Journal of Molecular Structure 1004 (2011) 163–173

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Journal of Molecular Structure

journal homepage: www.elsevier .com/ locate /molst ruc

Interaction of curcumin with Al(III) and its complex structures basedon experiments and theoretical calculations

Teng Jiang a, Long Wang a, Sui Zhang a, Ping-Chuan Sun b, Chuan-Fan Ding c, Yan-Qiu Chu c, Ping Zhou a,⇑a The Key Laboratory of Molecular Engineering of Polymers, Ministry of Education, Department of Macromolecular Science, Fudan University, Shanghai 200433, Chinab Key Laboratory of Functional Polymer Materials, Ministry of Education, College of Chemistry, Nankai University, Tianjin 300071, Chinac Physical Chemistry Institute, Department of Chemistry, Fudan University, Shanghai 200433, China

a r t i c l e i n f o

Article history:Received 30 May 2011Received in revised form 28 July 2011Accepted 28 July 2011Available online 12 August 2011

Keywords:CurcuminAluminum ionDensity functional theoryAlzheimer’s diseaseSpectroscopyNMR

0022-2860/$ - see front matter � 2011 Elsevier B.V. Adoi:10.1016/j.molstruc.2011.07.059

Abbreviations: AD, Alzheimer’s disease; Ab, amyloispectroscopy; NMR, nuclear magnetic resonance; DFMALDI-TOF, matrix assisted laser desorption ionizdimethyl sulfoxide; TMS, tetramethylsilane; TD-DFfunctional theory; PCM, polarizable continuum modatomic orbital; HOMO, highest occupied molecuunoccupied molecular orbital.⇑ Corresponding author. Tel./fax: +86 21 55664038

E-mail address: pingzhou@fudan.edu.cn (P. Zhou).

a b s t r a c t

Curcumin has been recognized as a potential natural drug to treat the Alzheimer’s disease (AD) by che-lating baleful metal ions, scavenging radicals and preventing the amyloid b (Ab) peptides from the aggre-gation. In this paper, Al(III)–curcumin complexes with Al(III) were synthesized and characterized byliquid-state 1H, 13C and 27Al nuclear magnetic resonance (NMR), mass spectroscopy (MS), ultravioletspectroscopy (UV) and generalized 2D UV–UV correlation spectroscopy. In addition, the density func-tional theory (DFT)-based UV and chemical shift calculations were also performed to view insight intothe structures and properties of curcumin and its complexes. It was revealed that curcumin could interactstrongly with Al(III) ion, and form three types of complexes under different molar ratios of [Al(III)]/[cur-cumin], which would restrain the interaction of Al(III) with the Ab peptide, reducing the toxicity effect ofAl(III) on the peptide.

� 2011 Elsevier B.V. All rights reserved.

1. Introduction

Amyloidosis is a family of pathologies induced by the transitionof endogenous proteins and peptides from a physiological globularconformation to a pathological fibrillar state [1]. The term summa-rizes a heterogeneous group of diseases which have more than 20species, characterized by extra-cellular deposition of fibrillar mate-rial [2]. Among those diseases, Alzheimer’s disease (AD) is aprogressive and devastative neurodegenerative pathology charac-terized by the presence of amyloid plaques [3]. AD has becomean important disease affecting the elderly life in the developedcountries [4]. The principle component of the AD amyloid plaquesis the amyloid b peptide with 42 amino acids, called Ab42 peptide[5,6]. Aggregation of the Ab42 peptide into insoluble fibrils is a keypathological event in AD [7,8]. The aggregation is induced by manyfactors [9–13] including oxidative stress, free-radicals,

ll rights reserved.

d b; UV, ultraviolet; MS, massT, density functional theory;ation-time of flight; DMSO,

T, time-dependant densityel; GIAO, gauge-independentlar orbital; LUMO, lowest

.

carbohydrates, lipid membranes, pH and metal ions, but the etiol-ogy is still unclear yet.

There are many investigations to demonstrate some metal ele-ments involved in the AD development. These metal elements in-clude Fe [14], Zn [15], Al [16], Cu [17], etc. Among those metals,Al(III), a component in the senile plaques, is an important elementimpacting on the aggregation and toxicity of Ab peptides [18,19]. Itis reported that Al(III) can inhibit the proteolytic degradation of Abpeptides by cathepsin D, triggering the intracellular accumulationof Ab peptides [20]. In addition, Ab peptide, when binding withAl(III), becomes more hydrophobic, resulting in the Ab peptideaggregation [21]. Furthermore, the prolonged Al(III) exposurecould induce the oxidative stress and increase Ab peptide expres-sion level in vivo [22,23]. Therefore, one of approaches for the ADtreatment is searching for the agents that can chelate metal ions[24], preventing metal ions from the interaction with Ab peptidesas well as the redox reaction which leads to the oxidative stress.So far, some chelating agents and antioxidants have been reportedto treat AD [25–27].

Recently, curcumin (1,7-bis[4-hydroxy-3-methoxy-phenyl]-1,6-heptadiene-3,5-dione) (Fig. 1), which is a main substance ofthe turmeric [28], has been demonstrated beneficial in preventingthe AD on the basis of its ability across blood brain barrier (BBB)and non-toxicity [29]. The 1,3-di-keto form of curcumin (Fig. 1A)can transform reversibly to the keto-enol tautomeric form(Fig. 1B), and the later is more stable [30] and readily chelates

Fig. 1. Di-keto (A) and keto-enolic tautomeric (B) conformers of curcumin. Thenumbers are symbols of the C or H atoms.

164 T. Jiang et al. / Journal of Molecular Structure 1004 (2011) 163–173

the metal ions to form the complexes, which can inhibit the amy-loid aggregation, reduce the oxidative neurotoxicity, and scavengethe active free-radicals [22,31,32]. In addition, by interacting withAb peptides directly, curcumin can also inhibit the Ab peptideaggregation and deposition [33], even disrupt the existing plaques,and partially restore the distorted neuritis [34,35].

Despite the detailed information from previous studies on cur-cumin structure and function, the interaction, components andstructures of Al(III)–curcumin complexes have not been investi-gated clearly. Thus the aim of this work is attempted to characterthose properties, using 1H, 13C, 27Al nuclear magnetic resonance(NMR), mass spectroscopy (MS), ultraviolet (UV) and the general-ized two-dimensional (2D) UV–UV correlation spectroscopy, aswell as the density functional theory (DFT). The work would pro-vide favorable structure evidences of the Al(III)–curcumin com-plexes and be helpful to understand the possible biologicalactivity of curcumin in the AD treatment when the metal ions pres-ent. Moreover, the complexes investigated in this work may serveas the models for the similar studies.

2. Experimental section

2.1. Chemicals and sample preparations

Al(III)–curcumin complexes were synthesized at room temper-ature in methanol solution since curcumin (molecular weight of368.39, purity of 95%, Fluka) has low solubility in water. The syn-thesis was carried out by adding various concentrations ofAlCl3�6H2O aqueous solution (0.5 mL) to curcumin methanol solu-tion (12 mmol, 3 mL) from absence of Al(III) to [Al(III)]/[curcumin]molar ratio of 4:1 for the UV analysis. The pH of the resulting com-plexes solution is 2.30. The sinapinic acid methanol solutions(10 mg mL�1, 0.1 mL) were added into 0.2 mL [Al(III)]/[curcumin]mixture solutions with the molar ratios of 1:1, 2:1 and 3:1 individ-ually. 0.5 lL of the solution was dropped on the stainless steelplate and air-dried at atmosphere for the MS analysis. The mixturesolutions of [Al(III)]/[curcumin] with molar ratios of 1:1, 2:1 and3:1 were air-dried and re-dissolved in methanol solvent for the27Al NMR analysis.

2.2. Solution 1H and 13C NMR

1H and 13C NMR spectra of curcumin in DMSO-d6 solvent wererecorded by Bruker DMX-500 spectrometer. 1H NMR spectrum wasrecorded with resonance frequency of 500.1 MHz, 1H pulse widthof 5 ls, pulse repeat delay of 3.0 s, and the scanning accumulationsof 136. 13C NMR spectrum was recorded with resonance frequency

of 125.8 MHz, 13C pulse width of 6 ls, pulse repeat delay of 3.0 s,and the scanning accumulations of 1024. The tetramethylsilane(TMS) was used as an external reference with its 1H and 13C chem-ical shifts set at 0.00 ppm.

2.3. Solution 27Al NMR

27Al NMR spectra of Al(NO3)3 and Al(III)–curcumin complexesin methanol were recorded by Varian Unity-400 plus spectrometerwith 27Al resonance frequency of 104.2 MHz, 27Al 90� pulse widthof 3.0 ls, pulse repeat delay time of 3.0 s, sampling time of 0.2 s,spectrum width of 100 kHz and scanning accumulations of 128.1.0 mol L�1 Al(NO3)3 aqueous solution was used as an external ref-erence with 27Al chemical shift set at 0.00 ppm.

2.4. Mass spectroscopy

Mass spectra of Al(III)–curcumin complexes were recorded bymatrix assisted laser desorption ionization with time of flight(MALDI-TOF) mass spectrometer (Voyager DE STR from AppliedBiosystems) and with sinapinic acid (SA, molecular weight of224) as a matrix.

2.5. UV spectroscopy and its generalized 2D UV–UV correlationspectroscopy

UV spectra were recorded by Perkin Elemer Lambda 35 UV/Visspectrometer. The absorption was scanned within the wavelengthof 300–600 nm with resolution of 1 nm. The generalized 2D corre-lation function for a perturbation-based spectrum is defined as[36]:

Uðv1; v2Þ ¼1

Tmax � Tmin

Z T max

T min

~yðv1; tÞ � ~yðv2; tÞdt ð1Þ

Wðv1;v2Þ ¼1

Tmax � Tmin

Z T max

T min

~yðv1; tÞ � ~hðv2; tÞdt ð2Þ

where Uðv1;v2Þ and Wðv1;v2Þ are called, respectively, the general-ized synchronous and asynchronous 2D correlation spectra. ~yðv1; tÞand ~yðv2; tÞ are the spectral profiles perturbed by the external phys-ical variable, t. ~hðv2; tÞ is the Hilbert–Noda transformation of~yðv2; tÞ. The detailed theory was explained in the Refs. [36,37].When ~yðv1; tÞ and ~yðv2; tÞ are a series of UV absorption spectra per-turbed by the variable, t, herein, the molar ratio of [Al(III)]/[curcu-min], and the vi, the UV absorption band, the two generalized 2DUV–UV correlation spectra, synchronous and asynchronous 2D cor-relation ones, are thus established based on the Eqs. (1) and (2). Thegeneralized 2D correlation spectrum was constructed by a 2D Shigesoftware (http://sci-tech.ksc.kwansei.ac.jp/’ozaki/e_2D.htm) com-posed by Shigeaki Morita (Department of Chemistry, KwanseiGakuin University). In the contour maps, the red-color regions aredefined as the negative correlation, while the black-color ones asthe positive correlation.

2.6. DFT calculation

The structure geometry optimization for each molecule, includ-ing curcumin and Al(III)–curcumin complexes, was carried outusing the density functional theory. The B3LYP correlation func-tional (a combination of three-parameter nonlocal exchange ofBecke [38] with the correlation function of Lee et al. [39]) andthe basis sets of 6-311G for all atoms in the studied molecule wereused. The UV calculation was carried out using time-dependentDFT (TD-DFT) with correlation functional of B3LYP and the basisset of 6-311G for all atoms. Polarizable continuum model (PCM)

T. Jiang et al. / Journal of Molecular Structure 1004 (2011) 163–173 165

was used for considering the solvent effect [40,41]. The UV calcu-lation was executed on a PC-GAMESS program package [42–44].The 27Al chemical shift calculation was carried out based on thegauge-independent atomic orbital (GIAO) packaged in Gaussian03 program [45]. Based on our previous work about the chemicalshift calculation [46], the correlation functional of B3LYP and thebasis sets of 6-311G for 13C and 27Al atoms, and 6-31G for otheratoms were used in our studied molecules, which is time-savingand efficient. All of the calculations were performed by 16 CPUsof Inter Xeon 3.06 GHz on High-End Computing Centre in FudanUniversity.

3. Results and discussion

3.1. Structure of curcumin

1H and 13C NMR spectra of curcumin in DMSO solvent areshown in Fig. 2A and B, respectively. There is an obvious 1H NMRpeak at 16.3 ppm in Fig. 2A, which is a typical active enol-H signal(H-11 in Fig. 1B) in enol tautomeric conformer [47–49], demon-strating that the keto-enolic conformer (Fig. 1B) of curcumin in

18 16 14 12 10 8 6 4 2 0chemical shift (ppm)

20

A B

Fig. 2. 1H NMR (A) and 13C NMR (B) spe

Trans

Fig. 3. Three conformers of curcumin. The term ‘‘Trans’’ indicates that two phenol–OCH3

they are on the same flank. ‘‘Cis-up’’ means two phenol–OCH3 groups are toward up thcurcumin backbone.

nature is more stable than di-keto conformer (Fig. 1A). In addition,our calculations based on the DFT geometry optimization showthat there are three keto-enolic tautomeric conformers of curcu-min with lower energy as in Fig. 3, which was also proved by Kolevand Balasubramanian [42,50]. The term ‘‘Trans’’ in Fig. 3 indicatesthat the two phenol–OCH3 groups are on the opposite flanks of thecurcumin backbone and ‘‘Cis’’ indicates those two groups are onthe same flank. ‘‘Cis-up’’ means two phenol–OCH3 groups are to-ward up the curcumin backbone, while ‘‘Cis-down’’ toward downthe curcumin backbone. The energy comparison of those threeketo-enolic conformers is listed in Table 1 referred to the ‘‘Trans’’energy set at 0.0 J mol�1. Among those conformers, ‘‘Trans’’ con-former has the lowest energy. However, the bond connecting thearomatic ring with the dienic backbone could readily rotate, lead-ing to the transition between ‘‘Trans’’ and ‘‘Cis-up’’ conformerswith a low rotational energy barrier of DE = 48 J mol�1 shown inTable 1, which was reported similarly by Benassi [30], conse-quently, ‘‘Trans’’ and ‘‘Cis-up’’ conformers have the similar popula-tion of 0.368 and 0.361, respectively, calculated in our previouspaper [51]. In addition, we correlated the experimental liquid-state13C NMR chemical shift with the theoretical ones (in Table 1) forthose three conformers. The regression coefficient of R2 is 0.9886,

0 180 160 140 120 100 80 60 40 20 0chemical shift (ppm)

ctra of curcumin in DMSO solvent.

Cis-up

Cis-down

groups are on the opposite flanks of the curcumin backbone and ‘‘Cis’’ indicates thate flank of the curcumin backbone, while ‘‘Cis-down’’ toward down the flank of the

Table 1Energy and 13C chemical shift assignments based on experiment and DFT-calculation for curcumin at its various conformers.

Items Atomsa Exp. Calc.

Cis-up Trans Cis-down

DE (J mol�1) 48 0 769Chemical shifts (ppm) —OCH3=—OCH03 56.2 52.9 50.6 52.8

52.9 50.9 52.9C1 101.3 108.0 106.3 107.6C2/20 183.6 186.9 182.2 187.4

187.4 182.6 186.8C3/30 123.5 126.1 126.0 124.9

126.1 125.1 125.0C4/40 141.1 142.5 142.0 143.3

142.9 141.7 142.9C5/50 126.8 130.8 129.0 130.8

130.9 129.6 130.8C6/60 111.9 114.1 103.8 105.9

114.2 112.9 105.9C7/70 148.5 149.7 147.0 150.3

149.7 146.7 150.3C8/80 149.8 153.0 151.1 153.1

153.1 150.5 153.1C9/90 116.2 116.8 115.3 116.1

116.8 116.1 116.1C10/100 121.6 121.7 129.5 130.5

121.7 120.3 130.4Populationb – – 0.361 0.368 0.270R2 – – 0.9967 0.9892 0.9884

a Atoms numbered as in Fig. 1.b Refer to our previous work [51].

166 T. Jiang et al. / Journal of Molecular Structure 1004 (2011) 163–173

0.9964 and 0.9844, respectively, for Trans, Cis-up and Cis-downconformers. The Cis-up conformer has the highest R2 value, consis-tent to our previous results [51]. Taking account for the X-ray re-sults [52,53] of the Cis-up and Trans conformers and the similarpopulation between them, we will adopt the Cis-up conformerwhich is maybe a mostly possible curcumin conformer [51] studiedin following work.

3.2. Chelating site of curcumin

Benassi reported that the molecular electrostatic potentialmaps of curcumin in the keto-enolic conformer showed the nega-tive charge localized on the oxygen atoms [30], therefore, curcu-min has three possible sites, ‘‘a’’, ‘‘b’’ and ‘‘c’’ (as shown in Fig. 4),chelating metal ions. The 1H NMR signal of enol-H at 16.3 ppm dis-appeared when Al(III) reacted with curcumin, which suggests thatthe site of ‘‘a’’ is most likely to join the reaction [54].

3.3. Complex components

Fig. 5A shows the mass spectra of Al(III)–curcumin complexesunder different molar ratios of [Al(III)]/[curcumin] with sinapinic

Fig. 4. Three chelating metal ion site

acid (SA) as the matrix. The peaks at m/z of 207 and 224 are as-signed to the fragment peaks of SA. The peaks at m/z of 471, 617,761 and 841 are assigned to the fragments of the Al(III)–curcumincomplexes. Among them, the peak at 841 in Fig. 5A is assigned tothe complex of C42H44O15Al2 formed by two curcumin anions (afterlosing the active enol H+ (H-11)), two Al3+ cations, three OH�, oneCH3OH, along with losing one formaldehyde (CH2O) molecule. Thepeak at 761 is assigned to the complex of C42H38O12Al formed bytwo curcumin anions and one Al3+ cation. The peak at 617 is as-signed to the complex of C25H39O14Al2 formed by one curcumin an-ion, two Al3+ cations, four OH� and four CH3OH. The peak at 471 isassigned to the complex of C21H21O9Al2 formed by one curcuminanion, two Al3+ cations, four OH�, along with losing one H2O. Inaddition, it is found that the peak at 471 comes from the peak at617 after losing one H2O and four CH3OH. Therefore, the m/z at471 and 617 are attributed to the same complex. To reveal thequantitative changes of the different peaks, the intensity ratios ofthe peaks at 471, 617, 761 and 841 to the total intensity of the fourpeaks from absence of Al(III) to [Al(III)]/[curcumin] molar ratios of3:1 are showed in Fig. 5B. The intensity ratio at m/z of 761 in-creases firstly and then decreases as the molar ratio of [Al(III)]/[curcumin] increases (Fig. 5B-a). Moreover, when the molar ratio

s of (a), (b) and (c) in curcumin.

0

15

30

45

60

75

(c)

m/z=617 or 471

A

0

15

30

45

60

75

(b)

inte

nsity

ratio

(%)

m/z=842

0.0 0.5 1.0 1.5 2.0 2.5 3.00

15

30

45

60

75

[Al(III)]/[curcumin]

m/z=761(a)

A B

Fig. 5. (A) MALDI-TOF mass spectra of Al(III)–curcumin complexes under different molar ratios of [Al(III)]/[curcumin] at 1:1, 2:1, 3:1. (B) Dependence of intensity ratios of m/z = 761 (a), 841 (b) and 617 or 471 (c) to the total intensity of those four peaks on the different molar ratios of [Al(III)]/[curcumin].

T. Jiang et al. / Journal of Molecular Structure 1004 (2011) 163–173 167

of [Al(III)]/[curcumin] reaches to 1:1, the intensity ratio at m/z of761 reaches maximum equal to 67%, which suggests that the cor-responding complex is more easily formed at low molar ratio of[Al(III)]/[curcumin], named as [Al(III)][curcumin]2. The intensityratio at m/z of 841 also increases firstly, and then decreases(Fig. 5B-b) but slower than that at m/z of 761, therefore corre-sponding to the formed complex, named [Al(III)]2[curcumin]2. Fur-thermore, when the molar ratio increases from 1:1 to 3:1, theintensity ratio of peak at 471 or 617 increases considerably(Fig. 5B-c), higher than that of other two peaks at 761 and 841,and reaches to 71% as the molar ratio of [Al(III)]/[curcumin] isequal to 3:1, therefore corresponding to the formed complex,named [Al(III)]2[curcumin]. Based on the above analysis, as themolar ratio of [Al(III)]/[curcumin] increases, the complex[Al(III)][curcumin]2 corresponding to m/z of 761 is a dominantcomponent at low molar ratio of [Al(III)]/[curcumin]. The complex

300 350 400 450 500 550

3 : 1

4 : 13.5 : 1

2.7 : 12.3 : 1

2 : 11.7 : 11.3 : 1

1 : 10.8 : 10.7 : 10.5 : 10.3 : 10.2 : 1

0 : 1

wavelength (nm)

[Al]:[curcumin]

300

A B

Fig. 6. (A) UV spectra of Al(III)–curcumin complexes in methanol solvent from absencespectra of (A) as the molar ratio of [Al(III)]/[curcumin] is 0.5:1, 0.8:1, 1.3:1, 1.7:1 and 2.

[Al(III)]2[curcumin] corresponding to m/z of 471 or 617 is more sta-ble than other two complexes at higher molar ratio of [Al(III)]/[curcumin].

Fig. 6A is the UV spectra of Al(III)–curcumin complexes in meth-anol solvent from absence of Al(III) to [Al(III)]/[curcumin] molar ra-tios of 4:1, and Fig. 6B is the second order derivative spectra ofFig. 6A, which can indicates clearly the multi-peak positions embed-ded in a broad absorption band. In Fig. 6B, there are two bands atabout 432 and 465 nm moving to 445 and 478 nm, respectively, asthe molar ratios of [Al(III)]/[curcumin] increase. In order to correlatethe UV absorption bands and complex components, the generalized2D UV–UV correlation spectra of Al(III)–curcumin complexes areconstructed in Fig. 7, which can reveal the detailed evolution infor-mation of different complex components with the change in the mo-lar ratio of [Al(III)]/[curcumin]. Fig. 7A and B are the synchronous andasynchronous 2D UV–UV correlation spectra, respectively,

350 400 450 500 550

478445

432

2.3:1

1.7:1

1.3:1

0.8:1

Wavelength (nm)

[Al]:[curcumin]

0.5:1

465

of Al(III) to [Al(III)]/[curcumin] molar ratios of 4:1. (B) The second order derivative3:1.

350 400 450 500 550

-0.008

-0.004

0.000

0.004

0.008

0.012

398

478

465

445

445

inte

nsity

wavelength (nm)

slice at λ2 of 398 nm in synchronous spectrumslice at λ2 of 398 nm in asynchronous spectrum

465

300 350 400 450 500 550

-0.002

0.000

0.002

0.004

432in

tens

ity

wavelength (nm)

slice at λ2 of 432 nm in synchronous spectrum

398

465

350 400 450 500

-0.01

0.00

0.01

0.02

0.03

0.04

478465

inte

nsity

wavelength (nm)

slice at λ2 of 465 nm in synchronous spectrum

398

300 350 400 450 500 550

-0.003

0.000

0.003

0.006

0.009

0.012

465

inte

nsity

wavelength (nm)

slice at λ2 of 445 nm in synchronous spectrum

445

398

C

E

D

F

A B

Fig. 7. Synchronous (A) and asynchronous (B) generalized 2D UV–UV correlation spectra of Al(III)–curcumin complexes from absence of Al(III) to [Al(III)]/[curcumin] molarratios of 1.3:1. Black regions indicate the positive correlation contours, while red regions indicate the negative correlation contours. Slices at k2 of 398 (C), 432 (D), 465 (E) and445 nm (F) along the k1-axis of the 2D spectra. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

168 T. Jiang et al. / Journal of Molecular Structure 1004 (2011) 163–173

established when the component intensities at the given absorptionbands either increase or decrease monotonously from absence ofAl(III) to [Al(III)]/[curcumin] molar ratios of 1.3:1 in Fig. 6, as re-quired by the theory of the generalized 2D correlation spectroscopy[36]. The averaged 1D UV spectrum profiles are shown at both rightand top sides of the 2D maps.

In this study, a pair of 2D spectra, synchronous and asynchro-nous maps, are constructed by two independent wavelength axes(k1, k2) and the correlation contour lines. 2D synchronous spectrumis symmetric with respect to the diagonal. Peaks appearing on thediagonal are autopeaks same as the peaks appearing in 1D

spectrum. The off-diagonal cross-peaks of U(k1, k2) are positiveor negative. The positive cross-peaks of U(k1, k2) indicate that bothintensities of peaks k1 and k2 increase or decrease simultaneouslyunder the perturbation, whereas the negative cross-peaks indicatethat the intensity of peak k1 changes opposite to that of peak k2, i.e.one increases while the other decreases. The 2D asynchronousspectrum is asymmetric with respect to the diagonal. Only off-diagonal cross-peaks of W(k1, k2) appear in the asynchronous spec-trum, and are either positive or negative. Basing on the signs of thecross-peaks appearing in both synchronous and asynchronousmaps, one can figure out the change orders of different peaks under

T. Jiang et al. / Journal of Molecular Structure 1004 (2011) 163–173 169

the perturbation. According to Noda’s rule [36,37], if cross-peaks ofU/W(k1, k2) in the synchronous and asynchronous maps have thesame signs, both positive or negative, it indicates that the peakk1 varies prior to the peak k2; while if cross-peaks of U/W(k1, k2)in the synchronous and asynchronous maps have different signs,it indicates that the peak k2 varies prior to the peak k1.

In the synchronous map (Fig. 7A), there are two strong autope-aks at U(398, 398) and U(465, 465) and one negative cross-peak atU(465, 398), besides a broad positive contours within 428–502 nmbelow the diagonal. In the asynchronous map (Fig. 7B), there arefive cross-peaks at W(445, 398), W(465, 432), W(465, 445),W(478, 398) and W(478, 465) below the diagonal. In order to viewclearly the signs of the cross-peaks, the slices at the wavelengthsk2’s of 398 (Fig. 7C), 432 (Fig. 7D), 465 (Fig. 7E) and 445 nm(Fig. 7F) along the k1-axis in the 2D maps are showed, which arehighly sensitive to the changes in the complex components. Eachpeak k1 on the slice k2 indicates there is a cross-peak of (k1, k2),and the sign of the peak k1 is that of the cross-peak (k1, k2). Forexample, there is a negative peak of 465 nm on the slice of398 nm in the synchronous spectrum (Fig. 7C), indicating thatthere is a negative cross-peak at U(465, 398). Similarly analyzingFig. 7C–F, we find both cross-peaks of (445, 398) and (465, 398)are positive (in black color) in the asynchronous map, but negative(in red color) in the synchronous map. Thus, according to Noda’srule [36,37], the band of 398 nm changes prior to the bands of445 and 465 nm. In addition, both cross-peaks of (465, 432) and(478, 465) are negative (in red color) in the asynchronous map,but positive in the synchronous map (in black color), thus, the bandof 432 nm changes prior to the band of 465 nm and the band of

Table 2TD-DFT-based UV calculation of curcumin.

Absorption bands (nm) Excitation orbitals

From To

446.1 HOMO LUMO396.1 HOMO-1 LUMO

350 400 450 500wavelength (nm)

A B

Fig. 8. (A) An example of the deconvoluted UV spectrum. (B) Changes in the absorption in[Al(III)] increases. The curve (d) was obtained after the intensity-normalized curve 445

465 nm changes prior to the band of 478 nm. Furthermore, thecross-peak of (465, 445) is positive in both synchronous and asyn-chronous maps, indicating that the band of 465 nm changes priorto the band of 445 nm.

Considering above orders, we suggest that the changing se-quence of those five bands is 398, 432 > 465 > 445, 478 nm (‘‘>’’means ‘‘change prior to’’) from absence of Al(III) to [Al(III)]/[curcu-min] molar ratios of 1.3:1. In addition, it is found that the absorp-tion intensities at bands of 432, 445, 465 and 478 nm change in thesame approach, different from that of the band at 398 nm.

To identify the absorption bands corresponding to the curcu-min, TD-DFT-based UV calculation was carried out. The resultsare shown in Table 2. There are two main theoretical absorptionbands at 446 and 396 nm. They are the electronic transitions fromthe electronic state of the highest occupied molecular orbital(HOMO) to that of the lowest unoccupied molecular orbital(LUMO), and electronic state of the secondary highest occupiedmolecular orbital (HOMO-1) to that of the LUMO, respectively.The two theoretical UV absorption bands are close to the experi-mental values of 445 and 398 nm, respectively, resolved from thederivative spectra and the generalized 2D UV–UV correlation spec-tra in Figs. 6B and 7. Therefore, the absorption bands at 445 and398 nm are assigned to the absorptions of curcumin.

Furthermore, for quantitatively analyzing the UV band changes,the spectra shown in Fig. 6 were deconvoluted into five bands at398, 432, 445, 465 and 478 nm based on the derivative spectraand the 2D UV–UV correlation analysis. An example of the decon-voluted spectrum of Fig. 6 is shown in Fig. 8A. Fig. 8B shows thechanges in the absorption intensities of different bands as the mo-lar ratio of [Al(III)]/[curcumin] increases. In Fig. 8B-a, the intensityof band at 398 nm decreases as the molar ratio of [Al(III)]/[curcu-min] increases, resulting from the curcumin reacted. Both intensi-ties of bands at 432 and 465 nm in Fig. 8B-b increase before themolar ratio of [Al(III)]/[curcumin] is equal to 1.2:1, identical tothe 2D UV–UV correlation results of 432 and 465 nm changing inthe same approach from absence of Al(III) to [Al(III)]/[curcumin]molar ratios of 1.3:1, and then decrease as the molar ratio of[Al(III)]/[curcumin] further increases, which indicates that the

0 10 20 30 40 50

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5

[Al(III)] (mmol)

432 nm465 nm

445 nm398 nm

478 nm

(d) 445 = (a)445 - (a)398

[Al(III)] / [curcumin]

(a)

(c)

(b)

(d)

tensity at bands of 398 and 445 nm (a), 432 and 465 nm (b) as well as 478 nm (c) asnm in (a) minus the intensity-normalized curve 398 nm in (a).

170 T. Jiang et al. / Journal of Molecular Structure 1004 (2011) 163–173

curcumin forms an unstable intermediate complex with Al(III) atfirst, and then the intermediate is dissociated at high concentrationof Al(III). Compared with MS results (Fig. 5B), the UV absorptions at432 nm and 465 nm are attributed to the intermediate complex of[Al(III)][curcumin]2 with m/z of 761. The complex was formed fromabsence of Al(III) to [Al(III)]/[curcumin] molar ratios of 1.2:1 andthen dissociated at high molar ratio of [Al(III)]/[curcumin] changedfrom 1.2:1 to 3:1. The intensity curve of the band at 478 nm lookslike a ‘‘S’’ shape (Fig. 8B-c), indicating that the complex corre-sponding to the band at 478 nm appears later than the complexof [Al(III)][curcumin]2 corresponding to the bands at 432 and465 nm, which is identical to the 2D UV–UV correlation results.In addition, in Fig. 8B-a the curve of 445 nm declines consistentlywith the curve of 398 nm before the molar ratio of [Al(III)]/[curcu-min] is equal to 1.2:1, and increases afterward, indicating that theUV band of 445 nm may also be contributed from one of the Al(III)–curcumin complexes in addition to curcumin. We normalized theintensity of the curves 398 and 445 nm in Fig. 8B-a with the inten-sity at the zero molar ratio of [Al(III)]/[curcumin] as unit, and thenallowed the intensity-normalized curve of 445 nm minus theintensity-normalized curve of 398 nm which is contributed fromcurcumin, Fig. 8B-d was thus obtained. The curve of 445 nm inFig. 8B-d has two ascending processes: one process goes throughas that of band at 478 nm (Fig. 8B-c) from absence of Al(III) to[Al(III)]/[curcumin] molar ratios of 2:1, which suggests that thecomplex corresponding to the process may have two absorptionbands at 445 and 478 nm; the other process goes up continuallyas molar ratio of [Al(III)]/[curcumin] increases from 2:1 to 4:1,maybe resulting from the other complex corresponding to the445 nm as well. Compared with the MS results (Fig. 5B), the UVabsorptions at 445 and 478 nm are suggested from two complexes:one is [Al(III)]2[curcumin]2 with m/z of 841, formed from absenceof Al(III) to [Al(III)]/[curcumin] molar ratios of 2:1; the other is[Al(III)]2[curcumin] with m/z of 471 and 617, formed dominantlyat high molar ratio of [Al(III)]/[curcumin] changed from 2:1 to4:1, because the absorption at 445 nm increases continually inFig. 8B-d while the absorption at 478 nm is saturated in Fig. 8B-cwhen the molar ratio of [Al(III)]/[curcumin] changes from 2:1 to4:1. Among the four bands of 432, 445, 465 and 478 nm, two bandsat 432 and 465 nm change prior to the bands at 445 and 478 nmfrom absence of Al(III) to [Al(III)]/[curcumin] molar ratios of1.2:1, identical to the 2D UV–UV results, therefore, it is suggestedthat the complex [Al(III)][curcumin]2 is most easily formed at lowmolar ratio of [Al(III)]/[curcumin], and then the complex[Al(III)]2[curcumin]2 formed. At high molar ratio of [Al(III)]/[curcu-min], those two complexes are dissociated partially, leading to thecomplex [Al(III)]2[curcumin] present dominantly. The assignmentsof the UV absorption bands for those three Al(III)–curcumin com-plexes are suggested in Table 3.

To clarify further the above analysis, 27Al NMR spectra of thecomplexes were investigated in Fig. 9. 27Al NMR spectrum of

Table 3Summary of experimental and theoretical UV, MS and NMR results.

Curcumin [Al(III)][curcumin]2 [Al(III)]2[c

Composite Curcumin 2Curcumin� + Al(III) + OH� + MeOH 2CurcuminMolecular

formulaC21H20O6 C43H43O14Al C45H53O18

Molecularweight

368 810 970

Mass (m/z) 369 (368 + H+) 761 (810 �MeOH � OH�) 841 (970 �

UV (nm) 398, 445 (396,446)a

432, 465 (436, 451)a 445, 478 (

27Al NMR(ppm)

– 4.2 (30)a 0.9, 2.5 (6

a The data in parentheses are heoretical results.

Al(NO3)3 in aqueous solution is shown in Fig. 9A and its deconvo-lution is shown in Fig. 9B, demonstrating the chemical shifts at�2.0 ppm and 0.9 ppm from two hydrolytic species of 27Al(III) ions[55,56]. Based on the MS and UV results of three Al(III)–curcumincomplexes of [Al(III)][curcumin]2, [Al(III)]2[curcumin]2 and[Al(III)]2[curcumin] present from absence of Al(III) to [Al(III)]/[cur-cumin] molar ratios of 4:1, 27Al NMR spectra of those complexeswere deconvoluted as shown in Fig. 9C under different molar ratiosof [Al(III)]/[curcumin]. In addition to the free 27Al(III) ion signals at�2.0 and 0.9 ppm, other four signals at chemical shifts of 1.9, 2.5,3.5 and 4.2 ppm are observed (Fig. 9C). To reveal the changes ofcomplex components under different molar ratios of [Al(III)]/[cur-cumin], the contents of those five signals at chemical shifts of 0.9,1.9, 2.5, 3.5 and 4.2 ppm, respectively, are showed in Fig. 9D. Thepeak at 4.2 ppm increases firstly from absence of Al(III) to[Al(III)]/[curcumin] molar ratios of 1:1 and then decreases as themolar ratio of [Al(III)]/[curcumin] increases further from 1:1 to3:1 (Fig. 9D-a). In addition, when the molar ratio of [Al(III)]/[curcu-min] reaches to 1:1, the peak at 4.2 ppm has the highest content of65%. The peak at 2.5 ppm increases from absence of Al(III) to[Al(III)]/[curcumin] molar ratios of 2:1, but slower than the peakat 4.2 ppm, meanwhile decreases slower than the peak at4.2 ppm as molar ratio of [Al(III)]/[curcumin] increases furtherfrom 2:1 to 3:1 (Fig. 9B-b). Moreover, it is also observed that thecurve of 0.9 ppm changes similarly to that of 2.5 ppm (Fig. 9B-c).If 0.9 ppm were only assigned to the free 27Al(III) ion, the signalwould increase monotonously from absence of Al(III) to [Al(III)]/[curcumin] molar ratios of 3:1; however, the monotonic changewas not observed, which implies that the chemical shift at0.9 ppm is not only contributed from the free 27Al(III) ion, but alsofrom the complex at the chemical shift of 2.5 ppm. In addition,when the molar ratio of [Al(III)]/[curcumin] increases from 1:1 to3:1, the contents of peaks at 1.9 (Fig. 9B-d) and 3.5 ppm (Fig. 9B-e) increase consistently, higher than that of other three peaks.Therefore, these two peaks are maybe contributed from the samecomplex. When the molar ratio of [Al(III)]/[curcumin] increasesto 3:1, the total area ratio of the two peaks at 1.9 and 3.5 ppmreaches to 67%. Comparing with UV and MS results, we assignthe peak at 4.2 ppm to complex [Al(III)][curcumin]2, the peaks at0.9 and 2.5 ppm to complex [Al(III)]2[curcumin]2, and the peaksat 1.9 and 3.5 ppm to complex [Al(III)]2[curcumin]. The complexesof [Al(III)]2[curcumin]2 and [Al(III)]2[curcumin] have two chemicalshifts possibly due to two species of Al(III) present in those twocomplexes. Furthermore, the intensity ratios of the MS signals ofthose three complexes are basically identical to the area ratios ofthe 27Al NMR signals, proving the assignments.

3.4. Complex structures

27Al chemical shift can reveal Al(III) complex structure. Theexperimental results demonstrate that the observed 27Al chemical

urcumin]2 [Al(III)]2[curcumin]

� + 2Al(III) + 3OH� + 3MeOH + Cl� Curcumin� + 2Al(III) + 4OH� + 4MeOH + Cl�

Al2Cl C25H39O14Al2Cl

652

Cl� � 2MeOH � CH2O) 617 (652 � Cl�), 471(652 � Cl� � 4MeOH � H2O)

445, 455)a 445, 478 (444, 453)a

, 15)a 1.9, 3.5 (13, 27)a

15 10 5 0 -5 -10 -15-500

0

500

1000

1500

2000

2500

3000

3500

4000

inte

nsity

chemical shift (ppm)15 10 5 0 -5 -10 -15

-500

0

500

1000

1500

2000

2500

3000

3500

4000

inte

nsity

chemical shift (ppm)

experimental curve fitted curve

-2.0

0.9

10 5 0 -5

10 5 0 -5

10 5 0 -5

[Al(III)]/[curcumin] =1:1

chemical shift (ppm)

0.91.93.5

4.2

2.5

[Al(III)]/[curcumin] =2:1

0.91.92.5

3.5

4.2

[Al(III)]/[curcumin] =3:1

4.2

3.5

2.51.9

0.9

0 1 2 30

1530456075

area

ratio

%

[Al(III)]:[curcumin]

(a) about 4.2 ppm

06

121824

about 0.9 ppm(c)

048

1216

about 2.5 ppm

(b)

07

14212835

about 1.9 ppm

(d)

07

14212835

about 3.5 ppm

(e)

A B

C D

Fig. 9. (A) 27Al NMR spectrum of Al(NO3)3 in aqueous solvent. (B) Deconvolution of spectrum (A). (C) Deconvolution of 27Al NMR spectra of complexes under different molarratios of [Al(III)]/[curcumin] at 1:1, 2:1, 3:1. (D) The content dependence of the complex components corresponding to the chemical shifts of 4.2 (a), 0.9 (b), 2.5 (c), 1.9 (d), and3.5 (e) ppm on the molar ratio of [Al(III)]/[curcumin] increase. The 27Al chemical shift of Al(NO3)3 aqueous solvent was used as the external reference of 0.0 ppm.

T. Jiang et al. / Journal of Molecular Structure 1004 (2011) 163–173 171

shifts result from the octahedral Al(III) [56,57]. Accordingly, the six-coordination models are designed for the geometry-optimization ofthe Al(III)–curcumin complexes on the basis of the MS results byadding methanol and/or OH� as the counterparts. Therefore, thecomplex of [Al(III)][curcumin]2 is formed by one Al3+ cation, twocurcumin anions, one OH� and one CH3OH molecule; the complexof [Al(III)]2[curcumin]2 is formed by two Al3+ cations, two curcuminanions, three OH� and three CH3OH molecules; the complex of[Al(III)]2[curcumin] is formed by two Al3+ cations, one curcuminanion, four OH� and four CH3OH molecules. To keep the electricneutrality of the three complexes, we added one Cl� to [Al(III)]2

[curcumin]2 and [Al(III)]2[curcumin], individually, in the secondarycoordination sphere of Al(III).

Based on the experimental and calculated results, the most pos-sible stereo structures of those three Al(III)–curcumin complexesby DFT calculation are shown in Fig. 10. For the [Al(III)][curcumin]2

complex, a six-coordination Al(III) complex is constructed with anapproximately equatorial planar by two oxygens from the twoketo-enol oxygens in one curcumin, one oxygen from one of

keto-enol oxygens in the second curcumin and one oxygen fromCH3OH molecule, as well as with an axis by two more ligands ofone OH� and the other keto-enol oxygen in the second curcumin.For the [Al(III)]2[curcumin]2 complex, one of six-coordinationAl(III) ions is coordinated with an approximately equatorial planarby two oxygens from two keto-enol oxygens in one curcumin, oneoxygen from one of keto-enol oxygens in the second curcumin andone oxygen from OH�, as well as with an axis by two more ligandsof one OH� and the other keto-enol oxygen in the second curcu-min; the other Al(III) ion is coordinated with an approximatelyequatorial planar by two oxygens from two CH3OH molecules,one oxygen from OH� and one oxygen from the OH� which is alsoshared on the axis by the first Al(III) ion, as well as with an axis bytwo more ligands of one CH3OH and one oxygen from the OH�

which is also shared on the planar by first Al(III) ion. For the[Al(III)]2[curcumin] complex, one of six-coordination Al(III) ionsis coordinated with an approximately equatorial planar by oneoxygen from one of keto-enol oxygens in curcumin, two oxygensfrom two CH3OH molecules, and one oxygen from OH�, as well

[Al(III)][curcumin]2

[Al(III)]2[curcumin]

2

[Al(III)]2[curcumin]

Fig. 10. Three theoretical structural models of complexes of [Al(III)][curcumin]2, [Al(III)]2[curcumin]2 and [Al(III)]2[curcumin]. The pink, red, black and gray balls indicateAl(III), oxygen, carbon and hydrogen atoms, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of thisarticle.)

172 T. Jiang et al. / Journal of Molecular Structure 1004 (2011) 163–173

as with an axis by two more ligands of one OH� and the other keto-enol oxygen from the curcumin; the other Al(III) ion is coordinatedwith an approximately equatorial planar by two oxygens from twoOH�, one oxygen from CH3OH and one oxygen from OH� which isshared on the axis by the first Al(III), as well as with an axis by twomore ligands of one CH3OH and one oxygen from the OH� which isalso shared on the planar by the first Al(III).

On the basis of the optimized structures of those three com-plexes, the chemical shifts of Al(III) as well as the UV absorptions

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5

5

10

15

20

25

30

[Al(III)][curcumin]2

[Al(III)]2[curcumin]

Theo

retic

al c

hem

ical

shi

ft (p

pm)

Experimental chemical shift (ppm)

R = 0.987

[Al(III)]2[curcumin]2

A

Fig. 11. Correlation between experimental and DFT-calculated 27Al NMR chemical shifts (and 0.968, respectively, in (A) and (B).

of the complexes were calculated. Comparison of the Al(III) chem-ical shifts from the DFT calculation with those from the experi-ments for those three complexes shows a good correlation with aregression coefficient of R = 0.987 in Fig. 11A. Moreover, compari-son of the UV absorption from the TD-DFT-based calculation withthose from the experiments also shows a good correlation with aregression coefficient of R = 0.968 in Fig. 11B. The systemic devia-tions between the calculation and the experiment are most possi-bly resulted from the methodology used in the calculation. The

430 440 450 460 470 480

435

440

445

450

455 [Al(III)]2[curcumin]2

[Al(III)][curcumin]2

[Al(III)]2[curcumin]

Theo

retic

al a

bsor

ptio

n ba

nds

(nm

)

Experimental absorption bands (nm)

R = 0.968B

A) and UV (B) of Al(III)–curcumin complexes. The regression coefficients of R = 0.987

T. Jiang et al. / Journal of Molecular Structure 1004 (2011) 163–173 173

calculations confirms the experimental assignments of Al(III) NMRand UV for the three complexes.

The complex components and properties are summarized in Ta-ble 3, based on the experimental MS, UV, 27Al NMR results and thetheoretical ones.

4. Conclusion

In this paper, on the basis of detailed experimental and theoret-ical methods, it is demonstrated that curcumin interacts stronglywith Al(III), and forms three types of complexes of [Al(III)][curcu-min]2, [Al(III)]2[curcumin]2 and [Al(III)]2[curcumin], implying thatcurcumin is capable of scavenging Al(III) and preventing Al(III)from the interaction with proteins like Ab, therefore, weakeningthe Ab toxicity and oxidative stress. The results enhance ourcomprehension about the curcumin roles in the AD treatment.Interestingly, this work would shed some light on the way to sys-tematically track the Al(III) during its interaction with curcuminand give some guidance to screen the potential drugs for treatmentof AD in future.

Acknowledgements

The work was supported by Natural Science Foundation of Chi-na (Nos. 10475017, 20673022 and 21074025). We also cordiallythank Prof. He-Yong He and Tai-Liu Wu in the Department ofChemistry, Fudan University for kindly helps in some NMRmeasurements.

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