Introduction

Traditional medicine has utilized herbs and their extracts since ancient times. Because it includes natural medicinal components, over 80% of people still rely on herbal therapy. Examples include, but are not limited to, medicinal substances that can reduce the growth of various cancer cell types and impede the proliferation of various pathogenic microorganisms (Azmir et al., 2013; Tsiaka et al., 2017). Herbal extracts also have antioxidant properties, which are crucial for lowering oxidative stress (Elshaer et al., 2022). In addition to the medical sector, the fields of dietary intake, nutritional supplements, and cosmetics also use herbs and their extracts (Ahmed et al., 2022). Almost 85,000 species of plants for used for medicinal purposes globally. However, many of these species with many phytochemicals are still unexplored; Sausurea costus (S. costus), belonging to the Asteraceae family, is one of these unexplored pharmaceutical plants (Elshaer et al., 2024).

Saussurea costus, a plant of medicinal importance, has been used for long in conventional Saudi Arabian medicine. Being a threatened species, it is thoroughly studied for its chemical formula, importance, and therapeutic value for conventional phytomedicine. It exhibits a variety of biological activities (Elnour and Abdurahman, 2024; Mujammami, 2020).

The goal of nanotechnology is to create nanoparticles (NPs) with controlled dimensions, form, and chemical makeup that could be used for the health benefits of humans. Although some of the physical and chemical methods (e.g., ball milling, chemical vapor deposition) can successfully manufacture well-defined nanoparticles, they are expensive and may present environmental hazards (Bhattacharya and Rajinder, 2005; Sastry et al., 2004). Nanoparticles are produced using a variety of methods, the most popular of which are chemical ones (Mohanpuria et al., 2008). Green synthesis methods provide a viable option by producing nanoparticles from sustainable resources, such as bio-based substances (Almayouf et al., 2024; Karim et al., 2023; Xu et al., 2023).

The possible use of metal nanoparticles as antibacterial substances has generated a lot of interest (Alshahrani et al., 2021; Rezaei et al., 2021). The high biocompatibility and low cytotoxicity of selenium nanoparticles (SeNPs) have made them popular and are widely used in food science and pharmaceuticals (Pon Matheswari et al., 2022). High absorption, low toxicity, and strong antibacterial and antioxidant activity are reported for SeNPs (Mellinas et al., 2019). A viable substitute for physical and chemical methods for the synthesis of SeNPs is the incorporation of microbes, plant extracts, and green chemistry (Adibian et al., 2022). There are a number of benefits for using biological or environment-friendly methods to produce inorganic nanoparticles. For example, biosynthetic methods are low-cost, environmentally benign, one-step, and clean processes that reduce or completely eradicate pollutants and ecological harm (Esmaili et al., 2022). Through the provision of reducing/stabilizing agents and herbal capping, plant extract-mediated nanoparticles enhance the formation of SeNPs. Furthermore, the formation of SeNPs can be accelerated in a single step by the bioactive ingredients present in plant extracts (Korde et al., 2020; Shayan et al., 2024). There is a great need to apply green synthesized nanoparticles in many medical applications. S. costus is used to prepare many nanoparticles, but few studies are conducted to prepare SeNPs and test its variable applications (Ao et al., 2023). There is a recent trend to investigate the various possible biological activities of plant extract-mediated nanoparticles. The objective of the current work was to biosynthesize SeNPs from the root extract of S. costus, characterization of the prepared nanoparticles, and comparing some of the in vitro biological activities of prepared extract and green synthesized SeNPs.

Materials and Methods

Plant and chemicals

The chemicals used in the study were acquired from Sigma Co. Ltd. (St. Louis, MO, USA). Fresh roots of S. costus were acquired from a local specialty herbal shop in Saudi Arabia. The identification of roots was confirmed at Al-Azhar University’s Faculty of Science in Cairo, Egypt with reference No. AZH-E-226. The sample’s certificate of authenticity was recorded at the faculty’s public herbarium.

Preparation of S. costus extract

In all, 50 g of dry roots of S. costus were homogenized, kept in a secured vessel, and mixed with 0.5 L of 90% ethanol. The mixed contents were left for 3 days in glass jars at an ambient temperature. The extract was kept for 60 min in a disruptor that was set at 40°C for traditional extraction method. After filtration and spinning the extract in a rotatory evaporator at 40°C, crude extract was created (Marinova et al., 2005).

High-performance liquid chromatography (HPLC) examination of S. costus extract

A 10 µL aliquot of the S. costus extract was injected into the HPLC system (Agilent Technologies, CA, USA) equipped with a C18 column (4.7 mm × 250 mm, 10 µm particle size). The column temperature was maintained at 46 °C. The mobile phase consisted of (a) water and (b) acetic acid (0.05%) in acetonitrile at a flowing rate of 0.99 mL per minute. The mobile phase was set up as follows: 0 (82% A), 0–1 (85% A), 1–11 (70% A), 11–18 (65% A), 18–22 (80% A), and 22–24 (80% A). Additionally, the multi-wavelength detector was recorded at 280 nm. An injection volume of 5.0 μL was used for every sample solution (Gupta et al., 2023).

Preparation of SeNPs using S. costus extract

In all, 80-µL aliquot of S. costus extract (50 mg/mL) was incorporated into Na₂SeO₃ solution at the final level of 10 mM in order to synthesize SeNPs. The biosynthesis process was performed by continuous stirring at 1,400 rpm for 35 min at 42°C. The reaction was completed at an ambient temperature in 24 h with continuous stirring in the dark. The solution with colloidal particles was kept for further examination and use at 5°C (Garza-García et al., 2023).

Characterization of SeNPs

The produced SeNPs were analyzed at 200–600 nm using a UV spectrophotometer (Drawell, China). The field emission scanning electron microscope (SEM; JEOL, Japan) was used to investigate the produced particles. Energy dispersing X-ray (EdX) was used to analyze various percentages of elements in the prepared SeNPs. Besides, SeNPs and S. costus extract spectra were produced and the functional groups were examined at a range of 400–4,000 cm^–1 using Fourier-transform infrared spectroscopy (FTIR; L160001J, PerkinElmer, USA). Additionally, X-ray diffraction (XRD) structures of SeNPs at a potential of 40 kV and an output current of 30 mA, together with an inversely opposite Ni-filter Cu-Kk energy, were used to assess the crystallographic features of the resulting SeNPs (Soliman et al., 2024).

Detection of anti-Helicobacter pylori activity and determination of minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC)

To evaluate the effects of the produced SeNPs and the S. costus extract on Helicobacter pylori, a standard H. pylori strain was cultured and used for antimicrobial testing (ATCC 43504) was provided by the Microbiology Department of Assuit University, Assiut, Egypt.

The anti-H. pylori properties were tested in vitro through the well agar diffusion method. In brief, Mueller–Hinton agar plates supplemented with 10% sheep blood were inoculated with 100 μL of Helicobacter pylori suspension (1.0 × 10⁸ CFU/mL). Wells of 6 mm diameter were then aseptically created in the agar using a sterile cork borer. Subsequently, 100 μL of each test sample (the S. costus extract or SeNPs) was added to the respective wells to evaluate antibacterial activity. Dimethyl sulfoxide (DMSO) was used as a negative control, while the antibiotics amoxicillin (0.06 mg/mL) and clarithromycin (0.06 mg/mL) were used as positive controls. The inhibition area’s dimensions were measured after 72-h incubation at 35°C in a moist microaerophilic device (Santiago et al., 2022).

For minimal inhibitory levels: Using nutritious broth for bacteria, the micro-dilution broth technique was used to determine the samples’ MIC. Each of the samples under investigation was diluted for two times to determine ultimate levels, which varied between 0.98 µg/mL and 1,000 µg/mL. To prepare 96-well microtitrate plate, 200 µL of the sample dilution under investigation in broth medium was placed in each well. The inoculum was generated using fresh bacterial cultures that satisfied the turbidity requirements of the 1.0 McFarland standard. To reach a level of 3.0 × 10⁶ CFU/mL, 2.0 µL of sterilized 0.9% NaCl was added to each well. After that, the bacteria were cultured at 35°C for 72 h. Concentrations of the items at which development of the standard strain was totally inhibited were visibly measured to determine MICs. Positive control (inoculum containing the subjected samples) and a negative reference (estimated samples containing no inoculum) were present on each microplate (Huang et al., 2021).

To determine the minimal bactericidal concentration (MBC), 100 μL from each well that showed complete inhibition of bacterial growth in the MIC assay was subcultured onto Mueller–Hinton agar plates supplemented with 10% sheep blood. The plates were then incubated at 35 °C for 72 hours. The MBC was defined as the lowest concentration of the sample that produced no visible bacterial colonies after incubation. The MBC–MIC ratios were calculated to determine whether the bactericidal or bacteriostatic activity of the investigated substance inhibited the microbial growth (Huang et al., 2021).

Assessment of antioxidant activity

To determine the antioxidant activity of the examined specimens, 0.10 mM 2,2-diphenyl-1- picrylhydrazyl (DPPH)-containing ethanol solution was utilized. Different concentrations of the samples in ethanol (ranging from 3.9 µg/mL to 1,000 µg/mL) were mixed with 3 mL of the prepared reagent solution. The mixtures were thoroughly shaken and left to stand for 30 minutes at room temperature to allow color development. The absorbance of each sample was then measured at 520 nm using a UV–Vis spectrophotometer to determine the antioxidant activity (Gueffai et al., 2022).

Elucidation of anti-inflammatory activity

To ascertain the anti-inflammatory capabilities of both specimens, membrane stabilization testing was performed. A range of sample concentrations, from 100 to 1,000 µg/mL, was created. The acquired samples were infused with hypotonic liquid. As negative and positive standards, indomethacin and purified water were used, respectively. Following the addition of 500 µL of samples to new erythrocyte suspension (3.0%) in 0.8 mL of saline, the mixture was incubated for 2 h at 35°C. At 7°C, the mixture was spun for 20 min at 14,000 ×g. Sample absorption was measured at 580 nm (Amina et al., 2023).

Determination of antidiabetic impact

Alpha-glucosidase testing

The α-glucosidase activity of specimens was measured. For 30 min at 30°C, 50-μL samples containing various amounts (1.97–1,000 μg/mL) were maintained with 10.0 μL of α-glucosidase enzyme solution (1.0 U/mL) and 125.0 μL of 0.12-M phosphate buffer (pH 7.3). To start the reaction after 20 minutes, 20 μL of 1-M pNPG (substrate) was added and the mixture was allowed to stand for 35 min. The reaction was stopped with the addition of 50 μL of 0.1-N Na₂CO₃, and absorption at 415 nm was measured using a spectrophotometer (Biosystm 310 plus, USA) (Taher et al., 2016).

Alpha amylase assay

The test was conducted using the 3,5-dinitrosalicylic acid (DSNA) technique. To get values ranging from 1.9 to 1,000 μg/mL, the extract was initially dispersed in 10% DMSO and then in buffer (0.020-M NaH₂PO₄/NaH₂PO₄ and 0.0090-M NaCl at pH 7.3). In total, 200 μL of each sample was mixed with 2.0 units/mL of α-amylase solution, and the mixture was incubated at 35 °C for 12 minutes

After that, each tube received 200 μL of 1.0% starch in water (w/v) solution, and the tubes were allowed to stand for 3 min. A water bath was used to heat the mixture for 12 min at 70°C after adding 200 μL of DNSA reagent (12.0 g of sodium potassium tartrate tetrahydrate in 8.0 mL of 2-M sodium hydroxide and 20 mL of 96-mM 3,5- dinitrosalicylic acid solution) to stop the reaction. Once the mixture had reached ambient temperature, it was diluted with 5 mL of deionized water and its absorption at 580 nm was measured using a UV-visible (Biosystem 310, USA) spectrophotometer (Oboh et al., 2012).

Cytotoxicity by MTT testing

The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was conducted to investigate the cytopathic influence on human epithelial Caco-2 cell line as cancer cells (a cell line originally derived from colon carcinoma) and Wi-38 as normal cells for the prepared samples after it became dispersed in DMSO. The result was a blue color that was directly proportional to the quantity of the living cells when evaluated with conventional phases. Absorbance was measured at 560 nm by utilizing a computerized microplate analyzer (BMG, LabTech, Australia). After the cells were allowed to adhere and form a confluent monolayer for 24 h, samples containing 1,000–31.25 µg/mL were added, and the cells were subsequently incubated for another 24 h at 35°C. MTT solution, 100 µL (5.0 mg/mL), was incorporated along with the fresh media, and it was kept at 35°C for 4 h. A CCD camera and a microscope (Accu-Scope INC., USA) were used to view the cells (Alsanosi et al., 2022).

Flow cytometry analysis of cell cycle

Using flow cytometry, the impact of SeNPs on Caco-2 cells’ cell cycle phase was investigated. Prior to being subjected to the SeNP’s half-maximal inhibitory concentration (IC₅₀), Caco-2 cells were first cultivated for 24 h at a density of 1.6×10⁶ cells/mL in a tissue culture plastic plate. Following trypsinization, the cells were washed with phosphate-buffered saline (PBS) and fixed at -20°C with ice-cold 70% ethanol. After that, the cells were treated with propidium iodide (PI) solution (Cytoflex Beckman, Germany) for 30 min at 5°C. The cell cycle phases were investigated by using flow cytometry (Cytexpert Software, Germany) (Cháirez-Ramírez et al., 2021).

Statistics for the results

The results were displayed as mean±SD values for each experiment, which was conducted for three times. With the Graph Pad Prism V5 (San Diego, CA, USA) software, t-test was used to analyze disparity in mean values. Significant variation was defined as results with P < 0.05.

Results

Determination of various bioactive compounds in S. costus extract

Different phenolic and flavonoids were observed as bioactive molecules in the ethanol extract of S. costus roots upon separation by HPLC (Supplementary Figure S1 and Table 1). In all, 15 compounds were detected in the S. costus extract, with seven major compounds, which were rutin, chlorogenic acid, coumaric acid, gallic acid, coffeic acid, naringenin, and syringic acid. Eight compounds present in low levels were daidzein, rosmarinic acid, ferulic acid, quercetin, cinnamic acid, methyl gallate, kaempferol, and hesperetin (Table 1).

Examination of biosynthesized SeNPs

A distinctive peak for the prepared SeNPs was observed at 275 nm upon screening using UV spectrophotometer (Figure 1A). Besides, transmission electron microscopic (TEM) analysis revealed the structure of prepared nanoparticles with a mean size of 73.7±0.7 nm (see Figure 1B). Furthermore, EdX analysis showed various proportions of elements in the prepared form, with selenium having a percentage of 23.7% as illustrated in Figure 1C. FTIR analysis of S. costus extract was performed and compared with the peaks of SeNPs. A characteristic peak at 3,389 cm^–1 was observed in S. costus extract that become shaper in SeNPs at 3,419 cm^–1 for O-H stretching vibrations, 1,418 cm^–1 for COO-group, and 719 cm^–1 for C-H stretching vibrations. A peak at 462 cm^–1 for the SeNP pattern showed the existence of selenium in nano-solution, which revealed production by S. costus extract as depicted in Figure 2A. XRD testing at a range of 10°–80° showed various characterized peaks as observed at angles of 100°, 101°, 111°, 201°, 210°, and 213°, and followed the Miller indices as shown in Figure 2B.

Anti-H. pylori for S. costus extract and SeNPs, MICs, and MBCs

Both S. costus extract and prepared SeNPs were tested for H. pylori as shown in Figure S2. It was observed that SeNPs had the highest inhibition zone of 30.7±0.2 mm, while the inhibition zones for S. costus extract and the control drug were 27.3±0.2 mm and 23.7 ±0.2 mm, respectively, as illustrated in Table 2. The MIC and MBC determined for SeNPs was 15.62±0.2 µg/mL, while the MICs and MBCs for S. costus extract and the control drug were 31.25±0.1 µg/mL and 62.5±0.1 µg/mL, respectively, as shown in Table 2.

omparing of antioxidant activity of S. costus extract and SeNPs

The DPPH scavenging activity of the produced SeNPs was 9.42±0.1 µg/mL, while the DPPH scavenging activity of S. costus extract was 18.0±0.2 µg/mL. In addition, the antioxidant value of ascorbic acid (control) was 2.36±0.2 µg/mL. There was a significant difference between S. costus extract, SeNPs, and the control (P ≤ 0.05) as depicted in Figure 3.

Comparative assessment of anti-inflammatory effect of S. costus extract and SeNPs

The anti-inflammatory activity of the synthesized SeNPs was 8.33 ±0.4 µg/mL, while the anti- inflammatory activity of S. costus extract was 16.46 ±0.3 µg/mL. In addition, the anti-inflammatory value of diclofenac as a control was 6.05±0.2 µg/mL. There was a striking difference between S. costus extract, SeNPs, and the control (P ≤ 0.05), as depicted in Figure 4.

Comparative assessment of antidiabetic activity for S. costus extract and SeNPs

The evaluation of alpha-glucosidase enzyme level in S. costus extract reveled its IC₅₀ = 23.63±0.6 µg/mL, while for the alpha-glucosidase enzyme level in SeNPs, IC₅₀ = 12.55±0.8 µg/mL. On the other hand, for level for the control, IC₅₀ = 6.27±0.4 µg/mL. There was a significant difference between S. costus extract, SeNPs, and the control (P ≤ 0.05), as shown in Figure 5A.

Determination of the level of alpha-amylase enzyme in S. costus extract showed its IC₅₀ =21.18±0.2 µg/mL. On the other, SeNPs showed a promising alpha-amylase activity with IC₅₀ = 8.75±0.3 µg/mL, whereas the level for the control was detected at IC₅₀ = 4.51±0.4 µg/mL. These results revealed a significant variation between two treatments and the control, as illustrated in Figure 5B.

Assessment of cytopathic impact of S. costus extract and SeNPs

The S. costus extract showed a promising anticancer effect toward Caco-2 cells with IC₅₀ = 82.77±1.02 µg/mL. There is a significant rise (P ≤ 0.05) in anticancer effect toward Caco-2 cells upon using the prepared SeNPs with IC₅₀ = 34.09±2.49 µg/mL, as shown in Figure 6 and Supplementary Table S1. On the other hand, both S. costus extract and biosynthesized SeNPs had a minimal cytotoxic activity toward Wi-38 cells with IC₅₀ = 146.5±0.52 and 87.91±0.45 µg/mL, respectively, as depicted in Figure 7 and Supplementary Table S2.

Comparative assessment of cell cycles

There was a dramatic reduction (P ≤ 0.05) in percentage in the G0 phase (resting phase) of Caco-2 cells upon treatment with S. costus extract and SeNPs, while a nonsignificant difference was observed in the proportions of S and G1 phases upon using various treatments, compared to the control. In addition, there was a significant reduction in the proportion of M phase upon using S. costus extract (P ≤ 0.05), compared to the control. The proportion of M phase reduced significantly upon using SeNPs (P ≤ 0.05), compared to S. costus extract, revealing that SeNPs had the best anticancer impact (see Table 3 and Figure 8).

Different superscript letters in the same row reveal significant differences at P ≤ 0.05.

Discussion

Numerous studies have documented the application of plant chemicals to combat various bacterial infections (Kukić et al., 2008). New and effective anticancer and antidiabetic medications with fewer adverse effects are also needed, and herbal remedies have shown promise as a source of these substances (DeSantis et al., 2014). On the other hand, since selenium is an essential part of antioxidant enzymes and may play a crucial role in maintaining health, it has gained greater interest in recent years (Puri et al., 2023). Traditional selenium substances are applied minimally because of their low safe dose level, which limits their applicability. However, materials at the nanometer scale exhibit physical and chemical properties that differ significantly from their bulk counterparts, enabling novel applications in various biomedical and technological fields. One such material is elemental selenium (Filipović et al., 2021), while SeNPs are observed to be a more efficient and secure form of selenium with low adverse effects and high accessibility (AlBasher et al., 2020).

In the present investigation, S. costus root extract showed a group of polyphenolic compounds and flavonoids upon HPLC testing, with rutin, chlorogenic acid, and coumaric acid being the most relevant compounds. Many studies showed that phenolic compounds could cause a variety of biological functions, and their ability of antioxidant effect was typically investigated in relation to their ability to eliminate free radicals’ assessments (Singh et al., 2016).

The TEM analysis revealed SeNPs as smooth and spherical particles with a relatively tiny shape, with an average diameter of 73.7±0.7 nm and a peak at 278 nm. Investigators had reported that the prepared SeNPs could have a size within a range of 50–400 nm, and changes in diameter could impact the stability and properties of nanoparticles (Mikhailova, 2023). Additionally, the presence of selenium in nano-solution, compared to S. costus extract, was demonstrated by FTIR, which displayed a distinctive peak at 462 cm^–1 for SeNPs pattern. Decrease in the transmittance percentage of nearly all functional groups in FTIR showed strong evidence of the production of metal nanoparticles (Al-Brakati et al., 2021). Other investigators used plant extracts to prepare metal nanoparticles (Abadi et al., 2025; Mohammadi-Aghdam et al., 2024).

The Gram -tive microaerobic bacterium H. pylori colonize in the stomach of humans causing chronic gastritis. Additionally, there is mounting evidence linking H. pylori infection to various illnesses, especially those involving mental, physiological, and cardiac processes (Pop et al., 2022). Regarding the investigated substances’ antibacterial activity, SeNPs demonstrated an anti-H. pylori effect with a MIC of 15.62±0.2 µg/mL and an inhibition diameter of 30.7±0.2 mm. Al-Saggaf et al. (2020) reported that selenium nanoparticles (SeNPs) synthesized using Saussurea costus extract exhibited anti-Helicobacter pylori activity, with a minimum inhibitory concentration (MIC) of 40 µg/mL.

The investigation and creation of antibacterial substances that can take the place of antibiotics in H. pylori medication has grown in popularity. Nanomaterials have attracted greater interest in health care because of their benefits, especially the superiority of metal-based nanoparticles over other nanoparticles (Yin et al., 2023). Furthermore, Shirzadi-Ahodashti et al. (2021) reported the antibacterial activity of eco-friendly synthesized AgNPs against multi-drug-resistant bacteria.

The study’s findings demonstrated that SeNPs’ anti-inflammatory, antioxidant, antidiabetic, and anticancer properties outperformed those of S. costus extract. The elemental form of selenium is discovered to have a lower antioxidant property than its nanoform (Binsuwaidan et al., 2024). Numerous investigations have demonstrated that SeNPs have more antioxidant capacity than the plant extract that was used to synthesize them (Bardaweel et al., 2018; Barzegarparay et al., 2024). Furthermore, studies showed the antioxidant level for S. costus at IC₅₀ = 12.32 µg/mL (Mammate et al., 2022), similar to the level discovered in the present work, that is 18.0±0.2 µg/mL. Thus, it was established that synthesized SeNPs could neutralize hydroxyl radicals. Since OH radicals make up the majority of reactive oxygen species (ROS), this is a crucial component. ROS can cause oxidative stress in living beings, an unbalance between the systemic generation of ROS and cells’ capacity to immediately detoxify reactive intermediates or repair the resulting damage (Di Meo et al., 2016). A new class of antioxidant therapeutics for the avoidance and management of oxidative stress-related disorders are nanoparticle antioxidants. They are stronger compared to the damage caused by free radicals, since they exhibit robust and long-lasting connections to structures (Sentkowska and Pyrzyńska, 2023). The data obtained validated SeNPs’ excellent antioxidant activity.

The ability of a product to stabilize membranes suggests that it could be used to protect cell membranes, which are essential for numerous biological procedures (Errasti-Murugarren et al., 2021). Cell membranes are made up of proteins and lipids and serve a variety of purposes, including cellular signaling, transportation of waste and nutrients, and cell death (Zhai et al., 2024). In the present study, both S. costus extract and the prepared SeNPs showed a promising anti-inflammatory impact, with SeNPs having a higher level of effect than S. costus extract. The present results revealed that SeNPs had an anti-inflammatory impact at IC50 = 8.33±0.4 µg/mL. On the other hand Almayouf et al. (2024) reported a DPPH value for AgNPS prepared from S. costus extract at IC₅₀ = 120 µg/mL.

Diabetes mellitus is a common endocrine condition that ranks as the fourth most common cause of death worldwide. This ailment has not yet been satisfactorily cured by allopathic medication. Innovative antidiabetic strategies with improved management and fewer adverse effects and less expenses are therefore desperately needed (Ahmed et al., 2017). The present study highlights the antidiabetic effect of SeNPs, which was higher than that of S. costus extract, similar to the results showed by Ahmed et al. (2017) and Karas et al. (2024)

Conclusion

The current research reports on the anti-H. pylori activity of SeNPs obtained from the root extract of S. costus. The findings showed that SeNPs had strong anti- inflammatory and antioxidant effects. They showed a promising antidiabetic effect that was corroborated by alpha glucosidase and alpha amylase activity. SeNPs showed substantial anticancer effects toward Caco-2 cells, confirmed by enhancing early apoptosis in cell cycle. Overall, the biosynthesized SeNPs demonstrated strong antioxidant, anti-inflammatory, antidiabetic, and anticancer activities, with minimal cytotoxicity toward normal cells. These findings suggest their potential for safe therapeutic use; however, further in vivo animal studies are required to confirm their safety and efficacy before large-scale or clinical application