Biomaterials 23 (2002) 28412847

Study on the stabilisation of collagen with vegetable tannins

in the presence of acrylic polymer
B. Madhan, C. Muralidharan*, R. Jayakumar
Central Leather Research Institute, Council of Scientific and Industrial Research, Adyar, Chennai 600 020, India
Received 30 May 2001; accepted 2 December 2001

Abstract
Collagen, a unique connective tissue protein nds extensive application as biocompatible biomaterial in wound healing, as drug
carriers, cosmetics, etc. A study has been undertaken to stabilise Type-I collagen of rat-tail tendon using plant polyphenol (Acacia
Mollissima) in the presence of an acrylic polymer. It has been found that collagen bres pre-treated with acrylic polymer followed by
the treatment with Acacia Mollissima exhibited an increase in hydrothermal stability by 251C. Infrared spectroscopic studies display
the changes in the spectral characteristics of native and treated collagen lms. Transmission electron microscopic and circular
dichroic studies provide an insight into the understanding of the improved stabilisation of collagen, due to treatment with acrylic
polymer and plant polyphenols. The study is expected to enhance the biomaterial applications of collagen tissues. r 2002 Elsevier
Science Ltd. All rights reserved.
Keywords: Vegetable tannins; Polyphenols; Acrylics; Collagen; Stabilisation; Biomaterial

1. Introduction
Collagen is the major structural component of
connective tissues. It is an important biomaterial nding
several applications as prosthesis, articial tissue, drug
carrier and cosmetics. Vegetable tannins are polyphenolic compounds present in the plant extracts. These are
compounds with molecular weights in the range of
5003000 Da [1,2]. The chromatographic studies indicate that vegetable tannin extracts are heterogeneous
polyphenolic species [35]. In the process of leather
making, vegetable tannins are used in the stabilisation of
skin [6]. Skin predominantly contains collagen, the
abundant protein in animals. There are 19 different
types of collagen [7], of which the type I collagen is the
main component of skin, tendon, bone and other tissues.
This protein is present as chains wound in tight triple
helical form [8], which are organised into brils of great
strength and stability [9]. The structure of collagen is
stabilised by inter- and intra-chain hydrogen bonds [10]
and by water-mediated hydrogen bonds [11,12]. Some of
the oligomers of chromium are known to interact with
*Corresponding author. Tel.: +91-44-491-5730; fax: +91-44-4911589.
E-mail address: cmurali62@yahoo.com (C. Muralidharan).

collagen [1315] bringing about irreversible matrix

changes and long-range ordering, thus imparting higher
hydrothermal stability [16].
Polyphenols in the form of vegetable tannin extracts
are being used in the process of stabilisation of skin
tracing back to the history of mankind. Polyphenols
present in extracts of green tea are known to possess
anti-carcinogenic activity. Extracts of green tea mostly
contain derivatives of catechins [17,18]. Pharmacological activity of tannins has been investigated, and their
antitumorantiviral effects have been revealed earlier
[19]. Polyphenols of vegetable tannin extracts are
capable of cross-linking with collagen through the
formation of multiple hydrogen bonds [20,21]. In our
earlier work, we were able to obtain leather of higher
hydrothermal stability by the treatment with vegetable
tannins in the presence of acrylic polymer [22]. Collagen
is the major leather-making protein [23]. Hence, it was
felt necessary to study the stabilisation of collagen
brought about by the usage of vegetable tannins in the
presence of an acrylic polymer. An attempt has been
made to understand the process of stabilisation of
collagen. Rat-tail tendon (RTT) which predominantly
contains type I collagen has been used in this study.
Wattle or Mimosa (Accasia Millissima) has been chosen
as the vegetable tannin source for the present study.

0142-9612/02/$ - see front matter r 2002 Elsevier Science Ltd. All rights reserved.
PII: S 0 1 4 2 - 9 6 1 2 ( 0 1 ) 0 0 4 1 0 - 0

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B. Madhan et al. / Biomaterials 23 (2002) 28412847

Mimosa belongs to a group of condensed tannins, which

basically contains molecules with avanoid structure
[2426].

tannins as mentioned in Section 2.3. The treated

collagen lms were washed with water air-dried and
characterised using FT-IR.

2. Materials and methods

2.6. Circular dichroic (CD) spectroscopic studies

2.1. Materials

Collagen solution extracted from RTT by a known

procedure [28] was used for CD studies. The collagen
content of the solution was estimated by standard
procedure [29]. Conformation of native collagen in 5 mm
acetic acid was recorded in the far UV region using
Jasco 715 CD spectropolarimeter. The collagen solution
(0.3 mg/ml) was treated with acrylic polymer (3 mg/ml),
and vegetable tannins (3 mg/ml), and the reaction
mixture was investigated for the conformational
changes.

Mimosa tannin extract from Tanzanian source was

used as a source of polyphenol. Commercial acrylic
polymer with an average molecular weight of 8000 was
employed for the treatment of collagen along with the
polyphenol.
2.2. Structural elucidation of acrylic polymer
The commercial acrylic polymer chosen for the study
was characterised using FT-IR, C13 NMR and H1 NMR
spectroscopic analysis.
2.3. Treatment of RTT fibres
Collagen bres teased from the tails of 6-weeks old
Sprague-Dawley rats were used for the study. Teased
collagen bres were washed with 0.9% NaCl at 41C, to
remove the adhering soluble proteins. The RTT was
washed extensively in double-distilled water at 41C.
Three different experiments were carried out with these
bres. One set of the tendons was treated with 1%
acrylic polymer for 12 h and another set was treated with
5% vegetable tannins (Acacia Mollissima) for 12 h.
Another experiment was carried out by treating the
tendons initially with 1% acrylic polymer for 1 h,
followed by treatment with 5% vegetable tannins for
12 h. Acrylic polymer and vegetable tannins used for the
treatment were weakly acidic.
2.4. Shrinkage temperature determination
The shrinkage temperature of RTT bres treated with
acrylics and vegetable tannins were measured using a
micro-shrinkage meter as described below using the
standard method [27]. A small strip of bre was cut and
placed on a water-grooved microscope slide. The slide in
turn was placed on a heating stage along with a
microscope mounted above the heating stage. The rate
of heating was maintained at 21C/min. The temperature
at which the bre shrinks to one-third of its length was
taken as the shrinkage temperature.
2.5. Interaction studies with collagen film
The collagen solution prepared from RTT [28] was
cast on a glass and airdried in a laminar ow hood. The
collagen lms were treated with acrylic, vegetable

2.7. Electron microscopic studies

The native and the treated RTT samples were
examined under a transmission electron microscope
(JEOL-1200EX) to identify the topological distribution
of vegetable tannins by analysing the change in banding
patterns of the native collagen.

3. Results
3.1. Characterisation of acrylic polymer
The FT-IR spectrum of the acrylic polymer is shown
in Fig. 1a. The carbonyl (C O) stretching vibration at
1708 cm1 is due to the carboxylic group; following this
signal, there is a shoulder signal at 1635 cm1 and this
may be due to the carboxyl group of carboxylate anion
(Fig. 1a). A series of signals noticed in the range of
14791178 cm1 support the structure of polymethacrylic acid. The conrmation of the sample being
polymethacrylic acid is established using H1 and C13
NMR spectroscopy shown in Fig. 1b and c. The H1
NMR shows four signals in the range of 1.072.07 ppm,
which are due to a-methyl and methylene proton of the
backbone. The C13 NMR shows a group of signals in
the range of 183185 ppm, which are due to carbonyl
carbons of the carboxylic group and its anion. This is
further supported by the signals observed at 5557, 47,
48 and 1920 ppm, respectively. The presence of a signal
at 1920 ppm clearly indicates the presence of a-methyl
group thereby establishing the polymethacrylic acid
structure. Additional signals as observed in the spectrum
could possibly be due to other additives present in the
commercial polymer. No attempt has been made to
elucidate these signals, as they were not expected to
inuence any major change in the collagen matrix.

B. Madhan et al. / Biomaterials 23 (2002) 28412847

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Table 1
Shrinkage temperature (1C) of native and treated collagen bres
RTT collagen bres

Shrinkage temperature (1C)

Native
Acrylic polymer
Vegetable tannins
Acrylic+vegetable tannins

58
68
76
86

found to be 581C. The RTT bres treated with the

acrylic polymer and vegetable tannin extract exhibit
shrinkage temperatures of 681C and 761C, respectively.
The RTT bre treated with acrylic followed by vegetable
tannin treatment exhibits a hydrothermal shrinkage
temperature of 861C.
3.3. Interaction of collagen with acrylic polymer and
vegetable tannins

Fig. 1. (a) FT-IR spectrum of the acrylic polymer; (b) H1 NMR

spectrum of the acrylic polymer; and (c) C13 NMR spectrum of the
acrylic polymer.

3.2. Hydrothermal stability of collagen fibres

The shrinkage temperatures measured for the native
and treated RTT bres are shown in Table 1. The
shrinkage temperature for native collagen bre has been

The IR spectra of the untreated collagen lm and

lms treated with acrylic polymer, vegetable tannins and
a combination of acrylic with vegetable tannins are
shown in Fig. 2ad, respectively. The major feature of
the IR spectrum of collagen lm (Fig. 2a) is the amide I
band between 1640 and 1660 cm1, which arises from
the stretching vibration of C O groups of amide
groups in protein. The intense absorption between 1500
and 1600 cm1 is due to the amide II mode, observed at
1550 cm1 in the spectrum for collagen (Fig. 2a), which
arises from NH stretching vibration strongly coupled
to the CN stretching vibration of collagen amide
groups. Collagen has high proportion of glycine and
proline, which makes it unique compared to other
proteins. These two amino acids might be responsible
for some of the spectral characteristics between 1200
and 1400 cm1. Signals in the spectral region of 1200
1400 cm1 absorption are generally attributed to the
amide III, arising due to the CN stretching and NH in
plane bending from amide linkages. The absorption seen
at 1238 cm1 is attributed to amide III vibration. In
addition, signicant absorption due to CH2 wagging
vibrations from the glycine backbone and proline side
chains are also seen in this region. The absorption seen
at 1340 cm1 is attributed to CH2 wagging vibration of
the proline side chain. The CN stretching vibration of
the cyclic proline may also contribute absorption
between 1200 and 1350 cm1.
The vibrations due to amide I, II and III correspond
to peaks at 1654, 1550, 1238 cm1, respectively. All the
three characteristic peaks are seen in the case of all other
spectrums (Fig. 2bd), where the collagen lm has been
subjected to treatment. The characteristic acrylic,
carbonyl stretching vibration at 1708 cm1 is also seen
in the case of the collagen lm treated with acrylic
polymer (Fig. 2b), but the peak is not sharp since it is

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B. Madhan et al. / Biomaterials 23 (2002) 28412847

coupled with the peak of amide I vibration, whereas this

characteristic acrylic peak is missing in the case of the
collagen spectrum treated with acrylic and vegetable
tannins (Fig. 2d). This is an interesting observation,
which shows a possibility of the carboxyl group of the
acrylic polymer being changed by the inuence of
vegetable tannins. The peaks in the region of 1000
1350 cm1, which are seen in the case of the collagen lm
treated with vegetable tannins (Fig. 2c and d), are due to
the bending of aromatic groups of the vegetable tannins.
In the untreated collagen lm, stretching of the NH of
the amino acids is observed at 3324 cm1, whereas in the
case of the collagen lm treated with vegetable tannins,
the peak is shifted towards the higher frequency range
around 3350 cm1 and broadened. This stretching
cannot be due to free hydroxyl stretching, because the
hydroxyl stretching vibrations appear only above
3550 cm1. Therefore, this characteristic peak should
be possibly because of the hydrogen bonding of the

vegetable tannins with the collagen. In the case of

collagen lm treated with a combination of acrylic and
vegetable tannins, the broadening is higher than that in
the earlier case.
Transmission electron photomicrographs of RTT
collagen treated with vegetable tannins, acrylic polymer
and acrylic followed by vegetable tannins are presented
in Fig. 3. The photomicrograph of RTT treated with
vegetable tannins (Fig. 3a) does not show a clear
banding pattern. This could probably be due to coating
of the bres with tannins. In the case of the photomicrograph of RTT treated with acrylic polymer alone
(Fig. 3b) and vegetable tannins in the presence of acrylic
polymer (Fig. 3c), clear banding patterns are observed.
In the photomicrograph of RTT treated with acrylic
polymer, a higher order of bre splitting is observed as
reported earlier [30]. Addition of vegetable tannins to
the acrylic-polymer-treated RTT also brings about a
certain degree of orderliness in the bre packing. This is
supported by a higher shrinkage temperature observed
in the case of RTT treated with acrylic polymer followed
by vegetable tannins, where the shrinkage temperature is
found to increase from 681C (RTT treated with acrylic
polymer) to 861C. The observed increase in shrinkage
temperature can be explained through long-range
ordering and cross-linking of bres [31] treated with
acrylic polymer and vegetable tannins.
The CD spectra of native and treated collagen
solutions are shown in Fig. 4. CD spectrum of native
collagen is dominated by the p2p amide transition at
196.5 nm and a positive n2p transition peak at 220 nm.
The CD spectrum of the collagen treated with acrylic
shows an increase in molar ellipticity at 196.5 nm, which
indicates that there is a moderate unfolding of collagen
on the introduction of acrylic polymer. When vegetable
tannins are added to the collagen solution after treating
with acrylic polymer, a change in amplitude of the
spectrum, which orients towards the spectrum of native
collagen, is observed.

4. Discussions

Fig. 2. FT-IR spectrum of the collagen lm: (a) Native collagen lm;
(b) collagen lm treated with acrylic polymer; (c) collagen lm treated
with vegetable tannins; and (d) collagen lm treated with acrylic
polymer followed by vegetable tannins.

Vegetable tannins, as a result of their general chemical

characteristics, impart hydrothermal stability to collagen brils predominantly by interaction with basic
groups like lysine and argentine [32], which are located
at the band region in the collagen structure [33]. The
band structure of collagen contains sterically bulky
acidic and basic amino acid side chains, which are open
and accessible for interactions with vegetable tannins.
Thus, the reaction of vegetable tannins with the collagen
brings about orderliness, which necessitates higher heat
energy for the denaturation to take place.
Polymers may increase the shrinkage temperature of
tendon by increasing the volume fraction of collagen

B. Madhan et al. / Biomaterials 23 (2002) 28412847

2845

Fig. 4. CD spectrum of collagen solutions: (FF) native collagen

solution (0.3 mg/ml); (F F) collagen solution treated with acrylic
polymer; (- - - - - ) collagen solution treated with vegetable tannins; and
( - ) collagen solution treated with acrylic polymer followed by
vegetable tannins.

Fig. 3. Transmission electron photomicrographs of RTT collagen

bres: (a) collagen bre treated with vegetable tannins; (b) collagen
bre treated with acrylic polymer; and (c) collagen bre treated with
acrylic polymer followed by vegetable tannins.

within the brils [34,35]. It therefore appears that

vegetable tannins in the presence of acrylic polymer
increase the shrinkage temperature by shifting the
equilibrium between native and denatured forms of
collagen towards a more compact native form by steric
exclusions. A similar proposal has been made to explain
the increased melting temperature of DNA in the
presence of polymer [36]. The acrylic polymer used for
the treatment of collagen bre was predominantly
polymethacrylicacid, which is likely to open up the
collagen molecule through electrostatic forces [37], as
they contain active sites for charge interactions. Such an
opening up is likely to facilitate vegetable tannin
molecules to diffuse into the collagen matrix and bring
about enhanced ordering, which is responsible for
improved hydrothermal stability of collagen, as seen in
the case of RTT collagen bre treated with acrylic
followed by the treatment with vegetable tannins. A
schematic representation of the interaction of collagen
with acrylic followed by vegetable tannins is mentioned
in Fig. 5. The reaction scheme mentioned in the gure is
a representation of the interactions taking place at the
inter-brillar level in the collagen tendons. The acrylics
behave as negatively charged molecules under the
applied conditions and hence can interact with positively
charged regions of the collagen. The collagen matrix
opens up because of such interaction. On the addition of
vegetable tannins, compactness of the collagen matrix
increases due to the interaction of vegetable tannins with
collagen. Vegetable tannins used in the study contain
polyphenolics such as catechin, gallic acid, etc. as main
constituents. The hydroxyls are the main functional
groups of interaction for these tannins. These hydroxyl
groups can form a hydrogen bond with the side chain

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B. Madhan et al. / Biomaterials 23 (2002) 28412847

carrying the presented work. Thanks are also due to the

members of the Chemical Laboratory of CLRI for their
support.

References

Fig. 5. Schematic representation of the interaction of collagen with

acrylic and vegetable tannins: (a) polypeptide chain of collagen
especially representing the basic amino acid residues; (b) side chains
of basic amino residues charged positively at weakly acidic conditions;
(c) acrylic polymerFnegatively charged because of the presence of
carboxylic groups; (d) complex of acrylic polymer and polypeptide
chain of collagen, exhibiting charged interactions; (e) representative
plant polyphenolic molecule; and (f) complex of collagenacrylic
polyphenolic indicating the multipoint electrostatic interactions
between hydroxyl group of the polyphenolics and the polar amino
acid residue between neighbouring polypeptide chains of collagen at
the inter-brillar level; a similar dipoledipole interaction is exhibited
between polyphenolics of vegetable tannins and acrylic polymer.

groups of polar amino acids like lysine, arginine,

aspartic acid and glutamic acid. The other amino acids
like serine and threonine can also involve in the
hydrogen bond formation with the polyphenolic molecules [38]. All these amino acid residues can act as
hydrogen bond donors and acceptors. Since these
polyphenolics have several hydroxyl and carboxyl
groups, they can form hydrogen bonds at multiple
points, imparting additional stability to the bre matrix.

Acknowledgements
The authors wish to thank Dr. T. Ramasami,
Director, CLRI for his interest and encouragement.
The authors also thank Dr. T. Narasimhaswamy and
Dr. A. Rajaram, scientists, CLRI for their help in

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