Synthesis, characterization, and antimicrobial activity of kojic acid grafted chitosan oligosaccharide

Article abstract of DOI:10.1021/jf404026f

A novel water-soluble chitosan oligosaccharide (COS) derivative, chitosan oligosaccharide/kojic acid grafts assigned as COS/KA, was prepared by using the selective partial alkylation of N-benzylidene COS and chlorokojic acid in the presence of dimethyl sulfoxide (DMSO) and pyridine (Py). The derivative was characterized by UV-vis spectroscopy, FTIR, ¹H NMR, TGA, SEM, and XRD techniques, which showed that the alkylation reaction took place at the C-6 and C-3 positions of COS. The results showed that the degree of substitution (DS) for COS/KA was from 0.38 to 1.21, and the product exhibited an excellent solubility in organic solvents and distilled water. The antibacterial results indicated that the antibacterial activity of COS/KA was strengthened relative to COS with the increase of DS for Staphylococcus aureus, Escherichia coli, Aspergillus niger and Saccharomyces cerevisiae. These findings provide important supports for developing new antibacterial agents and expand the scope of application of COS in the food industry.

Full text of DOI:10.1021/jf404026f

Article
pubs.acs.org/JAFC
Synthesis, Characterization, and Antimicrobial Activity of Kojic Acid
Grafted Chitosan Oligosaccharide
Xiaoli Liu, Wenshui Xia,* Qixing Jiang, Yanshun Xu, and Peipei Yu
State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Lihu Road
1800, Wuxi, 214122 Jiangsu, People’s Republic of China
*
S Supporting Information
ABSTRACT: A novel water-soluble chitosan oligosaccharide (COS) derivative, chitosan oligosaccharide/kojic acid grafts
assigned as COS/KA, was prepared by using the selective partial alkylation of N-benzylidene COS and chlorokojic acid in the
1
presence of dimethyl sulfoxide (DMSO) and pyridine (Py). The derivative was characterized by UV−vis spectroscopy, FTIR, H
NMR, TGA, SEM, and XRD techniques, which showed that the alkylation reaction took place at the C-6 and C-3 positions of
COS. The results showed that the degree of substitution (DS) for COS/KA was from 0.38 to 1.21, and the product exhibited an
excellent solubility in organic solvents and distilled water. The antibacterial results indicated that the antibacterial activity of
COS/KA was strengthened relative to COS with the increase of DS for Staphylococcus aureus, Escherichia coli, Aspergillus niger and
Saccharomyces cerevisiae. These findings provide important supports for developing new antibacterial agents and expand the scope
of application of COS in the food industry.
KEYWORDS: chitosan oligosaccharide, kojic acid, antimicrobial activity
1
4
15
INTRODUCTION
antidiabetic, and skin-whitening activities. Recently, meth-
ods for the synthesis of various kojic acid derivatives, such as
kojic acid esters kojic acid laureate and kojic acid dipalmitate,
■
Many researchers have focused on chitosan as a source of
potential bioactive material during the past few decades.
However, it has several drawbacks for its utilization in biological
applications, including poor solubility and absorption under
1
6,17
have been reported in many studies.
Moreover, kojic acid
provides a promising skeleton for development of new more
potent derivatives such as chlorokojic acid (2-chloromethyl-5-
hydroxy-4H-pyran-4-one), which is a good ligand for the
nucleophilic and electrophilic substitution reaction depending
on the reagent type and can inhibit Aeromonas aerogenes,
Micrococcus pyogenes var. aureus, Salmonella typhosa, Penicillium
digitalum, Russula nigricans, and Saccharomyces cerevisiae
1
physiological conditions. Unlike chitosan, chitosan oligosac-
charide (COS), the hydrolyzed product of chitosan, is a mixture
of oligomers of β-1,4-linked D-glucosamine residues that have
better biocompatibility and solubility due to their shorter chain
2
lengths and free amino groups in D-glucosamine units. Many
of the biological activities reported for COS, such as
1
0
3
,4
5
6
growth.
Marwaha and co-workers reported that
antifungal, antibacterial, and antitumor, are dependent on
their physicochemical properties, which allow COS to be
considered as a potential novel functional food ingredient,
organomercury(II) complexes of kojic acid and maltol
demonstrated greater antibacterial activity than kojic acid.
Nevertheless, at present, there is little information available
regarding the synthesis of COS/KA graft complexes and their
antibacterial activity.
18
7
particularly in the preparation of low-calorie foods. Recently, it
has been shown that COS and its derivatives exert antimicrobial
effects against different groups of microorganisms such as
bacteria, fungi, and yeast. Therefore, it has also been used as an
7
Both COS and kojic acid are good natural food preservatives
for improving food quality and safety. Herein we report the
preparation of a derivative of COS, N-benzylidene COS, by
alkylation with chlorokojic acid. The chemical structure and
physical properties of products were characterized by UV−vis
antibacterial agent and additive to improve the shelf life of food
8
products. There are three types of reactive functional groups
present in COS (an amino group as well as both primary and
secondary alcoholic OH groups at the C-2, C-3, and C-6
positions, respectively), which can be readily subjected to
chemical derivatization, allowing its antibacterial activity to be
1
spectroscopy, FTIR, H NMR, TGA, SEM, and XRD
techniques. Besides, the solubility and the antibacterial activity
against Staphylococcus aureus, Escherichia coli, Aspergillus niger,
and Saccharomyces cerevisiae were also studied. These two
chemical groups are expected to supplement each other for
their antibacterial activity in the preparation of new effective
and environmentally friendly biocides.
9
increased. However, the antibacterial activity of COS is low,
and chemical modification may lead to enhancement of its
antibacterial activity.
Kojic acid (KA), 2-hydroxymethyl-5-hydroxy-4H-pyran-4-
one, which is an organic acid, is a metabolic compound
produced by several species of economically valuable fungi,
1
0
such as Aspergillus, Acetobacter, and Penicillium. At present,
kojic acid and its derivatives have drawn attention because they
have been shown to possess various bioactivities such as
13
Received: September 13, 2013
Revised: November 18, 2013
Accepted: December 12, 2013
Published: December 23, 2013
11
12
antimicrobial and antiviral, anti-inflammatory, antitumor,
©
2013 American Chemical Society
297
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Journal of Agricultural and Food Chemistry
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Figure 1. Reaction scheme for the synthesis of COS/KA.
FTIR Spectroscopy. Fourier transform infrared (FTIR) spectrum
was recorded on a Nicolet NexuS470 instrument (Nicolet Instrument,
Thermo Co., Madison, WI, USA). Samples were prepared as KBr
pellet and scanned against a blank KBr pellet background at
MATERIALS AND METHODS
■
Materials. Chitosan oligosaccharide (MW = 1 kDa), with a degree
of deacetylation of 90%, was made from crab shell and obtained from
Zhejiang Jinke Biochemical Co. Ltd. (Zhejiang, China). Kojic acid (2-
hydroxymethyl-5-hydroxy-4H-pyran-4-one) was purchased from
Wuhan Weisheng Biochemical Co. Ltd. (Hubei, China). Chlorokojic
acid (2-chloromethyl-5-hydroxy-4H-pyran-4-one) was prepared by the
reaction of kojic acid with thionyl chloride in chloroform according to
−1
−1
wavenumber range 4000−400 cm with resolution of 4.0 cm .
1
1
H NMR Spectroscopy. H NMR spectra were obtained on a
Bruker AV400 MHz (Bruker, Rneinstetten, Germany). Samples were
dissolved in D O with tetramethylsilane (TMS) as internal standard.
2
Thermogravimetric Analysis (TGA). The TGA for the samples
was performed on a Mettler Toledo TGA/SDTA851 thermogravim-
eter (Zurich, Switzerland) at the heating rate of 10 °C/min under N2
atmosphere in the temperature range of 25−500 °C. STARe software
1
9
the method of Uher and Hudecova.
́
Chlorination of the 2-
hydroxymethyl moiety of kojic acid using thionyl chloride at room
temperature produced chlorokojic acid, with the ring alcoholic OH
being unaffected. Dimethyl sulfoxide (DMSO), deuterium oxide
(version 9.01) was used to analyze the thermal stability of the samples.
(
D O), pyridine, and benzaldehyde were purchased from Sigma-
2
X-ray Diffraction (XRD). Crystallinity measurements were made
Aldrich (St. Louis, MO, USA). Staphylococcus aureus, Escherichia coli,
Aspergillus niger, and Saccharomyces cerevisiae used for the antibacterial
assay were provided by the School of Food Science and Technology of
Jiangnan University. All of the reagents used were of a highly pure
grade and were used without further purification, and deionized water
was used for all reagent solutions.
using a Bruker D8 Advance diffractometer with an area detector
operating at a voltage of 40 kV and a current of 50 mA at Cu Kα
radiation of k = 0.154 nm. The scanning rate was 2°/min, and the
scanning scope was set from 5° to 80° at room temperature.
Scanning Electron Microscopy (SEM). The morphological
characteristics of COS and KA1−3 were checked using SEM (Hitachi
S-4800, Hitachi Co., Japan). The samples were attached to SEM stubs
using two-sided adhesive tape and spray-coated with gold powder
Synthesis of Chitosan Oligosaccharides/Kojic Acid (COS/
KA). Three chitosan oligosaccharide/kojic acid grafts designated
COS/KA1−3 were prepared by the reaction of N-benzylidene COS
with chlorokojic acid in DMSO and pyridine (Py) (Figure 1). Reaction
conditions are summarized in Supplemental Table 1 of the Supporting
Information. COS (ca. 6 g) was dissolved in 70 mL of 1% acetic acid
and diluted with 120 mL of methanol. Then 6 mL of benzaldehyde
was slowly added into the COS solution over 30 min to obtain N-
benzylidene COS, and the reaction was conducted at 60 °C for 12 h to
protect amino groups of COS as Schiff base. After the reaction was
completed, the reaction mixture was filtered using a sintered glass
filter, and the residue was washed three times sequentially with ethanol
and water to remove unreacted benzaldehyde and finally freeze-dried
for 24 h. Chlorokojic acid (2.16−1.07 mol) was dissolved in DMSO.
By stirring, N-benzylidene COS (1 mol) was added to this solution,
and then Py was added dropwise at room temperature. Both N-
benzylidene COS and chlorokojic acid dissolved completely in DMSO,
so the reaction was homogeneous in this case. The reaction mixtures
were stirred at different temperatures for 6 h and cooled to room
temperature. Then an excess of acetone was poured into the DMSO
solution, and the product was precipitated. The solid was recovered by
filtration, washed repeatedly with acetone, Soxhlet-extracted with
ethanol for 24 h, and finally oven-dried at 40 °C for 16 h to obtain N-
benzylidene COS/KA. Then, the N-benzylidene COS/KA was
suspended in 1000 mL of 0.25 mol/L hydrochloric acid solution at
room temperature for 12 h, followed by filtration and three washings
with ethanol and water, respectively, and oven-dried at 60 °C for 12 h
to obtain the product.
(<10 nm) to improve the conductivity of the surface.
Solubility Test. The solutions of COS, KA, and COS/KA 1−3
were evaluated at a concentration of 5 mg/mL in different solvents
including distilled water, anhydrous ethanol, acetone, diethyl ether,
ethyl acetate, and glacial acetic acid at room temperature (25 °C).
Antimicrobial Activity. The antibacterial activities of the COS,
KA, and COS/KA1−3 were tested using a modified colony counting
method on bacteria selected for their resistance to common
antimicrobial agents. A representative microbe colony was picked off
with a wire loop, placed in nutrient broth, and then incubated at 37 °C
overnight. By dilution with sterile normal saline (0.9%) solution, the
7
cultures of Staphylococcus aureus and Escherichia coli containing 10
CFU/mL were prepared and used for the antibacterial test. The
bacterial suspension (0.1 mL) was inoculated under aseptic condition
into 10 mL of liquid peptone medium containing COS, KA, and COS/
KA1−3. Each sample contained test materials at a concentration of 1.0,
1.25, 1.5, 2.0, 2.5, 3.0, 4.0, 5.0, 6.0, or 8.0 mg/mL, whereas a blank
without test materials was prepared for comparison. All of the samples
were incubated at 37 °C under constant agitation. During incubation,
0.1 mL of each suspension was taken to determine the bacteria count
by serial dilution with triplicate plating on agar plates. Then the plates
were taken out of the incubator, and the inhibitory activity was
calculated.
The inhibitory activity (I) was calculated using eq 1 and expressed
as percentage:
UV−Vis Spectroscopy. UV−vis absorption spectra of 0.2% (w/v)
solutions were obtained using a UV1000 spectrophotometer
I (%) = N − N /N × 100
(1)
1
2
1
(
Techcomp Ltd., China) in the spectral region of 190−600 nm and
N is the initial cell number (CFU/mL), and N is the cell number
after treatment (CFU/mL).
1
2
with a beam width of 2 nm.
2
98
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Journal of Agricultural and Food Chemistry
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2923.83 cm− was attributed to the −CH− stretch of COS. The
1
In the determination of the inhibition rate of COS, KA, and COS/
KA1−3 on the growth of Aspergillus niger and Saccharomyces cerevisiae
by the same method, the differences were that all of the samples were
incubated at 28 °C under constant agitation. The cultures were
filtered, the pellet was washed with distilled water and dried at 80 °C
to constant weight, and then measured the dry cell weight and the
inhibitory activity were calculated.
characteristic peaks at 1628.76, 1519.37, and 1322.79 cm−
were assigned to the carbonyl stretching ν
bending δN−H (amide II), and amide three absorption band of
1
(amide I), amine
CO
−1
COS, respectively. The absorption band at 1155.97 cm was
the asymmetric stretching of the C−O−C bridge. In addition,
the adsorption band at 1071.52 and 1034.16 cm− corre-
sponded to the involved skeletal vibration of the C−O
stretching and the band at 890.44 cm− had been assigned to
the β (1→4) glycoside linkage. The FTIR spectra of the COS/
KA1−3 samples were quite similar to each other. The intense
band at 1216.72 cm and the shoulder at 911.09 cm were
broadened and shifted from the corresponding features of
chlorokojic acid that could be due to covalent bonding of the
1
The inhibitory activity (I) was calculated using eq 2 and expressed
as percentage:
1
I(%) = W − W /W × 100
(2)
1
2
1
W1 is the dry cell weight of Aspergillus niger and Saccharomyces
−1
−1
cerevisiae in the control culture medium, and W is the dry cell weight
2
of Aspergillus niger and Saccharomyces cerevisiae in the COS, KA, and
COS/KA1−3 culture medium, respectively.
18
The 50% inhibitory concentrations (IC ) against microorganisms
kojic acid residues onto the COS. Compared with the NH2
characteristic absorption band of COS and the absorption band
50
were determined for COS, KA, and COS/KA1−3. Fitting of the data
was performed by linear regression using the concentration versus
percent inhibition. The calculated IC50 was submitted to analysis of
variance, and the means were compared by the Tukey test (p ≤ 0.05)
using SPSS 20.0.
−1
of COS/KA1−3, the characteristic peak at 1519.37 cm for the
NH group is present in both COS and COS/KA1−3, but the
2
−1
absorption peaks at 1030 and 1074 cm , which were attributed
to νC−O of C6−OH and C3−OH of COS, respectively, were
substantially weakened. These results indicate that the
alkylation reaction mainly occurred not on the amine group
but on the alcoholic OH groups of COS.
HNMR Spectroscopy Analysis of COS/KA. The
HNMR spectra of COS and COS/KA1 in D O solvent are
shown in Figure 3. In Figure 3A, the peak at 1.98 ppm existed
because of the presence of −CH3 of the N-alkylated
glucosamine residue. A singlet at 2.98 ppm was assigned to
H of glucosamine and N-acetylated GlcN, and multiplets from
.24 to 3.94 ppm corresponded to H , H , H , and H of the
RESULTS AND DISCUSSION
■
UV−Vis Spectroscopy Analysis of COS/KA. In the UV−
vis spectra of COS, KA, and COS/KA1−3 (Supporting
Information, Supplemental Figure 1), broad absorption bands
between 200 and 300 nm were observed for all five samples. On
the basis of the data reported in the published literature, the
broad absorption band of COS might be ascribed to the CO
1
1
2
2
0
group. In the UV spectrum of KA, obvious absorption peaks
appeared at 219 and 273 nm, ,whereas the UV−vis absorption
spectra of COS/KA1−3 obtained at different reaction
conditions were quite similar to each other. Two absorption
bands of COS/KA1−3 at 207 and 271 nm indicated the
presence of a 5-hydroxypyranone group. These bands were
assigned to intraligand n→π* and π→π* transitions of the
2
3
3 4 5 6
methine protons of glucosamine and N-acetylated glucosamine.
1
8,21
chromophoric CO group,
indicating that KA had been
substituted onto the COS.
FTIR Spectroscopy Analysis of COS/KA. FTIR spectros-
copy of COS and COS/KA1−3 is shown in Figure2. From the1
FTIR spectrum of COS, a characteristic peak at 3422.36 cm−
appeared, and this can be attributed to the −NH and −OH
group stretching vibration, as well as inter- and extramolecular
hydrogen bonding of COS molecules. The weak band at
1
Figure 2. FTIR spectra of COS, KA, and COA/KA1−3.
Figure 3. H NMR spectrum of COS (A) and COS/KA1 (B).
2
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The peak at 4.5 ppm was attributed to H of glucosamine, N-
1
acetylated glucosamine. The solvent proton resonates at 4.7
2
2,23
1
ppm.
For the H NMR spectrum of COS/KA1, the signals
of COS protons at 1.98 and 2.98 and from 3.24 to 3.94 ppm
did not change significantly their chemical shifts. In Figure 3B,
signals of the newly formed three resonance signals appeared at
4
.19 (CH ), 6.59 (H-3′), and 8.07 (H-6′) ppm. These new
2
peaks were assigned to the protons of kojic acid residues bound
to the polysaccharide.
1
0,11
In free kojic acid, the signals due to
CH , H-3′, and H-6′ have been reported in 4.52, 6.45, and 7.92
2
18
ppm regions, respectively. Therefore, compared with kojic
acid the signals of aromatic proton H-3′ and H-6′ in COS/KA1
were upfield shifted by 0.14 and 0.15 ppm, respectively,
whereas the CH signal was downfield shifted by 0.33 ppm.
2
Hence, the observed shifts confirm that the kojic acid moiety is
bound to COS via the CH group.
2
COS contains two hydroxyl groups in every monosaccharide
residue and potentially is able to undergo many reactions of
alcohols. One of the most common reactions of alcohols is
etherification, which takes place between OH groups of
alcohols or between an OH group of an alcohol and a halogen
24
group of an alkyl halide. Aromatic protons of the 4-pyrone
ring are much less sensitive to the chlorine substitution than the
methylene protons. Hence, the KA moiety is covalently bound
to GlcN of COS via the CH group.
2
TGA of COS/KA. TGA and the first derivative of weight loss
DTG) have been widely used to evaluate the thermal
(
properties of materials and to show the mechanism by which
25
a material loses weight as a result of controlled heating. Figure
shows the TGA and DTG curves of COS and COS/KA1−3.
4
Despite some differences in terms of the presence of bound
water and residual mass, similar profiles in the curves of mass
loss were observed. Both COS and COS/KA1−3 involved two
steps of weight loss. In the case of COS, the first stage of weight
loss of about 10% between 30 and 100 °C was observed
corresponding to the water content. The weight loss of about
Figure 4. Thermogravimetric curves of COS (solid) and the modified
COS: COS/KA1 (short dot), COS/KA2 (short dash dot), COS/KA3
(short dash).
47% in the second step was attributed to a complex process
including degradation of saccharide rings and disintegration of
macromolecule chains in the sample. It had a rapid
decomposition between 150 and 320 °C, reaching its peak
temperature at 214 °C. As for COS/KA1−3, the first stage,
with weight loss of about 6% before 100 °C, was ascribed to the
volatile low molecular products and water. The second event,
approximately from 160 to 350 °C, refers to a complex process
including dehydration of the saccharide rings, depolymeriza-
tion, and decomposition of the alkylated units of the polymer
26
with weight loss of about 50%. COS/KA1, COS/KA2, and
COS/KA3 had similar curves, which showed a maximum
degradation temperature (Tmax) at about 198 °C. This indicates
that they have similar thermal stabilities. The Tmax values of
COS and COS/KA1−3 were about 214 and 198 °C,
respectively. It was suggested that the thermal stability of
COS/KA1−3 decreased compared with COS, which might be
attributed to the disruption of crystalline structure. For COS a
slight change in crystalline structure could cause significant
Figure 5. XRD patterns of COS and COS/KA1.
and weaker, indicating that COS/KA1 was considerably more
amorphous than COS.
27
alteration in thermal stability.
SEM Analysis of COS/KA. Surface morphological studies of
COS and COS/KA1 have been investigated using SEM
(Supporting Information, Supplemental Figure 2). Because
the SEM images of COS/KA1−3 were quite similar, only the
image of COS/KA1 is presented here for comparison with
COS. The SEM image of COS shows a smooth surface and a
more or less spherical shape, because there is stronger
XRD Analysis of COS/KA. The XRD patterns of COS and
COS/KA1 are presented in Figure 5. The COS exhibited two
peaks at around 2θ = 11° and 2θ = 21°, respectively,
characteristic of crystallinity of COS, as has been shown
2
7,28
previously.
In contrast, the peak at 2θ = 11° completely
disappeared in COS/KA1, and the peak at 2θ = 21° was wider
3
00
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Journal of Agricultural and Food Chemistry
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interaction between the COS molecules, whereas the particles
of COS/KA1 were observed to be in the form of irregular flakes
and the surfaces of COS/KA1 had many indentations. This
could be attributed to effects of drying rate. High drying rates,
associated with small particles, usually led to rapid wall
solidification, and thus dent smoothing cannot occur. This
indicated that the alkylate reaction changed the fiber’s
microstructure and surface morphology.
excellent antifungal activity and expanded the antimicrobial
spectrum.
It can be observed that the antimicrobial activities of COS/
KA1−3 were enhanced with increasing of DS. COS/KA1 (DS
= 1.21) displayed the highest antibacterial activity followed by
COS/KA2 (DS = 1.13) and COS/KA3 (DS = 0.38) against the
four microorganisms with IC50 values from 1.04 to 2.15 mg/
mL, from 1.15 to 2.37 mg/mL, and from 1.24 to 2.46 mg/mL,
respectively. However, COS and KA possessed a weaker
antimicrobial activity (IC50 from 6.30 to 7.58 mg/mL)
compared to COS/KA1−3. The IC50 values of COS/KA
against antimicrobial activities were 3−7 times lower than those
of KA and COS. Hence, the introduction of a 5-
hydroxypyranone group to the COS chain greatly enhanced
the antimicrobial activity of the COS/KA.
29
Solubility Test of COS/KA. The solubilities of COS, KA,
and COS/KA1−3 were investigated in various solvents
(Supporting Information, Supplemental Table 1). The results
showed that COS did not dissolve in the acetone and diethyl
ether, but did dissolve in the other four solvents (distilled
water, anhydrous ethanol, ethyl acetate, and glacial acetic acid).
KA has better solubility compared with COS. However, COS/
KA1−3 could be dissolved in all of the solvents except acetone.
In these solvents, COS/KA1−3 showed improved solubility as
compared with COS. This can be explained by the contribution
of decreased crystallinity confirmed by XRD and TGA.
There were also some differences in the inhibition activity
against Staphylococcus aureus and Escherichia coli for COS, KA,
and COS/KA1−3. Both COS and COS/KA1−3 showed higher
activity against Staphylococcus aureus (IC50 = 6.30, 1.04−1.24
mg/mL) than against Escherichia coli (IC50 = 7.39, 1.23−1.58
mg/mL). On the contrary, KA showed higher activity against
Escherichia coli (IC50 = 6.04 mg/mL) than against Staph-
ylococcus aureus (IC50 = 6.63 mg/mL). This result accorded
COS/KA1−3 with different DS values were synthesized by
adjusting the molar ratio of chlorokojic acid to COS and the
reaction temperature. The DS of derivatives increased when the
molar ratio of chlorokojic acid to COS was increased and
decreased when the reaction temperature was increased. This
might be due to the introduction of a 5-hydroxypyranone
group, which disturbed the formation of the ordered structure
of COS and hindered the formation of inter- and intra-
3
3,34
with the previous studies
and may be attributed to their
different cell walls. The cell wall of Gram-positive bacteria
(Staphylococcus aureus) is fully composed of peptide poly-
glycogen. The peptidoglycan layer is composed of networks
with plenty of pores, which allow COS and COS/KA molecules
to come into the cell without difficulty and allow more rapid
absorption of COS and COS/KA into the cell. However, the
cell wall of Gram-negative bacteria (Escherichia coli) is made up
of a thin membrane of peptide polyglycogen and an outer
membrane constituted of lipopolysaccharide, lipoprotein, and
phospholipids. Because of the complicated bilayer cell structure,
the outer membrane is a potential barrier against COS and
COS/KA molecules. Therefore, the samples have different
effects on the two kinds of bacteria.
As discussed above, the probable antimicrobial mechanisms
of COS/KA are suggested as being, on one hand, the positive
charge of the amino group at the C-2 in the glucosamine
monomer of COS/KA allows interactions with negatively
charged microbial cell membranes that lead to the leakage of
intracellular constituents. On the other hand, the 5-hydrox-
ypyranone group was introduced along the molecular chain,
which can form complexes with various metal ions. The
hydroxyl group at the carbon 5 position acting as a weak acid is
capable of forming salts with a few metals such as sodium, zinc,
copper, calcium, nickel, and cadmium, which is important in
cell membranes. The catechol groups readily interact with
phospholipids of the cell membrane subsequently disrupting
the microbial membrane and may also denature proteins.
Hence, we can deduce that COS/KA showed a synergistic
effect against four microorganisms. More work is needed to
confirm this hypothesis.
18
molecular hydrogen bonds. Hence, it was helpful to dissolve.
Antimicrobial Activity Assessment. The antimicrobial
effect of chitosans has been reported to be dependent on
2
,30
31
MW
and degree of deacetylation, which suggested that
chitosans and highly deacetylated COS were more effective at
inhibiting the growth of Staphylococcus aureus, Escherichia coli,
Aspergillus niger, and Saccharomyces cerevisiae than COS with
low deacetylation. The COS used in this study was highly
deacetylated (90%). The antibacterial activities of COS, KA,
and COS/KA1−3 against Staphylococcus aureus, Escherichia coli,
Saccharomyces cerevisiae, and Aspergillus niger are shown in
Table 1. At tested conditions, COS/KA1−3 could inhibit the
Table 1. Antimicrobial Activity of COS, KA, and COS/KA1-
3
against Staphylococcus aureus, Escherichia coli, Aspergillus
niger, and Saccharomyces cerevisiae
IC50 (mg/mL)
COS/KA1 COS/KA2 COS/KA3
COS
KA
Staphylococcus aureus 6.30
6.63
6.04
7.58
1.04
1.23
1.56
1.15
1.38
1.63
1.24
1.58
1.75
Escherichia coli
7.39
7.37
Saccharomyces
cerevisiae
Aspergillus niger
2.15
2.37
2.46
growth of all four tested microorganisms, whereas COS and KA
could not inhibit Aspergillus niger. The result indicated no
noticeable antimicrobial activity when COS and KA were tested
against Aspergillus niger at concentrations up to 8.0 mg/mL.
However, COS/KA1−3 markedly inhibited growth of
Aspergillus niger with IC values ranging from 2.15 and 2.46
In this study, the COS/KA derivatives with different DS
values from 0.38 to1.21 were successfully prepared by alkylation
reaction of chitosan oligosaccharide and chlorokojic acid in the
presence of DMSO and Py. The product was characterized by
1
UV, FTIR, and H NMR, and XRD confirmed that the amino
5
0
mg/mL. Aspergillus niger belongs to the fungus category,
whereas the fungal cell wall contains chitosan. Therefore,
Aspergillus niger has a certain resistance to the antifungal
groups of COS reacted with chlorokojic acid to form the
alkylate chitosan. TGA showed that the thermal stability of
COS/KA was lower than that of COS. COS/KA exhibited an
excellent solubility in organic solvents and distilled water. The
32
performance of chitosan. However, COS/KA1−3 showed an
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Journal of Agricultural and Food Chemistry
Article
antimicrobial activity of COS was significantly enhanced by the
kojic acid derivative of COS, and it increased with DS for
Staphylococcus aureus, Escherichia coli, Aspergillus niger, and
Saccharomyces cerevisiae. Thus, COS/KA may have potential as
an antimicrobial agent in food applications. However, more
biological studies are needed to understand their mechanisms
of action, to improve their activity by other modifications of
those molecules, and to determine their safety for food
applications.
(10) Aytemir, M. D.; Ozcelik, B. Synthesis and biological activities of
new Mannich bases of chlorokojic acid derivatives. Med. Chem. Res.
2
(
011, 20, 443−452.
11) Aytemir, M. D.; Ozcelik, B. A study of cytotoxicity of novel
chlorokojic acid derivatives with their antimicrobial and antiviral
activities. Eur. J. Med. Chem. 2010, 45, 4089−4095.
(12) Blumenthal, C. Z. Production of toxic metabolites in Aspergillus
niger, Aspergillus oryzae, and Trichoderma reesei: justification of
mycotoxin testing in food grade enzyme preparations derived from
the three fungi. Regul. Toxicol. Pharmacol. 2004, 39, 214−228.
(13) Higa, Y.; Kawabe, M.; Nabae, K.; Toda, Y.; Kitamoto, S.; Hara,
ASSOCIATED CONTENT
T.; Tanaka, N.; Kariya, K.; Takahashi, M. Kojic acid-absence of tumor-
initiating activity in rat liver, and of carcinogenic and photo-genotoxic
potential in mouse skin. J. Toxicol. Sci. 2007, 32, 143−159.
■
*
S
Supporting Information
UV−vis absorption spectra of COS, KA, and COS/KA1−3,
SEM image of COS and COS/KA1, and reaction conditions for
N-benzylidene COS with chlorokojic acid, DS, yield and
solubility for the derivatives. This material is available free of
charge via the Internet at http://pubs.acs.org.
(
14) Xiong, X.; Pirrung, M. C. Modular synthesis of candidate indole-
based insulin mimics by Claisen rearrangement. Org. Lett. 2008, 10,
1151−1154.
(15) Burdock, G. A.; Soni, M. G.; Carabin, I. G. Evaluation of health
aspects of kojic acid in food. Regul. Toxicol. Pharmacol. 2001, 33, 80−
1
01.
(
16) Khamaruddin, N. H.; Basri, M.; Lian, G.; Salleh, A. B.; Abdul-
AUTHOR INFORMATION
■
Rahman, R.; Ariff, A.; Mohamad, R.; Awang, R. Enzymatic synthesis
and characterization of palm-based kojic acid ester. J. Oil Palm Res.
2008, 20, 461−469.
Corresponding Author
(W.X.) Phone: +86 510 85919121. Fax: +86 510 85919121.
E-mail: xiaws@jiangnan.edu.cn.
*
(
17) Lee, Y. S.; Park, J. H.; Kim, M. H.; Seo, S. H.; Kim, H. J.
Synthesis of tyrosinase inhibitory kojic acid derivative. Arch. Pharm.
006, 339, 111−114.
18) Marwaha, S. S.; Kaur, J.; Sodhi, G. S. Organomercury(II)
Funding
2
(
This research was financially supported by the Twelfth Five
Year National Science and Technology Plan of rural field
research mission contract (2011BAD23B00), and the Fund
Project for Transformation of Scientific and Technological
Achievements of Jiangsu Province (BA2009082).
complexes of kojic acid and maltol: synthesis, characterization, and
biological studies. J. Inorg. Biochem. 1994, 54, 67−74.
́ ́ ̌
(19) Uher, M.; Hudecova, D.; Brtko, J.; Dobias, J.; Kovac, J.;
̌
SturdíkE. Antifungal preparation. CS Patent 259592, 1989.
20) Cai, Q.; Gu, Z.; Chen, Y.; Han, W.; Fu, T.; Song, H.; Li, F.
(
Notes
Degradation of chitosan by an electrochemical process. Carbohydr.
Polym. 2010, 79, 783−785.
The authors declare no competing financial interest.
(
21) Sima, J.; Chochulova,
M.; Bradiakova,
complexes with kojic acid derivatives. Pol. J. Chem. 1993, 67, 1369−
374.
22) Fernandez-Megia, E.; Novoa-Carballal, R.; Quin
́ ́ ́
B.; Veverka, M.; Makanova, J.; Hajselova,
́
A. Efficiency of the photoreduction of iron (III)
ACKNOWLEDGMENTS
■
We thank the staff at our laboratory for the assistance they have
provided in this study.
1
(
̃
́
oa, E.; Riguera,
R. Optimal routine conditions for the determination of the degree of
1
REFERENCES
acetylation of chitosan by H-NMR. Carbohydr. Polym. 2005, 61, 155−
■
1
61.
(
1) Rinaudo, M. Chitin and chitosan: properties and applications.
Prog. Polym. Sci. 2006, 31, 603−632.
2) Jeon, Y. J.; Kim, S. K. Production of chitooligosaccharides using
an ultrafiltration membrane reactor and their antibacterial activity.
Carbohydr. Polym. 2000, 41, 133−141.
3) Xia, W.; Liu, P.; Zhang, J.; Chen, J. Biological activities of
chitosan and chitooligosaccharides. Food Hydrocolloids 2011, 25, 170−
79.
4) Xu, J.; Zhao, X.; Han, X.; Du, Y. Antifungal activity of
(23) Lavertu, M.; Xia, Z.; Serreqi, A.; Berrada, M.; Rodrigues, A.;
1
Wang, D.; Buschmann, M.; Gupta, A. A validated H NMR method for
the determination of the degree of deacetylation of chitosan. J. Pharm.
Biomed. Anal. 2003, 32, 1149−1158.
(
(24) Natalija, G.; Ricardas, M. Synthesis and study of water-soluble
(
chitosan-O-poly(ethylene glycol) graft copolymers. Eur. Polym. J. 2004,
0, 685−691.
25) Hong, J. H.; Choi, Y. H. Physico-chemical properties of protein-
4
(
1
(
oligochitosan against Phytophthora capsici and other plant pathogenic
fungi in vitro. Pestic. Biochem. Physiol. 2007, 87, 220−228.
bound polysaccharide from Agaricus blazei Murill prepared by
ultrafiltration and spray drying process. Int. J. Food Sci. Technol.
2007, 42, 1−8.
(
5) Kendra, D. F.; Hadwiger, L. A. Characterization of the smallest
chitosan oligomer that is maximally antifungal to Fusarium solani and
́
(26) Peniche, C.; Zaldivar, D.; Bulay, A.; Roman, J. S. Study of the
elicits pisatin formation in Pisum sativum. Exp. Mycol. 1984, 8, 276−
thermal degradation of poly(furfuryl methacrylate) by thermogravim-
etry. Polym. Degrad. Stab. 1993, 40, 287−295.
(27) Qin, C.; Du, Y.; Xiao, L.; Li, Z.; Gao, X. Enzymic preparation of
water-soluble chitosan and their antitumor activity. Int. J. Biol.
Macromol. 2002, 31, 111−117.
(28) Rhim, J. W.; Hong, S. I.; Park, H. M.; Ng, P. K. Preparation and
characterization of chitosan-based nanocomposite films with anti-
microbial activity. J. Agric. Food Chem. 2006, 54, 5814−5822.
(29) Rosenberg, M.; Kopelman, I. J.; Talmon, Y. A scanning electron
microscopy study of microencapsulation. J. Food Sci. 1985, 50, 139−
144.
(30) No, H. K.; Young Park, N.; Ho Lee, S.; Meyers, S. P.
Antibacterial activity of chitosans and chitosan oligomers with different
molecular weights. Int. J. Food Microbiol. 2002, 74, 65−72.
281.
(
6) Qin, C.; Zhou, B.; Zeng, L.; Zhang, Z.; Liu, Y.; Du, Y.; Xiao, L.
The physicochemical properties and antitumor activity of cellulase-
treated chitosan. Food Chem. 2004, 84, 107−115.
(
7) Jeon, Y. J.; Kamil, J. Y.; Shahidi, F. Chitosan as an edible invisible
film for quality preservation of herring and Atlantic cod. J. Agric. Food
Chem. 2002, 50, 5167−5178.
(
8) Kim, S.-K.; Rajapakse, N. Enzymatic production and biological
activities of chitosan oligosaccharides (COS): a review. Carbohydr.
Polym. 2005, 62, 357−368.
(
9) Can, Z.; Ya, D.; Yu, L.; Qi, P. Polymeric micelle systems of
hydroxycamptothecin based on amphiphilic N-alkyl-N-trimethyl
chitosan derivatives. Colloids Surf. B: Biointerfaces 2007, 50, 192−199.
3
02
dx.doi.org/10.1021/jf404026f | J. Agric. Food Chem. 2014, 62, 297−303

Journal of Agricultural and Food Chemistry
Article
(
31) Chung, Y. C.; Su, Y. P.; Chen, C. C.; Jia, G. W.; Huey, W.
Relationship between antibacterial activity of chitosan and surface
characteristics of cell wall. Acta Pharmacol. Sinica 2004, 25, 932−936.
(
32) Wang, J.; Wang, H. Preparation of soluble p-aminobenzoyl
chitosan ester by Schiff’s base and antibacterial activity of the
derivatives. Int. J. Biol. Macromol. 2011, 48, 523−529.
(
33) Xing, K.; Chen, X. G.; Kong, M.; Liu, C. S.; Cha, D. S.; Park, H.
J. Effect of oleoyl-chitosan nanoparticles as a novel antibacterial
dispersion system on viability, membrane permeability and cell
morphology of Escherichia coli and Staphylococcus aureus. Carbohydr.
Polym. 2009, 76, 17−22.
̈
34) Mutlu, D. A.; Berrin, O. Original article, A study of cytotoxicity
(
of novel chlorokojic acid derivatives with their antimicrobial and
antiviral activities. Eur. J. Med. Chem. 2010, 45, 4089−4095.
3
03
dx.doi.org/10.1021/jf404026f | J. Agric. Food Chem. 2014, 62, 297−303