48
N. Pravin, N. Raman / Inorganic Chemistry Communications 36 (2013) 45–50
efficiency of complexes is usually dependent on activators [38], the
cleavage activity is significantly enhanced by the activator ascorbic
acid. These phenomena imply that Cu(II), Co(II), Ni(II) and Zn(II) com-
plexes induce intensively the cleavage of circular pBR322 DNA in the
presence of AH2 (Fig. 4). In order to clarify the cleavage mechanism of
pBR322 DNA introduced by metal(II) complexes, the investigation has
been carried out further on adding DMSO (hydroxyl radical scavenger).
It reveals that Cu(II) and Zn(II) complexes promote the cleavage of
pBR322 DNA more efficiently than Co(II) and Ni(II) complexes.
The synthesized ligand and its complexes have been tested for their
in vitro antimicrobial activity. They were tested against the bacteria
Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Bacillus
subtilis, and Salmonella typhi by paper disc method. The antibacterial
activity of the newly synthesized compounds is presented in Table 3.
The results indicate that the ligand exhibits moderate antibacterial
activity with respect to the complexes against the same microorganisms
under identical experimental conditions. Further, the antibacterial
action of Schiff base ligand may be significantly enhanced on the pres-
ence of azomethine groups which have chelating properties. These
properties may be used in metal transport across the bacterial mem-
branes or to attach to the bacterial cells at a specific site from which it
can interfere with their growth. Ligand exhibited MIC in the range of
(16.7–15.4 μg/mL) against all the pathogens. The copper complex
showed better antibacterial activity (MIC, 1.7–2.8 μg/mL) against the
tested microorganisms than the other complexes which have MIC
values in the range 3.1–4.7 μg/mL. It may be attributed to the atomic ra-
dius and the electronegativity of Cu(II) ion. Current studies reveal that
the high atomic radius and electronegative metal ions in their metal
complexes exhibit high antimicrobial activity. Higher electronegativity
and large atomic radius decrease the effective positive charges on the
metal complex molecules which facilitates their interaction with the
highly sensitive cellular membranes towards the charged particle [39].
The Schiff base and its metal complexes were screened for their an-
tifungal activity against Aspergillus niger, Fusarium solani, Curvularia
lunata, Rhizoctonia bataticola and Candida albicans. The minimum inhib-
itory concentration (MIC) values of the investigated compounds are
summarized in Table 4. A comparative study of MIC values of ligand
(11.6–15.8 μg/mL) and its complexes (2.0–6.7 μg/mL) against all the
fungi indicates that the metal complexes exhibit higher antifungal activ-
ity than the ligand. Such increased activity on metal chelation can be
explained on the basis of Tweedy's chelation theory [40]. Chelation re-
duces the polarity of the metal ion considerably because of the partial
sharing of its positive charge with the donor groups and also due to π-
electron delocalization on the whole chelating ring. The lipids and poly-
saccharides are some important constituents of the cell wall and mem-
branes which are preferred for metal ion interaction. Apart from this,
the cell wall also contains many phosphates, carbonyl and cystenyl
ligands which maintain the integrity of the membrane by acting as a
diffusion barrier and also provide suitable sites for binding. Further-
more, increased lipophilicity enhances the penetration of the complexes
into lipid membrane and blocking of the metal binding sites in the en-
zymes of microorganisms. These complexes also disturb the respiration
process of the cell and thus block the synthesis of the proteins which
R
Fig. 3. Effect of increasing amounts of [CuL(ox)]Cl2
[ZnL(ox)]Cl2
(
), [CoL(ox)]Cl2 ( ), [NiL(ox)]Cl2 ( ),
( ) on the viscosity of DNA and [EB] ( ).
an obvious influence on the relative viscosity of DNA, suggesting the ab-
sence of intercalation of between the two species. However, when the
copper(II) complex was added, the relative viscosity of DNA increased
gradually, which is typical characteristic of intercalation. The classical
intercalators like ethidium bromide are known to increase the base
pair separation resulting in an increase in the relative viscosity of the
DNA. This effect of the metal(II) complexes is far less than that observed
for an intercalator such as EB indicating that there exists a moderate
intercalative interaction between the complexes and CT DNA. These re-
sults also suggest that the coordination geometry has great impact on
the binding mode of the small molecules with DNA. Compared with
the free ligand, the copper complex has a larger rigid planar structure,
which is more favorable for intercalation into DNA. Meanwhile, the neg-
ative charge of the free ligand is neutralized by coordinating with Cu(II)
ion, reducing the electrostatic repulse of the molecule to DNA.
The presence of bioactive ligand and DNA binder in the metal(II)
complexes is essential for observing efficient DNA cleavage activity. All
the complexes are found to exhibit nuclease activity. Fig. 4 shows the re-
sult of gel electrophoretic separations of pBR322 DNA induced by an ad-
dition of metal(II) complexes in the presence of AH2 (ascorbic acid).
Under the same conditions, free AH2 produces no cleavage of pBR322
DNA. During electrophoresis, while scission occurs on one strand
(nicking), the supercoiled form relaxes to generate nicked form (Form
II) [35]. When cleavage occurs on both the strands, a linear form
(Form III) is generated which migrates between Forms I and II [36,37].
For this case, all supercoiled (Form I) DNAs are cleaved to form the mix-
ture of Form II with the addition of the complexes. Since the nuclease
1
2
3
4
5
6
Form II
Form I
Table 3
The in vitro antibacterial activity of Schiff base and its metal complexes (MIC in μg/mL).
Complex
S. aureus
P. aeruginosa
E. coli
B. subtilis
S. typhi
[L]
16.7
1.7
3.2
3.7
3.5
1.7
–
15.4
2.4
3.6
3.3
4.6
1.9
–
16.3
2.3
2.9
3.9
3.4
2.0
–
15.7
2.6
3.1
4.2
3.8
1.8
–
14.6
2.8
3.4
4.6
4.7
2.4
–
[CuL(ox)]Cl2
[NiL(ox)]Cl2
[CoL(ox)]Cl2
[ZnL(ox)]Cl2
Ciprofloxacina
DMF
Fig. 4. Gel electrophoresis diagram showing the cleavage of pBR322 DNA (10 μM) by
the Cu(II), Ni(II), Co(II) and Zn(II) complexes in a buffer containing 50 mM Tris-HCl
and 50 mM NaCl in the presence of ascorbic acid (AH2, 10 μM) and DMSO (4 μL) at
37 °C. Lane 1, DNA control; lane 2, DNA + ligand + AH2 + DMSO (4 μL); lane 3,
DNA + AH2 + [CuL(ox)]Cl2 + DMSO (4 μL); lane 4, DNA + AH2 + [NiL(ox)]
Cl2 + DMSO (4 μL); lane 5, DNA + AH2 + [CoL(ox)]Cl2 + DMSO (4 μL); lane 6,
DNA + AH2 + [ZnL(ox)]Cl2 + DMSO (4 μL).
a
Ciprofloxacin is used as the standard. MIC (μg/mL) minimum inhibitory concentration,
i.e. the lowest concentration to completely inhibit the bacterial growth.