Distribution of Polyphenolic and Isoprenoid Compounds and Biological Activity Differences between in the Fruit Skin + Pulp, Seeds, and Leaves of New Biotypes of Elaeagnusmultiflora Thunb

antioxidants Article Distribution of Polyphenolic and Isoprenoid Compounds and Biological Activity Differences between in the Fruit Skin + Pulp, Seeds, and Leaves of New Biotypes of Elaeagnus multiflora Thunb Sabina Lachowicz-Wi´sniewska 1,*, Ireneusz Kapusta 2, Carla M. Stinco 3, Antonio J. Meléndez-Martínez 3, Anna Bieniek 4, Ireneusz Ochmian 5and Zygmunt Gil 1   Citation: Lachowicz-Wi´sniewska, S.; Kapusta, I.; Stinco, C.M.; Meléndez-Martínez, A.J.; Bieniek, A.; Ochmian, I.; Gil, Z. Distribution of Polyphenolic and Isoprenoid Compounds and Biological Activity Differences between in the Fruit Skin + Pulp, Seeds, and Leaves of New Biotypes of Elaeagnus multiflora Thunb. Antioxidants 2021,10, 849. https://doi.org/10.3390/ antiox10060849 Academic Editor: María Pilar Almajano Pablos Received: 6 April 2021 Accepted: 20 May 2021 Published: 26 May 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2021 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 (https:// creativecommons.org/licenses/by/ 4.0/). 1 Department of Fermentation and Cereals Technology, Wrocław University of Environmental and Life Science, Chełmo´nskiego 37, 51-630 Wroclaw, Poland; [email protected] 2Department of Food Technology and Human Nutrition, Faculty of Biology and Agriculture, Rzeszow University, Zelwerowicza 4, 35-601 Rzeszow, Poland; [email protected] 3Food Colour and Quality Laboratory, Area of Nutrition and Food Science, Universidad de Sevilla, 41012 Seville, Spain; [email protected] (C.M.S.); [email protected] (A.J.M.-M.) 4 Department of Horticulture, University of Warmia and Mazury, Prawoche´nskiego 21, 10-720 Olsztyn, Poland; [email protected] 5Department of Horticulture, West Pomeranian University of Technology in Szczecin, Słowackiego 17, 71-434 Szczecin, Poland; [email protected] *Correspondence: [email protected] Abstract: The purpose of this study was to determine the distribution of polyphenolic and isoprenoid compounds and organic acids in the fruit skin + pulp, seeds, and leaves of six new biotypes of Elaeagnus multiflora Thunb., as well as their in vitro biological potency. The polyphenols and isoprenoids were determined with UPLC-PDA-MS/MS (ultra-performance liquid chromatography coupled to photodiode array detection and electrospray ionization tandem mass spectrometry) and RRLC-MS/MS (rapid resolution liquid chromatography/tandem mass spectrometry) methods, the organic acid with HPLC-RID (high-performance liquid chromatography coupled to a Refractive Index Detector), and the antioxidant capacity using ABTS and FRAP assays. Enzymatic activity was established as the ability to inhibit α -amylase, α -glucosidase, and pancreatic lipase. Owing to such an effective technique, 88 compounds were recorded, with 17 polyphenolic compounds and 3 isoprenoids identified for the first time in the seeds and leaves of cherry silverberry. In total, 55 compounds were identified in the leaves, 36 in the seeds, and 31 in the fruit skin + pulp. The predominant polyphenol was polymeric procyanidin (66–95% of total polyphenolics), whereas the predominant isoprenoids were chlorophyll b and (all-E)-lycopene. The results of our work noted that there are significant differences in the profiles of several secondary metabolites between the analyzed parts of the plant, and depending on the need, the compounds can be used to develop different innovative food or cosmetic products. Keywords: carotenoids; cherry silverberry; seeds, polyphenolic compounds; antioxidative capacity; anti-diabetic activity; organic acids 1. Introduction Elaeagnus multiflora Thunb., appearing in the literature as goumi, cherry silverberry, or cherry elaegnus, belonging to the family Oleaster (Eleagnaceae), like the popular Hippopha ё rhamnoides L. [ 1 ], is a thorny, broadleaved shrub reaching 3 m in height. It produces yellow or orange-red elliptical 1-cm-long fruits, which are juicy and have an astringent taste resembling that of red currant. The fruits are suitable for direct consumption. In addition to lipids, organic acids, and macroand microelements, they contain Antioxidants 2021,10, 849. https://doi.org/10.3390/antiox10060849 https://www.mdpi.com/journal/antioxidants Antioxidants 2021,10, 849 2 of 18 significant amounts of lycopene. This carotenoid is responsible for their red color and has been extensively studied as one of the major dietary carotenoids for humans [ 2 ]. Lee et al. [ 3 ] demonstrated that in addition to chlorophylls, the leaves of cherry silverberry have high contents of sugars, fatty acids, and phytosterols. However, the polyphenolic profile of cherry silverberry fruits has not yet been thoroughly recognized. Only 14 compounds belonging to phenolic acids and flavan-3-ols have been identified so far [ 3 ]. Polyphenols are important compounds due to their beneficial effects, including anticarcinogenic, antiinflammatory, antioxidant, antitumor, and antidiabetic, as well as anti-atherosclerotic, activities [ 4 , 5 ]. In addition, they are the focus of interest in the food industry due to their high nutritive value [3]. E. multiflora Thunb. is known in Chinese medicine as a therapeutic plant [ 6 ]. Its fruits, in both fresh and processed forms, are used to treat ailments such as cough, diarrhea, pruritus, gastrointestinal tract disorders, and even cancer [ 4 ]. E. multiflora Thunb. fruits are also used to make juices, jams, compotes, or jellies [ 1 ], while in Asia and eastern Europe, they are used to produce herbal teas, wines, desserts, soups, ice cream glazes, and even candies and puddings [7]. The cherry silverberry shrub is a native plant of China, Japan, and Korea. Not long ago, this species also appeared in Russia, Ukraine, and the United States, while since the 1990s, it has been investigated at the Department of Horticulture of the University of Warmia and Mazury in Olsztyn (Poland). Studies conducted at the department have mainly focused on the selection and development of novel cultivars perfectly coping with the moderate climate in Poland during cultivation [ 1 ]. The environmental factors are inseparable elements that affect the contents of biologically active components. The accumulation of polyphenolic, terpenoid, or polysaccharide compounds in a plant can be effective only upon sufficient access of light, phosphorus content in the soil, and—last but not least—soil organic matter [ 8 ]. As demonstrated by Lachowicz et al. [ 5 ], contents of the basic physicochemical compounds were significantly affected by the novel cultivars analyzed. In addition, these cultivars can be excellent components of health-promoting and other beneficial products intended for human consumption (functional foods, nutraceuticals, supplements, nutricosmetics, etc.) [ 9 ]. Recently, foods rich in polyphenols have attracted great interest due to their potential benefits for human health. Being present not only in fruits and vegetables but also in seeds, olive oil, and beverages such as coffee and tea, raw materials are fine elements of healthy, nutritional patterns. The latest evidence suggests that a high intake of polyphenols with a diet may be negatively correlated with the general mortality rate associated with diseases of the circulatory system, certain types of cancer, cardiovascular diseases, adverse anthropometric changes, and mood disorders [ 10 ]. All these traits make polyphenols potential elements in designing functional food and in developing prophylactic strategies for lifestyle diseases. The positive impact of polyphenolic compounds on health is due to several factors, the major ones of which include their content in diet and their bioavailability, which may differ significantly. The content of polyphenols in food may be influenced by genetic, environmental, and technological factors; some of these factors can be controlled to optimize the polyphenol content in food. One of the means to increase the polyphenol content in food may involve the appropriate choice of fruit and vegetable cultivars, which differ significantly in terms of their polyphenol content. Biologically active compounds found in the cultivars (Jahidka and Sweet Scarlet) of cherry silverberry fruits have been identified [ 7 ]. However, little is known about the bioactive potential of the fractions of six novel biotypes of E. multiflora Thunb. cultivated in Poland. These studies tried to determine the versatile use of cherry silverberry in various areas of human life. Therefore, the objective of the present study was to characterize and determine the location of polyphenolic, carotenoid, chlorophyll, and tocopherol compounds (with ultra-performance liquid chromatography coupled to photodiode array detection and electrospray ionization tandem mass spectrometry and rapid resolution liquid chromatography/tandem mass spectrometry methods) and organic acids (with the high-performance liquid chromatography coupled to a Refractive Index Detector method) Antioxidants 2021,10, 849 3 of 18 and their biological activity in the fruit skin + pulp, seeds, and leaves of six new biotypes of E. multiflora Thunb. cultivated in Poland. Results from this study (1) can be used in plant genetics to achieve new suitable biotypes, (2) determine the location of individual compounds in tested materials and, depending on the need, develop novel products for human consumption (fortified foods, functional foods, (nutri)cosmetics, etc.), and (3) could help in the management of by-products. 2. Materials and Methods 2.1. Plant Materials Cherry silverberry plants were collected from the University of Warmia and Mazury in Olsztyn, Poland (53 ◦ 50 0 N, 20 ◦ 31 0 E), in June, 2019. The study used three vegetatively propagated biotypes (9-19-1996 (si1), 9-24-1996 (si2), and 9-34-1996 (si3)) obtained from the Institute for Fruit Growing in Samokhvalovitchy from the E-2 breeding farm and two biotypes (01-1999 (si0): 9-44-1996 (si4) and 9-84-1996 (si5)) obtained from the seeds of the 01-1999 (si0) biotype in 2005. The comparison of the biotypes also included the evaluation of the 01-1999 (si0) biotype obtained from seeds originating from a shrub cultivated in Olsztyn in 1999. All the plants were adults. All plants were planted with 4 × 2 m 2 spacing in Albic Luvisolx (Arenic) soil, which was deeply flattened and produced from clays of pH 6.8 in KCl [ 5 ]. The shrubs started to fructify in the third year after planting. This region of Poland is one of the coldest, and despite this, the studied biotypes adapted to this climate; therefore, it can be suggested that they would be suitable for cultivation throughout Europe. In the course of the tests, 3 replications (for each 25 randomly chosen leaves and about 2.5 kg of fruit) from 3 shrubs of each biotype were determined. The powders were stored in a refrigerator ( − 26 ◦ C) until extract preparation. The raw material was directly frozen in liquid nitrogen and freeze-dried (24 h; Christ Alpha 1-4 LSC; Osterode am Harz, Germany). The homogeneous dry material was obtained by crushing the dried tissues using a closed laboratory mill (IKA A.11; Darmstadt, Germany). The powders were kept in a refrigerator ( − 80 ◦ C) until extract preparation [7]. 2.2. Identification and Quantification of Polyphenols The extraction and measurement of polyphenolic compounds by the UPLC-PDAMS/MS technique were performed according to the method by Kapusta et al. [ 11 ]. Separation was carried out using a BEH C18 column (100 mm × 2.1 mm i.d., 1.7 µ m; Waters) kept at 50 ◦ C. For polyphenolic compounds, a lower concentration of formic acid was used (0.1% v/v). The gradient program was set as follows: 0 min, 5% B; from 0 to 8 min linear to 100% B; and from 8 to 9.5 min for washing and back to initial conditions. The injection volume of the samples was 5 µ L (partial loop with needle overfill), and the flow rate was 0.35 mL/min. The following parameters were used for TQD: capillary voltage, 3.5 kV; con voltage, 30 V in positive and negative modes; source temperature, 250 ◦ C; desolvation temperature, 350 ◦ C; con gas flow, 100 L/h; and desolvation gas flow, 800 L/h. Argon was used as a collision gas at a flow rate of 0.3 mL/min. Polyphenolic detection and identification were based on specific PDA spectra, massto-charge ratio, and fragment ions obtained after collision-induced dissociation (CID). Quantitative analysis was based on specific MS transitions in multiple reaction monitoring (MRM) mode. The MRM transitions, cone voltage, and collision energy of each individual polyphenolic compound were set manually with a dwell time of at least 25 ms. All measurements were noted three times and expressed as mg/100 g dry matter (d.w.). 2.3. Determination of Procyanidins by the Phloroglucinolysis Method The extraction and measurement of procyanidins by the phloroglucinol test were performed according to the method by Lachowicz et al. [ 7 ]. Phloroglucinolysis was analyzed using a liquid chromatograph (Waters, Milford, MA, USA) consisting of a diode array, scanning fluorescence detectors, and a column manager. Separation was carried out Antioxidants 2021,10, 849 4 of 18 using a Cadenza CD C18 column (75 mm × 4.6 mm, 3 µ m) kept at 15 ◦ C. For phloroglucinolysis investigation, the following solvent system (mobile phase A (25 mL of acetic acid and 975 mL of water) and mobile phase B (acetonitrile)) was applied. Fluorescence was recorded at emission wavelength 360 nm and excitation wavelength 278 nm. The calibration curves and quantification were evaluated using standards: ( − )-epicatechin, (+)-catechin, ( − )-epicatechin-phloroglucinol, and (+)-catechins-phloroglucinol. The degree of polymerization was analyzed by evaluating the molar ratio of all the flavan-3-ol units. All measurements were noted three times and expressed as mg/100 g d.w. 2.4. Determination of Isoprenoids (Carotenoids, Tocopherols, and Chlorophylls) The extraction and analysis of isoprenoids (carotenoids, chlorophylls, and tocopherols) were performed according to the method by Stinco et al. [ 12 ]. Isoprenoid analyses were performed by RRLC on an Agilent 1260 system (Agilent, Waldbronn, Germany) equipped with a UV–VIS diode array detector, which was set at 285 nm for phytoene and tocopherols, 350 nm for phytofluene, 410 nm for ζ -carotene and pheophytin A, 430 nm for chlorophyll A and pheophytin B, 472 nm for lycopene, and 450 nm for the rest of the CARS ( α -carotene, β -carotene, β -cryptoxanthin, capsanthin, lutein, violaxanthin, and zeaxanthin) and chlorophyll b. Separation was accomplished on a C30 column (150 mm × 4.6 mm I.D. 3 µ m particle size; YMC Europe, Dinslaken, Germany) kept at 28 ◦ C with a guard precolumn (10 mm × 4.0 mm I.D. 3 µ m particle size; YMC Europe, Dinslaken, Germany). The analysis was performed in triplicate and expressed as mg/100 g d.w. 2.5. Determination of Organic Acids The organic acid content was tested by the HPLC-RI method [ 11 ]. The chromatographic equipment SYKAM (Eresing, Germany) consisting of sample injector S5250, pump system S1125, column oven S4120, and RI detector S3590 was used. Separation was carried out using a Polymer IEX H column (6 µ m, 250 mm × 8 mm; SETREX). Separation was achieved with a mobile phase of 1.5 mM sulfuric acid in water in isocratic mode. The flow rate was 0.5 mL/min at a column temperature set at 90 ◦ C. The volume of the injected sample was 20 µ L, and 30 min was needed to complete the analysis. The analysis was performed in triplicate and expressed as g/100 g d.w. 2.6. Analysis of Antioxidant Activity Antiradical activity (ABTS) and reducing power (FRAP) tests were performed, as previously described by Re et al. [ 13 ] and Benzie and Strain [ 14 ]. Briefly, 10 µ L of the supernatant was mixed with 990 µ L of ABTS or FRAP. After 10 min of reaction, absorbance was measured at 734 nm for ABTS and 593 nm for FRAP. Determinations by ABTS and FRAP methods were performed using a UV-2401 PC spectrophotometer (Shimadzu, Kyoto, Japan) [ 5 ]. All antioxidant assays were performed in triplicate and expressed as mmol of TE per 100 g d.w. 2.7. Inhibitory of Biological Activity Antihyperglycemic activity, α -glucosidase and α -amylase inhibitory activity, and antiobesity pancreatic lipase inhibitory activity of the sample were determined, as described previously by Pods˛edek et al. [ 15 ] and Nickavar et al. [ 16 ]. The IC 50 of the material was obtained from 1 mL of the reaction substance relative to the percentage inhibition. All results were presented as the average of three replicates. 2.8. Statistical Analysis Statistical analysis, two-way ANOVA, Tuckey’s test, and PCA were performed using Statistica version 13.3 (StatSoft, Kraków, Poland), and significant differences (p< 0.05) were determined between mean parameters values. Antioxidants 2021,10, 849 5 of 18 3. Results and Discussion 3.1. Evaluation of Organic Acids The analysis of organic acids allows the determination of sensory attributes and health benefits of raw materials and food products. We investigated the contents of oxalic, tartaric, citric, isocitric, malic, quinic, and succinic acids in the fruit skin + pulp, seeds, and leaves of six biotypes of cherry silverberry (Figure 1). The analysis of organic acids indicated that succinic acid predominated in the leaves, whereas malic acid predominated in the fruit skin + pulp and seeds (with their contents accounting for 57%, 50%, and 75% of total organic acids, respectively), which is consistent with the results reported by other authors for cherry silverberry wine [ 17 ]. Both malic and succinic acids are natural metabolites involved in the Krebs cycle. They are used as acidity regulators and are responsible for the enhancement of the anti-oxidative potential of the raw materials they occur in. The average total organic acid content ranged from 1.24 to 12.83 g/100 g d.w. and was 10.0 and 7.0 times lower in the fruit skin + pulp than in the seeds and leaves, respectively, of the six cherry silverberry biotypes tested. Similar tendencies were found by Kolniak-Ostek [ 18 ] in pear fruit components. In addition, the contents of these organic acids in cherry silverberry wine were 17.0, 11.0, and 1.6 times lower than in the seeds, leaves, and fruit skin + pulp of E. multiflora Thunb., respectively [ 17 ]. The average amount of acids in the seeds of cherry silverberry was similar to that in red, white, and black currant and gooseberry and was 1.3, 1.6, 2.0, 2.7, and 5.6 times higher than in chokeberry, elderberry, bilberry, blackberry, and goji berry, respectively [ 19 ]. In addition, this amount in fruit skin + pulp was similar to that in goji berry and black mulberry [ 19 ]. Strong correlations were confirmed between the contents of organic acids and the anti-oxidative activity at r= 0.756 in ABTS assay and r= 0.748 in FRAP assay, as well as between antidiabetic activity and α -glucosidaseinhibiting activity (r= 0.657). In the case of compounds with antioxidative potential, organic acids showed a lower correlation, reaching r= 0.415. The results enable concluding that seeds, i.e., waste material being a component of pomace left after processing, represent the best source of organic acids among all studied components of cherry silverberry. This indicates that cherry silverberry by-products can be a good source of health-promoting compounds and an excellent additive to newly designed functional food. Antioxidants 2021, 10, x FOR PEER REVIEW 5 of 18 2.8. Statistical Analysis Statistical analysis, two-way ANOVA, Tuckey’s test, and PCA were performed using Statistica version 13.3 (StatSoft, Kraków, Poland), and significant differences (p < 0.05) were determined between mean parameters values. 3. Results and Discussion 3.1. Evaluation of Organic Acids The analysis of organic acids allows the determination of sensory attributes and health benefits of raw materials and food products. We investigated the contents of oxalic, tartaric, citric, isocitric, malic, quinic, and succinic acids in the fruit skin + pulp, seeds, and leaves of six biotypes of cherry silverberry (Figure 1). The analysis of organic acids indicated that succinic acid predominated in the leaves, whereas malic acid predominated in the fruit skin + pulp and seeds (with their contents accounting for 57%, 50%, and 75% of total organic acids, respectively), which is consistent with the results reported by other authors for cherry silverberry wine [17]. Both malic and succinic acids are natural metabolites involved in the Krebs cycle. They are used as acidity regulators and are responsible for the enhancement of the anti-oxidative potential of the raw materials they occur in. The average total organic acid content ranged from 1.24 to 12.83 g/100 g d.w. and was 10.0 and 7.0 times lower in the fruit skin + pulp than in the seeds and leaves, respectively, of the six cherry silverberry biotypes tested. Similar tendencies were found by Kolniak-Ostek [18] in pear fruit components. In addition, the contents of these organic acids in cherry silverberry wine were 17.0, 11.0, and 1.6 times lower than in the seeds, leaves, and fruit skin + pulp of E. multiflora Thunb., respectively [17]. The average amount of acids in the seeds of cherry silverberry was similar to that in red, white, and black currant and gooseberry and was 1.3, 1.6, 2.0, 2.7, and 5.6 times higher than in chokeberry, elderberry, bilberry, blackberry, and goji berry, respectively [19]. In addition, this amount in fruit skin + pulp was similar to that in goji berry and black mulberry [19]. Strong correlations were confirmed between the contents of organic acids and the anti-oxidative activity at r = 0.756 in ABTS assay and r = 0.748 in FRAP assay, as well as between antidiabetic activity and α-glucosidase-inhibiting activity (r = 0.657). In the case of compounds with antioxidative potential, organic acids showed a lower correlation, reaching r = 0.415. The results enable concluding that seeds, i.e., waste material being a component of pomace left after processing, represent the best source of organic acids among all studied components of cherry silverberry. This indicates that cherry silverberry by-products can be a good source of healthpromoting compounds and an excellent additive to newly designed functional food. Figure 1. Organic acid profile and amount in fruit skin + pulp, seeds, and leaves of cherry silverberry biotypes. Explanation: si0P-si5P, fruit skin + pulp; si0S-si5S, seeds; and si0L-si5L, leaves. Figure 1. Organic acid profile and amount in fruit skin + pulp, seeds, and leaves of cherry silverberry biotypes. Explanation: si0P-si5P, fruit skin + pulp; si0S-si5S, seeds; and si0L-si5L, leaves. 3.2. Identification of Polyphenolic Compounds Identification of polyphenols in six new cherry silverberry biotypes was performed with the LC-MS-TQD method (Table 1) in multiple reaction monitoring (MRM) mode on specific MS transitions. Identification allowed for tentative recognition of up to 63 compounds and 2 unidentified compounds as quercetin and kaempferol derivatives: Antioxidants 2021,10, 849 6 of 18 38 polyphenolic compounds were identified in the leaves; 14 in the seeds (3 less because they were repeated), and 11 in fruit skin + pulp. Owing to such an effective technique, all compounds were identified for the first time in the tested biotypes, and 17 polyphenolic compounds were identified for the first time in the seeds and leaves of cherry silverberry. Tentative identification was carried out in negative ion mode, whereas qualitative analysis of compounds was based on the reference standard, MS data, fragmentation patterns (MS/MS), and literature data. 3.2.1. Phenolic Acids Peaks 1 and 2 were identified in the leaves as quinic acid (m/z 191) and 3-p-coumaroylquinic acid (m/z 337), respectively, based on the standard and were previously reported in the fruits of pear [ 20 ], cherry elaegnus [ 7 ], and red grapes by Kapusta et al. [ 11 ]. Compound 14 was identified in the leaves and fruit skin + pulp as sinapic acid-O-hexoside, presented a neutral loss of 162 Da (hexose moiety), and was based on the main ion (m/z = 385) and MS/MS (m/z = 223). This compound was identified for the first time. 3.2.2. Flavonols In the present study, 52 flavonol derivatives were characterized as quercetin, isorhamnetin, and kaempferol flavonol derivatives. Among them, 12 compounds were quercetin derivatives (with a precursor ion at m/z = 301), 3 compounds were isorhamnetin derivatives (with the main fragment at m/z = 315), and 28 compounds were kaempferol derivatives (with the main fragment at m/z = 285). UV spectra of analyzed peaks demonstrated absorptions typical of these derivatives, where the maximum absorption at band I was 315–359 nm and at band II was 207–280 nm [ 21 ]. In turn, determination of sugar moieties was performed by classifying them as pentoside, hexoside, and/or deoxyhexoside, which corresponded to the losses of − 132, − 162, and/or − 308 units from the molecular ions [ 22 , 23 ]. Among the identified compounds, there were also acetylated compounds (42 units) and combinations with caffeic acid (136 Da) and p-coumaric acid (176 Da) [11]. In addition, 36 compounds showed the presence of precursors of kaempferol aglycone [ 7 , 24 – 27 ], and 7 compounds were detected for the first time in the fruits of cherry silverberry [ 24 – 27 ]. Moreover, 16 polyphenolic compounds showed the presence of precursors of quercetin and isorhamnetin aglycone, and 5 compounds were detected for the first time in the fruits of cherry silverberry [ 7 ]. Among the identified flavonols, 11 compounds and 2 unspecified compounds were recorded for the first time in E. multiflora Thunb., especially in the seeds and leaves. Based on mass fragmentation, UV spectra, and standards, as well as literature data concerning saffron [ 28 ], compound 41 was identified as kaempferol di-hexoside. In addition, compounds such as kaempferol 3-O-(6 00 -p-coumaryl)-galactoside and kaempferol 3-O-(6 00 -p-coumaryl)-glucoside were previously identified in Tiliae flos by Toker et al. [ 29 ] but for the first time in the seeds. Compound 5 detected in the seeds with the MS/MS fragment on the pseudomolecular ion present at m/z = 609, 447, and 285 was identified as kaempferol-tri-hexoside (m/z = 771) [ 28 ]. Compounds 57 and 59 lost 42 Da, pointing to their acetyl moiety, as implied by Kolniak-Ostek [ 18 ]. These compounds formed the [M-H]- ion at m/z = 581 and 547 with a single MS/MS ion at m/z = 285. These derivatives were tentatively identified in the leaves as eriodictyol glycoside-pentoside and kaempferol malonyl-glucuronide. Compounds 47 and 60 had the main ion at m/z = 603 and m/z = 891 and only a single base peak at m/z = 285 and m/z = 301. The second fragmentation path corresponded to the loss of three hexose residues. Compounds such as quercetin 3-O-rutinoside (m/z = 609), kaempferol rhamnoside-rutinoside (m/z = 739), and quercetin-rhamnoside-glucopyranoside-rhamnoside (m/z = 769) detected in the leaves were previously found in sea buckthorn [ 30 ], Tilia Americana [ 31 ], and Elaeagnus rhamnoides L. [ 32 ]. The signals are indicative of kaempferol derivatives (present in the leaves) and quercetin derivatives (present in the seeds). However, the exact identification of these compounds is impossible without thorough fragmentation. Antioxidants 2021,10, 849 7 of 18 Table 1. Tentative identification of polyphenolic compounds in the seeds, fruit skin + pulp, and leaves of cherry silverberry. Peak No. Tentative Identification Rt (min) [M-H]− (m/z) [M-H]− MS/MS (m/z) UV–VIS (nm) Leaves Seeds Fruit Skin + Pulp Literature 1 Quinic acid 1.05 191 172 262 X [7,11,20] 2 3-p-Coumaroyloqunic acid 1.33 337 191 276 X [7,11,20] 3 Methyl-quercetin 3-O-rhamnoside-pentoside 1.94 609 463/331/299 317 X [7] 4 Quercetin glycoside-pentoside-glycoside 2.53 757 595/463/301 255/352 X [7] 5 Kaempferol-tri-hexoside 2.57 771 609/447/285 267/350 X [28] 6 Kaempferol 3-O-rutinoside-7-O-glucoside 2.58 755 609/447/285 267/350 X [7,26,27] 7 Kaempferol-tri-hexoside-rhamnoside 2.65 917 771/609/285 269/350 X [7] 8 Kaempferol di-rhamnoside-di-glucoside 2.78 901 755/609/447/285 266/350 X [7] 9 Quercetin pentoside-rutinoside 2.8 741 609/463/301 255/350 X [7] 10 Kaempferol 7-O-pentoside 2.87 417 285 281/340 X [7,24,25] 11 Kaempferol 3-O-rhamnoside 3.14 431 285 267/325 X [7,24,25] 12 Kaempferol glucoside-rutinoside 3.17 755 431/285 266/319 X [7,26,27] 13 Kaempferol glucopyranoside-rhamnoside-deoxyhexose 3.30 915 593/285 267/350 X [7] 14 Sinapic acid-O-hexoside 3.36 385 223 325 X X 15 Kaempferol rhamnoside-dihexoside 3.37 917 771/285 274/322 X [7] 16 Quercetin rhamnoside-pentoside-rutinoside 3.39 887 609/579/301 255/352 X [7] 17 Kaempferol pentoside-rhamnoside-rutinoside 3.43 887 755/609/447/285 264/338 X [7] 18 Kaempferol glucoside-di-rhamnoside 3.43 755 593/431/285 266/347 X [7] 19 Quercetin 3-O-rutinoside 3.44 609 463/301 255/352 X [30–32] 20 Quercetin rhamnoside-pentoside-rhamnoside 5.45 887 741/595/433/301 255/355 X [7] 21 Quercetin 3-O-rhamnoside 3.49 447 301 255/326 X [7,26,27] 22 Kaempferol rhamnoside-rutinoside 3.53 739 593/447/285 265/326 X [30–32] 23 Trigalloyl-hexoside 3.53 635 421/169 274 X [33] 24 Kaempferol rhamnoside-pentoside-rutinoside 3.66 871 725/563/431/285 266/340 X [7] 25 Kaempferol pentoside-rutinoside 3.67 725 579/417/285 267/350 X [7] 26 Trigalloyl-hexoside 3.68 635 465/313 276 X [33] 27 Kaempferol pentoside-rutinoside 3.71 725 579/417/285 265/345 X [7] 28 Kaempferol hexoside-pentoside-rhamnose 3.76 725 563/431/285 267/350 X [7] 29 Kaempferol rhamnoside-pentoside 3.79 563 417/285 265/328 X [7] 30 Kaempferol 3-O-rutinoside 3.84 593 447/285 265/334 X [7,26,27] 31 Kaempferol glucopyranoside-dihexoside 3.86 785 609/447/285 267/350 X [7] 32 Quercetin pentoside-rutinoside 3.91 741 609/433 255/350 X [7] 33 Kaempferol di-rhamnoside-di-glycoside 3.97 901 755/609/447/285 264/339 X [7] 34 Trigalloyl-hexahydroxydiphenoyl 4.02 951 907/783 274 X [33] Antioxidants 2021,10, 849 8 of 18 Table 1. Cont. Peak No. Tentative Identification Rt (min) [M-H]− (m/z) [M-H]− MS/MS (m/z) UV–VIS (nm) Leaves Seeds Fruit Skin + Pulp Literature 35 Quercetin 3-O-rhamnoside-7-O-pentoside 4.12 595 433/301 255/350 X [7] 36 Digalloyl-gallagyl-hexoside 4.14 1085 765/633/451 272 X [33] 37 Quercetin-O-glucoside-O-pentoside 4.16 595 463/301 255/345 X [7] 38 Kaempferol di-rhamnoside-glucoside 4.19 739 593/447/285 264/345 X [7,26,27] 39 Tetragalloyl-hexoside 4.22 787 635/617/301 272 X [33] 40 Kaempferol rhamnoside-rutinoside 4.24 739 593/285 265/340 X [30–32] 41 Kaempferol-di-hexoside 4.31 609 447/285 267/350 X [28] 42 Quercetin-tri-rhamnoside 4.33 739 593/447/301 255/355 X [7] 43 Tetragalloyl-hexoside 4.37 787 635/617/301 277 X [33] 44 Kaempferol di-rhamnoside-glucoside 4.37 739 593/447/285 265/338 X [7,26,27] 45 Quercetin di-rhamnose 4.47 593 447/301 255/347 X [7] 46 Quercetin-rhamnoside-glucopyranoside-rhamnoside 4.48 769 593/447/301 255/352 X [30–32] 47 Unspecified quercetin derivative 4.48 603 301 252/366 X 48 Pentagalloyl-hexoside 4.57 939 787/635/301 269 X [33] 49 Kaempferol pentoside-rhamnoside-glucuronide 4.59 739 563/417/285 265/324 X [7] 50 Kaempferol di-rhamnoside-hexoside 4.66 739 593/447/285 265/319 X [7] 51 Kaempferol pentoside-di-rhamnoside 4.80 709 577/431/285 265/339 X [7] 52 Kaempferol di-rhamnose 4.89 577 431/285 264/341 X [7] 53 Pentagalloyl-hexoside 4.89 939 787/635/301 274 X [33] 54 Kaempferol-3-O-glucoside 5.01 447 285 264/319 X [7,24,25] 55 Isorhamnetin-7-O-rutinoside 5.09 623 477/315 253/358 X [7] 56 Kaempferol glucoside-glucuronide 5.12 623 447/285 264/317 X [7] 57 Eriodictyol glucoside-pentoside 5.19 581 285 265/315 X [18] 58 Isorhamnetin-3-O-glucoside 5.20 477 315 256/380 X [7] 59 Kaempferol malonyl-glucuronide 5.27 547 285 265/315 X [18] 60 Unknown derivative of Kaempferol 5.63 891 285 269/325 X 61 Isorhamnetin 3-O-(6”malonyl)-glucuronide-rhamnoside 5.71 723 491/315 270/350 X [7] 62 Kaempferol 3-O-rhamnoside 5.94 431 285 264/325 X [7,24,25] 63 Kaempferol 3-O-(6”-p-coumaryl)-galactoside 6.87 593 447/285 267/312 X X [29] 64 Kaempferol 3-O-(6”-caffeoyl)-glucoside 7.00 623 447/285 264/321 X X [7] 65 Kaempferol 3-O-(6”-p-coumaryl)-glucoside 7.11 593 447/285 267/315 X X [29] XIt is mean that the identified compounds are available in the tested fraction. Antioxidants 2021,10, 849 9 of 18 3.2.3. Hydrolysable Tannins Chromatographic analysis showed the presence of eight hydrolysable tannin derivatives determined just in the seeds. Compounds such as trigalloyl-hexoside were identified based on the main ion at m/z = 635 and a fragmentation ion at m/z = 421 and 169 and m/z = 465 and 331. The compound that had the [M-H] – ion at m/z = 951 and the MS/MS fragment ion at m/z = 907 and 783 was identified as trigalloyl-hexahydroxydiphenoyl. The compound digalloyl-gallagyl-hexoside was identified based on the major ion at m/z = 1085 and the MS/MS ion at m/z = 765, 633, and 451. Compounds whose Rt was 4.22 and 4.37 min had the [M-H] – ion at m/z = 787 and the fragment ion at m/z = 635, 617, and 301 and were identified as tetragalloyl-hexoside. The final compounds belonging to hydrolysable tannins were identified as pentagalloyl-hexoside with m/z = 939 and the fragment ion at m/z = 787, 635 and 301. These hydrolysable tannin derivatives have been previously noted in the literature [ 33 ], but they were determined here in cherry silverberry, especially in the seeds, for the first time. 3.3. Quantification of Polyphenols As presented in Figure 2and Supplementary Material Tables S1–S3, the greatest variety of polyphenols was noted in the leaves and seeds compared with the fruit skin + pulp . The content of polyphenols in the tested parts of cherry silverberry ranged from 171.57 (si5) to 1951.01 (si0) mg/100 g d.w. in the seeds, from 464.21 (si4) to 1142.03 (si0) mg/100 g d.w. in the leaves, and from 417.02 (si5) to 819.04 (si0) mg/100 g d.w. in the fruit skin + pulp . According to Yoon et al. [ 34 ], the content of polyphenols determined in the leaves of E.s multiflora Thunb. was 805.60 mg/100 d.w. and was similar to that assayed in our study. Further, the values of polyphenols in Jahidka and Sweet Scarlet cultivars of E. multiflora Thunb. were 904.65 and 1268.90 mg/100 g d.w., and the average content was similar and 1.5 times higher than that in the fruit skin + pulp of tested biotypes, respectively [ 7 ]. The total polyphenolic compound content determined in sea buckthorn fruits (Hippophae rhamnoides L.) and gummi (Elaeagnus indica) was, on average, 4.0 and 7.0 times lower, respectively, than in the fruit skin + pulp of cherry silverberry (E. multiflora Thunb.) [35,36] . Studies on polyphenolic compounds in pear parts proved that their content in the seeds is 978.50 mg/100 g d.w., in the fruit skin + pulp is 234.2 mg/100 g d.w., and in the leaves is 5326.70 mg/100 g d.w. These values are 1.3 and 2.5 times lower and 7.7 times higher, respectively, compared to the tested parts of the cherry silverberry [ 18 ]. However, the content of polyphenolic compounds tested in the fruit skin + pulp and leaves of cherry silverberry was 6.0 and 17.0 times lower than in the fruit and leaves of cranberry, respectively [37]. The main group of polyphenols in the tested fruit skin + pulp, leaves, and seeds of cherry silverberry was polymeric procyanidins [ 37 ]. This group accounted for over 66.0–95.0% of total polyphenols identified in cherry silverberry. The content of polymeric procyanidins in the analyzed seeds ranged from 160 (si5) to 1951 (si0) mg/100 g d.w. and was, on average, 2.8 times higher than in the leaves and fruit skin + pulp. The content of procyanidins in cherry silverberry was similar to that determined in pear leaves [ 38 ] and gummi fruits and leaves [ 7 ] and also significantly lower than in saskatoon berry and cranberry fruits [ 37 , 39 ]. The average degree of polymerization in the fruit skin + pulp was 12.0, which was 1.2 and 2.5 times lower than in the seeds and leaves, respectively, and also similar to the degree of polymerization determined in grape and gummi fruits [ 40 ]. However, procyanidins are the most desirable components of nutraceuticals due to their higher antioxidant activity compared to flavonols and phenolic acids and due to their positive impact on human health [7]. Antioxidants 2021,10, 849 16 of 18 4. Conclusions In summary, owing to such an effective technique as liquid chromatography, 88 compounds (including 7 organic acids, 1 tocopherol, 13 carotenoids, 4 chlorophylls, 52 flavonols, 8 hydrolysable tannins, and 3 phenolic acids) were recorded and 17 polyphenolic compounds and 3 isoprenoids were identified for the first time in the seeds and leaves of E. multiflora Thunb. In total, 55 compounds were identified in the leaves, 36 in the seeds, and 31 in the fruit skin + pulp. The predominant group of polyphenols was polymeric procyanidins (66–95% of total polyphenolics), whereas the predominant isoprenoids were chlorophyll b and (all-E)-lycopene. The leaves contained significant levels of phenolic acids, the sum of flavonols (kaempferol and quercetin), lycopene, β -carotene, α -carotene, and chlorophylls, as well as highly inhibited lipase activity. The seeds were rich in organic acids, hydrolysable tannins, and isorhamnetin and showed a high degree of polymerization, high antioxidant capacity, and high inhibition of α -amylase and α -glucosidase. In addition, the fruit skin + pulp had high contents of total polyphenolic compounds, carotenoids, and phytoene, as well as polymeric procyanidins. In vitro antidiabetic activity measured as α -glucosidase, α -amylase, and lipase inhibitory activity of cherry silverberry fractions was positively correlated with the contents of polyphenolic compounds, antioxidant activity, and isoprenoids. Given the results of studies on diverse bioactive compounds, fruit fractions can be used to develop products intended for human consumption, such as functional foods, nutritional supplements, and nutricosmetics, among others. Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/antiox10060849/s1, Table S1. Polyphenolic compound content in seeds of cherry silverberry (mg/100 g d.w.); Table S2. Polyphenolic compound content in fruit skin + pulp of cherry silverberry (mg/100 g d.w.); Table S3. Polyphenolic compound content in leaves of cherry silverberry (mg/100 g d.w.); Table S4. Antioxidant activity and in vitro enzymatic activity of new biotypes of cherry silverberry. Author Contributions: Conceptualization, S.L.-W.; material, A.B.; methodology, S.L.-W., I.K., A.J.M.-M. , C.M.S., and I.O.; formal analysis, S.L.-W.; investigation, S.L.-W.; writing—original draft preparation, S.L.-W. and A.J.M.-M.; writing—review and editing, S.L.-W.; project administra-tion, S.L.-W.; funding acquisition, S.L.-W.; data curation, Z.G. All authors have read and agreed to the published version of the manuscript. Funding: This publication was funded by the Minister of Science and Higher Education as part of the program entitled “Regional Initiative of Excellence” for the years 2019–2022 (project no. 010/RID/2018/19; amount of funding 12.000.000 PLN). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are available on request from the corresponding author. Acknowledgments: This work was supported by the NSC, Poland (DEC-2019/03/X/NZ9/00891). 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