Green Synthesis of Co3O4 Nanoparticles using Mappia Foetida Leaf Extract and its Antimicrobial Potential

Introduction

As cobalt oxide nanoparticles (Co₃O₄NPs) are antiferromagnetic p-type semiconductor they have great interest of researchers due to their various applications in different fields such as semiconductors¹, sensors¹, batteries¹, catalysis¹, storage devices¹ and capacitors¹. In Co₃O₄NPs Co³⁺ occupy the octahedral position and Co²⁺ occupy the tetrahedral position at cubic close packed arrangement of oxide ions in regular spinel structure¹.

Various chemical, physical and electrochemical methods have been reported for the synthesis of Co₃O₄NPs, but these methods are not eco-friendly as hazardous chemicals are used hence an alternative approach of green chemistry with minimum toxic chemicals and eco- friendly materials was used¹. Microorganisms^2-4 and plant extract⁵ can be used in green chemistry but use of plant extract is beneficial as use of microorganisms requires biohazards and elaborate process of maintaining the cell culture.

Mappia foetida or Nothapodytes nimmoniana is an Indian indigenous tree commonly known as Amruta, Kalgur or Narkya, belonging to the family Icacinaceae with anticancer, antiviral as well as anti HIV properties⁶. Mappia foetida contains various biomolecules and among these alkaloid Camptothecin (CPT) shows efficiency in animal tumour models but CPT showed cytotoxic nature and hence it is not used clinically but its water soluble derivatives are used in the treatment of cancer⁷ such as Topotecan, Irinotecan etc.

As per literature survey on the green synthesis of Co₃O₄NPs various plant extracts such as leaf extracts (Calotropis gigantea⁸, Aspalathus linearis⁵, Sageretia thea¹, Euphorbia heterophylla L.⁹, Helianthus annuus¹⁰, Moringa Oleifera¹¹), peel extracts (Punica granatum¹²) and fruit extracts (Terminalia chebula¹³ and Manihot esculenta Crantz¹⁴) were used for the synthesis of these particles but the green synthesis of Co₃O₄NPs using Mappia foetida leaf extract have not been reported which encouraged us to use it as a stabilizing agent for Co₃O₄NPs synthesis.

In the present study the green synthesis of Co₃O₄NPs by Mappia foetida leaf extract and its antimicrobial activity was studied and the structural and morphological properties were investigated by ultraviolet–visible (UV – Vis) spectroscopy, X – ray diffraction (XRD), scanning electron microscopy (SEM) and energy dispersive X – ray (EDX) analysis techniques.  

Materials and Method

Sample collection was done from the forest department of Shirala Tehsil (Sangli, Maharashtra, India). Green leaves were shed dried and crushed in mortar and pestel. The powder was stored in a desiccator at room temperature. Cobalt chloride (CoCl₂) was purchased from Merck specialities private limited, Mumbai. The solutions were prepared by using distilled water. All spectroscopic measurements were done at room temperature.

Preparation of nanoparticles

Brown coloured leaf extract of Mappia foetida was prepared by heating dried leaves powder into 100 mL of distilled water for 20 min. at 80⁰C which was filtered through Whatman filter no.1 and stored at 5⁰C. This leaf extract was then added to 0.01M CoCl₂ with constant stirring and heating at 60⁰C, which was further boiled and allowed to cool down before centrifuged, then after washing a black powder was obtained that was scraped out and dried in Muffle Furness for the further study. Chemically synthesized Co₃O₄NPs were prepared as per literature¹⁵.

Characterization of Co₃O₄NPs

UV-Vis double beam spectrophotometer of Equip – tronics UV – Visible spectrophotometer (EQ – 826) was used for the UV-Visible spectral analysis, and the baseline was adjusted by distilled water. EDX analysis was done on the EDX, EM912 model. X-ray diffractometer (Bruker, D2-Phaser) embedded with CuKα radiation at 30 mA current and 40 kV voltage was used for XRD analysis by using 2θ in the range of 0-90⁰. The JSM-6360 JEOL model was used for Scanning Electron Microscopy (SEM) analysis. Carbon coated copper grid was used to prepare sample film in which a small amount of sample was dropped on the grid and mercury lamp was used to dry this film on the grid after removal of extra solution by blotting paper.

Antimicrobial assay

Pure cultures of four human pathogenic bacteria in which Staphylococcus aureus, Bacillus subtilis as gram positive and Escherichia coli, Pseudomonas vulgaris as gram negative bacteria were produced from the Microbiology Department of “New Arts, Commerce and Science College” Ahmednagar, Maharashtra for investigation of the antibacterial activities of Co₃O₄NPs in triplicates by well diffusion method¹⁶.

Result and Discussion

In biological synthesis of Co₃O₄NPs black precipitate obtained after addition of brown coloured Mappia foetida leaf extract acting as capping and reducing agent confirms the formation of nanoparticles. The UV-Vis absorption spectrum of the solution was observed in the range of 200-800 nm where the characteristic absorption peak or surface plasmon resonance (SPR) peak was observed at 425 nm which was due to absorption of metal oxide. The SPR peaks were dependent on the size and shape of the particles and the type of solvent used for particle synthesis¹⁷.

Figure 1: UV – VIS spectra of biologically synthesized Co3O4NPs Click here to View figure

Chemical purity, stoichiometry and elemental phase was determined by Energy Dispersive X-ray¹⁸ (EDX) as indicated in figure 2 in between 0 to 10 keV. Obtained results shows strong signals at 0.8 keV, 7.0 keV and 7.6 keV were for Co and intense signal between 0.0-0.5 keV for O suggesting that Co and O were the major elements and formation of synthesis of cobalt oxide arise from the sample and other unexpected weak signals at 0.3 keV, 1.3 keV, 1.5 keV, 1.8 keV, 2.0 keV, 2.4 keV, 3.8 keV were from bio-compounds present in the leaf extract.

Figure 2: EDX spectra of biologically synthesized Co3O4NPs Click here to View figure

X-ray diffraction (XRD) technique was used to determine the purity and phase of the powdered Co₃O₄NPs. Figure 3 represented the typical diffraction pattern in which the peaks at 2θ were 31.28, 36.76, 59.28 A⁰ corresponds to Co₃O₄ having spinel structure and cubic close packed phase [JCPDS card no.–01-073-1701].  Insignificant peaks observed could be attributed to organic substances¹⁹. A shift in some peaks was due to the presence of impurities owing to the biomass residue²⁰. The presence of broad peaks suggests the synthesized particles to be very small in size in the nano dimensional state and amorphous in nature²¹. The average crystallite size determined by the Scherrer formula, D = 0.9λ / β cos θ using the half-width of the intense peak in the powder pattern. Where D is the crystallite size, λ is X – Ray wavelength which is 1.54 A⁰, β is full width at half maxima (FWHM) and θ is Bragg’s angle. The crystallite size of biologically synthesized Co₃O₄ NPs corresponding to the highest peak observed in XRD pattern was approximately 5 nm.

Figure 3: XRD of biologically synthesized Co3O4NPs. Click here to View figure

The surface morphology of the nanoparticles was determined by analysing the structure by the scanning electron microscopy. SEM images in figure 4 showed spherical shaped agglomerated surface morphology of Co₃O₄ NPs. Biomolecules from leaf extract acts as capping and stabilizing agents which forms coating on the individual nanoparticles and contains hydroxyl group which causes intermolecular hydrogen bonding resulting in agglomeration²². This agglomeration depends upon the nature and compounds present in the extract²³.

Figure 4: SEM images of biologically synthesized Co3O4NPs Click here to View figure

Eco toxic properties of transition metal oxide are due to shape, small size, high chemical reactivity, biological activity and agglomeration tendency which causes threat to the environment and human beings. The well diffusion method was used for antibacterial study against S. aureus, B. subtilis as Gram positive bacteria and E. coli, P. vulgaris as Gram negative bacteria. Here biologically synthesized Co₃O₄NPs showed relatively similar zone of inhibition as chemically synthesized Co₃O₄NPs (except for B. subtilis) and CoCl₂ (except for E. coli) and higher ZOI than that of positive control i.e. streptomycin. Hence antimicrobial activity of the biologically synthesized Co₃O₄NPs and chemically synthesized Co₃O₄NPs was significantly higher than that of streptomycin as antibiotics which indicate the development of resistance against the antibiotics. Our study showed different zone of inhibition for test bacteria indicating difference in sensitivity against Co₃O₄NPs due to difference in membrane stability as they belong to different genera and a thick peptidoglycan layer was present in gram positive bacteria while a rigid lipid and lipoproteins outer membrane is present in gram negative bacteria²⁴.

Figure 5: Antibacterial activity of Co3O4NPs against a), b) S. aureus, c), d) E. coli, e), f) B. subtilis,  g) and h) P. vulgaris. Click here to View figure

Table 1: Antimicrobial activity of Co₃O₄NPs (n =3).

Pathogens	Biological NPs	Chemical NPs	Plant extract	CoCl2	Positive Control
S. aureus	33.5	32.5	0	35.5	19.7
B. subtilis	33.5	29	0	32	12
E. coli	38.3	37.6	0	26	22.5
P. vulgaris	37.3	37.6	0	41.5	12

 

Conclusion

In present work biological synthesis of Co₃O₄NPs using Mappia foetida leaf extract provides an environmentally friendly route for the synthesis of nanoparticles by avoiding use of harmful and toxic chemicals. Spherical and agglomerated nanoparticles with an average size of 5 nm were synthesized.  Biologically synthesized Co₃O₄NPs showed relatively similar antimicrobial activity as chemically synthesized Co₃O₄NPs and higher antimicrobial activity than that of streptomycin as positive control and hence can be used as a strong antimicrobial agent.

Acknowledgment

The author would like to thank Department of Biotechnology and Department of Chemistry, Pune University Affiliated “New Arts, Commerce and Science College”, Ahmednagar who provided insight and expertise that greatly assisted the research and management of AJMVPS for their constant support and providing facilities for research.

Conflict of interest

The authors declare no conflict of interests.

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