# Synthesis and Characterization with Antineoplastic, Biochemical, Cytotoxic, and Antimicrobial Studies of Schiff Base Cu(II) Ion Complexes

Authors: M. M. Haque; Md. Kudrat-E-Zahan; Laila Arjuman Banu; Md. Shariful Islam; M. S. Islam
Journal: Bioinorganic Chemistry and Applications
 (2015)

---

## Abstract

Copper(II) complexes containing two Schiff base ligands derived from 2-hydroxybenzaldehyde with 2-aminophenol and 3-aminophenol have been synthesized and characterized by means of analytical, magnetic, and spectroscopic methods. Bacteria, fungus,Entamoeba histolytica, and antineoplastic activities of the synthesized complexes have been determined by monitoring the parameters cell growth inhibition, survival time of tumour mice, time-body relation, causing of intraperitoneal cells and macrophages, alkaline phosphatase activity, hematological effect, and biopsy of tumour.


---

## Body

## 1. Introduction

Schiff base metal complexes based research works have been widely carried out from 1930, because of their biological and industrial applications [1–5]. The use of metal complexes as pharmaceuticals has shown promise in recent years particularly as anticancer agents [6]. Previously, synthesis and properties of thiocyanato complex of low valent metal ions containing different monodentate auxiliary ligands have been reported from our laboratory [7–9]. Worldwide spread of drug-resistant bacteria is now a critical problem in global health. To find new drug, recently, we studied few mixed-ligand complexes containing heterocyclic amine as secondary ligands and few Schiff base containing complexes [10–12]. In present study, mixed-ligand complexes of Cu(II) containing the Schiff base ligand derived from 2-hydroxybenzaldehyde with 2-aminophenol/3-aminophenol and bidentate auxiliary ligands were synthesized and characterized. The auxiliary ligands used were potassium thiocyanato, 2-aminopyridine, and 2-phenylenediamine. Antineoplastic, biochemical, cytotoxic, and antimicrobial activities of the complexes were also studied.


## 2. Experimental

### 2.1. Physical Measurement

The weighing operation was performed on a METTLER PM-200 electronic balance. Conductivity measurements were carried out in dimethyl sulfoxide (DMSO) using a WPACMS 35 conductivity meter and dip-cell with platinized electrodes. The melting or decomposition temperatures of all the prepared metal complexes were observed in an electrothermal melting point apparatus model number AZ6512. The SHERWOOD SCIENTIFIC Magnetic Susceptibility Balance was used for the present investigation. Infrared spectra as KBr disc were recorded in a SIMADZU FTIR-8400 (Japan) infrared spectrophotometer, from 4000 to 400 cm−1. The absorbances of the complexes were recorded on SHIMUDZU Spectrophotometer. Analyses of the complexes for carbon, hydrogen, and nitrogen were carried out by Microanalytical Services at the University of St. Andrews, Scotland. Transplantable tumour (Ehrlich’s Ascites Carcinoma, EAC) used in this research was obtained from Indian Institute of Chemical Biology (IICB), Calcutta, India. In vivo antineoplastic activity of the test complexes was determined by measuring the effect of the test complexes on tumour cell growth inhibition, survival time of tumour bearing mice, hematological parameters, and serum alkaline phosphatase activity of tumour bearing mice. Tumour growth was monitored by recording daily weight change. The concentration of haemoglobin was measured by the usual procedure using Sahli’s haemometer.


### 2.2. Procedure of Preparation of Schiff Base (SB-1)

The Schiff bases were prepared by the condensation of 2-hydroxybenzaldehyde with 2-aminophenol. 2-Hydroxybenzaldehyde (1.7 g, 0.014 mol) in absolute ethanol (20 mL) was added to an ethanolic (30 mL) solution of 2-aminophenol (1.5 g, 0.014 mol). The mixture was heated to reduce the volume to 25 mL, and then it was cooled in an ice-bath. The black crystalline product was isolated and washed with hot ethanol. The structure of SB-1 is shown in Figure1.Figure 1
(2-Hydroxy-benzylidene)-(2-hydroxy-phenyl)-amine (SB-1). Yield: 3.0 g (78%). Anal. Calc. (%): C, 73.2; H, 5.2; N, 6.6. Found: C, 73.1; H, 5.1; N, 6.7. M.p. 160–162°C.


#### 2.2.1. Procedure of Preparation of Test Compound K[Cu(SB-1)(SCN)], SB-1 = C13H9NO2

An appropriate solution of CuCl2·H2O (0.005 mol) in absolute ethanol (25 mL) was added to an ethanolic (30 mL) solution of potassium thiocyanate (0.005 mol). The solution was filtered and the filtrate was added to the methanolic solution of C13H9NO2H2 (SB-1) (0.005 mol, 80 mL). The resulting mixture was boiled on a water bath for 5 minutes and cooled. The complexes were separated, washed with hot ethanol, and dried in vacuo over P4O10.


### 2.3. Procedure of Preparation of Schiff Base (SB-2)

The Schiff base was prepared by the condensation of 2-hydroxybenzaldehyde with 3-aminophenol. 2-Hydroxybenzaldehyde (1.7 g, 0.014 mol) in absolute methanol (20 mL) was added to an ethanolic solution (30 mL) of 3-aminophenol (1.5 g, 0.014 mol). The mixture was heated to reduce the volume to 25 mL and then it was cooled in an ice-bath. The black crystalline product was filtered and washed with hot ethanol. The structure of SB-2 is shown in Figure2.Figure 2
(3-Hydroxy-benzylidene)-(2-hydroxy-phenyl)-amine (SB-2). Yield: 3.1 g (82%). Anal. Calc. (%): C, 73.2; H, 5.2; N, 6.6. Found: C, 73.0; H, 5.2; N, 6.5. M.p. 160–162°C.


#### 2.3.1. Procedure of Preparation for [Cu(SB-2)(NN)] [NN = 2-Aminopyridine/2-Phenylenediamine]

25 mL of an ethanolic solution of the metal chloride (0.005 mol) [CuCl2·H2O] was added to 30 mL of an ethanolic solution of the above prepared Schiff base (1.05 g, 0.005 mol). Then 20 mL of an ethanolic solution of [NN] (0.005 mol) was added to the metal salt-Schiff base solution. The resulting mixture was boiled on a water bath for 5 minutes and after that it was cooled. The complexes that were separated were washed with hot ethanol and dried in vacuo over P4O10.


## 2.1. Physical Measurement

The weighing operation was performed on a METTLER PM-200 electronic balance. Conductivity measurements were carried out in dimethyl sulfoxide (DMSO) using a WPACMS 35 conductivity meter and dip-cell with platinized electrodes. The melting or decomposition temperatures of all the prepared metal complexes were observed in an electrothermal melting point apparatus model number AZ6512. The SHERWOOD SCIENTIFIC Magnetic Susceptibility Balance was used for the present investigation. Infrared spectra as KBr disc were recorded in a SIMADZU FTIR-8400 (Japan) infrared spectrophotometer, from 4000 to 400 cm−1. The absorbances of the complexes were recorded on SHIMUDZU Spectrophotometer. Analyses of the complexes for carbon, hydrogen, and nitrogen were carried out by Microanalytical Services at the University of St. Andrews, Scotland. Transplantable tumour (Ehrlich’s Ascites Carcinoma, EAC) used in this research was obtained from Indian Institute of Chemical Biology (IICB), Calcutta, India. In vivo antineoplastic activity of the test complexes was determined by measuring the effect of the test complexes on tumour cell growth inhibition, survival time of tumour bearing mice, hematological parameters, and serum alkaline phosphatase activity of tumour bearing mice. Tumour growth was monitored by recording daily weight change. The concentration of haemoglobin was measured by the usual procedure using Sahli’s haemometer.


## 2.2. Procedure of Preparation of Schiff Base (SB-1)

The Schiff bases were prepared by the condensation of 2-hydroxybenzaldehyde with 2-aminophenol. 2-Hydroxybenzaldehyde (1.7 g, 0.014 mol) in absolute ethanol (20 mL) was added to an ethanolic (30 mL) solution of 2-aminophenol (1.5 g, 0.014 mol). The mixture was heated to reduce the volume to 25 mL, and then it was cooled in an ice-bath. The black crystalline product was isolated and washed with hot ethanol. The structure of SB-1 is shown in Figure1.Figure 1
(2-Hydroxy-benzylidene)-(2-hydroxy-phenyl)-amine (SB-1). Yield: 3.0 g (78%). Anal. Calc. (%): C, 73.2; H, 5.2; N, 6.6. Found: C, 73.1; H, 5.1; N, 6.7. M.p. 160–162°C.


### 2.2.1. Procedure of Preparation of Test Compound K[Cu(SB-1)(SCN)], SB-1 = C13H9NO2

An appropriate solution of CuCl2·H2O (0.005 mol) in absolute ethanol (25 mL) was added to an ethanolic (30 mL) solution of potassium thiocyanate (0.005 mol). The solution was filtered and the filtrate was added to the methanolic solution of C13H9NO2H2 (SB-1) (0.005 mol, 80 mL). The resulting mixture was boiled on a water bath for 5 minutes and cooled. The complexes were separated, washed with hot ethanol, and dried in vacuo over P4O10.


## 2.2.1. Procedure of Preparation of Test Compound K[Cu(SB-1)(SCN)], SB-1 = C13H9NO2

An appropriate solution of CuCl2·H2O (0.005 mol) in absolute ethanol (25 mL) was added to an ethanolic (30 mL) solution of potassium thiocyanate (0.005 mol). The solution was filtered and the filtrate was added to the methanolic solution of C13H9NO2H2 (SB-1) (0.005 mol, 80 mL). The resulting mixture was boiled on a water bath for 5 minutes and cooled. The complexes were separated, washed with hot ethanol, and dried in vacuo over P4O10.


## 2.3. Procedure of Preparation of Schiff Base (SB-2)

The Schiff base was prepared by the condensation of 2-hydroxybenzaldehyde with 3-aminophenol. 2-Hydroxybenzaldehyde (1.7 g, 0.014 mol) in absolute methanol (20 mL) was added to an ethanolic solution (30 mL) of 3-aminophenol (1.5 g, 0.014 mol). The mixture was heated to reduce the volume to 25 mL and then it was cooled in an ice-bath. The black crystalline product was filtered and washed with hot ethanol. The structure of SB-2 is shown in Figure2.Figure 2
(3-Hydroxy-benzylidene)-(2-hydroxy-phenyl)-amine (SB-2). Yield: 3.1 g (82%). Anal. Calc. (%): C, 73.2; H, 5.2; N, 6.6. Found: C, 73.0; H, 5.2; N, 6.5. M.p. 160–162°C.


### 2.3.1. Procedure of Preparation for [Cu(SB-2)(NN)] [NN = 2-Aminopyridine/2-Phenylenediamine]

25 mL of an ethanolic solution of the metal chloride (0.005 mol) [CuCl2·H2O] was added to 30 mL of an ethanolic solution of the above prepared Schiff base (1.05 g, 0.005 mol). Then 20 mL of an ethanolic solution of [NN] (0.005 mol) was added to the metal salt-Schiff base solution. The resulting mixture was boiled on a water bath for 5 minutes and after that it was cooled. The complexes that were separated were washed with hot ethanol and dried in vacuo over P4O10.


## 2.3.1. Procedure of Preparation for [Cu(SB-2)(NN)] [NN = 2-Aminopyridine/2-Phenylenediamine]

25 mL of an ethanolic solution of the metal chloride (0.005 mol) [CuCl2·H2O] was added to 30 mL of an ethanolic solution of the above prepared Schiff base (1.05 g, 0.005 mol). Then 20 mL of an ethanolic solution of [NN] (0.005 mol) was added to the metal salt-Schiff base solution. The resulting mixture was boiled on a water bath for 5 minutes and after that it was cooled. The complexes that were separated were washed with hot ethanol and dried in vacuo over P4O10.


## 3. Results and Discussion

### 3.1. Elemental Analysis and Conductivity Measurement

The analytical data and physical properties of the synthesized complexes are given in Table1. The molar conductances in DMSO indicate that the complexes are all 1 : 1 electrolytes [13, 14].Table 1
Analytical dataa and physical properties.
Complex notation
Complex
Colour
Melting point (°C)
% M
% C
% H
% N
Molar conductance (Ohm1cm2mol−1)
Magnetic momentMeff (B.M)
A
K[Cu(SB-1)(SCN)]
Yellowish
175–177
45.22 (45.18)
2.42 (2.39)
7.53 (7.50)
29
1.95
B
[Cu(SB-2)NN]NN = aminopyridine
Greenish
228–230
17.14 (17.52)
58.27 (58.60)
4.05 (4.50)
11.33 (11.10)
35
1.90
C
[Cu(SB-2)NN]NN = phenylenediamine
Greenish
160–162
16.52 (16.05)
59.27 (58.89)
4.42 (4.10)
10.92 (10.50)
42
2.0
aThe values found are given in parenthesis.


### 3.2. IR Studies

Complex A. The infrared spectral data are shown in Table 2. The Schiff base C13H9NO2H2 [SB-1] behaves as tridentate dinegative ligand coordinating at the imino nitrogen and two oxygen atoms. In the complexes, the shift of ν(C=N) mode in a frequency 1605 cm−1 relative to the free ligand value 1610–1620 cm−1 (for ligand C13H9NO2) indicates that bond formation takes place through the imino nitrogen atom. The ν(OH) band observed in the free Schiff base (SB-1) disappears upon coordination, which indicates deprotonation and coordination at the oxygen sites. Furthermore, the presence of ν(M-O) and ν(M-N) linkages of bands at 455–535 cm−1, respectively, was observed for all the complexes (A) [15–17]. The ambidentate thiocyanate ligand can give either M-NCS or M-SCN bonding sequence, which nevertheless reveals the acidity of the metal ions. The complexes also display ν(CN) at 2100 cm−1 characteristic of S-bonded thiocyanato moieties. In Pearson’s terminology, these are soft acids. The ν(CS) modes appear at lower frequencies in the M-S-C=N complexes than those in the M-N-C=S complexes [8, 9, 18]. The band of ν(CS) at 750 cm−1 is characteristic of M-S-C=N bonding sequence. This is further apparent from the ν(M-S) modes at 350 cm−1 in the far infrared spectra of the complexes.Table 2
Selected IR spectral data of the complexes (band maxima, cm−1).
Compound
ν(C=N)
ν(NH2)
ν(CS)
ν(M-O)
ν(M-N)
ν(M-S-C=N)
Others
K[Cu(SB-1)(SCN)]
1605
750
535
415
340
ν(C=N) = 2100
Cu(SB-2)NN C18H17O2CuN3
32903150
537
295
ν(C=N) = 1555
Cu(SB-2)NN C19H17O2CuN3
33153232
535
285Compounds B and C. The free Schiff base ligand shows characteristic bands at 3530 cm−1 for ν(OH) and 1607 cm−1 for ν(C=N). In the complexes, ν(C=N) of the Schiff base ligand remains practically unchanged showing that the imino nitrogen does not participate in bonding. The IR spectrum of free 2-phenylenediamine shows ν(NH2) modes at 3400 and 3378 cm−1, respectively. These bands are also shifted to lower frequencies in the complexes (B, C) (3315, 3232 cm−1) indicating coordination by the amino nitrogen but appear at 3290 cm−1 and 3150 cm−1 in compound B. This is also evident from the appearance of bands at 285–310 cm−1 which are tentatively attributed to the ν(M-N) mode. Further, in complexes with 2-aminopyridine (B), the ν(C=N) mode appears at 1555 cm−1 indicating that the ring nitrogen is coordinated to the metal atom.


### 3.3. Magnetic Moment and Electronic Spectra

The effective magnetic moments and electronic spectral components are shown in Tables1 and 3. All the complexes are paramagnetic and show magnetic moment between 1.90 and 2.00 B.M corresponding to one unpaired electron. In electronic spectra three bands were observed at around 15455, 19500, and 22172 cm−1 corresponding to the transitions, B21g to A21g, B21g to E2g, and charge transfer, respectively. These bands are consistent with square planar geometry [19].Table 3
Electronic spectral data of the complexes (band maxima in cm−1).
Compound
Band I
Band II
Band III
K[Cu(SB-1)(SCN)]
15,267
19,157
22,000
Cu(SB-2)NN C18H17O2CuN3
15,395
19,500
24,000
Cu(SB-2)NN C19H17O2CuN3
15,400
19,890
23,995


## 3.1. Elemental Analysis and Conductivity Measurement

The analytical data and physical properties of the synthesized complexes are given in Table1. The molar conductances in DMSO indicate that the complexes are all 1 : 1 electrolytes [13, 14].Table 1
Analytical dataa and physical properties.
Complex notation
Complex
Colour
Melting point (°C)
% M
% C
% H
% N
Molar conductance (Ohm1cm2mol−1)
Magnetic momentMeff (B.M)
A
K[Cu(SB-1)(SCN)]
Yellowish
175–177
45.22 (45.18)
2.42 (2.39)
7.53 (7.50)
29
1.95
B
[Cu(SB-2)NN]NN = aminopyridine
Greenish
228–230
17.14 (17.52)
58.27 (58.60)
4.05 (4.50)
11.33 (11.10)
35
1.90
C
[Cu(SB-2)NN]NN = phenylenediamine
Greenish
160–162
16.52 (16.05)
59.27 (58.89)
4.42 (4.10)
10.92 (10.50)
42
2.0
aThe values found are given in parenthesis.


## 3.2. IR Studies

Complex A. The infrared spectral data are shown in Table 2. The Schiff base C13H9NO2H2 [SB-1] behaves as tridentate dinegative ligand coordinating at the imino nitrogen and two oxygen atoms. In the complexes, the shift of ν(C=N) mode in a frequency 1605 cm−1 relative to the free ligand value 1610–1620 cm−1 (for ligand C13H9NO2) indicates that bond formation takes place through the imino nitrogen atom. The ν(OH) band observed in the free Schiff base (SB-1) disappears upon coordination, which indicates deprotonation and coordination at the oxygen sites. Furthermore, the presence of ν(M-O) and ν(M-N) linkages of bands at 455–535 cm−1, respectively, was observed for all the complexes (A) [15–17]. The ambidentate thiocyanate ligand can give either M-NCS or M-SCN bonding sequence, which nevertheless reveals the acidity of the metal ions. The complexes also display ν(CN) at 2100 cm−1 characteristic of S-bonded thiocyanato moieties. In Pearson’s terminology, these are soft acids. The ν(CS) modes appear at lower frequencies in the M-S-C=N complexes than those in the M-N-C=S complexes [8, 9, 18]. The band of ν(CS) at 750 cm−1 is characteristic of M-S-C=N bonding sequence. This is further apparent from the ν(M-S) modes at 350 cm−1 in the far infrared spectra of the complexes.Table 2
Selected IR spectral data of the complexes (band maxima, cm−1).
Compound
ν(C=N)
ν(NH2)
ν(CS)
ν(M-O)
ν(M-N)
ν(M-S-C=N)
Others
K[Cu(SB-1)(SCN)]
1605
750
535
415
340
ν(C=N) = 2100
Cu(SB-2)NN C18H17O2CuN3
32903150
537
295
ν(C=N) = 1555
Cu(SB-2)NN C19H17O2CuN3
33153232
535
285Compounds B and C. The free Schiff base ligand shows characteristic bands at 3530 cm−1 for ν(OH) and 1607 cm−1 for ν(C=N). In the complexes, ν(C=N) of the Schiff base ligand remains practically unchanged showing that the imino nitrogen does not participate in bonding. The IR spectrum of free 2-phenylenediamine shows ν(NH2) modes at 3400 and 3378 cm−1, respectively. These bands are also shifted to lower frequencies in the complexes (B, C) (3315, 3232 cm−1) indicating coordination by the amino nitrogen but appear at 3290 cm−1 and 3150 cm−1 in compound B. This is also evident from the appearance of bands at 285–310 cm−1 which are tentatively attributed to the ν(M-N) mode. Further, in complexes with 2-aminopyridine (B), the ν(C=N) mode appears at 1555 cm−1 indicating that the ring nitrogen is coordinated to the metal atom.


## 3.3. Magnetic Moment and Electronic Spectra

The effective magnetic moments and electronic spectral components are shown in Tables1 and 3. All the complexes are paramagnetic and show magnetic moment between 1.90 and 2.00 B.M corresponding to one unpaired electron. In electronic spectra three bands were observed at around 15455, 19500, and 22172 cm−1 corresponding to the transitions, B21g to A21g, B21g to E2g, and charge transfer, respectively. These bands are consistent with square planar geometry [19].Table 3
Electronic spectral data of the complexes (band maxima in cm−1).
Compound
Band I
Band II
Band III
K[Cu(SB-1)(SCN)]
15,267
19,157
22,000
Cu(SB-2)NN C18H17O2CuN3
15,395
19,500
24,000
Cu(SB-2)NN C19H17O2CuN3
15,400
19,890
23,995


## 4. Antineoplastic Activity of the Test Compounds

### 4.1. The Effect of Test Compounds and Bleomycin on Ehrlich Ascites Carcinoma (EAC) Cell Growth Inhibition

Treatment with test compounds resulting in maximum cell growth inhibition on compounds A, B, and C as evident from 95.21%, 87.40%, and 89.49%, reduction of tumour cell, which was found to be equivalent to standard or nearly standard antitumour agent bleomycin, which shows that cell growth inhibition is 94.90%. The results are shown in Table4.Table 4
The effect of test compounds and bleomycin on EAC cell growth inhibition.
Experiment
Drugs
Dose
Number of EAC cells/mouse on 5th day after tumour (EAC) cell inoculation
% of cell growth inhibition
EAC
(9.61 ± 1.63) × 107
EAC + bleomycin
0.3 mg/kg
(0.49 ± 0.77) × 107
94.90
A
Synthetic
8 mg/kg
(0.46 ± 0.62) × 107
95.21
B
Synthetic
10 mg/kg
(1.21 ± 0.32) × 107
87.40
C
Synthetic
16 mg/kg
(1.01 ± 0.78) × 107
89.49
A = K[Cu(SB-1)(SCN)], B = [Cu(SB-2)NN], NN = 2-aminopyridine, and C = [Cu(SB-2)(NN)], NN = 2-phenylenediamine.


### 4.2. Effect of Test Compounds on Survival Time of EAC Cell Bearing Mice

It was found that treatment of tumour induced test animals with the compounds A, B, and C resulting in the increase of life span 35.70%, 16.98%, and 22.32%, respectively, when compared to control mice (life span 21.37 days). It was noticed that the anticancer antibiotic bleomycin increased the life span by 87.3% when compared to control. The results are shown in Tables5 and 6.Table 5
The effect of test compounds on survival time of EAC cell bearing mice.
Name of experiment
Drugs
Dose
Mean survival time
%  of increase life span
Control (EAC bearing mice)
(21.37  ±  1.7)
EAC + bleomycin
0.3 mg/kg
40  ±  1.2
87.17
A
Synthetic
8 mg/kg
29  ±  2.6
35.70
B
Synthetic
10 mg/kg
25  ±  1.4
16.98
C
Synthetic
16 mg/kg
26.14  ±  1.1
22.32
A = K[Cu(SB-1)(SCN)], B = [Cu(SB-2)NN], NN = 2-aminopyridine, and C = [Cu(SB-2)(NN)], NN = 2-phenylenediamine.Table 6
The effect of test compounds on survival time of EAC cell bearing mice.
Days
Control (EAC)
Bleomycin(0.3 mg/kg i.p.)
DMSO25 mg/kg
A8 mg/kg
B10 mg/kg
C16 mg/kg
0
00
00
00
00
00
00
2
0.77 ± 0.37
0.57 ± 0.17
0.76 ± 0.38
0.55 ± 0.13
0.64 ± 0.19
0.60 ± 0.26
4
0.98 ± 0.43
0.75 ± 0.22
0.97 ± 0.63
0.72 ± 0.22
0.84 ± 0.24
0.82 ± 0.17
6
1.30 ± 0.27
1.10 ± 0.24
1.31 ± 0.33
1.06 ± 0.24
1.21 ± 0.16
1.16 ± 0.31
8
1.54 ± 0.32
1.24 ± 0.14
1.57 ± 0.23
1.14 ± 0.12
1.37 ± 0.16
1.31 ± 0.14
10
1.78 ± 0.18
1.44 ± 0.30
1.76 ± 0.16
1.29 ± 0.19
1.48 ± 0.26
1.43 ± 0.22
12
2.13 ± 0.17
1.63 ± 0.16
2.14 ± 0.17
1.47 ± 0.22
1.77 ± 0.18
1.71 ± 0.14
14
2.55 ± 0.67
1.80 ± 0.23
2.55 ± 0.63
1.73 ± 0.32
1.93 ± 0.34
1.84 ± 0.18
16
3.94 ± 0.55
2.05 ± 0.27
3.93 ± 0.53
1.94 ± 0.14
2.17 ± 0.12
2.10 ± 0.21
18
4.44 ± 0.43
2.20 ± 0.15
4.40 ± 0.42
2.13 ± 0.12
2.47 ± 0.15
2.29 ± 0.23
20
5.13 ± 0.63
2.43  ± 0.11
5.14  ± 0.62
2.36 ± 0.16
2.73
2.49 ± 0.44
A = K[Cu(SB-1)(SCN)], B = [Cu(SB-2)NN], NN = 2-aminopyridine, and C = [Cu(SB-2)(NN)], NN = 2-phenylenediamine.


### 4.3. Effect of Test Compounds and Bleomycin on Average Tumour Weight

Treatment of the test animals with test compounds, previously incubated with EAC cells, resulted in the inhibition of tumour growth. In case of treated group the body weight was growing slowly and by 53.99%, 46.78%, and 51.46% less in compounds A, B, and C, respectively, compared to control group after 20 days of tumour inoculation. But in case of bleomycin, this value is 52.63% compared to control group. DMSO does not show any change of body weight compared to control group.


### 4.4. The Effect of Test Compounds on Hematological Parameters in Normal and Tumour Bearing Mice

Hematological parameters were studied in normal and tumour bearing mice. All were treated with test compounds for 12 days of tumour transplantation and after 12 days they were sacrificed and blood was collected for hematological examination. Number of mice were four. Results were shown in mean ± SEM and compared with normal (without EAC bearing mice) and control (EAC bearing mice) group as shown in Table7. The growth of tumour in mice induced by EAC cells effect in acute anemic condition as indicated by the significant decrease of the Hb% when compared to normal test animals under similar condition on day 12. The total white blood cell (WBC) count was also markedly decreased in the control group. In differential count of WBC, lymphocyte count was also found to be decreased and neutrophil count was increased on day 12 of tumour inoculation but no significant changes were observed in monocyte count on day 12 of the tumour inoculation as compared with normal mice. Effect of test compounds on hematological parameters of normal mice was also determined. No significant effect was found.Table 7
The effect of test compounds on hematological parameters in normal and tumour bearing mice.
Name of experiment
HB levelg/dL
RBCcell/mL
WBC (TC)cell/mL
Lymphocyte %
Neutrophil %
Monocyte %
Normal mice
13.65 ± 0.4
(7.96 ± 0.57) ×  109
(6.35 ± 0.26)  × 106
71.25 ± 0.91
19.19 ± 0.28
8.93 ± 0.23
N + A
12.25 ± 0.17
(6.16 ± 0.16) ×  109
(10.76 ± 0.17)  × 106
75.00 ± 0.83
21.79 ± 0.38
3.10 ± 0.34
N + B
10.23 ± 0.13
(5.93 ± 0.22) ×  109
(16 ± 0.23)  × 106
72.10 ± 0.34
26.23  ± 0.24
1.73 ± 0.17
N + C
11.89 ± 0.26
(6.33 ± 0.19) ×  109
(14 ± 0.23)  × 106
73.47 ± 0.83
24.21 ± 0.16
2 ± 0.21
EAC (mice)
7.11 ± 0.23
(2.40 ± 0.10) ×  109
(25.63 ± 0.18)  × 106
43.36 ± 0.43
38.23 ± 0.37
10 ± 0.28
EAC + A
11.25 ± 0.23
(6.04 ± 0.21) ×  109
(9.19 ± 0.36)  × 106
60.25 ± 0.68
26.65 ± 0.18
8 ± 0.86
EAC + B
10.14 ± 0.18
(5.62 ± 0.27) ×  109
(14.82 ± 0.41)  × 106
51.87 ± 0.78
34.11 ± 0.22
6.12 ± 0.92
EAC + C
10.34 ± 0.10
(5.89 ± 0.29) ×  109
(12.67 ±  1.40)  × 106
57.68 ± 1.37
33.20 ± 2.8
8.10 ± 3.10
A = K[Cu(SB-1)(SCN)], B = [Cu(SB-2)NN], NN = 2-aminopyridine, and C = [Cu(SB-2)(NN)], NN = 2-phenylenediamine.


### 4.5. The Effect of Test Compounds on Serum Alkaline Phosphate Activity

Serum alkaline phosphatase activities were studied in normal and tumour bearing mice. Tumour bearing mice were treated with test compounds for 5 days of tumour transplantation and after 5 days they were sacrificed and blood was collected for determination of serum phosphatase. Serum alkaline phosphatase activity level in tumour bearing was decreased due to tumorigenesis when compared to the normal. Treatment with the test compounds restores the enzyme activity towards normal significantly. The serum alkaline phosphatase activity of the test compounds is shown in Table8.Table 8
The effect of test compounds on serum alkaline phosphatase activity.
Name of experiment
ALKP activity (µmol of PNPP hydrolyzed/min/mL serum)
Name of experiment
ALKP activity (µmol of PNPP hydrolyzed/min/mL serum)
EAC
(8.56 ± 0.31)  ×  10−3
Normal value
(28.33 ± 0.71)  ×  10−3
EAC + DMSO
(28.96 ± 0.21)  ×  10−3
DMSO
(29.13 ± 0.14)  ×  10−3
EAC + A
(22.11 ± 0.46)  ×  10−3
N + A
(38.33 ± 0.27)  ×  10−3
EAC + B
(16.93 ± 0.49)  ×  10−3
N + B
(35.11 ± 0.45)  ×  10−3
EAC + C
(19.88 ± 0.88)  ×  10−3
N + C
(32.79 ± 0.67)  ×  10−3


### 4.6. Effect of Test Compounds on the Enhancement of Peritoneal Cells and Macrophages of Life

Treatment with the test compounds did not show any effect on the enhancement of number of peritoneal cells but the number of macrophages increased (Table9).Table 9
The effect of test compounds on the enhancement of peritoneal cells and macrophages of life.
Name of the experiment
Dosemg/kg
Total peritoneal macrophage cells(×106)/mouseMean ± SEM
Total peritoneal cells(×106)/mouseMean ± SEM
Normal
Tap water
3.23 ± 2.0
23.76 ± 0.321
A
8
5.23 ± 0.40
25.42 ± 0.66
B
10
4.10 ± 0.62
25.93 ± 0.19
C
16
4.91 ± 0.52
26.71 ± 0.44
A = K[Cu(SB-1)(SCN)], B = [Cu(SB-2)NN], NN = 2-aminopyridine, and C = [Cu(SB-2)(NN)], NN = 2-phenylenediamine.


### 4.7. The Effect of Test Compounds on Generation of MDA by Lipid Peroxidation in Serum of Normal Mice

Animals were treated with test compounds for 4 consecutive days. Sera from mice were collected on day 5 and malondialdehyde (MDA) concentration was measured. The dose of the test compounds A, B, and C was 8 mg/kg, 108 mg/kg, and 168 mg/kg, respectively. Effect of test compounds on normal mice showed that there was markedly increase in MDA, which indicated that there was release for free radical. The obtained data are shown in Table10.Table 10
The effect of test compounds on generation of MDA by lipid peroxidation in serum of normal mice.
Name of the experiment
mmol MDA/mL serum
Normal mice (control)
6.71  ±  0.32
A
11.43  ±  0.67
B
10.11  ±  0.20
C
10.94  ±  0.39
A = K[Cu(SB-1)(SCN)], B = [Cu(SB-2)NN], NN = 2-aminopyridine, and C = [Cu(SB-2)(NN)], NN = 2-phenylenediamine.


### 4.8. Histopathological Effect of Test Compounds

Ehrlich Ascites Carcinoma (EAC) cell inducing tumour at the site of injection was very prominent and showed fast growth, increased in size, and bulged out in skin. The histological feature shows necrosis at the centre and viable growing cells in the periphery. Some inflammatory reactions lymphocytic in nature with reduction of hair follicle were observed. The number of mitosis was observed which increases greatly. When treated with test compounds and bleomycin growth rate of tumour is reduced, inflammatory reaction has also increased to some extent (Table11). Necrotic area is increased and hair follicles show their normal appearance.Table 11
Histopathological effect of test compounds.
Name of the experiment
Observation
Size
Number of lymphocytes
Necrotic area
Inflammatory area
EAC
↑↑↑
↓↓↓
A
↓↓↓
↑↑↑
↑↑↑
↑↑↑
B
↓
↑
↑
↑
C
↓↓
↑↑
↑↑
↑↑
Bleomycin
↓↓↓
↑↑↑
↑↑↑
↑↑↑
A = K[Cu(SB-1)(SCN)], B = [Cu(SB-2)NN], NN = 2-aminopyridine, and C = [Cu(SB-2)(NN)], NN = 2-phenylenediamine.
↑ = increase, ↑↑ = moderately increase, and ↑↑↑ = greatly increase.
↓ = decrease, ↓↓  = moderately decrease, and ↓↓↓ = greatly decrease.


### 4.9. Effect of Test Compounds on Total Protein in Peritoneal Fluid

Inoculation of Ehrlich Ascites Carcinoma (EAC) cell in peritoneal cavity causes accumulation of fluid, which is rich in protein. But when treated with test compounds, the protein present in the peritoneal fluid is reduced and fluids accumulate in the peritoneal cavity very slowly (Table12).Table 12
Effect of test compounds on total protein in peritoneal fluid.
Name of the complex
Dose (mg/kg)
Total proteinmg/mL, P.F., mean ±  SEM
Control (EAC)
Tap water
174.73 ± 2.81
A
8
165.22 ± 2.93
B
10
167.33 ± 3.23
C
10
166.97 ± 3.20
A = K[Cu(SB-1)(SCN)], B = [Cu(SB-2)NN], NN = 2-aminopyridine, and C = [Cu(SB-2)(NN)], NN = 2-phenylenediamine.


## 4.1. The Effect of Test Compounds and Bleomycin on Ehrlich Ascites Carcinoma (EAC) Cell Growth Inhibition

Treatment with test compounds resulting in maximum cell growth inhibition on compounds A, B, and C as evident from 95.21%, 87.40%, and 89.49%, reduction of tumour cell, which was found to be equivalent to standard or nearly standard antitumour agent bleomycin, which shows that cell growth inhibition is 94.90%. The results are shown in Table4.Table 4
The effect of test compounds and bleomycin on EAC cell growth inhibition.
Experiment
Drugs
Dose
Number of EAC cells/mouse on 5th day after tumour (EAC) cell inoculation
% of cell growth inhibition
EAC
(9.61 ± 1.63) × 107
EAC + bleomycin
0.3 mg/kg
(0.49 ± 0.77) × 107
94.90
A
Synthetic
8 mg/kg
(0.46 ± 0.62) × 107
95.21
B
Synthetic
10 mg/kg
(1.21 ± 0.32) × 107
87.40
C
Synthetic
16 mg/kg
(1.01 ± 0.78) × 107
89.49
A = K[Cu(SB-1)(SCN)], B = [Cu(SB-2)NN], NN = 2-aminopyridine, and C = [Cu(SB-2)(NN)], NN = 2-phenylenediamine.


## 4.2. Effect of Test Compounds on Survival Time of EAC Cell Bearing Mice

It was found that treatment of tumour induced test animals with the compounds A, B, and C resulting in the increase of life span 35.70%, 16.98%, and 22.32%, respectively, when compared to control mice (life span 21.37 days). It was noticed that the anticancer antibiotic bleomycin increased the life span by 87.3% when compared to control. The results are shown in Tables5 and 6.Table 5
The effect of test compounds on survival time of EAC cell bearing mice.
Name of experiment
Drugs
Dose
Mean survival time
%  of increase life span
Control (EAC bearing mice)
(21.37  ±  1.7)
EAC + bleomycin
0.3 mg/kg
40  ±  1.2
87.17
A
Synthetic
8 mg/kg
29  ±  2.6
35.70
B
Synthetic
10 mg/kg
25  ±  1.4
16.98
C
Synthetic
16 mg/kg
26.14  ±  1.1
22.32
A = K[Cu(SB-1)(SCN)], B = [Cu(SB-2)NN], NN = 2-aminopyridine, and C = [Cu(SB-2)(NN)], NN = 2-phenylenediamine.Table 6
The effect of test compounds on survival time of EAC cell bearing mice.
Days
Control (EAC)
Bleomycin(0.3 mg/kg i.p.)
DMSO25 mg/kg
A8 mg/kg
B10 mg/kg
C16 mg/kg
0
00
00
00
00
00
00
2
0.77 ± 0.37
0.57 ± 0.17
0.76 ± 0.38
0.55 ± 0.13
0.64 ± 0.19
0.60 ± 0.26
4
0.98 ± 0.43
0.75 ± 0.22
0.97 ± 0.63
0.72 ± 0.22
0.84 ± 0.24
0.82 ± 0.17
6
1.30 ± 0.27
1.10 ± 0.24
1.31 ± 0.33
1.06 ± 0.24
1.21 ± 0.16
1.16 ± 0.31
8
1.54 ± 0.32
1.24 ± 0.14
1.57 ± 0.23
1.14 ± 0.12
1.37 ± 0.16
1.31 ± 0.14
10
1.78 ± 0.18
1.44 ± 0.30
1.76 ± 0.16
1.29 ± 0.19
1.48 ± 0.26
1.43 ± 0.22
12
2.13 ± 0.17
1.63 ± 0.16
2.14 ± 0.17
1.47 ± 0.22
1.77 ± 0.18
1.71 ± 0.14
14
2.55 ± 0.67
1.80 ± 0.23
2.55 ± 0.63
1.73 ± 0.32
1.93 ± 0.34
1.84 ± 0.18
16
3.94 ± 0.55
2.05 ± 0.27
3.93 ± 0.53
1.94 ± 0.14
2.17 ± 0.12
2.10 ± 0.21
18
4.44 ± 0.43
2.20 ± 0.15
4.40 ± 0.42
2.13 ± 0.12
2.47 ± 0.15
2.29 ± 0.23
20
5.13 ± 0.63
2.43  ± 0.11
5.14  ± 0.62
2.36 ± 0.16
2.73
2.49 ± 0.44
A = K[Cu(SB-1)(SCN)], B = [Cu(SB-2)NN], NN = 2-aminopyridine, and C = [Cu(SB-2)(NN)], NN = 2-phenylenediamine.


## 4.3. Effect of Test Compounds and Bleomycin on Average Tumour Weight

Treatment of the test animals with test compounds, previously incubated with EAC cells, resulted in the inhibition of tumour growth. In case of treated group the body weight was growing slowly and by 53.99%, 46.78%, and 51.46% less in compounds A, B, and C, respectively, compared to control group after 20 days of tumour inoculation. But in case of bleomycin, this value is 52.63% compared to control group. DMSO does not show any change of body weight compared to control group.


## 4.4. The Effect of Test Compounds on Hematological Parameters in Normal and Tumour Bearing Mice

Hematological parameters were studied in normal and tumour bearing mice. All were treated with test compounds for 12 days of tumour transplantation and after 12 days they were sacrificed and blood was collected for hematological examination. Number of mice were four. Results were shown in mean ± SEM and compared with normal (without EAC bearing mice) and control (EAC bearing mice) group as shown in Table7. The growth of tumour in mice induced by EAC cells effect in acute anemic condition as indicated by the significant decrease of the Hb% when compared to normal test animals under similar condition on day 12. The total white blood cell (WBC) count was also markedly decreased in the control group. In differential count of WBC, lymphocyte count was also found to be decreased and neutrophil count was increased on day 12 of tumour inoculation but no significant changes were observed in monocyte count on day 12 of the tumour inoculation as compared with normal mice. Effect of test compounds on hematological parameters of normal mice was also determined. No significant effect was found.Table 7
The effect of test compounds on hematological parameters in normal and tumour bearing mice.
Name of experiment
HB levelg/dL
RBCcell/mL
WBC (TC)cell/mL
Lymphocyte %
Neutrophil %
Monocyte %
Normal mice
13.65 ± 0.4
(7.96 ± 0.57) ×  109
(6.35 ± 0.26)  × 106
71.25 ± 0.91
19.19 ± 0.28
8.93 ± 0.23
N + A
12.25 ± 0.17
(6.16 ± 0.16) ×  109
(10.76 ± 0.17)  × 106
75.00 ± 0.83
21.79 ± 0.38
3.10 ± 0.34
N + B
10.23 ± 0.13
(5.93 ± 0.22) ×  109
(16 ± 0.23)  × 106
72.10 ± 0.34
26.23  ± 0.24
1.73 ± 0.17
N + C
11.89 ± 0.26
(6.33 ± 0.19) ×  109
(14 ± 0.23)  × 106
73.47 ± 0.83
24.21 ± 0.16
2 ± 0.21
EAC (mice)
7.11 ± 0.23
(2.40 ± 0.10) ×  109
(25.63 ± 0.18)  × 106
43.36 ± 0.43
38.23 ± 0.37
10 ± 0.28
EAC + A
11.25 ± 0.23
(6.04 ± 0.21) ×  109
(9.19 ± 0.36)  × 106
60.25 ± 0.68
26.65 ± 0.18
8 ± 0.86
EAC + B
10.14 ± 0.18
(5.62 ± 0.27) ×  109
(14.82 ± 0.41)  × 106
51.87 ± 0.78
34.11 ± 0.22
6.12 ± 0.92
EAC + C
10.34 ± 0.10
(5.89 ± 0.29) ×  109
(12.67 ±  1.40)  × 106
57.68 ± 1.37
33.20 ± 2.8
8.10 ± 3.10
A = K[Cu(SB-1)(SCN)], B = [Cu(SB-2)NN], NN = 2-aminopyridine, and C = [Cu(SB-2)(NN)], NN = 2-phenylenediamine.


## 4.5. The Effect of Test Compounds on Serum Alkaline Phosphate Activity

Serum alkaline phosphatase activities were studied in normal and tumour bearing mice. Tumour bearing mice were treated with test compounds for 5 days of tumour transplantation and after 5 days they were sacrificed and blood was collected for determination of serum phosphatase. Serum alkaline phosphatase activity level in tumour bearing was decreased due to tumorigenesis when compared to the normal. Treatment with the test compounds restores the enzyme activity towards normal significantly. The serum alkaline phosphatase activity of the test compounds is shown in Table8.Table 8
The effect of test compounds on serum alkaline phosphatase activity.
Name of experiment
ALKP activity (µmol of PNPP hydrolyzed/min/mL serum)
Name of experiment
ALKP activity (µmol of PNPP hydrolyzed/min/mL serum)
EAC
(8.56 ± 0.31)  ×  10−3
Normal value
(28.33 ± 0.71)  ×  10−3
EAC + DMSO
(28.96 ± 0.21)  ×  10−3
DMSO
(29.13 ± 0.14)  ×  10−3
EAC + A
(22.11 ± 0.46)  ×  10−3
N + A
(38.33 ± 0.27)  ×  10−3
EAC + B
(16.93 ± 0.49)  ×  10−3
N + B
(35.11 ± 0.45)  ×  10−3
EAC + C
(19.88 ± 0.88)  ×  10−3
N + C
(32.79 ± 0.67)  ×  10−3


## 4.6. Effect of Test Compounds on the Enhancement of Peritoneal Cells and Macrophages of Life

Treatment with the test compounds did not show any effect on the enhancement of number of peritoneal cells but the number of macrophages increased (Table9).Table 9
The effect of test compounds on the enhancement of peritoneal cells and macrophages of life.
Name of the experiment
Dosemg/kg
Total peritoneal macrophage cells(×106)/mouseMean ± SEM
Total peritoneal cells(×106)/mouseMean ± SEM
Normal
Tap water
3.23 ± 2.0
23.76 ± 0.321
A
8
5.23 ± 0.40
25.42 ± 0.66
B
10
4.10 ± 0.62
25.93 ± 0.19
C
16
4.91 ± 0.52
26.71 ± 0.44
A = K[Cu(SB-1)(SCN)], B = [Cu(SB-2)NN], NN = 2-aminopyridine, and C = [Cu(SB-2)(NN)], NN = 2-phenylenediamine.


## 4.7. The Effect of Test Compounds on Generation of MDA by Lipid Peroxidation in Serum of Normal Mice

Animals were treated with test compounds for 4 consecutive days. Sera from mice were collected on day 5 and malondialdehyde (MDA) concentration was measured. The dose of the test compounds A, B, and C was 8 mg/kg, 108 mg/kg, and 168 mg/kg, respectively. Effect of test compounds on normal mice showed that there was markedly increase in MDA, which indicated that there was release for free radical. The obtained data are shown in Table10.Table 10
The effect of test compounds on generation of MDA by lipid peroxidation in serum of normal mice.
Name of the experiment
mmol MDA/mL serum
Normal mice (control)
6.71  ±  0.32
A
11.43  ±  0.67
B
10.11  ±  0.20
C
10.94  ±  0.39
A = K[Cu(SB-1)(SCN)], B = [Cu(SB-2)NN], NN = 2-aminopyridine, and C = [Cu(SB-2)(NN)], NN = 2-phenylenediamine.


## 4.8. Histopathological Effect of Test Compounds

Ehrlich Ascites Carcinoma (EAC) cell inducing tumour at the site of injection was very prominent and showed fast growth, increased in size, and bulged out in skin. The histological feature shows necrosis at the centre and viable growing cells in the periphery. Some inflammatory reactions lymphocytic in nature with reduction of hair follicle were observed. The number of mitosis was observed which increases greatly. When treated with test compounds and bleomycin growth rate of tumour is reduced, inflammatory reaction has also increased to some extent (Table11). Necrotic area is increased and hair follicles show their normal appearance.Table 11
Histopathological effect of test compounds.
Name of the experiment
Observation
Size
Number of lymphocytes
Necrotic area
Inflammatory area
EAC
↑↑↑
↓↓↓
A
↓↓↓
↑↑↑
↑↑↑
↑↑↑
B
↓
↑
↑
↑
C
↓↓
↑↑
↑↑
↑↑
Bleomycin
↓↓↓
↑↑↑
↑↑↑
↑↑↑
A = K[Cu(SB-1)(SCN)], B = [Cu(SB-2)NN], NN = 2-aminopyridine, and C = [Cu(SB-2)(NN)], NN = 2-phenylenediamine.
↑ = increase, ↑↑ = moderately increase, and ↑↑↑ = greatly increase.
↓ = decrease, ↓↓  = moderately decrease, and ↓↓↓ = greatly decrease.


## 4.9. Effect of Test Compounds on Total Protein in Peritoneal Fluid

Inoculation of Ehrlich Ascites Carcinoma (EAC) cell in peritoneal cavity causes accumulation of fluid, which is rich in protein. But when treated with test compounds, the protein present in the peritoneal fluid is reduced and fluids accumulate in the peritoneal cavity very slowly (Table12).Table 12
Effect of test compounds on total protein in peritoneal fluid.
Name of the complex
Dose (mg/kg)
Total proteinmg/mL, P.F., mean ±  SEM
Control (EAC)
Tap water
174.73 ± 2.81
A
8
165.22 ± 2.93
B
10
167.33 ± 3.23
C
10
166.97 ± 3.20
A = K[Cu(SB-1)(SCN)], B = [Cu(SB-2)NN], NN = 2-aminopyridine, and C = [Cu(SB-2)(NN)], NN = 2-phenylenediamine.


## 5. Antifungal Activity of the Test Compounds

### 5.1. Zone of Inhibition of Antifungal Activity of Test Complexes

The antifungal activity of the test complexes against different fungi was investigated by using the doses of 80μg/disc, where standard antibiotic disc of Nystatin (45 μg/disc) was used for comparison purpose. The diameter was evaluated 4 mm, 2 mm, and 3 mm againsttinea pedis; 8 mm, 23 mm, and 10 mm againstAspergillus niger; 22 mm, 6 mm, and 8 mm againstConiothyrium sp., respectively, for test complexes A, B, and C whereas diameter of zone of inhibition of Nystatin was found to be 18 mm, 28 mm, and 20 mm, respectively, against the organism. The antifungal activity (zone of inhibition) of the test complexes against respective fungi is presented in Table 13. The minimum inhibitory concentration (MIC) of the test complex A is 64 μg/disc; for B and C it is 128 μg/disc as listed in Table 14.Table 13
Zone of inhibition of antifungal activity of test complexes.
Test fungus
Diameter of zone of inhibition (mm) of test complexes
Nystatin(45 µg/disc)
A
B
C
Tinea pedis
4
2
3
18
Aspergillus niger
8
23
10
28
Coniothyrium sp.
22
6
8
20
A = K[Cu(SB-1)(SCN)], B = [Cu(SB-2)NN], NN = 2-aminopyridine, and C = [Cu(SB-2)(NN)], NN = 2-phenylenediamine.Table 14
The summary of the results of minimum inhibitory concentration (MIC) of test complexes.
Name of fungus
MIC of test complex (µg/mL)
A
B
C
Aspergillus niger
64
128
128
Coniothyrium sp.
64
128
128
Tinea pedis
80
160
160
A = K[Cu(SB-1)(SCN)], B = [Cu(SB-2)NN], NN = 2-aminopyridine, and C = [Cu(SB-2)(NN)], NN = 2-phenylenediamine.


## 5.1. Zone of Inhibition of Antifungal Activity of Test Complexes

The antifungal activity of the test complexes against different fungi was investigated by using the doses of 80μg/disc, where standard antibiotic disc of Nystatin (45 μg/disc) was used for comparison purpose. The diameter was evaluated 4 mm, 2 mm, and 3 mm againsttinea pedis; 8 mm, 23 mm, and 10 mm againstAspergillus niger; 22 mm, 6 mm, and 8 mm againstConiothyrium sp., respectively, for test complexes A, B, and C whereas diameter of zone of inhibition of Nystatin was found to be 18 mm, 28 mm, and 20 mm, respectively, against the organism. The antifungal activity (zone of inhibition) of the test complexes against respective fungi is presented in Table 13. The minimum inhibitory concentration (MIC) of the test complex A is 64 μg/disc; for B and C it is 128 μg/disc as listed in Table 14.Table 13
Zone of inhibition of antifungal activity of test complexes.
Test fungus
Diameter of zone of inhibition (mm) of test complexes
Nystatin(45 µg/disc)
A
B
C
Tinea pedis
4
2
3
18
Aspergillus niger
8
23
10
28
Coniothyrium sp.
22
6
8
20
A = K[Cu(SB-1)(SCN)], B = [Cu(SB-2)NN], NN = 2-aminopyridine, and C = [Cu(SB-2)(NN)], NN = 2-phenylenediamine.Table 14
The summary of the results of minimum inhibitory concentration (MIC) of test complexes.
Name of fungus
MIC of test complex (µg/mL)
A
B
C
Aspergillus niger
64
128
128
Coniothyrium sp.
64
128
128
Tinea pedis
80
160
160
A = K[Cu(SB-1)(SCN)], B = [Cu(SB-2)NN], NN = 2-aminopyridine, and C = [Cu(SB-2)(NN)], NN = 2-phenylenediamine.


## 6. Antibacterial Activity of the Test Complexes

### 6.1. The Result of Antibacterial Activity of Test Compounds

The antibacterial activity of the test complexes was determined by using the dose of 80μg/disc. The results of antibacterial activity measured in terms of zone of inhibition are shown in Table 15. The complexes showed minimum sensitivity against the following number of both Gram-positive and Gram-negative bacteria and the results were compared with antibiotic disc of kanamycin.Table 15
Result of antibacterial activity of test compounds.
Test bacteria
Diameter of zone of inhibition (mm) of test complexes
Kanamycin (Ts/25µg/disc)
A
B
C
Bacillus subtilis
7
5
4
16
Streptococcus-β-haemolytica
13
9
7
25
Escherichia coli
9
11
7
17
Sarcina lutea
8
9
7
19
Klebsiella
11
14
7
17
Shigella flexneri
6
11
6
14
Shigella boydii
9
26
8
24
Shigella dysenteriae
12
10
9
14
Pseudomonas aeruginosa
12
7
10
13
A = K[Cu(SB-1)(SCN)], B = [Cu(SB-2)NN], NN = 2-aminopyridine, and C = [Cu(SB-2)(NN)], NN = 2-phenylenediamine.The histopathological investigation on the tumour showed a retardation of tumour growth, increase in the narcotic and inflammatory area, and increased hair follicles. The Schiff complexes showed significant antimicrobial activity compared to control. We definitely say that the synthesized complexes possess cytotoxic properties. Toxicological studies revealed that the complexes are much more toxic to liver and kidney. They altered all biochemical parameters of rat blood. The exact mode of action of the complexes is unknown to us. Further investigation is appreciated to investigate detailed mechanism of action and their effect in serum electrolyte before any clinical use, especially for the effective doses.


## 6.1. The Result of Antibacterial Activity of Test Compounds

The antibacterial activity of the test complexes was determined by using the dose of 80μg/disc. The results of antibacterial activity measured in terms of zone of inhibition are shown in Table 15. The complexes showed minimum sensitivity against the following number of both Gram-positive and Gram-negative bacteria and the results were compared with antibiotic disc of kanamycin.Table 15
Result of antibacterial activity of test compounds.
Test bacteria
Diameter of zone of inhibition (mm) of test complexes
Kanamycin (Ts/25µg/disc)
A
B
C
Bacillus subtilis
7
5
4
16
Streptococcus-β-haemolytica
13
9
7
25
Escherichia coli
9
11
7
17
Sarcina lutea
8
9
7
19
Klebsiella
11
14
7
17
Shigella flexneri
6
11
6
14
Shigella boydii
9
26
8
24
Shigella dysenteriae
12
10
9
14
Pseudomonas aeruginosa
12
7
10
13
A = K[Cu(SB-1)(SCN)], B = [Cu(SB-2)NN], NN = 2-aminopyridine, and C = [Cu(SB-2)(NN)], NN = 2-phenylenediamine.The histopathological investigation on the tumour showed a retardation of tumour growth, increase in the narcotic and inflammatory area, and increased hair follicles. The Schiff complexes showed significant antimicrobial activity compared to control. We definitely say that the synthesized complexes possess cytotoxic properties. Toxicological studies revealed that the complexes are much more toxic to liver and kidney. They altered all biochemical parameters of rat blood. The exact mode of action of the complexes is unknown to us. Further investigation is appreciated to investigate detailed mechanism of action and their effect in serum electrolyte before any clinical use, especially for the effective doses.



---

*Source: 923087-2015-07-29.xml*