Lycium barbarum polysaccharide peptide, preparation method therefor and use thereof

Abstract

The present invention relates to a Lycium barbarum polysaccharide peptide, a preparation method therefor and the use thereof. The Lycium barbarum polysaccharide peptide comprises monosaccharides including mannose, glucose, galactose, arabinose and xylose, has a protein content in the range of 25 wt%-40 wt%, and has a molecular weight in the range of 3-20 kDa. By means of experiments, it is verified that the Lycium barbarum polysaccharide peptide of the present application has significant amelioration and alleviation effects on the liver damage and the degree of liver fibrosis in mice with CCl4-induced chronic liver fibrosis. The Lycium barbarum polysaccharide peptide can be developed into a drug for treating liver fibrosis or into a liver health supplement.

Description

Lycium barbarum polysaccharide peptide, preparation method and use thereof Technical Field

[0001] The present application belongs to the field of pharmacy, and specifically relates to a wolfberry polysaccharide peptide, a preparation method thereof, and uses thereof. Background Art

[0002] Liver fibrosis is a pathological stage in which the liver is damaged by chronic injury, the damage-repair process becomes abnormal, and the extracellular matrix accumulates excessively, replacing damaged normal tissue to form fibrous scars, leading to organic liver lesions. Liver fibrosis is a common pathological process in many chronic liver diseases, mainly caused by hepatotoxic damage and cholestatic damage. The global incidence of liver fibrosis is high, with approximately 2 million people dying from liver disease each year. The prevalence of liver disease in my country has risen to approximately 30%. The continued progression of liver fibrosis to the end stage will lead to cirrhosis, which has a reduced chance of regression and becomes a fatal disease. Therefore, early intervention in the relatively reversible stage of liver fibrosis is of great significance.

[0003] According to the Shennong Bencaojing (Shennong's Classic of Materia Medica), the mature fruit of the Ningxia wolfberry (Lycium barbarum), a plant of the Solanaceae family, has the effect of nourishing the liver and kidneys, and is listed as a top-grade product. Clinically, Lycium barbarum is often used in decoctions to nourish the liver and kidneys. The Compendium of Materia Medica states that "long-term consumption of Lycium barbarum can strengthen bones and muscles, lighten the body and prevent aging, and provide resistance to cold and heat." The chemical components of Lycium barbarum mainly include Lycium barbarum polysaccharides (LBPs), Lycium barbarum polysaccharide peptides, flavonoid polyphenols, and carotenoids. LBPs have been reported to improve liver fibrosis. To further explore whether other biomacromolecules in Lycium barbarum decoctions have the same effect on improving liver fibrosis, thereby expanding the application of Lycium barbarum and improving the high-quality development of traditional Chinese medicine, it is necessary to develop new medicinal ingredients in Lycium barbarum. Summary of the Invention

[0004] This invention utilizes a simple and effective process and method for extracting and purifying plant polysaccharides. Using wolfberry fruit grown in Ningxia as raw material, a mixed polysaccharide fraction was obtained. This was then further purified to yield the polysaccharide peptide LBPW. Pharmacological experiments have shown that LBPW, administered via intraperitoneal injection or oral gavage, can alleviate liver damage and inhibit liver fibrosis in a carbon tetrachloride-induced chronic liver fibrosis model in mice. Therefore, LBPW has the potential to be developed into a glycopeptide drug for liver fibrosis or a liver health supplement.

[0005] Therefore, in one aspect, the present invention provides a neutral wolfberry polysaccharide peptide, the monosaccharides of which include mannose, glucose, galactose, arabinose and xylose, the protein content of which is in the range of 25wt%-40wt%, and the molecular weight of which is in the range of 3-20kDa.

[0006] In a specific embodiment, based on the total amount of sugars contained in the wolfberry polysaccharide peptide, the arabinose content in the wolfberry polysaccharide peptide is 10-40%, the xylose content is 3-15%, the mannose content is 5-30%, the glucose content is 15-45%, and the galactose content is 5-35%.

[0007] In a specific embodiment, the monosaccharide connection mode and ratio of the wolfberry polysaccharide peptide are:

[0008] The proportions of terminally linked and 1,5-linked arabinose are 8-20% and 10-20%, respectively;

[0009] terminally linked and 1,4-linked xylose in a ratio of 1-10% and 1-10%;

[0010] 1,4- and 1,4,6-linked mannose, in proportions of 1-15% and 1-15%, respectively;

[0011] End-linked, 1,4-, and 1,4,6-linked glucose in proportions of 1-10%, 3-20%, and 1-15%, respectively;

[0012] The proportions of 1,3- and 1,3,6-linked galactose are 3-15% and 5-20%, respectively.

[0013] In a specific embodiment, the protein in the wolfberry polysaccharide peptide is mainly composed of aspartic acid, serine, glutamic acid, alanine and proline, and the relative mass ratio thereof is 0.79:1.3:1.0:1.1:0.68.

[0014] In a specific embodiment, the connection between the sugar part and the protein part in the wolfberry polysaccharide peptide is O-linked.

[0015] In a specific embodiment, the wolfberry glycopeptide 13 C In the NMR spectrum, there are the following signal peaks in the anomeric carbon region: δ110.47, δ108.75, δ108.66, δ104.40, δ103.78, δ103.62, δ102.91, δ102.73, δ101.96, δ101.16, δ99.53, δ99.30, δ97.13, δ95.31, δ93.31.

[0016] On the other hand, the present invention also provides a method for preparing the wolfberry polysaccharide peptide, which comprises the following steps:

[0017] (1) Extraction of polysaccharides: Lycium barbarum was extracted with boiling water to obtain an extract, which was concentrated; the concentrate was added with 95% ethanol for precipitation, allowed to stand overnight, centrifuged, and the precipitate was freeze-dried to obtain crude Lycium barbarum polysaccharide;

[0018] (2) Purification of polysaccharide: The crude polysaccharide of wolfberry fruit prepared in step (1) was dissolved in water, centrifuged, and the supernatant was purified by DEAE Sepharose TM The Fast Flow anion exchange column was eluted with deionized water, and the eluate of deionized water was collected to obtain glycopeptides.

[0019] Preferably, in step (1), before the boiling water extraction, the wolfberry medicinal material is first soaked in ethanol for 3-10 days (such as 7 days) and then air-dried.

[0020] Preferably, in step (1), 15-30 times the weight of water is added, the temperature is raised to 100°C and boiling water extraction is performed for 3-5 hours, and extraction is performed 6-8 times in total. The filtrates are combined and concentrated, and dialyzed against water for 24-72 hours. The dialysis bag has a molecular weight cutoff of 1kDa-10kDa. After concentration and centrifugation, 5-10 times the volume of the supernatant is added with ethanol, and the precipitate is centrifuged to obtain a crude polysaccharide LBP. The crude polysaccharide LBP is obtained by vacuum drying or freeze drying.

[0021] Preferably, in step (1), 20 times the weight of water is added, the temperature is raised to 100°C and boiling water extraction is performed for 4 hours, a total of 7 extractions are performed, the filtrates are combined and concentrated, dialyzed against water for 48 hours, the dialysis bag has a molecular weight cutoff of 3.5 kDa, concentrated, centrifuged, and 4 times the volume of the supernatant is added with ethanol, centrifuged to obtain a precipitate, and vacuum dried or freeze-dried to obtain crude polysaccharide LBP.

[0022] Preferably, in step (2), the crude polysaccharide prepared in step (1) is taken, added to 3 to 6 times the weight of water to dissolve, centrifuged, the supernatant is passed through an anion exchange column, eluted with deionized water, the deionized water eluate is collected, concentrated, centrifuged, the supernatant is dialyzed against water, and freeze-dried to obtain polysaccharide peptide.

[0023] Preferably, in step (2), the crude polysaccharide prepared in step (1) is taken, added to 5 times the weight of water to dissolve, centrifuged, the supernatant is passed through a DEAE Sepharose Fast Flow anion exchange column, eluted with deionized water, the eluate with deionized water is collected and combined, concentrated, centrifuged, the supernatant is dialyzed against water, and freeze-dried to obtain polysaccharide peptide.

[0024] In another aspect, the present invention provides a pharmaceutical composition comprising the Lycium barbarum polysaccharide peptide and pharmaceutically acceptable excipients.

[0025] In a specific embodiment, the composition is in the form of an oral or injection solution. The excipients used can be selected from those commonly used in these preparations in the art without limitation.

[0026] In another aspect, the present invention also provides use of the Lycium barbarum polysaccharide peptide or the pharmaceutical composition in preparing a drug for treating liver fibrosis or a liver health product. Beneficial effects

[0027] The present invention extracts a new neutral polysaccharide peptide component from wolfberry. Experimental verification shows that it has a significant improvement and alleviation effect on liver damage and liver fibrosis in mice with chronic liver fibrosis induced by CCl4, and can be developed into a drug for treating liver fibrosis or a liver health product.

[0028] In addition, the preparation method of the polysaccharide peptides of the present application is simple and has great promotion value and market application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] FIG1 is a purity diagram (A) and an ultraviolet absorption curve (B) of the wolfberry polysaccharide peptide LBPW prepared in Preparation Example 1 by high performance liquid chromatography.

[0030] FIG2 is a protein detection standard curve (A) of the wolfberry polysaccharide peptide LBPW prepared in Preparation Example 1 and a diagram of the connection mode between the protein and the polysaccharide (B).

[0031] Figure 3 shows the polysaccharide peptide LBPW in preparation example 1. 13 C NMR spectrum.

[0032] FIG4 is a graph showing the effects of intraperitoneal injection and oral administration of Lycium barbarum polysaccharide peptide LBPW on liver damage in mice with chronic liver fibrosis induced by CCl4 in Test Example 1.

[0033] a. Experimental protocol for mice injected intraperitoneally with LBPW; b. Serum ALT levels in mice injected intraperitoneally with LBPW; c. Serum AST levels in mice injected intraperitoneally with LBPW; d. Appearance of livers and H&E staining of liver pathological sections in mice injected intraperitoneally with LBPW (scale bar, 50 μm); e. Experimental protocol for mice injected orally with LBPW; f. Serum ALT levels in mice injected orally with LBPW; g. Serum AST levels in mice injected orally with LBPW; h. Appearance of livers and H&E staining of liver pathological sections in mice injected orally with LBPW (scale bar, 50 μm). ip indicates intraperitoneal injection, ig indicates oral gavage, and qd indicates once daily.

[0034] One-way ANOVA was used for comparison among multiple groups, and p < 0.05 was considered to be a significant difference.

[0035] FIG5 is a graph showing the results of the effects of intraperitoneal injection and oral administration of Lycium barbarum polysaccharide peptide LBPW in Test Example 1 on the degree of liver fibrosis in mice with chronic liver fibrosis induced by CCl4.

[0036] a. Picrosirius red staining (scale bar, 100 μm) and immunohistochemical staining (α-SMA) (scale bar, 50 μm) of liver pathological sections from mice injected intraperitoneally with LBPW; b. Statistical graph of positive areas of IHC staining (α-SMA) in the livers of mice injected intraperitoneally with LBPW; c. Statistical graph of the ratio of picrosirius red-positive areas in the livers of mice injected intraperitoneally with LBPW; d. Western blotting evaluation of α-SMA expression in the livers of mice injected intraperitoneally with LBPW; e. Western blotting of liver tissues of mice injected intraperitoneally with LBPW Semi-quantitative results of α-SMA protein expression detected by blotting; f. Picrosirius red staining (scale length 100 μm) and immunohistochemistry (α-SMA) staining (scale length 50 μm) of liver pathological sections of LBPW-gavaged mice; g. Statistical graph of positive areas of IHC staining (α-SMA) in the livers of LBPW-gavaged mice; h. Statistical graph of the positive area ratio of Picrosirius red staining in the livers of LBPW-gavaged mice; i. Evaluation of α-SMA expression by Western blotting in the livers of LBPW-gavaged mice; j. Semi-quantitative results of α-SMA protein expression by Western blotting in the liver tissues of LBPW-gavaged mice.

[0037] One-way ANOVA was used for comparison among multiple groups, and p < 0.05 was considered to be a significant difference. DETAILED DESCRIPTION

[0038] Preparation Example 1: Extraction, separation, purification and structural characterization of Lycium barbarum polysaccharide peptide LBPW

[0039] (1) Extraction and separation of polysaccharides

[0040] Dried wolfberry (5 kg) was added to 20 volumes of deionized water and extracted with boiling water at 100°C for 4 hours each time for a total of 7 extractions. The extracts were combined and concentrated, and then dialyzed in running water for 2 days (dialysis bag with a molecular weight cut-off of 3.5 kDa). The dialyzed liquid was heated and concentrated, centrifuged and the precipitate discarded. The supernatant was added with stirring to four volumes of 95% v / v ethanol, allowed to precipitate overnight, and centrifuged. The resulting precipitate was vacuum dried at 60°C, redissolved in 500 mL of deionized water, and freeze-dried to obtain crude polysaccharide LBP (yield 1.35%).

[0041] (2) Purification of polysaccharides

[0042] Each time, 8 g of crude polysaccharide LBP prepared in the above preparation (1) was dissolved in 40 mL of deionized water, stirred overnight, centrifuged for 10 min to remove insoluble matter, and the supernatant was loaded on DEAE Sepharose. TMThe Fast Flow anion exchange column was eluted with deionized water and 0.05-0.4M NaCl solution in sequence, with the flow rate controlled at 13mL / 15min, and collected with an automatic collector. Each concentration of NaCl solution was eluted until there was no sugar component (no color was developed in the phenol-sulfuric acid method), and then eluted with the next concentration of NaCl solution. The samples in the collection tubes were sampled and tested in separate tubes (such as the 2nd, 4th, 6th tubes...), 100μL of sample solution was taken from each well, 400μL of 5% phenol solution was added, and then 2mL of concentrated sulfuric acid was added. The mixture was shaken and placed at room temperature for 15min, and then 100μL of the reaction solution was taken from each well and added to a 96-well plate. The OD value was detected using an enzyme-labeled instrument. 490 The elution curve was drawn. According to the elution curve, all sugar components came from the deionized water elution portion. Therefore, the deionized water elution portion was collected and combined, concentrated, and centrifuged. The supernatant was dialyzed against water to remove small molecules (the dialysis bag had a molecular weight cutoff of 3.5 kDa) and freeze-dried to obtain glycopeptide LBPW (144 mg, yield 1.8%).

[0043] (3) Structural identification of polysaccharide peptides

[0044] Polysaccharide peptide LBPW was connected to Sugar KS-802 (exclusion limit 1×10 4 Da) and KS-804 (exclusion limit 4×10 5 The characteristic spectrum of LBPW on a high-performance liquid gel permeation chromatography (HPGPC) column (Figure 1) is shown. Its molecular weight is 3-20 kDa. A clear signal is observed under a 280 nm UV detector, and the UV signal peak coincides with the peak of the polysaccharide signal, indicating that LBPW is a glycopeptide. Chromatographic conditions were: mobile phase: 0.1 M NaNO₃; flow rate: 0.5 mL / min; column temperature: 25°C; Agilent 1260 liquid chromatograph; detectors: differential detector and UV detector.

[0045] Bovine serum albumin (BSA) was used as the standard. Under alkaline conditions, divalent copper ions were reduced to monovalent copper ions by proteins. Monovalent copper ions and BCA solution can interact, that is, two molecules of BCA chelate one copper ion to form a purple complex. The complex showed strong absorbance at 562nm, and the absorbance value was positively correlated with the protein concentration. Different concentrations of BSA were taken as the standard and mixed with the quantitative BCA working solution to measure the OD value. 562 , reaching the protein standard curve, its R 2 =0.9972 (Figure 2A). Based on the absorbance value of LBPW under the same conditions, the protein content of LBPW was obtained by substituting it into the standard curve. The protein content was 25-40 wt%.

[0046] The β-elimination reaction was used to examine the type of linkage between the protein and polysaccharide in LBPW. LBPW (1 mg / mL) was dissolved in 0.1 M NaOH containing 1 M NaBH₄ and monitored at room temperature using UV light (200-400 nm). LBPW not treated with alkaline solution served as a control. The UV absorption results (Figure 2B) showed that the alkaline-treated LBPW exhibited a significant decrease in UV absorption relative to the original sugar, indicating that an elimination reaction occurred after alkaline treatment, forming α-aminoacrylic acid, confirming that the linkage between LBPW and the protein was an O-linkage.

[0047] The monosaccharide composition was determined by HPLC. The flow rate was set at 1 mL / min, the column temperature was 25°C, the UV detection wavelength was 254 nm, and the sample injection volume was 10 μL. The results showed that Lycium barbarum polysaccharide peptide LBPW is a neutral glycopeptide, mainly containing mannose, glucose, galactose, arabinose, and a small amount of xylose. The arabinose content was 10-40%, the xylose content was 3-15%, the mannose content was 5-30%, the glucose content was 15-45%, and the galactose content was 5-35%.

[0048] Methylation analysis was performed on the Lycium barbarum polysaccharide peptide component, LBPW. A 6-10 mg sample was weighed and placed in a desiccator overnight (to ensure the reaction was dry). The next day, the sample was completely dissolved in 2 mL of DMSO. 100 mg of ground sodium hydroxide powder was added and stirred for 2 hours. Over 30 minutes, 1 mL of iodomethane was added dropwise in an ice-water bath, followed by a 2-hour reaction in the dark. The reaction was quenched with 1 mL of deionized water. The solution was concentrated under reduced pressure to remove excess unreacted CH3I, dialyzed against water for 24-72 hours, and lyophilized. The reacted sample was subjected to the sugar alcohol acetate reaction and then transferred to a liquid chromatography vial. The polysaccharide linkage patterns were analyzed using GC-MS (Thermo Fisher ISQ7000).

[0049] Methylation results showed that the sugar residues in LBPW had the following linkage patterns: terminal-linked and 1,5-linked arabinose, with proportions of 8-20% and 10-20%, respectively; terminal-linked and 1,4-linked xylose, with proportions of 1-10% and 1-10%, respectively; mannose had 1,4- and 1,4,6-linked forms, with proportions of 1-15% and 1-15%, respectively; glucose had terminal, 1,4-, 1,4,6-linked forms, with proportions of 1-10%, 3-20% and 1-15%, respectively; galactose existed in 1,3- and 1,3,6-linked forms, with proportions of 3-15% and 5-20%, respectively.

[0050] The NMR spectrum of the polysaccharide peptide LBPW was measured on a Bruker AVANCE III 500M NMR spectrometer at 25°C and the polysaccharide peptide was analyzed by NMR. 35 mg of the glycopeptide LBPW was dissolved in 0.5 mL of D2O and 2.5 μL of acetone was added as an internal standard (δH = 2.29 ppm, δC = 31.5 ppm). The structure of the polysaccharide in the polysaccharide peptide LBPW was confirmed by referring to the NMR spectrum. 13 The C NMR results are shown in Figure 3. 13 In the C NMR spectrum ( Figure 3 ), in the anomeric carbon region, there are the following signal peaks: δ110.47, δ108.75, δ108.66, δ104.40, δ103.78, δ103.62, δ102.91, δ102.73, δ101.96, δ101.16, δ99.53, δ99.30, δ97.13, δ95.31, and δ93.31.

[0051] To test the amino acid composition of this polysaccharide peptide sample, weigh and mix an appropriate amount of LBPW. Add 10 mL of a 1:1 hydrochloric acid solution to a hydrolysis tube and mix thoroughly. Place the tube in an electric heated air-blast incubator at 110°C ± 1°C for 22 hours, then cool to room temperature. Open the tube, filter the hydrolyzate into a 25 mL volumetric flask, and dilute to the mark with water. After shaking, accurately pipette 0.5 mL of the filtrate into a 15 mL test tube, dry with nitrogen, and dilute to 10 mL with 0.02 mol / L hydrochloric acid. After vortexing and mixing, filter through a 0.22 μm microporous filter membrane for detection. The detection column is a sulfonic acid cationic resin with wavelengths of 570 nm and 440 nm.

[0052] After testing, it was found that LBPW is a polysaccharide peptide, among which serine and alanine are the highest in content. In addition, it also contains aspartic acid, threonine, glutamic acid, glycine, valine, isoleucine, leucine, tyrosine, phenylalanine, lysine, histidine, arginine, proline (content less than 1 mg / g) and trace amounts of cystine (less than 0.01 mg / g). The specific amino acid content is shown in Table 1 below.

[0053] Table 1

[0054] Test Example 1: Study on the anti-liver fibrosis activity of Lycium barbarum polysaccharide peptide component LBPW

[0055] The anti-fibrotic activity of LBPW, a polysaccharide peptide component of Lycium barbarum L., was detected in vivo in a chronic liver fibrosis model mouse model induced by carbon tetrachloride.

[0056] C57BL / 6J male mice, weighing 22 ± 1 g and aged 8 weeks, were purchased and housed in an SPF animal room maintained at a constant temperature of 22–24°C and a humidity of 60% ± 5%, with a 12-h light / 12-h dark cycle. All experimental protocols were approved by the Animal Care Committee of the Shanghai Institute of Materia Medica, Chinese Academy of Sciences. Mice were acclimated for one week before the experiments. For the LBPW intraperitoneal injection experiment (Figure 4a), mice were randomly divided into six groups, each containing seven mice: a negative control group (I); a CCl4-induced model group (II); a CCl4-induced model LBPW low-dose group (III) at a dose of 50 mg / kg; a CCl4-induced model LBPW intermediate-dose group (IV) at a dose of 100 mg / kg; a CCl4-induced model LBPW high-dose group (V) at a dose of 200 mg / kg; and a CCl4-induced model obeticholic acid (OCA) positive control group (VI) at a dose of 30 mg / kg. In the LBPW oral administration experiment (Figure 4e), mice were randomly divided into six groups: a negative control group (I); a CCl4-induced model group (II); a CCl4-induced model LBPW low-dose group (III) (100 mg / kg); a CCl4-induced model LBPW intermediate-dose group (IV) (200 mg / kg); a CCl4-induced model LBPW high-dose group (V) (300 mg / kg); and a CCl4-induced model obeticholic acid positive control group (VI) (30 mg / kg). Groups I, II, III, and VI consisted of six mice each, while groups IV and V consisted of seven mice each. In both animal studies, mice in groups II, III, IV, V, and VI were intraperitoneally injected with 10% CCl4 (CCl4 dissolved in olive oil, CCl4:olive oil volume ratio of 1:9, at a dose of 2 mL / kg, three times weekly for eight weeks) to induce chronic liver fibrosis. Mice in group I were intraperitoneally injected with the same volume of olive oil. After two weeks of CCl4 induction, mice in groups III, IV, and V were intraperitoneally injected or gavaged daily with LBPW (dissolved in saline). Mice in group VI were gavaged daily with obeticholic acid (a suspension prepared with 0.5% CMC-Na). Mice in groups I and II were intraperitoneally injected or gavaged daily with an equal amount of saline, with the administration volume being 10 mL / kg. The dosing cycle lasted for six weeks, and mice were sacrificed at the end of the experiment after fasting for 12 hours. Blood and liver samples were collected, and the liver tissue was photographed to observe the appearance of the lesions. After the blood was allowed to stand at room temperature for 30 minutes, it was centrifuged at 2000 rpm for 30 minutes at 4°C, and the upper serum layer was aspirated and stored at -80°C. A portion of the liver tissue was fixed with 4% paraformaldehyde, and a portion was snap-frozen in liquid nitrogen and stored at -80°C.

[0057] To evaluate the efficacy of LBPW in ameliorating liver damage in mice with hepatic fibrosis, mice were sacrificed, and images of the livers were collected and hematoxylin-eosin (H&E) stained to assess liver pathology. Liver appearance was observed in the normal group, which showed a smooth and shiny appearance, while the livers in the model group showed a gritty texture. Intraperitoneal and oral administration of LBPW at all three doses improved these appearances, with the liver surface becoming smoother with increasing dose concentration (Figures 4d and 4h). H&E results showed that in the negative control group, hepatocytes were densely packed, with no significant parenchymal lesions. In the model group, hepatocytes showed disorganized arrangement, increased vacuoles, increased inflammatory cell infiltration, and disrupted hepatocyte architecture. Liver damage was also observed in the positive control group, but it was significantly less severe than in the model group. All three doses of LBPW, administered intraperitoneally and orally, improved these pathological findings (Figures 4d and 4h). Serum transaminase levels are elevated in response to liver damage. Therefore, in the LBPW intraperitoneal injection experiment, ELISA kits (Nanjing Jiancheng) were used to detect the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), liver injury indicators in the serum; in the LBPW oral administration experiment, serum ALT and AST levels were measured using a biochemical analyzer to evaluate the effect of LBPW on the alleviation of liver injury indicators in mice. The results showed that ALT and AST levels in the model group were significantly increased, indicating that the liver function of the mice was impaired. However, the intraperitoneal injection of LBPW at all three doses significantly reduced the levels of these indicators (Figures 4b and 4c); and the oral administration of LBPW at all three doses also significantly reduced the serum AST and ALT levels (Figures 4f and 4g).

[0058] In summary, the three doses of LBPW administered by intraperitoneal injection and oral gavage all improved liver damage in CCl4-induced chronic liver fibrosis model mice.

[0059] To evaluate the effect of LBPW on the alleviation of the degree of fibrosis in mice with liver fibrosis, liver tissue was stained with Sirius red to assess collagen deposition; immunohistochemistry (IHC) for α-smooth muscle actin (α-SMA) was performed to assess hepatic stellate cell activation; 40 mg of liver tissue was extracted using an animal whole protein extraction kit (Shanghai Sangon Biotechnology) and 5× loading buffer was added. The protein was denatured by boiling at 100°C for 10 minutes, and the expression of α-SMA protein was detected by Western blotting.

[0060] The results of picrosirius red staining are shown in Figures 5a and 5f. Under an optical microscope, collagen fibers appeared red against a yellow background. No collagen deposition was observed in the negative control group mice, while collagen deposition was severe in the vascular and portal regions of the model group mice. LBPW administration significantly reduced collagen deposition after both routes (statistics shown in Figures 5c and 5h). Immunohistochemistry results, as shown in Figures 5a and 5f, showed brownish-yellow expression of α-SMA. α-SMA expression was elevated in the model group, while LBPW administration significantly decreased α-SMA expression in the liver tissue of mice after both routes (statistics shown in Figures 5b and 5g). Western blotting of liver tissue revealed that LBPW administration reduced α-SMA protein expression in both routes (Figures 5d, 5e, 5i, and 5j). Immunohistochemistry and Western blotting results indicate that LBPW inhibits hepatic stellate cell activation in vivo.

[0061] In summary, three doses of LBPW administered by intraperitoneal injection and oral gavage can significantly alleviate the degree of fibrosis in mice with liver fibrosis.

[0062] In summary, it can be seen from the examples that both intraperitoneal injection and oral administration of LBPW, a polysaccharide peptide component of wolfberry fruit, can alleviate liver fibrosis in mice.

[0063] Based on the above research results, it can be concluded that the polysaccharide peptide component LBPW of the present application can be developed into a potential glycopeptide drug or liver health product for the treatment of liver fibrosis.

Claims

1. A neutral wolfberry polysaccharide peptide, wherein the monosaccharide comprises: Mannose, glucose, galactose, arabinose and xylose, the protein content is in the range of 25wt%-40wt%, and its molecular weight is in the range of 3-20kDa.

2. The Lycium barbarum polysaccharide peptide according to claim 1, wherein Calculated based on the total amount of sugar contained in the wolfberry polysaccharide peptide, the molar ratios of various monosaccharides are: arabinose content is 10-40%, xylose content is 3-15%, mannose content is 5-30%, glucose content is 15-45%, and galactose content is 5-35%.

3. The Lycium barbarum polysaccharide peptide according to claim 1, wherein The monosaccharide connection mode and ratio of the wolfberry polysaccharide peptide LBPW are as follows: The proportions of terminally linked and 1,5-linked arabinose are 8-20% and 10-20%, respectively; terminally linked and 1,4-linked xylose in a ratio of 1-10% and 1-10%; 1,4- and 1,4,6-linked mannose, in proportions of 1-15% and 1-15%, respectively; End-linked, 1,4-, and 1,4,6-linked glucose in proportions of 1-10%, 3-20%, and 1-15%, respectively; The proportions of 1,3- and 1,3,δ-linked galactose are 3-15% and 5-20%, respectively.

4. The Lycium barbarum polysaccharide peptide according to claim 1, wherein The protein in the wolfberry polysaccharide peptide is mainly composed of aspartic acid, serine, glutamic acid, alanine and proline, and the relative mass ratio thereof is 0.79:1.3:1.0:1.1:0.

68.

5. The Lycium barbarum polysaccharide peptide according to claim 1, wherein The connection mode between the sugar part and the protein part in the wolfberry polysaccharide peptide is O-linked.

6. The Lycium barbarum polysaccharide peptide according to claim 1, wherein The wolfberry glycopeptide 13 C In the NMR spectrum, there are the following signal peaks in the anomeric carbon region: δ110.47, δ108.75, δ108.66, δ104.40, δ103.78, δ103.62, δ102.91, δ102.73, δ101.96, δ101.16, δ99.53, δ99.30, δ97.13, δ95.31, δ93.

31.

7. The method for preparing the Lycium barbarum polysaccharide peptide according to any one of claims 1 to 6, comprising the following steps: (1) Extraction of polysaccharides: Lycium barbarum was extracted with boiling water to obtain an extract, which was concentrated; the concentrate was added with 95% ethanol for precipitation, allowed to stand overnight, centrifuged, and the precipitate was freeze-dried to obtain crude Lycium barbarum polysaccharide; (2) Purification of polysaccharide: The crude polysaccharide of wolfberry fruit prepared in step (1) was dissolved in water, centrifuged, and the supernatant was purified by DEAE Sepharose TM The Fast Flow anion exchange column was eluted with deionized water, and the eluate of deionized water was collected to obtain glycopeptides.

8. The method according to claim 7, wherein: In step (1), 15-30 times the weight of water is added, the temperature is raised to 100° C., boiling water extraction is performed for 3-5 hours, and extraction is performed 6-8 times in total. The filtrates are combined and concentrated, dialyzed against water for 24-72 hours, the molecular weight cutoff of the dialysis bag is 1 kDa-10 kDa, concentrated, and centrifuged. After that, 5-10 times the volume of the supernatant is added with ethanol, centrifuged to obtain a precipitate, and vacuum dried or freeze-dried to obtain crude polysaccharide; Preferably, in step (1), 20 times the weight of water is added, the temperature is raised to 100°C and boiling water extraction is performed for 4 hours, a total of 7 extractions are performed, the filtrates are combined and concentrated, dialyzed against water for 48 hours, the molecular weight cut-off of the dialysis bag is 3.5 kDa, concentrated, centrifuged, 4 times the volume of the supernatant is added with ethanol, centrifuged to obtain a precipitate, and vacuum dried or freeze-dried to obtain crude polysaccharide LBP, and In step (2), the crude polysaccharide LBP prepared in step (1) is added to 3 to 6 times its weight of water for dissolution, centrifuged, and the supernatant is passed through an anion exchange column and eluted with deionized water. The deionized water eluate is collected, concentrated, centrifuged, and the supernatant is dialyzed against water and freeze-dried to obtain a polysaccharide peptide; Preferably, in step (2), the crude polysaccharide LBP prepared in step (1) is taken, added to 5 times the weight of water to dissolve, centrifuged, and the supernatant is passed through a DEAE Sepharose Fast Flow anion exchange column and eluted with deionized water. The eluate of deionized water is collected and combined, concentrated, centrifuged, and the supernatant is dialyzed against water with a dialysis bag having a molecular weight cutoff of 3.5 kDa, and freeze-dried to obtain a polysaccharide peptide.

9. A pharmaceutical composition comprising the Lycium barbarum polysaccharide peptide according to any one of claims 1 to 6, and pharmaceutically acceptable excipients.

10. Use of the Lycium barbarum polysaccharide peptide according to any one of claims 1 to 6 or the pharmaceutical composition according to claim 9 in the preparation of a medicament for treating liver fibrosis or a liver health product.

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