Nitazoxanide (NITA) was defined as a broad-spectrum anti-protozoal and anthelmintic agent (Hagras et al., 2025; Hagras et al., 2023, Amadi et al., 2002). NITA is heavily used for the treatment of many protozoa, such as cryptosporidiosis and giardiasis. NITA has been confirmed to possess a great efficiency against several helminthes (Anderson and Curran, 2007; Alonso and Farina, 2020; Colín-Lozano et al., 2017). NITA is extremely characterized with its immunomodulatory efficacy (Dupouy-Camet, 2004; Trabattoni et al., 2016; Elazar et al., 2009).
       
Recently, the process of drug discovery has led to novel chemical formulas that have more potent and highly promising activities against many biological pathogens. The main problem facing these novel formulations the poor cellular bioavailability and the poor solubility, which eventually leads to poor simulation (Basavaraj and Betageri, 2014). One of the most potent strategies to overcome this essential complication includes drug encapsulation and this occurs via the chelation with metals. It may also lower any side effects of the original drug and elevate and enhance the stability and the chemical absorbance of the original drug (Sharfalddin et al., 2021 and 2023).
       
Additionally, the used metals in chelation can elevate the cellular penetration of the drug complex via enhancing and elevation of its cellular penetration permeability (Kontoghiorghes, 2020). For example, Cu⁺² ions elevate the new properties of metformin to possess antidiabetic and anticancer capacities (Babak and Ahn, 2021; Müller et al., 2018).                      
       
Manganese (Mn) plays a crucial role in alleviating oxidative stress through its antioxidant properties. It  acts as a cofactor for several enzymes, including Mn SOD which is  involved in the detoxification of superoxide. Mn’s ability  to scavenge free radicals and reduce oxidative stress  is attributed to its reactive oxygen species (ROS)  production. This antioxidant function is essen tial for  maintaining homeostasis and preventing cellular damage  due to oxidative stress (Avila et al., 2014). 

     
Another example, “Mn /Minocycline formula”, which is a formula of metal ligand biological enhancing properties, as it is known that Minocycline is a broad-spectrum antimicrobial and it is considered a unique tetracycline derivative. But with this chelation with Mn, it produced a novel formula that is more effective as an antioxidant and antibacterial with expected high, potentially significant biological effects (Jastaniah et al., 2025).
       
Sigma receptors are widespread in the C.N.S and multiple tissues. Alpha sigma receptor consists of (σ1R) and (σ2R) and is expressed in numerous regions of the brain. The sigma receptor plays a more diverse role in cellular signaling, apoptosis and metabolic regulation. σ1Rs are intracellular receptors acting as chaperone proteins that modulate Ca²⁺ signaling via the IP3 receptor. The σ1R receptor, at the mitochondrial-associated endoplasmic reticulum membrane, is responsible for mitochondrial metabolic regulation and promotes mitochondrial energy depletion and apoptosis (Rousseaux and Greene, 2016).
       
There is an emerging indirect relationship between sigma receptors (particularly the sigma-1α receptor) and parasitic infections, primarily through modulation of the host immune response and inflammatory pathways rather than direct binding to the parasite itself. Recently, sigma-1 receptors act as intracellular chaperones, managing endoplasmic reticulum stress and modulating neuroinflammation, which is crucial during parasitic invasions (Tsai et al., 2009). Sigma-1 receptors are expressed in immune cells and can regulate the inflammatory response triggered by parasitic infection. Activation of sigma-1 receptors has been shown to reduce inflammatory cytokines in pathological conditions (Tsai et al., 2009).
       
Therefore, in this study, a NITA-based Mn (II) compound was fully synthesized and chemically characterized. Free radical-scavenging in vitro biological assays (ABTS, DPPH and metal chelation), besides molecular docking, were used to examine the antioxidant activity and docking efficacy with the sigma receptor for alleviation of both oxidative injury and inflammatory series using this novel complex, respectively.

Chemicals
 
Nitazoxanide (NITA) was purchased from a national pharmaceutical company, MnCl₂ salt was of pure grade and procured from Sigma-Aldrich Chemical Company.
 
Synthesis of manganese (Mn) nitazoxanide complex
 
Solid manganese nitazoxanide was prepared by mixing nitazoxanide (2 mmol, 0.614 g) in 30 mL of methanol as a solvent with (1 mmol) manganese chloride salt: in 20 mL of methanol. The reaction mixture was refluxed for 3 hr. The solid compound was filtered, washed with methanol and then dried under a vacuum over CaCl₂.
 
Chemical characterization methods
 
A Perkin Elmer CHN 2400 Elemental Analyzer (USA) was used to examine the amounts of C, H and N. The manganese (Mn) compound’s electrolytic or non-electrolytic properties were tested using the Jenway 4010 conductivity meter. Infrared spectra in the 4000-400 cm^-1 spectral range were obtained using a Bruker FT-IR spectrometer. UV-Vis spectra in DMSO between 800 and 200 nm were measured using the UV2 Unicam UV-Vis Spectrophotometer.
       
Magnetic moments were calculated using Sherwood Scientific’s Magnetic Susceptibility Balance. SEM images captured using the Quanta FEG 250 device.
       
TEM is utilized to explain structural and chemical properties in the nanoscale range through the imaging and diffraction of nanoscale materials. JEOL 100s microscopes were used for TEM studies.
 
Molecular docking
 
Molecular docking simulations were performed using AutoDock Vina (Trott and Olson, 2010). Grid boxes were configured to encompass the functional domains. The default exhaustiveness setting was used to ensure adequate conformational sampling.
 
HOMO-LUMO analysis
 
Density functional theory (DFT) calculations were performed to estimate the electronic properties and molecular stability or reactivity of Mn-NITA complex neutral singlet ground state, excluding solvent and charge effects. Geometry optimizations and frontier molecular orbital (FMO) analyses were carried out using the B3LYP functional with the 6-311G* (d,p) basis set in the ORCA software package. Molecular visualizations were generated using Avogadro and iBoView. Ionization potential (IP) and electron affinity (EA) were approximated from the negative of the HOMO and LUMO energies, respectively, while electronegativity (c), electronic chemical potential (μ), chemical hardness (η), softness (S) and electrophilicity index (ω) were derived from standard conceptual DFT equations. The calculated descriptors, including HOMO-LUMO gap (ΔE), IP, EA, χ, μ, η, S and ω, were used to evaluate the stability and reactivity of each compound, with results summarized in Table 1.


 
IR analysis
 
Infrared (IR) spectral analysis of Mn-NITA complex was performed using Density Functional Theory (DFT) at the B3LYP/6-311G*(d,p) level of theory. All calculations were conducted in the neutral singlet ground state without solvent effects. Frequency calculations were carried out to obtain vibrational wavenumbers and intensities, ensuring that no imaginary frequencies were present, thereby confirming the optimized structures as true minima. The simulated spectra were generated using the ORCA quantum chemistry package and visualization was performed with Avogadro and iBoView. Vibrational assignments were made by analyzing the characteristic regions of the spectra, with stretching, bending and deformation modes identified according to their typical frequency ranges.
 
Raman activity calculations
 
Raman vibrational properties of Mn-NITA complex computed using density functional theory (DFT). Geometry optimization was initially performed at the selected DFT level (e.g., B3LYP/6-31G (d,p) or your chosen functional/basis set), followed by a full harmonic frequency calculation to obtain vibrational normal modes. Raman activities were calculated from the derivatives of the polarizability tensor with respect to each normal coordinate, as implemented in the quantum chemical package employed for the study. No imaginary frequencies were observed, confirming that the optimised structures correspond to true minima on the potential energy surface. Each Raman-active vibrational mode was then converted into predicted Raman intensities.
 
Potential energy surface (PES) scanning
 
The conformational space of Mn-NITA complex was explored through a two-dimensional potential energy surface (PES) scan. Selected dihedral angles or structural coordinates (SC1 and SC2) were systematically varied in defined increments (commonly 10° or 15°), while all remaining internal coordinates were allowed to relax during constrained optimization. For each point on the PES grid, a restricted geometry optimization was performed at the same DFT level used for equilibrium structure determination (e.g., B3LYP/6-311G*(d,p), ensuring consistency across all computed energies. The total electronic energy at each grid point was recorded in atomic units (a.u.) and the resulting dataset was used to generate three-dimensional surface plots and corresponding contour maps. These visualizations allowed identification of energy minima, transition regions and conformational energy barriers. All calculations were conducted without imposing symmetry constraints and convergence thresholds followed default tight optimization settings to ensure numerical stability. The PES surfaces were rendered using scientific plotting software, with energy differences plotted relative to the global minimum to facilitate comparison of conformational stabilities.
 
Assessment of the antioxidant capacity
 
Assay of ABTS
 
Antioxidant activity of NITA and its (Mn) metal ion complex was performed according to Arnao et al., (2001) and Elkholy et al., (2023). ABTS reagent was dissolved in dis.H₂O. The previously prepared solution was added to K₂S₂O₈ and then it was left in the dark for about one day and further procedures. 3 replicates were approved for obtaining the results. The decline in ABTS absorbance was measured at ~734 nm. The results were recorded using a microplate reader, BMG LABTECH®-FLUOstar Omega microplate reader (Ortenberg, Germany).
 
Linearity of trolox in ABTS assay
 
Results of the samples are presented as μg TE/mg sample using the linear regression equation extracted from the following calibration curve (linear dose-inhibition curve of Trolox) (Fig 1).


 
DPPH assay
 
The antioxidant activity of NITA and its metal complex with (Mn) was measured by using DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) free radical assay (Boly et al., 2016 and Elkholy et al., 2023). The reactions were incubated for 30 min at 37°C in the dark. 3 replicates were approved for obtaining the results. The resulting reduction in DPPH colour intensity was measured at ~540 nm. The results were recorded using a microplate reader, BMG LABTECH®-FLUOstar omega microplate reader (Ortenberg, Germany) as shown in (Fig 2).


 
Metal chelation assay
 
Metal chelation assay was carried out according to Santos et al., (2017). Freshly prepared FeSO₄ was mixed with NITA and its metal complex with (Mn) ion in the sterilized well plates. Then, the addition of ferrozine. Subsequently, the well plate was incubated at 37°C for ~10 min. After incubation, the decline in the colorimetric intensity was immediately measured at ~562 nm (Santos et al., 2017 and Elkholy et al., 2023). The results were recorded using a microplate reader, BMG LABTECH®-FLUOstar Omega microplate reader (Ortenberg, Germany) (Fig 3).


 
Statistical analysis
 
The data were expressed statistically as (Mean±SE). One-way ANOVA and post hoc test were used. Valuation is significant at (p<0.05).

Elemental analysis and molar conductance
 
Manganese nitazoxanide complex is stable and insoluble in water. The molar conductance measured was Λm = 18 (Ω^-1 mol^-1 cm^-1), confirming the non-electrolytic behaviour as confirmed previously in a lot of studies (El-Megharbel et al., 2025; AlZahrani et al., 2025 and Al-Thubaiti et al., 2025). Elemental analyses (C, H and N) of nitazoxanide complex [Mn (NITA)₂(cl)₂] confirmed the 1:2 Mn⁺²: nitazoxanide ratio.
 
Infrared spectra
 
IR data for free nitazoxanide and its Mn (II) metal complex which are shown in (Fig 4). By comparing spectrum of IR for nitazoxanide with its Mn⁺² complex showed that the stretching vibration of NH amide group disappeared upon chelation to Mn (II) complex and shifted to higher frequencies, confirming that the NH amide group is involved in chelation (Ivana et al., 2010). The stretching vibration for ester group appeared at 1772 cm^-1 and 1160 cm^-1 due to C=O and C-O, respectively.


       
A shift in C=O carbonyl group value referred to it was involved in the chelation process. The binding effect to the ester oxygen caused the band, which first emerged in nitazoxanide at 1160 cm^-1, to move to a lower frequency with low intensity. The nitazoxanide molecule underwent chelation at these primary sites, undergoes blue shift for C=O amide and C=N modes of vibration in the thiazole ring following chelated to the Mn (II) ion.
       
For IR for the manganese complex there are new bands appeared at 699 cm^-1 and 530 cm^-1 range, which are referred to v(M-O) and a new band appeared at 490 cm^-1, referred to v(M-N) (Nakamoto, 1970; Bellamy, 1975).
 
UV-Vis spectra and magnetic measurements
 
Table 1 show the uv-vis spectra for free NITA and its Mn complex. For nitazoxanide two absorption maxima appeared at 225 and 410 nm. The band appearing at 410 nm is due to π → π* transitions, while for 225 nm is attributed to n → π* transitions. Weak bands at 280 and 240 nm for Manganese (II) complex, due to  to π → π* and n → π* transitions. The octahedral planner shape of the [Mn (NITA)₂(H₂O)₂Cl₂] complex was validated by the magnetic moment of 5.236 BM for the Mn (II) complex (Cotton et al., 1962; Figgis, 1967).
 
SEM and TEM investigations
 
SEM image for NITA and its [Mn (NITA)₂(H₂O)₂Cl₂] complex Fig 5. A tiny particle size with nano feature products. Using SEM analysis, the surface morphology of NITA and its Mn⁺² complex was detected. All particles were shown to have a high capacity to form agglomerates with variety of shapes. TEM images for NITA and its Mn⁺² complex Fig 5 shows a homogenous phase material that confirms the ordered arrangement for NITA metal chelate matrix and its Mn⁺² complex. A spherical black spot is appeared within nano-range particle sizes.


 
Molecular docking of NITA/Mn complex with sigma receptor
 
Sigma receptors are essentially expressed intracellularly in the nervous system, especially the glial cells and mainly regulate the functions of the ion channels. Additionally, sigma receptors mainly reside in (ER) “Endoplasmic Reticulum” and act as pharmacochaperones to modulate neurotransmission via different receptors and transporters. Thus, sigma receptors are thought to be involved in the effects of many drugs (Liechti et al., 2022).
       
Thus, Sigma α-1 receptors are unique, chaperone proteins located primarily in the endoplasmic reticulum (ER) membrane. They act as ligand-regulated molecular chaperones that modulate ion channels, calcium signaling and neurotransmitter systems, playing crucial roles in neuroprotection, cell survival and psychiatric disorders.  Sigma receptors act as intracellular chaperones, often regulating ER stress and calcium flow at the mitochondria-associated ER membrane (MAM). They are highly expressed in the central nervous system and other tissues. While sigma receptor originally thought to be opioid receptors, but the current metal complex NITA/Mn recently chelates with the sigma receptor via recording binding affinity with Med activity and the NITA/Mn complex showed a binding interaction energy ranging from -6.8 to -7.2 kcal/mol for sigma receptor with mean (-6.97) (Table 2)  and (Fig 6) which is Med which is a novel result that may open the gate for more prospective studies.




 
DFT analysis
 
HOMO-LUMO energy gap calculation and global descriptors
 
The frontier molecular orbital (FMO) analysis revealed distinct differences in the electronic properties of Mn-NITA complex. Mn-NITA exhibited a HOMO energy of -11.589 eV and a LUMO energy of -5.708 eV, resulting in a relatively large energy gap (ΔE) of 5.881 eV. The larger ΔE value of Mn-NITA indicates greater kinetic stability and lower chemical reactivity. The ionization potential (IP) and electron affinity (EA) further support this trend, with Mn-NITA demonstrating higher IP (11.589 eV), suggesting that Mn-NITA is less prone to electron donation.
       
The calculated global reactivity descriptors reinforce these observations. Mn-NITA exhibited higher chemical hardness (η = 2.941 eV). Additionally, Mn-NITA (12.72 eV), suggesting a stronger tendency to accept electrons. The chemical potential (μ) and electronegativity (χ) values also reveal that Mn-NITA possesses greater overall electronic stability. Overall, these results suggest that Mn-NITA is comparatively more stable and less reactive, which may influence its interaction behavior in chemical or biological systems, as shown in Table 3 and Fig 7.




 
IR plot
 
The IR spectra further confirm the vibrational characteristics of the complex. Strong absorption bands were observed in the 400-1800 cm^-1 region, with maximum molar absorptivity (ε) values reaching approximately 1100-1200 M^-1 cm^-1 for Mn-NITA. Prominent peaks near ~1600-1700 cm^-1 correspond to azomethine (C=N) stretching vibrations, while bands in the 1000-1300 cm^-1 region are associated with C-N and C-O stretching modes. The low-frequency region below 600 cm^-1 exhibits bands attributed to metal-ligand (M-N and M-Cl) vibrations, confirming successful coordination. Additionally, broad features around ~3400-3550 cm^-1 indicate O-H stretching vibrations. Notably, Mn-NITA demonstrates a slightly enhanced dipole strength (~1.8 × 10-40 esu²·cm²), suggesting stronger IR-active transitions in the Mn complex (Fig 8).


 
Raman activity
 
The Raman activity spectra of the Mn-NITA complex display prominent vibrational features across the 0-4000 cm^-1 region. For Mn-NITA complex, intense high-frequency bands were observed in the 3200-3550 cm^-1 range, corresponding to O-H and N-H stretching vibrations, with maximum intensities approaching ~10-12 arbitrary units and scattering activities nearing ~250 Εt /amu. In the fingerprint region (1000-1800 cm^-1), distinct strong peaks were recorded around ~1550-1700 cm^-1, attributable to C=N and C=S stretching modes. Mn-NITA exhibited comparatively sharper and slightly higher intensity peaks in this region, suggesting stronger metal-ligand coupling. Overall, Mn-NITA complex demonstrates significant Raman activity, with the high-frequency stretching region being the most dominant, as shown in Fig 9.


 
Potential energy surface (PES) analysis
 
The three-dimensional potential energy surface (PES) plots reveal the stability landscape of the Mn-NITA complex along the scanned coordinates (SC1). For Mn-NITA, the total energy varies within a narrow range of approximately -438.32 to -438.26 Hartree, showing multiple local minima and maxima, indicative of a rugged energy surface with several accessible conformational states. The deeper minima observed for Mn-NITA suggest greater thermodynamic stability. These findings correlate well with the previously calculated global reactivity descriptors, reinforcing that Mn-NITA is energetically more stable, as shown in Fig 10.



Antioxidant capacities of NITA and it’s Mn metal complex
 
The estimated percentages obtained of the chelating activity of NITA/Mn via using different assays of scavenging free radicals which are shown in Table 4. The assay of ABTS, Metal chelation and the assay of DPPH were used. The capacity of the NITA and NITA/Mn complex to scavenge the free radicals of ABTS was 230.51, which is more than NITA itself. Meanwhile, the metal chelating activity of NITA/Mn was higher than NITA itself by 72.41% (µM EDTA eq/mg), respectively.


         
Meanwhile, the scavenging ability of NITA/Mn was also the highest stable via the DPPH radical activity by 26.80 (µM trolox eq/mg). But, the metal complex (NITA/Mn) has a greater chelating capacity by 60.92 (µM trolox eq/mg) than NITA itself (Table 4).
       
NITA is a commonly used treatment for many types of intestinal protozoa. However, its low solubility is a real barrier for high treatment percentages in many protozoan infections. Thus, there is an urgent need for the development of new drugs to benefit from their tremendous advantages (Hagras et al., 2025; Hagras et al., 2023).
      
An innovative scientific trend in the drug delivery models is represented via the new formulation of metal drug complexes in order to overcome a lot of limitations regarding the commercial drugs (Hagras et al., 2019; Allam et al., 2022).
        
NPs with a very small size range have high potency to penetrate across the tissues. Consequently, this would increase the permeability of the new drug formulations and decrease the dose of the synthesized drugs. Additionally, these synthesized formulae offer a cost-effective strategy as the nano-encapsulation vehicles’ development is almost cheaper (Hagras et al., 2023).
       
Consequently, the excessive production of the oxidative injury markers due to a lot of parasitic infections is considered as areal and major reason for the incidence of tissue damage (Yan et al., 2015). Eventually, the oxidative injury markers of MDA were previously studied (Albogami, 2024 and Albogami, 2025). This oxidative injury is evidenced by the highest MDA levels in the parasitically infected groups. This observation reinforced the novel findings of the current study, which confirmed the antioxidant capacities of the novel complex NITA/Mn over the NITA drug only via in vitro antioxidant assays ABTS, DPPH and metal chelation.
       
Interestingly, the current findings are in great accordance with the study of Hagras et al., (2025), who proved that treated groups with NITA-loaded ZnO exhibited a reduction in the estimated oxidative biomarkers, which greatly confirmed the novel concept of the current study via the effectiveness of the novel synthesized complex NITA/MN via the high in vitro antioxidant capacities. These findings are essentially consistent with (Ashour et al., 2016; Nagajyothi et al., 2015) previous studies, which focused on NITA loaded with ZnO NPs effects in mitigating and declining the oxidative injury due to the parasitic infections.
       
This surprising decline in the oxidative markers after treatment with NITA new formulations further reinforces the therapeutic benefits of the novel metal complex formula in alleviating the oxidative injury markers associated with parasitic infections, which is in accordance with the previous potent effects of NITA as reported by (Hemphill et al., 2006).
       
The main aim of the current study was to synthesize a nano-sized novel formula of NITA/Mn to enhance its biological actions via measurement of the antioxidant capacities in vitro and DFT analysis of the utilized novel drug, which confirmed its high energy stability. The nano-formula was synthesized and characterized based in my knowledge for the 1^st time as a potent antioxidant via in vitro antioxidant assay. The results of the NITA/Mn characterization denoted that it lies in the nano-sized range. This smaller particle’s size saves high surface area as compared to the volume and eventually leads to higher penetration across the cellular membranes (Melk et al., 2021).
       
FTIR spectroscopy exhibited peaks that are attributed to (MnO stretching) the metal-oxygen vibration mode, while the clear shift that appeared in the FT-IR peaks indicates a high success of NITA/Mn as previously confirmed (Sawant et al., 2018).
       
Simulation docking with sigma receptor and DFT analysis revealed that NITA/Mn metal complex exhibited a Med docking force with Med energy stability. This is usually preferable as it offers a sufficient drug amount across the body tissues (El-Wakil et al., 2023).
       
There is an emerging indirect relationship between sigma receptors (particularly the sigma-1α receptor) and parasitic infections and inflammatory pathways, rather than direct binding to the parasite itself. Recently, sigma-1 receptors act as intracellular chaperones, managing endoplasmic reticulum stress and modulating neuroinflammation, which is crucial during parasitic invasions. This is in agreement with the current concept regarding a NITA-based Mn (II) compound with its activity in docking with this receptor and concurrently its potent antioxidant activities.
       
The mechanism of action of NITA is represented by inhibiting the pyruvate ferredoxin oxido-reductase enzyme, which can alter the parasitic metabolism, especially the respiratory system, besides causing a lot of cellular membrane lesions (Hagras et al., 2023). Despite the safe NITA nature in general, it still has decreased efficacy due mainly to its poor solubility as previously mentioned. Even NITA/Mn revealed the highest significant drug stability, which offered Med benefits via improving the drug solubility that can effectively enhance in prospective in vivo studies, the drug permeability and the efficacy.

NTZ/Mn complex was synthesized and it’s in vitro antioxidant activity was assessed. The complex proved to have a non-electrolytic nature. Its chemical structure was confirmed by IR and UV. SEM and TEM images showed small particles that agglomerated in different shapes. All DFT analysis reinforces that Mn-NITA is energetically more stable and less reactive. NITA/Mn chelates with the sigma receptor very effectively via high-forcing docking. The estimated scavenging capacities of NITA/Mn confirmed the potent capacity of NITA/Mn complex to scavenge the free radicals of ABTS, DPPH and metal chelation, confirming new potent antioxidant capacities of the synthesized complex.

The author declares that there is no conflict of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.