Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 117 (2014) 798–800

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Spectrochimica Acta Part A: Molecular andBiomolecular Spectroscopy

journal homepage: www.elsevier .com/locate /saa

Short Communication

Effect of Curcumin on the metal ion induced fibrillization of Amyloid-bpeptide

1386-1425/$ - see front matter � 2013 Elsevier B.V. All rights reserved.http://dx.doi.org/10.1016/j.saa.2013.09.064

⇑ Tel.: +91 7895635142; fax: +91 1332 27 3560.E-mail addresses: rona.banerjee@gmail.com, ronabsbt@iitr.ac.in

Rona Banerjee ⇑Department of Biotechnology, Indian Institute of Technology, Roorkee 247667, Uttaranchal, India

h i g h l i g h t s

� Curcumin can reduce the b-sheetcontent of Ab peptide in timedependent manner.� Metal ions like Cu(II) and Zn(II) cause

Ab fibrillization.� Curcumin can inhibit metal ion

induced fibrillization and aggregationof the peptide.

g r a p h i c a l a b s t r a c t

The reduction in b-sheet content of Amyloid-b peptide occurs in presence of Curcumin as a function oftime.

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20 Aβ+Curcumin (6hrs) Aβ+Curcumin (24 hrs)

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a r t i c l e i n f o

Article history:Received 20 May 2013Received in revised form 29 August 2013Accepted 18 September 2013Available online 27 September 2013

Keywords:CurcuminAbMetalsFibrillizationCDAFM

a b s t r a c t

The effect of Curcumin on Cu(II) and Zn(II) induced oligomerization and protofibrillization of theamyloid-beta (Ab) peptide has been studied by spectroscopic and microscopic methods. Curcumin couldsignificantly reduce the b-sheet content of the peptide in a time dependent manner. It also plays anantagonistic role in b-sheet formation that is promoted by metal ions like Cu(II) and Zn(II) as observedby Circular Dichroism (CD) spectroscopy. Atomic force microscopic (AFM) images show that spontaneousfibrillization of the peptide occurs in presence of Cu(II) and Zn(II) but is inhibited on incubation of thepeptide with Curcumin indicating the beneficial role of Curcumin in preventing the aggregation of Abpeptide.

� 2013 Elsevier B.V. All rights reserved.

Introduction

The fibrillation and aggregation of misfolded amyloid-beta (Ab)peptide leads to the formation of senile plaques that are the path-ological hallmarks in Alzheimer’s disease (AD). Ab peptide has avery high binding affinity to divalent metal ions like Zn(II) and

Cu(II) [1,2] that are found in excess in the AD brain and can actas agonist in the aggregation of Ab. The Fenton reaction involvingthe redox active metal generate the reactive oxygen species(ROS) causing severe oxidative stress and promotes protein aggre-gation [3,4].

Curcumin, a polyphenolic phytochemical obtained from the rhi-zome of Curcuma longa, is a potential anti-inflammatory, antioxi-dant and neuroprotective agent [5,6]. Curcumin has also beendemonstrated to inhibit the aggregation of Ab peptide in vitro

Fig. 1. Chemical structure of Curcumin.

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Fig. 2. CD spectra of Ab peptide (—), Ab with equimolar amount of Curcumin after30 min (� � �), 6 h (- �- �) and 24 h of incubation (---).

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Fig. 3. CD spectra of Ab peptide showing the effect of free metal ions and that incombination with Curcumin. Ab in 10 mM PBS (—), Ab with 20 lM CuCl2 (-- -), with20 lM ZnCl2 (� � �) with 20 lM Curcumin and 20 lM CuCl2 (- �- �), with 20 lMCurcumin and 20 lM ZnCl2 (- � �-).

R. Banerjee / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 117 (2014) 798–800 799

and in vivo [7]. By virtue of its chemical structure (Fig. 1) Curcumincan efficiently bind metal ions through its b-diketone moiety, andcan prevent free radical generation by metal ions in solution [8,9].

In the aggregation process of the peptide, the soluble oligomerscan act as the seeding factors for protofibril formation followed bymatured fibrils and aggregates. The accumulation of the aggregatesis considered as principally responsible for neurodegeneration [7].Recent reports also emphazise on the neurotoxicity imparted bythe high molecular weight oligomers that contain a substantialamount of b-sheet structure that accelerates the formation of pro-tofibrils. Therefore, inhibitors of b-sheets could play a crucial rolein preventing rapid oligomerization, fibrillization and cytotoxicity[10].

In this work, the effect of Curcumin on the secondary structureof the amyloid peptide and also the metal ion induced oligomeriza-tion and fibrillation have been investigated by biophysicalmethods.

Materials and methods

Curcumin (99% pure) from Merck (Germany) and synthetic hu-man Amyloid-b (1-42) peptide from Genscript (USA) were pro-cured and used without any further modification. Salts andbuffers were procured from Himedia and the solutions were pre-pared in Millipore water. Concentrated stock solution of Curcuminwas prepared in spectroscopic grade acetone and was diluted todesired concentration using 10 mM phosphate buffer of pH 7.4.The stability of Curcumin was checked before every experimentusing absorption spectroscopy. The peptide stock solution was pre-pared in 10 mM phosphate buffer saline (PBS) of pH 8 and stored at�20 �C.

Circular Dichroism (CD) studies were performed by Chirascanspectropolarimeter (Applied Photophysics, USA) using quartz cuv-ettes of 1 mm path length. The peptide samples were prepared in10 mM phosphate buffer of pH 7.4 and the experiments were car-ried out at a temperature of 25 �C. Atomic Force microscopy (AFM)has been employed to obtain information about the morphology ofthe peptide. The concentration of peptide was kept very low(10 lM) to ensure uniform spreading of the samples on the coverslips in thin monolayers for better visualization. The images wereacquired by the Atomic Force Microscope (AFM, NT-MDT modelNTEGRA T5-150) in the semi-contact mode at ambient air (RH50%) and temperature (23 �C) with a silicon cantilever at its reso-nance frequency five successive scans for each image.

Results and discussion

The CD spectroscopic studies show the effect of Curcumin onthe secondary structure of Ab peptide (Fig. 2) as a function of time.Incubation of the peptide with equimolar amount (50 lM) of Cur-cumin induces a significant decrease in the b-sheet content of thepeptide over an incubation period of 0–24 h showing a reduction in[h] value (mean residual molar ellipticity) at 220 nm. The appear-ance of isodichroic point around 210 nm for Curcumin containingsamples imply an equilibrium between b-sheet and the unstruc-tured coil structure of the peptide.

On addition of equimolar amount (20 lM) of CuCl2 and ZnCl2

separately in the buffer containing 20 lM peptide, the [h] valuesaround 218 nm were increased (Fig. 3) indicating a higher contentof b-sheets, whereas, on addition of equimolar amount of Curcu-min in the peptide solutions along with metal ions the reductionof [h] values signify a marked decrease in the same. It was alsoobserved that the reduction of b-sheet by Curcumin is morepronounced for Cu(II) containing peptide sample than with Zn(II)which could be explained by higher binding affinity of Curcuminto Cu(II) than Zn(II) [11]. As no precipitation of the peptideoccurred, therefore, it can be concluded that the reduction in thenegative ellipticity value implies less content of b-sheets in thepeptide for Curcumin containing samples where Curcumin couldinhibit the b-sheet structure formation.

The AFM images (Fig. 4) show the appearance of large oligo-meric aggregates and protofibrillar structures of Ab peptide(10 lM) on incubation for 6 h at room temperature. For peptidetreated with equimolar amount of Cu(II) in the buffer the presence

Fig. 4. AFM images acquired in semi-contact mode showing the oligomerization and fibrillization of (a) Ab peptide, Ab peptide treated with (b) Cu(II), (c) Zn(II), (d) Curcumin,(e) Cu(II) and Curcumin, (f) Zn(II) and Curcumin. The samples were incubated for 6hrs at room temperature.

800 R. Banerjee / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 117 (2014) 798–800

of beaded fibrillar structures was more pronounced and with Zn(II)treated sample, large oligomeric assemblies was observed. This isevident of the agonistic role of Cu(II) and Zn(II) in promoting fibril-lation and aggregation of the peptide. However, in the presence ofequimolar amount of Curcumin, considering the particle morphol-ogy and size, no fibrillar structures were observed in the peptide,only small oligomeric nano-aggregates were detected. Absence ofprotofibrillar structures also indicate that Curcumin could effec-tively inhibit the Cu(II) and Zn(II) induced fibrillization of thepeptide.

As Curcumin is a potential metal chelator, it can compete withthe peptide on complexing with metal ions thereby preventingpeptide–metal complex formation that produces degenerativeROS, promoting the fibrillation and aggregation of Ab. CD resultshave shown that Curcumin efficiently reduces the b-sheet contentof the peptide, which is the precursor for protofibrils and AFMimaging supports the observation as Curcumin could potentiallyinhibit fibrillation in vitro by arresting the polymerization of smalloligomers. These observations also agree with the moleculardynamics simulation studies recently reported by Zhao et al. [12]suggesting that Curcumin could reduce b-sheet content of amyloidpeptide without hindering the monomeric contacts. The presentwork also elucidates the novel property of Curcumin in inhibitingthe metal ion induced b-sheet formation of Ab peptide.

Conclusion

Curcumin inhibits the seeding of fibrillation by preventing thepeptide-metal complex formation with Cu(II) and Zn(II) as

observed by CD and AFM studies. Therefore, Curcumin, along withits many other beneficial properties can be considered as potentialtherapeutic agent for amyloidosis.

Acknowledgements

This work has been supported by the Department of Scienceand Technology, Govt. of India, under the SERC-Fast-track YoungScientist Scheme.

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