Green Synthesis of Co3O4 Nanoparticles using Mappia foetida leaf extract and its Antimicrobial Potential

In this paper the novel green synthesis of cobalt oxide nanoparticles (Co3O4NPs) from cobalt chloride (CoCl2) using Mappia foetida leaf extract was investigated. The characterization of the Co3O4NPs was done by using UV-Vis spectroscopy, EDX, XRD and SEM analysis techniques. Comparative antibacterial study was done against Gram-positive and Gram-negative bacteria by well diffusion method in which results revealed that the biologically synthesized Co3O4NPs showed relatively similar antibacterial potential as chemically synthesized Co3O4NPs and higher antibacterial potential than that of positive control.


INTROdUCTION
As cobalt oxide nanoparticles (Co 3 O 4 NPs) are antiferromagnetic p-type semiconductor they have great interest of researchers due to their various applications in different fields such as semiconductors 1 , sensors 1 , batteries 1 , catalysis 1 , storage devices 1 and capacitors 1 . In Co 3 O 4 NPs Co 3+ occupy the octahedral position and Co 2+ occupy the tetrahedral position at cubic close packed arrangement of oxide ions in regular spinel structure 1 .
Va r i o u s c h e m i c a l , p h y s i c a l a n d electrochemical methods have been reported for the synthesis of Co 3 O 4 NPs, 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 used 1 . Microorganisms 2-4 and plant extract 5 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.

M a p p i a f o e t i d a o r N o t h a p o d y t e s
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 properties 6 . 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 cancer 7 such as Topotecan, Irinotecan etc.
As per literature survey on the green synthesis of Co 3 O 4 NPs various plant extracts such as leaf extracts (Calotropis gigantea 8 , Aspalathus linearis 5 , Sageretia thea 1 , Euphorbia heterophylla L. 9 , Helianthus annuus 10 , Moringa oleifera 11 ), peel extracts (Punica granatum 12 ) and fruit extracts (Terminalia chebula 13 and Manihot esculenta crantz 14 ) were used for the synthesis of these particles but the green synthesis of Co 3 O 4 NPs using Mappia foetida leaf extract have not been reported which encouraged us to use it as a stabilizing agent for Co 3 O 4 NPs synthesis.
In the present study the green synthesis of Co 3 O 4 NPs 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 (CoCl 2 ) 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 minute. at 80 0 C which was filtered through Whatman filter no.1 and stored at 5 0 C. This leaf extract was then added to 0.01M CoCl 2 with constant stirring and heating at 60 0 C, 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 Co 3 O 4 NPs were prepared as per literature 15 .

Characterization of Co 3 O 4 NPs
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 Co 3 O 4 NPs in triplicates by well diffusion method 16 .

RESULT ANd dISCUSSION
In biological synthesis of Co 3 O 4 NPs 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 synthesis 17 . and amorphous in nature 21 . 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 Co 3 O 4 NPs corresponding to the highest peak observed in XRD pattern was approximately 5 nm.

Fig. 1. UV-VIS spectra of biologically synthesized Co 3 O 4 NPs
Chemical purity, stoichiometry and elemental phase was determined by Energy Dispersive X-ray 18 (EDX) as indicated in Fig. 6 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.

Fig. 2. EdX spectra of biologically synthesized Co 3 O 4 NPs
X-ray diffraction (XRD) technique was used to determine the purity and phase of the powdered Co 3 O 4 NPs. Fig. 3 represented the typical diffraction pattern in which the peaks at 2θ were 31.28, 36.76, 59.28 A 0 corresponds to Co 3 O 4 having spinel structure and cubic close packed phase [JCPDS card no.-01-073-1701]. Insignificant peaks observed could be attributed to organic substances 19 . A shift in some peaks was due to the presence of impurities owing to the biomass residue 20 . The presence of broad peaks suggests the synthesized particles to be very small in size in the nano dimensional state

. XRd of biologically synthesized Co 3 O 4 NPs
The surface morphology of the nanoparticles was determined by analysing the structure by the scanning electron microscopy. SEM images in Fig. 7 showed spherical shaped agglomerated surface morphology of Co 3 O 4 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 agglomeration 22 . This agglomeration depends upon the nature and compounds present in the extract 23 .

Fig. 4. SEM images of biologically synthesized Co 3 O 4 NPs
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 Co 3 O 4 NPs showed relatively similar zone of inhibition as chemically synthesized Co 3 O 4 NPs (except for B. subtilis) and CoCl 2 (except for E. coli) and higher ZOI than that of positive control i.e. streptomycin. Hence antimicrobial activity of the biologically synthesized Co 3 O 4 NPs and chemically synthesized Co 3 O 4 NPs 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 Co 3 O 4 NPs 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 bacteria 24 .

CONCLUSION
In present work biological synthesis of Co 3 O 4 NPs 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 5nm were synthesized. Biologically synthesized Co 3 O 4 NPs showed relatively similar antimicrobial activity as chemically synthesized Co 3 O 4 NPs and higher antimicrobial activity than that of streptomycin as positive control and hence can be used as a strong antimicrobial agent.

ACKNOWLEdGEMENT
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.