Structural characteristics of Brassica rapa L. Polysaccharide and its bioactivity on alcoholic liver injury

Graphical abstract

Abstract

The global prevalence of alcoholic liver disease (ALD) has shown a concerning upward trend, with an increasing incidence among younger demographics. In this study, ultrasound-assisted extraction was employed to isolate polysaccharides from Brassica rapa L (BRL). The purified polysaccharide (BRLPP) was structurally characterized, and its protective effects against alcoholic liver disease (ALD) were investigated. The optimal conditions, material-to-liquid ratio of 1:30 and 200 W ultrasonication for 3 h, yielded 6.90 % ± 0.39 % BRLP. Subsequent structural analysis revealed that BRLPP had an average molecular weight of 5.72 × 10⁴ Da and was primarily composed of glucose and arabinose in a molar ratio of 94.29:3.06. GC–MS analysis further identified three major glycosidic linkage patterns: →4)-Glcp-(1→, →4,6)-Glcp-(1→, and → 3,4)-Glcp-(1 → . Strong antioxidant activity was displayed by BRLPP, which showed notable scavenging effects on DPPH and ABTS⁺ radicals. Using 2 % ethanol exposure, a zebrafish ALD model was created to assess its hepatoprotective potential. The results indicated that BRLPP effectively attenuated ethanol-induced liver damage by reducing serum ALT (Alanine aminotransferase) and AST (Aspartate transaminase) levels. Additionally, BRLPP mitigated oxidative stress by modulating the activities of key biomarkers, including SOD (Superoxide dismutase), MDA (Malondialdehyde), and GSH-Px (Glutathione peroxidase). These results point to BRLPP’s potential as a bioactive component in functional foods for the prevention and treatment of ALD by indicating that it has noteworthy hepatoprotective and antioxidant qualities.

1. Introduction

Brassica rapa L. (BRL), commonly called Chimagu in China, has been deeply loved by the people of Xinjiang province for its distinctive flavor and nutritional properties, and it was known as “longevity holy fruit” and “natural lung treasure” [1]. In the Tibetan medicine classic Four Medical Classics, BRL was highly esteemed as a premier tonic with purported benefits for regulating body heat and qi circulation. In addition to being rich in carbohydrates, proteins, fats, minerals and vitamins, various classes of secondary metabolites have been previously identified from BRL, such as polysaccharides, flavonoids, and glucosinolates [[2], [3], [4]]. These bioactive compounds contribute to its broad spectrum of bioactivities, exhibiting anti-inflammatory, antioxidant, nephroprotective, antitumor, and hepatoprotective effects [[5], [6], [7], [8], [9]].

The liver serves as the body's primary metabolic organ, playing essential roles in human physiology. The pathological definition of alcoholic liver disease (ALD) is a range of progressive hepatic lesions caused by the intracellular accumulation of harmful metabolites derived from ethanol and the dysregulation of endogenous substance homeostasis after prolonged excessive alcohol consumption [10]. According to new research, alcohol and its metabolites prevent the production of hepatic proteins through immune-mediated, oxidative stress, and apoptosis-promoting pathways. Increased production of oxygen-free radicals results in hepatocyte necrosis and degeneration, which in turn causes liver damage [11]. Among them, oxidative stress mediated by alcohol and increased oxygen free radicals are the main causes of liver damage. Therefore, the key to avoiding and treating alcoholic liver disease is to reduce the oxidative stress injury caused by alcohol through antioxidant mechanisms.

The hot water extraction method is predominantly employed among polysaccharide extraction techniques due to its operational simplicity and minimal requirement for sophisticated equipment. However, polysaccharide breakdown put on by an excessively long extraction period may lower the yield. In contrast, the ultrasound-assisted extraction method is characterized by the cavitation-induced physical forces and hydrodynamic shear stress that destroy the cell wall and promote the release, dissolution, and diffusion of intracellular substances. It has been demonstrated to exhibit the advantages of short extraction time and fast extraction rate [[12], [13], [14]].

Zebrafish offer major advantages as experimental animals. Zebrafish are a small and fast-growing species, with early development completed in 24 h and liver maturity reached in 72 h. The zebrafish model system provides unparalleled experimental scalability through its exceptionally high fecundity and abbreviated reproductive cycle. Embryonic and larval stages exhibit complete optical transparency, making it possible to directly see the impact of medications on internal organs and embryo development under a microscope [15]. Meanwhile, the zebrafish model has been thoroughly verified in translation pathophysiology studies due to its 87 % genetic similarity to humans [16], signaling pathways that are essentially identical to those of humans, and biological structure and physiological functions that are highly similar to those of mammals, including organs like the liver and kidneys [17].

Previous studies have shown that various natural plant polysaccharides, such as Trametes orientalis polysaccharides [18], Polygala fallax Hemsl polysaccharides [19] and Ganoderma lucidum polysaccharides [20], exhibit a significant protective effect against alcoholic liver injury. These polysaccharides have the potential to mitigate the adverse effects that alcohol can inflict on the liver. Furthermore, anti-oxidation, lipid accumulation reduction, anti-inflammatory, and other processes can be used to support liver regeneration and repair [[21], [22], [23], [24]].

In this study, response surface methodology was used to optimize the polysaccharide extraction process of BRL polysaccharide, which led to a notable increase in polysaccharide production. Secondly, the novel polysaccharide BRLPP isolated from BRL was found to be a new polysaccharide by structural characterization. Concurrently, the zebrafish model was employed to demonstrate the hepatoprotective efficacy of turnip polysaccharides. The findings indicated that BRLPP manifested its hepatoprotective effect through the antioxidant mechanisms of MDA, SOD, and GSH-Px. This discovery provides a novel perspective for developing antioxidant-based therapies for alcoholic liver disease (ALD).

2. Materials and methods

2.1. Material and chemical reagents

BRL was purchased in Xinjiang Province, Cellulose DEAE-52 Cellulose column and Sephadex G-100 column were obtained from Shanghai Yuanye Biotechnology Co. (Shanghai, China). The monosaccharides (rhamnose, arabinose, xylose, mannose, glucose, galactose, and galacturonic acid) were purchased from Beijing Soleberg Technology Co. (Beijing, China). DPPH, ABTS⁺, •OH were purchased from Beijing YuanYe Technology Co. (Beijing, China). Kits for detecting ALT (Alanine aminotransferase), AST (Aspartate transaminase), MDA, SOD and GSH-Px were purchased from Nanjing Jiancheng Biotechnology Research Institute. (Nanjing, China). All chemicals and reagents were analytical grade.

2.2. Single factor experiments and optimized experimental design

The liquid–solid ratio, ultrasonic power, temperature, and extraction time were investigated. The liquid–solid ratio was set at 1:10, 1:20, 1:30, 1:40 and 1:50 g/mL. The experimental conditions for ultrasound power covered 100 W, 150 W, 200 W, 250 W, and 300 W, while ultrasound time was varied at 1 h, 2 h, 3 h, 4 h, and 5 h. The temperature range was 30 °C, 40 °C, 50 °C, 60 °C, and 70 °C. By applying response surface methodology (RSM) and Box-Behnken design (BBD), the extraction parameters were optimized by identifying the factors influencing the process.

2.3. Purification and content determination

100 g of BRL freeze-dried powder was weighed and added to water according to the material-liquid ratio of 1:30 (g/mL). The ultrasonic power was 200 W, the temperature was 60 °C, and the ultrasonic time was set at 3 h, then cooled to room temperature. After cooling to room temperature, centrifugation was carried out at 8,000 r/min for 15 min. The supernatant was collected and concentrated, and precipitated by adding 4 volumes of anhydrous ethanol. To separate the precipitate, the mixture was centrifuged once again after being chilled at 4 °C overnight. The Sevag method was used to extract the proteins from the polysaccharide and re-solubilize the precipitate [25]. The polysaccharide solution was subjected to 72 h (10,000 Da) dialysis to eliminate low-molecular-weight compounds. After lyophilization, the resulting product named as BRLP was weighed to determine the yield.

The BRLP was dissolved and underwent the first purification by using DEAE-52 Cellulose column. They were eluted with a concentration gradient of 0–0.5 mol/L NaCl. The collected fractions were assayed for their content by phenol sulphuric acid method [26]. Because the content of the water − washed fractions was higher, the polysaccharide fractions eluted with water were named as BRLP-1, and the yield of BRLP-1 was calculated by freeze-drying after removing NaCl by dialysis (500 Da). The Sephadex G-100 column was used to further isolate and purify BRLP-1. The major fraction, known as Brassica rapa L. pure polysaccharides (BRLPP), was collected, and the yield was determined by freeze-drying BRLPP.

2.4. Molecular weight (Mw) determination

The molecular weight distribution of BRLPP was detected by chromatography equipped with a multi-angle laser light scattering instrument and a refractive index detector (HPSEC-MALLS-RI, Wyatt Company, USA) [27]. A Shodex OHpak SB-806 M HQ column was used, with 0.1 M NaCL as the mobile phase. The column was heated to 35 °C, the injection volume was 100 µL, and the flow rate was 0.5 mL/min. 10 mg of BRLPP polysaccharides was weighed and dissolved in 10 mL of 0.1 M NaCl, filtered through an aqueous membrane of 0.45 µm and transferred to an injection vial for analysis.

2.5. Monosaccharide composition analysis

Ion chromatography (IC) was used to determine the composition of monosaccharides. A 10 mg portion of the polysaccharide was accurately measured to 4 mL of 4 mol/L Trifluoroacetic acid (TFA), the solution was filled with N₂, and hydrolysed was subjected to hydrolysis at 121°C for 2 h. After being hydrolyzed, the product was dried and redissolved in methanol. The material was fixed to 10 mL of sterile water following three iterations of this procedure. Following a 20-fold dilution, the sample was moved to an injection vial for examination after passing through a 0.22 μm membrane. Chromatographic separation was achieved under the following conditions: CarboPacTMPA20 3 × 150 mm column; eluent: ultrapure water, 250 mM NaOH and 1 M NaAC; detector: a pulsed amperometric detection system employing a gold working electrode; flow rate of 0.5 mL/min; injection volume: 10 µL; column temperature: 35 °C. The mobile phase consisted of water (A), 250 mM NaOH (B) and 1 M NaAC (C), and the column temperature was fixed at 35 °C. The injection volume was 10 μL and the flow rate was 0.5 mL/min. The mobile phase gradients were as follows: 0 min (97 % A, 3 % B), 20 min (92 % A, 3 % B), 35 min (77 % A, 3 % B), 35.1 min (20 % A, 80 % B), 45 min (20 % A, 80 % B), 45.1 min (97 % A, 3 % B) and 55 min (97 % A, 3 % B).

2.6. Fourier transform infrared spectrum (FT-IR) and UV analysis

The BRLPP and KBr powders were carefully mixed and ground, and then pressed into 1 mm thick transparent sheets, which were scanned 60 times within the wavenumber range of 4,000 cm⁻¹ to 400 cm⁻¹. The purpose of this scanning was to observe the characteristic absorption peaks of the infrared spectra, allowing for the determination of the types of functional groups present in the polysaccharides.

The BRLPP solution with a concentration of 0.1 mg/mL was analyzed by UV visible spectrophotometer (Shimadzu, Japan) within the wavelength range of 200–800 nm to detect whether BRLPP exhibited the characteristic absorption peaks of nucleic acids and proteins.

2.7. Methylation and GC–MS analysis

1 mL of DMSO was mixed with 5 mg of the weighed BRLPP sample and powdered NaOH. After sterilizing the mixture to dissolve it, CH₃I was added for a reaction, and the process was repeated until the O-H absorption vanished from the FT-IR spectrum (3,700–3,200 cm⁻¹). The methylated polysaccharide was taken and hydrolyzed by adding 1 mL TFA (2 M) at 120°C for 90 min. Then double-distilled water (2 mL) was added, 60 mg of NaBD₄ was added for overnight reduction, neutralized by adding glacial acetic acid, oven dried at 101 °C, then add acetic anhydride (1 mL) for the acetylation reaction, which was carried out at 100 °C for 1 h. Then 3 mL of toluene was added, and the resulting mixture was concentrated and evaporated under reduced pressure. This process was repeated 4–5 times. After adding water and dichloromethane to the product, mixing them, centrifuging them three times, and then collecting and drying the dichloromethane phase for analysis, Thermo Scientific 1,300–7,000 gas chromatography-mass spectrometry (GC–MS) device was employed to evaluate samples of the acetylation product

2.8. Scanning electron microscope (SEM) and atomic force microscope (AFM) analysis

In the SEM analysis, BRLPP was mounted on aluminum stubs and coated with a gold layer. A Hitachi S-3000 N scanning electron microscope was applied to view the cross-sectional microstructures and surface morphologies. A BRLPP solution with a concentration of 10 μg/mL was prepared for AFM analysis. A clean mica sheet was then covered with μL of the solution, which was then allowed to dry. Atomic force microscopy was utilized to scan the surface topology.

A 10 μg/mL BRLPP sample solution was prepared. Subsequently 15 μL was taken and added dropwise to the centre of the mica flake, which was dried naturally at room temperature. Afterward, it was subjected to AFM scanning, and the resulting image data were processed by the AFM accessory software.

2.9. X-ray Diffractometer (XRD) analysis

The BRLPP powder was evenly spread in the grooves of the sample plate. Subsequently, the excess sample was scraped off with a glass plate, and the surface of the grooves was flattened. The sample filled plate was then placed into the instrument for scanning. The 2θ scanning range was set from 5° to 90°, the scanning voltage was set at 40KV, and the scanning current was set at 20 mA.

2.10. Antioxidant activity detection

2.10.1. DPPH free radical scavenging activity

BRLPP aqueous solutions were produced in the following concentrations: 0.25, 0.5, 1.0, 2.0, and 4.0 mg/mL. After that, 1.0 mL of 0.2 M DPPH ethanol solution was added. The reaction was allowed to proceed at 25 °C for 30 min, and the absorbance of the reaction products was measured at 517 nm by means of an ultraviolet visible spectrophotometer. The DPPH free radical scavenging rate of the samples was calculated according to the following formula.

$$\mathrm{Y}(\%)=1-\frac{{\mathrm{A}}_1-{\mathrm{A}}_2}{{\mathrm{A}}_0}$$

Y was the radical-scavenging rate of DPPH of the sample, A₁ refers to the absorbance value of the sample, A₂ was defined as the absorbance value of the control solution and A₀ refers to the absorbance value of the blank solution.

2.10.2. ABTS⁺ free radical scavenging activity

The 7.0 mmol/L ABTS⁺ solution was combined with the 2.45 mmol/L K₂S₂O₈ solution in a volume ratio of 1:1. After 12 h of protect form light, the mixture was diluted and set aside. Subsequently, 400 μL of BRLPP solution was incorporated into 4 mL of ABTS⁺ solution. The reaction was allowed to proceed for 6 min, after which the absorbance was measured at 734 nm. The formula for calculating the ABTS⁺ radical − scavenging rate was as follows.

$$\mathrm{Y}(\%)=\frac{{\mathrm{A}}_1-{\mathrm{A}}_0}{{\mathrm{A}}_1}$$

Y indicates the clear ability of ABTS⁺, A₁ was the absorbance value of the blank solution and A₀ was the absorbance value of the sample.

2.10.3. •OH free radical scavenging activity

The Fenton reaction was utilized to generate •OH radicals [28]. The reactions were conducted in 0.2 M phosphate buffer (pH 7.4), in which 7.5 mM FeSO₄ was mixed with a 0.1 % aqueous solution of H₂O₂. The resultant mixture was incubated at 37°C in the dark for 1 h. After preparing polysaccharide samples in varying quantities, the polysaccharide solution described above was combined with a solution of 6 mM salicylic acid. An ultraviolet visible spectrophotometer (Perkin Elmer, USA) was used to test the product^’s absorbance at 5 min reaction. An equivalent volume of ultrapure water was employed to replace H₂O₂ in the blank sample. The formula for calculating the •OH radical − scavenging rate was as follows.

$$\frac{A-A1}{A0-A1}\times 100\%$$

Y represents the clear ability of •OH, A₀ represents the absorbance value for undamaged samples, A represents the absorbance value of the sample and A₁ represents the absorbance value of damaged samples.

2.11. Liver-protective effect of BRLPP

Wild type AB line zebrafish were sourced from the China Zebrafish Resource Centre. These zebrafish were propagated through natural pairing. Briefly, the zebrafish were reared under standard light/dark (14 h/10 h) conditions at a temperature of 28 ± 0.5 °C. Male and female zebrafish, in a ratio of 1:1, were placed in a breeding tank overnight. Embryos were obtained the next morning. After being cleaned and disinfected, these embryos were moved to an incubator for zebrafish embryo culture that was kept at 28 ± 0.5 °C. The following tests were performed using embryos that were three days post-fertilization.

Zebrafish at 3 days post-fertilization (3 dpf) were immersed in a 2 % alcohol solution for 32 h with daily water changes to establish a model of ALD. ALD fish were randomly divided into 5 groups (n = 30 each): the model group, the metadoxine group (149 μg/mL), and the BRLPP groups (125 μg/mL, 250 μg/mL, 500 μg/mL). The zebrafish in each group were cultured for 24 h [29].

2.11.1. The ameliorative effect of BRLPP on delayed yolk sac resorption

The ameliorative effect of BRLPP on delayed resorption of the yolk sac was examined using the Image J software on five randomly selected zebrafish that were photographed under a microscope. The calculation formula was as follows:

$$\frac{S1-S}{S1-S0}\times 100\%$$

Y was the ameliorative of yolk sac area, S₁ was the yolk sac area in the model group, S was the yolk sac area in the BRLPP group, S₀ was the yolk sac area in the control group.

2.11.2. Oil red O staining

Oil Red O is a strong lipid stainer that preferentially binds to lipids in tissues, thereby producing an orange-red coloration. Thus, the Oil Red O staining method can be utilized to detect fat buildup in liver tissue. The saturated Oil red O staining solution was diluted with purified water, allowed to stand at room temperature for 10 min, while being shielded from light, and then filtered prior to use. Zebrafish were placed into 2 mL centrifuge tubes, rinsed with phosphate − buffered saline (PBS), and then fixed in a refrigerator at 4 °C for 12 h with 4 % paraformaldehyde. Following a 30 min immersion in 60 % isopropanol and PBS wash, the zebrafish were stained with Oil red O in a dark setting. Finally, the background color of zebrafish was removed with 60 % isopropanol, and the magnification of the microscope as well as the exposure conditions were set, and the intensity of lipid droplets of zebrafish liver was observed under a stereomicroscope. Image J software was used to quantify the extent of Oil red O staining of the zebrafish liver.

2.11.3. Biochemical analyses of zebrafish

Zebrafish larvae (n = 30) were rinsed with PBS three times. After that, the larvae were homogenized and PBS was added. The mixture was then centrifuged for 10 min at 4 °C and 3,000 rpm to extract the liquid supernatant. The levels of ALT, AST, SOD, MDA, GSH-Px were determined by Reagent Kit (Nanjing Jiancheng, Nanjing, China), respectively, using a microplate reader in accordance with the kit instructions. Each sample was analyzed in triplicate.

2.12. Data statistics and analysis

Design Expert was applied to design the response surface optimization experiment for BRL extraction, conduct data analysis, and generate response surface diagrams. SPSS software was employed to perform multiple comparisons through one-way ANOVA, and p < 0.05 presented a significant difference. Microsoft Excel 2016 was used for basic data processing, and Origin 2021 was utilized for charting. All experiments were repeated three times and the data were expressed as mean ± standard deviation (SD).

3. Results and discussion

3.1. Single-factor extracting experiments of BRLP

The influence of liquid–solid ratio on polysaccharide yield was depicted in Fig. 1A. The yield gradually rose along with the liquid–solid ratio. The polysaccharide yield peaked at 6.26 % when the material-liquid ratio reached 1:30. This was because, as the material-liquid ratio increased gradually, the concentration of polysaccharides outside the cell decreased, which led to the establishment of a specific osmotic pressure both inside and outside the cell. Elevated osmotic pressure promoted enhanced polysaccharide exudation from the cells, which led to a progressive increase in yield. The yield tended to decrease as the material-liquid ratio increased because the extract became supersaturated, which limited the pace at which polysaccharides could move between the solutions [30].

Fig. 1.

The yield of BRLP extracted from different single factor experiments, solid–liquid ratio (A); extraction power (B); extraction temperature (C); extraction time (D). (Different letters represent significance between groups p > 0.05).

The influence of ultrasonic power on the polysaccharide yield was depicted in Fig. 1B. The yield peaked at 5.6 % when the ultrasonic power reached 250 W. Subsequently, as the ultrasonic power gradually increased, the polysaccharide yield demonstrated a gradual decreasing tendency. This was attributed to the fact that the ultrasonic process generates cavitation, which can disrupt the cell wall structure, thereby accelerating the dissolution of polysaccharides within the cells. However, excessive ultrasonic power may degrade polysaccharide structures, consequently compromising their biological activity. Additionally, the ultrasonic process exerts a potent mechanical cutting effect, which can cause the decomposition of polysaccharide components, resulting in a reduction in the yield [31].

Fig. 1C demonstrated a positive correlation between extraction temperature and polysaccharide yield, with progressively higher yields observed at elevated temperatures. Although significant differences were present among different temperatures, the overall difference was small. A portion of the acoustic energy was transformed into thermal energy under ultrasonic irradiation, resulting in a temperature rise within the system and a marginal enhancement in polysaccharide yield. Therefore, 60 °C was chosen as the ideal temperature for the ensuing extraction tests from the standpoint of energy conservation.

As shown in Fig. 1D, the polysaccharide yield was highly dependent on extraction time. Insufficient extraction time would prevent the polysaccharides in the material from being fully solubilized, leading to an interruption of the reaction. Conversely, low extraction efficiency and energy waste would result from an excessive extraction time. The yield of polysaccharides would typically decrease as the extraction time increased. This was probably because the prolonged ultrasonic treatment reduced the cavitation effect. Consequently, the adsorption capacity of the raw materials gradually enhanced, causing more polysaccharides to be adsorbed and thus leading to a decrease in the polysaccharide yield.

3.2. Response surface optimization for BRLP extraction

Three factors that had a greater impact on the yield were chosen for the design of a three-factor, three-level RSM experiment based on the findings of the single-factor experiment. The levels of the experimental factors were presented in Table 1. The polysaccharide yields obtained under different conditions were summarized in Table 2. Employing Design-Expert 11 software, the regression equation was derived as follows: Y = 7.06 + 0.12A + 0.15B + 0.19C + 0.22AB + 0.11AC + 0.13BC − 0.58A² − 0.565B² − 0.550C². The response surface analysis results were presented in Table 3.

Table 1.

Levels and factors of Box-Behnken test.

Factor	A: Liquid-solid (g/mL)	B: Ultrasonic power (W)	C: Extraction time (h)
−1	1:20	150	2
0	1:30	200	3
1	1:40	250	4

Table 2.

Experimental results of response surface.

Run	A: Liquid-solid (g/mL)	B: Ultrasonic power (W)	C: Extraction time (h)	Yield (%)
1	1:20	150	3	5.81
2	1:40	150	3	5.71
3	1:20	250	3	5.68
4	1:40	250	3	6.46
5	1:20	200	2	5.69
6	1:40	200	2	5.61
7	1:20	200	4	6.03
8	1:40	200	4	6.39
9	1:30	150	2	5.83
10	1:30	250	2	5.86
11	1:30	150	4	5.77
12	1:30	250	4	6.32
13	1:30	200	3	7.23
14	1:30	200	3	6.97
15	1:30	200	3	7.05
16	1:30	200	3	7.04
17	1:30	200	3	7.01

Table 3.

Results of regression statistical analysis.

Source	Sum of Squares	df	Mean Square	F-value	P-value	Significance
Model	5.40	9	0.6002	33.61	<0.0001	**
A	0.1152	1	0.1152	6.45	0.0387	*
B	0.1800	1	0.1800	10.08	0.0156	*
C	0.2888	1	0.2888	16.17	0.0050	**
AB	0.1936	1	0.1936	10.84	0.0133	*
AC	0.0484	1	0.0484	2.71	0.1437
BC	0.0676	1	0.0676	3.79	0.0928
A2	1.42	1	1.42	79.32	< 0.0001	**
B2	1.34	1	1.34	75.27	< 0.0001	**
C2	1.27	1	1.27	71.33	< 0.0001	**
Residual	0.1250	7	0.0179
Lack of Fit	0.0850	3	0.0283	2.83	0.1701
Pure Error	0.0400	4	0.0100
Cor Tatal	5.53	16

** indicates extremely significant difference (P < 0.01), * indicates significant difference (P < 0.05)

The model exhibited high predictive accuracy, with a predicted R² of 0.9774 and adjusted R2_adj of 0.9483 (Table 3), indicating reliable estimation of polysaccharide yield. The model was determined to be highly significant (p < 0.0001), while the lack of fit term was not significant, further corroborating the adequacy of the model. In conclusion, the proposed approach demonstrated both reliability and precision in predicting polysaccharide production across a range of conditions. The trend of the polysaccharide yield under the influence of two variables was illustrated in Fig. 2, which aligns with the pattern observed in the single-factor experiment results: an initial increase followed by a decline as the factor levels were elevated.

Fig. 2.

Response surface experiment results. 3D response surface (A, C and E) and contour plots (B, D and F) of liquid–solid ratio, extraction time and ultrasonic power and interaction on yield of water-soluble BRLP

The optimal extraction conditions were determined to be a material-liquid ratio of 1:31.6 (g/mL), a power of 209.4 W, and an extraction time of 3.2 h, resulting in a polysaccharide yield of 7.10 %. The liquid–solid ratio of 1:30 (g/mL) was selected for the laboratory extraction process, which took out for 3 h at 200 W. Subsequent analysis revealed that the polysaccharide yield was 6.90 ± 0.35 %, as compared with the theoretical value of 7.10 %. The observed error was 0.20 ± 0.35 %, indicating a close concordance between the experimental and theoretical values. These findings validated the model's predictive accuracy and reliability for polysaccharide yield.

3.3. Extraction and purification of BRLP

Deproteinization of the crude polysaccharide demonstrated that most of the protein content had been effectively eliminated. The 0–0.5 mol/L NaCl gradient was then employed to elute the crude polysaccharide after it had undergone hand chromatography on DEAE-52 Cellulose column. The elution fractions were analyzed by the phenol–sulfuric acid method, as illustrated in Fig. 3A and 3B. Water elution produced BRLP-1, which had a yield of 32.5 ± 1.54 % and a purity of 79.4 ± 4.38 %. Following further purification via Sephadex G-100 column chromatography, the yield of BRLPP was determined to be 68.5 ± 3.74 %, and its purity was enhanced to 92.50 ± 2.48 %.

Fig. 3.

Isolation and structural characterization of BRLPP. Elution curve of BRLP with DEAE-52 (A) Cellulose column, Sephadex G-100 column (B), Molecular weight measurement by an eighteen − angle laser instrument (C), UV–vis scanning spectrum (D), Monosaccharide composition analysis (E), FT-IR analysis (F) of BRLPP.

3.4. Molecular weight analysis

Extensive research has established a direct correlation between polysaccharide molecular weight and their corresponding biological activities. Since light scattering is considered to be a highly sensitive technique for detecting absolute Mw, it was frequently utilized for characterizing biological macromolecules. This allows it to address the restrictions associated with column calibration [32]. The HPSEC-MALLS-RI chromatogram of BRLPP was depicted in Fig. 3C, which reveals a single symmetrical peak in the refractive index (RI) signal. The molecular weight (Mw) of BRLPP was determined to be 5.72 × 10⁴ Da.

3.5. Monosaccharide composition

The results of the ion chromatographic analysis of the monosaccharide composition of the polysaccharide were presented in Fig. 3E. Through comparing the retention times of the peaks and analyzing the peak areas of the chromatograms, it was ascertained that BRLPP was composed of fucose, arabinose, galactose, glucose, xylose, and galacturonic acid, with molar ratios of 0.1: 3.06: 1.14: 94.29: 1.30: 0.1, respectively. The results demonstrate that BRLPP primarily consists of glucose and arabinose, while other monosaccharides were present in trace amounts and cannot be accurately quantified. These findings are largely consistent with the results reported by Chen et al. [33].

3.6. FT-IR and UV analysis

FT-IR is a widely used method for the preliminary characterization of polysaccharides. The FT-IR spectrum of BRLPP was depicted in Fig. 3F. The strong absorption peaks observed at 3,384.04 cm⁻¹ and 2,929.28 cm⁻¹ can be ascribed to the stretching vibrations of –OH groups and C-H bonds in –CH₂ groups, respectively. Asymmetric stretching vibrations of C O bonds and symmetric stretching vibrations were represented by additional absorption peaks at 1,649.06 cm⁻¹ and 1,369.5 cm⁻¹, respectively. Furthermore, the stretching vibration of the C-O ether bond in the pyranose glycan ring was linked to the absorption peak at 1,156.08 cm⁻¹ [34]. The absorption peak observed near 1,081.79 cm⁻¹ suggests that BRLPP contains a pyran type glycan ring [35]. The absorption band at 931.76 cm⁻¹ was characteristic of the furan ring structure [36]. The α-type glycosidic bond had an absorption peak at 850.93 cm⁻¹ [37]. Pyranose^’s distinctive absorption peak is located at 761.59 cm⁻¹ [38]. The infrared spectra showed that BRLPP polysaccharides contained α-glycosidic bonds, and the characteristic absorption peaks of acidic sugars did not appear, indicating that BRLPP is a neutral polysaccharide.

Ultraviolet (UV) detection is a critical method for identifying the presence of proteins and nucleic acids in samples. There were no absorption peaks at 260 or 280 nm in the BRLPP sample’s UV spectra (Fig. 3D), suggesting that proteins and nucleic acids were not present.

3.7. Methylation and GC–MS analysis

Methylation analysis was employed to determine the glycosidic linkage patterns. Following a series of methylation, hydrolysis, reduction, and acetylation, BRLPP was converted into partly methylated alcohol acetates (PMAAs), which were subsequently subjected to GC–MS analysis. As depicted in Fig. 4A, the disappearance of the absorption peak near 3,389 cm⁻¹ demonstrated that BRLPP had been completely methylated.

Fig. 4.

Structural characterization of BRLPP. FI-IR (A) and Total methylation ion flow diagram (B), SEM (C: 500×, D: 2000 × ), and AFM (E, F) of BRLPP.

The PMAAs obtained from GC–MS were identified by comparing their retention times and characteristic fragments with those reported in the literature. The sugar residues of BRLPP were tabulated in Table 4, and their corresponding mass spectra were illustrated in Fig. 4B.

Table 4.

Linkage analysis of BRLPP by methylation and GC–MS.

RT	Methylated sugar	Mass fragments (m/z)	Molar ratio	Type of linkage
16.238	2,3,5-Me3-Araf	43,71,87,101,117,129,143,161	0.022	Araf-(1→
27.216	2,3,4,6-Me4-Glcp	43,71,87,101,117,129,143,161,205	0.076	Glcp-(1→
29.866	2,3-Me2-Araf	43,71,87,99,101,117,129,161,189	0.034	→5)-Araf-(1→
41.521	2,3,6-Me3-Glcp	43,87,99,101,113,117,129,131,161,173,233	0.632	→4)-Glcp-(1→
49.259	2,6-Me2-Glcp	43,87,97,117,129,149	0.102	→3,4)-Glcp-(1→
54.248	2,3-Me2-Glcp	43,71,85,87,99,101,117,127,159,161,201,261	0.134	→4,6)-Glcp-(1→

3.8. The nuclear magnetic resonance (NMR)

NMR technique is an indispensable and powerful tool for the structural analysis of polysaccharides, due to the simplicity of the preliminary processing and its ability to accurately attribute glycosidic bonding types. The 1D NMR results of BRLPP were presented in Fig. S1 and S2. The positions of the anomeric carbons and anomeric hydrogens are labeled A-G. The ¹H spectra exhibit chemical shifts greater than 5.0 ppm, indicating that the anomeric proton of the polysaccharide adopts an α-configuration. The anomeric proton of a polysaccharide adopted a β-configuration when its chemical shift was below 5.0 ppm. As demonstrated in Fig. S1, the chemical shifts of the anomeric protons of BRLPP were observed to range from 4.46 to 4.82 ppm and from 5.04 to 5.27 ppm [39]. However, due to the limited resolution of 1D NMR and signal interference from residual water peaks, the anomeric proton signals exhibited partial overlap, complicating their assignment to specific hydrogen or carbon atoms. The ¹³C NMR spectra (Fig. S2) revealed characteristic chemical shifts for the anomeric carbons: α-configured sugar residues typically appear between 90–102 ppm, whereas β-configured anomeric carbons resonate downfield at 102–110 ppm [40,41]. The 1D NMR spectra thus confirm the coexistence of both α- and β-glycosidic linkages in BRLPP.

3.9. SEM and AFM analysis

Polysaccharides possess a complex spatial structure owing to their diverse monosaccharide compositions and linkages. Consequently, the microstructures of polysaccharides also vary. Therefore, SEM may be used to observe the size, micro-morphology, and porosity of polysaccharides. Fig. 4C, D depicts the scanning results of BRLPP under 500× and 2,000× magnifications. The results indicated that the polysaccharide exists in the form of flakes or crumbs, with a more dispersed and poorly –connected structure. At a magnification of 2,000×, it can be observed that the BRLPP polysaccharide has more pore structures.

The AFM sample was prepared in a facile manner that ensures minimal damage and conveniently reveals the most authentic sample morphology. AFM makes it possible to visualize the sample by manipulating the tiny, pointed probes that, through frictional engagement with the sample surface, produce surface contours [42]. From Fig. 4E, F, the average height of BRLPP was 4 nm. Given that this height was significantly lower than the range of 15–50 nm, it can be inferred that BRLPP does not possess a triple − helical structure [43].

3.10. XRD analysis

X-ray diffraction (XRD) is a technique employed for analyzing the crystal structure of a material. Certain X-rays scatter when they pass through a crystal. The X-rays were enhanced in specific directions as a result of this scattering phenomena [44]. These differences in diffraction phenomena can thus be utilized to mirror the diverse structures within the material. The more regular the crystal structure, the stronger and sharper the diffraction peaks emerge. The XRD was presented in Fig. 5A. This result indicated that the absence of a sharp diffraction peak at 2θ = 22°, with only broad features observed near 20°. This pattern suggested that BRLPP existed predominantly in an amorphous or semi-crystalline state.

Fig. 5.

Structural analysis and antioxidant studies of BRLPP. XRD (A), DPPH (B), ABTS⁺ (C), Hydroxyl radical (D). (Different letters represent significance between groups p > 0.05).

3.11. Antioxidant activity

The DPPH scavenging rate was dependent on the polysaccharide concentration in a dose response manner (Fig. 5B). As the BRLPP concentration increases, the scavenging ability also improves. The DPPH scavenging rate can reached 63.47 % ± 3.84 % at a polysaccharide concentration of 4 mg/mL. The ABTS⁺ (Fig. 5C) scavenging rate was measured at 36.44 % ± 1.59 % at a concentration of 2 mg/mL and increased to a maximum of 41.96 % ± 0.21 % with higher polysaccharide content. In contrast, the polysaccharide showed relatively lower efficiency in scavenging the hydroxyl radical (•OH) with a maximum value of only 13.60 % ± 0.99 % (Fig. 5D). Overall, the overall antioxidant effect of the polysaccharides was inferior to that of Vc.

3.12. Liver-protective effect of BRLPP

3.12.1. Effect of BRLPP on delayed yolk sac absorption and outcome of Oil red O

Researchers have investigated the processes of oxidative stress and apoptosis-related hepatotoxicity [45]. The embryonic stage was determined by the condition of the yolk sac, which was an essential source of protein and energy during zebrafish development [46]. As illustrated in Fig. 6A, BRLPP significantly enhanced yolk sac absorption, suggesting its potential role in mitigating developmental delays. In comparison with the normal controls, the juvenile fish treated with 2 % alcohol exhibited liver fat accumulation, spinal curvature, and impaired nutrient absorption from the yolk sacs. These manifestations indicated abnormal liver function. The zebrafish treated with 125 μg/mL of BRLPP demonstrated a significant reduction in the yolk-sac area and an improvement in yolk sac utilization (p < 0.01). When 500 μg/mL of BRLPP was administered, the delayed yolk sac uptake rose by 72.2 %. Moreover, BRLPP was capable of down regulating this abnormal liver function in a dose dependent manner. As shown in Fig. 6B, Oil Red O staining revealed reduced hepatic lipid accumulation in BRLPP-treated zebrafish. The low dosage BRLPP group demonstrated significantly fewer fat deposition (p < 0.001) than the model group, while the high dose group showed even more reduction of steatosis.

Fig. 6.

BRLPP improves delayed absorption of zebrafish (A), driving fat accumulation (B), ALT (C), AST (D) and Oil Red O staining of liver (E) (p < 0.05: *, p < 0.01: **, p < 0.001: ***, p < 0.0001: ****). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

3.12.2. Biochemical analyses of zebrafish

The impact of BRLPP on hepatic function and health status was evaluated through detection of activity changes in AST and ALT, the key enzymes involved in amino acid metabolism, and their levels are indicative of liver damage (Fig. 6C, 6D). These two enzymes were released into the bloodstream in significant quantities when the liver is injured [47]. When compared with the control group, the ALT and AST activities of zebrafish larvae in the model group were elevated to 70.47 ± 14.12 % and 44.65 ± 8.37 %, respectively, reaching significant differences (p < 0.05). The medium − dosage groups of BRLPP were capable of reducing the activities of ALT and AST in zebrafish with alcoholic liver (p < 0.05). By the intervention of the high-dose group, ALT and AST activities in ALD zebrafish were inhibited by 25.97 % (p < 0.001) and 20.95 % (p < 0.05), respectively. This indicated that BRLPP could effectively protect zebrafish larvae, repair liver injury, and reduce the levels of ALT and AST release into the serum.

3.12.3. Effects of BRLPP on liver SOD, MDA, and GSH-Px in zebrafish

Since liver injury induced by alcohol is usually attributed to oxidative damage, mitigating the harm inflicted by oxidative stress is the key to alleviating alcoholic liver injury. Therefore, SOD, MDA, and GSH-Px were examined in zebrafish, and the results were shown in Fig. 7A–C. SOD eliminates superoxide anion free radicals, safeguard the organism from injury, and plays a crucial role in the organism's oxidative–antioxidative balance [48]. MDA serves as an indicator for gauging the extent of damage caused by oxidative stress within the organism. When the organism is subjected to severe oxidative stress, it leads to an increase in the content of MDA [49].

Fig. 7.

Effects of BRLPP on early developmental and hepatic indices in ALD zebrafish. Antioxidant activity of BRLPP in ALD zebrafish. BRLPP effect on zebrafish SOD (A), MDA (B), GSH-Px (C) (p < 0.05: *, p < 0.01: **, p < 0.001: ***, p < 0.0001: ****).

SOD and GSH-Px levels were considerably lower in the model group than in the control group, whereas MDA levels were greater. The high-concentration BRLPP decreased MDA levels by 21.67 % (p < 0.0001) and increased the levels of SOD and GSH-Px by 15.47 % and 20.52 %, respectively (p < 0.001). These results were consistent with those of Yan et al., who investigated a mouse model of liver injury and found that plant polysaccharides could protect liver function by enhancing SOD activity [50]. Meanwhile, the study by Govindan and Gui confirmed that polysaccharides could alleviate hepatocellular injury by inhibiting lipid peroxidation, thus further supporting this mechanism [21,24]. The medium concentration BRLPP group exhibited some antioxidant capacity, although its effect on oxidative stress was not as significant as that of the high concentration group. However, the low concentration BRLPP group did not show significant improvement in the levels of MDA and GSH-Px. This might be ascribed to the fact that its concentration was too low to effectively trigger the antioxidant mechanism or achieve sufficient bioactivity.

In conclusion, the antioxidant capacity of zebrafish was affected by BRLPP to differing degrees. In particular, the strongest antioxidant benefits were obtained at high BRLPP concentrations. This suggests that BRLPP can effectively ameliorate ALD in zebrafish, and its underlying mechanism may be associated with the mitigation of oxidative stress. In order to further validate the hepatoprotective activity of BRLPP in mice and to explore the mechanism by which it exerts its effect, it is recommended that the relevant mouse experiments be followed up with reference to the method of Yan et al [50]. Furthermore, it would be advantageous to examine the oxidative pathway in order to achieve this. Consequently, BRLPP warrants further exploration as a potential hepatoprotective agent.

4. Conclusion

In this study, the BRLPP polysaccharide was efficiently extracted via the ultrasonic extraction method optimized through RSM. The optimal extraction conditions were determined as follows: a material– to–liquid ratio of 1:30, extraction duration of 3 h at a power of 200 W and a temperature of 60°C, yielding a polysaccharide content of 6.90 % ± 0.39 %. Subsequently, the extracted BRLPP polysaccharide underwent in-depth isolation and purification procedures to obtain the BRLPP fraction.

Structural characterization revealed that BRLPP consists primarily of glucose and arabinose in a molar ratio of 94.29:3.06, with an average molecular weight of 5.72 × 10⁴ Da. GC − MS analysis of BRLPP polysaccharide molecules disclosed the existence of notable glycosidic bond forms, such as → 4)-Glcp-(1→, →4,6)-Glcp-(1→, and → 3,4)-Glcp-(1 → .

This study employed in vitro antioxidant assays and a zebrafish ALD model to demonstrate that BRLPP significantly enhanced SOD and GSH-Px activity while reducing MDA levels, indicating its potent antioxidant and hepatoprotective effects. However, due to experimental limitations, only 1D NMR was used for polysaccharide structural analysis. Future studies could utilize 2D NMR techniques, such as HMBC, HSQC, and ¹H–¹H COSY, for more detailed structural characterization. The effect of BRLPP on ORAC can be examined in subsequent experiments. Additionally, in the follow-up experiments, mice models should be employed to further validate BRLPP's hepatoprotective activity. Western blot analysis and the assessment of other antioxidant enzymes (such as catalase, glutathione reductase, and glutathione-S-transferase) and non-enzymatic antioxidants (such as Vitamin C and glutathione), which were not examined in this study, could help elucidate the underlying mechanisms.

CRediT authorship contribution statement

Xin Xu: Writing – original draft, Visualization, Data curation. Xinxin Yang: Investigation, Formal analysis, Data curation. Meng Wang: Writing – review & editing, Validation. Jingrong Zhu: Supervision, Resources. Jing Li: Investigation, Data curation. Caiyue Chen: Software. Jiameng Liu: Software, Investigation. Jiahuan Zheng: Validation, Formal analysis. Bei Fan: Investigation, Funding acquisition. Fengzhong Wang: Supervision, Conceptualization. Jing Sun: Writing – review & editing, Funding acquisition, Conceptualization.

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.

Appendix A. Supplementary data

The following are the Supplementary data to this article:

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.