Radix scrophulariae polysaccharide, preparation method and application thereof

Abstract

The present application relates to a gardenia polysaccharide, a preparation method and uses thereof. The gardenia polysaccharide comprises or mainly consists of the following monosaccharide units: rhamnose, glucuronic acid, galacturonic acid, glucose, galactose and arabinose; and the molar ratio of rhamnose, glucuronic acid, galacturonic acid, glucose, galactose and arabinose in the gardenia polysaccharide is 3-15:1-8:4-18:1-6:15-35:25-70. The gardenia polysaccharide according to the present application can reduce FFA-induced lipid accumulation in mouse primary hepatocytes without obvious cytotoxicity, can reduce CDAHFD diet-induced liver fibrosis, and has a good effect of preventing and / or treating non-alcoholic steatohepatitis.

Description

Technical Field

[0001] This invention belongs to the field of polysaccharides, and relates to a polysaccharide derived from gardenia, its preparation method and uses. Specifically, it relates to a polysaccharide extracted from gardenia (Gardenia jasminoides), its preparation method, and its use in the preparation of reagents or drugs for inhibiting, preventing and / or treating non-alcoholic steatohepatitis (NAH). Background Technology

[0002] Nonalcoholic steatohepatitis (NAHH) is a group of liver abnormalities characterized by fat accumulation, liver damage, inflammation, and liver fibrosis. Progressive NHAH easily progresses to liver fibrosis, and may even develop into irreversible cirrhosis and eventually liver cancer. In 2024, the FDA approved Resmetirom as the world's first drug for the treatment of NHAH, marking the first glimmer of hope after more than 40 years of research and development stagnation. However, drug development in this area remains challenging; therefore, the development of active compounds and drugs targeting NHAH are of paramount importance.

[0003] Gardenia jasminoides is one of 250 species of flowering plants in the Rubiaceae family. Studies have found that gardenia contains a large number of chemical components, mainly including iridoids, triterpenes, flavonoids, volatile oils, organic acid esters, trace elements, and polysaccharides. Its dried fruit is used as a traditional medicine in Asian countries and has high medicinal value, possessing anti-inflammatory, diuretic, laxative, bile-regulating, and homeostatic effects. It can also relieve liver and abdominal pain. Currently, numerous systematic studies have been conducted on compounds derived from gardenia, examining their structure, function, and extraction methods, resulting in a large number of published articles. Research indicates that gardenia polysaccharides can improve cholestatic liver injury by alleviating intestinal flora imbalance; gardenia water extracts and their main components have anti-thrombotic effects; an iridoid glycoside derived from gardenia can protect mice from lipopolysaccharide-induced acute lung injury; gardenin, a traditional Chinese medicine extract from gardenia, can exert anti-inflammatory and cardioprotective effects; and gardenin can potentially exert antidepressant effects by regulating the hypothalamic-pituitary-adrenal axis, among others. In traditional Chinese medicine theory, gardenia belongs to the liver meridian. Gardenia has a long history of being used to treat liver diseases in traditional Chinese medicine. Clinically, gardenia is used in prescriptions (such as Yin Chen Hao Tang, Zhi Zi Da Huang Tang, Long Dan Xie Gan Tang, Zhi Zi Bai Pi Tang, etc.) and prepared Chinese medicines (such as Yin Zhi Huang oral liquid, Long Dan Xie Gan Wan, Qing Gan Li Dan oral liquid, Shu Gan Ning injection, etc.) for treating liver diseases. Based on this, the inventors attempted to explore the function of gardenia polysaccharides in non-alcoholic fatty liver disease. Summary of the Invention

[0004] This invention employs a simple and effective polysaccharide extraction method to obtain a homogeneous pectin-like polysaccharide, GJ005S, from gardenia fruit. Pharmacological experiments show that polysaccharide GJ005S can reduce lipid accumulation in primary hepatocytes and alleviate diet-induced fat accumulation and liver damage in NASH mice, inhibiting the development of fibrosis. Therefore, this polysaccharide has the potential to inhibit non-alcoholic steatohepatitis (NAH) and is expected to be developed into a glycoside drug for the treatment of NHA.

[0005] Based on this, one object of the present invention is to provide a gardenia polysaccharide.

[0006] Another object of the present invention is to provide a method for preparing the above-mentioned gardenia polysaccharide.

[0007] Another object of the present invention is to provide a composition comprising the gardenia polysaccharide.

[0008] Another object of the present invention is to provide the use of the gardenia polysaccharide in the preparation of a medicament or reagent for inhibiting non-alcoholic steatohepatitis.

[0009] Another object of the present invention is to provide the use of the gardenia polysaccharide in the preparation of medicaments for the prevention and / or treatment of non-alcoholic steatohepatitis.

[0010] Therefore, in one aspect of the present invention, a gardenia polysaccharide is provided, which comprises the following monosaccharide units: rhamnose, glucuronic acid, galacturonic acid, glucose, galactose and arabinose, or is mainly composed of them;

[0011] The main chain structure of the gardenia polysaccharide consists of alternating 1→2 linked α-rhamnose and 1→4 linked α-galacturonic acid; the side chains are linked at C-4 of the 1→2 linked α-rhamnose and C-3 of the 1→4 linked α-galacturonic acid in the main chain, including T-, 1,5-, 1,3-, and 1,3,5- linked α-arabinose; T-, 1,4-, 1,6-, and 1,3,6- linked β-galactose; 1,4- linked α-glucuronic acid; and α-rhamnose, α-galacturonic acid, and α-glucose linked at the ends;

[0012] The molar ratio of rhamnose, glucuronic acid, galacturonic acid, glucose, galactose and arabinose in the gardenia polysaccharide is 3-15:1-8:4-18:1-6:15-35:25-70.

[0013] In a specific embodiment, the weight-average molecular weight of the gardenia polysaccharide is in the range of 2 to 100 kDa; preferably 5 to 80 kDa; more preferably 10 to 60 kDa; even more preferably 10 to 25 kDa; even more preferably 15 to 20 kDa; and even more preferably 17.1 kDa.

[0014] In a specific embodiment, the molar ratio of rhamnose, glucuronic acid, galacturonic acid, glucose, galactose and arabinose is 7.6:3.7:9.4:2.2:26.8:50.3.

[0015] In a specific embodiment, the characteristic high-performance gel permeation chromatography (HPGPC) analysis of the gardenia polysaccharide is as follows: Figure 1 As shown; the chromatographic conditions for high-performance gel permeation chromatography were as follows: the chromatographic column was a Shodex SUGAR KS-804 and a Shodex SUGAR KS-802 used in series; the mobile phase was 0.1M NaNO3 aqueous solution; the flow rate was 0.5mL / min; the column temperature was 40℃; the detector was a differential detector (RID); and the detection temperature was 35℃.

[0016] In a specific embodiment, the gardenia polysaccharide is GJ005S, which... 1 In the 1H NMR spectrum, the signals in the anomeric hydrogen region are δ5.23, δ5.16, δ5.25, δ5.18, δ5.28, δ5.32, δ4.66, δ5.12, δ5.15, δ5.1, δ4.65, δ4.68, δ4.53, and δ4.54; corresponding to... 13 The characteristic signal peaks in the C NMR spectrum are: δ109.7, δ108.31, δ108.6, δ108.47, δ101.75, δ99.77, δ97.4, δ100.29, δ100.34, δ98.85, δ99.74, δ105.62, δ105.46, δ104.42, and δ104.33. The above chemical shifts have a deviation of no more than ±0.5. Preferably, the main signal values ​​of the one-dimensional NMR spectrum of the gardenia polysaccharide GJ005S are... Figure 2 As shown in A 1 H NMR spectra and / or Figure 2 As shown in B 13 The C NMR spectra are basically consistent.

[0017] In a specific embodiment, the gardenia polysaccharide has the following repeating structural units,

[0018]

[0019] In another aspect, the present invention provides a method for preparing gardenia polysaccharide, comprising the following steps:

[0020] (1) Extraction of polysaccharides: Gardenia was extracted with boiling water to obtain an extract. The extract was concentrated and precipitated with ethanol. The precipitate was collected to obtain crude gardenia polysaccharides.

[0021] (2) Purification of polysaccharides: The crude polysaccharide of gardenia was dissolved in water and eluted sequentially with deionized water and 0.05M NaCl solution through an anion exchange column. The eluent of 0.05M NaCl was collected as a secondary eluent. The secondary eluent was then purified by elution with a dextran gel column. The collected eluent was concentrated, dialyzed with water, and lyophilized to obtain gardenia polysaccharide.

[0022] In one embodiment, during the extraction in step (1), the amount of water used is 15 to 30 times that of gardenia, preferably 20 times.

[0023] In one embodiment, in step (1), the extraction time is 0.5 to 4 hours, preferably 1 to 3 hours; the number of extractions is 1 to 10 times, preferably 7 times.

[0024] In one embodiment, in step (1), the extract is concentrated and then dialyzed, with the dialysis bag having a molecular weight cutoff of 3500 Da.

[0025] In one embodiment, in step (1), the extract is concentrated and then dialyzed. After the dialysate is concentrated, centrifuged and the precipitate is discarded, 3 to 8 times (preferably 4 times) the volume of ethanol is added to the supernatant for alcohol precipitation.

[0026] In one embodiment, step (1) includes: extracting gardenia with boiling water, concentrating and dialyzing the obtained extract, with the molecular weight cutoff of the dialysis bag being 3500 Da, concentrating and centrifuging the dialysis fluid to discard the precipitate, precipitating the obtained supernatant with ethanol, and separating, washing and drying the precipitate to obtain crude gardenia polysaccharide.

[0027] In another embodiment, step (1) includes: taking dried gardenia, adding 15 to 30 times (preferably 20 times) of water by weight, extracting with boiling water for 0.5 to 4 hours (preferably 1 to 3 hours), extracting 1 to 10 times (preferably 7 times), filtering the extract, combining and concentrating the filtrates, dialysis, with the molecular weight cutoff of the dialysis bag being 3500 Da, after the dialysis fluid is concentrated, centrifuged and the precipitate is discarded, adding 3 to 8 times (preferably 4 times) of ethanol to the supernatant for alcohol precipitation, letting it stand overnight, centrifuging to obtain the precipitate, washing and drying to obtain crude gardenia polysaccharide.

[0028] In a specific embodiment, in step (1) above, the ethanol is an aqueous solution of ethanol with a content of 70% (v / v) or higher, preferably an aqueous solution of ethanol with a content of 85% (v / v) or higher, and more preferably an aqueous solution of ethanol with a content of 95% (v / v) or higher.

[0029] In one embodiment, in step (2), the gardenia crude polysaccharide prepared in step (1) is dissolved in 10 to 15 times (preferably 10 times) the weight of water.

[0030] In one embodiment, in step (2), the anion exchange column is a DEAE Sepharose. TM FastFlow.

[0031] In one embodiment, in step (2), the secondary elution fraction is concentrated, dialyzed (the molecular weight cutoff of the dialysis bag is 3500 Da), and freeze-dried to obtain the preliminarily separated secondary fraction;

[0032] In one embodiment, in step (2), the dextran gel column is a Sephacryl S-300HR gel column.

[0033] In one embodiment, in step (2), the secondary elution fraction is concentrated, centrifuged, and the supernatant is dialyzed (the molecular weight cutoff of the dialysis bag is 3500 Da). The supernatant is then freeze-dried to obtain the preliminarily separated secondary fraction. The secondary fraction is dissolved in 10 to 30 times (preferably 20 times) of its weight in 0.2 M NaCl aqueous solution, centrifuged, and the supernatant is eluted and separated by a Sephacryl S-300HR gel chromatography column with 0.2 M NaCl aqueous solution.

[0034] In one embodiment, in step (2), the secondary elution fraction is eluted and separated by a dextran gel column with a 0.2M NaCl aqueous solution, the separated fractions are collected and combined, concentrated, dialyzed (the molecular weight cutoff of the dialysis bag is 3500 Da), and freeze-dried.

[0035] In a specific embodiment, step (2) includes: dissolving the gardenia crude polysaccharide prepared in step (1) in 10 to 15 times (preferably 10 times) the weight of water, centrifuging, and using the supernatant to extract the supernatant using DEAE Sepharose. TM The Fast Flow anion exchange column was eluted sequentially with deionized water and 0.05M NaCl aqueous solution. The elution fractions of the 0.05M NaCl eluent were collected and combined, concentrated, centrifuged, and the supernatant was dialyzed (the molecular weight cutoff of the dialysis bag was 3500 Da). The supernatant was then freeze-dried to obtain the preliminarily separated secondary fraction. The secondary fraction was dissolved in 10 to 30 times its weight (preferably 20 times) of 0.2M NaCl aqueous solution, centrifuged, and the supernatant was eluted with 0.2M NaCl aqueous solution using a Sephacryl S-300HR gel chromatography column. The separated fractions were collected and combined, concentrated, dialyzed (the molecular weight cutoff of the dialysis bag was 3500 Da), and freeze-dried to finally obtain the dried powder of Gardenia polysaccharide.

[0036] The gardenia polysaccharide prepared according to the method of the present invention has the above-described structure and composition, which will not be repeated here.

[0037] Another aspect of the present invention provides a composition comprising the above-described gardenia polysaccharide or gardenia polysaccharide prepared by the above-described preparation method, or comprising a preventive and / or therapeutically effective amount of the above-described gardenia polysaccharide or gardenia polysaccharide prepared by the above-described preparation method as an active ingredient, wherein the composition optionally further comprises food, health product or pharmaceutically acceptable excipients.

[0038] In specific embodiments, the food, health product, or pharmaceutically acceptable excipients include, but are not limited to, carriers, excipients, adjuvants, and / or diluents.

[0039] In another aspect, the present invention provides the use of the above-mentioned gardenia polysaccharide or the gardenia polysaccharide prepared by the above-mentioned preparation method, or the above-mentioned composition, in the preparation of food, health products, or medicaments for the prevention and / or treatment of non-alcoholic steatohepatitis.

[0040] In this article, the concept of “prevention and / or treatment” means any measure applicable to the prevention and / or treatment of a state or disease associated with non-alcoholic steatohepatitis, or the prevention or treatment of such a state or disease or its symptoms, or the avoidance of further development of such a state or disease, such as the further development of the disease after the end of a treatment period or the treatment of symptoms of an already occurring disease, or the preemptive intervention to prevent, suppress or reduce the occurrence of such a disease or symptoms.

[0041] In a specific implementation, the non-alcoholic steatohepatitis is a disease related to lipid metabolism disorders caused by multiple factors such as liver cell damage.

[0042] In another aspect, the present invention provides the use of the above-described gardenia polysaccharide, or the gardenia polysaccharide prepared by the above-described preparation method, or the above-described composition, in the preparation of a drug or reagent having any one or more of the following effects:

[0043] (1) Inhibits lipid accumulation in hepatocytes;

[0044] (2) Inhibits damage to hepatocytes and ballooning lesions;

[0045] (3) Inhibit the fibrosis process in the later stage of non-alcoholic steatohepatitis.

[0046] In another aspect, the present invention provides a kit comprising the above-described gardenia polysaccharide, the gardenia polysaccharide prepared by the above-described preparation method, or the above-described composition. The kit can be used for:

[0047] (1) Inhibits lipid accumulation in hepatocytes;

[0048] (2) Inhibits damage to hepatocytes and ballooning lesions;

[0049] (3) Inhibit the fibrosis process in the later stage of non-alcoholic steatohepatitis.

[0050] In another aspect, the present invention provides a method for preventing and / or treating non-alcoholic steatohepatitis, the method comprising administering to a subject an effective amount of a composition selected from or containing the aforementioned polysaccharides.

[0051] The gardenia polysaccharide of the present invention can reduce lipid accumulation in primary hepatocytes of mice induced by FFA without significant cytotoxic effects, and can reduce liver fibrosis induced by CDAHFD diet, including restoring liver tissue morphology, reducing liver coefficient, and reducing the expression of proteins related to liver injury and fibrosis phenotype in animals, thus having a good effect in preventing and / or treating non-alcoholic steatohepatitis. Attached Figure Description

[0052] Figure 1 The characteristic high-performance gel permeation chromatogram (HPGPC) of gardenia polysaccharide GJ005S prepared in Example 1 (small peaks are solvent peaks).

[0053] Figure 2 The gardenia polysaccharide GJ005S prepared in Example 1 1 H NMR (A) and 13 C NMR (B) spectrum.

[0054] Figure 3 The structural repeating unit of gardenia polysaccharide GJ005S prepared in Example 1 is shown.

[0055] Figure 4 The images show the in vitro activity (A) and in vitro toxicity evaluation (B) of polysaccharide GJ005S in pharmacological examples 1 and 2.

[0056] Figure 5 The effects of Gardenia polysaccharide component GJ005S on liver tissue morphology (A) and liver tissue coefficient (B) in a non-alcoholic steatohepatitis model induced by a 60% high-fat, 0.1% methionine diet with choline deficiency (CDAHFD) in pharmacological Example 3 are shown; where *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

[0057] Figure 6 The following examples illustrate the effects of GJ005S on reducing liver fibrosis as observed by Sirius red staining in pharmacological example 4 (A), the effects of Gardenia polysaccharide GJ005S on the level of fibrosis-related proteins in mouse liver as detected by Western blotting in pharmacological example 5 (B), and the effects of Gardenia polysaccharide GJ005S on lipid accumulation in mouse liver as detected by Oil Red staining (C). Detailed Implementation

[0058] The present invention will be further described below with reference to embodiments. The following embodiments are described by way of example only. Obviously, those skilled in the art can make various modifications and variations to the present invention within the scope and spirit of the present invention.

[0059] In the following embodiments, high-performance gel permeation chromatography (HPGPC) was performed using Shodex SUGAR KS-804 (8.0 mm × 300 mm, Agilent Technologies, USA) and Shodex SUGAR KS-802 (8.0 mm × 300 mm, Agilent Technologies, USA) tandem columns to prepare standard curves using T-series standard dextran with different molecular weights;

[0060] High performance liquid chromatography (HPLC) was performed using an Agilent 1260 Seri high performance liquid chromatography system (Agilent Technologies, Inc., USA).

[0061] Nuclear magnetic resonance analysis was performed using a Brucker AM-500 nuclear magnetic resonance spectrometer (Brucker GmbH, Germany).

[0062] Molecular weight standards (Dextran T-700, T-580, T-500, T-110, T-80, T-70, T-40, T-11, T-9.3, T-6, T-4 series) were purchased from Pharmacia (Sweden); monosaccharide standards were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; 95% industrial ethanol, sulfuric acid, phenol, sodium chloride, sodium nitrate, anhydrous ethanol and acetone were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0063] Example 1: Extraction, isolation, purification and structural characterization of Gardenia polysaccharide GJ005S

[0064] (1) Extraction of polysaccharides

[0065] Two kg of dried gardenia fruit (purchased from Bozhou Traditional Chinese Medicine Slices Factory, Anhui Province) was extracted by soaking in 40 L of boiling water for 3 hours each time, for a total of 7 extractions. The extracts were combined, filtered through medical degreased gauze (China National Pharmaceutical Group Chemical Reagent Co., Ltd.), and the filtrates were combined and concentrated to a small volume. The solution was then dialyzed against running water for 48 hours (the molecular weight cutoff of the dialysis bag was 3500 Da). The dialysate was concentrated, centrifuged (8000 rpm, 10 min), and the precipitate was discarded. The extract was then precipitated with 95% ethanol at a volume ratio of 1:4 (v / v) and allowed to stand overnight. The excess ethanol on top was discarded, the precipitate was centrifuged, washed, and freeze-dried to obtain gardenia water-extracted crude polysaccharide GJ (yield 5.8%).

[0066] (2) Isolation and purification of polysaccharides

[0067] Each time, dissolve 8g of crude polysaccharide in 80mL of deionized water, stir overnight, centrifuge, and load the supernatant onto DEAE Sepharose. TM In a Fast Flow anion exchange column, elution was performed sequentially with deionized water and 0.05 M NaCl aqueous solution at a flow rate of 1 mL / min. 100 μL of the solution was subjected to sulfuric acid-phenol staining, and its absorbance was measured at 490 nm using a microplate reader. Elution curves were plotted using absorbance and elution volume. Based on the elution curves, the separated polysaccharides were collected, concentrated under reduced pressure, centrifuged, and the supernatant was dialyzed (molecular weight cutoff 3500 Da). Finally, the eluent was lyophilized to obtain the eluted fraction GJ005 (secondary polysaccharide, yield 2.16%) from the 0.05 M NaCl eluent.

[0068] Each time, 200 mg of the secondary polysaccharide GJ005 was dissolved in 4 mL of 0.2 M NaCl aqueous solution, centrifuged (4000 r / min), and the supernatant was loaded onto a Sephacryl S-300HR gel column. Elution was performed with 0.2 M NaCl eluent at a flow rate of 5 mL / 15 min using an automated collector. After colorimetric analysis using the sulfuric acid-phenol method, absorbance was measured using an ELISA reader, and elution curves were plotted. The desired fraction was collected, concentrated, and dialyzed (molecular weight cutoff 3500 Da). Finally, it was freeze-dried to obtain homogeneous gardenia polysaccharide, named GJ005S (yield of secondary fraction GJ005 was 29%).

[0069] (3) Structural identification of polysaccharides

[0070] A. In high-performance gel permeation chromatography (HPGPC) analysis, Shodex SUGAR KS-804 and Shodex SUGAR KS-802 columns were used in series. A 0.1M NaNO3 aqueous solution was used as the mobile phase, the flow rate was 0.5 mL / min, the column temperature was set to 40℃, and a differential detector (RID) with a detection temperature of 35℃ was used. The injection volume was 10 μL. The sample was dissolved in the mobile phase to obtain a 10 mg / mL polysaccharide solution for detection. HPGPC detection showed GJ005S as a symmetrical sharp peak (e.g., ...). Figure 1 As shown in the figure, its relative molecular mass is 17.1 kDa.

[0071] The monosaccharide composition was determined by HPLC. The flow rate was set at 1 mL / min, the column temperature at 25 °C, the UV detection wavelength at 254 nm, and the sample injection volume at 10 μL / sample. The results showed that the molar ratio of rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, and arabinose in the polysaccharide composition was 7.6:3.7:9.4:2.2:26.8:50.3.

[0072] B. Take 35 mg of Gardenia polysaccharide GJ005S sample, dissolve it in 0.5 mL of D2O, add 1.5 μL of acetone as an internal standard (δH = 2.29 ppm, δC = 31.5 ppm), and measure the one-dimensional NMR spectrum at 25 °C using a Bruker AVANCE III 500M NMR spectrometer. The results are as follows. Figure 2 As shown. Figure 2 In the middle, A represents polysaccharide. 1 H NMR spectrum, signals in the anomeric hydrogen region are δ5.23, δ5.16, δ5.25, δ5.18, δ5.28, δ5.32, δ4.66, δ5.12, δ5.15, δ5.1, δ4.65, δ4.68, δ4.53, and δ4.54 respectively belong to T-α-Araf; 1,5-α-Araf; 1,3-α-Araf; 1,3, 5-α-Araf; T-α-Rhap; 1,2 / 1,2,4-α-Rhap; T-β-GalpA; 1,4-α-GalpA; 1,3,4-α-GalpA; 1,4-α-GlcpA; T-α-Glcp; T-β-Galp; 1,4-β-Galp; 1,6-β-Galp and 1,3,6-β-Galp. These signals correspond to 13 C NMR spectrum ( Figure 2 Characteristic signal peaks in B): δ109.7, δ108.31, δ108.6, δ108.47, δ101.75, δ99.77, δ97.4, δ100.29, δ100.34, δ98.85, δ99.74, δ105.62, δ105.46, δ104.42 and δ104.33.

[0073] The above results indicate that the polysaccharide GJ005S is characterized by alternating 1→2 linked α-rhamnose and 1→4 linked α-galacturonic acid; the side chains are linked at C-4 of the 1→2 linked α-rhamnose (1,2-α-Rha) and C-3 of the 1→4 linked α-galacturonic acid (1,4-α-GalA) in the main chain. The side chains include T-, 1,5-, 1,3-, and 1,3,5-linked α-arabinose; T-, 1,4-, 1,6-, and 1,3,6-linked β-galactose; 1,4-linked α-glucuronic acid; and small amounts of terminally linked α-rhamnose, α-galacturonic acid, and α-glucose. Figure 3 The structural repeating unit of gardenia polysaccharide GJ005S is shown.

[0074] Pharmacological Example 1: Detection of the effect of Gardenia polysaccharide GJ005S on a cell model of free fatty acid (FFA)-induced non-alcoholic steatohepatitis using Oil Red staining.

[0075] Primary hepatocytes extracted from the livers of C57BL / 6 mice (purchased from Shanghai Lingchang Biotechnology Co., Ltd.) using collagenase digestion and density gradient centrifugation were divided into a control group, a model group, and a Gardenia polysaccharide GJ005S group. Cells treated with 1% fatty acid-free BSA served as the control group. The model group was treated with free fatty acids (FFA) (prepared by mixing palmitic acid and oleic acid in a 1:2 ratio to form a 0.3mM / 0.6mM solution) for 12 to 16 hours to induce a NASH cell model with lipid accumulation. The GJ005S group was treated with Gardenia polysaccharide GJ005S (concentrations of 0.1, 0.5, 0.75, and 1 mg / mL) for 16 hours, and neutral lipid droplets in the cells were stained in the dark using an Oil Red staining kit (purchased from Beyotime Biotechnology Co., Ltd.).

[0076] Oil red staining results showed that after treatment with gardenia polysaccharide GJ005S, the proportion of red-stained lipid droplets in the FFA-induced group cells was significantly reduced compared with the model group. Figure 4 (A) suggests that Gardenia polysaccharide GJ005S can significantly inhibit lipid accumulation in primary mouse hepatocytes.

[0077] Pharmacological Example 2: Safety test of Gardenia polysaccharide GJ005S on human hepatocytes

[0078] To confirm the safety of gardenia polysaccharide GJ005S, the effects of different concentrations of gardenia polysaccharide GJ005S on the proliferation of human hepatocytes (human hepatocyte cell line LO2 was purchased from the cell bank of the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences) were further investigated. Specifically, logarithmically growing human hepatocyte LO2 cells were seeded in 96-well plates at a density of 3 × 10⁶ cells per well. 3Cells were then treated with different concentrations of gardenia polysaccharide GJ005S for 24 hours. Cell proliferation was assessed using a CCK8 assay. The results showed that different concentrations of gardenia polysaccharide GJ005S did not reduce cell viability, indicating that the polysaccharide reduced lipid accumulation in hepatocytes without significant cytotoxicity. Figure 4 (B)

[0079] Pharmacological Example 3: Effects of Gardenia polysaccharide component GJ005S on liver morphology and liver tissue coefficient in a non-alcoholic steatohepatitis model induced by a 60% high-fat, 0.1% methionine diet with choline deficiency (CDAHFD).

[0080] This animal experiment followed the guidelines for the management and use of laboratory animals and was approved by the Animal Ethics Committee of the Shanghai Institute of Materia Medica, Chinese Academy of Sciences. Fifty-one 6-week-old male C57BL / 6 mice were purchased from Shanghai Lingchang Biotechnology Co., Ltd. The animals were randomly divided into three groups: a control group (n=8), a model group (n=8), and an experimental group (n=35). They were provided with free access to food and water, at a room temperature of 22±1℃, a relative humidity of 65±5%, and a 12-hour light cycle. The model and experimental groups were fed CDAHFD diet (purchased from Shuyishuer Biotechnology Co., Ltd.) for 8 weeks. Eight mice were administered an equal volume of 0.9% physiological saline by gavage as a normal control group (Chow). After 2 weeks of diet induction, the CDAHFD-fed animals were randomly divided into four groups: A, B, C, D, and E. Group A was the model group. Group B (n=8) received 6.25 mg / kg GJ005S polysaccharide, Group C (n=9) received 25 mg / kg GJ005S polysaccharide, Group D (n=9) received 100 mg / kg GJ005S polysaccharide, and Group E (n=9) received 5 mg / kg retinoic acid (purchased from MCE) as a positive control. All mice were administered the drugs daily by gavage for 6 weeks. The control and model groups received an equal volume of physiological saline. After 6 weeks of administration, the mice were sacrificed, and the morphology and weight of their liver tissue were observed. Results are as follows: Figure 5 As shown in Figures A and B, in mice with non-alcoholic steatohepatitis (NASH) treated with GJ005S polysaccharide, the liver tissue surface returned to a smooth, rosy, and shiny appearance, but the texture was not as soft and elastic as that of the control group. The liver coefficient (liver tissue weight / mouse body weight) was increased in the model group, while the liver coefficient decreased in the low (6.25 mg / kg), medium (25 mg / kg), and high (100 mg / kg) dose groups of GJ005S, showing significant differences compared to the CDAHFD-induced NASH model group.

[0081] Pharmacological Example 4: Sirius red staining to observe the effect of GJ005S on reducing liver fibrosis

[0082] The specific steps of the Sirius red staining experiment are as follows: ① Dewaxing paraffin sections to water: Immerse paraffin sections in xylene for 20 min, twice. Then immerse in anhydrous ethanol for 5 min, twice. Immerse in 75% ethanol for 5 min, and wash with tap water. ② Sirius red staining: Immerse sections in Sirius red staining solution for 8-10 min, and dehydrate with anhydrous ethanol 2-3 times. ③ Mounting: Immerse sections in xylene for 5 min to clear, and mount with neutral resin. ④ Use a tissue imaging analyzer to perform full-scan image acquisition and analysis. Under an optical microscope, collagen fibers appear red against a yellow background. The Sirius red staining results show that the negative control group mice had no collagen deposition, while the model group mice had severe collagen deposition in the vascular and portal areas. GJ005S administration significantly reduced collagen deposition. Figure 6 (A). This indicates that Gardenia polysaccharide GJ005S can effectively alleviate liver fibrosis lesions, reduce fibrosis, and slow down the progression of liver fibrosis.

[0083] Pharmacological Example 5: Western blot assay to detect the effect of Gardenia polysaccharide GJ005S on the level of fibrosis phenotype-related proteins in mouse liver.

[0084] Six weeks after drug administration, mice were sacrificed, and proteins were extracted from partial liver tissue using a tissue extraction kit (purchased from Sangon Biotech Co., Ltd.). After adding 5× loading buffer, the proteins were denatured at 95°C for 10 min in a metal heater, cooled, and stored at -80°C. Western blotting was used to detect α-smooth muscle actin protein expression. Results are as follows: Figure 6 As shown in Figure B, Gardenia polysaccharide GJ005S can inhibit the expression of proteins associated with the fibrosis phenotype.

[0085] Pharmacological Example 6: Oil Red Staining Experiment to Detect the Effect of Gardenia Polysaccharide GJ005S on Lipid Accumulation in Mouse Liver

[0086] The specific procedures for Oil Red O staining of liver fat sections are as follows: ① OCT-embedded liver tissue is prepared into 10μm thick sections using a cryostat. ② Preparation of Oil Red O staining working solution: Oil Red O isopropanol saturated stock solution and ddH2O are mixed at a ratio of 3:2 to prepare the working solution (prepare fresh before use). The frozen liver tissue sections are stained with Oil Red O staining working solution for 10-12 minutes. ③ Wash twice with double-distilled water for 20 seconds each time. ④ Wash with running water for 10 minutes. ⑤ Stain the nuclei with hematoxylin for 45 seconds. Repeat the rinsing with water. ⑥ Mount with 50% glycerol, observe the staining of the liver tissue sections under a microscope, and take photographs. Oil Red staining results show that in the negative control group, mouse liver cells are neatly arranged, with uniform cytoplasm and no lipid deposition. In the CDAHFD model group, mouse liver cells are swollen with a large number of lipid droplets deposited intracellularly. After GJ005S administration, the lipid droplet deposition in the liver is significantly reduced compared to the model group. Figure 6 (C)

[0087] In summary, Gardenia polysaccharide GJ005S reduced lipid accumulation in FFA-induced primary hepatocytes of mice without significant cytotoxicity. GJ005S also reduced CDAHFD-induced liver fibrosis, including restoring liver tissue morphology, reducing liver coefficient, and decreasing the expression of proteins associated with liver injury and fibrosis phenotypes in animals. Therefore, this polysaccharide component GJ005S may be a potential carbohydrate-based drug for the prevention and / or treatment of non-alcoholic steatohepatitis (NAH).

[0088] The above descriptions are merely embodiments of the present invention, and common knowledge regarding specific structures and characteristics is not elaborated upon here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the structure of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A gardenia polysaccharide comprising the following monosaccharide units: rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, and arabinose; The main chain structure of the gardenia polysaccharide consists of alternating 1→2 linked α-rhamnose and 1→4 linked α-galacturonic acid; the side chains are linked at C-4 of the 1→2 linked α-rhamnose and C-3 of the 1→4 linked α-galacturonic acid in the main chain. The side chains include T-, 1, 5-, 1, 3-, and 1, 3, 5- linked α-arabinose; T-, 1, 4-, 1, 6-, and 1, 3, 6- linked β-galactose; 1, 4- linked α-glucuronic acid; and α-rhamnose, α-galacturonic acid, and α-glucose linked at the ends. The molar ratio of rhamnose, glucuronic acid, galacturonic acid, glucose, galactose and arabinose in the gardenia polysaccharide is 3~15 : 1~8 : 4~18 : 1~6 : 15~35 : 50.

3.

2. The gardenia polysaccharide according to claim 1, wherein, The weight-average molecular weight of the gardenia polysaccharide is in the range of 2 to 100 kDa.

3. The gardenia polysaccharide according to claim 1, wherein, The weight-average molecular weight of the gardenia polysaccharide is in the range of 10~25 kDa.

4. The gardenia polysaccharide according to claim 1, wherein, The weight-average molecular weight of the gardenia polysaccharide is 17.1 kDa.

5. The gardenia polysaccharide according to claim 1 or 2, wherein, The molar ratio of rhamnose, glucuronic acid, galacturonic acid, glucose, galactose and arabinose is 7.6 : 3.7 : 9.4 : 2.2 : 26.8 : 50.

3.

6. The gardenia polysaccharide according to claim 1 or 2, wherein, The gardenia polysaccharide has the following repeating structural units: 。 7. A method for preparing gardenia polysaccharide according to any one of claims 1 to 6, comprising the following steps: (1) Extraction of polysaccharides: Gardenia was extracted with boiling water to obtain an extract. The extract was concentrated and then dialyzed. The molecular weight cutoff of the dialysis bag was 3500 Da. Ethanol was used for precipitation, and the precipitate was collected to obtain crude polysaccharides from gardenia. (2) Purification of polysaccharides: The crude polysaccharide of Gardenia is dissolved in water and eluted sequentially with deionized water and 0.05 M NaCl solution through an anion exchange column. The elution fraction of the 0.05 M NaCl eluent is collected as the secondary elution fraction. The secondary eluted fraction was then purified by elution using a dextran gel column. The collected eluted fraction was concentrated, dialyzed against water, and lyophilized to obtain gardenia polysaccharide.

8. The method for preparing gardenia polysaccharide according to claim 7, wherein, In step (1), During extraction, the amount of water used should be 15 to 30 times that of gardenia; and / or Extraction time is 0.5–4 hours; extraction times are 1–10; and / or The ethanol is an aqueous solution of ethanol with a concentration of 70% (v / v) or higher.

9. The method for preparing gardenia polysaccharide according to claim 7, wherein, In step (1), During extraction, the amount of water used is 20 times that of gardenia; and / or Extraction time is 1-3 hours; extraction times are 7 times; and / or The ethanol is an aqueous solution of ethanol with a concentration of 85% (v / v) or higher.

10. The method for preparing gardenia polysaccharide according to claim 9, wherein, The ethanol is an aqueous solution of ethanol with a concentration of 95% (v / v) or higher.

11. The method for preparing gardenia polysaccharide according to claim 7, wherein, Step (1) includes: taking dried gardenia, adding 15 to 30 times its weight of water, extracting with boiling water for 0.5 to 4 hours, extracting 1 to 10 times in total, filtering the extract, combining the filtrates and concentrating, dialysis, the molecular weight cutoff of the dialysis bag is 3500 Da, after the dialysis liquid is concentrated, centrifuged and the precipitate is discarded, 3 to 8 times the volume of the supernatant is added to the supernatant for alcohol precipitation, standing overnight, centrifuging to obtain the precipitate, washing and drying to obtain gardenia crude polysaccharide.

12. The method for preparing gardenia polysaccharide according to claim 7 or 8, wherein, In step (2), Dissolve the crude gardenia polysaccharide prepared in step (1) in 10-15 times its weight of water; and / or The anion exchange column is DEAE Sepharose™ Fast Flow; and / or The secondary elution fraction is concentrated, dialyzed, and freeze-dried to obtain a preliminarily separated secondary fraction; and / or The dextran gel column is a Sephacryl S-300 HR gel column; and / or The secondary elution fraction was eluted and separated by a dextran gel column with 0.2 M NaCl aqueous solution. The separated fractions were collected, concentrated, dialyzed, and freeze-dried.

13. The method for preparing gardenia polysaccharide according to claim 7 or 8, wherein, In step (2), Dissolve the crude gardenia polysaccharide prepared in step (1) in 10 times its weight of water; and / or The anion exchange column is DEAE Sepharose™ Fast Flow; and / or The secondary eluent was concentrated, dialyzed, with a molecular weight cutoff of 3500 Da in the dialysis bag, and freeze-dried to obtain the preliminarily separated secondary fraction; and / or The dextran gel column is a Sephacryl S-300 HR gel column; and / or The secondary elution fraction was eluted and separated by a dextran gel column with 0.2 M NaCl aqueous solution. The separated fractions were collected and combined, concentrated, dialyzed, and the molecular weight cutoff of the dialysis bag was 3500 Da. The fractions were then freeze-dried.

14. The method for preparing gardenia polysaccharide according to claim 7 or 8, wherein, Step (2) includes: dissolving the crude gardenia polysaccharide prepared in step (1) in 10-15 times its weight of water, centrifuging, and eluting the supernatant sequentially with deionized water and 0.05 M NaCl aqueous solution using a DEAE Sepharose™ Fast Flow anion exchange column. Collecting and combining the elution fractions of the 0.05 M NaCl eluent, concentrating them, centrifuging, dialyzing the supernatant, setting the molecular weight cutoff of the dialysis bag to 3500 Da, and freeze-drying to obtain the preliminarily separated secondary fractions. The secondary fractions are dissolved in 10-30 times their weight of 0.2 M NaCl aqueous solution, centrifuged, and the supernatant is eluted and separated using a Sephacryl S-300 HR gel chromatography column with 0.2 M NaCl aqueous solution. Collecting and combining the separated fractions, concentrating them, dialyzing, setting the molecular weight cutoff of the dialysis bag to 3500 Da, and freeze-drying to finally obtain the dried powder of the gardenia polysaccharide.

15. A composition comprising gardenia polysaccharide according to any one of claims 1 to 6 or gardenia polysaccharide prepared by the preparation method according to any one of claims 7 to 14, or comprising a preventive and / or therapeutically effective amount of gardenia polysaccharide according to any one of claims 1 to 6 or gardenia polysaccharide prepared by the preparation method according to any one of claims 7 to 14 as an active ingredient, the composition comprising pharmaceutically acceptable excipients.

16. Use of the gardenia polysaccharide according to any one of claims 1 to 6, or the gardenia polysaccharide prepared by the preparation method according to any one of claims 7 to 14, or the composition according to claim 15 in the preparation of a medicament for the prevention and / or treatment of non-alcoholic steatohepatitis.

17. The use of gardenia polysaccharide according to any one of claims 1 to 6, or gardenia polysaccharide prepared by the preparation method according to any one of claims 7 to 14, or the use of the composition according to claim 15 in the preparation of a drug or reagent having any one or more of the following effects: (1) Inhibits lipid accumulation in hepatocytes; (2) Inhibit the fibrosis process in the later stage of non-alcoholic steatohepatitis.

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