Biochem. J. (2008) 410, 147155 (Printed in Great Britain) doi:10.
1042/BJ20070891
Curcumin inhibits FtsZ assembly: an attractive mechanism for its
antibacterial activity
Dipti RAI*, Jay Kumar SINGH*, Nilanjan ROY and Dulal PANDA*1
*School of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Powai, Mumbai-400076, India, and Department of Biotechnology, National Institute of
Pharmaceutical Education and Research, Sector 67, S.A.S. Nagar, Punjab-160062, India
The assembly and stability of FtsZ protofilaments have been
shown to play critical roles in bacterial cytokinesis. Recent
evidence suggests that FtsZ may be considered as an important
antibacterial drug target. Curcumin, a dietary polyphenolic
compound, has been shown to have a potent antibacterial activity
against a number of pathogenic bacteria including Staphylococcus
aureus, Staphylococcus epidermidis and Enterococcus. We found
that curcumin induced filamentation in the Bacillus subtilis 168,
suggesting that it inhibits bacterial cytokinesis. Further, curcumin
strongly inhibited the formation of the cytokinetic Z-ring in
B. subtilis 168 without detectably affecting the segregation and
organization of the nucleoids. Since the assembly dynamics of
FtsZ protofilaments plays a major role in the formation and
functioning of the Z-ring, we analysed the effects of curcumin
INTRODUCTION
FtsZ, a prokaryotic homologue of eukaryotic cytoskeletal protein
tubulin, polymerizes to form a Z-ring at the mid cell that
orchestrates bacterial cell division [15]. FtsZ is shown to be
essential for bacterial cell division and viability (reviewed in
[5,6]). Previous studies have shown that the perturbation of FtsZ
functions by natural compounds and chemical agents leads to
inhibition of bacterial proliferation [712]. Most recently, totarol,
a naturally occurring diterpenoid phenol, has been found to inhibit
bacterial cytokinesis by perturbing Z-ring formation [13]. Totarol
has been shown to inhibit the assembly and GTPase activity of
Mycobacterium tuberculosis FtsZ in vitro. Several studies have
suggested that inhibitors of FtsZ assembly may be developed
into antibacterial drugs, including drugs against the multidrug-
resistant strains of pathogenic bacteria [8,10,13,14].
Curcumin, a naturally occurring polyphenolic compound, is
extracted from the rhizomes of Curcuma longa (Figure 1). It
has been used as an important dietary component for a long
time. It exhibits various biological activities including anti-
proliferative activity against various cancer cells, antioxidant
activity, wound healing ability and antimicrobial activity [15
19]. Recently, it has been found that curcumin binds to
tubulin and perturbs microtubule polymerization [19]. Curcumin
possesses antibacterial property against a number of Gram-
positive and Gram-negative bacteria [17]. It has been shown to kill
several pathogenic Gram-positive bacteria such as Staphylococcus
aureus, Staphylococcus epidermidis and Enterococcus that cause
infections such as skin diseases, pneumonia, meningitis and
urinary tract infections in human beings. In addition, curcumin in
Abbreviations used: Cy3, indocarbocyanine; DAPI, 4
,6-diamidino-2-phenylindole; DIC, differential interference contrast; IPTG, isopropyl -D-
thiogalactoside; LB, LuriaBertani; MIC, minimum inhibitory concentration.
1
To whom correspondence should be addressed (email panda@iitb.ac.in).
147
on the assembly of FtsZ protofilaments. Curcumin inhibited the
assembly of FtsZ protofilaments and also increased the GTPase
activity of FtsZ. Electron microscopic analysis showed that
curcumin reduced the bundling of FtsZ protofilaments in vitro.
Further, curcumin was found to bind to FtsZ in vitro with a
dissociation constant of 7.3 +
1.8 M and the agent also perturbed
the secondary structure of FtsZ. The results indicate that the
perturbation of the GTPase activity of FtsZ assembly is lethal to
bacteria and suggest that curcumin inhibits bacterial cell prolife-
ration by inhibiting the assembly dynamics of FtsZ in the Z-ring.
Key words: antibacterial activity, curcumin, filamentation, FtsZ
polymerization, GTPase activity, Z-ring.
combination with lactoferrin, N-acetylcysteine and pantoprazole
has recently been shown to significantly reduce the symptoms
caused by Helicobacter pylori infection in humans [20].
Further, curcumin is shown to have a potent phototoxic
effect against several bacteria such as Salmonella serotype
Typhimurium and Escherichia coli [21,22]. Curcumin also
displays antigenotoxic activity against DNA-damaging agents
such as mutagens, UV rays, -radiation etc. [2326]. Curcumin
inhibits SOS induction in Salmonella serotype Typhimurium and
E. coli [24]. The DMSO extracts of turmeric are shown to protect
DNA in E. coli cells against deleterious effects of X-rays [27].
Although curcumin is known for its antimicrobial and anti-
cancer activity for a long time, its therapeutic potential is severely
limited because of its poor bioavailability. Understanding the
mechanism of the antibacterial activity of curcumin will greatly
assist in designing potent analogues of curcumin, a natural remedy
for several bacterial diseases. In the present study, curcumin
has been found to induce filamentation in the B. subtilis 168,
suggesting that it inhibits bacterial cytokinesis. FtsZ plays an
important role in orchestrating bacterial cytokinesis. Curcumin
binds to tubulin and inhibits microtubule polymerization [19].
Since FtsZ is the prokaryotic homologue of tubulin, we sought to
determine the effects of curcumin on the assembly dynamics of
FtsZ. We found that curcumin increased the GTPase activity
of purified FtsZ and inhibited the assembly and bundling of FtsZ
protofilaments in vitro. To the best of our knowledge, curcumin
is the only inhibitor of FtsZ assembly found so far that increases
the GTPase activity of FtsZ. We provide evidence suggesting
that curcumin perturbs the Z-ring formation and inhibits bacterial
cytokinesis by inhibiting FtsZ assembly.

c The Authors Journal compilation 
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148 D. Rai and others
Figure 1 Structure of curcumin [1,7-bis-(4-hydroxy-3-methoxy-
phenyl)hepta-1,6-diene-3,5-dione]
MATERIALS AND METHODS
Materials
IPTG (isopropyl -D-thiogalactoside), Pipes, curcumin, glutam-
ate, GTP, ascorbic acid, DAPI (4
,6-diamidino-2-phenylindole)
and Cy3 (indocarbocyanine)-conjugated goat anti-rabbit second-
ary antibody were purchased from Sigma (St. Louis, MO, U.S.A.).
Primary polyclonal anti-FtsZ antibody developed in rabbit against
E. coli FtsZ was purchased from Bangalore Genei. All other
chemicals used were of analytical grade. B. subtilis 168 (trpC2),
E. coli K12 MG1655 and E. coli BL21 (DE3) are laboratory
collection strains.
Methods
Curcumin stocks were prepared in DMSO and then diluted in
aqueous buffer solution keeping the final concentration of DMSO
below 1 % (v/v) in all reaction milieu. The stability of curcumin
in aqueous buffers changes with various pH conditions. Curcumin
is highly unstable at neutralbasic pH conditions [28,29]. It has
been found that curcumin is quite stable at pH 6.5. Therefore all
in vitro experiments were performed at pH 6.5. It is also reported
that ascorbic acid helps to maintain curcumin stability [30];
therefore we incubated the bacterial culture with curcumin
supplemented with 1 mM ascorbic acid.
Determination of antibacterial activity of curcumin
The bacteria (B. subtilis 168, E. coli BL21 and E. coli K12
MG1655) were inoculated in LB (LuriaBertani) broth (10 g/l
casein enzymic hydrolysate, 5 g/l yeast extract and 5 g/l sodium
chloride) containing 1 mM ascorbic acid without or with different
concentrations of curcumin. The inhibition of bacterial growth
was determined by monitoring D600 , the attenuance at 600 nm, at
different time (0, 30, 60, 90, 120, 150, 180, 240 and 300 min)
intervals in the presence of different concentrations (20120 M)
of curcumin. The D600 of respective blanks having only curcumin
was subtracted to give the final D600 . The percentage inhibition of
bacterial growth was calculated by using the equation:
% Inhibition = [1 (X D600 /C D600 )] 100
where X D600 represents the final D600 value of X M curcumin-
treated culture at a particular time. C D600 represents the D600 of
the control culture at the same time. The percentage inhibition
of bacterial growth was determined 4 h after the addition of cur-
cumin to the bacterial culture. The IC50 of curcumin was deter-
mined by plotting the percentage inhibition of cell proliferation
against curcumin concentration. MIC (minimum inhibitory
concentration) of curcumin for B. subtilis 168 cells was deter-
mined by counting bacterial colonies as described earlier [13].
Briefly, B. subtilis 168 cells were grown for 4 h in the absence
and presence of different concentrations of curcumin. Appropriate

c The Authors Journal compilation 
c 2008 Biochemical Society
dilutions of bacterial culture were spread on to the LB agar plates
and incubated for an additional 12 h at 37 C. The number of
colony-forming units was calculated by counting the colonies on
each plate. The concentration of curcumin at which no colony was
detected in plates was considered as the MIC. The experiment was
performed three times.
Visualization of the morphology of bacteria
B. subtilis 168 was grown in the absence or presence of different
concentrations of curcumin for 4 h in LB broth supplemented with
1 mM ascorbic acid. The cells were fixed in 2.8 % formaldehyde
and 0.04 % glutaraldehyde at 25C. The bacterial cells were
finally collected, washed and resuspended in PBS (pH 7.4).
The cell morphology was examined under a DIC (differential
interference contrast) microscope (Nikon ECLIPSE TE2000-U)
at 40 magnification. The images were captured using a Cool
SNAP-Pro camera. The cell length was measured using Image-Pro
Plus software (Media Cybernetics, Silver Spring, MD, U.S.A.).
Immunofluorescence microscopy
The immunostaining of the Z-ring was performed using FtsZ
antibody as described earlier [11]. Briefly, the B. subtilis 168
cells were grown for 4 h in LB broth containing 1 mM ascorbic
acid without or with different concentrations of curcumin (25 and
50 M). The cells were fixed in 2.8 % formaldehyde and 0.04 %
glutaraldehyde for 45 min at 25 C. The cells were permeabilized
and then incubated with a polyclonal FtsZ antibody developed
in rabbit, followed by staining with a Cy3-conjugated goat anti-
rabbit secondary antibody. The nucleoids were stained with
1 g/ml DAPI. The cells were observed under a fluorescence
microscope (Nikon ECLIPSE TE2000-U) at 40 magnification.
The images were analysed using Image-Pro Plus software.
Purification of FtsZ
Recombinant E. coli FtsZ was overexpressed and purified from
E. coli BL21 strain as described previously [31]. The purity of
the protein was analysed by SDS/10 % PAGE and found to be
> 98 %. The concentration of purified FtsZ was determined by
the method of Bradford [32] using BSA as a standard. The protein
was aliquoted and stored at 80 C.
Light scattering assay
FtsZ (12.5 M) was incubated without or with different
concentrations (1040 M) of curcumin in 25 mM Pipes buffer
(pH 6.5) at 25 C for 10 min. Then, 50 mM KCl, 10 mM MgCl2
and 1 mM GTP were added to the reaction mixture. The kinetics
of the assembly of FtsZ was monitored by 90 light scattering
(550 nm) at 37 C using a JASCO 6500 spectrofluorimeter
equipped with a constant temperature water circulating bath. A
0.3 cm path length cuvette was used for all the measurements.
The appropriate blank was subtracted from all experimental data.
Electron microscopic analysis
FtsZ (12 M) was incubated in 25 mM Pipes buffer (pH 6.5)
without or with different concentrations (1050 M) of curcumin
at 25 C for 10 min. Then, 50 mM KCl, 5 mM MgCl2 and 1 mM
GTP were added to reaction mixtures and polymerization was
carried out at 37 C. After 15 min of polymerization, the protein
polymers were transferred to Formvar-carbon-coated copper
grids (300 meshes) for 45 s. The sample-containing grids were
stained with 1 % uranyl acetate for 35 s, air-dried and observed

under a transmission electron microscope (FEI TECNAI G2 12)
[31].
Measurement of GTPase activity of FtsZ
The amount of Pi released during the assembly of FtsZ was
measured using a standard Malachite Green/ammonium molyb-
date assay as described earlier [3335]. Briefly, FtsZ (12 M) was
incubated without or with different concentrations (5, 10, 20 and
30 M) of curcumin in 25 mM Pipes buffer (pH 6.5) containing
50 mM KCl and 5 mM MgCl2 on ice for 10 min. Then, 1 mM GTP
was added to the reaction mixtures and incubated at 37 C to start
the hydrolysis reaction. After 5 min of incubation, 40 l samples
were taken out from the reaction mixtures, and hydrolysis reaction
was quenched by addition of 10 % (v/v) 7 M HClO4 . The samples
were kept in ice for 2 min and then all the samples were incubated
at 25 C for 10 min. A 900 l portion of freshly prepared filtered
Malachite Green/ammonium molybdate solution was added to the
samples, and incubated at 25 C for 30 min in the dark. The Pi
release was determined by measuring the A at 650 nm. The
background A was subtracted from all the readings. A phosphate
standard curve was prepared using sodium phosphate. All the
solutions were prepared in deionized water.
Measurement of the dissociation constant of curcuminFtsZ
interaction
The increase in curcumin fluorescence upon binding to FtsZ was
used to determine the binding of curcumin to FtsZ [19]. FtsZ
(1 M) was incubated with different concentrations of curcumin
(1, 2, 3, 4, 5, 7 and 10 M) in 25 mM Pipes buffer (pH 6.5) at 25 C
for 30 min in the dark. The fluorescence spectra of the samples
were recorded using a ex of 425 nm. The fluorescence intensities
at 495 nm were used for determining the binding constant.
The dissociation constant (K d ) of curcuminFtsZ interaction
was calculated by plotting Lbound /Lfree against Lbound , where Lbound
represents the bound curcumin concentration and Lfree represents
the free curcumin concentration [36]. The Lbound was calculated
from the equation
L bound = F + C/Fmax
where F max is the fluorescence intensity of 1 M fully bound
curcumin. F max was calculated by plotting 1/fluorescence
intensity (495 nm) of curcumin against 1/[FtsZ]. For this, 1 M
curcumin was incubated without or with different concentrations
(1, 2, 4, 6, 8 and 12 M) of FtsZ in 25 mM Pipes buffer (pH 6.5)
at 25 C for 30 min in the dark. F was calculated by subtracting
F 0 from F, where F is the fluorescence intensity of the FtsZ
curcumin complex at a particular curcumin concentration and F 0
is the fluorescence intensity of only curcumin in the absence of
FtsZ.
The fluorescence intensities were corrected for the inner
filter effects using the following equation: F C =F
antilog[(A425 +A495 )/2]. F C represents the corrected fluorescence
intensity.
Analysis of secondary structural changes
FtsZ (4 M) was incubated without or with different concen-
trations of curcumin (20, 30 and 50 M) in 25 mM phosphate
buffer (pH 6.5) for 30 min at 25 C. The far-UV CD spectrum was
monitored over a wavelength range of 200250 nm using a JASCO
J-810 spectropolarimeter equipped with a Peltier temperature
controller using a 0.1 cm path length quartz cuvette. An average
Curcumin inhibits bacterial cytokinesis by inhibiting FtsZ assembly 149
of five scans was taken for each spectrum. Deconvolution and
statistical analysis of the CD spectra were performed using
CDNN, SSE software from Jasco and OriginPro 7.5 software
respectively.
RESULTS
Effect of curcumin on the proliferation of B. subtilis 168 and E. coli
K12 MG1655 cells
Curcumin inhibited the growth of B. subtilis 168 in a concen-
tration-dependent fashion (Figure 2A). The IC50 was found to
be 17 + 3 M for B. subtilis 168 (Figure 2B). The MIC of cur-
cumin for B. subtilis 168 cells was determined to be 100 M. Sim-
ilarly, curcumin also inhibited the growth of E. coli K12 MG1655
(see Supplementary Figure 1A at http://www.BiochemJ.org/
bj/410/bj4100147add.htm). The IC50 for E. coli K12 MG1655
was calculated to be 58 + 5 M, and 100 M curcumin inhibited
the growth of E. coli K12 MG1655 cells by 80 % (Supplementary
Figure 1B). The IC50 for E. coli BL21 was found to be 56 + 8 M
(results not shown).
Effect of curcumin on the morphology of B. subtilis 168
Curcumin induced filamentation in B. subtilis 168 cells (see
Supplementary Figure 2 at http://www.BiochemJ.org/bj/410/
bj4100147add.htm). The average length of B. subtilis 168 was
determined to be 3.3 + 1 m. The average cell length increased
by 2.5-fold from 3.3 + 1 to 8.6 + 5.3 m after 4 h of incubation
with 25 M curcumin. Also, the cell growth was inhibited by
81 +
11 % in the presence of 25 M curcumin (Figure 2B). In
the presence of 50 M curcumin, the average cell length of
B. subtilis 168 was increased by 6-fold from 3.3 + 1 to 20 +
16 m (Figure 2C) and the growth was inhibited by 90 + 9%
(Figure 2B).
Most of the curcumin-treated cells had strikingly larger cell
length than the control (vehicle-treated) cells (Figure 2C). In the
case of control, 33, 66 and 1 % of the cells had mean length in
the range of 13, 35 and 57 m respectively (Figure 2C).
In the presence of 50 M curcumin, 12, 4, 8 and 76 % of
the cells had mean length in the range of 35, 57, 79 and
> 9 m respectively. The results together indicate that curcumin
inhibits bacterial proliferation by inhibiting cytokinesis. Further,
curcumin did not perturb the membrane structure of B. subtilis
168.
Effect of curcumin on Z-ring formation and nucleoid segregation in
B. subtilis 168
B. subtilis 168 cells formed large filaments in the presence of
curcumin. Therefore we sought to find the effects of curcumin on
the nucleoids and as well as on the Z-ring in B. subtilis 168. In the
absence of curcumin, most of the B. subtilis 168 cells had a well-
defined Z-ring in between the two nucleoids (Figure 3). Curcumin
strongly inhibited the formation of Z-rings (Figures 3D, 3F, 3G
and 3I). For example, 63 % of the control cells had well-defined
Z-rings (Figures 3A and 3C), whereas only 20 and 7 % of the
curcumin-treated cells had Z-rings in the presence of 25 and
50 M curcumin respectively (Table 1). Further, the frequency
of occurrence of Z-rings/m of the bacterial cell length was
reduced strongly. For instance, the frequency of occurrence of the
Z-ring/m of the bacterial cell length was found to be 0.18 + 0.15,
0.03 +
0.07 and 0.01 +
0.03 in the absence and presence of 25 and
50 M curcumin respectively.

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150 D. Rai and others

Figure 2 Inhibition of growth of B. subtilis 168 cells by curcumin

B. subtilis 168 cells were grown in LB media supplemented with 1 mM ascorbic acid without or with different concentrations of curcumin. Growth curves for B. subtilis 168 cells in the absence ()
and presence of 20 M (), 30 M (), 60 M () and 100 M () curcumin are shown in (A). The results are the average of four independent experiments. (B) The Figure shows the inhibition
of growth of B. subtilis 168 in the presence of different concentrations of curcumin after 4 h of growth. The results are the means +
S.D. for eight experiments. The effects of 25 and 50 M curcumin
on the length of B. subtili s 168 cells are shown in (C). B. subtilis 168 cells were grown without or with different concentrations of curcumin supplemented with 1 mM ascorbic acid for 4 h, and the
cells were imaged using a DIC microscope.

Curcumin increased the number of nucleoids per cell, but the
frequency of occurrence of nucleoids/m of cell length was
similar to that of the control cells. For example, the percentage
of control cells having 1, 2 and 4 nucleoids per cell was 21, 75
and 4 % respectively. In the case of 25 and 50 M curcumin-
treated cells, the number of cells with 1 or 2 nucleoids reduced
considerably and the number of cells with 4 and 8 or more
nucleoids increased significantly. For example, 5, 37, 33 and
25 % of the 25 M curcumin-treated cells had 1, 2, 4 and 8
or more nucleoids respectively. Further, in the case of 50 M
curcumin-treated cells, the percentage of cells with 1, 2, 4 and 8
or more nucleoids was found to be 4, 14, 18 and 64 % respectively.
The frequency of occurrence of nucleoids/m of cell length was
found to be similar without or with curcumin. For example,
the frequency of occurrence of nucleoids/m of cell length was
determined to be 0.58 + 0.19, 0.52 + 0.16 and 0.53 + 0.11 in the
absence and presence of 25 and 50 M curcumin respectively.
The results showed that curcumin had no detectable effect on the
nucleoid segregation.
Out of the total control cells, 21 % of cells had a single nucleoid
with no Z-ring having a cell length of 2.5 + 0.5 m. This popu-
lation might reflect the neonate cells. The percentage of cells
with a single nucleoid decreased strongly in curcumin-treated
cells, indicating that curcumin inhibited cell division. Further, the
percentage of cells with two nucleoids reduced from 75 to 14 %
in the case of 50 M curcumin. Although approx. 80 % of the
control cells containing two nucleoids had a Z-ring, only a small
population (27 %) of the cells with two nucleoids had a Z-ring in
the presence of 25 M curcumin. None of the 50 M curcumin-
treated cells with two nucleoids had a Z-ring, indicating that
curcumin strongly inhibited the Z-ring formation. Taken together,
the results showed that curcumin inhibited the formation of the
Z-ring without affecting the nucleoid segregation detectably.
Effect of curcumin on FtsZ assembly and bundling in vitro
Since curcumin strongly induced filamentation in B. subtilis 168,
and inhibited the formation of the Z-ring in the bacteria, we
wanted to find out whether these effects are due to its interaction
with FtsZ. Therefore we polymerized FtsZ (12.5 M) in 25 mM
Pipes buffer containing 50 mM KCl, 10 mM MgCl2 and
1 mM GTP without or with different concentrations of curcumin
in vitro (Figure 4). FtsZ monomers assembled at a much
higher rate in the absence of curcumin than in its presence.
Further, curcumin reduced the extent of FtsZ polymerization
in a concentration-dependent fashion. For example, the light-
scattering intensity of FtsZ assembly was reduced by 17 % and
70 % in the presence of 10 and 40 M curcumin respectively
compared with that of the control, indicating that curcumin
inhibits the assembly of FtsZ (Figure 4). The IC50 of FtsZ assembly
occurred in the presence of 30 M curcumin.
The effect of curcumin on the assembly of FtsZ was also
analysed by transmission electron microscopy (Figure 5). The
control showed thick bundles of FtsZ polymers with an average
width of 71 + 36 nm (Figure 5A). The size and thickness of FtsZ
polymers reduced extensively with increasing concentrations


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 Curcumin inhibits bacterial cytokinesis by inhibiting FtsZ assembly 151

Figure 3 Curcumin inhibited Z-ring formation without affecting nucleoid segregation in B. subtili s 168
Bacterial cells were grown with different concentrations of curcumin supplemented with 1 mM ascorbic acid. The Z-rings were stained with polyclonal antibody developed in rabbit against FtsZ
followed by Cy3-conjugated goat anti-rabbit secondary antibody (red fluorescence). The nucleoids were stained with 1 g/ml DAPI (blue fluorescence). The cells were observed under a fluorescence
microscope. Shown are control cells [(A), (B) and overlay (C)] and 25 M [(D), (E) and overlay (F)] and 50 M [(G), (H) and overlay (I)] curcumin-treated cells. Scale bar, 10 m (AI).

Table 1 Effect of curcumin on the Z-ring and nucleoids of B. subtilis 168 of curcumin, the length of the FtsZ polymers was considerably
The B. subtilis 168 cells were grown without or with different concentrations of curcumin (25 and smaller compared with that of the control. For example, in
50 M) and incubated with polyclonal FtsZ antibody followed by staining with Cy3-conjugated the presence of 25 M curcumin, the average length of FtsZ
goat anti-rabbit secondary antibody. The nucleoids were stained with DAPI. The cells were polymers was 0.7 + 0.23 m, which was at least 75 % smaller
observed under a fluorescence microscope. Images were analysed using Image-Pro Plus than the control FtsZ polymers, which were larger than the
software. As many as 100 cells were examined for the control as well as for each of the field of view (3 4 m). Under the polymerization conditions,
concentrations of curcumin.
high concentrations of curcumin induced aggregation of FtsZ
monomers (Figures 5B5D, arrows).
Curcumin
Description Control 25 M 50 M
Effect of curcumin on the GTPase activity of FtsZ
Cell length (m) 3.3 +
1 8.6 +
5.3 20 +
16 The assembly dynamics of FtsZ is thought to be regulated by the
Proportion of cells with a Z-ring (%) 63 20 7 GTPase activity of FtsZ [37,38]. Curcumin was found to reduce
Frequency of occurrence of the 0.18 +
0.15 0.03 +
0.07 0.01 +
0.03 the assembly and bundling of FtsZ protofilaments (Figures 4 and
Z-ring/m of cell length
Frequency of occurrence of 0.58 + 0.52 + 0.53 + 5). Therefore the effect of curcumin on the GTPase activity of
0.19 0.16 0.11
nucleoids/m of cell length FtsZ was determined. Curcumin increased the GTPase activity
Proportion of cells having two 75 37 14 of FtsZ in a concentration-dependent manner (Figure 6A). For
nucleoids (%) example, when compared with the control, the GTPase activity of
Proportion of cells with two 80 27 None FtsZ was increased by 35 % in the presence of 30 M curcumin
nucleoids having a Z-ring (%)
(P < 0.0001), indicating that curcumin can perturb FtsZ assembly
dynamics.
GTP-bound FtsZ polymers are thought to be more stable than
of curcumin. For example, curcumin (10 M) reduced the the GDP-bound polymers [2,4,6,37,38]. Under challenged GTP
thickness of the bundles of FtsZ protofilaments by 60 % from conditions, FtsZ polymers are expected to disassemble once all
71 +
36 to 28 + 17 nm (Figure 5B). At a higher concentration available GTP molecules are exhausted. Curcumin increased the


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152 D. Rai and others
Figure 4 Curcumin reduced the rate and extent of FtsZ polymerization
FtsZ (12.5 M) was polymerized in 25 mM Pipes buffer (pH 6.5) containing 50 mM KCl, 10 mM
MgCl2 and 1 mM GTP without () and with 10 M (), 20 M (), 30 M () and 40 M
() of curcumin as described in the Materials and methods section. Appropriate blanks were
subtracted from all the traces. The experiment was performed four times.
GTP hydrolysis rate of FtsZ (Figure 6A). Therefore we argued
that under the challenged GTP conditions, FtsZ protofilaments
would disassemble at a higher rate in the presence of curcumin
than in its absence. To examine this possibility, the effects of
different concentrations of curcumin on the assembly of FtsZ
Figure 5 Electron micrographs of FtsZ polymers
FtsZ (12 M) was polymerized without or with different concentrations of curcumin at 37 C as described in the Materials and methods section. The FtsZ polymers were transferred to
Formvar-carbon-coated copper grids (300 meshes), negatively stained with 1 % uranyl acetate and observed under a transmission electron microscope (FEI TECNAI G2 12) at 20500 magnification.
Shown are the electron micrographs of FtsZ polymers in the absence (A) and presence of 10 M (B), 25 M (C) and 50 M (D) curcumin. The aggregates are shown by arrows (BD). The thickness
of a minimum of 100 polymers was measured in all cases. Scale bar, 2 m (AD).

c The Authors Journal compilation 
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(12.5 M) were analysed in the presence of a limited amount
(100 M) of GTP. The light scattering signal of FtsZ assembly
(control) decreased sharply after 300 s of assembly, indicating
the disassembly of FtsZ protofilaments (Figure 6B). As expected,
curcumin not only reduced the extent of FtsZ assembly but
also strongly reduced the steady-state duration of the assembly
(Figure 6B). Under the experimental conditions used, FtsZ proto-
filaments disassembled more quickly (within 200 s of assembly)
in the presence of curcumin than in the absence of curcumin
(Figure 6B). The results indicated that curcumin-induced increase
in the GTP hydrolysis rate might be the cause of the destabilization
of FtsZ protofilaments.
Curcumin binds to FtsZ in vitro
Curcumin (1 M) was incubated in the absence and presence
of different concentrations (112 M) of FtsZ for 30 min. The
fluorescence intensity of curcumin increased with increase in
concentration of FtsZ, indicating that it binds to FtsZ (Figure 7A).
The increase in the curcumin fluorescence at 495 nm upon binding
to FtsZ was used to determine the binding of curcumin to
FtsZ. FtsZ (1 M) was incubated with different concentrations
of curcumin for 30 min. The fluorescence intensity of the
curcuminFtsZ complex increased with increase in concentration
of curcumin (Figure 7B). The dissociation constant (K d ) of the
curcuminFtsZ interaction was determined to be 7.3 + 1.8 M
(Figure 7C). The number of binding sites of curcumin on FtsZ
was calculated to be 0.7 +
0.2 per FtsZ molecule.

Figure 6 Curcumin increased the GTPase activity of FtsZ
Effects of curcumin on the GTPase activity of FtsZ (A). FtsZ (12 M) was incubated without or
with different concentrations (5, 10, 20 and 30 M) of curcumin. Then, 1 mM GTP was added
to the reaction mixtures and the hydrolysis reaction was initiated immediately by putting the
reaction mixtures at 37 C. Samples were taken after 5 min of hydrolysis. The amount of Pi
released was measured using a standard Malachite Green/ammonium molybdate assay. The
results are the average of five experiments. Error bars represent S.D. The P values were from
< 0.01 to < 0.0001 for the different points. Curcumin reduced the steady-state duration of FtsZ
assembly (B). FtsZ (12.5 M) was polymerized in 25 mM Pipes buffer (pH 6.5) containing
50 mM KCl, 10 mM MgCl2 and 100 M GTP without () and with 10 M (), 20 M (),
30 M () and 40 M () curcumin as described in the Materials and methods section.
Appropriate blanks were subtracted from all the traces. The experiment was performed three
times.
The effects of curcumin on the secondary structures of FtsZ
were determined by monitoring far-UV CD spectra of FtsZ (Fig-
ure 8). Analysis of the CD spectra using deconvolution
software indicated that the secondary structure of FtsZ contained
31.4 +
0.8, 31.7 +
0.6, 9.4 +
0.3, 8.9 +
0.2 and 17.5 + 0.2 % -
helix, random coil, antiparallel, parallel and -turn respectively.
Curcumin perturbed the secondary structures of FtsZ (Figure 8).
For example, curcumin (50 M) reduced the helical content by
9 % from 31.4 + 0.8 % to 28.4 + 0.5 %, while it increased the
random coil content by 8.5 % from 31.7 + 0.6 % to 34.4 +
0.3 %.
The reduction in the -helix (P < 0.0001) and the increase in the
random coil (P < 0.00002) content of FtsZ in the presence of
50 M curcumin were found to be statistically significant. All
other secondary structures did not change significantly.
Curcumin inhibits bacterial cytokinesis by inhibiting FtsZ assembly 153
DISCUSSION
In the present study, we have found that curcumin induced
filamentation in B. subtilis 168, showing that it inhibits bacterial
cytokinesis. Curcumin was found to bind to FtsZ in vitro; it
increased the GTPase activity of FtsZ and inhibited the assembly
and bundling of FtsZ protofilaments. Curcumin had no detectable
effect on the segregation and organization of nucleoids in B.
subtilis 168, indicating that it does not inhibit bacterial division
by targeting DNA. We present evidence suggesting that curcumin
inhibited cytokinesis in B. subtilis 168 by inhibiting the Z-ring
formation through a direct interaction with FtsZ.
The assembly dynamics of FtsZ is considered to be regulated
by the GTPase activity of FtsZ [5,34,3741]. Curcumin decreased
the steady-state duration of FtsZ polymers (Figure 6B) and
curcumin was also found to increase the GTPase activity of
FtsZ (Figure 6A). GDP-bound FtsZ polymers are thought to
be less stable than the GTP-bound polymers and they readily
disassemble into FtsZ monomers [39,40]. In addition, GDP-FtsZ
monomers do not assemble as effectively as GTP-FtsZ monomers
[38]. The results indicated that curcumin decreases the stability
of FtsZ protofilaments by increasing the GTP hydrolysis rate of
FtsZ, which in turn inhibits FtsZ assembly. In addition, curcumin
reduced the -helix content, while it increased the random coil
content of FtsZ, suggesting that the perturbation in the secondary
structure of FtsZ might also be the cause of destabilization of FtsZ
protofilaments. Curcumin is the first small molecule inhibitor of
FtsZ assembly known to date that increases the GTPase activity
of FtsZ. Most of the inhibitors of FtsZ assembly have been
reported to suppress the rate of GTP hydrolysis [7,9,10,12,13]. For
example, 100 M SRI-3072 and SRI-7614 inhibited the GTPase
activity by 20 and 25 % respectively [7]. Similarly, PC58538 and
its analogues inhibited the GTPase activity of FtsZ with an IC50
value of 136 g/ml [12]. Zantrins inhibited GTP hydrolysis with
IC50 values ranging from 4 to 100 M [9]. Totarol suppressed
the GTPase activity of M. tuberculosis FtsZ [13]. For example,
24 % inhibition and 67 % inhibition of GTP hydrolysis were
found in the presence of 10 and 75 M totarol respectively.
Therefore curcumin perturbed the assembly dynamics of FtsZ
by a mechanism that is different from the other known assembly
inhibitors.
Curcumin decreased the light scattering intensity of FtsZ
protofilaments, indicating that it reduced the polymerized mass
of FtsZ protofilaments and/or decreased the bundling of FtsZ
protofilaments (Figure 4). Electron microscopic analysis of the
polymerization reaction showed that curcumin strongly reduced
the length of FtsZ protofilaments and the extent of bundling
of the protofilaments (Figure 5). The decrease in polymer length
and bundling of FtsZ protofilaments may be due to the binding
of bulky curcumin molecules on FtsZ. The presence of bulky
curcumin in the protofilaments may cause steric hindrance that
inhibits the assembly and bundling of FtsZ. Curcumin perturbed
the secondary structures of FtsZ (Figure 8). The decrease
in the polymerization and bundling of FtsZ may be due to
the conformational changes induced by curcumin in the FtsZ
monomers. The IC50 of FtsZ assembly occurred in the presence
of 30 M curcumin. Thus the inhibitory effects of curcumin
on FtsZ polymerization were comparable with that of other
known inhibitors of FtsZ assembly [7,9,11,13]. For example,
the polymerization ID50 (infectious dose to 50 % of exposed
individuals) values of SRI-3072 and SRI-7614 are shown to be
52 + 12 and 60 + 0 M respectively [7].
In the last few years, several natural and synthetic compounds
were shown to inhibit bacterial growth by targeting the assembly
dynamics of FtsZ [714]. For example, viriditoxin inhibited the

c The Authors Journal compilation 
c 2008 Biochemical Society
154 D. Rai and others

Figure 7 Binding of curcumin with FtsZ monitored by fluorescence spectroscopy

FtsZ (1 M) was incubated with different concentrations of curcumin (110 M) and the fluorescence spectra were monitored as described in the Materials and methods section. The ex and em
values were 425 and 495 nm respectively. (A) The Figure shows an increase in the fluorescence intensity of FtsZcurcumin complex in the absence () and presence of 1 M (), 2 M (),
4 M (), 6 M (), 8 M () and 12 M () FtsZ at constant curcumin concentration (1 M). (B) The Figure shows an increase in the fluorescence intensity of the FtsZcurcumin complex in
the absence () and presence of 1 M (), 2 M (), 3 M (), 4 M (), 5 M (), 7 M () and 10 M () curcumin at constant FtsZ concentration (1 M). (C) A plot of L bound /L free
against L bound of the binding of curcumin to FtsZ. The experiment was performed six times.

Similarly, in a recent study, totarol has been shown to inhibit

Figure 8 Effects of curcumin on the far-UV CD spectra of FtsZ
FtsZ (4 M) was incubated without or with different concentrations (2050 M) curcumin at
25 C. The Figure shows the far-UV CD spectra of FtsZ in the absence () and presence of
20 M (), 30 M () and 50 M () curcumin. The experiment was repeated five times.
E. coli wild-type cell division with an MIC of > 64 g/ml and
induced filamentation in the bacterial cell [8]. Zantrins are found
to inhibit the Z-ring formation in E. coli wild-type followed by
cell death with MIC values from 20 to 80 M or more, and only
zantrin Z5 induced filamentation in E. coli [9]. Further, 10 M
sanguinarine is shown to completely inhibit the proliferation
of B. subtilis 168 [11]. Sanguinarine induced filamentation in
bacteria and inhibited Z-ring formation in the B. subtilis 168.
the proliferation of B. subtilis 168 cells with an MIC value of
2 M [13]. Totarol is thought to inhibit the Z-ring formation
in bacteria by inhibiting FtsZ assembly. Curcumin inhibited the
proliferation of both Gram-positive (B. subtilis 168) and Gram-
negative (E. coli K12 MG1655 and E. coli BL21) bacteria with
an MIC of 100 M for B. subtilis 168 and induced filamentation
in B. subtilis 168 cells. The high MIC value for curcumin may
be due to the poor bioavailability of curcumin [42]. Several
bioconjugates of curcumin have been synthesized to enhance
the cellular uptake and targeted delivery of curcumin, thereby
increasing curcumins antibacterial potential, and they have shown
improved antibacterial activity [17,43].
Curcumin inhibited Z-ring formation in B. subtilis 168. Cur-
cumin is known to protect DNA against several DNA-damaging
agents like UV rays, X-rays, -radiation and chemical mutagens
[2325,27]. Further, curcumin is found to inhibit SOS induction
in Salmonella serotype Typhimurium and E. coli [24]. In the
present study, we found that curcumin did not affect the nucleoid
segregation and organization, further supporting the idea that
curcumin does not induce DNA damage detectably.
In summary, the results presented in this study suggest that
curcumin inhibits bacterial cell division, apparently by perturbing
the cytokinetic Z-ring through a direct interaction with FtsZ.
These findings may help to design potent curcumin analogues
with improved stability and bioavailability.
We thank Dr H.P. Erickson (Department of Cell Biology, Duke University Medical Center,
Durham, NC, U.S.A.) for providing us with the FtsZ clone, and SAIF (Sophisticated
Analytical Instrument Facility) IIT Bombay, Mumbai, India, for use of the electron-
microscopy facility. We thank Dr T.K. Beuria, Dr M. K. Santra and Renu Mohan of the


c The Authors Journal compilation 
c 2008 Biochemical Society

School of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Powai,
Mumbai, India, for their assistance in several experiments. We thank Richa Jaiswal, also
of the School of Biosciences and Bioengineering, for a critical reading of the manuscript
before its submission. This work was supported by a grant from the Department of Science
and Technology, Government of India. D. P. is supported by a Swarnajayanti fellowship.
J. K. S. acknowledges support from the CSIR (Council of Scientific and Industrial Research).
REFERENCES
1 Dai, K. and Lutkenhaus, J. (1991) ftsZ is an essential cell division gene in Escherichia
coli . J. Bacteriol. 173, 35003506
2 Nogales, E., Downing, K. H., Amos, L. A. and Lowe, J. (1998) Tubulin and FtsZ form a
distinct family of GTPases. Nat. Struct. Biol. 5, 451458
3 Errington, J., Daniel, R. A. and Scheffers, D.-J. (2003) Cytokinesis in bacteria.
Microbiol. Mol. Biol. Rev. 67, 5265
4 Romberg, L. and Levin, P. A. (2003) Assembly dynamics of the bacterial cell division
protein FtsZ: poised at the edge of stability. Annu. Rev. Microbiol. 57, 125154
5 Margolin, W. (2005) FtsZ and the division of prokaryotic cells and organelles. Nat. Rev.
Mol. Cell Biol. 6, 862871
6 Michie, K. A. and Lowe, J. (2006) Dynamic filaments of the bacterial cytoskeleton.
Annu. Rev. Biochem. 75, 467492
7 White, E. L., Suling, W. J., Ross, L. J., Seitz, L. E. and Reynolds, R. C. (2002)
2-Alkoxycarbonylaminopyridines: inhibitors of Mycobacterium tuberculosis FtsZ.
J. Antimicrob. Chemother. 50, 111114
8 Wang, J., Galgoci, A., Kodali, S., Herath, K. B., Jayasuriya, H., Dorso, K., Vicente, F.,
Gonzalez, A., Cully, D., Bramhill, D. et al. (2003) Discovery of a small molecule that
inhibits cell division by blocking FtsZ, a novel therapeutic target of antibiotics.
J. Biol. Chem. 278, 4442444428
9 Margalit, D. N., Romberg, L., Mets, R. B., Hebert, A. M., Mitchison, T. J., Kirschner, M. W.
and RayChaudhuri, D. (2004) Targeting cell division: small-molecule inhibitors of FtsZ
GTPase perturb cytokinetic ring assembly and induce bacterial lethality. Proc. Natl. Acad.
Sci. U.S.A. 101, 1182111826
10 Urgaonkar, S., La Pierre, H. S., Meir, I., Lund, H., RayChaudhuri, D. and Shaw, J. T.
(2005) Synthesis of antimicrobial natural products targeting FtsZ: (+/)-dichamanetin
and (+/)-2


-hydroxy-5

-benzylisouvarinol-B. Org. Lett. 7, 56095612
11 Beuria, T. K., Santra, M. K. and Panda, D. (2005) Sanguinarine blocks cytokinesis
in bacteria by inhibiting FtsZ assembly and bundling. Biochemistry 44,
1658416593
12 Stokes, N. R., Sievers, J., Barker, S., Bennett, J. M., Brown, D. R., Collins, I., Errington,
V. M., Foulger, D., Hall, M., Halsey, R. et al. (2005) Novel inhibitors of bacterial
cytokinesis identified by a cell-based antibiotic screening assay. J. Biol. Chem. 280,
3970939715
13 Jaiswal, R., Beuria, T. K., Mohan, R., Mahajan, S. K. and Panda, D. (2007) Totarol inhibits
bacterial cytokinesis by perturbing the assembly dynamics of FtsZ. Biochemistry 46,
42114220
14 Huang, Q., Kirikae, F., Kirikae, T., Pepe, A., Amin, A., Respicio, L., Slayden, R. A.,
Tonge, P. J. and Ojima, I. (2006) Targeting FtsZ for antituberculosis drug discovery:
noncytotoxic taxanes as novel antituberculosis agents. J. Med. Chem. 49,
463466
15 Ruby, A. J., Kuttan, G., Babu, K. D., Rajasekharan, K. N. and Kuttan, R. (1995)
Anti-tumour and antioxidant activity of natural curcuminoids. Cancer Lett. 94,
7983
16 Dorai, T. and Aggarwal, B. B. (2004) Role of chemopreventive agents in cancer therapy.
Cancer Lett. 215, 129140
17 Mishra, S., Narain, U., Mishra, R. and Misra, K. (2005) Design, development and
synthesis of mixed bioconjugates of piperic acidglycine, curcuminglycine/alanine and
curcuminglycinepiperic acid and their antibacterial and antifungal properties. Bioorg.
Med. Chem. 13, 14771486
18 Panchatcharam, M., Miriyala, S., Gayathri, V. S. and Suguna, L. (2005) Curcumin
improves wound healing by modulating collagen and decreasing reactive oxygen species.
Mol. Cell. Biochem. 290, 8796
19 Gupta, K. K., Bharne, S. S., Rathinasamy, K., Naik, N. R. and Panda, D. (2006) Dietary
antioxidant curcumin inhibits microtubule assembly through tubulin binding. FEBS J.
273, 53205332
Received 6 July 2007/11 October 2007; accepted 22 October 2007
Published as BJ Immediate Publication 23 October 2007, doi:10.1042/BJ20070891
Curcumin inhibits bacterial cytokinesis by inhibiting FtsZ assembly 155
20 Di Mario, F., Cavallaro, L. G., Nouvenne, A., Stefani, N., Cavestro, G. M., Iori, V.,
Maino, M., Comparato, G., Fanigliulo, L., Morana, E. et al. (2007) A curcumin-based
1-week triple therapy for eradication of Helicobacter pylori infection: something to learn
from failure? Helicobacter 12, 238243
21 Tonnesen, H. H., de Vries, H., Karlsen, J. and Beijersbergen van Henegouwen, G. (1987)
Studies on curcumin and curcuminoids. IX: investigation of the photobiological activity of
curcumin using bacterial indicator systems. J. Pharm. Sci. 76, 371373
22 Dahl, T. A., McGowan, W. M., Shand, M. A. and Srinivasan, V. S. (1989) Photokilling of
bacteria by the natural dye curcumin. Arch. Microbiol. 151, 183185
23 Azuine, M. A., Kayal, J. J. and Bhide, S. V. (1992) Protective role of aqueous turmeric
extract against mutagenicity of direct-acting carcinogens as well as benzo [alpha]
pyrene-induced genotoxicity and carcinogenicity. J. Cancer Res. Clin. Oncol. 118,
447452
24 Oda, Y. (1995) Inhibitory effect of curcumin on SOS functions induced by UV irradiation.
Mutat. Res. 348, 6773
25 Sharma, A., Gautam, S. and Jadhav, S. S. (2000) Spice extracts as dose-modifying factors
in radiation inactivation of bacteria. J. Agric. Food Chem. 48, 13401344
26 Shishu, Singla, A. K. and Kaur, I. P. (2002) Inhibitory effect of curcumin and its natural
analogues on genotoxicity of heterocyclic amines from cooked food. Indian J. Exp. Biol.
40, 13651372
27 Pal, A. and Pal, A. K. (2005) Radioprotection of turmeric extracts in bacterial system.
Acta Biol. Hung. 56, 333343
28 Wang, Y. J., Pan, M. H., Cheng, A. L., Lin, L. I., Ho, Y. S., Hsieh, C. Y. and Lin, J. K.
(1997) Stability of curcumin in buffer solutions and characterization of its degradation
products. J. Pharm. Biomed. Anal. 15, 18671876
29 Canamares, M. V., Garcia-Ramos, J. V. and Sanchez-Cortes, S. (2006) Degradation of
curcumin dye in aqueous solution and on Ag nanoparticles studied by ultraviolet-visible
absorption and surface-enhanced Raman spectroscopy. Appl. Spectrosc. 60, 13861391
30 Oetari, S., Sudibyo, M., Commandeur, J. N., Samhoedi, R. and Vermeulen, N. P. (1996)
Effects of curcumin on cytochrome P450 and glutathione S-transferase activities in rat
liver. Biochem. Pharmacol. 51, 3945
31 Santra, M. K., Dasgupta, D. and Panda, D. (2005) Deuterium oxide promotes assembly
and bundling of FtsZ protofilaments. Proteins 61, 11011110
32 Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram
quantities of protein utilizing the principle of proteindye binding. Anal. Biochem. 72,
248254
33 Geladopoulos, T. P., Sotiroudis, T. G. and Evangelopoulos, A. E. (1991) A malachite green
colorimetric assay for protein phosphatase activity. Anal. Biochem. 192, 112116
34 Beuria, T. K., Krishnakumar, S. S., Sahar, S., Singh, N., Gupta, K., Meshram, M. and
Panda, D. (2003) Glutamate-induced assembly of bacterial cell division protein FtsZ.
J. Biol. Chem. 278, 37353741
35 Santra, M. K. and Panda, D. (2003) Detection of an intermediate during unfolding of
bacterial cell division protein FtsZ: loss of functional properties precedes the global
unfolding of FtsZ. J. Biol. Chem. 278, 2133621343
36 Scatchard, G. (1949) The attraction of proteins for small molecules and ions. Ann. N.Y.
Acad. Sci. 51, 660672
37 Yu, X. C. and Margolin, W. (1997) Ca2+ -mediated GTP-dependent dynamic assembly of
bacterial cell division protein FtsZ into asters and polymer networks in vitro . EMBO J. 16,
54555463
38 Mukherjee, A. and Lutkenhaus, J. (1998) Dynamic assembly of FtsZ regulated by GTP
hydrolysis. EMBO J. 17, 462469
39 Scheffers, D. J., Blaauwen, T. d. and Driessen, A. J. M. (2000) Non-hydrolysable GTP- -S
stabilizes the FtsZ polymer in a GDP-bound state. Mol. Microbiol. 35, 12111219
40 Small, E. and Adinall, S. G. (2003) Dynamic FtsZ polymerization is sensitive to the GTP to
GDP ratio and can be maintained at a steady state using a GTP-regeneration system.
Microbiology 149, 22352242
41 Mukherjee, A. and Lutkenhaus, A. (1999) Analysis of FtsZ assembly by light scattering
and determination of the role of divalent metal cations. J. Bacteriol. 181, 823832
42 Aggarwal, B. B., Kumar, A., Aggarwal, M. S. and Shishodia, S. (2005) Curcumin derived
from turmeric (Curcuma longa ): a spice for all seasons. In Phytopharmaceuticals in
Cancer Chemoprevention (Preuss, H, ed.), pp. 349387, CRC Press, Boca Raton, FL
43 Kumar, S., Dubey, K. K., Tripathi, S., Fujii, M. and Misra, K. (2000) Design and synthesis
of curcumin-bioconjugates to improve systemic delivery. Nucleic Acids Symp. Ser. 44,
7576

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c 2008 Biochemical Society