Abstract
Methylated fatty acid (FA) compounds in the heartwood and bark of some softwood species, specifically Pinus sylvestris, Abies alba, Picea abies, and Larix decidua, grown in the Czech Republic were evaluated. Strong H2SO4 was used for methylation of the lipids. The highest content of lipid was found in P. abies bark (40.132 mg/g o.d. sample), and the lowest content was in A. alba wood (11.027 mg/g o.d. sample). The highest concentration of FAs was observed in L. decidua bark. The highest percentages of FAs in wood of P. sylvestris were arachidic acid and oleic acid. In bark, the highest percentages of FAs were stearic acid, palmitic acid, and oleic acid. The FAs with the highest concentrations in A. alba wood were arachidic acid, palmitic acid, pentadecanoic acid, and margarinic, and those in bark were behenic acid, lignoceric acid, and arachidic acid. P. abies wood FAs showed arachidic acid, palmitic acid, and margarinic acid, and the bark contained lignoceric acid and arachidic acid. The FAs of L. decidua wood were arachidic acid, palmitic acid, and stearic acid, and in bark they were pentacosylic acid, docosahexaenoic acid (DHA), lignoceric acid, arachidic acid, and behenic acid. The lack of typically dominant unsaturated fatty acids (e.g. 18:1, 18:2), compared to literature values were attributed to the application of strong acid for the hydrolysis.
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Methylated Fatty Acids from Heartwood and Bark of Pinus sylvestris, Abies alba, Picea abies, and Larix decidua: Effect of Strong Acid Treatment
Mohamed Z. M. Salem,a,* Ramadan A. Nasser,a,b Aleš Zeidler,c Hosam O. Elansary,d,e Ibrahim M. Aref,b Martin Böhm,f Hayssam M. Ali,g,h and Abdalla I. Ahmed b
Methylated fatty acid (FA) compounds in the heartwood and bark of some softwood species, specifically Pinus sylvestris, Abies alba, Picea abies, and Larix decidua, grown in the Czech Republic were evaluated. Strong H2SO4 was used for methylation of the lipids. The highest content of lipid was found in P. abies bark (40.132 mg/g o.d. sample), and the lowest content was in A. alba wood (11.027 mg/g o.d. sample). The highest concentration of FAs was observed in L. decidua bark. The highest percentages of FAs in wood of P. sylvestris were arachidic acid and oleic acid. In bark, the highest percentages of FAs were stearic acid, palmitic acid, and oleic acid. The FAs with the highest concentrations in A. alba wood were arachidic acid, palmitic acid, pentadecanoic acid, and margarinic, and those in bark were behenic acid, lignoceric acid, and arachidic acid. P. abies wood FAs showed arachidic acid, palmitic acid, and margarinic acid, and the bark contained lignoceric acid and arachidic acid. The FAs of L. decidua wood were arachidic acid, palmitic acid, and stearic acid, and in bark they were pentacosylic acid, docosahexaenoic acid (DHA), lignoceric acid, arachidic acid, and behenic acid. The lack of typically dominant unsaturated fatty acids (e.g. 18:1, 18:2), compared to literature values were attributed to the application of strong acid for the hydrolysis.
Keywords: Chemical composition; Fatty acids; Strong acid; Wood; Bark; Pinus sylvestris; Abies alba; Picea abies; Larix decidua
Contact information: a: Forestry and Wood Technology Department, Faculty of Agriculture (EL–Shatby), Alexandria University, Egypt; b: Range and Forestry Applied Research Unit, Plant Production Dept., Food and Agricultural Sciences Collage, King Saud University, Saudi Arabia; c: Department of Wood Processing, Faculty of Forestry and Wood Sciences, Czech University of Life Sciences Prague, Prague, Czech Republic; d: Department of Floriculture, Ornamental Horticulture and Garden Design, Faculty of Agriculture (EL-Shatby), Alexandria University, Alexandria, Egypt; e: Biodiversity Institute of Ontario, University of Guelph, Guelph, ON, Canada; f: Department of Wood Products and Wood Constructions, Faculty of Forestry and Wood Sciences, Czech University of Life Sciences Prague, Prague, Czech Republic; g: Timber Trees Research Department, Sabahia Horticulture Research Station, Horticulture Research Institute, Agriculture Research Center; Alexandria, Egypt; h: Botany and Microbiology Department, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia;*Corresponding author: zidan_forest@yahoo.com
INTRODUCTION
Biomass, mainly wood, is a great source for phytochemicals, which are useful in many areas of industries. The chemical composition of various softwood species has been the subject of numerous studies focused on North American and Central European species (Kubeczka and Schultze 1987; Sjödin et al. 1993; Hennig et al. 1994). Softwood species including pines (Pinus), spruces (Picea), larches (Larix), and Pseudotsuga are prolific resin producers and have well-established systems of horizontal and vertical ducts filled with resin (Parham and Gray 1984; Simoneit et al. 2000). The Pinaceae family contains 12 genera: Abies, Cathaya, Cedrus, Keteleeria, Larix, Nothotsuga, Picea, Pinus, Pseudolarix, Pseudotsuga, Tsuga, and Hesperopeuce; where Pinus is the largest, Abies ranked second, and Picea third (Page 1990; Wolff et al. 2001, 2002).
Fatty acids (FAs) grouped into saturated and unsaturated forms have been studied in many published works. The FAs and sterols, in contrast, are nutrients for attacking fungi; therefore, they reduce the durability of the wood (Fries et al. 2000). However, in the study of Salem et al. (2014a,b), some antifungal properties were found for the FAs. Additionally, the unsaturated FAs with additional reactions can form saturated FAs, which should decrease the relative amount of unsaturated FAs (Sjöström 1981).
Conifers are known for the presence of unusual FAs with the first site of unsaturation at the fifth carbon atom (∆5-UPIFAs) and also for cis (Z) configuration, such as cis-5,9-octadecadienoic (taxoleic), cis-5,9,12-octadecatrienoic (pinolenic), cis-5,11-octadecadienoic (ephedrenic), cis-5,11-eicosadienoic (keteleeronic), cis-5,11,14-eicosatrienoic (sciadonic), cis-5,9,12,15-eicosatetraenoic (coniferonic), and cis-5,11,14,17-eicosatetraenoic (juniperonic) acids (Takagi and Itabashi 1982; Piispanen and Saranpää 2002; Wolff et al. 2002; Lísa et al. 2007).
It has been reported previously that fatty acids are partially deposited in the cell lumen and pit membranes, where they decrease the permeability of wood during heartwood formation (Saranpää 1990). Moreover, secondary substances synthesized during heartwood formation must originate primarily from stored carbohydrates (Fischer and Höll 1992). The FAs (oleic, linoleic, α-linolenic, ∆5-taxoleic, pinolenic, and coniferonic acid) are valuable components in the family Pinaceae (Bağci and Karaağaçli 2004). FAs such as cis-5,9-octadecadienoic (taxoleic), cis-5,9,12-octadecatrienoic (pinolenic), cis-5,11-eicosadienoic (keteleeronic), and cis-5,11,14-eicosatrienoic acids (sciadonic) can be found in the coniferous trees (Lísa et al. 2007).
The free FA concentrations were negligible in the outer sapwood of Scots pine (Pinus sylvestris L.), but ranged between 5 and 18 mg/g (dm) in the heartwood (Piispanen and Saranpää 2002). The dominant FAs in all fractions analyzed by gas-liquid chromatography (GLC) sapwood were 16:0, 18:0, 18:1, and 18:2. 18:1 and 18:2 formed about 70% of the total triacylglycerol fatty acids (Saranpää and Nyberg 1987).
Most preceding studies have focused on specific components in the bark of Norway spruce (Picea abies (L.) H. Karst.), such as lipophilic extractives (Norin and Winell 1972; Ånäs et al. 1983). Larix species are widely distributed across Europe, North America, and Asia. The European Larch (Larix decidua Mill.) can grow up to 35 m high and is native to the higher regions of the Alps, Sudetes, and Carpathian mountains. Silver fir (Abies alba (Mill.)) is the most common tree species in Central Europe; therefore, it is important in wood production and is also very economically and environmentally significant (Ficko et al. 2011).
Most studies on the FA concentrations of softwood species have focused on the presence of FAs in seeds and leaves. Also, more details about the distribution and identification of FA composition of Pinaceae as taxonomic markers were reviewed by Wolff et al. (2001). As part of an extensive phytochemical analysis of some softwood species growing in the Czech Republic, the present work reports the authors’ first investigation toward the chemical constituents of the methylated fatty acids obtained from the heartwood and bark of Pinus sylvestris, Abies alba, Picea abies, and Larix deciduaas affected by strong acidic hydrolysis.
EXPERIMENTAL
Materials
Preparation of heartwood and bark samples
Freshly cut wood discs containing bark samples of Scots pine (Pinus sylvestris L.), European silver fir(Abies alba Mill.), Norway spruce (Picea abies (L.) H. Karst.), and European Larch (Larix decidua Mill. subsp. decidua), used in the present study were obtained at the end of 2014, by the Department of Wood Processing, Faculty of Forestry and Wood Sciences, Czech University of Life Sciences. The ages of the trees were 33, 34, 28, and 41 years for P. sylvestris, A. alba, P. abies, and L. decidua, respectively. Then, the discs were air-dried under shade at room temperature at the Laboratory of Wood Technology (Forestry and Wood Technology Department, Faculty of Agriculture (EL–Shatby, Alexandria University, Egypt). Heartwood samples were prepared with a particle size of 0.4 to 0.6 mm. Bark samples had a particle size of 0.1 to 0.2 mm.
Methods
Lipid extraction
Ten grams from each source of heartwood and bark were weighed out into a conical flask containing 10 mL of concentrated HCl and then boiled in a water bath until the entire sample had been dissolved. Then, 30 mL of diethyl ether was added to the solution to extract the fats through shaking. The extract was collected into a weighed flask after allowing the layers to separate. The extraction was repeated three more times and the solvent was distilled off. Then the fats were dried at 100 °C, cooled, and weighed using a balance (Kirk and Sawyer 1991). The lipid weighted based on one gram oven dry weight of the sample (mg lipid/gram o.d. material).
Methylation of lipid
Methylation of the lipids was carried out by weighing a sample of 50 mg of lipid from each source in a tube. The following chemicals were then added to each tube; 50 mL of the mixture from 1 mL concentrated sulfuric acid, 100 mL of methanol, and 2 mL of benzene. After the tube was sealed completely, the samples were placed in a water bath at 90 °C for 90 min. The tube was then cooled and 8 mL of water with 5 mL of petroleum ether was added. Then, the tube was thoroughly shaken and the ethereal layer was separated out and evaporated (Agoramoorthy et al. 2007). After methylation of the fatty acids (FAs), they were subjected to gas chromatography (GC) analysis.
Table 1. GC Condition for Analysis of Methylated Fatty Acids*
* Conditions used previously by Salem et al. (2014)
The compositions of the methylated FAs were identified by GC analysis using a HP (Hewlett Packard 6890 GC, USA) GC analyzer as shown in Table 1. This was done by matching their retention times with standard FAs (C4-C25) chromatographed under the same conditions (Mohamed and Awatif 1998).
Statistical Analysis
Analysis of variance (ANOVA) procedure (SAS version 8.2, 2001, USA) in a completely randomized design (CRD) was used to study the statistical differences among the studied parts of the tree species for the lipids and FAs concentrations. Fisher’s least significant difference (LSD) at a 5% level of probability was used to measure the differences among the mean values.
RESULTS AND DISCUSSION
Lipid and Fatty Acids Concentrations
Table 2 presents the concentration of lipids and fatty acids (FAs) found in the heartwood and bark of P. sylvestris, A. alba, P. abies, and L. decidua. Among the studied species, the highest content of lipid was found in the P. abies bark (40.132 mg/g o.d. sample), followed by P. sylvestris heartwood (31.649 mg/g o.d. sample), and the lowest content was present in L. decidua heartwood (11.548 mg/g o.d. sample) as well as in A. alba heartwood (11.027 mg/g o.d. sample). On the other hand, the highest concentration of FAs was observed in L. decidua bark (175.03 mg/g of lipid) followed by A. alba bark (155.695 mg/g of lipid). The lowest concentration observed in heartwood and bark of P. sylvestris was 7.153 and 6.968 mg/g of lipid, respectively. In other studies, the concentrations of fats were 10 times higher in Scots pine than in Norway spruce, grown in Finland, and the sapwood contained more lipophilic extractives than heartwood (Ekman 1979).
Table 2. Concentration of Lipid and Fatty Acids in Heartwood and Bark of the Studied Softwood Species
* Means with the same letters within the same column are not significantly different according to LSD0.05
Also, large differences in the FA compositions of different lipid classes in P. sylvestris and P. abieswoods have been demonstrated previously (Assarsson and Åkerlund 1967). Other works reported that the concentrations of neutral lipids and free FAs in the wood of Norway spruce were lower than the concentrations reported for Scots pine (Fischer and Höll 1992; Saranpää and Piispanen 1994).
The variation in the concentration between previous and present works could be related to reasons including that the amount of live cells in the xylem of fertilized trees was higher than in non-treated control trees, a high growth rate, tree vigor, nitrogen concentration in the stem wood, the carbon/nutrient balance hypothesis, and geographic and environmental locations (Kramer and Kozlowski 1979; Bryant et al. 1983; Stockfors and Linder 1998).
Methylated Fatty Acids in Heartwood and Bark of P. sylvestris, A. alba, P. abies, and L. decidua
Table 3 summarizes the methylated FAs and their concentration reported in the heartwood and bark of P. sylvestris, A. alba, P. abies, and L. decidua.
Fatty Acids in Pinus sylvestris Heartwood and Bark
The highest percentages of FAs in the heartwood of P. sylvestris were arachidic acid (55.100%), oleic acid (10.51%), tricosylic acid (9.862%), palmitic acid (8.816%), stearic acid (6.784%), linoleic acid (4.014%), and pentadecanoic acid (4.125%). The bark contained the following FAs; stearic acid (54.112%), palmitic acid (16.292%), oleic acid (14:500%), and pentadecanoic acid (7.973%).
According to the study of Saranpää and Nyberg (1987), the resin acids and FAs occur at low concentrations in the sapwood of P. sylvestris. In previous published works, the most abundant FAs of triacylglycerols were found to be oleic (18:1), linoleic (18:2ɷ6, 18:2∆5,9), linolenic (pinolenic, 18:3∆5,9,12 and 18:3ɷ3), and eicosatrienoic acid (20:3∆5,11,14 and 20:3ɷ6), also, the concentration of linoleic acid comprised 39% to 46% of the triacylglycerol fatty acids (Piispanen and Saranpää 2002) of P. sylvestris grown in Northern and Southern Finland. It was reported that the free FAs in heartwood are formed by the hydrolysis of sapwood triacylglycerols of Scots pine (Saranpää and Nyberg 1987). In another study, oleic and linoleic acid together comprised about 70% of the total triacylglycerol FAs, with linoleic acid making the major contribution of 39% to 46% (Saranpää and Nyberg 1987).
Fischer and Höll (1992) reported that oleic acid is the principal FA of triacylglycerols in stems of Scots pine trees grown in southern Germany. Sapwood fractions of Scots pine analyzed by GC observed the most important FAs as being 16:0 (palmitic acid), 18:0 (stearic acid), 18:1 (oleic acid), 18:2 (linoleic acid, the dominant fatty acid in all fractions), 18:3, and 20:3 (Saranpää and Nyberg 1987). In contrast, noticeable amounts of free FAs were present only in the heartwood.
Fatty acids in Abies alba Heartwood and Bark
The heartwood of A. alba was found to contain the following FAs: arachidic acid (37.417%), palmitic acid (22.019%), pentadecanoic acid (15.139%), margarinic acid (12.234%), stearic acid (5.831%), myristic acid (5.104%), and erucic acid (cis-13) (2.137%). The bark was found to contain behenic acid (41.777%), lignoceric acid (21.086%), arachidic acid (18.016%), tricosylic acid (4.522%), stearic acid (3.488%), heptadecenoic acid (2.548%), undecanoic acid (2.204%), and cis-10-pentadecenoic acid (2.066%).
Table 3. Concentration of the Identified Methyl Ester of Fatty Acids
Fatty Acids in Picea abies Heartwood and Bark
GC analysis of methylated FAs from heartwood of P. abies reported the following main FAs compounds: arachidic acid (58.319%), palmitic acid (11.197%), margarinic acid (8.135%), pentadecanoic acid (6.147%), and stearic acid (4.693%). In bark, the following FAs were found: lignoceric acid (29.513%), arachidic acid (24.809%), cis-13,16-docosadienoic acid (3.051%), pentacosylic acid (17.586%), cis-5,8,11,14,17-eicosapentaenoic acid (EPA) (3.633%), behenic acid (3.001%), stearic acid (2.742%), palmitic acid (2.629%), pentadecanoic acid (2.424%), and myristic acid (2.302%). FAs ranged from 1.5 to 0.27 mg/g of dry bark (Krogell et al. 2012) and from 0.009 to 0.011% DW of wood (Anttonen et al. 2002).
The free FAs have been reported as 0.6 to 1.3 mg/g of o.d. wood (Bertaud and Holmbom 2004). The spruce wood oleoresin also contains n-alkanes in homologous series ranging from C11 to C33, with C22 to C27 as the main constituents (Assarsson and Åkerlund 1967). In P. abies, pinolenic (Ekman 1980), taxoleic, coniferonic, sciadonic and juniperonic acids are found in many species of conifers (Jamieson and Reid 1972). Also, polyunsaturated acids (19:1, 5,9-19:2, 9,12-19:2, and 5,9,12-19:3 acids) have been detected in P. abies wood extracts (Ekman 1980).
Fatty Acids in Larix decidua Heartwood and Bark
The FAs components present in the heartwood of L. decidua were arachidic acid (66.744%), palmitic acid (9.340%), stearic acid (6.974%), margarinic acid (6.061%), and pentadecanoic acid (5.268%). The major FAs in bark were pentacosylic acid (22.759%), docosahexaenoic acid (DHA) (21.943%), lignoceric acid (14.413%), arachidic acid (13.347%), behenic acid (11.169%), cis-5,8,11,14,17-eicosapentaenoic acid (EPA) (6.753%), palmitic acid (2.441%), and stearic acid (2.414%).
All ∆5-UPIFAs (5,9-18:2 and 5,9,12-18:3 acids) have been characterized in the leaves of many Picea and Larix species (Jamieson and Reid 1972) and also in P. abies wood (Holmbom and Ekman 1978; Ekman 1980). It was reported that in most of the softwood species, the C18 ∆5-olefinic acids (5,9-18:2 and 5,9,12-18:3 acids) are present in considerably higher amounts than the C20 ∆5-olefinic acids (5,11-20:2 and 5,11,14-20:3 acids) (Wolff et al. 2001). Arachidonic acid was found in the cambium zone of L. sibirica (Groenewald and van der Westhuizen 1997), although thorough studies of P. abies wood extracts did not mention such an occurrence (Holmbom and Ekman 1978; Ekman 1980).
CONCLUSIONS
- The highest percentages of FAs in the heartwood of P. sylvestris were arachidic acid, oleic acid, tricosylic acid, and palmitic acid. In bark, stearic acid, palmitic acid, and oleic acid were the most abundant.
- The FAs most prominent in the heartwood of A. alba were arachidic acid, palmitic acid, pentadecanoic acid, and margarinic acid. In the bark of A. alba, the most prominent FAs presented were behenic acid, lignoceric acid, and arachidic acid.
- The heartwood of P. abies exhibited the following main FA compounds: arachidic acid, palmitic acid, and margarinic acid. In the bark of P. abies, the main FA compounds seen were lignoceric acid and arachidic acid.
- FAs in the heartwood of L. decidua were arachidic acid, palmitic acid, stearic acid, and margarinic acid. FAs in the bark of L. decidua were pentacosylic acid, docosahexaenoic acid (DHA), lignoceric acid, arachidic acid, behenic acid, and cis-5,8,11,14,17-eicosapentaenoic acid (EPA).
- The present results indicated changes in the heartwood and bark FAs from P. sylvestris, A. alba, P. abies, and L. decidua. Some of these changes may influence the suitability of such wood for different end-use purposes.
- The lack of typically dominant unsaturated fatty acids could be related to the use of strong acids in the present study.
ACKNOWLEDGMENTS
The authors would like to extend their sincerest appreciation to the Deanship of Scientific Research at King Saud University for the funding of this research through the Research Group Project No. RGP-VPP-226.
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Article submitted: May 20, 2015; Peer review completed: September 19, 2015; Revised version received and accepted: September 21, 2015; Published: September 30, 2015.
DOI: 10.15376/biores.10.4.7715-7724