2.1 Reagents and Chemicals

Anthrone reagent, Sulfuric acid, Methanol, Congo red, n-Butyl alcohol, Haematoxylin (Mayer’ reagent), Acetic anhydride, Chloroform, Potassium ferricyanide, Hydrochloric acid, DPPH (2,2-diphenyl-1-picrylhydrazyl) (HiMedia), (MTT) (Invitrogen), H2DCFDA (Invitrogen), MitoTracker and LysoTracker (Invitrogen), FBS and Antimycotic antibiotic solution (Gibco).

2.2 Cell line Procurement and Maintenance

HeLa cells were obtained from NCCS, Pune, and maintained in DMEM supplemented with 10 % FBS and 1 % Antibiotic-Antimycotic. Cells were kept at 37 °C in a 5 % CO₂ atmosphere in STERI-CYCLE i60 CO₂ Incubator, Thermo Fisher Scientific).

2.3 Extraction of P. Ostreatus derived β-Glucan particles

Extraction of β-Glucan particles from P. ostreatus was done using previously described methods (Rahar, 2011; Guizani, 2010). Fresh P. ostreatus was collected and washed. 100 g fruiting bodies were boiled at 100 °C for 30 min in 200 ml of distilled water. After boiling, the sample was centrifuged (9000 rpm) for 10 min, the supernatant was removed, and pellet was washed in 80 % ethanol. It was re-suspended in distilled water for 4 h heating at 90 °C centrifuged at 9,000 rpm for 10 min. The pellet was re-suspended in 50 ml Triple Distilled Water (TWD), with a pH between 4–5. The sample was further kept at 70 °C for 30 min and centrifuged. The supernatant was washed twice with Isopropyl alcohol and then with 5 ml acetone (twice) to evaporate the previous solvent and centrifuged. The obtained sample was lyophilized to collect β-Glucan particles.

2.4 Quantitative estimation of polysaccharide

Quantitative estimation of carbohydrate content in extracted β-Glucan particles was performed through the anthrone test. Briefly, Anthrone Reagent (0.2 gm) was dissolved in 100 ml of concentrated H₂SO₄ followed by the addition of anthrone reagent to each tube in the concentrations of 200–1000 μg/ml along with 1 unknown concentration of the β-Glucan particles followed by incubated for 30 min at 90 °C and absorbance was measured at 630 nm (Richards, 2020).

2.5 Size analysis

The size of the prepared particles was assessed by Malvern Zetasizer using the technique of Dynamic Light Scattering (DLS). Briefly, the suspension of particles was diluted in distilled water and further Dispersion Technology software was used to analyze the data and create histograms for particle size vs percent intensity.

2.6 Fourier-transform infrared (FTIR) analysis

FTIR was performed as per the previously published protocol (Zhong, 2019). In brief, 1 mg sample was loaded on the crystal of FTIR Bruker Alpha spectrometer, and % transmittance was measured from 4000-400 cm⁻¹.

2.7 Surface morphology assessment by scanning electron microscope

The surface morphological characteristics of the microstructural particles was analyzed using SEM in high vacuum conditions, utilizing an electron detector at an accelerated voltage of 15 kV along with high magnifications, no prior coats on the surface.

2.8 Atomic force microscopy (AFM) analysis

AFM analysis was carried out to get images of the prepared particles with enhanced spatiotemporal resolution, which were dispersed in distilled water at a concentration of 1 mg/mL. Following continuous stirring and incubation at 50 °C for 2  h, the solutions were adjusted to 10  μg/mL and subsequently deposited onto a freshly cleaved mica substrate for air drying. An atomic force microscope was utilized to get AFM images of the accessible samples (Yuan, 2022).

2.9 Heavy metal analysis

Heavy metal detection was performed in an Atomic Absorption Spectrophotometer. (AA 7000F Japan) for the determination of the heavy metals. The previously described protocol (Mkhize, 2021) was followed for the heavy metal determination. Briefly, the powdered sample was kept in a muffle furnace at 250 °C for 12 h. After 12 h, the sample was digested with aqua-regia solution, and the solution was mixed with distilled water and analyzed with the help of an atomic absorption spectrophotometer.

2.10 Antioxidant activity

DPPH assay was used to assess the antioxidant potential as per the previously described procedure with slight modifications (Lam and Okello, 2015). The procedure followed is described here. 0.2 mM DPPH was dissolved in methanol and added to different concentrations of β-Glucan particles (200, 400, 600, 800, 1000 µg/ml). Further, the mixture was incubated in the dark, and absorbance (in triplicate) was measured at 517 nm by using the synergy H1 multimode Elisa Plate Reader (BioTeK). Methanol was used as a control solution, and we have calculated the % radical scavenging activity using the below formula: $$\%\mathrm{R}\mathrm{S}\mathrm{A}=\frac{\mathrm{A}\mathrm{b}\mathrm{s}\mathrm{o}\mathrm{r}\mathrm{b}\mathrm{a}\mathrm{n}\mathrm{c}\mathrm{e}\mathrm{o}\mathrm{f}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}-\mathrm{A}\mathrm{b}\mathrm{s}\mathrm{o}\mathrm{r}\mathrm{b}\mathrm{a}\mathrm{n}\mathrm{c}\mathrm{e}\mathrm{o}\mathrm{f}\mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}}{\mathrm{A}\mathrm{b}\mathrm{s}\mathrm{o}\mathrm{r}\mathrm{b}\mathrm{a}\mathrm{n}\mathrm{c}\mathrm{e}\mathrm{o}\mathrm{f}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}}\times 100$$

2.11 Determination of antibacterial activity

We have used the agar well diffusion method according to the previously described protocol to determine the antibacterial activity (Sriramulu et al., 2020). The following bacterial strains were used for the test: Escherichia coli (E. coli) (MTCC No. 443), Pseudomonas aeruginosa (P. aeruginosa) (MTCC No. 1688), Staphylococcus aureus (S. aureus) (MTCC No. 96), and Streptococcus pyogenes (S. pyrogens) (MTCC No. 442). Sensitivity of the tested organisms against the β-Glucan particles derived from P. ostreatus were maintained. Cultures were inoculated on Muller Hinton agar plates using 100 μl of each strain. The organisms were distributed all over the surface of the medium using a sterile L-shaped spreader and allowed to dry for 5 min. The β-Glucan was taken in various concentrations (25 µg/ml-500 µg/ml) and added in each well on the medium received exactly 500 μl of each concentration and was left or about half an hour for appropriate diffusion for 24 h, and the antibiotic zone scale was used to assess the zone of inhibition in millimeters (mm) (Sriramulu et al., 2020).

2.12 Antifungal activity

The following fungus strains were used: Candida albicans (C. albicans) (MTCC No. 227), Aspergillus niger (A. niger) (MTCC No. 282), and Aspergillus clavatus (A. clavatus) (MTCC No. 1323). Nystatin and Griseofulvin were used as standard drugs in our antifungal activity related experiments.

The basic well diffusion method was employed to screen for antifungal activity according to the previously described protocol. 100 μl of cultures were inoculated on PDA plates. Using a sterile L-shaped spreader, the organism was distributed around the medium's surface and left to dry for roughly 5 min. The β-Glucan particles in various concentrations (25 µg/ml-500 µg/ml was added to the wells created using cork borer. Each well on the medium received exactly 50 μl of each concentration and the antifungal activity of β-Glucan particles was observed after 24–48 h at 28 °C. Zones of inhibition of fungal growth were measured to assess the fungal growth. The antibiotic zone scale in mm was used to measure the inhibition zones (Baskaran et al., 2012).

2.13 Cell viability assessment

The cell viability of HeLa cells against β-Glucan particles was examined by MTT assay. Assay was performed following the previously described protocol (Sriramulu et al., 2020). HeLa cells were seeded in 96 well plates and allowed for attachment, cells were then treated with different concentrations of sample particles (10–50 µg/ml) and incubated for 24 h. Post-treatment incubation, 10 µl of MTT dye was added for 4 h. Formazan crystals were dissolved using DMSO, and absorbance was measured at 490 nm. % viability of the treated and control cells was calculated using the given equation: $$\%\mathrm{V}\mathrm{i}\mathrm{a}\mathrm{b}\mathrm{i}\mathrm{l}\mathrm{i}\mathrm{t}\mathrm{y}=\left(\frac{\mathrm{A}\mathrm{b}\mathrm{s}\mathrm{o}\mathrm{r}\mathrm{b}\mathrm{a}\mathrm{n}\mathrm{c}\mathrm{e}\mathrm{o}\mathrm{f}\mathrm{t}\mathrm{r}\mathrm{e}\mathrm{a}\mathrm{t}\mathrm{m}\mathrm{e}\mathrm{n}\mathrm{t}}{\mathrm{A}\mathrm{b}\mathrm{s}\mathrm{o}\mathrm{r}\mathrm{b}\mathrm{a}\mathrm{n}\mathrm{c}\mathrm{e}\mathrm{o}\mathrm{f}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}}\right)\times 100$$

2.14 Hoechst staining

HeLa cells were treated with P. ostreatus derived β-Glucan particles at 10–50 µg/ml concentrations for 24 h and stained with 10 µl of Hoechst 33,342 for 15 min in the dark. Cells were imaged and morphological changes were observed using a fluorescence microscope (Owa et al., 2013).

2.15 Determination of intracellular ROS generation

The DCFH-DA dye was used to assess the level of intracellular ROS. It is a light reactive dye, 10 µL (working solution) was added to treated and control wells and incubated for 15 min in the dark inside the CO₂ incubator. It was rinsed with phosphate buffer saline (PBS) after incubation, and the results were visualized under fluorescence microscopy and photographed (Sankar, 2014).

2.16 Propidium iodide (PI) staining

The procedure followed was already discussed in the previous paper and is as follows: 1x10⁵ cells per well were seeded in 12 well plates treated with different concentrations (10–50 µg/ml) of sample particles after attachment and incubated for 24 h in a CO₂ incubator. After incubation, media was removed and 10 μl of PI dye was added in each well for staining and incubated at 37 °C in the dark for 20 min. Each well was washed with PBS, and the result was examined under fluorescence microscopy under a red-light filter (Brana et al., 2002).

2.17 LysoTracker red DND 99 staining

Cells were seeded in 12 well plates treated with the sample and incubated for 24 h. A 1 mM stock solution of LysoTracker Red DND-26 was diluted to a 1 µM working solution using warm incomplete DMEM, and the cells were stained with this solution. The cells were incubated for 30 min in the dark followed by PBS washing and images were captured using a fluorescence microscope (Owa et al., 2013).

2.18 MitoTracker red CMX ROS staining

MitoTracker red staining for the assessment of mitochondria was performed using a previous protocol (Kathayat and Dickinson, 2019). 10⁵ cells in 12 well plate was seeded and treated with various dosages of sample and again left for 24 h. MitoTracker red CMS ROS was used to stain the treated cells. A stock solution (1 mM) was made by adding DMSO (94 μl) to a vial (50 μg) of MitoTracker Red at room temperature. The working solution (10 μl) was made up by adding 1 μl of stock into complete DMEM media (9.99 mL) and stored in the dark at −20 °C. After staining with MitoTracker for 20 min the dye was removed and 10 μl Hoechst dye was added for 15 min at 37 °C in the dark and cells were visualized under a fluorescence microscope.

2.19 Measuring Caspase 3 and Caspase 9 activities using a flow cytometry

We utilized an established protocol to measure the activities of Caspase 3 and 9 (Ullah, 2020).

2.20 Statistical analysis

Statistical analysis was done using One Way ANOVA and p values less than 0.05 were considered significant.

3.1 Extraction of mushroom derived β-Glucan particles

After extraction, slightly brown-colored P. ostreatus mushroom derived β-Glucan particles were found in dried powder form after lyophilization as shown in Fig. 1-A.

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Fig. 1

A. Pleurotus ostreatus mushroom derived β-Glucan particles as dried powders. B. Figure depicts that mean absorbance was following a trend to increase in absorbance with concentration and maximum absorbance was found at 1000 μg/ml. Concentration of unknown sample was found to be 790 μg/ml.

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3.2 Quantitative estimation by anthrone test confirmed the presence of polysaccharide

The total carbohydrate/polysaccharide content of P. ostreatus mushroom derived β-Glucan particles (unknown) sample was determined using the anthrone method (Li, 2020). Fig. 1 B shows an Absorbance vs. Concentration graph, and total polysaccharide content was found to be 790 µg/ml.

3.3 Size analysis confirmed that particles are suitable for phagocytosis

The hydrodynamic size of particles was found to be in the phagocytosable range as shown in Fig. 2-A (Korotchenko, 2021).

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Fig. 2

Zeta potential (A) and particle size (B) of Pleurotus ostreatus mushroom derived β-Glucan particles.

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3.4 FTIR analysis showed the branching pattern of β-Glucan particles

IR spectra of the β-Glucan particles of P. ostreatus mushroom are shown in Fig. 3A. The spectra revealed the transmission peaks at 3300–3400, 2900, and 1650 cm⁻¹ for the OH, CH, And C = O respectively. The peaks in the 950–1200 cm⁻¹ region are indicative peaks of CH and CO stretching confirming that a major proportion of the extracted β-Glucan is polysaccharide-based. Our IR spectra were almost similar to the already reported spectra for P. ostreatus derive β-Glucan confirming the nature of our compound.

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Fig. 3

(A) Transmittance spectra of Pleurotus ostreatus mushroom derived particles confirming that the obtained spectra are of β-Glucan particles. (B) SEM image of the particles at the 100 nm resolution showed that particles having spherical morphology and porous in nature. (C) AFM imaging 2D and 3D of the prepared Pleurotus ostreatus mushroom derived β-Glucan particles as shown in figure A and B on different scale.

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3.5 Scanning electron microscopic analysis

SEM analysis of the prepared P. ostreatus mushroom-derived particles was performed to determine the surface morphology of the particles. The SEM image in Fig. 3B clearly showed the porous and smooth surface of the particles.

3.6 The atomic force microscope (AFM) analysis

AFM helped in the 3D characterization of particles and offered several advantages for particle characterization when compared to other characterization methods. Size measurement of prepared P. ostreatus mushroom-derived Glucan particles using atomic force microscopy as data shown in Fig. 3C.

3.7 Heavy metal analysis

An atomic absorption spectrophotometer was used to determine heavy metals and the analysis content is shown in Table S1. AA 7000F Japan model used for determination of the heavy metal in our mushroom derived β-Glucan particles.

3.8 P. Ostreatus mushroom derived β-Glucan particles showed efficient antioxidant activity

The DPPH radical scavenging used to asses antioxidant potential was associated with their ability to donate hydrogen. DPPH is a free radical that can be transformed into a stable molecule with the addition of an electron or hydrogen radical. Fig. 4 illustrates that the scavenging of DPPH radicals elevated with higher doses of β-Glucan particles derived from P. ostreatus (200–1000 µg/mL). The most significant increase in DPPH radicals (greater than 90 %) was recorded at a β-glucan concentration of 1000 µg/mL.

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Fig. 4

% Radical scavenging Activity of Pleurotus ostreatus mushroom derived β-Glucan particles was found to increase with concentration. The data are expressed as mean ± SEM from three independent experiments. All the values were significant with p < 0.0001.

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3.9 Antimicrobial activity of β-Glucan particles

P. ostreatus mushroom derived β-Glucan particles were tested for antimicrobial activities against selected bacterial strains. A clear zone of inhibition was shown against selected strains. Gentamycin, Ampicillin, Chloramphenicol, Ciprofloxacin, and Norfloxacin were used as standard drugs & Minimum Inhibitory Concentration (MIC) is shown in Table S2. Minimum Inhibitory Concentrations of P. ostreatus mushroom derived β-Glucan particles against selected bacterial strains have been shown in Table S2 and with their zone of inhibitions in Table S3.

P. ostreatus mushroom derived β-Glucan particles were tested for antifungal activities against selected fungal strains. C. albicans, A. niger, A. clavatus. Nystatin and Griseofulvin were used as standard drugs & with their MIC values in Table S4.

3.10 Effect of β-Glucan on cell viability

MTT assay has been versatile assay for the detection of cell viability or proliferation in in vitro conditions. Mitochondrial succinate dehydrogenase enzyme converts tetrazolium salt of MTT into formazan crystals by the process of cellular reduction and these crystals are dissolved with the help of DMSO for the measurement of absorbance to assess the cell viability (Xu, 2016). Cytotoxic effect β-Glucan particles derived from P. ostreatus was tested against HeLa cell line. The lowest cell death was observed at the minimum concentration of (10 μg/ml) while cell viability decreased to 37 % at the highest concentrations (50 μg/ml). Particles exhibited efficient toxicity against HeLa cells at 50 μg/ml. In many studies, it was found that Glucan polysaccharide halts the growth of human cancer cells and leads to cell apoptosis along with morphological changes (Gharibzahedi, 2022). Reported IC₅₀ was found to be 41.5 µg/ml as shown in Fig. 5A.

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Fig. 5

(A) Graphical representation of cell viability against cervical cancer cells at various concentrations of β-Glucan particles. The data are expressed as mean ± SEM from three separate experiments. All the values were significant with p < 0.0001. (B) Images of HeLa cells stained with Hoechst 33,342 dye. Morphological changes are indicated by arrows. (C) In untreated or control cells, nuclei are stained evenly and appear round in shape. In treated cells, the nuclei are not properly stained and are generally fragmented.

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3.11 Effect of β-Glucan on cellular morphology

P. ostreatus-derived β-Glucan particles showed significant visible changes in morphology and fragmentation of DNA in the HeLa cells. As the concentration of dosing increased, it showed a change in the morphology of the HeLa cell nuclei indicating apoptosis induction as shown in Fig. 5B. A broad view of morphological changes for easy visualization is shown in Fig. 5C.

3.12 Effect of β-Glucan on ROS generation

In our study, treatment of HeLa cells in concentrations of 10–50 µg/ml of P. ostreatus-derived β-Glucan particles showed the generation of ROS generation increased with concentration as shown in Fig. 6 A. A significant ROS generation in HeLa cells indicated that P. ostreatus-derived β-Glucan particles can suppress the growth of HeLa cells. One Way ANOVA analysis (shown in Fig. 6C) showed highly significant values with p < 0.0001.

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Fig. 6

(A) Intracellular ROS generation of HeLa cell lines against various concentrations of β-Glucan particles. In untreated (control) cells, there is no or little ROS generation while it significantly increased in treated cells. Maximum ROS generation was observed at 50 µg/ml concentration of β-Glucan particles. (B) In control cells, there is very low fluorescence indicating that most of the cells were live and dye was not able to permeate them. A significant increase in fluorescence upon the increase in concentration indicated that mortality of HeLa cells increased after treatment. (C) Statistical analysis of ROS showing % DCFDA fluorescence increasing with the concentration of β-Glucan with p < 0.001. (D) Propidium Iodide staining showed an increase in apoptotic cells upon treatment β-Glucan particles. All the values presented here are measured with the help of ImageJ software and analyzed with the help of a GraphPad prism. The data are expressed as mean ± SEM from three separate experiments. All the values were significant with p < 0.0001.

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3.13 Apoptosis detection by PI staining

PI staining showed that mushroom-derived β-Glucan particles affect HeLa cells and lead them to apoptosis at various concentrations (10–50 µg/ml). There is a successively increase in apoptotic cell as compared to the control which can be seen in red fluorescence as shown in Fig. 6-B. One Way ANOVA analysis showed p < 0.0001 indicating all the values are significant as shown in Fig. 6D.

3.14 Effect of β-Glucan on MMP

Mitochondrial membrane depolarization potential was analyzed in both untreated and β-Glucan particles-treated HeLa cells with MitoTracker Red and LysoTracker stain. The LysoTracker staining results showed that mushroom-derived β-Glucan particles affect HeLa cells and lead to a decrease in acidic organelles in a dose-dependent manner (10, 20, 30, 40, and 50 µg/ml) (Fig. 7A and C).

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Fig. 7

HeLa cells staining with LysoTracker and MitoTracker. (A) LysoTracker accumulation in acidic organelles resulted in intense red color fluorescence in untreated/control cells. Membrane disintegration occurred upon treatment with β-Glucan particles resulted in diminished fluorescence in treated cells. (B) Staining of HeLa cells with MitoTracker Red CMX ROS showed that β- Glucan particles increasing the mortality of HeLa cells with the increase in concentration. Dye accumulated well in the untreated cells and as treatment was given, mitochondrial membrane potential started to change into positive and resulted in less accumulation of dye in mitochondria resulting in reduced fluorescence indicating that cells are dying due to the treatment with β-Glucan particles. (C) Staining of HeLa cells with LysoTracker Red DND 99 showed dose-dependent accumulation of dye in cells and values were significant with p < 0.001. (D) MitoTracker Red CMX ROS analysis showed a decreased fluorescence upon the increase in the concentration of treatment.

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Furthermore, the MitoTracker staining also showed that mushroom-derived β-Glucan particles act in a concentration-dependent manner with a decrease of red fluorescence with the increase in concentration (10–50 µg/ml) as shown in Fig. 7-B. All the values were found significant with p < 0.0001 in statistical analysis as shown in Fig. 7D.

3.15 Effect of β-Glucan on Caspase 3 and Caspase 9 activities

The caspase family of cysteine proteases cleaves proteins at their aspartic acid residues, causing them to undergo apoptosis. To elucidate the process of apoptosis induction, we examined the role of caspases in HeLa cells by flow cytometry. The results demonstrated that β-glucan exposure greatly enhanced the activities of Caspase-3 and −9 after 24 h, as evidenced by the fluorescence intensity. The highest Caspase 3/9 activity was observed at 200 μM compared to the control as shown in Fig. 8.

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Fig. 8

Upon exposure to different doses of β-Glucan particles (100–200 μM) to HeLa cells Caspase 3 and Caspase 9 activities were found to be increased.

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