Use of a malus hallings polysaccharide in the preparation of a medicine for resisting liver fibrosis

Abstract

The application discloses application of a roxburgh rose polysaccharide in preparation of a medicine for resisting liver fibrosis, and belongs to the technical field of biological medicine. The roxburgh rose polysaccharide is roxburgh rose polysaccharide CLP2-2, a structural formula of which is [→4) alpha-GalAp-(1→] n , a relative molecular mass of which is 78.6 kDa, and a preparation method of which comprises the following steps: carrying out enzyme extraction on fresh mature roxburgh rose fruits by using papain and trypsin to obtain crude roxburgh rose polysaccharide, and then carrying out secondary purification to obtain the roxburg rose polysaccharide CLP2-2. The application further provides a medicine composition. It is verified through experiments that the roxburgh rose polysaccharide CLP2-2 has the effects of inhibiting activation of liver stellate cells and resisting liver fibrosis, can effectively improve chronic liver diseases, and has a wide application prospect in preparation of the medicine for resisting liver fibrosis or treating chronic liver diseases.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to application of a Rosa roxburghii polysaccharide in preparation of an anti-hepatic fibrosis drug. BACKGROUND

[0002] Fibrosis is the final pathway of common injuries and the basis of the pathogenesis of tissue damage in many chronic diseases. Hepatic fibrosis is a pathological process in which the excessive accumulation and nodule regeneration of extracellular matrix (ECM) components occur when various injury stimuli such as infection, toxicity and physical damage are encountered.

[0003] Polysaccharides are sugar chains combined by glycosidic bonds, and polymeric sugar macromolecular carbohydrates with complex structures and numerous types are composed of more than 10 monosaccharides. At present, there are hundreds of active polysaccharides discovered, which can be divided into five categories according to different sources, i.e. plant polysaccharides, fungal polysaccharides, algal polysaccharides, animal polysaccharides and bacterial polysaccharides. As an important life material, polysaccharides have rich biological activities, not only have immune regulation function, but also can resist tumors, viruses, inflammation, reduce blood sugar, blood lipids and blood pressure, resist radiation, etc., and have no toxic and side effects. Since 1986, when lentinan was approved for clinical use in Japan, dozens of polysaccharides have been approved for use in the treatment of diseases and health foods in China, the United States, South Korea, Japan and some European countries. At present, a variety of polysaccharides have been reported to have anti-fibrosis activity, for example: DOP has been proved to be able to regulate the activation of hepatic stellate cells and reduce liver fibrosis by inhibiting the SMO / Gli1 signaling pathway; a pectin-like polysaccharide (XHH2) derived from saffron has been proved to be able to inactivate hepatic stellate cells (HSC) by destroying the Gal-3 / integrin-β1 / FAK pathway, which helps to reduce liver injury and fibrosis in vitro and in vivo; arctiin (ALP) inhibits the activation of hepatic stellate cells (HSC) and reduces the accumulation of liver collagen by regulating the Wnt / β-catenin signaling pathway. Compared with existing anti-fibrosis drugs, these natural polysaccharides have the advantages of low toxicity and high stability, making them promising candidates for the development of anti-fibrosis therapies.

[0004] Rosa roxburghii is a plant of Rosaceae, which is a precious plant widely distributed in the mountainous areas of southwest and south-central China, and its fruit is called Rosa roxburghii. As early as 1640, there was a record of Rosa roxburghii in the book Guizhou Zhi written by Tian Wen of the Ming Dynasty, and its fruit is similar to pomegranate but smaller than pomegranate; in Bencao Gangmu Bu Bing, it is described as an edible wild fruit with a slightly sour taste that can help digestion. Moreover, other parts of Rosa roxburghii, such as flowers, leaves, roots and seeds, can be used as medicine to treat various diseases, and the fruit of Rosa roxburghii is rich in various nutritional components, including polysaccharides, ascorbic acid, phenols and superoxide dismutase.

[0005] In the prior art, a polysaccharide has been isolated from the roxburgh rose, for example, a roxburgh rose polysaccharide CLP2-2 is disclosed in a patent document (patent application number: 202410729810.4), which is identified as a homogalacturonan, the structural formula is [→4) α-GalAp-(1→] n , the relative molecular mass is 78.6 kDa, and has various biological activities, but its use for preparing a medicine for resisting liver fibrosis has not been reported. SUMMARY

[0006] Based on this, the purpose of the present application is to provide a use of a roxburgh rose polysaccharide in preparing a medicine for resisting liver fibrosis, the roxburgh rose polysaccharide is roxburgh rose polysaccharide CLP2-2, which is a homogalacturonan, the structural formula is [→4) α-GalAp-(1→] n , and the relative molecular mass is 78.6 kDa.

[0007] To achieve the above purpose, the present application adopts the following technical solutions:

[0008] The present application provides a use of a roxburgh rose polysaccharide in preparing a medicine for resisting liver fibrosis, the roxburgh rose polysaccharide is roxburgh rose polysaccharide CLP2-2, which is a homogalacturonan, the structural formula is [→4) α-GalAp-(1→] n , and the relative molecular mass is 78.6 kDa.

[0009] As a preference, the preparation method of the roxburgh rose polysaccharide CLP2-2 comprises the following steps:

[0010] a. Polysaccharide extraction: fresh mature roxburgh rose fruits are deseeded, washed clean, crushed, air-dried, defatted with anhydrous ethanol, air-dried, 30 times volume of deionized water is added, pH is adjusted, papain and trypsin are used for enzymatic extraction respectively, the supernatant is obtained by centrifugation, concentrated, freeze-dried, and roxburgh rose crude polysaccharide is obtained;

[0011] b. Polysaccharide purification: the roxburgh rose crude polysaccharide is dissolved in deionized water, centrifuged, the supernatant is separated by a DEAE cellulose anion column, eluted with distilled water, 0.1M NaCl and 0.2M NaCl in turn, detected by sulfuric acid-phenol, the 0.2M NaCl eluate is collected, concentrated, dialyzed, freeze-dried, and roxburgh rose polysaccharide CLP2 is obtained, which is separated and purified by a Bio Gel P-6 Gel gel chromatography column, and roxburgh rose polysaccharide CLP2-2 is obtained.

[0012] As a preference, the medicine comprises a therapeutically effective amount of roxburgh rose polysaccharide CLP2-2, and the concentration of the roxburgh rose polysaccharide CLP2-2 is 1-1000 μg / mL.

[0013] As preferred, the concentration of the Punica granatum polysaccharide CLP2-2 in the medicine is 200-800 μg / mL.

[0014] As preferred, the medicine further comprises a pharmaceutically acceptable carrier, and can be prepared into an injection, an emulsion, a tablet, a powder, a granule, an ointment, a liposome or an oral liquid.

[0015] The second aspect of the present application further provides a pharmaceutical composition comprising a therapeutically effective amount of the Punica granatum polysaccharide, which is the Punica granatum polysaccharide CLP2-2, a homogalacturonan, and has a structural formula of [→4) α-GalAp-(1→] n , and a relative molecular mass of 78.6 kDa.

[0016] As preferred, the concentration of the Punica granatum polysaccharide CLP2-2 in the medicine is 200-800 μg / mL.

[0017] As preferred, the concentration of the Punica granatum polysaccharide CLP2-2 in the medicine is 200-800 μg / mL.

[0018] As preferred, the medicine further comprises a pharmaceutically acceptable carrier, and can be prepared into an injection, an emulsion, a tablet, a powder, a granule, an ointment, a liposome or an oral liquid.

[0019] Compared with the prior art, the present application has the beneficial technical effects in that:

[0020] Based on the previous research, the present application further verifies through experiments that the Punica granatum polysaccharide CLP2-2 has the effects of inhibiting the activation of hepatic stellate cells and resisting liver fibrosis, and can effectively improve chronic liver diseases, so that the Punica granatum polysaccharide is used for preparing a medicine for resisting liver fibrosis or treating chronic liver diseases, and has a wide application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 The Punica granatum polysaccharide CLP2-2 in the examples 13 C NMR chart (A) and HSQC chart (B).

[0022] Figure 2 The Punica granatum polysaccharide CLP2-2 in the examples

[0023] Figure 3 The Punica granatum polysaccharide CLP2-2 in the examples

[0024] Figure 4 The Punica granatum polysaccharide CLP2-2 in the examples

[0025] Figure 5The figures show the expression levels of fibrosis-related proteins in LX-2 cells of each group in the examples.

[0026] Figure 6 The table shows the levels of AST, ALT, and HYP in mouse serum in this example.

[0027] Figure 7 The image shows the HE staining results of mouse liver tissue sections in the example.

[0028] Figure 8 The image shows the Masson staining results of mouse liver tissue sections in the example.

[0029] Figure 9 The above are the immunohistochemical results of mouse liver tissue sections in the example. Detailed Implementation

[0030] To more fully understand and demonstrate the technical solutions, objectives, and advantages of the present invention, the technical effects produced by the present invention will be described in further detail and completely below with reference to specific embodiments and accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. It should be noted that other embodiments obtained by those skilled in the art without departing from the concept of the present invention are all within the protection scope of the present invention.

[0031] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0032] Example 1: Preparation of Prickly Pear Polysaccharide CLP2-2

[0033] a. Polysaccharide extraction: Fresh prickly pear was crushed, air-dried, sieved, and then 10 times its volume of deionized water was added. The mixture was heated under reflux in a water bath at 70℃ for 3 hours. The residue was collected by centrifugation, and defatting was repeated once. The mixture was then freeze-dried to obtain defatted powder. 25 g of the defatted powder was added to 30 times its volume of deionized water, stirred until homogeneous, and the pH was adjusted to 6. Then, 2.5% (by weight of the defatted powder) of papain was added. Extraction was carried out at 60℃ for 12 hours with stirring. The solution was then inactivated at 100℃ for 15 minutes. After cooling, the pH was adjusted to 8, and 2.5% (by weight of the defatted powder) of trypsin was added. Extraction was carried out at 37℃ for 12 hours with stirring. The solution was again inactivated at 100℃ for 15 minutes. After cooling, the solution was centrifuged, the supernatant was collected, heated and concentrated, dialyzed, concentrated again, and finally freeze-dried to obtain 3.31 g of enzyme-extracted crude prickly pear polysaccharide.

[0034] b. Polysaccharide purification: 1 g of the enzyme-extracted crude prickly pear polysaccharide prepared above was dissolved in 30 mL of water, centrifuged to remove insoluble matter, and the supernatant was initially separated by passing it through a Cl-type DEAE-cellulose column. Elution was performed sequentially with distilled water, 0.1 M NaCl, and 0.2 M NaCl, with sulfuric acid-phenol detection. The combined 0.2 M NaCl eluent was collected, concentrated, dialyzed, and freeze-dried to obtain 125 mg of prickly pear polysaccharide CLP2. 100 mg of prickly pear polysaccharide CLP2 was dissolved in 2 mL of deionized water and centrifuged. The supernatant was passed through a mixed cellulose membrane and purified by passing it through a Bio Gel P-6 Gel column at a flow rate of 0.3 mL / min. An elution curve was plotted using the sulfuric acid-phenol method. The combined eluent was collected according to the elution curve, and this purification process was repeated to obtain 100 mg of prickly pear polysaccharide CLP2-2.

[0035] c. Polysaccharide structure analysis: High performance gel permeation chromatography (HPGPC) analysis showed that the molecular weight of prickly pear polysaccharide CLP2-2 was 78.6 kDa; monosaccharide composition analysis showed that prickly pear polysaccharide CLP2-2 mainly contained galacturonic acid (83.5%). 13 In C NMR and HSQC spectra ( Figure 1 The δ98.95u terminal carbon signal is the C1 signal of galacturonic acid and indicates that it is in the α configuration, while δ77.86 indicates that the C4 position of galacturonic acid is substituted, and δ175.29 is the signal of uronic acid substitution at the C6 position. The C / H hydrogen signals of prickly pear polysaccharide CLP2-2 were assigned, and the results are shown in Table 1.

[0036] Table 1

[0037]

[0038] Based on the monosaccharide composition, infrared spectroscopy, and NMR data, the prickly pear polysaccharide CLP2-2 is a homopolymeric galacturonic acid with the structure: [→4)α-GalAp-(1→] n ( Figure 2 ).

[0039] Example 2: The effect of prickly pear polysaccharide CLP2-2 on improving TGF-β1-induced fibrosis in LX-2 cells

[0040] I. Materials

[0041] 1. Instruments and reagents

[0042] Instruments: Clean bench, centrifuge (Thermo), cell incubator, Nanodrop micro spectrophotometer, gradient PCR instrument, fully automated real-time PCR instrument, etc.

[0043] Reagents: DMEM high glucose medium (Gibco), fetal bovine serum, trypsin digestion solution (Gibco), rat type I collagenase, prickly pear polysaccharide CLP2-2 prepared according to the method in Example 1 (purity >98%), SPARKeasy cell RNA rapid extraction kit (Cisco), reverse transcription reaction kit (Kangwei Century), real-time fluorescence quantitative qPCR reaction kit (Kangwei Century), ALT kit (Nanjing Jiancheng), AST kit (Nanjing Jiancheng), HYP kit (Nanjing Jiancheng), α-SMA (CST Corporation, USA), Fibronectin (proteintech), etc.

[0044] 2. Experimental cells and animals

[0045] Human hepatic stellate cells (LX-2) were purchased from Beijing Beina Chuanglian Biotechnology Research Institute.

[0046] C57BL / 6J mice, male, 8 weeks old, weighing 18-20g, SPF grade, purchased from Spiford (Suzhou) Biotechnology Co., Ltd.

[0047] II. Methods

[0048] 1. Establishment of a TGF-β1-induced liver fibrosis cell model

[0049] LX-2 cells were cultured in DMEM medium containing 10% fetal bovine serum, with the medium changed every two days. After the cells reached confluence, they were digested with trypsin and passaged. The passaged cells were then divided into groups of 5 × 10⁶ cells per cell line. 5 The cells were seeded into 6-well plates and divided into a control group (Ctrl), a model group (TGF-β1), and a treatment group (CLP2-2). When the cells reached approximately 70% confluence, the culture medium for the model group (TGF-β1) was replaced with one containing 10 ng / mL TGF-β1, and the culture medium for the treatment group (CLP2-2) was replaced with one containing 10 ng / mL TGF-β1 and 200 μg / mL, 400 μg / mL, or 800 μg / mL prickly pear polysaccharide CLP2-2. The culture medium for the control group (Ctrl) was replaced with fresh culture medium without TGF-β1 or prickly pear polysaccharide CLP2-2. The cells were cultured for another 24 h.

[0050] 2. PCR detection of changes in liver fibrosis marker genes

[0051] PCR experiments were used to detect changes in the levels of liver fibrosis-related marker genes α-SMA, Col1A1, and FN1. Adherent cells detached after digestion were collected by centrifugation, and the supernatant was discarded, leaving the cell pellet. 500 μL of Lysis Buffer was added to the prepared sample tube, and the mixture was immediately and vigorously vortexed or pipetted until no cell clumps were present, then allowed to stand for 1 min. The mixture was added to the adsorption column RA (placed in the collection tube), centrifuged at 12000 rpm for 30 s, and the filtrate was discarded as much as possible. 500 μL of Washing Bufer D2 was added to the adsorption column, centrifuged at 12000 rpm for 30 s, and the filtrate was discarded as much as possible; this process was repeated once. The adsorption column RA was placed back into the empty collection tube, centrifuged at 12000 rpm for 2 min, and the wash buffer was removed as much as possible. Remove the adsorption column RA and place it in a clean RNase-free centrifuge tube. Add 30 μL of RNase-free H2O to the middle of the adsorption membrane according to the expected RNA yield (pre-incubate at room temperature for 1 min, centrifuge at 10,000 rpm for 1 min, and keep the liquid in the collection tube for later use). Take 1 μL of RNA and use a Nanodrop micro spectrophotometer to measure the concentration and purity of RNA. Set the reverse transcription program according to the reverse transcription kit requirements, prepare the reverse transcription system, synthesize cDNA, and store it at -20℃.

[0052] Primers were designed using the Gene section of the NCBI website. The primer sequences are as follows:

[0053] Table 2

[0054]

[0055] Prepare the reaction mixture according to the recommended reaction ratios in the reagent instructions: 10 μL of 2×SuperStar Universal SYBR Master Mix, 0.4 μL of Forward Primer, 0.4 μL of Reverse Primer, 750 ng of total cDNA, and ddH2O to a final volume of 20 μL per well. After preparation, add this mixture to a PCR plate and perform RT-qPCR. Quantification is performed using the Threshold cycle (Ct) method, with GAPDH as an internal control. The expression level of the target gene is determined according to Formula 2. -∆∆Ct To calculate.

[0056] 3. Western blot observation of changes in protein levels in cells

[0057] Western blotting was used to detect changes in the expression levels of fibrosis-related proteins. Cells from each group were discarded from the culture medium, washed twice with PBS, and total protein was extracted using loading buffer or RIPA protein lysis buffer containing a protease phosphatase inhibitor. After quantification of total protein using the BCA method, an equal volume of boiled and cooled protein sample was loaded into polyacrylamide gel wells. Electrophoresis was performed at 80 V for 30 minutes in electrophoresis buffer, followed by 120 V for 1 hour. Then, the membrane was transferred at 350 mA for 70 minutes in wet transfer buffer. After transfer, the membrane was blocked at room temperature for 2 hours with blocking buffer containing 5% skim milk powder. After blocking, primary antibody was prepared according to the antibody manufacturer's instructions and incubated overnight at 4 °C. The next day, the membrane was washed three times at room temperature for 10 minutes each with TBST washing buffer. Secondary antibody was then added, and the membrane was incubated at room temperature for 2 hours, followed by three washes at room temperature for 10 minutes each with washing buffer. Finally, the membrane was developed after incubation with developing solution.

[0058] 4. Animal grouping, liver fibrosis model establishment, and drug administration

[0059] Male C57BL / 6J mice were randomly divided into 6 groups of 10 mice each. The specific groups were: blank control group (Control), CCl4 model group (Model), low-dose group of prickly pear polysaccharide CLP2-2 administration (CLP2-L), high-dose group of prickly pear polysaccharide CLP2-2 administration (CLP2-H), and positive control group (silymarin).

[0060] After one week of acclimatization feeding, mice in the model group and the drug treatment group were intraperitoneally injected with 20% CCl4 olive oil solution (0.10 mL·10 g). -1 Three times a week, the control group was injected intraperitoneally with the same volume of olive oil solution. Two weeks later, at the same time as the model was established, the mice were administered the drug by gavage at the set dose once a day for four weeks. In the seventh week, the mice were fasted for 12 hours but not allowed to drink, and then sacrificed. Blood and tissue samples were collected.

[0061] The dosage for each group is:

[0062] Normal control group: Mice were not given CCl4 injections, but were usually injected with an equal volume of solvent (olive oil).

[0063] Model group: Mice were injected with 20% CCl4 to induce liver fibrosis;

[0064] Low-dose group (50 mg / kg): Mice were treated by gavage after induction with 20% CCl4;

[0065] High-dose group (200 mg / kg): Mice were treated by gavage after induction with 20% CCl4;

[0066] Positive control group: Mice were treated with positive drug (silymarin 250 mg / kg) by gavage after induction with 20% CCl4.

[0067] 5. Determination of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and hydroxyproline (HYP) in mouse serum

[0068] After anesthetizing the mice in each group, blood was collected from the orbital cavity in an amount of approximately 200-300 μL. The blood was allowed to stand at room temperature for 1-2 hours, then centrifuged at 3500 rpm for 15 minutes. The supernatant was collected, and the levels of each indicator were determined according to the instructions of the biochemical reagent kit.

[0069] 6. Histopathological evaluation of liver tissue

[0070] Liver lobe sections were cut into pieces approximately 1 cm × 1 cm × 1.5 cm in size, and after routine dehydration and paraffin embedding, sections were prepared and stained with hematoxylin and eosin (HE) and Masson stain, and observed under a light microscope.

[0071] 7. Immunohistochemical analysis

[0072] Paraffin sections were dewaxed using standard procedures, then incubated with 3% hydrogen peroxide solution at room temperature in the dark for 25 min. They were washed three times with PBS, blocked for 1 h, and then incubated overnight at 4°C with α-SMA, COL1A1, and FN1 (1:500). Secondary antibody was added, and the sections were incubated at 37°C for 20 min. DAB staining was performed, followed by hematoxylin counterstaining for 3 min. The sections were then dehydrated using standard procedures, mounted, and observed and photographed using a digital panoramic scanner. Data were processed using NDP.view 2.9.22 software.

[0073] 8. Data Analysis

[0074] The data were plotted and statistically analyzed using GraphPad Prism and ImageJ software.

[0075] III. Results

[0076] 1. Results of toxicological experiments as follows Figure 3 As shown, the cell viability of LX-2 cells did not change significantly after treatment with different concentrations of prickly pear polysaccharide CLP2-2, indicating that prickly pear polysaccharide CLP2-2 has no toxic side effects on LX-2 cells.

[0077] 2. The expression levels of marker genes in LX-2 cells of each group are as follows: Figure 4 As shown, a representative change in fibrosis is the upregulation of expression of liver fibrosis-related genes in hepatic stellate cells, such as α-SMA, COl1A1, and FN1. From... Figure 4As can be seen, compared with the control group, the expression levels of α-SMA, COl1A1, and FN1 in the model group were significantly upregulated, indicating that the TGF-β1-induced liver fibrosis model was successfully established. Compared with the model group, the expression levels of α-SMA, COl1A1, and FN1 in the treatment group were significantly downregulated, indicating that prickly pear polysaccharide CLP2-2 significantly improved liver fibrosis.

[0078] 3. Protein expression levels in LX-2 cells of each group are as follows: Figure 5 As shown, the expression levels of α-SMA and FN1 are low in normal LX-2 cells. Figure 5 As can be seen, compared with the control group, the expression levels of α-SMA and FN1 proteins in the model group were significantly increased, indicating that LX-2 cells were activated and the TGF-β1-induced liver fibrosis model was successfully established. Compared with the model group, the expression levels of α-SMA and FN1 proteins in the treatment group were significantly downregulated, indicating that prickly pear polysaccharide CLP2-2 significantly improved liver fibrosis.

[0079] 4. The serum ALT, AST, and HYP levels in each group of mice were as follows: Figure 6 As shown, after CCl4 modeling, the levels of AST, ALT and HYP in mouse serum increased significantly, but decreased after treatment with prickly pear polysaccharide CLP2-2, indicating that prickly pear polysaccharide CLP2-2 can effectively reduce liver damage.

[0080] 5. HE staining of liver tissue sections from each group of mice as shown in the figure. Figure 7 As shown, the liver tissue of the control group mice remained intact, with regular lobular morphology and no obvious pathological changes. In contrast, the liver tissue of the model group mice exhibited significant pathological damage, including hepatocyte edema, vacuolation, and disordered cell arrangement. These pathological features indicate that the mouse liver tissue had suffered severe damage. In mice treated with different doses of prickly pear polysaccharide CLP2-2 and silymarin, although hepatocytes showed mild edema, no pathological manifestations such as lobular structural destruction or severe liver necrosis were observed. These results indicate that the CCl4-induced liver fibrosis (HF) mouse model exhibits severe liver damage, and the treatment in each group significantly reduced the degree of liver damage in the mice.

[0081] 6. Masson staining of liver tissue sections from each group of mice as follows: Figure 8As shown, collagen fibers are evenly distributed and few in number in normal liver tissue, while a large amount of blue collagen deposition is visible around the central vein and portal vein in the liver tissue of the model group mice, which is a typical manifestation of fibrosis. Treatment with different doses of prickly pear polysaccharide CLP2-2 showed a decrease in collagen fiber content compared to the model group. Quantitative analysis using Image J revealed that, compared to the model group, the degree of collagen fiber deposition in the livers of mice treated with different doses of prickly pear polysaccharide CLP2-2 and silymarin significantly decreased, all of which improved the degree of liver fibrosis in the model mice to varying degrees.

[0082] 7. Immunohistochemistry of liver tissue sections from each group of mice as follows: Figure 9 As shown, there were significant differences in the expression levels of α-SMA, COL1A1, and FN1 in the liver tissues of mice from different groups. In the model group mice, the expression levels of these fibrosis markers were significantly higher than those in the blank control group, indicating that the liver fibrosis model was successfully established. In contrast, the expression levels of liver fibrosis markers were significantly reduced in the liver tissues of mice treated with different doses of prickly pear polysaccharide CLP2-2, indicating that prickly pear polysaccharide CLP2-2 can effectively inhibit the activation of hepatic stellate cells and excessive deposition of extracellular matrix in the process of liver fibrosis.

[0083] The results showed that the prickly pear polysaccharide CLP2-2 provided in Example 1 possessed the biological activity of inhibiting hepatic stellate cell activation. HE staining and serum biochemical results revealed that prickly pear polysaccharide CLP2-2 had excellent efficacy in improving liver damage and could effectively improve chronic liver disease. Therefore, the prickly pear polysaccharide CLP2-2 of this invention has broad application prospects for the preparation of drugs for anti-liver fibrosis and / or treatment of chronic liver disease.

[0084] The above are merely preferred embodiments of the present invention and are not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. The application of a prickly pear polysaccharide in the preparation of a drug for treating liver fibrosis; The prickly pear polysaccharide is prickly pear polysaccharide CLP2-2, with the structural formula [→4)α-GalAp-(1→ n Its relative molecular mass is 78.6 kDa; The preparation method of the prickly pear polysaccharide CLP2-2 includes the following steps: a. Polysaccharide extraction: Fresh, ripe prickly pear fruits are seeded, washed clean, crushed and air-dried, sieved and defatted with anhydrous ethanol, air-dried, 30 times the volume of deionized water is added, pH is adjusted, and enzymatic extraction is performed using papain and trypsin respectively. The supernatant is collected by centrifugation, concentrated, and freeze-dried to obtain crude prickly pear polysaccharide extracted by enzyme. b. Polysaccharide purification: The enzyme-extracted crude prickly pear polysaccharide was dissolved in deionized water, centrifuged, and the supernatant was separated by passing it through a DEAE cellulose anion exchange column. It was eluted sequentially with distilled water, 0.1M NaCl, and 0.2M NaCl, and detected by sulfuric acid-phenol. The combined 0.2M NaCl eluent was collected, concentrated, dialyzed, and freeze-dried to obtain prickly pear polysaccharide CLP2. It was then separated and purified by Bio Gel P-6Gel gel chromatography column to obtain prickly pear polysaccharide CLP2-2. The drug contains a therapeutically effective amount of prickly pear polysaccharide CLP2-2, the concentration of which is 1-1000 μg / mL; The drug also includes a pharmaceutically acceptable carrier and can be prepared as an injection, emulsion, tablet, powder, granule, ointment, liposome or oral liquid.

2. The application according to claim 1, characterized in that, The concentration of the prickly pear polysaccharide CLP2-2 is 200-800 μg / mL.

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