Plant-Mediated Biological Synthesis of Ag-Ago-Ag2O Nanocomposites Using Leaf Extracts of Solanum Elaeagnifolium for Antioxidant, Anticancer, and DNA Cleavage Activities

The biogenic nanocomposite synthesis using a plant extract is rapid, simple, ecient, cost-effective, and eco-friendly. This study investigated selective pharmacological activities such as anticancer, antioxidant, and DNA cleavage activities of Solanum elaeagnifolium-mediated green synthesizing Ag-AgO-Ag 2 O nanocomposite. To the best of our knowledge, Solanum elaeagnifolium has been the rst time used to synthesize Ag-AgO-Ag 2 O nanocomposites. The synthesized nanocomposites were explored by using UV-Vis diffuse reectance spectroscopy, X-ray diffraction, Fourier transform infrared spectroscopy, scanning electron microscopy, high-resolution transmission electron microscopy, energy-dispersive X-ray spectroscopy, and photoluminescence analyses. Anticancer activity of Ag-AgO-Ag 2 O nanocomposites was tested on lung cancer cell lines (A-549) and showed activity at the IC 50 of 67.09 μg/mL. The maximum ABTS and DPPH scavenging activity were 25.78% and 20.86% at 100 µg/L, respectively. Moreover, Solanum elaeagnifolium-mediated green synthesized Ag-AgO-Ag 2 O nanocomposites also exhibited considerable DNA cleavage activity. These results assured that the synthesized Ag-AgO-Ag 2 O nanocomposites using Solanum elaeagnifolium leaves extract may have potential applications in biomedical engineering.

Noticeably, Solanum elaeagnifolium of the family Solanaceae, is a deep-rooted perennial plant that is found initially native to the Americas. As summarized in Fig. 1, Solanum elaeagnifolium extract contains several bioactive compounds, namely stigmasterol, kaempferol, C-glycoside, quercetin, mangiferin, rutin, chlorogenic acid, coumaroyl glycoside, dicaffeoyl quinic acid [45][46][47]. Also, leaves from Solanum elaeagnifolium have repellent and insecticidal characteristics towards various crop pests and possibly be used as an alternative for synthetic insecticides [48]. To the best of our knowledge, Solanum elaeagnifolium has been examined for its pharmacological effects, but it has never been employed to synthesize Ag-AgO-Ag 2 O nanocomposites.
Herein, this contribution reports on Ag-AgO-Ag 2 O nanocomposites engineered by an entirely green chemistry approach using Solanum elaeagnifolium natural extract as a fuel addition of any chemical additives. To further characterize the material, Ag-AgO-Ag 2 O nanocomposites were explored by various techniques. In addition, selective biomedical applications such as anticancer, antioxidant, and DNA cleavage activities were also investigated.

Collection of Solanum elaeagnifolium leaves and extracts preparation
The Solanum elaeagnifolium leaves were collected and appropriately washed using double distilled water. 5g of leaves were poured into 100 mL of distilled water and boiled for 15 min at 85-90 ºC. The extract obtained was ltered through ordinary lter paper and then, Whatmann No. 1 lter paper. The ltered Solanum elaeagnifolium leaves extract (SELE) was stored at 4°C for Ag-AgO-Ag 2 O NC synthesis.

Biosynthesis of Ag-AgO-Ag 2 O nanocomposites
Eco-benign synthesis of Ag-AgO-Ag 2 O nanocomposites involved adding 1.69 g silver nitrate to 100 mL of SELE, and then the reaction solution was stirred at 1100 rpm at room temperature with a magnetic stirrer. Initial con rmation of Ag-AgO-Ag 2 O nanocomposites synthesis is by a change in color of the reaction mixture from yellow to dark brown. Then, the resulting solution was then centrifuged at room temperature for 10 minutes at 3000 rpm. The black precipitates of material were dried at 200°C in a hot air oven. The obtained black color powder was stored in an airtight vial for further utilization.

Characterization of Ag-AgO-Ag 2 O nanocomposites
Various characterization tools were used to examine the chemical, optical, and physical properties of Ag-AgO-Ag 2 O nanocomposites. The XRD measurement of synthesized Ag-AgO-Ag 2 O nanocomposites was carried out using a diffractometer system (XPERT-PRO, PANalytical). The UVDRS of Ag-AgO-Ag 2 O nanocomposites were recorded using Jasco Spectrophotometer V-770. The functional groups analysis of biosynthesized Ag-AgO-Ag 2 O nanocomposites was using FT-IR-4600typeA. The morphological features and elemental composition of bio-fabricated Ag-AgO-Ag 2 O nanocomposites were studied by SEM equipped with EDX detector (VEGA3 TESCAN). Moreover, size and shape were studied using HRTEM (JEM-2100) operating at an accelerating 60-200 kV voltage. The photoluminescence nature of SELEmediated Ag-AgO-Ag 2 O nanocomposites was examined by using FP-8200 Spectro urimeter.

Anticancer activity of Ag-AgO-Ag 2 O nanocomposites
A549 lung cancer cells were procured from ATCC. Procured stock cells were grown in DMEM/RPMI supplemented with 10% inactivated Fetal Bovine Serum (FBS), penicillin (100 IU/ml), and streptomycin (100 µg/ml) in a humid environment of 5% CO 2 at 37 ºC. The cell was dissociated with cell dissociating solution (0.02 % EDTA, 0.2 % trypsin, and 0.05 % glucose in PBS). The vitality of the cells is tested, and the cells are centrifuged. In addition, 50,000 cells/well were seeded in a 96 well plate and incubated at 37 ºC under 5% CO 2 incubator for 24 hrs. Different concentrations of Ag-AgO-Ag 2 O nanocomposites (10, 20, 40, 80, 160, and 320 µg/mL) was added and incubated at 37 ºC for 48 h [49]. The resulting solutions in the wells were removed after incubation, and 100 µl of MTT (5 mg/10 ml of MTT in PBS) was mixed with every well. The cultured plates were incubated at 37°C for 4 hours under 5% CO 2 environment. The supernatant was discarded, and 100 µl of DMSO was mixed into the plates, which were gently agitated to dissolve the formed formazan [50]. The viability of cell lines was measure at 570 nm by an ELISA reader. Triplicates of experiments were carried out, and Doxorubicin standard drug was used in the study as a positive control. The cell viability percentage was estimated by using the formula, ascorbic acid as a reference antioxidant. The absorbance was measured to the respective blank solutions using spectrophotometry [51,52]. The following formula was used to compute the % inhibition: 2.5.1. DPPH radical scavenging assay Serial dilutions (20,40,60,80, and 100 µg/mL) of Ag-AgO-Ag 2 O nanocomposites were taken, and 50 µl of 0.659 mM 2,20-diphenyl-1-picrylhydrazyl (DPPH) dissolved in methanol was added, making up to one with distilled water. Thereafter, sample tubes were incubated for 20 minutes at 25°C [52]. A Shimadzu UV 1800 spectrophotometer was employed to record the absorbance at 510 nm.

Characterizations
The phase analysis, crystal structure, and composition of the SELE mediated Ag x O sample was analyzed through the XRD technique, and the result is evinced in Fig. 2(a). It may be observed through this gure that three different phases are present in the sample corresponding to Ag (marked by *), AgO (marked by # ), and Ag 2 O (marked by • ). This indicates that the current method of biosynthesis led to the formation of Ag-AgO-Ag 2 O heterostructured nanocomposite. The existence of these phases was identi ed based on ICDD card no. 04-0783 [55], 84-1108 [56], and 42-0874 for metallic Ag, AgO, and Ag 2 O, respectively [57][58][59]. This analysis, therefore, reveals that the three different phases are in the deposited form and not in the doped state since the diffraction peaks of all the phases are visible in the XRD spectrum.
Further, the unassigned peaks belong to AgNO 3 , which was used as the Ag-precursor in this study [60].
This means that the operating temperature was insu cient to eradicate the salt precursor. Nevertheless, based on the intensity of the diffraction peaks, it may be noted that the most dominant phase in this sample is that of AgO. The average crystallite size of the sample was ascertained using the Scherrer equation and found to be 69.4 nm. Based on the XRD result, it is clear that the biosynthesized Ag x O sample is composed of Ag-AgO-Ag 2 O nanocomposites.
It notes that the plant phytochemicals perform two primary functions: (i) bio-reduction of the metal precursor and (ii) control over the particle size and shape. Herein, the functionalization of Ag-AgO-Ag 2 O nanocomposites by these phytochemicals was con rmed from the FTIR studies. Fig. 2(b) represents the FTIR spectrum of the leaf extract of Solanum elaeagnifolium and Ag-AgO-Ag 2 O nanocomposites. On comparing these FTIR spectra, it may be revealed that the bands at 1648.8 cm −1 and 1037.8 cm −1 of plant extract have wholly lost their intensities after functionalizing the nanocomposites. This means that during the biosynthesis procedure, the functional groups associated with the corresponding phytochemicals were mainly involved in the reducing mechanism of the salt precursor (AgNO 3 ) [61].
Contrariwise, the rest of the assigned bands have shifted their positions in the nanocomposites, which may be attributed to their anchoring onto the surface of the nanoparticles. The FTIR bands observed at 1743.9 cm −1 in plant extract and 1762.6 cm −1 in the nanocomposites correspond to the alkaloid functional group [62]. The band at 1643.8 cm −1 may be ascribed to the C=O stretching vibrations of the amide group majorly because of protein molecules present in the leaf extract [63]. The highly intense bands at ~1381 cm −1 may be attributed to the lipid functional group [64]. The band at 1037.8 cm −1 represents polysaccharides because of O-substituted glucose residue [65]. The band at 783.3 cm −1 is due to the C-H out-of-plane bend of phenyl [65]. This band has shifted to 825.1 cm −1 in the nanocomposites.
Thus, based on the relative intensities of the prominent FTIR bands of plant extract and the assynthesized Ag-AgO-Ag 2 O nanocomposites, it may be concluded that protein and glucose metabolites were responsible for functioning as reductants, lipids, and alkaloid functional groups mainly exhibited the capping agent property. This means the latter functional groups have a more vital ability to bind with the Ag ions, and prevent their particles from the undesirable agglomeration phenomenon, thus, con rming the plant phytochemical screening test.
A typical SEM analysis was performed to study the morphological characteristic of the as-synthesized Ag-AgO-Ag 2 O nanocomposites, as shown in Fig. 2(c). In contrast, EDX analysis was employed to detect the elemental composition of this sample, and the results are depicted in Fig. 2(d). The SEM image (Fig. 2(c)) revealed a high density of nanoparticles. Although the particles have mostly agglomerated, it is still possible to clearly distinguish the boundaries between the individual particle grains. From this image, the morphology of the particles was observed to be quasi-spherical in shape. The EDX analysis of Ag-AgO-Ag 2 O nanocomposites (Fig. 2(d)) represents the existence of only Ag and O in the sample with no impurity peaks, indicating the method of biosynthesis employed in this study leads to the formation of impurity-free Ag-AgO-Ag 2 O nanocomposites. The EDX analysis evinced percentage relative elemental composition, such as Ag (17.76%) and O (82.24%), as presented in the inset table of Fig. 2(d).
The microstructural analysis and particle size determination of the as-synthesized sample were performed through HRTEM studies, and the results have been shown in Fig. 3. It may be observed from the TEM images in Fig. 3(a-b) that the particles have formed quasi-spherical microstructure, which is consistent with the morphology obtained through SEM analysis. However, the particles did not exhibit monodispersity, and hence, their average diameter was calculated to be in the range of 15 nm to 40 nm. From Fig. 3(c), which represents the HRTEM image of Ag-AgO-Ag 2 O nanocomposites, the appearance of criss-cross patterns is visible, further con rming the existence of different phases in this sample.
The optical absorbance of the as-synthesized sample was analyzed based on the UV-Vis absorbance data, and the corresponding spectra are presented in Fig. 4(a). This gure depicts the existence of two major absorbance bands at 295 nm and 455 nm. The band at 295 nm is due to the presence of the AgO component in the sample [55]. This means that AgO primarily absorbs in the UV-range. The broad absorbance maximum at 455 nm, as shown in Fig. 4(b), may be attributed to the absorbance contribution from the Ag and Ag 2 O [5,66] components present in the sample. As a result, the surface plasmon resonance (SPR) effect of Ag nanoparticles [55]. Generally, SPR for Ag nanoparticles is observed around 440 nm [65]. In this case, a shift in the SPR band may be attributed to the strong interfacial coupling of the Ag nanoparticles with the silver oxide components. Fig. 4(c) represents the Tauc plot tted using the Tauc equation [67] for obtaining the bandgap energy of the as-synthesized sample, which was calculated to be 3.3 eV. Fig. 4(d) corresponds to the PL spectra of the as-synthesized sample. A single emission band centered at 576 nm was observed. This photoluminescence peak corresponds to the bandgap of the as-synthesized nanocomposites as well as its exciting state transition [68].

Anticancer activity
The anticancer e cacy of SELE mediated Ag-AgO-Ag 2 O nanocomposites on human lung cancer cell line  (Fig. 6). When the concentration of Ag-AgO-Ag 2 O nanocomposites was gradually increased to 360 µg mL −1 on a human lung cancer cell line, the percentage of cell viability was reduced to 18.28 %.

Antioxidant activity
The scavenging ability of the Ag-AgO-Ag 2 O nanocomposites was evaluated using ABTS and DPPH scavenging assays. The radical scavenging potential of Ag-AgO-Ag2O nanocomposites was dependent on the concentration, increasing from 20 to 100 g µg mL −1 as the concentration of Ag-AgO-Ag2O nanocomposites (Fig. 7-ABTS and Fig. 8 Fig. 9. When compared to control DNA, there are changes in the bands of Lanes 2-5, as indicated in Fig. 9

Conclusion
The green chemistry approach was used for the production of Ag-AgO-Ag 2 O nanocomposites, and SELE was chosen as a natural reducing and/or stabilizing agent in our experiment. It was revealed that the SELE could be successfully employed for the facile synthesis of Ag-AgO-Ag 2 O nanocomposites at room temperature. SELE mediated green synthesizing Ag-AgO-Ag 2 O nanocomposites were explored using UVDRS, XRD, FTIR, SEM, HRTEM, EDX and PL analysis. In the HRTEM analysis of Ag-AgO-Ag 2 O nanocomposites, quasi-spherical shaped particles were obtained. The mean diameter of the Ag-AgO-Ag 2 O nanocomposites was found to be 69.4 nm. It was observed that synthesized Ag-AgO-Ag 2 O nanocomposites showed sound anticancer effects against A-549 lung cancer cell lines. However, antioxidant and DNA cleavage results have also been shown to be effective for Ag-AgO-Ag 2 O nanocomposites. The current study has revealed the possibility of executing SELE-mediated Ag-AgO-Ag 2 O nanocomposites, which might be exploited as an antioxidant, DNA cleavage, and anticancer agent.

Declarations
Con ict of interest The authors declare that there are no con icts of interest regarding the publication of this manuscript.   Antioxidant activities of Ag-AgO-Ag2O nanocomposites and ascorbic acid using ABTS assay Antioxidant activities of Ag-AgO-Ag2O nanocomposites and ascorbic acid using DPPH assay