XRD study. The poly-dispersed crystalline nano-material is revealed by the XRD spectrum. The XRD patterns of Co3O4 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. Co3O4 NPs show Bragg's reflections, as shown in Fig. 2a, (enlarged spectra on right side) with 2θ values at 31.382, 36.9599, 44.8974, 59.4242, and 65.3031 representing (220), (311), (400), (511), and (440) planes, respectively; these planes are due to the cubic Nano crystalline structure of Co3O4 NPs. The pattern was dependable, as evidenced by card No. 01-076-1802 in the JCPDS database 29.
The spectra of ZnO NRs (Fig. 2d) corresponds to JCPDS card No.03-065-3411, confirming the hexagonal wurzite-type structure. The observed 2θ values of ZnO NRs are: 31.8383, 34.4921, 36.3209, 47.5976, 56.6386, 62.8961, 66.4211, 67.9848, 69.1256, 72.6216, 76.992, 81.4166, and 89.631, which correspond to the (h k l) values (100), (002), (101), (102), (110), (103), (200), (112 (203). The JCPDS cards show that the synthesized nanomaterials are entirely crystalline in nature, with no adulterations 30–32. The spectra of Co 5% and Co 10% (Fig. 2b and c) give the conformation of sythesis of nanomaterials. This spectra corresponds to JCPDS cards 01-076-1802 (Co3O4 NPs) and 03-065-3411 (ZnO NRs) (enlarged spectrum on right side).
The Debye–Scherer’s formula (Eq. (4)) was used to compute crystallite size of the nanomaterials from the Full Width at Half Maxima (FWHM) denoted by β and Diffraction angle (θ),
$$D= \frac{0.9 }{\text{cos}}$$
4
………...
here in Eq. (4) where λ is the wavelngth of x-ray used for diffraction (0.1540 nm).
The crystallite size for various samples are calculated using above formala and represented in Table no. 1 for Co3O4 NPs and in Table no. 2 for ZnO NRs.
Morphology Index (MI). The interrelation between particle size and morphology determines the specific surface area of a NPs. FWHM is used to determine MI. MI is calculated using the following Eq. (5),
$$\text{M}\text{I}=\frac{\text{F}\text{W}\text{H}\text{M}\text{h}}{\text{F}\text{W}\text{H}\text{M}\text{h} + \text{F}\text{W}\text{H}\text{M}\text{p}}$$
5
………
The particulate FWHM value of a peak is FWHMp, and FWHMh is the highest FWHM value obtained from peaks.
The MI value of Co3O4 NPs ranges from 0.4998 to 0.6667 (Table no.1), whereas the estimated values of ZnO NRs also range from 0.4998 to 0.6667 (Table no.2). It is linked to the size of crystalline particles and the Specific Surface Area (SSA). Co3O4 NPs have an SSA value of 2.8543–29.8414 m2/g (Table no.1), while ZnO NRs have an SSA value of 12.6294–29.5411 m2/g (Table no.2). According to the estimated data, MI is directly proportional to particle size and inversely proportional to SSA with a minor fluctuation. Figure 3(a-b) and Fig. 4(a-b) show the results. The linear fit indicates the deviations and relationships between the figures.
Surface morphology. FESEM was used to study the shape and size of synthesized nanomaterials and their composites (Fig. 5). Furthermore, looking at the low magnification, it shows that particles grown at a high density are spherical shaped. On further magnification, it reveals that Co3O4 NPs tend to agglomerate. Most of the synthesized Co3O4 NPs lies in the size range of 100 nm – 400 nm. (Fig. 5.1). Apart from major spherical shape some irregular shaped nanomaterials are observed in FE-SEM imaging 33. The FESEM image (Fig. 5.2 and 5.3) of the synthesized nanocomposite flashes the formation of Co3O4 NPs anchored on the surface of the ZnO NRs. Speculation of the image reveals that the ZnO NRs are well aligned and the Co3O4 NPs are randomly dispersed in them. The FESEM images of the NRs are shown in Fig. 5.4 and 5.5. The FESEM image reveals that the diameters of the ZnO NRs lie in the range of 600–1000 nm, and the length of the nano rod is in the range of 2000–3000 nm.34. Furthermore, Fig. 5 also shows the elemental mapping of Co3O4 NPs, ZnO NRs and their composites. Firstly, in Fig. 5.1 a-c, 5.2 a-d, 5.3 a-d and 5.5 a-c, we can see the combined mapping of elements. Nevertheless, Fig. 5 consists of individual mappings of the elements Cobalt, Oxygen, and Zinc.
Energy Dispersive Spectroscopy (EDS) analysis. Elemental composition analysis of synthesized nanomaterials and their composites have been studied through the Energy Dispersive (ED) spectra. The characteristic ED spectra are shown in Fig. 6 and the analysis results are summarized in table. In the spectrum of Co3O4 NPs (Fig. 6a), 4 peaks are observed, which are identified as Cobalt and Oxygen however, in the spectrum of ZnO NRs (Fig. 6d) there are also 4 peaks with Zinc and Oxygen35. Even the traces of impurities and other elements are not observed. The observed composition ratios of Co3O4 and ZnO in the composite are consistent with expected composition ratio and it is shown in Fig. 6b and Fig. 6c. This indicates that the expected stoichiometry under preparation is well maintained in the samples prepared using the mortar-pestle36.
In order to explain the composition of material in wt%, in Co3O4 NPs, 73.40% out of total weight is cobalt and remaining is oxygen. Moreover, talking about composites in Co 5% and Co 10% the largest portion of total weight is acquired by Zinc i.e. 78.82% and 76.22% respectively. However, the smallest portion is made up of Cobalt contains 2.43% and 4.16% not to mention, remaining is Oxygen out of total weight. ZnO NRs consists 17.48% of Oxygen and 82.52% of Zinc.
FTIR Analysis. FT-IR spectrum of ZnO, Co3O4 and ZnO- Co3O4 Nano-composites were recorded in the range 4000 cm− 1 to 400 cm− 1. The plot (Fig. 7) illustrates the FT-IR spectrum of the Co3O4 NPs, Co 5%, Co 10% nanocomposite and ZnO NRs. Overall, this spectrum gives the information about different types of vibrations in these samples. not to mention, the data of Co3O4 NPs shown in Fig. 7a is the fingerprint of Co3O4. Shading the light, the spectrum of Co3O4 NPs, Co 5%, Co 10% and ZnO NRs, a broad peak centered at 3395, 3498, 3500 and 3390 cm− 1 is because of δ(H–O – H)37. In Fig. 7 (b-c), the Co 5% and Co 10% asymmetric stretching vibration –CH3 and –CH2 groups absorption bands, respectively, were observed at 2922 cm− 1 and 2923 cm− 1 while the ZnO stretching vibration of the –CH2 group is at 2810 cm− 1 and it is illustrated in Fig. 7d. 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 Nano rods, further next, these peaks show the presence of humidity. Besides these δN-H (amide II) group is confirmed due to presence of significant peak at 1530, 1521 and 1598 cm− 1in Co3O4, Co 5% and ZnO NRs respectively. In a same way, the transmittance band at 1383, 1385 and 1386 cm− 1 in Co 5%, Co 10% and ZnO NRs resulted into the presence of group νC-N (amide III) moreover, the band at 1103 cm− 1 in Co3O4 NPs, and 1168 (for Co 5% & 10%) and 1120 cm− 1 in ZnO NRs occurs because of the stretching vibrations C-C linkages correspondingly38. 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 900–920 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 − 700 cm− 1 are due to C-H bending, nevertheless, the peak in 700 − 630 cm− 1 range accounts for Co2+–O2− in tetrahedral coordination and the peak in 630 − 550 cm− 1 range stands for Co3+–O2− in octahedral coordination39. The transmittance peak at 495 cm− 1 is likewise ascribed to Zn-O vibrations 40.
Raman Spectroscopic Analysis. The optical properties of as synthesized Co3O4 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. The Fig. 8 illustrates the Raman Spectra of Co3O4 NPs, Co 5%, Co 10% Nano composite and ZnO NRs samples taken at RT in the range of 100–3250 cm− 1. It is observed that, in Fig. 8a, 8b and 8c there is a common peak at 691 cm− 1, which is because A1g phonon mode of Co3O4. However, there are two common peaks in Fig. 8b, 8c and 8d 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) (E21) mode, furthermore the peak at 360 cm− 1 represent the E2(High) (E2h) mode 42. The characteristic D and G bands for nanocomposites of Co 5% and Co 10% were D bands observed value is 1347 cm− 1 and G bands observed value is 1620 cm− 1, respectively.
Particle Size Distribution. Dynamic light scattering (DLS) can be used to determine the hydrodynamic diameter of produced nanoparticles, nanorods, and nanocomposites. Figure 9 shows the DLS, which reveals the hydrodynamic diameter of the produced nanomaterials.
When light passes through a colloidal solution, it bombards microscopic particles and scatters in every way possible (i.e. Rayleigh scattering). Even whether 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 Co3O4 NPs (Fig. 9a) was 729 nm, according to DLS. Biogenic Co3O4 NPs have a strong peak, indicating mono-dispersed nature. Figure 9d 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 poly-dispersed nature of ZnO Nanorods can be seen in their broad size distributions. Figure 9b and Fig. 9c 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 the Nano composites are poly-dispersed 43.
Electro kinetic Potential and Zeta Potential. To find out the stability of synthesized nanomaterial Electro kinetic Potential was used, additionally, it also shades light on the dispersion stability of colloidal solution and mobility of nanoparticles as well. As the value of zeta potential, either positive or negative are higher, the material is more stable.
The Fig. 10a consist the zeta potential value of the Co3O4 NPs however the value is -17.3 mV, Furthermore, looking at the Fig. 10d, ZnO NRs shows the zeta potential value − 30.5 mV. The high negative zeta potential (ξ) value supports long-term stability, good colloidal nature and high dispersity of ZnO NRs due to negative-negative repulsion. Figure 10b and Fig. 10c illustrate the zeta potential value of Co 5% and Co 10% and the values are − 30.0 mV and − 34.6 mV respectively. The synthesized nanomaterials have zeta potential values between − 35 to 0 mV have outstanding stability nevertheless, the dispersion stability also effects on the zeta potential values 44,45.
Thermogravimetric analysis. At a heating rate of 10°C/min, Fig. 11 (a-d) shows typical TGA/DSC curves of biosynthesized Co3O4 NPs, ZnO NRs, and their nanocomposites. The TGA profile of Co3O4 NPs, 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 463oC. However, due to the phase change of the material, there is a weight loss of 15.83% between 463o C and 1000oC. 29,46.
When it comes to ZnO NRs, annealing at temperature over 300°C appears to ensure the creation 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 up to 300oC. The conversion of Zn complex to Zinc hydroxide is responsible for the exothermic peak 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 after the last degradation, which takes place at 700°C and produces ZnO with a wurtzite-like structure that is stable up to 1000°C47. In short, ZnO NRs were more thermally stable as compared to the Co3O4 NPs.
Co 5% and Co 10% shows relevantly similar results, with the two endothermic peaks. The 1st peak is at 200oC and 181oC, respectively. These peaks are due to the different weight variation of the Co3O4 NPs and ZnO NRs. Moreover, the peak at 353 oC and 359 oC discretely, were present because of the conversion of the zinc hydroxide into zinc oxide. At last, both of the samples leave the 95.36% and 95.22% of residue 48.
Determination of antimicrobial Activities of ZnO NRs, Co 3 O 4 NPs and nanocomposite (Co 5% and Co 10%). The minimum inhibitory concentrations (MIC) of Co3O4 NPs, ZnO NRs and there nanocomposites were studied by turbidity measurement using spectrophotometric method at 625 nm 49. At lower concentrations of 50 to 200 g/mL, zero growth was detected under a spectrophotometer, indicating that this concentration has 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 no. 3, was demonstrated. Because of bacterial growth, lower concentrations of nanomaterials appear turbid. This suggests that NPs at lower concentrations have minimal antibacterial effect. Result also concludes that combination of 90% ZnO + 10% Co3O4 shows good potential bactericidal activity among all nanomaterials samples. 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 study by raj et al demonstrated MIC of zinc nanoparticle prepared from Brassica oleraceae leaves against similar type of bacteria50 .
Earlier report on different methodology for nanoparticle preparation and application from cow urine were detailed discussed by Dabhane et al 51. Previous literature on structural properties of ZnO NRs and antibacterial proficiency of based on four mechanisms for the production of reactive oxygen species (ROS) were studied by Bruna Lallo da Silva et al in-review study [14] which is similar relation with current study.
These antibacterial and antifungal strategies for nanomaterials prepared were assessed against a set of four bacterial and two fungi strains shown in table no. 4. The presence or absence of inhibition zones in mm was used to measure potency qualitatively. The observations are represented in table no. 4 indicates that, the concentrations of 200 µg/mL nanomaterials extract are showing higher significant antimicrobial activity against all gram-negative bacterial strains and are depicted in tabular form in table no. 4. The zone of inhibition observed is 17 ± 0.81 mm for microbe S. aureus which conclude to be better activity similar ZnO NRs using solanum nigrum leaf extract in both Gram positive (S. aureus) and Gram negative (S. paratyphi,V. cholerae, E. coli) bacteria were studied by Ramesh et al 52. In A. niger the minor antimicrobial spectrum of inhibition zone was found (15.66 ± 0.94) than Fusarium solani (14.66 ± 0.47) which indicate highest antifungal activity in Co 5% sample. A result clearly shows that, the combination method has more advantages compared with some other singular metallic cow urine nanoparticle preparation methods.
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 53. The DPPH and ABTS scavenging activities of nanomaterials are shown in Fig. 12 in comparison to standard antioxidant ascorbic acid, indicating maximum antioxidant activity, whereas the cumulative effect of Co3O4 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 100µg/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 the absorption intensity confirms the good scavenging activities of DPPH; this is due to its capability of good oxidant, electron loosing and capping agent present on the surface of different nanomaterials. Our results are similar contact with Ag2O and ZnO NRs using cow urine have different application in photoluminescence, photolytic, antibacterial and antioxidant activities are reported previously by Vinay et al 54 and Dabhane et al 51. Significantly, the biogenic synthesis of Co3O4 NPs and the hydrothermal synthesis of ZnO NRs exhibit a broad spectrum of antibacterial and antioxidant activity. As a result, it signifies promising antioxidants and antibacterial agents with potential use in the synthesis of pharmaceutical drugs. The order of maximum DPPH potential and ABTS potential is Co 10% ˃ Co 5% ˃ ZnO NRs ˃ Co3O4 NPs and Co 5% ˃ZnO NRs ˃ Co 10% ˃ Co3O4 NPs respectively. The order of value is 42.41 ± 0.18 ˃ 40.57 ± 0.58 ˃ 39.88 ± 0.43 ˃ 34.88 ± 0.48 and 36.19 ± 0.25 ˃ 34.97 ± 0.28 ˃ 29.89 ± 0.35 ˃ 26.66 ± 0.47.
Anti-inflammatory study. Previous literature by Agarwal et al clearly determines the mechanism-based anti-inflammatory properties of the NPs from several metal and metal oxide 55. NPs having promising anti-inflammatory properties due to their large surface area to volume ratio, which will be better at blocking inflammation enhancer’s 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 are summarized in Fig. 13. In a concentration-dependent manner, all tested doses effectively inhibited the denaturation of egg albumin. Whereas the max. BSA Denaturation % inhibition was 73.53 ± 0.14% observed at the concentration of 200µg/mL (highest) of Co 10%. The order of maximum was show below is Co 10% ˃ Co 5% ˃ ZnO NRs ˃ Co3O4 NPs and values are 67.46% ˃ 63.03% ˃ 66.40% ˃ 59.04%. While aspirin, used as standard drug exhibited an inhibition of 61.91 ± 0.24% at the concentration of 50 µg/mL.
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 drug possibly lyses and usually makes the reorganization of lymphocytes, that results in fast reduction in the peripheral blood lymphocyte number which cases longer term response. Because the erythrocyte membrane is comparable to the lysosomal 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. 13 indicate that water extract of Co 10% solution shave noteworthy anti-inflammatory action at various concentrations. Whereas of ZnO NRs and Co3O4 NPs separately gives lower RBC Stabilization of Leukocyte (%) which is 15.15 ± 0.24% and 13.65 ± 0.24% Respectively at concentration of 200 µg/mL. Where, Co 5% showed small lower anti-inflammatory potential when compared with the Co 10% and standard drug diclofenac have potential 27.52 ± 0.94% which is shown in Fig. 13. The given results are similar to previous literature of anti-inflammatory potential and antioxidant of zinc oxide nanoparticles synthesized using Polygala tenuifolia root extract 56 further study of anti-inflammatory and antinociceptive activities in the mice model were explained by liu et al 57.
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 Co3O4 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 Co3O4. In FTIR, we observed that both Co2+ and Co3+ species are present in our material, as well as the conformation of the Zn-O bond. The results demonstrated an expensive, straight forward, and eco-friendly method for synthesizing Co3O4 NPs, ZnO NRs, and their composites, which verified excellent antioxidant, antimicrobial, and anti-inflammatory activities. Thus, we believe that all of this new nanomaterial should be considered as a possible drug for management and treatment of various disorders. Furthermore, it was determined that the combination of ZnO NRs and Co3O4 NPs (10% Co + 90% ZnO, 5% Co + 95% ZnO) has greater in vitro anti-inflammatory and antioxidant potential than single ZnO NRs and Co3O4 NPs.