Chemical Characterisation, Antioxidant and Antibacterial Activities of Pinus pinaster Ait. and Pinus pinea L. Bark Polar Extracts: Prospecting Forestry By-Products as Renewable Sources of Bioactive Compounds

: Agroforestry by-products have gained rising attention in recent years as they represent inexpensive and abundant raw materials that are a source of added-value chemicals, e.g., for food and pharmaceutical applications, as well as for bioenergy generation. Pinus pinaster Ait. bark extracts are consumed worldwide for their cardiovascular beneﬁts, whilst the health potential of Pinus pinea L. bark has not yet been deeply exploited. Therefore, this study highlights the chemical characterisation of Portuguese P. pinaster Ait. and P. pinea L. bark polar extracts, via ultra-high performance liquid chromatography-diode array detection-tandem mass spectrometry (UHPLC-DAD-MS n ) analysis, and their antioxidant and antibacterial activities. Quinic acid, an A-type procyanidin dimer isomer, protocatechuic acid, and quercetin were identiﬁed for the ﬁrst time as P. pinea L. bark components. Moreover, this bark demonstrated a higher total content of identiﬁed polar compounds than P. pinaster Ait. bark, with quinic acid being the most abundant compound identiﬁed. Regarding antioxidant activity, the pine bark polar extracts exhibited strong reducing power and 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2-azinobis-(3-ethyl-benzothiazoline-6-sulfonic acid (ABTS) radical scavenging effects compared to natural antioxidants. Moreover, the bactericidal actions of pine bark extracts were shown against Staphylococcus aureus and Escherichia coli at a 3.13–25 mg mL − 1 range. Globally, these promising insights can boost the sustainable exploitation of P. pinea L. bark, as already occurs with P. pinaster Ait. bark, for the food and biomedical ﬁelds.


Pinus spp. Bark Samples
The P. pinaster Ait. and P. pinea L. barks were sampled in January 2017 from the nearby Aveiro region, Portugal. After 2 weeks of air-drying, these pine bark samples were milled using a coffee milling machine, and then, a fraction with particle size lower than 1 mm was selected. The samples were kept at room temperature and in the dark before analysis.

Preparation of Pinus spp. Bark Polar Extracts
Lipophilic compounds were previously extracted from the milled bark of Pinus spp. bark, as mentioned elsewhere [22]. Thereafter, 2 g of dry bark residue was submitted to a methanol/water/acetic acid (49.5:49.5:1, v/v/v) extraction for 24 h, at constant stirring (900 r.p.m.), in the dark. After separation of the extract from the biomass via vacuum filtration, the methanol was evaporated at low pressure using a rotative evaporator, and water was removed by freeze-drying. Pinus spp. bark polar extracts were obtained in triplicate, and the respective extractive yields (EYs) were expressed as the percentage of dry bark. The extracts were left in the dark, at room temperature, until their analyses were carried out.

Total Phenolic Content
The total phenolic content (TPC) of the Pinus spp. barks was evaluated using the Folin-Ciocalteu reagent, following a previously described procedure [23,26], with some adaptations [21]. The TPC was determined through a standard curve of gallic acid (5-100 µg mL −1 ) and expressed as mg of gallic acid equivalents (GAE) per g of extract and g GAE per kg of dw. Two aliquots of each pine bark extract were tested in triplicate.

Proanthocyanidins Content
The proanthocyanidins content (PC) was determined through the acid butanol assay, according to the method described elsewhere [27]. Three bark polar extracts of each pine species were dissolved in methanol/water (50:50, v/v) at 1 mg mL −1 . The absorbance of the cynanidin chromophore was recorded at 520 nm using the Thermo Scientific Multiskan TM FC microplate reader (Thermo Fisher Scientific Inc., Waltham, MA, USA). The PC was calculated via the linear regression equation of PACs purified from grape pomace (100-500 µg mL −1 ) and expressed as mg of PAC per g of extract and g of PAC per kg of bark dw. Two aliquots of each pine bark extract were tested in triplicate.

Qualitative Analysis of Polar Extracts by UHPLC-DAD-MS n Analysis
UHPLC-UV-MS n analysis of the Pinus spp. bark extracts was carried out in a UHPLC system equipped with an Accela 600 LC pump, an Accela autosampler (set at 16 • C), and an Accela 80 Hz photo diode array detector (DAD) (Thermo Fisher Scientific, San Jose, CA, USA). The extracts were prepared in methanol/water (50:50, v/v) at 10 mg mL −1 and the filtration process was carried out by tetrafluoroethylene filter having pore size 0.2 µm. The injection volume was 10 µL. The column used for separation was a Hypersil Gold RP C18 (100 × 2.1 mm; 1.9 µm packing) provided by Thermo Fisher Scientific (San Jose, CA, USA), with a C18 pre-column (2.1 mm i.d.) supplied by Thermo Fisher Scientific (San Jose, CA, USA); both were kept at 45 ºC. The mobile phase composed of water:acetonitrile (99:1, v/v) (A) and acetonitrile (B), each containing HCOOH (0.1% (v/v)) . Gradient elution was carried out at a flow rate of 0.45 mL min −1 for 32 min, as follows: 1% B kept from 0 to 3 min; 1-31% B over 27 min; and 31-100% B over 2 min, followed by reequilibration at 1% B for 4 min. The chromatograms were recorded at 280 and 370 nm, and UV spectra collected from 210 to 600 nm.
The UHPLC system was coupled to an LCQ Fleet ion trap mass spectrometer (Ther-moFinnigan, San Jose, CA, USA) with an electrospray ionization (ESI) source, conducted in the negative ionization mode, according to previously described conditions [21].

Quantitative Analysis of Polar Extracts by UHPLC-UV Analysis
Standard solutions of gallic and protocatechuic acids, catechin, naringenin, and quercetin were injected in the UHPLC to obtain standard curves. The solutions were prepared in HPLC grade methanol, with six concentrations ranging from 0.45 to 41.04 µg mL −1 . The compounds were quantified by using the linear regression equation (Table 1) of the most similar standard compound. The pine bark extracts were injected in triplicate. The reducing power was assayed according to the procedure described [28], with some modifications, and adapted to the 96-well microplate scale. Trolox was considered as the antioxidant reference. The stock solution of Trolox was prepared in methanol, whilst three bark polar extracts of each Pinus spp. were dissolved in methanol/water (50:50, v/v) at 1 mg mL −1 . Different volumes of extract and standard stock solutions were added to deionised water up to 1000 µL and then mixed with 1000 µL of 0.2 M phosphate buffer solution (pH 6.6) and 1000 µL of 1% (w/v) potassium ferrocyanide solution, obtaining the concentration ranges of 3.4-49.5 µg mL −1 and 3.6-71.1 µg mL −1 for the extracts and Trolox, respectively. The reactional mixture was kept at 50 • C for 20 min. Thereafter, 1000 µL of 10% (w/v) trichloroacetic acid solution was added to stop the redox reaction, and the mixture was left to cool to room temperature for 10 min. An aliquot of 125 µL of the mixture was transferred to the microplate well, and 125 µL of deionised water and 25 µL of 0.1% (w/v) ferric (III) chloride solution were added. Then, the absorbance was recorded against the blank at 690 nm in a Thermo Scientific Multiskan TM FC microplate reader. An increased absorbance means a higher reducing power of sample. The halfmaximal inhibitory concentration (IC 50 ) was considered as the sample concentration at which the absorbance was measured as 0.500, being calculated through the respective linear regression equations from the graph plotting absorbance at 690 nm against the sample concentration. Each concentration was tested in triplicate.

DPPH Free Radical Scavenging Effect
The antioxidant activity against DPPH of Pinus spp. bark extracts was approached as explained before [21,30] and adapted to the 96-well plate scale. Ascorbic acid was used as the antioxidant reference, being dissolved in methanol/water (50:50, v/v) at 1 mg mL −1 . Stock solutions (1 mg mL −1 ) of three polar extracts from the P. pinaster Ait. and P. pinea L. barks were also prepared in methanol/water (50:50, v/v). Briefly, in each well, 30 µL of 1 mM DPPH methanolic solution was mixed to 75 µL of sample and 195 µL of methanol, obtaining the concentration ranges of 1.0-43.2 µg mL −1 for the extracts and 0.5-15.9 µg mL −1 for ascorbic acid. The blank was composed of 30 µL of DPPH (1 mM) in methanol and 270 µL of methanol. After mixing, the microplate was protected from the light for 30 min. The absorbance of the resulting solution was measured at 520 nm in a microplate reader (Thermo Scientific Multiskan TM FC). The DPPH scavenging effect percentage was determined according to Equation (3).
DPPH scavenging e f f ect (%) = [ The IC 50 was determined by plotting the percentage of scavenged DPPH free radicals against the concentration logarithm. For comparison reasons, the AAI was calculated according to Equation (4) [31].
with the DPPH final concentration equal to 61.874 µg mL −1 . Each concentration was assayed in triplicate.

ABTS Radical Cation Scavenging Effect
The ABTS radical cation (ABTS •+ ) scavenging activity of the Pinus spp. bark extracts was measured according to a described procedure [23,32] and adapted to the 96-well plate scale. Firstly, stock solutions (1 mg mL −1 ) of three bark polar extracts from each Pinus species were prepared in methanol/water (50:50, v/v). Moreover, stock solutions (1 mg mL −1 ) of ascorbic acid and trolox (antioxidant standards) were prepared in methanol/water (50:50, v/v) and methanol, respectively. The ABTS radical cation was prepared by reacting 7 mM ABTS with 2.45 mM potassium persulfate. The reactional mixture was kept in the dark at room temperature for 16 h and then diluted with methanol, to give an absorbance value of 0.700 at 750 nm. In each microwell, 250 µL of ABTS •+ solution was mixed with 50 µL of sample, obtaining the 0.5-21.2, 0.5-15.9 , and 0.5-19.4 µg mL −1 concentration ranges for the extracts, ascorbic acid, and trolox, respectively. The control was constituted by 250 µL of ABTS •+ solution and 50 µL of methanol. After 30 min, protected from the light, the absorbance was recorded at 750 nm by a Thermo Scientific MultiskanTM FC microplate reader.
The IC 50 was calculated by plotting the percentage of scavenged ABTS radical cation as a function of the sample concentration logarithm. Each concentration was tested in triplicate.

Antibacterial Activity
The antibacterial effect of the Pinus spp. bark polar extracts was tested against the growth of S. aureus (ATCC ® 6538) and E. coli (ATCC ® 25922).
Before the antibacterial tests, a bacterial colony was inoculated in 30 mL of tryptic soy broth at 37 • C and 170 r.p.m. for 18 h. Later, the bacterial culture was diluted in phosphate buffered saline (PBS) solution (pH 7.4), to be adjusted to 0.5 McFarland standard, which corresponds to 10 8 -10 9 colony forming units (CFUs) per mL. The bacterial inoculum was added to the Pinus spp. bark polar extracts, obtaining the final concentrations of 0.39, 0.78, 1.56, 3.13, 6.25, and 12.5 e 25 mg mL −1 . Two bacterial controls were included, namely a bacterial growth control with only bacterial inoculum and a solvent control containing bacterial inoculum with 12.5% (v/v) ethanol, representing the maximum amount of ethanol added to the bacterial culture. The bacterial suspensions were incubated at 37 • C and 120 r.p.m. for 24 h. At 24 h of incubation, the aliquots of bacterial suspensions were sampled, serially diluted in PBS and plated in triplicate in tryptic soy agar. After the incubation at 37 • C for 18 h, the CFUs were calculated, and the viable bacterial density was determined as log CFU mL −1 and compared with the solvent control. The bacteriostatic and bactericidal effects were considered as the decrease of <3-log and ≥3-log in CFU mL −1 , respectively. The assays were performed as three independent experiments, with three replicates for each concentration.

Statistical Analysis
The statistical analysis was performed using the IBM ® SPSS ® Statistics Version 25 (IBM Corporation, New York, NY, USA). Tukey's Honestly Significant Difference (HSD) and Duncan Post Hoc Tests were used to determine the significance of difference (p < 0.05) for antioxidant and antibacterial activity data, respectively.

Extraction Yield, Total Phenolic, and Proanthocyanidins Contents
The polar extracts of the P. pinaster Ait. and P. pinea L. barks were obtained by solid liquid extraction with a mixture of methanol/water/acetic acid (49.5:49.5:1, v/v/v), which has been shown to be suitable for the efficient extraction of phenolic compounds from biomass, e.g., cultivated cardoon and willow bark [21,33]. The obtained extraction yields (EYs) were 18.3 ± 1.1% and 21.3 ± 0.7% w/w dry weight (dw) for the P. pinea L. and P. pinaster Ait. barks, respectively. These values were higher than those previously reported using ethanol (4.6-13.2% w/w dry P. pinaster Ait. bark and 12.1% w/w dry P. pinea L. bark) and water (2.4-3.2% w/w dry P. pinaster Ait. bark and 4.9% w/w dry P. pinea L. bark) [10,[34][35][36]. In addition to the extraction media, the geographical origin, edaphoclimatic conditions, pine tree age and cultivar, and sample conservation and extraction techniques can strongly affect EYs.
The P. pinaster Ait. and P. pinea L. bark polar extracts were evaluated regarding their TPC and PC, which are summarized in Table 2. The TPC of the studied Pinus spp. barks accounted for 103 g of gallic acid equivalents (GAE) kg −1 dw in the P. pinea L. bark and 116 g of GAE kg −1 dw in the P. pinaster Ait. bark. The TPC values reported here for the P. pinaster Ait. and the P. pinea L. bark extracts are within the described range for the aqueous and hydroethanolic extracts of P. pinaster Ait. bark collected in the north of Portugal (5.30-163.64 g GAE kg −1 bark) [10,37]. When the TPC was expressed as mg GAE per g of extract (Table 2), it ranged between 539 mg g −1 and 568 mg g −1 of the extracts derived from the P. pinaster Ait. and P. pinea L. barks, respectively. These values were in agreement with those described in the literature, regarding the ethanol, water, and alkaline extracts of P. pinaster Ait. bark (260-738.0 mg GAE g −1 of extract) [38][39][40], including Pycnogenol ® (572.28 mg GAE g −1 of extract) [41]. Furthermore, the TPCs of the studied extracts were concordant with those reported for the hot water extracts (HWE), derived from other Pinus spp. barks, ranging from a 111 mg g −1 extract of P. rigida x taeda to an 862 mg g −1 extract of P. koraiensis [42], as well as for the 80% (v/v) methanol extract of P. brutia bark (412.42 mg GAE g −1 extract) [43]. The values represent the mean ± standard deviation of two aliquots of each extract tested in triplicate. Abbreviations: GAE, gallic acid equivalents; PAC, proanthocyanidins purified from grape pomace; PC, proanthocyanidin content; TPC, total phenolic content.
Both Pinus spp. barks showed considerably high PCs (Table 2), accounting for 174 and 157 g PACs kg −1 dw in the P. pinaster Ait. and P. pinea L. barks, respectively. Interestingly, the PC of the studied pine bark extracts was much higher than those of the alkaline extracts from the P. pinaster Ait. bark (1.20-5.43 g cyanidin equivalents kg −1 bark) [44], despite the use of a different standard. Actually, the PC determined through the cyanidin standard curve has been considered as underestimated as not all the PCs are converted into anthocyanidins [45]. When the PCs of the Pinus spp. bark polar extracts were expressed as mg PAC g −1 of the extract (Table 2), it varied between 814 mg PAC g −1 of the P. pinaster Ait. bark extract and 857 mg g −1 of the P. pinea L. bark extract. These values are~22% higher than the value reported for Pycnogenol ® (700 mg g −1 extract) [8] and up to 6.7-fold higher than the values achieved for phloem extracts from P. pinaster and P. pinea [46].
Catechin (6) (Figure 2) was identified via the UV spectral data and the detection of the [M−H] − ion at m/z 289, as well as its MS 2 fragmentation [21,54], which agreed with those obtained for a commercial standard injected under the same experimental conditions. (+)-Catechin has been previously identified in the hydroethanolic extracts of P. pinaster Ait. bark [10,37], as well as in the commercial pine bark extracts [13,55,56]. This compound has also been detected in the ethyl acetate fraction of the aqueous extract of the P. pinea L. bark from Turkey [17].
Four B-type (epi)catechin dimers (4, 5, 7, and 12) (Figure 1) were identified according to the UV absorption data and the [M−H] − ion detected at m/z 577, as well as the characteristic product ions resulting from the MS 2 fragmentation of the ion at m/z 577 [21,47,48]. Furthermore, the (epi)catechin units present in compounds 4, 5, 7, and 12 can be bonded by C4→C6 or C4→C8 (B-type), but the obtained mass spectrometry data did not allow us to unequivocally distinguish between them ( Figure 2) [48,56]. Considering that the procyanidin dimers identified so far in nature range from B1 to B16 [48], the identity of compounds 4, 5, 7, and 12 may be attributed to four of those procyanidins, with the exception of either procyanidins B10, B12, B13, and B16, as these include an epigallocatechin monomer, or procyanidin B11, due to the presence of a galloylcatechin upper subunit. Moreover, procyanidins B1, B3, B6, and B7 have been already identified in Pycnogenol ® [8] and three B-type procyanidin isomers in P. pinea L. bark [17]. Appl The antibacterial activity of the P. pinaster Ait. and P. pinea L. bark polar extracts was assessed against two infectious agents of public health relevance, namely the Gram-positive S. aureus (ATCC ® 6538) and the Gram-negative E. coli (ATCC ® 25922), as illustrated in Figure 2. The P. pinaster Ait. bark polar extracts exhibited bactericidal action in comparison with the solvent control, at a 6.25-25 mg mL −1 range (Figure 2A), causing 8.3-, 6.3-, and 4.0-log (CFU mL −1 ) statistically significant decreases at 6.25, 12.5, and 25 mg mL −1 , respectively (p < 0.05) against the Gram-positive bacterium S. aureus. Furthermore, the P. pinea L. bark polar extracts showed a bactericidal effect at the same concentration range ( Figure  2A), revealing 8.3-, 6.0-, and 4.6-log (CFU mL −1 ) reductions in relation to the solvent control, at 6.25, 12.5, and 25 mg mL −1 , respectively (p < 0.05). On the one hand, P. pinea L. bark polar extracts even significantly suppressed S. aureus growth at 3.13 mg mL −1 compared to the solvent control (p < 0.05), via bactericidal action, resulting in a 3.15-log (CFU mL −1 ) loss; on the other hand, P. pinaster Ait. bark polar extracts decreased S. aureus growth, at that concentration, with no statistical difference in comparison to the solvent control (p > 0.05), leading to a 2.10-log (CFU mL −1 ) decrease.
Regarding the inhibitory effects of Pinus spp. bark polar extracts against the Gramnegative bacterium E. coli growth ( Figure 2B), with the 24 h incubation with 12.5 mg mL −1 , no statistical difference (p > 0.05) was observed between the extract and the solvent control. Surprisingly, when tested at 25 mg mL −1 , both extracts acted as strong bactericidal agents, with a significant 8.26-log (CFU mL −1 ) loss in relation to the solvent control (p < 0.05). The essay was repeated several times, corroborating the results reported here. In fact, a previous study has already pointed out that Gram-positive bacteria are more easily inactivated by pine extracts than Gram-negative ones [67]. To the best of our knowledge, the inhibitory effect of P. pinea L. bark polar extract against E. coli is revealed here for the first time.
Some studies have highlighted the antibacterial action of P. pinaster Ait. and P. pinea L. bark phenolic-rich extracts against S. aureus and E. coli. Recently, Ferreira-Santos et al. [10] have demonstrated the inhibitory action of 50 mg mL −1 aqueous and ethanolic extracts derived from P. pinaster Ait. bark against S. aureus (ATCC ® 6538), under the 24 h treatment, but they have not prevented E. coli (ATCC ® 9337) growth, whilst Douglas et al. [13] have also proved the suppressing capacity of Pycnogenol ® against methicillin-resistant S. aureus viability. Conversely, Torras et al. [20] evidenced bacteriostatic activity of 20 µ g mL −1 Pycnogenol ® for S. aureus (ATCC ® 6538) and E. coli (ATCC ® 9337) but not bactericidal effect. All these research groups have suggested that phenolic compounds may be involved in the antibacterial action of pine bark polar extracts, with particular attention to catechin and m/z 739 (heterocyclic C ring fission) were detected. The product ion at m/z 587 yielded from the product ion at m/z 739 after the retro Diels-Alder fission in the F ring. In the MS 2 [865] spectrum, the characteristic product ions at m/z 451 (heterocyclic F ring fission), m/z 425 (retro Diels-Alder fission in F ring), m/z 407 (loss of a water molecule from the product ion at m/z 425), and m/z 287 (trimer upper subunit after the quinone-methide fission between the C and D rings) were also found [47,48]. Interestingly, only one B-type procyanidin trimer isomer has already been found in commercial P. pinaster Ait. bark extracts [56,58], and two others in P. pinea L. bark [17].
Compound 9 was tentatively identified as an A-type procyanidin dimer ( Figure 2) as its UV molecular absorption spectrum, the detection of the [M−H] − ion at m/z 591, and the corresponding MS 2 spectrum (Table 3) were all concordant with the literature data [50,57,59]. The chemical structure of this compound can be envisaged as one of four hypotheses, as follows: (epi)gallocatechin-(2→O7, 4→8)-(epi)catechin; (epi)gallocatechin-(2→O5, 4→6)-(epi)catechin; (epi)catechin-(2→O7, 4→8)-(epi)gallocatechin; or (epi)catechin-(2→O5, 4→6)-(epi)gallocatechin [50,59]. Considering the detection of the product ions at m/z 301 and m/z 289 in the MS 2 spectrum of the ion at m/z 591, the former two hypotheses are more likely than the remaining two as these product ions represent, respectively, [(epi)gallocatechin−5H] − and [(epi)catechin−H] − ions formed by the methide fission between the C ring of the (epi)gallocatechin unit (upper unit) and the D ring of the (epi)catechin (terminal unit) [59] ( Figure S1). Furthermore, the MS 2 spectrum of the ion at m/z 591 presented a base peak at m/z 439, which resulted from the retro-Diels-Alder fission in the F-ring of the (epi)catechin unit, with the loss of 152 mass units ( Figure S1), strengthening the hypothesis of (epi)catechin being the terminal unit of compound 9 [59]. Nonetheless, it is not possible to distinguish the linkages of the units solely by mass spectrometry [59]. Thus, the chemical structure of compound 9 may be an isomer of the (epi)gallocatechin-(epi)catechin dimer. To the best of our knowledge, this procyanidin isomer is identified here for the first time in the P. pinaster Ait. and P. pinea L. barks.

•
Other compounds The identification of quinic acid (1) (Figure 2) was based on the characteristic UV molecular absorption spectrum, the detection of the [M−H] − at m/z 191, and the corresponding MS 2 spectrum, which showed a base peak at m/z 111 and the characteristic product ions at m/z 173 ([M−H−H 2 O] − ) and m/z 93 [13]. This compound has already been found in Pycnogenol ® [13], whereas it was identified for the first time, in this work, in P. pinea L. bark.
After confirmed by authentic standard, compound 2 was assigned as protocatechuic acid (Figure 2). In fact, this phenolic compound had been previously identified in P. pinaster Ait. [10] and P. pinaster Ait. var. moghrebiana barks [61], as well as in commercial pine bark extracts [13,55]. In contrast, protocatechuic acid was identified for the first time in P. pinea L. bark, to the best of our knowledge.
Compound 15 was identified as quercetin, whose retention time, molecular absorption spectrum, [M−H] − at m/z 301, and MS 2 spectrum were concordant with those of the authentic standard. This flavonol has been found in a commercial concentrated extract [56] and hydroethanolic extracts [10,37], derived from P. pinaster Ait. bark, but it has been reported here for the first time as a P. pinea L. component.

Quantitative UHPLC-UV Analysis of Pinus spp. Bark Polar Extracts
The fifteen compounds detected in the Pinus spp. bark extracts were quantified by HPLC-UV, and the individual contents of each component in the P. pinaster Ait. and P. pinea L. polar extracts are depicted in Table 4.
The total content of identified compounds varied between 14.81 g kg −1 of dry bark of P. pinaster Ait. and 18.56 g kg −1 of dry bark of P. pinea L. Quinic acid (1) represented the major compound identified in both Pinus spp. barks, accounting for 40% and 42% (w/w) of the total content of the identified compounds in the P. pinaster Ait. and P. pinea L. barks, respectively. The values represent the mean ± standard deviation obtained from Pinus spp. bark extracts injected in triplicate. Standard curves used for the quantification of the identified compounds: A gallic acid; B protocatechuic acid; C catechin; D naringenin; E quercetin. F The abundance is lower than the limit of quantification (LOQ). G The abundance of compounds 6 and 7 was determined at 280 nm, using the standard curve of catechin.
Flavan-3-ols constituted the major phenolic subclass detected in the studied pine bark polar extracts (Table 4), accounting for 47% and 48% (w/w) of the total identified content in the P. pinaster Ait. and P. pinea L. barks, respectively. Considering the abundance of flavan-3-ols expressed as mg of compound per g of extract, it was higher in the P. pinea L. bark extract (48.66 mg g −1 of extract) than in the P. pinaster Ait. Bark extract (33.03 mg g −1 of extract) (Table 4). Indeed, the flavan-3-ols contents of the Pinus spp. bark extracts analysed in the present work are concordant with that detected in Pycnogenol ® [13] but lower than that reported by Romani and colleagues [55] in a commercial pine bark extract (776.6 mg g −1 powder).
Moreover, catechin (6) and a B-type procyanidin dimer (7) were the main flava-3-ols present in both pine bark samples, representing 15% (w/w) of the total content of the identified compounds (Table 4). In particular, catechin and B-type procyanidin dimers accounted for 27.79 mg g −1 of the P. pinea L. extract and 20.06 mg g −1 of the P. pinaster Ait. extract, being slightly higher than that earlier evidenced for Pycnogenol ® (15.49 mg g −1 of extract) [13]. Catechin (6) has also been detected in P. pinea L. bark extracts, namely in an ethyl acetate fraction of HWE (35.8 mg g −1 of extract) [17]. B-type procyanidin trimers (8, 10, and 11) were also abundant in both pine barks, accounting for 14.2% (w/w) and 12.6% (w/w) of the total identified content in the P. pinea L. and P. pinaster Ait. barks, respectively. Their abundances agreed with those described for a commercial extract of P. maritima L. bark (21.1% (w/w) of total content) [55]. To the best of our knowledge, the abundances of procyanidins in P. pinea L. bark are exhibited, for the first time, in this study.
Flavanonols were also detected at considerable abundances in both pine barks, representing 9.8% (w/w) and 7.7% (w/w) of the total identified contents of the P. pinaster Ait. and P. pinea L. barks, respectively (Table 4). Taxifolin (14) was the major flavanonol, accounting for 7.66 mg g −1 of the P. pinea L. bark extract and 6.83 mg g −1 of the P. pinaster Ait. bark extract (Table 4). Taxifolin abundance in the P. pinaster Ait. bark extract is within the range reported for commercial P. pinaster Ait. bark extracts (1.20 mg g −1 extract-33.1 mg g −1 powder) [13,55]. However, the taxifolin abundance in the P. pinea L. bark polar extract was 4.2-fold lower than that reported for the ethyl fraction of HWE (28.7 mg g −1 fraction) [17]. It is noteworthy that both pine barks contained much higher taxifolin abundances than the respective Spanish species (up to 12.5-fold) [18]. Regarding the taxifolin-O-hexoside (13) content, the P. pinea L. bark demonstrated a much lower content than that of Pycnogenol ® (16.5-fold) [13].
Protocatechuic acid (2) was found in the P. pinaster Ait. and P. pinea L. barks, representing 2.4% (w/w) and 2.1% (w/w) of the respective biomasses (Table 4). Considering its concentration, expressed as mg per g of extract, the studied polar extracts evidenced similar values to the published data for the commercial pine bark extracts (1.81 mg g −1 of Pycnogenol ® -9.1 mg g −1 of P. maritima L. bark extract). Furthermore, the flavonol quercetin (15) was detected in both pine bark polar extracts, however in contents lower than the limit of quantification (LOQ) of the respective standard curve (Table 4).
Taking into account the antioxidant and antibacterial activities attributed to the quinic acid and phenolic compounds identified in the P. pinaster Ait. and P. pinea L. bark [62][63][64][65], the polar extracts were herein tested regarding their antioxidant activity, via reducing power, DPPH, and ABTS scavenging effects, as well as their antibacterial activities against E. coli and S. aureus.

Antioxidant Activity
P. pinaster Ait. and P. pinea L. bark polar extracts were evaluated regarding their antioxidant activities by three different methods, namely reducing power, DPPH and ABTS scavenging assays. Table 5 highlights the IC 50 values obtained for each extract. To the best of our current knowledge, the reducing power of the P. pinaster Ait. and P. pinea L. bark polar extracts was determined for the first time in the present study. The IC 50 of these extracts ranged between 21.21 µg mL −1 of P. pinea L. extract and 22.53 µg mL −1 of P. pinaster Ait. extract. Both pine bark polar extracts were slightly more efficient than the trolox (up to 1.4-fold) (p < 0.05). Although the Ferric Reducing Antioxidant Power (FRAP) of phenolic-containing extracts, derived from the P. pinaster Ait. bark, was previously addressed, the mechanisms underlying this assay differ from the reducing power approach used here. To the best of our current knowledge, only pine bark extracts from P. brutia (80% (v/v) methanol extracts) were assessed through the reducing power, evidencing a IC 50 range of 9.17-11.38 µg mL −1 [43]. Thereby, both the P. pinaster Ait. and the P. pinea L. bark polar extracts were slightly less effective as reducing agents than those of the aforementioned Pinus species (2.3-fold).
Moreover, the pine extracts tested herein have shown to be more active in scavenging the DPPH free radicals than the P. pinaster Ait. bark aqueous, 50% (v/v) ethanol, and ethanol extracts (82.24-108.74 mg AAE g −1 dry bark), achieving up to 1.5-fold [37]. Even considering the bark extracts of other pine species, the P. pinaster Ait. and P. pinea L. bark polar extracts characterised in the present work have shown better DPPH scavenging action than the extracts obtained by Soxhlet acetone/water (7:3, v/v) and microwave-assisted extraction of P. radiata bark, with AAI ranges of 1.71-1.81 and 2.27-3.37, respectively [66].
The ABTS scavenging effect of P. pinea L. bark extract was close to that of the P. pinaster Ait. bark extract (Table 6), showing promising IC 50 values of 3.89 and 3.95 µg mL −1 , respectively. Compared with the antioxidant standards, both extracts were as active as ascorbic acid in scavenging ABTS •+ but less active than Trolox in this assay (p < 0.05). Considering the IC 50 values expressed as mmol Trolox equivalents (TE) per g of extract, the data represented here are concordant with those described in the literature for P. pinaster Ait. bark water (4.35 mmol TE g −1 of extract) [19] and alkaline extracts (4.70-5.38 mmol TE per g −1 of extract) [40], but much stronger than ethanol extracts (28.8-fold) [10]. Moreover, both the studied extracts revealed similar ABTS scavenging actions compared to those of 80% methanol extracts, derived from P. brutia bark (0.65-1.12 mmol TE g −1 dw) [43]. Pearson correlations were determined between the total phenolic content, the total proanthocyanidins, and the abundances of identified compounds present in the studied pine bark polar extracts and the IC 50 values of the three antioxidant activity tests, as indicated in Table 6.
The highest Pearson correlation coefficient was observed between the quinic acid content and the IC 50 of the pine bark polar extracts in the reducing power assay (r = −0.856). There is an increasing attention on the antioxidant activity of cyclic polyols, as quinic acid, in addition to other health-promoting effects, such as the treatment of diabetic diseases [62], paving the way for plentiful applications for this compound. Strong correlations have been also found between the flavanonols and taxifolin contents and the IC 50 values of the reducing power (r values of −0.828 and −0.823, respectively). Although the remaining linear correlations presented in Table 6 did not have statistical significance (p > 0.05), the linear correlations between the taxifolin and flavanonols contents and the IC 50 of extracts in the ABTS scavenging effect assay (r values of −0.605 and −0.562, respectively), as well as the one between the C-type procyanidin trimer isomer 3 abundance and the IC 50 in the DPPH scavenging assay (r = −0.416), were moderate. Nevertheless, it is important to underline that other non-identified components in the analysed pine bark polar extracts may have interacted with the phenolic compounds.

Antibacterial Activity
The antibacterial activity of the P. pinaster Ait. and P. pinea L. bark polar extracts was assessed against two infectious agents of public health relevance, namely the Gram-positive S. aureus (ATCC ® 6538) and the Gram-negative E. coli (ATCC ® 25922), as illustrated in Figure 2.
Regarding the inhibitory effects of Pinus spp. bark polar extracts against the Gramnegative bacterium E. coli growth ( Figure 2B), with the 24 h incubation with 12.5 mg mL −1 , no statistical difference (p > 0.05) was observed between the extract and the solvent control. Surprisingly, when tested at 25 mg mL −1 , both extracts acted as strong bactericidal agents, with a significant 8.26-log (CFU mL −1 ) loss in relation to the solvent control (p < 0.05). The essay was repeated several times, corroborating the results reported here. In fact, a previous study has already pointed out that Gram-positive bacteria are more easily inactivated by pine extracts than Gram-negative ones [67]. To the best of our knowledge, the inhibitory effect of P. pinea L. bark polar extract against E. coli is revealed here for the first time.
Some studies have highlighted the antibacterial action of P. pinaster Ait. and P. pinea L. bark phenolic-rich extracts against S. aureus and E. coli. Recently, Ferreira-Santos et al. [10] have demonstrated the inhibitory action of 50 mg mL −1 aqueous and ethanolic extracts derived from P. pinaster Ait. bark against S. aureus (ATCC ® 6538), under the 24 h treatment, but they have not prevented E. coli (ATCC ® 9337) growth, whilst Douglas et al. [13] have also proved the suppressing capacity of Pycnogenol ® against methicillin-resistant S. aureus viability. Conversely, Torras et al. [20] evidenced bacteriostatic activity of 20 µg mL −1 Pycnogenol ® for S. aureus (ATCC ® 6538) and E. coli (ATCC ® 9337) but not bactericidal effect. All these research groups have suggested that phenolic compounds may be involved in the antibacterial action of pine bark polar extracts, with particular attention to catechin and proanthocyanidins [68,69]. Additionally, the hypothesis of complexation between phenolic compounds and bacterial proteins, through hydrogen bond and hydrophobic connections [20] and/or synergic effects between phenolic compounds and other extract components, should be posed. Furthermore, quinic acid present in the studied pine bark polar extracts may also have an important role in their antibacterial effect; Bai et al. [70] have recently demonstrated the antibacterial effect of quinic acid against ten foodborne pathogens, including S. aureus ATCC 6538 (minimum inhibitory concentration of 2.5 mg mL −1 ), with an ability to damage the normal function of the S. aureus cell membrane. This compound has also shown an inhibitory effect against E. coli ATCC ® 11229 growth (minimum inhibitory concentration of 5 mg mL −1 ) [70].
Considering the promising data revealed in the present study, polar fractions from Portuguese P. pinaster and P. pinea barks exhibit interesting antioxidant and antibacterial actions, which may be ascribed to quinic acid and phenolic compounds. Therefore, these forest by-products can be regarded as renewable raw materials of polar added-value bioactive compounds for food and nutraceutical and biomedical applications, such as skin care, in addition to the current energy generation, in accordance with the biorefinery concept.

Conclusions
The present study highlights promising insights on the chemical composition and related antioxidant and antibacterial activities of P. pinaster Ait. and P. pinea L. bark polar extracts. Indeed, the P. pinea L. bark polar fraction has demonstrated higher abundances of total phenolic and proanthocyanidins, as well as of quinic acid and phenolic compounds identified by UHPLC-DAD-MS n analysis, in comparison to that of P. pinaster Ait. Moreover, quinic acid (1) and three phenolic compounds, namely an isomer of (epi)gallocatechin-(epi)catechin dimer (9), protocatechuic acid (2), and quercetin (15), have been described herein, for the first time, as P. pinea L. bark components. Interestingly, both studied pine bark extracts have exhibited similarly effective reducing power and scavenging effects against DPPH and ABTS free radicals, being comparable to Trolox and ascorbic acid. Considering the current emergence in searching for alternative antibacterial agents, P. pinaster Ait. and P. pinea L. bark polar extracts have revealed bactericidal effects, at the 3.13-25 mg mL −1 concentration range and at 25 mg mL −1 , against S. aureus and E. coli growth, respectively. Synergisms between quinic acid and the phenolic compounds, or even with the components of the other extracts, may have promoted the bioactivities of the pine bark polar extracts. Although there are several commercial P. pinaster Ait. bark extracts with nutraceutical usage, this knowledge can provide prospective innovative food and cosmetic applications for the studied agroforest by-products, especially with regard to the P. pinea L. bark. Nevertheless, the use of alternative solvents, such as natural deep eutectic solvents, for the extraction of quinic acid and phenolic compounds from P. pinaster Ait. and P. pinea L. barks is a further important step forward for their sustainable exploitation.

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