atlantica ) CHEMICAL CHARACTERIZATION OF PINE BARK ( Pinus pinaster Aiton subsp . atlantica ) , ANTIOXIDANT PROPERTIES AND PHENOLIC PROFILE OF ITS EXTRACTS

Introduction: Pine bark is an agroindustrial residue from the timber industry and represents a source of phenolic compounds. These compounds have several beneficial properties being antioxidants, antimicrobial, anti-inflammatory, cardiovascular, among others. Objetives: The aim of this work was to study the chemical composition of the bark from Pinus pinaster Aiton subsp. atlantica and the phenolic profile of its aqueous, ethanolic and hydroethanolic extracts. Methods: The moisture content, ash, protein, crude fat and carbohydrates were analysed. The bark was extracted with water, ethanol or a mixture of both in a Soxhlet apparatus and the extraction yield, total phenolic content (TPC), antioxidant activity and phenolic profile by RP-HPLC-UV, in the extracts with higher TPC, were determined. Results: The results obtained for chemical composition were: 63.43 of carbohydrates, 2.81 of crude fat, 1.60 of proteins and 1.75 of ash, calculated in % w/w of dry bark. The extraction yield was greater for the ethanolic and the hydroethanolic extracts (17.08 and 17.55% w/w dry bark, respectively). The TPC and antioxidant activity were higher in the hydroethanolic extract (73.48 mg GAE/g and 108.74 mg AAE/g dry bark, respectively). Regarding the phenolic profile of the hydroethanolic extract, gallic acid, taxifolin, ferulic acid and quercetin were identified at 280 nm, and catechin was identified in the ethanolic extract at 320 nm. Conclusion: P. pinaster Aiton subsp. atlantica bark is mainly constituted by carbohydrates and it is rich in hydroethanolic and ethanolic extractives, being that these have high antioxidant activity. The ethanolic extract presents higher catechin amount when compared to the hydroethanolic extract.


INTRODUCTION
As a consequence of the growing world population, natural resources availability is of current concern.In this sense, the use of materials, such as biomass residues from forests and agriculture, formerly considered waste, appears as a new world tendency.The Portuguese pine sector represents an important component of the total forest economic value (around 17%), being the third most important species after eucalyptus and cork oak (Seabra, Dias, Braga, & de Sousa, 2012).Pine bark is an abundant residue of the wood industry, since it represents 10-20% of the pine tree trunk (Braga et al., 2008).Currently, more than half of the bark is incinerated or landfilled and the remainder is mainly used as a cheap source of energy in saw/pulp mills being that both destinations can lead to environmental problems (Jablonsky et al., 2017).It was observed that bark contains a large fraction of extractives, with some important phytochemical constituents, and lignin, which can be utilized as a renewable source following the worldwide tendency of recovering, recycling and upgrading wastes (Braga et al., 2008).Also, it has low price and long-term stability that together make the usage of this residue highly attractive (Seabra et al., 2012).P. pinaster bark extracts have been reported to have several bioactivities including antioxidants, cardiovascular benefits, and anti-diabetic effects (Aspé & Fernandez, 2011;Chupin et al., 2015).The most prominent feature of P. pinaster is that it can grow on poor soil that provides minimal nourishment (Tümen, Akkol, Taştan, Süntar, & Kurtca, 2018).Due to large availability of pine bark on a global scale, there is an increasing interest in its use (Ronda, Della Zassa, Biasin, Martin-Lara, & Canu, 2017).A lot of attention has been recently focused on the addition of polyphenols to foods and biological systems, due to their well-known abilities to scavenge free radicals, i.e. antioxidant power (Pinelo, Rubilar, Sineiro, & Núñez, 2004).They have received considerable attention in the fields of nutrition, health, and medicine owing to their physiological and biological activities, namely antibacterial, antiviral, anticarcinogenic, anti-inflammatory and cardiovascular system diseases' prevention (Seabra et al., 2012).The objective of extracting phenolic compounds from their plant sources is to release these compounds from the vacuolar structures where they are found, either by rupturing plant tissue or by a diffusion process.Usually, a high extraction yield is required for an efficient process, although it will not necessarily ensure a high concentration of bioactive components.Since some of these are very sensitive to oxygen and heat, more care should be taken to prevent their oxidation and thermal degradation.Therefore, the extraction yield and the bioactive components' characteristics should also be considered when an extraction method is selected (Aspé & Fernandez, 2011).Also, when the main goal is to apply these bioactive components in foods or nutraceutical products, the extraction solvent must be suitable for human consumption.Therefore, the aim of the present study was to assess the chemical composition of bark from Pinus pinaster Aiton subsp.atlantica and evaluate the antioxidant activity of the aqueous, hydroethanolic and ethanolic extracts.Furthermore, its chromatographic profile by reverse phase high-performance liquid chromatography (RP-HPLC) was studied.

Sample preparation
Pine bark (P.pinaster Aiton subsp.atlantica) was collected in Minho region, Northwest of Portugal, from trees aged 15 years.The inner bark was separated from the outer bark and the latter cut into pieces was oven dried to reach equilibrium humidity at 40 °C for 72 hours.The dried outer bark was ground by using a mixer (Termomix TM31, Vorwerk, Germany) for 20 s and sieved at an amplitude of 0.2 for 1 min to select the particles with a diameter between 200 and 850 µm.All analyses and extractions, except for moisture, were performed on outer dried pine bark.The inner bark was not analysed until the date of this article.

Chemical composition
Pine bark was analysed for moisture, ash, proteins, fat and carbohydrates contents using the AOAC procedures (AOAC, 1995).The moisture was determined by drying in an oven at 103 °C until constant weight (AOAC 930.04); the ash content was determined by incineration at 550 °C (AOAC 930.05); the crude protein content (N x 6.25) of the samples was estimated by Kjeldahl method (AOAC 978.04); the crude fat was determined by extracting a known weight of ground sample with petroleum ether, using a Soxhlet apparatus (AOAC 920.39) and the total carbohydrates were determined by the 3,5-dinitrosalicylic acid reaction with reducing sugars present in the sample after hydrolysis with hydrochloric acid and neutralization (Miller, 1959).

Extraction of pine bark and evaluation of its antioxidant properties
Different solvent types were tested with Soxhlet extraction.A total of 12.5 g of ground pine bark was put into a cartridge inside a Soxhlet apparatus.Then, 220 mL of solvent (water, hydroethanolic mixture (1+1) or ethanol) was added to the flask and refluxed over four hours as the minimal indicating time for the official AOAC method for crude fat.The extract was collected and completed to 250 mL with the respective solvent and named as PW, PWE and PE, corresponding to the aqueous, hydroethanolic and ethanolic extracts, respectively.

Extraction yield
Extraction yield (% w/w) is a measure of the solvent efficiency to extract specific components from the original material, defined as the amount of solid extract recovered in dry mass compared with the initial amount of dry bark.The extraction yield was calculated measuring an exact sample volume (V 1 =25 mL) from the total extract (V 2 =250 mL) and the volume was reduced at 35 °C until it was obtained a dry solid residue.Finally, it was dried at 103 °C until constant weight.

( )
Where w 1 is the empty recipient weight, w 2 is the recipient weight plus the dried extract weight and w sample is the sample amount weighed to perform the extraction.

Total phenolic content
Total phenolic content was estimated by Folin-Ciocalteu colorimetric assay according to the procedure previously described by Lafka, Sinanoglou, and Lazos (2007) based on Gutfinger (1981) and the results were expressed as mg of gallic acid equivalents (GAE) per g of dry sample.

Antioxidant activity
The antioxidant activity in the extracts was determined by the DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging method used by Deng, Penner, and Zhao (2011) based on Brand-Williams, Cuvelier, and Berset (1995).A volume of 2 mL of diluted extract stock solution (in methanol) was mixed with 6 mL of DPPH in methanol and allowed to stand at room temperature, in the dark, for 30 minutes prior to measuring the solution absorbance at 517 nm.The control was a DPPH solution containing absolute methanol instead of the sample.The antioxidant activity was based on the measurement of the reducing ability of pine extracts towards the radical DPPH.The results were obtained as mg of ascorbic acid equivalent (AAE) per g of dry bark.The standard curve was prepared with ascorbic acid at 0, 1, 2.5, 5, 7.5, 10, 20, 30, 35 and 40 mg/L.

Polyphenols identification (RP-HPLC-UV analysis)
Sample preparation A tenfold dilution (1/10) in methanol of the extract was performed.The solution was homogenized and filtered with disposable 0.2 µm syringe filters (Whatman, UK) prior to injection.
The experiments were performed on a Hewlett Packard Series 1100 HPLC system (Agilent Technologies, Waldbronn, Germany), equipped with a G1322A degasser, G1311A quaternary pump, G1314A UV-Vis detector and a G1328A manual injector.The software used was HP ChemSation for LC Rev. A.06.03 [509].

Statistical analysis
All the assays were carried out in triplicate.The results were expressed as mean values and standard deviation (SD).A correlation between the total phenolic content and antioxidant activity values was calculated and it was obtained a correlation coefficient (R 2 ).The results were analysed using one-way analysis of variance (ANOVA) followed by Tukey's HSD test (p<0.05).This treatment was carried out using IBM SPSS Statistics for Windows, version 25.0 (IBM Corp, New York, USA).

Chemical composition
There are little recent papers about the chemical composition of pine bark, namely the maritime pine grown in Portugal.Also, the composition of bark depends on the age of the tree, the location and the growing conditions, among other factors, as observed by Vázquez, Antorrena, and Parajó (1987).In this work the two parts of pine bark (outer and inner) were divided to be studied in separate.However, this work only presents the results for the outer pine bark fraction (Table 1).The moisture content of the pine bark studied was 12.82% w/w.The dry bark composition was: carbohydrates 63.43% w/w, fat 2.81% w/w, proteins 1.60% w/w and ash 1.75 % w/w.Vázquez et al. (1987) extracted dry P. pinaster bark with organic apolar solvents obtaining yields of 0.9% w/w with ether and 2.5% w/w with hexane and benzene extraction, which is similar to the fat content determined in this work (2.81% w/w).Other authors (Fradinho et al., 2002;Vázquez et al., 1987) studied dry maritime pine bark and obtained lower values for ash, namely 0.5 and 0.8% w/w, respectively.In this work the remaining value calculated by difference (30.41% w/w; Table 1) includes other components such as cellulose, lignin and suberin, among others.

Extraction of pine bark and evaluation of its antioxidant properties
In papers published to date different solvent systems and extraction techniques have been used for the extraction of polyphenols from pine bark.The yield, total phenolic content and antioxidant activity of the extracts is highly dependent on these factors.Water, ethanol, methanol, aqueous alkaline solutions, acetone, dichloromethane, are some of the solvents commonly used to extract phenolic compounds from pine bark (Aspé & Fernandez, 2011;Durkovid-Perica, Hrenovid, Kugler, Goid-Barišid, & Tkalec, 2015;Fradinho et al., 2002).
In Table 2 are presented results of extraction yield, total phenolic content and antioxidant properties of P. pinaster Aiton subsp.atlantica bark extracts.PWE and PE extracts did not show significant differences regarding extraction yield, with 17.55 and 17.08% w/w dry bark being obtained, respectively (p<0.05).Fradinho et al. (2002) have reported a similar value, namely 13.5% w/w dry bark for the successive Soxhlet extractions with ethanol and water in P. pinaster bark grown in Portugal.However, the PWE extract showed a higher total phenolic content (73.48±1.84mg GAE/g dry bark) and therefore, higher antioxidant activity (108.74±2.02mg AAE/g dry bark, Table 2, R 2 =0.983, p<0.05).Water extracts presented the lowest values for all the parameters analysed.Pinelo et al. (2004) observed the same behaviour when extracting P. pinaster sawdust with water, denoting that water was not a good solvent for extracting phenolics.However, mixing water and ethanol can improve the rate of extraction by causing the raw material to bloat, enabling the solvent to easily enter the solid particles (Gertenbach, 2002).Pine bark extracts showed an expressive antioxidant capacity when compared with red fruits.In a recent study (Seraglio et al., 2018), it was found that in Myrtaceae fruits, the highest level DPPH value is 50.66 mg AAE/g dry fruit, while in this study the hydroethanolic extract is 108.74 mg AAE/g dry pine bark.
Considering the results obtained in the 3.2 section, as the hydroethanolic and ethanolic extracts showed better antioxidant properties, they were studied by RP-HPLC-UV. Figure 1 shows the base-peak chromatogram of phytochemical compounds in pine bark hydroethanolic extract obtained by reverse phase with the corresponding retention times.Tentative assignment of these phytochemical compounds was obtained by comparing with the retention time of pure standards (Table 3).For each of the five phytochemicals presented in Table 3, a second chromatogram was obtained after addition of a known amount to the original pine bark extract.In the chromatogram with added standard it could be observed which peak increased its intensity.This methodology helps to clarify the assignment as for example, with compounds assigned with similar RT, taxifolin (22.9 min) and ferulic acid (23.6 min).
The HPLC-UV phytochemical profile recorded at 280 nm includes, by elution order: gallic acid, taxifolin, ferulic acid and quercetin.Catechin was detected at 320 nm and exhibits an intense signal in the ethanolic extract (Figure 2), but not in the hydroethanolic one.Braga et al. (2008) also observed an higher content of catechin in the ethanolic extract, however at 280 nm.
Although the pine bark water or ethanolic extracts have not been completely elucidated, the main constituents described in the literature for a commercialized standardized extract, Pycnogenol, indicates that it contains catechin, epicatechin and taxifolin (Packer, Rimbach, & Virgili, 1999).

CONCLUSIONS
The bark of P. pinaster Aiton subsp.atlantica is composed of carbohydrates (63.43% w/w), fat (2.81% w/w), proteins (1.60% w/w), ash (1.75% w/w) and other components (30.41% w/w).Pine bark is rich in hydroethanol and ethanol extractable phytochemicals, namely 73.48 and 63.38 mg GAE/g dry bark, respectively.The pine bark extracts have high levels of antioxidant properties (82-109 mg AAE/g dry pine bark), higher than some red fruits known to be rich in antioxidant properties.The ethanolic extract presents higher concentration of catechin compared to the hydroethanolic extract.Further studies should be developed to fully confirm the chemical composition of P. pinaster Aiton subsp.atlantica bark extracts.

-Figure 2 -
Figure 1 -Chromatogram of the pine bark hydroethanolic extract recorded at 280 nm, tenfold dilution in methanol.

Table 1 -
Chemical composition values of dry pine bark from P. pinaster Aiton subsp.atlantica.

Table 2 -
Extraction yield, total phenolic content and antioxidant activity of P. pinaster Aiton subsp.atlantica bark extracts.Means (n=3) with different uppercase letters in the same column are significantly different (p<0.05).GAE -Gallic acid equivalent, AAE -ascorbic acid equivalent.

Table 3 -
Tentative assignment of phytochemical compounds detected by HPLC-UV in pine bark hydroethanolic extracts.