Glucosidase Inhibitory Potential of Wild Arbutus Unedo L. Fruits from Central Italy: a Chemometric Approach

Article Phytochemical Proﬁle, Antiradical Capacity and α-Glucosidase Inhibitory Potential of Wild Arbutus unedo L. Fruits from Central Italy: A Chemometric Approach

Valentina Macchioni 1, Veronica Santarelli 2 and Katya Carbone 1,* 1 CREA Research Centre for Olive, Fruit and Citrus Crops, Via di Fioranello 52, 00134 Rome, Italy; [email protected] 2 Faculty of Bioscience and Technologies for Food, Agriculture and Environment, University of Teramo, Via Renato Balzarini 1, 64100 Teramo, Italy; [email protected] * Correspondence: [email protected]

Received: 28 November 2020; Accepted: 11 December 2020; Published: 16 December 2020

Abstract: Nowadays, there is a growing interest in botanicals for human nutrition and care. Arbutus unedo wild berries are edible and medicinal fruits that contain many healthy bioactive components, which can be considered a valuable resource for the food ingredient market and for nutraceutical and cosmetic sectors. In the present study, the polyphenols and in vitro antiradical and hypoglycemic activities of five wild Italian accessions of A. unedo were investigated, and their chemical profiles were treated by means of unsupervised chemometric techniques like the hierarchical and principal component analysis. Moreover, Fourier-transformed mid-infrared spectroscopy was used to provide a rapid assessment of the phytochemical composition of different accessions. Samples differed mainly in their anthocyanin content and overall nutraceutical potential. Anthocyanins were present mainly as glycosides of cyanidin and delphinidin, with delphinidin-3-O-glucoside being the most abundant one, ranging from 49 1 to 111 3 mg g 1 (for P1 and P2, respectively; ± ± − p < 0.05). Extracts were screened for their in vitro biological activities by using the 2,20-azino-bis + (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS• ), 2,2-diphenyl-1-picrylhydrazyl (DPPH•) antiradical tests, while their hypoglycemic activity was investigated by the α-glucosidase inhibition test. In both 1 in vitro antiradical tests, the highest capacity was recorded for P2 (EC50: 1.17 and 0.064 mg mL− , + for DPPH• and ABTS• , respectively), with values higher than those reported in the literature for A. unedo fruit extracts. P2 also showed the highest inhibition power towards α-glucosidase (about 70%). Moreover, the nonparametric correlation analysis pointed out a very high significant correlation between the percentage of α-glucosidase inhibition and cyanidin-3-O-rutinoside (r: 0.973; p < 0.01). Finally, the application of hierarchical analysis to samples analyzed provided three different clusters based on the average phytochemical content coded as low, medium and high. Moreover, principal component analysis made it possible to establish similarities among the accessions depending on their overall nutraceutical characteristics and on the relative anthocyanin content.

Keywords: A. unedo; strawberry tree fruit; polyphenols; nutraceuticals; wild berries; antiradical capacity; a-glucosidase inhibition

1. Introduction Arbutus unedo L. (Ericaceae family), commonly known as the strawberry tree, is a wild evergreen shrub native to the Mediterranean region, which grows spontaneously in other regions characterized by hot summers and mild and rainy winters [1]. It is a xerophilous plant that prefers siliceous soils

Plants 2020, 9, 1785; doi:10.3390/plants9121785 www.mdpi.com/journal/plants Plants 2020, 9, 1785 2 of 16 and grows at altitudes between 0 and 800 m. In Italy, its diffusion is continuous along the Ligurian, Sardinian, Sicilian, Tyrrhenian and Adriatic coasts. From an ecological point of view, A. unedo L. is a hardy plant, and this plays a key role in reforestation programs in southern European countries such as Greece, Italy, Portugal and Spain, where forest fires are common during the dry season [2]. A. unedo L. produces red spherical fruits, having a diameter of about 2–3 cm. These fruits are rarely eaten fresh, despite their pleasant taste when fully ripe, because they are rich in seeds and therefore not very appreciated by the modern consumer [3]. However, they have some importance in local agricultural communities, which use them for the production of alcoholic beverages, jams, jellies and marmalades [4]. Moreover, these fruits have been known since ancient times for their healthy properties, being used in folk medicine for diverse purposes [5] and the Food and Agriculture Organization (FAO) is currently looking for ways to increase the use of this precious wild species [6], with the aim to preserve plant biodiversity. Recent studies suggest that strawberry tree’s bioactive compounds are linked to a broad spectrum of human health benefits such as antidiabetic, antihypertensive, anti-inflammatory, antitumor, antioxidant and spasmolytic [5], antiseptic, diuretic and laxative properties [7]. The potential advantages of A. unedo fruits are related to the presence of polyphenolic compounds in its composition [5], capable of eliminating oxygen radicals and other reactive species. The main phenolic compounds in strawberry tree fruits are anthocyanins [8]. These bioactive substances are proved to be inhibitors of α-glucosidase, contributing to the reduction of type-2 diabetes [9,10]. Moreover, the European Union allows the use of anthocyanins as food colorants (namely E163) in acidic food or beverages, including soft drinks, fruit preserves, sugar confectioneries, dairy products, frozen products, dry mixes and alcoholic drinks. Anthocyanins have not only shown beneficial effects as antioxidants [11] but also, they have the ability to regulate the gene expression of adipocytokines [12]. Hence, there is little to no data in literature on the antioxidants present in the fruit of A. unedo L., especially those of spontaneous Italian flora, although its antioxidant content is very high compared to other fruits [13]. The formulation of phytoextracts rich in bioactive compounds is of fundamental importance to promote the use of these underutilized fruits in various industrial sectors, being a precious resource for the food ingredient market and for nutraceutical and cosmetic sectors. To the best of our knowledge, there is very limited information on the antiradical potential and nutraceutical attributes of wild A. unedo fruits growing in Italy [14] and on the relationships among these attributes and the biological activity linked to them, whereas the search for new resilient crops able to adapt to the strong climate change is increasingly pressing. In light of these considerations, the present study aimed to evaluate the content of phenols and anthocyanins in fruit extracts from five spontaneous accessions of Arbutus unedo L., growing in Central Italy, through a comparative analysis of their phenolic profile, antiradical potential and hypoglycemic activity. Finally, the relationships among analyzed parameters were carried out by using a chemometric approach based on hierarchical cluster analysis (HCA) and principal component analysis (PCA).

2. Materials and Methods

2.1. Chemicals

Folin–Ciocâlteu reagent, gallic acid, catechin, 2,2-diphenyl-1-picrylhydrazyl radical (DPPH•), + vanillin, 2,20-azinobis-(3-ethylbenzothiazolin-6-sulfonic acid) (ABTS• ), potassium persulfate, and vanillin were purchased from Sigma-Aldrich (Milan, Italy). Delphinidin-3-O-glucoside chloride, cyanidin 3-O-glucoside chloride, cyanidin-3-O-rutinoside chloride and malvidin-3-O-glucoside chloride standards used for identification and quantification purposes with high-performance liquid chromatography (HPLC) were purchased from Extrasynthese (Genay, France). Organic solvents used for chromatography were of HPLC ultragradient grade (Sigma-Aldrich, Milan, Italy), whereas distilled water was obtained by the Milli-Q system (Millipore, Milan, Italy). The 45 µm pore size membrane filters were purchased from Pall (Pall Corporation, Ann Arbor, MI, USA) and used for filtration of samples. Plants 2020, 9, 1785 3 of 16

2.2. Plant Material In the present study, fresh and healthy fruits of A. unedo, fully ripened (average total soluble solids: 17.1 0.7 Brix; skin fruit’s color: homogenous red-scarlet pigmentation), were collected randomly ± ◦ from plants (ﬁve accessions, indicated with the letter “P”) growing wild along the Lazio coast (Central Italy; 41◦52’47“N 12◦16’29” E; 70 m a.s.l.) and taxonomically identiﬁed. Fruits were kept in a cooler bag during the transport to the laboratory. Four lots of 30 fruits per accession were prepared and sorted based on homogeneous size and absence of physical injuries and/or infections. Soon after harvested, samples were frozen in liquid nitrogen, freeze-dried and ground prior to analysis.

2.3. Fourier–Transform Infrared Spectroscopy (FTIR) Analysis in Attenuated Total Reflectance (ATR) Mode ATR-FTIR spectra of A. unedo wild berries were acquired as described by Amoriello et al. [15] 1 without modifications. IR spectra (wavenumbers ranging from 4000 to 600 cm− ) were collected at room temperature with a FT-IR spectrometer (iS 50 FT-IR Nicolet Thermo Fisher Scientific Inc.,Milan, Italy) equipped with a single-reflection horizontal ATR cell with a diamond crystal. After the acquisition, processed spectral data were obtained with the OMNIC™ software (Thermo Fisher Scientific Inc., Milan, Italy).

2.4. Extraction of Bioactive Compounds Freeze-dried samples were extracted to determine their phytochemical composition, e.g., total polyphenol content (TPC), total flavan-3-ol content (FLC), total monomeric anthocyanins (TMA), HPLC with diode-array detection polyphenol profile, antiradical capacity (AC) and α-glucosidase inhibitory activity (AGA). 1.0 g of fruit powder was extracted with 15 mL of a hydroalcoholic solution (methanol: water = 80:20, v/v) acidified with 0.1% HCl (v/v), for 30 min, under continuous mechanical stirring at room temperature (shaking incubator mod. SKI 4; Argolab, Milan, Italy), followed by 30 min of ultrasound-assisted extraction, performed in a temperature-controlled sonication bath (UTA-200, Falc, Italy), operating at 40 kHz. The resulting extracts were then centrifuged at 6792 g for 15 min at 4 ◦C. The extraction procedure was repeated twice using 15 mL of fresh solvent, and the two extracts were combined and filtered through Whatman No. 1 paper immediately prior to all analyses.

2.5. Total Phenolic Content The total content of soluble polyphenols in the extracts was determined, according to Carbone et al. [16]. TPC was calculated using a calibration curve of gallic acid. The results were 1 expressed as milligrams of gallic acid (GAE) equivalents per gram of fruit powder (mg GAE g− ). All determinations were performed in triplicate.

2.6. Total Flavan-3-ol Content FLC was determined following the vanillin assay method, as reported by Carbone et al. [16]. FLC was calculated from a calibration curve, using catechin as a standard. Results were expressed as 1 milligrams of catechin equivalents per gram of fruit powder (mg CTE g− ). All determinations were performed in triplicate.

2.7. Total Monomeric Anthocyanin Content TMA content of the extracts was performed according to Ciccoritti et al. [10], without modiﬁcations. 1 Data were expressed as µg cyanidin-3-O-glucoside (cyd-3-glu) equivalents g− of fruit powder. All determinations were performed in triplicate.

2.8. HPLC-DAD Proﬁles of Single Phenols and Anthocyanins Anthocyanins were separated and identiﬁed by an analytical HPLC system (Agilent 1100 series, Agilent, Milan, Italy) equipped with a diode array detector (DAD) (Agilent Technologies, Milan, Italy). Plants 2020, 9, 1785 4 of 16

The separation was carried out on a Zorbax SB C18 column (Agilent, 4.6 250 mm; 5 µm particle size, × set at 30 ◦C), according to Ciccoritti et al. [10]. Single anthocyanins were identified at 520 nm by their retention times and spectral data as compared to individual standards and by the method of standard additions to the samples. The single phenols of the different extracts were separated and identified by an HPLC analytical system (Agilent 1100 series, Agilent, Italy) equipped with a DAD (Agilent Technologies, Italy), which was fixed simultaneously at 280 nm (benzoic acids and flavan-3-ols), 320 nm (hydroxycinnamic acids) and 370 nm (flavonols). In addition, UV-vis spectra were recorded in the range of 200–700 nm. The separation of single phenols was performed according to the protocol reported by Carbone and Mencarelli [17], without modifications, on a Luna C18 column (Phenomenex; 4.6 250 mm; particle × size 5 µm, set at 30 ◦C). The injection volume was 20 µL. All analytical data were evaluated using a chromatographic data management system (Chemstation 32.1, Agilent Technologies). Ten-point calibration curves based on external standard solutions (0–100 ppm) were obtained for quantification. 1 1 Results were expressed as mg g− (for phenolics) and as µg g− (for anthocyanins) of fruit powder.

2.9. In Vitro Bioactivity of A. unedo Fruit Extracts

2.9.1. Antiradical Capacity (AC) Assays The radical scavenging power of the samples was assessed by measuring their ability to scavenge + synthetic radicals (e.g., DPPH• and ABTS• ), according to Carbone et al. (2011) [16]. Results were expressed as EC50. All determinations were performed in triplicate.

2.9.2. α-Glucosidase Inhibition Assay The α-glucosidase activity of analyzed samples was evaluated according to the Sigma-Aldrich enzymatic assay for α-glucosidase [18], using p-nitrophenyl α-D-glucoside as substrate. The α-glucosidase activity was spectrophotometrically determined by measuring the release of p-nitrophenol from p-nitrophenyl α-D-glucopyranoside at 400 nm. Inhibition of the enzyme activity was expressed as percentage inhibition and calculated as follows:

α-glucosidase inhibition (%) = (Abs Abs )/(Abs ) 100. control − sample control × All determinations were performed in triplicate.

2.10. Statistical Analysis Statistical analysis was performed with SPSS 25.0 software (SPSS, Inc., Chicago, Illinois). Data were reported as means standard deviation (SD) of four independent experiments with three replicates. ± Prior to chemometric applications, all variables were auto-scaled (transformation into z-scores) to standardize the statistical importance of all responses. An exploratory data analysis was made to check the data-normal distribution (Kolmogorov–Smirnov test) and the homogeneity of variance (Levene’s test). Data violated ANOVA assumptions, even after their mathematical transformation. In light of the obtained results, we analyzed non-normally distributed data through the Kruskal–Wallis (K-W) nonparametric test, while significant mean differences were established using the Mann–Whitney test for independent and nonparametric procedures (p < 0.0167 for Bonferroni’s correction, where not differently specified; [19]). Correlations among all parameters in the dataset were analyzed using Spearman’s correlations (r; p < 0.05 and p < 0.01). Finally, principal component analysis (PCA) and hierarchical cluster analysis (HCA) were used as exploratory chemometric methods to study the data structure, investigating similarities and hidden patterns among the extracts analyzed. Both methods were applied to normalized data (z-scores) to retrieve all chemically relevant information systematically, using SPSS 25.0 software (SPSS, Inc., Chicago, IL). Clusters were computed by Ward’s method based Plants 2020, 9, 1785 5 of 16

Plantson Euclidean 2020, 9, x FOR distance, PEER REVIEW and the significant differences among clusters were investigated by K-W5 of test 16 followed by Dunn’s test for post-hoc analyses. 3. Results and Discussion 3. Results and Discussion 3.1. FTIR Spectra Analysis 3.1. FTIR Spectra Analysis In the present study, ATR-FTIR analysis of A. unedo wild berries was carried out, for the first In the present study, ATR-FTIR analysis of A. unedo wild berries was carried out, for the first time, time, to provide effective information about the qualitative composition of the plant matrix by to provide effective information about the qualitative composition of the plant matrix by analyzing the analyzing the relation between the spectral fingerprints and molecular structures of the samples. relation between the spectral fingerprints and molecular structures of the samples. Figure1a,b shows Figure 1a,b shows the absorbance (a) and first derivative spectra (b) of samples analyzed, acquired the absorbance (a) and first derivative spectra (b) of samples analyzed, acquired in the mid-infrared in the mid-infrared region (see also Table S1 in the electronic-supplementary material). The first region (see also Table S1 in the electronic-supplementary material). The first derivative IR spectra derivative IR spectra allowed the analysis of overlapped peaks. The spectra were analyzed with allowed the analysis of overlapped peaks. The spectra were analyzed with respect to the spectral respect to the spectral band positions in order to identify the signatures of the major functional groups band positions in order to identify the signatures of the major functional groups characterizing the characterizing the A. unedo berries. As can be seen (Figure 1a), the unique spectral patterns of extracts A. unedo berries. As can be seen (Figure1a), the unique spectral patterns of extracts from di fferent from different wild accessions reflected their compositional differences (different band positions and wild accessions reflected their compositional differences (different band positions and intensities), intensities), suggesting significant genetic differences. suggesting significant genetic differences.

(a)

(b)

Figure 1. AverageIR spectra of A. unedo wild berries analyzed: (a) raw spectra; (b) first derivative spectra. Figure 1. Average IR spectra of A. unedo wild berries analyzed: (a) raw spectra; (b) first derivative spectra. 1 1 Two characteristic spectral regions are clearly evident above 2800 cm− and below 1800 cm− (Figure1a); the latter indicative of the functional groups and fingerprinting of the plant matrix analyzed. −1 −1 Two characteristic spectral regions are clearly1 evident above 2800 cm and below 1800 cm The first broad band located at about 3300 cm− is attributed to the O-H stretching, which is associated (Figure 1a); the latter indicative of the functional groups and fingerprinting of the plant matrix with the presence of carboxyl groups in compounds such as polyphenol acids, as well as to the O-H analyzed. The first broad band located at about 3300 cm−1 is attributed to the O-H stretching, which stretching vibration of the phenolic groups, which are present in A. unedo wild berries [5]. A band is associated with the presence 1of carboxyl groups in compounds such as polyphenol acids, as well1 system around 3000–2800 cm− , with two weak and broad peaks centered at 2970 and 2935 cm− as to the O-H stretching vibration of the phenolic groups, which are present in A. unedo wild berries related to the symmetric and asymmetric stretching of -CH3 and -CH2- groups, characteristics of [5]. A band system around 3000–2800 cm−1, with two weak and broad peaks centered at 2970 and 2935 lipids and fatty acids, was also visible (Figure1b; inset c). In this region, the first derivative spectra −1 cm related to the symmetric and asymmetric1 stretching of -CH3 and -CH2- groups, characteristics of1 pointed out also a defined peak at 2859 cm− , preceded by a shoulder centered at about 2870 cm− , lipids and fatty acids, was also visible (Figure 1b; inset c). In this region, the first derivative spectra characterizing the presence of alkanes (C–H) and aldehydic groups (–CO–H), as reported by Umdale pointed out also a defined peak at 2859 cm−1, preceded by a shoulder centered at about 2870 cm−1, characterizing the presence of alkanes (C–H) and aldehydic groups (–CO–H), as reported by Umdale et al. [20]. Moreover, the derivative spectra pointed out the presence of a small but clear band to the left of the system of intense signals due to aliphatic stretching, i.e., just above 3000 cm−1 (3015 cm−1;

Plants 2020, 9, 1785 6 of 16

et al. [20]. Moreover, the derivative spectra pointed out the presence of a small but clear band to the 1 1 left of the system of intense signals due to aliphatic stretching, i.e., just above 3000 cm− (3015 cm− ; Figure1b, inset c), occurring by the presence of alkene terminal groups when a single H atom was bound to the double bond. These findings agree with those of Morgado et al. [5], who reported a high content of polyunsaturated fatty acids, representing about 60% of total fatty acids, in A. unedo berries. 1 The fingerprintingPlants 2020, 9, x FOR PEER region REVIEW between 1800 and 700 cm− showed significant differences6 of 16 between the samples analyzed (Figure1b, inset d). A well-defined peak, occurring at 1750 cm 1, related Figure 1b, inset c), occurring by the presence of alkene terminal groups when a single H atom was − to the carbonylbound to group the double (C= bond.O) stretchingThese findings vibrations agree with those ofthe of Morgado ester bond,et al. [5], was who reported present a high in all extracts 1 analyzed. Thecontent presence of polyunsaturated of esters fatty was acids, also repres confirmedenting about by the 60% strong of total absorptionfatty acids, in atA. unedo 1078 berries. cm− (stretching of COO-C) (FigureThe fingerprinting1b, inset d). region These between findings 1800 and are 700 in cm line−1 showed with significant the fact differences that phenolics, between especially the samples analyzed (Figure 1b, inset d). A well-defined peak, occurring at 1750 cm−1, related to the phenolic acids, occur naturally in combination with other compounds, usually in the form of esters [21]. carbonyl group (C=O) stretching vibrations of the ester bond, was present in all extracts analyzed. 1 The low-intensityThe presence absorption of esters was bands also in confirmed the range by 1500–1450the strong absorption cm− could at 1078 be cm attributed−1 (stretching to of C–C-O COO- stretching vibrationsC) [22 (Figure]. From 1b, inset derivative d). These findings spectrum are in (Figure line with1 theb; fact inset that d), phenolics, several especially peaks phenolic in the acids, spectral range occur 1naturally in combination with other compounds, usually in the form of esters [21]. The low- 1100–1000 cm− , due to the stretching of OH group, characteristic of phenolics, and to the aromatic intensity absorption bands in the range 1500–1450 cm−1 could be attributed to C–C-O stretching C–H-planevibrations deformation [22]. From vibrations, derivative werespectrum also (Figure observed 1b; inset [23 d),]. several Finally, peaks a well-defined in the spectral bandrange system in 1 the range 900–7501100–1000 cmcm−−1, duewas to the due stretching to the oscillationsof OH group, characteristic of the H atoms of phenolics, outside and the to the aromatic aromatic ring plane, dependingC–H-plane on the numberdeformation of vibrations, adjacent were H atoms also observ anded as [23]. a consequenceFinally, a well-defined on the band meta system substitution in of −1 aromatic protonsthe range [ 21900–750]. cm was due to the oscillations of the H atoms outside the aromatic ring plane, depending on the number of adjacent H atoms and as a consequence on the meta substitution of aromatic protons [21]. 3.2. Phytochemical Composition of the Extracts Analyzed 3.2. Phytochemical Composition of the Extracts Analyzed Figure2 shows the TPC of the di fferent extracts analyzed, which ranged from 17.6 0.3 to 23.3 Version December 16, 2020 submitted to Journal Not Specified 3 of 4 Version DecemberFigure 16,1 2020 2 shows submitted the TPC to Journal of the Notdifferent Specified extracts analyzed, which ranged from 17.6 ± 0.3 to 23.3± 3 of± 6 0.2 mg GAE g− (for P5 and P4, respectively). ± 0.2 mg GAE g−1 (for P5 and P4, respectively).

PlantsPlants 2020 2020, ,9 9, ,x x FOR FOR PEER PEER REVIEW REVIEW 10 of 16 PlantsPlants 2020 2020, ,9 9, ,x x FOR FOR PEER PEER REVIEW REVIEW 10 of 16

Figure 2. Total phenolic and flavan-3-ols content (mg g−1) 1of five A. unedo fruit extracts (mean ± SD). Figure 2. Total phenolic and flavan-3-ols content (mg g− ) of five A. unedo fruit extracts (mean SD). Different letters indicate significant differences in the mean (p < 0.05). TPC: total polyphenol content; ± Different lettersFLC: flavan-3-ols indicate content; significant GAE: digallicfferences acid equivalents; in the mean CTE: catechin (p < 0.05). equivalents. TPC: total polyphenol content; FLC: flavan-3-ols content; GAE: gallic acid equivalents; CTE: catechin equivalents. Based on the experimental results, the genotype significantly influenced the TPC of the extracts Based(p on < 0.05) the as experimental follows: P4~P2 results,> P3 > P1 the> P5.genotype These results significantly are in agreement influenced with those thereported TPC in of the the extracts literature((aa)) [7] [24] and higher than those reported by Doukani et al. [25], which ranged between 7 and (p < 0.05) as follows: P4~P2−1 > P3 > P1 > P5. These results are in agreement with−1 those reported in the 14( (amga)) GAE g . As regards flavan-3-ols, FLC ranged from 2.98 to 9.58 mg CTE g (for P1 and P2, literature [ 7][24] and higher than those reported by Doukani et al. [25], which ranged between 7 and 1 1 14 mg GAE g− . As regards flavan-3-ols, FLC ranged from 2.98 to 9.58 mg CTE g− (for P1 and P2, respectively; p < 0.05), being P2 > P5 > P4 > P3 > P1 (Figure2). Reported values are higher than those

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A 1-picrylhydrazylobtainobtain 50% 50% radical radical (DPPH scavenging).scavenging). test; ( bInIn) the,2the′-azino-bis histogram,histogram, (3-ethylbenzothiazoline-6-sulfonic differentdifferent letters indicate significant acid) differences (ABTS ()p test. < caption on a single line should be centered. μ −1 caption onIn a singleboth in linevitro shouldtests, antiradica be centered.l capacity expressed in terms of EC50 (μg mL−1 of extract required to In0.05).0.05). both in vitro tests, antiradical capacity expressed in terms of EC50 ( g mL of extract required to obtainobtain 50% 50% radical radical scavenging).scavenging). InIn thethe histogram,histogram, differentdifferent letters indicate significant differences (p < 0.05). 0.05).TheThe antiradical antiradical potentialpotential ofof thethe extractsextracts testedtested wass higherhigher than those reported by Tenuta et al. [40][40] forfor A.A. unedounedo berriesberries fromfrom SouthernSouthern Italy.Italy. NonparametriNonparametric correlation analysis, carried out on the entireThe dataset, antiradical pointed potential out the of highest the extracts significant tested correlation was higher between than those EC50DPPH reported and malvidin-3-O-by Tenuta et al. entireThe dataset, antiradical pointed potential out the of highest the extracts significant tested correlation was higher between than those EC50DPPH reported and malvidin-3-O-by Tenuta et al. [40] for A. unedo berries from Southern Italy.ρ Nonparametric correlation analysis, carried out on the [40]glucosideglucoside for A. contentunedocontent berries ofof extractsextracts from analyzedSouthernanalyzed (Italy.(ρ:: −−0.886;0.886; Nonparametri pp << 0.01),0.01), cwhilewhile correlation nono significant analysis, correlations carried out wereon the entirefound dataset,among EC pointed50ABTS and out the the variables highest analyzed.significant correlation between EC50DPPH and malvidin-3-O- entirefound dataset,among EC pointed50ABTS and out the the variables highest analyzed.significant correlation between EC50DPPH and malvidin-3-O- glucoside content of extracts analyzed (ρ: −0.886; p < 0.01), while no significant correlations were glucosideTypeType 2content 2 diabetes diabetes of isisextracts aa formform ofofanalyzed diabetesdiabetes ( thatρthat: − 0.886; isis characterizedcharacterized p < 0.01), bywhile high no blood significant sugar, insulin correlations resistance were foundand relative among lack EC of50ABTS insulin, and theand variables it represents analyzed. over 90% of diabetes cases worldwide. The reduction or foundand relative among lack EC of50ABTS insulin, and theand variables it represents analyzed. over 90% of diabetes cases worldwide. The reduction or inhibitionType 2of diabetes carbohydrate is a form absorption of diabetes by thatinhibiti is characterizedng digestive byenzymes high blood such sugar, as α-amylase insulin resistance and α- inhibitionType 2of diabetes carbohydrate is a form absorption of diabetes by thatinhibiti is characterizedng digestive byenzymes high blood such sugar, as α-amylase insulin resistance and α- andglucosidase relative islack one of of insulin, the most and wi itdely represents used strategies over 90% to ofreduce diabetes postprandial cases worldwide. hyperglycemia The reduction [10]. or andglucosidase relative islack one of of insulin, the most and wi itdely represents used strategies over 90% to ofreduce diabetes postprandial cases worldwide. hyperglycemia The reduction [10]. or inhibition of carbohydrate absorption by inhibiting digestive enzymes such as α-amylase and α- inhibition of carbohydrate absorption by inhibiting digestive enzymes such as α-amylase and α- glucosidase is one of the most widely used strategies to reduce postprandial hyperglycemia [10]. glucosidase is one of the most widely used strategies to reduce postprandial hyperglycemia [10].

Plants 2020, 9, x FOR PEER REVIEW 7 of 16

Plants 2020, 9, 1785 7 of 16 respectively; p < 0.05), being P2 > P5 > P4 > P3 > P1 (Figure 2). Reported values are higher than those found by Zitouni et al. [26] for wild Moroccan accessions. Moreover, it is interesting to note that the foundFLC of by the Zitouni P5 accession et al. [26 ]represented for wild Moroccan about accessions.50% of its TPC. Moreover, Such ithigh is interesting values of to flavans note that can the be FLC of ofconsiderable the P5 accession phytotherapeutic represented about importance; 50% of itsin TPC.fact, it Such is known high values that these of flavans compounds can be of are considerable associated phytotherapeuticwith a reduced risk importance; of stroke, inmyoc fact,ardial it is known infarction that theseand diabetes, compounds as well are associatedas with improvements with a reduced in risklipid of profiles, stroke, myocardialendothelium-depe infarctionndent and blood diabetes, flow as and well blood as with pressure, improvements insulin inresistance lipid profiles, and endothelium-dependentsystemic inflammation. blood flow and blood pressure, insulin resistance and systemic inflammation. InIn thethe presentpresent study,study, the the anthocyanin anthocyanin content content of of di differentfferent extracts extracts tested tested ranged ranged between between 219 219 8± ± and8 and 827 827 ±3 3µ gμg g g1−1fruit fruit (for (for P1 P1 and and P2, P2, respectively; respectively; Figure Figure3 ).3). ± −

1 FigureFigure 3. TotalTotal content content (μ (gµ g−1 g) −of) monomeric of monomeric anthoc anthocyaninsyanins of different of different A. unedoA. unedofruit extractsfruit extracts (mean (mean SD). Different letters indicate significant differences in the mean (p < 0.05). TMA: total ± SD). Different± letters indicate significant differences in the mean (p < 0.05). TMA: total monomeric monomericanthocyanin. anthocyanin.

OnOn average,average, thethe TMATMA ofof extractsextracts analyzedanalyzed was higherhigher than those obtained by Lopez et al.al. [[27]27] withwith optimizedoptimized ultrasoundultrasound extraction.extraction. Moreover, thethe anthocyaninanthocyanin contentcontent ofof thethe wildwild fruitfruit analyzedanalyzed waswas withinwithin thethe samesame rangerange ofof knownknown fruitsfruits characterizedcharacterized byby aa highhigh anthocyaninanthocyanin content,content, suchsuch asas µ 1 µ 1 NitrariaNitraria tangutorumtangutorum Bobr.Bobr. (~650(~650 μgg gg−−1 fruit dw)dw) [[28]28] andand AristoteliaAristotelia chilensischilensisL. L.(400–1500 (400–1500 μgg g g−−1 fruitfruit dw)dw) [[29].29]. These findings findings are of particular interest due to the role ofof anthocyaninsanthocyanins in the controlcontrol ofof obesity,obesity, thethe controlcontrol ofof diabetes,diabetes, thethe preventionprevention ofof cardiovascularcardiovascular diseasesdiseases andand thethe improvementimprovement ofof visualvisual andand brainbrain functions.functions. Overall,Overall, the didifferencesfferences foundfound inin thethe phytochemicalphytochemical parametersparameters analyzed could be duedue toto didifferencesfferences inin thethe geneticgenetic traitstraits ofof thethe didifferentfferent A. A. unedo unedoaccessions accessions analyzed. analyzed. 3.3. Chromatographic Profiling of Single Phenols 3.3. Chromatographic Profiling of Single Phenols As far as anthocyanins are concerned, A. unedo fruits are mainly characterized by the presence As far as anthocyanins are concerned, A. unedo fruits are mainly characterized by the presence of cyanidin and delphinidin glycosides [30]. In the present study, the content and abundance of cyanidin and delphinidin glycosides [30]. In the present study, the content and abundance of of different anthocyanins in the wild A. unedo fruit extracts, including delphinidin-3-O-glucoside, different anthocyanins in the wild A. unedo fruit extracts, including delphinidin-3-O-glucoside, cyanidin-3-O-glucoside, malvidin-3-O-glucoside and cyanidin-3-O-rutinoside, were determined by cyanidin-3-O-glucoside, malvidin-3-O-glucoside and cyanidin-3-O-rutinoside, were determined by HPLC-DAD (Figures4 and5). HPLC-DAD (Figures 4 and 5).

Plants 2020, 9, 1785 8 of 16 Plants 2020, 9, x FOR PEER REVIEW 8 of 16 Plants 2020, 9, x FOR PEER REVIEW 8 of 16

Figure 4. HPLC–DAD quantification of individual anthocyanins (μg g−1) of different extracts analyzed Figure 4. HPLC–DAD quantification of individual anthocyanins (μg g−1) of different extracts analyzed Figure(mean 4. ±HPLC–DAD SD). quantiﬁcation of individual anthocyanins (µg g− ) of diﬀerent extracts analyzed (mean ± SD). ±

FigureFigure 5. 5.A A typical typical HPLC-DAD HPLC-DAD chromatogram chromatogram (λ :(λ 520: 520 nm) nm) of of the the extracts extracts analyzed. analyzed. The The operating operating Figure 5. A typical HPLC-DAD chromatogram (λ: 520 nm) of the extracts analyzed. The operating conditionsconditions are are reported reported in in the the HPLC HPLC section. section. PeakPeak assignment:assignment: 1:1: delphi delphinidin-3-O-glucoside,nidin-3-O-glucoside, 2: conditions are reported in the HPLC section. Peak assignment: 1: delphinidin-3-O-glucoside, 2: 2: cyanidin-3-O-glucoside;cyanidin-3-O-glucoside; 3: cyanidin-3-O-rutinoside; 4: malvidin-3-O-glucoside. cyanidin-3-O-glucoside; 3: cyanidin-3-O-rutinoside; 4: malvidin-3-O-glucoside. The main anthocyanin found in all the extracts tested was delphinidin-3-O-glucoside, ranging The main anthocyanin found in all the extracts tested was delphinidin-3-O-glucoside, ranging fromThe 49 main1 to 111 anthocyanin3 µg g-1 (for found P1 andin all P2, the respectively; extracts testedp < 0.05), was followeddelphinidin-3-O-glucoside, by cyanidin-3-O-rutinoside, ranging from 49± ± 1 to 111± ± 3 µg g-1 (for P1 and P2, respectively; p < 0.05), followed by cyanidin-3-O-rutinoside, rangingfrom 49 ± from 1 to 111 9.5 ± 30.3 µg to g-1 26.2 (for P10.4 andµg P2, g respectively;1 (for P1 and p P2, < 0.05), respectively; followedp by< cyanidin-3-O-rutinoside,0.05). These features are ranging from 9.5± ± 0.3 to 26.2± ± 0.4 µg g−−1 (for P1 and P2, respectively; p < 0.05). These features are of ofranging particular from interest 9.5 ± 0.3 as to literature 26.2 ± 0.4 data µg g report−1 (for P1 cyanidins and P2,as respectively; the main components p < 0.05). These of strawberry features are fruit of particular interest as literature data report cyanidins as the main components of strawberry fruit extractsparticular [30 interest][31], highlightingas literature peculiardata report nutraceutical cyanidins traitsas the linked main tocomponents genetic features of strawberry of accessions fruit extracts [30] [31], highlighting peculiar nutraceutical traits linked to genetic features of accessions studied.extracts [30] Due [31], to their highlighting antioxidant pe potential,culiar nutraceutical delphinidin traits glucosides linked showedto genetic the features strongest of scavenging accessions studied. Due to their antioxidant potential, delphinidin glucosides showed the strongest scavenging activitystudied. againstDue to their superoxide antioxidant anion potential, and peroxynitrite delphinidin among glucosides several showed anthocyanins the strongest [32]. scavenging Moreover, activity against superoxide anion and peroxynitrite among several anthocyanins [32]. Moreover, delphinidinactivity against was superoxide reported to anion exert cytoprotectiveand peroxynitrite effects among for human several chondrocytes anthocyanins against [32]. Moreover, oxidative delphinidin was reported to exert cytoprotective effects for human chondrocytes against oxidative stressdelphinidin [33], as was well reported as inhibitory to exert eff cytoprotectiveects on oxidative effects stress for inhuman HepG2 chondrocytes cells [34]. To against the best oxidative of our stress [33], as well as inhibitory effects on oxidative stress in HepG2 cells [34]. To the best of our knowledge,stress [33], as this well is theas inhibitory first study effects reporting on oxidat malvidin-3-O-glucosideive stress in HepG2 in cellsA. unedo [34]. wildTo the fruit best extracts. of our knowledge, this is the first study reporting malvidin-3-O-glucoside in A. unedo wild fruit extracts. On Onknowledge, average, this extracts is the first analyzed study werereporting characterized malvidin-3-O-glucoside by a malvidin-3-O-glucoside in A. unedo wild fruit content extracts. of 9.1 On average, extracts analyzed were characterized by a malvidin-3-O-glucoside content of 9.1 ± 0.3 µg ±g−1, 0.3average,µg g extracts1, with P2analyzed showing were the characterized highest value by (11.1 a malvidin-3-O-glucoside0.4 µg g 1). From a nutritionalcontent of 9.1 point ± 0.3 of µg view, g−1, with P2− showing the highest value (11.1 ± 0.4 µg g−1±). From a nutritional− point of view, anthocyanins, anthocyanins,with P2 showing as the well highest as the value other (11.1 phenolic ± 0.4 classes, µg g−1). areFrom absorbed a nutritional at the point intestinal of view, tract anthocyanins, level, where as well as the other phenolic classes, are absorbed at the intestinal tract level, where they are theyas well are as hydrolyzed, the other andphenolic the aglycones classes, are (anthocyanidins) absorbed at the are intestinal subjected tract to phase level, II metabolismwhere they [35are]. hydrolyzed, and the aglycones (anthocyanidins) are subjected to phase II metabolism [35]. Among Amonghydrolyzed, anthocyanidins, and the aglycones delphinidin (anthocyanidins) and malvidin are were subjected found to to exhibit phase the II highestmetabolism growth-inhibitory [35]. Among anthocyanidins, delphinidin and malvidin were found to exhibit the highest growth-inhibitory potentialanthocyanidins, towards delphinidin neoplastic celland survival malvidin [36 ].were found to exhibit the highest growth-inhibitory potential towards neoplastic cell survival [36]. potential towards neoplastic cell survival [36].

Plants 2020, 9, 1785 9 of 16

According to literature studies, several classes of phenolic compounds were detected in strawberry tree extracts, including hydroxybenzoic acids (gallic, protocatechuic, syringic acids, hydroxybenzoic acids), hydroxycinnamic acids (chlorogenic, p-coumaric, ferulic and caffeic acids), flavanols (catechin and epigallocatechin) [37]. Significant variations in phenolic compounds were found at p < 0.001 1 among wild accessions analyzed. In detail, P2 showed the highest polyphenol content (4.46 mg g− ) 1 and P1 the lowest one (2.16 mg g− ) (Table1).

1 Table 1. Phenolic composition (mg g− ) of Arbutus unedo extracts determined by high-performance liquid chromatography (mean DS). ± Compound P1 P2 P3 P4 P5 Hydroxybenzoic acids (HBA) Gallic acid 0.65 0.01 b 0.74 0.01 b 0.63 0.01 b 1.29 0.02 c 0.31 0.03 a ± ± ± ± ± Syringic acid 0.09 0.01 a 0.14 0.02 a 0.11 0.02 a 0.26 0.01 b 0.47 0.02 c ± ± ± ± ± Protocatechuic acid 0.35 0.01 a 0.45 0.02 a 0.31 0.01 a 0.59 0.01 b 0.44 0.01 a ± ± ± ± ± p-Hydroxybenzoic acid 0.11 0.02 a 1.23 0.02 d 0.44 0.01 b 0.64 0.02 c 0.47 0.02 b ± ± ± ± ± Total HBA 1.2 0.1 2.56 0.07 1.49 0.05 2.78 0.06 1.69 0.08 ± ± ± ± ± Hydroxycinnamic acids (HCA) p-Coumaric acid n.d n.d n.d n.d n.d Caffeic acid n.d n.d n.d n.d n.d Chlorogenic acid 0.05 0.01 b 0.06 0.01 c 0.05 0.02 a 0.06 0.01 d 0.05 0.01 a ± ± ± ± ± Ferulic acid n.d n.d n.d n.d n.d Total HCA 0.05 0.01 0.06 0.01 0.05 0.02 0.06 0.01 0.05 0.01 ± ± ± ± ± Flavan-3-ols Catechin 0.82 0.02 a 1.72 0.01 d 1.04 0.02 b 1.34 0.01 c 0.87 0.01 a ± ± ± ± ± Epicatechin 0.09 0.01 a 0.12 0.02 a 0.11 0.02 a 0.16 0.02 b 0.11 0.01 a ± ± ± ± ± Total flavans 0.91 0.03 1.84 0.03 1.15 0.04 1.5 0.3 0.98 0.02 b ± ± ± ± ± n.d.: not detectable. Different letters in a row indicate significant differences in the mean (p < 0.001).

Among the phenolic classes analyzed, hydroxybenzoic acids (HBA) were the most abundant compounds, followed by flavan-3-ols and hydroxycinnamic acids (HCA), while no appreciable amounts of flavonols were found. These results are in agreement with literature data [38]. 1 The highest HBA content was found in P4 (2.78 0.06 mg g− ) and the lowest one in P1 (1.2 1 ± ± 0.1 mg g− ). Among the hydroxybenzoic acids, gallic acid was the most representative (ranging from 0.31 to 1.29 mg g-1, for P5 and P4, respectively) as also reported by Pimpão et al. [35]. Moreover, among HCA analyzed, only chlorogenic acid was found, but at much lower levels than those reported by Zitouni et al. [26] for wild Moroccan accessions. As far as flavan-3-ols are concerned, catechin was the most abundant compound found in all the extracts analyzed, being also the most representative phenolic compound in wild accessions studied. 1 Its content ranged from 0.82 to 1.72 mg g− for P1 and P2, respectively. This trend was similar to that previously observed for FLC, and obtained values were on average higher than those reported in literature studies [26] and in line with the values reported by Albuquerque et al. [39] under optimal extraction conditions. Flavonols are generally present in low amounts in A. unedo berries [26]. In the present study, myricetin was not found in any of the extracts tested, whereas other not identified flavonols were detected only in traces (data not shown).

3.4. In Vitro Biological Activities In order to investigate the radical scavenging properties of the diﬀerent extracts analyzed, two diﬀerent in vitro antiradical assays based on hydrogen and electron transfer were performed. Figure6 shows the antiradical potential of the extracts analyzed towards synthetic chromogenic + radicals DPPH• and ABTS• , expressed in terms of EC50: the lower this value, the higher the antiradical capacity of the extract. In both in vitro tests, the highest antiradical capacity values were recorded Plants 2020, 9, x FOR PEER REVIEW 6 of 16

Figure 1b, inset c), occurring by the presence of alkene terminal groups when a single H atom was bound to the double bond. These findings agree with those of Morgado et al. [5], who reported a high content of polyunsaturated fatty acids, representing about 60% of total fatty acids, in A. unedo berries. The fingerprinting region between 1800 and 700 cm−1 showed significant differences between the samples analyzed (Figure 1b, inset d). A well-defined peak, occurring at 1750 cm−1, related to the carbonyl group (C=O) stretching vibrations of the ester bond, was present in all extracts analyzed. The presence of esters was also confirmed by the strong absorption at 1078 cm−1 (stretching of COO- C) (Figure 1b, inset d). These findings are in line with the fact that phenolics, especially phenolic acids, occur naturally in combination with other compounds, usually in the form of esters [21]. The low- intensity absorption bands in the range 1500–1450 cm−1 could be attributed to C–C-O stretching vibrations [22]. From derivative spectrum (Figure 1b; inset d), several peaks in the spectral range 1100–1000 cm−1, due to the stretching of OH group, characteristic of phenolics, and to the aromatic C–H-plane deformation vibrations, were also observed [23]. Finally, a well-defined band system in the range 900–750 cm−1 was due to the oscillations of the H atoms outside the aromatic ring plane, depending on the number of adjacent H atoms and as a consequence on the meta substitution of aromatic protons [21].

3.2. Phytochemical Composition of the Extracts Analyzed VersionVersion December DecemberFigure 16, 16, 2020 2020 2 submittedshows submitted the TPC to toJournalJournal of the Not Notdifferent Speciﬁed Speciﬁed extracts analyzed, which ranged from 17.6 ± 0.3 to 23.3 3 of 64 ± 0.2 mg GAE g−1 (for P5 and P4, respectively).

Plants 2020, 9, 1785 10 of 16

PlantsPlants 2020 2020, ,9 9, ,x x FOR FOR PEER PEER REVIEW REVIEW 1 + 10 of 16 for sample P2 (EC50: 1.17 and 0.064 µg mL− , for DPPH• and ABTS• , respectively), according to its PlantsPlants 2020 2020, ,9 9, ,x x FOR FOR PEER PEER REVIEW REVIEW 10 of 16 highest polyphenolic content.

Figure 2. Total phenolic and flavan-3-ols content (mg g−1) of five A. unedo fruit extracts (mean ± SD). Different letters indicate significant differences in the mean (p < 0.05). TPC: total polyphenol content; FLC: flavan-3-ols content; GAE: gallic acid equivalents; CTE: catechin equivalents.

Based on the experimental results, the genotype significantly influenced the TPC of the extracts (p < 0.05) as follows: P4~P2 > P3 > P1 > P5. These results are in agreement with those reported in the literature((aa)) [7] [24] and higher than those reported by Doukani et al. [25], which ranged between 7 and −1 −1 14( (amga)) GAE g . As regards flavan-3-ols, FLC ranged from 2.98 to 9.58 mg CTE g (for P1 and P2,

((bb)) ((bb))

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FigureIn both 6.in In vitro vitro tests, biological antiradica activitiesl capacity of the expressed analyzed in samples: terms of antiradicalEC50 (μg mL capacity.−1 of extract (a) 2,2-diphenyl-required to FigureIn both 6.in In vitro vitro tests, biological antiradica activitiesl capacity of the expressed analyzed in samples: terms of antiradicalEC50 (μg mL capacity.−1 of extract (a1) 2,2-diphenyl-required to inIn the both secondin vitro panel.tests, Figures antiradical should•) capacity be placed expressed in the main in terms text near of EC to the(µg first mL timeof they extract•+ are required cited. A in the second1-picrylhydrazyl panel. Figures (DPPH should•) test; ( beb) ,2 placed′-azino-bis in the(3-ethylbenzothiazoline-6-sulfonic main text near to50 the first acid) time− (ABTS they•+) are test. cited. A 1-picrylhydrazylobtainobtain 50% 50% radical radical (DPPH scavenging).scavenging). test; ( bInIn) the,2the′-azino-bis histogram,histogram, (3-ethylbenzothiazoline-6-sulfonic differentdifferent letters indicate significant acid) differences (ABTS ()p test. < caption on a single line should be centered. μ −1 captionto obtain on 50%In a singleboth radical in linevitro scavenging). shouldtests, antiradica be centered. Inl capacity the histogram, expressed in di fftermserent of lettersEC50 (μg indicate mL−1 of extract significant required di toff erences In0.05).0.05). both in vitro tests, antiradical capacity expressed in terms of EC50 ( g mL of extract required to (p < 0.05).obtainobtain 50% 50% radical radical scavenging).scavenging). InIn thethe histogram,histogram, differentdifferent letters indicate significant differences (p < 0.05). 0.05).TheThe antiradical antiradical potentialpotential ofof thethe extractsextracts testedtested wass higherhigher than those reported by Tenuta et al. The antiradical[40][40] forfor A.A. unedounedo potential berriesberries fromoffrom the SouthernSouthern extracts Italy.Italy. tested NonparametriNonparametri was higherc correlation than those analysis, reported carried by out Tenuta on the et al. [40] entireThe dataset, antiradical pointed potential out the of highest the extracts significant tested correlation was higher between than those EC50DPPH reported and malvidin-3-O-by Tenuta et al. entireThe dataset, antiradical pointed potential out the of highest the extracts significant tested correlation was higher between than those EC50DPPH reported and malvidin-3-O-by Tenuta et al. for A. unedo[40]berries for A. unedo from berries Southern from Southern Italy. Nonparametric Italy.ρ Nonparametri correlationc correlation analysis, analysis, carried carried out on on the the entire [40]glucosideglucoside for A. contentunedocontent berries ofof extractsextracts from analyzedSouthernanalyzed (Italy.(ρ:: −−0.886;0.886; Nonparametri pp << 0.01),0.01), cwhilewhile correlation nono significant analysis, correlations carried out wereon the dataset, pointedentirefound dataset,among out the EC pointed50ABTS highest and out the significant the variables highest analyzed.correlationsignificant correlation between between EC50DPPH EC50DPPHand and malvidin-3-O-glucoside malvidin-3-O- entirefound dataset,among EC pointed50ABTS and out the the variables highest analyzed.significant correlation between EC50DPPH and malvidin-3-O- content ofglucoside extractsType 2content analyzed diabetes of is extracts (aρ form: 0.886; ofanalyzed diabetesp < (0.01),ρthatρ: − 0.886;is characterized while p < no0.01), significant bywhile high no blood significant correlations sugar, insulin correlations were resistance found were among glucosideType 2content diabetes of is extracts a form− ofanalyzed diabetes (that: − 0.886;is characterized p < 0.01), bywhile high no blood significant sugar, insulin correlations resistance were foundand relative among lack EC of50ABTS insulin, and theand variables it represents analyzed. over 90% of diabetes cases worldwide. The reduction or EC50ABTS foundandand relativethe among variables lack EC of50ABTS insulin, analyzed.and theand variables it represents analyzed. over 90% of diabetes cases worldwide. The reduction or inhibitionType 2of diabetes carbohydrate is a form absorption of diabetes by thatinhibiti is characterizedng digestive byenzymes high blood such sugar, as α-amylase insulin resistance and α- Typeinhibition 2 diabetesType 2of diabetes carboh is a formydrate is a form of absorption diabetes of diabetes thatby thatinhibiti is is characterized characterizedng digestive byenzymes by high high blood such blood sugar, as α sugar,-amylase insulin insulin resistance and α- resistance andglucosidase relative islack one of of insulin, the most and wi itdely represents used strategies over 90% to ofreduce diabetes postprandial cases worldwide. hyperglycemia The reduction [10]. or andglucosidase relative islack one of of insulin, the most and wi itdely represents used strategies over 90% to ofreduce diabetes postprandial cases worldwide. hyperglycemia The reduction [10]. or and relativeinhibition lack of of insulin, carbohydrate and absorption it represents by inhibiti over 90%ng digestive of diabetes enzymes cases such worldwide. as α-amylase Theand reductionα- inhibition of carbohydrate absorption by inhibiting digestive enzymes such as α-amylase and α- or inhibitionglucosidase of carbohydrate is one of the most absorption widely used by strategies inhibiting to reduce digestive postprandial enzymes hyperglycemia such as α [10].-amylase and glucosidase is one of the most widely used strategies to reduce postprandial hyperglycemia [10]. α-glucosidase is one of the most widely used strategies to reduce postprandial hyperglycemia [10].

In order to explore the hypoglycemic potential of A. unedo wild berries, all the extractsanalyzed 1 were evaluated for their inhibitory power against α-glucosidase in the range of 0–100 mg mL− . As can be seen from Figure7, the wild accessions studied showed a peculiar inhibition trend, with P2 and P5 samples exerting the highest inhibitory power (70 and 67%, respectively). Plants 2020, 9, x FOR PEER REVIEW 11 of 16

Plants 2020, 9, x FOR PEER REVIEW 11 of 16 In order to explore the hypoglycemic potential of A. unedo wild berries, all the extracts analyzed wereIn evaluated order to explorefor their the inhibitory hypoglycemic power potential against α of-glucosidase A. unedo wild in berries,the range all of the 0–100 extracts mg analyzedmL−1. As werecan be evaluated seen from for Figure their inhibitory7, the wild power accessions against studied α-glucosidase showed a in peculiar the range inhibition of 0–100 trend, mg mL with−1. AsP2 and P5 samples exerting the highest inhibitory power (70 and 67%, respectively). canPlants be2020 seen, 9, 1785from Figure 7, the wild accessions studied showed a peculiar inhibition trend, with11 of P2 16 and P5 samples exerting the highest inhibitory power (70 and 67%, respectively).

Figure 7. Percentage of inhibition of the a-glucosidase enzyme by extracts of A. unedo wild berries. Figure 7. PercentagePercentage of of inhibition inhibition of the a- a-glucosidaseglucosidase enzyme by extracts of A. unedo wild berries. Nonparametric correlation analysis pointed out very high significant correlations among ρ ρ percentageNonparametric of α-glucosidase correlation inhibition analysis and pointedcyanidin-3-O-rutinoside ou outt very high ( signiﬁcantsignificant: 0.973; p < correlations0.01), TMA ( among: 0.912; ρ percentagep < 0.01) and of αFLC-glucosidase ( : 0.912; inhibition p < 0.01), andand highlighting cyanidin-3-O-rutinoside the synergistic ((ρρ :effect: 0.973;0.973; of p

Figure 8. Hierarchical cluster analysis for the A. unedo accessions analyzed.

An overall separation was observed among the wild accessions at an average distance between 10 and 15 (two main clusters). Besides this, at an average distance ranging from 5 to 10, three clusters were Plants 2020, 9, 1785 12 of 16 observed, confirming that the nutraceutical composition and functional activities of the P1 accession were highly distinct from the remaining samples. HCA was then followed by K-W nonparametric analysis to highlight which variables contributed most to the separation. Where significant H-values occurred, Dunn’s post hoc comparisons were conducted to determine the simple effects between clusters. Significant differences (p < 0.05) were observed among the three clusters for TPC, FLC, TMA, anthocyanin compounds, phenolic acids, catechin, ACABTS + and ACDPPH (Table2). • • Table 2. Nonparametric analysis of functional traits of A. unedo extracts analyzed (mean values ± standard deviation).

Variable Cluster 1 Cluster 2 Cluster 3 TPC 18 2 ab 23.9 0.2 c 18.1 0.4 ba ± ± ± FLC 3.0 0.4 ab 8.4 0.2 ca 7.0 0.4 ba ± ± ± TMA 30.1 0.4 ab 63.1 0.4 ba 48.6 0.1 c ± ± ± DEL 49 1 abc 88 3 ca 86 1 ba ± ± ± CGC 6.3 0.1 ac 8.1 0.3 ca 7.5 0.1 b ± ± ± CRC 9.5 0.4 ac 21 1 ca 17.5 0.3 ba ± ± ± GA 0.60 0.06 b 1.00 0.04 cba 0.50 0.01 a ± ± ± pOH-B 0.10 0.01 abc 0.9 0.1 cba 0.40 0.02 bac ± ± ± SA 0.08 0.01 ac 0.20 0.01 ca 0.160 0.008 b ± ± ± pCA 0.35 0.01 ac 0.52 0.03 ca 0.38 0.01 b ± ± ± CAT 0.82 0.02 ac 1.53 0.06 cba 0.95 0.01 bc ± ± ± CA 1.6 0.2 abc 1.9 0.5 cba 1.7 0.6 bac ± ± ± ABTS 3.0 0.2 cba 2.4 0.1 bc 2.0 0.2 ac ± ± ± DPPH 109.6 0.3 ca 35 1 abc 100 1 ba ± ± ± TPC: total polyphenol content; FLC: flavan-3-ol content; TMA: total monomeric anthocyanins; pOH-B: p-hydroxybenzoic acid; CAT; catechin; CA: chlorogenic acid; DEL: delphinidin-3-O-glucoside; CGC: cyanidin-3-O-glucoside; CRC: cyanidin-3-O-rutinoside; MGC: malvidin-3-O-glucoside; DPPH: 2,2-diphenyl-1-picrylhydrazyl; GA: gallic acid; SA: syringic acid; pCA: protocatechuic acid; ABTS: 2,2’-azino-bis(3-ethylbenzothiazolin-6-sulfonic acid). Different letters in a row indicate significant differences in the mean (p < 0.001).

Based on the post hoc analysis (Table2), it was possible to group the accessions in the study into clusters with low (Cluster 1; P1), medium (Cluster 3; P3 and P5), and high (Cluster 2; P2 and P4) nutraceutical potential. Finally, PCA was used to visualize the hidden pattern in the experimental data and the relationships between data and samples. It was conducted on selected variables (Table3), chosen on the basis of the analysis of the correlation and anti-image correlation matrices obtained from the standardized z-scores [18], with orthogonal rotation (varimax model). The Kaiser–Meyer– Olkin measure verified the sampling adequacy for the analysis (KMO = 0.747; [19]). Bartlett’s test of sphericity (p < 0.001) showed that correlations between the considered items were sufficiently large for PCA. On the basis of eigenvalues > 1 (Kaiser’s criterion) and of the scree plot (not shown), two principal components (PCs), accounting for 93.52% of the total variance, were considered significant. Component loadings after rotation were reported in Table3, showing the main variables differentiating the extracts. As we can see, TPC, chlorogenic acid, catechin, malvidin-3-O-glucoside and antiradical potential (EC50DPPH) loaded highly on factor 1, explaining most of its variance (48.12%; rotated solution). Conversely, the remaining anthocyanins (mainly delphinidin) and TMA loaded highly on factor 2 (45.40%; rotated solution). Figure9 depicts the scores for samples analyzed in a two-dimensional plot. Across PC1, a broad separation was observed among P2 and P4 samples and the remaining ones, the former having an overall higher nutraceutical profile and significant antioxidant potential. A clear sample separation was also observed across PC2, highlighting the lower content of anthocyanins of P1 and P4 accessions compared to the other ones. Plants 2020, 9, x FOR PEER REVIEW 13 of 16

DEL 0.947 pOH-B 0.679 0.721 DPPH(EC50) −0.918 Eigenvalues 4.81 4.54 % of variance 48.12 45.40 Plants 2020* Rotation, 9, 1785 method: varimax with Kaiser normalization. Component loadings with absolute values13 of 16 less than 0.45 have been left out of the table for ease of comparison. TPC: total polyphenol content; TMA: total monomeric anthocyanins; pOH-B: p-hydroxybenzoic Acid; CAT; catechin; CA: Table 3. Loadings of the signiﬁcant measured variables on the three ﬁrst principal components (PCs) *. chlorogenic acid; DEL: delphinidin-3-O-glucoside; CGC: cyanidin-3-O-glucoside; CRC: cyanidin-3- O-rutinoside; MGC: malvidin-3-O-glucosideVariables ; DPPH: 2,2-diphenyl-1-picrylhydrazyl. 1 2 The Kaiser–Meyer– Olkin measure verified the sampling adequacy for the analysis (KMO = TPC 0.974 0.747; [19]). Bartlett’s test of sphericityTMA (p < 0.001) showed that correlations 0.825 between the considered items were sufficiently large for PCA.CAT On the basis 0.862of eigenvalues > 1 (Kaiser’s criterion) and of the scree plot (not shown), two principalCA components (PCs), 0.848 accounting for 93.52% of the total variance, MGC 0.805 0.547 were considered significant. ComponentCRC loadings after rotation 0.895 were reported in Table 3, showing the main variables differentiating theCGC extracts. 0.914 DEL 0.947 As we can see, TPC, chlorogenic acid, catechin, malvidin-3-O-glucoside and antiradical potential pOH-B 0.679 0.721 (EC50DPPH) loaded highly on factorDPPH(EC 1, explaining) most0.918 of its variance (48.12%; rotated solution). 50 − Conversely, the remaining anthocyaninsEigenvalues (mainly delphinidin) 4.81 and 4.54 TMA loaded highly on factor 2 (45.40%; rotated solution). % of variance 48.12 45.40 *Figure Rotation 9 depicts method: the varimax scores withfor samples Kaiser normalization. analyzed in a Componenttwo-dimensional loadings plot. with Across absolute PC1, values a broad separationless than was 0.45 observed have been among left out P2 of theand table P4 samples for ease ofand comparison. the remaining TPC: ones, total polyphenol the former content; having an TMA: total monomeric anthocyanins; pOH-B: p-hydroxybenzoic Acid; CAT; catechin; CA: chlorogenic overallacid; higher DEL: delphinidin-3-O-glucoside; nutraceutical profile and CGC: significant cyanidin-3-O-glucoside; antioxidant CRC:potential. cyanidin-3-O-rutinoside; A clear sample separation MGC: wasmalvidin-3-O-glucoside; also observed across DPPH: PC2, 2,2-diphenyl-1-picrylhydrazyl.highlighting the lower content of anthocyanins of P1 and P4 accessions compared to the other ones.

Figure 9. Score plot of the principal components PC1 and PC2. Figure 9. Score plot of the principal components PC1 and PC2. 4. Conclusions 4. Conclusions Recently, underutilized berry species have attracted growing interest as promising sources of bioactiveRecently, compounds, underutilized which, berry thanks species to ahave comprehensive attracted growing phytochemical interest as evaluation, promising could sources find of widerbioactive applications compounds, in various which, thanks fields suchto a comprehe as nutraceuticalnsive phytochemical and cosmeceutical evaluation, ones. could In the find present wider study,applications a phytochemical in various analysis fields such coupled as nutraceutica with unsupervisedl and cosmeceutical chemometric ones. tools In revealed the present the potential study, a ofphytochemical wild berries fromanalysis Italian coupledA. unedo with accessionsunsupervised as functionalchemometric ingredients. tools revealed The the study potential focused of wild on polyphenolberries from and Italian anthocyanin A. unedo accessions profiles as as well functional as in vitro ingredients.antiradical The and study hypoglycemic focused on activities polyphenol of fiveand accessions anthocyanin growing profiles in as central well as Italy. in vitro Moreover, antiradical FTIR and spectroscopy hypoglycemic was shownactivities to of rapidly five accessions provide valuablegrowing information in central onItaly. biochemical Moreover, composition FTIR spectr andoscopy genetic was di ffshownerences to among rapidly samples. provide Significant valuable diinformationfferences were on found biochemical among samples composition tested forand all genetic parameters differences analyzed, among being P2 samples. the most promisingSignificant accessiondifferences from were a phytochemical found among point samples of view. tested Furthermore, for all parameters HCA and PCA analyzed, allowed being differentiating P2 the most the accessions on the basis of their overall phytochemical profiles, to find out the variables that contribute most to this differentiation and to group the accessions according to low to high phytochemical content level. This study aims to open further opportunities for the enhancement of the wild berries of A. unedo, improving knowledge about their nutraceutical properties, whose cultivation by virtue of the resilience Plants 2020, 9, 1785 14 of 16 characteristics of the species could make an important contribution to the development of local rural areas facing the imminent problems of climate change.

Supplementary Materials: The following are available online at http://www.mdpi.com/2223-7747/9/12/1785/s1, Table S1: Spectral features of A. unedo wild berries analysed. Author Contributions: Conceptualization, K.C.; investigation, V.M. and V.S.; methodology, K.C. and V.M.; resources, K.C.; supervision, K.C.; validation; writing—review and editing, K.C. and V.M. All authors have read and agreed to the published version of the manuscript. Funding: This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Conflicts of Interest: The authors declare no conflict of interest.

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