Novel Chitosan Polymer Design, Synthesis Using Mentha piperita of ZnO NPs as a Catalyst: Antibacterial Evaluation against Gram-Negative Multidrug- Resistant Pathogens

The goal of this research is to create a novel Schiff base of chitosan polymer derivatives 1a-1j. Nanotechnology is a promising field since it avoids the usage of hazardous chemicals while also saving time. Using the leaf extract of the pharmacologically valuable herb Mentha piperita, we described a green synthesis of ZnO NPs. Zinc oxide ions may be easily reduced into ZnO NPs using a Mentha piperita extract. ZnO NPs were employed as a phytocatalyst in this investigation to make chitosan derivatives. The synthetic procedure is straightforward, with a short reaction time and a high yield. Our newly synthesized compounds have been characterized by FTIR and nuclear magnetic resonance spectroscopy (H NMR and C NMR), and morphology analysis was observed by XRD, SEM, and TEM. In addition, the antibacterial activity was also evaluated against gram-positive bacteria and gram-negative bacteria. Compound 1b is extremely active against gram-negative bacteria (4.0 μg/mL, E. coli), and compound 1h is highly active against gram-positive bacteria (6.0μg/mL, S. aureus) compared with standard erythromycin and other chitosan derivatives. As a result, compounds 1b and 1h could be a high crucial molecule in the development of antibacterial drugs.


Introduction
Antibacterial activity of biopolymers has been broadly studied for the last years. The (CH) (-1-4)-d-glucosamine) is the deacetylated structure of chitin, which is extracted from natural and marine animals [1,2]. Chitin can be isolated from microorgans [3], crustacean [4], and bug [5]. Chitin deacetylation improves its solubility in acid medium while additionally increasing its antibacterial motion [6,7]. Antimicrobial resistance is one of the third most serious concerns affecting human health, according to the WHO [8]. Recent studies have combined antibacterial nanoparticles with chitosan-linked ZnO composites, which have shown to have good antibacterial properties [9]. Chitosan, an adaptable hydrophilic polysaccharide derived from chitin, has a vast antibacterial range, making it prone to each gram-negative and gram-positive microorganism [10]. The biopolymer is widely employed in bioinformatics applications as a drug transporter [11], antibacterial [12], antioxidant [13], anticancer [14], and wound dressing agent [15,16] due to its unique qualities such as harmless, antibacterial activity, decomposable, and high biocompatibility [17,18]. Over the last decade, nanomedicine has developed rapidly, and it now provides promising treatments for bacterial infections. Because nanoparticles behave differently from their parent bulk, nanotechnology opens up new possibilities for medicinal applications [19][20][21][22]. The size and size distribution of nanoparticles, as well as their composition, shape, surface charge, surface chemistry, and surface functionalization, hydrophobicity, crystalline phase, crystallite size, and porosity, are all important factors that influence the physicochemical properties and, as a result, the activity of nanomaterials [23,24]. Nanoparticles with antibacterial properties can act as antibacterial agents or transporters, increasing antibiotic bioavailability and effectiveness [25]. Metallic nanoparticles, primarily composed of zinc, silver, or copper, have been found to have potent antibacterial properties [26]. The antibacterial drug was prepared using low cost, environmental friendliness, synthesizability in ambient atmosphere, and nontoxicity. A recent study has shown that green synthesis is the most effective method for synthesizing NPs [27]. Furthermore, ZnO NP is being used in biological and environmental sectors such as pharmaceutical administration, biological sensing, biological labelling, gene transfer, and nanomedicine [28][29][30][31]. ZnO NPs have also been reported to have antibacterial, antifungal, acaricidal, prediculicidal, and larvicidal properties [32][33][34][35][36]. Chitosan and its derivatives are referred to as ecological cleansing useful resources because they efficiently restrict the growth and reproduction of harmful bacteria as well as poisonous contaminants [37]. Physicochemical and biochemical features of chitosan derivatives include good antimicrobial activity, hygroscopic, and embolism. As a result, it has potential uses in a variety of disciplines, including agriculture, medicines, cosmetics, food processing, environmental protection, and biotechnology [38][39][40][41][42][43][44][45][46][47]. The chitosan molecule's containing enormous number of primary amine and hydroxyl groups allows for a wide range of chemical changes, resulting in a new class of biomaterials [48]. Chemical modifications to chitosan, such as sulfonation [49], amination [50], and carboxymethylation [51], allow for chemical transformation of the NH 2 and OH groups and the creation of different functional derivatives. Modified diisocyanate has been shown to have increased antibacterial activity [52][53][54][55][56][57]. The antibacterial activity of chitosan derivatives is shown in Figure 1. Similarly, the important functional groups in chitosan can readily react with amine groups to generate the appropriate chitosan Schiff base with imine characteristic group (-RC=N-). Chitosan Schiff base derivatives are one of the finest possibilities for improving antibacterial activity of chitosan [58]. Based on the above literature report, all the research work was done by using several amines but none of the researchers are not using the ethylenediamine react with C-2 position in chitosan, Schiff base formation in C-5 position, and green catalyst of ZnO NPs was used for our research work. The present work addresses the development of antibacterial aminoethyl chitosan derivatives and was characterized by Fourier transform infrared (FT-IR) and nuclear magnetic resonance ( 1 H NMR and 13 C NMR) spectroscopy, and morphology and its physical properties were observed by TEM, SEM, and X-ray diffraction (XRD). Additionally, their antibacterial activity was evaluated against a various pathogenic microorganisms.

Materials and Methods
2.1. General Methods. All the chemicals and reagents such as chitosan (degree of deacetylation-75%, Mw141 kDa), acetic acid, EDA, aromatic aldehydes, ethanol, and DMF are bought from Sigma Aldrich, India. Melting points of synthesized compounds were recorded in open capillary tubes and are uncorrected. On a Shimadzu 8201pc (4000-1000 cm -1 ), the FT-IR spectra were captured using the kBr disc approach. A Bruker DRX-300 MHz was used to record the 1 H NMR and 13 C NMR spectra. To obtain NMR spectra, the compound was dissolved in DMSO-d 6 . The morphology of zinc oxide nanoparticles is confirmed by using XRD, SEM, and TEM. The scanning electron microscope (SEM) model VP-1450 (LEO, Co., Germany), was used for SEM analysis. For transmission electron microscopy (TEM) analysis, an LEO 912 AB instrument was used. TLC was used to assess the purity of the compounds, with silica-gel as the adsorbent and 60F254 aluminium sheets as the adsorbent, and it was visualized by ultraviolet (E-Series UV hand lamp (254/365 nm wave length)).  [59]. The samples were withdrawn from the media, wiped with filter paper, and weighted once they had reached equilibrium swelling (1 day). Each sample's swelling ratio was calculated using the following equation: where W t and W 0 represent the weight of the swollen and dry samples, respectively.

Preparation of Mentha piperita Leaf Extract. 5 grams of
Mentha piperita leaves was rinsed thoroughly with distilled water and sanitized with alcohol using a delicate scouring technique. These leaves were warmed in 100 mL of distilled water at 60°C for 45 minutes. After that, Whatman 41 filter paper was used to sieve the extract. A cool and dry place was used to store the filtrate.

Synthesis of Zinc
Oxide Nanoparticles. 20 mL Mentha piperita leaf extract was mixed with 60 mL 20 percent NaOH solution to make zinc oxide nanoparticles via precipitation reaction procedure. Then, in a 250 ml measuring glass, 5 mL portions of that combination and 60 ml refined water               Step-1 Aceticacid, EDA, 4hr 100 0 C Scheme 1: Synthesis of chitosan derivatives 1a-1j using ZnO NPs as a catalyst. 6 Journal of Nanomaterials

In Vitro Antimicrobial
Screening. Using a previously reported approach [60], the antibacterial activity of the compounds 1a-1j was evaluated against the bacterial strains Staphylococcus aureus (ATCC-25923), Streptococcus pneumoniae, Escherichia coli (ATCC-25922), and Pseudomonas aeruginosa (ATCC-27853). The minimal inhibitory concentration for each of the produced substances was determined.
The test samples were dissolved in DMSO (dimethylsulfoxide) at a concentration of 64 μg/mL for each sample. Various dilutions (64, 32, and 0.5 μg/mL) were made using twofold dilutions. The microbe suspensions containing 106 CFU/ mL were injected into the matched wells and incubated at 36°C for 24 hours.

Results and Discussion
3.1. Characterization of Zinc Oxide Nanoparticles 3.1.1. Transmission Electron Microscopy (TEM). The size and morphology of the ZnO nanoparticles were determined using a TEM examination. Figure 3 shows a TEM image of both pure ZnO and ZnO NPs. In the range of 50 nm, the TEM image clearly shows the spherical shape of the ZnO NPs.

XRD (or) X-Ray Diffraction
Study. An X-ray diffraction investigation was carried out on chitosan and chitosan derivatives. Figure 5 [61] shows chitosan with very wide peaks at 2θ = 15°and 2θ = 30°. The chitosan derivatives displayed two faint peaks at 2θ of 15°and 30°in Figure 5. The peaks establish for chitosan at 2θ = 15°vanished in chitosan derivatives while the very broad peaks at 30°became weak, showing that chitosan has moral compatibility and good formation of porous xerogel network. According to the XRD results, the chitosan derivatives are amourphous in nature.

Recovery of Catalyst.
Catalyst recovery is important in the biosynthetic process. We tested their recyclability twice with essentially equal catalytic activity using the Schiff base of chitosan derivative (1a-1j) zinc oxide nanoparticle reaction. Following the addition of DMF, the ZnO NPs were centrifuged and washed with EtOH to remove any residual product. (1, 1a-1j). Chitosan (2 g, deactylated) was dissolved in 20 ml of 1% acetic acid solution (pH = 3:6 -3:7), and ethylenediamine (0.7 mL) was reacted at 100°C for 4 hrs using ZnO NPs as a green catalyst to get compound 1. After adding 1 M NaOH for adjusting the (pH = 4:0) of compound 1. A compound 1 (2.5 g dissolved in acetic acid with ethanol) and 4flurobenzaldehyde (1.2 g) in ethanol stirring for 8 hrs at 60°C with ZnO NPs used as a catalyst. The obtained white gel indicates the formation of compound 1a. The resulting product was precipitated in a solution of 5% sodium hydroxide. The precipitate was filtered and washed with ice-cold

Fourier Transform Infrared (FTIR) Spectroscopy.
In FTIR spectra of chitosan, NH peak was observed at 3674 cm -1which can be assigned to stretching vibration of amino group, and another peak observed at 3278 cm -1 can be attributed to hydroxyl group (O-H, str). Similarly, the stretching vibration of carbon and hydrogen in aromatic ring was observed at 2987 cm -1 . The weak band at 2900 cm -1 can be assigned to symmetric stretching of carbon-hydrogen (C-H, str), and a sharp characteristic peak at 1645 cm -1 can be attributed to stretching vibration of imine linkage (N=CH, str). In the chitosan-Schiff base, the bending vibration of amide observed at 1405 cm -1 can be assigned to (NH 2 , bending), and another sharp peak at 1393 cm -1 can be attributed to stretching vibration of (N-C, str). The FTIR spectrum of compound 1a is present in Figure 6.
3.3. In Vitro Antibacterial Activity Analysis. The antibacterial activity of chitosan derivatives of 1a-1j was tested in vitro against S. aureus (ATCC-25923), S. pneumoniae, E. coli (ATCC-25922), and P. aeruginosa (ATCC-27853). As a control, erythromycin was utilized. Compound 1b (4.0 μg/ mL, E. coli) is highly active, and compound 1h (6.0 μg/mL, S. aureus) is highly active compared with the standard drug. The antibacterial assay images are present in Figure 7, and MIC values are shown in Table 1. 3.4. Structure Activity Relationship. The structure activity relationship is a relationship that exists between a molecule chemical structure and its antibacterial activity. SAR analysis allows for the identification of chemical groups responsible for inducing antibacterial activity in the organism. The SAR was evaluated using the antibacterial activity of chitosan derivatives. The compounds 1b and 1h are highly active compared with control erythromycin and other compounds. Because of the reason due to the presence of chloro (Cl) and hydroxyl (-OH) groups at para position in phenyl moiety, it was exhibited to be highly active against 1b (4.0 μg/mL, E. coli, gram −ve) and 1h (6.0 μg/mL, S. aureus, gram +ve) bac-teria when compared to standard erythromycin. Figure 8 demonstrates the SAR of highly active compounds.

Conclusions
Chitosan possesses different functional groups (i.e., hydroxyl and amine groups) that help to prepare new chitosan derivatives. The present study intends to develop newly synthesized Schiff base of chitosan polymer derivatives 1a-1j so we are focused the green synthetic method for the synthesis of zinc oxide nanoparticles (ZnO NPs) using extract of Mentha piperita, which can reduce zinc oxide ions into ZnO NPs in a simple way. The newly formed Schiff bases 1a-1j were synthesized using ZnO NPs as a catalyst and were confirmed by FTIR, NMR, TEM, SEM, and XRD. Moreover, the antibacterial activity revealed that compound 1b (4.0 μg/ mL, E. coli) is highly active against gram-negative bacteria, and compound 1h (6.0 μg/mL, S. aureus) is moderate active against gram-positive bacteria. In future, compounds 1b and 1h can be developed as an antibacterial drug.

Data Availability
The supplementary file data used to support the findings of this study are included within the supplementary information files.