Synthesis, characterization and antimicrobial evaluation of two aromatic chitosan Schiff base derivatives

Accepted Manuscript Title: Synthesis, characterization and antimicrobial evaluation of two aromatic chitosan Schiff base derivatives Author: Tamer M. Tamer Mohamed A. Hassan Ahmed M. Omer Walid M.A. Baset Mohamed E. Hassan Muhammad E.A. El-Shafeey Mohamed S. Mohy Eldin PII: DOI: Reference:

S1359-5113(16)30317-8 http://dx.doi.org/doi:10.1016/j.procbio.2016.08.002 PRBI 10761

To appear in:

Process Biochemistry

Received date: Revised date: Accepted date:

5-5-2016 24-7-2016 2-8-2016

Please cite this article as: Tamer Tamer M, Hassan Mohamed A, Omer Ahmed M, Baset Walid MA, Hassan Mohamed E, El-Shafeey Muhammad EA, Eldin Mohamed S.Mohy.Synthesis, characterization and antimicrobial evaluation of two aromatic chitosan Schiff base derivatives.Process Biochemistry http://dx.doi.org/10.1016/j.procbio.2016.08.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Synthesis, characterization and antimicrobial evaluation of two aromatic chitosan Schiff base derivatives Tamer M. Tamer 1,*, Mohamed A. Hassan2,*, Ahmed M. Omer1,*, Walid M.A. Baset3, Mohamed E. Hassan4,5, Muhammad E.A. El-Shafeey6 and Mohamed S. Mohy Eldin7 1

Polymer Materials Research Department, Advanced Technologies, and New Materials Research Institute (ATNMRI), City of Scientific Research and Technological Applications (SRTA-City), New Borg ElArab

City,

P.O.

Box:

21934

Alexandria,

Egypt;

Emails:

[email protected];

[email protected] 2

Protein Research Department, Genetic Engineering and Biotechnology Research Institute (GEBRI), City of Scientific Research and Technological Applications (SRTA-City), New Borg El-Arab City, P.O. Box: 21934 Alexandria, Egypt; Email: [email protected]

3

National Organisation for drug control and Research (NODCAR), 51 Wezaret El-Zeraa st., Dokki, Cairo, Egypt; Email: [email protected]

4

Center of Excellence, Encapsulation & Nanobiotechnology Group, National Research Center, ElBehouth Street, Cairo 12311, Egypt; Email: [email protected]

5

6

Chemistry of Natural and Microbial Products Department, National Research Center, El-Behouth Street, Cairo, 12311, Egypt; Email: [email protected] Department of Medical Biotechnology, Genetic Engineering and Biotechnology Research Institute

(GEBRI), City of Scientific Research and Technology Applications (SRTA-City), New Borg El-Arab City, P.O. Box: 21934 Alexandria, Egypt; Emails: [email protected] 7 Chemistry Department, Faculty of Science, University of Jeddah, Osfan, P. O. Box: 80203, Jeddah 21589, Saudi Arabia; Email: [email protected]

*Corresponding author: 1.

Mohamed A. Hassan, Email ([email protected]), Tel: +2034593422

2.

Tamer M. Tamer, Email ([email protected]), Tel: +2034593414

3.

Ahmed M. Omer ([email protected]), Tel: +2034593414

1

Graphical abstract

Highlights •

4-chloro benzaldehyde was coupled with chitosan to prepare Schiff base I.

•

Benzophenone was coupled with chitosan to prepare Schiff base II.

•

The chemical structures of the Schiff bases verified through FT-IR, TGA and DSC.

•

The antimicrobial activities of the Schiff bases increased more than chitosan.

•

The new Schiff bases could be applied as antimicrobial wound dresser.

2

Abstract Recently, there have been significant scientific interests to scientists for the chemical modifications of chitosan to increase its applications. The main objective of this study was to prepare two aromatic chitosan Schiff bases (I and II) via coupling with 4-chloro benzaldehyde and benzophenone respectively for improvement the antimicrobial property of chitosan. The chemical structures of the prepared Schiff bases verified through FT-IR, TGA and DSC. However, degrees of substitution were estimated using potentiometric analysis, and they were 7.9% and 4.17% for Schiff bases (I and II) respectively. Antimicrobial activities evaluation were conducted against three Gramnegative bacteria (Escherichia coli, Pseudomonas aeruginosa and Salmonella sp.), two Gram-positive bacteria (Staphylococcus aureus and Bacillus cereus) and Candida albicans strain. The antimicrobial activities of the new derivatives increased significantly

more than chitosan in most microorganisms. The minimum inhibitory concentration (MIC) of Schiff base (I) with concentration (50 µg/ml) exhibited the highest activity against C. Albicans with growth inhibition up to 27.42%. While, 50 µg/ml of Schiff base (II) showed high activity against E. coli, Salmonella sp., S. aeureus and B. cereus more than chitosan. The results clearly suggested that the new Schiff bases could be applied as antimicrobial wound dressing agents to ameliorate wound healing. Keywords: Chitosan; Chitosan Schiff base; Antimicrobial polymer; 4-chloro benzaldehyde; Benzophenone

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1. Introduction Chitosan (deacetylated form of chitin) is one of the most common polymers found in nature, with chitin second only to cellulose regarding natural abundance [1, 2]. Both chitin and chitosan found in the shells of crustaceans and insects and certain other organisms, including many fungi, algae, and yeast. Chitosan is biopolymers composed of N-acetylated glucosamine and glucosamine units linked by β-(1–4) glycosidic bonds. Chitosan has been exhibiting various promising biological activities, including antimicrobial, antitumor, and hemostatic activity, and the acceleration of wound healing [3, 4]. The Unique properties of chitosan have driven the researchers to apply it in promising biomedical applications such as tissue engineering, wound dressing, gene and drug delivery, etc [5-9]. The antimicrobial activity of chiton saccharides is a function of several structural factors such as their degree of deacetylation (free amine groups), and their molecular weight. However, the environmental conditions can also have a significant effect such as the pH presence of some ions such as Ca2+, Mg

2+

ion [10–12]. It was reported that the

antimicrobial activity of chitosan has been linked to the glucosamine amino group so, the antimicrobial activity increase with decreasing pH [13-15]. Indeed, the reported antibacterial activities of chitosan saccharides showed a broad range of activity depending on the properties of the used material and the type of assay. The antimicrobial features of chitosan have not standardized, and, therefore, it is difficult to compare results from one study to another [16, 17]. Mutations of microorganisms are leading the scientists to increase antimicrobial activity of chitosan via modification of chemical structures. Chitosan has three reactive groups, i.e., primary (C-6) and secondary (C-3) hydroxyl groups on each repeated unit, and the amino groups (C-2) on each deacetylated unit. Therefore, these reactive groups of chitosan are readily subjected to 4

chemical modifications to alter its mechanical and physical properties. Also, the coupling of free amine groups on chitosan with the carbonyl group in aldehydes and ketones was very easy and common reaction to produce Schiff bases along polymer backbone (– RC=N–). Several chitosan Schiff bases have been prepared and published as chelating agents, antimicrobial and antioxidant materials, etc. [18-21]. Although chitosan was established in the various literatures as antimicrobial biopolymer, the continuous mutations of bacteria to resist and minimize action of antibiotics have driven scientists to develop and improve antimicrobial materials. This study concerned with synthesis and characterization of new derivatives of chitosan Schiff bases. Moreover, the antimicrobial activities of the obtained materials were evaluated to be used in the biomedical applications. 2. Materials and methods 2.1. Materials Shrimp shells collected from wastes of seafood restaurants in Alexandria. 4Chlorobenzaldehyde (purity 97% M.W., 140.57) obtained from Sigma-Aldrich (Germany). Benzophenone (purity 99 M.W., 182.22) obtained from Sigma-Aldrich (Germany), acetic acid (purity 99.8%), hydrochloric acid (purity 37%) and sodium hydroxide pellets (purity 99–100%) were purchased from Sigma-Aldrich (Germany). Sulfuric acid (Purity 98%, M.W., 96) purchased from Sigma-Aldrich (Germany) and Ethanol (Purity 99.9%, M.W.46.07) from International Co. for Supp. & Med. Industries (Egypt). 2.2. Microorganisms Five bacterial strains and one eukaryote strain were used for evaluating the antimicrobial activities of chitosan and its derivatives. The investigated microorganisms included three Gram-negative bacteria (E. coli, P. aeruginosa and Salmonella sp.) and two Gram5

positive bacteria (S. aureus and B. cereus) as well as, C. albicans strain. The strains were refreshed through inoculating in LB broth (peptone 1%, yeast extract 0.5%, NaCl 1%, and pH 7 ±0.2), and incubated overnight at 37°C and 150 rpm in a rotary shaker. 2.3. Methods 2.4. Extraction of chitin from shrimp shells According to the published procedure [23], the de-mineralization of shells was the main process for chitin preparation. In this step, the shells were dispersed in 5% (w/v) HCl at room temperature in the ratio of 1:14 (w/v) overnight. After 24 h, the shells were quite squishy and rinsed using water to remove acid and calcium chloride. The demineralized shells were treated with 5% (w/v) NaOH at room temperature for 24 h in the ratio of 12:1 (v/w). The residues were collected and washed to neutrality many times in running tap water and then; distilled water to obtain pure chitin. 2.5. Preparation of chitosan from chitin Preparation of chitosan is simply deacetylation of chitin in alkaline medium. Removal of acetyl groups from the chitin was achieved using 50% (w/v) NaOH with a solid to solution ratio of 1:50 (w/v) at 100-120°C for 12 h. The resultant chitosan washed to neutrality with distilled water; Fig (1) [24]. 2.6. Chitosan purification According to the previous method [25], chitosan sample was dissolved in 2% (w/v) acetic acid and was left overnight. Then, the chitosan solution was filtrated using cheesecloth to remove contaminants and undissolved particles. Finally, chitosan was precipitated with 5% (w/v) NaOH, collected and washed with distilled water to remove the excess of alkali. 2.7. Preparation of chitosan Schiff base derivatives According to our previous article [21], 1 g of chitosan was dissolved in 50 ml of 2%

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(w/v) acetic acid and stirred at room temperature for 6 hrs. Ten ml of ethanol contains (1.86 mM) of aldehyde or ketone (4-chloro benzaldehyde or benzophenone) were added drop wise to the solution. The mixture was kept under stirring for 6 h at 50°C. The formation of a deep yellow gel referred to the formation of the chitosan Schiff base. The resulting product was precipitated in a solution of 5% sodium hydroxide. The precipitate was filtered and washed with water and ethanol several times to remove unreacted aldehyde or ketone. The products were filtered and dried in a vacuum oven at 60°C overnight. The schematic diagram describes the proposed mechanistic pathway for synthesis of Schiff bases (I) and (II) is presented in Figure (2).

2.8. Characterization 2.8.1. Fourier Transform Infrared Spectrophotometer (FT-IR) analysis The structures of the chitosan and chitosan derivatives were investigated by FT-IR spectroscopic analyzes using FT-IR (Model 8400 S, Shimadzu, Japan). Samples (2–10 mg) were mixed thoroughly with KBr. The sample was pressed into pills with a Specac compressor (Specac Inc., Smyrna, USA) and the absorbance of samples scanned from 500-4000 cm-1. DD of chitosan material was calculated according to the following equation [26]:

Where, A is area of the peak 2.8.2. Determination of degree of acetylation using potentiometric titration 0.1 g of chitosan or chitosan derivatives was dissolved in 20 ml of 0.1 N HCl. The solution was kept under stirring overnight to provide polymer a sufficient time to be hydrated.

7

Titration was started by adding NaOH 0.1 N. pH values and the addition of NaOH recorded. The degree of deacetylation was calculated using the following formula [27]:

Where, V, C and Wdry stand for the volume of NaOH consumption between two abrupt changes of pH, concentration of NaOH, and the dry weight of chitosan sample (derived from the moisture content) respectively. NH2 and W(NH2) demonstrated as the percentage.

2.8.3. Gel permeation Chromatography (GPC) The molecular weight of chitosan and its Schiff base derivatives were measured by GPC (Waters® Alliance® GPC/V 2000 System, USA). Pullulans with a molecular weight range from 1.414 × 105 to 3.267 × 105 (American Polymer Standards Corporation, USA) were used as standard samples to carry out the universal calibration curve. Alliance® GPC/V 2000 System use both refractive index (RI) and viscometer detection, and UltrahydrogelTM 1000 Columns. The Chromatographic parameters were; flow rate of elution liquid (0.3 M CH3COOH + 0.2 M CH3COONa), 0.8 mL/min; column temperature, 40°C; and sample volume, 100 μL. All the solvents and solutions were filtered through a 0.45 μm filter (Whatman Inc., Clifton, NJ, USA). 2.8.4. UV-Vis Spectroscopic analysis 0.05 g of the polymer sample was dissolved in 25 ml of acetic acid (2%, v/v). The absorbance of each sample was scanned from 190-1000 nm and recorded [21]. 2.8.5. Thermogravimetric analysis (TGA) TGA analysis of chitosan and chitosan derivatives were carried out using

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Thermogravimetric Analyzer (Model 50/50H, Shimadzu, Japan) [28]. 2.8.6. Differential scanning calorimetric (DSC) DSC analysis of chitosan and chitosan derivatives were conducted using DSC Analyzer (Model 60A, Shimadzu, Japan). 2.9. Bio-evaluation studies 2.9.1. Antimicrobial activities of chitosan and the two derivatives 2.9.1.1. Preliminary screening using agar well diffusion method Agar-well diffusion method was applied for screening the antimicrobial activities of chitosan and chitosan derivatives against E. coli, P. aeruginosa, Salmonella sp., S. aureus, B. cereus and C. albicans as described by the reported method [29, 30]. Briefly, 50 µl of overnight cultures of the indicator microorganisms were swabbed on Luria Britani (LB) agar medium (1% peptone, 0.5% yeast extract, 1% NaCl and 1.5% agar). Cork borer was used for punching the agar to form wells of 6 mm in diameter and 30 µl of the tested materials were loaded. The plates were left in the refrigerator at 4°C for 2 h to allow to materials diffusion into the agar. The plates were incubated at 37°C for 24 h then, they were investigated and the bacterial inhibitions were photographed using gel documentation system. 2.9.1.2. Minimum inhibitory concentration (MIC) Determination The influence of different concentrations of chitosan and chitosan derivatives on the growth of E. coli, P. aeruginosa, Salmonella sp., S. aureus, B. cereus and C. albicans were estimated by the microtiter plate method as presented by the published method [31, 32]. Overnight cultures of strains were prepared by inoculating the strains in LB broth and they were incubated at 37°C and 150 rpm in shaking incubator. Bacterial cultures were diluted with the same broth medium 100 times to obtain optical densities (0.9) for all microorganisms at 600 nm. Then, 20 µl of diluted cultures were loaded into a sterile 9

96-wells microplate, and different of final concentrations of chitosan and chitosan derivatives (25, 50, 100, 150, 200 and 250 µg/ml) were added. The tested materials were passed through a syringe filter (0.22 µm) for sterilizing previous use. The wells were completed with LB broth up to 200 µl, agitated for 2 min at 100 rpm using bench shaker, and incubated at 37°C for 24 h. The positive and negative controls were prepared by mixing the materials only and diluted bacterial cultures only with free LB respectively. The microtiter plates were shaken for 30 seconds using a microplate reader, and measured at 600 nm to estimate the turbidity of bacterial cultures. The experiments were carried out in triplicate and recorded. The mean and standard deviation (SD) were calculated. The inhibition percentages of microbial growth were calculated using the following equation: Inhibition percentage (%) = [(normal activity - inhibited activity) / (normal activity)] * 100 2.9.1.3. Bactericidal and fungicidal activity The bactericidal activities of chitosan and its derivatives were performed using microtiter plate. The old-overnight cultures of different microorganisms were diluted with an autoclaved LB medium to obtain optical densities (1.8) at 600 nm for all cultures. One ml of each cells suspension was mixed with 1 ml of 1% (w/v) chitosan and chitosan derivatives. The samples were drawn after incubation at 37°C for different times (0, 1, 2, 3, 4 and 5 h), and ten µl from each sample was inoculated in 96-wells microplates followed by completing the wells with LB broth up to 200 µl. The microplates were agitated and incubated at 37°C for 24 h. All experiments were conducted in three determinations and the turbidities of incubated cultures were gauged at 600 nm using microplate reader as mentioned above. The mean and SD were calculated and recorded [33].

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2.10. Cytotoxicity studies in vitro The cell viability was carried out on NIH3T3 (mouse fibroblast cell line) and was evaluated using MTT [3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay [34]. The fibroblast cells were cultured in 50 cm2 culture flask including complete Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (DMEM supplemented with 10% fetal bovine serum) at 37°C, 5% CO2, and 85% humidity. The culture was observed until it became confluent and the cells were harvested by adding 0.05% trypsin/EDTA and incubated at 37°C, and 5% CO2 for 5 min for detaching the cells completely. The trypsin was neutralized using DMEM medium, and the cell suspension was centrifuged at 1200 rpm for 10 min and the cells were resuspended in DMEM. The cells were counted via light microscope using trypan blue and haemocytometer. Different concentrations (25, 50, 100, 150, and 200 mg) of chitosan and chitosan Schiff bases (1 & 2) Powder were applied for evaluating their cytotoxicity. The investigated materials were sterilized by embedding in 70% ethanol, and then washed four times with sterilized PBS. The materials were dried and exposed to UV in laminar flow for 30 min under sterilized conditions [35]. The fibroblast cells were seeded in a 96-wells tissue culture plate at numbers 4×103 cells/well. The total volume of each well was 200 µl with the tested material, but the control wells were containing the cells without material. The test was carried out in triple wells for each sample. The plate was incubated at 37°C, and 5% CO2 for 2 days. After incubation, the medium was removed from each well, and the wells were washed three times with sterilized PBS to remove the materials and cell debris. Twenty µl of MTT (5 mg m−1) was added to each well, and the plate was shaken for 5 min at 120 rpm for mixing quite. The plate was incubated at 37°C for 4 h. Following the incubation period, 200 µl dimethylsulfoxide (DMSO) was added to each well and shaken 11

again for 5 min at 120 rpm to dissolve the formed formazan crystals. The results were recorded, and the percentage of viable cells was calculated by comparing with control.

3. Results and discussions Increase resistance of bacteria to antibiotics stimulate scientist to develop new materials to confront this mutation. Several derivatives of chitosan were prepared to increase its antimicrobial activity such as amination, methylation, etc. [36-38]. In the current research, two new Schiff bases of chitosan were prepared by coupling chitosan with four chloro- benzaldehyde and benzophenone. The formation of Schiff bases verified through monitoring changes in chemical structure using FT-IR, TGA, DSC, spectroscopic analysis. 3.1. FT-IR analysis The FT-IR spectra of chitosan and two chitosan Schiff bases (I, II) shown in Fig. 3. The figure shows the basic characteristics of chitosan. Broadband at 3200-3600 cm-1 corresponds to the stretching vibration of –NH2 and OH groups. Bands at 2919 cm−1 (C-H stretching on methyl), 2879 cm−1 (C-H stretching in methylene). Bands appear at 1650, 1575, 1380 cm-1 attributed to stretching vibration of C=O of NH-C=O stretching (amide I), bending vibration of NH2 (amide II) and complex band, consisting of components from C-N stretching and N-H in plane bending from amide linkages ( amide (III). The bands at 1150 and 900 cm-1 attributed to the glycosidic bondings. Absorption bands at 1200–970 cm−1 are mainly due to C–C and C–O stretching in pyranose ring and to C–O–C stretching of glycosidic bonds [39]. FT-IR can be employed to determine DS by measuring chitosan DD. Absorption ratio A1655/A3450 was reported to use in measuring the degree of deacetylation. According to substation mechanism consumption of free amine groups can be estimated from the value

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of DD of new derivatives compared to parent chitosan. Table 1 summarized calculated DD and estimated DS using FT-IR. 3.2. Determination of free amines The degree of deacetylation (DD) of chitosan is the cornerstone of chitosan properties and applications. The degree of deacetylation (DD) influences chemical, physical and biological properties of chitosan [40]. Several methods were used to determination (DD) depend on free amine groups such as FT-IR [41], UV-spectrophotometry [42], NMR [43], colloidal titration [44], and potentiometric titration [45, 46]. Potentiometric titration proposed by Broussignac, 1968 is one of the simplest methods [47]. In this method, chitosan is dissolved in a known excess of hydrochloric acid, and the solution is then titrated potentiometrically with sodium hydroxide. This gives a titration curve having two inflection points (Figure 4). The first and second inflection points are the equivalence points of the titration of excessive hydrochloric acid and the titration of protonated chitosan respectively. In our test, we used simple potentiometric titration to estimated degree of substitution (DS). Table 1 summarized calculation of DD of chitosan and its two Schiff bases (I & II). Calculation showing calculated DS 7.9 and 4.17 for Schiff base I and Schiff base II respectively. 3.3. Gel permeation chromatography (GPC) Average molecular weight of chitosan and its two derivatives (Schiff bases 1 and II) were measured and recorded as shown in table 2. The obtained results show the moderate molecular weight of chitosan and its derivatives. 3.4. Electronic spectrum The visible and ultraviolet spectra of chitosan and its derivatives (Schiff bases I & II) measured on range from 200 to 500 nm. Normal chitosan showed absorbance band starting from 225 nm and maximum at 235 nm that corresponding to n-σ* transition of 13

amine free electrons [25, 26]. Coupling of chitosan with Phenolic aldehyde generate a new conjugation system that support transition with the steam of electrons lowering energy of transition (red shift) and increase that band intensity. In the other hand, the formation of Schiff base bond –N=C generate a new transition n-π* at a higher wavelength. 3.5. TGA analysis Thermal gravimetric analysis of chitosan and its two different Schiff bases (I & II) were performed as shown in Fig (5). Thermal decomposition of chitosan and its derivatives show three regular transitions. First depression may attribute to losing physically adsorbed moisture around 100°C that between 7-9% of polymer weight. A second depression of samples start from 220 to 350°C was attributed to the thermal decomposition of pyranose ring along polymer backbone to form complex adduct [4850]. The third decomposition attributed to thermal decomposition obtained from adducts. Chitosan shows slightly high thermal stability than its Schiff bases. It can show in the value of its T50. T50 represent the temperature at which sample loss half of its weight; T50 of chitosan and its Schiff bases (I & II) were 481.51, 445.47 and 386.75°C respectively. 3.6. DSC analysis DSC is efficient tools to characterized chitosan and its derivatives. DSC thermograms of chitosan and its Schiff base derivatives (I & II) shown in figure 6. DSC spectrum of chitosan shows a broad endothermic peak around 100°C that may attribute to evaporation of water content. The exothermic band at 245°C was corresponding to its thermal decomposition. Exothermic bands of chitosan Schiff bases exhibit some shift to a lower temperature that confirm its less thermal stability than chitosan.

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3.7. Antimicrobial evaluation Antimicrobial activity of chitosan won the attention of scientists over last Century Hence, several mechanisms proposed for the antimicrobial activity of chitosan and its derivatives. Three suggested mechanisms explained in previous literature; the first focuses on the interaction of positive charge of chitosan with a negative charge in the cell wall of microorganisms that causes leakage of intracellular substances. The second mechanism supposes the interaction of positive chitosan charge with DNA of microorganisms which inhibit RNA and protein synthesis. The last mechanism depends on the chelating activity of chitosan to assume that it chelates the essentiall elements for microbial growth such as Ca2+, Mg2+ etc. We expect that the above mechanisms can corporate to do the peculiar action of chitosan that depends on its source, molecular weight, deacetylation degree and recently modification [17]. In the current study, antimicrobial activities of chitosan and the two Schiff bases were carried out using agarwell diffusion method against five bacterial strains (two gram-positive; S. aureus and B. cereus and three gram-negative; E. coli, P. aeruginosa and Salmonella sp.) as well as fungal strain (C. albicans). This method applied for screening the effect of chitosan and the two its derivatives against the tested microorganisms before further investigations. All indicator microorganisms were sensitive to the tested materials as shown in figure 8. However, figure 8 shows the variety of the detected inhibition zones of chitosan and Schiff bases against the pathogenic strains. The obtained results demonstrated their importance to determine the MIC and bactericidal and fungicidal activities of chitosan and the two Schiff bases.

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3.7.1. MIC of chitosan and Schiff bases (I & II) MIC defined as the lowest concentration of an antimicrobial that will inhibit the visible growth of a microorganism after overnight incubation. MIC is useful as a practical indicator of primary activity against a selected pathogenic microorganism. Several studies reported on MIC of chitosan or their derivatives against different microorganism [51]. The MIC of chitosan and Schiff bases (I and II) investigated. MIC of our derivatives and chitosan were presented in details as in Table 3. Table 3 shows a dramatic increase of bacterial and fungal inhibition by increasing the polymer concentrations. MIC of Schiff base (I) was (50 µg/ml) against all pathogenic bacterial strains except E. coli and S. aeureus which were needed to 100 µg/ml for inhibiting their growth. Moreover, Schiff base (I) with concentration (50 µg/ml) exhibited the highest activity against Salmonella sp. and C. albicans strain with inhibition percentage up to 24.68% and 27.42%, respectively. These results are in contrast to chitosan and Schiff base (II) where, their activity started with 100 µg/ml in case of C. albicans. P. aeruginosa is an opportunistic human pathogen, and its inhibition must carry out using a combination of antibiotics as well as, it secrets alginate biofilm to defense against different antibiotics and free radicals of antibodies [52, 53]. Therefore, It is considered to mentioned that 250 µg/ml of Schiff base (I) showed complete inhibition of all bacterial growth with percentage up to 98.71% in case of P. aeruginosa in addition to its effect against C. albicans with percentage 79.63%. MIC of Schiff base (II) was observed at concentration of 50 µg/ml against all bacterial strains, and 100 µg/ml in case of C. albicans strain. Fifty µg/ml of Schiff base (II) showed high activity against E. coli, Salmonella sp., S. aeureus and B. cereus more than chitosan. On the other hand, 100 µg/ml of chitosan revealed as MIC against Gram-positive bacteria (S. aeureus and B. Cereus) as well as C. albicans strain. The lower concentration of 16

chitosan (50 µg/ml) presented growth inhibition against only Gramnegative bacteria and its activity was highest against P. aeruginosa compared to Schiff bases (I and II). It was reported that the MIC of the prepared chitosan were 125 and 500 µg/ml versus S. aeureus and E. coli respectively while, the MIC of the prepared hydrogel of chitosan and oxalyl bis 4-(2,5-dioxo-2H-pyrrol-1(5H)-yl) benzamide were 125 and 3.91 µg/ml [51]. In the current research, the antimicrobial activities of chitosan Schiff bases (I and II) proved that their influence was better than chitosan alone in a most microorganism that resulted from synergy effects of chitosan and Schiff bases and these results in agreement with the prvious reported results [54, 55]. The effect of chitosan and chitosan derivatives were strongest against Gram-negative more than Gram-positive bacteria, and this may attribute to the different in their cell walls structures. The cell wall in Gram-positive bacteria composed of thick layers of Peptidoglycan more than in Gram-negative and the chains Peptidoglycan are crosslinked to form rigid cell walls by a bacterial enzyme DD-transpeptidase. On the other hand, the cell wall in case of C. albicans as all fungal strains composed of chitin and glucan so, it is very rigid and the scientist still looking for more affected medical products have high affinity against fungal strains. The chitosan and its two derivatives can’t penetrate the bacterial cell wall referring to their molecular weight, so their activities could attribute to the first mechanism that depends on the interactions with the cell wall structure. 3.7.2. Bactericidal and fungicidal activity This test is important to know the behavior of microorganisms against the tested pharmaceutical products to limit the dose, intervals and duration time. Hassan et al., 2010 and abdou and Hassan, 2014 [35, 56] described the steps for applying the different medical and biotechnological products on mammalian cells and rats. After the Bactericidal and fungicidal test, the chitosan, and its derivatives should subject to further 17

investigations such as Hemocompatibility, cytotoxicity on mammalian cells before applying in vivo on animals. Bactericidal activity of chitosan and its Schiff base derivatives (I & II) were tested and presented in figures (9a-f). Contact of microorganism cultures with polymeric solutions before growing it was performed to measure inhibition activity of tested polymers. Figures show the varied behavior of bactericidal activities depends on the type of microorganism and polymer. Figure 9a shows the effect of polymer solution on E. coli chitosan and its derivatives exhibited almost same inhibition effect after one hour (from 60-65%). Inhibition of chitosan gives stable behavior than its derivatives. Schiff base (I) showing higher inhibition against B. cereus, P. aeruginosa and C. albicans than chitosan and Schiff base (II) (figure 9c, 9d and 9f). On the other hand, Schiff base (II) demonstrated higher inhibition with S. aureus (figure 9e). In the case of Salmonella sp., chitosan show inhibition activity higher than that of its new derivatives (Figure 9b). After 5 h, the chitosan had bactericidal effect against Salmonella sp. and S. aeureus while, it was bacteriostatic with other microorganisms and its effect was decreased significantly in the case of B. Cereus. Also, the effect of Schiff base (I and II) were bacteriostatic after 5 h except in the case of C. albicans strain with Schiff base (II). .

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3.8. Cytotoxicity studies in vitro The cytotoxicity of the prepared materials was carried out based on MTT assay. This reaction depends on the reduction of MTT by mitochondrial succinate dehydrogenase of viable cells. After incubation with cells, the MTT is metabolized and converted to insoluble formazan which was solubilized using DMSO to reveal the viable cells in the presence of materials. As shown in Table 4, the results of cytotoxicity of chitosan and chitosan Schiff bases (1 & 2) demonstrate slight differences between samples comparing to the control cells. Many previous articles proved that the chitosan and other chitosan Schiff bases have inconsiderable cellular toxicity, so chitosan has various applications in medical field [57-59]. The results revealed that the highest concentration (200 mg) of chitosan and chitosan Schiff bases (1 & 2) recorded 89%, 90%, and 91.1% of cell viability respectively. Zhang et al., demonstrated that 0.2 g of chitosan and modified chitosan were applied on fibroblast cells and showed non-toxicity with more than 90% cell viability [58]. On the other hand, the lowest concentration (25 mg) of the studied materials provokes a little cytotoxicity up to 2.5% which is highly acceptable in medical applications. Arachana et al., mentioned that the sample with cell viability more than 75% can be considered as non-cytotxic materials [59]. The cell viability evaluation confirms that the selection of these materials could depend only on the antimicrobial activities of each which boost the wound healing.

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4. Conclusions Two chitosan Schiff bases were successfully prepared via coupling with 4-chloro benzaldehyde and benzophenone. The structure of the prepared polymers was analyzed and confirmed using FT-IR spectra, TGA, and potentiometric analysis. The antimicrobial activities of the two chitosan derivatives were carried out throughout compared with chitosan alone. The MIC values were estimated and the bactericidal and fungicidal activities were determined as an important step to study the behavior of microorganisms versus the prepared Schiff bases. The prepared materials showed higher activity against Gram-negative bacteria than Gram-positive. moreover, their activities against fungal strain (C. albicans) were exhibited. The results of cell viability evaluation in vitro proved that these materials have inconsiderable cellular toxicity against fibroblast cell line. The results concluded that the two chitosan Schiff bases could be used as antimicrobial materials in medical applications such as wound dressing after conducting them in vivo on animals. 5. Conflict of interest The authors declare that there is no conflict of interest.

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27

OH

OH O O

HO

O

NaOH 50% n

12 hr

NH

O

HO

NH2

O

Figure 1: Preparation of chitosan.

OH OH

O O o

O O

HO NH2

n

6 hr / 50 C

+

n

O

HO N

Cl 4-Chlorobenzaldehyde

Chitosan

Cl Schiff base (I) OH OH

O O

HO

O o

O

6 hr / 50 C

n+

O

HO N

NH2

Chitosan

Benzophenone

Schiff base (II)

Figure 2: Preparation scheme of chitosan Schiff bases (I) and (II).

28

n

n

0.9

Chitosan Schiff base I Schiff base II

0.8 NH2 &

0.7 0.6

NH-C=O CH2

0.5

C=O CH

0.4 0.3 0.2 0.1 0 4000

3500

3000

2500 2000 Wavenumber (1/cm)

1500

1000

500

Figure 3: FT-IR of chitosan and its derivatives (Schiff bases I & II). 250

Chitosan Schiff base I

200

Schiff base II 150 100 50 0 15

20

25

volume add of 0.1 N NaOH

Figure 4: Potentiometric titration of chitosan and Schiff bases (I & II).

29

30

2.5

chitosan Schiff base (I) Schiff base (II)

2 1.5 1 0.5 0 200

250

300

350 400 wave length (nm)

450

500

Figure 5: Electronic spectrum of chitosan and its derivatives (Schiff bases I & II) (2 mg/ml). 120

Chitosan 100

Schiff base (I) Schiff base (II)

80

60

40 0

100

200 300 Temperature (oC)

400

Figure 6: TGA of chitosan and its derivatives (Schiff bases I & II).

30

500

Temperature ( oc ) DSC (mW)

0 0

50

100

150

200

250

300

350

400

-1 chitosan

-2

Schiff base (II) -3

Schiff base (I)

-4 -5 -6 -7 -8 -9 -10

Figure 7: DSC of chitosan and its derivatives (Schiff bases I & II).

31

Figure 8: Antimicrobial activities of chitosan (b), Schiff base I (c) and Schiff base II (d).

32

100

(A)

90 80 70 60 50 40 Chitosan Schiff base (I) Schiff base (II)

30 20 10 0 0

1

2

3

4

5

6

Time (h)

100

(B)

90 80 70 60 50 40

Chitosan Schiff base (I) Schiff base (II)

30 20 10 0 0

1

2

3 Time (h)

33

4

5

6

100

(C) 90 80 70 60 50 40 30 Chitosan Schiff base (I) Schiff base (II)

20 10 0 0

1

2

3

4

5

6

Time (h)

100

(D)

90 80 70 60 50 40 30 Chitosan Schiff base (I) Schiff base (II)

20 10 0 0

1

2

3 Time (h)

34

4

5

6

100

(E)

90 80 70 60 50 40 Chitosan Schiff base (I) Schiff base (II)

30 20 10 0 0

1

2

3

4

5

6

Time (h)

100

(F)

90 80

Chitosan Schiff base (I) Schiff base (II)

70 60 50 40 30 20 10 0

0

1

2

3

4

5

6

Time (h)

Figure 9: Bactericidal and fungicidal activity of chitosan and its derivatives Schiff bases (I & II) against (a) E. coli, (b) Salmonella sp., (c) P. aeruginosa, (d) B. cereus, (e) S. aureus and (f) C. albicans

35

Table 1: Degree of deacetylation calculated with different methods Sample

DD (FT-IR)

DS (FT-IR)

DD

DS

(Potentiometeric method) (Potentiometeric method)

Chitosan

94.4

---

93.15 %

---

Schiff base I

90.6

3.8

85.25 %

7.9 %

Schiff base II

88.6

5.8

88.98 %

4.17 %

Table 2: Average molecular weight of chitosan and its derivatives (Schiff bases 1 and II) Sample

Molecular weight (GPC)×105

Chitosan

2.03 ± 0.05

Schiff base I

1.98 ±0.11

Schiff base II

2.01 ±0.08

36

Table 3: MIC of chitosan and its Schiff base derivatives (I & II). (The data are the mean of three determinations. ± is standard deviation (SD))

Polymer Concentration (µg/ml)

25

0

0

0

0

0

0

0

0

0

0

0

0

0

0

50

15.75 ±1.8

0

36.63 ±1.3

23.20 ±0.65

6.11 ±2.3

15.98 ±1.8

21.03 ±2

24.68 ±1.5

22.79 ±0.86

12.09 ±2

100

29.68 ±2

13 ±1.5

63.22 ±2.9

32.24 ±1.1

22.41 ±1.8

24.34 ±3.9

40.25 ±1.3

51.72 ±2.7

26.27 ±1.5

19.90 ±1.3

36.91 ±1

17.67 ±1.3

150

32.32 ±1.5

67.16 ±2.2

64.54 ±2.7

34.59 ±1.2

79.76 ±2.9

53.27 ±2

39.81 ±0.98

62.86 ±3.1

31.59 ±1

36.03 ±1.1

73.43 ±1.6

200

57.52 ±2.6

87.72 ±3.4

65.45 ±3.2

39.77 ±1.5

97.24 ±1.5

57.54 ±2.6

47.14 ±1.6

81.08 ±2

40.98 ±2.8

51.23 ±2.6

052

57.84 ±3.1

93.62 ±1.2

67.73 ±1.6

49.32 ±2.7

98.71 ±3.6

63.64 ±3.2

73.21 ±3.5

90.07 ±1

44.82 ±2.3

55.68 ±2.4

0

0

0

0

0

16.43 ±1.9

19.69 ±0.96

39.87 ±3.1

38.71 ±2.5

20.07 ±1.7

20.24 ±1.6

30.77 ±1.3

19.37 ±2

35.82 ±1.9

51.87 ±2.5

67.25 ±3.1

32.42 ±3.6

24.86 ±3

71.55 ±1.5

19.79 ±3.2

99.22 ±1.8

36.24 ±1.4

57.54 ±2

80.98 ±2.2

45.04 ±3

52.40 ±2.8

78.43 ±2.4

19.88 ±1.8

99.29 ±2.7

39.20 ±3

75.13 ±1.2

96.95 ±4.5

56.39 ±2.6

57.42 ±3.7

79.63 ±0.75

15.45 ±3.25

0

0

27.42 ±0.8

0

0

Table 4: Cytotoxicity studies of chitosan and chitosan Schiff bases (1 & 2) on the viability of fibroblast cells Material Viable cells in the Viable cells in the Viable cells in the concentration presence of chitosan presence of Schiff base I presence of Schiff base II (mg) (%) (%) (%) 25

98±0.82

98±0.6

97.5±0.75

50

96±0.6

97.8±0.1.3

97.2±0.98

100

94.5±0.7

95.29±1.7

96.6±0.5

150

92.4±0.4

93.9±1.2

94.3±1.2

200

89±0.68

90±1.6

91.1±1.9

The data are the mean of three determinations and ± is standard deviation (SD).

41