Hawthorn leaf pectic polysaccharide with lipid-lowering activity as well as preparation method and application of hawthorn leaf pectic polysaccharide

Abstract

The invention discloses hawthorn leaf pectic polysaccharide with lipid-lowering activity and a preparation method and application thereof, and belongs to a preparation method of plant polysaccharide, the preparation method comprises the following steps: refluxing hawthorn leaves with ethanol, discarding monosaccharide and small molecule compounds, refluxing with hot water to extract medicinal materials, removing protein through an enzymolysis method and a Sevage reagent method, decolorizing with polyamide resin, and drying to obtain the hawthorn leaf pectic polysaccharide with lipid-lowering activity. The crude polysaccharide is separated through DEAE-52 column chromatography, and finally, the crude polysaccharide is further purified through Sephadex G-100, so that the uniform purified polysaccharide is obtained. Tests on pancreatic lipase inhibition, taurocholate binding capacity and glycocholate binding rate show that the compound has remarkable lipid-lowering activity and is expected to become a potential natural lipid-lowering preparation.

Description

Technical Field

[0001] The invention belongs to a method for preparing plant polysaccharides, and particularly relates to extracting a plant polysaccharide with lipid-lowering activity from hawthorn leaves and application of the polysaccharide in the fields of pharmaceuticals and food. Background Art

[0002] There are eighteen species of Crataegus in the Rosaceae family in my country, which can be roughly divided into four categories based on their distribution: northern hawthorn, southern hawthorn, Yunnan hawthorn, and Guangdong hawthorn. Hawthorn leaves, the dried leaves of Crataegus pinnatifida or Crataegus pinnatifida, have the effects of promoting blood circulation and removing blood stasis, dredge meridians and relieve pain, and clear turbidity and lower lipids. Hawthorn leaves have a complex chemical composition, with flavonoids and terpenes being the two most important classes of active substances. Furthermore, little research has been conducted on the structure and efficacy of hawthorn leaf polysaccharides. With the advancement of modern pharmaceutical research, it has been discovered that hawthorn leaf polysaccharides have lipid-lowering effects, but their structure remains unclear, and the mechanisms of lipid-lowering are also poorly understood.

[0003] Polysaccharides are a class of natural macromolecules with biological activity that have garnered widespread attention in recent years for their potential to regulate blood lipids. Studies have shown that natural polysaccharides can bind to substances such as bile salts and cholesterol in the blood, preventing their reabsorption and thus effectively lowering blood lipid levels. With increasing awareness of health, the development of natural, safe, and effective lipid-lowering drugs is becoming increasingly important. This study aims to identify polysaccharide components from hawthorn leaves with potential lipid-lowering properties, clarify the structure of the polysaccharides, and provide a theoretical basis for the development of polysaccharide lipid-lowering drugs. Summary of the Invention

[0004] The primary objective of the present invention is to provide a method for preparing a hawthorn leaf pectin polysaccharide with lipid-lowering activity. The method uses hot water extraction to obtain a crude polysaccharide aqueous extract. This extract is then deproteinized, depigmented, and subjected to vacuum rotary evaporation to obtain an extract. The extract is then separated by column chromatography to obtain a purified polysaccharide. The polysaccharide's structure is analyzed to reveal a pectin structure containing RG-I and XGA domains, demonstrating significant lipid-lowering activity.

[0005] The purpose of the present invention is achieved through the following technical solutions:

[0006] A method for preparing hawthorn leaf pectin polysaccharide with lipid-lowering activity comprises the following steps:

[0007] (1) After the dried hawthorn leaf raw material is pulverized, ethanol is added to perform reflux degreasing treatment;

[0008] (2) removing the alcohol-soluble supernatant, collecting the defatted residue, and air-drying;

[0009] (3) adding deionized water to cover the medicinal material and performing hot reflux extraction under boiling conditions;

[0010] (4) concentrating the extract under reduced pressure;

[0011] (5) Add ethanol to precipitate, centrifuge, and remove the supernatant;

[0012] (6) dissolving the precipitate with deionized water, adding a neutral enzyme for enzymatic hydrolysis, cooling after the enzymatic hydrolysis, centrifuging, and concentrating the supernatant;

[0013] (7) Sevag reagent was added to the supernatant, stirred, and centrifuged to concentrate the supernatant;

[0014] (8) decolorizing the concentrated supernatant using a polyamide resin, collecting and concentrating the eluate to obtain a crude polysaccharide extract;

[0015] (9) Using a DEAE-52 Cellulose column, the crude polysaccharide was separated using NaCl solution, collected, dialyzed, and freeze-dried to obtain the polysaccharide fraction YHLP-0.4;

[0016] (10) YHLP-0.4 was purified by Sephadex G-100 column chromatography, collected and lyophilized to obtain purified polysaccharide YHLP-2.

[0017] The above preparation method, wherein:

[0018] In the step (1), continuous reflux extraction is performed at 60° C. to 80° C. for 1 h to 4 h using 60% to 90% ethanol solution.

[0019] In the step (3), the reflux extraction time at 90° C.-100° C. is 2 h-3 h, and the extraction times are 2-3 times.

[0020] In the step (4), the extract is concentrated under reduced pressure at 35° C.-55° C. to a viscous extract.

[0021] In step (5), four times the volume of 60%-90% ethanol was added for precipitation, the mixture was kept at 4° C. overnight, centrifuged, and the supernatant was removed.

[0022] The enzymatic hydrolysis reaction conditions in step (6) are as follows: adding neutral protease at a ratio of 1%-3% (g / g), placing in a 50°C constant temperature water bath for 0.5h-3h, and then placing in an 80°C-90°C water bath for 5min-10min to inactivate the enzyme.

[0023] In step (7), a quarter volume of Sevag reagent (dichloromethane: n-butanol = 4:1, v / v) was added.

[0024] The concentration of the NaCl solution in step (9) is 0.3M-0.5M.

[0025] A hawthorn leaf polysaccharide (YHLP-2) with lipid-lowering activity was prepared using the above method. High-performance gel permeation chromatography determined the molecular weight of YHLP-2 to be 34.98 kDa. It is composed of Ara (22.4%), GalA (21.2%), Xyl (19.5%), Rha (19.4%), Gal (8.8%), Glu (3.4%), and Fuc (2.9%). The YHLP-2 polysaccharide includes RG-I and XGA domains. Ara represents arabinose, GalA represents galacturonic acid, Xyl represents xylose, Rha represents rhamnose, Gal represents galactose, Glu represents glucose, and Fuc represents fucose.

[0026] A hawthorn leaf polysaccharide (YHLP-2) with lipid-lowering activity was evaluated by its pancreatic lipase inhibition rate and bile salt binding effect. The lipid-lowering activity of this polysaccharide provides a new scientific basis for the functional efficacy of hawthorn leaves as a special lipid-lowering tea and health food, and for the in-depth development and utilization of special plant resources. It also provides more options for the development of lipid-lowering drugs.

[0027] A pharmaceutical composition comprising the hawthorn leaf polysaccharide (YHLP-2) with lipid-lowering activity described in the present invention and pharmaceutically acceptable excipients. The pharmaceutical composition is in the form of tablets, injections, capsules, granules, liquid preparations, and dry suspensions.

[0028] Beneficial effects of the present invention:

[0029] The polysaccharide preparation process of the present invention is simple, using primarily water, ethanol, and relatively low-toxic reagents such as neutral proteases. The resulting polysaccharide is highly pure and exhibits significant lipid-lowering activity. At a sample concentration of 16 mg / mL, YHLP-2 exhibited a pancreatic lipase inhibition rate of 91.13±2.13%. At 8 mg / mL, YHLP-2 exhibited a taurocholate binding capacity of 48.17±1.82%, and a glycocholate binding rate of 56.11±0.12%. Therefore, the polysaccharide preparation process described herein is environmentally friendly and has broad application prospects, providing a new scientific basis for the in-depth development and utilization of unique plant resources. Furthermore, zebrafish Oil Red O staining, BMI determination, and TC and TG content measurements demonstrated YHLP-2's ability to reduce hyperlipidemia and its potential to regulate obesity-related indicators, demonstrating its potential as a natural lipid-lowering agent.

[0030] The process of the method of the present invention is green and environmentally friendly and meets the safety requirements in the food field. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 is the infrared spectrum of YHLP-2 in the present invention;

[0032] Figure 2 is the YHLP-2 of the present invention 1 H NMR (600 MHz, D O) spectrum;

[0033] Figure 3 is the YHLP-2 of the present invention 13 C NMR (150 MHz, D O) spectrum;

[0034] Figure 4 is the YHLP-2 of the present invention 1 H- 1 H COSY (600 MHz, D O) spectrum;

[0035] Figure 5 HSQC (600 MHz, D2O) spectrum of YHLP-2 in the present invention;

[0036] Figure 6 is the HMBC (600 MHz, D2O) spectrum of YHLP-2 in the present invention;

[0037] Figure 7 Figure 2 shows the monosaccharide composition of the mixed standard and the monosaccharide composition test of YHLP-2 in the present invention (Standard sample: chromatographic peaks 1-10 are mannose, ribose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose, arabinose, and fucose, respectively);

[0038] Figure 8 This is a test chart of the inhibitory effect of YHLP-2 on pancreatic lipase in the present invention;

[0039] Figure 9 The figure shows the bile salt standard curve and the bile salt binding test of YHLP-2 in the present invention; wherein A is the bile salt standard curve and B is the bile salt binding test of YHLP-2;

[0040] Figure 10 Figure 2 is an Oil Red O staining image showing the effects of YHLP-2 on the liver and gastrointestinal tract of zebrafish; b, c, and d are the blank group, model group, and positive drug group, respectively; e, f, and g are the low-, medium-, and high-dose groups of YHLP-2 (50 μg / ml, 150 μg / ml, and 300 μg / ml, respectively);

[0041] Figure 11 This is the test chart of YHLP-2 on the BMI of each group of zebrafish in the present invention; *** p<0.001, ### p<0.001;

[0042] Figure 12This is a test chart of the YHLP-2 in the present invention on the total cholesterol and triglyceride levels in zebrafish; wherein A is the total cholesterol content and B is the triglyceride content; * p<0.05, ** p<0.01, *** p<0.001, ### p<0.001. DETAILED DESCRIPTION

[0043] The present invention is further illustrated with reference to specific examples. However, the present invention is not limited thereto. The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and biological materials described are commercially available unless otherwise specified.

[0044] Example 1

[0045] A method for preparing hawthorn leaf polysaccharide with lipid-lowering activity includes extraction, separation, and purification processes. The specific steps are as follows:

[0046] (1) The dried hawthorn leaves were pulverized, added with 75% ethanol solution for reflux degreasing, and subjected to continuous reflux extraction at 80°C for 3 h;

[0047] (2) Remove the alcohol-soluble supernatant, collect the defatted residue, and air-dry it in a fume hood until the solvent residue is completely removed;

[0048] (3) Add deionized water to cover the medicinal materials, perform hot reflux extraction at 100°C for 3 h, repeat 3 times, and combine the extracts;

[0049] (4) The composite extract was concentrated under reduced pressure on a rotary evaporator at 50°C to a viscous extract;

[0050] (5) Add four times the volume of 80% ethanol (v / v) for precipitation, incubate at 4°C overnight, centrifuge, and remove the supernatant;

[0051] (6) Dissolve the precipitate with deionized water, add neutral protease at a ratio of 2.5% (g / g), place in a 50°C constant temperature water bath for 3 h, then place in a 90°C water bath for 10 min to inactivate the enzyme, cool, centrifuge, and concentrate the supernatant;

[0052] (7) Add one-quarter volume of Sevag reagent (dichloromethane: n-butanol = 4:1, v / v) to the supernatant, stir thoroughly, and centrifuge to concentrate the supernatant;

[0053] (8) decolorizing the concentrated supernatant using a polyamide resin, eluting with distilled water, collecting and concentrating the eluate to obtain a crude polysaccharide extract;

[0054] (9) The crude polysaccharide was separated using a DEAE-52 Cellulose column with 0.4 M NaCl solution, collected, dialyzed, and freeze-dried to obtain a polysaccharide fraction named YHLP-0.4;

[0055] (10) YHLP-0.4 was purified by Sephadex G-100 column chromatography, eluted with distilled water, collected and lyophilized to obtain purified polysaccharide YHLP-2.

[0056] The polysaccharide YHLP-2 prepared above was tested, and its infrared spectrum was as follows Figure 1 Other tests include:

[0057] ①Determination of monosaccharide composition

[0058] Weigh 5.0 mg of polysaccharide sample YHLP-2, add 1-2 mL of purified water to dissolve, add 5 mL of TFA (2 M), react at 110 ° C for 6 h, cool to room temperature, add anhydrous methanol and evaporate under reduced pressure several times until there is no sour taste, transfer the sample to a centrifuge tube with a small amount of purified water, and store at -20 ° C in the dark.

[0059] Weigh 1 mg each of rhamnose, mannose, glucuronic acid, ribose, galacturonic acid, xylose, glucose, galactose, arabinose, and fucose, dissolve in purified water to prepare a 2 mg / mL stock solution, and take 100 μL of each monosaccharide stock solution and mix well to prepare a mixed standard solution.

[0060] Take 200 μL each of the monosaccharide mixed standard and the acid hydrolysis sample (YHLP-2), add 400 μL of NaOH solution (0.6 M), mix quickly, then add 800 μL of PMP-methanol solution (0.5 M), mix and react at 80 ° C for 100 min, cool to room temperature, add 400 μL of HCl (0.3 M) to adjust the pH to neutral, add an equal volume of chloroform, mix and centrifuge, discard the lower layer, and repeat 3-4 times until the chloroform layer is colorless.

[0061] Mobile phase: 0.05M phosphate buffer: acetonitrile = 83:17 (v / v); Chromatographic column: C 18 (4.6×250 mm, 5 μm); HPLC: Agilent 1260 high performance liquid chromatograph; detector: UV detector; detection wavelength: 254 nm.

[0062] YHLP-2 is composed of Ara (22.4%), Gal A (21.2%), Xyl (19.5%), Rha (19.4%), Gal (8.8%), Glu (3.4%), and Fuc (2.9%). Figure 7 shown.

[0063] ②Determination of molecular weight of hawthorn leaf polysaccharide

[0064] Dextran standards YHLP-1 and samples YHLP-2 with molecular weights of 4000 Da (T4), 10000 Da (T10), 20000 Da (T20), 40000 Da (T40), 200000 Da (T200), and 500000 Da (T500) were prepared into 5 mg / mL solutions, filtered through a 0.45 μm syringe filter, and set aside. An RID-20A differential refractive index detector (RID) was used, along with a TSK gel G5000PW column (ID = 7.5 mm, L = 300 mm). The mobile phase consisted of 0.02 mol / L KH2PO4 at a flow rate of 0.6 mL / min. A standard curve was constructed using the logarithm of the molecular weight (LgMr) of the dextran standards and the peak elution time.

[0065] According to the molecular weight and peak time of T4, T10, T20, T40, T200, and T500 standard dextran, the standard regression equation is: LgMr = -0.53805RT + 12.46281(R 2 =0.9805). The peak shape of YHLP-2 was symmetrical and sharp, indicating that it was a homogeneous polysaccharide. The molecular weight was calculated to be 34.98 kDa based on the peak time into the regression equation.

[0066] ③NMR spectrum analysis

[0067] The polysaccharide sample YHLP-2 (30 mg) was placed in a nuclear magnetic resonance tube and fully dissolved in 0.6 mL of 99.9% D2O. One-dimensional and two-dimensional nuclear magnetic resonance spectral data were collected and analyzed on a nuclear magnetic resonance instrument. Figure 2-Figure 6 The hydrogen signal of YHLP-2 is concentrated between 3.0-5.5ppm, and the carbon signal is concentrated between 60-110ppm, which are typical polysaccharide signals. H 5.69 and δ H The signal around 5.80 corresponds to the α-Araf residue, δ H 5.31 and δ H 5.10 can be assigned to α-Rhap residues and α-Gal A residues, and the rest are in δ H Those in the range of 4.10-4.60 can be classified as Galp, Xylp, etc. HThe signal of 1.32 is the CH3-6 signal of rhamnose. Combined with the HMBC results, it was confirmed that YHLP-2 is a pectin polysaccharide with a main chain structure of (→2)-α-L-Rhap-(1→4)-α-D-GalAp-(1→), and contains two side chain structures: AG-I side chain is (→4)-β-Galp-(1→) and AG-II side chain is (→3)-β-Galp-(1→). This indicates that YHLP-2 is a pectin polysaccharide with both RG-Ⅰ (Rhamnogalacturonan I) and XGA (Xylogalacturonan) domains.

[0068] ④ Pancreatic lipase inhibition

[0069] Pancreatic lipase (PL) was prepared into a 5 mg / mL solution in TBS (pH 8.0), the supernatant was centrifuged, and stored at -20°C. 9.6 mg of YHLP-2 was weighed and dissolved in deionized water at concentrations of 0.5, 1, 2, 4, 8, and 16 mg / mL. 1.6 mg of 4-nitrophenyl laurate (4-NL) was weighed and dissolved in a small amount of anhydrous methanol and the volume was made up to 4 mL with TBS. After all preparations were completed, 25 μL of TBS, 25 μL of PL solution, and 50 μL of sample solutions of varying concentrations were added to a 96-well plate. After mixing, the plate was incubated at 37°C for 30 minutes. Then, 25 μL of a 0.4 mg / mL 4-NL solution was added, and the plate was incubated at 37°C with shaking for 2 hours. The absorbance was measured at 405 nm using a microplate reader. Orlistat was used as a positive control. The inhibition rate was calculated as follows:

[0070]

[0071] Wherein: A1 is blank group (deionized water replaces sample); A2 is blank background group (deionized water replaces sample, TBS replaces pancreatic lipase); A3 is sample group; A4 is sample background group (TBS replaces pancreatic lipase).

[0072] Depend on Figure 8 It can be seen that when the concentration reaches 16 mg / mL, the inhibition rate of YHLP-2 on pancreatic lipase is 91.13±2.13%.

[0073] ⑤ Bile salt binding

[0074] a. Bile salt standard curve:

[0075] Standard curves for sodium taurocholate and sodium glycocholate were prepared separately. Two bile salt solutions with a concentration of 0.3 mM were prepared using PBS solution (0.1 M, pH = 6.3). 0, 0.1, 0.5, 1.0, 1.5, 2.0, and 2.5 mL of these solutions were respectively taken into stoppered test tubes, and the volume was made up to 2.5 mL with PBS solution. 7.5 mL of 60% sulfuric acid solution was added, and the mixture was heated in a 70°C water bath for 20 min, followed by an ice bath for 5 min. The absorbance was measured at 387 nm to establish standard curves of absorbance and different concentrations of sodium taurocholate and sodium glycocholate.

[0076] b.YHLP-2 bile salt binding:

[0077] YHLP-2 was prepared into an 8 mg / mL stock solution in two separate 50 mL plastic centrifuge tubes. 3 mL of pepsin solution and 1 mL of 0.01 M HCl were then added to the solution. The tubes were then placed in a 37°C shaker and shaken for 1 hour to simulate the gastric environment. 200 μL of 0.1 M NaOH was then added to adjust the pH to 6.3. 4 mL of trypsin solution was then added and the tubes were shaken at 37°C for 1 hour to simulate the intestinal environment. Add 4 mL of sodium glycocholate and sodium taurocholate solution (0.4 mM) to each of the two centrifuge tubes, shake them again at 37°C for 1 hour, and centrifuge them at 4000 rpm for 20 minutes. Take 2.5 mL of the supernatant and add 7.5 mL of 60% sulfuric acid solution. Incubate in a 70°C water bath for 20 minutes, then in an ice bath for 5 minutes. Measure the absorbance at 387 nm and substitute it into the standard curve. Use cholestyramine as a positive control. The formula for bile salt binding rate is as follows:

[0078]

[0079] Where: C0 is the initial concentration of sodium taurocholate / sodium glycocholate; C1 is the unbound concentration of sodium taurocholate / sodium glycocholate in the supernatant.

[0080] Depend on Figure 9 It can be seen that at 8 mg / mL, the binding capacity of YHLP-2 to taurocholate was 48.17±1.82%; the binding rate to glycocholate was 56.11±0.12%.

[0081] ⑥ Lipid-lowering activity experiment in zebrafish:

[0082] a. Zebrafish Modeling:

[0083] Wild-type zebrafish embryos were cultured in a medium containing methylene blue and placed in an incubator at 27°C. No additional feeding was required during this period, and the water was changed twice daily. Floating egg shells in the culture medium were removed with a pipette. Zebrafish fry were cultured to 5 dpf (5 days post-fertilization) before grouping and drug administration. This study selected 100 5 dpf zebrafish (Daniorerio) larvae of the AB strain as experimental subjects and randomly divided them into two groups. The control group (n=10) was cultured in embryo culture medium, while the experimental group (n=90) was cultured in a medium containing 1% (w / v) egg yolk powder. Intermittent feeding was used, with daily feeding for 12 hours for two consecutive days to establish the experimental model.

[0084] b. Binding of YHLP-2 to zebrafish:

[0085] After zebrafish modeling was completed, the experimental groups were randomly assigned to eight subgroups for subsequent treatment. The specific grouping scheme was as follows: the hyperlipidemia model group was transferred to conventional culture medium for continued culture; the positive control group was placed in culture medium containing 150 μg / mL simvastatin; and the remaining three experimental groups were exposed to low, medium, and high doses of YHLP-2 solutions at 50 μg / mL, 150 μg / mL, and 300 μg / mL, respectively. The animals were fed for 12 hours and then transferred to conventional culture medium after the administration was completed. The intervention effect of YHLP-2 on dyslipidemia was evaluated by Oil Red O staining, BMI measurement, and TC and TG levels.

[0086] Depend on Figure 10 It can be seen that YHLP-2 can effectively regulate zebrafish lipid metabolism and reduce the level of lipid accumulation in the zebrafish stomach and liver.

[0087] Depend on Figure 11 It can be seen that the low, medium and high dose groups of YHLP-2 significantly reduced the BMI of hyperlipidemic zebrafish.

[0088] Depend on Figure 12 It can be seen that YHLP-2 exhibits significant lipid-lowering activity in the hyperlipidemia zebrafish model.

[0089] The invention provides a method for extracting hawthorn leaf polysaccharides. The prepared hawthorn leaf polysaccharide extract has high purity and is subjected to structural analysis and lipid-lowering activity testing.

Claims

1. A method for preparing hawthorn leaf pectin polysaccharide with lipid-lowering activity, characterized in that: The steps include: (1) After the dried hawthorn leaf raw material is pulverized, ethanol is added to perform reflux degreasing treatment; (2) removing the alcohol-soluble supernatant, collecting the defatted residue, and air-drying; (3) adding deionized water to cover the medicinal material and performing hot reflux extraction under boiling conditions; (4) concentrating the extract under reduced pressure; (5) Add ethanol to precipitate, centrifuge, and remove the supernatant; (6) dissolving the precipitate with deionized water, adding a neutral enzyme for enzymatic hydrolysis, cooling after the enzymatic hydrolysis, centrifuging, and concentrating the supernatant; (7) Sevag reagent was added to the supernatant, stirred, and centrifuged to concentrate the supernatant; (8) decolorizing the concentrated supernatant using a polyamide resin, collecting and concentrating the eluate to obtain a crude polysaccharide extract; (9) Using a DEAE-52 Cellulose column, the crude polysaccharide was separated using NaCl solution, collected, dialyzed, and freeze-dried to obtain the polysaccharide fraction YHLP-0.4; (10) YHLP-0.4 was purified by Sephadex G-100 column chromatography, collected and lyophilized to obtain purified polysaccharide YHLP-2.

2. The method for preparing a hawthorn leaf pectin polysaccharide with lipid-lowering activity according to claim 1, characterized in that: In the step (1), continuous reflux extraction is performed at 60° C. to 80° C. for 1 h to 4 h using 60% to 90% ethanol solution.

3. The method for preparing a hawthorn leaf pectin polysaccharide with lipid-lowering activity according to claim 1, characterized in that: In the step (3), the reflux extraction time at 90° C.-100° C. is 2 h-3 h, and the extraction times are 2-3 times.

4. The method for preparing a hawthorn leaf pectin polysaccharide with lipid-lowering activity according to claim 1, characterized in that: In step (5), four times the volume of 60%-90% ethanol was added for precipitation, the mixture was kept at 4° C. overnight, centrifuged, and the supernatant was removed.

5. The method for preparing a hawthorn leaf pectin polysaccharide with lipid-lowering activity according to claim 1, characterized in that: The enzymatic hydrolysis reaction conditions in step (6) are as follows: adding neutral protease, placing in a constant temperature water bath at 50°C for 0.5h-3h, and then placing in a water bath at 80°C-90°C for 5min-10min to inactivate the enzyme; adding a quarter volume of Sevag reagent in step (7); and the concentration of NaCl solution in step (9) is 0.3M-0.5M.

6. A hawthorn leaf polysaccharide with lipid-lowering activity, characterized in that: The method is prepared according to any one of claims 1 to 5.

7. The hawthorn leaf polysaccharide with lipid-lowering activity according to claim 6, characterized in that: The polysaccharide has a molecular weight of 34.98 kDa and is composed of Ara, Gal A, Xyl, Rha, Gal, Glu and Fuc, including RG-Ⅰ and XGA domains.

8. Use of the hawthorn leaf polysaccharide with lipid-lowering activity according to claim 6 or 7 in the preparation of lipid-lowering health foods and lipid-lowering drugs.

9. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the hawthorn leaf polysaccharide with lipid-lowering activity according to claim 6 or 7 and pharmaceutically acceptable excipients, wherein the dosage form of the pharmaceutical composition is selected from tablets, injections, capsules, granules, liquid preparations, and dry suspensions.

10. Use of the pharmaceutical composition according to claim 9 in the preparation of lipid-lowering drugs.

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