Investigation of structural properties and antibacterial activity of AgO nanoparticle extract from Solanum nigrum/Mentha leaf extracts by green synthesis method

: Solanum nigrum and Mentha leaf extracts were used as reducing and stabilizing reagents in the green synthesis of silver oxide nanoparticles (AgO NPs), and their antibacterial e ﬃ cacy was subsequently evaluated. The structure and morphology of AgO NPs were evaluated using X-ray di ﬀ raction and ﬁ led emission scanning electron microscope. High-resolution transmission electron microscopy images were used to analyze the characteristics of certain particles with clearly discernible atomic structures. The functional group and elemental composition of AgO NPs were investigated using fourier transform infrared spectroscopy and energy dispersive X-ray spectroscopy. Ultraviolet – visible spectroscopy was used to determine the energy band gap ( E g ) of the sample. The dielectric constant of both samples was found to be inversely proportional to frequency, whereas the dielectric loss was found to be directly proportional to temperature but directly proportional to frequency. This suggests that the space charge has an e ﬀ ect on the mechanism of charge transfer as well as polarizability. AC conductivity rises and is inversely proportional to temperature increases. AgO NPs had a size range of around 56 nm and were mostly spherical. The antibacterial potential of the synthesized AgO NPs using both extracts was compared by the well-di ﬀ usion method. AgO NPs at 50 – 100 µg·mL − 1 concentration signi ﬁ - cantly inhibited the bacterial growth of Bacillus cereus


Introduction
Herbal extract synthesis of metallic nanoparticles (NPs) generated considerable interest in the dynasty of nanotechnology due to its cost-effective method of producing NPs in lofty quality and quantity with minimal time use and a decrease in toxicity levelsin the case of drug deliverycompared to chemical synthesis, which uses harmful chemicals with high consumption and sensitive to the environment [1][2][3].The field of nanotechnology has made amazing advances; however, researchers are turning to green metallic NPs to meet the need for ecologically friendly NP synthesis [4,5].Metallic NPs from the green synthesis clan can act as a capping and reducing agent [6,7].A considerable amount of studies have systematically investigated metal nanostructures because of their unique catalytic, electrical, and optical capabilities [8].In the province of metallic NPs, silver NPs fascinated the thoughts of researchers because of their idiosyncratic complexion in sensing, optoelectronic, catalysis, and drug delivery [9,10], and because of this, it is deemed that the production of silver NPs is of 500 tons every year and it may upswing in upcoming years [11].Additionally, its significant contributions to the fields of highsensitivity biomolecular detection, catalysis, medicine, and biosensors have suggested that it has inhibitory and bactericidal effects in combination with anti-angiogenesis, antiinflammatory, and anti-fungal activities [12,13].Significantly, silver oxide NPs (AgO NPs) have unique physical and chemical properties that are claimed to have great potential in medical applications.It can be non-toxic and environmentally friendly and reduce their size.NPs made using green synthesis are easier, more affordable, and more easily repeatable than those made using conventional techniques [14].The heterogeneous fashion of green synthesis includes microorganisms that give the formation of NPs but the pace of synthesis is sluggish collated to routes involving an herbal course of action and relatively reincarnated and habitually aggregates in more anchored materials [15].A large number of diseasecausing water and foodborne pathogenic bacteria are actively engaged in the antibacterial action of the silver NPs [16].Solanum nigrum and Mentha are significant homeopathic plants.Solanum nigrum and Mentha contain leaves that have rich amounts of calcium and minerals.The alkaloids and carbohydrates of the plant have a potent immune-stimulating impact.This was used to synthesize AgO NPs because it has anti-inflammatory and antibacterial effects [17].This herb is used to treat the common cold and cough and is used in the Siddha medicine system and treatment of liver diseases.This article is focused to study on the structural, optical, morphological, and dielectric characteristics of both AgO NPs synthesized using a simple and green synthesis method.The antimicrobial activity of the present samples is also examined for biological applications.

Chemicals and plant collection
The silver nitrate (AgNO 3 , manufactured by Merck with a high purity of 99%) was used to synthesize AgO NPs using a green manufacturing process.Solanum nigrum (black nightshade) and Mentha (mint) are the well-known herbal plants cultivated in good loam and black soil in Peramandapatti village, Karimangalam Taluk, Dharmapuri district, Tamil Nadu, India.Naturally, the soil of the district is relatively soft and fresh, varying in color from red to deep brown.Solanum nigrum plants were harvested for their tender, greenish-yellow leaves, which were then cleaned under running water, twice or three times, and then with distilled water.Each leaf was meticulously washed using a white Khadhi cloth before being dried for 3 hours at room temperature.

Synthesis of AgO NPs
Normal water, distilled water, and acetone were used to clean all glassware.In a 100 mL beaker, 10 g of Solanum nigrum and Mentha leaves was boiled with 50 mL of distilled water at 80°C for 20 min.To remove the particulate matter and obtain clear solutions, the various leaf extracts were filtered through the Whatman No. 1 filter paper in a 250 mL beaker, and 50 mL of 0.5 M AgNO 3 was placed and stirred for 20 min.Then, at room temperature, 10 mL of plant extracts (Solanum nigrum, Mentha) was added separately to it.For 20 min, the solution was vigorously stirred.When the reaction is initiated, the color of the solution changes from light pale yellow to dark brown.The final AgO NP product was dried in a hot air oven for 2 h at 80°C.Dried samples were characterized to analyze the properties of the prepared nanomaterials [18][19][20].The detailed flow chart of the synthesis method is given in Figure 1b.
As a multivalent element, silver interacts with oxygen to form a variety of phases, including Ag 2 O, AgO, Ag 3 O 4 , and Ag 2 O 3 .These phases include AgO NPs, a fascinating class of metal oxides.Due to the absence of annealing in the synthesis of silver oxide, Ag and AgO are the most observable phases in experiments.It has also been revealed that they decompose at temperatures lower than 250°C [21].

Characterization of AgO NPs
XRD is the most commonly used technique for investigating chemical compounds as well as determining the crystal structure and size of AgO NPs using Cu-K1 radiation with a wavelength of 1.5406 Å. Scanning electron microscopy (SEM) is an electron microscopy technique that provides the speed and high resolution of a direct surface image while also measuring dimensions (size and shape) at microand nanoscales.Filed emission scanning electron microscope (FESEM) (Hitachi 6600, Tokyo) was used to examine the particle aggregation degree and purity.The morphology, size, and crystallinity of the AgO NPs were examined using high-resolution transmission electron microscopy (HRTEM, JEOL-JEM-2100 PLUS, Japan) and a selected area electron diffraction (SAED) pattern.Fourier transform infrared spectroscopy (FTIR) is a reliable, simple, and widely used analytical technique for screening and determining whether or not biological molecules of NPs are involved in the synthesis.FTIR spectral analysis was used to identify the biomolecules that acted as reducing and capping agents during the AgONP synthesis process.UV-visible (UV-vis) absorption spectroscopy (Shimadzu instrument in diffuse reflectance spectra (DRS) mode) is a valuable technique for characterizing the absorbance bands and band gaps of NPs, particularly those of noble metals.Energy dispersive X-ray spectroscopy is a spectroscopic technique used to determine the elemental composition and purity of AgO NPs that have been synthesized.

Antimicrobial activity of AgO NPs
The given samples were tested for antimicrobial activity by the well-diffusion method.Liquid Mueller Hinton agar media and the Petri plates were sterilized by autoclaving at 121°C for about 30 min at 15 lbs pressure.Under aseptic conditions in the laminar airflow chamber, about 20 mL of the agar medium was dispensed into each Petri plate to yield a uniform depth of 4 mm.After solidification of the media, 18 h culture of Gram-positive microorganisms, such as Bacillus cereus (MTCC 430), Staphylococcus aureus (MTCC 3160), and Gram-negative microorganisms, such as Escherichia coli (MTCC 1698) and Klebsiella pneumoniae (MTCC10309), obtained from IMTECH, Chandigarh, were swabbed on the surface of the agar plates.The well was prepared by using a cork borer followed by loading of 50 and 100 µL of each sample to the distinct well with sterile distilled water as negative control and tetracycline (30 mcg•disk −1 ) as positive control.The sample-loaded plates were then incubated at 37°C for 24 h to observe the zone of inhibition.

Crystal structure analysis
The powder XRD of AgO NPs is depicted in Figure 2. The XRD spectra of the synthesized NP revealed that the four main prominent diffraction peaks were observed at 2θ = 38.07,44.24, 64.36, and 77.33 for Mentha and 38.06, 44.25, 64.35, and 77.32 for Solanum nigrum, which correspond to the (111), ( 200), (220), and (311) planes of silver and can be indexed as the cubic phase of Ag, and these results are well matching with JCPDS card No. 04-0783.The presence of strong peaks in the diffraction pattern confirms the high crystalline characteristics of the synthesized materials.There is a minor peak position at an angle of 27.76°(112), 32.17°(202), 46.17°(132), 54.74°(224), and 57.43°(402), 67.38°( 206), and 76.71°(136) for Mentha.The same planes were seen in Solanum nigrum-mediated AgONPs, with a slight change in 2θ values of 27.68°(112), 32.18°(202), 46.16°(132), 54.79°(224), and 57.28°(402), respectively, for Solanum nigrum, which represents the presence of other phases in the synthesized AgO NPs because of the incorporation of leaf extract, which is well matched with the standard JCPDS data of AgO (JCPDS No. 84-1108).According to the low-intensity peaks for AgO, the synthesized Ag/AgO sample primarily contains Ag NPs with very little AgO present [21].To determine the crystallite size of NPs, the Debye-Scherrer formula was applied and the results are shown in Table 1 [22,23].
where λ denotes the wavelength of X-ray in nm (Cu-Kα 1 , λ = 1.5406Å), β is the full-width-at-half-maximum of the highest peak intensity, θ is the Bragg angle in degree, k indicates the shape factor (k = 0.9), and the lattice constant (4.085Å) is calculated by using the following expression [24,25]: where hkl is the Miller index of the planes, d is the distance between the planes, and a is the lattice constant.

FTIR spectroscopy
The connection of the functional groups was examined by matching the FTIR spectra of AgO NPs from Solanum nigrum and Mentha leaf, as shown in Figure 3.The absorption peaks around 3,436 and 1,386 cm −1 correspond to COOH or OH groups.The peaks at the wave numbers 2,919 and 1,606 cm −1 are appropriate to the vibration of the C-H, C-C, and -C]C-stretching of covalent vibration bonds, respectively.The vibration absorption peak at 545 cm −1 is related to the Ag-O.Additionally, weak absorption peaks are observed in the spectra due to Ag ion bond [26,27].peaks that originated from the synthesis route [28].The corresponding weight and atomic percentage were also calculated, and the variation was observed in both samples [29].The results showed that Solanum nigrum has a high weight and atomic percentage.

UV-vis absorbance spectroscopy
The optical property of a metal NP was investigated using UV-vis DRS analysis.Although the peak was missing, the absorbance band edge of AgO NPs was observed around 397.55 nm (Mentha) and 293.50 nm (Solanum nigrum), respectively [30].Interestingly, the Solanum nigrum AgO NPs have very low absorbance when it is compared with Mentha AgO NPs.The expression for the Kubelka-Munk function is as follows: The optical band gap energy can be calculated by using the following equation [31].
where hν is the incident photon energy, C is the proportionality constant, E g is the band gap (eV), and n is the power factor representing allowed indirect and direct transitions for the n values of 0.  Green synthesis and structural properties of AgONPs  5 energy hν to determine the optical band gap E g from the point of interception with the X-axis.Band gap energy (E g ) of AgO NPs is calculated, using the Kubelka-Munk relationship [31], and the obtained band gap energy values are found to be 2.609 and 3.00 eV for Solanum nigrum and Mentha leaf extract, respectively (Figure 5).structures such as a flower and small spherical ball shapes on top of the flower with more voids.However, AgO NPs from Mentha leaf extract show a similar kind of flower structure with minimal voids.The result of minimal voids in the sample gives better antibacterial activity than other species [32,33].The structural morphology and particle size of AgO NPs were examined by transmission electron microscopy (TEM).Figures 7 and 8

Determination of antimicrobial activity by agar well diffusion method (AWDM)
The antibacterial activities of the synthesized AgO NPs were observed against Gram-positive (Staphylococcus aureus, Bacillus cereus) and Gram-negative bacteria (Klebsiella pneumonia, Escherichia coli) by AWDM.The various sizes (in mm) of the zone of inhibition results were compared with two different leaf extracts.Antibacterial activities of the green synthesized AgO NPs solution from Solanum nigrum leaf extract were tested against Mentha leaf, which was studied using bacteria such as Staphylococcus aureus,  The difference in the constrained diameter is assigned to the range of the bacteria cell zone [36].Thus, the basic principle of antibacterial activity in this study is ascribed to the high surface area-to-volume ratio between AgO NPs and bacteria.

Electrical analysis
The dielectric analysis is a useful tool for understanding the grain boundaries, the microstructure of the compound, its transport properties, charge-storage capacity, etc [37].At different temperatures, the dielectric properties, such as capacitance and the loss factor (or dissipation factor) of AgO NPs derived from leaf extract of Solanum nigrum/ Mentha, were measured in the frequency range of 4 Hz to 8 MHz. Figure 11(a and b) illustrates the dielectric permittivity (ε r ) and dielectric loss for both AgO NPs as a function of frequency at various temperatures.The value of ε r decreases fast as frequency increases and approaches a constant limiting point at which ε r essentially becomes frequency dependent, which is attributed to the reduction of domain walls, as can be seen from the frequency-dependent plot of ε r .In this case, the dipoles can simply align in the applied field direction.As a result of the dominance of space charge polarization in both samples, the dielectric constant of all samples has a greater value at low frequencies and practically reaches a constant value after 1 kHz.This distinctive behavior of polar dielectrics may be understood using the Koop and Maxwell Wagner theory, which makes the assumption that the dielectric medium is composed of conducting grains separated by relatively resistive grain boundaries [38,39].The nanocrystal sizes of the generated samples are another factor in the behavior of the samples.Dislocations, defects, and voids create declination in the dielectric constant at the nanoscale, which results in constant values of ε r [40].Figure 11(b) displays the variations in dielectric loss with frequency for both AgO NPs.The change in tan δ reached the constant value up to 10 kHz, and then the loss factor increases with increasing frequency.The synthesis process can have an impact on the behavior of leaf  extract, sample homogeneity, structural stoichiometry, and other factors that might cause changes in dielectric loss [41].Figure 12(a and b) indicates that both AgO NPs exhibit the greatest value of dielectric constant and δ, whereas the AgO NPs of Solanum nigrum leaf extract exhibit high values compared with the AgO NPs of Mentha leaf extract.Their resistance may play a part in the cause [42].Therefore, the energy loss of both AgO NPs cannot be influenced by Ag + ions.Previous studies have reported similar behaviors of ε r and tanδ for these samples.The tan δ curves of both samples showed some additional peaks.Particularly, the AgO NPs of Solanum nigrum leaf extract at 323 K explore the two peaks, which may be due to lack of resistance.In order to distinguish between the effects of intrinsic dielectric polarization and space charge polarization, the ε r and tan δ were plotted as a function of temperature at chosen frequencies of 1 kHz, 100 kHz, and 1 MHz, which are shown in Figure 13(a and b). Figure 13(a) shows that at all specified frequencies, the values of the dielectric constants rise with rising temperature.The dielectric constant changes with temperature at different frequencies as shown by the following.Since the thermal energy at low temperatures is insufficient to liberate the localized electric dipoles, the dielectric constant initially rises a little with increasing temperature.These dipoles make an effort to realign themselves toward the electric field being delivered outside.The direction of the dipoles in the presence of an external electric field determines the dipole polarization [43,44].The arrangement of the dipoles gets more challenging as the temperature rises due to an increase in entropy, which causes the dipole polarization to decrease as the temperature rises.
Figure 14(a and b) makes it very evident that the AgO NPs of Solanum nigrum leaf extract are high when compared to the AgO NPs of Mentha leaf extract and both the dielectric constant and dielectric loss rise.The study made on solids to characterize the resistance of the crystalline sample is AC conductivity, another significant attribute of the sample.The AC conductivity may be measured using a variety of techniques depending on the dielectric data [45,46].In Figure 15, the variation in AC electrical conductivity is shown as a function of temperatures for both AgO NPs and their embedding with various leaf extracts at different frequencies.The conductivity spectrum exhibits a unique conductivity dispersion in the frequency range of 100 Hz, 1 kHz, 10 kHz, 100 kHz, and 1 MHz.This graph shows the relationship between conductivity and frequency.As frequency decreases, there is an increasing charge accumulation at the electrode-sample contact, which causes this to happen.As a result, there are fewer mobile ions present, which reduces the conductivity at low frequencies.The conductivity of materials increases as frequency increases because charge carriers are more mobile at higher frequencies [47].Figure 16 depicts the ln σ ac versus 1/T graph, which follows the Arrhenius relationship.Electrical conductivity was improved by adding extract.The impurities in the material have an impact on the   activation energy decrease for the AgO NPs of Solanum nigrum leaf extract and increase for AgO NPs of Mentha leaf extract, which is illustrated in Figure 17.A little portion of the activation energy produced from the AC conductance calculation is useful for the production of electro-optical devices.

Conclusions
Green synthesis of AgO NPs using biological agents is of ecologically low cost and capable of producing NPs at room temperature.The cubic structure of the synthesis product was confirmed by XRD results with an average particle size of 28.71 nm.From a Touc plot, the optical band gap of the products is found to be 2.609 and 3.00 eV for Solanum nigrum and Mentha leaf extract, respectively.The FTIR spectra show the existence of a functional group, absorbance peaks at 3,436, 2,919, 1,606, 1,386, 1,041, and 545 cm −1 corresponding to -OH, -CH 2 , -COOH, C-C, C-C, C-H, and Ag-O groups, respectively.The SEM and TEM results show that the structural morphology of AgO NPs, which consists of varied shapes such as flower kind of structure with a small spherical on top of the flower surface, gives more bacterial activity.AgO NPs prepared from Solanum nigrum leaf extract and their dielectric dispersion behavior demonstrate that thermal hopping of space charges occurs in all of the samples.In contrast, the AgO NPs of Solanum nigrum leaf extract show higher values when compared to the AgO NPs of Mentha leaf extract.The examination of the dielectric reveals that at high frequencies, both dielectric dispersion components diminish, preventing interface dipoles from swiftly traveling to the alternate zone.A low dielectric dispersion is frequently preferred for a variety of applications, including high-speed integrated packages and satellite communication applications.Furthermore, the conductivity of both samples remains virtually constant up to a certain frequency, before rapidly rising.As a result, it has been demonstrated that the material is a good candidate for optoelectronic applications.In addition, the silver NPs synthesized using Mentha leaf extract exhibited better antibacterial activity than Solanum nigrum.This may be used as a potential candidate for antibacterial microbes.

Figure 1 :
Figure 1: (a) AgO NP synthesis process: a (0.5 M)AgNO 3 solution, (b) and (c) 10 mL Mentha and Solanum nigrum leaf extracts; (d) and (e) show the synthesized AgONPs from Mentha and Solanum nigrum leaf extracts.(b) Schematic diagram of AgO NPs extract from Solanum nigrum/Mentha leaf by the green synthesis method.

Figure 4 Figure 2 :
Figure4shows the EDX spectra of the green synthesized AgO NPs.In addition to Ag and O, images display the Cl

1 Figure 3 :
Figure 3: FTIR spectra of AgO NPs from the extract of Solanum nigrum and Mentha leaf.

Figure 4 :Figure 5 :
Figure 4: EDX images of AgO sample display the presence of Ag, oxygen, and chlorine (less percentage) in AgO NPs measured from EDX spectroscopy.

Figure 6 (
Figure 6(a) and (b) shows the FESEM image of 1 μm magnification for AgO NP synthesis from the extract of Solanum nigrum and Mentha leaves.It seems that AgO NPs from Solanum nigrum leaf extract have different kinds of

Figure 6 :
Figure 6: FESEM image for AgO NPs from the extract of (a) Solanum nigrum and (b) Mentha leaf.
display different magnifications of TEM bright field images, HRTEM, and SAED patterns for AgO NPs.Sample preparation for TEM analysis consists of AgO NPs from leaf extract dispersed in 1 mL of ethanol and sonicated for half an hour to obtain a well-mixed solution.The mixed solution was dropped on a copper grid and then dried at 60°C in an open-air atmosphere.The results revealed the clouds of spherical ball structures in different scale sizes.The AgO NPs indicate a size of 7-56 nm with an average size of 30 nm.Figures 7(c) and (d) and 8(c) and (d) display the interplanar spacing (fringe width) values of 0.20 and 0.220 nm of Solanum nigrum and Mentha AgO NP samples, which clearly indicates the lattice spacing of plane (200) and (111), respectively.The results are well coordinated with XRD results.

Figure 9 :
Figure 9: Antimicrobial activity of AgO NPs synthesized by Solanum nigrum leaf extract.

Figure 10 :
Figure 10: Antimicrobial activity of AgO NPs from Mentha leaf extract.

Figure 15 :
Figure 15: Variation of AC electrical conductivities with temperatures at different frequencies for Solanum nigrum/Mentha of leaf extract AgO NPs.

Figure 17 :
Figure 17: Variation of AC activation energy with log frequency for Solanum nigrum/Mentha of leaf extract AgO NPs.

Table 1 :
XRD raw data and average crystalline size of AgO NPs

Table 2 :
Maximum zone of inhibition by AgO NPs synthesized by Solanum nigrum and Mentha leaf extract