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Synthesis, characterization, cytotoxicity, DNA cleavage, and antimicrobial activity of lanthanide(III) complexes of a Schiff base ligand derived from glycylglycine and 4-nitrobenzaldehyde
⁎Corresponding author. Tel.: +91 94431 82502. skumarmsu@yahoo.com (S. Kumaresan)
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Received: ,
Accepted: ,
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.
Peer review under responsibility of King Saud University.
Abstract
Lanthanide complexes of Eu(III), Gd(III), Nd(III), Sm(III), and Tb(III) with the Schiff base derived from glycylglycine and 4-nitrobenzaldehyde were synthesized and characterized by elemental analysis, mass, IR, electronic spectra, molar conductance, TGA, and powder XRD. The results show that the Schiff base ligand acts as a tridentate monobasic donor coordinating through the azomethine nitrogen, deprotonated peptide nitrogen, and carboxylato oxygen atoms. Thermal decomposition profiles are consistent with the proposed formulations. The powder XRD studies show that all the complexes are amorphous in nature. Antimicrobial studies indicate that these complexes exhibit more activity than the ligand itself. The DNA cleavage activity of the ligand and its complexes were assayed on Escherichia coli DNA using gel electrophoresis in the presence of H2O2. The result shows that the Eu(III) and Nd(III) complexes have completely cleaved the DNA. The anticancer activities of the complexes have also been studied towards human cervical cancer cell line (HeLa) and Colon Cancer Cells (HCT116) and it was found that the Eu(III) and Nd(III) complexes were more active than the corresponding Gd(III), Sm(III), Tb(III) complexes and the free ligand on both the cancer cells.
Keywords
Lanthanide complexes
Glycylglycine
Antimicrobial
DNA cleavage
Anticancer
1 Introduction
Lanthanide complexes attract considerable interest in bioinorganic and coordination chemistry (Tircso et al., 2006; Samir et al., 2013; Shiju et al., 2013). Some of the lanthanide complexes are used in biomedical analysis as MRI contrast agents (Amie et al., 2006). Because of special, photophysical and biological properties, lanthanide complexes can be used as biological probes in the areas of clinical chemistry and molecular biology (Gassner et al., 2008). Due to their special electronic configuration, lanthanide complexes have inspired many efforts on the design and synthesis as potential anticancer and antibacterial agents (Eliseeva and Bunzli, 2010; Hermann et al., 2008; Gassner et al., 2008; Supkowski et al., 1999). Polydentate ligands such as Schiff bases, assisted by metal ions, provide highly organized supramolecular metal complexes. Such complexes possess binding sites and cavities for various cations, anions, and organic molecules (Dixit et al., 2009). Some of the Schiff base complexes containing N and O donor atoms are effective as stereospecific catalysts for oxidation (Kureshy et al., 1999), reduction (Aoyama et al., 1986), hydrolysis (Kelly et al., 1986), biocidal activity (Sengupta et al., 2001; Ramasubramaniana et al., 2012), and other organic and inorganic transformations. Schiff base complexes derived from amino acids are important due to their ability to possess unusual configurations and biological importance (Dhankar et al., 2012; Arish and Nair, 2010). Lanthanide Schiff base complexes have some advantages for luminescence research because of their special structures (Archer and Chen, 1998). Exhibiting outstanding optical properties and a broad spectrum of biological activities, rare-earth metal complexes of Schiff-base type ligands derived from amino acids attracted great interest of researchers in recent years (Danghui et al., 2006; Samir et al., 2013). In general, lanthanide complexes display varying cytotoxic effects (Kostova et al., 2011 and Kostova and Georgi, 2008; Hussain et al., 2012). Such properties of this type of complexes encouraged us to synthesize, characterize, and study the biological activity of new Eu(III), Gd(III), Nd(III), Sm(III), and Tb(III) complexes of the Schiff base ligand derived from glycylglycine and 4-nitrobenzaldehyde.
2 Experimental
2.1 Materials
4-Nitrobenzaldehyde and glycylglycine were purchased from Himedia. The human cervical cancer cell line (HeLa) and Colon Cancer Cells (HCT116) were obtained from National Centre for Cell Science (NCCS), Pune. Nitrates of Eu(III), Gd(III), Nd(III), Sm(III), and Tb(III) were purchased from Merck and Sigma–Aldrich. All other reagents and solvents were purchased from commercial sources and were of analytical grade.
2.2 Synthesis of Schiff base ligand (4-NBA-GG)
A mixture of glycylglycine (5 mmol) and KOH pellets (5 mmol) in MeOH (25 mL) was kept under continuous stirring in a 100 mL RB flask. A solution of 4-nitrobenzaldehyde (5 mmol) in MeOH (25 mL) was then added slowly to the flask. The reaction mixture was vigorously stirred at 60 °C for 8 h. The volume of the mixture was reduced to half of the initial volume under reduced pressure and an excess of anhydrous ether was added. An air sensitive yellow precipitate was formed, which was collected quickly by vacuum filtration and washed several times with anhydrous ether and then dried in vacuo over anhydrous CaCl2. The purity of the Schiff base ligand was checked by TLC. The yield of the isolated ligand was found to be 78%.
2.3 Synthesis of the Schiff base metal complexes
Glycylglycine (5 mmol), KOH pellets (5 mmol) and MeOH (25 mL) were taken in a 100 mL RB flask. A solution of 4-nitrobenzaldehyde (5 mmol) in MeOH (25 mL) was then added slowly to the flask. The reaction mixture was vigorously stirred at 60 °C for 8 h. A solution of lanthanide(III) nitrates (2.5 mmol) in aqueous MeOH (20 mL), was then added dropwise to the flask and the reaction mixture was stirred for 2 h. The precipitate was filtered off, washed several times with cold EtOH, ether, and then dried in vacuo over anhydrous CaCl2. The yield was found to be 61–66%.
2.4 Physical measurements
Elemental analysis was done using a Perkin–Elmer elemental analyzer. The metal contents in the complexes were determined by standard EDTA titration (Zhou and He, 2008). Molar conductance of the complexes was measured in DMF (10−3 M) solutions using a Coronation Digital Conductivity Meter. The mass spectra were recorded on a JEOL JMS600H mass spectrometer. IR(KBr) spectra were recorded on a JASCO FT/IR-410 spectrometer in the 4000–400 cm−1 region. The electronic spectra were recorded on a Perkin Elmer Lambda-25 UV–VIS spectrometer. Thermal analysis was carried out on SDT Q 600/V8.3 build 101 thermal analyzer with a heating rate of 20 °C/min using nitrogen atmosphere. Powder XRD was recorded on a Rigaku Dmax X-ray diffractometer with Cu–Kα radiation.
2.5 Antimicrobial activities
Antibacterial and antifungal properties of the ligand and its complexes were tested in vitro against the bacterial species Escherichia coli, Bacillus subtilis, Pseudomonas aeruginosa, and Staphylococcus aureus; fungal species, Aspergillus niger, Aspergillus flavus, and Candida albicans by the disc diffusion method. Amikacin, ofloxacin, and ciprofloxacin were used as standards for antibacterial activity and nystatin was used as a standard for antifungal activity. The test organisms were grown on nutrient agar medium in petri plates. The compounds were prepared in DMSO and soaked in filter paper discs of 5 mm diameter and 1 mm thickness. The discs were placed on the previously seeded plates and incubated at 37 °C and the diameter of inhibition zone around each disc was measured after 24 h for antibacterial and 72 h for antifungal activities. The minimum inhibitory concentration (MIC) was determined by ‘serial dilution technique’ (NCCLS, 1999).
2.6 DNA cleavage analysis
Cleavage reactions were run between the metal complexes and E. coli DNA, and the solutions were diluted with loading dye using 1% agarose gel. Then ethidium bromide (3 mL, 0.5 mg mL−1) was added to the above solution and mixed well. The warmed agarose was poured and clamped immediately with a comb to form sample wells. The gel was mounted onto an electrophoretic tank and enough electrophoretic buffers were added to cover the gel to a depth of about 1 mm. The DNA sample (30 mmol L−1), 50 mmol L−1 metal complex, and 500 mmol L−1 H2O2 in 50 mmol L−1 tris–HCl buffer (pH 7.1) were mixed with the loading dye and loaded into the well of the submerged gel using a micropipette. Electric current (50 mA) was passed into the running buffer. After 1–2 h, the gel was taken from the buffer and was photographed under a UV transilluminator and documented.
2.7 In vitro anticancer activity
The human cervical cancer cell line (HeLa) and Colon Cancer Cells (HCT116) were grown in Eagles Minimum Essential Medium (EMEM) containing 10% fetal bovine serum (FBS). The cells were maintained at 37 °C, 5% CO2, 95% air, and 100% relative humidity. Maintenance cultures were passaged weekly, and the culture medium was changed twice a week. The monolayer cells were detached with trypsin–ethylenediaminetetraacetic acid (EDTA) to make single cell suspension and viable cells were counted using a hemocytometer and diluted with a medium containing 5% FBS to give a final density of 1 × 105 cells/mL. One hundred microlitres per well of cell suspension were seeded into 96-well plates at a plating density of 10,000 cells/well and incubated to allow for cell attachment at 37 °C, 5% CO2, 95% air and 100% relative humidity. After 24 h, the cells were treated with serial concentrations of the test samples. They were initially dissolved in neat dimethylsulfoxide (DMSO) to prepare the stock (200 mM) and stored frozen prior to use. At the time of sample addition, an aliquot of frozen concentrate was thawed and diluted to twice the desired final maximum test concentration with serum free medium. Additional three, 2-fold serial dilutions were made to provide a total of five sample concentrations. Aliquots of 100 μL of these different sample dilutions were added to the appropriate wells already containing 100 μL of medium, resulting in the required final sample concentrations. Following sample addition, the plates were incubated for an additional 48 h at 37 °C, 5% CO2, 95% air and 100% relative humidity. The medium without samples served as control and a triplicate was maintained for all concentrations (Mosmann, 1983).
2.7.1 MTT assay
MTT is a yellow water soluble tetrazolium salt [(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide)]. Succinate-dehydrogenase, a mitochondrial enzyme in living cells cleaves the tetrazolium ring, converting the MTT to an insoluble purple formazan. Thus, the amount of formazan produced is directly proportional to the number of viable cells. After 48 h of incubation, 15 μL of MTT (5 mg/mL) in phosphate buffered saline (PBS) was added to each well and incubated at 37 °C for 4 h. The medium with MTT was then flicked off and formazan crystals obtained were solubilized in 100 μL of DMSO. The absorbance at 570 nm was measured using a micro plate reader (Monks, 1991). The % cell inhibition was determined using the following formula.
% cell inhibition = 100 − Abs (sample)/Abs (control) × 100.
Nonlinear regression graph was plotted between % cell inhibition and log10 concentration and IC50 was determined using GraphPad Prism software.
3 Results and discussion
3.1 Characterization of Schiff base ligand (4-NBA-GG)
The Schiff base ligand, 4-NBA-GG, is yellow in colour, air sensitive in nature, and soluble in all common organic solvents. The elemental analysis data (Table 1) of 4-NBA-GG are in good agreement with those calculated for the suggested formula. The mass spectrum of the ligand shows a well-defined molecular ion peak at m/z = 303 [M+ (17%)], which coincides with the formula weight of the Schiff base. In the 1H-NMR spectrum, the azomethine proton exhibits a singlet at 8.5 ppm and the aromatic ring protons appear at 7.4–8.3 ppm range. Both methylene protons appear at the range 4.1–4.6 ppm. In the 13C-NMR spectrum, the azomethine carbon and the carboxylato carbon peaks are displayed at 161 and 176 ppm, respectively. The aromatic carbons resonate at 120–150 ppm range. The methylene carbon adjacent to the carboxylato carbon appears at 43 ppm and the other methylene carbon adjacent to the azomethine nitrogen is displayed at 52 ppm. The amido carbonyl carbon appears at 171 ppm. The IR spectrum (Table 2) of the Schiff base ligand exhibits a band at 1652 cm−1 due to the azomethine group ν(C⚌N). The strong bands present in the region 1619 can be assigned to an asymmetric stretching frequency of the carboxylato group. The Schiff base ligand also displays bands at 1384, due to symmetric stretching vibration of the carboxylato group (Nakamoto, 1978). Sharp bands present in the spectrum of the Schiff base in the region ∼3362 and ∼1519 cm−1 are due to peptide N–H stretching frequencies (Nakamoto, 1978). In the UV spectrum of the ligand, intense absorption bands at 210 and 270 nm, are presumably from the π–π∗ and n-π∗ transitions of the Schiff base ligand respectively.
| Compound | Empirical formula | Colour | Yield | Elemental analysis Found (calcd) % | Λc (Ohm−1 cm2mol−1) | λmax (nm) | |||
|---|---|---|---|---|---|---|---|---|---|
| C | H | N | M | ||||||
| 4-NBA-GG (L) | C11H10KN3O5 | Yellow | 78 | 42.86 (43.56) | 3.62 (3.32) | 13.52 (13.85) | – | 210, 270 | |
| [EuL2(H2O)2]·4H2O | C22H31EuN6O16 | Yellow | 64 | 34.65 (33.55) | 4.48 (3.97) | 11.36 (10.67) | 18.46 (19.30) | 3.1 | 217, 276 |
| [GdL2(H2O)2]·4H2O | C22H31GdN6O16 | Yellow | 66 | 34.39 (33.33) | 4.63 (3.94) | 11.82 (10.60) | 18.74 (19.84) | 2.8 | 219, 278 |
| [NdL2(H2O)2]·4H2O9 | C22H31N6NdO16 | Light Yellow | 61 | 34.96 (33.89) | 4.92 (4.01) | 11.78 (10.78) | 19.14 (18.50) | 2.3 | 216, 276 |
| [SmL2(H2O)2]·4H2O | C22H31N6O16Sm | Light Yellow | 63 | 33.98 (33.62) | 4.24 (3.98) | 11.76 (10.69) | 18.59 (19.13) | 3.4 | 216, 277 |
| [TbL2(H2O)2]·4H2O | C22H31N6O16Tb | Yellow | 65 | 34.48 (33.26) | 4.33 (3.93) | 11.61 (10.58) | 19.38 (20.00) | 2.7 | 218, 276 |
| Compound | νazo.(C⚌N) | νasym.(COO−) | νsym.(COO−) | ν(H2O) | ν(M–O) | ν(M–N) |
|---|---|---|---|---|---|---|
| 4-NBA-GG (L) | 1652 | 1619 | 1345 | – | – | – |
| [EuL2(H2O)2]·4H2O | 1597 | 1521 | 1343 | 3363(b) | 532 | 419 |
| [GdL2(H2O)2]·4H2O | 1606 | 1525 | 1342 | 3356(b) | 541 | 421 |
| [NdL2(H2O)2]·4H2O | 1601 | 1519 | 1344 | 3362(b) | 528 | 423 |
| [SmL2(H2O)2]·4H2O | 1602 | 1519 | 1342 | 3363(b) | 523 | 419 |
| [TbL2(H2O)2]·4H2O | 1603 | 1520 | 1343 | 3352(b) | 537 | 424 |
3.2 Characterization of metal complexes
The analytical and physical characterization of Ln(III)–4-NBA-GG complexes are given in Table 1. The analytical data show that the metal to ligand ratio is 1:2 in all the complex systems. The composition of the complexes is [LnL2(H2O)2]·4H2O where L is the Schiff base ligand (4-NBA-GG). The Eu(III), Gd(III), Nd(III), Sm(III), and Tb(III) complexes are soluble in water, DMSO, and insoluble in other common organic solvents. The mass spectra of the Eu(III), Gd(III), Nd(III), Sm(III), and Tb(III) complexes show molecular ion peaks at m/z 789 (M+1, 16%) and 716 (M−4H2O, 14%); 793 (M+1, 13%) and 719 (M−4H2O 10%); 777 (M+1, 14%), and 705 (M−4H2O, 12%); 787 (M+1, 15%)], and 714 (M−4H2O, 21%); 794 (M+1, 17%) and 721 (M−4H2O, 18%) respectively, which coincide with the formula weights of the Schiff base complexes. The low molar conductance values (Table 1) of the metal complexes reveal their non-electrolytic nature (Geary, 1971).
3.3 IR spectra
The important IR stretching frequencies are given in Table 2. The band at 1652 cm−1 for the azomethine group of free ligand shifted to lower frequency in the range ∼1606–1597 cm−1 in the complexes is indicative of the coordination of the azomethine nitrogen atom to the metal ion. The new broad band that appeared at ∼3400 cm−1 can be attributed to the stretching vibration of the coordinated water molecules. A band at ∼850 cm−1 in the complexes is assigned to coordinated water molecule (Refat et al., 2009). On complexation, the asymmetric stretching bands of carboxylato groups are shifted to lower frequency in the range ∼1525–1519 for all the complexes indicating the formation of a linkage between the metal ion and carboxylato oxygen atom. Moreover, the difference (∼200 cm−1) between the asymmetric and symmetric stretching modes indicates the monodentate binding of the carboxylato group in the complexes (Deacon and Phillips, 1980). The spectrum of all the metal complexes show new bands in the 541–523 cm−1 and 424–419 cm−1 regions, which may probably be due to the formation of M–O and M–N bonds respectively (Majumder et al., 2006; Shelke et al., 2012).
3.4 Electronic spectra
The UV absorption spectra of the free ligand and the corresponding lanthanide complexes were measured in THF solution. The spectrum displayed an absorption maxima at 210 and 270 nm for the free ligand, which is attributable to the π–π∗ and n–π∗ absorptions of the Schiff base ligand. The absorption bands of Eu(III) (217, 276), Gd(III) (219, 278), Nd(III) (216, 276), Sm(III) (216, 277), and Tb(III) (218, 276) (Table 1) complexes are slightly red-shifted with respect to that of the free ligand (210, 270 nm). This is attributed to the alterations in the interelectronic repulsion parameter in the complexes. The extent of shift of the spectral lines represents ‘nephelauxetic’ effect (expansion of lanthanide orbital radius), which could be used as a measure of the metal–ligand covalent bonding (Jorgensen, 1980).
3.5 Thermal analysis
The thermal stability data of the complexes are listed in Table 3. The [EuL2(H2O)2]·4H2O, [GdL2(H2O)2]·4H2O, [NdL2(H2O)2]·4H2O, [SmL2(H2O)2]·4H2O, and [TbL2(H2O)2]·4H2O complexes undergo a similar decomposition mainly in three stages. The first stage taking place in the 70–110 °C range corresponds to the dehydration of four lattice water molecules. The second decomposition step is represented by the removal of two coordinated water molecules in the range 120–185 °C. The next is the decomposition of the organic ligand moiety in the 320–630 °C range with the formation of lanthanide oxide as the final product. The TG curve of the complex [EuL2(H2O)2]·4H2O shows a weight loss of 9.5% (calculated – 9.1%) in the temperature range 70–105 °C. This is due to the loss of four lattice water molecules. In the second step, two coordinated water molecules are liberated at the temperature range 120–165 °C. The third decomposition step of the complex is in the temperature range 400–500 °C bringing a weight loss of 40.9% (calculated 42%) which correlates with the loss of coordinated organic ligand. Above this temperature, a horizontal thermal curve has been observed due to the formation of the metal oxide. Similarly the TG curves of the complexes [GdL2(H2O)2]·4H2O, [NdL2(H2O)2]·4H2O, [SmL2(H2O)2]·4H2O, and [TbL2(H2O)2]·4H2O show a weight loss of 10.2% (calculated 9.0%), 11.4% (calculated 9.2%), 10.3% (calculated 9.1%), 9.4% (calculated 9.0%) in the temperature range 75–100, 70–110, 70–95, 65–90 °C respectively showing the elimination of four coordinated water molecules. The second decomposition is the liberation of two coordinated water molecules in the temperature range 120–185 °C for all the complexes. The third weight loss 41.4% (calculated 40.8%), 41.6% (calculated 43.1%), 41.4% (calculated 42.2%), 40.7% (calculated 40.5%) in the temperature range 390–530, 320–600, 380–540, 410–630 °C corresponds to the coordinated organic ligand in the complexes [GdL2(H2O)2]·4H2O, [NdL2(H2O)2]·4H2O, [SmL2(H2O)2]·4H2O, and [TbL2(H2O)2]·4H2O, respectively. Above this temperature, a horizontal curve has been observed for all the complexes due to the formation of a metal oxide. Based on the above studies, the proposed structure of the metal complex is shown in Fig. 1.
| Complex | Temperature range T (°C) | % Weight loss Obs.(calcd) |
Process |
|---|---|---|---|
| [EuL2(H2O)2]·4H2O | 70–105, 120–165, 400–500, >450 |
9.5(9.1), 5.3(4.5), 40.9(42.0), 44.3(44.5) |
–4H2O (lattice), –2H2O (coord), loss of organic moiety, Eu2O3 |
| [GdL2(H2O)2]·4H2O | 75–100, 130–170, 390–530 >550 |
10.2(9.0), 4.9(4.5), 41.4(40.8), 43.5(45.7) |
–4H2O (lattice), –3H2O (coord), loss of organic moiety, Gd2O3 |
| [NdL2(H2O)2]·4H2O | 70–110, 140–175, 320–600, >600 |
11.4(9.2), 5.8(4.6), 41.6(43.1), 41.2(43.1) |
–4H2O (lattice), –3H2O (coord), loss of organic moiety, Nd2O3 |
| [SmL2(H2O)2]·4H2O | 70–95, 120–165, 380–540, >550 |
10.3(9.1), 5.3(4.5), 41.4(42.2), 43.0(44.2) |
–4H2O (lattice), –3H2O (coord), loss of organic moiety, Sm2O3 |
| [TbL2(H2O)2]·4H2O | 65–90, 130–185, 410–630, >650 |
9.4(9.0), 5.1(4.5), 40.7(40.5), 44.8(46.0) |
–4H2O (lattice), –3H2O (coord), loss of organic moiety, Tb2O3 |
3.6 Powder XRD
Powder XRD pattern of the Eu(III), Gd(III), Nd(III), Sm(III), and Tb(III) complexes was recorded over the 2θ = 0–80 range. There was no well defined crystalline peak in the spectrum. From this it is observed that all these complexes are amorphous in nature.
4 Biological studies
4.1 Antimicrobial activity
The results of the antimicrobial activities are summarized in Table 4. The standard error for the experiment is ±0.001 cm and the experiment was repeated three times under similar conditions. DMSO was used as a negative control and amikacin, ofloxacin and ciprofloxacin were used as positive standards for antibacterial studies. Nystatin was used as a reference for antifungal studies. These compounds exhibit moderate to strong antimicrobial activity. Comparatively a better activity is found for the bacteria rather than the fungi. The Nd(III) complex exhibits a higher activity than the other metal complexes towards fungal species. The Nd(III) complex shows a good activity, especially against the Gram-negative bacteria such as E. coli and B. subtilis. The Nd(III) complex shows equal or better activity compared to the negative controls such as amikacin, ofloxacin, and ciprofloxacin. The Eu(III) and Gd(III) complexes display moderate activity against the bacteria. The antimicrobial activity of the complexes is greater than those of the free ligand, this indicates that the complexation to metal enhances the activity of the ligand. This is explained on the basis of Overtone’s concept and chelation theory (Priya et al., 2009). Chelation tends to make the ligand a more powerful and potent bacterial agent. A possible explanation for this increase in the activity upon chelation is that, in a chelated complex, the positive charge of the metal is partially shared with donor atoms present in the ligands and there is an electron delocalization over the whole chelated ring. This, in turn, increases the lipoid layers of the bacterial membranes. Generally, it is suggested that the chelated complexes deactivate various cellular enzymes, which play a vital role in various metabolic pathways of these microorganisms. Other factors such as solubility, conductivity, and dipole moment that are affected by the presence of metal ions may also be the possible reasons for increasing the biological activity of the metal complexes as compared to the ligand from which they are derived (Emara, 2010).
| Compound | Bacterial species | Fungal species | |||||
|---|---|---|---|---|---|---|---|
| E. coli | B. subtilis | P. aereuguioa | S. aureus | A. niger | A. flavus | C. albicans | |
| 4-NBA-GG (L) | 18 | >100 | 67 | 85 | 32 | 86 | >100 |
| [EuL2(H2O)2]·4H2O | 9 | 33 | 10 | 21 | 16 | 21 | 8 |
| [GdL2(H2O)2]·4H2O | 11 | 15 | 8 | 15 | 22 | 12 | 12 |
| [NdL2(H2O)2]·4H2O | 04 | 06 | 12 | 10 | 19 | 10 | 06 |
| [SmL2(H2O)2]·4H2O | 64 | 51 | 18 | 54 | 63 | 89 | 72 |
| [TbL2(H2O)2]·4H2O | 58 | 42 | 41 | 59 | >100 | 78 | 96 |
| Amikacina | 05 | 06 | 05 | 07 | – | – | – |
| Ciprofloxacinb | 04 | 05 | 05 | 05 | – | – | – |
| Ofloxacinc | 10 | 04 | 04 | 05 | – | – | – |
| Nystatind | – | – | – | – | 07 | 05 | 05 |
4.2 DNA cleavage analysis
Gel electrophoresis experiments using E. coli DNA were performed with the ligand and its complexes in the presence and absence of H2O2 as an oxidant. The results (Fig. 2) indicate that all the complexes can interact with E. coli DNA in the presence of H2O2. Eu(III) and Nd(III) complexes can cleave DNA completely compared to other systems. The lanthanide complexes can catalyze the production of highly reactive hydroxyl radicals from H2O2. These hydroxyl radicals participate in the oxidation of the deoxyribose moiety, followed by the hydrolytic cleavage of the sugar-phosphate backbone. Gd(III), Sm(III), and Tb(III) complexes partially cleaved the DNA. It was observed that most cleavage cases were due to the metal ions reacting with H2O2 to produce diffusible hydroxyl radicals or molecular oxygen, which might damage the DNA through Fenton type chemistry (Babu et al., 2007). In addition, the nuclease activity of the complexes was also investigated in the absence of the oxidant H2O2. That showed an insignificant effect.![DNA cleavage studies of Schiff base ligand and its lanthanide complexes. M – Marker, C – Control E. coli DNA (untreated sample), S1 – Ligand + DNA, S2 – [EuL2(H2O)2]·4H2O + DNA, S3 – [GdL2(H2O)2]·4H2O + DNA, S4 – [NdL2(H2O)2]·4H2O + DNA, S5 – [SmL2(H2O)2]·4H2O + DNA, S5 – [TbL2(H2O)2]·4H2O + DNA.](/content/184/2017/10/2_suppl/img/10.1016_j.arabjc.2013.09.036-fig2.png)
4.3 In vitro anticancer activity
The reliable criteria for judging the efficacy of any anticancer drug are prolongation of life span, improving the clinical, haematological, biochemical profile, and reduction in viable tumour cell count in the host (Mostafa, 2007). In order to evaluate the biological effects of the ligand, 4-NBA-GG and its Eu(III), Gd(III), Nd(III), Sm(III), and Tb(III) complexes on cancer cells, we used the compounds to treat HeLa (Human Cervical Cancer Cells) and HCT116 (Colon Cancer Cells) at the concentrations of 6.25, 12.5, 25, 50, and 100 μM for 48 h (Figs. 3a and b). The untreated cells were used as control. Cell growth inhibition was analyzed by the MTT assay and the results showed that the complexes and the ligand exhibited an inhibitory effect on the proliferation of HeLa and HCT116 cells in a dose-dependent manner (Table 5). Among them, Eu(III) and Nd(III) complexes showed the most potent inhibitory effect on the growth of both the cells compared to the Gd(III), Sm(III), and Tb(III) complexes and the free ligand. Values for Eu(III), Gd(III), Nd(III), Sm(III), and Tb(III) complexes for HeLa cancer cells are better than some previously reported values (Aguirre et al., 2009). The IC50 values for our metal complexes and the free ligand against HCT116 cancer cells show moderate activity compared to the IC50 value of the clinically used drug such as etoposide (29.6 μM) (LeBlanc et al., 2011). The activity of the metal complexes and the free ligand towards HeLa cancer cells is not much significant, compared to the known metal-free anticancer agents such as estramustine (IC50 ∼1.5–3.0 μM), (Nicholson et al., 2002) noscapine (IC50 ∼22 μM) (Zhou et al., 2003) as well as metal-bound anticancer reagents such as cisplatin (IC50 ∼8 μM) (Ray et al., 2007). These complexes display moderate cytotoxic effect compared to some previously reported lanthanide complexes (Akhtar and Akhil, 2012). From the IC50 values of Eu(III) and Nd(III) complexes on both the cancer cells, it is understood that these complexes are more active on HCT116 cancer cells than on the HeLa cancer cells.
| Compound | IC50 (μM) | |
|---|---|---|
| HeLa | HCT116 | |
| 4-NBA-GG (L) | 57.74 | 52.87 |
| [EuL2(H2O)2]·4H2O | 45.85 | 44.37 |
| [GdL2(H2O)2]·4H2O | >100 | 49.34 |
| [NdL2(H2O)2]·4H2O | 41.3 | 30.05 |
| [SmL2(H2O)2]·4H2O | 92.7 | >100 |
| [TbL2(H2O)2]·4H2O | >100 | 74.64 |
5 Conclusion
Eu(III), Gd(III), Nd(III), Sm(III), and Tb(III) complexes with the Schiff base ligand derived from glycylglycine and 4-nitrobenzaldehyde were synthesized and characterized by elemental analysis, mass, IR, electronic spectra, molar conductance, TGA, and powder XRD. From the IR data, it is understood that the Schiff base ligand acts as a tridentate monobasic donor coordinating through the azomethine nitrogen, deprotonated peptide nitrogen, and carboxylato oxygen atoms. A three-stage decomposition process is shown in the thermogravimetric spectra of all the complexes. Powder XRD results show that the complexes are amorphous in nature. The antimicrobial studies reveal that the complexes show a higher activity than the ligand. The Nd(III) complex shows better activity against gram negative bacteria such as E. coli compared to the other complexes and the free ligand. DNA cleavage studies indicate that the Eu(III) and Nd(III) complexes have completely cleaved the DNA. In vitro anticancer activity is not significant compared to the known anticancer agents such as estramustine, noscapine, and cisplatin (Suresh et al., 2008). The Eu(III) and Nd(III) complexes are more active than the other three complexes and the free ligand on both the cancer cells (HeLa and HCT116).
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