Green synthesis of nanosized N,N'-bis(1-naphthylidene)-4,4'-diaminodiphenylmethane and its metal (II) complexes and evaluation of their biological activity

Condensation of ecofriendly synthesized 4,4’-methanedianiline with 2-hydroxy-1-naphthaldehyde produced a (1:1) octopus-like Schiff base mixed ligand. Reaction with Co(OAc)2⋅H2O, NiCl2⋅6H2O, Cu(OAc)2⋅H2O and Zn(OAc)2⋅2H2O metals furnished their complexes in high yield and purity. All new structures were fully characterized by various spectroscopic and spectrometric measurements. The complexes exhibited high thermal stability up to 700 °C, leaving nearly 40% of their mass as residues. Antimicrobial screening results exhibited moderate activities towards all studied microbes. Antioxidant screening was concentration dependent, and their activities were in the order Ni(II) > Zn(II) > Cu(II) > Co(II) complexes. The NO inhibitory effect revealed that the nickel complex exhibited the highest activity, whereas the cobalt complex showed the lowest inhibition. All compounds showed a significant lipid peroxidation inhibitory effect against oxidative stress. The complexes significantly diminished the TBARS level, and the nickel complex exhibited the highest inhibition at p < 0.01. Antioxidants stress the oxidative damage induced by iron, indicating that the nickel complex has the highest reducing activity. The inhibitory effect against acetylcholine esterase showed that the copper complex has the highest activity. Membrane stabilization activities clearly indicated that most compounds can improve the integrity of the cells and stability of their membrane, and this result may be related to their antioxidant capacity to protect against cytotoxicity. The nickel complex exhibited a stronger total antioxidant capacity than the other complexes. The biological and antioxidant capacities of these complexes may make them promising candidates in pharmaceutical applications.


Synthesis of N,N'-bis(1-naphthylidene)4,4'-diaminodiphenylmethane
Synthesis of cobalt complex 6. Following the general method described above, a solution of Co(OAc) 2 .H 2 O was prepared (1.24 g, 5.0 mmol) in EtOH was added to the mixture of the prepared ligand (2.5 g, 5.0 mmol) and Et 3 N (2.0 mmol) in 1,4-dioxan (15 ml Antioxidant assay. DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging assay. The oxidative scavenging activity of the ligand and its complexes was evaluated by DPPH (4 mg/100 ml methanol) Zengin, et al. 16 . Briefly, equal volumes of different concentrations (0.5, 0.1, 0.2, 0.3, 0.4 and 0.5 mg/mL DMSO) of the tested components were added to DPPH reagent using ascorbic acid as a reference. All the investigated tests were shaken and incubated at room temperature in the dark for 30 min. The decrease in DPPH radicals was evaluated at 490 nm using an ELISA reader. Each test was performed in triplicate. The radical scavenging power was estimated as a percentage of DPPH inhibition using the equation: Nitric oxide scavenging assay. The nitric oxide scavenging activity of the ligand and its complexes was evaluated following a reported procedure Padmaja, et al. 17  Lipid peroxidation scavenging activity. Lipid peroxidation (LPO) is used as a marker of oxidative stress and tissue damage 19 . LPO was measured as thiobarbituric acid reactive substance (TBARS). Then, 0.5 mL of different concentrations of the tested samples (ligand and its metals 0.1, 0.2, 0.3, 0.4 and 0.5 mg/mL DMSO), distilled water (blank), DMSO (control) or ascorbic acid (reference standard) was added to 0.5 ml liver homogenate (10%, w/v) for 45 min at 37 °C. For peroxidation induction, ferrous sulfate and H 2 O 2 (0.5 mM and 1 mM, respectively) were added to all tubes except the blank tube and incubated for 30 min at 37 °C. Butylated hydroxyl toluene was added and centrifuged for 10 min at 3000 rpm. One milliliter of the supernatant was mixed with one mL of TCA (20%) and centrifuged at 3000 rpm for 15 min. Then, 0.5 mL of 0.7% thiobarbituric acid (TBA) was added to 1 mL of the supernatant and heated for 1 h at 100 °C in a boiling water bath. The color development was evaluated at 532 nm against the blank. The antioxidant activity of the tested compounds was assessed as the percentage inhibition of LPO in liver homogenate as follows: Determination of total antioxidants. The phosphomolybdate assay was used to evaluate the total antioxidant capacity of the ligand and its metal complexes. The assay is based on the reduction of molybdate(VI) to molybdate(V) by the samples and subsequent formation of a green phosphate Mo(V) complex at low pH using ascorbic acid as a standard. One hundred microliters of 0.5 mg/ml samples (Schiff base ligand and its metal complexes) or various concentrations of ascorbic acid was added to 1.9 ml antioxidant reagent (0.6 M H 2 SO 4 , 28 mM sodium dihydrogen phosphate and 4 mM ammonium molybdate) and incubated at 95 °C for 90 min. The samples were then cooled, and the absorbance was measured at 695 nm. Ascorbic acid was utilized as a reference standard 20,21 . The ascorbic acid standard curve was plotted to calculate the total antioxidant content in one mg.
Determination of membrane stabilization activities. Anti-inflammatory activities were evaluated through the antihemolytic membrane stabilization effect of the ligand and its metal complexes using a red blood cell (RBC) membrane stabilization method 22 . Fresh whole blood samples were collected in anticoagulant tubes from a healthy volunteer who was not treated with any nonsteroidal anti-inflammatory drugs (NSAIDs) for 2 weeks In the control tube, one distilled water was used instead of the tested compounds. Different concentrations of nonsteroidal anti-inflammatory drug (diclofenac potassium) were used as standards. Then, 100 µL of RBC suspension was added to each tube and incubated at room temperature for 60 min. After incubation, the samples were centrifuged for 10 min at 3000 rpm. The absorbance of the supernatant was detected at 540 nm using an ELISA reader. The inhibition of hemolysis in the samples was evaluated according to the equation: Determination of AChE inhibition. Acetylcholinesterase (AChE) activity was evaluated according to a previously reported method 23 . Briefly, 10 μL of different concentrations of the tested compounds (test) or organic solvent (control) was added to 130 μL of phosphate buffer (pH 7.4, 0.1 M) and 20 μL of brain homogenate and then incubated at 37 °C for 45 min. Then, 5 μL of acetylcholine iodide (75 mM) was added and incubated for 15 min at 37 °C. After incubation, 60 mL of 0.32 mM DTNB was added. The absorbance was recorded at 405 nm after 5 min using donepezil (AChEI) as a standard. The percent inhibition of AChE was calculated as follows: Antimicrobial activity. The ligand and its metal(II) complexes were evaluated for antimicrobial activity against two strains, gram-positive bacteria (Staphylococcus aureus and Staphylococcus faecalis), gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa) and pathogenic fungi (Candida albicans), using DMSO as a negative control. Tetracycline was used as a positive standard for antibacterial activities, and amphotericin B was used as a positive standard for antifungal activities. The antimicrobial activity of the tested samples was determined using a modified Kirby-Bauer disc diffusion method 24 . All the synthesized compounds were dissolved to prepare a stock solution of 1 mg/mL using DMSO. Stock solution was aseptically transferred, and twofold diluted to have solutions of different concentrations. The antibacterial and antifungal activities of test compounds were done by filter paper disc method 21 and the activities were determined by measuring the diameters of the inhibition zone (mm). Media with DMF was set up as a control. All cultures were routinely maintained on NA (nutrient agar) and incubated at 37 °C. The inoculums of bacteria were performed by growing the culture in NA broth at 37 °C for overnight. Approximately, 0.1 mL of diluted bacterial or fungal culture suspension was spread with the help of spreader on NA plates uniformly. Solutions of the tested compounds and reference drugs were prepared by dissolving 10 mg of the compound in 10 mL DMF. A 100 μL volume of each sample was pipetted into a hole (depth 3 mm) made in the center of the agar. Sterile 8 mm discs (Himedia Pvt. Ltd.) were impregnated with test compounds. The disc was placed onto the plate. Each plate had one control disc impregnated with solvent. The plates were incubated at 37 °C for 18-48 h. Standard discs of Tetracycline (Antibacterial agent; 10 μg/disc) and Amphotericin B (Antifungal agent; 10 μg/disc) served as positive controls for antimicrobial activity while filter discs impregnated with 10 µL of solvent DMSO were used as a negative control. All the experiments were performed at least in triplicate and the outcomes were averaged.

Results and discussions
Green synthesis of 4,4'-methylenedianiline 1. A modified green synthesis of the ligand 4,4'-methanedianiline was conducted 15 . The reaction of aniline (2 equivalents) and formaldehyde solution (34-38%, 1 equivalent) mediated by natural kaolinite was performed in an ultrasonic bath at 42 kHz in a water bath for 20 min. Kaolinite is a 1:1 layer phyllosilicate clay mineral, and its chemical formula is Si 2 Al 2 O 5 (OH) 4 Reaction mechanism. The kaolinite particles possess oppositely charged surface regions in aqueous media due to bonding of the silicate oxygen with OH's octahedral sheet. The protonation pH (≅ 7)/deprotonation (> 7) in the aqueous phase develops charges on the edges and faces of the octahedral-oxygen sheets, causing surface charge heterogeneity. In aqueous medium, kaolinite acidic sources developed by protonation of aluminol Al-OH sites (Al-OH + H + → Al-OH 2 + ), silanol (Si-OH) sites (Si-OH + H + → Si-OH 2 + ) and coordinated water molecules formed via a prototropy process (proton migration) of two hydroxyl units ( 25 . Figure 2 depicts the suggested reaction mechanism for diamine 1 formation. In aqueous medium, the kaolinite acidic sources are different from both the basic aniline nitrogen and the partially negatively charged oxygen of formaldehyde. In the former case, the reaction would give the Al-OH 2 + /Si-OH 2 + sites, while the latter would give the speculated carbocation, releasing hydrated Al-OH and Si-OH sites in both cases. Electrophilic attack of the aldehyde carbocation at the para position of a hydrated anilinium molecule would give the intermediate

Synthesis of N,N'-bis(1-naphthylidene)4,4'-diaminodiphenylmethane 3.
Condensation of the synthesized methanedianiline 1 with two equivalents of the commercial 2-hydroxy-1-naphthaldehyde 2 in boiling EtOH for 1 h produced a (1:1) mixture of the targeted bis-imine 3 and the Schiff base 4 as a homogenous yellow-orange solid, Fig. 3. Several trials to separate these two analogs by recrystallization and/or chromatography failed.    H-4), a doublet signal at δ 6.47 ppm (J 8.6 Hz) due to one proton (Ar H-4'), a broad singlet signal at δ 4.86 ppm due to two protons (NH 2 ); a singlet signal at δ 4.00 ppm due to two aliphatic protons) CH 2 ); and a singlet signal at δ 3.75 ppm due to two aliphatic protons 3.75 (CH 2 ). The 13  A scanning electron microscopy (SEM) photograph of the ligand mixture, Fig. 4, indicated that the aggregated particles that appeared as an Octopus-like morphology were self-assembled from spherical nanosized particles with an average diameter of 40 nm. Such morphology could be attributed to colloidal self-assembly, which relied solely on particle surface chemistry, based on both the hydrophobic-hydrophilic interaction mechanism and the presence of water 25 (Fig. 5).
The synthesized metal (II) complexes are air-stable at room temperature, insoluble in water, chloroform, and most organic solvents but freely soluble in DMSO and DMF. The observed molar conductivity values for the 1.00 × 10 -3 M DMSO solution at 25 ± 1 °C for zinc and cobalt complexes are found to be 30 S⋅cm 2 ⋅mol −1 and 33 S⋅cm 2 ⋅mol −1 , respectively, indicating a 1:1 electrolyte. The molar conductance was calculated using the equation: Λm = K/C, where K = specific Conductivity, C = concentration in mole per liter. The molar conductivity value of 90 S⋅cm 2 ⋅mol −1 for the copper complex revealed a 1:2 electrolyte. However, the detected lower molar conductivity value of 14 S⋅cm 2 ⋅mol -1 for the nickel complex estimated its nonelectrolyte nature 26 . The IR spectra of the ligand and its complexes showed broad bands in the range υ 3466-3452 cm −1 assignable to the phenolic OH, the nonacoordinate NH 2 's or water molecules associated with the complexes. The υ C=N str of the ligand appeared at υ 1625 cm −1 . This band was slightly shifted to a lower wavenumber at υ 1617 cm −1 in all metal complexes, confirming the participation of the azomethine' nitrogen in chelation. The complexes of copper 6, cobalt 7 and nickel 8 each showed strong bands at υ 1602 cm −1 , υ 1601 cm −1 and υ 1601 cm −1 , respectively, attributed to the keto group. However, in the case of zinc complex 5, u C=O was not observed, indicating the participation of the azomethine's nitrogen in chelation, as suggested in the structure. Moreover, the bands at approximately u 1534-u 1536 cm −1 found only in complexes 6-8 were attributed to the υ NH-C=C-C=O str tautomer. The prepared ligand band at υ 1322 cm −1 assigned to υ (C-O) was shifted to a lower wavenumber ranging in all complexes at υ 1302-1309 cm −1 . Bands associated with M-N and M-O bonds were assigned, respectively, at u 449 cm −1 , υ 553 cm −1 for complex 5, u 417 cm −1 , u 554 cm −1 for complex 6, at υ 452 cm −1 , υ 557 cm −1 for complex 7 and υ 417 cm −1 , υ 554 cm −1 for complex 8 27 .
Magnetic moment and electronic absorption spectra. Magnetic susceptibility measurements were carried out on the complexes in the solid-state at room temperature. The mass susceptibility, χ g , was calculated using the equation: χ g = C bal .l.(R − R 0 )/10 9 m, where C bal is the balance calibration constant (= 2.086); l is the sample length (cm); m is the sample mass (gm); R is the reading for tube plus sample; R 0 is the empty tube. The magnetic μ eff moment was calculated using the equation: μ eff = √χ g × MWt × t (°C) 28  Electron paramagnetic resonance spectra. The X-band EPR spectrum of Cu(II) complex 7 at room temperature ( Fig. 6) is anisotropic with a parallel and perpendicular spin being assignable. The copper complex exhibited a g∥ value of 2.373 and g⊥ value of 2.077. The axial pattern with g∥ > g⊥. implying that the unpaired electron resides in d x2-y2 with 2B1 g as the ground state. This spectral feature is consistent with the octahedral arrangement around Cu(II) 34 . The complex exhibited a value of g av = 2.17, and deviation from g av suggested the high covalence property of the complexes with distorted symmetry. The parameter G was found to be higher than 4 (G = 4.84), indicating negligible exchange interaction of Cu-Cu in the complex 35     Antimicrobial activity. The prepared ligand and its metal (II) complexes were evaluated for antimicrobial activity against two strains, gram-positive bacteria (S. aureus and S. faecalis), gram-negative bacteria (E. coli and P. aeruginosa) and pathogenic fungi (C. albicans), using DMSO as a negative control. Tetracycline was used as a positive standard for antibacterial activities, and amphotericin B was used as a positive standard for antifungal activities. The obtained antimicrobial results are presented in Table 2. The data showed that the prepared ligand and the Co(II) complex have no efficacy against these microbes, while the Cu(II) complex showed reasonable activity against only E. coli. Both Zn(II) and Ni(II) complexes exhibited comparable moderate activities towards all studied microbes. Notably, the octahedral Ni(II) complex exhibited a sole moderate antifungal activity, whereas all other samples had no activities towards the studied fungus. The increased activity of the metal chelates can be explained based on the overtone concept and chelation theory 37 , in which metal chelates deactivate various cellular enzymes that play a vital role in various metabolic pathways of these microorganisms. Nevertheless, the variation in the activity of different metal complexes against different microorganisms depends on the impermeability of the cells of the microbes or differences in ribosomes in microbial cells 38 . The higher antimicrobial activity of the nickel(II) complex relative to other metal complexes may be due to its structure, where the octahedral nickel(II) complex is formed from the coordination of the bis ligand 3 only to the nickel(II) center, as shown in Fig. 5. However, the other investigated octahedral metal(II) complexes are formed from both ligand mixtures, and this may form nickel(II)-ligand bonds stronger than other M(II)-ligand bonds, which in turn increases the lipophilic character of nickel(II) complexes and favors permeation through the microbial cell  www.nature.com/scientificreports/ membrane, thus destroying them more aggressively. In conclusion, the less bulky octahedral nickel(II) complex enhances its rate of uptake/entrance and thus increases its antimicrobial activity.

Antioxidant activities.
Oxidative stress is a result of a free radical/antioxidant imbalance that negatively deregulates a cascade of cellular reactions leading to tissue injury and various pathological disorders. This imbalance can damage vital biomolecules, such as carbohydrates, lipids, proteins, nucleic acids and DNA, accelerating cellular death as the basis of several pathological consequences 39,40 . Antioxidants have a crucial role in the human body to slow oxidative stress and its harmful effects. Antioxidant compounds can scavenge of free radicals and lipid peroxidation repairing the cell damage and retarding the progress of various diseases induced by oxidative damage 41 . In this study, the antioxidant capacities of the ligand and its metal complexes were measured using vitamin C as a standard to evaluate the antioxidant properties of these synthesized compounds.
DPPH radical scavenging activity. The percentage of the radical scavenging activity of the ligand and its metal complexes (Cu, Zn, Ni, Co) were evaluated using vitamin C as a standard (  42,43 . The oxidant activity was reversed by these Schiff base complexes due to their ability to reduce the radicals, preventing their harmful effect. The capacity of antioxidants depends on their way to neutralize the radicals that are produced in biological systems by donating an electron 44,45 . Nitric oxide scavenging activities. Nitric oxide (NO) plays an essential bioregulatory role in several biochemical processes, such as the immune response and neural signal transmission. However, the excessive production of NO is cytotoxic and induces various physiopathological conditions, including cancer. It reacts with superoxide radicals to form highly reactive peroxynitrite anions, which can induce lipid peroxidation and interfere with cellular signaling, causing damage to cellular proteins 46,47 . Additionally, NO is involved in apoptosis induction, cell cycle interruption, DNA disruption and protein modification 47 . The NO inhibitory effect of the ligand and its metal complexes was detected using ascorbic acid as a standard (Table 4 and Fig. 8). The scavenging effect of the metal complexes (Co, Cu, Zn, Ni) was more significant (p < 0.01) than that of the free ligand. The inhibition ratio  Figure 7. DPPH IC50 values for vitamin C, the ligand, and its metal complexes. Lipid peroxidation (TBARS). Lipids play an essential role in cell membrane structure and function. All body biochemical, immunological, and physiological processes are associated with structural and functional biological membranes. The peroxidative reaction of the lipid component of cellular membranes by free radicals results in lipid peroxidation 48,49 . LPO has a serious role in triggering many pathological disorders by degrading cellular membrane integrity and leakage of cytoplasmic components. Free radical scavenging is a common system that inhibits lipid peroxidation in the body by antioxidants 50 . The TBARS assay is the most widely used method for determining the lipid peroxidation process. MDA is produced by the degradation of polyunsaturated fatty acids, which react with TBA 51 . A TBARS assay was performed to detect the capacity of the free ligand and its complexes to inhibit lipid peroxidation using ascorbic acid as a standard. All components showed a significant inhibitory effect against oxidative stress at p < 0.01. All the complexes significantly diminished the TBARS level compared to their parent ligand ( Table 5, Fig. 9), explaining the ability of these compounds to reverse oxidative stress. The ligand, metal complexes and ascorbic acid exerted their radical inhibitory effects in a concentrationdependent manner. Our results showed that chelation with metal ions is effective in the termination of lipid peroxidation. Nickel complexes exhibited the highest inhibition of the TBARS ratio (IC50 = 0.26 mg/mL), while cobalt showed the lowest percentage (IC50 = 0.48 mg/mL) at p < 0.01. The inhibition in the levels of TBARS may reflect the antioxidant capacity of these compounds.

Scientific Reports
Reducing power. Iron plays an essential role in several biochemical processes, including drug metabolism, cell respiration and oxygen transport. However, iron is also involved in various biochemical oxidation reactions, which are implicated in pathological disorders such as atherosclerosis and neurodegeneration. Therefore, any compound that interacts with iron and stops its oxidative reactions with biological molecules can be used as an antioxidant agent. Compounds that have iron reducing power and act as iron chelating agents can be used for the treatment of ferric-induced diseases such as hemochromatosis, which results in Fe 3+ accumulation. The Schiff base ligand and its metal complexes could be used as antioxidants to stress the oxidative damage induced by iron 44 . The reducing power reflects the capacity of compounds to donate electrons, modulate the oxidation/ reduction reaction of the radicals and reflect their antioxidant activity. In the ferric ion reducing antioxidant www.nature.com/scientificreports/ power assay, the increase in the absorbance indicates an increase in the reducing capacity of the antioxidant compounds 41 . Generally, the reducing properties depend on the presence of the reductant. The ferric reducing power mechanism responsible for antioxidant properties explained the effect of the compound on the reduction of Fe(III) to Fe(II) to evaluate the antioxidant capacity 52,53 . The Schiff bases had a potent Fe 3+ reducing activity and electron donor properties for stabilizing and neutralizing free radicals and reactive oxygen species 54 . Figure 10 shows that the synthesized compounds and ascorbic acid changed the ferric yellow color to various shades of blue at 700 nm, depending on the reducing capacity of each compound. The higher absorbance indicates the stronger reducing abilities and antioxidant activity of the samples. The Schiff base ligand exerted a significantly (p < 0.01) lower reducing power than metal complexes, Table 6. Among the complexes, the nickel metal complex showed the highest significant reducing activity (better Fe 2+ -chelator) at p < 0.01. The increase in absorbance of our compounds indicates their ability to reduce Fe 3+ ions, which may be due to their ability to donate electrons. According to the results, ascorbic acid showed the highest reducing activity when compared to the Schiff base ligand and its complexes. All the components exhibited strong concentration-dependent antioxidant scavenging properties in agreement with those reported in the literature 55 , where Schiff base metal complexes have a stronger reducing capacity than the ligand depending on their concentration.  Figure 9. TBARS IC50 values for vitamin C, its ligand, and its metal complexes. hydrolyzes the neurotransmitter acetylcholine (ACh) to choline. The hyperactivation of AChE and ACh deficiency is associated with cholinergic neuron dysfunction and abnormal neurotransmission. Therefore, AChE hyperactivity plays a pathogenic role in the induction of many neurodegenerative disorders such as Alzheimer's disease (AD). Pharmacological research for drug screening to resist AD pathogenesis has focused on AChE suppression to improve neurotransmission and cholinergic deficits. Some Schiff bases can inhibit AChE, improving neurotransmission. Acetylcholine esterase inhibitors boost memory and cognitive functions and have been considered a strategy for the therapy of dementia and AD 56 . The free ligand and its complexes showed a significant (p < 0.01) inhibitory effect against AChE (Table 7, Fig. 11). All the tested compounds inhibited the enzyme in a concentration-dependent manner. The copper complex exhibited the strongest inhibitory effect with IC50 = 0.34 mg/mL, while cobalt showed a lower AChE inhibitory effect with IC50 = 0.56 mg/mL. These results are in concordance with the literature 57 , where the Schiff base ligand and its complexes (Fe, Ru, Co and Pd) have inhibitory effects against AChE activity.

Membrane stabilization activities.
In several pathological disorders, such as thalassemia, sickle cell anemia and malaria, RBC membranes are hemolyzed, releasing their hemoglobin 58,59 . Erythrocyte is very sensi-  www.nature.com/scientificreports/ tive to oxidative stress and hemolysis due to its high concentration of oxygen and high polyunsaturated content. Therefore, antioxidant supplementation might strengthen the radical defense system of RBCs 60 . In this result, the Schiff base ligand and its metal complexes significantly (p < 0.01) suppressed the erythrocyte membrane lysis induced by hypotonic solution, offering strong protection against RBC hemolysis and cell damage induced by inflammatory agents. The nickel complex showed the highest antihemolytic activity toward RBCs (IC50 = 0.21 mg/mL). The standard NSAID showed a significantly higher antihemolytic activity (IC50 = 0.12 mg/ mL) than the Schiff base compounds. All the compounds exhibited concentration-dependent effects. Our synthetic compounds can improve the integrity of the cells and stability of their membranes. The membrane stabilizing activity of these compounds may be related to their antioxidant capacity to protect against cytotoxicity.
Total antioxidant capacity. The antioxidant substances are capable of counteracting the damage caused by oxidative stress due to free radical propagation. Natural and synthetic antioxidants are used to protect against oxidant molecules and delay their deterioration. Additionally, antioxidants repair the risk of several diseases, including cancer, atherosclerosis, diabetes, eye disorders, autoimmune diseases, neurodegenerative disorders, and aging diseases 53 . In the current study, the total antioxidant activity of the Schiff base ligand was significantly (p < 0.01) lower than that of all the complexes (Fig. 12), presenting protection against oxidative stress-induced tissue damage. Among these complexes, the Ni complex exhibited a stronger total antioxidant capacity (66.28 μg/ mg ± 2.51) than the other complexes (Zn, Cu and Co) at p < 0.01. These results are in line with those reported in the literature 61 , which demonstrated that the total antioxidant activity of Schiff base ligands was lower than that of Cu complexes through the phosphor molybdenum experiment and that the total antioxidant ability of Schiff base ligands and their metal complexes (Ni, Co, Cu, and Zn) was dose-dependent in the molybdenum assay at different concentrations. The existence of a Schiff base and -OH groups also has an impact on the DPPH radical scavenging efficiency. Antioxidant mensuration from the attended compounds explained that the OH functional groups as well the existence of electron donation significantly impacted the radical scavenging efficiency from phenolic Schiff bases. The higher antioxidant activity of the complexes is due to the acquisition of additional superoxide dismutation centers, which causes an increase in the molecule's ability to stabilize unpaired electrons and therefore to scavenge free radicals. Cu +2 and Zn +2 , Co +2 ions coordinated to the keto enol functionality of the prepared ligand alter the antioxidant activity of the prepared ligand. Spectroscopic investigations of the complexes point towards metal coordination at the keto enol moiety of the prepared ligand. Thus, the hydroxy groups can participate in free radical scavenging activity. Moreover, vitamin C has the highest total antioxidant capacity compared to Schiff base ligands and their complexes 61 .
In comparison with other related analogs, the antimicrobial activities of transition metal complexes with N,N'-bis (1-naphthaldimine)-p-oxydianiline (H 2 L) were tested against the same examined pathogenic microorganisms 10 . H 2 L proved to be inactive, while zinc, copper, and nickel complexes exhibited moderate antibacterial activity compared to the standard antibiotic. Furthermore, the zinc complex specifically displayed high activity toward P. aeruginosa, S. aureus and S. faecalis, while the copper complex was only active against E. coli and S. aureus. The cobalt complex has no detectable efficacy against all investigated microbes. The variation in activity of different metal (II) complexes against organisms depends either on the impermeability of the cells or differences in the ribosome. The octahedral nickel complex showed obvious inhibition of C. albicans, whereas other complexes were inactive. The antifungal activity may be dependent on both the geometry and the bulk of the complex. On the other hand, the antioxidant activity studies indicated that free radical scavenging of the ligand and its complexes with the DPPH radical revealed the weak antioxidant power of the ligand. However, metal complexation significantly enhanced scavenging, and the activity was concentration dependent. The scavenging potential may be related to the reducing action of the free radical to convert it into a nonreactive species. The NO scavenging effects clearly indicated that complexes were more significant than the ligand, and the suppression ratio increased with increasing concentration. The zinc complex was the most effective among all the complexes and showed slightly less antioxidant activity than the standard ascorbic acid. The reducing www.nature.com/scientificreports/ power effect indicated that the zinc complex exhibited the highest activity, while cobalt metal showed the lowest reducing ability, and the power increased with increasing concentration. The reducing ability of a compound generally depends on the development of reductones, which display antioxidative potential by terminating the free radical chain reaction. The inhibitory effects against AChE indicated that all compounds inhibited the enzyme in a concentration-dependent manner at p < 0.005. The nickel complex exhibits the strongest inhibition compared to other compounds. All complexes exhibited high antihemolytic activity toward human erythrocytes, and the fraction of hemolysis inhibition was concentration dependent. In comparison to other complexes, the copper complex exhibited the highest activity. In view of these findings, the antioxidant activity of the complexes depends on both the oxidation state of the metal and the ligand type. The redox properties of such complexes depend on several factors, such as the degree of unsaturation and the size of the chelate ring. Antimicrobial studies of transition metal complexes with N,N'-bis (1-naphthaldimine)-p-sulphonyldianiline (H 2 L) 7 proved that the ligand has no efficacy against investigated microbes. The tetrahedral Ni(II) complex exhibited moderate activity towards all the studied microbes, while other complexes showed a negative effect. The increased activity of nickel chelate can be explained on the basis of the overtone concept and chelation theory, in which nickel chelate deactivates various cellular enzymes that play a vital role in various metabolic pathways of these microorganisms. However, the variation in activity between the tetrahedral nickel complex and other inactive metal(II) complexes may be related to the nature and structure, where only one nickel center is present, and this may increase the lipophilic nature and allow it to react easily with the bacterial cell wall. The complexes exhibited potent antioxidant and anti-inflammatory activities in the order [Cu 2 L 2 ]0.4H 2 O > [Zn 2 L 2 ] > [Ni (HL) 2 ] > [Co 2 L 2 ] > H 2 L. The DPPH scavenging activity indicated that metal chelation significantly enhanced the antioxidant activity. The copper complex exhibited high scavenging activity, whereas cobalt showed low activity. The antioxidant activity of the complexes was low compared to that of ascorbic acid, except in the case of the copper complex, which exhibited activity similar to that of vitamin C. The complexes showed powerful inhibitory action against oxidative stress in liver tissue. All the complexes significantly reduced the TBARS level compared to their parent ligand. Notably, the copper complex exhibited the highest TBARS activity, whereas the TBARS activity of the cobalt complex was low. TBARS inhibition in the liver tissue explained the reduction in oxidative stress, which may be related to the antioxidant effect of these compounds. The increase in TBARS levels is associated with a reduction in antioxidant enzymes such as superoxide dismutase (SOD), glutathione peroxidase (GPX), catalase (CAT) and glutathione reductase (GR). SOD is considered the first line of defense against free radicals through catalyzing the dismutation of superoxide anion radical (O 2 -) into molecular oxygen. Numerous types of SOD are found in mammalian cells and have metals in their active sites, such as Cu/Zn, Ni, Fe or Mn. With respect to normal cells, cancer cells produce a large amount of O 2 ⋅− and the activity of SOD in cancer cells is lower than that in normal cells. The studies of the complexes to activate hepatocyte SOD suggested that these compounds acted as SOD activators and that the complexes manifested SOD activity better than the ligand. The SOD activity of H 2 L and the complexes was correlated with the concentration, and the results showed that the inhibition of the superoxide anion ratio increased with increasing sample concentration. Both zinc and copper complexes exhibited better superoxide anion scavengers than nickel and cobalt complexes. The inhibitory effect of the complexes on hepatocyte lipid peroxidation stimulated by superoxide anion is useful to develop a new antioxidant generation for liver diseases.

Conclusions
We report a modified ecofriendly synthesis and a proposed reaction mechanism of the ligand 4,4'-methanedianiline mediated by natural acidic kaolinite clay. Condensation of the diamine with two equivalents of the commercial 2-hydroxy-1-naphthaldehyde produced a (1:1) inseparable mixed Schiff base ligand that appeared as an Octopus-like morphology as judged by the SEM image. The obtained mixed ligand imine was reacted with four transition metal salts, namely, Co(OAc) 2 ⋅H 2 O, NiCl 2 ⋅6H 2 O, Cu(OAc) 2 . H 2 O and Zn(OAc) 2 ⋅2H 2 O furnished their corresponding complexes in high yield and purity. The structures of the ligand and its metal complexes were fully characterized by spectroscopic and spectrometric measurements. All complexes exhibited high thermal stability up to 700 °C, leaving, in most cases, approximately 40% of their mass as residues. Antimicrobial screening results of the ligand and its metal(II) complexes indicated that zinc and nickel complexes exhibited moderate activities towards all studied microbes, while the ligand and the cobalt complex had no efficacy. Antioxidant screening was concentration dependent, and their activities were in the order Ni(II) > Zn(II) > Cu(II) > Co(II) complexes. The NO inhibitory effect of the ligand and its metal complexes was concentration dependent, and the nickel complex exhibited the highest activity, whereas the cobalt complex showed the lowest inhibition. All components showed a significant lipid peroxidation inhibitory effect against oxidative stress at p < 0.01. All the complexes significantly diminished the TBARS level, and the nickel complex exhibited the highest inhibition (IC50 = 0.26 mg/mL), while the cobalt complex showed the lowest percentage (IC50 = 0.48 mg/mL) at p < 0.01, and this inhibition level reflects the antioxidant capacity of these complexes. Evaluation of the ligand and its complexes as antioxidants for stressing the oxidative damage induced by iron indicated that the ligand exerted a significantly lower reducing power than the complexes and that the activities were concentration dependent. Among the complexes, the nickel complex showed the highest reducing activity. The free ligand and its complexes showed an interesting inhibitory effect against acetylcholine esterase in a concentration-dependent manner. The copper complex exhibited the highest activity, whereas the cobalt complex showed the lowest inhibition. Screening of membrane stabilization activities of the ligand and its complexes clearly indicated that most compounds can improve the integrity of the cells and stability of their membrane, and this result may be related to their antioxidant capacity to protect against cytotoxicity. In general, the antioxidant activity of the ligand was lower than that of its complexes. The nickel complex exhibited a stronger total antioxidant capacity than the other complexes.