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Assessment of total phenols and extractives of mahogany wood by near

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Holzforschung, Vol. xx, pp. xxx–xxx, 2012 • Copyright © by Walter de Gruyter • Berlin • Boston. DOI 10.1515/hf-2011-0207
Assessment of total phenols and extractives of mahogany
wood by near infrared spectroscopy (NIRS)
Allan Ribeiro da Silva1, Tereza Cristina Monteiro
Pastore2,*, Jez Willian Batista Braga1, Fabrice
Davrieux3, Esmeralda Yoshico Arakaki Okino2,
Vera Teresinha Rauber Coradin2, José Arlete Alves
Camargos2 and Alexandre Gustavo Soares do Prado
(In memoriam)1
1 Chemistry
Institute, University of Brasilia, Brasília, DF,
Brazil
2 Forest Products Laboratory, Brazilian Forest Service,
Brasilia, DF, Brazil
3 French Agricultural Research Centre for International
Development, CIRAD-UMR Qualisud, Montpellier,
France
* Corresponding author.
Laboratório de Produtos Florestais
Serviço Florestal Brasileiro, SCEN trecho 2
Bloco D 70818900, Brasilia, DF, Brazil
E-mail: tereza.pastore@florestal.gov.br
Abstract
The resistance to decomposition of mahogany wood
(Sweitenia macrophylla King) ranges from high to moderate
level. Wood extractives, mainly due to the presence of phenol
compounds are related to the natural durability of wood. The
technique of near infrared spectroscopy (NIRS) coupled with
multivariate analysis has been applied to assess the extrac-
tives and phenols of 41 samples of mahogany in powder
form. The hot water-soluble extractives were quantitatively
determined, and total phenol content was measured with the
Folin-Denis colourimetric reagent. Models were developed
with the NIRS data for each of the two variables. The results
indicated that NIRS can be a useful tool to a rapid evaluation
of the extractive contents and total phenolic compounds of
mahogany wood. The method was able to predict the interest-
ing properties with errors lower than 10% (w/w) and had the
capability of detecting samples that have a minimum concen-
tration of 2.4% (w/w) of extractives and total phenolic com-
pounds, respectively.
Keywords: extractive content; mahogany; NIRS; total phe-
nol content.
Introduction
Swietenia macrophylla King (Meliaceae), also called maho-
gany, caoba or bigleaf mahogany, is one of three tree spe-
cies, regarded as “genuine mahogany” and is distributed from
tropical Central to South America. Valuable mahogany wood
is used for many purposes, such as fine furniture, cabinet
making, civil constructions, boats, ships, interior trim, panel-
ling, veneers, musical instruments, turnery and carving. Its
characteristics are a glossy pink-red colour, medium specific
gravity (0.50 to 0.72 g cm-3), good wood machining (Torelli
and Čufar 1996; Coradin et al. 2009) and dimensional stabil-
ity (Melo et al. 1989; Torelli and Gorisek 1995). Mahogany
bark (Falah et al. 2008) and seeds (Bourdy et al. 2000) are
also valuable due to their biological activity. The chemical
composition of wood of S. macrophylla is still not well inves-
tigated though the natural durability of its wood is directly
related to the kind and amount of its extractives.
Extractives are organic substances of low molecular
weight that are not components of the cell wall matrix. They
are responsible for colour and resistance to fungi, insects
and marine borers. They may also act as toxins, as well as
repellents to insects, and have antioxidant properties (Fengel
and Wegener 1984; Venäläinen et al. 2006; Lim et al. 2007;
Mburu et al. 2007; Binbuga et al. 2008; Borges et al. 2008;
Gao et al. 2008; Donoso-Fierro et al. 2009; Little et al. 2010).
Chemically, the extractives vary between species, genera, and
various anatomical parts of wood may differ in terms of con-
centration and composition. In the last decade, the research
on extractives of softwoods has advanced a lot. Especially,
the discovery that the knots contain commercially attractive
components was a stimulus for this research area (Eaton and
Hale 1993; Willför et al. 2003a,b, 2005; Smeds et al. 2011).
Hardwoods may also contain terpenes, fats, waxes, phenols,
lignans, quinones, tannins, flavonoids and other compounds.
Interestingly, hardwoods contain waxes more frequently
than fatty acids. Tannins from woods and barks are phenolic
compounds of great economic interest and have toxic effects
against microorganisms and radical scavenging properties
(Monteiro et al. 2005; Makino et al. 2011).
The natural durable tropical woods contain more extrac-
tives than the woods from temperate climate zones. Déon et
al. (1980) noted that most research about the role of extrac-
tives in the natural durability of wood was focussed on
woods from temperate climates. So far, scientific contribu-
tions concerning extractives from Brazilian forest species
are rare. Jesus et al. (1998) studied the resistance of wood
decay organisms of 46 species of trees selected based on the
occurrence and availability in three regions of the Amazonas
state. Garlet (2010) investigated the same and other tropical
species for approximately 25 years in field tests installed in
a forest in Pará state. Carneiro et al. (2009) tested the natural
resistance of 28 Amazon tropical wood species against five
types of fungi. The authors introduced the parameter “resis-
tance potential”, which was obtained by multiplying the
2011/0207
 Article in press - uncorrected proof Page 2 of 31

2 A. Ribeiro da Silva et al.

wood densities by their extractive contents. Thus, it was pos- predict the levels of total phenolics and extractives of the
sible to verify a trend of direct correlation between weight wood of Larix decidua, Larix leptolepis and a hybrid (Larix
losses (WLs), extractive content and the “natural resistance” x eurolepis). The quoted authors tested 175 trees of different
parameter. They asserted that the tropical woods contain- provenances. Taylor et al. (2008) constructed a NIRS-based
ing 7% or more extractives had a higher natural resistance prediction model with 57 samples of mahogany collected
as indicated by less WL in decay tests. In the list of natural in Central America and South America; they considered the
durability, developed by Scheffer and Morrell (1998), the total volumetric shrinkage, the amount of extractive and spe-
decay resistance of S. macrophylla was classified in the range cific gravity as parameters. The extractives were quantified
from high to moderate. Torelli and Čufar (1994) reported by successive extraction with toluene/ethanol (2:1), 95%
that Trametes versicolor causes 36% WL and Gleophyllum ethanol and hot water. The average content of total extrac-
trabeum 16% WL with S. macrophylla. Mahogany wood is tives found was 18.1%, with values ranging from 11.8% to
also highly resistant against the attack of dry wood termites; 28.1%. Reasonable predictions could be done with the mod-
however, its durability is low in contact with moisture soil els developed, especially in cases when the improved statisti-
(Embrapa, 2011). cal techniques (“kernel and wavelet”) were applied. Zahri et
The water soluble extractive content of Brazilian mahog- al. (2008) assessed quantitatively the total phenol contents of Q1:
any was found to be 8.6% and 7.3%; alcohol-benzene solubles European oak by means of NIRS. Please supply
The objective of the present study was to correlate the total manufacturer’s
were detectable (Brazilian Technical Association of Pulp and
name,
Paper, ABCP Technical Standards; Jankowsky and Galvão content of phenolic compounds and the concentrations of hot city, state,
1979). For mahogany, native to Mexico, Rutiaga-Quiñones water solubles of mahogany wood with NIRS data for a rapid country for
et al. (1998a) tested the solubility with petrol ether (0.8%), prediction of the extractive contents of unknown mahogany all reagents,
acetone (3.2%), methanol (3.4%) and hot water (1.1%) and samples. devices,
cold water (2.1%) (standard test DIN 4188) and found in total software
mention
10.6% extractives. Rutiaga-Quiñones et al. (1998b) analy-
Material and methods throughout
sed the cell wall components by total hydrolysis and found
rhamose (0.4%), mannose (1.1%), arabinose (0.4%), galac-
tose (1.4%), xylose (12.3%) and glucose (55.0%), accounting Thirty-two samples of mahogany from Mato Grosso state in Brazil
were cut from the tips of boards stored under dry conditions. Nine
for 63.3% of total polysaccharides. The content of inorganic
other samples were prepared by mixing calculated amounts of two
compounds amounts to 0.36% and the lignin content to 31.1% powder samples with known content of hot water soluble extractives.
(Mejía-Díaz and Rutiaga-Quiñones 2008). Torelli and Čufar Thus, 41 samples were obtained at concentrations of extractives dis-
(1995a) detected traces of silica in the ash (0.32%) of wood tributed between the maximum and the minimum values found in
collected from Mexico. The pH values varied from 5.3 to the original samples (Table 1). The intentions were to reduce the
5.0 from the heartwood to the sapwood (Torelli and Čufar problem of low number of samples and to obtain a set of concen-
1995b). trations of extractives and total phenol uniformly distributed. The
Vegetable tannins can be quantified via precipitation of anatomical identification of woods was carried out at Forest Products
Q2: Please
proteins and metals or by colourimetric methods. Among Laboratory in Brasilia, registered as FPB in the Index Xylariorum
indicate
them, the Folin-Denis method has been widely used for the (Stern 1988). Each sample was air dried until reaching around 9%
clearly that
moisture content (MC), and part of it was chipped and milled in a
determination of total phenolics. However, the selectivity the use of
Wiley mill. The fraction of saw mill that passed through a 0.40-mm bold/italic
of this method is limited, as it can also react with oxidising (40 meshes) screen was used for analysis. The MC and hot water
and reducing agents present in the samples and as it inter- and roman
solubles were obtained according to TAPPI standards T 264 om-88 fonts for vari-
feres with the spectrometric reading. The tannins have a great and T 207 om-93, respectively. ables have
variety of structures and form readily complex products with Folin-Denis (FD) colourimetric reagent was purchased from Fluka been applied
a variety of oxidative materials; that is the reason why the Analytical. A standard calibration curve was constructed with tannic corrrectly
quantification process is controversially discussed in the lit- acids: 6 ml of distilled water was added to a 10-ml volumetric flask, throughout in
erature (Monteiro et al. 2005). followed by 0.1 ml of wood extractives and 0.5 ml of FD reagent. equations/text
Near infrared spectroscopy (NIRS) is a routine technique After 3 min, 1 ml of saturated solution of Na2CO3 was added to the and figures.
with little sample preparation for a rapid quality control of flask, and the volume was filled up. The mixture was centrifuged and
various complex products (Hein et al. 2010). NIRS is also
useful in wood science; it has been successfully tested for
predicting decay resistance (Fackler et al. 2007; Leinonen et Table 1 Average results for extractives and total phenols of
al. 2008), detecting inorganic preservatives (So et al. 2004; mahogany wood samples.
Taylor and Lloyd 2007) and, recently, discriminating tropical
wood species that are anatomically similar (Braga et al. 2011; Extractives Total phenols
Pastore et al. 2011). content (%) content (%)
Several authors estimated the extractive contents of vari- Average 8.38 6.81
ous woods (Terdwongworakul et al. 2005; Tsuchikawa Maximum 14.83 13.96
2007; Zahri et al. 2008; Schimleck et al. 2009). Gierlinger Minimum 4.01 2.25
et al. (2002) showed that NIRS can substitute time-consum- Standard 2.9 2.72
ing conventional methods of analysis and is able to quickly Deviation
 Page 3 of 31 Article in press - uncorrected proof
Q3: filtered after 40 min of reacting. The UV-Vis spectra were collected
after 1 h of adding saturated Na2CO3, and the λmax at 725.0 nm was
“UV/Vis” was
originally set
with a hyphen
measured. Instrument: UV/Vis FEMTO 800XI spectrophotometer
equipped with 1 cm quartz cells; scanning range: 490–850 nm.
between
“UV” and
Near infrared spectroscopy (NIRS)
“Vis”. Please
adopt one to
Instrument: Tensor 37 FT-IR spectrometer (Bruker Optics, Germany)
be consistent
throughout equipped with the Easi DiffTM diffuse reflectance accessory (Pike
the article Technologies Inc.). The spectra were collected at 4 cm-1 intervals
over 12,000 to 4000 cm-1. The instrument reference was a standard
mirror provided by the equipment manufacturer. A total of 64 scans
were accumulated for each spectrum, and three spectra were aver-
aged for data collection. Climate during spectroscopy: relative hu-
midity < 60% at 20°C.
Data analysis and model development
NIRS data were evaluated by means of a partial least square regres-
sion model (PLSR), which is a statistical method for calculating mod-
els for quantitative analysis in several areas (Geladi and Kowalski
1986; Haaland and Thomas 1988; Martens and Naes 1989; Brereton
2000). Shortly: the data matrix X is formed by the NIR spectra of
wood, whereas vector y is composed by the concentration of phe-
nolics and solubles in hot water. Thus, two different PLSR models
were developed with one dependent variable for each. The QUANT
2 routine provided by the OPUS software version 6.5 was applied to
this purpose (Bruker Optics, Germany).
The data were split into calibration and validation sets. In the
first one, 26 samples of mahogany were considered; the number of
latent variables was detected by means of the “leave one out cross-
validation procedure” (Thomas 1994). In the validation set, 12
independently prepared samples were included. The outlier identifi-
cation was performed by the test for extreme leverage, unmodelled
residuals in spectra and unmodelled residuals in dependent variables
(Valderrama et al. 2007). The remaining three samples were not con-
sidered because they showed inconsistent results for extractives and
total phenols obtained with the reference method.
That spectral range was chosen as the best one, which provided
the lowest “root mean square error of cross-validation” (RMSECV),
or the lowest number of latent variables, after the RMSECV had
reached an approximately constant value. The validation of the NIR
method was performed by the determination of the figures of merit.
To evaluate the accuracy of the method, the root mean square error
of calibration and prediction (RMSEP and RMSEC, respectively)
(Brereton, 2000) were calculated, and a paired t-test was performed
with the reference and estimated values.
By the definition of the RMSEP, a relative error of prediction
(REP) can be obtained according to Miller-Ihli et al. (1984):
I (y i − yˆ i )2 (1)
REP = ∑ 2
100
n=1 I cal y i
Sensitivity is an important parameter to compare the performance
of two independent methods. In inverse multivariate calibration
models, such as PLSR, it is determined by the inverse of the norm
of the regression coefficients (Lorber et al. 1997). However, the ca-
pability of detection (CCβ) described in the ISO 11843-2 provides
an easier and a direct comparison of the detectable concentrations of
the methods. Ortiz et al. (2003) have extended the CCβ application
for multivariate calibration methods such as PLSR. Accordingly, the
CCβ can be determined as:
Extractive content of mahogany 3
s 1 1 y2 (2)
CCβ= δa ,β + + Ical
b m I cal
∑ ( yi − y )2
i =1
where, s is the standard deviation of the residuals of the regression of
the estimated concentrations (ŷi) against the reference concentration
of calibration samples (y) with the intercept “a” and slope “b”, m is
the number of replicate measurements performed on each sample,
ȳ is the mean concentration of the standards and δα,β,ν is the non-
centrality parameter from the noncentral t-distribution with Ical-2
degrees of freedom.
Results and discussion
Wet chemical analyses
The contents of water-soluble and total phenols are statistically
variable (Table 1). Figure 1 is the corresponding in-detail plot
for 41 mahogany samples determined by conventional meth-
ods. The average content of extractives was 8.4% ranging from
4.0% to 14.8% with a standard deviation of 2.9%. Our results
corroborate the findings of Taylor et al. (2008). The authors
also found a wide variation in levels of total extractives of
S. macrophylla of Central America and South America, rang-
ing from 11.8% to 28.1% with a standard deviation of 3.1%.
The variability of extractive content between trees of a given
species has been demonstrated by several authors (Hillis 1962;
Giergeling 2002) and was even observed in a single individual
tree, when collecting samples of oak disks at different dis-
tances from the pith (Taylor et al. 2011).
The concentration of soluble phenols in hot water of
mahogany samples also varied widely as shown in Figure 1.
The mean average found for all samples was 6.8%, and the
standard deviation was 2.7%. For the European oak, Zahri
et al. (2008) found values varying from 1.3% to 7.0%, and
they also demonstrated that this class of compounds decreased
with age of the tree. The interpretation was that phenolics
bind with time irreversibly to the cell wall matrix and become
insoluble. The age of the mahogany woods collected for the
experiment of the present work is unknown. The variability of
polyphenol concentrations collected from woods with equal
age may be lower.
As illustrated in Figure 1, the data of samples 16, 20 and
34 are outliers, i.e., they contain essentially more extractives
than the others. The Folin-Dennis reagent is one of the most
widely used in colourimetric procedures to determine total
soluble phenolics. Nevertheless, the method fails sometimes
(Reed 1995). The extinction coefficient of chromophores pro-
duced with the tannic acid may have different values in some
wood samples. The literature may contain overestimated or
underestimated data for the total phenol contents.
Spectroscopic characterization and determination
As usual, visual comparison of the NIR spectra of an extracted
and not extracted wood sample do not reveal any obvious
spectral differences (Figure 2a). The insect in Figure 2a
 Article in press - uncorrected proof Page 4 of 31

4 A. Ribeiro da Silva et al.

25 Hot water extractives Total phenols

20
Concentration (%)

15

10

0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41
Sample number

Figure 1 Mean values and standard deviations of the contents of hot water extractives and total phenols of 41 Brazilian mahogany tree
samples. Samples 1– 33 – each value represents the average of a sample taken from the edge of a board of a tree. Samples 34–41 – each value
represents a mixture of powders of various trees. All determinations were made in triplicate.

a 0.001
1st Derivative

0
2 -0.001 1410

-0.002 Unextracted sample

-0.003 1900

1.8 1300 1400 1500 1600 1700 1800 1900 2000

Wavelength (nm)

Hot water extracted sample

1.6
Absorbance

1.4
1000 1200 1400 1600 1800 2000 2200 2400

b
1st Derivative

0
2 2136
-0.001 1410 2084

-0.002 Sample 1
-0.003 1900
1.8 1400 1600 1800 2000 2200 2400
Wavelength (nm)

Sample 2
1.6

1.4
1000 1200 1400 1600 1800 2000 2200 2400
Wavelength (nm)

Figure 2 Original NIR spectra of one mahogany (S. macrophylla) set of samples (a) with and without hot water extractives, (b) with the maxi-
mal and minimal concentration of hot water extractives. The spectra in the first derivative mode of wood powder are presented in the inset.
Page 5 of 31 Article in press - uncorrected proof
Extractive content of mahogany 5

Table 2 Model parameters. Table 3 Figures of merit for the PLS model.

Hot water Extractives Total phenols

Parameter extractives Total phenols Parameter (%) (%)
Number of latent 4 3 RMSECa 0.46 0.48
variables RMSECVa 0.63 0.66
Spectral region (nm) 1639.1–1731.6 1639.1–1731.6 RMSEPa 0.62 0.72
Pre-processing 1st derivate +vector 1st derivate +vector Relative error of predictionb 8.9 9.9
normalisation + mean normalisation + mean Sensitivity ( × 10 -3) 1.4 2.0
centre centre Capability of detectiona 2.4 2.4
Outliers Calibration 2 Estimated t value 0.013 0.9714
Validation 0 Noise level ( × 10 -4) 1.1 1.4
aPercentage (w/w), bpercentage.

presents the first-derivative spectra, which minimises the

effects resulting from particle size and other physical factors
caused by the reflectance measurement. The bands at 1410
and 1900 nm are elevated by hot water extraction. These
bands may be attributed tentatively to the first overtone of
the O-H and the combination bands of O-H and C = O bonds,
respectively, as interpreted by Michell and Schimleck (1996)
based on the experiments with water extraction of Eucalyptus
globulus.
Samples illustrated in Figure 2b represent the maximum
and minimum content of wood extractives observed in the
dataset. The inset of Figure 2b shows more spectral differ-
ences at 2084 and 2136 nm, which correspond to the region
of the O-H and C-H combination bands in the first-derivative
NIR spectra of wood samples. Michell and Schimleck (1996)
and Schawanninger et al. (2011) attributed these vibrations to
cellulose and xylan (2084 nm) and to lignin and extractives
(2136 nm).
The parameters used for the development of the two PLSR
models for the extractives and total phenols are summarised
in Table 2. The selected region for model development was
for extractives (Figure 3a) and total phenols (Figure 3b) are
shown. The absence of overfitting is obvious based on the
scattering data of the calibration and validation samples in
these figures. Considering the amplitude of the variation of
the extractive and total phenols, the relative error of predic-
tion (REP) of both models was almost 10%. However, it is
important to note that this error can be highly affected by only
a few samples presenting a large error. For example, for total
phenol determination, only 2 of the 12 samples present errors
larger than 10%. If these samples were considered outliers,
the new RMSEP and REP values would be 0.42% (w/w) and
15
a
3
13
11
9
Estimated concentration, % (w/w)

7
1639 to 1731 nm, which corresponds to the first overtone of
the C-H bonds in wood and provided the lowest RMSECV 5
but not the largest spectral differences. This suggests that the
3
main spectral differences may be caused by other wood com- 3 4 5 6 7 8 9 10 11 12 13 14
ponents and that all chemical information about the total phe-
nols and extractive content are spread in the entire spectrum, b
11.5
minimizing the influence of the other sources of variation.
The best pre-processing method was obtained by combin- 9.5
ing the first derivate of the NIR spectra, vector normalisation
and mean centreing. Table 2 also indicates that two and three 7.5
outliers were observed in the calibration set. All outliers were
5.5
identified by high prediction errors (residuals in dependent
variables). 3.5
Table 3 presents some figures of merit of the method.
The RMSEs represent the average error of the method. 1.5
Approximately, the same RMSEP values were observed for 1.5 3.5 5.5 7.5 9.5 11.5
Reference concentration, % (w/w)
the determination of the extractives and total phenol contents
indicating the same accuracy for both properties investigated.
Figure 3 Plots’reference concentrations vs. estimated concentra-
Comparison of the RMSEP and RMSEC by an F-test showed tions obtained by PLS model. (a) determination of extractive content,
no significant difference for both models (95% of confi- ( ) calibration samples, ( ) test samples, (—) ideal correlation line.
dence), indicating that no overfitting occurred. Plots “refer- (b) determination of total phenol content, ( ) calibration samples, (Δ)
ence vs. estimated values” for the calibration and test samples test samples, (—) ideal correlation line.
 Article in press - uncorrected proof
6 A. Ribeiro da Silva et al.
6.6%, respectively. The paired t-test performed with the vali-
dation samples shows that no systematic errors are present in
the model.
The noise level, estimated based on the spectral residu-
als of the PLSR model for the calibration samples, is con-
sistent with the instrumental variation in the NIR data. This
means that all systematic variation in the data was modelled
by PLSR. The sensitivity of the method is also illustrated in
Table 3. However, the capability of detection expresses the
sensitivity as well, but this parameter allows an easier inter-
pretation by presenting the same unit of the properties deter-
mined. According to the results, the lowest concentration that
can be determined by the model for both extractives and total
phenols is 2.4% (w/w). This makes possible the determina-
tion of the extractives and the total phenols in almost every
concentration range observed in the samples.
Conclusions
The results of the NIR method for determination of extrac-
tives and total phenol content are in good agreement with
the reference method. The accuracy and the detection results
demonstrated that the model has an appropriate sensitivity
and that the prediction performance for the determination of
the properties in focus is acceptable for a routine analysis.
The NIR methodology has advantages in comparison to the
reference method, such as an easy sample preparation (if
any) and short analysis times ( < 5 min). Disadvantage is the
careful calibration work needed before spectroscopy and the
necessity for a regular control of stability of the calibration
system.
Acknowledgements
The authors would like to thank FAPDF for the financial support
as well as the fellowship of Allan R. da Silva. The endeavours of
Dr. Paulo J.P. Fontes and Mr. Waldemar V. Lopes were essential to
obtain the necessary wood.
References
Binbuga, N., Ruhs, C., Hasty, J.K., Henry, W.P., Schultz, T.P. (2008)
Developing environmentally benign and effective organic
wood preservatives by understanding the biocidal and non-bi-
ocidal properties of extractives in naturally durable heartwood.
Holzforschung 62:264–269.
Borges, L.M.S., Cragg, S.M., Bergot, J., Williams, J.R., Shayler,
B., Sawyer, G.S. (2008) Laboratory screening of tropical hard-
woods for natural resistance to the marine borer Limnoria
quadripunctata: the role of leachable and non-leachable factors.
Holzforschung 62:99–111.
Bourdy, G., DeWalt, S.J., Chávez de Michel, L.R., Roca, A., Deharo,
E., Muñoz, V., Balderrama, L., Quenevo, C., Gimenez, A. (2000)
Medicinal plants uses of the Tacana, an Amazonian Bolivian eth-
nic group. J. Ethnopharmacol. 70:87–109.
Braga, J.W.B., Pastore, T.C.M., Coradin, V.T.R., Camargos, J.A.A.,
Silva, A.R. (2011) The use of near infrared spectroscopy to
Page 6 of 31
identify solid wood specimens of Swietenia macrophylla. IAWA
J. 32:285–296.
Brereton, R.G. (2000) Introduction to multivariate calibration in ana-
lytical chemistry. Analyst 125:2125–2154.
Carneiro, J.S., Emmert, L., Sternadt, G.H., Mendes, J.C., Almeida,
G.F. (2009) Decay susceptibility of Amazon wood species from
Brazil against white rot and brown rot decay fungi. Holzforschung
63:767–772.
Coradin, V.T.R., Camargos, J.A.A., Marques, L.F., Silva Jr E.R.
Madeiras similares ao mogno (Swietenia macrophylla King):
Q4:
chave ilustrada para identificação anatômica em campo. Serviço
Are the
Florestal Brasileiro/LPF, Brasilia, 2009.
reference
Déon, G., Chadenson, M., Hauteville, M. (1980) Influence des
“Embrapa”,
extraits naturels du bois sur sa resistance a la pourriture. Bois.
“Garlet
For. Trop. 191:75–89. (2010)”,
Donoso-Fierro, C., Becerra, J., Bustos-Concha, E., Silva, M. (2009) “ISO
Chelating and antioxidant activity of lignans from Chilean woods 111843-2
(Cupressaceae). Holzforschung 63:559–563. standared
Eaton, R.A., Hale, M.D.C. Wood: Decay, Pest, and Protection. (2000)”
Chapman & Hall, Great Britain, 1993. “Tappi
Embrapa (http://www.cnpf.embrapa.br/publica/circtec/edicoes/ standards
Circular140.pdf), accessed June 20, 2011. (1988)”
Fackler, K., Schwanninger, M., Gradinger, C., Srebotnik, E., and “Tappi
Hinterstoinsser, B., Messner, K. (2007) Fungal decay of spruce standards
and beech wood assessed by near-infrared spectroscopy in com- (1993)”
bination with uni- and multivariate data analysis. Holzforschung formatted
correctly?
61:689–687.
Falah, S., Suzuki, T., Katayama, T. (2008) Chemical constituents Q5:
from Swietenia macrophylla bark and their antioxidant activity. Please
Pak. J. Biol. Sci. 11:2007–2012.
confirm the
Fengel, D., Wegener, G. Wood – Chemistry, Ultrastructure, Reactions.
initial for
De Gruyter, Berlin, 1984.
Gao, H., Obanda, D.N., Shupe, T.F., Hse, C.Y., Ring, D.R. (2008) "schwan-
Antifungal activities of heartwood extracts of Port-Orford cedar ningu M"
extractives. Holzforschung 62:620–623. in ref.
Garlet (2010) http://www.noticiasdaamazonia.com.br/13093-estudo- Gierlinger
do-servico-florestal-revela-madeiras-mais-resistentes). et al (2002)
Geladi, P., Kowalski, B.R. (1986) Partial least-squares regression – a
tutorial. Anal. Chim. Acta 185:1–17.
Gierlinger, N., Schwanninger, M., Hinterstoisser, B., Wimmer, R.
(2002) Rapid determination of heartwood extractives in Larix
sp by means of Fourier transform near infrared spectroscopy. J.
Near Infrared Spec. 10:203–214.
Haaland, D.M., Thomas, E.V. (1988) Partial least-squares for spec-
tral analyses. 1. Relation to other quantitative calibration meth-
ods and the extraction of qualitative information. Anal. Chem.
60:1193–1202.
Hein, P.R.G., Lima, J.T., Chaix, G. (2010) Effects of sample prepa-
ration on NIR spectroscopic estimation of chemical proper-
ties of Eucalyptus urophylla S.T. Blake wood. Holzforschung
64:45–54.
Hillis, W.E. Wood Extractives and their Significance to the Pulp
and Paper Industries. Academic Press, New York and London,
1962.
ISO 11843-2 standard (2000) Capability of detection.
Jankowsky, I.P., Galvão, A.P.M. (1979) Influência do teor de extrati-
vos na umidade de equilíbrio da madeira. IPEF 18:1–33.
Jesus, M.A., Morais, J.W., Abreu, R.L.S., Cardias, M.F.C. (1998)
Durabilidade natural de 46 espécies de madeira amazônica em
contato com o solo em ambiente florestal. IPEF 54:81–92.
Leinonen, A., Harju, A.M. Venäläinen, M., Saranpää, P., Laako, T.
(2008) FT-NIR spectroscopy in predicting the decay resistance
related characteristics of solid scots pine (Pinus sylvestris L.)
heartwood. Holzforschung 62:284–288.
Page 7 of 31 Article in press - uncorrected proof
Lim, Y.W., Chedgy, R.J., Amirthalingam, S., Breuil, C. (2007)
Screening fungi tolerant to Western red cedar (Thuja plicata
Donn) extractives. Part 2. Development of a feeder strip assay.
Holzforschung 61:195–200.
Little, N.S., Schultz, T.P., Nicholas, D.D. (2010) Termite-resistant
heartwood. Effect of antioxidants on termite feeding deterrence
and mortality. Holzforschung 64:395–398.
Lorber, A., Faber, K., Kowalski, B.R. (1997) Net analyte signal cal-
culation in multivariate calibration. Anal. Chem. 69:1620–1626.
Makino, R., Ohara, S., Hashida, K. (2011) Radical scavenging
characteristics of condensed tannins from barks of various tree
species compared with quebracho wood tannin. Holzforschung
65:651–657.
Martens, H., Naes, T. Multivariate Calibration. John Wiley & Sons,
New York, 1989.
Mburu, F., Dumarçay, S., Gérardin, P. (2007) Evidence of fungicidal
and termicidal properties of Prunus africana heartwood extrac-
tives. Holzforschung 61:323–325.
Mejía-Díaz, L.A., Rutiaga-Quinõnes, J.G. (2008) Chemical compo-
sition of Schinus molle L. wood and kraft pulping process. Rev.
Mex. Ing. Quim. 7:145–149.
Melo, J.E., Carvalho, G.M., Martins, V.A. Espécies de madeiras sub-
stitutas do mogno. IBAMA, Brasília, 1989.
Michell, A.J., Schimleck, L.R. (1996) NIR spectroscopy of woods
from Eucaliptus globulus. Appita 49:23–26.
Miller-Ihli, N.J., O’Haver, T.C., Harnly, J.M. (1984) Calibration
and curve fitting for extended range AAS. Spectrochim. Acta B
39:1603–1614.
Monteiro, J.M., de Albuquerque, U.P., Araújo, E.L. (2005)
Taninos: uma abordagem da química à ecologia. Quim. Nova
28:892–896.
Ortiz, M.C., Sarabia, L.A., Herrero, A., Sánchez, M.S., Sanz, M.B.,
Rueda, M.E., Giménez, D., Meléndez, M.E. (2003) Capability of
detection of an analytical method evaluating false positive and
false negative (ISO 11843) with partial least squares. Chemometr.
Intell. Lab. Syst. 69:21–33.
Pastore, T.C.M., Braga, J.W.B., Coradin, V.T.R., Magalhães, W.L.E.,
Okino, E.Y.A., Camargos, J.A.A., Muñiz, G.I.B., Bressan,
O.A., Davrieux, F. (2011) Near infrared spectroscopy (NIRS)
as a potential tool for monitoring trade of similar woods: dis-
crimination of true mahogany, cedar, andiroba, and curupixá.
Holzforschung 65:73–80.
Reed, J.D. (1995) Nutritional toxicology of tannins and related poly-
phenols in forage legumes. J. Anim. Sci. 73:1516–1528.
Rutiaga-Quiñones, J.G., Windeisen, E., Strobel, C. (1998a)
Extraktstoffgehalt von Swietenia macrophylla King. Roh-
Werkst. 56:228.
Rutiaga-Quiñones, J.G., Windeisen, E., Strobel, C. (1998b)
Polysaccharide von Swietenia macrophylla King. Roh- Werkst.
56:234.
Schawanninger, M., Rodrigues, J.C., Fackler, K. (2011) A review
of band assignments in near infrared spectra of wood and wood
components. J. Near Infrared Spec. 19:287–308.
Scheffer, T.C., Morrell, J.J. Natural durability of wood: a worldwide
checklist of species. Research contribution 22. Forest Research
Laboratory. Oregon State University, 1998.
Schimleck, L.R., Espey, C., Mora, C.R., Evans, R., Taylor, A., Muniz,
G. (2009) Characterization of the wood quality of pernambuco
Q6: (Caesalpinia echinata Lam) by measurements of density, extrac-
Please update tives content, microfibril angle, stiffness, color, and NIR spec-
the ref. Smeds troscopy. Holzforschung 63:457–463.
et al (2011) Smeds, A.I., Eklund, P.C., Monogioudi, E., Willför, S.M. (2011)
appropriately Chemical characterization of polymerized products formed in the
Extractive content of mahogany 7
reactions of matairesinol and pinoresinol with the stable radical
2,2-diphenyl-1-picrylhydrazyl. Holzforschung DOI: 10.1515/
HF.2011.151.
So, C.-L., Lebow, S.T., Groom, L.H., Rials, T.G. (2004) The applica-
tion of near infrared (NIR) spectroscopy to inorganic preserva-
tive-treated wood. Wood Fiber Sci. 36:329–336.
Stern, W.L. (1988) Index xylariorum. 3. Institutional wood collec-
tions of the world. IAWA Bull. 9:209–210.
Tappi standards (1993) T 207 om-93. Water solubility of wood and
pulp.
Tappi standards (1988) T 264 om-88. Preparation of wood for chemi-
cal analysis.
Taylor, A., Lloyd, J. (2007) Potential of near infrared spectroscopy
to quantify boron concentration in treated wood. Forest Prod. J.
57:116–117.
Taylor, A.M., Baek, S.H., Jeong, M.K., Nix, G. (2008) Wood
shrinkage prediction using NIR spectroscopy. Wood Fiber Sci.
40:301–307.
Taylor, A.M., Labbé, N., Noehmer, A. (2011) NIR-based prediction
of extractives in American white oak heartwood. Holzforschung
65:185–190.
Terdwongworakul, A., Punsuwan, V., Thanapase, W., Tsuchikawa, S.
(2005) Rapid assessment of wood chemical properties and pulp
yield of Eucalyptus camaldulensis in Thailand tree plantations by
near infrared spectroscopy for improving wood selection for high
quality pulp. J. Wood Sci. 51:167–171.
Thomas, E.V. (1994) A primer on multivariate calibration. Anal.
Chem. 66:795A–804A.
Torelli, N., Čufar, K. (1994) Comparative decay resistance of 43
Mexican tropical hardwoods. Holz Roh- Werkst. 52:394–396.
Torelli, N., Čufar, K. (1995a) Mexican tropical hardwoods.
Comparative study of ash and silica content. Roh- Werkst.
53:61–62.
Torelli, N., Čufar, K. (1995b) Mexican tropical hardwoods. Ph-value.
Roh- Werkst. 53:133–134.
Torelli, N., Čufar, K. (1996) Mexican tropical hardwoods: machin-
ability, nailing and screwing Roh- Werkst. 54:69–71.
Torelli, N., Gorisek, Z. (1995) Mexican tropical hardwoods – dimen-
sional stability. Roh- Werkst. 53:277–280.
Tsuchikawa, S. (2007) A review of recent near infrared
research for wood and paper. Appl. Spectrosc. Rev. 42:
43–71.
Valderrama, P., Braga, J.W.B., Poppi, R.J. (2007) Variable selec-
tion, outlier detection, and figures of merit estimation in a partial
least-squares regression multivariate calibration model. A case
study for the determination of quality parameters in the alcohol
industry by near-infrared spectroscopy. J. Agric. Food Chem.
55:8331–8338.
Venäläinen, M., Harju, A.M., Terziev, N., Laakso, T., Saranpää, P.
(2006) Decay resistance, extractive content, and water sorption
capacity of Siberian larch (Larix sibirica Lebed.) heartwood tim-
ber. Holzforschung 60:99–103.
Willför, S., Hemming, J., Reunanen, M., Eckerman, C., Holmbom
B. (2003a) Lignans and lipophilic extractives in Norway spruce
knots and stemwood. Holzforschung 57:27–36.
Willför, S., Eklund, P., Sjöholm, R., Reunanen, M., Sillanpää, R.,
von Schoultz S., Hemming, J., Nisula, L., Holmbom, B. (2005)
Bioactive phenolic substances in industrially important tree
species. Part 4: Identification of two new 7-hydroxy divanil-
lyl butyrolactol lignans in some spruce, fir, and pine species.
Holzforschung 59:413–417.
Willför, S., Nisula, L., Hemming, J., Reunanen, M., Holmbom
B. (2003b) Bioactive phenolic substances in industrially
 Article in press - uncorrected proof Page 8 of 31

8 A. Ribeiro da Silva et al.

important tree species. Part 2: Knots and stemwood of fir spe-
cies. Holzforschung 58:650–659.
Zahri, S., Moubarik, A., Charrier, F., Chaix, G., Baillères, H., Nepveu,
G., Charrier, B. (2008) Quantitative assessment of total phenol
contents of European oak (Quercus petrae and Quercus robur)
by diffuse reflectance NIR spectroscopy on solid wood surfaces.
Holzforschung 62:679–687.
Received October 10, 2011; accepted May 30, 2012
Page 9 of 31

Running title: Extractives content of mahogany

ASSESSMENT OF TOTAL PHENOLS AND EXTRACTIVES OF MAHOGANY WOOD BY

NEAR INFRARED SPECTROSCOPY (NIRS)

Allan Ribeiro da Silva1, Tereza Cristina Monteiro Pastore2*, Jez Willian Batista Braga1, Fabrice
Davrieux3 , Esmeralda Yoshico Arakaki Okino2 , Vera Teresinha Rauber Coradin2, José Arlete Alves
Camargos2, Alexandre Gustavo Soares do Prado (In memorium)1
1
Chemistry Institute, University of Brasilia, Brasília, DF, Brazil
2
Forest Products Laboratory, Brazilian Forest Service, Brasilia, DF, Brazil
3
French Agricultural Research Centre for International Development, CIRAD-UMR Qualisud,
Montpellier, France

* Corresponding author.
Laboratório de Produtos Florestais,
Serviço Florestal Brasileiro, SCEN trecho 2, Bloco D
70818900, Brasilia, DF, Brazil
E-mail: tereza.pastore@florestal.gov.br

Abstract

The resistance to decomposition of mahogany wood (Swetenia macrophylla King.) ranges from

high to moderate level. Wood extractives, mainly due to the presence of phenol compounds are

related to the natural durability of wood. The technique of near infrared spectroscopy (NIRS)

coupled with multivariate analysis was applied to assess the extractive and phenols of 41 samples of

mahogany in powder form. The hot-water-soluble extractives were quantitatively determined and

total phenol content was measured using the Folin-Denis colorimetric reagent. Models were

developed with the NIRS data for each of the two variables. The results indicated that NIRS can be

a useful tool to quickly evaluate the content of extractives and total phenolic compounds of

mahogany wood. The method was able to predict the interesting properties with errors lower than

10% (w/w) and had the capability of detecting samples that present a minimum concentration of

2.4% (w/w) of extractives and total phenolic compounds, respectively.

Keywords: extractives content, mahogany, total phenols content, NIRS.

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 Page 10 of 31

Introduction

Swietenia macrophylla King. (Meliaceae), also called mahogany, caoba or bigleaf mahogany, is one

of three tree species, regarded as "genuine mahogany" and is distributed from tropical Central to

South America. Valuable mahogany wood is used for many purposes, such as fine furniture, cabinet

making, civil constructions, boats, ships, interior trim, panelling, veneers, musical instruments,

turnery and carving. Its characteristics are a glossy pink-red colour, medium specific gravity (0.50

to 0.72 g/cm3), good wood machining (Coradin et al 2009; Torelli and Čufar 1996) and dimensional

stability (Torelli and Gorisek 1995; Melo et al. 1989). Mahogany bark (Falah et al. 2008) and seeds

(Bourdy et al. 2000) are also valuable due to their biological activity. The chemical composition of

wood of S. macrophylla is still not well investigated though the natural durability of its wood is

directly related to the kind and amount of its extractives.

Extractives are organic substances of low molecular weight that are not components of the

cell wall matrix. They are responsible for colour and resistance to fungi, insects and marine borers.

They may also act as toxins as well as repellents to insects, and have antioxidant properties (Fengel

and Weneger 1984; Venäläinen et al. 2006; Mburu et al. 2007; Lim et al. 2007; Binbuga et al. 2008;

Borges et al. 2008; Gao et al. 2008; Donoso-Fierro et al. 2009; Little et al. 2010). Chemically, the

extractives vary between species, genera, and various anatomical parts of wood may differ in terms

of concentration and composition. In the last decade the research on extractives of softwoods has

advanced a lot. Especially, the discovery that the knots contain commercially attractive components

which was a stimulus for this research area (Eaton and Hale 1993; Willför et al. 2003a,b; 2005;

Smeds et al. 2011).

Hardwoods may also contain terpenes, fats, waxes, phenols, lignans, quinones, tannins,

flavonoids and other compounds. Interestingly, hardwoods contain waxes more frequently than fatty

acids. Tannins from woods and barks are phenolic compounds of great economic interest and have

toxic effects against microorganisms and radical scavenging properties (Monteiro 2005, Makino et

al. 2011).
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The natural durable tropical woods present levels of extractives higher than the woods from

temperate climate zones. Déon et al. (1980) noted that most research about the role of extractives in

the natural durability of wood was focused on wood from temperate climates. So far, scientific

contributions concerning extractives from Brazilian forest species are rare. Jesus et al. (1998)

studied the resistance of wood decay organisms of 46 species of trees selected based on the

occurrence and availability in three regions of the Amazonas state and Garlet (2010) investigated

the same and others tropical species for approximately 25 years in field tests installed in a forest in

Pará state. Carneiro et al. (2009) tested the natural resistance of 28 Amazon tropical wood species

against five types of fungi. The authors introduced the parameter ‘resistance potential’, which was

obtained by multiplying the wood densities by their extractive contents. Thus it was possible to

verify a trend of direct correlation between weight losses (WL), extractives content, and the "natural

resistance" parameter. They asserted that the tropical woods containing 7% or more extractives had

a higher natural resistance as indicated by less WL in decay tests. In the list of natural durability,

developed by Scheffer and Morrell (1998), the decay resistance of S. macrophylla was classified in

the range from high to moderate. Torelli and Cufar (1994) reported that Trametes versicolor causes

36% WL and Gleophyllum trabeum 16% WL with S. macrophylla. Mahogany wood is also highly

resistant to attack by dry-wood termites; however, its durability is low when in contact with

moisture soil (Embrapa, 2011).

The water soluble extractives content of Brazilian mahogany was found to be 8.6% and

7.3% alcohol-benzene solubles were detectable (Brazilian Technical Association of Pulp and Paper,

ABCP Technical Standards, Jankowsky and Galvão 1979). For mahogany, native to Mexico,

Rutiaga-Quinõnes et al. (1998a) tested the solubility with petrol ether (0.8%), acetone (3.2%),

methanol (3.4%), and hot water (1.1%) and cold water (2.1%) (standard test DIN 4188) and found

in total 10.6% extractives. Rutiaga-Quinõnes et al. (1998b) analysed the cell wall components by

total hydrolysis and found rhamose (0.4%), mannose (1.1%), arabinose (0.4%), galactose (1.4%),

xylose (12.3%) and glucose (55.0%), accounting for 63.3% of total polysaccharides. The content of

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inorganic compounds amounts to 0.36%, and the lignin content to 31.1% (Mejia-Díaz and Rutiaga-

Quinõnes 2008). Torelli and Čufar (1995a) detected traces of silica in the ash (0.32%) of wood

collected from Mexico. The pH values varied from 5.3 to 5.0 from the heartwood to the sapwood

(Torelli and Čufar 1995b).

Vegetable tannins can be quantified via precipitation of proteins and metals or by

colorimetric methods. Among them, the Folin-Denis method has been widely used for the

determination of total phenolics. However, the selectivity of this method is limited once it can also

react with oxidizing and reducing agents present in the samples, and also interfere with the

spectrometric reading. As the tannins have a great variety of structures and form readily complex

products with a variety of oxidative materials, the quantification process is complex and

controversially discussed in the literature (Monteiro et al. 2005).

Near infra-red spectroscopy (NIRS) is a routine technique with little sample preparation for

a rapid quality control of various complex products (Hein et al. 2010). NIRS is also useful in wood

science; it has been successfully tested for predicting decay resistance (Fackler et al. 2007;

Leinonen et al. 2008), detecting inorganic preservatives (So et al. 2004; Taylor and Lloyd 2007)

and, recently, discriminating tropical wood species that are anatomically similar (Pastore et al. 2011,

Braga et al. 2011).

Several authors estimated the extractive contents of various woods (Terdwongworakul et al.

2005; Tsuchikawa 2007, Zahri et al. 2008; Schimleck et al. 2009). Gierlinger et al. (2002) showed

that NIRS can substitute time-consuming conventional methods of analysis, and is able to quickly

predict the levels of total phenolics and extractives of the wood of Larix decidua, L. leptolepis, and

a hybrid (Larix x eurolepis). The quoted authors tested 175 trees of different provenances. Taylor et

al. (2008) constructed a NIRS based prediction model with 57 samples of mahogany collected in

Central America and South America; they considered the total volumetric shrinkage, the amount of

extractive, and specific gravity as parameters. The extractives were quantified by successive

extraction: toluene/ethanol (2:1), 95% ethanol, and hot water. The average content of total

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Page 13 of 31

extractives found was 18.1%, with values ranging from 11.8 to 28.1%. Reasonable predictions

could be done with the models developed, especially in cases when the improved statistical

techniques (‘kernel and wavelet’) were applied. Zahri et al. (2008) assessed quantitatively the total

phenol contents of European oak by means of NIRS.

The objective of the present study was to correlate the total content of phenolic compounds

and the concentrations of hot water solubles of mahogany wood with NIRS data for a rapid

prediction of the extractive contents of unknown mahogany samples.

Material and methods

32 samples of mahogany from Mato Grosso state in Brazil were cut from the tips of boards stored

under dry conditions. Nine other samples were prepared by mixing calculated amounts of two

powders samples with known content of hot water soluble extractives. Thus, 41 samples were

obtained at concentrations of extractives distributed between the maximum and the minimum

values found in the original samples (Table 1). The intentions were to reduce the problem of low

number of samples and to obtain a set of concentrations of extractives and total phenol uniformly

distributed. The anatomical identification of woods was carried out at Forest Products Laboratory in

Brasilia, registered as FPB in the Index Xylariorum (Stern 1988). Each sample was air dried until

reaching around 9% moisture content (MC) and part of it was chipped and milled in a Wiley mill.

The fraction of saw mill that passed through a 0.40 mm (40 meshes) screen was used for analysis.

The MC and hot water solubles were obtained according to TAPPI standards T 264 om-88 and T

207 om-93, respectively.

The Folin-Denis (FD) colorimetric reagent was purchased from Fluka Analytical. A standard

calibration curve was constructed with tannic acids: 6 mL of distilled water was added to a 10 mL

volumetric flask, followed by 0.1 mL of wood extractives and 0.5 mL of FD reagent. After 3 min, 1

mL of saturated solution of Na2CO3 was added to the flask and the volume was filled up. The

mixture was centrifuged and filtered after 40 min of reacting. The UV-Vis spectra were collected
5
 Page 14 of 31

after 1 h of adding saturated Na2CO3, and the λmax at 725.0 nm was measured. Instrument: UV/Vis

FEMTO 800XI spectrophotometer equipped with 1 cm quartz cells; scanning range: 490-850 nm.

Near infrared spectroscopy (NIRS)

Instrument: Tensor 37 FT-IR spectrometer (Bruker Optics, Germany) equipped with the Easi DiffTM

diffuse reflectance accessory (Pike Technologies Inc.). The spectra were collected at 4 cm-1

intervals over 12000 to 4000 cm-1. The instrument reference was a standard mirror provided by the

equipment manufacturer. A total of 64 scans were accumulated for each spectrum and 3 spectra

were averaged for data collection. Climate during spectroscopy: relative humidity <60% at 20ºC.

Data analysis and model development

NIRS data were evaluated by means of a partial least square regression model (PLSR), which is a

statistical method for calculating models for quantitative analysis in several areas (Geladi and

Kowalski 1986; Haaland and Thomas 1988; Brereton 2000; Martens and Naes 1989). Shortly: the

data matrix X is formed by the NIR spectra of wood, whereas vector y is composed by

concentration of phenolics and solubles in hot water. Thus two different PLSR models were

developed with one dependent variable for each. The QUANT 2 routine provided by the OPUS

software version 6.5 was applied to this purpose (Bruker Optics, Germany).

The data were split into calibration and validation sets. In the first one, 26 samples of

mahogany were considered; the number of latent variables was detected by means of the ‘leave one

out cross-validation procedure’ (Thomas 1994). In the validation set, 12 independently prepared

samples were included. The outlier identification was performed by the test for extreme leverage,

unmodeled residuals in spectra, and unmodeled residuals in dependent variables (Valderrama et al.

2007). The remaining 3 samples were not considered because they showed inconsistent results for

extractives and total phenols obtained with the reference method.

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Page 15 of 31

That spectral range was chosen as the best one, which provided the lowest ‘root mean square

error of cross-validation’ (RMSECV), or the lowest number of latent variables, after the RMSECV

had reached an approximately constant value. The validation of the NIR method was performed by

the determination of the figures of merit. To evaluate the accuracy of the method the the root mean

square error of calibration and prediction (RMSEP and RMSEC, respectively) (Brereton, 2000)

were calculated and a paired t-test was performed with the reference and estimated values.

By the definition of the RMSEP, a relative error of prediction (REP) can be obtained

according to Miller-Ihli et al. (1984):

I
(y i − ŷ i ) 2
REP = ∑
n =1 I cal y i2
100 (1)

Sensitivity is an important parameter to compare the performance of two independent

methods. In inverse multivariate calibration models, such as PLSR, it is determined by the inverse

of the norm of the regression coefficients (Lorber et al. 1997). However, the capability of detection

(CCβ) described in at the ISO 11843-2 provides an easier and a direct comparison of the detectable

concentrations of the methods. Ortiz et al. (2003) have extended the CCβ application for

multivariate calibration methods such as PLSR. Accordingly, the CCβ can be determined as:

s 1 1 y2
CCβ = δα,β + + Ical (2)
b m I cal
∑ (yi − y )2
i=1

where, s is the standard deviation of the residuals of the regression of the estimated concentrations

( ) against the reference concentration of calibration samples (y) with the intercept ‘a’ and slope

‘b’, m is the number of replicate measurements performed on each sample, is the mean

concentration of the standards and δα,β,ν is the noncentrality parameter from the noncentral t-

distribution with Ical-2 degrees of freedom.

Results and discussion

7
 Page 16 of 31

Wet chemical analyses

The content of water soluble and total phenols are statistically variable (Table 1). Figure 1 is the

corresponding in-detail plot for 41 mahogany samples determined by conventional methods. The

average content of extractives was 8.38% ranging from 4.01% to 14.8% with a standard deviation

of 2.9%. Our results corroborate Taylor et al. (2008) findings. The authors also found a wide

variation in levels of total extractives of S. macrophylla of Central America and South America,

ranging from 11.8% to 28.1% with a standard deviation of 3.1%. The variability of extractive

content between trees of a given species has been demonstrated by several authors (Hillis, 1962;

Giergeling, 2002) and was even observed in a single individual tree, when collecting samples of oak

disks at different distances from the pith (Taylor et al. 2011).

The concentration of soluble phenols in hot water of mahogany samples also varied widely

as showed in Figure 1. The mean average found for all sample was 6.81% and the standard

deviation was 2.72%. For the European oak, Zahri et al. (2008) found values varying from 1.3% to

7.0 % and they also demonstrated that this class of compounds decreased with age of the tree. The

interpretation was that phenolics bind with time irreversibly to the cell wall matrix and become

insoluble. The age of the mahogany woods collected for the experiment of the present work is

unknown. The variability of polyphenol concentrations collected from woods with equal age may

be lower.

As illustrated in Figure 1, the data of samples 16, 20, and 34 are outliers (data are too

elevated). The Folin-Dennis reagent is one of the most widely used in colorimetric procedures to

determine total soluble phenolics. Nevertheless, the method sometimes fails (Reed 1995). The

extinction coefficient of chromophores produced with the tannic acid may have different values in

some wood samples. The literature may contain overestimated or underestimated data for the total

phenol contents.

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Page 17 of 31

Spectroscopic characterization and determination

As usual, visual comparison of the NIR spectra of an extracted and not extracted wood samples do

not reveal any obvious spectral differences (Figure 2a). The insect in Figure 2a presents the 1st

derivative spectra, which minimizes the effects resulting from particle size and other physical

factors caused by the reflectance measurement. The bands at 1410 and 1900 nm are elevated by hot

water extraction. These bands may be attributed tentatively to the 1st overtone of the O-H and the

combination bands of O-H and C=O bonds, respectively, as interpreted by Mitchel and Schimleck

(1996) based on the experiments with water extraction of Eucalyptus globulus.

Samples illustrated in Figure 2b represent the maximum and minimum content of wood

extractives observed in the dataset. The insect of Figure 2b shows more spectral differences at 2084

and 2136 nm, which correspond to the region of the O-H and C-H combination bands in the 1st

derivative NIR spectra of wood samples. Mitchel and Schimleck (1996) and Schwanninger et al.

(2011) attributed these vibrations to cellulose and xylan (2084 nm) and to lignin and extractives

(2136 nm).

The parameters used for the development of the two PLSR models for the extractives and

total phenols are summarized in Table 2. The selected region for model development was 1639 to

1731 nm, which corresponds to the 1st overtone of the C-H bonds in wood and presented the lowest

RMSECV but not the largest spectral differences. This suggests that the main spectral differences

may be caused by other wood components and that all chemical information about the total phenols

and extractives content are spread in the entire spectrum, minimizing the influence of the other

sources of variation.

The best pre-processing method was obtained by combining the 1st derivate of the NIR

spectra, vector normalization and mean-centering. Table 2 also indicates that 2 and 3 outliers were

observed in the calibration set. All outliers were identified by high prediction errors (residuals in

dependent variables).

9
 Page 18 of 31

Table 3 presents some figures of merit of the method. The root mean square errors (RMSE)

represent the average error of the method. Approximately the same RMSEP values were observed

for the determination of the extractives and total phenols content indicating the same accuracy for

both properties investigated. Comparing the RMSEP and RMSEC by an F-test showed no

significant difference for both models was observed with 95 % of confidence, meaning no

overfitting. Graphics of the reference vs. estimated values for the calibration and test samples for

extractives (Fig. 3a) and total phenols (Fig. 3b) respectively are shown. The absence of over fitting

can also be observed by comparing the dispersions of the calibration and test samples in these

figures. Considering the amplitude of the variation of the extractive and total phenols, the relative

error of prediction (REP) of both models was almost 10 %. However, it is important to note that this

error can be highly affected by only a few samples presenting a large error. For example, for total

phenols determination only two of the twelve samples present errors larger than 10 %. If these

samples were considered outliers the new RMSEP and REP values would be 0.42 % (w/w) and 6.6

% respectively. The paired t-test performed with the validation samples shows that no systematic

errors are present in the model.

The noise level, estimated based on the spectral residuals of the PLSR model for the

calibration samples, presented consistent values with the instrumental variation in the NIR data.

This means that all systematic variation in the data was modeled by PLSR. The sensitivity of the

methods is also shown in Table 3. However, the capability of detection expresses the sensitivity as

well, but this parameter allows an easier interpretation by presenting the same unit of the interested

properties determined. According to the results the lowest concentration that can be determined by

the model for both extractives and total phenols is 2.4 % (w/w). This makes possible the

determination of the extractives and the total phenols in almost every concentration range observed

in the samples.

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Page 19 of 31

Conclusions

The results of the NIR method for determination of extractives and total phenols content presented

good agreement with the reference method. The accuracy and the capability of detection results has

shown that the model presented an appropriate sensitivity and prediction performance for the

determination of the interest properties in routine analysis. The NIR methodology presents many

advantages in comparison to the reference method, such as it does not need any sample preparation,

the analysis can be performed in less than 5 minutes and no chemical residuals are generated.

Acknowledgements

The authors would like to thank FAPDF for the financial support as well as the fellowship of Allan

R. da Silva. The endeavours of Dr. Paulo J.P. Fontes and Mr. Waldemar V. Lopes were essential to

obtain the necessary wood.

11
 Page 20 of 31

References

Binbuga, N., Ruhs, C., Hasty, J.K., Henry, W.P., Schultz, T.P. (2008) Developing environmentally
benign and effective organic wood preservatives by understanding the biocidal and non-
biocidal properties of extractives in naturally durable heartwood. Holzforschung 62(3):264–
269.
Borges, L.M.S. Cragg, S.M., Bergot, J., Williams, J.R., Shayler, B., Sawyer, G.S. (2008) Laboratory
screening of tropical hardwoods for natural resistance to the marine borer Limnoria
quadripunctata: The role of leachable and non-leachable factors. Holzforschung 62(1):99–111.
Bourdy, G., DeWalt, S.J., Chávez de Michel, L.R., Roca, A., Deharo, E., Muñoz, V., Balderrama, L.,
Quenevo, C., Gimenez, A. (2000) Medicinal plants uses of the Tacana, an Amazonian
Bolivian ethnic group. J..Ethnopharmacology 70:87-109
Braga, J. W. B., Pastore, T. C. M., Coradin, V. T. R., Camargos, J. A. A., Silva, A. R. (2011) The use
of near infrared spectroscopy to identify solid wood specimens of Swietenia macrophylla.
IAWA J. 32: 285-296
Brereton, R. G.(2000) Introduction to multivariate calibration in analytical chemistry. Analyst 125:
2125-2154
Carneiro, J.S., Emmert, L., Sternadt, G.H., Mendes, J.C., Almeida, G.F. (2009) Decay susceptibility
of Amazon wood species from Brazil against white rot and brown rot decay fungi.
Holzforschung 63: 767-772
Coradin, V.T.R, Camargos, J.A.A, Marques, L.F., Silva Jr, E.R. Madeiras similares ao mogno
(Swietenia macrophylla King.): chave ilustrada para identificação anatômica em campo.
Serviço Florestal Brasileiro/LPF, Brasilia, 2009
Déon, G., Chadenson, M., Hauteville, M. (1980) Influence des extraits naturels du bois sur sa
resistance a la pourriture. Bois. For.. Trop. 191:75-89
Donoso-Fierro, C., Becerra, J., Bustos-Concha, E., Silva, M. (2009) Chelating and antioxidant
activity of lignans from Chilean woods (Cupressaceae). Holzforschung 63(5):559–563.
Eaton, R.A., Hale, M.D.C. Wood: Decay, pest, and protection. Chapman & Hall, Great Britain,
1993
Embrapa (http://www.cnpf.embrapa.br/publica/circtec/edicoes/Circular140.pdf), accessed June 20,
2011
Fackler, K., Schwanninger, M., Gradinger, C., Srebotnik, E., Hinterstoinsser, B., Messner (2007)
Fungal decay of spruce and beech wood assessed by near-infrared spectroscopy in
combination with uni- and multivariate data analysis. Holzforschung 61: 689-687
Falah, S., Suzuki, T., Katayama, T. (2008) Chemical constituents from Swietenia macrophylla bark
and their antioxidant activity. Pak. J. Biol. Sci. 11: 2007-2012
Fengel, D., Wegener, G. Wood – Chemistry, Ultrastructure, Reactions. De Gruyter, Berlin, 1984
Gao, H., Obanda, D.N., Shupe, T.F., Hse, C.Y., Ring, D.R. (2008) Antifungal activities of
heartwood extracts of Port-Orford cedar extractives. Holzforschung 62(5):620–623.
Garlet (2010) http://www.noticiasdaamazonia.com.br/13093-estudo-do-servico-florestal-revela-
madeiras-mais-resistentes)
Geladi, P., Kowalski, B.R. (1986) Partial Least-squares regression – A tutorial. Anal. Chim. Acta
185:1-17
Gierlinger, N., Schwanninger, Hinterstoisser, B., Wimmer, R. (2002) Rapid determination of
heartwood extractives in Larix sp by means of Fourier transform near infrared spectroscopy. J.
Near Infrared Spec.. 10:203-214
Haaland, D. M., Thomas, E. V. (1988) Partial least-squares for spectral analyses. 1. Relation to
other quantitative calibration methods and the extraction of qualitative information. Anal.
Chem. 60: 1193-1202

12
Page 21 of 31

Hein, P.R.G., Lima, J.T., Chaix, G. (2010) Effects of sample preparation on NIR spectroscopic
estimation of chemical properties of Eucalyptus urophylla S.T. Blake wood. Holzforschung
64: 45-54
Hillis, W.E. Wood extractives and their significance to the pulp and paper industries. Academic
Press. New York and London, 1962.
ISO 11843-2 standard (2000) Capability of detection.
Jankowsky, I.P., Galvão, A.P.M. (1979) Influência do teor de extrativos na umidade de equilíbrio da
madeira. IPEF 18:1-33
Jesus, M.A., Morais, J.W., Abreu, R.L.S., Cardias, M.F.C. (1998) Durabilidade natural de 46
espécies de madeira amazônica em contato com o solo em ambiente florestal. IPEF 54:81-92
Leinonen, A., Harju, A.M. Venäläinen, M., Saranpää, P., Laako, T. (2008) FT-NIR spectroscopy in
predicting the decay resistance related characteristics of solid scots pine (Pinus sylvestris L.)
heartwood. Holzforschung 62:(284-288)
Lim, Y.W., Chedgy, R.J., Amirthalingam, S., Breuil, C. (2007) Screening fungi tolerant to Western
red cedar (Thuja plicata Donn) extractives. Part 2. Development of a feeder strip assay.
Holzforschung 61(2):195–200.
Little, N.S., Schultz, T.P., Nicholas, D.D. (2010) Termite-resistant heartwood. Effect of antioxidants
on termite feeding deterrence and mortality. Holzforschung 64(3):395–398.
Lorber, A, Faber, K, Kowalski, B.R. (1997) Net analyte signal calculation in multivariate
calibration. Anal. Chem. 69:1620-1626
Makino, R., Ohara, S., Hashida, K. (2011) Radical scavenging characteristics of condensed tannins
from barks of various tree species compared with quebracho wood tannin. Holzforschung,
65(5):651-657.
Martens, H., Naes, T. Multivariate Calibration. John Wiley & Sons, New York, 1989.
Mburu, F., Dumarçay, S., Gérardin, P. (2007) Evidence of fungicidal and termicidal properties of
Prunus africana heartwood extractives. Holzforschung 61(3):323–325.
Mejía-Díaz, L.A., Rutiaga-Quinõnes, J.G. (2008) Chemical composition of Schinus molle L. wood
and kraft pulping process. Rev. Mex. Ing. Quim. 7:145-149
Melo, J.E., Carvalho, G.M., Martins, V.A. Espécies de madeiras substitutas do mogno. IBAMA:
Brasília, 1989
Michell, A.J., Schimleck, L.R. NIR spectroscopy of woods from Eucaliptus globulus. Appita 49:
23-26
Miller-Ihli, N.J.; O’Haver ,T.C.; Harnly ,J.M. (1984) Calibration and curve fitting for extended
range AAS. Spectrochim. Acta B 39:1603-1614
Monteiro, J.M., Albuquerque, U.P. de, Araújo, E.de L. (2005) Taninos: uma abordagem da química
à ecologia. Quim. Nova 28:892-896
Ortiz, M.C., Sarabia, L.A., Herrero, A., Sánchez, M.S., Sanz, M.B., Rueda, M.E., Giménez, D.,
Meléndez, M.E. (2003) Capability of detection of an analytical method evaluating false
positive and false negative (ISO 11843) with partial least squares. Chemometr. Intell. Lab.
Syst. 69:21-33
Pastore, T.C.M., Braga, J.W.B., Coradin, V.T.R., Magalhães, W.L.E., Okino, E.Y.A., Camargos,
J.A.A., Muñiz, G.I.B., Bressan, O.A., Davrieux, F. (2011) Near infrared spectroscopy (NIRS)
as a potential tool for monitoring trade of similar woods: Discrimination of true mahogany,
cedar, andiroba, and curupixá. Holzforschung 65:73-80
Reed, J.D. (1995) Nutritional toxicology of tannins and related polyphenols in forage legumes. J.
Anim. Sci. 73:1516-1528
Rutiaga-Quiñones, J.G., Windeisen, E., Strobel, C. (1998a) Extraktstoffgehalt von Swietenia
macrophylla King. Roh- Werkst. 56:228
Rutiaga-Quinõnes Quiñones, J.G., Windeisen, E., Strobel, C. (1998b) Polysaccharide von Swietenia
macrophylla King. Roh- Werkst. 56:234

13
 Page 22 of 31

Scheffer, T.C., Morrell, J.J. Natural durability of wood: A worldwide checklist of species. Research
contribuition 22. Forest Research Laboratory. Oregon State University 1998
Schimleck, L.R., Espey, C., Mora, C.R., Evans, R., Taylor, A., Muniz, G. (2009) Characterization of
the wood quality of pernambuco (Caesalpinia echinata Lam) by measurements of density,
extractives content, microfibril angle, stiffness, color, and NIR spectroscopy. Holzforschung
63:457-463
Schawanninger, M., Rodrigues, J.C., Fackler (2011) A review of band assignments in near infrared
spectra of wood and wood components. J. Near Infrared Spectrosc. 19:287-308
Smeds, A.I., Eklund, P.C., Monogioudi, E., Willför, S.M. (2011) Chemical characterization of
polymerized products formed in the reactions of matairesinol and pinoresinol with the stable
radical 2,2-diphenyl-1-picrylhydrazyl. Holzforschung DOI: 10.1515/HF.2011.151
So, C.-L., Lebow, S.T., Groom, L.H., Rials, T.G. (2004) The application of near infrared (NIR)
spectroscopy to inorganic preservative-treated wood. Wood Fiber Sci. 36:329-336
Stern, W.L. (1988) Index Xylariorum3. Institutional wood colections of the world. IAWA Bull.
9:209-210
Tappi standards (1988) T 264 om-88. Preparation of wood for chemical analysis.
Tappi standards. (1993) T 207 om-93. Water solubility of wood and pulp.
Taylor, A., Lloyd, J. (2007) Potential of near infrared spectroscopy to quantify boron concentration
in treated wood. Forest Prod. J. 57:116-117
Taylor, A.M., Baek,S.H., Jeong, M.K., Nix, G. (2008) Wood shrinkage prediction using NIR
spectroscopy. Wood Fiber Sci. 40:301-307
Taylor, A.M., Labbé, N., Noehmer, A. (2011) NIR-based prediction of extractives in American
white oak heartwood. Holzforschung 65:185-190
Terdwongworakul, A., Punsuwan, V., Thanapase, W., Tsuchikawa, S. (2005) Rapid assessment of
wood chemical properties and pulp yield of Eucalyptus camaldulensis in Thailand tree
plantations by near infrared spectroscopy for improving wood selection for high quality pulp.
J. Wood Sci. 51:167-171
Thomas, E.V. (1994) A primer on multivariate calibration. Anal. Chem. 66:795A–804A
Torelli, N., Čufar, K. (1994) Comparative decay resistance of 43 Mexican tropical hardwoods. Holz
Roh- Werkst. 52:394-396 .
Torelli, N., Čufar, K. (1995a) Mexican tropical hardwoods. Comparative study of ash and silica
content. Roh- Werkst. 53:61-62
Torelli, N., Čufar, K. (1995b) Mexican tropical hardwoods. Ph-value. Roh- Werkst. 53:133-134
Torelli, N., Čufar, K. (1996) Mexican tropical hardwoods: Machinability, nailing and screwing Roh-
Werkst. 54:69-71
Torelli, N., Gorisek, Z. (1995) Mexican tropical hardwoods – dimensional stability. Roh- Werkst.
53:277-280
Tsuchikawa, S. (2007) A review of recent near infrared research for wood and paper. Appl.
Spectrosc. Rev. 42:43-71
Valderrama, P., Braga, J. W. B., Poppi, R. J. (2007) Variable selection, outlier detection, and figures
of merit estimation in a partial least-squares regression multivariate calibration model. A case
study for the determination of quality parameters in the alcohol industry by near-infrared
spectroscopy. J. Agric. Food Chem. 55:8331–8338
Venäläinen, M., Harju, A.M., Terziev, N., Laakso, T., Saranpää, P. (2006) Decay resistance,
extractive content, and water sorption capacity of Siberian larch (Larix sibirica Lebed.)
heartwood timber. Holzforschung 60(1):99–103.
Willför, S. Hemming, J. Reunanen, M. Eckerman, C. Holmbom B. (2003a) Lignans and Lipophilic
Extractives in Norway Spruce Knots and Stemwood. Holzforschung 57(1):27–36.
Willför, S., Eklund, P., Sjöholm, R., Reunanen, M., Sillanpää, R., von Schoultz S., Hemming, J.,
Nisula, L., Holmbom, B. (2005) Bioactive phenolic substances in industrially important tree

14
Page 23 of 31

species. Part 4: Identification of two new 7-hydroxy divanillyl butyrolactol lignans in some
spruce, fir, and pine species. Holzforschung 59(4):413–417.
Willför, S., Nisula, L., Hemming, J., Reunanen, M., Holmbom B., (2003b) Bioactive phenolic
substances in industrially important tree species. Part 2: Knots and stemwood of fir species.
Holzforschung 58(6):650–659.
Zahri, S., Moubarik, A., Charrier, F., Chaix, G., Baillères, H., Nepveu, G., Charrier, B. (2008)
Quantitative assessment of total phenol contents of European oak (Quercus petrae and
Quercus robur) by diffuse reflectance NIR spectroscopy on solid wood surfaces.
Holzforschung 62:679-687.

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Captions for figures:

Figure 1. Mean values and standard deviations of hot water extractives content and total phenols

content of 41 Brazilian mahogany tree samples. Samples 1 to 33 - each value represents the average

of a sample taken from the edge of a board of a tree. Samples 34 to 41 - each value represents a

mixture of powders of various trees. All determinations were made in triplicate.

Figure 2. Original NIR spectra of one mahogany (Swietenia macrophylla) set of samples (a) with

and without hot water extractives, (b) with the maxima and minimum concentration of hot water

extractives. The spectra in the first derivative mode of wood powder has been shown on the insect

to highlight the differences between them.

Figure 3. Reference concentrations against the estimated concentrations by the PLS model for the

(a) determination of extractives content. () Calibration samples, (■) test samples, (—) ideal

straight and (b) determination of total phenols content. (∆) Calibration samples, (■) test samples,

(—) ideal straight.

16
Page 25 of 31

Figure 1

17
 Page 26 of 31

Figure 2a e 2b

18
Page 27 of 31

Figure 3a e 3b

19
 Page 28 of 31

Table 1 – Average results for extractives and total phenols of mahogany wood samples.

Extractives Total phenols

content content
(%) (%)
Average 8.38 6.81
Maximum 14.83 13.96
Minimum 4.01 2.25
Standard 2.9 2.72
deviation

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Page 29 of 31

Table 2 – Model parameters

Hot water
Parameter Total phenols
extractives
Nº of latent
4 3
variables
Region 1639.1 – 1639.1 –
selectiona 1731.6 1731.6
1st derivate 1st derivate
Preprocessing
+ +
Vector Vector
normalization normalization
+ +
mean center mean center
Outliers Calibration 2
Validation 0
a
expressed in nm

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 Page 30 of 31

Table 3 – Figures of merit for the PLS model.

Parameter Extractives Total phenols

(%) (%)
RMSECa 0.46 0.48
RMSECVa 0.63 0.66
RMSEPa 0.62 0.72
Relative error of
8.9 9.9
predictionb
-3
Sensitivity (x10 ) 1.4 2.0
Capability of
2.4 2.4
detection a
Estimated t value 0.013 0.9714
Noise level (x10-4) 1.1 1.4
a
expressed as percentage (w/w), b percentage.

22
Page 31 of 31

Dear Dr. Faix,

Ref. "ASSESSMENT OF TOTAL PHENOLS AND EXTRACTIVES OF MAHOGANY WOOD BY

NEAR INFRARED SPECTROSCOPY (NIRS)"
Reg. : ID HOLZ.2011.0207
Holzforschung

REVIEWERS' COMMENTS:

General comment:

Reviewer 1:
It is mandatory to submit also the corresponding Word document, otherwise small corrections
cannot be made. Thank you very much in advance for your cooperation.

Answer: As requested, please find attached the manuscript file in Word.

Best regards,

Tereza C M Pastore

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