Gardenia polysaccharide as well as preparation method and application thereof

Abstract

The invention relates to gardenia polysaccharide as well as a preparation method and application thereof. The gardenia polysaccharide comprises the following monosaccharide units: rhamnose, glucuronic acid, galacturonic acid, glucose, galactose and arabinose, or mainly comprises rhamnose, glucuronic acid, galacturonic acid, glucose, galactose and arabinose; the molar ratio of rhamnose to glucuronic acid to galacturonic acid to glucose to galactose to arabinose in the gardenia polysaccharide is (3-15): (1-8): (4-18): (1-6): (15-35): (25-70). According to the gardenia polysaccharide, FFA induced mouse primary parenchymal hepatic cell lipid accumulation can be reduced, no obvious cytotoxic effect exists, CDAHFD diet induced hepatic fibrosis can be reduced, and the gardenia polysaccharide has a good effect of preventing and / or treating non-alcoholic steatohepatitis.

Description

Technical Field

[0001] The present invention belongs to the field of polysaccharides, and relates to a polysaccharide derived from gardenia, a preparation method and use thereof, and specifically relates to a polysaccharide extracted from gardenia jasminoides, a preparation method thereof, and use thereof in preparing an agent or a drug for inhibiting, preventing and / or treating non-alcoholic fatty hepatitis. Background Art

[0002] Nonalcoholic steatohepatitis is a type of liver abnormality characterized by liver fat accumulation, liver damage, inflammation, and liver fibrosis. Progressive nonalcoholic steatohepatitis can easily develop into liver fibrosis, and 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 nonalcoholic steatohepatitis, marking the first ray of light after more than 40 years of research and development. However, its drug development is still in the cold winter, so the development of active compounds and drug development around nonalcoholic steatohepatitis is very important.

[0003] Gardenia jasminoides is one of the 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 acids, trace elements and polysaccharides. Its dried fruits are used as traditional medicines in Asian countries and have high medicinal value. They have anti-inflammatory, diuretic, laxative, bile and homeostatic effects, and can also relieve liver and abdominal pain. At present, many systematic studies have been conducted on compounds derived from gardenia from the aspects of structure, function and extraction methods, and many related articles have been published. Studies have shown that gardenia polysaccharides can improve cholestatic liver injury by alleviating intestinal flora imbalance; gardenia water extract and its main components can resist thrombosis; an iridoid glycoside derived from gardenia can protect mice from lipopolysaccharide-induced acute lung injury; gardenia's traditional Chinese medicine extract gardenia glycoside can play an anti-inflammatory and protective role on cardiomyocytes; gardenia glycoside can play a potential antidepressant role by regulating the hypothalamus-pituitary-adrenal axis, etc. In traditional Chinese medicine theory, Gardenia belongs to the Liver Meridian. Gardenia has a long history of being used to treat liver disease in Chinese medicine theory. Prescriptions (such as Artemisia Capillaris Decoction, Gardenia and Rhubarb Decoction, Longdan Xiegan Decoction, Gardenia and Baipi Decoction, etc.) and Chinese patent medicines (such as Yinchenhuang Oral Liquid, Longdan Xiegan Pills, Qinggan Lidan Oral Liquid, Shuganning Injection, etc.) used clinically to treat liver diseases all contain Gardenia. On this basis, the inventors attempted to explore the function of Gardenia polysaccharides in non-alcoholic fatty hepatitis. Summary of the invention

[0004] The present invention adopts a simple and effective polysaccharide extraction method to obtain a uniform pectin-like polysaccharide GJ005S using Gardenia jasminoides as raw material. Pharmacological experiments show that polysaccharide GJ005S can reduce lipid accumulation in primary hepatocytes, alleviate fat accumulation and liver damage in diet-induced NASH mice, and inhibit the development of fibrosis. Therefore, the polysaccharide has the potential to inhibit non-alcoholic fatty hepatitis and is expected to be developed into a carbohydrate drug for treating non-alcoholic fatty hepatitis.

[0005] Based on this, an 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 jasminoides polysaccharide.

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

[0009] Another object of the present invention is to provide the use of the Gardenia jasminoides polysaccharide in the preparation of a medicament for preventing and / or treating non-alcoholic fatty liver disease.

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

[0011] The main chain structure of the gardenia polysaccharide is that α-rhamnose and α-galacturonic acid connected in 1→2 manner are connected alternately; the side chain is connected to C-4 of α-rhamnose connected in 1→2 manner and C-3 of α-galacturonic acid connected in 1→4 manner in the main chain, and the side chain includes T-, 1,5-, 1,3-, 1,3,5-connected α-arabinose; T-, 1,4-, 1,6- and 1,3,6-connected β-galactose; 1,4-connected α-glucuronic acid; and the terminal is connected with α-rhamnose, α-galacturonic acid and α-glucose;

[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 further 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 of the high performance gel permeation chromatography are: the chromatographic columns are Shodex SUGAR KS-804 and ShodexSUGAR KS-802 used in series; the mobile phase is 0.1M NaNO3 aqueous solution; the flow rate is 0.5mL / min, the column temperature is 40°C, the detector is a differential detector RID, and the detection temperature is 35°C.

[0016] In a specific embodiment, the Gardenia polysaccharide is GJ005S, which 1 In the H 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; 13 Characteristic signal peaks in the C NMR spectrum: 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 nuclear magnetic spectrum of the Gardenia polysaccharide GJ005S are consistent with Figure 2 As shown in A 1 H NMR spectroscopy and / or Figure 2 As shown in B 13 The C NMR spectra were basically consistent.

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

[0018]

[0019] Another aspect of the present invention provides a method for preparing Gardenia jasminoides polysaccharide, comprising the following steps:

[0020] (1) Extraction of polysaccharides: extracting gardenia with boiling water to obtain an extract, concentrating the extract, precipitating with ethanol, and collecting the precipitate to obtain crude gardenia polysaccharides;

[0021] (2) Purification of polysaccharides: The crude Gardenia polysaccharide is dissolved in water, and eluted with deionized water and 0.05 M NaCl solution in sequence through an anion exchange column, and the elution fraction of the 0.05 M NaCl eluate is collected as a secondary elution fraction; the secondary elution fraction is then eluted and purified through a dextran gel column, and the collected elution fractions are concentrated, dialyzed against water, and freeze-dried to obtain Gardenia polysaccharide.

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

[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, and the molecular weight cutoff of the dialysis bag is 3500Da.

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

[0026] In one embodiment, the step (1) comprises: extracting Gardenia jasminoides with boiling water, concentrating and dialyzing the obtained extract, the molecular weight cut-off of the dialysis bag is 3500Da, concentrating and centrifuging the dialyzed liquid to discard the precipitate, precipitating the obtained supernatant with ethanol, separating, washing and drying the precipitate to obtain Gardenia jasminoides crude polysaccharide;

[0027] In another embodiment, the step (1) comprises: 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) in total, filtering the extract, combining and concentrating the filtrates, dialyzing, the molecular weight cutoff of the dialysis bag is 3500Da, concentrating the dialyzed liquid, centrifuging and discarding the precipitate, adding 3 to 8 times (preferably 4 times) of ethanol in the volume of the supernatant to the supernatant for alcohol precipitation, standing overnight, centrifuging to obtain the precipitate, washing and drying to obtain crude gardenia polysaccharide.

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

[0029] In one embodiment, in step (2), the crude Gardenia jasminoides 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 DEAE Sepharose TM FastFlow.

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

[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 component is concentrated and centrifuged, the supernatant is dialyzed (dialysis bag molecular weight cutoff is 3500Da), and freeze-dried to obtain a preliminarily separated secondary component, the secondary component is dissolved in 10 to 30 times (preferably 20 times) the weight of 0.2M NaCl aqueous solution, centrifuged, and the supernatant is passed through a Sephacryl S-300HR gel chromatography column and eluted and separated with 0.2M NaCl aqueous solution.

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

[0035] In a specific embodiment, the step (2) comprises: adding 10 to 15 times (preferably 10 times) the weight of the Gardenia jasminoides polysaccharide prepared in step (1) to dissolve it in water, centrifuging it, and separating the supernatant with DEAE Sepharose TM The Fast Flow anion exchange column is eluted with deionized water and 0.05M NaCl aqueous solution in sequence, and the eluted components of the 0.05M NaCl eluate are collected and combined, concentrated, centrifuged, and the supernatant is dialyzed (the molecular weight cutoff of the dialysis bag is 3500Da), and freeze-dried to obtain a preliminarily separated secondary component; the secondary component is dissolved in 10 to 30 times (preferably 20 times) the weight of a 0.2M NaCl aqueous solution, centrifuged, and the supernatant is passed through a Sephacryl S-300HR gel chromatography column, eluted and separated with a 0.2M NaCl aqueous solution, and the separated components are collected and combined, concentrated, dialyzed (the molecular weight cutoff of the dialysis bag is 3500Da), and freeze-dried to finally obtain the dry powder of the gardenia polysaccharide.

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

[0037] Another aspect of the present invention provides a composition, which comprises the above-mentioned Gardenia polysaccharide or the Gardenia polysaccharide prepared by the above-mentioned preparation method, or comprises a preventive and / or therapeutic effective amount of the above-mentioned Gardenia polysaccharide or the Gardenia polysaccharide prepared by the above-mentioned preparation method as an active ingredient, and the composition optionally further comprises a food, health product or pharmaceutically acceptable excipient.

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

[0039] Another aspect of 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 care products or drugs for preventing and / or treating non-alcoholic fatty hepatitis.

[0040] The concept of "prevention and / or treatment" herein refers to any measure applicable to the prevention and / or treatment of non-alcoholic fatty liver disease-related conditions or diseases, or the prevention or treatment of such conditions or diseases or the symptoms manifested, or the avoidance of further development of such conditions or diseases, such as the further development of the disease after the end of the treatment period or the treatment of the symptoms of an already developed disease, or the pre-intervention prevention, inhibition or reduction of the occurrence of such diseases or symptoms.

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

[0042] In another aspect of the present invention, there is provided use of the Gardenia polysaccharide or the Gardenia polysaccharide prepared by the preparation method or the composition in preparing a drug or reagent having any one or more of the following effects:

[0043] (1) Inhibit lipid accumulation in hepatic parenchymal cells;

[0044] (2) Inhibit liver parenchymal cell damage and ballooning lesions;

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

[0046] In another aspect of the present invention, a kit is provided, which comprises the above-mentioned Gardenia polysaccharide, the Gardenia polysaccharide prepared by the above-mentioned preparation method, or the above-mentioned composition. The kit can be used for:

[0047] (1) Inhibit lipid accumulation in hepatic parenchymal cells;

[0048] (2) Inhibit liver parenchymal cell damage and ballooning lesions;

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

[0050] In another aspect of the present invention, a method for preventing and / or treating non-alcoholic steatohepatitis is provided, which comprises administering to a subject an effective amount of the polysaccharide selected from the above polysaccharide or a composition containing the polysaccharide.

[0051] The Gardenia polysaccharide according to the present invention can reduce the lipid accumulation of primary hepatocytes of mice induced by FFA without obvious cytotoxic effect, and can reduce the liver fibrosis induced by CDAHFD diet, including restoring the morphology of liver tissue, reducing the liver coefficient, and reducing the expression of proteins related to animal liver damage and fibrosis phenotype, and has a good effect in preventing and / or treating non-alcoholic fatty hepatitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 This is the characteristic high performance gel permeation chromatogram (HPGPC) of the Gardenia polysaccharide GJ005S prepared in Example 1 (the small peak is the solvent peak).

[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 the Gardenia polysaccharide GJ005S prepared in Example 1 is shown.

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

[0056] Figure 5 The effect of Gardenia polysaccharide component GJ005S on mouse liver tissue morphology (A) and liver tissue coefficient (B) in the non-alcoholic fatty hepatitis model induced by a 60% high-fat, 0.1% methionine, choline-deficient (CDAHFD) diet in pharmacological example 3 is shown; where *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.

[0057] Figure 6 The results show the effect of GJ005S on reducing liver fibrosis observed by Sirius red staining in Pharmacological Example 4 (A), the effect of Gardenia polysaccharide GJ005S on the level of proteins related to fibrosis phenotype in mouse liver detected by protein immunoblotting experiment in Pharmacological Example 5 (B), and the effect of Gardenia polysaccharide GJ005S on lipid accumulation in mouse liver detected by Oil red staining experiment (C). DETAILED DESCRIPTION

[0058] The present invention is further described below in conjunction with the embodiments, and the following embodiments describe the present invention only by way of example. Obviously, those skilled in the art can make various modifications and variations to the present invention within the scope and essence of the present invention.

[0059] In the following embodiments, high performance gel permeation chromatography (HPGPC) uses Shodex SUGAR KS-804 (8.0 mm×300 mm, Agilent, USA) and Shodex SUGAR KS-802 (8.0 mm×300 mm, Agilent, USA) columns connected in series to prepare a standard curve using T-series standard dextran (Dextran) of different molecular weights;

[0060] High performance liquid chromatography (HPLC) was determined using Agilent 1260Seri high performance liquid chromatography system (Agilent, USA);

[0061] The nuclear magnetic resonance analysis was performed using a Brucker AM-500 nuclear magnetic resonance instrument (Brucker, 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, separation, purification and structural characterization of Gardenia polysaccharide GJ005S

[0064] (1) Extraction of polysaccharides

[0065] 2kg of dried Gardenia (purchased from Anhui Bozhou Chinese Medicinal Materials Decoction Factory) was soaked and extracted with 40L boiling water, each extraction was 3h, and a total of 7 extractions were performed. The extracts were combined, filtered with medical degreased gauze (Sinopharm Group Chemical Reagent Co., Ltd.), and the filtrates were combined and concentrated to a small volume, and then dialyzed against flowing water for 48h (the molecular weight cutoff of the dialysis bag was 3500Da). The dialyzed liquid was concentrated and centrifuged (8000rpm, 10min) to discard the precipitate, and then precipitated with 95% ethanol in a volume ratio of 1:4 (v / v) to the extract and ethanol, and allowed to stand overnight. The excess ethanol in the upper layer was discarded, and the precipitate was obtained by centrifugation. After washing and freeze-drying, the crude polysaccharide GJ extracted from Gardenia was obtained (the yield was 5.8%).

[0066] (2) Separation and purification of polysaccharides

[0067] Each time, 8 g of crude polysaccharide was dissolved in 80 mL of deionized water, stirred overnight, centrifuged, and the supernatant was loaded onto DEAE Sepharose. TM In the Fast Flow anion exchange column, deionized water and 0.05M NaCl aqueous solution were used for elution in turn, the flow rate was controlled at 1mL / min, 100μL was taken for color development using the sulfuric acid-phenol method and its absorbance was detected at 490nm using an ELISA reader, and the elution curve was drawn using the absorbance and elution volume. According to the elution curve, the separated polysaccharides were collected and concentrated under reduced pressure, centrifuged, the supernatant was dialyzed (molecular weight cutoff 3500Da), and freeze-dried, and finally the elution component GJ005 (secondary polysaccharide, yield 2.16%) of the 0.05M NaCl eluent was obtained.

[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 on a Sephacryl S-300HR gel column, eluted with 0.2 M NaCl eluent, the flow rate was controlled at 5 mL / 15 min, and collected by an automatic collector. After color development using sulfuric acid phenol method, absorbance detection by an enzyme marker, and elution curve plotted, the desired separation components were collected for concentration and dialysis (molecular weight cutoff 3500 Da), and finally freeze-dried to obtain a uniform Gardenia polysaccharide, named GJ005S (the yield of the secondary component GJ005 was 29%).

[0069] (3) Structural identification of polysaccharides

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

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

[0072] B. Take 35 mg of Gardenia polysaccharide GJ005S sample, add 0.5 mL of D2O to dissolve, add 1.5 μL of acetone as internal standard (δH=2.29 ppm, δC=31.5 ppm), and measure the one-dimensional NMR spectrum at 25°C on a Bruker AVANCE III 500M NMR instrument. The results are as follows: Figure 2 shown. Figure 2 A in the formula 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 show that the polysaccharide GJ005S is connected alternately by 1→2-linked α-rhamnose and 1→4-linked α-galacturonic acid; the branches are connected to the C-4 of the 1→2-linked α-rhamnose (1,2-α-Rha) and the C-3 of the 1→4-linked α-galacturonic acid (1,4-α-GalA) of the main chain. The branches include T-, 1,5-, 1,3-, 1,3,5-linked α-arabinose; T-, 1,4-, 1,6- and 1,3,6-linked β-galactose; 1,4-linked α-glucuronic acid; a small amount of terminally linked α-rhamnose, α-galacturonic acid and α-glucose ( Figure 3 The structural repeating unit of Gardenia jasminoides polysaccharide GJ005S is shown).

[0074] Pharmacological Example 1 Oil red staining to detect the effect of Gardenia polysaccharide GJ005S on the cell model of non-alcoholic steatohepatitis induced by free fatty acids (FFA)

[0075] Primary hepatocytes extracted from the liver of C57BL / 6 mice (purchased from Shanghai Lingchang Biotechnology Co., Ltd.) by collagenase hydrolysis 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 were used as the control group; the model group used free fatty acids (FFA) (palmitic acid and oleic acid (ratio of 1:2) were prepared into a mixture of 0.3mM / 0.6mM concentration) to treat mouse primary hepatocytes for 12 to 16 hours to induce the cells to form a lipid-accumulating NASH cell model; the GJ005S group used Gardenia polysaccharide GJ005S (concentrations of 0.1, 0.5, 0.75, and 1 mg / mL) to treat the above model cells for 16 hours, and the neutral lipid droplets in the cells were stained in the dark using an Oil Red staining kit (purchased from Beyotime Biotechnology Co., Ltd.).

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

[0077] Pharmacological Example 2 Safety Test of Gardenia Polysaccharide GJ005S on Human Hepatocytes

[0078] In order to confirm the safety of Gardenia polysaccharide GJ005S, the effects of different concentrations of Gardenia polysaccharide GJ005S on the proliferation of human hepatocytes were further studied (human hepatocyte cell line LO2 was purchased from the cell bank of Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences). Specifically, logarithmically growing human hepatocyte LO2 cells were inoculated in 96-well plates at a density of 3×10 3Then, the cells were treated with different concentrations of Gardenia polysaccharide GJ005S for 24 hours. The CCK8 kit was used to detect the changes in cell proliferation. It was found that different concentrations of Gardenia polysaccharide GJ005S did not reduce cell viability, indicating that the polysaccharide had no obvious cytotoxicity while reducing lipid accumulation in hepatocytes ( Figure 4 (middle B).

[0079] Pharmacological Example 3 The effect of the Gardenia polysaccharide component GJ005S on the liver tissue morphology and liver tissue coefficient of mice was detected in the non-alcoholic steatohepatitis model induced by a 60% high-fat 0.1% methionine choline-deficient (CDAHFD) diet

[0080] This animal experiment complied with the Guide for the Care 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, 8 in the control group, 8 in the model group, and 35 in the experimental group. Free diet and water, room temperature of 22±1℃, indoor relative humidity of 65±5%, and 12h cycle light. The model group and the experimental group were fed with CDAHFD feed (purchased from Mouse One Mouse Two Biotechnology Co., Ltd.) for 8 weeks. Eight mice were gavaged with an equal volume of 0.9% saline as the normal control group (Chow). After 2 weeks of feed 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 was given 6.25 mg / kg GJ005S polysaccharide, a total of 8 mice, Group C was given 25 mg / kg GJ005S polysaccharide, a total of 9 mice, Group D was given 100 mg / kg GJ005S polysaccharide, a total of 9 mice, and Group E was given 5 mg / kg remedirol (purchased from MCE) as a positive drug control, a total of 9 mice, and was given by gavage every day for 6 weeks. The control group and the model group were given an equal volume of normal saline. After 6 weeks of administration, the weight of the mice was recorded and the mice were sacrificed. The liver tissue morphology of the mice in each group was observed and the liver tissue was weighed. The results are shown in Figure 5 As shown in A and B, the liver tissue surface of mice with non-alcoholic fatty liver disease after intervention with GJ005S polysaccharide returned to a smooth, ruddy and bright color, but the texture was not as soft and elastic as the liver tissue of mice in the control group. The liver coefficient (liver tissue weight / mouse body weight) of mice in the model group increased, and the liver coefficient of the low (6.25 mg / kg), medium (25 mg / kg) and high (100 mg / kg) dose groups of GJ005S decreased, which was significantly different from the NASH model group induced by CDAHFD.

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

[0082] The specific operation steps of Sirius red staining experiment (Sirius red) are as follows: ① Dewax the paraffin sections to water: Put the paraffin sections in xylene for 20 minutes, a total of 2 times. Then put them in anhydrous ethanol for 5 minutes, a total of 2 times. Put them in 75% alcohol for 5 minutes and wash with tap water. ② Sirius red staining: Put the sections in Sirius red staining solution for 8-10 minutes, and dehydrate with anhydrous ethanol 2-3 times. ③ Sealing: Put the sections in xylene to make them transparent for 5 minutes, and use neutral gum to seal the sections. ④ Use a tissue imaging analyzer to perform full scan acquisition and analysis of the image. Under an optical microscope, collagen fibers appear red and the background appears yellow. The results of Sirius red staining showed that there was no collagen deposition in the negative control group mice, and the model group mice had severe collagen deposition in the vascular area and portal area. GJ005S administration significantly reduced collagen deposition ( Figure 6 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 Protein immunoblotting experiment to detect the effect of Gardenia polysaccharide GJ005S on the level of fibrosis phenotype-related proteins in mouse liver

[0084] Six weeks after administration, the mice were sacrificed, and proteins were extracted from part of the 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 in a metal heater for 10 min, cooled, and stored at -80°C. Immunoblotting was used to detect the expression of α-smooth muscle actin protein. Figure 6 As shown in B, Gardenia polysaccharide GJ005S can inhibit the expression of proteins related to 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 operation of oil red staining of liver fat sections is as follows: ① Place the OCT-embedded liver tissue on a freezing microtome to make 10μm thick tissue sections. ② Preparation of Oil Red O staining working solution: Prepare the working solution of Oil Red O isopropanol saturated storage solution and dd H2O in a ratio of 3:2 (prepare immediately before use). The frozen sections of liver tissue are stained with Oil Red O staining working solution for 10-12 minutes. ③ Wash twice with double distilled water, 20 seconds each time. ④ Wash with running water for 10 minutes. ⑤ Stain the nucleus with hematoxylin for 45 seconds. Repeat the washing with clean water. ⑥ Seal the sections with 50% glycerol, observe the staining of the liver tissue sections under a microscope, and take pictures. The results of Oil Red staining showed that the liver cells of mice in the negative control group were neatly arranged, the cytoplasm was uniform, and there was no lipid deposition in the cells. The liver cells of mice in the CDAHFD model group were swollen, and a large number of lipid droplets were deposited in the cells. After the administration of GJ005S, the deposition of lipid droplets in the liver was significantly reduced compared with the model group ( Figure 6 Middle C).

[0087] In summary, Gardenia polysaccharide GJ005S reduced lipid accumulation in primary hepatocytes of mice induced by FFA without obvious cytotoxicity. Gardenia polysaccharide GJ005S can reduce liver fibrosis induced by CDAHFD diet, including restoring liver tissue morphology, reducing liver coefficient, and reducing the expression of proteins related to animal liver damage and fibrosis phenotype. Therefore, this polysaccharide component GJ005S can become a potential carbohydrate drug for the prevention and / or treatment of non-alcoholic fatty hepatitis.

[0088] The above is only an embodiment of the present invention, and the common knowledge such as the known specific structure and characteristics in the scheme is not described in detail here. It should be pointed out that for those skilled in the art, several deformations and improvements can be made without departing from the structure of the present invention, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records 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 is that α-rhamnose and α-galacturonic acid connected in 1→2 manner are connected alternately; the side chain is connected to C-4 of α-rhamnose connected in 1→2 manner and C-3 of α-galacturonic acid connected in 1→4 manner in the main chain, and the side chain includes T-, 1,5-, 1,3-, 1,3,5-connected α-arabinose; T-, 1,4-, 1,6- and 1,3,6-connected β-galactose; 1,4-connected α-glucuronic acid; and the terminal is connected with α-rhamnose, α-galacturonic acid and α-glucose; 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.

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, 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 further preferably 17.1 kDa.

3. 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.

4. The Gardenia polysaccharide according to any one of claims 1 to 3, wherein The gardenia polysaccharide has the following structural repeating units:

5. A method for preparing Gardenia polysaccharide, comprising the following steps: (1) Extraction of polysaccharides: extracting gardenia with boiling water to obtain an extract, concentrating the extract, precipitating with ethanol, collecting the precipitate, and obtaining crude gardenia polysaccharides; (2) Purification of polysaccharides: The crude Gardenia jasminoides polysaccharide was dissolved in water, and eluted with deionized water and 0.05 M NaCl solution through an anion exchange column, and the elution fraction of the 0.05 M NaCl eluate was collected as a secondary elution fraction; The secondary elution components are then purified by elution through a dextran gel column, and the collected elution components are concentrated, dialyzed against water, and freeze-dried to obtain Gardenia polysaccharide.

6. The method for preparing Gardenia polysaccharide according to claim 5, wherein: In step (1), During extraction, the amount of water used is 15 to 30 times, preferably 20 times, of the amount of gardenia; and / or 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; and / or The extract is concentrated and then dialyzed, and the molecular weight cut-off of the dialysis bag is 3500Da; and / or The ethanol is an ethanol aqueous solution of 70% (v / v) or more, preferably an ethanol aqueous solution of 85% (v / v) or more, and more preferably an ethanol aqueous solution of 95% (v / v) or more; or, The step (1) comprises: 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) in total, filtering the extract, combining and concentrating the filtrate, dialyzing, the molecular weight cutoff of the dialysis bag is 3500Da, concentrating the dialyzed liquid, centrifuging and discarding the precipitate, adding 3 to 8 times (preferably 4 times) of ethanol in the volume of the supernatant to the supernatant for alcohol precipitation, standing overnight, centrifuging to obtain the precipitate, washing and drying to obtain the crude gardenia polysaccharide.

7. The method for preparing Gardenia polysaccharide according to claim 5 or 6, wherein: In step (2), Dissolving the crude Gardenia jasminoides polysaccharide prepared in step (1) in 10 to 15 times (preferably 10 times) the weight of water; and / or The anion exchange column is DEAE Sepharose TM Fast Flow; and / or The secondary elution components are concentrated, dialyzed (dialysis bag molecular weight cut-off is 3500Da), and freeze-dried to obtain a preliminarily separated secondary component; and / or The dextran gel column is a Sephacryl S-300HR gel column; and / or The secondary elution fraction was separated by elution through a dextran gel column with a 0.2 M NaCl aqueous solution, and the separated fractions were collected, concentrated, dialyzed (dialysis bag with a molecular weight cut-off of 3500 Da), and freeze-dried; or The step (2) comprises: adding 10 to 15 times (preferably 10 times) the weight of the Gardenia jasminoides polysaccharide prepared in step (1) to water to dissolve the polysaccharide, centrifuging the polysaccharide, and separating the supernatant with DEAE Sepharose. TM The Fast Flow anion exchange column is eluted with deionized water and 0.05M NaCl aqueous solution in sequence, and the eluted components of the 0.05M NaCl eluate are collected and combined, concentrated, centrifuged, and the supernatant is dialyzed (the molecular weight cutoff of the dialysis bag is 3500Da), and freeze-dried to obtain a preliminarily separated secondary component; the secondary component is dissolved in 10 to 30 times (preferably 20 times) the weight of a 0.2M NaCl aqueous solution, centrifuged, and the supernatant is passed through a Sephacryl S-300HR gel chromatography column, eluted and separated with a 0.2M NaCl aqueous solution, and the separated components are collected and combined, concentrated, dialyzed (the molecular weight cutoff of the dialysis bag is 3500Da), and freeze-dried to finally obtain the dry powder of the gardenia polysaccharide.

8. A composition comprising the gardenia polysaccharide according to any one of claims 1 to 4 or the gardenia polysaccharide prepared by the preparation method according to any one of claims 5 to 7, or comprising a preventive and / or therapeutic effective amount of the gardenia polysaccharide according to any one of claims 1 to 4 or the gardenia polysaccharide prepared by the preparation method according to any one of claims 5 to 7 as an active ingredient, and the composition optionally further comprises a food, a health product or a pharmaceutically acceptable excipient.

9. Use of the gardenia polysaccharide according to any one of claims 1 to 4 or the gardenia polysaccharide prepared according to the preparation method according to any one of claims 5 to 7, or the composition according to claim 8 in the preparation of food, health products or drugs for preventing and / or treating non-alcoholic steatohepatitis.

10. Use of the Gardenia polysaccharide according to any one of claims 1 to 4, or the Gardenia polysaccharide prepared by the preparation method according to any one of claims 5 to 7, or the composition according to claim 8 in the preparation of a drug or reagent having any one or more of the following effects: (1) Inhibit lipid accumulation in hepatic parenchymal cells; (2) Inhibit liver parenchymal cell damage and ballooning lesions; (3) Inhibit the fibrosis process in the later stage of non-alcoholic fatty liver disease.

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