2.2 ESTUDIO DE REMUNERACIONES
2.2.5 Resultado Estudio Remuneraciones
Figure 8.4.a shows the anisaldehyde sprayed TLC plate performed for the analysis of the volatile fraction in the CO2 FSFE extracts (1st step), obtained at 303 K. The same zones appeared and with approximately similar intensities in the extracts obtained at different pressures, showing that pressure did not have a significant effect on the lipophilic extracts composition. Similar behavior was observed at 313 K and 323 K (results not shown).
Temperature also did not have a major effect on the compositions. The TLC plates were characterized by intense colored zones at Rf 0, which are indicative of the presence of high polarity substances. The standards used (rutin and quercetin) also remained at the origin, and their color (light orange/brown) is an indication that they were probably present in the extracts. There were also zones that migrated with the solvent, indicating that this extraction methodology conduced to the presence of lipophilic/low polarity compounds in the extracts, associated with the lipophilic nature of the solvent (CO2). There were two stronger violet zones, with Rf 0.05-0.15, which may correspond to terpene alcohols, and three intense blue zones, with Rf 0.4-0.6, that may correspond to terpenes hydrocarbons, according to Wagner et al. (1984). The concentration of terpenes hydrocarbons was lower in the solvent front.
This TLC analysis was an indication that 1st step CO2 FSFE was capable of extracting lipophilic/low polarity compounds, which resulted in the concentration of phenolic
compounds and of other polar substances in the vegetal matrix for the subsequent extraction Figure 8.4. TLC analysis of pine bark FSFE extracts obtained at 303 K. Results were drawn using
ACD/TLC Plate Tool for ChemSketch, Freeware version 10.02: (a) Anisaldehyde sprayed TLC plate for analysis of volatile compounds in 1st step CO2 FSFE extracts; (b) NP sprayed TLC plate, observed at 365 nm, for analysis of phenolic compounds in 2nd step CO2 + EtOH FSFE extracts: (1) 10 MPa; (2) 15 MPa; (3) 20 MPa; (4) 25 MPa; (5) 30 MPa; Standards: R Rutin and Q Quercetin.
NP sprayed TLC plates, observed at 365 nm, were done for the analysis of phenolic compounds extracted at the 2nd step CO2 + EtOH FSFE. The obtained plate (at 303 K) is presented in Figure 8.4.b. Similarly to the TLC plate for volatile compounds, the same zones appeared, and with approximately the same intensity, for the extracts obtained at different pressures and temperatures (results not shown), showing that neither pressure nor temperature had a significant effect on the phenolic CO2 + EtOH extracts composition. All chromatograms were characterized by four light blue fluorescent zones at Rf 0.05, 0.17, 0.63 and 1 (in the solvent front), which may be assigned to phenol carboxylic acids (according to Wagner et al., 1984), and a red zone, at Rf 0.45, that was not identified. The standards used in the analysis appeared at Rf 0 and were not identified in extracts.
8.4.3. GC analysis
Chromatograms corresponding to the volatile oil composition profile of 1st step CO2
FSFE, HD and SoE extracts are presented in Figure 8.5. Three groups of compounds, with retention times in the ranges of 10-25, 25-45 and 45-60 minutes, can be distinguished in all the samples.
The first group of compounds, which corresponds to the most volatile substances, is strongly represented in the HD sample, presenting many compounds and high intensities, and
weakly in the 1st step FSFE and SoE extracts, with few compounds with low intensities. The second group also appears with higher intensities for the HD sample and, for the SoE extract, there are two non identified pronounced compounds. The third group, corresponding to the heavier compounds, appears with more peaks with better resolution for the 1st step FSFE extracts, followed by the SoE sample. These compounds were also observed (without high resolution) in the HD extract chromatogram (Figure 8.5).
As already referred, the volatile oil composition profiles of the extracts are reported in Table 8.2. Nine compounds were identified and they were present in most of the samples, being diterpenes, oxygenated sesquiterpenes, fatty acids (saturated and unsaturated) and esters of fatty acids (Figure 8.6). The identified compounds quantities accounted for just 8-28% of total contents and the highest one corresponded to the SoE extract. For the 1st step FSFE, the non identified compounds had higher retention times (~38-57 min) (Table 8.2).
Among the identified compounds, the ones obtained in higher percentages were palmitic acid, (z)-9-octadecenoic acid, ethyl palmitate, and abietatriene. The CO2 solubilities (molar fractions) of myristic and palmitate acids are reported in literature (Iwai et al., 1991) for operational conditions close to the ones used in this study, and are 4.03×10-3 (19.7 MPa and 308 K) and 4.82×10-4 (20.6 MPa and 308 K), respectively.
For CO2 FSFE, different fractions were collected at different extraction periods (6 fractions, collected at 15 min interval time) and were analysed by GC. For all assays, there was a general increment tendency in the retention indexes of the extracted compounds (for each successive fraction recovered) which is associated with higher diffusion times of higher molecular weight substances.
In general terms, for the 1st step FSFE extracts obtained at 303 and 313 K, it can be observed a pronounced effect of extraction pressure on the composition profile of the identified compounds (with retention times lower than ~32 minutes) as well as on the non-identified separated compounds after ~40 minutes (Table 8.2). At 323 K, the pressure effect was not so pronounced and so, the differences in extracts compositions were not so marked, which is in accordance with the mass ratio of solute in the solvent phase data reported in the kinetic parameters discussion. With the temperature increment, there was a decreasing tendency of the relative amounts of the heavier compounds in the 1st step FSFE extracts, which are associated with the decrease of the solvent’s density and which shows a higher ability of a liquid solvent in solubilizing heavier compounds, as reported by Mukhopadhyay (2000).
0
Figure 8.5. GC chromatograms obtained for pine bark extract samples: hydrodistillation (a); 1st step CO2-FSFE, 303 K/10 MPa (b) and Soxhlet (c).
No additional phytochemical studies have been reported on volatile oil composition of Pinus pinaster bark. Other parts of the tree, such as needles, branches and cones have essential oil contents of 0.1-0.2% (w/w) (Macchioni et al., 2003) and wood has an oleoresin having approximately 30% of monoterpenes, 3% of sesquiterpenes and ~60% of resin acids (Arrabal et al., 2005).
It can also be seen that, for some operational conditions, 1st step FSFE was selective for some compounds like ethyl palmitate, abietatriene, palmitic acid and myristic acid. These substances are known to have some biological properties with industrial pharmaceutical applications: ethyl palmitate is used for the treatment of some diseases including cancer (Mann and Staba (1986) cited by Kumar et al., 2004); myristic acid revealed to be a potent enzyme inhibitor (Paige et al., 1990); abietatriene diterpenes were also extracted from Salvia and showed antibacterial and antioxidant activities (Marrero et al., 2002). Although fatty acid
esters, through esterification, may be transformed into nonionic surfactants to be used in cosmetic, pharmaceutical and food industries as emulsifiers (Sabeder et al., 2005; Otake et al., 2004), the oil separation and fractionation steps, with sequential recovering of palmitic acid (as antioxidant) is important for some industries, like the palm oil production (Gordillo et al., 2004).
Oxygenated sesquiterpenes
Myristic acid
Palmitic acid Ethyl palmitate
Abietatriene
14-hydroxy-9-epi-(E)-caryophyllene
0 20 40 60 80 100
14 19 24 29 34 39
Time, min
Voltage, mV
Figure 8.6. Zoomed GC chromatogram obtained for pine bark extract sample obtained by 1st step CO2 -FSFE, at 303 K and 10 MPa.
The presence of fatty acids in the 1st step FSFE extracts volatile oil may be an indication that the raw material was obtained from an old pine tree, or that there was a somehow process inefficiency on the recovering of the lighter extract fractions. It can also explain the obtained low extract yield for HD (0.02%) and explain its composition profile (shown in Figures 8.5 and 8.6). This low volatile oil yield indicates that the higher yields obtained for FSFE and for SoE extracts are essentially composed by high molecular weight substances. According to the literature (Fradinho et al., 2002), these substances may be holocellulose, hemicellulose (A and B), klason lignin, dioxane lignin, neutral sugars as arabinose, mannose and uronic acids.
Furthermore, some procyanidins, as tannins, are found in pine bark extracts, mainly to be applied in the tanning and adhesives industries (Fradinho et al., 2002; Seabra et al., 2007).
These monomers, oligomers and polymers are essentially formed by units of catechin, epicatechin, gallic acid and other phenolic compounds (Selga and Torres, 2005; Fradinho et al., 2002).