## 3 Results and Discussion
### The Extraction Yield

Table 1: The chemical composition of the lipophilic bark fractions from _P. pinaster_ and _P. pinea_ cultivated in Portugal (semi-quantitative data expressed in mg kg\({}^{-1}\) dw).\({}^{1}\)With respect to the identification of 7\(\beta\)-hydroxy-DHAA (7) and 7\(\alpha\)-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]. Most of the identified diterpene compounds, such as DHAA (5), abiotic acid (6), 7-oxo-DHAA (9), 15-hydroxy-DHAA (10), pimaric acid (1), sandaracopimaric acid (2), and isopimaric acid (3), have already been reported to be components of _P. nigra_ bark [21], Turkish _P. pinea_ bark [23], and Pakistani _P. wallichiana_, _P. roxburghii_, and _P. gerardiana_ bark [25]. However, to the best of our knowledge, their presence in Portuguese _P. pinaster_ bark and _P. pinea_ bark has not been previously reported. \begin{table}
\begin{tabular}{l c c c} \hline
**RT (min)** & **No.** & **Compound** & _P. pinaster_ & _P. pinea_ \\ \hline  & & _Unsaturated fatty acids_ & 75 & _80_ \\
39.38 & 47 & (92,122)-Octadeca-9,12-dienoic acid & 22 & 11 \\
39.58 & 48 & (92)-Octadec-9-enoic acid & 48 & 39 \\
39.79 & 49 & (92)-Octadec-9-enoic acid & 5 & 6 \\
43.83 & 50 & (112)-Eicos-11-enoic acid & n.d. & 24 \\  & & _Diacids_ & 4 & 8 \\
29.42 & 51 & Nonanedioic acid & 4 & 8 \\  & & **Long-chain aliphatic alcohols** & **124** & **219** \\
33.94 & 52 & Hexadecan-1-ol & 11 & 8 \\
37.86 & 53 & Octadec-9-en-1-ol & 11 & 8 \\
38.57 & 54 & Octadecan-1-ol & 6 & 8 \\
46.78 & 55 & Docosan-1-ol & 34 & 71 \\
50.47 & 56 & Tetracosan-1-ol & 62 & 124 \\  & & **Sterols** & **587** & **242** \\  & & **\(\Delta^{5}\)-_Sterols_ & **467** & **242** \\
60.66 & 57 & Campesterol & 33 & 18 \\
62.73 & 58 & \(\beta\)-Sitosterol & 434 & 224 \\  & & **\(\Delta^{4}\)-3-keto-steroids** & **120** & _n.d._ \\
64.08 & 59 & Stigmast-4-en-3-one & 120 2 & n.d. \\  & & **Others** & **55** & **285** \\
14.22 & 60 & Glycerol & 3 & 7 \\
59.42 & 61 & 2,3-Dihydroxypropyl docosanoate & 52 & 91 \\
64.08 & 62 & 2,3-Dihydroxypropyl tetracosaonate & 2 & 187 \\  & & **Total** & **4746** & **2910** \\ \hline \end{tabular} \({}^{1}\) The results represent the average of the concordant values 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).  
\({}^{2}\) The sum of the content of stigmast-4-en-3-one (59) and 2,3-dihydroxypropyl tetracosaoate (62). Abbreviations: n.d., not detected; tr, traces.  
\end{table} 
Table 1: _Cont._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**-**19**) and three sesquiterpenes (**20**-**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.