## 2 Materials and Methods
### Chemical Characterisation of Pinus spp. Bark Polar Extracts
#### 3.2.1 Qualitative Analysis
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. pinac_ 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\(\rightarrow\)C6 or C4\(\rightarrow\)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,  
Figure 1: Chemical structures of the compounds identified in _Pinus pinaster_ Ait. and _Pinus pinac_ L. bark polar extracts. procyanidins B1, B3, B6, and B7 have been already identified in Pycnogenol(r)[8] and three B-type procyanidin isomers in _P. pinea_ L. bark [17]. Compounds **3**, **8**, **10**, and **11** were tentatively identified as B-type (epi)catechin trimers as their molecular absorption spectra, the detection of the [M\(-\)H]\({}^{-}\) ion at \(m/z\) 865, and the MS\({}^{2}\) fragmentation of the [M\(-\)H]\({}^{-}\) ion (Table 3) were in agreement with the literature data [48, 57]. Indeed, the MS\({}^{2}\) spectrum of the [M\(-\)H]\({}^{-}\) ion at \(m/z\) 865 yielded the product ions at \(m/z\) 577 ([M\(-\)H\(-\)288]\({}^{-}\)) and \(m/z\) 287 ([(epi)catechin\(-\)3H]\({}^{-}\)), resulting from the quinone-methide fission between the C and D rings, with the loss of the extension unit [48, 57]. Furthermore, the retro Diels-Alder fission in the C ring of the [M\(-\)H]\({}^{-}\) ion at \(m/z\) 865 originated the product ion at \(m/z\) 713, which yielded the product ion at \(m/z\) 695, after the loss of a water molecule [48]. Additionally, the product ions at \(m/z\) 847 ([M\(-\)H\(-\)H\({}_{2}\)O]\({}^{-}\)) 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].