Schiff bases (SBs) are chemical compounds displaying a significant pharmacological potential. They are able to modulate the activity of many enzymes involved in metabolism and are found among antibacterial, antifungal, anti-inflammatory, antioxidant, and antiproliferative drugs. A new thiazolyl-triazole SB was obtained and characterized by elemental and spectral analysis. The antibacterial and antifungal ability of the SB was evaluated against Gram-positive and Gram-negative bacteria and against three
Aerobic organisms have antioxidant defense systems against reactive oxygen species- (ROS-) induced damage produced in various stress conditions. ROS are also involved in the innate immune system and have an important role in the inflammatory response; they attract cells, by chemotaxis, to the inflammation site. Nitric oxide (NO) is another important intracellular and intercellular signaling molecule involved in the regulation of multiple physiological and pathophysiological mechanisms. It acts as a biological modulator. NO is able to regulate vascular tone and can function as a host defense effector. Also, it can act as a cytotoxic agent in inflammatory disorders. NO synthase (NOS) enzyme family catalyzes NO production. Inhibition of inducible NOS (iNOS) might be beneficial in the course of treatment of certain inflammatory diseases [
Recent studies showed that exogenous NO, produced by bacterial NOS, protects Gram-positive and Gram-negative bacteria (
Schiff bases (SBs) are chemical structures that have a significant pharmacological potential. SBs contain an azomethine group obtained through the condensation of primary amines with carbonyl compounds [
Multiple studies showed the ability of SBs to act as antiproliferative and antitumoral agents [
The current study is aimed at testing a newly synthetized heterocyclic SB in terms of antimicrobial activity against Gram-positive and Gram-negative bacteria and antifungal effects against
All reagents and solvents used were purchased from Sigma-Aldrich and were used without further purification. The starting compound was previously reported and was synthesized by us according to methodologies described in the literature [
The synthesis of Schiff base (SB) 4-(3-bromobenzylideneamino)-5-(4-methyl-2-phenylthiazol-5-yl)-4H-1,2,4-triazole-3-thiol was made using a general procedure (Scheme
Synthesis of the Schiff base (SB).
SB was dissolved in DMSO, at a final concentration of 100
Antimicrobial activity was tested
Minimum inhibitory concentrations (MICs), minimum bactericidal concentrations (MBCs), and minimum fungicidal concentrations (MFCs) were determined by an agar dilution method. Strains of microorganisms used were as follows:
The DPPH antioxidant activity assay was done as previously described, with minor modification. SB was dissolved in DMSO (1 mg/mL). DPPH∙ radical was dissolved in methanol (0.25 mM). Equal volumes (1.0 mL) of methanolic DPPH solution and sample solution (or standard) in methanol at different concentrations have been used. The mixtures were incubated for 30 min at 40°C in a thermostatic bath; absorbance was measured at 517 nm. The percent DPPH scavenging ability was calculated as follows:
Human umbilical vein endothelial cells (HUVECs, Promocell, Hamburg, Germany) were used. The cells were grown in RPMI medium supplemented with 5% fetal calf serum, 50
Cells cultured at a density of 104/well on ELISA 96-well plaques (TPP, Switzerland) were settled for 24 hours, then exposed to different concentrations of the substance ranging from 0.001 to 200
Four groups were made: (1) control cells treated only with medium, (2) cells exposed to a high-glucose (4.5 g/L) medium, (3) cells treated with SB 0.001
The cell lysates used in the following experiments were prepared as previously described [
Quantification of malondialdehyde (MDA) a marker for the peroxidation of membrane lipids was performed by spectrophotometry, as previously described [
TNF-
Lysates (20
Phalloidin-FITC 50
The statistical significance of the differences between the control group and the treated groups was assessed with the nonparametric Kruskal-Wallis test for multiple groups, followed by a post hoc analysis using the Conover test. Correlation coefficients between parameters have been calculated using Spearman’s correlation coefficient for ranks (rho). Statistical tests were performed using MedCalc version 18.11.3 and GraphPad Prism Software version 8.0.2. The results were considered statistical significant at
The SB structure was confirmed by elemental analysis and on the basis of its mass spectrum (MS), infrared spectrum (IR), and nuclear magnetic resonance (1H NMR and 13C NMR) spectra [
Results obtained by measuring the diameters of growth inhibition zones of the tested microorganisms, compared to ciprofloxacin and fluconazole, used as standard reference drugs, are presented in Table
Inhibition zone diameters on tested microorganisms.
Samples | Diameter of the inhibition zone (mm) | ||||
---|---|---|---|---|---|
SA | LM | EC | ST | CA | |
14 | 14 | 14 | 18 | 18 | |
Ciprofloxacin | 28 | 18 | 27 | 22 | — |
Fluconazole | — | — | — | — | 25 |
SA:
MIC, MBC, and MFC values of the new compound are presented in Tables
Minimum inhibitory concentrations (MIC).
Samples | Minimum inhibitory concentration (MIC ( | |||||
---|---|---|---|---|---|---|
SA | LM | PA | ST | CA (ATCC 10231) | CA (ATCC 18804) | |
31.25 | 1.95 | 1.95 | 62.5 | 62.5 | 31.25 | |
Ciprofloxacin | 1.95 | 3.9 | 3.9 | 0.97 | — | — |
Fluconazole | — | — | — | — | 62.5 | 62.5 |
SA:
Minimum bactericidal (MBC) and minimum fungicidal concentrations (MFC).
Samples | MBC ( |
MFC ( | |||||
---|---|---|---|---|---|---|---|
SA | LM | PA | ST | CA (ATCC 10231) | CA (ATCC 18804) | CK (ATCC 6258) | |
62.5 | 3.9 | 3.9 | 125 | 125 | 62.5 | 62.5 | |
Ciprofloxacin | 3.9 | 7.8 | 7.8 | 1.95 | — | — | — |
Fluconazole | — | — | — | — | 125 | 125 | 125 |
SA:
The antioxidant capacity of the SB was determined by the DPPH bleaching method, and BHT and trolox were used as positive controls. The results are displayed in Table
Antioxidant capacity using the DPPH method.
Samples | IC50 ( |
---|---|
BHT | |
Trolox |
SB did not lead to significant changes in HUVEC viability for doses lower than 0.1
Cell viability testing. Schiff base (SB) was tested for multiple concentrations (0.01-200
Lipid peroxidation level (MDA), the ability to modulate inflammatory response (TNF-
The effect of the newly synthetized compound on lipid peroxidation (MDA level) was assessed. SB administration decreased the MDA level compared with both control and glucose-enriched medium, thus reducing the lipid peroxidation in endothelial cells (Figure
Lipid peroxidation levels (MDA) in endothelial cells exposed to medium (control), glucose (G), Schiff base (SB), and combination treatment (SB+G). Each bar represents the
The TNF-
TNF-
The same SB concentration (0.001
An inflammatory marker (COX2) and antioxidant enzyme (constitutive SOD1 and inducible NOS2) expression was quantified by Western Blot (Figure
Protein levels of COX2, SOD1, and NOS2 in endothelial cells exposed to medium (control), glucose (G), Schiff base (SB), and combination treatment (SB+G). Comparative Western blot images showing expressions of COX2, SOD1, and NOS2 in HUVECs (b, c, d). Image analysis of Western blot bands (a) was performed by densitometry; results were normalized to GAPDH. Each bar represents the
COX2, an inflammatory marker, significantly decreased after both glucose and SB treatments, compared to control. Combined exposure (SB+G) strongly decreased the protein level of COX2 (Figure
Correlation analysis, using Spearman’s coefficient for rank correlation (Table
Spearman’s coefficient of rank correlation (rho) between the oxidative stress and inflammation markers in HUVECs.
MDA | TNF- |
COX2 | SOD1 | NOS2 | |
---|---|---|---|---|---|
MDA | 1.00 | -0.776 |
0.699 |
0.462 | 0.595 |
TNF- | 1.00 | -0.818 |
-0.566 | -0.455 | |
COX2 | 1.00 | 0.755 |
0.431 | ||
SOD1 | 1.00 | -0.144 | |||
NOS2 | 1.00 |
Cell morphology does not seem to be affected by exposure to the Schiff base compared to control. When exposed to high-glucose concentration, cells had a tendency to conglomerate and to form multilayered spherical bodies, with alteration of the actin filament disposition. The aspect of the cells receiving combination treatment was similar to those of controls (Figure
Images of HUVECs treated with medium (control), glucose (G), Schiff base (SB), and combination treatment (SB+G), stained with phalloidin-FITC; the same microscopic field is presented as phase contrast images (a) and fluorescence images (b) for comparison (pictures taken through an Olympus BX inverted microscope, original magnification 20x).
The structure of the Schiff base was established by elemental analysis and on the basis of its mass spectrum (MS), infrared spectrum (IR), and nuclear magnetic resonance (1H-NMR and 13C-NMR) spectra. The results of the C, H, N, S quantitative elemental analysis were in agreement with the calculated values, within ±0.4% of the theoretical values. The spectral data confirmed the formation of the SB. The recorded mass spectrum revealed the correct molecular ion peak (
The aim of the present study was to evaluate the antibacterial and antifungal activity of a new SB as well as its ability to modulate oxidative stress.
The new thiazolyl SB exerted moderate to good antibacterial activity against tested strains (Tables
High-carbohydrate intake, impaired glucose tolerance, and diabetes mellitus lead to hyperglycemia and chronic inflammatory status. Endothelial lesions are often involved in the pathology of these conditions [
A glucose-enriched environment also triggers the release of proinflammatory cytokines, such as tumor necrosis factor alpha (TNF-
The results obtained by the DPPH study showed that the SB exhibited antioxidant activity. The low IC50 value, similar to the positive control (BHT), reflects a strong antioxidant activity
The ability of the SB, in low concentrations, to decrease lipid peroxidation, might be explained by its capacity to form complexes with the bivalent and trivalent metal ions located in the active center of the enzymes involved in the onset of the oxidative stress or in the scavenging of the prooxidant molecules [
Considering antibacterial activity, especially against
Nitric oxide synthase (NOS) inhibitor NO-donating drugs were reported to inhibit IL-1
Recent studies showed that the antibacterial and antifungal activity in general and antibiofilm activity of some newly identified classes seem to correlate with their ability to induce ROS synthesis [
Additional studies are needed in order to clarify the effect of such compounds as SB and their role as adjuvant antioxidant, antimicrobial, and local immune response modulators (TNF-
The new Schiff base exhibited antibacterial effects on both Gram-positive and Gram-negative bacteria, as well as antifungal activity against
The data used to support the findings of this study are included within the article.
The authors declare that there is no conflict of interest regarding the publication of this paper.
Cristian Cezar Login, Şoimiţa Suciu, Ioana Bâldea, and Brînduşa Tiperciuc conceived and planned the experimental design. Brînduşa Tiperciuc performed the chemical synthesis and the characterization of the compounds. Dan Cristian Vodnar performed the antibacterial and antifungal investigation. Daniela Benedec performed the
This research was funded by “Iuliu Hațieganu” University of Medicine and Pharmacy Cluj-Napoca internal research grant No. 4944/23/08.03.2016 (Cristian Cezar Login).