key: cord-0881634-enek88ao authors: Długosz, Olga; Ochnik, Michał; Sochocka, Marta; Franz, Dominika; Orzechowska, Beata; Anna, Chmielowiec-Korzeniowska; Agata, Drabik; Banach, Marcin title: Antimicrobial and antiviral activity of selenium sulphide nanoparticles synthesised in extracts from spices in natural deep eutectic solvents (NDES) date: 2022-04-11 journal: Sustainable Materials and Technologies DOI: 10.1016/j.susmat.2022.e00433 sha: 7e7f60688024269121b58b0e7a4a8c16b4f31e01 doc_id: 881634 cord_uid: enek88ao Selenium sulphide is a well-known bioactive chemical, but its preparation in nanometric form stabilised in water has not been widely reported. In the article, extracts of cinnamon, curcumin, and pepper obtained using natural deep eutectic solvents (NDES) were used to obtain stable selenium sulphide nanoparticles. The analysis confirmed that selenium sulphide nanoparticles with an average crystallite size of 28–44 nm and a particle size of approximately 500 nm were successfully synthesised. The use of NDES stabilised the SeS2 nanoparticles and increased their bioactivity towards microorganisms. The obtained systems revealed high biocidal and antiviral activity against S. aureus, E. coli, P. aeruginosa, and C. albicans strains, Human influenza virus A/H1N1, and Betacoronavirus 1 (Human coronavirus HCoV-OC43). The SeS2 nanoparticles obtained in the NDES extract of curcuma strongly inhibited the growth of pathogenic fungi and bacteria with minimum biocidal concentration (MBC) values of 117.2, 117.2, 117.2, and 468.8 mg/dm3 against E. coli, P. aeruginosa, S. aureus, and C. albicans, respectively. The suspensions containing selenium sulphide nanoparticles stabilised by spice extracts were also highly active against influenza viruses and B-coronavirus, showing a reduction of over 99%. The additive of inorganic nanoparticles, including metals, non-metals, metal oxides, salts, and many others, provides materials with bioactive properties that protect humans from bacteria, fungi, or even viruses [1, 2] . At the same time, substances of natural origin, which are known for their antiseptic activity, have long been used. Combining inorganic nanoparticles with natural substances in a synergistic manner acts on a number of microorganisms [3, 4] . However, ongoing extraction of active compounds. The use of NDES can prevent solubility problems of compounds, which increases the bioactivity of substances [23, 24] . For example, cinnamon contains a number of active compounds such as cinnamaldehyde and polyphenols, i.e., bioactive compounds that have anti-cancer, anti-inflammatory, anti-diabetic, hyperglycaemic, and antioxidant properties. However, polyphenol-rich cinnamon extract is poorly soluble in water and, therefore, has low bioavailability [25] . Therefore, the aim of this study was to investigate the functionality of selenium sulphide nanoparticles obtained in the presence of cinnamon, curcumin, and cayenne pepper extracts. The novelty of this work was the stabilisation of Se2S nanoparticles in extracts obtained from deeply eutectic solvents, increasing the bioavailability of selenium sulphide while introducing additional active compounds into the system. Literature sources have shown that selenium sulphide is highly effective against bacteria and fungi, but information on its antiviral activity still needs to be clarified [26] . Both selenium and sulphur can act on pathogens through different pathways, and the combination of the two elements can create new properties unique to the combination [10] . The deep eutectic solutions used present two functions. On the one hand, NDES prove to be an excellent extractant for active substances found in spices, which have limited solubility in water. On the other hand, the spice extracts stabilise the selenium sulphide nanoparticles, enabling them to disperse better in water and to better contact microorganisms. In this way, the material has a biocompatible effect and all the ingredients act synergistically. The obtained materials are characterised by high biocidal activity. Cinnamon, curcumin, mixtures of curcumin and black pepper and cayenne pepper were selected as natural compounds. The use of NDES as the extractant is preferred, as it allows for a higher recovery efficiency of active compounds, especially those with limited solubility in water. In this study, the bactericidal and fungicidal properties and viral activity of selenium sulphide nanoparticle suspensions of spice extracts in NDES were evaluated. In the synthesis of selenium sulphide nanoparticles, the following reagents were used: selenium chloride (SeCl4, 35 .0-36.5% Se), sodium sulphide (NaS2•9H2O, ≥ 98.0%), and sodium hydroxide (NaOH, ≥ 99.0%). The preparation of selenium sulphide nanoparticles was carried out directly in the presence of extracts from cinnamon, curcumin, black pepper, and cayenne pepper, which allowed stable nanoparticles with smaller sizes to be obtained compared to nanoparticles obtained without stabilising additives. Natural deep eutectic solvents (NDES) were prepared by mixing citric acid (C6H8O7, ≥ 99.0%), propanediol (C3H8O2, 99.0%), proline (C5H9NO2, 99.0%), lactic acid (C3H6O3, 80%), glucose (C6H12O6, 99.5%), and betaine (C5H11NO2, 98.0%). All reagents were from Sigma Aldrich. One of the applications of deep eutectic solutions is the extraction of natural compounds. In the study, four types of natural deep eutectic solvents (NDES) were prepared. As donors of hydrogen bonds, the solutions proline and betaine were chosen. As acceptors of hydrogen bonds, mixtures of organic acids and carbohydrates were chosen. The total mass of each NDES was 50 g and the molar ratios of the individual components were set at 2:2:1 ( Table 1 ). In addition, 10 ml (20% mass) of deionised water was added to the mixtures. All components were mixed together at 150 rpm and the mixtures were heated to 80 o C to obtain transparent solutions. The mixtures were then cooled to obtain the finished solutions (NDES1-NDES4). The composition of the each NDES and the content of the active substances are presented in Table 1 . Lactic acid-Glucose-Betaine 2:2:1 NDES4 Lactic acid -Glucose-Proline 2:2:1 The preparation of the spice extract involved the addition of 1 g of selected spices in powder form to 20 g of the previously prepared NDES. The suspension was shaken for 1 h in a ball mill. The shaking frequency was 10 s -1 . After completion of the processes, the suspension was centrifuged (5000 rpm, 10 min) to obtain clear extracts. The content of flavonoids, polyphenols, and components contained in the spices, i.e., curcumin, cinnamal, eugenol, and coumarin, was determined in the prepared extracts. Based on the obtained results, the NDES with the highest extraction content of active substances from the spices were selected for further studies. The total phenolic content was determined by the Folin-Ciocalteu method. The method is based on the reversible reduction of molybdenum(VI) to molybdenum (V) contained in the Folin-Ciocalteu reagent. The process takes place in an alkaline medium in the presence of polyphenols. First, 0.05 ml of each extract was mixed with 1 ml of a 10% Folin-Ciocalteu reagent and 2 ml of a sodium carbonate solution (75 g/l). The mixture was left for 120 min, and the absorbance was measured at 725 nm. From a calibration curve showing the dependence of the absorbance of a gallic acid solution on its concentration in the solution, the total phenolic content was determined. The polyphenol content was converted to gallic acid content per gram of natural material. Determination of free radical scavenging was performed using the reagent DPPH (1,1-diphenyl-2-picrylhydrazyl). The reduction of stable free radical violet DPPH to yellow diphenyl-2-picrylhydrazine allows for the calculation of the percentage of inhibition. The absorbance of the DPPH solution was read at 517 nm after 30 min. The antioxidant activity of the extracts was expressed as the percentage reduction of the DPPH radical with the extract relative to the sample without the antioxidant active ingredient: where I is the percentage inhibition of DPPH, Ac is the absorbance of the control sample, and As is the absorbance of the extract samples. For the determination of curcumin in the extracts, a standard curve was prepared as a first step. For this purpose, curcumin stock solutions containing 1 to 7 µg/ml were prepared in methanol. The absorbance at 421 nm was then measured. The curcumin content of the extracts was determined by dissolving 0.5 ml of the extracts in methanol in a 10 ml flask [27] . To determine the cinnamaldehyde content of the extracts, ethanolic solutions of cinnamal were prepared with concentrations ranging from 0.5 to 2.5 µg/ml. In parallel, 0.5 ml of the spice extracts was added to 9.5 ml of ethanol. Absorbance was measured at 286 nm against ethanol. The compound content was calculated from the calibration curve [28] . hydroxide solution was added and the solution was made up to the mark with distilled water, mixed, and its absorbance was measured at 450 nm [29] . The coumarin content was determined from the prepared coumarin standard solutions. From a stock solution of 10 mg coumarin in 50 ml of a 1:1 v/v methanol:acetone solution, solutions of 10, 15, and 25 µg/ml were prepared. Additionally, 0.5 ml of the extract was prepared in a 10 ml flask and diluted with a methanol:acetone mixture. The determination of total coumarins was calculated from the calibration curve for pure coumarin at 327 nm [30] . To determine the total flavonoid content, 0.5 ml of the extract and 2% AlCl3 were added to a 10 ml flask and made up to the mark with methanol. At the same time, 2% AlCl3 was added to a 10.0 ml volumetric flask and made up to the mark with methanol; this served as a standard solution. After 25 min, the absorbance of the test solutions was measured at 430 nm against the blank solution. The flavonoid content of the spices was calculated in terms of the quercetin content. For this purpose, a calibration curve was prepared using quercetin by preparing a solution of 0.050 mg/ml quercetin, which was diluted to obtain concentrations of 0.001, 0.006, 0.011, 0.016, and 0.021 mg/ml. The results were expressed as the amount of flavonoids (mg/g) of the herbal raw material [31] . Selenium sulphide nanoparticles were synthesised by the precipitation method. The addition of the spice extract in NDES served as a stabiliser of the SeS2 NPs. For this purpose, 0.2 M Na2S•H2O was dropped into the mixture of the 20 ml of the 0.2 M SeCl4 with the 10 ml of the extract. During the addition of Na2S a red selenium sulphide was obtained. The study examined the effect of the molar ratio of sulphide ions to selenium, ranging from 1.6 to 4.0, and the pH of the system, ranging from 8 to 10, which was determined by adding 0.5 M NaOH. The size of the selenium sulphide crystallites and the crystalline composition of the material were determined using X-ray diffraction (XRD) analysis (Philips X'Pert camera with monochromator PW 1752/00 CuKα). The content of the active ingredients was analysed using an UV-VIS spectrophotometer (Rayleigh 1800). The shape and morphology of the particles were studied using a scanning transmission electron microscope (STEM) (FEI NOVA NanoSEM). The characterisation of the selenium sulphide nanoparticles was also performed using FTIR analysis (Nicolet 380). The evaluation of the antimicrobial activity of the tested solutions was performed using the suspension method against indicator microorganisms, including Gram-positive bacteria (Staphylococcus aureus), Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa), and fungi (Candida albicans). The study used test strains from MicroBioLogics, Inc. (St. Cloud, USA). Inocula with a density of 0.5 McFarland and their dilutions Z -1 , Z -2 , and Z -3 were prepared from fresh (24 h) cultures of microorganisms. Then, 1 ml of the suspension from the Z -2 dilution was added to the test tubes that each contained 9 ml of the test preparation. The Z -3 suspension in Ringer's liquid served as a control. From each tube, 0.1 ml of the suspension (TSA for S. aureus and Mc Conkey for E. coli, enriched agar for P. aeruginosa, Sabouraud for C. albicans) was inoculated onto agar plates. The plates were incubated for 24 h at 37 °C, after which time the colonies were counted. On their basis, the logarithmic reduction index was calculated. Measurements were performed in triplicate. After incubation, the number of living organisms was counted and the results expressed in CFU/cm 2 . Three tests were performed for each microorganism. Finally, the decimal logarithmic reduction in the number of microorganisms present in the test dish in control samples (Nc) and after treatment with the nanoparticle suspension (N) was calculated for each strain according to the formula: where R is the reduction in cell count of the microorganisms tested, Ni is the number of microorganisms present in the control sample, and N is the number of microorganisms treated with the selected compounds. The minimum inhibitory concentration (MIC) and the minimum biocidal concentration (MBC) were determined by serial twofold dilutions. Test strains used in the study were S. aureus, E. coli, P. aeruginosa, and C. albicans from MicroBioLogics, Inc. (St. Cloud, USA). The MIC of the preparations was determined on liquid media: Mueller-Hinton broth (Merck, Germany) for bacteria and Sabouraud broth (BTL, Poland) for Candida albicans. 0.1 ml of inoculum (24-hour strains of 0.5 McFarland density) was introduced into prepared tubes with the medium and tested preparation. The control was a tube of Mueller-Hinton broth (10 ml) without any preparation (blank test). Samples were incubated at 37 °C for 24 h (bacteria) and 72 h (yeast fungi). The MIC value was taken as the concentration of the preparation that completely inhibited the growth of microorganisms (no turbidity in the tube). In order to determine the MBC from cultures of bacteria and fungi considered negative, cultures were performed on solid Triptic Soy Agar and Sabouraud medium (BTL, Poland). After 24 h and 72 h (bacteria and fungi) of incubation at 37 °C, a culture determination (macroscopic evaluation) was performed. MBC was defined as the J o u r n a l P r e -p r o o f lowest concentration of the formulation resulting in complete inhibition of bacterial/fungal growth. Testing of antiviral properties was performed using viruses and cell lines: Virus es titre was expressed with reference to the TCID50 (tissue culture infectious dose) value, based on the cytopathic effects (CPE) caused by this virus in approximately 50% of infected cells. 24-hour cultures of MDCK and HCT-8 cell lines were treated with several concentrations of selenium sulphide (0-5%) suspensions and incubated for 72 h in a humidified atmosphere incubator with 5% CO2 at 37 °C. Morphological changes of the cells (cytotoxic effects, CTE) were observed every day under an inverted microscope, and after 72 h of incubation, cell viability was calculated based on CTE (0 -lack of cytotoxic effects, 1 -CTE in 25% of cells, 2 -CTE in 50% of cells, 3 -CTE in 75% of cells, and 4 -100% of the cells affected with CTE). Inhibition of A/H1N1 and HCoV-OC43 replication was examined by the early viral entry assay. Serial concentrations of selenium sulphide were incubated with viruses for 2 h at room temperature. Next, virus-tested compound mixtures were diluted and adsorbed to MDCK cells at a density of 3 x 10 5 cells/ml (A/H1N1) or to HCT-8 cells at a density of 3.2 x 10 5 cells/ml (HCoV-OC43) in 96-well plates. Plates were incubated in 34 o C in 5% CO2. After 72 h (A/H1N1) or 96 h (HCoV-OC43) of incubation, viral cytopathic effects (CPE) were observed and evaluated under an inverted microscope (0 -lack of viral replication (lack of CPE), 1 -viral CPE in 25% of cells, 2 -viral CPE in 50% of cells, 3 -viral CPE in 75% of cells, and 4 -100% of the cells affected with CPE). Viral titre was expressed with reference to the TCID50. Viruses incubated with 50% ethanol served as positive controls; viruses incubated in J o u r n a l P r e -p r o o f an appropriate culture medium served as negative controls. The protocol for the inactivation assay was prepared with reference to Tai et al. [32] . Calculation of the virus inactivation induced by the tested compounds was carried out following the Spearman-Kärber method [33] . Table 2 shows the characteristics of the spice extracts obtained using NDESs of different compositions. The systems extracting active compounds from spices to the highest extent were mixtures of lactic acid, glucose, and betaine and mixtures of citric acid, propanediol, and betaine. The use of NDES as solvents results in a significantly higher content of active compounds compared to systems where water is the solvent. In selecting the NDES for further studies, the system that extracted the spicespecific compounds to the highest degree was selected. In the extraction of cinnamon, the highest cinnamal content was obtained for the solvent NDES3, which indicated a higher extraction of the compound in comparison to other active substances. The compounds with a high content of curcuma are curcumin and coumarin; hence, the solvent NDES1 was selected for further study. Cayenne pepper contains eugenol, among other compounds; hence, the extract obtained in NDES1 was selected for further studies. The compounds in black pepper catalyse the activity of certain compounds in curcumin; hence, an extract combining both spices was obtained. The highest coumarin content was again obtained in NDES1. J o u r n a l P r e -p r o o f In the first step, pure SeS2 nanoparticles were obtained. The effect of the pH of the system and the molar ratio of sulphur to selenium was investigated to obtain pure SeS2 nanoparticles and those with the smallest particles. The size results of the obtained materials are presented in Table 3 . The nanoparticles are characterised by a bimodal distribution with a narrow range (Fig. 1) Table 4 shows the phase composition of the obtained materials. The obtained Se2S6 materials adopt a monoclinic crystalline form with space group P 1 2/c 1 (13) . By increasing the molar ratio of nS/nSe, the formation of an additional SeS7 phase was observed. The values of bond lengths and angles for the different materials obtained under different conditions are summarised in Table 4 . The bond lengths and angles in the samples vary with the pH of the system and with the change in the molar ratio of sulphur to selenium. This is due to the change in the distribution of individual elements and the change of individual atoms in the sulphide structure. The fit of the individual experimental patterns to the Rietveld fit was also determined. The 2 error values range from 1.9 to 4.0. By analysing the effect of pH on the size of the obtained SeS2 nanoparticles, it was found that increasing the pH to a value of 10 and increasing the molar ratio of sulphur to selenium results in particles with smaller crystallites. The overall size of the SeS2 nanoparticles is affected to a greater degree by the nS/nSe ratio. It is necessary to increase the nS/nSe ratio, but note that increasing nS/nSe to a value of 4.0 results in an increase in the proportion of the SeS7 phase (Fig. 2) . Selenium sulphide nanoparticles were examined by FTIR spectroscopy (Fig. 3) . The peaks obtained at 1032 and 1647 cm -1 indicate the presence of CdS nanoparticles. At about 1250 cm -1 , the band indicates the presence of a thiol group at 2560 cm -1 . Selenium sulphide shows an absorption maximum at about 450 cm -1 , which is related to the presence of S-S bonds. The absorption peak at 616 cm -1 may correspond to Na2Se nanoparticles. In the study by Asghari-Paskiabi et al., the band at about 620 cm -1 was attributed to the occurrence of CdSe quantum dots [11] . It has been confirmed in the literature that bands in the range of 240 to 280 cm -1 correspond to stretching Se-Se bonds, and Se-S bonds exhibit an oscillation band in the range of 320-390 cm -1 [34, 35] . Pure selenium sulphide nanoparticles displayed an irregular particle shape. Figure 4 confirms the superstructure of successive layers of the particles. The average particle size of SeS2 was about 500 nm. The EDS analysis confirmed the uniform presence of sulphur and selenium. SeS2 nanoparticles obtained in the presence of spice extracts in NDES show a similar size, and the particles exhibit strongly irregular shapes (Fig. 5 ). The selected selenium sulphide nanoparticles (2-Se2S6), due to their strongly hydrophobic nature, significantly increased their dispersibility in water when added to solution systems of selected NDES-based extracts. The study compared which extracts and compounds contained in the spices had the highest biocidal effect. When comparing the activity of the extracts on the basis of the reduction in the number of cells of the microorganisms tested, the material with the highest activity was the curcuma extract ( Table 5 ). The presence of pepper in the curcuma extract slightly deteriorated the activity, which is due to the reduced content of curcumin itself. The high biocidal activity is due to the presence of curcumin, a compound with high biocidal activity, as confirmed for SeS2 in the curcumin solution. The obtained selenium sulphide nanoparticles stabilised by the curcuma extract and stabilised by the curcuma extract with pepper showed high biocidal activity (Fig. 6) . The compounds in pepper act synergistically with the active substances in turmeric, improving the biocidal activity of the materials. Considering that commercial selenium sulphide dissolves in carbon sulphide, which is a toxic compound, the use of NDES as a dispersing medium for nanoparticles is a safer alternative for future applications. Dispersed selenium sulphide nanoparticles in NDES with extracted compounds from spices have more than twice the effect on microorganisms than selenium sulphide nanoparticles suspended in water. The presence of extracts increases the adhesion of Se2S6 nanoparticles, enhancing the ability of the nanoparticles to interact with microbial cells. Selenium sulphide nanoparticles synthesised in the presence of curcuma extract, curcuma extract with pepper and pepper and selenium sulphide obtained with pure solutions of curcumin and coumarin active compounds in the range of 0.001-0.15% were nontoxic for MDCK and HCT-8 (CTE = 0). Slight cytotoxic effects were observed for every preparation at a concentration of 0.3% (CTE = 1-2). High cytotoxicity for MDCK and HCT-8 (CTE = 3-4) was observed for concentrations ≥ 0.6%. In the next step, selenium sulphides without cytotoxic effects were tested for antiviral activity against human influenza virus A and human betacoronavirus. The early viral entry assay (inactivation assay) was used. The test was performed to examine the ability of the tested compounds to inactivate viruses in a cell-free state (free virus particles). Nontoxic concentrations ≤ 0.15% expressed no antiviral activity. Based on this result, 10x higher concentrations of all compounds were used for the antiviral activity test. Finally, nontoxic concentrations ≤ 0.15% were incubated with MDCK and HCT-8 cells. SeS2, SeS3, SeS4, SeS6, and SeS7 in the range of 0.15-1.5% were incubated with A/H1N1 and HCoV-OC43 for 2 h and next titrated in MDCK and HCT-8. Viral titre was calculated with reference to the TCID50. Experiments were performed three times with two independent repetitions each. The results showed that selenium sulphide nanoparticles synthesised in the presence of curcuma extract, curcuma extract with pepper and pepper and selenium sulphide combined with solutions of the pure active compounds curcumin and coumarin at a concentration of 1.5% had a direct impact on the free A/H1N1 particles by inactivating them and neutralizing their infectivity (Table 1, Fig. 7) . Selenium sulphide nanoparticles declined A/H1N1 titre over 4 log TCID50, which means a 99.99% decrease in the virus infectivity. In addition, selenium sulphide nanoparticles combined with curcuma extract and with curcuma-pepper extract reduced the viral titre ≥ 2 log at a concentration of 0.75%, which means a 99% decrease in the virus infectivity. In the case of HCoV-OC43, antiviral activity was found for Se2S6 NPs with the curcuma extract, curcuma extract with pepper, and NDES solution with J o u r n a l P r e -p r o o f curcumin. These compounds reduced the HCoV-OC43 titre in the range of 2.9 to 3.4 log TCID50, which means a 99.9% decrease in the virus infectivity (Table 7) . Antiviral activity against A/H1N1 (virus that caused the "Spanish flu") also shows effectiveness in relation to other Influenza A subtypes, e.g., human A/H3N2 or bird flu viruses H5N8 and H5N1. Antiviral activity against HCoV-OC43 also shows effectiveness in relation to other β-coronaviruses, e.g., SARS-CoV-2. SeS2 nanoparticles were synthesised by precipitation from aqueous solutions of sodium sulphide and selenium chloride in the presence of spice extracts extracted in NDES based on citric acid propanediol and betaine (Fig. 8) . Taking into account the varying ratio of selenium to sulphur ions and the effect of the mixture pH, it was found that the number of nuclei forming initially was higher in systems with a higher sulphur content compared to selenium. This resulted in lower particle sizes at the nS/nSe content of 2.8 and 4.0. Particles with sizes below the critical diameter were re-dissolved in solution to then crystallize on already existing nanoparticle nuclei, causing them to gradually build up [9] . One of the factors determining the speed of these processes is the diffusion of single SeS2 molecules to the surface of growing nanoparticles [7] . In the presence of extracts, the contact between individual SeS2 nanoparticles is limited, preventing coalescence of particles and their aggregation (steric stabilisation). The presence of organic molecules ensures steric stabilisation of nanoparticles, i.e., by adsorption of polysaccharide molecules, curcumin, polyalcohols, and carboxylic acids on their surface. In this case, a stable hybrid system dispersed in water is formed: a core based on SeS2 NPs with an organic envelope. Selenium sulphide is a proven cytostatic agent with antimitotic, antibacterial, and mild antifungal activity. The action of SeS2 consists, among others, of reducing the rate of incorporation of thymidine, one of the four nucleosides of which the DNA of skin epithelial cells is composed [36] . In this article, selenium sulphide nanoparticles combined with NDES extract of curcumin were confirmed to exhibit high bactericidal, fungicidal, and virucidal activity. The high antifungal and antibacterial activity of the nanoparticles was enhanced by improving the dispersion of SeS2 by using NDES. The results confirmed that selenium sulphide exhibited high bactericidal as well as antifungal activity. In the study by Asghari-Paskiabi et al., selenium sulphide nanoparticles are able to inhibit the growth of, among others, A. flavus, A. fumigatus, A. alternata, T. rubrum, M. canis, and C. krusei [11] . Mavandadnejad [37] . Selenium compounds are thought to protect healthy cells from oxidative damage caused by ROS (reactive oxygen species) and RNS (reactive nitrogen species). At the same time, recent studies have suggested that selenium may have a dual role in oxidative stress, acting as a prooxidant or antioxidant [38] . Th1 lymphocytes, producing mainly interferon y and IL-2, trigger the overproduction of ROS and RNS in host cells as a result of cytokines. Increased oxidative and nitrosative stress acts in two ways. Firstly, it can enhance viral replication, and secondly, it increases the rate of mutation of the viral RNA genome [36] . Both factors additionally lead to a further increase in cytokine production and subsequent tissue destruction [38] . Selenium contained in sulphide is a trace element involved in the regulation of the immune response, oxidative stress, and chronic inflammation. The antiviral effect of selenium is based on interference with the attachment of the virus to the host by inhibiting the disulphide exchange reaction. Disulphide exchange is a key step that enables the transition of inactive forms of compounds to active forms that promote ligand binding to the cell membrane surface. Disulphide exchange reactions reduce protein disulphides in the viral glycoprotein. This results in the cleavage of hydrophobic epitopes, i.e., antigen fragments that bind directly to the free antibody, which can perforate the host membrane and facilitate viral entry into cells [36] . SARS-CoV-2 is an enveloped virus whose genome is single-stranded RNA with a positive polarity. Each SARS-CoV-2 virion is spherical in shape with a diameter of approximately 60-140 nm. Like other coronaviruses, SARS-CoV-2 has four structural proteins: protein S (spike) -fusion protein or surface glycoproteinresponsible for interaction with the receptor on the cell surface, protein E (envelope)coat protein -responsible, among other things, for virion formation, protein M (membrane) and N protein (nucleocapsid) -protein, which protects a large RNA molecule and participates in the modification of cellular processes and virus replication [39] [40] [41] . The N protein maintains the RNA genome and the S, E, and M proteins together form the virus envelope. The S protein is responsible for binding to the membrane of the host cell. Glycoprotein S, consisting of subunits S1 and S2, plays a key role in the process of virus entry into host cells. Glycoprotein S1 is responsible for the first stage of viral entry into the cell, and glycoprotein S2 can be used during the second phase of fusion of the viral envelope and host cell. The known mechanisms of antiviral action include inhibition of viral attachment to the infected cell membrane, blocking viral entry (viral adsorption), direct destruction or deformation of viral surface proteins (virucidal action), and inhibition of viral replication. Antiviral drugs usually act by inhibiting viral replication, allowing the immune system to eliminate the pathogen. The compounds in curcuma, mainly curcumin, have a number of bioactive properties, including antioxidant, antiseptic, anti-inflammatory, and anticarcinogenic effects against HIV, norovirus, Herpes Simplex virus (HSV), influenza A viruses, SARS-CoV-2 viruses, and hepatitis viruses, among others. Studies have shown that curcumin and its analogues significantly inhibit the production and release of proinflammatory cytokines in vitro and in vivo [42] . Curcumin has been shown to have a destructive effect on the steps of the virus replication cycle and virus attachment and penetration. In the work of Tomo et al., an aqueous extract of curcumin with piperine had a destructive effect on the SARS-CoV-2 virus. The authors suggested that the viral effect was due to the strong attraction of the extract to the virus spikes present on the surface of SARS-CoV-2 [38] . In studies of Sagar & Kumar, natural compounds from Tinospora cordifolia showed a high binding efficiency against the SARS-CoV-2 virus. The active compounds in the plant, mainly berberine and isocolumbine, were involved in virus attachment and replication. These compounds had a strong binding affinity for the SARS-CoV-2 surface glycoprotein (6VSB) as well as for the RNA polymerase (6M71) [43] . Mahmoud et al. obtained an ethanolic extract of Cuphea ignea leaves. The content of compounds from the phenolic group was p-coumaric acid -5.43 mg/g, quercetin -0.28 mg/g, and cinnamic acid -0.07 mg/g, among others. In silico studies demonstrated the effective ability of gallic acid, quercetin, resveratrol, and naringenin to inhibit SARS-CoV-2 internalisation by binding to its cellular receptor, angiotensin-converting enzyme 2 (ACE 2) [44] . The study presents the antimicrobial and antiviral properties of selenium sulphide nanoparticles stabilised by solutions of spice extracts in NDES. In contrast to commercial selenium sulphide, which is soluble only in CS2, the use of NDES allowed for the formation of a homogeneous suspension. The selenium sulphide nanoparticles obtained were dispersed in a solution of NDES based on citric acidpropanediol-betaine and lactic acid-glucose-betaine, the use of which resulted in the extraction of the highest content of active substances from the spices curcumin, cinnamon, and pepper. On the basis of particle size analysis, i.e., the influence of the pH of the environment and the molar ratio of selenium to sulphur, nanoparticles with the smallest size were obtained. The study showed that at pH 8 and nSe/nS equalled 2.8, nanoparticles with the smallest crystallite size of 28.72 nm and particle size of 830 nm were achieved. High antimicrobial and antiviral activity was confirmed for selenium sulphide nanoparticles stabilised by cinnamon extract, curcumin, and a mixture of curcumin and black pepper. The cinnamon extract showed a cinnamal content of 78-161 mg/g, the curcumin extract contained 14-74 mg/g of curcuma, while the curcumin and J o u r n a l P r e -p r o o f cayenne pepper extract contained 6-91 mg/g of curcumin. Moreover, the extracts showed a high polyphenol and coumarin content. The activity of SeS2 nanoparticles stabilized with spice extracts was 117.2 mg/dm 3 against bacteria and 117.2 to 468.8 mg/dm 3 against fungi. SeS2 nanoparticles with the addition of pure components found in spices, i.e., cinnamal, curcumin, and coumarin, also showed high bioactivity, confirming that these compounds are associated with high material activity. Virus titre reduction logTCID50 against the A/H1N1 virus was 5.5, while virus titre reduction logTCID50 against the HCoV-OC43 virus was 3.2 for selenium sulphide from turmeric extract. 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Efficacy of natural compounds from Tinospora cordifolia against SARS-CoV-2 protease, surface glycoprotein and RNA polymerase Delineating a potent antiviral activity of Cuphea ignea extract loaded nano-formulation against SARS-CoV-2: In silico and in vitro studies The authors report no declarations of interest. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sector.