Bos taurus (A-2) urine assisted bioactive cobalt oxide anchored ZnO: a novel nanoscale approach

In this study, a novel synthetic method for cobalt oxide (Co3O4) nanoparticles using Bos taurus (A-2) urine as a reducing agent was developed. In addition to this ZnO nanorods were produced hydrothermally and a nanocomposite is formed through a solid-state reaction. The synthesized materials were characterized through modern characterization techniques such as XRD, FE-SEM with EDS, DLS, zeta potential, FT-IR, Raman spectroscopic analysis, and TGA with DSC. The free radical destructive activity was determined using two different methods viz. ABTS and DPPH. The potential for BSA denaturation in vitro, which is measured in comparison to heat-induced denaturation of egg albumin and results in anti-inflammatory effects of nanomaterial was studied. All synthesized nanomaterials have excellent antibacterial properties, particularly against Salmonella typhi and Staphylococcus aureus. The composite exhibits excellent antioxidant and anti-inflammatory activities in comparison to pure nanomaterials. This reveals that these nanomaterials are advantageous in medicine and drug administration.

Biomimetic-synthesis of Co 3 O 4 NPs. Cobalt (II) Chloride was procured from Sigma Aldrich and used without further purification. Then we prepared 0.1 M CoCl 2 ·6H 2 O solution in 100 mL of distilled water. To stabilize the NPs, a cationic surfactant, CTAB (0.1% w/v), was used. The solution was kept on a magnetic stirrer. Then cow urine was added dropwise in the beaker containing cobalt (II) chloride solution. After the addition of a sufficient amount of cow urine (i.e. 25 mL) in the cobalt (II) chloride solution, a whitish brown colored precipitate appeared in the solution.
Bos taurus indicus (A-2) urine contains urea and it acts as a reducing agent in this reaction. CTAB provides additional stability. When cow urine reacts with Co 2+ ions, it gets converted into Co 3 O 4 . Thus, there is the formation of Co 3 O 4 NPs. Once the Co 3 O 4 nanomaterials were formed in the solution, it was centrifuged and the precipitate was separated and dried. Then the dried precipitate was kept in the furnace for about 2 h at 900 °C for annealing. The final product, which appeared dark blue, was collected and milled using the mortar and pestle into a fine granular powder. Lastly, the synthesized material was stored and used for further purposes like characterizations and applications.
Plausible mechanism of action. The literature survey reveals that the liquid metabolic waste of Bos taurus indicus (A-2) contains urea, creatinine, aurum hydroxide, carbolic acid, phenol, calcium, and magnesium 6,12,17 . After photoactivation, a few biogenic volatile inorganics and organic compounds like CO 2 , NH 3 , CH 4, methanol, propanol, acetone, and some secondary nitrogenous products are also formed. The possible reaction mechanism for the Co 3 O 4 NPs is given below: The chemical formula of urea is CO(NH 2 ) 2 . Here, the carbonyl group, i.e. C=O, is directly connected to two -NH 2 groups. Actually, due to the presence of a lone pair of electrons on the nitrogen atom, urea seems to be a base. However, because of the electronegative character of the carbonyl group, it behaves as a neutral molecule. However, when urea is treated with the enzyme urease or at a high temperature, urea is converted to ammonia through hydrolysis. Urea is broken down into ammonia and isocyanate ions as a byproduct in the first step of the reaction. At pH levels of less than 5 and greater than 12, this reaction is reversible. Isocyanate is hydrolyzed to form ammonia in the second step (Shown in Fig. 1), and carbon dioxide is produced as a byproduct. The rate of urea hydrolysis is faster at 35 °C than at 15 °C. The pH impact is only noticeable between pH 6 and pH 8 6 . www.nature.com/scientificreports/ prepared by dissolving the precursor in distilled water. In this experiment, 200 mL of zinc nitrate solution was taken in a 500 mL capacity borosilicate beaker. A magnetic needle was inserted into the beaker and kept on a magnetic stirrer. The magnetic needle was rotated at a high speed, around 700 rpm. Meanwhile, the burette was filled with 30 wt% ammonia solution, which was then dropped into the beaker while continuously stirring. Initially, a white precipitate was formed. However, after the addition of a few drops of ammonia, the solution turned colorless. When the solution became colorless, the ammonia addition was stopped, and the solution was transferred to an autoclave for hydrothermal treatment at 120 °C for 90 min. When the system was cooled to room temperature, the resultant product was collected and washed twice with distilled water followed by ethanol. In addition, the product was dried using the air-drying method at room temperature for 2 h, followed by a furnace for about 2 h at 450 °C for annealing. It was stored for further use 3 . Characterization study. After the successful synthesis of nanomaterials, it becomes very important to ensure their size, shape, surface charge, morphology, etc. As the materials at the nanoscale are beyond the perception of human eyes, we require advanced characterization techniques to reveal them. The products were characterized by simultaneous thermal analysis, X-ray diffraction, scanning electron micrographs, etc. To illustrate the crystalline structure of the Co 3 O 4 NPs, ZnO NRs, and Co 3 O 4 -ZnO nanocomposites, an X-ray diffractometer equipped with an irradiation line Kα of copper (Bruker D8 advanced, Germany) was used to record the XRD spectrum and their corresponding size was calculated using the Sherer equation. Furthermore, a Field Emission Scanning Electron Microscope (FESEM) is used to study the surface morphology of nanomaterials. Here, we used a TESCON MIRA-3 FESEM equipped with an Energy Dispersive Spectroscopy (EDS) detector to characterize Co 3 O 4 NPs, ZnO NRs, and Co 3 O 4 -ZnO nanocomposites. The in-situ investigation of the interface was done using FT-IR, which revealed diverse functional groups adsorbed on the synthesized nanomaterials. The material was put on KBr pellets and we used an ALPHA Bruker FT-IR spectrometer. When using a Ranishaw Raman spectroscope, the Raman analysis ranges from 100 to 1000 cm −1 . The particle size and charge were analyzed using Nano ZS 90 (Malvern, UK).

The biomedical potential of nanoparticles. Minimal inhibitory concentration (MIC).
The antibacterial potential of synthesized Co 3 O 4 NPs, ZnO NRs, and nanocomposites of both was evaluated by MICs 18 . The stocks of (1 mg/mL) nanomaterials were used after sonication, whereas different working solutions of nanomaterials (50, 100, 150, and 200 µg/mL) were made. In this experiment, new inoculums of gram-positive and gram-negative bacterial strains were used. All bacterial strains were inoculated individually in 100 mL of various nanomaterial concentrations and cultured for 20-24 h in a shaking incubator (REMI) at 100 rpm at 37 °C. Using a UV-Vis spectrophotometer, we evaluated the absorbance of each tube at 625 nm to see if microbial growth was inhibited or stimulated. A negative control as distilled water was maintained along with positive control of Streptomycin and Fluconazole (as standard reference substances) for antibacterial and antifungal potential, respectively, at a concentration of 1 mg/mL. MIC was defined as the lowest nanomaterial concentration at which the growth of bacterial cells was inhibited.
Agar well diffusion method for antibacterial and antifungal potential. The antibacterial and antifungal activities of the compounds are assessed using the standard agar well diffusion technique. With slight modifications, standard agar well diffusion procedures were used to determine the antibacterial efficiency of Co 3 O 4 NPs and ZnO NRs against four distinct microorganisms 19 . 100 µL of different nanomaterial concentrations were placed into wells on agar plates, which were then kept at 4 °C for 30-40 min before being moved to an incubator for www.nature.com/scientificreports/ overnight storage at 37 °C. The plates were observed and the inhibitory zones were seen after 48 h. After this medium-range antibiotic, Streptomycin of a concentration of 100 µg/mL was used as a reference substance. In similar consent, the PDA plates were prepared using submerged inoculation of two fungal strains: Aspergillus niger (NCIM 1456) and Fusarium solani JALPK 16 . The different concentrations of nanomaterials were introduced into the wells of PDA agar plates. These agar plates were then incubated at a temperature of 37 °C and the zone of inhibition was well measured in mm.
Antioxidant activity by ABTS and DPPH radical scavenging assay. Anti-oxidants are compounds having the potential to reduce the effects of free radicals produced by oxidative stress in the body, which are generated in various diseases. By performing the commonly used ABTS and DPPH methods, Co 3 O 4 NPs prepared from cow urine and hydrothermally synthesized ZnO NRs are analyzed for their antioxidant potential by performing the commonly used ABTS and DPPH methods. The ABTS radical scavenging assay was performed as described by Re et al. 20 with a few modifications 21 . 50 μL of nanomaterial water extract with 1 μg/mL of concentration was mixed with 2950 μL of ABTS reagent. Thereafter, the absorbance of the aliquot was measured after 2 h of incubation under dark conditions and measured at 734 nm to produce a sample. To produce a control, methanol (99.5%) was used as a blank solution and its absorbance was recorded. Positive control was ascorbic acid.
The DPPH assay was carried out using the method as described by Brand Williams 22 with slight modification 23 . The reaction was done by mixing 40 μL (1 μg/mL) nanomaterials with 3 mL of DPPH reagent, and incubation was done by avoiding light oxidation for 30 min. The spectrophotometric absorbance at 517 nm was used to determine the decrease of the DPPH radical. The radical-scavenging activity (RSA) is calculated using Eq. (1), where Asample is the absorbance of the solution when the sample has been added at a particular level, Acontrol is the absorbance of the DPPH or ABTS solution.
In vitro assays for anti-inflammatory study. Protein denaturation assay. The denaturation of protein causes changes in the physiochemical properties of the protein, which are caused by inflammatory agents. This method is explained by Grant et al. 24 with slight modification 25 . The 50 μL nanomaterials working solution was diluted with 450 μL of 5% w/v BSA before being incubated at 37 °C for 20 min and heated at 57 °C for 3 min. Tubes were cooled under running water and diluted with 2.5 mL phosphate buffer saline and absorbance was measured at 660 nm. The percent of inhibition of the Fetal Bovine Serum (BSA) protein is calculated by Eq. (2), where Acontrol is absorbance of the control, Asample is the absorbance of the test sample.
Leukocyte membrane stabilization test. This experiment is depending on hypotonicity-induced hemolysis of human red blood cells (HRBC) and the measurement of Hemoglobin content was measured at 560 nm. The experiment was described by Bhurat et al. 26 with the small modification described 27 . This study used diclofenac (50 µg/mL) as standard medicine. Measurements of percent stabilization of Leukocytes [Eq. (3)] by considering control as 100%.
where Acontrol is the absorbance of the control, Asample is the absorbance of the test sample.
Statistical analysis. Experiments were set up in a completely randomized block design and each experiment was repeated thrice. Results were expressed as Mean ± SD. Statistical analysis was carried out using Graph Pad Prism 5 software. The significance of the experiment was estimated by determining the p value (p < 0.05) by oneway ANOVA Dunnett's multiple comparison test.

Results and discussion
XRD study. The poly-dispersed crystalline nano-material is revealed by the XRD spectrum. The XRD patterns of Co 3 O 4 NPs, ZnO NRs, and their composites are shown in Fig. 2. The peak position denotes the unit cell's translational symmetry, i.e., its size and shape, whereas the peak intensities denote the electron density within the unit cell. Co 3 O 4 NPs show Bragg's reflections, as shown in Fig. 2a [29][30][31] . The spectra of Co 5% and Co 10% (Fig. 2b,c) give the  (4) where λ is the wavelength of the X-ray used for diffraction (0.1540 nm).
The crystallite size for various samples is calculated using the above formula and is represented in Table 1 for Co 3 O 4 NPs and in Table 2 for ZnO NRs.
Morphology Index (MI). The interrelation between particle size and morphology determines the specific surface area of NPs. FWHM is used to determine MI. MI is calculated using the following Eq. (5),   Table 2). According to the estimated data, MI is directly proportional to particle size and inversely proportional to SSA with a minor fluctuation. Figures 3a,b and 4a,b show the results. The linear fit indicates the deviations and relationships between the figures.
Surface morphology. FE-SEM was used to study the shape and size of synthesized nanomaterials and their composites (Fig. 5). Furthermore, looking at the low magnification shows that particles grown at a high density are relatively spherical shaped. On further magnification, it reveals that Co 3 O 4 NPs tend to agglomerate. Most of the synthesized Co 3 O 4 NPs lie in the size range of 100-400 nm. (Fig. 5a). Apart from the major spherical shape, some irregularly shaped nanomaterials are observed in FE-SEM imaging 32 . The FESEM image (Fig. 5b,   www.nature.com/scientificreports/ Fig. 6.1a-c,6.2a-d,6.3a-d,6.5a-c, we can see the combined mapping of elements. Nevertheless, Fig. 6 consists of individual mappings of the element's cobalt, oxygen, and zinc. Energy dispersive spectroscopy (EDS) analysis. Elemental composition analysis of synthesized nanomaterials and their composites has been studied through the Energy Dispersive (ED) spectra. The characteristic ED spectra are shown in Fig. 7 and the analysis results are summarized in the table. In the spectrum of Co 3 O 4 NPs (Fig. 7a), four peaks are observed, which are identified as cobalt and oxygen. However, in the spectrum of ZnO NRs (Fig. 7d), there are also 4 peaks with Zinc and oxygen 34 . Even the traces of impurities and other elements are not observed. The observed composition ratios of Co 3 O 4 and ZnO in the composite are consistent with the expected composition ratio and are shown in Fig. 7b,c. This indicates that the expected stoichiometry under preparation is well maintained in the samples prepared using the mortar-pestle 35 .
To explain the composition of the material in wt%, in Co 3 O 4 NPs, 73.40% of the total weight is cobalt and the remaining is oxygen. Moreover, talking about composites in Co 5% and Co 10%, the largest portion of the total weight is acquired by zinc, i.e., 78.82% and 76.22%, respectively. However, the smallest portion is made up of cobalt, which contains 2.43% and 4.16%, not to mention, that the remaining oxygen is out of total weight. There is 17.48% oxygen and 82.52% zinc in ZnO NRs.   1b-c, 6.2b-d, 6.3b-d, 6.5b-c 36 . In Fig. 8b,c, the Co 5% and Co 10% asymmetric stretching vibration -CH 3 and -CH 2 groups absorption bands, respectively, were observed at 2922 cm −1 and 2923 cm −1 while the ZnO stretching vibration of the -CH 2 group is at 2810 cm −1 and it is illustrated in Fig. 8d. However, -OH groups of water molecules are responsible for a small peak centered at 1629, 1635 and 1623 cm −1 in spectrum of all samples except ZnO nanorods. Furthermore, these peaks show the presence of humidity. Besides, this δN-H (amide II) group is confirmed due to the presence of significant peaks at 1530, 1521, and 1598 cm −1 in Co 3 O 4 , Co 5%, and ZnO NRs, respectively. In  The stretching vibrations of C-O stretching cause the band to appear at 1068-1020 cm −1 in Co 5%, Co 10%, and ZnO. The bands between 900 and 920 cm −1 are due to H-C-N functional group. The peak at 840 cm −1 in ZnO shows there is C=C bending. The peak ranges from 780 to 700 cm −1 due to C-H bending. Nevertheless, the peak in the 700-630 cm −1 range accounts for Co 2+ -O 2− in tetrahedral coordination, and the peak in the 630-550 cm −1 range stands for Co 3+ -O 2− in octahedral coordination 38 . The transmittance peak at 495 cm −1 is likewise ascribed to Zn-O vibrations 39 .
Raman spectroscopic analysis. The optical properties of as-synthesized Co 3 O 4 NPs, ZnO NRs and their composites were characterized using Raman spectroscopy. The presence of defects was detected using Raman spectroscopy, which was utilized to detect the disorder caused by dopant incorporation in the host lattice. Figure 9 illustrates the Raman Spectra of Co 3 O 4 NPs, Co 5%, Co 10% Nanocomposite, and ZnO NRs samples taken at RT in the range of 100-1000 cm −1 . It is observed that in Fig. 9a,b,c there is a common peak at 691 cm −1 , which is because of the A 1 g phonon mode of Co 3 O 4 . The peak at 580 cm −1 is attributed to the B1 (high) phonon and the peak at 380 cm −1 is attributed to the A1 (TO) mode in the ZnO, Co 5% and Co 10% 40 . However, there are two common peaks in Fig. 9b,c,d at 101 and 436 cm −1 respectively 41 . The nonpolar modes (E2) are Raman active and have two frequencies, E2 (high) and E2 (low), associated with the vibration of the oxygen atom and vibration of Zn atoms, The peak at 101 cm −1 represents the E2 (Low) (E 21 ) mode, and the peak at 360 cm −1 represents the E2 (High) (E2 h ) mode 42 .
Particle size distribution. Dynamic light scattering (DLS) can be used to determine the hydrodynamic diameter of produced nanoparticles, nanorods, and nanocomposites. Figure 10 shows the DLS, which reveals the hydrodynamic diameter of the nanomaterials. When light passes through a colloidal solution, it bombards microscopic particles and scatters them in every way possible (i.e. Rayleigh scattering). Even if the incident light is monochromatic or laser, we see a fluctuation in the intensity of light. This fluctuation in light intensity is caused by Brownian motion in solution, which is constantly occurring. DLS, also known as photon correlation scattering, is a common name for this approach.
The average particle size of biogenic Co 3 O 4 NPs (Fig. 10a) was 729 nm, according to DLS. Biogenic Co 3 O 4 NPs have a strong peak, indicating their mono-dispersed nature. Figure 10d shows the distribution of ZnO NRs by size, which ranges from 1000 to 3000 nm. ZnO NRs have an average particle size of 1733 nm. The polydispersed nature of ZnO Nanorods can be seen in their broad size distributions. Figure 10b,c show the particle size distribution of nanocomposites. Ball milling lowers the particle size of both nanocomposites when compared to ZnO NRs. The particle size of Co 5% is 810 nm. Co 10% has a diameter of 1156 nm. Both nanocomposites are polydispersed 43 .
Electro kinetic potential and zeta potential. To find out the stability of synthesized nanomaterial, the electrokinetic potential was used. Additionally, it also sheds light on the dispersion stability of colloidal solutions and the mobility of nanoparticles as well. The material is more stable as the value of zeta potential, either positive or negative, is higher.
The zeta potential of Co 3 O 4 NPs is shown in Fig. 11a, and it is − 17.3 mV. Furthermore, looking at Fig. 11d, ZnO NRs show a zeta potential value of − 30.5 mV. The high negative zeta potential (ξ) value supports the long-term stability, good colloidal nature, and high dispersion of ZnO NRs due to negative-negative repulsion. Thermogravimetric analysis. Figure 12a- (Fig. 12a), which show a larger weight loss than others, shows a steady weight reduction with two quasi-sharp shifts at 463 °C and 917 °C, followed by a practically constant plateau. The solvent is to blame for the weight loss. The evaporation of water molecules and nitrogen causes a 4.49% weight loss from 100 to 463 °C. Nitrogen loss leading to nitrate breakdown causes the peak at 463 °C. However, due to the phase change of the material, there is a weight loss of 15.83% between 463 and 1000 °C 28,46 .
When it comes to ZnO NRs (Fig. 12d), annealing at a temperature of over 300 °C appears to ensure the production of stable ZnO NRs. The loss of volatile surfactant molecules adsorbed on the surface of Zn complexes during synthesis conditions might account for the weight of up to 300 °C. The conversion the of Zn complex to zinc hydroxide is responsible for the exothermic peak of about 333 °C. The creation of ZnO NRs and the degradation of organic molecules might be attributed to the 2nd exothermic peak at 535 °C. A 96.53% residual is left  www.nature.com/scientificreports/ after the last degradation, which takes place at 700 °C and produces ZnO with a wurtzite-like structure that is stable up to 1000 °C 47 . In short, ZnO NRs were more thermally stable as compared to the Co 3 O 4 NPs. Co 5% and Co 10% (Fig. 12b,c) show relevantly similar results, with the two endothermic peaks. The 1st peak is at 200 °C and 181 °C, respectively. These peaks are due to the different weight variations of the Co 3 O 4 NPs and ZnO NRs. Moreover, the peaks at 353 °C and 359 °C discretely were present because of the conversion of the zinc hydroxide into zinc oxide. Finally, both samples left 95.36% and 95.22% of residue, respectively. Finally, both samples left 95.36% and 95.22% of residue, respectively 48 . Co 10%). The MIC of Co 3 O 4 NPs, ZnO NRs, and their nanocomposites were studied by turbidity measurement using a spectrophotometric method at 625 nm 49 . At lower concentrations of 50 to 200 μg/mL, no visible growth was observed under a spectrophotometer, indicating that this concentration has a strong bactericidal action, which is essential in the manufacture of antibacterial compounds. The existence or absence of turbidity, which was evaluated by + or − in Table 3, was demonstrated. Because of bacterial growth, lower concentrations of nanomaterials appear turbid. This suggests that NPs at lower concentrations have minimal antibacterial effects. The results also show that the combination of 90% ZnO + 10% Co 3 O 4 has the highest potential bactericidal activity of any nanomaterial sample. The results also showed that bacterial strains like Staphylococcus aureus and Salmonella typhi were extremely susceptible to nanomaterials, but Bacillus cereus and Escherichia coli had reduced bactericidal efficacy as seen by observable growth. Previous studies by Raj et al. 50 demonstrated the MIC of zinc nanoparticles prepared from Brassica oleraceae leaves against similar types of bacteria.

Determination of antimicrobial activities of Co 3 O 4 NPs, ZnO NRs, and nanocomposite (Co 5% and
An earlier report on different methodologies for nanoparticle preparation and application from cow urine was detailed and discussed by Dabhane et al. 51 . Previous literature on structural properties of ZnO NRs and antibacterial proficiency based on four mechanisms for the production of reactive oxygen species (ROS) were studied by Bruna Lallo da Silva et al. 52 in a review study, which is similar to the current study.
These antibacterial and antifungal strategies for nanomaterials prepared were assessed against a set of four bacterial and two fungal strains shown in Table 4. The presence or absence of inhibition zones in mm was used to measure potency qualitatively. The observations are represented in Table 4 and indicate that the concentrations www.nature.com/scientificreports/  www.nature.com/scientificreports/ of 200 µg/mL nanomaterials extract show higher significant antimicrobial activity against all gram-negative bacterial strains and are depicted in tabular form in Table 4. The zone of inhibition observed is 17 ± 0.81 mm for the microbe S. aureus which is concluded to be better activity than similar ZnO NRs using solanum nigrum leaf extract in both Gram-positive (S. aureus) and Gram-negative (S. paratyphi, V. cholera, E. coli) bacteria were studied by Ramesh et al. 53 . In A. niger the minor antimicrobial spectrum of the inhibition zone was found (15.66 ± 0.94) than Fusarium solani (14.66 ± 0.47) which indicates the highest antifungal activity in the Co 5% sample. A result clearly shows that the combination method has more advantages than another singular metallic cow urine nanoparticle preparation method.
Antioxidant activity. Antioxidants are free radical molecules that are created by a variety of systems that have the potential to harm biological cellular processes. ABTS and DPPH are two methods that are often used for measuring free radical destructive activity 54 . The DPPH and ABTS scavenging activities of nanomaterials are shown in Fig. 13 in comparison to standard antioxidant ascorbic acid, indicating maximum antioxidant activity, whereas the cumulative effect of Co 3 O 4 NPs shows maximum potential, which is 42.41 ± 0.18% in DPPH radical savaging activity and 42.41 ± 0.18% in ABTS radical scavenging activity at 100 µg/mL, whereas standard ascorbic acid shows 75.68 ± 0.47% activity. The dark violet color of the DPPH was gradually decreasing over a time interval, and a decrease in absorbance was also recorded. The decrease in absorption intensity confirms DPPH's good scavenging activities, which are due to its ability to be a good oxidant, electron-losing, and capping agent on the surface of various nanomaterials. Our results show similar contact with Ag 2 O and ZnO NRs using cow urine have different applications in Figure 13. Comparative study antioxidant of activity using (A) DPPH and (B) ABTS radical scavenging potential with different concentrations of Standard, ZnO NRs, Co 3 O 4 NPs, Co 10% and Co 5% nanocomposite. (n = 3). Data represent the mean ± SD; N = 3. *P < 0.01, **P < 0.001, ***P < 0.0001 compared with control.  56 clearly determines the mechanism-based antiinflammatory properties of the NPs from several metals and metal oxides. NPs have promising anti-inflammatory properties due to their large surface area to volume ratio, which will be better at blocking inflammation enhancers e.g. cytokines and inflammation-assisting enzymes. The in vitro assessment of BSA denaturation potential, which results in anti-inflammatory effects of nanoparticles assessed against heat-induced egg albumin denaturation, is summarized in Fig. 14. In a concentration-dependent manner, all tested doses effectively inhibited the denaturation of egg albumin. Whereas the maximum BSA Denaturation % inhibition 7 of 3.53 ± 0.14% was observed at the highest concentration of Co 10%of 200 μg/mL. The order of maximum shown below is Co 10% ˃ Co 5% ˃ ZnO NRs ˃ Co 3 O 4 NPs and values are 67.46% ˃ 63.03%˃ 66.40% ˃59.04%. At the concentration of 50 μg/mL, aspirin, used as a standard drug, inhibited the enzyme by 61.91 ± 0.24%. RBC Stabilization of Leukocyte is one of the methods used to measure inflammatory response by measuring the hemoglobin absorbance spectrophotometrically at 560 nm. Anti-inflammatory drugs may lyse and usually cause lymphocyte reorganization, resulting in a rapid decrease in the number of lymphocytes in the peripheral blood, which causes a longer-term response. Because the erythrocyte membrane is comparable to the lysosomal Figure 14. Anti-inflammatory effects of ZnO NRs, Co 3 O 4 NPs, Co 10%, and Co 5% nanocomposite assessed against (A) Heat-induced egg albumin denaturation and (B) RBC Stabilization of Leukocyte (%). Data represent the mean ± SD; N = 3. *P < 0.01, **P < 0.001, ***P < 0.0001 compared with control. www.nature.com/scientificreports/ membrane, the HRBC technique was chosen for in vitro assessment of anti-inflammatory efficacy. Its stabilization means that the NPs may just as well stabilize lysosomal membranes. Results demonstrated in Fig. 14 indicate that water extract of Co 10% solution has noteworthy anti-inflammatory action at various concentrations. Whereas ZnO NRs and Co 3 O 4 NPs separately provide lower RBC Stabilization of Leukocyte (%) values of 15.15 ± 0.24% and 13.65 ± 0.24%, Respectively at a concentration of 200 μg/mL. Whereas Co 5% had a slightly lower antiinflammatory potential than Co 10%, and standard drug diclofenac had a potential of 27.52 ± 0.94%, as shown in Fig. 14. The given results are similar to previous literature on anti-inflammatory potential and antioxidant of zinc oxide nanoparticles synthesized using Polygala tenuifolia root extract 57 further studies of anti-inflammatory and antinociceptive activities in the mice model were explained by Liu et al. 58 .

Conclusions
The results confirm that the biomolecules present in the physiologically processed liquid metabolic waste of Indian cows are responsible for the successful formation of cobalt oxide nanomaterials. When we analyze these materials, we discover that they have unique characterization results. We have the composite's conformation by XRD spectra and EDX analysis. Because of the low zeta potential value, the morphology of Co 3 O 4 NPs is aggregation form as compared to others. This suggests that the substance isn't very stable. But the stability is increased by making a composite of ZnO and Co 3 O 4 . In FTIR, we observed that both Co 2+ and Co 3+ species are present in our material, as well as the conformation of the Zn-O bond. The results demonstrated an inexpensive, simple, and eco-friendly method for synthesizing Co 3 O 4 NPs, ZnO NRs, and their composites, which verified excellent antioxidant, antimicrobial, and anti-inflammatory activities. In addition to that, the nanocomposite shows excellent antioxidant and anti-inflammatory properties as compared to the pure nanomaterial. Look into the details, according to ABTS and DPPH methods Co 10% and Co 5% exhibit high antioxidant potential respectively. Nevertheless, as compared to others the Co 10% shows greater anti-inflammatory potential as well as bactericidal activity potential. Moreover, all these nanomaterials are vulnerable to S. aureus and S. typhi. Thus, we believe that all of these new nanomaterials should be considered as possible drugs for the management and treatment of various disorders.