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**[CSN/KN24/V0105]** **GREEN SYNTHESIS OF COBALT NANOPARTICLES AND INVESTIGATION OF ITS ANTIOXIDANT AND ANTIBACTERIAL PROPERTIES** Fatima Muhammad Balarabe^1^, Safiya Yusuf Zubairu^1^ and Sahal Abdullahi^1^ ^1^Department of Chemistry and Biochemistry, School of Applied Sciences, Nuhu Bamalli Poly...

**[CSN/KN24/V0105]** **GREEN SYNTHESIS OF COBALT NANOPARTICLES AND INVESTIGATION OF ITS ANTIOXIDANT AND ANTIBACTERIAL PROPERTIES** Fatima Muhammad Balarabe^1^, Safiya Yusuf Zubairu^1^ and Sahal Abdullahi^1^ ^1^Department of Chemistry and Biochemistry, School of Applied Sciences, Nuhu Bamalli Polytechnic, Zaria \*Corresponding Author: (08032845249) **ABSTRACT** In this study, Cobalt nanoparticles (Co NPs) was synthesized using mango leaves extract as a reducing, stabilizing and capping agent. The Co NPs was characterized with the aid of Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), Scanning electron microscope- energy dispersive X-ray spectroscopy(SEM-EDX) and UV-Vis spectroscopy to determine their functional groups, crystallinity, morphology (size and shape) and optical properties. The UV-Vis spectra measurement was 306 nm. FTIR spectra showed the presence of various functional groups however, the peak at 783.9 cm^-1^ can be attributed to Co-O stretching vibrations of cobalt oxide NPs. The SEM images were irregular in shape and size, the EDS results showed the elemental compositions; cobalt, oxygen and other trace elements. XRD patterns of Co NPs showed high crystallinity, also it revealed that the NPs were cubic and tetragonal, the size was calculated using Debye Scherer's equation with an average size of 53.87 nm. The antioxidant potential of Co NPs of mango leaves extract was assessed using DPPH radical scavenging method. The Co NPs exhibited significant antioxidant activities which ranged between 56-82 %. An antibacterial activity of the NPs was conducted using agar diffusion method against *Salmonella typhi*, *Staphylococcus aureus* and *Escherichia* *coli*. The results showed remarkable activity which may be used in further therapeutic and biomedical aspects. This study not only synthesizes biocompatible cost effective and eco-friendly nanoparticles but can also be effectively used as a multifunctional agent in medical field. **Key words: Cobalt, nanoparticles, antioxidant, antibacterial, mango leaves.** **INTRODUCTION** Nanotechnology has a strong effect in every field of life. Synthesis of metallic nanoparticles has been a choice of interest over the few decades by many researchers as they provide a vast variety of applications. Apart from interests on this research, nanoparticles are very important due to their unusual properties and prospective uses in optical, electronic, catalytic, magnetic materials, thermal properties with consistent bulk materials (Eluri and Paul, 2012). **MATERIALS AND METHODS** **Collection and pretreatment of samples** Mango leaves was collected within Kaduna metropolis. The sample was washed with tap water several times, then with distilled water to remove dirt and was dried in room temperature. The leave sample was grinded using an electric blender to fine powder and kept ready for extraction. 20g of the dried leaf was weighed and placed in a 250ml beaker and diluted with 100ml distilled water. The mixture obtained was boiled for 35 minutes with stirring at 80^0^C. After boiling it was cooled, filtered and the filtrate (extract) was stored at 5^0^C for further use (Chandran *et al.,* 2006). **Solutions used were prepared according to standard method.** **Synthesis of Cobalt Nanoparticles** The metal nanoparticle was synthesized using bio reduction method as described by Geethalakshmi *et al., (*2010) with slight modifications Co-NPs was synthesised by mixing 10 ml of mango leaf extract with 100 ml of 0.5M aqueous cobalt chloride in 250 ml beaker and stirred. The mixtures was allowed to settle at room temperature and the bio reduction of Co^2+^ ion in the solution was monitored using a UV-visible spectrophotometer at different intervals from 0 to 24 hours. CoCl~2~.6H~2~O + Plant metabolites Co^0^NPs + byproducts **Characterization of the Synthesized nanoparticle** The synthesised cobalt nanoparticle (NPs) was characterized using UV-visible spectroscopy, Fourier Transform Infrared spectroscopy (FTIR), Scanning Electron Microscopy (SEM) and **X**- Ray Diffraction Analysis. **Determination of Antioxidant activity (DPPH Assay)** The antioxidant activities of the synthesised nanoparticle was determined using DPPH scavenging assay as described by Liyana-Pathiranan and Shahidi (2005). A solution of 0.135 mM of DPPH in methanol was prepared and 1.0 ml of the extract prepared in methanol containing 0.025- 0.5 mg of the cobalt NPs and standard drugs (ascorbic acid). The reaction mixture was vortexed thoroughly and left in the dark room, at room temperature for 30 minutes. The absorbance of the mixture was measured spectrophotometrically at 517nm, the percentage of DPPH radical scavenging assay of the MNps and standard compounds was calculated as: \%scavenging \[DPPH\] = \[(Ac -As/Ac)\] ×100 Where;, As - is the absorbance of the sample. Ac - is the absorbance of standard. **Antibacterial Screening Of the synthesised Cobalt Nanoparticles** **Test organisms** Three (3) clinical bacterial isolates (*Salmonella typhi, Staphylococcus aureus and, Escherichia coli)* were obtained from the Department of Microbiology, Faculty of Life science, Ahmadu Bello University Zaria for the antibacterial screening of the biosynthesised Nanoparticle (Co-Nps ). **Sensitivity Test (Zone of Inhibition Measurement) Agar diffusion method** The biosynthesised nanoparticle was screened for in vitro antibacterial activities against *S.typhi, E.coli and S. aureus.* Sterile swab sticks were immersed into the standardized inoculum and swabbed onto the prepared medium (Mueller Hinton agar) plates. Five wells were made in each plate using a sterile corn borer. Aliquots of 50 micro-liters of various concentrations of the sample (Co-Nps) were placed into the wells using a micro pipette. These were allowed to stand in room temperature for an hour for it to diffuse the agar. Afterwards, it was incubated for 24 hours at 37^0^ C. The sensitivity test was determined by measuring the diameter of the inhibition zones in millimetre produced against the test bacterial isolates. The experiments were conducted in replicates and the mean values were noted (Garba *et al.,* 2019). **Minimum Inhibitory Concentrations (MIC) and Minimum Bactericidal Concentration (MBC)** The minimum inhibitory concentrations of the biosynthesised nanoparticle was determined from the lowest concentrations that showed activity on the plate. Four different concentrations (mg/ml) were prepared; 200, 100, 50 and 25 of the biosynthesised cobalt nanoparticle in 2 ml peptone water in test tubes. These were inoculated with the bacterial isolates then, incubated for 24 hours at 370 C. For each experiment, two control tubes were made with the Biosynthesised nanoparticle and growth medium without the standardised inoculum in test tubes and tube with the growth medium and the inoculum (organism control). (Garba *et al.,* 2009). The MBC of the biosynthesised nanoparticle were determined by sub culturing all tubes that exhibited no visible bacterial growth from the MIC on the fresh solid media, then incubated for 1 day at 37^0^ C (Garba *et al.,* 2009). **RESULTS**  Fig 2: FTIR spectra of cobalt nanoparticles synthesized using mango leave extract Plate 1a: SEM image of Cobalt nanoparticles synthesized using mango leave extract  Plate 1b: SEM image of Cobalt nanoparticles synthesized using mango leave extract **Energy dispersive X-ray Spectroscopy Analysis** **Table 1**: EDS analysis of cobalt nanoparticles synthesized using mango leave extract -- -- -- -- -- -- -- -- -- -- **Fig 3:** Diffractogram of XRD analysis of cobalt nanoparticles synthesized using mango leave extract Table 2: Percentage DPPH Radical Scavenging Assay **EXTRACT CONC. (µL/mL)** **% DPPH RSA FOR MANGO COBALT** --------------------------- --------------------------------- 62.5 56.84±2.908 125 76.973±1.617 250 80.603±0.255 500 82.43±0.226 **Table 3: Sensitivity Test (Zone of inhibition)** +-----------------+-----------------+-----------------+-----------------+ | Test | Conc (mg/ml | Co-Np | Control | | microorganismsm | | | | | | | | CPX | +=================+=================+=================+=================+ | *S.aureus* | 200 | 34 | 35 | +-----------------+-----------------+-----------------+-----------------+ | | 100 | 29 | | +-----------------+-----------------+-----------------+-----------------+ | | 50 | 17 | | +-----------------+-----------------+-----------------+-----------------+ | | 25 | \- | | +-----------------+-----------------+-----------------+-----------------+ | | 12.5 | \- | | +-----------------+-----------------+-----------------+-----------------+ | *E.coli* | 200 | 40 | 40 | +-----------------+-----------------+-----------------+-----------------+ | | 100 | 30 | | +-----------------+-----------------+-----------------+-----------------+ | | 50 | 25 | | +-----------------+-----------------+-----------------+-----------------+ | | 25 | 18 | | +-----------------+-----------------+-----------------+-----------------+ | | 12.5 | 14 | | +-----------------+-----------------+-----------------+-----------------+ | *S.typhi* | 200 | 40 | 45 | +-----------------+-----------------+-----------------+-----------------+ | | 100 | 31 | | +-----------------+-----------------+-----------------+-----------------+ | | 50 | 28 | | +-----------------+-----------------+-----------------+-----------------+ | | 25 | 22 | | +-----------------+-----------------+-----------------+-----------------+ | | 12.5 | \- | | +-----------------+-----------------+-----------------+-----------------+ Key: CPX = Ciproflaxin (control) - = No zone of inhibition **Table 4: Minimum Inhibitory Concentrations (MIC) and Minimum Bactericidal Concentrations (MBC)** +-----------------------+-----------------------+-----------------------+ | Test | MMC | Co-Np | | | | | | MO | | (M) | +=======================+=======================+=======================+ | *S.aureus* | MIC | 50 | +-----------------------+-----------------------+-----------------------+ | | MBC | 200 | +-----------------------+-----------------------+-----------------------+ | *E.coli* | MIC | 12.5 | +-----------------------+-----------------------+-----------------------+ | | MBC | 50 | +-----------------------+-----------------------+-----------------------+ | *S.typi* | MIC | 25 | +-----------------------+-----------------------+-----------------------+ | | MBC | 100 | +-----------------------+-----------------------+-----------------------+ Key: MMC = Minimum Microbial Concentrations MIC = Minimum Inhibitory Concentrations MBC = Minimum Bactericidal Concentrations **DISCUSSION** The UV-visible absorbance spectra of the cobalt nanoparticles synthesized using mango leave extract showed peak at 306 nm as presented in (Fig. 1) indicates the formation of cobalt oxide nanoparticles (Cuenca *et al*., 2022). This peak corresponds to the band gap energy of cobalt oxide nanoparticles, which is influenced by the size and shape of the nanoparticles (Moumen *et al*., 2019; Cuenca *et al*., 2022). FTIR spectra of Co NPs synthesized from mango leaf extract were shown in Fig 2. Several functional groups were identified; peak at 783.9 cm^-1^ can be attributed to the Co-O stretching vibrations of cobalt oxide nanoparticles (Hafeez *et al*., 2020; Rajeswar *et al*., 2023). The peak at 1599 cm^-1^ corresponds to the C=C stretching vibration of the aromatic rings present in the mango leaf extract (Rajeswar *et al*., 2023). Additionally, the peak at 2322.1 cm^-1^ indicates the C≡N stretching vibration of nitriles or cyanides in the mango leaf extract (Singh *et al*., 2023). The peak at 3183.1 cm^-1^ is assigned to the O-H stretching vibration of hydroxyl groups in the mango leaf extract (Hafeez *et al*., 2020). Moreover, the peak at 3395.6 cm^-1^ is associated with the N-H stretching vibration of amides or amines in the mango leaf extract (Singh *et al*., 2023). Finally, the peak at 3526.1 cm^-1^ is linked to the O-H stretching vibration of carboxylic acids or esters in the mango leaf extract (Farhadi *et al.,* 2016). Based on the FTIR spectrum, it can be inferred that the cobalt nanoparticles are coated with the mango leaf extract, which may serve as a stabilizing or reducing agent during the synthesis process. Furthermore, the mango leaf extract might impart biological properties to the cobalt nanoparticles, including antioxidant, anti-inflammatory, and antibacterial activities (Naseri *et al*., 2010; Mugundan *et al*., 2015). The SEM images on Plate 4.1a and 4.1b shows a scanning electron microscope (SEM) view of cobalt nanoparticles synthesized from mango leaves at a magnification of 500 and 2000 respectively. [The images show that the nanoparticles are irregularly shaped and vary in size](https://www.academia.edu/104061295/Biological_Synthesis_of_Cobalt_Nanoparticles_from_Mangifera_indica_Leaf_Extract_and_Application_by_Detection_of_Manganese_II_Ions_Present_in_Industrial_Wastewater). The surface of the nanoparticles appears rough and uneven, with some particles clustered together and some separated. This may indicate different degrees of aggregation and dispersion of the nanoparticles in the solution (Okwunodulu *et al*., 2019). However, there are varying degrees of particle aggregation, with some areas showing densely packed particles while others are sparser. This may affect the magnetic and catalytic properties of the nanoparticles (Samari *et al*., 2018). The EDS result of the elemental composition of the synthesized cobalt nanoparticles using mango leave extract is presented in table 1. The prominent elements are Chlorine, Oxygen, and Cobalt with atomic concentrations of 25.93, 57.09, and 14.55% respectively. The result is similar to the findings of [Shahzadi *et a*l., (2019) who synthesized cobalt nanoparticles using *Celosia argentea* plant extract and reported the presence of chlorine, oxygen, and cobalt with atomic concentrations of 28.14, 54.32, and 17.54%, respectively](https://link.springer.com/article/10.1007/s13369-019-03937-0). It is also consistent with the results of [Ali *et al*.](https://portlandpress.com/bioscirep/article/43/7/BSR20230151/233156/Biosynthesis-and-characterization-of-cobalt)[,(2023) who biosynthesized cobalt nanoparticles using a combination of garlic and onion peels and detected chlorine, oxygen, and cobalt with atomic concentrations of 26.63, 56.21, and 17.16%, respectively](https://link.springer.com/article/10.1007/s13369-019-03937-0). (Govindasamy *et al*., 2022). It is also in contrast with the data of [Ahmed *et al*.](https://pdfs.semanticscholar.org/9ae5/f2092955a2dbc3c751221e20d0c883701bb0.pdf) (2021). Hence, the presence of other elements indicates that the cobalt nanoparticles are not pure, but contain some impurities from the mango leave extracts or the synthesis process (Vodyashkin *et al*., 2022). The presence of chlorine and oxygen may indicate the formation of cobalt chloride and cobalt oxide, which are common phases of cobalt nanoparticles (Osorio-Cantillo *et al*., 2012). Some possible sources of these impurities may include the use of cobalt chloride as the cobalt precursor, which may not be completely reduced to metallic cobalt by the mango leave extract (Abd Elkodous *et al*., 2022). The exposure of the cobalt nanoparticles to air or water, which may cause oxidation or hydrolysis of the cobalt surface (Wang *et al*., 2017). The presence of other elements in the mango leave extract, such as carbon, sulfur, phosphorus, silicon, calcium, manganese, iron, and zinc, which may act as dopants or contaminants in the cobalt nanoparticles (Shi *et al*., 2021). The Impurities may affect the properties of the cobalt nanoparticles, such as their size, shape, crystallinity, magnetic behavior, catalytic activity, and biocompatibility (Shi *et al*., 2021). XRD pattern revealed that the cobalt nanoparticles synthesized from mango leave extract displayed distinct diffraction peaks with 2θ values of 16.45, 22.12, and 34.15 correspond to the (111), (200), and (220) planes of the cubic lattice as presented in Fig 3. The average crystal size of 53.87 nm was calculated from the derby Scherer equation, which relates the width of the XRD peak to the size of the crystallite. The equation is; [*D* = *K* \* *λ*/(*β* \* *cos* *θ*)]{.math.inline}D=βcosθKλ​ where D is the average crystal size, K is a shape factor (usually 0.9), λ λ is the wavelength of the x-ray, 𝓑β is the full width at half maximum of the peak, and thetaθ is the Bragg angle. [The smaller the crystal size, the broader the peak width](https://en.wikipedia.org/wiki/Marialite). The radical scavenging activity of NPs synthesized using mango leave extract was evaluated using DPPH scavenging assay as shown in tables 2. The results exhibited an increase (% DPPH radical scavenging assay) with increase in the concentrations of cobalt NPs made. Thereby, revealed that Co NPs possesses significant antioxidant potential. The biosynthesized nanoparticles (Co-Nps) was investigated for sensitivity test against *Salmonella typhi,, Staphylococcus aureus and, Escherichia coli.* Table 4.8 shows the antibacterial activities (sensitivity tests), which indicated the diameter of the zone of inhibition of the test isolates (microorganisms) at different concentrations (200 mg/ml -- 25 mg/ml) of the biosynthesised nanoparticle. Co-Nps derived from mango leaves against *Salmonella typhi,, Staphylococcus aureus and, Escherichia coli had* zone of inhibition; 40 mm, 34 mm and 40 mm. The results inferred that Cobalt biosynthesised nanoparticle demonstrated antibacterial properties against *Salmonella typhi,, Staphylococcus aureus and, Escherichia coli* which may be used in further therapeutic and biomedical aspect. Based on this study, the most susceptible bacteria is *Salmonella typhi* followed by *Escherichia coli,* then *Staphylococcus aureus.* The zone of inhibition formed by the ciproflaxin were slightly greater with zone of inhibition between 25 -- 40 mm. CLSI considered the diameter of the zone of inhibition for the tested bacterial strains which is said to be susceptible was \> or = 18 mm, intermediate (between 13 mm -- 17 mm) and 12 mm is said to be resistant. The greater the zone of inhibition, the more sensitive the organism is to the nanoparticle. Table 4 show the results of the minimum inhibition concentrations (MIC) and minimum bactericidal concentration (MBC) of the biosynthesized nanoparticles against the test microorganisms were determined using broth dilution. The MIC of the nanoparticle was from 12.5 -- 100 mg/ml. Co-Nps of mango showed the low MIC of 12.5 mg/ ml against *E.* *coli*. **Conclusion and Recommendations** **Conclusion** Co Nps were prepared using green synthesis and was characterized using Uv-vis, FTIR, XRD and SEM for their crystallinity, morphology and functional groups. The evaluation of antioxidant activity using DPPH method indicated that Cobalt Nps of both mango leave extract showed a potential antioxidant capacity. The antibacterial assay demonstrated that the nanoparticle showed a remarkable activity against *Salmonella typhi* and inhibited growth of bacterial strains of *Salmonella typhi E.coli* and *S. aureus* with Co-Nps. Hence, it was inferred that cobalt nanoparticles has potential as multifunctional agents for biomedical use. **5.2 Recommendations** Based on the results of this study, the following recommendations were made: 1. 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