2.1 Chemicals

ABTS (2,2′-azinobis-3-ethylbenzothiazoline-6-sulfonic acid), DPPH, p-nitrophenyl-a-d-glucopyranoside, α-amylase and α-glucosidase were purchased from Sigma–Aldrich Chemical Corporation, St. Louis, Mo, USA. DNS (dinitro salicylic acid) was obtained from Himedia chemicals, India, and all other chemicals were of analytical grade.

2.2 Synthesis and characterization of bis(3-acetyl-5-methyl-pyran-2,4-dione)ethylenediimine Zn(II) complex complex (ZnL)

The Schiff base ligand, bis(3-Acetyl-5-methyl-pyran-2,4-dione)ethylenediimine (H₂L) and its Zinc complex (ZnL) were synthesized according to the procedure of Ponnam deshmukg et al., 2010 with slight modification. H₂L was prepared from the condensation of dehydroacetic acid and ethylene diamine in ethanol medium at 70 °C. The Zn(II) Schiff base complex (ZnL) was synthesized from the ligand (Fig. 1). Color: Pale yellow; Yield: 62%; Melting point: 163 °C.

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Figure 1 Structure of N₂O₂ Schiff base Zn complex.

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In the next step, the ligand (1.1232 g, 4 mmol) was dissolved in ethanol (20 ml) by heating at 80 °C. The obtained solution was mixed with ethanol containing zinc chloride dehydrate drop by drop with continuous stirring and the resulting reaction mixture was further refluxed for 5 h. On cooling the reaction mixture, a solid product gets settled. The product was filtered, washed with absolute ethanol followed by petroleum ether and dried in vacuum. Color: Brown; Yield: 74%; Melting point: 226 °C.

2.3 Antioxidant activity

2.4 Antibacterial analysis

Antibacterial activity was performed according to the method of Threlfall et al. (1999) and Walker et al. (2000) using 10 g peptone, 10 g NaCl and 5 g yeast, 20 g agar in 1000 ml of distilled water. The stock cultures of bacteria were revived by inoculating in broth media and grown at 37 °C for 18 h. Agar plates were prepared and wells were made in the plate. Each plate was inoculated with 18 h old cultures (100 μl, 10⁻⁴ cfu) and spread evenly on the plate. After 20 min, the wells were filled with compound of different concentrations. The control wells with gentamicin were also prepared. The plates were incubated at 37 °C for 24 h and all the tests were performed in triplicate and the average was taken as final reading. The inhibition zone diameters were measured in millimeters.

2.5 α-Amylase inhibition assay

α-Amylase inhibitory activity was determined by the method described by the Worthington Enzyme Manual (Worthington Biochemical Corp., 1993a) with slight modification. A total of 500 μl of ZnL complex and 500 μl of 0.02 M sodium phosphate buffer (pH 6.9 with 0.006 M NaCl) containing α-amylase solution (1.0 U/ml) was incubated at 25 °C for 10 min. After pre incubation, 500 μl of 1% starch solution was added to 0.02 M sodium phosphate buffer (pH 6.9 with 0.006 M NaCl). The reaction mixture was then incubated at 25 °C for 10 min. The reaction was stopped by adding 1.0 ml of DNS color reagent. The test tubes were then incubated in a boiling water bath for 5 min and cooled to room temperature. The reaction mixture was then diluted by adding 10 ml of distilled water, and absorbance was measured at 540 nm. The α-amylase inhibitory activity was calculated according to the equation given below: $$\text{Inhibition}(\%)=(A_{\text{control}}-A_{\text{sample}})/A_{\text{control}}\times 100$$ where A_control was the absorbance of the control (without ZnL complex); A_sample was the absorbance in the presence of ZnL complex.

2.6 α-Glucosidase inhibition assay

A modified version of the assay described by the Worthington Enzyme Manual was followed (Worthington Biochemical Corp., 1993b). A volume of 500 μl of ZnL complex was diluted with 100 μl of 0.1 M potassium phosphate buffer (pH 6.9) containing α-glucosidase solution (1.0 U/ml) and was incubated in 96-well plates at 25 °C for 10 min. After pre incubation, 50 μl of 5 mM p-nitrophenyl-α-D-glucopyranoside solution in 0.1 M potassium phosphate buffer (pH 6.9) was added to each well at timed intervals. The reaction mixtures were incubated at 25 °C for 5 min. The α-glucosidase inhibitory activity was expressed as percentage of inhibition and was calculated as follows: $$\text{Inhibition}(\%)=(A_{\text{control}}-A_{\text{sample}})/A_{\text{control}}\times 100$$ where A_control was the absorbance of the control (without ZnL complex); A_sample was the absorbance in the presence of ZnL complex.

2.7 Kinetics of inhibition

The mode of inhibition of α-amylase and α-glucosidase by ZnL complex was determined by using Dixon and Lineweaver–Burk plots. Starch in the concentration range 0.5–2%, and p-nitrophenyl-α-d-glucopyranoside in the range 0.5–2 mM, were used as substrates for α-amylase and α-glucosidase respectively. The inhibitions of enzyme activities are determined in the presence and absence of ZnL complex at different concentrations for α-amylase (50–200 μg/ml) and α-glucosidase (20–80 μg/ml). The values of kinetic parameters (K_m and K_i) were determined according to the type of inhibition for each enzyme reaction with ZnL complex by both LB and Dixon plots (Bowden, 1974; Dixon, 1953).

2.8 Statistical analysis

Analysis at every time point from each experiment was carried out in triplicates. The results were statistically analyzed by ANOVA. Statistical significance was accepted at a level of p < 0.05.

3.1 Synthesis and characterization

IR spectrum of ligand (H₂L) exhibits characteristic peaks at 3282, 2818, 1638, and 1616 cm⁻¹ which correspond to phenolic (–O–H) stretching, C–H stretching, carbonyl (C⚌O) stretching and azomethine (C⚌N) stretching vibration frequencies, respectively. Phenolic (O-H) stretching disappeared in the IR spectrum of complexes which indicates the binding of ligand with ‘Zn’ ion via deprotonation. The IR spectrum of ZnL exhibits C⚌O stretching at 1633 cm⁻¹, almost similar to that of ligand spectrum. This shows that the carbonyl group remains free and does not involve in any new bonding. Whereas the azomethine frequency undergoes a red shift (1607 cm⁻¹) in complexes. This clearly shows the binding of azomethine nitrogen with Zn ion. Thus, the IR spectra of H₂L and ZnL clearly exhibit the binding of ligand with metal through two azomethine nitrogens and two phenolic oxygens. UV–Vis spectrum was recorded for the complex ZnL, which exhibits three peaks at wavelengths (λ_max) 224, 286 and 424 nm. Bands at 224 and 286 nm were attributed to intra ligand electronic transitions. Peaks at 424 nm can be due to M-L charge transfer.

¹H-NMR spectra of H₂L and ZnL further confirm the binding of ligand with Zn ion. Phenolic OH proton exhibits a peak at the chemical shift of 11.4 ppm. This peak disappear in the ¹HNMR spectra of ZnL confirms the deprotonation due to the binding of phenolic oxygen with the metal group. The peaks corresponding to free methyl groups have been observed at similar region (2.1–2.5 ppm) in both the spectrum of ligand and complex. Methylene (–CH₂–) protons and ring (C–H) protons exhibit their peaks around 2.8 and 3.6 ppm, receptively. The spectral results confirm that H₂L acts as a binegative quadridentate ligand in nature.

3.2 Hydroxyl radical scavenging activity

In the present study the ZnL complex exhibited a significant strongest hydroxyl radical scavenging activity as compared with ascorbic acid Fig. 2. IC₅₀ values of ZnL complex and ascorbic acid were found to be 0.21 and 0.18 μg/ml, respectively (Table 1). This result shows that ZnL complex exhibited a dose dependent antioxidant activity. The free radicals such as hydroxyl and superoxide radical are formed in biological systems and they have been associated as an extremely damaging species in free radical pathology, capable of injurious to almost every molecule found in living cells (Gulcin, 2006). Hydroxyl radicals are very strongly reactive oxygen species and there is no specific enzyme to defend against them in humans (Liu et al., 2005).

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Figure 2 Hydroxyl radical scavenging activity of the ZnL complex. Mean inhibition (%) ± SDⁿ significantly different (p < 0.05), n = values are given as mean of three replicates.

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3.3 Inhibition of DPPH radical scavenging activity (1-1-diphenyl 2-picryl hydrazyl)

The highest DPPH scavenging activity was found in ZnL complex. The scavenging activity of ZnL complex was compared with that of ascorbic acid. The results showed that, ZnL complex had significant scavenging effects with increasing concentration in the range of 100–500 μg/ml. When compared to ascorbic acid; the scavenging effect of ZnL complex was lower. The IC₅₀ values were found to be 0.25 and 0.22 μg/ml for ZnL complex and ascorbic acid, respectively (Table 1). DPPH scavenging activity of the ZnL complex is a dose dependent in vitro antioxidant activity. The method was based on the reduction of alcoholic DPPH solution in the presence of a hydrogen-donating antioxidant due to the formation of the non-radical DPPH (Soares et al., 1997). Fig. 3 displays the DPPH activity of ZnL complex in percentage of free radical scavenging activity. The higher DPPH radical-scavenging activity is associated with the lower IC₅₀ value. The DPPH was determined by the decrease in its absorbance at 517 nm. The DPPH scavenging method has been used to evaluate the antioxidant activity of compounds due to its simplicity, rapidity, sensitivity and reproducibility (Ozcelik et al., 2003). The absorbance decreased when the DPPH is scavenged by an antioxidant through donation of hydrogen to form a stable DPPH radical molecule. In the radical form, this molecule has an absorbance at 517 nm, which disappears after acceptance of an electron or hydrogen radical from an antioxidant compound to become a stable diamagnetic molecule (Matthaus, 2002). When DPPH reacts with suitable reducing agents, the electrons become paired off and the solutions lose color stoichiometrically depending upon the incoming electron (Rock et al., 1996; Sies and Stahl 1995).

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Figure 3 The DPPH radical scavenging activity of ZnL complex. Mean inhibition (%) ± SDⁿ significantly different (p < 0.05), n = values are given as mean of three replicates.

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3.4 ABTS (2,2′-azinobis-3-ethylbenzothiazoline-6-sulfonic acid) radical scavenging assay

ABTS radical-scavenging is a common method used to assess the antioxidant activity of the ZnL complex compared to that of ascorbic acid. In the present study the reduction of ABTS by ZnL complex was determined and the reducing capacity of ZnL complex was noted. ZnL complex and ascorbic acid scavenged the ABTS radicals at a concentration of 100–500 μg/ml. IC₅₀ value of ZnL complex and ascorbic acid was 0.1 and 0.19 μg/ml, respectively. The result is shown in Table 1. As shown in Fig. 4, the ZnL complex displayed potential scavenging effect of ABTS radicals. ABTS radical cation decolorization technique was a widely accepted method/assay (Greene et al., 1987). ABTS radical is a blue chromophore produced by the reaction of ABTS and potassium persulfate after incubation in the dark environment. The reaction of ZnL complex with this preformed radical cation decolorized the blue chromophore with increasing concentration (Igbinosa et al., 2011).

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Figure 4 The ABTS scavenging activity of ZnL complex. Mean inhibition (%) ± SDⁿ Significantly different (p < 0.05), n = values are given as mean of three replicates.

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3.5 Antibacterial activity

ZnL complex was screened for their antibacterial activity against S. aureus and S. typhi. Table 2 shows the antibacterial activity of ZnL complex. The results showed that ZnL complex exhibited a potential antibacterial activity against the standard drug gentamicin (S. aureus and S. typhi), respectively (Table 2).The ZnL complex exhibited significant activity and the zone of inhibition was up to 24–28 and 21–24 mm at a concentration of 800 μg/ml against S. aureus and S. typhi respectively. Schiff base derived from furylglyoxal and p-toluidene showed antibacterial activity against E. coli, S. aureus, Bacillus subtilis and Proteus vulgaris. Metal complexes of Mo(IV) and Mn(II) with ligands hydrazine carboxamide and hydrazine carbothiamide showed antibacterial activity against S. aureus and Xanthomonas compestris (Bhardwaj and Singh 1994). Bharti et al. (2010) reported that 2,4-disubstituted thiazole ring which was carrying imino-1,2 diphenylethanol substituent appeared to exhibit the highest anti-bacterial activity against S. aureus and Vibrio cholera with the MIC of 12.5 and 25 μg/ml. The 6,8-dibromo-4(3H) quinazolinone derivatives were screened for their antibacterial activity against S. aureus and it exhibited the MIC of 25 μg/ml (Mosaad Mohamed et al., 2010).

3.6 Inhibition of α-amylase and α-glucosidase activity

The α-amylase and α-glucosidase inhibitory properties of the ZnL complex are presented in Figs. 5 and 6. The ZnL complex inhibited both α-amylase and α-glucosidase in a dose dependent manner. The IC₅₀ values for α-amylase and α-glucosidase were found to be 0.18 and 0.23 mg ZnL complex, respectively (Table 3). Inhibition of enzymes involved in the hydrolysis of carbohydrates such as α-amylase and α-glucosidase has been exploited as a therapeutic approach for controlling postprandial hyperglycemia (Shim et al., 2003). The inhibition activity of α-amylase was extended and might be responsible for decreasing the rate of glucose absorption and concentration of postprandial serum glucose (Chau et al., 2003; Kwon et al., 2007). This effect would delay the degradation of starch and oligosaccharides, which would in turn cause a decrease in the absorption of glucose and consequently inhibit the increase in postprandial blood glucose (Lee et al., 2007). In the human species, α-amylase is present in both salivary and pancreatic secretions. This enzyme is responsible for cleaving large malto-oligosaccharides to maltose, which is then a substrate for intestinal α-glucosidase (Ramasubbu et al., 2004).

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Figure 5 Inhibition of α-amylase activities by ZnL complex.

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Figure 6 Inhibition of α-glucosidase activities by ZnL complex.

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3.7 Mode of inhibition

ZnL complex is likely to influence the α-amylase and α-glucosidase in quite a number of ways, for instance it competes with the substrate to bind the active site of the enzyme or it disrupts the catalytic process irreversibly. Kinetic studies were carried out to identify the mode of inhibition of ZnL complex using the LB plot. Fig. 7A and B shows the straight lines intercepting at a single point in the second quadrant indicating mixed noncompetitive and noncompetitive inhibition for α-amylase and α-glucosidase with inhibitory constant (K_i) value of ZnL complex of 77.8 and 31.6 μg, respectively (Fig. 8A and B). Kinetic constants (K_i) for the inhibition of α-glucosidase and α-amylase are listed in Table 3. Apostolidis et al. (2007) reported that kinetic inhibition of porcine pancreatic α-amylase by acarbose, maltose and maltotriose was mixed non-competitive type inhibition.

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Figure 7 LB plot analysis of the inhibition kinetics of (A) α-amylase and (B) α-glucosidase effects by ZnL complex.

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Figure 8 Dixon plot analysis of kinetic constants of (A) α-amylase and (B) α-glucosidase effects by ZnL complex.

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