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Curcumin inhibits influenza virus infection and haemagglutination activity

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Curcumin (diferuloylmethane) is a widely used spice and colouring agent in food. Accumulated evidence indicates that curcumin is associated with a great variety of pharmacological activities, including an anti-microbial effect. In this study, the anti-influenza activity of curcumin was evaluated. Our results demonstrated that treatment with 30 μM curcumin reduced the yield of virus by over 90% in cell culture. The EC50 determined using plaque reduction assays was approximately 0.47 μM (with a selective index of 92.5). Time of drug addition experiments demonstrated curcumin had a direct effect on viral particle infectivity that was reflected by the inhibition of haemagglutination; this effect was observed in H1N1 as well as in H6N1 subtype. In contrast to amantadine, viruses did not develop resistance to curcumin. Furthermore, by comparison of the antiviral activity of structural analogues, the methoxyl groups of curcumin do not play a significant role in the haemagglutinin interaction.
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Curcumin inhibits influenza virus infection and haemagglutination activity
Da-Yuan Chen
a
, Jui-Hung Shien
b
, Laurence Tiley
c
, Shyan-Song Chiou
a
, Sheng-Yang Wang
d
,
Tien-Jye Chang
b
, Ya-Jane Lee
b,e
, Kun-Wei Chan
b
, Wei-Li Hsu
a,*
a
Graduate Institute of Microbiology and Public Health, National Chung Hsing University, Taichung 402, Taiwan
b
Department of Veterinary Medicine, National Chung Hsing University, Taichung 402, Taiwan
c
Department of Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge CB3 OES, UK
d
Department of Forestry, National Chung Hsing University, Taichung 402, Taiwan
e
Teaching Hospital of Veterinary Medicine, National Chung Hsing University, Taichung 402, Taiwan
article info
Article history:
Received 4 March 2009
Received in revised form 2 September 2009
Accepted 4 September 2009
Keywords:
Curcumin
Influenza
Plaque reduction assay
Inhibition of haemagglutination
abstract
Curcumin (diferuloylmethane) is a widely used spice and colouring agent in food. Accumulated evidence
indicates that curcumin is associated with a great variety of pharmacological activities, including an anti-
microbial effect. In this study, the anti-influenza activity of curcumin was evaluated. Our results demon-
strated that treatment with 30
l
M curcumin reduced the yield of virus by over 90% in cell culture. The
EC
50
determined using plaque reduction assays was approximately 0.47
l
M (with a selective index of
92.5). Time of drug addition experiments demonstrated curcumin had a direct effect on viral particle
infectivity that was reflected by the inhibition of haemagglutination; this effect was observed in H1N1
as well as in H6N1 subtype. In contrast to amantadine, viruses did not develop resistance to curcumin.
Furthermore, by comparison of the antiviral activity of structural analogues, the methoxyl groups of cur-
cumin do not play a significant role in the haemagglutinin interaction.
Ó2009 Elsevier Ltd. All rights reserved.
1. Introduction
Curcumin (diferuloylmethane), a nature polyphenolic com-
pound derived from turmeric (Curcuma longa), is a widely used
spice and colouring agent in food (Goel, Kunnumakkara, & Aggar-
wal, 2007). Traditionally, curcumin is commonly applied in many
therapeutic remedies, either alone or in conjunction with other
natural substances (Araujo & Leon, 2001). Accumulated evidence
indicates that curcumin is associated with a great variety of phar-
macological activities, such as anti-inflammatory (Brouet & Ohshi-
ma, 1995), antioxidant (Sreejayan & Rao, 1997), and anti-microbial
(Jagannath & Radhika, 2006; Kutluay, Doroghazi, Roemer, & Trie-
zenberg, 2008; Si et al., 2007). Curcumin also inhibits a number
of tumours in vitro and in animal models (Anand, Kunnumakkara,
Newman, & Aggarwal, 2007; Maheshwari, Singh, Gaddipati, & Sri-
mal, 2006). Such effects have been attributed to the interaction of
curcumin with a diverse range of molecular targets involved in cell
growth, metastasis, tumourangiogenesis and apoptosis; for in-
stance, nuclear factor
j
B (NF-
j
B), cyclooxygenase-2, matrix
metalloproteinase, vascular cell adhesion molecule-1, and p53
(Goel et al., 2007). By inhibiting I
j
B phosphorylation by I
j
B kinase,
curcumin effectively suppressed NF-
j
B signalling, which regulates
the expression of genes contributing to tumourigenesis and cell
survival (Aggarwal & Shishodia, 2004; Bharti, Donato, Singh, &
Aggarwal, 2003; Kumar, Dhawan, Hardegen, & Aggarwal, 1998).
Influenza A virus (IAV) caused three pandemics in the 20th cen-
tury. In 1997, a highly pathogenic strain, H5N1, emerged in Hong
Kong. Worldwide attention was drawn to avian influenza for the
first time, due to the devastating outbreaks in domestic poultry
and sporadic human infections with a high fatality rate (Webster
& Govorkova, 2006). The IAV genome consists of eight negative-
stranded RNA segments encoding 11 viral proteins; among those,
the major glycoproteins on the viral surface, haemagglutinin
(HA) and neuraminidase (NA), are two of the main target antigens
of the host immune system (Fiers, De Filette, Birkett, Neirynck, &
Min Jou, 2004; Nicholls, 2006). Outbreaks of avian H5N1 pose a
public health risk of potentially pandemic proportions; however,
the pre-existing antiviral resistance to amantadine and the emer-
gence of H5N1 variants resistant to oseltamivir and zanamivir,
highlight the need for developing new antiviral therapeutic
strategies.
One of the hallmark cellular responses to influenza virus infec-
tion is the activation of transcription factor NF-
j
B signalling (Lud-
wig, Planz, Pleschka, & Wolff, 2003; Ludwig, Pleschka, Planz, &
Wolff, 2006; Shin, Liu, Tikoo, Babiuk, & Zhou, 2007) by the action
of double-stranded viral RNA, and viral proteins (Bernasconi, Ami-
ci, La Frazia, Ianaro, & Santoro, 2005; Wurzer et al., 2004; Zhirnov &
Klenk, 2007). Recently, several reports demonstrated that NF-
j
B
inhibitors efficiently blocked propagation of influenza, suggesting
0308-8146/$ - see front matter Ó2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2009.09.011
*Corresponding author. Tel.: +886 4 22840694; fax: +886 4 22852186.
E-mail address: wlhsu@dragon.nchu.edu.tw (W.-L. Hsu).
Food Chemistry 119 (2010) 1346–1351
Contents lists available at ScienceDirect
Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem
that modulation of NF-
j
B signalling may be a target for anti-influ-
enza intervention (Mazur et al., 2007; Nimmerjahn et al., 2004).
Our study is based on the observation that curcumin is a strong
inhibitor of NF-
j
B signalling and may therefore impact upon IAV
propagation. We demonstrated that treatment of cells with curcu-
min greatly reduced the yield of IAV at sub-cytotoxic doses. Pre-
incubation of virus with curcumin pronouncedly inhibited influ-
enza virus plaque formation. Thus, in addition to its potential ef-
fects on cellular function, curcumin also acts through direct
interaction with viral particles that interrupts an early stage(s) of
IAV infection. In addition, we confirmed that curcumin interferes
with HA receptor binding activity. To our knowledge, this is the
first report demonstrating that curcumin exerts anti-influenza
activity, and the anti-influenza effect is via a mechanism that abol-
ishes virus-cell attachment.
2. Materials and methods
2.1. Cell culture and virus infection
Madin-Darby canine kidney (MDCK) cells were passed in mini-
mal-essential medium (MEM) with 10% foetal bovine serum (FBS),
penicillin 100 U/ml, and streptomycin 10
l
g/ml. Before infection,
cells were washed with PBS and cultured in infection medium
(MEM without FBS) supplemented with antibiotics and 1 mg/ml
of trypsin (Gibco; Invitrogen, Carlsbad, CA).
Human influenza virus PR8, A/Puerto Rico/8/34 (H1N1), and
avian influenza virus A/chicken/Taiwan/NCHU0507/99 (H6N1),
kindly provided by Paul Digard (Cambridge) and H.-K. Shieh (Lee
et al., 2006), were propagated in MDCK cells.
2.2. Compounds
Curcumin, obtained from Sigma–Aldrich, was dissolved in
DMSO at a stock concentration of 100 mM and stored at 80 °C.
2.3. Antiserum
The PR8 antiserum used in western blot was prepared from two
six-week-old BALB/c mice initially immunised with PR8 virus (2
10
HA units, HAU) followed by two boosters (same dose) at two-week
intervals. Two weeks after the second booster, the serum was
collected.
2.4. Cytotoxicity test
MDCK cells (1 10
5
) grown in 24-well plates for 24 h were
washed twice with PBS and then were treated with curcumin at
the indicated concentrations or mock control solutions (DMSO)
at 37 °C and 5% CO
2
for 24 h. Proliferation of cells was measured di-
rectly by total cell counts and the survival rate was estimated as
the ratio of living cells/total cell counts after staining with 0.4% try-
pan blue. Cytotoxicity of the compounds was estimated by com-
parison of the cell survival rate of curcumin-treated cells with
that of mock-treated. The mock-treatment control was arbitrarily
set as 100%.
2.5. Viral infections and curcumin treatment
MDCK cells (4 10
4
/well) were seeded in 48-well plates 16 h
before infection. Cell monolayers were infected with 2000 pfu (pla-
que forming units) of A/PR/8/34 virus. Supernatant from infected
cells was collected at 12, 18, 24, and 30 h post-infection (hpi)
and the yield of virus progeny was measured by plaque assay.
For time of addition experiments, the indicated concentrations
of curcumin or mock treatment (DMSO) were added to the med-
ium at various times of infection. Briefly, (1) pre-treatment: curcu-
min was included in the cell culture medium for 8 h and was
removed prior to virus infection; (2) simultaneous: curcumin
mixed with virus in the infection medium was added simulta-
neously to the cells and left on the cells throughout; (3) post-infec-
tion: curcumin was added to cells at 2 hpi and remained
throughout the time of infection.
2.6. Plaque assay
MDCK cell monolayers in 12-well plates (2 10
5
cells/well)
were washed twice with PBS followed by infection with serial dilu-
tions of virus. After 2 h absorption at 37 °C, unbound viruses were
removed and cells were then cultured for 2 days with 1 ml/well
MEM supplemented with 0.6% agarose at 37 °C and 5% CO
2
. Viral
plaques were visualised by staining with Giemsa (Sigma, St. Louis,
MO).
2.7. Plaque reduction assay
Five thousand pfu of virus were pre-incubated with 30
l
M (un-
less otherwise stated) of curcumin or various concentrations of re-
lated compounds for one hour. MDCK cells seeded in 12-well
plates were washed twice with PBS followed by infection with se-
rial dilutions of the curcumin-treated viruses. After 2 h absorption
at 37 °C, the virus inoculum was removed and cells were then cul-
tured for 2 days with 1 ml/well MEM supplemented with 0.6% aga-
rose at 37 °C and 5% CO
2
. Viral plaques were visualised by staining
with crystal violet (Sigma).
2.8. Haemagglutination inhibition (HI) test
The haemagglutination (HA) titres of virus stocks were initially
determined by standard HA assay. HI tests were subsequently per-
formed using 4 HA units (HAU) of virus per reaction. Twofold serial
dilutions of curcumin were prepared in round-bottomed 96-well
micro-plates. An equal volume (25
l
l/well containing 4 HAU) of
virus stock was added into each well and incubated at room tem-
perature for 1 h. Subsequently, 50
l
l of chicken erythrocytes (di-
luted to 0.75% v/v in Hank’s buffered saline) were added to each
well. The haemagglutination reaction was observed after 30 min
incubation.
2.9. High performance liquid chromatography (HPLC)
HPLC was employed to isolate the curcuminoid components of
curcumin. The HPLC system consisted of an Agilent quaternary
HPLC, Model 1100 series (Agilent, Waldbronn, Germany), fitted
with a COSMOSIL 5SL-II Waters (Milford, MA) silica column
(10 250 mm i.d.). An Intelligent UV–Vis detector (Agilent 1100)
used at a wavelength of 280 nm was used for detection. Curcumin
prepared as a 5 mg/ml stock dissolved in ethyl acetate (EA) was ap-
plied to the column and the three distinct fractions of curcumi-
noids were eluted individually with EA/Hexane (50/50 v/v). The
solvent from HPLC elutes was then removed using a rotatory vac-
uum evaporator. For identification, the purified compounds were
subjected to
1
H NMR spectral analysis.
1
H NMR spectra were re-
corded at 200 MHz on an INOVA 200 instrument (Varian, Palo Alto,
CA).
2.10. Western blot analysis
Sodium dodecyl sulphate–polyacrylamide gel electrophoresis
(SDS–PAGE; 10%) was performed with a MINI-PROTEAN III appara-
D.-Y. Chen et al. / Food Chemistry 119 (2010) 1346–1351 1347
tus (Bio-Rad; Hercules, CA), and then the proteins were electropho-
retically transferred to PVDF membranes according to the manu-
facturer’s recommendations. After a blocking step in PBS
containing 0.1% Tween-20 (PBS-T) and 5% dried milk for 1 h at
room temperature, the filter was incubated with the primary anti-
body (mouse anti-PR8 serum) diluted in PBS-T containing 5% dried
milk at room temperature for 2–3 h. Subsequently, the filter was
washed with PBS-T at least four times, followed by incubation with
1:5000 diluted secondary antibody conjugated with alkaline phos-
phatase (goat anti-mouse antibody; Sigma) for 1 h. After extensive
washes with PBS-T, the signal was revealed using BCIP/NBT re-
agent (Sigma).
2.11. Drug resistance test
Amantadine was used as a control for the drug resistance test.
In detail, 10
l
M of curcumin or amantadine were included in med-
ium of MDCK cell monolayers infected with 5000 pfu of PR8
viruses in 6-well plates. Supernatants were collected at 18 hpi
and the titre of progeny virions was determined by standard pla-
que assay. Subsequently, 5000 pfu of the passaged PR8 viruses
were taken for the next round of infection. Procedures were re-
peated for five rounds. The yield of progeny virus was monitored.
2.12. Statistical analysis
All data were calculated by Microsoft Excel and analysed with
SAS statistical software (Cary, NC). Results were reported as mean
values ± standard deviation (SD). For the anti-influenza efficacy
study, the chi-square test was used and p-values less than 0.05
were considered to be statistically significant.
3. Results and discussion
3.1. Treatment of curcumin reduces influenza A viruses replication
The initial goal of our study was to determine whether curcu-
min (also designated curcumin I elsewhere) has anti-influenza
virus activity in cell culture. Firstly, cytotoxicity to MDCK cells
was measured based on cell proliferation and viability. The CC
50
(drug concentration inhibiting cell growth by 50% relative to un-
treated control) was approximately 43
l
M and no significant cellu-
lar toxic effect was observed below 30
l
M(Supplementary Fig. S1).
To evaluate the effect of curcumin on influenza virus replica-
tion, cell culture medium was supplemented with various concen-
trations of curcumin at 8 h prior to infection and then maintained
for the duration of the experiment. The yield of virus was deter-
mined at 12, 18, 24 and 30 hpi. As shown in Fig. 1A, the production
of virus was significantly reduced upon treatment with curcumin
in a dose-dependent manner; in the presence of 30
l
M curcumin,
the titre of virus was less than 5% of that in mock-treated cells at all
time points of infection analysed (Fig. 1A). Noticeably, the synthe-
sis of viral proteins, such as haemagglutinin (HA), neuraminidase
(NA), and matrix protein (M1) was affected by curcumin treatment
(Fig. 1B). However, virus protein production was delayed rather
than abrogated; substantial amounts of virus protein were pro-
duced between 24 and 30 hpi, although virus yields were reduced
by over 95% at these time points (compare with Fig. 1A, 30 hpi/
30
l
M). This phenomenon is consistent with a previous study
showing that inactivation of NF-
j
B signalling by aspirin impaired
viral RNP export and subsequent virus multiplication, but did not
significantly affect viral protein accumulation (Mazur et al., 2007).
3.2. Curcumin affects an early stage of virus infection
Time of addition experiments were performed to determine the
stage(s) at which curcumin exerted its inhibitory effects. Curcumin
was added to MDCK cells at three distinct time points: prior to
infection (pre-treatment), at the same time as virus infection
(simultaneously), or at 2 hpi (after entry). As shown in Fig. 2, MDCK
cells pre-treated with curcumin 8 h prior to infection (but removed
just before virus infection) reduced the production of virus to 20%
at 12, 18, and 24 hpi (possibly through effects on NF-
j
B, although
this is not addressed by this study). The addition of curcumin
simultaneously with virus resulted in a much stronger inhibition
than that of cells pre-treated with curcumin at 18 and 24 hpi
(Fig. 2, with significance p< 0.05). Importantly, addition of curcu-
min at 2 h after infection reduced the degree of inhibition (in the
A
B
0%
20%
40%
60%
80%
0 uM
10 uM
20 uM
30 uM
0%
20%
40%
60%
80%
100%
0 uM
10 uM
20 uM
30 uM
9
200
10
3
9168
2583
750
218
34168
13250
2250
1183
70000
35000
3500
625
0 uM
10 uM
20 uM
30 uM
0 uM
10 uM
20 uM
30 uM
12hr
18hr 24h 30hr
Hours post infection (hpi)
983
200
10
3
6
2583
750
218
4
13250
2250
1183
35000
3500
625
Virus Titer (% of Mock Control)
HA
NA
M1
β
-actin
-
+-+-+-+-
curcumin (30
μ
M)
12 hpi 18 hpi 24hpi 30hpi mock
β
-
+-+-+-+-
μM)
Fig. 1. Treatment of curcumin reduces influenza A viruses replication. (A) MDCK cells were pre-incubated with curcumin 8 h prior to and throughout the time of PR8
influenza virus infection (MOI = 0.05). The yield of virus progeny was determined by plaque assay as shown in the top of each column and plotted as a percentage of the
untreated control. (B) Accumulation of viral proteins as determined by Western Blot of infected cell extracts taken at 12, 18, 24, and 30 h post-infection (hpi).
1348 D.-Y. Chen et al. / Food Chemistry 119 (2010) 1346–1351
case of the 18 and 24 h time points back to the pre-treatment lev-
els). This suggested that curcumin may directly interfere with a
very early stage (possibly directly with the virus particle), to pre-
vent infection. We therefore performed plaque reduction assays
to measure the plaque formation ability of IAV particles pre-incu-
bated with curcumin. As indicated in Fig. 3A, the minimal concen-
tration for complete inhibition was 6
l
M(Fig. 3A) and the EC
50
(i.e.
the concentration of curcumin that reduced the plaque formation
by 50%, relative to the control without test compound) was
0.47
l
M. Given the CC
50
of curcumin was 43
l
M, the selectivity in-
dex (SI) value (CC
50
/EC
50
) of curcumin is approximately 92.5, high-
er than several anti-influenza agents published elsewhere (Song
et al., 2007).
Since the inhibitory effect was observed when virions were pre-
exposed to curcumin prior to infection, whereas when it was intro-
duced into the cell culture medium after virus attachment, a mod-
erate inhibitory effect was observed in the yield of progeny viruses
(Fig. 2), these results led us to suspect that the main target of cur-
cumin is at the early stage of virus infection, most likely virus
attachment. Therefore, we used a plaque reduction assay to evalu-
ate whether curcumin affected attachment or not. Binding of IAV
was carried out at 4 °C, the temperature that permits attachment
but not endocytosis and membrane fusion, for 1 h in the presence
of curcumin. Unbound viruses were then removed by cold buffer
wash, and the quantity of bound virus was determined by counting
the subsequent formation of plaques. The results indicate that
incubation of curcumin with virus prior to (Fig. 3B-I), or upon
(Fig. 3B-II) virus attachment completely abolished plaque
formation.
To assess the effect of curcumin on penetration, viral attach-
ment was synchronised at 4 °C without curcumin, unbound viruses
were removed by washing and virus penetration was carried out at
37 °C for 30 min with curcumin treatment, after which the curcu-
min was removed. Noticeably, the plaque formation in cells treated
with curcumin after virus attachment (Fig. 3B-III) displayed a sim-
ilar infection rate to that of mock-treated cells (Fig. 3B-IV), indicat-
ing the curcumin-mediated antiviral activity acts on viral
attachment but not penetration.
3.3. Curcumin blocks haemagglutinating activity of IAV virus particles
Previous results indicated that treatment with curcumin, prior
to, or upon virus entry completely abrogated virus infectivity (Figs.
2 and 3B); hence, it is likely that the action of curcumin may be
through the interference with binding of virus particles to the sialic
acid receptor at the cell surface. To determine whether this was the
case, a HA inhibition (HI) assay was employed to evaluate whether
curcumin is able to inhibit haemagglutination by IAV. Four HAU of
IAV were incubated with various concentrations of curcumin for
60 min at room temperature, followed by detection of RBC aggluti-
nation. Results demonstrated that curcumin pre-treatment
(31.2
l
M) prevented the binding of PR8 virus to chicken RBCs, as
indicated by the spot-like appearance of non-haemagglutinated
cells (Fig. 4A). This concentration is markedly higher than the
EC
50
against virus in the plaque reduction assay, but this may re-
flect the different assay parameters (4 HAU is many orders of mag-
nitude more virus than is used in any of the tissue culture assays).
The development of effective compounds that block virus infectiv-
ity by inhibition of the receptor binding or membrane fusion activ-
ities of HA has been limited due to the lead compounds acting
against only certain subtypes of HA. Interestingly, curcumin also
prevented the binding of another subtype of influenza virus (strain
H6N1) to RBC; a concentration as low as 15.6
l
M was sufficient to
interfere with HA activity (Fig. 4A).
Loss of the HA activity of curcumin-treated influenza viruses
suggests curcumin interrupts the link between the viral HA mole-
cule and its cellular receptor by preoccupying the binding site on
HA protein or by modification of the virus envelope. Increasing evi-
dence indicates many proteins are influenced by curcumin, for in-
stance, epidermal growth factor receptor (Chen, Xu, & Johnson,
2006), P-glycoprotein (Anuchapreeda, Leechanachai, Smith,
Ambudkar, & Limtrakul, 2002), etc. Nevertheless, to date no direct
0%
20%
40%
60%
80%
100%
12hr 18hr 24hr 30hr
Control
pre- trea tment
After entry
Simutaneously
Hours post infection
Virus Titer (% of Mock Control)
*
*
*
*
*
*
*
Fig. 2. The effect of curcumin on different stages of virus infection. About 30
l
M
curcumin was added to cells at three distinct time points: 8-h prior to infection
(pre-treatment), at the same time as virus infection (simultaneously), or at 2 hpi
(after entry). The yield of progeny viruses in supernatant was determined at 12, 18,
24, and 30 hpi. * indicates the p-value < 0.05.
BI II III IV
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
0.1 1 10 100
0
plaque formation (% of mock control)
Curcumin Concentration (
μ
M)
0
μ
10
-1
10
-2
10
-3
curcumin + + + -
Timing prior to upon after after
infection binding entry entry
A
Fig. 3. Curcumin reduces plaque formation activity. Data are from three independent experiments. The dose-dependent effect of curcumin treatment was observed and the
dotted line shows the EC
50
of 0.47 ± 0.05
l
M (A). (B) Evaluation of the effect of curcumin on various stages, such as virus attachment (I, II as labelled on top of panel B) or on
penetration (III, IV).
D.-Y. Chen et al. / Food Chemistry 119 (2010) 1346–1351 1349
binding interaction with curcumin has been identified for any of
these proteins. It was proposed that curcumin associates with
membranes and high concentration of curcumin (>100
l
M) leads
to the alteration of erythrocyte cell membrane integrity (Jaruga,
Sokal, Chrul, & Bartosz, 1998). However, the HA inhibitory effect
is not an artifact resulting from curcumin-induced disruption of
RBC because the minimal concentration required for HA inhibition
was under 15
l
M, which is not toxic to MDCK cells (Supplemen-
tary Fig. S1) and haemolysis was not observed at a concentration
as high as 250
l
M in the HA test (Fig. 4A). In addition, pre-treat-
ment of RBC cells with curcumin followed by its removal did not
affect the HA activity of influenza viruses (data not shown), indi-
cating the membranes of RBC were intact at the concentrations
used. Taken together, the HA inhibitory effect is primarily due to
the interaction of curcumin with virus particles, not via an effect
on RBC cells.
3.4. Characterising the pharmacophore of curcumin involved in HA
interference
Commercially available curcumin consists of three major com-
ponents: curcumin (curcumin I; 77%), demethoxycurcumin (cur-
cumin II; 17%), and bisdemethoxycurcumin (curcumin III; 3%)
(Goel et al., 2007). The structure of curcuminoids differs only by
the number of methoxyl groups (Fig. 4B). Curcumin has been
shown to exert various biological effects; bisdemethoxycurcumin
appeared to be the most active scavenger of superoxide radicals
and inhibition of Ehrlich ascites tumour in mice (Ruby, Kuttan,
Babu, Rajasekharan, & Kuttan, 1995). Another goal of this study
was to define the structure/activity relationship (SAR) of curcumin;
more specifically, to determine whether the methoxyl groups con-
tribute to the antiviral effect by comparing the anti-influenza
activities among three curcuminoids. Consequently, the curcumi-
noid components were separated by HPLC (Fig. 4C) and the
authenticity of the three individual constituents was confirmed
by NMR (Supplementary Fig. S2).
The antiviral activity of the three purified curcuminoids was ini-
tially confirmed by plaque reduction assay (data not shown). The
structure of curcumin is symmetric with two phenolic groups,
two methoxyl groups and two adjacent carbonyl/enol groups that
give rise to an active methylene, which act as potential active sites
for chemical modification and covalent linking with biomolecules.
As indicated in Fig. 4D, in the HA interference assays the com-
pounds lacking one methoxyl group (curcumin II), or both methox-
yl groups (curcumin III) exhibited similar potency to curcumin I.
This indicates that the presence of the methoxyl groups does not
play a significant role in the HAI interaction. However, chemical
synthesis of a series of curcumin analogues is required for a more
detailed SAR assessment of the functional groups involved in its
anti-influenza activity.
3.5. Curcumin treatment does not elicit viral resistance
Currently, two classes of antiviral drugs are available to treat
influenza A infection: the inhibitors of M2, amantadine and riman-
tadine, and the neuraminidase inhibitors, zanamivir and oseltami-
vir (Monto, 2003). In light of the recent evidence for the emergence
Curcumin (CI)
Demethoxycurcumin (CII)
Bisdemethoxycurcumin (CIII)
62.5 μΜ
31.2 μΜ
3.9 μΜ
Curcumin concentration
250 μΜ
125 μΜ
15.6 μΜ
7.8 μΜ
Curcumin: + -+ -+
Virus: PR8 PR8 H6N1 H6N1 -
Curcumin mix
Curcumin III
Curcumin II
Curcumin I
62.5 μ
31.2 μ
3.9 μ
Curcumin concentration
250 μ
125 μ
15.6 μ
7.8 μ
Curcumin mix
Curcumin III
Curcumin II
Curcumin I
μΜ
μΜ
μΜ
μΜ
μΜ
μΜ
μΜ
ABC
Fig. 4. Haemagglutination inhibitory activity of curcumin and other structure analogues. (A) 4 HA units of influenza viruses strain PR8 or H6N1 were incubated with twofold
diluted curcumin or PBS (negative control) and the HA activity tested by incubation with chicken RBC (cRBC). The concentrations of curcumin necessary to completely inhibit
haemagglutination (MIC) were approximately 26.04 and 15.63
l
M for H1N1 and H6N1, respectively. The HI activities of three curcuminoids (B), isolated by HPLC (C), were
analysed by same protocol as described in experiment A.
Contr ol curcumin amantadine
100
80
60
40
20
0
1
st
2
nd
3
rd
4
th
5
th passage
Fig. 5. Curcumin treatment does not elicit viral resistance. Virus yield of mock-
treated cells was arbitrarily set as 100%.
1350 D.-Y. Chen et al. / Food Chemistry 119 (2010) 1346–1351
of resistance to both classes of drugs, it is of importance to evaluate
whether curcumin has the potential to induce viral resistance. To
do so, a multi-passage experiment was performed, in which
MDCK-passaged viruses that lacked the drug pressure were used
as sensitive controls and amantadine treatment as the parallel con-
trol for resistant virus development. As shown in Fig. 5, the titre of
progeny virions from cells treated with amantadine increased sig-
nificantly after the fourth passage. In contrast to amantadine, the
inhibition of virus passage remained throughout the time of the
experiment and the vial yield did not rise even after the fifth pas-
sage, indicating treatment of curcumin is not prone to emerging of
resistant viruses (Fig. 4).
4. Conclusions
Results from the plaque reduction test and HI test clearly show
that curcumin interrupts virus-cell attachment, which leads to
inhibition of influenza virus propagation, although it is not known
yet whether curcumin directly interacts with the viral HA protein
or with other viral surface components. With an established safety
profile and high SI value of 92.5, curcumin has promising potential
for using as an anti-influenza drug.
Acknowledgements
We thank Professor Min-Liang Wong (Department of Veterinary
Medicine, National Chung Hsing University, Taichung 402, Taiwan)
for helpful comments on the manuscript. This study was supported
partially by National Science Council, Taiwan (NSC96-2313-B-005-
024, NSC97-2313-B-005-001).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.foodchem.2009.09.011.
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