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Article

The Chemical and Cytotoxic Properties of Sambucus nigra Extracts—A Natural Food Colorant

by
Mariusz Banach
1,2,
Barbara Khaidakov
3,
Daria Korewo
3,
Magdalena Węsierska
3,
Wojciech Cyplik
1,
Joanna Kujawa
2,
Lilia M. Ahrné
4 and
Wojciech Kujawski
2,*
1
Greenvit Ltd., 27A Wojska Polskiego Avenue, 18-300 Zambrów, Poland
2
Faculty of Chemistry, Nicolaus Copernicus University in Toruń, 7 Gagarina Street, 87-100 Toruń, Poland
3
AronPharma Ltd., 3 Trzy Lipy Street, 80-172 Gdańsk, Poland
4
Department of Food Science, University of Copenhagen, Rolighedsvej 26, DK-1958 Frederiksberg, Denmark
*
Author to whom correspondence should be addressed.
Sustainability 2021, 13(22), 12702; https://doi.org/10.3390/su132212702
Submission received: 10 October 2021 / Revised: 8 November 2021 / Accepted: 10 November 2021 / Published: 17 November 2021
(This article belongs to the Special Issue Sustainable Food Waste Valorisation by Membrane Technology)
Browse Figures
Versions Notes

Abstract

:
Elderberry fruits contain valuable components that are beneficial to human health. Owing to the high content of anthocyanins, elderberry extracts can be used as natural food colorants with health-promoting properties. Moreover, the development of new natural food dyes enables the reduction in the use of synthetic ones. Anthocyanins-rich elderberry dry extracts (EDE) were prepared from the same batch of frozen fruits applying water extraction, followed by membrane separation (batch B1) or purification by column chromatography (batch B2) and then spray-dried. Subsequently, the content of anthocyanins, flavonols, and polyphenols was determined. The extract obtained with the application of column chromatography (B2) contained 33% anthocyanins, which is more than typical market standards, whereas the extract B1 contained 14% anthocyanins. The color properties of both extracts were also determined. Since water was used as an extractant, the extracts are well soluble in water and can therefore be used as a natural food colorant. The cytotoxic activity of both extracts was additionally determined using the MTT test and the tumor cells of the A-549, A-2780, MCF-7, Caco-2 line, and Peripheral blood mononuclear cells. It was revealed that both EDEs inhibit the proliferation of cancer cells, except those of the lung cancers. Extract B2 showed a much stronger cytotoxic effect. Additionally, both extracts stimulate the proliferation of peripheral blood mononuclear cells since they may have immunostimulatory properties.

1. Introduction

Nowadays, synthetic dyes are increasingly being replaced by natural ones. The plant-based dyes are not only harmless to humans but often exhibit health-promoting properties [1,2]. One of the dyes used in the food industry is anthocyanins (E163). These dyes are used to stain the products such as drinks, jams, fruit preparations, and candies. The advantages of anthocyanins as food colorants include high color intensity and health-promoting properties. The disadvantages are the color’s dependence on the pH and the low stability of anthocyanins at neutral pH. Anthocyanins are unstable and susceptible to degradation in solutions. The major internal factors that affect anthocyanin stability are pH, temperature, oxygen, metal ions, and enzymes [3,4]. Anthocyanins are the most stable and have the most intense red color under acidic conditions [5,6,7,8]. In products where anthocyanins are not stable, it is possible to use a dye based on pyranoanthocyanins (PACN). PACNs can be produced from berry extracts by heating extracts with a suitable cofactor, e.g., caffeic acid [9].
Elderberry (Sambucus nigra L.) is a species of large shrub in the Adoxaceae family. The fruits and flowers are traditionally used parts of this plant. Elderberry fruits are characterized by a high content of anthocyanins. The content of anthocyanins varies between 0.45 and 1.4%, depending on the variety, climatic conditions, and growing conditions. The elderberry anthocyanin profile is very distinctive. The vast majority are 3-O-sambubioside and cyanidin 3-O-glucoside. There are also small amounts of cyanidin-3-O-sambubioside-5-O-glucoside and cyanidin-3,5-di-O-glucoside [10,11,12,13].
Elderberry is traditionally used to treat colds and flu but also has a beneficial effect on the circulatory system. Sambucus nigra fruits are also helpful in diabetes. The elderberry extract and also purified anthocyanins stimulate glucose and fatty acid uptake in the skeletal muscle cells, which is suppressed in type 2 diabetes. Moreover, the anti-cancer effect of elderberry fruit has also been investigated [14,15,16,17].
Elderberry fruit extracts are typically standardized to the high content of anthocyanins and polyphenols. The manufacturing of such extracts is not possible without the use of separation methods.
Moreover, the fruit extracts are characterized by a high content of mono- and disaccharides. These substances cause a low glass transition temperature (Tg) and low sticky point temperature of the feed in a dryer. For that reason, the dried extract sticks to the walls of the dryer. In order to dry the extract efficiently, it is necessary to reduce the sugar content or to add a significant amount of a high molecular weight carrier, e.g., maltodextrin [18,19,20].
The most important methods of reducing the content of sugars, salts, and low molecular weight organic acids are the use of adsorption chromatography or membrane separations. The use of these techniques also enables manufacturers to greatly increase the content of polyphenols, including anthocyanins, in the extract.
Adsorption chromatography uses beds of various structures without ion exchange groups. Chromatography can be used as the only method of separation. The mechanisms of adsorptive macroporous resins include hydrophobic interactions, electrostatic forces, hydrogen bonding, complex formation, and π–cation interactions. In this technique, the column is fed with a large amount of extract, usually much greater than the volume of the bed. The column is washed with a suitable solvent (commonly water) to remove unbounded compounds. Substances bounded to the resin are eluted with a suitable solvent, usually a mixture of ethanol and water [21]. The extract can be dried after the evaporation of ethanol in a vacuum evaporator. The advantages of preparative chromatography include a high adsorptive capacity, the relatively low cost of resins, easy regeneration, and easy scale-up. The disadvantage is a high production cost due to the need of the use and regeneration of high-percentage ethanol [22,23,24].
Membrane techniques such as ultrafiltration allow the fractionation of extract components according to their molecular weight. Smaller molecules such as salts, mono- and disaccharides, and most organic acids pass through the membrane pores, while larger ones are concentrated in the retentate. This allows extracts to be enriched with specific groups of substances. In this case, the liquid flow is tangential to the surface of the membrane, which reduces the possibility of clogging the membrane. Depending on the size of the pores and the pressures used, membrane techniques are divided by microfiltration, ultrafiltration, nanofiltration, and reverse osmosis. Microfiltration is usually used for solid particle separation, ultrafiltration for the separation and purification of macromolecules. Nanofiltration membranes are characterized by small pore sizes, retaining molecules with molecular weights bigger than ca. 120 Da. Reverse osmosis, on the other hand, allows only water to pass through and concentrates almost all dissolved substances. The smaller pores of the membrane require applying the higher operating pressure.
Membrane techniques do not require significant heating and are therefore characterized by low energy consumption. Chemicals are used only for cleaning the installation—there is no need to add any chemical additives during the process itself. The advantages of membrane techniques are also relatively low production costs, convenient operation, and high efficiency [25,26,27,28,29].
The purpose of this study was the preparation, analysis, and evaluation of anti-cancer properties of the new on the market and commercially available anthocyanin-rich Sambucus nigra dry water-soluble extracts. The extracts were produced from the same raw material but using two different separation technologies—chromatography and membrane separation. To the best of our knowledge, it is the first comparison of the properties of S. nigra extracts produced from the same batch of raw material using different methods.

2. Materials and Methods

2.1. Chemicals

Catechin, chlorogenic acid, lipopolysaccharide (LPS from E. coli 0111:B4), Dulbecco′s Modified Eagle Medium (DMEM), heat-inactivated fetal bovine serum (FBS), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA), and cyanidin-3-O-glucoside was provided by Labmix24 (Hamminkeln, Germany). Acetonitrile and sodium carbonate were delivered from Honeywell (Morris Township, NJ, USA), and formic acid was provided by Merck (Darmstadt, Germany). The Folin–Ciocâlteu reagent and methanol were purchased from Chempur (Piekary Śląskie, Poland). All reagents and solvents were of analytical or HPLC grade and were used as received. Dimethyl sulfoxide (DMSO) was purchased from VWR International (Radnor, PA, USA). 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and phosphate-buffered saline (PBS) were purchased from Sigma-Aldrich (Saint Louis, MO, USA). For viability assay, stock solutions of two Sambucus nigra extracts were dissolved in DMSO (100 mg/mL).

2.2. Extracts Used for Research

The elderberry dry extracts were produced according to the technology developed at Greenvit Ltd. This technology is the company′s intellectual property, and therefore some information, e.g., important details about the extraction process or the type of resin used for the separation of extracts, cannot be disclosed.
The general scheme of extraction and separation is shown in Figure 1. Frozen elderberry was thawed, extracted with water, and filtered.
Batch B1: 20 L of the extract was subjected to membrane filtration. Subsequently, the extract was concentrated on a vacuum evaporator (Hei-vap value Digital, Heidolph Instruments, Schwabach, Germany) to 25% of dry weight. The membrane filtration process was carried out in a batch mode.
Batch B2: The extract containing ca. 400 g of anthocyanins was loaded on a chromatography column (Megan S. C. Gliwice, Poland) filled with 20 L of adsorption resin. Then, the column was washed with 120 L of water, and the adsorbed compounds were eluted using 40 L of 75% ethanol. The main fraction from the column was concentrated by applying a vacuum evaporator to 25% of dry weight.
Afterward, both extracts were dried on a disk spray dryer (Changzhou Fengqi Drying Equipment Co. Ltd., Zhenglu Town, China) without any carrier.

2.3. Identification and Quantification of Anthocyanins by HPLC-DAD Method

The identification and quantification of anthocyanins and flavonols of elderberry extract were carried out using a Shimadzu High-Performance LC Prominence system equipped with a photodiode array detector (Shimadzu Corp., Kyoto, Japan). The solid extracts were dissolved in 40% methanol acidified with 1% formic acid to obtain the concentration of ca. 1 mg/mL. The separations were carried out by injecting a 10-µL sample onto a Kinetex C18 column (Phenomenex Torrance, CA, USA) at 25 °C.
The gradient, phases, and separation conditions were taken from the United States Pharmacopoeia (USP). The anthocyanin content was calculated as the cyanidin 3-glucoside equivalent. The mobile phase consisted of solvent A (water, acetonitrile, and trifluoroacetic acid—19:1:0.06, v/v) and solvent B (methanol, acetonitrile, water, and trifluoroacetic acid—7:8:5:0.06 v/v). The isocratic elution started with 20% of solvent B (0–5 min), and then a linear gradient 20–40% B in 5–20 min, 40–90% B in 20–30%, and isocratic elution (90% B) in 30–35 min was applied. From 35 to 36 min, the gradient returned to the initial composition (20% B), and subsequently, it was held constant for the additional 4 min to equilibrate the column. The flow rate was equal to 0.8 mL/min. Anthocyanins and phenolic acids were identified on the basis of the characteristic UV-Vis spectrum. Anthocyanins have an absorbance maximum at about 520 and about 280 nm. The peaks were identified by the characteristic anthocyanin profile found in the USP.

2.4. Color Measurement

Color measurements were performed with a CR-5 desktop colorimeter (Konica Minolta, Tokyo, Japan). The extracts were dissolved at a concentration of 0.01% (w/v) in the following buffers:
(a)
0.025 M potassium chloride buffer, pH = 1.0;
(b)
0.4 M sodium acetate buffer, pH = 4.5;
(c)
0.03 M phosphate buffer, pH = 7.0.
The measurements were performed in a cuvette with an optical path length of 20 mm. Subsequently, the hue angle plot was prepared using the SpectraMagic NX software (Konica Minolta, Tokyo, Japan).

2.5. Cell Lines and Culture Conditions

2.5.1. Adherent Cells

The human lung epithelial carcinoma cell line (A-549), human ovarian cancer cell line (A-2780), human breast cancer cell line (MCF-7), and human colon carcinoma cell line (Caco-2) were purchased from the European Collection of Authenticated Cell Cultures (ECACC, Porton Down, England, UK). The human colon cancer cell line (HCT-116) was received from American Type Culture Collection (ATCC, Manassas, VA, USA).
A-549 cells were cultured in Nutrient Mixture Kaighn’s Modification medium, F12K (Gibco, Waltham, MA, USA) containing 10% FBS (Corning, New York, NY, USA) and 1% antibiotic-antimycotic solution (A/A; Sigma-Aldrich). A-2780 cells were cultured in Roswell Park Memorial Institute medium (RPMI 1640; Corning) supplemented with 10% FBS and 1% A/A, MCF-7 cells were cultured in Modified Eagle’s Medium (MEM; Corning) containing 10% FBS and either 1% A/A, or no antibiotics, respectively. Caco-2 cells were cultured in high glucose Dulbecco’s Modified Eagle’s Medium (DMEM; ATCC, Manassas, MD) containing 10% FBS, 1% MEM nonessential amino acids (NEAA; Biowest, Nuaillé, France), 1% penicillin and streptomycin solution (P/S; BioShop, Burlington, Ontario, Canada). HCT-116 were grown in McCoy’s 5A medium (Biowest) supplemented with 10% FBS and 1% P/S. All cell lines were incubated at 37 °C in an atmosphere of 5% CO2.

2.5.2. Non-Adherent Cells

Peripheral blood mononuclear cells (PBMC) were isolated from buffy coats (BC) obtained from the Regional Center of Blood Donation and Blood Therapy in Gdansk (Poland) according to standard procedure described elsewhere [1]. Briefly, BC was diluted, subsequently, the suspension was layered onto the Ficoll (1.077 g/L) and centrifuged at 800× g for 20 min at room temperature. The interface containing mononuclear cells was collected. PBMC were washed twice with PBS. The viability of the cells was measured by the trypan blue exclusion staining and was about 99%. The final concentration of the lymphocytes was adjusted to 106 cells/mL by adding complete RPMI 1640 (supplemented with 10% FBS and 1% antibiotic) to the single cell suspension.

2.5.3. Viability Assay

Cell viability was determined spectrophotometrically using the thiazolyl blue tetrazolium bromide (MTT) assay. The relative number of viable cells that are able to convert yellow soluble MTT to purple formazan crystals. The viable cell number was proportional to the production of water-insoluble formazan. The absorbance was measured spectrophotometrically at 563 nm after the dissolution of formazan crystals in DMSO [30]. Briefly, cells were seeded in a 96-well plate at the following density: A-549, MCF-7, and HCT-116 cells −9 × 103 cells/well for 24 h incubation and 7 × 103 cells/well for 72, 96, and 24 + 96 h. A-2780 cells −8 × 103 cells/well (24 h), 4 × 103 cells/well (72, 96 and 24 + 96 h) and Caco-2 cells −1 × 104 cells/well (24 h), 5 × 103 cells/well (72 h), 4 × 103 cells/well (96 and 24 + 96 h). PBMCs were seeded on a 96-well plate at a density of 2 × 105 cells/well (24, 72, 96 h). After overnight incubation, the cells were treated with various concentrations (1–400 µg/mL) of two Sambucus nigra extracts (dissolved in DMSO) and incubated for an indicated time. For 24 + 96 h incubation time, cells were first treated with extracts for 24 h, and the medium containing extracts was aspirated. The medium was replaced with a fresh one, without EDE, and incubated for an additional 96 h. Next, 20 µL of filter sterilized MTT (5 mg/mL) in phosphate buffered saline (PBS) was added to each well and incubated at 37 °C for 3 h. The medium with MTT was removed, and the formed formazan crystals were solubilized by the addition of 100 µL DMSO (VWR International). The absorbance was measured at 570 nm using EnVision 2103 Multilabel Reader (PerkinElmer, Waltham, MA, USA). Results were expressed as a % of untreated control. The IC50 values were calculated using a non-linear regression curve in the GraphPad Prism statistical software (San Diego, CA, USA). The results are the mean values of two different experiments performed in triplicates.

3. Results and Discussion

3.1. Analysis of Elderberry Extracts

A quantitative analysis of the extracts revealed the content of anthocyanins in both tested extract batches (B1, B2) at the level of 14.47% and 33.13%, respectively (Table 1). The polyphenols content was 20.52 and 48.55, respectively.
Regardless of the analytical method used (UV-VIS or HPLC), the results of the quantitative analysis of anthocyanins are similar for a given batch. The different content of anthocyanins in the extracts from the B1 and B2 batches results only from a different production method as the raw material used to produce both batches of extracts was exactly the same. Denev et al., using amberlite XAD-7 adsorption resin, obtained black elderberry dry extract with an anthocyanin content of 24.6% and polyphenols of 28.3% [31]. Apart from having a lower anthocyanin content, this extract was characterized by a different proportion of polyphenols to anthocyanins.
The anthocyanin and the flavonol glycosides profiles are consistent with literature data [1,13,31]. The chromatographic profile of anthocyanins was in both cases characteristic for the S. nigra anthocyanins and showed the presence of four cyanidin glycosides, i.e., cyanidin-3-O-sambubioside-5-O- glucoside, cyanidin-3,5-di-O-glucoside, cyanidin-3-O-sambubioside, and cyanidin-3-O-glucoside (Figure 2a). The extracts also contained the flavonols glycosides-rutin and quercetin-3-O-glucoside (Figure 2b), which are the main polyphenols of various cultivars and wild S. nigra [31,32,33,34]. The content of anthocyanins and polyphenols in extract B1 is similar to other commercially available extracts also obtained using membrane techniques. This extract contains ca. 15% anthocyanins and ca. 22% polyphenols [34]. To the best of our knowledge, this one is the only commercially available elderberry extract obtained using membrane techniques.
The extract obtained using adsorption chromatography contains more anthocyanins as well as flavonol glycosides than the extract obtained using membrane filtration (Table 1). The extract B2 contains 2.29 times more anthocyanins than the extract from batch B1, whereas the difference in the content of flavonol glycosides is greater −5.27 times for rutin and 4.23 times for quercetin 3-O-glucoside. The preferred adsorption of flavonol glycosides compared to anthocyanins on a nonpolar adsorption resin may be due to the lower polarity of flavonol glycosides compared to the polarity of anthocyanins.

3.2. Color Properties of EDEs

The use of elderberry fruit preparations as a natural food coloring is known, and the color properties of both extracts were determined. As shown in Figure 3 and Table 2, the color angle values are more similar between extracts from the same batches at pH 4.5 and 7.0 than between different batches at the same pH. The values at pH 1.0 deviate from the rest of the results and differ between batch 1 and batch 2, mainly in the value of the parameter b. The differences in the colorimetric data, primarily L* (luminosity/lightness) and C*ab, should be affected by the differences in the concentrations of anthocyanins [12,35]. The color of anthocyanin-containing extracts is also influenced by copigmentation with other polyphenols. This is the phenomenon occurring when the colorless copigment (usually other polyphenols) and anthocyanin molecules are combined by noncovalent interactions. This causes a change, usually an increase, in the color of the solution, as well as a slight shift in the absorbance maximum [36,37].

3.3. Effect of Elderberry Extracts on Cell Viability of Cultured Human Cancer Cell Lines

The effect of two batches of elderberry dry extract (EDE) on the proliferation of different cell lines was evaluated using tetrazolium assay (MTT). The multiple concentrations of extracts were used, and effective doses, expressed as IC50 value, were determined from the dose–response curve. As shown in Figure 4, Figure 5, Figure 6 and Figure 7, batches B1 and B2 of elderberry extract exhibited different cytotoxic potential towards the tested cell lines.
For A-549 cells, both batches showed no cytotoxic effect in all used concentration ranges, even after 96 h of incubation (Figure 4, Table 3).
In contrast to A-549 cells, for A-2780 and MCF-7 cells, both extracts demonstrated a dose-dependent decrease in the cell viabilities (Figure 5 and Figure 6). The IC50 values indicated stronger cytotoxic properties in the B2 extract, as evidenced by a lower IC50 value −147 µg/mL vs. 247 µg/mL, and 140 µg/mL vs. 268 µg/mL for A-2780 and MCF-7 cells, respectively, when incubated with the extracts for 72 h (Table 3). For other incubation times, the B1 extract showed no, or only slight, cytotoxic effect for both ovarian and breast cancer cells. Interestingly, the B2 extract decreased A-2780 and MCF-7 cell viabilities starting from 24 h of incubation (Figure 5 and Figure 6), showing a stronger inhibitory effect towards ovarian cancer cells, where the viability above 72 h of incubation dropped down to about 10%.
The most prominent difference in the cytotoxic properties of both extracts was demonstrated for colon cancer cells, HCT-116 (Figure 7). While the B1 extract had no inhibitory effect in all incubation times and concentration ranges, the B2 extract strongly inhibited cell proliferation to about 45% starting from 24 h of incubation. What is more, the B1 extract exhibited proliferation stimulatory properties towards HCT-116, starting from 50 µg/mL after 24 h and 72 h of incubation. Even though after 72 h and 96 h the B2 extract also slightly stimulated HC-116 cells proliferation in concentrations from 50–200 µg/mL, prolonged incubation resulted in cells viability decrease. What is worth noting is the fact that washing out a media with the extract B2 after 24 h of initial incubation and replacing it with a fresh media without extract, followed by 96 h of incubation, resulted in similar activity as constant incubation with the extract for 96 h. This indicates that the cytotoxic effect of the extracts starts within the first 24 h of incubation and a longer incubation only potentiates this effect. Further studies need to be conducted to explain this phenomenon as it may be related to the metabolic transformation of the extract in the cells and the induction/inhibition of membrane transport proteins.
The anti-cancer properties of anthocyanins and extracts with high anthocyanin content have been extensively researched [38,39]. Among the many beneficial properties of anthocyanins are anti-inflammatory, apoptotic, and antioxidant ones [40,41,42,43]. An example of the extract being a subject of anticancer studies is the hibiscus flower extract [44]. The main anthocyanin present in hibiscus flower extract, cyanidin 3-O-sambubioside, is also present in a significant amount in the elderberry fruit. In the studies of Maciel et al. [45], it was shown that the growth of HepG-2, Caco-2, and A-549 cells was inhibited by both hibiscus extract and anthocyanin fraction. However, no activity of elderberry extract towards A-549 cells was found in that study. This indicates the selectivity of natural extracts towards particular cancer cell lines. The elderberry extracts developed in our study also exhibited selectivity in their cytotoxic potential. Ovarian, breast, and colon cancer cells were more sensitive to both elderberry extracts than lung cancer cells. Moreover, it was observed the significant difference between the two batches of elderberry extracts regarding their cytotoxic properties. The extract from batch 2 exhibited a stronger cytotoxic potential towards A-2780, MCF-7, and HCT-116 than the extract from batch 1. This is explained by the ratio and number of active compounds in each extract as batch 1 extract contained 20.52% of total polyphenols and 15.24% of anthocyanins (UV-Vis method) compared to 48.55% and 34.28% for the batch 2 extract (Table 1). The selectivity of natural extracts towards different cancer cell lines was also found by Pereira et al. [46]. They showed that fractions of Sambucus nigra L. flower extracts exhibited cytotoxic activity in bladder carcinoma cells (T24) but not in MRC-5 normal human lung epithelial cells. The cytotoxicity of anthocyanin-rich extracts has also been described in other cell lines [47,48,49]. Triterpenoid acids, e.g., ursolic acid, are the components of elderberry fruit that also cause a cytotoxic effect on cancer cells [50]. However, these substances are practically insoluble in water, so the extracts should not contain these substances. Lectins are also listed as active substances in elderberry extracts [51]. The extract B2 should not contain lectins in their native form since proteins, including lectins, are denatured with high concentrations of alcohols [52]. In the case of the B2 extract, high-percentage ethyl alcohol was used for the elution.

3.4. Effect of Elderberry Extracts on Cell Viability of Cultured Peripheral Blood Mononuclear Cells

In order to evaluate the sensitivity of normal immunocompetent cells involved in the antitumor immune response, the cytotoxicity of elderberry extracts was also tested against healthy peripheral blood mononuclear cells (PBMC). Data presented in Figure 8 show that both extracts stimulated proliferation of the PBMC cells starting from a concentration of 100 µg/mL. Stronger stimulatory properties were observed for the B2 extract, where after 72 h of incubation, viability reached about 230% against about 170% for the B1 extract. Interestingly, for both extracts, incubation with cells for 96 h had almost no effect on cell proliferation.
PBMCs are a mixture of different normal blood cells, among which are lymphocytes, monocytes or macrophages, i.e., cells of the immune system. The stimulating effect of EDE extracts on PBMCs’ proliferation may confirm the stimulating effect on the immune system [53,54]. Barak et al. [55] showed that Sambucol Elderberry Extract activated the healthy immune system by significantly increasing inflammatory cytokine production (IL-1 beta, TNF-alpha, IL-6, IL-8). Elderberry extracts might therefore be beneficial to the immune system activation in the inflammatory process in healthy individuals or in patients with various diseases, including cancer. In studies on polyphenols from red and white wine, cytotoxic activity was found against both cancer cells and PBMC cells. Interestingly the wine polyphenolic extracts and resveratrol showed a weaker cytotoxic effect on PBMCs than on cancer cell lines [43]. Moreover, Ampasavate et al. [55] showed that different fruit extracts with high antioxidant potential had a potent cytotoxic effect on leukemia HL60 cells but was non-toxic to normal PBMCs. Fan et al. [56] demonstrated, in in vivo studies that the black rice anthocyanin extract promoted the PBMC cells population. In another research, Decendit et al. showed that malvidin-3-O-b glucoside (Malbg), the major grape anthocyanin, is bioactive with no toxicity on human PBMC cells [57].
In our study, it was demonstrated that both elderberry extracts, did not only show any cytotoxic properties towards PMBC cells, but they strongly stimulated their proliferation with a greater extent to the batch 2 extract. This indicates that the extracts can act as a cytoprotectant towards normal, healthy blood cells, and what is more, it can stimulate the immune response in order to protect other cells. In light of the obtained results, an interesting approach would be to test purified compounds as this will help to determine the specific chemicals responsible for the observed biological effects on cancer and normal cells.

4. Conclusions

It is known that anthocyanin-rich extracts possess many health-promoting properties, inhibit the proliferation of cancer cells, and stimulate immune cells. This work compares, for the first time, the properties of two extracts from Sambucus nigra fruits obtained by using two different technologies—adsorption chromatography and membrane filtrations—from the same raw material. The resulting extracts differ not only in the content of anthocyanins and other polyphenols but also in the ratio of anthocyanins to polyphenols and flavonols. Both extracts inhibit the proliferation of the A-2780 ovarian cancer cell line, the MCF-7 breast cancer cell line, and the HCT-116 colon cancer cell line but do not change the proliferation of the A-549 lung cancer cell line.
A-459 cells are not sensitive even to high extract concentrations or long incubation times. This indicates that the extracts show different cytotoxic activity depending on the origin of the cells.
This paper shows that in addition to their health-promoting properties, both extracts, owing to their strong colorant properties, can be also used as a natural colorant.

Author Contributions

Conceptualization, M.B., B.K. and W.K.; data curation, M.B. and W.C.; formal analysis, B.K., J.K. and W.K.; funding acquisition, M.B., W.C. and W.K.; investigation, M.B., B.K., D.K. and M.W.; methodology, M.B., B.K., D.K., M.W. and W.C.; supervision, W.K.; visualization, M.B. and J.K.; writing—original draft, M.B. and B.K.; writing—review and editing, M.B., J.K., L.M.A. and W.K. All authors have read and agreed to the published version of the manuscript.

Funding

This study was supported by the Ministry of Science and Higher Education (Implementation Doctorates Project 1st Edition—Doktorat Wdrożeniowy), National Centre for Research and Development POIR.01.01.01-00-1206/20. The financial support from the Polish National Agency for Academic Exchange (NAWA) under Grant PPI/APM/2018/1/00036/U/001 is kindly acknowledged.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data supporting the findings of this study are available within the article.

Acknowledgments

Natalia Sosnowska from BART, 05-250 Słupno, Poland is acknowledged for color analyses.

Conflicts of Interest

M.B. and W.C. are employees of GreenVit Ltd.

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Figure 1. Scheme of elderberry extracts manufacturing.
Figure 1. Scheme of elderberry extracts manufacturing.
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Figure 2. Composition of elderberry dry extract from batch B1, separated by using HPLC recorded at (A) 520 nm and (B) 353 nm.
Figure 2. Composition of elderberry dry extract from batch B1, separated by using HPLC recorded at (A) 520 nm and (B) 353 nm.
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Figure 3. Hue angle plot of analyzed extracts at different pH: A—EDE B1 at pH 7.0; B—EDE B2 at pH 7.0; C—EDE B1 at at pH 4.5; D—EDE B2 at pH 4.5; E—EDE B1 at pH 1.0; F—EDE B 2 at pH 1.0.
Figure 3. Hue angle plot of analyzed extracts at different pH: A—EDE B1 at pH 7.0; B—EDE B2 at pH 7.0; C—EDE B1 at at pH 4.5; D—EDE B2 at pH 4.5; E—EDE B1 at pH 1.0; F—EDE B 2 at pH 1.0.
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Figure 4. Cell viability evaluated by the MTT assay: the influence of EDEs on human cancer A-549 cells. Data are expressed as means ± SD from three independent experiments.
Figure 4. Cell viability evaluated by the MTT assay: the influence of EDEs on human cancer A-549 cells. Data are expressed as means ± SD from three independent experiments.
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Figure 5. Cell viability evaluated by the MTT assay: the influence of EDEs on human cancer A-2780 cells. Data are expressed as means ± SD from three independent experiments.
Figure 5. Cell viability evaluated by the MTT assay: the influence of EDEs on human cancer A-2780 cells. Data are expressed as means ± SD from three independent experiments.
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Figure 6. Cell viability evaluated by the MTT assay: the influence of EDEs on human cancer MCF-7 cells. Data are expressed as means ± SD from three independent experiments.
Figure 6. Cell viability evaluated by the MTT assay: the influence of EDEs on human cancer MCF-7 cells. Data are expressed as means ± SD from three independent experiments.
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Figure 7. Cell viability evaluated by the MTT assay: the influence of EDEs on human cancer HCT-116 cells. Data are expressed as means ± SD from three independent experiments.
Figure 7. Cell viability evaluated by the MTT assay: the influence of EDEs on human cancer HCT-116 cells. Data are expressed as means ± SD from three independent experiments.
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Figure 8. Cell viability evaluated by the MTT assay: the influence of batch 1 (B1) and batch 2 (B2) of elderberry extract on human peripheral blood mononuclear cells (PBMC) proliferation. Data are expressed as means ± SD from three independent experiments.
Figure 8. Cell viability evaluated by the MTT assay: the influence of batch 1 (B1) and batch 2 (B2) of elderberry extract on human peripheral blood mononuclear cells (PBMC) proliferation. Data are expressed as means ± SD from three independent experiments.
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Table
Table 1. Antioxidant activity and the content of marked components of Sambucus nigra extracts.
Table 1. Antioxidant activity and the content of marked components of Sambucus nigra extracts.
Batch IDTotal Polyphenols Calculated as Catechin (UV_VIS) [wt.%]Anthocyanins Calculated as Cy-3-Glu Chloride (HPLC) [wt.%]Anthocyanins Calculated as Cy-3-Glu (UV-VIS) [wt%]Rutin (HPLC) [wt%]Quercetin-3-O Glucoside [wt%]
B120.52 +/− 0.5314.47 +/− 0.3915.24 +/− 0.390.87 +/− 0.090.13 +/− 0.03
B248.55 +/− 1.7833.13 +/− 0.6234.28 +/− 0.784.59 +/− 0.160.55 +/− 0.08
Table
Table 2. CIELAB color coordinates of Sambucus nigra extracts at different pH for B1 and B2 batches.
Table 2. CIELAB color coordinates of Sambucus nigra extracts at different pH for B1 and B2 batches.
pHLaB
B1B2B1B2B1B2
7.056.9026.8415.8934.98−2.279.90
4.584.6274.3018.4732.651.302.01
1.065.6356.7763.2969.3738.8772.61
Table
Table 3. IC50 values for S. nigra dry extracts on analyzed cancer cell lines.
Table 3. IC50 values for S. nigra dry extracts on analyzed cancer cell lines.
24 h72 h96 h24 + 96 h
B1B2B1B2B1B2B1B2
A-549--------
A-2780--247147-132-
MCF-7--268140299--330
HCT-116-372-347-354-324
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Banach, M.; Khaidakov, B.; Korewo, D.; Węsierska, M.; Cyplik, W.; Kujawa, J.; Ahrné, L.M.; Kujawski, W. The Chemical and Cytotoxic Properties of Sambucus nigra Extracts—A Natural Food Colorant. Sustainability 2021, 13, 12702. https://doi.org/10.3390/su132212702

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Banach M, Khaidakov B, Korewo D, Węsierska M, Cyplik W, Kujawa J, Ahrné LM, Kujawski W. The Chemical and Cytotoxic Properties of Sambucus nigra Extracts—A Natural Food Colorant. Sustainability. 2021; 13(22):12702. https://doi.org/10.3390/su132212702

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Banach, Mariusz, Barbara Khaidakov, Daria Korewo, Magdalena Węsierska, Wojciech Cyplik, Joanna Kujawa, Lilia M. Ahrné, and Wojciech Kujawski. 2021. "The Chemical and Cytotoxic Properties of Sambucus nigra Extracts—A Natural Food Colorant" Sustainability 13, no. 22: 12702. https://doi.org/10.3390/su132212702

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