In Situ Green Synthesis of Co3O4@ZnO Core-Shell Nanoparticles Using Datura stramonium Leaf Extract: Antibacterial and Antioxidant Studies

Investigating and synthesizing potent antibacterial NPs using biological methods is highly preferred, and it involves nontoxic, cost-effective, and environmentally friendly chemicals and methods. Antibiotic drug resistance and oxidative stress have become a serious public health issue worldwide. Hence, the key objective of this study was to biologically synthesize and characterize the potent antibacterial Co3O4@ZnO core-shell nanoparticles for the antibacterial application. The radical scavenging ability of green synthesized Co3O4@ZnO core-shell nanoparticles was also determined. In this study, Co3O4@ZnO core-shell nanoparticles (CZCS NPs) have been synthesized using three different core to shell materials ratios of Co3O4 to ZnO (0.5 : 0.25 CZCS (1), 0.5 : 0.5 CZCS (2), and 0.5 : 0.75 M CZCS (3)) by employing Datura stramonium leaf extract. The polycrystalline nature of Co3O4@ZnO core-shell nanoparticles was investigated using the XRD and SAED characterization techniques. The investigated nanostructure of Co3O4@ZnO core-shell nanoparticles appeared with Co3O4 as the core and ZnO as an outer shell. Additionally, a variety of physicochemical properties of the nanoparticles were determined using various characterization techniques. The average crystallite sizes of CZCS (1), CZCS (2), and CZCS (3) were found to be 24 ± 1.4, 22 ± 1.5, and 25 ± 1.5 nm, respectively. The band gap energy values for CZCS (1), CZCS (2), and CZCS (3) determined from the UV-DRS data were found to be 2.75, 2.76, and 2.73 eV, respectively. The high inhibition activities against S. aureus, S. pyogenes, E. coli, and P. aeruginosa bacterial strains were obtained for the small size CZCS (2) nanoparticles at the concentration of 100 mg/mL with 22 ± 0.34, 19 ± 0.32, 18 ± 0.45, and 17 ± 0.32 mm values, respectively. The high inhibition performance of CZCS (2) nanoparticles against Gram-positive and Gram-negative bacteria which is even above the control drug ampicillin is because of its small size and synergistic effect. The percentage scavenging activity of Co3O4@ZnO core-shell nanoparticles was also studied and CZCS (2) nanoparticles showed a good scavenging capacity (86.87%) at 500 μg/mL with IC50 of 209.26 μg/mL.


Introduction
Te current development of nanotechnology has encouraged researchers in the feld to design and investigate novel nanomaterials with properties that signifcantly address the increasing demand for their application towards biological and environmental remediation.Among the recently fabricated nanomaterials, metal nanoparticles (NPs) are highly interesting, due to their unique and modifed physicochemical properties, such as catalytic activity, stability, size, shape, surface energy, reactivity, surface area, sensitivity, chemical composition, porosity, crystallinity, electrical, magnetic, and optical [1][2][3][4].Te particle size and surface area to volume ratio of NPs are the vital properties that determine other characteristics of the NPs.Researchers have reported the use of various methods and approaches to develop metal NPs with a small size and a large surface area to volume ratio [5][6][7][8].For more than half a century, single NPs have been studied widely.However, over the past few decades, hybrid NPs such as core-shell structures have become more attractive from a technological viewpoint associated with the unique properties that enable them to be used in several applications [9][10][11].In core-shell NPs, one or more core materials are coated by the other organic or inorganic shell materials which enhance the properties of the core materials [12][13][14].
Te core-shell nanostructures are preferable due to the synergistic efect of the core and shell materials [15].Te core-shell metal oxide nanostructures are highly functional with enhanced physicochemical properties.Te core-shell nanostructures have unique properties such as thermal stability, biocompatibility, selectivity, dispersibility, and reactivity than those of their single nanoparticle counterparts [16,17].Te biggest advantage of core-shell NPs is enhancing the low potential that is not well possessed by single NPs and minimizing the side efects of their parent materials.As reported in some of the studies, even at high concentrations, core-shell NPs were almost nontoxic, as compared to the corresponding monometallic NPs [18][19][20][21][22].
Among inorganic oxides, Co 3 O 4 is an attractive oxide because of its mechanical strength, availability, and its cost in contrast to other noble materials such as Ag, Au, and Pt [23][24][25].Te nanostructure of Co 3 O 4 has several properties that enable it to be used in diferent felds of study.Te special characteristics of Co 3 O 4 NPs such as catalytic properties, sensors, magnetic behavior, bioavailability, storage, and reactivity attract the intention of numerous researchers.Te high surface area because of its small size empowers it to be used in a variety of applications such as photocatalysts, capacitors, feld emission materials, sensing, drug delivery, magnetic resonance imaging, biomedical, and antimicrobial agents [26][27][28].Co 3 O 4 , a white powder having a band gap of 1.48 eV, is an antiferromagnetic p-type semiconductor.It has a spinel crystal structure and is widely used in diferent applications and is developed as the core nanostructure [29,30].In addition to cobalt oxide NPs, zinc oxide nanostructure has also received the attention of researchers.Due to their unique properties, ZnO NPs are at the crown of the research and widely used as a pigment, ointment, adhesive, food, cosmetics, sunscreen, lubricants, paints, etc. Te special characteristics of ZnO NPs are bioavailability and low toxicity to normal cell, which promote its activity in disease treatment such as cancer, oxidative stress, bacterial infections, and diabetes [28,31,32].
Additionally, ZnO NPs have good antibacterial property and are widely used in drug delivery.Te n-type semiconductor ZnO has a large band gap than Co 3 O 4 NPs with a value of 3.37 eV and is known for its unique properties such as bioavailability, biocompatibility, low toxicity, and high solubility.Due to their novel properties, the hybridized structure called core-shell nanoparticles of Co 3 O 4 and ZnO NPs has been designed to investigate their synergistic efects specifcally on antibacterial strains.Based on their novelty and physicochemical properties, Co 3 O 4 has been designated as the core of the nanoparticles whereas ZnO NPs serves as the shell in this particular study [24,33,34].
In Co 3 O 4 @ZnO core-shell NPs, the core material Co 3 O 4 was coated by ZnO which acts as a shell to control its reactivity and enhance thermal stability.Additionally, the shell decreases the toxicity of Co 3 O 4 NPs and increases the dispersibility [35][36][37].
As mentioned in previous reports, several physical and chemical techniques were used to prepare metal oxide nanoparticles.Te top-down is when particles are broken down from macro to nanoscale and bottom-up builds up the nanoparticles from atoms and molecules.Te bottom-up method is more chosen because of the difculties in achieving a uniform shape, desired size, and perfect surface of nanoparticles [38][39][40].Nowadays, the most widely used techniques in the synthesis of the nanoparticle are chemical methods and biological methods.Te most common chemical methods such as vapor deposition, sol-gel, thermal evaporation, microwave-assisted, pulsed laser deposition, electrochemical reaction, sputtering, hydrothermal, solvothermal, and microemulsion are used in the fabrication of nanoparticles [41][42][43][44].Similarly, Co 3 O 4 @ZnO core-shell NPs have been synthesized by using the chemical and physical methods.Te chemically synthesized Co 3 O 4 @ZnO core-shell NPs appeared to possess a particle size of 12 nm and had a band gap value of 4.97 eV which was larger than their parent materials.Te hydrothermal/sol-gel method synthesized Co 3 O 4 @ZnO core-shell NPs with an average inner core material size of 22 nm, and outer shell with 56 nm particle size from the TEM image was also reported [17,[45][46][47].However, chemically synthesized Co 3 O 4 @ZnO NPs are not environmentally friendly but afect both human and environmental health.Green synthesis methods for the preparation NPs has become a promising alternative to chemical methods because they require low energy, involve low cost, low temperature, and low pressure, are environmentally friendly, and have a one-pot synthesis.Green synthesis route involves the use of diferent parts of plants, bacteria, fungi, and algae.Te biomolecules present in these sources are used to reduce and stabilize the NPs by preventing the agglomeration of grains during the preparation of NPs [48][49][50].In this work, the green synthesis method which is an environmentally friendly, costefective, and nontoxic method in which Datura stramonium 2 Bioinorganic Chemistry and Applications leaf extract has been applied for the synthesis of Co 3 O 4 @ZnO core-shell NPs as illustrated in Figure 1.
Datura stramonium is a fowering weed commonly called Astenager in Amharic or Menji in Afaan Oromo and it belongs to the family Solanaceae.Several phytochemical constituents of Datura stramonium such as alkaloids, favonoids, tannins, saponins, phenol, proteins, glycosides, and steroids have been studied.Phytochemicals such as alkaloids, favonoids, tannins, saponins, terpenoids, and steroids found in Datura stramonium leaf were extracted using petroleum ether, chloroform, and methanol solvents, and the positive screening test results obtained confrmed the presence of the aforementioned phytochemicals [51][52][53].
Tere exists a report of the development of a series of facilely accessible quinoline derivatives that display potent antibacterial activity against a panel of multidrug-resistant Gram-positive bacterial strains, especially C. difcile [32,54,55].However, there is no report regarding the use of Datura stramonium plant extract for the synthesis of NPs.
Nowadays, bacterial infection and oxidative stress have become priorities among the global health threats.Te bacterial infectious diseases are caused by the potential microorganisms that are unfriendly to human beings [56,57].Recent reports have revealed that the bacterial resistance against the antibiotics has become a serious health issue, posing a global risk [58].Te lack of development of new drugs for the bacteria-caused diseases, features of multidrug-resistant bacteria, and misusing antibiotics enable the bacteria to be resistant to the drug [59][60][61].It is estimated that by 2050, antibiotic resistance will have caused approximately 300 million deaths, with an economic loss of $100 trillion, and according to the World Health Organization (WHO) report, antibiotic resistance is one of the major health problems of the century.Tese concerns have initiated a search for innovative strategies in antimicrobial therapies [62].Among the strategies that have been under investigation, the use of antimicrobial peptides, phage therapy, therapeutic antibodies, quorum sensing inhibitors, and antimicrobial NPs could be mentioned [54,63].As alternatives for overcoming the problems due to drugresistant bacteria, several NPs such as CuO, ZnO, NiO, Ag, TiO 2 , and Co 3 O 4 NPs have been widely studied [64,65].Te advantage of using NPs over conventional drugs is the high efcacy in treatment because of their small size, specifcity, and low side efect on the host.In this work, the antibacterial activities of Co 3 O 4 @ZnO core-shell NPs have been studied against two Gram-positive (S. aureus ATCC25923 and S. pyogenes ATCC19615) and Gramnegative (E. coli ATCC25922 and P. aeruginosa ATCC27853) strains [66].
In addition to drug-resistant bacteria, the oxidative stress that is resulting from the disproportion between fabrication and accumulation of reactive oxygen species (ROS) has been noticed also as a critical health issue.In normal cellular respiration under both physiological and pathological conditions, mitochondria produce ROS such as superoxide (O •− 2 ), singlet oxygen ( 1 O 2 ), hydrogen peroxide (H 2 O 2 ), and hydroxyl radicals (•OH).Natural antioxidants such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) found in the human body are known to scavenge ROS molecules to prevent the adverse efects on health [61,66,67].However, the overproduction of ROS molecules induces oxidative stress.Hence, oxidative stress leads to several diseases such as cancer, diabetes, infammation of diferent organs, and acute diseases.To scavenge ROS, a variety of conventional antioxidants and NPs such as Fe 2 O 3 , Ag, Cu, Co 3 O 4 , and ZnO NPs have been widely studied [68][69][70][71].In this work, the antioxidant activities of biologically synthesized Co 3 O 4 @ZnO core-shell NPs were also studied.

Collection and Preparation of Plant Extract.
Te leaves of the Datura stramonium plant used in this study were collected from around Adama Science and Technology University, East Shewa, Ethiopia.Te specimen was identifed and authenticated at the Addis Ababa University Herbarium (Voucher No. AUGH016) and documented for reference purposes along with the medicinal plants.Te leaves were washed three times using distilled water to remove the dust particles from the surface of the leaves.Te contaminant-free Datura stramonium leaves were dried under shadow at room temperature for 15 days and ground using a micro-grinding machine.30 g of Datura stramonium leaf powder was added to a 1000 mL conical fask containing 500 mL distilled water.Using a hotplate, the mixture was heated to 40 °C and stirred for 90 min.Te fltrate was collected using Whatman Number 1 flter paper from the boiled colloidal solution cooled at room temperature.Finally, the fltrate was stored in the refrigerator at 4 °C for the synthesis of the NPs.Te procedure involved is presented in Figure 2.  precipitation of the NPs.Te precipitate was kept in a refrigerator overnight.Te precipitate was centrifuged at 2000 rpm for 20 min and washed three times using ethanol and distilled water.Te washed CZCS (2) was collected on a ceramic crucible and dried in an oven at 100 °C and fnally calcined at 360 °C using a mufe furnace, and the obtained crystalline nanostructure was stored for the purpose of the characterization [44].Te same procedure was repeated for the synthesis of CZCS (1) and CZCS (3).

Antibacterial Activities.
Te antibacterial activities of the synthesized NPs, CZCS (1), CZCS (2), and CZCS (3), were evaluated using the disc difusion method.Te inhibition efciency of the biologically synthesized Co 3 O 4 @ ZnO core-shell NPs was studied for Gram-positive (S. aureus ATCC25923 and S. pyogenes ATCC19615) and Gram-negative (E. coli ATCC25922 and P. aeruginosa ATCC27853) bacterial strains.In the disc difusion method, 12 g of broth agar was prepared in 200 mL of distilled water.Te solution of nutrient agar was dispensed onto the Petri dish.Te poured liquid nutrient agar was solidifed on the Petri dish and well homogenized, and the grown culture of the four bacteria was inoculated and kept on a shaker at 35 °C for 24 h at 200 rpm.Te standard drug ampicillin (positive control) was used in the analysis for determining the antibacterial activities of biologically synthesized Co 3 O 4 @ZnO core-shell NPs.In addition to this, DMSO was used as solvent and negative control.Ten, the biologically synthesized CZCS (1), CZCS (2), and CZCS (3) were applied to the Gram-positive and Gram-negative strains in four different concentrations (25,50,75, and 100 mg/mL).Te plates were incubated at 37 °C for about 24 h and checked for the zone of inhibition.Te scale of the image was determined in millimeters using ImageJ software.
2.5.Antioxidant Activities.Te stable purple 2, 2-diphenyl-1-picrylhydrazyl (DPPH) free radical was utilized to investigate the radical scavenging ability of biologically synthesized CZCS NPs.Te activity was measured following the modifed procedure used in the previous work report [54,72].A 4 mL of 100 μM DPPH was prepared in DMSO and added to methanolic 1000 μL CZCS NPs with 50, 100, 200, 300, and 500 μg/mL.Te mixture was sonicated and kept in the dark chamber for 30 min, followed by incubation at 37 ± 2 °C for the same time.Te UV-vis absorbance of positive control (ascorbic acid) of the same concentration, DPPH, and the incubated sample was measured at 517 nm.All the experiments were performed in triplicate, and the average absorbance for each sample was considered.Finally, the percentage scavenging capacity of NPs was determined using the following equation [73].

Result and Discussion
3.1.Phytochemical Analysis.In this study, ten selected phytochemical constituents of Datura stramonium leaf were screened using the methods mentioned in Table 1.Te phytochemical screening showed a positive result.Among the screened phytochemicals, only steroids show negative tests [52].Te selected phytochemicals are mostly phenolic and carboxylic groups containing molecules which are used to reduce the metal to its nanoscale size.In addition to this, these bioactive molecules are utilized as a capping agent to prevent the agglomeration of nanoparticles during synthesis [75].Te change in physical properties of the CZCS (2) was monitored as a function of controlled temperature change.In this study, 10 mg of CZCS (2) was analyzed within a temperature range of 29-800 °C and the sample started to decompose as the temperature rose.At a temperature of 360 °C and onwards, the core-shell nanostructures became stable.Some molecules such as ethanol, water, and organic bioactive molecules from the plant extract were decomposed before the calcination point of core-shell nanostructures.Te TGA curves of CZCS ( 2) which run at a heating rate of 10 °C min −1 have shown the decomposition change at four diferent steps.Te frst two weight losses, 4.95 % and 6.77 %, recorded were due to the decomposition of ethanol and water, respectively, whereas, the remaining two steps (9.01 and 17.2%) were due to the loss of organic molecules from the plant extract.In general, about 37.93% weight of the sample was lost in the thermal decomposition of CZCS (2) within the temperature range of 30-360 °C.Te diference in steps of weight loss is because of the gelly-like property of CZCS (2) NPs which highly incorporate organic molecules and decomposed at diferent temperatures.Tis corroborated that CZCS (2) was reduced and capped by the phytochemicals of the plant extract [56].As shown in Figure 4, from the TGA/DTA curve, it can be noted that above a temperature of 360 °C, CZCS (2) NPs were found to be thermally stable.101), ( 012), ( 110), (112), and (013) planes, respectively.As shown in Figures 5(a) and 5(b), all the difraction peaks of the Co 3 O 4 @ZnO core-shell NPs within CZCS (1), CZCS (2), and CZCS (3) have ftted the peaks of the corresponding parent particles which is similar to the previous work report [29].

X-Ray
In addition to this, the formation of all CZCS (1), CZCS (2), and CZCS (3) correctly matched the standard database of JCPDS card no.01-079-5606.Te average crystalline size of in situ green synthesized Co 3 O 4 @ZnO core-shell NPs has been calculated using the following formula: where D stands for average crystallite size (nm), k is Scherrer's constant with a 0.154 nm value, λ is the wavelength of the X-ray source which is CuK α with 1.5406 Å value and β is the full-width at half-maximum (FWHM) of the difraction peak appeared at 2θ in radian, and θ is half of the angle between transmission and difraction [52].Te average crystal sizes of CZCS (1), CZCS (2), and CZCS (3) were found to be 24 ± 1.4, 22 ± 1.5, and 25 ± 1.5 nm, respectively.Relatively, the calculated average crystalline size of CZCS (2) was found to be 22 ± 1.5 nm which is smaller than the two ratios.Te diference in the average crystalline size of Co 3 O 4 @ZnO core-shell NPs is due to the variation in ZnO NPs in their concentration.In the case of CZCS (1), the excess amount of phytochemicals present in the leaf extract is believed to compete with each other than reduce the NPs.But, in CZCS (3), the number of phytochemicals present in the extract was believed to be less enough to reduce and cap the NPs which resulted in the agglomeration of NPs.

FT-IR Spectral Analysis.
Te variety of Datura stramonium functional groups of plant leaf powder and that of the synthesized NPs were determined using FTIR.Te FTIR analysis indicated the functional groups of diferent phytochemical constituents that were involved in the reduction during the synthesis of core-shell nanostructures.As shown in Figure 6(b), the functional groups seen in the FTIR spectra of the plant have not appeared in the spectra of Co 3 O 4 @ZnO core-shell NPs.Te absence of functional groups in the spectra of Co 3 O 4 @ZnO core-shell NPs indicated that these functional groups were used in the reduction process.As depicted in Figure 6(a), in the spectra of plant powder, a variety of functional groups absorbed the IR radiation at 3448, 2925, 2852, 2107, 1637, 1390, 1245, 1043, and 556 cm −1 .Similarly, the Datura stramonium leaf Bioinorganic Chemistry and Applications extract-mediated core-shell nanostructures have shown absorption bands at 940, 664, 556, and 399.416 cm −1 .Te broad absorption band at ∼3448 cm −1 is believed to be due to the stretching of O-H of a phenolic group.Te absence of this broad absorption band in the spectra of NPs was due to the calcination of CZCS (2) at a high temperature that degrades and eliminates.Te spectral bands observed at 2925 and 2852 cm −1 were because of C−H stretching vibration of alkane and alkene groups, respectively [76].Te bands that appeared at 2107, 1637, and 1390 cm −1 were due to the presence of C ≡ C, C � C,andC � N functional groups, respectively.Additionally, the absorption bands of C-O vibrations of the ester group and the C-N vibration of amide groups were found at 1245 and 1045 cm −1 , respectively [51,77].
In FTIR analysis of Co 3 O 4 @ZnO core-shell NPs, almost all the functional groups present in the phytochemicals of D. stramonium leaf powder were not seen.Figure 6(b) depicts the absorption band formed as a result of the stretching vibration of Zn-OH.Te stretching modes of Zn-O were also found at 940 and 399.98 cm −1 .Te analysis also showed O-Co-O and Co-O stretching vibration at 664 and 556 cm −1 , respectively.

UV-DRS Analysis.
UV-difused refectance spectroscopy has been used to determine the optical band gap energy of Co 3 O 4 @ZnO core-shell NPs.As depicted in Figures 7(a) and 7(b), the percentage of refectance and optical band gap energies (E g ) of biologically synthesized Co 3 O 4 @ZnO coreshell NPs have been found, respectively.Te band gap energy of CZCS (1), CZCS (2), and CZCS (3) was calculated by converting the generated refectance using the Kubelka-Munk formula [74].
where F (R) is equivalent to absorption values and R is difuse refectance.As shown in Figure 7(b), the direct band gap energy from the Tauc plot for CZCS (1), CZCS (2), and CZCS (3) was deduced to be 2.75, 2.76, and 2.73 eV, respectively.Tese values were obtained from the plot of (F(R)h]) 2 versus 1240/λeV by extrapolating within the linear range of the graph.Te E g value of CZCS ( 2) is greater than CZCS (1) and CZCS (3) due to its small size which matches with XRD results and assured that the smaller the nanosize is, the larger the band gap energy will be.Additionally, the E g values of all the three ratios showed smaller values than their bulk parents [78].

SEM-EDAX Analysis.
Te shape and size of Co 3 O 4 @ ZnO core-shell NPs were analyzed using scanning electron microscopy (SEM).In scanning electron microscopy, a packet of the electron beam interacts with the surface of samples and the shape of CZCS NPs was determined from the scanned surface.Figure 8(a) depicts the rod-like and spherical shape of Co 3 O 4 @ZnO core-shell NPs.Te spherically shaped NPs are highly branched from the same point which seems most likely shell material that surrounded the core NPs.Te overall shape of CZCS NPs shows agglomeration due to the adhesion force developed from the electrostatic interaction between the oxygen of core NPs and hydroxide of shell NPs.From SEM images, the diameters of the particles were generated in nanometers using ImageJ software.As shown in Figure 8(b), the average crystalline size of the particles was found to be 63.40 ± 1.602 nm.
Te elemental composition of Co 3 O 4 @ZnO NPs was also identifed using energy dispersion X-ray spectroscopy within the 0 to 10 keV range.From the EDAX analysis indicated in Figure 8(c), the synthesized NPs were found to contain Co, Zn, O, and C elements.An intense peak of ∼ 500 at 1 KeV was attributed to the elemental composition of shell NPs whereas those making up the core NPs exhibited ∼100 counts at 0.8 KeV.As an impurity, carbon element is believed to be from three diferent sources.Most likely, it is from the atmosphere in the form of carbon dioxide which is absorbed on the surface of NPs, formed during the calcination of synthesized materials in a mufe furnace, and from specimen holders which were used for coating nonconducting materials for analysis.Te atomic percentages of Co, Zn, and O were found to be 8.4, 20.6, and 49.9%, respectively.Te atomic percentage of shell NPs is more than twice the core NPs for the materials synthesized from the equal molarity of precursor salts.Te weight percentage Co, Zn, and O obtained from EDAX was 17.6, 46.5, and 27.6%, respectively.

TEM/HR-TEM and SAED Analysis.
Te cuboid and spherical shapes were obtained for Co 3 O 4 NPs and the ZnO NPs from the TEM analysis, as shown in Figures 9(a) and 9(b), respectively.Te average particle sizes of Co 3 O 4 and ZnO NPs were found to be 15 nm and 17 nm, respectively.Te Co 3 O 4 @ZnO NPs were characterized by using TEM/ HR-TEM technique.In TEM/HR-TEM, the detailed analysis was carried out to investigate the shape, structure, size, and As depicted in Table 2 (lower panel), the particle size of Co 3 O 4 core particle was found to be 10.952 ± 0.27 nm.Te size of the core material was obtained using the normal      Bioinorganic Chemistry and Applications (Gaussian) distribution formula mentioned in (5).Te value of the size of core particle is less than that of the overall size of the core-shell nanostructure.Tis indicated that the size of the shell material coating the inner core was larger than the core.
where y0 is ofset, xc is the center of size distribution, w is the width of the curve, and A is the amplitude of the curve.Te normal Gaussian distribution plot in terms of particle size showed that the value of xc was 10.68 to 11.22 nm which is the size of the Co 3 O 4 core nanoparticle.Te average particle size CZCS NPs found from the TEM image was 21.945 nm which is determined under the nonlinear curve Gaussian ft.Te particle size obtained from the TEM image was matched with the average crystalline size calculated for X-ray difraction peaks (22.35 nm).
Te TEM and HR-TEM analysis explored the grain distribution of synthesized NPs as depicted in Figures 10(a) and 10(b), respectively.Te distance between fringes (dspacing) was determined from HR-TEM using Gatan software.Te d-spacing for Co 3 O 4 @ZnO NPs obtained from IFFT of Gatan software and that determined from X-ray difraction peak using Origin 2023 were almost equal.As indicated in Figures 10(c) and 10(d), the d-spacing for the ZnO (002) and Co 3 O 4 (220) planes was 0.259 and 0.28 nm, respectively.
Te crystalline structure of the Co 3 O 4 @ZnO NPs was revealed using selected area electron difraction (SAED).Figure 11(c) demonstrates the polycrystalline structure of Co 3 O 4 @ZnO NPs from the SAED image.Unlike that of XRD,   Bioinorganic Chemistry and Applications electron beams were difracted from the highly selected area.
In SAED analysis, the atoms in the core-shell nanostructure difracted by the electrons resulted in small bright spots made up of a full ring-like pattern.In XRD analysis, the crystallinity was confrmed by the difraction peaks of X-ray from the atoms found on the planes, whereas SAED used difraction of electrons that showed the regularly ordered spots.Te bright ring of SAED refers to the fringe alignment which is equivalent to the plane in which its atoms difract X-ray.Te circular electron difraction depicted in Figure 11(d) has resulted from many crystalline arrangements.In addition to its crystalline nature, SAED is also used for determining the distance between each ring which corresponds to the plane represented by Miller indexes (hkl).Te SAED pattern of Te average value of the inhibition zone was taken for each concentration applied to both Gram-positive and Gramnegative bacteria.Te antibacterial activities of the Co 3 O 4 @ ZnO NPs were found to have increased with increase in the concentration [4].
Te highest inhibition zones of CZCS (1) samples against the studied bacterial strains were 17 ± 0.2 (S. aureus), 16 ± 0.32 (S. pyogenes), and 15 ± 0.32 (E.coli), and the CZCS (2) sample exhibited highest inhibition zones of 14 ± 0.32 (P.aeruginosa), 22 ± 0.34 (S. aureus), 13 ± 0.32-19 ± 0 .32(S. pyogenes), 10 ± 0.32-18 ± 0 .45(E.coli), and 11 ± 0.22-17 ± 0.32 (P.aeruginosa).Similarly, CZCS (3) sample had inhibition zones of 10 ± 0.43-16 ± 0.38 (S. aureus), 9 ± 0.32-15 ± 0.43 (S.pyogenes), 8 ± 0.32-14 ± 0.31 (E.coli), and 9 ± 0.32-14 ± 0 .43mm (P.aeruginosa).Among all the samples, CZCS (2) was highly efective even at lower concentration in contrast to the others.Te potential activities of CZCS (2) against both Grampositive and Gram-negative were due to its nanoscale which enables it to penetrate the cell wall of bacteria.Te observed diferences in the inhibition zones between the Gram-positive and Gram-negative bacteria were believed to be due to the anatomical structural nature of bacterial strains.Te small-size core-shell NPs showed high inhibition values even at low concentrations against both Gram-negative and Gram-positive bacteria.As reported in the earlier work [73], the green synthesized Co 3 O 4 @ZnO NPs showed more potent antibacterial activities than individual Co 3 O 4 and ZnO NPs of the previous study.Te red algae extract-mediated Co 3 O 4 NPs showed good inhibition activity against S. aureus, E. coli, and P. aeruginosa bacterial strains.Te report also indicated that the inhibition activity of Co 3 O 4 NPs increased with the increase in the concentration.Other reports also showed that the antibacterial activities of Co 3 O 4 and ZnO NPs for E. coli which is drug resistant were almost similar with standard drug [73].However, the novel green synthesized Co 3 O 4 @ZnO NPs in this work inhibit drug-resistant E. coli than standard drug.Te potent synergistic efect of core and shell was also  supported by previous work such that the percentage of dead bacteria at the same concentration by Au@Ag NPs was better than that of their single nanomaterials [6].
In Gram-positive bacteria, the positively charged cobalt and zinc ions from core-shell nanostructures interact with the thick cell wall of bacteria which are negatively charged and cause necrotic damage.In addition to this, the glycerol phosphate and glucosyl phosphate of peptidoglycan which is an anionic polymer trapped by the cobalt and zinc ions of biologically synthesized core-shell nanostructures cleaved the glycoside bond of disaccharide peptide.Te main feature that made the Gram-positive bacteria to be susceptible to Co 3 O 4 @ZnO NPs was the absence of an outer membrane [68].
Unlike Gram-positive bacteria, Gram-negative bacteria are highly resistant to antibacterial drugs.However, the small size of Co 3 O 4 @ZnO core-shell NPs good inhibitory activities against Gram-negative bacteria than standard drugs (ampicillin).For example, the zone of inhibition measured for CZCS (2) and ampicillin against the drug-resistant E. coli was found to be 18 ± 0.45 and 15 ± 0.51 mm, respectively, at 100 mg/mL.However, CZCS (3) and CZCS (1) exhibited inhibition zones of about 14 ± 0.31 and 15 ± 0.32 mm at 100 mg/mL respectively, against E. coli, which is less than the standard drug used as control.It has been observed that, at low concentrations, the small-size CZCS (3) showed good performance against Gram-negative bacteria than the large-size CZCS (1).Te other reason that Gram-negative bacteria cannot resist the toxicity of Co 3 O 4 @ZnO NPs is the less thickened cell wall, the pore on the outer membrane, and the negatively charged lipopolysaccharide molecules as reported [17,79].

Mechanistic Interaction of Co 3 O 4 @ZnO NPs.
As reported elsewhere [71], the potential electrical charge of NPs developed the adhesion force which enables it to stick on the surface of the bacterial cell wall.In a similar sense, cobalt and zinc ions from the solution of Co 3 O 4 @ZnO NPs interact with the cell wall of bacteria which causes shrinkage and results in rupturing that leads to cell death.Additionally, the ions of Co 3 O 4 @ZnO NPs bound to the bacterial cell and congested the electron transport chain.On the other hand, as shown in the report of a mechanistic study of Ag NPs [72], the small-size NPs can cross the cell wall of the bacteria.In similar mechanisms, the cobalt and zinc ions of Co 3 O 4 @ZnO NPs can cross the outer membrane through its pore or interact with the thiol groups and deactivated proteins [32].Te Co 3 O 4 @ZnO NPs entered the membrane, which can damage the DNA, deactivate the enzymes, and generate the reactive oxygen species molecules.Te free radical oxygen reactive molecules such as hydrogen peroxide (H 2 O 2 ), superoxide anion (O .− 2 ), hydroxyl (HO .), peroxyl (RO .−), and alkoxy radicals (RO .) are generated by ions of Co 3 O 4 @ZnO NPs within the bacterial cell [74,80].Tis reactive oxygen produced by Datura stramonium leaf extract-mediated Co 3 O 4 @ZnO NPs caused an oxidative stress which inhibits the growth of both Gram-negative and Gram-positive bacteria as shown in Figure 13.3.9.Antioxidant Study.Te antioxidant activity of Co 3 O 4 @ ZnO core-shell NPS was measured by utilizing the stable free radical DPPH as shown in Table 4. Te addition of methanolic Co 3 O 4 @ZnO NPs to the purple DPPH solution gradually changed to yellow color.In addition to physical visualization, the scavenging capacity of CZCS (1), CZCS (2), and CZCS (3) was determined from the UV-absorbance conducted at 517 nm using (1) mentioned under Section 2.5.As shown in Table 4 and Figure 14(a), the percentage scavenging activities of CZCS (1), CZCS (2), CZCS (3), and ascorbic acid (AA) at 500 μg/mL were found to be 68.63,86.87, 67.16, and 98.24%, respectively.Among the three ratios, CZCS (2) exhibited a high-efciency performance in scavenging the DPPH free radical.Te half-maximal inhibitory concentration (IC50) of CZCS (2) was also less than that of the other two ratios that assured the good capacity of these NPs at low concentrations.Te half-maximal inhibitory concentrations of CZCS (1), CZCS (2), and CZCS (3) were 267.54, 209.26, and 278.2 μg/mL, respectively, which were obtained by using a mathematical linear equation from Y � mx ± c, where m is the slope, x is the concentration of the sample, y is the percentage scavenging   14 Bioinorganic Chemistry and Applications activity, and c is constant.Te linear regression value also showed a consistent increment of percentage scavenging activity with increasing concentration.Te absorbance of free radicals was decreased at 517 nm as the concentration of Co 3 O 4 @ZnO core-shell NPs increased which increased scavenging capacity.As shown in Figure 14(b), the change in color of DPPH is due to the reduction of the nitrogen atom in the molecule by an electron from the oxygen atom on the Co 3 O 4 @ZnO core-shell NPs.Te electron transition is believed to be from n oxygen orbital to antibonding nitrogen orbital (π)(n ⟶ π * ) [19].

Conclusion
In this work, the Co 3 O 4 @ZnO core-shell NPs were synthesized using three diferent concentration ratios of core metal to shell precursor salt CZCS (1), CZCS (2), and CZCS (3) with the help of Datura stramonium leaf extract as reducing and/or capping agent and calcined at 360 °C for 5 h.Te formation of all three biologically synthesized core-shell NPs was confrmed using characterization techniques such as FTIR, XRD, UV-DRS, SEM-EDAX, TEM-HRTEM, and SAED.Te arrangement of Co 3 O 4 NPs and ZnO NPs as a core and shell layer, respectively, was confrmed using XRD, TEM, and SAED techniques.Te average crystallite sizes of Co 3 O 4 @ZnO core-shell NPs, synthesized within the concentration ratios CZCS (1), CZCS (2), and CZCS (3), were found to be 25 ± 1.4, 22 ± 1.5, and 25 ± 1.5 nm, respectively.Among the three Co 3 O 4 @ZnO core-shell NPs, CZCS (2) possess a small size (22.35nm) and large band gap energy (2.76 eV).Te particle size of core particle is 10.952 ± 0.27 nm which is less than half of the average crystallite size of core-shell NPs, and this was matched with data from a previous study in which the size of the core must be less than the size of shell NPs.Te rod-like shape and spherical shape of Co 3 O 4 @ZnO NPs were revealed from SEM and TEM characterization techniques.In this study, the high inhibition performance of Co 3 O 4 @ZnO NPs was evaluated against Gram-positive and Gram-negative bacteria.Te average inhibition zones of nanosize CZCS (2) at 100 mg/mL against S. aureus, S. pyogenes, E. coli, and P. aeruginosa were found to be 22 ± 0.34, 19 ± 0.32, 18 ± 0.45, and 17 ± 0.32 mm, respectively, which are greater than those of standard drug ampicillin.Te high-performance antibacterial activities of core-shell NPs against Gram-positive and Gramnegative pathogens are due to the synergistic efect developed from the Co 3 O 4 core and ZnO shell structures.Similarly, the small-size CZCS (2) showed 86.87% scavenging capacity and IC50 of 209.26 μg/mL.Te synthesized Co 3 O 4 @ZnO NPs have the potentiality to be better antibacterial and antioxidant material.Finally, we conclude that the Co 3 O 4 @ZnO NPs were biologically synthesized and showed good antibacterial activities against drug-resistant human pathogens which are a public health challenge worldwide.

3. 2 .
Termal (TGA/DTA) Analysis.Te thermal stability of CZCS (2) sample is presented as shown in Figure 4. Te TGA-DTA plots showed the degradation pattern of the NPs and were used to determine the calcination temperature at Bioinorganic Chemistry and Applications which synthesized NPs become thermally stable.Te percentage decomposition of several chemical constituents has been determined from the curve of TGA.Te endothermic and exothermic energy changes of the sample were determined by DTA.

Co 3 O 4 @
ZnO NPs revealed the existence of two rings; the frst inner ring with a d-spacing of 0.275 nm and the second with d-spacing value of 0.258 nm.Tese results are in good agreement with the d-spacing values obtained from XRD analysis attributable to the miller indexes 220 and 002.Te plane with miller index 220 belongs to Co 3 O 4 NPs whereas the 002 plane corresponds to ZnO NPs confrming that Co 3 O 4 NPs is located at the core and the ZnO NPs exist on the shell of Co 3 O 4 @ZnO core-shell NPs.Tis investigation indicated good agreement with the core-shell nanostructure formation obtained from TEM image and its particle size distribution shown in Figures 11(a) and 11(b), respectively.3.8.Antibacterial Study.Te inhibition efciency of Co 3 O 4 @ ZnO NPs against Gram-negative (Escherichia coli and Pseudomonas aeruginosa) and Gram-positive (Staphylococcus aureus and Streptococcus pyogenes) bacterial strains has been evaluated using the difusion method.Table 3 and Figure 12 depict the inhibition zones of the Gram-negative (S. aureus and S. pyogenes) and Gram-positive (E. coli and P. aeruginosa) strains by CZCS (1), CZCS (2), and CZCS (3).All three Co 3 O 4 @ZnO core-shell NPs have been applied to the Gramnegative and Gram-positive bacteria within the concentration of 25, 50, 75, and 100 mg/mL.Te zone of inhibition was determined by measuring the diameter of the spot four times.

Figure 11 :
Figure 11: (a) Core-shell image obtained from TEM analysis, (b) histogram graph showing particle size distribution, (c) SAED image, and (d) a number of planes and corresponding Miller index obtained from SAED of Co 3 O 4 @ZnO core-shell NPs.

Figure 13 :
Figure 13: Mechanism of Co 3 O 4 @ZnO NPs action in the bacterial cell.
).In all the NPs, the concentration of Co 3 O 4 core nanoparticle was kept constant throughout the work.In XRD analysis, the electron of both the core and shell nanostructure scattered the X-rays and produced diferent constructive interference patterns with diferent intensities.Te difraction peaks observed in separate Co 3 O 4 NPs and ZnO NPs have appeared in the biosynthesized core-shell NPs which are consistent with other previous reports.Te less intense difraction peaks were observed at 31.86, 56.66, 59.12, 65.14, and 68.06 of 2θ values which were assigned to the crystal planes with miller indices (220), (442), (511), (440), and (522), respectively, which correspond to Co 3 O 4 NPs [73].Te intense peak of ZnO shell NPs was seen at 2θ values of 34.52, 36.34,47.71, 56.68, 62.96, and 68.06 which correspond to (002), (

Table 1 :
Some selected phytochemicals screened from Datura stramonium leaf extract.

Table 2 :
Te particle size distribution of Co 3 O 4 core particle (lower panel) and the d-spacing of CZCS obtained from XRD patterns and HR-TEM images (upper panel).

Table 3 :
Te inhibition zone of Co 3 O 4 @ZnO core-shell NPs against bacterial strain.