A multidisciplinary approach to green nanotechnology: Synthesis, characterization, molecular docking, and biological applications of CuO/TiO₂ and CuO/TiO₂@Chitosan nanocomposites derived from M. chamomilla

2.4.1. Synthesis of CuO/TiO₂ NC

Green synthesis of CuO/TiO₂ NC was carried out using the M. chamomilla extract, following previously reported green synthesis methods [12]. Initially, 1 mM copper sulfate solution (30 mL) was prepared in deionized water. To this, the M. chamomilla extract (30 mL, 4.86 mg/mL) was added dropwise slowly under stirring at 30°C. After complete addition, the temperature was raised to 70°C, which caused a change in color to dark grey or black, indicating the formation of copper oxide nanoparticles. In another experiment, 1 mM TiO₂ aqueous solution was sonicated at 60°C for 1 h and added dropwise to the CuO nanoparticle solution. The achieved mixture, turned grey, was sonicated for 4 h at 80°C and then again sonicated for 3 h at the same temperature. The resulting nanocomposite was centrifuged at 15,000 rpm for 15 min to precipitate it, washed with 80% ethanol, and dried at 100°C to use it in additional spectroscopic characterization.

2.4.2. Synthesis of CuO/TiO₂@Chitosan NC

A modified synthesis of CuO/TiO₂@Chitosan NC followed the procedures described in previous literature publications [12]. The production of a 3% (w/v) chitosan suspension started by combining deionized water at 25°C while it was agitated. Afterward, the solution received 1 mL of glycerol (75%) along with glacial acetic acid (98%). A stirred solution of CuO/TiO₂ nanocomposites received the formed chitosan solution while both solutions remained at 25°C. The mixture received 60°C heat stimulation during 4 h of stirring that followed by 2 h of sonication under identical temperature. The established product required 15-min centrifugation at 15,000 rpm, followed by ethanol washing, before heat drying at 100°C to obtain a dry NC.

3.1.1. Formation of CuO/TiO₂ NC

Previous studies have established that M. chamomilla contains a variety of bioactive molecules with considerable reducing and stabilizing capacities, crucial to nanocomposite development. Moricz et al. [16] extracted antibacterial compounds such as flavonoids and phenolic acids using the assistance of over-pressured liquid chromatography (OPLC) and gas chromatography-mass spectrometry (GC-MS), while while they provided evidence of antifungal compounds revealed by GC-MS that provided additional proof of the presence of phenolic derivatives. They demonstrated that the flowers of chamomile are rich in polyphenolic compounds with respect to extraction efficiency, and they emphasized essential oils and phenolic metabolites as being accountable for antioxidant and antimicrobial activities. Of these, rosmarinic acid and related phenolics are particularly noteworthy due to their electron donation, metal ion chelation, and metal oxide surface strong binding properties. These biological molecules are likely the prime reducing and capping agents in the fabrication of CuO/TiO₂@Chitosan NC, therefore determining their stability and bioactivity.

The aqueous M. chamomilla extract contains various phytochemicals, including phenolics and flavonoids, as well as tannins and terpenoids that enable its reducing and stabilizing capabilities in green nanomaterial production. As shown in Figure 1, the phenolic compounds chlorogenic acid, caffeic acid, and ferulic acid lead the group with their multiple hydroxyl groups that enable metal ion reduction by creating Cu²⁺ to CuO. The flavonoids apigenin, quercetin, and luteolin donate electrons through their keto and hydroxyl function groups during bioreduction processes. On the other hand, TiO₂ has favorable characteristics, including a large surface area and excellent electron mobility, together with high chemical stability, which allows it to effectively support CuO nanoparticles and drive efficient charge separation through catalysis or photocatalysis. TiO₂ particles in the NC perform the critical function of attaching CuO nanoparticles while also stopping clustering effects, which improves particle distribution.

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Figure 1.

The anticipated mechanism for the formation of CuO/TiO₂ NC.

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The phytochemical-rich extract from M. chamomilla shows a strong impact on the process of converting bulk TiO₂ into well-distributed TiO₂ nanoparticles during green synthesis. Phytoconstituents with phenolics together with flavonoids, tannins, and terpenoids interact with TiO₂ precursors or bulk particles by using their reactive functional groups featuring hydroxyl (-OH), carbonyl (C=O), and carboxyl (-COOH) moieties. Phenolic acids, along with flavonoids, preserve nanoparticle separation stability as they simultaneously accept Ti⁴⁺ ions to start TiO₂ nanomaterial synthesis. The increased reactivity of such bio-fabricated nanoparticles enhances CuO/TiO₂ nanocomposite performance characteristics by forming synergistic interactions with CuO when co-synthesized, especially in catalytic reactions with the presence of antimicrobial functionality and photocatalytic action. The phytochemical blend promotes the process that lowers TiO₂ to nanometer levels and simultaneously lowers copper ion concentration [13].

3.1.2. Chitosan interaction with CuO/TiO₂ NC

The binding process between chitosan molecules and CuO/TiO₂ NC influences the whole material performance by strengthening its structure and improving its operational capabilities (Figure 2). Chitosan’s glucosamine units contain free amino (-NH₂) and hydroxyl (-OH) groups that work well with metal oxide surfaces. Chitosan amino groups become protonated into -NH₃⁺, so they form powerful electrostatic bonds with negative TiO₂ and CuO nanoparticle surfaces when the solution remains acidic. The interactions stop nanoparticles from clumping while they create a better distribution of particles to achieve a more homogeneous nanocomposite structure. Chitosan also forms hydrogen and coordinate bonds with surface oxygen and metal atoms. The strong network that binds polymer molecules to oxide nanoparticles is established through these interactions, which create a stable interface. Chitosan caps the nanoparticle surface, controlling growth and stabilizing suspensions. Chitosan also contributes antimicrobial and pollutant-binding properties [13]. Improved photocatalytic activity comes from the ability of chitosan to enhance these nanocomposites’ charge transfer activity between TiO₂ and CuO surfaces. The addition of chitosan into CuO/TiO₂ NC creates a green multifunctional material that has high potential in biomedical, environmental, and catalytic applications.

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Figure 2.

The projected interactions between chitosan and CuO/TiO₂ NC.

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3.2.1. UV-visible spectroscopy

UV-Vis spectroscopy served as a method to verify the production of CuO/TiO₂@Chitosan NC while evaluating their optical characteristics. The UV-Vis absorption spectra showed unique peaks which revealed the specific electronic transitions of M. chamomilla extract as well as CuO/TiO₂ NC and CuO/TiO₂@Chitosan NC (Figure 3, Table S1). Strong absorbance at 394 nm (0.540) in M. chamomilla extract indicates bioactive components such as flavonoids and polyphenols that potentially aided in nanoparticle synthesis. These phytochemicals that contain aromatic rings exhibit delocalized electrons responsible for generating π-π* transitions in addition to potential n-π* transitions from oxygen-containing non-bonding electrons such as hydroxyls and carbonyls.

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Figure 3.

(a) UV-Vis absorption data of (1) M. chamomilla extract, (2) CuO/TiO₂ NC, and (3) CuO/TiO₂@Chitosan NC. (b) FTIR spectra of M. chamomilla extract, chitosan, and nanocomposites.

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The absorbance spectrum for CuO/TiO₂ NC showed two distinct peaks appearing at 392 nm with a value of 0.412 and 725 nm reaching 0.263 (Figure 3a). The 392 nm peak demonstrates the O^2- → Ti⁴⁺ charge transfer transition that matches the properties of TiO₂ by showing electrons from oxygen 2p orbitals rising to titanium 3d orbitals. Systematically studied frequency peaks identify d-d electronic transitions in Cu²⁺ ions that exist within CuO nanoparticles. CuO acts as a p-type semiconductor material that shows light absorption in visible and near-infrared wavelengths because of its ligand field state transitions and the localized surface plasmon resonance effect, which causes collective electron oscillation with light exposure. The electronic environment changed when chitosan was added to the solution, which resulted in new absorption peaks at 757 nm and 377 nm, where the absorbance reached 0.114 and 0.263. The observed redshift at 757 nm implies stronger electronic interactions between CuO and the chitosan framework, probably because the increased electron mobility causes changes to LSPR. A blueshift at 377 nm suggests changes in TiO₂’s bandgap, possibly from coordination between chitosan’s surface groups and TiO₂. CuO/TiO₂ NCs display O^2- → Ti⁴⁺ charge transfer and d-d transitions, while the chitosan-functionalized composite shows redshift and blueshift patterns that reflect improved electronic stabilization via charge transfer mechanisms [17].

3.2.2. Fourier transform infrared (FTIR)

The FTIR spectroscopic analysis evaluated functional groups alongside intermolecular bonds in M. chamomilla extract and all other fabricated nanocomposites, along with pure chitosan (Figure 3b, Table S2). The FTIR spectrum of M. chamomilla extract showed absorption bands at 3342 cm^-1 attributed to the O-H stretching vibration. The FTIR spectrum shows C=O stretching vibrations at 1728 and 1676 cm^-1 related to carbonyl groups that probably exist in flavonoids and polyphenols. The FTIR spectrum of CuO/TiO₂ NC contained absorption bands that revealed the existence of both metal oxide materials as well as organic bond interactions through their appearance at 3311, 2940, 2883, 1662, and 1636 cm^-1. C=O stretching vibrations appear at 1662 and 1636 cm^-1, which point to organic functional groups and synthesis-related organic residues. The chitosan FTIR spectrum displayed absorption bands including 3291 and 2869 cm^-1 and several other peaks at 2113, 1984, and 1662 cm^-1. N-H stretching from the amine group appears at 3291 cm^-1, and C-H stretching occurs at 2869 cm^-1. The 1662 cm^-1 peak indicates acetylated chitosan through C=O stretching vibration, which shows the structure of the chitosan polymer. The N-H and C=O groups of chitosan indicate its capability to bond through electrostatic forces or hydrogen bonding with metal oxides.

The FTIR spectrum of CuO/TiO₂@Chitosan NC demonstrated absorption bands at 3213, 2932, 2879, 1646, 1599, and 1522 cm^-1, which confirmed the successful addition of chitosan. The O-H stretching vibration at 3213 cm^-1 can be found in both the metal oxides and chitosan through hydroxyl groups. FTIR spectroscopy showed the 2932 and 2879 cm^-1 peaks, which affirm the occurrence of alkyl groups present in chitosan. C=O stretching appears as a peak at 1646 cm^-1, and C=C stretching arises from the aromatic rings at 1599 cm^-1 in the chitosan structure. C-N stretching vibrations of chitosan amine group produce the 1522 and 1459 cm^-1 bands, which strengthen the bond between chitosan matrix and metal oxides. Depiction of Cu-O and Ti-O bonds with chitosan through new peaks at 793, 647, and 613 cm^-1 leads to the successful combination of biopolymer and metal oxides.

The FTIR spectrum shows that chitosan successfully binds with CuO/TiO₂ NC through peaks that distinguish the CuO/TiO₂@Chitosan NC spectrum. Studies of characteristic absorption bands show that chitosan-metal oxide intermolecular bonds exist because of the O-H stretching band redshift and new C-N and C=O bond formation. The strong bonds between chitosan functional groups and the surfaces of metal oxides stem from three possible types of interactions: hydrogen bonding, electrostatic forces, and coordination bonds. Stabilization, together with aggregate-prevention and improved biocompatibility of the nanocomposite, results from the influence of O-H, C-N, and C=O functionalities found in chitosan. The O-H stretching band at 3311 cm^-1 in CuO/TiO₂ NC matches the reported results by Raut et al. [18], indicating the presence of hydroxyl groups that exist on TiO₂ and CuO surfaces. The FTIR spectral results are matched with the results reported by Mohamed et al. [19], who detected bands identifying organic groups and metal oxide components in CuO/TiO₂-chitosan NC.

In addition, comparison with the reported FTIR of pure rosmarinic acid also validates its role in stabilizing the prepared NCs. Rosmarinic acid typically exhibits characteristic bands associated with O-H stretching, C=O stretching for carboxyl and ester groups, and aromatic C=C vibrations [20]. In the FTIR spectrum of CuO/TiO₂@Chitosan NC, the broad O-H stretching band at 3213 cm^-1, C=O stretching vibration at 1646 cm^-1, and aromatic C=C band at 1599 cm^-1 agree with reported characteristics, and it is concluded that rosmarinic acid (of the M. chamomilla extract) is actively involved in the capping and stabilization process. This observation is in accordance with the molecular docking results, which revealed that rosmarinic acid has the highest binding affinity towards the nanocomposite surface, further corroborating its dual role as a reducing and stabilizing agent in the synthesis. The FTIR study proves that chitosan successfully integrates into the CuO/TiO₂ NC framework, which ensures its potential for use in both antimicrobial and biomedical applications.

3.2.3. High-resolution transmission electron microscopy (HR-TEM)

The micrographs of biosynthesized CuO/TiO₂ and CuO/TiO₂@Chitosan NCs have been presented in Figure 4. Both CuO/TiO₂ and CuO/TiO₂@Chitosan nanoparticles have a similar range of sizes from 10 to 30 nanometers, but show slightly smaller sizes of 10-25 nanometers in the presence of organic coating and particle aggregation. The CuO/TiO₂ NC shows varying contrast, indicating the coexistence of two distinct regions with different electron densities.

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Figure 4.

HR-TEM micrographs of biosynthesized nanocomposites. (a) CuO/TiO₂ NC: HR-TEM micrograph taken at 200,000× magnification reveals spherical and quasi-spherical nanoparticles with lattice fringes, indicating a crystalline nature. Lattice spacing corresponds to the (111) plane of monoclinic CuO and the (101) plane of anatase TiO₂. (b) CuO/TiO₂@Chitosan NC: HR-TEM image captured at 200,000× magnification shows aggregated particles enveloped by an amorphous organic matrix representing the chitosan shell. Particle size range: 10-25 nm. Clear phase contrast indicates successful integration of CuO and TiO₂ within the chitosan matrix.

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The regions showing darker contrast represent CuO nanoparticles because their atomic number is higher, and the electron density exceeds that of the TiO₂ regions. The obtained micrographs of lattice fringes further validated that the NC had crystalline properties while matching the (111) plane of monoclinic CuO and the (101) plane of anatase TiO₂.

The CuO/TiO₂@Chitosan NC features diffused contrast patterns surrounding the nanoparticles because of its outer amorphous chitosan shell. The chitosan successfully prevents particle clumping and disperses them evenly, which establishes its function as a stabilizing protection agent [13]. Chitosan serves a dual role by enhancing particle stability and providing biofunctional groups for further interactions. These properties establish a beneficial combination for biomedical applications because they allow maximum utilization of the antibacterial properties from CuO with the photocatalytic activity of TiO₂ and bio-functional capabilities of chitosan. The CuO/TiO₂ NC exhibits a crystalline structure with good integration between materials, while the chitosan-coated version demonstrates enhanced dispersion and better stability and biocompatibility.

3.2.4. Zeta potential analysis

The surface charge properties together with colloidal stability in aqueous solutions were evaluated through zeta potential analysis for all biosynthesized CuO/TiO₂ and CuO/TiO₂@Chitosan NCs. The CuO/TiO₂ NC had a mean zeta potential of +48.9 mV, which produced an electrophoretic mobility value of 0.000378 cm²/Vs (Figure 5a, Table S3). The stable nanostructure of the particles can be attributed to their strong electrostatic repulsion since the positive zeta potential indicates effective anti-agglomeration properties. The mean zeta potential value of +27.5 mV was obtained for CuO/TiO₂@Chitosan NC while showing a mean electrophoretic mobility of 0.000212 cm²/Vs (Figure 5b, Figure S1). The chitosan component forms electrostatic bonds with metal oxide nanoparticles while covering their surface, which leads to reduced total surface charge. The chitosan matrix enhances steric stabilization, compensating for the reduced electrostatic repulsion between particles [21]. The electrostatic stability of CuO/TiO₂ NC is better than CuO/TiO₂@Chitosan NC, but the latter obtains stability via both electrostatic and steric stabilization mechanisms. Zeta potential measurements indicate that both NCs carry a positive surface charge and exhibit adequate colloidal stability.

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Figure 5.

Zeta potential and DLS analyses of nanocomposites. Zeta potential analyses of (a) CuO/TiO₂ NC, and (b) CuO/TiO₂@Chitosan NC. DLS analyses of (c) CuO/TiO₂ NC, and (d) CuO/TiO₂@Chitosan NC.

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3.2.5. DLS analysis

The DLS technique provided information about the synthesized NC hydrodynamic diameters as well as their polydispersity index (PDI) to analyze their dispersion behavior in aqueous solutions. The mean particle size of the CuO/TiO₂ NC measured by DLS reached 1.1 nm, while the PDI value amounted to 0.491 (Figure 5c, Table S4). The particle size distribution exhibits moderate polydispersity according to the relatively high PDI value because aggregation phenomena and cluster-sized differences may be the underlying cause. The mean average size of the CuO/TiO₂@Chitosan NC reached 2514 nm while the corresponding PDI value stood at 0.420 (Figure 5d, Figure S2).

The PDI value of the chitosan-coated CuO/TiO₂ NC appears slightly lower than the bare CuO/TiO₂ NC, even though the system has been characterized by larger size measurements. The formation of polymer-nanoparticle hybrid structures through chitosan affects NC size and dispersion properties due to differences in hydrodynamic size measurements [22]. Although the DLS analysis showed an average hydrodynamic size of approximately 1.1 nm for CuO/TiO₂ NCs, this size appears considerably smaller relative to particle sizes seen from TEM and SEM imaging, which were in the range of 10-30 nm. The cause of this discrepancy may be the existence of ultrafine particles, the sensitivity of the equipment utilized, or agglomeration behavior upon imaging in the dry state. Further optimization or different size analysis techniques may be necessary, so a reconciliation of this discrepancy may be made.

3.2.6. Scanning electron microscopy (SEM)

The SEM microscopy images presented in Figure 6 show crucial morphological and structural information about the biosynthesized CuO/TiO₂ and CuO/TiO₂@Chitosan NCs. A SEM micrograph (7.50 kx) (CuO/TiO₂ NC) shows that densely packed granules create a rough, porous surface. The observed microstructure contains irregular-shaped particles that show uniform distribution throughout the entire matrix. Its porous micro-granular structure and well-determined surface texture make this material ideal for catalytic and adsorptive use because of its high surface area potential. The SEM micrograph of the CuO/TiO₂@Chitosan NC shows agglomeration along with porosity throughout a network, but the surface appears rougher and less uniform than the unmodified CuO/TiO₂ NC.

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Figure 6.

SEM micrographs of nanocomposites at 7.5 kx magnification. (a) CuO/TiO₂ NC: The image reveals a densely packed, rough, porous surface composed of granular particles. The uniform distribution of irregular-shaped particles suggests a micro-granular network ideal for catalytic and adsorptive applications. (b) CuO/TiO₂@Chitosan NC: A rougher, more porous morphology with visible agglomeration within an organic matrix. The embedded spherical particles within the chitosan framework indicate improved porosity and nanoparticle dispersion due to biopolymer functionalization.

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The NC structure contains spherical particles embedded in organic material that create voids and channels that enhance the overall porosity. The chitosan matrix prevents extreme nanoparticle aggregation through hydrogen bonds and electrostatic attraction, but does not fully eliminate the collective behavior of the particles. Because of its chitosan composition, the structure enables better nanoparticle dispersion, which benefits from the abundant amino and hydroxyl functional groups [23]. The porous structure combined with high surface area properties from both NCs leads to better performance in photocatalysis and antimicrobial coatings, as well as adsorption and drug delivery applications. CuO/TiO₂ NC displays a micro-granular morphology with uniformly distributed metal oxide phases. The materials show promise for different uses since their morphological properties indicate their ability to support applications demanding stable surfaces with high surface area and biofunctionality. Regardless of not applying FESEM here, one should also realize that this technique can provide valuable data on possible alterations of the 2D spacing of nanomaterials in water conditions. Previous studies have revealed that interlayer spacings in compounds such as graphene oxide are altered upon exposure to water and have direct effects on their functional properties [24].

3.2.7. Energy-dispersive X-ray (EDX)

The EDX analysis identified elemental composition in both CuO/TiO₂ and CuO/TiO₂@Chitosan NCs samples to determine how important elements are distributed within these nanocomposites (Figures 7a-d, Table S5). The nanocomposite contained CuO particles that showed 13.26% mass, whereas the atomic percentage was 4.3%. The addition of chitosan in the CuO/TiO₂@Chitosan NC changed the composition of elemental constituents. The mass ratio of titanium dropped to 21.47% while its atomic ratio fell to 7.76% which indicates that TiO₂ experienced some structural modification through chitosan interaction. The analysis detected nitrogen from the chitosan particles, which play a crucial role in nanocomposite stabilization through their amino groups (-NH₂). The detection showed mass percentages of 8.21% and atomic percentages of 6.11%. The incorporation of chitosan increased the carbon content to 11.68% by mass, confirming its successful integration. The disruption of metal oxide content through chitosan addition demonstrates better nanoparticle distribution and stability owing to the binding interaction of chitosan’s functional groups with metal oxides [13]. Elemental analysis demonstrates that both NC assemblies carry their designated metal oxide elements CuO and TiO_2, while the detection of nitrogen together with elevated carbon levels verifies chitosan in the CuO/TiO₂@Chitosan NC structure.

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Figure 7.

(a) EDX elemental analysis of CuO/TiO₂ NC. (b) EDX elemental analysis of CuO/TiO₂@Chitosan NC. (c) The mass ratio and atom ratio for each element of CuO/TiO₂ NC. (d) The mass ratio and atom ratio for each element of CuO/TiO₂@Chitosan NC. (e) XRD patterns of CuO/TiO₂ NC. (f) XRD patterns of CuO/TiO₂@Chitosan NC.

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3.2.8. X-ray diffraction (XRD)

XRD pattern of CuO/TiO₂ NC (Figure 7e, Table S6) identifies specific peaks that match with the crystalline phases of both CuO and TiO₂. These results align closely with findings reported by Elderdery et al. [9], where the same peak confirmed CuO crystallinity in CuO/TiO₂-Chitosan-Escin NCs. The d-spacing values of 2.387 Å and 1.899 Å also confirm the crystalline structure of CuO in addition to the 37.68° peak. The anatase phase of TiO₂ can be identified through XRD peaks at 47.88° (d-spacing of 1.899 Å) and 53.81° (d-spacing of 1.703 Å), and 62.55° (d-spacing of 1.484 Å). The composite structure contains anatase TiO₂ elements because the two principal XRD peaks exist at 37.68° and 47.88°, which match the (101) and (004) planes of anatase TiO₂. The width at half maximum of X-ray peak reflections reveals both NC crystallinity characteristics and particle lengths. Our results are similar to those of Elderdery et al. [9], who identified anatase contributions. The NC contains crystalline structures of CuO and TiO₂ since lower FWHM values indicate larger crystallite dimensions. The broad peak width at higher 2θ positions of 70.24° and 74.93° demonstrates small crystallite dimensions and possible structural irregularities caused by heterojunction formation between CuO and TiO_2, as well as amorphous domains at the interface.

The XRD patterns of CuO/TiO₂@Chitosan NC (Figure 7f, Table S7) display a crystalline (002) plane of CuO, having 25.30° 2θ equals 3.520 Å, which dominates the diffraction pattern, showing the presence of CuO nanoparticles in the composite. The reflections at 37.77° (d-spacing of 2.382 Å and 48.04° (d-spacing of 1.894 Å together with 53.85° (d-spacing of 1.703 Å confirm the presence of TiO₂ and the characteristic planes of CuO. The XRD analysis demonstrates the presence of both metal oxides through peaks at 37.77° and 53.85°, which match the (101) and (004) planes of anatase TiO₂. The crystallinity of the material seems to be affected by chitosan presence according to XRD results, which show diminished peak intensity with broadening most prominently in TiO₂-related peaks. The TiO₂ structure in the nanocomposite adopts a more amorphous appearance and a lower crystalline state because of the chitosan matrix composition.

XRD data show that both CuO/TiO₂ NC and CuO/TiO₂@Chitosan NC display crystalline arrangements consisting of CuO and TiO₂ components, revealed through well-defined (002) and (101) plane reflections from CuO and TiO₂. XRD analysis shows strong signs of both metal oxide presence, which indicates a well-formed crystal structure in the CuO/TiO₂ NC. When chitosan is incorporated into the CuO/TiO₂@Chitosan NC, the XRD pattern alters through both changes in peak intensity and peak spreading, which shows modified crystallinity of the metal oxides.

3.3.1. Phenolic, flavonoid, and tannin contents

The analysis of M. chamomilla extract phytochemicals demonstrates that phenolic compounds together with flavonoids and tannins actively contribute to the bioreduction of CuO/TiO₂ and CuO/TiO₂@Chitosan NCs during synthesis (Figure 8a, Table S8). The formation of metal oxide nanoparticles through bioreduction occurs because phenolic and flavonoid components donate electrons to metal ions by releasing their hydroxyl groups (-OH) during the process. The phytochemical content experienced a marked decline in the synthesis of CuO/TiO₂ NC since phenolics reduced by 64.81 mg, and flavonoids measured 49.75 mg, while tannins reached 15.23 mg. A portion of bioactive compounds was utilized during copper and titanium reduction into CuO and TiO₂ nanoparticles due to their participation in the synthesis. Extracted phenolics, flavonoids, and tannins from petroleum oil treatment reached 49.89 mg, 39.37 mg, and 14.56 mg after adding the CuO/TiO₂@Chitosan NC. The combination of chitosan with the NC improved particle stability and dispersion, but this was correlated with phytochemical reductions because of binding interactions between compounds and chitosan and metal oxide substances [13]. The amino and hydroxyl functional groups in chitosan potentially bind with the phytochemicals, thereby affecting their accessibility and lowering their levels in the produced nanocomposite [12]. The metal reduction capabilities and stabilizing properties of these phytochemicals within the nanocomposites improve their functionality for antimicrobial treatments and biomedical functions [11-14].

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Figure 8.

(a) The results of phytochemical analysis. (b) The % scavenging activity plotted versus the sample concentration (mg/mL).

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3.3.2. Carbohydrate content

Anthrone colorimetric assay was employed to estimate the carbohydrate levels in M. chamomilla extract and both CuO/TiO₂ and CuO/TiO₂@Chitosan NCs. The research data appear in Figure 8(a) & Table S9. The M. chamomilla extract absorbed 1.605 when its concentration reached 8.48 mg/mL, which translates to 0.391 mg/mL carbohydrate levels at 92.326 mg/g DW. The absorbance measurement of 1.543 obtained from the CuO/TiO₂ NC solution containing 12.36 mg/mL determined its carbohydrate content at 0.376 mg/mL and 60.897 mg/g DW. The highest carbohydrate level was measured in the tested samples through the CuO/TiO₂@Chitosan NC. The absorbance reading at 14.44 mg/mL concentration was 2.787, which led to the identification of 0.680 mg/mL carbohydrate content and 94.149 mg/g DW.

M. chamomilla extract contained high concentrations of carbohydrates at 92.326 mg/g DW because it has naturally occurring sugars together with polysaccharides and glycosides in its plant material. The bioactive compounds found in these substances play an essential role in biological functions by functioning as antioxidants and for antimicrobial, immunomodulatory actions [13]. The nanocomposite surface might retain phytochemicals from plant extract use in biosynthesis since they could have adsorbed onto the surface. The CuO/TiO₂@Chitosan NC contained the largest amount of carbohydrate at 94.149 mg/g DW. Chitosan serves as the main cause of this major increase because this biopolymer possesses abundant amino and hydroxyl groups that add polysaccharide content to the final NC structure [11-14].