Green Synthesis of silver nanoparticles using Amomum nilgiricum leaf extracts: Preparation, physicochemical characterization and ameliorative effect against human cancer cell lines

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Kariyappa, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5197419/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 10 Dec, 2024 Read the published version in Cytotechnology → Version 1 posted 19 You are reading this latest preprint version Abstract The present study to production of silver nanoparticles (AgNPs) by leaf extracts of A. nilgiricum and to evaluate the activity of anticancer by using AgNPs against cancer cell lines such as MCF-7, HEPG2, H9C2, HEK293 and H1975. The synthesized AgNPs were characterized by using UV–Vis spectroscopy, EDS, FT-IR, XRD, DLS, SEM and HRTEM with SAED patterns. The surface plasmon resonance (SPR) of AgNPs formed a peak centered at 427 nm by UV–Vis analysis. FTIR analysis reveals that existence of functional groups subjected to silver ions reduction to metallic silver. Crystalline form of the AgNPs was assessed by XRD analysis, four spectral peaks at 111, 200, 220, and 311 were formed and zeta potential peak was found at 28.5 mV indicating the higher stability. The size average diameter of the AgNPs was between 27–30 nm by TEM and SEM analysis was reveals the morphology of AgNPs as elongated, irregular and aggregated and some particles are spherical. EDX analysis confirmed the elemental composition of AgNPs with 81.43% Ag. The average diameter of AgNPs was found 21.49 nm in diameter and width was about 12.01nm by DLS analysis. Cytotoxicity of AgNPs was investigated by using MTT, SRB assay and comet assay was performed as a genotoxicity. The results revealed that AgNPs decreased the viability of cancer cells in a concentration dependent pattern (50 to 350µg/ml). The influence of AgNPs on cell cycle stop was studied on H1975, HEP-G2 and MCF-7 cells and found that AgNPs could induce sub G0 cell cycle arrest. The AgNPs was also induced DNA fragmentation confirms the DNA damage in nanoparticles treated cell lines. The anticancer action of nanoparticles was analyzed using proapoptotic and antiapoptotic caspase 8 and caspase 3 mRNA expression levels. Finally the results suggested that AgNPs is an effective anticancer agent which induces apoptosis in H1975, HEP-G2 and MCF-7 cells. Based on our studies, further identification of the major compounds of leaf extracts is acceptable. Amomum nilgiricum silver nanoparticles cancer cell lines anticancer activity Apoptosis Caspases Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Introduction Globally, people face various healthcare challenges and the devastating pandemic outbreaks affecting the lives and socioeconomic status of people. Cancer still remains a major disease affecting people and a leading cause of death among the developed and developing countries. With the ever-increasing cancer cases worldwide, the occurrence of new cases is expected to be 23.6 million reports of cancer per year by 2030 (Bray et al. 2018 ). The existing treatments for cancer include the application of chemotherapeutics, radiotherapy or surgery which support the patient’s health and survival but have adverse side effects, toxic effects on non-target tissues, drug resistance as well as lead to reappearance of cancer affecting the life of the individual and the family (Choudhari et al. 2020 ). Although intensive efforts are in progress, but cancer still persists as an aggressive killer worldwide. Presently, the various synthetic chemotherapeutic agents employed for treatment of cancer have not been effective in treatment in spite of the considerable cost of their development. Therefore there is a continuous demand to develop novel, effective, and reasonable anticancer drugs. The burden exerted by the disease demands the search for novel and efficient drugs against cancer from natural sources (Cabral et al. 2018 ). Plants have been used conventionally in the treatment of several diseases as they are sources for potent bioactive metabolites of pharmaceutical significance (Elrayess and El-Hak 2019 ). Despite advances in development of synthetic drugs, plant based drugs have played a central part in the presence of potent main molecules. Among the FDA approved anti-infectious and anticancer drugs, natural origin of drugs have a share of 60 to 75% from natural products or their derivatives (Sahoo et al. 2010 ).World Health Organization (WHO) reported, the primary health care still 80% of the population in some countries depend upon traditional medicines (Khazir et al. 2014 ). Herbal remedies are the best popular form of traditional medicine, and are extremely productive in the worldwide market. National Cancer Institute has selected about 114,000 plant extracts from 35,000 samples of plant identified from 20 nations for their anticancer potential (Kaur et al. 2011 ). The discovery of novel anti-cancer biomolecules from higher plants by phytochemical research based on ethnopharmacological information is normally considered as a valuable approach (Mani et al. 2020 ). The family Zingiberaceae encompasses rhizomatous herbs that are rich sources of valuable products used as food, spices, flavouring agents, traditional medicine, dyes, aromatic products and so on (Tan et al. 2018 ). This family comprises of around 52 genera and over 1300 species of aromatic plants many of them with therapeutic and ethanomedicinal value (Kress et al. 2002 ). Members of this family including turmeric, ginger and several others have proven to be potential as antioxidant, antimicrobial, analgesic, antiobesity, anti-angiogenic, proapoptotic, anti-inflammatory, immunomodulatory, antitumor, agents (Chumroenphat et al. 2019 ; Konappa et al. 2019 , 2020 ). Intensive botanical assessment of the forests of Western Ghats of South India led to the detection of a wild ginger A. nilgiricum , an interesting species of the family Zingiberaceae (Thomas et al. 2012 ). In current years, the interest in the synthesis and properties of metal nanoparticles (NPs) like copper, silver, palladium, zinc, gold, nickel, titanium, aluminum, chromium, iron, and cobalt, platinum. The silver (Ag) has been attractive attention in nanomedicine because of their catalytic, optical and physical properties and has been used in varied arenas such as electronics and therapeutics (Lee and Jun 2019 ). The nanotechnology includes by synthesis of NPs size distending from 1–100 nm. The NPs are broadly used for disease control, medical drive, and environment protection (Ebrahimzadeh et al. 2020 ). The related with chemical and physical production, green production ensure several benefits because it needs fewer chemicals, barren of long procedure and necessity of massive energy and fewer contaminant that evade lavish refinements (Awwad et al. 2013 ). Natural based techniques by plant aqueous or extracts of microbes are chosen (Rafique et al. 2017 ). Medicinal plant based production of NPs has several biotic advantages then NPs have nontoxic substances and bioactive molecules play a significant role in stabilizing, and capping materials (Shah et al. 2019 ). The proteins, amino acids, phenolics, alkaloids, flavones terpenoids, and polysaccharides are existence in extracts of plant can act as capping and reducing constituents (Naikoo et al. 2021 ). The production of AgNPs by easy, less toxicity, cost effective, defensible, compatibility and lengthier production period with adequate particle size dissemination (Hema et al. 2016 ). The AgNPs have been widely useful in different arenas such as drug delivery, fabric, farming, parasitology, catalysis, food, biomedical, water management, cosmetics, etc. (Divya et al. 2018 ). Plant centered produced silver NPs are having great antagonistic action against microbes and NPs are extensively used as an constituent in the medicinal production for ready of human healthiness care drugs mainly revealed favorable outcomes for anti-inflammatory, wound healing and anticancer activities (Rashid et al. 2019 ). The AgNPs can decrease the liberal growth of cancer cells through delaying several signaling cascades accountable for the growth and tumors pathogenesis. Numerous investigation results shown that AgNPs can kill human cancer cells with very little harm to normal cells (Sayed et al. 2019 ). The silver NPs are interrelating with cancer cells and control passive and active cellular reactions also chromosomal abnormalities and damage of DNA at lesser dosage deprived of noxiousness, particularly not at all genotoxicity action on cells of human (Gurunathan et al. 2018 ). Meanwhile the addition of silver NPs as a drug transferor in the treatment of cancer has presently increased significant consideration (Brahmbhatt et al. 2013 ). According to this, the present research was designed to synthesis of AgNPs from A. nilgiricum leaf extracts and activity on different cancer cell lines. The physic-chemical characteristics of the freshly produced AgNPs were studied by several methods such as UV–vis, DLS, XRD, FT-IR, SEM with EDAX and TEM studies. Further the bioactivity was evaluated by measuring cell viability, assessment of the DNA damage and genotoxicity, apoptosis induction in cancerous cells, cell cycle arrest and the expression levels of apoptosis connected genes in treated with AgNPs. Further apoptotic potential of AgNPs was proven by staining and cell cycle analysis. Therefore, plant based production of AgNPs creates a substitute to control the cancer to evade difficulties worried with conventional chemical treatment. To the greatest of our data, this research is the novel report of anticancer activity of synthesized AgNPs from A. nilgiricum leaf extracts. Materials and Methods Cell Lines Human hepatocarcinoma cells (HEPG2), rat cardio myoblast cells (H9C2), human breast cancer cells (MCF-7), human embryonic cells (HEK293) and Lung cancer cell lines (H1975) were obtained from the national center for cell science (Pune, India). All obtained cells lines were cultured and maintained from DMEM media added with heat inactivated fetal bovine serum (FBS) (10%) and streptomycin/ penicillin (50 U/ml). A humidified incubator holding 95% air and 5% CO2 was used to grow the cells at 37°C with media replenishment every two days. Collection of plant and preparation of leaf extracts Healthy leaves of A. nilgiricum was collected from Palakkad district, Kerala, India, at 11°03'15.46" N, 076°32'23.58" E and at an elevation of 1150 m above the sea level. The collected leaves were washed with tap water to eliminate the external contaminants and soil on leaves, followed by sterilized water; shade dried and prepared to fine powder. About 50g of air dried leaf powder of A. nilgiricum was extracted in100ml distilled water for 72 h. The leaf extract was filtered through Whatman no.1 filter paper and leaf filtrate was used for production of silver nanoparticles. Biosynthesis of silver nanoparticles For the AgNPs biosynthesis, 10 ml of leaf filtrate A. nilgiricum was transferred into 90 ml of AgNO 3 (10 mM) solution and the reaction mixture was kept overnight at 25°C with 100 rpm allowed them to mix appropriately. The AgNO 3 was used as a control. After incubation, observed for the reduction of AgNO 3 to silver ions (Ag + into Ag 0 nanoparticle) was confirmed by the light colored change from yellowish color to dark brown color (Fig. 1 ). The reaction mixture was used to centrifuge at 15,000 rpm for 25 min, the supernatant solution was removed and the residual solid was washed 5–6 times with distilled water. The produced AgNPs was studied by UV–vis spectroscopy. The obtained AgNPs was dehydrated at 60°C for overnight and stored until for characterization analysis. The dried AgNPs were further characterized using FTIR, XRD, DLS, SEM, EDS and TEM studies (Karuppiah et al. 2014 ; Konappa et al. 2021 ). Characterization of synthesized silver nanoparticles Development of silver NPs was observed by UV-vis spectroscopic analysis. The absorbance of AgNPs attained at maximum was detected by spectral scan at range of 200–800 nm by using UV–vis spectrophotometer (Hitachi, U-2800).The fourier transform infrared spectroscopy (FTIR) analysis was studied by Perkin Elmer Spectrum 1000 with the spectral range of 4000 − 400 cm -1 at resolution at 4 cm -1 to determine the potential functional groups in bioactive compounds existence in the leaf extract. Biomolecules are accountable for the reduction of ions and capping agents accountable for the strength of NPs. The study of DLS was done to detect the dispersal pattern, size of produced AgNPs and understand the size distribution pattern of very small NPs existing in solution (Microtrac /FLEX 11.0.0.2). The patterns of XRD with AgNPs was identified with Cu Kα radiation by using X-ray powder diffractometer (Rigaku Desktop Miniflex II) (λ = 1.5406 Ao) is the source of energy. The particle size and nature of the AgNPs was analyzed by using XRD. The diffracted intensities were noted at 2θ angles from 20–80º. The place of the maximum peak was related with standard libraries to identify phases of crystalline. The size of the particles of the AgNPs was determined by using Debye Sherrer's formula. D = Kλ/βcosθ Where D is the size of the particle (nm), β is the full line width at half maximum elevation of the main peak, λ is the X-ray wavelength, K is the shape and θ is the refractive angle. The scanning electron microscopy (SEM) analysis was studied by a tiny film of AgNPs prepared, dropped on the carbon coated copper grid film and dried with mercury lamp for 5 min. The morphological structures of the produced AgNPs were identified (Hitachi, S-3400N, Japan). The EDS assay was conducted using about 0.2g of AgNPs crystals to identify the existence of Ag ions in the solution (Hitachi Noran System 7, USA). Transmission electron microscopy (TEM) with selective area electron diffraction (SAED) was assessed to describe the size of the synthesized AgNPs (Hitachi H7500) (Konappa et al. 2021 ). In vitro anticancer activity of AgNPs by MTT assay For anticancer activity, MCF-7, HEPG2, H9C2, HEK293 and H1975 cells were inoculated in 96-well plate at 1x10 4 cells/well with DMEM complete media and kept for 24 h incubation. After the cells were totally attached to the wells and the media was removed. The cancer cells were added with AgNPs with different concentrations such as 50, 100, 150, 200, 250, 300 and 350µg/ml and incubated for 48 h. Later, 200µl of freshly prepared MTT (3-(4, 5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reagent (5 mg/ml of phosphate buffer solution) was incorporated and incubated at 37 ºC for 4–6 h. Afterward incubation, the MTT reagent was removed and dimethyl sulfoxide (DMSO) was mixed to dissolve the formazan crystals produced with live cells and measured absorbance at 570 nm by using Monochromator Microplate Reader (Mode Tecan 1650). IC50 value was calculated by linear regression equation. The results were calculated in percentage of reduction of MTT compared to the control cells absorbance. The tests were repeated three and plot graphs (Mosmann 1983 ; Hartman 2003). In vitro anticancer activity of AgNPs by Sulforhodamine B (SRB) assay Cytotoxic effect of silver NPs was studied by colorimetric Sulforhodamine B (SRB) assay (Voigt 2005 ). The cancer cells were inoculated in 96 wells plate at 1x10 4 cells/well and incubation for 24 h. Later the cancer cells were totally bounded to the wells, the media was detached. The cells were added with numerous concentrations ranging from 50 to 350 µg/ml of silver NPs and kept for 48 h. Cell fixation were prepared using 100µl/well with 10% tricholoroacetic acid (TCA) for 1 h at 4°C. The culture plates were washed and dried for 1h at room temperature. Further the cells were stained with SRB (0.02%) with 1% acetic acid and incubated at room temperature for 1h. Then the culture plates were washed thrice with 1% of 200 µl/ well acetic acid and dried. The 10 mM Tris-HCl (pH 10.5) (200 µl) was added to every well to remove SRB after placing in an orbital shaker for 1h. Microplate reader (Thermo Scientific) was used to determine the absorbance at 510 nm. Dual acridine orange/ethidium bromide fluorescent staining The cancerous cells were subjected to AgNPs treatment at several concentrations from 50 to 350µg/ml and stained with mixture of acridine orange (AO) and ethidium bromide (EB) dye (1:1) (Yashaswee and Trigun 2020). Detection of fragmentation of nuclei by using fluorescence microscope (Carl Zeiss, Germany) study. The AO intercalates between the double standard DNA and emits green fluorescence. The AO penetrates both live and dead cancerous cells. Instead, EB permeates only the dead cells and emits the red fluorescence indicating membrane damage. The images were collected using digital camera (Olympus). Single cell gel electrophoresis assay The single cell gel electrophoresis technique was studied to evaluate genotoxicity and DNA destruction induced by AgNPs (Alkaline Comet Assay) (Singh et al. 1988 ). The experiments were conducted in dark condition. The MCF-7 cells at 1×10 5 were seeded into a six well plate and kept overnight for cells attachment. Further the cells were added with many concentrations (250 µg/ml and 350 µg/ml) of silver NPs and kept for 24 h and (0.1%) DMSO was used as solvent control. The negative control (untreated cells grown in media) and positive control (cells treated with 20 mM hydrogen peroxide) were used. After incubation, the cells were trypsinized (cell dissociation using trypsin), centrifuged and the cell suspension was rinsed with sterilized Phosphate buffered saline (PBS). Further, 75 µl of pre-heated low melting agarose (1%) was added to the treated and control cell suspension and poured onto the super frost glass slide pre-coated with 1% normal melting agarose and covered by cover slip. Afterward solidification of the agarose at 0°C for 5 min and removed the cover slip. The cancer cells were kept in tank filled with cold lysis buffer at 4°C (300 mM NaOH, 2% DMSO and NaCl at 1.2M and 1% of Triton X-100). Then the slides were shifted to the electrophoretic tank filled with alkaline buffer for 30 min to allow the DNA unwinding. Later the electrophoresis, the slides were gently washed with neutralization buffer twice for 5 min. The cells were fixed onto the slides by submerging slides in 70% ethanol for 20 min and stained with 1X ethidium bromide. The DNA damage was analyzed using a fluorescence microscope. The amount of DNA destruction was calculated by evaluating the DNA movement distance and the transferred DNA percentage using Image J software with Open Comet. Randomly selected 50 to 100 cells were examined by the software. DNA fragmentation assay The two MCF-7 and HEPG-2 cells at 1x10 6 cells/ml were inoculated in 35 mm petridish containing complete DMEM media and kept for 24h and further added with various concentrations of AgNPs (250µg/ml and 350µg/ml) for 24 h. The genomic DNA of MCF-7 and HEPG2 were isolated by lysis buffer, imperiled to 1.5% of agarose gel electrophoresis. Finally the fragmented DNA bands were visualized using ethidium bromide staining under UV- transilluminator along with DNA ladder (1kb) as control (Kumar et al. 2017). Cell cycle analysis For determining the cell cycle halt properties of AgNPs, the cancer cell lines were inoculated into six well plates at 1x10 6 cells/well and kept for 24 h at 37°C. Then the cell lines were added with AgNPs for 48 h at 250µg/ml and 350µg/ml. The cancer cell lines were collected, washed two times with ice cold phosphate buffer saline and fixed by using 70% of chilled ethanol at 4°C for 4 h. Then the cell lines were suspended with 0.5ml propidium iodide (PI) containing with 50 µg/ml PI, 50µg/ml RNase, 0.2 mM EDTA and Triton X-100 (0.1%). The cell samples were kept for 30 min in dark. Further cell cycle evaluation was carried out by fluorescence-activated cell sorting (FACS) apparatus (Becton Dickinson Heidelberg, Germany). RNA isolation, cDNA synthesis and RT-PCR method The MCF-7 cells at 5x10 5 cells/well were inoculated into 6-well plate and subjected to AgNPs treatment of two different concentrations (250µg/ml and 350µg/ml) for 48h. From the treated and untreated cell samples the total RNA was isolated by Trizol method. Nanodrop spectrophotometer (Thermo Scientific, USA) was used to evaluate the concentration and quality of RNA. The Verso cDNA synthesis kit was used for the formation of cDNA (Thermo Fisher Scientific). The PCR mixture include 10 µl of Red Taq Master Mix, 1 µl of cDNA, nuclease free water and 1µl of each complementary primer specific for β-actin sequence, Caspase 8 and Caspase 3. The mixture was run for 30 amplification cycles. The positive control was used as normalization β-actin. The study of PCR yields were studied by electrophoresis using 1% of agarose gel and 1X TAE buffer. Then mRNA expression was enumerated by using image study software. Results Characterization of synthesized AgNPs The SPR-surface plasmon resonance of AgNPs made a peak centered nearby 427nm. The AgNPs absorbance was occupied primarily the color of sample was yellowish and afterward the color of sample was turned to the dark brown color (Fig. 2 a). The visible region of the peak shows the reduction of the Ag + and the production of AgNPs. The FTIR spectrum was used to identify the purity and existence of functional groups in the produced AgNPs. The existing main functional groups from biomolecules from leaf extract acts as reducing agents of Ag + to Ag o . The FTIR spectrum attained between the wavelength ranges of 400–4000 cm -1 , shows major peaks were observed including peaks range at 3462, 2971, 1637, 1584, 1381, 1264, 1093 and 805 cm -1 . A strong peak at 3462 cm -1 shows the N-H stretching of NH 2 group; moreover specify the O-H bond existing in polyphenol protein, phenol and polysaccharides. The peak at 2971 cm -1 characterizes the aliphatic C-H stretching, 1637cm -1 represents to (NH) = O bond, 1584 cm -1 represents the existence of C = C stretch as alkenes, 1381 cm -1 designates C = O stretch and 1093 cm -1 for C-O-C bond stretching of terpenoids and flavonoids. The band at 805 cm -1 and 1264 cm -1 endorses the existence of alkyl halides (Fig. 2 b). The synthesized Ag NPs was confirmed as crystalline form by XRD analysis. The 3 main peaks were identified in the XRD analysis and displays distinct peaks at 2θ = 38.23°, 44.42°, 64.44° and 77.39°, which related to (111), (220), (200) and (311) respectively (Fig. 2 d). The diffraction data were compared with standard AgNPs published in joint committee on powder diffraction standards (JCPDS), silver file No. 04-0783. The normal diameter of the produced AgNPs is between 27 and 30 nm. The DLS analysis of AgNPs was identified the size of the particle distribution in the solution. The average diameter of silver NPs was found 21.49 nm in diameter and width was about 12.01nm (Fig. 2 c). The produced AgNPs size was detected by using SEM analysis noted as 87 nm and it shows the AgNPs morphology, as extended, collected and irregular and some nanoparticles were rounded (Fig. 2 e). This outcomes were strongly confirms that A. nilgiricum leaf extract might main role in reducing and capping agents in the synthesis of silver NPs. Energy Dispersive Spectroscopy (EDS) analysis was analyzed to identify the elements in the biosynthesized AgNPs. The EDS result displays that the produced AgNPs comprises the 81.43% followed by carbon etc. (Fig. 2 f). The metallic AgNPs usually shows a distinctive signal at 3 KeV regions showing the surface plasmon resonance. The HRTEM image confirmed the NPs were spherical shape (Fig. 2 g). The diameter of the silver NPs was average between 27 and 30 nm. Slight percentage of silver NPs were partly collected, but was even in size and non-aggregated form. The interplanar spacing which was observed and refers to the face centered cubic crystalline structure and crystalline form of the silver NPs. In vitro anticancer activity of synthesized AgNPs The in vitro anticancer activity of synthesized AgNPs from A. nilgiricum leaf extract was determined by using MTT and SRB assays. Five cell lines were added with numerous concentrations of AgNPs varies from 50–350µg ml -1 for 48 h. From MTT assay, the results were done in dose dependent inhibition of cell growth by synthesized AgNPs (Fig. 3 ). Among the concentrations of AgNPs, 350µg ml -1 showed maximum cytotoxic effect followed by 300 250, 200, 150, 100 and 50 µg ml -1 and 350 µg ml -1 . At 350 µg ml -1 of synthesized AgNPs was showed maximum percentage of cytotoxicity activity against all cell lines such as MCF-7, HEPG-2, H9C2, HEK293 and H1975 up to 89.58 ± 1.86%, 86.45 ± 1.73%, 83.53 ± 2.35%, 79.15 ± 2.31% and 87.12 ± 2.42% respectively (Fig. 3 a). At 250 µg ml -1 of AgNPs was showed percentage of cytotoxicity activity against cell lines such as MCF-7, HEPG-2, H9C2, HEK293 and H1975 up to 67.66 ± 1.96%, 65.73 ± 1.61%, 58.54 ± 2.11%, 56.88 ± 1.93% and 59.54 ± 2.16% respectively. While 50 µg ml -1 of AgNPs was showed percentage of cytotoxicity activity against cell lines such as MCF-7, HEPG-2, H9C2, HEK293 and H1975 up to 39.65 ± 1.23%, 36.32 ± 1.36%, 32.76 ± 1.46%, 32.77 ± 1.63% and 38.23 ± 2.12% respectively (Fig. 3 ). The IC50 value of AgNPs was found at 40µg ml -1 for MCF-7, 68µg ml -1 for HEPG-2, 105µg ml -1 for H9C2, for HEK293 95µg ml -1 and 62µg ml -1 for H1975 cells line. Similar results were observed in SRB assay, at 350 µg ml -1 of AgNPs from A. nilgiricum leaf extract was showed maximum percentage of cytotoxicity activity against all cell lines such as MCF-7, HEPG-2, H9C2, HEK293 and H1975 cells up to 82.69 ± 1.83%, 75.92 ± 2.36%, 69.62 ± 2.11%, 60.91 ± 2.23% and 74.59 ± 3.16 respectively (Fig. 3 b). At 250 µg ml -1 of AgNPs was showed percentage of cytotoxicity activity against cell lines such as MCF-7, HEPG-2, H9C2, HEK293 and H1975 of 64.54 ± 1.93%, 65.69 ± 2.25%, 53.89 ± 2.13%, 46.75 ± 2.89% and 61.45 ± 2.23% cells respectively. While 50 µg ml -1 of AgNPs was showed percentage of cytotoxicity activity against cell lines such as MCF-7, HEPG-2, H9C2, HEK293 and H1975 of 36.85 ± 1.76%, 37.57 ± 1.63%, 33.65 ± 1.43%, 32.97 ± 1.76% and 35.54 ± 2.12% cells respectively. The IC50 value of AgNPs was found at 42 µg ml -1 for MCF-7, 85 µg ml -1 for HEPG-2, 98 µg ml -1 for H9C2, 110 µg ml -1 for HEK29 375 and 76 µg ml -1 for H1975. The AgNPs from A. nilgiricum leaf extract induced morphological changes of cell lines. The cells were added with AgNPs at 350µg ml -1 , 250µg ml -1 and 50µg ml -1 against MCF-7, HEPG2, H9C2, HEK293 and H1975 cells for 48h. The morphology of cells was intensely changed afterward the addition with AgNPs was observed as related to control cells (Fig. 4 ). Apoptotic assay Further AO/EB dual staining study was assessed using two concentrations of AgNPs (250 µg ml -1 and 350 µg ml -1 ) were selected based on the in vitro cytotoxic activity assays. To evaluate apoptosis, AO/EB double staining procedure was used. The cancer cells H1975, HEPG2, MCF-7, HEK293 and H9C2 were added with 2 concentrations (250µg ml -1 and 350µg ml -1 ) of AgNPs from A. nilgiricum leaf extract for 24h and stained by AO/EB. The control cells were appeared bright green color with spherical nucleus distributed uniformly in the middle of the cells. While the AgNPs treated cells at 250 µg ml -1 concentration showed early stage apoptosis, marked by membrane blebbing, granular or crescent-shaped green-yellow AO nuclear staining (Fig. 5 ). But, the cells treated with 350µg ml -1 of AgNPs showed late apoptotic stage with bright orange patches of condensed chromatin in the nucleus characterized by asymmetrically localized orange nuclear EB staining (Fig. 5 ). This indicates AgNPs induce apoptosis in the cancer cells. Comet assay if AgNPs from A. nilgiricum leaf extract According to the outcomes of MTT and SRB assays maximum cytotoxicity was observed in, MCF-7 cells showed IC50 value at 40 µg ml -1 and 42 µg ml -1 respectively. Hence MCF-7 cells cancer cell line was selected for comet assay. In this assay was performed to study the genotoxicity effect in MCF-7 cells. For this assay, AgNPs from leaf extract of 250µg ml -1 and 350µg ml -1 concentrations having cytotoxic effect were used (Fig. 6 ). The DNA damage was expressed as olive moments, AgNPs showed DNA damage in both the concentrations. When related to control, AgNPs at 350µg ml -1 showed maximum DNA damage than 250µg ml -1 . The H 2 O 2 was used as positive control which exhibited maximum DNA damage (Fig. 7 ). Cell cycle analysis by flow cytometry method The cell cycle of the cancer cells were deliberated using flow cytometry. The cell lines were added with 2 doses of silver NPs at 250 µg ml -1 and 350 µgml -1 for 48 h, the cells were removed and static in ice-cold ethanol. After subjected to PI staining for 15–20 min and examined for cell cycle halt at different phases. The flow cytometry study outcomes shown that the AgNPs treatment on H1975, HEP-G2 and MCF-7 cells at 250 µg mL -1 has no significant effect at G2M phase arrest when related to control cells, 21.40%, 21.04% and 16.42% respectively. But the higher concentration at 350 µg ml -1 AgNPs was arrested the cells at SubG0 phase of cell cycle as compared to control cells (Fig. S1 -S3). The flow cytometry analysis suggests induction of SubG0 phase of cell cycle arrest from AgNPs treatments. Caspase-3 and Caspase-8 in MCF-7 cells expression analysis Further we studied the anticancer activity of silver NPs from A. nilgiricum leaf extract upon caspases activation, which is a key effector protein of apoptosis. Semi quantitative RT-PCR was used to study the expression of caspase-3 and caspase-8 gene in MCF-7 cells. Overall, the expressions of Caspase-3, Caspase-8 genes were up controlled in AgNPs (250 µg ml -1 and 350 µg ml -1 ) added cells as related to untreated cells (Fig. 8 ). The up regulation of these apoptotic genes in cells indicates the anticancer activity of AgNPs from A. nilgiricum leaf extract (Fig. 9 ). DNA fragmentation study for detection of apoptosis The DNA fragmentation was confirmed by DNA ladder technique to support the initiation of apoptosis by AgNPs from A. nilgiricum leaf extract against MCF-7 and HEP-G2 cell lines which showed greater cytotoxic response. DNA fragmentation was noticed that the AgNPs (250µgml -1 and 350µg ml -1 ) treated cells were revealed dose dependent DNA laddering pattern as compare to control cells (Fig. 10 ). The results suggested that the AgNPs induced DNA ladder formation is a symbol of apoptosis. Discussion Cancer is a major serious health challenge worldwide with huge implications to public health and several exertions were made for exploring and developing innovative treatment strategies. Even though, for cancer treatment several therapies like endocrine therapy, chemotherapy and targeted therapy have been clinically recommended, still numerous patients still suffer from reversion because of the heterogeneity of tumor. However, these therapies exhibit many side effects and the treatment cost is also high. The formation of chemo resistance in tumor cells is another hurdle for the control of cancer by pharmacological approach (Singh et al. 2020 ). The traditional knowledge of medicinal plants offers an alternative method for developing anticancer medicines with enhanced compatibility, cost-effectiveness and lesser toxicity. The metabolites produced by plants have molecular targets for stimulating the apoptosis in various cancer cell lines (Masih et al. 2012). The numerous metallic NPs, particularly silver were extensively existence verified for remedial applications in cancer study. We synthesized the AgNPs from A. nilgiricum leaf extract and evaluated their antitumor activities. Many researchers have described the usage of nanoparticles for controlling the growth of cancer from in vitro studies. Numbers of investigators have been described about production of AgNPs by using different plant extracts exhibited unique anticancer activity (Kummara et al. 2016 ). The Amomum genus belongs to Zingiberaceae family. Globally, it includes around 150 species and are mainly scattered in tropical regions of Oceania and Asia (Cai et al. 2021 ). The seeds, leaves and fruits of Amomum were used in the preparation of traditional medicines. It has been used for controlling various ailments such as gastric disorders, inflammation, digestive disorder, cancer, dental infections and malaria (Dahigaonkar et al. 2018 ). There is no research has been studied on the anti-cancer activity of Amomum nilgiricum and their nanoparticles on any cancer cell lines. The most basic technique is to observe AgNPs synthesis by directly noticing the conversion in the shade of the sample from yellow color to dark brown color. Spectrophotometric method can be endorse the tracking manner and identify NPs peaks in the visible area from UV–vis spectrum with between 200 and 800 nm wavelength (Balashanmugam et al. 2013 ). The produced peak in the visible area specifies the Ag + reduction and production of silver NPs from A. nilgiricum leaf extract. The conversion of color through the production of silver NPs is linked to the excitation outcome of SPR (Balaraman et al. 2020 ). Other methods, comprising FTIR, DLS, XRD, SEM, EDAX and TEM, were used to analyze the dispersion, morphology, size, and composition of NPs. In the present study, the SPR of silver NPs created a peak centered nearby 427nm, because of the transition of electrons. The spectroscopic results of AgNPs are valuable methods for characterizing produced silver NPs (Elamawi et al. 2018 ). The similar reports supported that the silver NPs from UV absorption peak at 456 nm wavelength. Mtambo et al. ( 2019 ) was reported from the extract of Bidens pilosa was used to synthesis of silver NPs exhibited peak by UV-absorption at 410 nm. The silver NPs production in several plants extracts for example Clonorchis sinensis, Ocimum tenuiflorum, Centella asiatica and detected OD at 420 nm by UV-spectrophotometer (Moodley et al. 2018 ). In the present study, absorbance was observed at 440 nm from NPs production by using Plumbago zeylanica extract and from Catharanthus roseus extract at 400 nm (Nayak et al. 2016 ). In the current research, the FTIR spectrum obtained between the wavenumber ranges of 400–4000 cm -1 , shows major peaks were observed including peaks at 3462, 2971, 1637, 1584, 1381, 1264, 1093 and 805 cm -1 . A strong peak at 3462 cm -1 signifies the N-H bond vibration of NH 2 group, moreover showed the O-H stretch or H-bond existent in protein, polyphenol, polysaccharides and phenol. The peaks denotes the (NH) = O stretching, aliphatic C-H bond vibrations, C = C stretch in alkenes, C = O stretching modes and C-O-C stretching manners of terpenoids and flavonoids. The sharp intense band confirms the presence of alkyl halides and corresponding with the previous reports (Pushparaj et al. 2023 ). The FTIR spectroscopy method can be used to identify the functional groups accountable for producing silver NPs. The present major functional groups in the biomolecules from extract acted as reducing agents of Ag + to Ag◦ and stabilizing the AgNPs synthesized (Ajaykumar et al. 2023 ). The important functional groups for example methyl, alkanes, aliphatic and halides, amides, alcohol formed their existence of silver NPs (Ajaykumar et al. 2023 ). Hence, the FTIR analysis is an important and inexpensive method to describe the part of biomolecules from production, stability of produced silver NPs (Soliman et al. 2023 ). The A. nilgiricum leaf extract was described to comprise various biomolecules like, flavonoids, alkaloids, saponins, steroids, phenolics, tannins etc. (Konappa et al. 2017 ).The presence of those peaks, with a minor change in the wavenumber, earlier reports described to the synthesis of AgNPs (Wang and Wei 2021 ). The existence of various IR bands connected to presence of several functional groups in A. nilgiricum leaf extract. A similar outcome was reported by Fafal et al. ( 2017 ), the Asphodelus aestivus plant interceded production of silver NPs by FTIR spectrum analysis signified the accessibility of bioactive molecules responsible to reducing and capping. Noorbazargan et al. ( 2021 ) confirmed the spectra of FTIR showing leaf extracts and produced silver NPs that comprise certain bioactive molecules. The XRD study was used from several studies to define the synthesized AgNPs was confirmed as crystallinity. This method is valuable for identifying the purity as it can definitely show whether the sample is contains impurities or pure (Alharbi et al. 2022 ). The three main peaks were detected from XRD spectrum from AgNPs and displays distinct peaks at 2θ = 44.42°, 38.23°, 64.44°, and 77.39°, which match to (220), (111), (200) and (311) respectively. The average diameter of the biosynthesized Ag NPs is between 27 and 30 nm. The study of material using XRD subjected to the diffraction patterns for every sample has a distinctive diffraction beam (Hembram et al. 2018 ). Also, the XRD method has been used to estimate the nanoparticle crystallinity, size and check the crystallinity of materials. In, same outcome was identifying AgNPs from leaf extract of Pedalium murex presented peaks at 64.56°, 38.19°, 44.37° and 77.47° structures to the crystalline plane of 220, 111, 200, and 311 with normal size of 14nm (Anandalakshmi et al. 2016 ). Likewise, the XRD spectrum of AgNPs prepared from Sargassum myriocystum plant extract (Balaraman et al. 2020 ). Vetrivel et al. ( 2019 ) synthesized crystalline silver NPs by green synthesis from Ceropegia bulbosa Roxb root tuber powder extract and detected XRD distinct peaks from the planes of 111, 220, 200 and 311, these represented to the silver NPs. From earlier study, the silver NPs from Carmona retusa leaf extract of exhibited four peaks by XRD method (Rajkumar et al. 2018 ). In the present study, the size of crystal NPs quantity by Debye Scherrer’s formula and size of NPs was 22.6nm. The (111), (200), (220) and (311) diffraction peaks signify face centered cubic silver, whereas the sharpness of those peaks shows the creation of nanosized material (Deivanathan and Prakash, 2023 ). The DLS can be used to detect the size, particle size distribution and surface charge of synthesized AgNPs in the s colloidal suspension. This method is subject to on the interface of the Brownian motion of spherical material with the light pass over a colloidal solution (Bamal et al. 2021 ). In the current investigation, the average diameter of AgNPs was showed 21.49nm in diameter and width was about 12.01nm. Vanin dos et al. (2022) reported that the concentration of the plant extract increases, increase the normal size between 70 to 144 nm of silver NPs synthesized from Ilex paraguariensis extract. Likewise, AgNPs produced by plant extract of Salvia miltiorrhiz a exhibited the size of particle was 128 nm (Zhang et al. 2016 ). In the current research, the SEM analysis outcome of synthesized AgNPs from leaf extract of A. nilgiricum was showed morphological structure in extended, collected, asymmetrical and some NPs are circular with size was 87 nm. This outcome strongly confirms that A. nilgiricum leaf extract act as a reducing and capping agent in the synthesis of Ag NPs. Similarly previous reports were observed AgNPs from Ajug abracteosa; Cinnamomum tamala (Choi et al. 2021 ). Ghabban et al ( 2022 ) reported that the silver NPs synthesized in Astragalus spinosus was showed circular and size between from 30–40 nm by SEM analysis. The AgNPs produced in Allium cepa L. was cubical shape (Abdellatif et al. 2022 ). A study described that silver NPs produced by using extracts of Z. officinale was circular and size was between 30–50 nm (Gurunathan et al. 2013 ). Furthermore, silver metal dispersal of biogenic AgNPs was confirmed by EDS (Dimitrijevic et al. 2013 ). Present study EDS result shows that the biosynthesized Ag NPs contain 81.43% followed by carbon, etc. The metallic AgNPs usually displays a distinct at 3 KeV regions representing the surface plasmon resonance. The similar type of elemental analysis was made in the production of AgNPs from neem leaf ( Azadirachta indica ) extract (Ahmed et al. 2016 ). The EDS is a method that describes the metallic conformation of the solution used to check the existence of silver metal from synthesized Taxus wallichiana AgNPs. The Huong and Nguyen ( 2021 ) was analyzed the silver NPs from leaves extract of Brassica oleracea , from EDS spectrum presented the occurrence of silver. The property of AgNPs that were produced by ecologically friendly approaches has been previous reports (Mohammadi et al. 2019 ). In the current research, the result of HRTEM method was showed circular in shape of produced silver NPs and average diameter in between 27 to 30 nm. TEM gives better results related with SEM analysis and allows a more in-depth study of NPs (Sreelekha et al. 2021 ). The produced silver NPs have been described and observed by TEM by numerous investigators. The extracts of C. longa and Z. officinale were reduction of reducing agents, which clue to varied size of silver NPs (Dubey et al. 2010 ). Rather et al ( 2022 ) reported that the TEM determines the silver NPs synthesized from the Cuphea carthagenensis leaf extracts, particles were identified as circular and between 4 to 18 nm in size. Extract of Rubus ellipticus and Lysiloma acapulunsis AgNPs, TEM study exhibited the crystalline structure with noticeable lattice fringes particles were circular and size from 13.85 to 34.30 nm (Garibo et al. 2020 ; Khanal et al. 2022 ). The silver NPs synthesized from C. guianensis leaf extracts, V. lantana , and M. capitata exhibited size between 25–40 nm, 30–35 nm and 20–70 nm, respectively and have mainly spherical shape (Srirangam and Rao, 2017 ). Also Amaliyah et al. ( 2022 ) used TEM analysis of AgNPs from Piper retrofractum revealed the NPs were mainly circular and between from 1–40 nm diameter. In the current study, the cancer cells with numerous concentrations of AgNPs from A. nilgiricum leaf extract was assessed by MTT and SRB assays for 48h about the anticancer activities on MCF-7, HEPG-2, H9C2, HEK293 and H1975 showing that the AgNPs could be an alternative of conventional drugs against cancer. The AgNPs at 350µg/ml showed maximum inhibition of all cancer cell lines proliferation in comparison with other concentrations ranging from 97.13% − 85.87% from MTT assay and 95.58% − 93.12% from SRB assay. The IC 50 value of AgNPs was showed 40µg/ml against MCF-7. The outcomes of these assesses shown that the AgNPs concentration increases, decreased viability of the cancer cells. The cancer cells added with silver NPs revealed the reduced metabolic actions, reliant on the form of cancer cells and the size of the NPs (Hussein and Abdullah, 2022 ). In present study, the cytotoxicity activity results was connected to the outcome of Sabah et al. ( 2020 ) study, leaf extract of B. oleracea produced silver NPs showed anticancer activity at IC 50 of 55µg/ml. Singh et al. ( 2017 ) described the related effects for silver NPs produced from leaf extract of Borago officinalis . Similar outcomes were deliberated from Murraya koenigii leaf extract of AgNPs (Roshni et al. 2018 ). Similar results were deliberated the outcomes of experiment by cytotoxicity influence on growth of cell was detected at different concentrations (10–100µg/ml) of AgNPs in Mallus domestica against MCF-7 cells (Mariadoss et al. 2019 ). In dose-dependent manner, the AgNPs from Ananas comosus were showed greater anticancer action against HepG2 cells (Ahmad and Sharma 2012 ). The Dehghanizade et al. ( 2018 ) studied that, the silver NPs from leaf extract of Anthemisa tropatana was revealed great anticancer activity to HT-29. The synthesized silver NPs was observed as mutagenic and genotoxic because of existence of alkaloids and flavonoids (Ghramh et al. 2020 ). In the current investigation, AgNPs induce apoptosis in H1975, HEPG2, MCF-7, HEK293 and H9C2 cells were measured by dual staining by AO/EB. The control cells appeared bright green in color with spherical nucleus distributed uniformly in the middle of the cells. While the AgNPs added cells showed in early phase apoptotic cells develops membrane blebbing, granular or crescent-shaped greenish yellow and late phase develops orange because of necrosis in AO nuclear staining. The late apoptotic phase with bright orange patches of condensed chromatin in the nucleus characterized by asymmetrically contained orange nuclear EB staining (Roy et al. 2019 ). These results revealed the primary characteristic feature of apoptotic cell death (Singh et al. 2020 ). In the current study outcomes of cell cycle study shown that the cancer cells were showed using flow cytometry. This method to regulate the metabolic activity by using AgNPs prevent cell development, the flow cytometry study was used to describe distribution of cell cycle (Chan et al. 2011). The flow cytometer study results shown that this AgNPs treatment on H1975, HEP-G2 and MCF-7 cells at 250 µg/ml has no significant effect at G2M phase arrest when related to control cells, 21.40%, 21.04% and 16.42% respectively. But the higher concentration at 350µg/ml has stopped the cells at SubG0 phase of cell cycle related to control cells. In the current study, the treatment with silver NPs, from the cell cycle was found to be stopped in S-phase and expressively decreases the cell growth. The results of present study agrees with those previously reports (Mishra et al. 2021 ). The stop the cell cycle was supposed to be produced normally damage of DNA (Akter et al. 2018). The AgNPs added on A549 cells shows the up regulation of p53 which indicates the stop the cell cycle at G0-G1 stage, halts cell proliferation (Nair et al. 2012). Earlier reports were confirmed that the oxidative stress indications to damage of DNA and abnormalities of chromosomes, and apoptosis of cells adding with silver NPs (Kai et al. 2011). Numerous cancer cells exhibit sub-G1 stop and apoptosis after being exposed to AgNPs. Furthermore, by decreasing tumor cell development and angiogenesis, AgNPs can prevent distant metastasis (Mishra et al. 2021 ). In A549 cells, the silver NPs are down regulated the protein kinase C which detect the cell cycle stop at G2/M phase (Jain et al. 2021 ). In the present study, results showed in the DNA fragmentation assay, AgNPs (250µg/ml and 350µg/ml) treated cells was shown dosage dependent DNA laddering pattern on compare to control cells. The overall results suggest that the AgNPs induced DNA ladder formation is a hallmark of apoptosis. The fragmentation of DNA was detected in the AgNPs treated cells which were continual by equivalent study. The treatment with silver NPs particularly improved length and creation of number of tail DNA from cell lines (Bin-Jumah et al. 2020 ). Genes accountable for regulating the cell cycle, caspase-3, CAT genes, P53, pointedly prevent the cell development and generate apoptosis (Datkhile et al. 2020 ). The apoptosis can be induced by death receptors or through the mitochondrial pathway. It is an intrinsic programmed cell death mediated by major downstream initiation of caspase cascade which is generally followed by recruitment of caspase family of proteins including caspase-3 and caspase-8 (Kanamori et al. 2021 ). The AgNPs were examined for cytotoxic action on MCF-7 cells and detected to initiate stress on endoplasmic reticulum over unfolded protein response and improves initiation of caspase 9 and 7 producing cell death (Simard et al. 2016 ). Rageh et al. ( 2018 ) reported that the silver NPs was exerting their anticancer activity by producing DNA destruction from cells. The AgNPs from extract of Taxus brevifolia exhibited anticancer effect on MCF-7 at 25mM exhibited 75% death rate (Sarli et al. 2020 ). Lee et al. ( 2011 ) was revealed that the mechanism of anticancer activity comprises the antiapoptotic protein down-regulation, like BCL-2 and upregulation of P53 proteins, caspase 3, ROS (Chen and Wen 2022 ). Conclusions In the current research, produced AgNPs from leaf extract of A. nilgiricum and discovered their application against medically significant to cancer cell lines like MCF-7, H1975, HEPG2, H9C2, HEK293. Different methods were used to assess the morphology of produced silver NPs in A. nilgiricum leaf extract showing distributions of sizes and shape. In current study, the NPs synthesis technique was a very easy, rapid, simple, clean, reliable and ecologically friendly without any involvement of energy usage steps for producing AgNPs using A. nilgiricum leaf extract. Further the synthesized AgNPs was induced anticancer effect which is recognized to inhibited the cell cycle development was enumerated by using flow cytometer to determine the mechanism with AgNPs prevent the cancer cell development. Also these AgNPs were induced cell apoptosis and eventually cell death confirmed by AO/EB staining, comet assay and mRNA expression of caspases. Moreover, the DNA damage in cancer cells because of silver NPs was examined in fragmentation of DNA and apoptosis of cancer cells were also observed. These results recommend that AgNPs might have promising anticancer activity and used as therapeutic material for cancer therapy. This biosynthesis of AgNPs could also work as a benefit for the cancer treatment. Therefore, future study is required to completely understand the mechanism and the strength of the AgNPs in terms of stops the growth of cancer cells should be studied under in vivo . To conclude, the toxicity of synthesized NPs to normal cells permits broad examinations to launch the potential medical uses of AgNPs. Declarations Acknowledgments We are gratefully acknowledging the financial assistance granted by Post-Doctoral Fellowship (No. F. /PDFSS-2014-15-ST-KAR-7487), University Grant Commission (UGC), New Delhi, for carrying out this research. The authors are also thankful to Department of forests and wildlife, Govt. of Kerala, for giving necessary forest permission for sample collection. Author Contributions: Conceptualization, validation, formal analysis, investigation and data curation, N.K., S.K., C.S. and N.S.R. writing original draft preparation, N.K., C.S. and R.K., writing review and editing, N.K, R.P., A.S.K., R.K., and N.S.R. All authors have read, reviewed and agreed to the published version of the manuscript. Funding This research was funded by University Grant Commission (UGC), New Delhi, No. F. /PDFSS-2014-15-ST-KAR-7487. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5197419","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":381044345,"identity":"e8db9c7e-00b2-4fe3-9309-c6eb12971cca","order_by":0,"name":"Narasimhamurthy Konappa","email":"","orcid":"","institution":"Bangalore University","correspondingAuthor":false,"prefix":"","firstName":"Narasimhamurthy","middleName":"","lastName":"Konappa","suffix":""},{"id":381044346,"identity":"2274f64b-86fd-4ba6-a4f6-8c92252073e1","order_by":1,"name":"Rajeshwari H Patil","email":"","orcid":"","institution":"Bangalore University","correspondingAuthor":false,"prefix":"","firstName":"Rajeshwari","middleName":"H","lastName":"Patil","suffix":""},{"id":381044349,"identity":"3d016a8d-4a2c-449e-b579-299d6b7e8898","order_by":2,"name":"Anupama S. 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The data are presented as mean ± SD of three independent experiments were analyzed.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5197419/v1/9ceab70e9de6b3b9f33801b9.png"},{"id":70565859,"identity":"316426af-6b84-4c25-856c-9703d80cda2d","added_by":"auto","created_at":"2024-12-04 12:46:14","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":847166,"visible":true,"origin":"","legend":"\u003cp\u003eAgNPs induced morphological changes in cancerous cell lines MCF-7, HEPG2, H9C2, HEK293 and H1975 cells were treated with AgNPs (a: 350μg/ml, b: 250μg/ml and c: 50μg/ml) for 48 h.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5197419/v1/80123216c6f55a47615d4d5c.png"},{"id":70567371,"identity":"867b1be8-b04b-460d-a582-975380b95bd7","added_by":"auto","created_at":"2024-12-04 13:02:14","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":628079,"visible":true,"origin":"","legend":"\u003cp\u003eAgNPs induced morphological changes in cancerous cell lines MCF-7, HEPG2, H9C2, HEK293 and H1975 cells were treated with AgNPs (a: 350μg/ml, b: 250μg/ml and c: 50μg/ml) for 48 h.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5197419/v1/cb0b91eb925cf744ad10e8e8.png"},{"id":70565865,"identity":"89bec122-39dd-4d30-81cc-f8b0b0d1f800","added_by":"auto","created_at":"2024-12-04 12:46:14","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":158249,"visible":true,"origin":"","legend":"\u003cp\u003ePhotomicrographs of stained DNA from comet assay for 24h, DNA damage in comet tail of MCF-7 cell line treatment with AgNPs. Comet images of control MCF-7 cells (a), positive control H2O2 at 150 µM (b), AgNPs at 250 μg/ml of AgNPs (c) and 350 μg/ml of AgNPs from A. nilgiricum leaf extract.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-5197419/v1/fdd00cd67d1d36a438229f26.png"},{"id":70565858,"identity":"ea2a393a-72c5-4088-8245-c34bf43b871e","added_by":"auto","created_at":"2024-12-04 12:46:14","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":14831,"visible":true,"origin":"","legend":"\u003cp\u003eGraphical representation of olive moments by DNA damage expression.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-5197419/v1/b87b4ece17ea77bc70c15aaa.png"},{"id":70567373,"identity":"96dd811d-7df7-4b59-8685-f54d9b00fb87","added_by":"auto","created_at":"2024-12-04 13:02:20","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":44602,"visible":true,"origin":"","legend":"\u003cp\u003eRelative mRNA expression of (a) Caspase-3 and (b) Caspase-8 in untreated control and treated MCF-7 cell line. Results are stated as mean ± SD (n=3).\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-5197419/v1/81141586e93e0f73d1e717fe.png"},{"id":70565868,"identity":"7bb442d2-a3bd-4872-92af-8e5065b2e7e9","added_by":"auto","created_at":"2024-12-04 12:46:14","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":134132,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of AgNPs from \u003cem\u003eA. nilgiricum\u003c/em\u003e leaf extract on the mRNAs expression of Caspase-3 and Caspase-8 in MCF-7 cells. a. 2% agarose gel electrophoresis of the RT-PCR products of β-actin. Lane 1: DNA ladder (100 bp), lane 2: An untreated control cell, lane 3 and 4 represents cells treated with AgNPs at 250 µg/ml and 350 µg/ml respectively. b. RT-PCR products of Caspase-3 and Caspase-8 in MCF-7 cells. Lane 1: 100 bp DNA ladder, lane 2: control cells (caspase-3), lane 3 and 4 represents cells added with AgNPs at 250 µg/ml and 350 µg/ml respectively. Lane 5: control cells (Caspase-8), lane 6 and 7 represents cells added with AgNPs at 250 µg/ml and 350 µg/ml respectively.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-5197419/v1/636ff38f2fc4596ed08bb548.png"},{"id":70565863,"identity":"724a1ef8-c2e1-45c0-b84e-ecf1524f3867","added_by":"auto","created_at":"2024-12-04 12:46:14","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":86004,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of AgNPs on DNA fragmentation in MCF-7 and HepG2 cells lines. Cells treated with AgNPs at 250μg/ml and 350μg/ml. Where: Lane 1- MCF-7 untreated control, Lane 2-MCF7 250 μg/ml, Lane 3- MCF-7 350 μg/ml, Lane 4-HepG2 untreated control, Lane 5- HepG2 250 μg/ml and Lane 6-HepG2 350 μg/ml.\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-5197419/v1/1674de225632a3898eb78920.png"},{"id":71552574,"identity":"0173a002-002d-4064-bd86-a3c57aae8f2c","added_by":"auto","created_at":"2024-12-16 16:07:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4010600,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5197419/v1/b1e4748a-19a3-4787-b7ae-9f00a7fc8ff1.pdf"},{"id":70566801,"identity":"9008f658-5a0f-4acd-a9cb-48ee709b8bd5","added_by":"auto","created_at":"2024-12-04 12:54:14","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":1897170,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-5197419/v1/5df71351635baf5844b8206a.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Green Synthesis of silver nanoparticles using Amomum nilgiricum leaf extracts: Preparation, physicochemical characterization and ameliorative effect against human cancer cell lines","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eGlobally, people face various healthcare challenges and the devastating pandemic outbreaks affecting the lives and socioeconomic status of people. Cancer still remains a major disease affecting people and a leading cause of death among the developed and developing countries. With the ever-increasing cancer cases worldwide, the occurrence of new cases is expected to be 23.6\u0026nbsp;million reports of cancer per year by 2030 (Bray et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The existing treatments for cancer include the application of chemotherapeutics, radiotherapy or surgery which support the patient\u0026rsquo;s health and survival but have adverse side effects, toxic effects on non-target tissues, drug resistance as well as lead to reappearance of cancer affecting the life of the individual and the family (Choudhari et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Although intensive efforts are in progress, but cancer still persists as an aggressive killer worldwide. Presently, the various synthetic chemotherapeutic agents employed for treatment of cancer have not been effective in treatment in spite of the considerable cost of their development. Therefore there is a continuous demand to develop novel, effective, and reasonable anticancer drugs. The burden exerted by the disease demands the search for novel and efficient drugs against cancer from natural sources (Cabral et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Plants have been used conventionally in the treatment of several diseases as they are sources for potent bioactive metabolites of pharmaceutical significance (Elrayess and El-Hak \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDespite advances in development of synthetic drugs, plant based drugs have played a central part in the presence of potent main molecules. Among the FDA approved anti-infectious and anticancer drugs, natural origin of drugs have a share of 60 to 75% from natural products or their derivatives (Sahoo et al. \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).World Health Organization (WHO) reported, the primary health care still 80% of the population in some countries depend upon traditional medicines (Khazir et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Herbal remedies are the best popular form of traditional medicine, and are extremely productive in the worldwide market. National Cancer Institute has selected about 114,000 plant extracts from 35,000 samples of plant identified from 20 nations for their anticancer potential (Kaur et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). The discovery of novel anti-cancer biomolecules from higher plants by phytochemical research based on ethnopharmacological information is normally considered as a valuable approach (Mani et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe family \u003cem\u003eZingiberaceae\u003c/em\u003e encompasses rhizomatous herbs that are rich sources of valuable products used as food, spices, flavouring agents, traditional medicine, dyes, aromatic products and so on (Tan et al. \u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). This family comprises of around 52 genera and over 1300 species of aromatic plants many of them with therapeutic and ethanomedicinal value (Kress et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Members of this family including turmeric, ginger and several others have proven to be potential as antioxidant, antimicrobial, analgesic, antiobesity, anti-angiogenic, proapoptotic, anti-inflammatory, immunomodulatory, antitumor, agents (Chumroenphat et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Konappa et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Intensive botanical assessment of the forests of Western Ghats of South India led to the detection of a wild ginger \u003cem\u003eA. nilgiricum\u003c/em\u003e, an interesting species of the family \u003cem\u003eZingiberaceae\u003c/em\u003e (Thomas et al. \u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn current years, the interest in the synthesis and properties of metal nanoparticles (NPs) like copper, silver, palladium, zinc, gold, nickel, titanium, aluminum, chromium, iron, and cobalt, platinum. The silver (Ag) has been attractive attention in nanomedicine because of their catalytic, optical and physical properties and has been used in varied arenas such as electronics and therapeutics (Lee and Jun \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The nanotechnology includes by synthesis of NPs size distending from 1\u0026ndash;100 nm. The NPs are broadly used for disease control, medical drive, and environment protection (Ebrahimzadeh et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe related with chemical and physical production, green production ensure several benefits because it needs fewer chemicals, barren of long procedure and necessity of massive energy and fewer contaminant that evade lavish refinements (Awwad et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Natural based techniques by plant aqueous or extracts of microbes are chosen (Rafique et al. \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Medicinal plant based production of NPs has several biotic advantages then NPs have nontoxic substances and bioactive molecules play a significant role in stabilizing, and capping materials (Shah et al. \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The proteins, amino acids, phenolics, alkaloids, flavones terpenoids, and polysaccharides are existence in extracts of plant can act as capping and reducing constituents (Naikoo et al. \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The production of AgNPs by easy, less toxicity, cost effective, defensible, compatibility and lengthier production period with adequate particle size dissemination (Hema et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The AgNPs have been widely useful in different arenas such as drug delivery, fabric, farming, parasitology, catalysis, food, biomedical, water management, cosmetics, etc. (Divya et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Plant centered produced silver NPs are having great antagonistic action against microbes and NPs are extensively used as an constituent in the medicinal production for ready of human healthiness care drugs mainly revealed favorable outcomes for anti-inflammatory, wound healing and anticancer activities (Rashid et al. \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The AgNPs can decrease the liberal growth of cancer cells through delaying several signaling cascades accountable for the growth and tumors pathogenesis. Numerous investigation results shown that AgNPs can kill human cancer cells with very little harm to normal cells (Sayed et al. \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe silver NPs are interrelating with cancer cells and control passive and active cellular reactions also chromosomal abnormalities and damage of DNA at lesser dosage deprived of noxiousness, particularly not at all genotoxicity action on cells of human (Gurunathan et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Meanwhile the addition of silver NPs as a drug transferor in the treatment of cancer has presently increased significant consideration (Brahmbhatt et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). According to this, the present research was designed to synthesis of AgNPs from \u003cem\u003eA. nilgiricum\u003c/em\u003e leaf extracts and activity on different cancer cell lines. The physic-chemical characteristics of the freshly produced AgNPs were studied by several methods such as UV\u0026ndash;vis, DLS, XRD, FT-IR, SEM with EDAX and TEM studies. Further the bioactivity was evaluated by measuring cell viability, assessment of the DNA damage and genotoxicity, apoptosis induction in cancerous cells, cell cycle arrest and the expression levels of apoptosis connected genes in treated with AgNPs. Further apoptotic potential of AgNPs was proven by staining and cell cycle analysis. Therefore, plant based production of AgNPs creates a substitute to control the cancer to evade difficulties worried with conventional chemical treatment. To the greatest of our data, this research is the novel report of anticancer activity of synthesized AgNPs from \u003cem\u003eA. nilgiricum\u003c/em\u003e leaf extracts.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell Lines\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eHuman hepatocarcinoma cells (HEPG2), rat cardio myoblast cells (H9C2), human breast cancer cells (MCF-7), human embryonic cells (HEK293) and Lung cancer cell lines (H1975) were obtained from the national center for cell science (Pune, India). All obtained cells lines were cultured and maintained from DMEM media added with heat inactivated fetal bovine serum (FBS) (10%) and streptomycin/ penicillin (50 U/ml). A humidified incubator holding 95% air and 5% CO2 was used to grow the cells at 37\u0026deg;C with media replenishment every two days.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCollection of plant and preparation of leaf extracts\u003c/h3\u003e\n\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eHealthy leaves of \u003cem\u003eA. nilgiricum\u003c/em\u003e was collected from Palakkad district, Kerala, India, at 11\u0026deg;03'15.46\" N, 076\u0026deg;32'23.58\" E and at an elevation of 1150 m above the sea level. The collected leaves were washed with tap water to eliminate the external contaminants and soil on leaves, followed by sterilized water; shade dried and prepared to fine powder. About 50g of air dried leaf powder of \u003cem\u003eA. nilgiricum\u003c/em\u003e was extracted in100ml distilled water for 72 h. The leaf extract was filtered through Whatman no.1 filter paper and leaf filtrate was used for production of silver nanoparticles.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003eBiosynthesis of silver nanoparticles\u003c/h3\u003e\n\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eFor the AgNPs biosynthesis, 10 ml of leaf filtrate \u003cem\u003eA. nilgiricum\u003c/em\u003e was transferred into 90 ml of AgNO\u003csub\u003e3\u003c/sub\u003e (10 mM) solution and the reaction mixture was kept overnight at 25\u0026deg;C with 100 rpm allowed them to mix appropriately. The AgNO\u003csub\u003e3\u003c/sub\u003e was used as a control. After incubation, observed for the reduction of AgNO\u003csub\u003e3\u003c/sub\u003e to silver ions (Ag\u003csup\u003e+\u003c/sup\u003e into Ag\u003csup\u003e0\u003c/sup\u003e nanoparticle) was confirmed by the light colored change from yellowish color to dark brown color (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The reaction mixture was used to centrifuge at 15,000 rpm for 25 min, the supernatant solution was removed and the residual solid was washed 5\u0026ndash;6 times with distilled water. The produced AgNPs was studied by UV\u0026ndash;vis spectroscopy. The obtained AgNPs was dehydrated at 60\u0026deg;C for overnight and stored until for characterization analysis. The dried AgNPs were further characterized using FTIR, XRD, DLS, SEM, EDS and TEM studies (Karuppiah et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Konappa et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eCharacterization of synthesized silver nanoparticles\u003c/h3\u003e\n\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eDevelopment of silver NPs was observed by UV-vis spectroscopic analysis. The absorbance of AgNPs attained at maximum was detected by spectral scan at range of 200\u0026ndash;800 nm by using UV\u0026ndash;vis spectrophotometer (Hitachi, U-2800).The fourier transform infrared spectroscopy (FTIR) analysis was studied by Perkin Elmer Spectrum 1000 with the spectral range of 4000\u0026thinsp;\u0026minus;\u0026thinsp;400 cm\u003csup\u003e-1\u003c/sup\u003e at resolution at 4 cm\u003csup\u003e-1\u003c/sup\u003e to determine the potential functional groups in bioactive compounds existence in the leaf extract. Biomolecules are accountable for the reduction of ions and capping agents accountable for the strength of NPs. The study of DLS was done to detect the dispersal pattern, size of produced AgNPs and understand the size distribution pattern of very small NPs existing in solution (Microtrac /FLEX 11.0.0.2).\u003c/p\u003e \u003cp\u003eThe patterns of XRD with AgNPs was identified with Cu Kα radiation by using X-ray powder diffractometer (Rigaku Desktop Miniflex II) (λ\u0026thinsp;=\u0026thinsp;1.5406 Ao) is the source of energy. The particle size and nature of the AgNPs was analyzed by using XRD. The diffracted intensities were noted at 2θ angles from 20\u0026ndash;80\u0026ordm;. The place of the maximum peak was related with standard libraries to identify phases of crystalline. The size of the particles of the AgNPs was determined by using Debye Sherrer's formula.\u003c/p\u003e \u003cp\u003eD\u0026thinsp;=\u0026thinsp;Kλ/βcosθ\u003c/p\u003e \u003cp\u003eWhere D is the size of the particle (nm), β is the full line width at half maximum elevation of the main peak, λ is the X-ray wavelength, K is the shape and θ is the refractive angle. The scanning electron microscopy (SEM) analysis was studied by a tiny film of AgNPs prepared, dropped on the carbon coated copper grid film and dried with mercury lamp for 5 min. The morphological structures of the produced AgNPs were identified (Hitachi, S-3400N, Japan). The EDS assay was conducted using about 0.2g of AgNPs crystals to identify the existence of Ag ions in the solution (Hitachi Noran System 7, USA). Transmission electron microscopy (TEM) with selective area electron diffraction (SAED) was assessed to describe the size of the synthesized AgNPs (Hitachi H7500) (Konappa et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003eanticancer activity of AgNPs by MTT assay\u003c/b\u003e\u003c/p\u003e \u003cp\u003eFor anticancer activity, MCF-7, HEPG2, H9C2, HEK293 and H1975 cells were inoculated in 96-well plate at 1x10\u003csup\u003e4\u003c/sup\u003e cells/well with DMEM complete media and kept for 24 h incubation. After the cells were totally attached to the wells and the media was removed. The cancer cells were added with AgNPs with different concentrations such as 50, 100, 150, 200, 250, 300 and 350\u0026micro;g/ml and incubated for 48 h. Later, 200\u0026micro;l of freshly prepared MTT (3-(4, 5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reagent (5 mg/ml of phosphate buffer solution) was incorporated and incubated at 37 \u0026ordm;C for 4\u0026ndash;6 h. Afterward incubation, the MTT reagent was removed and dimethyl sulfoxide (DMSO) was mixed to dissolve the formazan crystals produced with live cells and measured absorbance at 570 nm by using Monochromator Microplate Reader (Mode Tecan 1650). IC50 value was calculated by linear regression equation. The results were calculated in percentage of reduction of MTT compared to the control cells absorbance. The tests were repeated three and plot graphs (Mosmann \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e1983\u003c/span\u003e; Hartman 2003).\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003eanticancer activity of AgNPs by Sulforhodamine B (SRB) assay\u003c/b\u003e\u003c/p\u003e \u003cp\u003eCytotoxic effect of silver NPs was studied by colorimetric Sulforhodamine B (SRB) assay (Voigt \u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). The cancer cells were inoculated in 96 wells plate at 1x10\u003csup\u003e4\u003c/sup\u003e cells/well and incubation for 24 h. Later the cancer cells were totally bounded to the wells, the media was detached. The cells were added with numerous concentrations ranging from 50 to 350 \u0026micro;g/ml of silver NPs and kept for 48 h. Cell fixation were prepared using 100\u0026micro;l/well with 10% tricholoroacetic acid (TCA) for 1 h at 4\u0026deg;C. The culture plates were washed and dried for 1h at room temperature. Further the cells were stained with SRB (0.02%) with 1% acetic acid and incubated at room temperature for 1h. Then the culture plates were washed thrice with 1% of 200 \u0026micro;l/ well acetic acid and dried. The 10 mM Tris-HCl (pH 10.5) (200 \u0026micro;l) was added to every well to remove SRB after placing in an orbital shaker for 1h. Microplate reader (Thermo Scientific) was used to determine the absorbance at 510 nm.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003eDual acridine orange/ethidium bromide fluorescent staining\u003c/h3\u003e\n\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe cancerous cells were subjected to AgNPs treatment at several concentrations from 50 to 350\u0026micro;g/ml and stained with mixture of acridine orange (AO) and ethidium bromide (EB) dye (1:1) (Yashaswee and Trigun 2020). Detection of fragmentation of nuclei by using fluorescence microscope (Carl Zeiss, Germany) study. The AO intercalates between the double standard DNA and emits green fluorescence. The AO penetrates both live and dead cancerous cells. Instead, EB permeates only the dead cells and emits the red fluorescence indicating membrane damage. The images were collected using digital camera (Olympus).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eSingle cell gel electrophoresis assay\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe single cell gel electrophoresis technique was studied to evaluate genotoxicity and DNA destruction induced by AgNPs (Alkaline Comet Assay) (Singh et al. \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e1988\u003c/span\u003e). The experiments were conducted in dark condition. The MCF-7 cells at 1\u0026times;10\u003csup\u003e5\u003c/sup\u003e were seeded into a six well plate and kept overnight for cells attachment. Further the cells were added with many concentrations (250 \u0026micro;g/ml and 350 \u0026micro;g/ml) of silver NPs and kept for 24 h and (0.1%) DMSO was used as solvent control. The negative control (untreated cells grown in media) and positive control (cells treated with 20 mM hydrogen peroxide) were used. After incubation, the cells were trypsinized (cell dissociation using trypsin), centrifuged and the cell suspension was rinsed with sterilized Phosphate buffered saline (PBS). Further, 75 \u0026micro;l of pre-heated low melting agarose (1%) was added to the treated and control cell suspension and poured onto the super frost glass slide pre-coated with 1% normal melting agarose and covered by cover slip. Afterward solidification of the agarose at 0\u0026deg;C for 5 min and removed the cover slip. The cancer cells were kept in tank filled with cold lysis buffer at 4\u0026deg;C (300 mM NaOH, 2% DMSO and NaCl at 1.2M and 1% of Triton X-100). Then the slides were shifted to the electrophoretic tank filled with alkaline buffer for 30 min to allow the DNA unwinding. Later the electrophoresis, the slides were gently washed with neutralization buffer twice for 5 min. The cells were fixed onto the slides by submerging slides in 70% ethanol for 20 min and stained with 1X ethidium bromide. The DNA damage was analyzed using a fluorescence microscope. The amount of DNA destruction was calculated by evaluating the DNA movement distance and the transferred DNA percentage using Image J software with Open Comet. Randomly selected 50 to 100 cells were examined by the software.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eDNA fragmentation assay\u003c/h3\u003e\n\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe two MCF-7 and HEPG-2 cells at 1x10\u003csup\u003e6\u003c/sup\u003e cells/ml were inoculated in 35 mm petridish containing complete DMEM media and kept for 24h and further added with various concentrations of AgNPs (250\u0026micro;g/ml and 350\u0026micro;g/ml) for 24 h. The genomic DNA of MCF-7 and HEPG2 were isolated by lysis buffer, imperiled to 1.5% of agarose gel electrophoresis. Finally the fragmented DNA bands were visualized using ethidium bromide staining under UV- transilluminator along with DNA ladder (1kb) as control (Kumar et al. 2017).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003eCell cycle analysis\u003c/h3\u003e\n\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eFor determining the cell cycle halt properties of AgNPs, the cancer cell lines were inoculated into six well plates at 1x10\u003csup\u003e6\u003c/sup\u003e cells/well and kept for 24 h at 37\u0026deg;C. Then the cell lines were added with AgNPs for 48 h at 250\u0026micro;g/ml and 350\u0026micro;g/ml. The cancer cell lines were collected, washed two times with ice cold phosphate buffer saline and fixed by using 70% of chilled ethanol at 4\u0026deg;C for 4 h. Then the cell lines were suspended with 0.5ml propidium iodide (PI) containing with 50 \u0026micro;g/ml PI, 50\u0026micro;g/ml RNase, 0.2 mM EDTA and Triton X-100 (0.1%). The cell samples were kept for 30 min in dark. Further cell cycle evaluation was carried out by fluorescence-activated cell sorting (FACS) apparatus (Becton Dickinson Heidelberg, Germany).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eRNA isolation, cDNA synthesis and RT-PCR method\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe MCF-7 cells at 5x10\u003csup\u003e5\u003c/sup\u003ecells/well were inoculated into 6-well plate and subjected to AgNPs treatment of two different concentrations (250\u0026micro;g/ml and 350\u0026micro;g/ml) for 48h. From the treated and untreated cell samples the total RNA was isolated by Trizol method. Nanodrop spectrophotometer (Thermo Scientific, USA) was used to evaluate the concentration and quality of RNA. The Verso cDNA synthesis kit was used for the formation of cDNA (Thermo Fisher Scientific). The PCR mixture include 10 \u0026micro;l of Red Taq Master Mix, 1 \u0026micro;l of cDNA, nuclease free water and 1\u0026micro;l of each complementary primer specific for β-actin sequence, Caspase 8 and Caspase 3. The mixture was run for 30 amplification cycles. The positive control was used as normalization β-actin. The study of PCR yields were studied by electrophoresis using 1% of agarose gel and 1X TAE buffer. Then mRNA expression was enumerated by using image study software.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eCharacterization of synthesized AgNPs\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe SPR-surface plasmon resonance of AgNPs made a peak centered nearby 427nm. The AgNPs absorbance was occupied primarily the color of sample was yellowish and afterward the color of sample was turned to the dark brown color (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). The visible region of the peak shows the reduction of the Ag\u0026thinsp;+\u0026thinsp;and the production of AgNPs. The FTIR spectrum was used to identify the purity and existence of functional groups in the produced AgNPs. The existing main functional groups from biomolecules from leaf extract acts as reducing agents of Ag\u0026thinsp;+\u0026thinsp;to Ag\u003csup\u003eo\u003c/sup\u003e. The FTIR spectrum attained between the wavelength ranges of 400\u0026ndash;4000 cm\u003csup\u003e-1\u003c/sup\u003e, shows major peaks were observed including peaks range at 3462, 2971, 1637, 1584, 1381, 1264, 1093 and 805 cm\u003csup\u003e-1\u003c/sup\u003e. A strong peak at 3462 cm\u003csup\u003e-1\u003c/sup\u003e shows the N-H stretching of NH\u003csub\u003e2\u003c/sub\u003e group; moreover specify the O-H bond existing in polyphenol protein, phenol and polysaccharides. The peak at 2971 cm\u003csup\u003e-1\u003c/sup\u003e characterizes the aliphatic C-H stretching, 1637cm\u003csup\u003e-1\u003c/sup\u003e represents to (NH)\u0026thinsp;=\u0026thinsp;O bond, 1584 cm\u003csup\u003e-1\u003c/sup\u003e represents the existence of C\u0026thinsp;=\u0026thinsp;C stretch as alkenes, 1381 cm\u003csup\u003e-1\u003c/sup\u003e designates C\u0026thinsp;=\u0026thinsp;O stretch and 1093 cm\u003csup\u003e-1\u003c/sup\u003e for C-O-C bond stretching of terpenoids and flavonoids. The band at 805 cm\u003csup\u003e-1\u003c/sup\u003e and 1264 cm\u003csup\u003e-1\u003c/sup\u003e endorses the existence of alkyl halides (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). The synthesized Ag NPs was confirmed as crystalline form by XRD analysis. The 3 main peaks were identified in the XRD analysis and displays distinct peaks at 2θ\u0026thinsp;=\u0026thinsp;38.23\u0026deg;, 44.42\u0026deg;, 64.44\u0026deg; and 77.39\u0026deg;, which related to (111), (220), (200) and (311) respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). The diffraction data were compared with standard AgNPs published in joint committee on powder diffraction standards (JCPDS), silver file No. 04-0783. The normal diameter of the produced AgNPs is between 27 and 30 nm. The DLS analysis of AgNPs was identified the size of the particle distribution in the solution. The average diameter of silver NPs was found 21.49 nm in diameter and width was about 12.01nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe produced AgNPs size was detected by using SEM analysis noted as 87 nm and it shows the AgNPs morphology, as extended, collected and irregular and some nanoparticles were rounded (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee). This outcomes were strongly confirms that \u003cem\u003eA. nilgiricum\u003c/em\u003e leaf extract might main role in reducing and capping agents in the synthesis of silver NPs. Energy Dispersive Spectroscopy (EDS) analysis was analyzed to identify the elements in the biosynthesized AgNPs. The EDS result displays that the produced AgNPs comprises the 81.43% followed by carbon etc. (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef). The metallic AgNPs usually shows a distinctive signal at 3 KeV regions showing the surface plasmon resonance. The HRTEM image confirmed the NPs were spherical shape (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg). The diameter of the silver NPs was average between 27 and 30 nm. Slight percentage of silver NPs were partly collected, but was even in size and non-aggregated form. The interplanar spacing which was observed and refers to the face centered cubic crystalline structure and crystalline form of the silver NPs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eIn vitro anticancer activity of synthesized AgNPs\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe \u003cem\u003ein vitro\u003c/em\u003e anticancer activity of synthesized AgNPs from \u003cem\u003eA. nilgiricum\u003c/em\u003e leaf extract was determined by using MTT and SRB assays. Five cell lines were added with numerous concentrations of AgNPs varies from 50\u0026ndash;350\u0026micro;g ml\u003csup\u003e-1\u003c/sup\u003e for 48 h. From MTT assay, the results were done in dose dependent inhibition of cell growth by synthesized AgNPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Among the concentrations of AgNPs, 350\u0026micro;g ml\u003csup\u003e-1\u003c/sup\u003e showed maximum cytotoxic effect followed by 300 250, 200, 150, 100 and 50 \u0026micro;g ml\u003csup\u003e-1\u003c/sup\u003e and 350 \u0026micro;g ml\u003csup\u003e-1\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAt 350 \u0026micro;g ml\u003csup\u003e-1\u003c/sup\u003e of synthesized AgNPs was showed maximum percentage of cytotoxicity activity against all cell lines such as MCF-7, HEPG-2, H9C2, HEK293 and H1975 up to 89.58\u0026thinsp;\u0026plusmn;\u0026thinsp;1.86%, 86.45\u0026thinsp;\u0026plusmn;\u0026thinsp;1.73%, 83.53\u0026thinsp;\u0026plusmn;\u0026thinsp;2.35%, 79.15\u0026thinsp;\u0026plusmn;\u0026thinsp;2.31% and 87.12\u0026thinsp;\u0026plusmn;\u0026thinsp;2.42% respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). At 250 \u0026micro;g ml\u003csup\u003e-1\u003c/sup\u003e of AgNPs was showed percentage of cytotoxicity activity against cell lines such as MCF-7, HEPG-2, H9C2, HEK293 and H1975 up to 67.66\u0026thinsp;\u0026plusmn;\u0026thinsp;1.96%, 65.73\u0026thinsp;\u0026plusmn;\u0026thinsp;1.61%, 58.54\u0026thinsp;\u0026plusmn;\u0026thinsp;2.11%, 56.88\u0026thinsp;\u0026plusmn;\u0026thinsp;1.93% and 59.54\u0026thinsp;\u0026plusmn;\u0026thinsp;2.16% respectively. While 50 \u0026micro;g ml\u003csup\u003e-1\u003c/sup\u003e of AgNPs was showed percentage of cytotoxicity activity against cell lines such as MCF-7, HEPG-2, H9C2, HEK293 and H1975 up to 39.65\u0026thinsp;\u0026plusmn;\u0026thinsp;1.23%, 36.32\u0026thinsp;\u0026plusmn;\u0026thinsp;1.36%, 32.76\u0026thinsp;\u0026plusmn;\u0026thinsp;1.46%, 32.77\u0026thinsp;\u0026plusmn;\u0026thinsp;1.63% and 38.23\u0026thinsp;\u0026plusmn;\u0026thinsp;2.12% respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The IC50 value of AgNPs was found at 40\u0026micro;g ml\u003csup\u003e-1\u003c/sup\u003e for MCF-7, 68\u0026micro;g ml\u003csup\u003e-1\u003c/sup\u003e for HEPG-2, 105\u0026micro;g ml\u003csup\u003e-1\u003c/sup\u003e for H9C2, for HEK293 95\u0026micro;g ml\u003csup\u003e-1\u003c/sup\u003e and 62\u0026micro;g ml\u003csup\u003e-1\u003c/sup\u003e for H1975 cells line.\u003c/p\u003e \u003cp\u003eSimilar results were observed in SRB assay, at 350 \u0026micro;g ml\u003csup\u003e-1\u003c/sup\u003e of AgNPs from \u003cem\u003eA. nilgiricum\u003c/em\u003e leaf extract was showed maximum percentage of cytotoxicity activity against all cell lines such as MCF-7, HEPG-2, H9C2, HEK293 and H1975 cells up to 82.69\u0026thinsp;\u0026plusmn;\u0026thinsp;1.83%, 75.92\u0026thinsp;\u0026plusmn;\u0026thinsp;2.36%, 69.62\u0026thinsp;\u0026plusmn;\u0026thinsp;2.11%, 60.91\u0026thinsp;\u0026plusmn;\u0026thinsp;2.23% and 74.59\u0026thinsp;\u0026plusmn;\u0026thinsp;3.16 respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). At 250 \u0026micro;g ml\u003csup\u003e-1\u003c/sup\u003e of AgNPs was showed percentage of cytotoxicity activity against cell lines such as MCF-7, HEPG-2, H9C2, HEK293 and H1975 of 64.54\u0026thinsp;\u0026plusmn;\u0026thinsp;1.93%, 65.69\u0026thinsp;\u0026plusmn;\u0026thinsp;2.25%, 53.89\u0026thinsp;\u0026plusmn;\u0026thinsp;2.13%, 46.75\u0026thinsp;\u0026plusmn;\u0026thinsp;2.89% and 61.45\u0026thinsp;\u0026plusmn;\u0026thinsp;2.23% cells respectively. While 50 \u0026micro;g ml\u003csup\u003e-1\u003c/sup\u003e of AgNPs was showed percentage of cytotoxicity activity against cell lines such as MCF-7, HEPG-2, H9C2, HEK293 and H1975 of 36.85\u0026thinsp;\u0026plusmn;\u0026thinsp;1.76%, 37.57\u0026thinsp;\u0026plusmn;\u0026thinsp;1.63%, 33.65\u0026thinsp;\u0026plusmn;\u0026thinsp;1.43%, 32.97\u0026thinsp;\u0026plusmn;\u0026thinsp;1.76% and 35.54\u0026thinsp;\u0026plusmn;\u0026thinsp;2.12% cells respectively. The IC50 value of AgNPs was found at 42 \u0026micro;g ml\u003csup\u003e-1\u003c/sup\u003e for MCF-7, 85 \u0026micro;g ml\u003csup\u003e-1\u003c/sup\u003e for HEPG-2, 98 \u0026micro;g ml\u003csup\u003e-1\u003c/sup\u003e for H9C2, 110 \u0026micro;g ml\u003csup\u003e-1\u003c/sup\u003e for HEK29 375 and 76 \u0026micro;g ml\u003csup\u003e-1\u003c/sup\u003e for H1975. The AgNPs from \u003cem\u003eA. nilgiricum\u003c/em\u003e leaf extract induced morphological changes of cell lines. The cells were added with AgNPs at 350\u0026micro;g ml\u003csup\u003e-1\u003c/sup\u003e, 250\u0026micro;g ml\u003csup\u003e-1\u003c/sup\u003e and 50\u0026micro;g ml\u003csup\u003e-1\u003c/sup\u003e against MCF-7, HEPG2, H9C2, HEK293 and H1975 cells for 48h. The morphology of cells was intensely changed afterward the addition with AgNPs was observed as related to control cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eApoptotic assay\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eFurther AO/EB dual staining study was assessed using two concentrations of AgNPs (250 \u0026micro;g ml\u003csup\u003e-1\u003c/sup\u003e and 350 \u0026micro;g ml\u003csup\u003e-1\u003c/sup\u003e) were selected based on the \u003cem\u003ein vitro\u003c/em\u003e cytotoxic activity assays. To evaluate apoptosis, AO/EB double staining procedure was used. The cancer cells H1975, HEPG2, MCF-7, HEK293 and H9C2 were added with 2 concentrations (250\u0026micro;g ml\u003csup\u003e-1\u003c/sup\u003e and 350\u0026micro;g ml\u003csup\u003e-1\u003c/sup\u003e) of AgNPs from \u003cem\u003eA. nilgiricum\u003c/em\u003e leaf extract for 24h and stained by AO/EB. The control cells were appeared bright green color with spherical nucleus distributed uniformly in the middle of the cells. While the AgNPs treated cells at 250 \u0026micro;g ml\u003csup\u003e-1\u003c/sup\u003e concentration showed early stage apoptosis, marked by membrane blebbing, granular or crescent-shaped green-yellow AO nuclear staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). But, the cells treated with 350\u0026micro;g ml\u003csup\u003e-1\u003c/sup\u003e of AgNPs showed late apoptotic stage with bright orange patches of condensed chromatin in the nucleus characterized by asymmetrically localized orange nuclear EB staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). This indicates AgNPs induce apoptosis in the cancer cells.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eComet assay if AgNPs from \u003cem\u003eA. nilgiricum\u003c/em\u003e leaf extract\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eAccording to the outcomes of MTT and SRB assays maximum cytotoxicity was observed in, MCF-7 cells showed IC50 value at 40 \u0026micro;g ml\u003csup\u003e-1\u003c/sup\u003e and 42 \u0026micro;g ml\u003csup\u003e-1\u003c/sup\u003e respectively. Hence MCF-7 cells cancer cell line was selected for comet assay. In this assay was performed to study the genotoxicity effect in MCF-7 cells. For this assay, AgNPs from leaf extract of 250\u0026micro;g ml\u003csup\u003e-1\u003c/sup\u003e and 350\u0026micro;g ml\u003csup\u003e-1\u003c/sup\u003e concentrations having cytotoxic effect were used (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The DNA damage was expressed as olive moments, AgNPs showed DNA damage in both the concentrations. When related to control, AgNPs at 350\u0026micro;g ml\u003csup\u003e-1\u003c/sup\u003e showed maximum DNA damage than 250\u0026micro;g ml\u003csup\u003e-1\u003c/sup\u003e. The H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e was used as positive control which exhibited maximum DNA damage (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eCell cycle analysis by flow cytometry method\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe cell cycle of the cancer cells were deliberated using flow cytometry. The cell lines were added with 2 doses of silver NPs at 250 \u0026micro;g ml\u003csup\u003e-1\u003c/sup\u003e and 350 \u0026micro;gml\u003csup\u003e-1\u003c/sup\u003e for 48 h, the cells were removed and static in ice-cold ethanol. After subjected to PI staining for 15\u0026ndash;20 min and examined for cell cycle halt at different phases. The flow cytometry study outcomes shown that the AgNPs treatment on H1975, HEP-G2 and MCF-7 cells at 250 \u0026micro;g mL\u003csup\u003e-1\u003c/sup\u003e has no significant effect at G2M phase arrest when related to control cells, 21.40%, 21.04% and 16.42% respectively. But the higher concentration at 350 \u0026micro;g ml\u003csup\u003e-1\u003c/sup\u003e AgNPs was arrested the cells at SubG0 phase of cell cycle as compared to control cells (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e-S3). The flow cytometry analysis suggests induction of SubG0 phase of cell cycle arrest from AgNPs treatments.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eCaspase-3 and Caspase-8 in MCF-7 cells expression analysis\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eFurther we studied the anticancer activity of silver NPs from \u003cem\u003eA. nilgiricum\u003c/em\u003e leaf extract upon caspases activation, which is a key effector protein of apoptosis. Semi quantitative RT-PCR was used to study the expression of caspase-3 and caspase-8 gene in MCF-7 cells. Overall, the expressions of Caspase-3, Caspase-8 genes were up controlled in AgNPs (250 \u0026micro;g ml\u003csup\u003e-1\u003c/sup\u003e and 350 \u0026micro;g ml\u003csup\u003e-1\u003c/sup\u003e) added cells as related to untreated cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). The up regulation of these apoptotic genes in cells indicates the anticancer activity of AgNPs from \u003cem\u003eA. nilgiricum\u003c/em\u003e leaf extract (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eDNA fragmentation study for detection of apoptosis\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe DNA fragmentation was confirmed by DNA ladder technique to support the initiation of apoptosis by AgNPs from \u003cem\u003eA. nilgiricum\u003c/em\u003e leaf extract against MCF-7 and HEP-G2 cell lines which showed greater cytotoxic response. DNA fragmentation was noticed that the AgNPs (250\u0026micro;gml\u003csup\u003e-1\u003c/sup\u003e and 350\u0026micro;g ml\u003csup\u003e-1\u003c/sup\u003e) treated cells were revealed dose dependent DNA laddering pattern as compare to control cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e). The results suggested that the AgNPs induced DNA ladder formation is a symbol of apoptosis.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eCancer is a major serious health challenge worldwide with huge implications to public health and several exertions were made for exploring and developing innovative treatment strategies. Even though, for cancer treatment several therapies like endocrine therapy, chemotherapy and targeted therapy have been clinically recommended, still numerous patients still suffer from reversion because of the heterogeneity of tumor. However, these therapies exhibit many side effects and the treatment cost is also high. The formation of chemo resistance in tumor cells is another hurdle for the control of cancer by pharmacological approach (Singh et al. \u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The traditional knowledge of medicinal plants offers an alternative method for developing anticancer medicines with enhanced compatibility, cost-effectiveness and lesser toxicity. The metabolites produced by plants have molecular targets for stimulating the apoptosis in various cancer cell lines (Masih et al. 2012).\u003c/p\u003e \u003cp\u003eThe numerous metallic NPs, particularly silver were extensively existence verified for remedial applications in cancer study. We synthesized the AgNPs from \u003cem\u003eA. nilgiricum\u003c/em\u003e leaf extract and evaluated their antitumor activities. Many researchers have described the usage of nanoparticles for controlling the growth of cancer from \u003cem\u003ein vitro\u003c/em\u003e studies. Numbers of investigators have been described about production of AgNPs by using different plant extracts exhibited unique anticancer activity (Kummara et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The \u003cem\u003eAmomum\u003c/em\u003e genus belongs to \u003cem\u003eZingiberaceae\u003c/em\u003e family. Globally, it includes around 150 species and are mainly scattered in tropical regions of Oceania and Asia (Cai et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The seeds, leaves and fruits of Amomum were used in the preparation of traditional medicines. It has been used for controlling various ailments such as gastric disorders, inflammation, digestive disorder, cancer, dental infections and malaria (Dahigaonkar et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). There is no research has been studied on the anti-cancer activity of \u003cem\u003eAmomum nilgiricum\u003c/em\u003e and their nanoparticles on any cancer cell lines.\u003c/p\u003e \u003cp\u003eThe most basic technique is to observe AgNPs synthesis by directly noticing the conversion in the shade of the sample from yellow color to dark brown color. Spectrophotometric method can be endorse the tracking manner and identify NPs peaks in the visible area from UV\u0026ndash;vis spectrum with between 200 and 800 nm wavelength (Balashanmugam et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The produced peak in the visible area specifies the Ag\u003csup\u003e+\u003c/sup\u003e reduction and production of silver NPs from \u003cem\u003eA. nilgiricum\u003c/em\u003e leaf extract. The conversion of color through the production of silver NPs is linked to the excitation outcome of SPR (Balaraman et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Other methods, comprising FTIR, DLS, XRD, SEM, EDAX and TEM, were used to analyze the dispersion, morphology, size, and composition of NPs.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003eIn the present study, the SPR of silver NPs created a peak centered nearby 427nm, because of the transition of electrons. The spectroscopic results of AgNPs are valuable methods for characterizing produced silver NPs (Elamawi et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The similar reports supported that the silver NPs from UV absorption peak at 456 nm wavelength. Mtambo et al. (\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) was reported from the extract of \u003cem\u003eBidens pilosa\u003c/em\u003e was used to synthesis of silver NPs exhibited peak by UV-absorption at 410 nm. The silver NPs production in several plants extracts for example \u003cem\u003eClonorchis sinensis, Ocimum tenuiflorum, Centella asiatica\u003c/em\u003e and detected OD at 420 nm by UV-spectrophotometer (Moodley et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In the present study, absorbance was observed at 440 nm from NPs production by using \u003cem\u003ePlumbago zeylanica\u003c/em\u003e extract and from \u003cem\u003eCatharanthus roseus\u003c/em\u003e extract at 400 nm (Nayak et al. \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eIn the current research, the FTIR spectrum obtained between the wavenumber ranges of 400\u0026ndash;4000 cm\u003csup\u003e-1\u003c/sup\u003e, shows major peaks were observed including peaks at 3462, 2971, 1637, 1584, 1381, 1264, 1093 and 805 cm\u003csup\u003e-1\u003c/sup\u003e. A strong peak at 3462 cm\u003csup\u003e-1\u003c/sup\u003e signifies the N-H bond vibration of NH\u003csub\u003e2\u003c/sub\u003e group, moreover showed the O-H stretch or H-bond existent in protein, polyphenol, polysaccharides and phenol. The peaks denotes the (NH)\u0026thinsp;=\u0026thinsp;O stretching, aliphatic C-H bond vibrations, C\u0026thinsp;=\u0026thinsp;C stretch in alkenes, C\u0026thinsp;=\u0026thinsp;O stretching modes and C-O-C stretching manners of terpenoids and flavonoids. The sharp intense band confirms the presence of alkyl halides and corresponding with the previous reports (Pushparaj et al. \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The FTIR spectroscopy method can be used to identify the functional groups accountable for producing silver NPs. The present major functional groups in the biomolecules from extract acted as reducing agents of Ag\u003csup\u003e+\u003c/sup\u003e to Ag◦ and stabilizing the AgNPs synthesized (Ajaykumar et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The important functional groups for example methyl, alkanes, aliphatic and halides, amides, alcohol formed their existence of silver NPs (Ajaykumar et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Hence, the FTIR analysis is an important and inexpensive method to describe the part of biomolecules from production, stability of produced silver NPs (Soliman et al. \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe \u003cem\u003eA. nilgiricum\u003c/em\u003e leaf extract was described to comprise various biomolecules like, flavonoids, alkaloids, saponins, steroids, phenolics, tannins etc. (Konappa et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).The presence of those peaks, with a minor change in the wavenumber, earlier reports described to the synthesis of AgNPs (Wang and Wei \u003cspan citationid=\"CR94\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The existence of various IR bands connected to presence of several functional groups in \u003cem\u003eA. nilgiricum\u003c/em\u003e leaf extract. A similar outcome was reported by Fafal et al. (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), the \u003cem\u003eAsphodelus aestivus\u003c/em\u003e plant interceded production of silver NPs by FTIR spectrum analysis signified the accessibility of bioactive molecules responsible to reducing and capping. Noorbazargan et al. (\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) confirmed the spectra of FTIR showing leaf extracts and produced silver NPs that comprise certain bioactive molecules.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eThe XRD study was used from several studies to define the synthesized AgNPs was confirmed as crystallinity. This method is valuable for identifying the purity as it can definitely show whether the sample is contains impurities or pure (Alharbi et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The three main peaks were detected from XRD spectrum from AgNPs and displays distinct peaks at 2θ\u0026thinsp;=\u0026thinsp;44.42\u0026deg;, 38.23\u0026deg;, 64.44\u0026deg;, and 77.39\u0026deg;, which match to (220), (111), (200) and (311) respectively. The average diameter of the biosynthesized Ag NPs is between 27 and 30 nm. The study of material using XRD subjected to the diffraction patterns for every sample has a distinctive diffraction beam (Hembram et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Also, the XRD method has been used to estimate the nanoparticle crystallinity, size and check the crystallinity of materials.\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eIn, same outcome was identifying AgNPs from leaf extract of \u003cem\u003ePedalium murex\u003c/em\u003e presented peaks at 64.56\u0026deg;, 38.19\u0026deg;, 44.37\u0026deg; and 77.47\u0026deg; structures to the crystalline plane of 220, 111, 200, and 311 with normal size of 14nm (Anandalakshmi et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Likewise, the XRD spectrum of AgNPs prepared from \u003cem\u003eSargassum myriocystum\u003c/em\u003e plant extract (Balaraman et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Vetrivel et al. (\u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) synthesized crystalline silver NPs by green synthesis from \u003cem\u003eCeropegia bulbosa\u003c/em\u003e Roxb root tuber powder extract and detected XRD distinct peaks from the planes of 111, 220, 200 and 311, these represented to the silver NPs. From earlier study, the silver NPs from \u003cem\u003eCarmona retusa\u003c/em\u003e leaf extract of exhibited four peaks by XRD method (Rajkumar et al. \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In the present study, the size of crystal NPs quantity by Debye Scherrer\u0026rsquo;s formula and size of NPs was 22.6nm. The (111), (200), (220) and (311) diffraction peaks signify face centered cubic silver, whereas the sharpness of those peaks shows the creation of nanosized material (Deivanathan and Prakash, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eThe DLS can be used to detect the size, particle size distribution and surface charge of synthesized AgNPs in the s colloidal suspension. This method is subject to on the interface of the Brownian motion of spherical material with the light pass over a colloidal solution (Bamal et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In the current investigation, the average diameter of AgNPs was showed 21.49nm in diameter and width was about 12.01nm. Vanin dos et al. (2022) reported that the concentration of the plant extract increases, increase the normal size between 70 to 144 nm of silver NPs synthesized from \u003cem\u003eIlex paraguariensis\u003c/em\u003e extract. Likewise, AgNPs produced by plant extract of \u003cem\u003eSalvia miltiorrhiz\u003c/em\u003ea exhibited the size of particle was 128 nm (Zhang et al. \u003cspan citationid=\"CR96\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eIn the current research, the SEM analysis outcome of synthesized AgNPs from leaf extract of \u003cem\u003eA. nilgiricum\u003c/em\u003e was showed morphological structure in extended, collected, asymmetrical and some NPs are circular with size was 87 nm. This outcome strongly confirms that \u003cem\u003eA. nilgiricum\u003c/em\u003e leaf extract act as a reducing and capping agent in the synthesis of Ag NPs. Similarly previous reports were observed AgNPs from \u003cem\u003eAjug abracteosa; Cinnamomum tamala\u003c/em\u003e (Choi et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Ghabban et al (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) reported that the silver NPs synthesized in \u003cem\u003eAstragalus spinosus\u003c/em\u003e was showed circular and size between from 30\u0026ndash;40 nm by SEM analysis. The AgNPs produced in \u003cem\u003eAllium cepa\u003c/em\u003e L. was cubical shape (Abdellatif et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). A study described that silver NPs produced by using extracts of \u003cem\u003eZ. officinale\u003c/em\u003e was circular and size was between 30\u0026ndash;50 nm (Gurunathan et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eFurthermore, silver metal dispersal of biogenic AgNPs was confirmed by EDS (Dimitrijevic et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Present study EDS result shows that the biosynthesized Ag NPs contain 81.43% followed by carbon, etc. The metallic AgNPs usually displays a distinct at 3 KeV regions representing the surface plasmon resonance. The similar type of elemental analysis was made in the production of AgNPs from neem leaf (\u003cem\u003eAzadirachta indica\u003c/em\u003e) extract (Ahmed et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The EDS is a method that describes the metallic conformation of the solution used to check the existence of silver metal from synthesized \u003cem\u003eTaxus wallichiana\u003c/em\u003e AgNPs. The Huong and Nguyen (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) was analyzed the silver NPs from leaves extract of \u003cem\u003eBrassica oleracea\u003c/em\u003e, from EDS spectrum presented the occurrence of silver. The property of AgNPs that were produced by ecologically friendly approaches has been previous reports (Mohammadi et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn the current research, the result of HRTEM method was showed circular in shape of produced silver NPs and average diameter in between 27 to 30 nm. TEM gives better results related with SEM analysis and allows a more in-depth study of NPs (Sreelekha et al. \u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The produced silver NPs have been described and observed by TEM by numerous investigators. The extracts of \u003cem\u003eC. longa\u003c/em\u003e and \u003cem\u003eZ. officinale\u003c/em\u003e were reduction of reducing agents, which clue to varied size of silver NPs (Dubey et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Rather et al (\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) reported that the TEM determines the silver NPs synthesized from the \u003cem\u003eCuphea carthagenensis\u003c/em\u003e leaf extracts, particles were identified as circular and between 4 to 18 nm in size. Extract of \u003cem\u003eRubus ellipticus\u003c/em\u003e and \u003cem\u003eLysiloma acapulunsis\u003c/em\u003e AgNPs, TEM study exhibited the crystalline structure with noticeable lattice fringes particles were circular and size from 13.85 to 34.30 nm (Garibo et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Khanal et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The silver NPs synthesized from \u003cem\u003eC. guianensis\u003c/em\u003e leaf extracts, \u003cem\u003eV. lantana\u003c/em\u003e, and \u003cem\u003eM. capitata\u003c/em\u003e exhibited size between 25\u0026ndash;40 nm, 30\u0026ndash;35 nm and 20\u0026ndash;70 nm, respectively and have mainly spherical shape (Srirangam and Rao, \u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Also Amaliyah et al. (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) used TEM analysis of AgNPs from \u003cem\u003ePiper retrofractum\u003c/em\u003e revealed the NPs were mainly circular and between from 1\u0026ndash;40 nm diameter.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eIn the current study, the cancer cells with numerous concentrations of AgNPs from \u003cem\u003eA. nilgiricum\u003c/em\u003e leaf extract was assessed by MTT and SRB assays for 48h about the anticancer activities on MCF-7, HEPG-2, H9C2, HEK293 and H1975 showing that the AgNPs could be an alternative of conventional drugs against cancer. The AgNPs at 350\u0026micro;g/ml showed maximum inhibition of all cancer cell lines proliferation in comparison with other concentrations ranging from 97.13% \u0026minus;\u0026thinsp;85.87% from MTT assay and 95.58% \u0026minus;\u0026thinsp;93.12% from SRB assay. The IC\u003csub\u003e50\u003c/sub\u003e value of AgNPs was showed 40\u0026micro;g/ml against MCF-7. The outcomes of these assesses shown that the AgNPs concentration increases, decreased viability of the cancer cells. The cancer cells added with silver NPs revealed the reduced metabolic actions, reliant on the form of cancer cells and the size of the NPs (Hussein and Abdullah, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eIn present study, the cytotoxicity activity results was connected to the outcome of Sabah et al. (\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) study, leaf extract of \u003cem\u003eB. oleracea\u003c/em\u003e produced silver NPs showed anticancer activity at IC\u003csub\u003e50\u003c/sub\u003e of 55\u0026micro;g/ml. Singh et al. (\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) described the related effects for silver NPs produced from leaf extract of \u003cem\u003eBorago officinalis\u003c/em\u003e. Similar outcomes were deliberated from \u003cem\u003eMurraya koenigii\u003c/em\u003e leaf extract of AgNPs (Roshni et al. \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Similar results were deliberated the outcomes of experiment by cytotoxicity influence on growth of cell was detected at different concentrations (10\u0026ndash;100\u0026micro;g/ml) of AgNPs in \u003cem\u003eMallus domestica\u003c/em\u003e against MCF-7 cells (Mariadoss et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In dose-dependent manner, the AgNPs from \u003cem\u003eAnanas comosus\u003c/em\u003e were showed greater anticancer action against HepG2 cells (Ahmad and Sharma \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The Dehghanizade et al. (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) studied that, the silver NPs from leaf extract of \u003cem\u003eAnthemisa tropatana\u003c/em\u003e was revealed great anticancer activity to HT-29. The synthesized silver NPs was observed as mutagenic and genotoxic because of existence of alkaloids and flavonoids (Ghramh et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn the current investigation, AgNPs induce apoptosis in H1975, HEPG2, MCF-7, HEK293 and H9C2 cells were measured by dual staining by AO/EB. The control cells appeared bright green in color with spherical nucleus distributed uniformly in the middle of the cells. While the AgNPs added cells showed in early phase apoptotic cells develops membrane blebbing, granular or crescent-shaped greenish yellow and late phase develops orange because of necrosis in AO nuclear staining. The late apoptotic phase with bright orange patches of condensed chromatin in the nucleus characterized by asymmetrically contained orange nuclear EB staining (Roy et al. \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). These results revealed the primary characteristic feature of apoptotic cell death (Singh et al. \u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eIn the current study outcomes of cell cycle study shown that the cancer cells were showed using flow cytometry. This method to regulate the metabolic activity by using AgNPs prevent cell development, the flow cytometry study was used to describe distribution of cell cycle (Chan et al. 2011). The flow cytometer study results shown that this AgNPs treatment on H1975, HEP-G2 and MCF-7 cells at 250 \u0026micro;g/ml has no significant effect at G2M phase arrest when related to control cells, 21.40%, 21.04% and 16.42% respectively. But the higher concentration at 350\u0026micro;g/ml has stopped the cells at SubG0 phase of cell cycle related to control cells. In the current study, the treatment with silver NPs, from the cell cycle was found to be stopped in S-phase and expressively decreases the cell growth. The results of present study agrees with those previously reports (Mishra et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The stop the cell cycle was supposed to be produced normally damage of DNA (Akter et al. 2018). The AgNPs added on A549 cells shows the up regulation of p53 which indicates the stop the cell cycle at G0-G1 stage, halts cell proliferation (Nair et al. 2012). Earlier reports were confirmed that the oxidative stress indications to damage of DNA and abnormalities of chromosomes, and apoptosis of cells adding with silver NPs (Kai et al. 2011).\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eNumerous cancer cells exhibit sub-G1 stop and apoptosis after being exposed to AgNPs. Furthermore, by decreasing tumor cell development and angiogenesis, AgNPs can prevent distant metastasis (Mishra et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In A549 cells, the silver NPs are down regulated the protein kinase C which detect the cell cycle stop at G2/M phase (Jain et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In the present study, results showed in the DNA fragmentation assay, AgNPs (250\u0026micro;g/ml and 350\u0026micro;g/ml) treated cells was shown dosage dependent DNA laddering pattern on compare to control cells. The overall results suggest that the AgNPs induced DNA ladder formation is a hallmark of apoptosis. The fragmentation of DNA was detected in the AgNPs treated cells which were continual by equivalent study. The treatment with silver NPs particularly improved length and creation of number of tail DNA from cell lines (Bin-Jumah et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Genes accountable for regulating the cell cycle, caspase-3, CAT genes, P53, pointedly prevent the cell development and generate apoptosis (Datkhile et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The apoptosis can be induced by death receptors or through the mitochondrial pathway. It is an intrinsic programmed cell death mediated by major downstream initiation of caspase cascade which is generally followed by recruitment of caspase family of proteins including caspase-3 and caspase-8 (Kanamori et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe AgNPs were examined for cytotoxic action on MCF-7 cells and detected to initiate stress on endoplasmic reticulum over unfolded protein response and improves initiation of caspase 9 and 7 producing cell death (Simard et al. \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Rageh et al. (\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) reported that the silver NPs was exerting their anticancer activity by producing DNA destruction from cells. The AgNPs from extract of \u003cem\u003eTaxus brevifolia\u003c/em\u003e exhibited anticancer effect on MCF-7 at 25mM exhibited 75% death rate (Sarli et al. \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Lee et al. (\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) was revealed that the mechanism of anticancer activity comprises the antiapoptotic protein down-regulation, like BCL-2 and upregulation of P53 proteins, caspase 3, ROS (Chen and Wen \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eIn the current research, produced AgNPs from leaf extract of \u003cem\u003eA. nilgiricum\u003c/em\u003e and discovered their application against medically significant to cancer cell lines like MCF-7, H1975, HEPG2, H9C2, HEK293. Different methods were used to assess the morphology of produced silver NPs in \u003cem\u003eA. nilgiricum\u003c/em\u003e leaf extract showing distributions of sizes and shape. In current study, the NPs synthesis technique was a very easy, rapid, simple, clean, reliable and ecologically friendly without any involvement of energy usage steps for producing AgNPs using \u003cem\u003eA. nilgiricum\u003c/em\u003e leaf extract. Further the synthesized AgNPs was induced anticancer effect which is recognized to inhibited the cell cycle development was enumerated by using flow cytometer to determine the mechanism with AgNPs prevent the cancer cell development. Also these AgNPs were induced cell apoptosis and eventually cell death confirmed by AO/EB staining, comet assay and mRNA expression of caspases. Moreover, the DNA damage in cancer cells because of silver NPs was examined in fragmentation of DNA and apoptosis of cancer cells were also observed. These results recommend that AgNPs might have promising anticancer activity and used as therapeutic material for cancer therapy. This biosynthesis of AgNPs could also work as a benefit for the cancer treatment. Therefore, future study is required to completely understand the mechanism and the strength of the AgNPs in terms of stops the growth of cancer cells should be studied under \u003cem\u003ein vivo\u003c/em\u003e. To conclude, the toxicity of synthesized NPs to normal cells permits broad examinations to launch the potential medical uses of AgNPs.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;We are gratefully acknowledging the financial assistance granted by Post-Doctoral Fellowship (No. F. /PDFSS-2014-15-ST-KAR-7487), University Grant Commission (UGC), New Delhi, for carrying out this research. The authors are also thankful to Department of forests and wildlife, Govt. of Kerala, for giving necessary forest permission for sample collection.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u0026nbsp;\u003c/strong\u003eConceptualization, validation, formal analysis, investigation and data curation, N.K., S.K., C.S. and N.S.R. writing original draft preparation, N.K., C.S. and R.K., writing review and editing, N.K, R.P., A.S.K., R.K., and N.S.R. All authors have read, reviewed and agreed to the published version of the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by University Grant Commission (UGC), New Delhi, No. F. /PDFSS-2014-15-ST-KAR-7487.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis study did not require ethics approval.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis is not applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbdellatif AA, Mahmood A, Alsharidah M, Mohammed HA, Alenize SK, Bouazzaoui A, Abdulla MH (2022) Bioactivities of the green synthesized silver nanoparticles reduced using \u003cem\u003eAllium cepa\u003c/em\u003e L aqueous extracts induced apoptosis in colorectal cancer cell lines. 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AMB Express 11:61. https://doi.org/10.1186/s13568-021-01216-6\u003c/li\u003e\n\u003cli\u003ePushparaj K, Balasubramanian B, Kandasamy Y, Arumugam VA, Kaliannan D, Arumugam M, Alodaini HA, Hatamleh AA, Pappuswamy M, Meyyazhagan A (2023) Green synthesis characterization of silver nanoparticles using aqueous leaf extracts of \u003cem\u003eSolanum melongena\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e evaluation of antibacterial pesticidal and anticancer activity in human MDA-MB-231 breast cancer cell lines. J King Saud Uni Sci 35:102663. https://doi.org/10.1016/j.jksus.2023.102663\u003c/li\u003e\n\u003cli\u003eRafique M, Sadaf I, Rafique MS, Tahir MB (2017) A review on green synthesis of silver nanoparticles and their applications. Artif Cells Nanomed Biotechnol\u003cem\u003e \u003c/em\u003e45(7):1272\u0026ndash;1291. https://doi.org/10.1080/21691401.2016.1241792\u003c/li\u003e\n\u003cli\u003eRageh MM, El-Gebaly RH, Afifi MM (2018) Antitumor activity of silver nanoparticles in Ehrlich carcinoma-bearing mice Naunyn-Schmiedeberg\u0026rsquo;s. Archives of Pharmacology 391:1421\u0026ndash;1430. https://doi.org/10.1007/s00210-018-1558-5 \u003c/li\u003e\n\u003cli\u003eRajkumar R, Shivakumar MS, Senthil Nathan S, Selvam K (2018) Pharmacological and larvicidal potential of green synthesized silver nanoparticles using \u003cem\u003eCarmona retusa\u003c/em\u003e (Vahl) Masam leaf extract. J Cluster Sci\u003cem\u003e \u003c/em\u003e29:1243\u0026ndash;1253. https://doi.org/10.1007/s10876-018-1443-x\u003c/li\u003e\n\u003cli\u003eRashid S, Azeem M, Khan SA, Shah MM, Ahmad R (2019) Characterization and synergistic antibacterial potential of green synthesized silver nanoparticles using aqueous root extracts of important medicinal plants of Pakistan. Colloids and Surfaces B Biointerfaces 179:317\u0026ndash;325. https://doi.org/10.1016/j.colsurfb.2019.04.016\u003c/li\u003e\n\u003cli\u003eRather MA, Deori PJ, Gupta K, Daimary N, Deka D, Qureshi A, Dutta TK, Joardar SN, Mandal M (2022) Ecofriendly phytofabrication of silver nanoparticles using aqueous extract of \u003cem\u003eCuphea carthagenensis\u003c/em\u003e and their antioxidant potential and antibacterial activity against clinically important human pathogens. Chemosphere 300:134497. https://doi.org/10.1016/j.chemosphere.2022.134497\u003c/li\u003e\n\u003cli\u003eRoshni K, Younis M, Ilakkiyapavai D, Basavaraju P, Puthamohan VM (2018) Anticancer activity of biosynthesized silver nanoparticles using \u003cem\u003eMurraya koenigii \u003c/em\u003eleaf extract against HT-29 colon cancer cell line. Sci World J Cancer Sci Ther 10:72\u0026ndash;75. doi: 10.4172/1948-5956.1000521\u003c/li\u003e\n\u003cli\u003eRoy A, Bulut O, Some S, Mandal AK, Yilmaz MD (2019) Green synthesis of silver nanoparticles:biomolecule-nanoparticle organizations targeting antimicrobial activity RSC Adv 9: 2673\u0026ndash;2702. doi: 10.1039/c8ra08982e \u003c/li\u003e\n\u003cli\u003eSabah A, Hajera T, Norah SMA, Mir NA, Basmah A, Salma A, Manal ANB, Roua A (2020) Ecofriendly silver nanoparticles synthesis by \u003cem\u003eBrassica oleracea\u003c/em\u003e and its antibacterial anticancer and antioxidant properties Sci Rep 10:18564. https://doi.org/10.1038/s41598-020-74371-8\u003c/li\u003e\n\u003cli\u003eSahoo N, Manchikanti P, Dey S (2010) Herbal drugs: standards and regulation. Fitoterapia 81(6):462-71. https://doi.org/10.1016/j.fitote.2010.02.001\u003c/li\u003e\n\u003cli\u003eSarli S, Kalani MR, Moradi AA (2020) Potent and safer anticancer and antibacterial taxus-based green synthesized silver nanoparticle. Int J Nanomed 15:3791. doi: 10.2147/IJN.S251174\u003c/li\u003e\n\u003cli\u003eSayed R, Sabry D, Hedeab G, Ali H (2019) \u003cem\u003eIn vitro\u003c/em\u003e characterization and evaluation of silver nanoparticles cytotoxicity on human \u0026ldquo;liver and breast\u0026rdquo; cancer cells versus normal melanocytes Egypt J Histol 42:755\u0026ndash;66. https://doi:10.21608/EJH.2019.6981.1058\u003c/li\u003e\n\u003cli\u003eShah A, Haq S, Rehman W, Waseem M, Shoukat S, Rehman MU (2019) Photocatalytic and antibacterial activities of \u003cem\u003epaeonia emodi\u003c/em\u003e mediated silver oxide nanoparticles. Mat Res Express 6(4):045045. https://doi 10.1088/2053-1591/aafd42\u003c/li\u003e\n\u003cli\u003eSimard JC, Durocher I, Girard D (2016) Silver nanoparticles induce irremediable endoplasmic reticulum stress leading to unfolded protein response dependent apoptosis in breast cancer cells Apoptosis 21:1279-1290. https://doi.org/10.1007/s10495-016-1285-7\u003c/li\u003e\n\u003cli\u003eSingh H, Du J, Yi TH (2017) Green and rapid synthesis of silver nanoparticles using \u003cem\u003eBorago officinalis\u003c/em\u003e leaf extract: anticancer and antibacterial activities. Artif Cells Nanomed Biotechnol\u003cem\u003e \u003c/em\u003e45:1310-1316. https://doi.org/10.1080/21691401.2016.1228663\u003c/li\u003e\n\u003cli\u003eSingh NP, McCoy MT, Tice RR, Schneider EL (1988) A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res 1751:184\u0026ndash;191. https://doi.org/10.1016/0014-4827(88)90265-0\u003c/li\u003e\n\u003cli\u003eSingh RK, Ranjan A, Srivastava AK, Singh M, Anil Kumar S, Atri N, Mishra A, Singh AK, Kumar Singh S (2020) Cytotoxic and apoptotic inducing activity of Amoora rohituka leaf extracts in human breast cancer cells J Ayurveda and Integrative Med 11:383-390. https://doi.org/10.1016/j.jaim.2018.12.005\u003c/li\u003e\n\u003cli\u003eSoliman MK, Salem SS, Abu-Elghait M, Azab MS (2023) Biosynthesis of silver and gold nanoparticles and their efficacy towards antibacterial antibiofilm cytotoxicity and antioxidant activities. Appl Biochem Biotechnol 195:1158-1183. https://doi.org/10.1007/s12010-022-04199-7\u003c/li\u003e\n\u003cli\u003eSreelekha E, George B, Shyam A, Sajina N, Mathew BA (2021) Comparative study on the synthesis characterization and antioxidant activity of green and chemically synthesized silver nanoparticles Bio Nano Science 11:489-496. https://doi.org/10.1007/s12668-021-00824-7\u003c/li\u003e\n\u003cli\u003eSrirangam GM, Rao KP (2017) Synthesis and characterization of silver nanoparticles from the leaf extract of \u003cem\u003eMalachra capitata\u003c/em\u003e (l). Ras J Chem 10:46\u0026ndash;53.\u003c/li\u003e\n\u003cli\u003eTan JW, Israf, DA, Tham, CL (2018) Major bioactive compounds in essential oils extracted from the rhizomes of \u003cem\u003eZingiber zerumbet\u003c/em\u003e (L) Smith: A Mini-Review on the anti-allergic and immunomodulatory properties Front Pharmacol 9: 652. https://doi.org/10.3389/fphar.2018.00652\u003c/li\u003e\n\u003cli\u003eThomas VP, Sabu M, Prabhu Kumar KM (2012) \u003cem\u003eAmomum nilgiricum\u003c/em\u003e (\u003cem\u003eZingiberaceae\u003c/em\u003e) a new species from Western Ghats India. Phyto Keys 8:99-104. \u003c/li\u003e\n\u003cli\u003eVanin dos Santos Lima M, Beloni de Melo G, Gracher Teixeira L, Grella Miranda C, Hermes de Ara\u0026uacute;jo PH, Sayer C Hess Gon\u0026ccedil;alves O (2022) Green synthesis of silver nanoparticles using Ilex paraguariensis extracts: antimicrobial activity and acetilcolinesterase modulation in rat brain tissue. Green Chem Lett Rev 15:128-138. https://doi.org/10.1080/17518253.2021.2024896\u003c/li\u003e\n\u003cli\u003eVetrivel C, Balamuralikrishnan B, Durairaj K, Sungkwon P, Velmurugan P, Ragavendran C, Sigamani S, Maruthupandian A (2019) Fabrication and characterization of noble crystalline silver nanoparticles from \u003cem\u003eCeropegia bulbosa\u003c/em\u003e Roxb root tuber extract for antibacterial larvicidal and histopathology applications. Nanosci Nanotechnol Lett 11:11\u0026ndash;21. https://doi.org/10.1166/nnl.2019.2845.\u003c/li\u003e\n\u003cli\u003eVoigt W (2005) Sulforhodamine B assay and Chemosensitivity. Methods Mol Med 110:39\u0026ndash;48.\u003c/li\u003e\n\u003cli\u003eWang Y, Wei S (2021) Green fabrication of bioactive silver nanoparticles using \u003cem\u003eMentha pulegium\u003c/em\u003e extract under alkaline: An enhanced anticancer activity. ACS omega 7:1494-1504. https://doi.org/10.1021/acsomega.1c06267\u003c/li\u003e\n\u003cli\u003eYashaswee S, Surendra Kumar T (2020) Cytotoxicity and induction of apoptosis in melanoma (MDA MB 435S) C ells by Emodin. J Scientific Res Institute of Sci Banaras Hindu University Varanasi India 64:158-166. https://doi:10.37398/JSR.2020.640223\u003c/li\u003e\n\u003cli\u003eZhang X-F, Liu Z-G, Shen W, Gurunathan S (2016) Silver Nanoparticles: synthesis characterization properties applications and therapeutic approaches. Int J Mol Sci 17:15\u0026ndash;34. https://doi.org/10.3390/ijms17091534\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cytotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cyto","sideBox":"Learn more about [Cytotechnology](http://link.springer.com/journal/10616)","snPcode":"10616","submissionUrl":"https://submission.nature.com/new-submission/10616/3","title":"Cytotechnology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Amomum nilgiricum, silver nanoparticles, cancer cell lines, anticancer activity, Apoptosis, Caspases","lastPublishedDoi":"10.21203/rs.3.rs-5197419/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5197419/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe present study to production of silver nanoparticles (AgNPs) by leaf extracts of \u003cem\u003eA. nilgiricum\u003c/em\u003e and to evaluate the activity of anticancer by using AgNPs against cancer cell lines such as MCF-7, HEPG2, H9C2, HEK293 and H1975. The synthesized AgNPs were characterized by using UV\u0026ndash;Vis spectroscopy, EDS, FT-IR, XRD, DLS, SEM and HRTEM with SAED patterns. The surface plasmon resonance (SPR) of AgNPs formed a peak centered at 427 nm by UV\u0026ndash;Vis analysis. FTIR analysis reveals that existence of functional groups subjected to silver ions reduction to metallic silver. Crystalline form of the AgNPs was assessed by XRD analysis, four spectral peaks at 111, 200, 220, and 311 were formed and zeta potential peak was found at 28.5 mV indicating the higher stability. The size average diameter of the AgNPs was between 27\u0026ndash;30 nm by TEM and SEM analysis was reveals the morphology of AgNPs as elongated, irregular and aggregated and some particles are spherical. EDX analysis confirmed the elemental composition of AgNPs with 81.43% Ag. The average diameter of AgNPs was found 21.49 nm in diameter and width was about 12.01nm by DLS analysis. Cytotoxicity of AgNPs was investigated by using MTT, SRB assay and comet assay was performed as a genotoxicity. The results revealed that AgNPs decreased the viability of cancer cells in a concentration dependent pattern (50 to 350\u0026micro;g/ml). The influence of AgNPs on cell cycle stop was studied on H1975, HEP-G2 and MCF-7 cells and found that AgNPs could induce sub G0 cell cycle arrest. The AgNPs was also induced DNA fragmentation confirms the DNA damage in nanoparticles treated cell lines. The anticancer action of nanoparticles was analyzed using proapoptotic and antiapoptotic caspase 8 and caspase 3 mRNA expression levels. Finally the results suggested that AgNPs is an effective anticancer agent which induces apoptosis in H1975, HEP-G2 and MCF-7 cells. Based on our studies, further identification of the major compounds of leaf extracts is acceptable.\u003c/p\u003e","manuscriptTitle":"Green Synthesis of silver nanoparticles using Amomum nilgiricum leaf extracts: Preparation, physicochemical characterization and ameliorative effect against human cancer cell lines","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-04 12:46:09","doi":"10.21203/rs.3.rs-5197419/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-11-21T16:35:38+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-11-21T16:25:51+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"203265450953667739685530160088538619005","date":"2024-11-21T09:22:03+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"291310809951413292291006610312900008184","date":"2024-11-20T14:19:47+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"333265153249166479943028650953133000853","date":"2024-11-20T08:50:09+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"59848422654763413449436620203081276584","date":"2024-11-20T07:36:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"52575661448669709151114591224657945572","date":"2024-11-19T15:40:32+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"280855602566474727999461529417945916802","date":"2024-11-19T07:04:02+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"318603642551664929521550106371175917205","date":"2024-11-17T15:54:30+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"105833576011094944929536966112238132468","date":"2024-11-16T06:16:51+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"124599360499310123326404332375131711112","date":"2024-11-15T15:31:54+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"43426817727910110621621707950552903132","date":"2024-11-15T15:03:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"60007890948251699839492474490635901028","date":"2024-11-15T14:45:15+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-11-15T10:38:45+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"336165230067839884289962972601735629807","date":"2024-11-15T08:58:39+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-11-15T08:45:04+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-10-03T14:48:22+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-10-03T14:47:29+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cytotechnology","date":"2024-10-03T09:39:41+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cytotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cyto","sideBox":"Learn more about [Cytotechnology](http://link.springer.com/journal/10616)","snPcode":"10616","submissionUrl":"https://submission.nature.com/new-submission/10616/3","title":"Cytotechnology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"b60310fb-16f9-4c74-91ef-4988a776948a","owner":[],"postedDate":"December 4th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-12-16T16:05:37+00:00","versionOfRecord":{"articleIdentity":"rs-5197419","link":"https://doi.org/10.1007/s10616-024-00674-7","journal":{"identity":"cytotechnology","isVorOnly":false,"title":"Cytotechnology"},"publishedOn":"2024-12-10 15:57:24","publishedOnDateReadable":"December 10th, 2024"},"versionCreatedAt":"2024-12-04 12:46:09","video":"","vorDoi":"10.1007/s10616-024-00674-7","vorDoiUrl":"https://doi.org/10.1007/s10616-024-00674-7","workflowStages":[]},"version":"v1","identity":"rs-5197419","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5197419","identity":"rs-5197419","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}