Hybrid Chitosan/CaO-Based Nanocomposites Doped with Plant Extracts from Azadirachta indica and Melia azedarach: Evaluation of Antibacterial and Antibiofilm Activities

This study examines substances from plant leaves of Azadirachta indica and Melia azedarach extracted with methanol (80%) and then successively fractionated with increasing gradient polarity solvents. The methanol extract from the two plant leaves was selected to be incorporated with nanocomposites because of their high antimicrobial activities. HPLC analysis was performed for the determination of total phenolics and flavonoids. LC–ESI–MS positive ion acquisition mode revealed the presence of four main chemical classes: limonoids, triterpenes, fatty acids, and phenolics. Sol–gel processes were adapted to prepare CaO/chitosan composite loaded with the methanol extract of the two plant leaves, acting as innovative antibacterial agents. The fabricated nanocomposites were characterized by XRD, TEM, SEM, and FTIR methods. Minimal bactericidal concentrations (MBC) were established by agar plating using bacteria treated with MIC and at least 2 concentrations double higher than the MIC. Biofilm was cultivated in the presence of sub-MICs of the extracts and the nanocomposites, followed by quantification with the crystal violet assay. The examined chitosan–CaO-based hybrid nanocomposites loaded with the methanol fractions from the leaves of A. indica and M. azedarach had an excellent performance as biofilm inhibitors. This indicates their good potential for biomedical applications.


Introduction
Azadirachta indica L. and Melia azedarach L. are members of the Meliaceae family (the mahogany family). They have an essential role as health-promoting agents due to their richness of a variety of antioxidant compounds. They have been widely used in traditional Chinese and Ayurvedic medicines worldwide, especially in India, to treat and prevent various diseases. A. indica is traditionally known as neem. It has one of the two species in the genus of Azadirachta. It is native to India, Thailand, and Nepal. It cultivates in tropical and sub-tropical areas. Such plant neem is considered the most important medicinal plant, declared worldwide as the "Tree of the twenty-first century" by the United Nations. It is called "Divine Tree" and "Nature's Drugstore" [1]. The chemical components found in the neem leaves encompassing were nimbin, nimbanene, 6-desacetylnimbinene, nimbandiol, nimbolide, ascorbic acid, n-hexacosanol, and amino acid, 7-desacetyl-7-benzoylazadiradione, 7-diacetyl-7-benzoylgedunin, 17-hydroxyazadiradione, and nimbiol. Due to its medicinal importance, neem is used to prepare formulated medicals to treat various human ailments [2]. From a therapeutic point of view, the crude extracts from the bark and leaves were utilized in folk medicine to prevent Tsvetelina Paunova-Krasteva and Bahaa A. Hemdan contributed equally to this work infectious diseases such as leprosy and intestinal and respiratory worms. Besides, many other reports on biological and pharmacological actions include antiviral, antibacterial, antifungal, anti-inflammatory, antipyretic, antiseptic, and cytotoxic activities [3].
Melia azedarach L. is a botanical species commonly named as China tree, chinaberry tree, Indian lilac, and white cedar. It is native to Asia and some regions of Northern Australia and Africa. It is regularly utilized as an antiparasitic and antifungal agent. Still, many of its constituents have played a significant role in the free radical scavenging and prevention of disease pathogenesis. The major bioactive constituents are recognized as limonoids [4].
Nanotechnology is considered one of the major significant sophisticated research technologies dealing with synthesizing, engineering, and applying nanoparticle (NPs) structures between 1 and 100 nm [5][6][7]. Nanobiotechnology is a division of modern nanotechnology and an unfettered novel generation of material science receiving worldwide attention owing to its practically various applications [8]. The nanobiotechnology approach serves as an essential technique related to chemistry, physics, biology, biochemistry, and medicine. It aims to exploit nontoxic, eco-friendly, stable, and reactive procedures for the assemblage of NPs possessing the intrinsic ability to decrease metals by particular metabolic pathways.
A naturally available polymeric biological material, chitosan (Cs), is recognized. It is generated by hydrolytic deacetylation from chitin material. Biopolymer Cs has been employed as a biocomposite super-disintegrant substance to produce a variety of drug, gene, protein, and protein and other substance delivery systems because of its highly desired medical and biological properties, such as renewability, biocompatibility, environmental friendliness, chemical versatility, capability to produce hydrogel and films, and high adsorption ability. Further, tissue engineering has made substantial use of nanocomposites derived from chitosan. Due to their antibacterial qualities, chitosan-based hydrogels, sponges, and nanocomposite films have been widely applied in healing [9]. One of the chitosan's appealing qualities is its antimicrobial characteristics towards various bacterial, fungal, and viral diseases. This enables chitosan to be beneficial for biological applications like fabrics, medicine, biomedical engineering, farming, and ecological sustainability [10].
Nanoparticles of CaO are essential materials because they have several applications as bio-integrated systems, biocatalysts, and chemisorbents for toxic gases. CaO nanoparticles were prepared by sol-gel method using Ca(NO3)2.H2O as Ca precursor. So, controlled sol-gel manufacture of Ca oxide nanoparticles is influential for highly effective applications [11,12]. It is reported that the sol-gel solution method provides a high degree of control through the fabrications yields [13,14]. Furthermore, the low-temperature sol-gel fabrication and the ability of organic molecule loading significantly affect the size, uniformity, and shape properties of the formed particles [15]. The facility of chitosan to link various metal ions is mainly due to its primary and secondary amine groups, which could serve as good coordination sites for different metal ions [16].
The amplitude of metal adsorption depends on the ease of the metal ion, the chitosan source, the level of deacetylation, and the blend conditions such as metal type and pH. Thus, the metal adsorption on the chitosan matrix is highly applied in many experimental procedures to assess its adsorption capacity under various conditions due to possessing carboxyl, amine, and hydroxyl functional groups, which chelate with metals, pollutants, and abolish toxically materials [17,18]. The availability of blends, chemical composition, nanoparticles, and polymers are potential sources of filler for chitosan composites appropriate for bio-application. Chitosan with CaO nanoparticles represents a current class of high-performance composites and is of outstanding academic and manufacturing attention [19]. In the present work, we prepared CaO/chitosan bio-nanocomposite loaded with two extracts, P3 and P6, and evaluated their impact. In detail, the prepared nanocomposites were characterized by XRD, TEM, SEM, and FTIR. To estimate the potential of the nanocomposites for biomedical applications, the antimicrobial and antibiofilm properties of the nanocomposites were characterized.

Chemicals
Chitosan (deacetylation rate 95%, the average molecular weight of (6.0 × 105) g mol −1 ), calcium nitrate (Ca(NO 3 ) 2 -4H 2 O), acetic acid (ACS reagent, 99.7%), ethylene glycol, and distilled water were used. All chemicals were procured from Sigma-Aldrich Ltd. and used as received. All solvents used in this present study, such as petroleum ether (40-60 °C), chloroform, ethyl acetate, and methanol, were obtained from BDH, UK. The DPPH (2,2-diphenyl-1-picrylhydrazyl) was obtained from Sigma-Aldrich. Targeted reference microbial strains used in the current study were obtained from American Type Culture Collection (ATCC, USA). Further, all culture media in powder form were purchased from HIMEDIA, India.

Plant materials
Leaves of Azadirachta indica L., and Melia azedarach L., were collected from Orman Garden in Giza, Egypt. The collected leaves of the two plants were air-dried, separately powdered, and kept in tightly closed containers.

Extraction procedure
The powdered leaves (500 g) from each plant were extracted with methanol solvent (80%) using a cold extraction method. After complete extraction, it was adequately filtered, and the methanol solvent was evaporated entirely using a rotary evaporator. The remaining extract was dissolved in distilled water (100 ml). The suspension was transferred into a separating funnel. Then it was extracted successively with petroleum ether (40-60 °C) and chloroform, respectively [20]. As shown in Table 1, different extraction solvents were applied for the perspective extraction process.
Evaluation of antimicrobial activities of all plant extracts was employed against four microbial species. The antimicrobial action was performed using an agar diffusion assay (El Nahrawy et al. 2020b). Afterward, P3 and P6 plant extracts, which have promising antimicrobial potential, were selected according to the antimicrobial activity results.

Determination of total phenolic and flavonoid content
The total phenolic content was determined by the Folin-Ciocalteu reagent (FCR) method. The aluminum chloride method determined the total flavonoid content of leaves crude extracts and fractions based on the quercetin standard curve, and readings of UV absorbance were performed by spectroscopy at 450 nm wavelength [21].

Liquid chromatography-mass spectrometry (LC-MS) studies
ESI-MS positive ion acquisition mode was carried out on a XEVO TQD triple, quadruple instrument (Waters Corporation, Milford, MA 01,757 USA, mass spectrometer, ACQUITY UPLC-BEH C18 as column 1.7 µm, 2.1 × 50 mm column); the flow rate was adjusted at 0.2 mL/min using mobile gradient phase comprising two eluents: eluent A is H 2 O acidified with 0.1% formic acid, and eluent B is MeOH acidified with 0.1% formic acid. Mass spectra were detected in the ESI between 100 and 1000 m/z. The peaks and spectra were processed using the Maslynx 4.1 software and tentatively identified by comparing its retention time (R t ) and mass spectrum with reported data [22].

Preparation of chitosan/CaO nanocomposites
A solution-casting technique prepared the chitosan-based solution. Initially, 3 g of chitosan powder was diluted in 1.0% acetic acid/100 ml distilled water using a stirrer to obtain a homogeneous CS solution at ambient temperature.

Characterization
The XRD pattern investigations of chitosan/CaO composites were carried out on a Diano X-ray diffractometer using Cu (Kα 1 /Kα 2 ) radiation source energized at 45 kV and a Philips X-ray diffractometer (PW 1930 generator, PW 1820 goniometer). The XRD patterns were recorded in a diffraction angle range from 5 to 50°. We used Tecnai-20 transmission electron microscope (TEM) with an acceleration voltage of 200 kV. The surface morphology of chitosan/CaO and loaded with P3 and P6 composites was analyzed using scanning electron microscopy (SEM; JEOL/Noran). Fourier transform infrared (FTIR) spectra chitosan/CaO and loaded with P3 and P6 composites were recorded with a FTIR spectrometer (Nicolet Impact-400 FTIR spectrophotometer) in the range of 200-4000 cm −1 .

Examination of antibacterial activities
The synthesized nanocomposites' antimicrobial efficacy was assessed using the Kirby-Bauer agar diffusion assay against the above mention species [25]. To determine MIC and MBC, the microdilution plate assay was performed on U-shaped 96-well microtiter plates. The test was applied with modification. This comprised the addition of 10% of Alamar Blue reagent (Thermo Fisher), which allowed the estimation of the bacterial viability in the wells. This allowed to measure the amounts of the plant extracts applied. Initially, the weight of 10 µl from each liquid sample was measured. The plant extract samples were then suspended in MHB, and serial dual dilutions were prepared to start from 30 µg/ml as the highest concentration of the samples. Where the MIC could not be determined within this range, the test was repeated with an increase up to 90 µg/ml of the starting concentration. The nanocomposites were initially dissolved as 1 g/ml stocks in DMSO, from which the test suspensions in MHB were prepared. The nanocomposites were tested at serial dual dilutions starting from 10 mg/ml. The wells were inoculated with 20 µl of 1 × 10 4 bacterial cells/ml calibrated by Densilameter II using the McFarland standard, and the plates were incubated for 24 h at 37 °C. The results were red by visual estimation of the change of color from blue to pink. The concentration at the highest dilution that resulted in no modification of the blue color was accepted as MIC. The negative control for the antimicrobial study was sterile distilled water, and the reference drug ciprofloxacin (10 µL) was applied as a positive control [26]. The control for MIC estimation compared the cultivation of the strains in MHB containing DMSO equal to its amount in the corresponding test samples.
To determine the minimal bactericidal concentration (MBC), samples taken with cotton swabs from three sequential wells from the microtiter plate were applied on MHA plates. The wells comprised the one with the established MIC and two wells containing 2 × MIC and 4 × MIC, respectively. The lowest amount of the substances that totally eliminated the presence of viable bacteria, registered as a lack of bacterial growth on the MHA, was accepted as MBC.

Examination of antibiofilm activities
Biofilm growth of the bacteria was checked on 96-well polystyrene U-shaped plates. The tested substances were applied as 1/2 MIC or where MICs could not be strictly determined, at the corresponding highest concentration that was involved in the microtiter plate assay. The nanocomposites were initially dissolved as 1 g/ml stocks in DMSO, from which the test suspensions in MHB were prepared. Overnight, bacterial cultures in MHB were added in the final 1:100 dilution, and the suspensions were applied as a 150 µl quota in the wells, 5 wells per variant. Controls comprised biofilms cultivated in MHB, in MHB plus DMSO in the amount used within the test samples, and negative controls, wells containing aliquots of the tested substance suspensions but with no bacteria. The plates were incubated for 24 h at 37 °C. Then the OD 620 nm of the microplates was determined to register the bacterial growth in the wells' liquid phase (planktons). After that, the plankton was removed, and the wells were washed with PBS to remove non-adherent bacteria. The crystal violet (CV) assay was performed as described in detail elsewhere [27].

Statistical analysis
The experimental data gained were represented as the mean ± standard deviation (SD). The experimental trials were performed three times separately with a significance level of p ≤ 0.05. The quantitative data of biofilm experiments were calculated as % of control, processed on MS Excel software, and represented as average ± standard deviation.

Extraction and characterization of plant extract
The yield of methanol extract of A. indica and M. azedarach was 24% and 27%, respectively. Moreover, the yield of petroleum ether and chloroform fractions of A. indica was 8% and 4% while petroleum ether and chloroform fractions of M. azedarach was 12% and 5%, respectively.
As shown in Table 2, the results of the antimicrobial activities indicated that no inhibition zone appeared in plant extracts P1, P2, P4, and P5. However, the plant extracts P3 and P6 had potential antimicrobial action against all tested pathogenic microorganisms. For this reason, both plant extracts, P3 and P6, were chosen to be loaded in different nanocomposites. Moreover, the results displayed that the antimicrobial ability of both P3 and P6 was more elevated compared to the reference drug ciprofloxacin used as a positive control.

Determination of total phenolic and flavonoid content
The results of total phenolics and flavonoids in the crude extract and different fractions of A. indica and M. azedarach leaves are presented in Table 3, where the highest amount of total phenolics and flavonoids was obtained in the crude extract of the two plants (68.12 mg/g and 381.02 mg/100 g in A. indica and 85.14 mg/g and 390.12 mg/100 g for M. azedarach, respectively). At the same time, the lowest concentrations were observed in petroleum ether and chloroform fractions. Moreover, ten phenolic compounds were identified from A. indica, representing 29.04%, and thirteen were present in M. azedarach crude extracts yielding 27.03%. The primary fatty acids and esters in A. indica and M. azedarach were characterized as palmitic acid (9.27%) and 9,10-dihydroxy-12-octadecenoic acid (11.67%), respectively. Nevertheless, protocatechuic acid (7.64%) and dicaffeoylquinic acid (4.46%) were the main phenolic compounds identified in A. indica and M. azedarach.

LC-ESI-MS analysis of the crude methanol extracts of
Limonoids, a group of highly oxygenated and modified nortriterpenoids, are characteristic components of the plants of the Meliaceae family that exhibit broad antimicrobial activity against Gram-positive and Gram-negative bacteria [28]. Throughout the literature review, a significant antimicrobial effect was proved for fatty acid compositions and steroidal containing extract of many medicinal and edible plants, and the activity of these classes was attributed to their presence. The reported action mode was increasing cell membrane permeability [29]. Previous findings proved that flavonoids exhibited potent antimicrobial activity by inhibiting biofilm formation, membrane disruption, cell envelope synthesis, nucleic acid synthesis, and bacterial motility inhibition [30]. Petroleum ether and chloroform fractions extracted only the non-polar compounds such as sterols, terpenes, and fatty acids. But no extract phenolic and flavonoids have potent and significant activities. The crude methanol extract has all nonpolar and polar compounds, including sterols, terpenes, fatty acids, limonoids, phenolic, and flavonoids, and all these compounds play their antimicrobial activity in synergism pattern. In addition, natural organic bioflavonoids, which have antibacterial features, are a noteworthy ingredient of nutritional supplements. Many bioflavonoids have antitumor, antibacterial, antifungal, and anti-inflammatory impacts, but their efficacy is insufficient due to toxicity at the molecular level, and their antiviral characteristics are negligible. By producing hybrid nanocomposites, the synergistic relationship between nanoscience and flavonoid chemistry improves the epidemiological properties of flavonoids while also lowering antimicrobial resistivity (AMR). For biocidal flavonoids and their administration as antimicrobial agents, nanocomposites and nanomaterials employ various nanocomposites [31].

Sol-gel chitosan/Ca loaded with P3 and P6 reactions
This research investigates bio-sol-gel reactions in nanocomposites using two different natural extracts, P3 and P6. Both extracts contained many compounds with terminal -OH groups and hydrogen bonds that catalyze their linkage in the chitosan matrix in the presence of Ca during the sol-gel production of chitosan-calcium in normal conditions [32]. The Ca-O bonds can be capable hydrogen bond acceptors and could link with other hydrogen bonds from P3 and P6 [24]. Likewise, the presence of chitosan facilitates the hydrolysis of P3 and P6 feedstocks to link through the production of bio-chitosan composite [33,34]. Figure 1 shows the XRD spectra for chitosan/calcium (CS/ Ca) and CS/Ca-P6 nanocomposites dried at 40 °C. The XRD spectra of the CS/Ca illustrate an amorphous structure with some characteristic peaks at 2 theta 8.2-18°, which belong to the pure chitosan structure. Figure 1b shows that the CS/ Ca nanocomposite is partially suppressed upon loading P6.

XRD analysis
In the XRD of CS-Ca nanocomposite, the most substantial  [35]. The XRD shows the grafted CaO being presented into the chitosan matrix and also both P3 and P6. The calcium (CaO) diffraction peaks are narrower than the peak for chitosan, signifying that as CS/CaO and CS/CaO-P6 are formed, smaller particles break off in CS/CaO (definite by TEM), resulting in a fine CaO crystallite size. These results are because of the lower concentration of Ca (low for X-ray detection) embedded in the CS matrix and also the fine size.

TEM
The TEM images of CS/CaO nanocomposite and loaded with P6 are given in Fig. 2a and b. Figure 2 confirms that the nanocomposites are both composed of a mass of irregular CS chains linked to Ca nanoparticles. Figure 2b demonstrates that the addition of the extract P6 inside the chitosan/ Ca relatively changes the morphology of CS/CaO nanocomposite. The TEM images of CS/CaO nanocomposites recorded show their overall amorphous behavior with the presence of small CaO nanoparticles distributed during the nanocomposite formation. Hence, these small CaO NPs are not clearly detected from the XRD patterns.    Figure 4 illustrates the FTIR spectra of chitosan-calcium nanocomposite and loaded with two extracts P3 and P6 samples formed at 30 °C. The bands in the range 449 cm −1 to 990 cm −1 are characteristic for the Ca-O-Ca and Ca-OH bending vibration in chitosan composite [36,37]. The band 801 cm −1 in the spectra of chitosan-calcium was due to CaCO 3 [38]. The formation of the unloaded sample is shown in the range of 800-1200 cm −1 by a complex group of absorption bands. The strong peak at 1059 cm −1 appearing in both CS-Ca and loaded nanocomposites was generally ascribed to the saccharide framework of the chitosan, also coincided with the vibration of Ca-O linkage through the chitosan matrix [39].

FTIR study
The change in FTIR of the chitosan-calcium nanocomposite after P3 and P6 adsorption (Fig. 4) implies a direct shift in some of the characteristic peaks of chitosan and Ca functional groups with the adsorption of P3 and P6. The new peaks intensities at 841 cm −1 , 1341 cm −1 , and 1463 cm −1 in the loaded CS-Ca nanocomposites suggest involving new functional groups in the chitosan-calcium nanocomposite aggregates. The medium bands from 1200 to 1631 cm −1 are assigned to the internal vibration of Ca-OH, water molecules, and chitosan amino group characteristic of these composites [40]. Bands around 2750-3350 cm −1 in chitosan-calcium-based changed after P3 and P6 loading. The FTIR characteristic chitosan peaks in these nanocomposites are 3390 cm −1 (-OH/-NH moiety), 2900 cm −1 (-CH moiety), and 1643 cm −1 (amide I); their intensity increased with loading the Velosef [41]. The bands in this range have resulted from imbricating the stretching vibrations of -NH and -OH functional groups through the hydrogen bonds [42,43]. FTIR of sol-gel chitosan-calcium nanocomposites can be classified as successful adsorption nanocomposite for the removal of cationic moieties, sulfonate group, dyes, and cleaning of wastewater, due to versatile, functional groups capability to compose hydrogen bonds.

Disc diffusion assay
In this study, the antibacterial properties of three examined nanocomposites (CS/CaO, CS/CaO loaded with P3, and CS/CaO loaded with P6) was performed in vitro against E. coli O157, P. aeruginosa, S. aureus, and L. monocytogenes using the agar diffusion assay. The recorded results indicated that the newly synthesized nanocomposites loaded with P3 and P6 of medicinal plant extracts displayed significantly higher potential for inhibiting a broad spectrum of examined bacterial species ( Table 6). The ZOI of the unloaded Cs/ CaO nanocomposite was smaller than the other loaded nanocomposite as the ZOI diameters were 10 ± 0.22, 12 ± 0.24, 9 ± 0.23, and 8 ± 0.14 mm, respectively, for E. coli O157, P. aeruginosa, S. aureus, and L. monocytogenes.

Determination of MIC and MBC values
In the present study, MIC was determined based on the capacity of the strains to reduce resazurin, the active substance in the Alamar Blue kit that was used, to resorufin. This approach providing information for suppressing bacterial metabolic activity was suggested as a test of antibiotic activities [44]. It is now gaining a more comprehensive application, also in estimating the antibacterial potential of a variety of substances, including plant-derived ones [45]. MIC and MBC values are summarized in Table 7. In our experiments, the samples were serially dissolved from a DMSO stock. In these experiments, we had to prepare the solutions in sucs so that the final concentration of DMSO equals or less than 2%. Higher concentrations have been shown to suppress bacterial growth [46].
The microdilution assay test in the presence of the resazurin reagent showed that P3 from A. indica had MIC of 3.75 µg/µl and MBC of 7.5 µg/µl when applied to S. aureus 29,213. However, this extract did not effectively suppress E. coli 25,922 metabolic activity within the used concentration range, up to 90 µg/µl. Previous studies on ethanol and methanol extracts, from the leaves of A. indica have shown promising antimicrobial results on various Grampositive and Gram-negative bacterial strains, including clinical isolates. Substantial differences exist between the MIC values reported in the different publications. Microdilution assays for drug-resistant strains of S. aureus have shown MIC values from 128 to 256 µg/µl and more than 250 µg/ µl [47,48]. The data registered in one of the studies for clinically isolated E. coli strains varied from 64 to 128 µg/µl. Such strain-to-strain differences are not unexpected, and variations in the microbial world's susceptibility or resistance to antibacterials are widespread. Compared to the above-published data, the MIC and MBC data obtained in the present study characterized the Gram-positive model strain S. aureus 29,213 as sensitive and susceptible to the active substances in A. indica methanol extract E. coli 25,922 appeared more resistant.
The results from the microdilution assay showed that the P6 sample from M. azedarach leaves had wider antimicrobial potential than P3 against the model strains. This is in agreement with the trend noted in the disc diffusion assay (Table 2), which was performed with other species or strains of bacteria, and it can thus be summarized that the observation of the higher antibacterial potential of P6 is valid for even more comprehensive range of microorganisms.
A MIC of 5 µg/µl and an MBC of 20 µg/µl could be determined for E. coli 25,922. For S. aureus 29,213, the corresponding values were 2.5 µg/µl (MIC) and 10 µg/µl (MBC). However, despite the extensive common use of M. azedarach in ethnomedicine, the antibacterial activities of the substances isolated from it have only rarely been examined. Previous studies by disc diffusion assays of leaves [48] and seed extracts [49] in different organic solvents confirmed the antibacterial potential against Gram-positive and Gram-negative bacterial strains. In a more detailed study, including MIC and MBC evaluation, Hadadi et al. [50] compared the effects of methanolic and chloroform extracts from the fruits of M. azedarach. Out of the tested by them model bacterial strains, they found out that the methanol extract was effective only against Pseudomonas aeruginosa. Chloroform extracts in the cited study were much more effective; for example, the determined MIC and MBC values were 12.5 µg/µl, respectively, 25 µg/ µl for E. coli PTCC 1330, and 50 µg/µl, respectively, 100 µg/ µl for S. aureus PTCC 1431. Together with possible strainto-strain variations, such differences between the above-cited data obtained with fruit extracts and the present results on leaf extracts could indicate specific characteristics in the amounts and/or nature of the antibacterial substances accumulated in the different parts of the plant. The antibacterial activities of the CS/CaO-based nanocomposites were tested by the microdilution assay in the presence of Alamar Blue, with amounts starting from 10 mg/ ml. The chitosan/CaO base was adequate at equal values of MIC and MBC, 1.25 mg/ml, to E. coli; the MIC determined for S. aureus was 0.156 mg/ml, with the respective MBC of 0.312 mg/ml. The different outcomes of the antibacterial treatments of the CS/CaO base in the absence and the presence of P3 and P6 could be explained by the changes in the structure and the physicochemical characteristics during the formation of the hybrid nanocomposites. However, the experiments to determine the MICs and MBCs of the nanocomposites were unsuccessful within the tested concentration range. The amounts of nanocomposites in the MIC test could not be increased further due to their poor solubility. On the other hand, applying the nanocomposites during the disc diffusion assay (performed on a solidified agar surface) indicated good antibacterial potential. Therefore, it can be summarized that the proven antibacterial potential of the nanocomposites can be successfully used for purposes that do not require solubility, e.g., antibacterial coating of surfaces used in the medicine or food processing industry, or also as ingredients of formulations for the treatment of skin lesions.

Suppression of growth and biofilm formation by sub-MIC of the tested nanocomposites
There is recently growing concerned about the spread and importance of biofilm-related food contamination, biofilmrelated nosocomial infections, and the so-called biofilm infections [51]. The structure of the biofilm communities comprises bacteria that are attached to surfaces and surrounded by extensive amounts of extracellular material. This structure determines a high antibiotic tolerance even if the biofilm bacteria may be antibiotic-sensitive [52,53]. These peculiarities of attached bacterial consortia necessitate the search of novel substances capable to suppress biofilm growth [54].
To check the effects of the tested samples on biofilm growth, it would be essential to differentiate between biofilm suppression per se and impacts due to the inhibition of bacterial growth. To solve this, the approach chosen in the present study was to perform the biofilm experiments under conditions that would not wholly eliminate bacterial growth, i.e., in the presence of sub-MICs. The active samples were applied at 1/2 MIC, where MIC values could be determined. Where MIC determination was not successful, the nanocomposites were added at the highest concentration that was used in the microdilution test experiments.
The effects of the tested solutions on biofilm growth were examined while parallelly taking into account the outcome of the treatments on the development of the unattached bacteria (plankton). For this reason, the optical density of the wells was measured at 620 nm wavelength to gain an idea of the plankton amount; before its removal and performance, the CV staining of the cells attached to the wells that formed the biofilm. The relative effects of the treatments on bacterial growth as unattached plankton cells and on biofilm production, presented as % of the control (strains growing in MHB without test substances), are illustrated in Fig. 5. The 1/2 MICS of P3, P6. CS/Ca did not seem to affect the plankton growth of the two strains substantially. As MIC for the hybrid nanocomposites was not established, 10 mg/ml amounts were used. This quantity of CS/Ca-P6 suppressed plankton growth to 76 ± 9% of E. coli and 45 ± 11% of S. aureus. The exact amount of CS/Ca-P3 was less effective against E. coli (32 ± 7% suppression) and more effective against S. aureus plankton I (74 ± 8% suppression). This indicated that the two nanocomposites' 10 mg/ml concentration could be accepted as sub-MICs.
Regarding the effects of the plant extracts not included in nanocomposites, P3 and P6 had only insignificant impacts on E. coli biofilm. However, notable was the significant stimulation instead of inhibition of the attached growth of S. aureus by P3 and P6 (Fig. 5B). This was not wholly unexpected. It has been shown that subinhibitory amounts of various antibacterial substances (among which antibiotics and plant substances) may have diverse effects on biofilm growth, from suppression to stimulation [55,56]. Previous studies on the antibiofilm effects of plant substances derived from A. indica are scarce. Extracts from A. indica were shown to interfere with the biofilm growth of P. aeruginosa [57] and MRSA [58]. Supplementation of mouthwash preparations with A. indica extracts had positive effects in dental plaque reduction [59]. The present observation for the increase of S. aureus biofilm indicates that these extracts should be applied with caution in cases where there is a hazard of developing infectious biofilm. However, such a stimulation effect was no longer noted when these extracts were incorporated into the nanocomposites. For these, the reduction of biofilm growth was significant in both model strains: for sample CS/Ca-P6, the biofilm was inhibited 73 ± 7% with E. coli and 93 ± 3% with S. aureus. The inhibition values for CS/Ca_P3 were 62 ± 12% for E. coli and 89 ± 4% for S. aureus.
Overall, this is a good study regarding the antibacterial and antibiofilm activities of CaO/chitosan nanocomposites doped with plant extracts. From the results, it can be assumed that the nanocomposites loaded with plant extract certainly have some inhibitory effects on newly formed biofilm. In my opinion, for better consistency of the results, it would be advisable to try the effects of the nanocomposites on more structured biofilms (3gg, 7gg) [60].

Conclusion
Two plant extracts (A. indica P3 and M. azedarach P6) were selected to be loaded onto chitosan-calcium nanocomposite (Cs-Ca). Total phenolics and flavonoids were evaluated by HPLC analysis. The existence of four major classes of organic compounds was identified using the LC-ESI-MS positive ion acquisition mode: limonoids, triterpenes, fatty acids, and phenolics. The Cs-Ca nanocomposite was successfully tested as a housing and stabilizing agent for loading P3 and P6 formed by sol-gel polymerization at 30 °C. SEM was employed to demonstrate the morphological features. The fabrication and characterization of fabricated nanocomposites were substantiated using an XRD spectral pattern with an abnormally high value. The structure of these nanocomposites was highly dependent on the linkage molecules. Notably, from FTIR analysis, the higher availability of the chitosan-calcium structure to capture various molecules due to their free amino groups and terminal Ca-O-, where S. aureus in broth and biofilm formation within a 24-h interval. The samples were applied as 1/2 MIC (P3, P6, and CS/Ca) and in 10 mg/ ml concentration for CS/Ca-P3 and CS/Ca-P6. The data from plankton growth registered at OD 620 nm are included in the same plots to compare with the possible contribution of bacterial growth suppres-sion. The two datasets are presented as % of controls to make the two datasets comparable. Each bar represents the average ± standard deviation from 5 wells per variant. The results showed that the inclusion of the plant extracts, samples P3 and P6, in CS/Ca-based nanocomposites resulted in very prospective materials as inhibitors of biofilm growth and can be recommended antibiofilm agents the band range was 449 cm −1 to 990 cm −1 . The fabricated nanocomposites loaded with plant extract (P3 and P6) could be applied as a potent inhibitor for pathogenic microbes and could prevent the development of bacterial biofilm by Grampositive and Gram-negative bacterial strains. Uses coating medical surfaces or surfaces in the food industry to keep biofilms from forming or adding them to medical formulations to treat biofilm-infected skin lesions. This could be useful for them.