Biogenic synthesis of selenium nanoparticles using Hibiscus esculentus L. extract: Catalytic degradation of organic dye and its anticancer, antibacterial and antifungal activities European Journal of Chemistry 14 (1) (2023) 144-154 European Journal of Chemistry ISSN 2153-2249 (Print) / ISSN 2153-2257 (Online) – Copyright © 2023 The Authors – Atlanta Publishing House LLC – Printed in the USA. This work is published and licensed by Atlanta Publishing House LLC – CC BY NC – Some Rights Reserved. https://dx.doi.org/10.5155/eurjchem.14.1.144-154.2401 European Journal of Chemistry View Journal Online View Article Online Biogenic synthesis of selenium nanoparticles using Hibiscus esculentus L. extract: Catalytic degradation of organic dye and its anticancer, antibacterial and antifungal activities Mohammad Ali Ebrahimzadeh , Mina Moradsomarein , Fatemeh Sadeghi Lalerdi and Seyedeh Roya Alizadeh * Department of Medicinal Chemistry, School of Pharmacy and Pharmaceutical Sciences Research Center, Mazandaran University of Medical Sciences, Sari, 4815733971, Iran * Corresponding author at: Department of Medicinal Chemistry, School of Pharmacy and Pharmaceutical Sciences Research Center, Mazandaran University of Medical Sciences, Sari, 4815733971, Iran. e-mail: r.alizadeh.2019@gmail.com (S.R. Alizadeh). 10.5155/eurjchem.14.1.144-154.2401 Received: 24 December 2022 Received in revised form: 21 January 2023 Accepted: 28 January 2023 Published online: 31 March 2023 Printed: 31 March 2023 In this work, we develop the synthesis of selenium nanoparticles (B@SeNPs) using a green method using the aqueous extract of Hibiscus esculentus L. Various techniques were used to characterize bio-synthesized B@SeNPs. The mixture color was clearly changed to reddish at 45-50 °C and the extract pH = 6. According to Fourier transform infrared spectroscopy (FT- IR), the B@SeNPs were produced, capped, and stabilized using biomolecules found in plant extracts. The energy dispersive X-ray (EDX) analysis profile revealed an atomic Se signal (1.39 mV). The powder X-ray diffraction (PXRD) pattern confirmed the hexagonal phase crystalline form of B@SeNPs. The zeta potential for SeNPs was determined to be -51.3 mV. Scanning electron microscope (SEM) and transmission electron microscopy (TEM) micrographs revealed spherical Se particles with sizes of roughly 62 nm. Furthermore, B@SeNPs can degrade methylene blue dye by 98.3% at 21 min with a rate constant of 0.1023 min-1 in the presence of NaBH4. In biological evaluation, the synthesized nanoparticles have been proven to be effective against two human cancers (AGS and MCF-7 cells) with IC50 values of 20.46 and 88.43 µg/mL, respectively. Additionally, B@SeNPs showed high safety in the Beas cell line (normal) at 123 µg/mL as the highest concentration. The biofabricated SeNPs had a moderate antibacterial effect against ATCC and multidrug-resistant clinical isolates. They had no antifungal activity against the tested fungus strains except C. albicans (IFRC 1873), with a MIC value of 138.75 µg/mL. Finally, the green-synthesized B@SeNPs could be a contender for further testing as a chemotherapeutic agent in the treatment of some human cancers. Catalytic effect Methylene blue Green synthesis Anticancer activity Antibacterial activity Selenium nanoparticles Cite this: Eur. J. Chem. 2023, 14(1), 144-154 Journal website: www.eurjchem.com 1. Introduction The dyeing and textile industries are to blame for water contamination. The removal of organic contaminants from industrial wastewater is critical for environmental technology [1,2]. Methylene blue (MB), a heterocyclic aromatic dye, is commonly used to colour cotton, wool, and silk in the textile industry. It reduces the amount of dissolved oxygen in water and releases toxic substances as a result of chemical or biolo- gical interactions, putting aquatic life in danger [3]. Various approaches can be used to degrade organic dye pollution. However, the expensive expense of some of these techniques, as well as their inability to remove soluble dyes and the use of chemicals, limit them. Of all dye removal techniques available, adsorption is one of the most efficient and affordable. Activated carbon is an effective adsorbent to separate dyes from indust- rial wastewater effluents, but its high cost prevents it from being used on a large scale [4]. In addition, photocatalysis is a method for purifying water that employs light to produce active species on light-sensitive molecules. Photocatalysis also has the advantage of being low cost to operate and can work in any environment. Furthermore, degradation products are generally nontoxic and environmentally beneficial [1]. Throughout the past decade, nanotechnology has resulted in the discovery of materials with improved physicochemical features and applica- tions ranging from nanoelectronics to nanomedicine [5]. Metallic and semiconductor nanoparticles are efficient dye degradation catalysts and photocatalysts in the aquatic environment [6-10]. Selenium nanoparticles (SeNPs) could be employed in a variety of applications. Selenium is a trace element that is vital for the nutritional and growth of the human body [11]. Therefore, as a result, they have received a great deal of attention in recent years and many synthetic methods have been used. A green method with plant extracts that requires nontoxic solvents and mild temperatures has gained favour as a cost-effective, environmentally friendly, and safe approach. Furthermore, it employs a biodegradable and readily available reducing agent [12,13]. Due to the high surface-to-volume ratio of selenium nanoparticles produced using plant extracts, they ABSTRACT RESEARCH ARTICLE KEYWORDS https://dx.doi.org/10.5155/eurjchem.14.1.144-154.2401 https://www.eurjchem.com/ https://dx.doi.org/10.5155/eurjchem.14.1.144-154.2401 mailto:r.alizadeh.2019@gmail.com http://www.eurjchem.com/ https://crossmark.crossref.org/dialog/?doi=10.5155/eurjchem.14.1.144-154.2401&domain=pdf&date_stamp=2023-03-31 Ebrahimzadeh et al. / European Journal of Chemistry 14 (1) (2023) 144-154 145 2023 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.14.1.144-154.2401 Figure 1. The picture of Hibiscus esculentus L. are also particularly effective materials for photocatalytic dye degradation [3,14]. SeNPs synthesized by leaf extract of Fiscus benglalensis degrade methylene blue dye at around 60% in 40 min [3]. In another study, bromothymol blue dye was removed using biogenic SeNP under UV illumination after 60 min at 62.3% [15]. Furthermore, biofabricated selenium nanoparticles demonstrated antibacterial, anti-diabetic, and cytotoxic pro- perties [16]. In various studies, SeNPs have been shown to be effective against a variety of cancer cell lines [17-21], justifying the investigation of various physical, chemical, and biological manufacturing pathways. SeNPs are increasingly important in biomedical applications due to their anticancer characteristics. The action of colloidal SeNPs in human breast cancer cells was also established [1]. In this work, selenium nanoparticles are synthesized using an aqueous extract of Abelmoschus esculentus L. Okra (Hibiscus esculentus L.) is known as a common vegetable in Bangladesh and the Indo-Pak subcontinent. It belongs to the Malvaceae family. It is the most consumed food in India, but originated in Ethiopia and Sudan. Furthermore, it is one of the oldest agricultural crops in the world, growing in numerous countries from Africa to Asia, southern Europe, and America [22]. In addition, it is named "bamye" in Persian [22]. Although it is grown throughout the year, most of its production occurs during summer [23]. This fruit contains 86.1% water, 2.2 % protein, 0.2% fat, 9.7% carbohydrate, 1.0% fiber, and 0.8% ash [24], and also calcium, potassium, vitamins and other minerals are bound in this fruit. High levels of phenols and flavonoids were present in the extract [25]. In addition, efficient antihemolytic, antioxidant [25], and antihypoxic [22] activities of H. esculentus were confirmed. In this study, the production of selenium nanoparticles using Hibiscus esculentus L. extract was described. Our research team has already demonstrated that H. esculentus extract is a valuable source for the fabrication of biogenic nanomaterials. The synthesized nanoparticles were characterized using various analytical methods. The anticancer, antibacterial, and antifungal activities of the synthesized SeNPs were investigated and used for photocatalytic degradation of MB under visible light and NaBH4. 2. Experimental Sodium selenite and sodium borohydride were provided by Sigma Aldrich (USA) and Merck (Germany). Methylene blue and other chemicals were bought from Merck India Ltd. 2.1. Synthesis of selenium nanoparticles The H. esculentus plant (Figure 1) was dried in the sun and cut into small sizes (2-3 mm). The plant (10 g) was added to 100 mL of deionized water, heated for 1 h at 50 °C, sonicated for 30 min, and filtrated by Whatman filter paper (No 1). The filtrate was used to synthesize SeNPs. 10 mL of aqueous extract was added to the stock solution Na2SeO3 (0.01 M) at 45-50 °C and 500 rpm. After two days, the reaction color shifted from colorless to reddish, indicating that the Se ions had been reduced. After that, the solution containing SeNPs was washed four times with water and methanol, centrifuged, and dried in an oven at 60 °C. The dried product was used for subsequent analyses. 2.2. Characterization of the synthesized SeNPs The validation of prepared B@SeNPs was performed using UV-Vis spectrophotometer, T80 UV-Vis spectrophotometer PGI (Beijing, China) to evaluate the Surface Plasmon Resonance (SPR). The size and morphology of B@SeNPs were estimated by TEM Philips EM 208S and SEM TESCAN BRNO-Mira3 LMU. In addition, the EDX identified the purity and elemental composition of the fabricated B@SeNPs that was carried out along with SEM analysis. Next, the DLS study evaluated their average size dispersion. Following that, the crystallinity of SeNPs was determined by XRD analysis using PANalytical X- PERT PRO diffractometer (2θ = 10-80°) with CuKα source (λ = 1.5406 Å). To approve the capped SeNPs, the FT-IR analysis was applied to determine the potential molecules involved in the reduction of selenium ions (ATR, Agilent, Cary 630, FT-IR Spectrometer, equipment in 4000 and 650 cm-1). 2.3. Antibacterial activity of synthesised SeNPs The antibacterial activity of bioproduced SeNPs using H. esculentus extract was tested against bacteria from the American Type Culture Collection (ATCC), comprising: Proteus mirabilis, Klebsiella pneumoniae, Pseudomonas aeruginosa, Escherichia coli, Enterococcus faecalis, Staphylococcus aureus, and Acinetobacter baumannii. In addition, the synthesized SeNPs were examined against eight multidrug resistant (MDR) bacteria isolated from clinical including: P. mirabilis, K. pneumoniae, P. aeruginosa, E. coli, E. faecalis, S. aureus (two strains), and A. baumannii. The minimum inhibitory concent- ration (MIC) of biosynthesized SeNPs was assessed using the microdilution method. In this experiment, positive control (conventional antibiotic ciprofloxacin) and negative control (medium and SeNPs) were used. One hundred microliters of Mueller-Hinton broth (MHB) with different amounts of SeNPs (277.5-0.27) were loaded into all wells. Subsequently, each well was filled with 100 µL of dilute bacterial suspension (0.5 McFarland turbidity standards) and the plate was incubated at 37 °C. After that, the growth of bacteria in the wells was visually checked and the lowest concentration of SeNPs, which prevented the visible growth of bacteria in MHB, was deter- mined as MIC. The minimum bactericidal concentration (MBC) was measured using Muller-Hinton agar media (MHA) on different plates according to the MIC value; 10 µL from MIC well and three concentrated wells were cultured in MHA for 24 h at 37 °C. After 24 h, the MBC value was calculated as the lowest concentration of SeNPs that did not show observable growth in MHA [26-29]. 2.4. Antifungal activity of synthesized SeNPs Based on the M27-A3 method from the Clinical and Laboratory Standards Institute (CLSI), formerly NCCLS, the broth microdilution method was used to estimate the MIC value. 146 Ebrahimzadeh et al. / European Journal of Chemistry 14 (1) (2023) 144-154 2023 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.14.1.144-154.2401 Figure 2. UV-vis spectra of B@SeNPs and H. esculentus L. The tested strains contained Candida albicans (IFRC 1873), Candida albicans (IFRC 1874), Aspergillus fumigatus (IFRC 1649), Aspergillus fumigatus (IFRC 1505), Trichophyton mentagrophytes (FR1 22130), Trichophyton mentagrophytes (FR5 22130), Fusarium proliferatum (IFRC 1871), Fusarium equiseti (IFRC 1872). In summary, 100 μL of B@SeNPs (555- 0.54 μg/mL) were added to the microplate wells and serially diluted with 100 μL of RPMI media. Then 100 μL of cell suspensions were introduced into each well. Visible growth was checked after 24 h (C. albicans) and 48 h (other strains) under incubation conditions. Itraconazole was considered a reference antifungal agent [30,31]. 2.5. Anticancer activity of synthesized SeNPs The protocols mentioned earlier were used to determine cytotoxicity [32]. From the pasture Institute in Iran, human gastric cancer (AGS), human breast adenocarcinoma (MCF-7), and human non-tumorigenic lung epithelial cell line (Beas) cells were generated. The dimethyl thiazolyl tetrazolium bromide (MTT) assay was used to test the cytotoxicity of the produced SeNPs on three tested cell lines. The cells were cultured in RPMI-1640 with 10% FCS and penicillin/streptomycin; 6- 10×103 cells were seeded in 96-well plates and incubated for 24 h at 37 °C under CO2. Then, supernatant from the grown cells was collected after 24 h, and different quantities of SeNPs (10 to 240 µg/mL) were diluted with the growth medium and added to each well. After 48 h of incubation, 20 µL of MTT (5 mg/mL) was added to each well and incubated for 4 h. Finally, the purple-colored formazan crystals were dissolved in a 200 µL DMSO solution. A spectrophotometer (Biotek Instruments; USA) was used to measure the optical density of each well at 590 nm. The % cell viability was assessed by the following equation [28,33,34]: Cell viability = The optical density of the sample well The optical density of control well (1) 2.6. Catalytic activity of synthesized B@SeNPs Metal nanoparticles could be used to catalyze chemical reactions that would otherwise be impossible to achieve. The catalytic effect of the B@SeNPs produced was tested on methylene blue as a toxic dye using NaBH4; 30 µL of 10 mM MB solution was mixed with 5.77 mL of H2O and 200 µL of freshly made 0.1 M NaBH4 solution. Then 70 µL of colloidal SeNPs (1110 µg/mL) was added to the prepared mixture containing MB and NaBH4. There was also a blank sample made without SeNPs. The color of the SeNPs-containing sample progressively faded from deep to light blue before becoming colorless. The MB reduction was measured using a UV-vis spectrophotometer at regular time intervals. The test was carried out at room temperature. The pseudo-first-order equation (2) was used to monitor the MB’s degradation rate; At and A0 were the absorbance at interval times and time 0, respectively [31]. 𝐿𝐿𝐿𝐿 � 𝐴𝐴 𝐴𝐴0 � = −𝑘𝑘𝑘𝑘 (2) 2.7. Statistical analysis All experiments were carried out in triplicate and data were represented as (mean value±SD). All statistical analyses were performed by one-way ANOVA at p < 0.05 using GraphPad prism software. 3. Results and discussion 3.1. Characterization of green synthesized B@SeNPs Biomolecules in the aqueous extract of H. esculentus reduced the Na2SeO3 salt solution to SeNPs. The solution color changed from colorless to reddish after 48 h; this is the surface plasmon resonance effect (SPR). Various temperatures (25, 45, and 75 °C) and pH values (6 (extract pH), 10, and 12) were studied to optimize the synthesis conditions of SeNPs using H. esculentus; Based on the results, the reddish color of the mixture occurred at 45-50 °C and the extract pH (pH = 6), as demonstrated in Figure 2. Other conditions did not affect the color change. Furthermore, the absorbance of the solution was measured using a UV-visible spectrophotometer with a wavelength range of 200 to 800 nm (Figure 2). Cassia auriculata-mediated SeNPs had a UV-intensive peak at 252 nm [18]. Ramamurthy and colleagues used Fenugreek extract to make SeNPs, with a distinctive peak between 200 and 400 nm [35]. The solution containing SeNPs was centrifuged after the washing process and dried in an oven before being used for additional analysis. The particle length and form of SeNPs have been predicted with the aid of TEM images. Figure 3 indicates the implied length of SeNPs (62 nm), with sphere polydispersed debris; this is attributed to the biomolecules in the plant extract; that perform as capping, reducing, and stabilizing agents. SEM images were used to examine the shape and surface charac- teristics of SeNPs. The SEM picture of SeNPs with variable size and homogeneous rounded form is shown in Figure 4. Additionally, the average size obtained from the SEM image was 50.1 nm; the histogram is presented in Figure 5. The SeNPs synthesized by Allium paradoxum extract displayed semi- spherical SeNPs with an average size of 37.5 nm [31]. Ebrahimzadeh et al. / European Journal of Chemistry 14 (1) (2023) 144-154 147 2023 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.14.1.144-154.2401 Figure 3. TEM image of green synthesized B@SeNPs. Figure 4. The SEM image of green synthesized B@SeNPs. Figure 5. Histogram obtained from the SEM image of green synthesized B@SeNPs. SEM and TEM images of SeNPs using Mucuna pruriens seed powder showed that the NPs produced had a spherical shape and a homogeneous dispersion in a size range of about 100-110 nm [36]. Green SeNPs produced by the Solanum nigrum fruit extract were spherical with a particle diameter of 87 nm [37]. TEM micrographs of the biogenic SeNPs of Spirulina platensis presented spherical shapes with a mean size of 79.40±44.26 nm [38]. As seen in Figure 6, EDX analysis was used to verify the elemental summary of SeNPs and its ability to indicate the purity of the offered SeNPs. SeNPs exhibited typical Se absorp- tion maxima at 1.39 keV. The high amount of selenium demonstrates the purity of this element (Se = 81.37 %). The presence of carbon at 0.29 keV and oxygen at 0.54 keV indicates the existence of alkyl chain stabilizers. SeNPs were discovered to have a particle size and distribution of 266.3 nm (Figure 7). The DLS size range of the synthesized SeNPs was found to be significantly larger than the TEM size range. Because DLS measures the hydrodynamic diameter of the SeNPs when they are surrounded by water molecules, it is possible that this is the source of the enormous size of the capped formulation. The zeta potential that showed the stability of SeNPs was found to be -51.3 mV (Figure 8). The zeta potential of S. platensis-mediated SeNPs was -32.9±8.12 mv [38]. Furthermore, SeNPs synthesized by aqueous extract of Portulaca oleracea indicated a zeta potential of −43.8 mV [39]. The synthesized SeNPs were crystallized according to the XRD pattern of the sample (Figure 9). The peaks at 23.45, 29.65, 41.20, 43.70, 45.35, 51.75, 56.20, 61.65, and 65.45 ° were labelled in Bragg’s appearances as (100), (101), (110), (102), (111), (201), (112), (202), and (210), respectively. All peaks can be matched with SeNPs JCPDS (Joint Committee on Powder Diffraction Standards) (JCPDS File No. 06-0362). The result indicates that the bioprepared SeNPs produced the hexagonal phase crystalline form [40]. The determined size by the Debye- Scherrer equation was 34.8 nm. 148 Ebrahimzadeh et al. / European Journal of Chemistry 14 (1) (2023) 144-154 2023 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.14.1.144-154.2401 Figure 6. EDX analysis of green prepared B@SeNPs. Figure 7. DLS graph of the synthesized B@SeNPs. Figure 8. Zeta potential of the synthesized B@SeNPs. Figure 9. The XRD pattern of the synthesized B@SeNPs. Ebrahimzadeh et al. / European Journal of Chemistry 14 (1) (2023) 144-154 149 2023 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.14.1.144-154.2401 Table 1. The MIC and MBC values obtained from biosynthesized B@SeNPs against several ATCC strains. Bacteria ATCC B@SeNPs Extract Ciprofloxacin MIC (µg/mL) MBC (µg/mL) MIC (µg/mL) MIC (µg/mL) S. aureus ATCC 29213 69.35 138.75 >2500 0.21 E. faecalis ATCC 29212 17.34 138.75 >2500 0.21 P. aeruginosa ATCC 27853 138.75 1110 >2500 0.3 A. baumannii ATCC 19606 34.7 1110 >2500 0.25 E. coli ATCC 25922 138.75 555 >2500 0.1 K. pneumoniae ATCC 700603 69.35 555 >2500 0.1 P. mirabilis ATCC 25933 17.34 277.5 >2500 0.1 Table 2. The MIC and MBC values obtained from biosynthesized B@SeNPs against several clinically isolated strains *. Bacteria B@SeNPs Extract Susceptibility to antibacterial agents MIC (µg/mL) MBC (µg/mL) MIC (µg/mL) M V O T G P CI CE E CM AK S. aureus (VRSA) 138.75 555 >2500 R R R S R R R R R R R S. aureus (MRSA) 138.75 555 >2500 R R R S R R R R R R R E. faecalis 138.75 555 >2500 S R R R R R S R R R R P. aeruginosa 277.5 1110 >2500 R R R R R R R R R R R A. baumannii 277.5 1110 >2500 R R R R R R R R R R R E. coli 277.5 1110 >2500 R R R R R R R R R R R K. pneumoniae 277.5 555 >2500 R R R R R R R R R R R P. mirabilis 277.5 1110 >2500 R R R R R R R R R R R * R, resistant; S, susceptible; M, Meticillin; V, Vancomycin; O, Oxacillin; T, Tetracycline; G, Gentamicin; P, Penicillin; CI, Ciprofloxacin; CE, Ceftazidime; E, Erythromycin; CM, Clindamycin; A, Amikacin; MRSA, methicillin resistant Staphylococcus aureus; VRSA, Vancomycin resistant Staphylococcus aureus. Figure 10. The FT-IR of the synthesized B@SeNPs. These results were consistent with the XRD pattern of SeNPs prepared using Diospyros montana extract, which presented the hexagonal structure of SeNPs [41]. FT-IR analysis was used to identify the molecular interac- tion between the extract and G@SeNPs. As shown in Figure 10, the FT-IR spectrum of the extract exhibited absorption bands at 3267, 2927, 1618, 1405, 1030, 922, and 776 cm-1. Moreover, the spectrum of synthesized SeNPs showed absorption peaks at 3269, 2926, 1628, 1527, and 1032 cm-1. The broad peak at 3267 and 2927 cm-1 corresponded to the stretching of O-H and the asymmetric stretching of the C-H bonds, respectively [42]. The peaks at 1618 and 1405 cm-1 are assigned to carbonyl stretching bands (amides) [43] and -OH bonds of carboxylates [18,42,44,45]. Another peak at 1030 cm-1 was related to C-N (stretching vibration of primary amines) [18,42]. According to the FT-IR data, the intensity of all bonds had decreased in the SeNPs spectrum, which could be attributed to the binding of SeNPs to the functional groups of the extract. 3.2. Antibacterial activity of SeNPs against ATCC strains and clinically isolated strains This study used two series of ATCC bacteria and clinically obtained bacteria to test their susceptibility to prepared B@SeNPs (Tables 1 and 2). The antibacterial properties of B@SeNPs produced in the presence of H. esculentus extract were tested against Gram positive and Gram-negative bacteria. Various concentrations of B@SeNPs (277.5 to 0.27 µg/mL) and H. esculentus extract (2500 to 312.5 µg/mL) were used to treat test pathogens. The MIC assay was performed using the broth dilution method for both MDR and ATCC strains listed in Tables 1 and 2. The antibacterial activity of B@SeNPs revealed that when the concentration of B@SeNPs increased, the MIC value of bacteria decreased. As shown, E. faecalis (ATCC) and P. mirabilis (ATCC) were found to be the most sensitive to B@SeNPs, with a MIC value of 17.34 µg/mL. Furthermore, clinically isolated strains E. faecalis, S. aureus (VRSA), and S. aureus (MRSA) exhibited the lowest MIC value of 138.75 µg/mL. S. nigrum-mediated SeNPs did not inhibit S. aureus, while it showed a MBC value of 50±1.76 mg/mL against E. coli [37]. In another study, green SeNPs from Lysinibacillus sp. NOSK inhibited the biofilm of P. aeruginosa compared to untreated samples [46]. The antibacterial investigation of SeNPs from Vitamin C showed a dose-dependent manner against E. coli, P. aeruginosa, S. aureus, and S. epidermidis [47]. According to many sources, the antimicrobial activity of SeNPs is primarily due to the generation of reactive oxygen species (ROS), which causes the phospholipid bilayer to be disrupted, allowing the SeNPs to interact with intracellular proteins and inactivate them or react with the sulphydryl and thiol groups that exist in membrane proteins, causing them to denaturise. Furthermore, ROS causes cell death by disrupting the protein synthesis cycle, intervening in the respiratory or food metabolic pathways, and affecting DNA replication [48,49]. 3.3. Antifungal activity of SeNPs against fungi strains resistant to itraconazole The antifungal effect of SeNPs was evaluated against eight fungus strains resistant to itraconazole by the micro broth dilution method. 150 Ebrahimzadeh et al. / European Journal of Chemistry 14 (1) (2023) 144-154 2023 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.14.1.144-154.2401 Table 3. The MIC value achieved from biosynthesized B@SeNPs against several fungi isolates. The fungi isolates tasted B@SeNPs Itraconazole Extract MIC (µg/mL) MIC (µg/mL) MIC (µg/mL) Aspergillus fumigatus (IFRC 1649) R ≥16 >2500 Aspergillus fumigatus (IFRC 1505) R ≥16 >2500 Trichophyton mentagrophytes (FR1 22130) R ≥16 >2500 Trichophyton mentagrophytes (FR5 22130) R ≥16 >2500 Fusarium proliferatum (IFRC 1871) R ≥16 >2500 Fusarium equiseti (IFRC 1872) R ≥16 >2500 Candida albicans (IFRC 1873) 138.75 ≥16 >2500 Candida albicans (IFRC 1874) R ≥16 >2500 Figure 11. In-vitro cytotoxicity of B@SeNPs at different concentrations against AGS cell line. Figure 12. In-vitro cytotoxicity of B@SeNPs at different concentrations on MCF-7 cell line. The minimal inhibitory concentration (MIC) was visually determined, which means it cannot be quantified. Our synthe- sized SeNPs at concentrations 555-0.54 µg/mL did not show antifungal activity against tested fungi strains except C. albicans (IFRC 1873), which had a MIC value of 138.75 µg/mL (Table 3). Furthermore, the antifungal activity of the extract was examined at concentrations of 2500 to 312.5 µg/mL, showing growth in all wells. The reference antibiotic was itraconazole. The MIC values of the Allium paradoxum-mediated SeNPs were 0.68 μg/mL against all fungal strains similar to those tested in our study, except for two strains of C. albicans. In fact, SeNPs were resistant to C. albicans [31]. In another investigation, the SeNPs manufactured by Bacillus species Msh-1 displayed MIC values of 70 μg/ml and 100 μg/ml against C. albicans and A. fumigatus, respectively [50]. 3.4. Anticancer activity of SeNPs Chemotherapy is a well-known cancer treatment method, and employing targeted NPs to deliver chemotherapeutic medications to cancer patients has several advantages. Because drugs associated with NPs can penetrate deeper into organs, NPs are used in cancer drug delivery. Interestingly, various research organizations have concentrated on developing a promising source of new therapeutic molecules for cancer treatment [1]. In this work, the in vitro cytotoxicity of SeNPs was investigated against human gastric cancer (AGS), human breast adenocarcinoma (MCF-7), and human non-tumorigenic lung epithelial cell line (Beas) using the MTT assay. Cells were treated with five different concentrations of SeNPs (123, 61.7, 30.8, 15.4, and 7.7 µg/mL) at 48 h. The results of the MTT test revealed that SeNPs had significant effects on AGS and MCF-7; The IC50 values on AGS and MCF-7 were found to be 20.46 and 88.43 µg/mL, respectively (Figures 11 and 12). Furthermore, our synthesized nanoparticles exhibited excellent safety in the normal cell line, Beas, at the highest concentration (123 µg/mL) (Figure 13). Therefore, the prepared B@SeNPs showed high cytotoxicity against the AGS and MCF-7cell lines. This synthetic method provides many advantages, including simplicity, cost- effectiveness, and compatibility for pharmaceutical and medical applications. In a study in 2019, In a study in 2019, IC50 values of 150.87, 392.57, and 252.44 µg/mL were discovered on the Caco2, HepG-2 and MCF-7 cell lines for M. Oleifera leaves mediated SeNPs, respectively [1]. The IC50 value for SeNPs produced by Vitamin C on MCF-7 cancer cells was calculated at 23.20 µg/mL [51]. Ebrahimzadeh et al. / European Journal of Chemistry 14 (1) (2023) 144-154 151 2023 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.14.1.144-154.2401 Figure 13. In-vitro cytotoxicity of B@SeNPs at different concentrations on normal cell lines of Beas. Figure 14. The catalytic reduction of MB using 70 µL B@SeNPs (1110 µg/mL) in the presence of NaBH4. Figure 15. The reduction of MB using NaBH4. 3.5. Catalytic effect of B@SeNPs on methylene blue The methylene blue reduction process by NaBH4 was used as a model reaction to evaluate the catalytic activity of biosynthesised B@SeNPs. This dye is a basic aniline dye, also identified as methylthioninium chloride, which has a wide range of applications in biology and chemistry, as well as as a stain and medication. When MB is oxidized, it is blue, but when it is reduced, it becomes colorless leucomethylene blue [52]. In an aqueous solution, MB shows an absorption maxima band around 665 nm due to the π → π* and n → π* transitions. Based on the result, the UV-Vis spectra of MB reduction by NaBH4 in the presence of produced catalyst SeNPs are shown in Figure 14. The MB reduction by NaBH4 in the lack of the SeNPs was shown in Figure 15. Obviously, in the absence of B@SeNPs, the dye reduction rate was substantially slower than that in their presence. The reduction was accomplished in 21 min in the presence of 70 µL of B@SeNPs. Additionally, the degradation percentage of MB in time intervals was presented in Figure 16. This result demonstrated that adding B@SeNPs in the right amount to the MB reduction process made it more efficient. This reduction reaction was found to be pseudo-first order, and the rate constant was calculated as 0.1023 min-1. The spectra in Figure 17 exhibited ln At/A0 versus time for MB reduction using B@SeNPs at room temperature. The biosynthesized SeNPs using the leaf extract of Ficus benghalensis exhibited a degradation of 57.63% in 40 min with a rate constant of 0.02162 s−1 against the MB dye [3]. In another study, eco- friendly and rapidly fabricated SeNPs through aqueous extracts of Ceropegia bulbosa tuber presented effective photocatalytic activity against MB using a halogen lamp with 96% degradation [53]. In addition, Withania somnifera-mediated SeNPs decreased the MB peak intensity during 30 min under solar light [14]. 152 Ebrahimzadeh et al. / European Journal of Chemistry 14 (1) (2023) 144-154 2023 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.14.1.144-154.2401 Figure 16. Degradation percentage of MB reduction using B@SeNPs and NaBH4. Figure 17. The calculation of the rate constant by plotting ln(At/A0) vs time. Based on the data, biosynthesized colloidal B@SeNPs were applied as an excellent green catalyst, mediating electron transfer during the MB reduction by NaBH4. The bond dissociation energy (BDE) is a significant factor in the breaking and/or production of new bonds during chemical reactions. In the interaction between MB and NaBH4, NaBH4 operates as a donor and MB as an acceptor, resulting in electron transfer. The introduction of selenium nanocatalysts to the reaction mixture acted as a possible intermediary between the MB and BH4 ions. It initially reduced the BDE and increased the efficiency of electron transport between them. In the presence of SeNPs, the rate of MB reduction by NaBH4 was enhanced. In the catalytic reduction of organic color, colloidal SeNPs produced from readily available extracts exhibited encouraging results [52]. 4. Conclusion This work used an aqueous extract of H. esculentus to produce SeNPs without the use of harmful or dangerous chemicals. In this green synthesis process, the extract of H. esculentus serves as a natural reducing, capping, and stabilizing agent. The existence of various functional groups in H. esculentus was indicated by FT-IR findings, which influenced the synthesis and stabilization of B@SeNPs. The color of the reaction medium changed from colorless to brick red, showing the synthesis of Se0. The production of crystalline B@SeNPs with an average size of 34.8 nm was confirmed using the XRD pattern. The produced NPs were found to be of atomic Se using EDX. SEM and TEM studies were used to document the morphology of the synthesized B@SeNPs. In addition, they have a spherical shape with an average size of 62 nm in the TEM image. Catalytic analysis revealed that B@SeNPs are capable of degrading methylene blue dye in the presence of NaBH4 and visible light. As a result, B@SeNPs could be used in water treatment. Furthermore, the anticancer activity of B@SeNPs generated in an environmentally friendly manner using the plant-mediated method was investigated. B@SeNPs have been shown to be effective against two types of human cancer cells (AGS and MCF-7 cells) while remaining relatively safe for normal cell Beas. The IC50 values of 20.46 and 88.43 µg/mL suggest that B@SeNPs are powerful anticancer agents with high safety in normal cells that suppress the growth of both types of cancers. Furthermore, the biofabricated SeNPs revealed moderate antibacterial activity against two types of bacterial strains (ATCC and clinically isolated), and they did not show antifungal activity against some fungi strains. The green synthesis of B@SeNPs could be a useful method in biomedical applications. Acknowledgements This research was supported by a grant from the research council of Mazandaran University of Medical Sciences, Iran (Grant No. 10555). Disclosure statement Conflict of interest: The authors declare that they have no conflict of interest. Ethical approval: All ethical guidelines have been adhered. Sample availability: Samples of the compounds are available from the author. Declaration of competing interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. CRediT authorship contribution statement Conceptualization: Mohammad Ali Ebrahimzadeh; Methodology: Mohammad Ali Ebrahimzadeh, Fatemeh Sadeghi Lalerdi, Mina Moradsomarein; Formal analysis: Mohammad Ali Ebrahimzadeh; Investigation: Mohammad Ali Ebrahimzadeh, Fatemeh Sadeghi Lalerdi; Resources: Seyedeh Roya Alizadeh; Funding: Seyedeh Roya Alizadeh; Supervision: Seyedeh Roya Alizadeh; Ebrahimzadeh et al. / European Journal of Chemistry 14 (1) (2023) 144-154 153 2023 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.14.1.144-154.2401 Writing - Original Draft: Mohammad Ali Ebrahimzadeh, Fatemeh Sadeghi Lalerdi, Mina Moradsomarein, Seyedeh Roya Alizadeh; Review and Editing: Seyedeh Roya Alizadeh. All authors have read and agreed to the published version of the manuscript. ORCID and Email Mohammad Ali Ebrahimzadeh zadeh20@gmail.com https://orcid.org/0000-0002-8769-9912 Mina Moradsomarein minamrds97@gmail.com https://orcid.org/0000-0002-5374-1940 Fatemeh Sadeghi Lalerdi fatemehsadeghi132@yahoo.com https://orcid.org/0000-0001-5518-0051 Seyedeh Roya Alizadeh r.alizadeh.2019@gmail.com ro.alizadeh@mazums.ac.ir https://orcid.org/0000-0001-7435-4635 References [1]. Hassanien, R.; Abed-Elmageed, A. A. I.; Husein, D. Z. Eco-friendly approach to synthesize selenium nanoparticles: Photocatalytic degradation of sunset yellow azo dye and anticancer activity. ChemistrySelect 2019, 4, 9018–9026. [2]. Vuppala, V.; Motappa, M. G.; Venkata, S. S.; Sadashivaiah, P. H. Photocatalytic degradation of methylene blue using a zinc oxide- cerium oxide catalyst. Eur. J. Chem. 2012, 3, 191–195. [3]. Tripathi, R. M.; Hameed, P.; Rao, R. P.; Shrivastava, N.; Mittal, J.; Mohapatra, S. 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This work is published and licensed by Atlanta Publishing House LLC, Atlanta, GA, USA. The full terms of this license are available at http://www.eurjchem.com/index.php/eurjchem/pages/view/terms and incorporate the Creative Commons Attribution-Non Commercial (CC BY NC) (International, v4.0) License (http://creativecommons.org/licenses/by-nc/4.0). By accessing the work, you hereby accept the Terms. This is an open access article distributed under the terms and conditions of the CC BY NC License, which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited without any further permission from Atlanta Publishing House LLC (European Journal of Chemistry). No use, distribution, or reproduction is permitted which does not comply with these terms. Permissions for commercial use of this work beyond the scope of the License (http://www.eurjchem.com/index.php/eurjchem/pages/view/terms) are administered by Atlanta Publishing House LLC (European Journal of Chemistry). http://www.eurjchem.com/index.php/eurjchem/pages/view/terms http://creativecommons.org/licenses/by-nc/4.0 http://www.eurjchem.com/index.php/eurjchem/pages/view/terms 1. Introduction 2. Experimental 2.1. Synthesis of selenium nanoparticles 2.2. Characterization of the synthesized SeNPs 2.3. Antibacterial activity of synthesised SeNPs 2.4. Antifungal activity of synthesized SeNPs 2.5. Anticancer activity of synthesized SeNPs 2.6. Catalytic activity of synthesized B@SeNPs 2.7. Statistical analysis 3. Results and discussion 3.1. Characterization of green synthesized B@SeNPs 3.2. Antibacterial activity of SeNPs against ATCC strains and clinically isolated strains 3.3. Antifungal activity of SeNPs against fungi strains resistant to itraconazole 3.4. Anticancer activity of SeNPs 3.5. Catalytic effect of B@SeNPs on methylene blue 4. Conclusion Acknowledgements Disclosure statement CRediT authorship contribution statement ORCID and Email References PrintField10: PrintField11: PrintField12: PrintField13: PrintField14: PrintField15: PrintField16: PrintField17: PrintField18: PrintField19: PrintField110: PrintField20: PrintField21: PrintField22: PrintField23: PrintField24: PrintField25: PrintField26: PrintField27: PrintField28: PrintField29: PrintField210: