In vitro evaluation of the antitumor and antioxidant effects of purified and characterized polysaccharides from Ganoderma applanatum

Abstract

Background

In this study, the chemical properties of Ganoderma applanatum polysaccharides (GAP) were systematically investigated, followed by a comprehensive analysis of their antitumor and antioxidant capabilities.

Methods

Ultrasonic circulation technology was employed for the extraction of GAP, facilitating the procurement of crude polysaccharides through the Sevag method, dialysis, and sequential alcohol precipitation. The chemical constituents of these polysaccharides were subsequently analyzed utilizing Fourier-transform infrared spectroscopy and gas chromatography-mass spectrometry. The antitumor and antioxidant properties of the polysaccharide components were assessed utilizing 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and free radical scavenging methods, respectively.

Results

Gradient ethanol precipitation yielded three principal polysaccharide fractions: GAP-40, GAP-60 and GAP-80. Analysis revealed glucose as the predominant component in these fractions, with average molecular weights determined to be 77.75, 9.25 and 1.03 kDa, respectively. The antitumor activity of GAP-40, GAP-60 and GAP-80 against MCF-7 cells demonstrated both time and concentration dependence. Significantly, GAP-40, at a concentration of 1000 μg/mL over 48 h, presented a notable inhibition rate of 56.77%, outperforming GAP-60 and GAP-80. Furthermore, the antioxidant capacities of GAP-40, GAP-60 and GAP-80 were comparably significant to that of vitamin C, with detailed analysis revealing marked differences in antioxidant activity among the GAP variants. Specifically, GAP-40 exhibited superior efficacy in scavenging 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS+) radicals relative to GAP-60 and GAP-80. In contrast, GAP-80 was distinguished by its exceptional hydroxyl radical scavenging capacity, surpassing that of both GAP-40 and GAP-60.

Conclusion

These results substantiate the potential of GAPs as viable and effective antitumor agents, additionally suggesting their utility as functional foods endowed with antioxidant attributes.

1. Background

Polysaccharides, recognized as fundamental constituents of fungi, are attributed with a wide spectrum of pharmacological properties. These encompass antitumor activities [1,2], antioxidant capabilities [1,3], immunomodulatory effects [4], anti-diabetic, lipid-lowering and anti-aging properties [5,6], as well as antibacterial and antiviral properties [7,8]. Their multifaceted therapeutic potential has garnered considerable attention from biochemists and pharmacologists, driven by their potent healing properties and low toxicity profile. As a result, polysaccharides are emerging as notable substances for crafting safe antitumor drugs and as functional food additives endowed with antioxidant advantages.

Ganoderma applanatum, a medicinal fungus of notable importance within the Ganoderma genus, has been esteemed in traditional Chinese medicine for centuries for its diverse therapeutic properties. These include analgesic, hepatoprotective, immunomodulatory and antitumor effects [2]. Polysaccharides, as one of its prominent active constituents, are distinguished by their abundance and physiological effectiveness. However, conventional extraction techniques for these polysaccharides typically necessitate significant volumes of organic solvents, raising concerns regarding environmental pollution. Consequently, contemporary methodologies for the extraction and purification of polysaccharides, including ultrasonic-assisted extraction, supercritical fluid extraction and membrane separation techniques, have been adopted to mitigate environmental impact. Despite the extensive focus of research on the structural analysis and structure–activity correlations of polysaccharides, there remains a scarcity of data pertaining to the application of gradient ethanol concentration techniques for the polysaccharide separation process.

In this study, ultrasonic circulating extraction technology coupled with gradient ethanol purification methods was utilized for the preparation of G. applanatum polysaccharides (GAPs), culminating in the derivation of three distinct polysaccharide fractions: GAP-40, GAP-60 and GAP-80. An exhaustive characterization of these polysaccharides was conducted, encompassing analyses of molecular weight, monosaccharide composition, and infrared spectral properties. Furthermore, the antitumor efficacy of these polysaccharide fractions was evaluated using the MTT assay, alongside assessments of their antioxidant capabilities via in vitro experiments that included DPPH, ABTS+, and hydroxyl radical scavenging assays.

2. Material and method

2.1. Materials and chemicals

Ganoderma applanatum was obtained from a local drugstore (Mudanjiang, Heilongjiang Province, China), and ground into powder. d-glucose, d-galactose, d-galactose, d-xylose, d-rhamnose, and d-mannose were all purchased from Sigma Chemical Co. (St. Louis, MO, USA). MTT, DPPH and dimethyl sulfoxide (DMSO) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Fetal bovine serum and RPMI 1640 medium were purchased from Invitrogen (Carlsbad, CA, USA). All other reagents and chemicals were of analytical grade.

2.2. Polysaccharide extraction and isolation from G. applanatum

The flat G. lucidum underwent extraction utilizing 95% ethanol in four separate two-hour sessions, which facilitated the removal of pigments and oils. The polysaccharide component of G. lucidum was extracted using distilled water under specific conditions: ultrasonic power of 100 W, ultrasonic temperature of 50 °C, liquid-to-solid ratio of 25 mL/g and ultrasonic duration of 50 min. To eliminate associated proteins, the Sevag method was employed, and the process was repeated three times on the extracted substances [9]. The ethanol solutions were then adjusted to concentrations of 40, 60 and 80% for the purpose of fractional precipitation, aiming to fractionate the deproteinized polysaccharides, which were subsequently allowed to settle overnight at a temperature of 4 °C. The resulting precipitates were centrifuged at 3000×g for 10 min until they were completely dissolved. Following this, the precipitates underwent dialysis against distilled water (with a molecular weight cut-off of 3500 Da) for 48 h to eliminate small molecular weight compounds. The final step involved freeze-drying the samples using a lyophilizer, resulting in the acquisition of refined polysaccharides, which were designated as GAP-40, GAP-60 and GAP-80.

2.3. Characterization of GAPs

In this experiment, we used glucose as the reference standard, and employed the phenol–sulfuric acid method to estimate the total sugar content [10]. Then, the regression equation was compared with the standard curve (y = 9.33x − 0.0145, R² = 0.9991), and the total sugar content of GAPs was calculated. Bradford’s method was used to determine protein content [11].

2.4. Monosaccharide composition analysis

Gas chromatography-mass spectrometry (GC-MS) was used to determine the monosaccharide composition of polysaccharides [12]. Trifluoroacetic acid (TFA) at a concentration of 2 M was employed to hydrolyze samples of polysaccharides (2 mg) for a duration of 90 min, followed by the removal of excess TFA through decompressive evaporation. The resultant residue was redissolved in water, and any remaining sodium borohydride was neutralized via the addition of acetic acid. This mixture was then subjected to a rotary evaporator and subsequently oven-dried at a temperature of 110 °C. For the process of acetylation, 1 mL of acetic anhydride was introduced to the sample at 100 °C and maintained for 1 h. To facilitate cooling, 3 mL of toluene was added to the mixture. Vacuum concentration was applied to the product to achieve a dry state, a procedure that was repeated five times to ensure the removal of unreacted acetic anhydride. The resultant product was dissolved in 3 mL of chloroform and subsequently introduced into a separating funnel containing 10 mL of distilled water for the purpose of fractionation. The mixture underwent vigorous shaking, post which the upper layer was discarded, and the chloroform layer was dried using anhydrous sodium sulfate. For the analysis by gas chromatography-mass spectrometry (GC-MS), an RXI-5 SIL MS column (30 m × 0.25 mm × 0.25 μm) was utilized.

The specified experimental parameters for the procedure entailed a helium flow rate set at 1.0 mL/min, accompanied by both an injection and detector temperature maintained at 250 °C. The temperature of the column was methodically programmed to escalate at a consistent rate of 3 °C/min, initiating from a baseline of 120 °C and progressing to 250 °C, whereupon it was sustained for a duration of 5 min.

2.5. Molecular weight analysis

For high-performance gel permeation chromatography (GPC) analysis, a Shodex SUGAR KS-805 column (7.8 × 300 mm, SHODEX, Japan) was integrated with the Agilent high-performance gel permeation chromatography system, operating at a temperature of 40 °C. Prior to analysis, the column was calibrated using standard dextran solutions. A polysaccharide sample with a concentration of 2 mg/mL was prepared, of which 20 µL was injected into the column. The sample was then eluted with a 20 mM CH₃COONH₄ solvent at a flow rate of 0.8 mL/min. The detection of the eluted sample was conducted using a 1260 Infinity Evaporative Light Scattering Detector (ELSD) (Agilent, Santa Clara, California, USA).

2.6. IR spectroscopy analysis

The infrared spectra of the polysaccharide samples were recorded using a Scimitor 800 spectrophotometer (VARIAN, USA). Each sample was mixed with KBr powder and pressed into particles for measurement. The spectral range for this process was set between 400 and 4000 cm⁻¹.

2.7. Anti-proliferation activity assay

2.7.1. Cell culture

MCF-7 (human breast cancer) cell lines were provided by Harbin Medical University. RPMI-1640 medium with 10% fetal bovine serum was used to culture cells. Cell culture environment was 37 °C with 5% CO₂ in a humidified atmosphere.

2.7.2. Inhibition of cell proliferation assay

For the MCF-7 cell line, the inhibitory effects of GAP-40, GAP-60 and GAP-80 on cell growth were evaluated using the MTT assay [13]. Initially, 200 μL of MCF-7 cells at a concentration of 5 × 10³ cells/mL were seeded into individual wells of a 96-well plate and allowed to incubate for 12 h. Subsequently, polysaccharide solutions at varying concentrations (62.5, 125, 250, 500 and 1000 μg/mL) were prepared in the cell culture medium, and 200 μL of each solution was added to separate wells. The control group received no polysaccharide solution. After incubating for 24 or 48 h, 20 μL of MTT solution (0.5 mg/mL) was added to each well and further incubated for 4 h at 37 °C. Following this incubation period, the supernatant was carefully removed, and 200 μL of DMSO was added to dissolve the formazan crystals. The absorbance of the cells was measured at 570 nm using an enzyme labeling instrument (Bio-Rad, USA) to calculate the IC50 values for the different samples."

The following formula was used to calculate the inhibition rate: inhibition (%) = (1 − absorbance of experimental group/absorbance of blank control group)×100%.

2.8. Antioxidant activity assay

2.8.1. DPPH radical scavenging activity

The DPPH radical scavenging activities of GAP-40, GAP-60, and GAP-80 were determined using the method described by Braca et al. [14] Different concentrations of polysaccharide solutions (0.2, 0.4, 0.6, 0.8 and 1 mg/mL) were prepared and mixed with 3 mL of DPPH ethanol solution. After 30 min of incubation in the dark at 25 °C, the absorbance values were measured at 517 nm, with vitamin C (VC) used as the reference. The following formula was used to calculate the scavenging activity of the DPPH radical. Scavenging rate (%) = [1−(Ai−Aj)/A0]×100, wherein, the absorbance of the reference (without sample) was A0, that of the absorbance in the presence of samples and DPPH was Ai, and that of absorbance of blank sample (without DPPH) was Aj. The scavenging activities of DPPH radical in different samples were evaluated using the IC₅₀ value.

2.8.2. ABTS+ radical scavenging activity

Adapting a previously described methodology, we evaluated the ABTS+ radical scavenging capacities of GAP-40, GAP-60 and GAP-80. [15] The ABTS+ solution was prepared and diluted to achieve an absorbance of 0.70 ± 0.02 at 734 nm, and it was preserved for subsequent use. Employing VC as a reference, 1 mL of the polysaccharide solution at varying concentrations (0.2, 0.4, 0.6, 0.8 and 1 mg/mL) was combined with 2 mL of the ABTS+ solution. This mixture was stirred briefly for 30 s, then incubated at 37 °C for 30 min, after which the absorbance was measured at 734 nm. The following formula was used to calculate the scavenging activity of ABTS+. Scavenging rate (%) = [1−(Ai−Aj)/A0]×100, in which the absorbance of the reference (without sample) was A0, that of the absorbance in the presence of samples and ABTS+ was Ai, and that of absorbance of blank sample (without ABTS+) was Aj. The scavenging activities of ABTS+ radicals in different samples were evaluated by IC₅₀ value.

2.8.3. Hydroxyl radical scavenging activity

Modifications were applied to the methodology originally outlined by Chen, Wu, and Huang for assessing the hydroxyl radical scavenging activities of GAP-40, GAP-60, and GAP-80 [16]. The adjusted reaction mixture comprised 1 mL of hydrogen peroxide (H₂O₂) at a concentration of 6 mmol/L, 1 mL of ferrous sulfate (FeSO₄) at 18 mmol/L, 1 mL of salicylic acid in ethanol at 10 mmol/L, and 1 mL of the polysaccharide solution, which was tested at various concentrations (0.2, 0.4, 0.6, 0.8 and 1 mg/mL). The final mixture was ­incubated at 37 °C for 30 min. VC was employed as the reference, deionized water served as the blank, and the absorbance of the mixture was measured at 510 nm. The following formula was used to calculate the hydroxyl radical scavenging activity. Scavenging rate (%) = [1−(Ai−Aj)/A0]×100, in which the absorbance of the reference (without sample) was A0, absorbance in the presence of samples and hydroxyl radicals was Ai, and absorbance of blank sample (without hydroxyl radicals) was Aj. The scavenging activities of hydroxyl radicals in different samples were evaluated using the IC₅₀ value.

2.9. Statistical analysis

Each sample was measured in triplicate, and the resulting data were presented as the mean ± standard deviation (SD). Statistical evaluations were executed utilizing IBM SPSS Statistics software (Version 20.0, USA). A one-way analysis of variance (ANOVA) was performed to determine statistical significance, which was established at a threshold of p < 0.05.

3. Results

3.1. Characteristics of GAPs

GAPs were extracted from G. applanatum using ultrasonic extraction, with protein impurities removed via the Sevag method. The application of gradient ethanol concentrations at 40%, 60%, and 80% facilitated the fractional precipitation approach, culminating in the procurement of three polysaccharide variants designated as GAP-40, GAP-60 and GAP-80. As shown in Table 1, the carbohydrate content in GAP-40, GAP-60 and GAP-80 was 80.23, 83.33 and 84.51%, respectively. The protein content in GAP-40, GAP-60 and GAP-80 was 0.54, 0.36 and 0.28%, respectively. The average molecular weight of GAP-40, GAP-60 and GAP-80 was 77.75, 9.25 and 1.03 kDa. The average molecular weight of GAP-80 was the lowest among the three kinds of GAPs.

Table 1.

Physicochemical properties of GAPs.

Graded precipitation	Yield (%)	Carbohydrate (%)	Protein (%)	Average molecular weight (kDa)
GAP-40	44.5	80.23	0.54	77.75
GAP-60	35.7	83.33	0.36	9.25
GAP-80	14.6	84.51	0.28	1.03

3.2. Monosaccharide compositions of GAPs

As shown in Figure 1, GAP-40 was composed of glucose and galactose with a molar ratio of 98.9:1.1, both GAP-60 and GAP-80 were composed of glucose. Our results showed that GAP-40, GAP-60, and GAP-80 were rich in glucose.

Figure 1.

HPLC Chromatograms of PMP derivatives of 10 standard monosaccharides (A) and component monosaccharides of GAP-40 (B), GAP-60 (C), and GAP-80 (D). Peaks: (1) rhamnose; (2) fucose; (3) arabinose; (4) xylose; (5) mannose; (6) glucose; (7) galactose.

3.3. Fourier-transform infrared (FT-IR) spectroscopy analysis of GAPs

The infrared spectra of GAP-40, GAP-60 and GAP-80 ranged from 4000 to 400 cm⁻¹, as depicted in Figure 2. The broad and strong absorption bands at 3384–3404 cm⁻¹ were attributable to O–H stretching vibration [17]. The small absorption bands at around 2900 cm⁻¹ (GAP-40: 2925.22 cm⁻¹, GAP-60: 2926.23 cm⁻¹, and GAP-80: 2928.50 cm⁻¹) were attributable to the C–H stretching vibration of methyl and methylene groups [18]. The absorption peaks of 1641–1653 cm⁻¹ and 1412–1419 cm⁻¹ were characterized by carbonyl groups and carboxyl groups, which indicated that GAPs had uronic acid [19]. The bands at 1153, 1079 and 1024 cm⁻¹ were the characteristic absorption peaks of the pyranose unit, and were assigned to the skeletal modes of pyranose rings in the monosaccharide of GAP-40, GAP-60 and GAP-80 [20]. The absorptions at 931–934 cm⁻¹, 849–853 cm⁻¹, and 761–762 cm⁻¹, indicated that GAP-40, GAP-60 and GAP-80 were rich in glucose.

Figure 2.

FT-IR spectra of GAP-40 (A), GAP-60 (B), and GAP-80 (C).

3.4. Growth inhibition on MCF-7 cells

The MTT method was used to detect the inhibitory effect of GAP on the growth of MCF-7 cells for 24 h or 48 h in vitro (Figure 3). The results showed that the inhibitory effects of GAP-40, GAP-60 and GAP-80 on the growth of MCF-7 cells were time and dose-dependent. Additionally, compared with GAP-60 and GAP-80, the inhibitory effect of GAP-40 on MCF-7 cells was more obvious. GAP-40, GAP-60 and GAP-80 exhibited maximum growth inhibition against MCF-7 cells at 48 h incubation at 1000 μg/mL (56.77, 45.13 and 44.55%, respectively).

Figure 3.

Inhibition rate of GAP-40, GAP-60 and GAP-80 on MCF-7 cells with different concentrations at 24 h (A) and at 48 h (B).

3.5. Antioxidant activity of GAPs

DPPH, ABTS+, and hydroxyl radical scavenging tests were used to detect the antioxidant activities of three polysaccharide components; the results are displayed in Figure 4. As a very stable free radical, DPPH is widely used in studies on the scavenging ability of various antioxidant [21,22]. The scavenging activities of GAP-40, GAP-60 and GAP-80 on DPPH radical are depicted in Figure 4A. At a concentration range of 0.2–1.0 mg/mL, GAP-40, GAP-60 and GAP-80 showed obvious dose-dependent ability for scavenging free radicals. Compared with GAP-60 and GAP-80, GAP-40 had stronger scavenging capacity. At 1.0 mg/mL, the scavenging rate of GAP-40, GAP-60 and GAP-80 was 65.83, 51.46 and 43.29%, respectively, comparable to VC (74.61%). IC₅₀ values were 0.73, 0.96 and 1.18 mg/mL, respectively, and indicated a decreasing trend in hydroxyl radical scavenging activity as follows: GAP-40 > GAP-60 > GAP-80.

Figure 4.

Scavenging activities of GAPs on DPPH radical (a), ABTS+ radical (B), and hydroxyl radical (C) in vitro.VC was used as a positive reference.

ABTS+ radicals are blue-green. When antioxidants combine with ABTS+ radicals, they cause changes in color. The scavenging ability of antioxidants on ABTS+ radicals can be evaluated by measuring color changes under specific absorbance [23]. The ABTS+ radical scavenging activities of GAP-40, GAP-60 and GAP-80 are illustrated in Figure 4B. Across a concentration spectrum of 0.8 mg/mL to 1.6 mg/mL, an incremental enhancement in the scavenging impact on ABTS+ radicals was observed for GAP-40, GAP-60 and GAP-80. At the concentration of 1.6 mg/mL, the scavenging efficacies of these polysaccharide fractions were recorded at 78.53, 69.48 and 50.43%, respectively, demonstrating comparability to the effect observed with VC, which exhibited a scavenging ability of 99.82%. GAP-40 exhibited superior scavenging ability against ABTS+ radicals compared to GAP-60 and GAP-80, with IC50 values of 1.24, 1.35 and 1.57 mg/mL respectively. Additionally, the scavenging effects of GAP-40, GAP-60 and GAP-80 on hydroxyl radicals gradually diminished.

Hydroxyl radicals are the most active free radicals and can induce oxidative damage in adjacent macromolecules (carbohydrates, proteins, lipids, etc.) through the cell membrane, and cause tissue damage or apoptosis [24,25]. As depicted in Figure 4C, GAP-40, GAP-60 and GAP-80 effectively removed hydroxyl radicals in the concentration range of 0.2 mg/mL to 1 mg/mL, outperforming VC. Specifically, the scavenging rates for GAP-40, GAP-60 and GAP-80 were 53.74, 40.00 and 63.92% respectively. Notably, GAP-80 exhibited superior hydroxyl radical scavenging ability in comparison to GAP-40 and GAP-60. The IC50 values for GAP-40, GAP-60 and GAP-80 were 0.95, 1.34 and 0.77 mg/mL, respectively, indicating that the hydroxyl radical scavenging activity followed the order of GAP-80 > GAP-40 > GAP-60.

4. Discussion

In this study, the determined average molecular weights for GAP-40, GAP-60 and GAP-80 were found to be 77.75, 9.25 and 1.03 kDa, respectively. Analyses through both component composition and FT-IR spectroscopy identified glucose as the predominant component in all three polysaccharide variants. Moreover, our results highlighted that GAP-40 displayed a significantly stronger antitumor activity against MCF-7 cells in comparison to GAP-60 and GAP-80, with the observed inhibitory effects varying temporally. The disparities in growth inhibition efficacy among GAP-40, GAP-60 and GAP-80 may be ascribed to their unique charge characteristics, monosaccharide profiles, and molecular weights, as suggested in previous studiesechoing the findings of prior research [26].

Additionally, our extensive evaluation of antioxidant actions uncovered differential scavenging capabilities of the GAPs across various oxidative systems, demonstrating an increase in activity correlating with higher concentrations. It is important to note, however, that this observed trend was limited to the specific concentration range examined in our experiments. These outcomes suggest the antioxidative potential of GAP-40, GAP-60 and GAP-80, with variances in their efficacies across distinct oxidative reactions likely due to their inherent structural attributes, such as variations in molecular weights, monosaccharide content, and structural configurations, in alignment with assertions made by Cheng et al. (2013) [27–29].

Owing to their varied origins and multifaceted properties, natural polysaccharides possess the capacity to act as bioactive agents that modulate physiological functions with minimal toxicological profiles and side effects. Consequently, these substances are widely utilized in clinical medicine, attracting substantial interest from the research community. Both traditional Chinese medicine and contemporary scientific investigations affirm the significant potential of mushrooms as valuable resources for pharmaceutical applications [30]. G. applanatum, a species of wild mushroom found predominantly on wood, is renowned for its considerable medicinal properties. It has been extensively utilized in traditional medicinal practices for the management and prevention of a diverse range of illnesses [31]. In-depth research into the chemical composition of G. applanatum has uncovered a wide array of chemical compounds, including but not limited to polysaccharides, sterols, proteins, fatty acids [32], flavonoids [33], saponins, and phenolic compounds. The spectrum of biological effects attributed to G. applanatum spans immunomodulatory [34], antitumor [35], hypoglycemic [36], anti-inflammatory, hepatoprotective [37], antioxidant and antibacterial [38] activities.

Polysaccharides, recognized as active macromolecular substances, have garnered significant attention within the research community. It has been elucidated that the biological efficacy of polysaccharides is closely linked to their molecular weight, functional groups, monosaccharide composition, and additional structural features [39]. Despite this, there exists a paucity of data concerning the antitumor and antioxidant capabilities of GAPS. The objective of this study is to furnish a theoretical foundation and framework for the exploration and utilization of polysaccharides derived from G. applanatum.

The present study concentrated on the isolation and purification of GAP-40, GAP-60 and GAP-80 from G. applanatum. Analysis revealed that the carbohydrate content of GAP-40, GAP-60 and GAP-80 was 80.23, 83.33 and 84.51%, respectively. Furthermore, the average molecular weights were established as 77.75 kDa for GAP-40, 9.25 kDa for GAP-60, and 1.03 kDa for GAP-80, identifying GAP-80 as the variant with the lowest average molecular weight among the trio. Differences in the biological activity of polysaccharides are often attributed to their molecular weights, which vary according to their sources. Moreover, the molar ratio and types of monosaccharides comprising polysaccharides partially dictate their biological efficacy [40].

Based on antitumor activity assessments, GAP-40, GAP-60, and GAP-80 demonstrated significant growth inhibition on MCF-7 cells, with maximum inhibitory effects observed after 48 h of incubation at a concentration of 1000 μg/mL, amounting to 56.77, 45.13 and 44.55%, respectively. Additionally, at a concentration of 1.0 mg/mL, the scavenging effects on the DPPH radical by GAP-40, GAP-60 and GAP-80 were recorded at 65.83, 51.46 and 43.29%, respectively. For the ABTS+ radical, the scavenging activities of GAP-40, GAP-60 and GAP-80 at 1.6 mg/mL were 78.53, 69.48 and 50.43%, respectively. The hydroxyl radical scavenging activities of GAP-40, GAP-60 and GAP-80 were measured at 53.74, 40.00 and 63.92%, respectively, at a concentration of 1.0 mg/mL.

It is widely acknowledged that both the growth inhibition and radical scavenging activities of polysaccharides are determined by an interplay of various factors, including the monosaccharide composition, molecular weight, types of glycosidic linkages, and the content of uronic acids [27]. The findings from this investigation offer a theoretical framework and practical guidance for the utilization of polysaccharides derived from G. applanatum.

5. Conclusion

This paper provides a detailed examination of the purification processes and physicochemical characteristics of polysaccharides extracted from G. applanatum. Additionally, it explores the in vitro antitumor and antioxidant capabilities of these polysaccharides. The study specifically targeted the isolation of three polysaccharide fractions from G. applanatum, namely GAP-40, GAP-60 and GAP-80. Throughout the experimental phase, structural attributes of GAP-40, GAP-60 and GAP-80, such as their average monosaccharide compositions, molecular weights, and infrared spectral profiles, were meticulously analyzed.

In this study, the activities of polysaccharide fractions were evaluated, focusing on their in vitro antitumor and antioxidant capabilities. Notably, GAP-40 demonstrated the most potent antitumor effect on MCF-7 cells. Additionally, our analyses indicated that GAP-40 outperformed GAP-60 and GAP-80 in scavenging DPPH and ABTS+ radicals. However, it was observed that GAP-80 possessed a remarkable ability to scavenge hydroxyl radicals, exceeding the capacities of both GAP-40 and GAP-60. These findings underscore the significant potential of GAPs for applications in the fields of medicine, nutrition, and cosmetic industry. However, to fully elucidate the mechanisms underlying their antitumor efficacy and radical scavenging activities, further comprehensive studies are warranted.

Funding Statement

This work was supported by Science Fund Torch Program project of Mudanjiang Medical College (2022-MYHT-016), Doctoral Research Foundation project of Mudanjiang Medical College (2021-MYBSKY-048), Research Project of the Heilongjiang Health Commission (20221313050608), Research project on Chinese medicine in Heilongjiang (ZHY2024-293), Heilongjiang Province education science planning key topic (GJB1423366), Heilongjiang Province graduate course ideological and political teaching case construction project (HLJYJSZLTSGC-KCSZAL-2023-099).

Authors’ contributions

Conception and design of the research: Jia-Wei Liu. Acquisition of data: Hai-Yan Li. Analysis and interpretation of the data: Hai-Yan Li, Zhi-Ping Li. Statistical analysis: Zhi-Ping Li. Obtaining financing: Jia-Wei Liu, Dan-Rong Ni. Writing of the manuscript: Jia-Wei Liu. Critical revision of the manuscript for intellectual content: Dan-Rong Ni. All authors read and approved the final draft.

Disclosure statement

The authors declare that they have no competing interests.

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.