Rheum officinale polysaccharide as well as preparation method and application thereof

Abstract

The invention discloses rhubarb polysaccharide as well as a preparation method and application thereof, the total sugar content of the rhubarb polysaccharide is 76.25%, the protein content is 3.4%, and the rhubarb polysaccharide contains active components (such as glucose, arabinose and galacturonic acid) in a specific proportion. The preparation method disclosed by the invention can be used for rapidly and efficiently extracting the rhubarb polysaccharide, and is low in energy consumption, simple and convenient to operate, low in process cost and suitable for large-scale industrial production. Meanwhile, the process ensures the stability and activity of the polysaccharide, and a wide prospect of providing drugs or food additives for preventing pancreatitis and regulating intestinal bacteria for the development and application of the rhubarb polysaccharide is provided. The rhubarb polysaccharide can obviously regulate intestinal flora disorder of mice with pancreatitis induced by arginine so as to up-regulate tight junction protein and promote tissue repair and regeneration, and has a great application prospect in the fields of regulating intestinal bacteria, preventing and treating hypertriglyceridemia type acute pancreatitis, metabolism-related new drugs and special medical food development and the like. Wide development and application prospects are realized.

Description

(I) Technical Field

[0002] This invention belongs to the field of biomedical technology, specifically relating to a rhubarb polysaccharide, its preparation, and its application in the preparation of drugs for treating hypertriglyceridemia-type acute pancreatitis and / or regulating intestinal flora structure. (II) Background Technology

[0004] Hypertriglyceridemic acute pancreatitis (HTGAP) is one of the most common types of acute pancreatitis in clinical practice, and its onset is closely related to significantly elevated serum triglyceride levels. In my country, with changes in dietary structure, the incidence of HTGAP has jumped to the second leading cause of various acute pancreatitis (AP) cases, with approximately 5%–20% of AP patients also having hypertriglyceridemia. Its overall mortality rate is 5%–10%, and the condition is more likely to progress to severe acute pancreatitis (SAP), with a mortality rate as high as 30%, accompanied by multiple organ dysfunction and a poor clinical prognosis. Currently, the clinical treatment strategies for HTGAP mainly include fasting, fluid replacement, lipid-lowering therapy (such as plasma exchange and insulin administration), and symptomatic supportive care. However, there is a lack of specific targeted therapies, and the treatment effects aimed at directly relieving pancreatic inflammation and improving prognosis remain unsatisfactory.

[0005] Rhubarb, a traditional Chinese medicine, possesses purgative, heat-clearing, fire-purging, blood-cooling, detoxifying, blood-activating, and menstruation-regulating effects. Modern pharmacological studies have shown that rhubarb and its active ingredients (such as anthraquinones and tannins) have demonstrated potential in the treatment of acute pancreatitis, and their mechanism of action may be related to inhibiting pancreatic enzyme activity and reducing inflammatory responses. However, current research and applications have largely focused on the small-molecule chemical components of rhubarb, while relatively little attention has been paid to its large-molecule components—rhubarb polysaccharides.

[0006] Known studies have shown that plant polysaccharides possess a wide range of biological activities, including immunomodulation, anti-inflammation, and lipid-lowering effects. However, whether rhubarb polysaccharides have a therapeutic effect on acute pancreatitis, especially hypertriglyceridemia-related acute pancreatitis closely related to lipid metabolism disorders, remains a blank. Furthermore, existing extraction and preparation processes for rhubarb polysaccharides often suffer from low extraction rates, activity degradation, or difficulty in industrial production, which limits the in-depth development and clinical application of rhubarb polysaccharides.

[0007] Therefore, there is an urgent need in this field for a method that can efficiently and stably prepare bioactive rhubarb polysaccharides and explore their new uses in the treatment of HTGAP, in order to provide a new, safe and effective treatment option for clinical practice. (III) Summary of the Invention

[0009] The purpose of this invention is to provide a rhubarb polysaccharide, its preparation method, and its application in the preparation of drugs for treating hypertriglyceridemia-type acute pancreatitis and / or regulating intestinal flora structure. The rhubarb polysaccharide has significant anti-inflammatory effects, effectively reducing pancreatic inflammation and promoting pancreatic tissue repair, and has no toxic side effects. It can be used as a main ingredient in functional foods or medicines for the prevention and treatment of pancreatitis.

[0010] The technical solution adopted in this invention is:

[0011] In a first aspect, the present invention provides a rhubarb polysaccharide, wherein the main monosaccharides of the rhubarb polysaccharide are glucose, arabinose, galacturonic acid, galactose, mannose, and glucuronic acid.

[0012] Furthermore, the mass ratio of glucose, arabinose, galacturonic acid, galactose, mannose, and glucuronic acid is 36:24:6:6:3:1.

[0013] Furthermore, the rhubarb polysaccharide is prepared according to the following method:

[0014] (1) Raw material processing: Select rhubarb rhizome slices, wash and dry them to obtain rhubarb rhizome slices; the best rhubarb rhizome slices are those with full shape, yellow-brown surface with brocade pattern, light red cross-section with star spots, firm texture with sufficient powder, and bitter taste with sticky texture.

[0015] (2) Degreasing treatment: Add rhubarb root and stem slices to an ethanol aqueous solution, soak overnight at room temperature, filter, and let the filter residue air dry to obtain defatted rhubarb slices;

[0016] (3) Water extraction and alcohol precipitation: Add water to defatted rhubarb slices and decoct, filter, and repeat the decoction of the residue 1-3 times. Combine the filtrates, and concentrate the extract after filtration with medium-speed filter paper to 1 / 2 to 3 / 4 of the original volume under reduced pressure. Then add an ethanol aqueous solution (preferably 4 times the volume of the concentrate), stir and mix well, and then let stand overnight to collect the precipitate.

[0017] (4) Drying and preservation: The precipitate was washed 1-3 times with anhydrous ethanol and anhydrous ether, and then dried to obtain rhubarb polysaccharide.

[0018] Furthermore, in step (2), the volume concentration of the ethanol aqueous solution is 80-95% (preferably 95%); the volume of the ethanol aqueous solution used is 1-5 mL / g based on the mass of the rhubarb root slices, preferably 1.5 mL / g.

[0019] Furthermore, in step (2), the rhubarb rhizome slices are soaked for 24 hours, filtered through two layers of gauze, and spread out to dry.

[0020] Furthermore, in step (3), the volume of water used is 5-15 mL / g based on the mass of defatted rhubarb slices, preferably 10 mL / g; the decoction temperature is 50-70℃ (60℃), and the decoction time is 1-3h (preferably 1h).

[0021] Furthermore, after the first decoction in step (3) is cooled to room temperature, it is first filtered with four layers of gauze. The dregs are then decocted twice more. The three filtrates are combined and filtered again with medium-speed filter paper (pore size 30-50μm) to obtain the extract.

[0022] Furthermore, in step (3), the vacuum concentration is carried out using a rotary evaporator with a gas pressure of 60~70 hPa, a temperature of 40~45℃, and a rotation speed of 80~100 rpm.

[0023] Furthermore, in step (3), the volume concentration of the ethanol-water solution is 80-95%, preferably 95%; the volume ratio of the concentrated solution after vacuum concentration to the ethanol-water solution is 1:4; stirring refers to stirring with a glass rod for 3 minutes to mix evenly; standing overnight means standing at room temperature for 24 hours to precipitate.

[0024] Furthermore, in step (4), the drying conditions are 80℃ for 12 hours.

[0025] Furthermore, the room temperature is 20~25℃.

[0026] Secondly, the present invention provides the use of the rhubarb polysaccharide in the preparation of drugs for the prevention and / or treatment of pancreatitis.

[0027] Furthermore, the drug is a drug for the prevention and / or treatment of hypertriglyceridemia-type acute pancreatitis.

[0028] Furthermore, the drug also includes pharmaceutically acceptable excipients, including fillers and binders.

[0029] Furthermore, the drug dosage forms include injections, oral preparations, ointments, and suppositories.

[0030] Thirdly, the present invention provides a medicine or food additive prepared from the rhubarb polysaccharide for the prevention and treatment of hyperlipidemic pancreatitis.

[0031] Fourthly, the present invention provides a composition for the prevention and treatment of pancreatitis, the composition comprising rhubarb polysaccharide and other drugs for the prevention and treatment of pancreatitis.

[0032] Fifthly, the present invention provides an application of the rhubarb polysaccharide in the preparation of an intestinal flora regulator.

[0033] Furthermore, the regulator is a preparation that regulates intestinal flora dysbiosis in mice with arginine-induced pancreatitis.

[0034] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0035] (1) The rhubarb polysaccharide described in this invention is obtained through a specific extraction and purification process and has high purity. The total sugar content is 76.25%, the protein content is 3.4%, and it contains a specific proportion of active ingredients (such as glucose, arabinose, and galacturonic acid).

[0036] (2) The preparation method of this invention can rapidly and efficiently extract rhubarb polysaccharides, with low energy consumption, simple operation, and low process cost, making it suitable for large-scale industrial production. At the same time, this process ensures the stability and activity of the polysaccharides, providing a broad prospect for the development and application of rhubarb polysaccharides as drugs or food additives for the prevention of pancreatitis and the regulation of intestinal flora.

[0037] (3) The rhubarb polysaccharide described in this invention can significantly regulate the intestinal flora disorder in mice with arginine-induced pancreatitis, thereby upregulating tight junction proteins and promoting tissue repair and regeneration. It has broad development and application prospects in the fields of regulating intestinal flora and preventing and treating hypertriglyceridemia-type acute pancreatitis and metabolic-related new drugs and special medical foods. (iv) Description of the attached drawings

[0039] Figure 1 This is a structural analysis diagram of rhubarb polysaccharides. In the diagram, A represents Fourier transform infrared spectroscopy (FTIR), with the x-axis representing wavenumber and the y-axis representing transmittance; B represents LC-MS monosaccharide composition analysis (LC-MS), with the x-axis representing retention time and the y-axis representing signal intensity; C represents high-performance gel permeation chromatography (HPLC), with the x-axis representing retention time and the y-axis representing signal response; and D represents ultraviolet-visible spectroscopy (UV-Vis), with the x-axis representing wavelength and the y-axis representing absorbance.

[0040] Figure 2 This diagram shows the pathological effects of rhubarb polysaccharide on mice with hypertriglyceridemia-induced acute pancreatitis. A represents the therapeutic effect of rhubarb polysaccharide on acute pancreatitis in the study / a comparative study of the effects of different treatment groups on the acute pancreatitis model; B shows sections of pancrease tissue from experimental animals in different treatment groups; C shows a comparative analysis of lipase activity in mice across groups, with the x-axis representing different groups and the y-axis representing mouse lipase activity; D shows a comparative analysis of amylase activity in mice across groups, with the x-axis representing different groups and the y-axis representing mouse amylase activity; E shows sections of colon tissue from experimental animals in different treatment groups; F shows histological scores on the left and ZO-1 mRNA expression levels on the right; G shows histological scores on the left and Occludin mRNA expression levels on the right. The NC group represents the normal diet group, the HTGAP group represents the control group for hyperlipidemic pancreatitis, and the RP group represents the rhubarb polysaccharide experimental group. p≤0.05; p≤0.01; p≤0.001; p≤0.0001.

[0041] Figure 3 This diagram illustrates the regulatory effect of rhubarb polysaccharides on the gut microbiota structure of mice with hypertriglyceridemia-predominant acute pancreatitis. A represents Pielou diversity analysis of the gut microbiota in each group; B represents Simpson diversity analysis; C represents the top 9 phyla-level composition analysis of the gut microbiota heatmap in each group; D represents the top 10 genera-level composition analysis of the phylogenetic tree constructed from the gut microbiota in each group; and E represents the principal component analysis diagram of the gut microbiota in each group. F represents the content of *Muribaculum* in each group of mice; G represents the content of *Lawsonibacter* in each group of mice; H represents the content of *Anaerotignum_189163* in each group of mice; I represents the content of *Ruminococcus_F* in each group of mice; J represents the content of *UBA9414* in each group of mice; K represents the content of *CAG_485* in each group of mice; L represents the content of *Erysipelatoclostridium* in each group of mice; and M represents the content of *Poseudobutyricicoccus* in each group of mice. p≤0.05; p≤0.01; p≤0.001; p≤0.0001.

[0042] Figure 4 This image shows the composition of intestinal metabolites in mice with hypertriglyceridemia-induced acute pancreatitis induced by rhubarb polysaccharides. A represents the volcano plots of the HTGAP group vs. the NC group; B represents the volcano plots of the RP group vs. the HTGAP group; C represents the content of Tryptophol tryptophan metabolites; D represents the interaction network analysis of microorganism-metabolite construction; E represents the content of 3-hydroxyanthranilic acid (3-HAA) tryptophan metabolites; F represents the content of 5-Hydroxyindole-3-Acetic Acid (5-HIAA) tryptophan derivatives; G represents the content of Pyruvic Acid tryptophan derivatives; and H represents the content of Tryptamine tryptophan derivatives. (V) Detailed Implementation Methods

[0044] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:

[0045] Example 1: Preparation and structural analysis of rhubarb polysaccharides

[0046] 1. Extraction of rhubarb polysaccharides

[0047] (1) Raw material processing: Select rhubarb rhizome slices with yellow-brown appearance with brocade pattern, light red cut surface with star-like spots, firm texture with a sandy feel and slightly bitter taste. Wash and dry in an oven at 40℃ to obtain rhubarb rhizome slices.

[0048] (2) Degreasing treatment: Mix 100g of rhubarb rhizome slices with 150mL of 95% ethanol aqueous solution, soak at room temperature (20~25℃, the same below) for 24h, filter with two layers of gauze, spread out and air dry to obtain 98g of defatted rhubarb slices;

[0049] (3) Water extraction and alcohol precipitation: Add 1000mL of water to 98g of defatted rhubarb slices, decoct at 60℃ for 1h, cool to room temperature, filter first with 4 layers of gauze, decoct the residue twice more, combine the filtrates from the three decoctions, and filter with medium-speed filter paper (pore size 30-50 μm) to obtain the extract. Concentrate the extract to 3 / 4 of the original volume using a rotary evaporator under reduced pressure at 60-70 hPa, 40-45℃ and 80-100 rpm. Add 4 times the amount of 95% ethanol to the concentrate, stir with a glass rod for 3min to mix evenly, let stand at room temperature for 24h to precipitate, and collect the precipitate.

[0050] (4) Drying and preservation: Wash all the precipitate from step (3) three times with 10 mL of anhydrous ethanol and 10 mL of anhydrous ether respectively, place it in an oven and dry at 80℃ for 12 hours to obtain 3 g of rhubarb polysaccharide, which is denoted as RP.

[0051] 2. Polysaccharide structure identification

[0052] The physicochemical properties of the rhubarb polysaccharide prepared in step 1 were analyzed, including the determination of total sugar content, molecular weight distribution and monosaccharide composition.

[0053] (1) The total sugar content was determined by the phenol-sulfuric acid method.

[0054] 0.1 mg / mL glucose standard solution: Weigh 10 mg of anhydrous glucose and dilute to 100 mL with water. 6% phenol solution: Accurately weigh 0.6 g of phenol, dissolve in water and dilute to 10 mL (9.4 mL), mix well, and prepare fresh before use. Construction of the glucose standard curve: Take 6 test tubes, three replicates per group, numbered 0, 1, 2, 3, 4, and 5. Add 0, 0.1, 0.3, 0.5, 0.7, and 0.9 mL of glucose standard solution to each tube, respectively. Add distilled water to make up to 1 mL of the system. Add 1 mL of 6% phenol solution to each tube, then add 2.5 mL of concentrated sulfuric acid (98%), mix well, and let stand for 20-30 minutes. Measure the absorbance at 490 nm using an ELISA reader. A standard curve was plotted with glucose content on the x-axis and absorbance on the y-axis. The curve equation was y = 0.7629x + 0.2451, R² = 0.99386, where y is the absorbance at 490 nm and x is the glucose concentration in mg / mL.

[0055] Accurately weigh an appropriate amount of rhubarb polysaccharide sample, prepare a 0.1 mg / mL polysaccharide solution with deionized water, accurately pipette 1 mL of the polysaccharide solution, and measure the absorbance value according to the above operation steps. Calculate the total sugar content in the polysaccharide sample using a standard curve, which is 76.25%.

[0056] (2) Molecular weight distribution

[0057] The average molecular weight (Mw) of the polysaccharides was determined by high-performance gel permeation chromatography (HPGPC) equipped with a refractive index detector (RID), and the results are shown in [Figure number missing]. Figure 1 High-performance gel permeation chromatogram of medium C. The column used was a TSK-GEL G3000 PWXL (7.8 × 300 mm).

[0058] The rhubarb polysaccharide prepared in step 1 was dissolved in an aqueous solution of 10 mg / mL and filtered through a 0.45 µm water membrane to obtain the test sample. Dextran standards of different molecular weights (4 kDa, 12.6 kDa, 60.6 kDa, 420 kDa, 820 kDa) were dissolved in aqueous solutions of 10 mg / mL and filtered through a 0.45 µm water membrane to obtain standard samples. The chromatographic column was maintained at 40℃, with an injection of 10 µL, and elution using H2O at a flow rate of 0.5 mL / min. A calibration curve was plotted based on the relationship between the logarithm of different molecular weights of the dextran standards and retention time. The curve equation is y = -0.3458x + 9.2937, R0. 2 =0.9871, where y is the logarithm of molecular weight and x is the retention time (minutes). The molecular weight of the sample was calculated to be 26 kDa based on the standard curve.

[0059] (3) Monosaccharide composition

[0060] Monosaccharide analysis was performed using the PMP derivatization method. 1–2 mg of rhubarb polysaccharide sample was placed in an ampoule, and 1 mL of 4 mol / L trifluoroacetic acid (TFA) aqueous solution was added. The ampoule was then sealed with an alcohol burner and hydrolyzed at 110 °C for at least 4 h to obtain the sample hydrolysate. The hydrolysate was then dried using nitrogen blowing, and 200 µL of methanol was added. This process was repeated 2–3 times, followed by further drying. 100 µL of 0.1 M NaOH aqueous solution was added, and the volume was brought to 1 mL with 900 µL of deionized water. This process was repeated 2–3 times to remove residual TFA. 400 µL of the sample was mixed with 450 µL of a 0.5 M 1-phenyl-3-methyl-5-pyrazolone methanol solution and 450 µL of a 0.3 M NaOH aqueous solution. Derivatization was performed at 70 °C for 30–60 min, followed by cooling to room temperature. Subsequently, the reaction was terminated by adjusting the pH to neutral by adding 450 µL of 0.3M HCl aqueous solution. After adding 1–2 mL of chloroform and vortexing for 2 min, the mixture was extracted three times to remove the lower chloroform layer. The upper aqueous phase was collected and filtered through a 0.45 µm water membrane. The PMP-derived rhubarb polysaccharide sample was then obtained.

[0061] Simultaneously, eight 0.02 M monosaccharide standards—Man (mannose), Gal (galactose), Glc (glucose), Rha (rhamnose), GlcA (glucuronic acid), Ara (arabinose), and Fuc (fucose)—were prepared with water to a concentration of 1 mg / mL. 100 µL of the 1 mg / mL monosaccharide standard was mixed with 450 µL of a 0.5 M methanol solution of 1-phenyl-3-methyl-5-pyrazolone and 450 µL of a 0.3 M NaOH aqueous solution. Derivatization was carried out at 70 °C for 30–60 min, followed by cooling to room temperature. The reaction was then terminated by adjusting the pH to neutral by adding 450 µL of a 0.3 M HCl aqueous solution. After adding 1–2 mL of chloroform and vortexing for 2 min, extraction was performed three times. The lower chloroform layer was removed, and the upper aqueous phase was collected and filtered through a 0.45 µm water membrane to obtain the PMP-derived monosaccharide standard. The derivatized monosaccharide standard was centrifuged and diluted 100-fold before analysis.

[0062] Using an Eclipse XDB-C18 column at 40 ℃, the mobile phase consisted of a mixture of 95% ultrapure water containing 25 mM ammonium acetate and 5% acetonitrile, and an organic phase of 95% acetonitrile aqueous solution. An initial mobile phase ratio of 90:10 (V / V) was set, starting elution with a lower organic phase ratio. The organic phase ratio was gradually increased based on the polarity and retention time of the monosaccharide standards, and the optimal elution conditions were determined by adjusting the mobile phase ratio to separate the eight monosaccharide standards. The sample loading volume was 1 µL. The mass-to-charge ratio and elution time of each monosaccharide in rhubarb polysaccharide were determined by LC-MS mass spectra. Comparison with the mass spectra of the standard monosaccharides confirmed that the main monosaccharide composition of rhubarb polysaccharide was glucose:arabinose:galacturonic acid:galactose:mannose:glucuronic acid. The mass ratio of each monosaccharide is calculated based on the peak area or peak height. For example, the mass ratio of glucose:arabinose:galacturonic acid:galactose:mannose:glucuronic acid is 36:24:6:6:3:1. Figure 1 LC-MS monosaccharide composition analysis of B.

[0063] Example 2: Rhubarb polysaccharides can alleviate pathological symptoms in HTGAP mice.

[0064] 1. Animal grouping and construction of HTGAP mouse model

[0065] Reference Figure 2 Eighteen male C57BL / 6 mice (6 weeks old, 20-22 g) were purchased from Qizhen Laboratory Animal Technology Co., Ltd. All animals underwent a 12-hour light-12-hour dark cycle at 24±1℃, provided with normal feed and sterilized water. After 2 days of acclimatization, all mice were divided into three groups: a normal diet group (NC), a hyperlipidemic pancreatitis control group (HTGAP), and a rhubarb polysaccharide treatment experimental group (RP), with 6 mice in each group.

[0066] Mice in the NC group were injected with saline for 8 days, every other day, at a dose of 0.2 mL / kg. Mice in the HTGAP and PR groups were injected with P407 (poloxam 407) for 7 days, every other day, at a dose of 0.2 mL / kg. On day 8, mice in the HTGAP group were injected with P407 (poloxam 407) + 20% Arg (arginine) once, at a dose of 0.2 mL / kg. Mice in the RP group were injected with P407 + 20% Arg and gavaged with a 1 mg / mL rhubarb polysaccharide aqueous solution prepared in Example 1, twice daily, at a dose of 0.2 mL / kg. All groups were provided with normal feed and normal sterilized water. On day 8, the modeling was completed, and blood, pancreas, colon tissue, and cecal contents were collected from the mice.

[0067] 2. Histopathological analysis

[0068] Pancreatic and colonic tissues were rapidly removed from dissected mice and fixed in 4% paraformaldehyde to maintain tissue morphology. Subsequently, the tissues were dehydrated with graded alcohols, cleared with xylene, impregnated with paraffin, and embedded into paraffin blocks. The paraffin blocks were then sliced ​​into 4-5 μm thin sections using a microtome, stained with hematoxylin and eosin (HE), mounted with neutral resin, and finally observed and imaged under a microscope. Results are shown below. Figure 2 As shown in Figures B and E, Figure B displays pathological sections of pancreatic tissue, from left to right: NC (control group), HTGAP (hyperlipidemic acute pancreatitis group), and RP (rhubarb polysaccharide treatment group). Figure E displays pathological sections of colonic tissue, with the same groupings as in Figure B. The results indicate that, as shown in Figure B, the HTGAP group exhibited significant pathological changes in pancreatic tissue, such as acinar cell necrosis and inflammatory cell infiltration (indicated by arrows), while the NC group showed normal pancreatic tissue structure. The pathological changes in the RP group were less pronounced than those in the HTGAP group. As shown in Figure E, the HTGAP group also showed some pathological changes in colonic tissue, such as mucosal damage and inflammatory cell infiltration (indicated by arrows). The NC group showed normal colonic tissue structure, while the pathological changes in the RP group were less pronounced than those in the HTGAP group.

[0069] 3. Lipase analysis

[0070] Serum lipase activity was measured using a lipase (LPS) assay kit (A054-2-1, methyl halogen substrate method, microplate method, 96T). Results are shown below. Figure 2 Image C shows serum lipase activity. The LPS activity in the HTGAP group was significantly higher than that in the NC group, while the LPS activity in the RP group was significantly lower than that in the HTGAP group.

[0071] 4. Amylase analysis

[0072] Serum amylase activity was measured using an amylase (AMS) test kit (catalog number C016-1-2, iodine-starch colorimetric method, microplate method, 96T). Results are shown below. Figure 2 Image D shows the activity of amylase (AMY) in mouse serum. The AMY activity in the HTGAP group was significantly higher than that in the NC group, while the AMY activity in the RP group was significantly lower than that in the HTGAP group.

[0073] 5. Histological scoring

[0074] Figure 2The left-hand figures (F and G) show the histological scores of the pancreas and colon in different experimental groups (NC control group, HTGAP hyperlipidemic acute pancreatitis group, and RP rhubarb polysaccharide treatment group), respectively. In the pancreatic tissue, the NC group had extremely low scores and normal structure, the HTGAP group had significantly higher scores, and the RP group had significantly lower scores than the HTGAP group but higher scores than the NC group. Similarly, in the colonic tissue, the NC group also had extremely low scores and normal structure, the HTGAP group had significantly higher scores, and the RP group had significantly lower scores than the HTGAP group but higher scores than the NC group. These results indicate that hyperlipidemic acute pancreatitis (HTGAP) causes significant pathological damage to the pancreas and colon, specifically manifested as a significantly elevated histological score. Rhubarb polysaccharide treatment (RP) effectively alleviated these two types of tissue damage caused by HTGAP, resulting in a significant reduction in histological scores. This suggests that rhubarb polysaccharide has a protective effect against tissue damage associated with hyperlipidemic acute pancreatitis. p≤0.05; p≤0.01; p≤0.001; p≤0.0001.

[0075] 6. Molecular protein expression

[0076] Figure 2 The right-hand sides of F and G plots show the expression levels of tight junction proteins ZO-1 and occludin in pancreatic tissue from different experimental groups (NC control group, HTGAP hyperlipidemic acute pancreatitis group, and RP rhubarb polysaccharide treatment group), respectively. The mRNA expression levels of ZO-1 and occludin were significantly lower than those in the normal control group (NC group). After treatment with rhubarb polysaccharide (RP group), the mRNA expression levels of ZO-1 and occludin were significantly higher than those in the HTGAP group. These results indicate that rhubarb polysaccharide can, to some extent, reverse the abnormal expression of intestinal tight junction protein genes caused by hyperlipidemic acute pancreatitis, and plays a positive role in maintaining intestinal barrier integrity.

[0077] 7. Animal experiment results:

[0078] like Figure 2As shown in the diagram (pathological effects of rhubarb polysaccharide on mice with hypertriglyceridemia-type acute pancreatitis), the experimental results show that RP treatment effectively reduced serum amylase (AMY) and lipase (LPS) levels. Regarding pancreatic protection, histopathological analysis revealed a significant improvement in the typical pathological features of the model group, such as widened interlobular spaces, acini swelling, and inflammatory infiltration (P<0.01). Furthermore, in terms of intestinal barrier function, RP treatment not only significantly reduced villous edema but also repaired epithelial cell damage, while simultaneously upregulating the mRNA expression of tight junction proteins ZO-1 and Occludin. These findings, at the histopathological and molecular levels, confirm that RP has a significant protective effect against pancreatic and intestinal damage in HTGAP mice through a multi-target mechanism.

[0079] Example 3: Rhubarb polysaccharides can improve intestinal flora imbalance in HTGAP mice

[0080] 1. High-throughput sequencing

[0081] To investigate the changes in gut microbiota in HTGAP mice after RP administration, the composition of gut microbiota in the three groups of mice in Example 2 was studied by 16S rRNA high-throughput sequencing.

[0082] Cecal contents of mice from each group in Example 2 were collected in sterile 1.5 mL EP tubes and immediately frozen at -80°C. Total genomic DNA was extracted from the samples using a kit, and its quality was assessed by agarose gel electrophoresis and micro-spectrophotometry. Subsequently, using qualified DNA as a template, PCR amplification was performed on the V3-V4 hypervariable region of the bacterial 16S rRNA gene using specific primers (338F / 806R) with sequencing adapters. After purification, the amplified products were precisely quantified using quantitative fluorescence methods and mixed in equimolar proportions to construct sequencing libraries. Finally, the libraries were sequenced on the Illumina Miseq PE300 high-throughput sequencing platform (Hangzhou Qingke Biotechnology Co., Ltd.) to generate raw sequencing data for subsequent bioinformatics analysis. After sequencing, species composition analysis was performed on the QIIME2 multi-omics analysis platform, and data analysis was conducted using STAMP and GraphPad plotting software to reveal information such as the diversity, abundance, and evolutionary relationships of the microbial community. Results are shown below. Figure 3 .

[0083] The results showed that the HTGAP group increased gut microbiota α-diversity, while mice fed rhubarb polysaccharide showed a decreasing trend in gut microbiota α-diversity. Figure 3 (A). Significant changes occurred in the gut microbiota of the three groups of mice at the phylum level, manifested as follows: Firmicutes and Desulfobacterota Increase, ProteobacteriaThe decrease was reversed by RP intervention. The mean relative abundance in the three groups was shown on a phylogenetic tree constructed based on representative 16S rRNA gene sequences of each species. The results showed significant differences in the abundance of 37 bacterial species between the NC and HTGAP groups, with 16 species showing significant improvement after RP treatment. Simultaneously, at the species level, the abundance of 8 bacterial species changed significantly compared to the control group, with HTGAP mice showing a significantly higher abundance in their gut. Lachnoclostridium, Alistipes indistinctus, Desulfovibrio, Oscillospiraceae, Tuzzerella, Bilophila, GCA-900066575 and Muribaculum The abundance of these bacteria increased, while RP intervention decreased their abundance.

[0084] Example 4: Rhubarb polysaccharides can regulate the composition of intestinal metabolites in HTGAP mice

[0085] First, the frozen cecal contents sample from Example 3 was rapidly extracted on ice using a pre-cooled solvent (such as a methanol-water system). After centrifugation, the supernatant was collected and subjected to vortexing and sonication to fully release metabolites, followed by nitrogen blowing concentration and reconstitution. The extract was then separated using an ultra-high performance liquid chromatography (UHPLC) system and analyzed by a high-resolution mass spectrometer in both positive and negative ion modes. The raw data were processed using specialized software for peak extraction, alignment, normalization, and compound identification. Finally, multivariate statistical analysis was used to screen for differentially expressed metabolites and explore related metabolic pathways. Figure 4 The diagram shown (composition of intestinal metabolites in mice with hypertriglyceridemia-induced acute pancreatitis) reveals the regulatory role of rhubarb polysaccharide (RP) on the metabolic profile of HTGAP mice and its interaction mechanism with the gut microbiota through metabolomics analysis. Volcano plots show that 490 metabolites were upregulated and 1350 were downregulated in the HTGAP group compared to the NC group, indicating that RP treatment significantly reversed this metabolic disorder. Key analyses revealed characteristic changes in gut microbiota-derived tryptophan metabolites: tryptophol was abnormally elevated in the HTGAP model but was corrected by RP; elevated 3-hydroxyanthranilic acid (3-HAA) suggests its potential involvement in inflammatory responses through the gut-pancreas axis; and differential changes in tryptophan derivatives such as 5-Hydroxyindole-3-Acetic Acid (5-HIAA), Pyruvic Acid, and Tryptamine confirm that RP has a broad regulatory role in the tryptophan metabolic pathway. The microbial-metabolite interaction network constructed by Spearman correlation analysis elucidates the multi-target mechanism by which RP improves HTGAP through the "microbiome-metabolite" axis from a systems biology perspective.

[0086] The implementation of the present invention is not limited to the embodiments described above. Any changes, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and are included within the protection scope of the present invention.

Claims

1. A rhubarb polysaccharide, characterized in that, The main monosaccharides in the rhubarb polysaccharide are glucose, arabinose, galacturonic acid, galactose, mannose, and glucuronic acid.

2. The rhubarb polysaccharide as described in claim 1, characterized in that, The mass ratio of glucose, arabinose, galacturonic acid, galactose, mannose, and glucuronic acid is 36:24:6:6:3:

1.

3. The rhubarb polysaccharide as described in claim 1, characterized in that, The rhubarb polysaccharide was prepared according to the following method: (1) Raw material processing: Select rhubarb rhizome slices, wash and dry them to obtain rhubarb rhizome slices; (2) Degreasing treatment: Add rhubarb root and stem slices to an ethanol aqueous solution, soak overnight at room temperature, filter, and let the filter residue air dry to obtain defatted rhubarb slices; (3) Water extraction and alcohol precipitation: Add water to defatted rhubarb slices and decoct, filter, and decoct the dregs again 1-3 times. Combine the filtrates, and concentrate the extract after filtration with medium-speed filter paper under reduced pressure to 1 / 2 to 3 / 4 of the original volume. Add ethanol aqueous solution, stir and mix well, and then let stand overnight to collect the precipitate. (4) Drying and preservation: The precipitate was washed 1-3 times with anhydrous ethanol and anhydrous ether, and then dried to obtain rhubarb polysaccharide.

4. The rhubarb polysaccharide as described in claim 3, characterized in that, In step (3), the decocting temperature is 50-70℃ and the decocting time is 1-3h.

5. The rhubarb polysaccharide as described in claim 3, characterized in that, In step (3), after the first decoction is cooled to room temperature, the residue is first filtered with four layers of gauze, and then the dregs are decocted twice. The filtrates from the three decoctions are combined and then filtered with medium-speed filter paper to obtain the extract.

6. The use of the rhubarb polysaccharide of claim 1 in the preparation of a medicament for the prevention and / or treatment of pancreatitis.

7. The application as described in claim 6, characterized in that, The drug is for the prevention and / or treatment of acute pancreatitis with hypertriglyceridemia.

8. A pharmaceutical or food additive prepared from rhubarb polysaccharide as described in claim 1 for the prevention and treatment of hyperlipidemic pancreatitis.

9. A composition for the prevention and treatment of pancreatitis, characterized in that, The composition includes the rhubarb polysaccharide of claim 1 and other drugs used to prevent and treat pancreatitis.

10. The use of the rhubarb polysaccharide of claim 1 in the preparation of an intestinal flora regulator.

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