Synthesis and Characterization of Pyridine Acetohydrazide Derivative for Antimicrobial Cotton Fabric

An increase in textile resistance to antimicrobial agents has posed a pressing need for the development of new antimicrobials. Therefore, the antimicrobial characteristics of thiophene and pyridine acetohydrazide derivatives have been developed as novel textile-modified complexes exhibiting antibacterial agents. Synthesis and characterization of pyridyl-thienyl acetohydrazide derivative (AHZ) using NMR (13C and 1H) and FTIR. Modification of cotton fabric (CF) with acetohydrazide (AHZ) and metal chlorides of divalent Cr, Mn, Co, Ni, Cu, and Zn and trivalent Fe, and Cr. SEM-EDX and Fourier-transform infrared were utilized to characterize cellulose-based cotton fabric (CF) attached to AHZ and their metal (M) complexes. Antimicrobial activity was examined against two types of bacteria, namely S. aureus and E. coli, and two types of fungi, namely C. albicans and A. flavus. All modified samples exhibited higher efficiency towards bacterial strains than fungal strains. In addition, cellulose modified with Ni (II) confers the most antibacterial protection efficiency.


Introduction
Due to the increasing number of microorganisms that are resistant to antibiotics, there is a significant need to develop novel antibacterial agents other than commercially available compounds to combat this [1]. Cellulose, which is the main component of cotton fabric, is the most common type of natural polysaccharide derived from algae, trees, plants, and bacteria. Cotton is a biodegradable, natural, hydrophilic cellulose-based fiber with OH functional groups that has numerous benefits in textile and biomedical engineering [2,3]. Cotton fabrics encourage the growth of germs like bacteria and fungi [4]. The most promising area for new textile materials is medical fabrics with antibacterial properties. Significant efforts are being made to enhance materials and techniques that could provide secure and efficient defense from various bacteria [5]. Therefore, it is essential to understand cotton fabrics' (CF) antibacterial properties. Numerous antibacterial materials have been imparting antibacterial properties and improving them, including nanomaterials [6], reduced graphene oxide/silver nano complex [7], chitosan [8], and curcumin/titanium dioxide nanocomposite [5].
There is a lot of curiosity about the biological activity of hydrazides (R-CO-NH-NH 2 ), such as their antibacterial, antifungal, and antitumoral effects [9]. Many ligands of amino acids hydrazide derivatives have been reported to act as models for biologically significant species such as metalloenzymes, making them vital in the advancement of bioinorganic chemistry. These complexes are well-known for their outstanding functions in biological, analytical, therapeutic, and industrial applications in addition to their vital roles in catalysis, drug dealing, and chemical synthesis [10]. Recently the ability of complexes of acetohydrazide derivatives to interact with DNA more than the free ligands has been demonstrated [11].
3rd step: preparation of AHZ ligand (3) Hydrazine hydrate (99%) was added to a solution of the ester 2 (5 mmol) in absolute EtOH (25 mL), and the solution was refluxed for 4 h. The solvent was removed using a Hydrazine hydrate (99%) was added to a solution of the ester 2 (5 mmol) in absolute EtOH (25 mL), and the solution was refluxed for 4 h. The solvent was removed using a rotary evaporator. The resulting precipitate was filtered, dried, and crystallized to produce the yellow crystals of AHZ compound 3. Scheme 1. preparation of pyridyl-thienyl acetohydrazide derivative.

Coating of Cotton Fabric (CF) with AHZ and M-AHZ
For 3-4 min at (50 °C, 40 kHz), 0.1 g of the prepared ligand was sonicated in thirty ml of DMF. In total, 1 g of cotton textile was soaked in the previous solution and sonicated for 30 min. Samples were then dried and produced cotton fabric-based cellulose modified with acetohydrazide (AHZ-CF). After that, 0.1 g of metal salt (Zn, Ni, Cu, Co, Cr, Mn, or Fe) was added to the mixture. Once again, the AHZ-CF sample was submerged in solution while being stirred constantly under sonication for 30 min. After removal, washing in distilled water, and drying, the resulting cotton fabric (CF) treated with AHZ metal complexes was obtained (M-AHZ-CF); M is Co, Cr, Mn, Cu, Fe, Ni, or Zn) [17].

Instruments
The 1 H and 13 C NMR spectroscopies were carried out using a Bruker spectrometer at 850 MHz (Billerica, MA, USA). The solid-state Fourir-transform infrared (FTIR) spectra were carried out using an Agilent spectrometer (Cary 600) in the 4000-400 cm −1 wavenumber range (Santa Clara, CA, USA). An SEM analysis was implemented by means of a VEGA3 (Tescan, Brno, Czechia). An energy-dispersive X-ray (EDX) was achieved by using JEOL JSM-7100F (EDX, Oxford X-act, Tokyo, Japan).

Tensile Strength
The test method (ASTM D-1682-94, (1994) [22] was utilized to determine the tensile strength of the fabric specimens. Two specimens were examined in the warp direction to measure the breaking load (Lb) of each modified fabric, and the average value was reported. Scheme 1. Preparation of pyridyl-thienyl acetohydrazide derivative.

Coating of Cotton Fabric (CF) with AHZ and M-AHZ
For 3-4 min at (50 • C, 40 kHz), 0.1 g of the prepared ligand was sonicated in thirty ml of DMF. In total, 1 g of cotton textile was soaked in the previous solution and sonicated for 30 min. Samples were then dried and produced cotton fabric-based cellulose modified with acetohydrazide (AHZ-CF). After that, 0.1 g of metal salt (Zn, Ni, Cu, Co, Cr, Mn, or Fe) was added to the mixture. Once again, the AHZ-CF sample was submerged in solution while being stirred constantly under sonication for 30 min. After removal, washing in distilled water, and drying, the resulting cotton fabric (CF) treated with AHZ metal complexes was obtained (M-AHZ-CF); M is Co, Cr, Mn, Cu, Fe, Ni, or Zn) [17].

Instruments
The 1 H and 13 C NMR spectroscopies were carried out using a Bruker spectrometer at 850 MHz (Billerica, MA, USA). The solid-state Fourir-transform infrared (FTIR) spectra were carried out using an Agilent spectrometer (Cary 600) in the 4000-400 cm −1 wavenumber range (Santa Clara, CA, USA). An SEM analysis was implemented by means of a VEGA3 (Tescan, Brno, Czechia). An energy-dispersive X-ray (EDX) was achieved by using JEOL JSM-7100F (EDX, Oxford X-act, Tokyo, Japan).

Tensile Strength
The test method (ASTM D-1682-94, (1994) [22] was utilized to determine the tensile strength of the fabric specimens. Two specimens were examined in the warp direction to measure the breaking load (Lb) of each modified fabric, and the average value was reported.

The Add-On (%) Loading
The equation was used to calculate the add-on where (W 1 ) and (W 2 ) are the pre and post treatment weights of specimens of the CF, respectively.

Antimicrobial Efficiency
The coated fabrics with the AHZ and M-AHZ were tested for antimicrobial activity against various strains of Gram-negative bacteria Escherichia coli (E. coli) and Grampositive bacteria Staphylococcus aureus (S. aureus), as well as fungal strains Candida albicans (C. albicans) and Aspergillus flavus (A. flavus) using the disk diffusion method [5]. where (W1) and (W2) are the pre and post treatment weights of specimens of the CF, respectively.

Antimicrobial Efficiency
The coated fabrics with the AHZ and M-AHZ were tested for antimicrobial activity against various strains of Gram-negative bacteria Escherichia coli (E. coli) and Gram-positive bacteria Staphylococcus aureus (S. aureus), as well as fungal strains Candida albicans (C. albicans) and Aspergillus flavus (A. flavus) using the disk diffusion method [5].

FTIR Spectra of AHZ
The ligand's significant FT-IR spectral bands are shown in Figure 3. The spectrum of the AHZ appeared medium band in the range of (3183-3324 cm −1 ), which is conformable to NH 2 and NH groups, and a strong band at 2218 cm −1 concerning the C≡N group [23], whilst the strong band at 1676 cm −1 concerning the C=O group of acetohydrazide [9], and strong bands at 1610, 1021 cm −1 concerning C=N and N-N of the pyridine ring and acetohydrazide [24,25], respectively. At 819 cm −1 , a strong band induced by (C-S) thiophene stretching appeared [26].

FTIR Spectra of AHZ
The ligand's significant FT-IR spectral bands are shown in Figure 3. The spectrum of the AHZ appeared medium band in the range of (3183-3324 cm −1 ), which is conformable to NH2 and NH groups, and a strong band at 2218 cm −1 concerning the C≡N group [23], whilst the strong band at 1676 cm −1 concerning the C=O group of acetohydrazide [9], and strong bands at 1610, 1021 cm −1 concerning C=N and N-N of the pyridine ring and acetohydrazide [24,25], respectively. At 819 cm −1 , a strong band induced by (C-S) thiophene stretching appeared [26].

FTIR Spectra of AHZ
The ligand's significant FT-IR spectral bands are shown in Figure 3. The spectrum of the AHZ appeared medium band in the range of (3183-3324 cm −1 ), which is conformable to NH2 and NH groups, and a strong band at 2218 cm −1 concerning the C≡N group [23], whilst the strong band at 1676 cm −1 concerning the C=O group of acetohydrazide [9], and strong bands at 1610, 1021 cm −1 concerning C=N and N-N of the pyridine ring and acetohydrazide [24,25], respectively. At 819 cm −1 , a strong band induced by (C-S) thiophene stretching appeared [26].

The Proposed Mechanism between CF and AHZ Derivative
Scheme 2 shows the suggested interaction between the cellulose CF and the acetohydrazide complex. The acetohydrazide ligand and cotton fabric cellulose molecules interact primarily through H-bonding and a weak Vander-Wall interaction between the amino group of the AHZ and the OH groups of the cellulose molecules. Upon complexation, the participation of the C=O, NH 2 of the AHZ molecule, and OH group of cellulose chains in binding to the metal ions. Coordination bonds are created between the OH groups of cellulose chains and metal ions. As a consequence, a complex is created between AHZ and the cellulose structure, using the metal ions attached in the cellulose chain via coordination bond.

The Proposed Mechanism between CF and AHZ Derivative
Scheme 2 shows the suggested interaction between the cellulose CF and the acetohydrazide complex. The acetohydrazide ligand and cotton fabric cellulose molecules interact primarily through H-bonding and a weak Vander-Wall interaction between the amino group of the AHZ and the OH groups of the cellulose molecules. Upon complexation, the participation of the C=O, NH2 of the AHZ molecule, and OH group of cellulose chains in binding to the metal ions. Coordination bonds are created between the OH groups of cellulose chains and metal ions. As a consequence, a complex is created between AHZ and the cellulose structure, using the metal ions attached in the cellulose chain via coordination bond. Scheme 2. The proposed interaction mechanism between the cellulose-based CF and metal complexes. Scheme 2. The proposed interaction mechanism between the cellulose-based CF and metal complexes.

FT-IR Spectra of the Modified Cotton
The adsorption of AHZ and M-AHZ on the surface of CF is studied using the FT-IR spectral method. Figure 4 and Table 1 show the spectra of blank cotton fabric, as well as AHZ and complex modified CFs. A peak appeared in the (3200-3500 cm −1 ) range for unmodified CF, which can be assigned to O-H stretching. The cellulose vibrations of C-H stretching and C-H bending were attributed to the weaker peaks at 2904 cm −1 and 1371 cm −1 [27]. While an appearance of O-H, C-O-C, and C-O vibrations caused the cellulose bands to appear in the 1500-800 cm −1 range [7]. Moreover, the spectra of modified CFs showed characteristic peaks related to cellulose structure, in addition to the peaks of the AHZ. Figure 4 shows that AHZ-CF has a new peak at 1652 cm −1 , which corresponds to the stretching C=O group of the ligand. Furthermore, AHZ-CF has broadband around 3468 cm −1 and 3191 cm −1 [28], which could be due to vibrational (NH) stretching vibration for a ligand latent behind the hydroxyl band CF. In this instance, hydrogen bonding dominates the interaction between the acetohydrazide ligand and the cellulose molecule, with a minor Van-der-Wall interaction between the NH 2 group of the AHZ and the hydroxyl groups of the cellulose structure. C=O, NH 2 , and OH groups are shifted to lower wavenumbers due to complexation with metal ions [29,30]. The participation of the NH 2, C=O of the AHZ, and the OH group of cellulose in binding to the metal ion is supported by these shifts. Furthermore, all metal complexes have peaks M-Cl, M-O, and M-N in the range of 592 to 434 cm −1 which overlap with peaks of CF [31,32].
nor Van-der-Wall interaction between the NH2 group of the AHZ and the hydroxyl groups of the cellulose structure. C=O, NH2, and OH groups are shifted to lower wavenumbers due to complexation with metal ions [29,30]. The participation of the NH2, C=O of the AHZ, and the OH group of cellulose in binding to the metal ion is supported by these shifts. Furthermore, all metal complexes have peaks M-Cl, M-O, and M-N in the range of 592 to 434 cm −1 which overlap with peaks of CF [31,32].

SEM/EDX Analysis
The surface morphology of cotton fabrics (CF) with acetohydrazide ligand and its metal complexes were evaluated using scanning electron microscopy (SEM). Figure 5a displays the SEM of the blank cotton fabric, which reveals the appearance of a smooth surface [33,34], whereas Figure 5b-i shows agglomerated particles on the coated cellulosic fiber surface (CF) with acetohydrazide (AHZ) and the CF modified with acetohydrazide metal complexes (M-AHZ-CF; M is Cu, Ni, Zn, Co, Cr, Mn, or Fe) as a result of the modification of CF. Figure 6 also shows the results of an EDX analysis of coated cellulose cotton fabric (CF) with acetohydrazide ligand (AHZ) and AHZ metal complexes. The untreated fabric (Figure 6a) demonstrates that only carbon and oxygen are present in the fabric's composition. The deposition of the acetohydrazide on the cellulosic fiber is  Figure 6b. Figure 6b-i show Zn, Ni, Cu, Co, Cr, Mn, and Fe metals with nitrogen and sulfur, which is evidence that the CF was successfully modified with AHZ metal complexes [34].

Antimicrobial Studies
Using the disk diffusion method, the microbial activity of unmodified and modified CFs were tested against representative pathogens, including Gram+ (S. aureus) and Gram-(E. coli). Also, all samples were tested against a variety of fungal strains, namely (A. flavus) and (C. albicans). As shown in Table 2 and Figure 7, all treated samples of cotton fabric had an antibacterial action with a zone of inhibition ranging from 10 to 21 mm. It is clear from Table 2 that all modified CFs with AHZ and its M-AHZ complexes had almost a greater response to Gram-positive than Gram-negative, and variance in bacterial cell wall organization structure may account for this. It was noticed that the ligand modified cotton fabric was found to have moderate activity against Gram-positive (S. aureus) bacteria, whereas low activity against Gram-negative (E. coli) bacteria. When compared to the AHZ-CF, cotton fabric modified with metal complexes was found to have remarkable antimicrobial activity. The Ni-AHZ-CF complex demonstrated the highest antibacterial activity against both Gram-and Gram+ bacteria, followed by the Zn-AHZ-CF, as well as the Fe-AHZ-CF displayed the lowest antibacterial activity when compared to the other M−AHZ-CF. The order of antibacterial effects is as follows: Ni-AHZ-CF > Zn-AHZ-CF > Cu-AHZ-CF > Co-AHZ-CF > Cr-AHZ-CF, Mn-AHZ-CF > Fe-AHZ-CF, as presented in Figure 8. The improved performance of M-AHZ complexes can be demonstrated by the chelation theory [35][36][37]. This indicated that upon complexation, the polarity of the metal ion is diminished as a result of its positive charge being shared partially with donor groups. In addition, it boosts the complex's lipophilicity and increases the delocalization of π-electrons across the entire chelate ring. This increased lipophilicity improves the complexes' ability to penetrate the bacterial cell membrane's lipid layer and hinders the M-binding sites on the enzymes of microorganisms. On the other hand, Table 2 also shows the antifungal activity of all treated samples. Ligand-modified cotton fabric has no activity against both fungal strains, (A. flavus) and (C. albicans). Metal complexes modified cotton fabric were found to have a higher activity against C. albicans than A. flavus. All complexes had no activity against A. flavus. Zn-AHZ-CF was found to possess higher antifungal activity against C. albicans compared with all the studied samples of cotton fabric-modified complexes (Figure 7). The add-on values represent the amount of chemicals used during the modification process that were deposited on the CF. Table 3 shows add-on percentage results. The outcomes presented that the add-on values for modified CF with AHZ were 2.1% while the M-AHZ modified fabrics show much larger add-on values ranging from 4.5% to 9.4%. In contrast, the treated samples' tensile strength values have significantly decreased. This could be explained by the various modifications made to cotton fabric.  The add-on values represent the amount of chemicals used during the modification process that were deposited on the CF. Table 3 shows add-on percentage results. The outcomes presented that the add-on values for modified CF with AHZ were 2.1% while the M-AHZ modified fabrics show much larger add-on values ranging from 4.5% to 9.4%. In contrast, the treated samples' tensile strength values have significantly decreased. This could be explained by the various modifications made to cotton fabric.  The add-on values represent the amount of chemicals used during the modification process that were deposited on the CF. Table 3 shows add-on percentage results. The outcomes presented that the add-on values for modified CF with AHZ were 2.1% while the M-AHZ modified fabrics show much larger add-on values ranging from 4.5% to 9.4%. In contrast, the treated samples' tensile strength values have significantly decreased. This