) and prunasin (Figure 2). It was discovered that the altitude at which the fruits grow can influence the sambunigrin content in them because the low temperature and the high level of solar radiation stimulate the biosynthesis of this compound. Furthermore, unripe EDB contains zierin and holocalin (m-hydroxysubstituted glycosides), which can be transformed into cyanide by hydrolysis. Applying a heat treatment can degrade these compounds [9,47].

At doses of 0.5–3.5 mg/kg body weight (bw), cyanogenic glycosides can be toxic to animals and humans, but processes such as boiling and fermentation can reduce the increased level of these harmful compounds to a level where the body is able to carry out the metabolization of cyanide [48]. EDB also contains terpenes and lectins, free and conjugated forms of amino acids, proteins, and fiber [49]. Jiménez et al. [50] discovered that the risk of allergenicity due to lectins can be reduced by immersing the fruit in boiled water for 5–10 min, so that the hydrolytic enzymes will react with the lectins.

The excessive consumption of EDB and the lack of fruit processing or inadequate processing (to reduce the toxic effect) can lead to the appearance of some diseases of the digestive system [5,9].

EDBs have a characteristic aroma due to the presence of the following compounds: (E)-β-damascenone, dihydroedulan, ethyl-9-decenoate, 2-phenyl-ethanol, phenylacetaldehyde, and nonanal. A series of volatile compounds (alcohols, esters, and aldehydes) were also identified [49].

The detailed knowledge of the chemical composition of EDB can be a solid scientific argument for the use of extracts for the development of new food products, functional foods, food supplements, pharmaceutical and cosmetic products. This is especially important because there are large compositional variations, depending on many factors.

3. Elderberries in Food Products

Elderberries are used to obtain food products due to their pleasant taste, specific aroma, and complex chemical composition. Since phenolic compounds are the main compounds responsible for the beneficial effects on the body, their presence in the final product is important. There are some studies that have focused on the chemical changes that occur after the processing of elderberries.

Senica et al. [51] studied the presence of phenolic compounds and cyanogenic glycosides in various elderberry products (juice, tea, liquor, and spread). They reported that the following anthocyanins were the majority in the analyzed products: cyanidin 3-O-sambubioside (100 mg/kg in elderberry liqueur, 77 mg/kg spread, 48 mg/kg juice, and 21 mg/kg tea) and cyanidin-3-O-glucoside (88 mg/kg liqueur, 42 mg/kg juice, and 35 mg/kg tea). These compounds were the main anthocyanins in elderberries [30,44] and were quantified in three times lower amounts in the products compared to fresh EDB. The most important factors that affected the stability of anthocyanins were time, temperature (temperatures above 60 °C can reduce the level of anthocyanins), pH, and type of solvent [52]. High temperatures, correlated with a long time of exposure, resulted in a lower level of anthocyanins in elderberry juice and tea. Moreover, the extraction of anthocyanins was more efficient in organic solvents than in water, which is why the non-alcoholic drinks presented a lower level of anthocyanins. Quercetin-3-O-rutinoside was the main flavonol compound identified in all the analyzed products. The highest level was found in spread and liquor (65.0 mg/kg and 38 mg/kg, respectively), and the lowest in EDB juice (2 mg/kg). As in the case of anthocyanins, the organic solvent contributed to the increased level of this compound. Similar results were obtained in the case of flavanones. Regarding the content of toxic compounds, the highest level of cyanogenic glycosides was found in EDB juice (11 mg/kg), and the lowest in liquor and spread (0.8 mg/kg), a reduction of 96% compared to the control sample [51]. Akande and Fabiyi [53] showed that a reduction in the level of glycosides and cyanogenic alkaloids can be made by heating for 60 min. However, the best solution for preserving the nutritional value and avoiding the ingestion of large amounts of toxic compounds is to obtain products from ripe fruits, because they contain a low level of cyanogenic glycosides [54].

Schmitzer et al. [42] used elderberries to obtain wine. They also quantified the total content of phenolic compounds in the EDB must and wine, the antioxidant activity, the pH, and the changes that occur with the aging of the wine. The results showed that the pH of the EDB must was much higher compared to that of the EDB wine. Compared to red wine, which had a pH between 3.0 and 3.7 [55,56], the acidity of EDB wine was lower (pH between 3.9 and 4.17). After three years of aging in bottles, the pH of the EDB wine increased due to esterification of organic acids. Withal, the wine lost its color intensity after the aging process because the phenolic compounds (mainly anthocyanins) underwent a series of transformations due to polymerization, condensation, and oxidation reactions, with the main cause being the increase in pH. The content of phenolic compounds in wine depends on a series of factors such as the initial level of phenolic compounds in the fruit, the techniques applied for winemaking, the age of the wine, and the storage conditions [57]. The EDB wine had a higher content of anthocyanins and total phenolic compounds than the EDB must and the wine aged three years (with 6% more anthocyanins) and 2 times more anthocyanins than the red wines [42,58], while the total content of phenolic compounds decreased by 20% after aging (1714.53 mg Gallic Acid Equivalent L⁻¹ in EDB must, 2004.13 mg GAE L⁻¹ in EDB wine, and 1584.99 mg GAE L⁻¹ in aged wine). The main anthocyanin compounds identified were cyanidin-3 sambubiosides (in EDB must) and cyanidin-3-glucosides (in EDB wine). The EDB wine had the highest antioxidant activity (9.95 mM trolox L⁻¹), followed by the EDB must (8.18 mM trolox L⁻¹) and the aged wine (6.13 mM trolox L⁻¹). Among quercetins, quercetin-3-rutinoside and quercetin-3-glucoside were the most abundant, especially in EDB wine [42]. Consuming grape wine can have positive effects on the body, especially due to the phenolic compounds it contains [59]. Since elderberry wine also has a high content of phenolic compounds, we can say that its consumption can have positive effects on human health.

Cordeiro et al. [60] studied the changes in anthocyanin content depending on several cooking methods (boiling, steaming, and baking) and depending on the product obtained (jam, crumble, muffin, and mousse). The results obtained showed that the loss of anthocyanins was 60% in the case of steaming, 45% in boiling, and 35% in baking, compared to the content of anthocyanins in fresh fruits. It should be mentioned that boiling water and steaming water contain 20% anthocyanins. In the case of desserts, the loss of anthocyanins was 60% for jam, 35% for muffins, 35% for crumble, and 10% for mousse. Therefore, the cooking process led to the loss of large amounts of anthocyanins. In the case of jam, it can be about the creation of complex networks between sugars and anthocyanins; thus, the alcoholic extraction of anthocyanins is reduced [61]. In the case of muffins, the addition of sodium bicarbonate could be the factor that led to the decrease in the stability of anthocyanins [62]. In the case of the crumble, the low water activity and the high temperature contributed to the decrease in the anthocyanin content. The content of anthocyanins in the mousse decreased due to their interaction with the other components of the product, especially the sugars and fats from the condensed milk [60]. Sun-Waterhouse et al. [63] showed that the fiber-enriched pasta in which concentrated EDB juice was added had a higher nutritional value compared to the control samples. Moreover, the content of phenolic compounds and antioxidant activity were higher. The croissants in which EDB juice was added (2, 4, and 8% juice) showed a higher content of bioactive compounds and a higher antioxidant activity [64]. Różyło et al. [65] showed that the gluten-free wafers in which different percentages of EDB powder were added (1, 2, 3, 4, and 5% powder) had a higher degree of acceptance by consumers, especially the wafers in which the highest percentage of elderberry powder was added.

The results of using elderberries in dairy products have been observed in several studies. The addition of 10% fruit juice increased the antioxidant capacity and antidiabetic properties of yogurt. The addition of 25% restructured juice (obtained from 85% EDB juice, 14% sugar, and 1% alginate) was more easily accepted by consumers from a sensory point of view (it increased the consistency of the yogurt and led to a change in texture) [66]. Najgebauer-Lejko et al. [67] showed that the yogurt obtained with 10% EDB puree had, compared to the regular yogurt, a higher antioxidant capacity and higher amounts of total phenolic compounds and total anthocyanins. Obtaining kefir by using 10% commercial EDB juice determined a slight increase in the level of phenolic compounds and anthocyanins. The same percentage of fresh EDB juice determined the increase in the content of both phenolic compounds and anthocyanins, as well as the antioxidant capacity, of kefir [68].

The use of elderberries in meat products also had positive effects. The addition of 1% elderberry powder in pork patties had positive effects because it determined the decrease in the appearance of substances reactive to thiobarbituric acid (decrease in the formation of secondary oxidation products) [69]. Cordeiro et al. [70] conducted a study in which they sprayed pork loin with elderberry vinegar before grilling. The results showed an 82% inhibition of polycyclic aromatic hydrocarbons (PAHs). Since the formation of PAHs involves the generation of free radicals, the phenolic compounds in elderberry vinegar contributed to the elimination of free-radical precursors and inhibited the formation of PAHs.

Therefore, the food products based on EDB lost some of the bioactive compounds, but they remained in notable quantities. Factors such as temperature, time, and pH are important in the preservation of potentially therapeutic compounds. The products in which EDB or fruit preparations were added had an improved nutritional value; the preservation of the bioactive compounds depended, in addition to the factors mentioned above, on the interaction with the other compounds in the product. The extracts could be used to diversify the range of food products. They can be used in products intended for certain categories of population with special needs (children, the elderly, athletes, and people exposed to a toxic environment).

4. Health Benefits

Phenolic compounds are secondary plant metabolites, and their chemical structure includes a hydroxylated aromatic ring. The biological activities that phenolic compounds can have are antioxidant, immunostimulatory, anti-inflammatory, anti-allergic, anticancer, antibacterial, and antiviral properties [71]. The beneficial properties of phenolic compounds can be altered if they are degraded following exposure to oxygen, light, enzymatic activities, unfavorable conditions of temperature and pH, metal ions, and water [72,73].

4.1. Antioxidant Capacity

The excessive accumulation in the human body of some products derived from the reactions between oxygen and nitrogen can cause oxidative stress, affecting DNA, blood vessel walls, proteins, and lipids. As a result of these processes, dangerous diseases, such as neurodegenerative ones (Alzheimer and Parkinson), diabetes, or premature aging, can appear. Cell development can be affected by the cleavage of DNA molecules, leading to the formation of cancer cells and the appearance of cardiovascular diseases [74].

Oxidative reactions in the human body can be reduced by antioxidants, which act as protective agents. The presence of phenolic compounds in plant extracts gives them antioxidant and pharmacological properties [44,75,76]. Oxidation reactions are initiated by free radicals, and flavonoids can interact with them, preventing the occurrence of oxidation. They can also interact with reactive oxygen species (ROSs), which are produced during chain reactions [44]. In normal homeostatic conditions, ROSs have an important role in many physiological processes, having the ability to regulate key cellular functions, such as proliferation, differentiation, and apoptosis, being also involved in the defense against pathogens. They act as second messengers for multiple signal transduction pathways and gene transcription [77,78].

Elderberries can modulate the concentration of ROS in intestinal contents as well as in intestinal epithelial cells due to the increased content of bioactive compounds with antioxidant potential [79]. Numerous in vitro antiradical activity tests confirmed the antioxidant capacity of S. nigra fruits [80]. The hydrolysis and degradation of anthocyanins in the small and large intestine can be caused by the activity of the microflora in the colon and their instability in an alkaline environment [81]. In addition, they pass from an acidic pH (at the stomach level) to a slightly neutral or neutral pH (at the intestinal level) [82,83,84]. It was shown that, in the small intestine, the antioxidant activity of EDB extract decreased from 15.71 ± 1.09 µM Trolox/mL/50 mg (in non-digested EDB, dried by lyophilization) to 9.89 ± 0.39 µM Trolox/mL. This decrease in antioxidant activity was due to the transformation of the main polyphenolic compounds, including anthocyanins, which have a major contribution to the antioxidant effect. Therefore, the obtained results indicated that, when passing through the gastrointestinal tract, the EDB extract reaches the compartments of the small intestine in a modified but still active form [85]. Following the simulated digestion in an artificial digestive tube (stomach, small intestine, and large intestine), an 88.4% reduction was observed in the content of cyanidin-3-O-sambubioside and cyanidin-3-O-glucoside. There were also compounds more resistant to the intestinal digestion process (cyanidin-3,5-O-diglucoside and cyanidin-3-O-sambubiosyl-5-O-glucoside). In the colon, the loss of anthocyanins from the EDB extract was 44%. Small amounts of pelargonidin- and peonidin-derivatives were present in the undigested EDB extract and absent in the colon-digested extract. The phenolic compounds (other than anthocyanins) were relatively stable in the upper section of the intestine. The increase in the amount of quinic acid confirmed the hydrolysis of chlorogenic acid. Additionally, the degradation of EDB tannins was observed, probably due to the activity of colonic microflora. However, it was observed that the colon-digested EDB extract contained a breakdown product of rutin and isoquercetin, called ‘quercetin’. Simulated gastrointestinal digestion at the level of the stomach and the small and large intestine showed a loss of 80.5% of the total phenolic compounds quantified in the elderberry extract [79,85]. The digested EDB extract contains sufficient amounts of antioxidants to have a positive action on the cells of the intestinal mucosa by protecting them against oxidants that cause mutagenesis and against DNA oxidative stress and limiting the hyperproduction of intracellular ROS. The use of EDB fruits as functional food components could be postulated due to the non-genotoxic, non-cytotoxic, and non-mutagenic effects that the fruit extract has on the body [79].

Numerous human diseases have among the main factors of their occurrence the inflammation process. Among these diseases are some types of cancer, hypertension, cardiovascular diseases, dyslipidemia, metabolic syndrome and insulin resistance, allergies, inflammatory bowel disease, chronic asthma, neurodegenerative disorders, rheumatoid arthritis, and multiple sclerosis [86,87]. Extracts from berries have shown anti-inflammatory properties (inhibition of pro-inflammatory prostaglandins) due to the large amounts of anthocyanins that they contain [88]. Anthocyanins had increased antioxidant activity, leading to a downregulation of the redox-sensitive nuclear factor—κB signaling pathway—from which the anti-inflammatory potential resulted. Other pathways that may play a role in the inflammatory response are the mitogen-activated protein kinase pathways [89]. The bacterium Escherichia coli was used to stimulate bone-marrow-derived dendritic cells, and anti-inflammatory effects of the EDB extracts were observed [90]. It was also found that the antioxidant activity increases directly proportional to the concentration of anthocyanins in EDB extracts, but only up to a certain level because, above this level, the antioxidant activity starts to decrease [91].

4.2. Antibacterial Properties

The antibacterial activity of EDB extracts was associated with the content of phenolic acids and flavonoids. The compounds that have the greatest influence on the antibacterial activity are kaempferol, apigenin, ferulic acid, protocatechuic, and p-coumaric acids. The bacteria that are most sensitive to the extract of phenolic compounds from EDB are Micrococcus luteus, Proteus mirabilis, Pseudomonas fragi, and Escherichia coli. Previous research showed that only selected tannins derived from hydroxycinnamic, gallic and caffeic acids, and triterpenes (oleanic acid; α- and β-amarin) were considered antimicrobial substances. The studies showed that selected phenolic compounds and their esters inhibited the growth of bacteria of the following genera: Yersinia, Bacillus, Corynebacterium, Proteus, Staphylococcus, Enterococcus, Klebsiella, Micrococcus, Escherichia, and Pseudomonas [25,92,93,94]. Bacillus subtilis and Bacillus cereus bacteria are responsible for food poisoning. Their growth was inhibited by caffeic, ferulic, and protocatechuic acids from the EDB extract. The growth of Yersinia enterocolitica rods was also inhibited by phenolic acids [95].

Hearst et al. [96] used aqueous ethanol extract obtained from EDB concentrates dried by lyophilization to study the antibacterial effects on 13 nosocomial pathogens, including methicillin-resistant Staphylococcus aureus (MRSA). Most of the analyzed bacteria were inhibited by the EDB extract, including Staphylococcus sp. or Bacillus cereus (Gram-positive), Salmonella poona, and Pseudomonas aeruginosa (Gram-negative). Chatterjee et al. [97] showed that elderberries had an inhibition activity of 30% against Helicobacter pylori. To demonstrate the antibacterial effect of elderberry extract on bacteria infecting the respiratory tract, Krawitz et al. [98] used 10% standardized liquid extract of EDB for liquid bacterial cultures. The results showed a 70% inhibition (compared to control samples) of the development of bacterial strains of Streptococcus pyogenes, Streptococcus Group C and G (Gram-positive bacteria), and Branhamella catarrhalis (Gram-negative bacteria). Moreover, the inhibition percentage of 99% was obtained by adding EDB extract in a concentration of 20%.

4.3. Antiviral Properties

It was found that the compounds kaempferol 3-rutin, isorhamnetin 3-rutin, and isorhamnetin 3-glucoside and cyanidin derivatives are antiviral chemicals [99]. The influence of the EDB extract on the human influenza A (H1N1) virus applied to infect the Madin–Darby canine kidney (MDCK) NBL-2 cells was studied, and it was discovered that, by using a concentration of 252 µg/mL, a 50% inhibition (IC₅₀) can be obtained. After applying a concentration of 1000 µg/mL, a 100% inhibition of viral infection was obtained [100].

After the addition of the EDB extract, the H1N1 virions lost their ability to infect host cells because the phenolic compounds in the extract are bound to the virions. The polyphenolic compounds bound to H1N1 virions are 5,7,3,4′-tetra-O-methylquercetin and 5,7-dihydroxy-4-oxo-2-(3,4,5-trihydroxyphenyl)chroman-3-yl-3,4,5-trihydroxycyclohexanecarboxylate [101]. Both in vitro and in vivo experiments demonstrated the ability of elderberry extract to inhibit the development of influenza A and B viruses [101,102,103]. Moreover, following some experiments carried out in vivo, it was demonstrated that elderberries stimulated the immune response and effectively suppressed viral replication [104].

Besides phenolic compounds, lectins are another class of compounds commonly found in plant extracts. They can have antiviral activity by binding to host receptors or viral proteins, preventing their interaction [105]. In addition, anthocyanins are another class of compounds abundant in elderberries which have shown antiviral activity [15]. Elderberry extracts are also characterized by a high content of quercetin, a known in vitro inhibitor of the bovine coronavirus [106]. EDB extracts obtained by crude ethanol extraction of fruit compounds inhibited IBV (avian infectious bronchitis virus, a gamma-coronavirus that infects the respiratory tract of chickens) by several orders of magnitude. Viral inhibition decreased in direct proportion to decreasing EDB extract concentrations and increased with decreasing virus concentrations. In order to inhibit the development of the virus, the treatment with EDB extracts was applied before infection. However, this treatment was not sufficient to completely inhibit the virus. Electron microscopy of virions treated with EDB extracts showed the appearance of membrane vesicles and the compromise of viral envelopes. The obtained results demonstrated that the EDB extract can inhibit IBV at an early stage of the infection because the virion structure is compromised [99]. The host-cell receptors used by IBV were blocked by elderberry lectins. There is the possibility that the lectins will bind directly to the viral proteins, thus inhibiting the infection. Alpha-coronavirus and beta-coronavirus virions were inhibited by direct binding of lectins. On the other hand, IBV proteins, such as the spike protein, contain several sequences that signal the addition of N-linked oligosaccharides [107]. The effect of the S. nigra extract on the structure of the IBV virion may be due either to a substance that has not yet been discovered in it or to the synergistic effect of the compounds in the extract. Moreover, another option could be the presence of cholesterol chelators. The effect of losing the infectivity of other viruses has been demonstrated, acting on the viral membrane. [108]. The inhibitory activity given by the synergistic action of the substances was demonstrated with various combinations of treatments with EDB extract. When virus pretreatment was combined with post-infection treatment, complete virus inhibition occurred [99].

Human immunodeficiency virus (HIV) belongs to the genus Lentivirus in the family Retroviridae. Viral inhibition due to EDB extract was analyzed by using extract prepared in two different dilutions, which were incubated with the virus before being added to the tested cells. A significant decrease in virus infectivity was observed, and no viral load was detected after four to nine days of incubation. Other studies have shown that viral load in HIV patients can be reduced by a combination of elderberry extract and thymus extract, but further studies are needed. Flavonoids are the main compounds in elderberry extract that inhibit HIV infection. These could be a new alternative used in HIV-1/AIDS (acquired immunodeficiency syndrome) therapy, especially for simultaneous use with existing treatments [109,110].

Given the effectiveness of S. nigra products in the treatment of cold and influenza symptoms, recent studies have shown that they can be a potential adjunctive treatment for the COVID-19 virus, based on randomized placebo-controlled studies and meta-analyses [111,112,113]. The inhibition given by both the extract of wild elderberry fruits and the extract obtained from ‘Haschberg’ fruits was analyzed. The first of these showed a higher inhibitory capacity toward ACE2 and SARS-CoV2 RBD binding than the extract obtained from cultivated fruits. The promising results encourage further studies for new industrial anti-SARS-CoV2 applications of S. nigra [92].

Although additional studies are needed, considering the results obtained in numerous studies, it seems that S. nigra represents a promising treatment for the prevention and treatment of infections caused by viral and non-viral pathogens. In addition to the antibacterial and antiviral potential, the parasiticidal effect has also been studied. The efficacy of methanolic extracts of S. nigra fruits and leaves against Toxoplasma gondii tachyzoite was investigated by Daryani et al. [114]. The results showed that S. nigra has an acceptable efficacy in vitro and the parasiticidal effect of the fruit extract was significantly better than that of the leaf extract. EDB also exhibits low extraction costs in contrast to synthetic drugs [109].

4.4. Antidiabetic Properties

It has been found that glucose uptake in human skeletal muscle cells can be increased by anthocyanins in EDB extracts, mainly cyanidin-3-glucoside and cyanidin-3-sambubioside, procyanidins, and their metabolites. The inhibition of fat and sugar absorption from the intestinal tract, produced by EDB extract, could represent anti-obesity and antidiabetic effects for the human body, being a remedy for metabolic dysfunctions [12,115]. The increased content of dietary lipids can cause the occurrence of oxidative stress, which is involved in the occurrence of comorbidities related to cardiovascular complications and obesity, including insulin resistance and diabetes, even in the occurrence of cancer [116]. During the excess of nutrients (in obese organisms), macrophages and adipocytes generate large amounts of ROS at the level of visceral adipose tissue, which accumulates in the adipose tissue of the body. Nicotinamide adenine dinucleotide phosphate oxidases (NADPH oxidases, NOX) are activated following the release of large amounts of fatty acids from adipose tissue lipids. They can induce or stimulate the production of ROS. Moreover, chronic inflammation, hyperleptinemia, and low antioxidant defense contribute to obesity and oxidative stress [117]. The generation of ROS could be diminished following the use of EDB extract by decreasing the expression of NOX4, the major isoform of NOX in adipocytes. After using EDB extract to treat hypertrophied 3T3-L1 adipocytes, there was a significant decrease in NOX4 messenger RNA (mRNA) expression. Furthermore, the increase in the antioxidant defense efficiency of adipocytes could be achieved by mRNA regulation of the expression of antioxidant enzymes, such as SOD (superoxide dismutase) and GPx (glutathione peroxidase) [6].

EDB extract has been shown to be rich in anthocyanins, which have the ability to alleviate systemic inflammation and insulin resistance. Administration of EDB extract to obese mice fed a high-fat diet had an effect of reducing metabolic disorders by decreasing triglycerides and reducing systemic inflammation and insulin resistance. The extract also produced a reduction in serum monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor-α (TNF-α), which are pro-inflammatory markers of low-grade chronic inflammation [118]. It has been proven that polyphenolic compounds and triterpenic acids from elderberry extract are responsible for decreasing insulin resistance in diabetic rats. The correction of hyperglycemia or, respectively, the reduction of insulin secretion represent the mechanisms by which the extracts modulated glucose metabolism [119]. The progression of atherosclerosis was attenuated in mice fed with EDB extract because it contains large amounts of anthocyanins, which are able to decrease the total cholesterol of the aorta [120].

The study by Opriș et al. [121] showed that the blood glucose level can be decreased by using photosynthesized gold nanoparticles (NPs), using EDB extract (administered at doses of 0.3 mg/kg bw), and the most important effect is the reduction of inflammation and oxidative stress induced by hyperglycemia. Karthick et al. [122] also demonstrated that gold NPs biosynthesized by using natural extracts exhibit antidiabetic properties and also reduce inflammation at a dose of 0.5 mg/kg bw.

In vitro studies have shown that aqueous extracts of EDB have an insulin-like effect on glucose absorption. Moreover, the natural phenolic compounds extracted from EDB had positive effects on the regression of osteoporosis in male diabetic rats by improving bone mineral density, which is extremely low in diabetic rats. Diabetic rats showed low percentages of body fat, and the administration of phenolic compounds led to their improvement. After extending the results obtained on animals to human diabetic patients, it was concluded that a dietary intake rich in natural phenolic compounds could lead to the regression of diabetes complications and osteoporosis. The regression of osteoporosis due to the administration of EDB extract demonstrates the benefits of phenolic compounds used in the treatment of complications of chronic diabetes [123,124].

Along with other berries, S. nigra fruits can be considered effective inhibitors of pancreatic lipase, α-amylase, and α-glucosidase activity. Obesity and obesity-related disorders accompanied by oxidative stress, inflammation, and insulin resistance could be prevented or treated with elderberry extract. Moreover, the treatment of obesity and metabolic comorbidities could be performed through the ability of EDB extract to reduce the intestinal absorption of dietary lipids and carbohydrates by inhibiting the activity of digestive enzymes. Following in vitro and in vivo studies, researchers found that S. nigra fruit extract could be used for nutraceutical applications [6].

Although there are numerous studies, they are not exhaustive in terms of the effects of EDB on human health. New studies should be conducted to confirm the obtained results and investigate new effects (for example, studies performed on other pathogenic strains and other viral entities).

5. Extraction of Bioactive Compounds

The recovery of bioactive compounds from natural sources has been investigated by using numerous extraction methods because the extraction step is very important. The choice of extraction method depends on the extraction yield and the desired phenolic compounds’ purity because each method has its advantages and limitations. In general, extraction involves the sequential and systematic release of polyphenols from plant material. Soluble compounds are extracted by using an aqueous organic solvent. However, there are also insoluble bound polyphenol complexes, which are not extractable by organic solvents. These phenolic compounds are coupled to cell-wall polymers by ether, ester, and glycosidic bonds. As for bound phenolic acids, they are usually released by acid hydrolysis, alkaline hydrolysis, or both [125,126].

Anthocyanin extraction involves the use of slightly acidified alcoholic solvent (usually HCl), followed by concentration in vacuo, purification, and separation of pigments. It is desired to extract the compounds without the appearance of chemical changes. Because anthocyanins are sensitive to high temperatures and light, temperatures above 40 °C were avoided [127].

5.1. Conventional Solvent Extractions

In most cases, conditioning the fruit before extraction involves washing, drying, and obtaining the fruit powder. Turek and Cisowski [128] introduced a precursor step before extraction to remove lipid constituents. This consisted of extraction with petroleum ether on a water bath for 6 h, after which the plant material was drained and dried to apply the solvent extraction. Several solvents can be used to extract phenolic compounds: ethanol (most commonly used), methanol, isopropanol, acetone, diethyl ether, ethyl acetate, and mixtures thereof with water, which increase the extraction efficiency by increasing the contact surface between the solvent and the extracted phenolic compounds (solute). To obtain a high extraction yield from using conventional extraction, a large amount of solvent is required [129]. The conventional extraction technique uses high temperatures to obtain faster kinetics. This is obtained following the decrease in the viscosity of the extraction medium, a process that favors the penetration of the solvent into the plant’s matrix. Heat is applied with continuous stirring in the incubator or water bath, allowing unattended operation in a temperature-controlled environment [130,131].

The reported results showed that the solvent used can influence the efficiency of the extraction process (because the type of solvent and its polarity influence the solubility of phenolic compounds). Moreover, other important factors are the concentration of the solvent, ratio between sample and solvent, temperature used, pH of the solution, extraction time, degree of polymerization of phenolic compounds, and the interaction of phenolic compounds with other constituents of the plant matrix [14,132,133]. Using different extraction parameters on EDB samples, the overall extraction yields obtained had values between 38.21 and 45.25%. By varying the percentage of ethanol and the temperature, an inverse correlation was obtained with the ethanolic solvent concentration. The highest yield (>44%) was obtained at low ethanol concentration, and the extraction temperature did not influence the process. Low or high pH values improved the yield by 45% [14]. Vatai et al. [134] showed that the extraction of total phenols from EDB was more efficient by using 50% ethanol–water and acetone–water solutions as solvents and an increased temperature (to 60 °C). Therefore, the results are contradictory, and more studies are needed to optimize the extraction methods.

Anthocyanins are extracted by using a mixture of water and organic compounds as solvent because these compounds contain both hydrophilic and hydrophobic groups. Researchers [135] found that the use of 60% methanol allowed the extraction of a higher percentage of procyanidins and flavonoids, while changing the extraction solvent to aqueous acetone allowed higher yields of anthocyanins, hydroxycinnamic acids, and ellagitannins. They also studied what is the optimal concentration of ethanol to obtain a maximum extraction efficiency of anthocyanins from EDB, the effect of different acids (citric, hydrochloric, oxalic, and succinic) for solvent acidification, the optimal extraction time, and the number of extraction cycles. At a low pH, anthocyanins are in the stable red flavylium cation form. In contrast, anthocyanins are not stable in neutral or alkaline solutions. Aqueous acid solvents are used to break down the cell membranes in order to dissolve the pigments and favor the extraction. The results showed that the use of three extraction cycles (30 min extraction time) with 70% aqueous ethanol and 1% citric acid solvent allowed the extraction of 0.98% anthocyanins. Levels between 0.664 and 1.816% anthocyanins in EDB have been reported in the literature [136].

5.2. Soxhlet Extraction

Soxhlet extraction is one of the first techniques used for the extraction of phenolic compounds from plants. The principle of the method involves introducing the sample in a thimble-holder, and the phenolic compounds are extracted with the condensed solvent. The process is repeated several cycles. One of the biggest disadvantages of this method is the high cost, given by the large amount of solvent used. Moreover, the extraction is performed slowly, at the boiling temperature of the solvent, and this implies a thermal degradation of the extracted compounds. Moreover, excessive exposure to light and oxygen of the extract leads to the degradation of the thermal-sensitive compounds [132,137,138].

Paes et al. [139] applied Soxhlet extraction on blueberries, using different solvents. Acetone has a low selectivity and a high solvating power. The use of acetone in the extraction process led to the highest overall yield, while extraction with methanol and ethanol had similar yields. Regarding anthocyanins, Soxhlet extraction with ethanol allowed researchers to obtain the highest concentration.

5.3. Pressurized Liquid Extraction

The pressurized liquid extraction (PLE) method allows for the rapid extraction of target compounds and involves the use of a small amount of solvent [139,140]. PLE increases the extraction efficiency compared to conventional methods performed at room temperature and atmospheric pressure because high temperatures (50–200 °C) and pressures (3.5–20 MPa) are used [141]. Using high pressures, the temperature is maintained at values higher than the boiling point of the solvent. This allows easier penetration of the matrix and improved solubility of the phenolic compounds (mass transfer to the extraction solvent) [132,142,143].

Regarding the extraction of anthocyanins using the PLE method, the high temperatures could lead to the degradation of the anthocyanins, being thermally unstable. However, their extraction from blueberries, sweet potatoes, and red onions has been reported [144].

5.4. Microwave-Assisted Extraction

Microwave-assisted extraction (MAE) involves mixing the sample with solvent and heating it with microwave energy. The result is represented by an increase in the intracellular temperature, which results in the breaking of the cell walls, followed by the penetration of the solvent into the sample matrix. In this way, the molecules of interest are transferred into the solvent. The advantages that MAE has are low costs (requires a small amount of solvent), short extraction time, energy saving, temperature control, and obtaining an extract with higher antioxidant activity (compared to conventional methods) [129,132,145,146].

As with the PLE method, too-high temperatures can cause problems in the extraction of anthocyanins, being unstable compounds. To avoid overheating, small-to-moderate powers are used, but this increases the extraction time. There are data showing that an increase in temperature and/or microwave exposure time may decrease the yield of anthocyanins extracted from cherries [147], but there are also reports of increased yields of anthocyanins extracted from grape-skin peels [148]. Therefore, the matrix from which the extraction is performed is an important factor.

5.5. Ultrasound-Assisted Extraction

Ultrasound-assisted extraction (UAE), or sonication, is among the most efficient processes for extracting phenolic compounds from plants, whether we are talking about vegetables, fruits, herbs, or spices [149]. The hydration of the cell wall can be facilitated by ultrasound by widening its pores. There are also situations in which a mass transfer occurs following the rupture of the cell wall, allowing the extraction efficiency to increase [144].

The rupture of the plant matrix is caused by the phenomenon of acoustic cavitation. Briefly, the application of ultrasonic waves to a liquid matrix leads to the formation of bubbles. The bubbles continue to absorb energy up to their maximum limit, and further exposure to ultrasonic waves causes the bubbles to grow and collapse (cavitation). The energy produced is high and uncontrolled in every direction in the ultrasonic bath. During extraction, the release of energy produces temperature and pressure changes in the bubbles. Increasing the contact surface between solvent and matrix and improving mass transfer are favored by ultrasonic waves, which break up solid particles (disruption) and remove layers of inert material that can cause passivation. The main advantages of UAE method are the low costs due to the simplicity, rapidity, and low volume of solvents required; it is also an environmentally friendly method [129,150].

It has been found that the extraction yield of phenolic compounds from various plant sources can be improved by up to 35% by using this method [151]. UAE is a process that can be integrated with other extraction methods to improve extraction efficiency [152]. Azari et al. [153] compared the UAE and Soxhlet methods for extracting phenolic compounds from S. nigra leaves. The results showed that the UAE method allows for the extraction of a larger amount of total phenolic compounds than the Soxhlet method. Moreover, Oniszczuk et al. [154] showed that the most efficient method of extraction of phenolic compounds from functional foods with EDB was ultrasonic-assisted extraction at 60 °C. Dos Santos Nascimento et al. [155] showed that the UAE ethanolic extracts of EDB had higher antioxidant activity than the fermented ones (p < 0.01); the UAE extracts showed to be around 2 to 5 times more effective as antioxidants than those obtained by fermentation. In contrast, the EDB extracts obtained by fermentation had higher amounts of anthocyanins compared to those obtained by UAE.

The particle size of plant material did not significantly affect the efficiency of polyphenol extraction, since ultrasonic waves induced a reduction in particle size (approximately 5%) by changing the cell wall, leading to increased solvent penetration into the plant matrix and greater release of phenolic compounds, regardless of the initial size of plant particles. At the same time, the presence of a larger amount of plant particles can contribute to the attenuation of ultrasound waves, which can result in the restriction of the active part of the ultrasound inside the area located in the vicinity of the ultrasound emitter. Another disadvantage of this method could be the fact that the kinetics of extraction depend on the characteristics of the plant material [130].

5.6. Supercritical Fluid Extraction

Supercritical fluid extraction (SFE) is a process that uses fluids brought to the vapor-liquid critical point (supercritical point) from matrices that can be both solid and liquid [144]. SFE has been widely used to extract compounds from solid matrices such as food ingredients and phytopharmaceuticals. Carbon dioxide and water are the most widely used supercritical fluids. Water becomes supercritical fluid at 101.1 °C and a pressure of 217.6 atm, while CO₂ becomes a supercritical fluid (scCO₂) at 31.2 °C and a pressure of 72.9 atm. In addition, scCO₂ is generally considered safe (GRAS) and does not leave residue after extraction, simply by depressurization at room temperature. Since the polarity of CO₂ is lower than that of flavonoids (highly polar compounds), scCO₂ is not suitable for extraction, and this is why cosolvents (modifiers) such as ethanol and methanol are used for the extraction of polar compounds, so as to increase the efficiency of the extraction process [132]. However, Henriques et al. [156] showed that the yields of extraction were relatively low and ranged from 0.8 ± 0.1% (m/m) for fresh EDB with ethanol 96% (v/v) to 9.3 ± 0.1% (m/m) for dried EDB with the same solvent. In general, the results showed that scCO₂ pretreatment of elderberry and grape berries did not significantly influence the amounts of anthocyanins extracted but improved the extraction of total phenols [134]. Because anthocyanins are polar molecules, often the extraction solvent was a mixture of CO₂, ethanol and, in some cases, water in varying proportions, depending on the tissue and/or plant material. The mixture had to be supercritical, and the ethanol/water fraction was added in proportions that were soluble in scCO₂ at the pressure and temperature used. If larger amounts were used, the two phases (liquid and supercritical) coexisted, and the yield and recovery of the target compounds were affected. Following the contact between CO₂ and the water contained in the extraction matrix, carbonic acid was formed (which lowered the pH). Unlike solid–liquid extractions, solvent acidification in SFE extractions has been described as having a significant positive effect on the recovery of compounds of interest. The SFE process is applied mainly because the amount of organic solvent used is very small or absent (even residual solvents are removed from the products); additional reasons are that the extraction time and temperature time are reduced, oxidation processes during extraction are avoided, and selectivity is high. The biggest disadvantage of SFE is the high cost involved in the process. ScCO₂ pretreatment can remove non-polar components, and polar phenolic compounds can become more accessible [139,144].

5.7. Pulsed Electric Field Extraction

Extraction in pulsed electric field (PEF) is a recently studied non-thermal process on a large scale. This pretreatment of plant material involves the electro-permeabilization (also known as electroporation) of plant cells, leading to increased mass transfer through the extraction of molecules, even at a lower electric field. PEF uses a biological membrane to amplify the electrical signal because it is more conductive compared to the extracellular environment and cytoplasm [132,157].

5.8. High Hydrostatic Pressure Extraction

High hydrostatic pressure (HHP) is a process governed by Le Chatelier’s principle. The process can be thermal or non-thermal, and it involves applying an instantaneous and uniform pressure (100–1000 MPa) regardless of the size and geometry of the matrix. It is often used on flexible packaging materials filled with liquid or solid food products. Moreover, a transmission fluid (usually water) is used to transmit the pressure [132]. Currently, a large number of studies have focused on improving the extraction of phenolic compounds and other innovative applications of HHP.

5.9. Enzymatic Extraction

This technique has recently been used to extract phenolic compounds from several plant materials, especially for the production of new cosmeceutical and pharmaceutical agents. The method uses water as a solvent in which a series of enzyme-catalyzed reactions occur, thus resulting in enzymatic degradation of plant tissues. Because cell-wall digestion is desired, fermentation is based on the action of various ligninolytic and carbohydrate-metabolizing enzymes, as well as microorganisms, which facilitate the extraction of phenolic compounds and several other compounds [155]. To extract anthocyanins from inside the cellular vacuole, solvents must be able to cross the cell wall, membrane, cytoplasm, and vacuolar membrane before reaching them. For this, commercially available enzyme complexes composed of pectinases and cellulases have been used in various ratios [35].

The methods of extracting bioactive compounds show numerous variations, depending on the main targeted compounds. The factors that have a major contribution in the obtained yields are the method used, the solvent, the concentration, the ratio between the solvent and the plant material, the temperature, the extraction time, and the pH. Optimization is necessary to obtain high yields in the shortest possible time and with low costs.

6. Separation and Purification

The extraction of the bioactive compounds is followed by the separation and purification steps because the composition of the extracts is complex, and a selective extraction cannot be achieved. In addition, extraction processes often involve the use of organic solvents, which are toxic to human health. The use of EDB extracts in pharmaceutical preparations requires their high purification and the concentration of the main bioactive compounds.

6.1. Membrane Based Process

Since the phenolic compounds that must be concentrated have different molecular weights (290–1200 g/mol), the membrane process can be more or less efficient from the point of view of purification and separation of these compounds. Other factors that can contribute to the increase/decrease in efficiency are the membrane-molecular-weight cutoff (MWCO) dimensions and the driving force (transmembrane pressure) applied in the system. Ultrafiltration, nanofiltration, and reverse-osmosis methods are most often used. In general, the membrane process is successfully applied to aqueous solutions. In particular, the membrane process is effective for the separation and concentration of phenolic compounds from fruit juices [158,159]. The biggest disadvantages presented by membrane processes are represented by the reduced selectivity for phenolic compounds and the possibility of fouling the membrane (reduction of the size of the pores or their complete obturation). Membrane fouling with phenolic compounds was studied by Susanto et al. [160]. They proposed that there are different interaction mechanisms between the membrane materials and polyphenol molecules or polyphenol aggregates: hydrophobicity, hydrogen bonds, and the interaction of the benzene ring through π–π stacking when the membrane contains benzene rings (polyethersulfone).

6.2. Electrodialysis with Ultrafiltration Membrane

In comparison to pressure-assisted filtration (a process in which membrane pore clogging occurs frequently), in this method, an electric field is applied to facilitate the migration of ionic species from one solution to another, avoiding/reducing the occurrence of clogging during microfiltration. The separation of polyphenols by electrodialysis was first applied by Bazinet et al. [161] on tobacco extract. The method is effective only if the optimization is carried out. The factors that have a major contribution to the process are the material from which the membrane is made, the size of the membrane pores, and the electrodialytic parameters.

6.3. Precipitation of Anthocyanins

The purification of anthocyanins is mostly performed by precipitation in aqueous solutions with bivalent lead. The pH has a major influence on the precipitation process because anthocyanins are compounds with an amphoteric character. Unlike acidic media, neutral and alkaline media allow for better precipitation. To dissociate lead salts from anthocyanins, a lead salt was precipitated, which was later removed. For this, an alcoholic solution was used (either with HCl or with H₂SO₄), finally obtaining an alcoholic suspension of anthocyanins. The disadvantage is that there are other chemical species that precipitate in the presence of Pb²⁺ (organic acids, fatty, flavonoids, amino, phenolic, and tannins), which is why precipitation is only a stage of purification (sugars are not precipitated) [144].

6.4. Countercurrent Chromatography

Countercurrent chromatography (CCC) is a technique that can separate relatively large amounts of sample, using two immiscible liquid phases. High-speed countercurrent chromatography (HSCCC) is increasingly used to isolate anthocyanins from elderberries; a series of cyanidin derivatives has been obtained on a preparative scale. Compared to preparative HPLC (High-Performance Liquid Chromatography), HSCCC offers several advantages: it allows for a higher sample loading, the cost of separation is reduced because the solvents used are cheaper compared to solid-phase columns, and the method has mild operating conditions and can even ensure the isolation of labile compounds [144,162].

7. Extract Characterization

7.1. Determination of the Total Phenolic Content (TPC)

This method is based on the reducing power of the phenolic hydroxyl groups, which react with the Folin–Ciocâlteu phenol reagent to form chromogens that can be detected spectrophotometrically [153].

Domínguez et al. [14] determined the content of total phenolic compounds in EDB extracts obtained by several methods. Three best extracts were obtained through extraction with 50% of ethanol (3157 mg GAE/g, 3090 mg GAE/g, and 3000 mg GAE/g).

This is a rapid and inexpensive method, but it is not specific for phenolic compounds because other substances from the plant extract may interfere during the reaction (reducing sugars and ascorbic acid). Improvement could be made by extract purification prior to analysis by correcting the TPC value by subtracting the amount of the other components, or by using oxidative agents at a low concentration.

7.2. Determination of the Total Flavonoid Content (TFC)

The quantification of flavonoid compounds is performed according to their reaction with aluminum trichloride (AlCl₃), and the results are expressed as mg of quercetin/rutin equivalents per 1.0 g of a dry extract weight [163]. With this method, Goud and Prasad [164] showed that the TFC was recorded as 15 ± 1.12 mg rutin equivalents/g DW EDB.

This is a quick and inexpensive method, but in the extracts, there could be flavonoids that react with ACl₃ and have absorption maxima below 400 nm, or flavonoids that have absorption maxima above 440 nm

7.3. Determination of Total Anthocyanin Content (TAC)

The total anthocyanin content of EDB extracts can be measured spectrophotometrically by the differential method, using extraction at two buffer solutions (pH = 1 and pH = 4.5). The results were expressed in mg cyanidin-3-glucoside/100 g DW [165]. Stănciuc et al. [34] obtained TAC of 1652.92 ± 7.41 mg cyanidin-3-glucoside/g DW.

This is a rapid, simple, and inexpensive method, but it is subjected to interferences given by other light-absorption impurities that are present in the plant extract

7.4. Determination of Phenolic Profile/Phytochemical Composition

The phenolic compounds known for their biological activity were identified and quantified in Sambucus sp. extracts, using Liquid Chromatography (LC). UV–VIS or photo-diode array (PDA) detection may be used, but there are some inconveniences regarding the sensitivity and the compounds that may be eluted at the same time. Improved identification, selectivity, and sensitivity may be achieved with LC coupled with mass spectrometry (LC–MS) methods. Separation may be carried out with HPLC or Ultra Performant Liquid Chromatography (UPLC) systems with an RP–C18 column, at 25–40 °C. The composition of the mobile phase is usually a mixture of 0.1% formic acid/H₂O (solvent A) and 0.1–0.2% formic acid/acetonitrile or methanol (solvent B). A gradient elution is performed with a flow rate between 0.25 and 0.4 mL/min. A single-quadrupole or triple-quadrupole mass analyzer may be used for detection, operating in electrospray ionization (ESI)-negative mode.

The advantages of LC–MS methods are good recoveries (80–105%), low limits of detection (0.0005–0.05 μg/mL, and rapid and precise identification of the phenolic compounds [154,166,167]. The main disadvantages are high costs per analysis due to expensive equipment, solvents, reference substances, and highly qualified specialists.

7.5. Determination of the Antioxidant Activity (AA)

The antioxidant potential of food is an important parameter because the compounds responsible for it show the health benefits. Various phytochemical components have been identified as being responsible for the antioxidant properties [168]. A study by Dawidowicz et al. [44] showed that there is no direct correlation between the level of flavonoids in the extracts and their AA. However, the results presented by Seabra et al. [11,169] are contradictory because they showed that the antioxidant activity is due to the presence of flavonoids, anthocyanins (mainly Cyanidin-3-O-glucosides), and rutin. The amount of each compound strongly depends on the extraction conditions used.

7.5.1. ABTS—2,2-Azino-Bis(3-ethylbenzothiazoline-6-sulfonic Acid)

Trolox Equivalent Antioxidant Capacity (TEAC) is determined with 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical cation, produced by reacting ABTS solution with potassium persulphate (K₂S₂O₈). The results were expressed in Trolox equivalents (mmol Trolox/100 g FW), using a Trolox calibration curve. The TEAC result obtained by this method was 10.9 mmol Trolox/100 g [35].

7.5.2. DPPH—(2,2′-Diphenyl-picrylhydrazyl) Radical-Scavenging Activity

The DPPH method is considered to be simple and very fast. The principle of the method is based on the decolorizing action of antioxidant substances on the stable radical, colored violet. Antioxidants will react with DPPH due to their ability to donate hydrogen at a very rapid rate. Substances that are able to carry out this reaction can be considered radical scavengers and can cause a decrease in absorbance. Vitamin C, butylhydroxyanisole (BHA), and quercetin were used as reference substances [153,170]. The DPPH method can be used in aqueous and nonpolar organic solvents to screen both hydrophilic and lipophilic antioxidants [171].

Goud and Prasad [166] reported that the result of antioxidant activity of EDB extract determined by DPPH assay was 62.56 ± 1.12%. To avoid thermal degradation of the antioxidant compounds, the antioxidant efficiency was measured at ambient temperature.

7.5.3. ORAC Assay

The ORAC test depends on the damage of free radicals, in the presence of a fluorochrome, by changing the intensity of its fluorescence, which is an index of the degree of free-radical damage. In the presence of antioxidants, protection against fluorescence changes occurs. Azo initiators are thought to produce peroxyl radicals by heating, leading to loss of fluorescence. The inhibition capacity is expressed as concentration Trolox equivalents per 100 g/DW, and this is quantified by integration of the area under the fluorescence decay curve [172,173].

7.5.4. FRAP Assay

The principle of the FRAP method is based on the reduction of a ferric-tripyridyltriazine complex (Fe³⁺, yellow color) to its ferrous form, Fe²⁺ (blue color), in the presence of antioxidants, due to the electrons they donate. The FRAP reagent contained TPTZ (2,4,6-tripyridy-s-triazine) solution in HCl plus FeCl₃ and acetate buffer (pH 3.6). The results are expressed as the concentration of antioxidants having a ferric-reducing ability equivalent to that of 1 mmol/L FeSO₄. Adequate dilution is needed if the FRAP value measured is over the linear range of the standard curve [174].

When performing ABTS, DPPH, ORAC, and FRAP tests to measure the antioxidant activity of the methanol extract of guava fruits, comparable results were obtained. However, the FRAP technique showed the highest correlation with both ascorbic acid and total phenolic compounds. The reproducibility of the method was high, and other advantages are the simplicity and speed of testing [173]. Dominguez et al. [14] used these methods in the analysis of extracts obtained from elderberries. The results obtained were ABTS 5840 mg ascorbic acid/g DW, DPPH 56.0 mg Trolox/g DW, ORAC 193.4 mg Trolox/g DW, and FRAP 9786 µmol Fe²⁺/100 g DW.

All three methods are simple and fast but have a disadvantage in the sense that each one has a specific radical that reacts with antioxidants; therefore, some antioxidant compounds may not have the same activity for all of these radicals.

7.5.5. Cellular Antioxidant Activity Assay

The cellular antioxidant activity (CAA) assay is used to quantify the antioxidant activity of the extracts. Human HepG2 liver cancer cells are cultured in a 96-well plate. Subsequently, cells are pre-incubated with a fluorescent probe dye 2′,7′-dichlorofluorescein (DCFH), which can be readily oxidized to fluorescent dichlorofluorescein (DCF). The free-radical initiator 2,2′-azobis(2 amidinopropane) dihydrochloride (ABAP) is then added. Antioxidants prevent the formation of DFC by peroxyl radicals. Fluorescence is measured, and a decrease in it compared to the control sample indicates the antioxidant capacity of the compounds in the extract. This analysis method is important because, unlike the usual methods of antioxidant capacity analysis, it is representative from a biological point of view [175].

The use of EDB extracts in food products or to obtain food supplements or pharmacological preparations is conditioned by a good characterization of the extracts. Simple characterization methods are useful to follow the synergistic effect of the compounds in the extract, while advanced methods seek to know each component in the extract (especially for the pharmaceutical industry).

8. Conclusions

The methods of extraction and analysis of bioactive compounds have been investigated in numerous studies, but there are also conflicting results. This is mainly due to the fact that the chemical composition of the fruits can vary a lot, depending on many factors. Therefore, the optimization of the extraction method is necessary for the capitalization of these fruits in order to use the bioactive compounds as an additional source of phenolic compounds. The optimization of extract analysis methods could represent a basis for the development of new food products, functional foods, food supplements, and pharmaceutical and cosmetic products. In addition, more studies are needed to track which compounds have the most important role in both preventing and combating certain diseases, as well as their synergistic effect.

Although more research is needed, due to their complex composition, S. nigra fruits have many beneficial constituents to general health, implicated in the relief of a wide variety of health disorders. The results of the studies showed that not only raw fruits had bioactive compounds. The products obtained from elder fruits have preserved an important part of these useful substances (depending on the product obtained), and their introduction into the diet could have positive effects. The extracts could be used in a wider range of products, including products intended for certain categories of the population with special needs (children, the elderly, athletes, and people exposed to a toxic environment).