research article the study of naphthoquinones and their

Research ArticleThe Study of Naphthoquinones and TheirComplexes with DNA by Using Raman Spectroscopyand Surface Enhanced Raman Spectroscopy New Insight intoInteractions of DNA with Plant Secondary Metabolites

Veronika Vaverkova1 Oldrich Vrana2 Vojtech Adam3

Tomas Pekarek4 Josef Jampilek5 and Petr Babula1

1 Department of Natural Drugs Faculty of Pharmacy University of Veterinary and Pharmaceutical Sciences Palackeho 1-3612 42 Brno Czech Republic

2 Institute of Biophysics Academy of Sciences of the Czech Republic Kralovopolska 135 C612 65 Brno Czech Republic3 Department of Chemistry and Biochemistry Faculty of Agronomy Mendel University in Brno Zemedelska 1613 00 Brno Czech Republic

4 Zentiva ks Development Department U Kabelovny 130 102 37 Praha 10 Czech Republic5 Department of Chemical Drugs Faculty of Pharmacy University of Veterinary and Pharmaceutical Sciences Palackeho 1-3612 42 Brno Czech Republic

Correspondence should be addressed to Petr Babula petr-babulaemailcz

Received 16 February 2014 Accepted 21 May 2014 Published 22 June 2014

Academic Editor Vicky Kett

Copyright copy 2014 Veronika Vaverkova et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Naphthoquinones represent the group of plant secondary metabolites with cytotoxic properties based on their ability to generatereactive oxygen species and interfere with the processes of cell respiration Due to this fact the possible cytotoxic mechanisms oncellular and subcellular levels are investigated intensively There are many targets of cytotoxic action on the cellular level howeverDNA is a critical target of many cytotoxic compounds Due to the cytotoxic properties of naphthoquinones it is necessary tostudy the processes of naphthoquinones DNA interactions (14-naphthoquinone binapthoquinone juglone lawsone plumbagin)especially by usingmodern analytical techniques In our work the Raman spectroscopy was used to determine the possible bindingsites of the naphthoquinones on the DNA and to characterize the bond of naphthoquinone to DNA Experimental data reveals therelationships between the perturbations of structure-sensitive Raman bands and the types of the naphthoquinones involved Themodification of DNA by the studied naphthoquinones leads to the nonspecific interaction which causes the transition of B-DNAinto A-DNA conformationThe change of the B-conformation of DNA for all measured DNAmodified by naphthoquinones exceptplumbagin is obvious

1 Introduction

Naphthoquinones are naturally widespread secondary met-abolites the products of some actinomycetes (StreptomycesWaksman and Henrici) fungi (Fusarium Link MarasmiusFr and Verticillium Nees) lichens algae and plantsThe most important naphthoquinones-containing plantsbelong to the group of phylogenetically heterogeneous plantfamilies Avicenniaceae Endl ex Schnizl Balsaminaceae

DC Bignoniaceae Juss Boraginaceae Juss DroseraceaeSalisb Ebenaceae Gurke Juglandaceae A Rich ex KunthNepenthaceae Dum and Plumbaginaceae Juss [1ndash4] Thechemical structure of the monomeric naphthoquinones isbased on the bicyclic systemmdashnaphthalene skeleton substi-tuted in the positions C(1) and C(4) (14-naphthoquinones)or C(1) and C(2) (12-naphthoquinones) There are dimericand trimeric naphthoquinones which evidence significantcytotoxic properties on different tumour cell lines including

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 461393 12 pageshttpdxdoiorg1011552014461393

2 BioMed Research International

prostate androgen-dependent cell lines and breast tumourcell lines [5 6] Naphthoquinones have many physiologicalroles the most important ubiquitous naphthoquinones arevitamins of the K groupmdashphylloquinone and menaquinone[7ndash9] Plenty of naphthoquinones are important phytoalex-ins so they play an important role in ecological relationships[10] Naphthoquinones have found many possibilities forusemdashantimicrobial antifungal antiviral and antiprotozoalproperties have been identified In traditional medicinesparticularly in Asia (China) and South America plants withnaphthoquinones have a wide range of application especiallyin the treatment of various types of cancer [2] The cytotoxicproperties of the naphthoquinones are in the focus of interestof many scientists They are predominantly based on theability of naphthoquinones to generate reactive oxygenspecies (ROS) which are involved in many cellular processesand serve as signal molecules in apoptosisprogrammed celldeath and are partially responsible for cytotoxic propertiesdemonstrated in many in vitro tumour cell line systems[11ndash14] The ability of naphthoquinones to interfere withthe respiratory chain poses the second possible cytotoxicmechanism [15ndash17] DNA is the critical target of the activityof many compounds including plant secondary metabolitesThere is a large group of natural origin compounds withinteresting cytotoxic properties Their cytotoxic effect isbased on their interactions with DNA especially on thepossibility of forming various types of covalent adductswith DNA The most important compounds whose effectis based on DNA interaction are anthracyclines cytotoxicantibiotics and platinum-based cytostatics [18ndash21]

DNA-metal and DNA-metal based complexes interac-tions and their effect on DNA structure are already known[22ndash29] Cisplatin as an important and frequently used anti-cancer drug forms covalent bonds with nucleophilic sites onguanine residues present in DNA As a cisplatin is a bifunc-tional agent it is able to bind to one (monofunctional adduct)or two (intrastrand crosslinks interstrand crosslinks) placesin a DNA chain [30ndash32] All of these adducts characteristi-cally distort the DNA conformation [33 34] The main prob-lem in the curing process with many anticancer complexessuch as cisplatin is the reduced sensitivity of the tumourtowards this drug when the drug is repeated in the treatmentA medical treatment is connected with many inadvisableeffects [35 36] Therefore new structures with reducedtoxicity often of natural origin are intensively searchedDue to significant cytotoxic properties of the selectednaphthoquinones (see Figure S1 in Supplementary Materialavailable online at httpdxdoiorg1011552014461393) wedecided to study the naphthoquinones-DNA interactions asa possible mechanism of their cytotoxicity In recent yearsnaphthoquinone derivatives have been intensively studiedin connection with their cytotoxic and antibacterial effectsand subsequently possible usage in cancer treatment [37ndash41] Despite the above-mentioned mechanisms of naphtho-quinones cytotoxicity their interactions with DNA have notyet been establishedDue to this fact we focused on the inves-tigation of naphthoquinones-DNA interactions in our workThe identification of the binding sites of naphthoquinoneson DNA and the determination of how the binding may

affect secondary and tertiary structures is still lacking Somenaphthoquinones have been identified as potent inhibitors oftopoisomerases I and II this activity may contribute to theircytotoxic effect [2 37 42] In addition newly synthesizedcomplexes of lawsone andmetal ions are recently investigatedas cytotoxic compounds with the cytotoxic effect based onthe DNA interactions [43] The vibrational spectra [44 45]optical absorption and fluorescence emission spectra [4647] and SERS studies [44 45 48ndash51] of some naphtho-quinone derivatives have also been reported However thenaphthoquinones-DNA interactions can play the crucial rolein their cytotoxic effectThe vibrational spectroscopy (Ramanspectroscopy) can help making unambiguous identificationof vibrational modes of biological molecular systems [24 32]We used Raman spectroscopy to assess the binding sitesof the naphthoquinones to DNA and to characterize andcompare the perturbations of DNA after its modification bynaphthoquinones in our workWe used Raman spectroscopyand SERS in the analysis of DNA modified by the selectednaphthoquinones 14-naphthoquinone binaphthoquinonejuglone lawsone and plumbagin (Figure S1) The spectraobtained were analyzed and characteristic spectral changeswere described with the aim of determining interactionsbetween selected naphthoquinones and DNA and analyzingDNAstructural changes under naphthoquinones-DNA inter-actions In addition the linear dichroism spectroscopy wasused to verify these results

2 Materials and Methods

21 Chemicals Material and pH Measurements 14-Naph-thoquinone binaphthoquinone [22-bi(3-hydroxy-14-naph-thoquinone] juglone [5-hydroxy-14-naphthoquinone] law-sone [2-hydroxy-14-naphthoquinone] and plumbagin [5-hydroxy-2-methy-14-naphthoquinone] as well as all otherchemicals of ACS purity unless noted otherwise werepurchased from Sigma Aldrich Chemical Corp (Sigma-Aldrich USA) Deionised water underwent demineralizationby reverse osmosis by using an instrument Aqua Osmotic02 (Aqua Osmotic Tisnov Czech Republic) and was subse-quently purified by using a Millipore RG (Millipore CorpUSA 18M1015840Ω)mdashMiliQ water The pH value was measured byusing the WTW inoLab pH meter (Weilheim Germany)

22 Preparation of DNA DNA was isolated from the calfthymus in accordance with the published work of Brabecet al [33] DNA was dissolved in 05M NaCl and wasthen sonicated for 20 minutes by using a Sonicator 3000(Giltron USA) and subsequently analyzed The first step ofdenatured DNA analysis was the electrophoresis by using thesource Standard Power Pack P25 by (Biometra Germany)The gel was photographed after the backlighting the gel byUV on a transilluminator TVX-20M (312 nm) (AlysLabwareSwitzerland) The next step was the polarography on ananalyzer EGampG PARC Model 384B (LabX Canada) Theconcentration of DNAwasmeasured on a BeckmanDU 7400(Pall Gelman Germany) For the dialysis a SpectraPor 12-14000 (Cole-Palmer USA) was used The lyophilization was

BioMed Research International 3

performed on a Labconco 45 (LabX Canada) To removethe proteins which cause the fluorescence of DNA in Ramanspectroscopy phenol and chloroform extractions were usedThe final DNA concentration was 2 times 10minus1M

Naphthoquinones were dissolved in a volume of 3mLin the purified Milli-Q water The final concentrations ofthe selected naphthoquinones were 14-naphthoquinonemdash32times10

minus1Mbinaphthoquinonemdash31times10minus1M juglonemdash29times10minus1M lawsonemdash32times10minus1M andplumbaginmdash28times10minus1M

23 Raman Spectroscopy Spectra were recorded on theRaman spectrometer Jobin-Yvon T64000 (Horiba France)

24 Surface-Enhanced Raman Spectroscopy The preparationof Ag colloid component Amdash515mg of hydroxylaminehydrochloride was added to 5mL of redistilled water andcomponent Bmdash600mg of sodium hydroxide was also addedto 5mL of redistilled water Both these components weremixed and immediately added to the solution of silvernitrate (848mg silver nitrate in 45mL of redistilled water)The colloid was measured using UV spectroscopy and thencompared with previous studies [33 52 53] The silver(Ag) colloid complex (of naphthoquinones) was prepared bymixing equal volumes of the solution of naphthoquinones orDNA with the Ag colloid to obtain the final concentration of5times10minus7Mor 5times10minus6MThefinal concentration of the colloid

was 9 times 10minus4M The spectra of the prepared samples weremeasured immediately after mixing the naphthoquinone orDNA with Ag colloid at 25∘C Samples (10 120583L) were sealedin a glass capillary The measurement of the whole spectrumwas divided into two parts and each part consisted of severalaccumulations (16 times 2 s)

25 Preparation of DNA-Naphthoquinone Complexes To pre-pare each naphthoquinone-DNA complex DNA was incu-bated with the naphthoquinones in the solution of 001MNaClO4 at 37

∘C in the dark for 24 hours

26 Instrumentation The complexes (naphthoquinones +DNA) were dissolved to a final concentration of 30mgmLin 01M NaCl (pH 70) Aliquots (4120583L) of such preparedcomplexes were sealed in a glass capillary Kimax 34507(Kimble products USA) Spectra of these aliquots wereexcited at 488 nm using an argon ion laser (Innova 90C FredCoherent USA) The radiant power at the sample was set to100mW and it was monitored by a Broadband Power Meter(MellesGriotte USA)Measurementswere performed at 25∘Cin back scattering geometry Rayleigh line was removed fromthe scattered light by a Kaiser Optical notch filterThe Ramansignal was detected in a single mode using 1200 linesnmgrating entrance slit of 01mm wide and a spectrum OneCCD3000 LN2 cooled detector Spectrometer and detectorwere controlled by JobinYvonLabSpec software version 303t(Horiba France)

The Raman spectra of individual naphthoquinones usedfor complexation were measured prior to the colloid spectralanalysis Spectra of these molecules were acquired directly

in the solid phase in the same instrumental settings asmentioned above The laser power was set to 10mW

27 Data Processing Some spectra were disturbed by asignificant fluorescent background It was approximated bythe polynomial function and subtracted by using ldquobaselinecorrectionrdquo functionality of the LabSpec software For thenormalization of the different spectra the Raman band near1092 cmminus1 assigned to the phosphate group vibration wasused as an internal intensity standard The intensity of thisband remains the same regardless of the A-DNA or B-DNAconformation [54] The processing of the data measured wascarried out by using the LabSpec software (Horiba France)Spectra were further converted to Omnic 62 (Thermo USA)format

28 Linear Dichroism (LD) LD was used to confirm theintercalation of the naphthoquinones to DNA LD spectrawere recorded by using a flow Cuvette cell in a Jasco J-720 spectropolarimeter adapted for LDmeasurements (JascoAnalytical Instruments Japan) Longmolecules such as DNAcan be orientated in the flow Cuvette cell The flow cellconsists of a fixed outer cylinder and a rotating solid quartzinner cylinder separated by a gap of 05mm giving a totalpath length of 1mm [55] LD spectra of ctDNA modifiedby the selected naphthoquinones were recorded at 25∘C in10mM NaClO4

3 Results

31 Control of DNA Denaturation DNA was sonicated andthe possible denaturation of DNA was checked (see Supple-mentary Material)

32 Raman Spectra of DNA Modified by Selected Naph-thoquinones (Solid Phase Solutions) The overview of theselected naphthoquinonesrsquo Raman spectra is given in FigureS3 These spectra are used further for the evaluation ofthe Raman spectra of the colloid-DNA complexes in thefollowing chapters

33 The Analysis of DNA Modified by 14-NaphthoquinoneWe used the main bands in the spectra for the comparisonof the changes in the cases of the modified DNA by selectednaphthoquinones These bands are used for description ofinteraction in cases of different compounds [1 31 45 56ndash59] Only the most important spectral features are discussedin the following chapters particular bands assignment ispresent in Table 2 The bands near 670 and 683 cmminus1 area marker of C31015840-endoanti-conformation in case of A-formDNA (A-DNA) The intensity of the band near 670 cmminus1decreases together with the conformation of the sugarpart of deoxyriboguanosine conversion from C21015840-endoanti-conformation to C31015840-endo conformation [31 60ndash62] Theratio of bandsrsquo intensities 683669 cmminus1 gt 1 indicates thatthe DNA modified by the complex is in the B-conformationHigher intensity of the band at 669 cmminus1 the higher theportion of the modified DNA in the A-form Thus the final


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Figure 1 Raman spectra (a) (1) difference Raman spectrum of DNA and 14-naphthoquinone (spectrum 2-spectrum 3) (2) Raman spectrumof DNA and 14-naphthoquinone and (3) control ctDNA (the areas with the differences in spectra are in the border) (b) (1) difference Ramanspectrum of DNA and binaphthoquinone (spectrum 2-spectrum 3) (2) Raman spectrum of DNA and binaphthoquinone and (3) controlctDNA (the areas with the differences in spectra are in the border)

ratio of the bandsrsquo intensities 683669 cmminus1 lt 1 proves the A-conformation of the modified DNA by 14-naphthoquinone(Figure 1(a)) There is not an intensive band at 834 cmminus1 inDNAmodified by 14-naphthoquinone and this fact supportsthe assumption of the transition from B-DNA to A-DNAThe increase of the band at 804 cmminus1 in DNA modified by14-naphthoquinone indicates the local structural changesand it is the next evidence of A-form presence The bandat 788 cmminus1 in the DNA is a combination band of cytosinethymine and the phosphodiester bandThis band is thereforevery intensive also in the modified DNA The bands of DNA(modified and unmodified) in the region 800ndash1050 cmminus1 cor-respond to the vibrations of the sugar-phosphate backboneskeleton The intensity change of the band near 1078 cmminus1is influenced by the contribution of 14-naphthoquinone(Figure 1(a)) The band at 1340 cmminus1 corresponds to thevibration of adenine and guanine Changes of the intensityof this band indicate changes in the pairing between GCand AT bases In the DNA modified by 14-naphthoquinonestacking interactionwas interrupted and 14-naphthoquinonewas fastened onto adenine in DNA structure The bandat 1377 cmminus1 in the spectrum of DNA modified by 14-naphthoquinone is influenced by the thymine vibration Dueto changes of adenine-thymine pairing the intensity of thisband is changed in themodified DNA spectrum compared tonative DNAThe band 1504 cmminus1 (in B-DNA is located in thearea of 1511 cmminus1) in DNA modified by 14-naphthoquinoneis assigned to the vibration of imidazole ring of adenineand the increasing intensity of this band indicates a partialmodification of adenine and guanine The band in DNAmodified by 14-naphthoquinone near 1574 cmminus1 correspondsto the overlapping contributions from the dG and dA This

band is located at 1577 cmminus1 in the spectrum of B-DNAThe increasing intensity of this band in the case of DNAmodification by 14-naphthoquinone results in disruption ofhydrogen bonds in GC and AT pairs This fact is relatedwith the predenaturation and denaturation changes in DNAmodified by 14-naphthoquinone The band at 1688 cmminus1corresponds to the carbonyl group stretching vibration of the(C=O) group of the thymine and its width is influenced bythe hydrogen bonds of thymine and adenine

34 Analysis of DNA Modified by Binaphthoquinone Theratio of intensities at 683670 cmminus1 is 07 which indicatesthe transition from the B-conformation in A-conformation(Table 3) in the spectrum of DNA modified by binaphtho-quinone (Figure 1(b)) like in the case of DNA modifiedby 14-naphthoquinone Furthermore changes in the DNAmodified by binaphthoquinone in the intensity of the bandat 804 cmminus1 are observed This is in concordance withchanges from the B-conformation (834 cmminus1) into the A-conformation (807 cmminus1) and this change coincides with anincrease in the intensity of band 670 cmminus1 The band at1339 cmminus1 in the spectrum of native DNA is a combinationband of adenine guanine and thymine In the spectrumof DNA modified by binaphthoquinone this band indicateshigh distorsions in AT and GC base pairs and large localdenatruration was obvious and the large local denaturationwas obvious

35 Analysis of DNA Modified by Juglone DNA modifiedby juglone was changed in the areas around 670 cmminus1 and683 cmminus1 compared to the nativeDNAThe ratio of the bandsrsquo


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Figure 2 Raman spectra (a) (1) difference Raman spectrum of DNA and juglone (spectrum 2-spectrum 3) (2) Raman spectrum of DNAand juglone and (3) control ctDNA (the areas with the differences in spectra are in the border) (b) (1) difference Raman spectrum of DNAand lawsone (spectrum 2-spectrum 3) (2) Raman spectrum of DNA and lawsone and (3) control ctDNA (the areas with the differences inspectra are in the border)

intensities 683670 cmminus1 is 09 (Table 3) which indicatesthe transition from the B-conformation in A-conformation(Figure 2(a)) as it was in the case of DNA modified by14-naphthoquinone and binaphthoquinone This fact is alsosupported by the highest increasing intensity of the bandat 807 cmminus1 in the case of DNA modified by juglone Theband at 1237 cmminus1 represents the vibrations of cytosineand thymine in the native DNA and its increase in thecase of DNA modified by juglone corresponds to the localdenaturation In DNA modified by juglone the increaseof intensity near 1343 cmminus1 is related to the changes inthe deoxyribonucleoside conformation in the GC and ATpairs which is significant for the local denaturation (aswell as the band at 1237 cmminus1) In the difference Ramanspectrum of DNA modified by juglone the increase ofthe band at 1503 cmminus1 is clearly observable this intensitychange is related to the hydrogen bond of adenine andthymine This means that the AT hydrogen bonds have beenbroken and juglone was fastened to the adenine in the DNAstructure

36 Analysis of DNA Modified by Lawsone From the dif-ference Raman spectrum of DNA modified by lawsone it isclear that there are changes in the areas near 670 cmminus1 andnear 683 cmminus1 (Figure 2(b)) In theDNAmodified by lawsonethe intensity of the band at 670 cmminus1 increased more thanthe intensity of the band near 683 cmminus1 The ratio of bandsrsquointensities at 683670 cmminus1 is 07 (Table 3) which indicatesthe transition from the B-conformation of native DNA to A-conformation of the modified DNA similarly to DNA mod-ified 14-naphthoquinone binaphthoquinone and juglone

We also identified changes in the spectra of DNAmodified bylawsone near 807 cmminus1 which also indicates changes from B-conformation (834 cmminus1) in the A-conformation (807 cmminus1)and this change coincides with an increase in the intensityof band at 670 cmminus1 The band at 1343 cmminus1 is slightly shiftedin DNA modified by lawsone comparing to native DNA andit corresponds to the vibrations of adenine guanine andthymineThere have been high distortions inAT andGCbasepairs and therefore there was a large local denaturation in ATand GC base pairs In DNA modified by lawsone the band at1577 cmminus1 increased its intensity bymore than 18 than in thecase of DNA modified by 14-naphthoquinone and binaph-thoquinone This change indicates much more noticeabledisruption of the hydrogen bonds inGCandTApairs (similarto the band at 1338 cmminus1) and greater predenaturation anddenaturation effects than if it was in the case ofDNAmodifiedby 14-naphthoquinone and binaphthoquinone

37 Analysis of DNA Modified by Plumbagin From theRaman spectra of DNA modified by plumbagin it is obvi-ous that modified DNA was not transformed in the areasaround 670 cmminus1 and 683 cmminus1 (Figure 3(a)) The ratio ofthe bandsrsquo intensities 683670 cmminus1 is 13 (Table 3) whichdoes not indicate the transition from the B-conformationDNA in A-conformation In DNA modified by plumbaginthe band at 1236 cmminus1 corresponds to vibrations of cytosineand thymine and in this area the local denaturation isobvious There were some denaturation changes in the DNAstructure when modified by plumbagin but these changes donot inevitably indicate that plumbagin was intercalated intoDNA


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(6)

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(1)

Wavelength (nm) (1) 14-Naphthoquinone (2) Lawsone (3) DNA

(4) Binaphthoquinone (5) Juglone (6) Plumbagin

(b)

Figure 3 (a) Raman spectra (1) difference Raman spectrum of DNA and plumbagin (spectrum 2-spectrum 3) (2) Raman spectrum of DNAand plumbagin and (3) control ctDNA (the areas with the differences in spectra are in the border) (b) linear dichroism (LD) spectra of calfthymus DNA modified by (1) 14-naphthoquinone (2) lawsone (3) control nonmodified DNA (4) binaphthoquinone (5) juglone and (6)plumbagin

Table 1 The increase (decrease) of the bands in the Raman spectrum of DNA after the modification by the studied naphthoquinones Thechanges are expressed in the percentages (lowastthe observation of Raman intensity of calf thymus DNA represents 100 the change is countedseparately for each band)

DNA-naphthoquinone DNAlowast 14-Naphthoquinone Binaphthoquinone Juglone Lawsone Plumbagin670 cmminus1 vibrations of thymine 100 181 191 190 186 95683 cmminus1 vibrations of guanine 100 112 101 118 101 120834 cmminus1 B-conformation phosphodiester bond 100 96 98 98 98 991237 cmminus1 vibrations of cytosine and thymine 100 85 147 129 144 1021256 cmminus1 vibrations of cytosine and thymine 100 81 116 95 113 1001338 cmminus1 vibrations of adenine and guanine 100 102 115 97 114 1051376 cmminus1 vibrations of thymine 100 78 109 101 107 861511 cmminus1 vibrations of adenine 100 108 118 119 116 1161578 cmminus1 vibrations of adenine and guanine 100 125 126 123 125 123

38 Results Summary of All Measured Raman Spectra ofDNA Modified by Selected Naphthoquinones (Solid PhaseSolutions) The results obtained by Raman spectroscopywere compared with previously published studies wherethe DNA interactions with some substances were studied[31 54 60 62ndash74] The increase (decrease) of the selectedbands intensity in the Raman spectrum of DNA aftermodification by the studied naphthoquinones is summa-rized in Table 1 All the changes of the Raman band posi-tions and intensities after the modification of DNA by theselected naphthoquinones are introduced in Table 2 Table 3shows changes in DNA conformation after its modificationby naphthoquinones It is obvious that after modificationof DNA by all complexes DNA was changed from B-conformation to A-conformation except of DNAmodified byplumbagin

39 SummaryResults of All theMeasured SERS Spectra ofDNAModified by Selected Naphthoquinones The SERS methodwas used to confirm the hypothesis that substances bind toDNA and to confirmwhichmain bases aremodified after thisbinding to DNA (see Supplementary Material)

310 Linear Dichroism Spectroscopy Long molecules suchas DNA show a signal in the LD spectra (if it is orientedspecifically) Small unbound molecules show no signal Theintensity of the LD signal of DNA modified by binaphtho-quinone and lawsone had been decreased in the area of260 nm A smaller decrease in this intensity of LD signalwas observed in the case of DNA modified by juglone and14-naphthoquinone and the lowest decrease in intensityof LD signal in the case of DNA modified by plumbagin


Table 2The changes of the Raman intensities after themodification of DNAby the selected naphthoquinones w-weak m-medium s-strongsh-shoulder br-broad as-asymmetric

Peak (cmminus1) Assignment 14-Naphthoquinone Binaphthoquinone Juglone Lawsone Plumbagin670 dT dA 670 m 667 m 667 m 667 m 667 w683 dG mdash mdash mdash mdash mdash729 A mdash mdash mdash mdash mdash750 dT 740 w mdash 741 w mdash mdash787 bk O-P-O str + dT 805 m 804 m br 807 m br 807m 807 m834 bk O-P-O B-DNA 827 w mdash 821 w mdash mdash894 dr C21015840H2 rock mdash mdash mdash mdash mdash922 dr ring str mdash mdash mdash mdash 916 m970 T C6H op-def bk mdash mdash mdash mdash mdash1014 T CH3 rock mdash mdash mdash mdash 1018 sh1054 bk C-O str 1079 m-s 1038 m br 1076 m-s 1039 m br 1073 s br1093 POsym str 1110 w mdash mdash mdash mdash1143 dT mdash mdash 1142 w mdash 1147 m1178 dT dG dC mdash mdash mdash mdash 1176 w1217 dT dA dG mdash mdash 1215 sh mdash 1216 m as1237 dT dC mdash 1239 m-s br 1237 m 1240 m br mdash1256 dC dA dT (dG) mdash mdash mdash mdash mdash1307 dA dT mdash mdash 1281 w 1304 w mdash1338 dA dG mdash mdash 1323 w 1322 sh mdash1376 T CH3def 1377 s 1398 w 1350 m br 1359 m 1355 m br 1384 w1421 dr C51015840H2def mdash mdash mdash mdash mdash1446 1448 w 1452 w 1448 w 1448 w 1442 w1462 mdash mdash mdash mdash mdash1490 G im ring dA dT 1490 s as 1490 s 1490 s 1490 s as 1490 s1511 1509 w 1502 w 1502 w 1503 w sh 1507m1578 dG dA 1574 s as 1578 s as 1578 s as 1579 s as 1576 s1669 T dG 1688 w 1688 m as 1685 m as 1689 w br 1680 m-s br as

Table 3 The ratio of the size of the peak intensities at 683670 cmminus1with a peak intensity of 683 cmminus1

DNA-naphthoquinone 683670 cmminus1

14-Naphthoquinone 08Binaphthoquinone 07Juglone 09Lawsone 07Plumbagin 13

This decreasing intensity of the peak 260 nm means theintercalation of the studied naphthoquinones into DNAThedecreases of the intensities of the LDband (220ndash300 nm) con-firm that DNA modified by all studied naphthoquinones isshifted fromB-DNA to A-DNA [55]The largest intercalationmode of naphthoquinone to DNA is observed in the caseof DNA modified by binaphthoquinone and lawsone wherethe decrease of LD band at 260 nm is the most significantand the molecule of DNA by this modification is the mostrigid The results measured on the LD spectroscopy con-firm the assumption that all the selected naphthoquinones

intercalate into DNA in the range of lawsone gt binaph-thoquinone gt juglone gt 14-naphthoquinone gt plumbagin(Figure 3(b))

4 Discussion

It was necessary to reflect on the dramatic changes and tocompare these changes in the cases of the modified DNA by14-naphthoquinone binaphthoquinone juglone lawsoneand plumbagin We use the main bands in the spectra forthese comparisons These bands are widely used for descrip-tion of interaction in cases of different compounds with DNA[1 31 45 56ndash59] The bands at 670 cmminus1 and 683 cmminus1 aremarkers of A or B-conformation inDNAThese two peaks areassociated with the vibration of deoxynucleosides dT (mainly670 plusmn 3 cmminus1) and dG (683 cmminus1) These markers are used inthe studies of interactions between proteins and DNA [75]Changes in the band near 670 cmminus1 indicate the changes inA-B-DNA conformation (see Table 3) However they cannotbe interpreted independently but in the comparison of thechanges of the intensities of the band at 683 cmminus1 The bandnear 670 cmminus1 is a marker of C31015840-endoanti-conformation incase of A-form DNA and in the B-form it is primarily due to


dT Decrease of the intensity of this peak is observed whenconformation of the sugar moiety of deoxyriboguanosineis converted from C21015840-endoanti-conformation to C31015840-endoconformation [31 60 61] It is important to note that Ramanband near 660ndash685 cmminus1 is also sensitive to the sugar puckerand glycosyl torsion of deoxyguanosine nucleoside residues[31 60 61]The signals in the area 800ndash1100 cmminus1 aremarkersof changes in backbone geometry and DNA secondary struc-ture [54] All the selected naphthoquinones are responsiblefor the transition of DNA from B to A conformation exceptthe DNAmodified by plumbaginThe band at 807plusmn6 cmminus1 isin the A-conformation DNA (A-form DNA phosphodiestermarker) counterpart of the intensive band 834 cmminus1 in B-conformation (phosphodiester B-form marker) [54] Theband 807 cmminus1 is thus the marker of A-conformation (ifthe nucleoside conformation does not remain in the C21015840-endo conformation) and its intensity increased the mostfor DNA modified by binaphthoquinone and plumbaginBy contrast the intensity increased less for DNA modifiedby 14-naphthoquinone juglone and lawsone The band at834 cmminus1 is located in the area of 800ndash1000 cmminus1 Bands inthis area are sensitive to changes in the secondary structureof DNA and the changes in the sugar-phosphate backboneskeleton The band at 834 cmminus1 is one of the characteristicmarkers of B-DNA conformation and the correspondingvibration of the phosphodiester links [45 76ndash78] In the A-conformation of DNA this band is shifted to lower values tothe area from 807 to 811 cmminus1 as already was mentioned Inthe spectrum of modified DNA by 14-naphthoquinone andplumbagin this band is slightly lower than in the comparisonin the spectrum of the DNA modified by binaphthoquinonejuglone and lawsone which is in good agreement with thedata above-mentionedTheband at 1073 cmminus1 is partly relatedto the vibration of the sugar-phosphate backbone (probablyelectrostatic interaction of the phosphate group) [79ndash82]but it especially corresponds with the vibrations to thecontribution of the naphthoquinones measured in the solidphasemdashin the case of juglone and plumbagin The area ofband near 1237 cmminus1 (1100ndash1400 cmminus1) is in the spectrum ofa very rich DNA The changes in the area of 1200ndash1600 cmminus1correspond mostly to the changes of the vibration of purineand pyrimidine so the bands in this area are associated withchanges in the structure of the aromatic ring of purine andpyrimidine which are sensitive to the binding of metal atomsto the aromatic [60 65 83] Intensity of the band at 1237 cmminus1which is related to the vibration of thymine and cytosineincreased mostly for DNA modified by juglone (129 ofcontrol) and lawsone (144 of control) The significantincrease of the intensity of the band results is the contributionof the cytosine and thymine vibration in DNA it is associatedwith the bond of the studied naphthoquinones on the DNAand is a significant feature of the local denaturation Bandat 1237 cmminus1 was not used only for description of in vitrosystems but also in systems represented by the cell lines [84]The vibrationalmode at 1338 cmminus1 is assigned to the vibrationof adenine and guanine and signified the local denaturationin AT and GC base pairs [85] On the other hand this bandindicates formation of triple helices [86]The band is sensitive

to the changes in the electron structure of aromatic ring ofpurine and pyrimidine In the case of DNA modificationby 14-naphthoquinone there is a slight increase in thepeak affected by 14-naphthoquinone contribution measuredin the solid phase and this contribution is predominatedby the conformation changes in GC and AT pairs Thesignificant increase in this peak was observed also for DNAmodified by binaphthoquinone and lawsone In contrast tothis only a small decrease of 1338 cmminus1 was observed inthe DNA modified by juglone This result indicates the roleof substitution of the naphthoquinone skeleton by hydroxylgroups The bands at 1376 cmminus1 and 1352 cmminus1 confirm thelocal denaturation and they are related to the vibration ofthymine especially its methyl group [80 87ndash89] The bandnear 1376 cmminus1 is linked in the case of DNA modified bybinaphthoquinone juglone lawsone and plumbaginwith thecontribution of these complexes measured in the solid phaseIt can be explained that in the case of DNAmodified by thesenaphthoquinones the band intensity increased not only bythe contribution of the local denaturation but also by thecontribution of these complexes themselves The change ofthe band at 1490 cmminus1 is assigned to the vibration of guanineand especially adenine [62 90 91] Changes in the differenceRaman spectra of this signal were observed for the DNAmodified by all studied naphthoquinones When there is theobvious decrease in the difference Raman spectrum in thisarea it is related to the modification of N(7) guanine TheN(7) site of guanine is recognized as an important site ofthe identification of some metal interaction with DNA [92]The size of the band at 1490 cmminus1 decreases after electrophilebinding agents to the N(7) After the modification of DNAby selected naphthoquinones this decrease is related withthe guanine vibration not with the metal interaction withDNA

The band at 1511 plusmn 4 cmminus1 is the vibration mode of theimidazolersquos ring of adenine particularly adenine stretchingvibration [93] This band intensity increased and it is slightlyshifted in case of DNA modification by 14-naphthoquinonebinaphthoquinone juglone lawsone and plumbagin Thesechanges indicate the significant disruption of the hydrogenbonds between adenine and thymine Similar results areshown in the work of Iyandurai and Sarojini who focused onthe interactions between spermine and DNA [93] Moreoverin the case ofDNAmodified by the selected naphthoquinonesthe band intensity at 1537 cmminus1 increasedThis is linked to thearomatic ring vibration of cytosine (in the B-conformationof DNA this band is in the area 1531 cmminus1) and this changetherefore well coincides with the changes from B-DNA toA-DNA The characteristic point of the difference spectrumof DNA modified by all the naphthoquinones studied is thedecrease and subsequent increase in the intensity at 1578 plusmn4 cmminus1 and 1586 plusmn 4 cmminus1 respectively Both of these bandsare related to the vibration of adenine and guanine Thesignificant increase of the band intensity at 1578plusmn4 cmminus1 canbe interpreted as a pronounced disruption of the hydrogenbonds in GC and TA pairs Similar results have been estab-lished for the investigation of alive and dead cells (MLE-12cell line) where dead cells demonstrated significant increase


of this peak [94] This fact is related to the predenaturationand denaturation effect occurring in DNA modified by allmeasured naphthoquinonesThe band at 1578 cmminus1 increasedthe most in the case of DNAmodified by binaphthoquinoneThis change indicates very high disruption of the hydrogenbonds in GC and TA pairs and large predenaturation anddenaturation effects in DNAmodified by binaphthoquinoneThe opposite results were demonstrated in the case of pacli-taxel and 5-fluorouracil where significant decrease of bandnear 1578 cmminus1 was detectable [85 95] On the other handthese two studies were carried out on the individual tumourcells From the changes mentioned above it is clear that allthe measured naphthoquinones except plumbagin transferDNA from B-conformation to A-conformation In the caseof DNA modified by plumbagin some local denaturationchanges were observed thus some interactions betweenDNAand plumbagin take place but it is not obvious if plumbaginintercalated into DNA By using SERS and LD spectroscopythe interaction was confirmed In the SERS spectra thechanges of the spectra included changes in the intensities andin the positions of the bands too This analysis demonstratesthat naphthoquinones bind specifically to DNA and cause aspecific distortion in the DNA

5 Conclusions

All the studied DNA modifications by the selected naphtho-quinones induced changes in the spectral part which corre-sponds to the vibration of the sugar-phosphate backbone inthe areas of vibration of the bases and also in the areas ofvibration of each naphthoquinoneThemodification of DNAby the studied naphthoquinones leads to the nonspecificinteraction which causes the distortion of DNA from B-conformation of DNA to A-conformation The change of theB-conformation of DNA for all measured DNA modifiedby naphthoquinones except plumbagin is obvious It is alsoevident from the SERS spectra that the interaction betweenDNA and complexes takes places at the bases and not atthe sugar-phosphate backbone In the case of modificationof the bases the adenine and thymine were modified inall DNA modified by naphthoquinones The area around1200 plusmn 5 cmminus1 was affected by the local denaturationThe areaaround 1600 plusmn 5 cmminus1 exhibits no significant changes Foreach DNAmodified by all studied complexes the intensity ofthe band near 1277 cmminus1 increased which is affected by thevibration of adenine and guanine It is obvious from theseresults that all naphthoquinones are bound to DNA except ofplumbagin In the case of plumbagin there were only somedenaturation effects after the modification of DNA due tothe methyl group in the naphthalene skeleton The analysisof the difference spectra leads to the clear conclusions thatbases have been modified and these changes have predenat-uration and denaturation character Nevertheless the resultsdemonstrate that naphthoquinones bind specifically to DNAand cause a specific distortionThe cytotoxic activity of somenaphthoquinones may be clarified in this way

Abbreviations

A-DNA Right-handed double helix shortermore compact helical structure

B-DNA Right-handed double helixbp Base pairbk BackboneCisplatina cis DDP cis-[(NH3)2Cl2Pt]ct DNA Thyme DNAds DNA Double-strand DNAss DNA Single-strand DNAdA DeoxyadenosinedC DeoxycytidinedG DeoxyguanosineDNA Deoxyribonucleic acidDPP Difference pulse polarographydT DeoxythymidineLD Linear dichroismWavenumber (cmminus1)

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

Financial support from the following Grants GACR P304112246 IGA VFU 422011FaF and IGA VFU 432011FaF ishighly acknowledged

References

[1] A Sakunphueak and P Panichayupakaranant ldquoEffects of donorplants and plant growth regulators on naphthoquinone produc-tion in root cultures of Impatiens balsaminardquo Plant Cell Tissueand Organ Culture vol 102 no 1 pp 9ndash15 2010

[2] P Babula V Adam L Havel and R Kizek ldquoNoteworthy sec-ondary metabolites naphthoquinonesmdashtheir occurrence phar-macological properties and analysisrdquo Current PharmaceuticalAnalysis vol 5 no 1 pp 47ndash68 2009

[3] P Babula D Huska P Hanustiak et al ldquoFlow injection analysiscoupled with carbon electrodes as the tool for analysis ofnaphthoquinones with respect to their content and functions inbiological samplesrdquo Sensors vol 6 no 11 pp 1466ndash1482 2006

[4] P Babula R Mikelova V Adam et al ldquoChromatographicanalysis of naphthoquinones in plantsrdquoChemicke Listy vol 100no 4 pp 271ndash276 2006

[5] A Emadi A Le C J Harwood et al ldquoMetabolic and electro-chemicalmechanisms of dimeric naphthoquinones cytotoxicityin breast cancer cellsrdquo Bioorganic and Medicinal Chemistry vol19 no 23 pp 7057ndash7062 2011

[6] A E Ross A Emadi L Marchionni et al ldquoDimeric naph-thoquinones a novel class of compounds with prostate cancercytotoxicityrdquo BJU International vol 108 no 3 pp 447ndash4542011

[7] K Nakagawa ldquoNew developments in research on vitamin Kbiosynthesisrdquo Journal of Health Science vol 56 no 6 pp 623ndash631 2010


[8] J Oldenburg M Marinova C Muller-Reible and M WatzkaldquoThe vitamin K cyclerdquo Vitamins and Hormones vol 78 pp 35ndash62 2008

[9] DW Stafford ldquoThe vitamin K cyclerdquo Journal ofThrombosis andHaemostasis vol 3 no 8 pp 1873ndash1878 2005

[10] P Babula V Adam R Kizek Z Sladky and LHavel ldquoNaphtho-quinones as allelochemical triggers of programmed cell deathrdquoEnvironmental and Experimental Botany vol 65 no 2-3 pp330ndash337 2009

[11] B K Aithal M R S Kumar B N Rao N Udupa and B S SRao ldquoJuglone a naphthoquinone from walnut exerts cytotoxicand genotoxic effects against cultured melanoma tumor cellsrdquoCell Biology International vol 33 no 10 pp 1039ndash1049 2009

[12] K Gong and W Li ldquoShikonin a Chinese plant-derived naph-thoquinone induces apoptosis in hepatocellular carcinomacells through reactive oxygen species a potential new treat-ment for hepatocellular carcinomardquo Free Radical Biology andMedicine vol 51 no 12 pp 2259ndash2271 2011

[13] V Klaus T Hartmann J Gambini et al ldquo14-Naphthoquinonesas inducers of oxidative damage and stress signaling in HaCaThuman keratinocytesrdquo Archives of Biochemistry and Biophysicsvol 496 no 2 pp 93ndash100 2010

[14] R R Kitagawa C Bonacorsi L M da Fonseca W Vilegasand M S G Raddi ldquoAnti-Helicobacter pylori activity andoxidative burst inhibition by the naphthoquinone 5-methoxy-34-dehydroxanthomegnin from Paepalanthus latipesrdquo RevistaBrasileira de Farmacognosia vol 22 no 1 pp 53ndash59 2011

[15] G S Levin G Tremasova Ya and S V Kostova ldquoOn themechanism of action of a synthetic 14-naphthoquinone deriva-tive on the respiratory chain of liver and heart mitochondriardquoBiokhimiya vol 54 no 10 pp 1630ndash1637 1989

[16] A G Medentsev Y A Arinbasarova and V K AkimenkoldquoRespiratory activity and naphthoquinone synthesis in thefungus Fusarium decemcellulare exposed to oxidative stressrdquoMikrobiologiya vol 71 no 2 pp 176ndash182 2002

[17] A GMedentsev A NMaslov and V K Akimenko ldquoNaphtho-quinonemetabolites of the fungi Fusarium andVerticillium themechanism of phytotoxic effectrdquo Biokhimiya vol 55 no 10 pp1766ndash1772 1990

[18] G L Beretta and F Zunino ldquoMolecular mechanisms of anthra-cycline activityrdquo in Anthracycline Chemistry and Biology IIMode of Action Clinical Aspects and New Drugs vol 283 pp1ndash19 Springer Berlin Germany 2008

[19] M Masarik D Huska V Adam et al ldquoDoxorubicin andgenomic DNA new insights in drug-nucleic acids interactionsrdquoInternational Journal of Molecular Medicine vol 24 pp S49ndashS49 2009

[20] R Prusa D Huska V Adam et al ldquoIn vivo and in vitro elec-trochemical investigation of doxorubicinellipticine interactionwith DNArdquo Clinical Chemistry vol 55 no 6 pp A217ndashA2172009

[21] L Trnkova M Stiborova D Huska et al ldquoElectrochemicalbiosensor for investigation of anticancer drugs interactions(doxorubicin and ellipticine) with DNArdquo in IEEE SensorsConference (SENSORS rsquo09) vol 1ndash3 pp 1200ndash1203 New YorkNY USA October 2009

[22] Z-Y Hao Q-W Liu J Xu L Jia and S-B Li ldquoSynthesischaracterization antioxidant activities andDNA-binding stud-ies of (E)-N1015840-[1-(pyridin-2-yl)ethylidene]isonicotinohydrazideand its Pr(III) and Nd(III) complexesrdquo Chemical and Pharma-ceutical Bulletin vol 58 no 10 pp 1306ndash1312 2010

[23] T M Jovin L P McIntosh and D J Arndt-Jovin ldquoLeft-handedDNA from synthetic polymers to chromosomesrdquo Journal ofBiomolecular Structure and Dynamics vol 1 no 1 pp 21ndash571983

[24] N-U H Khan N Pandya M Kumar et al ldquoInfluence ofchirality using Mn(iii) salen complexes on DNA binding andantioxidant activityrdquo Organic and Biomolecular Chemistry vol8 no 19 pp 4297ndash4307 2010

[25] A M Pizarro A Habtemariam and P J Sadler ldquoActivationmechanisms for organometallic anticancer complexesrdquo Topicsin Organometallic Chemistry vol 32 pp 21ndash56 2010

[26] E F Sachs A Nadler and U Diederichsen ldquoA DNA and metalbinding hybrid based on triostin Ardquo Journal of Peptide Sciencevol 16 pp 86ndash86 2010

[27] H A Tajmirriahi M Langlais and R Savoie ldquoA comparative-study of DNA interaction with divalent metal-ionsmdashmetal-ion binding-sites and DNA conformational-changes studiedby laser Raman-spectroscopyrdquo in Metal Ions in Biology andMedicine pp 561ndash563 1990

[28] A Woisard G V Fazakerley and W Guschlbauer ldquoZ-DNAis formed by poly(dC-DG) and poly (dm5C-dG) at microor nanomolar concentrations of some zinc(II) and copper(II)complexesrdquo Journal of Biomolecular Structure and Dynamicsvol 2 no 6 pp 1205ndash1220 1985

[29] C Zimmer and G Luck ldquoConformation and reactivity of DNAVI Circular dichroism studies of salt induced conformationalchanges of DNAs of different base compositionrdquo Biochimica etBiophysica Acta vol 361 no 1 pp 11ndash32 1974

[30] A Brozovic J Damrot R Tsaryk et al ldquoCisplatin sensitivity isrelated to late DNA damage processing and checkpoint controlrather than to the early DNA damage responserdquo MutationResearch-Fundamental and Molecular Mechanisms of Mutagen-esis vol 670 no 1-2 pp 32ndash41 2009

[31] O Vrana V Masek V Drazan and V Brabec ldquoRamanspectroscopy of DNA modified by intrastrand cross-links ofantitumor cisplatinrdquo Journal of Structural Biology vol 159 no 1pp 1ndash8 2007

[32] R Zhang Y Niu and Y Zhou ldquoIncrease the cisplatin cyto-toxicity and cisplatin-induced DNA damage in HepG2 cells byXRCC1 abrogation relatedmechanismsrdquo Toxicology Letters vol192 no 2 pp 108ndash114 2010

[33] V Brabec O Vrana and V Boudny ldquoSuperhelical torsioncontrols DNA interstrand cross-linking by antitumor cisplatinrdquoProgress in Biophysics andMolecular Biology vol 65 pp PB113ndashPB113 1996

[34] Y Katsuda Y Yoshikawa T Sato et al ldquoCisplatin and itsanalogues induce a significant change in the higher-orderstructure of long duplex DNArdquo Chemical Physics Letters vol473 no 1ndash3 pp 155ndash159 2009

[35] H Wermann H Stoop A J M Gillis et al ldquoGlobal DNAmethylation in fetal human germ cells and germ cell tumoursassociationwith differentiation and cisplatin resistancerdquo Journalof Pathology vol 221 no 4 pp 433ndash442 2010

[36] J J Yu X B Liang Q W Yan et al ldquoCHK2 and ERCC1 in theDNA adduct repair pathway that mediates acquired cisplatinresistancerdquo in Platinum and Other Heavy Metal Compoundsin Cancer ChemotherapymdashMolecular Mechanisms and ClinicalApplications pp 189ndash194 Humana Press Totowa NJ USA2009


[37] M N da Silva V F Ferreira and M C B V De SouzaldquoAn overview of the chemistry and pharmacology of naph-thoquinones with emphasis on 120573-Lapachone and derivativesrdquoQuimica Nova vol 26 no 3 pp 407ndash416 2003

[38] CWei G Jia J Yuan Z Feng and C Li ldquoA spectroscopic studyon the interactions of porphyrin with G-quadruplex DNAsrdquoBiochemistry vol 45 no 21 pp 6681ndash6691 2006

[39] A Kawiak A Krolicka and E Łojkowska ldquoIn vitro culturesof Drosera aliciae as a source of a cytotoxic naphthoquinoneramentaceonerdquo Biotechnology Letters vol 33 no 11 pp 2309ndash2316 2011

[40] K N Kontogiannopoulos A N Assimopoulou K Dimas andV P Papageorgiou ldquoShikonin-loaded liposomes as a new drugdelivery system physicochemical characterization and in vitrocytotoxicityrdquo European Journal of Lipid Science and Technologyvol 113 no 9 pp 1113ndash1123 2011

[41] R R Kitagawa W Vilegas I Z Carlos and M S G RaddildquoAntitumor and immunomodulatory effects of the naphtho-quinone 5-methoxy-34-dehydroxanthomegninrdquo Revista Bras-ileira de Farmacognosia vol 21 no 6 pp 1084ndash1088 2011

[42] Z F Plyta T Li V P Papageorgiou et al ldquoInhibition oftopoisomerase I by naphthoquinone derivativesrdquo Bioorganicand Medicinal Chemistry Letters vol 8 no 23 pp 3385ndash33901998

[43] P Babula J Vanco L Krejcova et al ldquoVoltammetric charac-terization of Lawsone-Copper(II) ternary complexes and theirinteractions with dsDNArdquo International Journal of Electrochem-ical Science vol 7 no 8 pp 7349ndash7366 2012

[44] A BulovasNDirvianskyte Z Talaikyte et al ldquoElectrochemicaland structural properties of self-assembled monolayers of2-methyl-3-(120596-mercaptoalkyl)-14-naphthoquinones on goldrdquoJournal of Electroanalytical Chemistry vol 591 no 2 pp 175ndash188 2006

[45] D Sajan K P Laladhas I Hubert Joe and V S JayakumarldquoVibrational spectra and density functional theoretical calcu-lations on the antitumor drug plumbaginrdquo Journal of RamanSpectroscopy vol 36 no 10 pp 1001ndash1011 2005

[46] C E M Carvalho I M Brinn A V Pinto and M D CF R Pinto ldquoFluorescent symmetric phenazines from naph-thoquinones 3 Steady-state spectroscopy and solvent effect ofseven phenazine derivatives structure-photophysics correla-tionsrdquo Journal of Photochemistry and Photobiology A vol 136no 1-2 pp 25ndash33 2000

[47] C E M Carvalho N C de Lucas J O M Herrera AV Pinto M C F R Pinto and I M Brinn ldquoFluorescentsymmetric phenazines from naphthoquinones 4 Solvent effecton time-resolved fluorescencerdquo Journal of Photochemistry andPhotobiology A vol 167 no 1 pp 1ndash9 2004

[48] G Balakrishnan A Babaei A J McQuillan and S UmapathyldquoResonance Raman and infrared spectral studies on radicalanions of model photosynthetic reaction center quinones(naphthoquinone derivatives)rdquo Journal of Biomolecular Struc-ture and Dynamics vol 16 no 1 pp 123ndash131 1998

[49] J-R Burie A Boussac C Boullais et al ldquoFTIR spectroscopy ofUV-generated quinone radicals evidence for an intramolecularhydrogen atom transfer in ubiquinone naphthoquinone andplastoquinonerdquo Journal of Physical Chemistry vol 99 no 12 pp4059ndash4070 1995

[50] M Kazemekaite V Railaite A Bulovas et al ldquoSynthesisself-assembling and redox properties of 2-[(120596-sulfanylalk-yl)amino]-14-naphthoquinonesrdquo Collection of CzechoslovakChemical Communications vol 71 no 9 pp 1383ndash1391 2006

[51] M Umadevi A Ramasubbu P Vanelle and V Ramakrish-nan ldquoSpectral investigations on 2-methyl-14-naphthoquinonesolvent effects host-guest interactions and SERSrdquo Journal ofRaman Spectroscopy vol 34 no 2 pp 112ndash120 2003

[52] N Leopold and B Lendl ldquoA newmethod for fast preparation ofhighly surface-enhanced raman scattering (SERS) active silvercolloids at room temperature by reduction of silver nitrate withhydroxylamine hydrochloriderdquo Journal of Physical Chemistry Bvol 107 no 24 pp 5723ndash5727 2003

[53] C H Munro W E Smith M Garner J Clarkson and P CWhite ldquoCharacterization of the surface of a citrate-reducedcolloid optimized for use as a substrate for surface-enhancedresonance raman scatteringrdquo Langmuir vol 11 no 10 pp 3712ndash3720 1995

[54] B Prescott W Steinmetz and G J Thomas Jr ldquoCharac-terization of DNA structures by laser Raman spectroscopyrdquoBiopolymers vol 23 no 2 pp 235ndash256 1984

[55] A Rodger ldquoLinear dichroismrdquoMethods in Enzymology vol 226pp 232ndash258 1993

[56] T Bugarcic O Novakova A Halamikova et al ldquoCytotoxicitycellular uptake and DNA interactions of new monodentateruthenium(II) complexes containing terphenyl arenesrdquo Journalof Medicinal Chemistry vol 51 no 17 pp 5310ndash5319 2008

[57] L Dalla Via O Gia S M Magno A Dolmella D Marton andV Di Noto ldquoSynthesis characterization and biological activityof platinum(II) complexes with l- and d-ornithine ligandsrdquoInorganica Chimica Acta vol 359 no 13 pp 4197ndash4206 2006

[58] L Giovagnini C Marzano F Bettio and D Fregona ldquoMixedcomplexes of Pt(II) and Pd(II) with ethylsarcosinedithiocar-bamate and 2-3-picoline as antitumor agentsrdquo Journal ofInorganic Biochemistry vol 99 no 11 pp 2139ndash2150 2005

[59] R Zaludova G Natile and V Brabec ldquoThe effect of antitumortrans-[PtCl2(E-iminoether)2] on BrarrZ transition in DNArdquoAnti-Cancer Drug Design vol 12 no 4 pp 295ndash309 1997

[60] J Duguid V A Bloomfield J Benevides and G J Thomas JrldquoRaman spectroscopy of DNA-metal complexes I Interactionsand conformational effects of the divalent cations Mg Ca SrBa Mn Co Ni Cu Pd and Cdrdquo Biophysical Journal vol 65 no5 pp 1916ndash1928 1993

[61] L G Marzilli J S Saad Z Kuklenyik K A Keating and Y XuldquoRelationship of solution and protein-bound structures of DNAduplexes with the major intrastrand cross-link lesions formedon cisplatin binding to DNArdquo Journal of the American ChemicalSociety vol 123 no 12 pp 2764ndash2770 2001

[62] D Serban J M Benevides and G J Thomas Jr ldquoDNA second-ary structure andRamanmarkers of supercoiling in Escherichiacoli plasmid pUC19rdquo Biochemistry vol 41 no 3 pp 847ndash8532002

[63] J M Benevides J Kawakami and G J Thomas Jr ldquoMech-anisms of drug-DNA recognition distinguished by Ramanspectroscopyrdquo Journal of Raman Spectroscopy vol 39 no 11 pp1627ndash1634 2008

[64] J M Benevides S A Overman and G J Thomas Jr ldquoRa-man polarized Raman and ultraviolet resonance Raman spec-troscopy of nucleic acids and their complexesrdquo Journal ofRaman Spectroscopy vol 36 no 4 pp 279ndash299 2005

[65] H Deng V A Bloomfield J M Benevides and G J ThomasldquoDependence of the raman signature of genomic B-DNA onnucleotide base sequencerdquo Biopolymers vol 50 no 6 pp 656ndash666 1999

[66] J G Duguid V A Bloomfield J M Benevides and G JThomas Jr ldquoRaman spectroscopy of DNA-metal complexes II


The thermal denaturation of DNA in the presence of Sr2+ Ba2+Mg2+ Ca2+ Mn2+ Co2+ Ni2+ and Cd2+rdquo Biophysical Journalvol 69 no 6 pp 2623ndash2641 1995

[67] J G Duguid V A Bloomfield J M Benevides and G JThomas Jr ldquoDNAmelting investigated by differential scanningcalorimetry and Raman spectroscopyrdquo Biophysical Journal vol71 no 6 pp 3350ndash3360 1996

[68] C I Morari and C M Muntean ldquoNumerical simulations ofRaman spectra of guanine-cytosine Watson-Crick and proto-natedHoogsteen base pairsrdquoBiopolymers vol 72 no 5 pp 339ndash344 2003

[69] CMMuntean and I Bratu ldquoRaman spectroscopic study on thesubpicosecond dynamics in calf-thymus DNA upon loweringthe pH and in the presence of Mn2+ ionsrdquo Spectroscopy vol 22no 6 pp 475ndash489 2008

[70] C M Muntean L Dostal R Misselwitz and H Welfle ldquoDNAstructure at low pH values in the presence of Mn2+ ions aRaman studyrdquo Journal of Raman Spectroscopy vol 36 no 11 pp1047ndash1051 2005

[71] C MMuntean and GM J Segers-Nolten ldquoRamanmicrospec-troscopic study of effects of Na(I) and Mg(II) ions on low pHinduced DNA structural changesrdquo Biopolymers vol 72 no 4pp 225ndash229 2003

[72] W L Peticolas Z Dai and G A Thomas ldquoThe use of Ramanspectroscopy to characterize double BZ conformational junc-tions in DNArdquo Journal of Molecular Structure vol 242 pp 135ndash141 1991

[73] C Rajani J R Kincaid andDH Petering ldquoThepresence of twomodes of binding to calf thymus DNA bymetal-free bleomycina low frequency Raman studyrdquo Biopolymers vol 52 no 3 pp129ndash146 1999

[74] M Schmitt and J Popp ldquoRaman spectroscopy at the beginningof the twenty-first centuryrdquo Journal of Raman Spectroscopy vol37 no 1ndash3 pp 20ndash28 2006

[75] E M Evertsz G AThomas andW L Peticolas ldquoRaman spec-troscopic studies of the DNA Crobinding site conformationfree and bound to cro proteinrdquo Biochemistry vol 30 no 4 pp1149ndash1155 1991

[76] V M Dadarlat and V K Saxena ldquoStability of triple-helicalpoly(dT)-poly(dA)-poly(dT) DNA with counterionsrdquo Biophys-ical Journal vol 75 no 1 pp 70ndash91 1998

[77] P Grof D Aslanian andG Ronto ldquoChanges of phage T7 nucle-oprotein structure at low ionic strength ARaman spectroscopicstudyrdquoBiochimica et Biophysica Acta vol 1289 no 1 pp 95ndash1041996

[78] C M Muntean G J Puppels J Greve G M J Segers-Noltenand S Cinta-Pinzaru ldquoRaman microspectroscopic study onlow-pH-induced DNA structural transitions in the presence ofmagnesium ionsrdquo Journal of Raman Spectroscopy vol 33 no 10pp 784ndash788 2002

[79] K L Aubrey S R Casjens and G J Thomas Jr ldquoSecondarystructure and interactions of the packaged dsDNA genomeof bacteriophage P22 investigated by Raman difference spec-troscopyrdquo Biochemistry vol 31 no 47 pp 11835ndash11842 1992

[80] C M Muntean and I Bratu ldquoMolecular relaxation processesin calf-thymus DNA in the presence of Mn2+ and Na+ ionsa Raman spectroscopic studyrdquo Spectroscopy vol 22 no 5 pp345ndash359 2008

[81] J Stangret andR Savoie ldquoVibrational spectroscopic study of theinteraction of metal ions with diethyl phosphate a model forbiological systemsrdquo Canadian Journal of Chemistry vol 70 no12 pp 2875ndash2883 1992

[82] S Mukherjee and D Bhattacharyya ldquoEffect of phosphoroth-ioate chirality on the grooves of DNA double helices a molec-ular dynamics studyrdquo Biopolymers vol 73 no 2 pp 269ndash2822004

[83] M Shanmugasundaram and M Puranik ldquoComputational pre-diction of vibrational spectra of normal and modified DNAnucleobasesrdquo Journal of Raman Spectroscopy vol 40 no 12 pp1726ndash1748 2009

[84] J Qi B Liu Y Li D Wu and W Tang ldquoRaman spectroscopicstudy on Hela cells irradiated by X rays of different dosesrdquoChinese Optics Letters vol 7 no 8 pp 734ndash737 2009

[85] D Lin J Lin Y Wu et al ldquoInvestigation on the interactionsof lymphoma cells with paclitaxel by Raman spectroscopyrdquoSpectroscopy vol 25 no 1 pp 23ndash32 2011

[86] A Gfrorer M E Schnetter J Wolfrum and K O GreulichldquoDouble and triple helices of nucleic-acid polymers studiedby Uv-resonance Raman-spectroscopyrdquo Berichte der Bunsenge-sellschaft fur Physikalische Chemie vol 97 no 2 pp 155ndash1621993

[87] C Krafft W Hinrichs P Orth W Saenger and H WelfleldquoInteraction of Tet repressor with operator DNA and withtetracycline studied by infrared and Raman spectroscopyrdquoBiophysical Journal vol 74 no 1 pp 63ndash71 1998

[88] L Movileanu J M Benevides and G J Thomas Jr ldquoTem-perature dependence of the Raman spectrum of DNA PartImdashRaman signatures of premelting and melting transitions ofPoly(dA-dT) sdot poly(dA-dT)rdquo Journal of Raman Spectroscopyvol 30 no 8 pp 637ndash649 1999

[89] C M Muntean and I Bratu ldquoMolecular dynamics in calf-thymus DNA at neutral and low pH in the presence ofNa+ Ca2+ and Mg2+ ions a Raman microspectroscopic studyrdquoSpectroscopy vol 21 no 4 pp 193ndash204 2007

[90] C M Muntean K Nalpantidis I Feldmann and V DeckertldquoZn2+-DNA interactions in aqueous systems a Raman spectro-scopic studyrdquo Spectroscopy vol 23 no 3-4 pp 155ndash163 2009

[91] A Toyama A Matsubuchi N Fujimoto and H TakeuchildquoIsotope-editedUVRaman spectroscopy of protein-DNA inter-actions binding modes of cyclic AMP receptor protein to anatural DNA recognition siterdquo Journal of Raman Spectroscopyvol 36 no 4 pp 300ndash306 2005

[92] R M Clement J Sturm and M P Daune ldquoInteraction ofmetallic cations with DNA VI Specific binding of Mg++ andMn++rdquo Biopolymers vol 12 no 2 pp 405ndash421 1973

[93] N Iyandurai and R Sarojini ldquoSelenomethionine inducedchanges on the binding of spermine with DNA a study byFourier transform Raman and Fourier Transform infra redspectroscopyrdquo International Journal of Pharmacology vol 5 no2 pp 126ndash136 2009

[94] I Notingher S Verrier H Romanska A E Bishop J M Polakand L L Hench ldquoIn situ characterisation of living cells byRaman spectroscopyrdquo Spectroscopy vol 16 no 2 pp 43ndash512002

[95] H Yao Z Tao M Ai et al ldquoRaman spectroscopic analysisof apoptosis of single human gastric cancer cellsrdquo VibrationalSpectroscopy vol 50 no 2 pp 193ndash197 2009

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

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StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of



Advances in Pharmacological Sciences

Tropical MedicineJournal of


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AddictionJournal of



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Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Autoimmune Diseases


Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of


Pharmaceutics


MEDIATORSINFLAMMATION

of


prostate androgen-dependent cell lines and breast tumourcell lines [5 6] Naphthoquinones have many physiologicalroles the most important ubiquitous naphthoquinones arevitamins of the K groupmdashphylloquinone and menaquinone[7ndash9] Plenty of naphthoquinones are important phytoalex-ins so they play an important role in ecological relationships[10] Naphthoquinones have found many possibilities forusemdashantimicrobial antifungal antiviral and antiprotozoalproperties have been identified In traditional medicinesparticularly in Asia (China) and South America plants withnaphthoquinones have a wide range of application especiallyin the treatment of various types of cancer [2] The cytotoxicproperties of the naphthoquinones are in the focus of interestof many scientists They are predominantly based on theability of naphthoquinones to generate reactive oxygenspecies (ROS) which are involved in many cellular processesand serve as signal molecules in apoptosisprogrammed celldeath and are partially responsible for cytotoxic propertiesdemonstrated in many in vitro tumour cell line systems[11ndash14] The ability of naphthoquinones to interfere withthe respiratory chain poses the second possible cytotoxicmechanism [15ndash17] DNA is the critical target of the activityof many compounds including plant secondary metabolitesThere is a large group of natural origin compounds withinteresting cytotoxic properties Their cytotoxic effect isbased on their interactions with DNA especially on thepossibility of forming various types of covalent adductswith DNA The most important compounds whose effectis based on DNA interaction are anthracyclines cytotoxicantibiotics and platinum-based cytostatics [18ndash21]

DNA-metal and DNA-metal based complexes interac-tions and their effect on DNA structure are already known[22ndash29] Cisplatin as an important and frequently used anti-cancer drug forms covalent bonds with nucleophilic sites onguanine residues present in DNA As a cisplatin is a bifunc-tional agent it is able to bind to one (monofunctional adduct)or two (intrastrand crosslinks interstrand crosslinks) placesin a DNA chain [30ndash32] All of these adducts characteristi-cally distort the DNA conformation [33 34] The main prob-lem in the curing process with many anticancer complexessuch as cisplatin is the reduced sensitivity of the tumourtowards this drug when the drug is repeated in the treatmentA medical treatment is connected with many inadvisableeffects [35 36] Therefore new structures with reducedtoxicity often of natural origin are intensively searchedDue to significant cytotoxic properties of the selectednaphthoquinones (see Figure S1 in Supplementary Materialavailable online at httpdxdoiorg1011552014461393) wedecided to study the naphthoquinones-DNA interactions asa possible mechanism of their cytotoxicity In recent yearsnaphthoquinone derivatives have been intensively studiedin connection with their cytotoxic and antibacterial effectsand subsequently possible usage in cancer treatment [37ndash41] Despite the above-mentioned mechanisms of naphtho-quinones cytotoxicity their interactions with DNA have notyet been establishedDue to this fact we focused on the inves-tigation of naphthoquinones-DNA interactions in our workThe identification of the binding sites of naphthoquinoneson DNA and the determination of how the binding may

affect secondary and tertiary structures is still lacking Somenaphthoquinones have been identified as potent inhibitors oftopoisomerases I and II this activity may contribute to theircytotoxic effect [2 37 42] In addition newly synthesizedcomplexes of lawsone andmetal ions are recently investigatedas cytotoxic compounds with the cytotoxic effect based onthe DNA interactions [43] The vibrational spectra [44 45]optical absorption and fluorescence emission spectra [4647] and SERS studies [44 45 48ndash51] of some naphtho-quinone derivatives have also been reported However thenaphthoquinones-DNA interactions can play the crucial rolein their cytotoxic effectThe vibrational spectroscopy (Ramanspectroscopy) can help making unambiguous identificationof vibrational modes of biological molecular systems [24 32]We used Raman spectroscopy to assess the binding sitesof the naphthoquinones to DNA and to characterize andcompare the perturbations of DNA after its modification bynaphthoquinones in our workWe used Raman spectroscopyand SERS in the analysis of DNA modified by the selectednaphthoquinones 14-naphthoquinone binaphthoquinonejuglone lawsone and plumbagin (Figure S1) The spectraobtained were analyzed and characteristic spectral changeswere described with the aim of determining interactionsbetween selected naphthoquinones and DNA and analyzingDNAstructural changes under naphthoquinones-DNA inter-actions In addition the linear dichroism spectroscopy wasused to verify these results

2 Materials and Methods

21 Chemicals Material and pH Measurements 14-Naph-thoquinone binaphthoquinone [22-bi(3-hydroxy-14-naph-thoquinone] juglone [5-hydroxy-14-naphthoquinone] law-sone [2-hydroxy-14-naphthoquinone] and plumbagin [5-hydroxy-2-methy-14-naphthoquinone] as well as all otherchemicals of ACS purity unless noted otherwise werepurchased from Sigma Aldrich Chemical Corp (Sigma-Aldrich USA) Deionised water underwent demineralizationby reverse osmosis by using an instrument Aqua Osmotic02 (Aqua Osmotic Tisnov Czech Republic) and was subse-quently purified by using a Millipore RG (Millipore CorpUSA 18M1015840Ω)mdashMiliQ water The pH value was measured byusing the WTW inoLab pH meter (Weilheim Germany)

22 Preparation of DNA DNA was isolated from the calfthymus in accordance with the published work of Brabecet al [33] DNA was dissolved in 05M NaCl and wasthen sonicated for 20 minutes by using a Sonicator 3000(Giltron USA) and subsequently analyzed The first step ofdenatured DNA analysis was the electrophoresis by using thesource Standard Power Pack P25 by (Biometra Germany)The gel was photographed after the backlighting the gel byUV on a transilluminator TVX-20M (312 nm) (AlysLabwareSwitzerland) The next step was the polarography on ananalyzer EGampG PARC Model 384B (LabX Canada) Theconcentration of DNAwasmeasured on a BeckmanDU 7400(Pall Gelman Germany) For the dialysis a SpectraPor 12-14000 (Cole-Palmer USA) was used The lyophilization was















3 Results





400 600 800 1000 1200 1400 1600

minus3000

minus2000

minus1000

0

0

2000

4000

6000

8000

6000

8000

10000

12000

14000

Ram

an in

tens

ity


(1)

(2)

(3)

(a)

400 600 800 1000 1200 1400 1600

minus3000

minus2000

minus1000

0

0

2000

4000

6000

8000

6000

8000

10000

12000

Ram

an in

tens

ity


(1)

(2)

(3)

(b)







400 600 800 1000 1200 1400 1600


minus3000

minus2000

minus1000

0

0

2000

4000

6000

8000

6000

8000

10000

12000

Ram

an in

tens

ity

(1)

(2)

(3)

(a)

Ram

an in

tens

ity

400 600 800 1000 1200 1400 1600


minus3000

minus2000

minus1000

0

2000

4000

6000

8000

6000

8000

10000

12000

(1)

(2)

(3)

(b)







400 600 800 1000 1200 1400 1600

minus7000

minus6000

minus5000

minus40000

2000

4000

6000

8000

6000

8000

10000

12000Ra

man

inte

nsity


(1)

(2)

(3)

(a)

200 250 300 350 400 450 500

(6)

(5)

(4)

(3)

(2)

(1)



(b)















4 Discussion








5 Conclusions


Abbreviations





Acknowledgments


References








































































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of















3 Results





400 600 800 1000 1200 1400 1600

minus3000

minus2000

minus1000

0

0

2000

4000

6000

8000

6000

8000

10000

12000

14000

Ram

an in

tens

ity


(1)

(2)

(3)

(a)

400 600 800 1000 1200 1400 1600

minus3000

minus2000

minus1000

0

0

2000

4000

6000

8000

6000

8000

10000

12000

Ram

an in

tens

ity


(1)

(2)

(3)

(b)







400 600 800 1000 1200 1400 1600


minus3000

minus2000

minus1000

0

0

2000

4000

6000

8000

6000

8000

10000

12000

Ram

an in

tens

ity

(1)

(2)

(3)

(a)

Ram

an in

tens

ity

400 600 800 1000 1200 1400 1600


minus3000

minus2000

minus1000

0

2000

4000

6000

8000

6000

8000

10000

12000

(1)

(2)

(3)

(b)







400 600 800 1000 1200 1400 1600

minus7000

minus6000

minus5000

minus40000

2000

4000

6000

8000

6000

8000

10000

12000Ra

man

inte

nsity


(1)

(2)

(3)

(a)

200 250 300 350 400 450 500

(6)

(5)

(4)

(3)

(2)

(1)



(b)















4 Discussion








5 Conclusions


Abbreviations





Acknowledgments


References








































































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


400 600 800 1000 1200 1400 1600

minus3000

minus2000

minus1000

0

0

2000

4000

6000

8000

6000

8000

10000

12000

14000

Ram

an in

tens

ity


(1)

(2)

(3)

(a)

400 600 800 1000 1200 1400 1600

minus3000

minus2000

minus1000

0

0

2000

4000

6000

8000

6000

8000

10000

12000

Ram

an in

tens

ity


(1)

(2)

(3)

(b)







400 600 800 1000 1200 1400 1600


minus3000

minus2000

minus1000

0

0

2000

4000

6000

8000

6000

8000

10000

12000

Ram

an in

tens

ity

(1)

(2)

(3)

(a)

Ram

an in

tens

ity

400 600 800 1000 1200 1400 1600


minus3000

minus2000

minus1000

0

2000

4000

6000

8000

6000

8000

10000

12000

(1)

(2)

(3)

(b)







400 600 800 1000 1200 1400 1600

minus7000

minus6000

minus5000

minus40000

2000

4000

6000

8000

6000

8000

10000

12000Ra

man

inte

nsity


(1)

(2)

(3)

(a)

200 250 300 350 400 450 500

(6)

(5)

(4)

(3)

(2)

(1)



(b)















4 Discussion








5 Conclusions


Abbreviations





Acknowledgments


References








































































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


400 600 800 1000 1200 1400 1600


minus3000

minus2000

minus1000

0

0

2000

4000

6000

8000

6000

8000

10000

12000

Ram

an in

tens

ity

(1)

(2)

(3)

(a)

Ram

an in

tens

ity

400 600 800 1000 1200 1400 1600


minus3000

minus2000

minus1000

0

2000

4000

6000

8000

6000

8000

10000

12000

(1)

(2)

(3)

(b)







400 600 800 1000 1200 1400 1600

minus7000

minus6000

minus5000

minus40000

2000

4000

6000

8000

6000

8000

10000

12000Ra

man

inte

nsity


(1)

(2)

(3)

(a)

200 250 300 350 400 450 500

(6)

(5)

(4)

(3)

(2)

(1)



(b)















4 Discussion








5 Conclusions


Abbreviations





Acknowledgments


References








































































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


400 600 800 1000 1200 1400 1600

minus7000

minus6000

minus5000

minus40000

2000

4000

6000

8000

6000

8000

10000

12000Ra

man

inte

nsity


(1)

(2)

(3)

(a)

200 250 300 350 400 450 500

(6)

(5)

(4)

(3)

(2)

(1)



(b)















4 Discussion








5 Conclusions


Abbreviations





Acknowledgments


References








































































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of









4 Discussion








5 Conclusions


Abbreviations





Acknowledgments


References








































































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of







5 Conclusions


Abbreviations





Acknowledgments


References








































































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

































































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

research article the study of naphthoquinones and their

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