Unravelling the biological potential of Pinus pinaster bark extracts

antioxidants Article Unravelling the Biological Potential of Pinus pinaster Bark Extracts Pedro Ferreira-Santos 1,* , Zlatina Genisheva 1, Cláudia Botelho 1, Joana Santos 2, Carla Ramos 2, JoséA. Teixeira 1and Cristina M.R. Rocha 1,* 1 CEB—Centre of Biological Engineering, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal 2CISAS—Centro de Investigação e Desenvolvimento em Sistemas Agroalimentares e Sustentabilidade, Escola Superior de Tecnologia e Gestão, Instituto Politécnico de Viana do Castelo, Rua Escola Industrial e Comercial de Nun’Álvares, 4900-347 Viana do Castelo, Portugal *Correspondence: [email protected] (P.F.-S.); cmr[email protected] (C.M.R.R.); Tel.: +351-253-604-426; (P.F.-S.); +351-253-604-423 (C.M.R.R.) Received: 18 March 2020; Accepted: 17 April 2020; Published: 20 April 2020   Abstract: Natural compounds from agro-food by-products have fostered interest in food industries. The aim of this study was to unravel potential uses for Pinus pinaster bark extracts (PBE). As functional features of this type of extracts are usually attributed to phenolic compounds, the extraction process was studied. Different PBEs were achieved, with high content in phenolic compounds, using different water/ethanol combinations as a solvent. These PBEs were chemically characterized, and their bioactivity and in vitro cell viability were evaluated. Extracts obtained with hydroethanolic solvents had higher content in phenolic and flavonoid compounds. All the PBEs presented high antioxidant, antibacterial and antihyperglycemic activities. Moreover, PBEs have low cytotoxicity and a selective activity against cancer cells as these were negatively affected. These features may allow the extracts to be used in food formulation and processing (as preservatives, antioxidants or bioactive ingredients), but they showed also potential for the pharmaceutical or nutraceutical sectors. Keywords: Pinus pinaster bark; extraction; pine bark extracts; phenolic compounds; flavonoid compounds; antioxidant activity; antihyperglycemic activity; antimicrobial activity; cells’ metabolism 1. Introduction Natural compounds, such as phenolics, flavonoids, proteins, carotenoids, among others, have fostered interest in different industries including paints, fertilizers, surfactants, textiles, rubbers, pharmaceuticals, etc. [ 1 ]. Additionally, in food industry technology, they are used as natural preservatives against oxidation and microorganisms (bacterial and fungal contaminations), and in the development of functional food ingredients [2]. The biological activity of plant extracts can be attributed to secondary metabolites such as phenolic acids, flavonoids and other phenolic compounds. The popularity of these extracts is linked to their biological properties, such as antioxidant, anti-inflammatory, antimicrobial, antiviral, antiatherogenic, etc. [ 3 – 7 ]. In particular, the extracts of Pinus pinaster (a conifer plant found in some Mediterranean countries and used in afforestation of Africa, New Zealand and Australia), are rich in phenolic acids, flavanols and flavonoids (e.g., cinnamic acid, hydroxybenzoic acid, catechin, quercetin and taxifolin) with a potent antioxidant activity [ 5 , 8 ]. These extracts have demonstrated beneficial effects for the treatment of several diseases, such as cardiovascular, metabolic, neurological, etc. [ 8 – 11 ]. The activities reported for P. pinaster extracts make this underexploited by-product (bark) of the wood industry of high interest for the pharmaceutical and food industries. There are only a few studies about the potential bioactivities and toxicity of the extracts from P. pinaster bark. However, the existent Antioxidants 2020,9, 334; doi:10.3390/antiox9040334 www.mdpi.com/journal/antioxidants Antioxidants 2020,9, 334 2 of 21 studies are focused on the commercial product Picnogenol ® , an aqueous extract used as an active supplement [10,11]. The extracts from P. pinaster bark are predominantly obtained by conventional solvent extraction, Soxhlet, microwaves, supercritical CO 2 and, more recently, by ohmic heating assisted extraction [5,12–15] . However, the variety of extraction conditions (such as type of solvent, solid-liquid ratio, time and temperature) may potentially affect the processes’ yield and the phenolic profile of the extracts. To prevent the environmental impact and reduce the bio-wastes and by-products of the agro-food industry, it is necessary to optimize the recovery of bioactive compounds with high added value and to enable their re-introduction in the market [ 16 – 18 ]. In addition, the applied methodology needs to be sustainable and “green” by using alternative and highly clean and nontoxic solvents [ 19 ]. It is known that polar solvents, such as ethanol or aqueous mixtures containing ethanol, are frequently used for the recovery of phenolic compounds from plant tissues [ 16 ]. In this sense, the aim of this research work was to study the functional potential of phenolic-rich extracts from pine bark (PB) using water and water-ethanol in different ratios as a solvent. The pine bark extracts (PBE) with the highest phenolic compounds’ content and highest in vitro antioxidant activity were chemically characterized, and its bioactivity (antioxidant, antimicrobial and antidiabetic) and in vitro cell viability (in normal and cancer cell lines) were evaluated. 2. Materials and Methods 2.1. Chemicals Folin-Ciocalteu reagent, 2,2 0 -Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), 2,2-Di(4-tert-octylphenyl)-1-picrylhydrazyl (DPPH), 2,4,6-Tris(2-pyridyl)-s-triazine (TPTZ), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), porcine pancreatic α -amylase (EC 3.2.1.1, type VI), Saccharomyces cerevisiae α -Glucosidase (EC 3.2.1.20, type I), p-nitrophenyl-R-d-glucopyranoside (pNPG), aluminium chloride (AlCl 3 ), acarbose, Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin solution, resazurin sodium salt, dimethyl sulfoxide (DMSO, ≥ 99.9%) and all standard markers for HPLC were obtained from Sigma-Aldrich (St. Louis, MO, USA). Other reagents were analytical grade, and ultra-pure water was used throughout the experiments. 2.2. Raw Material Preparation and Characterization Bark from P. pinaster (approximate age 15 years) was collected in Ponte de Lima, Portugal (April 2016). Firstly, the bark was washed with distilled water and dried at 40 ◦C for 48 h and subsequently milled in a cutting mill (Retsch SM 2000) to a granulometry of 0.1–0.45 mm for general chemical composition and 1–1.6 mm for extraction process. Chemical summative analyses were determined in accordance to the National Renewable Energy Laboratory (NREL) official protocols, and included ethanol extractives (NREL/TP-510-42619), structural carbohydrates (namely cellulose and hemicellulose), klason and acid soluble lignin (NREL/TP-510-42618) and ash content (NREL/TP-510-42622). The mineral content was determined by inductively coupled plasma atomic emission spectrometry (ICP-AES), after PB digestion with HNO 3 . Fat content was determined according to the official AOAC method (n º 920.39). Total proteins content estimated by using the N × 6.25 conversion factor, was performed using a Kjeldahl distillator (Kjeltec 8400 Analyzer, FOSS, Hilleroed, Denmark) by quantification of Nitrogen after PB digestion. PB moisture was determined gravimetrically using a moisture analyzer (MAC 50/1/NH, RADWAG, Radom, Poland). All experiments were performed in triplicate. 2.3. Extraction Conditions and Extracts Preparation In the first part of the work, the procedure to study the influence of the extraction parameters of phenolic compounds present in PB was carried out using 100 mL cylindrical reactors duly protected Antioxidants 2020,9, 334 3 of 21 from light in thermostatized water bath with shaking (170 rpm). The volume of extraction for all experiments was 40 mL. Experiments were performed using response surface methodogy RSM (2 3 central composite design) for solid-liquid extraction with water or hydroethanolic solvent (30–90% (v/v)). The levels of independent variables were selected based on the results obtained from our preliminary experiments (data not shown) and data from the literature [ 14 , 15 ]. The five levels of each of the three variables were coded in 18 runs (including four replicates of the center point) and were performed in a random order. Independent variables for extraction were time (min, x 1 ), temperature ( ◦ C, x 2 ) and solid: liquid ratio (g/mL, x 3 ). Dependent variables (Y 1 and Y 2 ) were total phenolic content (TPC, mg GAE/g PB) and ferric reducing antioxidant power (FRAP, mmol Fe 2+ /g PB), respectively. Coded and actual values of the independent variables together with data of dependent variables are given in Table 1. Data were correlated following the polynomial Equation (1). Yi=β0i+β1ix1+β2ix2+β3ix3+β11ix2 1+β22ix2 2+β33ix2 3+β12ix1x2+β13ix1x3+β23ix2x3(1) where, Y i correspond to the dependent variables; x 1 , x 2 and x 3 value of independent variables; β0i , β1i , β2i , β3i , β11i , β22i , β33i , β12i , β13i and β23i are regression coefficients calculated from experimental data by multiple regression using the least-squares method. Antioxidants 2020,9, 334 4 of 21 Table 1. Experimental runs using coded levels of time (min, x 1 ), temperature ( ◦ C, x 2 ) and solid: liquid ratio (g/mL, x 3 ) according to the 2 3 full factorial central composite design and data of total phenolic content (TPC) and reducing antioxidant activity (FRAP) of extracts obtained under those conditions for the tested experimental model (EtOH 0%, EtOH 30%, EtOH 50%, EtOH 70% and EtOH 90%). Runs Time (min) x1 Temperature (◦C) x2 Solid-Liquid Ratio (g/mL) x3 EtOH (v/v) 0% (H2O) 30% 50% 70% 90% TPC FRAP TPC FRAP TPC FRAP TPC FRAP TPC FRAP 135 (−1) 38 (−1) 0.05 (−1) 18.70 0.13 20.81 0.29 68.56 0.42 60.94 0.44 59.74 0.34 235 (−1) 38 (−1) 0.15 (1) 31.77 0.26 93.44 0.93 99.04 1.15 97.34 1.06 91.54 0.83 335 (−1) 82 (1) 0.05 (−1) 20.18 0.17 60.94 0.35 69.04 0.48 71.04 0.50 61.74 0.34 435 (−1) 82 (1) 0.15 (1) 37.59 0.33 106.14 0.99 119.44 1.39 126.24 1.15 98.84 0.97 5115 (1) 38 (−1) 0.05 (−1) 19.86 0.16 58.14 0.32 61.34 0.43 63.44 0.45 60.64 0.37 6115 (1) 38 (−1) 0.15 (1) 31.76 0.26 86.84 0.94 99.04 1.40 108.54 1.11 99.94 0.61 7115 (1) 82 (1) 0.05 (−1) 22.68 0.16 75.74 0.41 93.24 0.58 82.04 0.60 62.44 0.43 8115 (1) 82 (1) 0.15 (1) 48.13 0.35 120.14 1.35 163.64 1.50 136.54 1.44 123.84 1.14 98 (−1.682) 60 (0) 0.1 (0) 5.30 0.08 72.24 0.49 77.24 0.61 76.34 0.55 75.64 0.53 10 142 (1.682) 60 (0) 0.1 (0) 30.59 0.25 83.14 0.71 102.54 1.21 100.24 0.96 89.34 0.94 11 75 (0) 23 (−1.682) 0.1 (0) 20.31 0.18 69.24 0.49 79.54 0.66 81.54 0.57 73.64 0.51 12 75 (0) 97 (1.682) 0.1 (0) 35.61 0.29 95.14 1.08 115.94 1.07 124.34 1.43 104.24 0.96 13 75 (0) 60 (0) 0.016 (−1.682) 9.08 0.09 19.38 0.17 59.69 0.21 57.64 0.38 25.35 0.17 14 75 (0) 60 (0) 0.184 (1.682) 36.09 0.33 118.64 1.30 138.84 1.34 133.04 1.40 115.64 1.07 15 75 (0) 60 (0) 0.1 (0) 27.97 0.25 83.64 1.01 92.54 1.09 106.94 0.94 89.14 0.81 16 75 (0) 60 (0) 0.1 (0) 29.24 0.26 94.94 0.79 92.74 0.98 99.24 1.01 85.04 0.78 17 75 (0) 60 (0) 0.1 (0) 28.28 0.25 91.34 1.00 94.94 1.16 95.24 0.95 79.44 0.81 18 75 (0) 60 (0) 0.1 (0) 27.87 0.25 86.74 0.80 94.14 1.15 103.24 1.24 80.24 0.95 TPC, Total Phenolic Content (mg GAE/g PB); FRAP, Ferric Reducing Antioxidant Power (mmol Fe2+/g PB). Antioxidants 2020,9, 334 5 of 21 The experimental data were fitted to the proposed model using Statistica software (Statistica 8.0). The statistical analysis was performed using ANOVA, which established the model significance, the significance for each polynomial coefficient, and the determination coefficient R2. For the best conditions selected in the previous stage, new experimental assays were carried out in order to characterize and evaluate the bioactive potential and cell viability of extracts. The obtained extracts were dried by freeze drying and keep at 4 ◦C for further analysis. 2.4. Chemical Analysis of Extracts 2.4.1. Total Phenolic Content (TPC) The total content of phenolic compounds was measured by the Folin–Ciocalteu method that was based on the colorimetric reduction/oxidation reaction of phenols [ 5 , 20 ]. Gallic acid was used to perform the standard curve (R 2 =0.996) and the results were expressed as milligram gallic acid equivalents (GAE) per gram of pine bark (optimization process) or dry extract (extract characterization). 2.4.2. Total Flavonoid Content (TFC) Theappliedmethodforthedeterminationoftotalflavonoidscontenthasbeenpreviously described by Barros et al. [ 21 ]. An aliquot (500 µ L) of the PBE solution was mixed with distilled water and NaNO 2 solution (5%). After 6 min, AlCl 3 solution (10%) was added and allowed to stand further 6 min; thereafter, NaOH solution (4%) was added to the mixture. Then, the mixture was properly mixed and allowed to stand for 15 min, and the absorbance was measured at 510 nm. (+)-Catechin was used to calculate the standard curve (R 2 =0.997) and the results were expressed as mg of catechin equivalents (CE) per g of extract (mg CE/g extract). 2.4.3. UPLC Chromatography Identification and quantification analysis of phenolic presents in PBE were performed as described previously [ 5 ] using a Shimatzu Nexpera X2 UPLC chromatograph equipped with Diode Array Detector (DAD) (Shimadzu, SPD-M20A, Columbia, MA, USA). Separation was performed on a reversed-phase Aquity UPLC BEH C18 column (2.1 mm × 100 mm, 1.7 µ m particle size; from Waters, Milford, MA, USA) and a pre-column of the same material at 40 ◦ C. The HPLC grade solvents used were water/formic acid (0.1%) and acetonitrile as eluents and the flow rate was 0.4 mL/min. Phenolic compounds were identified by comparing their UV spectra and retention times with that of corresponding standards. Quantification was carried out using calibration curves for each compound analyzed using concentrations between 250–2.5 mg/mL (250, 125, 100, 50, 25, 10, 5, 2.5 mg/mL). In all cases, the coefficient of linear correlation was R 2 >0.99. Compounds were quantified and identified at different wavelengths (209–370 nm). 2.4.4. ATR-Fourier Transform Infrared Spectroscopy Chemical groups and bonding arrangement of constituents present in the PBE dried samples were determined by Fourier Transform Infrared Spectroscopy (FTIR) using an ALPHA IIBruker spectrometer (Ettlingen, Germany) with a diamond-composite attenuated total reflectance (ATR) cell. The measurements were recorded with a wavenumber range from 4000 to 400 cm −1 , with a resolution of 4 cm−1and 24 scans per sample. 2.5. Evaluation of in Vitro Bioactivities 2.5.1. Antioxidant Activity Three different methods of measuring the antioxidant activity were used: DPPH, ABTS and FRAP, as previously described by Ferreira-Santos et al. [5]. Antioxidants 2020,9, 334 6 of 21 Free radical scavenging assay (DPPH assay) consists in the reduction of the 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH • ) radical in the presence of hydrogen-donating antioxidant, and in the formation of the non-radical DPPH-H form at the end of the reaction. Scavenging activity of 2,2 0 -azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) radical cation (ABTS •+ ) (ABTS assay) is based on interaction between antioxidant and ABTS radical, that, in the presence antioxidant compounds, the ABTS •+ nitrogen atom quenches the hydrogen atom, causing the solution decolorization. The concentration of the tested PBE and Trolox as a standard compound ranged between 1 and 250 µ g/mL. The lyophilized extracts were re-suspended in the respective solvent used in the extraction process, and the Trolox prepared in absolute ethanol. A corresponding control was used for each solvent. The radical scavenging activity for DPPH and ABTS methods (% inhibition) was calculated as Equation (2). % Inhibition =Ac −As Ac ×100 (2) where As represents the sample absorbance and Ac the control sample absorbance. The results were expressed as the sample concentration ( µ g/mL) required to inhibit 50% of the activity (IC 50 ) calculated from a dose response curve using GraphPad software (San Diego, CA, USA). Ferric reducing antioxidant power (FRAP assay) consists in the ability of extracts to reduce ferric ions (Fe 3+ to Fe 2+ ), in the form of ferric 2,4,6-tripyridyl-s-triazine (TPTZ). FRAP values are expressed as micromoles of ferrous equivalent per g of dry weight (µmol Fe2+/g PBE). 2.5.2. Antihyperglycemic Activity Two different methods of measuring the potential antihyperglycemic activity were used: α -Amylase and α -Glucosidase inhibition assays. These methodologies were previously reported by Irondi and coworkers [ 22 ], and used with some minors modifications. The concentration of the tested PB extracts (dissolved in 20% DMSO and 80% water) were between 50–1000 µ g/mL for α -Amylase assay (1000, 750, 500, 250, 125, 50 µ g/mL) and 1–250 µ g/mL for α -Glucosidase assay (250, 200, 150, 100, 50, 25, 10, 1 µg/mL, prepared by successive dilutions). For the α -Amylase inhibition assay, different concentrations of PBE were incubated with α -amylase (0.5 mg/mL) and 1% of starch solution for 15 min at 37 ◦ C. Afterwards, dinitrosalicylic acid color reagent (96 mM 3,5-dinitrosalicylic acid, 5.31 M sodium potassium tartrate in 2 M NaOH) was added to the reaction and placed 10 min in a boiling water bath. This heating step also allowed to stop the reaction (inactivate the enzyme). Finally, the mixture was diluted 10 times in distilled water. In these conditions, the reduction of 3,5-dinitrosalicylic acid to 3-amino-5-nitrosalicylic acid allows to quantify maltose which being detectable at 540 nm. Acarbose was used as a reference control, and the concentration used for a-amylase assay is between 1–200 µ g/mL (200, 100, 50, 25, 10, 1 µ g/mL). The a-amylase inhibition (%) was calculated using the same equation (Equation (2)) used for DPPH assay. For α -Glucosidase inhibition assay, α -Glucosidase solution (10 U/mL) was incubated with different concentrations of PBE and p-nitrophenyl-R-d-glucopyranoside (pNPG, 3 mM). The mixture was then incubated at 37 ◦ C for 15 min, and the reaction was stopped by adding Na 2 CO 3 solution (1M). The activity of α -glucosidase was determined by measuring the absorbance of p-nitrophenol released at 400 nm. Acarbose was used as a reference control, and the concentration used for α -glucosidase assay is between 2500–15,000 µ g/mL (15,000, 12,500, 10,000, 7500, 5000, 2500 µ g/mL). The α -glucosidase inhibitory activity was calculated using the same equation (Equation (2)) used for DPPH assay. The results were expressed as the sample concentration ( µ g/mL) required to inhibit 50% of the activity (IC50) calculated from a dose response curve using GraphPad software. Antioxidants 2020,9, 334 7 of 21 2.5.3. Antimicrobial Activity For this assay, a disk diffusion method was used to determine the diameter of the inhibition zone of tested extracts (50 mg/mL of each PBE ´ s reconstituted in DMSO ( ≥ 99.9%)) and was performed following the protocols established by the Clinical and Laboratory Standards Institute (2012) [ 23 ]. The PBE concentration was selected based on the results obtained from preliminary experiments (data not shown) and data from the literature in which extracts rich in polyphenols were used [24]. Strains ofBacilluscereus ATCC11778, ClostridiumperfringensATCC 13124, EscherichiacoliATCC 25922, Listeria monocytogenes ATCC 13932, Staphylococcus aureus ATCC 25923, Salmonella enterica serovar Enteritidis ATCC 25928, Aspergillus brasiliensis ATCC 16404, Saccharomyces cerevisiae NCTC 10716 and Candida albicans ATCC 10231 were inoculated in Columbia Agar +5% Sheep Blood (COS, Biom é rieux, Craponne, France). Active cultures (0.5 McFarland) were spread onto Mueller-Hinton Agar (MHA) (Oxoid, Basingstoke, England) for general bacteria and MHA +0.2% glucose for fungus. Blank disks 6 mm in diameter were placed onto inoculated plates and impregnated with 10 µ L of PBE or controls (DMSO and commercial solution of sodium hypochlorite (LX)). In this study, a solution of sodium hypochlorite was used as positive control instead of commercial antibiotics, in order to control the sensitivity of the microbial test and reduce antibiotic use in basic research studies. Afterwards, the plates were incubated for 24 h at 37 ◦ C (for bacteria) and 48 h (for yeasts and fungi). Zones of inhibition were measured in mm with the help of ImageJ software (US National Instiutes of Health, http://rsb.info.nih.gov/ij/). 2.5.4. Cell Viability In vitro cell metabolic activity of the PB extracts was assessed in different cell lines: normal mouse fibroblast (L929-ATCC ® CCL -1), human embryonic kidney (HEK293TATCC ® CRL-11268) and human lung cancer (A549ATCC ® CCL-185); these cell lines were kindly provided by Andreia Gomes (Department of Biology, University of Minho). The metabolic activity of each cell line was evaluated by the resazurin reduction assay [ 25 ]. Cells were grown Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin, at 37 ◦ C in a humidified atmosphere with 5% CO 2 . When the cell culture reached 70–80% of confluence, the cells were trypsinized and seeded in a 96-well plate at a density of 1 × 10 5 cells per well. The different cell lines were incubated with supplemented DMEM and PBE in a concentration ranging from 75 to 1000 µ g/mL for 24 h. After incubation, cell viability was measured using the resazurin assay (7-Hydroxy-3H-phenoxazin-3-one-10-oxide sodium salt). The supernatant was replaced by 200 µ L culture media containing resazurin (0.5 mM in PBS). After 2 h of incubation at 37 ◦ C, 150 µ L of the supernatant were transferred to a new 96-well microplate and the pink fluorescent resultant product (resorufin) was detected at 560 nm ( λ ex) and 590 nm ( λ em) using a microplate reader (Cytation 3, BioTek Instruments, Inc., Winooski, VT, USA). The % cell viability was calculated correcting blank values (cell-free medium) and related to untreated controls (0.5% DMSO). IC 50 values were calculated from a dose response curve using GraphPad software. 2.6. Statistical Analysis All experiments were performed in triplicate and the data are presented as mean ± standard deviation (SD) values. GraphPad Prism ® software (version 6.0; San Diego, CA, USA) was used for statistical analyses. The analysis of variance (ANOVA) and Tukey’s multiple comparisons test were used to determine statistically different values at a significance level of p<0.05. Antioxidants 2020,9, 334 8 of 21 3. Results and Discussion 3.1. Chemical Characterization of PB The chemical composition of the PB is summarized in Table 2. The results show that the majority fractions of the total bark composition are lignin, representing 41.6% (being 41.0% klason and 0.6% acid soluble lignin). The monomeric composition of polysaccharides, which correspond to an average of approximately 30% of pine bark, shows a predominance of the cellulose fraction (17.4% of glucose content). In the hemicellulose fraction, xylans are most representative (10.9%), being arabinan and mannan groups present in 1.4% of total monosaccharides. In this work, the ethanol soluble extractives represent 13.2% of total PB composition. The inorganic substances represent 2.6% of total bark composition, and the most relevant minerals, determined by plasma atomic emission spectrometry, are potassium, magnesium, calcium and iron. Other constituents such a lipid fraction (fat) and protein content represent 2.5% and 1.6% of total composition of PB, respectively. Table 2. Chemical composition of Pinus pinaster bark, expressed as percentage of dry raw material weight (composition by 100 g). Composition (%) Cellulosea17.39 ±0.37 Hemicellulose 12.31 ±0.20 Xylose 10.92 ±0.19 Arabinose +manose 1.39 ±0.01 Acetyl group n.d. Lignin 41.65 ±0.24 Klason 41.05 ±0.24 Acid soluble 0.60 ±0.00 Fat 2.54 ±0.26 Protein 1.64 ±0.03 Ash 0.87 ±0.00 Moisture 8.15 ±0.02 Ethanol extractives 13.20 ±0.31 Inorganic substances 2.56 ±0.33 Macro minerals (Na, K, Ca, Mg, Fe) 2.54 ±0.33 Micro minerals (Zn, Mn, Cu) 0.02 ±0.00 aestimated from the glucan content; n.d.: not detected. 3.2. Solid–Liquid Extraction Influence of Variables on the TPC and Antioxidant Activity Assays were performed using water and ethanol (30%, 50%, 70% and 90% (v/v)) as solvents, in order to select the best extraction conditions for each solvent. In this way, five different extracts were generated with different polarities and features presumably representative of the pine bark potential. These extracts were used in the subsequent analyzes. The solvents used (water and ethanol) are considered natural, environmentally friendly (green solvents), nontoxic and food grade [ 16 , 26 ]. This is very important, as the extracts obtained will be studied as possible food and nutraceutical applications for human or animal consumption. The influence of independent variables, such as time, temperature and solid-liquid ratio for different ethanol concentration on the TPC and antioxidant activity (FRAP), was evaluated by the models shown in Table 1and in Supplementary Materials Figure S1 (Contour line plots for FRAP variable) and S2 (Pareto chart). The significance of each coefficient was also determined using F-value and the corresponding p-value (Supplementary Materials Table S1). Antioxidants 2020,9, 334 9 of 21 Statistical analysis for each solvent (water (EtOH 0%) or water-ethanol mixtures) is represented in Supplementary Materials, Figure S2 and Table S1. The experimental variables were correlated following Equation (1) (quadratic model). Theproposed mathematicalmodelsdescribingtheextractiontime(x 1 ), temperature (x 2 )andsolid-liquid ratio (x 3 ) as function, and using normalized values of regression coefficient for TPC and FRAP, are presented in the Supplementary Materials Table S2. In addition, the validation experiments performed under the predicted conditions derived from the experimental design demonstrated that the experimental values were close to the predicted values (data not shown), confirming the validity and adequacy of the proposed mathematical models. The conditions of test number 8 of all experimental models, which extraction conditions of 115 min, 82 ◦ C and a solid-liquid ratio of 0.15 g/mL (6 g /40 mL), showed a higher TPC (48.1 mg GAE/g PB for EtOH 0%, 120.1 mg GAE/g PB for EtOH 30%, 163.6 mg GAE/g PB for EtOH 50%, 136.5 mg GAE/g PB for EtOH 70%, 123.8 mg GAE/g PB for EtOH 90%, respectively) compared to the other conditions tested. In addition, the antioxidant activity evaluated by the FRAP method was also in agreement with the TPC of the obtained extracts. Taking into account these results to obtain extracts with high content of bioactive polyphenols, the conditions mentioned above were selected in order to evaluate the chemical profile, potential bioactivity and cytotoxicity of the extracts. 3.3. Phenolic and Flavonoid Contents of PBE The spectrophotometric determination of total phenolic content (TPC) and total flavonoid content (TFC) of aqueous and hydroethanolic extracts from P. pinaster bark are presented in Figure 1. Moreover, the individual phenolic compounds were identified and quantified using liquid chromatography, and the results are presented in Table 3. Antioxidants 2020, 9, x FOR PEER REVIEW 5 of 21 the experimental values were close to the predicted values (data not shown), confirming the validity and adequacy of the proposed mathematical models. The conditions of test number 8 of all experimental models, which extraction conditions of 115 min, 82 °C and a solid-liquid ratio of 0.15 g/mL (6 g /40 mL), showed a higher TPC (48.1 mg GAE/g PB for EtOH 0%, 120.1 mg GAE/g PB for EtOH 30%, 163.6 mg GAE/g PB for EtOH 50%, 136.5 mg GAE/g PB for EtOH 70%, 123.8 mg GAE/g PB for EtOH 90%, respectively) compared to the other conditions tested. In addition, the antioxidant activity evaluated by the FRAP method was also in agreement with the TPC of the obtained extracts. Taking into account these results to obtain extracts with high content of bioactive polyphenols, the conditions mentioned above were selected in order to evaluate the chemical profile, potential bioactivity and cytotoxicity of the extracts. 3.3. Phenolic and Flavonoid Contents of PBE The spectrophotometric determination of total phenolic content (TPC) and total flavonoid content (TFC) of aqueous and hydroethanolic extracts from P. pinaster bark are presented in Figure 1. Moreover, the individual phenolic compounds were identified and quantified using liquid chromatography, and the results are presented in Table 3. Figure 1. Total phenolic content (TPC) and total flavonoid content (TFC) of aqueous and hydroethanolic extracts from Pinus pinaster bark. Values are expressed as mean ± SD of 3–4 experiments; GAE: gallic acid equivalents; CE: catechin equivalents. Different letters show significant differences (p < 0.05) between groups for the same experiment. The use of specific solvents is responsible for the selectivity of the compounds in the extract, and the dissolution of intracellular compounds of the raw material, i.e., plants or by-products [27]. Important metabolites with antioxidant properties of natural resources, such as phenolic compounds, are more soluble in polar solvents, due to the presence of a hydroxyl group [27,28]. Therefore, in this work, water and ethanol were used as effective environmentally friendly polar solvents [16]. Our results show that the PB extracts have a high content of phenolic compounds. The phenolic content was determined by the Folin–Ciocalteu method, ranging from 460 to 675 mg of GAE/g PBE. The extracts obtained using 50% and 70% ethanol have higher concentration of TPC (674.5 ± 23 and 626.8 ± 2 mg GAE/g, respectively) when compared to aqueous, 30% and 90% hydroalcoholic extracts (462.5 ± 11, 586.5 ± 11 and 530.8 ± 15 mg GAE/g, respectively). Even so, water extracts present a very Pine Bark Extract (PBE) PB 0% PB 30% PB 50% PB 70% PB 90% 0 200 400 600 800 TPC (mg GAE/g PBE) TFC (mg CE/g PBE) b b b d bc c a a aab Figure 1. Total phenolic content (TPC) and total flavonoid content (TFC) of aqueous and hydroethanolic extracts from Pinus pinaster bark. Values are expressed as mean ± SD of 3–4 experiments; GAE: gallic acid equivalents; CE: catechin equivalents. Different letters show significant differences (p<0.05) between groups for the same experiment. The use of specific solvents is responsible for the selectivity of the compounds in the extract, and the dissolution of intracellular compounds of the raw material, i.e., plants or by-products [ 27 ]. Antioxidants 2020,9, 334 16 of 21 Generally, Gram-negative bacteria are more resistant than Gram-positive bacteria. Therefore, it was important to evaluate the antimicrobial properties of PBEs obtained under different conditions and the in presence of different of bacteria, yeast and fungi strains. The results of PBEs demonstrate a potent antibacterial activity against Gram-positive bacteria. At the concentration of 50 mg/mL, the highest inhibition zones were achieved against C. perfringens, and the most resistant bacteria was L. monocytogenes. These extracts did not show activity for Gram-negative bacteria, nor antifungal activity against C. albicans,S. cerevisiae and A. brasiliensis. It is apparent that the extracts obtained by hydroethanolic extraction have a higher antibacterial activity than the aqueous extracts. This may be due to the higher contents of phenol and flavonoids in these extracts (Figure 1and Table 3). While plants serve as rich, natural and safer sources of antimicrobials, the rapid incidences of increased resistance to available antibiotics worldwide have turned the attention of researchers and the pharmaceutical industries to plants in search of viable alternatives. There are different studies that demonstrates the antimicrobial activity of the phenolic compounds [ 55 ], more specifically, plant bark extracts used as a natural preservatives. In a previous study, different polyphenolic bark extracts from Canadian forest species and a commercial product from pine bark were tested against two non-pathogenic bacteria strains (E. coli and L. ivanovii). Their results show that these extracts were more active for Gram-positive bacteria [ 30 ]. These results are consistent with the data obtained in our study. Natural extracts, such as PBEs, rich in antioxidant bioactive compounds like phenolics (phenolic acids and flavonoids) can be a viable alternative to the serious problem of microbial resistance to antibiotics. In this sense, PBEs are a good option to be considered as a complement to bio-preservation for the food industry, particularly in foods where Gram-positive bacteria are important and common contaminants. 3.8. Cell Viability As demonstrated, the PBE has enormous potential to be used for human consumption and as a nutraceutical component. Therefore, the next step is to verify in which concentrations the extract is safe to be used. For that three different cell lines, one normal mouse cell line (L929) and two human cell lines (normal—HEK293T—and derived from cancer tissue—A549) were used. Several authors have used similar cell lines to evaluate the potential toxicity of other plant extracts and isolated products [ 56 , 57 ]. Different concentrations of active extracts were placed in contact with the cells and their ability to metabolize resazurin into resorufin in the presence of PBE was used as a measurement of cells metabolic activity. A decreased of resazurin conversion indicates impairment of cellular metabolism [ 25 ], being an indication of the PBE toxicity. The cells were exposed to different concentrations of each extract for 24 h (0 to 1000 µ g/mL). The three cells lines demonstrated a dose-dependent effect. However, the behavior of non-tumor cells was different from the behavior of tumor cells. The presence of low concentrations of all extracts (75 and 125 µ g/mL) stimulated their metabolic activity, while, for the same concentration, the tumor cells had a decrease on their metabolic activity. Though the variability of results is high (which is common in these type of tests), this effect is statistically different and this result is in accordance with the literature, where it is described that naturally available extracts of different sources selectively inhibit abnormal cell proliferation without interrupting normally functioning cells [ 58 ]. Therefore, this finding is the ideal condition for a potential anti-cancer effect, as it would only affect the cancer cells and not the healthy cells. However, this is still a very preliminary indicator (useful in a first potential screening) and further tests at different levels ( in vitro , ex vivo and in vivo ) are needed to validate (or not). When analyzing the IC 50 , it is clear that the action mechanism of the compounds present in the PBE extracts is different for the three cells lines. As it can be seen on Figure 3the IC 50 decreases with the increase of ethanol on the extraction solvent for the mouse cells line (L929) and for the Antioxidants 2020,9, 334 17 of 21 human cancer cell line (A549), on the hand, there is no clear relationship between the IC 50 and the solvent extraction composition for the human normal cells line (HEK 293T). As it is known the solvent extraction composition influences the extracted compounds. The highest IC 50 for the non-tumor cells was observed for the PBE extracted using 30% of ethanol (PB 30%), while for the tumor cell the highest is for aqueous (PB 0%), followed by PB 30%. Interestingly the extract PB 30% does not have the highest content in TPC or TFC, which indicates that the total amount of these compounds is not the main reason the cellular mechanisms observed. Even when comparing with the DPPH, ABTS and FRAP, there is no clear evidence. The extracts PB 0% and PB 90% present similar anti-oxidative activity. Moreover, these extracts showed the lowest anti-oxidative activity comparatively to the rest of the extracts. Therefore, it is not clear which mechanism was responsible for the observed cell behavior. Moreover, the antioxidant effect of PB extracts on the oxidative status of these cancer cell lines should be studied in the future. Antioxidants 2020, 9, x FOR PEER REVIEW 13 of 21 Figure 3. Cellular viability (%) of aqueous and hydroethanolic extracts from Pinus pinaster bark against normal mouse fibroblast (L929) (A), human embryonic kidney (HEK293T) (B), human lung cancer (A549) (C) cell lines, and the respective IC50 values (D). Values are expressed as mean ± SD of 4-5 experiments. Different letters show significant differences (p < 0.05) between groups for the same experiment. When analyzing the composition of the extracts, it is possible to observe that gallocatecchin is only present in PB 0% and PB 30% and quercetin is absent (or undetected), only being detected on the PB 50%, 70% and 90% that induced the highest cytotoxicity (lower IC50), except for the nontumoral cell line HEK293T. It has been described that gallocathecin can inhibit the tumor cell line HCT-116 growth up to 57%, and that quercetin can also induce selective growth inhibition and apoptosis in hepatic tumor cells, but not in normal cells [59]. Also, the quercetin showed the strongest dose-dependent antiproliferative activities to colon cancer cells (HT-29) and liver cancer cells (HepG2) [46]. Taking in consideration the results obtained, it is hypothesised that the selectivity of the PBE obtained are related to a synergetic effect of several molecules, in particular to gallocathecin and quercetin. Recently, Gascón and coworkers [47] described the antiproliferative, apoptotic and redox system controlling effects of bark extracts from three pine species, including Pinus pinaster, on Caco-2 cells. Touriño and collaborators [60] reported that P. pinaster bark extracts have a high antioxidant activity and can control proliferation in a human melanoma cell line. Additionally, Mao et al. [57] demonstrated that extracts from the Pinus massoniana bark inhibit migration of the lung cancer A549 cell line. % of viability % of viability % of viability IC50 (µg/mL) A C B D Figure 3. Cellular viability (%) of aqueous and hydroethanolic extracts from Pinus pinaster bark against normal mouse fibroblast (L929) ( A ), human embryonic kidney (HEK293T) ( B ), human lung cancer (A549) ( C ) cell lines, and the respective IC 50 values ( D ). Values are expressed as mean ± SD of 4-5 experiments. Different letters show significant differences (p<0.05) between groups for the same experiment. When analyzing the composition of the extracts, it is possible to observe that gallocatecchin is only present in PB 0% and PB 30% and quercetin is absent (or undetected), only being detected on the PB 50%, 70% and 90% that induced the highest cytotoxicity (lower IC 50 ), except for the non-tumoral cell line HEK293T. It has been described that gallocathecin can inhibit the tumor cell line HCT-116 growth up to 57%, and that quercetin can also induce selective growth inhibition and apoptosis in hepatic tumor cells, but not in normal cells [ 59 ]. Also, the quercetin showed the strongest dose-dependent anti-proliferative activities to colon cancer cells (HT-29) and liver cancer cells (HepG2) [46]. Antioxidants 2020,9, 334 18 of 21 Taking in consideration the results obtained, it is hypothesised that the selectivity of the PBE obtained are related to a synergetic effect of several molecules, in particular to gallocathecin and quercetin. Recently, Gasc ó n and coworkers [ 47 ] described the antiproliferative, apoptotic and redox system controlling effects of bark extracts from three pine species, including Pinus pinaster, on Caco-2 cells. Touriño and collaborators [ 60 ] reported that P. pinaster bark extracts have a high antioxidant activity and can control proliferation in a human melanoma cell line. Additionally, Mao et al. [ 57 ] demonstrated that extracts from the Pinus massoniana bark inhibit migration of the lung cancer A549 cell line. These results demonstrate that the aqueous and hydroethanolic extracts of P. pinaster bark at the tested concentrations present low cytotoxicity, and may have the potential to inhibit the tumor cell growth to some extent, though this has to be further validated. 4. Conclusions The present study revealed a wide range of phytochemicals in P. pinaster extracts, belonging to different chemical groups: phenolic acids, flavonoids, flavonols and stilbens. Extracts showed different phenolic profiles, depending on the solvent used. However, all demonstrated high potential antioxidant, antidiabetic and antimicrobial activities. The extracts from intermediate ethanol concentrations (50% and 70%) showed the highest bioactivities. These features demonstrate the potential of these extracts to be used in food formulation and processing, either with a technologic function (such as preservative or antioxidant) or as a bioactive ingredient. Moreover, the PBEs have low cytotoxicity, but most importantly, they act selectively on cancer cells, as these are negatively affected and the non-tumor cells are not. These results unravel the potential use of PBE in the medical or nutraceutical sectors. Supplementary Materials: The following are available online at http://www.mdpi.com/2076-3921/9/4/334/s1, Figure S1: Pareto chart. Figure S2: Contour line plots representing the antioxidant activity (FRAP assays) of the tested experimental models. Table S1: Factors and interaction effects of the tested experimental model. Table S2: Quadratic models describing the responses variation of the tested experimental model and their correspondent R2coefficients. Author Contributions: Conceptualization: P.F.-S., J.A.T. and C.M.R.R.; resources and investigation: P.F.-S., J.S., J.A.T. and C.M.R.R.; methodology and investigation: P.F.-S, Z.G., C.B., J.S. and C.R.; data curation: P.F.-S., Z.G., C.R. and C.B.; writing–original draft: P.F.-S. and C.B.; writingreview and editing: P.F.-S., Z.G., J.S., C.R., J.A.T. and C.M.R.R.; funding acquisition: J.A.T, J.S. and C.M.R.R. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund (FEDER) under the scope of Norte2020—Programa Operacional Regional do Norte, by program INTERREG V-B Sudoe (REDVALUE, SOE1/P1/E0123) and by project MOBFOOD (POCI-01-0247-FEDER-024524), cofounded by PORTUGAL2020, Lisb@a2020, COMPETE 2020 and the European Union. Zlatina Genisheva is supported by the project OH2O (POCI-01-0145-FEDER-029145) funded by FCT and FEDER under the scope of Programa Operacional de Competividade e Internacionalizaçao (POCI)-COMPETE 2020 and PORTUGAL2020. Pedro Santos is recipient of a fellowship supported by a doctoral advanced training (call NORTE-69-2015-15), funded by the European Social Fund under the scope of Norte2020 (NORTE-08-5369-FSE-000036). Conflicts of Interest: The authors declare no conflict of interest. References 1. Raza, W.; Lee, J.; Raza, N.; Luo, Y.; Kim, K.-H.; Yang, J. Removal of phenolic compounds from industrial waste water based on membrane-based technologies. J. Ind. Eng. Chem. 2019,71, 1–18. [CrossRef] 2. Mark, R.; Lyu, X.; Lee, J.J.L.; Parra-Sald í var, R.; Chen, W.N. Sustainable production of natural phenolics for functional food applications. J. Funct. Foods 2019,57, 233–254. [CrossRef] 3. Calvo Torras, M.A.; Faura, C.A.; Schönlau, F.; Rohdewald, P. Antimicrobial activity of Pycnogenol. Phyther. Res. 2005,19, 647–648. [CrossRef] 4. Sharma, A.; Goyal, R.; Sharma, L. Potential biological efficacy of Pinus plant species against oxidative, inflammatory and microbial disorders. BMC Complement. Altern. Med. 2016 ,16, 1–11. [CrossRef] [PubMed] Antioxidants 2020,9, 334 19 of 21 5. Ferreira-Santos, P.; Genisheva, Z.; Pereira, R.N.; Teixeira, J.A.; Rocha, C.M.R. Moderate Electric Fields as a Potential Tool for Sustainable Recovery of Phenolic Compounds from Pinus pinaster Bark. ACS Sustain. Chem. Eng. 2019,7, 8816–8826. [CrossRef] 6. Salehi, B.; Vlaisavljevic, S.; Adetunji, C.O.; Adetunji, J.B.; Kregiel, D.; Antolak, H.; Pawlikowska, E.; Uprety, Y.; Mileski, K.S.; Devkota, H.P.; et al. Plants of the genus Vitis: Phenolic compounds, anticancer properties and clinical relevance. Trends Food Sci. Technol. 2019,91, 362–379. [CrossRef] 7. Lee, K.J.; Baek, D.; Lee, G.; Cho, G.; So, Y.; Lee, J. Phytochemicals and Antioxidant Activity of Korean Black Soybean (Glycine max L.) Landraces. Antioxidants 2020,9, 213. [CrossRef] 8. M á rmol, I.; Quero, J.; Jim é nez-Moreno, N.; Rodr í guez-Yoldi, M.J.; Anc í n-Azpilicueta, C. A systematic review of the potential uses of pine bark in food industry and health care. Trends Food Sci. Technol. 2019 ,88, 558–566. [CrossRef] 9. Maimoona, A.; Naeem, I.; Saddiqe, Z.; Jameel, K. A review on biological, nutraceutical and clinical aspects of French maritime pine bark extract. J. Ethnopharmacol. 2011,133, 261–277. [CrossRef] 10. Rohdewald, P. A review of the French maritime pine bark extract (Pycnogenol), a herbal medication with a diverse clinical pharmacology. Int. J. Clin. Pharmacol. Ther. 2002,40, 158–168. [CrossRef] 11. Iravani, S.; Zolfaghari, B. Pharmaceutical and nutraceutical effects of Pinus pinaster bark extract. Res. Pharm. Sci. 2011,6, 1–11. [PubMed] 12. Chupin, L.; Maunu, S.L.; Reynaud, S.; Pizzi, A.; Charrier, B.; Charrier-EL Bouhtoury, F. Microwave assisted extraction of maritime pine (Pinus pinaster) bark: Impact of particle size and characterization. Ind. Crops Prod. 2015,65, 142–149. [CrossRef] 13. Braga, M.E.M.; Santos, R.M.S.; Seabra, I.J.; Facanali, R.; Marques, M.O.M.; De Sousa, H.C. Fractioned SFE of antioxidants from maritime pine bark. J. Supercrit. Fluids 2008,47, 37–48. [CrossRef] 14. Jerez, M.; Pinelo, M.; Sineiro, J.; N ú ñez, M.J. Influence of extraction conditions on phenolic yields from pine bark: Assessment of procyanidins polymerization degree by thiolysis. Food Chem. 2006 ,94, 406–414. [CrossRef] 15. Mellouk, H.; Meullemiestre, A.; Maache-Rezzoug, Z.; Bejjani, B.; Dani, A.; Rezzoug, S.A. Valorization of industrial wastes from French maritime pine bark by solvent free microwave extraction of volatiles. J. Clean. Prod. 2016,112, 4398–4405. [CrossRef] 16. Chemat, F.; Abert-Vian, M.; Fabiano-Tixier, A.S.; Strube, J.; Uhlenbrock, L.; Gunjevic, V.; Cravotto, G. Green extraction of natural products. Origins, current status, and future challenges. TRAC Trends Anal. Chem. 2019 , 118, 248–263. [CrossRef] 17. Torres-Valenzuela, L.S.; Ballesteros-G ó mez, A.; Rubio, S. Green Solvents for the Extraction of High Added-Value Compounds from Agri-food Waste. Food Eng. Rev. 2019,12, 83–100. [CrossRef] 18. Herrero, M.; Ibañez, E. Green extraction processes, biorefineries and sustainability: Recovery of high added-value products from natural sources. J. Supercrit. Fluids 2018,134, 252–259. [CrossRef] 19. Lourenço, S.C.; Mold ã o-Martins, M.; Alves, V.D. Antioxidants of natural plant origins: From sources to food industry applications. Molecules 2019,24, 4132. [CrossRef] 20. Singleton, V.L.; Orthofer, R.; Lamuela-Ravent ó s, R.M. Analysis of total phenols and other oxidation substrates and antioxidants by means of folin-ciocalteu reagent. Methods Enzymol. 1999,299, 152–178. 21. Barros, L.; Oliveira, S.; Carvalho, A.M.; Ferreira, I.C.F.R. In vitro antioxidant properties and characterization in nutrients and phytochemicals of six medicinal plants from the Portuguese folk medicine. Ind. Crops Prod. 2010,32, 572–579. [CrossRef] 22. Irondi, E.A.; Akintunde, J.K.; Agboola, S.O.; Boligon, A.A.; Athayde, M.L. Blanching influences the phenolics composition, antioxidant activity, and inhibitory effect of Adansonia digitata leaves extract on α -amylase, α-glucosidase, and aldose reductase. Food Sci. Nutr. 2017,5, 233–242. [CrossRef] [PubMed] 23. Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Disk Susceptibility Tests: Approved Standard, 11th Ed. ed; Clinical and Laboratory Standards Institute: Wayne, PA, USA, 2012; Volume 32, pp. 1–58. 24. Gontijo, D.C.; Gontijo, P.C.; Brand ã o, G.C.; Diaz, M.A.N.; De Oliveira, A.B.; Fietto, L.G.; Leite, J.P.V. Antioxidant study indicative of antibacterial and antimutagenic activities of an ellagitannin-rich aqueous extract from the leaves of Miconia latecrenata. J. Ethnopharmacol. 2019,236, 114–123. [CrossRef] [PubMed] Antioxidants 2020,9, 334 20 of 21 25. Helm, K.; Beyreis, M.; Mayr, C.; Ritter, M.; Jakab, M.; Kiesslich, T.; Plaetzer, K. In Vitro Cell Death Discrimination and Screening Method by Simple and Cost-Effective Viability Analysis. Cell. Physiol. Biochem. 2017,41, 1011–1019. [CrossRef] 26. Prat, D.; Wells, A.; Hayler, J.; Sneddon, H.; McElroy, C.R.; Abou-Shehada, S.; Dunn, P.J. CHEM21 selection guide of classicaland less classical-solvents. Green Chem. 2015,18, 288–296. [CrossRef] 27. Aryal, S.; Baniya, M.K.; Danekhu, K.; Kunwar, P.; Gurung, R.; Koirala, N. Total Phenolic Content, Flavonoid Content and Antioxidant Potential of Wild Vegetables from Western Nepal. Plants 2019,8, 96. [CrossRef] 28. Rafi´nska, K.; Pomastowski, P.; Rudnicka, J.; Krakowska, A.; Maru´ska, A.; Narkute, M.; Buszewski, B. Effect of solvent and extraction technique on composition and biological activity of Lepidium sativum extracts. Food Chem. 2019,289, 16–25. [CrossRef] 29. Chupin, L.; Motillon, C.; Charrier-El Bouhtoury, F.; Pizzi, A.; Charrier, B. Characterisation of maritime pine (Pinus pinaster) bark tannins extracted under different conditions by spectroscopic methods, FTIR and HPLC. Ind. Crops Prod. 2013,49, 897–903. [CrossRef] 30. Royer, M.; Prado, M.; Garc í a-P é rez, M.E.; Diouf, P.N.; Stevanovic, T. Study of nutraceutical, nutricosmetics and cosmeceutical potentials of polyphenolic bark extracts from Canadian forest species. PharmaNutrition 2013,1, 158–167. [CrossRef] 31. Bocalandro, C.; Sanhueza, V.; G ó mez-Caravaca, A.M.; Gonz á lezá lvarez, J.; Fern á ndez, K.; Roeckel, M.; Rodr í guez-Estrada, M.T. Comparison of the composition of Pinus radiata bark extracts obtained at benchand pilot-scales. Ind. Crops Prod. 2012,38, 21–26. [CrossRef] 32. Jim é nez-Moreno, N.; Volpe, F.; Moler, J.A.; Esparza, I.; Anc í n-Azpilicueta, C. Impact of extraction conditions on the phenolic composition and antioxidant capacity of grape stem extracts. Antioxidants 2019 ,8, 597. [CrossRef] [PubMed] 33. Jesus, M.S.; Genisheva, Z.; Roman í , A.; Pereira, R.N.; Teixeira, J.A.; Domingues, L. Bioactive compounds recovery optimization from vine pruning residues using conventional heating and microwave-assisted extraction methods. Ind. Crops Prod. 2019,132, 99–110. [CrossRef] 34. Tanase, C.; Cosarc ă , S.; Muntean, D.L. A critical review of phenolic compounds extracted from the bark of woody vascular plants and their potential biological activity. Molecules 2019 ,24, 1182. [CrossRef] [PubMed] 35. Lantto, T.A.; Dorman, H.J.D.; Shikov, A.N.; Pozharitskaya, O.N.; Makarov, V.G.; Tikhonov, V.P.; Hiltunen, R.; Raasmaja, A. Chemical composition, antioxidative activity and cell viability effects of a Siberian pine (Pinus sibirica Du Tour) extract. Food Chem. 2009,112, 936–943. [CrossRef] 36. Yesil-Celiktas, O.; Otto, F.; Parlar, H. A comparative study of flavonoid contents and antioxidant activities of supercritical CO2 extracted pine barks grown in different regions of Turkey and Germany. Eur. Food Res. Technol. 2009,229, 671–677. [CrossRef] 37. Karapandzova, M.; Stefkov, G.; Cvetkovikj, I.; Stanoeva, J.P.; Stefova, M.; Kulevanova, S. Flavonoids and Other Phenolic Compounds in Needles of Pinus peuce and Other Pine Species from the Macedonian Flora. Nat. Prod. Commun. 2015,10, 987–990. [CrossRef] 38. Talmaciu, A.I.; Ravber, M.; Volf, I.; Knez, Ž.; Popa, V.I. Isolation of bioactive compounds from spruce bark waste using suband supercritical fluids. J. Supercrit. Fluids 2016,117, 243–251. [CrossRef] 39. Yen, G.C.; Duh-Der, P.; Huang, D.W.; Hsu, C.L.; Fu, T.Y.C. Protective effect of pine (Pinus morrisonicola Hay.) needle on LDL oxidation and its anti-inflammatory action by modulation of iNOS and COX-2 expression in LPS-stimulated RAW 264.7 macrophages. Food Chem. Toxicol. 2008,46, 175–185. [CrossRef] 40. Sasidharan, S.; Chen, Y.; Saravanan, D.; Sundram, K.M.; Yoga Latha, L. Extraction, isolation and characterization of bioactive compounds from plants’ extracts. Afr. J. Tradit. Complement. Altern. Med. 2011 , 8, 1–10. [CrossRef] 41. Cuevas-Valenzuela, J.; Gonz á lez-Rojas, Á .; Wisniak, J.; Apelblat, A.; P é rez-Correa, J.R. Solubility of (+)-catechin in water and water-ethanol mixtures within the temperature range 277.6-331.2K: Fundamental data to design polyphenol extraction processes. Fluid Phase Equilib. 2015,382, 279–285. [CrossRef] 42. Ko, M.J.; Cheigh, C.I.; Chung, M.S. Relationship analysis between flavonoids structure and subcritical water extraction (SWE). Food Chem. 2014,143, 147–155. [CrossRef] [PubMed] 43. Coates, J. Interpretation of Infrared Spectra, A Practical Approach. Encycl. Anal. Chem. 2006, 10815–10837. 44. Ricci, A.; Olejar, K.J.; Parpinello, G.P.; Kilmartin, P.A.; Versari, A. Application of Fourier Transform Infrared (FTIR) Spectroscopy in the Characterization of Tannins. Appl. Spectrosc. Rev. 2015,50, 407–442. [CrossRef] Antioxidants 2020,9, 334 21 of 21 45. Carocho, M.; Ferreira, I.C.F.R. A review on antioxidants, prooxidants and related controversy: Natural and synthetic compounds, screening and analysis methodologies and future perspectives. Food Chem. Toxicol. 2013,51, 15–25. [CrossRef] [PubMed] 46. Fan, M.; Chen, G.; Zhang, Y.; Nahar, L.; Sarker, S.D.; Hu, G.; Guo, M. Antioxidant and anti-proliferative properties of Hagenia abyssinica roots and their potentially active components. Antioxidants 2020 ,9, 143. [CrossRef] [PubMed] 47. Gasc ó n, S.; Jim é nez-Moreno, N.; Jim é nez, S.; Quero, J.; Rodr í guez-Yoldi, M.J.; Anc í n-Azpilicueta, C. Nutraceutical composition of three pine bark extracts and their antiproliferative effect on Caco-2 cells. J. Funct. Foods 2018,48, 420–429. [CrossRef] 48. Maqsood, S.; Adiamo, O.; Ahmad, M.; Mudgil, P. Bioactive compounds from date fruit and seed as potential nutraceutical and functional food ingredients. Food Chem. 2019,308, 125522. [CrossRef] 49. Chen, X.; Xiong, J.; Huang, S.; Li, X.; Zhang, Y.; Zhang, L.; Wang, F. Analytical profiling of proanthocyanidins from Acacia mearnsii bark and in vitro assessment of antioxidant and antidiabetic potential. Molecules 2018 , 23, 2891. [CrossRef] 50. Bezerra, F.W.A.; Fechine, L.M.U.D.; Lopes, K.P.S.; De Sousa, A.F.; Do Nascimento, G.O.; Amaral, H.H.; Leal, L.K.A.M.; Trevisan, M.T.S.; Ribeiro, M.E.N.P.; Ricardo, N.M.P.S. α -Glucosidase inhibitory activity of mangiferin-loaded F127/PEG micellar system. Mater. Lett. 2019,255, 126522. [CrossRef] 51. Schäfer, A.; Högger, P. Oligomeric procyanidins of French maritime pine bark extract (Pycnogenol) effectively inhibit α-glucosidase. Diabetes Res. Clin. Pract. 2007,77, 41–46. [CrossRef] 52. Liu, X.; Wei, J.; Tan, F.; Zhou, S.; Würthwein, G.; Rohdewald, P. Antidiabetic effect of Pycnogenol ® French maritime pine bark extract in patients with diabetes type II. Life Sci. 2004 ,75, 2505–2513. [CrossRef] [PubMed] 53. Kim, Y.M.; Jeong, Y.K.; Wang, M.H.; Lee, W.Y.; Rhee, H.I. Inhibitory effect of pine extract on α -glucosidase activity and postprandial hyperglycemia. Nutrition 2005,21, 756–761. [CrossRef] [PubMed] 54. Deng, Y.; Zhao, Y.; Padilla-Zakour, O.; Yang, G. Polyphenols, antioxidant and antimicrobial activities of leaf and bark extracts of Solidago canadensis L. Ind. Crops Prod. 2015,74, 803–809. [CrossRef] 55. Daglia, M. Polyphenols as antimicrobial agents. Curr. Opin. Biotechnol. 2012,23, 174–181. [CrossRef] 56. Delazar, A.; Asnaashari, S.; Nikkhah, E.; Asgharian, P. Phytochemical analysis and antiproliferative activity of the aerial parts of Scrophularia subaphylla. Res. Pharm. Sci. 2019,14, 263–272. [CrossRef] 57. Mao, P.; Zhang, E.; Chen, Y.; Liu, L.; Rong, D.; Liu, Q.; Li, W. Pinus massoniana bark extract inhibits migration of the lung cancer A549 cell line. Oncol. Lett. 2017,13, 1019–1023. [CrossRef] 58. Abraham, M.; Augustine, D.; Rao, R.S.; Sowmya, S.V.; Haragannavar, V.C.; Nambiar, S.; Prasad, K.; Awan, K.H.; Patil, S. Naturally Available Extracts Inhibiting Cancer Progression: A Systematic Review. J. Evid. Based Complement. Altern. Med. 2017,22, 870–878. [CrossRef] 59. Du, G.J.; Zhang, Z.; Wen, X.D.; Yu, C.; Calway, T.; Yuan, C.S.; Wang, C.Z. Epigallocatechin gallate (EGCG) is the most effective cancer chemopreventive polyphenol in green tea. Nutrients 2012 ,4, 1679–1691. [CrossRef] 60. Touriño, S.; Selga, A.; Jim é nez, A.; Juli á , L.; Lozano, C.; Liz á rraga, D.; Cascante, M.; Torres, J.L. Procyanidin fractions from pine (Pinus pinaster) bark: Radical scavenging power in solution, antioxidant activity in emulsion, and antiproliferative effect in melanoma cells. J. Agric. Food Chem. 2005 ,53, 4728–4735. [CrossRef] © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).