In vitro bioactivity and antimicrobial activity of Picea abies and Larix decidua wood and bark extracts

Abstract

Full Article

In vitro Bioactivity and Antimicrobial Activity of Picea abies and Larix decidua Wood and Bark Extracts

Mohamed Z. M. Salem,^a,* Hosam O. Elansary,^b Amr A. Elkelish,^c Aleš Zeidler,^d Hayssam M. Ali,^e,f Mervat EL-Hefny,^b and Kowiyou Yessoufou ^g

Picea abies and Larix decidua were subjected to GC/MS analyses, and antimicrobial (fungi and bacteria) assays of their stem wood and bark extracts were investigated. L. decidua bark extract exhibited the highest antifungal and antibacterial activities against the microorganisms that were screened. The microbes Penecillium ochrochloron and Aspergillus ochraceus were the most sensitive to the extracts, whereas Candida albicans was the most resistant fungus. L. deciduawood and bark did not exhibit much variation in their antibacterial activities, except against Micrococcus flavus and Pseudomonas aeruginosa. The bacterium most sensitive to the extracts was Escherichia coli, whereas the most resistant was M. flavus. 13-epimanool and α-cedrol were the main components of P. abies wood extract. The main components in its bark were abietic acid, astringin, dehydroabietic acid, and α-terpineol. The main chemical compounds in L. deciduawood extract were abietic acid, oleanolic acid, duvatrienediol, and larixol. The main chemical compounds in its bark were (-)-2,9–dihydroxyverrucosane and larixol. The study revealed that P. abies and L. decidua stem wood and bark extracts contain several compounds that have antimicrobial activities towards diverse human pathogenic, food, and agricultural microbes. These results might guide in future searches for novel natural products with chemotherapeutic uses.

Keywords: Picea abies; Larix decidua; Antifungal activity; Antibacterial activity; GC/MS

Contact information: a: Department of Forestry and Wood Technology, Faculty of Agriculture (El-Shatby), Alexandria University, Alexandria, Egypt; b: Molecular Biology Laboratory, Floriculture, Ornamental Horticulture and Garden Design Department, Faculty of Agriculture (El-Shatby), Alexandria University, Alexandria, Egypt; c: Department of Botany, Faculty of Science, Suez Canal University, Ismailia 41522, Egypt; d: Department of Wood Processing, Faculty of Forestry and Wood Sciences, Czech University of Life Sciences Prague, Prague, Czech Republic; e: Department of Botany and Microbiology, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia; f: Department of Timber Trees Research, Sabahia Horticulture Research Station, Horticulture Research Institute, Agriculture Research Center, Alexandria, Egypt; g: Department of Environmental Sciences, University of South Africa, Florida campus, Florida 1710, South Africa;

* Corresponding author: zidan_forest@yahoo.com; mohamed-salim@alexu.edu.eg

INTRODUCTION

Forestry residues such as tree barks and branches are important but underexplored supplies of biologically or pharmaceutically active compounds (Liimatainen et al. 2012; Si et al. 2012; Patinha et al. 2013). Tree bark, for example, provides not only a physical protective barrier for plants, but also an important chemical defense mediated through the secondary compounds they produce (Horsfall 1980; Merrill 1992; Alfredsen et al. 2008). Numerous studies recently conducted on bark extracts focus on tropical tree species (da Silva et al. 2013; Kusumoto et al.2014; Salem et al. 2015a; Yessoufou et al. 2015). In the present study, we shift the focus onto Central European softwood species in the Pinaceae family (Salem et al. 2015b). This family is a taxonomic group of high economic importance in the production of paper, timber, construction materials, chemicals, and essential oils used for perfume or pharmaceutical and aromatherapy purposes (Salem et al. 2015b).

Previous studies have been dedicated to analyzing the chemical composition of the essential oils and the antimicrobial activities of several parts of Pinaceae members (needles, branches, cones, seeds, and bark) (Grassmann et al. 2003; Nikolić et al. 2007; Tumen et al. 2010; Bier et al. 2016; Fattahi et al. 2016; Salem et al. 2016). Nevertheless, the majority of studies regarding the chemical composition of softwood species have related to North American and Central European species (Hennig et al. 1994; Danneyrolles et al. 2016; Greenberg et al. 2016; Krieg et al. 2016).

European larch (Larix decidua L.) is a deciduous tree that grows to 35 m and is indigenous to the Alps, the Sudetes, and the Carpathian mountains (von Bruchhausen et al. 1993; Pferschy-Wenzig et al. 2008). Larch wood is strong, water-resistant, and durable, but especially bendable in thin strips. The heartwood is extremely weather-proof due to its excessive resin content and hard machinability, and it is usually used as building timber (Pferschy-Wenzig et al. 2008). Larch sawdust is principally utilized in the production of pellet fuels, and the wood contains phenolic compounds such as lignans and flavonols (mainly dihydroquercetin and dihydrokaempferol) (von Bruchhausen et al. 1993). These compounds possess antioxidant and anti-inflammatory effects (Kolhir et al. 1996; Pietarinen et al. 2006). Polysaccharides such as arabinogalactan (also found in European larch) have been approved by the U.S. FDA as a source of fibers, have immunostimulant effects, and may be used in cancer therapy (Kelly 1999). Turpentine in L. decidua, often known as Venice turpentine (Pferschy-Wenzig et al. 2008), is made up of 15% essential oil, 50 to 65% resin acids, and approximately 15% non-saponifiable resin. The principal component of the resin fraction is the labdane type diterpene larixyl acetate (Kolhir et al. 1996). Larixol and larixyl acetate are distinctive compounds for larch resin, as they have been specifically found in L. decidua and L. gmelinii.

Norway spruce (Picea abies) is indigenous to the European Alps and ranges north towards Scandinavia and North Russia (Salem et al. 2013; Bianchi et al. 2014). P. abies wood is robust, soft, straight- and fine-grained, and simply worked (Cope et al. 2002). It is commonly used for building, pulp, furniture, and musical instrument manufacturing (Cope et al. 2002). The older trees are normally heavy with algae and possess shallow rounded scales which can be easily shed (Cope et al. 2002). In P. abies cells, the different soluble glycoside-bound types of phenolic acids make up ~ 85% of the overall content of the methanol extract, accompanied by the methanol-insoluble cell wall-bound phenolic esters (7 to 8%) (Malá et al. 2011). Some soluble methanol esters and free phenolic acids are very low, comprising ~2 and 4 to 5% of the entire phenolic content, respectively (Malá et al. 2011).

Additional information regarding the chemical structure of the bark tannins from P. abies and L. decidua is needed (Bianchi et al. 2014). Spruce bark tannins are recognized as procyanidins, with few incidences of prodelphinidins, having a mean polymerization degree of about 4.6 (Matthews et al. 1997). Similar results were reported for root bark (Pan and Lundgren 1995) and needle extracts (Behrens et al. 2003). Lignans and stilbenes, as well as their derivatives, were found in the oligomeric structure of condensed tannins of spruce (Steynberg et al. 1983; Bianchi et al.2014). However, no characterization of condensed tannins from phloem or xylem tissues has yet been reported.

In the present study, the chemical composition of wood and bark extracts of P. abies and L. decidua were identified using GC-MS, and their biological activities on diverse bacteria, and fungi were investigated to elucidate the biochemical composition and the antifungal and antibacterial potentials for human health benefits, agricultural pest control, and wood industry enhancement.

EXPERIMENTAL

Plant Materials

Wood and bark samples of Larix decidua and Picea abies were supplied by the Department of Wood Processing (Czech University of Life Sciences, Czech Republic, February 2015), and vouchered (No. Zidan00971 to Zidan00974) at the Division of Forestry and Wood Technology, Alexandria University. The laboratory work was completed in March 2016. The ages of the trees were 28 and 41 years for P. abies and L. decidua, respectively. Heartwood and bark samples were air-dried at the laboratory conditions, and then the samples were prepared with a particle size of 0.4 to 0.6 mm.

Chemicals and cell cultures

Analytical/HPLC grade chemicals were bought from Sigma-Aldrich, Alexandria, Egypt. Fungi and bacteria were supplied by the Department of Plant Pathology and Department of Floriculture (Faculty of Agriculture, Alexandria University, Egypt).

Methods

Preparation of extracts

The extracts were prepared from ground air-dried (40 to 60 mesh) wood and bark samples, and extracted three times using 95% methanol over a water bath for one day at room temperature. All extracts were combined and then evaporated until dry in a vacuum at 40 °C. The yield of methanol extracts for L. decidua was 6.42% (heartwood), and 10.36% (bark) and for P. abies was 7.2% (heartwood), and 4.2% (bark), according to oven dry weight.

GC-MS analysis of extracts

The chemical composition of the wood and bark extracts of L. decidua and P. abies were analyzed using a Trace GC Ultra-ISQ mass spectrometer (Thermo Scientific, USA) with a TG-5MS capillary column (30 m x 0.25 mm x 0.25 µm film thickness) apparatus at Atomic and Molecular Physics Unit, Experimental Nuclear Physics Department, Nuclear Research Centre, Egyptian Atomic Energy Authority, Inshas, Cairo, Egypt. Helium was used as the carrier gas (flow rate of 1 mL/min), and the oven temperature program was set as follows: 45 °C to 165 °C (4 °C/min) and 165 °C to 180 °C (15 °C/min) with a post run (off) at 180 °C. Samples (1 μL) were injected at 250 °C using a split/split-less injector (50:1 split ratio) and a splitless mode flow of 10 mL/min. The solvent delay was 2 min, and diluted samples of 1 µL were injected automatically using an Auto-sampler AS3000 coupled with GC in the split mode. EI mass spectra were collected at 70 eV ionization voltages in the range of 40 to 550 m/z in full scan mode. The ion source and transfer line temperatures were set at 200 °C and 250 °C, respectively. The components were identified by comparing their retention times and mass spectra with those of the Wiley 09, mainlib, replib, and NIST 11 mass spectral database (Adams 2007).

Antifungal activities

The antifungal activities of wood and bark extracts were examined for several fungi, including Penicillium funiculosum (ATCC 56755), P. ochrochloron (ATCC 48663), Aspergillus niger(ATCC 6275), A. flavus (ATCC 9643), A. ochraceus (ATCC 12066), and Candida albicans(ATCC 12066). The cultures were renewed monthly and stored at 4 °C. The microdilution method (Espinel-Ingroff 2001) was used to determine the minimum inhibitory (MIC) and minimum fungicidal (MFC) concentrations using a spore suspension concentration of (1.0 × 10⁵) dilutions in 96-well microtiter plates. Wood and bark extracts and isolated compounds were diluted to the desired concentrations in microplates containing Malt medium broth mixed with inoculum. The microplates were incubated at 28 °C for 72 h on a rotary shaker.

The lowest concentration that inhibits fungi growth at the binocular microscope level was defined as the MIC. The MFC was defined as the minimum concentration exhibiting no visible growth, indicating a 99.5% killing of the original inoculum. Serial sub-cultivations (2 µL) of wood and bark extracts incubated at 28 °C for 72 h in microtiter plates containing 100 µL of broth and inoculum were used to calculate the MIC. Positive controls were used, including fluconazole (FLZ) and ketoconazole (KLZ) (1 to 3500 µg/mL). The experiments were performed in triplicate.

Antibacterial activities

Gram-positive and Gram-negative bacteria were used for analyses. The Gram-positive bacteria included Micrococcus flavus (ATCC 10240), Bacillus cereus (clinical isolate), Listeria monocytogenes (ATCC 19113), and Staphylococcus aureus (ATCC 6538). The Gram-negative bacteria were Pseudomonas aeruginosa (D s0432-1), Escherichia coli (ATCC 35210), Pectobacterium atrosepticum (ATCC 33260), Pectobacterium carotovorum subsp. carotovorum(ATCC 15713), and Dickeya solani (D s0432-1). The micro-dilution method (Espinel-Ingroff 2001) was used to determine the minimum inhibitory (MIC) and minimum bactericidal (MBC) concentrations. The concentration of the bacteria was adjusted to 1.0 × 10⁵ CFU/mL using sterile saline, and then stored at 4 °C. The wood and bark extracts were added to 100 μL Triptic Soy broth (TSB) containing a bacteria inoculum (1.0 × 10⁵ CFU per well) in a microtitre plate, then the minimum inhibitory concentrations (MICs) and the minimum bacterial concentrations (MBCs) were determined. The microplates were incubated at 37 °C for 24 h in a rotary shaker.

A serial sub-cultivation of 2 μL was placed in microtitre plates containing 100 μL of TSB for each well and incubated for 24 h to determine the MIC and MBC. The optical density was measured using a microplate manager at 655 nm. Experiments were completed in triplicate. DMSO (5%) and streptomycin + ampicillin (1 mg/mL) were used as negative and positive controls, respectively.

RESULTS AND DISCUSSION

Chemical Composition of Wood and Bark Extracts of Picea abies and Larix decidua

P. abies wood extract contained the following main compounds: 13-epimanool (12.48%), α-cedrol (10.60%), 2,6-di-t-butyloctahydroazulene-3a,8-diol (8.32%), astringin (7.68%), (Z,Z,Z)-9,12,15-octadecatrienoic acid-methyl ester (7.12%), and (1,5,5,8-tetramethylbicyclo[4.2.1]non-9-yl)acetic acid (6.07%) (Table 1). The main chemical compounds found in bark extract were abietic acid (26.80%), astringin (15.00%), dehydroabietic acid (5.90%), α-terpineol (4.67%), methyl sandaracopimarate (4.07%), and 9-desoxo-9-x-acetoxy-3,8,12-tri-o-acetylingol (3.79%) (Table 2).

Table 1. Suggested Chemical Composition of Methanol Extract of Picea abies Wood Analyzed Using GC/MS

Note: RT, retention time

Table 2 (Part 1). Suggested Chemical Composition of Methanol Extract of Picea abies BarkAnalyzed Using GC/MS

Table 2 (Part 2). Suggested Chemical Composition of Methanol Extract of Picea abies BarkAnalyzed Using GC/MS

Note: RT, retention time

Table 3. Suggested Chemical Composition of Methanol Extract of Larix decidua Wood Analyzed Using GC/MS

Note: RT, retention time

The main chemical composition of L. decidua wood extract was abietic acid (30.50%), oleanolic acid (15.80%), duvatrienediol (15.79%), larixol (14.55%), squalene (9.13%), and 13-epimanool (5.07%) (Table 3). The main chemicals in the methanol extract of L. decidua bark were (-)-2,9-dihydroxyverrucosane (17.48%), larixol (15.80%), nonacosane (11.03%), cholan-24-oic acid,3,12-dihydroxy-(3α,5α,12α)- (8.77%), decane (8.63%), and 5-hydroxy-2,3,3-trimethyl-2-(3-methyl-buta-1,3-dienyl)-cyclohexanone (5.95%) (Table 4).

Table 4. Suggested Chemical Composition of Methanol Extract of Larix decidua Bark Analyzed Using GC/MS

Note: RT, retention time

Previously, five benzoic acid derivatives (syringic acids, p-hydroxybenzoic, vanillic, and anisic), two cinnamic acid derivatives, p-coumaric, and ferulic acids were identified in P. abies cells (Metsämuuronen and Siren 2014). Native ferulic acid and p-hydroxybenzoic acid glucoside were found in root extracts (Münzenberger et al. 1990). Various stilbenes and stilbenes glucosides have been previously observed in different parts of P. abies (Metsämuuronen and Siren 2014). Isorhapontin and astringin are the major constitutive stilbenes glycosides in P. abies (Viiri et al.2001; Zeneli et al. 2006; Malá et al. 2011). Zeneli et al. (2006) found that astringin and isorhapontin represent 20.2% and 71.8% of sapwood phenolics, and 38.8% and 46.5% of bark phenolics in P. abies trees growing in Norway, respectively. The standard amount of extractable phenolic compound in P. abies wood is about 15% (w/w) (Metsämuuronen and Siren 2014), but higher values have also been reported up to 30% (w/w) (Eklund et al. 2004).

Wood phenolics also contain 5.1% piceid and bark phenolics, 7.7% piceid, and 0.4% piceatannol (Metsämuuronen and Siren 2014). These types of stilbenes aglycones and glucosides have previously been discovered in bark extracts (Pietarinen et al. 2006). Moreover, piceoside, piceatannol and its glucoside, and isorhapontin have been detected in P. abies roots (Münzenberger et al. 1990; Latva-Mäenpää et al. 2014). However, various reports did not find stilbenes in the hydrophilic knotwood extractives of P. abies (Willför et al. 2003). Prior research discovered that more than a half of the knotwood extracts are lignans, with the remaining mostly oligolignans (Metsämuuronen and Siren 2014). One of the most substantial lignans was hydroxymatairesinol (Willför et al. 2003; Pietarinen et al. 2006), which comprised around 70% of the total lignans (Willför et al. 2003).

Diterpene acids from conifers are well known to provide several bioactivities (Rauha et al. 2000; Keeling and Bohlmann 2006). Many abietane acids exhibit antimicrobial activity against a wide range of microorganisms. Dehydroabietic acid derivatives have been proved to act as antiulcer agents (Sepúlveda et al. 2005), and several abietane acids have shown cardiovascular effects. Furthermore, several side effects of diterpene acids, such as resin acids within pulp and paper mill effluents, are well known to be noxious to aquatic organisms (Rissanen et al. 2003; Kamaya et al.2005).

The oxidation products of resin acids appear to have a dermal allergenic potential (San Feliciano et al. 1993; Eriksson et al. 2004). Moreover, several studies have investigated the anti-inflammatory potential of diterpene acids. A combination of laevopimaric, abietic, neoabietic, palustric, and isopimaric acids along with triglycerides, have been prescribed for the external therapy of chronic diseases such as rheumatism and gout (Khare 2004). Abietic acid, which is also considered to be present in larch resin, has been identified as an inhibitor of soybean 5-lipoxygenase (Ulusu et al. 2002). The compound appears to inhibit PGE2 generation in lipopolysaccharide-treated macrophages in vitro, and prevent rat-paw and mouse-ear edema following oral or topical application in vivo (Fernández et al. 2001).

Antifungal and Antibacterial Activities

The MIC and MFC values of P. abies and L. decidua wood extracts were higher than their bark extracts (Table 5). P. abies wood extract showed the highest MIC and MFC for Aspergillus ochraceus, Candida albicans, and Penicillium ochrochloron, whereas the L. decidua wood extract showed the highest MIC and MFC for A. flavus, A. niger, and P. funiculosum. MIC from both commercial antifungal agents were lower than the examined wood and bark extracts, and ranged from 0.14 to 0.32 mg/mL and 0.11 to 3.71 mg/mL for FLZ and KTZ, respectively. The most sensitive fungus was A. flavus towards P. abies bark extract, and the most resistant fungus towards all wood and bark extracts was C. albicans.

In addition, the methanol extract of P. abies and L. decidua wood and bark were screened for their antibacterial activities against Gram-positive and Gram-negative bacteria. All of the examined wood or bark extracts for P. abies and L. decidua exhibited antibacterial activities against all bacteria. However, the antibacterial potential of the bark was much higher than the wood extract in general (Table 6). The MIC value of P. abies and L. decidua bark ranged from 0.008 to 1.15 mg/mL, whereas the MBC ranged from 0.19 to 2.30 mg/mL. While the MIC value of P. abies and L. decidua wood extract ranged from 0.06 to 1.20 mg/mL, the MBC ranged from 0.27 to 2.31 mg/mL. The extract that showed the highest antibacterial activity was found in L. decidua bark, with an MIC and MBC of 0.11 to 0.54 mg/mL and 0.36 to 0.96 mg/mL, respectively. This was followed by L. decidua wood extract, which exhibited an MIC and MBC of 0.13 to 0.54 mg/mL and 0.33 to 1.1 mg/mL, respectively. P. abies bark and wood did not exhibit much variation in antibacterial activities except against S. aureus and E. coli, where P. abies bark had the highest MIC in the whole data set (0.37 mg/mL). Furthermore, only P. abies wood had higher antibacterial activities than antibiotics (0.06 mg/mL), and most P. abies and L. decidua wood and bark extracts were only slightly higher than antibiotics. In general, the most sensitive bacterium was E. coli, and the most resistant was P. aeruginosa.

These results indicated that the bark and wood extracts of P. abies and L. decidua represent potential antibacterial and antifungal resources and that the antibacterial activities of the bark and wood compounds are much higher than their antifungal activities. However, the bark of L. decidua demonstrated the highest antibacterial and antifungal activities.

The fungicidal activity of different components of the bark of coniferous trees has attracted much attention. Recently, Minova et al. (2015) confirmed that ethanol extracts of the bark of P. abies inhibit mycelial growth of B. cinerea, C. acutatum, P. cactorum, and M. fragariae. Bark extracts can reduce the sporulation of B. cinerea, C. acutatum, and P. cactorum (Minova et al. 2015). The bark extract of P. abies has the most efficient antioxidant activity within lipid peroxidation tests (Pietarinen et al. 2006). The extract of this bark is made up of stilbenes and stilbene glycosides, which are efficient in preventing lipid peroxidation (Mérillon et al. 1997). Similar to flavonoids, the glycosidation of stilbenes lowers antioxidant potency. Packer et al. (1999) evaluated the well-known antioxidative properties and chemical composition of Pycnogenol. The principle components could be divided into flavonoids (catechin, epicatechin, and taxifolin) and condensed tannins. Pycnogenol has been shown to have a higher biological capability as a mixture than its purified components, implying that its components act synergistically (Packer et al. 1999). The antioxidant potencies of B. pendula, Pycnogenol, and P. menziensii are most likely the result of condensed tannins (Pietarinen et al. 2006).

Five types of flavonoids (flavones, flavonols, flavanones, dihydro-flavonols, and flavans), reported by Rauha et al. (2000), were found in P. abies. Most antibacterial activity data concerns aglycones, and only small amounts of data on glycosides can be found (Metsämuuronen and Siren 2014). Out of all these flavonoids, aglycones, quercetin (Rice-Evans et al. 1996; Ibewuike et al.1997; Puupponen-Pimiä et al. 2001), kaempferol (Rauha et al. 2000), and myricetin (Puupponen-Pimiä et al. 2001) have already been recognized to possess antibacterial activity. However, quercetin-3-glucoside (quercitrin) (Puupponen-Pimiä et al. 2001) has actually been found to be inactive. Zhou (2013) discovered that quercetin and myricetin-3-rhamnosides are inactive on Proteus mirabilis and E. coli at a concentration of 500 mg/L, and S. aureus, S. epidermidis, and S. haemolyticus at a concentration of 350 mg/L. This could be because the glycosylation of flavonoids minimizes their antibacterial activity compared to related aglycones.

The quantitative assay validated that the intensity of the antimicrobial activities of P. abies (wood and bark) are unique, and are determined by the tested microbial strain (Radulescu et al. 2011). It is possible that the most susceptible strains could be the Gram-positive ones (Bacillus cereus and S. aureus). If this is the case, it would confirm the suggested hypothesis that the outer membrane of Gram-negative bacteria could be an efficient barrier for the internalization of the active compounds of the wood and bark extract. The greater sensitivity of Gram-positive bacteria might be related to their outer layer chemical structure (peptidoglycan), which is not an efficient permeable barrier (Radulescu et al. 2011). The outer membrane of Gram-negative bacteria is normally negatively charged and hydrophilic, and because of its structural lipopolysaccharide components, may be less permeable to lipophilic substances. Although porins characterize a selective shield to high molecular weight hydrophilic compounds (Kaur and Arora 2009).

The in vitro study of Rautio et al. (2012) demonstrated that resin purified from the trunk of P. abies clearly shows a wide spectrum of antifungal activities. Moreover, the resin was highly microbicidal against all dermatophytes, but not against yeasts and opportunistic fungi. The authors suggested that the mechanisms through which the resin inhibits the development of the microbes are “specific” (Rautio et al. 2012). Similarly, extracts of P. abies show microbicidal activity against Gram-positive bacteria, but not against Gram-negative bacteria (Rautio et al.2007). Nevertheless, a different pattern was reported in the European Pharmacopoeia, with P. abies exerting a microbicidal activity against the Gram-negative bacteria, E. coli, and Pseudomonas aeruginosa (Sipponen and Laitinen 2011). This study showed the importance of using the extracts from heartwood and bark of P. abies and L. decidua as bioactive agents against the growth of some plant and human pathogens. Environmentally, the study suggested the save way in how to get rid of the woodworking residues (wood shavings and sawdust) resulted from the production of boards from the commercial softwoods to produce a bioactive extracts.

CONCLUSIONS

1. In this study, the methanol extracts from the wood and bark of P. abies and L. decidua were evaluated for their antibacterial and antifungal activity. Their chemical compositions were then analyzed using GC/MS.
2. The main chemical compounds of P. abies wood extract were 13-epimanool, α-cedrol, 2,6-di-t-butyloctahydroazulene-3a,8-diol, astringin, (Z,Z,Z)-9,12,15-octadecatrienoic acid-methyl ester, and (1,5,5,8-tetramethylbicyclo[4.2.1]non-9-yl)acetic acid. The main chemical compounds of P. abies bark were abietic acid, astringin, dehydroabietic acid, α-terpineol, methyl sandaracopimarate, and 9-desoxo-9-x-acetoxy-3,8,12-tri-o-acetylingol.
3. The main chemical compounds of L. decidua wood extract were abietic acid, oleanolic acid, duvatrienediol, larixol, squalene, and 13-epimanool. The main chemical compounds of L. decidua bark were (-)-2,9-dihydroxyverrucosane, larixol, nonacosane, cholan-24-oic acid,3,12-dihydroxy-(3α,5α,12α)-decane, and 5-hydroxy-2,3,3-trimethyl-2-(3-methyl-buta-1,3-dienyl)-cyclohexanone.
4. P. abies wood extract showed the highest MIC and MFC for Aspergillus ochraceus, Candida albicans, and Penicillium ochrochloron, whereas the L. decidua wood extract showed the highest MIC and MFC for A. flavus, A. niger, and P. funiculosum.
5. The examined wood or bark extracts for P. abies and L. decidua exhibited antibacterial activities against all bacteria. However, the antibacterial potential of the bark was much higher than the wood extract.
6. The results suggest that the wood and bark extracts from P. abies and L. decidua have a potential effect for use in food and/or pharmaceutical industries. The results showed that extracts have a good potential or human health benefits, agricultural pest control, and wood industry enhancement. However, further studies need to be undertaken to ascertain fully the bioactivity of the extracts.

ACKNOWLEDGMENTS

This project was supported by the King Saud University, Deanship of Scientific Research, College of Science Research Center.

Table 5. Minimum Inhibitory (MICs) and Fungicidal (MFC) Concentration of Wood and Bark Extracts (mg/mL) of Picea abies and Larix decidua

Note: FLZ, Fluconazole; KLZ, Ketoconazole

Table 6. Minimum Inhibitory (MICs) and Bactericidal (MBCs) Concentrations of Wood and Bark Extracts (mg/mL) of Picea abies and Larix decidua

REFERENCES CITED

Adams, R. P. (2007). Identification of Essential Oil Components by Gas Chromatography/mass Spectroscopy (4^th Ed.), Allured Publishing, Carol Stream, IL, USA, p. 804.

Alfredsen, G., Solheim, H., and Slimestad, R. (2008). “Antifungal effect of bark extracts from some European tree species,” Eur. J. For. Res. 127(5), 387-393. DOI: 10.1007/s10342-008-0222-x

Behrens, A., Maie, N., Knicker, H., and Kögel-Knabner, I. (2003). “MALDI-TOF mass spectrometry and PSD fragmentation as means for the analysis of condensed tannins in plant leaves and needles,” Phytochemistry 62(7), 1159-1170. DOI: 10.1016/S0031-9422(02)00660-X

Bianchi, S., Gloess, A. N., Kroslakova, I., Mayer, I., and Pichelin, F. (2014). “Analysis of the structure of condensed tannins in water extracts from bark tissues of Norway spruce (Picea abies[Karst.]) and Silver fir (Abies alba [Mill.]) using MALDI-TOF mass spectrometry,” Ind. Crops Prod. 61(November), 430-437. DOI: 10.1016/j.indcrop.2014.07.038

Bier, M. C. J., Medeiros, A. B. P., de Oliveira, J. S., Côcco, L. C., da Luz Costa, J., de Carvalho, J. C., and Soccol, C. R. (2016). “Liquefied gas extraction: A new method for the recovery of terpenoids from agroindustrial and forest wastes,” J. Supercrit Fluids 110(April), 97-102. DOI: 10.1016/j.supflu.2015.12.016

Cope, J. A., Winch, F. E., and Cope, E. A. (2002). Know Your Trees, Cornell University Media and Technology Services Resource Center, Ithaca, NY, USA.

Danneyrolles, V., Arseneault, D., and Bergeron, Y. (2016). “Pre-industrial landscape composition patterns and post-industrial changes at the temperate-boreal forest interface in western Quebec, Canada,” J. Veg. Sci. 27(3), 470-481. DOI: 10.1111/jvs.12373

da Silva, A. R., Monteiro Pastore, T. C., Batista Braga, J. W., Davrieux, F., Arakaki Okino, E. Y., Rauber Coradin, V. T., Alves Camargos, J. A., and do Prado, A. G. S. (2013). “Assessment of total phenols and extractives of mahogany wood by near infrared spectroscopy (NIRS),” Holzforschung 67(1), 1-8. DOI: 10.1515/hf-2011-0207

Eklund, P. C., Willför, S. M., Smeds, A. I., Sundell, F. J., Sjöholm, R. E., and Holmbom, B. R. (2004). “A new lariciresinol-type butyrolactone lignan derived from hydroxymatairesinol and its identification in spruce wood,” J. Nat. Prod. 67(6), 927-931. DOI: 10.1021/np0340706

Eriksson, K., Wiklund, L., and Larsson, C. (2004). “Dermal exposure to terpenic resin acids in Swedish carpentry workshops and sawmills,” Ann. Occup. Hyg. 48(3), 267-275. DOI: 10.1093/annhyg/meh013

Espinel-Ingroff, A. (2001). “In vitro fungicidal activities of voriconazole, itraconazole, and amphotericin B against opportunistic moniliaceous and dematiaceous fungi,” J. Clin. Microbiol.39(3), 954-958. DOI: 10.1128/JCM.39.3.954-958.2001

Fattahi, B., Nazeri, V., Kalantari, S., Bonfill, M. and Fattahi, M. (2016). “Essential oil variation in wild-growing populations of Salvia reuterana Boiss. collected from Iran using GC-MS and multivariate analysis,” Ind. Crops Prod. 81(March), 180-190. DOI: 10.1016/j.indcrop.2015.11.061

Fernández, M. A., Tornos, M. P., Garcia, M. D., Heras, B. D. L., Villar, A. M., and Saenz, M.T. (2001). “Anti-inflammatory activity of abietic acid, a diterpene isolated from Pimenta racemosavar. grissea,” J. Pharm. Pharmacol. 53(6), 867-872. DOI: 10.1211/0022357011776027

Grassmann, J., Hippeli, S., Vollmann, R., and Elstner, E. F. (2003). “Antioxidative properties of the essential oil from Pinus mugo,” J. Agric. Food Chem. 51(26), 7576-7582. DOI: 10.1021/jf030496e

Greenberg, C. H., Collins, B. S., McNab, W. H., Miller, D. K., and Wein, G. R. (2016). “Introduction to natural disturbances and historic range of variation: Type, frequency, severity, and post-disturbance structure in central hardwood forests,” in: Natural Disturbances and Historic Range of Variation, C. H Greenberg and B. S. Collins (eds.), Springer International Publishing, Cham, Switzerland, pp. 1-32.

Hennig, P., Steinborn, A., and Engewald, W. (1994). “Investigation of the composition of Pinus peuce needle oil by GC-MS and GC-GC-MS,” Chromatographia 38(11), 689-693. DOI: 10.1007/BF02269622

Horsfall, J. G. (ed.) (1980). “5: How plants defend themselves,” in: Plant Disease: An Advanced Treatise, Academic Press, New York, NY.

Ibewuike, J. C., Ogungbamila, F. O., Ogundaini, A. O., Okeke, I. N., and Bohlin, L. (1997). “Antiinflammatory and antibacterial activities of C-methylflavonols from Piliostigma thonningii,” Phytother. Res. 11(4), 281-284. DOI: 10.1002/(SICI)1099-1573(199706)11:4<281::AID-PTR281>3.0.CO;2-9

Kamaya, Y., Tokita, N., and Suzuki, K. (2005). “Effects of dehydroabietic acid and abietic acid on survival, reproduction, and growth of the crustacean Daphnia magna,” Ecotoxicol. Environ. Saf.61(1), 83-88. DOI: 10.1016/j.ecoenv.2004.07.007

Kaur, G. J., and Arora, D. S. (2009). “Antibacterial and phytochemical screening of Anethum graveolens, Foeniculum vulgare and Trachyspermum ammi,” BMC Complement. Altern. Med. 9, 30. DOI: 10.1186/1472-6882-9-30

Kelly, G. S. (1999). “Larch arabinogalactan: Clinical relevance of a novel immune-enhancing polysaccharide,” Altern. Med. Rev. 4(2), 96-103.

Keeling, C. I., and Bohlmann, J. (2006). “Diterpene resin acids in conifers,” Phytochemistry67(22), 2415-2423. DOI: 10.1016/j.phytochem.2006.08.019

Khare, C. P. (ed.) (2004). Indian Herbal Remedies, Springer, Berlin, Germany.

Kolhir, V. K., Bykov, V. A., Baginskaja, A. I., Sokolov, S. Y., Glazova, N. G., Leskova, T. E., Sakovich, G. S., Tjukavkina, N. A., Kolesnik, Y. A., and Rulenko, I. A. (1996). “Antioxidant activity of a dihydroquercetin isolated from Larix gmelinii (Rupr.) Rupr. wood,” Phytother Res.10(6), 478-482. DOI: 10.1002/(SICI)1099-1573

Krieg, O. D., Schwinn, T., and Menges, A. (2016). “Integrative design computation for local resource effectiveness in architecture,” in: Urbanization and Locality, F. Wang and M. Prominski (eds.), Springer, Berlin, Germany, pp. 123-143.

Kusumoto, N., Zhao, T., Swedjemark, G., Ashitani, T., Takahashi, K., and Borg‐Karlson, A. K. (2014). “Antifungal properties of terpenoids in Picea abies against Heterobasidion parviporum,” For. Pathol. 44(5), 353-361. DOI: 10.1111/efp.12106

Latva-Mäenpää, H., Laakso, T., Sarjala, T., Wähälä, K., and Saranpää, P. (2014). “Root neck of Norway spruce as a source of bioactive lignans and stilbenes,” Holzforschung 68(1), 1-7. DOI: 10.1515/hf-2013-0020

Liimatainen, J., Karonen, M., Sinkkonen, J., Helander, M., and Salminen, J. P. (2012). “Characterization of phenolic compounds from inner bark of Betula pendula,” Holzforschung66(2), 171-181. DOI: 10.1515/HF.2011.146

Münzenberger, B., Heilemann, J., Strack, D., Kottke, I., and Oberwinkler, F. (1990). “Phenolics of mycorrhizas and non-mycorrhizal roots of Norway spruce,” Planta 182(1), 142-148. DOI: 10.1007/BF00239996

Malá, J., Hrubcová, M., Máchová, P., Cvrcková, H., Martincová, O., and Cvikrová, M. (2011). “Changes in phenolic acids and stilbenes induced in embryogenic cell cultures of Norway spruce by two fractions of Sirococcus strobilinus mycelia,” J. For. Sci. 57(1), 1-7.

Matthews, S., Mila, I., Scalbert, A., and Donnelly, D. M. X. (1997). “Extractable and non-extractable proanthocyanidins in barks,” Phytochemistry 45(2), 405-410. DOI: 10.1016/S0031-9422(96)00873-4

Mérillon, J. M., Fauconneau, B., Teguo, P. W., Barrier, L., Vercauteren, J., and Huguet, F. (1997). “Antioxidant activity of the stilbene astringin, newly extracted from Vitis vinifera cell cultures,” Clin. Chem. 43(6), 1092-1093.

Merrill, W. (1992). “Mechanisms of resistance to fungi in woody plants: A historical perspective,” in: Defense Mechanisms of Woody Plants against Fungi, R. A. Blanchette, A. R. Biggs (eds). Springer, Berlin, Germany, pp. 1-12.

Metsämuuronen, S., and Siren, H. (2014). “Antibacterial compounds in predominant trees in Finland: Review,” J. Bioprocess Biotech. 4(5), 167. DOI: 10.4172/2155-9821.1000167

Minova, S., Sešķēna, R., Voitkāne, S., Metla, Z., Daugavietis, M., and Jankevica, L. (2015). “Impact of pine (Pinus sylvestris L.) and spruce (Picea abies (L.) Karst.) bark extracts on important strawberry pathogens,” Proc. Latv. Acad. Sci. Sect. B. Nat. Exact. Appl. Sci. 69(1-2), 62-67. DOI: 10.1515/prolas-2015-0008

Nikolić, B., Ristić, M., Bojović, S., Marin, P. D. (2007). “Variability of the needle essential oils of Pinus heldreichii from different populations in Montenegro and Serbia,” Chem. Biodivers. 4(5), 905-916. DOI: 10.1002/cbdv.200790079

Packer, L., Rimbach, G., and Virgili, F. (1999). “Antioxidant activity and biologic properties of a procyanidin-rich extract from pine (Pinus maritima) bark, pycnogenol,” Free Radic. Biol. Med.27(5-6), 704-724. DOI: 10.1016/S0891-5849(99)00090-8

Pan, H., and Lundgren, L. N. (1995). “Phenolic extractives from root bark of Picea abies,” Phytochemistry 39(6), 1423-1428. DOI: 10.1016/0031-9422(95)00144-V

Patinha, D. J. S., Domingues, R. M. A., Villaverde, J. J., Silva, A. M. S., Silva, C. M., Freire, C. S. R., Neto, C. P., and Silvestre, A. J. D. (2013). “Lipophilic extractives from the bark of Eucalyptus grandis x globulus, a rich source of methyl morolate: Selective extraction with supercritical CO₂,” Ind. Crops Prod. 43(May), 340-348. DOI: 10.1016/j.indcrop.2012.06.056

Pferschy-Wenzig, E. M., Kunert, O., Presser, A., and Bauer, R. (2008). “In vitro anti-inflammatory activity of larch (Larix decidua L.) sawdust,” J. Agric. Food Chem. 56(24), 11688-11693. DOI: 10.1021/jf8024002

Pietarinen, S. P., Willför, S. M., Ahotupa, M. O., Hemming, J. E., and Holmbom, B. R. (2006). “Knotwood and bark extracts: Strong antioxidants from waste materials,” J. Wood Sci.52(March), 436-444. DOI: 10.1007/s10086-005-0780-1

Puupponen‐Pimiä, R., Nohynek, L., Meier, C., Kähkönen, M., Heinonen, M., Hopia, A., and Oksman‐Caldentey, K. M. (2001). “Antimicrobial properties of phenolic compounds from berries,” J. Appl. Microbiol. 90(4), 494-507. DOI: 10.1046/j.1365-2672.2001.01271.x

Radulescu, V., Saviuc, C., Chifiriuc, C., Oprea, E., Ilies, D. C., Marutescu, L., and Lazar, V. (2011). “Chemical composition and antimicrobial activity of essential oil from shoots spruce (Picea abies L.),” Revista De Chimie –Bucharest 62(1), 69-72.

Rauha, J. P., Remes, S., Heinonen, M., Hopia, A., Kähkönen, M., Kujala, T., Pihlaja, K., Vuorela, H., and Vuorela, P. (2000). “Antimicrobial effects of Finnish plant extracts containing flavonoids and other phenolic compounds,” Int. J. Food Microbiol. 56(1), 3-12. DOI: 10.1016/S0168-1605(00)00218-X

Rautio, M., Sipponen, A., Lohi, J., Lounatmaa, K., Koukila-Kähkölä, P., and Laitinen, K. (2012). “In vitro fungistatic effects of natural coniferous resin from Norway spruce (Picea abies),” Eur. J. Clin. Microbiol. Infect. Dis. 31(8), 1783-1789. DOI: 10.1007/s10096-011-1502-9

Rautio, M., Sipponen, A., Peltola, R., Lohi, J., Jokinen, J. J., Papp, A., Carlson, P., and Sipponen, P. (2007). “Antibacterial effects of home-made resin salve from Norway spruce (Picea abies),” APMIS 115(4), 335-340. DOI: 10.1111/j.1600-0463.2007.apm_548.x

Rice-Evans, C. A., Miller, N. J., and Paganga, G. (1996). “Structure-antioxidant activity relationships of flavonoids and phenolic acids,” Free Radic. Biol. Med. 20(7), 933-956. DOI: 10.1016/0891-5849(95)02227-9

Rissanen, E., Krumschnabel, G., and Nikinmaa, M. (2003). “Dehydroabietic acid, a major component of wood industry effluents, interferes with cellular energetics in rainbow trout hepatocytes,” Aquat. Toxicol. Amst. Neth. 62(1), 45-53. DOI: 10.1016/S0166-445X(02)00066-8

Salem, M. Z. M., Zeidler, A., Böhm, M., and Srba, J. (2013). “Norway spruce (Picea abies [L.] Karst.) as a bioresource: Evaluation of solid wood, particleboard and MDF technological properties and formaldehyde emission,” BioResources 8(1), 1199-1221. DOI: 10.15376/biores.8.1.1199-1221

Salem, M. Z. M., Nasser, R. A., Zeidler, A., Elansary, H. O., Aref, I. M., Böhm, M., Ali, H. M., and Ahmed, A. I. (2015a). “Methylated fatty acids from heartwood and bark of Pinus sylvestris, Abies alba, Picea abies, and Larix decidua: Effect of strong acid treatment,” BioResources 10(4), 7715-7724. DOI: 10.15376/biores.10.4.7715-7724

Salem, M. Z. M., Zeidler, A., Böhm, M., Mohamed, M. E., and Ali, H. M. (2015b). “GC/MS analysis of oil extractives from wood and bark of Pinus sylvestris, Abies alba, Picea abies, and Larix decidua,” BioResources 10(4), 7725-7737. DOI: 10.15376/biores.10.4.7725-7737

Salem, M. Z. M., Zidan, Y. E., Mansour, M. M., El Hadidi, N. M., and Elgat, W. A. A. (2016). “Antifungal activities of two essential oils used in the treatment of three commercial woods deteriorated by five common mold fungi,” Int. Biodeterior. Biodegrad. 106(January), 88-96. DOI: 10.1016/j.ibiod.2015.10.010

San Feliciano, A., Gordaliza, M., Salinero, M. A., and Miguel del Corral, J. M. (1993). “Abietane acids: Sources, biological activities, and therapeutic uses,” Planta Med. 59(6), 485-490. DOI: 10.1055/s-2006-959744

Sepúlveda, B., Astudillo, L., Rodríguez, J. A., Yáñez, T., Theoduloz, C., and Schmeda-Hirschmann, G. (2005). “Gastroprotective and cytotoxic effect of dehydroabietic acid derivatives,” Pharmacol. Res. 52(5), 429-437. DOI: 10.1016/j.phrs.2005.06.004

Si, C. L., Jiang, J. Z., Liu, S. C., Hu, H. Y., Ren, X. D., Yu, G. J., and Xu, G. H. (2012). “A new lignan glycoside and phenolics from the branch wood of Pinus banksiana Lambert,” Holzforschung 67(4), 357-363. DOI: 10.1515/hf-2012-0137

Sipponen, A., and Laitinen, K. (2011). “Antimicrobial properties of natural coniferous rosin in the European Pharmacopoeia challenge test,” APMIS 119(10), 720-724. DOI: 10.1111/j.1600-0463.2011.02791.x

Steynberg, J. P., Ferreira, D., and Roux, D. G. (1983). “The first condensed tannins based on a stilbene,” Tetrahedron Lett. 24(38), 4147-4150. DOI: 10.1016/S0040-4039(00)88284-2

Tumen, I., Hafizoglu, H., Kilic, A., Dönmez, I. E., Sivrikaya, H., and Reunanen, M. (2010). “Yields and constituents of essential oil from cones of Pinaceae spp. natively grown in Turkey,” Molecules 15(8), 5797-5806. DOI: 10.3390/molecules15085797

Ulusu, N. N., Ercil, D., Sakar, M. K., and Tezcan, E. F. (2002). “Abietic acid inhibits lipoxygenase activity,” Phytother. Res. 16(1), 88-90. DOI: 10.1002/ptr.983

Viiri, H., Annila, E., Kitunen, V., and Niemelä, P. (2001). “Induced responses in stilbenes and terpenes in fertilized Norway spruce after inoculation with blue-stain fungus, Ceratocystis polonica,” Trees 15(2), 112-122. DOI: 10.1007/s004680000082

von Bruchhausen F., Ebel S., and Hackenthal E., et al. (eds) (1993). Hagers Handbuch der Pharmazeutischen Praxis, Springer Berlin Heidelberg, Berlin, Heidelberg.

Willför, S. M., Ahotupa, M. O., Hemming, J. E., Reunanen, M. H., Eklund, P. C., Sjöholm, R. E., Eckerman, C. S., Pohjamo, S. P., and Holmbom, B. R. (2003). “Antioxidant activity of knotwood extractives and phenolic compounds of selected tree species,” J. Agric. Food Chem. 51(26), 7600-7606. DOI: 10.1021/jf030445h

Yessoufou, K., Elansary, H. O., Mahmoud, E. A., and Skalicka-Woźniak, K. (2015). “Antifungal, antibacterial, and anticancer activities of Ficus drupacea L. stem bark extract and biologically active isolated compounds,” Ind. Crops Prod. 74(November), 752-758. DOI: 10.1016/j.indcrop.2015.06.011

Zeneli, G., Krokene, P., Christiansen, E., Krekling, T., and Gershenzon, J. (2006). “Methyl jasmonate treatment of mature Norway spruce (Picea abies) trees increases the accumulation of terpenoid resin components and protects against infection by Ceratocystis polonica, a bark beetle-associated fungus,” Tree Physiol. 26(8), 977-988. DOI: 10.1093/treephys/26.8.977

Zhou, Y. (2013). “Mathematical modelling of protein precipitation based on the phase equilibrium for an antibody fragment from E. coli lysis,” J. Bioprocess. Biotech. 3(2), 129. DOI: 10.4172/2155-9821.1000129

Article submitted: June 28, 2016; Peer review completed: September 4, 2016; Revised version received and accepted: September 7, 2016; Published: September 21, 2016.

DOI: 10.15376/biores.11.4.9421-9437.