Determination of Protein Binding Affinities and Investigation into the Antimicrobial Activities of Cu(II), Co(II) and Ni(II) Mixed Ligand Complexes

 

Sameena Mehtab, Harpreet Parmar, Tanveer Irshad Siddiqi,

Atanu Singha Roy

Department of Chemistry, Lovely Professional University, Jalandhar-Delhi G.T. Road, NH1, Phagwara,

Punjab 144411, India

*Corresponding Author E-mail: sameena.17937@lpu.co.in

The mixed ligand complexes (MLC) of Cu(II), Co(II) and Ni(II) ions with 1,10-phenanthroline (phen), glycine (gly) and tryptophan (trp) are synthesized and characterized. The interactions of these mixed ligand complexes with bovine serum albumin (BSA) have been investigated by fluorescence spectroscopy in combination with UV-VIS spectroscopy under physiological conditions. [Cu(gly)2phen] complex is able to quench to the fluorescence of bovine serum albumin (BSA) indicating the complexation between protein and ligand. The anti-microbial activities of all these complexes have been executed and good anti-microbial activities of the complexes over the ligands have been observed against the gram positive, gram negative bacteria and fungi.

 

 

 

1. INTRODUCTION:

The understanding of how the compounds are transported in the blood is essential to evaluate their bioavailability. Serum proteins plays key roles in the pharmacokinetic properties of drugs as they control their distribution and hence their bioavailability [1, 2]. These remain an important agent for the solubilization of poorly water-soluble drugs. It also transports steroid hormones, billirubin, bile salts, hematin, tryptophan, vitamins and metal ions (like those of Ni, Zn, Co, Cd, Ca) [3-5]. As a buffer in blood, its function is to contribute to the maintenance of blood pH [6].  The intensity of bovine serum albumin (BSA) binding has made it necessary for pharmaceutical companies to screen potential drugs at the early stages of drug development. A huge number of drug discovery projects had to be abandoned as a result of extremely strong binding or weak binding [7-9].

 

1,10-Phenanthroline and its derivatives are classed with π-electron-deficient N-hetero-arenes. They are efficient chelating ligands for most d-element ions and act as fairly powerful σ-donors and π-acceptors. These ligands can stabilize low oxidation degrees of d-elements in complexes and metal clusters and endow complexes with hydrophobic properties [10,11].

 

The specific pharmacologic activity of transition element complexes with 1,10-phenanthrolines and amino acid ligands, shows antibacterial, antifungal, antiviral and antitumor effects. These biochemical activities are associated with the complexes due to a high chelating power of 1,10-phenanthroline, as well as a planar geometry and π-electron deficiency of these ligands [12,13].

 

1,10-Phenanthroline is a biochemically active compound, inhibitor of metallo proteases and mitochondrial and chloroplast functions. At a concentration of 10^–5 М it decreases by 50% release of O₂ in photosynthesis, at concentrations of 10^–8 М it inhibits certain Fe-containing enzymes and glutamate hydrogenase, at a concentration of ~ 0.5 × 10^–3 М it inhibits by 28% ubiquinol-cytochrom-c reductase [14,15] and inhibits formation of hydroperoxide-induced single-stranded DNA cleavage in HL-90 cell lines [16] and growth of lactate bacteria [17]. 1,10-phenanthroline and its derivatives were tested for mutagenicity on Salmonella bacteria [18]. It was founds that 1,10-phenanthroline exhibits tuberculostatic activity with respect to Mycobacterium phlei and Mycobacterium bovis bacteria [19,20].

 

Metal ions that bind to amino acids have attracted the attention of many researchers due to relevance in the processes of adhesion, structural support and catalytic roles of metal ions that interact with proteins, enzymes and other biomolecules produced by amino acids [21,22]. An investigation was, therefore, undertaken to determine which structural features of tryptophan and glycine are responsible for its binding and to elucidate the nature of the binding site of the protein [23-25]. Mixed ligand transition metal complexes have attracted much attention due to their anticarcinogenic [26], anti-inflammatory [27], antibacterial [28] and antifungal [29] activities and other effects. Their pharmacokinetic and bioavailability can be evaluated on the basis of their interaction with serum proteins (albumin) [30, 31]. Therefore, understanding the nature of interaction of transition metal complexes with serum proteins, specifically serum albumin, is of utmost importance [32].

 

In present study interaction of synthesized compounds are checked with bovine serum albumin by UV titration and fluorescence experiments. Complexes are also studied for antibacterial and antifungal activities. The compounds tested against some pathogenic microorganisms like, gram-positive bacteria and gram-negative bacteria by well diffusion method, and they showed comparable results with standard drugs.

 

2. EXPERIMENTAL:

2.1. Materials

L-Tryptophan, 1,10-Phenonthroline, Tris buffer, Cobalt chloride, Nickel chloride, Copper chloride, potassium hydroxide  were purchased from Loba Chemie and glycine  was obtained from Central Drug House (P) Ltd. BSA was obtained from SDFCL. Tris buffer solution was prepared using double distilled water. 

 

2.2. Physical Measurements

Elemental analyses for Carbon, hydrogen, nitrogen and oxygen content were performed on a Perkin-Elmer 2400 II analyzer. IR spectra were obtained with KBr pellets on a Nicolet Shimadzu  FT-IR spectrometer in the range of 4,000–400 cm^-1 using KBr pellets. UV–visible absorption spectra were recorded on a Shimadzu UV2600 (200-800).

Fluorescence measurements were carried out on a Horiba Jobin Yvon FL-1057 Tau 3 spectrofluorometer. The experiments were carried out at room temperature. For steady-state fluorescence intensity measurements excitation was performed at 295 nm (selective excitation of Trp 213).

 

2.3. Synthesis of metal complexes

2.3.1 Synthesis of [Cu(gly)₂(phen)]complex

The complexes were prepared by reacting metal salts with the ligands using 1:2:1 mole ratio, i.e. one mole of copper (II) chloride with two mole of glycine and one mole of 1,10-phenanthroline [33]. [Cu(phen)(gly)₂] was prepared by adding glycine (0.30 g, 4 mmol) and potassium hydroxide (0.22 g, 4 mmol) in 20 ml hot  methanol. The mixture was then stirred at room temperature for 1h at 40^-50^°C. A white homogeneous solution was obtained. Homogenous mixture copper chloride was added (0.34 g, 2.0 mmol) in 2 ml of methanol dropwise and stirred for 2 h. After that 1,10-phen (0.39 g, 2 mmol) dissolved in 5 ml methanol and added dropwise to the solution and reflux for 5 h with constant stirring. The resultant solution was filtered and kept at room temperature for 10 days, where upon green block-shaped crystals suitable for X-ray diffraction were obtained. Monoclinic prism Yield: 78%.

 

2.3.2 Synthesis of [Cu(trp)₂(phen)]complex

[Cu(phen)(trp)₂] was prepared by adding tryptophan (0.204 g, 1mmol) and potassium hydroxide (0.056 g, 1mmol) in 20 ml hot methanol. The mixture was then stirred at room temperature for 1h at 40-50^°C_. A homogeneous solution was obtained. To the above homogenous solution Copper chloride was added (0.085 g, 0.0005mmol) in 2 ml of methanol dropwise and stirred for 2 h. 1,10-Phen (0.099 g, 0.0005 mmol) dissolved in 5 ml methanol and added dropwise to the solution and reflux for 5 h with constant stirring. The resultant solution was filtered and kept at room temperature for 12 days, where upon dark brown powder was obtained. Yield 58%.

 

2.3.3 Synthesis of [Ni(gly)₂(phen)]complex

[Ni(phen)(gly)₂] complex was prepared by adding Glycine (0.30 g, 4 mmol) and potassium hydroxide (0.22 g, 4 mmol) in 20 ml hot  methanol. The mixture was then stirred at room temperature for 1 h at 40^-50^OC_. A white homogeneous solution was obtained. To the above homogenous solution nickel chloride was added (0.47 g, 2.0 mmol) in 2 ml of methanol dropwise and stirred for 2 h. Next, a methanol solution (5 mL) of 1,10-phenanthroline (0.39 g, 2 mmol) was added dropwise and stirred for 5 h with constant stirring. The resultant solution was filtered and kept at room temperature for 15 days, where upon shiny green needle shape crystals suitable for X-ray diffraction were obtained. Yield 74%.

 

2.3.4  Synthesis of [Ni(trp)₂(phen)] complex

The complex was prepared by adding tryptophan (0.204 g, 1 mmol) and potassium hydroxide (0.056 g, 1mmol) in 20 ml hot methanol. The mixture was then stirred at room temperature for 1 h at 40-50°C_. A homogeneous solution was obtained.  To the above homogenous solution nickel chloride was added (0.118 g, 0.5 mmol) in 2 ml of methanol dropwise and stirred for 2 h. In that mixture 1,10-phen (0.099 g, 0.5 mmol) dissolved in 5ml methanol and added dropwise to the solution and reflux for 5 h with constant stirring. The resultant solution was filtered and kept at room temperature for 10 days, where light brown shiny powder was obtained Yield 73%.

 

2.3.5 Synthesis of [Co(gly)₂(phen)] complex

[Co(gly)₂(phen)] complex was prepared by adding Glycine (0.30 g, 4 mmol) and potassium hydroxide (0.22 g, 4 mmol) in 20 ml hot methanol. The mixture was then stirred at room temperature for 1h at 40^-50^°C. A white homogeneous solution was obtained.  To the above homogenous solution Cobalt chloride was added (0.47 g, 2.0 mmol) in 2 ml of methanol dropwise and stirred for 2 h. Then a methanol solution 5 mL of 1,10-phenanthroline (0.39 g, 2 mmol) was added dropwise and stirred for 5 h with constant stirring. The resultant solution was filtered and kept at room temperature for 10 days, where upon dark brown precipitate was obtained. Yield 79%.

 

2.3.6 Synthesis of [Co(trp)₂(phen)]

The complex was prepared by adding Tryptophan (0.204 g, 1 mmol) and potassium hydroxide (0.056 g, 1mmol) in 20 ml hot methanol. The mixture was then stirred at room temperature for 1h at 40^-50°C_. A homogeneous solution was obtained. To this homogenous solution Cobalt chloride was added (0.118 g, 0.5 mmol) in 2 ml of methanol dropwise and stirred for 2 h. In that mixture 1,10-phen (0.099 g, 0.5 mmol) dissolved in 5 ml methanol and added dropwise to the solution and reflux for 5 h with constant stirring. The resultant solution was filtered and kept at room temperature for 10 days, where upon brown precipitate were obtained. Yield was 65%.

 

2.3.7 Synthesis of [Cu(gly)(phen)]

The complexes were prepared by reacting the respective metal salts with the ligands using 1:1:1 mole ratio, i.e. one mole of Copper chloride: one mole of 1,10-phenanthroline : one mole of glycine. The [Cu(phen)(gly)] was prepared by adding glycine (0.30 g, 4 mmol) and potassium hydroxide (0.22 g, 4 mmol) in 20 ml hot  methanol. The mixture was then stirred at room temperature for 1h at 40^-50^°C_. A white homogeneous solution was obtained. To the above homogenous solution copper chloride was added (0.68 g, 4.0 mmol) in 2 ml of methanol dropwise and stirred for 2 h. To the above mixture 1,10-phen (0.79 g, 4mmol) dissolved in 5 ml methanol and added dropwise to the solution and reflux for 5 h with constant stirring. The resultant solution was filtered and kept at room temperature for 10 days. Green needle like crystal was obtained. Yield was 84%. 

 

2.3.8 Synthesis of [Ni(gly)(phen)]

The complex was synthesized by adding glycine (0.30 g, 4 mmol) and potassium hydroxide (0.22 g, 4 mmol) in 20 ml hot methanol. The mixture was then stirred at room temperature for 1h at 40^-50^°C. A white homogeneous solution was obtained.  To the above homogenous solution Nickel chloride was added (0.95 g, 4.0 mmol) in 2 ml of methanol dropwise and stirred for 2 h. Then 1,10-phen (0.79 g, 4 mmol) dissolved in 5 ml methanol was added dropwise to the solution and reflux for 5 h with constant stirring. The resultant solution was filtered and kept at room temperature for 10 days. Pink powered precipitates were obtained. Yield was 82%.

 

2.3.9 Synthesis of [Ni(trp)(phen)]

The complex of [Ni(trp)(phen)] was prepared by adding tryptophan (0.204 g, 1 mmol) and potassium hydroxide (0.056 g, 1 mmol) in 20 ml hot methanol. The mixture was then stirred at room temperature for 1h at 40^-50°C._. A white homogeneous solution was obtained.  To this homogenous solution nickel chloride was added (0.170 g, 1.0 mmol) in 2 ml of methanol dropwise and stirred for 2 h. Then mixture 1,10-phen (0.198 g, 1 mmol) dissolved in 5 ml methanol and added dropwise to the solution and reflux for 5 h with constant stirring. The resultant solution was filtered and kept at room temperature for 10 days. Shiny green precipitates were obtained.  

 

2.3.10 Synthesis of [Co(gly)(phen)]

The complex was synthesized by adding Glycine (0.30 g, 4 mmol) and potassium hydroxide (0.22 g, 4 mmol) in 20 ml hot methanol. The mixture was then stirred at room temperature for 1h at 40^-50°C_. A white homogeneous solution was obtained. To the above homogenous solution cobalt chloride was added (0.95 g, 4.0 mmol) in 2 ml of methanol dropwise and stirred for 2 h. In that solution 1,10-phen (0.79 g, 4 mmol) dissolved in 5ml methanol and added dropwise to the solution and reflux for 5 h with constant stirring. The resultant solution was filtered and kept at room temperature for 10 days. Cylindrical shape blocked crystal was obtained. Yield was 58%.

 

2.3.11 Synthesis of [Co(phen)] (trp)

The complex of [Co(trp)(phen)] was prepared by adding tryptophan (0.204 g, 1 mmol) and potassium hydroxide (0.056 g, 1 mmol) in 20 ml hot methanol. The mixture was then stirred at room temperature for 1 h at 40^-50^°C_. A white homogeneous solution was obtained.

 

Homogenous solution of cobalt chloride was added (0.237 g, 1.0 mmol) in 2 ml of methanol dropwise and stirred for 2 h. To the above mixture 1,10-phen (0.198 g, 1 mmol) dissolved in 5 ml methanol and added dropwise to the solution and reflux for 5 h with constant stirring. The resultant solution was filtered and kept at room temperature for 10 days. Shiny brown precipitates were obtained.

 

Detail of structures and physical/analytical data is provided in Fig.1 and Table 1 respectively.

 

 

Fig.1. Structure of different complexes [M(phen)_n(trp)_n] and [M(phen)_n(gly)_n], where M=Cu(II),Co(II) and Ni(II). In structure (A) Phen:Gly:M (1:1:1), structure (B) Phen:Gly:M (1:2:1) structure (C) Phen:Try:M (1:1:1), structure (D) Phen:Try:M (1:2:1).

 

 

3. RESULTS AND DISCUSSIONS:

3.1 UV-Vis, IR spectra and elemental analysis

All obtained complexes exhibit similar electronic absorption spectra, which indicate that the central ions and ligands are coordinated in a similar mode. The UV-VIS spectrum of different complexes shows absorptions in the range 215-230 nm at low concentrations in Tris buffer (pH 7.4). These bands indicates n to π and π to π* transitions which confirm binding of 1,10- Phenonthroline with metal center. While at high complex concentration one broad band at around 1600 nm appears which indicate d-d transition and coordination of metals with ligands (Table 2).

 

The IR spectra of the ligands (1,10-phenonthroline, glycine and tryptophan)  and its complexes are given in figures. The significant regions of IR spectra of all complexes are very similar and the following observations are of interest. Comparison of IR spectra of   ligand with that of its metal complexes has been adopted to determine the coordinating atoms of the ligand to metal ions. From the IR spectrum of the ligand, the absorption band at 3200-3300 cm^-1 is due to the absorption of N-H group stretching vibration. The absorption bands at 680-1100 cm^-1 are due to the vibration of 3-phenonthroline ring, C-H stretching frequency. The absorption bands at 1400-1550 cm^-1 are due to COO asymmetric and symmetric vibrations frequency. The FT–IR spectrum of the ligand is compared with the spectra of the complexes. The characteristic absorption bands 450-750 cm^-1 suggesting coordination (M-N and M-O bonds) of both nitrogen and oxygen atoms to metal center in complex. Thus A comparison of IR spectra of the complexes obtained with those of free additional ligands helps to distinguish their coordination mode (Table 2).

 

The elemental analysis data (C,H,N,O) are in close agreement to the predicted data, and suggested that all the complexes are mononuclear with the ligand coordinated to the central metal atom.

 

 

Table 1 Physical and analytical data of mixed ligand complexes.

S.No	Complexes	Colour	M.P (◦C)	Yield (%)	Elemental analysis % Found (% Cal.)
					C	N	H	M	O
1.	[Cu(gly)2(phen)] C16H14CuN4O	Green	232-235	78	49.29 (48.26)	14.37 (15.23)	3.63 (3.89)	16.32 (16.32)	16.45 (16.30)
2.	[Cu(trp)2(phen)] C34H28CuN6O4	Dark Brown	235	58	63.00 (63.03)	9.80 (9.76)	4.35 (4.39)	12.97 (12.94)	9.88 (9.88)
3.	[Ni(gly)2(phen)] C16H14N4NiO4	Green	108	74	48.91 (48.88)	14.55 (14.53)	3.67 (3.69)	15.25 (15.23)	17.62 (17.67)
4.	[Ni(trp)2(phen)] C34H28N6NiO4	Light Brown	232	73	62.48 (62.48)	13.06 (13.06)	4.39 (4.40)	9.12 (9.18)	10.95 (10.88)
5.	[Co(phen)(gly)2] C34H30N6NiO4	Dark brown	238	79	62.28 (62.36)	12.02 (14.05)	4.69 (4.57)	9.10 (9.11)	8.92 (9.91)
6.	[Co(trp)2(phen)] C16H16CoN4O4	Brown	216	65	50.72 (50.70)	14.29 (14.31)	4.20 (4.19)	15.26 (15.29)	16.53 (16.50)
7.	[Cu(gly)(phen)] C14H12CuN3O2	Green	180	84	51.95 (51.94)	13.32 (13.31)	3.81 (3.83)	19.99 (19.95)	11.93 (11.97)
8.	[Ni(gly)(phen)] C14H12N3NiO2	Pink	260	82	52.73 (52.76)	13.67 (13.68)	3.86 (3.83)	18.70 (18.70)	11.11 (11.03)
9.	[Ni(trp)(phen)] C23H19N4NiO2	Green	NA*	NA*	62.48 (62.50)	12.67 (12.65)	4.33 (4.38)	13.28 (13.27)	7.24 (7.20)
10.	[Co(gly)(phen)] C14H12CoN3O2	Brown	234	58	52.69 (52.70)	13.42 (12.43)	3.86 (3.85)	18.82 (19.74)	10.48 (11.28)
11.	[Co(trp)(phen)] C23H19CoN4O2	Brown	NA*	NA*	62.45 (62.44)	11.67 (11.66)	4.33 (4.31)	13.6 (13.8)	7.83 (7.86)

*Due to viscous nature of product MP and yield were not observed.

 

Table 2 Selected UV (nm) and IR frequencies (cm^-1) of the mixed ligand complexes.

 

Fig. 2. Absorbance spectra of complex-BSA system in 0.1 M Tris buffer pH 7.4 for BSA (1 µM) with successive increasing amount (0.5 to 3.6 µM) of [Cu(gly)₂phen].

 

 

3.2 Binding with BSA

The binding constants of BSA-complexes were calculated [34]. It is assumed that the interaction between the ligand L and the substrate S is 1:1; for this reason a single complex SL (1:1) is formed. The relationship between the observed absorbance change per centimeter and the system variables and parameters is as follow.

 

               (1)

 

 

Where ΔA = A-A₀ from the mass balance expression S_t = [S] + [SL], we get [S] = S_t/(1 + K₁₁[L]). Eq. (1) is the binding isotherm, which shows the hyperbolic dependence on free ligand concentration. The double-reciprocal form of plotting the rectangular hyperbola 1/y = f/d× 1/x + e/d is based on the linearization of Eq. (1) according to the following equation 2.

              (2)

 

Thus, the double reciprocal plot of 1/ΔA versus 1/[L] is linear and the binding constant can be estimated from the following equation.

                                              (3)

BSA was reported to have two binding sites for metals. One site is the MBS which is the principal binding site, while the second site is the site B primary site. The UV-visible  spectra observed after titrating 1 µM of BSA  with increasing mol equivalents (0.5 to 4 µM) of complex showed that initially upto around 2.5 µM complexes concentration BSA-complex adduct showed increasing absorption. While on further increase in complex concentration BSA-complex adduct band disappears which indicate saturation at BSA binding site. Two main bands, (~216 nm) and (~230 nm), showed up which globally increase in intensity with increasing complexes concentration. From these BSA-complex titration binding constant were also determined. The plot of 1/(A-A_o) versus 1/([Cu(gly)₂phen] concentration for BSA and its drugs adducts, where A_o is the initial absorption band of free BSA (267 nm) and A is the recorded absorption at different complex and BSA adduct concentrations ( 0.5-3.6µM complex and 1 µM). Binding constants indicate high-affinity (Ka ∼ 10⁶ M⁻¹) BSA binding site of the complex corresponds to the complex binding area in subdomain IIIA (Table 3).

 

Table 3 Binding constant of selected mixed ligand complexes with BSA, calculated from UV titration spectra.

 

3.3 Fluorescence quenching of BSA by [Cu(gly)₂phen]

Fluorescence is a useful technique for the characterization of protein-ligand binding. In this work the steady-state fluorescence measurements has been performed on the BSA-[Cu(gly)₂phen] system. Fluorescence emission spectra of BSA in absence and presence of [Cu(gly)₂phen] are provided in Fig. 3a and it has been observed that the intrinsic fluorescence intensity of BSA is quenched in presence of the copper complex. The results indicate that the complex is able to quench the fluorescence and a decrease of ~ 70% for 1:4 protein-ligand ratio was observed.

 

The Stern‑Volmer plot is used to evaluate the mechanism of quenching involved in the interaction.

 

              (1)

 

All the signs in the equation 1 have their standard meanings in fluorescence spectroscopy. We can conclude the mode of fluorescence quenching from the data obtained by the plot of F₀/F versus [Q] (Fig. 3b) by calculating the value of K_SV. The upward curvature nature of the Stern-Volmer plot (Fig. 3b) indicates the combination of both static and dynamic quenching processes [35]. The value of K_SV (4.082×10⁴ M^-1) is calculated from the slope of regression plot of linear portion of Stern-Volmer plot (inset of Fig. 3a) and the bimolecular quenching constant (K_q) is also estimated from the expression, K_SV = K_qτ₀. The value of K_q (8.164×10¹² M^-1 s^-1) is larger than the maximum value of 10¹⁰ M^-1 s^-1, [35] which is the largest plausible value in water for dynamical quenching mode. In case where both the quenching modes are involved the equation 1 can be written as follows (equation 2) and on rearrangement it changes to equation 3.

 

                 (2)

 

         (3)

 

The notations, K_S and K_D indicate the static and dynamical quenching constant and [Q] represents the quencher concentration. The values of K_S and K_D are determined from the slope and intercept of the plot of  versus [Q] (Fig. 3c). The K_D value calculated is found to be imaginary, suggested that our assumption of combined static and dynamic quenching in the present context is wrong. Hence the non-linearity along with the upward curvature of the Stern-Volmer plot is possibly due to the presence of either more than one binding site or a high quencher concentration surround the fluorophore [35]. A similar kind of result was also found in the case of interaction of the mixed ligand-Cu(II) complex with serum albumins [36].

 

 

The binding parameters (K_b, the equilibrium binding constant and n, number of binding sites) for the interaction of copper complex with BSA have been estimated from the following equation using the fluorescence quenching data.

 

                               (4)

Where ΔF = F₀ – F; F₀ and F are the fluorescence intensities of BSA in absence and presence of the copper complex respectively. The value of K_b is estimated from the intercept of the plot of equation 4 (Fig. 3d.) The values of K_b and n are found to be 1.718 × 10⁴ M^-1 and 1.471 respectively. The higher number of binding sites may be one reason for the upward curvature nature.

 

Fig. 3. (a) Fluorescence emission spectra of BSA (5 µM) in the absence and in presence of [Cu(gly)₂phen] complex (0 to 20 µM) in Tris buffer of pH 7.4. (b) The Stern-Volmer plot of the binding of BSA and [Cu(gly)₂phen] complex; Inset: Linear regression plot. (c) The plot of K_app versus [Q] for the determination of quenching mode. (d) The binding plot for the interaction of [Cu(gly)₂phen] complex with BSA.  λ_ex = 295 nm.

 

Table 4 Antimicrobial activities of complexes.

Name of Complexes	Diameter of zone inhibition in (mm)
	Fungi						Bacteria
	A. Femigotus			P. Chrysogenum			E. Coli			S. Typhimurium
	50	100	200	50	100	200	50	100	200	50	100	200
[Cu(trp)2(phen)]	3	4	6	4	6	9	0	1	6	0	3	5
[Cu(gly)2(phen)]	0	2	3	1	2	4	0	1	2	1	3	4
[Ni(gly)2(phen)]	3	5	7	0	2	3	0	1	1	0	3	5
[Cu(gly)(phen)]	2	3	4	0	2	5	0	1	1	0	5	7
[Ni(gly)(phen)]	0	2	6	0	2	5	0	1	2	0	2	3
[Co(gly)2(phen)]	2	4	5	1	2	2	1	1	1	0	1	2
[Co(gly)(trp)]	2	4	2	2	2	2	2	2	3	2	2	3
[Ni(trp)2(phen)	2	2	4	2	4	6	2	2	4	2	2	4

 

Fig. 4. Showing Anti-microbial activity of [Cu(trp)₂(phen)], [Cu(gly)(phen)], [Ni(gly)(phen)].

3.4. Antimicrobial screening of all the complexes

The minimum inhibitory concentrations (MIC) of complexes/products were investigated with bacteria and fungi seeded in tubes with nutrient broth (NB) [37]. In the laminar chamber the autoclaved homogenous suspensions of NB poured into petri dishes and allow for solidification. After cooling as well as solidification of the NB in petri plates the paper disc of different concentration, 0.005 mM, 0.0025 mM and 0.00125 mM of the investigated compound applied using a sterilized forceps. After incubation for 48h in an incubator at 37°C and 28°C for bacteria and fungi, respectively, the inhibition zone diameters were measured and expressed in mm. Filter discs impregnated with 10 mm³ solvent (water/methanol etc.) were used as negative control. The percentage of inhibition was calculated by using the formula: % inhibition = [((A-B)/A)*100]; here A = inhibition (in mm) by solvent (water) and B = inhibition (in mm) by compound at above mentioned concentrations [38].

 

Table 4 shows that the complexes exhibits significant activity against fungus and bacteria (Fig 4-5). The complexes were screened in vitro for its microbial activity against certain pathogenic bacterial and fungal species using disc diffusion method. In our biological experiments using Copper, Cobalt and Nickel complex, we observed considerable antifungal and anti bacterial activity against Aspergillus fermigatus, Penicillum chrysogenum and E.coli, Salmonella typhimurium. The Nickel complex has shown a good activity against A. fermigatus than copper complexes. But in case of anti bacterial activity against E. coli and S. typhimurium Copper complexes show good activity against it. It may be concluded that our Cu (II) complex inhibits the growth of bacteria and Ni(II) complex inhibit the fungi growth to a good extent than cobalt complexes.

 

4. CONCLUSION:

In this paper, we have described the synthesis of new copper(II), cobalt(II) and nickel(II) mixed ligand complexes, with 1,10-phenonthroline, glycine and tryptophan. These complexes [Cu(trp)₂(phen)], [Cu(gly)₂(phen)], [Ni(gly)₂(phen)], [Cu(gly)(phen)], [Ni(gly)(phen)], [Co(gly)₂(phen)], [Co(gly)(trp)], [Ni(trp)₂(phen)] were characterized by elemental analysis (C,H,N,O), IR and UV-Vis spectra. The presence of NH₂, C-H, C=O, M-N and M-O peaks confirm the complex synthesis. The n to π and n to π* transitions in the UV spectra further confirm the presence of phenonthroline binding with metals. Some complexes were crystallized and under consideration for crystal structure determination by X-ray crystallography. The interactions of bovine serum albumin with different complexes were studied and their properties have been investigated by using UV absorption titration spectra in aqueous solution at physiological conditions using Tris buffer (pH = 7.4). All the results indicated that the complexes bind moderately with binding constant value in the range of 10⁶ M^-1.It is important to note here that the low affinity binding is consistent with the role of serum proteins as carrier molecules for the delivery of the parent drug and its derivatives to target tissues. Antimicrobial and antifungal studies of complexes were performed on A. Femigatus, P. Chrysogenum, E. Coli, and S. Typhimurium. The anti-bacterial and anti-fungal data given for the compounds presented in this paper allowed us to state that the metal complexes generally have better activity than phenanthroline ligands and comparable with standards.

 

Fig. 5. Anti-microbial studies of mixed ligand metal complexes of copper (a) [Cu(trp)₂(phen)] (b) [Cu(gly)₂(phen)].

 

5. ACKNOWLEDGEMENT:

Author’s are grateful to Lovely Professional University, India for providing financial assistance for this work.

 

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Received on 01.12.2014         Modified on 20.12.2014

Accepted on 05.01.2015         © AJRC All right reserved