foods

Article Valorization of European Cranberry Bush (Viburnum opulus L.) Berry Pomace Extracts Isolated with Pressurized Ethanol and Water by Assessing Their Phytochemical Composition, Antioxidant, and Antiproliferative Activities

Lijana Dienaite˙ 1, Milda Pukalskiene˙ 1, Carolina V. Pereira 2, Ana A. Matias 2 and Petras Rimantas Venskutonis 1,* 1 Department of Food Science and Technology, Kaunas University of Technology, Radvilenu˛pl.˙ 19, Kaunas LT-50254, Lithuania; [email protected] (L.D.); [email protected] (M.P.) 2 IBET—Instituto de Biologia Experimental e Tecnológica, Food & Health Division Apartado 12, 2780-901 Oeiras, Portugal; [email protected] (C.V.P.); [email protected] (A.A.M.) * Correspondence: [email protected]; Tel.: +370-699-40978 or +370-37-456647



Received: 9 September 2020; Accepted: 2 October 2020; Published: 6 October 2020


Abstract: Defatted by supercritical CO2, Viburnum opulus berry pomace (VOP) was subjected to consecutive extraction with pressurized ethanol (E) and water (W) and yielded 23% of VOP-E and 8% of VOP-W, respectively. The major phytochemical groups covering 42 identified and quantified constituents in VOP extracts were organic and phenolic acids, iridoids, quercetin and (epi)catechin derivatives, flavalignans, procyanidins, and anthocyanins. The on-line HPLC-DPPH•-scavenging assay revealed the presence of numerous antioxidants. VOP-E had a higher total phenolic content, was a stronger antioxidant (equivalent to 0.77, 0.42, and 0.17 g trolox/g in oxygen radical absorbance capacity (ORAC), ABTS, and DPPH assays, respectively), and recovered the major part of phenolics from the pomace; however, both extracts demonstrated similar antioxidant activity in the cellular assay. VOP-E inhibited HT29 cancer cells at non-cytotoxic concentrations. The results of this study revealed that VOP contains valuable phytochemicals possessing antioxidant and antiproliferative activities. Consequently, extracts from VOP substances may be of interest in developing functional ingredients for healthy foods, nutraceuticals, and cosmeceuticals.

Keywords: guelder rose berries; pomace; pressurized liquid extraction; antioxidant capacity; antiproliferative activity; flavonoids

1. Introduction Many berry varieties are well-known fruits and are consumed as fresh foods or used as a raw material for various processed products such as jams, juices, beverages, and others. In addition to systematically studied, comprehensively valorized, and widely commercialized berries, there are many underutilized species, which produce the fruits with specific, rather unusual sensory quality and are therefore less acceptable by the consumers as fresh commodities. However, more recently, some of the underutilized berry species have attracted an increasing interest as promising sources of health beneficial bioactive compounds, which, after comprehensive evaluation and systematic valorization, might find wider applications in the production of both regular and, particularly, rapidly developing functional foods, nutraceuticals, and cosmeceuticals [1–5]. European cranberry bush (Viburnum opulus L.) berries, also called guelder rose berries, may be assigned to the underutilized fruits and emerging source of food grade products and ingredients [1].

Foods 2020, 9, 1413; doi:10.3390/foods9101413 www.mdpi.com/journal/foods Foods 2020, 9, 1413 2 of 23

The Viburnum genus contains 160–170 species, while guelder rose classified in the subsection Opulus contains five species. Many Viburnum spp. have become popular as garden or landscape plants, while some of them produce edible red colored fruits with specific astringent taste and unique, although not very pleasant, odor [2]. Some of the sweet cultivars may be consumed as fresh fruits; however, most of the guelder rose berries are mixed with other berries and processed into jams and juice [1]. Some berry phenolic compounds are responsible for organoleptic characteristics such as color and flavor; however, their health promoting properties have been in the focus of numerous studies during the last decades. The major class of phenolic compounds in V. opulus is represented by flavonoids including catechins, quercetin glycosides [1,3], proanthocyanidins [4], and phenolic acids [3]. Chlorogenic acid is present as the main phenolic acid [3], while ascorbic acid is another important guelder rose berry bioactive compound [5]. Beneficial effects of dietary phytochemicals have been reported in numerous studies. For instance, the diets rich in flavonoids and other phenolic compounds possessing antioxidant and anti-inflammatory activities have been associated with the reduced risk of various factors associated with metabolic syndrome [6], cancer [7], macular degeneration and eye-related diseases [8], as well as obesity and diabetes [9]. Therefore berry-rich diets are considered as effective means in disease prevention and even their treatment. Earlier studies on V. opulus reported its antioxidant activity [5] and strong radical scavenging capacity, mainly due to a high phenolic concentration [10], as well as anticancer activity [11–13]. For instance, Ceylan et al. [14] reported that the extracts of V. opulus fruits exhibit in vitro and in vivo anticancer activity against Ehrlich ascites carcinoma (EAC) bearing mice, and these effects were related to alkaloids, phenolic compounds, flavonoids, and/or their synergistic effects. Ulger with co-authors [13] demonstrated that the total number of tumor lesions were reduced in mice with 1,2-dimethylhydrazine-induced colon cancer when treated with drinking water enriched with guelder rose juice at the initiation stage of carcinogenesis. Zakłos-Szyda and Pawlik [11] reported anticancer properties of methanol and acetone extracts from the pomace, the juice, and polyphenolic fraction of V. opulus fruits in human breast (MCF-7) and cervical (HeLa) cancer cell lines. They suggested that chlorogenic acid, catechin, epicatechin, rutin, quercetin flavonoids, procyanidin B2, procyanidin trimer, and proanthocyanidin dimer monoglycoside present in the obtained preparations may contribute to anticancer activity. Koparal [12] revealed anticancer properties of guelder rose juice, which inhibited Caco-2 and HeLa cancer cell lines but did not have a significant effect in normal and A549 cancer cell lines; anticancer activity was related to the bioactive compounds such as chlorogenic acid and anthocyanins. Sustainability and waste reduction are among the most challenging issues in fruit processing. For instance, winemaking or pressing juice generates 10–35% of berry pomace, consisting mainly of skins, seeds, and pulp residues. Currently large amounts of berry pomace are discarded as a waste or inefficiently used as a by-product for composting and animal feeding [15]. Considering that polyphenols are distributed in berries unevenly, i.e., approx. 10% of the total polyphenols are present in pulp, 28–35% in skin, and 60–70% in seeds [16], a larger fraction of these bioactive compounds is lost with the pomace after pressing the juice. Therefore, valorization of berry pomace has become a topical issue of research [15]. For instance, the application of high pressure fractionation of dried guelder rose berries and its pomace gave several valuable fractions, namely, polyunsaturated fatty acid rich lipids, higher polarity polyphenolic, and insoluble dietary fiber products [17]. Consequently, berry pomace containing valuable nutrients and being exposed to minimal pesticide levels have been recognized as an attractive source of new food-grade ingredients. Dried and ground pomace powder can be used directly as food ingredient or processed into higher added value bioactive phytochemical preparations for functional foods, nutraceuticals, and other applications. In the latter case, green and preferably fast extraction techniques have become among the most important research trends for producing higher quality products (extracts) for wider and safer uses [18]. Supercritical fluid extraction with CO2 (SFE-CO2) and pressurized liquid extraction (PLE) Foods 2020, 9, 1413 3 of 23 with environment friendly solvents, usually ethanol and water, have been widely used for biorefining berry pomace during last few years [15]. So far, as development of health beneficial ingredients is one of the most important tasks in valorizing berry pomace, comprehensive evaluation of their bioactivities, including antioxidant potential, is a very important issue. Bioactivities related to the antioxidant properties are usually measured by extracellular and cellular methods. A wide range of the in vitro assays have been proposed for this purpose and therefore their selection should be well justified. Determination of + DPPH•/ABTS• -scavenging and oxygen radical absorbance capacity (ORAC) have been frequently used chemical extracellular assays, while the cellular antioxidant activity (CAA) assay was more recently introduced. Chemical methods, although not always correlating with the in vivo results, are useful for fast screening [19], while CAA is more relevant to the processes occurring in vivo [20]. It involves the studies of phenolic compounds nature, their absorption, distribution, metabolism, and synergistic effect between plant-derived secondary metabolites. Previous studies demonstrated that berry processing by-products consisting of fruit skin, pulp, and seeds contain high amounts of dietary fiber, vitamins, minerals, phytochemicals, and antioxidants, i.e., natural nutrients, which may play an important role in preventing various degenerative diseases and improving human wellbeing [15]. For instance, there is an increasing evidence supporting the hypothesis that dietary antioxidants may assist the endogenous antioxidative system of protection against oxidative stress by mitigating damaging effects of excessive reactive oxygen species, which may form due to the adverse impact of environmental pollution, inflammatory processes, and other factors. Considering the increasing interest in guelder rose berry cultivation and processing and still limited studies on biochemical composition and bioactivities of its by-products, this study aimed at a more systematic valorization of bioactive ingredients recovered from berry pomace by PLE with green solvents, ethanol, and water. The following characteristics were determined: (1) total phenolic and + anthocyanin content; (2) DPPH•/ABTS• -scavenging and ORAC; (3) composition of phytochemicals and their radical scavenging capacities by HPLC-DPPH•-scavenging on-line; (4) CAA and cytotoxicity in Caco-2 cells; (5) cell growth activity in a panel of human cancer cells HT29.

2. Materials and Methods

2.1. Chemicals and Cells

Folin–Ciocalteu reagent, Trolox, DPPH• (98%), gallic acid, KH2PO4, KCl, NaCl, formic acid (98%), ABTS, (98%), K2S2O8, ammonium hydroxide, AAPH, HPLC grade and LS-MS grade acetonitrile, and HPLC grade formic acid (98%) were obtained from Sigma-Aldrich (Darmstadt, Germany). Disodium fluorescein, Na HPO 2H O, and ethanol (99.9%) were from TCI Europe (Antwerp, 2 4· 2 Belgium), Riedel-de-Haen (Seelze, Germany), and Scharlau (Barcelona, Spain), respectively. Na2CO3, 20,70-dichlorofluorescin diacetate (DCFH-DA), and quercetin (95%) were from Sigma-Aldrich (St. Quentin Fallavier, France). Ultra-pure water was produced in a Simplicity 185 system (Millipore, Billerica, MA, USA). Analytical grade methanol was purchased from StanLab (Lublin, Poland). The standards used for UPLC analysis (malic acid, fructose, glucose, sucrose, quinic acid, rutin, citric acid, chlorogenic acid) were from Supelco Analytical (Bellefonte, PA, USA); catechin, proanthocyanidin B2, and quercetin-3-O-glucoside were from Extrasynthese (Genay Cedex, France). Human Caco-2 and HT29 cell lines were purchased from DSMZ (Braunschweig, Germany) and ATCC (Manassas, VA, USA), respectively. The cell culture medium and supplements were purchased from Invitrogen (Gibco, Paisley, UK). Phosphate-buffered saline was obtained from Sigma-Aldrich (St. Louis, MO, USA) and cell viability was assessed using a CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega, Madison, WI, USA). Foods 2020, 9, 1413 4 of 23

2.2. Plant Material and Extraction Procedure Dried Viburnum opulus berry pomace (VOP) was kindly donated by BestBerry (Auce, Latvia). According to the information provided by the company, mature berries were pressed in order to remove the juice within 4 h after harvesting; the residue was immediately subjected to drying. Similar preparatory and extraction procedures were applied for VOP as previously reported [17,21]. Briefly, dry VOP was grounded in a laboratory mill Vitek (An-Der, Austria) by using a 0.5 mm size sieve. VOP powder was extracted by SFE-CO2 in a 500 mL extractor (Applied Separations, Allentown, PA, USA) to remove lipophilic fraction. For PLE, 10 g of defatted pomace powder was mixed with 4 g of diatomaceous earth, placed in 66 mL extraction cells, and consecutively extracted with ethanol (VOP-E) and water (VOP-W) in an accelerated solvent extraction apparatus ASE350 (Dionex, Sunnyvale, CA, USA) at a constant 10.3 MPa pressure and temperature (70 ◦C for VOP-E and 120 ◦C for VOP-W) using 15 min static and a 90 s purge time for each extraction cycle (total 3 cycles, in one run, 120 mL H2O and EtOH were used). Ethanol was evaporated in a Rotavapor R-114 (Büchi, Flawil, Switzerland), while residual water was removed by freeze-drying in a Maxi Dry Lyo (Hetto-Holton AIS, Allerod, Denmark). The extracts were weighed and stored at 18 C in a freezer until further analysis. − ◦ 2.3. Proximate Analysis The chemical composition of VOP was determined according to the procedures established by the Association of Official Analytical Chemists (AOAC, 1990) [22]: moisture by drying at 105 ◦C to constant weight; ash by mineralizing in a muffle furnace F-A1730 (Thermolyne Corp., Dubuque, IA, USA) at 500 ◦C for 3 h; proteins by the Kjeldahl method in a nitrogen analyzer (Leco Instruments Ltd., Mississauga, ON, Canada) using a conversion factor of 6.25; crude lipids by Soxhlet extraction with hexane for 6 h. The rest of the dry matter was assigned to carbohydrates. Each determination was carried out in triplicate.

2.4. Total Phenolic Content (TPC) and Antioxidant Capacity Folin–Ciocalteau (TPC), DPPH, ABTS, and ORAC assays were used for evaluating antioxidant potential of VOP extracts [22]. TPC was performed as described by Singleton and Rossi (1965) with some modifications [23]. A total of 30 µL of extract solutions was mixed with 150 µL Folin–Ciocalteu reagent (1:10 in distilled water) with 120 µL 7% Na2CO3 solution in a 96-well microplate and the absorbance was measured at 765 nm after 30 min in a FLUOstar Omega Reader (BMG Labtech, Offenburg, Germany). Gallic acid solutions (10–250 µg/mL) were used for the calibration curve. TPC was expressed in mg of gallic acid equivalents in g of extract and pomace, GAE/g DWE (dry weight of extract) and DWP (dry weight of pomace), respectively. + Re et al.’s (1999) method with some modifications was used for ABTS• decolourisation assay [24]. + + A total of 6 µL of sample was added to 294 µL of ABTS• working solution. The ABTS• solution was prepared by mixing 50 mL of ABTS (2 mM) with 200 µL of potassium persulfate (70 mM); the mixture was kept in the dark for 15–16 h before use. The working solution was prepared by diluting with PBS (prepared from 8.18 g of NaCl, 0.27 g of KH PO , 1.78 g of Na HPO 2H O, and 0.15 g of KCl in 1 L 2 4 2 4 × 2 of distilled water) to obtain the absorbance of 0.800 0.030 at 734 nm. The absorbance was measured ± in a 96-well microplate using a FLUOstar Omega Reader during 30 min at 734 nm. A series of Trolox solutions (399–1198 µM/L) were used for calibration. The results were expressed as µM TE/g DWE and DWP. Brand-Williams et al.’s (1995) method was applied for the DPPH•-scavenging assay with some modifications [25]. A total of 8 µL of sample was mixed with 292 µL of DPPH• methanolic solution (0.0059 g/250 mL). The working solution was prepared by diluting with methanol to obtain the absorbance of 0.7 at 515 nm. After mixing, the microplate was placed in a reader, shaken for 30 s, incubated for 60 min, and the absorbance read at 515 nm. The results were calculated as in the ABTS assay. Foods 2020, 9, 1413 5 of 23

ORAC assay was performed by using fluorescein as a fluorescent probe and AAPH as a peroxyl radical generator [26]. A total of 25 µL of sample was pipetted into 150 µL (14 µM) fluorescein solution, and after incubation during 15 min at 37 ◦C, 25 µL of AAPH (240 mM) were added. The fluorescence was recorded every cycle (in total, 120 cycles) using 485 excitation and 530 emission fluorescence filters. Antioxidant curves (fluorescence versus time) were first normalized, and from the normalized curves, the net area under the fluorescein decay curve (AUC) was calculated by the following formula: AUC = (1 + f f + f f ... fi f ) CT (1), where f is the initial fluorescence reading at 0 min, f is the 1 0 2 0 0 × 0 i fluorescence reading at time i, and CT is cycle time in minutes. The final ORAC values were calculated by using the regression equation between the Trolox concentration and the net AUC. Trolox solutions (0–250 µM) were used for the calibration. The results were expressed as µM TE/g DWE and DWP. Each measurement was carried out in six replicates.

2.5. HPLC-DPPH•-Scavenging On-Line

HPLC-DPPH•-scavenging online analysis was carried out on a Waters HPLC system (Waters Corporation, Milford, MA, USA) as previously described [21] with minor changes. The Hypersil C18 analytical column (250 0.46 cm, 5 µm; Supelco Analytical, Bellefonte, PA, USA) was used for × compound separation. The mobile phase consisted of 0.4% formic acid in water (A) and acetonitrile (B). The elution profile was at the start point 90% A, then changing to 60% in 45 min, after that, in 50 min A was decreased to 5%, then in 53 min increased to 90% A, then it was held at 90% for 3 min, and in 2 min it was returned to initial conditions and the column was equilibrated for 5 min. The decrease of absorbance after the reaction of radical scavengers with DPPH• was measured at 515 nm with the variable-wavelength Shimadzu SPD–20A UV detector (Shimadzu Corporation, Kyoto, Japan), while the identification of compounds was performed by using the Waters Acquity UPLC system (Milford, MA, USA).

2.6. UPLC-ESI-QTOF-MS Analysis Quantitative and qualitative analysis was performed with an ultra-performance liquid chromatograph (Waters Acquity UPLC system, Milford, MA, USA) equipped with a quadrupole time-of-flight tandem mass spectrometer (MaXis 4G Q-TOF-MS). Data acquisition and instrument control were performed by using HyStar 3.2 (SR2 software, Bruker Daltonics, Bremen, Germany) [21]. Briefly, samples were eluted with a gradient of solvent A (1% formic acid in ultrapure water) and B (acetonitrile) on a 1.7 µm, 100 mm 2.1 mm i.d. Acquity BEH C18 column (Waters) over 14 min at a × flow rate of 0.4 mL/ min. The injection volume was 1 µL and column temperature was maintained at 40 ◦C. Gradient elution was performed as follows: 95% A in 0–4 min, 95–90% A in 4–6 min, 90–70% A in 6–10 min, 70–5% A in 10–12 min, and 5–95% A in 12–14 min. The Q-TOF mass spectrometer was equipped with an electrospray ionization (ESI) source in a negative-ion mode. ESI-Q-TOF-MS spectra were recorded in the mass range of 80–1200 m/z. Two scan events were applied, namely, full-scan analysis followed by data-dependent MS/MS of the most intense ions. The data-dependent MS/MS used 20–30.0 eV as collision energies (fragmentation was performed by using collision induced dissociation (CID)); the capillary voltage was +4000 V; end plate offset 500 kV; flow rate of drying (N2) gas 10.0 L/min; nebulizer pressure 2.5 bar. The same column was used for sugars, while separation was performed using 75% acetonitrile, 25% water, and 0.1% ammonia hydroxide as mobile phase at isocratic conditions at 0.35 mL/min, 10 min, and 35 ◦C. The method for anthocyanins is described elsewhere [27]. The quantification was performed using cyanidin-3-glucoside as an external standard. The amounts of individual identified compounds were expressed as mg/100 g DWP. A quantitative analysis of other compounds was performed by using calibration curves (0.1–10 µg/mL) of available standards (malic acid, rutin, quinic acid, citric acid, catechin, proanthocyanidin B2, chlorogenic acid, quercetin-3-O-glucoside; for sugars glucose, fructose and sucrose were used). Concentrations of phytochemicals were determined from the peak areas, using the linear regression equations of calibration curves obtained from six concentrations of each standard. Quantification trace for quantified Foods 2020, 9, 1413 6 of 23 compounds were detected by calculating limit of detection (LOD) and limit of quantification (LOQ) values [28]. The results were expressed as mg/100 g DWE and g/100 g DWP.

2.7. Cell Culture and Sample Preparation Water and ethanol VOP extracts were solubilized in DMSO (200 mg/mL) and ethanol (100 mg/mL), respectively, and stored at 20 C protected from light. Cell-based assays were performed using a − ◦ maximum concentration of solvents, 1% and 5% for DMSO and ethanol, respectively. Cell lines were cultured in RPMI-1640 medium supplemented with 10% of heat-inactivated fetal bovine serum (FBS) and 1% penicillin-streptomycin (PS), in the case of Caco-2. Cells were maintained at 37 ◦C with 5% CO2 in a humidified incubator and routinely grown as a monolayer in 75 mL culture flasks.

2.8. Cytotoxicity Assay in Caco-2 Cell Monolayer Cytotoxicity was assessed using confluent and non-differentiated Caco-2 cells as a model of the human intestinal epithelium, as previously described [29]. Briefly, Caco-2 cells were incubated with the samples diluted in the culture medium (RPMI supplemented with 0.5% FBS). Cell viability was assessed using CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega, Madison, WI, USA) containing the MTS reagent, according to the manufacturer’s instructions. The absorbance was measured at 490 nm using a Spark® 10 M Multimode Microplate Reader (Tecan Trading AG, Männedorf, Switzerland) and cell viability was expressed in terms of percentage of living cells relative to the control. The experiments were performed in triplicate.

2.9. Cellular Antioxidant Activity (CAA) Assay The CAA assay was carried out by the procedure of Wolfe and Liu (2008) [20]. In Caco-2 monolayers, 50 µL of PBS, sample and standard (quercetin, 2.5–20 µM) solution, and 50 µL of DCFH-DA solution (50 µM) was added and incubated (60 min at 37 ◦C, 5% CO2). Afterwards, 100 µL of AAPH (12 mM) solution was added to each well containing PBS/quercetin standards/samples, while 100 µL of PBS was added to the blank wells. Fluorescence kinetics was recorded every 5 min during 60 min by using a Microplate Fluorimeter FLx800 (Bioteck Instruments, VT, USA) (excitation and emission wavelengths of 485 nm and 540 nm, respectively). CAA values were expressed as µM of QE/g of extract of independent experiments performed in triplicates.

2.10. Antiproliferation Assay The antiproliferative effect of VOP and standard compounds was evaluated in HT29 cells, as described elsewhere [30]. Briefly, cells were seeded at a density of 1 104 cells/well in 96-well culture × plates. After 24 h, cells were incubated with different concentrations of the samples diluted in the culture medium. Cell proliferation was measured after 24 h using the MTS reagent, as mentioned above. Results were expressed in terms of percentage of living cells relatively to the control. Three independent experiments were performed in triplicate.

2.11. Statistical Data Handling All experiments were repeated at least three times and the results obtained are presented as means standard deviations (SD). Statistical unpaired t test with p < 0.05 was used to identify ± significant differences.

3. Results and Discussion

3.1. Proximate Composition, Total Yield, and Antioxidant Capacity Berry pomace are highly heterogeneous materials, which are composed of macronutrients (mainly proteins, fats, and carbohydrates) and a large variety of micronutrient phytochemicals such as phenolic acids, flavonoids, and glycosides. The latter compounds belong to the class of polyphenols and are Foods 2020, 9, 1413 7 of 23 promising substances for developing natural antioxidants and functional food ingredients with various bioactivities. In this study, a proximate analysis of VOP was performed. The content of protein, fat, water, and ash in VOP was 16.82 1.54%, 26.24 0.59%, 7.76 0.16%, and 1.21 0.03%, respectively. ± ± ± ± Other macrocomponents should consist of various carbohydrates. For comparison, Polka et al. [31] reported approximately 3-fold and 2.5-fold lower amounts of protein and fat, respectively, and a 2.4-fold higher amount of ash in dried guelder rose berries. Polyphenolic antioxidants and ascorbic acid, which are abundant in various fruits, are the most valuable constituents of berries, firstly, as health beneficial compounds and, secondly, as natural additives, which in some cases may be used to replace synthetic preservatives and colors. Therefore, the evaluation of an antioxidant potential of VOP was among the main objectives of this study. The antioxidant capacity of extracts (expressed for DWE) and the recovery of antioxidants from the dry VOP (expressed for DWP) were determined for each assay for this purpose. Both characteristics are important for pomace valorization, the former evaluates antioxidant properties of the product (extract, fraction), while the latter evaluates the efficiency of the extraction process. For instance, a low yield extract may possess stronger antioxidant activity, while in the case of high yields, better recovery of antioxidants from the plant may be achieved, although the extracts may show lower antioxidant capacity due to the dilution of active constituents with the neutral ones. An extraction with ethanol resulted in an approximately three times higher total extract yield comparing to water (Table1); furthermore, antioxidant capacity values of VOP-E were significantly higher than those of VOP-W in the all assays.

Table 1. Yield, total phenolic content, and antioxidant capacity of guelder rose berry pomace extracts.

Characteristic Material VOP-E VOP-W DWE 3085 55.53 a 2544 33.30 b ORAC, µM TE/g ± ± DWP 730.1 10.41 a 209.1 6.75 b ± ± a b + DWE 1667 32.12 1282 19.33 ABTS• , µM TE/g ± ± DWP 394.7 13.22 a 105.4 8.36 b ± ± DWE 667.5 20.74 a 496.9 18.58 b DPPH•, µM TE/g ± ± DWP 158.0 9.51 a 40.85 3.21 b ± ± DWE 154.2 3.33 a 131.6 2.18 b TPC, mg GAE/g ± ± DWP 36.50 1.91 a 10.82 0.95 b ± ± Yields,% 23.67 0.11 a 8.22 0.13 b ± ± Values represented as mean standard deviation (n = 6); a, b: the mean values in the rows for Viburnum opulus pomace ethanol (VOP-E) and water± (VOP-W) extracts (dry weight of extract (DWE)) and initial material (dry weight of pomace (DWP)) followed by different letters are significantly different (p < 0.05).

In addition, ethanol, due to remarkably higher yield, recovered a 3.4–3.8 times higher amount of polyphenolics from the pomace (DWP). This could be explained by the higher content and wider spectra of various soluble components in ethanol during the first step of PLE (Table2). Despite the differences in antioxidant capacity values determined by the applied methods, which may be explained by different chemical structures of used radicals for the assay and the peculiarities of reaction mechanisms, the scavenging capacity and TPC of VOP-W were also quite high. Foods 2020, 9, 1413 8 of 23

Table 2. Identification and quantification of individual phenolic compounds of guelder rose berry pomace extracts.

Molecular t m/z, VOP-E VOP-W VOP-E VOP-W Peak No. Compound R MS/MS (min) [M–H] Formula − mg/100 g DWP mg/100 g DWE 1 Malic acid a,d,m,2 C H O 0.4 133.0144 71; 115 304.2 0.25 a 156.1 0.003 b 1285 5.6 a 1899 10.88 b 4 6 5 ± ± ± ± 2 Citric acid a,d,s,2 C H O 0.4 191.0199 87; 111; 127; 173 - 9.95 0.001 - 121.0 0.45 6 8 7 ± ± Dihydroferulic acid 3 C H O 0.6 371.0982 175 - 3.61 0.003 - 43.92 0.21 4-glucuronide b,c,d,l 16 20 10 ± ± 4 Procyanidin derivative b,c,d,p C H O 0.7 593.1297 289; 305; 423; 467 - 1.36 0.001 - 16.55 0.10 30 26 13 ± ± 5 Quinic acid a,d,q,2 C H O 0.8 191.0564 59; 85; 93; 127 0.24 0.002 a 2.39 0.001 b 1.01 0.001 a 29.08 0.03 b 7 12 6 ± ± ± ± Dihydroxybenzoic acid 6 C H O 0.9 153.0191 109 - 1.35 0.001 - 16.42 0.20 derivative b,c,d,m 7 6 4 ± ± 113; 145; 173; 199; 217; 7 Iridoid derivative b,c,d,g C H O 1.0 441.1248 2.53 0.001 - 10.69 0.01 - 16 26 14 275; 395 ± ± Dihydroxybenzoic acid 8 C H O 1.0 153.0191 109 - 2.37 0.01 - 28.83 0.03 derivative b,c,d,m 7 6 4 ± ± Malic acid dimethyl ester 9 C H O 1.0 161.0453 133; 143 - 2.63 0.01 - 31.99 0.20 c,d,m 6 10 5 ± ± 10 1-caffeoylquinic acid b,d,l C H O 1.2 353.0875 133; 135; 161; 179; 191 - 2.95 0.002 - 35.89 0.02 16 18 9 ± ± 125; 135; 161; 179; 201; 11 NI g C H O 1.2 421.1354 1.04 0.001 a 0.02 0.0001 b 4.39 0.01 a 0.24 0.01 b 17 26 12 215; 357; 375 ± ± ± ± 12 Hydroxybenzoic acid b,c,d,m C H O 1.5 137.0243 93 - 2.38 0.004 - 28.95 0.03 7 6 3 ± ± Procyanidin dimer I (B2) 13 C H O 1.6 577.1346 245; 289; 407; 425; 451 2.63 0.003 a 2.42 0.0001 a 11.11 0.04 a 29.44 0.03 b a,b,d,p 30 26 12 ± ± ± ± Scopoletin-7-O-sophoroside 14 C H O 1.6 515.1414 179; 191; 341; 353 1.96 0.01 - 8.28 0.01 - b,d,p 22 28 14 ± ± 15 (Epi)catechin-dihexoside b,d,r C H O 1.7 613.1785 271; 289; 451; 503 1.06 0.003 a 3.09 0.001 b 4.48 0.001 a 37.59 0.02 b 27 34 16 ± ± ± ± 16 Catechin a,b,d,k,2 C H O 2.0 289.0716 203; 205; 245 32.81 0.02 a 25.58 0.54 b 138.6 0.01 a 311.19 0.55 b 15 14 6 ± ± ± ± 17 Gambiriin b,d,p C H O 2.0 579.1515 205; 245; 289; 425 3.01 0.01 a 0.72 0.003 b 12.72 0.01 a 8.76 0.02 b 30 28 12 ± ± ± ± 18 Chlorogenic acid a,b,d,l C H O 2.2 353.0879 135; 161; 179; 191 214.8 0.02 a 99.20 0.01 b 907.5 1.45 a 1206 4.89 b 16 18 9 ± ± ± ± 19 (Epi)catechin hexoside b,d,g C H O 2.5 451.1245 245; 289; 341 1.74 0.001 a 1.29 0.001 a 7.35 0.01 a 15.69 0.51 b 21 24 11 ± ± ± ± 20 Procyanidin dimer II b,d,p C H O 2.5 577.1347 245; 289; 407; 425; 451 1.97 0.004 - 8.32 0.002 - 30 26 12 ± ± 21 Cryptochlorogenic acid b,d,l C H O 2.6 353.0877 135; 161; 173; 191 0.49 0.003 a 9.53 0.43 b 2.07 0.001 a 115.94 0.23 b 16 18 9 ± ± ± ± 22 Procyanidin dimer III b,d,p C H O 2.8 577.1348 245; 289; 407; 425; 451 8.89 0.01 a 8.23 0.01 a 37.56 0.04 a 100.1 0.76 b 30 26 12 ± ± ± ± 125; 161; 245; 289; 407; 23 NI p,1 C H O 3.4 579.1350 - 2.54 0.21 - 30.90 0.01 b 26 28 15 409; 427; 453 ± ± 24 Epicatechin b,d,k,2 C H O 3.5 289.0714 203; 205; 245 8.52 0.01 a 16.08 0.80 b 67.93 0.11 a 195.6 0.48 b 15 14 6 ± ± ± ± 25 Neochlorogenic acid b,d,l C H O 3.5 353.0875 135; 161; 191 14.52 0.02 a 3.34 0.002 b 61.34 0.12 a 40.63 0.57 b 16 18 9 ± ± ± ± 26 NI l C H O 3.6 373.1503 - 3.37 0.01 - 14.24 0.01 - 17 26 9 ± ± Foods 2020, 9, 1413 9 of 23

Table 2. Cont.

Molecular t m/z, VOP-E VOP-W VOP-E VOP-W Peak No. Compound R MS/MS (min) [M–H] Formula − mg/100 g DWP mg/100 g DWE 287; 289; 407; 425; 451; 27 Procyanidin C1 b,d,r C H O 4.4 865.1984 2.20 0.003 - 9.29 0.01 - 45 38 18 577; 695; 739 ± ± 289; 325; 435; 451; 569; 28 Cinchonain IIx b,d,r C H O 4.6 739.1665 2.66 0.002 - 11.24 0.02 - 39 32 15 587; 629 ± ± 29 Dihydromyricetin b,d,l C H O 5.3 319.0459 125; 153; 165; 193; 301 3.85 0.004 - 16.27 0.01 - 15 12 8 ± ± Quercetin pentoside 30 C H O 6.5 595.1304 151; 179; 301; 447 1.45 0.21 a 0.57 0.003 b 6.13 0.01 a 6.93 0.02 a hexoside I b,d,p 26 28 16 ± ± ± ± 31 Cinchonain Ix b,d,g C H O 7.0 451.1032 177; 189; 217; 231; 341 0.70 0.001 - 2.96 0.001 - 24 20 9 ± ± 32 Rutin a,b,d,r C H O 7.4 609.1453 151; 179; 301; 463 1.33 0.002 - 5.62 0.03 - 27 30 16 ± ± Quercetin-3-O-glucoside 33 C H O 7.5 463.0879 151; 179; 301 1.85 0.001 - 7.82 0.02 - a,b,d,g 21 20 12 ± ± 34 Procyanidin dimer IV b,d,p C H O 7.6 577.1348 245; 289; 407; 425; 451 0.48 0.003 - 2.03 0.001 - 30 26 12 ± ± 35 Ethylchlorogenate b,d,l C H O 8.0 381.1195 135; 161; 179 0.41 0.006 - 1.73 0.001 - 18 22 9 ± ± 36 Cinchonain Ix b,d,g C H O 8.3 451.1033 177; 189; 217; 231; 341 59.84 1.41 a 15.53 0.66 b 252.8 0.63 a 188.9 1.79 b 24 20 9 ± ± ± ± 37 Cinchonain Ix b,d,g C H O 8.7 451.1034 177; 189; 217; 231; 341 3.42 0.01 a 0.10 0.0001 b 14.45 0.88 a 1.22 0.001 b 24 20 9 ± ± ± ± Quercetin pentoside 38 C H O 8.9 595.2670 151; 179; 301 0.77 0.001 - 3.25 0.01 - hexoside II b,d,p 28 36 14 ± ± 39 Secologanate b,d,l C H O 8.9 373.1141 123; 149; 167; 193; 211 2.77 0.04 - 11.70 0.01 - 16 22 10 ± ± (Epi)catechin dimer 40 C H O 9.0 739.2665 177; 289; 339; 451; 577 1.28 0.01 - 5.41 0.56 - monoglycoside b,d,r 31 48 20 ± ± 41 Cinchonain Ix b,d,g C H O 9.1 451.1038 177; 189; 217; 231; 341 1.98 0.001 a 3.45 0.001 b 8.37 0.04 a 41.97 1.34 b 24 20 9 ± ± ± ± Viburtinoside derivative 42 C H O 9.2 607.2240 231; 339; 441; 501; 561 3.56 0.01 - 15.04 0.03 - b,d,e,r 26 40 16 ± ± Viburtinoside derivative 43 C H O 9.3 607.2242 231; 339; 441; 501; 561 1.21 0.01 - 5.11 0.01 - b,d,e,r 26 40 16 ± ± 453; 513; 573; 615; 633; 44 Opulus iridoid III b,d,e,r C H O 10.0 781.2775 4.11 0.001 - 17.36 0.12 - 33 50 21 675; 735 ± ± 45 Opulus iridoid II b,d,e,r C H O 10.2 649.2346 381; 483; 501; 543; 603 10.51 0.53 - 44.40 0.99 - 28 42 17 ± ± Superscript letters with the compound name: a confirmed by a standard; b confirmed by a reference; c confirmed by parent ion mass using a free chemical database (Chemspider, PubChem, Metlin); d confirmed by MS/MS fragmentation; e detected as formic acid adduct; m, s, q, p, k, l, r, and g based on the calibration curve obtained by using malic (LOD 2.30; LOQ 6.98), citric (LOD 4.57; LOQ 13.84), quinic (LOD 1.48; LOQ 4.47), proanthocyanidin B2 (LOD 16.24; LOQ 49.20), catechin (LOD 15.33; LOQ 46.46), chlorogenic acid (LOD 14.97; LOQ 45.38), rutin (LOD 0.58; LOQ 1.77), and quercetin-3-O-glucoside (LOD 0.81; LOQ 2.45), respectively; 1 collision energy 20 V, 2 collision energy 25 V; a, b the mean values in the rows for V. opulus pomace ethanol (VOP-E) and water (VOP-W) extracts (DWE) followed by different letters are significantly different (p < 0.05); NI-: not identified. Foods 2020, 9, 1413 10 of 23

Most likely, the increased temperature in PLE with water promotes the recovery of polyphenols from the used residue after extraction with ethanol due to the breakdown of the cell walls and increase of membrane permeability. In addition, heating might soften plant tissue and weaken the interactions between phenols and biopolymers present in the raw material, namely, proteins and polysaccharides. Consequently, the diffusion of phenolics into the solvent would increase. Some data on antioxidant capacity of guelder rose berry juice and pomace extracts are available from the previously performed studies. Significantly lower TPC values were determined in different juice samples (5.47–10.61 mg GAE/g) while the antioxidant capacity varied from 127.37 to 260.38 and from 31.95 to 109.81 µmol TE/g in the ORAC and ABTS assays, respectively [1]. Zakłos-Szyda et al. [11] produced a polyphenol-rich fraction from the guelder rose berries by solid phase extraction under pressure; it contained 361.52 mg GAE/g compared to 5.98 mg GAE/g in juice. TPC values of methanol and acetone extracts of VOP reported in the aforementioned study agreed with our results. Kraujalis et al. [17] reported TPC in different VOP extracts from 88.6 to 74.9 mg GAE/g DW and it was higher in the water extract. The same tendencies were observed for the antioxidant capacity determined by the DPPH, ABTS, and ORAC methods. Comparing with similar products obtained from sea buckthorn berry pomace [21], guelder rose berry pomace extracts were remarkably stronger antioxidants in the all applied assays and exhibited larger TPC values. It may be also noted that the concentration of secondary metabolites as well as some macronutrients is often larger in the berry pomace compared to the fruit pulp [11,31].

3.2. Characterization and Quantification of Phenolic Compounds in VOP Extracts The identification and quantification of phenolic acids and flavonoids in the extracts was of major interest for valorizing their potential to provide health benefits. It is well-known that the type and the structural peculiarities of phenolics may predestine extract’s antioxidant and other activities. The composition of VOP extracts was analyzed by UPLC-QTOF-MS, while the radical scavenging capacity of the separated compounds was additionally monitored by the HPLC-DPPH•-scavenging on-line method. As a result, 45 phytochemical compounds were detected of which 42 were identified and 45 quantified using calibration curves produced with various analytical standards. It may be observed that most of them belong to flavonoids (Figure1A,B, Table2). Combined UV (positive signals) and DPPH• quenching (negative signals) chromatograms of VOP extracts isolated from SFE-CO2 residue are presented in Figure1(C: VOP-W; D: VOP-E). In the latter assay, the HPLC-separated antioxidants react post-column with DPPH•, the solution of which is circulating in the coil. The induced bleaching of a radical is detected as a negative peak photometrically at 515 nm. Phenolic acids (3, 6, 8, 10, 12, 18, 21, 25, 35), iridoids (7, 39, 42–45), flavalignans (28, 31, 36, 37, 41), (epi)catechin derivatives (4, 13, 15, 16, 19, 20, 22, 24, 27, 34, 40), quercetin derivatives (30, 32, 33, 38), and other flavonoids (14, 17, 29) were found to be active radical scavengers in the investigated extracts. The VOP-E extract was a stronger radical scavenger than the VOP-W extract. The activity of VOP-W was comparable with that of phenolic acids, especially chlorogenic and hydroxybenzoic, and catechin derivatives (13, 15, 16, 22, 24), while the activity of VOP-E was considerably higher, most likely due to the presence of other constituents such as iridoids (7, 39, 42–45), flavalignans (28, 31), quercetin derivatives (33, 38), and other flavonoids (14, 29, 32, 35). The compounds 6, 8, 10, 12, 16, 18, 24, 25, 36, 41 and 7, 13, 14, 16, 18, 20, 24, 25, 28, 30, 33, 36, 41, 42, and 45 in VOP-W and VOP-E, respectively, were detected as the main antioxidants responsible for extracts antioxidant capacity. Chlorogenic acid was the strongest radical scavenger, while the input of other constituents, as it may be observed from the negative peaks in the chromatograms, was remarkably lower. Chlorogenic acid and rutin were identified based on retention time and MS of authentic standards. When the standards were not available, the identification was performed by comparing the exact mass of the parent ion (M-H) and typical MS fragmentation pattern with available data in previously published articles and PubChem, Chemspider, and Metlin databases. Foods 2020, 9, 1413 11 of 23

1

2 3 Fig. 1. Figure 1. Representative UPLC-QTOF-MS chromatograms of water (A) and ethanol (B) extracts of VOP. HPLC-UV-DPPH•-scavenging chromatograms of water (C) and ethanol (D) extracts of VOP. Foods 2020, 9, 1413 12 of 23

Malic (1), citric (2), and quinic (5) acids were identified by using standards. Citric acid was detected only in VOP-W. These acids were reported in V. opulus previously [1,32,33]. Perova et al. [33] in 100 g of eleven tested guelder rose berry varieties collected from different locations determined 578.0–2090, 279.0–1630, and 52.0–346.0 mg of malic, citric, and quinic acids, respectively. It is not surprising—the major part of soluble organic acids is transferred to the juice during pressing. Based on deprotonated ion m/z 371 and a fragment m/z 175, corresponding to the glucuronide moiety, compound 3 was tentatively identified as dihydroferulic acid 4-O-glucuronide. This compound was identified and quantified for the first time as a minor constituent in VOP-W extract. Compound 4 generated the fragment m/z 423 by RDA (retro-Diels-Alder), while other two fragments, m/z 467 and 305, were originated due to HRF (heterocyclic ring fusion) and QM (quinone methide cleavage) fragmentation, respectively. The fragment m/z 289 was formed due to the cleavage of the interflavan C–C bond corresponding to the (epi)catechin. Therefore, this compound was assigned to the procyanidin derivative. A small amount of this compound was detected only in the VOP-W extract. Three benzoic acid derivatives were detected in VOP extracts. The compounds 6 and 8 gave similar [M-H]− ion m/z 153 and fragment ion m/z 109 (the loss of [M–H–CO2]−), while the compound 12 with parent ion m/z 137 gave a fragment m/z 93 (the loss of CO2), which coincided with the fragmentation pattern of hydroxybenzoic acid derivatives containing two and one hydroxyl groups, respectively. Structurally, hydroxybenzoic acids may contain as many as four hydroxyl groups surrounding a single benzene ring. Hydroxyl groups in natural compounds are usually attached to the 3, 4, and 5 positions which cannot be exactly determined by MS. Therefore, on the basis of the mass spectra and previously published data, the peaks 6, 8, and 12 were tentatively identified as di- and mono-hydroxybenzoic acid derivatives [34]. All three compounds were detected and quantified in VOP recovered with water, the amount of 12 was eight-fold lower than reported by Velioglu et al. [3]. The peaks 7, 39, 42, 43, 44, and 45 were identified as iridoids (Table3, Figure2). The compound 39 produced a molecular ion m/z 373 [M H]− in MS spectra and the fragments m/z 123, 149, 167, 193, and 2 − 211 in the MS spectra. The fragment ion 211 [M–H–162]− corresponded to the loss of glucose residue, while further loss of H2O produced the fragment m/z 193. The ion m/z 149 could be formed due to the loss of H2O and CO2 from m/z 167 and m/z 193, respectively. While the ion m/z 123 could originate from m/z 149 and m/z 167 by the loss of C2H2 and CO2, respectively. As compared with the MS fragments and literature, peak 39 was plausibly characterized as secologanate [35]. This compound was not reported in V. opulus previously. Peak 7 gave pseudomolecular ion m/z 441 and several fragment ions listed in Table2. The fragmentation pathway was similar to iridoids via formation of acetic and valeric acid moieties; however, MS data was not sufficient to elucidate the exact structure of this iridoid. Other compounds, based on the previously reported in V. opulus fruits data and fragmentation patterns, were assigned to viburtinoside and opulus iridoids [32,36]. For example, MS2 fragments of viburtinoside derivatives (42, 43) indicate the sequential neutral losses of two acetic and one (iso)valeric acid groups, while opulus iridoid III (44) had three acetic and one (iso)valeric acid moieties. Opulus III (45) contains additional pentose moiety. Bock et al. [37] and Bujor et al. [32] reported the detailed fragmentation pathways for these compounds. In our study, iridoids constituted one of the major parts of all quantified phytochemicals, and they were recovered only in ethanol. According to the recovered amount, their content in 100 g of dried VOP was from 1.21 0.01 mg (43) to 10.51 0.53 mg (45). These ± ± constituents were quantified for the first time in VOP. Foods 2020, 9, 1413 13 of 23 Foods 2020, 9, x FORFoods PEER 2020 REVIEW, 9, x FOR PEER REVIEWFoods 2020 , 9, x FOR PEER REVIEWFoods 2020, 9, x FOR PEER REVIEW13 of 22 13 of 22 13 of 22 13 of 22 FoodsFoods 2020 2020,Foods 9, x, 9FOR ,2020 x FOR PEER, 9, PEERx FOR REVIEWFoods REVIEW PEER 2020 REVIEW, 9Foods, x FOR 2020 PEER, 9, x REVIEW FOR PEERFoods REVIEW 2020Foods, 9 , 2020 xFoods FOR, 9, 2020 PEERx FOR, 9 REVIEW, PEERx FOR REVIEW PEER REVIEW 13 of13 22of 2213 of 22 13 of 22 13 of 22 13 of 2213 of 2213 of 22 Foods 2020Foods, 9, x FOR2020 ,PEER 9, x FOR REVIEW PEERFoods 2020REVIEW, 9, x FOR PEER REVIEWFoods 2020 , 9, x FORFoods PEER 2020 REVIEW, 9, x FOR PEER REVIEW 13 of 22 13 of 22 13 of 22 13 of 22 13 of 22 Table 3. Structures of some bioactive compounds identified in guelder rose berry pomace extracts. Table 3. StructuresTable of some3. Stru bioactivectures of compoundssomeTable bioactive 3. identifiedStru compoundsctures inof gueldersome identified bioactive rose berryin gueldercompounds pomace rose extracts. identifiedberry pomace in guelder extracts. rose berry pomace extracts. TableTable 3. StructuresStru Table3. Structuresctures 3. Structures of some of someTable bioactive of bioactivesome 3. StructuresStruTable bioactivecompoundsctures compounds 3. Structuresof compounds some identified identifiedbioactive ofTable some identified in guelder Table3.compoundsin bioactive Structures Struguelder 3.Tableinctures rose Structuresguelder compoundsrose identifiedberry3.of Structures someberry rose pomace of bioactivepomaceberrysome inidentified guelder of extracts. bioactive pomacesome extracts. compounds inrose bioactive guelderextracts. compounds berry identified compoundsrosepomace berry identified extracts. in pomace guelderidentified in guelderextracts. rose in guelderberry rose pomaceberry rose pomaceberry extracts. pomace extracts. extracts. Table 3. StructuresTable 3. Structures of some Table bioactiveof some 3. Stru bioactive compoundsctures compoundsof someidentifiedTable bioactive identified in3. Structuresguelder compounds in roseTableguelder of berrysome identified3. Structuresrose pomacebioactive berryViburtinoside in extracts.guelderpomaceof compounds some roseextracts.bioactive derivatives berry identified compoundspomace in guelder extracts. identified rose berry in guelder pomace rose extracts. berry pomace extracts. Viburtinoside derivativesViburtinoside derivativesVibu rtinoside derivatives ViburtinosideVibuViburtinosidertinosideViburtinoside derivatives derivatives derivatives ViburtinosideVibu rtinoside Viburtinoside derivatives derivatives ViburtinosideViburtinoside ViburtinosideViburtinoside derivatives derivatives derivatives RRViburtinosideViburtinoside1 derivatives derivatives Viburtinoside derivativesR2 Viburtinoside derivativesViburtinoside derivatives R3 R4 R R1 R R2 R1 R3 R2 R4 R3 R4 R R R 1 R RR12 R1 R RR2 3R 2 R1 RR34 R3 RR2 R4 R4 R1R3 R2R4 R3 R4 Foods 2020,, 9,, xx FORFOR PEERPEER REVIEWREVIEW R R R1 RR1 R2 R 13R R 21of 22 R3 R1 RRR 23 R R4 R2R R1RR 43 R1 R R31 R2R 4 R2 RR42 R3 R3 R3 R4 R4 R4 4 4 4 4 R4 4 4 OH R R4 ROH R1 R1OH R R2 OH4 R2OH R41 R34 R R3R 2 R4 OH RR1 R4 R3 R R2 1 R4 RR32 RR4 3 R4 R R R R R OHOH 4 OH OHR OHOH OH OHOH OH OH OH 4 R 4 IV 4 R OH R 4OH OHR OHR4 OHR OHOH OH OH OH OH OH OH R R R3 RIV IVR 3 OH OHOH IVOH R OH OH OHROH OH OHOH OH OH OH OH OH OH 3 R3 R3 R IVR IV3 IV IVOH OH R 3 OH R OHIV OHOH IVOH OHOH OHOHOH OH OH OH OHOH OH R3 3 OH O IVR 3 OR3 OH IVOHO R3 IVO R3 O 3 OHIV OHIVOH O OH OHOH OH OH OH OH OH OH OH OH TableOHR 3. Structures of OHsomeOH bioactiveOHIV compoundsO identifiedOOH O Rin guelderOH rose berryOR pomaceOIVO OHextracts. O OHIV OO OH OH O OH O OH O OHOH OH R3 3 OHIV R3 O OH IVOH 3 OH O 3 O OHOHO OH OH OH OHOO O OHO OH O O OH OH O OHO OH OHO OOH OH O OH OHO O O O O O O OH OOHOHOH OH OHOH OH O OH OH OH R O O O R OViburtinosideOH O derivativesOH OH O OOH OHO OH OH OH OHOH OH OH R R R V R V V R OH ViburtinosideOH OH derivativesV OHOH OHR OHOHR OH OHOH OHOHOH OH OH OH OH OH RO O V RV O V OR V O OR VO R OHOH O OHO V OHVOH OHOHV OHOH OHOH OHOH OH OH OHOH OH OH OHOH OH OH R R O OR R O OO R1 O R R2 O O O R3 RO4 O O O O O V R V O O R1 V O R2 O OH O OR3 OHV O OHRO4 OHOHVO OHO OOHOH O O OHO OHO OH OHOH OH 4 O O O O O O O 4 OHO O O OHO O O O OO O OR O OHO OHO O IV OH Opulus iridoidsOpul OHus iridoids OH OpulOpulusus iridoids iridoids R3 IV OH OpulusOpulOpulusOHus iridoids Opulus iridoids iridoidsOH OpulusOpulus iridoids OpulusOpulus iridoidsOpulus iridoids 3 R OH RO O Opulus iridoids Opulus iridoids Opulus iridoids R1 R1 R OH RO1 OOpulusR iridoidsOpulus iridoids Opulus iridoids Opulus iridoids Opulus iridoids R1 1 R1 R1 R OH R1 R OHR1 OH OH OH OH O1 O O O OH1 OH 1 OHO OH 1OH 1 OH OOHOHOH OHOH OHOH OH OH OH R1 R1 O O R1 O OHO R1 OHO R1 OH OOH OHO OHO OH OHOHOH OH OHOH OH OHOH OH OH OH OH OH R R V II R II OH OHIIOH OH OH OH OH OHOH OH OHOH OH OH OH OH OH OH OH R2 R2 R O II OV R2II II OHO OH OHIIOH ROH O OHOH OOHOHOH II OH OHOH OH OH OH OH RO2 2CHOHR2 O CH RO2II OHRIIO O CH IIOH R2 ROHIIO RO2 OH II O IIOH OII OH OHOHO OH OH OH OH OH OH OH O O2 OH3 OHO OOH CH3OCHO2 OH 2OOO O CH3 O 2 OHO2 OOHOOO OHCH3 O O OO O O O R R O RIICH O3 3 II CH3 OO OH RCHOHII OO3 OHCH R O O O IIOH OCH 3 OOCH OCHII 3 OHOO O OHO OHO O O OH OHO O O O O OHO O OH 2 OH2 OH 2 3 OH 2 OH 3 2 OH 3 HO OH OH OH O CHO CH3 OO CHO 3 O O CH3 O O O CHO3 OO O HOOH HO HOO OH O O HO CH 3O CH3 O CHOpulus 3 iridoids HO HO HO HO HO O 3 O OCH3CH3 CH O CHOpulus3 iridoids O CH3 CH O CH3 OOH 3 OOHCH3 O CH3OH OH O OHOHCHO3 CHO3 OH3 HOOH HO OH OH HO HO R1 OHOH OH OHOH OHOH OHOHO OH O OH OH R1 O CH O CH3 O CH3OH OH O CH3OH OH OOHCHOH3 O OH OHOHOHO OH OHOH OH OHO OH OH OH OH OH OOH O 3 OH IIIOH OH OH O O O O O O O O O III III III IIIOH OHOH IIIOH OHOH OH OH OH OHOH III OH OHOH OH OHOH OH OHOH OH R II III IIIOH IIIOH IIIOHO OHOH III IIIOH OOH OIII O OHOHOHO OHO OH OH O OOH R2 OH II O OOHO O O OOO OH O O OO OH O OH O OH OOH OH OH OH 2 OH O CH3 III O III O IIIOH O O OH O O OIIIOH O O OIII OO O OHO OO O OH O O O O O O O O CH3 O O O OH OH OH OH OH OH OH O O O O O OH O O OOOH O OHO OH O OHO O OH HO OH OH OH OH OH OH OH O CH3 O O O O O O O O O 3 OH O O O OH O O O OHO O O O O OHO OHO O O OH O O OHO OH OH O OH O O OH OO O O O OH O O O O O O HOO OH R O R HOOH HOOH O R1 OH OH OH OH OH OH O III O O O HOOH HOHOO 1HO O R1OROOH OHOHO HOOHOOOR1HOHOO OHOH O OHOOOHHOOOHO OHOHR1 O O HOO HO OHO OH III O OHO HO OH OHO OH OR1 H1O OHR1 OHHO OHO RO1OHOHOHOOHHORHOHOOH HO OHOH O OHOHOR1 ROH 1OH R1 OH O OHOH OH OH OOH O OHOH OOH OHOHOHOOH OHHO HOHO OHOH OHH1OOH HO OHHOHO OHO OH OH OHOHOHOOH OHHO OOH HO OH O OHOHHO OHHO HOOOH O O HO OH O O OHOH O HO O R1 O OH R 1 OO R1 O OHHO R1 R1 HOOH HO HO R-ROH OH OH R-R OHOH OHOH HO OH OHOHHOHO OH HO O OHOHOOH OH OHO OH HO OH O HO O HO R-RR 3 R-RRHOR-R3 R-RHO R-RR 3 HO HO R-RROH OH OH OH R-RR R3 3 R 3 HO R-RR 3 R-R R-RR 3 R-RR R-RR 3 OH OH OH OH OH OH HO HO OH O3 O 3 ROHO3 3 3 OH OH OH OH OH R-RR R-RR 3 R-RR R-RR 3 R R-RR 3 R3 R3 OH O 3 HO R 3 HO OH3 R3 R OH R OH R3 OH R3 OH OH O HO OH OHR R1 OHOH HOR OHOH OHHO OH O OH OHR3 3 3 OH R3 R R3 R3 R3 HO HOOH HO R OH OHOHR RHO 1 OHOHOHOHHOOH OH R OHOHOHOHHOR2 OH O OHOHROH ROH2OH OH OH OH OH R3 OH HO HO HO R HO R OH HOOHHHOOR2 HHOOOHHROOHROH2 RH2O OHR OHROHROH ROHR3 ROH2OHR OH OHHO OHR3OHHO HOHOHO OHOHR2 OHR R3 OHR OH OHROH3 HO R-R 2 2 3HO OH2 ROH2 OH 2 OH2 2 OH OH OH HO HO RR-R3 OH R R OH HOHO RHO OH HOOHOHOH OH R OHR OHOH OH OHR R HO OHOHOH HO R OH OH R OH R 3 OH OH HOHO OH OHR2 2 OH OHOH OH2 OH OH OH OH OH2 OH2 OH OH OHOH OH OHOHOH OH OH OHOHOH OH OH OH OHOH OH OH OH OH OH OH O O R O R OH R3 R O O OR OHR O OH OH R OH OHOHO OHOH OHR O OHR3 OHOH OH OHOHOH OH OH OH HORa-b O Ra-bR RR O OH OH ORHOHa-bO R OH OHRO2 O R R R OH OH OH HO Ra-b a-b ChlorogenicRR OH Chlorogenic1-RHa-bOO-caffeoylquinic OH 1-O -caffeoylquinicR2CryptochlorogenicChlorogenicOHOHRa-b CryptochlorogenicR 1-OHONeochlorogenicR-caffeoylquinicChlorogenic Neochlorogenic Cryptochlorogenic 1-O -caffeoylquinic NeochlorogenicCryptochlorogenic Neochlorogenic O O Ra-b O a-b R Chlorogenica-bChlorogenicChlorogenicO R a-b 1-O1--caffeoylquinicR O-caffeoylquinic1-OO-caffeoylquinica-b CryptochlorogenicR a-bCryptochlorogenicOH a-bCryptochlorogenic NeochlorogenicNeochlorogenic ChlorogenicNeochlorogenic 1-O-caffeoylquinic Cryptochlorogenic Neochlorogenic R R OH OHOHR ChlorogenicOH OH 1-O-caffeoylquinicR ChlorogenicOH Chlorogenic CryptochlorogenicROH 1-O-caffeoylquinic 1-OOH-caffeoylquinicChlorogenic Neochlorogenic ChlorogenicCryptochlorogenic 1-CryptochlorogenicO -caffeoylquinic1-O -caffeoylquinicNeochlorogenic CryptochlorogenicNeochlorogenic Cryptochlorogenic Neochlorogenic Neochlorogenic OOOOa-b a-b ChlorogenicOHOOa-b Chlorogenic 1-OHO-caffeoylquinic 1-OChlorogenic-caffeoylquinica-b OH Cryptochlorogenic 1- OCryptochlorogenic-caffeoylquinica-b Chlorogenic NeochlorogenicCryptochlorogenic Neochlorogenic1-ChlorogenicO-caffeoylquinic 1-Neochlorogenic O-caffeoylquinicCryptochlorogenic Cryptochlorogenic Neochlorogenic Neochlorogenic O OOOOOOOOOH Chlorogenic OH OOacid acid acid acid O OOR OH OH acid OOOH OH OOacidacid OH OOacid acidOO OOacid acidOHacid acid acid acid acid acid acid acid RRa-b OH OH OHacidacid acid OH acidacid OHacid 1-acidO -ca ffeoylquinicacidacidacidOH acid acidOH acidOH acidacid acidacid acid Cryptochlorogenic acid acidacid acidacid acid Neochlorogenicacid acid acid OOROOa-b OOChlorogenic 1-Oacid-caffeoylquinic OO Cryptochlorogenicacid OO Neochlorogenicacid acid acid acidacid acid acid acid acid acid OH ChlorogenicOHacid 1-Oacidacid-caffeoylquinic OH acid Cryptochlorogenicacidacid acidOHNeochlorogenic acidacid acid OHacid acidacid acid acidacid acid acidacid acid OO OH OH Cinchonain I Cinchonain I Cinchonain I OOOH OH OH OH acid acidOH acid CinchonainCinchonainCinchonainOHacid I I I Cinchonain I Cinchonain I OH OH acidOH acidOH OH acid CinchonainOH acidI OH OH CinchonainCinchonain I I CinchonainCinchonain I Cinchonain I I OH OH Cinchonain I CinchonainCinchonain I I Cinchonain I HO HOOH HOHO OH OH OH CinchonainOH I OH Cinchonain I HO OH HO a HO a HOb Cinchonain I b HO ca HO HO c b d d c d OH OH a a Cinchonaina OH I b b a b c cb c a d dc a d b db c c d d HO HO OH OH OH OH a HOOH b a HO a c OHb ba da c b c b d c d c d d OHHO OH OH OHOHaOH OH bOH OH OH OH c d HO OH OH OH a a OHa b b ab c OH c b d c OHa d c da b db c c d d OH OH OH a OH b OH OH c OH OH d OH OH OH OH OH OH OH OH HO OH OH HO OH OH OH OH OH OH OH OH HOOH HO HO OH HOHO OH HO HOHO OH HO HO HO HO OH OHHO OH OHHO HO OH OH HHOOOH HO OH HO HOHO HO HOH O OH HO HOHO HO OH OH HOOHOHHO HO OHOHOH OH OHOH OH OOO OOO OOOHOH OOOHOHOHOH OH OH OH O O O OOOOOOO OOOH OHOH OHO OOOHOOOH OOOH OH OHOH OH OH OH OH OH O OOO O O OH O OOOH O O O O OOOOOOOOOH OH OOOH OHOHOOOH OH OHOH OH O O O O HOO OH OH OH OH HO HOOH HOHO HO HOOH HOOH OH OH HO OH HO HOHO HO HO HO HO HO HHOO HO HO HO HO HHOO HO OH HO OH HO OH OH OH HO OH HO HO HO OH OH HO HOHOOH HOHOHO HOOH HOOH OH OH HO OH R OHR OH R OH OH RR OH HO OH HOHO HO HO H O HO HO HHOO HO HO HO HO R R OHRRc-dR OH OH RRc-d OH OH RR OH OH OH c-d Rc-d RRc-dRc-d RR R c-d R R c-d R HORR HO OH HO HO HO HO HO c-d Rc-d OH c-d Rc-d R OH Rc-d Rc-d OH c-d HO OH R OH R R OH c-d RR R Rc-d Rc-d Rc-d c-d Rc-d CinchonainCinchonainCinchonain IIII II Cinchonain II Cinchonain II OH CinchonainCinchonainCinchonain II II II Cinchonain II Cinchonain IICinchonain II OH OH OHa-b OH Cinchonain II CinchonainCinchonain II II CinchonainCinchonain II II OHR a-bOH OH OH HO a-b OHR R O OH a-b OH a a-b Cinchonain OH CinchonainOH IIb OH II Cinchonain II Cinchonain II Cinchonain II HO R R O R a-b a-b O a ROHa-b O b a-b HO ROH O R a-b R O a-bOH a R a-b O a a-b R a-b ba a-bO a-b b b OH HOOH HO OH R R OOHR OH O O R OOHaR a OH a OH R a R O R bO b b a b b HO O HO O R a-b O HO a-bHO aR HO O a-bHO HO a Ra-b Oa b a a b a b b b b O a-b O R R R HO R OHOR O RHO O HO a R HO OHaO R O b a b a b b HO O O O OH OH O HO O a O HO b O O OO O O O O O O OH O OH O OH HO O OO OHOH O O O OH O O O OOH OH HO OH OH OHHO OH HOHOOH HO HO HOOH OH HOOH HO OH OH OH HO O OO O OH HO O HOHO O HOOH HO O OHO OH O HOOH HOOH HO HO HO HOHO HO HO HO HO OH OH OH OOH OH OH OH OH OH HOOHOH HO OH OHOH HOOHOH HO HO OH HO HO HO HO HO HOOH HOHO OHOH OH HOOH HO OH OH HOOH OHOH OH OH OH OH OH HO HO HO HO HO HO HO HOOH HHO HO HO HOHO HO HO HO HO HO OH OH OHHO OH HO HO HO HO HO OHO OHOH OHOH OOHHO OH OH HOOH HO HO O HOH O OOH OH OH HO OOH HOOH OH HO OHO HO OH HOOH OH HO HO HO HOOH HOOH O O O OH O OH O O O HOOH OH HOOH HO OH H O O OHHO HO HO OH OH O O O O HO OH HOO OH HO HO OH OH OH Quertecin-3-O-glucoside SecologanateOH OH OH GambiriinOH Scopoletin-7- O-sophorosideOH OH Quertecin-3-O-glucosideOH SecologanateOH OH GambiriinOH OH Scopoletin-7-O-sophorosideOH OH OH OH HOOH OH OHOH OH OHOHHO O OH OH OH O OH OH OH HO O HOOH OH OH OH OH OH O CH3 OH OH OHO OH OH OHCH3 OH OH O OH Quertecin-3-O-glucoside Quertecin-3-SecologanateO-glucoside OH Secologanate Gambiriin Scopoletin-7- Gambiriin O-sophoroside Scopoletin-7- O-sophoroside Quertecin-3-O-glucosideQuertecin-3-Quertecin-3- O-glucosideOSecologanate-glucoside Quertecin-3- SecologanateSecologanateO-glucoside Gambiriin OH Quertecin-3-Secologanate GambiriinO GambiriinO Scopoletin-7-O -glucoside O O-sophoroside Scopoletin-7- Gambiriin Scopoletin-7-Secologanate O-sophorosideO -sophoroside Scopoletin-7- GambiriinO-sophoroside Scopoletin-7-O-sophoroside OQuertecin-3-Quertecin-3-O-glucosideO-glucoside Secologanate OHSecologanate OH GambiriinO Quertecin-3- Gambiriin O OH O-glucoside Scopoletin-7- Scopoletin-7- O-sophorosideSecologanateO-sophoroside Gambiriin Scopoletin-7-O-sophoroside HO OQuertecin-3-O O-glucosideO Quertecin-3-SecologanateQuertecin-3-O-glucoside O -glucosideOH Quertecin-3-SecologanateOH GambiriinQuertecin-3-O SecologanateO -glucoside O-glucosideOHOH Scopoletin-7-OH Gambiriin SecologanateSecologanate O Gambiriin-sophoroside Scopoletin-7- Gambiriin Scopoletin-7-O Gambiriin-sophoroside O -sophoroside Scopoletin-7- Scopoletin-7- O-sophorosideO-sophoroside HO O O HOOH OHO OH OH OH OH O OH O OH O O O HO OH OHO O HO HO OHHO OH OH O OH O OH OH O OH HO O Quertecin-3-Quertecin-3-O-glucosideO O-glucoside Quertecin-3-Secologanate OSecologanate-glucoside HO HOOHO Quertecin-3-HO SecologanateOH GambiriinO OOH-glucosideOH GambiriinQuertecin-3- OOH OO Scopoletin-7--glucosideSecologanateHO GambiriinO Scopoletin-7-OH HOO -sophorosideOHSecologanateOOHOOCH-sophorosideO Scopoletin-7- O Gambiriin HO O HO OH-sophorosideO GambiriinOHCH Scopoletin-7-OH O-sophoroside Scopoletin-7- O-sophoroside OH O HO OH OH HO OH HO O CHOH3 HOHO O CHH3O OHO O CHOH3 O O CH O O OH OH OH O H C O OH OHO O OH O OH CH3 CH3 CH 3 OH 3 CH OH OHHO OH O O 2 O OHOHOHO O O HOHO OH OH OH OHO OH O OH HOCH3 OOHOH 3 HO OHCHOH3 OH CH CH3 CH3 3 OH OH OH HHO2C O HO OH O OO OH O OHOH OH HO OH O OOHHO O 3 O O HO HO HO O OH OHO OHO O OH O O O OH O HO OH OH OH OHHOHO OH O OH O OHOH CH O CH3 O OH CH O OH OH CH3 CH3 HO O HO OH OH OH OH O OHHOHO OH OH OO OH O 3 OH OHOH OH O 3 OHOH OH O O OHO OH HO OH O O O OH O O OH OH OHO OHOO O O OOH OO O O OH OH O O OH HO OH OHO O O O OH O O OHO OH O O OH OHOOH O O O OO OHOH OHO OOH OH O O OHO OH O OH O OO O O O HOOOH HO OHO O HO OH O O O O OH OHOH OH OOHO OH O OHO O OH O OH O OH OH OH O OH O OH OH OH O O OH OH OH HO HO O O O HO HO HO OO O OH HOHO OH OOHHO HO HO OHOHOH OHO O O OH OH OH O O O O O HOO OH O HOOH OH OO OOHO OHHOO OH O OH O O OH O O OH OOH OH O O OHOH O O O O O O HO O O O HO HO HOO OHO O OH OHO OH O OH OOH O OH OH OH O HOO OHOH OH OHOH O OH HO O HO OH O OHO OH O O O O O HOOHOHOH O O OHHO O HO O O OH O O HO O OHHOO OO OHO O OH HO OH HOO O OH O O OH O O OHO OH OO O CH OH OO 2CH O OH O O O O OH OHO OH O O O OO OO O OH OH HO OH H2COH OH OOH O HO2CCH O OH O OH OH O O CH OH OHOH OH OH OHO O OH CH HO OH OH O OH OOH OH OH OH OH O OH OH O HOOH HO OOHO O HH2CO H2C O HOH C OH HHOHOOH OOHOO O OHHO2C OO O OH O OH OH O O O OH 2C O O H C OH HO O OH OH OH HOOH OHHO H2C 2 HO HO OHHOO2 OH OH2COH HHOO HO O OHO 2 OOH2C 2 HOO O O HO O O O O O O HO O HO OH O OHOH OH OH OH O O OH OH OH O OH HO OH HOHOO OH O HHOHOOO O OH O OHOH HOHOHO OH O OH OH OH HO OHHOO O HO HOOH HO OH O O O OOH H C OH O CH O OH HO OH O HOC OHHOHO HOHO O OH CO OH OH HOHO O OH HOHO O OH OHHO O OH HO O HOOH HOHO HHOOHOOH OH2C HOHHOO 2O OH HO OHOH HOOHO 2 O OHHO O OHO OHHO OHOOH HO O 2 HOHO HOHO HOHOHOO2 OH O OHHOHHOO OOHOHO OH HOHO HOHO O HOHO OH O OH HO HO OH OH OH OH OH OH OH HO OH OH OH OH OH O OH O OH O OH HO HOOH HO OH HOOH HO OHHO OHOH HOHO OH HO OH OH OH OH OH HO O HO O OH HO HO OH OHO HO OH OHOH HO OHO HOHO HO OHOHHO O OHHO OHHO HO OH HOHHHOO HOHOHOOHO OHHOOHOHOH O OH HOHO O OH OHOH HOHO O OHHOHO O OH HO OH OH OH OH OH HO OH OH OH OH OH OH OH HO HOOH HOOH OH OH OH OH OH HO OH OH OH HO OH OHHO HO OHOH HOHO OH OHOH OH HO HO HO HO OH HO OH HO OH HO HOOH OH HO HO OHHO HO HOHO OH OH OH HO OH HO OH HO HOHO OH OH OH OHOH OH OH OH OH OH OH OHOH HO OH H O HO HO OH HO OH OH OH HO OH HO OH HO OH OH OH OH OH

Foods 2020, 9, x FOR PEER REVIEW 13 of 22 Foods 2020Foods, 9, x FOR 2020 PEER, 9, x FOR REVIEW PEER REVIEWFoods Foods2020 , 20209, x FOR, 9, x PEERFOR PEER REVIEW REVIEW Foods Foods 2020 ,2020 9, x ,FOR 9, x FORPEER PEER REVIEW REVIEW 13 of 22 13 of 22 13 of 1322 of 22 13 of13 22 of 22

Table 3.Table StructuresTable 3. 3.Stru Structures ofctures some of bioactive ofsome someTable bioactive bioactivecompoundsTable 3. Structures compounds 3. compounds Stru identifiedctures of some identified of identified in some bioactive guelder bioactivein inguelderTable rose compoundsguelderTable berry3.compounds rose Structures rose3. pomace berryStructures identified berry pomaceidentified of extracts. pomace some of in some guelderextracts. bioactive inextracts. guelderbioactive rose compounds berryrose compounds berry pomace identified pomace identifiedextracts. extracts. in guelder in guelder rose roseberry berry pomace pomace extracts. extracts.

ViburtinosideVibuViburtinosidertinoside derivatives derivatives derivatives Viburtinoside Viburtinoside derivatives derivatives ViburtinosideViburtinoside derivatives derivatives

R R1 R2 R3 R4 R R R1 R1 R2R R R2 R R3 1 R1 R 3 RR4 2 RR 2 RR4 R3 R1R 3 R1 R4 R2R 4 R2 R3 R3 R4 R4 4 4 R 4 4 OH4 OH4 4 R R OHR ROH OHOH OHR OHR OHOHOHOH OH OH R3 IVIV R OHOH IV R R OHOHOHIV IV OHOH OHOH OH OH OH OH OH OH R3 R3 OHIV R3 3 O OH IV 3 O3 OHOH OH OH OH OH OH O O OH OH OO O O OH OH O OO O O O O OH OHOH OH OHOH OH OH OH OH OH OH OH OH R O O O O O O R R V V V R R V V OHR OHROH OH OHOHV V OHOH OHOHOH OH OH OHOH OH OH OH OH OH OH O O O O O O O O O OO O O O O O O O O O O

OpulusOpul iridoidsOpulusus iridoids iridoids OpulusOpul iridoidsus iridoids OpulusOpulus iridoids iridoids R R 1R R R R R 1 1 O 1 1 OH 1 1 OH OH O O OH OOH O OHOH OHOHO OOH OH OHOHOHOH OH OH OH OH OH OH R II OH OH R 2R OH II R II R2 OH II OHII R R OH OH II II OH OH OH OH OH OH OH OH 2 OH 2 OHO CH3 2 OOH 2 2 OHO OH O O CHO CH O OH OO O CH CH3 OO O O O OO CH3 CHO3 O OO O O O O O O O 3 3 3 OH HO HO HO OH HO HO O CH3 CH CH CH O CH3 O CH3 OHO CHO3 3 OH O O 3 3 OH OH OH OHOH OHOH OH OH OHO OHOHOH OH OH OH OH OH OH III OH O O O O O O III III OH III OHIII OH OHIII III OH OH OH O O O OO O O O O O O OOHO O OH O O O OHO OH O O OH OH OH OH OH OH OH OH OH O O O O O O O O O O O O O O OO O O O O O OH O OH R1 OH OH HO HO O O HO HO OHO R O R HOOHOH HO HO OROHOR1 OH OHHO HHOO OHO R R1 OH OH OH OH OH OH 1 OH1 OH OH OHOH 1 HOHO OH HOHO O OH OOHOH OH H1O OH OH OH O O OH OH HO HO O O OH HO HO R-RHO OH HO HO R R-R3 R-R R-RR-R OH R-RR 3 R 3 R-RR 3R 3 R 3R 3 OH OH OH OH OH OH R3R R R R OH R OH OH OHR3 3 R3 3 3 3 HO HO RHO OH R OH HOOH OH HO OHR HORHROHO OHR2ROHHOHO OH OH OHROHROHR R OH OHOHHO OHHO OH OH R R OH OH 2 2 OH 2OH 2 OH OH 2 2 OH OH OH OH OH OH OH OH OH OH OHOH OH OH OH OH OHOH OH OH OH OH OH O R O O R OOH O R O O R R R a-bR OH OH ChlorogenicR R 1-OOH-caffeoylquinicOH CryptochlorogenicR R OH OHNeochlorogenic a-b a-b ChlorogenicChlorogenica-b a-b1-O-caffeoylquinic 1-O-caffeoylquinicChlorogenic CryptochlorogenicChlorogenic a-bCryptochlorogenic1-a-b O-caffeoylquinic1- O-caffeoylquinicNeochlorogenic NeochlorogenicChlorogenicCryptochlorogenicChlorogenic Cryptochlorogenic 1-O-caffeoylquinic 1-O-caffeoylquinicNeochlorogenic Neochlorogenic Cryptochlorogenic Cryptochlorogenic Neochlorogenic Neochlorogenic OO OOOOOHOOOOacid acid OOOOacid acid OH OHacid acid acidOH OHacid acid acidacid acidacid acidOH acid OH acidacid acidacid acid acid acidacid acid acid acid acid acid

OH Cinchonain I OH OH OH OH CinchonainCinchonain I I OH OH CinchonainCinchonain I I CinchonainCinchonain I I OH HO HO HO OH a b HO HO c d OH a a b b a a c c b b d a d a c c b b d d c c d d OH OH OH OH OH OH OH OH OH OH OH OH OH

OH OH OH HO OH OH HO HO HO HO Foods 2020, 9, 1413 OH OHHO HO HO OH OH HO HO OH OH 14 of 23 OH OH O OO OH OH OH OH OH OH OH OH OH OH OH OH O OOO OOO OOOOO O OOOOO OH HO HO HO OH HOHO OH HO OH HO HO HO HO OH Table 3. Cont. OH H O HO OH HOHO OH OH HO HO HO HO OHRR OH OH OH OH OH HO HO RR c-dRR RR RR RR RR c-d c-d c-d c-d c-d c-d Cinchonain II CinchonainCinchonainCinchonain II II II Cinchonain II CinchonainCinchonain II II OH Cinchonain II OH OH a-b OH OH OH OH a b R a-b a-b a-b OH Ra-b R Ra-b O Ra-b R a R R b HO HO R OR OHO OH R a R O a O HO HO Ra Ra b O O b a ab b b b O O O OO OOH O O O OH O O O O O OH OH OH HO OHHO OH HO OH HO OH OHHO HO HHOO OH HO HO OH OH OH OH OH OH OH OH OH OH HO HO HO HOOH HO HOHOHO OH HO HO OH HO HO OH HO HO HO OH O OH OH OH OH OH OH O O O O HO HOO HO OH O HO HO

OH OH OH OH OH OH OH OH OH OH OH OH OH OH Quertecin-3-O-glucoside Secologanate Gambiriin Scopoletin-7-O-sophoroside Quertecin-3-Quertecin-3-Quertecin-3-O-glucosideO-glucosideO-glucoside Secologanate Quertecin-3-SecologanateQuertecin-3-Secologanate O-glucosideO -glucoside Gambiriin Secologanate GambiriinQuertecin-3- GambiriinSecologanate Quertecin-3- O Scopoletin-7--glucosideO-glucoside Scopoletin-7- Scopoletin-7- GambiriinO -sophoroside GambiriinSecologanateO -sophorosideSecologanateO-sophoroside Scopoletin-7- Scopoletin-7- GambiriinO-sophoroside GambiriinO-sophoroside Scopoletin-7- Scopoletin-7-O-sophorosideO-sophoroside O OH OH OH HO HO OH HO OH OH O HO HO OH OH HO O HO O OH HO OHOOH O OHO CHOHO HOO O O O O O OH3 OH O OH OH CH3 OH CH3 CH3 CHOH3 OH CH3 CH3 OH OH O OH OH O OH O OHO OH O O OH OH OH OH OH OH OH O O O OH OH O OH OH OOH O OO O OH OH O O O O O OH OH OH O O O O O O O O O O O O O O OH OH OH O OH OH OH OH OH HO HO HO O OH O O OH OH HOO HO O OHO OOH O O O OO O OH OH OH OH O O O O HO HO OH OHO OHO OH O O OH HO HO OH O OH O O O O O O OH OH O CH OH OH O OH OOH O O OHO O OH O O OH OH OH OH OH OOH O O O 2 OH O O O O CH O O H OC H C OH OH HO HO H2COH OH H2C HO HO HOH OO H2OC OH2 OH HO HOH O OHO O 2 2 HO HO O O OH O HOHO HO OH OH OH HO HO O OH HO O HO O OH HO OH HO O O HO OH OH O O HO HO OH HOHOHO OHOHO OH O OH HOHO HOHO O O OH OH HOHO HOHO O O OH OH OH OH OH OH HO HO HO OHHO OH HO OH HO OH OH OHOH HO HO HO HO OH OH OH OH OH OH OH OH OH OH OH HO HO OH OH HO HO HOHO HO OH HO OH OH OH HO HO OH OH OH HO OH OH OH OH OH

FoodsFoods 20202020, 9,, 9x, FOR 1413 PEER REVIEW 14 15of of22 23

FigureFigure 2. 2.HypotheticHypothetic structures structures of selected of selected daughter daughter ions resulting ions resulting from MS-MS from MS-MSfragmentation fragmentation of the compoundof the compound 13 procyanidin 13 procyanidin dimer B2 (A dimer), 45 opulus B2 (A ),iridoid 45 opulus II (B), and iridoid 28 cinchonain II (B), and IIx 28 (C cinchonain). HRF— heterocyclicIIx (C). HRF—heterocyclic ring fussion; PC—phenyl ring fussion; cleavage; PC—phenyl RDA—retro-Diels-Alder cleavage; RDA—retro-Diels-Alder reaction; IF—interflavan reaction; fussion.IF—interflavan fussion.

PeakPeak 9 9waswas detected detected only only in in VOP-W VOP-W and and exhibited exhibited the the molecular molecular ion ion m/zm /161z 161 and and fragment fragment ions ions m/zm /z143143 (–18 (–18 Da) Da) and and 133 133 (–28 (–28 Da), Da), which which suggested suggested a adimethyl dimethyl ester ester derivative derivative of of malic malic acid. acid. Four Four derivativesderivatives (10 (10, 18, 18, 21, 21, and, and 2525) )of of chlorogenic chlorogenic acid acid were were detected detected in in VOP VOP (Table (Table 3,3, Figure Figure 22).). An interpretationinterpretation of of their their MS MS2 spectra2 spectra using using previously previously developed developed hierarchical hierarchical keys keys enabled enabled to to assign assign thesethese peaks peaks to to a particular a particular positional positional derivative derivative [3 [8–40].38–40 ].Furthermore, Furthermore, it itwas was found found that that the the fragment fragment ionion type type and and ion ion intensity intensity were were in in a agood good correlati correlationon with with the the position position of of the the caffeoyl caffeoyl group. group. The The characteristiccharacteristic fragments fragments m/zm /z133,133, 135, 135, 161, 161, 173, 173, 179, 179, and and 191 191 were were formed formed due due to to the the cleavage cleavage of of an an esterester bond bond between between caffeoyl caffeoyl and and quinic quinic acid acid groups, groups, quinic quinic acid acid dehydration, dehydration, along along with with caffeoyl caffeoyl decarboxylation.decarboxylation. The The compounds compounds 1010 andand 2525 sharedshared a asimilar similar fragmentation fragmentation pattern pattern (Table (Table 2);2); these these moleculesmolecules produced produced a abase base ion ion m/zm/ z191191 ([quinic ([quinic acid acid −H]H]−) by) by the the loss loss of ofa caffeoyl a caffeoyl residue, residue, an an ion ion m/zm /z − − 161161 ([caffeoyl–H ([caffeoyl–H−HH2O]O]−) by) bythe the loss loss of ofC7H C12HO6, Oanother, another ion ionm/z m135/z 135 ([caffeoyl–H ([caffeoyl–H−COCO2]−) by] )the by loss the lossof − 2 − 7 12 6 − 2 − C8ofH10 COH7, andO ,finally and finally one more one moreion m/z ion 133m/z ([caffeoyl–H133 ([caffeoyl–H−H2O−HCO]O−).CO] ). 8 10 7 − 2 − − TheThe absence absence or orlow low intensity intensity of m/z of m179/z 179([caffeoyl ([caffeoyl−H]−) H]enabled) enabled to separate to separate them from them 3-O- from − − caffeoylquinic3-O-caffeoylquinic acid and acid to andidentify to identify as 1- and as 1-5-caffeoylquinic and 5-caffeoylquinic acids [40]. acids Thus, [40 ].compound Thus, compound 10 was identified10 was identified as 1-caffeoylquinic as 1-caffeoylquinic acid, while acid, the while absence the absence of m/z 179 of m for/z 179 peak for 25 peak let 25uslet to usidentify to identify it as it neochlorogenicas neochlorogenic acid. acid. Compound Compound 18 was18 wasidentified identified as 3-O-caffeoylquinic as 3-O-caffeoylquinic acid acid(chlorogenic (chlorogenic acid); acid); it yieldedit yielded the thebase base peak peak m/zm 191/z 191 (deprotonated (deprotonated quinic quinic acid) acid) and and also also gave gave an anion ion m/zm /179z 179 [M–caffeic [M–caffeic acid–H]– with half of the intensity of the base peak. Moreover, it was confirmed by the analytical standard. Compound 21 gave characteristic ion m/z 173 [M–quinic acid–H–H2O]– and was identified

Foods 2020, 9, 1413 16 of 23 acid–H]– with half of the intensity of the base peak. Moreover, it was confirmed by the analytical – standard. Compound 21 gave characteristic ion m/z 173 [M–quinic acid–H–H2O] and was identified as 4-O-caffeoylquinic acid (cryptochlorogenic acid) according to the previously described fragmentation pattern [39,40]. Chlorogenic acid, as the main phenolic compound in fresh berries of V. opulus, has been reported previously [1,3,33]. Chlorogenic acid and its derivatives are among the major constituents in VOP extracts as well. The content of these phenolic acids recovered from 100 g of VOP varied from 0.49 0.00 to 214.8 0.02 mg. Chlorogenic acid (18) was dominant in VOP-W, while 1-caffeoylquinic acid ± ± (10) was detected and quantified only in VOP-W. For comparison, the content of chlorogenic acid was similar to the previously reported in guelder rose berry juice by Velioglu et al. [3]; however, 1.16–2.7 times lower than reported by Perova et al. [33] in guelder rose fruits. MS data for compounds 11 and 26 were not sufficient for their identification. Despite that, these compounds were quantified. Moreover, spectral information of these compounds are presented in the Supplementary Materials; the content of 11 was 52-fold higher in VOP-E, than in VOP-W, while compound 26 was determined and quantified only in VOP-E. The compounds 13, 20, 22, and 34 gave m/z 577 corresponding to a molecular ion [M–H]– of a procyanidin dimer with the MS fragments m/z 451, 425, 407, 289, and 245 (Figure2). The fragment m/z 425 is a result of RDA (retro-Diels–Alder) fission of flavonoid nucleus, which gives rise to m/z 407 after the loss of H2O. An ion m/z 451 indicates the loss of the A-ring (126 Da) [41]. The cleavage of the interflavan C–C bond produced m/z 289 corresponding to the catechin (epicatechin) unit, while further loss of 44 amu (C2H4O) in the benzofuran skeleton yielded m/z 245. Consequently, the extract had some procyanidin dimers, which have different retention times due to different bonding of catechin and epicatechin moieties. In addition, compound 13 was confirmed by the authentic standard as proanthocyanidin B2. The content of recovered procyanidin dimers varied from 0.48 0.00 to 8.89 ± 0.01 mg/100 g DWP. The recovered content of 13 and 22 was similar by both solvents, while the ± compounds 20 and 34 were found only in VOP-E with an approximately five-fold higher content of 20 than 34. Procyanidin dimers were reported previously without quantification [1,32,41]; the concentration of procyanidin dimer determined in guelder rose berry juice [3] was similar to its content in VOP determined in our study. The deprotonated ion [M-H]– m/z 515 of 14, as well as MS2 fragments m/z 353 [M–scopoletin-hexose–H]–, 341 [M–sophoroside–H]–, 191 [scopoletin-H]–, and 179 [M–hexose–H]– indicate on the structure of scopoletin-7-sophoroside [42]. To best of our knowledge, this compound was not previously reported in VOP (Table3). This compound was identified and quantified only in VOP-E, which recovered 1.96 0.11 mg/100 g DWP. ± Compound 15 was tentatively assigned to (epi)catechin-dihexoside on the basis of its fragment ions m/z 271, 289, 451, and 503. The loss of the B ring (110 amu) from the parent ion could explains the fragment m/z 503. The sequential loss of two hexosyl moieties from m/z 613 produced m/z 451 [M–hexosyl–H]– and 289 [M–2 hexosyl–H]–. Elimination of H O from aglycone generated the fragment × 2 m/z 271. This compound was not identified previously in V. opulus. Moreover, compound 19 has one sugar moiety less than compound 15. Previously, this compound was identified in V. opulus fruits [32]. The typical (epi)catechin aglycone (m/z 289) fragmentation was proved by the MS2 spectra by showing the loss of 162 Da, and further loss of 44 Da (C2H4O) in the benzofuran skeleton yielding m/z 245. Finally, loss of the B ring (110 amu) from the parent ion yielded an ion m/z 341. The recovered content of 19 was similarly recovered by both solvents, while the content of compound 15 was eight-fold higher in VOP-W. These compounds were reported previously without their quantification [32,41]. The compounds 16 and 24 eluting at 2.0 and 3.4 min, respectively, displayed the same molecular 2 ion m/z 289 (C15H13O6). Furthermore, they gave the same characteristic MS fragments m/z 245 (loss of CO2), 205, and 203 (cleavage of the A-ring of flavan-3-ol). The identity of compound 16 was finally proved using the catechin analytical standard. The hydrophobicity of epicatechin is higher than catechin; therefore, 24 eluted later than 16 and was assigned to epicatechin [32,43]. The contents of catechin and epicatechin in V. opulus berry juice were 29.04 and 2.69 mg/100 g, respectively. Comparing Foods 2020, 9, 1413 17 of 23 to Velioglu et al.’s [3] study, ethanol recovered higher contents of catechin, while water produced lower amounts. Recovered epicatechin values were three- to six-fold higher than reported by Velioglu et al. [3]. The compounds 17 and 23 yielded molecular ion m/z 579, but exactly suggested two different molecular formulas: C30H28O12 and C26H28O15, respectively. Compound 17 was tentatively identified as gambiriin based on the characteristic MS2 fragmentation pattern consisting of base m/z 289, which fragmented into m/z 245 and 205 after RDA and QM [44]. To the best of our knowledge, this compound was identified and quantified for the first time in VOP. Compound 23 generated several fragments in negative ionization mode (Table2); however, this information was not su fficient for its tentative identification. Furthermore, spectral information of this compound are presented in the Supplementary Materials. The latter compound was detected and quantified only in VOP-W, while the calculated concentration of gambiriin was approximately two times higher in VOP-E than in VOP-W. The precursor ion m/z 865 was detected for compound 27. The fragment m/z 739 was caused by HRF reaction of the upper unit, while m/z 713 occurred due to a RDA reaction of (epi)catechin unit, while m/z 695 indicates the loss of H2O. The fragment m/z 577 corresponds to the (epi)catechin dimer and originates from a QM reaction. Furthermore, the fragment ion of the dimer was observed following the RDA-based reaction (m/z 425), which yielded m/z 407 owing to the subsequent loss of H2O. The HRF reaction was also observed for the dimer, generating m/z 451. Finally, a QM reaction with the dimer formed at single quinone, resulting in m/z 289 and 287 ions. Therefore, this compound was tentatively assigned to procyanidin C1 [1,32,41]. This compound was quantified only in VOP-E with the recovery value of 2.20 mg/100 g DWP. Five flavalignans in the form of flavanols substituted with phenylpropanoids were found in VOP (Table3, Figure2). The molecular ion of 31, 36, 37, and 41 with m/z 451 (C24H19O9) produced the fragments m/z 341, 231, 217, 189, and 177, while the molecular ion of 28 with m/z 739 (C39H31O15) typically fragmented to m/z 629, 587, 569, 451, 435, 325, and 289. Based on this information, the compounds 31, 36, 37, and 41 were identified as cinchonains Ix (x = a, b, c, or d), while compound 28 was assigned to one of cinchonains IIx (x = a, b). The structures of these compounds together with the fragmentation pathways were proposed by Hokkanen et al. [45]. Purification and NMR would be required for more precise identification of compounds 28, 31, 36, 37, and 41. Cinchonains were previously found in lingonberries and bilberries [45], while their presence in V. opulus has not been reported. Flavalignans constituted another large group of phytochemicals quantified in VOP; their concentration varied from 0.10 0.00 to 59.84 1.41 mg/100 g DWP. Quantitatively, compound 36 was ± ± dominant in both extracts with higher value in VOP-E. The compounds 28 and 31 were quantified only in VOP-E, while the content of 37 and 41 was higher in VOP-E and VOP-W, respectively. Quercetin-3-O-glucoside (32) and rutin (33) were unambiguously identified by the obtained MS data and by using available standards. These compounds were quantified in VOP-E. Both of them were reported previously [3,11,32,33,41]. The amount of 33 quantified in guelder rose berry juice by Velioglu et al. [3] was approximately three-fold lower compared to our study, while the content of 32 determined by Zakłos-Szyda et al. [11] varied from 0.01 to 1.06 mg/g of extract depending on the type of sample preparation. Compound 29 present in VOP-E gave m/z 319 ion (C15H12O8) and, based on the detailed fragmentation pattern reported by Fan et al. [46], was tentatively identified as dihydromyricetin. This compound has not been reported in V. opulus previously. The compounds 30 and 38 eluting at 6.5 and 8.9 min with a similar pseudomolecular ion [M–H]– m/z 595 were assigned to quercetin pentoside hexoside. The identity is supported by the characteristic fragment ions, especially the diagnostic ion m/z 301.0356 of deprotonated quercetin aglycon, the loss of hexose (162 Da) and pentose (132 Da) [32,41]. Compound 38 was quantified only in VOP-E, while the amount of 30 was similar in both extracts. Compound 35 detected in the VOP-E produced m/z 381 (C18H21O9); based on MS, it was tentatively identified as ethylchlorogenate. This compound was identified and quantified for the first time in VOP. The most intense transition used for quantification of the compound 40 was the fragment m/z Foods 2020, 9, 1413 18 of 23

739, which yielded m/z 289 due to the well-known quinone methide cleavage (QM). Fragments ions m/z 577, 451, 339, and 177 were formed due to the loss of hexose (162 Da), heterocyclic ring fusion (–287 Da) of ring C of upper unit, the loss of ring B from the upper unit after the 739 ion QM cleavage, and finally, the loss of hexose (162 Da) from the m/z 339. Based on this and reported data, compound 40 was tentatively identified as (epi)catechin dimer monoglycoside [41,47]; it was detected and quantified only in VOP-E. The most important soluble sugars were quantified by using authentic standards: their amounts in 100 g DWP varied from 0.87 0.12 to 2.98 0.51 g of glucose, from 1.09 0.11 to 5.48 1.0 g of fructose, ± ± ± ± and from 1.17 0.54 to 2.69 0.50 g of sucrose, in VOP-E and VOP-W, respectively. Approximately ± ± 4.5–6-fold higher levels of quantified sugars were determined in VOP-W. Perova et al. [33] reported 2.3–4.0 g/100 g fructose and 2.8–4.6 mg/100 g glucose in 11 varieties of guelder rose berries collected from different locations; while sucrose was determined only in three samples in the range of 0.04–0.14 mg/100 g. In total, three anthocyanins were quantified in VOP-E and one in VOP-W (0.090 0.02 mg/100 g ± DWP). Cyanidin-3-O-gliucoside (15.18 0.08 mg/100 g DWP) was the dominant anthocyanin in VOP-E ± followed by cyanidin-3-rutinoside (2.12 0.17 mg/100 g DWP) and cyanidin-3-xylosyl-rutinoside (0.14 ± ± 0.05 mg/100 g DWP). Perova et al. [33] tested 11 V.opulus berry varieties and detected eight anthocyanins. Total anthocyanin amounts were in the range of 11.0–31.0 mg/100 g of fresh berries. The relative content of cyanidin-3-xylosyl-rutinoside was the highest (in some varieties constituted up to 95.7%), followed by cyanidin glucoside (up to 77%) and cyanidin-3-rutinoside (up to 20.6%). Zakłos-Szyda et al. [11] reported anthocyanins in V. opulus berry juice and extracts (in mg/g): cyanidin-3-glucoside 0.20 0.01–17.08 0.01, cyanidin-3-rutinoside 0.06 0.0–5.21 0.01, and cyanidin-3-sambubioside ± ± ± ± 0.85 0.0–55.78 0.69. The highest values were observed in the polyphenol rich fraction (78.06 ± ± ± 0.68 mg/g), while the lower ones in juice (1.12 0.0 mg/g). Our results, in general, are in the rage of the ± reported values. Anthocyanins, as the compounds demonstrating antioxidant, anti-inflammatory, and anti-apoptotic effects, have been proposed for the prevention and treatment of various diseases and disorders [48]. Our study demonstrated that ethanol effectively extracts phenolic compounds; however, PLE with water revealed that residual amounts of various phytochemicals, particularly higher polarity hydrophilic compounds, still remain in the residue after PLE with ethanol. On the other hand, it should be noted that high PLE temperature may increase the risk of decomposition of some thermolabile components, while some of them may undergo partial hydrolysis. In general, the results showed that VOP is rich in a large diversity of polyphenols, most of which were shown to possess strong antioxidant activity. It is known, that hydroxyl position in the molecule and some other structural properties determine antioxidant properties of flavonoids; in general, they depend on the ability to donate hydrogen or electron to a free radical. In addition, the variations in the content of reported compounds could also occur due to genotype, origin, harvesting time, and juice processing parameters.

3.3. Antiproliferative Activity and Cytotoxicity Effects of VOP Extracts In order to evaluate the antiproliferative activity of VOP extracts, HT29 cells were used at the exponential grow phase, while cytotoxicity assessment used Caco-2 cell line as the best accepted intestinal model. When confluent, Caco-2 cells share some characteristics with crypt enterocytes and, therefore, have been a widely implemented cell model to assess the effect of chemicals, food compounds, and nano/microparticles on the intestinal function [49]. Thus, HT-29 and Caco-2 were selected as convenient cell models to evaluate the anticancer effects of VOP extracts. To the best of our knowledge, the antiproliferative activity and cytotoxicity of VOP extracts in this study are reported for the first time. Both extracts were diluted in the solvents at their maximum acceptable concentration levels; VOP-E strongly inhibited cancer cell growth with an EC50 value of 0.39 0.03 mg/mL (Figure3A), without adverse e ffects on normal epithelia once no cytotoxicity ± was observed on Caco-2 (Figure3B). VOP-W did not exert an antiproliferative e ffect at 2 mg/mL Foods 2020, 9, x FOR PEER REVIEW 18 of 22 attributed to the presence of active phytochemicals and/or their interactions (Table 2). Procyanidins, catechin, epicatechin, hydroxybenzoic acids, anthocyanins, quercetin derivatives, as well as chlorogenic acid and its derivatives strongly suppressed cancer cell growth [11]. VOP-E contained higher amounts of gambiriin (17), cinchonains (36, 37), and anthocyanins. In addition, the antiproliferative effect of VOP-E could be attributed to the presence of other phenolic compounds, which were not found in VOP-W, e.g., cinchonains (28, 31), iridoids (7, 39, 42– 45), (epi)catechin derivatives (20, 27, 34, 40), quercetin derivatives (32, 33, 38), scopoletin-7-O- sophoroside (14), and dihydromyrecitin [50] (29) and ethylchlorogenate (35). The bioactivities of most of these phytochemicals have not been reported previously. The strong anticancer compound scopoletin [51] is an aglycone of scopoletin-7-O-sophoroside (14), which might exert anticancer Foodsactivity2020 ,after9, 1413 hydrolysis. Moreover, the information on V. opulus iridoids and their bioactivities19 of 23is rather scarce. Some iridoids demonstrated remarkable effects against various cancer cells [52]. It is concentrations,also worth mentioning while higher that the doses compounds were not showin applied.g other The antiproliferativebioactivities such e ffasect antimicrobial of VOP may and be attributedantiviral usually to the presenceare promising of active candidates phytochemicals for anticancer and/or their agents. interactions For instance, (Table 2Ming). Procyanidins, et al. [53] catechin,reported epicatechin,antibacterial, hydroxybenzoic antifungal, antiviral, acids, anthocyanins, and hepatoprotective quercetin derivatives, properties as of well cinchonains. as chlorogenic In acidgeneral, and itsVOP-E derivatives showed strongly an antiproliferative suppressed cancer effect cell against growth HT-29 [11]. VOP-E cells containedin vitro and higher could amounts be a ofpromising gambiriin substance (17), cinchonains for expanding (36, 37 its), andtesting anthocyanins. using experimental animal models.

(A) (B)

(C)

Figure 3.3. Dose–response curvescurves of ofViburnum Viburnum opulus opulusberry berry pomace pomace (VOP) (VOP) extracts. extracts. Antiproliferative Antiproliferative (A) and(A) and cytotoxicity cytotoxicity (B) e(Bff)ects effects using using HT29 HT29 and and Caco-2 Caco-2 cell cell lines, lines, respectively. respectively. Results Results are are expressed expressed in termsin terms of of mean mean SD± SD performed performed in in triplicate. triplicate. Antioxidant Antioxidant activityactivity ofof VOPVOP extractsextracts evaluatedevaluated by the ± cellular antioxidant activityactivity (CAA) assay (C). Results are expressed as µμmol QE per mg of dry extract of mean ± SDSD performed performed in triplicate. Unpaired Unpaired tt testtest was was assessed assessed with with pp < 0.05.0.05. ± 3.4. CellularIn addition, Antioxidant the antiproliferative Activity (CAA) eofff VOPect of Extracts VOP-E could be attributed to the presence of other phenolic compounds, which were not found in VOP-W, e.g., cinchonains (28, 31), iridoids (7, 39, 42–45), In contrast to antiproliferative and cytoprotective properties, both VOP extracts showed strong (epi)catechin derivatives (20, 27, 34, 40), quercetin derivatives (32, 33, 38), scopoletin-7-O-sophoroside and similar antioxidant activity with CAA values of 27.23 ± 9.01 and 30.36 ± 4.82 μmol QE/mg DWE (14), and dihydromyrecitin [50](29) and ethylchlorogenate (35). The bioactivities of most of these for VOP-W and VOP-E, respectively (Figure 3 C). It is interesting noting that despite no phytochemicals have not been reported previously. The strong anticancer compound scopoletin [51] is antiproliferative activity of VOP-W in HT29 cells its antioxidant activity in Caco-2 cells was similar. an aglycone of scopoletin-7-O-sophoroside (14), which might exert anticancer activity after hydrolysis. It may be assumed that the antiproliferative effects of antioxidant phytochemicals of VOP, due to Moreover, the information on V. opulus iridoids and their bioactivities is rather scarce. Some iridoids structural peculiarities, may be achieved by different mechanisms, which are not directly related to demonstrated remarkable effects against various cancer cells [52]. It is also worth mentioning that the compounds showing other bioactivities such as antimicrobial and antiviral usually are promising candidates for anticancer agents. For instance, Ming et al. [53] reported antibacterial, antifungal, antiviral, and hepatoprotective properties of cinchonains. In general, VOP-E showed an antiproliferative effect against HT-29 cells in vitro and could be a promising substance for expanding its testing using experimental animal models.

3.4. Cellular Antioxidant Activity (CAA) of VOP Extracts In contrast to antiproliferative and cytoprotective properties, both VOP extracts showed strong and similar antioxidant activity with CAA values of 27.23 9.01 and 30.36 4.82 µmol QE/mg DWE for ± ± VOP-W and VOP-E, respectively (Figure3C). It is interesting noting that despite no antiproliferative Foods 2020, 9, 1413 20 of 23 activity of VOP-W in HT29 cells its antioxidant activity in Caco-2 cells was similar. It may be assumed that the antiproliferative effects of antioxidant phytochemicals of VOP, due to structural peculiarities, may be achieved by different mechanisms, which are not directly related to their antioxidant capacity measured by the CAA assay. For example, phenolic hydroxyl groups at ortho or para positions with respect to each other increase the antioxidant activity due to additional resonance stabilization and o-quinone or p-quinone formation [54]. Two ortho hydroxyl groups contribute more to the radical-scavenging capacity than do the two hydroxyl groups in meta position. For example, protocatechuic acid has two hydroxyl groups in the ortho position and possesses higher antioxidant activity than γ-resorcylic acid with two hydroxyls in meta position [25]. Furthermore, higher amounts of procyanidin dimers (13, 22), (epi)catechin derivatives (15, 19, 41), catechin (16), epicatechin (24), chlorogenic (18), and cryptochlorogenic acids (25) had influence for high cellular antioxidant activity of the VOP-W extract. Additionally, the extracts demonstrated similar activity in CAA, while VOP-E showed stronger antioxidant capacity in non-cellular chemical-based assays. The CAA assay using a reduced DCFH-DA probe takes into account the bioavailability, distribution, and metabolism of antioxidants within the cell and reflects the activity of the tested extracts in the biological system [55]. It could be related to the structure of bioactive compounds; for instance, flavonoids with 2,3-double bond and 4-oxo group such as quercetin usually possessed high CAA activity [55]. Zakłos-Szyda and co-authors [56] evaluated chemoprotective activity of V. opulus fruit juice, acetone and methanol extracts of pomace, and polyphenol-rich fraction in Caco-2 cells using a strong pro-oxidant and in vitro oxidative stress inducer tert-butylhydroperoxide (t-BOOH). The extracts and especially the polyphenol-rich fraction strongly protected the cells from oxidative damage. Therefore, it was suggested that an antioxidant activity of extracts depends on the composition of polyphenolic compounds such as catechin, epicatechin, anthocyanins, procyanidin B1 and B2, neo and chlorogenic acid, rutin other quercetin derivatives, and hydroxycinnamic acids. Pre-incubation with guelder rose fruit extract at 0.365 mg/mL also protected βTC3 cells against oxidative damage induced by t-BOOH [57]. Consequently, the extracts of previously studied V. opulus fruits and in our study investigated pomace contain valuable bioactive compounds with cytoprotective properties.

4. Conclusions Forty-two bioactive phytochemicals were characterized in defatted guelder rose berry pomace extracts recovered by consecutive extraction with pressurized ethanol and water. Dihydroferulic acid 4-O-glucuronide, scopoletin-7-O-sophoroside, gambiriin, five flavalignans, dihydromyrecitin, and ethylchlorogenate, as well as antiproliferative and antioxidant activity of V. opulus berry pomace (VOP) extracts are reported for the first time. Ethanol extract showed higher antiproliferative activity with no cytotoxicity at the applied doses. Both extracts showed strong antioxidant activity in Caco-2 cells protecting them upon stress stimuli. The differences in the antioxidant power and antiproliferative activity of extracts may be related to a phytochemical composition and structural peculiarities of the compounds, particularly to the distribution of hydroxyl groups in the molecules. Considering strong antiproliferative and antioxidant properties, guelder rose berry pomace extracts, especially isolated by ethanol, may find promising applications in functional foods and nutraceuticals. However, technological upscaling and economic issues should be further evaluated in order to assess the possibilities of industrial production of ingredients from V. opulus berry pomace.

Supplementary Materials: The following are available online at http://www.mdpi.com/2304-8158/9/10/1413/s1. Author Contributions: Conceptualization, P.R.V. and L.D.; methodology, L.D., M.P., and C.V.P.; software: L.D.; validation, L.D., M.P., and C.V.P.; formal analysis, L.D.; investigation, L.D., M.P., and C.V.P.; resources, P.R.V., A.A.M.; data curation, P.R.V.; writing—original draft preparation, L.D.; writing—review and editing, P.R.V., A.A.M.; visualization, L.D.; supervision, P.R.V.; project administration, P.R.V. and A.A.M.; funding acquisition, P.R.V. and A.A.M. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the European Regional Development Fund according to the supported activity Research Projects Implemented by World-class Researcher Groups’ under Measure No. 01.2.2-LMT-K-718, grant no. 01.2.2-LMT-K-718-01-0017. Foods 2020, 9, 1413 21 of 23

Conflicts of Interest: The authors declare no conflict of interest.

References

1. Kraujalyte,˙ V.; Venskutonis, P.R.; Pukalskas, A.; Cesonienˇ e,˙ L.; Daubaras, R. Antioxidant properties and polyphenolic compositions of fruits from different European cranberrybush (Viburnum opulus L.) genotypes. Food Chem. 2013, 141, 3695–3702. [CrossRef][PubMed] 2. Kraujalyte,˙ V.; Leitner, E.; Venskutonis, P.R. Chemical and sensory characterisation of aroma of Viburnum opulus fruits by solid phase microextraction–Gas chromatography–olfactometry. Food Chem. 2012, 132, 717–723. [CrossRef] 3. Velioglu, Y.S.; Ekici, L.; Poyrazoglu, E.S. Phenolic composition of European cranberrybush (Viburnum opulus L.) berries and astringency removal of its commercial juice. Int. J. Food Sci. Technol. 2006, 41, 1011–1015. [CrossRef] 4. Zayachkivska, O.S.; Gzhegotsky, M.R.; Terletska, O.I.; Lutsyk, D.A.; Yaschenko, A.M.; Dzhura, O.R. Influence of Viburnum opulus proanthocyanidins on stress-induced gastrointestinal mucosal damage. J. Physiol. Pharmacol. 2006, 57, 155–167. [PubMed] 5. Rop, O.; Reznicek, V.; Valsikova, M.; Jurikova, T.; Mlcek, J.; Kramarova, D. Antioxidant properties of European cranberrybush fruit (Viburnum opulus var. edule). Molecules 2010, 15, 4467–4477. [CrossRef][PubMed] 6. Sivamaruthi, B.S.; Kesika, P.; Chaiyasut, C. the influence of supplementation of anthocyanins on obesity-associated comorbidities: A concise review. Foods 2020, 9, 687. [CrossRef][PubMed] 7. Batiha, G.E.; Beshbishy, A.M.; Ikram, M.; Mulla, Z.S.; Abd El-Hack, M.E.; Taha, A.E.; Algammal, A.M.; Elewa, Y.H.A. The pharmacological activity, biochemical properties, and pharmacokinetics of the major natural polyphenolic flavonoid: Quercetin. Foods 2020, 9, 374. [CrossRef] 8. Khoo, H.E.; Ng, H.S.; Yap, W.S.; Goh, H.J.H.; Yim, H.S. Nutrients for prevention of macular degeneration and eye-related diseases. Antioxidants 2019, 8, 85. [CrossRef] 9. Del Rio-Celestino, M.; Font, R. The health benefits of fruits and vegetables. Foods 2020, 9, 369. [CrossRef] 10. Sagdic, O.; Aksoy, A.; Ozkan, G. Evaluation of the antibacterial and antioxidant potentials of cranberry (gilaburu, Viburnum opulus L.) fruit extract. Acta Aliment. 2006, 35, 487–492. [CrossRef] 11. Zakłos-Szyda, M.; Pawlik, N. The influence of Viburnum opulus polyphenolic compounds on metabolic activity and migration of HELA and MCF cells. Acta Innov. 2019, 31, 33–42. [CrossRef] 12. Koparal, A.T. In vitro evaluation of gilaburu (Viburnum opulus L.) juice on different cell lines. AJESI 2019, 9, 549–571. [CrossRef] 13. Ulger, H.; Ertekin, T.; Karaca, O.; Canoz, O.; Nisari, M.; Unur, E.; Elmal, F. Influence of gilaburu (Viburnum opulus) juice on 1,2-dimethylhydrazine (DMH)-induced colon cancer. Toxicol. Ind. Health. 2013, 29, 824–829. [CrossRef] 14. Ceylan, D.; Aksoy, A.; Ertekin, T.; Yay, H.A.; Nisari, M.; Karatoprak, ¸S.G.;Ülger, H. The effects of gilaburu (Viburnum opulus) juice on experimentally induced Ehrlich ascites tumor in mice. J. Cancer Res. Ther. 2018, 14, 314–320. [PubMed] 15. Venskutonis, P.R. Berries. In Valorization of Fruit Processing By-Products, 1st ed.; Galanakis, C., Ed.; Academic Press: London, UK, 2020; pp. 95–126. 16. Heinonen, M. Antioxidant activity and antimicrobial effect of berry phenolics a Finnish perspective. Mol. Nutr. Food Res. 2007, 51, 684–691. [CrossRef] 17. Kraujalis, P.;Kraujaliene,˙ V.;Kazernaviˇciut¯ e,˙ R.; Venskutonis,P.R.Supercritical carbon dioxide and pressurized liquid extraction of valuable ingredients from Viburnum opulus pomace and berries and evaluation of product characteristics. J. Supercrit. Fluids 2017, 122, 99–108. [CrossRef] 18. Belwal, T.; Cravotto, G.; Chemat, F.; Venskutonis, P.R.; Bhatt, I.; Luo, Z. Recent advances in scaling-up of non-conventional extraction techniques: Learning from successes and failures. Trends Anal. Chem. 2020, 127, 115895. [CrossRef] 19. Granato, D.; Shahidi, F.; Wrolstad, R.; Kilmartin, P.; Melton, L.D.; Hidalgo, F.J.; Miyashitag, M.; van Camph, J.; Alasalvari, C.; Ismail, A.B.; et al. Antioxidant activity, total phenolics and flavonoids contents: Should we ban in vitro screening methods? Food Chem. 2018, 264, 471–475. [CrossRef] 20. Wolf, K.L.; Liu, R.H. Cellular antioxidant activity of common fruits. J. Agric. Food Chem. 2008, 56, 8418–8426. [CrossRef] Foods 2020, 9, 1413 22 of 23

21. Dienaite,˙ L.; Pukalskiene,˙ M.; Matias, A.A.; Pereira, C.V.; Pukalskas, A.; Venskutonis, P.R. Phytochemical composition, antioxidant and antiproliferative activities of defatted sea buckthorn (Hippophaë rhamnoides L.) berry pomace fractions consecutively recovered by pressurized ethanol and water. Antioxidants 2020, 9, 274. [CrossRef] 22. Helrich, K. Official Methods of Analysis, 15th ed.; The Association of Official Analytical Chemists: Arlington, VA, USA, 1990; ISBN 0-935584-42-0. 23. Singleton, V.L.; Rossi, J.A. Colorimetry of total phenolics with phosphomolybdic–phosphotungstic acid reagents. Am. J. Enol. Viticult. 1965, 16, 144–158. 24. Re, R.; Pellegrini, N.; Proteggente, A.; Pannala, A.; Yang, M.; Rice-Evans, C. Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radic. Biol. Med. 1999, 26, 1231–1237. [CrossRef] 25. Brand-Williams, W.; Cuvelier, M.E.; Berset, C. Use of a free radical method to evaluate antioxidant activity. LWT Food Sci. Technol. 1995, 28, 25–30. [CrossRef] 26. Huang, D.; Ou, B.; Hampsch-Woodill, M.; Flanagan, J.A.; Prior, R.L. High-throughput assay of oxygen radical absorbance capacity (ORAC) using a multichannel liquid handling system coupled with a microplate fluorescence reader in 96-well format. J. Agric. Food Chem. 2002, 50, 4437–4444. [CrossRef][PubMed] 27. Grunovaite,˙ L.; Pukalskiene,˙ M.; Pukalskas, A.; Venskutonis, P.R. Fractionation of black chokeberry pomace into functional ingredients using high pressure extraction methods and evaluation of their antioxidant capacity and chemical composition. J. Funct. Foods 2016, 24, 85–86. [CrossRef] 28. Shrivastava, A.; Gupta, V.B. Methods for the determination of limit of detection and limit of quantitation of the analytical methods. Chron. Young Sci. 2011, 2, 21–25. [CrossRef] 29. Pereira, C.V.; Duarte, M.; Silva, P.; Bento da Silva, A.; Duarte, C.M.M.; Cifuentes, A.; García-Cañas, V.; Bronze, M.R.; Albuquerque, C.; Serra, A.T.; et al. Polymethoxylated flavones target cancer stemness and improve the antiproliferative effect of 5-fluorouracil in a 3D cell model of colorectal cancer. Nutrients 2019, 11, 326. [CrossRef] 30. Silva, I.; Estrada, M.F.; Pereira, C.V.; da Silva, A.B.; Bronze, M.R.; Alves, P.M.; Duarte, C.M.M.; Brito, C.; Serra, A.T. Polymethoxylated flavones from orange peels inhibit cell proliferation in a 3D cell model of human colorectal cancer. Nutr. Cancer 2018, 70, 257–266. [CrossRef] 31. Polka, D.; Pods˛edek,A.; Koziołkiewicz, M. Comparison of chemical composition and antioxidant capacity of fruit, flower and bark of Viburnum opulus. Plant. Foods Hum. Nutr. 2019, 74, 436–442. [CrossRef] 32. Bujor, A.; Miron, A.; Luca, S.V.; Skalicka-Wozniak, K.; Silion, M.; Ancuceanu, R.; Dinu, M.; Girard, C.; Demougeot, C.; Totoson, P. Metabolite profiling, arginase inhibition and vasorelaxant activity of Cornus mas, Sorbus aucuparia and Viburnum opulus fruit extracts. Food Chem. Toxicol. 2019, 133, 110764. [CrossRef] 33. Perova, I.B.; Zhogova, A.A.; Cherkashin, A.V.; Éller, K.I.; Ramenskaya, G.V.; Samylina, I.A. Biologically active substances from European guelder berry fruits. Pharm. Chem. J. 2014, 48, 332–339. [CrossRef] 34. Hong, C.; Chang, C.; Zhang, H.; Jin, Q.; Wu, G.; Wang, X. Identification and characterization of polyphenols in different varieties of Camellia oleifera seed cakes by UPLC-QTOF-MS. Food Res. Int. 2019, 126, 108614. [CrossRef][PubMed] 35. Zhang, Y.D.; Huang, X.; Zhao, F.L.; Tang, Y.L.; Yin, L. Study on the chemical markers of Caulis Lonicerae japonicae for quality control by HPLC-QTOF/MS/MS and chromatographic fingerprints combined with chemometrics methods. Anal. Methods 2015, 7, 2064–2076. [CrossRef] 36. Tomassini, L.; Foddai, S.; Nicoletti, M.; Cometa, M.F.; Palazzino, G.; Galeffi, C. Iridoid glucosides from Viburnum ayavacense. Phytochemistry 1997, 46, 901–905. [CrossRef] 37. Bock, K.; Jensen, S.R.; Nielsen, B.J.; Norn, V. Iridoid allosides from Viburnum opulus. Phytochemistry 1978, 17, 753–757. [CrossRef] 38. Wianowska, D.; Gil, M. Recent advances in extraction and analysis procedures of natural chlorogenic acids. Phytochem. Rev. 2019, 18, 273–302. [CrossRef] 39. Clifford, M.N.; Johnston, K.L.; Knight, S.; Kuhnert, N. Hierarchical scheme for LC-MSn identification of chlorogenic acids. J. Agric. Food Chem. 2003, 51, 2900–2911. [CrossRef] 40. Clifford, M.N.; Knight, S.; Kuhnert, N. Discriminating between the six isomers of dicaffeoylquinic acid by LC-MSn. J. Agric. Food Chem. 2005, 53, 3821–3832. [CrossRef] 41. Karaçelik, A.A.; Küçük, M.; Ískefiyeli, Z.; Aydemir, S.; de Smet, S.; Miserez, B.; Sandra, P. Antioxidant components of Viburnum opulus L. determined by on-line HPLC-UV-ABTS radical scavenging and LC-UV-ESI-MS methods. Food Chem. 2015, 175, 106–114. [CrossRef] Foods 2020, 9, 1413 23 of 23

42. Mohamed, M.A.; Marzouk, M.S.A.; Moharram, F.A.; El-Sayed, M.M.; Baiuomy, A.R. Phytochemical constituents and hepatoprotective activity of Viburnum tinus. Phytochemistry 2005, 66, 2780–2786. [CrossRef] 43. Singh, A.; Kumar, S.; Kumar, B. LC-MS Identification of proanthocyanidins in bark and fruit of six Terminalia species. Nat. Prod. Commun. 2018, 13, 555–560. [CrossRef] 44. Shrestha, A. Phytochemical Analysis of Rhododendron Species. Ph.D. Thesis, Information Resource Center der Jacobs University Bremen, Bremen, Germany, 2016. 45. Hokkanen, J.; Mattila, S.; Jaakola, L.; Pirttila, A.M.; Tolonen, A. Identification of phenolic compounds from lingonberry (Vacciniumvitis-idaea L.), bilberry (Vaccinium myrtillus L.) and hybrid bilberry (Vaccinium intermedium Ruthe, L.) Leaves. J. Agric. Food Chem. 2009, 57, 9437–9447. [CrossRef][PubMed] 46. Fan, L.; Tong, Q.; Dong, W.; Yang, G.; Hou, X.; Xiong, W.; Shi, C.; Fang, J.; Wang, W. Tissue distribution, excretion, and metabolic profile of dihydromyricetin, a flavonoid from vine tea (Ampelopsis grossedentata) after oral administration in rats. J. Agric. Food Chem. 2017, 65, 4597–4604. [CrossRef][PubMed] 47. Zerbib, M.; Mazauric, J.P.; Meudec, E.; le Guerneve, C.; Lepak, A.; Nidetzky, B.; Cheynier, V.; Terrier, N.; Saucier, C. New flavanol O-glycosides in grape and wine. Food Chem. 2018, 266, 441–448. [CrossRef] 48. Eker, M.E.; Aaby,K.; Budic-Leto, I.; Brncic, S.R.; Nehir, E.; Karakaya, S.; Simsek, S.; Manach, C.; Wiczkowski, W.; de Pascual-Teresa, S. A review of factors affecting anthocyanin bioavailability: Possible implications for the inter-individual variability. Foods 2020, 9, 2. [CrossRef] 49. Sambruy, Y.; Ferruzza, S.; Ranaldi, G.; de Angelis, I. intestinal cell culture models. In Cell Culture Methods for in Vitro Toxicology; Stacey, G., Doyle, A., Ferro, M., Eds.; Springer Science+Business Media: Dordrecht, Germany, 2001; p. 113. 50. Li, H.; Li, Q.; Liu, Z.; Yang, K.; Chen, Z.; Cheng, Q.; Wu, L. The versatile effects of dihydromyricetin in health. Evid. Based Complement. Alternat. Med. 2017, 2017, 1053617. [CrossRef] 51. Seo, E.J.; Saeed, M.; Law, B.Y.; Wu, A.G.; Kadioglu, O.; Greten, H.J.; Efferth, T. Pharmacogenomics of scopoletin in tumor cells. Molecules 2016, 21, 496. [CrossRef] 52. Hussain, H.; Green, I.R.; Saleem, M.; Raza, M.L.; Nazir, M. Therapeutic potential of iridoid derivatives: Patent review. Inventions 2019, 4, 29. [CrossRef] 53. Ming, D.S.; López, A.; Hillhouse, B.J.; French, C.J.; Hudson, J.B.; Towers, G.H.N. Bioactive constituents from Iryanthera megistophylla. J. Nat. Prod. 2002, 65, 1412–1416. [CrossRef] 54. Chen, J.H.; Ho, C.T. Antioxidant activities of caffeic acid and its related hydroxycinnamic acid compounds. J. Agric. Food Chem. 1997, 45, 2374–2378. [CrossRef] 55. Wolfe, K.L.; Liu, R.H. Cellular antioxidant activity (CAA) Assay for assessing antioxidants, foods, and dietary supplements. J. Agric. Food Chem. 2007, 55, 8896–8907. [CrossRef][PubMed] 56. Zakłos-Szyda, M.; Pawlik, N.; Polka, D.; Nowak, A.; Koziołkiewicz, M.; Podsedek, A. Viburnum opulus fruit phenolic compounds as cytoprotective agents able to decrease free fatty acids and glucose uptake by Caco-2 cells. Antioxidants 2019, 8, 262. [CrossRef][PubMed] 57. Zakłos-Szyda, M.; Majewska, I.; Redzynia, M.; Koziołkiewicz, M. Antidiabetic effect of polyphenolic extracts from selected edible plants as α-amylase, α-glucosidase and PTP1B inhibitors, and β pancreatic cells cytoprotective agents—A comparative study. Curr. Top. Med. Chem. 2015, 15, 2431–2444. [CrossRef] [PubMed]

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).