Comparative evaluation of extraction methods for Scorias spongiosa polysaccharides: Yield, bioactivity, and anti-aging potential

Graphical abstract

Abstract

Scorias spongiosa, a highly nutritious edible fungus native to bamboo forests, is colloquially known as “Bamboo bird’s nest”. Polysaccharides are its primary bioactive ingredients, but comparative studies on their structural characteristics, in vitro antioxidant activities and anti-aging effects across different extraction methods remain scarce. In this study, Scorias spongiosa polysaccharides (SSPs) were extracted using five distinct techniques, including room-temperature water extraction (RWE, 37°C), hot water extraction (HWE), ultrasound-assisted water extraction (UAE), ultrasound-assisted acid extraction (UAE-H), and ultrasound-assisted alkali extraction (UAE-OH). Among these techniques, UAE-OH was identified as the most efficient approach for SSPs preparation, mainly owing to its higher polysaccharide yield (25.4 ± 3.77 %), improved purity (85.25 ± 4.13 %), and enhanced antioxidant activity. In addition, SSPs-UAE-OH possessed the robust anti-senescence effect by mitigating H₂O₂-induced oxidative damage, reactive oxygen species (ROS) accumulation, malondialdehyde (MDA) production and SA-β-gal expression in H9C2 cells. Structural analysis revealed that SSPs-UAE-OH primarily consists of glucose, with a molecular weight of 490 KDa, and FT-IR spectroscopy suggested its potential classification as an α/β-dextran. Furthermore, ultrasonic power was optimized to 500 W through a yield- and activity-guided approach, achieving an enhanced SSPs-UAE-OH yield (32.57 ± 1.57 %) and significantly improved the viability of H9C2 cells (82.33 ± 3.74 %). This study not only established an efficient extraction protocol for SSPs but also elucidated the structural basis of their antioxidant and anti-aging properties in vitro. These findings provide critical insights for developing SSPs-based functional foods.

1. Introduction

Aging, an inevitable and complex biological process, entails progressive alterations at the cellular, tissue, organ, and systemic levels, increasing susceptibility to numerous age-related pathologies [1,2]. A hallmark of aging is cellular senescence, a state of irreversible cell cycle arrest triggered by a variety of endogenous and exogenous factors [3]. Notably, senescent cells exhibit apoptotic resistance, leading to their abnormal accumulation, a phenomenon strongly correlated with advancing age [4]. Beyond cellular dysfunction, senescence plays a pivotal role in the pathogenesis and progression of multiple age-associated disorders. With the rapidly aging global population, cardiac aging and its associated diseases have emerged as a pressing public health challenge [5]. Substantial evidence underscores cellular senescence as a critical contributor to cardiovascular diseases (CVDs), where the establishment and persistence of senescent cells exacerbate disease severity and accelerate pathological progression [6,7]. Consequently, there is an urgent need for appropriate interventions to delay and reduce cardiac aging as well the incidence of age-related CVDs.

Oxidative stress has been strongly implicated in both aging and the pathogenesis of age-related CVDs [8]. The imbalance between reactive oxygen species (ROS) generation and antioxidant defenses leads to the development of pathological processes in CVDs [9]. Sustained oxidative stress triggers aberrant activation of multiple signaling pathways in cardiac tissue, accelerating age-dependent structural and functional deterioration [10]. Experimental evidence from stress-induced myocardial remodeling murine models demonstrated that uncontrolled ROS accumulation results in pronounced ventricular dilation, pathological hypertrophy and contractile dysfunction [11]. Furthermore, excessive ROS production promotes cardiomyocyte injury through apoptotic signaling pathways and other forms of programmed cell death [12]. Given its central role in driving cardiomyocyte death, oxidative stress is widely regarded as a unifying mechanism underlying diverse aging-associated CVDs [13]. Therefore, targeting oxidative stress-mediated damage represents a pivotal therapeutic strategy for mitigating age-related CVDs.

Accumulating evidence suggests that the use of antioxidants can effectively mitigate myocardial remodeling and improve cardiac function [14,15]. Antioxidants based on natural polysaccharides have attracted widespread attention, such as polysaccharides derived from Indian seaweeds [16] and Zizania latifolia [17]. However, polysaccharides face challenges in high-yield and efficient extraction from natural materials, which greatly limits their industrial applications [18]. In order to overcome these drawbacks, modern auxiliary technologies have been exploited for efficient extraction of polysaccharides in recent years. In contrast, ultrasonic-assisted extraction (UAE) is a promising non-thermal extraction technique, offering advantages such as low energy consumption, reduced processing time, and enhanced extraction efficiency [19]. These benefits stem from its cavitation effects to promote the release and diffusion of polysaccharides from plant material [[20], [21], [22]]. More importantly, proper ultrasonication can improve the bioactivity of polysaccharides by modifying their structures [23,24]. In addition, acidic extraction has been shown to promote the effective cleavage of glycosidic bonds in polysaccharides and enable higher yields of bioactive low-molecular-weight polysaccharides [25,26]. Meanwhile, alkaline extraction has proved effective in obtaining polysaccharide-rich extracts with enhanced bioactivity [[26], [27], [28]]. Thus, the method of combining auxiliary extraction technology with acid or alkaline solutions has emerged. For example, arabinoxylan from the corn bran achieved 27.78 % yield using ultrasonic-microwave assisted alkali extraction [29].

Scorias spongiosa, known as Bamboo bird's nest, is an economically valuable mushroom species that grows in bamboo forest [30]. S. spongiosa grows mainly in Sichuan province, China, and has not yet been artificially cultivated, although it was discovered by Professor He Xinsheng in 2011 [31], which lead to its high price due to edible and medicinal values. Polysaccharides have been widely recognized as key bioactive components, demonstrating diverse biological activities [32]. At present, polysaccharides from S. spongiosa (SSPs) have been shown to have antioxidant and anti-inflammatory abilities to maintain the integrity of the gut barrier [33,34]. To date, the researches on SSPs remain scarce, with scant information available regarding extraction methodologies and their capacity to attenuate oxidative injury in myocardial tissue.

In this study, five extraction methods, including 37°C water extraction (RWE), hot water extraction (HWE), ultrasound-assisted water extraction (UAE), ultrasound-assisted acid extraction (UAE-H), ultrasound-assisted alkali extraction (UAE-OH), were used to examine their effects on the polysaccharides derived from S. spongiosa. The yield, chemical compositions and structural characteristics were analyzed. And the biological properties of SSPs were evaluated, specifically the antioxidant capacity and protective effects against H₂O₂-induced oxidative damage in H9C2 cardiomyocytes. Furthermore, a yield- and activity-guided UAE was conducted to extract SSPs to optimize the extraction parameters. This is the first attempt to explore the efficient acquisition method on SSPs extracted by the co-treatment of ultrasound with either acid or alkali solutions. The findings offer valuable guidance for the targeted preparation of SSPs that deliver high yields and potent protection against oxidative stress and cardiomyocyte senescence.

2. Materials and methods

2.1. Reagents

S. spongiosa were collected from Yibin Bamboo Forest Base (Yibin, Sichuan, China) on November 3, 2024. Monosaccharide standards were sourced from Sigma Aldrich (Sigma, St Louis, MO, USA), Aladdin Bio-Chem Technology Co., Ltd. (Shanghai, China), Honeywell Fluka^TM (Seelze, Germany), and Vetec^TM (Sigma-Aldrich, USA), respectively. National Institute for Food and Drug Control (Beijing, China) offered a series of dextran standards with different molecular weight (Mw). Solarbio Science & Technology Co., Ltd. (Beijing, China) provided 1,1-diphenyl-2-picrylhydrazyl (DPPH).

H9C2 rat cardiomyocyte line was provided by CoBioer Biosciences Co., Ltd (Nanjing, Jiangsu, China). Dulbecco's Modified Eagle's medium (DMEM), fetal bovine serum (FBS), penicillin, and streptomycin were purchased from Gibco (Grand Island, NY, USA). Beyotime Institute of Biotechnology (Shanghai, China) supplied Cell Counting Kit-8 (CCK-8), ROS Assay Kit, and Lipid Peroxidation (Malondialdehyde, MDA) Assay Kit.

All other chemicals and solvents were of analytical grade.

2.2. Preparation of SSPs by different extraction methods

Fresh S. spongiosa was dried at 45°C, ground into powders, passed through a 60-mesh sieve, and degreased with 5 ∼ 6 times ethanol. At a solid–liquid ratio of 1:30 g/mL, the defatted powder was immersed in the water for 72h. Five techniques were used to prepare the SSPs, including RWE, HWE, UAE, UAE-H, and UAE-OH. The experimental flow chart was depicted in Scheme. 1.

Scheme. 1.

. Roadmap for SSPs preparation.

RWE was conducted in a 37 °C water bath with continuous magnetic stirring for 4 h after adding an equal volume of water. Hot water extraction was performed similarly at 80 °C with magnetic stirring for 2 h after adding an equal volume of water [35]. For the remaining three extraction methods, equal volume of distinct solvent systems (water, 1 % citric acid solution and 1 % sodium hydroxide solution) were introduced, resulting in pH values of approximately 5.0–5.3, 3.1–3.4, and 12.0–12.3, respectively. SSPs were subsequently prepared using an XH-2008D ultrasonic extraction system (XiangHu Technologies, Beijing, China) at 60 °C for 30 min [17,26]. After centrifugation, the supernatant was concentrated to 1/10 of its original volume and precipitated with three volumes of 95 % ethanol. The resulting precipitates were collected, reconstituted in ultrapure water, dialyzed against deionized water, and lyophilized to obtain crude polysaccharide fractions, named as SSPs-RWE, SSPs-HWE, SSPs-UAE, SSPs-UAE-H, and SSPs-UAE-OH, respectively.

The yield of crude polysaccharide was calculated using equation (1).

$$Y(\%)=m/M\times 100\%$$ (1)

Here, Y was the yield of crude polysaccharide (%), m was the weight of crude polysaccharide (g), M was the weight of extracted material for Scorias spongiosa (g).

2.3. Surface morphology analysis of extraction residues for Scorias spongiosa

The untreated S. spongiosa powders and dried residuals after extraction were evenly sprayed with gold powders under vacuum provided. Surface morphology was observed with a Hitachi Regulus^TM 8100 scanning electron microscope (Tokyo, Japan).

2.4. Chemical composition analysis of SSPs

The total polysaccharide content of SSPs was quantified by the phenol–sulfuric acid method with glucose as the reference standard [36,37], and the calibration curve was Y = 1.013X (R² = 0.9976). The protein content in SSPs was determined by Bradford's assay [38], with the standard curve following the equation Y = 7.8029X-0.00395 (R² = 0.9983). The polyphenol content in SSPs was analyzed using the Folin-Ciocalteu method with gallic acid as the standard [39], yielding a linear regression of Y = 12.089X (R² = 0.9927). Uronic acid content in SSPs was measured via the carbazole-sulfuric acid method using galacturonic acid as the standard [40], with the calibration curve demonstrating good linearity (Y = 25.7823X-0.02448, R² = 0.9915).

2.5. Structural characterization of SSPs

Mw was analyzed using an Agilent 1260 high-performance liquid chromatograph (Agilent, USA) equipped with a TSKgel GMPWxl gel column (300 × 7.8 mm i.d.) and a refractive index detector (RID). The analytical conditions were as follows: mobile phase 0.1 mol/L Na₂SO₄ solution, flow rate 0.5 mL/min, column temperature 35 °C [41].

Trifluoroacetic acid (TFA, 2 mol/L) was used to hydrolyze SSPs into monosaccharides at 110 °C for 4 h. After removing the residual TFA, the derivatization reaction was carried out with PMP-methanol solution under alkaline conditions at 70 °C. Subsequently, monosaccharides composition was analyzed using Agilent 1260 high-performance liquid chromatograph (Agilent, USA) equipped with a Zorbax KP-C18 column (250 mm × 4.6 mm, 5 μm) and a UV detector. The analytical conditions were as follows: mobile phase consisted 0.1 mol/L phosphate buffer (pH 7.0) and acetonitrile in a ratio of 83:17 (v/v, %), column temperature 30 °C, flow rate 0.8 mL/min, detector wavelength 254 nm [41].

For UV spectral analysis, the polysaccharide was prepared as a 0.5 mg/mL aqueous solution, and the UV spectra were scanned at 190 ∼ 800 nm with the instrument UV–visible spectrophotometer (T600, Pulsar) [17].

The fourier transform infrared spectroscopy (FT-IR) of SSPs was tested by a Nicolet Nexus 470 infrared spectrometer in the range of 400 to 4000 cm⁻¹ with a KBr pellet as previously described [41].

The Congo red experiment was used to evaluate the triple helix conformation of SSPs. Briefly, Congo red (0.2 mmol/L) was mixed with SSPs solution (2 mg/mL). Then, NaOH solution (0 ∼ 0.8 mol/L) was gradually added to the system, and the maximum absorption wavelength of the mixture was measured in the range of 200 ∼ 800 nm [42]. Distilled water instead of the polysaccharide solution was used as a blank control group.

2.6. Antioxidant activity

2.6.1. DPPH radical scavenging assay

The SSPs solution (0.5 ∼ 5 mg/mL) was mixed with an equal volume of 0.1 mM DPPH ethanolic solution and allowed to react in the dark for 30 min. The absorbance was then measured at 517 nm (A₁) [43,44]. Two control groups were established: (1) a blank control (A₀) using distilled water instead of SSPs solution, and (2) a normal control (A₂) consisting of the reaction system without DPPH. The DPPH radical scavenging rate of SSPs was calculated using Equation (2).

2.6.2. Hydroxyl radical scavenging assay

The SSPs solutions (0.5 ∼ 5 mg/mL) extracted by the five methods were mixed with equal volumes of 6 mM FeSO₄ solution, 6 mM ethanol salicylic acid solution, and 6 mM H₂O₂ solution. The absorbance at 510 nm was read after incubation for 30 min at 37 °C (A₁) [43,44]. Distilled water was used instead of SSP solution as a blank control (A₀) and the lack of H₂O₂ in the reaction system was used as a normal control (A₂).

Equation (2) was used to calculate the free radical scavenging rate.

$$\mathit{Freeradicalscavengingrate}=[1-(A_1-A_2)/A_0]\times 100\%$$ (2)

2.7. Protective effects against oxidative damage in cardiomyocytes

H9C2 cardiomyocytes were maintained in complete DMEM medium supplemented with 10 % FBS and 1 % penicillin–streptomycin, and cultured in a standard incubator (37 °C, 5 % CO₂). Cells in the logarithmic growth phase were seeded in cell culture plates at a density of 3 × 10⁴ cells/well. After 12 h of adherent culture, cells were pretreated with various concentrations of SSPs for 2 h, followed by induction of oxidative injury through addition of a specific concentration of H₂O₂. The cells were harvested for subsequent experimental analyses [7].

2.7.1. Cell counting kit-8 (CCK-8) assay

After removing the culture medium, 100 μL of CCK-8 working solution (cell culture medium and CCK-8 reagent were mixed in the ratio of 9:1) were added to each well. Following incubation at 37 ℃ for 2 h, the absorbance at 450 nm (A₄₅₀) was detected using the Biotek Synergy 2 plate reader. Cell viability was calculated according to Equation (3).

$$Cellviability\left(\%\right)=A_{450}oftreatmentgroup/A_{450}ofcontrolgroup\times 100\%$$ (3)

2.7.2. Cellular ROS assay

After aspiration of the culture medium, serum-free DMEM medium containing 10 μmol/L DCFH-DA were added to each well. Following incubation at 37 ℃ for 20 min, cells were washed for three times with serum-free DMEM medium. The immunofluorescence images were photographed using an Olympus fluorescence microscope (Olympus) and analyzed with Image Pro Plus 6.0 software.

2.7.3. Measurement of senescence-associated β-galactosidase (SA-β-gal) activity

After treatment, H9C2 cells were rinsed with PBS and then incubated with 1 mL SA-β-gal staining fixing solution at room temperature for 15 min. After washing three times with PBS, the cells were stained using 1 mL SA-β-gal dyeing working solution at 37 °C overnight. During incubation overnight, the 6-well plate was sealed with parafilm to prevent evaporation. Subsequently, images were obtained using an inverted microscope. The percentage of SA-β-gal-positive cells was quantified from light microscopy images of 5 randomly chosen fields.

2.7.4. MDA assay

After aspiration of the culture medium, cells were washed with PBS and collected by centrifugation. Cells were then lysed by ultrasonication, and MDA content was determined according to the manufacturer's protocol.

2.8. Yield- and anti-cardiomyocyte senescence-guided UAE-OH of SSPs

The defatted powder of Scorias spongiosa was immersed in the water for 72h at a solid–liquid ratio of 1:30 g/mL. After adding an equal volume of 1 % sodium hydroxide, samples were prepared by UAE using an XH-2008D ultrasonic extraction system (XiangHu Technologies, Beijing, China) at 60 °C for 30 min at the powers spanning from 300 to 700 W. According to section “2.2”, SSPs was obtained by centrifugating, ethanol precipitating, dialyzing and lyophilizing. The yield of SSPs was calculated using equation (1).

To evaluate the anti-aging potential for cardiomyocytes, the cell viability under H₂O₂-induced oxidative damage in H9C2 cells was tested. According to section “2.7.1”, H9C2 cells were pretreated with 100 μg/mL SSPs for 2 h, followed by induction of oxidative injury through addition of 400 μmol/L H₂O₂. And the cells were harvested after 12 h to test the cell viability using the CCK-8 assay. Cell viability was calculated according to Equation (3).

2.9. Statistical analysis

All results were expressed as means ± standard deviation (SD). Statistical differences were analyzed using one-way analysis of variance (ANOVA) with GraphPad Prism software (version 6.0.1), and p < 0.05 was considered statistically significant. Correlation analysis was performed using OriginPro 2021 software via Pearson’s correlation test.

3. Results

3.1. Extraction yields

As exhibited in Fig. 1A, the extraction yield of SSPs-RWE was the lowest (2.54 ± 0.75 %). Compared with SSPs-RWE, the higher temperature significantly increased the extraction efficiency, increasing the yield to 6.90 ± 1.32 %. The application of UAE demonstrated superior efficiency, achieving 11.6 ± 2.44 % yield through enhanced cell wall disruption via cavitation effect [45,46]. Notably, the extraction yield of SSPs was further improved by the co-treatment of ultrasound with either acid or alkali solutions, of which the SSPs-UAE-H yield was highest (27.7 ± 4.81 %), followed by SSPs-UAE-OH (25.4 ± 3.77 %).

Fig. 1.

The effects of different extraction methods on the yield (A) and chemical compositions (C) of SSPs, SEM images of S. spongiosa powders (B). (B) SEM images of unextracted powders (a) and extracted residues by HWE, RWE, UAE, UAE-H and UAE-OH (b-f). The scale was 10 μm in a-f, and the scale was 3 μm in a₁-f₁.

To explore the contribution of different extraction methods on SSPs yields, SEM analysis was carried out on the unextracted and extracted S. spongiosa powders. As presented in Fig. 1B, distinct morphological differences were observed between unextracted powders and extracted residues. Unextracted S. spongiosa exhibited intact cell walls with scattered fibrous structures, while RWE-treated samples showed destruction of the stirring paddle, resulting in roughened surface. Residues were in flake form and had numerous fissures after extraction of HWE and UAE, indicating that the extractions were insufficient. However, compared with HWE, UAE brought more fine cracks of cell walls because of cavitation bubbles, which result in a higher yield of SSPs-UAE [47]. It can be seen to have a high degree of breakage in the extraction residues of UAE-H and UAE-OH. Comparatively, the UAE-H treatment had a more typical catheter-like residue and larger pores, indicating a more thorough extraction of SSPs. These findings suggested that the co-treatment of ultrasound with either acid or alkali solutions exerted great damage to the microstructure of S. spongiosa powders and promoted the outflow of SSPs, thereby obtaining substantially higher yield.

3.2. Chemical composition

Fig. 1C showed the chemical composition of SSPs extracted using different methods. All five types of SSPs contained extremely low levels of proteins (<0.3 %) and polyphenols (<0.4 %). Among them, SSPs-UAE-OH and SSPs-UAE-H demonstrated the highest total polysaccharide content of 85.25 ± 4.13 % and 84.34 ± 7.61 %, respectively, followed by SSPs-UAE (81.63 ± 0.36 %). The total polysaccharide content of SSPs-HWE showed the lowest purity (69.98 ± 0.84 %) and SSPs-RWE showed comparable results (71.00 ± 2.82 %). In addition, the uronic acid content of SSPs ranged from 1.74 % and 2.54 %. The enhancement of polysaccharide purity contributed to the elevated uronic acid levels. Notably, SSPs-UAE-OH, SSPs-UAE-H and SSPs-UAE exhibited higher uronic acid content compared to the other samples.

3.3. Basic characteristics of SSPs

From the HPLC chromatograms (Fig. 2A), different extraction methods had little effect on the monosaccharide composition of SSPs. As shown in Table 1, SSPs were mainly composed of glucose (≥ 90 %), and contained a small amount of mannose, rhamnose and galactose. In addition, temperature, pH and applied force all increased the percentage content of glucose in the structure of SSPs and reduced the presence of other monosaccharides. Among them, SSPs-UAE-H and SSPs-UAE-OH were the most obvious, with a glucose content of 96.90 % and 97.61 %, respectively. Previous studies indicate that most edible fungal polysaccharides are heteropolysaccharides primarily consisting of glucose [48]. In contrast, lentinan and Ganoderma lucidum polysaccharides were composed entirely of glucose, and their structures were mainly β-glucan linked by β-(1,6)- and β-(1,3)-glycosidic bonds, which allow them effectively exert the immune antioxidant activities [49,50]. Therefore, it was speculated that SSPs may be an excellent active polysaccharide.

Fig. 2.

HPLC chromatograms (A) and HPGPC chromatograms (B) of SSPs extracted by different methods. (A) Peaks 1 ∼ 11 stand for mannose, glucosamine, rhamnose, glucuronic acid, galacturonic acid, galactosamine, glucose, galactose, xylose, arabinose, fucose in turn.

Table 1.

The monosaccharide composition and Mws of SSPs extracted by different methods.

	Monosaccharide composition (%)						Mw (Da)
	Man	Rha	GlcA	GalA	Glu	Gal
SSPs-RWE	4.20	0.10	0.56	0.65	91.93	2.56	7.31 × 105
SSPs-HWE	1.32	0.20	0	1.10	96.35	1.03	1.38 × 106
SSPs-UAE	2.81	0.40	0.56	0.80	93.82	1.61	6.39 × 105
SSPs-UAE-H	0.46	0.30	0.48	0.95	97.61	0.20	1.90 × 104
SSPs-UAE-OH	0.55	0.65	0.6	1.05	96.9	0.25	4.90 × 105

Chromatograms of High performance gel permeation chromatography (HPGPC) (Fig. 2B) revealed that SSPs was a monodisperse, single-component polysaccharide whose Mws nonetheless varies markedly with the extraction protocol employed. Table 1 showed that SSPs-HWE possessed the highest Mw of 1.38 × 10⁶ Da, suggesting that the elevated temperature not only destroy the cell wall but also dramatically boosts the solubilization of polymeric constituents relative to RWE. Ultrasound treatment and extreme pH conditions (either too high or too low) were found to partially destroy the glycosidic linkages, resulting in a lower Mw of SSPs-UAE than SSPs-HWE, and a further decrease in the Mw of SSPs-UAE-H and SSPs-UAE-OH. Similar observations have been reported in previous studies by Nuerxiati et al. [51] and Sun et al. [26].

From the FT-IR spectrums (Fig. 3A), the characteristic absorption peaks of SSPs extracted by different methods were similar, including peaks around 3394.35 cm⁻¹ (stretching vibration of O-H), 2932.52 cm⁻¹ (stretching vibration of C-H), 1619.39 cm⁻¹ (stretching vibration of −C=O), and 1424.48 cm⁻¹ (band vibration of C-H) [52]. In addition, the signals at 1159.19 cm⁻¹ and 1026.19 cm⁻¹ were attributed to the bending vibration of C–O–C, confirming the presence of pyranose ring [53]. The absorption peaks at 931.71 cm⁻¹ and 848.56 cm⁻¹ were indicative of C-H deformation vibration corresponding to β-glycosidic and α-glycosidic linkages, respectively [54]. An ester carbonyl stretching vibration was observed at 1732.19 cm⁻¹, confirming the presence of esterification in SSPs-RWE, SSPs-HWE and SSPs-UAE [55]. But SSPs-UAE-H exhibited a lower esterification degree, with SSPs-UAE-OH showing minimal to no ester substitution.

Fig. 3.

The FT-IR spectra (A), UV spectra (B) and triple-helical configurational analysis (C) of SSPs extracted by different technologies.

The UV spectroscopic analysis (Fig. 3B) revealed no characteristic absorption peaks at approximately 260 nm, demonstrating the absence of significant protein impurities in the SSPs. This finding corroborated the quantitative protein assay results presented in Fig. 1C. In addition, SSPs-UAE-H exhibited a new UV absorption peak possibly relating to acid degradation at 206 nm, which caused the cleavage of glycosidic bonds to expose new reducing ends, or partial sugar ring dehydration to form unsaturated bonds (C=C or C=O). In comparison, this peak of SSPs-UAE-OH was not obvious, indicating minimal alkali-induced degradation effect.

The Congo red test (Fig. 3C) showed that all the SSPs prepared by five extraction methods had the presence of an ordered triple-helical configuration due to a red-shift in the peak absorption wavelength of the SSPs-Congo red complex across NaOH concentrations ranging from 0 to 0.8 M [42]. In contrast, the hydrogen bonds of SSPs-UAE-OH and SSPs-UAE-H were more easily broken in alkaline environments, resulting in the transformation of the triple helix structure into random coiling, which led to a decrease in the intensity of the red shift. The other three polysaccharides obtained a relatively stable triple helix.

3.4. Antioxidant activity of SSPs

Oxidative stress is an important driver of aging, and its essence is excessive oxidative damage caused by an imbalance between free radical production and scavenging in the body [56]. As a key component of the endogenous antioxidant defense system, antioxidants play a crucial role in eliminating these radicals to delay the heart aging process [57]. The DPPH radical scavenging curves were presented in Fig. 4A. A significant concentration-dependent manner was observed for DPPH radical scavenging capacity of all SSPs extracts across 0.5 to 5 mg/mL range (p < 0.05 or p < 0.01). Statistical analysis of IC₅₀ values (Fig. 4B) demonstrated the following potency ranking for DPPH radical scavenging efficacy: SSPs-UAE-OH > Vc ≈ SSPs-UAE-H > SSPs-UAE > SSPs-HWE > SSPs-RWE. As shown in Fig. 4C, a similar concentration-dependent pattern was also seen in hydroxyl radical reducing power within the concentration range from 0.5 to 5 mg/mL. According to the statistical analysis of IC₅₀ values (Fig. 4D), the reducing power ranks as follows: Vc > SSPs-UAE ≈ SSPs-UAE-H ≈ SSPs-UAE-OH > SSPs-HWE > SSPs-RWE.

Fig. 4.

The scavenging capacities of SSPs against DPPH (A and B) and hydroxyl radicals (C and D). Different lowercase letters in the same color denote statistical differences (p < 0.05 or p < 0.01).

Overall, SSPs-UAE-OH exhibited the strongest antioxidant activity, followed by SSPs-UAE-H and SSPs-UAE, and the antioxidant activity of SSPs-RWE was weakest. Specifically, the ability of all SSPs extracts scavenging DPPH radical was higher than that for hydroxyl radical. For example, the IC₅₀ value of SSPs-UAE-H for scavenging DPPH radical was 0.14 ± 0.06 mg/mL, significantly lower than that for scavenging hydroxyl radical (IC₅₀ = 0.72 ± 0.30 mg/mL, p < 0.05). The same findings have been found in other studies [58]. Furthermore, although the hydroxyl radical-scavenging capacity of all SSPs extracts was weaker than that of Vc (IC₅₀ = 0.14 ± 0.06 mg/mL), the DPPH radical-scavenging capacity of SSPs-UAE-H (IC₅₀ = 0.14 ± 0.07 mg/mL) had no significant difference with Vc (IC₅₀ = 0.14 ± 0.07 mg/mL), and SSPs-UAE-OH (IC₅₀ = 0.013 ± 0.011 mg/mL) possessed a stronger DPPH scavenging ability than Vc. The results indicated that ultrasound promoted the antioxidant activity of SSPs by changing the structure, and the synergy between ultrasound and alkaline environment was more beneficial. In addition, SSPs-UAE-OH exhibited a anti-aging potential due to its excellent antioxidant activity.

3.5. Protective effect of SSPs against cardiomyocyte oxidative damage and cardiomyocyte senescence

Oxidative stress is a key mechanism underlying cardiac aging [56]·H₂O₂, a exogenous oxidant, is widely employed to induce cellular senescence through ROS-mediated oxidative stress [59,60]. In this study, we systematically optimized the experimental conditions for establishing a reliable H₂O₂-induced oxidative stress model in H9C2 cardiomyocytes. As illustrated in Fig. 5A, H9C2 cardiomyocytes were exposed to increasing concentrations of H₂O₂ (0, 100, 200, 400 and 800 μmol/L) for 12 h, followed by assessment of cell viability using the CCK-8 assay·H₂O₂ treatment induced a concentration-dependent reduction in cell activity. Compared with untreated control group (0 μmol/L H₂O₂), exposure to 100 μmol/L H₂O₂ caused only a marginal decreased in cardiomyocyte viability (p > 0.05), whereas 200 μmol/L H₂O₂ treatment elicited a statistically significant reduction in cell viability (p < 0.05). The most pronounced effect was observed at 400 μmol/L H₂O₂, which markedly suppressed cell viability to approximately 50 % (p < 0.01). Exposure to 800 μmol/L H₂O₂ resulted in obvious cell death. Based on these dose–response finding, 400 μmol/L H₂O₂ was selected as the optimal concentration for establishing the oxidative stress model. As shown in Fig. 5B, time-course analysis revealed that treatment with 400 μmol/L H₂O₂ led to a time-dependent decline in H9C2 cardiomyocyte viability. Prolonged exposure (> 12 h) significantly exacerbated cellular damage. Consequently, a 12 h treatment period was determined to be the appropriate duration for H₂O₂ treatment in this model.

Fig. 5.

Assessment of H9C2 cardiomyocyte viability using the CCK-8 assay. (A-B) Dose-dependent and time-dependent effects of H₂O₂ on H9C2 cells viability. (C) Cytotoxicity of SSPs on H9C2 cells. (D) Effect of SSPs against H₂O₂-induced oxidative damage in H9C2 cells. Data are presented with mean ± SEM (n = 5). *p < 0.05 and ^**p < 0.01 versus control; ^#p < 0.05 and ^##p < 0.01 versus model group.

Based on the findings presented in Fig. 4, we evaluated the protective effects of SSPs, including SSPs-UAE, SSPs-UAE-H and SSPs-UAE-OH against H₂O₂-induced oxidative stress in H9C2 cardiomyocytes. Firstly, CCK-8 assays showed that all three SSPs samples exhibited no obvious cytotoxicity across a wide concentrations range of 0 ∼ 1000 μg/mL (Fig. 5C). In the established H₂O₂-induced oxidative stress model, all three SSPs samples demonstrated significant cytoprotective effects at concentrations of 100–200 μg/mL (Fig. 5D).

Oxidative stress arises from an imbalance between pro-oxidant and antioxidant systems, leading to excessive generation of ROS and subsequent elevated lipid peroxidation [61,62]. Evidence suggests an inverse correlation between intracellular ROS levels and organismal lifespan. Comparative studies have demonstrated that short-lived species exhibit significantly higher ROS levels than their long-lived counterparts [63,64]. To further evaluate the impact of SSPs on oxidative stress in H₂O₂-treated H9C2 cardiomyocytes, intracellular ROS generation was quantitatively analyzed using DCFH-DA fluorescent staining. As demonstrated in Fig. 6A and 6B, the fluorescent intensity of three SSPs samples exhibited a biphasic response within the concentration range of 50–200 μg/mL, showing an initial decrease followed by subsequent increase in fluorescence intensity. Among the tested SSPs, SSPs-UAE-OH demonstrated a significant inhibition on H₂O₂-induced ROS production (p < 0.05 and p < 0.01), whereas SSPs-UAE-H showed no obvious effects. Although SSPs-UAE treatment at 100 and 200 μg/mL significantly attenuated fluorescence intensity compared to the model group (p < 0.05), its relative fluorescence values (81.76 ± 24.47 % and 103.89 ± 9.40 %, respectively) remained higher than those of SSPs-UAE-OH (78.50 ± 8.41 % and 84.69 ± 10.81 %, respectively).

Fig. 6.

Effect of SSPs against H₂O₂-induced oxidative stress and senescence in H9C2 cells. (A) ROS production was detected using the DCFH-DA fluorescent staining. (B) quantification of DCFH-DA fluorescence. Bar = 50 μm. (C) MDA level was evaluated by the Elisa kit. (D) Senescence was analyzed by SA-β-gal staining of H9C2 cells. Bar = 25 μm. (E) quantification of SA-β-gal staining. Data are presented with mean ± SEM (n = 3). *p < 0.05 and ^**p < 0.01 versus control; ^#p < 0.05 and ^##p < 0.01 versus model group.

MDA, a well-established biomarker of oxidative stress, serves as a key indicator of lipid peroxidation in biological systems [8]. As illustrated in Fig. 6C, SSPs-UAE-OH (50 μg/mL) significantly attenuated H₂O₂-induced MDA formation (p < 0.05), while neither SSPs-UAE nor SSPs-UAE-H exhibited significant effects at this concentration. At 100 μg/mL, SSPs-UAE-OH demonstrated the most potent antioxidant activity, reducing MDA content to 1.63 ± 0.35 nmol/g (p < 0.01). Although SSPs-UAE and SSPs-UAE-H also significantly decreased MDA levels (p < 0.05), their effects were less pronounced (1.91 ± 0.33 nmol/g and 2.15 ± 0.32 nmol/g, respectively). In addition, at the highest tested concentration (200 μg/mL), SSPs-UAE-OH maintained its antioxidant activity, lowering MDA to 2.03 ± 0.29 nmol/g (p < 0.05). This reduction remained significantly greater than that achieved by either SSPs-UAE or SSPs-UAE-H treatment at the same concentration.

In summary, comparative analysis revealed that SSPs-UAE-OH exhibited the most potent antioxidant activity, offering significant protective effect against H₂O₂-induced oxidative damage in H9C2 cardiomyocytes, while SSPs-UAE showing intermediate efficacy. Furthermore, SA-β-gal staining analysis revealed that SSPs-UAE-OH effectively attenuated H₂O₂-induced cellular senescence in H9C2 cardiomyocytes across 50 ∼ 200 μg/mL concentration range (Fig. 6D-E), with optimal anti-senescence activity observed at 100 µg/mL. These results were consistent with established mechanistic evidence that implicates ROS accumulation and subsequent oxidative damage were primary mediators of cellular senescence processes [65]. Particularly, bioactive polysaccharides have been well-documented to mitigate H₂O₂-induced senescence in H9C2 cardiomyocytes through modulation of cellular redox homeostasis [7]. These findings collectively suggested that SSPs-UAE-OH represents a promising therapeutic candidate for attenuating cardiomyocyte senescence.

Nonetheless, it is important to acknowledge the limitations of the H₂O₂-treated H9C2 cells model. As a cell line derived from rat embryos, H9C2 cells may not fully recapitulate the biological characteristics of adult cardiomyocytes in vivo. Furthermore, the present model reflects aging induced by acute oxidative stress, the drivers and molecular mechanisms of which may differ from those underlying the physiological aging process. To further validate the anti-aging efficacy of SSPs, future studies employing animal models and human-derived cells are warranted to elucidate the associated molecular mechanisms in a more physiologically relevant context.

3.6. Correlation analysis

Pearson correlation analysis were employed to investigate the relationships between extraction methods, yield and physicochemical properties and protective effects against H₂O₂-induced oxidative damage in H9C2 cells of SSPs (Fig. 7). It was observed that the enhanced DPPH and hydroxyl radicals reducing powers, along with improved viability of H₂O₂-induced senescent H9C2 cells were attributed to the increased polysaccharides and uronic acid content of SSPs achieved through the ultrasonic technique. Polysaccharides were the main components that exerted the antioxidant properties and anti-cardiomyocyte senescence, and the nucleophilic carbonyl groups in uronic acids boost the interactions of polysaccharides with free radicals [66,67]. Additionally, the inhibition of H₂O₂-induced ROS and MDA production in H9C2 cells depended not only on polysaccharide content but also Rha levels of SSPs. This observation aligned with previous finding by Xie et al., who reported that rhamnose consumption is particularly effective in reducing ROS levels and enhancing oxidative stress resistance compared to other sugars [68]. In Table 1, Rha levels of five SSPs samples (SSPs-RWE, SSPs-HWE, SSPs-UAE, SSPs-UAE-H, SSPs-UAE-OH) were 0.1 %, 0.2 %, 0.4 %, 0.3 % and 0.65 %, respectively. The polysaccharide content ranks as follows: SSPs-UAE-OH ≈ SSPs-UAE-H > SSPs-UAE > SSPs-RWE ≈ SSPs-HWE, and the content of uronic acid ranks as follows: SSPs-UAE-H > SSPs-UAE-OH > SSPs-UAE > SSPs-RWE > SSPs-HWE (Fig. 1). These findings collectively demonstrate that SSPs obtained by UAE, especially SSPs-UAE-OH, exhibited superior anti-age-related CVDs potentials, owing to their higher polysaccharide, uronic acid and Rha content (Fig. 1, Fig. 4 ∼ 6).

Fig. 7.

Pearson correlation analysis between extraction methods, yield and physicochemical properties, anti-aging effect. Red symbols denote positive correlations, while blue symbols represent negative correlations. Abbreviations: PSs, polysaccharide content. PPs, polyphenol content. CV, cell viability.

Fig. 7 demonstrated that polysaccharide content was positively correlated with uronic acid content, while uronic acid content was inversely proportional to Mw. And the Mw of SSP did not show a linear relationship with biological activities. As shown in Table 1, the Mw of SSPs-UAE-OH was 4.90 × 10⁵ Da, which falls between that of SSPs-UAE (6.39 × 10⁵ Da) and SSPs-UAE-H (1.90 × 10⁴ Da). It is widely recognized that low-Mw polysaccharides are more likely to approach and bind with free radicals but are less capable of forming complex spatial structures due to the reduction of reducing terminal groups and insufficient contact area with free radical [69]. Correspondingly, SSPs-UAE-OH possessed the strongest scavenging ability on DPPH radicals and protective effects against H₂O₂-induced oxidative damage in H9C2 cells from Fig. 4 ∼ 6, which was related with its appropriate Mw.

As shown in Fig. 7, the pH change (< 5 or > 9) of the extraction solution significantly contributed to the yield, protein and polyphenol contents of SSPs. The highest yield of SSPs was found in UAE-OH (25.4 ± 3.77 %) and SSPs-UAE-H (27.7 ± 4.81 %), no significant differences were observed (p > 0.05). The protein and polyphenol content of five SSPs samples were very low and did not significantly affect the purity of polysaccharides. These results indicated that UAE-OH was the suitable extraction strategies to prepare SSPs with high yields and excellent desired anti-cardiomyocyte senescence activity.

3.7. Effect of ultrasonic frequency on yield and anti-aging

Ultrasonic power is a critical process parameter in UAE, significantly influencing the extraction outcome and biological activities of plant extracts [[70], [71], [72], [73]]. To obtain SSPs-UAE-OH with high yield and strong activity, this work further explore the proper power of UAE using a dual-parameter strategy based on yield and anti-aging activity.

As represented in Fig. 8, the yield of SSPs-UAE-OH increased with ultrasonic power from 300 W to 500 W, followed by a decline (p < 0.05). A minor rebound was observed at 700 W, though the difference from yields at 500 W and 600 W was not statistically significant (p > 0.05), potentially due to considerable data variability. The maximum yield was achieved at 500 W. Mechanistically, appropriate ultrasonic power promotes cavitation bubble formation and implosion, generating high-shear microjets that enhance cell wall disruption and improve solvent access to intracellular components [74,75]. Conversely, excessive power leads to cavitation overload, which attenuates acoustic energy transmission into the extraction medium, thereby reducing extraction efficiency [76].

Fig. 8.

Effects of ultrasonic power on the yield and anti-aging of SSPs-UAE-OH. Different letters above the error bar represented statistical difference within group.

In parallel, the protective effect of SSPs prepared by UAE-OH at different power levels against H₂O₂-induced cardiomyocyte injury was assessed using the CCK-8 assay. Results demonstrated a progressive increase in cell viability as ultrasonic power rose from 300 W to 700 W, indicating that ultrasonic energy enhances the anti-senescence properties of SSPs-UAE-OH. Although the highest cell viability was recorded at 700 W, the differences among 500 W, 600 W, and 700 W were not statistically significant (p > 0.05). Previous studies have indicated that ultrasonic treatment can reduce the molecular weight of nearly all plant and microbial polysaccharides through ultrasonic degradation [77]. Lower molecular weight and smaller particle size are known to facilitate cellular uptake of polysaccharides and potentiate their bioactivity [78,79]. This bioactivity modulation may be attributed to the ability of ultrasonication to selectively degrade or structurally modify polysaccharides through mechanical scission and cavitation, thereby altering their physicochemical properties and bioactivity profiles [80,81].

Collectively, these findings validate the advantages of UAE in enhancing both extraction yield and bioactivity. Therefore, 500 W was identified as the optimal ultrasonic power for SSPs-UAE-OH, balancing production efficiency and pharmacological potential. Under this condition, the polysaccharide yield reached 32.57 ± 1.57 %, and H9C2 cell viability was maintained at 82.33 ± 3.74 %.

4. Conclusion

This study presented a comprehensive comparative analysis of five extraction methods for SSPs. Pearson correlation analysis observed that the pH change (< 5 or > 9) of the extraction solution significantly promoted the dissolution of SSPs. The yield of SSPs-UAE-OH was comparable to that of SSPs-UAE-H, however, both were significantly higher than the other extraction methods. And ultrasound-assisted extraction improved polysaccharide content, uronic acids content and biological activities. Among them, SSPs-UAE-OH elicited strongest DPPH radicals-scavenging capacity. Meanwhile, SSPs-UAE-OH exerted promising potency for antioxidant and anti-aging because of the obvious inhibition on H₂O₂-induced oxidative stress in H9C2 cells. Furthermore, a yield- and activity-guided strategy was used to identify that 500 W was the proper ultrasonic power. These results indicated that UAE-OH was an effective extraction technique for SSPs preparation with a high yield and strong effect against cardiomyocyte senescence, which provided a reasonable preparation method for the application of SSPs in cardiac aging.

CRediT authorship contribution statement

Yiting Xue: Conceptualization, Project administration, Supervision, Writing – original draft. Jun Zhang: Data curation, Methodology, Validation. Ji Yan: Investigation, Methodology. Junhao Qiu: Data curation, Methodology. Jiaxin Zeng: Investigation, Methodology. Yixin Li: Data curation, Methodology. Xiaoren Huang: Data curation, Methodology. Xinjue Li: Resources, Supervision. Lingyuan Guo: Methodology. Lingtian Wu: Funding acquisition, Writing – review & editing. Qian Wang: Funding acquisition, Writing – review & editing.

Funding

This work was supported by grants from the National Natural Science Foundation of China (82370317 and 81900293), the Fundamental Research Funds for the Central Universities (YG2023QNB12). The Jiangsu Agricultural Science and Technology Innovation Funds (CX(23)3100), the China Postdoctoral Science Foundation (2023 M742570), the Natural Science Research Project of Jiangsu Higher Education Institutions (24KJB210001, 24KJB550001), the Project of Gusu Leading talents of Innovation and Entrepreneurship Science and Technology (ZXL2022003).

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

Data will be made available on request.