Eco-friendly synthesis and biomedical potential of silver nanoparticles using Pandanus dubius extract

Abstract

This study highlights a novel, eco-friendly approach for synthesizing silver nanoparticles (AgNPs) using the Pandanus dubius extract. This study characterized the resulting Pds-AgNPs and evaluated their antioxidant, antibacterial, and anticancer properties. The characterization of Pds-AgNPs used various methods UV-Vis spectroscopy confirmed the absorption peak at 458 nm, while FT-IR analysis identified the functional groups responsible for reduction and stabilization. X-ray diffraction (XRD) analysis provided insights into the crystallographic features of the nanoparticles, and scanning electron microscopy (SEM) and transmission electron microscopy (TEM) analyses revealed their spherical shape. The biomedical potential of Pds-AgNPs was thoroughly investigated. Antioxidant activity, significant free radical scavenging capabilities, with the Pds-AgNPs outperforming the methanol extract. The antibacterial activity of Pds-AgNPs was tested against human pathogens, and the Pds-AgNPs exhibited strong antibacterial efficacy, surpassing the performance of the methanol extract alone. Furthermore, the anticancer properties of Pds-AgNPs were assessed using in vitro cytotoxicity assays on A549 human lung cancer cells. The IC₅₀ values of 40.84 ± 0.092 µg/mL for the Pds-AgNPs and 54.88 ± 0.105 µg/mL for the extract, highlighting the enhanced therapeutic potential of the nanoparticles (NPs). These findings underscore the potential of green-synthesized Pds-AgNPs as multifunctional therapeutic agents with robust antioxidant, antimicrobial, and anticancer properties. This study is the first to report the analysis of phytochemicals and the sustainable, eco-friendly synthesis of Pds-AgNPs using a P. dubius plant-mediated approach. These findings provide a basis for further research on the use of Pds-AgNPs in biomedical applications.

Introduction

Nanotechnology, a rapidly expanding field of interdisciplinary study, is at the forefront of merging nanoscience with the life sciences. This domain focuses on the manipulation and utilization of materials at the nanoscale, where they exhibit distinct physical and chemical properties, offering novel solutions to challenges in health, technology, and environmental science. Central to nanotechnology are NPs, which are broadly categorized into organic and inorganic types¹. Organic NPs, primarily made of carbon-based structures, are celebrated for their compatibility with biological systems and their potential in drug delivery, biosensing, and other bio-interactive applications². Inorganic NPs, encompassing a variety of materials such as magnetic particles, noble metals, and semiconductors, are particularly notable for their exceptional properties, including high surface-area-to-volume ratios, adjustable optical properties, and enhanced catalytic activities. These inorganic metal NPs are indispensable in medical imaging, targeted therapies, environmental clean-up, and energy production^3,4.

Inorganic AgNPs are incredibly versatile, playing a crucial role across various sectors. In medicine, they are vital for their antimicrobial properties, leading to innovations in wound care, drug delivery, and diagnostics. For environmental clean-up, AgNPs are utilized in water purification systems, acting as catalysts to degrade pollutants and remove heavy metals. Furthermore, their application in energy production is emerging, particularly in enhancing the efficiency of solar cells and other renewable energy technologies. These diverse applications highlight the indispensable nature of AgNPs in addressing critical global challenges^5,6. As research advances, the development and functionalization of both organic and inorganic NPs continue to evolve, leading to innovative applications that could transform medicine, materials science, and other fields^2,7.

Nanoparticles can adopt various shapes, such as cylinders, tubes, triangles, and spheres, which are exploited across physical, chemical, and biological disciplines. Their application in drug delivery systems is especially significant, as they can improve the effectiveness and accuracy of treatments^8,9. Recently, 4 d transition metals such as silver, gold, palladium and platinum have garnered significant attention for their catalytic potential, owing to their high surface-to-volume ratios and exclusive physicochemical characteristics. Among these AgNPs stand out due to their remarkable optical and electronic properties. AgNPs have found diverse applications, including photoreactions, sensing, larvicidal activity, seed germination enhancement, electro-chromic devices, paints, dye-sensitized solar cells, air purification systems, cancer therapy, and antimicrobial treatments¹⁰.

The green synthesis of AgNPs has transformed their production by avoiding toxic chemicals and energy-intensive methods. This shift focuses on environmentally friendly, cost-effective, and sustainable practices that utilize natural resources. Plant-based synthesis is the most explored technique, using various plant parts, such as leaves, stems, roots, flowers, fruits, and even waste materials like citrus peels. Plants contain phytochemicals (polyphenols, flavonoids and terpenoids) that serve as reducing agents to convert silver ions into nanoparticles and as capping agents to stabilize the AgNPs, preventing aggregation^11,12. AgNPs have strong applications in various fields. In targeted therapies, AgNPs are engineered to specifically reach diseased cells, minimizing harm to healthy tissue. Once inside, they can directly kill cells by generating reactive oxygen species or act as nanocarriers, delivering chemotherapy drugs directly to tumor’s, boosting efficacy and cutting side effects. Beyond cancer, AgNPs are also used for antimicrobial drug delivery, using their natural antimicrobial traits, sometimes with antibiotics, to fight drug-resistant infections by targeting bacteria^13,14. AgNPs are integrated into filters and coatings because of their powerful antimicrobial action against bacteria, viruses, and fungi, which effectively removes microbial contaminants from drinking water and wastewater^15,16.

Cancer is a highly dangerous disease that significantly contributes to a high rate of deaths worldwide¹⁷. Various treatments are available for different types of cancer, with chemotherapy being one of the most commonly used methods. This approach involves the use of cytotoxic drugs to target and destroy cancer cells^18,19. Chemotherapy can have substantial side effects. To counteract this, medical NPs such as AgNPs have been researched to reduce these negative effects^20,21. The synthesis of AgNPs can be achieved through a variety of methods, including chemical vapor deposition, sol-gel techniques, UV-irradiation, aerosol technology, photochemical reduction, microwave irradiation, and electrochemical approaches²². In contrast, plant-based synthesis is gaining popularity due to its use of less toxic reducing agents, faster processing, scalability, and ease of handling. Plant extracts, rich in bioactive compounds, act as both reducing and capping agents, influencing the size and shape of the resulting nanoparticles²³.

The Pandanus genus, with around 700 species and economic importance in Asia, is known for its bioactive compounds, including flavonoids and phenols, which show potent anticancer properties²⁴. While research on the Pandanus genus for AgNPs synthesis remains limited, studies involving P. odorifer, P. amaryllifolius, P. tectorius and P. canaranus have demonstrated its significant potential. Leaf extracts from these species effectively produce spherical AgNPs, typically ranging from 17 to 40 nm in size. The resulting nanoparticles exhibit valuable biological activities, including potent antimicrobial, antioxidant, anti-cancer, and catalytic properties, underscoring the genus’s promise for green nanotechnology applications^10,25–27. In this research mainly focuses on utilizing Pandanus dubius leaf extract for the synthesis of Pds-AgNPs, aims to enhance the reduction process effectively. The investigation of morphological characteristics of the synthesized Pds-AgNPs and evaluating their antioxidant, antibacterial, and anticancer properties. Our findings reveal that these synthesized nanoparticles exhibit significant effects in these areas. Notably, to the best of our knowledge, this study is the first to report the phytochemicals analysis and synthesis of Pds-AgNPs using P. dubius leaf extract, marking a novel contribution to the field.

Materials and methods

Chemicals

Silver nitrate (AgNO₃, 99.0%) was obtained from Sigma-Aldrich Chemicals (USA). All the chemicals (99%) and biological media were sourced from Hi-Media (India). Double-distilled water (ddH₂O) was employed for all experiments.

Plant collection and identification

Pandanus dubius leaves were collected in June from the Periyar University campus in Salem District, Tamil Nadu (Fig. 1), and identified in Department of Botany, Periyar University, Salem, Tamil Nadu, India. After collection, the leaves were thoroughly washed with fresh water. They were then shade-dried for one week at room temperature (34^∘C) and 76% humidity. The dried leaves were ground into a fine powder and stored in sterilized containers at 4°Cfor future experiments.

Fig. 1.

Habit of Pandanus dubius spreng.

Extract preparation

Maceration extraction was carried out on the dried leaf powder samples. Specifically, 20 g of the powdered plant material were submerged in methanol at 1:10ratio. The mixture was then agitated for 24 h on an orbital shaker set to 125 rpm. After extraction, the solvent was removed by rotary vacuum evaporation at 50 °C. The obtained extract was subsequently lyophilized and stored in a deep freezer at −80 °C until further biochemical analyses.

Quantification of total phenol

The Folin-Ciocalteu technique was used to measure the total phenolic content²⁸. In test tubes, 200 µL of the methanol extract was diluted with distilled water to a final volume of 1 mL, and a distilled water blank was prepared concurrently. 500 µL of 1 N Folin-Ciocalteu reagent was added to each tube, and it was then incubated for five minutes. After adding 2.5 mL of a 20% sodium carbonate solution, the solutions were violently vortexed. The blue colour that developed after 40 min of dark incubation at room temperature was measured at 725 nm using a spectrophotometer, in comparison to the blank. Gallic acid equivalents (GAE, mg/g) were used to express the results of the quantification, which was carried out using a gallic acid standard curve. Every analysis was carried out three times.

Estimation of flavonoids

Total flavonoid content was calculated using Zhishen et al.²⁹ methodology. In test tubes, 200 µL of the methanol extract was diluted with distilled water to a final volume of 2.5 mL, and a distilled water blank was prepared concurrently. After adding 150 µL of 5% sodium nitrite to each tube, the tubes were incubated at room temperature for six minutes. After that, all tubes-including the blank-were filled with 150 µL of 10% aluminium chloride, and they were incubated for an additional six minutes. The final volume was then adjusted to 5 mL with distilled water after adding 2 mL of 4% sodium hydroxide. After thorough mixing and 15-minute incubation at room temperature, a pink colour formed, signifying the presence of flavonoids. Its absorbance was measured at 510 nm using a spectrophotometer, against a blank. The findings of the triple analysis of each sample were reported as rutin equivalents (RE, mg/g).

Analysis of gas chromatography – mass spectrometry (GC-MS)

GC-MS analysis was used to identify the volatile components in the methanol extract. A Clarus 680 GC system with an Elite-35 MS fused silica column (30 m × 0.25 mm ID × 250 μm df, 5% biphenyl/95% dimethylpolysiloxane) was used for the analysis. The carrier gas was helium, which flowed at a steady 1 mL/min. A 1 µL aliquot of the extract was injected while the injector temperature was kept at 260 °C. The temperature schedule for the GC oven was set to start at 60 °C and hold it there for two minutes, then ramp up to 300 °C at a rate of 10 °C per minute, and finally hold it there for six minutes. The ion source and transfer line were both adjusted to 240 °C, and the mass spectrometer was run in electron impact (EI) mode at 70 eV. With a scan time of 0.2 s and an inter-scan delay of 0.1 s, data acquisition was carried out in full-scan mode, encompassing a mass range of 50–600 Da. The mass spectra of the discovered compounds were compared to the NIST (2017) library database in order to identify them.

Antioxidant assays

Estimation of DPPH assay

The DPPH (2, 2 – diphenyl – 1 – picrylhydrazyl) potentials of solvent extracts were assessed using the procedure described by Braca et al.³⁰. The free radical scavenging activity of the extracts were determined based on the scavenging activity of the stable 1,1-diphenyl-2-picrylhydrazyl test. In short, 3 mL of a 0.004% methanolic DPPH solution was combined with various concentration of Pds-AgNPs or leaf methanol extract 100 to 500 µg/mL. Absorbance at 517 nm was determined after 30 min and the percentage inhibition activity was calculated from.

Percentage Inhibition = [(A₀–A₁)/A₀] x100,

Where A₀ is the absorbance of the control, and A₁ is the absorbance of the extract/standard. The inhibition curves were prepared and IC₅₀ values were obtained.

Determination of metal chelating assay

The chelating activity for ferrous ions Fe²⁺ was measured according to the method described by Dinis et al.³¹. In short, 0.05 mL of 2 mM FeCl₂ was combined with 1.6 mL of deionized water and various concentration of Pds-AgNPs or leaf methanol extract 100 to 500 µg/mL. After 30 s, 0.1mLferrozine (5 mM) was added. Ferrozine reacted with the divalent iron to form stable magenta complex species that were very soluble in water. After 10 min at room temperature, the absorbance of the Fe²⁺–Ferrozine complex was measured at 562 nm. EDTA was used as the standard and the results were expressed as mg EDTA equivalents mg/g, with EDTA serving as the reference standard.

Estimation of antibacterial activity

The antibacterial activity of P. dubius leaf extract and its bio-synthesized Pds-AgNPs was evaluated against a panel of Gram-positive and Gram-negative bacterial pathogens. This assessment was performed via the conventional agar disk diffusion method, utilizing various concentrations from 50, 75 to 100 µg/mL that compared to positive control (Cefotaxime, 25 µg/mL) and negative control (Dimethyl sulfoxide). Gram-positive Enterococcus faecalis, Staphylococcus aureus, as well as Gram-negative Escherichia coli and Pseudomonas aeruginosa, were among the test organisms. Appropriate antibiotic standards were used as controls. The produced NPs’ antibacterial properties were examined in aqueous solutions. Samples were aseptically applied to sterile disks, and the infected plates were then incubated for 24 h at 37 °C. By measuring the inhibitory zones (including disk diameter) that developed surrounding the disks, antibacterial efficacy was objectively evaluated; larger zones denoted higher antimicrobial activity.

Determination of anticancer activity

Cell culture condition

The National Centre for Cell Science (NCCS, Pune, India) provided the A549 human lung adenocarcinoma cells. The cells were cultivated in Dulbecco’s Modified Eagle Medium (DMEM), which was enhanced with 5 mM glutamine, 10% fetal bovine serum, and 1% antibiotics (50 mg/L streptomycin and 50,000 U/L penicillin). They were kept in tissue culture flasks at 37 °C with 95% relative humidity and 5% CO₂ in a humidified environment. At least twice a week, the medium was changed to guarantee ideal growth circumstances.

Cytotoxic activity

The cytotoxic effect of leaf extract and Pds-AgNPs on human lung cancer cell lines was evaluated according to the methodology²⁴. To put it briefly, cells were planted at a density of 1 × 10³ cells per well in a 96-well plate, and they were left to adhere for a whole day. Following treatment with varying quantities of Pds-AgNPs and leaf extract (6.25, 12.5, 25, 50 and100 µg/mL), they were incubated for 24 h at 37 °C in a humidified environment with 5% CO₂. Instead of test chemicals, DMSO (Dimethyl sulfoxide) was added to the control wells. Following treatment, each well received 25 µL of MTT solution (5 mg/mL), and the plate was incubated for four hours. After that, the medium was gently shaken for 15 min and replaced with 100 µL of DMSO to dissolve the formazan crystals. Using an ELISA microplate reader, absorbance was measured at 570 nm. After calculating cell viability (%) in relation to the untreated control, the IC₅₀ value was established for further examination.

Pandanus dubius leaf extract preparation and synthesis of AgNPs

First, freshly collected P. dubius leaves were thoroughly cleaned with running tap water and then distilled water. Ten grams of these leaves were cut into small pieces (0.5 × 0.5 cm) and then transferred to a 250 mL conical flask containing 100 mL of boiled water for 10 min. The resulting boiled leaf extract was filtered through Whatman No. 1 filter paper. Subsequently, 10 mL of this leaf extract was added to 100 mL of 1 mM AgNO₃ solution in a separate container. Each mixture was stirred at 250 rpm for 16 h at room temperature (37 ^◦C). The initial colorless AgNO₃ solution gradually changed to a brown color, which served as the first confirmation of Ag-NPs synthesis¹⁰.

Characterizations of synthesized Pds-AgNPs

UV-visible spectroscopy analysis of synthesized Pds-AgNPs

The successful bio-reduction and formation of Pds-AgNPs was initially indicated by a visual color shift in the reaction mixture from pale yellow to dark brown. This was further confirmed by UV-Vis spectroscopic analysis (Shimadzu UV-1800, Japan), which identified a distinct absorption peak within the 200–800 nm range. All measurements were taken using pure water as a blank reference.

FTIR analysis of synthesized Pds-AgNPs

To identify the functional groups involved in capping and stabilizing the silver nanoparticles, FTIR analysis was carried out. The Pds-AgNPs were dehydrated at 45 °C for 24 h to prevent spectral interference from water. Subsequently, the samples were homogenized with potassium bromide (KBr) and pressed into pellets. Analysis was performed on a Thermo Nicolet-380 spectrometer (Madison, USA), with spectra collected in transmittance mode from 400 to 4000 cm⁻¹.

X-ray diffractometric analysis of Pds-AgNPs

The crystalline structure, lattice parameters, and crystallite size of the synthesized Pds-AgNPs were determined by X-ray diffractometry (Rigaku Miniflex 600, Austin, TX, USA). The powdered sample was packed onto a hollow slide and flattened for analysis. XRD spectra were collected in the 2θ range of 10–80° using a CuKα radiation source (λ = 0.15418 nm) in 2θ/θ scanning mode at a rate of 1.2° per minute. The crystallite size was calculated from the Scherrer equation:

D = 0.9λ/βcosθ.

where D is the average crystallite size (nm), λ is the X-ray wavelength (nm), β is the full width at half maximum (FWHM) of the diffraction peak (in radians), and θ is the Bragg angle.

SEM and EDAX analysis of Pds-AgNPs

The morphology, structure, and distribution pattern of the synthesized Pds-AgNPs were characterized by scanning electron microscopy (SEM) using a JSM-IT 500LA instrument (Tokyo, Japan). The presence of elemental silver was verified by energy dispersive X-ray spectroscopy (EDX) attached to the SEM. Sample preparation involved depositing a small quantity of Pds-AgNPs onto an aluminum stub fitted with conductive carbon tape, followed by gold sputtering for 3 min to create a conductive surface for imaging.

TEM analysis of Pds-AgNPs

The morphology and size of the synthesized Pds-AgNPs were characterized by transmission electron microscopy (TEM) using a Zeiss EM10C instrument operated at an accelerating voltage of 200 kV.

Statistical analysis

For each treatment, data were collected in triplicate, and the experiment was replicated. The results are expressed as the mean ± standard deviation (SD). Statistical analysis was performed using one-way ANOVA to determine significant differences among the treatment groups. Duncan’s Multiple Range Test (DMRT) was used for post-hoc comparisons, with a significance level set at P < 0.05. All statistical analyses were performed by means of IBM SPSS 20 software³². Additionally, Origin pro version 2023 was used to draw the graph³³.

Result

Characterization of Pds-AgNPs

The aqueous extraction of P. dubius leaves proved to be an effective reducing agent for silver nitrate, leading to the successful synthesis of Pds-AgNPs. This reduction process was notably swift, with Pds-AgNPs forming in just 16 h at room temperature, as indicated by the solution’s color change from colorless to brown. The presence of Pds-AgNPs was further validated through UV-visible spectral analysis, which showed a distinct absorption peak at 458 nm in the reaction mixture (Fig. 2). In the synthesis mediated by plant extracts, the reduction of silver ions (Ag⁺) to elemental silver (Ag⁰) occurs through electron transfer from various biomolecules, including polyphenols, flavonoids, terpenes, sugars, and alkaloids, which serve as natural reducing agents. The Hexadecanoic acid and Octadecanoic acid, both saturated fatty acids, possess a carboxylic acid functional group. This group readily undergoes oxidation, donating electrons to silver ions (Ag⁺) from the AgNO₃ solution, thereby reducing them. The accumulation of these reduced silver atoms then initiates the nucleation of NPs. The phytochemicals found in P. dubius leaf extract, such as Stigmasterol, Octadecanoic acid, and Hexadecanoic acid, play crucial roles in the green synthesis of AgNPs by acting as both reducing agents and stabilizing/capping agents.

Fig. 2.

UV–visible spectra of P. dubius leaves extract mediated synthesis Pds-AgNPs.

FTIR identified functional groups in the P. dubius aqueous extract that facilitated silver nitrate reduction and Ag-NP capping. Hydroxyl (-OH) groups, likely from alcohols, phenols, and catechins, were observed at 3751 cm⁻¹. Aliphatic C-H bending vibrations appeared at 2927 cm⁻¹. Carbon dioxide (O = C = O stretching) was detected in both the extract (2352 cm⁻¹) and Ag-NPs (2354 cm⁻¹). Strong fluoro compounds (C-F stretching) were also present, with peaks at 1033 cm⁻¹ (extract) and 1000 cm⁻¹ (Ag-NPs). Alkyl halide (C-Cl stretching) presence in Ag-NPs was indicated by a broad peak at 664 cm⁻¹ (Fig. 3).

Fig. 3.

FTIR spectrum of P. dubius leaves extract mediatd synthesized Pds-AgNPs.

XRD analysis was employed to assess the crystalline structure, size, and purity of the synthesized Pds-AgNPs. The XRD pattern exhibited four prominent diffraction peaks at 2θ = 37.99, 44.19, 64.39, and 77.27, corresponding to the (111), (200), (220), and (311) planes, respectively (Fig. 4). These peaks confirmed the crystalline nature of the Ag-NPs, with the intense peak for the (111) plane and its narrow full width at half maximum, indicating high crystalline quality. The crystallite sizes, determined by the Scherrer equation, was shown in Table 1. The elemental composition of the synthesized Pds-AgNPs was determined using EDX. Results showed the NPs contained 19% carbon, 24% oxygen, 13% chlorine, and 43% silver. The relatively high percentages of carbon and oxygen indicate that organic compounds from the plant extract effectively acted as capping agents during nanoparticle synthesis (Fig. 5).

Fig. 4.

XRD pattern of P. dubius leaves extract mediated synthesized Pds-AgNPs.

Table 1.

Crystallite size analysis of Pds-AgNPs using the scherrer formula from XRD data.

Peak position (2 Theta)	FWHMβ (°)	Crystallite size D (nm)	D nm (average)
37.99	0.41287	21.2549988	17.13521064
44.19	0.64427	13.898231
64.39	0.59014	16.61286626
77.27	0.43566	24.38855835
77.31022	1.11592	9.521398782

Fig. 5.

EDX spectrum of P. dubius leaves extract mediated synthesized Pds-AgNPs.

The SEM images reveal that the Pds-AgNPs have a tendency to combine and agglomerate, resulting in larger clusters. Despite this agglomeration, the individual nanoparticles within these clusters largely retain a spherical morphology (Fig. 6). HR-TEM imaging revealed the morphology and size of Pds-AgNPs. SEM showed spherical aggregates, while HR-TEM confirmed an average particle size of 17.13 ± 5.880 nm, indicating successful synthesis of relatively small NPs (Fig. 7).

Fig. 6.

Scanning electron microscopic images obtained from P. dubius leaves extract mediated synthesized AgNPs. ((A), (B), (C) and (A) various magnification of SEM images).

Fig. 7.

HR-TEM images of P. dubius leaves extract mediadet synthesized Pds-AgNPs. (A), (B), (C) various magnification of HR-TEM images, (D) SAED pattern of Pds-AgNPs.

GC-MS analysis

GC-MS analysis of the P. dubius methanol extract revealed the presence of a diverse range of phytocompounds. The identification of these compounds was thoroughly confirmed through the analysis of peak area, retention time, and molecular formula. A total of thirty phytochemicals were identified within the P. dubius methanol extract, based on their relative peak area percentage and retention time. These findings are comprehensively documented in Table 2, which provides detailed information about each identified compound. The corresponding GC-MS chromatograms, illustrating the separation and detection of these compounds, are presented in Fig. 8. Notably, n-Hexadecanoic acid, also known as palmitic acid, was identified as the most abundant compound, exhibiting the highest peak area percentage. This observation suggests a potential significant contribution of n-Hexadecanoic acid to the overall bioactivity of the P. dubius extract.

Table 2.

GC-MS analysis of P. dubius leaf methanol extract.

S. No	RT	Peak area%	Molecular formula	MW	Name of the compound	Biological activities
1	16.758	0.62	C16H34O2	242	1-Hexadecanoic acid	Antimicrobial and Antioxidant
2	19.030	1.27	C18H36	252	1-Octadecene	Antibacterial, Antioxidant and Anticancer properties
3	19.887	0.97	C18H36O	268	8-Octadecanone	Antibacterial activity
4	20.401	9.63	C17H34O2	270	Hexadecanoic acid	Anti-inflammatory, Antibacterial, Antioxidant, Cancer prevention and Neurotrophic
5	20.772	12.23	C16H32O2	256	n-Hexadecanoic acid	Anti-inflammatory, Antibacterial, Antioxidant, Cancer prevention and Neurotrophic
6	21.086	0.73	C20H42O	298	1-Eicosanol	Antioxidant activity
7	22.051	11.00	C19H34O2	294	9,12-Octadecadienoic acid	Anti-inflammatory, Antibacterial, hypocholesterolemic, and hepatoprotective properties
8	22.108	7.26	C21H36O2	320	11,14,17-Eicosatrienoic acid	Cardiovascular protection and Protection from ischemic injury
9	22.340	2.57	C19H38O2	298	Methyl stearate	Anti-inflammatory, Antioxidant, Antifungal and Antinociceptive
10	22.431	9.20	C21H38O4	354	9,12-Octadecadienoic acid	Anti-inflammatory, Antibacterial, hypocholesterolemic, and hepatoprotective properties
11	22.484	6.64	C19H34O	278	(Z, Z)−6,9-CIS-3,4-EPOXY-NONADECADIENE	----
12	22.595	0.98	C57H104O6	884	9-Octadecenoic acid	Antioxidant and antimicrobial activities
13	22.676	2.17			Octadecanoic acid	Antioxidant and antimicrobial activities
14	23.747	0.92	C20H41NO2	327	Dimethylaminoethyl palmitate	Anti-inflammatory, antioxidant and antimicrobial activities
15	23.860	2.85	C19H36O3	312	Glycidyl palmitate	----
16	23.915	1.42	C29H48O	412	Stigmasterol	Anti-inflammatory, antioxidant, anticancer, and neuroprotective effects
17	24.034	0.67	C20H35NO	305	2-((8Z,11Z)-Heptadeca-8,11-dien-1-yl)−4,5-dihydrooxazole	Antimicrobial activity and anti-inflammatory properties.
18	24.118	1.27	C21H42O2	326	Methyl Icosanoate	----
19	25.178	1.37	C22H43NO2	353	2-(Dimethylamino) ethyl vaccenoate	----
20	25.323	1.10	C9H20O2Si	188	Cyclohexanol	Antimicrobial and analgesic
21	25.464	0.79	C16H38O2Si2	318	Decane, 1,9-bis[(trimethylsilyl)oxy]-	----
22	25.659	5.51	C17H34O2	270	Hexadecanoic acid	Anti-inflammatory, Antibacterial, Antioxidant, Cancer prevention and Neurotrophic
23	25.759	1.76	C23H46O2	354	Methyl 20-methyl-heneicosanoate	----
24	25.813	2.90	C28H48O2	416	(R)−6-Methoxy-2,8-dimethyl-2-((4R,8R)−4,8,12-trimethyltridecyl) chroman	Anti-inflammatory, neuroprotective, Antioxidant and Anticancer
25	26.241	4.19	C29H50O	414	gamma. -Sitosterol	Cholesterol-Lowering Effects, Antidiabetic activity, Anti-inflammatory, Antibacterial, Antioxidant and Anticancer
26	26.726	0.95			beta. -Eudesmol	Antioxidant, Anticancer, stress reduction, and neurological protection
27	26.859	0.78	C19H32O2	292	9,12,15-Octadecatrienoic acid	Anti-inflammatory, hypo-cholesterolemic, antioxidant, and anti-cancer properties
28	27.150	5.21	C22H40O2	336	Butyl 9,12-octadecadienoate	Anti-inflammatory properties
29	27.224	2.14	C22H38O2	334	Butyl 9,12,15-octadecatrienoate	Anti-inflammatory properties
30	27.404	0.91	C21H42O4	358	2,3-dihydroxypropyl ester	Antimicrobial properties

RT Retention Time, WM Molecular Weight.

Fig. 8.

GC-MS analysis of P. dubius leaf methanol extract.

Quantification of secondary metabolites

The levels of total phenolics and flavonoids in the methanol extract of P. dubius leaves are quantified in Fig. 9. The analysis revealed a substantial presence of secondary metabolites within this plant extract. Notably, the total flavonoids were measured at 6.703 ± 0.115, which was significantly higher than the total phenolic content, recorded at 1.422 ± 0.659. These values indicate that the methanol extract of P. dubius contains a greater concentration of flavonoids compared to phenolics when compared to standard equivalents.

Fig. 9.

Estimation of total phenol and total flavonoid from P. dubius leaves methanol extract.

Antioxidant activity

This study investigated the antioxidant properties of P. dubius leaf extract and synthesized Pds-AgNPs using DPPH and metal chelating assays. We compared their effectiveness against standard antioxidants. Both the Pds-AgNPs and the methanol extract were tested at concentrations from 100 to 500 µg/mL, revealing a dose-dependent increase in radical scavenging activity. For DPPH radical scavenging activity, the IC₅₀ values were 37.175 µg/mL for the leaf extract, 27.496 µg/mL for Pds-AgNPs and 36.807 µg/mL for the standard vitamin-C. Notably, Pds-AgNPs consistently exhibited superior maximum scavenging activity compared to the leaf extract in both the DPPH radical scavenging assay (Fig. 10A) and the metal chelating assay (Fig. 10B). It is probable that the observed enhanced activity is a result of the free radical scavenging mechanism mediated by the phenolic and flavonoid compounds in P. dubius extract. The synthesis of Pds-AgNPs may effectively enhance the obtainability of these secondary metabolites, contributing to their antioxidant capabilities. This suggests that as the concentration of Pds-AgNPs increases, their ability to neutralize free radicals also intensifies, further supporting their potential as effective antioxidant agents. The study revealed that the bio-based synthesis of AgNPs exhibited dose-dependent effectiveness in reducing DPPH and metal chelating activities, with ascorbic acid used as the reference standard.

Fig. 10.

Antioxidant activities of P. dubius leaves methanol extract and synthesized Pds-AgNPs. (A) DPPH free radical scavenging activity (B) Metal chelating activity.

Antibacterial activity

This study evaluated the in vitro antibacterial activity of P. dubius methanol extract and Pd-Ag-NPs against four pathogenic bacterial strains (Gram-positive, Staphylococcus aureus, Enterococcus faecalis; Gram-negative: Escherichia coli, Pseudomonas aeruginosa) using the agar disc diffusion method. Zones of inhibition were measured at 25, 50, and 100 µg/mL concentrations and compared to a Cefotaxime positive control. Pds-AgNPs exhibited significantly stronger antibacterial activity than the P. dubius methanol extract across all tested strains. Notably, Pds-AgNPs showed the largest zones of inhibition against S. aureus (1.53 ± 0.41 cm), E. faecalis (1.40 ± 0.20 cm), E. coli (1.40 ± 0.40 cm) and P. aeruginosa (1.46 ± 0.30 cm) the results were shown Figs. 11 and 12.

Fig. 11.

Antibacterial activity of P.dubius leaves methanol extract against some baterial pathogens. (A) Escherichia coli, (B) Staphylococcus aureus, (C) Pseudomonas aeruginosa, (D) Enterococcus faecalis.

Fig. 12.

Antibacterial activity of P. dubius leaves methanol extract against different pathogens, used for Zone of inhibition for various concentrations 50, 75 and 100 µg/mL against bacteria.

The P. dubius methanol extract also demonstrated antibacterial activity, though to a minor level. Zones of inhibition were observed for E. faecalis (1.36 ± 0.14 cm), S. aureus (1.33 ± 0.11 cm), E. coli (1.13 ± 0.11 cm) and P. aeruginosa (1.06 ± 0.11 cm). No inhibition was observed with the DMSO negative control (Figs. 13 and 14). These results indicate that Pds-AgNPs possess potent antibacterial properties against a range of pathogenic bacteria, particularly S. aureus. The P. dubius methanol extract also exhibits significant antibacterial activity, although with varying efficacy across different strains. These findings suggest that both Ag-NPs and P. dubius methanol extracts have potential applications as antibacterial agents in medicine and food preservation.

Fig. 13.

Antibacterial activity of P.dubius leaves extract mediated synthesis Pd-Pds-AgNPs against some baterial pathogens. (A) Escherichia coli, (B) Staphylococcus aureus, (C) Pseudomonas aeruginosa, (D) Enterococcus faecalis.

Fig. 14.

Antibacterial activity of Pd-Pds-AgNPs against selected pathogens. Zone of inhibition for various concentrations 50, 75 and 100 µg/mL against pathogen bacterias.

Cytotoxicity activity

This study evaluated the cytotoxic effects of a P. dubius leaf methanol extract and biosynthesized Ag-NPs against A549 lung cancer cells. Both the extract and Ag-NPs induced dose-dependent cell death, evidenced by morphological changes like cell shrinkage and necrosis (Figs. 15 and 16). Notably, Ag-NPs exhibited a lower IC₅₀ (40.84 ± 0.092 µg/mL) compared to the methanol extract (54.88 ± 0.105 µg/mL) after 24 h, indicating more inhibitory activity. Ag-NPs and plant derived methanol extracts both exhibit anticancer activity, but they do so through different mechanisms. Ag-NPs primarily act as inorganic agents that physically and chemically disrupt cancer cells, while methanol extracts contain complex mixtures of organic phytochemicals that modulate intracellular signaling pathways. Ag-NPs, due to their small size and positive surface charge, can bind to the negatively charged cell membrane of cancer cells. This interaction can physically disrupt the membrane’s integrity, creating pores or holes. The loss of membrane integrity leads to an uncontrolled influx of ions and water and the leakage of essential cellular components, ultimately causing the cell to burst. Ag-NPs and the silver ions (Ag⁺) they release can generate an excessive amount of ROS. The ROS can also directly attack and damage cellular macromolecules like DNA, lipids, and proteins. DNA damage can activate cell cycle checkpoints and trigger cell death. The phytochemicals in P. dubius, particularly the alkaloid and phenolic compounds, can enhance ROS-mediated cytotoxicity by directly increasing ROS generation, disrupting mitochondrial function, and depleting the cell’s antioxidant defense systems. This creates a state of severe oxidative stress that cancer cells cannot tolerate, leading to their selective destruction. These findings suggest that both P. dubius leaf-derived Ag-NPs and the methanol extract hold promise as cytotoxic agents against lung cancer, with Ag-NPs demonstrating greater effectiveness. AgNPs have demonstrated significant cytotoxic effects across a wide range of human cancer cell lines, highlighting their potential as a promising therapeutic agent.

Fig. 15.

In vitro cytotoxicity microcopic images (A549) humen lung cancer cells at 24 h treatment of Pds-AgNPs and methanol extract. (A) Control, (B) 6.25 µg/mL, (C) 25 µg/mL, (D) 50 µg/mL and E- 100 µg/mL, used various concentration of treatments.

Fig. 16.

In vitro cytotoxicity of cell viability (%) of A549 cells after 24 h treatment. (A) Pds-AgNPs (B) methanol extract.

Discussion

The functional groups such as hydroxyl (-OH) and carboxyl (-COOH) play a crucial role by donating electrons to Ag⁺ ions, thereby facilitating their conversion into Ag⁰. This process highlights the effectiveness of plant extracts in the green synthesis of AgNPs⁶². A maximum absorbance peak at 456 nm, observed for the AgNPs, suggests a red shift from the typical surface plasmon resonance range of 400–450 nm^34,35. This shift is consistent with variations reported in other green synthesis methods, such as the 427 nm peak obtained using Berberis asiatica root extract³⁶. UV-Vis spectroscopy tracks the size evolution of AgNPs by analyzing shifts in their characteristic localized surface plasmon resonance bands. These distinct absorption peaks directly reflect changes in the AgNPs size and morphology³⁷. Green tea-synthesized silver nanoparticles exhibit UV-vis absorption peaks in the range of 400–490 nm. The precise position of the peak depends on factors such as particle size, shape, and synthesis conditions. This absorption arises from surface plasmon resonance (SPR), a phenomenon in which incident light at specific wavelengths excites collective oscillations of conduction electrons on the nanoparticle surface. Variations in particle morphology and size alter these electron oscillations, resulting in shifts of the SPR peak within the observed wavelength range³⁸. UV-Vis spectroscopy, spanning 200–800 nm, was used to monitor the formation and optimization of AgNPs. The initial color change visually indicated AgNPs formation, which was then confirmed by a characteristic surface plasmon resonance peak between 400 and 500 nm, typical for AgNPs³⁹. Denser surface charges on AgNPs lead to increased aggregation and larger particle sizes. This aggregation, along with shifts in surface plasmon resonance peaks, likely causes the observed decrease in AgNPs absorbance⁴⁰. UV-Vis spectroscopy confirmed the successful synthesis of AgNPs, showing a characteristic absorption peak at 435 nm. This falls within the typical 400–500 nm range for silver⁴¹. The flavonoids, tannins, saponins, glycosides, phenol, and alkaloids identified in Sonchus arvensis are proposed to facilitate the green synthesis of SA-AgNPs by reducing Ag⁺ to Ag⁰ and subsequently acting as effective capping and stabilizing agents⁴². The formation and characteristics of these AgNPs are significantly influenced by bioactive compounds like vitamins, flavonoids, tannins, phenolic acids, and proteins, which contribute to the reduction of Ag⁺ ions and control nanoparticle properties⁴³. Aqueous extract of Pandanus tectorius aerial root efficiently reduced silver nitrate, yielding Pt-Ag-NPs within 65 min. The color change from colorless to brown signaled successful nanoparticle formation. UV-Vis analysis confirmed the presence of Ag-NPs, showing a distinct absorbance peak at 415 nm¹⁰. The presence of AgNPs can be detected through UV-Vis spectroscopy. This method relies on the excitation of surface plasmon vibrations in colloidal AgNPs, leading to light absorption. Such absorption is typically observed in the wavelength range of 400–450 nm, generating a unique absorption band that is characteristic of AgNPs⁴⁴. Initial confirmation of nanoparticle synthesis was provided by UV-visible spectroscopy, which revealed a surface plasmon resonance effect, evidenced by a visual color change from light to dark brown⁴⁵. Potential AgNPs binding to phytochemicals was suggested by peaks at 488 and 407 cm⁻¹⁴⁶. A shift from 3462 cm⁻¹ (extract) to 3242 cm⁻¹ (Ag-NPs) for O-H or N-H stretching vibrations of phenolic compounds was also observed⁴⁷. The presence of aromatic C = C stretching vibrations was confirmed by a peak at 1634 cm⁻¹. Further characteristic aromatic features were observed at 1390 cm⁻¹, corresponding to aromatic C–H bending. The spectrum also displayed a peak at 1054 cm⁻¹, indicative of C–O stretching vibrations⁴⁸.

The crystallite size of the AgNPs was significantly influenced by the XRD peak values, which were indirectly impacted by the reducing and capping properties of the phytochemicals present in the plant extracts used during their synthesis⁴⁹. The AgNPs synthesized from Pisum sativum were characterized using EDX spectroscopy, which identified their elemental composition. A distinct peak at 3 keV was observed, confirming the presence of Ag⁵⁰. The EDX spectrum of the green-synthesized AgNPs revealed a prominent peak at 3.0 keV⁵¹.

The similarly reported that Malus domestica extract-synthesized AgNPs displayed a predominantly spherical morphology and a polydisperse size distribution between 40 and 100 nm. The bioactive compounds within the extract played a dual role, acting as both reducing and stabilizing agents, thus determining the NPs’ shape and size range. The polydispersity observed in the NPs highlights the natural variability in the synthesis process mediated by plant extracts, which can influence the size and uniformity of the resulting NPs⁵². The synthesized nanoparticles had a crystalline morphology, as confirmed by SEM, TEM, and XRD analyses⁵³.

SEM micrographs confirmed the synthesis of polydisperse AgNPs, primarily spherical in shape, that tended to form small clusters. These images provided a clear morphological characterization of the nanoparticles⁵⁴. The agglomerated particles exhibited a surface topography dotted with several medium-sized micro-particles⁵⁵. Comprehensive insights into AgNPs shape, size, and internal structure were obtained through TEM analysis. High-resolution TEM images confirmed nanoscale dimensions and a predominantly spherical morphology, with a few oval shapes. The majority of AgNPs were synthesized within a 20 nm size range. This level of detail is essential for verifying size distribution and morphological consistency⁵⁶. The D. indica extract produced predominantly spherical silver nanoparticles ranging from 10.0 to 23.24 nm in size. The phytochemicals in the extract likely acted as reducing agents to form the AgNPs and also served as capping agents, which stabilized a significant portion of the nanoparticles and kept them well-dispersed, despite some observed aggregation. This dual role of the extract’s compounds contributed to the moderate size variation and overall morphology⁵⁴. TEM analysis showed the synthesized AgNPs were mostly spherical, with some oval shapes. Their size distribution was up to 20 nm^57,58. The morphology and size of the biosynthesized RC@AgNPs were characterized by TEM, which illustrated isotropic spherical particles with an average size of 20.98 nm based on the particle size distribution histogram⁵⁹.

The major component of Pandanus odoratissimus essential oil, as determined by GC-MS, was 2-phenyl ethyl methyl ether, while terpinene-4-ol was also a significant constituent^60,61. GC-MS analysis of Pandanus conoideus red fruit oil identified 35 volatile compounds, with 1,3-dimethyl-benzene, N-glycyl-L-alanine, trichloromethane, and ethane as the primary components⁶². The ethanolic extract of Pandanus odorifer flowers was analyzed using GC-MS to identify its bioactive compounds. Out of the total compounds detected, 22 bioactive compounds were selected for further analysis. Among these, 9,12-Octadecadienoic acid (Z, Z) exhibited the highest peak area percentage, indicating its predominance in the extract⁶³. The phytochemical profile of Pandanus odorifer leaf extracts is analyzed by a significant presence of 28-demethyl-beta-amyrone (39.24%), gamma-sitosterol (38.17%), stigmasterol (37.00%), trans-geranylgeraniol (30.27%), 3-cyclohexenecarboxylic acid, 6,6-dimethyl-4-(4-morpholyl)−2-oxo-methyl ester (27.64%), and 5-methyl-Z-5-docosene (27.15%)⁶⁴. Conversely, GC-MS analysis of Pandanus amaryllifolius leaf ethanol extract identified n-hexadecanoic acid (19.31%), 9,12,15-octadecatrienoic acid (17.82%), and benzofuran, 2,3-dihydro- (6.84%) as the major constituents⁶⁵.

The leaves of Pandanus tectorius are rich in bioactive phytochemicals such as alkaloids, steroids, phenols, tannins, terpenes, flavonoids, saponins, and glycosides. These compounds give the leaves significant therapeutic potential, including antioxidant, anti-inflammatory, antimicrobial, and cardioprotective effects⁶⁶. The leaf extract of P. amaryllifolius is rich in glycosides, alkaloids, and saponins, which contribute significantly to its therapeutic potential. Glycosides offer cardiovascular and antimicrobial benefits, alkaloids provide analgesic and anti-inflammatory effects, and saponins exhibit immune-modulating and cholesterol-lowering properties. These bioactive compounds underscore the extract’s value as a natural source for diverse health applications⁶⁷. The high flavonoid and phenol content in Pandanus canaranus leaf ethyl acetate and methanol extracts, which possess strong antioxidant properties, suggests their potential as natural agents against oxidative stress²⁴. This is consistent with findings in Pandanus amaryllifolius leaf ethanol extract, which also demonstrates high concentrations of flavonoids and polyphenols, alongside moderate levels of alkaloids and terpenoids⁶⁸.

The observed antioxidant activity is primarily a function of the high concentration of phytochemicals, including proteins, flavonoids, carbohydrates, and phenolic compounds, all characterized by an abundance of hydroxyl (-OH) functional groups. The mechanism of action involves the donation of hydrogen atoms from these hydroxyl groups, leading to the stabilization and neutralization of DPPH radicals. The substantial presence of hydroxyl groups within the stabilized AgNPs contributes significantly to their antioxidant properties, enabling efficient scavenging of free radicals and a reduction in oxidative stress^10,69. The antioxidant potential of Cocos nucifera meat-derived Cm-AgNPs was evaluated using DPPH, ABTS, hydroxyl radical scavenging, and reducing power assays. Cm-AgNPs showed concentration-dependent activity, with 300 µg/mL exhibiting maximum scavenging. However, their activity was slightly lower than ascorbic acid; for instance, 80% DPPH scavenging versus 87% for ascorbic acid at 300 µg/mL⁷⁰. In comparison, Lactobacillus plantarum extract-synthesized AgNPs showed stronger antioxidant activity, with 82.92% DPPH and 85.44% H₂O₂ scavenging⁷¹. Sargassum polycystum extract-synthesized AgNPs demonstrated 59% total antioxidant activity, marginally higher than the extract alone (54%) at 500 µg/mL^72,73. AgNPs biosynthesized using Costusafer leaf extract showed significantly better antioxidant activity than the raw leaf extract. Remarkably, their antioxidant capacity was on par with ascorbic acid, a common antioxidant standard^74,75. AgNPs likely scavenge DPPH and ABTS free radicals by donating electrons, leading to their neutralization⁷⁶. The antibacterial properties of metal NPs are likely attributed to mechanisms such as the release and absorption of free metal ions, which can infiltrate bacterial cells, disrupt cell membrane integrity, and damage nucleic acids, ultimately resulting in cell death⁷⁷. The powerful antibacterial effects of Ag-NPs arise from their capacity to interrupt bacterial cell walls, inhibit with cellular metabolic processes and inhibit the formation of new microbial cells. These extraordinary properties are made possible through the advanced use of nanotechnology⁷⁸. Ag-NPs synthesized using Stachys inflata exhibit impressive antibacterial activity, particularly against multi-drug-resistant Gram-positive and Gram-negative bacteria pathogens, effectively inhibiting their growth⁷⁹. Considering the influence of strain variations in bacterial species and the differences in the shape and structure of synthesized AgNPs, it is reasonable to conclude that these factors may contribute to variations in their susceptibility to antimicrobial agents⁸⁰. AgNPs, at 100 µg/mL, yielded a 16.9 ± 0.10 mm inhibition zone against Salmonella, demonstrating significant antimicrobial activity. Similarly, AgNPs from water, methanol, and acetone extracts effectively suppressed microbial growth⁸¹. Aloe vera leaf extract-mediated synthesis of AgNPs resulted in potent antibacterial activity against various bacterial strains, enhanced by the high surface area and the acemannan compound dual role⁸². However, AgNPs showed a smaller inhibition zone of 9 mm against E. coli and S. aureus⁸³. The enhanced antibacterial activity observed is due to the small size of the AgNPs synthesized using Urticadioica. leaf extract. These diminutive AgNPs possess a high surface-to-volume ratio, which significantly increases their interaction with bacterial cells. This amplified contact enables more effective disruption of bacterial functions, leading to superior antimicrobial effects compared to larger particles^84,85. Potent antimicrobial efficacy against Salmonella enterica (Gram-negative bacterium) and Rhizopus oryzae (fungus) was demonstrated by both crude extracts of Lycium shawii and their mediated AgNPs⁸⁶. Surface charge, colloidal stability, shape, size, and concentration are the key physicochemical properties governing the antibacterial efficacy of AgNPs, powerful antimicrobial agents of growing interest⁸⁷. AgNPs effectively induce cell death and suppress proliferation in colon, breast, liver, and lung cancer cells, highlighting their broad anticancer potential. This activity is linked to their high surface area and ROS generation. The consistent cytotoxic effects across cancer cell lines suggest AgNPs may be valuable in targeted cancer therapies⁸⁸. A cell viability assay was conducted on human lung cancer cell lines (A549) treated with varying concentrations of AgNPs and AuNPs (20, 40, 60, 80, and 100 µg/mL) for 24 h. The results demonstrated that AgNPs exerted a stronger inhibitory effect on cancer cell growth compared to AuNPs, even at lower concentrations. The study further revealed that AgNPs displayed significantly higher cytotoxic activity than AuNPs in a dose-dependent manner. The IC₅₀ values were calculated to be 76.90 µg/mL for AgNPs and 102.30 µg/mL for AuNPs, highlighting the superior effectiveness of AgNPs against lung cancer cells⁸⁹.

The anticancer effectiveness of AuNPs against A549 lung cancer cells is significantly influenced by their concentration. Research indicates that as the concentration of AuNPs increases, there is a consistent rise in cell death among the A549 cells. This relationship suggests that higher concentrations of AuNPs can enhance their cytotoxic effects, leading to more effective induction of apoptosis in tumor cells²⁷. AgNPs were observed to induce DNA damage, which intensified over time, particularly after 24 h of exposure. This progression indicates the disruption of cellular self-regulatory mechanisms and the accumulation of persistent DNA damage. Although no notable rise in intracellular reactive oxygen species (ROS) levels was detected, the cytotoxic effects were linked to the rate at which silver ions (Ag⁺) were released intracellularly. This suggests that the toxicity of AgNPs is primarily driven by the sustained release of Ag⁺ ions within the cells, rather than ROS-mediated pathways, highlighting a distinct mechanism of action underlying their damaging effects on cellular DNA⁹⁰. AgNPs have been shown to activate the apoptotic pathway in vitro by stimulating the generation of ROS. This induction of apoptosis enhances their multifaceted anticancer capabilities, which include anti-proliferative properties that hinder cancer cell growth and anti-angiogenic effects that block the development of new blood vessels critical for tumor progression. These findings emphasize the potential of AgNPs as a therapeutic agent that targets cancer cells through ROS-mediated mechanisms, ultimately leading to cell death and the inhibition of tumor growth⁹¹.

The ethanol extract of Allium sativum was employed as both a reducing and stabilizing agent in the synthesis of Ag-NPs. The biosynthesized Ag-NPs exhibited remarkable cytotoxic activity against A549 human lung cancer cells. Experimental results revealed that 50% inhibition of cell proliferation (IC₅₀) occurred at a concentration of 22 µg/mL. This suggests that the Ag-NPs effectively penetrated cancer cell membranes, disrupted critical cellular processes, and induced cell death, potentially through mechanisms such as oxidative stress, DNA damage, and apoptosis. These findings highlight the potential of Allium sativum-mediated Ag-NPs as a promising candidate for cancer treatment^92,93. AgNPs exhibit a dose-dependent inhibition of cell proliferation. After 24 h of treatment, an IC₅₀ value of 47.58 µg/mL was observed, in contrast to the untreated control group. These findings indicate that Ag-NPs effectively target HeLa cancer cells, significantly diminishing their growth and viability under in vitro conditions. This highlights the potential of Ag-NPs as a promising therapeutic opportunity for cervical cancer treatment¹⁰. Reactive oxygen species (ROS), such as superoxide (O₂•⁻), hydrogen peroxide (H₂O₂), and hydroxyl radicals (•OH), are highly reactive molecules that play a crucial role in normal cellular physiology, including signal transduction and differentiation. A shift in the redox balance towards overproduction or impaired antioxidant defense results in oxidative stress. This pathological state represents a fundamental principle underlying several strategies for cancer treatment⁹⁴. BP-AgNP treatment induced a concentration-dependent decrease in the viability of A431 and B16F10 cancer cells line over 24 h and 48 h. Notably, after 24 h, cell viability remained largely unaffected at lower concentrations (10–50 µg/mL) but was significantly reduced at 100 µg/mL⁹⁵.

Conclusion

A sustainable, eco-friendly, and cost-effective biological method has been developed for synthesizing Pds-AgNPs. FT-IR analysis identified the functional groups involved in the reduction and stabilization processes, confirming the presence of phytochemicals such as flavonoids and phenolics. The leaf extract of P. dubius, rich in surface-active molecules, played a crucial role in stabilizing the NPs. These phytochemicals interacted with the silver surface, enhancing the stability of the Pds-AgNPs. XRD analysis demonstrated that the NPs exhibited a face-centered cubic structure and crystalline nature, as evidenced by the intensity profiles of all recorded peaks. SEM and TEM imaging revealed that the Pds-AgNPs were spherical in shape. The synthesized Pds-AgNPs showed notable antioxidant activity, effectively scavenging free radicals in DPPH and metal chelating assays, outperforming the P. dubius leaf extract. Furthermore, the particle size of the Pds-AgNPs significantly influenced their strong antibacterial activity. The study also highlighted the dose-dependent anticancer potential of the Pds-AgNPs against A549 lung cancer cell lines, underscoring their promising therapeutic applications.

Author contributions

S.V., and V.B., conceptualized the idea, conducted plant extraction, and prepared the original draft. V.B., E.N.S., P.R., S.K., and M.A.T., performed all biological investigations, including molecular docking studies, and contributed to the original draft and its review. All authors participated in data analysis, wrote their respective sections, and approved the final submitted version of the paper.

Data availability

The corresponding author can provide the current research’s datasets upon reasonable request.

Declarations

Competing interests

The authors declare no competing interests.