International Journal of Biological Macromolecules 101 (2017) 179–186

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International Journal of Biological Macromolecules

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

Exploiting oleuropein for inhibiting collagen fibril formation

H. Bharathy, N. Nishad Fathima ∗
Inorganic and Physical Chemistry Laboratory, CSIR-Central Leather Research Institute, Adyar, Chennai 600020, India

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

Article history: Collagen fibrils accumulate in excessive amounts and impair the normal functioning of the organ; there-
Received 26 September 2016 fore it stimulates the interest for identifying the compounds that could prevent the formation of fibrils.
Received in revised form 19 January 2017 Herein, inhibition of self-assembly of collagen using oleuropein has been studied. The changes in the
Accepted 10 March 2017
physico-chemical characteristics of collagen on interaction with increasing concentration of oleuropein
Available online 12 March 2017
has been studied using techniques like viscosity, UV–vis, CD and FT-IR. The inhibitory effect of oleuropein
on fibril formation of collagen was proved using SEM. Circular dichroism and FT-IR spectra elucidates the
Keywords:
alterations in the secondary structure of collagen suggesting non-covalent interactions between oleu-
Collagen fibril formation
Oleuropein
ropein and collagen. The decreased rate of collagen fibril formation also confirms the inhibition in the
Self-assembly self-assembly of collagen. Hence, our study suggests that inhibition of the self-assembly process using
FT-IR oleuropein may unfold new avenues to treat fibrotic diseases.
SEM © 2017 Elsevier B.V. All rights reserved.

1. Introduction Chung and coworkers employed antibody-based inhibitors to

Collagen is the most dominant protein in the extracellular
matrix characterized by the unique triple-helical structure [1].
Fibril-forming collagen plays a crucial role in maintaining the
integrity of tissues and organs by providing physical strength.
Triple-helical structure of the collagen has intrinsic propensity to
self-assemble, forming well-ordered fibrils [2]. Collagen fibril for-
mation is a spontaneous and thermally driven process, which is
favored by a large positive entropy contribution due to the dis-
placement of structured water around collagen molecules [3].
In native tissues, a precise balance between the process of
biosynthesis and degradation of collagen maintains the physiolog-
ical homeostasis. The excessive accumulation of collagen fibrils is
a hallmark of number of localized fibrotic diseases such as cardiac
fibrosis, keloids and systemic scleroderma. In cardiac fibrosis, the
accumulation of collagen fibrils on the blood vessels leads to thick-
ening of the arteries and alters the cardiac architecture and function
[4]. Various antifibrotic treatments have been suggested to target
proteins involved in the network of myofibroblast differentiation
but clear evidence on preventing or inhibiting the cardiac fibrosis
has not been briefed yet [5]. Therefore, inhibition of collagen fibrils
is recognized as a promising mechanism for the development of
antifibrotic agents.
effectively limit in vitro fibril formation in engineered tissue models
of localized fibrosis [6]. Cisplatin, an anticancer drug has been found
to inhibit the fibril formation in vitro without affecting the triple
helical structure of collagen [7]. Capsaicin has been also reported
as a potential candidate to suppress collagen fibril formation [8].
Hence, there is a need for identifying small molecules, which could
inhibit the collagen fibril formation.
Oleuropein, a phenolic compound found in olive has sev-
eral pharmacological properties viz., anti-oxidant [9], anti-
inflammatory [10], anti-atherogenic [11], anti-microbial [12] and
anti-tumor effects [13]. It has been employed as a cross linking
agent for fabrication of collagen scaffolds for bone tissues [14].
It shows cardioprotective action against diabetes induced car-
diomyopathy [15] and also against acute adriamycincardiotoxicity
by inhibiting the products of lipid peroxidation [16]. Oleuropein
undergoes deglucosidation upon enzyme activity to produce an
active form, oleuropeinaglyconeto crosslink collagen [17].
Oleuropein has been explored as a potential candidate for
inhibiting collagen fibril formation. In this study, a detailed investi-
gation has been carried out to find the inhibiting effect of oleuropein
on the collagen fibril formation. Further, physico-chemical interac-
tion studies have also been performed by various characterization
techniques viz., Fourier transform infrared spectroscopy (FT-IR),
circular dichroic studies (CD), viscosity analysis and UV–vis studies.

∗ Corresponding author.
E-mail addresses: nishad@clri.res.in, nishad.naveed@gmail.com (N.N. Fathima).

http://dx.doi.org/10.1016/j.ijbiomac.2017.03.050
0141-8130/© 2017 Elsevier B.V. All rights reserved.
180 H. Bharathy, N.N. Fathima / International Journal of Biological Macromolecules 101 (2017) 179–186
2. Experimental
2.1. Materials
Rat tail tendons was collected from animal house, CSIR-CLRI
and were used for extraction of type I collagen. Oleuropein was
procured from Sigma-Aldrich Ltd, India and used without further
purification. All experiments were done using millipore water.
2.2. Isolation of acid-soluble collagen
Rat tail tendons were teased from 6-month old male albino
rats at 4 ◦ C, washed with 0.9% saline and dissolved in 0.5 M acetic
acid followed by NaCl precipitation. Dialysis was carried out using
0.02 M phosphate buffer and 0.05 M acetic acid and purified colla-
gen was obtained [18]. The purity of collagen was resolved using 8%
sodium dodecyl sulfate- poly acrylamide gel electrophoresis (SDS-
PAGE) with collagen depicting bands of two ␣-chains at ∼130 KDa
and one ␤-chain at ∼250 KDa (Fig. S1). The concentration of the
collagen was estimated using Woessner method [19].
2.3. Preparation of collagen-oleuropein composite
The collagen solution and oleuropein were blended in acetate
buffer of pH 4 in different molar ratios of collagen-oleuropein viz.,
1:0(C), 1:1(C O1), 1:10 (C O2), 1:50 (C O3), 1:100 (C O4) under
continuous stirring at 4 ◦ C for three hours. The working concentra-
tion of collagen was taken as 0.8 ␮M. The solutions were incubated
at 4 ◦ C for 24 h and further characterization was performed. The
samples were also lyophilized under pressure of 6.4 Pa at −40 ◦ C
and used for FT-IR studies.
2.4. Physico-chemical characterization of the collagen-oleuropein
composite
2.4.1. UV–vis studies
Collagen-oleuropein composite was analyzed using UV-1800
Shimadzu (UV–vis spectrophotometer). A spectrum ranging from
200 nm to 400 nm was recorded using a quartz cuvette of 1.0 cm
path length, assuring the proper determination of the baseline.
2.4.2. Circular dichroic studies
Circular dichroic studies were carried out using Jasco 815 circu-
lar dichroism spectropolarimeter at 25 ◦ C with a quartz cell having
a path length of 1 mm. The CD spectra was recorded from 190 to
260 nm under nitrogen atmosphere and scanned at 0.2 nm inter-
vals. The data was obtained in milli degrees and was converted to
Molar Ellipticity (deg cm2 dmol−1 ). The results were plotted with
molar-residue-weight ellipticity (deg cm2 dmol−1 ) versus wave-
length in nanometers (nm).
2.4.3. Viscosity measurements
Viscosity measurements were performed using an Ostwald type
viscometer with a capacity of 5 ml at 20 ◦ C with the help of a ther-
mostat. The flow time of each sample was recorded three times and
the average value was taken. The viscosity () was calculated using
the relation,
rel = /0
where,  is the time flow of buffer and 0 is the time flow of
sample. The relative viscosity (/0 ) was plotted against the 1/R
(R = Concentration of collagen/Concentration of oleuropein).
2.4.4. Dynamic light scattering measurements
Dynamic light scattering (DLS) was performed on a high perfor-
mance Zetasizer Nano Series, Malvern at 25◦ C using a 4 mW He/Ne
laser with a scattering angle of 175◦ .
2.4.5. FT-IR studies
The wavelength dependent transmission intensity was char-
acterized to observe the interactions between collagen and
oleuropein using Jasco FT-IR-4200 (Fourier Transform Infrared
Spectrophotometer) in the range of 4000–400 cm−1 at 25 ◦ C with a
resolution of 4 cm−1 .
2.4.6. Turbidity assay
Turbidity assay was carried out using Cary series UV–vis-NIR
(UV–vis-Near Infrared) spectrophotometer. In vitro fibril formation
can be initiated by providing physiological conditions using the
50 mM phosphate buffered saline at pH 7.4 and 0.8N NaOH. The
collagen solution was pre-treated with oleuropein at various molar
ratios. After 24 h, the oleuropein treated collagen solution was
allowed to form fibrils under physiological conditions. The turbidity
was monitored at 313 nm as absorbance versus time graph portray-
ing crystal-growth kinetics with a lag phase or nucleation phase
followed by a rapid interface-controlled growth phase, where the
nuclei develops into fibrils and finally reaches plateau phase with
a span of 20 min. The rate of collagen fibril formation (1/t1/2 ) was
estimated by measuring the increase in opacity of the solution as
the collagen precipitates, using the Boltzmann-Sigmoidal fit.
2.4.7. SEM analysis
The collagen fibrils formed during the turbidity assay were
dried and coated onto the microscopic glass slides. The samples
were sputter coated with gold and viewed under scanning elec-
tron microscope TESCAN VEGA-3SBU with an accelerating voltage
of 15 kV.
3. Results and discussion
3.1. UV–vis spectroscopy accrediting the changes in absorption
spectra of protein
UV–vis studies were carried out to check the sensitivity of aro-
matic amino acids, thus exploiting the spectroscopic behavior of
protein and using it as a probe to study the conformations and
dynamics [20,21]. Fig. 1a shows the absorption spectra for native
collagen and collagen-oleuropein composite. The peptide absorp-
tion peak was evident at 226 nm in control and red shift in the
absorbance spectra was observed for C O3 and C O4, respectively.
The peaks for oleuropein (Fig. 1b) was observed at 287 nm, however
there were no prominent changes observed for collagen-oleuropein
composite (C O1–C O4).
The tyrosine and phenylalanine peaks were identified at 275 nm
and 258 nm, respectively (Fig. 1c). The lowest molar ratio (C O1)
showed higher absorption than control (C), which might be due
to the least interaction of collagen with oleuropein. There was a
decreasing trend in the absorbance intensity as the concentration of
oleuropein increased. C O4 with the highest concentration of oleu-
ropein showed a very low absorption peaks at 275 nm and 258 nm.
It might be due to the microenvironmental changes in the hydration
network of the protein, which may further lead to the movement
of aromatic residues to interior of collagen molecule thus leading
to aggregation of the protein. Similar effect of small molecules or
additives on collagen has been reported earlier [22,23].
 H. Bharathy, N.N. Fathima / International Journal of Biological Macromolecules 101 (2017) 179–186 181

Fig. 1. (a) UV–Vis absorption spectra of collagen and collagen-oleuropein composite (C Control, C O1 (1:1), C O2 (1:10), C O3 (1:50) and C O4 (1:100)). (b) UV–vis
absorption spectra of oleuropein. (c) Bcar chart of tyrosine (275 nm) and phenylalanine (258 nm) residues.

3.2. Circular dichroic studies accrediting the changes in
secondary structure of collagen
Circular dichroic spectra would depict the changes in secondary
structure of collagen if any, when treated with oleuropein at dif-
ferent molar ratios. Oleuropein alone shows CD signal at 194 nm
(Fig. 2b), albeit it doesn’t show signal at 222 nm, which is hallmark
for type II Polyproline structure of collagen and the triple helical
structure of collagen can be identified by its absorption maxima
at 222 nm and absorption minima at 197 nm in the far UV region
as shown by the control (C) in Fig. 2a. At 222 nm, the molar ellip-
ticity values (deg cm2 dmol−1 ) were plotted (Fig. 2a (inset)), which
serves as a characteristic helical content of type II Polyproline struc-
ture of collagen [24]. A decrease in the molar ellipticity values with
the increase in oleuropein concentration was observed when com-
pared to control. The decrease in the molar ellipticity might be due
to the aggregation of collagen caused by oleuropein.
The parameter Rpn, denotes the ratio of positive peak intensity
to negative peak intensity as an indicator for the conformational
changes in collagen [25]. The Rpn values were found to decrease
with the increasing concentration of the oleuropein (Fig. 2c). This
again indicates minor changes occurring in the secondary structure
of collagen on interaction with oleuropein. Collagen on denatu-
ration leads to red shift of the negative band and disappearance
of the positive band [26]. No such effect is observed with oleu-
ropein treated collagen thus confirming that the change in the CD
spectra of collagen in the presence of increasing concentration of
oleuropein was not due to the loss of triple helicity or denaturation.
3.3. Viscosity studies accrediting the changes in the flow rate of
collagen
In order to understand the influence of oleuropein on the rhe-
ological property of collagen viscosity measurements were carried
out. Relative viscosity was measured for collagen in the pres-
ence of varying concentration of oleuropein. The plot of relative
viscosity indicates the changes in the inter-molecular as well as
intra-molecular forces of collagen [27]. The plot (Fig. 3) shows that
the viscosity of collagen increases with the increasing concentra-
tion of oleuropein. Such behavior is indicative of aggregation of
protein [28]. Thus, it can be said that collagen aggregates with
increasing concentration of oleuropein thereby resisting the flow
and C O1 was found to have less aggregation, which also substan-
tiates with UV–vis results. The size of the aggregates was studied
using dynamic light scattering and the results are given in sup-
plementary section (Fig. S3). It was observed that with increasing
concentration of oleuropein, the size of the aggregates increased.
182 H. Bharathy, N.N. Fathima / International Journal of Biological Macromolecules 101 (2017) 179–186

Fig. 2. (a) CD spectra of collagen and collagen-oleuropein composite (C Control, C O1 (1:1), C O2 (1:10), C O3 (1:50) and C O4 (1:100)). (b) CD spectra of oleuropein.
(c) Rcpn values from the CD spectrum of collagen and collagen-oleuropein composite (C Control, C O1 (1:1), C O2 (1:10), C O3 (1:50) and C O4 (1:100)).

3.4. FT-IR studies accrediting the changes in the functional groups

of collagen

Infrared (IR) spectroscopy is a classical method for investigating

Fig. 3. A plot of relative viscosity (/o ) ( and o are the viscosity of collagen
in the presence and absence of oleuropein), Conditions: temperature = 25 ± 1 ◦ C,
(C Control, C O1 (1:1), C O2 (1:10), C O3 (1:50) and C O4 (1:100)).
the protein structure, molecular mechanisms of protein reactions,
protein folding, unfolding and misfolding [29,30]. Amide bands of
FT-IR spectra are sensitive to the secondary structure of collagen.
Collagen shows the characteristic peak at 3300 cm−1 for amide
A, 2948 cm−1 for amide B, 1640 cm−1 for amide I, 1560 cm−1 for
amide II and 1240 cm−1 for amide III. Oleuropein shows characteris-
tic bands at 3561 cm−1 and 3477 cm−1 corresponds to free hydroxyl
O–H stretching, 3280 cm−1 for phenolic O–H stretching, 3106 cm−1
accounts for aromatic C–H stretching, 1705 cm−1 reads for carbonyl
(C O) stretching and 1524 cm−1 for C C stretching.
The N–H stretching vibrations were identified for amide A and
amide B with a frequency of 3285 cm−1 and 3170 cm−1 , respec-
tively. These bands are mainly localized on NH groups and depend
mainly on the strength of hydrogen bonding. Fig. 4 discloses
the negligible shifts in peaks for amide A from 3285 cm−1 to
3280 cm−1 . The frequency of amide B decreased gradually from
C O1 (3175 cm−1 ) to C O4 (3170 cm−1 ), indicating a plausible
interaction of oleuropein with NH groups of the collagen with an
 H. Bharathy, N.N. Fathima / International Journal of Biological Macromolecules 101 (2017) 179–186
Fig. 4. FT-IR spectra of oleuropein, collagen and collagen-oleuropein(C Control, C O1 (1:1), C O2 (1:10), C O3 (1:50) and C O4 (1:100)).
effect on the strength of hydrogen bonding. The peptide carbonyl
group (C O) stretching vibrations are exhibited for amide I, which
depends on the backbone structure of collagen. It shows a band
ranging from 1640 to 1650 cm−1 , thus proving the shifts in bands
at 1635 cm−1 for all the samples when compared with the control
(1640 cm−1 ). Amide II band provides information for C N stretch-
ing vibration with smaller contributions from C O in plane bend,
C C and N C stretching vibrations. A frequency of 1561 cm−1 was
exhibited for native collagen; there was a gradual shift in bands
to 1554 cm−1 with the increasing order of molar ratios. An in-
phase combination of NH bending and CN stretching vibrations
with small contributions from C O plane bending and C C stretch-
ing vibrations, gives rise to Amide III band which was identified at
1240 cm−1 . There was shift in peaks for amide III from 1240 cm−1 to
1230 cm−1 . The peak at 1705 cm−1 indicates the presence of C O
(carbonyl) group for all increasing concentrations (C O1, C O2 and
C O3), which is a characteristic of oleuropein compound. For C O4,
peaks for oleuropein were observed at 3561 cm−1 and 3455 cm−1
indicating the presence of free hydroxyl O H groups. A shift in peak
from 3106 cm−1 to 3044 cm−1 was seen for C O4 recording the aro-
matic C H stretching and1705 cm−1 for carbonyl (C O) stretching
and 1520 cm−1 for C C stretching. Thus, it explicates the strong
interaction of collagen with the highest concentration of oleuropein
(C O4).
From the experimental observations, it was concluded that oleu-
ropein strongly interacts with collagen suggesting the changes in
the microenvironment, which lead to collagen aggregation as seen
from viscosity studies as well. These shifts in bands are indicative
of the non-covalent interactions viz., hydrogen bonding and van-
183
der Waal interactions, which are possible between collagen and
oleuropein.
3.5. Examining the rate of fibril formation using turbidity assay
It is well known that purified collagen molecules in solution in
vitro would spontaneously self-assemble at neutral pH and room
temperature to form fibrils, which appear to be same as that of in
vivo. In order to monitor the rate of fibril formation, turbidity assay
was performed, which was characterized with a lag, log, growth and
a plateau phase [31]. The interaction of oleuropein with collagen at
various molar ratios is shown in Fig. 5a, which demonstrates a short
lag phase at 37 ◦ C followed by a growth phase similar to that of the
control.
The efficiency of collagen fibril formation in the presence of
an inhibitor may be expressed in terms of the apparent rate of
fibril formation. The apparent rate of fibril formation (1/t1/2 ) was
obtained by taking the reciprocal value of time taken to reach the
middle point of final opacity [31]. The apparent rate of fibril forma-
tion gradually decreases with increasing oleuropein concentration
and it is shown in Fig. 5b. Control (C) showed highest rate of fibril
formation when compared to oleuropein treated collagen. It was
also found that for C O3, there was less formation of collagen
fibrils when compared to native collagen. The highest concentra-
tion (C O4) recorded negligible fibril formation. This might be due
to the presence of oleuropein, which strongly binds to the colla-
gen molecules thereby resisting their self-assembly process and
ultimately decreasing the rate of fibril formation. Further to com-
184 H. Bharathy, N.N. Fathima / International Journal of Biological Macromolecules 101 (2017) 179–186

Fig. 5. (a) Taurbidity measurements plots of collagen and collagen with increasing concentration of oleuropein (C Control, C O1 (1:1), C O2 (1:10), C O3 (1:50) and
C O4 (1:100)). (b) Rate of fibril formation for collagen and collagen with increasing concentration of oleuropein (C Control, C O1 (1:1), C O2 (1:10), C O3 (1:50) and
C O4 (1:100)).

plement turbidity assay, SEM analysis was carried out to visualize It was evident from turbidity measurements that there was a
the formation of collagen fibrils. decrease in the formation of fibrils with the increasing concentra-
tion of oleuropein. It might be due to the competitive hydrogen
bonding, where a decrease in the formation of fibrils was observed
(C O1–C O4).
3.6. SEM analysis

The effect of oleuropein on fibril formation of collagen was 4. Conclusion

also studied using SEM by coating the fibrils formed on a glass
plate. Type I collagen forms fibrils in vitro under the physiologi-
cal conditions [32] (Temperature and pH). No significant difference
was observed for lower concentration of oleuropein (C O1), when
compared to control, whereas at higher concentration a significant
decrease in the formation of fibrils (C O4) is observed as seen from
Fig. 6. Histograms of the fibril diameter are shown in Fig. 7. Thus,
it is concluded that oleuropein at higher concentrations has great
effect on collagen by decreasing the fibril formation, which can act
as potential inhibitor for cardio vascular diseases.
The current work investigates the inhibition of self-assembly
of collagen with the complementing physico-chemical interac-
tions for collagen-oleuropein composite. In this study, there was
an increasing aggregation of collagen molecules with the increas-
ing concentration of oleuropein. Collagen shows great influence of
oleuropein at higher levels with the increase in rheology, with the
decrease in the molar ellipticity values in circular dichroic studies
to the tenuous shifts in bands in FT-IR spectra. UV–vis studies also
suggest changes in the microenvironment of collagen. There was a
 H. Bharathy, N.N. Fathima / International Journal of Biological Macromolecules 101 (2017) 179–186 185

Fig. 6. SEM images for collagen and collagen oleuropein composite for native collagen (Control), C O1 (1:1), C O2 (1:10), C-03 (1:50) and C-04 (1:100).

Fig. 7. Histograms of diameter for collagen fibrils and collagen-oleuropein composite, Native collagen (Control), C O1 (1:1), C O2 (1:10), C-03 (1:50) and C-04 (1:100).
186 H. Bharathy, N.N. Fathima / International Journal of Biological Macromolecules 101 (2017) 179–186
drastic decrease in the formation of fibrils suggesting the effect of
oleuropein on collagen self-assembly. Thus, it clearly implies that
the collagen fibril formation is inhibited with the increasing con-
centration of oleuropein. This step may initiate new avenues in the
field of drug discovery targeting fibrotic diseases.
Acknowledgement
Indian National Science Academy (INSA) start-up research grant
for young scientists (SP/YSP/108/2015/1039) for funding this work
Appendix A. Supplementary data
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.ijbiomac.2017.
03.050.
References
[1] D. Whitford, Proteins: Sructure and Function, John Willet and Sons Ltd.,
England, 2005.
[2] J.P. Orgel, J.D. San Antonio, O. Antipova, Molecular and structural mapping of
collagen fibril interactions, Connect. Tissue Res. 52 (2011) 2–17.
[3] K. Kadler, Y. Hojima, D. Prockop, Assembly of collagen fibrils de novo by
cleavage of the type I pC-collagen with procollagen C-proteinase, J. Biol.
Chem. 260 (1987) 15696–16003.
[4] G. Krenning, E.M. Zeisberg, R. Kalluri, The origin of fibroblast and mechanism
of cardiac fibrosis, J. Cell Physiol. 225 (2010) 631–637.
[5] A. Leask, Potential therapeutic targets for cardiac fibrosis −Tgf␤, angiotensin,
endothelin, CCN2 and PDGF, partners in fibroblast activation, Circ. Res. 106
(2010) 1675–1680.
[6] H.J. Chung, A. Steplewski, K.Y. Chung, J. Uitto, A. Fertala, Collagen fibril
formation: a new target to limit cardiac fibrosis, J. Biol. Chem. 283 (2008)
25879–25886.
[7] M. Zenda, H. Yasui, S. Oishi, R. Masudal, N. Fujji, T. Koide, A cisplatin derivative
that inhibits collagen fibril formation In vitro, Chem. Biol. Drug Des. 85 (2015)
519–526.
[8] S. Perumal, K. Dubey, R. Badhwar, K.J. George, R.K. Sharma, G. Bagler, B.
Madhan, K. Kar, Capsaicin inhibits collagen fibril formation and increases the
stability of collagen fibers, Eur. Biophys. J. 44 (2014) 69–76.
[9] S. Bulotta, M. Celano, S.M. Lepore, T. Montalcini, A. Pujia, D. Russo, Beneficial
effects of the olive oil phenolic components oleuropein and hydroxytyrosol:
focus on protection against cardiovascular and metabolic diseases, J. Transl.
Med. 12 (2014) 2–9.
[10] F. Visioli, S. Bellosta, C. Galli, Oleuropein, the bitter principle of olives,
enhances nitric oxide production by mouse macrophages, Life Sci. 62 (1998)
541–546.
[11] M.A. Carluccio, L. Siculella, M.A. Ancora, M. Massaro, E. Scoditti, C. Storelli, F.
Visioli, A. Distante, R.D. Caterina, Olive oil and red wine antioxidant
polyphenols inhibit endothelial activation: anti-atherogenic properties of
mediterranean diet phytochemicals, Arterioscler. Thromb. Vasc. Biol. 23
(2003) 622–629.
[12] E. Tripoli, M. Giammanco, G. Tabacchi, D.D. Majo, S. Giammanco, M.L. Guardia,
The phenolic compounds of olive oil: structure, biological activity and
beneficial effects on human health, Nutr. Res. Rev. 18 (2005) 98–112.
[13] H.K. Hamdi, R. Castellon, A non-toxic olive iridoid, is an anti-tumor agent and
cytoskeleton disruptor, Biochem. Biophys. Res. Commun. 334 (2005) 769–778.
[14] H. Fan, J. Hui, Z. Duan, D. Fan, Y. Mi, J. Deng, H. Li, Novel scaffolds fabricated
using oleuropein for bone tissue engineering, BioMed. Res. Int. (2014) 1–11.
[15] A.A. Nekooeian, A. Khalili, M.B. Khosravi, Oleuropein offers cardioprotection
in rats with simultaneous type 2 diabetes and renal hypertension, Indian J.
Pharmacol. 46 (2014) 398–403.
[16] I. Andreadou, F. Sigala, E.K. Iliodromitis, M. Papaefthimiou, C. Sigalas, N.
Aligiannis, P. Savvari, V. Gorgoulis, E. Papalabros, D.T. Kremastinos, The olive
constituent oleuropein exhibits anti-ischemic, antioxidative and
hypolipidemic effects in anesthetized rabbits, J. Nutr. 136 (2006) 2213–2219.
[17] A.P.M. Antunes, G. Attenburrow, A.D. Covington, J. Ding, Utilisation of
oleuropein as a crosslinking agent in collagenic films, J. Leather Sci. 2 (2008)
17.
[18] G. Chandrakasan, G.A. Torchia, K.A. Piez, Preparation of inact monomeric
collagen from rat tail tendon and skin and the structure of nonhelical ends in
solution, J. Biol. Chem. 251 (1976) 6062–6067.
[19] J.F. Woessner, The determination of hydroxyproline in tissue and protein
samples containing small proportions of this imino acid, Arch. Biochem.
Biophys. 93 (1961) 440–447.
[20] F. Schmid, Encyclopedia of Life Sciences, Macmillan Publishers Ltd., 2001.
[21] N.O. Metrevel, K.K. Jariashvili, O. Namicheishvilli, D. Svintradze, E.N.
Chicvaidze, A. Sionkowsa, UV-Vis and FT-IR spectra of ultraviolet irradiated
collagen in the presence of antioxidant ascorbic acid, Ecotoxicol. Environ. Saf.
73 (2010) 448–455.
[22] A. Mehta, I. Kanungo, J.R. Rao, N.N. Fathima, Microenvironmental changes in
collagen/polyvinyl alcohol blends in the presence of ionic liquid: a
spectroscopic analysis, J. Bioact. Compat. Polym. (2015) 1–11.
[23] I. Kanungo, N.N. Fathima, J.R. Rao, B.U. Nair, Go natural and smarter fenugreek
as a hydration designer of collagen biomaterials, Phys. Chem. Chem. Phys. 17
(2015) 2778–2793.
[24] N.J. Greenfield, Methods to estimate the conformation of proteins and
polypeptides from circular dichroism data, Anal. Biochem. 235 (1996) 1–10.
[25] N.N. Fathima, B. Madhan, J.R. Rao, B.U. Nair, T. Ramasami, Interaction of
aldehydes with collagen: effect on thermal, enzymatic and conformational
stability, Int. J. Biol. Macromol. 34 (2004) 241–247.
[26] N.N. Fathima, B. Madhan, J.R. Rao, B.U. Nair, Effect of zirconium (IV)
complexes on the thermal and enzymatic stability of type I collagen, J. Inorg.
Biochem. 95 (2003) 47–54.
[27] H.Y. Srivastava, B.U. Nair, Structural modification and aggregation of mucin by
chromium (III) complexes, J. Biomol. Struct. Dyn. 20 (2003) 575–587.
[28] I. Kanungo, N.N. Fathima, J.R. Rao, B.U. Nair, Hydration dynamics of
Collagen/PVA composites: thermoporometric and impedance analysis, Mater.
Chem. Phys. 140 (2013) 357–364.
[29] A. Barth, Infrared spectroscopy of proteins, Biochim. Biophys. Acta 1767
(2007) 1073–1101.
[30] B.C. Vidal, M.L.S. Mello, Collagen type I amide I band and infrared
spectroscopy, Micron 42 (2011) 283–289.
[31] T. Hayashi, Y. Nagai, Factors affecting the interaction of collagen molecules as
observed by in vitro fibril formation: effects of small molecules, especially
saccharides, J. Biochem. 72 (1972) 749–758.
[32] A. Mehta, J.R. Rao, N.N. Fathima, Electrostatic forces mediated by choline
dihydrogen phosphate stabilize collagen, J. Phys. Chem. B 119 (2015)
12816–12827.