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Synthesis of Schiff Bases of Acetophenone with Aniline and its Different Chloro-Substituted Derivatives, and Study of their Antioxidant, Enzyme Inhibitory and Antimicrobial Properties.

Byline: Hira Mir and Dildar Ahmed

Summary: In the present work, five different Schiff bases of acetophenone were synthesized with aniline (AL1) and its mono- and di-chloro derivatives including ortho-chloroaniline (AL2), meta- chloroaniline (AL3), para-chloroaniline (AL4), and 2,4-dichloroaniline (AL5) and were characterized using GC-MS (gas chromatograph-mass spectrometer), 1H-NMR and 13C-NMR spectral data. Antioxidant properties of the bases were evaluated using DPPH radical scavenging, FRAP (Ferric Reducing Antioxidant Potential), reducing power, phosphomolybdate and lipid peroxidation assays. None of the synthesized compounds responded to DPPH assay. All synthesized compounds responded moderately to FRAP, reducing power and phosphomolybdate assay, and had effective lipid peroxidation inhibition power. Since few prepared bases inhibits Urease and Protease, hence it shows Schiff Bases have such properties.Prepared Schiff Bases show low to moderate antibacterial activity against most of the bacterial strains.

Keywords: Acetophenone, Aniline Schiff bases, Antioxidant, Anti-enzymatic, Antimicrobial.

Introduction

Efforts to produce new therapeutic substances, on the part of chemists, are continuously required in order to provide more effective, safer and more affordable drugs with properties such as antioxidant, anti-enzymatic and anti-microbial. Antioxidants are required to combat free radicals such as reactive oxygen species (ROS), which are produced in our body as part of biological processes or under the induction of exogenous factors [1,2]. Free radicals are highly reactive species and may cause oxidative stress [3] stimulating the process of aging and promoting various age related disorders such as cataracts, cancer, cardiovascular diseases, atherogenesis [4], Alzheimer's [5] and Parkinson's diseases [6]. Schiff bases have been reported to possess significant lipid peroxidation inhibiting and free radical scavenging properties [7-10].

Urease is an enzyme that converts urea into ammonia. Urease inhibitors are required for medicinal and agricultural purposes. The bacterium Helicobacter pylori, which is a major cause of gastro- intestinal ulcer, can live in acute acidic medium of human stomach by releasing urease that produces basic ammonia, which neutralizes the acid around the pathogen and thereby provides shelter to it [11,12]. In agriculture, excessive urease activity - conversion of fertilizer urea into ammonia - can pose serious environmental and economic problems [13, 14]. Hence, urease inhibition can be considered as the first line of treatment for urease causing infections by bacteria.

Proteases are the enzymes that cut peptide bonds and therefore are involved in post-translational modifications. Abnormalities in protease catalysis can cause degenerative diseases like cancer, AIDS, arthritis, and cardiovascular disorders [15,16]. Thus, protease inhibition can serve as a mechanism to cure or avoid such ailments [17,18]. Schiff bases and their metal complexes have a promising history in protease inhibition [19]. Some substituted Schiff bases, such as N4-arylideneaminotriazole derivatives, exhibited anti-HIV activity [20] by targeting its serin-protease.

As the old antibiotics are losing efficacy due to the emergence of multidrug resistance in both Gram positive and Gram negative bacteria [21,22], an immediate remedial attention is needed to control the increasing rate of morbidity and mortality [23]. Schiff bases have been shown to possess potential for new antibiotic medicines. They have structural compatibility with many natural biological systems and can combat infections caused by microorganisms [24-26].

As part of our quest for new therapeutic agents, the present work aimed to synthesize Schiff bases of acetophenone with aniline and its mono- and di-chloro derivatives and compare their bio-activities. The study would be interesting to establish structure- activity relationship and contribute towards rational drug discovery.

Experimental

Chemicals

Most of the solvents used were of LR grade and were purified before use in different reactions. Ascorbic acid and DPPH (1,1-diphenyl-2- picrylhydrazyl) radical were purchased from MPBiomedicals (France). Folin-Ciocalteu reagent, ammonium molybdate, iron(II) sulfate, iron(II)chloride and butylated hydroxyanisole (BHA) werepurchased from Merck (Germany) and gallic acidfrom Scharlau (Switzerland). Linoleic acid and TPTZ(2,4,6-Tripyridyl-s-triazine) were obtained from Sigma-Aldrich (Germany). Potassium ferricyanideand trichloroacetic acid were of Unichem (China).Urease (Jack Bean) was purchased from Avonchem.All the reagents and solvents employed in this workwere of analytical grade. To record absorbance, UV-visible spectrophotometer UVD-3200 Labomed,

Synthesis of Schiff Bases

Acetophenone was treated with each of the amines to produce corresponding Schiff bases following a previously reported protocol [38]. The concentration of each reactant used was 10 mM. Thus, acetophenone (1.16 mL) was reacted with aniline (0.90 mL) for AL1, 2-chloroaniline (1.04 mL) for AL2, 3-chloroaniline (1.05 mL) for AL3, 4- chloroaniline (1.27 g) for AL4, and 2,4-dichloroanile (1.62 g) for AL5. The reactants in each case were placed in a round bottom flask using methanol (20 mL) as a solvent along with 0.5 mL glacial acetic acid. The mixture was then refluxed on a hot plate at 40 oC for about 3 h. The progress of the reaction was monitored by running TLC (thin layer chromatography) of the reaction mixture from time to time. The products were purified by crystallization from ethanol with the aid of glass beads. Small sharp crystals were obtained at low temperature by placing the container in a refrigerator.

The solvents used throughout the experiment for running TLC were chloroform and methanol in the ratio of 8:12; toluene, ethyl acetate and formic acid in ratio of 5:3:2 (T:E:F) and benzene and ethanol in the ratio of 8:2. Iodine chambers were used for visualization of TLC spots.

Antioxidant Assays

DPPH (2,2-diphenyl-1-picrylhydrazyl) Radical Scavenging Assay

The DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical scavenging assay was conducted using the method reported by Brand-Williams et al. [27]. Briefly, the stock solution of the radical was prepared by dissolving 24 mg of DPPH in 100 mL of methanol and kept in a refrigerator all covered with foil until used. The working solution was prepared by taking 20 mL of the DPPH stock solution and diluting it with methanol to obtain the absorbance of about 0.97 ( 0.03) at 517 nm. Different dilutions of each synthesized Schiff base were prepared in methanol.

Ascorbic acid was used as a standard antioxidant. In a glass vial, 3 mL DPPH working solution was mixed with 100 uL of a Schiff base solution and incubated at 37 degC for 30 min. Absorbance was measured against blank (methanol) at 517 nm. The antioxidant activity of a sample was calculated by using the following formula:

%Antioxidant Activity = [(1 - (As/Ac)] x 100%

where, As (sample absorbance) is the absorbance by 3 mL DPPH working solution + 100 uL Schiff base, while As (control absorbance) is the absorbance by 3 mL DPPH working solution + 100 uL methanol.

where,

DPPH = 2,2-diphenyl-1-picrylhydrazyl

Ac = Control Assorbance

As = Sample Absorbance

FRAP (Ferric Reducing Antioxidant Potential) Assay

The FRAP assay was conducted as per the method of Benzie and Strain [28]. For the preparation of 10 mM TPTZ solution, 0.031 g of TPTZ was dissolved in 10 mL HCl solution (40 mM). The solution was heated at 50 degC on a water bath for 5 min. Fresh FRAP reagent was prepared by mixing 2.5 mL TPTZ solution (10 mM) with 2.5 mL ferric chloride solution (20 mM) in 25 mL acetate buffer. The mixture was then incubated at 37 degC for 15 min. Different dilutions of a given Schiff base were prepared in methanol. Aqueous solution of ascorbic acid was used as a standard. In a test tube, 2.9 mL FRAP reagent and 100 uL Schiff base solution were mixed and incubated for 30 min in the dark. The absorbance of the mixture was then noted against the blank (2.9 mL FRAP reagent in 100 uL methanol) at 593 nm. The FRAP value of samples were expressed as micrograms per milliliter of Ascorbic Acid equivalents (ug/mL of AAE).

Reducing Power Assay

The reducing power assay was carried out by the method of Oyaizu [29]. Briefly, three dilutions of each base were prepared with concentrations of 1, 0.5 and 0.25 mg/mL. The solution of a base or gallic acid standard (2.5 mL) was mixed with 2.5 mL sodium phosphate buffer (0.2 M) and 2.5 mL potassium ferricyanide (1%). The mixture was incubated at 50 degC for 20 min and 2.5 mL trichloroacetic acid solution (100 mg/L) was added. The mixture was centrifuged at 650 rpm for 10 min, and 5 mL supernatant was mixed with 5 mL distilled water and 1 mL ferric chloride solution (0.1%). The absorbance was measured at 700 nm. An increased absorbance of the reaction mixture indicates increased reducing power. A standard with BHT at various concentrations was also made in a similar manner for comparison. All experiments were done in triplicate. The result is expressed in percentage as compared to the standard.

Phosphomolybdate Antioxidant Assay

The phosphomolybdate antioxidant assay was carried out using a reported method [30]. Phosphomolybdate reagent was prepared by mixing equal volumes of 0.6 M sulfuric acid, 4 mM ammonium molybdate and 28 mM sodium phosphate solutions. In a test tube, 3 mL phosphomolybdate reagent was mixed with 300 uL base solution. The test tube was capped with silver foil and incubated in water bath at 95 degC for 90 min. After the contents of the test tubes were cooled down, the absorbance of the test tube contents were measured at 765 nm against a blank. Ascorbic acid was used as a standard, and antioxidant activity of a sample was expressed as micrograms per millilitre of Ascorbic Acid Equivalents (ug/mL of AAE).

Lipid Peroxidation Assay

The lipid peroxidation inhibiting ability of bases were determined following the method reported by Mitsuda [31]. An emulsion of linoleic acid was prepared by mixing 175 ug Tween 20 and 155 uL linoleic acid and the volume was made 50 mL by adding 0.05 M of potassium phosphate buffer (pH 7). Different concentrations (0.5, 1.0, 1.5 and 2.0 mg/mL) of sample solution and BHA were prepared. In a test tube, 100 uL sample solution was mixed with 2.4 mL potassium phosphate buffer and 2.5 mL linoleic acid emulsion. The mixture was incubated at 37 degC for 25 min. Then, 100 uL of this solution was regularly taken at 24 h intervals and dissolved in 3.7 mL ethanol. It was reacted with 100 uL ferrous chloride solution (20 mM) and then 100 uL potassium thiocyanate solution (30%) was added and thoroughly mixed to obtain a clear solution.

Absorbance was measured at 500 nm each time. A 5mL solution consisting of linoleic acid emulsion (2.5 mL) and potassium phosphate buffer (2.5 mL) was used as blank. BHA was used as a standard antioxidant. All experiments were done in triplicate.

Where: As,t-X h and As,t-0 h are the absorbances of the sample at X h and 0 h, respectively; and A0,t-X h and A0,t-0 h are the absorbances of the blank at X h and 0 h, respectively. Plots were made of scavenging activity against the concentration of the samples at 96h, Fig. 2.

Assay for Urease Inhibitory Activity

The urease inhibiting activity of the synthesized Schiff bases was determined using a reported protocol [32].

In the assay, the enzyme urease is allowed to catalyze the conversion of urea to ammonia, and the released ammonia is made to react with a mixture of salicylate, hypochlorite and nitroprusside to yield a blue-green dye called indophenol whose production is monitored spectrophotometrically at 625 nm.

The reagent R1 is made by mixing 5 different solutions in equal volume, which are 0.12 M phosphate buffer (pH 7, prepared by dissolving 1.62 g potassium dihydrogen phosphate in 100 mL distilled water and adjusting its pH by the addition of 25% potassium hydroxide solution), 0.06 M aqueous sodium salicylate, 0.005 M aqueous sodium nitroprusside (0.148 g sodium nitroprusside dissolved in 100 mL distilled water), 0.001 M aqueous EDTA (0.037 g EDTA dissolved in 100 mL distilled water) and the enzyme (prepared by dissolving 0.0005 g urease in 100 mL distilled water). The reagent R2 was prepared by mixing 0.12 M phosphate buffer, 0.4 M sodium hydroxide solution and 0.01 M sodium hypochlorite in equal volume.

Different dilutions of each Schiff base and thiourea (standard) were prepared in 0.12 M phosphate buffer. To a dilution of a Schiff base in phosphate buffer (total volume 5 mL), 15 uL urea solution, 0.485 mL phosphate buffer and 2.5 mL R1 were mixed in a test tube and incubated at 37 degC for 5 min. Then, 2.5 mL R2 was mixed and the mixture was allowed to incubate for further 10 min at 25 degC. The absorbance was measured at 625 nm. For blank, 5.5 mL buffer, 2.5 mL R1 and 2.5 mL R2 were added. For control, 15 uL urea solution, 5.485 mL buffer and 2.5 mL R1were added and incubated at 37 degC for 5 min. Then, 2.5 mL R2 was added and incubated for further 10 min at 25 degC. Following formula was used to calculate % inhibitory activity of a sample:

% Inhibitory activity = 100 x [(Control Absorbance - Test Absorbance)/Control Absorbance]

Assay for Protease Inhibitory Activity

A reported protocol was used to determine protease inhibitory activity of the prepared Schiff bases [33]. Casein was used as a substrate. Upon action with protease, casein undergoes hydrolysis to produce tyrosine along with other amino acids. The released tyrosine is allowed to react with the Folin- Ciocalteu reagent which results in the formation of a coloured product which can be monitored by measuring the absorbance at 660 nm. Casein solution (0.65 % w/v) was prepared in potassium phosphate buffer (50 mM, pH 7.5). The enzyme diluent consisted of sodium acetate-calcium acetate buffer (pH 7.5) was prepared mixing sodium acetate (10 mM) and calcium acetate (5 mM) solutions and adjusting the pH with 0.1 M acetic acid or sodium hydroxide. The protease solution (10 mg/mL) was prepared immediately before use in the enzyme diluent. Solutions of different dilutions of a given Schiff base were prepared in methanol.

A drug called Ritonavir (in methanol) was used as a standard. To 5 mL pre-incubated casein (37 degC), a solution of the given Schiff base (5 mL) was added. The mixture was incubated at 37 degC for 15 min followed by the addition of enzyme solution (1 mL). After incubating the mixture for another 15 min at 37 degC, trichloroacetic acid (5 mL, 110 mM) was added to stop the reaction. The mixture was allowed to stand for 15 min at 37 degC and then filtered. To 2 mL of the filtrate, 5 mL of sodium carbonate solution (500 mM) and 1 mL Folin-Ciocalteu reagent (2 M) was added. After final incubation for 30 min at 37 degC, absorbance was noted at 660 nm against a black. The blank contained the same reagents except that it was added 1 mL enzyme diluent instead of the enzyme solution. The control contained 5 mL methanol in place of the inhibitor (Schiff base).

The percent inhibitory activity was calculated with the following formula:

% Inhibitory activity = 100 x [(Control Absorbance- Test Absorbance)/Control Absorbance]

Antimicrobial Analysis

Agar well diffusion method was employed for antimicrobial screening in terms of ZOI (zones of inhibition, mm) [13], while agar dilution method was executed to evaluate MIC (minimum inhibitory concentrations, mg/mL) of synthesized compounds and standard antibiotics [34].

Preparation of Test Solutions and Standard Antibiotics

Solution of each Schiff base was prepared in DMSO with concentration of 2 mg/mL. Three antibiotics (Levofloxacin, Cefixime and Amoxylin) were used as standards, and their solutions were prepared in a similar way (2 mg/mL in DMSO).

Test Microbes

The microorganisms Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Pseudomonas aurantiaca , Salmonella typhi, Stenotroph maltophilia, Enterobacter cloacae, Enterobacter aerogenes, Klebsiella pneumoniae, Staphylococcus epidermidis, Bacillus subtilis and Bacillus megaterium were obtained from Children Hospital, Lahore, while Achromobacter xylosoxidans, Azospirillum lipoferum and Citrobacter freundii, were obtained from biotechnology laboratory, FC College, Lahore.

Preparation of Inoculums

All microbial cultures were re-isolated thrice successively on Mueller-Hinton agar (Merck) with incubation period of 24 h at 37 degC and identity confirmed by standard bacteriological methods [35]. A loop full of bacterial suspension was then diluted with sterile physiological solution to standardize inoculum density up to 108 CFU/mL of bacterial cells (equivalent to turbidity of McFarland, barium sulfate standard 0.5) followed by 24 h incubation at 37 degC.

Antimicrobial Screening (Zones of Inhibition)

The bacterial inoculums were uniformly swabbed on prepared Mueller-Hinton agar plates followed by a session of drying, after which three wells of 7 mm diameter each were dug in the agar gel 33 mm apart from one another using a sterile cork borer. A 100 uL volume of test compound dilutions were pipetted into the triplicate wells and allowed to stand for an hour for diffusion to take place. Finally, plates were incubated at 37 degC for 24 h after which zones of inhibition were measured.

Determination of Minimum Inhibitory Concentration (MIC)

The minimum inhibitory concentration (MIC) is defined as the lowest concentration of an anti-microbial agent which can inhibit the bacterial growth. Stock solution (0.004 g/mL ) of each of the test compounds were prepared in DMSO, from which various dilutions were made with concentrations of 0.02, 0.04, 0.08, 0.12, 0.16, 0.20, 0.24, 0.28, 0.32, 0.36 and 0.40 mg/mL in total 20 mL of Mueller- Hinton agar plates by varying the volume of agar and test stock solutions. Prepared plates were spotted with 0.1 mL overnight activated cultures of the microbes and incubated at 37 degC for 24 h. The results were interpreted in accordance with NCCLS [36].

Characterization of Schiff Bases

Structures of the synthesized Schiff bases were confirmed by GC-MS screening and 1H-NMR and 13C-NMR spectral data. The GC-MS was Agilent GC 7890A/MS 5975. The gas chromatography equipped with a HP-5 MS. The carrier gas was helium at flow 0.8 mL/min. The temperature of the column was 60degC for zero min 5degC/min to 70degC and 10degC/min to 310deg C for 4 min.

N-(1-phenylethylidene)aniline (AL1)

GC retention time: 19.288 min; MS m/z 195 (M+), 180 (base peak), 77, 51. 1H-NMR(CD3OD, 300MHz) d 1.82(s, 3H, CH3), 7.22 (m, 3H, ArHe=e',f), 6.78(d, 2H, ArHa=a', 3J=7.2Hz), 6.90(t, 1H, ArHc, 3J=7.2Hz), 7.95(d, 2H, ArHd=d', 3J=7.5Hz), 6.65(t, 2H, ArHb=b', 3J=7.2Hz); 13C-NMR(CD3OD, 75MHz) d 18.6(1C, CH3), 129.1(2C), 128.8(2C), 127.5(1C), 123.2(1C), 119.4(2C), 130.4(2C), 139.2(1C), 152.4(1C, C-N), 164.3(1C, C=N).

2-Chloro-N-(1-phenylethylidene)aniline (AL2)

GC retention time: 20.846 min; MS m/z 231 ([M+2]+), 229 (M+), 216 (base peak + 2), 214 (base peak), 152, 111, 75, 51. 1H-NMR(CD3OD, 300MHz) d 2.28 (s, 3H, CH3), 7.19 (m, 3H, ArHf=f', g), 7.07(d, 1H, ArHd, 3J=7.2Hz), 7.13(t, 1H, ArHc, 3J=7.2Hz), 7.41(d, 2H, ArHe=e', 3J=7.5Hz), 7.19(t, 1H, ArHb, 3J=7.2Hz), 7.29(d, 1H, ArHa, 3J=7.2Hz); 13C- NMR(CD3OD, 75MHz) d 24.4(1C, CH3), 126.2(2C), 126.8(2C), 130.0(1C), 135.7(1C), 126.6(1C), 129.01(1C), 127.6(1C), 126.1(1C), 126.2(1C), 144.2(1C, C-N), 165.3(1C, C=N).

3-Chloro-N-(1-phenylethylidene)aniline (AL3)

GC retention time: 21.315 min; MS m/z 231 ([M+2]+), 229 (M+), 216 (base peak +2), 214 (base peak), 152, 111, 75, 51.

H-NMR(CD3OD, 300MHz) d 2.28 (s, 3H, CH3), 7.20 (m, 3H, ArHf=f', g), 6.96(d, 1H, ArHd, 3J=7.2Hz), 7.14(t, 1H, ArHc, 3 3J=7.2Hz), 7.94(d, 2H, ArHe=e', J=7.5Hz), 7.25(d, 1H, ArHb, 3J=7.2Hz), 7.82(s, 1H, ArHa); 13C-NMR(CD3OD, 75MHz) d 24.4(1C, CH3), 128.3(2C), 128.6(2C), 131.1(1C), 138.4(1C), 122.1(1C), 134.7(1C), 124.2(1C), 131.8(1C), 121.5(1C), 152.1(1C, C-N), 164.0(1C, C=N).

4-Chloro-N-(1-phenylethylidene)aniline (AL4)

GC retention time: 21.496 min; MS m/z 231 ([M+2]+), 229 (M+), 216 (base peak +2), 214 (base peak), 152, 111, 75, 51. 1H-NMR(CD3OD, 300MHz) d 2.27 (s, 3H, CH ), 7.02(d, 2H, ArH), 7.22(m, 5H, ArH, a,a',d,d'), 7.52(d, 2H, ArHc=c , 3J=7.5Hz); 13C NMR(CD3OD, 75MHz) d 24.2(1C, CH3), 117.2(2C), 123.3(2C), 123.6(2C), 129.8(2C), 131.1(1C), 132.5(1C), 139.4(1C), 147.1(1C, C-N), 167.4(1C, C=N).

2,4-dichloro-N-(1-phenylethylidene)aniline (AL5)

GC retention time: 22.641 min; MS m/z 265 ([M+2]+), 263 (M+), 250 (base peak +2), 248 (base peak), 186, 145, 109, 77, 51. 1H-NMR(CD3OD, 300MHz) d 2.20 (s, 3H, CH3), 7.48 (m, 3H, ArHe=e',f), 7.36(s, 1H, ArHa), 6.98(d, 1H, ArHb, 3J=7.2Hz), 7.89(d, 2H, ArHd=d', 3J=7.5Hz), 6.64(d, 1H, ArHc, 3J=7.2Hz); 13C-NMR(CD3OD, 75MHz) d 24.2(1C, CH3), 128.1(2C), 128.4(2C), 131.2(1C), 138.7(1C), 126.2(1C), 132.6(1C), 133.2(1C), 127.2(1C), 125.3(1C), 142.3(1C, C-N), 165.3(1C, C=N).

Results and Discussion

Antioxidant Activities

DPPH (2,2-diphenyl-1-picrylhydrazyl) Radical Scavenging Activity

DPPH is a stable free radical, which has the ability to capture a H atom or an electron from an antioxidant. None of the seven Schiff bases displayed any antioxidant activity in DPPH assay. Probably, it is because of non-availability of hydrogen in these bases that can readily be donated as H radical to DPPH. This support the idea that DPPH works through HAT (hydrogen atom transfer) and not ET (electron transfer) mechanism. This observation is of considerable significance with reference to the mechanism of DPPH free radical scavenging activity. It is in agreement with our previous findings [37,38], with the implication that at least with Schiff bases, DPPH follows HAT mechanism.

The concentration of antioxidant that causes a 50% decrease in DPPH concentration is defined as IC50. Ascorbic acid was very reactive with IC50 of 8ug/mL.

FRAP Activities

The FRAP assay is based on reduction of ferric to ferrous by an antioxidant to form a colored ferrous-tripyridyltriazine complex. Ascorbic acid was used as standard, and FRAP values were calculated using the equation from the standard curve of ascorbic acid.

Absorbance = 0.0026 (Concentration of ascorbic acid equivalent) + 0.0123

Concentration of ascorbic acid equivalent = (Absorbance - 0.0123)/ 0.0026

The results are displayed in Table 1. The FRAP assay involves the electron transfer (ET) mechanism. In this assay, the antioxidant activities of the Schiff bases AL1, AL2, AL3, AL5, were, respectively 24.11, 25.26, 23.73, and 25.26 ug/mL AAE, which are very close to one another. AL4, however, showed exceptionally high activity. It may be because chloro group is attached para to the azomethine group. Since it is higher than AL2 and AL3, the ortho- and meta-chloro isomers of AL4, the effect seems to be steric in nature rather than electronic.

Reducing Power Assay

In the reducing power assay, the sample antioxidants reduces Fe3+ to Fe2+ by donation of an electron resulting in the formation of a blue coloured iron(II) complex. It was carried out at three different concentrations where 1 mg/mL was serially diluted to 0.5 mg/mL and 0.25 mg/mL, and the results are shown in Fig. 1. An increased absorbance of the reaction mixture indicates increased reducing power. It was highest for AL1, the base having no chloro substituent, followed by AL5 and AL4. AL5 is ortho/para dichloro substituted, while AL4 has only one chloro substituent at position para. It can, thus, be concluded that presence of a chloro group at meta position lowers the free radical scavenging activity of a base, while its presence at para position enhances the activity. The same trend was found in FRAP assay, another iron based assay discussed above.

Table-1: Antioxidant activities of acetophenone derived Schiff bases determined by different methods.

###FRAP Assay###+PM Assay ug/mL of AAE*

###ug/mL of AAE*

AL1###24.11+-0.01###46.50+-0.02

AL2###25.26+-0.02###49.40+-0.03

AL3###23.73+-0.02###50.25+-0.02

AL4###30.29+-0.01###46.04+-0.01

AL5###25.26+-0.02###55.25+-0.01

Antioxidant activity by Phosphomolybdate Assay

The phosphomolybdate antioxidant assay follows the reduction of Mo(VI) to Mo(V) by the antioxidant sample and is detected by the formation of green coloured molybdenum(V) complex at an acidic pH. Table 1 shows the results. Of the monochloro substituted bases, AL2 and AL3 showed almost equal and good activity, while AL4 was least active. Of dichloro substituted compounds, AL5, which has chloro substituents at ortho/para positions, exhibited the highest activity. This followed the trend of activity of these bases in FRAP and reducing power assays. Notable exception was AL4, which showed high activity in iron based assays, was here least active.

Lipid Peroxidation Inhibitory Assay

The findings of this assay are shown in Fig. 2. The activity was determined as a function of time for a period of 96 h. BHA was used as standard antioxidant for comparison. In general, lipid peroxidation inhibitory activities of these bases decrease with time. However, AL3 and AL4 exhibited a sharp increase in activity after 48 h. They were distinctively more potent inhibitors than BHA at the end of 96 h. AL5 was least potent.

Urease and Protease Inhibitory Activities

Urease and protease inhibitory activities of the acetophenone derived Schiff bases were measured using reported protocols and the results are displayed in Table 2. The bases demonstrated a varying degree of anti-enzymatic properties at different concentrations. As the table shows, AL1 and AL2 showed no activity at all against both the enzymes. AL3 remained highly inactive against the enzymes within a broad concentration range. It suggests, monochloro substituted bases either showed no activity at all or were only poorly active.

Table-2: Urease and protease EC50 (ug/mL) values of acetophenone derived Schiff bases.

Schiff Base###Urease###Protease

AL1###nd###nd

AL2###nd###nd

AL3###75.13+-0.02###400+-0.01

AL4###36.47+-0.02###310+-0.02

AL5###42.62+-0.01###385+-0.01

Standard###9.66+-0.02###36.5+-0.01

Table-3: Zones of inhibition (mm) of acetophenone derived Schiff bases against thirty different bacterial strains and their comparison with three standard antibiotics.

###Bacterial strains###Zones of inhibition (mm)

Sr. No###Antibiotics

###Schiff Bases (2 mg/mL)

###(2 mg/mL)

###Gram-negative bacteria

###L###C###A###AL1###AL2###AL3###AL4###AL5

###1###Escherichia coli###36###20###17###16+-0.01###25+-0.01###18+-0.03###16+-0.01 22+-0.01

###2###Pseudomonas aeruginosa###40###20###15###14+-0.02###14+-0.01###11+-0.03###--###17+-0.02

###3###Pseudomonas aurantiaca###32###20###16###14+-0.02###13+-0.03###10+-0.01###17+-0.01 15+-0.01

###4###Salmonella typhi###28###16###10###15+-0.02###--###13+-0.01###14+-0.02 18+-0.01

###5###Stenotroph maltophilia###38###27###24###14+-0.01###9+-0.01###11+-0.02###23+-0.02 19+-0.03

###6###Enterobacter cloacae###32###21###11###--###--###--###20+-0.03 10+-0.01

###7###Enterobacter aerogenes###33###16###14###--###13+-0.03###21+-0.01###16+-0.01 12+-0.01

###8###Klebsiella pneumoniae###38###30###25###8+-0.01###18+-0.01###21+-0.02###25+-0.01 18+-0.02

###9###Achromobacter xylosoxidans###38###22###15###--###12+-0.01###32+-0.01###--###15+-0.01

###10###Azospirillum lipoferum###35###28###20###--###--###11+-0.02###10+-0.01 15+-0.01

###11###Citrobacter freundii###32###16###14###13+-0.01###11+-0.01###15+-0.02###10+-0.01 15+-0.01

###Gram-positive bacteria

###1###Staphylococcus aureus###35###22###16###17+-0.01###--###17+-0.01###21+-0.02 30+-0.01

###2###Bacillus subtilis###35###20###18###12+-0.01###17+-0.01###11+-0.01###15+-0.03 16+-0.01

###3###Bacillus megaterium###37###26###22###--###--###--###13+-0.02 11+-0.01

###4###Staphylococcus epidermidis###36###20###16###13+-0.02###--###--###9+-0.01 28+-0.02

Table-4: MIC (ug/mL) of prepared Schiff bases against different bacterial strains.

###Bacterial strains###MIC (ug/mL)

Sr. No.

###Gram negative bacteria###AL1###AL2###AL3###A4###AL5

###1###Escherichia coli###200###80###160###200###120

###2###Pseudomonas aeruginosa###200###200###240###320###160

###3###Pseudomonas aurantiaca###200###200###240###160###200

###4###Salmonella typhi###200###320###200###200###160

###5###Stenotroph maltophilia###200###280###240###80###160

###6###Enterobacter cloacae###320###320###360###120###240

###7###Enterobacter aerogenes###360###200###120###200###240

###8###Klebsiella pneumoniae###280###160###120###80###160

###9###Achromobacter xylosoxidans###320###240###20###260###200

###10###Azospirillum lipoferum###320###320###240###240###200

###11###Citrobacter freundii###200###240###200###240###200

###Gram positive bacteria

###1###Staphylococcus aureus###160###320###160###120###20

###2###Bacillus subtilis###240###160###240###200###200

###3###Bacillus megaterium###320###360###320###200###240

###4###Staphylococcus epidermidis###200###320###360###280###40

Antimicrobial Assay

Antimicrobial assay results (Tables 3 and 4) showed a wide range of activity of prepared Schiff bases against different bacterial strains. The number of chloro group/s and its/their position on the ring affects the antimicrobial activity of theses bases. AL1, the base with no chloro substitution, proved to be least active. Dichloro substituted bases were, in general, more lethal towards most of the test bacteria than monochloro substituted. AL3 had high antibacterial activity against P. aeruginosa. AL5 displayed significant activity against S. aureus with MIC as low as 20 ug/mL, while AL3 was most active against A. xylosoxidans (MIC, 20 ug/mL). The presence of chloro groups at meta position, therefore, made the bases more active than their other isomers.

Conclusion

Synthesized Schiff bases differed only with respect to the chloro group substitution which was also depicted in the assays carried out. It was seen that reactivity pattern of synthesized Schiff bases mostly remained same in different activity analysis. Schiff Bases possess Urease and Protease inhibition properties. These compounds are also effective against most of the bacterial strains ranging from innocuous to strongly pathogenic. Furthermore, this paper also supports our hypothesis that DPPH assay is based on the ability to donate proton and not electron.

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