The leaves of B. pinnatum were collected during January 2019 near the Kapil Dhara fall of the Amarkantak region under the Anuppur district of Madhya Pradesh, India. Plant species authentication was carried out by the subject experts, and the voucher specimen (DOB/19/BP/044) was deposited in the herbarium of Botany department, IGNTU, Amarkantak, India.

Silver nitrate (AgNO₃; AR grade), calf thymus DNA sodium salt (CT-DNA), 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) were purchased from Himedia Laboratories Pvt. Ltd., Mumbai, India. Acetocarmine, Hydrogen peroxide (H₂O₂), Methylene blue, and Rhodamine-b were obtained from CDH Laboratories Pvt. Ltd. Mumbai.

The collected fresh leaves were washed thoroughly twice with double distilled water (DDW) to eliminate dust and natural impurities. The B. pinnatum leaf extract (BPLE) was made with 25 g of fresh leaves, which were cut into equal size. The sliced leaves were boiled in 100 mL of DDW for 20 min at 65°C, and after cooling, the leaves extract was filtered twice with whatman No. 1 filter paper and kept at 4°C.

Preliminary phytochemical screening was carried out using the standard protocol followed by Ali et al. (2018) and Chandraker et al. (2020) to identify the phytoconstituents present in BPLE.

Respective volumes (0.5, 1.0, 1.5, and 2.0 mL) of BPLE were added individually to 24.5, 24.0, 23.5, and 23 mL of 1 mM AgNO₃ solutions for a final volume of 25.0 mL in 50 mL Erlenmeyer flasks. Similarly, 23 mL solutions of AgNO₃ were arranged with four concentrations (0.1, 0.25, 0.5, and 1 mM) and mixed with 2.00 mL BPLE. Further, the effect of pH on the synthesis of BP-AgNPs was analyzed. For this study, 2 mL of BPLE was separately added to 23 mL AgNO₃ (1 mM) having variable pH viz., 2, 4, 6, and 8. The pH was adjusted using sulfuric acid, and sodium hydroxide solutions drop by drop. To evaluate the effect of temperature on the green synthesis of BP-AgNPs, the reaction mixture was kept at variable temperatures (27, 40, 60, and 80°C) in a hot air oven. The color shift from colorless solution to yellowish/dark brown solution suggesting BP-AgNPs synthesis and confirmed the reduction from Ag⁺ to Ag⁰.

The synthesis of BP-AgNPs were determined by UV-visible spectroscopy (Shimadzu UV-1800). Fourier Transform Infra-Red spectra obtained with FTIR (Bruker, Germany. Model: Vertex 70) using KBr at a resolution of 0.5 cm⁻¹ in the diffuse reflectance mode and within a range of 500–4,000 cm⁻¹. Studies of X-Ray Diffraction (XRD) were performed using Bruker D8 at 30 kV and 20 mA current with Cu K (I = 1.54 A) to test the crystallinity of BP-AgNPs. Using Scanning electron microscopy (EVO 18; Carl Zeiss, Germany), the surface morphology and elemental composition (Energy Dispersive X-ray Analysis) of BP-AgNPs were determined. Transmission electron microscopy (Technai G20 FEI) operated at 200 kV, and a 104 beam current was used to determine the size of NPs.

Antimitotic, antioxidant and MTT assays were performed in triplicate and their data expressed in mean ± SE following OriginPro 8.5 software. For MTT assay, data were analyzed by Student's t-test, where P < 0.05 was considered statistically significant.

The occurrence of flavonoids, terpenoids, saponins, alkaloids, and tannins in BPLE was confirmed by various qualitative tests (Table 1). Such phytochemicals might be adsorbed on the surface of Ag+ causing their reduction to Ag⁰, and further, prevent their agglomeration. The previous studies also reported the presence of the same compounds in B. pinnatum (Uchegbu et al., 2017). Among several metabolites, flavonoids were explored to share a significant role in the ethnomedicinal importance of B. pinnatum (Chibli et al., 2014; Fernandes et al., 2019). Besides, pure flavonoids are said to be mainly compound for the transformation of metallic ions to their nanostructures with enhanced anti-microbial and anti-cancer activities (Jain and Mehata, 2017). Similarly, alkaloids, tannins, and saponins have also been reported as green reducing agents to develop novel nanomaterials with versatile applications (Ahmad, 2014; Almadiy and Nenanaah, 2018; Choi et al., 2018). The schematic of BP-AgNPs synthesis is shown in Figure 1.

UV-visible spectroscopy is a relible and basic technique for characterizing nanoparticles. Metallic NPs exhibit absorption bands in the visible region because of surface plasmon resonance (SPR) band (Lopes et al., 2018). Therefore, the synthesis of BP-AgNPs was confirmed by spectroscopic analysis through their distinctive SPR peak at 412 nm, as the band is typical of the nanoscale silver. Whereas, no absorption spectra was detected with an aqueous solution of BPLE and suspension of AgNO₃ salt, separately (Figure 2). Green synthesis of BP-AgNP tends to be highly selective and relies on some essential factors, i.e., extract concentration, pH, temperature, and AgNO₃ concentration. Because of their distinctive properties, these parameters regulate the size, shape, yield, stability, and agglomeration of the BP-AgNPs.

A concentration-dependent effect of leaf extract was found in BP-AgNPs synthesis (Figure 3A). The green synthesis of BP-AgNPs is very low at 0.5 mL concentration of BPLE. This is because of the lesser availability of phytochemicals like phenols, triterpenes, glycosides, alkaloids, flavonoids, steroids, lipids, and organic acids, which act as capping, reducing, and stabilizing agent in phytosyntesis process (Nabikhan et al., 2010). The optimal level of leaf extract was found to be 2.0 mL for green synthesis nanoparticles.

pH plays a major role in green synthesis of BP-AgNPs. It was confirmed that the size and shape of the NPs could be regulated by adjusting the pH of the solution media (Patra and Baek, 2014). In Figure 3B, the characteristic SPR peak of Ag⁰ was not observed at acidic pH. However, a slightly basic medium (pH 8) supports the formation of BP-AgNPs. The obtained result is similar to earlier observations of Gurunathan (2019) and Vanaja et al. (2013) where acidic pH either suppresses NPs formation or gives low absorbance band, whereas, negative ions (OH⁻) after dissociation of NaOH in basic medium causes much reduction of Ag⁺ into AgNPs.

By taking variable concentrations of AgNO₃ in the optimization experiment, it was found that on lower concentrations (0.12–0.5 mM), the absorption peak for AgNPs was not observed (Figure 3C). This may be due to the very low availability of Ag⁺ ions in the reaction mixture. At 1.0 mM AgNO₃, significant absorption spectra (412 nm) was found in the UV-vis spectrophotometer. Hence, 1 mM AgNO₃ is the optimal concentration for green synthesis of AgNPs. However, in some other reports, a further increase in concentration up to 5 mM showed more absorbance (hyperchromic shift) at the same wavelength and produced larger NPs (Vanaja et al., 2013).

Temperature is also a relevant parameter for NPs synthesis. The physical and chemical methods require respective temperatures >350 and <350°C, whereas, green synthesis approach follows ambient to 100°C (Patra and Baek, 2014). In the present study, the synthesis of BP-AgNPs increases with increasing the reaction temperature. The UV-visible spectra (Figure 3D) showed that 60°C is the most suitable condition for BP-AgNP synthesis. At room temperatureand 40°C, synthesis of NPs is very low to moderate, whereas, at 80°C, nanoparticle synthesis is high but not favored due to aggregation. A similar result was obtained using olive leaf mediated NPs at different temperatures (Khalil et al., 2014).

The stability of BP-AgNPs was checked regularly following 8 months of green synthesis. The spectral data suggested adequate stability of NPs with a slight increase in the SPR peak, which may be due to proximity effect or agglomeration.

The FT-IR spectrum of BP-AgNPs is shown in Figure 4a, where noticed peaks denote functional groups in various chemical compounds, such as flavonoids, polysaccharides, polyphenols, and triterpenoids. The FTIR bands at 3,000–3,300, 2904.33, 2358.56, 1594.12, 1385.12, 1021.45, and 669.19 cm⁻¹. The peak centered on ~3,300 cm⁻¹ corresponding to O-H stretching, whereas, 2904.33 cm⁻¹ suggests amide C–C stretching. The peak observed at 2358.95 cm⁻¹ may be due to C–H stretching of a methylene group, and the peak at 1594.12 cm⁻¹ corresponds to -C=C- and -C=N stretching. The band at 1385.12 cm⁻¹ corresponds to O-H bend, and 1021.45 cm⁻¹ can be attributed to the stretching vibration of the O–C bend. The final peak observed at 669.17 cm⁻¹ may be due to the N–H stretching of the amide group (Nabikhan et al., 2010).

XRD profile of phytosynthesized BP-AgNP is shown in Figure 4b, confirming the presence of Ag in the sample. In the XRD graph at 2θ = 28, 32, and 46, the Bragg reflections were observed that noticeably shows the existence of (111), (200), and (220) lattice plane, respectively. The peak pattern can be an indexed face-centered cubic (fcc) silver (JCPDS, File No. 893722) (Sudhakar et al., 2015). From the analysis of XRD Pattern it was confirmed that green synthesized particles had a nano-size with crystalline nature. Certain extra unattributed peaks near typical peaks were also recorded, showing the crystallization of the bioorganic stage on the NP's surface.

The topology and morphology of BP-AgNPs were visualized through SEM assessment. The synthesis of consistent and comparatively orbicular BP-AgNPs is confirmed in Figure 4c. Energy-dispersive X-ray analysis (EDX) reveals the composition of elementals in BP-AgNPs. The EDX spectrum, which shows the major elementary peak at 3 keV, indicates metallic silver in Figure 4e. During the capping of AgNP by biomolecules of BPLE, other small peaks of K, Cl, Ca, and O were also created. The quantitative estimate shows that elemental Ag has a maximum weight percentage of 81.77%, while O, Cl, Ca, and K having had 11.43, 5.91, 0.27, and 0.63%, respectively Figure 4f.

Figure 4d displays the TEM image which elucidates the formation of isotropic, nearly spherical NPs. The average particle size was measured 15 ± 5 nm, following the particle size determined from the XRD.

MTT assay was performed to determine the cytotoxic property of BP-AgNPs. It is a colorimetric measurement that analyses the emergence of purple-blue formazan crystals by reduction of yellow color dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) by the mitochondrial enzyme succinate dehydrogenase. Based on the viability of cancer cells (human squamous cell carcinoma A431 and mouse melanoma B16F10), the effect of NPs was analyzed. In vitro results displayed a decrease in viability of both A431 and B16F10 cells with an increase in the concentration of BP-AgNP_S after 24 h and 48 h of treatment. After 24 h of treatment with BP-AgNPS, there was not much decrease in cell viability at the lower concentrations of 10, 25 and 50 μg/ml, but at higher concentration of 100 μg/ml, cell viability was reduced significantly by ~50% in A431 cells (p < 0.01) and 80% in B16F10 cancer cells (p < 0.005) in comparison to untreated control cells. Similar results were found after 48 h of treatment, and there was a concentration-dependent decrease in viability of A431 and B16F10 cancer cells. Cell viability was ~46% in A431 cells (p < 0.01) and 23% in B16F10 cells (p < 0.01) compared to control. BP-AgNPs showed significant cytotoxicity toward skin cancer cells (Figures 5A,B). BP-AgNPs were found to be more effective against B16F10 cell lines with IC₅₀: 59.5 μg/ml, rather than A431 cell line showing IC₅₀: 96.61 μg/ml after 24 h of treatment. In earlier reports, Butea monosperma mediated Ag and AuNPs were found to be a biocompatible vehicle for chemotherapeutic drugs but did not exhibit cytotoxic effects against the B16F10 cell line (Patra et al., 2015). However, phytosynthesized AgNPs using Impatiens balsamina flowers showed less cytotoxicity and increased IC₅₀ (196.5 μg/ml) than our report against B16F10 cell line (Nalavothula et al., 2015). Likewise, several research groups documented cytotoxic activity of metallic-NPs against various cancer cell lines in a dose-dependent manner, however, biocompatibility and non-toxicity against normal cells (Ribeiro et al., 2018). In separate experiments, Balasubramani et al. (2015) and Farah et al. (2016) observed a dose-dependent decrease in viability of MCF-7 breast cancer cells with 217 and 257.8 μg/ml IC₅₀ when treated with Adenium obesum mediated AgNPs and Antigonon letopus mediated AuNPs, respectively. The definite mechanism of NPs' anti-cancer exercise is not yet fully presumed. However, the generation of reactive oxygen species, up-regulation of p53 protein, and expression of caspases are considered to be the major anti-cancer mechanisms (Barabadi et al., 2017). According to Rai et al. (2016) nanoparticle generated ROS promotes caspase-3 activation that is responsible for cell apoptosis by arresting the G2/M phase of the cancerous cell cycle. Additionally, increased oxidative stress causes oxidation of antioxidant glutathione to glutathione disulfide. Hence, ROS damages not prevented in the cells (Ovais et al., 2017). Besides, AgNPs have shown to downregulate the action of DNA-dependent protein kinase, a damage repair enzyme. Jeyaraj et al. (2013) reported that AgNPs mediated Bcl-2 and Bax gene regulation, which further activates the cascade and controls the caspases 3, 8, and 9 are responsible for the apoptosis of HeLa cell line.

Allium test is suggested by UNEP, IPCS (International Programme on Chemical Safety), IPPB (International Programme on Plant Bioassay), and WHO as a typical test in cytogenetic monitoring of environmental hazards (Maity et al., 2020). The experiment is also suggested to evaluate the genotoxic effects of novel nanomaterials and considered as a substitute to animal tests (Klančnik et al., 2011). Although severalphysico-chemical mutagens, metal-complexes, and Cu, CdS-NPs have been reported to induce chromosomal (Kumbhakar et al., 2016; Sharma et al., 2018), the cytotoxic effects of Ag-NPs are poorly reported. Root meristem of A. cepa was used to detect the cytotoxic effect of BP-AgNP_S at three different concentrations (10, 20, and 30 μg/mL). The present investigation showed that BP-AgNPs decrease the mitotic index (MI) (Table 2) and induce significant alterations in the treated roots relative to the control samples (DDW and AgNO3 treated roots). BP-AgNPs might have been prompted cytotoxicity in treated roots of meristematic cells, causing DNA damage or cell death (Lateef et al., 2016). The values of MI for BP-AgNPs were found 45.0 ± 0.28, 41.66 ± 0.44, and 39.96 ± 0.08 after 12 h which significantly decreased to 40.63 ± 0.27, 38.70 ± 0.60, and 34.53 ± 0.26 after 24 h at 10, 20, and 30 μg/ml, respectively. However, in the control set and AgNO₃ solution (30 μg/ml), the MI was found to be 55.6 ± 0.4 and 52.76 ± 1.38, respectively. The experiment demonstrated that the cell division was inversely proportional to the BP-AgNP concentrations. The findings were statistically significant (p < 0.05) in all the treated concentrations as compared to the control. Chromosomal abnormalities were severely observed in treated meristematic cells with C-metaphase, anaphase with lagging chromosome, stickiness telophase, vagrants chromosomes, anaphase with chromosome bridge, anaphase with lagging chromosome, sticky metaphase, etc. (Figure 6). Our observations are supported by earlier findings where NPs decrease MI with the increase in mitotic anomalies in Allium cepa (Nagaonkar et al., 2015; Kumbhakar et al., 2016). Few reports advocate that AgNPs might interfere with the normal cycle of mitotic cells causing decreased gene expression encoding cyclin-dependent kinase 2, slower advancement of cells to S—phase, and blockage of G2—phase, leading to cell death. AgNPs are found to alter cytoplasm viscosity which leads to atypical behavior of the spindle causing chromosomal abnormalities and formation of micronuclei (Daphedar and Taranath, 2018; Fouad and Hafez, 2018). NPs are also responsible for the breakage and reunion of chromosomal material resulting in structural and numerical changes in chromosomes (Chromosomal abbreviations).

The UV-vis spectroscopy is one of the most significant techniques for determining the efficiency of the compounds to interact with DNA. Before the addition of BP-AgNP, the purity and sustainability of CT-DNA were tested at room temperature showing a high absorption peak at 264 nm. The concentration of BP-AgNPs was made constant and CT-DNA was added gradually for the study. Figure 7 displayed a decrease in the absorption of NPs (i.e., hypochromic) with a minor bathochromic or red-shift in spectra (420–424 nm) preferably indicating intercalation of BP-AgNPs with the corresponding DNA (Komal and Kaushik, 2019). The absorption spectra of CT-DNA demonstrated a minor blue shift or hypsochromic effect (264–260 nm) with the isosbestic point showing a strong interaction of NPs with CT-DNA. There are a few reports which have mentioned such kind of interaction between nanostructures and DNA as providing such an interactive feature. Three possible modes for these interactions are electrostatic linking, groove linking, and intercalation (Ribeiro et al., 2018).

Two different types of dyes (thiazine: MB and azo: Rh-B) were selected as model pollutants to study the dye degradation capabilities of BP-AgNPs under solar irradiation. Both the dyes are positively charged (basic). The distinctive absorption maxima for MB and Rh-B were peaked at 663 and 554 nm, respectively (Figures 8A,B). MB and Rh-B are gradually degraded which is expressed by a significant decrease in peak strength with time increase. Until the end of the exposure period, there was no color change in the control set of both the dyes in the absence of BP-AgNPs (Figures 8C,D). The efficacy of BP-AgNPs in dye degradation was determined as follows (Alshehri et al., 2017).

Where C₀ is the initial concentration of the dye solution at t = 0 and Ct is the concentration of the dye solution after a specific exposure to sunlight. In the present work, it is represented in plot that MB and Rh-B were degraded 100% within 110 and 100 min, respectively. However, in similar reports, MB was found to be degraded significantly, after 6 and 72 h from Cordia dichotoma and Morinda tinctoria mediated NPs, respectively (Vanaja et al., 2014; Kumari et al., 2016). Green synthesized NPs from Zanthoxylum armatum were also reported for noteworthy degradation of toxic dyes Safranine-O, Methyl-red, Methyl-orange, and MB after 24 h (Jyoti and Singh, 2016). Thus, the biofabricated BP-AgNPs can serve as a stable and effective green catalyst for the nano-degradation of MB and Rh-B under visible light.

The kinetic study is among the most relevant approaches from which reaction mechanisms are described. In Figures 8E,F, a linear plot (with slope: −0.022 for MB and −0.033 for Rh-B) of ln(C_t/C₀) vs. time for photocatalytic degradation of MB and Rh-B by BP-AgNPs, shows pseudo-first -order kinetics. The rate constant value of MB degradation is calculated 4.9 × 10⁻⁴ sec⁻¹ and for Rh-B 4.5 × 10⁻⁴ sec⁻¹. The regression coefficient (for MB R2 = 0.98 and Rh-B R2 = 0.80) revealed that the degradation rate of MB and Rh-B were following the Langmuir-Hinshelwood kinetic model.

BP-AgNPs was evaluated with potential H₂O₂ sensing capacity in the present report. The comparative spectra at regular interval is shown in Figure 9. The brown color (vial a) constantly diminished and finally appeared colorless (vial b) after 14 min (Figure 9ii). In the control set without H₂O₂, no changes in the color and intensity of the SPR absorbance were observed. Mohan et al. (2014) proposed that the inclusion of AgNPs to H₂O₂ causes the creation of reactive oxygen species, that trigger the degeneration AgNPs. Figure 9(i) indicates a linear plot (slope: 0.021) of C_t/C₀ (1-ln) vs. time for BP-AgNPs mediated H₂O₂ sensing, exhibiting pseudo-first-order kinetics. The sensing rate constant value for H₂O₂ is estimated at 3.8 × 10–3 s⁻¹. The regression coefficient of H₂O₂, R² = 0.88 indicated that the sensing capacity of BP-AgNPs has followed the kinetic model of Langmuir—Hinshelwood.

These results indicate that BP-AgNPs can be used successfully to identify H₂O₂ concentration in several unknown samples or environmental effluents. Similar NPs-based H₂O₂ sensors were also reported using Bacillus subtilis and Calliandra haematocephala (Mohan et al., 2014; Raja et al., 2017).

The following mechanism shows the probable H₂O₂ decomposition reaction process by BP- AgNPs (Ghosh et al., 2019).

DPPH and ABTS assays are relatively quick and sensitive techniques used to analyze the antioxidant activity of different compounds. DPPH is a strong free radical that transforms into a diamagnetic compound (bright yellow) when counter with electron or hydrogen molecules. The magnitude of the color transition of DPPH from violet-blue to bright-yellow depends on the nature and amount of the substance. Figures 10A,B are showing dose-dependent free radical scavenging activity of BP-AgNPs. The concentration of BP-AgNPs ranging from 31.25 to 500 μg/mL showed 60.50–87.57% DPPH scavenging activity with 89.05 μg/mL IC₅₀. However, for the ascorbic acid 75.64–98.94% scavenging activity reported with 41.5 μg/mL IC₅₀ at the same concentrations (Figure 10A). Similarly, ABTS free radical scavenging was observed from 9.16 to 79.32% at 31.25–500 μg/mL BP-AgNPs with an IC₅₀ value of 259.14 μg/mL, whereas, ascorbic acid displayed 38.39–88.09% inhibition with 173.12 μg/mL IC₅₀ (Figure 10B). Similar observations were found with leaf extracts of Prunus japonica and Desmostachya bipinnata showing antioxidant activity in terms of free radical inhibition (Saravanakumar et al., 2017; Guntur et al., 2018). The results firmly advocate the utilization of BP-AgNPs as natural antioxidants to preserve health against oxidative stresses affiliated with degenerative diseases.