Mechanism and Kinetics of the Crosslinking Reaction

between Biopolymers Containing Primary Amine Groups

and Genipin

MICHAEL F. BUTLER, YIU-FAI NG, PAUL D. A. PUDNEY

Unilever R&D Colworth, Sharnbrook, Bedfordshire, MK44 1LQ, United Kingdom

Received 27 June 2003; accepted 14 August 2003

ABSTRACT: The reaction mechanism of chitosan, bovine serum albumin (BSA), and
gelatin with genipin (a natural crosslinking reagent) was examined with infrared,
ultraviolet–visible, and 13C NMR spectroscopies; protein-transfer reaction mass spec-
trometry; photon correlation spectroscopy; and dynamic oscillatory rheometry. Two
reactions that proceeded at different rates led to the formation of crosslinks between
primary amine groups. The fastest reaction to occur was a nucleophilic attack on
genipin by a primary amine group that led to the formation of a heterocyclic compound
of genipin linked to the glucosamine residue in chitosan and the basic residues in BSA
and gelatin. The second, slower, reaction was the nucleophilic substitution of the ester
group possessed by genipin to form a secondary amide link with chitosan, BSA, or
gelatin. A decreased crosslinking rate in the presence of deuterium oxide rather than
water suggested that acid catalysis was necessary for one or both of the reactions to
proceed. The behavior of the gel time with polymer concentration was consistent with
second-order gelation kinetics resulting from an irreversible crosslinking process, but
was complicated by the oxygen radical-induced polymerization of genipin that caused
the gels to assume a blue color in the presence of air. The lower elastic modulus attained
after a given time during crosslinking of the globular protein BSA as compared to the
coiled protein gelatin, despite possessing more crosslinkable basic residues, demon-
strated the importance of protein secondary and tertiary structures in determining the
availability of sites for crosslinking with genipin in protein systems. © 2003 Wiley
Periodicals, Inc. J Polym Sci Part A: Polym Chem 41: 3941–3953, 2003
Keywords: chitosan; bovine serum albumin; gelatin; genipin; covalent crosslinking;
hydrogels; biopolymers; crosslinking

INTRODUCTION low pH, with good biocompatibility and low cytotox-

Chitosan is the partially deacetylated from of
chitin [poly(N-acetyl-D-glucosamine)], which is a
structural polysaccharide found in insects, crus-
tacea, and some fungi.1 Its component residues, glu-
cosamine and acetyl-D-glucosamine, are shown in
Figure 1(a,b), respectively. As a commercially avail-
able natural cationic polyelectrolyte that swells at
Correspondence to: M. F. Butler (E-mail: michael.butler@
unilever.com)
Journal of Polymer Science: Part A: Polymer Chemistry, Vol. 41, 3941–3953 (2003)
© 2003 Wiley Periodicals, Inc.
icity, it has recently attracted much interest as a
stimulus-responsive, mucoadhesive material1–11 for
the controlled delivery of active molecules in phar-
maceutical applications. In the area of foods, chi-
tosan is considered to be a dietary fiber with several
beneficial health properties.12 These include anti-
cholesterolaemic, antiulcer, and antiuricemic prop-
erties13 stemming from its ability to bind fatty ac-
ids, bile acids, phospholipids, and uric acid.
Chitosan dissolves readily in acidic conditions
(below ca. pH 6.5) to form a viscous solution. In
the presence of phosphate ions, thermogels can be
formed.14 –16 Covalently crosslinked hydrogels
3941
3942 BUTLER, NG, AND PUDNEY
Figure 1. Chemical formulas for (a) glucosamine, (b) acetyl-glucosamine, (c) genipin,
and (d) geniposide, where glu represents glucose.
may also be formed after reaction with various
chemicals such as glutaraldehyde, diglycidyl ethers,
epoxides,3,4 and ␤-cyclodextrin.17 These reagents
are relatively cytotoxic. An alternative natural
crosslinking reagent does exist, however, called ge-
nipin. It has recently provoked interest18 –29 for its
ability to crosslink chitosan and certain proteins
containing residues with primary amine groups,
particularly, gelatin and soya protein isolates.
Genipin is obtained from its parent compound,
geniposide, via enzymatic hydrolysis with ␤-glu-
cosidase. The formulas for genipin and geniposide
are depicted in Figure 1(c,d), respectively. Geni-
poside is isolated from the fruits of Genipa ameri-
cana and Gardenia jasminoides Ellis and consti-
tutes about 4 – 6% of dried fruit. Genipa ameri-
cana is found in tropical America, from Mexico
and the Caribbean to Argentina, where it is
widely planted for its shade and fruit. The fruits,
with a taste similar to quinces, are eaten raw,
used to make a sour, refreshing drink, cooked
with sugar to flavor liquor, and used as a diuretic
and as a remedy for respiratory ailments. Garde-
nia jasminoides plants are grown in the Far East,
and their fruits have long been used in Chinese
medicine for their antiphlogistic, anti-inflamma-
tory, diuretic, choleretic, and haemostatic proper-
ties. Extracts from the fruits have also been used
to form brilliant blue pigments via the reaction of
genipin with primary amines in the presence of
oxygen,30 –35 which are used as a food dye com-
monly known as gardenia blue. These blue pig-
ments are of particular importance because they
are highly stable to heat, pH, and light.35
Initial observation of the formation of dimers of
genipin in the presence of glycine led to the sug-
gestion that genipin could be used to covalently
crosslink proteins containing residues with pri-
mary amine groups.18 Subsequently, it has been
used in studies of tissue fixation to crosslink col-
lagen and gelatin,21,25 in foodstuff to crosslink soy
protein isolates,28,29 and in studies of drug deliv-
ery, where it has been used to crosslink chi-
tosan.18 –20,22–24,26,27 In all of these applications,
genipin was chosen because of its markedly lower
cytotoxicity as compared with alternative
crosslinkers such as glutaraldehyde.20,21,24,27
Physiological studies have shown that geniposide,
which is highly abundant in certain edible fruits,
is converted into genipin in the gastrointestinal
tract, with no adverse effects.36,37
The aim of this work was to extend the under-
standing of the reaction mechanism between chi-
tosan, as an example of a biopolymer containing
primary amine groups, and genipin by combining
measurements of the gelation kinetics with those
of the reaction mechanism. Other biopolymers,
namely, bovine serum albumin (BSA) and gelatin,
that possess basic residues (e.g., lysine or argi-
nine) with primary amine groups were also exam-
ined to extend the relevance of the findings from
the study of chitosan and genipin.
EXPERIMENTAL
Materials and Sample Preparation
Chitosan was supplied by Primex and was 90%
deacetylated. Glucosamine and acetyl-glu-

cosamine were obtained from Sigma Chemical Co.
and were reagent grade. Genipin was supplied by
Challenge Bioproducts, Ltd. (Taiwan). Gelatin
(porcine, bloom 175) and BSA were obtained from
Sigma Chemical.
Two percent (weight/volume) stock solutions of
chitosan, glucosamine, and acetyl-glucosamine
were prepared by dissolving the powder in 1%
(v/v) acetic acid. Genipin solutions of the required
concentration were prepared by dissolving the ge-
nipin powder in 1% (v/v) acetic acid for reaction
with chitosan and in deionized, distilled water for
the BSA and gelatin solutions. To test for the
effect of isotopic subsitution, a genipin solution
was made in 1% (v/v) acetic acid with deuterium
oxide as the solvent rather than water. Twenty
percent (weight/volume) gelatin and BSA solu-
tions were prepared by dissolving the powder in
deionized, distilled water. The gelatin was dis-
solved at 60 °C, and the BSA was dissolved at
room temperature.
For all experiments, 15 mL of the chitosan,
glucosamine, acetyl-glucosamine, gelatin, or BSA
stock solution were pipetted into a glass vial and
mixed with 5 mL of the required genipin solution
to produce a sample with a final chitosan, glu-
cosamine, or acetyl-glucosamine concentration of
1.5% (w/v), a final gelatin or BSA concentration of
15% (w/v), and a genipin concentration of 1, 5, 10,
25, or 100 mM.
Experiments were also conducted by reacting
10 mM of genipin with amidated pectin, low-me-
thoxy pectin, skim milk powder, sodium casein-
ate, whey protein, soy isolate, maltodextrin, gum
arabic, and glutamine at 40 °C.
Ultraviolet–Visible (UV–Vis) Spectroscopy
UV–vis spectroscopy experiments were per-
formed with a PerkinElmer Lambda40 spectro-
photometer on the individual components and on
chitosan, glucosamine, and acetyl-glucosamine
samples containing an overall genipin concentra-
tion of 1 mM. The samples were placed in a quartz
cell with a path length of 5 mm. A layer of mineral
oil was placed on top of the sample, and the cell
was sealed with a plastic lid to limit solvent evap-
oration and oxygen ingress during the kinetic
runs. Experiments were performed at 20 °C.
Spectra were obtained every 20 min over a
period of 48 –96 h in the wavelength range from
900 to 220 nm, at 2 nm resolution.
Fourier Transform Infrared (FTIR) Spectroscopy
FTIR spectra were performed with a Bio-Rad
FTS6000 FTIR spectrometer equipped with a di-
CROSSLINKING REACTION 3943
amond attenuated total reflectance accessory
(Golden Gate, Specac, Ltd.). The solutions of chi-
tosan (1.5% w/v), genipin (10 mM), and chitosan
(1.5% w/v) containing an overall genipin concen-
tration of 10 mM were placed onto the diamond
surface and covered with a small Petri dish,
which was sealed with Vaseline petroleum jelly to
limit evaporation of water from the sample during
the experiment. Spectra were obtained every
minute over a period of 24 h.
13
C NMR Spectroscopy
13
C NMR spectra were acquired with a Bruker
AMX400 spectrometer at 100 MHz with 4260
scans and a relaxing delay of 1 s. The spectra
were thus acquired every 2 h and 31 min. The
sample was maintained at a temperature of about
2.5 °C during the course of the experiment. Line
broadening of 30 Hz was used to process the spec-
tra. ACD/CNMR software (version 3.5) was used
to generate a predicted spectrum for genipin to
assist with the interpretation of the experimental
results.
Protein-Transfer Reaction-Mass Spectrometry (PTR-
MS)
PTR-MS experiments were performed on chi-
tosan, glucosamine, and acetyl-glucosamine in
the presence of genipin (10 mM) at 20 °C. The
genipin was introduced after approximately 30
min to ascertain the response of the chitosan,
glucosamine, and acetyl-glucosamine solutions
without crosslinker. The experiments were per-
formed over a time period of about 12 h, and
volatile organic compounds with a molecular
weight range from 30 to 150 were detected.
Photon Correlation Spectroscopy (PCS)
PCS was used to measure the change in molecu-
lar size during the reaction of BSA and genipin.
Measurements were made on a mixture contain-
ing 15 wt % BSA and 25 mM of genipin with a
Malvern Instruments Autosizer 4700 equipped
with a Uniphase Cyonics argon ion laser operat-
ing at a wavelength of 488 nm and power output
of 20 mW. Measurements of the scattering inten-
sity fluctuations were made at a scattering angle
of 90°. Particle sizes were obtained from the mea-
sured field correlation function with the Contin
analysis method.
3944 BUTLER, NG, AND PUDNEY
Rheology
The development of the oscillatory rheological be-
havior of chitosan, gelatin, and BSA solutions
containing genipin was characterized with a Cou-
ette geometry in a dynamic stress rheometer sup-
plied by Rheometric. A sample volume of about 20
mL was used, and a layer of mineral oil was
placed on top of the sample to minimize solvent
evaporation and oxygen ingress during the course
of the experiments, which lasted between 48 and
96 h.
The autostress facility was used to ensure that
the correct stress was applied throughout the
crosslinking reaction to yield accurate and reli-
able values of the modulus, which changed by
several orders of magnitude as the sample
changed from a relatively nonviscous liquid to a
stiff gel. The rheological behavior of chitosan so-
lutions (1.5% w/v) and chitosan– genipin gels was
thoroughly characterized before the collection of
the kinetic data.
An initial stress of 0.025 Pa was applied to the
sample, rising to 2 Pa when a strong gel had
formed after several days. The storage and loss
moduli were measured at an applied frequency of
0.1 Hz, and data were collected every minute. To
investigate the effect of crosslinker concentration
on the chitosan– genipin crosslinking kinetics,
samples with genipin concentrations of 1, 5, 10,
25, and 100 mM were examined at a fixed tem-
perature (20 °C). To investigate the effect of tem-
perature, samples containing 10 mM of genipin
were analyzed at 20, 40, 60, and 80 °C. The rhe-
ology of gelatin and BSA samples with a protein
concentration of 15% (w/v) reacting with 10 mM of
genipin was measured at a temperature of 40 °C,
where both proteins were still in solution and not
aggregated.
RESULTS
Observations
Glucosamine, acetyl-glucosamine, and genipin so-
lutions were initially clear and colorless, and the
chitosan, BSA, and gelatin solutions were ini-
tially clear and slightly yellow. After a time pe-
riod that was dependent on the temperature and
amount of genipin, for concentrations exceeding
0.1 mM, the solutions containing glucosamine or
chitosan with genipin first became green, then
blue. At very low genipin concentrations (0.01
mM), glucosamine solutions became clear and
Figure 2. UV spectra of glucosamine, acetyl-glu-
cosamine, chitosan, and genipin.
slightly yellow, whereas the chitosan solutions
were not discernibly altered in appearance. The
solution containing acetyl-glucosamine and geni-
pin remained clear and colorless, even after sev-
eral weeks at room temperature or after heating
to 80 °C for several hours. No qualitative differ-
ences were observed between mixtures of chi-
tosan and genipin (at sufficient concentrations to
form a gel) that were left in the dark and those
that were left in daylight or artificial light. The
exposure of the mixture to air had a significant
influence on the development of the blue color,
however. In all of the chitosan gels, the blue col-
oration was deepest near the surface exposed to
air, and gradually moved further into the sample.
The BSA and gelatin gels became uniformly blue,
however. In the presence of deuterium oxide, the
crosslinking reaction between chitosan and geni-
pin was much slower than when the solvent was
water. The mixture of chitosan and genipin in
deuterium oxide did not form a gel or develop any
blue coloration until several days had elapsed, in
contrast to several hours in the presence of water.
BSA and gelatin went blue when mixed with
solutions containing all concentrations of genipin,
but took longer and required larger quantities of
polymer and genipin to form gels. BSA and gelatin
solutions with polymer concentrations in excess of
about 10 wt % and genipin concentrations in excess
of about 15 mM were required to form a gel.
UV–Vis Spectroscopy
The UV–vis spectra of glucosamine, acetyl-glu-
cosamine, chitosan, and genipin are depicted in
Figure 2. Glucosamine possessed major absorp-
 CROSSLINKING REACTION 3945

Figure 3. Evolution of the UV–vis spectrum of glu- Figure 4. Evolution of the UV–vis spectrum of acetyl-
cosamine after mixing with genipin, with selected re- glucosamine after mixing with genipin, with selected
gions of the spectrum expanded. regions of the spectrum expanded.

tion peaks at 240 and 280 nm, and acetyl-glu- mixture additionally displayed an increase in the
cosamine possessed a UV absorption peak at 275 intensity of the peaks at 240 and 280 nm from the
nm. The spectrum of chitosan was a superposition start of the reaction.
of the contributions from glucosamine and acetyl-
glucosamine, but was dominated by the spectrum
of glucosamine. Genipin possessed a major UV
absorption at 240 nm.
The development of the UV spectra of glu-
cosamine, acetyl-glucosamine, and chitosan mixed
with genipin is illustrated in Figures 3–5, re-
spectively. For the spectrum of the glucosamine–
genipin mixture, the intensity of the genipin peak
at 240 nm decreased slightly at the start of the
reaction before reaching a constant value. After a
certain time, a new peak appeared at 605 nm that
continued to increase in intensity throughout the
course of the reaction. For the acetyl-glucosamine/
genipin mixture, there was also an initial decrease
in the intensity of the genipin peak at 240 nm [Fig.
4(b)]. However, no new peak appeared around 605
nm. The results for chitosan mixed with genipin
were partially similar to those for glucosamine, in
that an initial decrease in the 240-nm genipin peak
was measured as well as the delayed appearance
and subsequent continual increase in intensity of Figure 5. Evolution of the UV–vis spectrum of chi-
the peak at 605 nm. However, the chitosan– genipin tosan after mixing with genipin.
3946 BUTLER, NG, AND PUDNEY

Figure 6. Evolution of the FTIR spectrum of the chi- Figure 7. Change in the ratio of FTIR absorbances
tosan– genipin mixture, with decreasing absorbance of 1092:1076 cm⫺1 after mixing chitosan and genipin.
the peaks at 1550 and 1414 cm⫺1 and increasing ab-
sorbance of the peak at 1092 cm⫺1 indicated by arrows.
8(a,b), respectively. There was agreement be-
FTIR Spectroscopy tween the positions and relative intensities of the
peaks in the experimental and predicted spectra.
IR spectra measured at different times during the
reaction between chitosan and genipin are dis-
played in Figure 6. Significant absorbance was
observed at 1710, 1550, 1414, 1280, 1155, 1092,
1076, and 1022 cm⫺1. The peaks at 1022 and 1155
cm⫺1 were assigned to vibrational modes associ-
ated with saccharide units in chitosan, and the
peak at 1280 cm⫺1 was due to the chitosan hy-
droxyl group.22,23 The peaks at 1076 and 1092
cm⫺1 resulted from C–O and/or C–N stretching.22
The band at 1414 cm⫺1 was assigned to a ring
stretching mode in the genipin molecule.22 The
peaks at 1550 and 1710 cm⫺1 were due to proton-
ated amine groups on the chitosan molecule22,23
and carboxylic acid groups in the acetic acid sol-
vent, respectively.
The absorbance peak at 1092 cm⫺1 gradually
increased in intensity from the onset of addition
of genipin to the chitosan solution, accompanied
by a slower increase in intensity of the peak at
1076 cm⫺1. Significant decreases in absorbance
were also measured for the peaks at 1550 and
1414 cm⫺1, and a new peak appeared at about
1630 cm⫺1 toward the later stages of the reaction
(apparent after 7920 s but not at 3960 s in Fig. 6).
No changes were measured in the peaks at 1700,
1300, 1150, and 1050 cm⫺1. Figure 7 shows that
the increase in intensity of the 1076-cm⫺1 peak
was directly proportional to that of the 1092-cm⫺1
peak.
Figure 8. (a) Predicted 13C NMR spectrum for geni-
13
C NMR Spectroscopy pin, showing the peak assignments for the carbon at-
oms marked in Figure 1. (c) and (b) experimentally
Experimentally measured and predicted 13C measured 13C NMR spectrum for genipin dissolved in
NMR spectra of genipin are portrayed in Figure 1% w/v acetic acid.

Figure 9. Experimentally measured 13C NMR spec-
tra for (a) chitosan and (b) chitosan– genipin mixture.
The peaks in the experimental genipin spectrum
in Figure 8, and therefore some of the peaks in the
chitosan– genipin mixture, were thereby assigned
to the carbon atoms in the genipin molecule, as
numbered in Figure 1. The spectra for chitosan
and a chitosan– genipin mixture are shown in Fig-
ure 9, with the assigned genipin peaks marked.
The spectrum of the chitosan– genipin mixture
was a combination of the spectra from the indi-
vidual components.
The change in intensity of each of these peaks
was measured, and plots of the peak intensity
versus time for selected peaks are shown in Fig-
ure 10. The intensity of the peak at 155 ppm, from
the C3 carbon atom on the unreacted genipin
molecule, decreased from the start of the reaction
between chitosan and genipin. No change in in-
tensity was measured for the peak at 172 ppm
from the C11 carbon atom on the unreacted geni-
pin molecule. Decreases in intensity were mea-
sured for the peaks at 112, 36, 130, and 62 ppm
from the genipin C4, C6, C7, and C10 carbon
atoms, respectively. For these four carbon atoms,
CROSSLINKING REACTION 3947
the rate of decrease of the intensity became
greater toward the later stages of the experiment.
PTR-MS
RH⫹ species with masses of 61 and 43, which
were identified as signatures from the acetic acid
present in the solvent,38 were detected as soon as
the glucosamine and acetyl-glucosamine solu-
tions were placed in the reaction vessel, and
reached a constant concentration that was main-
tained throughout the experiment. No immediate
changes were detected upon addition of the geni-
pin solution to either sample, but for the solution
containing glucosamine, shown in Figure 11(a),
an RH⫹ species with a mass of 33 was detected
after about 150 min. This species was interpreted
as methanol.38 The concentration of this species
then increased throughout the remainder of the
experiment. For the solution containing acetyl-
glucosamine, also shown in Figure 11(a), the RH⫹
species with a mass of 33 was not detected at any
stage of the experiment. Similarly, a delayed pro-
duction of the RH⫹ species with a mass of 33
(methanol) was detected in the chitosan sample,
shown in Figure 11(b), several minutes after the
addition of genipin. The time at which this oc-
curred coincided with the time at which a signif-
icant increase in elastic modulus was detected
[marked by the arrow in Fig. 11(b)] during rheo-
logical measurements on a separate chitosan– ge-
nipin sample reacted under the same conditions.
PCS
At the start of the experiment, PCS detected a
single particle diameter at about 7.5 nm for BSA.
During the course of the experiment, and shown
in Figure 12, the particle diameter continually
increased. After about 3 h, the signal was too
weak to obtain a reliable particle size because of
the significant amount of absorption that oc-
curred as a result of the change in color of the
sample. Measurements were not made on the chi-
tosan– genipin system because unambiguous in-
terpretation of the correlation function for the
chitosan solutions could not be made.
Rheology
The evolution of the elastic modulus (G⬘) with
time after mixing chitosan with genipin is dis-
played in Figure 13 for chitosan– genipin mix-
tures with a genipin concentration of 10 mM that
was reacted at different temperatures. An initial
3948 BUTLER, NG, AND PUDNEY

Figure 10. Change in peak height with time after mixing the 13C NMR peaks from
significant genipin carbon atoms in a chitosan– genipin mixture [see Fig. 1(c) for
assignment]: (a) C11, (b) C3, (c) C4, (d) C6, (e) C7, and (f) C10.

induction time, during which no increase in the
value of the G⬘ was observed, was followed by a
sharp increase in the G⬘. Although the G⬘ did not
reach a plateau within the timescale of the exper-
iment, the values of the G⬘ measured at the dif-
ferent temperatures appeared to be asymptoti-
cally heading toward similar values (of the order
of 1000 Pa). Increasing the reaction temperature
decreased the induction time before significant
increases in the gel strength were measured. A
logarithmic plot of the inverse of the gel time,
measured as the time at which the value of the G⬘
exceeded those of the loss modulus (G⬙) versus the
inverse of the absolute temperature is shown in
Figure 14. An apparent Arrhenius relationship
was observed.
The evolution of the G⬘ with time after mixing is
portrayed in Figure 15 for chitosan– genipin mix-
tures reacting at 40 °C and containing different
concentrations of genipin. With the exception of the
sample containing 1 mM of genipin, the G⬘ did not
reach a plateau. Increasing the genipin concentra-
tion decreased the induction time during which no
increase in the value of the G⬘ was measured and
enabled higher gel strengths to be reached in a
given time after mixing. The value of the G⬘ for the

Figure 11. PTR-MS measurements for (a) glu-
cosamine and acetyl-glucosamine mixed with genipin
and (b) chitosan mixed with genipin.
sample containing 100 mM of genipin had reached
10,000 Pa and was still increasing at the same
(power law) rate after 48 h, when measurements
Figure 12. PCS measurements of BSA molecular di-
ameter after mixing with genipin.
CROSSLINKING REACTION 3949
Figure 13. Evolution of the elastic modulus for for
chitosan (1.5% w/v)/genipin (10 mM) mixtures reacting
at different temperatures.
ceased. Likewise, the sample containing 25 mM of
genipin possessed an G⬘ in excess of 1000 Pa after
the same time that showed no indication of a de-
crease in its (power-law) rate of increase. The sam-
ples containing lower concentrations of genipin
showed signs of tending toward a plateau G⬘, how-
ever. Figure 16 is a double logarithmic plot of the
inverse of the gel time against genipin concentra-
tion. A straight line was obtained, indicating an
inverse power-law relationship.
Figure 17 shows the cure curves of BSA and
gelatin reacting with 10 mM of genipin at 40 °C,
which exhibited similar behavior to those of chi-
tosan. The increase in G⬘ was less rapid for the
protein samples, and the modulus at a given time
after mixing was lower than that achieved for
chitosan reacting with genipin under the same
conditions.
Figure 14. Arrhenius plot for the inverse of the gel
time for 10 mM of genipin reacting with chitosan at
different temperatures.
3950 BUTLER, NG, AND PUDNEY
Figure 15. Evolution of the elastic modulus for chi-
tosan (1.5% w/v)/genipin mixtures reacting at 40 °C for
different genipin concentrations.
DISCUSSION
Previous studies20,22–24,26 have demonstrated
that genipin reacts with materials containing pri-
mary amine groups, such as chitosan and some
peptides and polypeptides, to form covalently
crosslinked networks. It is believed that the
crosslinks are formed via two reactions involving
different sites on the genipin molecule. From pre-
vious FTIR and 13C NMR studies of chitosan–
genipin mixtures,22 the two reactions involving
genipin that are believed to result in crosslinking
of polymers containing primary amine groups are
shown in Figure 18.
Reaction scheme 1 in Figure 18 is an SN2 nu-
cleophilic substitution reaction that involves the
replacement of the ester group on the genipin
Figure 16. Variation of the inverse of the gel time
with genipin concentration at 40 °C for chitosan– geni-
pin mixtures.
Figure 17. Evolution of the elastic modulus for gela-
tin (15% w/v)/genipin (10 mM) and BSA (15% w/v)/
genipin (10 mM) mixtures.
molecule by a secondary amide linkage. In this
study, its occurrence was identified by the evolu-
tion of methanol that was detected in chitosan–
genipin and glucosamine– genipin mixtures with
PTR-MS. Because methanol was only evolved for
glucosamine and chitosan, but not for acetyl-glu-
cosamine, we can conclude that only the glu-
cosamine residues in chitosan undergo this reac-
tion. The nucleophile was, therefore, the primary
amine group. Additional evidence for the conver-
sion of primary amine groups on chitosan to sec-
ondary amide linkages between chitosan and ge-
nipin was provided by the decrease in absorbance
of the protonated amine IR peak at 1550 cm⫺1
combined with the appearance of the secondary
amide absorbtion at 1630 cm⫺1 at the later stages
of the reaction.23
Because the G⬘ began to increase at the same
time as the first traces of methanol were detected,
we can conclude that the nucleophilic subsitution
of the ester group on the genipin molecule was the
second reaction of the crosslinking process. The
constant intensity of the 13C NMR peak due to the
C11 carbon atom in the genipin molecule, which
is the carbon atom linked to the ester group, pro-
vided further evidence that nucleophilic subsitu-
tion of the ester group occurred during the later
stages of the crosslinking reaction. If it were the
first stage, then a change in intensity from the
onset of the reaction would have occurred.
The other half of the crosslink, which must
have already formed by the time that the ester
substitution occurred, is believed to form via re-
action scheme 2 in Figure 18. This reaction begins
with an initial nucleophilic attack of the genipin
C3 carbon atom from a primary amine group to

Figure 18. Crosslinking reactions involving genipin.
form an intermediate aldehyde group. Opening of
the dihydropyran ring is then followed by attack
on the resulting aldehyde group by the secondary
amine formed in the first step of the reaction. A
heterocyclic compound of genipin linked to the
glucosamine residue in chitosan and the residues
containing primary amine groups in BSA and gel-
atin, via the primary amine group, is thereby
formed. In this study, the immediate increase in
the intensity of an IR band at 1092 cm⫺1 at the
expense of the 1076-cm⫺1 band, combined with
the decrease in intensity of the protonated amine
band at 1550 cm⫺1 upon mixing chitosan and
genipin, is interpreted as the formation of C–N
bonds at the expense of C–O bonds during the
formation of the heterocyclic genipin– chitosan
compound. Because this occurred as soon as the
genipin and chitosan were mixed, this must have
been the first reaction to occur. Additional evi-
dence for the immediate occurrence of this reac-
tion was provided by the immediate decrease in
intensity of the 13C NMR peak that was attrib-
uted to the C3 carbon atom in the genipin mole-
cule and the immediate increase in intensity of
the UV–vis absorption at 280 nm that is believed
to be due to the presence of a heterocyclic geni-
pin– glucosamine/chitosan compound.22 The ne-
cessity for acid catalysis of at least the SN2 ester
substitution reaction and possibly the ring-open-
ing reaction39 explains the much slower rate of
CROSSLINKING REACTION 3951
crosslinking in the presence of deuterium oxide.
The constant absorbance of the IR peaks at 1710,
1280, 1155, and 1022 cm⫺1 justifies their assign-
ment to bonds present in the chitosan or acetic
acid solvent that did not participate in the
crosslinking reaction.
The formation of blue pigments suggests that,
in addition to the reactions involved in crosslink-
ing, other more complex reactions occurred. Pre-
vious studies of the blue pigments obtained in the
reaction of genipin with amino acids found that
they were formed from the oxygen radical-in-
duced polymerization of genipin and dehydroge-
nation of intermediate compounds, following the
ring-opening reaction because of attack of genipin
by a primary amine group.22,34,40,41 The change in
intensity of the 13C NMR peaks due to the genipin
C6, C8, and C10 carbon atoms that were mea-
sured during the reaction provided evidence for
the occurrence of reactions other than those in-
volving the C3 and C11 carbon atoms that were
directly involved in crosslinking. These results
support the suggestion that the polymerization
reactions are induced by the presence of oxygen
radicals because the blue coloration was initially
more pronounced at the interface of the gelled
samples and gradually moved down through the
sample with time. These results also suggest that
the polymerization reactions could only occur
once one of the crosslinking reactions had taken
3952 BUTLER, NG, AND PUDNEY
place. No blue pigments formed when genipin was
mixed with acetyl-glucosamine because it was un-
able to initiate either of the crosslinking reac-
tions.39 Moreover, the continual increase in BSA
particle size measured by PCS may be interpreted
as the polymerization of genipin attached to BSA
molecules. If the increase in particle size was due
to BSA molecule crosslinking, then it would be
expected to occur at a later time once the ester
substitution reaction occurred, and furthermore
to occur in units of the unreacted BSA molecule
size. From this interpretation, polymerization
was therefore induced by the formation of the
heterocyclic genipin compound rather than the
ester substitution reaction.
Crosslinking of chitosan molecules by genipin
polymers of different lengths can explain the sus-
tained increase in G⬘ and the high G⬘ that were
achieved for the higher genipin concentrations in
the chitosan– genipin system. If crosslinks were
formed from single genipin molecules, gelation
would eventually be slowed because of steric con-
straints on the number of genipin molecules that
could link separate network polymer molecules.
Crosslinks formed from several genipin molecules
will be more flexible, allowing network polymer
molecules to be linked that are further apart.
Analysis of rheologically obtained gel time may
be performed. In the simplest interpretation, the
reciprocal of the gel time, 1/tc, is predicted to be
related to the temperature, T, and the concentra-
tion of the components, C, by the following equa-
tion:42
1
tc
⬁C m⫺1 exp 冉 冊
Ea
RT
(1)
where m is the order of the crosslinking reaction,
and Ea is the activation energy of the rate-limit-
ing step in the crosslinking process. From these
results for chitosan, at fixed concentration an Ar-
rhenius relationship was observed. The value of
Ea that was measured from this plot was 39 kJ
mol⫺1. This value must relate to the rate-limiting
step in the formation of the gel, which is the
nucleophilic substitution of the ester group on the
genipin molecule by the secondary amide linkage
between chitosan and genipin. At fixed tempera-
ture, m ⫺ 1 was measured to be 0.78, which gave
a value of m of 1.78. This value was close to 2,
which is the value expected from a simple irre-
versible gelation process. The occurrence of the
various polymerization reactions that gave the
blue color to the gels may be the reason for the
deviation from second-order reaction kinetics.
The lower values of G⬘ that were attained at a
given time for BSA and gelatin as compared to
chitosan, along with the higher polypeptide con-
centrations required to form gels, was due to the
lower number of amine groups available for par-
ticipating in the crosslinking reaction in these
systems. For a globular protein such as BSA, the
secondary and tertiary structures are also impor-
tant because it may be assumed that the lysine or
arginine residues involved in the crosslinking re-
action must be near the outside surface of the
molecule for them to be effective. Gelatin pos-
sesses a coiled conformation at the temperature
at which it was crosslinked. The greater availabil-
ity of the crosslinkable residues in gelatin as com-
pared to those in BSA, even though BSA pos-
sesses a greater number of basic residues,43– 46
may therefore explain the higher modulus ob-
tained in the crosslinked gelatin sample as com-
pared with BSA.
CONCLUSIONS
The reaction mechanism between biopolymers
containing primary amine groups (chitosan, BSA,
and gelatin) and genipin was investigated with
IR, UV–vis, and 13C NMR spectroscopies; PTR-
MS, PCS, and dynamic oscillatory rheometry.
Two separate reactions led to the formation of
crosslinks between primary amine groups. The
fastest, and therefore first, reaction to occur was a
nucleophilic attack of the genipin C3 carbon atom
from a primary amine group that led to the for-
mation of a heterocyclic compound of genipin
linked to the glucosamine residue in chitosan and
the basic residues in BSA and gelatin. The slow-
est second reaction was the nucleophilic substitu-
tion of the ester group possessed by genipin to
release methanol and form a secondary amide
link with chitosan, BSA, or gelatin. A decreased
reaction rate in the presence of deuterium oxide
rather than water suggested that acid catalysis
was necessary for one or both of the reactions
to proceed. The behavior of the gel time with
polymer concentration was consistent with sec-
ond-order gelation kinetics resulting from an ir-
reversible crosslinking process. It was, however,
complicated by the oxygen radical-induced poly-
merization of genipin that occurred once the het-
erocyclic genipin compund linked to chitosan,
BSA, or gelatin had formed and caused the gels to
assume a blue color in the presence of air. Protein
secondary and tertiary structures were important
in determining the availability of sites for

crosslinking in protein systems because a lower
G⬘ was attained after a given time during
crosslinking of the globular protein BSA than the
coiled protein gelatin, despite possessing more
crosslinkable basic residues.
Alan Peilow, of Unilever R&D Colworth, is thanked for
his assistance with the PTR-MS measurements. Don
Farrer, retired, formerly of Unilever R&D Colworth, is
gratefully acknowledged for his many useful discus-
sions.
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