Inorganica Chimica Acta 496 (2019) 119048

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Inorganica Chimica Acta

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

Research paper

DNA binding interaction studies of flavonoid complexes of Cu(II) and Fe(II) T

and determination of their chemotherapeutic potential
Erum Jabeena,b, , Naveed Kausar Janjuab, Safeer Ahmedb, Iftikhar Tahiric, Muhammad Kashif
⁎

(DUHS)d, Aneela Javede

a
Department of Chemistry, Allama Iqbal Open University, Islamabad 44400, Pakistan
b
Department of Chemistry, Quaid-i-Azam University, Islamabad 45320, Pakistan
c
Department of Chemistry, Federal Urdu University of Arts, Science and Technology, Karachi 75300, Pakistan
d
ICCBS-Department of Chemistry, University of Karachi, Karachi 75300, Pakistan
e
Healthcare Biotechnology ASAB-National University of Science and Technology, Islamabad, Pakistan

ARTICLE INFO ABSTRACT

Keywords: The present study is based on molecular level investigation of DNA binding of three flavonoids (Fls), morin
Metal flavonoid complexes (mor), quercetin (quer) and primuletin (prim) and their metal (Cu (II) and Fe (III)) complexes through UV–Vis
ds-DNA spectroscopy, fluorescence spectroscopy, thermal analysis and cyclic voltammetry (CV). The spectroscopic ex-
Binding constant periments were performed by titrating compounds with DNA and the results were in good agreement with those
Cancer cell lines
obtained from titration of DNA by the compounds. The Fe-mor, Cu-quer, Fe-quer and prim which exhibited
Cytotoxicity
hypsochromic/hyperchromic shifts in UV–Vis spectra, showed negligible effects over florescent spectra of NR-
Cytostatic activity
DNA (intercalating Neil red dye–DNA complex), intact Tm (thermal melting temperature) of DNA and the shift of
peak potential (Ep) towards less positive value were designated to exhibit electrostatic mode of binding with
dsDNA having binding constant, Kb ≈ 102 M−1. The mor, Cu-mor, Cu-prim and Fe-prim showed intercalating
mode of interaction with dsDNA which was recognized from hypochromic/bathochromic shifts in the spectra, Ep
shifts towards higher potential in CV, significant decay in fluorescence intensity, distinct shift in Tm of DNA and
binding constant (Kb) of the order of 104 M−1. The mor exhibited mixed binding mode (intercalation and groove
binding) at different concentrations. The quer displayed increase in peak current and hyperchromism thus de-
picted groove binding into dsDNA (Kb ≈ 103 M−1). The binding modes were found to be correlated with the
apoptotic activity of the Fls and M-Fls (flavonoids and metal-flavonoids) assayed against human breast cancer
cell lines (MCF-7) and human uterine cervical cancer cell lines (HeLa). The correlation is interpreted in terms of,
the intercalation mode as cytostatic activity and the groove binding with no apoptosis. However, electrostatic
binding of (M-)Fls to dsDNA did not produce significant anti-proliferation activity against Hela cell lines with
exception of Fe-mor (cytostatic activity) and Cu-quer (cytotoxicity) against MCF-7 cell line.

1. Introduction flavones have been reported to be mutagenic against different bacterial

One of the important pharmacological effects assigned to flavonoids
(Fls) is associated with their anti-cancerous activity [1,2]. Reactive
oxygen species’, (ROS) induced oxidative stress can degenerate DNA
leading to mutagenesis. Flavonoids can inhibit the ROS-induced da-
mages to DNA because of their radical scavenging ability [2]. Thus, the
flavonoids can be regarded as potential chemo-preventive agents
against certain carcinogens. Despite their commonly accepted role of
safeguarding against carcinogens, flavonoids are also considered as
mutagenic and have ability to damage DNA [3–6]. For example 3-hy-
droxyflavone, 5-hydroxyflavones, 7-hydroxyflavone and polyhydroxy
stain under certain conditions [7]. Flavonoids have ability to bind to
DNA and this can hinder the DNA replication under physiological
conditions [8]. However, this DNA damage resulting from resonance
stabilized flavonoid radicals is expected to be less than that of ROS [9].
Many flavonoid metal complexes ((M-)Fls) bind to DNA more
strongly than their flavonoid counterparts. Some literature reports state
that metal-flavonoid can intercalate into DNA strands, whereas flavo-
noids alone bind to DNA in other ways or intercalate into DNA to a
lesser extent [10]. For example, quercetin has been reported to bind to
DNA electrostatically while the La-quercetin intercalates into DNA
under the same conditions [10]. Same is the case with La-chrysin [11]

⁎
Corresponding author. Tel.: +92 03315693504.
E-mail address: erum.jabeen@aiou.edu.pk (E. Jabeen).

https://doi.org/10.1016/j.ica.2019.119048
Received 10 June 2019; Received in revised form 2 August 2019; Accepted 2 August 2019
Available online 06 August 2019
0020-1693/ © 2019 Published by Elsevier B.V.
E. Jabeen, et al.
Fig. 1. Effect of incremental addition of dsDNA on 5 µM mor spectrum (a) and of mor on 5 µM dsDNA spectrum (b) in 20% methanol PBS (pH = 7.4) 310 K.
and Eu-quercetin [12]. The Zn (II) [13], Cu (II) [14] and Bi (III) [15]
complexes of morin can bind to DNA mainly by intercalation, whereas
free morin interacts with DNA via a non-intercalating mode [16]. The
availability of planner and non-planner parts within a single molecule
(metal complex) imparts higher intercalative binding affinity to the
complexes with additional electrostatic interactions between the metal
cation and the negatively charged phosphate backbone [10]. Thus, in
general, it can be inferred that the respective binding mode in (M-)Fls
imparts strength to the flavonoid-DNA binding. Considering this very
effect of the metal in (M-)Fls and the ability of flavonoids to act as anti-
cancerous species, an interesting study could be the correlation be-
tween anti-cancerous potential and the mode of interaction (and
binding strength) of Fls or (M-)Fls with dsDNA.
Most of the literature on anti-cancerous activity of flavonoids or
their metal flavonoid complexes presents either binding modes or
binding constants [17,18] but the role of the binding mode in defining
the cytotoxicity or cytostatic activity has not been explored so far. Al-
though the cellular mode of action is likely to be more complex and the
compound does not interact with cellular DNA with sole mode of action
as observed for isolated DNA, establishing a correlation between
binding mode and their anti-proliferation of cancer cells will be helpful
in screening new compounds before cancer cell activity studies in ra-
tional drug designing.
In the present work, the DNA binding of the three flavonoids (Fls)
namely morin (mor), quercetin (quer) and primuletin (prim) and their
metal (Cu(II) and Fe(III)) complexes (M-Fls) have been investigated
quantitatively which was subsequently considered purposefully for the
correlation studies of the interaction mode with anti-proliferation ac-
tivity. Further polyhydroxy flavone exhibits different metal complexion
sites in their structure, therefore can form more than one type of
complexes in different ratios [19,20]. The current study is limited to
those complexes of quercetin and morin where metal atom has been
coordinated with C]O at C4 position and –OH group at C3 position and
the only chelation site available in primuletin (structure of these
compounds have already been reported by our group and also given in
Supplementary data Fig. S1) [21].
2. Materials and methods
2.1. Materials
Quercetin dihydrate (C15H10O7·2H2O) and morin dihydrate
(C15H10O7·2H2O) were purchased from Merck, Germany while primu-
letin used was of Sigma Aldrich, Germany. Cu (II) and Fe (III) com-
plexes of flavonoids were synthesized using reported method [22] and
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Inorganica Chimica Acta 496 (2019) 119048
their characterization is reported in our previous work [21]. Sodium
phosphate buffer, which also acted as supporting electrolyte in cyclic
voltammetry (CV) experiments, was prepared by mixing 190 mL 0.1 M
NaH2PO4 with 810 mL of 0.1 M Na2HPO4. All the reagents used were of
analytical grade.
2.2. Methods
2.2.1. UV–Vis measurements of dsDNA interaction
5 mM stock solutions of the (M-)Fls in methanol were diluted by
0.1 M buffer (phosphate buffer-pH 7.4) to prepare 5 μM test solution.
Calf thymus blood double stranded DNA (dsDNA) was extracted by
Falcon method [23] and its purity was judged from absorbance ratio at
260 nm and 280 nm. The ratio (A260/A280) > 1.8 indicated that dsDNA
was sufficiently pure and free from protein [24]. This dsDNA was then
stored at 4 °C. A 20 µM stock solution of dsDNA was prepared in doubly
distilled water and its exact concentration was calculated by noting its
absorbance at 260 nm (the molar extinction coefficient (ε) of dsDNA is
taken to be 6600 M−1cm−1).
For dsDNA interaction study, the spectrum was recorded at the λmax
of Fls by keeping the concentration of (M-)Fls constant and by varying
dsDNA and vice versa. The absorbance variation was used to get
binding constant Kb from Benesi-Hildebrand relation [25–27]:
A0 1
= G
+ G
.
A A0 Kb [DNA] (1a)
H G G H G G
where A0 and A are the absorbance’s of free and bound flavonoid, εG
and εH–G are their molar extinction coefficients, respectively (H and G
correspond to host and guest, respectively).
When traditional Benesi-Hildebrand equation was exploited for vice
versa data that was the titration of constant excess dsDNA concentra-
tion with variable (M-)Fls concentration, it was found that the data
yielded straight line plots in accordance with following equation;
A0 G G 1
= + .
A A0 H G G H G G Kb [M Fls] (1b)
The derivation of this form exactly follows the derivation and as-
sumption of classical form of Benesi-Hildebrand equation [28] with the
condition that dsDNA was in excess and constant amount (we are not
mentioning its derivation here). Therefore, we are reporting the re-
ciprocal method/experiment for dsDNA binding studies (part (b) of
Figs. 1, 4 and 5) which yield same Kb values (Table 1) as obtained from
classical method (part (a) of Figs. 1, 4 and 5).
E. Jabeen, et al. Inorganica Chimica Acta 496 (2019) 119048

Table 1
Binding constants (Kb) and -ΔG for (M-)Fls interaction with dsDNA obtained from UV–Vis spectroscopy and cyclic voltammetry at 310 K in PBS (pH-7.4).
(M-)Fls Obtained through

UV–Vis spectroscopy Cyclic voltammetry Fluorescence spectroscopy

[DNA] constant [DNA] variable [DNA] variable [DNA] constant

Kb · 10−3 −ΔG Kb × 10−3 −ΔG Kb × 10−3 −ΔG Kb × 10−3 −ΔG

(M−1) (kJ/mol) (M−1) (kJ/mol) (M−1) (kJ/mol) (M−1) (kJ/mol)

mor 9.94 23.72 9.98 23.73 9.01 23.22 9.84 23.70

Cu-mor 11.0 23.98 11.0 23.98 12.0 24.21 10 23.74
Fe-mor 0.96 17.70 0.94 17.64 0.53 16.17 0.97 17.72
quer 4.21 21.51 4.18 21.49 4.82 21.86 3.64 21.13
Cu-quer 0.98 17.75 0.95 17.67 0.92 17.59 0.99 17.79
Fe-quer 0.82 17.29 0.81 17.26 0.89 17.50 0.62 16.56
prim 0.89 17.50 0.85 17.38 0.88 17.47 0.87 17.45
Cu-prim 17.61 25.20 17.60 25.20 18.02 25.26 15.5 24.86
Fe-prim 9.98 23.73 9.91 23.71 9.89 23.71 9.96 23.73

2.2.2. Electrochemical measurements of dsDNA interaction time zero plates in order to fix the cells. 100 µL of either (M-)Fls or
Cyclic voltammetry titration was performed using three electrode standard anticancer drug doxorubicin were added in experimental wells
system consisting of glassy carbon working, platinum counter and sa- and were incubated for 48 h at 37 °C in CO2 incubator. After 2 days
turated calomel reference (SCE) electrode to study the interaction of incubation, the experimental plates were fixed with 50% TCA and in-
(M-)Fls with dsDNA. The binding constant (Kb) was calculated by ex- cubated at room temperature for 30 min. The plates were turned upside
ploiting cyclic voltammograms of metal-flavonoid in the presence of down over the sink and was flicked sharply two or three times to shake
dsDNA using [29]: out the fluid from well, rinsed with distilled water and air dried until no
moisture was visible [30].
1 Kb (1 A) The plates were then stained by adding 100 µL of 0.4% SRB stain
= Kb solution for 30 min at room temperature, washed with 1% acetic acid
[DNA]
{ 1
i
i0 } (2) and were air dried. 100 µL of 10 mM tris base was added to solubilize
the SRB stain and plates were kept at plate shaker for 5–10 min fol-
where i and io are respective peak currents of (M-)Fls complex in the
lowed by optical density (OD) measurement at 515 nm.
absence and presence of dsDNA.
The % growth inhibition was calculated as;

2.2.3. Fluorescence spectroscopy Percent growth inhibition = 100% {100[(T Tz)/(C Tz)]} (3)
The Neil red dye was used as florescent probe. Neil red is an in- where C indicates control optical density (OD) and Tz and T indicate OD
tercalating agent. It’s intercalation into DNA results in generating an values at time of addition of drugs and after treatment for 48 h, re-
intense fluorescence peak at 256 nm. This florescent intensity will de- spectively
crease if any ligand displaces the probe form DNA base pairs which can
be only done by an intercalator. Therefore, the decrease in fluorescence 3. Result and discussion
intensity of NR-DNA at 256 nm can be monitored as indicator of in-
tercalation. The fluorescence experiments were conducted by titrating The interaction of (M-)Fls (flavonoids or metal-flavonoids) with
NR-DNA against variable concentrations (10–80 μM) of (M-)Fls and dsDNA was studied at molecular level using UV–Vis spectroscopy and
recording absorbance at 256 nm. The decrease in absorbance was then cyclic voltammetry followed by anti-cancerous potential study using
plotted according to Stern Volmer equation; two different cancel cell lines.
(F0 F)
log = logKb + nlog[(M )Fls]
F (3) 3.1. Binding mode investigations of (M-)mor

The Fig. 1 shows the effect of incremental addition of mor over

2.2.4. Thermal melting
The thermal melting of DNA was noticed in the absence and pre-
sence of (M-)Fls by recording the absorbance (at 256 nm) vs. tem-
perature plots for a temperature range of 40–90 °C at a rate of
1 °C min−1 on Perkin Elmer Lambda 35 UV–Vis spectrometer equipped
with temperature controlling programmer (0.2 °C).
The ΔG of (M-)Fl-DNA complex was calculated from the van’t Hoff
equation [29] using Kb calculated from Eqs. (1a) and (1b), (2) and (3).
2.2.5. Cancer cell lines study
The sulforhodamine (SRB) assay was started in 96-well plates by
adding 100 µL of plain RPMI medium in control wells of 96-well plates,
whereas cancer cells (10,000 per well) of HeLa and MCF-7 were placed
in treatment wells in the 100 µL culture medium. Two time zero plates
(time zero 1 and 2) and experimental plates were incubated for 24 h at
37 °C in CO2 incubator (5%, CO2) followed by addition of 50 µL of ice-
cold 50% trichloroacetic acid (TCA) on top of the culture medium in
dsDNA spectrum and vice versa.
When 5 μM mor was titrated with variable concentration of dsDNA,
a decrease in absorption was reported without shift in λmax (Fig. 1(a)).
Another set of experiments was performed with addition of variable
concentrations of mor to constant concentration of dsDNA in order to
verify previous results. A hypochromic blue shift in dsDNA spectrum by
mor was reported (Fig. 1(b)). This indicated that mor and dsDNA may
have interacted through mixed mode of binding including intercalation
and groove binding [31,32].
The mode of binding was then tested through fluorescence spec-
troscopy and thermal melting experiments. The results for both com-
plementary experiments are given in Fig. 2. In fluorescence, the inter-
calator would be able to replace intercalating florescent probe,
therefore, the fluorescence intensity would decrease and the groove
binder and electrostatic binder will not affect the fluorescence of in-
tercalating probe. In fluorescence experiments, the fluorescence in-
tensity NR-DNA was monitored where NR is intercalating Neil red dye.

3
E. Jabeen, et al.
Fig. 2. (a) Fluorescence spectra of NR-DNA (10 µM) in the presence of different concentrations of mor in 20% methanol PBS (pH = 7.4) 310 K. (b) Thermal melting
plots for DNA and mor-DNA.
The Fig. 2(a) reveals that NR-DNA intensity decreased significantly
with incremental addition of mor. This depicted that the mode of mor-
DNA binding is intercalation. In thermal melting, the intercalator re-
sults in stabilizing dsDNA double strands, thus, the thermal melting
temperature for 50% denaturation will be 5-8 °C higher than the cor-
responding Tm for dsDNA. The mor resulted in increase in Tm by 5.1 °C
as an indicator of intercalation.
The Ep of mor shifted towards positive value upon slight addition of
dsDNA. It can be suggested that intercalation takes place between mor
and dsDNA at lower concentrations of dsDNA up to slight addition of
dsDNA [33]. With the increase in concentration of dsDNA the Ep
shifting becomes less pronounced however Ep for all mor-DNA mixtures
were higher (more positive value) that of mor only (0 μΜ DNA)
Fig. 3(a). This interesting trend confirms the intercalation [33,34].
Similarly, slight red shift (3 nm) was observed in the spectrum of
Cu-mor when dsDNA was added to it (Fig. 4(a)) along with decrease in
absorbance. This red shift hypochromism generated isosbestic points at
287 nm and 439 nm Fig. 4(a). The parallel experiment revealed hypo-
chromism with red shift (bathochromic) in the spectrum of dsDNA by
Cu-mor Fig. 4(b). This decrease in absorbance with slight red shift in
both titrations pointed towards intercalation of Cu-mor into dsDNA
[35]. The fluorescence spectra of titration of NR-DNA with Cu-mor
revealed that slight red shift observed in UV–Vis spectra corresponded
to intercalation of Cu-mor in dsDNA Fig. 5(a). The thermal melting plot
supported the intercalation with 5.2 °C increase in Tm Fig. 5(b).
Similarly, dsDNA shifted Ep of Cu-mor towards higher (more posi-
tive) value which can be regarded as a sign of intercalation of Cu-mor
into dsDNA Fig. 3(b). Therefore, combining bathochromic shift in
UV–Vis spectroscopy, decay in fluorescence intensity, positive Ep shift
in cyclic voltammetry and shift in Tm; intercalation mode of binding can
be safely assigned to Cu-mor-DNA interaction.
The differential spectrum of Fe-mor exhibited an interesting trend
that was a decrease in absorbance without a shift in wavelength of
maximum absorption (λmax) when 3 μM dsDNA was added in excess of
Fe-mor (15 μM) increase Fig. 6(a). Further addition of dsDNA resulted
in hyperchromism. This contradicting trend may be an indicator of non-
covalent binding (intercalation/groove or electrostatic binding) [36].
Similarly, the variation in dsDNA spectrum was recorded by keeping
the concentration of dsDNA constant and varying the concentration of
Fe-mor. The Fe-mor caused hyperchromic blue shift in dsDNA spectrum
(Fig. 6(b)). Hyperchromic blue shift caused by dsDNA in Fe-mor spec-
trum and vice versa suggested electrostatic mode of binding for Fe-mor-
DNA interaction [31,32].
The fluorescence spectra of NR-DNA did not undergo any significant
peak current decrease by added Fe-mor. However when Fe-mor was
4
Inorganica Chimica Acta 496 (2019) 119048
added in excess, distinct decrease in fluorescence intensity was ob-
served Fig. 7(a). There was no significant change in Tm at 1:1 Fe-
mor:DNA depicting non-intercalating mode of binding however when
thermal analysis was conducted for Fe-mor concentration 40 μM against
10 μM dsDNA (e.g. 4:1 Fe-mor:DNA) then clear increase of 4.1 °C in Tm
was observed Fig. 7(b). Therefore, it can be said that in Fe-mor-DNA
mixtures intercalation is observed only for higher (almost 4 times) Fe-
mor content.
In electrochemical measurements, the cyclic voltammograms of Fe-
mor exhibited a shift in Ep towards lower Ep values (towards less po-
sitive potential-negative shift) with current decay on addition of dsDNA
Fig. 3(c). This negative shift in peak potential is an indicator of elec-
trostatic mode of mor-DNA interaction. Thus, electrochemical data
trend is in agreement with UV–Vis spectroscopy results.
Therefore, combining UV–Vis spectroscopy, fluorescence spectro-
scopy, thermal melting and cyclic voltammetry; mixed mode of binding
(electrostatic and intercalation) was assigned to mor (hypochromic blue
photometric shift and positive shift in Ep), Cu-mor was categorized as
intercalator (bathochromic λmax shift plus positive Ep shift) while Fe-
mor (blue λmax shift with negative Ep shift) was labeled as electro-
statically binding agent.
3.2. Binding mode investigations of (M-)quer
The UV–Vis spectrum of quer depicted an increase in absorbance
without change in λmax (hyperchromic shift) when dsDNA was added to
it. Same results were obtained when dsDNA spectra were recorded by
adding quer to it (results not shown here). This indicates binding of
quer into grooves of dsDNA. The exceptional behavior was in cyclic
voltammograms where the current increased without Ep shift. This re-
vealed that quer exhibited groove binding mode. The fluorescence
quenching depicted non-intercalative mode of binding as fluorescence
intensity did not vary Fig. S2(a) and thermal melting supported the
observation in terms of intact Tm Fig. S2(b).
Therefore, the groove binding was assigned to quer-DNA binding
Fig. 8.
The hyperchromism with distinct blue shift in UV–Vis spectrum of
Cu-quer was observed by adding dsDNA to it Fig. 9(a) which indicated
either electrostatic mode of binding. There was no intercalation. The
fluorescence spectral did not change to larger extent and Tm remained
intact revealing that Cu-quer did not undergo intercalation Fig. S3. The
cyclic voltammogram exhibited minute negative shift in Ep and de-
crease in ip was reported in of Cu-quer (Fig. 9(b)) suggesting electro-
static mode of Cu-quer-DNA binding [35–38].
Similarly, Fe-quer exhibited hyperchromic blue shift on dsDNA
E. Jabeen, et al. Inorganica Chimica Acta 496 (2019) 119048

Fig. 3. Effect of incremental addition of dsDNA on cyclic voltammograms of 25 µM of (a) mor, (b) Cu-mor, (c) Fe-mor in 20% methanol-PBS (pH = 7.4) at 0.1 V/s vs.
SCE at 310 K.

Fig. 4. Effect of incremental addition of dsDNA on 5 µM Cu-mor spectrum (a) and of Cu-mor on 5 µM dsDNA spectrum (b) in 20% methanol PBS (pH = 7.4) 310 K.

5
E. Jabeen, et al.
Fig. 5. (a) Fluorescence spectra of NR-DNA (10 µM) in the presence of different concentrations of Cu-mor in 20% methanol PBS (pH = 7.4) 310 K (b) thermal melting
plots for dsDNA and Cu-mor-DNA.
spectrum (Fig. 10(a)) as an indicator of electrostatic mode of binding.
Again the fluorescence spectroscopy and thermal melting analysis re-
vealed non-intercalative mode of binding Fig. S4. In electrochemical
measurements, the electrochemical peak current decay was reported in
cyclic voltammograms of Fe-quer (Fig. 10(b)). There was minute ne-
gative shift in Ep potential at 0.952 V while shoulder peak at 0.623 V
displayed significant shift towards lower potential with dsDNA addi-
tion. Thus, Fe-quer can bind to dsDNA through electrostatic interactions
with DNA base pairs.
3.3. Binding mode investigations of (M-)prim
The Fig. 11 reveals hypochromic blue shift for prim addition to
dsDNA and peak current decrease with negative shift in peak potential
which was assisted by indication of non-intercalation form Fig. S5.
Thus, electrostatic mode of interaction was suggested for prim-DNA
interaction (Fig. 11).
The Fig. 12 revealed hypochromism in dsDNA spectra by Cu-prim
and significant positive shift in peak potential of Cu-prim by dsDNA.
Both observation supported intercalation. This intercalation was con-
firmed through fluorescence intensity decay (a) and a shift of 5.5 °C in
Tm of dsDNA Fig. S6.
Fe(III) complexes of prim interact with dsDNA through intercalation
as it was associated with hypochromism and pronounced
Fig. 6. Effect of incremental addition of dsDNA on 15 µM Fe-mor spectrum (a) and of Fe-mor on 15 µM dsDNA spectrum (b) in 20% methanol PBS (pH = 7.4) 310 K.
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Inorganica Chimica Acta 496 (2019) 119048
bathochromism (Fig. 13 (a)) in UV–Vis spectroscopy and significant Ep
shift towards higher value along with peak current decease in cyclic
voltammetry in (Fig. 13(b)). This was assisted by fluorescence and
thermal analysis Fig. S7.
3.4. Evaluation of binding strength and kinetic parameters
The shift in the UV–Vis spectra in double titrations confirmed the
dsDNA binding with mor, quer, prim and their metal complexes which
was further verified through the electrochemical and fluorescence data.
The peak current decay was marked as the index of (M-)Fl-DNA inter-
action which was then exploited to calculate binding constants. All the
(M-)Fls under investigation resulted in straight line Benesi-Hildebrand
plots (Fig. S8) confirming 1:1 Fl-DNA interaction which also yielded
binding constants. The Table 1 reveals that the binding constant ob-
tained from four different experiments were in good agreement with
each other. Further, the Kb calculated in both UV–Vis spectroscopic
experiments were same which depicts that titrating DNA and compound
in either way will not affect the results form Benesi-Hildebrand equa-
tion. These calculations were also confirmed by the cyclic voltammetry
where the Kb calculated by electrochemical data plots (Fig. S8(c))
complemented spectroscopic results. The negative value of ΔG in-
dicated the dsDNA binding process is spontaneous. The value of Kb
gives the quantitative evaluation of the strength of interaction, and is
E. Jabeen, et al.
Fig. 7. (a) Fluorescence spectra of NR-DNA (20 µM) in the presence of different concentrations of Fe-mor in 20% methanol PBS (pH = 7.4) 310 K. (b) Thermal
melting plots for dsDNA and Fe-mor-DNA.
also useful to interpret the binding mode. The Kb value of ≈104 M−1
defines the intercalation of complex into dsDNA while smaller value of
Kb corresponds to electrostatic binding mode. Kb ≈ 103–104 M−1 de-
fined mixed mode of binding ranging from groove to intercalation
[35–38]. After going through Table 1, it can be stated that prim, Fe-
mor, Cu-quer and Fe-quer exhibited electrostatic modes of binding
(Kb ≈ 102) while quer with Kb ≈ 103 is expected to have groove
binding. The Cu-prim, Fe-prim, Cu-mor and mor can intercalate into
dsDNA (Kb ≈ 104). It is also clear from Table 1 that the metal com-
plexes of prim exhibit stronger interaction with dsDNA than prim. The
Kb values are well in agreement with the observed variation in UV–Vis
spectrum and cyclic voltammograms and clearly suggested that metal
complexation has modified their dsDNA binding mode hence affecting
their binding strength (Table 1).
The Table 2 shows the diffusion coefficient and electron transfer
rate constants obtained from plots in Fig. S9. The diffusion coefficient
and electron transfer rate constants of (M-)Fls are lower in the presence
of dsDNA as compared to the corresponding values in the absence of
dsDNA, Table 2. This evidently reflects the binding of dsDNA with (M-)
Fls which has reduced their mobility as a result of bulky structure
formation. Further, (M-)Fls-DNA binding has also affected the rate of
electron transfer (ET) process.
Kinetic data obtained from cyclic voltammetry suggested that
dsDNA also stemmed the decrease in diffusion coefficients of all the Fls
and their Cu (II) or Fe (III) complexes.
Fig. 8. Effect of incremental addition of; (a) quer on 5 µM dsDNA spectrum, (b) dsDNA on cyclic voltammograms of 19 µM quer; in 20% methanol PBS (pH = 7.4)
310 K.
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Inorganica Chimica Acta 496 (2019) 119048
3.5. Cancer cell lines study of (M-)Fls
In-vitro study was conducted over two types of cell lines; human
breast cancer cell line (MCF-7) and human uterine cervical cancer cell
line (HeLa). For a compound to be anti-cancerous, it should be either
cytotoxic or cytostatic. Cytotoxicity means the ability of the drug to
destroy tumor by killing its cells while cytostatic activity is its ability to
stop tumor growth which is also termed as cytostatic therapy. The cy-
totoxic activity is labeled by negative sign (decrease in number of
cancer cells) and cytostatic has been labeled as potential positive sign
(positive response in stopping growth rate of tumor). Therefore, in
Fig. 14 positive sign on a single scale stands for cytostatic while ne-
gative sign stands for cytotoxicity activity.
The results obtained against human breast cancer cell line (MCF-7)
and human uterine cervical cancer cell line (HeLa) showed that the Cu
(II) and Fe(III) complexes of prim and mor exhibited significant cyto-
static activity against MCF-7 cell line while Cu-quer revealed significant
cytotoxicity against this cell line. Similarly, for HeLa cancer cell line,
Cu-mor, Cu-prim and Fe-quer are cytostatic while Cu-quer and prim are
cytotoxic against it. In case of MCF-7 cell line, metal complexation has
granted the anti-cancerous ability to the Fls which themselves are not
anti-cancerous against it except Fe-quer. Thus, M-Fls can be a good
cancer stopping agent for human breast cancer subject to their suc-
cessful clinical trials. The M-Fls, Cu-prim and Cu-mor can be tested
clinically for human uterine cervical cancer as they displayed high
E. Jabeen, et al. Inorganica Chimica Acta 496 (2019) 119048

Fig. 9. Effect of incremental addition of; (a) Cu-quer on 5 µM dsDNA spectrum, (b) dsDNA on cyclic voltammograms of 19 µM Cu-quer; in 20% methanol PBS
(pH = 7.4) 310 K.

Fig. 10. Effect of incremental addition of; (a) Fe-quer on 5 µM dsDNA spectrum, (b) dsDNA on cyclic voltammograms of 19 µM Fe-quer; in 20% methanol PBS
(pH = 7.4) 310 K.

Fig. 11. Effect of incremental addition of; (a) prim on 5 µM dsDNA spectrum, (b) dsDNA on cyclic voltammograms of 19 µM prim; in 20% methanol PBS (pH = 7.4)
310 K.

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E. Jabeen, et al. Inorganica Chimica Acta 496 (2019) 119048

Fig. 12. Effect of incremental addition of; (a) Cu-prim on 5 µM dsDNA spectrum, (b) dsDNA on cyclic voltammograms of 19 µM Cu-prim; in 20% methanol PBS
(pH = 7.4) 310 K.

Fig. 13. Effect of incremental addition of; (a) Fe-prim on 5 µM dsDNA spectrum, (b) dsDNA on cyclic voltammograms of 19 µM Fe-prim; in 20% methanol PBS
(pH = 7.4) 310 K.

Table 2
Kinetic data for (M-)Fls alone and in complex form with dsDNA, (M-)Fl-DNA at
310 K obtained from cyclic voltammetry in PBS (pH-7.4).
(M-)Fls D0 × 106 (cm2 s−1) in the presence Ks,h × 103 (cm s−1) in the presence
of of

Nothing dsDNA Nothing dsDNA

mor 16.80 1.51 10.38 3.10

Cu-mor 3.65 0.46 4.12 2.60
Fe-mor 0.46 0.08 2.60 2.42
quer 4.77 1.19 4.65 2.95
Cu-quer 1.19 0.67 2.95 2.70
Fe-quer 0.11 0.17 2.43 2.46
prim 29.80 1.86 16.57 3.27
Cu-prim 7.45 0.91 5.93 2.81
Fe-prim 1.86 0.30 3.27 2.52

potential (about 80% cytostatic activity) in cell line trials which is
comparable to standard drugs Doxorubicin (for breast cancer cell lines)
(Fig. 14) and 5-Fluorouracil [39].
The LC50 (for cytotoxicity) and GI50 (for cytostatic activities) are
given in Table 3. The Table 1 reveals that Cu-quer exhibited LC50 52 μM
depicting that 52 μM Cu-quer can kill 50% of MCF-7 cancerous cells.
Fig. 14. Effect of Fls or M-Fls over the human breast cancer cell line (MCF-7)
(blue) and human uterine cervical cancer cell line (HeLa) (red) at 100 µM dose
as compare to 10 µM doxorubicin using SRB assay. (For interpretation of the
references to colour in this figure legend, the reader is referred to the web
version of this article.)

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E. Jabeen, et al. Inorganica Chimica Acta 496 (2019) 119048

Table 3 The Table 4 above reveals that the Fe-mor, Cu-quer, Fe-quer and
LC50 (for cytotoxicity) and GI50 (for cytostatic activities) for (M-)Fls and stan- prim which are assigned electrostatic mode of binding through both
dard drug. spectrophotometry and cyclic voltammetry exhibit no apoptotic effects
Flavonoid LC50 (μM) GI50 (μM) over Hela cancer cell lines. The electrostatic binding of Fe-mor with
dsDNA exhibited cytostatic activity against MCF-7. On the other hand,
HeLa MCF-7 HeLa MCF-7 electrostatic binding of Cu-quer with dsDNA can be lethal enough to kill
the cells, hence, imparting cytotoxic behavior to Cu-quer in the list of
Prim 109 – – –
Cu-prim – – 32 37 compound under investigation. Similarly, intercalation of the (M-)Fls
Fe-prim 124 – – 28 into dsDNA imparted cytostatic activity to them. The Cu-mor, Cu-prim
Mor – 116 196 – and Fe-prim displayed intercalation mode of interaction and exhibited
Cu-mor – – 31 34
cytostatic activity against MCF-7 call lines. The Cu(II) complexes of mor
Fe-mor – – 203 29
Quer – – 198 – and prim, with intercalation mode of binding, were cytostatic against
Cu-quer 104 52 – – Hela cell lines while intercalation Fe-prim did not affect Hela cell lines.
Fe-quer – – 100 – The compounds which bind to the grooves of dsDNA (mor and quer) did
Doxorubicin 10 15 – – not displayed any cell killing activity against any of studied cell lines.
Thus, intercalation of (M-)Fls into dsDNA may be effective in stopping
Bold value denotes significant cytotoxicity.
the growth of tumor. Therefore, it is difficult to establish a specific
correlation between dsDNA binding mode and their apoptotic activity,
This activity was equivalent to the 15 μM of standard drug. Although
however, it can generally be inferred from current study that inter-
the LC50 of Cu-quer > LC50 of doxorubicin, the Cu-quer being metal
calating (M-)Fls might be expected to have cytostatic activity.
complex of natural antioxidant could be administrable at higher doses
Selectivity between cancer and healthy cells is a crucial feature of
than traditional drug. Due to non-availability of the normal cell lines for
potential antitumor agents and the anti-proliferative properties of (M-)
HeLa or MCF-7 the compounds were tested for the lethal doses against
Fls make them good candidates for becoming cytostatic anti-neoplastic
normal cells “hek’. The cell viability of normal HeK cells was fond to be
drugs. One of the causes of the cytotoxicity of flavonoid complexes is
85.5% for Cu-quer (100 μM). This revealed that Cu-quer was 14.5%
their ability to cause oxidative damage to dsDNA by generating ROS.
cytotoxic against HeK normal cells while it was 81% cytotoxic against
This feature is strongly associated with the induction of strand breaks
MCF-7 cell line.
which directly impairs cell proliferation and may induce apoptosis
The 50% growth inhibition was reported against MCF-7 cell line for
[46]. So, future perspective of the work includes investigating the
Cu-mor, Fe-mor, Cu-prim and Fe-prim in the concentration range of
mechanism of cytotoxicity of electrostatically bound Cu-quer (which is
28–37 μΜ. Among these cytostatic agents, the Cu-mor and Fe-mor were
beyond the scope of current studies) leading to in-vivo investigations of
tested against HeK cells and the % HeK cell viability was found to be
their anti-cancerous potential over diseased models.
100%. The dose higher than standard drug is acceptable for natural
antioxidants, as reported in literature [40–43], because natural anti-
oxidants, particularly flavonoids are chemo-preventive in nature and
4. Conclusions
are safe to administer at high doses. For example, the previous studies
on some flavonoids have shown them to protect normal tissues from
In the present study, Fe-mor, Cu-quer, prim and Fe-quer bound to
chemotherapy-induced damage without decreasing oncological efficacy
dsDNA through electrostatic mode of binding while Cu-mor, mor, Cu-
at a dose greater than 100 μM [40,41,44,45]. Further, the strong anti-
prim and Fe-prim intercalated into dsDNA. The (M-)Fls exhibited dif-
oxidant potential of these (M-)Fls has already been established [2].
ferent binding modes from corresponding Fls. The quer displayed
groove binding with dsDNA. The metal complexation of prim has re-
3.6. Binding modes vs. anti-cancerous activity sulted in rise in its dsDNA binding strength. The apoptotic activity of
the flavonoids and their metal complexes were affected by binding
The Table 4 reveals the relation of assigned binding modes with modes. Intercalation of (M-)Fls prompted cytostatic activity while the
observed cytotoxic/cytostatic activity. It is clear from the results of involvement groove binding decreased anti-cancerous potential drasti-
dsDNA interaction study of (M-)Fls and cancer cell lines study that cally. Electrostatic binding of (M-)Fls to dsDNA did not produce sig-
binding modes have affected their cytotoxic or cytostatic activity of (M- nificant anti-proliferation activity except for Cu-quer where it displayed
)Fls to certain extent. Further, the (M-)Fls exhibit different binding significant cytotoxicity.
modes from corresponding Fls.

Table 4
Binding modes vs. anti-cancerous activity of (M-)Fls.
(M-)Fl Binding mode evaluated form Binding Strength Anti-cancerous activity

−1
UV–Vis spectroscopy Cyclic Voltammetry Kb (M ) of the order of Against Hela cell line Against MCF-7 cell line

mor Intercalation + Groove Intercalation 103–104 – –

Cu-mor Intercalation Intercalation 104 Cytostatic Activity Cytostatic Activity
Fe-mor Electrostatic Electrostatic 102 – Cytostatic Activity
quer Groove Groove 103 – –
Cu-quer Electrostatic Electrostatic 102 – Cytotoxic activity
Fe-quer Electrostatic Electrostatic 102 – –
prim Electrostatic Electrostatic 102 – –
Cu-prim Intercalation Intercalation 104 Cytostatic Activity Cytostatic Activity
Fe-prim Intercalation Intercalation 104 – Cytostatic Activity

10
E. Jabeen, et al.
Acknowledgement
We acknowledge Quaid-i-Azam University Islamabad for the pro-
vision of funds as URF and HEC Pakistan for funding through HEC re-
search project-No.20/1718/R&D. Authors acknowledge Department of
Chemistry, FUUAST, Pakistan.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.ica.2019.119048.
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