1. Introduction
The use of bark evolved from ancient times to present day, expanding according to the different socioeconomic contexts, as well as the scientific and technological advances. Barks show a large diversity and have a high chemical compound richness, namely regarding extractives such as sterols, terpenes, and a large number of different phenolic compounds, allowing application in medicine and pharmacy, adhesives, formaldehyde scavengers, and antioxidants.
Quercus rotundifolia Lam., generally known as holm oak (“azinheira” in Portuguese and “encina” in Spanish) due to its leaf’s resemblance to
Ilex aquifolium L. (the common European holly used in Christmas), is taxonomically complex and either recognized as a separate species or subspecies (
Q. ilex subesp.
ballota (Desf.) Samp. or
Q. ilex subesp.
rotundifolia (Lam.) O. Schwarz ex Table Morais) belonging to the subsection
Sclerophyllodrys O. Schwartz [
1,
2]. It is naturally distributed in southern Europe (Portugal, south and southeast Spain) and northwestern Africa (mainly Morocco) in the western Mediterranean basin.
Quercus rotundifolia is the main evergreen oak, besides
Q. suber, which is characteristic of the Mediterranean typical agrosilvopastoral system in Portugal (“montado”) and Spain (“dehesa”), that populates these savanna-like ecosystems.
Quercus rotundifolia is found in a wide variety of soils and its drought-tolerant capacity has been well emphasized in literature due to a xylem plasticity showing greater resistance than the co-occurring evergreen
Q. ilex L. or
Q. ilex subsp.
ilex L. and deciduous
Q. faginea species (e.g., [
3,
4]). Several ecophysiological and biochemical studies have referred to the seedling and root performances, the stomatal responses, and the antioxidant systems as combined strategies to increase drought-tolerance of the holm oak under the Mediterranean-type climate (e.g., [
5,
6,
7]). Those studies are important for the species’ conservation and sustainability due to climate change and the potential decline of this forest system. However, most of the studies have dealt with
Q. ilex or
Q. ilex subesp.
ilex, which are morphologically and genetically distinct from
Q. rotundifolia and differently distributed [
1,
8,
9].
Most reports on
Q. rotundifolia have been historically related with the establishment, management, and maintenance of the agrosilvopastoral system. Its ecological importance is well acknowledged, while the economic importance is mainly related with acorn production for animal grazing. In fact, the
Q. rotundifolia acorns are described as the sweetest among oaks, already used since the 18th century in the Iberian Peninsula as nuts associated with poor diets, and now gaining high value as a source of consumable flour within the local economies [
10]. Another main economic interest of
Q. rotundifolia, one that has prevailed for a long time, is related with the high density and the excellent calorific properties of the wood for charcoal production and firewood uses, for which the wood obtained from thinning or pruning operations is almost exclusively used [
11].
Considering
Quercus barks as potential resources, the case of
Q. suber bark is highlighted due to the extensive cork layer in its periderm, which allows for sustainable cork production with major economic importance [
12]. Cork has a unique set of properties, given by the material’s structural and chemical features, which are at the base of a dedicated industrial sector producing known cork products worldwide [
13].
Quercus cerris [
14] and
Q. variabilis [
15], among other cork rich species [
16], are also potential sources of cork, even if structural inhomogeneity must be taken into consideration before processing. However, the barks of the majority of the other oaks are not cork-rich and are referred in the literature as polyphenol-rich materials, showing excellent antioxidative capacity, namely with antimicrobial, anti-inflammatory, and antitumoral properties. Yet, little has been studied for most of the species, with some exceptions for
Q. infectoria,
Q. coccifera, and
Q. crassifolia [
17]. In general, most interests are related with the bark phenolic compounds, such as flavonoids and tannins [
18]. In fact, the first and ancient sources for tanning included
Quercus spp. barks in western Europe, before being replaced by non-natural agents [
1,
19]. While for the most valued oaks,
Q. petraea and
Q. robur, from which wood is the main product, a high phenolic content in bark extracts was confirmed (e.g., [
20,
21,
22]) and the potential of other lesser-known oaks, such as
Q. faginea, has also been acknowledged [
23]. The lipophilic content of bark is also of high interest due to its application in food- and medicine-related areas, and compounds such as sterols and triterpenes were also found, for example, in
Q. faginea bark [
23]. Furthermore, recent studies on the potential for biorefineries of oak barks highlighted
Q. rubra, Q. laurina, and
Q. crassifolia due to their chemical compositions (e.g., [
24,
25,
26]). This diversity of organic and inorganic components is related with their structural and physiological functions, namely the phenol-based compounds for oak wood durability and biotic defense of oak trees [
27].
For the valorization and assessment of the potential of barks, it is important to have specific information on their structure and anatomy, in the line of what has been studied by this research group for
Q. faginea [
28] and
Q. cerris [
29]. Regarding holm oak, its bark anatomy was not studied yet, with exceptions for the first works of the periderms of
Q. ilex x
Q. suber hybrids [
30], describing a rhytidome with successive and thin phellems. Studies dealing with the chemical properties are not available, with the exception of studies for the bark of
Q. ilex L. reporting its antibacterial activity [
31] and suitability for pollution biomonitoring [
32]. The bark potential of
Q. rotundifolia has not yet been analyzed and, to our knowledge, the present study is the first to characterize, in detail, the holm oak bark structure, anatomy, chemical composition, and antioxidant properties, also calling attention to the anatomical and chemical variability of the bark of different trees at two sites. These results will contribute to more knowledge-based decision-making on future
Q. rotundifolia management within its natural and geographic distribution range as part of the
montado.
2. Material and Methods
2.1. Sites and Sampling
The bark samples were obtained from
Q. rotundifolia trees selected along the species’ natural distribution in Portugal—at the Perímetro Florestal da Contenda (38°03′ N, 07°06′ W; 450 m altitude; site 1), a stand under the management of the public institute ICNF (Instituto da Conservação da Natureza e das Florestas), and at Mora (38°56′ N, 8°7′ W; 130 m; site 2), a privately owned stand. For each site, legal permission was given to the sampling by ICNF. At both sites, the holm oak trees (hereafter referred to
Q. rotundifolia, except if the opposite is mentioned) are sparse, the stands are unevenly aged and holm oaks are mixed with the dominant
Q. suber trees. At each site, five trees were randomly selected and were sampled during February 2018 at site 1 and during October 2017 at site 2. The sampled trees are characterized in
Table 1. The trees showed the characteristic holm oak architecture with an average of 7 m of tree height, a clear stem below branching of 1.6 m, and a 26.5 cm diameter at breast height (b.h., i.e., at 1.30 m above ground). Stand year plantation was not known and annual rings were not easily distinguishable in stem cross-sections (data not published). However, tree age was approximately estimated at 50–60 years and deemed to be similar at both sites. A 2 cm thick cross-sectional disc was cut from each tree at b.h. Bark thickness was measured along two cross-diameters. The samples of bark were manually removed, air-dried, and separated in two sets, one for anatomical characterization of the wood and the other for chemical analysis.
2.2. Cellular Structure Characterization
The macroscopic observations were made on the bark sample cross-section after surface sanding (P 1000) using a modular stereomicroscope (Leica MZ6, Leica Microsystems Ltd., Heerburg, Germany) coupled to a digital camera (Leica DC320, Leica Microsystems Ltd., Heerburg, Germany). For the microscopic observations, the bark samples were impregnated with polyethylene glycol (DP 1500), and transverse and longitudinal microscopic sections of approximately 17 μm thickness were prepared with a sliding microtome (Leica SM 2400, Leica Microsystems Nussloch GmbH, Nussloch, Germany) using adhesive for sample retrieval. The sections were stained with a double staining of chrysodine/astra blue and Sudan 4 was used for selective staining of suberin. Individual bark specimens were also macerated for observation. Slide preparation and maceration followed standard procedures described in previous works [
28]. Phloem and rhytidome were measured at two random intact points using image analysis systems (Leica Qwin Pro, v 3.5.0). Qualitative and quantitative observations were made using light microscopy (Nikon Microphot-FXA, Nikon, Japan). Bark terminology followed the IAWA List of Microscopic Bark Features [
33].
2.3. Chemical Summative Analysis
The bark samples of each tree were ground separately in a cutting mill (Retsch SM 2000, Retsch GmbH, Haan, Germany) using an output sieve of 10 mm × 10 mm, followed by one of 2 mm × 2 mm, and fractionated with a vibratory system with standard sieves (Retsch AS 200, Retsch GmbH, Haan, Germany). The 40–60 mesh (0.425–0.250 mm) fractions were used for chemical analysis. The summative chemical analysis included determination of ash; extractives in dichloromethane, ethanol, and water; suberin; Klason and acid-soluble lignin; and the monomeric composition of polysaccharides. Determinations were made in duplicate samples. The methods followed the procedures adopted in our laboratory for bark chemical characterization (e.g., [
23,
34]) and can be briefly described as follows. The ash content was determined by incinerating 2.0 g of each sample at 525 °C overnight and weighing the residue, reported as percentage of the original samples (Tappi 211 om-02). The extractives were determined with procedures adapted from Tappi 204 cm-97, performed in a Soxhlet system with dichloromethane, ethanol, and water under reflux successively, after which the content was calculated for each solvent by mass difference of the oven-dried solid residue and reported as a percentage of the original sample. The suberin content was determined by methanolysis for depolymerization using 1.5 g of the sample of extractive-free material and is expressed as a percentage of the initial dry mass [
35]. The lignin content was analyzed from the extracted and desuberinized samples by acid hydrolysis. Klason lignin was determined as the mass of the solid residue after drying at 105 °C (Tappi T 222 om-02). The acid-soluble lignin was determined by measuring the UV absorbance (TAPPI Useful Method UM 250). The remaining acid solution was kept for sugar analysis. The carbohydrates were calculated based on the amount of the neutral sugar monomers (rhamnose, arabinose, xylose, galactose, mannose, and glucose) and uronic acids (galacturonic and glucuronic acids) released by total hydrolysis, after derivatization as alditol acetates and separation by high-pressure ion-exchange chromatography with a pulsed amperometric detector (HPIC-PAD). The content of acetic acid was also determined in the hydrolysate using high-pressure ion-exclusion chromatography with a UV/visible detector (HIPCE-UV).
2.4. Composition and Antioxidant Activity of Polar Extracts
The ethanol and water extracts were obtained by successive Soxhlet extraction and analyzed in relation to total phenolics (TPC), flavonoids (FC), and condensed tannin (TC) content, as previously described [
23]. Each assay was performed at least three times and at least three independent replicates were prepared for each standard and sample. The Folin–Ciocalteu method was used for TPC determination, using gallic acid as standard, and the results were reported as mg gallic acid equivalents (GAE)/g of dried bark extract. The AlCl
3 colorimetric assay was used for the FC determination, using catechin as standard, with the results expressed as mg of catechin equivalents (CE)/g of dried bark extract. TC was determined by the vanillin-sulfuric acid assay, using catechin as standard, and the results are expressed as mg catechin equivalents (CE)/g of dried bark extract. The antioxidant activity of the ethanol and water extracts was determined using two methods—ferric reducing/antioxidant power (FRAP), which measures the sample’s ferric reducing power, and 2,2-diphenyl-1-picryhydrazyl (DPPH), which measures the free radical scavenging capacity, as previously described [
23]. FRAP is expressed as Mmol Trolox equivalents/g dry mass and the DPPH is expressed in terms of the amount of extract required to reduce 50% of the DPPH concentration (IC
50) and Trolox equivalents on a dry extract base (TEAC).
2.5. Composition of Lipophilic Extracts
Aliquots of the dichloromethane (DCM) extracts (5 mL) were taken, evaporated under N2 flow, and dried at room temperature under vacuum overnight. For the GC-MS analysis, one aliquot from the DCM extracts (2 mg) was derivatized to trimethylsilyl ethers/esters (TMS) with 100 μL of pyridine (Sigma–Aldrich, St. Louis, MO, USA) and 100 μL of BSTFA (N,O-bis(trimethylsilyl)trifluoroacetamide with 1% trimethylchlorosilane, Sigma–Aldrich, St. Louis, MO, USA) at 60 °C for 30 min. After cooling at room temperature, the extracts were injected in splitless mode in a GC-MS (Agilent 7890 A-5975C MSD, Santa Clara, CA, USA) with an autoinjector and a high-temperature capillary column (Zebron 5 H T, 30 m × 0.25 mm x 0.1 μm, Anaheim, CA, USA) using He as carrier gas at a constant flow of 1 mL/min. The injector temperature was 280 °C and the oven was programmed with an initial temperature of 100 °C (1 min), 10 °C/min to 250 °C (1 min), 8 °C/min to 350 °C (5 min), and 8 °C/min to 380 °C (5 min). The MS source conditions were MSD transfer-line temperature maintained at 330 °C, the MS source at 230 °C, the quadrupole at 150 °C, and the electron ionization energy at 70 eV. The electronic impact mass spectra (EIMS) were taken over a range of m/z 40–950. For semi-quantification analysis, the GC-MS was externally calibrated with standards (hexadecanoic acid and asiatic acid) that are representative of the major families of the lipophilic extracts (saturated fatty acids and triterpenes, respectively). The respective multiplication factors needed to acquire a correct quantification of the peak areas were calculated as an average of three GC-MS runs. The compounds are expressed as a % of each peak in TIC. Each aliquot was injected and duplicated. The identification of the compounds (as TMS derivatives) was based on comparisons with standards, Wiley 6 and NIST libraries data, and interpretation of mass spectrometric fragmentation patterns.
2.6. Composition of Suberin
Aliquots of the dichloromethane extracts (5 mL) from the suberin depolymerization reaction were taken, evaporated under N2 flow, and dried at room temperature under vacuum overnight. For the GC-MS analysis, one aliquot from the DCM extracts (1 mg) was derivatized to trimethylsilyl ethers/esters (TMS) with 100 μL of pyridine (Sigma-Aldrich, St. Louis, MO, USA) and 100 μL of BSTFA (N,O-bis(trimethylsilyl) trifluoroacetamide with 1% trimethylchlorosilane, Sigma-Aldrich, St. Louis, MO, USA) at 60 °C for 30 min. The subsequent procedures are described above.
2.7. Statistical Analysis
All results are expressed as mean and standard deviation. The significance of differences (p < 0.05) among the corresponding mean values was determined by one-way analysis of variance (ANOVA) using the SPSS statistical software (version 26).