Plant polysaccharide, and preparation method and application thereof

Abstract

The application discloses a plant polysaccharide and a preparation method and application thereof, wherein monosaccharides in the jasmine flower polysaccharide are composed of rhamnose, arabinose, galactose, glucose, xylose, mannose and galacturonic acid; and the molar ratio of the rhamnose, arabinose, galactose, glucose, xylose, mannose and galacturonic acid is (14-22):(100-120):(160-185):(200-240):(16-25):(18-27):(400-450). The jasmine flower polysaccharide component in the scheme has the characteristics of inhibiting inflammatory reaction and enhancing antioxidant activity, can effectively improve APAP-induced acute liver injury, and provides a certain theoretical basis for development and utilization of the jasmine flower and research and development of liver protection products.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a plant polysaccharide and a preparation method and application thereof. Background Art

[0002] Drug-induced liver injury (DILI) is a leading cause of idiosyncratic or intrinsic liver disease worldwide. Acetaminophen (APAP) is a widely used over-the-counter analgesic and antipyretic. Hepatotoxicity caused by APAP overdose is a leading cause of acute liver failure (ALF) in the United States and parts of Europe, limiting its clinical application.

[0003] When taken at recommended doses, nearly 90% of APAP is metabolized through glucuronidation or sulfation and then excreted through the bile, approximately 2% is excreted unmetabolized in the urine, and less than 8% is metabolized to the reactive NAPQI via CYP450-mediated oxidases. Under normal circumstances, NAPQI is detoxified by rapid conjugation to glutathione (GSH). However, excessive APAP metabolism depletes glutathione and increases N-acetyl-p-benzoquinone imide (NAPQI) levels, leading to oxidative stress, DNA damage, and hepatocyte necrosis, ultimately causing liver damage.

[0004] N-acetyl-L-cysteine ​​(NAC) is currently the only drug approved for the treatment of APAP poisoning. However, due to its narrow therapeutic window for treating acute liver injury and certain toxic side effects, its clinical application is greatly limited. Therefore, finding a more effective treatment with fewer side effects is crucial. Summary of the Invention

[0005] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention provides a frangipani flower polysaccharide.

[0006] The present invention also provides a preparation method of the frangipani flower polysaccharide.

[0007] The present invention also provides the application of the frangipani flower polysaccharide.

[0008] The present invention also provides a medicine.

[0009] According to one aspect of the present invention, a frangipani polysaccharide is provided, wherein the monosaccharides in the frangipani polysaccharide are composed of rhamnose, arabinose, galactose, glucose, xylose, mannose and galacturonic acid; and the molar ratio of the rhamnose, arabinose, galactose, glucose, xylose, mannose and galacturonic acid is (14-22): (100-120): (160-185): (200-240): (16-25): (18-27): (400-450).

[0010] In some embodiments of the present invention, the molar ratio of rhamnose, arabinose, galactose, glucose, xylose, mannose and galacturonic acid is (17-21):(105-115):(170-180):(210-230):(18-23):(19-25):(430-440).

[0011] In some embodiments of the present invention, the molar ratio of rhamnose, arabinose, galactose, glucose, xylose, mannose and galacturonic acid is 19:109:174:220:21:22:434.

[0012] In some embodiments of the present invention, the molecular weight of the frangipani polysaccharide is 69445 Da.

[0013] According to a second aspect of the present invention, a method for preparing the above-mentioned frangipani polysaccharide is provided, comprising the following steps:

[0014] The frangipani flower sample was subjected to defatting, water extraction, alcohol precipitation, protein removal and dialysis in sequence to obtain frangipani flower polysaccharide.

[0015] In some embodiments of the present invention, the defatting is performed using anhydrous ethanol.

[0016] In some embodiments of the present invention, in the defatting step, the solid-to-liquid ratio of the frangipani flower sample to anhydrous ethanol is 1 g: 8-12 mL.

[0017] In some embodiments of the present invention, the solid-liquid ratio is 1 g:10 mL.

[0018] In some embodiments of the present invention, degreasing is performed by heating under reflux.

[0019] In some embodiments of the present invention, the heating reflux is performed at a temperature of 70 to 90° C. for 1 to 2 hours, and the heating reflux step is repeated 2 to 3 times.

[0020] In some embodiments of the present invention, in the water extraction step, the solid-to-liquid ratio of the defatted frangipani flower sample to water is 1 g: 10-20 mL.

[0021] In some embodiments of the present invention, the solid-liquid ratio is 1 g:15 mL.

[0022] In some embodiments of the present invention, water extraction is performed by combining ultrasonic extraction and water bath extraction.

[0023] In some embodiments of the present invention, the ultrasonic extraction is performed under the condition of ultrasonic power of 90-110w for 15-25min.

[0024] In some embodiments of the present invention, the water bath extraction is carried out at 95-105° C. for 2-3 hours.

[0025] In some embodiments of the present invention, the water extraction further includes a step of solid-liquid separation and collecting the separated liquid phase.

[0026] In some embodiments of the present invention, the alcohol precipitation uses ethanol with a mass fraction of 95% or more.

[0027] In some embodiments of the present invention, in the alcohol precipitation step, the volume ratio of the water extract to ethanol is 1:(3-5).

[0028] In some embodiments of the present invention, the alcohol precipitation temperature is 0-4° C., and the alcohol precipitation time is 8-12 h.

[0029] In some embodiments of the present invention, the step of washing the precipitate obtained by alcohol precipitation with anhydrous ethanol, ether and acetone in sequence is further included.

[0030] In some embodiments of the present invention, the protein removal is performed using Sevag reagent.

[0031] In some embodiments of the present invention, the Sevag reagent is a mixed solution of chloroform and n-butanol in a volume ratio of 4:1.

[0032] In some embodiments of the present invention, the volume ratio of the Sevag reagent to the polysaccharide solution obtained by dissolving the precipitate obtained by washing with anhydrous ethanol, ether and acetone in water is 1:4-6.

[0033] In some embodiments of the present invention, the dialysis is performed using a dialysis bag with a molecular weight cut-off of >8000Da.

[0034] In some embodiments of the present invention, the dialysis time is 1 to 3 days.

[0035] According to a third aspect of the present invention, a use of the above-mentioned frangipani polysaccharide is proposed, and the use is in the preparation of a medicine for treating acute liver injury.

[0036] In some embodiments of the present invention, the application is application in preparing functional food for assisting in improving acute liver injury.

[0037] In some embodiments of the present invention, the acute liver injury includes APAP-induced acute liver injury.

[0038] In some embodiments of the present invention, the frangipani polysaccharide has the function of inhibiting the expression level of inflammatory factors.

[0039] In some embodiments of the present invention, the inflammatory factors include IL-1β, IL-6, and TNF-α.

[0040] In some embodiments of the present invention, the frangipani polysaccharide has the function of reducing serum alanine aminotransferase (ALT) and / or aspartate aminotransferase (AST) levels.

[0041] In some embodiments of the present invention, the frangipani polysaccharide has the function of enhancing antioxidant activity.

[0042] In some embodiments of the present invention, the enhancing antioxidant activity comprises increasing the activities of catalase (CAT), superoxide dismutase (SOD), and glutathione (GSH).

[0043] In some embodiments of the present invention, the enhancing antioxidant activity comprises reducing the content of malondialdehyde (MDA) and reactive oxygen species (ROS).

[0044] According to a fourth aspect of the present invention, a medicine is provided, comprising the above-mentioned frangipani flower polysaccharide.

[0045] In some embodiments of the present invention, the medicament is used to treat acute liver injury.

[0046] In some embodiments of the present invention, the acute liver injury includes APAP-induced acute liver injury.

[0047] In some embodiments of the present invention, the drug further comprises a pharmaceutically acceptable excipient.

[0048] In some embodiments of the present invention, the pharmaceutically acceptable excipients include at least one of a diluent, an excipient, a filler, a binder, a disintegrant, an absorption enhancer, a surfactant, an adsorption carrier, a lubricant, a sweetener, and a flavoring agent.

[0049] In some embodiments of the invention, the excipient comprises water.

[0050] In some embodiments of the present invention, the filler comprises at least one of starch and sucrose.

[0051] In some embodiments of the present invention, the binder comprises at least one of a cellulose derivative, alginate, gelatin, and polyvinyl pyrrolidone.

[0052] In some embodiments of the invention, the humectant comprises glycerin.

[0053] In some embodiments of the present invention, the disintegrant comprises at least one of agar, calcium carbonate and sodium bicarbonate.

[0054] In some embodiments of the present invention, the absorption enhancer comprises a quaternary ammonium compound.

[0055] In some embodiments of the invention, the surfactant comprises cetyl alcohol.

[0056] In some embodiments of the present invention, the adsorption carrier includes at least one of kaolin and bentonite.

[0057] In some embodiments of the present invention, the lubricant includes at least one of talc, calcium stearate, magnesium stearate and polyethylene glycol.

[0058] In some embodiments of the present invention, the dosage form of the drug is in the form of solid, semi-solid or liquid, and can be an aqueous solution, non-aqueous solution or suspension.

[0059] In some embodiments of the present invention, the dosage form of the drug is tablets, capsules, soft capsules, granules, pills, oral liquids, dry suspensions, pellets, dry extracts, injections or infusions, transdermal agents, and transdermal microneedles.

[0060] In some embodiments of the present invention, the drug may be administered by conventional methods in the art, including but not limited to injection or oral administration.

[0061] In some embodiments of the present invention, the injection administration can be intravenous injection, intramuscular injection, intraperitoneal injection, intradermal injection or subcutaneous injection.

[0062] In some embodiments of the present invention, the mass fraction of the frangipani polysaccharide in the medicine is 0.01% to 100%.

[0063] According to some preferred embodiments of the present invention, the mass fraction of the frangipani polysaccharide in the medicine is 0.05% to 95%.

[0064] According to some preferred embodiments of the present invention, the mass fraction of the frangipani polysaccharide in the medicine is 0.05% to 50%.

[0065] According to some embodiments of the present invention, at least the following beneficial effects are achieved: the present invention extracts and separates a frangipani polysaccharide component from the frangipani flower. The frangipani polysaccharide component obtained by the scheme of the present invention has the characteristics of inhibiting inflammatory response and enhancing antioxidant activity, and can also effectively improve APAP-induced acute liver injury, providing a certain theoretical basis for the development and utilization of frangipani flowers and the research and development of liver protection products. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] The present invention will be further described below with reference to the accompanying drawings and embodiments, in which:

[0067] Figure 1 This is a glucose standard curve diagram in Example 1 of the present invention;

[0068] Figure 2 This is the ultraviolet spectrum analysis diagram in Example 1 of the present invention;

[0069] Figure 3 This is an infrared spectrum analysis diagram in Example 1 of the present invention;

[0070] Figure 4 This is a high performance gel permeation chromatography analysis chart in Example 1 of the present invention;

[0071] Figure 5 This is a diagram showing the monosaccharide composition of the frangipani polysaccharide in Example 1 of the present invention;

[0072] Figure 6 Figures 2 and 3 show the test results of mouse body weight, liver index, and spleen index in Example 2 of the present invention, wherein A shows the test results of mouse body weight, B shows the test results of spleen index, and C shows the test results of liver index. ##p<0.01, ####p<0.0001 compared with the blank group; *p<0.05 compared with the model group (n=7);

[0073] Figure 7 Figures 2 show the liver phenotype and liver histopathological analysis results in Example 2 of the present invention, where A shows the liver morphology and B shows the HE pathological section staining results; ####p<0.0001 compared with the blank group; **p<0.01, ***p<0.001, ****p<0.0001 compared with the model group (n=7);

[0074] Figure 8 Graphs showing the test results of serum biochemical indicators of mice in Example 2 of the present invention, wherein A is a graph showing the test results of AST, B is a graph showing the test results of ALT, C is a graph showing the test results of IL-6, D is a graph showing the test results of IL-1β, and E is a graph showing the test results of TNF-α. #p<0.05, ##p<0.01, ###p<0.001, ####p<0.0001 are compared with the blank group; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 are compared with the model group (n=7);

[0075] Figure 9The figures are the test results of mouse liver biochemical indicators in Example 2 of the present invention, wherein A is the SOD test result, B is the MDA test result, C is the GSH test result, D is the CAT test result, and E is the ROS content test result. ##p<0.01, ###p<0.001, ####p<0.0001 compared with the blank group; *p<0.05, **p<0.01 compared with the model group (n=7). DETAILED DESCRIPTION

[0076] The following will clearly and completely describe the concept and technical effects of the present invention in conjunction with the embodiments to fully understand the purpose, features and effects of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0077] Example 1 Preparation of Frangipani Polysaccharide

[0078] This example prepared a frangipani polysaccharide, and the specific preparation and verification methods are as follows:

[0079] 1. Extraction of Frangipani Polysaccharide

[0080] (1) Crushing: crushing the fully dried frangipani flowers, sieving, and drying to obtain frangipani pollen;

[0081] (2) Degreasing: Jasmine pollen was degreased with 10 times the amount of anhydrous ethanol (i.e., the ratio of Jasmine pollen to anhydrous ethanol was 1 g: 10 mL) at 85°C and refluxed for 90 min. The reflux was repeated once, and the residue was filtered and evaporated to obtain the residue, which was then dried.

[0082] (3) Ultrasonic-assisted extraction: The defatted dry powder was mixed with distilled water at a material-liquid ratio of 1 g:15 mL, ultrasonic power 100 W, ultrasonication for 20 min, then placed in a 100 °C water bath for 2.5 h, centrifuged and filtered to obtain the aqueous extract;

[0083] (4) Alcohol precipitation: Take the filtrate and evaporate it to 20% of the original volume using a rotary evaporator. Then add 4 times the amount of anhydrous ethanol to the concentrate, refrigerate it in a refrigerator overnight, collect the precipitate by suction, and evaporate the ethanol on the surface in a fume hood to obtain the precipitate;

[0084] (5) Washing: The precipitate was washed twice with anhydrous ethanol, ether, and acetone in sequence, and dried to obtain the crude polysaccharide extract I;

[0085] (6) Protein removal: Dissolve the crude polysaccharide extract I in an appropriate amount of water, add Sevag reagent according to the ratio of crude polysaccharide to Sevag reagent 5:1, shake thoroughly for 30 minutes, and centrifuge at 6000 r / min for 15 minutes; carefully aspirate the upper polysaccharide solution, remove the organic phase and precipitate, and then add Sevag solvent equivalent to 1 / 5 of its volume, repeat several times until no obvious precipitation is produced, and store at 4°C to prevent deterioration for subsequent impurity removal;

[0086] (7) Impurity removal: Use a ready-to-use dialysis bag with a diameter of >8000D to remove impurities. First, cut the dialysis bag into a suitable length, soak the preservation solution with deionized water, place the deproteinized frangipani polysaccharide solution into the bag, seal it with a dialysis bag clip, and place a weight at the bottom to make it vertical to the bottom. Then, immerse the dialysis bag in a beaker filled with deionized water and a magnet, and then place the entire beaker on a magnetic stirrer to accelerate the dialysis rate, thereby assembling a complete dialysis device. After dialysis for 48 hours, remove the liquid in the dialysis bag and centrifuge to obtain a precipitate. After freeze-drying the precipitate, obtain the crude frangipani polysaccharide extract II (i.e., purified frangipani polysaccharide).

[0087] 2. Determination of Frangipani Polysaccharide Content

[0088] (1) Glucose standard curve drawing and polysaccharide determination method

[0089] Standard curve preparation: The purified frangipani polysaccharide was determined using the phenol-sulfuric acid method. Accurately weigh 100 mg of glucose standard into a 100 mL volumetric flask and dilute to 100 mL with distilled water to prepare a 0.1 mg / mL glucose standard solution. Transfer the prepared glucose standard solution and dilute to 5 mL with distilled water to prepare concentrations of 0.02 mg / mL, 0.04 mg / mL, 0.06 mg / mL, 0.08 mg / mL, 0.10 mg / mL, 0.12 mg / mL, and 0.14 mg / mL, respectively. Take 200 μL of each dilution and place it in a 2 mL test tube. Add 100 μL of 6% phenol and 500 μL of concentrated sulfuric acid to each test tube, let it stand for 10 minutes, shake it well, and let it stand at room temperature for 20 minutes. Punch 200 μL into each well of a 96-well plate, and punch 3 wells into each test tube. Use a microplate reader to measure the absorbance at a wavelength of 490 nm, using distilled water as a blank control, to obtain a standard curve (such as Figure 1 shown).

[0090] Determination of polysaccharide content: Prepare a 1 mg / mL solution of total polysaccharides from Jasmine flower with distilled water. Dilute 10-fold with distilled water, pipette 200 μL of the solution, and place it into three wells of a 96-well plate. Add phenol and sulfuric acid in the appropriate proportions. Let the plate rest for 10 minutes, shake well, and let it stand at room temperature for 20 minutes. Measure the absorbance at 490 nm using a microplate reader. Calculate the polysaccharide content (%) using the following formula: (C × V2 × D) / (W × V1 × 10) × 100% (Formula 2); where C is the sugar content calculated using the standard curve (mg); V2 is the total volume of the extract (mL); V1 is the volume used for measurement (mL); D is the dilution factor; and W is the sample weight (g).

[0091] The glucose standard curve was obtained by phenol-sulfuric acid method. Figure 1 As shown, the regression equation is y = 6.8588x-0.0177; the goodness of fit of the equation is R 2 = 0.999. In the range of 0-0.14 mg / mL, there is a good linear relationship between glucose concentration and absorbance.

[0092] The absorbance of total polysaccharides was measured and substituted into the regression equation to determine the polysaccharide concentration. Substituting this into (Equation 2) yielded a total polysaccharide content of 66.03%. Many factors affect polysaccharide concentration and purity, primarily including: higher extraction temperatures can cause polysaccharide decomposition, and more Sevage deproteinization cycles also result in polysaccharide loss.

[0093] 3. Ultraviolet spectrum analysis of frangipani polysaccharide

[0094] The purified frangipani polysaccharide was dissolved in ultrapure water to prepare a polysaccharide solution with a concentration of 1 mg / mL, and the ultraviolet absorption spectrum of the polysaccharide solution was scanned (200-600 nm) using a UV-visible spectrophotometer.

[0095] The results are as follows Figure 2 As shown in the figure, it can be seen that aromatic amino acids in proteins (such as tryptophan, tyrosine, and phenylalanine) have characteristic absorption peaks near 280 nm, and frangipani polysaccharide has a small absorption peak near 280 nm, indicating that it contains a small amount of protein; chlorophyll, carotenoids, etc. usually have characteristic absorption peaks near 380 nm, and frangipani polysaccharide has no absorption peak at 380 nm, indicating that it does not contain these pigments or the pigment content is extremely low.

[0096] 4. Infrared spectroscopy analysis of frangipani polysaccharides

[0097] Take a certain amount of the dried frangipani polysaccharide sample after purification, mix it with dry KBr powder and press it into tablets. -1Perform infrared spectrum scanning within the range and record the infrared spectrum.

[0098] The results are as follows Figure 3 As shown in the figure, it can be seen that the frangipani polysaccharide is at 3414.351cm -1 The strong absorption peak is caused by the stretching vibration of OH, which is a characteristic peak of sugars; it proves that the substance is sugar. -1 The absorption peak at wave number 1748.156 cm is the characteristic peak of CH stretching vibration; -1 This is the characteristic absorption peak of carboxyl group, indicating that frangipani polysaccharide contains carboxyl group. -1 The observed absorption peaks are associated with the stretching of COC and COH, indicating the possible presence of a pyranose ring.

[0099] 5. Determination of relative molecular weight of frangipani polysaccharide

[0100] The molecular weight of the purified frangipani polysaccharide was determined by high-performance gel permeation chromatography (HPGPC). The chromatographic conditions were as follows: mobile phase: 0.05 M NaCl solution; column: BRT105-103-101 tandem gel column (8 × 300 mm); flow rate: 0.7 mL / min; column temperature: 40°C; injection volume: 25 μL; detector: differential refractive index detector (RID-20A); and analysis time: 70 min.

[0101] The molecular weight spectrum of frangipani polysaccharide is as follows Figure 4 As shown in the figure, it can be seen that the frangipani polysaccharide peaks at 34.879 min, its relative peak area is 100%, and its molecular weight is 69445 Da.

[0102] 6. Determination of monosaccharide composition of frangipani polysaccharide

[0103] Monosaccharide composition was determined using ion chromatography. Accurately weigh 5 mg of the purified frangipani polysaccharide sample and place it in an ampoule. Add 2 mL of 3M TFA and hydrolyze at 120°C for 3 h. Accurately transfer the acid hydrolysis solution to a tube, blow dry with nitrogen, add 5 mL of water, and vortex to mix. Aspirate 100 µL of the solution and add 900 µL of deionized water. Centrifuge at 12,000 rpm for 5 min. The supernatant was then analyzed by IC.

[0104] The results are shown in Table 1 and Figure 5As shown, it can be seen from the peak time of standard polysaccharides and polysaccharide samples that the main monosaccharide composition and molar percentage of frangipani polysaccharide are rhamnose (Rha): arabinose (Ara): galactose (Gal): glucose (Glc): xylose (Xyl): mannose (Man): galacturonic acid (GalA) = 0.019: 0.109: 0.174: 0.220: 0.021: 0.022: 0.434; among them, the monosaccharides with the highest content are galacturonic acid (GalA), glucose (Glc), galactose (Gal) and arabinose (Ara).

[0105] Table 1

[0106]

[0107] Example 2 Application of Jasmine Flower Polysaccharide in the Development of Drugs for Treating APAP-Induced Acute Liver Injury

[0108] This example verifies the effect of frangipani polysaccharide on APAP-induced liver injury in BALB / c mice. The specific verification method is as follows:

[0109] 1. Animals and groups

[0110] Animal model: 35 commercially available SPF-grade BALB / c male mice, aged 6-8 weeks and weighing 20-24 g were used. After one week of adaptive feeding, the mice were randomly divided into five groups of seven mice each, with an average weight of 24 g per group.

[0111] Groups: (1) blank control group (CON); (2) model group (APAP, i.e., MOD group); (3) positive control group (200 mg / kg·BW, NAC); (4) low-dose jasmine flower polysaccharide group (60 mg / kg·BW, SP-L); (5) high-dose jasmine flower polysaccharide group (120 mg / kg·BW, SP-H).

[0112] 2. Experimental methods

[0113] 1) Establishment of APAP-induced acute liver injury model in BALB / c mice

[0114] The mice were gavaged continuously for 14 days at a dose of 0.1 mL / 10 g. The blank control group and the model group were gavaged with pure water, and the other treatment groups were gavaged with the above dosage. Two hours after gavage on the 14th day, the mice except the normal group were intraperitoneally injected with APAP at a dose of 300 mg / kg to induce an acute liver injury model. The normal group mice were injected with pure water. To stabilize the model and eliminate the influence of diet on modeling, the mice were fasted for 15 hours before the injection of APAP. 18 hours after injection, the body weight was recorded before death. The eyeballs were removed and blood was collected. The blood was placed in a 1.5 mL ordinary centrifuge tube and placed at room temperature for more than 2 hours. The supernatant was placed in another new centrifuge tube as serum and stored at -80°C until use. After blood collection, mice were sacrificed, and the liver and spleen were removed and washed in physiological saline to remove excess blood. Liver morphology was photographed, and the liver and spleen were weighed for calculation of organ coefficients: a portion of the liver tissue was fixed in 4% paraformaldehyde solution, and the remaining portion was placed in a cryovial, rapidly frozen in liquid nitrogen, and stored at -80°C. This was used to calculate the liver and spleen organ coefficients.

[0115] 2) Measurement of mouse body weight and liver and spleen indices

[0116] Body weight was recorded every two days and before death, and the liver and spleen were weighed. The organ coefficient was calculated according to the following formula: organ coefficient = liver or spleen mass (mg) / mouse body weight (mg) × 100%.

[0117] The results of mouse body weight and liver and spleen indices are as follows Figure 6 As shown, from Figure 6 As shown in Figure A, the body weight of mice in each group did not change significantly during the 14 days of preventive administration, indicating that frangipani polysaccharide has no toxic effect on mice. However, after the injection of APAP, the weight of the mice decreased significantly within a short period of time, indicating that the mouse model was successfully established.

[0118] from Figure 6 It can be seen from the BC graph and weight graph in the figure that the liver and spleen indexes of the model group were significantly increased. After treatment with frangipani polysaccharide, the liver index and spleen index decreased to varying degrees.

[0119] 3) Liver phenotype and liver histopathological analysis

[0120] Experimental method: The liver tissue was removed from 4% paraformaldehyde and subjected to dehydration, paraffin embedding, sectioning, dewaxing, rehydration, HE staining, and hematoxylin-eosin staining. Finally, the slides were sealed with neutral gum and dried in a fume hood overnight. The slides were observed and photographed using an upright bright-field microscope.

[0121] Liver phenotype and liver histopathological analysis results are as follows Figure 7 As shown, from Figure 7 As shown in Figure A, liver morphology shows that in the APAP-induced liver injury model group, the liver is clearly congested and enlarged, with a noticeable graininess. However, after pretreatment with frangipani polysaccharide, the dark red congestion on the liver surface is effectively alleviated, and the graininess is significantly reduced.

[0122] from Figure 7 As shown in Figure B, HE staining of the pathological sections shows that APAP intoxication causes severe hepatocyte necrosis, nuclear condensation, and massive inflammatory cell infiltration. In contrast, the livers of mice in the CON group showed no significant abnormalities. Pretreatment with NAC, SP-L, and SP-H alleviated APAP-induced liver damage, with a significant decrease in necrotic area and inflammatory cell infiltration.

[0123] 4) Detection of mouse serum biochemical indicators

[0124] Experimental methods: Mouse serum was taken out and diluted with normal saline for a certain multiple before direct testing. AST and ALT were determined according to the operating instructions of the Nanjing Jiancheng kit, and inflammatory factors (IL-6, IL-1β, TNF-α) in the serum were detected using the Xinbosheng QuantiCyto○RELISA kit.

[0125] Serum biochemical index test results such as Figure 8 As shown, from Figure 8 As can be seen from the AB graph in the figure, compared with the normal group, the ALT and AST levels of the MOD group were significantly increased (P<0.0001), but the ALT and AST levels were significantly decreased after intervention with frangipani polysaccharide (the results are shown), indicating that frangipani polysaccharide can improve APAP-induced acute liver injury in mice.

[0126] from Figure 8 Results from serum inflammatory cytokine measurements (Figure CE) show that compared with the CON group, the MOD group had significantly elevated serum TNF-α, IL-1β, and IL-6 levels (p < 0.01). However, pretreatment with SP-L and SP-H reversed these elevated inflammatory factors, suggesting that frangipani polysaccharides may have a protective effect against APAP-induced liver injury by inhibiting inflammatory responses.

[0127] 5) Detection of biochemical indicators in mouse liver

[0128] Experimental method: 20-50 mg of liver tissue was cut and placed in a 1.5 ml centrifuge tube. Normal saline was added as a homogenization medium at a ratio of 1:9 between tissue and normal saline. A 10% liver homogenate was prepared using a manual tissue homogenizer. The homogenate was diluted with normal saline a certain multiple to obtain the optimal detection concentration, and then used for the detection of biochemical indicators. The contents of SOD, MDA, GSH, CAT, and ROS were measured in sequence according to the operating instructions of the kit.

[0129] The results are as follows Figure 9 As shown in the figure, compared with the CON group, the MOD group showed significantly decreased levels of SOD, CAT, and GSH in the liver (P<0.01), while significantly increased levels of MDA and ROS (P<0.01). Pretreatment with frangipani polysaccharide significantly increased the enzyme activities of SOD, GSH, and CAT, and decreased the levels of MDA and ROS. These results suggest that frangipani polysaccharide can enhance antioxidant activity and protect the liver from damage.

[0130] Jasmine is a winding, vine-like evergreen ornamental shrub of the genus Jasmine (Oleaceae). It has a bitter taste, is neutral in nature, and is non-toxic. It enters the liver meridian and has traditional benefits of soothing the liver, relieving depression, promoting qi circulation, and relieving pain. Modern medicinal chemistry studies have revealed that Jasmine contains iridoids, triterpenes, flavonoids, and lignans. Among these are monomeric compounds such as oleuropein, hydroxytyrosol, hydroquinone, kaempferol, ethyl gallate, salidroside, and quercetin. This plant is a rich source of active ingredients for both medicinal and edible uses. The research team previously discovered that Jasmine extracts exhibit significant anti-inflammatory and antioxidant activities. This study is the first to demonstrate that Jasmine polysaccharides can improve APAP-induced acute liver injury.

[0131] While the embodiments of the present invention have been described in detail above with reference to the accompanying drawings, the present invention is not limited to the embodiments described above. Various modifications may be made within the scope of knowledge possessed by a person skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof may be combined with one another unless there is a conflict.

Claims

1. A frangipani polysaccharide, characterized in that: The monosaccharides in the frangipani polysaccharide are composed of rhamnose, arabinose, galactose, glucose, xylose, mannose and galacturonic acid; the molar ratio of rhamnose, arabinose, galactose, glucose, xylose, mannose and galacturonic acid is (14-22): (100-120): (160-185): (200-240): (16-25): (18-27): (400-450); The preparation method of the frangipani polysaccharide comprises the following steps: The frangipani flower sample was subjected to defatting, water extraction, alcohol precipitation, protein removal and dialysis in sequence to obtain frangipani flower polysaccharide; The water extraction is carried out by combining ultrasonic extraction and water bath extraction; In the alcohol precipitation step, the volume ratio of the water extract to ethanol is 1:5; The dialysis was performed using a dialysis bag with a molecular weight cut-off of >8000Da.

2. The frangipani polysaccharide according to claim 1, characterized in that The molecular weight of the frangipani polysaccharide is 69445 Da.

3. The preparation method according to claim 2, characterized in that The dialysis time is 1-3 days.

4. Use of the frangipani flower polysaccharide according to claim 1 or 2 in the preparation of medicines for treating acute liver injury.

5. Use of the frangipani polysaccharide according to claim 1 or 2 in preparing functional foods for assisting in improving acute liver injury.

6. The use according to claim 4 or 5, characterized in that The acute liver injury includes acute liver injury caused by APAP.

7. The use according to any one of claims 4 to 6, characterized in that The frangipani polysaccharide has the function of inhibiting the expression of inflammatory factors; The inflammatory factors include IL-1β, IL-6, and TNF-α.

8. The use according to any one of claims 4 to 6, characterized in that: The frangipani flower polysaccharide can reduce serum ALT and AST levels.

9. The use according to any one of claims 4 to 6, characterized in that: The frangipani polysaccharide can enhance the activities of catalase, superoxide dismutase and / or glutathione.

10. The use according to any one of claims 4 to 6, characterized in that: The frangipani polysaccharide can reduce the content of malondialdehyde and / or active oxygen.

11. A medicine, characterized in that: The medicine comprises the frangipani polysaccharide according to claim 1 or 2.

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