N-aryl maleimides. Similarly, the MOE software was used for the molecular docking study to explore the binding mode of the potent compounds against different enzymes. In the anti-cholinesterase activity, the compounds MSJ2 and MSJ10 exhibited outstanding activity against acetylcholinesterase (AChE), i.e., 91.90, 93.20%, and against butyrylcholinesterase (BChE), i.e., 97.30, 91.36% inhibitory potentials, respectively. The compounds MSJ9 and MSJ10 exhibited prominent α-glucosidase inhibitory potentials, i.e., 87.63 and 89.37 with IC50 value of 32 and 28.04 μM, respectively. Moreover, the compounds MSJ2 and MSJ10 revealed significant scavenging activity against DPPH free radicals with IC50 values of 2.59 and 2.52, while against ABTS displayed excellent scavenging potential with IC50 values 7.32 and 3.29 μM, respectively. The tentative results are added with molecular docking studies in the active sites of enzymes to predict the theoretical protein-ligand binding modes. Further detailed mechanism-based studies in animal models are essential for the in vivo evaluation of the potent compound.Based on the diverse pharmacological potency and the structural features of succinimide, this research considered to synthesize succinimide derivatives. Moreover, these compounds were estimated for their biological potential in terms of anti-diabetic, anti-cholinesterase, and anti-oxidant capacities. The compounds were synthesized through Michael addition of various ketones to  In the  concern . Nature  concern , 4. Rece concern , 6. Day  concern . Continu concern . There i concern , e.g., s concern .Diabetes mellitus (DM) can cause illness for prolonged time due to which the premature mortality ratio is high as compared to HIV-AIDS with approximately one death in every ten seconds across the globe . This pa2), hydroxyl radicals (OH), hydrogen peroxide (H2O2), and/or decrease in the anti-oxidant defense system. In the pathogenesis of DM (diabetes mellitus), the effect of oxidative stress is not only due to the presence of free radicals but also due to decreased anti-oxidant enzyme, glucose auto-oxidation, non-enzymatic protein glycosylation, and formation of peroxides [Free radicals have been involved in several diseases such as in cancer, liver cirrhosis, diabetes mellitus, and atherosclerosis , and thoeroxides , 25.β-secretase activities as well oxidative stress-reducing capability [In the progression of Alzheimer's disease, oxidative stress is one of the initial events before the formation of AD plaques; to counteract this effect with interrelated factors is to raise the conation of ACH through the inhibition of AChE (acetylcholinesterase) and BChE (butyrylcholinesterase), which are responsible for the breakdown of acetylcholine in the synaptic clefts . On the pability .α-amylase, α-glucosidase, and anti-oxidant activities. The as acquired derivatives were then in vitro screened in enzymes assays for AChE, BChE, ABTS, DPPH, alpha glucosidase, and alpha amylase inhibition. The enzyme interactions were then further validated using molecular docking studies.Succinimides have been observed to play a significant role in therapeutic strategies . Howeverα-glucosidase , KOH , α-amylase , creatinine , phosphate buffer, glibenclamide, and other important chemicals were purchased from the standard quality supplier.All of the chemicals, solvents, and drugs used in the analyses were obtained from Sigma Aldrich's local seller. Tween 80 , Alloxan (CAS no: 50-71-5), E.A , maleimides , chloroform , silica gel , n-hexane , MSJ 1—10) was carried out by organocatalytic Michael addition. Various cyclic and acyclic ketones were added to N-substituted aryl-maleimides. Initially, 2.0 equivalent of ketone was added to chloroform (1 M) in the presence of catalytic amounts of L-isoleucine and KOH  in a small reaction vial. This was stirred for a short time to produce the nucleophilic enamine species of the respective ketones. Afterwards, the maleimides were added to the respective reaction as the limiting reagent  to precede the reaction. The reaction was routinely checked by TLC analysis. The reaction was quenched with distilled water when the limiting reagent disappeared on the TLC analysis. The upper layer (organic) was separated 3 times from the water layer. The materials were dried and subjected to flash chromatography for the isolation of pure compounds [The synthesis of the compounds (ompounds .µL of 1.0 × 10−3 M), was prepared freshly and then added to CH3OH . A solvent like methanol was used as a control group. The mixture was then incubated for 30 minutes at 25°C. A spectrophotometer is used to calculate the DPPH free radicals at specific wavelength like 517 nm. After the incubation period, Trolox (drug) was used as the control group (positive). The anti-oxidant activity was determined using the following formula:A previously reported method for DPPH radical scavenging activity was used to determine the anti-oxidant activity of the synthetic compounds with insignificant modification . A solutµl) was mixed with the synthesized compounds (50 µl) and then reserved in darkness for about 10 minutes. The same procedure was repeated 3 times and then the results were verified using mean ± standard deviation. A solvent, e.g., methanol, was used as blank and the standard (butyl hydroxytoluen in methanol) were run at the same time. The absorbance was checked at a wavelength of 734 nm by utilizing a microplate reader.A previously reported method for ABTS radical scavenging assay was used to determine the anti-oxidant activity of the synthetic compounds with negligible modification . The stoIn Ellman's test, enzymes like (BChE) butyrylcholinesterase from equine serum and (AChE) acetylcholinesterase from electric eel were designated for studying the enzyme inhibitory capacity of the new synthesized compounds , 34. Thiµg/ml, KH2PO4 (13.6 g/L) and K2HPO4 (17.4 g/L), were dissolved in 0.1 M phosphate buffer solution. After mixing, they were combined in 6 and 94% ratio, to make 0.1 M and 8.0 phosphate buffer solutions having 0.1 pH. Potassium hydroxide was used to change the pH. The freshly prepared buffer pH 0.8, AChE (518 U/mg solid), and BChE (7–16 U/mg) were diluted to achieve final absolute concentrations of 0.01 U/ml and 0.03 U/ml, respectively. In distilled water, BChI (0.0005 M), DTNB iedithio bis-nitrobenzoic acid solution (0.0002273 M), and AChI were prepared and then kept in the refrigerator [Synthetic compounds in concentration ranging from 31.25 to 500 igerator .μl of the enzyme solution into a cuvette which has 205 μl of the synthesized compound solution; then, add 5 μl of the DTNB iedithio bis-nitrobenzoic acid reagent. After that, the substrate solution (5 μl) was added into the solution present in mixture form and was held in a water bath for 15 minutes at 30°C. Absorbance was measured at 412 nm using a double beam spectrophotometer [V = Abs/t)In this assay, introduce 5 otometer . In thisµg/ml were used for preparing the sample solutions. Glucosidase in distilled water (0.5 µg/ml) was added to the above mixture. This mixture was then incubated for 20 minutes at 37°C [In this assay, glucopyranoside is added to the solvent (phosphate buffer). Various concentrations of the synthetized compounds like 500, 250, 125, 62.5, and 31.25  at 37°C . After tµg/ml, 250 µg/ml, 125 µg/ml, 62.5 µg/ml, and 31.25 µg/ml) were mixed in this solution. Starch solution was then mixed with the above solution and then incubated for about 20 minutes at 37°C. After incubation, the reactant mixture was then kept in a water bath for some time at 100°C. Color intensity was measured using a microplate reader at 656 nm. To determine percentage inhibition, the following formula was used:In this assay, previously documented protocols were used . For preMSJ (1-10) inside the protein pockets of selected targeted macromolecules. Docking studies were performed through Autodock Vina 1.1.2 interlinked with PyRx Software that has excellent authenticity to perform. The Pdb IDs of all targeted protein moieties  were downloaded from an online data bank server https://www.rcsb.org/pdb and saved in the Pdb format. LGA  and the empirical energy-free function were employed for docking scoring through PyRx. The three-dimensional structures of all the synthesized compounds were generated through the latest version 20.0.0.41 of PerkinElmer Chem Draw professional software. Files were saved in the mol. Format. Furthermore, these structures were converted to the Pdb format through BioVia Discovery Studio Visualizer 20.0 after the addition of polar hydrogen. Meanwhile, the Pdb IDs of all targeted proteins were purified by removing the co-crystallized ligands and adding polar hydrogen, in addition to energy minimization. Docking runs were generated by adjusting the grid box with dimensions (Å) X: 52.21, Y: 51.98, Z: 48.12. Out of top-10 postures generated, the one having a greater negative binding energy was selected for each of the synthesized compounds and analyzed to understand the nature of the binding interactions.Docking studies of the synthesized compounds were carried out to analyze the stabilizing interactions of all synthesized compounds MSJ 1—10) have been synthesized and evaluated in this study as shown in MSJ1 and MSJ2 have been synthesized with cyclohexanone additions to N-phenyl and N-benzylmaleimides with isolated yields of 90 and 69%, respectively. MSJ1 was a white solid while MSJ2 was half white. The compounds MSJ4 and MSJ5 are the extended structures/modifications of MSJ1 and 2 where a 4-methylcyclohexanone has been used in the synthesis of both. The isolated yields for MSJ4 and 5 were 79 and 63%, respectively. MSJ4 was a white solid while MSJ5 was yellowish in color. The compound MSJ3 is synthesized by reacting cyclohexanone with N-(4-bromo)phenylmaleimide with 75% isolated yield and with yellowish color. The compound MSJ6 has been synthesized with a derivative of cyclohexanone having a heteroatom (oxygen) at the para position. The compound was half white in color with 73% isolated yield. Similarly, in structures MSJ7 and 8, the ketone ring is increased and decreased by a methylene unit, respectively. The isolated yields of compounds MSJ7 (white) and 8 (yellowish) were 78 and 74%, respectively. The last two compounds (MSJ9 and MSJ10) have been synthesized with acyclic ketones, i.e., acetone (for MSJ9) and 3-methyl-2-butanone (for MSJ10). Both of these compounds were yellowish in color with isolated yields of 76 (MSJ9) and 70% (MSJ10). The 1H and 13C NMR spectra are provided in the supplementary material, Figures Ten different ketone derivatives of succinimides (MSJ (1–10) were tested at different concentrations of 31.25, 62.5, 125, 250, and 500 μmol/mL, respectively. In this assay, the most potent compound was MSJ10 which caused a percent radical scavenging of 93.03 ± 0.48, 90.90 ± 0.48, 85.79 ± 0.63, 79.67 ± 0.61, and 75.69 ± 0.77 (IC50 2.52 µM), the second highest activity was displayed by MSJ2 causing 86.47 ± 0.70, 84.47 ± 0.46, 81.50 ± 0.61, 78.23 ± 0.44, and 73.45 ± 0.65 (IC50 2.59 µM), respectively. All the other compounds like MSJ1, MSJ3, MSJ4, MSJ5, MSJ6, MSJ7, MSJ8, and MSJ9 also exhibited significant results causing IC50 34.57 µM, 33.47 µM, 31.96 µM, 39.01 µM, 44.86 µM, 31.44 µM, 29.23 µM, and 53.02 µM, respectively. The standard drug, ascorbic acid, displayed 98.65 ± 1.32, 93.56 ± 0.45, 91.52 ± 0.66, 88.22 ± 1.28, and 86.42 ± 0.43 at various concentrations with IC50 6.25 µM as shown in μmol/ml of the various synthesized compounds are exhibited in In the DPPH scavenging assay, the succinimide derivatives µmo/mL). In the DPPH anti-radical assay, again, the compounds MSJ10 and MSJ2 had the highest activity with percent inhibitions of 93.03 ± 0.48, 90.90 ± 0.48, 85.79 ± 0.63, 79.67 ± 0.61, and 75.69 ± 0.77 (IC50 3.29 µM), and 90.09 ± 0.32, 88.67 ± 1.20, 83.40 ± 0.25, 78.58 ± 1.12, and 74.65 ± 1.34 (IC50 7.32 µM) correspondingly (50 4.66 µM). All the other compounds also displayed good to moderate activity . They provide an accurate approach of identification of ligand behavior inside the binding pockets of macromolecules (Ref). All the synthesized compounds MSJ2 and MSJ10, showing increased binding affinities with the protein moieties. These results were validated through comparison with co-crystallized ligands of each targeted macromolecule. The results were compared in terms of RMSD values, binding posture of ligands, and interactions like conventional hydrogen bonds, pi-pi bonds, pi-sigma interaction, carbon-hydrogen bonds, and others appearing. Regarding the binding behavior of MSJ2 with acetylcholinesterase, it gave −8.7 Kcal/mol binding energy in its best pose, while in the case of other targets, it gave −8.9 Kcal/mol, −8.2 Kcal/mol, and −8.5 Kcal/mol when interacting with butyryl cholinesterase, α-amylase, and α-glucosidase, respectively, a satisfactory comparison with in vitro results. All the visual parameters have been described in All of the synthesized compounds performed satisfactorily with the targeted macromolecules, but the results were excellent in the case of α-amylase and α-glucosidase were PHE A: 229, PHE A: 252, PHE A: 335, ARG A: 398, TYR A: 360, ARG A: 594, and HIS A: 717.When interacting with acetylcholinesterase, the cyclohexanone side chain established strong pi-sigma bond with TYR A: 334, while the aromatic ring observed a carbon-hydrogen bond with TRP A: 84. This interaction becomes more prominent in the case of butyrylcholinesterase, in which the cyclohexanone formed four alkyl and pi-alkyl linkages with TRP A: 231, LEU A: 286, PHE A: 329, and PHE A: 398. Amide pi-stacked was observed with GLY A: 115. One strong conventional hydrogen bond with ALA A: 199 further stabilized the linkages. The other prominent amino acid residues at the active site that were involved in the interaction in the case of α-amylase PDB ID: 5U3A with a bond length of 2.24 Å. The aromatic ring in this ligand formed pi-sigma linkages with LEU A: 701 and pi-alkyl linkages with VAL A: 816 and LEU A: 775.MSJ10 with all the targeted proteins including acetylcholinesterase, butyrylcholinesterase, α-amylase, and α-glucosidase displayed interesting features with binding affinities of −9.5 Kcal/mol, −9.1 Kcal/mol, −8.2 Kcal/mol, and −8.8 Kcal/mol, respectively The protein-ligand complex of the synthesized chemical moiety α-amylase, it formed two conventional hydrogen bonds with ARG A: 195 and HIS A: 299 through the cyclopentane-1,3-dione ring. Pi-anion linkage provided more stability through ASP A: 197 interaction with an aromatic ring. This structural unit formed conventional hydrogen bonds with ILE A: 780 and GLN A: 776 through the side chain 3,3-dimethylbutan-2-one in the case of the α-glucosidase macromolecular structure. The interaction of the standard drug galantamine with the active site of butyrylcholinesterase has been elaborated in This compound consists of 3,3-dimethylbutan-2-one side chain that provides a prominent interaction in the form of pi-sigma with PHE A: 331 and TYR A: 334 in the case of acetylcholinesterase. Conventional hydrogen bonds were formed with TYR A: 121 through the cyclopentane-1,3-dione structure. This complex established three conventional hydrogen bonds in butyrylcholinesterase with GLY A: 117, Gly A: 116, and ALA A: 119 through the cyclopentane-1,3-dione ring. The aromatic ring in this ligand formed pi-pi T-shaped linkages with PHR A: 329 and TRP A: 231, and pi-alkyl linkages with LEU A: 286 with a bond length of 5.25 Å, 6.21 Å, and 5.05 Å, respectively. Furthermore, TRP A: 82 formed pi-sigma linkages with 3,3-dimethylbutan-2-one. Further visualizing the binding affinity and interaction of this compound in ++ radical cation [In pharmacological research, the most interesting fields are the discovery and development of multi-target drug-able moieties. Oxygen is an important component of the aerobic life, but it also has a negative effect on our health by initiating the development of free radicals like ROS (reactive oxygen species), which are responsible for diseases like cancer, diabetes, inflammation, neurodegenerative disorders (Alzheimer's and dementia), ulcers, immune suppression, aging, and atherosclerosis , 39. Thel cation .50 of the compounds displayed virtuous enzyme inhibition. MSJ10, MSJ2, and MSJ7 had the final IC50 value, representing that they have good inhibition of the enzymes.The anti-cholinesterase enzyme presents a striking targeting moiety for normal drugs as well as for finding the mechanism's base inhibitors it plays a part in the breakdown of neurotransmitters like acetylcholine. The synthesis of AChE inhibitors presents an efficient approach to diagnose the mental symptoms of AD (Alzheimer disorder) and other promising therapeutic applications in the management of ataxia, senile dementia, and Parkinson's disease . Furtherα-amylase inhibition of the tested compounds was evaluated [α-amylase is present in pancreatic juice and saliva which can convert larger polysaccharides into smaller particles [α-glucosidase was present in the small intestine which can convert disaccharides into monosaccharides. The metabolism of carbohydrates was delayed due to the inhibitory action on α-glucosidase and α-amylase, which decreased the postprandial blood glucose level but also has adverse drug reactions (ADRs) like diarrhea and intestinal problems [α-amylase inhibition effect of MSJ10 shows an IC50 value of 16.62 µM and MSJ9 has 27.24 µM. For α-glucosidase, MSJ10 shows an IC50 of 28.04 µM while MSJ9 exhibited 32 µM. The results are shown in The in vitro valuated . Howeverarticles . Similarproblems , 54. At problems , 56. Theα-amylase, and α-glucosidase potentials of these compounds and reveals their central role in AD and diabetes. Furthermore, these compounds also scavenge ABTS and DPPH free radicals. Due to the background information and present studies of succinimide derivatives, it may be assumed that compound MSJ10 is a potentially active compound as compared to the other derivatives and could be a significant drug against DM (diabetes mellitus) and AD (Alzheimer's disease), subsequently going through additional screening and evaluations.In conclusion, we synthesized ketone succinimide derivatives. All these molecules were formed in a single step with excellent isolated produces. The finding of the current research work shows the devastating AChE, BChE,


Then, six samples of the control group and NPs GTE groups were thawed at 37°C or 39°C sterile water for 30 s and submitted to sperm quality evaluations. The sperm viability, motility, and intact of the plasma membrane (IPM) were higher (p<0.05) in NPs GTE group than control group. In contrast, the NPs GTE group presented lower (p<0.05) malondialdehyde levels and sperm DNA fragmentation (SDF) compared with the control group. The catalase levels were not significantly different (p > 0.05) between the control and NPs GTE groups. Thawing at 39°C resulted in higher (p<0.05) sperm viability, motility, and IPM than thawing at 37°C. However, thawing at 39°C group presented lower (p<0.05) malondialdehyde levels compared with thawing at 37°C. SDF and catalase levels were similar (p>0.05) between thawing at 37°C and thawing at 37°C. In conclusion, supplementation of 0.001 mg/mL of NPs GTE in SM-EY extender and thawing temperature of 39°C resulted in a better quality of frozen-thawed Kacang goat semen.Kacang goats are small ruminants produced by low-income households in smallholder and farm to reduce poverty and prevent undernutrition. Studies to find a cryopreservation protocol for Kacang goat semen are expected to multiplication of genetically superior animals selected by the paternal lineage. This study evaluated the effect of thawing temperature and supplementation of the green tea extract nanoparticle in skim milk-egg yolk (SM-EY) extender on post-thaw sperm quality of Kacang goat semen. Six ejaculates of Kacang goat were diluted in SM-EY supplemented or not (control group) with 0.001 mg/mL NPs GTE. The diluted semen was packaged with 0.25 mL straws (insemination dose: 60x10 Unfortunately, frozen semen of Kacang goat is not commercially available. Several studies conducted to date have not found appropriate protocol for freezing goat semen and the right temperature for thawing. The main problem is the susceptibility of Kacang goat sperm to cryodamage  techniques can increase yodamage  than thayodamage . Based oyodamage . Highly yodamage . Researcyodamage , which dyodamage . The redyodamage , reduce yodamage , and speyodamage . Our preyodamage . The smayodamage  makes ityodamage  and peneyodamage . Other pyodamage . These pyodamage . TherefoSperm thawing is needed to return the sperm cell to its physiologic temperature at 37°C and reactivate its metabolism . An usuaThis study was performed in 24 April - 28 December 2021 at the AI Center, Airlangga University, in the Tanjung village of Gresik District, East Java, Indonesia. It is located at coordinates 7° 19′ 25′′ S and 112° 32′ 54′′ E.The procedure of this study has been examined and approved by the Animal Care and Use Committee of Airlangga University with reference number 520/HRECC.FODM/VII/2021.The green tea leaves were extracted using ethanol solvent as conducted in the previous study , and thead libitum. Semen was collected using an artificial vagina twice a week to obtain six ejaculate samples processed into frozen semen. The ejaculates were pooled to eliminate the individual variation.This study used three Kacang bucks aged 2 to 3 years, weighing 35 to 40 kg. The goats were fed with approximately 4 kg forage crop and 3.5 kg animal feed concentrate (16% to18% crude protein) daily with drinking water Skim milk powder (Merck 115338) 10 g was added in 100 mL distilled water followed by heated to 92°C-95°C for 10 min, then cooled to 37°C. Egg yolk (derived from laboratory chicken eggs) 5 mL was added to 95 ml of skim milk solution. A 1 I.U./mL penicillin  and 1 mg/mL streptomycin  was then added . The ali6 spermatozoa/mL. The second volume was added with glycerol to reach 16% (w/v), then added to the first mixture to obtain 240 x106 spermatozoa/mL. The extended semen was cooled from 25° to 5°C for 1 hour, then filled in 0.25 ml French straws  at 60 x106 sperm/straw and sealed. The filled straws were placed on steel racks  held in liquid nitrogen vapor for 10 min (the temperature decreased from 5°C to − 140°C), and stored in liquid nitrogen (−196°C) for seven days before being evaluated (The first dilution of semen into the extender was conducted at 25°C (laboratory room temperature). Each group of the SM-EY extender was divided equally into two volumes portion. The first volume was added to fresh semen to reach 480x10valuated .Evaluation of semen before freezing was performed at the post-equilibrated step on sperm viability, motility, and IPM. Meanwhile, MDA, SDF, and catalase measurements before feezing were not performed for technical reasons. Semen must be frozen immediately; whereas, the measurements of MDA, SDF, and catalase require varying amounts of time, respectively. Post-thawed sperm quality evaluation was conducted as follows. The straws were thawed at 37°C or 39°C sterile water for 30 s, for semen quality evaluation. Sperm viability, sperm motility, IPM, MDA levels, SDF, and catalase level were evaluated .A drop of sample semen and nigrosine (Sigma-Aldrich) was mixed and smeared on a glass slide, then dried over a bunsen flame. A hundred sperm cells were classified as viable and nonviable sperm under a light microscope  at 400x magnification. The live sperm appeared with transparent heads, whereas the dead sperm were red-colored .).Sample semen of 10 µL was mixed homogenously with 1 ml of 0.9% (w/v) NaCl solution, then dropped on a glass slide and covered. The number of progressively motile sperm was counted for 100 sperm in 400× magnification  under a A 0.1 mL of semen sample was added to hypoosmotic solution 1 mL then incubated at 37°C for 30 min. A hypoosmotic solution contained 7.35 g of sodium citrate dihydrate (Sigma-Aldrich) and 13.52 g of fructose (Sigma-Aldrich) dissolved in 1 L of distilled water. The sperm IPM was assessed for 100 sperm under a light microscope  at 400× magnification. Sperm with intact plasma membrane showed a curved tail, whereas those with a damaged plasma membrane showed a straight tail .Malondialdehyde levels in the semen were determined using the thiobarbituric acid (Sigma-Aldrich) method. Semen sample100 µL, malondialdehyde kits containing 0, 1, 2, 3, 4, 5, 6, 7, and 8 µg/mL of malondialdehyde were added to 550 µL of distilled water, and 100 µL of 20% trichloroacetic acid respectively. The mixtures were homogenized for 30 s, added with 250 µL of 1 N HCl, homogenized, added with 100 μl of 1% sodium thiobarbiturate, and homogenized again. The final mixture was centrifuged at 28 G for 10 min, and then the supernatant was incubated in the 100°C water bath for 30 min and left at room temperature. Color absorption was determined in a spectrophotometer (Thermo Fisher Scientific) at a wavelength of 533 nm. The malondialdehyde levels (ng/mL) were obtained based on sample’s absorbance values extrapolated against the standard malondialdehyde curve .Acridine orange staining was used for SDF assessment. A drop of semen sample was placed on a glass slide, smeared, air dried, and fixed in 96% ethanol at 4°C for 30 min. Furthermore, the slide was hydrolyzed in 0.1 N HCl at 4°C for 5 min, rinsed with distilled water twice, and then stained with 0.05% toluidine blue (Sigma-Aldrich) for 10 min. The slide was washed in distilled water, dehydrated using t-butanol (Sigma-Aldrich), and cleaned twice with xylol (Sigma-Aldrich). The percentage DNA fragmentation was examined on 100 sperm under a light microscope  at 400× magnification. The head of sperm with intact DNA was yellow-colored, whereas the head of sperm with fragmented DNA was green-colored .2O2 phosphate-buffered saline (30 mM) 1 ml was added with 2 mL semen sample (came from 8 straws) at room temperature against a blank containing the enzyme solution. The buffered H2O2 solution (30 mM) made of 0.34 ml 30% H2O2 was diluted with fresh phosphate buffer . The UV spectrophotometric absorbance method at 240 nm wavelength measured the catalase activity  software v.23 . Data were subjected to analysis of variance and to Tukey 6/mL, viability 86.17% ± 0.98%, and progressive motility 82.50% ± 2.74%.The mean ± standard deviation particle size of NPSs GTE was 208.00 ± 26.33 nm. The mean values ± standard deviation of the semen evaluation are: volume 1.48 ± 0.10 mL, pH 6.33 ± 0.52, sperm concentration 1956.00 ± 4.38 x10p < 0.05) sperm viability, motility, and IPM (p < 0.05) MDA levels and DNA fragmentation of post-thawed sperm. However, the catalase levels were not significantly different (p > 0.05) between groups (The supplementation of green tea extract nanoparticles (GTE NPs) in the SM-EY extender increased  was smaller than the GTE's mean microparticle size (40.43-225.64 µm) . NanoparSemen has antioxidants themselves, i.e., glutathione peroxidase, superoxide dismutase, and catalase . The supKacang goat frozen semen was better thawed at 39°C for 30 s. There is some variation in temperature and duration of thawing among different species. The ram’s sperm motility, viability, the functional and structural of the plasma membrane integrity were higher when straws were thawed at 39°C for 120 s or 50°C for 30 s compared with 90°C for 2 s, 75°C for 5 s, or 75°C for 10 s . TraditiSupplementation of 0.001 mg/mL of NPs GTE in SM-EY extender resulted in higher post-thawed sperm viability, progressive motility, IPM, and lower MDA and SDF levels. Thawing temperature of 39°C resulted in a better quality of frozen-thawed Kacang goat semen.


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.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) Kaolinite is a 1:1 layer phyllosilicate clay mineral, and its chemical formula is Si2Al2O5 (OH)4, Al2O5Si2.2H2O or Al2O3.2Si4.2H2O1 along with other minor metal oxide impurities2. Because of its acidic properties, natural kaolinite has been used in many chemical and industrial reactions1. 4,4’-Methylenedianiline has been extensively used in many applications, including curing agents and chain extenders in polymers. In this work, we highlight green heterogeneous catalysts for methylenedianiline synthesis and report, for the first time, plausibly the reaction mechanism. The insertion of a methylene group, as in the case of methylenedianiline, causes the dilution of aromatic nuclei, a source of polar forces, and improves processability and properties, such as solubility and melting of the product containing such a moiety3.Clay minerals are an efficient new green technology in a variety of industrial processes because of their different set of properties, such as being inexpensive, environmentally friendly, nontoxic, reusable, and mid-catalyst. They are also very effective catalysts for a wide variety of regio- and selective organic reactions6. Copper is an essential element for life because it is associated with several copper-dependent enzymes that are key in biological processes in mammals. Elevated copper levels in plasma can be important for the etiology of some illness. For example, copper ions are closely involved in neurodegenerative disorders, especially in Parkinson's disease. Copper (II) complexes possessed various activities such as antiulcer, antiamoebic, antidiabetic, anticonvulsant, anti-inflammatory, antimicrobial and antitumor7. Cobalt like copper is an essential trace element for higher organisms. It is required in the active center of coenzymes, the so-called cobalamins, especially Vitamin B12 which regulates indirectly the synthesis of DNA. Moreover, there are at least eight cobalt-dependent proteins. Cobalamins alone are pharmaceutical agents and are treated in pathologies arising from a lack of vitamin B12. Compared to copper complexes, the cobalt complexes are limited in medical usage. Such complexes found activity against leukemia and lymphoma cell lines and bacteria strains. Further biological activity of cobalt complexes involves insulin-like properties, antifungal and antioxidant activity7. Zinc is an essential trace element which is found throughout the human body in a variety of tissues, such as skin, bone, liver, muscle, or brain. This element is the most abundant transition metal in the brain after iron, where its concentrations reach 0.1–0.5 mM. Is it essential for the folding of DNA-binding domains of transcription factors and has a variety of effects on the nervous system. It plays a crucial role in regulating the aspects of cellular metabolism, including protein, hormone, transcription, and replication functions. However, overabundant levels of zinc can lead to apoptosis and neuronal death, therefore It is important to regulate the zinc balance to maintain the homeostasis. Elevated extracellular levels of zinc lead to the breakdown of the zinc transporting system of the plasma membrane. Zinc complexes exhibited interesting biological activity such as antimicrobial, anti-inflammatory agents, successfully tested for healing zinc-deficient, chronic, and surgical wounds by local administration7. In contrast to other transition metals, only trace concentrations of manganese are found in human serum (< 10 nM) and tissue (< 4 μM). Only a handful of strictly manganese-dependent enzymes are known in both eukaryotes and prokaryotes because manganese in metalloenzymes appears to be readily interchangeable with other divalent cations1. Manganese is the oxygen-evolving complex of photosynthetic plans. Its large amounts (0.5 mg/L) and apparently with far greater effectiveness through inhalation, it can cause a poisoning syndrome in mammals with neurological damage which is sometimes irreversible8.Transition metal ions are involved in many biological processes crucial for sustaining life. These metals can serve as cofactors in proteins, enabling their biological function, regulating their activity, and/or stabilizing their structure14, we investigated a 2:1 Schiff base green synthesis derived from the reaction of 2-hydroxy-1-naphthaldehyde and the readily available 4,4’-methylenedianiline and its metal (II) complexes with four transition metal salts, namely, zinc-, cobalt- and copper acetates and nickel chloride. The diamine used was synthetic using a modified eco-friendly kaolinite clay catalyst as an acid source in aqueous medium with ultrasonic irradiation15. Bearing in mind the pronounced biological activity of Schiff base metal complexes, we examined the biological properties of the titled targeted ligand and its metal(II) complexes against the growth of bacteria and pathogenic fungi. The potential antioxidant ability has been evaluated with respect to 1,1-diphenyl-2-picrylhydrazyl (DPPH), reducing power and nitric oxide scavenging ability as well as acetylcholine esterase (AChE) inhibition. Antihemolytic membrane stabilization activities were also tested.In continuation of our interest in the chemistry of biologically active Schiff bases and their metal complexes2-Hydroxy-1-naphthaldehyde, aniline (Aldrich), formaldehyde , 4,4’-diaminodiphenylmethane (Aldrich), ethanol, 1,4-dioxane, dimethyl sulfoxide (DMSO) and diethyl ether were purchased from Aldrich. The inorganic acetate salts of zinc (II), copper (II), cobalt (II) and nickel (II) chloride were obtained from BDH. All the reagents used were of reagent grade quality and used as received without further purification. The natural kaolinitic clay was obtained from the Department of Geology, Faculty of Science, Alexandria University. Distilled water was used in the whole experiments.–3 M solution in DMSO. Magnetic susceptibility measurements were carried out at room temperature in powder form on a Magway Sherwood product Model MK1 magnetic susceptibility balance using a sealed off sample of MnCl2 solution as a calibrant. Absorption spectra were measured with a UV 500 UV‒vis spectrometer at room temperature. Infrared spectra  were recorded on a Perkin-Elmer Infrared Spectrophotometer (FTIR 1650). All spectra were recorded within the wavenumber range of 500–4000 cm−1 at 25 °C. The 1H-NMR spectra were recorded on a JEOL 500 spectrometer in DMSO-d6 solution with TMS as an internal standard. Electron impact mass spectrometry (EI-MS) measurements were determined using a Finnigan SSQ 7000 spectrometer attached to a digital DEC 300 workstation at the central scientific services unit, National Research Center, Dokki, and Cairo, Egypt. The X-band ESR spectra of the polycrystalline samples were recorded at room temperature using a Varian E-12 X-band spectrometer with 100 kHz modulation frequency in the presence of DPPH as an external standard. TGA/DTG analyses were carried out in the temperature range from 25 to 700 °C under nitrogen employing a Shimadzu DTG 60H thermal analyzer. The experimental conditions were a platinum crucible, nitrogen stream with a 30 mL min−1 flow rate and a heating rate of 20 °C min−1. The analyses were carried out using SDT-Q600-V20.5-Build-15 at the Institute of Graduate Studies and Research, Alexandria University. The morphologies of the nanosized materials were observed by scanning electron microscopy (SEM) (JEOL-JSM5300) and transmission electron microscopy (TEM) (JTM-1400 Plus) at the E-Microscope Unit, Faculty of Science, Alexandria University. The samples were sonicated in deionized water for 5 min, deposited onto carbon-coated copper mesh and allowed to air-dry before examination.Melting points were measured on a Stuart SMP10 digital melting point apparatus and are uncorrected. Elemental analyses were performed at the Micro Analytical Unit, Cairo University. Metal analysis was determined by the atomic absorption technique at the Faculty of Science, Alexandria University. The molar conductivity measurements were carried out using an HI 8033 HANNA conduct meter at 25 °C for a 10Bahulayan et al.15, of the ligand 4,4’-methanedianiline 1 was conducted. In an ultrasonic bath at 42 kHz in a water bath, 1 g of the sieved kaolinite powder through two layers of cheesecloth, to ensure particle homogeneity, was added to distilled water (200 ml) followed by successive addition of freshly distilled aniline  and dropwise formaldehyde solution . The mixture was further ultrasonicated for a period of 30 min and stirred using glass rode from time to time. The obtained yellowish-white precipitate was filtered by an air pump, and contaminated crude sample 1 was recrystallized by EtOH from many other byproducts, m.p. 98 °C (Lit.15 96 °C). Unfortunately, the obtained low yield of pure diamine by this method has turned our direction to use commercial 1 in the next step.A modified green synthesis, reported by 2  dissolved in 15 ml ethanol was added to a warm stirred solution of 4,4'-methanedianiline 1  in ethanol (15 ml), and the mixture was refluxed for 1 h. The resulting yellow–orange precipitate was filtered, subsequently washed with EtOH and Et2O (20 ml each) and then dried in a vacuum oven at 60 °C. Yield: 3.85 g (88%), mp: 275 °C. Anal. Calc. for C35H26N2O2 (%) (507); C, 82.98; H, 5.17; N, 5.52. Found; C, 82.53; H, 5.47; N, 6.67. FT-IR ν(cm): 3466 (OH), 1625 (C=N), 1545, 1511, 1350, 1322, 1212, 1140, 970, 827. UV–Vis in DMSO: λmax nm (log ɛ) L mol−1 cm−1; 274 nm (4.51), 323 nm (4.72), 339 nm (3.74), 388 nm (4.41), 444 nm (4.46), 468 nm (4.28). 1H-NMR (500 MHz): d 15.83 ; 9.60 ; 8.44 ; 7.88 ; 7.74 ; 7.55 ; 7.50 ; 7.35 ; 6.95 ; 6.86 ; 6.47 ; 4.86 ; 4.00 ; 3.75 . 13C-NMR (125 MHz): 171.53 (−C=N), 155.59, 155.36, 142.22, 140.29, 137.32, 137.32, 133.70, 130.11, 129.61, 127.06, 123.26, 122.59, 121.18, 120.98, 120.71, 108.88, 90.68, 88.84, 40.31, 40.14. MS (EI); m/z (intensity): 491 M-16 (4), 369 (29.6), 368 (100), 353 (18.5), 339 (11), 314 (19), 283(20), 274 (18), 255 (19.5), 213 (11), 160 (10), 145 (18.5), 131 (13.5), 123 (34.9), 107 (19.5), 105 (35.9), 91 (25.8), 81 (22.5), 69 (10.2), 55 (13.2), 43 (13.3), 29 (2.5).2-Hydroxy-1-naphthaldehyde 3N (2.0 mmol) in 1,4-dioxan (15 mL). The mixture was refluxed for 2 h, whereupon the complex was precipitated. It was filtered off, washed with MeOH and Et2O and dried in a vacuum oven at 60 °C.A solution of the appropriate metal salt (5.0 mmol) dissolved in EtOH (15 mL) was added to a warm stirred mixture of the ligand (5.0 mmol) and Et2.2H2O  in EtOH was added to the ligand  and Et3N (2.0 mmol) in 1,4-dioxan (15 ml). The yellow precipitate was collected in 75% (0.76 g) yield; m.p. > 300 °C, mol. wt. (980.29); Anal. Calc. for C61H48N4O5Zn (%); C, 74.58; H, 4.92; N, 5.70; Zn, 6.65. Found %, C, 74.11; H, 4.52; N, 5.23; Zn, 6.60. IR : 3452, 2921, 2852, 1617, 1577, 1502, 1456, 1427, 1391, 1362, 1303, 1249, 1182, 1161, 1143, 1119, 1043, 1017, 984, 872, 553, 495, 449. UV–Vis in DMSO: λmax nm (log ɛ) L mol−1 cm−1 = 324 nm (3.47), = 427 nm (3.47). 1H-NMR : 10.49 , 9.58 ; 8.50 ; 7.90 ; 7.74 ; 7.55 ; 7.50 ; 7.35 ; 6.95 ; 6.86 ; 6.47 ; 4.86 ; 4.00 ; 3.75 , 2.4 (buried) . MS (EI); m/e (intensity): 1178 (3), 1147 (36), 1125 (23), 1076 (13), 998 (9), 947 (9), 906 (24), 815 (6), 729 (8), 671 (2), 611 (10), 555 (4), 481(21), 424 (2), 389 (12), 336 (7), 291 (6), 249 (12), 176 (9), 58 (11), 33 (1). Molar conductance (Ω−1 cm2 mol−1): 30, µeff (B.M.): diamagnetic.Following the general procedure given above, a solution of Zn(OAc)2.H2O was prepared  in EtOH was added to the mixture of the prepared ligand  and Et3N (2.0 mmol) in 1,4-dioxan (15 ml). The reddish-brown precipitate obtained gave the following physical results: Yield: 79%, m.p. > 300 °C. Mol. wt.; 993; Anal. calc. for C61H50N4O6Co (%); C, 73.71; H, 5.07; N, 5.64; Co, 5.93 Found %, C, 74.12; H, 5.54; N, 5.02; Co, 5.88, IR : 3466, 1617, 1602, 1571, 1534, 1502, 1454, 1425, 1388, 1360, 1302, 1250, 1183, 1162, 1143, 1119, 980, 872, 557, 498, 452. MS (EI); m/z (intensity): 618 M/2 (18.2), 588 (14.4), 517 (2.4), 438 (1.8), 412 (6.5), 393 (18.8), 378 (11.3), 366 (21.8), 333 (10.6), 322 (99.9), 323 (27.1), 283 (7.4), 250 (17.7), 228 (11.8), 209 (13), 180 (16.7), 136 (8.9), 111 (6.2), 95 (11), 89 (13), 69 (7), 59 (40.3), 57 (24.4), 41 (13.9), 30 (37.7), 18 (22.2). UV–Vis in DMSO: λmax nm (log ɛ) L mol−1 cm−1; 323 nm (3.63), 361 nm (2.69), 442 nm (2.65), 470 nm (2.39), 992 nm (0.61). Molar conductance (Ω−1 cm2 mol−1): 33, µeff (B.M.): 5.06.Following the general method described above, a solution of Co(OAc)2. H2O  in EtOH (15 mL) was added to a mixture of the prepared ligand  and Et3N (2.0 mmol) in 1,4-dioxin (15 mL). The light brown precipitate was isolated in 82% yield, m.p. > 300 °C, Mol. Wt. 1074.69; Anal. Calc. for C63H54N4O9Cu (%); C, 70.41; H, 5.06; N, 5.22; Cu, 5.91. Found %, C, 70.83; H, 3.65; N, 4.27; Cu, 5. 88. IR : 3465, 1617, 1601, 1577, 1536, 1501, 1456, 1431, 1394, 1365, 1309, 1252, 1185, 1162, 1142, 1094, 1017, 983, 854, 554, 496, 417. UV–Vis in DMSO: λmax nm (log ɛ) L mol−1 cm−1 = 324 nm (3.60), 424 nm (3.53), 470 nm (3.59). MS : 1105 (2), 1077 (13), 998 (9), 977 (8), 946 (13), 906 (2), 889 (5), 859 (5), 815 (6), 759 (5), 729 (8), 671 (28), 641 (2), 611 (10), 555 (4), 496 (4), 481 (21), 467 (2), 400 (7), 321 (6), 291 (6), 249 (12), 175 (9), 147 (2), 81 (2), 64 (1), 62 (2), 61 (1), 60 (37), 31 (1). Molar conductance (Ω−1cm2 mol−1): 90, µeff (B.M.): 2.16.Following the general method, a solution of Cu(OAc)2.6H2O  in EtOH was added to a mixture of the ligand  and Et3N (2.0 mmol) in 1,4-dioxan. The olive-green precipitate of the complex was obtained with 75% yield, m.p. > 300 °C. The following physical data are collected: mol. wt. 688. Anal. calc. for C35H32N2Cl2O5Ni (%); C, 60.90; H, 4.67; N, 4.06; Ni, 8.53. Found %, C, 61.07; H, 1.27; N, 4.48; Ni, 8.30. IR : 3465, 1617, 1601, 1577, 1536, 1501, 1456, 1431, 1394, 1365, 1309, 1252, 1185, 1162, 1142, 1094, 1017, 983, 854, 554, 496, 417. UV–Vis in DMSO: λmax nm (log ɛ) L mol−1 cm−1; 323 nm (2.60), 340 nm (4.57), 361 nm (4.53), 442 nm (3.11), 471 nm (2.60). MS (EI); m/e (intensity): 977 (12), 946 (20), 919 (23), 906 (37), 889 (8), 859 (9), 831 (8), 815 (100), 801 (46), 759 (8), 729 (12), 671 (4), 611 (15), 555 (6), 496 (6), 481 (32), 467 (3), 400 (11), 349 (5), 321 (9), 291 (9), 263 (8), 249 (18), 175 (13), 121 (3), 58 (46). Molar conductance: (Ω−1 cm2 mol−1): 14, µeff (B.M.): 4.06.Following the general method described above, a solution of NiClZengin, et al.16. Briefly, equal volumes of different concentrations  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:The oxidative scavenging activity of the ligand and its complexes was evaluated by DPPH (4 mg/100 ml methanol) Padmaja, et al.17. Sodium nitroprusside  in phosphate buffer  was added to 50 µL of different concentrations of compounds  and incubated for 150 min at 25 °C. The samples were added to 150 µL Griess reagent (1% sulfanilamide and 0.1% naphthyl ethylenediamine hydrochloride in 2.5% orthophosphoric acid) and incubated at 25 °C for 30 min. The absorbance (A) was read at 540 nm using an ELISA reader. The experiment was repeated in triplicate, and ascorbic acid was used as a standard. The scavenged activity of NO was calculated using the equationThe nitric oxide scavenging activity of the ligand and its complexes was evaluated following a reported procedure 18. Then, 0.5 mL of different concentrations  was added to 0.5 mL of sodium phosphate buffer  and 0.5 mL of 1% potassium ferricyanide. The samples were incubated at 50 °C for 20 min. Then, 0.5 mL trichloroacetic acid (10%) was added and centrifuged at 3000 rpm for 15 min. The supernatant (1.0 mL) was incubated with 1.0 mL H2O2 and 0.2 mL of 1% ferric chloride for 30 min. The absorbance of the samples was measured at 700 nm. Ascorbic acid was used as a standard. An increase in the absorbance value indicates a higher reducing power.The reducing power capacity of the investigated compounds was evaluated by the method of Oyaizu19. LPO was measured as thiobarbituric acid reactive substance (TBARS). Then, 0.5 mL of different concentrations of the tested samples , distilled water (blank), DMSO (control) or ascorbic acid (reference standard) was added to 0.5 ml liver homogenate  for 45 min at 37 °C. For peroxidation induction, ferrous sulfate and H2O2  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:Lipid peroxidation (LPO) is used as a marker of oxidative stress and tissue damage2SO4, 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 standard21. The ascorbic acid standard curve was plotted to calculate the total antioxidant content in one mg.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  or various concentrations of ascorbic acid was added to 1.9 ml antioxidant reagent  for 2 weeks before the experiment. Erythrocytes were extracted by centrifugation at 3000 rpm for 5 min. The anticoagulated cells were washed 3 times with 0.9% saline and centrifuged at 3000 rpm for 10 min. An RBC suspension (40% v/v) was prepared using isotonic saline phosphate buffer . One milliliter of phosphate buffer  was added to one mL of various concentrations of the tested compounds  and 2 mL sodium chloride solution (0.25%). 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: 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 method23. Briefly, 10 μL of different concentrations of the tested compounds (test) or organic solvent (control) was added to 130 μL of phosphate buffer  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:Acetylcholinesterase (AChE) activity was evaluated according to a previously reported methodStaphylococcusaureus and Staphylococcusfaecalis), gram-negative bacteria (Escherichiacoli and Pseudomonasaeruginosa) and pathogenic fungi , 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 method24. All the synthesized compounds were dissolved to prepare a stock solution of 1 mg/mL using DMSO. Stock solution was aseptically transferred, and two-fold diluted to have solutions of different concentrations. The antibacterial and antifungal activities of test compounds were done by filter paper disc method21 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  and Amphotericin B  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.The ligand and its metal(II) complexes were evaluated for antimicrobial activity against two strains, gram-positive bacteria  and formaldehyde solution  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 Si2Al2O5(OH)4, Al2O5Si2.2H2O or Al2O3.2Si4.2H2O)2 along with other minor metal oxide impurities. Its structure possesses a sheet of octahedral Al(OH)4 bonded via H-bonds to a sheet of combined tetrahedral SiO4. Notably, no reaction occurs using other carbonate-containing clays, such as limestone or dolomite, as catalysts. According to the EDX analysis shown in Fig. 2O3 (40.62 ± 0.51), SiO2 (55.32 ± 0.71), TiO2 (2.86 ± 0.17) and FeO (1.20 ± 0.12).A modified green synthesis of the ligand 4,4’-methanedianiline was conducted+  → Al-OH2+), silanol (Si–OH) sites (Si–OH + H+  → Si-OH2+) and coordinated water molecules formed via a prototropy process (proton migration) of two hydroxyl units 25. Figure 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-OH2+/Si-OH2+ 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 (4-aminophenyl) methanol. The reaction of the kaolinite acidic sources with the alcoholic intermediate would give the corresponding 4-aminobenzyl cation and release the hydrated Al–OH sites. Reaction of the carbocation with a second hydrated anilinium molecule would give the expected target hydrated methylenedianiline. Notably, in the presence of such a hydrated and variable acidic source medium, another reaction of the acidic-trapped electron-rich amino-nitrogen center in either step was not expected.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  mixture of the targeted bis-imine 3 and the Schiff base 4 as a homogenous yellow‒orange solid, Fig. Condensation of the synthesized methanedianiline 3 and 4 exhibited a broad band at υ 3466 cm−1 corresponding to the phenolic OH groups, a strong band at υ 1625 cm−1 due to the imine bonds CH=N– and bending bands at υ 1545, 1511, 1350, 1322, 1212, 1140, 970, and 827 cm−1. The 1H-NMR spectrum exhibited a broad singlet signal at δ 15.83 ppm , a multiplet signal at δ 9.60 ppm corresponding to the two imine protons (H-1), a doublet signal at δ 8.44 ppm (J 7.65 Hz) corresponds to two protons , a doublet signal at δ 7.88 ppm (J 9.55 Hz) due to two protons , a doublet signal at δ 7.74 (J 7.65 Hz) due to two protons , a doublet signal at δ 7.55 ppm (J 7.65 Hz) due to two protons , a multiplet signal at δ 7.50 ppm for four protons , a multiplet signal at δ 7.35 ppm due to four protons , a doublet signal at δ 6.95 ppm (J 9.55 Hz) corresponds to two protons , a doublet signal at δ 6.86 ppm (J 7.65 Hz) due to one proton (Ar 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 (NH2); a singlet signal at δ 4.00 ppm due to two aliphatic protons) CH2); and a singlet signal at δ 3.75 ppm due to two aliphatic protons 3.75 (CH2). The 13C-NMR spectrum exhibited three imines (–C=N) with distinguished signals at δ 171.53 ppm, 155.59 ppm, and 155.36 ppm. Other aromatic carbons resonated at δ 142.22 ppm, δ 140.29 ppm, δ 137.32 ppm, δ 137.32 ppm, δ 133.70 ppm, δ 130.11 ppm, δ 129.61 ppm, δ 127.06 ppm, δ 123.26 ppm, δ 122.59 ppm, δ 121.18 ppm, δ 120.98 ppm, δ 120.71 ppm and δ 108.88 ppm. The carbon atoms of the two aliphatic (-CH2-) atoms resonated at δ 40.31 ppm and δ 40.14 ppm. Electron ionization mass spectrometry analysis showed a fragment peak at m/z 491 corresponding to , and the rest of the fragmentation peaks appeared at m/z 369, 368 (base peak), 353 , 339, 314, 283, 274, 255, 213, 160, 145, 131, 123, 107, 105, 91, 81, 69, 55, 43, and 29. Anal. Calc. for C59H46N4O3 (%) (858.36); C, 82.49; H, 5.40; N, 6.52. Found; C, 82.53; H, 5.47; N, 6.67.IR analysis of ligand mixtures 25.A scanning electron microscopy (SEM) photograph of the ligand mixture, Fig. 2. H2O, NiCl2.6H2O, Cu(OAc)2. H2O and Zn(OAc)2. 2H2O was heated with a mixture of the ligand and Et3N in 1,4-dioxan for 2 h, and the corresponding formed complex (5–8) was filtered and worked up  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 827.An ethanolic solution of the appropriate metal salt, namely, Co(OAc) complex –8 was fig, was calculated using the equation: χg = Cbal.l.(R − R0)/109 m, where Cbal 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; R0 is the empty tube. The magnetic μeff moment was calculated using the equation: μeff = √χg × MWt × t (°C)28. The UV–Vis spectra of the complexes were recorded in DMSO solution. The electronic spectrum of the prepared ligand in DMSO displayed intense bands at 274, 323 (sh), 339 (sh), 388 (sh), 444 and 468 nm. The former two bands could be assigned to π–π* transitions, while the latter could be assigned to charge transfer transitions. The effective magnetic moment of zero for zinc complex 5 confirms its diamagnetic nature, while the single high-intensity band at 468 nm could be assigned to charge transfer rather than a d–d transition. The Zn(II) complex is found to be diamagnetic, as expected for the d10 configuration, and an octahedral geometry is proposed for this complex. The electronic spectrum of copper complex 7 exhibited bands at 690 and 550 nm, which may be assigned to 2B1g → 2Eg and 2B1g → 2A1g, respectively29. These bands favored distorted octahedral geometry around the Cu(II) ion and were supported by the magnetic moment value of 2.16 BM30. Moreover, the meff value of nickel complex 8 is 4.06 B.M. which agrees with the reported values for octahedral, tetrahedral, or high spin five coordinate nickel(II) complexes. The electronic spectrum of nickel complex 8 in DMSO shows absorption bands at 323, 340, 361, 442, and 471 nm. Transitions assigned to 3A2g → 3T1g (F) and 3A2g (F) → 3T1g (P) are hidden by the very intense charge transfer and ligand absorption bands. The μeff value of cobalt(II) complex 6 is 5.06 B.M., suggesting an octahedral environment for Co(II)32. The electronic spectrum of the cobalt complex in DMSO shows absorption bands at 323, 361, 442, 470, and 992 nm. The latter d–d transition in the visible region is assigned to 4T1g (F) → 4A2g(P)33.Magnetic susceptibility measurements were carried out on the complexes in the solid-state at room temperature. The mass susceptibility, χx2–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 gav = 2.17, and deviation from gav 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 complex35. Thus, based on the EPR analysis of the investigated Cu(II) complex 7, the greater gav value indicated the presence of Cu–O and Cu–N bonds in these chelates36.The X-band EPR spectrum of Cu(II) complex 7 at room temperature Fig.  is aniso5–8 in the range of 25–700 °C under N2 and their stepwise thermal postulated degradation data are compiled in Table 11H9 as a residue. The TGA profile of zinc complex 5 showed two decomposition steps; the first process at 150 °C may be related to dehydration of water molecules and elimination of nonacoordinate -OAc groups and NH2 groups . The second decomposition step at 450 °C may correspond to the cleavage of the diphenyl-methylene fragments , leaving mass residue assigned as C35H21N2Zn . The thermal profile of complex 6 exhibited three significant thermal events within the temperature range of 25–700 °C. The first revealed an exothermic peak with a mass loss of 9.19%  at 216 °C, corresponding to the elimination of adsorbed water, the coordinated OAc group and the NH2 group. The second decomposition step occurred at 450 °C (mass loss not determined), and the major fragmentation step took place up to 690 °C with a mass loss of 45.94%  attributed to the loss of C35H24N. The residual mass could be assigned to C24H20N22O3 Calc 44.40%; Found 44.48%). Decomposition of copper complex 7 proceeded in one main broad step occurring in the temperature range 311–690 °C with a total mass loss of 54.63%, attributed to the loss of the hydrated bis-ligand moiety leaving a remaining mass residue C26H21N2O2Cu . The thermal profile of complex 8 exhibited four thermal fragmentation steps within the temperature range of 25–700 °C. The first revealed an exothermic peak with a mass loss of 8.63%  at 200 °C, corresponding to the dehydration of adsorbed water molecules. The successive second and third decomposition steps occurred at 460 °C and 550 °C (mass loss not determined), and the major fragmentation step took place at 690 °C with a mass loss of 55.93%  attributed to the loss of the dehydroxylated bis-ligand moiety. The residual mass was 34.83% assigned to C10H8ClO2Ni .The thermogravimetric (TGA) and derivative thermogravimetric (DTG) plots of the prepared ligand and its investigated metal complexes S. aureus and S. faecalis), gram-negative bacteria (E. coli and P. aeruginosa) and pathogenic fungi , 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 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 cells38. 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. The prepared ligand and its metal (II) complexes were evaluated for antimicrobial activity against two strains, gram-positive bacteria  were evaluated using vitamin C as a standard Table , Fig. 7.47. Additionally, NO is involved in apoptosis induction, cell cycle interruption, DNA disruption and protein modification47. The NO inhibitory effect of the ligand and its metal complexes was detected using ascorbic acid as a standard , while Cobalt exhibited the lowest effect (IC50 = 0.38 mg/m). The complexes may have the ability to counteract the harmful effect of NO formation by repairing the damaging effects of excessive NO generation, which may be important for protecting human health.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 proteinsrd Table . The sca49. 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 antioxidants50. 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 TBA51. 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  to Fe(II) to evaluate the antioxidant capacity53. The Schiff bases had a potent Fe3+ reducing activity and electron donor properties for stabilizing and neutralizing free radicals and reactive oxygen species54. Figure 2+-chelator) at p < 0.01. The increase in absorbance of our compounds indicates their ability to reduce Fe3+ 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 literature55, where Schiff base metal complexes have a stronger reducing capacity than the ligand depending on their concentration.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 Fe56. The free ligand and its complexes showed a significant (p < 0.01) inhibitory effect against AChE  have inhibitory effects against AChE activity.Acetylcholine esterase (AChE) is mainly found in the central nervous system (CNS) and 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 ADhE Table , Fig. 1159. Erythrocyte is very sensitive 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 RBCs60. 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.In several pathological disorders, such as thalassemia, sickle cell anemia and malaria, RBC membranes are hemolyzed, releasing their hemoglobin53. 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  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 complexes61.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 diseasesxes Fig. , presentN,N’-bis -p-oxydianiline (H2L) were tested against the same examined pathogenic microorganisms10. H2L 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 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.In comparison with other related analogs, the antimicrobial activities of transition metal complexes with N,N’-bis -p-sulphonyldianiline (H2L)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 [Cu2L2]0.4H2O > [Zn2L2] > [Ni (HL)2] > [Co2L2] > H2L. 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 (O2-) 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 O2⋅− 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 H2L 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.Antimicrobial studies of transition metal complexes with 2⋅H2O, NiCl2⋅6H2O, Cu(OAc)2. H2O and Zn(OAc)2⋅2H2O 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.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)Supplementary Figures.