Research ArticleDichotomous Effect of Caffeine Curcumin and Naringenin onGenomic DNA of Normal and Diabetic Subjects

Debarati Chattopadhyay Ashok SomaiahDivya Raghunathan and Kavitha Thirumurugan

Structural Biology Lab Centre for Biomedical Research School of Bio Sciences amp TechnologyVIT University Vellore Tamil Nadu 632014 India

Correspondence should be addressed to Kavitha Thirumurugan kavithirugmailcom

Received 3 January 2014 Accepted 3 March 2014 Published 30 March 2014

Academic Editors G Garibotto and J Salas-Salvado

Copyright copy 2014 Debarati Chattopadhyay et alThis is an open access article distributed under the Creative CommonsAttributionLicense which permits unrestricted use distribution and reproduction in anymedium provided the originalwork is properly cited

Nutraceutical compounds show antioxidant and prooxidant properties under stress conditions like cancer diabetes and otherdiseasesThe objective of this study is to find the dichotomic behavior of caffeine curcumin and naringenin onDNAof diabetic andnormal subjects in the presence and absence of copper hydrogen peroxide and complex of copper-hydrogen peroxide Hydrogenperoxide releases hydroxyl free radicals (∙OH) on oxidation of Cu (I) toCu (II) through Fenton-type reaction to causeDNAdamageIn the results agarose gel electrophoretic pattern speculates the prooxidant effect of caffeine and antioxidant effect of curcuminon DNA in the presence of copper and hydrogen peroxide UV-Vis spectral analysis shows hyperchromism on addition of DNAto caffeine hypochromism with curcumin and subtle changes with naringenin The chosen nutraceuticals act as inducers andquenchers of oxidative free radicals arising from diabetes

1 Introduction

Modern sedentary life-style coupled to high calorie dietaggravates oxidative stress level culminating in avoidable dis-eases like obesity diabetes hypertension cataract and can-cer The oxidative stress caused by diabetes is accompaniedby production of reactive oxygen species (ROS) (hydroxylssuperoxides peroxides) which can generate potential dam-age to mammalian deoxyribonucleic acid (DNA) [1] Oxida-tive stress arises due to an imbalance between productionand consumption of ROS and presence of metals exacerbatesthe generation of free radicals Copper is one of the mostredox-active metal ions present in cells and closely associatedwith chromatin Copper concentration in tissues increasesin various malignancies [2] Hydroxyl radicals ( ∙OH) arereleased in the superoxide driven Fenton reaction whereCu (I) gets oxidized to Cu (II) in the presence of hydrogenperoxide [3] and causes DNA damage Because copper ionscatalyze hydroxyl radical formation in vivo the copper levelis maintained low to protect the cell Hydrogen peroxide haspoor radical activity but at higher concentration (gt50120583M)through the formation of hydroxyl radical it can cause

DNA strand breakage and base modification [4] The extentof hydroxyl radicals causing damage to DNA of diabeticpatients is higher compared to healthy humans [5] ROS isinvolved in blocking the pathway between insulin-receptorsubstrate (IRS-1) and phosphatidylinositol-45-bisphosphate3-kinase to result in high insulin resistance [6] Antioxi-dant enzymes (superoxide dismutase glutathione peroxidasecatalase) and nonenzymatic antioxidants (vitamin E vitaminC glutathione) offer detoxification to a certain extent Whenexcess free radicals are produced these antioxidant systemsare not adequate to meet the requirement necessitatingconsumption of natural food source rich in polyphenolsPolyphenol compounds are routinely studied for their radicalscavenging activity [7] There is a growing interest to find themode of interaction between phytochemical compounds andDNA In our study the hypothesis was proposed to know thepotential of caffeine curcumin and naringenin to protect theDNA damage that may arise from stress related to diabetes

Coffee is the most happily consumed beverage world-wide Hence knowing the beneficial and harmful effectof its intake on human health is crucial Caffeine (137-trimethylxanthine) is a bitter white crystalline alkaloid

Hindawi Publishing CorporationScientificaVolume 2014 Article ID 649261 7 pageshttpdxdoiorg1011552014649261

2 Scientifica

present as a legal stimulant in coffee Caffeine contributesto the antioxidant value of coffee and removal of caffeineresulted in 25ndash30 reduction in antioxidant value [8] Regu-lar intake of coffee (mean daily intake sim480mL) contributesto 64 of total antioxidant capacity in humans [9] Presenceof caffeine reduces insulin sensitivity in humans [10 11] Incontrast there are reports on the beneficial effect of caffeineconsumption on type 2 diabetic patients where it lowersthe diabetes risk [12ndash15] Caffeine ingestion reduced whole-body glucose disposal to 24 and carbohydrate storageby 35 compared to placebo administration [16] Thereis a marked reduction in blood glucose level of diabeticpatients consuming caffeinated instant coffee for 16 weeks[17]

Curcumin (diferuloylmethane) is a natural polyphenolcompound present in turmeric Presence of ortho-methoxyphenolic OH and 120573-diketone moiety of curcumin offers theability to directly quench ROS and prevent oxidative damage[18] Curcumin can exist in many tautomeric forms themost commonly occurring forms are the 13-di-keto formand two enol forms The di-keto form is primarily involvedin deprotonation and enolates thereby contributing to itsactive antioxidative capabilities The ability of curcumin toreduce blood sugar level dates back to early years [19] andcurcuminoids (NCB-02) intake (150mgtwice a day) by dia-betic patients improved endothelial function and reduced theoxidative stress and inflammatory markers [20] Curcuminbinds with DNA along the ldquominor grooverdquo and it may causeDNA damage in the cell at high concentration [21]

Naringenin (4101584057-trihydroxyflavanone) is a flavanonepresent in many citrus fruits for example grapefruit [22]Peak plasma concentration for naringenin following inges-tion of grapefruit juice indicates its beneficial effect [23]Naringenin improved glucose stimulated insulin secretionand glucose sensitivity in INS-1E cells [24] Naringenin showsanti-inflammatory effect in STZ-induced diabetic rats byblocking NF-120581B pathway [25]

In our study we have investigated the effect of threenutraceutical compounds (caffeine curcumin and narin-genin) on DNA in the presence and absence of coppersulphate through agarose gel electrophoresis and UV-visiblespectrophotometry

2 Materials and Methods

21 Isolation of Genomic DNA Genomic DNA was isolatedfrom whole blood sample of diabetic patients and healthyindividuals using phenolchloroform method The researchfollowed the ethical standards formulated in the HelsinkiDeclaration of 1964 revised in 2000 and written consent wasobtained from the human subjects before collecting the bloodsamples The clinical characteristics of the studied subjectswere shown in Table 1 Though the number of subjectsstudied is limited volume of blood collected was enough toisolate required genomic DNA Each vial containing DNAwas dissolved in 1X TBE buffer and stored in minus20∘C forfurther use The purity of DNA measured using UV-visiblespectrophotometer was in the range of 19-20

22 Stock Solutions Caffeine (137-trimethylxanthine)(HiMedia RM6753) curcumin (diferuloylmethane) (SigmaC-1386) and naringenin (4101584057-trihydroxyflavanone)(Sigma N-5893) were all diluted to 10mM stock solutionswith deionized water just prior to use To avoid the radicalscavenging activity of solvents such as DMSO or ethanoladdition of a small amount of NaOH (20ndash100120583L of 1MNaOH per 10mL polyphenol stock solution) was sufficient toquickly and completely dissolve curcumin and naringeninStock solution of hydrogen peroxide [10mM] was preparedfresh in PBS solution [10mM] and copper sulphate [10mM]was dissolved in water before use The phytochemicals werestored in 4∘C and wrapped with aluminum foil to reducetheir sensitivity to light

23 Induction of DNADamage The reactionmixture (20 120583L)for agarose gel electrophoresis consists of genomic DNA(15 120583g) H

2

O2

phytochemical compounds (caffeine cur-cumin and naringenin) and CuSO

4

at equimolar concen-trations (005mMndash6mM) in phosphate buffered saline atpH 74 Incubation was carried out at 37∘C for 30 minutesfollowed by exposure to ultraviolet radiation for 30 minutesto ensure maximum damage After incubation samples wereloaded onto agarose gel electrophoresis (12) Untreatedgenomic DNA served as a control Each sample was run threetimes to ensure reproducibility and reliability The gels wereplaced in transilluminator to observe DNA damage

24 UV-Vis Absorbance Studies UV-visible spectra of theinteraction between phytocompounds (final concentration50 120583M) and genomic DNA at various final concentrations(18 120583g 36 120583g 72 120583g) in PBS buffer (700120583L) were measuredafter incubation for 1 h at 37∘C The interaction betweenthese phytochemical compounds with normal and diabeticgenomic DNA in the presence of CuSO

4

(50120583M) was alsodetected after 1 h incubation at 37∘C DNA was allowedto interact with phytocompounds for 5 minutes to ensurecomplex formation before it was exposed to copper sul-phate Spectral reading was performed using Evolution 260Bio Thermo Scientific spectrophotometer in the scan rangewavelength of 200ndash500 nm

3 Results

31 Agarose Gel Electrophoresis It has been known that redoxmetals like iron zinc and copper play a major role inexacerbating the DNAdamage along with hydrogen peroxideby Haber-Weiss and Fenton reaction [26] Polyphenols havebeen found to mobilize endogenous copper and bring aboutprooxidant activity on reduction of Cu (II) to Cu (I) Threepolyphenol compounds (caffeine curcumin and naringenin)were evaluated for their potential to cause or prevent damageon normal and diabetic genomic DNA In the case ofnondiabetic DNA (Figures 1(a) 1(b) and 1(c)) hydrogenperoxide or copper sulphate alone cannot cause the damage(L2 L3) but they can cause marked damage (shearing) whenused in combination (L4) At low concentration of caffeinecurcumin (50 120583M) with equimolar hydrogen peroxide and

Scientifica 3

Table 1 Clinical characteristics of studied subjects

Subjects Postprandial bloodglucose level (mgdL) Age (Year) Smoking habits Other symptoms

Patient 1 452 38 Chain smoker Delayed wound healing boils high blood pressure (140100)Patient 2 379 55 Nonsmoker Knee pain poor eyesightPatient 3 337 59 Nonsmoker Knee and back pain numbness of feetPatient 4 308 51 Nonsmoker NonePatient 5 411 50 Nonsmoker Poor eyesight high blood pressure (15090)Patient 6 427 50 Nonsmoker Poor eye sight boilsAll the patients were newly diagnosed with Type 2 Diabetes Mellitus Therefore none of the patients were on any drug administration and insulin treatment

L1 L2 L3 L4 L5 L6 L7 L8 L9 L10

(a)

L1 L2 L3 L4 L5 L6 L7 L8 L9 L10

(b)

L1 L2 L3 L4 L5 L6 L7 L8 L9 L10

(c)

L1 L2 L3 L4 L5 L6 L7 L8 L9 L10

(d)

L1 L2 L3 L4 L5 L6 L7 L8 L9 L10

(e)

L1 L2 L3 L4 L5 L6 L7 L8 L9 L10

(f)

Figure 1 Agarose gel electrophoresis (12) of genomic DNA from nondiabetic human ((a) (b) (c)) and diabetic subject ((d) (e) (f))L1 Genomic DNA (untreated control 15120583g) L2 DNA (15 120583g) + H

2

O2

(4mM) L3 DNA (15120583g) + CuSO4

(4mM) L4 DNA (15120583g) +H2

O2

(4mM) + CuSO4

(4mM) L5 DNA (15120583g) + H2

O2

(50 120583M) + lowastPhytocompound (50 120583M) + CuSO4

(50120583M) L6 DNA (15120583g) + H2

O2

(500 120583M) + lowastPhytocompound (500 120583M) + CuSO4

(500 120583M) L7 DNA (15120583g) + H2

O2

(1mM) + lowastPhytocompound (1mM) + CuSO4

(1mM)L8 DNA (15120583g) +H

2

O2

(2mM) + lowastPhytocompound (2mM) +CuSO4

(2mM) L9 DNA (15120583g) +H2

O2

(4mM) + lowastPhytocompound (4mM)+ CuSO

4

(4mM) L10 DNA (15120583g) + H2

O2

(6mM) + lowastPhytocompound (6mM) + CuSO4

(6mM) lowastCaffeine curcumin and naringenin

copper sulphate the damage was not observed (Figures1(a) and 1(b) L5) but they displayed shearing at higherconcentrations (4mM 6mM) (Figures 1(a) and 1(b) L9 L10)Caffeine showed the damage even at 2mM concentration(Figure 1(a) L8) Surprisingly naringenin offers protectionat higher concentrations (Figure 1(c) L9 L10) but it causesdamage from 500 120583M to 2mM (Figure 1(c) L6ndashL8) In theDNA sample obtained from diabetic patients (Figures 1(d)1(e) and 1(f)) adding either hydrogen peroxide or coppersulphate resulted in shearing of DNA (Figures 1(d) and 1(f)L2 L3) In combination they had displayed the damage(Figures 1(d) and 1(e) L4) suggesting the significant roleplayed by copper in mediating havoc There is a pronouncedshearing effect when caffeine curcumin and naringenin usedequimolar alongwith hydrogen peroxide and copper sulphateat concentration from 500120583M to 6mM (Figures 1(d) 1(e)and 1(f) L6ndashL10) Caffeine starts to show damage even at low

concentration of 50120583M (Figure 1(d) L5) This is in contrastto the observed behaviour in nondiabetic DNA

32 UV-Vis Absorbance Studies Caffeine exhibited anabsorption maximum at 272 nm On addition of DNAsample at varying concentrations to caffeine the absorbanceincreased linearly in proportion to increase in DNAconcentration (Figures 2(a) and 2(d)) The same pattern wasobserved in the presence of copper Curcumin displayed anabsorption maximum at 430 nm When DNA was addedto curcumin the spectral absorbance reduced linearly withincreasing DNA concentration (Figures 2(b) and 2(e)) Thisis in contrast to the absorbance pattern of caffeine Onaddition of copper to DNA-curcumin the peak disappearedNaringenin showed an absorption maximum at 321 nm Onaddition of diabetic DNA sample at varying concentrations to

4 Scientifica

Wavelength (nm)

Abso

rban

ce (A

)1009080706050403020100

200 220 240 260 280 300 320 340

Nondiabetic DNA+ caffeine plusmn copper

(a)

18161412100806040200

Nondiabetic DNA+ curcumin plusmn copper

200 240 280 320 360 400 440 480

Wavelength (nm)

Abso

rban

ce (A

)

(b)

18161412100806040200

Abso

rban

ce (A

)

Nondiabetic DNA+ nar plusmn copper

360

Wavelength (nm)200 240 280 320

(c)

14

12

Diabetic DNA + caffeine plusmn copper

Wavelength (nm)

Abso

rban

ce (A

)

10

08

06

04

02

00

200 220 240 260 280 300 320 340

Caffeine 50120583MDNA 18120583g + caff 50120583MDNA 36120583g + caff 50120583MDNA 72120583g + caff 50120583MDNA 18120583g + caff 50120583M + Cu 50120583MDNA 36120583g + caff 50120583M + Cu 50120583MDNA 72120583g + caff 50120583M + Cu 50120583M

DNA alone 461120583g

(d)

08

09

06

07

04

05

02

03

01

00

Diabetic DNA+ curcumin plusmn copper

200 240 280 320 360 400 440 480

Wavelength (nm)

Abso

rban

ce (A

)

DNA alone 461120583gCurcumin 25120583MDNA 18120583g + cur 50120583MDNA 36120583g + cur 50120583MDNA 72120583g + cur 50120583MDNA 18120583g + cur 50120583M + Cu 50120583MDNA 36120583g + cur 50120583M + Cu 50120583MDNA 72120583g + cur 50120583M + Cu 50120583M

(e)

Diabetic DNA + nar plusmn copper16

14

12

10

08

06

04

02

00

200 240 280 320 360

Wavelength (nm)

Abso

rban

ce (A

)

DNA alone 461120583g

DNA 18120583g + nar 50120583MDNA 36120583g + nar 50120583MDNA 72120583g + nar 50120583MDNA 18120583g + nar 50120583M + Cu 50120583MDNA 36120583g + nar 50120583M + Cu 50120583MDNA 72120583g + nar 50120583M + Cu 50120583M

Naringenin 57120583M

(f)

Figure 2 Spectra of caffeine curcumin and naringenin with DNA from nondiabetic ((a) (b) (c)) and diabetic subject ((d) (e) (f)) in thepresence and absence of copper

naringenin the absorbance increased linearly with increasein DNA concentration and new peak emerged at 270 nmboth in the presence and absence of copper (Figure 2(f))The characteristic peak of naringenin alone at 321 nm wasnot observed in DNA-naringenin with and without copper(Figures 2(c) and 2(f))

4 Discussion

41 AgaroseGel Electrophoresis When genomicDNAof non-diabetic subjects was treated with equimolar concentrationsof hydrogen peroxide or copper sulphate (4mM each) therewas no damage observed (Figures 1(a) 1(b) and 1(c) L2L3) but marked shearing was observed in the presence ofhydrogen peroxide and copper sulphate together at 4mM(Figures 1(a) and 1(b) L4) These results imply that copperions might react with hydrogen peroxide to generate DNA-damaging hydroxyl free radicals Use of caffeine and cur-cumin failed to rescue the damage at the same concentration(4mM)but offered protection at the concentrations of 50120583M500120583M and 1mM Polyphenols when used on their ownmay not have the strong ability to bind DNA and causedamage but in the presence of copper these compounds

confer damage through redox-cycling pathway of copper ionsin a concentration dependent manner In the case of DNAsamples obtained from diabetic subjects hydrogen peroxideor copper sulphate on their own and in combination pro-duced damage (Figures 1(d) and 1(f) L2ndashL4) This speculatesthat DNA of diabetic sample is much prone to be attackedreadily by stress inducing chemicals And on treatment withcaffeine or naringenin at the same concentration (4mM)these phytocompounds failed to relieve the damage (Figures1(d) and 1(f) L9) Curcumin behaves as antioxidant at50 120583M concentration (Figure 1(e) L5) whereas caffeine actsas prooxidant at the same concentration (Figure 1(d) L5)but all the three phytocompounds produced severe damageat higher concentrations starting from 500120583M to 6mMThis suggests that caffeine and curcumin confer dichotomouseffect (prooxidant and antioxidant) against DNA damageinduced in the presence of copper and hydrogen peroxideThe reason for observing DNA damage at lower concentra-tions might be due to the insufficient number of availablecaffeinemolecules to scavenge hydroxyl radicals so that theseradicals are left free to cause damage to DNA

Hyperglycemia induced oxidative stress promotes pro-duction of reactive oxygen species (ROS) [27] Like Janus

Scientifica 5

the two-faced roman God ROS gives beneficial and harmfuleffect when being present in low and high concentrationsrespectively At low concentrations they help in combatinginfectious agents thus protecting the cell and at high con-centrations they cause oxidative stress Overproduction ofROS in the presence of limited availability of antioxidantsaugments the oxidative stress induced damage on proteinslipids andDNA impairing their function [28]This demandsthe need for maintaining ldquoredox equilibriumrdquo between pro-oxidant system and antioxidant system which plays a keyrole in various diseases like aging obesity diabetes andcancer Prooxidant activity of polyphenols in the presence ofcopper has been studied [29] According to the structure-activity relationship of compounds in polyphenols com-pounds bearing hydroxyl groups are found to be most activein inducing DNA damage in the presence of copper [30]This high prooxidant activity of polyphenols is due to thepresence of hydroxyl groups which are capable of chelatingwith copper to form polyphenol-copper complex that enablesintramolecular electron transfer to form semiquinone radicalanion Reacting with O

2

the radical undergoes a secondelectron transfer to form quinones and O2minus At neutralpH O2minus protonates and forms H

2

O2

H2

O2

may also beformed by dismutation of O2minus Thus H

2

O2

can immediatelyparticipate in Fenton-type ∙OH formation andDNA cleavagereaction In addition quinones are reportedly involved inDNA damage by forming covalent adducts with DNA [31]In brief the formation of the hydroxyl radical and thecopper-redox cycle play a key role in inducing DNA damageAmong the oxygen radicals the hydroxyl group is the mostelectrophilic radical with high reactivity and possesses a smalldiffusion radius Thus to cleave DNA the hydroxyl radicalshould be produced in the vicinity of DNA [32] Green teapolyphenols have the ability to cause oxidative DNA damagein the vicinity of Cu (II) [33]

42 UV-Vis Absorbance Studies Our spectral results showthe increased absorbance (hyperchromism) observed onaddition of DNA to caffeine both in the presence andabsence of copper (Figures 2(a) and 2(d)) We speculate thismight be due to molecular aggregation and hydrogen bonddamage There is a gradual reduction in spectral absorbance(hypochromism) on binding of curcumin to DNA (Figures2(b) and 2(e)) This implies the possibility of intercalationamong the bases and 120587-120587lowast interaction between DNA andcurcumin [34] Disappearance of peak on addition of copperto DNA-curcumin complex suggests weaker affinity betweenoxygen ligand of curcumin and copper ion (soft Lewis acid)[35] Effect of copper on phytocompounds interaction withDNA is important to know the extent of metal influence onstability It was expected that DNA being a negatively chargedpolyanion could attract cations thereby favouring the stronginteraction There is a conflicting report on the function ofcurcumin-copper complex Curcumin-Cu (II) complex hadshown prooxidant effect [36] Curcumin-copper complex(1 1 and 1 2) displayed in vitro antioxidant activity andsuperoxide dismutase (SOD) activity [37] There is a markedhypochromic shift in the presence of progressively increasingconcentration of DNA due to close connection of 120587 electron

cloud of curcumin with DNA base pairs [38] They didnot observe any unwinding of DNA and conformationalchanges on binding to curcumin and hydrophobic forcesgovern the stability of interaction and not electrostatic forcesIn the interaction studies of DNA with naringenin there isincrease in absorbance (Figures 2(c) and 2(f)) This mightbe due to unwinding of DNA and exposure of the basesThe disappearance of peak at 321 nm and emergence of newpeak at 270 nm in DNA of diabetic subject with naringenin(with and without copper) implies complex formation Effectof various metals (Cu2+ Co2+ Ni2+ K+) on binding ofnaringenin to DNA did not show change in binding constant[39] Spectral studies of Cu (II)-naringin complex and DNAdisplayed intercalative mode of interaction through N(7) ofguanine site [39]

From the gel studies we observe that the complex DNA-caffeine acts as prooxidant even at lower concentration(50 120583M) and the complex DNA-curcuminnaringenin actsas antioxidants This shows the dichotomous behaviour ofphytocompounds Presence of copper enhances the damagein DNA obtained from blood sample of diabetic subjectsSpectral absorbance studies display increase in absorbancewith increasing DNA concentration on binding to caffeinenaringenin and hypochromism on binding to curcumin Wespeculate that unwinding of DNA makes it vulnerable forattack by themetal-aided free radicals and intercalation offersprotection against attack To conclude nutraceuticals likecaffeine curcumin and naringenin may act as inducers aswell as quenchers of oxidative free radicalsThis dichotomousbehaviour can be applied wisely to combat oxidative stressarising from diabetes and cancer

Conflict of Interests

The authors declare that they have no conflict of interests

Authorsrsquo Contribution

Debarati Chattopadhyay Ashok Somaiah andKavithaThiru-murugan conceived and designed the experiments carriedout the analysis and interpreted the results Kavitha Thiru-murugan drafted the paper and others have contributedsignificantly in the refinement Divya Raghunathan carriedout parts of the experiments Debarati Chattopadhyay andAshok Somaiah contributed equally to the paper

Acknowledgments

The authors thank VIT University for providing requiredinfrastructure facilities Thanks are also due to Dr EThirumurugan (Palar Hospital Senthil Clinic amp PharmacyGudiyattam) for helping us to get blood samples of diabeticpatients

References

[1] M S Cooke M D Evans M Dizdaroglu and J LunecldquoOxidative DNA damage mechanisms mutation and diseaserdquoThe FASEB Journal vol 17 no 10 pp 1195ndash1214 2003

6 Scientifica

[2] M Ebara H Fukuda R Hatano et al ldquoRelationship betweencopper zinc and metallothionein in hepatocellular carcinomaand its surrounding liver parenchymardquo Journal of Hepatologyvol 33 no 3 pp 415ndash422 2000

[3] M R Gunther P M Hanna R P Mason and M S CohenldquoHydroxyl radical formation from cuprous ion and hydrogenperoxide a spin-trapping studyrdquo Archives of Biochemistry andBiophysics vol 316 no 1 pp 515ndash522 1995

[4] P A Hyslop D B Hinshaw W A Halsey Jr et al ldquoMech-anisms of oxidant-mediated cell injury The glycolytic andmitochondrial pathways of ADP phosphorylation are majorintracellular targets inactivated by hydrogen peroxiderdquo TheJournal of Biological Chemistry vol 263 no 4 pp 1665ndash16751988

[5] B Halliwell ldquoAntioxidants in human health and diseaserdquoAnnual Review of Nutrition vol 16 pp 33ndash50 1996

[6] A Bloch-Damti R Potashnik P Gual et al ldquoDifferential effectsof IRS1 phosphorylated on Ser307 or Ser632 in the induction ofinsulin resistance by oxidative stressrdquo Diabetologia vol 49 no10 pp 2463ndash2473 2006

[7] N R Perron J N Hodges M Jenkins and J L BrumaghimldquoPredicting how polyphenol antioxidants prevent DNA damageby binding to ironrdquo Inorganic Chemistry vol 47 no 14 pp 6153ndash6161 2008

[8] X Shi N S Dalal and A C Jain ldquoAntioxidant behaviour ofcaffeine efficient scavenging of hydroxyl radicalsrdquo Food andChemical Toxicology vol 29 no 1 pp 1ndash6 1991

[9] A Svilaas A K Sakhi L F Andersen et al ldquoIntakes ofantioxidants in coffee wine and vegetables are correlated withplasma carotenoids in humansrdquo The Journal of Nutrition vol134 no 3 pp 562ndash567 2004

[10] G B Keijzers B E de Galan C J Tack and P Smits ldquoCaffeinecan decrease insulin sensitivity in humansrdquo Diabetes Care vol25 no 2 pp 364ndash369 2002

[11] A Reunanen M Heliovaara and K Aho ldquoCoffee consumptionand risk of type 2 diabetes mellitusrdquo The Lancet vol 361 no9358 pp 702ndash703 2003

[12] R M van Dam and E J M Feskens ldquoCoffee consumption andrisk of type 2 diabetes mellitusrdquo The Lancet vol 360 no 9344pp 1477ndash1478 2002

[13] R M van Dam and F B Hu ldquoCoffee consumption and risk oftype 2 diabetes a systematic reviewrdquo Journal of the AmericanMedical Association vol 294 no 1 pp 97ndash104 2005

[14] N M Wedick A M Brennan Q Sun F B Hu C SMantzoros and R M van Dam ldquoEffects of caffeinated anddecaffeinated coffee on biological risk factors for type 2 diabetesa randomized controlled trialrdquo Nutrition Journal vol 10 article93 2011

[15] X Jiang D Zhang and W Jiang ldquoCoffee and caffeine intakeand incidence of type 2 diabetes mellitus a meta-analysis ofprospective studiesrdquo European Journal of Nutrition vol 53 no1 pp 25ndash38 2014

[16] F Greer R Hudson R Ross and T Graham ldquoCaffeine inges-tion decreases glucose disposal during a hyperinsulinemic-euglycemic clamp in sedentary humansrdquo Diabetes vol 50 no10 pp 2349ndash2354 2001

[17] K Ohnaka M Ikeda T Maki et al ldquoEffects of 16-weekconsumption of caffeinated and decaffeinated instant coffee onglucosemetabolism in a randomized controlled trialrdquo Journal ofNutrition andMetabolism vol 2012 Article ID 207426 9 pages2012

[18] U SinghA Barik BG Singh andK I Priyadarsini ldquoReactionsof reactive oxygen species (ROS) with curcumin analoguesstructure-activity relationshiprdquo Free Radical Research vol 45no 3 pp 317ndash325 2011

[19] M Srinivasan ldquoEffect of curcumin on blood sugar as seen in adiabetic subjectrdquo Indian Journal of Medical Sciences vol 26 no4 pp 269ndash270 1972

[20] P Usharani A A Mateen M U R Naidu Y S N Rajuand N Chandra ldquoEffect of NCB-02 atorvastatin and placeboon endothelial function oxidative stress and inflammatorymarkers in patients with type 2 diabetes mellitus a randomizedparallel-group placebo-controlled 8-week studyrdquoDrugs in R ampD vol 9 no 4 pp 243ndash250 2008

[21] A Kumar and U Bora ldquoInteractions of curcumin and itsderivatives with nucleic acids and their implicationsrdquo Mini-Reviews inMedicinal Chemistry vol 13 no 2 pp 256ndash264 2013

[22] C Felgines O Texier C Morand et al ldquoBioavailability ofthe flavanone naringenin and its glycosides in ratsrdquo AmericanJournal of PhysiologymdashGastrointestinal and Liver Physiologyvol 279 no 6 pp G1148ndashG1154 2000

[23] I Erlund E Meririnne G Alfthan and A Aro ldquoPlasmakinetics and urinary excretion of the flavanones naringeninand hesperetin in humans after ingestion of orange juice andgrapefruit juicerdquoThe Journal of Nutrition vol 131 no 2 pp 235ndash241 2001

[24] S Bhattacharya N Oksbjerg J F Young and P B JeppesenldquoCaffeic acid naringenin and quercetin enhance glucose stim-ulated insulin secretion and glucose sensitivity in INS-1E cellsrdquoDiabetes Obesity and Metabolism 2013

[25] T Annadurai P A Thomas and P Geraldine ldquoAmeliora-tive effect of naringenin on hyperglycemia-mediated inflam-mation in hepatic and pancreatic tissues of Wistar ratswith streptozotocin-nicotinamide-induced experimental dia-betes mellitusrdquo Free Radical Research vol 47 no 10 pp 793ndash803 2013

[26] R Meneghini ldquoIron homeostasis oxidative stress and DNAdamagerdquoFree Radical BiologyampMedicine vol 23 no 5 pp 783ndash792 1997

[27] T V Fiorentino A Prioletta P Zuo and F FollildquoHyperglycemia-induced oxidative stress and its role indiabetes mellitus related cardiovascular diseasesrdquo CurrentPharmaceutical Design vol 19 no 32 pp 5695ndash5703 2013

[28] G D Stoner L-S Wang and B C Casto ldquoLaboratory andclinical studies of cancer chemoprevention by antioxidants inberriesrdquo Carcinogenesis vol 29 no 9 pp 1665ndash1674 2008

[29] N Schweigert A J B Zehnder and R I L Eggen ldquoChemicalproperties of catechols and their molecular modes of toxicaction in cells from microorganisms to mammalsrdquo Environ-mental Microbiology vol 3 no 2 pp 81ndash91 2001

[30] L-F Zheng Q-Y Wei Y-J Cai et al ldquoDNA damage inducedby resveratrol and its synthetic analogues in the presence of Cu(II) ions mechanism and structure-activity relationshiprdquo FreeRadical Biology amp Medicine vol 41 no 12 pp 1807ndash1816 2006

[31] AM Samuni E Y ChuangM C Krishna et al ldquoSemiquinoneradical intermediate in catecholic estrogen-mediated cytotoxi-city and mutagenesis chemoprevention strategies with antiox-idantsrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 100 no 9 pp 5390ndash5395 2003

[32] A S Azmi S H Bhat and S M Hadi ldquoResveratrol-Cu(II)induced DNA breakage in human peripheral lymphocytesimplications for anticancer propertiesrdquo The FEBS Letters vol579 no 14 pp 3131ndash3135 2005

Scientifica 7

[33] A Malik S Azam N Hadi and S M Hadi ldquoDNA degradationby water extract of green tea in the presence of copper ionsimplications for anticancer propertiesrdquo Phytotherapy Researchvol 17 no 4 pp 358ndash363 2003

[34] Z Li X Yang S Dong and X Li ldquoDNa breakage inducedby piceatannol and copper(II) mechanism and anticancerpropertiesrdquo Oncology Letters vol 3 no 5 pp 1087ndash1094 2012

[35] M H Leung D T Pham S F Lincoln and T W KeeldquoFemtosecond transient absorption spectroscopy of copper(II)-curcumin complexesrdquo Physical Chemistry Chemical Physics vol14 no 39 pp 13580ndash13587 2012

[36] A Barik B Mishra A Kunwar et al ldquoComparative study ofcopper(II)-curcumin complexes as superoxide dismutase mim-ics and free radical scavengersrdquo European Journal of MedicinalChemistry vol 42 no 4 pp 431ndash439 2007

[37] A Basu and G S Kumar ldquoBiophysical studies on curcumin-deoxyribonucleic acid interaction spectroscopic and calori-metric approachrdquo International Journal of Biological Macro-molecules vol 62 pp 257ndash264 2013

[38] A H Hegde S N Prashanth and J Seetharamappa ldquoInterac-tion of antioxidant flavonoids with calf thymus DNA analyzedby spectroscopic and electrochemical methodsrdquo Journal ofPharmaceutical and Biomedical Analysis vol 63 pp 40ndash462012

[39] L D Mello R M S Pereira A C H F Sawaya M NEberlin and L T Kubota ldquoElectrochemical and spectroscopiccharacterization of the interaction between DNA and Cu(II)-naringin complexrdquo Journal of Pharmaceutical and BiomedicalAnalysis vol 45 no 5 pp 706ndash713 2007

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

2 Scientifica

present as a legal stimulant in coffee Caffeine contributesto the antioxidant value of coffee and removal of caffeineresulted in 25ndash30 reduction in antioxidant value [8] Regu-lar intake of coffee (mean daily intake sim480mL) contributesto 64 of total antioxidant capacity in humans [9] Presenceof caffeine reduces insulin sensitivity in humans [10 11] Incontrast there are reports on the beneficial effect of caffeineconsumption on type 2 diabetic patients where it lowersthe diabetes risk [12ndash15] Caffeine ingestion reduced whole-body glucose disposal to 24 and carbohydrate storageby 35 compared to placebo administration [16] Thereis a marked reduction in blood glucose level of diabeticpatients consuming caffeinated instant coffee for 16 weeks[17]

Curcumin (diferuloylmethane) is a natural polyphenolcompound present in turmeric Presence of ortho-methoxyphenolic OH and 120573-diketone moiety of curcumin offers theability to directly quench ROS and prevent oxidative damage[18] Curcumin can exist in many tautomeric forms themost commonly occurring forms are the 13-di-keto formand two enol forms The di-keto form is primarily involvedin deprotonation and enolates thereby contributing to itsactive antioxidative capabilities The ability of curcumin toreduce blood sugar level dates back to early years [19] andcurcuminoids (NCB-02) intake (150mgtwice a day) by dia-betic patients improved endothelial function and reduced theoxidative stress and inflammatory markers [20] Curcuminbinds with DNA along the ldquominor grooverdquo and it may causeDNA damage in the cell at high concentration [21]

Naringenin (4101584057-trihydroxyflavanone) is a flavanonepresent in many citrus fruits for example grapefruit [22]Peak plasma concentration for naringenin following inges-tion of grapefruit juice indicates its beneficial effect [23]Naringenin improved glucose stimulated insulin secretionand glucose sensitivity in INS-1E cells [24] Naringenin showsanti-inflammatory effect in STZ-induced diabetic rats byblocking NF-120581B pathway [25]

In our study we have investigated the effect of threenutraceutical compounds (caffeine curcumin and narin-genin) on DNA in the presence and absence of coppersulphate through agarose gel electrophoresis and UV-visiblespectrophotometry

2 Materials and Methods

21 Isolation of Genomic DNA Genomic DNA was isolatedfrom whole blood sample of diabetic patients and healthyindividuals using phenolchloroform method The researchfollowed the ethical standards formulated in the HelsinkiDeclaration of 1964 revised in 2000 and written consent wasobtained from the human subjects before collecting the bloodsamples The clinical characteristics of the studied subjectswere shown in Table 1 Though the number of subjectsstudied is limited volume of blood collected was enough toisolate required genomic DNA Each vial containing DNAwas dissolved in 1X TBE buffer and stored in minus20∘C forfurther use The purity of DNA measured using UV-visiblespectrophotometer was in the range of 19-20

22 Stock Solutions Caffeine (137-trimethylxanthine)(HiMedia RM6753) curcumin (diferuloylmethane) (SigmaC-1386) and naringenin (4101584057-trihydroxyflavanone)(Sigma N-5893) were all diluted to 10mM stock solutionswith deionized water just prior to use To avoid the radicalscavenging activity of solvents such as DMSO or ethanoladdition of a small amount of NaOH (20ndash100120583L of 1MNaOH per 10mL polyphenol stock solution) was sufficient toquickly and completely dissolve curcumin and naringeninStock solution of hydrogen peroxide [10mM] was preparedfresh in PBS solution [10mM] and copper sulphate [10mM]was dissolved in water before use The phytochemicals werestored in 4∘C and wrapped with aluminum foil to reducetheir sensitivity to light

23 Induction of DNADamage The reactionmixture (20 120583L)for agarose gel electrophoresis consists of genomic DNA(15 120583g) H

2

O2

phytochemical compounds (caffeine cur-cumin and naringenin) and CuSO

4

at equimolar concen-trations (005mMndash6mM) in phosphate buffered saline atpH 74 Incubation was carried out at 37∘C for 30 minutesfollowed by exposure to ultraviolet radiation for 30 minutesto ensure maximum damage After incubation samples wereloaded onto agarose gel electrophoresis (12) Untreatedgenomic DNA served as a control Each sample was run threetimes to ensure reproducibility and reliability The gels wereplaced in transilluminator to observe DNA damage

24 UV-Vis Absorbance Studies UV-visible spectra of theinteraction between phytocompounds (final concentration50 120583M) and genomic DNA at various final concentrations(18 120583g 36 120583g 72 120583g) in PBS buffer (700120583L) were measuredafter incubation for 1 h at 37∘C The interaction betweenthese phytochemical compounds with normal and diabeticgenomic DNA in the presence of CuSO

4

(50120583M) was alsodetected after 1 h incubation at 37∘C DNA was allowedto interact with phytocompounds for 5 minutes to ensurecomplex formation before it was exposed to copper sul-phate Spectral reading was performed using Evolution 260Bio Thermo Scientific spectrophotometer in the scan rangewavelength of 200ndash500 nm

3 Results

31 Agarose Gel Electrophoresis It has been known that redoxmetals like iron zinc and copper play a major role inexacerbating the DNAdamage along with hydrogen peroxideby Haber-Weiss and Fenton reaction [26] Polyphenols havebeen found to mobilize endogenous copper and bring aboutprooxidant activity on reduction of Cu (II) to Cu (I) Threepolyphenol compounds (caffeine curcumin and naringenin)were evaluated for their potential to cause or prevent damageon normal and diabetic genomic DNA In the case ofnondiabetic DNA (Figures 1(a) 1(b) and 1(c)) hydrogenperoxide or copper sulphate alone cannot cause the damage(L2 L3) but they can cause marked damage (shearing) whenused in combination (L4) At low concentration of caffeinecurcumin (50 120583M) with equimolar hydrogen peroxide and

Scientifica 3

Table 1 Clinical characteristics of studied subjects

Subjects Postprandial bloodglucose level (mgdL) Age (Year) Smoking habits Other symptoms

Patient 1 452 38 Chain smoker Delayed wound healing boils high blood pressure (140100)Patient 2 379 55 Nonsmoker Knee pain poor eyesightPatient 3 337 59 Nonsmoker Knee and back pain numbness of feetPatient 4 308 51 Nonsmoker NonePatient 5 411 50 Nonsmoker Poor eyesight high blood pressure (15090)Patient 6 427 50 Nonsmoker Poor eye sight boilsAll the patients were newly diagnosed with Type 2 Diabetes Mellitus Therefore none of the patients were on any drug administration and insulin treatment

L1 L2 L3 L4 L5 L6 L7 L8 L9 L10

(a)

L1 L2 L3 L4 L5 L6 L7 L8 L9 L10

(b)

L1 L2 L3 L4 L5 L6 L7 L8 L9 L10

(c)

L1 L2 L3 L4 L5 L6 L7 L8 L9 L10

(d)

L1 L2 L3 L4 L5 L6 L7 L8 L9 L10

(e)

L1 L2 L3 L4 L5 L6 L7 L8 L9 L10

(f)

Figure 1 Agarose gel electrophoresis (12) of genomic DNA from nondiabetic human ((a) (b) (c)) and diabetic subject ((d) (e) (f))L1 Genomic DNA (untreated control 15120583g) L2 DNA (15 120583g) + H

2

O2

(4mM) L3 DNA (15120583g) + CuSO4

(4mM) L4 DNA (15120583g) +H2

O2

(4mM) + CuSO4

(4mM) L5 DNA (15120583g) + H2

O2

(50 120583M) + lowastPhytocompound (50 120583M) + CuSO4

(50120583M) L6 DNA (15120583g) + H2

O2

(500 120583M) + lowastPhytocompound (500 120583M) + CuSO4

(500 120583M) L7 DNA (15120583g) + H2

O2

(1mM) + lowastPhytocompound (1mM) + CuSO4

(1mM)L8 DNA (15120583g) +H

2

O2

(2mM) + lowastPhytocompound (2mM) +CuSO4

(2mM) L9 DNA (15120583g) +H2

O2

(4mM) + lowastPhytocompound (4mM)+ CuSO

4

(4mM) L10 DNA (15120583g) + H2

O2

(6mM) + lowastPhytocompound (6mM) + CuSO4

(6mM) lowastCaffeine curcumin and naringenin

copper sulphate the damage was not observed (Figures1(a) and 1(b) L5) but they displayed shearing at higherconcentrations (4mM 6mM) (Figures 1(a) and 1(b) L9 L10)Caffeine showed the damage even at 2mM concentration(Figure 1(a) L8) Surprisingly naringenin offers protectionat higher concentrations (Figure 1(c) L9 L10) but it causesdamage from 500 120583M to 2mM (Figure 1(c) L6ndashL8) In theDNA sample obtained from diabetic patients (Figures 1(d)1(e) and 1(f)) adding either hydrogen peroxide or coppersulphate resulted in shearing of DNA (Figures 1(d) and 1(f)L2 L3) In combination they had displayed the damage(Figures 1(d) and 1(e) L4) suggesting the significant roleplayed by copper in mediating havoc There is a pronouncedshearing effect when caffeine curcumin and naringenin usedequimolar alongwith hydrogen peroxide and copper sulphateat concentration from 500120583M to 6mM (Figures 1(d) 1(e)and 1(f) L6ndashL10) Caffeine starts to show damage even at low

concentration of 50120583M (Figure 1(d) L5) This is in contrastto the observed behaviour in nondiabetic DNA

32 UV-Vis Absorbance Studies Caffeine exhibited anabsorption maximum at 272 nm On addition of DNAsample at varying concentrations to caffeine the absorbanceincreased linearly in proportion to increase in DNAconcentration (Figures 2(a) and 2(d)) The same pattern wasobserved in the presence of copper Curcumin displayed anabsorption maximum at 430 nm When DNA was addedto curcumin the spectral absorbance reduced linearly withincreasing DNA concentration (Figures 2(b) and 2(e)) Thisis in contrast to the absorbance pattern of caffeine Onaddition of copper to DNA-curcumin the peak disappearedNaringenin showed an absorption maximum at 321 nm Onaddition of diabetic DNA sample at varying concentrations to

4 Scientifica

Wavelength (nm)

Abso

rban

ce (A

)1009080706050403020100

200 220 240 260 280 300 320 340

Nondiabetic DNA+ caffeine plusmn copper

(a)

18161412100806040200

Nondiabetic DNA+ curcumin plusmn copper

200 240 280 320 360 400 440 480

Wavelength (nm)

Abso

rban

ce (A

)

(b)

18161412100806040200

Abso

rban

ce (A

)

Nondiabetic DNA+ nar plusmn copper

360

Wavelength (nm)200 240 280 320

(c)

14

12

Diabetic DNA + caffeine plusmn copper

Wavelength (nm)

Abso

rban

ce (A

)

10

08

06

04

02

00

200 220 240 260 280 300 320 340

Caffeine 50120583MDNA 18120583g + caff 50120583MDNA 36120583g + caff 50120583MDNA 72120583g + caff 50120583MDNA 18120583g + caff 50120583M + Cu 50120583MDNA 36120583g + caff 50120583M + Cu 50120583MDNA 72120583g + caff 50120583M + Cu 50120583M

DNA alone 461120583g

(d)

08

09

06

07

04

05

02

03

01

00

Diabetic DNA+ curcumin plusmn copper

200 240 280 320 360 400 440 480

Wavelength (nm)

Abso

rban

ce (A

)

DNA alone 461120583gCurcumin 25120583MDNA 18120583g + cur 50120583MDNA 36120583g + cur 50120583MDNA 72120583g + cur 50120583MDNA 18120583g + cur 50120583M + Cu 50120583MDNA 36120583g + cur 50120583M + Cu 50120583MDNA 72120583g + cur 50120583M + Cu 50120583M

(e)

Diabetic DNA + nar plusmn copper16

14

12

10

08

06

04

02

00

200 240 280 320 360

Wavelength (nm)

Abso

rban

ce (A

)

DNA alone 461120583g

DNA 18120583g + nar 50120583MDNA 36120583g + nar 50120583MDNA 72120583g + nar 50120583MDNA 18120583g + nar 50120583M + Cu 50120583MDNA 36120583g + nar 50120583M + Cu 50120583MDNA 72120583g + nar 50120583M + Cu 50120583M

Naringenin 57120583M

(f)

Figure 2 Spectra of caffeine curcumin and naringenin with DNA from nondiabetic ((a) (b) (c)) and diabetic subject ((d) (e) (f)) in thepresence and absence of copper

naringenin the absorbance increased linearly with increasein DNA concentration and new peak emerged at 270 nmboth in the presence and absence of copper (Figure 2(f))The characteristic peak of naringenin alone at 321 nm wasnot observed in DNA-naringenin with and without copper(Figures 2(c) and 2(f))

4 Discussion

41 AgaroseGel Electrophoresis When genomicDNAof non-diabetic subjects was treated with equimolar concentrationsof hydrogen peroxide or copper sulphate (4mM each) therewas no damage observed (Figures 1(a) 1(b) and 1(c) L2L3) but marked shearing was observed in the presence ofhydrogen peroxide and copper sulphate together at 4mM(Figures 1(a) and 1(b) L4) These results imply that copperions might react with hydrogen peroxide to generate DNA-damaging hydroxyl free radicals Use of caffeine and cur-cumin failed to rescue the damage at the same concentration(4mM)but offered protection at the concentrations of 50120583M500120583M and 1mM Polyphenols when used on their ownmay not have the strong ability to bind DNA and causedamage but in the presence of copper these compounds

confer damage through redox-cycling pathway of copper ionsin a concentration dependent manner In the case of DNAsamples obtained from diabetic subjects hydrogen peroxideor copper sulphate on their own and in combination pro-duced damage (Figures 1(d) and 1(f) L2ndashL4) This speculatesthat DNA of diabetic sample is much prone to be attackedreadily by stress inducing chemicals And on treatment withcaffeine or naringenin at the same concentration (4mM)these phytocompounds failed to relieve the damage (Figures1(d) and 1(f) L9) Curcumin behaves as antioxidant at50 120583M concentration (Figure 1(e) L5) whereas caffeine actsas prooxidant at the same concentration (Figure 1(d) L5)but all the three phytocompounds produced severe damageat higher concentrations starting from 500120583M to 6mMThis suggests that caffeine and curcumin confer dichotomouseffect (prooxidant and antioxidant) against DNA damageinduced in the presence of copper and hydrogen peroxideThe reason for observing DNA damage at lower concentra-tions might be due to the insufficient number of availablecaffeinemolecules to scavenge hydroxyl radicals so that theseradicals are left free to cause damage to DNA

Hyperglycemia induced oxidative stress promotes pro-duction of reactive oxygen species (ROS) [27] Like Janus

Scientifica 5

the two-faced roman God ROS gives beneficial and harmfuleffect when being present in low and high concentrationsrespectively At low concentrations they help in combatinginfectious agents thus protecting the cell and at high con-centrations they cause oxidative stress Overproduction ofROS in the presence of limited availability of antioxidantsaugments the oxidative stress induced damage on proteinslipids andDNA impairing their function [28]This demandsthe need for maintaining ldquoredox equilibriumrdquo between pro-oxidant system and antioxidant system which plays a keyrole in various diseases like aging obesity diabetes andcancer Prooxidant activity of polyphenols in the presence ofcopper has been studied [29] According to the structure-activity relationship of compounds in polyphenols com-pounds bearing hydroxyl groups are found to be most activein inducing DNA damage in the presence of copper [30]This high prooxidant activity of polyphenols is due to thepresence of hydroxyl groups which are capable of chelatingwith copper to form polyphenol-copper complex that enablesintramolecular electron transfer to form semiquinone radicalanion Reacting with O

2

the radical undergoes a secondelectron transfer to form quinones and O2minus At neutralpH O2minus protonates and forms H

2

O2

H2

O2

may also beformed by dismutation of O2minus Thus H

2

O2

can immediatelyparticipate in Fenton-type ∙OH formation andDNA cleavagereaction In addition quinones are reportedly involved inDNA damage by forming covalent adducts with DNA [31]In brief the formation of the hydroxyl radical and thecopper-redox cycle play a key role in inducing DNA damageAmong the oxygen radicals the hydroxyl group is the mostelectrophilic radical with high reactivity and possesses a smalldiffusion radius Thus to cleave DNA the hydroxyl radicalshould be produced in the vicinity of DNA [32] Green teapolyphenols have the ability to cause oxidative DNA damagein the vicinity of Cu (II) [33]

42 UV-Vis Absorbance Studies Our spectral results showthe increased absorbance (hyperchromism) observed onaddition of DNA to caffeine both in the presence andabsence of copper (Figures 2(a) and 2(d)) We speculate thismight be due to molecular aggregation and hydrogen bonddamage There is a gradual reduction in spectral absorbance(hypochromism) on binding of curcumin to DNA (Figures2(b) and 2(e)) This implies the possibility of intercalationamong the bases and 120587-120587lowast interaction between DNA andcurcumin [34] Disappearance of peak on addition of copperto DNA-curcumin complex suggests weaker affinity betweenoxygen ligand of curcumin and copper ion (soft Lewis acid)[35] Effect of copper on phytocompounds interaction withDNA is important to know the extent of metal influence onstability It was expected that DNA being a negatively chargedpolyanion could attract cations thereby favouring the stronginteraction There is a conflicting report on the function ofcurcumin-copper complex Curcumin-Cu (II) complex hadshown prooxidant effect [36] Curcumin-copper complex(1 1 and 1 2) displayed in vitro antioxidant activity andsuperoxide dismutase (SOD) activity [37] There is a markedhypochromic shift in the presence of progressively increasingconcentration of DNA due to close connection of 120587 electron

cloud of curcumin with DNA base pairs [38] They didnot observe any unwinding of DNA and conformationalchanges on binding to curcumin and hydrophobic forcesgovern the stability of interaction and not electrostatic forcesIn the interaction studies of DNA with naringenin there isincrease in absorbance (Figures 2(c) and 2(f)) This mightbe due to unwinding of DNA and exposure of the basesThe disappearance of peak at 321 nm and emergence of newpeak at 270 nm in DNA of diabetic subject with naringenin(with and without copper) implies complex formation Effectof various metals (Cu2+ Co2+ Ni2+ K+) on binding ofnaringenin to DNA did not show change in binding constant[39] Spectral studies of Cu (II)-naringin complex and DNAdisplayed intercalative mode of interaction through N(7) ofguanine site [39]

From the gel studies we observe that the complex DNA-caffeine acts as prooxidant even at lower concentration(50 120583M) and the complex DNA-curcuminnaringenin actsas antioxidants This shows the dichotomous behaviour ofphytocompounds Presence of copper enhances the damagein DNA obtained from blood sample of diabetic subjectsSpectral absorbance studies display increase in absorbancewith increasing DNA concentration on binding to caffeinenaringenin and hypochromism on binding to curcumin Wespeculate that unwinding of DNA makes it vulnerable forattack by themetal-aided free radicals and intercalation offersprotection against attack To conclude nutraceuticals likecaffeine curcumin and naringenin may act as inducers aswell as quenchers of oxidative free radicalsThis dichotomousbehaviour can be applied wisely to combat oxidative stressarising from diabetes and cancer

Conflict of Interests

The authors declare that they have no conflict of interests

Authorsrsquo Contribution

Debarati Chattopadhyay Ashok Somaiah andKavithaThiru-murugan conceived and designed the experiments carriedout the analysis and interpreted the results Kavitha Thiru-murugan drafted the paper and others have contributedsignificantly in the refinement Divya Raghunathan carriedout parts of the experiments Debarati Chattopadhyay andAshok Somaiah contributed equally to the paper

Acknowledgments

The authors thank VIT University for providing requiredinfrastructure facilities Thanks are also due to Dr EThirumurugan (Palar Hospital Senthil Clinic amp PharmacyGudiyattam) for helping us to get blood samples of diabeticpatients

References

[1] M S Cooke M D Evans M Dizdaroglu and J LunecldquoOxidative DNA damage mechanisms mutation and diseaserdquoThe FASEB Journal vol 17 no 10 pp 1195ndash1214 2003

6 Scientifica

[2] M Ebara H Fukuda R Hatano et al ldquoRelationship betweencopper zinc and metallothionein in hepatocellular carcinomaand its surrounding liver parenchymardquo Journal of Hepatologyvol 33 no 3 pp 415ndash422 2000

[3] M R Gunther P M Hanna R P Mason and M S CohenldquoHydroxyl radical formation from cuprous ion and hydrogenperoxide a spin-trapping studyrdquo Archives of Biochemistry andBiophysics vol 316 no 1 pp 515ndash522 1995

[4] P A Hyslop D B Hinshaw W A Halsey Jr et al ldquoMech-anisms of oxidant-mediated cell injury The glycolytic andmitochondrial pathways of ADP phosphorylation are majorintracellular targets inactivated by hydrogen peroxiderdquo TheJournal of Biological Chemistry vol 263 no 4 pp 1665ndash16751988

[5] B Halliwell ldquoAntioxidants in human health and diseaserdquoAnnual Review of Nutrition vol 16 pp 33ndash50 1996

[6] A Bloch-Damti R Potashnik P Gual et al ldquoDifferential effectsof IRS1 phosphorylated on Ser307 or Ser632 in the induction ofinsulin resistance by oxidative stressrdquo Diabetologia vol 49 no10 pp 2463ndash2473 2006

[7] N R Perron J N Hodges M Jenkins and J L BrumaghimldquoPredicting how polyphenol antioxidants prevent DNA damageby binding to ironrdquo Inorganic Chemistry vol 47 no 14 pp 6153ndash6161 2008

[8] X Shi N S Dalal and A C Jain ldquoAntioxidant behaviour ofcaffeine efficient scavenging of hydroxyl radicalsrdquo Food andChemical Toxicology vol 29 no 1 pp 1ndash6 1991

[9] A Svilaas A K Sakhi L F Andersen et al ldquoIntakes ofantioxidants in coffee wine and vegetables are correlated withplasma carotenoids in humansrdquo The Journal of Nutrition vol134 no 3 pp 562ndash567 2004

[10] G B Keijzers B E de Galan C J Tack and P Smits ldquoCaffeinecan decrease insulin sensitivity in humansrdquo Diabetes Care vol25 no 2 pp 364ndash369 2002

[11] A Reunanen M Heliovaara and K Aho ldquoCoffee consumptionand risk of type 2 diabetes mellitusrdquo The Lancet vol 361 no9358 pp 702ndash703 2003

[12] R M van Dam and E J M Feskens ldquoCoffee consumption andrisk of type 2 diabetes mellitusrdquo The Lancet vol 360 no 9344pp 1477ndash1478 2002

[13] R M van Dam and F B Hu ldquoCoffee consumption and risk oftype 2 diabetes a systematic reviewrdquo Journal of the AmericanMedical Association vol 294 no 1 pp 97ndash104 2005

[14] N M Wedick A M Brennan Q Sun F B Hu C SMantzoros and R M van Dam ldquoEffects of caffeinated anddecaffeinated coffee on biological risk factors for type 2 diabetesa randomized controlled trialrdquo Nutrition Journal vol 10 article93 2011

[15] X Jiang D Zhang and W Jiang ldquoCoffee and caffeine intakeand incidence of type 2 diabetes mellitus a meta-analysis ofprospective studiesrdquo European Journal of Nutrition vol 53 no1 pp 25ndash38 2014

[16] F Greer R Hudson R Ross and T Graham ldquoCaffeine inges-tion decreases glucose disposal during a hyperinsulinemic-euglycemic clamp in sedentary humansrdquo Diabetes vol 50 no10 pp 2349ndash2354 2001

[17] K Ohnaka M Ikeda T Maki et al ldquoEffects of 16-weekconsumption of caffeinated and decaffeinated instant coffee onglucosemetabolism in a randomized controlled trialrdquo Journal ofNutrition andMetabolism vol 2012 Article ID 207426 9 pages2012

[18] U SinghA Barik BG Singh andK I Priyadarsini ldquoReactionsof reactive oxygen species (ROS) with curcumin analoguesstructure-activity relationshiprdquo Free Radical Research vol 45no 3 pp 317ndash325 2011

[19] M Srinivasan ldquoEffect of curcumin on blood sugar as seen in adiabetic subjectrdquo Indian Journal of Medical Sciences vol 26 no4 pp 269ndash270 1972

[20] P Usharani A A Mateen M U R Naidu Y S N Rajuand N Chandra ldquoEffect of NCB-02 atorvastatin and placeboon endothelial function oxidative stress and inflammatorymarkers in patients with type 2 diabetes mellitus a randomizedparallel-group placebo-controlled 8-week studyrdquoDrugs in R ampD vol 9 no 4 pp 243ndash250 2008

[21] A Kumar and U Bora ldquoInteractions of curcumin and itsderivatives with nucleic acids and their implicationsrdquo Mini-Reviews inMedicinal Chemistry vol 13 no 2 pp 256ndash264 2013

[22] C Felgines O Texier C Morand et al ldquoBioavailability ofthe flavanone naringenin and its glycosides in ratsrdquo AmericanJournal of PhysiologymdashGastrointestinal and Liver Physiologyvol 279 no 6 pp G1148ndashG1154 2000

[23] I Erlund E Meririnne G Alfthan and A Aro ldquoPlasmakinetics and urinary excretion of the flavanones naringeninand hesperetin in humans after ingestion of orange juice andgrapefruit juicerdquoThe Journal of Nutrition vol 131 no 2 pp 235ndash241 2001

[24] S Bhattacharya N Oksbjerg J F Young and P B JeppesenldquoCaffeic acid naringenin and quercetin enhance glucose stim-ulated insulin secretion and glucose sensitivity in INS-1E cellsrdquoDiabetes Obesity and Metabolism 2013

[25] T Annadurai P A Thomas and P Geraldine ldquoAmeliora-tive effect of naringenin on hyperglycemia-mediated inflam-mation in hepatic and pancreatic tissues of Wistar ratswith streptozotocin-nicotinamide-induced experimental dia-betes mellitusrdquo Free Radical Research vol 47 no 10 pp 793ndash803 2013

[26] R Meneghini ldquoIron homeostasis oxidative stress and DNAdamagerdquoFree Radical BiologyampMedicine vol 23 no 5 pp 783ndash792 1997

[27] T V Fiorentino A Prioletta P Zuo and F FollildquoHyperglycemia-induced oxidative stress and its role indiabetes mellitus related cardiovascular diseasesrdquo CurrentPharmaceutical Design vol 19 no 32 pp 5695ndash5703 2013

[28] G D Stoner L-S Wang and B C Casto ldquoLaboratory andclinical studies of cancer chemoprevention by antioxidants inberriesrdquo Carcinogenesis vol 29 no 9 pp 1665ndash1674 2008

[29] N Schweigert A J B Zehnder and R I L Eggen ldquoChemicalproperties of catechols and their molecular modes of toxicaction in cells from microorganisms to mammalsrdquo Environ-mental Microbiology vol 3 no 2 pp 81ndash91 2001

[30] L-F Zheng Q-Y Wei Y-J Cai et al ldquoDNA damage inducedby resveratrol and its synthetic analogues in the presence of Cu(II) ions mechanism and structure-activity relationshiprdquo FreeRadical Biology amp Medicine vol 41 no 12 pp 1807ndash1816 2006

[31] AM Samuni E Y ChuangM C Krishna et al ldquoSemiquinoneradical intermediate in catecholic estrogen-mediated cytotoxi-city and mutagenesis chemoprevention strategies with antiox-idantsrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 100 no 9 pp 5390ndash5395 2003

[32] A S Azmi S H Bhat and S M Hadi ldquoResveratrol-Cu(II)induced DNA breakage in human peripheral lymphocytesimplications for anticancer propertiesrdquo The FEBS Letters vol579 no 14 pp 3131ndash3135 2005

Scientifica 7

[33] A Malik S Azam N Hadi and S M Hadi ldquoDNA degradationby water extract of green tea in the presence of copper ionsimplications for anticancer propertiesrdquo Phytotherapy Researchvol 17 no 4 pp 358ndash363 2003

[34] Z Li X Yang S Dong and X Li ldquoDNa breakage inducedby piceatannol and copper(II) mechanism and anticancerpropertiesrdquo Oncology Letters vol 3 no 5 pp 1087ndash1094 2012

[35] M H Leung D T Pham S F Lincoln and T W KeeldquoFemtosecond transient absorption spectroscopy of copper(II)-curcumin complexesrdquo Physical Chemistry Chemical Physics vol14 no 39 pp 13580ndash13587 2012

[36] A Barik B Mishra A Kunwar et al ldquoComparative study ofcopper(II)-curcumin complexes as superoxide dismutase mim-ics and free radical scavengersrdquo European Journal of MedicinalChemistry vol 42 no 4 pp 431ndash439 2007

[37] A Basu and G S Kumar ldquoBiophysical studies on curcumin-deoxyribonucleic acid interaction spectroscopic and calori-metric approachrdquo International Journal of Biological Macro-molecules vol 62 pp 257ndash264 2013

[38] A H Hegde S N Prashanth and J Seetharamappa ldquoInterac-tion of antioxidant flavonoids with calf thymus DNA analyzedby spectroscopic and electrochemical methodsrdquo Journal ofPharmaceutical and Biomedical Analysis vol 63 pp 40ndash462012

[39] L D Mello R M S Pereira A C H F Sawaya M NEberlin and L T Kubota ldquoElectrochemical and spectroscopiccharacterization of the interaction between DNA and Cu(II)-naringin complexrdquo Journal of Pharmaceutical and BiomedicalAnalysis vol 45 no 5 pp 706ndash713 2007

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Scientifica 3

Table 1 Clinical characteristics of studied subjects

Subjects Postprandial bloodglucose level (mgdL) Age (Year) Smoking habits Other symptoms

Patient 1 452 38 Chain smoker Delayed wound healing boils high blood pressure (140100)Patient 2 379 55 Nonsmoker Knee pain poor eyesightPatient 3 337 59 Nonsmoker Knee and back pain numbness of feetPatient 4 308 51 Nonsmoker NonePatient 5 411 50 Nonsmoker Poor eyesight high blood pressure (15090)Patient 6 427 50 Nonsmoker Poor eye sight boilsAll the patients were newly diagnosed with Type 2 Diabetes Mellitus Therefore none of the patients were on any drug administration and insulin treatment

L1 L2 L3 L4 L5 L6 L7 L8 L9 L10

(a)

L1 L2 L3 L4 L5 L6 L7 L8 L9 L10

(b)

L1 L2 L3 L4 L5 L6 L7 L8 L9 L10

(c)

L1 L2 L3 L4 L5 L6 L7 L8 L9 L10

(d)

L1 L2 L3 L4 L5 L6 L7 L8 L9 L10

(e)

L1 L2 L3 L4 L5 L6 L7 L8 L9 L10

(f)

Figure 1 Agarose gel electrophoresis (12) of genomic DNA from nondiabetic human ((a) (b) (c)) and diabetic subject ((d) (e) (f))L1 Genomic DNA (untreated control 15120583g) L2 DNA (15 120583g) + H

2

O2

(4mM) L3 DNA (15120583g) + CuSO4

(4mM) L4 DNA (15120583g) +H2

O2

(4mM) + CuSO4

(4mM) L5 DNA (15120583g) + H2

O2

(50 120583M) + lowastPhytocompound (50 120583M) + CuSO4

(50120583M) L6 DNA (15120583g) + H2

O2

(500 120583M) + lowastPhytocompound (500 120583M) + CuSO4

(500 120583M) L7 DNA (15120583g) + H2

O2

(1mM) + lowastPhytocompound (1mM) + CuSO4

(1mM)L8 DNA (15120583g) +H

2

O2

(2mM) + lowastPhytocompound (2mM) +CuSO4

(2mM) L9 DNA (15120583g) +H2

O2

(4mM) + lowastPhytocompound (4mM)+ CuSO

4

(4mM) L10 DNA (15120583g) + H2

O2

(6mM) + lowastPhytocompound (6mM) + CuSO4

(6mM) lowastCaffeine curcumin and naringenin

copper sulphate the damage was not observed (Figures1(a) and 1(b) L5) but they displayed shearing at higherconcentrations (4mM 6mM) (Figures 1(a) and 1(b) L9 L10)Caffeine showed the damage even at 2mM concentration(Figure 1(a) L8) Surprisingly naringenin offers protectionat higher concentrations (Figure 1(c) L9 L10) but it causesdamage from 500 120583M to 2mM (Figure 1(c) L6ndashL8) In theDNA sample obtained from diabetic patients (Figures 1(d)1(e) and 1(f)) adding either hydrogen peroxide or coppersulphate resulted in shearing of DNA (Figures 1(d) and 1(f)L2 L3) In combination they had displayed the damage(Figures 1(d) and 1(e) L4) suggesting the significant roleplayed by copper in mediating havoc There is a pronouncedshearing effect when caffeine curcumin and naringenin usedequimolar alongwith hydrogen peroxide and copper sulphateat concentration from 500120583M to 6mM (Figures 1(d) 1(e)and 1(f) L6ndashL10) Caffeine starts to show damage even at low

concentration of 50120583M (Figure 1(d) L5) This is in contrastto the observed behaviour in nondiabetic DNA

32 UV-Vis Absorbance Studies Caffeine exhibited anabsorption maximum at 272 nm On addition of DNAsample at varying concentrations to caffeine the absorbanceincreased linearly in proportion to increase in DNAconcentration (Figures 2(a) and 2(d)) The same pattern wasobserved in the presence of copper Curcumin displayed anabsorption maximum at 430 nm When DNA was addedto curcumin the spectral absorbance reduced linearly withincreasing DNA concentration (Figures 2(b) and 2(e)) Thisis in contrast to the absorbance pattern of caffeine Onaddition of copper to DNA-curcumin the peak disappearedNaringenin showed an absorption maximum at 321 nm Onaddition of diabetic DNA sample at varying concentrations to

4 Scientifica

Wavelength (nm)

Abso

rban

ce (A

)1009080706050403020100

200 220 240 260 280 300 320 340

Nondiabetic DNA+ caffeine plusmn copper

(a)

18161412100806040200

Nondiabetic DNA+ curcumin plusmn copper

200 240 280 320 360 400 440 480

Wavelength (nm)

Abso

rban

ce (A

)

(b)

18161412100806040200

Abso

rban

ce (A

)

Nondiabetic DNA+ nar plusmn copper

360

Wavelength (nm)200 240 280 320

(c)

14

12

Diabetic DNA + caffeine plusmn copper

Wavelength (nm)

Abso

rban

ce (A

)

10

08

06

04

02

00

200 220 240 260 280 300 320 340

Caffeine 50120583MDNA 18120583g + caff 50120583MDNA 36120583g + caff 50120583MDNA 72120583g + caff 50120583MDNA 18120583g + caff 50120583M + Cu 50120583MDNA 36120583g + caff 50120583M + Cu 50120583MDNA 72120583g + caff 50120583M + Cu 50120583M

DNA alone 461120583g

(d)

08

09

06

07

04

05

02

03

01

00

Diabetic DNA+ curcumin plusmn copper

200 240 280 320 360 400 440 480

Wavelength (nm)

Abso

rban

ce (A

)

DNA alone 461120583gCurcumin 25120583MDNA 18120583g + cur 50120583MDNA 36120583g + cur 50120583MDNA 72120583g + cur 50120583MDNA 18120583g + cur 50120583M + Cu 50120583MDNA 36120583g + cur 50120583M + Cu 50120583MDNA 72120583g + cur 50120583M + Cu 50120583M

(e)

Diabetic DNA + nar plusmn copper16

14

12

10

08

06

04

02

00

200 240 280 320 360

Wavelength (nm)

Abso

rban

ce (A

)

DNA alone 461120583g

DNA 18120583g + nar 50120583MDNA 36120583g + nar 50120583MDNA 72120583g + nar 50120583MDNA 18120583g + nar 50120583M + Cu 50120583MDNA 36120583g + nar 50120583M + Cu 50120583MDNA 72120583g + nar 50120583M + Cu 50120583M

Naringenin 57120583M

(f)

Figure 2 Spectra of caffeine curcumin and naringenin with DNA from nondiabetic ((a) (b) (c)) and diabetic subject ((d) (e) (f)) in thepresence and absence of copper

naringenin the absorbance increased linearly with increasein DNA concentration and new peak emerged at 270 nmboth in the presence and absence of copper (Figure 2(f))The characteristic peak of naringenin alone at 321 nm wasnot observed in DNA-naringenin with and without copper(Figures 2(c) and 2(f))

4 Discussion

41 AgaroseGel Electrophoresis When genomicDNAof non-diabetic subjects was treated with equimolar concentrationsof hydrogen peroxide or copper sulphate (4mM each) therewas no damage observed (Figures 1(a) 1(b) and 1(c) L2L3) but marked shearing was observed in the presence ofhydrogen peroxide and copper sulphate together at 4mM(Figures 1(a) and 1(b) L4) These results imply that copperions might react with hydrogen peroxide to generate DNA-damaging hydroxyl free radicals Use of caffeine and cur-cumin failed to rescue the damage at the same concentration(4mM)but offered protection at the concentrations of 50120583M500120583M and 1mM Polyphenols when used on their ownmay not have the strong ability to bind DNA and causedamage but in the presence of copper these compounds

confer damage through redox-cycling pathway of copper ionsin a concentration dependent manner In the case of DNAsamples obtained from diabetic subjects hydrogen peroxideor copper sulphate on their own and in combination pro-duced damage (Figures 1(d) and 1(f) L2ndashL4) This speculatesthat DNA of diabetic sample is much prone to be attackedreadily by stress inducing chemicals And on treatment withcaffeine or naringenin at the same concentration (4mM)these phytocompounds failed to relieve the damage (Figures1(d) and 1(f) L9) Curcumin behaves as antioxidant at50 120583M concentration (Figure 1(e) L5) whereas caffeine actsas prooxidant at the same concentration (Figure 1(d) L5)but all the three phytocompounds produced severe damageat higher concentrations starting from 500120583M to 6mMThis suggests that caffeine and curcumin confer dichotomouseffect (prooxidant and antioxidant) against DNA damageinduced in the presence of copper and hydrogen peroxideThe reason for observing DNA damage at lower concentra-tions might be due to the insufficient number of availablecaffeinemolecules to scavenge hydroxyl radicals so that theseradicals are left free to cause damage to DNA

Hyperglycemia induced oxidative stress promotes pro-duction of reactive oxygen species (ROS) [27] Like Janus

Scientifica 5

the two-faced roman God ROS gives beneficial and harmfuleffect when being present in low and high concentrationsrespectively At low concentrations they help in combatinginfectious agents thus protecting the cell and at high con-centrations they cause oxidative stress Overproduction ofROS in the presence of limited availability of antioxidantsaugments the oxidative stress induced damage on proteinslipids andDNA impairing their function [28]This demandsthe need for maintaining ldquoredox equilibriumrdquo between pro-oxidant system and antioxidant system which plays a keyrole in various diseases like aging obesity diabetes andcancer Prooxidant activity of polyphenols in the presence ofcopper has been studied [29] According to the structure-activity relationship of compounds in polyphenols com-pounds bearing hydroxyl groups are found to be most activein inducing DNA damage in the presence of copper [30]This high prooxidant activity of polyphenols is due to thepresence of hydroxyl groups which are capable of chelatingwith copper to form polyphenol-copper complex that enablesintramolecular electron transfer to form semiquinone radicalanion Reacting with O

2

the radical undergoes a secondelectron transfer to form quinones and O2minus At neutralpH O2minus protonates and forms H

2

O2

H2

O2

may also beformed by dismutation of O2minus Thus H

2

O2

can immediatelyparticipate in Fenton-type ∙OH formation andDNA cleavagereaction In addition quinones are reportedly involved inDNA damage by forming covalent adducts with DNA [31]In brief the formation of the hydroxyl radical and thecopper-redox cycle play a key role in inducing DNA damageAmong the oxygen radicals the hydroxyl group is the mostelectrophilic radical with high reactivity and possesses a smalldiffusion radius Thus to cleave DNA the hydroxyl radicalshould be produced in the vicinity of DNA [32] Green teapolyphenols have the ability to cause oxidative DNA damagein the vicinity of Cu (II) [33]

42 UV-Vis Absorbance Studies Our spectral results showthe increased absorbance (hyperchromism) observed onaddition of DNA to caffeine both in the presence andabsence of copper (Figures 2(a) and 2(d)) We speculate thismight be due to molecular aggregation and hydrogen bonddamage There is a gradual reduction in spectral absorbance(hypochromism) on binding of curcumin to DNA (Figures2(b) and 2(e)) This implies the possibility of intercalationamong the bases and 120587-120587lowast interaction between DNA andcurcumin [34] Disappearance of peak on addition of copperto DNA-curcumin complex suggests weaker affinity betweenoxygen ligand of curcumin and copper ion (soft Lewis acid)[35] Effect of copper on phytocompounds interaction withDNA is important to know the extent of metal influence onstability It was expected that DNA being a negatively chargedpolyanion could attract cations thereby favouring the stronginteraction There is a conflicting report on the function ofcurcumin-copper complex Curcumin-Cu (II) complex hadshown prooxidant effect [36] Curcumin-copper complex(1 1 and 1 2) displayed in vitro antioxidant activity andsuperoxide dismutase (SOD) activity [37] There is a markedhypochromic shift in the presence of progressively increasingconcentration of DNA due to close connection of 120587 electron

cloud of curcumin with DNA base pairs [38] They didnot observe any unwinding of DNA and conformationalchanges on binding to curcumin and hydrophobic forcesgovern the stability of interaction and not electrostatic forcesIn the interaction studies of DNA with naringenin there isincrease in absorbance (Figures 2(c) and 2(f)) This mightbe due to unwinding of DNA and exposure of the basesThe disappearance of peak at 321 nm and emergence of newpeak at 270 nm in DNA of diabetic subject with naringenin(with and without copper) implies complex formation Effectof various metals (Cu2+ Co2+ Ni2+ K+) on binding ofnaringenin to DNA did not show change in binding constant[39] Spectral studies of Cu (II)-naringin complex and DNAdisplayed intercalative mode of interaction through N(7) ofguanine site [39]

From the gel studies we observe that the complex DNA-caffeine acts as prooxidant even at lower concentration(50 120583M) and the complex DNA-curcuminnaringenin actsas antioxidants This shows the dichotomous behaviour ofphytocompounds Presence of copper enhances the damagein DNA obtained from blood sample of diabetic subjectsSpectral absorbance studies display increase in absorbancewith increasing DNA concentration on binding to caffeinenaringenin and hypochromism on binding to curcumin Wespeculate that unwinding of DNA makes it vulnerable forattack by themetal-aided free radicals and intercalation offersprotection against attack To conclude nutraceuticals likecaffeine curcumin and naringenin may act as inducers aswell as quenchers of oxidative free radicalsThis dichotomousbehaviour can be applied wisely to combat oxidative stressarising from diabetes and cancer

Conflict of Interests

The authors declare that they have no conflict of interests

Authorsrsquo Contribution

Debarati Chattopadhyay Ashok Somaiah andKavithaThiru-murugan conceived and designed the experiments carriedout the analysis and interpreted the results Kavitha Thiru-murugan drafted the paper and others have contributedsignificantly in the refinement Divya Raghunathan carriedout parts of the experiments Debarati Chattopadhyay andAshok Somaiah contributed equally to the paper

Acknowledgments

The authors thank VIT University for providing requiredinfrastructure facilities Thanks are also due to Dr EThirumurugan (Palar Hospital Senthil Clinic amp PharmacyGudiyattam) for helping us to get blood samples of diabeticpatients

References

[1] M S Cooke M D Evans M Dizdaroglu and J LunecldquoOxidative DNA damage mechanisms mutation and diseaserdquoThe FASEB Journal vol 17 no 10 pp 1195ndash1214 2003

6 Scientifica

[2] M Ebara H Fukuda R Hatano et al ldquoRelationship betweencopper zinc and metallothionein in hepatocellular carcinomaand its surrounding liver parenchymardquo Journal of Hepatologyvol 33 no 3 pp 415ndash422 2000

[3] M R Gunther P M Hanna R P Mason and M S CohenldquoHydroxyl radical formation from cuprous ion and hydrogenperoxide a spin-trapping studyrdquo Archives of Biochemistry andBiophysics vol 316 no 1 pp 515ndash522 1995

[4] P A Hyslop D B Hinshaw W A Halsey Jr et al ldquoMech-anisms of oxidant-mediated cell injury The glycolytic andmitochondrial pathways of ADP phosphorylation are majorintracellular targets inactivated by hydrogen peroxiderdquo TheJournal of Biological Chemistry vol 263 no 4 pp 1665ndash16751988

[5] B Halliwell ldquoAntioxidants in human health and diseaserdquoAnnual Review of Nutrition vol 16 pp 33ndash50 1996

[6] A Bloch-Damti R Potashnik P Gual et al ldquoDifferential effectsof IRS1 phosphorylated on Ser307 or Ser632 in the induction ofinsulin resistance by oxidative stressrdquo Diabetologia vol 49 no10 pp 2463ndash2473 2006

[7] N R Perron J N Hodges M Jenkins and J L BrumaghimldquoPredicting how polyphenol antioxidants prevent DNA damageby binding to ironrdquo Inorganic Chemistry vol 47 no 14 pp 6153ndash6161 2008

[8] X Shi N S Dalal and A C Jain ldquoAntioxidant behaviour ofcaffeine efficient scavenging of hydroxyl radicalsrdquo Food andChemical Toxicology vol 29 no 1 pp 1ndash6 1991

[9] A Svilaas A K Sakhi L F Andersen et al ldquoIntakes ofantioxidants in coffee wine and vegetables are correlated withplasma carotenoids in humansrdquo The Journal of Nutrition vol134 no 3 pp 562ndash567 2004

[10] G B Keijzers B E de Galan C J Tack and P Smits ldquoCaffeinecan decrease insulin sensitivity in humansrdquo Diabetes Care vol25 no 2 pp 364ndash369 2002

[11] A Reunanen M Heliovaara and K Aho ldquoCoffee consumptionand risk of type 2 diabetes mellitusrdquo The Lancet vol 361 no9358 pp 702ndash703 2003

[12] R M van Dam and E J M Feskens ldquoCoffee consumption andrisk of type 2 diabetes mellitusrdquo The Lancet vol 360 no 9344pp 1477ndash1478 2002

[13] R M van Dam and F B Hu ldquoCoffee consumption and risk oftype 2 diabetes a systematic reviewrdquo Journal of the AmericanMedical Association vol 294 no 1 pp 97ndash104 2005

[14] N M Wedick A M Brennan Q Sun F B Hu C SMantzoros and R M van Dam ldquoEffects of caffeinated anddecaffeinated coffee on biological risk factors for type 2 diabetesa randomized controlled trialrdquo Nutrition Journal vol 10 article93 2011

[15] X Jiang D Zhang and W Jiang ldquoCoffee and caffeine intakeand incidence of type 2 diabetes mellitus a meta-analysis ofprospective studiesrdquo European Journal of Nutrition vol 53 no1 pp 25ndash38 2014

[16] F Greer R Hudson R Ross and T Graham ldquoCaffeine inges-tion decreases glucose disposal during a hyperinsulinemic-euglycemic clamp in sedentary humansrdquo Diabetes vol 50 no10 pp 2349ndash2354 2001

[17] K Ohnaka M Ikeda T Maki et al ldquoEffects of 16-weekconsumption of caffeinated and decaffeinated instant coffee onglucosemetabolism in a randomized controlled trialrdquo Journal ofNutrition andMetabolism vol 2012 Article ID 207426 9 pages2012

[18] U SinghA Barik BG Singh andK I Priyadarsini ldquoReactionsof reactive oxygen species (ROS) with curcumin analoguesstructure-activity relationshiprdquo Free Radical Research vol 45no 3 pp 317ndash325 2011

[19] M Srinivasan ldquoEffect of curcumin on blood sugar as seen in adiabetic subjectrdquo Indian Journal of Medical Sciences vol 26 no4 pp 269ndash270 1972

[20] P Usharani A A Mateen M U R Naidu Y S N Rajuand N Chandra ldquoEffect of NCB-02 atorvastatin and placeboon endothelial function oxidative stress and inflammatorymarkers in patients with type 2 diabetes mellitus a randomizedparallel-group placebo-controlled 8-week studyrdquoDrugs in R ampD vol 9 no 4 pp 243ndash250 2008

[21] A Kumar and U Bora ldquoInteractions of curcumin and itsderivatives with nucleic acids and their implicationsrdquo Mini-Reviews inMedicinal Chemistry vol 13 no 2 pp 256ndash264 2013

[22] C Felgines O Texier C Morand et al ldquoBioavailability ofthe flavanone naringenin and its glycosides in ratsrdquo AmericanJournal of PhysiologymdashGastrointestinal and Liver Physiologyvol 279 no 6 pp G1148ndashG1154 2000

[23] I Erlund E Meririnne G Alfthan and A Aro ldquoPlasmakinetics and urinary excretion of the flavanones naringeninand hesperetin in humans after ingestion of orange juice andgrapefruit juicerdquoThe Journal of Nutrition vol 131 no 2 pp 235ndash241 2001

[24] S Bhattacharya N Oksbjerg J F Young and P B JeppesenldquoCaffeic acid naringenin and quercetin enhance glucose stim-ulated insulin secretion and glucose sensitivity in INS-1E cellsrdquoDiabetes Obesity and Metabolism 2013

[25] T Annadurai P A Thomas and P Geraldine ldquoAmeliora-tive effect of naringenin on hyperglycemia-mediated inflam-mation in hepatic and pancreatic tissues of Wistar ratswith streptozotocin-nicotinamide-induced experimental dia-betes mellitusrdquo Free Radical Research vol 47 no 10 pp 793ndash803 2013

[26] R Meneghini ldquoIron homeostasis oxidative stress and DNAdamagerdquoFree Radical BiologyampMedicine vol 23 no 5 pp 783ndash792 1997

[27] T V Fiorentino A Prioletta P Zuo and F FollildquoHyperglycemia-induced oxidative stress and its role indiabetes mellitus related cardiovascular diseasesrdquo CurrentPharmaceutical Design vol 19 no 32 pp 5695ndash5703 2013

[28] G D Stoner L-S Wang and B C Casto ldquoLaboratory andclinical studies of cancer chemoprevention by antioxidants inberriesrdquo Carcinogenesis vol 29 no 9 pp 1665ndash1674 2008

[29] N Schweigert A J B Zehnder and R I L Eggen ldquoChemicalproperties of catechols and their molecular modes of toxicaction in cells from microorganisms to mammalsrdquo Environ-mental Microbiology vol 3 no 2 pp 81ndash91 2001

[30] L-F Zheng Q-Y Wei Y-J Cai et al ldquoDNA damage inducedby resveratrol and its synthetic analogues in the presence of Cu(II) ions mechanism and structure-activity relationshiprdquo FreeRadical Biology amp Medicine vol 41 no 12 pp 1807ndash1816 2006

[31] AM Samuni E Y ChuangM C Krishna et al ldquoSemiquinoneradical intermediate in catecholic estrogen-mediated cytotoxi-city and mutagenesis chemoprevention strategies with antiox-idantsrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 100 no 9 pp 5390ndash5395 2003

[32] A S Azmi S H Bhat and S M Hadi ldquoResveratrol-Cu(II)induced DNA breakage in human peripheral lymphocytesimplications for anticancer propertiesrdquo The FEBS Letters vol579 no 14 pp 3131ndash3135 2005

Scientifica 7

[33] A Malik S Azam N Hadi and S M Hadi ldquoDNA degradationby water extract of green tea in the presence of copper ionsimplications for anticancer propertiesrdquo Phytotherapy Researchvol 17 no 4 pp 358ndash363 2003

[34] Z Li X Yang S Dong and X Li ldquoDNa breakage inducedby piceatannol and copper(II) mechanism and anticancerpropertiesrdquo Oncology Letters vol 3 no 5 pp 1087ndash1094 2012

[35] M H Leung D T Pham S F Lincoln and T W KeeldquoFemtosecond transient absorption spectroscopy of copper(II)-curcumin complexesrdquo Physical Chemistry Chemical Physics vol14 no 39 pp 13580ndash13587 2012

[36] A Barik B Mishra A Kunwar et al ldquoComparative study ofcopper(II)-curcumin complexes as superoxide dismutase mim-ics and free radical scavengersrdquo European Journal of MedicinalChemistry vol 42 no 4 pp 431ndash439 2007

[37] A Basu and G S Kumar ldquoBiophysical studies on curcumin-deoxyribonucleic acid interaction spectroscopic and calori-metric approachrdquo International Journal of Biological Macro-molecules vol 62 pp 257ndash264 2013

[38] A H Hegde S N Prashanth and J Seetharamappa ldquoInterac-tion of antioxidant flavonoids with calf thymus DNA analyzedby spectroscopic and electrochemical methodsrdquo Journal ofPharmaceutical and Biomedical Analysis vol 63 pp 40ndash462012

[39] L D Mello R M S Pereira A C H F Sawaya M NEberlin and L T Kubota ldquoElectrochemical and spectroscopiccharacterization of the interaction between DNA and Cu(II)-naringin complexrdquo Journal of Pharmaceutical and BiomedicalAnalysis vol 45 no 5 pp 706ndash713 2007

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

4 Scientifica

Wavelength (nm)

Abso

rban

ce (A

)1009080706050403020100

200 220 240 260 280 300 320 340

Nondiabetic DNA+ caffeine plusmn copper

(a)

18161412100806040200

Nondiabetic DNA+ curcumin plusmn copper

200 240 280 320 360 400 440 480

Wavelength (nm)

Abso

rban

ce (A

)

(b)

18161412100806040200

Abso

rban

ce (A

)

Nondiabetic DNA+ nar plusmn copper

360

Wavelength (nm)200 240 280 320

(c)

14

12

Diabetic DNA + caffeine plusmn copper

Wavelength (nm)

Abso

rban

ce (A

)

10

08

06

04

02

00

200 220 240 260 280 300 320 340

Caffeine 50120583MDNA 18120583g + caff 50120583MDNA 36120583g + caff 50120583MDNA 72120583g + caff 50120583MDNA 18120583g + caff 50120583M + Cu 50120583MDNA 36120583g + caff 50120583M + Cu 50120583MDNA 72120583g + caff 50120583M + Cu 50120583M

DNA alone 461120583g

(d)

08

09

06

07

04

05

02

03

01

00

Diabetic DNA+ curcumin plusmn copper

200 240 280 320 360 400 440 480

Wavelength (nm)

Abso

rban

ce (A

)

DNA alone 461120583gCurcumin 25120583MDNA 18120583g + cur 50120583MDNA 36120583g + cur 50120583MDNA 72120583g + cur 50120583MDNA 18120583g + cur 50120583M + Cu 50120583MDNA 36120583g + cur 50120583M + Cu 50120583MDNA 72120583g + cur 50120583M + Cu 50120583M

(e)

Diabetic DNA + nar plusmn copper16

14

12

10

08

06

04

02

00

200 240 280 320 360

Wavelength (nm)

Abso

rban

ce (A

)

DNA alone 461120583g

DNA 18120583g + nar 50120583MDNA 36120583g + nar 50120583MDNA 72120583g + nar 50120583MDNA 18120583g + nar 50120583M + Cu 50120583MDNA 36120583g + nar 50120583M + Cu 50120583MDNA 72120583g + nar 50120583M + Cu 50120583M

Naringenin 57120583M

(f)

Figure 2 Spectra of caffeine curcumin and naringenin with DNA from nondiabetic ((a) (b) (c)) and diabetic subject ((d) (e) (f)) in thepresence and absence of copper

naringenin the absorbance increased linearly with increasein DNA concentration and new peak emerged at 270 nmboth in the presence and absence of copper (Figure 2(f))The characteristic peak of naringenin alone at 321 nm wasnot observed in DNA-naringenin with and without copper(Figures 2(c) and 2(f))

4 Discussion

41 AgaroseGel Electrophoresis When genomicDNAof non-diabetic subjects was treated with equimolar concentrationsof hydrogen peroxide or copper sulphate (4mM each) therewas no damage observed (Figures 1(a) 1(b) and 1(c) L2L3) but marked shearing was observed in the presence ofhydrogen peroxide and copper sulphate together at 4mM(Figures 1(a) and 1(b) L4) These results imply that copperions might react with hydrogen peroxide to generate DNA-damaging hydroxyl free radicals Use of caffeine and cur-cumin failed to rescue the damage at the same concentration(4mM)but offered protection at the concentrations of 50120583M500120583M and 1mM Polyphenols when used on their ownmay not have the strong ability to bind DNA and causedamage but in the presence of copper these compounds

confer damage through redox-cycling pathway of copper ionsin a concentration dependent manner In the case of DNAsamples obtained from diabetic subjects hydrogen peroxideor copper sulphate on their own and in combination pro-duced damage (Figures 1(d) and 1(f) L2ndashL4) This speculatesthat DNA of diabetic sample is much prone to be attackedreadily by stress inducing chemicals And on treatment withcaffeine or naringenin at the same concentration (4mM)these phytocompounds failed to relieve the damage (Figures1(d) and 1(f) L9) Curcumin behaves as antioxidant at50 120583M concentration (Figure 1(e) L5) whereas caffeine actsas prooxidant at the same concentration (Figure 1(d) L5)but all the three phytocompounds produced severe damageat higher concentrations starting from 500120583M to 6mMThis suggests that caffeine and curcumin confer dichotomouseffect (prooxidant and antioxidant) against DNA damageinduced in the presence of copper and hydrogen peroxideThe reason for observing DNA damage at lower concentra-tions might be due to the insufficient number of availablecaffeinemolecules to scavenge hydroxyl radicals so that theseradicals are left free to cause damage to DNA

Hyperglycemia induced oxidative stress promotes pro-duction of reactive oxygen species (ROS) [27] Like Janus

Scientifica 5

the two-faced roman God ROS gives beneficial and harmfuleffect when being present in low and high concentrationsrespectively At low concentrations they help in combatinginfectious agents thus protecting the cell and at high con-centrations they cause oxidative stress Overproduction ofROS in the presence of limited availability of antioxidantsaugments the oxidative stress induced damage on proteinslipids andDNA impairing their function [28]This demandsthe need for maintaining ldquoredox equilibriumrdquo between pro-oxidant system and antioxidant system which plays a keyrole in various diseases like aging obesity diabetes andcancer Prooxidant activity of polyphenols in the presence ofcopper has been studied [29] According to the structure-activity relationship of compounds in polyphenols com-pounds bearing hydroxyl groups are found to be most activein inducing DNA damage in the presence of copper [30]This high prooxidant activity of polyphenols is due to thepresence of hydroxyl groups which are capable of chelatingwith copper to form polyphenol-copper complex that enablesintramolecular electron transfer to form semiquinone radicalanion Reacting with O

2

the radical undergoes a secondelectron transfer to form quinones and O2minus At neutralpH O2minus protonates and forms H

2

O2

H2

O2

may also beformed by dismutation of O2minus Thus H

2

O2

can immediatelyparticipate in Fenton-type ∙OH formation andDNA cleavagereaction In addition quinones are reportedly involved inDNA damage by forming covalent adducts with DNA [31]In brief the formation of the hydroxyl radical and thecopper-redox cycle play a key role in inducing DNA damageAmong the oxygen radicals the hydroxyl group is the mostelectrophilic radical with high reactivity and possesses a smalldiffusion radius Thus to cleave DNA the hydroxyl radicalshould be produced in the vicinity of DNA [32] Green teapolyphenols have the ability to cause oxidative DNA damagein the vicinity of Cu (II) [33]

42 UV-Vis Absorbance Studies Our spectral results showthe increased absorbance (hyperchromism) observed onaddition of DNA to caffeine both in the presence andabsence of copper (Figures 2(a) and 2(d)) We speculate thismight be due to molecular aggregation and hydrogen bonddamage There is a gradual reduction in spectral absorbance(hypochromism) on binding of curcumin to DNA (Figures2(b) and 2(e)) This implies the possibility of intercalationamong the bases and 120587-120587lowast interaction between DNA andcurcumin [34] Disappearance of peak on addition of copperto DNA-curcumin complex suggests weaker affinity betweenoxygen ligand of curcumin and copper ion (soft Lewis acid)[35] Effect of copper on phytocompounds interaction withDNA is important to know the extent of metal influence onstability It was expected that DNA being a negatively chargedpolyanion could attract cations thereby favouring the stronginteraction There is a conflicting report on the function ofcurcumin-copper complex Curcumin-Cu (II) complex hadshown prooxidant effect [36] Curcumin-copper complex(1 1 and 1 2) displayed in vitro antioxidant activity andsuperoxide dismutase (SOD) activity [37] There is a markedhypochromic shift in the presence of progressively increasingconcentration of DNA due to close connection of 120587 electron

cloud of curcumin with DNA base pairs [38] They didnot observe any unwinding of DNA and conformationalchanges on binding to curcumin and hydrophobic forcesgovern the stability of interaction and not electrostatic forcesIn the interaction studies of DNA with naringenin there isincrease in absorbance (Figures 2(c) and 2(f)) This mightbe due to unwinding of DNA and exposure of the basesThe disappearance of peak at 321 nm and emergence of newpeak at 270 nm in DNA of diabetic subject with naringenin(with and without copper) implies complex formation Effectof various metals (Cu2+ Co2+ Ni2+ K+) on binding ofnaringenin to DNA did not show change in binding constant[39] Spectral studies of Cu (II)-naringin complex and DNAdisplayed intercalative mode of interaction through N(7) ofguanine site [39]

From the gel studies we observe that the complex DNA-caffeine acts as prooxidant even at lower concentration(50 120583M) and the complex DNA-curcuminnaringenin actsas antioxidants This shows the dichotomous behaviour ofphytocompounds Presence of copper enhances the damagein DNA obtained from blood sample of diabetic subjectsSpectral absorbance studies display increase in absorbancewith increasing DNA concentration on binding to caffeinenaringenin and hypochromism on binding to curcumin Wespeculate that unwinding of DNA makes it vulnerable forattack by themetal-aided free radicals and intercalation offersprotection against attack To conclude nutraceuticals likecaffeine curcumin and naringenin may act as inducers aswell as quenchers of oxidative free radicalsThis dichotomousbehaviour can be applied wisely to combat oxidative stressarising from diabetes and cancer

Conflict of Interests

The authors declare that they have no conflict of interests

Authorsrsquo Contribution

Debarati Chattopadhyay Ashok Somaiah andKavithaThiru-murugan conceived and designed the experiments carriedout the analysis and interpreted the results Kavitha Thiru-murugan drafted the paper and others have contributedsignificantly in the refinement Divya Raghunathan carriedout parts of the experiments Debarati Chattopadhyay andAshok Somaiah contributed equally to the paper

Acknowledgments

The authors thank VIT University for providing requiredinfrastructure facilities Thanks are also due to Dr EThirumurugan (Palar Hospital Senthil Clinic amp PharmacyGudiyattam) for helping us to get blood samples of diabeticpatients

References

[1] M S Cooke M D Evans M Dizdaroglu and J LunecldquoOxidative DNA damage mechanisms mutation and diseaserdquoThe FASEB Journal vol 17 no 10 pp 1195ndash1214 2003

6 Scientifica

[2] M Ebara H Fukuda R Hatano et al ldquoRelationship betweencopper zinc and metallothionein in hepatocellular carcinomaand its surrounding liver parenchymardquo Journal of Hepatologyvol 33 no 3 pp 415ndash422 2000

[3] M R Gunther P M Hanna R P Mason and M S CohenldquoHydroxyl radical formation from cuprous ion and hydrogenperoxide a spin-trapping studyrdquo Archives of Biochemistry andBiophysics vol 316 no 1 pp 515ndash522 1995

[4] P A Hyslop D B Hinshaw W A Halsey Jr et al ldquoMech-anisms of oxidant-mediated cell injury The glycolytic andmitochondrial pathways of ADP phosphorylation are majorintracellular targets inactivated by hydrogen peroxiderdquo TheJournal of Biological Chemistry vol 263 no 4 pp 1665ndash16751988

[5] B Halliwell ldquoAntioxidants in human health and diseaserdquoAnnual Review of Nutrition vol 16 pp 33ndash50 1996

[6] A Bloch-Damti R Potashnik P Gual et al ldquoDifferential effectsof IRS1 phosphorylated on Ser307 or Ser632 in the induction ofinsulin resistance by oxidative stressrdquo Diabetologia vol 49 no10 pp 2463ndash2473 2006

[7] N R Perron J N Hodges M Jenkins and J L BrumaghimldquoPredicting how polyphenol antioxidants prevent DNA damageby binding to ironrdquo Inorganic Chemistry vol 47 no 14 pp 6153ndash6161 2008

[8] X Shi N S Dalal and A C Jain ldquoAntioxidant behaviour ofcaffeine efficient scavenging of hydroxyl radicalsrdquo Food andChemical Toxicology vol 29 no 1 pp 1ndash6 1991

[9] A Svilaas A K Sakhi L F Andersen et al ldquoIntakes ofantioxidants in coffee wine and vegetables are correlated withplasma carotenoids in humansrdquo The Journal of Nutrition vol134 no 3 pp 562ndash567 2004

[10] G B Keijzers B E de Galan C J Tack and P Smits ldquoCaffeinecan decrease insulin sensitivity in humansrdquo Diabetes Care vol25 no 2 pp 364ndash369 2002

[11] A Reunanen M Heliovaara and K Aho ldquoCoffee consumptionand risk of type 2 diabetes mellitusrdquo The Lancet vol 361 no9358 pp 702ndash703 2003

[12] R M van Dam and E J M Feskens ldquoCoffee consumption andrisk of type 2 diabetes mellitusrdquo The Lancet vol 360 no 9344pp 1477ndash1478 2002

[13] R M van Dam and F B Hu ldquoCoffee consumption and risk oftype 2 diabetes a systematic reviewrdquo Journal of the AmericanMedical Association vol 294 no 1 pp 97ndash104 2005

[14] N M Wedick A M Brennan Q Sun F B Hu C SMantzoros and R M van Dam ldquoEffects of caffeinated anddecaffeinated coffee on biological risk factors for type 2 diabetesa randomized controlled trialrdquo Nutrition Journal vol 10 article93 2011

[15] X Jiang D Zhang and W Jiang ldquoCoffee and caffeine intakeand incidence of type 2 diabetes mellitus a meta-analysis ofprospective studiesrdquo European Journal of Nutrition vol 53 no1 pp 25ndash38 2014

[16] F Greer R Hudson R Ross and T Graham ldquoCaffeine inges-tion decreases glucose disposal during a hyperinsulinemic-euglycemic clamp in sedentary humansrdquo Diabetes vol 50 no10 pp 2349ndash2354 2001

[17] K Ohnaka M Ikeda T Maki et al ldquoEffects of 16-weekconsumption of caffeinated and decaffeinated instant coffee onglucosemetabolism in a randomized controlled trialrdquo Journal ofNutrition andMetabolism vol 2012 Article ID 207426 9 pages2012

[18] U SinghA Barik BG Singh andK I Priyadarsini ldquoReactionsof reactive oxygen species (ROS) with curcumin analoguesstructure-activity relationshiprdquo Free Radical Research vol 45no 3 pp 317ndash325 2011

[19] M Srinivasan ldquoEffect of curcumin on blood sugar as seen in adiabetic subjectrdquo Indian Journal of Medical Sciences vol 26 no4 pp 269ndash270 1972

[20] P Usharani A A Mateen M U R Naidu Y S N Rajuand N Chandra ldquoEffect of NCB-02 atorvastatin and placeboon endothelial function oxidative stress and inflammatorymarkers in patients with type 2 diabetes mellitus a randomizedparallel-group placebo-controlled 8-week studyrdquoDrugs in R ampD vol 9 no 4 pp 243ndash250 2008

[21] A Kumar and U Bora ldquoInteractions of curcumin and itsderivatives with nucleic acids and their implicationsrdquo Mini-Reviews inMedicinal Chemistry vol 13 no 2 pp 256ndash264 2013

[22] C Felgines O Texier C Morand et al ldquoBioavailability ofthe flavanone naringenin and its glycosides in ratsrdquo AmericanJournal of PhysiologymdashGastrointestinal and Liver Physiologyvol 279 no 6 pp G1148ndashG1154 2000

[23] I Erlund E Meririnne G Alfthan and A Aro ldquoPlasmakinetics and urinary excretion of the flavanones naringeninand hesperetin in humans after ingestion of orange juice andgrapefruit juicerdquoThe Journal of Nutrition vol 131 no 2 pp 235ndash241 2001

[24] S Bhattacharya N Oksbjerg J F Young and P B JeppesenldquoCaffeic acid naringenin and quercetin enhance glucose stim-ulated insulin secretion and glucose sensitivity in INS-1E cellsrdquoDiabetes Obesity and Metabolism 2013

[25] T Annadurai P A Thomas and P Geraldine ldquoAmeliora-tive effect of naringenin on hyperglycemia-mediated inflam-mation in hepatic and pancreatic tissues of Wistar ratswith streptozotocin-nicotinamide-induced experimental dia-betes mellitusrdquo Free Radical Research vol 47 no 10 pp 793ndash803 2013

[26] R Meneghini ldquoIron homeostasis oxidative stress and DNAdamagerdquoFree Radical BiologyampMedicine vol 23 no 5 pp 783ndash792 1997

[27] T V Fiorentino A Prioletta P Zuo and F FollildquoHyperglycemia-induced oxidative stress and its role indiabetes mellitus related cardiovascular diseasesrdquo CurrentPharmaceutical Design vol 19 no 32 pp 5695ndash5703 2013

[28] G D Stoner L-S Wang and B C Casto ldquoLaboratory andclinical studies of cancer chemoprevention by antioxidants inberriesrdquo Carcinogenesis vol 29 no 9 pp 1665ndash1674 2008

[29] N Schweigert A J B Zehnder and R I L Eggen ldquoChemicalproperties of catechols and their molecular modes of toxicaction in cells from microorganisms to mammalsrdquo Environ-mental Microbiology vol 3 no 2 pp 81ndash91 2001

[30] L-F Zheng Q-Y Wei Y-J Cai et al ldquoDNA damage inducedby resveratrol and its synthetic analogues in the presence of Cu(II) ions mechanism and structure-activity relationshiprdquo FreeRadical Biology amp Medicine vol 41 no 12 pp 1807ndash1816 2006

[31] AM Samuni E Y ChuangM C Krishna et al ldquoSemiquinoneradical intermediate in catecholic estrogen-mediated cytotoxi-city and mutagenesis chemoprevention strategies with antiox-idantsrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 100 no 9 pp 5390ndash5395 2003

[32] A S Azmi S H Bhat and S M Hadi ldquoResveratrol-Cu(II)induced DNA breakage in human peripheral lymphocytesimplications for anticancer propertiesrdquo The FEBS Letters vol579 no 14 pp 3131ndash3135 2005

Scientifica 7

[33] A Malik S Azam N Hadi and S M Hadi ldquoDNA degradationby water extract of green tea in the presence of copper ionsimplications for anticancer propertiesrdquo Phytotherapy Researchvol 17 no 4 pp 358ndash363 2003

[34] Z Li X Yang S Dong and X Li ldquoDNa breakage inducedby piceatannol and copper(II) mechanism and anticancerpropertiesrdquo Oncology Letters vol 3 no 5 pp 1087ndash1094 2012

[35] M H Leung D T Pham S F Lincoln and T W KeeldquoFemtosecond transient absorption spectroscopy of copper(II)-curcumin complexesrdquo Physical Chemistry Chemical Physics vol14 no 39 pp 13580ndash13587 2012

[36] A Barik B Mishra A Kunwar et al ldquoComparative study ofcopper(II)-curcumin complexes as superoxide dismutase mim-ics and free radical scavengersrdquo European Journal of MedicinalChemistry vol 42 no 4 pp 431ndash439 2007

[37] A Basu and G S Kumar ldquoBiophysical studies on curcumin-deoxyribonucleic acid interaction spectroscopic and calori-metric approachrdquo International Journal of Biological Macro-molecules vol 62 pp 257ndash264 2013

[38] A H Hegde S N Prashanth and J Seetharamappa ldquoInterac-tion of antioxidant flavonoids with calf thymus DNA analyzedby spectroscopic and electrochemical methodsrdquo Journal ofPharmaceutical and Biomedical Analysis vol 63 pp 40ndash462012

[39] L D Mello R M S Pereira A C H F Sawaya M NEberlin and L T Kubota ldquoElectrochemical and spectroscopiccharacterization of the interaction between DNA and Cu(II)-naringin complexrdquo Journal of Pharmaceutical and BiomedicalAnalysis vol 45 no 5 pp 706ndash713 2007

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Scientifica 5

the two-faced roman God ROS gives beneficial and harmfuleffect when being present in low and high concentrationsrespectively At low concentrations they help in combatinginfectious agents thus protecting the cell and at high con-centrations they cause oxidative stress Overproduction ofROS in the presence of limited availability of antioxidantsaugments the oxidative stress induced damage on proteinslipids andDNA impairing their function [28]This demandsthe need for maintaining ldquoredox equilibriumrdquo between pro-oxidant system and antioxidant system which plays a keyrole in various diseases like aging obesity diabetes andcancer Prooxidant activity of polyphenols in the presence ofcopper has been studied [29] According to the structure-activity relationship of compounds in polyphenols com-pounds bearing hydroxyl groups are found to be most activein inducing DNA damage in the presence of copper [30]This high prooxidant activity of polyphenols is due to thepresence of hydroxyl groups which are capable of chelatingwith copper to form polyphenol-copper complex that enablesintramolecular electron transfer to form semiquinone radicalanion Reacting with O

2

the radical undergoes a secondelectron transfer to form quinones and O2minus At neutralpH O2minus protonates and forms H

2

O2

H2

O2

may also beformed by dismutation of O2minus Thus H

2

O2

can immediatelyparticipate in Fenton-type ∙OH formation andDNA cleavagereaction In addition quinones are reportedly involved inDNA damage by forming covalent adducts with DNA [31]In brief the formation of the hydroxyl radical and thecopper-redox cycle play a key role in inducing DNA damageAmong the oxygen radicals the hydroxyl group is the mostelectrophilic radical with high reactivity and possesses a smalldiffusion radius Thus to cleave DNA the hydroxyl radicalshould be produced in the vicinity of DNA [32] Green teapolyphenols have the ability to cause oxidative DNA damagein the vicinity of Cu (II) [33]

42 UV-Vis Absorbance Studies Our spectral results showthe increased absorbance (hyperchromism) observed onaddition of DNA to caffeine both in the presence andabsence of copper (Figures 2(a) and 2(d)) We speculate thismight be due to molecular aggregation and hydrogen bonddamage There is a gradual reduction in spectral absorbance(hypochromism) on binding of curcumin to DNA (Figures2(b) and 2(e)) This implies the possibility of intercalationamong the bases and 120587-120587lowast interaction between DNA andcurcumin [34] Disappearance of peak on addition of copperto DNA-curcumin complex suggests weaker affinity betweenoxygen ligand of curcumin and copper ion (soft Lewis acid)[35] Effect of copper on phytocompounds interaction withDNA is important to know the extent of metal influence onstability It was expected that DNA being a negatively chargedpolyanion could attract cations thereby favouring the stronginteraction There is a conflicting report on the function ofcurcumin-copper complex Curcumin-Cu (II) complex hadshown prooxidant effect [36] Curcumin-copper complex(1 1 and 1 2) displayed in vitro antioxidant activity andsuperoxide dismutase (SOD) activity [37] There is a markedhypochromic shift in the presence of progressively increasingconcentration of DNA due to close connection of 120587 electron

cloud of curcumin with DNA base pairs [38] They didnot observe any unwinding of DNA and conformationalchanges on binding to curcumin and hydrophobic forcesgovern the stability of interaction and not electrostatic forcesIn the interaction studies of DNA with naringenin there isincrease in absorbance (Figures 2(c) and 2(f)) This mightbe due to unwinding of DNA and exposure of the basesThe disappearance of peak at 321 nm and emergence of newpeak at 270 nm in DNA of diabetic subject with naringenin(with and without copper) implies complex formation Effectof various metals (Cu2+ Co2+ Ni2+ K+) on binding ofnaringenin to DNA did not show change in binding constant[39] Spectral studies of Cu (II)-naringin complex and DNAdisplayed intercalative mode of interaction through N(7) ofguanine site [39]

From the gel studies we observe that the complex DNA-caffeine acts as prooxidant even at lower concentration(50 120583M) and the complex DNA-curcuminnaringenin actsas antioxidants This shows the dichotomous behaviour ofphytocompounds Presence of copper enhances the damagein DNA obtained from blood sample of diabetic subjectsSpectral absorbance studies display increase in absorbancewith increasing DNA concentration on binding to caffeinenaringenin and hypochromism on binding to curcumin Wespeculate that unwinding of DNA makes it vulnerable forattack by themetal-aided free radicals and intercalation offersprotection against attack To conclude nutraceuticals likecaffeine curcumin and naringenin may act as inducers aswell as quenchers of oxidative free radicalsThis dichotomousbehaviour can be applied wisely to combat oxidative stressarising from diabetes and cancer

Conflict of Interests

The authors declare that they have no conflict of interests

Authorsrsquo Contribution

Debarati Chattopadhyay Ashok Somaiah andKavithaThiru-murugan conceived and designed the experiments carriedout the analysis and interpreted the results Kavitha Thiru-murugan drafted the paper and others have contributedsignificantly in the refinement Divya Raghunathan carriedout parts of the experiments Debarati Chattopadhyay andAshok Somaiah contributed equally to the paper

Acknowledgments

The authors thank VIT University for providing requiredinfrastructure facilities Thanks are also due to Dr EThirumurugan (Palar Hospital Senthil Clinic amp PharmacyGudiyattam) for helping us to get blood samples of diabeticpatients

References

[1] M S Cooke M D Evans M Dizdaroglu and J LunecldquoOxidative DNA damage mechanisms mutation and diseaserdquoThe FASEB Journal vol 17 no 10 pp 1195ndash1214 2003

6 Scientifica

[2] M Ebara H Fukuda R Hatano et al ldquoRelationship betweencopper zinc and metallothionein in hepatocellular carcinomaand its surrounding liver parenchymardquo Journal of Hepatologyvol 33 no 3 pp 415ndash422 2000

[3] M R Gunther P M Hanna R P Mason and M S CohenldquoHydroxyl radical formation from cuprous ion and hydrogenperoxide a spin-trapping studyrdquo Archives of Biochemistry andBiophysics vol 316 no 1 pp 515ndash522 1995

[4] P A Hyslop D B Hinshaw W A Halsey Jr et al ldquoMech-anisms of oxidant-mediated cell injury The glycolytic andmitochondrial pathways of ADP phosphorylation are majorintracellular targets inactivated by hydrogen peroxiderdquo TheJournal of Biological Chemistry vol 263 no 4 pp 1665ndash16751988

[5] B Halliwell ldquoAntioxidants in human health and diseaserdquoAnnual Review of Nutrition vol 16 pp 33ndash50 1996

[6] A Bloch-Damti R Potashnik P Gual et al ldquoDifferential effectsof IRS1 phosphorylated on Ser307 or Ser632 in the induction ofinsulin resistance by oxidative stressrdquo Diabetologia vol 49 no10 pp 2463ndash2473 2006

[7] N R Perron J N Hodges M Jenkins and J L BrumaghimldquoPredicting how polyphenol antioxidants prevent DNA damageby binding to ironrdquo Inorganic Chemistry vol 47 no 14 pp 6153ndash6161 2008

[8] X Shi N S Dalal and A C Jain ldquoAntioxidant behaviour ofcaffeine efficient scavenging of hydroxyl radicalsrdquo Food andChemical Toxicology vol 29 no 1 pp 1ndash6 1991

[9] A Svilaas A K Sakhi L F Andersen et al ldquoIntakes ofantioxidants in coffee wine and vegetables are correlated withplasma carotenoids in humansrdquo The Journal of Nutrition vol134 no 3 pp 562ndash567 2004

[10] G B Keijzers B E de Galan C J Tack and P Smits ldquoCaffeinecan decrease insulin sensitivity in humansrdquo Diabetes Care vol25 no 2 pp 364ndash369 2002

[11] A Reunanen M Heliovaara and K Aho ldquoCoffee consumptionand risk of type 2 diabetes mellitusrdquo The Lancet vol 361 no9358 pp 702ndash703 2003

[12] R M van Dam and E J M Feskens ldquoCoffee consumption andrisk of type 2 diabetes mellitusrdquo The Lancet vol 360 no 9344pp 1477ndash1478 2002

[13] R M van Dam and F B Hu ldquoCoffee consumption and risk oftype 2 diabetes a systematic reviewrdquo Journal of the AmericanMedical Association vol 294 no 1 pp 97ndash104 2005

[14] N M Wedick A M Brennan Q Sun F B Hu C SMantzoros and R M van Dam ldquoEffects of caffeinated anddecaffeinated coffee on biological risk factors for type 2 diabetesa randomized controlled trialrdquo Nutrition Journal vol 10 article93 2011

[15] X Jiang D Zhang and W Jiang ldquoCoffee and caffeine intakeand incidence of type 2 diabetes mellitus a meta-analysis ofprospective studiesrdquo European Journal of Nutrition vol 53 no1 pp 25ndash38 2014

[16] F Greer R Hudson R Ross and T Graham ldquoCaffeine inges-tion decreases glucose disposal during a hyperinsulinemic-euglycemic clamp in sedentary humansrdquo Diabetes vol 50 no10 pp 2349ndash2354 2001

[17] K Ohnaka M Ikeda T Maki et al ldquoEffects of 16-weekconsumption of caffeinated and decaffeinated instant coffee onglucosemetabolism in a randomized controlled trialrdquo Journal ofNutrition andMetabolism vol 2012 Article ID 207426 9 pages2012

[18] U SinghA Barik BG Singh andK I Priyadarsini ldquoReactionsof reactive oxygen species (ROS) with curcumin analoguesstructure-activity relationshiprdquo Free Radical Research vol 45no 3 pp 317ndash325 2011

[19] M Srinivasan ldquoEffect of curcumin on blood sugar as seen in adiabetic subjectrdquo Indian Journal of Medical Sciences vol 26 no4 pp 269ndash270 1972

[20] P Usharani A A Mateen M U R Naidu Y S N Rajuand N Chandra ldquoEffect of NCB-02 atorvastatin and placeboon endothelial function oxidative stress and inflammatorymarkers in patients with type 2 diabetes mellitus a randomizedparallel-group placebo-controlled 8-week studyrdquoDrugs in R ampD vol 9 no 4 pp 243ndash250 2008

[21] A Kumar and U Bora ldquoInteractions of curcumin and itsderivatives with nucleic acids and their implicationsrdquo Mini-Reviews inMedicinal Chemistry vol 13 no 2 pp 256ndash264 2013

[22] C Felgines O Texier C Morand et al ldquoBioavailability ofthe flavanone naringenin and its glycosides in ratsrdquo AmericanJournal of PhysiologymdashGastrointestinal and Liver Physiologyvol 279 no 6 pp G1148ndashG1154 2000

[23] I Erlund E Meririnne G Alfthan and A Aro ldquoPlasmakinetics and urinary excretion of the flavanones naringeninand hesperetin in humans after ingestion of orange juice andgrapefruit juicerdquoThe Journal of Nutrition vol 131 no 2 pp 235ndash241 2001

[24] S Bhattacharya N Oksbjerg J F Young and P B JeppesenldquoCaffeic acid naringenin and quercetin enhance glucose stim-ulated insulin secretion and glucose sensitivity in INS-1E cellsrdquoDiabetes Obesity and Metabolism 2013

[25] T Annadurai P A Thomas and P Geraldine ldquoAmeliora-tive effect of naringenin on hyperglycemia-mediated inflam-mation in hepatic and pancreatic tissues of Wistar ratswith streptozotocin-nicotinamide-induced experimental dia-betes mellitusrdquo Free Radical Research vol 47 no 10 pp 793ndash803 2013

[26] R Meneghini ldquoIron homeostasis oxidative stress and DNAdamagerdquoFree Radical BiologyampMedicine vol 23 no 5 pp 783ndash792 1997

[27] T V Fiorentino A Prioletta P Zuo and F FollildquoHyperglycemia-induced oxidative stress and its role indiabetes mellitus related cardiovascular diseasesrdquo CurrentPharmaceutical Design vol 19 no 32 pp 5695ndash5703 2013

[28] G D Stoner L-S Wang and B C Casto ldquoLaboratory andclinical studies of cancer chemoprevention by antioxidants inberriesrdquo Carcinogenesis vol 29 no 9 pp 1665ndash1674 2008

[29] N Schweigert A J B Zehnder and R I L Eggen ldquoChemicalproperties of catechols and their molecular modes of toxicaction in cells from microorganisms to mammalsrdquo Environ-mental Microbiology vol 3 no 2 pp 81ndash91 2001

[30] L-F Zheng Q-Y Wei Y-J Cai et al ldquoDNA damage inducedby resveratrol and its synthetic analogues in the presence of Cu(II) ions mechanism and structure-activity relationshiprdquo FreeRadical Biology amp Medicine vol 41 no 12 pp 1807ndash1816 2006

[31] AM Samuni E Y ChuangM C Krishna et al ldquoSemiquinoneradical intermediate in catecholic estrogen-mediated cytotoxi-city and mutagenesis chemoprevention strategies with antiox-idantsrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 100 no 9 pp 5390ndash5395 2003

[32] A S Azmi S H Bhat and S M Hadi ldquoResveratrol-Cu(II)induced DNA breakage in human peripheral lymphocytesimplications for anticancer propertiesrdquo The FEBS Letters vol579 no 14 pp 3131ndash3135 2005

Scientifica 7

[33] A Malik S Azam N Hadi and S M Hadi ldquoDNA degradationby water extract of green tea in the presence of copper ionsimplications for anticancer propertiesrdquo Phytotherapy Researchvol 17 no 4 pp 358ndash363 2003

[34] Z Li X Yang S Dong and X Li ldquoDNa breakage inducedby piceatannol and copper(II) mechanism and anticancerpropertiesrdquo Oncology Letters vol 3 no 5 pp 1087ndash1094 2012

[35] M H Leung D T Pham S F Lincoln and T W KeeldquoFemtosecond transient absorption spectroscopy of copper(II)-curcumin complexesrdquo Physical Chemistry Chemical Physics vol14 no 39 pp 13580ndash13587 2012

[36] A Barik B Mishra A Kunwar et al ldquoComparative study ofcopper(II)-curcumin complexes as superoxide dismutase mim-ics and free radical scavengersrdquo European Journal of MedicinalChemistry vol 42 no 4 pp 431ndash439 2007

[37] A Basu and G S Kumar ldquoBiophysical studies on curcumin-deoxyribonucleic acid interaction spectroscopic and calori-metric approachrdquo International Journal of Biological Macro-molecules vol 62 pp 257ndash264 2013

[38] A H Hegde S N Prashanth and J Seetharamappa ldquoInterac-tion of antioxidant flavonoids with calf thymus DNA analyzedby spectroscopic and electrochemical methodsrdquo Journal ofPharmaceutical and Biomedical Analysis vol 63 pp 40ndash462012

[39] L D Mello R M S Pereira A C H F Sawaya M NEberlin and L T Kubota ldquoElectrochemical and spectroscopiccharacterization of the interaction between DNA and Cu(II)-naringin complexrdquo Journal of Pharmaceutical and BiomedicalAnalysis vol 45 no 5 pp 706ndash713 2007

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

6 Scientifica

[2] M Ebara H Fukuda R Hatano et al ldquoRelationship betweencopper zinc and metallothionein in hepatocellular carcinomaand its surrounding liver parenchymardquo Journal of Hepatologyvol 33 no 3 pp 415ndash422 2000

[3] M R Gunther P M Hanna R P Mason and M S CohenldquoHydroxyl radical formation from cuprous ion and hydrogenperoxide a spin-trapping studyrdquo Archives of Biochemistry andBiophysics vol 316 no 1 pp 515ndash522 1995

[4] P A Hyslop D B Hinshaw W A Halsey Jr et al ldquoMech-anisms of oxidant-mediated cell injury The glycolytic andmitochondrial pathways of ADP phosphorylation are majorintracellular targets inactivated by hydrogen peroxiderdquo TheJournal of Biological Chemistry vol 263 no 4 pp 1665ndash16751988

[5] B Halliwell ldquoAntioxidants in human health and diseaserdquoAnnual Review of Nutrition vol 16 pp 33ndash50 1996

[6] A Bloch-Damti R Potashnik P Gual et al ldquoDifferential effectsof IRS1 phosphorylated on Ser307 or Ser632 in the induction ofinsulin resistance by oxidative stressrdquo Diabetologia vol 49 no10 pp 2463ndash2473 2006

[7] N R Perron J N Hodges M Jenkins and J L BrumaghimldquoPredicting how polyphenol antioxidants prevent DNA damageby binding to ironrdquo Inorganic Chemistry vol 47 no 14 pp 6153ndash6161 2008

[8] X Shi N S Dalal and A C Jain ldquoAntioxidant behaviour ofcaffeine efficient scavenging of hydroxyl radicalsrdquo Food andChemical Toxicology vol 29 no 1 pp 1ndash6 1991

[9] A Svilaas A K Sakhi L F Andersen et al ldquoIntakes ofantioxidants in coffee wine and vegetables are correlated withplasma carotenoids in humansrdquo The Journal of Nutrition vol134 no 3 pp 562ndash567 2004

[10] G B Keijzers B E de Galan C J Tack and P Smits ldquoCaffeinecan decrease insulin sensitivity in humansrdquo Diabetes Care vol25 no 2 pp 364ndash369 2002

[11] A Reunanen M Heliovaara and K Aho ldquoCoffee consumptionand risk of type 2 diabetes mellitusrdquo The Lancet vol 361 no9358 pp 702ndash703 2003

[12] R M van Dam and E J M Feskens ldquoCoffee consumption andrisk of type 2 diabetes mellitusrdquo The Lancet vol 360 no 9344pp 1477ndash1478 2002

[13] R M van Dam and F B Hu ldquoCoffee consumption and risk oftype 2 diabetes a systematic reviewrdquo Journal of the AmericanMedical Association vol 294 no 1 pp 97ndash104 2005

[14] N M Wedick A M Brennan Q Sun F B Hu C SMantzoros and R M van Dam ldquoEffects of caffeinated anddecaffeinated coffee on biological risk factors for type 2 diabetesa randomized controlled trialrdquo Nutrition Journal vol 10 article93 2011

[15] X Jiang D Zhang and W Jiang ldquoCoffee and caffeine intakeand incidence of type 2 diabetes mellitus a meta-analysis ofprospective studiesrdquo European Journal of Nutrition vol 53 no1 pp 25ndash38 2014

[16] F Greer R Hudson R Ross and T Graham ldquoCaffeine inges-tion decreases glucose disposal during a hyperinsulinemic-euglycemic clamp in sedentary humansrdquo Diabetes vol 50 no10 pp 2349ndash2354 2001

[17] K Ohnaka M Ikeda T Maki et al ldquoEffects of 16-weekconsumption of caffeinated and decaffeinated instant coffee onglucosemetabolism in a randomized controlled trialrdquo Journal ofNutrition andMetabolism vol 2012 Article ID 207426 9 pages2012

[18] U SinghA Barik BG Singh andK I Priyadarsini ldquoReactionsof reactive oxygen species (ROS) with curcumin analoguesstructure-activity relationshiprdquo Free Radical Research vol 45no 3 pp 317ndash325 2011

[19] M Srinivasan ldquoEffect of curcumin on blood sugar as seen in adiabetic subjectrdquo Indian Journal of Medical Sciences vol 26 no4 pp 269ndash270 1972

[20] P Usharani A A Mateen M U R Naidu Y S N Rajuand N Chandra ldquoEffect of NCB-02 atorvastatin and placeboon endothelial function oxidative stress and inflammatorymarkers in patients with type 2 diabetes mellitus a randomizedparallel-group placebo-controlled 8-week studyrdquoDrugs in R ampD vol 9 no 4 pp 243ndash250 2008

[21] A Kumar and U Bora ldquoInteractions of curcumin and itsderivatives with nucleic acids and their implicationsrdquo Mini-Reviews inMedicinal Chemistry vol 13 no 2 pp 256ndash264 2013

[22] C Felgines O Texier C Morand et al ldquoBioavailability ofthe flavanone naringenin and its glycosides in ratsrdquo AmericanJournal of PhysiologymdashGastrointestinal and Liver Physiologyvol 279 no 6 pp G1148ndashG1154 2000

[23] I Erlund E Meririnne G Alfthan and A Aro ldquoPlasmakinetics and urinary excretion of the flavanones naringeninand hesperetin in humans after ingestion of orange juice andgrapefruit juicerdquoThe Journal of Nutrition vol 131 no 2 pp 235ndash241 2001

[24] S Bhattacharya N Oksbjerg J F Young and P B JeppesenldquoCaffeic acid naringenin and quercetin enhance glucose stim-ulated insulin secretion and glucose sensitivity in INS-1E cellsrdquoDiabetes Obesity and Metabolism 2013

[25] T Annadurai P A Thomas and P Geraldine ldquoAmeliora-tive effect of naringenin on hyperglycemia-mediated inflam-mation in hepatic and pancreatic tissues of Wistar ratswith streptozotocin-nicotinamide-induced experimental dia-betes mellitusrdquo Free Radical Research vol 47 no 10 pp 793ndash803 2013

[26] R Meneghini ldquoIron homeostasis oxidative stress and DNAdamagerdquoFree Radical BiologyampMedicine vol 23 no 5 pp 783ndash792 1997

[27] T V Fiorentino A Prioletta P Zuo and F FollildquoHyperglycemia-induced oxidative stress and its role indiabetes mellitus related cardiovascular diseasesrdquo CurrentPharmaceutical Design vol 19 no 32 pp 5695ndash5703 2013

[28] G D Stoner L-S Wang and B C Casto ldquoLaboratory andclinical studies of cancer chemoprevention by antioxidants inberriesrdquo Carcinogenesis vol 29 no 9 pp 1665ndash1674 2008

[29] N Schweigert A J B Zehnder and R I L Eggen ldquoChemicalproperties of catechols and their molecular modes of toxicaction in cells from microorganisms to mammalsrdquo Environ-mental Microbiology vol 3 no 2 pp 81ndash91 2001

[30] L-F Zheng Q-Y Wei Y-J Cai et al ldquoDNA damage inducedby resveratrol and its synthetic analogues in the presence of Cu(II) ions mechanism and structure-activity relationshiprdquo FreeRadical Biology amp Medicine vol 41 no 12 pp 1807ndash1816 2006

[31] AM Samuni E Y ChuangM C Krishna et al ldquoSemiquinoneradical intermediate in catecholic estrogen-mediated cytotoxi-city and mutagenesis chemoprevention strategies with antiox-idantsrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 100 no 9 pp 5390ndash5395 2003

[32] A S Azmi S H Bhat and S M Hadi ldquoResveratrol-Cu(II)induced DNA breakage in human peripheral lymphocytesimplications for anticancer propertiesrdquo The FEBS Letters vol579 no 14 pp 3131ndash3135 2005

Scientifica 7

[33] A Malik S Azam N Hadi and S M Hadi ldquoDNA degradationby water extract of green tea in the presence of copper ionsimplications for anticancer propertiesrdquo Phytotherapy Researchvol 17 no 4 pp 358ndash363 2003

[34] Z Li X Yang S Dong and X Li ldquoDNa breakage inducedby piceatannol and copper(II) mechanism and anticancerpropertiesrdquo Oncology Letters vol 3 no 5 pp 1087ndash1094 2012

[35] M H Leung D T Pham S F Lincoln and T W KeeldquoFemtosecond transient absorption spectroscopy of copper(II)-curcumin complexesrdquo Physical Chemistry Chemical Physics vol14 no 39 pp 13580ndash13587 2012

[36] A Barik B Mishra A Kunwar et al ldquoComparative study ofcopper(II)-curcumin complexes as superoxide dismutase mim-ics and free radical scavengersrdquo European Journal of MedicinalChemistry vol 42 no 4 pp 431ndash439 2007

[37] A Basu and G S Kumar ldquoBiophysical studies on curcumin-deoxyribonucleic acid interaction spectroscopic and calori-metric approachrdquo International Journal of Biological Macro-molecules vol 62 pp 257ndash264 2013

[38] A H Hegde S N Prashanth and J Seetharamappa ldquoInterac-tion of antioxidant flavonoids with calf thymus DNA analyzedby spectroscopic and electrochemical methodsrdquo Journal ofPharmaceutical and Biomedical Analysis vol 63 pp 40ndash462012

[39] L D Mello R M S Pereira A C H F Sawaya M NEberlin and L T Kubota ldquoElectrochemical and spectroscopiccharacterization of the interaction between DNA and Cu(II)-naringin complexrdquo Journal of Pharmaceutical and BiomedicalAnalysis vol 45 no 5 pp 706ndash713 2007

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Scientifica 7

[33] A Malik S Azam N Hadi and S M Hadi ldquoDNA degradationby water extract of green tea in the presence of copper ionsimplications for anticancer propertiesrdquo Phytotherapy Researchvol 17 no 4 pp 358ndash363 2003

[34] Z Li X Yang S Dong and X Li ldquoDNa breakage inducedby piceatannol and copper(II) mechanism and anticancerpropertiesrdquo Oncology Letters vol 3 no 5 pp 1087ndash1094 2012

[35] M H Leung D T Pham S F Lincoln and T W KeeldquoFemtosecond transient absorption spectroscopy of copper(II)-curcumin complexesrdquo Physical Chemistry Chemical Physics vol14 no 39 pp 13580ndash13587 2012

[36] A Barik B Mishra A Kunwar et al ldquoComparative study ofcopper(II)-curcumin complexes as superoxide dismutase mim-ics and free radical scavengersrdquo European Journal of MedicinalChemistry vol 42 no 4 pp 431ndash439 2007

[37] A Basu and G S Kumar ldquoBiophysical studies on curcumin-deoxyribonucleic acid interaction spectroscopic and calori-metric approachrdquo International Journal of Biological Macro-molecules vol 62 pp 257ndash264 2013

[38] A H Hegde S N Prashanth and J Seetharamappa ldquoInterac-tion of antioxidant flavonoids with calf thymus DNA analyzedby spectroscopic and electrochemical methodsrdquo Journal ofPharmaceutical and Biomedical Analysis vol 63 pp 40ndash462012

[39] L D Mello R M S Pereira A C H F Sawaya M NEberlin and L T Kubota ldquoElectrochemical and spectroscopiccharacterization of the interaction between DNA and Cu(II)-naringin complexrdquo Journal of Pharmaceutical and BiomedicalAnalysis vol 45 no 5 pp 706ndash713 2007

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom