Schiff bases are known to possess anticancer, antibacterial, antifungal, antitubercular, anti-inflammatory, antimicrobial and antimalarial properties. In this paper antibacterial studies against variety of plants and human pathogenic bacteria with eight newly synthesized Schiff bases and several intermediate silyl compounds have been reported. The antibacterial activities of the synthesized compounds were primarily determined by paper disc diffusion method. The minimum inhibitory concentration (MIC) of each compound was also determined by tube dilution process. Seven different human pathogenic bacteria and eighteen different plant pathogenic bacteria were used for the antibacterial activity studies. While all synthesized compounds have shown significant antibacterial activity, one intermediate silyl compound has shown remarkably high antibacterial property. 5-substituted derivatives have shown relatively higher activity than non-substituted compounds. Polar substituent which increases hydrophilicity may have a positive impact on the antibacterial property.
Schiff base, named after Hugo Schiff, is another name of iminie functional groups or azomethine groups (-C=N) that are classically formed by condensation of a primary amine with an aldehyde or ketone [1] . Since various Schiff bases and their complexes have been formed and exhibited promising activity against bacteria and other microorganism, biochemists are interested to study Schiff bases for their medical importance and use in design of medicinal compounds [2] [3] . Many Schiff bases have antimicrobial and antifungal activities [4] . Additionally, aryl groups or heterocyclic residues containing Schiff bases possess excellent biological activities [5] [6] .
Phenanthroline and its derivatives have significantly antibacterial, antifungal and anticancer activity [7] . Phenanthroline metal complex interacts with DNA by cleavage of the DNA strand involving interaction of the metal center with a phosphate group and cleavage of the phosphate ester bond [8] [9] . Also, some phenanthroline Schiff bases and their complexes are found to be photocleavers of pUC19 DNA in visible light and cytotoxic in HeLa (human cervical cancer) and MCF-7 (human breast cancer) cells in visible light [10] . Phenanthroline complexes exhibited high cytotoxicity in cancer cell lines, even sometimes higher activity than cis-platin and substitution at 5- or 5,6-position increases cytotoxicity such as the S, S isomer of complex is 100-fold more cytotoxic than of cis-platin in L2110 murine leukemia cell lines [11] .
Schiff bases of thiosemicarbazone are very important due to their antimicrobial action [12] . Also, Schiff bases and their metal complexes having amino thiol group (HN2-NH-CS-R) show various biological activities. Thiosemicarbazone containing compounds can be used as drug against tuberculosis, leprosy [13] and tumor [14] .
The biological study of Schiff bases containing 5-substituted-1,10-phenanthroline-2,9-dicarboxaldehyde with amines containing thione (C=S) groups has not been studied precisely. We, therefore, report here the antibacterial study of eight new Schiff bases containing 5-subntituted-1,10-phenanthroline moiety with different thiosemicarbazones. The intermediate silyl and aldehyde compounds are also studied against these bacteria.
2. Materials and Methods
Preparation of test microorganism
The sterilized (autoclaved for 30 min) medium was poured into sterilized petri dish (500 ml for 20 petri dishes) and solidified (1.5%, w/v agar) at room temperature for 30 min. 18 different types of plant pathogenic and 7 human pathogenic bacterial strains were used in this study of both gram positive and gram negative bacteria. A small amount of bacteria was collected from bacteria seed and streaked it onto an appropriate media plate for the bacteria. The plate was placed in a incubator for 24 h at 28˚C for plant pathogenic bacteria and at 37˚C for human pathogenic bacteria.
2.1. Preparation of Reagents
Fifteen compounds including eight new Schiff bases, four intermediates and one starting molecule were used for antibacterial screening test by using disk diffusion method (Table 1). The eight new Schiff bases were synthesized and characterized by using different spectroscopic methods including IR, 1H NMR, 13C NMR and Mass spectroscopy [15] . The concentration of the test compounds was 20 mg/ml. The concentration for standard, tetracycline (TC), was 10 mg/ml for screening test. Test compounds were dissolved in dimethylsulphoxide (DMSO). The name of the compounds and their structure are given in Table 1.
2-3 bacteria colonies were taken in appropriate liquid media and shook it in an incubator shaker for 5 h at 28˚C for plant pathogenic bacteria and at 37˚C for human pathogenic bacteria. The spinning speed maintained at 180 ppm. The bacteria solution was then diluted four times and spread 0.15 ml of this fresh pure culture on media plate with a help of spreader under aseptic condition. Sterile 6 mm whatman filter paper was used which was impregnated with 10 µl of sterile stock solution (20 mg/ml) of the compound. The filter paper with solution was
Names of the compounds that are used in antibacterial study
placed in the middle of the bacteria spread media plate. The plates were incubated at appropriate temperature for 24 h. The diameter of the inhibition zone observed was measured in mm scale. Disc impregnated with sterile DMSO was used as a control and with sterile tetracycline (TC) was used as antibacterial reference standard. All tests were performed under sterile conditions in duplicate and repeated three times.
2.3. Determination of Minimum Inhibitory Concentration (MIC)
Minimum Inhibitory Concentration (MIC) indicates the lowest concentration of an antimicrobial agent that inhibits the visible growth of a microorganism after overnight incubation. To determine MIC, different variable concentrations of the compounds were used. Media plate and bacteria culture were prepared the same way that were done before in the disk diffusion method. After spreading about 0.15 ml diluted bacteria on the media plate, seven holes were punched on the plate for seven different solutions. 25 µL of each solution (Tc, T0, T1, T2, T3, T4, T5) was put in each hole according to the concentration and kept it in incubator at appropriate temperature for 1 day. The diameter of the inhibition zone was measured in mm scale and recorded the data. All the tests were repeated three times.
3. Results and Discussion3.1. Antibacterial Activity Screening Test
The antibacterial study is based upon a comparison of inhibition of growth of bacteria by test compounds with that produced by standard antibiotic, Tetracycline (TC). Inhibition zone is the clear region around the paper disc saturated with an antimicrobial agent. The screening result showed that all the compounds exhibited noticeable antibacterial activity against different bacteria. The following Tables show the name of the compounds and antibacterial activity according to the inhibition zones in mm scale.
The antibacterial activity of compounds against 7 human pathogenic bacteria is presented in Table 2. The bar charts represent a comparison of the activities of 5-substituted Schiff bases with similar Schiff bases without the substituents. All compounds found to possess antibacterial activity against all human pathogenic bacteria. In fact, compounds showed higher activity for human pathogenic bacteria than plant pathogenic bacteria. Also, it is observed very high activity for silyl derivative, N (Figure 1 and Figure 4). The silyl derivative showed higher activity than tetracycline (TC) in case of Staphylococcus aures, Streptococcus pyogenes, Bacillus subtilus and Listeria monocytogenes. Figure 2 and Figure 3 show graphical representations of antibacterial activity against human pathogenic bacteria. These bacterias are E. Coli MC4100, E. Coli DH5a, Staphylococcus aures, Streptococcus pyogenes, Yersinia enterocoitica 27729, Listeria monocytogenes and Bacillus subtilus.
The antibacterial activity of compounds against 18 plant pathogenic bacteria is presented in Table 3 and Figures 4-6. While all compounds exhibited significant activity, the silyl derivative, N has higher activity in case of Pectobacterium atosepticum SCR1043, Serratia odorifera 33077, Xanthomonous campestris pv. Pelargonii Xcp42, Xanthomonous campestris pv. Pelargonii Xcp58 and Pantoea sterwartii DC28.
Nine different bacteria of Pseudomonas genre were tested. The result is presented in Table 4. In this case, nitro and bromo aldehyde (A and F) showed higher activity against Pseudomonus Cichorii 302959, Pseudomonas syringae 728α and Ps.Pv. Morspruorum 218-01 (Figures 7-9).
Antibacterial activity of the compounds against human pathogenic bacteria
Compound
Zone of Inhibition (mm)
E. Coli MC4100
E. Coli DH5α
Staphylococcus aureus
Streptococcus pyogenes
Yersinia enterocolitica 27729
Listeria monocytogenes
Bacillus subtilus
Medium
LB
LB
TSB
TSB
LB
LB
TSB
Std. (TC)
24
24
29
30
40
33
37
A
15
14
15
16
15
18
27
B
7
9
8
8
11
9
11
C
9
10
01
9
11
9
12
D
8
10
8
10
11
12
11
E
7
9
7
8
11
9
11
F
10
10
14
15
12
16
29
G
7
9
7
10
10
--
8
H
8
9
8
11
10
8
8
I
7
10
8
11
11
11
11
J
8
9
9
11
10
16
10
K
10
9
15
15
13
30
29
L
10
10
9
10
9
22
14
M
10
10
12
12
11
14
11
N
9
10
21
32
10
29
58
O
8
8
10
12
10
21
14
Antibacterial activity of nine plant pathogenic bacteria
Compound
Zone of Inhibition (mm)
Pectobacterium carotovorum Eec7
Dickeya dadantii 3937
Pectobacterium atosepticum SCR 1043
Serratia odorifera 33077
Pantoea sterwartii DC283
Xanthomonous campestris pv. Pelargonii Xcp42
Xanthomonous campestris pv. Pelargonii Xcp58
Pseudomonas corrugata 301678
Pseudomonas syringae tomatto DC3000
Medium
LB
LB
LB
LB
LB
NY
NY
NY
KB
Std. (TC)
29
33
28
23
39
41
40
21
31
A
15
10
16
15
19
19
17
14
11
B
11
10
10
8
10
10
10
7
10
C
11
11
11
10
12
10
11
9
10
D
11
14
12
8
10
10
10
10
12
E
10
11
12
8
10
9
10
8
14
F
12
11
11
9
14
16
16
10
12
G
10
11
10
10
10
10
11
7
10
H
11
10
11
8
11
9
10
9
9
I
12
10
11
7
10
11
15
11
11
J
10
10
10
10
12
11
10
9
10
K
10
10
10
8
15
12
10
10
9
L
11
9
10
9
13
11
11
10
10
M
10
10
10
7
11
8
14
9
8
N
12
13
15
11
65
35
38
11
12
O
11
10
9
12
14
12
12
9
10
Antibacterial screening test of nine bacteria of Pseudomonas genre
Compound
Zone of Inhibition (mm)
P. syringae morspruorum302756
P. cichorii 302959
P. aeruginosa PAO1
P. Savastanoi
P. viridiflava
P. cichorii 302699
P. syringae FF5
P. syringae 728α
Ps.Pv. Morspruorum 218-01
Medium
KB
KB
KB
KB
KB
KB
KB
KB
KB
Std. (TC)
32
27
10
40
25
29
27
26
25
A
13
50
12
15
13
12
11
13
15
B
7
7
7
10
7
7
7
8
7
C
7
7
7
10
9
8
10
8
8
D
7
7
7
10
7
9
11
7
8
E
7
--
7
10
--
7
7
--
11
F
13
37
10
12
11
10
12
24
19
G
7
7
7
9
8
8
8
7
8
H
7
7
8
19
--
7
7
9
7
I
7
7
7
10
7
8
8
8
11
J
8
7
8
10
7
7
8
7
10
K
--
--
7
9
7
8
7
7
--
L
9
8
7
9
8
8
7
8
8
M
8
8
7
9
8
8
8
8
8
N
9
9
--
9
9
7
11
11
12
O
7
8
7
9
7
8
9
7
9
Photos of the antibacterial test of compound N and standard
Antibacterial activity test against four human pathogenic bacteria
Antibacterial activity test against three human pathogenic bacteria
Some real pictures of the high activity of silyl methyl phenanthroline compound, N against Pantoea sterwartii DC283, Xanthomonous campestris pv. Pelargonii Xcp42, Xanthomonous campestris pv. Pelargonii Xcp58.
Antibacterial activity test against four plant pathogenic bacteria
Antibacterial activity test against five plant pathogenic bacteria
Photo of antibacterial test of bromo, F and nitro aldehyde, A against Pseudomonas cichorii
Antibacterial activity test against four plant pathogenic bacteria
Antibacterial activity test against five plant pathogenic bacteria
3.2. Minimum Inhibitory Concentration (MIC)
Minimum inhibitory concentration (MIC) is the minimum concentration of the test compound that inhibits the visual growth of the microorganism. MIC was determined for those compounds that showed higher activity on some bacteria. 5 Different concentrated solutions of the compounds were used to test MIC starting from 100 μg/ml to 5 mg/ml. In all cases, the MIC of silyl methyl phenanthroline, N (Figure 10 and Figure 11) was found to be 100 μg/ml toward Streptococcus pyogenes, Listeria monocytogenes, Bacillus subtilus, Xanthomonous campestris pv. Pelargonii Xcp42, Xanthomonous campestris pv. Pelargonii Xcp58, Pantoea sterwartii DC283 (Table 5). Also, for nitro aldehyde, A, 100 μg/ml was the MIC for Streptococcus pyogenes, Listeria monocytogenes, Xanthomonous campestris pv. Pelargonii Xcp42, Xanthomonous campestris pv. Pelargonii Xcp58, E. coli MC4100, Pseudomonas syringae Morspruorum 302756 and 200 μg/ml was the MIC for Bacillus subtilus (Figure 12 and Figure 13). All the results are shown in Table 6 and Table 7. Moreover, 100 μg/ml was the MIC for bromo aldehyde, F against Streptococcus pyogenes, Bacillus subtilus, Listeria monocytogenes, Xanthomonous campestris pv. Pelargonii Xcp42, Xanthomonous campestris pv. Pelargonii Xcp58, Pseudomonus Cichorii 302959 (Figure 14 and Figure 15). Table 8 and Figure 16 show summary of results.
Pictures of minimum inhibitory concentration (MIC) test for the Compound N
MIC of Silyl compound, N against different bacteria
MIC of Nitro-aldehyde, A against five bacteria
MIC of Nitro-aldehyde, A against six bacteria
MIC of Bromo-aldehyde, F against four bacteria
MIC of Bromo-aldehyde, F against four bacteria
Overall summery of MIC test
Minimum inhibitory concentration (MIC) of Silyl methyl Phenanthroline compound, N
Name of Bacteria
Zone of Inhibition (mm) of Silyl Methyl Phenanthroline, N
5 mg/ml
2 mg/ml
500 μg/ml
200 μg/ml
100 μg/ml
Std. TC (5 mg/ml)
Streptococcus pyogenes
17.17 ± 0.62
13.17 ± 0.24
8.83 ± 0.24
6.83 ± 0.23
5.83 ± 0.24
34.83 ± 0.23
Listeria monocytogenes
26.67 ± 0.94
22.33 ± 1.25
15.67 ± 1.7
11.17 ± 1.18
8.67 ± 0.47
29 ± 2.16
Bacillus subtilus
21.33 ± 0.47
17.33 ± 0.47
13.17 ± 0.24
10.5 ± 0.41
8.67 ± 0.24
30.33 ± 0.47
Xanthomonous campestris pv. pelargonii Xcp42
10.17 ± 0.24
9.17 ± 0.24
8.17 ± 0.24
7.17 ± 0.24
6.33 ± 0.24
38.67 ± 1.25
Xanthomonous campestris pv. pelargonii Xcp58
31.67 ± 0.47
22 ± 0.82
14.5 ± 0.41
10.17 ± 0.24
6.83 ± 0.24
40.33 ± 0.47
Pantoea sterwartii DC283
8.17 ± 0.24
7.17 ± 0.24
6.17 ± 0.24
0 ± 0
0 ± 0
34.67 ± 0.94
Minimum inhibitory concentration (MIC) of nitro aldehyde, A compound
Name of Bacteria
Zone of Inhibition (mm) of Nitro Aldehyde, A
5 mg/ml
2 mg/ml
500 μg/ml
200 μg/ml
100 μg/ml
Std. TC (5 mg/ml)
Streptococcus pyogenes
18.67 ± 0.47
14.83 ± 0.24
9.67 ± 0.47
7.67 ± 0.24
7 ± 0.41
37 ± 0.82
Listeria monocytogenes
14.33 ± 0.47
10.33 ± 0.62
8 ± 0.41
7 ± 0
6.83 ± 0.24
30 ± 0.82
Bacillus subtilus
11.33 ± 0.62
8.67 ± 0.24
6.5 ± 0
5.67 ± 0.24
0 ± 0
28.33 ± 0.47
Xanthomonous campestris pv. Pelargonii Xcp42
13.5 ± 0.41
11 ± 0.41
8.17 ± 0.62
7 ± 0
6.33 ± 0.47
40.33 ± 0.47
Xanthomonous campestris pv. Pelargonii Xcp58
12.83 ± 0.24
10 ± 0
7.83 ± 0.24
6.33 ± 0.24
6 ± 0
39.33 ± 0.94
Pantoea sterwartii DC283
16 ± 0.41
12.17 ± 0.24
7.67 ± 0.47
0 ± 0
0 ± 0
36.67 ± 1.25
E. coli MC4100
10.33 ± 0.24
8.17 ± 0.62
6.67 ± 0.24
6 ± 0
0 ± 0
24 ± 0.82
Pseudomonas corrugata 301678
9.67 ± 0.24
7.17 ± 0.24
0 ± 0
0 ± 0
0 ± 0
23.67 ± 0.47
Pseudomonus cichorii 302959
26.33 ± 2.62
17.67 ± 2.05
0 ± 0
0 ± 0
0 ± 0
34 ± 0.82
Pseudomonas syringae morspruorum 302756
14.67 ± 0.94
12.17 ± 0.24
9 ± 0
8 ± 0.41
7 ± 0
27.33 ± 0.94
Minimum inhibitory concentration (MIC) of bromo aldehyde, F compound
Name of Bacteria
Zone of Inhibition (mm) of Bromo Aldehyde, F
5 mg/ml
2 mg/ml
500 μg/ml
200 μg/ml
100 μg/ml
Std. TC (5 mg/ml)
Streptococcus pyogenes
15.17 ± 0.24
13 ± 0.41
10.5 ± 0.41
8.83 ± 0.24
8.5 ± 0.41
37 ± 1.63
Listeria monocytogenes
12.17 ± 0.62
9.83 ± 0.24
8.5 ± 0
7.33 ± 0.24
6.83 ± 0.24
29 ± 0.82
Bacillus subtilus
11.5 ± 0.41
9.33 ± 0.47
7.5 ± 0.41
6.83 ± 0.24
6.67 ± 0.24
29 ± 0
Xanthomonous campestris pv. pelargonii Xcp42
14.17 ± 0.24
12 ± 0.41
10.17 ± 0.24
8.5 ± 0.71
7.5 ± 0.41
40.67 ± 0.47
Xanthomonous campestris pv. pelargonii Xcp58
13.83 ± 0.24
12.67 ± 0.47
10.5 ± 0.41
8.17 ± 0.62
6.83 ± 0.24
41 ± 0.82
Pantoea sterwartii DC283
9.33 ± 0.85
7.5 ± 0.41
6.67 ± 0.47
0 ± 0
0 ± 0
36 ± 0.82
Pseudomonus cichorii 302959
9.17 ± 0.62
7.5 ± 1.08
6.5 ± 0.41
6.17 ± 0.24
5.83 ± 0.24
33.33 ± 0.47
Pseudomonas syringae morspruorum 302756
9.33 ± 0.62
6.33 ± 0.24
5.5 ± 0.41
0 ± 0
0 ± 0
26.33 ± 1.7
Overall summary of MIC
Name of the Bacteria
Minimum Inhibitory Concentration (µg/mL)
N
A
F
Streptococcus pyogenes
100
100
100
Listeria monocytogenes
100
100
100
Bacillus subtilus
100
200
100
Xanthomonous campestris pv. pelargonii Xcp42
100
100
100
Xanthomonous campestris pv. pelargonii Xcp58
100
100
100
Pantoea sterwartii DC283
500
500
500
E. coli MC4100
--
100
--
Pseudomonas corrugata 301678
--
2000
--
Pseudomonus cichorii 302959
--
2000
100
Pseudomonas syringae morspruorum 302756
--
100
500
4. Conclusion
Antibacterial studies of 15 different compounds including intermediate silyl and aldehyde compounds were carried out. These compounds were tested on 18 plants and 7 human pathogenic bacteria. The antibacterial activity screening test of the synthesized compounds was initially examined by paper disc diffusion technique. In screening test, the compounds showed higher antibacterial activity for human pathogenic bacteria. High activity was observed for silyl derivative as well. Compound N was even more active than the standard tetracycline (TC). Moreover, it has been found that the activity of the 5-substituted Schiff bases has shown higher activity than their non- substituted members. Finally, the minimum inhibitory concentrations (MIC) of the compounds were determined by tube dilution method. For most of the compounds MIC was 100 μg/ml.
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