A polysaccharide extraction and purification method derived from traditional chinese medicine cat ginseng and application thereof

Abstract

This invention discloses a method for extracting and preparing polysaccharides from the traditional Chinese medicine *Cat Ginseng* and its applications. The process involves drying and pulverizing the raw material, defatting it with 95% ethanol, and then using ultrasound-assisted extraction. The liquid-to-solid ratio, extraction time, and ultrasonic power are optimized using response surface methodology. The extract is then centrifuged, concentrated, precipitated with alcohol, deproteinized and decolorized using macroporous resin, dialyzed, and freeze-dried to obtain crude polysaccharides. These crude polysaccharides are then purified by DEAE-52 and Sephadex G-150 column chromatography to obtain three homogeneous new polysaccharide fractions: AvPs1-1, AvPs2-1, and AvPs2-2. The polysaccharide extract, dominated by AvPs2-1 and AvPs2-2, not only exhibits protective effects on zebrafish embryonic development but also demonstrates significant anti-fibrotic activity in a liver injury model. Due to its natural non-toxicity, water solubility, and multiple functions including embryonic development protection and anti-fibrosis, this polysaccharide shows broad application prospects in functional foods, health products, and therapeutic drugs.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and food technology, specifically to a method for extracting and purifying polysaccharides derived from the traditional Chinese medicine *Cat Ginseng* and its application. Background Technology

[0002] Actinidia chinensis (Kiwifruit) Actinidia valvata Actinidia yunnanensis (Dunn) is a plant belonging to the genus Actinidia in the family Actinidiaceae, possessing unique dual value as both food and medicine. Its fruit has a delicious flavor and is widely loved by consumers; its root, known as "cat ginseng" in traditional Chinese medicine, has shown significant pharmacological activities such as anti-tumor, anti-inflammatory, antioxidant, and immune-enhancing effects in clinical practice, and is widely used to treat major diseases such as gastric cancer, esophageal cancer, lung cancer, and multiple myeloma.

[0003] Current modern pharmacological research on ginseng (Panax quinquefolius) focuses primarily on small molecule compounds. However, most of these discovered small molecule compounds are alcohol-soluble. In daily diets and clinical drug use, water is the primary extraction solvent, indicating that identifying effective chemical components from its water extracts has greater practical significance.

[0004] Plant polysaccharides are among the main water-soluble active ingredients, possessing advantages such as wide availability, low toxicity, and diverse biological activities. Notably, root polysaccharides from plants within the same genus have shown remarkable biological activity. For example, *Actinidia chinensis* (Chinese kiwifruit)... A. chinensis Polysaccharides can inhibit the invasion and metastasis of gastric cancer cells, while hairy kiwifruit (Actinidia chinensis) A. eriantha Root polysaccharides can regulate macrophage function by activating the TLRs / NF-κB signaling pathway and demonstrate therapeutic potential against gastric cancer. However, systematic research on polysaccharides from the root of Actinidia cuspidatum (cat ginseng) has long been lacking. This serious deficiency in understanding hinders its development and utilization as a valuable natural resource and is one of the main bottlenecks to its commercial application.

[0005] Furthermore, current technologies for extracting active ingredients from cat ginseng mostly employ traditional methods such as thermal reflux. These methods generally suffer from significant drawbacks, including low extraction efficiency, high energy consumption, long processing times, and the tendency for high temperatures to degrade polysaccharide activity. This not only results in low yields of cat ginseng polysaccharides (AvPs), making it difficult to meet the raw material requirements for subsequent research, but also severely restricts the progress of research on their mechanism of action and industrial applications. Therefore, developing a new technology for the efficient, rapid, and environmentally friendly extraction of AvPs is an urgent need to realize its value transformation.

[0006] In summary, the existing technologies face the following critical issues that urgently need to be addressed: 1) Insufficient raw material exploration: The abundant polysaccharide resource of cat ginseng has been severely neglected, and its structure, function, and applications remain largely unknown. 2) Outdated extraction technology: Traditional extraction methods are inefficient, energy-intensive, and prone to destroying activity, resulting in low yields and weak activity of AvPs, failing to meet subsequent research and development needs. 3) Gaps in applied research: There is a lack of systematic research from extraction and purification to structural analysis and activity evaluation, especially a lack of activity verification of AvPs in areas such as developmental protection and anti-liver fibrosis (particularly in biological models), making it difficult to fully realize and apply its functional value. Summary of the Invention

[0007] The purpose of this invention is to provide an extraction and purification method for polysaccharides derived from the traditional Chinese medicine cat ginseng and its application, in order to overcome the above-mentioned defects, and to elucidate its structural characteristics, developmental protective effects and anti-liver fibrosis activity, so as to provide a solid scientific basis and technical support for its development as a novel functional food or drug.

[0008] To achieve the above objectives, the present invention provides the following technical solution: a method for extracting and purifying polysaccharides derived from the traditional Chinese medicine *Panax ginseng*, comprising the following steps: First, the *Panax ginseng* raw material is dried and pulverized, and pretreated with 95% ethanol for defatting; then, using water as the extraction solvent, ultrasonic-assisted extraction is performed under the conditions of a liquid-to-solid ratio of 10-30 mL / g, ultrasonic power of 200-600 W, and extraction time of 30-60 min; after centrifugation, the supernatant of the extract is concentrated, ethanol is added to precipitate crude polysaccharide, which is then subjected to deproteinization and decolorization treatment with macroporous adsorption resin, followed by dialysis, and finally freeze-drying to obtain crude *Panax ginseng* polysaccharide; the crude polysaccharide is further separated and purified by DEAE-52 ion exchange column and Sephadex G-150 gel column chromatography, finally obtaining three homogeneous polysaccharide components AvPs1-1, AvPs2-1, and AvPs2-2.

[0009] Preferably, the drying temperature is 50-60℃, and the material is pulverized through a 60-mesh sieve; the degreasing process is as follows: add 95% ethanol at a ratio of 1:20 (w / v), and perform ultrasonic degreasing 3 times, each time for 1-2 hours.

[0010] Preferably, the ultrasonic extraction conditions are optimized using a response surface methodology (RSM) to obtain the optimal parameters: the liquid-to-solid ratio is preferably 15 mL / g, the ultrasonic power is preferably 370 W, and the extraction time is preferably 46 min.

[0011] Preferably, the separation and purification of the polysaccharide includes the following steps: the crude polysaccharide is purified sequentially by DEAE-Cellulose-52 ion exchange chromatography (gradient elution: deionized water, 0.15-0.6 M NaCl) and Sephadex G-150 gel chromatography, during which the elution curve is plotted by the phenol-sulfuric acid method and the fractions are combined, and the mixture is treated with a 3500 Da dialysis membrane to finally obtain three components: AvPs1-1, AvPs2-1 and AvPs2-2.

[0012] Preferably, the total content of the AvPs2-1 component and the AvPs2-2 component accounts for more than 70% of the total polysaccharide extract; AvPs2-1 and AvPs2-2 are highly similar in structural features, while AvPs1-1 retains the core structural features of both and is a product of partial degradation of AvPs2-1 and AvPs2-2.

[0013] Preferably, the three homogeneous polysaccharides after purification are all non-protein-bound polysaccharides with a triple helix conformation. AvPs1-1 is a white powder with an average molecular weight of approximately 1.93 kDa; while AvPs2-1 and AvPs2-2 are both pale yellow flocculent substances with very large molecular weights, both exceeding 2000 kDa. kDa; the monosaccharide composition of all three is mainly composed of galactose and arabinose, but the proportions of each monosaccharide (mannose:rhamnose:galacturonic acid:glucose:galactose:arabinose:fucose) are different, specifically 0.81:4.25:1.89:7.11:43.60:31.13:11.21, 3.82:8.41:1.49:4.36:42.80:29.40:9.72 and 2.73:7.16:2.95:6.75:47.77:23.22:9.43.

[0014] Preferably, the three homogeneous polysaccharide components of AvPs1-1 have a highly branched structure with a branching degree of 65.13%, and their monosaccharide linkages include terminal arabinofuranose, terminal fucopyranose, terminal galactopyranose, 5-linked arabinofuranose, 2-linked glucose, 3-linked galactose, 2-linked galactose, 4-linked galactose, 4-linked glucose, 2,3-linked mannose, 6-linked galactose, and 2,3-linked galactose.

[0015] This invention also provides applications of polysaccharides derived from cat ginseng. The polysaccharides, after separation and purification, yield three homogeneous components that possess in vivo developmental protective activity, protecting zebrafish embryos from normal development under adverse conditions. They also exhibit in vivo anti-liver fibrosis activity, effectively inhibiting the process of liver fibrosis by exerting liver-protective effects, demonstrating broad application prospects.

[0016] Preferably, the polysaccharide AvPs2-1 component has a protective effect against H2O2-induced oxidative stress in a zebrafish model. This effect is manifested in its ability to comprehensively alleviate the damage of oxidative stress to the embryo by increasing the hatching rate of zebrafish embryos, reducing their heart rate, improving their body length, reducing their pericardial edema area, and inhibiting the excessive generation of reactive oxygen species. The polysaccharide AvPs2-1 component plays a role in preventing and / or treating liver injury and liver fibrosis by inhibiting oxidative stress (manifested as increasing glutathione and decreasing malondialdehyde) and inhibiting the activation of hepatic stellate cells and extracellular matrix deposition (manifested as decreasing α-smooth muscle actin and type I collagen).

[0017] Preferably, the extracted cat ginseng polysaccharide or its components are used in food, medicine or health products.

[0018] Compared with the prior art, the beneficial effects of the present invention are: (1) Highly efficient and green extraction technology significantly improves product quality and yield. This invention employs ultrasound-assisted extraction combined with response surface methodology to precisely optimize the extraction process. This technology utilizes the ultrasonic cavitation effect to efficiently break down cell walls, increasing the yield of cat ginseng polysaccharides to a maximum of 2.84%, an improvement of approximately 40% compared to the previous method. Simultaneously, the optimized process achieves a lower liquid-to-material ratio, significantly reducing energy consumption. Furthermore, it uses environmentally friendly macroporous adsorption resin instead of traditional chemical methods for decolorization and deproteinization. The entire process is highly efficient, precise, and leaves no solvent residue, ensuring the product's high purity and eco-friendly characteristics.

[0019] (2) The first systematic analysis of fine structure fills a research gap in key areas. This invention marks the first successful separation of three high-purity, homogeneous components (purity >95%) from *Gynostemma pentaphyllum* using dual-column chromatography (DEAE-52 & Sephadex G-150). Furthermore, by comprehensively utilizing various cutting-edge analytical techniques, the molecular weight, monosaccharide composition, linkage mode, and triple helix conformation of *Gynostemma pentaphyllum* polysaccharides were fully revealed, filling a gap in the fundamental structural research of *Gynostemma pentaphyllum* polysaccharides. Simultaneously, the structural relationships between the different components were elucidated, providing a solid theoretical foundation for understanding their functions.

[0020] (3) It has multiple remarkable effects and broad and huge application prospects. Studies have found that this polysaccharide extract possesses a wide range of biological activities. It not only effectively protects cells but also exhibits powerful organ protection and anti-fibrotic functions, demonstrating significant effects in liver injury models comparable to classic drugs. As a natural, safe, and non-toxic substance, cat ginseng polysaccharide combines multiple benefits, providing a solid scientific basis and broad market prospects for the development of next-generation functional foods, health products, and therapeutic drugs. Attached Figure Description

[0021] Figure 1 The effects of different liquid-to-solid ratios, ultrasonic time, and ultrasonic power on the yield of cat ginseng polysaccharides were investigated.

[0022] Figure 2 The 3D surface plot (ac) and 2D contour plot (df) show the interaction between the liquid-to-material ratio, ultrasonic time, and ultrasonic power in pairs on the early yield of cat ginseng polysaccharide.

[0023] Figure 3 Elution patterns of crude ginseng polysaccharide on a DEAE-cellulose column (a) and on a Sephadex G-150 gel filter column (b, c), cover image of the lyophilized polysaccharide fraction (d), and ultraviolet spectra in the 200-400 nm range (e).

[0024] Figure 4 The HPGPC-ELSD chromatograms of AvPs1-1(a), AvPs2-1(b), and AvPs2-2(c) are shown, as are the chromatograms of the degradation products after partial acid hydrolysis of AvPs1-1(d), AvPs2-1(e), and AvPs2-2(f).

[0025] Figure 5 The HPLC-DAD chromatogram of the hydrolysis product of cat ginseng polysaccharide after PMP derivatization is shown.

[0026] Note: 1. Mannose; 2. Rhamnose; 3. Galacturonic acid; 4. Glucose; 5. Galactose; 6. Arabinose; 7. Fucose.

[0027] Figure 6 The FT-IR spectra of AvPs1-1, AvPs2-1, and AvPs2-2 are shown.

[0028] Figure 7 The images show the 1H NMR spectra of AvPs1-1, AvPs2-1, and AvPs2-2 acquired using an Agilent 400M NMR spectrometer.

[0029] Figure 8 The total ion chromatogram of PMAAs derivatives after methylation of AvPs1-1 is obtained by GC-MS.

[0030] Figure 9 MS ion fragmentation diagram of PMAAs derivatives after methylation of AvPs1-1.

[0031] Figure 10 The chemical shift of AvPs1-1 in the NMR spectrum. (a) 1 H NMR spectrum, (b) 13(c) C NMR spectrum; (d) COSY spectrum; (e) HSQC spectrum; (f) HMBC spectrum; and (c) NOESY spectrum. Gly, residues linked by glycosidic bonds.

[0032] Figure 11 Eight possible segment structures for AvPs1-1 repeating units.

[0033] Figure 12 The scanning spectra of AvPs1-1, AvPs2-1 and AvPs2-2 after reaction with I2-KI are shown in (a) using a UV-Vis spectrophotometer; and the maximum absorption wavelength of the polysaccharide-Congo red complex in the NaOH concentration range of 0-0.5 mM is shown in (b).

[0034] Figure 13 Scanning electron microscope images of AvPs1-1 (a1 and a2), AvPs2-1 (b1 and b2), and AvPs2-2 (c1 and c2). a1, b1, and c1 are 500x SEM magnification images, and a2, b2, and c2 are 10000x SEM magnification images.

[0035] Figure 14 To investigate the protective effect of AvPs2-1 against H2O2-induced oxidative stress in zebrafish embryos. (a) Hatching rate of AvPs2-1 during 96–112 hpf, (b) Heartbeats in 20 seconds at 104 hpf, (c) Body length of larvae at 104 hpf, (e) Pericardial edema area of ​​zebrafish embryos at 104 hpf. Results were normalized to the control group. (d) Lateral view of embryos exposed to different concentrations of AvPs2-1 at 96 hpf. All experiments were performed three times. *p<0.05 was considered statistically significant compared to the untreated group.

[0036] Figure 15 The effect of AvPs2-1 on ROS levels induced by H2O2 in zebrafish embryos. (a) Zebrafish embryos under a fluorescence microscope; (b) ROS production levels in zebrafish embryos treated with H2O2 and co-treated with AvPs2-1. Fluorescence intensity (representing ROS content) was calculated using Image-Pro Plus (version 6.0). Experiments were repeated three times, and data are expressed as mean ± standard error. p <0.01, *** p <0.001.

[0037] Figure 16 shows the therapeutic effect of AvPs2-1 on CCl4-induced physiological parameters in C57BL / 6J mice. (a) Dynamic changes in body weight, (b) liver-to-body ratio, (c) serum aspartate aminotransferase (AST) level, (d) serum alanine aminotransferase (ALT) level, (e) malondialdehyde (MDA) content in liver tissue, and (f) glutathione (GSH) content in liver tissue. Data are expressed as mean ± standard deviation (n ≥ 4). Statistical significance: *p<0.05, **p<0.01, ***p<0.001 vs. control group; #p<0.05, ##p<0.01, ###p<0.001 vs. model group.

[0038] Figure 17 shows the histopathological assessment of collagen deposition in liver tissue. (a) Hematoxylin-eosin (H&E) staining, (b) Masson's trichrome staining, (d) Sirius red staining. Collagen was quantified using ImageJ by (c) Masson's trichrome staining and (e) Sirius red staining, where collagen fibers were stained blue (Mason's) or red (Sirius red), cell nuclei were blue-black, and cytoplasmic components were red. Data are expressed as mean ± standard deviation (n = 5). ***p < 0.001. Detailed Implementation

[0039] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0040] Example 1: A polysaccharide extraction process from cat ginseng 1. Raw material processing Cat ginseng was dried and pulverized at 60℃ and passed through a 60-mesh sieve to obtain powder. 100 g of powder was taken and refluxed with 95% ethanol (1:10 w / v) three times for 2 h each time to remove fat. The ethanol solution was discarded and the residue was evaporated to dryness for later use.

[0041] 2. Single-factor experimental verification 1) Effect of liquid-to-material ratio With a fixed ultrasonic power of 600 W and an extraction time of 30 min, the effect of liquid-to-solid ratios (10, 20, 30, 40, and 50 mL / g) on ​​polysaccharide yield was investigated. The results showed that the highest yield (2.6%) was achieved at a liquid-to-solid ratio of 20 mL / g. Beyond this ratio, the yield decreased due to solvent dilution and reduced ultrasonic penetration. Figure 1 a).

[0042] 2) Impact of extraction time With a fixed liquid-to-solid ratio of 20 mL / g and ultrasonic power of 600 W, the effect of time (20, 30, 40, 50, and 60 min) was investigated. The yield reached its peak at 50 min (2.78%), and decreased after 50 min due to ultrasonic degradation. Figure 1 b).

[0043] 3) Effect of ultrasonic power With a fixed liquid-to-solid ratio of 20 mL / g and an extraction time of 50 min, the effect of power (120, 240, 360, 480, 600 W) was investigated. The highest yield (2.72%) was achieved at 480 W; yields decreased beyond 480 W due to mechanical shear force damaging the polysaccharide structure. Figure 1 c).

[0044] 3. Response Surface Optimization Experiment Based on the single-factor results, a Bx-Behnken experiment was designed using Design-Expert 13 software. The three-factor, three-level experimental setup is shown in Table 1, the results of the 17 groups are shown in Table 2, the statistical analysis data are shown in Table 3, and the response surface visualization results are shown in [Table 1]. Figure 2 The model equation obtained through regression analysis is: Yield = -26.14250 + 0.063250A + 0.652250B + 0.073375C + 0.002950AB - 0.000150AC - 0.000333BC - 0.004925A² - 0.006225B² - 0.000076C². ANOVA analysis showed that the model was highly significant (p < 0.0001), with significant interactions AB (p = 0.0002) and BC (p < 0.0001), and no significant lack-of-fit term (p = 0.2042), indicating a high model fit (R² = 0.9955). Optimal conditions were obtained through model optimization: liquid-to-solid ratio 14.55 mL / g, time 45.93 min, power 369.97 W, with a predicted yield of 2.87%. Subsequently, the polysaccharides were extracted using optimized conditions, and the yield of polysaccharides was calculated to be 2.84% ± 0.08% (n=3), with a deviation of only 1.05% from the response surface prediction (2.87%), verifying the reliability of the process.

[0045] Table 1. Variable coding level and actual level in the Box-Behnken design.

[0046] Table 2. RSM experimental design and results of AvPs extracted using ultrasound-assisted extraction.

[0047] Table 3. Analysis of Variance for Regression Models

[0048]

[0049] Example 2: Extraction, separation, preparation, and structural characterization of a polysaccharide derived from cat ginseng. 1. Extraction of cat ginseng polysaccharides Same as Example 1.

[0050] 2. Isolation and preparation of polysaccharides 1) Preliminary separation by ion exchange chromatography Weigh an appropriate amount of crude ginseng polysaccharide, dissolve it in distilled water to a concentration of 0.1 g / mL, filter through a 0.45 μm filter membrane, and load the sample onto a DEAE-52 cellulose column (65 × 100 mm). Elute sequentially with deionized water, 0.15 M, 0.3 M, 0.45 M, and 0.6 M NaCl solutions at a flow rate of 1.0 mL / min, collecting 10 mL of fraction from each tube. Detect the absorbance of each tube at 490 nm using the phenol-sulfuric acid colorimetric method, and plot the elution curve as shown below. Figure 3 As shown in Figure a, the fractions were combined based on the position of the main peak to obtain components AvPs1 (tubes 27 to 38) and AvPs2 (tubes 44 to 59).

[0051] 2) Dialysis desalting and molecular weight cutoff The combined components AvPs1 and AvPs2 were dialyzed in deionized water for 72 h through a dialysis membrane with a molecular weight cutoff of 3500 Da to remove small molecule salts and impurities. The dialysate was then freeze-dried for later use.

[0052] 3) Size exclusion chromatography for further purification The lyophilized sample after dialysis was dissolved in distilled water and loaded onto a Sephadex G-150 column (26×310 mm). Elution was performed at a flow rate of 0.5 mL / min, collecting 3 mL of fraction from each tube. The absorbance of each tube was measured at 490 nm using the phenol-sulfuric acid colorimetric method, and elution curves were plotted. The elution curves are shown below. Figure 3 b, Figure 3 As shown in c. After purification, AvPs1 yielded a single symmetrical peak (tubes 18 to 31), named AvPs1-1. Figure 3 b); After purification, AvPs2 yielded two peaks (tubes 7 to 18 and tubes 20 to 34), which were named AvPs2-1 and AvPs2-2, respectively. Figure 3 c).

[0053] 3. Homogeneity and molecular weight analysis 1) Homogeneity analysis The phenotypic characteristics of freeze-dried AvPs1-1 (white powder), AvPs2-1, and AvPs2-2 (pale yellow flocculent matter) were identified and their appearance was recorded. Figure 3 d); UV spectroscopy analysis showed that after preparing a 1 mg / mL solution of the three substances and scanning at 200-700 nm, no significant absorption was observed at 260 nm (characteristic peak of nucleic acid) and 280 nm (characteristic peak of protein), indicating that there were very few pigment, protein, and nucleic acid residues. Figure 3 e); High performance size exclusion chromatography-evaporative light scattering detection (HPSEC-ELSD) homogeneity analysis (using a TSK-GEL GMPWxl column, deionized water as the mobile phase, flow rate 0.7 mL / min, ELSD parameters: drift tube temperature 115℃, nitrogen pressure 360-380 kPa). The results showed that AvPs1-1 exhibited a single symmetrical peak ( Figure 4 a), AvPs2-1 and AvPs2-2 also showed symmetrical single chromatographic peaks ( Figure 4 b, 4c (different retention times) indicate that all three are high-purity homogeneous polysaccharides. In summary, the results of phenotypic properties, ultraviolet spectroscopy, and homogeneity analysis confirm that AvPs1-1, AvPs2-1, and AvPs2-2 are all high-purity homogeneous polysaccharides.

[0054] 2) Molecular weight determination and comparison The HPGPC-ELSD method was used in conjunction with the dextran standard curve (ln(Mw) = -22.04ln(RT) + 64.46). R² The molecular weights of each component were determined using a molecular weight of 0.9913. The results showed that the retention time of AvPs1-1 was 13.122 min ( ). Figure 4 a), with an average molecular weight (Mw) of approximately 1.93 kDa. In comparison, AvPs2-1 (RT: 6.229 min, Figure 4 b) and AvPs2-2 (RT: 6.716 min, Figure 4 c) The retention time of AvPs1-1 was much earlier than that of standard T-200 (Mw of T-200 is 2000 kDa), and their average molecular weights all exceeded 2000 kDa (the maximum molecular weight of the selected standard). This indicates that the molecular weight of AvPs1-1 is significantly smaller than that of AvPs2-1 and AvPs2-2. Notably, in this embodiment, AvPs1-1 was not detected in the crude polysaccharide chromatogram, while the elution times of AvPs2-1 and AvPs2-2 were consistent with those of the crude polysaccharide, suggesting that some polysaccharide chains may have degraded during the separation and purification process.

[0055] 4. Partial acid hydrolysis and analysis of its products To investigate the structural relationships among the components, AvPs1-1, AvPs2-1, and AvPs2-2 were partially acid-hydrolyzed, and the degradation products were analyzed by HPGPC. The hydrolysis results showed that ( Figure 4 The retention times of the degradation products of AvPs1-1 (d-4f) (12.917 min) were similar to those of the original component (13.122 min). However, the retention times of the degradation products of AvPs2-1 and AvPs2-2 (13.220 min and 13.369 min, respectively) were significantly different from their respective parent components (6.229 min and 6.716 min). This result indicates that after hydrolysis, AvPs2-1 and AvPs2-2 generated a low molecular weight polysaccharide component with a similar molecular weight to AvPs1-1. Therefore, it can be preliminarily inferred that AvPs1-1 is likely an incomplete degradation product of AvPs2-1 or AvPs2-2. In addition, combined with the elution patterns previously obtained on the Sepharse G-150 gel filtration column ( Figure 3 (b, 3c) AvPs2-1 and AvPs2-2 originate from the same subfraction, while AvPs1-1 originates from another subfraction. This suggests that AvPs2-1 and AvPs2-2 may have the same or similar ion exchange properties, further providing evidence that they have similar structural features.

[0056] 5. Chemical property analysis The contents of total carbohydrates, total protein, and total uronic acid were determined by the phenol-sulfuric acid method, the BCA method, and the m-hydroxydiphenol method, respectively. For the phenol-sulfuric acid method, glucose standards or polysaccharide samples (100 μL) of different concentrations (0.1–1.0 mg / mL) were added to a mixture containing 60 μL of phenol and 1.5 mL of concentrated sulfuric acid. After incubation at 25°C for 60 minutes, the absorbance was measured at 490 nm. For the total protein method, 20 μL of polysaccharide solution or bovine serum albumin standard was added to a 96-well plate, along with 20 μL of BCA working solution. After incubation at 37°C for 30 minutes, the absorbance was measured at 560 nm. For the total uronic acid method, the absorbance of the solution at a specific wavelength was measured. The contents of each component were calculated using their respective calibration curves.

[0057] The results showed that AvPs1-1 was almost entirely composed of carbohydrates (102.42 ± 4.00%), with extremely low levels of uronic acid (2.11 ± 1.07%) and protein (0.03 ± 0.62%), indicating that it is a neutral polysaccharide. In contrast, AvPs2-1 and AvPs2-2 had similar chemical compositions, both consisting of high levels of carbohydrates (95.85 ± 3.09% and 89.24 ± 3.24%), small amounts of uronic acid (3.16 ± 0.41% and 2.76 ± 0.20%), and protein (2.44 ± 0.10% and 2.46 ± 0.28%). This result further confirms that AvPs1-1 may be derived from the degradation of AvPs2-1 or AvPs2-2, and that the two have similar chemical properties. At the same time, it is speculated that during the partial degradation of AvPs1-1, the proteins and uronic acids it carries may be removed as free small molecules during the dialysis step.

[0058] Table 4 Monosaccharide composition and chemical characteristics of AvPs1-1, AvPs2-1, and AvPs2-2

[0059] Note: a The molar percentage of sugar residues in the obtained polysaccharide; Man, mannose; Rha, rhamnose; GalA, galacturonic acid; Glc, glucose; Gal, galactose; Ara, arabinose; Fuc, fuc.

[0060] 6. Monosaccharide composition After acid hydrolysis and PMP derivatization, the monosaccharide composition of the polysaccharide sample was analyzed using high-performance liquid chromatography-diode array detector (PMP-HPLC-DAD). Specifically, 2.5 mg of the polysaccharide sample was hydrolyzed with 3 M trifluoroacetic acid at 90°C for 8 hours in an argon-filled sealed tube; excess acid was then removed by centrifugation, and residual acid was completely removed by methanol co-distillation; 200 μL of the hydrolysate was mixed with 300 μL of 0.3 M sodium hydroxide and 300 μL of 0.5 M PMP solution, and incubated at 70°C for 1 hour for derivatization. Finally, the obtained monosaccharide derivatives were separated and detected on a C18 column using acetonitrile / phosphate buffer (pH 7.2) as the mobile phase and 245 nm as the detection wavelength.

[0061] The results are as follows Figure 5As shown, the chromatograms revealed clear peak shapes of the monosaccharide derivatives, and the molar percentage of each monosaccharide was calculated by integrating the peak areas. The results showed that the main monosaccharides of the three polysaccharides (AvPs1-1, AvPs2-1, and AvPs2-2) were galactose (Gal) and arabinose (Ara). Specifically, the molar ratio of monosaccharides in AvPs1-1 was Man:Rha:GalA:Glc:Gal:Ara:Fuc = 0.81:4.25:1.89:7.11:43.60:31.13:11.21; in AvPs2-1 it was 3.82:8.41:1.49:4.36:42.80:29.40:9.72; and in AvPs2-2 it was 2.73:7.16:2.95:6.75:47.77:23.22:9.43. Comparative analysis revealed that the contents of mannose (Man) and rhamnose (Rha) in AvPs2-1 and AvPs2-2 were significantly higher than those in AvPs1-1, while the chromatographic peaks of other monosaccharides were similar. This result suggests that AvPs2-1 and AvPs2-2 have similar structural characteristics, and that AvPs1-1 may be a degradation product resulting from chain scission at the Man and Rha residue sites.

[0062] 7. FT-IR spectral analysis The structure of polysaccharide samples was characterized using a Fourier transform infrared spectroscopy (FT-IR) instrument (Niclet iS10, Thermo Fisher Scientific, USA). The experimental method involved mixing the polysaccharide samples with potassium bromide (KBr) and pressing them into slides, then scanning within the wavenumber range of 4000–400 cm⁻¹. FT-IR is an effective tool for analyzing the major functional groups of biological macromolecules. Figure 6 As shown, the infrared spectral characteristics of the three polysaccharides, AvPs1-1, AvPs2-1, and AvPs2-2, are basically consistent. The main absorption peaks include: a broad peak at 3420 cm⁻¹ attributed to the stretching vibration of OH (hydroxyl group and water molecule); absorption peaks at 2934 and 1412 cm⁻¹ corresponding to the stretching and bending vibrations of CH, respectively; peaks at 1610-1630 cm⁻¹ originating from the asymmetric stretching vibration of bound water or carboxyl groups (-COO); signals at 1143 (AvPs1-1), 1121 (AvPs2-1), and 1138 cm⁻¹ (AvPs2-2) are trans-bridging oxygen COC stretching vibrations, while peaks at 1073, 1076, and 1072 cm⁻¹ are attributed to CO stretching vibrations, indicating the presence of a pyranose ring; and a weak absorption signal at 817 cm⁻¹ indicates the presence of an α-configuration D-mannopyranose unit. These characteristic peaks all conform to the infrared spectral characteristics of typical polysaccharides.

[0063] 8. Comparative Analysis of the Proton NMR Spectra of Three Homogeneous Polysaccharides To further compare the structural similarities and differences among the three homogeneous polysaccharides, proton nuclear magnetic resonance (¹H NMR) spectroscopy was used for analysis. The experimental procedure was as follows: Approximately 35 mg of dried polysaccharide sample was accurately weighed, dissolved in 2.0 mL of D₂O, and then freeze-dried to fully exchange the solvated water in the sample for deuterated water. Finally, the freeze-dried sample was redissolved in 0.6 mL of D₂O and NMR was performed at room temperature. The ¹H NMR signals were measured on an Agilent-400 MHz NMR spectrometer. Data processing and structural analysis of the spectra were performed using MestReNova® software.

[0064] like Figure 7 As shown, the ¹H NMR spectrum of the purified cat ginseng polysaccharide exhibited multiple characteristic signals in the δ 3.0–5.8 ppm region, consistent with typical polysaccharide characteristics. Among these, the chemical shift of the terminal hydrogen atoms (H1) is crucial for determining the α- or β-configuration. In the ¹H NMR spectrum of AvPs1-1, nine signals (δ 5.76, 5.48, 5.36, 5.24, 5.21, 5.19, 5.15, 5.02, and 4.89 ppm) were observed, representing α-configuration glycosidic bonds; several other signals (δ 4.68, 4.66, 4.64, 4.62, 4.58, 4.56, 4.50, 4.48, 4.39, 4.33, and 4.31 ppm) represented β-configuration glycosidic bonds. These numerous terminal proton signals indicate that AvPs1-1 possesses a complex and diverse polysaccharide structure. Notably, the ¹H NMR spectra of AvPs2-1 and AvPs2-2 show highly similar signals in the δ 5.76–4.31 ppm region to AvPs1-1, indicating that the sugar chains of the three polysaccharides may share the same or similar sugar residue types and linkage patterns. Other signals in the δ 4.50–3.10 ppm region are attributed to H2–H6 protons of the sugar residues, while the peak around δ 4.79 ppm represents deuterated heavy water (HOD). Based on the above multidimensional structural analysis data, it can be concluded that AvPs2-1 and AvPs2-2 have similar structural features, and their partial degradation can yield the AvPs1-1 component.

[0065] 9. Methylation analysis Based on previous chromatographic, molecular weight, monosaccharide composition, chemical composition, and FT-IR analysis results, it was determined that AvPs1-1 retains the main structural features of AvPs2-1 and AvPs2-2, and has a relatively small molecular weight (approximately 1.93 kDa), making it suitable for further structural analysis. Therefore, methylation analysis was performed on AvPs1-1: the sample was dissolved in DMSO and then completely methylated using a DMSO / NaOH-CH3I system; the hydrolysis product was reduced with NaBD4 and acetylated with acetic anhydride to obtain a partially methylated ethyl aldehyde (PMAA) derivative. Linkage bond analysis was performed using an Agilent 6890A-5975C gas chromatograph-mass spectrometer (BPX70 column) with high-purity helium as the carrier gas.

[0066] GC-MS total ion chromatogram ( Figure 8 ) and the corresponding ion spectrum ( Figure 9 The data shows that AvPs1-1 contains 12 monosaccharide linkages, including terminal arabinofuranose (t-Ara(f)), terminal fucopyranose (t-Fuc(p)), terminal galactopyranose (t-Gal(p)), 5-linked arabinofuranose (5-Ara(f)), 2-linked glucose (2-Glc(p)), 3-linked galactose (3-Gal(p)), 2-linked galactose (2-Gal(p)), and 4-linked galactose (...). The molar ratios of 4-Gal(p)), 4-linked glucose (4-Glc(p)), 2,3-linked mannose (2,3-Man(p)), 6-linked galactose (6-Gal(p)), and 2,3-linked galactose (2,3-Gal(p)) were 10.35:16.90:22.93:2.03:4.82:12.78:2.62:3.86:5.26:2.61:3.51:12.34 (Table 5). Analysis showed that the polysaccharide backbone was composed of multiple linkages, with galactose (Gal) having the highest proportion. Major residues included t-Ara (…). f ), t-Fuc( p ), t-Gal( p ), 3-Gal ( p ) and 2,3-Gal( p ), of which 2,3-Gal( pThe presence of galactose at C-2 indicates a branching point. The presence of arabinose, fucose, and galactose as terminal reducing sugars suggests that AvPs1-1 has a highly branched structure. Using the formula DB=(Nt+Nb) / (Nt+Nb+Nl) (where Nt, Nb, and Nl represent the number of terminal residues, branched residues, and linear residues, respectively), the branching degree is calculated to be 65.13%. Notably, the proportion of terminal groups is nearly 50%, while branching points account for only 15%. This may be attributed to AvPs1-1 being a partial degradation product (low molecular weight) of AvPs2-1 or AvPs2-2, resulting in a high concentration of terminal sugars.

[0067] Table 5. Methylation analysis data of AvPs1-1

[0068] 9. Multidimensional nuclear magnetic resonance spectroscopy analysis To elucidate the fine structure of AvPs1-1, we employed multidimensional nuclear magnetic resonance spectroscopy for detailed analysis. The experimental procedure was as follows: Approximately 35 mg of dried polysaccharide sample was accurately weighed, dissolved in 2.0 mL of D₂O, and then freeze-dried to fully exchange the solvated water in the sample for deuterated water. Finally, the freeze-dried sample was redissolved in 0.6 mL of D₂O and subjected to NMR spectroscopy at room temperature. The measurements were performed on a Bruker AVANCE NEO 500 M spectrometer, acquiring ¹H NMR (500 MHz), ¹³C NMR (125 MHz), ¹H-¹H COSY, HSQC, NOESY, and HMBC spectra. Figure 10 ).

[0069] Detailed identification of the chemical shifts of each glycosidic bond in AvPs1-1 is summarized in Table 6. In ¹H NMR spectra (… Figure 10 In a), 12 terminal proton signals were identified: seven of these signals (δ 5.54, 5.47, 5.45, 5.40, 5.30, 5.09, and 4.94 ppm) were attributed to β-configured sugar residues G, A, L, J, B, D, and I; the other five signals (δ 4.72, 4.65, 4.57, 4.42, and 4.39 ppm) were attributed to α-configured sugar residues E, F, C, H, and K.

[0070] However, the ¹³C NMR spectrum signal was very weak, with only five distinct terminal carbon signals observed. Figure 10 (b) These are identified as the terminal carbons of sugar residues H, E, I, L, and C, with chemical shifts of δ 104.03, 102.95, 100.75, 97.90, and 96.58 ppm, respectively. The C2-C6 signals of other sugar residues are mainly distributed in the δ 60-85 ppm region.

[0071] Building upon this, we further utilized HSQC and COSY spectra to determine the carbon and hydrogen chemical shifts of each sugar residue. Taking sugar residue C as an example, in the COSY spectrum (… Figure 10 In c), based on its H1 signal (δ 4.57 ppm), the chemical shifts of its H2 to H6 can be deduced to be δ 3.54, 3.78, 3.90, 3.93, and 3.69 ppm, respectively. In HSQC spectra (… Figure 10 In d), the corresponding C2-C6 chemical shifts can be deduced from the H2-H6 signals as δ 70.44, 72.03, 72.78, 73.82, and 61.14 ppm. Combining these results with the methyl analysis data, we infer that the structure of sugar residue A is T-β-D-Gal. p -(1→). Using the same method, we determined the structures of the remaining eleven sugar residues: B is T-α-L-Fuc p -(1→), D is T-β-D-Gal p -(1→), E is →5)-α-L-Ara f -(1→), F is →2)-β-D-Glc p -(1→), G is →3)-β-D-Gal p -(1→), H is →2)-α-D-Gal p -(1→), I is →4)-β-D-Gal p -(1→), J is →4)-α-D-Glc p -(1→), K is→2,3)-α-D-Man p -(1→), L is →6-β-D-Gal p -(1→). The internal linkages of some sugar residues were further confirmed by NOESY spectra. For example, in the NOESY spectra, the intra-residue cross-peaks at δH / δH 5.54 / 3.96 and 5.54 / 4.23 ppm were attributed to the H1-H3 and H1-H5 related signals of sugar residue G, respectively. Figure 10 f).

[0072] To elucidate the interrelationships between sugar residues, we focused on analyzing coupling signals in HMBC long-range correlation and NOESY spectra. HMBC spectra correlate hydrogen nuclei with their long-range coupled carbon nuclei, revealing the connections between residues. Although the HMBC spectrum showed relatively weak signals due to the low abundance of carbon signals, we still observed two key interresidue cross-peaks: δH / δC 5.47 / 79.25 ppm (corresponding to the correlation between H1 of residue A and C3 of residue J) and 4.41 / 76.45 ppm (corresponding to the correlation between H1 of residue H and C3 of residue F). Figure 10 e), which confirms the linkage modes of A-(1→3)-J and H-(1→3)-F, respectively. The associations between these residues are also strongly supported in the NOESY spectrum. For example... Figure 10 As shown in f, the NOESY spectrum reveals more connectivity information, such as: the correlation between A H1 and L H2 confirms the A-(1→2)-L connection; the correlation between L H1 and I H4 confirms the L-(1→4)-I connection; and a series of connections such as A-(1→3)-J, G-(1→4)-H, J-(1→4)-H, I-(1→6)-K, D-(1→3)-J, I-(1→2)-L, F-(1→2)-G, C-(1→2)-G and H-(1→3)-F.

[0073] Due to the missing connection signals, the complete main chain repeating unit cannot yet be obtained. However, based on the above comprehensive analysis, we successfully identified eight possible structural segments in the repeating unit of AvPs1-1, such as... Figure 11 As shown in the figure. These results lay the foundation for understanding the complex structure and function of AvPs family polysaccharides.

[0074] 10. I2-KI Analysis To preliminarily identify whether the polysaccharide possesses an amyloid structure, an equal volume of I₂-KI solution was added to a 1 mg / mL polysaccharide solution and mixed, with starch used as a positive control. Observation revealed that the mixed solution exhibited a blue reaction similar to the positive control, indicating that the polysaccharide may contain a helical chain structure linked by α-1,4-glycosidic bonds, thereby forming a complex with iodine molecules and producing the characteristic color. Figure 12 As shown in figure a, all purified polysaccharides (AvPs1-1, AvPs2-1, and AvPs2-2) were detected using a UV-Vis spectrophotometer. No absorption peak was observed at 565 nm, indicating that these purified polysaccharides have long sugar chains and numerous side chains. Furthermore, this result is consistent with the methylation analysis results.

[0075] 11. Congo Red Analysis The Congo red experiment was used to investigate the higher-order structure of polysaccharides, particularly the stability of their triple-helix conformation. The experimental results are as follows: Figure 12 As shown in b, when the polysaccharide binds to Congo red in a weakly alkaline environment of 0.1 M, the maximum absorption wavelength (λmax) of the complex shifts from 492 nm (the original Congo red concentration) to 504-505 nm. This directly confirms that AvPs1-1, AvPs2-1, and AvPs2-2 can all form Congo red complexes, providing strong evidence that they possess a triple-helix conformation. Notably, with increasing NaOH concentration, this λmax value gradually undergoes a blue shift, indicating that the increased alkalinity disrupts the helical structure of the polysaccharide. Combined with the result of no absorption at 565 nm, it can be concluded that these polysaccharides have a relatively long main chain and numerous branches, and their biological activity is likely closely related to their ability to maintain a stable triple-helix conformation in a weakly alkaline environment.

[0076] 12. Scanning electron microscopy morphology analysis The microstructures of the three polysaccharides were observed by scanning electron microscopy, and the results clearly revealed their significantly different phenotypic structures. Figure 13 AvPs1-1 exhibits an irregular, fragmented blocky shape with a surface covered in large wrinkles; while AvPs2-1 and AvPs2-2 are both tightly packed granules, with AvPs2-1 being regular polyhedral grains and AvPs2-2 being ellipsoidal particles. This significant difference in morphology is fundamentally attributed to their different molecular weights. AvPs1-1 has a smaller molecular weight, making its sugar chains more prone to breakage during extraction, forming larger branched structures and resulting in its rough, irregular surface. Conversely, AvPs2-1 and AvPs2-2 have larger molecular weights, allowing the chains to arrange themselves more effectively and form relatively stable crystalline or quasi-crystalline structures, thus exhibiting a more regular, dense granular morphology. This indicates that molecular weight is the key factor determining the final physical form of these three chemically similar polysaccharides.

[0077] Implementation Case 3: Antioxidant Activity Against H2O2-Induced Oxidative Stress in a Zebrafish Model 1. Extraction of polysaccharides Extraction was performed using the optimized extraction parameters from Implementation Case 1.

[0078] 2. Isolation and purification of polysaccharides The extraction scheme used in Implementation Case 2 was adopted.

[0079] 3. Evaluate the protective effect against H2O2-induced toxicity. AB-strain zebrafish were provided by Shanghai Fish Biotechnology Co., Ltd. (Shanghai, China). The zebrafish were reared in aerated and UV-sterilized water at a temperature of 26±1°C and a light / dark cycle of 14 / 10 hours. They were fed twice daily a formulated diet consisting of Antarctic krill, cod, salmon, and spirulina. Zebrafish in a 1:1 female-to-male ratio were placed in isolated spawning tanks, and spawning was induced the following morning when the lights were turned on. One hour later, zebrafish embryos were collected and rapidly transferred to embryo culture medium within 30 minutes, and cultured in sterile plastic bottles at 28°C. Healthy embryos were selected for subsequent experiments.

[0080] Starting approximately 48 hours post-fertilization (hpf), zebrafish embryos were transferred to individual wells of 6-well plates (at least 15 embryos per well) and cultured at 28°C with 2 mL of embryo culture medium. Embryos were treated with 5 mM H2O2, followed by the addition of polysaccharides at final concentrations of 20, 100 μg / mL, and 500 μg / mL, and incubated at 28 ± 0.2°C until 48 hpf. Embryos treated with only 5 mM H2O2 were designated as the H2O2 model group, while those without treatment (H2O2 and / or polysaccharides) were designated as the control group. The exposure solution was changed every 24 hours. Embryonic development was monitored, and mortality at 104 hpf, presence of 20-second heartbeats at 104 hpf, hatching rates at 96, 100, 104, 108, and 112 hpf, and pericardial edema area in hatched larvae at 104 hpf were examined or observed using an inverted microscope (Olympus IX73, Tokyo, Japan). Pericardial edema area was measured using Scpepht software (Chengdu Jingcheng Co., Ltd., China). To prevent movement of the zebrafish during microscopic observation, phenoxyethanol (0.05%) was used for anesthesia.

[0081] 4. Determination of ROS and apoptosis in zebrafish larvae ROS accumulation in zebrafish was analyzed using the oxidation-sensitive fluorescent probe dye 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA). Specifically, zebrafish larvae were incubated with DCFH-DA (2 μm) at 30°C in the dark for 10 minutes at 104 hpf. After incubation, the larvae were washed with fresh embryo culture medium and anesthetized with phenoxyethanol before observation. The larvae were then observed and photographed using a fluorescence microscope (Olympus IX73, Tokyo, Japan) at 4 × 10 magnification. The fluorescence intensity of each larva, representing ROS content, was analyzed using Image-Pro Plus (version 6.0). 5. Protective effect of AvPs2-1 against H2O2-induced oxidative stress in zebrafish embryos Excessive accumulation of reactive oxygen species (ROS) can have detrimental effects on biomolecules and cause damage to cell structures. Embryonic development is a highly vulnerable stage, characterized by rapid metabolism and extreme susceptibility to adverse environmental disturbances. Harmful substances in the environment can often affect the epigenetic regulation of organogenesis and even lead to developmental abnormalities. Therefore, the search for embryonic development protectants is an important need.

[0082] To assess the protective activity of AvPs2-1, a zebrafish embryo injury model was constructed using H2O2, and the developmental phenotypes of zebrafish embryos under additional ROS stress were analyzed, including hatching rate, heart rate, body length, and pericardial area change rate. Figure 14 As shown in Figure a, the hatching rate of zebrafish was significantly reduced when only H2O2 was added, compared to the blank control group. For example, after 96 hpf, almost no embryos hatched in the H2O2-treated group, while the hatching rate of the blank control group reached 50%. After 104 hpf, the hatching rate of the H2O2-treated group was 30%, while that of the blank control group was 80%. Therefore, H2O2 severely inhibited zebrafish embryo development due to oxidative stress. However, when AvPs2-1 was added, the inhibitory effect of H2O2 on zebrafish embryo development was significantly reduced, and the effect was directly related to the dosage. For example, as shown in Figure a... Figure 14 As shown in Figure a, at a concentration of 20 μg / mL, the developmental rate of zebrafish embryos at the same developmental time point showed almost no difference. However, when the concentration increased to 100 μg / mL, the hatching rate significantly increased, reaching 50% after 104 hpf. At a concentration of 500 μg / mL, the reversal effect of AvPs2-1 on the inhibition of zebrafish embryonic development by H2O2 was even more significant, almost approaching the hatching rate of the blank control group. Therefore, AvPs2-1 can resist the inhibitory effect of H2O2 on zebrafish embryonic development, and its effect is directly proportional to the concentration. Figure 14 As shown in b, after 104 hpf, the addition of H2O2 significantly increased the heart rate of zebrafish (p<0.05). When AvPs2-1 was added at concentrations of 100 or 20 μg / mL, there was no significant difference compared to the H2O2 group or the blank control group. However, when the concentration was increased to 500 μg / mL, the heart rate showed no significant difference compared to the blank control group, demonstrating a significant reversal effect of H2O2 on the heart rate of zebrafish. Figure 14 As shown in Figure c, after 104 hpf, H2O2 significantly affected the body length of zebrafish (p<0.05), and the addition of a low concentration of AvPs2-1 (20 μg / mL) did not improve the body length of zebrafish affected by H2O2. However, when the concentration was 100 μg / mL or 500 μg / mL, the body length of zebrafish co-hatched with H2O2 was not significantly different from that of the blank control group, indicating that AvPs2-1 can resist the effect of H2O2 on the body length of zebrafish at higher concentrations. Figure 14As shown in d, during zebrafish embryo hatching, the addition of H2O2 significantly increased the pericardial area (p<0.05). The addition of low concentrations of AvPs2-1 did not reverse pericardial effusion, but when the concentration was increased to 100 μg / mL or 500 μg / mL, the reduction in pericardial area was significantly improved. Figure 14 (e) There was no significant difference compared to the blank control group. In summary, H2O2 caused a series of abnormalities in zebrafish embryos, including hatching rate and pericardial edema, indicating its toxicity. However, zebrafish embryos exposed to H2O2 with the addition of 500 μg / mL AvPs2-1 showed no significant difference compared to the control group, suggesting that AvPs2-1 has a protective effect against the toxicity of H2O2 to zebrafish embryos.

[0083] 6. Inhibitory effect of AvPs2-1 on H2O2-induced ROS production in zebrafish embryos In vitro antioxidant activity studies showed that AvPs2-1 exhibited good free radical scavenging activity and had a protective effect against H2O2-induced oxidative stress in zebrafish embryos. To further analyze whether the protective effect of AvPs2-1 on zebrafish was related to the scavenging of reactive oxygen species (ROS), we tested the scavenging effect of AvPs2-1 on H2O2-induced ROS. Figure 15 As shown in a and 15b, H2O2 treatment significantly increased ROS production in zebrafish embryos compared to untreated embryos (p<0.001). However, in zebrafish embryos treated with a low concentration of AvPs2-1 (20 μg / mL), the amount of ROS induced by H2O2 was not significantly reduced compared to the blank control group. However, when the concentration of AvPs2-1 was increased to 100 or 500 μg / mL, there was no significant difference compared to the blank control group, indicating a significant reduction in ROS production. Excessive intracellular or intercellular ROS can not only interfere with normal physiological functions but also induce programmed cell death, leading to a range of diseases. Although *Gynostemma pentaphyllum* has good anti-tumor effects, current focus is mainly on its small molecule compounds. We first elucidate that *Gynostemma pentaphyllum* polysaccharides have a protective effect on zebrafish embryo development by scavenging ROS in vivo.

[0084] Implementation Case 4: Antioxidant Activity Against H2O2-Induced Oxidative Stress in a Zebrafish Model 1. Extraction of polysaccharides Extraction was performed using the optimized extraction parameters from Implementation Case 1.

[0085] 2. Isolation and purification of polysaccharides The extraction scheme used in Implementation Case 2 was adopted.

[0086] 3. AvPs2-1 alleviates CCl4-induced liver injury in C57BL / 6J mice Previous studies have confirmed that CCl4 can be metabolized into trichloromethyl free radicals (CCl3•), which induce lipid peroxidation of hepatocyte membranes and mitochondrial dysfunction, leading to severe hepatocyte damage and acute liver reactions, such as hepatomegaly, liver fibrosis, and weight loss in mice. In this study, mice were randomly divided into a normal control group, a model group, an AvPs2-1 treatment group, and a positive control group. A liver fibrosis model was established by intraperitoneal injection of 20% CCl4 / olive oil solution (2 mL / kg) twice a week. During the modeling period, the normal control group and the model group were administered an equal volume of physiological saline by gavage daily, the AvPs2-1 treatment group was administered 200 mg / kg AvPs2-1 polysaccharide solution by gavage daily, and the positive control group was administered 50 mg / kg silymarin solution by gavage daily for six weeks.

[0087] The results showed that although the average weight of the AvPs2-1 group increased compared to the model group in the later stages of treatment, no significant difference was observed (p > 0.05). Figure 16 a). Subsequently, we examined other indicators of liver injury. Liver enzyme release is an indicator of liver injury and hepatotoxicity. We collected blood samples from different treatment groups to measure the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in mouse plasma. The results showed that the levels of both enzymes were significantly elevated in the model group (a). p The result < 0.001 confirmed the successful establishment of the model. Compared with the model group, both the AvPs2-1 polysaccharide group and the positive control group (silymarin) significantly reduced CCl4-induced ALT levels in mouse plasma. p < 0.001, Figure 16 b) and AST p < 0.01, Figure 16 c) Levels. Specifically, compared to the model group, the AvPs2-1 treatment group showed a reduction of approximately 55% in ALT levels and approximately 23% in AST levels. Furthermore, CCl4 treatment often leads to hepatomegaly and an abnormal increase in liver weight, which can be indicated by liver indices. Figure 16 As shown in d, after CCl4 treatment, the liver index (liver weight / body weight ratio) in the model group was significantly higher than that in the normal control group. p < 0.05). However, compared with the model group, treatment with silymarin and AvPs2-1 significantly reduced liver index ( p The value <0.05 indicates that AvPs2-1, like silymarin, can significantly improve liver weight changes induced by CCl4 treatment.

[0088] 4. Effects of AvPs2-1 on enzymatic and non-enzymatic antioxidant activities Oxidative stress is one of the core mechanisms of liver fibrosis progression. Glutathione (GSH) and malondialdehyde (MDA) serve as markers of the non-enzymatic antioxidant system and lipid peroxidation, respectively, dynamically reflecting the liver's redox state. As a major intracellular antioxidant, GSH depletion can lead to mitochondrial dysfunction and apoptosis, thereby exacerbating the fibrosis process. In this study, as... Figure 16 As shown in e, compared with the normal control group, the level of GSH in the liver of the model group mice was significantly reduced ( p < 0.001). Treatment with AvPs2-1 significantly increased the GSH content in the liver of mice ( p < 0.001), approximately 2.38 times higher, even exceeding the level in the silymarin group, and comparable to the normal control group. Similarly, as a product of lipid peroxidation, MDA levels in the model group were also significantly higher than in the normal control group ( p < 0.01), indicating that CCl4 can induce severe lipid peroxidation damage in C57BL / 6J mice. After gavage administration of AvPs2-1, MDA levels were significantly reduced by 44%, and the levels were comparable to those in the positive control group. These results are consistent with the ROS scavenging capacity of cat ginseng polysaccharides observed in Case Study 3, suggesting that AvPs2-1 can maintain oxidative balance by directly scavenging reactive oxygen species (ROS) or activating endogenous antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT). This also demonstrates that antioxidant activity is one of the reasons why AvPs2-1 helps maintain normal liver function to some extent.

[0089] 5. Liver histopathological analysis Histopathological sections and staining of liver tissue were performed. For example... Figure 17 As shown in Figure a, the hepatocytes in the normal control group had uniform morphology, clear nuclei, and orderly structure. In contrast, the liver tissue in the CCl4 model group exhibited typical pathological features, including hepatocyte ballooning degeneration, inflammatory cell infiltration, and necrotic foci. After AvPs2-1 treatment, the hepatocytes recovered, with clear cell boundaries, visible nuclei, and reduced cell swelling.

[0090] To further investigate changes in collagen fibers within liver tissue, this study employed two staining methods: Masson's trichrome staining and Sirius red staining. Figure 17 As shown in b, Masson's trichrome staining revealed blue collagen fibers, indicating that liver fibrosis leads to the production and accumulation of collagen bundles. The results showed a significant increase in collagen fibers in the model group's liver, forming fibrous septa. In contrast, the polysaccharide treatment group showed well-organized hepatocytes, reduced inflammatory cells, decreased hepatocyte necrosis, and a significant reduction in collagen fibers, alleviating fibrosis and essentially restoring normal liver tissue structure. Figure 17 As shown in c, the semi-quantitative results of Masson's trichrome staining showed that, compared with the model group, both the AvPs2-1 treatment group and the positive control group (silymarin) significantly reduced the degree of liver fibrosis. p < 0.001). After staining with Sirius red, collagen fibers appear bright red or pink, while other tissue components such as cytoplasm and nuclei are stained yellow or green, creating a striking contrast with the red collagen fibers. For example... Figure 17 As shown in Figure d, collagen fibers significantly increased and formed fibrous septa in CCl4-induced mouse liver tissue; however, AvPs2-1 treatment reduced collagen deposition, with an effect almost comparable to the positive control group. Semi-quantitative analysis of Sirius red staining images showed that the degree of fibrosis in the model group was significantly higher than that in the normal control group. p < 0.001). After AvPs2-1 treatment, the degree of liver fibrosis was significantly reduced ( p < 0.001), the effect was comparable to the positive control group ( Figure 17 e).

[0091] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for extracting and purifying polysaccharides derived from the traditional Chinese medicine *Cat's Ginseng*, characterized in that: Includes the following steps: First, the raw material of cat ginseng was dried and pulverized, and pretreated with 95% ethanol for defatting. Then, using water as the extraction solvent, ultrasonic-assisted extraction was performed under the conditions of a liquid-to-solid ratio of 10-30 mL / g, ultrasonic power of 200-600 W, and extraction time of 30-60 min. After centrifugation, the supernatant of the extract was concentrated, and ethanol was added to precipitate crude polysaccharide. The crude polysaccharide was then subjected to deproteinization and decolorization treatment with macroporous adsorption resin, followed by dialyzing, and finally freeze-drying to obtain the crude polysaccharide of cat ginseng. The crude polysaccharide was further purified by DEAE-52 ion exchange column and Sephadex G-150 gel column chromatography to finally obtain three homogeneous polysaccharide fractions AvPs1-1, AvPs2-1, and AvPs2-2.

2. The method for extracting and purifying polysaccharides derived from the traditional Chinese medicine *Cat's Ginseng* according to claim 1, characterized in that: The drying temperature is 50-60℃, and the powder is pulverized through a 60-mesh sieve. The degreasing process is as follows: add 95% ethanol at a ratio of 1:20 (w / v), and ultrasonically degrease 3 times, each time for 1-2 hours.

3. The method for extracting and purifying polysaccharides derived from the traditional Chinese medicine *Cat's Ginseng* according to claim 1, characterized in that: The optimal parameters for ultrasonic extraction were obtained by optimization using a response surface methodology (RSM): the liquid-to-solid ratio was preferably 15 mL / g, the ultrasonic power was preferably 370 W, and the extraction time was preferably 46 min.

4. The method for extracting and purifying polysaccharides derived from the traditional Chinese medicine *Cat's Ginseng* according to claim 1, characterized in that: The separation and purification of the polysaccharide includes the following steps: the crude polysaccharide is purified sequentially by DEAE-Cellulose-52 ion exchange chromatography (gradient elution: deionized water, 0.15-0.6 M NaCl) and Sephadex G-150 gel chromatography. During the process, the elution curve is plotted by the phenol-sulfuric acid method and the fractions are combined. At the same time, the polysaccharide is treated with a 3500 Da dialysis membrane to finally obtain three components: AvPs1-1, AvPs2-1 and AvPs2-2.

5. The method for extracting and purifying polysaccharides derived from the traditional Chinese medicine *Panax ginseng* according to claim 4, characterized in that: The total content of AvPs2-1 and AvPs2-2 components accounts for more than 70% of the total polysaccharide extract; AvPs2-1 and AvPs2-2 are highly similar in structural features, while AvPs1-1 retains the core structural features of both and is a product of partial degradation of AvPs2-1 and AvPs2-2.

6. The method for extracting and purifying polysaccharides derived from the traditional Chinese medicine *Cat's Ginseng* according to claim 1, characterized in that: The three homogeneous polysaccharides after purification are all non-protein-bound polysaccharides with a triple helix conformation. Among them, AvPs1-1 is a white powder with an average molecular weight of approximately 1.93 kDa; while AvPs2-1 and AvPs2-2 are both pale yellow flocculent substances with huge molecular weights, both exceeding 2000 kDa. The monosaccharide composition of the three is mainly composed of galactose and arabinose, but the proportions of each monosaccharide (mannose:rhamnose:galacturonic acid:glucose:galactose:arabinose:fucose) are different, specifically 0.81:4.25:1.89:7.11:43.60:31.13:11.21, 3.82:8.41:1.49:4.36:42.80:29.40:9.72 and 2.73:7.16:2.95:6.75:47.77:23.22:9.

43.

7. The method for extracting and purifying polysaccharides derived from the traditional Chinese medicine *Cat's Ginseng* according to claim 1, characterized in that: The three homogeneous polysaccharide components of AvPs1-1 have a highly branched structure with a branching degree of 65.13%. Their monosaccharide linkages include terminal arabinofuranose, terminal fucopyranose, terminal galactopyranose, 5-linked arabinofuranose, 2-linked glucose, 3-linked galactose, 2-linked galactose, 4-linked galactose, 4-linked glucose, 2,3-linked mannose, 6-linked galactose, and 2,3-linked galactose.

8. The application of the polysaccharide derived from the traditional Chinese medicine *Cat's Ginseng* according to any one of claims 1-7, characterized in that: The polysaccharide, after separation and purification, yields three homogeneous components that possess in vivo developmental protective activity, enabling zebrafish embryos to develop normally under adverse conditions. It also exhibits in vivo anti-hepatic fibrosis activity, effectively inhibiting the process of liver fibrosis by exerting liver-protective effects, thus demonstrating broad application prospects.

9. The application of the polysaccharide derived from the traditional Chinese medicine *Cat Ginseng* according to claim 8, characterized in that: The polysaccharide AvPs2-1 component exhibits a protective effect against H2O2-induced oxidative stress in a zebrafish model. This effect is manifested in its ability to comprehensively alleviate the damage of oxidative stress to embryos by increasing the hatching rate of zebrafish embryos, reducing their heart rate, improving their body length, reducing their pericardial edema area, and inhibiting the excessive generation of reactive oxygen species. The polysaccharide AvPs2-1 component also plays a role in preventing and / or treating liver injury and liver fibrosis by inhibiting oxidative stress (manifested as increasing glutathione and decreasing malondialdehyde) and inhibiting hepatic stellate cell activation and extracellular matrix deposition (manifested as decreasing α-smooth muscle actin and type I collagen).

10. The application of the polysaccharide derived from the traditional Chinese medicine *Cat Ginseng* according to claim 9, characterized in that: The application of the extracted cat ginseng polysaccharide or its components in food, medicine or health products.

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