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International Biodeterioration & Biodegradation 64 (2010) 711e715

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International Biodeterioration & Biodegradation

journal homepage: www.elsevier .com/locate/ ibiod

Natural resistance of five woods to Phanerochaete chrysosporium degradation

Luciana S. Oliveira a, Andréa L.B.D. Santana a, Cláudia A. Maranhão a, Rita de Cássia M. de Miranda b,Vera Lúcia A. Galvão de Lima c, Suzene I. da Silva d, Márcia S. Nascimento b,*, Lothar Bieber a

aDepartamento de Química Fundamental e CCEN, Universidade Federal de Pernambuco, Cidade Universitária, 50670-901 Recife, Pernambuco, BrazilbDepartamento de Antibióticos e CCB, Universidade Federal de Pernambuco, Cidade Universitária, 50670-901 Recife, Pernambuco, BrazilcDepartamento de Economia Doméstica, Universidade Federal Rural de Pernambuco, Dois Irmãos, 52171-030 Recife, Pernambuco, BrazildDepartamento de Botânica, Universidade Federal Rural de Pernambuco, Dois Irmãos, 52171-030 Recife, Pernambuco, Brazil

a r t i c l e i n f o

Article history:Received 6 May 2010Received in revised form5 August 2010Accepted 6 August 2010Available online 28 September 2010

Keywords:Lignin contentExtractivesHymenaea stigonocarpaAnadenanthera colubrinaCaesalpinia ferreaManilkara huberiDelonix regiaPhanerochaete chrysosporium

* Corresponding author. Tel.: þ55 8121268347; faxE-mail address: msn@ufpe.br (M.S. Nascimento).

0964-8305/$ e see front matter � 2010 Elsevier Ltd.doi:10.1016/j.ibiod.2010.08.001

a b s t r a c t

This research evaluated the natural resistance of five woods to the white-rot wood-destroying fungusPhanerochaete chrysosporium under laboratory conditions and in nature. The studied species wereHymenaea stigonocarpa, Anadenanthera colubrina, Caesalpinia ferrea, Manilkara huberi and Delonix regia.The natural resistance to decay is one of the most important properties of wood, mainly assigned tolignin and extractives of wood. A. colubrina has the highest content of extractives and M. huberi thehighest content of lignin; both are known as resistant to xylophagous organisms and were also mostresistant to the tested fungus. C. ferrea has the lowest content of extractives and D. regia of lignin; bothspecies did not inhibit the fungus Phanerochaete chrysosporium. H. stigonocarpa occupies an intermediateposition in content of extractives and lignin as well in resistance to P. chrysosporium.

� 2010 Elsevier Ltd. All rights reserved.

1. Introduction

The chemical composition of wood is complex. The two majorchemical components in wood are the macromolecular cell wallcomponents, carbohydrate (65e75%) and lignin (18e35%), and anarray of low-molecular-mass compounds as extractives (4e10%)(Pettersen,1984). Hydrophilic extractives comprise a great diversityof compounds, such as flavonoids (anthocyanins, flavanols, flavo-nols and flavones) and several classes of non-flavonoids (phenolicacids, tannins, stilbenes) (Harborne, 1989). Wood extractives arealso lipophilic substances consisting mainly of triglycerides, fattyacids, diterpenoid resin acids, sterols, waxes and steryl esters(Fengel and Wegener, 1989).

Natural durability or decay resistance is the ability of wood toprevent biological degradation (Eaton and Hale, 1993). Aftercellulose, lignin is the second most abundant type of biopolymerson the earth and provides plant resistance to microbial

: þ55 8121268346.

All rights reserved.

degradation, markedly influencing the natural durability of wood(Syafh et al., 1988; Tuomela et al., 2000).

Although extractives contribute merely a few percent to theentire wood composition, they are very important to trees asdefense mechanisms against microbial attack (Silva et al., 2007).According to Amusant et al. (2007) there is no doubt that extrac-tives are the most significant factor influencing the durability ofwood due to their fungicidal and antioxidant activity. Among theextractives, phenolic compounds are important to the plants fornormal growth development and defense against infection andinjury (Jerez et al., 2007). They play a key role as antioxidants due tothe presence of aromatic hydroxyl groups, which enable them toscavenge free radicals.

Different organisms can deteriorate wood, but the greatestdamage is caused by fungi. White-rot basidiomycete fungi are theonly known microorganisms in nature that are capable of degrad-ing lignin completely. Phanerochaete chrysosporium has been wellstudied as a model strain because of its specialized ability todegrade lignin, while leaving the white cellulose nearly untouched(Kersten and Cullen, 2007; Hu et al., 2009).

The aim of this work was to investigate the natural resistance offive different tropical woods to P. chrysosporium and to observe

L.S. Oliveira et al. / International Biodeterioration & Biodegradation 64 (2010) 711e715712

whether resistance correlates with the extractive- or lignin contentof the wood.

2. Materials and methods

2.1. Wood samples

The wood from five different species, namely Hymenaea stigo-nocarpa, Anadenanthera colubrina, Caesalpinia ferrea, Delonix regiaandManilkara huberi were investigated. The information about thetrees and their habitats was provided in Table 1.

2.2. Chemical analysis of wood

2.2.1. Extractives contentThe rotor-milled wood samples (1 g) were extracted with

cyclohexane and thenwith ethanol (500 ml) in a soxhlet apparatusfor 4 h to remove extractives. The lower polarity solvent, cyclo-hexane, was used for the first step to remove lipophilic compounds,and the more polar ethanol was used for the second extraction stepto remove hydrophilic compounds. The extract solutions wereconcentrated in a rotary evaporator. The wood samples free ofextractives were dried at 37 �C, weighed and used for the deter-mination of lignin.

2.2.2. Phytochemical tests of the plantsThe wood samples were air dried at room temperature,

powdered and subsequently subjected to phytochemical screening,using procedures described by Costa (1982), to identify the mainclasses of secondary metabolites (Table 3).

2.2.3. Lignin contentThe lignin content was evaluated following the TAPPI method

(Tappi, 1996), which is based on the isolation of Klason lignin afterhydrolysis of the polysaccharides (cellulose and hemicellulose) byconcentrated sulfuric acid (72%). After filtering of the insolublelignin the hydrolyzed samples (triplicate) were put in a water bathfor 2 h at 30 � 1 �C and later were transferred to an Erlenmeyerflask and diluted to 4% acid concentration with distilled water.

Table 1Key characteristics of tested wood species.

Wood Characteristic

Hymenaea stigonocarpaOccurrence: Bolívia, BrazilFamily: CaesalpinioideaeCommon name: jatobá-do-cerrado

Natural resistfurniture, use

Anadenanthera colubrinaOccurrence: Bolivia, Brazil, PeruFamily: MimosoideaeCommon name: Angico-branco, angico-de-caroço

Natural resistused in furnitand naval ind

Caesalpinia ferreaOccurrence: BrazilFamily: CaesalpinioideaeCommon name: jucá, pau-ferro

Durable woodand manufactfiddles and p

Manilkara huberiOccurrence: Brazil, Venezuela, French GuianaFamily: SapotaceaeCommon name: maçaranduba, beefwood,bulletwood, sapotilla

High durabilion fences, pomarket, Japan

Delonix regiaOccurrence: native to Madagascar butintroduced to tropical areas.Family: CaesalpinioideaeCommon name: Flamboyant, flame of theforest, poinciana

Soft and weasubject to ter

These samples were autoclaved for 1 h at 121 � 3 �C. The acid-soluble lignin was determined spectrophotometrically at 280 nmwavelength.

2.2.4. Total phenol contentsThe total phenolic content was estimated by the FolineCiocalteu

colorimetric method, based on the procedure of Waterman andMole (1994), and the results are expressed as gallic acid equiva-lents (GAE). Standard concentrations of gallic acid between 0.78and 50 mg ml�1 were used to prepare calibration curves. 0.5 ml ofwood extract methanol/water (8/2) or gallic acid (standardphenolic compound) was mixed with FolineCiocalteu reagent(2.5 ml, 10% diluted with distilled water) and aqueous solutionsodium carbonate (2.0 ml of 7.5%). The mixture was kept for 5 minat 50 �C; the absorbance at 760 nm was measured. The analyseswere done in triplicate and the mean value was calculated.

2.2.5. DPPH radical scavenging assayDPPH (1,1-Diphenyl-2-picryl-hydrazyl) scavenging activity of

wood extracts was determined according to the method describedby Brand-Willians et al. (1995) with slight modifications. 0.2 ml ofthe ethanolic extract (100 mg ml�1 in methanol) was added to 4 mlof DPPHmethanolic solution (43 mg ml�1). This method is based onthe reduction of a methanol solution of DPPH in the presence ofa hydrogen donating antioxidant due to the formation of the non-radical form DPPH-H. The antioxidanteradical reactions wereconducted for 30 min in the dark at ambient temperature. Thistransformation results in a change of color from purple to yellow,which is measured spectrophotometrically by the disappearance ofthe purple color at 515 nm. Ascorbic acid was used as standard.

2.3. Fungi

The white-rot fungus P. chrysosporium from Collection of Trop-ical Culture (CCT 1999) Fundação André Tosello, was used in thisstudy. Cultures weremaintained onmalt extract agar at 30 �C for 10days. The fungus was then incubated on solid culture mediumcontaining 15 g of agar, 15 g of yeast extract and 1000 ml distilledwater.

and use References

ance soft-rot fungi and termites,d in building and naval industries.

Lorenzi, 1992Santana et al., 2010

ance soft-rot fungi and termites,ure, leather tanning, buildingustries.

Lorenzi, 1998Santana et al., 2010

is used in building industryure of furniture, guitars,ipes.

Lorenzi and Matos, 2002

ty natural, indicated for usests and floors. Exported to U.S.and some European countries

Lorenzi, 1998

k wood limiting its use in carpentry,mite attack.

Schütt and Lang, 2004Lorenzi et al., 2003

Table 2Percent extractives in the studied wood species.

Extraction procedure (%) Hymenaea stigonocarpa Anadenanthera colubrina Caesalpinia ferrea Manilkara huberi Delonix regia

Cyclohexane 1.5 � 0,6 0.33 � 0.08 1.0 � 0.5 0.6 � 0.2 0.8 � 0.4Ethanol 5 � 2 8 � 2 3.2 � 0,3 7.1 � 0,5 4 � 1Total 7 � 2 9 � 2 4.3 � 0.4 7.7 � 0,4 5 � 1

L.S. Oliveira et al. / International Biodeterioration & Biodegradation 64 (2010) 711e715 713

2.4. Laboratory test on the susceptibility of woods to the white-rotfungus Phenerochaete chrysosporium

The laboratory tests to evaluate the natural durability of wood todegradation by P. chrysosporium were carried out based on theprocedure of Kamida et al. (2005) with slight modifications. 5 g offinely powdered wood samples were weighed in Erlenmeyer flasks(125 ml) and humidified with 1 ml of tap water. Three replicateswere used for extracted and unextracted woods. Non-inoculated,sterilized wood was used as a biotic control. Three disks (�6 mm indiameter) from solid culture medium inoculated with the funguswere transferred to each Erlenmeyer flask. The growth of P. chrys-osporium mycelium was evaluated after 4 weeks at 30 �C.

3. Results and discussion

Table 2 displays the extractives of woods. The amount ofcyclohexane-extractives was always lower than ethanol-extrac-tives. The total content of extractives (extracted gradually withcyclohexane and ethanol) was the greatest in A. colubrina (9% of thetotal dry weight of wood), followed by M. huberi (7.7%) andH. stigonocarpa (7%), D. regia (5%) and C. ferrea (4.3%). These valuesagree rather well with other woods reported in the literature. Astudy carried out by Santana and Okino (2007) with 36 Braziliantropical timbers found extractive content between 17.3% and 0.9%,but only eight of the thirty six species showed extractive contenthigher than 10%. In that study M. huberi had an extractives contentof 8% so is comparable with our results. Species with high contentsof extractives are of interest for future studies because chemicalcompounds from the crude extracts can be used for pharmaceuti-cals, dyes, cosmetics, perfumes and natural antioxidants (Santanaand Okino, 2007). High extractive content is known to result ingood natural durability (Windeisen et al., 2002).

The result of the preliminary phytochemical analysis of theextract of wood species is shown in Table 3. The phytochemicalstudies revealed the presence of flavonoids, terpenes and steroidsin all analyzed woods. Tannin presence was very strong inA. colubrina. Saponins were found only in M. huberi indicating that,among the studied woods, there were significant differences in theamount and in the composition of extractives. Phenoliccompounds, such as tannins and flavonoids, have antimicrobial andantioxidative properties and are involved in the defense againstfungi and other microorganisms (Boudet, 2007). Saponins havebeen reported to prevent antimicrobial activity (Sparg et al., 2004).Secondary metabolites (extractives) are present in all plants,generally as mixtures that can be highly diverse. A large number ofstudies have demonstrated the importance of these metabolites as

Table 3Preliminary phytochemical analysis of extractives detected in the woods.

Classes of extractives Extraction procedure Hymenaea stigonocarpa An

Saponins Frothing � �Tannins Ferric chloride þ þþAlkaloids Dragendorff, Mayer þ �Flavonoids Shinoda Oxalo-boric acid þþ þþSteroids and terpenoids LiebermanneBuchard þþ þþ

(�) not detected; (þ) detected. The number of positive signs indicates the intensity of th

plant defense compounds. A high diversity of extractives in highconcentration provides a more effective protection against herbi-vores than single compounds or low diversity in both low and highconcentrations (Castellanos and Espinosa-Garcia, 1997). Thediversity (Table 2) as well as the concentration of phenoliccompounds (Table 3) was more pronounced in A. colubrina andM. huberi in sharp contrast to D. regia and C. ferrea.

Table 4 provides the quantification of lignin. According to Syafhet al. (1988) lignin is themost important non-toxic factor that limitsthe growth of the microorganisms in wood biodegradation.M. huberi presents the highest content of total lignin (30%). In thisstudy, D. regia had the lowest concentration of lignin (22.3%), thussuggesting that lignin may be the main factor for the low resistanceof this wood. Among 36 woods analyzed, Santana and Okino (2007)found an acid-soluble lignin (ASL) content of 0.7e1.8% and an acid-insoluble lignin (AIL) of 26.7e37%. The values for M. huberi were34% AIL and and 0.9% ASL. The lignin content for all woods wassimilar and typical for tropical hardwoods, except for D. regia. Theseresults suggest that differences in the content and structure oflignin may influence the decay resistance caused by wood-decaying fungi (Syafh et al., 1988).

The content of phenolics (mg g�1) in ethanol extracts wasdetermined from a regression equation of the calibration curve(y ¼ 0.004663x þ 0.0565, R2 ¼ 0.99) and expressed in Gallic AcidEquivalents (GAE). As shown in Table 5, there is a close correlationbetween the phenolic content and the % DPPH radical quenched.The ethanol extract from H. stigonocarpa contained the highestamount of total phenolics and, consequently, the highest antioxi-dant activity, followed by A. colubrina, C. ferrea, D. regia, andM. huberi. Although D. regia has higher phenolic contents thanM. huberi, it displays the lowest antioxidant activity. These resultsindicate that phenolic composition among both woods differwidely in terms of chemical composition.

The influence of extractives on the growth of P. chrysosporium inthe tested woods is shown in Table 6. The extracted and unex-tracted wood of M. huberi inhibited fungal growth completely,confirming that lignin plays an important role in natural resistanceof this wood (Syafh et al., 1988; Tuomela et al., 2000). This obser-vation suggests that the content of saponins is not responsible forthe natural resistance ofM. huberi against fungal attack since decayresistance was not affected by their extraction (Table 3).

Among all the studied woods, A. colubrina was the only specieswhere the influence of extractives was very well observed. Fungalgrowth was entirely inhibited by unextracted wood but intensegrowth formed in the presence of extracted wood (Fig. 1). Rowellet al. (2005) states that wood resistance to fungal attack is attrib-uted mainly to the presence of extractives that are toxic to

adenanthera colubrina Caesalpinia ferrea Manilkara huberi Delonix. regia

� þþþ �þ þ � �

� þ �þ þ þþ þ

þ þþ þþe reactions (Costa, 1982).

Table 5Total phenolic content and DPPH scavenging activity of phenolic extractives from analyzed woods.

Methanol/water extract Hymenaea stigonocarpa Anadenanthera colubrina Caesalpinia ferrea Manilkara huberi Delonix regia

mg GAE g�1 of extracts 0.248 � 0.009 0.231 � 0.003 0.151 � 0.005 0.069 � 0.004 0.092 � 0.007% DPPH radical quenched 91.00 � 0.050 71.90 � 0.099 21.51 � 0.358 5.99 � 0.050 3.07 � 0.14

Table 6Phanerochaete chrysosporium growth in the studied woods species.

Woodsamples

Hymenaeastigonocarpa

Anadenantheracolubrina

Caesalpiniaferrea

Manilkarahuberi

Delonixregia

Unextractedwood

þ � þþþ � þþþ

Extractedwood

þþ þþ þþþ � þþþ

(�) no growth; (þ) weak, (þþ) medium and (þþþ) abundant growth of Phaner-ochaete chrysosporium.

Table 4Lignin content of the studied woods species.

Lignin (%) Hymenaea stigonocarpa Anadenanthera colubrina Caesalpinia ferrea Manilkara huberi Delonix regia

Acid-soluble (ASL) 0.6 � 0.4 1.81 � 0.03 1.7 � 0.3 1.40 � 0.06 0.3 � 0.1Acid-insoluble (AIL) 27 � 1 25.9 � 0.9 26 � 4 28.6 � 0.4 22 � 1Total 28 � 1 27.6 � 0.9 28 � 4 30.0 � 0.4 22.3 � 1

L.S. Oliveira et al. / International Biodeterioration & Biodegradation 64 (2010) 711e715714

xylophagous organisms, thus providing the wood with naturaldurability. In addition, A. colubrina contains a high content ofphenolic compounds and high antioxidant activity (Table 5) and alsohas good natural resistance to termites (Silva et al., 2007; Santanaet al., 2010). According to Schultz and Nicholas (2000), the radical

Fig. 1. The growth of Phanerochaete chrysosporium showing on extracted and unextrac

Fig. 2. The growth of Phanerochaete chrysosporium showing on extracted and

scavenging activity of phenolic compounds may further acceleratefungal death by scavenging the radicals produced by fungus andreducing fungal nutrition necessary for repairing cell wall injuries,thus resulting in the strong synergy of antifungal activity.

As can be seen in Fig. 1, unextracted wood of H. stigonocarpastimulates only weak growth of P. chrysosporium, whereas fungalgrowth in the presence of extracted wood is far more pronounced,confirming that the extractives could not completely inhibit thegrowth of the fungus but acted as a primary barrier to preventcolonization. Although H. stigonocarpa extractives did not inhibitcompletely fungus growth, this wood is known for its naturalresistance (Table 1). According to Rudman (1965), heartwoodextractives from durable species are not always toxic againsta broad spectrum of wood-destroying fungi but are often specific toa limited number of species. Apparently, the extractives ofH. stigonocarpa do not exhibit strong antifungal activity againstP. chrysosporium used in this study.

ted woods from Hymenaea stigonocarpa (left) and Anadenanthera colubrina (right).

unextracted woods from Delonix regia (left) and Caesalpinia ferrea (right).

L.S. Oliveira et al. / International Biodeterioration & Biodegradation 64 (2010) 711e715 715

Fig. 2 shows similar behavior for C. ferrea and D. regia in respectto fungal growth. The extractives from both woods showed weakantifungal activities. Usually flavonoids and tannins are responsiblefor antioxidant activity, which sequester the free radicals producedby fungi during the decay process (Schultz and Nicholas, 2000).C. ferrea andD. regiahave a lower content of extractives (Table 2) andphenolic compounds (Table 5) than other woods. These factors mayexplain the low resistance of both woods against P. chrysosporium.

4. Conclusion

The durability of A. colubrina can be attributed mainly to thepresence of phenolic compounds, particularly tannins and flavo-noids that inhibit fungal growth. Although the wood of M. hubericontains large quantities of extractives, especially saponins, theyarenot the only compounds responsible for antifungal activity. The highlignin content and the chemical composition of the lignin seem tobethe major reasons for the high decay resistance of this wood. Thenaturally low durability of C. ferrea and D. regia wood against P.chrysosporiummaybe the result of various factors, including the lowquantity of extractives, the low antioxidant activity of the phenolicextractives, and the structural composition of the lignin.

Acknowledgements

The authors are grateful to Coordenação de Aperfeiçoamento dePessoal de Nível Superior (CAPES) and Conselho Nacional deDesenvolvimento Científico e Tecnológico (CNPq) for grants andfellowships.

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