High antimicrobial, cytotoxicity, and catalytic activities of biosynthesized selenium nanoparticles using Crocus caspius extract

2.1 Synthesis of selenium nanoparticles (C@SeNPs) using C. caspius aqueous extract

The 10 g dried C. caspius was cut into smaller pieces and heated at 45–50 °C for 1 hr with 80 ml deionized water. Then, the flask was sonicated for 30 min. The obtained extract was filtered by filter paper (Whatman No. 1) and the filtrate was used to prepare selenium nanoparticles.

For the biosynthesis of SeNPs using C. caspius extract, Na₂SeO₃ (17.3 mg) dissolved in 10 ml deionized water was shaken at 45–50 °C and 500 rpm, and then, C. caspius extract (5 ml) was added into the mixture. The reaction color changed from colorless to red or reddish-orange after 2 days, indicating that the Se ions had been reduced.

2.2 High-pressure liquid chromatography (HPLC)

UV–Vis spectrophotometric detector (model K-2600) and solvent delivery system with Rheodyne injection valve (20 µl sample loop) were used in the HPLC system. To elute the sample, an isocratic solvent system with acetonitrile: water: formic acid (17:82.6:0.4) mobile phase was employed with a flow rate of 1 ml/min and a run time of 40 min at 25 °C via an “ODS-C18 column (Shim-pack VP-ODS: 250 mm × 4.6 mm id, 5 mm)”. Detection was also done with a UV–Vis detector (280 nm). The current technique was utilized to determine the phenol and flavonoid content of C. caspius extract.

2.3 Characterization of green synthesized C@SeNPs

The synthesized C@SeNPs were analyzed using UV–Visible spectroscopy “T80 UV–Vis spectrophotometer PGI, Beijing, China” in 200–800 nm. The surface morphology of C@SeNPs was detected by SEM (“TESCAN BRNO-Mira3 LMU”) and their elemental composition was identified by EDX. Transmission electron microscopes (TEM) analysis (“TEM Philips EM 208S”) exhibited the exact morphology of the synthesized SeNPs. The nature of C@SeNPs was defined by X-ray diffraction (XRD) under “PANalytical X-PERT PRO diffractometer (2θ = 10° to 80°) with Cu Kα (1.54 °A) source”. The average size of C@SeNPs was measured by Scherrer’s formula D = 0.9λ/βcosθ (Shirmehenji et al., 2020). The functional groups involved in SeNPs synthesis were studied by FT-IR analysis. The FT-IR spectrum was recorded by “ATR, Agilent, Cary 630, FT-IR Spectrometer, equipment in 4000 and 650 cm⁻¹”. The size and distribution of the C@SeNPs were determined using the DLS particle size analyzer (“Horiba-SZ-100-Z”). The thermal stability of the biosynthesized SeNPs was assessed by TGA and DTA (55–900 °C) at a heating rate of 0.01–100 K/min an autonomic TG/DTA (“STA 503, BAHR, Germany”).

2.4 DPPH assay (“2,2-diphenyl-1-picryl-hydrazyl-hydrate”)

The free radical scavenging activity of C@SeNPs was evaluated using the stable DPPH radical (Ebrahimzadeh et al., 2010; Hashemi et al., 2020; Nabavi et al., 2013). Different quantities of C@SeNPs (17.06, 34.125, 68.25, 196.5, and 273 µg/ml) were added to 1 ml of DPPH methanolic solution (4 mg/100 ml MeOH) and shacked in the dark at 25 °C. As a conventional control, BHA was utilized. The absorbance was determined at 517 nm after 15 min. The sample concentration necessary to scavenge 50 % of DPPH free radicals is known as the IC₅₀ value. The following formula (1) was used to determine the sample's ability to scavenge the DPPH radical:

2.5 Iron chelation activity (Ferrozine assay)

We assessed the ability of the C@SeNPs to chelate Fe²⁺ ions according to our previous published (Ebrahimzadeh et al., 2008; Ebrahimzadeh et al., 2010; Eslami et al., 2016; Hashemi et al., 2020). The fabricated C@SeNPs (2.034–130.16 μg/ml) mixed with 3 ml of deionized water were added to a solution containing FeCl₂ (50 µl × 2 mM) and ferrozine (100 µl × 5 mM) and shaken dynamically for 10 min. The solution's absorbance was taken spectrophotometrically at 562 nm. The standard compound was EDTA. The inhibition percentage of ferrozine-Fe²⁺ complex formation was calculated by equation (2):

2.6 Antibacterial activity

A micro-broth dilution method was used to test the antibacterial impact of C@SeNPs against “several American Type Culture Collection (ATCC) bacteria including, Staphylococcus aureus, Enterococcus faecalis (gram-positive bacteria), Proteus mirabilis, Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumannii, and Escherichia coli (gram-negative bacteria)”. For the micro-broth dilution tests based on (Chowdhury et al., 2016), Muller-Hinton broth (MHB,100 µl), prepared C@SeNPs (0.133–136.67 µg/ml), and the diluted bacterial suspension (100 µl, 0.5 McFarland turbidity standards) were supplemented to each well of the 96 well plate and incubated for 24 hrs at 37 °C. The MIC was determined as the lowest concentration of C@SeNPs that caused no observable bacterial growth. To determine the MBC, a nutrient agar plate was filled with 10 µl of MIC well and three concentrated wells, which were incubated for 24 hrs. The MBC of the prepared C@SeNPs was determined after 24 hrs based on the lowest concentration that displayed no bacterial growth on the Muller Hinton agar surface. Pure bacterial suspensions (without any C@SeNPs) and MHB (without any bacterial suspension) were studied as positive and negative controls, respectively. The reference antibiotic was ciprofloxacin. All experiments were carried out in safe conditions ( Mortazavi-Derazkola et al., 2020).

2.7 Antileishmanial activity

Leishmania major promastigotes (MRHO/IR/75/ER) were grown in RPMI-1640 medium. They were mixed with “10 % fetal bovine serum (FBS) and 100 µg/ml streptomycin/ penicillin at 24 °C”. The media comprising promastigotes was sub-cultured every 72 hrs (Akhtari et al., 2019; Faridnia et al., 2018). Serial dilutions of C@SeNPs were prepared at concentrations of 273.5–8.547 µg/ml in 100 µl of RPMI-1640 medium. Each well of plates received 100 µl of the promastigotes medium (1 × 10⁵) and incubation was performed for 72 hrs at 24 °C. After 72 hrs, 10 µl of MTT solution (2.5 mg/5 ml PBS) was added to each well and incubated for 5 hrs at 37 °C and were centrifuged (3000 rpm, 5 min). After this, obtained suspensions were dissolved in isopropyl alcohol. The absorbance of the microplate was measured by ELISA reader “Synergy H1 hybrid multi-model microplate reader (Biotek Instruments, Winooski, Vermont, NE, USA” at 492 nm. Negative controls were three untreated wells comprising the parasite. The positive controls, Amphotericin B exhibited an IC₅₀ value of 28.7 µg/ml (Hashemi et al., 2021).

2.8 Anticancer activity

The effect of C@SeNPs on “human gastric cancer cell line (AGS) and human breast adenocarcinoma cell line (MCF-7) ”was assessed by MTT assay according to our previous research (Hashemi et al., 2020). In the living cells, “3- (4,5- dimethylthiazol-2-yl) 2.5- diphenyltetrazolium bromide” (yellow dye) was reduced to formazan crystal (purple) that is related to the activity of mitochondrial enzymes. The cells (6–10 × 10³ cells/well) were placed in a 96-well and incubated for 24 hrs at 37 °C in a 5 % CO₂. Then, the medium was removed and 100 µl of C@SeNPs (1, 10, 25, 50, and 100 µg/ml) was added to each well. After 24 hrs incubation, the C@SeNPs were removed, and 20 µl of MTT solution (5 mg/ml PBS) was loaded to each well and incubated in the dark for 4 hrs under standard conditions. After 4 hrs, DMSO (100 µl) was added to each well to dissolve the formazan crystals, recording the absorbance of the solution at 570 nm by “Synergy H1 hybrid multi-model microplate reader (Biotek Instruments, Winooski, Vermont, NE, USA”. The percent cells viability was calculated by equation (3) (Al Jahdaly et al., 2021; Hashemi et al., 2020; Hashemi et al., 2022). The same assay was conducted using fibroblast cell lines to investigate the cytotoxic effects of C@SeNPs on noncancer cells. The experiments were validated thrice.

2.9 Antifungal activity

The MIC value was estimated by broth micro-dilution method as described in “method M27-A3 from the Clinical and Laboratory Standards Institute (CLSI) formerly NCCLS” [15]. In the process, 200 μl of C@SeNPs was loaded to the first well of the 96-well plate, then 100 μl of RPMI media was entered into the wells of 2 to12 at the 96-well plate. In the following, 100 μl of the first well was taken and added to the second well, then serially diluted, 100 μl of cell suspensions were added to each well and after 24 hrs for two Candida albicans strains ((IFRC1873) and (IFRC1874)), and 48 hrs for Aspergillus fumigatus (IFRC 1649), Aspergillus fumigatus (IFRC 1505), Trichophyton mentagrophytes (FR1_22130), Trichophyton mentagrophytes (FR5_22130), Fusarium proliferatum (IFRC 1871), Fusarium equiseti (IFRC 1872), observable growth of wells were examined and the MIC value was defined. The references antibiotic was Itraconazole.

2.10 Catalytic reduction of MB dye using C@SeNPs

Methylene blue (MB) degradation under visible light was used to test the catalytic activity of biosynthesized C@SeNPs. Fresh NaBH₄ (200 μl × 0.1 M) was mixed with 5.77 ml H₂O and 30 μl MB solution (10 mM) during the process. After that, the prepared solution was treated with 40, 50, and 60 μl of C@SeNPs solution (546.67 μg/ml). Absorbance measurements in the region of 200–800 nm were taken at various time intervals. At 665 nm, the maximum absorption of MB was recorded. The pseudo-first-order reaction (4) was used to follow the MB degradation rate; At and A0 were presented as the absorbance at interval times and time 0, respectively (Nadaf and Kanase, 2019).

2.11 Statistical analysis

The biological analysis was performed in triplicate. One-way analysis of variance (ANOVA) was accomplished, followed by Tukey’s test using the GraphPad Prism 9.0 software. Statistical significance was defined as p < 0.05.

3.1 Preparation and characterization of C@SeNPs

The color of the mixture solution comprising sodium selenite and C. caspius aqueous extract changed from light brown to red or reddish-orange after completing the SeNPs production reaction due to SPR of the produced SeNPs, indicating that Se ions were reduced into SeNPs using functional groups (natural reductants) in C. caspius extract (Hatami et al., 2020). Fig. 1 displays the red color of the solution with synthesized C@SeNPs after 48 hrs. Different pH levels (6 (extract pH), 10, 12) and temperatures (25–30, 45–50, 75–80 °C) were evaluated to optimize the synthesis condition of SeNPs using C. caspius; the results showed that the solution color was changed from colorless to red or reddish-orange at the 45–50 °C and extract pH (pH = 6); however, this color change did not occur in other conditions. In addition, UV–vis spectrophotometry was used to characterize C@SeNPs by reporting the absorption of colloidal solution at 200–800 nm (Fig. 1). Fardsadegh and coworkers effectively prepared SeNPs with a particle size of 136 nm using the leaf extract of Pelargonium zonale and microwave heating (Fardsadegh et al., 2019).

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Fig. 1

The UV–vis spectra of synthesized SeNPs by C. caspius aqueous extract.

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The size, shape, and distribution of C@SeNPs were investigated using the TEM image, as shown in Fig. 2a. C@SeNPs have a semi-spherical shape, and their average size is about 20.34 nm in the provided image. The image proved the fabrication of NPs that were capped with their biomoites. In addition, the SEM image (Fig. 2b) revealed a semi-spherical shape for the surface morphology of C@SeNPs with an average particle size of around 23.47 nm, which is very close to the TEM results. Furthermore, synthesized NPs possess a good polydispersity. The histogram from the SEM image demonstrated that about 90 % of the fabricated C@SeNPs possess a size range of 10–40 nm (Fig. 2c). Keskin et al. synthesized SeNPs using Lysinibacillus sp. NOSK and the SEM analysis showed a spherical shape around 130 nm (Keskin et al., 2020). In a study in 2019, according to the SEM data, the prepared SeNPs were spherical shape with a size range of approximately 100–150 nm (Menon et al., 2019).

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Fig. 2

A) the tem image with 20.34 nm average size with semi-spherical shape; b) the SEM image with 23.47 nm average size; c) the histogram obtained from SEM image; d) the EDX analysis containing C, O, and Se bonds; e) The DLS graph with 266.3 nm average size; f) zeta potential analysis with −44.75 mV; g) XRD data of green synthesized C@SeNPs; h) FT-IR graph of C@SeNPs and C-Extract; i) TG/DTA data of C@SeNPs.

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EDX analysis of the extract-mediated SeNPs from the portion of SEM images has confirmed that the majority of the detected elements in the synthesized sample is Se (Fig. 2d). However, similar to what is reported in the literature, the presence of some amount of carbon and oxygen (and some other elements) might be inevitable, which stems from the natural substances (Tripathi et al., 2020). Keskin et al. displayed Se signal along with C and O group peaks using the elemental analysis (Alipour et al., 2021).

The size distribution of C@SeNPs was measured by DLS particle size analyzer, and the average size of the NPs was determined to be 266.3 nm by number (Fig. 2e); due to the aggregation of C@SeNPs in the solution, some of the large particles appeared in the DLS graph. The particle size distribution of ginger mediated-SeNPs was obtained in the range of 100–120 nm by Menon et al (Menon et al., 2019). The obtained size of C@SeNPs using two methods, zetasizer and TEM, differed; because TEM determines the actual particle radius while zetasizer determines the average hydrodynamic radius. In the green synthesis, the intrinsic capping provided an additional benefit of stability (Mittal et al., 2014).

The surface electric charge of the prepared C@SeNPs is defined by Zeta potential; the NPs stability in colloids possess a direct relationship with the ZP value (Hatami et al., 2020). The zeta potential of synthesized C@SeNPs was −44.75 ± 0.95 mV, indicating the nanoparticles were stable (Fig. 2f). Thus, the phytochemicals as capping agents can be induced repulsive forces on the surface of nanoparticles, making them more stable.

The XRD analysis is conducted to investigate the phase composition and crystalline nature of the plant-mediated SeNPs, which is presented in Fig. 2g. The result shows several Bragg’s reflections at (1 0 0), (1 0 1), (1 1 0), (1 0 2), (1 1 1), (2 0 1), (1 1 2), (2 0 2), (2 1 0), (2 1 1), and (1 1 3) ascribing 23.61, 29.85, 41.36, 43.83, 45.52, 51.83, 56.20, 61.67, 65.11, 67.95, and 71.75 of trigonal selenium crystallographic planes (Menon et al., 2021). The green synthesis of SeNPs using Withania somnifera, Emblica oficinalis, and Pseudomonas aeruginosa (Amiri et al., 2021) was consistent with our study. The crystallite size is evaluated using the well-known Debye-Scherrer equation (Menon et al., 2020). The calculated crystallite size for our synthesized SeNPs is about 33.42 nm.

The FT-IR analysis is performed to explore the effect of the organic moieties corresponding to the C. caspius. In Fig. 2h, the spectrum of the extract is compared with that of plant-mediated NPs. The broad and rich hydroxyl band at 3585 cm⁻¹ of C-Extract attests to this fact that there are plenty of phenolic compounds and flavones. The symmetric and antisymmetric stretching vibrational of C—H group are also observed at 2854 cm⁻¹ and 2923 cm⁻¹, respectively (Boroumand et al., 2019). The 1708 cm⁻¹ peak is evidence of the presence of carbonyl functional group in the C-Extract (Nasrollahzadeh and Sajadi, 2015). The C⚌O stretching feature, which appeared around 1636 cm⁻¹ in the FT-IR of C@SeNPs, has shown a small shift toward higher wavenumbers in the case of C-Extract (Hamelian et al., 2018). Anu et al. have ascribed a similar observation to the formation of Se nanoparticles as a result of reduction of the carbonyl groups presented in the natural plant (Anu et al., 2020). The band at 1040 cm⁻¹ is assigned to C—N stretching vibrations of aliphatic amines (Salem et al., 2021). The peak at lower wavenumbers is expected to be as a result of either ν(C—O) or ν(C—C) vibrations.

The above results testified the involvement of the functional groups such as OH, C⚌O, and C—H. This could be easily observed by the significant vanishing of the OH group in the case of FT-IR spectra of the plant-mediated SeNPs. Hence, the presented FT-IR has vividly identified the interaction between Se and metabolites of plant extract, which is responsible for capping and reducing intermediate for as-synthesized SeNPs.

TG/DTA curve obtained from C@SeNPs (Fig. 2i) confirmed about 60 % weight loss between 260 and 500 °C in TGA analysis, suggesting that biomolecules surround the metallic core (Nasrollahzadeh et al., 2016). At around 100 °C, no significant weight loss showed the lake or low content of moisture at the product. Furthermore, there was no considerable weight loss (about 5 %) at temperatures about 500–850 °C, indicating the stability of C@SeNPs.

In the DTA curve of C@SeNPs, one endothermic peak at about 110 °C is related to the loss of low content of water molecules (Elahi et al., 2020; Sabouri et al., 2019). One endothermic peak about 220 °C is related to the melting point of SeNPs (Ezhuthupurakkal et al., 2017), and two weak endothermic peaks around 350 and 400 may be responsible for the decomposition of organic biomolecules. An exothermic peak of about 720 possibly corresponds to the boiling point of Se.

3.2 HPLC analysis

The HPLC method was selected for the direct detection of phenolic acids in a C.caspius extract. In this investigation, it was obvious that phenolic acids and flavonoids in C.caspius extract could be separated using an isocratic solvent system. According to HPLC analysis, the main constituents existing in this extract are phenolic acid and flavonoid compounds, such as catechin, gallic acid, caffeic acid, p-coumaric acid, and rutin, (Table. 1). Fig. 3 is suggested as a mechanism for creating SeNPs in the presence of C.caspius extract. Based on the HPLC data, catechin (8.403 mg/g plant) was the major antioxidant among the other injected standards. Generally, the ortho-hydroxyl groups of these compounds are involved in the synthesis of SeNPs. Se⁴⁺ is reduced to Se⁰ when occurs releasing two electrons in the flavonoid ring. The catechol ring is oxidized into a 3́,4́-quinone ring as a stable end product. Se undergoes additional aggregation to larger clusters as the process progresses, eventually forming SeNPs. Quinone form of catechin is also linked to the surface of NPs, resulting in less Se⁰ aggregation at the nanoscale.

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Fig. 3

The possible mechanism of the biosynthesized selenium nanoparticles using Crocus caspius extract.

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3.3 DPPH assay

The DPPH assay is a quick and easy approach to evaluate a compound's capacity to quench free radicals (Nakkala et al., 2016). Due to the transfer of free electrons, when an antioxidant compound reacts with this reagent, the violet color of the solution converts to colorless, demonstrating that the antioxidant agent with hydrogen-donating activity has taken up the free radicals. In our study, the antioxidant activity of various concentrations of C@SeNPs (17.06–273 µg/ml) was investigated by DPPH assay. The absorbance was recorded by helping spectrophotometry. In this assay, the IC₅₀ (“concentration required to inhibit 50 % of free radicals”) of C@SeNPs has obtained 123.9 ± 2.5 µg/ml. The standard compound was Butylated Hydroxyanisole (BHA) with an IC₅₀ value of 53.96 µg/ml. As a result, the antioxidant activity increased with increasing the concentration of the C@SeNPs. In a study (2019) was reported that ginger extract mediated-SeNPs with spherical shape and 100–150 nm presented an IC₅₀ value of 125 µg/ml (Menon et al., 2019). In another investigation, biosynthesized SeNPs with 4–16 nm were shown to have an IC₅₀ of 0.225 µg/ml (Kokila et al., 2017).

3.4 Metal chelating activity

The iron chelating activity of our synthesized C@SeNPs (2.034–130.16 µg/ml) was studied, and it was discovered that they could chelate ferrous (Fe²⁺) ion. The IC₅₀ value was determined by the concentration required to inhibit 50 % of the Ferrozine-Fe²⁺ complex was known as; C@SeNPs had an IC₅₀ value of 67.63 ± 1.11 µg/ml. In addition, IC₅₀ value for positive control (EDTA) was obtained 18.27 ± 0.59 µg/ml. Therefore, prepared C@SeNPs revealed remarkable iron chelation activity.

3.5 Antibacterial activity

Antibacterial potential of C@SeNPs green synthesized from C. caspius extract was assessed against the ATCC bacterial strains using micro-broth dilution method (Table. 2). The antibacterial potential of fabricated C@SeNPs exhibited remarkable antibacterial activity against all tested bacterial strains, according to the findings. Maximum antibacterial activity was seen against S. aureus and A. baumannii at a MIC value of 17.08 µg/ml, and MBCs of 273.33 and 68.33 µg/ml. Synthesized C@SeNPs also exhibited moderate activity against P. aeruginosa, E. coli, and K. pneumoniae with MICs of 34.17, 68.33, and 68.33 µg/ml. In addition, E. faecalis and Proteus mirabilis presented MIC of 136.66 µg/ml. The SeNPs biosynthesized by bee propolis ethanol extract revealed antimicrobial and antioxidant potential (Shubharani et al., 2019). An efficient antimicrobial activity was reported for the SeNPs synthesized using Allium sativum extract against S. aureus and B. subtilis (Jay and Shafkat, 2018). Diospyros montana mediated-SeNPs display potent antimicrobial activity against S. aureus, E. coli, and A. niger (Kokila et al., 2017). Therefore, our prepared SeNPs can be considered as an effective antibacterial agent.

3.6 Anticancer activity against MCF-7 and AGS cell lines

The in vitro cytotoxic effects of biosynthesized C@SeNPs were checked on two cancer cell lines, MCF-7 and AGS, and normal cell (HEK) using the MTT assay (Fig. 4). Based on the result, the growth of cells decreased with increasing the content of C@SeNPs. Synthesized selenium nanoparticles exhibited capacity to limit the growth of AGS and MCF-7 cell lines by 18 % and 22 % at lowest concentration (1 µg/ml). The IC₅₀ values of 47.59, 50.7, and 53.33 µg/ml were found on MCF-7, AGS, and HEK cells, respectively. Besides, 5-FU as standard anticancer demonstrated IC₅₀ values of 62.73, 44.25, and 37.39 µg/ml against MCF-7, AGS, and HEK cell lines, respectively. Therefore, C@SeNPs presented a strong growth inhibitory impact that was close to standard anticancer. SeNPs prepared by the biomolecules existing in M. Oleifera extract has revealed to be effective against MCF-7 cells, Caco-2 cells, and HepG2 cells with IC₅₀ values of 252.44, 150.87, and 392.57 µg/ml (Hassanien et al., 2019). Withania somnifera leaves extract mediated-SeNPs indicated antiproliferative activity against A549 cells (IC₅₀ = 25 μg/ml) (Alagesan and Venugopal, 2019). In another study, SeNPs produced using Acinetobacter displayed effective anticancer activity against “4T1, MCF-7 and NIH/3T3, HEK293” (Wadhwani et al., 2017). In various studies, potential anticancer mechanisms of SeNPs have all been proposed the formation of reactive oxygen species (ROS), the activation of apoptotic pathways, cellular homeostasis disruption, mitochondrial malfunction, cell cycle arrest, and DNA fragmentation (Vahidi et al., 2020).

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Fig. 4

Effect of biosynthesized C@SeNPs on viability of AGS, MCF-7, and HEK cells determined by MTT assays.

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3.7 Antileishmanial activity

Due to the importance of discovering antileishmanial drugs, fabricated C@SeNPs (8.547–273.5 µg/ml) were assessed for their antileishmanial activity (in vitro) and showed an IC₅₀ value of 42.81 µg/ml. The amount of IC₅₀ for amphotericin B (positive control) was obtained 28.7 µg/ml. In comparison, green synthesized C@SeNPs diplayed antileishmaniasis potency very close to positive control. Hence, our C@SeNPs can be discussed as promising antileishmanial agents. In the research by Mahmoudvand et al. was presented that SeNPs showed the IC₅₀ values of 2.7 and 3.6 μg/ml against “sensitive and glucantime-resistant strains” (Mahmoudvand et al., 2014). In another study, SeNPs mediated Bacillus sp. MSh-1 demonstrated an IC₅₀ of 1.62 ± 0.6 μg/ml against the promastigote (Beheshti et al., 2013). According to the researchers, DNA fragmentation could be the main mechanism of cellular apoptosis in promastigotes exposed to SeNPs (Ahmad et al., 2020).

3.8 Antifungal activity

The antifungal activity of fabricated C@SeNPs was evaluated by standard microdilution protocol. Based on the result, the C@SeNPs (273.5–0.27 µg/ml) inhibited the tested fungi strains, containing Candida albicans (IFRC1873), Candida albicans (IFRC1874), Aspergillus fumigatus (IFRC 1649), Aspergillus fumigatus (IFRC 1505), Trichophyton mentagrophytes (FR1_22130), Trichophyton mentagrophytes (FR5_22130), Fusarium proliferatum (IFRC 1871), and Fusarium equiseti (IFRC 1872), with low MIC values (Table. 3). C@SeNPs exhibited MIC values of 0.53 µg/ml against all tested strains. The MIC of the reference antibiotic (Itraconazole) was found around 16 µg/ml that was higher than C@SeNPs (0.53 µg/ml). In addition, the two tested C. albicans were fully resistance against fluconazole with an MIC value of around 64 µg/ml. Shakibaie and coworkers found MIC values of 70 μg/ml and 100 μg/ml for the biosynthesized SeNPs by Bacillus species Msh-1 against C. albicans and A. fumigatus (Shakibaie et al., 2015). Bacillus megaterium-Mediated SeNPs showed antifungal activity against Rhizoctonia solani with minimum inhibition of 0.0625 mM (Hashem et al., 2021). As a result, our synthesized C@SeNPs can be considered as promising antifungal agents because of their strong inhibitory potential against several fungi strains.

Selenium has been discovered to promote the production of ROS; both elements are able to react with intracellular thiols and create intermediates that lead to oxidative stress by producing superoxide radicals. Nanoparticles can affect the integrity of cell membrane or wall and lead to their functional damages (Ismail et al., 2016).

3.9 Catalytic reduction of MB by C@SeNPs

Removing organic contaminants from industrial wastewaters is an essential part of environmental technology (Hassanien et al., 2019). This work was designed to study the reduction of MB dye using biosynthesized C@SeNPs (nanocatalyst) and NaBH₄ (reducing agent) (Fig. 5a-f). When dye reduction was tried without C@SeNPs, the procedure did not demonstrate any progress until after 24 hrs test period. The reduction could not be completed with NaBH₄; the absorbance peak at 665 nm remained unchanged, as seen in Fig. 5e. Therefore, 40, 50, and 60 µl of our SeNPs (546.67 µg/ml) was added to the MB and NaBH₄ solution (Fig. 5a-c). The disappearance of the solution was observed after 15, 13, and 11 min, respectively. The MB reduction time was shown to be faster when the concentration of NPs was increased (Fig. 5d). The reduction rate was obtained 0.1474, 0.1538, and 0.1625 min⁻¹ for 40, 50, and 60 µl of C@SeNPs (Fig. 5f). Nanoparticles provide a larger surface area; therefore, the reduction rate increases after adding C@SeNPs to the solution (MB and NaBH₄). This data showed that the biosynthesized C@SeNPs indicated good catalytic activity in the reducing MB that the action mechanism represented in Fig. 6.

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Fig. 5

a-c) UV–vis spectra of MB reduction reaction after adding 40, 50, and 60 µl C@SeNPs; d) the comparison of three volumes of C@SeNPs in the reduction of MB dye; e) reaction mixture without any nanocatalyst C@SeNPs until 24 hrs; f) reaction constant calculated for three reduction reactions.

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Fig. 6

The mechanism of action of biofabricated C@SeNPs in the reduction of methylene blue (MB) in the presence of NaBH₄.

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