Chemical Composition of Lipophilic Bark Extracts from Pinus pinaster and Pinus pinea Cultivated in Portugal

The chemical composition of lipophilic bark extracts from Pinus pinaster and Pinus pinea cultivated in Portugal was evaluated using gas chromatography-mass spectrometry. Diterpenic resin acids were found to be the main components of these lipophilic extracts, ranging from 0.96 g kg−1 dw in P. pinea bark to 2.35 g kg−1 dw in P. pinaster bark. In particular, dehydroabietic acid (DHAA) is the major constituent of both P. pinea and P. pinaster lipophilic fractions, accounting for 0.45 g kg−1 dw and 0.95 g kg−1 dw, respectively. Interestingly, many oxidized compounds were identified in the studied lipophilic extracts, including DHAA-oxidized derivatives (7-oxo-DHAA, 7α/β-hydroxy-DHAA, and 15-hydroxy-DHAA, among others) and also terpin (an oxidized monoterpene). These compounds are not naturally occurring compounds, and their formation might occur by the exposure of the bark to light and oxygen from the air, and the action of micro-organisms. Some of these compounds have not been previously reported as lipophilic constituents of the bark of the referred pine species. Other constituents, such as aromatic compounds, fatty acids, fatty alcohols, and sterols, are also present in the studied extracts. These results can represent an opportunity to valorize P. pinaster and P. pinea by-products as a primary source of the bioactive resin acids that are integrated into the current uses of these species.


Introduction
Pinus pinaster and Pinus pinea are the two dominant pine species in Portugal, occupying ca. 714,000 and 176,000 ha, which represent 23% and 6% of the total forest area, respectively [1]. These conifers have an important impact on the national economy due to their vast applicability in timber and resin industries and also in pulp fibers production [2]. P. pinea is also cultivated because of the high commercial value of its edible seeds, which have high nutritional value and are a traditional component of the Mediterranean diet [2].
The bark of these two Pinus species consists in an interesting raw material, since it is produced in large amounts (6481 and 253 k ton year −1 of P. pinaster and P. pinea bark, respectively [3]) as a by-product of the abovementioned industries. These bark residues are simply burnt to produce energy or used as an organic substrate for plant nurseries after composting. An interesting alternative is their exploitation as a source of added-value chemicals, such as bioactive ingredients with potential nutraceutical applications, which ultimately can be integrated into the referred traditional applications in a biorefinery perspective [4] in order to maximize the biomass value.
weighting the extract's mass and expressed as a percentage of dry biomass (% w/w). The extracts were prepared in duplicate.

The GC-MS Analysis
Before the GC-MS analysis, nearly 20 mg of each dried sample was trimethylsilylated in 250 µL pyridine containing 0.3 mg of tetracosane (internal standard) through the addition of 250 µL of N,O-bis(trimethylsilyl)trifluoroacetamide and 50 µL of chlorotrimethylsilane. The mixture was kept at 70 • C for 30 min [32].
GC-MS analyses were carried out in a GC-MS-QP2010 Ultra (Shimadzu, Japan), and compounds were separated in a DB-1 J&W capillary column (30 m × 0.32 mm inner diameter, 0.25 µm film thickness), using helium as the carrier gas (40 cm s −1 ). The chromatographic conditions were as follows: initial temperature, 80 • C for 5 min; temperature rate, 4 • C min −1 up to 260 • C, 2 • C min −1 up to 285 • C, which was maintained for 10 min, and 2 • C min −1 up to 295 • C, which was maintained for 4 min; injector temperature, 250 • C; transfer-line temperature, 290 • C; split ratio, 1:60. The mass spectrometer was operated in the electron impact mode with an energy of 70 eV, and data were collected at a rate of 1 scan s −1 over a range of m/z 33-700. The ion source was kept at 250 • C [33].
For the quantitative analysis, the GC-MS apparatus was calibrated with pure reference compounds that were representative of the major lipophilic extracts' components, namely α-terpineol, dehydroabietic acid, vanillin, hexadecanoic acid, nonadecan-1-ol, stigmasterol, and betulinic acid, in relation to tetracosane (the internal standard), which allowed us to determine the respective response factors. Compounds were quantified using tetracosane as an internal standard, and their abundance is expressed as mg kg −1 dw of bark.
Two aliquots of each extract were analyzed, and the results are represented as the average of the concordant values that were obtained for the two aliquots of the two extracts in each sample (less than 5% variation between aliquots of the same sample, and between extracts of the same sample).

The Chemical Characterization of Lipophilic Fractions Derived from the P. pinaster and P. pinea Barks
Five major families of lipophilic components were identified and quantified in the lipophilic extracts from the bark of P. pinaster and P. pinea by GC-MS. These were terpenic compounds, aromatic compounds, fatty acids, long-chain aliphatic alcohols (LCAAs), and sterols, as demonstrated in Figures 1 and 2 and detailed in Table 1. The total content of identified compounds varied between 2910 mg kg −1 dw in P. pinea bark and 4746 mg kg −1 dw in P. pinaster bark. Terpenes are the main family present in these extracts, ranging from 965 mg kg −1 dw in P. pinea bark to 2865 mg kg −1 dw in P. pinaster bark, which accounted for 33% and 60% w/w of the total content of identified compounds, respectively.

Terpenic Compounds
Resin acids represent the major terpenic subclass present in the P. pinaster and P. pinea bark lipophilic fractions (Table 1), contributing 50% and 33% w/w of the total content of identified compounds, respectively. At a considerably lower abundance, betulin was the only pentacyclic triterpene identified in the P. pinaster bark extracts. Monoterpenes were also found in minor amounts, followed by sesquiterpenes.
Two groups of tricyclic diterpenic compounds were found in the studied Pinus spp. bark lipophilic extracts (Table 1), namely nine abietane-type and three pimarane-type resin acids ( Figure  3). In fact, abietane-type resin acids were the most abundant components in the bark of the studied Pinus species, followed by pimarane-type resin acids. DHAA (5) was found to be the most abundant component in bark of the studied Pinus species, representing 49% and 57% w/w of the total content of abietane-type resin acids in the P. pinaster and P. pinea bark samples, respectively (Table 1). Interestingly, this compound is only a minor constituent of fresh conifer resin; however, its abundance increases with aging, which results from the oxidation pathways of the abietadiene-type precursors (Scheme 1) [6,38,44]. Furthermore, the formation of DHAA might result from exposure to light and oxygen from the air. In addition, other DHAA oxidation derivatives were identified in the studied Pinus spp. extracts, namely 7α/β-hydroxy-DHAA (7 and 8), 7-oxo-DHAA (9), and other oxidation products (4 and 10-12) (Figure 3).

Terpenic Compounds
Resin acids represent the major terpenic subclass present in the P. pinaster and P. pinea bark lipophilic fractions (Table 1), contributing 50% and 33% w/w of the total content of identified compounds, respectively. At a considerably lower abundance, betulin was the only pentacyclic triterpene identified in the P. pinaster bark extracts. Monoterpenes were also found in minor amounts, followed by sesquiterpenes.
Two groups of tricyclic diterpenic compounds were found in the studied Pinus spp. bark lipophilic extracts (Table 1), namely nine abietane-type and three pimarane-type resin acids ( Figure  3). In fact, abietane-type resin acids were the most abundant components in the bark of the studied Pinus species, followed by pimarane-type resin acids. DHAA (5) was found to be the most abundant component in bark of the studied Pinus species, representing 49% and 57% w/w of the total content of abietane-type resin acids in the P. pinaster and P. pinea bark samples, respectively (Table 1). Interestingly, this compound is only a minor constituent of fresh conifer resin; however, its abundance increases with aging, which results from the oxidation pathways of the abietadiene-type precursors (Scheme 1) [6,38,44]. Furthermore, the formation of DHAA might result from exposure to light and oxygen from the air. In addition, other DHAA oxidation derivatives were identified in the studied Pinus spp. extracts, namely 7α/β-hydroxy-DHAA (7 and 8), 7-oxo-DHAA (9), and other oxidation products (4 and 10-12) (Figure 3).

Terpenic Compounds
Resin acids represent the major terpenic subclass present in the P. pinaster and P. pinea bark lipophilic fractions (Table 1), contributing 50% and 33% w/w of the total content of identified compounds, respectively. At a considerably lower abundance, betulin was the only pentacyclic triterpene identified in the P. pinaster bark extracts. Monoterpenes were also found in minor amounts, followed by sesquiterpenes.
Two groups of tricyclic diterpenic compounds were found in the studied Pinus spp. bark lipophilic extracts (Table 1), namely nine abietane-type and three pimarane-type resin acids ( Figure 3). In fact, abietane-type resin acids were the most abundant components in the bark of the studied Pinus species, followed by pimarane-type resin acids. DHAA (5) was found to be the most abundant component in bark of the studied Pinus species, representing 49% and 57% w/w of the total content of abietane-type resin acids in the P. pinaster and P. pinea bark samples, respectively (Table 1). Interestingly, this compound is only a minor constituent of fresh conifer resin; however, its abundance increases with aging, which results from the oxidation pathways of the abietadiene-type precursors (Scheme 1) [6,38,44]. Furthermore, the formation of DHAA might result from exposure to light and oxygen from the air. In addition, other DHAA oxidation derivatives were identified in the studied Pinus spp. extracts, namely 7α/β-hydroxy-DHAA (7 and 8), 7-oxo-DHAA (9), and other oxidation products (4 and 10-12) (Figure 3). With respect to the identification of 7β-hydroxy-DHAA (7) and 7α-hydroxy-DHAA (8), both TMS derivatives exhibit a small molecular ion at m/z 460 and a fragment ion at m/z 445 in their MS spectra, corresponding to the loss of a methyl radical from the ionized TMS groups. Further ions at m/z 191, 234, 237, 252, 299, 370, and 417 are also observed. The relative abundances of these fragments differ strongly between the two isomers, which allows for the unambiguous assignment of their structures [38]. The chromatographic elution order was also crucial together with the comparison between the obtained MS spectra and the ones found in the literature [38].
Additionally, seven monoterpenes (13)(14)(15)(16)(17)(18)(19) and three sesquiterpenes (20)(21)(22) were detected at low abundances in the Portuguese P. pinaster bark, representing 1.9% and 1.1% w/w of the total terpene contents, respectively (Table 1). Terpin (19) was found to be the most abundant monoterpene (29% w/w of P. pinaster bark monoterpene content), whereas longifolene (20) was found to be the major sesquiterpene (66% w/w of total sesquiterpene content). The identified mono-and sesquiterpenes are reported in this study for the first time to be constituents of P. pinaster bark. Monoterpenes are common components of pine wood turpentine [46], and, in particular, α-terpineol (18) has been identified in P. pinea needles [20], whereas longifolene (20) has been identified in P. pinea wood [23]. As in the case of DHAA and its oxidized derivatives, terpin is a dehydrated monoterpene that is not naturally found in wood. Therefore, its abundance in bark extracts should be associated with exposure to an external environment. Finally, the low abundance of monoterpenes that was observed in both studied Pinus spp. can be justified by their possible loss during the drying and grinding processes. The methodology employed in the present work is not the most suitable for the characterization of their volatile fraction, which is beyond the scope of this work. The high abundance of DHAA and its oxidation derivatives adds value to these extracts, since they display important biological properties. For instance, DHAA (5) exhibits antiulcer, antimicrobial, antitumor, and anti-inflammatory effects [45]. Moreover, 7-oxo-DHAA (9) has shown contact allergenic properties, and 15-hydroxy-DHAA (10) and 15-hydroxy-7-oxo-DHAA (12) have shown anti-inflammatory activity.
Additionally, seven monoterpenes (13)(14)(15)(16)(17)(18)(19) and three sesquiterpenes (20)(21)(22) were detected at low abundances in the Portuguese P. pinaster bark, representing 1.9% and 1.1% w/w of the total terpene contents, respectively (Table 1). Terpin (19) was found to be the most abundant monoterpene (29% w/w of P. pinaster bark monoterpene content), whereas longifolene (20) was found to be the major sesquiterpene (66% w/w of total sesquiterpene content). The identified mono-and sesquiterpenes are reported in this study for the first time to be constituents of P. pinaster bark. Monoterpenes are common components of pine wood turpentine [46], and, in particular, α-terpineol (18) has been identified in P. pinea needles [20], whereas longifolene (20) has been identified in P. pinea wood [23]. As in the case of DHAA and its oxidized derivatives, terpin is a dehydrated monoterpene that is not naturally found in wood. Therefore, its abundance in bark extracts should be associated with exposure to an external environment. Finally, the low abundance of monoterpenes that was observed in both studied Pinus spp. can be justified by their possible loss during the drying and grinding processes. The methodology employed in the present work is not the most suitable for the characterization of their volatile fraction, which is beyond the scope of this work. The high abundance of DHAA and its oxidation derivatives adds value to these extracts, since they display important biological properties. For instance, DHAA (5) exhibits antiulcer, antimicrobial, antitumor, and anti-inflammatory effects [45]. Moreover, 7-oxo-DHAA (9) has shown contact allergenic properties, and 15-hydroxy-DHAA (10) and 15-hydroxy-7-oxo-DHAA (12) have shown anti-inflammatory activity.
Additionally, seven monoterpenes (13)(14)(15)(16)(17)(18)(19) and three sesquiterpenes (20)(21)(22) were detected at low abundances in the Portuguese P. pinaster bark, representing 1.9% and 1.1% w/w of the total terpene contents, respectively (Table 1). Terpin (19) was found to be the most abundant monoterpene (29% w/w of P. pinaster bark monoterpene content), whereas longifolene (20) was found to be the major sesquiterpene (66% w/w of total sesquiterpene content). The identified mono-and sesquiterpenes are reported in this study for the first time to be constituents of P. pinaster bark. Monoterpenes are common components of pine wood turpentine [46], and, in particular, α-terpineol (18) has been identified in P. pinea needles [20], whereas longifolene (20) has been identified in P. pinea wood [23]. As in the case of DHAA and its oxidized derivatives, terpin is a dehydrated monoterpene that is not naturally found in wood. Therefore, its abundance in bark extracts should be associated with exposure to an external environment. Finally, the low abundance of monoterpenes that was observed in both studied Pinus spp. can be justified by their possible loss during the drying and grinding processes. The methodology employed in the present work is not the most suitable for the characterization of their volatile fraction, which is beyond the scope of this work.
Betulin (23) was the single pentacyclic triterpene present in the P. pinaster bark lipophilic fraction, representing 15% w/w of the total terpene content (Table 1). Its identification was achieved through the detection of the molecular ion at m/z 586 from the corresponding TMS derivative, and the product ions common to the mass fragmentation of trimethylsilyl derivatives of lupane-type pentacyclic triterpenes, namely at m/z 571 ([M-CH 3 ] + ), 496 ([M-TMSOH] + ), 279, 190, and 189 as the base peak [47]. To the best of our knowledge, betulin (23) has been described herein for the first time in the bark of P. pinaster. Nevertheless, this compound has recently been detected in both the outer and inner bark of Pinus merkusii and the outer bark of Pinus montezumae [48]. The presence of this compound in P. pinaster bark can be an additional factor for exploiting this forest by-product, as it can be converted into betulinic acid, which exhibits several interesting biological properties, including Human Immunodeficiency Virus (HIV) inhibition, antimicrobial, and anti-inflammatory activity [49].

Sterols
Two ∆ 5 -sterols, namely campesterol (57) and β-sitosterol (58), and a ∆ 4 -3-keto-steroid, named stigmast-4-en-3-one (59), were found, at lower abundances, in the P. pinaster and P. pinea bark lipophilic extracts (Table 1). Among these, β-sitosterol was found to be the major component in the studied Pinus bark samples. To the best of our knowledge, these sterols were also identified here for the first time in P. pinaster bark. On the other hand, campesterol (57) and β-sitosterol (58) have already been described as lipophilic components of P. pinea bark and wood [23].

Long-Chain Aliphatic Alcohols and Minor Compounds
Low abundances of five LCAAs (52-56) were identified in the analyzed P. pinaster and P. pinea lipophilic extracts, representing 2.6% and 7.5% w/w of the total content of identified constituents, respectively (Table 1). The chain length of these compounds ranged from 16 to 24 carbon atoms, with octadec-9-en-1-ol (53) as the only unsaturated component of this subclass. Tetracosan-1-ol (56) was the major constituent of this lipophilic class in both of the Pinus lipophilic fractions. With the exception of hexadecan-1-ol (52) [50], the remaining LCAAs have been mentioned in the present work for the first time as constituents of bark from the Portuguese P. pinaster. For the P. pinea bark, the LCAAs hexadecan-1-ol (52) and octadec-9-en-1-ol (53) were herein found for the first time.
Finally, glycerol (60) and two monoglycerides (61 and 62) were found in the P. pinaster and P. pinea bark samples, representing 1.2% and 9.8% w/w of the total content of identified compounds, respectively (Table 1).

Conclusions
In conclusion, the Portuguese P. pinaster and P. pinea barks demonstrated a high abundance of diterpenic compounds, with abietic-type resin acids as the main constituents of this family. In particular, dehydroabietic acid was found to be the major resin acid component of these by-products, together with several oxidized DHAA derivatives, suggesting that extensive oxidation/dehydrogenation of naturally occurring abietane-type resin acids had taken place. In addition, aromatic compounds, long-chain aliphatic alcohols, fatty acids, and sterols were also identified at lower amounts. This study suggests that P. pinaster and P. pinea barks can be a good source of valuable diterpenic compounds for further nutraceutical applications. Finally, there is a commercially available bark extract from French maritime pine (Pycnogenol ® ) that is rich in polyphenolic compounds, such as catechin, taxifolin, procyanidins, and phenolic acids. Due to the importance of phenolic compounds, new studies involving polar extracts from Portuguese P. pinaster and P. pinea bark will be performed next.