Synthesis, characterization, antioxidant and anticancer potential of Kalanchoe pinnata green synthesized silver nanoparticles targeting p53/MDM2 nexus in hepatic cancer: integrated in vitro and in silico study.

Introduction
Recent advancements in nanotechnology have established it as an effective and versatile platform for the nanoscale fabrication of various materials, resulting in the emergence of distinctive physicochemical characteristics, applicable across various domains of environmental, industrial and biomedical sciences [1]. Among these, green- and/or bio- synthesized silver nanoparticles (AgNPs) have recently attracted considerable attention owing to their multifaceted properties, including antioxidant, catalytic, antimicrobial, and biomedical attributes [2, 3]. Recent studies have further elucidated the potential anticancer effects of AgNPs through exploration of interlinked molecular mechanisms such as oxidative stress induction, apoptosis and alteration of cancer-specific signaling pathways [4, 5]. Besides this, integration of AgNPs into specifically engineered biological and therapeutic platforms, illustrates their proficiency in targeted drug delivery and cancer therapy [6], Furthermore, the challenges behind using conventional chemotherapy with detrimental effects, highlights the need for developing nanotechnology- based anticancer therapeutics intended to enhance the therapeutic efficacy and curtail systemic cytotoxicity.
Primary liver cancer or hepatocellular carcinoma (HCC) is one of the leading causes behind cancer associated mortality across the world. High incidence rates of more than 75% were reported from Asian and African continents alone [7]. Reports have outlined that HCC is ranked as third global cause of cancer associated death and is the seventh leading cause cancer related deaths in USA [8]. The most commonly associated risk factor behind development of HCC includes chronic hepatitis B virus (HBV) or hepatitis C virus (HCV) infection followed by excessive alcohol usage, steatotic hepatic disorders often associated with metabolic syndrome, obesity and exposure to aflatoxins [9, 10]. Recent advancements in therapeutic modalities, transplantation, ablation and surgical resection have undoubtedly increased the prognosis of patients. Nevertheless, non-specificity of therapeutic modalities, onset of drug related adverse effects and recurrence of disease still obstruct effective clinical management of HCC [11, 12]. Among other mutations, alteration in TP53 tumor suppressor gene with elevated MDM2 (negative regulator of tumor suppressor p53 gene) expression has been reported in HCC [13, 14]. Indeed, this nexus is pivotal for progression of several cancers including HCC, and thus, a targeted approach towards this holds substantial potential in redefining the clinical outcomes [15].
Since prehistoric times, plants have been a valuable asset for human civilization. Efficient chemotherapeutics used clinically for the management of different ailments have found their origin in plants [16, 17]. Compelling evidence from several preclinical and clinical studies has established various mechanisms, by which the bioactive compounds in plants exert their pharmacological effects [16, 18–22]. Plants also intrinsically possess the ability to reduce certain metals like Au2+, Ag3+ etc. into nanoparticles [23]. Indeed, the synthesis of metallic nanoparticles using ‘green method’ is not only eco-friendly but also augments the safety and therapeutic efficacy of the nanoparticles. Owing to these benefits, various plant extracts have been reported for synthesizing nanoparticles in place of comparatively toxic chemicals as reducing agents.
Kalanchoe pinnata also known as Bryophyllum pinnatum is a member of Kalanchoe genus, representing a succulent ornamental plant cultivated either as a shrub or herb [24]. Morphologically, the leaves of K. pinnata are compound and simple with characteristic red to dark purple crenate margins. K. pinnata has also been documented for its use as traditional medicine for treating various ailments, including cancer [25, 26]. K. pinnata extract from different plant parts has been reported to possess various bioactive compounds including sterols, alkaloids terpenes, flavonoids, nitrates, β-carotenes and tannins among others, which are responsible for the cytotoxic effects [27, 28].
Metallic nanoparticles have been delineated as potent free radical scavengers [29]. Subsequently, green synthesis of silver nanoparticles (AgNPs) is mediated through reduction of Ag+ into Ag0, by various bioactive compounds such as saponins, phenols, alkaloids, and quinines [30]. Intriguingly, AgNPs synthesized using phytochemicals often possess augmented antioxidant potency as compared to the extract individually. Additionally, reports have also shown that green synthesis of AgNPs also elevates the antibacterial potential [31–33]. Moreover, a plethora of scientific evidence has outlined the efficacy of green synthesized AgNPs as plausible therapeutics against various forms of cancer [34–36]. Furthermore, low cytotoxicity, elevated drug delivery, and biocompatibility with novel optical properties make AgNPs a more suitable therapeutic candidate, among several other metallic nanoparticles [37].
To the best of the author’s knowledge, until recently there has been a lacuna in exploring the biogenic synthesis of AgNPs using K. pinnata, despite the benefits of green synthesized AgNPs [6]. Thus, the present research focuses on synthesizing AgNPs using Kp-EtOH, and further assessing its potential biological efficacy. Initially, various bioactive constituents of Kp-EtOH were identified using GC-MS analysis. Subsequently, Kp-EtOH was used for fabrication of silver nitrate (AgNO3) solution to yield Kp-AgNPs, which were further characterized using DLS, zeta-potential and electron microscopy. Subsequently, pharmacological attributes of Kp- AgNPs such as its anti-oxidant and anti-cancer potential against hepatocellular carcinoma HepG2 cells were also investigated.

Materials and methods

Materials
2-Azino-bis (3- ethylbenzthia-zoline-6-sulphonic acid) (ABTS), α, α-Diphenyl-picrylhydrazyl (DPPH), silver nitrate salt along with DAPI stain was obtained from Sigma–Aldrich (St. Louis, MO, USA). Growth media for bacterial (nutrient broth medium, Mueller–Hinton agar medium) and cell culture (fetal bovine serum known as FBS, DMEM-high glucose medium, antibiotic- antimycotic solution) along with ascorbic acid were obtained from HiMedia, Maharashtra, India.

Collection of plant material and its authentication
Entire Kalanchoe pinnata plant was collected from a residential garden area of New Delhi city (Latitude: 28.615018° and Longitude: 77.056823°) India, with due permission of the landowner. Collection of Kalanchoe pinnata plant was in compliance with the relevant national and international guidelines and legislations. The authentication of the plant was undertaken by Dr. Rohini M.R., Scientist, Flower and Medicinal Crop Division, Indian Council of Agricultural Research-Indian Institute of Horticulture Research (ICAR-IIHR), Bengaluru, Karnataka. The specimen was identified and submitted in the institutional herbarium under Voucher No. CP/WS/101. The leaves from K. pinnata were then collected and rinsed with distilled water and left to shade dry at room temperature of 25 °C. The dried leaves were crushed in liquid N2 and then stored at 4 °C in sterile containers.

Methods

Preparation of K. pinnata leaves ethanolic extract
K. pinnata leaves were gently washed in running tap water and were crushed in liquid N2 using a pestle mortar [38]. Resulting powder was then utilised for Soxhlet extraction for 4 h at 60 °C, using 70% analytical grade ethanol. The obtained extract solution was filtered using Whatman filter and was subsequently subjected to vacuum drying. The resulting Kp-EtOH was placed at 4 °C in sterile eppendorf for further investigations.

Characterization of bioactive compounds
To identify the presence of bioactive constituents in Kp-EtOH GC–MS analysis (Shimadzu; QP 2010 Plus, Japan) was carried out, using pre-set calibrations of the instrument as described previously [39]. During the analysis 2 mL of Kp-EtOH (1 mg/mL) was injected through Hamilton syringe into the instrument for ion chromatographic analysis. The retention indices and patterns of several compounds present within Kp-EtOH, were compared with 62,000 compounds available in the library of National Institute Standard and Technology (NIST).

Kp-EtOH mediated synthesis of Kp-AgNPs
Kp-AgNPs were synthesized using silver nitrate (AgNO3) solution as described previously. [40]. Briefly, 0.1 mM AgNO3 (100 mL) was mixed with freshly extracted Kp-EtOH (20 mL) in 5:1 ratio. The reaction was left undisturbed for 48 h, at 500 rpm and 37°C in a shaker incubator. Change in the color of Kp-EtOH-AgNO3 reaction mixture from dark green to yellowish was assessed so as to determine the reduction of Ag+ to Ag0.

Characterization of synthesized AgNPs

Dynamic light scattering, stability and transmission electron microscopy (TEM) analysis of Kp-AgNPs
The average hydrodynamic radii also referred to as Zavg of the synthesized Kp-AgNPs along with its zeta (ζ)-potential was measured using Zeta Sizer (Malvern, UK). Kp-AgNPs were diluted with deionized water in 1:1 ratio, and recorded for Zavg and ζ-potential. During each measurement of Zavg and ζ-potential, a minimum of three individual runs of 10 s each at 25 °C was recorded. TEM was further used to investigate the exact diameter/size and morphology of the synthesized nanoparticles. TEM analysis was carried out using FEI-Titan G2 60-300 KV TEM, FEI, USA. Prior to the analysis, Kp-AgNPs were fixed through air drying on carbon coated grid.

FT-IR analysis
The presence of bioactive secondary metabolites within Kp-EtOH and functional groups on Kp-AgNPs were further determined through ATR-FTIR. During the analysis, 10 mg of Kp-EtOH and Kp-AgNPs each were uniformly dispersed and treated with 100 mg of potassium bromide (KBr). Subsequently, both the extract and the synthesized AgNPs were scanned from 5100 to 600 cm–1 with a resolution of 4 cm–1 using Cary630 FTIR, Agliant Technologies, USA. In order to further identify the distinguishing functional groups in the sample, the spectra was measured by Agilent MicroLab Software®.

Antioxidant effects

DPPH assay
Free radical scavenging efficacy of Kp-AgNPs was quantified through DPPH assay [41]. 1 mL of the stated concentrations of Kp-AgNPs (50, 100, 200, 300 and 400 μg/mL), was mixed with 3 mL of DPPH (100 mM). The reaction was incubated in dark for 30 min after vigorous shaking. Eventually, the optical density of Kp-AgNPs was read at 517 nm through a spectrophotometer (BioRad, USA). Ascorbic acid (Vitamin C) was taken as positive control, whereas the optical density of the solvent was used as blank. The observation was expressed as stated
Here, A0 represented the absorbance of blank, A1 represented the absorbance of DPPH with varying stated Kp-AgNPs concentrations or ascorbic acid (60 μg/mL).

ABTS assay
The antioxidant potential of Kp-AgNPs was also reaffirmed through ABTS assay [42]. Kp-AgNPs at various concentrations were taken in 1 mL aliquot and supplemented with ABTS (3 mL) and left undisturbed in dark for 10 min. The absorbance was recorded at 734 nm. The blank and positive controls were the same as stated above. The results were expressed as radical scavenging percentage as mentioned above.

Anticancer studies

Maintenance of liver cancer cells
Human liver cancer HepG2 and murine alveolar macrophage (J774A.1) cells were commercially procured from NCCS, Pune India. The media used for proliferating HepG2 and J774A.1 cells were Minimum Essential Media (MEM) and Dulbecco’s Modified Eagle Medium (DMEM) respectively. Each medium was supplemented with 10% v/v fetal bovine serum (FBS) and 1% v/v solution of antibiotic-antimycotic mixture. The cells were continuously maintained in CO2 incubator at 37 °C with 5% CO2.

Cell viability assay
The cytotoxic effects of Kp-EtOH and Kp-AgNPs were assessed against HepG2 cells as per the protocol described earlier [43]. 5 × 103 cells were treated with 20, 30 and 40 μg/mL of Kp-EtOH and Kp-AgNPs separately for 24 h. These HepG2 cells were then stained for 30 min using 5 mg/mL of MTT stain (10 μL/well). Next, DMSO (100 μL/well) was supplemented to allow the dissolution of formazan crystals from viable cells. Subsequently, absorbance of each well representing a particular dosed group was recorded using BioRad spectrophotometer (USA). The cytotoxic efficacy of Kp-AgNPs and Kp-EtOH were interpolated as cell viability percentage (%) in comparison with standard drug doxorubicin using the formula

Morphological assessments
1 × 104 HepG2 cells treated for 24 h with 20, 30, and 40 μg/mL Kp-AgNPs were visualized for altered morphological characteristics. Cells exposed to Kp-AgNPs were compared with the positive control (doxorubicin) for attributes such as cell shrinkage, compromised plasma membrane and floating cells using FLoid imaging station, Thermo-Fischer Scientific, USA. Kp-EtOH and Kp-AgNPs were also evaluated for their cytotoxic effects against murine alveolar macrophages (J774A.1) cells. The cells were individually treated with the highest concentration of Kp-EtOH and Kp-AgNPs i.e. 40 μg/mL and incubated for 24 h. The cells were further visualized for any morphological alterations using bright field microscopy as stated above.

Evaluation of nuclear morphology
The presence of condensed and fragmented nuclei within Kp-AgNPs treated HepG2 cells was assessed using DAPI as described earlier [44]. 5 × 103 HepG2 cells were treated with 20, 30 and 40 μg/mL Kp-AgNPs for 24 h. The cells were then fixed using cold methanol (100 μL) followed by exposure to DAPI stain (2 μg/mL) for 30 min at 37 °C. Finally, the images of Kp-AgNPs treated HepG2 cells were captured through blue fluorescence filter.

Oxidative stress
The generation of oxidative stress within HepG2 was evaluated using DCFH-DA stain as described previously [45]. For this assay, 1 × 105 cells of HepG2 were exposed to Kp-AgNPs as stated above for 6 h. For positive control, similar count of HepG2 cells were treated with doxorubicin (1.1 μM) for the same incubation period. The cells were then pelleted, and exposed for 30 min to 10 μM DCFH-DA in dark. Finally, the photomicrographs of Kp-AgNPs treated HepG2 cells were captured and compared with negative control for their DCF-DA mediated green fluorescence.
Quantification of ROS was also done by treating HepG2 cells with Kp-AgNPs at their respective concentrations for 6 h. The cells were then centrifuged and the pellets were exposed to the same DCFH-DA concentration in dark for 30 min. Thereafter, HepG2 cells were recorded for their absorbance in treated and/or untreated group at 485:528 nm using fluorescent spectrophotometer (BioTek, Vermont, USA). The observations were expressed by comparing DCF-DA intensity percentage (%) of Kp-AgNPs treated HepG2 cells with the positive control.

Assessment of apoptotic cell death
Kp-AgNPs exposure mediated induction of apoptosis in HepG2 cells was studied as described earlier using AO/EtBr dual staining [46]. For the assay, 5 × 105 cells in each treatment group were treated with Kp-AgNPs for 24 h. The cells were centrifuged and the pellets were treated with equal volumes of AO and EtBr (100 μL) for 15 min. The photomicrographs of treated and control cells were then evaluated for viable and apoptotic cells by relative fluorescence of stated dyes. Further, both viable and apoptotic HepG2 cells were quantified using ImageJ software (NIH, USA).

In silico studies
Molecular docking serves as a method to ascertain the optimal ligand-protein interaction. Among the various bioactive compounds identified from Kp-EtOH, β-amyrin was chosen for docking analysis. The target proteins MDM2, p53, and caspase-3 were docked with β-amyrin using BIOVIA, Discovery Studio (version 2021), and AutoDock Vina software version 4.1, with subsequent calculations of binding energies. The resulting binding energies, along with the binding interactions of each ligand and the docked data, were meticulously analyzed utilizing Discovery Studio Visualizer.

Retrieval of 3D protein structure
The crystal structure of p53 (PDB: 2OCJ), MDM2 (PDB: 1YCR) and caspase-3 (PDB:IQX3) used in this study was extracted from Brookhaven Protein Data Bank (http://www.rcsb.org/pdb). The structure of p53, MDM2 and caspase-3 used for docking analysis lacked all the heteroatoms such as ions, water, etc.

Retrieval and preparation of β-amyrin (ligand)
The 2D/3D structures of GC-MS-identified bioactive compound (β-amyrin) with anti-HCC potential was retrieved through PubChem (http://pubchem.ncbi.nlm.nih.gov/) and saved in SDF format. Structural details of β-amyrin were further collected through Simplified Molecular Input Line Entry Specification submitted in CORINA software [47]. The coordinates of binding pocket and the cubic box within the grid was set to 40×40×40 [48]. Non-polar hydrogen atoms along with Gasteiger charges were also supplemented whereas the rotational interactions were determined and changed.

Docking
The ligands that were meticulously prepared, along with the corresponding target proteins, a comprehensive analysis was done utilizing AutoDock Vina version 4.1 so as to facilitate the docking process. A variety of conformational states for the ligand were meticulously generated during the docking protocol, followed by an exhaustive energy refinement of the ligand’s pose. The docking score for the most favorable pose within the target proteins for all evaluated bioactive compounds was systematically calculated. The most optimal docked position was selected based on the presence of interacting residues, including hydrogen bonds that exhibited a high binding affinity measured in kcal/mol. The interactions between the protein and ligand within the docked complexes were visualized in two dimensions using LigPlot, while Maestro 12.5 was employed for the generation of all binding pockets.

Statistical estimations
In the present study, measurement of data was represented as the mean ± SEM of at least three individual experiments performed in triplicates. All the statistical estimations were performed using GraphPad Prism (ver. 5.0) software through one-way analysis of variance (ANOVA), and Dunnett’s post-hoc test. *Represents p<0.05, **p<0.01, and ***p<0.001.

Results

Bioactive compounds in Kp-EtOH
During the GC-MS analysis, several bioactive compounds (Fig. 1) were found to be present in Kp-EtOH as listed in Table 1. The GC-MS chromatogram identified 33 phytoconstituents, including aromatic hydrocarbons, diallyl ethers, vitamins, alcohols, carbodiimides, cycloalkanes, carboxylic esters, triterpenes, pentacyclic terpenes, alkanes, lactones and alkylamines among several others. Undoubtedly, these bioactive compounds were found to correlate with various pharmacological activities of K. pinnata leaves. In fact, compounds detected in Kp-EtOH namely Squalene has been reported previously to possess antioxidant, emollient and anticancer properties [49, 50]. Similarly, β-Amyrin present in Kp-EtOH has also been reported to possess significant analgesic, antioxidant and anti-inflammatory effects [51].

Biosynthesis and characterization of Kp-AgNPs
The biosynthesis of Kp-AgNPs was initially evaluated by visible change in the color of Kp-EtOH. As shown in Fig. 2, Kp-EtOH exhibited typical dark green color, which after the stated incubation with AgNO3 salt, changed to yellow. This change in color indicated the synthesis of Kp-AgNPs from Kp-EtOH [52]. Subsequently, Kp-AgNPs were also assessed for their mean hydrodynamic radii (Zavg) through DLS. The results showed that Kp-AgNPs had Zavg of 44.74 ± 1.41 nm. The polydispersity index (PdI) of Kp-AgNPs was found to be 0.474 ± 0.09. Intriguingly, maximum Kp-AgNPs were found to have Zavg of ~ 50 nm as shown in Fig. 3A. To assess the stability of synthesized Kp-AgNPs, zeta (ζ)-potential of these particles was also assessed. As shown in Fig. 3B, the ζ-potential of Kp-AgNPs was found to be –23.91 ± 0.07 mV. However, the ζ-potential of the majority of Kp-AgNPs was found to be ~ –25 mV.
As per the electron microscopy images [Fig. 4A], the synthesized Kp-AgNPs were found to possess a nearly spherical morphology, with an average size of 90.78nm, as shown in the size distribution graph (Fig. 4B). Further evaluation of Kp-EtOH and Kp-AgNPs was carried out using the FTIR spectra (Fig. 5) which explicitly revealed a broad O–H/N–H stretching band in the range of 3400–3200 cm⁻1, which was intensified at the Kp-AgNPs peak, specifying the involvement of flavonoids, proteins and phenols in reducing Ag+ ions. The peaks at ~2974 and ~2933 cm⁻1 represented aliphatic C-H stretching with concomitant shifts within the 1650–1500 cm⁻1 (amide I/II region) range further validating the participation of terpenoids and proteinaceous residues in capping the synthesized Kp-AgNPs. Obvious changes were also seen in the fingerprint region in between 1100–1000 cm⁻1 corresponding to C–O stretching of polysaccharides, which may have contributed to surface stabilization of Kp-AgNPs. Conclusively, the above findings confirmed that carbonyl, hydroxyl, C–O and amide groups present in Kp-EtOH served as natural reducing and capping agents during the biogenic synthesis of Kp-AgNPs [53].

Kp-AgNPs showed significant anti-oxidant potential
DPPH is a widely used organic and stable free radical with characteristic absorption band at 512-528 nm commonly employed for screening the free radical scavenging potential of different compounds [54]. In principle, DPPH assay uses the reducing capabilities of alcoholic DPPH in the presence of a stimulant (characteristically a hydrogen donating agent). During the study, Kp-AgNPs exhibited a dose-dependent free radical scavenging potential. The percentage neutralization of DPPH free radical by synthesized Kp-AgNPs was found to be 18.48 ± 0.94 (50 μg/mL), 34.29 ± 0.99 (100 μg/mL), 59.20 ± 2.61 (200 μg/mL), 73.24 ± 2.93 (300 μg/mL) and 86.37 ± 1.93 (400 μg/mL) with an average EC50 value of ~163 μg/mL.
ABTS radical based anti-oxidative assay has characteristic short reaction time and absorbance at 734 nm. Kp-AgNPs exhibited ABTS radical scavenging potential of 13.56 ± 1.26 (50 μg/mL), 46.72 ± 1.39 (100 μg/mL), 55.39 ± 2.72 (200 μg/mL), 69.56 ± 2.83 (300 μg/mL) and 79.21 ± 1.89 (400 μg/mL) with an average EC50 value of ~138 μg/mL. These observations reaffirmed the strong antioxidant potential of Kp-AgNPs.

In vitro anticancer effects of Kp-AgNPs

Kp-AgNPs reduced the viability of HepG2 cells
Kp-EtOH and Kp-AgNPs induced cytotoxic effects on hepatocellular carcinoma HepG2 cells was quantified through formazan based MTT assay. HepG2 cells were exposed to various specified concentrations of Kp-EtOH and Kp-AgNPs as well as Doxorubicin (positive control) for 24 h. It was observed that the number of viable HepG2 cells reduced significantly to 89.92 ± 2.63, and 78.61 ± 5.78%, post-exposure to 20 μg/mL each of Kp-EtOH and Kp-AgNPs respectively (Fig. 6A). The alleviation in viability of HepG2 cells continued further, and was found to be 77.15 ± 3.43 (30 μg/mL; p<0.001) and 52.81 ± 2.25 (40 μg/mL; p<0.001) post-Kp-EtOH treatment. Intriguingly, the cell viability further lowered to 46.56 ± 3.97% (30 μg/mL; p<0.001) and 26.42 ± 2.69% (40 μg/mL; p<0.001) (Fig.6B). Thus, it was deduced that Kp-AgNPs hold the potential of exerting significant cytotoxic activity against hepatocellular carcinoma HepG2 cells, in comparison to Kp-EtOH. The IC50 of Kp-AgNPs against HepG2 cells was found to be 26.56 ± 1.42 μg/mL, whereas the IC50 of Kp-EtOH against HepG2 cells was found to be 34.85 ± 1.54 (Fig. 6C and D). During the assay, it was further observed that both Kp-EtOH and Kp-AgNPs failed to induce any considerable cytotoxic effects against normal murine alveolar macrophage (J774A.1) cells (Supplementary file 1).

Kp-AgNPs altered appearance of HepG2 cells
The morphology of cancerous cells post-exposure with plausible therapeutics gets altered, and this provides an insight into the functioning of the drug/intervention. Therefore, an investigation into the morphological alterations of HepG2 cells was undertaken, post-exposure to Kp-AgNPs. As presented in Fig. 7, the phase contrast photomicrographs exhibited profound swelling, rupturing and withering of HepG2 plasma membrane post-Kp-AgNPs exposure (indicated by arrows). Contrastingly, no such changes were recorded in negative control HepG2 cells.

Kp-AgNPs alter homeostatic nuclear morphology
Condensation and fragmentation of cellular nuclei are the crucial attributes of apoptosis. In order to analyze the alterations of HepG2 nuclei, after treatment with various concentrations of Kp-AgNPs, DAPI assay was performed. The captured fluorescent micrographs revealed that Kp-AgNPs were able to instigate a dose-dependent condensation, and fragmentation in HepG2 nuclei as against the untreated cells (Fig. 8).

Kp-AgNPs escalated ROS production
DCFH-DA mediated fluorescence was evaluated to estimate the effect of Kp-AgNPs on ROS production within the treated HepG2 cells. The fluorescent photomicrographs as shown in Fig. 9A, showed an increase in the stain-specific green fluorescence in HepG2 cells, in proportionality with the increase in concentration of the Kp-AgNPs. The increased fluorescence is a direct indication of the augmented ROS production in liver cancer HepG2 cells.
Besides this, the quantitative assessment of intracellular ROS was also performed, to reaffirm the qualitative results. In case of HepG2 cells, at varying doses of 20 μg/mL, 30 μg/mL and 40 μg/mL, the level of intracellular ROS was found to be 27.38% ± 4.62%, 56.68% ± 3.13% and 112.88% ± 5.94%, respectively, in comparison to the untreated cells (Fig. 9B). These results clearly suggest that Kp-AgNPs elevate the levels of ROS in a dose-dependent manner.

Kp-AgNPs induced apoptosis
AO/EtBr dual staining was used, to delineate the apoptosis inducing effects of Kp-AgNPs against HepG2 cells. Exposure to Kp-AgNPs substantially induced apoptosis in HepG2 cells. As shown in Fig. 10, the viable cells (VCs) showed bright green fluorescence (AO stained), whereas the HepG2 cells undergoing apoptosis in their early and late stages showed reddish orange fluorescence (EtBr stained) with increasing intensity coinciding with the increase in Kp-AgNPs concentration. By quantifying the early apoptotic (EA) and late apoptotic (LA) cells it was found that 9.92% ± 0.71% of EA and 13.56% ± 1.84% of LA cells were present in the group treated with 20 μg/mL Kp-AgNPs. Similarly, 19.01% ± 3.10% and 30.21% ± 3.01%, EA cells were present in HepG2 cells treated with 30 and 40 μg/mL Kp-AgNPs respectively. At the same concentration, LA HepG2 cells were found to be 21.09% ± 1.89% and 41.50% ± 3.18% respectively (Fig. 11).

Molecular docking analysis
Among the bioactive compounds identified by GC–MS analysis from K. pinnata, β-amyrin was selected and subjected to molecular docking process with caspase-3, p53 and MDM2 proteins (Fig. 12a, b and c). Doxorubicin was used as a positive control in the anticancer studies. The 2D structure of β-amyrin was first retrieved from the PubChem database.
The structures of ligands β-amyrin, doxorubicin and Z-DEVD-FMK and structures of p53, MDM2 and caspase-3 are shown in Fig. 12A-C. The binding analysis of β-amyrin with p53, MDM2 and capspase-3 proteins revealed that the binding pattern varied with the nature of the ligands. The docking results of β-amyrin are shown in Fig. 13A-F. The molecular docking studies revealed that the binding energy scores of β-amyrin docked with MDM2 protein (–6.73 kcal/mol) was higher than doxorubicin (–8.5 kcal/mol) (Table 1). The residues involved in hydrophobic interaction of β-amyrin with MDM2 were Met50, Leu54, Ile61, Met62, Tyr67, Val75, Val93, Ile99, Tyr100, Ile103 whereas Gln72 engaged itself in hydrogen bonding. On the other side, the hydrophobic residues involved in interaction of β-amyrin with p53 were Met62, Leu66, Tyr67, Tyr76 whereas Thr63 engaged in hydrogen bonding.
Furthermore, the binding energy scores of β-amyrin docked with p53 protein (–6.9 kcal/mol) was comparable with doxorubicin (–7.1 kcal/mol) (Table 2). The residues involved in hydrophobic interaction of β-amyrin with p53 were Leu111, Phe113, Tyr126, Trp146, Phe270 whereas Ser269 engage in hydrogen bonding. On the other side the hydrophobic residues involved in interaction of doxorubicin with p53 were Leu130, Pro250, Val274, Leu289 whereas Lys132 engage in hydrogen bonding. In addition, the hydrophobic residues involved in molecular interaction of caspase-3 with β-amyrin were Cys163, Tyr204, Trp206, Phe250, Phe252, Phe256 respectively.

Discussion
Recently, there has been a global shift in investigating the pharmacological potential of various herbal plants owing to the presence of different bioactive compounds. Undoubtedly, these bioactive compounds possess the potential of various pharmacological attributes including anti-oxidant, anti-inflammatory, anti-pesticidal anti-cancer and neuroprotection all of which are indispensable for the human health [54, 55]. Furthermore, green synthesis approach for synthesis of AgNPs offers distinct advantages over conventional chemical and physical methods by employing a single-step, eco-friendly process, that avoids hazardous reagents and high-energy requirements. In the present study, the use of Kp-EtOH serves a dual role in nanoparticle reduction and stabilization, resulting in intrinsically biofunctionalized AgNPs. Such surface modification, mediated by plant-derived phytochemicals, may enhance biological interactions, thereby extending the applicability of these nanoparticles in therapeutic contexts. This integration of sustainable synthesis with functional bioactivity highlights the significance of the present method within the broader scope of nanotechnology research.
Recent reports have established AgNPs as promising anticancer therapeutic agents. They selectively induce cytotoxicity, through oxidative stress-mediated activation of apoptotic pathways in different cancer cell lines, often at low effective concentrations [56]. Green- and/or bio-synthesized AgNPs have further enhanced their anticancer potential by improving biocompatibility and bio functionalization, which has intensified selective cytotoxicity towards the malignant cells [57]. Furthermore, AgNPs are being extensively explored for their role as radiosensitizers, to increase the efficacy of radiotherapy, by promoting oxidative stress and hampering the cell repair mechanism specifically in tumor cells [58]. Collectively, these features augment the versatile applicability of AgNPs in cancer therapy, and their potential to overcome the limitations of conventional anticancer strategies.
The present study was undertaken to explore the therapeutic potential of green synthesized Kp-AgNPs using Kp-EtOH. K. pinnata is a well reputed pharmacological plant which till dill date remains less explored in nanomedicine. Although, K. pinnata has been significantly explored for its pharmacological attributes, still there is a paucity on its role in facilitating the green synthesis of silver nanoparticles. Consequently, the biological activities of Kp-AgNPs including its cytotoxicity against hepatocellular carcinoma has not yet been established. By combining the apoptosis inducing effects of Kp-EtOH with nanotechnology, this present study introduces a plausible new approach for addressing hepatocellular carcinoma. Indeed, the ability to synergize the intrinsic bioactive potential of K. pinnata leaf extract along with increased nanoparticle properties is a new avenue for developing natural-based nanomedicines.
The GC-MS chromatogram identified 33 phytoconstituents, including various aromatic hydrocarbons, alkylamines, alcohols, alkanes, diallyl ethers, carbodiimides, pentacyclic terpenes, vitamins, cycloalkanes, carboxylic esters, triterpenes, lactones. Indeed, the presence of these groups of bioactive compounds can be correlated with various pharmacological activities associated with K. pinnata leaves. Compounds such as squalene and β-amyrin detected in Kp-EtOH are reported previously to possess antioxidant, emollient, analgesic, anti-inflammatory and anticancer properties [59–61].
Computational analysis through appropriate in silico methods such as molecular docking have played a vital role in the process of developing new drugs [62]. Recently, due to increased cases of resistance towards various chemotherapeutics, plant-based therapeutics have emerged as a potential alternative owing to their nominal side effects, in comparison to the chemotherapeutics [63]. In view of this the binding affinity of the bioactive compound β-amyrin found within the Kp-EtOH was assessed. β-amyrin was selected in this study, owing to its well documented antioxidant and anti-inflammatory activities among other pharmacological attributes. Also, previous reports documented the impeding effect of β-amyrin on the progress of hepatocellular carcinoma, by activating apoptosis and disruption of cell cycle progression [64]. Intriguingly, reports have also outlined the potential efficacy of β-amyrin against some key targets in cancer therapeutic. For the very first time, our in-silico studies have validated that β-Amyrin can effectively bind to key cancer targets such as p53, MDM2 and caspase-3. A high negative docking score of β-amyrin indicates its high binding affinity with the above stated target proteins.
Generation of free radicals is a homeostatic physiological process. However, the imbalance between generation and neutralization of these free radicals has serious consequences. Free radicals have been shown to impede the structural integrity of several biomolecules, including DNA, lipids and proteins. These radicals further alter the normal functioning of the immune system resulting in several chronic metabolic and associated ailments [65]. Antioxidants serve to be a natural source of countering the detrimental effects of the free radicals generated, as they hold the intrinsic efficacy of neutralizing the free radicals within the cells. At cellular level, certain antioxidant defence mechanism acts naturally, however under certain disease conditions, it fails to neutralize the free radicals being generated by the cells. Therefore, administration of external antioxidant source has become a necessity in the diseased state. Recent scientific literature has highlighted the emerging role of biosynthesized AgNPs for their plausible antioxidant effects [66–68]. In this study too Kp-AgNPs have exhibited their efficacy in scavenging DPPH and ABTS•+ radicals. Therefore, it can be inferred that Kp-AgNPs do hold the potential of inducing significant antioxidant potential which can further be refined so as to increase its potency.
The present investigation was also directed towards gaining insights into the anticancer effects of Kp-AgNPs against hepatocellular carcinoma. Recently, a detailed mechanistic investigation of K. pinnata extract mediated anticancer efficacy against various cancers such as breast, prostate and colorectal cancers was reported by Faun-des-Gandolfo and group [25]. Previously, the cytotoxic effects of K. pinnata extract were screened only against lung cancer, mesothelioma and hepatocellular carcinoma [23]. Nevertheless, till date, no study has reported the synthesis and a detailed insight into the anticancer effects of AgNPs from Kp-EtOH against hepatocellular carcinoma. This study, for the first time has reported the development and characterization of Kp-AgNPs along with the assessment of its pharmacological relevance in treating HCC through their anticancer effect against liver cancer HepG2 cells. The investigation explicitly details the cytotoxic effect of Kp-AgNPs against HepG2 cells. Kp-AgNPs have also been successful in altering the morphological characteristics, including the blebbing of plasma membrane, and shrinkage of HepG2 cells.
The initial stage of apoptotic cell death is characterized by the loss of integrity of the nucleus. Fragmentation and condensation of nucleus has been reported as an important impetus for apoptosis [69–71]. Subsequently, cell death and condensation of nuclear matter, in correlation with the oxidative stress positively reenforce apoptosis. Indeed, oxidative stress is accumulation of free radicals due to a deficient antioxidant mechanism [72]. Among several other free radicals, ROS generated by highly reactive singlet oxygen, superoxide anion radicals and hydroxyl ions. It is now established that ROS mediated oxidative stress activates apoptosis by impeding the functionality of key cellular components [73, 74]. The present study explicitly demonstrates that Kp-AgNPs increased the intracellular ROS levels in hepatic cancer HepG2 cells. Thus, it can be inferred that Kp-AgNPs not only have the intrinsic property of increasing acute ROS in liver cancer HepG2 cells, but also have an effective anti-oxidant potential. Caspases (cysteine proteases) are also well-known to play an imperative role in apoptotic pathway. Recently, it has been reported that activation of apoptotic cell death through green synthesized nanoparticles via enhanced ROS production, resulted in the downstream activation of caspase-3 [75], which could additionally contribute towards induction of apoptosis in HepG2 cells.
In summary, the present report enhances our present understanding of bioactivities of K. pinnata particularly, by providing an insight into the role of bioactive molecules, in not only reducing Ag+ but also showing a strong binding affinity towards p53/MDM2 complex. Secondly, the computational modeling and biological assays carried out indicate the anticancer activity of Kp-AgNPs by modulating the p53 reactivation pathway, which has not been previously reported for K. pinnata-derived nanoparticles. Thus, the combination of structural docking, ATR-FTIR functional group mapping, nanoparticle characterization, ROS modulation analysis, and cytotoxicity assays provides a comprehensive dataset that is lacking in earlier green synthesized silver nanoparticle studies. Indeed, due to a paucity in the toxicological and in vivo data, applicability of Kp-AgNPs remains uncertain. Nevertheless, the promising data reported herewith might prove itself useful for instigating future studies on Kp-AgNPs.

Conclusions
In summary, the present study validates the multifaceted bioactivities of Kp-AgNPs, synthesized using Kp-EtOH. Among its various bioactive constituents, β-amyrin was, for the first time, shown to exhibit significant docking scores towards key molecular targets including p53, MDM2 and caspase-3. Subsequent results revealed Kp-AgNPs exhibit potent antioxidant, and anticancer effects in appropriate in vitro model systems. Collectively, these results confirmed the therapeutic potential of Kp-AgNPs, and opened the possibilities of their development in nano-therapeutics, specifically for treatment of hepatocellular carcinoma.

Supplementary Information
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