Ultrasonic-Assisted Hot-Water Extraction Optimization, Preliminary Characterization and Antioxidant Activity of Polysaccharides from Zingiber striolatum

Abstract

Polysaccharides serve as the main bioactive compounds of Zingiber striolatum (Z. striolatum). However, their structures and functions require further research. The present work used response surface methodology (RSM) for optimizing polysaccharide extraction conditions from Z. striolatum through ultrasonic-assisted hot-water extraction and evaluating the antioxidant properties of Z. striolatum polysaccharides. According to our findings, optimal Z. striolatum polysaccharide extraction conditions included ultrasonic power of 200 W, extraction temperature of 79 °C, extraction duration of 2.5 h, and ultrasonic time of 20 min, with the extraction yield reached 19.96% ± 0.18%. The molecular weight (MW) of the purified polysaccharide ZS-P1-1 isolated from Z. striolatum was 5.63 × 10⁴ kDa, mainly composed of galactose (Gal), galacturonic acid (GalA) and glucuronic acid (GlcA) in a 0.453:0.162:0.114 molar ratio. Typically, 2,2-diphenyl-1-picrylhydrazyl radical (DPPH·) and hydroxyl radical (·OH) scavenging capacities and total reducing ability reached 30.49% ± 1.82%, 28.51% ± 1.40% and 0.64 ± 0.07 of ZS-P1-1 at 0.5 mg/mL, respectively. Moreover, ZS-P1-1 increased the reduced glutathione (GSH) level, enhanced the total superoxide dismutase (T-SOD), reduced the malondialdehyde (MDA) level, and alleviated the liver and kidney damage caused by oxidative stress in mice. Our results suggest that ZS-P1-1 shows excellent antioxidant properties and provides a certain theoretical foundation for developing and utilizing Z. striolatum polysaccharides.

1. Introduction

Zingiber striolatum (Z. striolatum) is a perennial herbaceous plant with valuable edible and medicinal properties belonging to the phylum Angiospermae, class Monocotyledonae, order Scitaminales, family Zingiberaceae, and genus Zingiber. Z. striolatum is frequently used in traditional Chinese herbal medicines and nutraceutical foods, with a variety of biological activities such as promoting blood circulation to relieve pain, dispelling wind, and eliminating dampness, as well as antioxidant, antimicrobial, anti-gout, antifungal, anti-inflammatory and anticancer activities [1,2]. Z. striolatum is rich in various kinds of active components such as red pigments, flavonoids, essential oil, polysaccharides, dietary fiber, amino acids, vitamins and minerals [3,4]. However, as one of the main active ingredients, a systemic investigation on the biological activities of Z. striolatum polysaccharide (ZS-P) has not yet been reported.

The extraction methods of plant polysaccharides usually include solvent extraction, acid/alkali extraction, enzymatic hydrolysis, and physically assisted extraction [5]. Hot water extraction represents an easy, cost-effective, and eco-friendly method, suitable for the lab-scale extraction of most plant polysaccharides [6]. However, this method has several drawbacks, such as a relatively extended extraction duration, high operating temperature, and decreased extraction efficacy [7]. Ultrasonic-assisted extraction can reduce processing time and solvent usage while achieving a higher extract yield through the ultrasonic cavitation effect, which disrupts cell walls [8]. A high ultrasound frequency can accelerate cell wall disruption via thermal effects, thereby improving penetration, enhancing adhesion and mixing between the extraction liquid, and promoting the diffusion of target compounds into the solvent [9,10,11,12]. Moreover, optimized ultrasonic-assisted extraction parameters enhance polysaccharide structural integrity and bioactivity [13]. However, excessively high ultrasonic extraction power and prolonged extraction time may lead to the breakage of glycosidic bonds in polysaccharides and structure disruption, and it difficult to control the temperature during the extraction process [14]. Furthermore, ultrasonic-assisted extraction could potentially degrade polysaccharides, modify their molecular weight or physicochemical characteristics, and consequently impact biological activities [15]. Therefore, optimizing the conditions for ultrasonic-assisted extraction is crucial for enhancing the efficiency of polysaccharide extraction. Currently, the investigation of ultrasonic-assisted extraction and antioxidant bioactivity of polysaccharide from Z. striolatum lacks systematic investigation, and key operational parameters remain undefined.

For plant polysaccharides, their bioactivities are tightly associated with the structures. There were four water-soluble polysaccharides (ALP-1, ALP-2, ALP-3, ALP-4) derived from Arctium lappa L. roots, and ALP-1, which had the lowest molecular weight, possessed the highest antioxidant effect in vivo [16]. Three burdock-derived polysaccharide components, RF50, RF30, and EF30, contained arabinose (Ara), fucose (Fuc), Gal, GalA, glucose (Glc), mannose (Man), rhamnose (Rha), and xylose (Xyl). However, RF30 has higher anti-edema effect than RF50 and EF30, probably because of its greatest Rha level [17]. Moreover, the polysaccharides with a β-(1 → 2)-linked fructan structure exhibit significant anti-inflammatory activity, indicating that polysaccharides’ backbone and branched chains are also closely related to their biological activities [18]. Currently, the isolation and characterization of ZS-P have rarely been reported; therefore, further research is needed to analyze its structure and composition.

A Box–Behnken design (BBD) of RSM was employed in this work to optimize the extraction parameters of polysaccharides from Z. striolatum using ultrasonic-assisted hot-water extraction. Furthermore, we investigated the structural characteristics and antioxidant activity of Z. striolatum polysaccharides. This study aims to provide a theoretical basis for the development and utilization of polysaccharides isolated in Z. striolatum for use in functional foods, health products, and pharmaceuticals.

2. Materials and Methods

2.1. Z. striolatum Polysaccharide Preparation

After drying and crushing, the powders of Z. striolatum were filtered with an 80-mesh screen. Powders were suspended in distilled water (1:20 solid–liquid ratio) and heated within the 80 °C water bath for 2 h. This resultant mixed sample was subsequently sonicated (200 W, 15 min) in an ultrasound-microwave synergistic extraction instrument (XO-SM100, ATPIO, Nanjing, China) with an ultrasound frequency of 25 MHz and an ultrasound probe diameter of Φ 10, followed by 15 min of centrifugation (4000× g). Supernatants were subjected to rotary evaporation (SENCO, Shanghai, China) for concentration and overnight anhydrous ethanol precipitation (1:4, v/v) under 4 °C. The precipitate was collected using centrifugation (4000× g, 20 min) to obtain crude Z. striolatum polysaccharide (ZS-P). The concentration of ZS-P was determined using the phenol–sulfuric acid method [19]. The extraction yield was calculated according to Equation (1):

$$\mathrm{yield}\left(\%\right)=c·v·d/m\times 100$$

(1)

where c is the ZS-P concentration (g/mL), v is the extract volume (mL), d is the dilution factor, and m is the weight of Z. striolatum powders (g).

The polysaccharides were re-dissolved with distilled water and deproteinated using the Sevag method [20]. ZS-P underwent dialysis with a cut-off of 3500 Da (MW) for 48 h at 4 °C, followed by lyophilization for 48 h. Subsequently, 300 mg of ZS-P was dissolved in 5 mL of deionized water and filtered through a 0.45 μm membrane filter (Yuanye, Shanghai, China). The filtrate was then subjected to separation on a DEAE-52 column (2.6 cm × 30 cm; Henghuibio, Beijing, China) and washed using stepwise elution with distilled water containing increasing concentrations of NaCl (0, 0.1, 0.3, and 0.5 mol/L) at a flow rate of 1.0 mL/min. Eluent fractions (10 mL each tube) were collected, with 40 tubes collected for each concentration. The eluent was detected and fractions containing identical components were pooled. Each pooled eluent was concentrated and purified using a Sephadex G-100 column (1.5 cm × 80 cm; Shanghai, China). The Sephadex G-100 column was eluted with distilled water at a flow rate of 0.5 mL/min, and a total of 60 tubes (5 mL each tube) were collected. The purified fraction was lyophilized for subsequent analyses.

2.2. Optimization of Extraction Conditions Using RSM

Extraction temperature (°C), extraction duration (h), ultrasound time (min), and ultrasound power (W) were used, with the extraction yield of ZS-P as the dependent variable, to evaluate the range of the independent variables using a single-factor experiment. Table S1 shows the single-factor experimental design. This single-factor experiment was performed to analyze how the above-mentioned four variables affected the response. Moreover, the best extraction conditions were determined using a BBD of RSM for enhancing polysaccharide production from Z. striolatum.

2.3. Structural Characterization

2.3.1. Mw Distribution

In brief, 5 mg polysaccharide samples were added into NaCl solution (1 mL, 0.5 mol/L, Kermel, Tianjin, China) and then centrifuged (12,000× g, 10 min). After filtration through a 0.22 μm aqueous microporous membrane, the Mw of the polysaccharide was determined using high-performance gel permeation chromatography (UltiMate3000, Thermo, MA, USA) equipped with a series gel column (BRT105-103-101, 8 mm × 300 mm) and a refractive index detector (RID-20A, Shimadzu, Kyoto, Japan). The injection volume, flow rate and column temperature were 25 μL, 0.7 mL/min and 40 °C, separately.

2.3.2. Monosaccharide Composition

Briefly, 5 mg polysaccharides were subjected to 3 h of hydrolysis using trifluoroacetic acid (2 mL, 3 mol/L, ANPEL, Shanghai, China) under 121 °C. After drying under nitrogen and washing with methanol (ANPEL, Shanghai, China), the samples were dissolved with 5 mL deionized water. Subsequently, 50 µL of the mixed fluid was added to 950 µL of deionized water followed by centrifugation at 8000× g for 5 min. The supernatant was collected and determined monosaccharide composition using high-performance ion chromatography with CarboPac PA-20 column (3 mm × 150 mm; Dionex, Sunnyvale, CA, USA). The mobile phase consisted of double-distilled H₂O (A), NaOH (15 mmol/L; Alfa Aesar, Heysham, UK) (B), and a mixture of NaOH (15 mmol/L)/NaAc (100 mmol/L; Thermo Fisher, Waltham, MA, USA) (C). The flow rate was 0.3 mL/min, the injection volume 5 μL, and the column temperature 30 °C. The elution gradient is detailed in Table S2.

2.3.3. Fourier Transform Infrared (FT-IR) Spectroscopy

To be specific, 2 mg polysaccharide was introduced into KBr powder (200 mg) and prepared in 1 mm pellets. Then, the FT-IR spectra of polysaccharides were recorded within 4000–400 cm⁻¹ using a spectrometer (FT-IR650, Tianjin, China).

2.3.4. Scanning Electron Microscopy (SEM)

Polysaccharides (5 mg) after freeze-drying were adhered to the conductive carbon film coated with a double-sided adhesive, followed by spraying using gold powder using an ion sputtering instrument for 40 s, and were put into the observation chamber under a scanning electron microscope (SU8600, HITACHI, Tokyo, Japan) to examine their surface microstructure at a 5 kV acceleration voltage.

2.4. Antioxidant Property Analysis In Vitro

2.4.1. DPPH· Scavenging Capacity

Polysaccharides were mixed with distilled water for preparing sample solutions with varying concentrations (0.1, 0.2, 0.3, 0.4, 0.5 mg/mL). Thereafter, every sample solution (2 mL) was added into a DPPH-ethanol solution (2 mL, 0.2 mmol/L), followed by 30 min of reaction under 37 °C, and absorbance values were then read at 532 nm. With vitamin C (Vc) being the positive control, scavenging capacity (%) could be determined as follows:

$$\mathrm{DPPH}·\mathrm{scavenging} \mathrm{rate} (\%)=(1-{\displaystyle \frac{A_1-A_2}{A_0}})\times 100$$

(2)

where A₁ is the absorbance of the sample with the DPPH-ethanol solution, A₂ is the absorbance of the sample without the DPPH, and A₀ is the absorbance of DPPH-ethanol solution without the sample.

2.4.2. ·OH Radical Scavenging Capacity

Polysaccharide samples (2 mL) of diverse concentrations (0.1, 0.2, 0.3, 0.4, 0.5 mg/mL) were added into FeSO₄ and salicylic acid (both 2 mL, 9 mmol/L). After the subsequent addition of H₂O₂ (2 mL, 8.8 mmol/L), the resultant mixture underwent 30 min of incubation under 37 °C, and its absorbance was read at 510 nm, with Vc and distilled water being positive and blank controls, separately. The scavenging capacity (%) could be determined as follows:

$$·\mathrm{OH} \mathrm{scavenging} \mathrm{rate} (\%)=(1-{\displaystyle \frac{A_1-A_2}{A_0}})\times 100$$

(3)

in which A₁ refers to sample absorbance, A₂ stands for the H₂O₂-free sample absorbance, and A₀ indicates blank control absorbance.

2.4.3. Total Reducing Capacity

Polysaccharide solutions (2 mL) of diverse concentrations (0.1, 0.2, 0.3, 0.4, 0.5 mg/mL) were added into phosphate buffer (2 mL, 0.2 mol/L, pH = 6.6) as well as 1% potassium ferricyanide (2 mL). This resultant mixture later underwent 20 min of incubation under 50 °C, followed by the addition of 10% trichloroacetic acid (1 mL) for reaction termination. The mixture later received centrifugation (4000× g, 5 min), with supernatants (1 mL) being mixed with ultrapure water (1 mL) and 1% FeCl₃ (0.2 mL). Following 10 min of reaction under 37 °C, absorbance was read at 700 nm. The total reducing capacity of the sample was calculated, with Vc being the positive control. A greater absorbance suggested a more potent reducing capacity.

2.5. Antioxidant Activity Analysis In Vivo

The 8-week-old male-specific pathogen-free Kunming mice provided by Pengyue Experimental Animal Breeding Co., Ltd. (Jinan, China), were acclimated for one week and could take water and food unrestrictedly. The mice were then randomly divided into the control group (CG), model group (MG), positive group (PG), and ZS-P1-1 group (ZG), with 5 mice in each group. Except for the mice in the CG, the other groups received intraperitoneal injection of 5% D-galactose (1000 mg/kg·BW) daily to induce an oxidative stress injury model. Meanwhile, NC and MC mice received saline administration, PG mice received administration of V_C (200 mg/kg·BW), whereas ZG mice underwent 30 days of intragastric administration of ZS-P1-1 (200 mg/kg·BW). After this experiment, the animals received anesthesia using ether. Later, their blood was extracted for obtaining serum samples through 15 min of centrifugation (3000× g). The activities of T-SOD, as well as GSH and MDA content, were analyzed using the corresponding assay kits (Nanjing, China), as recommended. Tissue samples were collected, fixed, dehydrated, embedded, and sliced into sections of 5 μm before hematoxylin and eosin (H&E) staining. All experiments gained approval from Experimental Animal Ethics Committee of Xuzhou Medical University (approval number: L20210226457) and were performed following ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines.

2.6. Statistical Analysis

All results were represented by mean ± S.D. Response surface analysis was completed with Design Expert (Version 13, Minneapolis, MN, USA). GraphPad Prism (version 8.0.2, San Diego, CA, USA) was employed for statistical analysis with one-way ANOVA with Dunnett’s multiple comparison test. The p < 0.05 stood for statistical significance.

3. Results

3.1. Single-Factor Analysis

When the temperature elevated from 60 to 80 °C, the ZS-P production elevated from 16.84% ± 0.09% to 17.26% ± 0.08%. The ZS-P production started to decline after the temperature further elevated beyond 80 °C (Figure 1a). In addition, ZS-P production elevated with the extension of extraction duration from 1 to 2.5 h and peaked at 2.5 h (20.15% ± 0.98%), but it began to decrease as the extraction duration further increased (Figure 1b). Furthermore, ZS-P production elevated from 14.40% ± 0.25% to 17.05% ± 0.45%, with ultrasonic time extending from 5 to 20 min. However, the ZS-P production later decreased as the ultrasonic time increased (Figure 1c). Finally, ZS-P production elevated from 15.06% ± 0.95% to 21.63% ± 0.81% as ultrasonic power elevated at 100–200 W, and it began to decrease with increasing ultrasonic power (Figure 1d).

3.2. Extraction Condition Optimization Using BBD

The range and the level of each independent factor were obtained based on a single-factor experimental analysis (Table S3). The effects of the independent variables at different experimental combinations on the values of ZS-P yield are listed in

Table 1. Effects of the factors at different combinations on the ZS-P yield.

Std	Ultrasonic Time (min)	Ultrasonic Power (W)	Extraction Time (h)	Extraction Temperature (°C)	Yield
1	15	150	2.5	80	16.45 ± 0.74
2	25	150	2.5	80	16.02 ± 0.26
3	15	250	2.5	80	14.37 ± 0.44
4	25	250	2.5	80	16.44 ± 0.22
5	20	200	2	70	15.67 ± 0.56
6	20	200	3	70	13.69 ± 0.32
7	20	200	2	90	13.16 ± 0.25
8	20	200	3	90	14.91 ± 0.35
9	15	200	2.5	70	15.29 ± 1.04
10	25	200	2.5	70	15.74 ± 0.37
11	15	200	2.5	90	14.60 ± 0.54
12	25	200	2.5	90	13.38 ± 0.39
13	20	150	2	80	15.31 ± 0.10
14	20	250	2	80	13.68 ± 0.34
15	20	150	3	80	13.5 ± 0.26
16	20	250	3	80	14.79 ± 0.45
17	15	200	2	80	14.82 ± 0.23
18	25	200	2	80	14.37 ± 0.22
19	15	200	3	80	14.61 ± 0.38
20	25	200	3	80	15.7 ± 0.19
21	20	150	2.5	70	13.89 ± 0.28
22	20	250	2.5	70	15.74 ± 0.32
23	20	150	2.5	90	14.41 ± 0.12
24	20	250	2.5	90	13.03 ± 0.30
25	20	200	2.5	80	19.74 ± 0.43
26	20	200	2.5	80	20.3 ± 0.50
27	20	200	2.5	80	19.82 ± 0.38
28	20	200	2.5	80	19.62 ± 0.22
29	20	200	2.5	80	19.44 ± 0.13

. A total of 29 experimental combinations including those four individual variables obtained using the BBD were used to determine the optimal conditions.

Multiple regression was conducted on experimental results, and regression equations between ZS-P yield and each independent variable were obtained. The fitted model equation is as follows:

Y = 19.78 + 0.1258A − 0.1275B + 0.0158C − 0.5442D + 0.625AB + 0.385AC − 0.4175AD + 0.73BC − 0.8075BD + 0.9325CD − 1.9A² − 2.42B² − 2.85C² − 2.94D²

Experimental results were assessed by analyzing variance, and the significance of regression coefficients were evaluated by their corresponding p-values (

Table 2. Analysis of variance of regression model for ZS-P yield.

Source	Sum of Squares	df	Mean Square	F-Value	p-Value
Model	126.01	14	9	30.80	<0.0001	significant
A—ultrasound time (min)	0.19	1	0.19	0.65	0.43
B—ultrasonic power (W)	0.20	1	0.20	0.67	0.43
C—extraction time (h)	0.003	1	0.003	0.01	0.92
D—extraction temperature (°C)	3.55	1	3.55	12.16	0.0036
AB	1.56	1	1.56	5.35	0.0365
AC	0.59	1	0.59	2.03	0.18
AD	0.70	1	0.70	2.39	0.14
BC	2.13	1	2.13	7.30	0.017
BD	2.61	1	2.61	8.93	0.0098
CD	3.48	1	3.48	11.9	0.0039
A2	23.42	1	23.42	80.17	<0.0001
B2	38	1	38	130.04	<0.0001
C2	52.61	1	52.61	180.04	<0.0001
D2	55.89	1	55.89	191.27	<0.0001
Residual	4.09	14	0.29
Lack of Fit	3.68	10	0.37	3.55	0.12	not significant
Pure Error	0.41	4	0.1
Cor Total	130.1	28
R2	0.97
Adj R2	0.94
Pred R2	0.83
C.V. %	3.46

). A p-value < 0.0001 and an F-value of 30.8 indicate that the model was more significant. A lack-of-fit F-value of 3.55 and a corresponding p-value of 0.12 indicate that the model adequately fit the observed data. The determination coefficient (R² = 0.97) of the quadratic regression model suggests that this model did not explain 3.14% of overall variation. Moreover, both the determination coefficient and adjusted determination coefficient (Adj R² = 0.94) were highly correlated, implying that these models can be effectively applied to optimize the ZS-P yield. Meanwhile, the lower coefficient of variation (CV% = 3.46) represents the higher precision level and higher experimental data reliability. From the p-values, an independent variable (D), four interaction terms (AB, BC, BD, and CD) and all the quadratic terms significantly affected the ZS-P yields.

Contour plots, which can graphically represent regression equation, can visually interpret between-variable relationships (Figure 2). Circular contour plots suggest insignificant between-variable interactions. However, elliptical plots demonstrate significant between-variable interactions. Therefore, the contour plots of this study indicate that the order of the interaction strength among the corresponding variables interactions were as follows: CD > BD > BC > AB > AD > AC.

According to the RSM analysis, the ZS-P extraction conditions were optimized as follows: ultrasonic power of 199.70 W, extraction duration of 2.49 h, extraction temperature of 79.03 °C, and ultrasonic time of 20.21 min, and the predicted maximum ZS-P yield was 19.81%. The real value was 19.96% ± 0.18% under the following adjusted conditions: ultrasonic power of 200 W, extraction duration of 2.5 h, extraction temperature of 79 °C, and ultrasonic time of 20 min. These predicted values were closely consistent with the experimental ones, indicating that this model can be accurately used for optimizing the extraction conditions of ZS-P.

3.3. Isolation and Purification of Polysaccharides from Z. striolatum

The crude polysaccharide ZS-P was separated into five distinct peaks, designated as ZS-P1, which was eluted with water, and ZS-P2, ZS-P3, ZS-P4, and ZS-P5, which were eluted with different concentrations of NaCl using a DEAE-52 column. Because the yield of ZS-P1 was much higher than that of other components (Figure 3a), ZS-P1 was collected and purified using a Sephadex G-100 column. The main fraction, designated as ZS-P1-1, was collected for structural and activity analyses (Figure 3b).

3.3.1. Mw and Monosaccharides in ZS-P1-1

In line with the retention time–Mw standard curve, ZS-P1-1 had a Mw of 5.63 × 10⁴ kDa (Figure 3c). Monosaccharide components of ZS-P1-1 were analyzed, which comprised Rha, Ara, glucosamine (GlcN), Gal, Glc, Xyl, Man, GalA and GlcA at a 0.021:0.062:0.002:0.453:0.077:0.098:0.011:0.162:0.114 molar ratio (Figure 3d,e).

3.3.2. FT-IR Spectral Analysis

FT-IR spectra for ZS-P1-1 revealed an absorption band at 3423 cm⁻¹ within 3600–3200 cm⁻¹, which is the typical peak of O-H stretching vibration absorption of polysaccharides [21]. Meanwhile, peaks at 2924 cm⁻¹ and 2854 cm⁻¹, within 2800–3000 cm⁻¹, corresponded to C–H stretching vibration in the methyl group of the sugar ring, which are both representative of polysaccharide peaks [22,23]. The absorption peak at around 1741 cm⁻¹ was due to the stretching vibration of C=O in the ester or carboxyl groups, indicating the presence of uronic acids [24]. The characteristic absorptions at 1610 cm⁻¹ and 1421 cm⁻¹ was ascribed to the asymmetric and symmetric stretching vibrations of the –C=O in –COOH for GalA [25]. The band at approximately 1242 cm⁻¹ represented the valence vibration of the C–O vibration of the O-acetyl groups [26]. Additionally, absorption bands at 1155 cm⁻¹ and 1039 cm⁻¹ were associated with C-O-C asymmetric stretching vibrations, confirming that a pyranose ring existed in the sugar residues [27] (Figure 3f).

3.3.3. SEM Analysis

The SEM images show that ZS-P1-1 exhibited a predominantly flaky morphology, with both smooth and rough surface regions. Some areas displayed irregular blocky attachments and cracks at magnifications of 500(×) (Figure 4a). Multiple layers of flaky cracks can be seen on the surface of ZS-P1-1 at magnifications of 1000(×) and 2000(×), respectively (Figure 4b,c). At a higher magnification of 5000(×), the lamellar cracks and granular attachments become more pronounced, indicating a complex surface microstructure (Figure 4d).

3.4. Antioxidant Activity of ZS-P1-1 In Vitro

The DPPH· scavenging capacity, ·OH scavenging capacity and total reducing capacity of ZS-P1-1 all increased in a dose-dependent manner within 0.1–0.5 mg/mL, even though ZS-P1-1 had a decreased antioxidant capacity compared with Vc at the same concentration (Figure 5). At 0.5 mg/mL, the DPPH· and ·OH scavenging and total reducing capacities reached 30.49% ± 1.82%, 28.51% ± 1.40% and 0.64 ± 0.07, respectively (Figure 5). The results demonstrate that ZS-P exhibited potent DPPH· and ·OH scavenging effects, as well as total reducing capacity, which increased linearly with concentration, suggesting that ZS-P1-1 possesses excellent antioxidant properties in vitro.

3.5. Antioxidant Activity of ZS-P1-1 In Vivo

Compared to the CG, the T-SOD activity (33.88 ± 13.21 vs. 95.06 ± 12.20 U/mL, p < 0.001) and GSH content (0.19 ± 0.09 vs. 1.26 ± 0.48 μmol/L, p < 0.001) markedly declined, whereas the serum MDA level in the MG (45.62 ± 6.23 vs. 12.66 ± 1.90 nmol/mL, p < 0.001) significantly increased (Figure 6a–c). However, ZS-P1-1 significantly increased T-SOD activity (0.64 ± 0.12 vs. 0.19 ± 0.09 U/mL, p < 0.01) and GSH content (0.64 ± 0.12 vs. 0.19 ± 0.09 μmol/L, p < 0.01) and reduced MDA content (29.62 ± 4.73 vs. 45.62 ± 6.23 nmol/mL, p < 0.01) (Figure 6a–c) relative to the MG. T-SOD has the ability to clear superoxide anions and inhibit free radicals, which are important indicators of antioxidant capacity [28]. A reduction in GSH levels enhances intracellular reactive oxygen species (ROS) generation [29]. MDA is the metabolic product of oxygen free radical damage and lipid peroxidation, and serum MDA levels can reflect the metabolic status of free radicals [30]. Moreover, the liver and kidney organ coefficients of the MG were reduced compared to the mice in the NG, and ZS-P1-1 increased mouse liver and kidney organ indexes, although it did not reach a significant difference (Figure 6d,e). Histopathological analysis revealed that ZS-P1-1 alleviated abnormal lipid metabolism in liver cells, reduced lipid accumulation, decreased the number and volume of lipid vacuoles, and reduced the number of inflammatory cells (Figure 6f). Moreover, ZS-P1-1 reduced glomerular volume and mesangial cell proliferation (Figure 6f). The results indicate that ZS-P1-1 exert antioxidant activity in vivo, indicating its potential as a promising natural antioxidant.

4. Discussion

The extraction methods of polysaccharides are crucial factors that affect their yield, physicochemical properties, and structural characteristics, which in turn largely influence their biological activities [31]. Therefore, selecting a suitable extraction technique is important for achieving better production and keeping integral polysaccharide structures and functions in practical applications [32]. Traditional extraction methods, including hot-water extraction, despite simple operation and minimal equipment requirements, usually present several limitations, including high operating temperatures, long extraction times, and low extraction efficiency [33]. This highlights the need for developing efficient extraction methods that can achieve both high yield and preserved bioactivity. The ultrasonic-assisted extraction of polysaccharides has remarkable advantages in enhancing extraction efficiency, reducing processing time, and lowering solvent consumption [34]. Moreover, a combination of multiple extraction methods is significantly more efficient than a single method in enhancing extraction efficiency while minimizing the degradation of bioactive constituents [35]. Li et al. found that the extraction rate of ultrasound-assisted enzymatic method to isolate Tremella fuciformis polysaccharides was higher than that of ultrasound or enzyme extraction methods individually [36]. The current work adopted an ultrasonic-assisted hot water technique for extracting Z. striolatum polysaccharides while optimizing extraction conditions by the BBD of RSM. Notably, those optimized extraction conditions included ultrasonic power of 200 W, extraction duration of 2.5 h, extraction temperature of 79 °C, and ultrasonic time of 20 min, and the extraction yield reached 19.96% ± 0.18% under these conditions. However, the difference between ultrasound-assisted hot water extraction of Z. striolatum polysaccharides and the individual ultrasound or hot water methods still need to be elucidated.

Naturally derived polysaccharides have shown promising antioxidant potential with high safety and minimal side effects. Heteropolysaccharides isolated from the brown algae Cystoseira amentacea had potent antioxidant capacity, with IC50 values of DPPH· scavenging and iron reduction capacities of 446.00 ± 1.41 and 1142.33 ± 1.44 μg/mL, respectively [37]. Moreover, Momordica charantia polysaccharides dramatically increase SOD and catalase contents, and partially decrease MDA levels within serum, spleen and liver of mice [38]. The polysaccharide isolated from Laowuzeng Jiuzao exhibited strong DPPH·, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), hydroxyl radical/super-oxide anion scavenging capacities dose-dependently, and protected from H₂O₂-mediated ROS production, efficiently preventing cell death of zebrafish [39]. Similarly, in this study, ZSP-1-1 presented potent DPPH· and ·OH scavenging abilities, as well as a high total reducing capacity. Moreover, ZS-P1-1 also improved T-SOD activity, GSH and MDA levels, and alleviated the tissues damages caused by peroxidation stress in mice. The above findings lay the theoretical foundation for developing functional foods, natural medicines, and antioxidants of polysaccharides from Z. striolatum.

Despite these promising results, several limitations remain. First, more technologies are needed to extract Z. striolatum polysaccharides and compare their advantages and disadvantages with the ultrasound-assisted hot water method, providing a theoretical basis for screening more suitable extraction methods for polysaccharides in the future. Second, the molecular weight, monosaccharide composition and functional groups significantly affect polysaccharides’ bioactivities. However, this was only a preliminary study on the structure of ZS-P1-1; hence, it is necessary to adopt more research methods, such as methylation analysis and nuclear magnetic resonance, are needed to determine the specific chain linkage structure information of ZS-P1-1 in the future. Moreover, the structure–activity relationship that affects the antioxidant activity of ZS-P1-1 has not been fully elucidated. Lastly, although the antioxidant activity of ZS-P1-1 was validated using in vitro and in vivo methods, it is still necessary to explore the key signaling pathway of ZS-P1-1 to explain the specific antioxidant regulatory mechanism of ZS-P1-1.

5. Conclusions

The present work adopted RSM for optimizing conditions for extracting polysaccharides from Z. striolatum using the ultrasonic-assisted hot water method. By analyzing impacts of extraction duration, extraction temperature, ultrasound time, and ultrasound power on ZS-P production, extraction conditions were optimized as follows: ultrasonic power of 200 W, extraction duration of 2.5 h, extraction temperature of 79 °C, and ultrasonic time of 20 min, and extraction yield reached 19.96% ± 0.18% under these conditions. At 0.5 mg/mL, DPPH and hydroxyl radical scavenging capacities and total reducing power reached 30.49% ± 1.82%, 28.51% ± 1.40% and 0.64 ± 0.07, respectively. After separation and purification with DEAE-52 and Sephadex G-100 columns, the uniform polysaccharide ZS-P1-1, whose Mw was 5.63 × 10⁴ kDa, was obtained. It mostly comprised Gal, GalA and GlcA at a 0.453:0.162:0.114 molar ratio. ZS-P1-1 also exerted an antioxidant effect by improving T-SOD activity, GSH, and MDA levels and alleviating the liver and kidney damage caused by peroxidation stress. The present work lays a theoretical foundation and reference for developing functional foods, natural medicines and antioxidants of polysaccharides from Z. striolatum.