Collecting the material

The materials studied were wood and bark samples from the forest use of the indigenous community at Nuevo San Juan Parangaricutiro, Michoacan, Mexico (19° 21’ 00” and 19° 34’ 45”, 102° 08’ 15” and 102° 17’ 30”). Sapwood and heartwood were separated from the wood. Sapwood, heartwood, and bark samples from the three oak species were dried outside in the shade. Afterwards, the material was crushed using a Wiley mill (^{TAPPI, 2000}), it was sieved with a 40 mesh (420 mμ), and the wood dust obtained was used to carry out this study.

Reagents

Chemical solvents with analytical degree were used during the Soxhlet extraction process. They were obtained from J. T. Baker (Mexico). All the reagents used to determine the antioxidant capacity and the standards used in the HPLC analysis were obtained from Sigma-Aldrich (Mexico).

Extractive content

The extractives were obtained through a successive 6 h extraction using solvents with increasing polarity (cyclohexane, acetone, methanol) in a Soxhlet equipment, before subjecting to hot water under reflux (^{Mejía-Díaz and Rutiaga-Quiñones, 2008}). Organic solvents were recovered in a rotary evaporator (using vacuum), while the aqueous extracts were recovered using a lyophilization equipment. For this study, only the methanolic and aqueous extracts were studied, as explained below.

Inhibition of lipid oxidation test

This test was carried out using the method described by ^{Huang et al. (2005)} and the antioxidant activity percentage was calculated using the following equation:

% of the reduction of lipid peroxidation = (A_c - A_m)/A_c *100

where A_c is the witness’ absorbance (distilled water) and A_m is the sample’s absorbance (^{Ruiz-Martínez et al., 2011}).

HPLC (high pressure liquid chromatography)

The phenolic profile of the methanolic and aqueous extracts -taken from the wood and bark of the three oaks used in the antioxidant activity- was evaluated according to the methodology proposed by ^{Ruiz-Martínez et al. (2011)}, using a HPLC equipment (Varian Pro-Star 330, model 0128). The 10-μL samples were injected into a varian pursuit Xrs C₁₈ column (150 x 4.6 mm). The standard solutions were cinnamic acid, chlorogenic acid, coumaric acid, hydrocinnamic acid, catechin, gallic acid, methyl gallate, quercetin, pyrogallol, and resorcinol.

Statistical analysis

All the analyses were carried out in triplicate and the results included the average ± standard deviation.

Content of extractives

The total content of compounds extracted from the tree oak species was classified in ascending order as follows: sapwood > heartwood > bark. Their values varied from 8.72 % (sapwood, Q. candicans) to 19.39 % (bark, Q. rugosa) (Table 1). According to ^{Fengel and Wegener (1984}), bark had a great content of extractives and their concentration in heartwood is greater than in sapwood. The findings of our study match those results. With regard to the solvents used, methanol had the highest solubility; polyphenolic substances are extracted with high polarity solvents (such as alcohols and water), and these phenolic compounds show good antioxidant activity (^{Salazar et al., 2008}; ^{Wong-Paz et al., 2015a}). The overall quantity of compounds extracted from the heartwood of Q. candicans is similar to the percentage (10.2 %) obtained from the same species by ^{Rutiaga et al. (2000)}. The overall output of extractives in Q. laurina in this study was higher than the percentage reported for sapwood (5.29 %) and heartwood (5.35 %), while bark had a slightly lower percentage than the one reported for the inner bark (14.9 %) of the same oak species (^{Ruiz-Aquino et al., 2015}). The overall content of compounds extracted from Q. laurina in this study was higher than the content obtained from the sapwood (4.91 %) and heartwood (5.02 %) of the same oak species, at the State of Puebla, by ^{Honorato and Hernández (1998)}. The variation of extract percentage in timber-yielding species does not only depend on the solvents that are used, but also on the species itself. Additionally, environmental and genetic factors, the age of the tree, weather conditions, available nutrients, and the time when the cut is made can influence the result (^{Hillis, 1971}).

Inhibition of lipid oxidation

Inhibiting the extracts against the perodixation of the linoleic acid caused by FeCl₂ did not modify the antioxidant activity of any of the tissues of the three oaks, regardless of the solvent used to extract them (Table 2). The methanolic extract taken from the sapwood of Q. rugosa had the highest percentage of antioxidant activity (94.09 %). In contrast, the methanolic extract taken from the sapwood of Q. laurina had the lowest percentage (42.54 %). The inhibition against the peroxidation of the linoleic acid from the methanolic extract taken from the sapwood of Q. rugosa had a similar activity to gallic acid (95 %), when the same technique was applied (^{Sánchez-Aldama et al., 2013}). The antioxidant activity of the aqueous extracts taken from the sapwood and bark of the three oaks studied in our research is greater than the 65 % (average) obtained from ethanol-aqueous extracts of plants located in semiarid regions of Mexico (Flourensia cernua, Eucalyptus camaldulensis, and Turnera diffusa), when the same methodology is used (^{Wong-Paz et al., 2015a}). The inhibition values of the methanolic extracts taken from the bark of Q. laurina and from the sapwood of Q. rugosa are higher than the 76.86 % of the ethanolic extract taken from Laurus nobilis (^{Muñiz-Márquez et al., 2014}). The number of phenolic compounds in the extracts correlates with their antioxidant activity (^{Wong-Paz et al., 2015b}) which confirms that the variation of antioxidant activity depends on the polarity of the solvents used and on the phenolic compounds that contain the extracts from each plant.

HPLC analysis

The compounds identified by the qualitative HPLC analysis of the methanolic and aqueous extracts are shown in Table 3. The compound most frequently found in the sapwood, heartwood, and bark of the three oaks was chlorogenic acid which is a powerful antioxidant compound (^{Kamiyama et al., 2015}). Additionally, it is the main component of the methanolic and ethanolic extracts found in the plants used in traditional Chinese medicine (^{Wu, 2007}). Methanolic extracts taken from the sapwood of Q. laurina and Q. rugosa have the highest content of phenolic compounds (six compounds each); resorcinol was frequently found in the methanolic extracts taken from heartwood of the three oak species (Table 3), and it is used as a chemical intermediate for the synthesis of pharmaceutical products (^{Ruiz-Martínez et al., 2011}). Gallic acid -a chemical compound with powerful antioxidant, antiviral, antibacterial, and anticarcinogenic properties- was identified in the wood of Q. rugosa (^{Lekha and Lonsane, 1997}; ^{Shahrzard et al., 2001}; ^{Ruiz-Martínez et al., 2011}). Catechin contributes to the antimicrobial and antioxidant properties which are important to prevent such pathologies as diabetes (^{Ruiz-Martínez et al., 2011}; ^{Alam et al., 2014}; ^{Huang et al., 2015}). This compound was detected in the methanolic extract taken from the bark of Q. candicans, as well as from the sapwood and bark of Q. laurina. Studies about other oak species have identified gallic acid and catechin in the acetone-aqueous extract (70 %) taken from the bark of Quercus sideroxyla (^{Rosales-Castro et al., 2012}), while coumaric acid, gallic acid, catechin, and chlorogenic acid have been identified in infusions made from the leaves of Q. resinosa (^{Rocha-Guzmán et al., 2009}). In both cases, they were detected through a HPLC analysis.

Methyl gallate was identified in the sapwood and bark of Q. laurina and this compound has a strong antimicrobial and antioxidant activity (^{Choi et al., 2009}; ^{Cheng et al., 2009}). Quercetin is a powerful antioxidant, antihistamine, anti-inflammatory, and antidiabetic agent (^{Ruiz-Martínez et al., 2011}; ^{Alam et al., 2014}). This phenol was found in the methanolic extract taken from the heartwood of Q. rugosa. Using an IR analysis, ^{Kim et al. (2008)} found methyl gallate and quercetin in the methanolic extract taken from the trunk of this species. Cinnamic acid was identified in the methanolic extracts taken from the bark of Q. candicans and from the heartwood of Q. rugosa; this phenol is a powerful antimicrobial agent, it inhibits certain fungi species and is responsible for antitubercular activity (^{Guzmán, 2014}). Based on this discussion, wood and bark extracts from the three oaks are certainly rich in phenolic compounds with a powerful antioxidant activity; besides, they might prevent and inhibit certain human pathologies. Nevertheless, further studies are necessary to determine conditions for a better use.