The 119 dried samples of O. sinensis natural fruiting bodies were collected from various regions in China, including Yunnan and Gansu (20 samples), Qinghai (20 samples), Sichuan (39 samples), and Tibet (40 samples). Each sample of O. sinensis fruiting body was carefully washed with sterile water and then soaked in a 1% sodium hypochlorite solution (Merck, Germany) for one min. Next, the sample was cut into small pieces, measuring 2 - 5 mm in length, and then inoculated onto SDA agar. The inoculated samples were then cultured aseptically at 16°C for 7 days. Finally, the obtained colonies were observed, and only white colonies were selected. Fungal colonies were subcultured and repeated several times until completely bacteria-free. Stock cultures of pure fungal strains were maintained at a temperature of -20°C, using 10% glycerol (v/v) as a preservative.

The fungal morphology was macroscopically studied by observing the color, shape, size, and hyphae colony features. For microscopic examination, the slide culture technique and staining with lactophenol cotton blue along with fungal culture on SDA for 5 - 7 days were performed (15).

The fungal isolates’ strain-specificity was confirmed using the method employed by Cao et al. (16). The method involved extracting total genomic DNA from the mycelium and isolating the nuclear ribosomal DNA, including ITS1-5.8S-ITS2, using DreamTaq DNA polymerase and primers ITS4 and ITS5. The PCR amplification was done using a thermal cycler, and the resulting products were analyzed via agarose gel electrophoresis, purified, and sequenced for comparison against the GenBank data set.

A new fungus of O. sinensis OS8 was applied in this investigation. The fungal culture was preserved on PDA slants at a temperature of 4°C, after which it was regularly sub-cultured every four weeks. The submerged culture was used for the biomass production of O. sinensis OS8 using a 24-oz culture bottle. Submerged culture media of 30 ml/bottle was used. The media composition (g/l) by Leung et al. (9) included 200 g of potato, 40 g of glucose (Merck, Germany), 10 g of yeast extract (Becton, USA), 5 g of peptone (Becton, USA), 1 g of KH₂PO₄ (Merck, Germany), and 0.5 g of MgSO₄ (Ajax, Australia). The stock mycelial inoculum of 1x1 cm size in PDA was used and placed in the center of the culture bottle. The culture was maintained in darkness at 16°C for 14 days. Afterward, the mycelial biomass was centrifuged at 3,000 g for 15 min, washed with sterile water, filtered, and then dried at 60°C for 24 h in a hot air oven.

The extraction methods described by Huang et al. (17) were utilized to extract adenosine from O. sinensis OS8 mycelia, as follows: the mycelia of O. sinensis OS8, which had been dried, were pulverized into a powder using liquid nitrogen. Approximately 1.0 g of the powder was combined with 10 ml of a 50/50 (v/v) mixture of ethanol and water in a 50 ml centrifuge tube. The samples were subsequently placed in an ultrasonic machine to extract adenosine at a power of 75 W at 50°C for 60 min. The sample extraction procedures were repeated twice. Prior to HPLC analysis, the samples’ supernatants were filtered using a 0.45 µm filter. The investigations were performed in triplicate.

HPLC (Agilent, HP 1100 series, USA) analysis was conducted with an auto-injector and a reverse phase analytical column (Hypersil ODS C18 4 × 250 mm, 5 µm, UK). The standard of adenosine (Sigma, USA) was injected to generate a calibration curve. The injection volumes were 12.5, 25, 50, 100, and 200 µg/ml. The mobile phase was a mixture of 0.05 M potassium dihydrogen phosphate, water/methanol (85:15, v/v), and water/acetonitrile (95:5, v/v). The conditions for HPLC analysis included an injection volume of 5 µl, and the analysis was performed with a flow rate of 0.5 ml/min, a detection wavelength of 242 nm, and the column temperature maintained at 20°C.

The extraction methods of Wang et al. (14) were used to extract O. sinensis OS8 polysaccharide (OS8P). To prepare the extract, 10 g of mycelium powder was dissolved in 100 ml of distilled water for 2 h, followed by ultrasonic extraction at 50°C for 15 min. The sample was mixed with 300 ml of 95% ethanol, stirred, and settled at 4°C for 18 h. It was then separated via centrifugation at 3,000 g for 15 min and the resulting OS8P precipitate was rapidly pre-frozen at -35°C for 1 h before undergoing vacuum freeze-drying. Finally, the obtained OS8P was further investigated for anticancer, antioxidant, and immunomodulatory activities.

The bioactive glucan compounds were quantified using a mixed-linkage β-D-glucan assay kit obtained from Megazyme International (Ireland), as per the method described by McCleary and Draga (18).

McCleary and Draga’s method (18) was used to determine the total D-glucan and D-glucose in free D-glucose and sucrose. The process involved weighing 10 mg of OS8P sample powder and adding it to each tube containing 2.0 ml of ice-cold 12 M sulfuric acid. The tubes were then placed in an ice-water bath for 2 h before adding distilled water, boiling in a water bath, and incubating for 2 h. The contents were transferred to a 100 ml volumetric flask, mixed, and centrifuged. Then, 0.1 ml of sample solution was mixed with exo-1,3-β-glucanase and β-glucosidase and incubated at 40°C for 60 min. GOPOD reagent was added, and the absorbance was measured at 510 nm.

To begin the process (18), 10 mg of OS8P powdered sample was mixed with 2 ml of 1.7 M NaOH in a tube and stirred in an ice-water bath for 20 min. Then, 8 ml of 1.2 M sodium acetate buffer (pH 3.8) was added to each tube with stirring, followed immediately by the addition of 0.2 ml of amyloglucosidase (1,630 U/ml) and invertase (500 U/ml). The tubes were then incubated in a 40°C water bath for 30 min. Samples containing less than 10% α-glucan content were centrifuged at 3,000 g for 5 min, and 0.1 ml of the resulting solution was mixed with 0.1 ml of sodium acetate buffer (200 mM, pH 4.5) and 3.0 ml of GOPOD reagent. The tube contents were incubated for 20 min at 40°C before measuring the absorbance at 510 nm against the blank reagent. The calculation for glucan is as follows:

where: ΔA = reaction absorbance – blank absorbance.

F = a factor to convert absorbance to μg of D-glucose. = 100 (μg of the D-glucose standard) GOPOD absorbance for 100 μg of D-glucose standard.

W = weight of sample.

The chemical components of OS8P were analyzed using a gas chromatograph with mass spectrometry (GC-MS), following the modified method of Chit-aree et al. (19). Sample preparation involved mixing 0.1 mg of OS8P with 1 ml of anhydrous ether (Merck, Germany). GC-MS analysis was conducted using an Agilent HP 5890 gas chromatography instrument coupled with an Agilent MSD HP 5970 mass selective detector and HP 59970 MS Chemstation with a 59973 NBS mass spectral library (NBS_REVF). A fused silica column of 25 m × 0.20 mm × 0.25 µm film thickness (Varian Inc., Agilent Technologies, USA) was used for separation, and each extracted sample of 1 µl was injected in an injector operation. High-purity helium gas (99.999%) was used as a carrier gas at a 1.0 ml/min flow rate. The ion source and quadrupole mass analyzer were kept at 230 and 150°C, respectively, and the analysis was conducted for 60 min per sample. The MSD Chemstation software (Agilent Technologies) and commercial libraries (NIST02.L, NIST05.L, and Wiley7Nist05.L) were used to identify the chemical compounds of OS8P.

HT-29 human colon cancer cells (ATCC) were cultured in a 25 cm³ flask (Nunc, Denmark) with Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, USA) containing 10% fetal bovine serum (FBS) (Gibco, USA), 1% non-essential amino acid (Gibco, USA), and 1% penicillin-streptomycin (Gibco, USA). The cells were incubated at 37°C under a 5% CO₂ atmosphere in a CO₂ incubator (Forma Scientific, 3111, USA) and subcultured after trypsinization once they reached confluence. These colon cancer cells were used for subsequent investigations of antiproliferation and apoptosis.

The MTT assay (20, 21) was utilized to evaluate cell survival. Cells (100 µl at 10⁶ cells/ml density in DMEM medium) were seeded in a 96-well plate (Nunc, Denmark), incubated for 24 h, washed, and exposed to different concentrations of OS8P (0, 25, 50, 100, 200, and 400 µg/ml) for 48 h. Following incubation, the cells were washed twice with PBS, and MTT (Sigma, USA) solution (0.5 mg MTT/ml in DMSO) was added to each well. The plate was then incubated in the dark at 37°C for 4 h, and absorbance was measured at 595 nm using a microplate reader after solubilizing the formazan precipitates with DMSO (Sigma, USA). This experiment was repeated three times with eight replicates per observation, and the inhibition rate of cell proliferation was calculated.

DNA fragmentation in HT-29 cells treated with OS8P at 200 µg/ml was assessed using a modified method based on Wasunan et al. (24). Cells were seeded in 6-well plates, treated with either 1 nM Paclitaxel or 200 µg/ml of OS8P, and incubated for 48 h. Genomic DNA was extracted, and electrophoresis was performed on a 1.5% agarose gel for 45 minutes at 80 V.

HT-29 colon cancer cells were cultured in DMEM supplemented with FBS and antibiotics, and were incubated under standard conditions. After attaching to 24-well plates, the cells were treated with OS8P at its IC₅₀ concentration for 24 h. The fixed cells were washed and dehydrated with a series of ethanol solutions, then coated with gold for scanning electron microscopy (SEM; HV SEM; TESCAN, Vega III, Czech Republic) imaging, following previous protocols (25).

The scavenging activity of OS8P against DPPH radical was determined using a modified version of the procedure by Wang et al. (26). Different concentrations (0.1-0.7 mg/ml) of OS8P solution were mixed with DPPH-methanol solution and incubated for 30 minutes. Absorbance was measured at 517 nm using a microplate reader, and positive controls of α-tocopherol and Trolox were used as reference standards. The scavenging percentage was calculated using the equation:

where the absorbance of the DPPH reagent in the occurrence of the solutions is represented as Aa, the absorbance of the solutions is represented as Ab, and the absorbance of the DPPH as the blank control in the absence of the solutions is represented as Ac.

The method in this study was modified from Xiao et al. (27). To prepare ABTS•+ (Sigma, USA), 20 ml of 7.4 mM ABTS solution was mixed with 13.2 mg K₂S₂O₈. Then, 150 µl of OS8P solution at different concentrations (0.10 to 1.25 mg/ml) was added to 50 µl of the diluted ABTS•+ solution in a 96-well plate, which was kept in the dark for 6 min. Absorbance was measured at 734 nm using a microplate reader. Positive controls, such as α-tocopherol and trolox, were used. The ability of OS8P to scavenge ABTS•+ was calculated using the following equation:

when the absorbance of the solutions is denoted as Ab, the absorbance of ABTS•+ in the presence of the solutions and the absorbance of ABTS•+ as the blank control in the absence of the solutions are denoted respectively as Aa and Ac.

The MTT colorimetric method was used to determine spleen cell proliferation, following the protocol by Lee et al. (29). Spleen cell suspensions at 2.0 × 10⁵ cells/ml were added to 96-well plates with 50 µl of B or T lymphocyte activator (LPS or ConA, 10 µg/ml) and 50 µl of OS8P at various concentrations (0 - 200 µg/ml). After 72 h of incubation, MTT (5 mg/ml) was added to each well and incubated for 4 h. Then, formazan crystals were dissolved in DMSO, and the absorbance at 595 nm was measured using a microplate reader (20).

The mean value with standard deviation was used to present the results, which were analyzed using IBM SPSS. Multiple comparisons between groups were conducted with one-way ANOVA and Duncan’s multiple range test. A significance level of P< 0.05 was considered statistically significant.

The process of isolating fungal mycelia from O. sinensis fruiting bodies involves a series of steps that are crucial in ensuring successful fungal growth. The use of dried fruiting bodies is a common method of extracting mycelia since it reduces the risk of contamination by other microorganisms. The fact that most of the dried O. sinensis fruiting bodies did not exhibit any mycelial growth suggests that the isolation process is highly dependent on a number of factors, such as the quality of the fruiting bodies, environmental conditions, and the techniques employed during isolation. This highlights the importance of standardizing the isolation process to ensure consistency in the results obtained. The 14 dried O. sinensis fruiting bodies that showed positive signs of fungal mycelia development were found to have distinct morphologies, such as a white or cream center. These variations in morphology may be attributed to differences in the genetic makeup of the fungal mycelia, which can impact their growth characteristics.

The successful cultivation of these mycelial colonies is a significant step towards further analysis and understanding of their properties. These properties may include bioactive compounds, which are of great interest in pharmaceutical and medicinal research. However, it is important to note that the results obtained from this study are specific to the O. sinensis species and may not necessarily apply to other fungal species. The isolation of fungal mycelia from O. sinensis fruiting bodies is a crucial step towards further analysis and understanding of their properties. The process is highly dependent on several factors, and standardization is necessary to ensure consistency in results. The distinct morphologies exhibited by the mycelial colonies provide valuable insight into their genetic makeup and growth characteristics, which may have important implications in various fields, including medicine and biotechnology.

Identifying fungal genera is a complex task that requires thorough analysis of various species. In this study, Table 1 presents the different species used to identify the genera of fungi. The most frequently identified genus was Penicillium roqueforti, while Nigrospora oryzae had a lower prevalence but has been found to produce bioactive compounds. The remaining genera, including Penicillium soppii, Megasporoporia minor, Ophiocordyceps sinensis, and Truncatella angustata, were identified in only one colony each, suggesting their potential uniqueness. Further studies are needed to fully understand their properties and benefits.

Moreover, O. sinensis OS8 was investigated against several aspects, including its morphological characteristics. A colony of O. sinensis OS8 exhibited white, plumose, downy, and dense aerial mycelium and hyphal strands, with interwoven hyphae, rod-shaped spores, and were mostly 5 – 10 µm long. O. sinensis is a well-known medical mushroom used in traditional medicine, highlighting the potential for further investigation of O. sinensis OS8 in various fields.

In this investigation, the O. sinensis OS8 strain was chosen as the source for biomass production, due to its potential for high yields. The biomass obtained from this strain was then subjected to a polysaccharide extraction, followed by an examination of its bioactivities. To obtain the mycelial biomass, O. sinensis OS8 was cultured in liquid media for a period of 14 days. The resulting mycelial biomass was then harvested and dried, yielding a total of 23.61 g/l of dried weight, as shown in Table 2 . The high yield of mycelial biomass obtained from O. sinensis OS8 is a promising indication of its potential as a source of bioactive compounds.

The dried mycelial biomasses of O. sinensis OS8 were analyzed using HPLC to determine the adenosine compound present. The results showed a significant adenosine content of 306.1 mg per 100 g of dried mycelial biomass ( Table 2 ).

Polysaccharide extraction from dried mycelial biomass of O. sinensis OS8 was further performed using ultrasonic instrument. As a result, obtained polysaccharide yield was at the rate of 3.22 g/100 g of dried weight ( Table 2 ).

The ratios of polysaccharide compositions as D-glucan, α-, and β-D-glucan from dried mycelial biomass of O. sinensis OS8 polysaccharides (OS8P) are shown in Figure 1 . Of our results, with the new findings, it showed that these polysaccharides of OS8P were enriching β-D-glucan at a percentage of 56.92% ( Figure 1B ). Also, another type of α-D-glucan was found at a percentage of 35.32% ( Figure 1B ). The total polysaccharide D-glucan in the OS8P was obtained at a rate of 92.24% ( Figure 1A ).

The GC-MS analysis identified the compositions of OS8P, as shown in Table 3 . The main components of OS8P were dodecamethyl pentasiloxane, 2,6-bis (methylthiomethyl) pyridine, 2-(4-pyrimidinyl)-1H-Benzimidazole, and 2-Chloro-4-(4-nitroanilino)-6-(O-toluidino)-1,3,5-triazine at the rates of 32.5, 20.0, 17.5, and 16.25%, respectively.

This study investigated the potential action of OS8P on anticancer activity against colon cancer. OS8P challenge with various concentrations of 25, 50, 100, and 200 µg/ml in colon cancer cells resulted in a dose-dependent concentration on inhibition of colon cancer cell proliferation. In this growth inhibition, all concentrations of OS8P significantly inhibited HT-29 cells (P< 0.05) compared to the control group. The growth inhibition of colon cancer cells at the concentration of OS8P with 25, 50, 100, and 200 µg/ml were 8.20, 20.81, 30.23, and 48.00%, respectively ( Figure 2 ). The IC₅₀ value of OS8P efficacy on antiproliferation of colon cancer cells was 202.98 µg/ml.

The apoptotic involvement in the OS8P-induced colon cancer cell death was further investigated. The cell apoptosis could be distinguished by simultaneously staining with AO/PI and visualized by fluorescence microscope. The morphology change of the OS8P-treated HT-29 cells differed from those of the untreated cells. The cell membrane and nucleus of treated cells were stained orange or red, indicating that they were apoptotic-induced ( Figure 3B ), whereas the control cells were not ( Figure 3A ).

Exposure of the colon cancer cells with OS8P for 24 h resulted in apoptotic effects in HT-29 cells after being visualized by a fluorescence microscope. Cell morphological change of the OS8P-treated HT-29 cells was different from the untreated cells. A blue glow of the OS8P-treated cell nucleus appeared under fluorescence microscopy, which indicated that they were apoptotic-induced ( Figure 4B ), whereas the control cells were blue non-glow ( Figure 4A ).

The occurrence of cell death apoptosis in colon cancer cells was seen through DNA fragmentation, as shown in Figure 5 . The non-fragmented DNA appeared in the untreated HT-29 cells (lane 1), as indicated in living cells. After being challenged with Paclitaxel or OS8P for 48 h, the small DNA fragments occurred in both groups of Paclitaxel (positive control, lane 2) or OS8P (lane 3), as evidenced in colon cancer cell death apoptosis.

The morphological changes of colon cancer cells induced by OS8P, HT-29 cell deaths were observed under SEM. As illustrated in Figure 6 , the cells in the control group showed a spherical shape, numerous microvilli on the cell surface, and were generally kept intact ( Figure 6A ). At the same time, morphological changes of apoptotic cells characterized by cell lysis, loss of microvilli, blebbing formation, and appearance of apoptotic bodies were found after treatment with Paclitaxel ( Figure 6B ) as positive control and challenged with 200 µg/ml OS8P ( Figure 6C ).

The DPPH and ABTS aspects were performed in this study. A DPPH scavenging activity of OS8P was at the IC₅₀ value of 0.52 mg/ml, which was not different with α-tocopherol as a positive control at the 0.48 mg/ml rate. However, the IC₅₀ value of Trolox as another positive control at the rate of 0.23 mg/ml was significantly lower (P< 0.05) than that of OS8P ( Figure 7 ).

To correlate to the ABTS scavenging activity, the antioxidant property was found in the OS8P with the IC₅₀ value of 2.07 mg/ml. However, it was significantly higher (P< 0.05) as compared to the positive controls of α-tocopherol and trolox at the rates of 0.47 and 0.77 mg/ml, respectively ( Figure 8 ).

Cell immune induction of rat spleen lymphocyte cell proliferation treated with OS8P is shown in Figures 9 , 10 . The OS8P induced the spleen cell proliferation with the dose responses and were significantly higher (P< 0.05) than the control group of LPS (B lymphocyte cell stimulation) alone, except for the low concentration of OS8P at 25 µg/ml. These proliferation rates of spleen cells in the concentration of 25, 50, 100, and 200 µg/ml were 114.3, 115.8, 117.2, and 118.6%, respectively ( Figure 9 ).

The spleen cells were also treated with OS8P and restimulated by ConA (T lymphocyte cell stimulation) in a dose-dependent concentration. It showed a significant increase in splenocyte proliferation (P< 0.05) in only high doses of 50, 100, and 200 µg/ml compared to spleen cells treated with ConA alone as the control group. The growth rates of spleen cells in the concentration of 25, 50, 100, and 200 µg/ml were 113.1, 126.1, 123.0, and 121.4%, respectively ( Figure 10 ).