Antioxidant activity and electrochemical elucidation of the enigmatic redox behavior of curcumin and its structurally modified analogues

Article abstract of DOI:10.1016/j.electacta.2014.11.026

Here, we report studies on the antioxidant activity and redox behavior of curcumin and its structurally modified synthetic analogues. We have synthesized a number of analogues of curcumin which abrogate its keto-enol tautomerism or substitute the methylene group at the centre of its heptadione moiety implicated in the hydride transfer and studied their redox property. From cyclic voltammetric studies, it is demonstrated that H- atom transfer from CH₂ group at the center of the heptadione link also plays an important role in the antioxidant properties of curcumin along with that of its phenolic -OH group. In addition, we also show that the conversion of 1, 3- dicarbonyl moiety of curcumin to an isosteric heterocycle as in pyrazole curcumin, which decreases its rotational freedom, leads to an improvement of its redox properties as well as its antioxidant activity.

Full text of DOI:10.1016/j.electacta.2014.11.026

Accepted Manuscript
Title: Antioxidant activity and electrochemical elucidation of
the enigmatic redox behavior of curcumin and its structurally
modified analogues
Author: Niki S. Jha Satyendra Mishra Shailendra K. Jha
Avadhesha Surolia
PII:
S0013-4686(14)02221-X
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.electacta.2014.11.026
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7-8-2014
5-11-2014
5-11-2014
Please cite this article as: Niki S.Jha, Satyendra Mishra, Shailendra K.Jha, Avadhesha
Surolia, Antioxidant activity and electrochemical elucidation of the enigmatic redox
behavior of curcumin and its structurally modified analogues, Electrochimica Acta
http://dx.doi.org/10.1016/j.electacta.2014.11.026
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Antioxidant activity and electrochemical elucidation of the
enigmatic redox behavior of curcumin and its structurally modified
analogues
Niki S. Jhaa,1, Satyendra Mishraa,1, Shailendra K. Jhab and Avadhesha Suroliaa,*
aMolecular Biophysics Unit, Indian Institute of Science, Bangalore 560012, India
bCSIR - Central Electrochemical Research Institute, Karaikudi, Tamil Nadu, India
*Corresponding author. Tel.: +91-80-23602763; fax: 91-80-23600535, 23600683
E. mail: surolia@mbu.iisc.ernet.in
1 Equal Contribution
Graphical abstract
Highlights
 Structural analogues of curcumin have been synthesized.
 Confirmation of redox behaviour emanates from H- shift from central methylene group in
curcumin.
 Mechanism of curcumin oxidation has been proposed.
 Correlation between redox behavior and antioxidant activity has been established.
Abstract
Here, we report studies on the antioxidant activity and redox behavior of curcumin and its
structurally modified synthetic analogues. We have synthesized a number of analogues of
curcumin which abrogate its keto-enol tautomerism or substitute the methylene group at the
centre of its heptadione moiety implicated in the hydride transfer and studied their redox
property. From cyclic voltammetric studies, it is demonstrated that H- atom transfer from CH2

group at the center of the heptadione link also plays an important role in the antioxidant
properties of curcumin along with that of its phenolic –OH group. In addition, we also show that
the conversion of 1, 3- dicarbonyl moiety of curcumin to an isosteric heterocycle as in pyrazole
curcumin, which decreases its rotational freedom, leads to an improvement of its redox
properties as well as its antioxidant activity.
Keywords: Curcumin; Antioxidant; Free radical; Cyclic voltammetry; Alkoxyl radical;
Hepatadione
1. Introduction
Curcumin, a yellow colored phenolic compound isolated from the roots and rhizomes of
Curcuma longa has been used since centuries as a spice, dietary pigment and traditional
medicine in India and China [1-3]. It displays a wide spectrum of medicinal properties ranging
from anti – bacterial, anti-viral, anti-protozoal, anti-fungal and anti – inflammatory to anti-cancer
activity [4-7]. The ability of curcumin to neutralize chemical carcinogens such as superoxide,
peroxyl, hydroxyl radical and nitric oxide radical constitutes a major subject of interest. While
mode of its action is still under exploration, it is proposed to act through a number of targets and
metabolic pathways involving transcription, cell growth, apoptosis etc [6, 7]. Despite its
invocation of a number of biologic targets it is practically non-cytotoxic to normal human cells
but cytotoxic for cancer cells. It inhibits oncogenic cell proliferation by arresting cell cycle
progression and induction of apoptosis [6, 7]. However curcumin's low solubility, high rate of
metabolism and decomposition, poor bioavailability and pharmacokinetics are responsible for its
limited efficacy [8-10].
Chemically curcumin is (1E, 6E)-1,7-Bis(4-hydroxy-3-methoxyphenyl) hepta-1,6-dienne-3,5-
dione.
Curcumin

Curcumin is a multifunctional molecule and it has three functional groups that can contribute to
its biologic activity, namely an aromatic o-methoxy phenolic group, α, β-unsaturated β-diketo
moiety and a seven carbon linker. Its redox behavior is expectedly more complex and has led to
conflicting views on the contribution of its key constituents [11-19].
The redox behavior of curcumin has led to proposal of two divergent mechanisms. Jovonoic et
al, have proposed that its antioxidant mechanism involves H-atom abstraction from CH2 group
of the heptadione link and the H- atom donation from the phenol group makes merely 15%
contribution towards it [13,14]. On the other hand, Barclay et al’s proposal that it’s a chain
breaking antioxidant, donating H-atom from its phenolic group which discounts a role of CH2
group of the heptadione link in its redox properties has received a much wider acceptance [15].
Litwinienko and Ingold later observed that curcumin donates hydrogen atom from the phenolic
or enolic hydroxyl group via sequential proton loss electron transfer (SPLET) or hydrogen atom
transfer (HAT) and hence, reconciled the earlier differing opinions [18,19]. Currently accepted
mechanism of the antioxidant action of curcumin lays much of its emphasis on the chain
breaking ability of its methoxyphenol group (s) that donates one H-atom and discounts a
significant role of its methylene group in the process.
Here we have explored voltammetric analysis to understand the redox behavior and its
correlation to phenolic O-H and methylene hydrogen attached to β-diketone of curcumin and its
various synthesized analogues. These studies have investigated for the first time, systematically
the role of the methylene radical at the center of curcumin’s has crucial involvement in its redox
behavior as its Knoevenagel condensate (viz. 4-(4-Hydroxy-3-methoxybenzylidene)) which lacks
the ability of hydride transfer exhibits redox potential at a significantly higher potential (viz.
decreased antioxidant ability) despite possessing an additional ortho-methoxy phenyl group. This
in turn implies a significant role for the heptadione methylene group in manifesting the redox
potential of its ortho-methoxy phenol group at a lower potential and higher intensity. That the
redox behavior of curcumin emanates from an H-shift from its central methylene group was
confirmed further by the diminution in the antioxidant abilities of butylidene and benzylidene
derivatives of this methylene moiety.

2. Experimental
2.1. Chemicals
3-(3,4-Dihydroxyphenyl) prop-2-enoic acid (Caffeic acid) and (3-(3-hydroxy-4-methoxyphenyl)
prop–2-enoic acid (ferulic acid), vanillin, isobutyraldehyde, benzaldehyde, 4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid (HEPES) buffer and others reagent were purchased from Sigma-
Aldrich. All solvents used were of spectral grade or distilled prior to use.
2.2. General Procedures Used for the Synthesis and Characterization of Curcumin
Analogues
Reaction progress was monitored by TLC using Merck silica gel 60 F254 with detection by UV.
Column chromatography was performed using Merck silica gel 230–400 mesh. Melting points
1
were determined in Pyrex capillary tube using Büchi Melting Point B-540 apparatus. H NMR
spectra was recorded on 300 and 400 MHz Bruker NMR spectrometers using tetramethylsilane
as an internal standard and the chemical shifts are reported in (δ) units. Coupling constants are
reported as a J value in Hertz (Hz).The sample concentration in each case was approximately
10 mg in chloroform-d/methanol-d4/DMSO-d6 (0.6 mL). Mass spectra were recorded on an
Electrospray–MS (Bruker Daltonis) instrument.
2.3. General Procedure for the Preparation of Curcumin Pyrazole, N-(-Substituted) Phenyl
Pyrazoles and Knoevengel Condensates of Curcumin
Curcumin pyrazole, and N-(substituted) phenyl pyrazoles derivatives of curcumin were prepared
according to previously reported procedure by us and others with some modifications (Scheme 1
& 2) [1, 20-21]. Curcumin (1 mmol) was dissolved in glacial acetic acid (5 mL), hydrazine
hydrate and the various phenyl substituted hydrazine hydrochlorides (1.2 mmol) were added to
the solution. The solution was refluxed for 8 -24 hr, and then the solvent was removed in
vacuum. Residue was dissolved in ethyl acetate and washed with water. Organic portion was
collected, dried over sodium sulfate, and concentrated in vacuum. Crude product was purified by
column chromatography.
Knoevenagel condensates of curcumin was prepared by treatment of curcumin with aromatic
aldehyde (4-hydroxy-3-methoxybenzaldehyde; vanillin) in presence of piperidine (as catalyst)
and anhydrous DMF (as solvent) to yield 4-(4-Hydroxy-3-methoxybenzylidene) curcumin.
Likewise, butylidiene curcumin and benzylidiene curcumin were prepared in good yields using

the above procedure wherein isobutyraldehyde and benzyldehyde were used instead of (4-
hydroxyl -3-methoxy benzaldehyde) (Scheme 3) [1, 22-25]. Reaction mixture was diluted with
ethyl acetate and washed with water and saturated brine. The organic phase was dried over
Na2SO4 concentrated under vacuum, and purified by chromatography with EtOAC/hexanes
mixtures to provide pure compound.
2.4. Electrochemical Studies
Voltammetric experiments were performed with a potentionstat PGSTAT 30, Autolab (ECO
CHEMIE Ltd., The Netherlands) driven with GPES software (Eco Chemie). A conventional
three- electrode cell consisting of a gold electrode, a platinum wire as counter electrode and
Ag/AgCl (saturated KCl) as a reference electrode were used. For characterization of gold
electrode, we have used Ag/AgCl (saturated KCl) as a reference electrode. The gold (Au)
electrode was carefully polished with 1.0, 0.3 and 0.05 μm Al2O3 slurry, and cleaned by brief
ultrasonication. Cyclic voltammetry experiment was performed in 10 mM HEPES buffer, where
Ag/AgCl has been used as a reference electrode. For electrochemical studies, a stock solution of
2 x 10 -3 M curcumin was prepared in DMSO just before experiment and then these solutions
were diluted with buffer to the convenient concentration after mixing with 10 mM HEPES as
buffer supporting electrolyte. The 10 mM HEPES buffer solution has been made in 0.1 M KCl
solution. The UV-vis absorption spectra of curcumin and its various analogues were collected
after 15-20 min subsequent to its solubilization in buffer to estimate degradation of curcumin.
Slight degradation of curcumin was noted at 15-20 min subsequent to dissolution. Pyrazole
curcumin and its analog were stable at physiological pH and no significant degradation was
observed even after 30 min subsequent to dissolution. Our observation is similar to finding of
Griesser el.al. [26] and Soumyananda et.al [27]. The data were analyzed using origin 8 software
for obtaining the value of current (I) and oxidation potential (V).
2.5. Evaluation of Antioxidant Activity
The antioxidant activities of curcumin analogues were determined in terms of radical scavenging
activity using stable radical DPPH. Stock solution of each compound was diluted to variable
concentration from 25-150 µM of each compound. 60 µM of freshly prepared methanolic
solution DPPH solution (1 mM) was added to each sample solution to make final volume 1ml.

The mixture was shaken vigorously and allowed to stand at room temperature for 30 min. In the
radical form, DPPH∙ shows a maximum absorbance at 517 nm, but on reduction by an
antioxidant, the absorption disappears and the pale yellow non radical form is produced. The
lower absorbance of the reaction mixture indicates higher free radical scavenging activity.
Methanol was used as a blank. The antioxidant effect was expressed in terms of the reduction
percentage of the DPPH absorbance, refereed as scavenging effect was calculated using
following equation [28]
A0  AT
Scavenging Effect =
100
A0
Where A0 is the absorbance of the control reaction and AT is the absorbance in the presence of
the sample.
The assay was carried out using curcumin and its various analogues as antioxidant and in
triplicate. The absorbance was measured in spectrophometer UV/vis from JASCO V-530
spectrophotometer.
3. Results and Discussion
3.1. Synthesis and Characterization of Curcumin Analogues
Curcumin pyrazole, and N-(substituted) phenyl pyrazole derivatives of curcumin were prepared
according to previously reported procedure by us and others [1, 20-21] with some modifications
(Schemes 1& 2). Curcumin (1 mmol) was dissolved in glacial acetic acid (5 mL), hydrazine
hydrate and the various phenyl substituted hydrazine hydrochlorides (1.2 mmol) were then added
it in solution. The solution was refluxed for 8 -24 hr, and then the solvent was removed in
vacuum. Residue was dissolved in ethyl acetate and washed with water. Organic portion was
collected, dried over sodium sulfate, and concentrated in vacuum. Crude product was purified by
column chromatography. The 1H NMR spectra showed singlet at ꢀ 6.52–6.62 ppm
corresponding to CH=C proton (pyrazole); multiplet at ꢀ 6.81–7.83 ppm corresponding to
aromatic protons; broad singlet at ꢀ 9.23–9.80 ppm corresponding to OH and disappearance of
peak of the methylene proton (ꢀ 6.02 ppm) confirm the formation of curcumin pyrazole (2) and
derivatives of curcumin pyrazole (3-8). Mass spectral studies of the compound 2 (ESI-MS)
exhibited molecular ion at 365.0 (M+H)+ corresponding to curcumin pyarazole. The derivatives

of curcumin pyrazole (3-8) were also further characterized by ESI-MS. [see Supporting
Information MS spectral data Fig.S5-Fig.S16]. Knoevenagel condensates of curcumin was
prepared by treatment of curcumin with aromatic aldehyde (4-hydroxy-3-methoxybenzaldehyde;
vanillin) in presence of piperidine (as catalyst) and anhydrous DMF (as solvent) to yield 4-(4-
Hydroxy-3-methoxybenzylidene) curcumin, likewise, butylidiene curcumin and benzylidiene
curcumin were prepared in good yields using the above procedure wherein isobutyraldehyde and
benzyldehyde were used instead of (4- hydroxyl -3-methoxy benzaldehyde) (Scheme 3) [1, 25-
28] [see Supporting Information Experimental]. The formation of compounds 9-10 via
Knoevengel’s condensation was validated by the disappearance of aldehydic proton (ꢀ 9.80-
10.10 ppm) and appearance of singlet proton at 8.0 ppm and 7.85 ppm corresponds to
benzylidene (=CH-Ar) ; indicates the structure of desired compounds 9 and 10. Similiarly
appearance of alkylidene proton (=CH-CH2) at ꢀ 7.83 showed the formation of compound 11.
Furthermore all the Knoevengel condensates of curcumin were characterized by ESI-MS also
[Supporting Information MS spectral data Fig.S17-Fig.S22]. The structure of dimethyl curcumin
(compound 12) was confirmed by the presence of 12 protons peak at ꢀ 3.74 ppm (singlet; 4 ×
OCH3) in 1H NMR spectrum and ESI-MS demonstrated molecular ion at 419.9 (M+Na)+ validate
compound 12 [Supporting Information MS spectral data Fig.S23-Fig.S24]. Chemical structures
of curcumin and pyrazole derivative of curcumin are shown in Table 1.
3.2. Cyclic Voltammetry of Curcumin
Curcumin and its analogues belong to the class of polyphenols, their antioxidant activities are of
great interest because of their widespread use as nutraceuticals, therapeutics and as components
of industrial processes. How are the antioxidant activities of curcumin and its analogues
manifested about diverse free radical forms? It is widely believed that much of the redox
behavior of curcumin is a consequence of its two o-methoxy phenolic groups. In comparison the
contribution of the methylene group located at the centre of its heptadione link has received little
attention and its substituent have not been probed in the context of the overall anti-oxidant
activity of curcumin. We report cyclic voltammetric studies of curcumin and its analogues to
understand its redox behaviour comprehensively.
Fig. 1a shows representative cyclic voltammogram of a bare gold electrode in 0.5 M H2SO4. The
formation of gold oxides on electrode surface is clearly reflected in the potential region of 1.1 V
to 1.4 V. But when the potential scan is reversed then there is a sharp cathodic peak observed in

the range of 0.8 V to 1.0 V. vs. Ag/AgCl (saturated KCl), representing the reduction of gold
oxide on the electrode surface. The peak current of gold oxide reduction is 55 µA. This is
consistent with the formation of gold oxide and reduction of gold oxides on electrode surface
which are the characteristic features of a well cleaned polycrystalline bare gold electrode [29].
The cyclic voltammetry of bare gold in 10 mM HEPES buffer solution, pH 7.4 at 250C at a scan
rate of 20 mV s-1 shows an anodic peak at 0.23 V (Fig 1b) indicating that HEPES molecules are
getting oxidized at the gold surface consistent with the property of this salt as a reductant
[30,31]. Despite adsorption of HEPES on the gold surface observation of the redox peaks of gold
demonstrates that the oxidation and reduction of gold has occurred in a reversible manner.
To understand the redox behavior of curcumin, we have performed a cyclic voltammetric (CV)
study on a polycrystalline gold electrode in the absence and presence of various concentration of
curcumin. It has been elucidated in Fig 2a. CV of curcumin at pH 7.4 showed one anodic peak at
0.66 V. There is an increasing trend of anodic peak intensity upto 8 µM concentration of
curcumin whereas 10 µM onwards it does not follow linearity (Fig 2b). So, for further
electrochemical understanding, we have chosen 8 µM concentration of curcumin and its
modified analogues for these studies. Fig 2c illustrates the effect of scan rate on 8 µM curcumin
in 10 mM HEPES buffer (pH 7.4, 25 C) at gold electrode. The oxidation current of curcumin on
the Au electrode increased linearly with the square root of the scan rate (Fig. 2d), in the scan
range of 20 to 100 mV s-1, which indicates the existence of a mass transfer controlled process.
Since it does not emanate from the origin, it indicates curcumin electrooxidation is controlled by
diffusion which can be preceded by a chemical reaction. The value of electron transfer
coefficient for the reaction has been obtained from the following equation [32] which is valid for
a totally irreversible-diffusion controlled process.
Using the above equation a plot of anodic peak potential vs. ln (ν) yields a value of electron
transfer coefficient (α) of 0.27 ± 0.01. These electrochemical studies are consistent with the
involvement of two electrons per molecule of curcumin for its oxidation. The first one electron
oxidation produces the phenoxy radical and the second correspond to the formation of quinone

[33]. Thus during the oxidation of curcumin electron transfer occurs in concert with proton
transfer.
The diketo moiety in the heptadione link is known for its propensity for keto–enol tautomerism
in a pH dependent manner. Hence, we have examined the redox behavior of 8 µM curcumin in
10 mM HEPES buffer at different pH values by cyclic voltammetry [Fig 2e]. The oxidation
potential of curcumin is nearly the same (0.64 V-0.66 V) at various pH values examined while
the oxidation peak intensity increases with increase in pH. The reduction peak in the potential
range of 0.35 V to 0.55 V observed at acidic pH declines progressively as the pH is increased.
However, a shoulder adjacent to 0.66 V peak or an additional peak in the higher potential region
(̴ 1.0 V) ascribed to that of the CH2 group emanating from the centre of heptadione link is not
observed [16]. This in turn suggests that the oxidation peak for this methylene group and the o-
methoxy phenyl group overlap with each other in native curcumin. Hence, the result indicates
that at pH > 7 deprotonation of hydroxyl moiety generating a quinone facilitates electron transfer
boosting the antioxidant activity of curcumin at or above the physiological pH.
3.3. Redox Behavior of Diketone Anlogues of Curcumin
In the redox reaction of curcumin the alkoxy radical generated initially undergoes a rapid
intramolecular H- shift to generate a phenoxy radical. Introduction of pyrazole group at the
position of diketone moiety decreases the rotational freedom of the linker facilitating the
intramolecular radical shift from alkoxyl radical at the methylene group at the centre of the
heptadione link. This in turn leads to a substantial increase (60%) in intensity of redox potential
with a slightly decreased potential value, 0.62 V for pyrazole curcumin. This is in good
agreement with our previously reported results on the anti-oxidant properties of curcumin
pyrazole [23]. A substituent in the aromatic ring attached to pyrazole is expected to influence the
stability of the phenoxyl radical which in turn should perturb the redox behavior of the resulting
compound. Hence, a few substituted pyrazole containing derivatives of curcumin were
synthesized (Table 1, Ref 34) to understand the effect of introduction of a methoxy, halogen and
a nitro group in the aromatic ring attached to pyrazole in the formation of the phenoxy radical as
deduced from their electrochemical responses (Fig 3a – 3c and supporting information Fig S1).
Phenyl pyrazole curcumin has an oxidation potential close to that of pyrazole curcumin with
slight decrease in the intensity with respect to pyrazole curcumin [Table 1, Fig S1a]. A methoxy,

a moderate activating group substituted at meta position leads to a slight increase in the oxidation
potential (0.66 V) as compared to that of pyrazole curcumin [Fig S1b]. The presence of methoxy
at meta position of the phenyl ring decreases the resonance of the phenyl ring irrespective of its
inductive and ortho/para effect increasing the oxidation potential as observed experimentally.
Since halogens are weakly deactivating group, they perturb the electrochemical behaviour of
pyrazole analogues of curcumin as evident from Fig 3a. Thus, a decrease in the oxidation
potential of N-(3-fluorophenyl pyrazole) curcumin as compared to its pyrazole counterpart is
attributed to the increase in the electron withdrawing nature of the fluorine atom at meta position.
Since fluorine in ortho position of N-(2-Fluorophenyl pyrazole) can contribute to oxidation
potential through both inductive and resonance effect, it marginally increases the oxidation
potential with decrease in its intensity (supporting information Fig S1c). Fig. 3b clearly indicates
that the introduction of a nitro group at the same position decreases the intensity of the oxidation
potential as compared to its pyrazole counterpart and it is due to its meta directing nature.
Whereas the –CF3 group being a strong electron withdrawing group which increases the bond
dissociation energy of the phenoxy O-H group, as a result it gives rise to a shift of its redox
potential by +0.05 V with a 20% decrease in intensity for N-(3-Trifluoromethylphenyl)
curcumin as compared to pyrazole curcumin (Fig. 3c).
3.4. Substitutions at the Methylene Group at the Centre of the Heptadione Link to Probe
Its Role in the Redox Behaviour of Curcumin
There are several different interpretations on the mechanism of action of curcumin as an
antioxidant, for example; it has been proposed that the hydrogen abstraction from its central
methylene group is the major contributor of its redox properties [14, 15, 35]. But the theoretical
study of Sun et al [35] concluded that the antioxidant action of curcumin involves H-abstraction
from its phenolic groups, and not from its central CH2 group in agreement with the conclusion of
Barclay et al [15]. Later, Ingold et al. have proposed that in ionizing solvent curcumin undergoes
sequential proton loss electron transfer process [18].
Thus despite a number of studies on the redox properties of curcumin and its derivatives in the
past a systematic approach to delineate a role of the methylene moiety at the centre of its
heptadione link has been lacking. We have addressed this issue by substituing it by butyldehyde
or benzyldehyde or 3- methoxy–phenyl aldehyde and studied their redox behavior by cyclic

voltammetry. Fig. 4a shows a cyclic voltammogram of 8 µM of 4-(4-hydroxy-3-methoxy
benzylidene) curcumin in HEPES buffer (pH 7.0; 25 °C) and scan rate 20 mVs-1. It exhibits the
oxidation potential at +0.80 V with low current intensity of 5.6 µA. This value can be assigned to
the oxidation potential of the phenoxyl radical alone as probability of oxidation of methylene
radical in it has been completely eliminated. From the data, it is apparent that the values of
oxidation potential have become closer to those of phenol. It is evident from these results that the
probability of intramolecular H- atom transfer through β-alkoxyl radical is completely prohibited
in 4- (4-hydroxy-3-methoxybenzylidene) curcumin, which leads to increase in the oxidation
potential with decrease in anodic peak intensity. Hence, 4-(4-hydroxy-3-methoxybenzylidene)
curcumin has become poorer as an antioxidant as compared to curcumin. In other words, the
inability to form the β-alkoxyl radical has hindered the efficacy of the formation of phenoxy
radical. Further to rule out the contribution of the additional phenoxy group present in 4-
methoxy phenyl benzylidene curcumin, we have synthesized 4-benzylidene curcumin and 4-
butylidene curcumin which contain no additional –OH group at the central methylene. The
oxidation potential for 4-benzylidene curcumin and 4-butylidene curcumin, respectively, are 0.84
V and 0.85 V (Fig 4b) which are again much higher than that observed for curcumin. Because of
the substitution of the methylene group at the centre of curcumin these compound show a
oxidation potential that is close to the oxidation potential values observed for a typical phenoxy
radical (E6 = 0.87 V) [15].
To understand the role of the phenolic –OH of curcumin, we have also studied the redox
properties of half curcumin. 3-(3,4-dihydroxyphenyl) prop–2-enoic acids and 3-(3-hydroxy-4-
methoxyphenyl) prop–2-enoic acid (Dehydrozingerone) which show the value of oxidation
potential of +0.65 V and +0.79 V respectively ( Fig 4c and 4d). The observation of the anodic
peak at lower oxidation potential for 3-(3,4-dihydroxyphenyl) prop – 2-enoic acid i.e., at +0.65 V
compared to that for 3-(3-hydroxy-4-methoxyphenyl) prop–2-enoic acid, 0.79 V, can be ascribed
to ortho–hydroxyl phenoxy radical, which are more stable due to intramolecular hydrogen
bonding interaction [36] which in turn will facilitate the further oxidization ortho-hydroxy
phenoxy radical to form ortho-quinone.
In order to understand the contribution of the phenolic –OH in antioxidant activity in the
curcumin, we have also studied oxidation potential of dimethyl curcumin where both the

phenolic –OH groups have been blocked. The dimethyl curcumin shows peak at 0.87 V with a
peak intensity 4.4 µA (supporting information Fig S2). Thus the peak intensity in case of
dimethyl derivative is much less as compared to curcumin and exhibits a higher potential for its
oxidation. Since both the hydroxyl groups are blocked in dimethoxy curcumin, oxidation
potential observed at +0.87 V can be assigned to CH2 group at the centre of the hepatadione link.
Taken together our studies demonstrate that the oxidation potential of native curcumin seen at
+0.66 V is as a result of overlapping contributions from the central methylene group and o-
methoxy phenyl group. It is, therefore, apparent that the lack of an intramolecular H- shift from
the alkoxyl radical generated at the centre of dimethoxy curcumin to its termini shifts the
oxidation potential of the methylene group to a higher potential i.e from a value +0.66 V for
curcumin to +0.87 V in compound 12. It has been suggested earlier that ortho-methoxy phenol
group can form intramolecular hydrogen bond with phenolic hydrogen, making H- abstraction
from ortho-methoxy phenols easier [37,38].
Mechanism 1: Proposed mechanism of curcumin oxidation
Together, these data imply that the intramolecular H- atom transfer from β-alkoxyl radical plays
a vital role in the antioxidant function of curcumin as shown in mechanism 1. It supports
Jovanoic et al’s conclusions that the alkoxyl radical generated at the methylene group at the
centre of the heptadione link in curcumin undergoes an intramolecular H-shift towards the

phenoxy radical at its termini. Therefore, the presence of the –CH2 group at the centre of
curcumin and phenoxy hydroxyl groups at both of its termini are intrinsic and essential for the
antioxidant function of curcumin.
3.5. DPPH Assay for Antioxidant Activity
To further confirm the anti-oxidant properties of substituted curcumin used for deducing its anti-
oxidant mechanism, we have studied their free radical scavenging ability using DPPH radical
which reacts with an electron or hydrogen donor to become a stable diamagnetic molecule viz
hydrazine. The solution therefore loses colour depending on the number of electrons accepted.
Substances capable of donating electrons /hydrogen atoms are able to convert the purple colour
of DPPH to its non-radical yellow form; 1,1-diphenyl-2-picrylhydrazine, a reaction which can
be followed spectrophotometrically. Curcumin exhibits better free radical scavenging than the 4-
(4-hydroxy-3-methoxy benzylidene) curcumin (Fig 5). Structure- activity relationship showed
that the decreased antioxidant active of 4-(4-hydroxy-3-methoxy benzylidene) curcumin could be
attributed to absence of methylene hydrogen at the center of dicarbonyl moiety. In 4-benzylidene
curcumin, the delocalization is larger with a higher degree of conjugation. Apparently, the small
difference in delocalization has larger influence on antioxidant activity, as 4- benzylidene
curcumin shows a higher antioxidant activity than less conjugated counterpart, 4-butylidene
curcumin. These interpretations are strengthened further by the reduced free radical scavenging
activity of 4-benzylidene and 4- butylidene curcumin as compared to the parent compound.
4. Conclusions
The oxidation of the curcumin involves H- atom transfer from β-alkoxyl radical generated at the
centre of its heptadione link which undergoes molecular rearrangement to form the phenoxy
radical. Thus the central methylene group of the heptadione link is also playing a role along with
the hydroxyl group in the antioxidant activity of curcumin. A lower oxidation potential of
curcumin than that observed for a typical phenoxy radical provide a rationale for explaining
widely appreciated antioxidant properties of curcumin as compared to phenols. The pyrazole
curcumin shows better antioxidant properties (viz lower oxidation potential) than curcumin due
to the absence of keto–enol tautomerism and oxidation in the flexibility of its heptadione link. 4-
(4-Hydroxy-3-methoxybenzylidene) curcumin and other analogues of the central methylene
group eg. 4- butylidene and 4- benzylidene curcumin, fail to generate the β-alkoxy radical. Such

analogues of curcumin are therefore, poor as antioxidants. In other words, failure to generate β-
alkoxyl radical in them compromises an efficient formation of phenoxy radical. Also, an
oxidation potential that of alkyl peroxy radical (E7 = 1.06V), and the superoxide radical (E7 >
1.06 V) [39] makes curcumin an important nutritional antioxidant.
Acknowledgements
AS is a Bhatanagar fellow of the Council of Scientific and Industrial Research (CSIR), India
HRD/Bhatnagar/2011. This work is supported by a grant from CSIR to AS. SM is a Research
Associate of CSIR. NSJ gratefully acknowledge the financial support by Department of Science
and Technology (DST), India SR/WOS-A/CS-95/2012 as DST Woman Scientist. NSJ is also
thankful to Dr. Sheela Berchmans, CSIR-CECRI, Karaikudi for instrument access and support.
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Scheme 1 Synthesis of Pyrazole derivative of curcumin
Scheme 2 Synthesis of N-(substituted) phenylcurcumin pyrazole analogues
Scheme
3
Synthesis of Knoevenagel condensate of curcumin (4-(4-Hydroxy-3-
methoxybenzylidene) curcumin (10), 4-Benzylidene curcumin (11) and 4-Butylidene curcumin
(12).
Figure caption
Fig. 1 Characteristic cyclic voltammogram of a cleaned polycrystalline gold electrode in (a) 0.5
M H2SO4. Scan rate is 100 mV s-1 (b) 10 mM HEPES buffer. Scan rate is 20 mV s-1
Fig. 2 Cyclic voltammograms of (a) various concentrations of curcumin in 10 mM HEPES buffer
(pH 7.4, 25 °C). Scan rate 20 mV s-1 (b) plot of anodic peak current versus concentration of
curcumin, (c) various scan rate from 20 to 100 mV s-1 of 8 µM curcumin at pH 7.4, 25 °C (d)
plot of anodic peak current versus ln (ν) and plot of oxidation current versus the square root of
the scan rate as inset (2d), and (e) 8 µM curcumin at various pH value. Scan rate is 20 mV s-1.
Fig. 3 Cyclic voltammograms of pyrazole curcumin (-) with (a) 3-fluorophenyl pyrazole
curcumin(-), (b) 3-nitrophenylpyrazole curcumin(-) and (c) 3- trifluoromethyl phenyl pyrazole
curcumin(-) , concentration 8µM, at a scan rate of 20 mV s-1
Fig. 4 Cyclic voltammogram for 8 µM of (a) 4-(4-hydroxy-3-methoxy benzylidene) curcumin (-)
(b) 4-benzylidene curcumin (-) and 4- butyldiene curcumin (-), (c) 3-(3,4-dihydroxyphenyl)
prop–2-enoic acid(-) and (d) 3-(3-hydroxy-4-methoxyphenyl) prop–2-enoic acid (-), pH 7.4 at
250 C. Scan rate is 20 mV s-1

▼), 2
Fig. 5 Free radical scavenging activity of curcumin (
-methoxy 3-hydroxy benzyldiene
curcumin (▲), 4-benzyldiene curcumin (●) and 4-butylidene curcumin (■) measured by DPPH.
O
O
N
NH
H3CO
HO
OCH3
OH
H3CO
HO
OCH3
OH
NH2-NH2
2
Acetic acid, reflux, 6-8h
Scheme 1
Substituted hydrazine
AcOH, reflux, 8-24h
3-8
R2
Compound
R1
R3
3
4
H
H
H
OCH3
H
H
5
6
7
H
F
H
F
H
NO2
H
H
H
8
H
CF3
H
Scheme 2
10
Vanillin/benzaldehyde/
isobutyledhyde in anhydrous DMF
piperidine (cat), 48 h
11
12
Scheme 3
Fig. 1

Fig. 2

Fig. 3

Fig. 4
Fig. 5

Table 1
The oxidation potential of curcumin and its analogs at 8 µmol dm-3, pH 7.4, 250 C.

Compound
1.
Structures
Chemical Name
Curcumin
Electrochemical Current,
Oxidation
Potential, E0(V)
0.66
I (µA)
10.84
O
O
H3CO
HO
OCH3
OH
H3CO
HO
N
NH
OCH3
OH
2.
3.
Pyrazole curcumin
0.62
16.94
15.81
N-(Phenyl pyrazole) 0.63
curcumin
OCH3
OH
N
N
H3CO
HO
H3CO
4.
5.
N-(3-Methoxyphenyl 0.66
pyrazole) curcumin
16.81
16.11
OCH3
OH
H3CO
HO
N
N
F
N-(3-Fluorophenyl
pyrazole) curcumin
0.59
N
N
H3CO
HO
OCH3
OH
F
6.
7.
N-(2-Fluorophenyl
pyrazole) curcumin
0.64
0.59
15.28
13.59
OCH3
OH
N
N
H3CO
HO
O2N
N-(3-Nitrophenyl
pyrazole) curcumin
N
N
H3CO
HO
OCH3
OH
F
F
F
8.
9.
N-(3-trifluoromethyl 0.64
pyrazole) curcumin
15.21
5.60
N
N
H3CO
HO
OCH3
OH
O
O
H3CO
OCH3
OH
4-(4-Hydroxy-3-
methoxybenzylidene)
curcumin
0.80
HO
OH
OCH3
10.
11.
4-Benzylidene
curcumin
0.84
0.85
2.24
3.32
O
O
H3CO
HO
OCH3
OH
O
O
H3CO
HO
OCH3
OH
4-Butylidene
curcumin
12.
13.
Dimethyl curcumin
0.87
0.65
4.40
O
O
H3CO
H3CO
OCH3
OCH3
O
3-(3,4-dihydroxy
phenyl) prop–2-enoic
acid
12.31
HO
HO
OH
O
14.
3-(3-hydroxy-4-
methoxyphenyl)
prop–2-enoic acid
0.78
10.76
H3CO
HO
OH