Industrial Crops and Products 49 (2013) 897–903

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Industrial Crops and Products

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Characterisation of maritime pine (Pinus pinaster) bark tannins

extracted under different conditions by spectroscopic methods,
FTIR and HPLC
L. Chupin a,∗ , C. Motillon a , F. Charrier-El Bouhtoury a , A. Pizzi b , B. Charrier a
a
EPCP-IPREM, IUT des Pays de l’Adour, 371 Rue du Ruisseau, BP 201, 40004 Mont de Marsan, France
b
ENSTIB-LERMAB, University of Lorraine, 27 Rue Philippe Seguin, BP 1041, 88051 Epinal, France

a r t i c l e i n f o a b s t r a c t

Article history: For the first time an extensive characterisation of maritime pine (Pinus pinaster) bark condensed tannins
Received 18 March 2013 was conducted. Extractives were obtained from the bark of maritime pine from the south west of France
Received in revised form 26 June 2013 using 1% NaOH and 5% NaOH at 70 ◦ C and 80 ◦ C. For each extraction, extraction yield, total polyphenols
Accepted 27 June 2013
(Folin-Ciocalteu), condensed tannins and reactivity to formaldehyde (Stiasny number) were quantified.
The extracts were characterised by Fourier transformed infrared spectroscopy (FTIR) and reverse phase
Keywords:
high pressure liquid chromatography (RP-HPLC). The higher extraction yields originated from an extrac-
FTIR
tion with 5% NaOH at 80 ◦ C. The extracts obtained with 1% NaOH at 70 ◦ C had the highest amounts of total
Polyphenols
Condensed tannins
polyphenols, condensed tannins and reactivity to formaldehyde. Absorbance at 1260 cm−1 , 1440 cm−1
Pinus pinaster bark and 1495 cm−1 were only detected for extracts obtained with 1% NaOH. RP-HPLC revealed that catechin
RP-HPLC was the main condensed tannin in the extracts, lower quantities of epicatechin and epicatechin gallate
were also measured.
© 2013 Elsevier B.V. All rights reserved.

1. Introduction
Pinus pinaster (maritime pine) can be found in some Mediter-
ranean countries, such as France, Portugal and Spain, and in some
north western African countries (Jerez et al., 2007a; Navarrete et al.,
2010; Seabra et al., 2012). They are mostly cultivated for their tim-
ber. The pine bark is a lumber industry sub-product that is produced
when wood is transformed.
Pine bark is rich in phenolic compounds (Jerez et al., 2006).
The main tannin structures found in maritime pine bark are
catechin/epicatechin, epigallocatechin and epicatechin gallate
(Navarrete et al., 2010). Many possible applications for pine bark
are cited in the scientific literature. Its extracts have been reported
to have antiradical, antioxidant and anti-inflammatory properties
(Jerez et al., 2007a,b; Maimoona et al., 2011; Packer et al., 1999)
and are marketed as a food supplement and a herbal-based medi-
cation, Pycnogenol® . Tannins from many different plants have been
used to make wood adhesives, such as grape pomace (Ping et al.,
2012), chestnut (Vásquez et al. 2009), pecan nut (Gornik et al.,
2000), mimosa (Kim, 2009; Navarrete et al., 2012) and maritime
∗ Corresponding author. Tel.: +33 558513722; fax: +33 558513737.
E-mail address: lucie.chupin@univ-pau.fr (L. Chupin).
pine (Jorge et al., 2002). Tannins from pine bark have also been
used to make foams (Lacoste et al., 2013; Tondi et al., 2009).
Traditionally, tannins are used in the tanning process of leather.
The capacity of tannins to precipitate proteins such as collagen,
thus generating a non-putrescible material under moist and warm
conditions. The best procedure to obtain good quality tannins for
leather is with an acetone–water–bisulfite procedure (Falcão and
Araújo, 2011; Herrick, 1980).
In order to increase the extraction of procyanidins without
the use of too many organic solvents, research has been done to
find new environmentally-friendly extraction conditions such as
microwave assisted extraction, ultrasound assisted extraction or
maceration (Aspé and Fernández, 2011).
This study aims to optimise tannin extraction form mar-
itime pine bark and characterise these tannins in order to
make wood adhesives and foams. Three extraction conditions
were compared. For the first time an extensive characterisa-
tion of maritime pine bark condensed tannins extracted was
conducted. The total polyphenolics and condensed tannins of
the extracts are evaluated. The extracts are characterised by
several colorimetric analyses, by their reactivity to formalde-
hyde, by Fourier transformed infrared spectroscopy (FTIR) and
by reverse-phase high pressure liquid chromatography (RP-
HPLC).

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898 L. Chupin et al. / Industrial Crops and Products 49 (2013) 897–903
Table 1
Extraction methods.
Extraction method Solvent
1 Water + 1% NaOH + 0.25% Na2 SO3 + 0.25% NaHSO3
2 Water + 5% NaOH + 0.25% Na2 SO3 + 0.25% NaHSO3
3 Water + 1% NaOH + 0.25% Na2 SO3 + 0.25% NaHSO3
2. Materials and methods
2.1. Reagents
(+) Catechin hydrate, (–) epicatechin gallate and cysteamine
hydrochloride were purchased from Sigma–Aldrich (St. Louis, USA).
Gallic acid, trifluoroacetic acid and HPLC grade acetonitrile were
obtained from Fisher Scientific (Waltham, USA). (−) Epicatechin
was obtained from LGC Standard (Teddington, UK) and vanillin was
purchased from VWR (Radnor, USA).
2.2. Samples
Bark from five different trees of maritime pine was collected in
the Landes forest (Uchacq, 40). The trees’ diameters ranged from
31 to 35.5 cm and were 27–32 years old. The barks were air-dried
and ground in a mill (Retsch) and sieved to select particles smaller
than 1 mm. The ground pine bark was kept in sealed bags.
2.3. Extraction
Tannins were extracted in different conditions described in
Table 1. Bark, water, NaOH, Na2 SO3 and NaHSO3 were stirred to
the desired temperature. NaOH was used in the extraction in order
to be in high alkalinity conditions and increase the extraction yield.
Na2 SO3 and NaHSO3 were added to lessen the viscosity of the
extracts and to stabilise them. This was kept stirring and heating for
120 min. The supernatant was filtered through Whatman paper no.
1 and the residue was rinsed with water. The filtrates were dried
in an oven at 40 ◦ C. The extraction yield was calculated as the per-
centage of the amount of extract recovered in mass compared to
the initial mass of dry bark.
2.4. Determination of total polyphenolic content
Total polyphenolic content was determined with the Folin-
Ciocalteu method (Singleton and Rossi, 1965). 2.5 mL of Folin
reagent (diluted 10 times) was added to 0.5 mL of aqueous extract.
2 mL of sodium carbonate (75 g L−1 ) was then added. The mix-
ture was then put in a water bath at 50 ◦ C for 5 min before the
absorbance was read at 760 nm. A calibration curve was done with
a solution of gallic acid (80 ppm, Jenway 6300 Spectrophotometer).
The results were obtained as mg of gallic acid equivalent (GAE) per
g of dry bark (mg GAE/g bark).
2.5. Determination of condensed tannins
Condensed tannin content was determined with the vanillin
method as described by Broadhurst and Jones (1978). 3 mL of
vanillin (4% in methanol) was added to 0.5 mL of the aqueous
extract. 1.5 mL of highly concentrated HCl was then added. The mix-
ture was then kept in the dark for 15 min at 20 ◦ C. The absorbance
was read at 500 nm. A calibration curve was prepared with a solu-
tion of catechin (30 ppm, Jenway 6300 Spectrophotometer). The
results were obtained in mg of catechin equivalent per g of dry
bark (mg CE/g bark).
Temperature (◦ C) Ratio (s/l)
80 1/9
80 1/9
70 1/9
2.6. Proanthocyanidin content
Proanthocyanidin content was obtained with a BuOH/HCl test
as described by Scalbert et al. (1989). 5 mL of an acidic ferrous
solution (77 mg FeSO4 ·7H2 O in 500 mL HCl/BuOH (2/3)) was added
to 0.5 mL of the aqueous extract. The tubes were covered and
put in a water bath at 95 ◦ C for 15 min. The absorbance was
read at 530 nm (Jenway 6300 Spectrophotometer). Results were
expressed as mg of cyanidin per g of dry bark (mg Cya/g bark)
(εmol = 34,700 L mol−1 cm−1 ).
mg CyaE A × V × D × M × 1000
= (1)
g bark ε×l×m
where A is absorbance at 530 nm; V, volume of the reaction; D, dilu-
tion factor; M, cyanidin molar mass; ε, molar extinction coefficient;
l, the path length; and m, mass of dry bark.
2.7. Stiasny number determination
The reactivity of the extracts to formaldehyde was determined
by measuring the Stiasny number as described by Voulgaridis et al.
(1985). A solution of extract at a concentration of 4 g L−1 was pre-
pared. 25 mL of this solution was put in a round bottom flask and
5 mL of formaldehyde 37% and 2.5 mL of HCl 10 M were added. The
mixture was heated under reflux for 30 min. The residue was fil-
tered through a sintered glass n◦ 2 or 4. The precipitate was washed
with water and dried at 105 ◦ C until constant weight. The reactivity
was calculated with the formula:
A
SI = × 100
B
where SI is Stiasny number; A, dry weight of the precipitate; and B,
dry weight of extract.
2.8. FTIR spectroscopy
FTIR spectra were recorded on a Perkin Elmer Spectrum One
equipped with an ATR-FTIR unit. A few milligrams of ground extract
sample were placed on a crystal (diamond/ZnSe). The spectra were
obtained with a resolution of 4 cm−1 and 10 co-addition scans in
a wavelength range of 650–4000 cm−1 . The spectra were collected
and analysed using Spectrum software (Perkin Elmer).
2.9. Thiolysis and RP-HPLC
The composition of the proanthocynidins in the extracts was
determined by RP-HPLC. The extracts were dissolved in methanol
(10 g L−1 ). An aliquot (400 ␮L) was added to 400 ␮L of acidified
methanol (3.3% HCl) and 800 ␮L of cysteamine hydrochloride
(50 g L−1 in methanol). The mixture was put in a waterbath at
40 ◦ C for 30 min. The proanthocyanidins were depolymerised by
thiolysis with cysteamine as described by Torres and Selga (2003).
The samples were analysed by RP-HPLC-DAD (Ultimate 3000,
Thermo Scientific), equipped with a acclaim 120 C18, 250 × 4.6 mm,
5 ␮m column. Elution: [A] 0.1% (v/v) aqueous TFA, [B] acetonitrile.
The solvent gradient was as follow: 0–12.6 min, 97.7A/2.3B (v/v);
12.6–15.6 min, 97A/3B; 15.6–18 min, 92A/8B; 18–28 min, 84A/16B;
28–58 min, 100B; 58–83 min, 100B. Detection was done at 220,
 L. Chupin et al. / Industrial Crops and Products 49 (2013) 897–903
Table 2
Total polyphenolic content extracted with three different conditions.
Extraction method Extraction yield Total polyphenolics
(%)1,3 (mg GAE/g bark)2,3
1 22.06 ± 7.79a 62.21 ± 22.54a
2 31.30 ± 10.42b 22.01 ± 4.91b
3 22.07 ± 4.46a 54.23 ± 19.36a
a,b
Group of values with significant differences between each group for each parameters.
1
Standard deviations are of at least ten replicates.
2
Standard deviations are of at least eighteen replicates.
3
The means were statistically analysed with the Student test or the Welch test when required at P < 0.05 significance level.
254, 272 and 280 nm. The chromatograms were analysed with
Chromeleon software.
2.10. Statistical analysis
The data are presented as mean ± SD values. The extrac-
tion yield, the total polyphenolic content, the condensed tannins
content, the cyanidin equivalent content and the Stiasny num-
ber for the different trees and different extraction methods
were compared using Fisher Snedcor test, Aspin Welch test,
Mann–Whitney–Wilcoxon test, student test and the difference of
mean test used when required. All the statistical analyses were
carried out at P < 0.05 significance level.
3. Results and discussion
3.1. Extraction
Three extraction methods are performed on maritime pine bark.
For each method the extraction yield is presented in Table 2. The
most important yield is obtained with the highest concentration of
NaOH and the highest temperature (extraction method 2, Table 1).
We obtain the highest extraction yield, 31.30%, with 5% NaOH at
80 ◦ C. It is significantly higher than for the other protocols (Welch
and Student tests). With 1% NaOH we obtain 22.06% and 22.07%
at 80 ◦ C and 70 ◦ C (extraction method 1 and 3, Table 1). There is
no significant difference between the two (Welch test). The more
alkaline the solution is the more extracts are recovered. Similar
extraction yields are obtained by Yazaki and Collins (1994), whilst
extracting maritime pine bark tannins with a succession of water
extraction and water at pH 8.3 at 100 ◦ C for 15 min. Vásquez et al.
(1996, 2001) also find similar results when extracting maritime
pine bark tannins in water with 2% and 5% NaOH at 100 ◦ C and
90 ◦ C respectively for 30 min. Smaller amounts of extractives are
recovered when the solvent used are dichloromethane, ethanol and
urea (Bertaud et al., 2011; Fradinho et al., 2002; Seabra et al., 2012).
3.2. Total polyphenolic content
The total polyphenolic content for the different extraction meth-
ods and for different maritime pine trees are presented in Table 2.
The total phenol content does not significantly change between
maceration at 70 ◦ C and 80 ◦ C for the same amount of NaOH (54.23
and 62.21 mg GAE/g bark respectively) (extraction method 1 and
2, Table 1), (Welch and Student tests). However a lower amount
of polyphenols are extracted with 5% NaOH (22.01 mg GAE/g bark)
than with 1% NaOH. A significantly lower amount of total polyphe-
nols is extracted with the second extraction method than for
the first and third extraction method. For the second extraction
method, the total polyphenolic content is very low compared to
the extraction yield, which means with a higher amount of NaOH
an important quantity of non-tannin material is recovered.
Five different bark trees are submitted to an extraction at 70 ◦ C,
giving a range in polyphenolic content from 37.21 to 96.81 mg
899
Vanillin test (mg BuOH–HCl (mg Stiasny number pH1,3
CE/g bark)2,3 CyaE/g bark)2,3 (%)2,3
4.73 ± 1.67a 5.65 ± 3.15a 48.97 ± 9.56a 9.52 ± 0.33a
2.18 ± 0.57b 1.20 ± 0.44 b 17.92 ± 9.49b 10.87 ± 0.13b
5.15 ± 2.97a 5.43 ± 3.34a 53.98 ± 12.48a 9.89 ± 0.31a
GAE/g bark (Table 3). This can be due to the difference in age of
the trees. Alonso-Amelot et al. (2007) show the trees at the same
altitude do not have the same amount of phenols in their fronds. The
trees with greater exposure to the sun have higher concentrations
of phenols than the shaded trees.
3.3. Condensed tannins content
Results of the vanillin assay for the different extraction methods
are presented in Tables 2 and 3. The highest extractions of con-
densed tannins are obtained with 1% NaOH at 70 ◦ C and at 80 ◦ C
(5.15 mg CE/g bark and 4.73 mg CE/g bark respectively), (Welch
and Mann–Whitney–Wilcoxon tests). We extract the least amount
of condensed tannins at 80 ◦ C with 5% NaOH (2.18 mg CE/g bark).
Ku et al. (2007) find similar amounts of condensed tannins for
other pine species such as P. densiflora (19.6 mg CE/g bark), P. rigida
(70.4 mg CE/g bark) and P. radiata (93.7 mg CE/g bark).
The tree two has 10.62 mg CE/g bark. It is significantly higher
than the other trees when the extraction is carried out in the same
conditions (Mann–Whitney–Wilcoxon test). The condensed tan-
nins contents are statistically the same for the trees one, three
and five (Mann–Whitney–Wilcoxon and Student tests). There are
more condensed tannins in the fourth tree than for the first, third
and fifth trees (Student and Mann–Whitney–Wilcoxon tests). The
bark was obtained in winter; the tree two might have been less
lixiviated which explains the higher amount of condensed tannins
compared to the other trees. Saad et al. (2012) notice this lixiviation
of condensed tannins soluble in water for pomegranate peels. The
condensed tannins soluble in water from the trees one, three, four
and five have been partly lixiviated by the rain.
3.4. Cyanidin equivalent content
The BuOH–HCl assay gives similar results as the vanillin assay
(Tables 2 and 3). We have the same trend. The highest amounts of
cyanidin are obtained with the first and third method of extraction
(5.65 mg CyaE/g bark and 5.43 mg CyaE/g bark, respectively). The
second extraction method presents less cyaniding than the other
two (1.20 mg CyaE/g bark) (Mann–Whitney–Wilcoxon test). The
cyanidin contents are not statistically different for the extraction
protocols one and three (Welch test). The cyanidin content for the
extraction with 5% NaOH can be due to a mechanism of autocon-
densation of tannins. Merlin and Pizzi (1996) have noticed that the
procyanidins from P. radiata autocondensate in presence of silica
at high alkalinity. In this study silica is not added to the extraction
solvent however the presence of silica as sand attached to the bark
is possible.
There is significantly more cyanidin in the tree two than in
the others (Mann–Whitney–Wilcoxon, Student and Welch tests).
The trees one, four and five have significantly the same amount
of cyanidin (Mann–Whitney–Wilcoxon and Welch tests). The
third tree has the lowest amount of cyanidin for all the trees
(Mann–Whitney–Wilcoxon, Student and Welch tests).
900 L. Chupin et al. / Industrial Crops and Products 49 (2013) 897–903

Table 3
Total polyphenolic content extracted from the bark of five different maritime pine trees.

Extraction yield (%)1,3 Total polyphenolics Vanillin test (mg CE/g BuOH–HCl (mg Cya/g Stiasny Number (%)2,3 pH1,3
(mg GAE/g bark)2,3 bark)2,3 bark)2,3

P1T1 26.09 ± 4.02a 61.81 ± 11.62a 5.38 ± 0.84a 5.67 ± 1.62a 48.54 ± 11.71a 9.81 ± 0.33
P1T5 22.84 ± 12.31a 64.09 ± 31.60a 4.08 ± 2.08a 5.63 ± 4.30a 49.40 ± 7.53a 9.23 ± 0.08
P2T1 33.73 ± 3.4d 24.68 ± 3.34d 2.58 ± 0.30d 1.39 ± 0.32a 17.09 ± 11.42a 10.90 ± 0.03
P2T5 20.48 ± 5.44e 19.34 ± 4.90e 1.77 ± 0.48 1.01 ± 0.49a 18.75 ± 7.71a 10.75 ± 0.10
P3T1 18.39 ± 3.29d 48.50 ± 10.43a 3.42 ± 0.64a 3.86 ± 0.96a 48.82 ± 16.15a 10.36 ± 0.04
P3T2 26.94 ± 3.04a 96.81 ± 1.32d 10.62 ± 1.66d 11.73 ± 1.32d 47.51 ± 3.49a 9.65 ± 0.08
P3T3 19.86 ± 2.46b 37.21 ± 4.08e 2.81 ± 0.48a 3.00 ± 0.68e 58.13 ± 15.44abc 9.78 ± 0.08
P3T4 24.70 ± 2.31a 56.25 ± 3.35ab 4.78 ± 0.39e 4.74 ± 0.24a 52.74 ± 10.05ab 9.56 ± 0.09
P3T5 20.95 ± 4.11b 60.33 ± 8.54b 4.10 ± 0.67a 3.81 ± 0.66a 64.24 ± 5.42c 10.06 ± 0.08
a,b,c,d,e
Group of values with significant differences between each group for each parameters.
ab
No significant differences between a and b group.
abc
No significant differences between a, b and c group.
1
Standard deviations are of five replicates.
2
Standard deviations are of nine replicates.
3
The means were statistically analysed with the Student test, the Mann–Whitney–Wilcoxon test and the Welch test when required at P < 0.05 significance level.

Catechin and cyanidin levels are not significantly different,
when looking at the results obtained with the vanillin assay and
the BuOH–HCl assay (difference of mean test). The vanillin assay
measures condensed tannins and simple flavonoids and not only
condensed tannins like the BuOH–HCl assay (FAO/IAEA, 2000). This
can mean that we have extracted mostly condensed tannins and
very few simple flavonoids.
the trees come from. In this study, the trees come from the Landes
forest in France. Guilley et al. (2004) have noticed that oak trees had
different properties when grown in different regions of France. The
Stiasny number is significantly the same for the extraction at 70 ◦ C
and at 80 ◦ C (Student test). The Stiasny number seems independent
for this range of temperature. This supposition is confirmed with
the findings of Vásquez et al. (2001).

3.5. Stiasny number 3.6. FTIR spectroscopy

The results are presented in Tables 2 and 3. The Stiasny number
gives us the reactivity of our extracts to formaldehyde, this informa-
tion can help us determine if the extracts can be used as adhesives
(Vásquez et al., 2009; Vieira et al., 2011; Ping et al., 2011b). Yazaki
and Collins (1994) assessed that the minimum Stiasny value to pro-
duce high quality adhesives is 65%. However, Ping et al. (2011a)
have produced good quality adhesives while obtaining a Stiasny
number of 46%. In this study, we obtain a similar Stiasny value in the
case of the first extraction method and the third extraction method
(respectively 48.97% and 53.98%). The minimum value is obtained
for the second extraction method (17.92%), which is significantly
lower than the Stiasny number for the other two protocols (Student
test). The Stiasny number decreases for high amounts of NaOH, sim-
ilar results were obtained by Vásquez et al. (1996) and Voulgaridis
et al. (1985) for other pine species. Vásquez et al. (1996) obtained
more important Stiasny values for maritime pine bark from Spain
with similar extraction conditions; this could be due to the region
The IR spectra of the tannins extracted with different conditions
are recorded in the 650–4000 cm−1 region and are presented in
Fig. 1. These spectra show that there are clear differences between
the extraction method with 5% NaOH (P2T1) and the ones with
1% NaOH. The spectrum for the extraction with 1% NaOH at 80 ◦ C
(P1T1) is similar to the spectrum of the extracts obtained with 1%
NaOH at 70 ◦ C (P3T1). The analyses of the spectra are based on
the assignments given by Boeriu and Bravo (2004) and Ping et al.
(2012).
The band at 3300 cm−1 is assigned as OH stretch vibra-
tion in phenolic and aliphatic structures. Small peaks at 2930
and 2850 cm−1 originate from CH stretch vibration in aromatic
methoxyl groups and in methyl and methylene groups of side
chains. The band at 1575 cm−1 cannot be identified precisely; Soto
et al. (2005) suggest the peaks between 1400 and 2000 cm−1 show
the aromatic nature of the structure. The band at 1370–1380 cm−1
is attributed to phenolic stretch vibration of OH and aliphatic CH

Table 4
Composition of maritime pine bark extracts determined by RP-HPLC after thiolysis.

Catechin (mg/g bark)3 Epicatechin (mg/g bark)3 Epicatechin gallate Gallic acid (mg/g bark)3
(mg/g bark)3

P12 3.73 ± 1.46a 1.51 ± 0.78a 0.68 ± 0.91a 2.74 ± 1.37a

P22 4.78 ± 1.45a 1.75 ± 0.68a 0.78 ± 1.12a 5.20 ± 2.01a
P32 3.82 ± 0.81a 1.43 ± 0.68a 0.49 ± 0.13a 1.43 ± 0.42a
P1T11 3.86 ± 0.73a 1.87 ± 0.93a 0.99 ± 1.05a 2.77 ± 0.73a
P1T51 3.59 ± 1.80a 1.23 ± 0.30a 0.31 ± 0.10a 2.72 ± 1.69a
P2T11 5.05 ± 0.70a 1.92 ± 0.41a 0.67 ± 0.54a 5.50 ± 0.73a
P2T51 4.56 ± 1.70a 1.61 ± 0.75a 0.87 ± 1.33a 4.97 ± 2.43a
P3T11 3.02 ± 0.36a 0.84 ± 0.40ab 0.43 ± 0.14ab 2.16 ± 0.46a
P3T21 4.83 ± 0.42c 1.79 ± 0.41c 0.48 ± 0.04ab 3.31 ± 0.36b
P3T31 3.23 ± 0.42a 0.93 ± 0.18a 0.60 ± 0.13b 2.62 ± 0.38a
P3T41 4.05 ± 0.30b 1.56 ± 0.69b 0.51 ± 0.15ab 3.36 ± 0.27b
P3T51 3.67 ± 0.71ab 1.80 ± 0.71ab 0.40 ± 0.07ab 3.08 ± 0.66ab
a,b,c
Group of values with significant differences between each group for each parameters.
ab
No significant differences between a and b group.
1
Standard deviations are of at least three replicates.
2
Standard deviations are of at least nine replicates.
3
The means were statistically analysed with the Student test, the Mann–Whitney–Wilcoxon test and the Welch test when required at P < 0.05 significance level.
 L. Chupin et al. / Industrial Crops and Products 49 (2013) 897–903 901

Fig. 1. FTIR spectra of (a) P1T1 (1% NaOH, 80 ◦ C); (b) P2T1 (5% NaOH, 80 ◦ C); (c) P3T1 (1% NaOH, 70 ◦ C).

deformation in methyl groups. This band is common to lignins and
is more intense for P2T1. It can mean that more lignins are extracted
at high alkalinity. Aromatic CH bending in plane bending vibration
is detected at 1115 cm−1 and a CO stretch vibration is produced
at 1040 cm−1 .The lower intensity of the peaks at 1040 cm−1 and
1115 cm−1 at pH > 10 can be due to an opening of the cyclic ether
structure of polyflavonoids (Soto et al., 2005). For peaks at wave-
lengths smaller than 900 cm−1 , aromatic CH stretch vibration is
detected. Three main bands are detected only for P1T1 and P3T1 at
1495 cm−1 assigned to aromatic squeal vibration, 1440 cm−1 cor-
responding to CH deformation and aromatic ring vibration, and
at 1260 cm−1 . This last peak is harder to identify, Vásquez et al.
(2000) attribute it as C O stretch vibration, whereas Ping et al.
(2012) assign it to saturated C C stretch vibration attributed to
CR2 CHR CR(SO3 2− ). This structure can be due to the opening of
the pyran ring during sulphitation of flavanoid tannins.
The IR spectra of the tannins extracted with the third method
for five different trees are recorded in the 650–4000 cm−1 region
and are presented in Fig. 2. All the spectra present the same bands.
There is a band at 3300 cm−1 corresponding to an OH stretch
vibration in phenolic and aliphatic structures, two small peaks at
2950 and 2850 cm−1 assigned to CH stretch vibration in aromatic
methoxyl groups and in methyl and methylene groups, a band
at 1370–1380 cm−1 due to phenolic stretch vibration of OH and
aliphatic CH deformation in methyl groups, a peak at 1115 cm−1
attributed to aromatic CH bending in plane bending vibration and
a band at 1040 cm−1 corresponding to CO stretch vibration. Small
differences can be noted for P3T4 (Fig. 2d) and P3T5 (Fig. 2e). P3T4
has a very intense peak at 1115 cm−1 , which corresponds to aro-
matic CH bending vibration, compared to the spectra for the other
extracts of the other trees. P3T5 is the only tree to present a peak
at 1515 cm−1 originating from aromatic skeletal vibration.

Fig. 2. FTIR spectra of tannins extracted with the third method for five different trees; (a) P3T1; (b) P3T2; (c) P3T3; (d) P3T4; (e) P3T5.
902 L. Chupin et al. / Industrial Crops and Products 49 (2013) 897–903
Fig. 3. RP-HPLC chromatogram of P3T5 after thiolysis at 280 nm (Cat: catechin, ECG: epicatechin gallate, GA: gallic acid, EC: epicatechin).
3.7. RP-HPLC
The nature of the polyphenols extracted can be determined by
thiolysis followed by RP-HPLC (Guyot et al., 2001). Catechin, epi-
catechin, epicatechin gallate and gallic acid are detected (Fig. 3,
Table 4). These components are also found by Jerez et al. (2006,
2007a,b) and Navarrete et al. (2010). Catechin is the main con-
densed tannin present in the extracts followed by epicatechin
and then epicatechin gallate. When comparing the amounts mea-
sured by HPLC and with the vanillin assay for catechin, we find
that there are no significant differences between the two (differ-
ence of mean test). The same amounts of catechin, epicatechin,
epicatechin gallate are obtained for the three extraction meth-
ods (Student, Mann–Whitney–Wilcoxon and Welch tests). Extracts
obtained with the second method present more gallic acid than the
other extracts (Student and Welch tests).
4. Conclusion
In this study, the third extraction method, with 1% NaOH at 70 ◦ C,
presented the highest amounts of polyphenolics, of condensed tan-
nins and the highest reactivity to formaldehyde. Smaller amounts
of polyphenolics and condensed tannins were obtained when we
increased the extraction temperature or the concentration of NaOH.
High concentration of NaOH gave better extraction yields but less
tannin is extracted. The main condensed tannins units present in
the extracts were catechin units. Epicatechin, epicatechin gallate
and gallic acid were also detected but in lower quantities. Further
studies need to be conducted since all the components present were
not identified.
The FTIR spectra highlighted the presence of other polyphenolic
compounds in the extracts such as lignins. The second method
of extraction, with 5% NaOH at 80 ◦ C, had more lignins than the
other extraction methods. Therefore the higher the alkalinity of
the extraction solvent the more lignins were extracted.
Complementary studies will be needed in order to evaluate the
possibility of using the tannins extracted in this study to prepare a
wood adhesive.
Acknowledgements
We gratefully acknowledge the financial support of the “Conseil
Général des Landes” and this work was funded by ANR-10-EQPX-16
Xyloforest.
References
Alonso-Amelot, M.E., Oliveros-Bastidas, A., Calgagno-Pisarelli, M.P., 2007. Phenolics
and condensed tannins of high altitude Pteridium arachnoideum in relation to
sunlight exposure, elevation and rain regime. Biochem. Syst. Ecol. 35, 1–10.
Aspé, E., Fernández, K., 2011. The effect of different extraction techniques on extrac-
tion yield, total phenolic, and anti-radical capacity of extracts from Pinus radiata
bark. Ind. Crop. Prod. 34, 838–844.
Bertaud, F., Tapin-Lingua, S., Pizzi, A., Navarrete, P., Petit-Conil, M., 2011. Develop-
ment of green adhesives for fibreboards manufacturing, using tannins and lignin
from pulp mill residues. Rev. Assoc. Tech. Ind. Papet. 65, 6–12.
Boeriu, C.G., Bravo, D., 2004. Characterisation of structure-dependent functional
properties of lignin with infrared spectroscopy. Ind. Crop. Prod. 20, 205–218.
Broadhurst, R.B., Jones, W.T., 1978. Analysis of condesed tannins using acidified
vanillin. J. Sci. Food Agric. 29, 788–797.
Falcão, L., Araújo, M.E.M., 2011. Tannins characterisation in new and historical veg-
etable tanned leather fibres by spot tests. J. Cult. Herit. 12, 149–156.
FAO/IAEA, 2000. Quantification of tannins in tree foliage. FAO/IAEA working docu-
ment, IAEA, Vienna, 26.
Fradinho, D.M., Pascoal Neto, C., Evtugin, D., Jorge, F.C., Irle, M.A., Gil, M.H., Pedrosa
De Jesus, J., 2002. Chemical characterisation of bark and of alkaline bark extracts
from maritime pine grown in Portugal. Ind. Crop. Prod. 16, 23–32.
Gornik, D., Hemingway, R.W., Tišler, V., 2000. Tannin-based cold-setting adhesives
for face lamination of wood. Holz Roh-Werkst. 58, 23–30.
Guilley, E., Charpentier, J.P., Ayadi, N., Snakkers, G., Nepveu, G., Charrier, B., 2004.
Decay resistance against Coriolus versicolor in Sessile oak (Quercus petraea Liebl.):
analysis of the between-tree variability and correlations with extractives, tree
growth and other basic wood properties. Wood Sci. Technol. 38, 539–554.
Guyot, S., Marnet, N., Drilleau, J.-F., 2001. Thiolysis-HPLC characterization of apple
procyanidins covering a large range of polymerization states. J. Agric. Food Chem.
49, 14–20.
Herrick, F.W., 1980. Chemistry and utilization of western hemlock bark extractives.
J. Agric. Food Chem. 28, 228–237.
Jerez, M., Pinelo, M., Sineiro, J., Núñez, M.J., 2006. Influence of extraction conditions
on phenolic yields from pine bark: assessment of procyanidins polymerization
degree by thiolysis. Food Chem. 94, 406–414.
Jerez, M., Selga, A., Sineiro, J., Torres, J.L., Núñez, M.J., 2007a. A comparison between
bark extracts from Pinus pinaster and Pinus radiata: antioxidant activity and
procyanidin composition. Food Chem. 100, 439–444.
Jerez, M., Touriño, S., Sineiro, J., Torres, J.L., Núñez, M.J., 2007b. Procyanidins from
pine bark: relationships between structure, composition and antiradical activity.
Food Chem. 104, 518–527.
 L. Chupin et al. / Industrial Crops and Products 49 (2013) 897–903
Jorge, F.C., Pascoal Neto, C., Irle, M.A., Gil, M.H., Pedrosa De Jesus, J., 2002.
Wood adhesives derived from alkaline extracts of maritime Pine bark: prepa-
ration, physical characteristics and bonding efficacy. Holz Roh-Werkst. 60,
303–310.
Kim, S., 2009. Environment-friendly adhesives for surface bonding of wood-based
flooring using natural tannin to reduce formaldehyde and TVOC emission. Biore-
sour. Technol. 100, 744–748.
Ku, C.S., Jang, J.P., Mun, S.P., 2007. Exploitation of polyphenol-rich pine barks for
potent antioxidant activity. Jpn. Wood Res. Soc. 53, 524–528.
Lacoste, C., Basso, M.C., Pizzi, A., Laborie, M.-P., Celzard, A., Fierro, V., 2013. Pine
tannin-based rigid foams: mechanical and thermal properties. Ind. Crop. Prod.
43, 245–250.
Maimoona, A., Naeem, I., Saddiqe, Z., Jameel, K., 2011. A review on biological,
nutraceutical and clinical aspects of French maritime pine bark extract. J.
Enthnopharmcol. 133, 261–277.
Merlin, A., Pizzi, A., 1996. An ESR study of the silica-induced autocondensation of
polyflavonoid tannins. J. Appl. Polym. Sci. 59, 945–952.
Navarrete, P., Pizzi, A., Pasch, H., Rode, K., Delmotte, L., 2010. MALDI-TOF and 13C
NMR characterization of maritime pine industrial tannin extract. Ind. Crop. Prod.
32, 105–110.
Navarrete, P., Pizzi, A., Tapin-Lingua, S., Benjelloun-Mlayah, B., Pasch, H., Delmotte,
L., Rigolet, S., 2012. Low formaldehyde emitting biobased wood adhesives man-
ufactured from mixtures of tannin and glyoxylated lignin. J. Adhes. Sci. Technol.
26, 1667–1684.
Packer, L., Rimbach, G., Virgili, F., 1999. Antioxidant activity and biologic properties
of a procyanidin-rich extract from pine (Pinus maritima) bark. Pycnogenol. Free
Radic. Biol. Med. 27, 704–724.
Ping, L., Brosse, N., Chrusciel, L., Navarrete, P., Pizzi, A., 2011a. Extraction of con-
densed tannins from grape pomace for use as wood adhesives. Ind. Crop. Prod.
33, 253–257.
Ping, L., Pizzi, A., Guo, Z.D., Brosse, N., 2011b. Condensed tannins extraction from
grape pomace: characterization and utilization as wood adhesives for wood
particleboard. Ind. Crop. Prod. 34, 907–914.
Ping, L., Pizzi, A., Guo, Z.D., Brosse, N.N., 2012. Condensed tannins from
grape pomace: characterization by FTIR and MALDI-TOF and pro-
duction of environment friendly wood adhesive. Ind. Crop. Prod. 40,
13–20.
903
Saad, H., Charrier-El Bouhtoury, F., Pizzi, A., Rode, K., Charrier, B., Ayed, N., 2012.
Characterization of pomegranate peels tannin extractives. Ind. Crop. Prod. 40,
239–246.
Scalbert, A., Monties, B., Janin, G., 1989. Tannins in wood: comparison of different
estimation methods. J. Agric. Food Chem. 37, 1324–1329.
Seabra, I.J., Dias, A.M.A., Braga, M.E.M., De Sousa, H.C., 2012. High pressure solvent
extraction of maritime pine bark: study of fractionation, solvent flow rate and
solvent composition. J. Supercrit. Fluid. 62, 135–148.
Singleton, V.L., Rossi, J.A., 1965. Colorimetry of total phenolics with
phosphomolybdic-phosphotungstic acid reagents. Am. J. Enol. Vitic. 16,
144–158.
Soto, R., Freer, J., Baeza, J., 2005. Evidence of chemical reactions between di- and
poly-glycidyl ether resins and tannins isolated from Pinus radiata D. Don bark.
Bioresour. Technol 96, 95–101.
Tondi, G., Oo, C.W., Pizzi, A., Trosa, A., Thevenon, M.F., 2009. Metal adsorption of
tannin based rigid foams. Ind. Crop. Prod. 29, 336–340.
Torres, J.L., Selga, A., 2003. Procyanidin size and composition by thiolysis with cys-
teamine hydrochloride and chromatography. Chromatographia 57, 441–445.
Vásquez, G., Antorrena, G., González, J., Alvarez, J.C., 1996. Tannin-based adhesives
for bonding high-moisture Eucalyptus veneers: influence of tannin extraction
and press conditions. Holz Roh-Werkst. 54, 93–97.
Vásquez, G., Freire, S., González, J., Antorrena, G., 2000. Characterization of Pinus
pinaster bark and its alkaline extracts by diffuse reflectance Fourier transform
infrared (DRIFT) spectroscopy. Holz Roh-Werkst. 58, 57–61.
Vásquez, G., González-Álvarez, J., Freire, S., López-Suevos, F., Antorrena, G., 2001.
Characteristics of Pinus pinaster bark extracts obtained under various extraction
conditions. Holz Roh-Werkst. 59, 451–456.
Vásquez, G., González-Álvarez, J., Santos, J., Freire, M.S., Antorrena, G., 2009. Evalua-
tion of potential applications for chesnut (Castanae sativa) shell and eucalyptus
(Eucalyptus globulus) bark extracts. Ind. Crop. Prod. 29, 364–370.
Vieira, M.C., Lelis, R.C.C., Silva, B.C., Oliveira, G.L., 2011. Tannin extraction from the
bark of Pinus oocarpa var. oocarpa with sodium carbonate and sodium bisulfate.
Floresta e Ambiente 18, 1–8.
Voulgaridis, E., Grigoriou, A., Passialis, C., 1985. Investigation on bark extractives of
Pinus halepensis Mill. Holz Roh-Werkst. 43, 269–272.
Yazaki, Y., Collins, P.J., 1994. Wood adhesives based on tannin extracts from barks
of some pine and spruce species. Holz Roh-Werkst. 52, 307–310.