Preparation method and application of novel rhizoma bletillae glucomannan

Abstract

The invention relates to a preparation method and application of novel rhizoma bletillae glucomannan. The preparation method of the rhizoma bletillae glucomannan comprises the following steps: 1, drying, crushing and sieving rhizomes of a raw material rhizoma bletillae; 2, putting the crushed and sieved rhizoma bletillae into ethanol for degreasing; 3, obtaining crude polysaccharide through a distilled water ice bath ultrasonic extraction technology and an ethanol precipitation technology; 4, separating and purifying through a DEAE-52 cellulose column and a G-150 gel column to obtain the high-purity rhizoma bletillae homogeneous polysaccharide BsPsIII1-1, namely the rhizoma bletillae glucomannan; 5, carrying out semipermeable membrane dialysis on the BsPsIII1-1 to remove salt, and carrying out freeze drying; and 6, carrying out chemical analysis on the obtained BsPsIII1-1. According to the preparation method and the application of the novel rhizoma bletillae glucomannan, animal experiments show that BsPsIII1-1 can significantly improve CCl4 induced mouse liver tissue injury, reduce serum ALT and AST levels, effectively relieve oxidative stress by reducing MDA content and increasing GSH level, and significantly inhibit hepatic fibrosis by down-regulating expression of alpha smooth muscle actin (alpha-SMA) and collagen I (COL-1).

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of natural pharmaceutical chemistry, in particular to a preparation method and application of a new type of bletilla striata glucomannan. BACKGROUND

[0002] Liver fibrosis, characterized by the abnormal accumulation of collagen, glycoproteins, and proteoglycans in the extracellular matrix (ECM), is a common pathological process in chronic liver diseases such as viral hepatitis, drug-induced liver injury, and metabolic-related fatty liver disease. Its essence is the repair response of the liver to persistent injury, but excessive fibrosis will lead to destruction of liver tissue structure and functional failure, and significantly increase the risk of cirrhosis and hepatocellular carcinoma. Although early liver fibrosis is reversible, there is a lack of specific treatment drugs in clinical practice, and existing anti-fibrosis candidate compounds are difficult to be converted for application due to toxicity or insufficient efficacy, so there is an urgent need to develop safe and efficient new treatment strategies.

[0003] In recent years, natural polysaccharides have become a research hotspot in the field of anti-fibrosis due to their low toxicity, multi-target action, and biodegradability. For example, Belapectin, a galectin-3 inhibitor, has been verified for safety through phase I clinical trials and has been granted a fast track qualification for the treatment of non-alcoholic steatohepatitis (NASH) by the FDA, demonstrating the great potential of polysaccharide drugs in the field of anti-fibrosis. However, the structure-activity relationship between the complexity and functional diversity of polysaccharides has not been clearly elucidated, which has hindered the process of their directed development.

[0004] Bletilla striata glucomannan is a polysaccharide component extracted from Bletilla striata. As a traditional Chinese medicinal material in China, Bletilla striata is rich in bletilla striata polysaccharides (BsPs) in its dried rhizomes, which have been proven to have activities such as hemostasis, anti-inflammatory, and promotion of tissue repair. Our team's previous studies have shown that crude bletilla striata polysaccharides can significantly alleviate the progression of liver fibrosis and have excellent biocompatibility and cost-effectiveness, but the specific active ingredients and mechanisms of action have not been clearly elucidated. Notably, the existing reported BsPs are mainly dominated by high mannose content (e.g., mannose: glucose = 3:1), while the crude polysaccharides isolated from the cultivated Bletilla striata in Zunyi present a unique 1:1 mannose-glucose composition, suggesting that they may be structurally novel functional glucomannans. However, the fine structure analysis, large-scale preparation method, and anti-liver fibrosis activity of such 1:1 type BsPs are all in the blank field.

[0005] Therefore, developing efficient and green directional extraction and purification technology of active bletilla striata polysaccharides, elucidating their structural characteristics and anti-liver fibrosis mechanisms, has important scientific significance and clinical value for promoting the high-value application of bletilla striata resources in the prevention and treatment of chronic liver diseases. SUMMARY

[0006] The present application aims to provide a novel preparation method of bletilla striata glucosan and its application, so as to solve the problems of the fine structure analysis, large-scale preparation method and anti-hepatic fibrosis activity of bletilla striata glucosan in the current market.

[0007] To achieve the above-mentioned purpose, the present application provides the following technical scheme: a novel preparation method of bletilla striata glucosan, which comprises the following steps of the preparation method of bletilla striata glucosan:

[0008] 1. Dry, crush and sieve the rhizome of the raw material bletilla striata;

[0009] 2. Put the crushed and sieved bletilla striata into ethanol for defatting;

[0010] 3. Obtain crude polysaccharide by distillation water ice bath ultrasonic extraction technology and ethanol precipitation technology;

[0011] 4. Obtain high-purity bletilla striata uniform polysaccharide BsPsIII1-1, i.e. bletilla striata glucosan, by DEAE-52 cellulose column and G-150 gel column separation and purification;

[0012] 5. Desalt BsPsIII1-1 by semi-permeable membrane dialysis and freeze-drying;

[0013] 6. Perform chemical analysis on the obtained BsPsIII1-1.

[0014] Preferably, the ethanol concentration in step 2 is 95%, and the defatting treatment is performed three times.

[0015] Preferably, in step 3, the residue after defatting in step 2 is subjected to drying treatment, double distilled water is added to the residue at a solid-liquid ratio of 1:30, and ice bath ultrasonic extraction is performed for 2 hours; after repeating the water extraction three times, the filtrate is combined, concentrated under reduced pressure to 20%-30% of the original volume, 4 times volume of 95% ethanol is added for precipitation, and the precipitate is collected by centrifugation, resuspended, dialyzed to remove small molecular impurities, and then freeze-dried to obtain crude polysaccharide.

[0016] Preferably, in step 5, the freeze-dried BsPsIII1-1 is white or off-white cotton-like, the surface is partially smooth and transparent, and it is easily crushed into powder under pressure; under 1000 times magnification by scanning electron microscope (SEM), it presents a strip-shaped morphology coexisting with flat irregular sheet structure, the surface of some areas is sparse and wrinkled, and some areas are flat and smooth; under 5000 times magnification, the surface of the sheet structure is rough, shallow wrinkles and densely distributed small circular depressions can be seen; under 10000 times magnification, the edge of the depression structure is clear, and there are fine cracks.

[0017] Preferably, the determination of BsPsIII1-1 in step 6 includes total sugar content, protein content and uronic acid content to ensure its characteristics as neutral polysaccharide and not combined with protein.

[0018] Preferably, when BsPsIII1-1 is detected and analyzed, it is analyzed by HPGPC-ELSD technology, and a calibration curve is established by T series dextran standards to determine its average molecular weight, so as to obtain BsPsIII1-1 with specific homogeneity and average molecular weight (about 74.8 kDa), and BsPsIII1-1 has specific monosaccharide composition, mainly composed of mannose (Man), glucose (Glc) and galactose (Gal), with a molar ratio of about 100.00:88.17:2.32, wherein the proportion of Man and Glc is more than 98%; the connection mode and relative monosaccharide abundance of BsPsIII1-1 are determined by methylation analysis, and its main derivatives are 2,3,6-trimethyl-D-mannopyranose (43.77%) and 2,3,6-trimethyl-D-glucopyranose (46.60%), indicating that BsPsIII1-1 is mainly composed of →4)-Manp-(1→ and →4)-Glcp-(1→ residues, with a molar ratio close to 1:1, and a branching degree (DB) of about 9.62%, and shows a triple helix conformation; the fine structure of BsPsIII1-1 is determined by nuclear magnetic resonance (NMR) technology, including different types of glycosyl residues and their specific chemical shift assignment, and the connection sites and sequences between residues are determined by HMBC and ROESY spectra.

[0019] Preferably, the BsPsIII1-1 is used 1 H NMR, 13 C NMR, 1 H- 1 H COSY, 1 H- 13 CHSQC, 1 H- 13 CHMBC, 1 H- 1 H ROESY and other technologies, combined with methylation analysis results, to speculate that the main chain repeating unit of the polysaccharide is:

[0020] →{4)-β-D-Glcp-(1} x →{4)-β-D-Manp-(1} n →{4)-β-D-Glcp-(1} y →4)-β-D-2Ac-Manp-(1→4)-β-D-3Ac-Manp-(1→4)-β-D-2Ac-Manp-(1→{4)-β-D-Manp-(1}m → 4) - β-D-3Ac-Manp-(1→ {4) - β-D-Manp-(1} i →, (wherein x+y=8, n+m+i=4).

[0021] Preferably, the BsPsIII1-1 is applied in the application of anti-hepatic fibrosis, and animal experiments show that BsPsIII1-1 has significant liver protection activity in vivo, can improve the liver tissue morphology and reduce liver damage in a dose-dependent manner in a CCl4-induced C57BL / 6J mouse liver injury model, and in particular, the high-dose (400 mg / kg) group can significantly reduce the weight loss of mice caused by CCl4 and significantly reduce the alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in the serum, and the effect is comparable to that of the positive control group (silymarin); through H&E, Masson and Sirius Red staining observation, BsPsIII1-1 can effectively reduce the degree of CCl4-induced liver fibrosis in mice, inhibit collagen fiber deposition, and the high-dose group (400 mg / Kg) effect is close to the positive control group (silymarin), and has a significant anti-hepatic fibrosis effect; in addition, BsPsIII1-1 can down-regulate the expression of fibrosis-related proteins alpha smooth muscle actin (alpha-SMA) and collagen I (COL-1) in a dose-dependent manner, and play an anti-hepatic fibrosis role by inhibiting the activation of the TGF-beta1 / SMAD2 / 4 / 7 signaling pathway.

[0022] Preferably, the BsPsIII1-1 has antioxidant stress capacity, can effectively alleviate the oxidative stress of mouse liver caused by CCl4, significantly reduce the content of malondialdehyde (MDA) in the liver, and increase the level of glutathione (GSH), thereby helping to maintain the normal function of the liver, and showing antioxidant stress capacity.

[0023] Preferably, the application range of the BsPsIII1-1 includes but is not limited to the preparation of anti-hepatic fibrosis drugs, functional foods or health products.

[0024] Compared with the prior art, the beneficial effects of the present application are:

[0025] 1. The preparation method and application of the novel white and glucosan show that BsPsIII1-1 has great potential for the preparation of anti-hepatic fibrosis drugs, functional foods or health products, and provides a new choice for the development of natural anti-hepatic fibrosis drugs.

[0026] 2、The preparation method and application of the novel bletilla striata polysaccharide show that, through animal experiments, BsPsIII1-1 can significantly improve the liver tissue injury of CCl4-induced mice, reduce the serum ALT and AST levels, effectively alleviate oxidative stress by reducing the MDA content and increasing the GSH level, and significantly inhibit liver fibrosis by down-regulating the expression of alpha smooth muscle actin (alpha-SMA) and collagen I (COL-1). The mechanism of action is to significantly inhibit the secretion of TGF-beta1, reduce the level of p-SMAD2, reduce the expression of SMAD4, and up-regulate the SMAD7 negative regulator, thereby blocking the activation of the TGF-beta / SMAD signaling pathway. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 The key step structure diagram of bletilla striata polysaccharide extraction, purification and separation;

[0028] Figure 2 The elution chromatogram of crude BsPsIII on DEAE-cellulose column and Sepharose G-150 gel filtration column, and the ultraviolet-visible spectrum and HPGC-ELSD spectrum of BsPsIII1-1;

[0029] Figure 3 The PMP pre-column derivatization high performance liquid chromatography (HPLC) chromatogram of hydrolyzed monosaccharides, the gas chromatography-mass spectrometry (GC / MS) chromatogram of PMAAs of BsPsIII1-1, and the mass spectrometry fragment and deduced residue diagram of PMAAs of BsPsIII1-1;

[0030] Figure 4 The spectrum of BsPsIII1-1 structure analysis by nuclear magnetic resonance (NMR);

[0031] Figure 5 The maximum absorption wavelength of BsPsIII1-1-congo red complex in the range of 0-0.5M NaOH concentration, the freeze-dried form of BsPsIII1-1, and the micrograph of BsPsIII1-1 (freeze-dried) recorded by scanning electron microscope at 1.0k, 5.0k and 10.0k magnification;

[0032] Figure 6 The results of BsPsIII1-1 on animal experiment design, liver morphology, body weight, liver index, ALT and AST content in serum, and the effect of CCl4-induced liver MDA and GSH content in C57BL / 6J mice;

[0033] Figure 7 The H&E, Masson and Sirius red staining results of mouse liver tissue;

[0034] Figure 8 BsPsIII1-1 on CCl4-induced liver fibrosis in mice fibrosis marker relief effect results chart;

[0035] Figure 9 BsPsIII1-1 on CCl4-induced liver fibrosis in mice fibrosis marker relief effect results chart;

[0036] Figure 10 BsPsIII1-1 on CCl4-induced liver fibrosis in mice fibrosis marker relief effect results chart;

[0037] Figure 11 BsPsIII1-1 on CCl4-induced liver fibrosis in mice fibrosis marker relief effect results chart. DETAILED DESCRIPTION

[0038] The technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the scope of the present application.

[0039] Please refer to Figures 1-11 The present application provides a technical solution: a preparation method of a new type of bletilla striata glucomannan and its application, which comprises the following steps of the preparation method of bletilla striata glucomannan:

[0040] 1. Dry, crush, and sieve the rhizomes of the raw material bletilla striata;

[0041] 2. Put the crushed and sieved bletilla striata into ethanol for degreasing;

[0042] 3. Obtain crude polysaccharides by distillation water ice bath ultrasonic extraction technology and ethanol precipitation technology;

[0043] 4. Obtain high-purity bletilla striata uniform polysaccharide BsPsIII1-1, i.e. bletilla striata glucomannan, by DEAE-52 cellulose column and G-150 gel column separation and purification;

[0044] 5. Perform semi-permeable membrane dialysis desalination on BsPsIII1-1, and freeze-dry;

[0045] 6. Perform chemical analysis on the obtained BsPsIII1-1.

[0046] Example 1: Extraction and purification of a novel glucosan from Bletilla striata

[0047] 1. Extraction of crude polysaccharides

[0048] Fresh rhizomes of Bletilla striata (Thunb. ex Murray) Rchb. f. from Zheng'an County, Zunyi City, Guizhou Province, China were used as raw material. After drying at 45°C, the rhizomes were ground and sieved. The powder was defatted with 95% ethanol three times. The residue was dried and then extracted with double-distilled water at a solid-to-liquid ratio of 1:30 for 2 hours in an ice bath with ultrasonic assistance. The extraction was repeated three times, and the combined filtrate was concentrated under reduced pressure to 20%-30% of the original volume. Four times the volume of 95% ethanol was added to precipitate the polysaccharides. The precipitate was collected by centrifugation, resuspended in water, and dialyzed against a dialysis bag with a molecular weight cutoff of 3500 Da for 72 hours to remove small molecular impurities. The crude polysaccharides (BsPsIII) were obtained by freeze-drying, with an extraction rate of about 38%( Figure 1 ). This process, which involves low-temperature ultrasonic-assisted extraction and ethanol precipitation, meets the standards for green pharmaceutical and food processing.

[0049] 2. Isolation and purification of polysaccharides

[0050] The crude polysaccharides BsPsIII were dissolved in water to a concentration of 50 mg / mL. The solution was loaded onto a DEAE-52 cellulose column (60 x 270 mm) and eluted with pure water and 0.15 M NaCl solution (flow rate: 1.0 mL / min). The absorbance at 490 nm was detected by the phenol-sulfuric acid method, and the main peak fractions (tube numbers 16-29, designated BsPsIII1) were collected. After dialysis and desalting, the main peak fractions were further purified by Sephadex G-150 gel column chromatography (3.5 x 60 cm) using deionized water as the eluent (flow rate: 0.3 mL / min). The main peak fractions (tube numbers 21-42, designated BsPsIII1-1) were collected and freeze-dried for storage( Figure 2 A-2B).

[0051] 3. Detection of polysaccharide homogeneity

[0052] High-performance gel permeation chromatography (HPGPC) analysis was performed using a TSK-GEL GMPWxl chromatographic column (7.8 mm x 30.0 cm) and an evaporative light scattering detector (ELSD). The results showed that the chromatogram of BsPsIII1-1 exhibited a single symmetrical peak( Figure 2 C), indicating that it was a polysaccharide with uniform molecular weight. The average molecular weight was calculated to be 74.8 kDa based on the standard curve.

[0053] 4. Detection of polysaccharide purity

[0054] (1) Ultraviolet spectroscopy analysis: BsPsIII1-1 was prepared as a 1.0 mg / mL aqueous solution. Ultraviolet full-wavelength scanning showed no characteristic absorption peaks at 260 nm and 280 nm.Figure 2 D), indicating no nucleic acid or protein residues;

[0055] (2) Determination of total sugar content: Phenol-sulfuric acid method (calibration curve Y = 1.1183X + 0.2525, R 2 =0.9953), the total carbohydrate content was measured to be 93.80±4.65%;

[0056] (3) Protein content determination: by BCA method (calibration curve Y = 0.9708X + 0.1946, R 2 =0.9986), and the protein content was -0.37±4.63%. The above results confirm that BsPsIII1-1 is a high-purity, protein-free neutral polysaccharide.

[0057] Example 2: Extraction, purification and structural characterization of a novel glucomannan from Bletilla striata

[0058] 1. In this example, the extraction and purification of the novel glucomannan were the same as in Example 1 and will not be repeated here. Through this step, a high-purity uniform polysaccharide BsPsIII1-1 was finally obtained.

[0059] 2. Analysis of Monosaccharide Composition of BsPsIII1-1

[0060] Monosaccharide composition was determined by high-performance liquid chromatography (HPLC) coupled with pre-column derivatization with 1-phenyl-3-methyl-5-pyrazolone (PMP). The following steps were performed: 2.0 mg of BsPsIII1-1 was accurately weighed and added to 0.5 mL of 3 M trifluoroacetic acid (TFA) in a sealed ampoule. The mixture was hydrolyzed at 95°C for 6 hours (under nitrogen atmosphere). After deacidification by centrifugation at 12,000 × g, 200 μL of the hydrolyzate was derivatized with monosaccharide standards (mannose, glucose, galacturonic acid, galactose, arabinose, and fucose) using PMP: 300 μL of 0.3 M NaOH and 300 μL of 0.5 M PMP methanol solution were added and the mixture was reacted at 70°C for 1 hour. The derivatized products were analyzed by Agilent 1260 HPLC system (Supersil AQ-C18 column, 250×4.6 mm, 5 μm) with a mobile phase of 17% acetonitrile-83% phosphate buffer (0.05 mM, pH 7.2) at a flow rate of 1 mL / min, a detection wavelength of 245 nm, and an injection volume of 10 μL.

[0061] like Figure 3As shown in Table 1, the monosaccharide composition of BsPsIII1-1 is mannose (Man), glucose (Glc) and galactose (Gal) with a molar ratio of 100.00:88.17:2.32, in which the ratio of Man and Glc is more than 98%. This result is consistent with the characteristics of white Bupleurum polysaccharides reported in the literature, which are mainly composed of Man and Glc. However, the ratio of Man:Glc in this study is approximately 1:1, which is significantly different from the traditional high-mannose type BsPs (such as 3:1 or 2:1), and the presence of Gal residues in the homogeneous polysaccharide is confirmed for the first time, which is consistent with the previous findings of the research team on the composition characteristics of crude polysaccharides (see Pan F, et al. Journal of Chinese Pharmaceutical Sciences, 2024, Vol 33, Issue 8, p686).

[0062] Chemical properties and monosaccharide composition of BsPsIII1-1

[0063]

[0064] Table 1

[0065] 3. Methylation analysis of BsPsIII1-1

[0066] 2.0 mg of dried polysaccharide was treated with 0.5 mL of anhydrous DMSO under nitrogen. Subsequently, 0.5 mL of NaOH-DMSO suspension and 0.5 mL of iodomethane were added to the mixture. After the reaction, 4 mL of deionized water was added to terminate the methylation reaction. Then, the mixture was extracted with 1:1 v / v chloroform. The chloroform layer was collected and concentrated. To ensure complete methylation, the process was repeated three times. Subsequently, the methylated polysaccharide was hydrolyzed. Sodium borohydride (NaBH4) was used to reduce the monosaccharides after hydrolysis to form sugar alcohols. Subsequently, acetic anhydride and pyridine were added to acetylate the hydroxyl groups of these sugar alcohols to form partially methylated sugar alcohol acetates (PMAA). After the PMAA sample was dried, it was dissolved in 0.3 mL of chloroform to obtain partially methylated sugar alcohol acetates for gas chromatography / mass spectrometry (GC / MS) analysis. At the same time, the same operation was performed on the blank sample without adding polysaccharide.

[0067] The ISQ-TRACE 1310-AI 1310 GC / MS system from Thermo Fisher Scientific was used, equipped with an electron impact ionization source (EI). The chromatographic column was a J&W DB-5ms gas chromatographic column (30 m x 0.25 mm x 0.25 pm, Agilent, USA). The injection volume was 1 pL, the split ratio was 15:1, the carrier gas was helium, and the flow rate was 1.0 mL / min. The column temperature program was as follows: the initial temperature was 100 °C, maintained for 3.0 min, increased to 140 °C at 10 °C / min, maintained for 2 min, then increased to 230 °C at 3 °C / min, maintained for 3 min. The analytes were detected in the full scan (SCAN) mode using an electron impact ionization source (EI mode, 70 eV), and the mass spectrometry scan range (m / z) was 50-400. The individual peaks and fragmentation patterns of PMAA were identified by mass spectrometry and relative retention time.

[0068] The GC / MS data (Table 2, Figure 3 B-3F) showed that the main derivatives of BsPsIII1-1 were 2,3,6-trimethyl-D-mannopyranose (43.77%) and 2,3,6-trimethyl-D-glucopyranose (46.60%), indicating that the backbone was composed of →4)-Manp-(1→ and →4)-Glcp-(1→ residues, with a molar ratio close to 1:1, consistent with the monosaccharide composition analysis results. In addition, a small amount of 2,3,4,6-tetramethyl-Manp (4.26%) and 2,3,4,6-tetramethyl-Glcp (5.36%) were detected, suggesting the presence of terminal Manp and Glcp residues. Combined with the branching degree (DB) calculation formula (DB = (Nt + Nb) / (Nt + Nb + Ni)), the DB value of BsPsIII1-1 was about 9.62%, confirming that it was a lowly branched linear polysaccharide.

[0069] It is worth noting that the existing literature reports that the uniform BsPs mainly present a high mannose ratio (such as Man:Glc = 3:1), while the Man:Glc ≈ 1:1 characteristic in this study is only found in a small amount of crude polysaccharide research. For example, the uniform polysaccharide (Man:Glc ≈ 1:1) isolated from Bletilla striata by Liu et al. contains →4)-a-D-Glcp (50.15%) and →4)-a-D-Manp (22.61%) (Liu et al. International Journal of Biological Macromolecules 205 (2022) 553-562.), but its glycosidic bond configuration (a type) is significantly different from that of BsPsIII1-1 (b type). The above differences indicate that BsPsIII1-1 may be a novel glucosan.

[0070] Gas chromatography-mass spectrometry (GC / MS) analysis of methylated BsPsIII1-1

[0071]

[0072] Table 2

[0073] Note: a , based on derivatized O-methyl alditol acetates; b , by gas chromatography-mass spectrometry (GC / MS) analysis, after full O-methylation, complete acid hydrolysis, reduction and acetylation treatment, 2,3,4,6-Me4-Manp = 1,5-di-O-Actyl-2,3,4,6-tetra-O-methyl-mannose, otherwise identical.

[0074] After lyophilization replacement, 25 mg of BsPsIII1-1 was dissolved in 0.7 mL of D2O, and the Agilent-400MHz or Bruker AVANCE NEO 600MHz nuclear magnetic resonance instrument was used for acquisition 1 HNMR (400 / 600MHz), 13 C NMR (125.48MHz), COSY, HSQC, HMBC and ROESY spectra Figure 4 ).

[0075] 1 H NMR spectrum Figure 4 A) showed that there were more than 10 anomeric proton signals in the range of δ4.3-5.5ppm, indicating that BsPsIII1-1 contained at least 10 different monosaccharide residues. 13 C NMR spectrum Figure 4 B) showed that the anomeric carbon signals were concentrated in δ90-110ppm, and the C6 signal at δ65-70ppm indicated that there was no substitution feature, which was consistent with the results of methylation analysis. 1 H- 13 C HSQC spectrum Figure 4 D) showed that the cross peaks at δ1.88-2.20 / 20.00-22.30ppm and δ1.98-2.20 / 174.10-174.85ppm corresponded to acetyl methyl and carbonyl signals, respectively, confirming the presence of acetylation modification.

[0076] Combining the methylation analysis results, the backbone was composed of→4)-β-D-Manp-(1→(residue A, δ4.68 / 100.28 ppm) and→4)-β-D-Glcp-(1→(residue B, δ4.44 / 102.51 ppm), whose C1 and C4 chemical shifts (residue A: δ100.28 ppm, 76.52 ppm; residue B: δ102.51 ppm, 78.33 ppm) were significantly low field shifts compared with the free monosaccharides, which confirmed the C1 and C4 glycosidic bond connection. In addition, the signals of δ5.42 / 71.38 ppm (residue C) and δ5.04 / 73.60 ppm (residue F) indicated the presence of→4)-β-D-2Ac-Manp-(1→and→4)-β-D-3Ac-Manp-(1→acetylated residues, and the cross peaks in the HMBC and ROESY spectra (such as δ4.85 / 71.38 ppm, δ5.42 / 3.95 ppm) further verified their connection sites.

[0077] The signals of δ5.10 / 93.89 ppm (residue D) and δ4.81 / 93.62 ppm (residue E) in the HSQC spectrum were the signals of the reducing terminal of mannose, corresponding to→4)-α-D-Manp and→4)-β-D-Manp, respectively; the signals of δ5.15 / 92.36 ppm (residue J) and δ4.57 / 95.38 ppm (residue K) were the signals of the reducing terminal of glucose, indicating that there was a difference in the α / β configuration at the end of the sugar chain. The non-reducing terminal residues T-D-Manp-(1→(residue G, δ4.66 / 99.90 ppm) and T-D-Glcp-(1→(residue H, δ4.44 / 104.12 ppm) were also identified, which was consistent with the results of the terminal residues in the methylation analysis.

[0078] The connection sequence between the sugar residues was analyzed by HMBC and ROESY spectra Figure 4 E-4F) The connection sequence between the sugar residues was analyzed by HMBC and ROESY spectra (Fig. 2E-4F). Residue A was connected to residues F, A, and B through 1,4-glycosidic bonds (such as the cross peaks of δ4.68 / 73.89 ppm, δ4.68 / 76.52 ppm in the HMBC); residue B was connected to residues B, A, and C, forming→4)-β-D-Glcp-(1→4)-β-D-Glcp-(1→and→4)-β-D-Glcp-(1→4)-β-D-Manp-(1→sequences. Combining the monosaccharide composition and molecular weight data, it was inferred that the repeat unit structure of BsPsIII1-1 was

[0079] →{4)-β-D-Glcp-(1}x→{4)-β-D-Manp-(1}n→{4)-β-D-Glcp-(1}y→4)-β-D-2Ac-Manp-(1→4)-β-D-3Ac -Manp-(1→4)-β-D-2Ac-Manp-(1→{4)-β-D-Manp-(1}m→4)-β-D-3Ac-Manp-(1→{4)-β-D-Manp-(1}i→( Figure 4 G), where x+y=8, n+m+i=4. 5. Conformation and morphological characterization of BsPsIII1-1

[0080] Congo red binding assay was used to analyze the conformation of polysaccharide chains: BsPsIII1-1 solution (2.5 mg / mL) was mixed with NaOH solution containing 0.08 mmol / L Congo red, and the final concentration was adjusted to 0-0.5 M. After incubation for 20 minutes, the maximum absorption wavelength (λmax) was measured. Figure 5 As shown in A, as the NaOH concentration increases (0-0.3M), the λmax of the BsPsIII1-1-Congo red complex significantly red-shifts, and shows a downward trend after 0.3M; this phenomenon does not occur in the control group without adding polysaccharide, confirming that BsPsIII1-1 has a triple helical structure.

[0081] After freeze-drying, BsPsIII1-1 is white or off-white cotton-like, with a smooth and translucent surface. Figure 5 B), which is easily broken into powder under pressure. The microscopic morphology was further observed using a Hitachi SU8010 scanning electron microscope (accelerating voltage 3kV): at 1000x magnification, the polysaccharide showed a coexistence of strip-like and flat irregular flake-like structures, with sparse wrinkles and smooth areas alternating on the surface ( Figure 5 C); 5000 times magnification shows that the surface of the flakes is rough, accompanied by shallow wrinkles and dense small circular depressions ( Figure 5 D); Under 10000 times magnification, the edge of the depression is clear and fine cracks are visible ( Figure 5 E) The above morphological characteristics are closely related to the limited branching structure of polysaccharides and the intermolecular hydrogen bonding.

[0082] Example 3: Application of a novel glucomannan derived from Bletilla striata in anti-liver fibrosis

[0083] 1. In this example, the extraction and purification of a new glucomannan from Bletilla striata was the same as in Example 1, which will not be repeated here. Through this step, a high-purity uniform polysaccharide BsPsIII1-1 was finally obtained.

[0084] 2. The structure of the new glucosaminoglycan from Bletilla striata in this example was characterized as in Example 2, which is not repeated here. Through this step, a new structure of Bletilla striata high-purity homogeneous polysaccharide BsPsIII1-1 was finally obtained.

[0085] 3. Experimental design and animal handling of BsPsIII1-1 anti-hepatic fibrosis

[0086] Fifty 6-8-week-old male C57BL / 6J mice weighing 18±2 g were purchased from Sifang Life Biotechnology Co., Ltd. (Beijing, License No. SCXK (Jing) 2024-0001). The experimental design is shown in Figure 6 A. After a week of adaptation, the mice were randomly assigned to the following groups: normal group, model group, positive control group (50 mg / kg of silymarin was given by gavage), and BsPsIII1-1 group (100 or 400 mg / kg, given by gavage). Except for the normal group, all mice were injected intraperitoneally with 20% carbon tetrachloride (CCl4) corn oil solution (5 mg / kg, twice a week for 6 weeks). The normal group and the model control group were given the same volume of normal saline by gavage every day. The body weight of mice in each group was measured and recorded at a fixed time every week. All experiments were approved by the Animal Research Ethics Committee of Zunyi Medical University (ZMU21-2303-017).

[0087] 4. Protective effect of BsPsIII1-1 on liver injury and oxidative stress

[0088] After treatment, the body weight and liver weight of mice were measured, and the liver index (liver weight / body weight x 100) was calculated. As shown in Figure 6 B, after CCl4 induction, the liver surface of the model group mice was rough and the elasticity was reduced, and the BsPsIII1-1 high-dose group (400 mg / kg) significantly improved the liver morphology and alleviated the decrease in body weight Figure 6 C).

[0089] Serum was obtained by centrifuging mouse blood at 3500 x g for 5 minutes, and AST and ALT levels were determined using commercial kits according to the manufacturer's instructions. The results of serum ALT and AST level detection showed that the model group ALT and AST increased by 2.33 times and 1.5 times, respectively (p<0.01), while the BsPsIII1-1 high-dose group reduced ALT by 50% and AST by 32%, which was comparable to silymarin Figure 6 E-6F).

[0090] An appropriate amount of mouse liver tissue was homogenized thoroughly on ice (1 : 10, w / v) and vortex mixed. The homogenate was centrifuged at 10,000 g for 10 min at 4 °C. The supernatant was stored at 4 °C for subsequent use in the kit for MDA and GSH content determination. The results showed that liver MDA (a marker of lipid peroxidation) was significantly increased in the model group (p < 0.01), and the BsPsIII1-1 low / high dose groups reduced by 50% and 58% (p < 0.01), respectively (G); at the same time, BsPsIII1-1 significantly restored GSH levels (p < 0.001), even higher than the normal group (H), indicating that it alleviated oxidative stress by scavenging ROS. Figure 6 G); at the same time, BsPsIII1-1 significantly restored GSH levels (p < 0.001), even higher than the normal group (H), indicating that it alleviated oxidative stress by scavenging ROS. Figure 6 H).

[0091] 5. Histopathological analysis of liver tissue after BsPsIII1-1 treatment

[0092] Liver mid-lobular tissue sections were fixed in 4% neutral buffered formaldehyde solution for 24 h, paraffin-embedded, and cut into 4-μm-thick sections. After deparaffination and hydration by ethanol gradient, the sections were stained with hematoxylin-eosin (H&E), Masson's trichrome staining, and sirius red staining according to the standard operating procedures of the manufacturers. The stained sections were dehydrated, mounted with neutral resin, and imaged under an optical microscope.

[0093] As shown in Figure 7 A, the normal control group liver showed uniform hepatocytes with clear nuclei and ordered structure. In contrast, CCl4 treatment induced significant pathological changes in the mouse liver, including hepatocyte enlargement, cytoplasmic rarefaction with cytoplasmic vacuolization (ballooning degeneration of cell swelling, extensive cell contraction, fragmentation, or dissolution indicating cell death, fat accumulation, massive inflammatory cell infiltration, and unclear cell edges). After treatment with different concentrations of BsPsIII1-1, the hepatocytes were restored, with clear cell boundaries, visible nuclei, and reduced cell swelling, especially in the 400 mg / kg dose group, which was almost equivalent to the positive control silymarin. These findings indicated that BsPsIII1-1 pretreatment significantly improved the CCl4-induced pathological changes in liver tissue.

[0094] Figure 7 B shows that Masson staining showed blue-stained collagen fibers and indicated that liver fibrosis resulted in the production and accumulation of collagen bundles. The results revealed that the collagen fibers in the liver of the model group were significantly increased, forming fibrous septa, accompanied by hepatocyte enlargement and nuclear shrinkage, and staining deepened. In contrast, the hepatocytes in the polysaccharide treatment groups were arranged in an orderly manner, the fibrous septa were narrowed and significantly reduced. Notably, the high-dose group showed improved cell morphology, reduced inflammatory cells, significantly reduced hepatocyte necrosis and collagen fibers, reduced fibroplasia, and restored normal liver tissue morphology. In Figure 7In D, the semi-quantitative results of Masson's trichrome staining showed that the degree of liver fibrosis in the BsPsIII1-1 treatment groups was reduced in a dose-dependent manner compared with the model group (p < 0.001). Notably, no significant difference was observed between the high concentration BsPsIII1-1 group and the silymarin positive control group.

[0095] After staining with Sirius red, collagen fibers appear bright red or pink, while other tissue components such as cytoplasm and nuclei are stained in different colors, such as yellow or green, which contrasts sharply with the red collagen fibers. As shown in Figure 7 C, in CCl4-induced mouse liver tissue, collagen fibers were significantly increased and formed fibrous septa; however, treatment with different concentrations of BsPsIII1-1 reduced collagen deposition, with high-dose BsPsIII1-1 leading to a significant reduction, almost comparable to the effect of the positive control. Semi-quantitative analysis of Sirius red-stained images showed that the fibrosis level of the model group was significantly higher than that of the normal control group (p < 0.001). After polysaccharide treatment, the degree of liver fibrosis in all groups was reduced, with the high-dose group showing a significant reduction (p < 0.001), comparable to the liver fibrosis level of the positive control Figure 7 E). Combined with the results of Masson staining and Sirius red staining, it can be clearly seen that collagen fiber deposition is significantly reduced, effectively reversing liver fibrosis.

[0096] 6. Detection of BsPsIII1-1 anti-fibrosis related protein expression regulation based on immunofluorescence technology

[0097] Antigen retrieval was performed on tissue sections using sodium citrate buffer. Endogenous peroxidase activity was blocked by treatment with 3% H2O2 for 15 minutes. The sections were incubated with primary antibody for 70 minutes at room temperature, washed with PBS for 3 times (3 minutes each), and then incubated with secondary antibody for 40 minutes at room temperature. After color development with diaminobenzidine (DAB) (5 minutes) and hematoxylin re-staining, the sections were differentiated, dehydrated and mounted for microscopic observation. Alpha-SMA is a key mediator of extracellular matrix (ECM) synthesis such as type I / III collagen and fibronectin, playing a crucial role in the progression of liver fibrosis. Its expression level is related to the severity of fibrosis and serves as a biomarker for the activation of hepatic stellate cells (HSCs). In addition, type I collagen (COL-1) is a major component of the extracellular matrix, and due to its excessive deposition, it is an important pathological indicator of liver fibrosis. Elevated COL-1 expression reliably indicates fibrosis progression. Therefore, alpha-SMA and COL-1 were detected according to this procedure.

[0098] As Figure 8As shown in A, immunohistochemical staining showed that the a-SMA positive area of CCl4-treated mice was significantly enlarged compared with the normal control group. BsPsIII1-1 treatment dose-dependently reduced the a-SMA positive area of the model group. Semi-quantitative analysis confirmed that BsPsIII1-1 significantly improved the expression of a-SMA in CCl4-induced mouse liver (p<0.001, Figure 8 B).

[0099] As Figure 8 As shown in C, in the normal control group, COL-1 was expressed in a small amount in the portal area, and no expression was observed in other areas. CCl4-treated mice showed significant COL-1 deposition in a star-shaped and diffuse manner around the sinusoids and central veins. BsPsIII1-1 or silymarin treatment significantly reduced the COL-1 positive area Figure 8 C). Semi-quantitative analysis showed that the inhibitory effect of BsPsIII1-1 on COL-1 was dose-dependent, with a significant difference compared with the model group (p<0.01), and the high-dose group showed a more significant effect (p<0.001), with no significant difference between the positive control group Figure 8 C).

[0100] 7. Detection of BsPsIII1-1 expression regulation of liver fibrosis-related proteins based on Western blot (WB) technology

[0101] The liver tissue was homogenized in ice-cold RIPA buffer (containing protease / phosphatase inhibitors), lysed on ice for 30 minutes, and centrifuged at 12000 x g at 4°C for 15 minutes. The protein concentration was determined by the BCA method, and equal amounts of protein samples were separated in 10% SDS-PAGE. The proteins were transferred to a PVDF membrane, blocked at room temperature for 15 minutes, and then incubated with the primary antibody at 4°C overnight. After PBST washing, the membrane was incubated with the secondary antibody at room temperature for 2 hours. The protein bands were detected using the no-dye rapid protein blotting system, with GAPDH as the internal reference. Quantitative analysis was performed using ImageJ software. According to this procedure, two key liver fibrosis-related proteins, a-SMA and COL-1, were detected.

[0102] The results showed that the expression of a-SMA in mice treated with silymarin (positive control) and different concentrations of BsPsIII1-1 was significantly reduced compared with the model group with the highest a-SMA expression in mouse liver tissue (p<0.05, Figure 8 E and Figure 8 F), consistent with the results of immunohistochemistry. These findings suggest that BsPsIII1-1 alleviates liver fibrosis by inhibiting a-SMA.

[0103] Similarly, COL-1 expression was significantly reduced (p < 0.001, Fig. 6B) in the Silymarin and high concentration of BsPsIII1-1 treated groups compared to the model group, Figure 8 E and Figure 10 G), consistent with the immunohistochemistry data. This indicates that BsPsIII1-1 attenuates fibrosis by inhibiting the production of COL-1. In summary, both immunohistochemistry and WB confirmed that BsPsIII1-1 effectively down-regulated a-SMA and COL-1 in CCl4-induced fibrotic livers, thereby reducing collagen accumulation, exerting a significant anti-fibrotic effect. In combination with other hepatoprotective evidence in this study, we identified BsPsIII1-1 as a novel B. striata polysaccharide with significant anti-fibrotic activity.

[0104] Example 4: Mechanism of a novel glucosaminoglycan from Bletilla striata against liver fibrosis

[0105] 1. In this example, the extraction and purification of a novel glucosaminoglycan from Bletilla striata is the same as in Example 1, which is not repeated here. Through this step, a high-purity homogeneous polysaccharide BsPsIII1-1 of a novel structure was finally obtained.

[0106] 2. In this example, the structural characterization of a novel glucosaminoglycan from Bletilla striata is the same as in Example 2, which is not repeated here. Through this step, a high-purity homogeneous polysaccharide BsPsIII1-1 of a novel structure from Bletilla striata was finally obtained.

[0107] 3. In this example, the determination of the anti-liver fibrosis activity of a novel glucosaminoglycan from Bletilla striata is the same as in Example 3, which is not repeated here. Through this step, a high-purity homogeneous polysaccharide BsPsIII1-1 of a novel structure from Bletilla striata with obvious application potential for anti-liver fibrosis was finally obtained.

[0108] 4. Mechanism of BsPsIII1-1 against liver fibrosis

[0109] Transforming growth factor-beta (TGF-β) is a multifunctional cytokine that plays a central role in the process of tissue fibrosis. TGF-β1 is the most extensively studied isoform of this family and the most common in human cancers, which plays a key role in TGF-β signaling activation and fibrosis process. Autocrine / paracrine TGF-β1 can activate hepatic stellate cells (HSCs) and induce their phenotypic transition characterized by proliferation, fibrosis and contraction. In the fibrosis signaling pathway, TGF-β1 / SMAD axis is a key regulatory factor. TGF-β receptor (serine / threonine kinase) combined with ligand can phosphorylate SMAD2 / 3, and then these phosphorylated SMAD2 / 3 form a complex with SMAD4 and transfer to the nucleus to drive the transcription of fibrosis genes (e.g., collagen type I (COL-1) and alpha-smooth muscle actin (a-SMA)). This pathway is negatively regulated by SMAD7, which initiates a feedback loop to attenuate signaling. The whole process is shown in Figure 9 .

[0110] To detect the expression of the key proteins in the above signaling pathway, we detected them by WB technique as described in Example 3. To evaluate the protein levels of TGF-β, SMAD2, phosphorylated SMAD2 (p-SMAD2), SMAD4 and SMAD7 in the livers of mice in different treatment groups, the results are shown in Figure 9 . After CCl4 treatment, the production of TGF-β1 was significantly increased (p<0.001), while the phosphorylation ratio of SMAD2 (p-SMAD2 / SMAD2) (p<0.01) and SMAD4 (p<0.001) were also increased, and the level of SMAD7 was significantly decreased (p<0.01). Combined with the data of a-SMA / COL-1, these results indicate that CCl4 activates HSCs to secrete TGF-β1, propagate SMAD signal, and drive the overexpression of fibrosis proteins and severe fibrosis. However, after the gavage treatment of different doses of BsPsIII1-1, BsPsIII1-1 showed a significant inhibitory effect on the TGF-β / SMAD signaling pathway, and this effect was obviously dose-dependent. After the treatment of high-dose BsPsIII1-1, the secretion of TGF-β1 (p<0.01), the phosphorylation ratio of SMAD2 (p<0.05) and the expression of SMAD4 (p<0.001) were significantly decreased, thereby inhibiting the activation of the SMAD pathway. In addition, BsPsIII1-1 can also increase the level of negative regulatory factor SMAD7 (p<0.01), further inhibit the activation of TGF-β / SMAD signaling pathway, and down-regulate the expression of key fibrosis proteins such as a-SMA and COL-1 ​). Therefore, we determined that the TGF-β1 / SMAD pathway is the key signal axis of BsPsIII1-1 to reverse or alleviate CCl4-induced liver fibrosis in mice, and plays an important role in its hepatoprotective effect.

[0111] Although the present application has been described in detail with reference to the foregoing embodiments, the technical solutions recorded in the foregoing embodiments can be modified by those skilled in the art, or some technical features thereof can be replaced by equivalents, and any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for preparing a novel Bletilla striata glucomannan, characterized in that: The preparation method of the Bletilla striata glucomannan comprises the following steps:

1. Dry, crush and sieve the rhizomes of Bletilla striata; 2. Put the crushed and sieved Bletilla striata into ethanol to defatted; 3. Obtain crude polysaccharides through distilled water ice bath ultrasonic extraction technology and ethanol precipitation technology; 4. The high-purity homogeneous polysaccharide BsPsIII1-1 from Bletilla striata was obtained by separation and purification using DEAE-52 cellulose column and G-150 gel column; 5. Perform desalination treatment on BsPsIII1-1 by semipermeable membrane dialysis and freeze-drying; 6. Perform chemical analysis on the obtained BsPsIII1-1.

2. The method for preparing the novel Bletilla striata glucomannan according to claim 1, wherein: In the step 2, the ethanol concentration is 95%, and the degreasing treatment is performed three times.

3. The method for preparing the novel Bletilla striata glucomannan according to claim 2, wherein: During the operation of step 3, the residue after defatting in step 2 is dried, double distilled water is added to the residue at a material-liquid ratio of 1:30, and ice bath ultrasonic extraction is performed for 2 hours. The water extraction is repeated three times, and the filtrate is combined and concentrated under reduced pressure to 20%-30% of the original volume. Then, 4 times the volume of 95% ethanol is added for precipitation, and the precipitate is collected by centrifugation, redissolved, dialyzed to remove small molecular impurities, and then freeze-dried to obtain crude polysaccharide.

4. The method for preparing the novel Bletilla striata glucomannan according to claim 3, wherein: In the step 5, BsPsIII1-1 is white or grayish cotton-like after freeze-drying, with a locally smooth and translucent surface and easily broken into powder under pressure; through scanning electron microscopy (SEM), at 1000-fold magnification, it presents a strip-like morphology coexisting with a flat irregular sheet structure, with some areas sparsely wrinkled and some areas flat and smooth; when magnified to 5000 times, the surface of the sheet structure is rough, with shallow wrinkles and densely distributed small circular depressions visible; after further magnification to 10,000 times, the edges of the depression structure are clear, accompanied by fine cracks.

5. The method for preparing the novel Bletilla striata glucomannan according to claim 4, wherein: In step 6, the determination of BsPsIII1-1 includes total sugar content, protein content and uronic acid content to ensure its characteristics as a neutral polysaccharide and not bound to proteins.

6. The method for preparing the novel Bletilla striata glucomannan according to claim 5, wherein: When the BsPsIII1-1 is detected and analyzed, it is analyzed by HPGPC-ELSD technology, and a calibration curve is established by T-series dextran standards to determine its average molecular weight, thereby obtaining BsPsIII1-1 with specific homogeneity and average molecular weight (about 74.8 kDa), and BsPsIII1-1 has a specific monosaccharide composition, mainly composed of mannose (Man), glucose (Glc) and galactose (Gal), with a molar ratio of about 100.00:88.17:2.32, of which Man and Glc account for more than 98%; the BsPsIII1-1 connection pattern and relative monosaccharide abundance are determined by methylation analysis. The main derivatives of BsPsIII1-1 were 2,3,6-trimethyl-D-mannopyranose (43.77%) and 2,3,6-trimethyl-D-glucopyranose (46.60%), indicating that BsPsIII1-1 was mainly composed of →4)-Manp-(1→) and →4)-Glcp-(1→) residues with a molar ratio close to 1:

1. The degree of branching (DB) was about 9.62%, and it showed a triple helical conformation. The fine structure of BsPsIII1-1, including its different types of glycosyl residues and their specific chemical shift assignments, was determined by nuclear magnetic resonance (NMR) technology. The connection sites and sequences between the residues were determined by HMBC and ROESY spectra.

7. The use of the novel Bletilla striata glucomannan according to claim 6, characterized in that: The BsPsIII1-1 is utilized 1 HNMR, 13 C NMR, 1 H- 1 H COSY, 1 H- 13 C HSQC, 1 H- 13 C HMBC, 1 H- 1 H ROESY and other techniques, combined with methylation analysis results, it is speculated that the main chain repeating unit of the polysaccharide is: →{4)-β-D-Glcp-(1} x →{4)-β-D-Manp-(1} n →{4)-β-D-Glcp-(1} y →4)-β-D-2Ac-Manp-(1→4)-β-D-3Ac-Manp-(1→4)-β-D-2Ac-Manp-(1→{4)-β-D-Manp-(1} m →4)-β-D-3Ac-Manp-(1→{4)-β-D-Manp-(1} i →, (where x + y = 8, n + m + i = 4).

8. The use according to claim 7, characterized in that: The BsPsIII1-1 is used in anti-liver fibrosis applications. Animal experiments have shown that BsPsIII1-1 has significant hepatoprotective activity in vivo and can improve liver tissue morphology and alleviate liver damage in a CCl4-induced C57BL / 6J mouse liver injury model in a dose-dependent manner. In particular, the high-dose (400 mg / kg) group can significantly reduce the weight loss of mice caused by CCl4 and significantly reduce the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the serum, which is equivalent to the positive control group (silymarin); H&E, Masson and Sirius Red staining observation showed that BsPsIII1-1 could effectively reduce the degree of liver fibrosis induced by CCl4 in mice and inhibit collagen fiber deposition. The effect of the high-dose group (400 mg / Kg) was close to that of the positive control group (silymarin), and it had a significant anti-liver fibrosis effect. In addition, BsPsIII1-1 could dose-dependently downregulate the expression of fibrosis-related proteins α-smooth muscle actin (α-SMA) and collagen I (COL-1), and exert its anti-liver fibrosis effect by inhibiting the activation of the TGF-β1 / SMAD2 / 4 / 7 signaling pathway.

9. The use according to claim 8, characterized in that: The BsPsIII1-1 has the ability to resist oxidative stress, can effectively alleviate the oxidative stress in the liver of mice caused by CCl4, significantly reduce the malondialdehyde (MDA) content in the liver, and increase the level of glutathione (GSH), thereby helping to maintain the normal function of the liver and showing the ability to resist oxidative stress.

10. The use according to claim 9, characterized in that: The application scope of the BsPsIII1-1 includes but is not limited to the preparation of anti-liver fibrosis drugs, functional foods or health products.

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