Electron Paramagnetic Resonance Study of the Free Radical Scavenging Capacity of Curcumin and Its Demethoxy and Hydrogenated Derivatives

Abstract

The quantitative free radical scavenging capacity of curcumin and its demethoxy derivatives (demethoxycurcumin (Dmc) and bisdemethoxycurcumin (Bdmc)) and hydrogenated derivatives (tetrahydrocurcumin (THC), hexahydrocurcumin (HHC) and octahydrocurcumin (OHC)) towards 1,1-diphenyl-2-picryl hydrazyl (DPPH), nitric oxide radical (NO), hydroxyl radical (HO^·) and superoxide anion radical (O₂^·) were investigated by electron paramagnetic resonance (EPR) spectroscopy. One mole of the hydrogenated derivatives scavenged about 4 mol of the DPPH radical, while curcumin and Dmc scavenged about 3 mol of the DPPH radical. Curcumin and THC showed moderate scavenging activity towards NO, yielding 200 mmol of NO scavenged per 1 mol of the scavenger. In contrast, curcumin and its derivatives showed very low scavenging activity towards HO^· and O₂^·, yielding approximately only 3–12 mmol scavenged per 1 mol of the tested compounds. Our results suggest that curcumin and its derivatives principally act as chain breaking antioxidants rather than as direct free radical scavengers. Furthermore, we showed that the ortho-methoxyphenolic group and the heptadione linkage of these molecules greatly contributed to their DPPH and NO scavenging activity.

Curcumin [1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione] is the primary biologically active constituent that is isolated from the spice, turmeric (Curcuma longa LINN). It shows various therapeutically beneficial activities, including anti-oxidant, anti-inflammatory, anti-cancer, and anti-viral activities^1,2) mediated through its interactions with several molecular targets such as enzymes, receptors, and transcription factors.^3,4) As a potent anti-oxidant, several mechanisms have been proposed which describe the direct interaction of curcumin with reactive oxygen species (ROS) as well as its involvement in ROS-independent mechanisms (i.e., induction of antioxidant enzymes).^5,6)

The structure–antioxidant activity relationship of curcumin and the compound it reduces has been demonstrated in both in vitro and in vivo models, for example, 1,1-diphenyl-2-picryl-hydrazyl (DPPH) kinetic analysis, γ-radiolysis of rat liver microsomes,⁷⁾ 2,2′-azobis(2-amidinopropane)dihydrochloride (AAPH)-induced low density lipoprotein (LDL) oxidation,⁸⁾ AAPH-induced linoleic oxidation.⁹⁾ Lastly, the protective effects of curcumin on oxidative injury have been demonstrated in vivo in several animal diseases models.^10–12)

Free radicals are normally formed by the physiological processes of the cell. For example superoxide anion radical (O₂^−·) is produced during the mitochondrial respiratory chain reaction and the nitric oxide radical (NO) is generated by the activation of nitric oxide synthase. In some circumstances, the overproduction of those radicals, as well as the formation of toxic radicals such as the hydroxyl radical (HO^·) leads to the damage of biological molecules, thus resulting in many disease conditions. Increased free radical generation causes oxidative stress and has been implicated in inflammatory processes. Superoxide and nitric oxide are key culprits in the pathogenesis of cardiovascular diseases as well as in chronic inflammatory diseases,¹³⁾ while the hydroxyl radical is involved in diseases related to abnormal metal metabolism such as iron overload conditions and various neurological disorders.¹⁴⁾ Thus, compounds with radical scavenging activity could reduce the amount of free radical formation, as well as retard the progression of such diseases.

The scavenging activity of curcumin has been analyzed using stable radicals such as DPPH radical and 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid (ABTS) radical.¹⁵⁾ Singh et al.¹⁶⁾ has demonstrated effective scavenging activity of curcumin and its analogues towards superoxide anion radicals, and single oxygen and peroxyl radicals and performed kinetic analyses of the radical reaction. These studies have provided informative data regarding the structure–activity relationship (SAR) of curcumin and its analogues. However, the qualitative scavenging activity of curcumin and its analogues towards other radicals has not yet been demonstrated.

Electron paramagnetic resonance (EPR) spectroscopy is a unique technique for the detection of species with unpaired electrons. EPR is both a quantitative and qualitative technique. Together with spin trapping, various free radicals could be specifically identified from their EPR spectrum. Therefore, EPR has wide application for the analysis of free radical generation in vitro, and also for the measurement of radical scavenging activity of plant extracts in aqueous solution without color interference.¹⁷⁾

In this study, the free radical scavenging activity of curcumin and its demethoxy derivatives (demethoxycurcumin (Dmc) and bisdemethoxycurcumin (Bdmc)) and hydrogenated derivatives (tetrahydrocurcumin (THC), hexahydrocurcumin (HHC) and octahydrocucumin (OHC)) towards DPPH radical, nitric oxide, hydroxyl radical, superoxide anion radical, was investigated. As EPR spectroscopy only detects molecules or atoms with unpaired electrons, the concentrations of free radicals (or spin) could be simply calculated from the area under the peak of the EPR spectrum. Thus, the radical scavenging capacity and potency of curcumin and its derivatives towards different radicals could be measured by a loss of this spin concentration.

MATERIALS AND METHODS

Curcumin and Its Derivatives

Curcumin, Dmc and Bdmc were separated from curcuminoids (Government Pharmaceutical Organization, Bangkok, Thailand) as described previously.⁹⁾ Curcumin was converted to THC by hydrogenation with palladium–carbon as a catalyst.¹⁸⁾ HHC and OHC were synthesized from THC by reduction with sodium borohydride.¹⁹⁾ The identity and purity of all of the products were confirmed using mass spectroscopy (MS) and NMR analysis. MS and NMR analysis of curcumin and its derivatives were consistent with the results of our previous report.⁹⁾ The chemical structures of curcumin and its derivatives are shown in Fig. 1.

Fig. 1. Chemical Structures of Curcumin, and Its Demethoxy and Hydrogenated Derivatives

DPPH Radical Scavenging Assay

The hydrogen donating or radical scavenging ability of curcumin and its derivatives were evaluated using a stable radical, 1,1-diphenyl-2-picryl hydrazyl (DPPH, Sigma, St. Louis, MO, U.S.A.). The reaction mixture was composed of 50 µL of 0.25 mM DPPH solution and 50 µL of the test compound in a total volume of 200 µL of methanolic solution. Trolox (Sigma) was used as a standard antioxidant. Stock solutions of test compounds were prepared in 100% methanol and the final concentrations ranged from 2.5–80 µM. The reaction mixtures were transferred to capillary tubes and fitted into the cavity (ELEXSYS Super High Sensitivity Probehead Cavity, Bruker, U.S.A.) of the EPR spectrometer (E500, Bruker). The EPR spectra were recorded 60 s after adding the test compound into the cavity. EPR measurement conditions were as follows: central field 3505±50 g, modulation frequency 100 kHz, modulation amplitude 2.0 g, microwave power 1.02 mW, gain 65 dB, scan time 20.97 s and time constant 40.96 ms.

Nitric Oxide (NO) Radical Assay

Nitric oxide radical was generated from S-nitroso-N-acethylpenicillamine (SNAP), and an iron–dithiocarbamate complex [(MGD)₂–Fe²⁺] was used as the trapping agent. Spin trap iron–dithiocarbamate complex [(MGD)₂–Fe²⁺] was prepared by reacting 25 mM of MGD (Fluka, Switzerland), with 5 mM of Fe₂SO₄.²⁰⁾

The reaction mixture was mixed in sequence starting with 5 µL of 5 mM SNAP (Sigma), 5 µL of [(MGD)₂–Fe²⁺], and 80 µL of deionized water, followed by addition of 10 µL of test compound to give a total volume of 100 µL. Stock solutions of the test compounds were prepared in 100% methanol and the final concentrations were 50–400 µM. After mixing, the reactions were incubated at 37°C in a water bath for 10 min and the mixture was then transferred to a tube and fitted into the cavity of the EPR spectrometer. Hemoglobin (Sigma) was used as a reference antioxidant. The EPR spectra was recorded at ambient temperature with the following settings: central field 3510±50 g, modulation frequency 100 kHz, modulation amplitude 3 g, microwave power 1.75 mW, gain 70 dB, scan time 41.94 s and time constant 1.28 ms.

Hydroxyl Radical Scavenging Assay

Hydroxyl radical was generated by a Fenton reaction using ferrous sulfate (Fe₂SO₄) and hydrogen peroxide (H₂O₂). 5,5-Dimethyl-L-pyrroline-N-oxide (DMPO, Sigma) was used as the trapping agent. DMPO was purified prior to use by adding activated carbon into the DMPO solution followed by centrifugation at 12000 rpm for 20 min. The process was repeated until a clear solution was obtained.

The reaction mixtures were mixed in sequence, starting with 20 µL of 0.25 mM freshly prepared ferrous sulfate (Fe₂SO₄), 40 µL of deionized water, 10 µL of 1.12 M DMPO, 10 µL of test compound, followed by 20 µL of 0.25 mM H₂O₂ to give a total volume of 100 µL. Final concentrations of the test compounds ranged from 50–200 µM. Ascorbic acid was used as a reference antioxidant. The EPR spectra of the DMPO–OH adduct were recorded 60 s after the addition of 0.25 mM H₂O₂ at ambient temperature. EPR measurement conditions were as follows: central field 3505±50 g, modulation frequency 100 kHz, modulation amplitude 1.25 g, microwave power 10.10 mW, gain 70 dB, scan time 41.49 s and time constant 5.12 ms.

Superoxide Anion Radical Scavenging Assay

Superoxide anion was generated with a hypoxanthine/xanthine oxidase system.²¹⁾ DMPO was used as the trapping agent. The reaction mixture was mixed in sequence starting with addition of 80 µL of 4 mM hypoxanthine, followed by 10 µL of 10 mM diethylenetriamine pentaacetic acid (DTPA, Sigma), 10 µL of 1.12 M DMPO, 10 µL of test compound, and 10 µL of 0.8 unit/mL xanthine oxidase (Sigma). Final concentrations of the tested compounds were 80 to 300 µM. Superoxide dismutase was used as a reference antioxidant. The EPR spectra of the DMPO–OOH adduct were recorded 120 s after the addition of the xanthine oxidase at ambient temperature. EPR settings used were the same as for the hydroxyl radical, with the exception of microwave power 1.75 mW, gain 78 dB and time constant 1.28 ms.

Data Analysis

The area under the peak of the EPR spectra was calculated by double integrations (BRUKER Xepr, Bruker, U.S.A.). The scavenging activity of curcumin and its derivatives was calculated as percentage of inhibition (% inhibition) by comparing the area under the peak of the EPR spectrum of the control reaction in the presence or absence of the test compound. The IC₅₀ value was obtained from the plot of % inhibition vs. the concentration of the antioxidant.

In order to estimate the scavenging capacity or the loss of radical (or spin) per mole of test compound, the total spin concentration of the control and test reactions were calculated using a standard curve constructed from the area under the peak of the EPR spectra of various concentrations of hydroxyl-TEMPO (Sigma-Aldrich, Steinheim, German) recorded under the same EPR settings for each radical. Since DPPH itself is a stable radical, the total spin concentration was calculated directly from the area under the peak of the DPPH spectra.

The slope obtained from a plot of total spin concentration versus the concentration of the test compounds is defined as the “scavenging capacity.” For hydroxyl radical and superoxide anion radical, the scavenging capacity was estimated at maximum test concentration of 200 and 300 µM, respectively.

RESULTS AND DISCUSSION

DPPH Scavenging Activity

DPPH radical is widely used as a radical molecule to evaluate the free radical scavenging ability of various compounds. The DPPH radical is characterized by a strong EPR signal intensity, and it is able to accept an electron or hydrogen atom (proton) to become a stable diamagnetic molecule.²²⁾ The effect of antioxidants on the scavenging of the DPPH radical was thought to be due to the ability of the antioxidants to donate hydrogen to the DPPH. Thus, the loss of the DPPH EPR signal intensity in the presence of antioxidants could be directly proportional with the concentration (or number) of protons accepted.

EPR spectra of DPPH radical in methanol are shown in Fig. 2a. Its EPR signal intensity was stable but it was rapidly reduced after addition of the test compound in concentration dependent manner. In this present study, trolox was used as a reference hydrogen donor molecule, as it is known that trolox has the capacity to donate two hydrogen atoms.²³⁾ Our calculations showed that 1 molecule of trolox donated 1.7 hydrogen atoms to DPPH which was nearly the theoretical number of donating hydrogen atoms (Table 1). Therefore, the hydrogen donating capability of curcumin and its derivatives can be determined by analysis of the EPR spectra of the DPPH radical. Antioxidant activity presented as IC₅₀ and scavenging capacity are shown in Table 1.

Fig. 2. EPR Spectra of DPPH Radical (a), Nitric Oxide Radical (b), DMPO–OH Adduct (c) and DMPO–OOH Adduct (d)

Table 1. The IC₅₀ and Scavenging Capacity Values of DPPH by Curcumin and Its Derivatives

Test compound	DPPH
	IC50 (µM)	Scavenging capacity (mol/mol test compound)
Curcumin	38.4±1.4	3.4
Demethoxycurcumin	20.9±1.6	3.1
Bisdemethoxycurcumin	85.6±6.0	1.7
Tetrahydroxycurcumin	20.7±2.1	4.1
Hexahydroxycurcumin	23.4±1.7	3.7
Octahydroxycurcumin	14.7±1.9	3.8
Trolox	39.3±2.1	1.7

Data are mean±S.D. of three independent experiments.

Through comparison of the IC₅₀ values, curcumin had comparable scavenging activity as the reference antioxidant, trolox. All other derivatives, except Bdmc, showed significantly higher efficacy compared to curcumin and trolox. The relative rank order of the DPPH scavenging potency of curcumin and its derivatives is as follows: OHC>Dmc≥THC ≥HHC>curcumin=trolox≫Bdmc.

Our results demonstrated that THC and the other hydrogenated derivatives (HHC and OHC) had higher capacities than curcumin and the demethoxy derivatives. One molecule of hydrogenated derivatives scavenged about 4 mol of DPPH, while 1 mol of curcumin and Dmc scavenged about 3 mol, and Bmc scavenged 1.7 mol of DPPH. This may be related primarily to the differences in the ability to donate hydrogen atoms from a phenolic vs. enolic group due the differences in their chemical structure.^{7–9,15,16,24,25)} The additional hydroxyl group in OHC and the hydrogen atom from the central methylenic group may also contribute to the hydrogen donating ability of these hydrogenated derivatives.²⁶⁾

In addition, the results confirmed that loss of the ortho-methoxyphenolic group in Bdmc caused a greater loss of hydrogen donating activity, whereas the molecule with an ortho-methoxyphenolic group and β-diketone moiety showed the comparable activity to trolox.

NO Scavenging Activity

NO radical was spontaneously released from a nitric oxide donor, SNAP. NO was trapped with iron–dithiocarbamate to produce the [(MGD)₂–Fe²⁺–NO] adduct which gave a triplet-line EPR signal, as shown in Fig. 2b. Hemoglobin, but not methanol, inhibited the EPR signal, confirming that this spin adduct was produced from NO. Curcumin and its derivatives showed moderate scavenging activity towards NO. The IC₅₀ values and scavenging capacities of curcumin and its derivatives are shown in Table 2. Among the test compounds, curcumin and THC had the most potent NO scavenging activity. OHC showed only limited potency towards NO; therefore, an IC₅₀ value could not be obtained. The relative rank order of NO scavenging potency according to the IC₅₀ values is as follows: curcumin=THC>Dmc>Bdmc=HHC≫OHC.

Table 2. The IC₅₀ and Scavenging Capacity Values of NO by Curcumin and Its Derivatives

Test compound	NO
	IC50 (µM)	Scavenging capacity (×10−3 mol/mol test compound)
Curcumin	100.4±7.6	266
Demethoxycurcumin	164.5±2.4	142
Bisdemethoxycurcumin	227.4±85.8	98
Tetrahydroxycurcumin	104.2±5.7	204
Hexahydroxycurcumin	277.9±13.8	24
Octahydroxycurcumin	Nd	11

Data are mean±S.D. of three independent experiments. Hemoglobin was the reference compound for the NO assay. Nd=Not detectable.

The calculated scavenging capacity indicated that 1 mol of curcumin or THC scavenged about 200 mmol of NO. Demethoxy derivatives, Dmc and Bdmc scavenged about 100 mmol of NO per mole. Hydrogenated derivatives, HHC and OHC, had the lowest scavenging capacity for NO. Our results suggested that the structure of the heptadione linkage is important for NO scavenging activity, as loss of the double bond in this structure markedly reduced the activity as is the case for HHC and OHC.

Overproduction of NO is implicated in various inflammatory-related pathologies including cardiovascular and neurodegenerative diseases. The cytotoxicity of NO is mediated by the formation of peroxynitrite (ONOO⁻) through chemical interaction of NO with superoxide anion radical.²⁷⁾ Therefore, removing NO may reduce and delay the toxic effects of NO. The beneficial properties of curcumin as an anti-inflammatory agent has previously been reviewed.^3,28) It is well known that curcumin inhibits NO production indirectly by inhibition of inducible nitric oxide synthase (iNOS) though suppression of nuclear factor-kappa B (NF-κB).^10,29) Our EPR study suggests that the direct interaction of curcumin and THC with NO may partially contribute to their anti-inflammatory activity. Despite only moderate NO scavengers, this mechanism of direct interaction should be considered in the development or use of curcumin analogues as anti-inflammatory agent. Furthermore, the effect of curcumin on physiologic levels and functions of NO should be clarified.

Hydroxyl Radical Scavenging Activity

Hydroxyl radicals were generated by a Fenton reaction and trapped with DMPO. The typical 1 : 2 : 2 : 1 quarted-line EPR spectra of the DMPO–OH adduct are shown in Fig. 2c. The EPR signal intensity of the DMPO–OH adduct was reduced in the present of methanol. Therefore, the same concentrations of methanol as in the tested reaction with no test compound were used as the respective controls.

Curcumin and its derivatives showed weak hydroxyl radical scavenging activity. The maximum % inhibition of the tested compounds, with the exception of THC, ranged from 10–40%. Therefore, instead of IC₅₀ values, the % inhibition and scavenging capacity at the maximum tested concentration (200 µM) are presented in Table 3 for these compounds. THC was the most potent hydroxyl radical scavenger and its IC₅₀ was approximately 138 µM. The scavening capacity of THC for hydroxyl radicals was approximately 12 mmol per mol THC.

Table 3. The Percentage of Inhibition and Scavenging Capacity Value towards Hydroxyl Radical by Curcumin and Its Derivatives

Test compound	HO·
	% Inhibition	Scavenging capacity (×10−3 mol/mol test compound)
Curcumin	28.6±0.9	4.8
Demethoxycurcumin	35.8±4.9	9.5
Bisdemethoxycurcumin	16.2±2.4	2.7
Tetrahydroxycurcumin	77.4±3.6	12.7
Hexahydroxycurcumin	9.6±0.9	1.6
Octahydroxycurcumin	23.4±2.1	3.9

Data are mean±S.D. of three independent experiments. The % inhibition of hydroxyl radical at the test concentration of 200 µM.

To the best of our knowledge, there have been no reports on the hydroxyl radical scavenging activity of curcumin and its derivatives. The hydroxyl radical scavenging activity of curcumin and its derivatives may be secondary to their properties as iron chelators. Curcumin is able to bind ferrous and ferric ions through its hydroxyl and methoxyl group on the phenolic ring, and through its keto–enol moiety.^15,30) In terms of chelation ability, we noted that the number of double bonds in the heptadione linkage may influence the ligand conformation and binding ability of the molecules. The SAR for these compounds as pertains to the hydroxyl scavenging and iron chelating abilities still needs further investigation.

Superoxide Anion Radical Scavenging Activity

Superoxide anion radicals were generated by enzymatic reaction of hypoxantine and xanthine oxidase. The EPR spectra of the DMPO–OOH adduct are shown in Fig. 2d. The determined maximum inhibition values and superoxide scavenging capacities are shown in Table 4. Curcumin and its derivatives showed very low potency for superoxide anion radical scavenging. Less than 10 mmol of the radical could be scavenged by 1 mol of the test compounds. Although they had very low scavenging potency, curcumin, Dmc and THC were the most effective for superoxide anion radical scavengers out of the test compounds.

Table 4. The Percentage of Inhibition and Scavenging Capacity Value of Superoxide Anion Radicals by Curcumin and Its Derivatives

Test compound	O2−·
	% Inhibition	Scavenging capacity (×10−3 mol/mol test compound)
Curcumin	39.1±10.2	10.3
Demethoxycurcumin	32.2±8.2	7.8
Bisdemethoxycurcumin	16.1±9.6	4.4
Tetrahydroxycurcumin	20.4±4.3	8.9
Hexahydroxycurcumin	22.9±4.5	3.2
Octahydroxycurcumin	21.0±7.6	5.0

Data are mean±S.D. of three independent experiments. The % inhibition of superoxide anion radical at the test concentration of 300 µM.

A previous study on the SAR of superoxide anion scavenging demonstrated that superoxide anion radical reacted preferably at the keto–enol moiety but was not influenced by the presence of a phenolic OH group. The methoxy substitution on phenyl ring did not significantly increase the reaction rate constant with superoxide anion radical.¹⁶⁾ Our findings supported the importance of the keto–enol moiety. However, the decrease scavenging activity of Dmc and Bdmc may imply the effect of the ortho-methoxyphenolic group.

Among the studied free radicals, curcumin and its derivatives were not effective hydroxyl radical or superoxide anion radical scavengers. On the other hand, they are potent hydrogen donors. Therefore, the chain breaking mechanism is the primary contributor to the protective effects of curcumin and its derivatives in biological systems, rather than direct interaction with the initial radical such as the hydroxyl radical.

THC is a reduced derivative of curcumin that is rapidly formed by conversion of curcumin during intestinal absorption in vivo. In this study, THC showed higher or comparable scavenging potency as curcumin towards all of the tested free radicals. Several studies have demonstrated potent antioxidant activities of THC and revealed other biological activities of this compound as well.^9,31) Hydrogenation of the two double bonds conjugated to the β-diketones was suggested to be important for THC antioxidant activity in addition to the phenolic hydroxyl group in the structure.^24,25)

In conclusion, our EPR with spin trapping studies provided qualitative analysis of the free radical scavenging activity of curcumin and its derivatives. Although this method is an end-point analysis, the estimated number of scavenged radicals still provides information regarding the chemical mechanism of this scavenging activity as well as some information on the structure–antioxidant activity of the test compounds. The method could apply for other free radicals using various radical generating system and spin trapping agents.

Acknowledgments

A part of this research work was supported by the Government Pharmaceutical Organization of Thailand, and by Thailand Research Funds and the Commission on Higher Education (RMU 5080058).

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

The online version of this article contains supplementary materials.

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