## 2 Materials and Methods
### Chemical Characterisation of Pinus spp. Bark Polar Extracts
#### 3.2.1 Qualitative Analysis
The chemical structure of this compound can be envisaged as one of four hypotheses, as follows: (epi)gallocatechin-(2\(\rightarrow\)O7, 4\(\rightarrow\)8)-(epi)catechin; (epi)gallocatechin-(2\(\rightarrow\)O5, 4\(\rightarrow\)6)-(epi)catechin; (epi)catechin; (epi)catechin-(2\(\rightarrow\)O7, 4\(\rightarrow\)8)-(epi)gallocatechin; or (epi)catechin-(2\(\rightarrow\)O5, 4\(\rightarrow\)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  
Figure 2: Inhibitory effect of _Pinus pinaster_ Ait. and _Pinus pinea_ L. bark polar extracts against the growth of (**A**) _S. aureus_ (ATCC® 6538) and (**B**) _E. coli_ (ATCC® 25922). The solvent control represents the bacterial growth under 12.5% (\(\sigma/\sigma\)) ethanol. The columns and bars depict, respectively, the mean and the mean-standard of the error of three independent experiments performed in triplicate. Columns with different letters are statistically different from the solvent control (unidirectional ANOVA followed by Duncan’s Test; \(p<0.05\)). CFU, colony forming units. (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. pinaea_ L. barks. * Flavononols  
Compound **13** was assigned as taxifolin-O-hexoside (Figure 2), based on the UV molecular absorption spectrum, the [M\(-\)H]\({}^{-}\) ion at \(m/z\) 465, and the corresponding MS\({}^{2}\) spectrum [13; 49]. Actually, this MS\({}^{2}\) spectrum presented the product ion at \(m/z\) 285 due to the loss of a water molecule from the product ion at \(m/z\) 303 ([taxifolin\(-\)H]\({}^{-}\)) [13; 51; 52]. Additionally, taxifolin-O-hexoside was eluted before taxifolin (**14**), as reported elsewhere [13]. Compound **14** was identified as taxifolin (Figure 2) as its retention time, the UV spectrum, the detection of the [M\(-\)H]\({}^{-}\) ion at \(m/z\) 303, and the respective MS\({}^{2}\) and MS\({}^{3}\)[303\(\rightarrow\)285] data matched with those of the commercial standard injected under the same experimental conditions. Regarding the fragmentation data profile of this compound, the MS\({}^{2}\) spectrum of the [M\(-\)H]\({}^{-}\) ion at \(m/z\) 303 depicted the base peak at \(m/z\) 285 ([M\(-\)H\(-\)H\({}_{2}\)O]\({}^{-}\)), whilst the MS\({}^{3}\)[303\(\rightarrow\)285] fragmentation yielded the product ions at \(m/z\) 243 (loss of C\({}_{2}\)H\({}_{2}\)O) and \(m/z\) 217 (loss of C\({}_{3}\)O\({}_{2}\)), due to the presence of 4\({}^{\prime}\)-hydroxyl groups at C4\({}^{\prime}\) in the B ring and at C5 or C7 in the A ring, respectively [60]. Indeed, taxifolin has also been identified in commercial [13; 55] and hydroethanolic [10; 37] extracts of _P. pinaster_ Ait. bark. Two taxifolin isomers have also been described in _P. pinaea_ L. bark [17].