A polysaccharide with auxiliary lipid-lowering and weight-reducing effects, and a preparation method and application thereof

Abstract

The application discloses a polysaccharide with the functions of assisting in reducing fat and losing weight, and a preparation method and application thereof. The polysaccharide is obtained by extracting a crude extract from plant raw materials and performing multi-stage separation and purification, and has the functions of assisting in reducing fat and losing weight. The polysaccharide can reduce or control weight gain, reduce fat content or lipid load, improve or restore liver morphology, inhibit or reduce liver weight gain, inhibit or reverse liver steatosis, improve sugar and lipid metabolism, improve intestinal flora structure and the like.

Description

Technical Field

[0001] This invention belongs to the fields of biomedicine and food technology, and in particular relates to a polysaccharide with auxiliary lipid-lowering and weight-loss effects, its preparation method and application. Background Technology

[0002] Obesity is a chronic metabolic disease caused by long-term energy intake exceeding energy expenditure, and has been listed by the World Health Organization as one of the leading preventable risk factors for death worldwide. Statistics show that the global overweight and obese population has exceeded 2 billion and is continuing to rise. Obesity not only significantly increases the incidence of metabolic syndrome-related diseases such as type 2 diabetes, non-alcoholic fatty liver disease, hypertension, and atherosclerosis, but also impairs the function of multiple organs through a chronic low-grade inflammatory state, severely affecting patients' quality of life and life expectancy.

[0003] However, commonly used weight-loss drugs such as orlistat often have adverse reactions such as diarrhea, steatorrhea, and liver and kidney damage, and their long-term use has obvious limitations.

[0004] Therefore, finding safe, efficient, and multi-target synergistic novel anti-obesity active substances from natural products has become a technical problem that urgently needs to be solved in food nutrition and medicinal chemistry research. Summary of the Invention

[0005] To address at least some of the technical problems in the prior art, this invention provides a polysaccharide with auxiliary lipid-lowering and weight-loss effects, its preparation method, and its applications. Specifically, this invention includes the following:

[0006] In a first aspect, the present invention provides a polysaccharide with an auxiliary lipid-lowering and weight-loss effect, comprising a main chain and side chains. The main chain is formed by α-D-pyranose glucose residues linked by (1→6) glycosidic bonds, and the side chains are formed by two β-D-pyranose glucose residues linked by (1→4) glycosidic bonds. The side chains are connected to the main chain through the C4 position of the glucose residues in the main chain. The polysaccharide has the structure shown in Formula I. Formula I; Where n is selected from natural numbers from 2 to 20.

[0007] In some embodiments, the polysaccharide with lipid-lowering and weight-loss-enhancing effects according to the present invention includes at least one of the following: (1) Reduce or control weight gain; (2) Reduce fat content or lower lipid load; (3) Improve or restore liver morphology; (4) Inhibit or reduce liver weight gain; (5) Inhibit or reverse hepatic steatosis; (6) Improves glucose and lipid metabolism; (7) Improve the structure of gut microbiota.

[0008] A second aspect of the present invention provides a composition having an auxiliary effect in lowering lipids and reducing weight, comprising the polysaccharide described in the first aspect of the present invention.

[0009] In some embodiments, the composition according to the present invention having an auxiliary lipid-lowering and weight-loss effect is wherein the composition is a pharmaceutical composition or a food composition.

[0010] A third aspect of the present invention provides a method for preparing a polysaccharide with an auxiliary lipid-lowering and weight-loss effect according to the first aspect of the present invention, comprising the following steps: (1) Use an aqueous solvent to extract the plant material at 60-100℃ for 30-120 min to obtain an extract, add a precipitant to precipitate and remove the protein in the precipitate to obtain crude polysaccharide; (2) The crude polysaccharide is separated and purified to obtain the polysaccharide.

[0011] In some embodiments, according to the method for preparing polysaccharides with auxiliary lipid-lowering and weight-loss effects according to the present invention, the ratio of the plant raw material to the aqueous solvent is 1:(0.5-5).

[0012] In some embodiments, according to the method for preparing polysaccharides with auxiliary lipid-lowering and weight-loss effects according to the present invention, proteins in the precipitate are removed by the Sevag method.

[0013] In some embodiments, the method for preparing polysaccharides with lipid-lowering and weight-loss effects according to the present invention includes separation and purification comprising ion exchange chromatography and non-ionic gel chromatography.

[0014] In some embodiments, according to the method for preparing polysaccharides with adjuvant lipid-lowering and weight-loss effects according to the present invention, the separation and purification includes: The crude polysaccharide was separated by ion exchange chromatography, and the elution fraction corresponding to the first peak was collected. Using water as the mobile phase, the elution fraction corresponding to the first peak is passed through a non-ionic gel filter column to obtain the polysaccharide with the function of assisting in lowering lipids and losing weight.

[0015] A fourth aspect of the present invention provides the use of the polysaccharide according to the first aspect of the present invention in the preparation of a medicament or health food for assisting in lowering lipids and losing weight.

[0016] This invention extracts crude extracts from plant materials and performs multi-stage separation and purification to screen and obtain polysaccharides with single-component effects in assisting lipid-lowering and weight loss. The polysaccharides of this invention can improve metabolic disorders by improving glucose tolerance, correcting dyslipidemia, protecting the liver, and reversing the hypertrophy of fat deposits and adipocytes. They can also reshape the dysbiosis caused by obesity, achieving an auxiliary effect in lowering lipids and losing weight without suppressing appetite and energy intake. Furthermore, the polysaccharides of this invention have advantages such as high safety and good efficacy. Attached Figure Description

[0017] Figure 1 A is the elution curve of ion exchange chromatography, B is the elution curve of Sephacryl S-300 HR gel purification, C is the molecular weight determination result of the polysaccharide PASP of the present invention, D is the monosaccharide composition determination result of the polysaccharide PASP of the present invention, E is the ultraviolet spectral scanning result of the polysaccharide PASP of the present invention, F is the infrared spectral analysis result of the polysaccharide PASP of the present invention, and G is the Congo red test result of the polysaccharide PASP of the present invention.

[0018] Figure 2 The screening process for polysaccharide PASP, which has the effect of improving weight loss, is as follows: A represents the change in body weight of mice in each treatment group over time, and B and E represent the effects of each treatment group on body weight, liver weight, epididymal fat weight, and subcutaneous fat weight, respectively.

[0019] Figure 3 The polysaccharide of this invention 13 C NMR spectral analysis results.

[0020] Figure 4 The polysaccharide of this invention 1 H- 1 HCl COSY spectral analysis results.

[0021] Figure 5 The results are shown in the HSQC spectral analysis of the polysaccharide of this invention.

[0022] Figure 6 The results of HMBC spectral analysis of the polysaccharide of this invention are shown.

[0023] Figure 7 The results of TOCSY spectral analysis of the polysaccharide of this invention are shown.

[0024] Figure 8 A represents the drug administration diagram of the model mice, B and K represent the effects of different treatment groups on body weight, ALBII, ALT, AST, CREA-S, UREA, RBC, WBC, HGB, and PLT, respectively, and L represents the H&E staining histological results.

[0025] Figure 9A is a schematic diagram of drug administration; B is the effect of different treatment groups on body weight; C is the effect of different treatment groups on liver weight; D is the morphological observation results of different treatment groups; E and F are the effects of different treatment groups on tissue weight and food intake, respectively; G is the fatty liver disease activity score; H is the quantitative result of Oil Red O staining; and I is the H&E staining and Oil Red O staining results of the liver.

[0026] Figure 10 A represents the glucose tolerance test results of each treatment group; B represents the results of PASP improving glucose clearance in a dose-dependent manner; C represents the effects of each treatment group on serum parameters; DE represents the effects of each treatment group on serum ALT and AST; FG represents the effects of each treatment group on liver TG and TC; H represents the H&E staining results of adipose tissue; and IL represents the statistical analysis results of the effects of each treatment group on epididymis, subcutaneous tissue, perirenal tissue, and brown adipose tissue.

[0027] Figure 11 The regulatory effect of PASP on gut microbiota structure is shown in the figure. A is the Venn analysis plot, B is the Chao1 index box plot, C is the Simpson index box plot, D is the principal coordinate analysis plot, EJ is the relative abundance analysis results of phylum-level microbiota, KM is the relative abundance analysis results of key genera at the genus level, and O is the heatmap correlation analysis results of gut microbiota and metabolic phenotypic indicators. Detailed Implementation

[0028] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0029] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that the upper and lower limits of the range and each intermediate value between them are specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0030] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention.

[0031] polysaccharides In one aspect, the present invention provides a polysaccharide with an auxiliary lipid-lowering and weight-loss effect, comprising a main chain and side chains. The main chain is formed by α-D-pyranose glucose residues linked by (1→6) glycosidic bonds, and the side chains are formed by two β-D-pyranose glucose residues linked by (1→4) glycosidic bonds. The side chains are connected to the main chain through the C4 position of the glucose residues in the main chain. The polysaccharide has the structure shown in Formula I. Formula I; Wherein, n is selected from natural numbers from 2 to 20, preferably from 3 to 19, even more preferably from 4 to 18, further preferably from 5 to 17, more preferably from 6 to 16, and even more preferably from 7 to 15, for example 7, 8, 9, 10, 11, 12, 13, 14, 15. In a specific embodiment, n is 11.

[0032] In this invention, the auxiliary lipid-lowering and weight-loss effects include, but are not limited to, the following: reducing or controlling weight gain, reducing fat content or lipid load, improving or restoring liver morphology, inhibiting or reducing liver weight gain, inhibiting or reversing hepatic steatosis, improving glucose and lipid metabolism, and improving intestinal flora structure.

[0033] The present invention provides structural analysis and characterization of the obtained polysaccharide PASP. In a specific embodiment, high-performance gel permeation chromatography (HP-GPC) was used to determine the molecular weight, and the relative molecular weight of the polysaccharide PASP of the present invention was 2.63 kDa.

[0034] In the specific implementation plan, the polysaccharide PASP is detected by infrared spectroscopy analysis using IR mode, with a scanning wavenumber range of 4000 cm⁻¹. -1 -400 cm -1 The number of scans was 32. The polysaccharide PASP of this invention was measured at 3389 cm⁻¹. -1 The peak at 2927 cm⁻¹ represents the OH stretching vibration of carbohydrate molecules; -1 The peak at 1421 cm⁻¹ represents the CH stretching vibration of carbohydrate molecules. -1 It has a CH bending vibration peak at 1047 cm⁻¹; -1 The COH deformation vibration peak with a pyranose ring structure is present.

[0035] In the specific implementation plan, the specific chemical structure of the polysaccharide PASP was determined by nuclear magnetic resonance analysis, and β-D-Glc was found to be present in the polysaccharide PASP. p -(1→and→4)-β-D-Glc p -(1→、→4)-β-D-Glc p -(1→and→4,6)-α-D-Glc p -(1→、→6)-α-D-Glcp -(1→and→4,6)-α-D-Glc p -(1→ connection method, exists →6)-α-D-Glc p -(1→6)-α-D-Glc p -(1→ repeating structural unit.

[0036] Composition In one aspect, the present invention provides a composition having an auxiliary lipid-lowering and weight-loss effect, comprising the polysaccharide described in the present invention, wherein the polysaccharide may be the sole active ingredient having the auxiliary lipid-lowering and weight-loss effect.

[0037] In a preferred embodiment, the composition of the present invention is a pharmaceutical composition, further comprising a pharmaceutically acceptable carrier that participates in the transport or delivery of the drug from one organ or part of the body to another organ or part of the body. Each carrier is "acceptable," meaning it is compatible with other components of the formulation (e.g., the polysaccharides of the present invention) and does not harm the patient.

[0038] In this invention, the pharmaceutically acceptable carrier includes, but is not limited to, buffers, emulsifiers, colorants, diluents, fillers, wetting agents, binders, lubricants, sweeteners, and antioxidants. Examples of buffers include, but are not limited to, citrates, histidine, and succinates; examples of emulsifiers include, but are not limited to, polysorbates; examples of colorants include, but are not limited to, sodium copper chlorophyllin, betaine, curcumin, β-carotene, and anthocyanins; examples of diluents include, but are not limited to, physiological saline, aqueous buffer solutions, solvents, and dispersion media; fillers include, but are not limited to, sucrose, trehalose, and xylitol; wetting agents include, but are not limited to, water; binders include, but are not limited to, hydroxypropyl methylcellulose and povidone; lubricants include, but are not limited to, magnesium stearate and micronized silica; sweeteners include, but are not limited to, sucralose, acetylsupan, saccharin, sucrose, xylitol, mannitol, sorbitol, and aspartame; and antioxidants include, but are not limited to, ascorbic acid, sodium ascorbate, and tea polyphenols.

[0039] In a preferred embodiment, the composition of the present invention is a food composition, which further includes excipients. The excipients include, but are not limited to, thickeners, emulsifiers, stabilizers, sweeteners, acidity regulators, nutritional fortifiers, and preservatives. Examples of thickeners include, but are not limited to, konjac gum, carrageenan, agar, sodium alginate, etc.; examples of emulsifiers include, but are not limited to, glyceryl monostearate, glyceryl distearate, sucrose fatty acid esters, soybean lecithin, etc.; examples of stabilizers include, but are not limited to, sodium carboxymethyl cellulose, microcrystalline cellulose, propylene glycol alginate, etc.; examples of sweeteners include, but are not limited to, fructose syrup, xylitol, erythritol, maltitol, sorbitol, steviol glycosides, etc.; examples of acidity regulators include, but are not limited to, citric acid, lactic acid, malic acid, tartaric acid, fumaric acid, sodium citrate, sodium bicarbonate, etc.; examples of nutritional fortifiers include, but are not limited to, vitamin C, vitamin E, B vitamins, calcium carbonate, calcium lactate, ferrous sulfate, etc.; examples of preservatives include, but are not limited to, potassium sorbate, sodium benzoate, sodium dehydroacetate, etc.

[0040] Preparation method In one aspect, the present invention provides a method for preparing a polysaccharide with an auxiliary lipid-lowering and weight-loss effect according to the present invention, comprising steps (1) and (2), which are described in detail below.

[0041] Step (1) of the present invention is to extract the plant raw material with an aqueous solvent at 60-100℃ for 30-120 min to obtain an extract, add a precipitant to precipitate and obtain a precipitate, remove the protein in the precipitate and obtain crude polysaccharide.

[0042] In this invention, the aqueous solvent includes, but is not limited to, distilled water, deionized water, reverse osmosis water, and ultrapure water; the plant raw materials include, but are not limited to, dragon fruit, bird's nest fruit, custard apple, snake fruit, jaboticaba, cactus, oats, longan, goji berries, red dates, yam, and monk fruit; and the precipitant includes, but is not limited to, ethanol, propanol, isopropanol, and acetone.

[0043] In a preferred embodiment, step (1) of the present invention includes extracting the plant material using an aqueous solvent at 60-100°C (preferably 65-100°C, more preferably 70-100°C, further preferably 75-100°C, even more preferably 80-100°C, for example 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100°C) for 30-120 min (preferably 30-110 min, more preferably 30-100 min, even more preferably 30-90 min, even more preferably 30-80 min, for example 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80 min) to obtain an extract, and then rubbing the extract at 1000-5000 rpm (preferably 1200-4800 rpm, even more preferably 1400-4600 rpm, even more preferably 1600-4400 rpm). Centrifuge at rpm, more preferably 1800-4200 rpm, even more preferably 2000-4000 rpm, such as 2000, 2200, 2400, 2600, 2800, 3000, 3200, 3400, 3600, 3800, 4000 rpm, for 5-30 min (preferably 5-25 min, even more preferably 5-20 min, more preferably 5-15 min, such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 rpm, more preferably 1800-4200 rpm, even more preferably 2000-4000 rpm, for example 2000, 2200, 2400, 2600, 2800, 3000, 3200, 3400, 3600, 3800, 4000 rpm) for 5-30 min (preferably 5-25 min, even more preferably 5-20 min, more preferably 5-15 min, for example 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 rpm). The supernatant was obtained by adding a precipitant (e.g., ethanol with a final concentration of 70-90%, preferably 72-88%, more preferably 74-86%, even more preferably 76-84%, such as 76, 77, 78, 79, 80, 81, 82, 83, 84%) to precipitate at 0-10°C (preferably 1-9°C, even more preferably 2-8°C, such as 2, 3, 4, 5, 6, 7, 8°C) for 5-15 h (preferably 6-14 h, even more preferably 7-12 h, such as 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12) h) A precipitate is obtained, and the protein in the precipitate is removed by the Sevag method to obtain crude polysaccharide. The material-to-liquid ratio of the plant material to the aqueous solvent is 1:(0.5-5) g / mL, preferably 1:(0.6-4.8) g / mL, even more preferably 1:(0.7-4.6) g / mL, further preferably 1:(0.8-4.4) g / mL, more preferably 1:(0.9-4.2) g / mL, and even more preferably 1:(1-4) g / mL, for example 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4 g / mL. It is understood that, in order to better extract the active ingredients, the extraction process can be repeated several times, and conventional operations such as concentrating the supernatant or freeze-drying the crude polysaccharide can be performed; all of these are within the scope of protection of this invention.

[0044] Step (2) of the present invention is to separate and purify the crude polysaccharide to obtain the polysaccharide PASP.

[0045] In a preferred embodiment, step (2) of the present invention includes loading the crude polysaccharide aqueous solution onto an ion exchange chromatography column (e.g., but not limited to, a DEAE-52 anion exchange resin column), and performing gradient elution sequentially using 0 M NaCl, 0.1 M NaCl, 0.2 M NaCl, 0.3 M NaCl, and 0.5 M NaCl solutions as eluents at an elution rate of 0.1-5 mL / min (preferably 0.2-4 mL / min, more preferably 0.3-3 mL / min, further preferably 0.4-2 mL / min, and more preferably 0.5-1.5 mL / min, for example 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, and 1.5 mL / min), collecting the elution fraction corresponding to the 0 M NaCl; and loading the elution fraction corresponding to the 0 M NaCl onto a non-ionic gel filtration column (e.g., but not limited to, Sephacryl S-300). An HR column was used as the eluent, and water was used as the eluent at a rate of 0.1-2 mL / min (preferably 0.1-1.8 mL / min, more preferably 0.1-1.6 mL / min, even more preferably 0.1-1.4 mL / min, more preferably 0.2-1.2 mL / min, and even more preferably 0.3-0.6 mL / min, for example 0.3, 0.4, 0.5, 0.6 mL / min). The solution was then freeze-dried to obtain the polysaccharide PASP.

[0046] application In one aspect, the invention provides the use of the polysaccharide described in the invention in the preparation of a medicament or health food for assisting in lowering lipids and losing weight.

[0047] In this invention, lipid-lowering and weight loss are achieved by administering a therapeutically effective amount of the drug to the subject. Subjects include, but are not limited to, mammals, including but not limited to, humans, mice, rabbits, cats, dogs, cattle, sheep, and pigs.

[0048] The therapeutically effective dose described in this invention refers to a pharmaceutically recognized effective dosage, that is, the amount of the active compound (i.e., the polysaccharide of this invention) is sufficient to significantly improve the condition without causing serious side effects. The daily dosage of the drug is typically 10-5000 mg / kg, preferably 100-4500 mg / kg, even more preferably 200-4000 mg / kg, further preferably 300-3500 mg / kg, and more preferably 400-3000 mg / kg, for example 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000 mg / kg. It can be administered as a single dose once daily, or divided into multiple doses daily, or used at intervals.

[0049] In this invention, there are no particular limitations on the method of administration of the drug. Representative methods of administration include, but are not limited to, oral, rectal, and parenteral (intravenous, intramuscular, or subcutaneous) administration. Accordingly, the drug of this invention can be formulated into various clinically acceptable dosage forms, examples of which include, but are not limited to, decoctions, powders, pills, ointments, tablets, capsules, granules, and oral liquids.

[0050] The daily intake of the health food of the present invention is typically 10-5000 mg / kg, preferably 100-4500 mg / kg, even more preferably 200-4000 mg / kg, further preferably 300-3500 mg / kg, and more preferably 400-3000 mg / kg, for example 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, and 3000 mg / kg. It can be consumed once daily or divided into multiple servings throughout the day. The health food of the present invention can be made into various forms, such as, but not limited to, tablets, capsules, powders, oral liquids, candies, jellies, beverages, etc.

[0051] Example The following shows the preparation, characterization, and application of polysaccharides that have auxiliary lipid-lowering and weight-loss effects.

[0052] 1. Materials and Methods 1.1 Materials Fresh white-fleshed dragon fruit was purchased from a farmers' market in Changchun, Jilin Province, China. DEAE-cellulose and monosaccharide standards were purchased from Shanghai Yuanye (China). Sephacryl S-300 HR was purchased from GE Healthcare (USA).

[0053] 1.2 Extraction and purification of PASP 1.2.1 Extraction of PASP After washing and peeling, white-fleshed dragon fruit was homogenized using a homogenizer. Distilled water was added at a material-to-liquid ratio of 1:1 (m / v), and the mixture was boiled for 1 hour. The extract was centrifuged at 3000 rpm for 10 minutes to obtain the supernatant. 95% ethanol was slowly added to the supernatant for alcohol precipitation until the final ethanol concentration reached 80%. The mixture was allowed to stand at 4°C overnight for alcohol precipitation. The next day, the supernatant was collected, and the ethanol was recovered by rotary evaporation. The obtained extract was redissolved in distilled water, and the protein was removed using the Sevag method. An equal volume of Sevag reagent (chloroform: n-butanol = 4:1, v / v) was added, and the mixture was shaken thoroughly and centrifuged. The upper aqueous phase was collected, and the operation was repeated 5 times until no obvious protein layer was observed. The protein-removed aqueous solution was concentrated by rotary evaporation, and 95% ethanol was added for alcohol precipitation again. The supernatant was collected by centrifugation, and the ethanol was recovered by rotary evaporation. The obtained extract was freeze-dried to obtain crude dragon fruit polysaccharide.

[0054] 1.2.2 Ion exchange column chromatography The obtained crude polysaccharides were dissolved in distilled water and loaded onto a DEAE-52 cellulose column (2.5 × 40 cm). Gradient elution was performed with 0, 0.1, 0.2, 0.3, and 0.5 M NaCl solutions at a rate of 1 mL / min. The collected volume per tube was approximately 10 mL. After dialyzing, the polysaccharides were lyophilized to obtain dragon fruit polysaccharides (PASP-0, PASP-0.1, PASP-0.2, PASP-0.3, and PASP-0.5). The polysaccharide components were detected using the phenol-sulfuric acid method, and protein concentrations were determined using the Bradford assay.

[0055] 1.2.3 Screening of anti-obesity active polysaccharides Forty-two male C57BL / 6J mice were acclimatized for one week and then randomly divided into seven groups of six each: a normal control group (K), a high-fat model group (M), and dragon fruit polysaccharide groups (PASP-0, PASP-0.1, PASP-0.2, PASP-0.3, and PASP-0.5). Except for the normal control group, the other groups were fed a high-fat diet (60% fat content) to induce an obesity model in mice for eight consecutive weeks. Simultaneously, the polysaccharide groups were administered 1000 mg / kg of the corresponding polysaccharide extracts daily by gavage, while the normal control and high-fat model groups were administered the same volume of physiological saline daily by gavage. Weight changes, food intake, and water intake were monitored weekly. On the last day of the experiment, mice were fasted for 12 hours after the last administration. Liver, epididymal white adipose tissue (eWAT), inguinal white adipose tissue (iWAT), perirenal white adipose tissue (pWAT), and brown adipose tissue (BAT) were dissected, weighed, and stored at -80℃ for later use.

[0056] 1.2.4 Gel column chromatography The active ingredient was purified using a Sephacryl S-300 HR column with water as the eluent. The main elution peaks were collected, and the resulting polysaccharide was obtained after lyophilization. The total sugar content was determined using the phenol-sulfuric acid method, and the protein concentration was determined using the Bradford method.

[0057] 1.3 Structural Characterization 1.3.1 Molecular weight determination The purity and molecular weight of PASP were determined by high-performance gel permeation chromatography (HP-GPC). The PASP sample was dissolved in distilled water at a concentration of 5 mg / mL and filtered through a 0.22 μm filter. HP-GPC analysis was then performed using a TSK-gelG4000 PWXL F3393 column and a differential detector. The injection volume was set to 20 μL, the mobile phase to be 0.1 M NaNO3, and the flow rate to be 0.7 mL / min.

[0058] 1.3.2 Monosaccharide Composition Analysis To perform monosaccharide composition analysis, 2 mg of PASP sample was weighed and added to 2 mL of trifluoroacetic acid (2 mol / L) in a hydrolysis tube, and hydrolyzed at 110 °C for 2 h. After hydrolysis, the sample was transferred to an evaporating dish, incubated in a water bath at 70 °C, and repeatedly evaporated to dryness until pH=7. The hydrolysis product was dissolved in 1.5 mL of ultrapure water and centrifuged, then filtered through a 0.22 μm aqueous filter membrane. The monosaccharide composition of the hydrolysis product was then determined using high-performance anion exchange chromatography-pulse amperometric detection (HPAEC-PAD). A Thermo Scientific Dionex CarboPac™ PA100 column was used, with gradient elution using 20 mM NaOH and 100 mM NaOH + 500 mM NaAc as the eluent. The flow rate was set to 0.4 mL / min. Weigh out 5 mg each of fucose (Fuc), rhamnose (Rha), arabinose (Ara), glucosamine (GlcN), galactose (Gal), glucose (Glc), mannose (Man), xylose (Xyl), fructose (Fru), and glucuronic acid (GlcA), dissolve them in 1 mL of ultrapure water, filter through a 0.22 μm aqueous filter membrane, and dilute to prepare a 10 μg / mL standard solution.

[0059] 1.3.3 Ultraviolet (UV) Structure Analysis The PASP sample was dissolved in distilled water to prepare a 0.5 mg / mL solution, and its ultraviolet spectrum was recorded in the range of 190-800 nm using a NanoDrop One spectrometer (Thermo Fisher Scientific).

[0060] 1.3.4 Infrared (FT-IR) structural analysis The PASP sample was mixed with potassium bromide (KBr) at a mass ratio of 1:100 and ground into powder in an agate mortar. The powder was then compressed into tablets using a tablet press and detected using IR mode with a scanning wavenumber range of 4000 cm⁻¹. -1 up to 400 cm -1 The number of scans was 32.

[0061] 1.3.5 Congo Red Test A 2 mg / mL PASP solution was mixed with an 80 μM Congo red solution at a 1:1 volume ratio. NaOH solutions with concentrations ranging from 0 to 0.6 M were added to the reaction mixture. The solution was analyzed using UV-Vis spectrophotometry, scanning in the 300–600 nm range to determine the peak absorption wavelength.

[0062] 1.3.6 Methylation Analysis For methylation analysis, 2 mg of PASP sample was dissolved in 0.5 mL of DMSO. Under nitrogen protection, 20 mg of dry NaOH was added, and the mixture was sonicated until the polysaccharide was completely dissolved. Then, under nitrogen protection, 0.3 mL of iodomethane solution was added, and the mixture was stirred at room temperature in the dark for 2.5 h. The reaction was terminated by adding 0.3 mL of ultrapure water and stirring for 0.5 h. Subsequently, 1 mL of dichloromethane was added to wash the reaction tube, and the entire solution was transferred to a clean glass tube. 3 mL of ultrapure water was added to the glass tube for extraction. The lower organic phase was collected, and the extraction was repeated three times with ultrapure water. Excess NaSO4 was added to the lower layer to adsorb excess water, and the mixture was then transferred to a clean glass tube and dried under nitrogen. This reaction was repeated three times until the polysaccharide hydroxyl groups were completely substituted.

[0063] 1 mL of 2 M TFA was added to the methylated sample, and the reaction was carried out at 110 °C for 2 h. After hydrolysis, the sample was placed in an evaporating dish and heated to dryness in a water bath at 70 °C. A small amount of methanol was added and evaporated to dryness. This process was repeated 5 times until the pH of the aqueous solution was neutral. Then, 0.3 mL of ultrapure water was added, and 1 drop of 1% ammonia was added to adjust the pH to alkaline. 5 mg of NaBD4 was added to the hydrolysis product and mixed well. The mixture was stirred at room temperature and the reduction reaction was carried out for 12 h. After the reaction was completed, the pH was adjusted to neutral with acetic acid, and the reaction solution was evaporated to dryness. Methanol was added and the solution was washed repeatedly and evaporated to dryness. This process was repeated three times. Then, 1 mL of acetic anhydride:pyridine (1:1 = v / v) was added and the reaction was carried out at 90 °C for 2 h. 1 mL of ultrapure water was added to terminate the reaction. The sample was then extracted twice with 1 mL of dichloromethane. After thorough shaking, the dichloromethane phase was collected and evaporated to dryness.

[0064] Finally, 0.6 mL of dichloromethane was added for reconstitution, filtered through a 0.22 μm organic filter membrane, and analyzed by GC-MS. An Agilent 8890-7250 GC-MS system was used for detection, with an HP-5MS capillary column (0.25 μm, 30 m × 0.25 mm). The temperature program was as follows: initial temperature 80 °C, held for 2 min. Then, the temperature was increased to 320 °C at a rate of 15 °C / min and held for 2 min. High-purity helium was used as the carrier gas at a flow rate of 1 mL / min.

[0065] 1.3.7 Nuclear Magnetic Resonance Analysis 20 mg of PASP was dissolved in 0.5 mL of D₂O, and one-dimensional NMR spectroscopy was performed using an AVANCE III HD 700 MHz liquid NMR spectrometer. 1 H NMR, 13 C NMR and two-dimensional HSQC, COSY and HMBC spectra. Chemical shifts are expressed in parts per million (ppm), with the chemical reference shift for 1H set at 4.78 ppm (D2O).

[0066] 1.4 Animal Experiments Male C57BL / 6J mice aged 6-8 weeks were purchased from Changchun Yisi Animal Hospital (License No.: 2023-0002). The mice were housed at the Experimental Animal Center of Changchun University of Traditional Chinese Medicine under the following conditions: temperature 22-25℃, relative humidity 40%-60%, 12-hour light / dark cycle, and free access to water and food. All animal experimental procedures were conducted in accordance with the guidelines approved by the Experimental Animal Ethics Committee of Changchun University of Traditional Chinese Medicine (Approval No.: 20251002).

[0067] 1.4.1 Safety Experiment After a week of acclimatization, mice were randomly divided into two groups (n=8) to evaluate the safety of PASP. A blank control group and a high-dose PASP group were included, with administration continued for 8 weeks. Body weight was recorded daily. At the end of the experiment, mice were euthanized by cervical dislocation, and blood, liver, heart, spleen, lung, and kidney samples were collected for further analysis.

[0068] 1.4.2 Obesity Experiment After a week of acclimatization, mice were randomly divided into two groups: one group was fed a normal diet (CD, n=10), and the other group was fed a high-fat diet (HFD, n=50; 60% fat content, 12492M; Beijing Boai Port). Simultaneously, HFD mice were orally administered saline (model group M), 20 mg / kg orlistat (positive control group Y), 700 mg / kg PASP (PASP-L), 1400 mg / kg PASP (PASP-M), and 2100 mg / kg PASP (PASP-H), respectively, for 8 weeks. The control group was given saline. Body weight and food intake were recorded weekly for each group, and fecal samples were collected starting in week six. Blood was collected at the end of the experiment for biochemical analysis. Epididymal white adipose tissue (eWAT), subcutaneous white adipose tissue (iWAT), perirenal white adipose tissue (pWAT), brown adipose tissue (BAT), and liver tissue were photographed, weighed, and stored at -80℃ for further analysis.

[0069] 1.5 Oral Glucose Tolerance Test (OGTT) In week 7 of the experiment, an oral glucose tolerance test (OGTT) was conducted. Mice were fasted overnight for 14 hours (with free access to water). The following day, they were orally administered D-glucose solution (2 g / kg body weight) via gavage. Blood samples were collected via tail vein at time points before gavage (0 minutes) and at 30, 60, 90, and 120 minutes after gavage. Blood glucose concentrations were measured immediately using a glucometer to assess the glucose metabolism function of the mice. The ambient temperature was kept stable throughout the experiment to minimize stress.

[0070] 1.6 Complete Blood Count Analysis A fully automated blood cell analyzer was used to analyze whole blood cells in mice.

[0071] 1.7 Biochemical Analysis Serum levels of total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatinine (CREA), and urea (UREA) were measured using a fully automated biochemical analyzer.

[0072] 1.8 Liver TC and TG Measurement According to the manufacturer's instructions, the total cholesterol (TC) and triglyceride (TG) levels in liver tissue were determined using a commercial test kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

[0073] 1.9 Histopathological staining Fresh tissue was fixed, embedded, and sectioned, and then stained with hematoxylin and eosin (H&E). Oil Red O staining was used to detect lipid droplet deposition; the specific procedure was as follows: frozen sections were prepared, and then oil red staining and hematoxylin counterstaining were performed sequentially.

[0074] 1.10 Gut microbiota 16S rRNA sequencing analysis Cecal contents were collected from mice in the blank control group (K), model group (M), and high-dose group (PASP-H). Total microbial DNA was extracted and quality-checked by UV quantitative PCR and 0.8% agarose gel electrophoresis, then stored at -20°C for later use. PCR amplification was performed using primers 338F and 806R, which are specific to the V3-V4 region of the bacterial 16S rRNA gene. The barcodes in the primers were oligonucleotide sequences of 7-10 bases used to distinguish different samples. PCR was performed using NEB Q5 high-fidelity polymerase. A 25 μL reaction system contained: 0.25 μL Q5 enzyme, 5 μL 5× reaction buffer, 5 μL 5× high GC buffer, 2 μL dNTP (10 mM), 2 μL template DNA, 1 μL each of forward and reverse primers (10 μM), and 8.75 μL water. The amplification program was 98°C pre-denaturation for 5 min, followed by 25 cycles (98°C for 30 s, 52°C for 30 s, 72°C for 70 s). The amplified products were purified by magnetic beads, quantified using a kit, and mixed in equimolar amounts for library construction. After quantification with Qubit 4 and quality control by a bioanalyzer, the samples were sequenced on an Illumina NovaSeq platform (2×250 bp paired-end sequencing). The raw data underwent Demux decoding, primer removal with Cutadapt, quality filtering with the DADA2 plugin, noise reduction, splicing, and chimera removal to generate ASV characteristic sequences and abundance tables. Species annotation was performed using a Naive Bayes classifier based on the database, and bioinformatics analyses such as Alpha diversity, Beta diversity, and LEfSe differential analysis were conducted. This sequencing was provided by Shanghai Paisenno Biotechnology Co., Ltd.

[0075] 1.11 Statistical Methods Statistical analysis was performed using software. All experimental results are expressed as mean ± standard deviation (mean ± SD). One-way ANOVA was used for comparisons of multiple groups, while Student's t-test was used for comparisons of two groups. Furthermore, Spearman rank correlation analysis was used to assess the correlation between obesity-related indicators and metabolites or gut microbiota. Intergroup comparisons were performed using... P <0.05 indicates a statistically significant difference.

[0076] 2. Results 2.1 Isolation and purification of PASP First, crude polysaccharide was obtained from dragon fruit by hot water and ethanol extraction, with a yield of 0.99 ± 0.03% (w / w). Subsequently, PASP was purified using a DEAE-52 ion-exchange chromatography column. Figure 1The yields of PASP-0 (A) were 73.53 ± 1.84%, PASP-0.1 were 3.93 ± 0.17%, PASP-0.2 were 4.44 ± 0.83%, PASP-0.3 were 2.27 ± 0.46%, and PASP-0.5 were 0.18 ± 0.02%. To screen for the polysaccharide fraction with the best activity, the anti-obesity effects of five different DEAE-52 eluted polysaccharide fractions on high-fat diet-induced obese mice were investigated. The results are as follows: Figure 2 As shown in the AE, compared with the normal control group (K), the high-fat model group (M) mice had significantly increased body weight, liver weight, epididymal fat weight and subcutaneous fat weight (p<0.0001), showing a clear obesity phenotype; compared with the high-fat model group, the PASP-0 group significantly reduced the increase in mouse body weight (p<0.0001), liver weight (p<0.0001), epididymal fat weight (p<0.0001) and subcutaneous fat weight (p<0.0001), and the effect was better than other polysaccharide components (PASP-0.1, PASP-0.2, PASP-0.3, PASP-0.5).

[0077] Based on the combined yield and activity screening results, PASP-0 was ultimately selected as the component for subsequent research and named PASP. Further purification using Sephacryl S-300 HR gel filtration yielded purified PASP. Figure 1 B), after lyophilization, was a white powder with a yield of 57.89±2.81%. The total sugar content of PASP was determined to be 99.25±0.55% using the phenol-sulfuric acid method. Furthermore, no protein was detected in PASP.

[0078] 2.2 Structural Characterization of PASP 2.2.1 Molecular weight and monosaccharide composition of PASP The high-performance gel permeation chromatography (HP-GPC) chromatogram of PASP showed a single symmetrical peak, indicating its high homogeneity; GPC analysis revealed that the molecular weight of PASP is 2.63 kDa. Figure 1 (C); Monosaccharide composition analysis showed that glucose accounted for more than 98%, with small amounts of fucose and fructose as well. Figure 1 (D).

[0079] 2.2.2 Infrared and Ultraviolet Analysis No characteristic absorption peaks were observed at 260 nm and 280 nm in the ultraviolet spectrum. Figure 1 The presence of E indicates that PASP does not contain protein or nucleic acid.

[0080] In addition, the infrared spectral results of PASP are as follows: Figure 1As shown in F, at 3389 cm -1 The strong absorption peak at 2927 cm⁻¹ is caused by the stretching vibration of the OH bonds in the polysaccharide backbone; -1 The absorption peak at 1421 cm⁻¹ corresponds to the stretching vibration of CH in the polysaccharide functional group; -1 The absorption peak at 1047 cm⁻¹ is related to the bending vibration of CH₄; -1 The strong absorption peak at the point represents the deformation vibration of COH in the pyranose ring structure, indicating that pyranose, as the main monosaccharide, constitutes the dragon fruit pulp polysaccharide.

[0081] 2.2.3 Congo Red Test The Congo red assay results showed that the maximum absorption wavelength (λmax) of the complex formed by PASP and Congo red did not undergo a significant red shift as the NaOH concentration increased from 0.1 M to 0.5 M. Furthermore, λmax gradually shifted to blue with increasing alkali concentration, indicating that PASP does not possess a triple helix structure, suggesting that the PASP molecular chain may exist in a random coil or single-chain conformation. Figure 1 (G).

[0082] 2.2.4 Methylation and NMR Analysis Comprehensive application 13 C 1 H- 1 The fine structure of PASP was systematically characterized using HCOSY, HSQC, HMBC, and TOCSY nuclear magnetic resonance techniques. Combined with the results of monosaccharide composition and methylation analysis (Table 1), the structure of each sugar residue was further analyzed. 1 H and 13 The C chemical shifts were assigned in detail (Table 2).

[0083] Table 1. Methylation analysis of PASP Table 2 PASP 1 H NMR and 13 C10 NMR chemical shift 13 C-NMR spectrum ( Figure 3 The results showed that the anomeric carbon signals, ranked from highest to lowest chemical shift, were 98.2 ppm (residue B), 95.7 ppm (residue A), 91.9 ppm (residue D), and 91.9 ppm (residue C). These signals were observed in the HSQC spectrum. Figure 5 The corresponding cross peaks in the COSY spectrum were 4.55 / 98.2 ppm, 4.56 / 95.7 ppm, 5.14 / 91.9 ppm, and 5.14 / 91.9 ppm, respectively. Subsequently, combined with the COSY spectrum... Figure 4 The H1-H2 related peaks (4.56 / 3.19 ppm) and TOCSY spectrum in the image () Figure 7 Based on this, the H-2 chemical shift of residue A is estimated to be 3.19 ppm, and the H3-H6 / C3-C6 signals are 3.30 / 75.7 ppm, 3.61 / 72.2 ppm, 3.37 / 75.6 ppm, and 3.79 / 60.5 ppm, respectively. Therefore, residue A is assigned as β-D-Glc. p -(1→。Analysis of COSY and TOCSY results showed that the H2-H6 chemical shifts of residue B were 3.19 ppm, 3.62 ppm, 3.73 ppm, 3.32 ppm, and 3.62 ppm, respectively; combined with HSQC spectra, the corresponding C2-C6 chemical shifts were 73.4 ppm, 72.4 ppm, 80.5 ppm, 75.6 ppm, and 60.4 ppm, respectively. Among them, the C4 signal showed a significant low-field shift (approximately 10 ppm), indicating that substitution occurred at this position. Therefore, residue B was assigned to →4)-β-D-Glc p Similarly, based on the combined analysis of COSY, TOCSY, and HSQC spectra, the H2 / C2-H6 / C6 chemical shifts of residue C are 3.44 / 71.1 ppm, 3.65 / 72.3 ppm, 3.31 / 75.7 ppm, 3.96 / 72.0 ppm, and 3.79 / 69.5 ppm, respectively. Among these, the C6 signal shows a significant low-field shift (approximately 8-10 ppm), suggesting substitution at this position. Therefore, residue C is assigned to →6)-α-D-Glc p -(1→. Joint analysis of the COSY, TOCSY, and HSQC spectra of residue D revealed its H2 / C2-H6 / C6 chemical shifts to be 3.45 / 72.2 ppm, 3.78 / 72.5 ppm, 3.37 / 75.7 ppm, 3.96 / 72.0 ppm, and 3.74 / 71.2 ppm, respectively. Compared with unsubstituted α-D-Glc... p Compared to other residues, the C4 and C6 signals are both shifted to a lower field by approximately 10 ppm, indicating that both sites are substituted. Therefore, residue D is assigned to →4,6)-α-D-Glc p -(1→. Based on HMBC spectrum( Figure 6 The key long-range correlation signals observed in β-D-Glc further clarified the connection mode between residues: the correlation peak AH1-BC4 (4.56 / 80.5 ppm) indicates that β-D-Glc p -(1→and→4)-β-D-Glc p -(1→Connected via C4 bit; Signal BH1-DC4 (4.55 / 75.7 ppm) Description→4)-β-D-Glcp -(1→and→4,6)-α-D-Glc p -(1→connected at C4; related peak CH1-DC6 (5.14 / 71.2 ppm) suggests →6)-α-D-Glc p -(1→and→4,6)-α-D-Glc p -(1→ is connected at C6; while the signal CH1-CC6 (5.14 / 69.5 ppm) indicates that PASP contains →6)-α-D-Glc p -(1→6)-α-D-Glc p -(1→repeated structural fragments).

[0084] Based on the above analysis and the molar ratio of residues obtained by methylation, the structure of PASP is shown in Formula I, n=11.

[0085] 2.3 Animal Experiment Results 2.3.1 Evaluation of chronic toxicity of PASP in C57BL / 6J mice To systematically evaluate the chronic safety of PASP, this study administered a high dose of PASP (2100 mg / kg body weight) to male C57BL / 6J mice via drinking water and continued this administration by gavage for 8 weeks. Figure 8 Group A and the control group (Group K) were given water. Weight was monitored weekly during the experiment. The weight gain trends in both groups were consistent, with no statistically significant differences at any time point. Figure 8 The results (B) indicate that PASP has no adverse effect on normal growth. Serum biochemical analysis showed that albumin (ALB) concentrations in both groups remained within the normal range of 28-32 g / L. Figure 8 (C); There were no significant differences in ALT and AST activities between groups, and the PASP group showed a slight upward trend but did not reach statistical significance. Figure 8 The levels of D and E indicate no hepatotoxicity. Creatinine (CREA-S) and blood urea nitrogen (UREA) were stable and comparable in both groups. Figure 8 The results (F and G) indicate that long-term PASP exposure does not impair renal function. A complete blood count showed that red blood cells (RBCs) (F and G) were present. Figure 8 H), hemoglobin (HGB) Figure 8 J) and platelets (PLT) Figure 8 K) showed no significant changes. White blood cell (WBC) counts showed a moderate increasing trend in the PASP group, with increased dispersion. Figure 8 The I value was [not specified], but it did not reach statistical significance. H&E staining histological examination showed no morphological abnormalities in the liver, heart, spleen, lungs, and kidneys after PASP treatment. Figure 8(L). The liver lobule structure is normal, without fatty degeneration or inflammatory infiltration; the myocardial fiber striations are preserved; the white pulp / red pulp boundary of the spleen is clear; alveolar ventilation is good; there are no lesions in the glomeruli and renal tubules.

[0086] In summary, high-dose PASP gavage for 8 weeks demonstrated excellent safety profile in C57BL / 6J mice, with no organ-specific toxicity observed in growth parameters, serum biochemistry, hematology, or histopathology.

[0087] 2.3.2 PASP intervention alleviates high-fat diet-induced obesity and hepatic steatosis in mice To evaluate the therapeutic effect of PASP on diet-induced obesity, C57BL / 6J mice were fed a high-fat diet for 8 weeks, and were simultaneously treated with different doses of PASP. Figure 9 (A). The model group mice progressively gained weight to approximately 30 g, while PASP dose-dependently inhibited weight gain. The PASP-H group maintained weight at the normal control level (approximately 23 g). Figure 9 (B). The liver weight of mice in the high-fat diet group was significantly increased, while PASP significantly reduced liver weight in a dose-dependent manner. Figure 9 (C). Morphological observation showed that the model group mice had enlarged livers and expanded adipose tissue; these changes were improved to some extent after PASP treatment. Figure 9 Quantitative analysis of liver and adipose tissue confirmed that the weight of liver, epididymis, and subcutaneous fat decreased in a dose-dependent manner. P <0.0001), the efficacy of the PASP-H group was comparable to that of orlistat ( Figure 9 The weekly food intake of mice in each group was maintained within the range of 2.5-4.0 g / day. Unlike orlistat, there was no significant difference in PASP intake among the groups. P >0.05), meaning that the weight loss effect of PASP is not achieved by suppressing appetite or reducing energy intake. Figure 9 The F). H&E and Oil Red O staining of liver tissue visually confirmed the above improvements, with the PASP group showing restoration of liver tissue structure and almost complete lipid clearance (F). Figure 9 I). H&E staining of mouse livers was used to score the fatty liver disease activity (NAS), and the results showed that HFD induced significant hepatic steatosis. P <0.0001), the model group score was close to 8 points (severe fatty degeneration), and both the Y group and the PASP-H group significantly reduced the fatty degeneration score to near normal levels. P <0.0001), and the PASP-L and PASP-M groups also showed some degree of improvement ( Figure 9 Quantitative analysis using Oil Red O staining revealed that the model group showed significant lipid accumulation in the liver (G). P<0.0001), PASP progressively reduces lipid load, decreasing to approximately 3% in the PASP-H group ( P <0.0001)( Figure 9 H).

[0088] In conclusion, PASP exerts a dose-dependent protective effect against obesity induced by a high-fat diet without suppressing appetite and energy intake, indicating that the mechanism of action of PASP differs from that of orlistat drugs, supporting its potential as a treatment for metabolic diseases.

[0089] 2.3.3 PASP intervention improves glucose and lipid metabolism disorders induced by a high-fat diet in mice Oral glucose tolerance test showed that mice in the high-fat diet group had severe glucose intolerance, with significantly elevated peak blood glucose levels and persistent hyperglycemia. Figure 10 PASP improves glucose clearance in a dose-dependent manner, with the AUC in the PASP-H group decreasing to near-normal levels. P <0.0001)( Figure 10 (B). Biochemical analysis showed that a high-fat diet led to elevated TG and TC and decreased HDL-C. PASP corrected these abnormalities in a dose-dependent manner: TG and TC decreased in all PASP groups, and PASP-H returned to near-normal levels compared to the model group. P <0.0001), indicating a favorable improvement in the lipid profile ( Figure 10 The hepatoprotective effect was confirmed by a dose-dependent decrease in serum ALT and AST. In the PASP-H group, ALT levels decreased to below those of the normal control group (C). P <0.0001), the AST-lowering effect is better than orlistat ( P <0.0001)( Figure 10 The levels of TG and TC in liver tissue also decreased in a corresponding dose-dependent manner, with the PASP-H group approaching normal values. Figure 10 (F and G). H&E staining of adipose tissue revealed characteristic pathological hypertrophy induced by a high-fat diet in white adipose tissue (epididymis, subcutaneous, and perirenal), with adipocytes enlarging into giant vacuoles, cytoplasmic edge compression, and perinuclear displacement. In the model group mice, the brown adipose tissue (BAT) in the scapular region showed prominent white vacuoles and large unilocular lipid droplets, suggesting possible thermogenesis dysfunction. PASP treatment reversed these pathological changes in all adipose tissues in a dose-dependent manner. Figure 10 Quantitative analysis of fat vacuolar area revealed that PASP significantly reduced the area of ​​epididymis, subcutaneous tissue, perirenal region, and brown adipocytes in a dose-dependent manner (all). P <0.0001), among which PASP-H completely returned to normal levels in subcutaneous and perirenal fat, and was more effective than orlistat in epididymal fat.P <0.01), suggesting that PASP can completely reverse high-fat diet-induced adipocyte hypertrophy ( Figure 10 (IL).

[0090] In conclusion, PASP comprehensively improves metabolic disorders by enhancing glucose tolerance, correcting dyslipidemia, protecting the liver, and reversing hypertrophy of all fat deposits and adipocytes, supporting its potential as a treatment for metabolic syndrome.

[0091] 2.3.4 PASP reshapes gut microbiota structure and regulates dysbiosis To investigate whether the anti-obesity effect of PASP is mediated by gut microbiota regulation, 16S rRNA gene sequencing was performed.

[0092] Venn diagram analysis showed significant differences in OTU composition, suggesting a partial recovery of the gut microbiota structure towards normality. Figure 11 (A). α-diversity indices confirmed the high-fat diet-induced gut microbiota dysbiosis. The Chao1 index, reflecting species richness, was significantly reduced in mice on a high-fat diet. P <0.0001), the PASP-H group showed a moderate increase, but still below the normal level ( Figure 11 The Simpson index, which reflects community evenness, was significantly reduced in mice on a high-fat diet. P <0.05), PASP-H improvement is limited ( Figure 11 Principal coordinate analysis (PCoA) showed clear separation among the three clusters, with PASP-H located between groups K and M, tending towards the normal control cluster, suggesting partial recovery of β-diversity. Figure 11 (D). At the phylum level, a high-fat diet induces characteristic dysbiosis: the Firmicutes / Bacteroidetes (F / B) ratio is significantly increased. P <0.0001), manifested as expansion of Firmicutes and contraction of Bacteroidetes; PASP-H significantly reversed these changes, and the F / B ratio was significantly reduced ( P <0.0001), but not fully recovered to normal ( Figure 11 (EG). Furthermore, the metabolic disease-associated phylum Dethiobacterium is a biomarker of metabolic-microbiota dysbiosis, and its metabolic imbalance may further indirectly participate in the inflammatory process through metabolites such as H2S. The level of Dethiobacterium was significantly increased in group M mice (EG). P <0.0001), suggesting that HFD-induced obesity may lead to intestinal inflammation, and PASP-H significantly inhibited its abundance ( P <0.0001)( Figure 11 Conversely, PASP-H reduces the number of wart microbes (H). P <0.01)( Figure 11I), while significantly expanding the Actinobacteria phylum ( P <0.05)( Figure 11 At the genus level, PASP exerts differential regulation on obesity-related microbiota. The rapidly expanding *Agniformes* genus in high-fat diet mice (…). P <0.0001), which showed some improvement after PASP treatment but did not return to normal levels. Figure 11 The genus *Melhera*, as sulfate-reducing bacteria of the phylum Dethiobacterium, participates in H2S production and its activity significantly expands in mice fed a high-fat diet. P <0.0001). PASP-H significantly inhibited the abundance of *Melherba* spp. ( P <0.05%, indicating that PASP partially inhibits H2S-producing bacteria ( Figure 11 (L). It is worth noting that *Desulfovibrio*, as a well-established pathogenic commensal bacterium in metabolic diseases, also belongs to the phylum Desulfobacteria and can participate in H2S production. Its levels are elevated in mice on a high-fat diet, and PASP-H reduces them below normal levels. Figure 11 H2S can disrupt intestinal barrier integrity, promote CD36-mediated hepatic steatosis, and exacerbate systemic inflammation; therefore, its clearance suggests a key role for PASP in liver protection and metabolic improvement. LEfSe analysis identified characteristic biomarkers in each group: the control group mice were predominantly Bacteroidetes; the model group mice shifted to Dethiobacterium predominance, enriched in Dethiovibrales, Dethiovibrium spp., and Melherella spp.—these taxa are associated with metabolic endotoxemia and inflammation; PASP-H specifically enriched in Firmicutes, Clostridium, Oscillatoria, and Rochetomyces spp., all known short-chain fatty acid (SCFA) producers with anti-inflammatory properties. Correlation analysis was performed on gut microbiota and metabolic phenotype (…). Figure 11 PASP-enriched Firmicutes A was significantly negatively correlated with serum ALT, TG, body weight, adipose tissue weight, and liver TG. P <0.01), which was positively correlated with HDL-C. Conversely, Firmicutes D and Dethiobacteria, which are enriched in high-fat diets, were significantly positively correlated with these metabolic dysfunction markers (respectively <0.01). P <0.01 and P <0.05).

[0093] In summary, PASP can selectively remodel HFD-induced dysbiosis, partially restore α-diversity, shift β-diversity towards normal controls, reduce the F / B ratio, and inhibit H2S sulfate-reducing bacteria. These factors collectively support the fact that the metabolic benefits of PASP are partly mediated by gut microbiota regulation.

[0094] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. These modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A polysaccharide with auxiliary lipid-lowering and weight-loss effects, characterized in that, It comprises a main chain and side chains. The main chain is composed of α-D-pyranose glucose residues linked by (1→6) glycosidic bonds, and the side chains are formed by two β-D-pyranose glucose residues linked by (1→4) glycosidic bonds. The side chains are connected to the main chain via the C4 position of the glucose residues in the main chain. The molecular weight of the polysaccharide is 2.63 kDa. The polysaccharide contains more than 98% glucose and less than 2% fucose and fructose. The polysaccharide has the structure shown in Formula I. Equation I; Where n is 11.

2. The polysaccharide with auxiliary lipid-lowering and weight-loss effects according to claim 1, characterized in that, The aforementioned auxiliary lipid-lowering and weight-loss effects include at least one of the following: (1) Reduce or control weight gain; (2) Reduce fat content or lower lipid load; (3) Improve or restore liver morphology; (4) Inhibit or reduce liver weight gain; (5) Inhibit or reverse hepatic steatosis; (6) Improves glucose and lipid metabolism; (7) Improve the structure of gut microbiota.

3. A composition having an auxiliary effect in lowering lipids and promoting weight loss, characterized in that, It includes the polysaccharide described in claim 1 or 2.

4. The composition with auxiliary lipid-lowering and weight-loss effects according to claim 3, characterized in that, The composition is a pharmaceutical composition or a food composition.

5. The method for preparing polysaccharides with auxiliary lipid-lowering and weight-loss effects according to claim 1 or 2, characterized in that, Includes the following steps: (1) Use an aqueous solvent to extract the plant material at 60-100℃ for 30-120 min to obtain an extract, add a precipitant to precipitate and remove the protein in the precipitate to obtain crude polysaccharide; (2) The crude polysaccharide is separated and purified to obtain the polysaccharide.

6. The method for preparing the polysaccharide with auxiliary lipid-lowering and weight-loss effects according to claim 5, characterized in that, The ratio of the plant material to the aqueous solvent is 1:(0.5-5).

7. The method for preparing the polysaccharide with auxiliary lipid-lowering and weight-loss effects according to claim 5, characterized in that, Proteins in the precipitate were removed using the Sevag method.

8. The method for preparing the polysaccharide with auxiliary lipid-lowering and weight-loss effects according to claim 5, characterized in that, The separation and purification include ion exchange chromatography and non-ionic gel chromatography.

9. The method for preparing the polysaccharide with auxiliary lipid-lowering and weight-loss effects according to claim 5, characterized in that, The separation and purification process includes: The crude polysaccharide was separated by ion exchange chromatography, and the elution fraction corresponding to the first peak was collected. Using water as the mobile phase, the elution fraction corresponding to the first peak is passed through a non-ionic gel filter column to obtain the polysaccharide with the function of assisting in lowering lipids and losing weight.

10. The use of the polysaccharide according to claim 1 or 2 in the preparation of drugs or health foods for assisting in lowering lipids and losing weight.

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