Acorus gramineus polysaccharide as well as preparation method and application thereof

Abstract

The invention discloses rhizoma acori graminei polysaccharide as well as a preparation method and application thereof. According to the method disclosed by the invention, the acorus tatarinowii polysaccharide is obtained from the root tuber of the acorus tatarinowii Schott by adopting the methods of water extraction, alcohol precipitation and column separation and purification. Cell experiment results show that the rhizoma acori graminei polysaccharide can significantly inhibit activation of TGF-beta induced hepatic stellate cells (LX-2), and carbon tetrachloride (CCl4) induced hepatic fibrosis mouse model experiments show that the rhizoma acori graminei polysaccharide can significantly reduce CCl4 induced collagen deposition and expression of liver tissue alpha-smooth muscle actin (alpha-SMA), and can significantly inhibit TGF-beta induced hepatic stellate cells (LX-2) activation. The content of asparaginic acid aminotransferase (AST), alanine aminotransferase (ALT) and the like in serum of a hepatic fibrosis model mouse is reduced, and the compound can be used for preparing candidate carbohydrate drugs or liver protection health care products for treating and / or treating hepatic fibrosis.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of polysaccharides, and particularly relates to Acorus tatarinowii Schott polysaccharide extracted from the rhizome of Acorus tatarinowii Schott, and a preparation method and uses thereof. BACKGROUND

[0002] Chronic liver disease (CLD) is a collective term for a group of diseases characterized by liver damage lasting more than six months, with complex and diverse causes, including viral hepatitis, long-term excessive alcohol consumption, autoimmune diseases, metabolic abnormalities, etc. Liver fibrosis is a key pathological process in the development of chronic liver disease to cirrhosis and even liver cancer, and is closely related to the abnormal activation of hepatic stellate cells and persistent inflammatory response, and is mainly characterized by excessive deposition of extracellular matrix (ECM) components. In the early stage of the disease, liver fibrosis can cause slight changes in liver structure, and as the fibrosis process accelerates, the liver gradually becomes hard, and normal liver tissue is replaced by fibrous tissue, eventually leading to a series of serious complications such as portal hypertension, esophageal variceal bleeding, ascites, and hepatic encephalopathy, which greatly affects the quality of life and survival rate of patients. Related studies have shown that drug intervention can alleviate liver fibrosis to some extent, but there is currently no specific anti-liver fibrosis drug on the market. Therefore, there is an urgent need to screen safe and effective anti-liver fibrosis drugs.

[0003] Traditional Chinese medicine Acorus tatarinowii Schott is the dried rhizome of Acorus tatarinowii Schott of Araceae. It was first recorded in Shennong Bencao Jing and was classified as a superior drug: "Acorus tatarinowii Schott, with a bitter and warm taste, is used to treat wind-cold-dampness, cough, open the heart orifice, tonify five zang organs, open nine orifices, brighten ears and eyes, and restore voice. Long-term use can light the body, not forget, not confused, or prolong life." The 2020 edition of Chinese Pharmacopoeia records Acorus tatarinowii Schott: bitter and warm, and belongs to the heart and stomach channels. The functions and indications are: opening orifice and resolving phlegm, refreshing and benefiting intelligence, eliminating dampness and promoting appetite. It is used for mania, epilepsy, forgetfulness, insomnia, tinnitus, deafness, abdominal distension, anorexia, and diarrhea. Modern pharmacological studies have shown that the main active ingredient of Acorus tatarinowii Schott is volatile oil, and there is less research on the macromolecular active ingredient Acorus tatarinowii Schott polysaccharide. Currently, there is no related research report on Acorus tatarinowii Schott polysaccharide for treating liver fibrosis. SUMMARY

[0004] The present application adopts a simple and effective process and method for extracting and purifying plant polysaccharides, and obtains a mixed polysaccharide component from Acorus gramineus Rhizoma as raw material, and further purifies to obtain a uniform polysaccharide. Cell experiments show that the polysaccharide can significantly inhibit the activation of hepatic stellate cells induced by TGF-β, and has no obvious cytotoxicity to liver parenchymal cells. Animal experiments show that the polysaccharide can significantly reduce the content of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the serum of liver fibrosis model mice, reduce the collagen deposition in the liver tissue of mice, and reduce the expression of fibrosis marker proteins in the liver tissue of mice: alpha-smooth muscle actin (alpha-SMA), type I collagen (Collagen I) and fibronectin 1 (Fibronectin 1); therefore, the polysaccharide has a potential effect of treating liver fibrosis, and is expected to be developed into a saccharide drug for treating chronic liver disease accompanied by liver fibrosis.

[0005] Based on this, one object of the present application is to provide an Acorus gramineus polysaccharide.

[0006] Another object of the present application is to provide a preparation method of the Acorus gramineus polysaccharide.

[0007] Another object of the present application is to provide a pharmaceutical composition containing the Acorus gramineus polysaccharide.

[0008] Another object of the present application is to provide the use of the Acorus gramineus polysaccharide or the pharmaceutical composition containing the Acorus gramineus polysaccharide in the preparation of a drug for inhibiting the activation of hepatic stellate cells.

[0009] Another object of the present application is to provide the use of the Acorus gramineus polysaccharide or the pharmaceutical composition containing the Acorus gramineus polysaccharide in the preparation of a drug for preventing and / or treating chronic liver disease accompanied by liver fibrosis, or the use of the Acorus gramineus polysaccharide in the preparation of a liver-protecting health product.

[0010] Therefore, in one aspect of the present application, an Acorus gramineus polysaccharide is provided, wherein the monosaccharide composition of the polysaccharide comprises glucose (40-50%), mannose (15-25%), galactose (10-20%), arabinose (10-20%), and xylose (5-15%), based on 100% of the total molar amount of glucose, mannose, galactose, arabinose, and xylose; preferably, the monosaccharide composition of the Acorus gramineus polysaccharide is glucose (41.62%), mannose (20.15%), galactose (12.13%), arabinose (16.84%), and xylose (9.26%), based on 100% of the total molar amount of glucose, mannose, galactose, arabinose, and xylose.

[0011] In some embodiments, the Acorus gramineus polysaccharide has a backbone consisting of 1,6-β-Galp, 1,3,6-β-Galp, 1,4-α-Glcp, 1,4,6-α-Glcp and 1,4-α-Manp, and side chains T-α-Ara and arabinobiose are linked to C-3 of 1,6-β-Galp of the backbone, and xylose-containing side chains are linked to C-6 of 1,4-α-Glcp of the backbone.

[0012] In some embodiments, the Acorus gramineus polysaccharide has a weight average molecular weight in the range of 1-30 kDa, preferably 3-10 kDa, such as 4.0, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0 kDa, etc.

[0013] In some embodiments, the Acorus gramineus polysaccharide has a molecular weight distribution PDI in the range of 1.0 to 2, preferably 1.0 to 1.5, such as 1.01, 1.05, 1.08, 1.10, 1.15, 1.20, 1.25, 1.30, 1.35, 1.40, etc. Here PDI refers to the ratio of weight average molecular weight Mw to number average molecular weight Mn.

[0014] In some embodiments, the Acorus gramineus polysaccharide has a characteristic high performance liquid chromatogram substantially as shown in Figure 1

[0015] In some embodiments, the Acorus gramineus polysaccharide has an infrared spectrum with absorption peaks at 3379.44 cm -1 , 2930.40 cm -1 , 1412.30-1601.56 cm -1 , 1153.81-1026.38 cm -1 , and the above absorption peak wave numbers can have a deviation of no more than 0.5%; preferably, the infrared spectrum of the Acorus gramineus polysaccharide is substantially consistent with the infrared spectrum shown in Figure 2

[0016] In another aspect of the present application, a preparation method of Acorus gramineus polysaccharide is provided, comprising the following steps:

[0017] a. polysaccharide extraction:

[0018] The Acorus gramineus tuber is extracted with boiling water to obtain an extract; the extract is concentrated, and the obtained concentrated solution is subjected to alcohol precipitation by adding an ethanol aqueous solution, then is left to stand, and the precipitate is separated and dried to obtain Acorus gramineus crude polysaccharide;

[0019] b. polysaccharide purification:

[0020] ​​b1. The crude polysaccharide prepared in step a is dissolved in water, centrifuged, and the supernatant is eluted through a cation exchange column with deionized water to obtain a preliminary purified crude polysaccharide;

[0021] b2. The preliminary purified crude polysaccharide is eluted through a gel chromatography column with 0.2M NaCl aqueous solution to obtain the Acorus gramineus polysaccharide.

[0022] In some embodiments, in step a:

[0023] Before the boiling water extraction, the Acorus gramineus tuber is soaked in an ethanol aqueous solution for 7-14 days, and then dried; and / or,

[0024] In the boiling water extraction of Acorus gramineus, the ratio of Acorus gramineus to water is 3:30-50 (unit: kg / L), preferably 3:40 (unit: kg / L); and / or,

[0025] The boiling water extraction is performed for 3-10 times, preferably 8 times, for 3-5 hours each time, preferably 4 hours; and / or,

[0026] The extraction liquid is concentrated to 1 / 400-1 / 250, preferably 1 / 320, of the original volume; and / or,

[0027] The ethanol aqueous solution has a volume fraction of 80-98%, preferably 95%; and / or,

[0028] The volume of the ethanol aqueous solution used for precipitation is 3-6 times that of the concentrated liquid; and / or,

[0029] Before the alcohol precipitation of the concentrated liquid, the concentrated liquid is centrifuged, and then the supernatant is dialyzed in water for 24-72 hours, and then the dialyzed liquid is added with the ethanol aqueous solution for alcohol precipitation; and / or,

[0030] The standing time is 6-24 hours; and / or,

[0031] The crude polysaccharide of Acorus gramineus obtained in step a is washed before the polysaccharide purification in step b. Preferably, the crude polysaccharide is alternately washed with ethanol and acetone for 2-4 times, preferably 3 times, and then vacuum dried or freeze-dried to obtain the crude polysaccharide of Acorus gramineus.

[0032] In some embodiments, step b1 comprises: dissolving the crude polysaccharide of Acorus gramineus prepared in step a in 10-15 times, preferably 12.5 times, of water by weight, centrifuging, eluting the supernatant through a cation exchange column, preferably a DEAE Sepharose FastFlow cation exchange column, with deionized water as the eluent, collecting the eluted components, concentrating, centrifuging, dialyzing the supernatant, and freeze-drying to obtain a preliminary purified crude polysaccharide;

[0033] In some embodiments, the step b2 comprises: dissolving the preliminarily purified crude polysaccharide in about 10-30 times, preferably about 20 times, weight of 0.2M NaCl aqueous solution, centrifuging, collecting the supernatant, eluting and separating the polysaccharide component through a gel chromatography column, preferably a Sephacryl HR S-200 gel chromatography column, using 0.2M NaCl aqueous solution, concentrating, dialyzing, and freeze-drying to obtain the Acorus gramineus polysaccharide.

[0034] Another aspect of the present application provides a pharmaceutical composition comprising a therapeutically effective amount of the Acorus gramineus polysaccharide prepared by the above method as an active ingredient.

[0035] In some embodiments, the pharmaceutical composition can further comprise pharmaceutically acceptable pharmaceutical adjuvants, such as carriers, excipients, adjuvants, and / or diluents, etc.

[0036] Still another aspect of the present application provides the use of the Acorus gramineus polysaccharide prepared by the above method, or the pharmaceutical composition containing the same, in the preparation of a medicament for treating and / or preventing chronic liver disease with liver fibrosis, and the use of the Acorus gramineus polysaccharide prepared by the above method in the preparation of a health care product for protecting liver and a health care product with auxiliary protection function for chemical liver injury. Here, the term "health care product" can also be referred to as "dietary supplement".

[0037] The concept of "treatment and / or prevention" herein means any measure suitable for treating chronic liver disease with liver fibrosis related diseases, or prophylactic treatment for such manifested diseases or symptoms, or avoiding further development of such diseases, such as further development of the disease after the end of the treatment period or treatment of symptoms of the disease that has already occurred, or preventive intervention to prevent or inhibit or reduce the occurrence of such diseases or symptoms.

[0038] In some embodiments, the chronic liver disease with liver fibrosis can be a disease caused by abnormal activation of hepatic stellate cells in the liver leading to excessive deposition of extracellular matrix, such as metabolic dysfunction related fatty liver disease with liver fibrosis.

[0039] Still another aspect of the present application also provides the use of the Acorus gramineus polysaccharide prepared by the above method, or the pharmaceutical composition containing the same, in the preparation of a medicament having any one or more of the following effects:

[0040] (1) inhibiting the activation of hepatic stellate cells;

[0041] (2) down-regulating the expression of α-smooth muscle actin (α-SMA);

[0042] (3) down-regulate the expression of Collagen I;

[0043] (4) down-regulate the expression of Fibronectin;

[0044] (5) down-regulate the content of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in serum. BRIEF DESCRIPTION OF DRAWINGS

[0045] Figure 1 Chromatogram of characteristic high-performance gel permeation chromatography of Acorus gramineus polysaccharide AW1 prepared in Example 1.

[0046] Figure 2 Characteristic infrared spectrum of Acorus gramineus polysaccharide AW1 prepared in Example 1.

[0047] Figure 3 Characteristic infrared spectrum of Acorus gramineus polysaccharide AW1 prepared in Example 1. 1 H NMR(A) and 13 C NMR(B) spectrum.

[0048] Figure 4 Structure diagram of Acorus gramineus polysaccharide AW1 prepared in Example 1.

[0049] Figure 5 Line graph of the effect of Acorus gramineus polysaccharide AW1 on the activity of hepatic stellate cells in Example 3.

[0050] Figure 6 Western blot experiment (A) and protein quantification graph (B) for detecting the effect of Acorus gramineus polysaccharide AW1 on the level of fibrosis phenotype related proteins in hepatic stellate cells in Example 4; wherein *, p<0.05, **, p<0.01, ***, p<0.001, ****, p<0.001, represent the degree of significant difference compared with the control group.

[0051] Figure 7 Flowchart (A) for testing the animal experiment in Example 5, and the effect of Acorus gramineus polysaccharide AW1 on serum liver injury indicators of CCl4-induced chronic liver fibrosis mice (B).

[0052] Figure 8 H&E staining (A) for observing the improvement of AW1 on CCl4-induced liver tissue injury of mice, and Sirius red staining (B) and Masson staining (Masson) for observing the reducing effect of AW1 on collagen deposition in mouse liver (B) and relative collagen staining area quantification graph (C) in Example 5.

[0053] Figure 9 Figure (A) and protein quantification figure (B) of the results of testing the effect of Acorus calamus polysaccharide AW1 gavage on the collagen-related protein content of liver tissue of CCl4-induced chronic liver fibrosis mice in the protein immunoblotting experiment in Example 5. DETAILED DESCRIPTION

[0054] The present application will be described in detail below by way of examples. In the present application, the examples described below are for better illustration of the present application and do not limit the scope of the present application. Various changes and modifications can be made to the present application without departing from the spirit and scope of the present application.

[0055] The raw materials and equipment used in the detailed description of the present application are conventional raw materials and reagents in the art, which are obtained by purchase from the market, unless otherwise specified.

[0056] In the following embodiments, high-performance gel permeation chromatography (HPGPC) uses Shodex SUGAR KS-804 (8.0 mm x 300 mm, Agilent, USA) and Shodex SUGAR KS-802 (8.0 mm x 300 mm, Agilent, USA) tandem columns, with a standard curve prepared using different molecular weight T-series dextran (Dextran);

[0057] High-performance liquid chromatography (HPLC) is determined by Agilent 1260 Seri high-performance liquid chromatography system (Agilent, USA);

[0058] Infrared analysis is determined by Perkin-Elmer 599B infrared spectrophotometer (Perkin-Elmer, USA);

[0059] Nuclear magnetic resonance analysis is determined by Brucker AM-500 nuclear magnetic resonance instrument (Brucker, Germany).

[0060] Example 1 Extraction, separation and purification of Acorus calamus polysaccharide AW1

[0061] a. Polysaccharide extraction:

[0062] Drying 2 kg of A. tuberosum tubers (origin: Zhejiang) in 95% ethanol for 14 days, and then drying in the air. Add 20 times the volume of deionized water, and extract 8 times with boiling water, each time for 4 hours. Test the sugar content of the extract with the phenol-sulfuric acid method until the reaction is not obvious. Combine the extracts, concentrate to 1 L, and centrifuge to remove the precipitate. Dialyze the supernatant with a flow of water for 2 days. Add 95% ethanol to the dialysate to precipitate the polysaccharides, and stir while adding the ethanol. The volume ratio of the extract to the added ethanol is 1:4 (v / v), and the mixture is left to stand overnight. Discard the excess ethanol, and centrifuge the precipitate. Wash the precipitate with ethanol and acetone alternately for 3 times, and then centrifuge to obtain the precipitate. Resuspend the precipitate in 500 mL of deionized water, and freeze-dry to obtain 36.47 g of crude A. tuberosum polysaccharides (yield: 1.82% compared to the weight of the dried medicinal material).

[0063] b. Purification of the polysaccharides:

[0064] Take 8 g of the crude A. tuberosum polysaccharides prepared above, and dissolve in 80 mL of deionized water. Centrifuge at 4000 r / min for 10 min to remove the insoluble matter. Separate the supernatant by DEAE Sepharose Fast Flow anion exchange column, and use deionized water as the eluent. Collect the samples eluted with deionized water by a partial automatic collector, at a flow rate of 1 mL / min, 15 min / tube, and elute until there is no sugar component (no color reaction with the phenol-sulfuric acid method). Take samples from the collection tubes (e.g., tubes 2, 4, 6, etc.), and take 100 μL of each sample, add 400 μL of 5% phenol solution, and then add 2 mL of concentrated sulfuric acid. Shake well, and let stand at room temperature for 15 min. Take 100 μL of each reaction solution, and add to a 96-well plate. Detect the OD value of each well with an enzyme label meter, and plot the elution curve. According to the elution curve, collect the sugar-containing components eluted with deionized water, and concentrate to a small volume in a water bath at 50°C under reduced pressure. Transfer to a centrifuge tube, centrifuge at 8000 rpm for 15 min at 4°C, discard the precipitate, and take the supernatant. Dialyze the supernatant in a dialysis bag with MWCO = 3500 Da for 48 hours (change the water every 6 hours), and then freeze at -20°C for 6 hours. Dry in a freeze dryer for 48 hours to obtain the preliminarily purified polysaccharides AW, about 867.2 mg (yield: 10.84%). 490

[0065] ​The 200 mg of the preliminary purified polysaccharide AW was dissolved in 4 mL of 0.2 M NaCl aqueous solution, centrifuged at 4000 r / min for 10 min, and the supernatant was eluted by a Sephacryl HR S-200 gel chromatography column with 0.2 M NaCl aqueous solution and a flow rate of 5 mL / 15 min / tube. Further, the elution curve was detected and drawn by the sulfuric acid-phenol method, and the polysaccharide AW1 component was collected according to the elution curve, concentrated to a small volume at 50°C under reduced pressure, transferred to a dialysis bag with MWCO = 3500 Da, and dialyzed with deionized water for 48 hours (every 6 hours, change water), and the dialysis product was collected, pretreated at -20°C for 6 hours, and then placed in a freeze dryer for drying for 48 hours, to obtain about 54 mg of uniform polysaccharide AW1 (yield 27%).

[0066] Example 2 Structural identification of polysaccharide

[0067] The structure of polysaccharide AW1 was determined by comprehensive analysis of monosaccharide composition, methylation, infrared and nuclear magnetic resonance, and the specific analysis method and results are as follows.

[0068] (1) Polysaccharide uniformity and molecular weight determination

[0069] In the high performance gel permeation chromatography (HPGPC) analysis, the chromatography column was Shodex SUGAR KS-804 and Shodex SUGAR KS-802 used in series, 0.1 mol / L NaNO3 aqueous solution was selected as the mobile phase, the flow rate was 0.5 mL / min, the column temperature was set to 40°C, the detector was ultraviolet detector UV (detection wavelength was 280 nm) and differential detector RID (detection temperature was 35°C), and the injection volume was 10 μL. The dextran standard and the sample AW1 to be tested were dissolved in 0.1 mol / L NaNO3 mobile phase to prepare a sample of 4 mg / mL, and then filtered through a 0.22 μm microporous filter before HPLC detection. The standard curve was drawn by the molecular weight of different dextrans and the retention time. The linear relationship of the peak retention time of AW1 in the dextran standard curve was used to calculate the result by software, which showed that the number average molecular weight (Mn) of polysaccharide AW1 was about 5.5 kDa, the weight average molecular weight (Mw) was about 6.2 kDa, and the molecular weight distribution PDI (Mw / Mn) was about 1.1. The characteristic spectrum is shown in Figure 1

[0070] (2) Determination of monosaccharide composition by PMP-HPLC derivatization method

[0071] ​Preparation of monosaccharide standards: The monosaccharide standards were D-mannose, L-rhamnose, D-glucuronic acid, D-galacturonic acid, D-glucose, D-galactose, L-xylose, L-arabinose and L-fucose, respectively. 9 mg of each monosaccharide was dissolved in 1 mL of deionized water to form a 9 mg / mL solution. 100 μL of each solution was mixed to form a standard solution with a concentration of 1 mg / mL. 50 μL of the standard solution was added to 50 μL of 0.6 M sodium hydroxide and 100 μL of 0.5 M PMP methanol solution (87.1 mg / mL). The mixture was derivatized at 70°C for 100 min, and then 100 μL of 0.3 M hydrochloric acid was added to neutralize the reaction. 700 μL of deionized water and 1 mL of chloroform were added, and the mixture was vortexed, centrifuged, and allowed to stand at room temperature for 90 min. 800 μL of the upper aqueous phase was transferred to a new 2 mL EP tube, and the chloroform layer was discarded. Chloroform was added again, and the extraction was repeated twice. The upper aqueous phase was collected, filtered through a 0.22 μm microporous filter, and then loaded into a liquid phase vial to obtain the standard solution.

[0072] Preparation of samples: 2-3 mg of dried Acorus gramineus polysaccharide AW1 sample was placed in a clean pear-shaped flask, 2 mL of deionized water and 2 mL of 4 M trifluoroacetic acid (TFA) were added, and the flask was sealed with a stopper. The mixture was completely acid hydrolyzed at 110°C for 4 h, cooled to room temperature, and then methanol was added to remove the excess TFA by rotary evaporation until no acid smell was detected. The residue was dissolved in 200 μL of deionized water, and 50 μL of the solution was transferred to a new 2 mL EP tube. The subsequent derivatization and extraction were performed as described above for the preparation of the standard solution.

[0073] Preparation of the mobile phase: 13.6 g of potassium dihydrogen phosphate and 1.8 g of sodium hydroxide were weighed and dissolved in 2 L of millii Q water. The solution was filtered through a water phase filter to obtain solution ①. Acetonitrile (HPLC grade) was mixed with solution ① at a ratio of 17:83 (v / v) and ultrasonicated for 30 min to obtain the mobile phase.

[0074] An Agilent 1260 high-performance liquid chromatography system was used to analyze the monosaccharide composition. The chromatographic column was Agilent-XDB-C18 (5 μm, 250 mm x 4.6 mm), the flow rate was set at 1 ml / min, the column temperature was 25°C, the ultraviolet detection wavelength was 254 nm, and the sample injection volume was 10 μL per injection. The retention time of each peak in the sample was determined by comparison with standard monosaccharides, and the types of monosaccharides were identified. The molar ratio of each monosaccharide was estimated by peak area normalization and molar mass conversion. The results showed that the monosaccharide composition of Acorus gramineus polysaccharide AW1 mainly included glucose, mannose, galactose, arabinose, and xylose.

[0075] The monosaccharide composition of the Acorus gramineus polysaccharide AW1 is glucose (41.62%), mannose (20.15%), galactose (12.13%), arabinose (16.84%), and xylose (9.26%) based on the total molar amount of glucose, mannose, galactose, arabinose, and xylose.

[0076] (3) Infrared spectrum

[0077] Figure 2 The peak at 3379.44 cm-1 is an O-H stretching vibration absorption peak, the peak at 2930.40 cm-1 is a C-H stretching vibration absorption peak, the peaks at 1412.30-1601.56 cm-1 are C-O and sugar ring vibration signals, the peaks at 1153.81-1026.38 cm-1 are C-O, C-C stretching vibrations, and the peaks at 1026.38-800.00 cm-1 are C-O, C-C, and C-O-C stretching vibrations. -1 -1 -1 -1

[0078] (4) NMR analysis

[0079] The polysaccharide AW1 30 mg was dissolved in D2O 0.5 mL, 1.5 μL of acetone was added as an internal standard (δH = 2.29 ppm, δC = 31.5 ppm), and one-dimensional and two-dimensional nuclear magnetic resonance spectra were measured at 25°C on a Bruker AVANCE III 500M nuclear magnetic resonance instrument. The structure of the polysaccharide AW1 was confirmed by reference to the nuclear magnetic resonance spectrum, and the results are shown in Table 1. Figure 3

[0080] 1 Figure 3 13 Figure 3

[0081] Assignment of H1 / C1 chemical shift signals of AW1 (ppm)

[0082]

[0083] According to the analysis, the Acorus gramineus polysaccharide AW1 has the structure shown in Table 1, the main chain is composed of 1,6-β-Galp, 1,3,6-β-Galp, 1,4-α-Glcp, 1,4,6-α-Glcp, and 1,4-α-Manp, the branched chain T-α-Ara and arabinobiose are connected to C-3 of the main chain 1,6-β-Galp, and the xylose branched chain is connected to C-6 of the main chain 1,4-α-Glcp. Figure 4

[0084] ​​​​​​​​​​Example 3 CCK8 experiment to detect the effect of Acorus gramineus polysaccharide AW1 on the growth of hepatic stellate cells

[0085] The hepatic stellate cells in the logarithmic growth phase (from Shangcheng Beinai Chuanlian Biotechnology Co., Ltd.) were seeded into a 96-well plate at a density of 3 x 10 3 给药孔 调零孔 空白孔 调零孔 6 The cells were cultured overnight in a culture box, the cell supernatant was then removed, and complete culture medium containing AW1 at concentrations of 0 mg / mL (i.e., the Control group), 0.0625 mg / mL, 0.125 mg / mL, 0.25 mg / mL, 0.5 mg / mL, 0.75 mg / mL, and 1.0 mg / mL was added, and a blank group (i.e., the zero hole) was set up, which did not contain cells and AW1. After 48 hours of continuous culture, 10 μL of CCK-8 solution (purchased from Shanghai Saiyin Biotechnology Co., Ltd.) was added to each well, and the cell culture solution in the well was removed after 1.5 hours of continuous culture. The absorbance was collected at 450 nm using an enzyme-labeled instrument. The cell survival rate was calculated according to the following formula:

[0086] Cell survival rate (%) = (OD 给药孔 - OD 调零孔 ) / (OD 空白孔 - OD 调零孔 ) x 100%.

[0087] The results are shown in Table 1. The cell survival rates of the various groups were all greater than 90%, indicating that the polysaccharide AW1 had no obvious cytotoxicity. Figure 5

[0088] Example 4 Acorus gramineus polysaccharide AW1 inhibits the activation of hepatic stellate cells

[0089] The hepatic stellate cells (from Shangcheng Beinai Chuanlian Biotechnology Co., Ltd.) were cultured in DMEM high-sugar culture medium (purchased from the United States Thermo Fisher Company) containing 10% fetal bovine serum (purchased from the United States Gibco Company), 100 U / mL penicillin, and 100 μg / mL streptomycin. When the cells reached 80%-95% confluence, they were seeded into a 6-well plate at a density of 3 x 10 6 给药孔 调零孔 空白孔 调零孔 6 After 1 hour, the model group, the experimental group, and the positive control group were each added with TGF-β protein (from Beijing Yiqiao God Science and Technology Co., Ltd.) to make the final concentration 10 ng / mL. After 48 hours, the total cell protein was extracted.

[0090] The total protein was extracted by lysing cells, and the protein concentration was determined by BCA method. The cell lysate was prepared as follows (add sterile purified water to a total volume of 120 μL)

[0091]

[0092] Western blotting was used to determine the expression of fibrosis-related proteins

[0093] A 9% SDS gel was prepared, and 20 μg of each sample was loaded. The electrophoresis program was 80 V for 30 minutes and 120 V for 1 hour. The membrane was transferred at 100 V for 1 hour for GADPH, 1 hour for α-SMA, 3 hours for Collagen I, and 3 hours for Fibronectin 1 (Fn1). The primary antibody was mouse-derived from Wuhan Abmabio Biotech Co., Ltd. for GADPH, rabbit-derived from Shanghai Lambden Biotech Co., Ltd. for α-SMA, rabbit-derived from Wuhan Abmabio Biotech Co., Ltd. for Collagen I, and rabbit-derived from Shanghai Youningwei Biotech Co., Ltd. for Fibronectin 1 (Fn1). The above primary antibodies were diluted 1:1000 and incubated at 4°C overnight. The secondary antibody was incubated at room temperature for 1 hour after recovering the primary antibody (anti-mouse secondary antibody from Wuhan Abmabio Biotech Co., Ltd. for GADPH, and anti-rabbit secondary antibody from Wuhan Abmabio Biotech Co., Ltd. for the rest of the proteins), and washed with TBST for 10 minutes three times. After washing three times, the membrane was developed.

[0094] The results are shown in Figure 6 Different concentrations of AW1 can significantly down-regulate the expression of fibrosis markers α-SMA, Fibronectin 1, and Collagen I in TGF-β-induced hepatic stellate cells, indicating that AW1 can inhibit TGF-β-induced activation of hepatic stellate cells.

[0095] Example 5: Evaluation of the in vivo anti-hepatic fibrosis activity of Acorus tatarinowii polysaccharide AW1 using a carbon tetrachloride-induced hepatic fibrosis model.

[0096] C57BL / 6J male mice (from Shanghai Slac Animal Limited Liability Company, license number: 2025-01-DK-145) weighing 22 ± 1 g and 8 weeks old were kept in a SPF animal room at a constant temperature of 22-24°C and humidity of 60% ± 5% with a 12-hour light / 12-hour dark cycle. The relevant experimental protocols were approved by the Animal Management Committee of the Shanghai Institute of Materia Medica, Chinese Academy of Sciences. The mice were adapted for one week before the experiment. In the AW1 gavage experiment, Figure 7), the mice were randomly divided into 6 groups, 7 in each group: negative control group (I); CCl4induced model group (II); CCl4induced model AW1 low-dose administration group (III), the dose was 12.5 mg / kg; CCl4induced model AW1 medium-dose administration group (IV), the dose was 25 mg / kg; CCl4induced model AW1 high-dose administration group (V), the dose was 50 mg / kg; CCl4induced model Obeticholic acid (OCA) positive control group (VI), the dose was 30 mg / kg. The mice in groups II, III, IV, V and VI were injected intraperitoneally with 10% CCl4(CCl4was dissolved in olive oil, the volume ratio of CCl4to olive oil was 1:9, the dose was 2 mL / kg, 3 times a week, and the injection was continued for 8 weeks) to induce chronic liver fibrosis, and the mice in group I were injected intraperitoneally with the same volume of olive oil. After 2 weeks of CCl4induction, the mice in groups III, IV and V were administered with AW1(dissolved in normal saline) by gavage every day, the mice in group VI were administered with Obeticholic acid (suspension prepared with 0.5% CMC-Na) by gavage every day, and the mice in groups I and II were administered with the same amount of normal saline by gavage every day, and the administration volume was 10 mL / kg. The administration period was 6 weeks, and the mice were sacrificed at the end of the experiment, and the mice were fasted for 12 hours before being sacrificed. Blood and liver were taken, and the liver tissue was first photographed to observe the appearance of the lesions. After standing at room temperature for 30 minutes, the blood was centrifuged at 2000 rpm for 30 minutes at 4°C, and the upper serum was collected and stored at -80°C. The content of serum liver damage indicators ALT, AST and LDH was detected by entrusting the national compound library for testing. Part of the liver tissue was fixed with 4% paraformaldehyde, and part of the liver tissue was quickly frozen in liquid nitrogen and stored at -80°C. The fixed liver tissue sections were sent to Wuhan Seville Company for hematoxylin-eosin staining (H&E staining), Masson staining and sirius red staining.

[0097] The serum results are shown in Table 2. Figure 7 As shown in Table 2, AW1 can significantly down-regulate the contents of liver damage indicators such as ALT, AST and LDH, indicating that AW1 can significantly improve the liver damage of liver fibrosis mice.

[0098] The staining results are shown in Table 3. Figure 8 As shown in Table 3, the H&E staining results show that Acorus polysaccharide AW1 can improve the cell morphology of liver tissue and reduce cell vacuolization. Masson staining and sirius red staining show that the Acorus polysaccharide AW1 can significantly reduce the collagen deposition in the liver tissue of liver fibrosis mice. It is indicated that AW1 can improve the damage of liver tissue and reduce collagen deposition.

[0099] Part of the liver tissue was extracted with a tissue extraction kit (from Shenguo Bioengineering Co., Ltd.) to extract protein, 5x loading buffer was added, and the protein was denatured at 95℃ in a metal heater for 10 min, and then stored at -80℃ after cooling. The effect of Acorus calamus polysaccharide AW1 on the expression of liver fibrosis-related proteins in liver tissue was detected by immunoblotting, and the results are shown in Figure 9 The expression of fibrosis-related proteins α-SMA, Fibronectin 1 and Collagen I in the model group was increased, while the expression of the above proteins in the liver tissue of mice after administration of Acorus calamus polysaccharide AW1 was significantly reduced, further indicating that AW1 has good anti-hepatic fibrosis activity in vivo.

[0100] In summary, through the above examples, it can be known that Acorus calamus polysaccharide AW1 can relieve liver fibrosis in mice and has an auxiliary protective function for chemical liver damage. Based on the above research results, it can be concluded that the Acorus calamus polysaccharide AW1 described in the present application can be used to prepare potential glycan drugs or liver-protecting health products for preventing / treating liver fibrosis.

Claims

1. A polysaccharide of Acorus calamus, characterized in that: Based on the total molar amount of glucose, mannose, galactose, arabinose and xylose as 100%, the monosaccharide composition of the polysaccharide includes 40-50% glucose, 15-25% mannose, 10-20% galactose, 10-20% arabinose and 5-15% xylose; Preferably, the structure of the Acorus calamus polysaccharide includes a main chain composed of 1,6-β-Galp, 1,3,6-β-Galp, 1,4-α-Glcp, 1,4,6-α-Glcp and 1,4-α-Manp, the side chains T-α-Ara and arabinobiose are connected to the C-3 position of the main chain 1,6-β-Galp, and the xylose-containing side chain is connected to the C-6 position of the main chain 1,4-α-Glcp. More preferably, the Acorus calamus polysaccharide has the structure shown in Figure 4.

2. The Acorus calamus polysaccharide according to claim 1, characterized in that Based on the total molar amount of glucose, mannose, galactose, arabinose and xylose being 100%, the monosaccharide composition of the polysaccharide is 41.62% glucose, 20.15% mannose, 12.13% galactose, 16.84% arabinose and 9.26% xylose.

3. The Acorus calamus polysaccharide according to claim 1 or 2, characterized in that The characteristic HPLC chromatogram of the polysaccharide is roughly as shown in FIG1 ; and / or, The weight average molecular weight range is 1-30 kDa, preferably 3-10 kDa, more preferably 6.2 kDa; and / or, The molecular weight distribution PDI ranges from 1.0 to 2, preferably from 1.0 to 1.5; and / or, In the infrared spectrum of the Acorus calamus polysaccharide, at 3379.44 cm -1 、2930.40cm -1 、1412.30-1601.56cm -1 、1153.81-1026.38cm -1 There is an absorption peak at , and the deviation of the absorption peak wave number does not exceed 0.5%; preferably, the infrared characteristic spectrum of the Acorus calamus polysaccharide is basically consistent with the infrared characteristic spectrum shown in Figure 2.

4. The method for preparing the Acorus calamus polysaccharide according to any one of claims 1 to 3, comprising the following steps: a. Polysaccharide extraction: The dried Acorus tatarinowii root tuber is extracted with boiling water to obtain an extract; the extract is concentrated, and the obtained concentrate is added with ethanol aqueous solution for alcohol precipitation, and then allowed to stand to separate the precipitate, and dried to obtain crude Acorus tatarinowii polysaccharide; b. Polysaccharide purification: b1. Acorus tatarinowii crude polysaccharide prepared in step a was dissolved in water and centrifuged. The supernatant was eluted with deionized water through an anion exchange column and the deionized water eluted fraction was collected to obtain a preliminary purified crude polysaccharide. b2. The crude polysaccharide was purified by gel chromatography and eluted with 0.2M NaCl aqueous solution to obtain Acorus tatarinowii polysaccharide.

5. The preparation method according to claim 4, characterized in that In the step a, Before the boiling water extraction, the Acorus calamus root tubers are soaked in an ethanol aqueous solution for 7 to 14 days and then dried; and / or, In the boiling water extraction of Acorus calamus, the ratio of Acorus calamus to water is 3:30 to 50, unit: kg / L, preferably 3:40, unit: kg / L; and / or, The number of boiling water extractions is 3 to 10 times, preferably 8 times, each time for 3 to 5 hours, preferably 4 hours; and / or, Concentrating the extract to 1 / 400 to 1 / 250, preferably 1 / 320, of its volume before concentration; and / or, The ethanol aqueous solution has a volume fraction of 80 to 98%, preferably a volume fraction of 95% ethanol aqueous solution; and / or, The volume of the ethanol aqueous solution used for precipitation is 3 to 6 times that of the concentrated solution; and / or Before the concentrate is subjected to alcohol precipitation, the concentrate is first centrifuged, and then the supernatant of the centrifugation is dialyzed in water for 24 to 72 hours; then the dialyzed liquid is added with the ethanol aqueous solution for alcohol precipitation; and / or, The standing time is 6 to 24 hours; and / or, The crude Acorus calamus polysaccharide obtained in step a is washed before the polysaccharide purification in step b. Preferably, ethanol and acetone are used for alternating washing, each washing 2 to 4 times, preferably 3 times, and then vacuum dried or freeze-dried to obtain the crude Acorus calamus polysaccharide.

6. The preparation method according to claim 4, characterized in that The step b1 comprises: taking the crude Acorus tatarinowii polysaccharide prepared in step a, adding 10 to 15 times, preferably 12.5 times, of water by weight to dissolve it, centrifuging it, passing the supernatant through an anion exchange column, preferably a DEAE Sepharose Fast Flow anion exchange column, eluting it with deionized water, collecting the eluted fractions, concentrating them, centrifuging them, dialyzing the supernatant, and freeze-drying it to obtain a preliminarily purified crude polysaccharide; and / or The step b2 comprises: dissolving the preliminarily purified crude polysaccharide in 10 to 30 times, preferably 20 times, by weight of a 0.2M NaCl aqueous solution, centrifuging, and eluting the supernatant with a 0.2M NaCl aqueous solution through a gel chromatography column, preferably a Sephacryl HR S-200 gel chromatography column, to separate the polysaccharide components, concentrating, dialysis, and freeze-drying to obtain the Acorus tatarinowii polysaccharide.

7. A pharmaceutical composition comprising a therapeutically effective amount of the Acorus calamus polysaccharide according to any one of claims 1 to 3 or the Acorus calamus polysaccharide prepared by the preparation method according to claims 4 to 6 as an active ingredient, Preferably, the pharmaceutical composition optionally includes pharmaceutically acceptable pharmaceutical excipients. Preferably, the pharmaceutical excipients include carriers, excipients, adjuvants and / or diluents.

8. Use of the Acorus calamus polysaccharide according to any one of claims 1 to 3 or the Acorus calamus polysaccharide prepared by the preparation method according to claims 4 to 6, or the pharmaceutical composition according to claim 7 in the preparation of a medicament for inhibiting hepatic stellate cell activation.

9. Use of the Acorus calamus polysaccharide according to any one of claims 1 to 3 or the Acorus calamus polysaccharide prepared by the preparation method according to claims 4 to 6, or the pharmaceutical composition according to claim 7 in the preparation of a medicament for treating and / or preventing chronic liver disease, or use of the Acorus calamus polysaccharide according to any one of claims 1 to 3 or the Acorus calamus polysaccharide prepared by the preparation method according to claims 4 to 6 in the preparation of a liver protection health product (a health product with auxiliary protective function against chemical liver damage); Preferably, the chronic liver disease is a disease caused by abnormal activation of hepatic stellate cells in the liver; more preferably, the chronic liver disease is accompanied by liver fibrosis.

10. Use of the Acorus calamus polysaccharide according to any one of claims 1 to 3 or the Acorus calamus polysaccharide prepared by the preparation method according to claims 4 to 6, or the pharmaceutical composition according to claim 7 in the preparation of a medicament having any one or more of the following effects: (1) Inhibit hepatic stellate cell activation; (2) downregulating the expression of α-smooth muscle actin; (3) downregulate the expression of type I collagen; (4) downregulating the expression of fibronectin; (5) Down-regulate the levels of aspartate aminotransferase and alanine aminotransferase in serum.

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