Kudzuvine root active polysaccharide as well as preparation method and application thereof

Abstract

The invention relates to the technical field of active polysaccharides, in particular to an arrowroot active polysaccharide as well as a preparation method and application thereof. In an infrared spectrum of the arrowroot active polysaccharide, the arrowroot active polysaccharide at least has characteristic absorption peaks at the following positions: 3317 cm, 2911 cm, 1404 cm <-1 >, 1021 cm <-1 > and 642 cm. The polysaccharide is extracted and separated from yam beans, and the polysaccharide has a protective effect in a mouse model with hepatic fibrosis induced by ligation of bile ducts, also has a regulating effect on intestinal microbiota, and exerts anti-fibrosis activity.

Description

Technical Field

[0001] This invention relates to the field of active polysaccharide technology, specifically to a jicama active polysaccharide, its preparation method, and its application. Background Technology

[0002] Liver fibrosis is a pathological condition caused by persistent liver damage, with diverse causes including hepatitis B virus (HBV) and hepatitis C virus (HCV) infection, alcoholic fatty liver disease, non-alcoholic steatohepatitis (NASH), and cholestasis. These diseases share common characteristics, including chronic liver tissue damage, secondary injury responses, inflammatory responses, and the formation of fibrosis. This process disrupts the dynamic balance between extracellular matrix (ECM) synthesis and degradation, leading to abnormal collagen accumulation and subsequent excessive fibrosis in the liver lobules and portal regions. Recent studies indicate that liver fibrosis is the initial stage of chronic liver damage and is reversible. The gut microbiota plays a crucial role in the occurrence and progression of diseases such as alcoholic liver disease, non-alcoholic liver disease, viral hepatitis, autoimmune hepatitis, and liver cancer. Impaired liver function increases the burden on the gut for uric acid excretion, leading to changes in the composition of the gut microbiota. Gut microecological imbalance can trigger local and systemic inflammatory responses, potentially exacerbating liver dysfunction. Comparative studies of fecal microbiota between healthy individuals and patients with cirrhosis have shown a significant decrease in the number of beneficial bacteria and a marked increase in harmful bacteria such as Enterobacteriaceae. These harmful bacteria can weaken the intestinal barrier function, reduce the production of short-chain fatty acids, and interfere with amino acid metabolism, potentially affecting the occurrence and development of liver diseases in multiple ways. Therefore, maintaining gut microbiota homeostasis and restoring its disordered metabolites may be a feasible strategy for preventing liver fibrosis.

[0003] Consuming foods rich in polysaccharides plays a crucial role in regulating the composition of the gut microbiota. Generally, diets rich in fruits, vegetables, dietary fiber, and various vitamins are closely associated with improved gut microbiota diversity. Jicama (Pachyrhizuserosus, PE) is a plant native to tropical and subtropical regions, also known as jicama or sweet potato. Its tuberous root is the main edible part. It is low in calories but rich in water, bioactive polysaccharides, and essential nutrients such as vitamin C, potassium, and antioxidants. Although jicama has a sweet taste, its glycemic index is low, making it an ideal food for diabetics. Furthermore, jicama polysaccharides have a certain effect on regulating the gut microbiota and possess potential anti-liver fibrosis effects. Based on this, this invention provides an active jicama polysaccharide, its preparation method, and its applications. Summary of the Invention

[0004] The present invention aims to provide an active polysaccharide of jicama, its preparation method and application.

[0005] This invention provides an active polysaccharide from jicama, the chemical formula of which is shown below: .

[0006] Furthermore, the infrared spectrum of the jicama active polysaccharide has characteristic absorption peaks at at least the following positions: 3317 cm⁻¹, 2911 cm⁻¹, and 1404 cm⁻¹. -1 1021cm -1 And 642cm⁻¹.

[0007] Furthermore, the ¹H and ¹³C NMR spectra of the active polysaccharide are located at C-2 carbon atoms in β-D-Fruf in the range of δ 104.01-103.23 ppm, H-4 in D-Fruf residues in the range of δ 4.00-4.15 ppm, and H-3 in D-Fruf residues in the range of 4.15-4.25 ppm; the ¹H NMR spectra are located at H-1 in α-D-glucopyranose (Glcp) in the range of δ 5.17-5.35 ppm and H-1 in β-D-Glcp in the range of δ 4.57-4.59 ppm.

[0008] On the other hand, the present invention also provides a method for preparing active polysaccharides from jicama, characterized in that the steps include: (1) Extraction and preliminary purification: After cutting the root of the jicama into sections, add boiling water for hot extraction; combine the extracts, concentrate and centrifuge to remove insoluble matter, and obtain supernatant A; (2) Deproteinization: Add Sevag reagent to supernatant A, shake and centrifuge to remove organic phase and interfacial protein. Repeat the operation until protein is completely removed to obtain deproteinized supernatant B. (3) Alcohol precipitation and crude polysaccharide acquisition: The supernatant B was dialyzed, and then ethanol was added to the dialysate for alcohol precipitation. The precipitate was collected, resuspended, and dried to obtain crude polysaccharide PEP. (4) Ion exchange chromatography: After dissolving and centrifuging the crude polysaccharide PEP, the supernatant was subjected to DEAE anion exchange column chromatography. Gradient elution was performed using deionized water and NaCl solutions of different concentrations. Each eluted fraction was collected and processed to obtain multiple polysaccharide subfractions PEP-W. (5) Gel filtration chromatography: After dissolving and centrifuging the polysaccharide subfraction PEP-W obtained in step (4), the supernatant was separated by passing it through a Sephacryl S-300 gel column with purified water as the mobile phase. The target elution peak was collected to obtain the purified jicama active polysaccharide fraction PEP-W-1.

[0009] Further, in step (1), the conditions for thermal extraction are: material-to-liquid ratio 1:(8-12) w / v, boiling water extraction, extraction for 1-3 hours each time, extraction 1-4 times; the concentration is to concentrate the combined extract to 1 / 8 to 1 / 12 of the original volume; the centrifugation is to centrifuge at 4000-6000×g for 10-20 minutes.

[0010] Further, in step (2), the Sevag reagent is prepared by mixing chloroform and n-butanol in a volume ratio of (2-4):1; the volume ratio of supernatant A to Sevag reagent is (3-5):1; the volume ratio of chloroform to n-butanol in the Sevag reagent is 3:1; the volume ratio of supernatant A to Sevag reagent is 4:1; the deproteinization operation is repeated 4-6 times.

[0011] Further, in step (3), the dialysis is performed using a dialysis bag with a molecular weight cutoff of 12-15 kDa, and the dialysis is carried out in flowing tap water and high-purity distilled water for 48-96 hours in sequence; the alcohol precipitation is performed by adding 2-4 times the volume of ethanol with a concentration of 70-90% v / v to the dialysis solution and letting it stand at 2-8℃ for 8-16 hours.

[0012] In step (3), a dialysis bag with a molecular weight cutoff of 14 kDa was used, and the total dialysis time was 72 hours. During alcohol precipitation, 3 times the volume of ethanol with a concentration of 80% v / v was added, and the mixture was left to stand overnight at 4°C.

[0013] In step (4), the DEAE anion exchange column is a DEAE Sepharose Fast Flow column; the concentration gradient of the NaCl solution includes 0.1 mol / L, 0.2 mol / L, and 0.3 mol / L; the polysaccharide subfraction PEP-W includes PEP-1, PEP-2, and PEP-3.

[0014] On the other hand, the present invention provides the application of the aforementioned active polysaccharide of jicama and the method for preparing the aforementioned active polysaccharide of jicama in the preparation of drugs and / or foods that regulate the intestinal microbiota and / or have potential anti-liver fibrosis effects.

[0015] The beneficial effects of this invention are as follows: This invention extracts and isolates polysaccharides from yam beans. These polysaccharides have a protective effect in a mouse model of liver fibrosis induced by bile duct ligation (BDL) and also have a regulatory effect on the gut microbiota (GM), thus exerting anti-fibrotic activity. Attached Figure Description

[0016] Figure 1The following are the separation characteristics of PEP-W-1: (A) Elution curve of crude PEP extracted via DEAE-Sepharose FastFlow column; (B) High-performance gel permeation chromatography (HPGPC) analysis of PEP-W-1; (C) High-performance anion exchange chromatography (HPAEC) chromatogram of standard monosaccharides; (D) FT-IR spectrum of PEP-W-1; (E) High-performance anion exchange chromatography (HPAEC) chromatogram of PEP-W-1; (F) GC-MS total ion chromatogram of partially methylated alcohol sugar acetates (PMAAs) of PEP-W-1. Figure 2 The diagram shows the structure of PEP-W-1, where (A) the structure of PEP-W-1 is shown using a structural formula; and (B) the structure of PEP-W-1 is shown using symbols. Figure 3 The image shows the partial protective effect of PEP-W-1 against BDL-induced liver fibrosis in mice. (A) Representative images of the liver appearance of mice in each group; (B) Weight of major organs / body weight ratio. Figure 4 The diagram shows the partial protective effect of PEP-W-1 against BDL-induced liver fibrosis in mice, specifically the serum ALT, ALP, and AST levels. Figure 5 The diagram shows the partial protective effect of PEP-W-1 against BDL-induced liver fibrosis in mice, specifically the changes in mouse body weight during the period. Figure 6 This image illustrates the partial protective effect of PEP-W-1 against BDL-induced liver fibrosis in mice. Specifically, it shows representative images of H&E, Masson, and EVG staining, as well as immunohistochemical staining of α-SMA and COL1A1 in R liver tissue, along with quantitative analysis of the corresponding fibrosis area. Detailed Implementation

[0017] The technical solution of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, 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.

[0018] Example 1: Preparation of PEP-W-1 Freshly harvested yam tubers were cut into approximately 3cm segments and extracted in boiling water at a material-to-liquid ratio of 1:10 (w / v). Each extraction lasted 2 hours and was repeated three times. The total volume of the three extracts was 5L, which was then concentrated by rotary evaporation at 40℃ to obtain 500mL of concentrate. The concentrate was then centrifuged at 5000×g for 15 minutes to remove impurities and obtain the supernatant.

[0019] The supernatant was deproteinized using the Sevag method. Specifically, Sevag reagent was prepared by mixing chloroform and n-butanol at a ratio of 3:1 (v / v), and the supernatant to Sevag reagent volume ratio was 4:1 (v / v), i.e., 125 mL of Sevag reagent was added to 500 mL of supernatant. After thorough shaking and mixing, the mixture was centrifuged to separate the layers, and the organic phase was discarded. This step was repeated 4–5 times until protein removal was complete.

[0020] The supernatant after treatment was concentrated again by rotary evaporation at 40°C for 30 min, and then dialyzed using a dialysis bag with a molecular weight cutoff of 14,000 Da in running water and distilled water for 72 h (with water changed 3–4 times daily). After dialysis, 3 times the volume of 80% ethanol was added to the dialysate to precipitate the polysaccharide, and the mixture was allowed to stand overnight at 4°C. The precipitate was collected by centrifugation, resuspended in a small amount of distilled water, and freeze-dried to obtain the crude polysaccharide PEP. For further purification, 10 g of crude polysaccharide was dissolved in 50 mL of distilled water, centrifuged at 10,000 × g for 10 minutes, and the supernatant was collected. Ion exchange separation was performed using a DEAE Sepharose Fast Flow column [GE Healthcare Ltd. (Stockholm, Sweden)], eluting sequentially with deionized water and NaCl aqueous solutions of different concentrations (0.1 mol / L, 0.2 mol / L, and 0.3 mol / L). After collecting each eluent, the fractions were concentrated, dialyzed, and freeze-dried to obtain multiple polysaccharide fractions: PEP-W, PEP-1, PEP-2, and PEP-3.

[0021] To further purify the main component, 100 mg of PEP-W was dissolved in purified water, centrifuged at 10,000 × g for 10 minutes, and the supernatant was collected and separated using a Sephacryl S-300 high-performance gel column with purified water as the mobile phase, finally obtaining the main polysaccharide component PEP-W-1.

[0022] PEP crude polysaccharide was obtained through water extraction, alcohol precipitation, and deproteinization. Subsequently, it was purified using DEAE-52 and Sephadex G-75 chromatography columns to finally obtain the single component PEP-W-1 (… Figure 1 The yield of polysaccharide A was 13.02±3.43%, compared to the crude polysaccharide extracted from yam and yam tubers; the purity (total sugar content) was 96.14±1.42%.

[0023] Example 2: Structural Characterization of PEP-W-1 (1) Molecular weight (Mw) analysis PEP-W-1 was dissolved in an aqueous solution containing 0.1 M sodium nitrate (NaNO3) and 0.02% (w / w) sodium azide (NaN3) to a final concentration of 1 mg / mL. The resulting solution was filtered through a membrane with a pore size of 0.45 μm before analysis. Its molecular weight was determined using gel permeation chromatography-differential refractive index detection-multi-angle laser light scattering (GPC-RI-MALS). The analytical system included a U3000 liquid chromatograph (Thermo, USA), an OptilabT-rEX differential refractive index detector (Wyatt Technology, CA, USA), and a DAWNHELEOSII multi-angle light scattering detector (Wyatt Technology, CA, USA).

[0024] The separation process was performed using two gel filter columns connected in series: Ohpak SB-805 HQ (300×8 mm) and Ohpak SB-803 HQ (300×8 mm). The column temperature was maintained at 45 °C, and the injection volume was 100 μL per injection. The mobile phase was a solution containing 0.02% (w / w) NaN3 and 0.1 M NaNO3 (denoted as mobile phase A), and the flow rate was set at 0.6 mL / min. The mixture was run in isocratic elution mode for 75 minutes.

[0025] The molecular weight distribution of PEP-W-1 was analyzed using high-performance gel permeation chromatography (HPGPC). The results showed that it exhibited a single symmetrical peak, indicating good homogeneity. Further analysis revealed that the average molecular weight (Mw) of PEP-W-1 was 261.63 kDa, and the polydispersity index (PDI) was 1.33. Figure 1 (B in the middle).

[0026] (2) Monosaccharide composition analysis 5 mg of PEP-W-1 sample was added to 1 mL of 2 M trifluoroacetic acid (TFA) and hydrolyzed at 121 °C for 4 hours. After the reaction was complete, the reaction solution was dried under a nitrogen stream. The residue was then dissolved in methanol and evaporated again under a nitrogen stream. This methanol dissolution-drying process was repeated three times to completely remove residual TFA. Finally, the reaction product was dissolved in distilled water and analyzed using a Thermo ICS 5000+ ion chromatography system (ICS 5000+, Thermo Fisher Scientific, USA) equipped with an electrochemical detector.

[0027] Chromatographic separation was performed using a Dionex™ CarboPac™ PA20 analytical column (150 × 3.0 mm, 10 μm particle size) with an injection volume of 5 μL. The mobile phase consisted of deionized water (elution A), 0.1 M sodium hydroxide (elution B), and a mixture of 0.1 M sodium hydroxide and 0.2 M sodium acetate (elution C). The column temperature was maintained at 30 °C, and the flow rate was set to 0.5 mL / min.

[0028] The monosaccharide composition of PEP-W-1 was analyzed using ion chromatography combined with a UV-Vis detector. By comparing the spectra with standard monosaccharides, PEP-W-1 showed two distinct characteristic peaks, indicating that it is composed of two monosaccharides. Figure 1 (C in the table). As shown in Table 1, the identified monosaccharides are glucose and fructose, with molar ratios of 95.16 and 2.68, respectively, with glucose being the main component.

[0029] (3) Methylation analysis First, 5 mg of dried polysaccharide sample PEP-W-1 was dissolved in 2 mL of anhydrous DMSO, followed by the addition of 50 mg of freshly ground NaOH powder. After stirring for 30 minutes under nitrogen protection, 0.5 mL of iodomethane was added dropwise. The reaction was continued at room temperature for 1 hour, then quenched with water, and the methylated product was extracted with chloroform. After methylation, the sample was hydrolyzed at 121 °C with a 2 mol / L trifluoroacetic acid solution for 1.5 hours. The solution was then evaporated, and the residue was dissolved in 100 μL of distilled water. Subsequently, the sample was reduced with sodium deuterated borohydride and acetylated at 100 °C with an acetic anhydride-trifluoroacetic acid (1:1 v / v) mixture for 2.5 hours.

[0030] The reacted acetate was dissolved in chloroform and analyzed using gas chromatography-mass spectrometry (GC-MS) with an Agilent 6890A-5977B instrument equipped with an Agilent BPX70 column (30m × 0.25mm × 0.25µm, SGE, Australia). High-purity helium was used as the carrier gas (split ratio 10:1) with an injection volume of 1μL. The experiment was performed by Shanghai Sanshu Biotechnology Co., Ltd.

[0031] The mass spectrometry analysis conditions were as follows: initial temperature of 140℃, held for 2.0 min; then increased to 230℃ at a rate of 3℃ / min, held for 3 min. The scanning mode was SCAN, and the scanning range was m / z 50-350.

[0032] To investigate the glycosidic bond types in PEP-W-1, the sample underwent three rounds of methylation to ensure complete methylation of the polysaccharide. Subsequently, the resulting partially methylated alcohol-glycosyl acetate (PMAA) was analyzed by gas chromatography-mass spectrometry (GC-MS), and a total ion current chromatogram (TIC) was plotted. Figure 1 (F in the text). The analysis results showed that seven different types of glycosidic bonds were detected in the PEP-W-1 derivative, and their corresponding molar ratios are detailed in Table 1.

[0033] Table 1. Methylation analysis of PEP-W-1 (4) Infrared spectroscopy and thermogravimetric analysis The functional groups in the samples were identified using a Fourier transform infrared spectrometer (IRTracer-100AH, Shimadzu Corporation, Japan); the main thermodynamic properties of PEP-W-1 were characterized using a thermogravimetric analyzer (TGA, PerkinElmer TGA-4000, Waltham, MA, USA).

[0034] like Figure 1 As shown in Figure D, PEP-W-1 exhibits typical polysaccharide absorption peaks at 3317 cm⁻¹ and 2911 cm⁻¹, corresponding to the stretching vibrations of the hydroxyl group (-OH) and the carbon-hydrogen bond (CH), respectively. 1404 cm⁻¹ -1 The absorption peak is attributed to the stretching vibration of the CO bond, 1021 cm⁻¹. -1 The absorption peak is related to bound water. In addition, the absorption peak at 642 cm⁻¹ indicates the presence of an α-configuration pyranose ring structure in PEP-W-1.

[0035] (5) Nuclear magnetic resonance spectroscopy (NMR) analysis The lyophilized PEP-W-1 was dissolved in heavy water (D2O) to a final concentration of 50 mg / mL. The sample was subjected to one-dimensional and two-dimensional nuclear magnetic resonance analysis, including ¹H NMR, ¹³CNMR, COSY, NOESY, HMBC and HSQC, using a Bruker AVANCE NEO 400 MHz nuclear magnetic resonance spectrometer (Bruker, Rheinstetten, Germany) at 25 °C.

[0036] The ¹H and ¹³C NMR spectra of PEP-W-1 exhibit typical fructan chemical shift characteristics. In the ¹³C NMR spectrum, two signal peaks in the δ 104.01–103.23 ppm range are attributed to the C-2 carbon atom in the β-D-fructofuranose residue (β-D-Fruf). Broader signal peaks were observed in the mid-to-low field regions (δ 4.00–4.15 ppm and 4.15–4.25 ppm), which are attributed to the H-4 and H-3 protons in the D-Fruf residues, respectively.

[0037] In the ¹H NMR spectrum, the signal at δ 5.17–5.35 ppm corresponds to the chemical shift of H-1 in α-D-glucopyranose (Glcp), while the signal at δ 4.57–4.59 ppm is attributed to the proton signal of H-1 in β-D-Glcp.

[0038] The HSQC spectrum of PEP-W-1 shows eight distinct cross peaks at δH / C chemical shifts of 5.34 / 92.1, 5.35 / 99.5, 4.57 / 95.8, 4.59 / 95.8, and 5.17 / 92.0 ppm, corresponding to residues A, B, D, E, F, and G, respectively. Based on methylation analysis, monosaccharide composition, and NMR data, the structures of the seven sugar residues in PEP-W-1 are: α-D-Glcp-(1→, →4)-α-D-Glcp-(1→, →2,4)-β-D-Glcp-(1→, →3,4)-β-D-Glcp-(1→, →4,6)-α-D-Glcp-(1→, β-D-Fruf-(2→) and →1,6)-β-D-Fruf-(2→), corresponding to residues A, B, D, E, G, I, and J, respectively (see Table 2).

[0039] Table 2 NMR Assignments of PEP-W-1 In the COSY spectrum, the cross peaks at 4.16 / 4.04 ppm, 4.04 / 3.76 ppm, 3.76 / 3.65 ppm, and 3.81 ppm are attributed to H3 / H4, H4 / H5, and H5 / H6 of residue I, respectively. Residue J also exhibits similar correlation signals, further supporting the determination of its structural characteristics.

[0040] By analyzing the relevant cross-peaks in the HMBC spectrum of PEP-W-1, the connection sites and their connection order between different sugar residues were further verified. The relevant peaks observed at δ5.34 / 76.2ppm (AH-1 / GC-4) indicate that the O-1 of residue A is linked to the C-4 of residue G. The cross peaks 4.57 / 76.5ppm (DH-1 / BC-4), 5.34 / 76.5ppm (AH-1 / DC-4), 5.34 / 76.2ppm (AH-1 / EC-4), 5.35 / 103.6ppm (BH-1 / JC-2), 3.62 / 63.7ppm (JH-1 / GC-6), 3.51 / 104.1ppm (JH-6 / IC-2), and 3.20 / 95.8ppm (DH-2 / EC-1) indicate that: the O-1 of residue D is linked to the C-4 of residue B; the O-1 of residue A is linked to the C-4 of residues D and E, respectively; the O-1 of residue B is linked to the C-2 of residue J; the O-1 of residue J is linked to the C-6 of residue G; the O-6 of residue J is linked to the C-2 of residue I; and the O-2 of residue D is linked to the C-1 of residue E.

[0041] Furthermore, the cross peak observed at δ 5.35 / 76.5 ppm (BH-1 / BC-4) indicates that the O-1 of residue B is linked to its own C-4, suggesting that residue B exists as a repeating unit in the PEP-W-1 structure. Similarly, the cross peaks at 4.57 / 75.6 ppm (DH-1 / DC-2), 4.59 / 75.8 ppm (EH-1 / EC-3), 3.63 / 92.0 ppm (GH-6 / GC-1), and 3.62 / 103.6 ppm (JH-1 / JC-2) further reveal that residues D, E, G, and J also exist in a repeating form in the PEP-W-1 structure.

[0042] Based on the molecular weight determination, monosaccharide composition analysis, GC-MS methylation analysis, and NMR data of PEP-W-1, its main structure is as follows: Figure 2 As shown.

[0043] Example 3 Animal Experiment (1) Laboratory animals Male C57BL / 6J mice aged 4-6 weeks and weighing 20-25g were selected. Mice were housed in a constant temperature (22±2℃) environment with 12-hour light / dark cycles and free access to food and water. All animal experimental procedures were conducted in strict accordance with the approval of the Animal Ethics Committee of Wenzhou Medical University (Approval No.: WYYY-IACUC-AEC-2025-092) and relevant animal ethics guidelines.

[0044] (2) Experimental design After a one-week acclimatization period, mice were weighed and randomly assigned to groups based on their body weight (average 22g). The experiment consisted of four groups: a control group (sham-operated group), a model group (BDL group), a low-dose PEP-W-1 group (LPEP, 50mg / kg), and a high-dose PEP-W-1 group (HPEP, 200mg / kg). In the sham-operated group, only a midline abdominal incision was made and the wound sutured; no bile duct ligation or other treatment was performed.

[0045] The specific procedure for bile duct ligation (BDL) surgery was as follows: Mice were anesthetized with a combination of ketamine (90 mg / kg), xylazine (10 mg / kg), and clofenamic acid (5 mg / kg). During the surgery, a midline incision was performed under sterile conditions to expose the common bile duct, which was then ligated using 0-5 silk sutures. In the sham surgery group, the bile duct was only lightly touched without ligation; the remaining procedures were the same as in the BDL group. Postoperatively, the abdominal wall was sutured, and the mice were placed on a heating pad for resuscitation. One day postoperatively, the appearance of darkened urine and yellowing of the ears indicated successful establishment of the cholestasis model.

[0046] The BDL group received surgery only without drug intervention; the LPEP and HPEP groups received daily intraperitoneal injections of PEP-W-1 (50 mg / kg and 200 mg / kg, respectively) starting from the second day after surgery, continuing until day 14 (e.g., Figure 1 (As shown). Mouse body weight was recorded daily throughout the administration period.

[0047] On day 14, blood samples were collected via the retroorbital venous plexus, and mice were euthanized by cervical dislocation. Major organs were then dissected, and the liver weight / body weight ratio (%LW / BW) was calculated. Liver tissue samples were stored at −80℃ for later use. Blood samples were centrifuged at 4℃ and 10,000 rpm for 10 minutes to separate plasma. The levels of bilirubin, aspartate aminotransferase (SGOT), alanine aminotransferase (SGPT), and alkaline phosphatase (ALP) in the plasma were measured using commercial kits. The remaining liver tissue was used for protein expression detection (Western blot), oxidative stress marker analysis, and histopathological observation.

[0048] (3) Detection of plasma liver function enzyme indicators To assess the extent of liver damage caused by bile duct ligation (BDL), blood samples were collected from mice. After centrifugation to separate the plasma, multiple liver function biomarkers, including alanine aminotransferase (ALT), alkaline phosphatase (ALP), aspartate aminotransferase (AST), and total bilirubin, were measured using enzymatic methods according to the kit instructions.

[0049] (4) Histopathological and immunohistochemical evaluation of liver tissue To perform histopathological analysis on liver tissue samples from mice in different experimental groups, the fixed tissue was first embedded in paraffin and sectioned to a thickness of 5 μm. After dewaxing and rehydration, the paraffin sections were stained with hematoxylin and eosin (H&E) for histological observation. Some liver tissue sections were further stained with Masson's trichrome to assess the degree of liver fibrosis; the number, length, and thickness of fibrous septa were measured under blinded conditions.

[0050] In immunohistochemical analysis, to detect α-SMA expression, tissue sections were first blocked with 5% bovine serum albumin (BSA) at room temperature for 1 hour, followed by overnight incubation with mouse anti-human α-SMA primary antibody (1:200, Abcam) at 4°C. After washing, horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG secondary antibody was added, and the reaction was carried out at room temperature for 30 minutes. A horseradish peroxidase-labeled streptavidin-biotin detection system (Biotechnology Co., Ltd., Fuzhou, China) was used.

[0051] The percentage area of ​​fibrotic regions in CLILA1 and α-SMA staining was analyzed and quantitatively assessed using ImageJ software (version 1.53k, National Institutes of Health, Bethesda, MD, USA).

[0052] (5) Immunoblot analysis of fibrosis markers Proteins were extracted from liver tissue homogenates using RIPA lysis buffer containing protease inhibitors and benzyl sulfonyl fluoride (PMSF). Western blot analysis was performed to detect the expression level of the target proteins. Extracted protein samples were separated by 10–15% SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The membranes were blocked with 5% bovine serum albumin (BSA) at room temperature for 1 hour to prevent non-specific binding, followed by incubation overnight at 4°C with a specific primary antibody (1:1000 dilution). After washing, HRP-labeled secondary antibody was added, and the reaction was carried out at room temperature for 1 hour.

[0053] Imaging was performed using a ChemiDoc imaging system (Bio-Rad Laboratories, Hercules, CA, USA) with enhanced chemiluminescence reagent (ECL, Bio-Rad) for color development. Protein band intensity was quantitatively analyzed using ImageJ software.

[0054] (6) Real-time quantitative PCR (RT-qPCR) analysis Total RNA was extracted from liver tissue following the kit instructions and administered using TRIzol reagent (Invitrogen, CA, USA). Reverse transcription was performed using the RevertAid First Strand cDNA Synthesis Kit (ThermoFisher Scientific, Waltham, MA, USA). Real-time quantitative PCR (qPCR) was performed using HOT FIREPol EvaGreen qPCR Mix Plus (Solis Biodyne, Tartu, Estonia) on a PikoReal Real-Time PCR system (Thermo Fisher Scientific).

[0055] The amplification program was as follows: initial pre-denaturation at 95℃ for 15 minutes, followed by 40 cycles, each cycle consisting of 95℃ for 15 seconds, 60℃ for 20 seconds, and 72℃ for 20 seconds. The relative expression levels of mRNA were determined using... The method was used for calculation, and β-Actin was used as an internal reference gene for standardization.

[0056] Primer sequences were designed using Primer3 software (version 0.4.0) and validated using Netprimer software (Premier Biosoft, CA, USA). Primer sequences are detailed in Table 1.

[0057] (7) Gut microbiota analysis On day 8, cecal contents were collected from mice and immediately stored at −80°C. Genomic DNA of the microbiota was extracted using the EZNA® Soil DNA Kit (Omega Bio-tek, Norcross, GA, USA) strictly following the kit instructions. The extracted DNA was used as a template to amplify the V3-V4 variable region sequence of the 16S rRNA gene using primers 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) (Liu et al., 2016). PCR amplification was performed on an ABI GeneAmp® 9700 thermal cycler (ABI, USA). After purification, the amplified products were used to construct libraries using the NEXTFLEX Rapid DNA-Seq Kit and then subjected to high-throughput sequencing on an Illumina MiSeq PE300 or NovaSeq PE250 platform.

[0058] To assess the differences in gut microbiota among groups, various methods, including Venn diagrams, α-diversity index, Bray-Curtis distance matrix, principal coordinate analysis (PCoA), and β-diversity analysis, were used for statistical analysis and visualization. Furthermore, linear discriminant analysis of effect size (LEfSe) and genus-level relative abundance heatmaps were used to identify significantly dominant bacterial species.

[0059] Meanwhile, the PICRUSt2 microbiome function prediction tool based on phylogenetic information was used to predict the function of 16S rRNA gene sequences, and the abundance of metabolic pathways was analyzed in conjunction with the KEGG (Kyoto Encyclopedia of Genes and Genomes) database to screen out metabolic pathways with significant differences among groups.

[0060] (8) Statistical analysis All data are expressed as mean ± standard deviation (mean ± SD), and all experiments were repeated three times. Statistical analysis was performed using GraphPad Prism 8 software. One-way ANOVA was used to compare differences between groups, and a p-value < 0.05 was considered statistically significant.

[0061] The test results are as follows: (1) PEP-W-1 alleviates BDL-induced liver fibrosis in mice To assess the progression of liver fibrosis, histopathological and biochemical analyses of liver tissue from mice in each experimental group were performed at week 4 post-treatment. Compared to the sham-operated group, mice in the BDL group exhibited typical pathological changes such as yellow ascites, fibrous nodules on the liver surface with irregular morphology, and disordered hepatic lobule ratios. Figure 3 (A in the text). In the groups treated with PEP-W-1 (50 mg / kg and 200 mg / kg), the aforementioned pathological damage was significantly improved, and showed a dose-dependent remission trend.

[0062] Considering that liver fibrosis is usually accompanied by increased liver weight and liver index, liver mass was measured after mouse sacrifice to assess the degree of fibrosis and the effect of treatment. Results showed that the liver index in the BDL group was approximately 1.4 times higher than that in the sham-operated group, indicating successful model establishment. Compared with the BDL group, the liver index in the PEP-W-1 treatment group was significantly lower (…). Figure 3 The quality of organs B was significantly different from that of other major organs.

[0063] BDL can cause significant hepatocellular damage and fibrosis, leading to elevated levels of various liver function indicators. Figure 4Compared with the sham-operated group, BDL significantly increased ALP and AST levels, indicating cholestasis and liver injury. Both low-dose and high-dose PEP-W-1 reduced ALP levels to varying degrees, although the reduction did not return to normal levels, indicating that PEP-W-1 has a certain protective effect against BDL-induced liver injury. Similarly, ALT levels were significantly elevated in the BDL group, while both doses of PEP-W-1 reduced ALT levels, with the high-dose group showing a more significant effect, suggesting that it can dose-dependently alleviate liver injury and hepatocellular damage.

[0064] In addition, mouse body weight was monitored daily throughout the experiment. The control group showed no significant change in body weight, while the BDL group experienced a significant decrease; whereas the PEP-W-1 treated group showed a gradual increase in body weight compared to the BDL group. Figure 5 ).

[0065] Histological observations after HE staining showed that the liver tissue structure of the control group mice was normal, while the BDL group showed obvious tissue damage and fibrosis. Figure 6 In contrast, PEP-W-1 treatment improved liver tissue structure in a dose-dependent manner, with the 200 mg / kg group showing the most significant reduction in fibrosis, demonstrating the effects of promoting tissue regeneration, protecting hepatocytes from necrosis and damage, and restoring normal liver structure.

[0066] This result was also confirmed by Masson's trichrome staining: almost no collagen deposition was found in the liver tissue of the control group, while a large amount of collagen deposition was found in the BDL group, indicating severe fibrotic lesions. PEP-W-1 treatment significantly reduced collagen deposition, and the effect decreased with increasing dose, with the 200 mg / kg group showing the smallest area of ​​fibrotic tissue.

[0067] Similarly, Elastin-Van Gieson (EVG) staining results showed extensive proliferation of elastic fibers in the liver tissue of the BDL group, indicating late-stage fibrosis. With increasing PEP-W-1 dosage, the content of elastic fibers gradually decreased, with the high-dose group showing the most significant inhibitory effect. These results further confirm the role of PEP-W-1 in alleviating BDL-induced liver fibrosis.

[0068] To further verify the anti-fibrotic effect of PEP-W-1 in vivo, we analyzed the expression of key fibrosis markers at the protein and mRNA levels. Figure 6 Collagen type I (COL1A1), a major component of the extracellular matrix, and α-smooth muscle actin (α-SMA), a marker of hepatic stellate cells (HSCs) activation, were detected by immunohistochemistry, Western blot, and real-time quantitative PCR (qPCR), respectively.

[0069] Immunohistochemical staining results showed that the expression of COL1A1 and α-SMA in the liver tissue of BDL group mice was significantly upregulated, indicating a significant fibrotic response and HSC activation. Figure 6 In the PEP-W-1 treatment group, the staining intensity of COL1A1 and α-SMA decreased significantly with increasing dose, with the 200 mg / kg group showing the most significant inhibitory effect, exhibiting a clear dose-dependent relationship.

[0070] Western blot analysis further confirmed the above results: compared with the control group, the levels of COL1A1 and α-SMA proteins in the BDL group were significantly increased; PEP-W-1, especially under high-dose treatment, significantly inhibited the expression of these two proteins, indicating that it can effectively block the accumulation of fibrosis-related proteins.

[0071] To further validate the anti-fibrotic effect of PEP-W-1 at the transcriptional level, qPCR was used to detect the mRNA expression levels of fibrosis-related genes Fn1, Acta2, COL1A1, and COL3A1. The results showed that the transcriptional levels of these genes were significantly upregulated in the BDL group, reflecting the active state of the fibrosis signaling pathway. In contrast, the PEP-W-1 treatment group significantly downregulated the expression of these genes in a dose-dependent manner, further supporting its anti-fibrotic potential at the transcriptional level.

[0072] In summary, the above results fully demonstrate that PEP-W-1 significantly alleviates the progression of liver fibrosis by inhibiting the expression of key fibrosis markers at both the transcriptional and translational levels.

[0073] (2) PEP-W-1 improves BDL-induced liver fibrosis through a gut microbiota-dependent mechanism. To investigate whether the anti-liver fibrosis effect of PEP-W-1 depends on the gut microbiota, this invention established a bile duct ligation (BDL) model and then used antibiotic pretreatment to create a pseudo-germ-free mouse model to evaluate its efficacy. In conventionally fed mice, PEP-W-1 treatment significantly alleviated the pathological changes such as liver swelling and cholestasis induced by BDL. However, in the antibiotic-treated group (Abx + PEP-W-1), its tissue protective effect was significantly weakened, suggesting that the gut microbiota plays a key regulatory role in the action of PEP-W-1.

[0074] Serum biochemical analysis showed that PEP-W-1 significantly reduced the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and total bilirubin (TBIL) in BDL mice, while this hepatoprotective effect was significantly weakened in the Abx+PEP-W-1 group. These results indicate that the liver-restorative effect of PEP-W-1 depends on the presence of gut microbiota.

[0075] Histopathological examination further supported the above findings. HE staining showed extensive hepatocellular necrosis and inflammatory cell infiltration in the liver tissue of the BDL group, while PEP-W-1 significantly improved the aforementioned pathological damage in conventionally fed mice. Masson trichrome staining and EVG staining results showed significant collagen and elastic fiber deposition in the liver tissue of BDL group mice, and PEP-W-1 significantly inhibited the formation of fibrous tissue, but this effect was significantly weakened in antibiotic-treated (Abx) mice.

[0076] Immunohistochemical staining further confirmed the above trend: in conventional mice, PEP-W-1 significantly inhibited the expression of α-SMA and COL1A1, suggesting that it can effectively block the activation of myofibroblasts and the excessive accumulation of collagen; however, in the Abx group, the inhibitory effect of PEP-W-1 on these two fibrosis markers was significantly weakened.

[0077] Western blot results also showed that, compared with the sham-operated group, the protein expression levels of COL1A1 and α-SMA were significantly increased in the BDL group. As expected, PEP-W-1 significantly reduced the expression levels of the above two proteins in conventional mice; while in antibiotic-pretreated mice, this inhibitory effect was significantly weakened, suggesting that the anti-fibrotic effect of PEP-W-1 is significantly reduced in the absence of gut microbiota.

[0078] Consistent with changes in protein levels, qPCR analysis showed that the transcriptional levels of fibrosis-related genes Fn1, Acta2, Col1a1, and Col3a1 were significantly upregulated in the BDL group. In mice with intact gut microbiota, PEP-W-1 significantly reversed the abnormal expression of these genes; however, in the antibiotic-treated group, PEP-W-1 failed to effectively downregulate the expression levels of these genes.

[0079] The above results further indicate that the anti-fibrotic effect of PEP-W-1 largely depends on the presence of gut microbiota.

[0080] In summary, the results indicate that the anti-fibrotic and hepatoprotective effects of PEP-W-1 are significantly gut microbiota-dependent, highlighting the key regulatory role of the gut-liver axis in its efficacy.

[0081] (3) PEP-W-1 regulates the gut microbiota in mice with BDL-induced liver fibrosis To investigate whether the effect of PEP-W-1 on liver fibrosis is related to gut microbiota regulation, this invention performed 16S rRNA gene sequencing analysis on fecal samples from mice in the control group, BDL group, and PEP-W-1 treatment group. α-diversity analysis showed that the abundance and diversity of microorganisms in the BDL group were significantly decreased, as evidenced by significantly lower Chao1, ACE, Shannon, and Simpson indices compared to the control group. However, after PEP-W-1 treatment, these diversity indices significantly rebounded, suggesting that PEP-W-1 can alleviate BDL-induced dysbiosis and enhance the richness and diversity of the gut microbiota.

[0082] β-diversity analysis based on Bray-Curtis distance, further demonstrated by PCoA and NMDS plots, revealed significant differences in gut microbiota composition among the three groups. Specifically, the gut microbiota structure of the BDL group deviated significantly from both the control and PEP-W-1 groups, while the gut microbiota composition of the PEP-W-1 treatment group partially recovered and approached that of the control group. This indicates that PEP-W-1 can remodel the gut microbiota dysbiosis caused by cholestatic injury.

[0083] In phylum-level taxonomic analysis, significant changes occurred in the gut microbiota composition among different treatment groups. BDL-induced liver fibrosis led to a significant increase in the relative abundance of Proteobacteria, while Bacteroidetes and Actinobacteria were significantly reduced. PEP-W-1 treatment significantly reversed these changes, particularly with a significant increase in Bacteroidetes abundance and a significant decrease in Proteobacteria (often considered a marker of dysbiosis and inflammation) even when the relative abundance of Firmicutes remained unchanged. This suggests that PEP-W-1 can restore microbial balance by inhibiting harmful bacteria and promoting the recovery of beneficial bacteria.

[0084] Furthermore, the Firmicutes / Bacteroidetes (F / B) ratio, an important indicator of gut microbiota homeostasis, was significantly elevated in the BDL group, but tended to return to normal levels after PEP-W-1 treatment, further supporting the role of PEP-W-1 in restoring gut microbiota balance.

[0085] In summary, these results suggest that the anti-hepatic fibrosis effect of PEP-W-1 may be closely related to its ability to alleviate BDL-induced gut microbiota dysbiosis, particularly in its role in inhibiting excessive Proteobacteria proliferation and regulating the F / B ratio. This highlights the potential regulatory mechanism of the gut-liver axis in the PEP-W-1-induced liver fibrosis process.

[0086] To further elucidate the microbiome alteration mechanism behind the effects of PEP-W-1, this invention analyzed the composition of the gut microbiota at the genus level. Compared with the control group, the BDL group mice showed a significant increase in potentially pathogenic genera and a significant decrease in beneficial commensal genera. PEP-W-1 treatment significantly restored the relative abundance of several key genera, suggesting its ability to reshape a dysregulated gut ecosystem.

[0087] Specifically, in the BDL group, the effects closely related to anti-inflammatory activity and mucosal barrier maintenance were observed. Lactobacillus , Muribaculum and Bacteroides The relative abundance of certain probiotic genera decreased significantly after administration of PEP-W-1, while it significantly increased after administration, suggesting that PEP-W-1 can enhance intestinal stability. Conversely, in the BDL group... Escherichia-Shigella , Desulfovibrio and Enterococcus The levels of pro-inflammatory or opportunistic pathogens were significantly increased, while PEP-W-1 significantly inhibited the abnormal proliferation of these bacteria, further verifying its role in regulating the structure of the intestinal flora.

[0088] PEP-W-1 almost completely eliminated typical endotoxin-producing bacteria. Escherichia-Shigella This suggests that it may effectively reduce the load of gut-derived lipopolysaccharide (LPS), thereby alleviating systemic inflammatory responses. In summary, these results indicate that PEP-W-1 not only restores overall gut microbiota diversity but also selectively enriches beneficial bacteria and inhibits potentially pathogenic bacteria, thus achieving a protective effect against liver fibrosis through a microbiota-mediated mechanism.

[0089] The aforementioned changes in the gut microbiota structure may help enhance intestinal barrier function, reduce endotoxemia levels, and inhibit abnormal activation of hepatic stellate cells in a bile duct ligation model, thereby alleviating the progression of liver fibrosis.

[0090] To identify specific bacterial species involved in the PEP-W-1 microbial-mediated effects, this invention employs species-level taxonomic analysis and differential abundance analysis. BDL-induced liver fibrosis leads to significant changes in the gut microbiota composition, characterized by a substantial reduction in beneficial symbiotic bacteria and a large proliferation of pro-inflammatory bacteria. PEP-W-1 treatment, to some extent, restores the gut microbiota ecological structure, specifically manifested in increased microbial diversity and a rebalancing of the abundance of several key bacterial groups.

[0091] Further statistical comparative analysis showed that, compared with the control group, the abundance of several probiotic species in the BDL group was significantly decreased, including Ligilactobacillusmurinus , Roseburiasp.1XD42-69 , Bacteroidesacidifaciens as well as BifidobacteriumpseudolongumThis suggests that its progression during fibrosis was inhibited. Simultaneously, BDL promotes the abnormal proliferation of certain pro-inflammatory or dysbiosis-related bacterial species, such as... Candidatus Amulumruptor caecigallinarus and Akkermansia muciniphila The latter has been reported to be closely related to the degradation of the intestinal mucus layer and the disruption of barrier function.

[0092] Importantly, comparing the BDL and PEP-W-1 treatment groups revealed that PEP-W-1 significantly reduced... Candidatus Amulumruptor caecigallinarus and Akkermansia muciniphila The relative abundance of [unclear] was increased, while the abundance of various probiotic strains was also upregulated, including [unclear]. Lactobacillus taiwanensis , Bifidobacterium pseudolongum and Roseburia sp. 1XD42-69 .

[0093] The above results indicate that PEP-W-1 helps improve the imbalance of the gut microbiota by inhibiting the proliferation of pathogenic bacteria and enriching specific probiotic species, thereby exerting its microbial-mediated anti-fibrotic effect.

[0094] The aforementioned changes in the microbiota indicate that PEP-W-1 can effectively reverse the dysbiosis associated with liver fibrosis. Further findings suggest that PEP-W-1 exhibits selective remodeling at the species level, enriching symbiotic bacteria that produce short-chain fatty acids (SCFAs) and possess anti-inflammatory properties, while inhibiting the proliferation of mucin-degrading bacteria and endotoxin-associated flora. This precise regulation of the core microbiota may help restore intestinal barrier integrity and, to some extent, alleviate the occurrence and progression of liver fibrosis.

[0095] (4) PEP-W-1 remodels the CAZyme functional spectrum of gut microbiota in mice with BDL-induced liver fibrosis. As mentioned earlier, polysaccharides such as PEP-W-1 are mainly fermented and utilized by gut microbes after oral ingestion, a process that depends on the participation of carbohydrate-active enzymes (CAZymes). CAZymes can degrade complex glycoside structures to generate fermentable monosaccharides and bioactive metabolites.

[0096] To investigate the regulatory role of PEP-W-1 on the functional potential of gut microbiota in a BDL-induced liver fibrosis model, this invention performed functional annotation on metagenomic sequencing data based on the CAZy database and compared the abundance distribution characteristics of the CAZyme family among different experimental groups.

[0097] PLS-DA analysis showed that the Control group, BDL group and PEP-W-1 treatment group exhibited a clear clustering and separation trend in the functional spectrum of microbial CAZyme (carbohydrate active enzyme), indicating that PEP-W-1 treatment can remodel the composition of carbohydrate degrading enzymes that were disordered due to BDL damage.

[0098] The PEP-W-1 treatment group showed significant enrichment of various CAZyme family enzymes, including glycoside hydrolases (GH77, GH57, GH3, GH13, GH4, GH32, GH5, GH116), glycosyltransferases (GT9, etc.), and carbohydrate-binding modules (CBM48, CBM6). According to annotations in the CAZy and CAZypedia databases, these enzymes are widely involved in the degradation of plant-derived polysaccharides.

[0099] For example, the GH13 and GH57 families encode various α-amylases, 4-α-glucantransferases, and pullulanases, which can hydrolyze α-1,4 and α-1,6 glycosidic bonds; the GH3, GH4, and GH116 families contain various β-glucosidases and MLG-terminal o-β-1,3 / 1,4-glucanases, targeting β-1,3, β-1,4, and mixed-bond glucan structures; GH31 contains α-glucosidases and dextranases, which act on α-1,6 glycosidic bonds in the dextran side chain; and the GH77 enzymes possess glucantransferase activity and can reconstruct the α-glucan backbone structure, indicating that they not only participate in terminal degradation but also have the ability to remodel structures.

[0100] In addition, GH32 family members such as inulinase and fructosylase can target β-2,1 and β-2,6 fructosyl bonds, suggesting that microorganisms may utilize fructan branches in PEP-W-1 or co-ferment dietary fiber.

[0101] To further explore the microbial origin of these enzymes, this invention analyzed the association between the enrichment of the CAZyme family and specific bacterial species. Gene layer prediction results based on metagenomic annotation showed that the abundance enrichment of CAZyme was closely related to the amplification of several key microbial species. In the PEP-W-1 group, Akkermansia muciniphila , Roseburia sp. 1XD42-69 , Bifidobacterium pseudolongum as well as Ruminococcus flavefaciens The abundance of certain strains increased significantly and they were predicted to encode enzymes related to the degradation of glucan and fructan, such as GH116, GH13, GH3, GH31 and GT4, which is highly consistent with the structural characteristics of PEP-W-1.

[0102] In summary, PEP-W-1 can promote the amplification of microbial groups with specific metabolic capabilities for their structural features, thereby driving the reconstruction of the CAZyme functional spectrum in the gut microbiota. This process may be one of the important mechanisms by which it mediates anti-liver fibrosis effects through gut microbiota.

[0103] (5) PEP-W-1 targets functional genes encoded by gut microbiota to enhance short-chain fatty acid (SCFA)-related metabolic pathways. To investigate whether PEP-W-1 exerts its anti-liver fibrosis effect by regulating molecular pathways derived from gut microbiota, this invention used metagenomic shotgun sequencing data and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database to annotate and analyze microbial functional genes. Functional annotation results showed that BDL significantly altered the expression of gut microbial functional genes, with 527 KEGG orthologs (KOs) showing significant changes in the BDL model (p<0.05). Among these, PEP-W-1 treatment selectively restored the expression of 73 KOs, indicating a targeted reprogramming effect on microbial function. These differentially expressed KO genes are mainly involved in functional pathways such as carbohydrate metabolism, energy metabolism, and cofactor biosynthesis.

[0104] Further functional annotation revealed that PEP-W-1 significantly enriched multiple KO genes associated with short-chain fatty acid (SCFA) synthesis, particularly concentrated in pathways such as glycolysis / gluconeogenesis, carbon metabolism, pyruvate metabolism, and butyrate metabolism. Specifically, KO genes K00929 and K01034, involved in butyrate synthesis, were significantly upregulated in the PEP-W-1 group, suggesting enhanced microbial butyrate synthesis capacity. Meanwhile, K01689, a multifunctional enzyme present in glycolysis / gluconeogenesis and carbon metabolism, was associated with the synthesis of acetic acid, propionic acid, and butyrate, reflecting the overall activation of carbohydrate fermentation degradation pathways.

[0105] Notably, K00627 (encoding pyruvate-flavin oxidoreductase and other pyruvate metabolism-related enzymes) was enriched in multiple pathways, including glycolysis, pyruvate metabolism, and the tricarboxylic acid cycle (TCA), revealing the linkage mechanism of central carbon flow to downstream SCFA products. Similarly, K00134, as one of the key enzymes in glycolysis and carbon metabolism, is also closely related to the synthesis of three major SCFAs (acetic acid, propionic acid, and butyric acid), indicating its central role in microbial energy metabolism.

[0106] The above results indicate that PEP-W-1 can reshape the gut microbiota functional map and enhance the synthesis of SCFAs, especially butyrate, which may help improve the host's metabolic state and intestinal barrier function, thereby exerting an anti-fibrotic effect.

[0107] To further clarify the microbial origin of these SCFA-related KO genes, the study combined metagenomic annotation results with the NCBI species taxonomy database for species-level analysis. The results showed that species enriched in the PEP-W-1 treatment group (such as...) Muribaculum intestinale , Acetatifactor muris and Lactobacillus taiwanensis It carries more functional genes related to the encoding of K00627, K00929, and K01689. Correlation analysis further indicates that... Ligilactobacillus murinus , Bacteroides acidifaciens and Schaedlerella arabinosiphila These bacterial species showed a significant positive correlation with the aforementioned KO, while those associated with liver fibrosis... Escherichia- Shigella and Enterococcus faecalis Then there is a negative correlation.

[0108] In summary, PEP-W-1 regulates the gut microbiota at the metabolic level by promoting the enrichment of functional microbial genes related to SCFA synthesis, and may thus exert its anti-liver fibrosis effect through microbial metabolic pathways. Its upregulation of key KOs in the acetic acid and propionic acid synthesis pathways suggests that PEP-W-1 can reprogram the metabolic potential of the gut microbiota, prompting it to produce more metabolites with anti-inflammatory and hepatoprotective effects.

[0109] (6) PEP-W-1 enhances tryptophan metabolism and promotes the biosynthesis of IPA / IAA through the gut microbiota-derived pathway. To investigate the functional interaction between PEP-W-1 and tryptophan metabolism from gut microbiota, this invention reconstructed the microbial tryptophan metabolic pathway using the KEGG database based on metagenomic KO annotation information. The results showed that the metabolic process of tryptophan conversion to indole-3-propionic acid (IPA) and indole-3-aceticacid (IAA) involves the synergistic action of multiple key enzymes, mainly encoded by K00466, K01667, K18116, K04103, and K10870, ultimately activating the aryl hydrocarbon receptor (AhR) signaling pathway.

[0110] Compared with the BDL group, the abundance of the aforementioned KO genes was significantly upregulated in the PEP-W-1 treatment group, suggesting that it enhances the metabolic potential of gut microbiota in synthesizing IPA / IAA. Among them, K01667 (aromatic amino acid transaminase) and K18116 (indolepyruvate decarboxylase) showed particularly significant increases in the PEP-W-1 group. These two enzymes are directly involved in the key steps of tryptophan to indolepyruvate and then to IPA, indicating that this metabolic pathway may be one of the important mechanisms by which PEP-W-1 exerts its effects.

[0111] To identify the key microbial sources involved in the aforementioned KO pathway, this invention employed the KEGG classification mapping method to classify and track the bacterial species carrying the relevant functional genes. In the PEP-W-1 treatment group, Eubacterium sp. 14-2 , Muribaculum intestinale , Bacteroides uniformis as well as Roseburia sp. 1XD42-69 The bacteria were significantly enriched and predicted to encode multiple KO genes related to IPA / IAA synthesis.

[0112] Consistent with the above results, the abundance of key enzymes related to tryptophan metabolism, including TnaA (tryptophanase), AAT (aromatic amino acid transaminase), and IPDC (indolepyruvate decarboxylase), was significantly higher in the PEP-W-1 group than in the BDL group, further supporting the mechanism by which PEP-W-1 promotes the synthesis of IPA / IAA through the microbial-mediated tryptophan metabolic pathway.

[0113] Furthermore, direct quantitative results of tryptophan metabolites from gut microbiota showed that, compared with the BDL group, the concentrations of IPA and IAA in mice treated with PEP-W-1 were significantly increased, further verifying that PEP-W-1 can enhance gut microbiota-mediated tryptophan metabolism, especially its activity along the IPA / IAA metabolic axis.

[0114] In summary, the results of this invention indicate that PEP-W-1 can selectively promote the enrichment of dominant bacterial communities and their functional genes involved in IPA / IAA synthesis, and may exert its immunomodulatory and antifibrotic effects by activating the aryl hydrocarbon receptor (AhR) signaling pathway.

[0115] Finally, it should be noted that the above embodiments and comparative examples are only used to illustrate the present invention and are not intended to limit the technical solutions described in the present invention; those skilled in the art should understand that modifications or equivalent substitutions can still be made to the present invention; and all technical solutions and improvements that do not depart from the spirit and scope of the present invention should be covered within the scope of the claims of the present invention.

Claims

1. A polysaccharide active from Sagella sp., characterized in that, The chemical formula of the active polysaccharide of Amorphophallus konjac is as follows: 。 2. The active polysaccharide of jicama according to claim 1, characterized in that, The active polysaccharide of jicama exhibits characteristic absorption peaks at least at the following positions in its infrared spectrum: 3317 cm⁻¹, 2911 cm⁻¹, and 1404 cm⁻¹. -1 1021cm -1 And 642cm⁻¹.

3. A polysaccharide according to claim 1, wherein the polysaccharide is a polysaccharide having a molecular weight of 100,000 to 1,000,000. The 1H and 13C nuclear magnetic resonance spectrum of the active polysaccharide of Amorphophallus konjac is as follows: C-2 carbon atom in β-D-Fruf in the range of δ 104.01-103.23 ppm, H-4 in D-Fruf residue in the range of δ 4.00-4.15 ppm, and H-3 in D-Fruf residue in the range of δ 4.15-4.25 ppm; 1H NMR spectrum is as follows: H-1 in α-D-Glcp in the range of δ 5.17-5.35 ppm, and H-1 in β-D-Glcp in the range of δ 4.57-4.59 ppm.

4. A process for the preparation of a polysaccharide active of Sargassum sp. according to any one of claims 1 to 3, characterized by the steps of It comprises: (1) Extraction and preliminary purification: after the Amorphophallus konjac tuber is cut into segments, hot extraction is performed by adding boiling water; The extraction liquid is combined, concentrated, and centrifuged to remove insoluble substances, and the supernatant A is obtained; (2) De-proteinization: Sevag reagent is added to the supernatant A, and after oscillation and centrifugation, the organic phase and the interface protein are removed, and the operation is repeated until the protein is completely removed, and the supernatant B after de-proteinization is obtained; (3) Alcohol precipitation and crude polysaccharide: the supernatant B is dialyzed, and then ethanol is added to the dialysate for alcohol precipitation, and the precipitate is collected, resuspended, and dried to obtain the crude polysaccharide PEP; (4) Ion exchange chromatography: after the crude polysaccharide PEP is dissolved and centrifuged, the supernatant is subjected to DEAE anion exchange column chromatography, and gradient elution is performed by using deionized water and NaCl solutions with different concentrations, and each elution component is collected and treated to obtain a plurality of polysaccharide sub-components PEP-W; (5) Gel filtration chromatography: after the polysaccharide sub-component PEP-W obtained in step (4) is dissolved and centrifuged, the supernatant is subjected to Sephacryl S-300 gel column chromatography with purified water as the mobile phase, and the target elution peak is collected to obtain the refined Amorphophallus konjac active polysaccharide component PEP-W-1.

5. A process for the preparation of active polysaccharides of Sargassum sp. as claimed in claim 4, wherein, In step (1), the hot extraction conditions are as follows: the solid-liquid ratio is 1:(8-12) w / v, boiling water extraction, each extraction for 1-3 hours, and extraction for 1-4 times; the concentration is to concentrate the combined extraction liquid to 1 / 8 to 1 / 12 of the original volume; and the centrifugation is at 4000-6000 x g for 10-20 minutes.

6. A process for the preparation of active polysaccharides of Sargassum sp. as claimed in claim 4, wherein, In step (2), the Sevag reagent is prepared by mixing chloroform and n-butanol at a volume ratio of (2-4):1; the volume ratio of the supernatant A to the Sevag reagent is (3-5):1; the volume ratio of chloroform to n-butanol in the Sevag reagent is 3:1; the volume ratio of the supernatant A to the Sevag reagent is 4:1; and the de-proteinization operation is repeated 4-6 times.

7. A process for the preparation of active polysaccharides of Sargassum sp. as claimed in claim 4, wherein, In step (3), the dialysis is performed by using a dialysis bag with a molecular weight cut-off of 12-15 kDa, and dialysis is performed in flowing tap water and high-purity distilled water for 48-96 hours; and the alcohol precipitation is performed by adding 2-4 times the volume of ethanol with a concentration of 70-90% v / v to the dialysate, and standing at 2-8°C for 8-16 hours.

8. A process for the preparation of active polysaccharides of Sargassum sp. as claimed in claim 4, wherein, In step (3), the dialysis bag with a molecular weight cut-off of 14 kDa was used, and the total dialysis time was 72 hours; 3 times the volume of 80% v / v ethanol was added for alcohol precipitation, and the mixture was allowed to stand at 4°C overnight.

9. A process for the preparation of active polysaccharides of Sargassum sp. as claimed in claim 4, wherein, In step (4), the DEAE anion exchange column is a DEAE Sepharose Fast Flow column; the concentration gradient of the NaCl solution comprises 0.1 mol / L, 0.2 mol / L and 0.3 mol / L; and the polysaccharide sub-fraction PEP-W comprises PEP-1, PEP-2 and PEP-3.

10. Use of the active polysaccharides of Sagittaria sagittifolia according to any one of claims 1-3 and the preparation method of the active polysaccharides of Sagittaria sagittifolia according to any one of claims 4-9 in the preparation of drugs and / or food for regulating intestinal microbiota and / or potential anti-liver fibrosis.

Images