Introduction

Phytomedicines, the medicines that are derived from one or more plants, are widely used across the world for medicinal purposes such as diagnosis, prevention, and treatment1. As per World Health Organization (WHO) reports, these phytotherapeutic agents contain active plant components, which are considered to be active ingredients, along with additional substances such as solvents or preservatives regarded as excipients2. They are mostly utilized for treating chronic, long-lasting, persistent diseases whose treatment comes with potential side effects and toxicity, or increased cost. Natural products have gained public interest due to their cost-effectiveness, comparatively fewer side effects, and maximum accessibility3. Natural products exhibit the potential to improve the effectiveness of chemotherapy by increasing the sensitivity of disease cells to certain treatments4. These drugs are commonly known as chemosensitizers, having natural elements like vincristine from Catharanthus roseus, as well as extracts from Centaurea albonitens and Solanum nigrum. They can also enhance the toxicity of chemotherapeutic drugs such as doxorubicin and 5-FU without causing any harm to normal healthy cells5. Curcumin has specifically displayed promising results in improving the effectiveness of chemotherapy by targeting NF-κB pathways6. Traditionally, natural medicines are used all around the world for their effectiveness in treating enormous health concerns. These contain some bioactive constituents like alkaloids, flavonoids, tannins, and polyphenols, known for their antimicrobial, antioxidant, antidiabetic, and anticancer activities7.

The phytochemicals found in different plants provide a variety of therapeutic alternatives and options. The Himalayas, a vast mountain range located in Asia, have long been recognized as a significant source of medicinal flora8. The genus Fagonia, belonging to the Zygophyllaceae family, comprises 34 species, distributed all over the world. The medicinal power of this whole plant genus can be attributed to its abundance of secondary metabolites9. One of the important plant species of this genus is Fagonica indica, which possesses numerous health benefits10. This plant is commonly recognized by names such as Dhamasa, Dhamana, and Shoka’a. In local terms, it’s recognized as “Sacchi boti,” meaning “True herb”, and is abundantly produced in Pakistan, Afghanistan, India, and Egypt11. The habitat of this plant is primarily desert or saline areas in temperate and tropical regions across the globe, with a significant presence in the deserts of Asia and Africa12. Numerous studies have found that F. indica contains various important chemical constituents like flavonoids, alkaloids, tannins, saponins, glycosides, and pectin. The medicinal benefits of this plant include anti-inflammatory, laxative, gastroprotective, hepatoprotective, anti-cancer, anti-leishmanial, antipyretic, and antioxidant activity13.

In recent years, there has been a rise in interest in the utilization and production of eco-friendly, less commercial nanoparticles due to notable usage in different processes like catalytic processes, sensing technologies, electronic devices, photonic systems, and medical applications14. Researchers have turned their attention to biological approaches for synthesizing nanoparticles attributed to natural reduction, capping, and stabilizing agents, as well as to avoid hazardous chemicals and excessive energy consumption15. A variety of approaches to producing nanoparticles have been commonly utilized, like chemical approaches using Metal precursors, along with both inorganic and organic stabilizers and reducing agents16. The primary physical method for creating NPs in the material is to break it into smaller pieces by employing various physicochemical techniques, such as electrochemical reactions, microwave (MW) irradiation, and ultrasonication17. The procedures for green synthesis are way more convenient for the formation of NPs and the possibility of minimum loss in terms of cost, precision, effectiveness, and complication18.

The potential advantages of green synthesis processes for synthesizing nanoparticles make them highly appealing19. Various studies have already shown that the biogenic reduction of metal precursors to respective NPs is eco-friendly highly sustainable20, free from any kind of chemical contamination, less expensive and can be used for mass production. Secondary metabolites, mostly proteins, sugars, and even whole cells like enzymes, that stabilize NPs, facilitate the interaction of NPs with other biomolecules, providing enhanced antimicrobial potency by facilitating improved interactions with microorganisms21,22. This study thus aims to explore and compare the antioxidant activities of the green synthesized nanoparticles from the F. indica methanol and water extracts with their respective nanoscale counterparts.

Methodology

Chemicals

Distilled Water (H₂O), Methanol (CH₃OH), Silver Nitrate (AgNO3), Lab prepared Phosphate buffer, Ascorbic acid (C6H8O6), 2,2-diphenyl-1-picrylhydrazyl (DPPH), sulfuric acid (H2SO4), monosodium phosphate (NaH2PO4·H2O), ammonium molybdate tetrahydrate (NH4)6Mo7O24·4H2O), disodium phosphate dihydrate (Na2HPO4·2H2O), monosodium phosphate (NaH2PO4), potassium ferricyanide (K3Fe(CN)6), trichloroacetic acid, ferric chloride (FeCl3),  dimethyl sulfoxide (DMSO). All the chemicals utilized were purchased from Sigma Aldrich, USA.

Methods

This research has been conducted after getting approval from the Institutional Review Board of Shifa Tameer-e-Millat University and Shifa International Hospital, Islamabad, Pakistan with the approval number IRB # 095–24.

Plant material identification

The plant material used in this study was formally identified by Dr. Muhammad Zafar, Department of Plant Sciences, Quaid-i-Azam University, Islamabad, Pakistan. A voucher specimen (PHM-010) has been prepared and deposited in the Herbarium of the Shifa College of Pharmaceutical Sciences, Shifa Tameer-e-Millat University, where it is publicly accessible for future reference. Permission to collect Fagonia indica was duly obtained from the relevant local authorities. All collection procedures were conducted following institutional, national, and international guidelines.

Collection and extraction of plant

The collection of Fagonia indica in Pakistan typically involves gathering the plant’s leaves from its natural habitats, which are arid and semi-arid regions. In this case, F. indica leaves were collected from Muzaffargarh, Pakistan, following proper botanical identification to ensure the authenticity of the species. After collecting, the leaves were air-dried in a shaded area to prevent deterioration of bioactive compounds caused by direct sunlight. Once thoroughly dried, the leaves were finely ground into a uniform powder for use in the extraction process from the plant.

For the aqueous and methanolic extraction of F. indica, the following criteria were used. After collecting and drying, the leaves of F. indica were homogenized into fine particles17. The powdered material (5 g) was soaked in 100 mL of distilled water and methanol each for 24 hours, with constant stirring at interval of 30 minutes. The mixtures were filtered next day and solvents were evaporated using rotary evaporator. The resultant extract was dried and afterwards kept at 4°C for further use. For preparation of aqueous nanoparticles, the powdered plant material was mixed with 100 mL of distilled water at a concentration of 5 mg, and the mixture was left to soak for 24 h. The solution was sonicated at every 30-min interval to maximize cell wall breakdown and facilitate the release of water-soluble bioactive compounds like alkaloids and tannins20. After sonication, it was allowed to rest overnight to ensure thorough extraction. The next morning, the solution was sonicated again to ensure uniformity21. The prepared extract was kept at 4°C for further use 22. For preparation of methanolic nanoparticles, 5 mg of powdered plant material was introduced into 100 mL of methanol for 24 h. The solution was sonicated every 30 min to facilitate cell wall breakdown and enhance the release of bioactive compounds. After sonication, the solution was allowed to rest overnight for complete extraction23. The solution was again sonicated the following morning to obtain uniformity.  Afterwards, the solution was kept at 4°C for further use. This process is preferred for the isolation of polyphenols, flavonoids, and other antioxidants from F. indica15. To make a 10 mM solution of silver nitrate (AgNO₃), 0.16987 g AgNO₃ was dissolved in 100 mL of distilled water. The preparation was made in a volumetric flask wrapped with aluminum foil to avoid any kind of deterioration from light. Once prepared, the solution was kept in the dark. It was ensured that the solution is thoroughly mixed before use24.

Nanoparticles formulation

To synthesize nanoparticles using the water extract, we took 10 mL out of the 100 mL water extract into a volumetric flask25. It was covered with aluminum foil, and it was left on a magnetic stirrer at 60°C, 350 rpm. Slowly, 90 mL of 10 mM AgNO₃ solution was added in a dropwise manner while stirring, and then stirred continuously for 2 h, during which the solution became a dark brown color, showing the formation of nanoparticles26 The same procedure was applied to the preparation of methanolic NPs. After observing a color change, UV–visible spectroscopy analysis was done in the range 300–700 nm, the graph was obtained, and readings were taken27. The nanoparticle solution was subsequently centrifuged at 13,000 rpm for 10 min. It was subsequently washed with the appropriate solvent and centrifuged for 10 min. Washing and centrifugation were repeated three times28. Then the supernatant was decanted, and the nanoparticle pellets were taken into a petri dish, which was put in a hot air oven at 25°C for 3 h. Once the initial drying period was complete, the petri dish was left in a dry environment to allow the nanoparticles to air dry for an additional 24 h (Table 1).

Table 1 Sample identification.
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Characterization of nanoparticles

Scanning electron microscopy(SEM)

SEM analysis was done by virtue of an electron microscope (Model: MIRA3, TESCAN, Czech Republic) to observe the microstructure and surface chemistry of the synthesized nanoparticles. SEM images of FIAqNps and FIMNps samples were taken using accelerated electrons with a 15 kV voltage at a nanometer scale resolution29.

Zeta potential

The zeta potential of the synthesized FIAqNps and FIMNps was determined using a zeta potential analyzer (MAL1168467). The measurements were conducted at room temperature with the nanoparticles dispersed in their respective solvents, i.e., distilled water for FIAqNps, and methanol for FIMNps. The stability of the nanoparticles was determined based on the magnitude of the zeta potential values30.

FTIR spectroscopy

FTIR analysis was performed with a Cary 630 FTIR (Agilent Technologies). All of the samples, FIAqE, FIME, FIAqNps, and FIMNps were transferred to the sample holder to be measured. Evaluations of the nanoparticles covered the 1000–3300 cm−1 wavelength. All of the samples were studied, and the records of the readings were taken to find out which functional groups took part in the synthesis of AgNPs31,32.

UV spectroscopy

To confirm the formation of AgNPs, the samples were examined with a UV–visible spectrophotometer (UV-1601 from Shimadzu, Japan). The spectrum was recorded for all four samples, FIAqNps, FIAqE, FIME, and FIMNps, from 300 to 700 nm33. At the beginning, all solutions were diluted 10 times, mixed in matching solvents, and the final sample was made up to 10 mL for each one. The analysis for the last 3 mL of the solution was done in a quartz cuvette. To perform the baseline correction, distilled water and methanol were used as the blank solution34.

X-ray diffraction

A usual powder x-ray diffraction setup primarily consists of four components, including: An X-ray source, a specimen stage, receiving optics, X-ray detector35. The X-ray source, detector, and their associated optics were arranged along the boundary of a focusing circle, while the sample stage was aligned at the center of the circle. Both the samples of Fagonia indica, including FIAqNps and FIMNps, were placed on a silicon holder. XRD data were collected in the range of 20–80°26. X-ray diffraction readings were collected on a Rigaku Smart lab diffractometer by the application of the planar monochromator method and Cu Kα radiation. Calculation of the cell parameters was performed through a Voigt Profile fitting procedure. The average crystalline size of the diffraction line widths was measured using the Scherrer formula. For calculation procedure, the line widths of the most intensive peak were utilized36.

Antioxidant assays

A total of four samples were prepared, each at a concentration of 4 mg/mL, using aqueous and methanolic extracts of F. indica and their corresponding nanoparticles37. These four samples were labeled accordingly and were utilized during the performance of all antioxidant assays. Ascorbic acid was taken as a positive control. 1 mg/mL positive control was prepared by dissolving 4 mg of ascorbic acid in 4 mL of dimethyl sulfoxide (DMSO). In all assays, DMSO and 10 mM AgNO3 salt solutions acted as the negative control38.

DPPH free radical-scavenging activity

Stock solution and procedure

The free radical-scavenging assay using DPPH was performed according to previously described protocols39. The stock solutions were prepared by taking 9.6 mg of DPPH reagent and dissolving it in 100 mL of methanol. The solution was then stirred on a magnetic stirrer for one hour at a speed of 350 rpm. 10 μL of all samples were added to the individual wells of a 96-well plate at the final concentrations of 100, 50, 25, and 12.5 μg/mL. For the positive control, the same serial dilutions were prepared. Once all dilutions were prepared, 190 μL of DPPH reagent (properly covered to avoid light exposure) was added to each well40. The plate was then covered and incubated for 1 h at 37 °C. After the incubation period, the absorbance was calculated at 517 nm by using a microplate reader to assess the antioxidant activity of the samples. The formula was used for calculation of %inhibition; %inhibition = (1-Absorbance of sample/Absorbance of control)X100.

Total antioxidant capacity

1.63 mL of sulfuric acid was mixed with 50 mL of distilled water in a 100 mL flask to prepare the reagent (0.6 M H2SO4, 28 mM NaH2PO4, 4 mM Ammonium molybdate)41. Then, 0.4918 g of monosodium phosphate (NaH₂PO₄·H₂O) (or 0.218 g of NaH₂PO₄·2H₂O) was added along with 0.247 g of ammonium molybdate to the solution. As following previously reported protocol, 100 μL of each sample, including FIAqE, FIME, FIAqNps, and FIMNps, was taken in four separate Eppendorf tubes42. Additionally, 100 μL of ascorbic acid was added to another Eppendorf tube as the positive control, while 100 μL of DMSO and 100 μL of AgNO₃ were added to two separate tubes as negative controls. Next, 900 μL of prepared reagent was added to each tube, and the initial color of the solution was noted. Then the tubes were incubated at 95 °C for 90 min in a water bath14. After incubation, color changes were observed, and the tubes were allowed to cool at room temperature. After this, 200 μL from each Eppendorf tube was transferred to a 96-well plate. The plate was then analyzed using a microplate reader, and the absorbance was measured at a wavelength of 630 nm. The results were presented as absorbance unit (a.u.) demonstrating the total antioxidant activity (TAA) of the test samples43.

Total reducing power assay

A 0.2 M phosphate buffer with a pH of 6.6 was prepared by mixing 1.42 g of Na₂HPO₄·2H₂O and 1 g of NaH₂PO₄ in a 100 ml flask. Then, 50 ml of distilled water was added to the flask. The pH of the solution was measured using a pH meter, and adjustments were made with the sodium hydroxide as needed and required14. The solution was then stirred on a magnetic stirrer for about ten minutes. A 1% potassium ferricyanide solution was prepared by the dissolution of 1 g of potassium ferricyanide in a 100 mL of distilled water, 10% trichloroacetic acid solution was made by dissolving 10 g of trichloroacetic acid in a 100 mL of distilled water, and finally a 0.1% ferric chloride solution was prepared by dissolving 0.1 g of ferric chloride in 100 mL of distilled water44. As per the protocols, 100 μL of each sample, including FIAqE, FIME, FIAqNps, and FIMNps, was taken into four separate Eppendorf tubes45. Along with that, 100 μL of ascorbic acid was taken into another Eppendorf tube, which served as the positive control, and negative controls were established by 100 μL of DMSO and 100 μL of AgNO₃ into two separate tubes. 200 μL of 0.2 M phosphate buffer (pH 6.6) and 250 μL of 1% potassium ferricyanide was then added to each Eppendorf tube, after which they were incubated at 50 °C for 20 min After incubation, 200 μL of 10% trichloroacetic acid was added to all the tubes, and then the tubes were centrifuged at 3000 rpm for 10 min46.

After centrifugation, 50 μL of a 0.1% ferric chloride solution was introduced into the designated wells of a 96-well plate. Then, 150 μL of the supernatant obtained from the centrifuged samples was carefully transferred into the appropriate wells. The contents of each well were mixed thoroughly to achieve a uniform distribution. The plate was then analyzed using a microplate reader, and the absorbance was measured at a wavelength of 630 nm to evaluate the outcomes. The results were demonstrated as absorbance unit (a.u.).

In Silico methodology

Ligand and protein selection and preparation

Four key phytoconstituents commonly reported in Fagonia indica, quercetin, kaempferol, apigenin, and luteolin, were selected for docking analysis. Their 3D structures were retrieved from the PubChem database, and 3D structures were generated using Open Babel and minimized using the MMFF94 force field. Three antioxidant-relevant enzymes were selected. Nrf2–Keap1 complex (PDB ID: 6FFS), which is responsible for regulating antioxidant genes. Cyclooxygenase-2 (COX-2, PDB ID: 5IKQ) is involved in oxidative inflammation, and Xanthine oxidase (XO, PDB ID: 3NRZ) are the key enzyme in ROS generation. Proteins were prepared by removing water molecules and co-crystallized ligands and adding hydrogen atoms, and assigning Gasteiger charges. Saving the structure in PDBQT format using AutoDockTools (v1.5.7).

Drug-likeness & ADMET profiling

Physicochemical and Lipinski’s Rule parameters were calculated using RDKit. ADMET (Absorption, Distribution, Metabolism, Excretion, Toxicity) predictions were carried out using SwissADME and pkCSM.

Results

Extract formation and nanoparticles yield

To synthesize the nanoparticles from aqueous and methanolic extracts of F. indica, the dried plant material was soaked in the respective solvents. After stirring for the designated period, the color change was observed in Fig. 1A. After the addition of salt solutions and centrifuging for a designated time, the mixture was centrifuged to separate the supernatant from the pellets of nanoparticles, which were settled at the bottom of the Falcon tubes, as presented in Fig. 1B. Overall, the resulting percent yield of both types of NPs has been presented in Table 2.

Fig. 1
figure 1

(A) Fagonia indica extracts before (a FIME, b FIAqE) and after treatment with AgNO3 solution (c FIMNPs, d FIAqNPs), showing a noticeable color change to darker tones. (B) The FIAqNps and FIMNps were separated after centrifugation at 13,000 rpm.

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Table 2 Obtained quantities and calculated percentage yields of silver nanoparticles of F. indica.
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Characterization

UV–visible spectra analysis

Green synthesized FIAqE and FIME AgNPs absorbed the radiation in a range between 300 and 700 nm. In case of aqueous extract, the maximum absorbance (λmax) was recorded at 328.5 nm (0.129 Å) however, in the case of methanol extract, the absorption maxima (λmax) were recorded at 323.5 nm (0.240 Å). In case of aqueous nanoparticles, the maximum absorbance (λmax) was recorded at 431.0 nm, with a correspondence of 0.227 Å, while in the case of methanolic nanoparticles, the absorption maxima (λmax) were recorded at 320.6 nm (0.222 Å). It is generally observed that the λmax was between 320 and 431 nm for the AgNPs to have the desired size. A detailed explanation of the results is being provided in Table 3.

Table 3 UV–visible absorbance values of FIAqE, FIME, FIAqNps, and FIMNPs.
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FTIR spectroscopy

FTIR spectroscopy was done for the qualitative analysis of the expected reducing and capping compounds/metabolites that are involved in the synthesis of FIAqNps and FIMNps and their comparison with extracts, FIAqE, and FIME. In this study, the FTIR spectrum of FIAqE showed major peaks at 3533.5 cm−1, 3391.9 cm−1, 2870.1 cm−1, 2206.6 cm−1, 1982.9 cm−1, 1684.8 cm−1, 1617.7 cm−1 and 1431.3 cm−1, 1095.8 cm−1, 1006.4 cm−1, 838.7 cm−1, and 670.9 cm−1. FIME showed at 3287.5 cm−1, 2922.2 cm−1, 1617.7 cm−1, 1513.3 cm−1, 1252.4 cm−1, 1408.9 cm−1, 1066.0 cm−1. FIAqNps showed major peaks at 3356.5 cm−1, 2372.5 cm−1, 2115.3 cm−1, 1869.3 cm−1, 1751.8 cm−1, 1623.3 cm−1, and 1287.8 cm−1 and FIMNps presented major peaks at 3345.3 cm−1, 2920.4 cm−1, 2372.5 cm−1, 2115.3 cm−1, 1869.3 cm−1, 1735.1 cm−1, 1640.0 cm−1, 1282.2 cm−1, 1041.8 cm−1, and 801.4 cm−1 (Fig. 2).

Fig. 2
figure 2figure 2

FTIR spectra of plant extracts and their corresponding nanoparticles. (A) Spectrum of FIAqE showing a major peak at 3533.5 cm⁻1, and (B) FIME exhibiting a peak at 3287.5 cm⁻1, both indicative of O–H stretching vibrations from hydroxyl groups. (C) Spectrum of FIAqNps with a key peak at 3356.5 cm⁻1, and (D) FIMNps showing a peak at 3345.3 cm⁻1, suggesting the presence of phenolic and alcohol groups involved in nanoparticle capping. The x-axis represents wavenumber (cm⁻1), and the y-axis shows transmittance (%). These spectral shifts confirm the involvement of phytochemicals in the reduction and stabilization of the nanoparticles.

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The presence of functional groups e.g. hydroxyl, carbonyl, and amine peaks in all four samples (FIAqE, FIAqNps, FIME, FIMNps) indicated that these played a crucial role in reducing silver ions and stabilizing the nanoparticles. Most importantly, the peaks observed in the 1600–3500 cm⁻1 range were seen in both FIAqNps and FIMNps samples, suggesting the involvement of phenolic compounds and proteins as capping agents, which justified the stability of nanoparticles formed from silver salt.

Zeta potential

A zeta potential analysis was conducted to determine the charge on the surfaces of green-synthesized FIAqNps and FIMNps. Good stability is indicated by zeta potential values greater than + 30 mV or less than − 30 mV. The zeta potential of FIAqNps values were − 11.8 mV, FIMNps values 1.72 mV, while the Polydispersity Index (PDI) of FIAqNps was 1.000 and FIMNps was 0.531 (Fig. 3).

Fig. 3
figure 3

Zeta potential distribution and size distribution by intensity of FIAqNps and FIMNps. (A) Zeta potential graph of FIAqNps showing a sharp peak at -11.8 mV, indicating low surface charge and limited colloidal stability. (a) Size distribution of FIAqNps with a narrow peak at214.8 nm, indicating monodispersity. (B) Zeta potential graph of FIMNps showing a high positive surface charge (+1.72 mV), suggesting enhanced colloidal stability. (b) Size distribution of FIMNps showing a sharp peak 28.18 nm, reflecting uniform and smaller particle size.

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Scanning electron microscopy

SEM was performed to determine the shape of FIAqNPS and FIMNps. Some aggregates were observed in the SEM images. The images also confirmed the non-spherical, irregular-shaped symmetry of synthesized nanoparticles in our colloidal suspension at the scale of 10 µm (Fig. 4).

Fig. 4
figure 4

Scanning Electron Microscopy (SEM) images of FIAqNps and FIMNps. (A) SEM image of FIAqNps at 2500 × magnification showing irregularly shaped, loosely agglomerated particles with rough surfaces. (B) SEM image of FIMNps at 1000 × magnification depicting more compact and clustered nanoparticles with relatively smoother and denser surface morphology. Both images were captured at an accelerating voltage of 20 kV with a scale bar of 10 µm.

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X-ray diffraction (XRD)

The XRD analysis of the synthesized FIANps and FIMNps confirmed the successful formation of crystalline nanoparticles. The diffraction patterns of both the samples showed characteristic peaks corresponding to the face-centered cubic (fcc) structure of metallic silver. The XRD pattern for Sample FIANps displayed distinct diffraction peaks at 2θ values around 38.1°, 44.3°, 64.4°, and 77.3°, which correspond to the (111), (200), (220), and (311) planes, respectively. These peaks align with standard JCPDS (Joint Committee on Powder Diffraction Standards) data for silver (File No. 04–0783), indicating the formation of crystalline silver nanoparticles. The broadness of the peaks suggested the nanoparticles were in the nanometer size range, with some degree of amorphous content likely due to organic stabilizers from the plant extract.

FIMNps exhibited similar diffraction peaks at approximately the same 2θ positions, indicating that both aqueous and methanolic syntheses led to the formation of fcc silver. However, the peaks for FIMNps were slightly sharper and more defined, suggesting a higher degree of crystallinity and potentially larger particle size compared to FIAqNps. Both XRD patterns confirmed the crystalline nature of the synthesized AgNPs, with no additional peaks detected that would indicate impurities or by-products. The average crystallite size, calculated using Scherrer’s equation, revealed that the particles for both samples were within the expected nanometer range, with FIAqNps showing smaller average crystallite sizes compared to FIMNps (Fig. 5).

Fig. 5
figure 5

X-ray diffraction (XRD) patterns of synthesized nanoparticles. (A) XRD profile of FIAqNps exhibits prominent peaks at 2θ = 38.1°, 44.3°, 64.5°, and 77.4°, corresponding to the (111), (200), (220), and (311) planes of the face-centered cubic (FCC) structure of silver nanoparticles, in accordance with JCPDS card No. 04–0783. The sharpness and intensity of the peaks indicate good crystallinity. (B) XRD pattern of FIMNps shows similar diffraction peaks with slightly increased intensity and reduced peak width, suggesting enhanced crystallinity and smaller crystallite size due to effective capping by methanolic phytoconstituents. These results confirm the successful biosynthesis of crystalline metallic silver nanoparticles in both aqueous and methanolic extracts.

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Antioxidant DPPH assay

In the DPPH experiment, FIMNps exhibited the highest percentage of inhibition, 18.93 ± 1.51%, indicating that it had a more powerful radical scavenging ability than FIME (16.32 ± 1.42%). However, when it comes to aqueous solvents, FIAqE exhibited a greater proportion of inhibition, 11.28 ± 1.53%, than FIAqNp 0%. The aqueous nanoparticle form may not perform well in this test for scavenging free radicals, as indicated by the 0% inhibition shown by FIAqNp (Fig. 6A and B).

Fig. 6
figure 6

(A): 96-well plate showing the colorimetric changes in the wells containing FIAqE, FIME, FAqNps, FIMNps, positive and negative controls. (B): DPPH radical sacvanging activity at 100 µg/mL. Bar chart depicting the percentage inhibition activity of aqueous extract (FIAqE), methanolic extract (FIME), and their respective silver nanoparticles (FIAqNp and FIMNp) of Fagonia indica. The assay was performed in triplicate (n = 3), and results are shown as mean ± SEM. FIMNp exhibited the highest inhibition, followed by FIME. FIAqNp showed no significant inhibition under the tested conditions, highlighting differences in nanoparticle efficacy based on extract type and phytochemical composition. The positive control showed 85.6 ± 2.51% inhibition.

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Total antioxidant capacity assay

The FIAqNps showed a higher antioxidant activity compared to the standard extract FIAqE, indicating enhanced antioxidant potential. In the case of methanol, FIMNps shows a higher AAE value than FIME, indicating increased bioavailability of non-polar antioxidant compounds (Fig. 7A and B).

Fig. 7
figure 7

(A) 96-well plate showing the colorimetric changes in the wells containing FIAqE, FIME, FAqNps, FIMNps, positive and negative controls (TAC Assay). (B) Total Antioxidant Capacity Assay: Bar graph showing total antioxidant activity of aqueous extract (FIAqE), methanolic extract (FIME), and their respective silver nanoparticles (FIAqNp and FIMNp) of Fagonia indica. Antioxidant capacity is expressed in absorbance units (a.u.). Data are presented as mean ± SEM (n = 3). Error bars represent the standard error of the mean. The FIAgNPs and FIMNPs exhibited significantly higher antioxidant activity compared to the plant extracts alone.

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Total reducing power assay

FIME has the highest AAE value as compared to FIMNps, demonstrating its ability to reduce oxidative species without nanoparticle enhancement. But in another case, FIAqNp showed a modest increase over FIAqE (Fig. 8A and B).

Fig. 8
figure 8

(A) 96 well plate showing the colorimetric changes in the wells containing FIAqE, FIME, FAqNps, FIMNps, positive and negative controls (TRP Assays). (B) The graph represents the results. Bar chart depicting the total reducing power of aqueous extract (FIAqE), methanolic extract (FIME), and their respective silver nanoparticles (FIAqNp and FIMNp) of Fagonia indica. The assay was performed in triplicate (n = 3), and results are shown as mean ± SEM.

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Targeted proteins and ligands 3D structures

The 3D structures of three protein Nrf2–Keap1 complex (PDB ID: 6FFS), Cyclooxygenase-2 (COX-2, PDB ID: 5IKQ) and Xanthine oxidase (XO, PDB ID: 3NRZ) which were taken from RCSB (Fig. 9: A–C). They were clean in Discovery Studio by removing water molecules, ligand or other unnessesory molecules. Then in Auto Dock Tools water molecules were added, and charges i.e. Kollman Charges were added to them and saved their file in PDBQT format for further docking.

Fig. 9
figure 9

Targeted Proteins and ligands 3D Structures. Proteins are (A) Nrf2–Keap1 complex (PDB ID: 6FFS), (B) Cyclooxygenase-2 (COX-2, PDB ID: 5IKQ), (C) Xanthine oxidase (XO, PDB ID: 3NRZ). Ligands are (D) Quercetin, (E) Kaempferol, (F) Apigenin, and (G) Luteolin.

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The compounds reported in Fagonia indica which are used as ligands in this study are quercetin, kaempferol, apigenin, and luteolin, were selected for docking analysis. Their 3D structures were retrieved from the PubChem database in SDF format minimized using the MMFF94 force field. The 3D structures of ligands by using Discovery studio used further for docking procedure (Fig. 9D–G).

Molecular docking binding affinities

Quercetin and luteolin showed the strongest binding across all targets, particularly with COX-2 and XO. Binding energies below − 8.0 kcal/mol indicate strong and stable interactions (Table 4).

Table 4 Target and protein interactions with binding energies kcal/mol.
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Drug-likeness summary (Lipinski’s rule of five)

Quercetin and luteolin are the most promising antioxidant agents, with high binding affinity and favorable pharmacokinetics. Methanolic extracts of Fagonia indica, which likely contain higher levels of these flavonoids, demonstrate enhanced antioxidant activity, supporting the experimental findings of your study. The drug-likeness properties of Quercetin, Kaempferol, Apigenin, and Luteolin were evaluated using Lipinski’s Rule of Five, and none of the compounds violated these rules, supporting their potential bioavailability. These compounds may act through multiple targets like Nrf2, COX-2, and XO, validating their multi-target antioxidant mechanism. All ligands satisfy Lipinski’s rule, indicating good oral drug-likeness (Table 5 and 6).

Table 5 Drug-likeness summary (Lipinski’s rule of five).
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Table 6 ADMET summary.
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Discussion

Natural products have become increasingly central to drug discovery, serving as key sources for new pharmacologically active compounds. Advances in modern research have expanded our understanding of the therapeutic potential, structural diversity, and biological activities of these natural substances46. Nowadays, bioactive natural products are often used as probes in biochemical and molecular studies to help identify specific therapeutic targets and mechanisms47. This progress has been facilitated by developing sensitive techniques for detecting and isolating biologically important compounds. With the use of modern methods to isolate, extract, purify, understand and analyze these ingredients, scientists have learned more benefits as well48. The use of Fagonia indica is well-documented in traditional medicine, mainly as an antidiabetic, against cancer, leishmaniasis, fevers, inflammation, helpful as a laxative, protects the gut and liver and has strong antioxidant properties16. Many bioassays were used in the study to test the antioxidant properties of F. indica. The plant material was harvested, washed and dried outside in shade to stop sunlight from harming the important ingredients. The method medicinal plants are dried, both indoors and outdoors, has a strong effect on their yield and the chemicals they contain49. Sun-drying results in photodecomposition which weakens the plant and leaves it open to being attacked by bacteria. Unlike other methods, air drying is not expensive, it’s better for the environment and it ensures the plant’s healing power is not lost. After drying, the leaves of F. indica were ground and their extracts and nanoparticles were tested for antioxidant activity50.

Solvents methanol and water were used to make extracts from F. indica. Because of this, the ability of each solvent to obtain various bioactive compounds could be evaluated51. Because water is safe for the environment and follows green chemistry ideas, it is labeled as the ‘greenest solvent’ and helps to thoroughly dissolve phytoconstituents with a low environmental impact52. Unlike ether, the solvent methanol is generally polar and can dissolve both polar and nonpolar chemicals which is useful for extracting more types of phytochemicals. The highest DPPH free radical scavenging activity was shown by the methanolic extract, indicating that compounds with varying polarity can act as effective free radical scavengers19. The choice of solvent had the influence on the chemical composition and biological activity of the synthesized silver nanoparticles applying green methods22. To improve extraction efficiency, sonication-assisted maceration was employed. Sonication helps in the disruption of plant cell walls by utilizing high-frequency sound waves, releasing intracellular compounds and increasing the yield of bioactive constituents53. The combined effect of the former technique along with conventional maceration enhanced solvent penetration and compound extraction54. After 24 h, extracts were filtered to remove plant residues, and the color of the extracts was noted as an indicator of phytochemical content55. UV-spectroscopy was then used to analyze the absorbance spectra, giving information about the chemical makeup and potential bioactivity of extracts and reaction process56.

Recent progress in nanotechnology has greatly impacted healthcare. In this study, a higher yield of F. indica nanoparticles was obtained from aqueous extracts compared to methanolic extracts, likely due to the polarity and hydrogen bonding of water, which promote better solubilization and uniform nanoparticle formation57. Low volatility of water also helps maintain stable reaction temperatures, reducing unwanted side reactions and by-products16. Using plant extracts as reducing agents allowed silver ions to turn into silver nanoparticles without releasing harmful chemicals, helping sustainability as well. Proper settings for extract and silver nitrate concentration, temperature, light exposure and pH were determined to manage the method of nanoparticles formation and their stability19. After the synthesis, the nanoparticles were separated in a centrifuge, cleaned and dried so a stable powder could be used for additional analysis58. Properties of silver nanoparticles made with aqueous and methanolic extracts were examined using FTIR, UV–visible spectroscopy, zeta potential analysis and SEM. This information on the size, shape, chemistry and stability of the nanoparticles was obtained by using these techniques59.

Absorbance peaks seen in both extracts at 320–431 nm proved that AgNPs were created successfully60. FTIR indicated that the nanoparticles contain hydroxyl, carbonyl and amine which help in their reduction and stabilization. Carbonyl bonds found at 1600–3500 cm⁻1 point to phenolic compounds and proteins covering the nanoparticles, which makes them more stable and ecologically safe61. Particles in water had a zeta potential of − 11.8 mV and the methanolic ones had 1.72 mV, showing that both were relatively stable because their zeta potential was below ± 30 Mv62. At the same time, the aqueous drugs formed a relatively stable colloidal mixture. On average, particles had a diameter of 104.3 nm. Based on the PDI, aqueous nanoparticles showed a wider range of sizes, while methanolic ones had a similar size and were more uniform63.

It was found using SEM that, despite nanoparticles clumping and not being clearly spherical, their size was still appropriate for those made from plants64. More uniformization of nanoparticles might be achieved by using dialysis or ultrafiltration Broad peaks in the XRD patterns suggested that the nanoparticles were crystalline but also amorphous because of plant substances covering them42. More crystalline or larger nanoparticles were found with methanol as the solvent, as demonstrated by wider peaks on the XRD graphs65. Methanolic nanoparticles (FIMNps) scavenged more free radicals than the normal methanolic extract (FIME) which demonstrates that adopting a nanoparticle formulation can improve antioxidant activity66. The reason the aqueous extract showed more inhibition than the nanoparticle form is that aggregation of nanoparticles may decrease the total surface area available for activity67. Methanol being water soluble and easy to absorb in the body kept the vital molecules available for use in the reaction. It was found that methanol extracts were most effective at reducing power because methanol can dissolve both polar and non-polar compounds, which include flavonoids and phenolics68. Using nanoparticles made the reduction process more effective, due to the extra surface area and closer interaction with the test liquids69. Interestingly, nanoparticles based on aqueous solutions (FIAqNps) performed better in reducing reactions than the normal particles, overcoming the issues that water causes. Alternatively, nanoparticle synthesis in methanol (FIMNps) did not cause a notable rise in reducing power, possibly because the particles aggregated, limiting their surface area70. All in all, nanoparticles of methanol extracts had more reducing power than aqueous extracts of plants and nanoparticle formation was key in improving their antioxidant effect.

These results were confirmed by the total antioxidant capacity (TAC) assay which proved that nanoparticles, mainly those from methanol, highly increase antioxidant activity by promoting the activity and absorption of key compounds71. Nanoparticles extracted with methanol (FIMNps) showed the highest TAC levels, due to the solvent’s ability to bring out multiple biological active molecules and the expanded surface area after nanosizing. There was a higher antioxidant activity with aqueous nanoparticles (FIAqNps) than with whole aqueous extracts, due to better stability and easier mixing of the hydrophilic antioxidants72. It’s noteworthy that the crude methanol extracts (FIME) displayed the weakest total antioxidant capacity (TAC), suggesting that the process of forming nanoparticles is key to unlocking the complete antioxidant potential of compounds soluble in methanol73. These findings underscore how the selection of the solvent and the application of nanoparticle synthesis are critical factors in achieving the highest antioxidant effectiveness from plant extracts74.

Conclusion

In this study, DPPH, Total Antioxidant Capacity (TAC) and Total Reducing Power assays were chosen to analyze the antioxidant performance of F indica extracts as well as its green-synthesized silver nanoparticles (AgNPs). According to the analysis, we infer the effect of both the solvent and the nanoparticles on antioxidant ability of F.indica. FIMNps demonstrated the greatest level of Ascorbic Acid Equivalency (AAE) in the DPPH radical scavenging assay, which suggests it is more effective at eliminating radicals. Unlike other materials, FIMNps did very well in the TAC test, proving that nanosizing chemicals from methanol enhances their stability and reaction. The observed increase in antioxidant capabilities could be attributed to methanol’s effectiveness in extracting a broader spectrum of active compounds, potentially including lipophilic antioxidants. These types of antioxidants may exhibit enhanced potency when formulated as nanoparticles.

Total Reducing Power studies showed that FIME is superior to FIAqE and its nanoparticle equivalent at donating electrons. It may be linked to methanol being able to dissolve a large variety of antioxidants, especially flavonoids and polyphenols which are strong reducers. Despite this, the aqueous nanoparticles demonstrated a respectable reducing power, suggesting that nanosizing may partially compensate for the typically lower reducing capacity of hydrophilic compounds by increasing their reactivity. UV–Vis spectral analysis further confirmed successful nanoparticle synthesis, with a notable λmax shift in aqueous nanoparticles, signifying characteristic surface plasmon resonance in silver nanoparticles. The study’s findings affirm that nanoparticle formulation, especially in methanol, substantially enhances antioxidant activity across assays, likely due to increased surface area and optimized compound stability. Meanwhile, aqueous-based nanoparticles offer a viable alternative for applications requiring high radical scavenging with excellent bioavailability.

Overall, this research provides a basis for the application of F. indica-derived nanoparticles as potent natural antioxidants, with implications for therapeutic use in oxidative stress-related conditions. Future studies could focus on further optimizing nanoparticle synthesis methods to refine antioxidant capacity and explore in vivo applications, offering a promising eco-friendly approach to developing plant-based antioxidant therapeutics.

Future recommendations

Future research can emphasize the practical knowledge and diverse uses of nanoparticles formulated through green synthesis methods, particularly those extracted from F. indica. Different classes of natural compounds with low or no toxicity and minimal side effects can be selected as promising agents in combating a range of diseases linked to oxidative stress. Consequently, the wide range of pharmaceutical applications of these natural compounds holds significant importance for targeting deadly diseases, including cancer, microbial infection, and inflammation. Future efforts should focus on human welfare by prioritizing the bioavailability, stability, and effectiveness of nanoparticles. However, a more detailed and comprehensive analysis of formulated nanoparticles can be done through Transmission Electron Microscopy (TEM). The future insight revolves around the understanding of the morphology of nanoparticles and their impact on the biological activity of nanoparticles.Finally, the synthesis of environmentally sound and easily scalable methods for producing nanoparticles will be crucial for widespread adoption and translation.