An extract of Astragalus complanatus and its preparation method

Abstract

This invention provides an extract of Astragalus complanatus and its preparation method. The extract comprises the following raw materials in molar percentages: Astragalus complanatus polysaccharide 31.79%–84.74%, protein 6.56%–34.35%, uronic acid 4.07%–27.11%, polyphenols 3.76%–10.83%, rhamnose 0.4%–3.35%, arabinose 0.97%–22.55%, xylose 0.30%–25.28%, mannose 0–58.37%, glucose 0–1.27%, galactose 17.38%–43.03%, and glucuronic acid 0.58%–55.71%. The Astragalus complanatus extract of this invention has the advantages of high purity, clearly defined active ingredients, and uniform stability. In vivo efficacy experiments using zebrafish have shown that the Astragalus complanatus extract of this invention has significant therapeutic effects on fatty liver, non-alcoholic steatohepatitis, alcoholic steatohepatitis, and hyperlipidemia.

Description

Technical Field

[0001] This invention relates to an extract of Astragalus complanatus and its preparation method, belonging to the field of traditional Chinese medicine separation and purification. Background Technology

[0002] Non-alcoholic fatty liver disease (NAFLD), also known as metabolic fatty liver disease (MAFLD), is a systemic liver syndrome characterized by widespread steatosis of hepatocytes. In recent years, with the continued rise in the global prevalence of obesity and type 2 diabetes, NAFLD has become the most common chronic liver disease worldwide, with an estimated prevalence exceeding 25% of the global population. Non-alcoholic steatohepatitis (NASH) is a subtype of NAFLD, also known as metabolic dysfunction-associated steatohepatitis (MASH). NASH, as a progressive form of NAFLD, is characterized by hepatocyte steatosis, lobular inflammation, and ballooning degeneration of hepatocytes, with or without fibrosis. NASH is a key driver of liver fibrosis, cirrhosis, and even hepatocellular carcinoma (HCC), posing a significant public health burden. Lifestyle interventions, such as dietary restrictions and increased physical activity to reduce weight, can alleviate NASH. However, maintaining these lifestyle changes long-term is extremely difficult for most patients, resulting in limited efficacy and significant individual variability. In terms of drug treatment, vitamin E and the insulin sensitizer pioglitazone have shown some efficacy only in specific patient populations, and long-term use of Western medicines carries certain hepatotoxicity risks. Currently, the US Food and Drug Administration has approved resimeltiro for the treatment of metabolic dysfunction-related steatohepatitis, but its overall efficacy is limited, with an effectiveness rate not exceeding 30%. Therefore, there is a need to develop a novel, safe, and effective drug for the treatment of NASH. Studies have shown that traditional Chinese medicine has significant advantages in alleviating the clinical symptoms of NAFLD and possesses broad application potential.

[0003] Astragalus complanati semen (ACS) is the dried, mature seed of Astragalus complanati, a legume. Also known as Sha Yuan Ji Li, it is a regional specialty medicinal herb from Qin (Shaanxi) medicine. It is warm in nature and sweet in taste, entering the liver and kidney meridians. It has the effects of tonifying the kidneys and strengthening yang, nourishing the liver and improving eyesight. ACS has a long history of medicinal use; the *Yuanhe Zhi* records that it was a tribute to the imperial court starting from Emperor Suzong. ACS has liver-protective and kidney-protective effects; the *Bencao Beiyao* records that it is "bitter and warm, tonifying the kidneys; pungent and warm, purging lung qi and dispersing liver wind; benefiting essence and improving eyesight." ACS has very low or even no toxicity. Its main components are polysaccharides, fatty acids, triterpenoid glycosides, amino acids, flavonoids, and trace elements. Clinically, ACS is mainly used to treat cardiovascular diseases, diabetes, and cancer, among other chronic diseases. Studies have shown that ACS has good lipid-lowering and liver-protective effects, and also has a protective effect against acute chemical liver injury. Its water extract, oil extract, 50% ethanol extract, and 95% ethanol extract all significantly reduced high-cholesterol diet-induced hyperlipidemia, helped reduce the fat content in the liver of hyperlipidemic rats, and protected the liver from CCl4 damage. Furthermore, it showed anti-liver cancer effects in both in vivo and in vitro studies. Astragalus complanatus polysaccharide is the main active ingredient of Astragalus complanatus, but there are currently no in-depth reports on its liver-protective effects. Previous studies by our research group found that Astragalus complanatus polysaccharide has a good therapeutic effect on high-fat diet-induced non-alcoholic fatty liver disease.

[0004] An analysis of patent literature on "Astragalus complanatus extract" reveals that CN118477112A discloses an Astragalus complanatus extract and its preparation method. This patent describes a water extraction process for Astragalus complanatus determined through orthogonal experiments, resulting in an extract with 37.15% total polysaccharides and 1.92% total flavonoids. This extract can reduce serum triglyceride and total cholesterol levels in mice, protect liver cells, and alleviate non-alcoholic fatty liver disease. Patent publication CN111135208A reports the use of ethanol extraction and petroleum ether and ethyl acetate extraction methods to remove pigments and lipids from Astragalus complanatus, yielding a defatted Astragalus complanatus ethanol extract. This extract can improve blood rheological parameters, has anti-aging, lipid-lowering and liver-protecting effects, enhances immune function, and has anti-inflammatory and analgesic properties.

[0005] CN106265867A discloses an ethanol extract of Astragalus complanatus and its application. The extract is obtained from Astragalus complanatus in 65-75% ethanol (v / v). It has therapeutic effects as an active ingredient in drugs for hyperlipidemia and fatty liver. CN105012375A discloses Astragalus complanatus pulverized and then defatted 2-3 times with petroleum ether. The defatted crude powder is extracted 2-3 times with 70%-90% ethanol at 60-70℃, concentrated, extracted 2-3 times with petroleum ether, diluted 2-3 times with distilled water, and purified with AB-8 macroporous resin. After loading, the mixture is allowed to stand for 2 hours, eluted with 50% ethanol, and the eluent is collected and concentrated under reduced pressure. The concentrated solution is purified with polyamide, eluted with 30% methanol, and the eluents are combined and concentrated under reduced pressure. The concentrated solution is passed through an LH-20 column, eluted with ethyl acetate, the eluents are collected, combined, concentrated, passed through an LH-20 column, eluted with petroleum ether, combined, concentrated under reduced pressure, and freeze-dried. This extract can treat Duchenne muscular dystrophy, cystic fibrosis, and hemophilia. The existing Astragalus complanatus extract mentioned above suffers from technical defects such as low enrichment of effective components and unclear active ingredients, resulting in weak pharmacological activity. Therefore, developing an Astragalus complanatus extract with clearly defined and highly enriched effective components is of great significance. Summary of the Invention

[0006] This invention provides an extract of Astragalus complanatus and its preparation method. The extract possesses advantages such as high purity, clearly defined and uniformly stable active ingredients. The preparation process is simple, efficient, stable, and feasible. In vivo efficacy experiments using zebrafish have demonstrated that the Astragalus complanatus extract exhibits significant therapeutic effects on fatty liver, non-alcoholic steatohepatitis, alcoholic steatohepatitis, and hyperlipidemia. This indicates that the Astragalus complanatus extract and its preparation method have broad prospects for industrial application.

[0007] This invention prepares ACP via water extraction and alcohol precipitation. Different components of ACP are obtained through fractional alcohol precipitation with different concentrations of ethanol, and their physicochemical properties are determined. The optimal active component of ACP for regulating NASH is screened using a zebrafish NASH model. This research helps to preliminarily reveal the relationship between ACP and NASH, broadens the drug treatment options for NASH, and injects new vitality into the modernization of traditional Chinese medicine research, providing new ideas and methods.

[0008] The technical solution of this invention patent application is as follows:

[0009] An extract of Astragalus complanatus, comprising the following molar percentages of active pharmaceutical ingredients: Astragalus complanatus polysaccharide 31.79%–84.74%, protein 6.56%–34.35%, uronic acid 4.07%–27.11%, and polyphenols 3.76%–10.83%.

[0010] Preferably, the Astragalus complanatus extract comprises the following molar percentages of active pharmaceutical ingredients: Astragalus complanatus polysaccharide 55.92%, protein 10.28%, uronic acid 4.07%, and polyphenols 4.98%.

[0011] Preferably, the Astragalus complanatus extract comprises the following molar percentages of active pharmaceutical ingredient: Astragalus complanatus polysaccharide 84.74%, protein 6.56%, uronic acid 4.49%, and polyphenols 3.76%.

[0012] Preferably, the Astragalus complanatus extract comprises the following molar percentages of active pharmaceutical ingredient: Astragalus complanatus polysaccharide 34.46%, protein 25.99%, uronic acid 27.11%, and polyphenols 8.11%.

[0013] Preferably, the Astragalus complanatus extract comprises the following molar percentages of active pharmaceutical ingredients: Astragalus complanatus polysaccharide 31.79%, protein 34.35%, uronic acid 15.80%, and polyphenols 10.83%.

[0014] As a further preferred embodiment of the present invention, the monosaccharides in the Astragalus complanatus extract comprise the following molar percentage components: rhamnose 0.4%–3.35%, arabinose 0.97%–22.55%, xylose 0.30%–25.28%, mannose 0–58.37%, glucose 0–1.27%, galactose 17.38%–43.03%, and glucuronic acid 0.58%–55.71%.

[0015] Preferably, the monosaccharides in the Astragalus complanatus extract include the following molar percentage components: arabinose 0.97%, xylose 0.30%, mannose 54.26%, galactose 43.03%, and glucuronic acid 1.44%.

[0016] Preferably, the monosaccharides in the Astragalus complanatus extract include the following molar percentage components: arabinose 0.99%, xylose 0.64%, mannose 58.37%, galactose 39.42%, and glucuronic acid 0.58%.

[0017] Preferably, the monosaccharides in the Astragalus complanatus extract include the following molar percentage components: rhamnose 3.15%, arabinose 9.36%, xylose 14.39%, galactose 39.42%, and glucuronic acid 55.71%.

[0018] Preferably, the monosaccharides in the Astragalus complanatus extract include the following molar percentage components: rhamnose 3.35%, arabinose 22.55%, xylose 25.28%, mannose 2.58%, glucose 1.27%, galactose 19.50%, and glucuronic acid 25.47%.

[0019] The preparation method of Astragalus complanatus extract of the present invention includes the following steps:

[0020] (1) Extraction of Astragalus complanatus seeds

[0021] After purification, the seeds of Astragalus complanatus are crushed and passed through a No. 2 sieve to obtain coarse powder. 200g of coarse powder is weighed and 4-6 times the amount of 95% ethanol is added. The mixture is heated under reflux at 75-85℃ for 2-4 hours, filtered, and the residue is dried at 55-65℃. 9-12 times the amount of pure water is added and the mixture is stirred in a water bath at 75-85℃ for 1-2 hours. The extraction is repeated 1-2 times. The mixture is filtered through gauze, and the filtrate is concentrated under reduced pressure at 60℃ to a suitable volume. 80% anhydrous ethanol of the concentrate is added and the mixture is allowed to stand overnight to precipitate all the polysaccharides from Astragalus complanatus.

[0022] (2) Fractional alcohol precipitation of polysaccharides from Astragalus complanatus

[0023] Different volumes of anhydrous ethanol (10%, 30%, 60%, 80%) were added sequentially, and the mixture was stirred slowly and quickly to obtain different alcohol precipitation fractions ACPI~ACPIV of Astragalus complanatus polysaccharide. The fractions were then freeze-dried under vacuum to obtain extracts of each component of Astragalus complanatus.

[0024] A further preferred embodiment of the method for preparing the extract of Astragalus complanatus of the present invention includes the following steps:

[0025] (1) Extraction of Astragalus complanatus seeds

[0026] After purification, the seeds of Astragalus complanatus were pulverized and passed through a No. 2 sieve to obtain coarse powder. 200g of coarse powder was weighed and 5 times the amount of 95% ethanol was added. The mixture was heated under reflux at 80℃ for 3 hours, filtered, and the residue was dried at 60℃. 10 times the amount of pure water was added and the mixture was extracted in a water bath at 80℃ with stirring for 1.5 hours. The extraction was repeated twice. The mixture was filtered through gauze and the filtrate was concentrated under reduced pressure at 60℃ to a suitable volume. 80% anhydrous ethanol of the concentrate was added and the mixture was allowed to stand overnight to precipitate all the polysaccharides from Astragalus complanatus.

[0027] (2) Fractional alcohol precipitation of polysaccharides from Astragalus complanatus

[0028] Different volumes of anhydrous ethanol (10%, 30%, 60%, 80%) were added sequentially, and the mixture was stirred slowly and quickly to obtain different alcohol precipitation fractions ACPI~ACPIV of Astragalus complanatus polysaccharide. The fractions were then freeze-dried under vacuum to obtain extracts of each component of Astragalus complanatus.

[0029] Preferably, the use of Astragalus complanatus extract of the present invention in the preparation of drugs for treating fatty liver, non-alcoholic steatohepatitis, alcoholic steatohepatitis, and hyperlipidemia.

[0030] Beneficial effects of the extract of Astragalus complanatus in this invention

[0031] (1) This invention explored the optimal extraction process of Astragalus complanatus extract and performed fractional alcohol precipitation. The physicochemical properties and monosaccharide composition of the crude polysaccharide and each fractional alcohol-precipitated component were determined. The physicochemical properties of ACPⅠ~ACPⅣ components were determined and preliminary structural analysis was performed. The optimal effective component ratio was obtained: ① Astragalus complanatus extract (ACPI) comprises the following molar percentages of raw material: Astragalus complanatus polysaccharide 55.92%, protein 10.28%, uronic acid content 4.07%, and polyphenols 4.98%.

[0032] ② The extract of Astragalus complanatus (ACPII) comprises the following molar percentages of active pharmaceutical ingredient: Astragalus complanatus polysaccharide 84.74%, protein 6.56%, uronic acid 4.49%, and polyphenols 3.76%.

[0033] ③ The extract of Astragalus complanatus (ACPIII) comprises the following molar percentages of active pharmaceutical ingredient: Astragalus complanatus polysaccharide 34.46%, protein 25.99%, uronic acid 27.11%, and polyphenols 8.11%.

[0034] ④ The extract of Astragalus complanatus (ACPIV) comprises the following molar percentages of active pharmaceutical ingredient: Astragalus complanatus polysaccharide 31.79%, protein 34.35%, uronic acid 15.80%, and polyphenols 10.83%.

[0035] (2) This invention uses a water extraction and alcohol precipitation method to extract ACP, and analyzes the ACP by alcohol precipitation, obtaining four alcohol precipitation fractions of Astragalus complanatus (ACPI, ACPII, ACPIII, ACPIV) with similar physicochemical properties. The physicochemical properties and preliminary structures of each fraction of ACP and ACPI-IV were determined. After extensive further experimental optimization, the polysaccharide active components in the Astragalus complanatus extract of this invention include the following: ① The monosaccharides in the Astragalus complanatus extract (ACPI) include the following molar percentage components: arabinose 0.97%, xylose 0.30%, mannose 54.26%, galactose 43.03%, and glucuronic acid 1.44%. ② The monosaccharides in the Astragalus complanatus extract (ACPII) include the following molar percentage components: arabinose 0.99%, xylose 0.64%, mannose 58.37%, galactose 39.42%, and glucuronic acid 0.58%. ③ The monosaccharides in Astragalus complanatus extract (ACPIII) include the following molar percentage components: rhamnose 3.15%, arabinose 9.36%, xylose 14.39%, galactose 39.42%, and glucuronic acid 55.71%. ④ The monosaccharides in Astragalus complanatus extract (ACPIV) include the following molar percentage components: rhamnose 3.35%, arabinose 22.55%, xylose 25.28%, mannose 2.58%, glucose 1.27%, galactose 19.50%, and glucuronic acid 25.47%.

[0036] Systematic analysis revealed significant differences in their physical morphology, chemical composition, and molecular structure, thus achieving efficient and targeted separation of complex Astragalus complanatus polysaccharide mixtures. Through fractional alcohol precipitation, neutral polysaccharides, acidic polysaccharides, and complex polysaccharides were separated from ACP. Ethanol fractionation not only separated them by molecular weight but also achieved fine separation by polysaccharide structural type. Low-concentration ethanol precipitation yielded mannogalactan, medium-concentration ethanol precipitation yielded pectin-like polysaccharides, and high-concentration ethanol precipitation yielded arabinoxylan and other complex polysaccharides. Fractional alcohol precipitation successfully separated ACP from the "mixture" into "characteristic components." The lightest-colored component, ACPⅡ, had the highest purity and fewest impurities; the darkest-colored component, ACPIV, had the highest protein and polyphenol content. ACPⅡ is a neutral sugar characterized by high purity and low uronic acid content; ACPⅢ is a pectin polysaccharide with high uronic acid content; ACPⅣ is a complex polysaccharide that more readily binds to proteins and polyphenols, forming dark-colored complexes. Ethanol fractionation not only separates polysaccharides by molecular weight but also achieves precise separation based on polysaccharide structure. Low-concentration ethanol precipitation yields mannogalactan, medium-concentration ethanol precipitation yields pectin-like polysaccharides, and high-concentration ethanol precipitation yields complex polysaccharides such as arabinoxylan. Infrared spectroscopy analysis shows that ACP and ACPⅢ–IV exhibit characteristic absorption peaks for polysaccharides.

[0037] (3) Animal experiments were conducted on zebrafish to simulate the liver-protective effects of oral administration, intestinal fermentation, and SCFA regulation in humans. The studies also assessed the comprehensive improvement of hepatic steatosis, inflammation, and fibrosis by polysaccharide components, more closely resembling the actual physiological state. This makes it an ideal model for screening the optimal active component for treating NASH. This study, through in vivo experiments on zebrafish using ALT, AST, TG, and TC as detection indicators, determined ACPⅠ as the optimal active component for treating NASH in zebrafish, further demonstrating the therapeutic efficacy of the Astragalus complanatus extract in treating hyperlipidemia.

[0038] Instruction manual illustrations

[0039] The accompanying drawings are provided to further explain the traditional Chinese medicine composition of the present invention and constitute a part of the present invention. The illustrative embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention.

[0040] Figure 1 Powder image of ACP and ACPI-IV components of Astragalus complanatus extract;

[0041] Figure 2Results of monosaccharide composition of ACP and ACP I-IV components in Astragalus complanatus extract; Note: Rha is rhamnose, Fuc is fucose, Ara is arabinose, Xyl is xylose, Man is mannose, Glc is glucose, Gal is galactose, GclA is galacturonic acid, and GalA is glucuronic acid.

[0042] Figure 3 Scanning electron micrographs of ACP and ACP I-IV components of Astragalus complanatus extract;

[0043] Figure 4 High performance liquid chromatography-gel chromatograms of ACP and ACP I-IV fractions of Astragalus complanatus extract;

[0044] Figure 5 Infrared spectra of ACP and ACP I-IV components of Astragalus complanatus extract;

[0045] Figure 6 The effect of different egg yolk powder molding times on the survival rate of zebrafish;

[0046] Figure 7 Zebrafish liver oil stained with Red O at different time points;

[0047] Figure 8 H&E staining images of zebrafish livers at different time points (100×).

[0048] Figure 9 Effects of different egg yolk powder molding times on transaminase and lipid metabolism in zebrafish;

[0049] Figure 10 The effect of different concentrations of egg yolk powder on the survival rate of zebrafish;

[0050] Figure 11 Images of zebrafish liver oil stained with Red O at different concentrations;

[0051] Figure 12 Images of zebrafish livers stained with H&E at different concentrations (100×).

[0052] Figure 13 Effects of different concentrations of egg yolk powder on survival rate, transaminase and lipid metabolism in zebrafish;

[0053] Figure 14 Survival curves of zebrafish after administration of ACP and ACP components I-IV;

[0054] Figure 15 Effects of ACP and ACP I-IV components on transaminase and lipid metabolism levels in zebrafish;

[0055] Figure 16 Correlation analysis of ACP and ACP I-IV components in the treatment of NASH;

[0056] Figure 17 Zebrafish liver oil red O staining image. Note: H represents the high-dose group (132.50 μg / mL); M represents the medium-dose group (66.25 μg / mL); L represents the low-dose group (33.13 μg / mL). Compared with the control group, *p < 0.05, **p < 0.01; compared with the model group, # p < 0.05 ## p < 0.01;

[0057] Figure 18 Image of zebrafish liver H&E staining (100×). Detailed Implementation

[0058] To better understand the implementation of this invention, the following components are included: ACP-Astragalus complanatus polysaccharide extract, NASH-nonalcoholic steatohepatitis, TG-triglycerides, AST-aspartate aminotransferase, TC-total cholesterol, and ALT-alanine aminotransferase. One or more experimental examples are listed below, and the invention will be further illustrated through typical embodiments.

[0059] Example 1

[0060] (1) Extraction of Astragalus complanatus seeds

[0061] After purification, the seeds of Astragalus complanatus were pulverized and passed through a No. 2 sieve to obtain coarse powder. 200g of coarse powder was weighed and 5 times the amount of 95% ethanol was added. The mixture was heated under reflux at 80℃ for 3 hours, filtered, and the residue was dried at 60℃. 10 times the amount of pure water was added and the mixture was extracted in a water bath at 80℃ with stirring for 1.5 hours. The extraction was repeated twice. The mixture was filtered through gauze and the filtrate was concentrated under reduced pressure at 60℃ to a suitable volume. 80% anhydrous ethanol of the concentrate was added and the mixture was allowed to stand overnight to precipitate all the polysaccharides from Astragalus complanatus.

[0062] (2) Fractional alcohol precipitation of polysaccharides from Astragalus complanatus

[0063] Different volumes of anhydrous ethanol (10%, 30%, 60%, 80%) were added sequentially, and the mixture was stirred slowly and quickly to obtain different alcohol precipitation fractions ACPⅠ~ACPⅣ of Astragalus complanatus polysaccharide. The fractions were then freeze-dried under vacuum to obtain extracts of each component of Astragalus complanatus.

[0064] Example 2

[0065] (1) Extraction of Astragalus complanatus seeds

[0066] After purification, the seeds of Astragalus complanatus were crushed and passed through a No. 2 sieve to obtain coarse powder. 200g of coarse powder was weighed and 6 times the amount of 95% ethanol was added. The mixture was heated to reflux at 85°C for 2 hours and filtered. The residue was dried at 65°C and 12 times the amount of pure water was added. The mixture was stirred in a water bath at 75°C for 2 hours and extracted once. The mixture was filtered through gauze and the filtrate was concentrated under reduced pressure at 60°C to a suitable volume. The concentrate was added to 80% anhydrous ethanol and allowed to stand overnight to precipitate all the polysaccharides from Astragalus complanatus.

[0067] (2) Fractional alcohol precipitation of polysaccharides from Astragalus complanatus

[0068] Example 3

[0069] (1) Extraction of Astragalus complanatus seeds

[0070] After purification, the seeds of Astragalus complanatus were pulverized and passed through a No. 2 sieve to obtain coarse powder. 200g of coarse powder was weighed and 4 times the amount of 95% ethanol was added. The mixture was heated to reflux at 75°C for 4 hours, filtered, and the residue was dried at 55°C. 9 times the amount of pure water was added and the mixture was stirred in a water bath at 85°C for 1 hour. The extraction was repeated twice. The mixture was filtered through gauze and the filtrate was concentrated under reduced pressure at 60°C to a suitable volume. 80% anhydrous ethanol of the concentrate was added and the mixture was allowed to stand overnight to precipitate all the polysaccharides from Astragalus complanatus.

[0071] (2) Fractional alcohol precipitation of polysaccharides from Astragalus complanatus

[0072] Different volumes of anhydrous ethanol (10%, 30%, 60%, 80%) were added sequentially, and the mixture was stirred slowly and quickly to obtain different alcohol precipitation fractions ACPⅠ~ACPⅣ of Astragalus complanatus polysaccharide. The fractions were then freeze-dried under vacuum to obtain extracts of each component of Astragalus complanatus.

[0073] Experimental Example 1

[0074] Accurately weigh appropriate amounts of ACP (Astragalus complanatus polysaccharide) and the lyophilized powders of each alcohol-precipitated fraction into a capped reaction flask. Add 2 mL of 2 mol / L trifluoroacetic acid (TFA) (0.3 mL TFA + 1.7 mL double-distilled water) and react in an oven at 121℃ for 2 h. Transfer the reaction solution to a sleeve, evaporate the solvent by rotation, add 2 mL of double-distilled water, and then dry under vacuum. Repeat 3-4 times. Add a trace amount of sodium hydroxide solution to neutralize, rinse the wall with water, and dry under vacuum. Add 500 μL of 0.5 mol / L sodium carbonate solution and incubate at 30℃ for 45 min. Add 0.5 mL of 4% sodium borohydride solution and let stand at room temperature for 1.5 h. Add 25% acetic acid dropwise until no more bubbles are produced. Pass the solution through a cation exchange column [(DOWEX) 50×8 400 mesh]. Evaporate the eluent under vacuum at 45℃, add 2 mL of methanol, and evaporate to dryness. Repeat 3 times. Heat under vacuum at 85℃ for 2 h to convert uronic acid into lactone. Dissolve the residue in 1 Add 1 mL of n-propylamine to pyridine, react at 55°C for 30 min, and after the solution cools to below 45°C, heat to 55°C and evaporate to dryness under vacuum. Add 1 mL of pyridine and acetic anhydride to the residue and react at room temperature overnight. After the reaction is complete, dry at 80°C, add 1 mL of dichloromethane and shake well, then add 1 mL of distilled water and extract at 6000 r / min for 3 min. Repeat the process 4 times.

[0075] II. Experimental Materials, Instruments and Animals

[0076] 1. Reagents and reagents, experimental instruments

[0077] The experimental animals were juvenile zebrafish, 7 dpf, purchased from Shanghai Feixi Biotechnology Co., Ltd., and raised in 6-well plates at a water temperature of 28℃, 30 fish / well.

[0078] III. Experimental Methods

[0079] 1. Preparation method of crude polysaccharide and fractionated alcohol precipitation components of ACP

[0080] 1.1 Preparation of crude polysaccharide from ACP

[0081] The *Astragalus complanatus* seed was pulverized and passed through a 20-mesh sieve. 100.0 g of coarse *Astragalus complanatus* powder was added to 17 times the amount of purified water and decocted three times, 1.5 h each time. The extract was passed through 200-mesh gauze, and the filtrates were combined and concentrated. Anhydrous ethanol was added to the concentrated solution until the alcohol content reached 80%, and the solution was precipitated overnight at 4°C. The precipitate was collected by suction filtration. The precipitate was reconstituted with distilled water, and the sample solution was treated using the Sevage method (dichloromethane: n-butanol = 4:1) to remove protein. The resulting supernatant was freeze-dried (pre-freezing temperature -30°C to -50°C, drying time 4 to 8 hours, vacuum degree 10 to 50 Pa, shelf temperature -30°C to -50°C, heating rate 0.5 to 1°C / h) to obtain ACP.

[0082] 1.2 Preparation of ACP fractionated alcohol precipitation components

[0083] A stepwise alcohol precipitation process was used to separate ACP from polysaccharides.

[0084] Weigh 1000 mg of ACP and dissolve it in an appropriate amount of double-distilled water to a mass concentration of 1%. Slowly add a certain volume of anhydrous ethanol to the ACP aqueous solution to make the ethanol mass fraction of the solution reach 10%. Let it stand at 4℃ for 12 h, then centrifuge at 10,000 r / min for 10 min. Collect the precipitate and supernatant I separately. Dissolve the precipitate in an appropriate amount of double-distilled water and centrifuge at 10,000 r / min for 10 min. Discard the precipitate and freeze-dry the supernatant to obtain the polysaccharide (ACP I) with a 10% ethanol concentration. Slowly add a certain volume of anhydrous ethanol to supernatant I to make the ethanol mass fraction of the solution reach 30%. After "standing at 4℃ for 12 h", repeat the above operation steps to sequentially precipitate ACP fractions with 10%, 30%, 60%, and 80% ethanol concentrations, and name them ACP I to IV, respectively.

[0085] 2. Determination of the physicochemical properties of ACP and ACP I-IV polysaccharides

[0086] 2.1 Determination of sugar content

[0087] The sugar content of ACP and ACP I-IV fractions was determined using the phenol-concentrated sulfuric acid method. The protein content of ACP and ACP I-IV fractions was determined using the BCA protein concentration assay kit. The uronic acid content of ACP and ACP I-IV fractions was determined using the m-hydroxybiphenyl method. The polyphenol content of ACP and ACP I-IV fractions was determined using the Folin-Ciocalteu reagent method.

[0088] 2.2 Determination of Monosaccharide Composition

[0089] The ACP and ACP I-IV components were determined according to Jacob Lehrfeld's monosaccharide composition method.

[0090] 2.2.1 Sample Preparation

[0091] Accurately weigh appropriate amounts of lyophilized ACP and ACP I-IV components into a capped reaction flask, add 2 mL of 2 mol / L trifluoroacetic acid (TFA) (0.3 mL TFA + 1.7 mL double-distilled water), and react in an oven at 121℃ for 2 h. Transfer the reaction solution to a sleeve, evaporate the solvent by rotation, add 2 mL of double-distilled water, and then dry under vacuum. Repeat 3-4 times. Neutralize with a trace amount of sodium hydroxide solution, rinse the walls with water, and dry under vacuum. Add 500 μL of 0.5 mol / L sodium carbonate solution and incubate at 30℃ for 45 min. Add 0.5 mL of 4% sodium borohydride solution and let stand at room temperature for 1.5 h. Add 25% acetic acid dropwise until no more bubbles are produced. Pass the solution through a cation exchange column [(DOWEX) 50×8 400 mesh], evaporate the eluent under vacuum at 45℃, add 2 mL of methanol, evaporate to dryness, and repeat 3 times. Heat under vacuum at 85℃ for 2 h to convert uronic acid into lactone. Dissolve the residue in 1 Add 1 mL of n-propylamine to pyridine, react at 55°C for 30 min, and after the solution cools to below 45°C, heat to 55°C and evaporate to dryness under vacuum. Add 1 mL of pyridine and acetic anhydride to the residue and react at room temperature overnight. After the reaction is complete, dry at 80°C, add 1 mL of dichloromethane and shake well, then add 1 mL of distilled water and extract at 6000 r / min for 3 min. Repeat the process of adding water 4 times.

[0092] 2.2.2 Determination of Monosaccharide Composition

[0093] Gas chromatography (GC) was used to determine the monosaccharide composition of ACP and ACP I-IV components. The GC column was an Rtx-50 (30.0 m × 0.25 mm × 0.25 μm), and the temperature program was 180℃ (2 min), 210℃ (6℃ / min), 215℃ (0.3℃ / min), and 240℃ (6℃ / min, 30 min).

[0094] 2.3 Molecular weight determination

[0095] The molecular weight of ACP and its components I-IV was determined by high-performance gel permeation chromatography (HPGPC).

[0096] 2.4 Observation of polysaccharide morphology

[0097] The morphology of ACP and ACP I-IV components was observed using scanning electron microscopy. Before measurement, the samples were freeze-dried for 48 h, and an appropriate amount was placed on the sample stage and deposited with a conductive gold film in an ion sputtering instrument for observation.

[0098] 2.5 Fourier Transmission Infrared Spectroscopy (FT-IR) Analysis

[0099] Weigh 2.0 mg of ACP and ACP I-IV polysaccharide samples, add 100 mg of dried KBr powder, mix well, grind thoroughly in an agate mortar, compress into tablets, and analyze using a Fourier transform infrared spectrometer at 4000-400 cm⁻¹. -1 The infrared spectra of each component sample were measured by scanning within the wavelength range.

[0100] 3. Screening of the optimal active components of ACP I-IV for the treatment of NASH

[0101] 3.1 Screening of modeling concentration

[0102] Forty-five healthy zebrafish juveniles aged 7 dpf were randomly selected and placed in clean 6-well plates. Four mass concentrations (0, 1, 2, 3, 4 mg / mL) were set up. 6 mL of egg yolk powder modeling solution was added to each well. Culture medium was used as a negative control. The treatment lasted for 72 h, and the medium was changed every 24 h. The toxic phenotype and mortality of zebrafish were observed and recorded. The mortality rate of zebrafish at each concentration was calculated. The contents of TG, TC, ALT, and AST were detected. The results of Oil Red O staining, HE staining, and Masson staining were observed to screen the optimal modeling concentration.

[0103] 3.2 Screening of Modeling Time

[0104] Forty-five healthy zebrafish juveniles with a 7 dpf growth factor were randomly selected and placed in a clean 6-well plate. Five time points were set (0 h, 24 h, 48 h, 72 h, and 96 h). 6 mL of egg yolk powder modeling solution was added to each well. System water was used as a negative control. The toxic phenotype and mortality of the zebrafish were observed and recorded. The mortality rate of the zebrafish at each concentration was calculated. The contents of TG, TC, ALT, and AST were detected. The results of Oil Red O staining, HE staining, and Masson staining were observed to screen the optimal modeling time.

[0105] 3.3 Acute toxicity test of Astragalus complanatus

[0106] Healthy zebrafish juveniles with a dpf of 7 were randomly selected and placed in clean 24-well plates, and randomly divided into groups of 10. A series of concentration gradient groups were set up to screen the drug concentration in all groups. After 72 h of drug administration, the survival rate was calculated as follows: survival rate (%) = (number of surviving embryos / total number of embryos) × 100%.

[0107] 3.4 Oil Red O staining

[0108] Sample collection and fixation: Zebrafish juveniles were placed in 6-well plates, washed 3 times with PBS solution, and fixed with 4% paraformaldehyde for 24 hours.

[0109] Dehydration: Wash three times with PBS solution, then permeate with a gradient of 20%, 40%, 80%, and 100% propylene glycol for 20 min each time.

[0110] Staining: Subsequently, immerse the sample in 0.5% Oil Red O solution at 65°C in the dark for 1 hour;

[0111] Background color removal: Wash the background color with 100%, 80%, 40%, and 20% propylene glycol in sequence, 20 min each time, and then rinse off the propylene glycol with PBS solution.

[0112] Observation: Six zebrafish were randomly selected from each group and observed under a stereomicroscope. Image J software was used to analyze the images and calculate the stained area.

[0113] 3.5 H&E staining

[0114] Liver tissue fixed with 4% paraformaldehyde was dehydrated, embedded in paraffin, and cut into 4 μm thick sections.

[0115] Hematoxylin staining: Immerse the sections in hematoxylin staining solution for 3 to 5 minutes, wash with tap water, differentiate with differentiation solution, wash with tap water, blue back solution, and rinse with running water.

[0116] Eosin staining: The sections were dehydrated in 95% alcohol for 1 min, and then stained in eosin staining solution for 15 s;

[0117] Dehydration and mounting: The sections were sequentially placed in anhydrous ethanol I (2 min) - anhydrous ethanol II (2 min) - anhydrous ethanol III (2 min) - n-butanol I (2 min) - n-butanol II (2 min) - xylene I (2 min) - xylene II (2 min) for clearing and mounting with neutral resin; microscopic examination and image acquisition and analysis.

[0118] 3.6 Detection of TG, TC, ALT, and AST efficacy indicators

[0119] Forty-five zebrafish juveniles were collected and placed in 2 mL centrifuge tubes. 300 μL of physiological saline was added as the homogenizing medium. Homogenization was performed at 4℃ and 1500 r / min, followed by centrifugation at 12000 r / min for 10 min. The supernatant was collected, and the protein concentration of the homogenate was determined according to the BCA protein concentration assay kit. The TG and TC contents in the zebrafish juveniles were determined according to the manufacturer's instructions. The AST and ALT contents in the zebrafish juveniles were also determined according to the manufacturer's instructions.

[0120] IV. Experimental Results

[0121] 1. Yields and physicochemical properties of ACP and ACP I-IV components

[0122] 1.1 Yields of ACP components I-IV

[0123] The polysaccharide yields of ACP components I through IV are as follows: ACP I yield 15.50%, ACP II yield 53.00%, ACP III yield 9.00%, and ACP IV yield 3.00%.

[0124] 1.2 Images of ACP components I-IV

[0125] As per the instruction manual Figure 1 As shown, the freeze-dried powders of ACPI-IV components range in color from light to dark: ACPI is light yellow with a uniform texture, appearing as a fine and loose powder; ACPII is off-white with a uniform and fine powder texture; ACPIII is brown; and ACPIV is brownish-brown.

[0126] 1.3 Physicochemical properties of ACP components I-IV

[0127] The basic physicochemical properties of ACP I~IV components are shown in Table 1. ACPI is a transitional component between crude polysaccharide and high-purity component; ACPII is a high-purity neutral polysaccharide; ACPIII is an acidic pectin polysaccharide; and ACPIV is a complex polysaccharide.

[0128]

[0129] 1.4 Monosaccharide composition of ACP and ACP I-IV components

[0130] The monosaccharide compositions of ACP and ACP I-IV fractions are shown in Table 2. Chromatograms are attached to the instruction manual. Figure 2 The results showed that ACP precipitation with low concentration ethanol yielded mannogalactan, precipitation with medium concentration ethanol yielded pectin-like polysaccharides, and precipitation with high concentration ethanol yielded arabinoxylan and other complex polysaccharides. ACP and ACP I-II were the polysaccharide types with the highest mannogalactan content; ACP II was a neutral sugar, characterized by high purity and low uronic acid content; ACP III was a pectin polysaccharide with high uronic acid content; and ACP IV was a complex complex polysaccharide.

[0131]

[0132] Note: Rha is rhamnose, Fuc is fucose, Ara is arabinose, Xyl is xylose, Man is mannose, Glc is glucose, Gal is galactose, GclA is galacturonic acid, and GalA is glucuronic acid.

[0133] 1.5 Morphological observation of ACP and ACP I-IV components

[0134] The microstructure of ACP and its components I-IV was observed using scanning electron microscopy (SEM), and the results are as follows: Figure 3As shown. ACP has a relatively smooth surface with slight wrinkles and cracks, but no obvious pores; ACPⅠ exhibits a filamentous and irregular sheet-like structure, with filaments and sheets interwoven, showing breakage and curling; ACPⅡ exhibits a porous, network-like structure composed of many interconnected fibers; ACPⅢ presents a rough, uneven, blocky or granular aggregate, with the surface composed of a large number of tiny, uneven spherical or granular materials, resulting in a dense structure; ACPⅣ presents a fluffy, cotton-like structure, with a loose, fragile network formed by extremely fine, coarse fibers intertwined, containing numerous irregular voids, and the fiber surfaces are rough with many small protrusions or particles. Compared to ACPⅢ, ACPⅣ has a more loose and disordered structure.

[0135] 1.6 Results of molecular weight determination of ACP and ACP I-IV components

[0136] Based on the molecular weight and retention time of the standards, calibration curves for LgMp-RT (peak molecular weight), LgMw-RT (weight-average molecular weight), and LgMn-RT (number-average molecular weight) were calculated. The equation for the LgMp-RT calibration curve is: y = -0.1402x + 9.4033, R 2 = 0.9942; The equation of the LgMw-RT correction curve is: y = -0.1399x + 9.3915, R 2 = 0.9933; The equation of the LgMn-RT correction curve is: y = -0.1391x + 9.3547, R 2 = 0.9948. The linear correlation coefficient R between the Lg values ​​of each parameter and retention time. 2 A value >0.99 indicates good linearity. Substituting these values ​​into the equation, the peak molecular weights (Mp) of ACP and ACPⅠ-Ⅳ are 577 171 Da, 513 523 Da, 436 700 Da, 50 907 Da, and 21 557 Da; the weight-average molecular weights (Mw) are 571 591 Da, 508 687 Da, 432 739 Da, 50 680 Da, and 21 501 Da; and the number-average molecular weights (Mn) are 550 579 Da, 490 420 Da, 417 711 Da, 49 721 Da, and 21 232 Da.

[0137] As the alcohol concentration increases, the polarity of the solvent system continuously decreases, and its solubility for polymers declines accordingly. Larger molecular weight polymers are more sensitive to changes in solvent polarity, and the interactions between molecular chains are more likely to exceed their interactions with the solvent, thus precipitating first at lower alcohol concentrations. By gradually increasing the alcohol concentration, fractional precipitation separation of the mixture can be achieved according to molecular weight from largest to smallest. Therefore, as the alcohol concentration increases, the molecular weight of each precipitated component decreases sequentially. From ACP to ACPⅠ~IV, the molecular weight of polysaccharides shows a decreasing trend. Simultaneously, the polydispersity index (PDI) of each component is close to 1, indicating that the polysaccharide molecules within each component are very uniform in size. This suggests that after fractional alcohol precipitation and merging, components with concentrated yet distinct molecular weight ranges were obtained. The results are shown in Table 3 and appendix. Figure 4 .

[0138]

[0139] 1.7 FT-IR identification of ACP and ACP I-IV components

[0140] The FT-IR spectra of ACP and its components I-IV are similar in main peak shape, exhibiting typical polysaccharide characteristic absorption peaks. However, significant differences exist in the relative intensities of the absorption peaks within a specific wavenumber range, indicating differences in the specific chemical structures of each component. For example... Figure 5 As shown, the broad absorption peak in the 3300 cm⁻¹ region is attributed to the OH stretching vibration; all components exhibit a strong and broad absorption peak at this region. The absorption peak near 2920 cm⁻¹ is attributed to the CH stretching vibration; these are all typical characteristic peaks of polysaccharides. 1600 cm⁻¹ -1 and 1400 cm -1 The nearby absorption peaks are attributed to the asymmetric and symmetric stretching vibrations of the carboxylate ion (COO⁻). This region is crucial for identifying acidic polysaccharides; ACPIII and ACPIV show significantly stronger absorption peaks here than other components, while the peaks of neutral polysaccharide components ACPI and ACPII are relatively weak. (1000 ~ 1200 cm⁻¹) -1 The strong and broad absorption peaks in the region are attributed to the stretching vibrations of COC and COH, which are characteristic fingerprint regions of the pyranose ring skeleton. All components have the strongest absorption here, indicating that ACP and ACP I-IV components of Astragalus complanatus extract are all pyranose polysaccharides.

[0141] 2. Results of the zebrafish experiment

[0142] 2.1 Screening of Modeling Time

[0143] 2.1.1 Effect of different egg yolk powder molding times on the survival rate of zebrafish juveniles

[0144] As per the instruction manual Figure 6 As shown, within a certain range, with the increase of exposure time to egg yolk powder solution, the mortality rate of zebrafish was 56.48% and the deformity rate was 40% after 96 hours of administration. When the administration time did not exceed 72 hours, the zebrafish were in good condition with no abnormalities, and the incidence of toxic phenotypes such as death and deformity did not exceed 10%.

[0145] 2.1.2 Results of Oil Red O staining

[0146] As per the instruction manual Figure 7 As shown, Oil Red O staining results indicated that, compared to 0 h, with increasing drug administration time, the zebrafish in the model group showed deeper staining, severe lipid deposition in the liver, and significant accumulation of lipid staining in the tail, subintestinal vessels, and intersegmental vessels. Oil Red O staining results showed that at 72 h, significant lipid deposition was observed in the zebrafish liver (p < 0.01).

[0147] 2.1.3 H&E staining results

[0148] As per the instruction manual Figure 8 As shown, with increasing modeling time, disordered hepatic cord structure was observed, some hepatocytes exhibited irregular shapes, cytoplasmic degeneration with balloon-like lesions, cell vacuolation, hepatocyte necrosis, and inflammatory cell infiltration. H&E staining results indicated that significant damage to the zebrafish liver was observed at 72 h.

[0149] 2.1.4 Effects of different egg yolk powder molding times on transaminase and lipid metabolism in zebrafish juveniles

[0150] As per the instruction manual Figure 9 As shown, within a certain range, the levels of TG, TC, ALT, and AST in zebrafish juveniles increased in a dose-dependent manner with increasing exposure time to egg yolk powder solution. At 72 h, all indicators of TG, TC, ALT, and AST showed significant differences compared to 0 h (p < 0.05, p < 0.01). Therefore, the administration time was determined to be 72 h.

[0151] 2.2 Screening of Modeling Concentration

[0152] 2.2.1 Effect of different concentrations of egg yolk powder on the survival rate of zebrafish juveniles

[0153] As per the instruction manual Figure 10 As shown, within a certain range, with the increase of egg yolk powder exposure concentration, the mortality rate of zebrafish was 100% when administered 4 mg / mL, 16% when administered 3 mg / mL, and the deformity rate was 50%.

[0154] 2.2.2 Results of Oil Red O staining

[0155] As per the instruction manual Figure 11 As shown, Oil Red O staining results indicated that, compared to 0 mg / mL, with increasing administration time, the zebrafish in the model group showed deeper staining, severe lipid deposition in the liver, and significant accumulation of lipid staining in the tail, subintestinal vessels, and intersegmental vessels. Oil Red O staining results showed that at 2 mg / mL, significant lipid deposition occurred in the zebrafish liver (p < 0.01).

[0156] 2.2.3 H&E staining results

[0157] As per the instruction manual Figure 12 As shown, with increasing concentrations, disordered hepatic cord structure was observed, some hepatocytes exhibited irregular shapes, cytoplasmic degeneration with ballooning lesions, cell vacuolation, hepatocyte necrosis, and inflammatory cell infiltration. H&E staining results indicated that at 2 mg / mL, zebrafish liver showed significant damage.

[0158] 2.2.4 Effects of different concentrations of egg yolk powder on transaminase and lipid metabolism in zebrafish

[0159] As per the instruction manual Figure 13 As shown, within a certain range, zebrafish TG, TC, ALT, and AST increased in a dose-dependent manner with increasing egg yolk powder exposure concentration. At 2 mg / mL, all TG and AST levels were significantly different from those at 0 mg / mL (p < 0.05).

[0160] 2.3 Determination of dosage for ACP and ACP components I-IV

[0161] As per the instruction manual Figure 14 The acute toxicity dose-mortality data for zebrafish exposed to ACP and ACP I-IV aqueous solutions are shown in the figure. (LC50 of ACP component aqueous solution) 50 The concentration of ACPⅠ component aqueous solution was 1325 μg / mL (LC50). 50 The concentration of ACPⅡ component aqueous solution was 1809 μg / mL (LC50). 50 The concentration of ACPⅢ component aqueous solution was 1988 μg / mL (LC50). 50 The concentration of ACP IV component aqueous solution was 1298 μg / mL (LC50). 50 It was 1118 μg / mL. [The following appears to be a separate, unrelated sentence:] Using 1 / 10 LC... 50 1 / 20 LC 50 1 / 40 LC 50 The high, medium, and low dosages for subsequent intervention in NASH zebrafish were determined. Therefore, the dosages for each fractionated alcohol-precipitated component and crude polysaccharide component are shown in Table 4 below.

[0162]

[0163] 2.4 ACP and ACP I-IV fractions' therapeutic activity against NASH

[0164] 2.4.1 Effects of ACP and ACP I-IV components on transaminase and lipid metabolism levels in zebrafish

[0165] As per the instruction manual Figure 15 As shown, compared with the blank group, the zebrafish in the model group showed a significant increase in TG, ALT, and AST (p < 0.01); compared with the model group, the ACP and ACPⅠ components could reduce their content to varying degrees after intervention, and combined with the correlation analysis results, the ACPⅠ-L group had a more significant effect (p < 0.05).

[0166] 2.4.2 Effects of ACP and ACP I-IV components on transaminase and lipid metabolism levels in zebrafish

[0167] As shown in the attached instruction manual Figure 16 As shown, Astragalus complanatus polysaccharides have a good intervention effect on NASH by reducing liver function indicators (ALT, AST) and improving lipid metabolism (TC, TG). Correlation heatmap analysis showed that the ACPⅠ(P)-L component exhibited the strongest negative correlation and statistical significance, indicating that it is the core effective component of ACP that exerts its anti-NASH effect. This provides a pharmacodynamic basis for further development of ACP components into granules for the treatment of NASH.

[0168] 2.4.3 Results of Oil Red O staining

[0169] Oil Red O staining results attached Figure 17 The results showed that, compared with the control group, the zebrafish in the model group had darker staining, severe lipid deposition in the liver, and significant accumulation of lipid staining in the tail, subintestinal vessels, and intersegmental vessels, indicating that the NASH zebrafish model was successfully established (p < 0.01). After intervention with ACP and various alcohol precipitation components, lipid accumulation in the liver was significantly reduced (p < 0.01), and the improvement effect of ACPⅠ-L was more significant. The statistical results of the staining area are shown in […]. Figure 17 .

[0170] 2.4.4 H&E staining results

[0171] As per the instruction manual Figure 18As shown, the liver structure of the control group zebrafish was good, with normal hepatocyte morphology, polygonal shape, normal cytoplasmic staining, and obvious cell nuclei. Compared with the control group, the model group showed disordered hepatic cord structure, some hepatocytes exhibited irregular shapes, cytoplasmic degeneration with ballooning lesions, cell vacuolation, hepatocyte necrosis, and inflammatory cell infiltration. After treatment with ACP and various alcohol precipitation components, the zebrafish liver showed reduced ballooning lesions and inflammatory damage compared to the model group, and the hepatocyte structure was restored, with a small amount of vacuolation remaining. After ACPI treatment, the morphology and basic structure of hepatocytes were restored, cytoplasmic staining was normal, inflammatory cell aggregation was reduced, and vacuolation was weakened.

[0172] V. Summary and Discussion

[0173] This study used water extraction and alcohol precipitation to extract ACP, and analyzed the ACP by alcohol precipitation, obtaining four alcohol-precipitated fractions (ACPI, ACPII, ACPIII, and ACPIV) with similar physicochemical properties. The physicochemical properties and preliminary structures of ACP and ACPI-IV fractions were determined. Systematic analysis revealed significant differences in their physical morphology, chemical composition, and molecular structure, thus achieving efficient and targeted separation of complex polysaccharide mixtures. Through fractional alcohol precipitation, neutral polysaccharides, acidic polysaccharides, and complex polysaccharides were separated from ACP. Ethanol fractionation not only separated by molecular weight but also achieved fine separation by polysaccharide structural type. Low-concentration ethanol precipitation yielded mannogalactan, medium-concentration ethanol precipitation yielded pectin-like polysaccharides, and high-concentration ethanol precipitation yielded arabinoxylan and other complex polysaccharides. Fractional alcohol precipitation successfully separated ACP from a "mixture" into "characteristic components." Neutral polysaccharides, acidic polysaccharides, and complex polysaccharides were separated from ACP through fractional alcohol precipitation. The lightest-colored component, ACPⅡ, has the highest purity and fewest impurities; the darkest-colored component, ACPIV, has the highest protein and polyphenol content. ACPⅡ is a neutral sugar, characterized by high purity and low uronic acid content; ACPⅢ is a pectin polysaccharide with high uronic acid content; ACPⅣ is a complex polysaccharide that more easily binds to proteins and polyphenols, forming dark-colored complexes. Ethanol fractionation not only separates by molecular weight but also achieves fine separation based on polysaccharide structure type. Low-concentration ethanol precipitation yields mannogalactan, medium-concentration ethanol precipitation yields pectin-like polysaccharides, and high-concentration ethanol precipitation yields complex polysaccharides such as arabinoxylan. Infrared spectroscopy analysis shows that ACP and ACPIV possess characteristic absorption peaks for polysaccharides.

[0174] Studies have shown that the human body lacks the enzymes to degrade most large polysaccharides, which are typically utilized by the gut microbiota through fermentation. As prebiotics, polysaccharides selectively promote the growth of beneficial bacteria (such as Bifidobacteria and Lactobacillus). Furthermore, through fermentation of polysaccharides by the gut microbiota, short-chain fatty acids (SCFAs) such as butyric acid, propionic acid, and acetic acid are produced, providing energy for intestinal epithelial cells and maintaining the integrity of the intestinal barrier. At the same time, SCFAs enter the bloodstream and act on cells of distant organs (such as immune cells, adipocytes, and hepatocytes), regulating gene expression and cellular function by inhibiting histone deacetylases or activating G protein-coupled receptors, thereby exerting systemic effects such as anti-inflammation and metabolic regulation. Zebrafish have become an important pharmaceutical tool for early drug discovery in recent years and are widely used as a crucial model organism for drug efficacy screening and toxicity evaluation. Current research indicates that zebrafish express a variety of drug-metabolizing enzymes, such as phase I metabolic enzyme cytochrome P450 (CYP450), and phase II metabolic enzymes uridine-diphospho-glucuronosyltransferase (UGT) and sulfo-transferase (SULT). The gene or amino acid sequences of these enzymes share more than 50% identity with those in humans. Furthermore, zebrafish possess a rich gut microbiota. Therefore, using zebrafish for in vivo pharmacodynamic experiments to screen for active components is highly effective in mimicking the hepatoprotective effects in humans from oral administration and intestinal fermentation to SCFA regulation. It can also assess the comprehensive improvement of hepatic steatosis, inflammation, and fibrosis by polysaccharide components, more closely approximating the actual physiological state. This makes zebrafish an ideal model for screening the optimal active components for treating NASH. This study used in vivo experiments in zebrafish, with ALT, AST, TG, and TC as detection indicators, to determine that ACPⅠ is the optimal active component for treating NASH in zebrafish.

[0175] Through systematic component separation and characterization, the scientific rigor and depth of the research were significantly enhanced, and key material basis and theoretical guidance were provided for the future development of Astragalus complanatus polysaccharide health products or drugs with clear targets and mechanisms.

Claims

1. A Syagrus vera extract, characterized by, The Astragalus complanatus extract comprises the following molar percentages of active pharmaceutical ingredients: Astragalus complanatus polysaccharides 31.79%–84.74%, protein 6.56%–34.35%, uronic acid 4.07%–27.11%, and polyphenols 3.76%–10.83%.

2. The extract of Salsola Kali as claimed in claim 1, wherein, The Astragalus complanatus extract comprises the following raw material components in molar percentage: Astragalus complanatus polysaccharide 55.92%, protein 10.28%, uronic acid 4.07%, and polyphenols 4.98%.

3. The extract of Salsola Kali as claimed in claim 1, wherein, The Astragalus complanatus extract comprises the following raw material components in molar percentages: Astragalus complanatus polysaccharide 84.74%, protein 6.56%, uronic acid 4.49%, and polyphenols 3.76%.

4. The extract of Salsola Kali as claimed in claim 1, wherein, The Astragalus complanatus extract comprises the following raw material components in molar percentage: Astragalus complanatus polysaccharide 34.46%, protein 25.99%, uronic acid 27.11%, and polyphenols 8.11%.

5. The extract of Salsola Kali as claimed in claim 1, wherein, The Astragalus complanatus extract comprises the following raw material components in molar percentage: Astragalus complanatus polysaccharide 31.79%, protein 34.35%, uronic acid 15.80%, and polyphenols 10.83%.

6. The extract of Salsola Kali as claimed in claim 1, wherein, The monosaccharides in the Astragalus complanatus extract include the following molar percentage components: rhamnose 0.4%–3.35%, arabinose 0.97%–22.55%, xylose 0.30%–25.28%, mannose 0–58.37%, glucose 0–1.27%, galactose 17.38%–43.03%, and glucuronic acid 0.58%–55.71%.

7. The extract of Salsola Kali as claimed in claim 6, wherein, The monosaccharides in the Astragalus complanatus extract include the following molar percentage components: arabinose 0.97%, xylose 0.30%, mannose 54.26%, galactose 43.03%, and glucuronic acid 1.44%.

8. The extract of Salsola Kali as claimed in claim 6, wherein, The monosaccharides in the Astragalus complanatus extract include the following molar percentage components: arabinose 0.99%, xylose 0.64%, mannose 58.37%, galactose 39.42%, and glucuronic acid 0.58%.

9. The extract of Salsola Kali as claimed in claim 6, wherein, The monosaccharides in the Astragalus complanatus extract include the following molar percentage components: rhamnose 3.15%, arabinose 9.36%, xylose 14.39%, galactose 39.42%, and glucuronic acid 55.71%.

10. The use of the extract of Astragalus complanatus as described in any one of claims 1 to 8 in the preparation of drugs for treating fatty liver, non-alcoholic steatohepatitis, alcoholic steatohepatitis, and hyperlipidemia.

Images