International Biodeterioration & Biodegradation 64 (2010) 711e715

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International Biodeterioration & Biodegradation

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

Natural resistance of ve woods to Phanerochaete chrysosporium degradation

Luciana S. Oliveira a, Andra L.B.D. Santana a, Cludia A. Maranho a, Rita de Cssia M. de Miranda b,
Vera Lcia A. Galvo de Lima c, Suzene I. da Silva d, Mrcia S. Nascimento b, *, Lothar Bieber a
a

Departamento de Qumica Fundamental e CCEN, Universidade Federal de Pernambuco, Cidade Universitria, 50670-901 Recife, Pernambuco, Brazil
Departamento de Antibiticos e CCB, Universidade Federal de Pernambuco, Cidade Universitria, 50670-901 Recife, Pernambuco, Brazil
c
Departamento de Economia Domstica, Universidade Federal Rural de Pernambuco, Dois Irmos, 52171-030 Recife, Pernambuco, Brazil
d
Departamento de Botnica, Universidade Federal Rural de Pernambuco, Dois Irmos, 52171-030 Recife, Pernambuco, Brazil
b

a r t i c l e i n f o

a b s t r a c t

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

This research evaluated the natural resistance of ve woods to the white-rot wood-destroying fungus
Phanerochaete chrysosporium under laboratory conditions and in nature. The studied species were
Hymenaea 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 to
lignin and extractives of wood. A. colubrina has the highest content of extractives and M. huberi the
highest content of lignin; both are known as resistant to xylophagous organisms and were also most
resistant to the tested fungus. C. ferrea has the lowest content of extractives and D. regia of lignin; both
species did not inhibit the fungus Phanerochaete chrysosporium. H. stigonocarpa occupies an intermediate
position in content of extractives and lignin as well in resistance to P. chrysosporium.
2010 Elsevier Ltd. All rights reserved.

Keywords:
Lignin content
Extractives
Hymenaea stigonocarpa
Anadenanthera colubrina
Caesalpinia ferrea
Manilkara huberi
Delonix regia
Phanerochaete chrysosporium

1. Introduction
The chemical composition of wood is complex. The two major
chemical components in wood are the macromolecular cell wall
components, carbohydrate (65e75%) and lignin (18e35%), and an
array of low-molecular-mass compounds as extractives (4e10%)
(Pettersen, 1984). Hydrophilic extractives comprise a great diversity
of compounds, such as avonoids (anthocyanins, avanols, avonols and avones) and several classes of non-avonoids (phenolic
acids, tannins, stilbenes) (Harborne, 1989). Wood extractives are
also lipophilic substances consisting mainly of triglycerides, fatty
acids, diterpenoid resin acids, sterols, waxes and steryl esters
(Fengel and Wegener, 1989).
Natural durability or decay resistance is the ability of wood to
prevent biological degradation (Eaton and Hale, 1993). After
cellulose, lignin is the second most abundant type of biopolymers
on the earth and provides plant resistance to microbial

* Corresponding author. Tel.: 55 8121268347; fax: 55 8121268346.

E-mail address: [email protected] (M.S. Nascimento).
0964-8305/$ e see front matter 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.ibiod.2010.08.001

degradation, markedly inuencing the natural durability of wood

(Syafh et al., 1988; Tuomela et al., 2000).
Although extractives contribute merely a few percent to the
entire wood composition, they are very important to trees as
defense mechanisms against microbial attack (Silva et al., 2007).
According to Amusant et al. (2007) there is no doubt that extractives are the most signicant factor inuencing the durability of
wood due to their fungicidal and antioxidant activity. Among the
extractives, phenolic compounds are important to the plants for
normal growth development and defense against infection and
injury (Jerez et al., 2007). They play a key role as antioxidants due to
the presence of aromatic hydroxyl groups, which enable them to
scavenge free radicals.
Different organisms can deteriorate wood, but the greatest
damage is caused by fungi. White-rot basidiomycete fungi are the
only known microorganisms in nature that are capable of degrading lignin completely. Phanerochaete chrysosporium has been well
studied as a model strain because of its specialized ability to
degrade 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 of
ve different tropical woods to P. chrysosporium and to observe

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

whether resistance correlates with the extractive- or lignin content

of the wood.

These samples were autoclaved for 1 h at 121  3  C. The acidsoluble lignin was determined spectrophotometrically at 280 nm
wavelength.

2. Materials and methods

2.1. Wood samples
The wood from ve different species, namely Hymenaea stigonocarpa, Anadenanthera colubrina, Caesalpinia ferrea, Delonix regia
and Manilkara huberi were investigated. The information about the
trees and their habitats was provided in Table 1.
2.2. Chemical analysis of wood
2.2.1. Extractives content
The rotor-milled wood samples (1 g) were extracted with
cyclohexane and then with ethanol (500 ml) in a soxhlet apparatus
for 4 h to remove extractives. The lower polarity solvent, cyclohexane, was used for the rst step to remove lipophilic compounds,
and the more polar ethanol was used for the second extraction step
to remove hydrophilic compounds. The extract solutions were
concentrated in a rotary evaporator. The wood samples free of
extractives were dried at 37  C, weighed and used for the determination of lignin.
2.2.2. Phytochemical tests of the plants
The wood samples were air dried at room temperature,
powdered and subsequently subjected to phytochemical screening,
using procedures described by Costa (1982), to identify the main
classes of secondary metabolites (Table 3).
2.2.3. Lignin content
The lignin content was evaluated following the TAPPI method
(Tappi, 1996), which is based on the isolation of Klason lignin after
hydrolysis of the polysaccharides (cellulose and hemicellulose) by
concentrated sulfuric acid (72%). After ltering of the insoluble
lignin the hydrolyzed samples (triplicate) were put in a water bath
for 2 h at 30  1  C and later were transferred to an Erlenmeyer
ask and diluted to 4% acid concentration with distilled water.

2.2.4. Total phenol contents

The total phenolic content was estimated by the FolineCiocalteu
colorimetric method, based on the procedure of Waterman and
Mole (1994), and the results are expressed as gallic acid equivalents (GAE). Standard concentrations of gallic acid between 0.78
and 50 mg ml1 were used to prepare calibration curves. 0.5 ml of
wood extract methanol/water (8/2) or gallic acid (standard
phenolic compound) was mixed with FolineCiocalteu reagent
(2.5 ml, 10% diluted with distilled water) and aqueous solution
sodium carbonate (2.0 ml of 7.5%). The mixture was kept for 5 min
at 50  C; the absorbance at 760 nm was measured. The analyses
were done in triplicate and the mean value was calculated.
2.2.5. DPPH radical scavenging assay
DPPH (1,1-Diphenyl-2-picryl-hydrazyl) scavenging activity of
wood extracts was determined according to the method described
by Brand-Willians et al. (1995) with slight modications. 0.2 ml of
the ethanolic extract (100 mg ml1 in methanol) was added to 4 ml
of DPPH methanolic solution (43 mg ml1). This method is based on
the reduction of a methanol solution of DPPH in the presence of
a hydrogen donating antioxidant due to the formation of the nonradical form DPPH-H. The antioxidanteradical reactions were
conducted for 30 min in the dark at ambient temperature. This
transformation results in a change of color from purple to yellow,
which is measured spectrophotometrically by the disappearance of
the purple color at 515 nm. Ascorbic acid was used as standard.
2.3. Fungi
The white-rot fungus P. chrysosporium from Collection of Tropical Culture (CCT 1999) Fundao Andr Tosello, was used in this
study. Cultures were maintained on malt extract agar at 30  C for 10
days. The fungus was then incubated on solid culture medium
containing 15 g of agar, 15 g of yeast extract and 1000 ml distilled
water.

Table 1
Key characteristics of tested wood species.
Wood

Characteristic and use

References

Hymenaea stigonocarpa
Occurrence: Bolvia, Brazil
Family: Caesalpinioideae
Common name: jatob-do-cerrado
Anadenanthera colubrina
Occurrence: Bolivia, Brazil, Peru
Family: Mimosoideae
Common name: Angico-branco, angico-de-caroo
Caesalpinia ferrea
Occurrence: Brazil
Family: Caesalpinioideae
Common name: juc, pau-ferro
Manilkara huberi
Occurrence: Brazil, Venezuela, French Guiana
Family: Sapotaceae
Common name: maaranduba, beefwood,
bulletwood, sapotilla
Delonix regia
Occurrence: native to Madagascar but
introduced to tropical areas.
Family: Caesalpinioideae
Common name: Flamboyant, ame of the
forest, poinciana

Natural resistance soft-rot fungi and termites,

furniture, used in building and naval industries.

Lorenzi, 1992
Santana et al., 2010

Natural resistance soft-rot fungi and termites,

used in furniture, leather tanning, building
and naval industries.

Lorenzi, 1998
Santana et al., 2010

Durable wood is used in building industry

and manufacture of furniture, guitars,
ddles and pipes.

Lorenzi and Matos, 2002

High durability natural, indicated for use

on fences, posts and oors. Exported to U.S.
market, Japan and some European countries

Lorenzi, 1998

Soft and weak wood limiting its use in carpentry,

subject to termite attack.

Schtt and Lang, 2004

Lorenzi et al., 2003

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713

Table 2
Percent extractives in the studied wood species.
Extraction procedure (%)

Hymenaea stigonocarpa

Anadenanthera colubrina

Caesalpinia ferrea

Manilkara huberi

Delonix regia

Cyclohexane
Ethanol
Total

1.5  0,6
52
72

0.33  0.08
82
92

1.0  0.5
3.2  0,3
4.3  0.4

0.6  0.2
7.1  0,5
7.7  0,4

0.8  0.4
41
51

2.4. Laboratory test on the susceptibility of woods to the white-rot

fungus Phenerochaete chrysosporium
The laboratory tests to evaluate the natural durability of wood to
degradation by P. chrysosporium were carried out based on the
procedure of Kamida et al. (2005) with slight modications. 5 g of
nely powdered wood samples were weighed in Erlenmeyer asks
(125 ml) and humidied with 1 ml of tap water. Three replicates
were used for extracted and unextracted woods. Non-inoculated,
sterilized wood was used as a biotic control. Three disks (6 mm in
diameter) from solid culture medium inoculated with the fungus
were transferred to each Erlenmeyer ask. The growth of P. chrysosporium mycelium was evaluated after 4 weeks at 30  C.
3. Results and discussion
Table 2 displays the extractives of woods. The amount of
cyclohexane-extractives was always lower than ethanol-extractives. The total content of extractives (extracted gradually with
cyclohexane and ethanol) was the greatest in A. colubrina (9% of the
total dry weight of wood), followed by M. huberi (7.7%) and
H. stigonocarpa (7%), D. regia (5%) and C. ferrea (4.3%). These values
agree rather well with other woods reported in the literature. A
study carried out by Santana and Okino (2007) with 36 Brazilian
tropical timbers found extractive content between 17.3% and 0.9%,
but only eight of the thirty six species showed extractive content
higher than 10%. In that study M. huberi had an extractives content
of 8% so is comparable with our results. Species with high contents
of extractives are of interest for future studies because chemical
compounds from the crude extracts can be used for pharmaceuticals, dyes, cosmetics, perfumes and natural antioxidants (Santana
and Okino, 2007). High extractive content is known to result in
good natural durability (Windeisen et al., 2002).
The result of the preliminary phytochemical analysis of the
extract of wood species is shown in Table 3. The phytochemical
studies revealed the presence of avonoids, terpenes and steroids
in all analyzed woods. Tannin presence was very strong in
A. colubrina. Saponins were found only in M. huberi indicating that,
among the studied woods, there were signicant differences in the
amount and in the composition of extractives. Phenolic
compounds, such as tannins and avonoids, have antimicrobial and
antioxidative properties and are involved in the defense against
fungi and other microorganisms (Boudet, 2007). Saponins have
been 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 of
studies have demonstrated the importance of these metabolites as

plant defense compounds. A high diversity of extractives in high

concentration provides a more effective protection against herbivores than single compounds or low diversity in both low and high
concentrations (Castellanos and Espinosa-Garcia, 1997). The
diversity (Table 2) as well as the concentration of phenolic
compounds (Table 3) was more pronounced in A. colubrina and
M. huberi in sharp contrast to D. regia and C. ferrea.
Table 4 provides the quantication of lignin. According to Syafh
et al. (1988) lignin is the most important non-toxic factor that limits
the growth of the microorganisms in wood biodegradation.
M. huberi presents the highest content of total lignin (30%). In this
study, D. regia had the lowest concentration of lignin (22.3%), thus
suggesting that lignin may be the main factor for the low resistance
of this wood. Among 36 woods analyzed, Santana and Okino (2007)
found an acid-soluble lignin (ASL) content of 0.7e1.8% and an acidinsoluble lignin (AIL) of 26.7e37%. The values for M. huberi were
34% AIL and and 0.9% ASL. The lignin content for all woods was
similar and typical for tropical hardwoods, except for D. regia. These
results suggest that differences in the content and structure of
lignin may inuence the decay resistance caused by wooddecaying fungi (Syafh et al., 1988).
The content of phenolics (mg g1) in ethanol extracts was
determined from a regression equation of the calibration curve
(y 0.004663x 0.0565, R2 0.99) and expressed in Gallic Acid
Equivalents (GAE). As shown in Table 5, there is a close correlation
between the phenolic content and the % DPPH radical quenched.
The ethanol extract from H. stigonocarpa contained the highest
amount of total phenolics and, consequently, the highest antioxidant activity, followed by A. colubrina, C. ferrea, D. regia, and
M. huberi. Although D. regia has higher phenolic contents than
M. huberi, it displays the lowest antioxidant activity. These results
indicate that phenolic composition among both woods differ
widely in terms of chemical composition.
The inuence of extractives on the growth of P. chrysosporium in
the tested woods is shown in Table 6. The extracted and unextracted wood of M. huberi inhibited fungal growth completely,
conrming that lignin plays an important role in natural resistance
of this wood (Syafh et al., 1988; Tuomela et al., 2000). This observation suggests that the content of saponins is not responsible for
the natural resistance of M. huberi against fungal attack since decay
resistance was not affected by their extraction (Table 3).
Among all the studied woods, A. colubrina was the only species
where the inuence of extractives was very well observed. Fungal
growth was entirely inhibited by unextracted wood but intense
growth formed in the presence of extracted wood (Fig. 1). Rowell
et al. (2005) states that wood resistance to fungal attack is attributed mainly to the presence of extractives that are toxic to

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

Extraction procedure

Hymenaea stigonocarpa

Anadenanthera colubrina

Caesalpinia ferrea

Manilkara huberi

Delonix. regia

Saponins
Tannins
Alkaloids
Flavonoids
Steroids and terpenoids

Frothing
Ferric chloride
Dragendorff, Mayer
Shinoda Oxalo-boric acid
LiebermanneBuchard





() not detected; () detected. The number of positive signs indicates the intensity of the reactions (Costa, 1982).

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

Table 4
Lignin content of the studied woods species.
Lignin (%)

Hymenaea stigonocarpa

Anadenanthera colubrina

Caesalpinia ferrea

Manilkara huberi

Delonix regia

Acid-soluble (ASL)
Acid-insoluble (AIL)
Total

0.6  0.4
27  1
28  1

1.81  0.03
25.9  0.9
27.6  0.9

1.7  0.3
26  4
28  4

1.40  0.06
28.6  0.4
30.0  0.4

0.3  0.1
22  1
22.3  1

Table 5
Total 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 g1 of extracts

% DPPH radical quenched

0.248  0.009
91.00  0.050

0.231  0.003
71.90  0.099

0.151  0.005
21.51  0.358

0.069  0.004
5.99  0.050

0.092  0.007
3.07  0.14

Table 6
Phanerochaete chrysosporium growth in the studied woods species.
Wood
samples

Hymenaea
stigonocarpa

Unextracted
wood
Extracted

wood

Anadenanthera Caesalpinia Manilkara Delonix

colubrina
ferrea
huberi
regia


() no growth; () weak, () medium and () abundant growth of Phanerochaete chrysosporium.

xylophagous organisms, thus providing the wood with natural

durability. In addition, A. colubrina contains a high content of
phenolic compounds and high antioxidant activity (Table 5) and also
has good natural resistance to termites (Silva et al., 2007; Santana
et al., 2010). According to Schultz and Nicholas (2000), the radical

scavenging activity of phenolic compounds may further accelerate

fungal death by scavenging the radicals produced by fungus and
reducing 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. stigonocarpa
stimulates only weak growth of P. chrysosporium, whereas fungal
growth in the presence of extracted wood is far more pronounced,
conrming that the extractives could not completely inhibit the
growth of the fungus but acted as a primary barrier to prevent
colonization. Although H. stigonocarpa extractives did not inhibit
completely fungus growth, this wood is known for its natural
resistance (Table 1). According to Rudman (1965), heartwood
extractives from durable species are not always toxic against
a broad spectrum of wood-destroying fungi but are often specic to
a limited number of species. Apparently, the extractives of
H. stigonocarpa do not exhibit strong antifungal activity against
P. chrysosporium used in this study.

Fig. 1. The growth of Phanerochaete chrysosporium showing on extracted and unextracted woods from Hymenaea stigonocarpa (left) and Anadenanthera colubrina (right).

Fig. 2. The growth of Phanerochaete chrysosporium showing on extracted and unextracted woods from Delonix regia (left) and Caesalpinia ferrea (right).

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

Fig. 2 shows similar behavior for C. ferrea and D. regia in respect

to fungal growth. The extractives from both woods showed weak
antifungal activities. Usually avonoids and tannins are responsible
for antioxidant activity, which sequester the free radicals produced
by fungi during the decay process (Schultz and Nicholas, 2000).
C. ferrea and D. regia have a lower content of extractives (Table 2) and
phenolic compounds (Table 5) than other woods. These factors may
explain the low resistance of both woods against P. chrysosporium.
4. Conclusion
The durability of A. colubrina can be attributed mainly to the
presence of phenolic compounds, particularly tannins and avonoids that inhibit fungal growth. Although the wood of M. huberi
contains large quantities of extractives, especially saponins, they are
not the only compounds responsible for antifungal activity. The high
lignin content and the chemical composition of the lignin seem to be
the major reasons for the high decay resistance of this wood. The
naturally low durability of C. ferrea and D. regia wood against P.
chrysosporium may be the result of various factors, including the low
quantity of extractives, the low antioxidant activity of the phenolic
extractives, and the structural composition of the lignin.
Acknowledgements
The authors are grateful to Coordenao de Aperfeioamento de
Pessoal de Nvel Superior (CAPES) and Conselho Nacional de
Desenvolvimento Cientco e Tecnolgico (CNPq) for grants and
fellowships.
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