Different curcuminoids inhibit T-lymphocyte proliferation independently of their radical scavenging activities

Article abstract of DOI:10.1007/s11095-008-9579-2

Purpose. We investigated the inhibitory effects of curcumin, curcumin derivatives and degradation products on OKT3-induced human peripheral blood mononuclear cell (PBMC) proliferation and the role of their radical scavenging activity. Methods. OKT3-induced human PBMC proliferation was determined by measuring ³H-thymidine incorporation. Radical scavenging activity was evaluated by using an in vitro DPPH assay. Results. OKT3-induced PBMC proliferation was inhibited by curcumin, isocurcumin, bisdesmethoxy-, diacetyl-, tetrahydro-, hexahydro-, and octahydrocurcumin as well as by vanillin, ferulic acid, and dihydroferulic acid with IC₅₀-values of 2.8, 2.8, 6.4, 1.0, 25, 38, 82, 729, 457, and >1,000 μM, respectively. The investigated substances with the strongest effect on radical scavenging were tetrahydro-, hexahydro-, and octahydrocurcumin with IC₅₀ values of 10.0, 11.7, and 12.3 μM, respectively. IC₅₀-values of dihydroferulic acid, ferulic acid, and curcumin were 19.5, 37, and 40 μM. The substances with the lowest radical scavenging activities were vanillin, isocurcumin, diacetylcurcumin, and bisdesmethoxycurcumin with IC₅₀ values higher than 100 μM each. Conclusions. Curcuminoid-induced inhibition of OKT3-induced PBMC proliferation depends on the number of carbon atoms and double bonds of the 1,6-heptadiene-3,5-dione structure as well as on the phenolic ring substitutes of the curcuminoids but is not correlated to their respective radical scavenging activity.

Full text of DOI:10.1007/s11095-008-9579-2

Pharmaceutical Research, Vol. 25, No. 8, August 2008 (# 2008)
DOI: 10.1007/s11095-008-9579-2
Research Paper
Different Curcuminoids Inhibit T-Lymphocyte Proliferation Independently
of Their Radical Scavenging Activities
1
1
1
1
2
Michael Deters, Heiko Knochenwefel, Daniel Lindhorst, Therese Koal, Hartmut H. Meyer,
3
1
1,4
Wolfram Hänsel, Klaus Resch, and Volkhard Kaever
Received November 6, 2007; accepted March 25, 2008; published online April 22, 2008
Purpose. We investigated the inhibitory effects of curcumin, curcumin derivatives and degradation
products on OKT3-induced human peripheral blood mononuclear cell (PBMC) proliferation and the role
of their radical scavenging activity.
3
Methods. OKT3-induced human PBMC proliferation was determined by measuring H-thymidine
incorporation. Radical scavenging activity was evaluated by using an in vitro DPPH assay.
Results. OKT3-induced PBMC proliferation was inhibited by curcumin, isocurcumin, bisdesmethoxy-,
diacetyl-, tetrahydro-, hexahydro-, and octahydrocurcumin as well as by vanillin, ferulic acid, and
dihydroferulic acid with IC50-values of 2.8, 2.8, 6.4, 1.0, 25, 38, 82, 729, 457, and >1,000 μM, respectively.
The investigated substances with the strongest effect on radical scavenging were tetrahydro-, hexahydro-,
and octahydrocurcumin with IC50 values of 10.0, 11.7, and 12.3 μM, respectively. IC50-values of
dihydroferulic acid, ferulic acid, and curcumin were 19.5, 37, and 40 μM. The substances with the lowest
radical scavenging activities were vanillin, isocurcumin, diacetylcurcumin, and bisdesmethoxycurcumin
with IC50 values higher than 100 μM each.
Conclusions. Curcuminoid-induced inhibition of OKT3-induced PBMC proliferation depends on the
number of carbon atoms and double bonds of the 1,6-heptadiene-3,5-dione structure as well as on the phe-
nolic ring substitutes of the curcuminoids but is not correlated to their respective radical scavenging activity.
KEY WORDS: antioxidants; curcumin; curcuminoids; immunosuppression; OKT3-induced human
PBMC proliferation; radical scavengers.
INTRODUCTION
consumed daily by approximately a quarter of the world’s
population (3). Curcuminoids are now being investigated in
Curcuminoids are low molecular weight phenolic com- more detail for use in Western medicine. Within the last
pounds derived from the rhizomes of turmeric (botanical 5 years more than 700 new publications concerning curcumin
name: Curcuma longa) a member of the ginger family are listed in the literature database MEDLINE. The most
Zingiberaceae (1). The major representative of these phyto- detailed studies using curcumin showed a plethora of
chemicals is curcumin (diferuloylmethane; Fig. 1) which has beneficial effects including choleretic, anti-inflammatory,
been used in traditional Indian and Chinese medicine for antioxidant, anticarcinogenic, antiviral, and anti-infectious ac-
centuries (2). As a principal ingredient in curry, curcumin is tivities (4,5) although the underlying molecular mechanism(s)
of action remain poorly defined. In experiments with human
low density lipoproteins curcumin and some analogues were
This work was part of HK’s thesis at the Hannover Medical School.
potent inhibitors of low density lipoprotein oxidation (6).
Curcumin has been described to attenuate the proliferation of
human lymphocytes and the production of several inflamma-
tory mediators including lipid mediators and cytokines (7–8).
Thus, it may be suited as a lead substance for the development
1
Institute of Pharmacology, Hannover Medical School, 30623, Hannover,
Germany.
2
Institute of Organic Chemistry, Leibnitz Universität Hannover, 30167,
Hannover, Germany.
3
Pharmaceutical Chemistry, Christian-Albrecht’s-University Kiel, 24118, of new immunosuppressive and anti-rheumatic drugs.
Kiel, Germany.
In vitro, curcumin is described to be an unstable
substance that decomposes to other substances like ferulic
acid and vanillin (9). The bioavailability of curcumin is low
and after resorption it is metabolized to a great extent in the
intestine and liver of humans and rats to curcumin glucur-
onides, sulfates, as well as tetrahydrocurcumin, and hexacur-
cumin (10). Whether curcumin itself or one of its metabolites
4
To whom correspondence should be addressed. (e-mail: kaever.
volkhard@mh-hannover.de)
ABBREVIATIONS: ABTS, 2,2-azino-bis(3-ethylbenzthiazoline-6-
sulphonic acid); DPPH, 1,1-diphenyl-2-picrylhydrazyl radical; HBC,
-hydroxypropyl-γ-cyclodextrin; HHC, hexahydrocurcumin; IC50,
halfmaximal inhibitory concentration; JNK, junN-terminal kinase;
NFκB, nuclear factor κB; OHC, octahydrocurcumin; OKT3, mouse
2
anti-human CD3 antibody; PBMC, peripheral blood mononuclear or degradation products mediates the numerous pharmaco-
cells; THC, tetrahydrocurcumin.
dynamic effects is still under discussion.
0
724-8741/08/0800-1822/0 # 2008 Springer Science + Business Media, LLC
1822

Inhibition of OKT3-induced Human PBMC Proliferation by Curcuminoids
1823
Curcumin
H
Therefore, we investigated the effects of various curcu-
minoids and curcumin derivatives (curcumin, isocurcumin,
bisdesmethoxy-, diacetyl-, tetrahydro-(THC), hexahydro-
O
O
H
3
CO
OCH
3
(
HHC), and octahydrocurcumin (OHC) as well as vanillin,
ferulic acid and dihydroferulic acid) on OKT3-induced
proliferation of human peripheral blood mononuclear cells
HO
OH
(
PBMC) in order to establish some first structure–activity rela-
Curcumin analogues
tionships. Additionally, we determined the radical scavenging
activities of these curcuminoids by using a chemical assay in
order to proof a possible relationship to their immunosup-
pressive effects.
H
isocurcumin
O
O
HO
OH
OCH
3
3
H CO
MATERIALS AND METHODS
Materials
H
bisdesmethoxy-
curcumin
O
O
Curcumin was obtained from Roth, Karlsruhe, Germany.
HO
OH
Ferulic acid, vanillin, 2-hydroxypropyl-γ-cyclodextrin, and
1
,1-diphenyl-2-picrylhydrazyl radical were purchased from
Sigma, Taufkirchen, Germany. Dihydroferulic acid was
obtained from Lancaster, Frankfurt, Germany and [ H]
H
diacetylcurcumin
O
O
3
H
O
3
CO
OCH
thymidine (specific activity 25 Ci/mmol) was from Amersham,
Braunschweig, Germany. Stock solutions of the curcuminoids
3
O
(
1 M) were prepared in DMSO except for tetrahydro-
H
3
C
O
O
CH
3
hexahydro-, octahydrocurcumin, and dihydroferulic acid,
where HBC (0.45%) was used as solvent. All further dilutions
were done in RPMI1640 medium/5% fetal calf serum
Curcumin biotransformation products
(
proliferation assay) or ethanol (radical scavenging).
H
tetrahydrocurcumin
O
O
H
3
CO
OCH
3
Synthesis of Curcuminoids
Isocurcumin and bisdesmethoxycurcumin were synthe-
sized at the Pharmaceutical Institute, University Kiel. Briefly,
to a solution of 0.04 mol of either isovanillin or 4-hydroxy-
benzaldehyde in tributyl borate (0.08 mol) the reaction
product of acetylacetone (0.02 mol) and boric (0.014 mol)
anhydride in dry ethyl acetate was added. Butylamine was
then added as a catalyst and the resulting mixture was stirred
at 40°C for 18 h. Diluted hydrochloric acid (0.4 N) was used
to free isocurcumin or bisdesmethoxycurcumin, respectively,
which were purified by recrystallization in ethanol and
HO
OH
O
OH
OH
hexahydrocurcumin
H
3
CO
OCH
3
HO
OH
OH
octahydrocurcumin
H
3
CO
OCH
3
1
HO
OH
characterized by combustion analysis, IR, UV and H-NMR
O
(data not shown).
dihydroferulic acid
The curcumin derivative diacetylcurcumin, as well as the
hydrocurcumins THC, HHC, and OHC used in this study
were synthesized at the Institute of Organic Chemistry,
University Hannover. Diacetylcurcumin was synthesized from
curcumin using acetanhydride and potassium carbonate in
waterfree acetone. Briefly, curcumin (0.30 mmol), acetanhy-
dride (0.75 mmol), and potassium carbonate (1.00 mmol) were
given to 3 ml waterfree acetone and stirred for 48 h at room
temperature. The reaction mixture was taken up in dichlor-
methane and water. After addition of diluted HCl the organic
phase was removed and extracted twice with dichlormethane.
The extracted organic phase was dried by sodium sulfate and
evaporated. The product was purified chromatographically on
a silica gel column (2.4×5.0 cm). As eluents hexane/methyl-t-
butylether, 1/2 (v/v), and pure methyl-t-butylether were used.
In the initial fraction diacetylcurcumin (light-yellow crystal-
H
3
CO
OH
HO
Curcumin chemical degradation products
O
ferulic acid
H
3
CO
OH
HO
vanillin
O
H
3
CO
H
HO
Fig. 1. Chemical structures of different curcuminoids and their loid substance) with a melting point of 172°C was gained. The
degradation products.
spectroscopic data were as described in the literature (11).

1
824
Deters et al.
The hydrocurcumins were synthesized according to curcumin concentration of 2.8 μM (Table I). Dissolving the
previously described methods (10,12–14) with variations of stock solution of curcumin in either NaOH or DMSO
the reaction conditions regarding the hydrogenation of revealed no difference in the IC50 value (data not shown).
curcumin by palladium/carbon in 10% acetic acid with the The commercial curcumin (Roth, Karlsruhe, Germany) con-
addition of 3% acetic acid and control of hydrogen uptake. tains 77% curcumin, 19% desmethoxy- and 4% bisdesme-
Hydrogenation was stopped immediately when the yellow thoxycurcumin. We compared the influence of purified
colouring of the educt disappeared. Because the velocity of curcumin to commercial curcumin on proliferation of
hydrogenation of THC is in the same range as the hydroge- OKT3-induced human PBMC. The IC50 values of purified
nation of curcumin always HHC and OHC (as diastereomere) and commercial curcumin were 2.8 and 3.7 μM, respectively.
can be found as by-products. The hydrogenation of curcumin Because this difference was statistically not significant we
should be finished totally to avoid problems of preparative used the commercial curcumin for further experiments.
chromatography. Briefly, curcumin (5 mmol, purified by
repeated crystallization) in ethyl acetate/acetic acid, proliferation of the chemical derivative diacetylcurcumin
0:1.6 ml (v/v), was hydrogenated until the yellow colour was three times higher and that of bisdesmethoxycurcumin
disappeared. The volume of the hydrogen taken up was 2.5 times lower than of curcumin (Fig. 2) with IC50 values of
4.5 mmol. The product was filtrated, evaporated and 1.0 and 6.4 μM, respectively, whereas the curcumin analogue
purified chromatographically on a silica gel column (3.8× isocurcumin showed the same inhibitory effect (IC50 2.8 μM)
2 cm). As eluents hexane/methyl-t-butylether mixtures and as curcumin itself (Table I).
pure methyl-t-butylether were used. The gain was as follows: The physiological metabolites of curcumin THC, HHC,
The inhibitory effect on OKT3-induced PBMC cell
6
1
1
THC, Fp=98°C (46% gain), HHC, Fp=80°C (32% gain), and OHC inhibited the proliferation of OKT3-stimulated
OHC, Fp=23°C (23% gain). All three substances showed the human PBMC less than 4.4 to 14.4 times compared to
same spectroscopic characteristics as described in the litera- curcumin (Fig. 2) with IC50 values of 25, 38, and 82 μM,
ture (10,12–14).
respectively (Table I).
Compared to curcumin, the effects of the curcumin
degradation products vanillin and ferulic acid on PBMC
proliferation were weak (Fig. 2). The IC50 of curcumin was
Proliferation Assay
Peripheral blood mononuclear cells were isolated from 163 times lower than the IC50 of ferulic acid (IC50 457 μM)
heparinized blood drawn from healthy adults by Ficoll/ and even 260 times lower than the IC50 of vanillin (729 μM;
Hypaque gradient centrifugation. After overnight culture
non-adherent lymphocytes were resuspended in RPMI1640
medium containing 5% fetal calf serum. Proliferation assays
5
were performed in 96-well microtiter plates (15). The cells (10
1
00
per well) were preincubated with the various curcuminoids
for 1 h and then activated by addition of the monoclonal anti
human T-cell receptor/CD3 complex antibody OKT3 (1 ng/
8
6
4
0
0
0
3
ml). After an incubation time of 44 h [ H]thymidine (0.5 mCi/
well) was added and the culture was carried on for further 4 h
prior to cell harvest by an automatic 12-well harvester.
Incorporated radioactivity was determined by liquid
scintillation counting.
20
0
Determination of Radical Scavenging Activity
of Curcuminoids
0
.1
1.0
10.0
100.0
1000.0
concentration [µM]
The radical scavenging activity of the various curcumi-
noids was investigated similar as described previously (16–
Curcumin
Isocurcumin
1
0
7). The curcuminoids were dissolved at concentrations of
.63–200 μM in ethanol and given 1:1 (v/v) to a 100 μM
Bisdesmethoxycurcumin
Diacetylcurcumin
Tetrahydrocurcumin
Hexahydrocurcumin
Octahydrocurcumin
Vanillin
radical solution stabilized by 1,1-diphenyl-2-picrylhydrazyl
DPPH). The reaction time was 20 min at room temperature.
The decrease in the optical density was measured photomet-
rically at 517 nm.
(
RESULTS
Ferulic acid
Dihydroferulic acid
Effect of Curcuminoids on Proliferation of OKT3-induced
Human PBMC
Fig. 2. Influence of curcuminoids and their degradation products on
proliferation of OKT3-stimulated human PBMC. Human PBMC of
different donors were stimulated by 1 ng/ml OKT3 and incubated
with different concentrations of the test substances for 48 h. Means of
Curcumin concentrations up to 0.63 μM showed no or
only a little effect on PBMC proliferation (Fig. 2). The at least three independent experiments each. Standard error of the
proliferation of PBMC was inhibited by 50% (IC ) at a means (S.E.M.) was ±11% in maximum and is not shown in the figure.
5
0

Inhibition of OKT3-induced Human PBMC Proliferation by Curcuminoids
1825
Table I. IC50 Values [μM] of Various Curcuminoids and their PBMC proliferation the number of carbon atoms of this
Degradation Products Concerning Inhibition of PBMC Cell Prolifer- structure and the presence of the aromatic ring structure are
ation and Scavenging DPPH Stabilized Radicals
IC50 [μM]
obviously important for the immunosuppressive effect of
curcumin.
In vivo, curcumin is not only degraded but also
metabolized rapidly, as well. The main metabolites are THC
and HHC as well as dihydroferulic acid. Curcumin and these
metabolites are conjugated to glucuronic acid (10,18).
Because of the short halftime of curcumin (less than 1 h)
the biological effects could be mediated by the metabolites
rather than by curcumin itself. We investigated the inhibitory
effects of tetra-, hexa-, and octahydrocurcumin, and dihydro-
ferulic acid on OKT3-induced proliferation of human PBMC.
Because of the low solubility, these substances had to be
dissolved in HBC. Dissolution of curcumin in HBC reduced
its inhibitory effect on cell proliferation statistically not
significant by the half (IC50 5.7 μM). However, the inhibitory
effects of THC (IC50 25 μM), HHC (IC50 38 μM), and OHC
Inhibition
of proliferation
Binding
of radicals
Curcumin
Isocurcumin
2.8
2.8
40
>100
>100
>100
10.0
11.7
12.3
>100
37
Bisdesmethoxycurcumin
Diacetylcurcumin
Tetrahydrocurcumin
Hexahydrocurcumin
Octahydrocurcumin
Vanillin
6.4
1.0
25
38
82
729
457
>1,000
Ferulic acid
Dihydroferulic acid
19.5
(
IC50 82 μM) were lower by a factor of 4.4, 6.7, and 14.4,
Table I). Dihydroferulic acid showed no effect on prolifera- respectively. For the curcumin metabolite dihydroferulic acid
tion of OKT3-stimulated human PBMC up to a concentration no IC50 value could be determined (Table I). The reduction
of 1,000 μM (Table I).
of curcumin to THC, HHC, and OHC was accompanied by a
loss of double bonds of the 1,6-heptadiene structure and a
loss of inhibitory effect on OKT3-induced PBMC prolifera-
tion. From these results we conclude that the intact 1,
Radical Scavenging Activity of Curcuminoids
To get further information about the mechanism of the 6-heptadiene structure is an important feature of the immu-
curcuminoid-mediated inhibition of PBMC proliferation we nosuppressive effect of curcumin. The remaining immuno-
investigated the effect on curcuminoids and their degradation suppressive effect of THC, HHC, and OHC was much higher
products on binding radicals stabilized by 1,1-diphenyl-2- than that of ferulic acid, dihydroferulic acid, and vanillin
picryl-hydrazol (DPPH; Fig. 3). The investigated substances
with the strongest effect on scavenging radicals were THC,
HHC, and OHC with IC50 values of 10.0, 11.7, and 12.3 μM,
1
00
respectively (Table I). The radical scavenging activity of
dihydroferulic acid, ferulic acid and curcumin were in the
middle range with IC -values of 19.5, 37 and 40 μM each.
8
6
4
2
0
0
0
0
0
5
0
The lowest ability as radical scavengers were observed in
experiments with vanillin, isocurcumin, diacetylcurcumin, and
bisdesmethoxycurcumin with all IC50 values higher than
1
00 μM (Fig. 3; Table I).
DISCUSSION
In our experiments it was shown that curcumin inhibited
OKT3-induced proliferation of human PBMC in a dose-
dependent manner with an IC50 of 2.8 μM. This value is
comparable to values observed in experiments with PMA-/
CD28-, and PHA-induced proliferation of human T-lympho-
cytes with IC50 of 3.5 and 7.7 μM, respectively (8). The
inhibitory effect of the commercially available curcumin
powder of Roth (77% curcumin, 19% desmethoxy-, 4%
bisdesmethoxycurcumin) was the same as of purified curcu-
min. Therefore, it was possible to use the commercial powder
for all further experiments.
The main degradation products of curcumin are vanillin
and ferulic acid. We studied the inhibitory effect of these
products on OKT3-induced cell proliferation of PBMC, as
well. The inhibitory effect of curcumin (IC50 2.8 μM) was 163
times stronger than of ferulic acid (IC50 457 μM) and 260
times stronger than of vanillin (IC50 729 μM). Because
0.1
1.0
10.0
100.0
concentration [µM]
Curcumin
Isocurcumin
Bisdesmethoxycurcumin
Diacetylcurcumin
Tetrahydrocurcumin
Hexahydrocurcumin
Octahydrocurcumin
Vanillin
Ferulic acid
Dihydroferulic acid
Fig. 3. Radical scavenging activities of different curcuminoids and
their degradation products. The radical scavenging activities were
determined in the established DPPH assay. Means of at least three
cleavage of the 1,6-heptadiene-3,5-dione moiety resulted in independent experiments each. Standard error of the means (S.E.M.)
a significant loss of the inhibitory effect on OKT3-induced was ±7% in maximum and is not shown in the figure.

1
826
Deters et al.
(
Table I). Therefore, the number of carbon atoms of the 1,6- (23), and bisdesmethoxycurcumin with IC50 values higher
heptadiene structure seemed to be even more important for than 100 μM (Table I). From these results we conclude that
the inhibitory effect than the loss its double bounds. After reduction of the 1,6-heptadiene structure (THC, HHC, and
reduction of the 3,5-dione structure (THC, HHC, OHC) a OHC) increases the radical scavenging effects of curcumin.
little decrease of the inhibitory effect on cell proliferation was Because the IC50 values of THC, HHC, and OHC radical
observed, as well (Table I) but the decrease of the immuno- scavenging activity were in the same range the loss of the 3,5-
suppressive effect was quite lower than after the loss of the dione structure seemed to have no influence on the antiox-
1
,6-heptadiene structure.
idative effect of curcumin. Sugiyama et al. (19) examined the
To get further information about the role of different inhibitory effects of curcumin and THC on the lipid peroxi-
substitutes and their position in the aromatic ring structure, dation of erythrocyte membrane ghosts induced by tertbu-
we investigated the inhibitory effect of the curcumin ana- tylhydroperoxide. Their results demonstrated that THC
logues iso-, diacetyl-, and bisdesmethoxycurcumin on OKT3- showed a greater inhibitory effect than curcumin, as well.
induced proliferation of human PBMC. The inhibitory effect On the other side, however, their results suggested that the
of isocurcumin was nearly the same as of curcumin (Table I). beta-diketone moiety of THC must exhibit antioxidative
The inhibitory effect of diacetylcurcumin, however, was three activity by cleavage of the C–C bond at the active methylene
times higher (IC50 1.0 μM) and that of bisdesmethoxycurcu- carbon between two carbonyls in the beta-diketone moiety. In
min 2.5 times lower than of curcumin (Table I). According to our present study, changes of the 4-hydroxy-3-methoxyphenyl
these observations, we hypothesize that the location of the 4- group position (isocurcumin), loss of the 3-methoxy group
hydroxy-3-methoxyphenyl group (Fig. 1) is not important for (bisdesmethoxycurcumin), and derivatisation of the 4-hydroxy
the immunosuppressive effect. The loss of the 3-methoxy group (diacetylcurcumin) significantly reduced the curcumin
group (bisdesmethoxycurcumin), however, reduces and acety- radical scavenging effect. This observation is in accordance
lation (diacetylcurcumin) of the 4-hydroxyl group (Fig. 1) with the results of Chen et al. (6) who also found that the 4-
increases the inhibitory effect of curcumin on OKT3-induced hydroxy-3-methoxyphenyl group is important for the antiox-
proliferation of human PBMC. The role of the in vivo idant activity of curcumin. In another study the antioxidant
observed glucuronidation of curcumin has to be investigated activity of curcumin and dimethoxycurcumin was tested
in further experiments.
by following radiation-induced lipid peroxidation in rat
The underlying molecular mechanism(s) of the immuno- liver microsomes. It was concluded that the phenolic OH
suppressive effects of curcumin remain poorly defined. group plays a major role in the activity of curcumin (20).
Curcumin and some synthetic symmetrical curcumin ana- Venkateswarlu et al. (21) evaluated the antioxidant activity of
logues have been described to show activity for scavenging polyhydroxycurcuminoids by superoxide radical and DPPH
free radicals (16–17,19–23) and in experiments with human free radical scavenging methods. In addition, they proofed
low density lipoproteins curcumin and some analogues were their cytotoxicity in lymphocytes from normal or patients with
potent inhibitors of low density lipoprotein oxidation (6). leukemia and antitumor activity on Dalton’s lymphoma
Curcumin is known to exert radical scavenging activities via ascites tumor cells. In their experiments the inhibitory effect
several mechanisms (21), due to its various functional groups, of curcumin on scavenging DPPH free radicals was compa-
i.e. the ß-diketone group, carbon–carbon double bonds, and rable with our results (IC50 21 μM). As in our experiments no
phenyl rings containing hydroxyl and methoxy substituents correlation between the cytotoxicity and the antitumor
(
Fig. 1). Therefore, we determined the radical scavenging activity of the curcuminoids on the one hand and their radical
activity of the curcuminoids by using an established DPPH scavenging effect on the other was observed. Venkateswarlu
assay and proofed a possible relationship to their immuno- et al. (21) found in their experiments that the superoxide as
suppressive effects. Venkatesan and Rao (16) found in their well as the DPPH radical scavenging activity of the curcumi-
experiments with inhibition of lipid peroxidation, scavenging noids increased with the number of hydroxyl groups of their
of DPPH and ABTS+ that the phenolic analogues of benzene ring. Furthermore, the substitution pattern of
curcumin were more active than the non-phenolic analogues, hydroxyls played an important role. In our experiments, no
some of which were inactive. The highest antioxidant activity radical scavenging activity of polyhydroxycurcuminoids was
was obtained when the phenolic group was sterically hindered investigated. The acetylation of the hydroxylated benzene
by the introduction of two methyl groups at the ortho rings of curcumin to diacetylcurcumin was accompanied by a
position. In our experiments we investigated only phenolic significant loss of DPPH free radicals scavenging activity
curcuminoids. The substances with the strongest effect on (Table I). This observation is in accordance with the
scavenging radicals were THC, HHC, and OHC with IC50 conclusion of Venkateswarlu et al. (21) that hydroxyl groups
values of 10.0, 11.7, and 12.3 μM, respectively (Table I). of the benzene rings are important in radical scavenging
Recently, Somparn et al. (17) also studied the effect of activity of curcuminoids.
curcumin, THC, HHC, OHC, and bisdesmethoxycurcumin
Comparing the influences of changes of the curcumin
on radical scavenging determined by the DPPH assay. In their molecule on its inhibitory effect on OKT3-induced prolifer-
experiments the scavenging activity decreased in the order ation of human PBMC and on its radical scavenging activity
THC > HHC = OHC > curcumin > bisdesmethoxycurcumin. the results can be summarized as follows: Decreasing the
In our present study, the radical scavenging activities of number of carbon atoms of the 1,6-heptadiene-3,5-dione
dihydroferulic acid, ferulic acid and curcumin were in the structure (vanillin, ferulic acid, and dihydroferulic acid;
middle range with IC50-values of 19.5, 37 and 40 μM each. Fig. 1) significantly reduces the inhibitory effect of curcumin
The lowest effects as radical scavengers were observed in on OKT3-induced proliferation of human PBMC but for
experiments with vanillin, isocurcumin, diacetylcurcumin example in the case of dihydroferulic acid the radical

Inhibition of OKT3-induced Human PBMC Proliferation by Curcuminoids
1827
5
.
.
R. K. Maheshwari, A. K. Singh, J. Gaddipati, and R. C. Srimal.
Multiple biological activities of curcumin: A short review. Life
Sci. 78:2081–2087 (2006).
scavenging activity was even increased (Table I). Reduction
of curcumin to THC, HHC, and OHC is accompanied by a
loss of double bounds of the 1,6-heptadiene structure and a
loss of inhibitory effect on OKT3-induced PBMC prolifera-
tion but by an increase of radical scavenging activity. After
reduction of the 3,5-dione structure (THC, HHC, OHC;
Fig. 1) a little decrease of the inhibitory effect on cell prolif-
eration but no significant change of the antioxidant activity
was observed (Table I). The position of the 4-hydroxy-3-
methoxyphenyl group (Fig. 1) is not important for the
inhibitory effect but changes in this position (isocurcumin)
resulted in a distinct loss of curcumin radical scavenging
activity (Table I). The loss of the 3-methoxy group (bisdes-
methoxycurcumin) only slightly decreases the immunosup-
pressive effect of curcumin but significantly reduces its
antioxidant activity (Table I). Acetylation of the 4-hydroxy
group increases the immunosuppressive activity of curcumin
but significantly diminishes its antioxidant activity (Table I).
Taken together, the correlation of the structural relation-
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pressive effect on the one hand and for the antioxidant
6
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radical scavenging activity of the curcuminoids. The role of
other mechanisms responsible for the immunosuppressive
effect of the curcuminoids like inhibition of CD28 costimula-
tory pathway (8) or of the transcription factors JNK and
NFκB, as described in literature (24,25) has to be examined in
further experiments.
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CONCLUSIONS
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loid sinomenine in combination with the immunosuppressive drugs
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Curcuminoid-induced inhibition of OKT3-induced
PBMC proliferation depends on the number of carbon atoms
and double bonds of the 1,6-heptadiene-3,5-dione structure as
well as on the phenolic ring substitutes of the curcuminoids
but is not correlated to their scavenging activity.
1
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ACKNOWLEDGEMENTS
18. J. N. Commandeur, and N. P. Vermeulen. Cytotoxicity and
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We thank Annette Garbe for excellent technical
assistance. This study was supported by the Deutsche
Forschungsgemeinschaft grant SFB265 A7.
1
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0. K. I. Priyadarsini, D. K. Maity, G. H. Naik, M. S. Kumar, M. K.
Unnikrishnan, J. G. Satav, and H. Mohan. Role of phenolic O–H
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