A raspberry polysaccharide and its use in preventing and treating obesity.

Abstract

The present application discloses a raspberry uniform polysaccharide, the monosaccharide component of the raspberry uniform polysaccharide includes fucose, rhamnose, arabinose, galactose, xylose, galacturonic acid, glucuronic acid and apiose; wherein, the main chain structure of the raspberry uniform polysaccharide is 1,4-connected galacturonic acid pyranose, and the weight average molecular weight is 8.54±5.0kDa.The present application discloses a raspberry uniform polysaccharide component for inhibiting dietary fat absorption and preventing and improving obesity, the uniform polysaccharide component is prepared by the steps of water extraction, alcohol precipitation, ion exchange chromatographic column elution and osmosis gel chromatographic column elution, and the structure is analyzed by high efficient gel permeation chromatography, infrared spectroscopy, ion chromatography, gas chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy.The uniform polysaccharide component can be used for improving obesity and fat tissue accumulation hypertrophy, and the effect is related to inhibiting intestinal dietary fat absorption and promoting dietary fat discharge to the outside.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a raspberry polysaccharide and its use in preventing and treating obesity. Background Technology

[0002] Obesity and overweight are diseases caused by long-term energy intake exceeding energy expenditure, leading to conditions such as hyperlipidemia, diabetes, and cardiovascular diseases. One approach to treating overweight and obesity is to restrict energy intake by controlling the digestion and absorption of nutrients such as fat. Currently, very few drugs are approved for market to restrict fat digestion and absorption, and they often have significant side effects. For example, orlistat is an anti-obesity drug approved by the US Food and Drug Administration. It works by forming a covalent bond with lipase to inhibit its activity, reducing the absorption of dietary fats in the intestines. It can reduce fat absorption by approximately 30%, promoting fat excretion and thus improving obesity, making it particularly suitable for obese individuals with high-fat diets. However, orlistat has been reported to have side effects including liver damage and steatorrhea. Raspberry is a traditional Chinese medicine with medicinal and edible properties. Its polysaccharide components can improve obesity induced by a high-fat diet in mice, but there are no reports on the RG-II type polysaccharide in raspberry inhibiting the digestion and absorption of dietary lipids in the intestines, thereby improving obesity. Summary of the Invention

[0003] Based on this, the present invention provides a raspberry homogeneous polysaccharide, the monosaccharide components of which include fucose, rhamnose, arabinose, galactose, xylose, galacturonic acid, glucuronic acid and celery.

[0004] The main chain structure of this raspberry homogeneous polysaccharide is 1,4-linked galacturonic acid pyranosan.

[0005] The weight-average molecular weight of this raspberry homogeneous polysaccharide is 8.54 ± 5.0 kDa.

[0006] Furthermore, the following seven monosaccharide molar ratios of the raspberry homogeneous polysaccharide are fucose: rhamnose: arabinose: galactose: xylose: galacturonic acid: glucuronic acid = (0.1-5): (0.1-5): (5-10): (0.1-5): (0.1-5): (70-90): (0.1-5).

[0007] Furthermore, the following seven monosaccharide molar ratios of the raspberry homogeneous polysaccharide are fucose: rhamnose: arabinose: galactose: xylose: galacturonic acid: glucuronic acid = (1-3): (3-4): (6-8): (1-3): (1-3): (75-88): (1-3).

[0008] Furthermore, the following seven monosaccharide molar ratios of the raspberry homogeneous polysaccharide are fucose: rhamnose: arabinose: galactose: xylose: galacturonic acid: glucuronic acid = approximately 1.2: approximately 3.4: approximately 7.2: approximately 2.0: approximately 1.6: approximately 83.2: approximately 1.4.

[0009] Preferably, the molar percentage of celery syrup in the homogeneous polysaccharide of raspberry is greater than 0.2%.

[0010] Furthermore, the raspberry homogeneous polysaccharide is a rhamnogalacturonic acid polysaccharide-II domain.

[0011] Furthermore, the weight-average molecular weight of this raspberry homogeneous polysaccharide is 8.54 ± 3.0 kDa.

[0012] Furthermore, the weight-average molecular weight of this raspberry homogeneous polysaccharide is 8.54 ± 1.0 kDa.

[0013] Furthermore, the weight-average molecular weight of this raspberry homogeneous polysaccharide is approximately 8.54 kDa.

[0014] Preferably, the glycosidic bonds of the homogeneous raspberry polysaccharide are selected from one or more of the following: terminal arabinofuranose (T-Araf), terminal xylopyranose (T-Xylp), terminal rhamnopyranose (T-Rhap), 1,5-linked arabinofuranose (1,5-Araf), terminal galactopyranose (T-Galp), terminal galacturonic acid pyranose (T-GalAp), 1,3,4-linked rhamnopyranose (1,3,4-Rhap), and 1,3,4-linked fucose. Pyranose (1,3,4-Fucp), 1,4-linked galacturonic acid pyranose (1,4-GalAp), 1,3,4-linked galacturonic acid pyranose (1,3,4-GalAp), 1,2,4-linked galacturonic acid pyranose (1,2,4-GalAp), 1,3,6-linked galactopyranose (1,3,6-Galp), 1,2-linked glucuronic acid pyranose (1,2-GlcAp), and 1,3-linked apigeninose (1,5-Apif).

[0015] Furthermore, the molar ratio of the following 12 glycosidic bonds in the homogeneous raspberry polysaccharide is as follows: T-Araf: T-Xylp: T-Rhap: 1,5-Araf: T-Galp: T-GalAp: 1,3,4-Rhap: 1,3,4-Fucp: 1,4-GalAp: 1,3,4-GalAp: 1,2,4-GalAp: 1,3,6-Galp = (0.1~5): (0.1~5): (0.1~5): (0.1~5): (0.1~5): (2~10): (0.1~5): (0.1~5): (70~90): (0.1~5): (0.1~5): (0.1~5).

[0016] Furthermore, the molar ratio of the following 12 glycosidic bonds in the homogeneous raspberry polysaccharide is as follows: T-Araf: T-Xylp: T-Rhap: 1,5-Araf: T-Galp: T-GalAp: 1,3,4-Rhap: 1,3,4-Fucp: 1,4-GalAp: 1,3,4-GalAp: 1,2,4-GalAp: 1,3,6-Galp = (1.5~4): (0.5~2): (0.1~1.5): (0.5~2): (0.5~2): (3~8): (0.5~2): (0.1~2): (75~88): (1~3): (0.5~2): (0.5~2).

[0017] Furthermore, the molar ratio of the following 12 glycosidic bonds in the homogeneous raspberry polysaccharide is as follows: T-Araf: T-Xylp: T-Rhap: 1,5-Araf: T-Galp: T-GalAp: 1,3,4-Rhap: 1,3,4-Fucp: 1,4-GalAp: 1,3,4-GalAp: 1,2,4-GalAp: 1,3,6-Galp = approximately 2.3: approximately 1.3: approximately 0.8: approximately 1.3: approximately 1.6: approximately 5.0: approximately 0.9: approximately 0.6: approximately 82.4: approximately 1.8: approximately 0.9: approximately 1.1.

[0018] According to another aspect of the present invention, a method for preparing the above-mentioned homogeneous raspberry polysaccharide is provided, the method comprising the following steps:

[0019] (1) Take an appropriate amount of raspberry fruit, crush it, add ethanol, let it stand at room temperature, and repeat twice.

[0020] (2) Add water to the raspberry powder after the ethanol has evaporated, soak at 90°C for 3 hours, repeat twice, combine the filtrates, concentrate, and obtain concentrated solution.

[0021] (3) Stir the concentrated liquid, add ethanol, let it stand to settle, filter, and obtain filter residue;

[0022] (4) The filter residue was evaporated to dryness with ethanol, dissolved in water, and dried to obtain crude raspberry polysaccharide; and

[0023] (5) The crude raspberry polysaccharide was separated by DEAE-Sepharose Fast Flow weak anion exchange chromatography to obtain a 0.1–0.3 M sodium chloride fraction. This 0.1–0.3 M sodium chloride fraction was then subjected to Superdex chromatography. TM The homogeneous polysaccharide from raspberries was purified by 200 g / L gel permeation chromatography.

[0024] Furthermore, the fruit is an immature fruit.

[0025] Furthermore, this raspberry is *Rubus chingii* Hu.

[0026] Furthermore, the volume percentage concentration of the ethanol is 80% to 100%, for example, about 95%.

[0027] Further, in step (1), the volume / mass (L / kg) ratio of the ethanol to the raspberry fruit is 1 to 10, for example, about 4;

[0028] Furthermore, in step (1), the settling time is 12 to 36 hours, for example, about 24 hours.

[0029] Further, in step (2), the ratio of the water to the volume / mass (L / kg) of the raspberry fruit is 1 to 60, for example, about 40;

[0030] Furthermore, in step (2), the temperature of the water is 60°C to 100°C, for example, about 90°C;

[0031] Furthermore, in step (2), the concentration is a vacuum rotary evaporation concentration.

[0032] Furthermore, in step (3), the volume ratio of the ethanol to the concentrate is 1 to 6, for example, about 4;

[0033] Furthermore, in step (3), the settling time is more than 4 hours, for example, about 12 hours.

[0034] Furthermore, in step (3), the filtration is vacuum filtration.

[0035] Furthermore, in step (4), the drying is vacuum freeze drying.

[0036] Furthermore, in step (5), the concentration of the sodium chloride portion is approximately 0.2 M.

[0037] According to another aspect of the present invention, the use of the above-mentioned raspberry homogeneous polysaccharide in the preparation of pharmaceuticals, foods and / or health products for improving obesity is provided;

[0038] This food is classified as a functional food.

[0039] According to another aspect of the present invention, the use of the above-described raspberry homogeneous polysaccharide in the preparation of a medicament for the prevention and / or adjunctive treatment of obesity is provided.

[0040] Furthermore, this obesity is caused by dietary habits, genetics, metabolism, and / or fat cell abnormalities.

[0041] Furthermore, the abnormality is an increase in the number of adipocytes and / or adipocyte hypertrophy.

[0042] Furthermore, the raspberry homogeneous polysaccharide has the effect of preventing and / or adjuvant treatment of obesity through one or both of the following mechanisms: inhibiting lipid absorption and promoting lipid and / or fat excretion.

[0043] Furthermore, the lipids include triglycerides, cholesterol, and / or low-density lipoprotein.

[0044] Furthermore, the oil contains long-chain fatty acids.

[0045] Furthermore, the long-chain fatty acid includes palmitic acid, oleic acid, linoleic acid, and / or stearic acid.

[0046] The beneficial effects of this invention are:

[0047] This invention discloses a homogeneous polysaccharide component from raspberry for inhibiting dietary fat absorption and preventing and improving obesity. This homogeneous polysaccharide component is prepared through water extraction and alcohol precipitation, ion exchange chromatography, and permeation gel chromatography. Its structure is determined by high-performance gel permeation chromatography, infrared spectroscopy, ion chromatography, gas chromatography-mass spectrometry, and nuclear magnetic resonance spectroscopy. This homogeneous polysaccharide component can be used to improve obesity and adipose tissue accumulation and hypertrophy, and its efficacy is related to inhibiting intestinal absorption of dietary fats and promoting their excretion from the body. Attached Figure Description

[0048] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without exceeding the scope of protection claimed by the present invention.

[0049] Figure 1This is a flowchart of the extraction, separation, and purification process of raspberry homogeneous polysaccharide RCP-2-3. In the flowchart, a represents the extraction, separation, and purification process of raspberry polysaccharide; b represents immature East China raspberry fruit; and c represents raspberry homogeneous polysaccharide RCP-2-3.

[0050] Figure 2 This is a schematic diagram illustrating the physicochemical properties of raspberry homogeneous polysaccharide RCP-2-3. In the diagram, a is a gel permeation chromatography chromatogram of raspberry homogeneous polysaccharide RCP-2-3; b is an HPSEC chromatogram of raspberry homogeneous polysaccharide RCP-2-3; and c is a scanning electron microscope image of raspberry homogeneous polysaccharide RCP-2-3.

[0051] Figure 3 This is a schematic diagram illustrating the structural deduction of Raspberry homogeneous polysaccharide RCP-2-3. In the diagram, a is the FT-IR spectrum of Raspberry homogeneous polysaccharide RCP-2-3; b is the ion chromatogram of the standard and Raspberry homogeneous polysaccharide RCP-2-3; cm is the gas chromatogram and secondary ion fragment mass spectrometry.

[0052] Figure 4 This is a schematic diagram illustrating the structural identification and structural reasoning of the homogeneous polysaccharide RCP-2-3 from raspberries. Where a represents... 1 1H NMR spectrum; b is... 13 C10 NMR spectrum; c represents DEPT135 13 C NMR spectrum; d is 1 H- 1 H COSY NMR spectrum; e is HSQC NMR spectrum; f is HMBC NMR spectrum; g is a fine structure deduction diagram of raspberry homogeneous polysaccharide RCP-2-3.

[0053] Figure 5 This is a schematic diagram illustrating the in vitro functional properties of different raspberry polysaccharides. In the diagram, a represents the oil-holding capacity; b represents the cholesterol adsorption capacity.

[0054] Figure 6 This diagram illustrates the results of a dietary fat intake experiment in mice. In the diagram: a) is the experimental flowchart; b) is a schematic diagram of the change in serum TG in mice stimulated with soybean oil after a meal over time; c) is a schematic diagram of the area under the serum TG curve; d) is a schematic diagram of TG content in cecal contents; e) is a schematic diagram of BODIPY fluorescent sections of the duodenum and proximal jejunum; f) is a schematic diagram of BODIPY fluorescence intensity in serum; g) is a schematic diagram of BODIPY fluorescence intensity in cecal contents; h) is a schematic diagram of BODIPY fluorescence intensity in the duodenum; i) is a schematic diagram of BODIPY fluorescence intensity in the proximal jejunum; j) is a schematic diagram of mouse feces collected in test tubes containing methanol / chloroform extract; k) is a schematic diagram of the content of four long-chain fatty acids in mouse feces.

[0055] Figure 7This diagram illustrates the results of an overfeeding experiment on mice with a high-fat diet. In the diagram: a) is the experimental flowchart; b) is the weight gain trend of mice over 28 days; c) is the serum TG content after fasting; d) is the serum TC content after fasting; e) is the serum LDL-C content after fasting; f) is the serum HDL-C content after fasting; g) is the adipose tissue weight; h) is the ratio of mouse weight gain to food intake; i) is the fecal weight excreted by mice within 12 hours after gavage; and j) is the TG content in the mouse feces. Detailed Implementation

[0056] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0057] Unless otherwise stated, all technical and scientific terms and abbreviations used herein have the meanings commonly understood by one of ordinary skill in the field of this invention or the field of application of such terms. While any methods, conditions, substances, or materials similar to or equivalent to those disclosed herein may be used in the practice of this invention, preferred methods, conditions, substances, or materials are described herein.

[0058] This invention is intended to cover all options, variations, and equivalents that may be included in the field of prior art as defined in the claims. Those skilled in the art will recognize many similar or equivalent methods and substances described herein that can be applied in the practice of this invention. This invention is by no means limited to the description of methods and substances.

[0059] The singular forms “a,” “an,” and “the” used in the specification and appended claims include plural indicators unless the context clearly specifies otherwise.

[0060] In this invention, the term "comprising" and "including" are synonymous. The terms "comprising," "including," "having," "containing," or any other variations thereof as used herein are intended to cover a non-exclusive inclusion. For example, a composition, step, method, article, or apparatus that includes the listed elements is not necessarily limited to those elements, but may include other elements not expressly listed or elements inherent to such a composition, step, method, article, or apparatus.

[0061] As described in the background section, very few drugs are currently approved for marketing to restrict fat digestion and absorption, and they often have significant side effects. To address these issues, this invention provides a homogeneous raspberry polysaccharide whose monosaccharide components include fucose, rhamnose, arabinose, galactose, xylose, galacturonic acid, glucuronic acid, and apiose.

[0062] The main chain structure of this raspberry homogeneous polysaccharide is 1,4-linked galacturonic acid pyranosan.

[0063] The weight-average molecular weight of this raspberry homogeneous polysaccharide is 8.54 ± 5.0 kDa.

[0064] In this invention, when ratios, molar ratios, concentrations, masses, times, temperatures, or other values ​​or parameters are expressed as ranges, preferred ranges, or a series of upper and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pairing of any upper or preferred value with any lower or preferred value, regardless of whether the range is disclosed individually. For example, when the range “5–10” is disclosed, the described range should be interpreted as including ranges “5–10”, “5–9”, “5–8”, “5–7”, “5–6”, “6–10”, “6–9”, “6–8”, “6–7”, “7–10”, “7–9”, “7–8”, “8–10”, “8–9”, “9–10”, etc. When numerical ranges are described herein, unless otherwise stated, the range is intended to include its endpoints and all integers and fractions within that range.

[0065] In a preferred embodiment, the following seven monosaccharides of the raspberry homogeneous polysaccharide are in the following molar ratio: fucose: rhamnose: arabinose: galactose: xylose: galacturonic acid: glucuronic acid = (0.1-5): (0.1-5): (5-10): (0.1-5): (0.1-5): (70-90): (0.1-5).

[0066] In a preferred embodiment, the following seven monosaccharide molar ratios of the raspberry homogeneous polysaccharide are fucose: rhamnose: arabinose: galactose: xylose: galacturonic acid: glucuronic acid = (1-3): (3-4): (6-8): (1-3): (1-3): (75-88): (1-3).

[0067] In a preferred embodiment, the following seven monosaccharide molar ratios of the raspberry homogeneous polysaccharide are fucose: rhamnose: arabinose: galactose: xylose: galacturonic acid: glucuronic acid = about 1.2: about 3.4: about 7.2: about 2.0: about 1.6: about 83.2: about 1.4.

[0068] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 1.2" includes ±5% of 1.2, or from 1.14 to 1.26; "about 3.4" includes ±5% of 3.4, or from 3.23 to 3.57; "about 7.2" includes ±5% of 7.2, or from 6.84 to 7.56; "about 2.0" includes ±5% of 2.0, or from 1.9 to 2.1; "about 1.6" includes ±5% of 1.6, or from 1.52 to 1.68; "about 83.2" includes ±5% of 83.2, or from 79.04 to 87.36; and "about 1.4" includes ±5% of 1.4, or from 1.33 to 1.47.

[0069] In a preferred embodiment, the mass percentage of the celery syrup in the raspberry homogeneous polysaccharide is greater than 0 and less than 2%.

[0070] In a preferred embodiment, the raspberry homogeneous polysaccharide is a rhamnogalacturonic acid polysaccharide-II domain.

[0071] In a preferred embodiment, the weight-average molecular weight of the raspberry homogeneous polysaccharide is 8.54 ± 3.0 kDa.

[0072] In a preferred embodiment, the weight-average molecular weight of the raspberry homogeneous polysaccharide is 8.54 ± 1.0 kDa.

[0073] In a preferred embodiment, the weight-average molecular weight of the raspberry homogeneous polysaccharide is about 8.54 kDa.

[0074] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 8.54" includes ±5% of 8.54, or from 8.113 to 8.967.

[0075] In a preferred embodiment, the glycosidic bonds of the raspberry homogeneous polysaccharide are selected from one or more of the following: terminal arabinofuranose (T-Araf), terminal xylopyranose (T-Xylp), terminal rhamnopyranose (T-Rhap), 1,5-linked arabinofuranose (1,5-Araf), terminal galactopyranose (T-Galp), terminal galacturonic acid pyranose (T-GalAp), 1,3,4-linked rhamnopyranose (1,3,4-Rhap), 1,3,4-linked... Fucopyranose (1,3,4-Fucp), 1,4-linked galacturonic acid pyranose (1,4-GalAp), 1,3,4-linked galacturonic acid pyranose (1,3,4-GalAp), 1,2,4-linked galacturonic acid pyranose (1,2,4-GalAp), 1,3,6-linked galactopyranose (1,3,6-Galp), 1,2-linked glucuronic acid pyranose (1,2-GlcAp), and 1,3-linked apiose furanose (1,5-Apif).

[0076] In a preferred embodiment, the molar ratio of the following 12 glycosidic bonds in the homogeneous raspberry polysaccharide is T-Araf:T-Xylp:T-Rhap:1,5-Araf:T-Galp:T-GalAp:1,3,4-Rhap:1,3,4-Fucp:1,4-GalAp:1,3,4-GalAp:1,2,4-GalAp:1,3,6-Galp = (0.1~5):(0.1~5):(0.1~5):(0.1~5):(0.1~5):(2~10):(0.1~5):(0.1~5):(70~90):(0.1~5):(0.1~5):(0.1~5).

[0077] In a preferred embodiment, the molar ratio of the following 12 glycosidic bonds in the homogeneous raspberry polysaccharide is T-Araf:T-Xylp:T-Rhap:1,5-Araf:T-Galp:T-GalAp:1,3,4-Rhap:1,3,4-Fucp:1,4-GalAp:1,3,4-GalAp:1,2,4-GalAp:1,3,6-Galp = (1.5~4):(0.5~2):(0.1~1.5):(0.5~2):(0.5~2):(3~8):(0.5~2):(0.1~2):(75~88):(1~3):(0.5~2):(0.5~2).

[0078] In a preferred embodiment, the molar ratio of the following 12 glycosidic bonds in the homogeneous raspberry polysaccharide is as follows: T-Araf: T-Xylp: T-Rhap: 1,5-Araf: T-Galp: T-GalAp: 1,3,4-Rhap: 1,3,4-Fucp: 1,4-GalAp: 1,3,4-GalAp: 1,2,4-GalAp: 1,3,6-Galp = about 2.3: about 1.3: about 0.8: about 1.3: about 1.6: about 5.0: about 0.9: about 0.6: about 82.4: about 1.8: about 0.9: about 1.1.

[0079] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 2.3" includes ±5% of 2.3, or from 2.185 to 2.415; "about 1.3" includes ±5% of 1.3, or from 1.235 to 1.365; "about 0.8" includes ±5% of 0.8, or from 0.76 to 0.84; "about 1.6" includes ±5% of 1.6, or from 1.52 to 1.68; "about 5.0" includes ±5% of 5.0, or from 4.75 to 5.25. "About 0.9" includes 0.9 ± 5%, or from 0.855 to 0.945; "About 0.6" includes 0.6 ± 5%, or from 0.57 to 0.63; "About 82.4" includes 82.4 ± 5%, or from 78.28 to 86.52; "About 1.8" includes 1.8 ± 5%, or from 1.71 to 1.89; "About 1.1" includes 1.1 ± 5%, or from 1.045 to 1.155.

[0080] According to another aspect of the present invention, a method for preparing the above-mentioned homogeneous raspberry polysaccharide is provided, the method comprising the following steps:

[0081] (1) Take an appropriate amount of raspberry fruit, crush it, add ethanol, let it stand at room temperature, and repeat twice.

[0082] (2) Add water to the raspberry powder after the ethanol has evaporated, soak at 90°C for 3 hours, repeat twice, combine the filtrates, concentrate, and obtain concentrated solution.

[0083] (3) Stir the concentrated liquid, add ethanol, let it stand to settle, filter, and obtain filter residue;

[0084] (4) The filter residue was evaporated to dryness with ethanol, dissolved in water, and dried to obtain crude raspberry polysaccharide; and

[0085] (5) The crude raspberry polysaccharide was separated by DEAE-Sepharose Fast Flow weak anion exchange chromatography to obtain a 0.1–0.3 M sodium chloride fraction. This 0.1–0.3 M sodium chloride fraction was then subjected to Superdex chromatography. TMThe homogeneous polysaccharide from raspberries was purified by 200 g / L gel permeation chromatography.

[0086] In a preferred embodiment, the fruit is an unripe fruit.

[0087] In a preferred embodiment, the raspberry is Rubus chingii Hu.

[0088] In a preferred embodiment, the volume percentage concentration of the ethanol is 80% to 100%, for example, about 95%.

[0089] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 95%" includes 95% ± 5%, or from 90.25% to 99.75%.

[0090] In a preferred embodiment, in step (1), the ratio of the volume / mass (L / kg) of the ethanol to the raspberry fruit is 1 to 10, for example, about 4.

[0091] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 4" includes ±5% of 4, or from 3.8 to 4.2.

[0092] In a preferred embodiment, in step (1), the settling time is 12 to 36 hours, for example, about 24 hours.

[0093] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 24" includes ±5% of 24, or from 22.8 to 25.2.

[0094] In a preferred embodiment, in step (2), the ratio of the water to the volume / mass (L / kg) of the raspberry fruit is 1 to 60, for example, about 40.

[0095] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 40" includes ±5% of 40, or from 38 to 42.

[0096] In a preferred embodiment, in step (2), the temperature of the water is 60°C to 100°C, for example, about 90°C.

[0097] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 90" includes ±5% of 90, or from 85.5 to 94.5.

[0098] In a preferred embodiment, in step (2), the concentration is a vacuum rotary evaporation concentration.

[0099] In a preferred embodiment, in step (3), the volume ratio of the ethanol to the concentrate is 1 to 6, for example, about 4.

[0100] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 4" includes ±5% of 4, or from 3.8 to 4.2.

[0101] In a preferred embodiment, in step (3), the settling time is more than 4 hours, for example, about 12 hours.

[0102] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 12" includes ±5% of 12, or from 11.4 to 12.6.

[0103] In a preferred embodiment, in step (3), the filtration is vacuum filtration.

[0104] In a preferred embodiment, in step (4), the drying is vacuum freeze drying.

[0105] In a preferred embodiment, in step (5), the concentration of the sodium chloride portion is about 0.2 M.

[0106] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.2" includes ±5% of 0.2, or from 0.19 to 0.21.

[0107] According to another aspect of the present invention, the use of the above-mentioned raspberry homogeneous polysaccharide in the preparation of pharmaceuticals, foods and / or health products for improving obesity is provided;

[0108] This food is classified as a functional food.

[0109] According to another aspect of the present invention, the use of the above-described raspberry homogeneous polysaccharide in the preparation of a medicament for the prevention and / or adjunctive treatment of obesity is provided.

[0110] In a preferred embodiment, the obesity is caused by dietary habits, genetics, metabolism, and / or fat cell abnormalities.

[0111] In a preferred embodiment, the abnormality is an increase in the number of adipocytes and / or adipocyte hypertrophy.

[0112] In a preferred embodiment, the raspberry homogeneous polysaccharide prevents and / or assists in the treatment of obesity through one or both of the following mechanisms: inhibiting lipid absorption and promoting lipid and / or fat excretion.

[0113] In a preferred embodiment, the lipid includes triglycerides, cholesterol, and / or low-density lipoprotein.

[0114] In a preferred embodiment, the oil comprises long-chain fatty acids.

[0115] In a preferred embodiment, the long-chain fatty acid includes palmitic acid, oleic acid, linoleic acid, and / or stearic acid.

[0116] The terms “food,” “health product,” “food product,” and “health product” in this invention are products intended to be ingested by animals (including humans) and to provide them with nutritional or health benefits.

[0117] According to another aspect of the present invention, the above-described raspberry homogeneous polysaccharide is provided for the prevention and / or adjunctive treatment of obesity in subjects.

[0118] According to another aspect of the invention, a method for preventing and / or adjunctive treating obesity in a subject is provided, comprising administering to the subject an effective amount of the aforementioned raspberry homogeneous polysaccharide.

[0119] In this invention, the terms "subject," "individual," or "patient" are used interchangeably and refer to a vertebrate, preferably a mammal. The mammal can be a human, a non-human primate, a mouse, a rat, a dog, a cat, a horse, or a cow, but is not limited to these examples. Mammals other than humans can advantageously be used as subjects representing obesity models. Preferably, the subject is a human. Such subjects typically suffer from or are susceptible to a condition that can be prevented or treated by administering the aforementioned raspberry homogeneous polysaccharide of the present invention.

[0120] The "effective amount" of the aforementioned homogeneous raspberry polysaccharide used in this invention can achieve the desired therapeutic and / or preventative effects. The effective amount for this purpose will depend on factors such as the route of delivery, the activity of the specific active substance or preparation used, the type of disease, the stage and severity of the disease being treated, the individual's weight and overall health status, and the prescribing physician's judgment. Dosage can be administered once a week, twice a week, daily, or even several times a day. Dosage units can be administered over a short period (e.g., weeks to months) or a longer period (months to years). Specifically, the "effective amount" refers to the amount of the aforementioned homogeneous raspberry polysaccharide that imparts a therapeutic effect (e.g., control, relief, improvement, mitigation, or slowing of progression) or preventative effect (e.g., delaying onset or reducing the risk of development) to the treated subject, disease, condition, or symptom thereof.

[0121] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or conditions recommended by the manufacturer.

[0122] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0123] The features mentioned above in this invention, or the features mentioned in the embodiments, can be combined arbitrarily. All features disclosed in this patent specification can be used in any compositional form, and each feature disclosed in the specification can be replaced by any alternative feature that provides the same, equivalent, or similar purpose. Therefore, unless otherwise specified, the disclosed features are merely general examples of equivalent or similar features.

[0124] Example

[0125] 1. Extraction, separation and purification of RCP-2-3

[0126] One kilogram of immature raspberry (Rubus chingii Hu.) fruits were pulverized and allowed to stand in 4 L of 95% ethanol for 24 h. This process was repeated twice to remove fat-soluble components. After the ethanol was naturally evaporated, 40 L of water was added to the remaining raspberry fruit powder, and the mixture was heated to 90 °C for extraction. This process was repeated twice. The extract was concentrated to 1 L by rotary evaporation under reduced pressure. 4 L of 95% ethanol was added while stirring continuously, and the mixture was allowed to settle naturally for 12 h. The mixture was then filtered, and the ethanol in the residue was evaporated. After dissolving in water, the residue was freeze-dried under vacuum to obtain crude raspberry polysaccharide (RCP). The crude raspberry polysaccharide was separated by DEAE-Sepharose Fast Flow weak anion exchange chromatography using water, 0.2 M sodium chloride, and 0.5 M sodium chloride solutions to obtain refined polysaccharides of different polar fractions. The refined polysaccharide of the 0.2 M sodium chloride fraction (RCP-0.2) was further analyzed by Superdex chromatography. TM The raspberry polysaccharide fraction (RCP-2-3) was purified by 200-gel permeation chromatography. Figure 1 and Figure 2 As shown in a.

[0127] 2. Chemical characterization of RCP-2-3

[0128] 2.1 Molecular weight determination

[0129] The weight-average molecular weight (Mw) of RCP-2-3 was determined using high-performance liquid chromatography (HPLC) with a differential detector and high-performance size exclusion chromatography (HPSEC). Chromatographic conditions: Shodex KS-804 and KS-802 tandem columns (300 mm × 8.0 mm, id 5 μm); mobile phase: 0.2 mol / L sodium chloride solution; injection volume: 20 μL; column temperature: 40 °C; flow rate: 0.8 mL / min; injection time: 30 min. Standards were dextran with different molecular weights (Mw: 0.66 × 10⁻⁶). 4 0.99×10 4 2.30×10 4 5.06×10 4 11.0×10 4 20.6×10 4 34.3×10 4 73.6×10 4 Dextran standard solution (Da) was prepared by dissolving it in the mobile phase to a concentration of 2.0 mg / mL. RCP-2-3 was prepared by dissolving it in the mobile phase to a concentration of 2.0 mg / mL and filtered through a 0.45 μm microporous membrane. A calibration curve was plotted using a cubic fitting of the retention time of the dextran standard and the corresponding weight-average molecular weight. The weight-average molecular weight of RCP-2-3 was calculated based on the fitting equation and the retention time of the sample.

[0130] 2.2. FT-IR analysis

[0131] The infrared spectral characteristics of RCP-2-3 were detected using Fourier transform infrared spectroscopy (FT-IR). RCP-2-3 was thoroughly mixed and ground with KBr dried to constant weight at a mass ratio of 1:100 (w / w), then pressed into thin sheets, and its infrared spectral characteristics were measured at frequencies from 4000 to 400 cm⁻¹. -1 The transmittance within the specified range was used to obtain infrared spectra, which were then analyzed.

[0132] 2.3 Scanning Electron Microscopy Analysis

[0133] The microstructure and morphological characteristics of RCP-2-3 were analyzed using field emission scanning electron microscopy. The sample was attached to a metal disk containing double-sided adhesive and coated with gold powder using a spray gun. The sample was then placed in a vacuum chamber and scanned using a field emission scanning electron microscope at an accelerating voltage of 3.0 kV to obtain and analyze microstructure images at different magnifications.

[0134] 2.4 Monosaccharide Composition Analysis

[0135] Accurately weigh fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, fructose, ribose, galacturonic acid, guluronic acid, glucuronic acid, and mannulic acid to prepare a 100 μg / mL working solution. Dilute to different concentrations and transfer to chromatographic vials for analysis. Accurately weigh 5 mg (±0.05 mg) of RCP-2-3 sample, add 1 mL of 2M TFA acid solution, and heat at 121℃ for 2 hours. Purge with nitrogen and dry. Wash with methanol, then dry again, repeating the methanol washing 2-3 times. Dissolve in sterile water and transfer to chromatographic vials for analysis.

[0136] The chromatographic system used is a Thermo ICS5000+ ion chromatography system (ICS5000+, Thermo Fisher Scientific, USA), equipped with a Dionex... TM CarboPac TM PA20 (150*3.0mm, 10μm) HPLC column, injection volume 5μL. Mobile phase A (H2O), mobile phase B (0.1M NaOH), mobile phase C (0.1M NaOH, 0.2M NaAc), column temperature 30℃, gradient elution (0–26.0 min, 95%–85% A, 5% B, 0%–10% C; 26.0–42.0 min, 85% A, 5% B, 10% C; 42.0–42.1 min, 85%–60% A, 5% B, 0% C). The flow rates were 0.5 mL / min. Monosaccharide components were analyzed using an electrochemical detector. Chromatographic data were processed using Chromeleon software. The retention time (time, min) was plotted on the x-axis, and the ion detection response value (Response, nC) was plotted on the y-axis. Standard sample ion chromatograms and sample ion chromatograms were used. Qualitative analysis was performed based on the retention time of the chromatographic peaks, and quantitative analysis was performed using the external standard method. The mass of different monosaccharides was determined, and the molar ratio of each monosaccharide was calculated based on its molar mass.

[0137] 2.5 Glycosidic bond linkage analysis

[0138] Weigh 10 mg of RCP-2-3 sample, dissolve in water, add 1 mL of 100 mg / mL carbodiimide, and react for 2 h. Add 1 mL of 2 M imidazole and 1 mL of 30 mg / mL NaBD4, and react for 3 h. Add 100 μL of glacial acetic acid to terminate the reaction, dialyze, and lyophilize. Weigh 1 mg of the sample to be tested and dissolve in 500 μL of DMSO. Add 1 mg of NaOH and incubate for 30 min. Add 50 μL of iodomethane solution and react for 1 h. Add 1 mL of water and 2 mL of dichloromethane, vortex to mix, centrifuge, and discard the aqueous phase. Repeat washing with water 3 times. Take the lower dichloromethane phase and evaporate to dryness. Add 100 μL of 2 M TFA and react at 121 °C for 90 min. Evaporate to dryness at 30 °C. Add 50 μL of 2 M ammonia and 50 μL of 1 M NaBD4, mix well, and react at room temperature for 2.5 h. The reaction was terminated by adding 20 μL of acetic acid, dried under nitrogen, washed twice with 250 μL of methanol, and dried under nitrogen again. 250 μL of acetic anhydride was added, vortexed, and the mixture was reacted at 100 °C for 2.5 h. 1 mL of water was added, and the mixture was allowed to stand for 10 min. 500 μL of dichloromethane was added, vortexed, centrifuged, and the aqueous phase was discarded. The washing process was repeated three times with water. The lower dichloromethane phase was collected and analyzed.

[0139] The chromatography system was an Agilent 7890A-5977B GC-MS system equipped with an HP-5MS capillary column (30m × 0.25mm × 0.25μm, Agilent J&W Scientific, Folsom, CA, USA). High-purity helium (purity not less than 99.999%) was used as the carrier gas at a flow rate of 1.0 mL / min and an injection port temperature of 260℃. The injection volume was 1 μL, split injection with a split ratio of 10:1 and a solvent delay of 2.2 min. The temperature program was: 50℃ for 1.0 min, ramped at 50℃ / min to 130℃, ramped at 3℃ / min to 230℃, and held for 2 min. An electron impact ionization (EI) source was used with an injection port temperature of 230℃, a quadrupole temperature of 150℃, and an electron energy of 70 eV. The scanning mode was full scan (SCAN), with a mass scan range (m / z) of 30-600.

[0140] 2.6 NMR Analysis

[0141] Take 100 mg of RCP-2-3 sample, dissolve it thoroughly in 550 μL D2O, freeze-dry under vacuum, reconstitute in 550 μL D2O, add 1 μL of acetone (internal standard), centrifuge at 10000 rpm for 10 min, take the supernatant and filter it through a 0.22 μm organic microporous membrane, transfer it to an NMR tube, and perform NMR analysis. 1 H NMR, 13 CNMR, DEPT 135NMR, 1 H- 1H COSY NMR, HSQC NMR, HMBC NMR.

[0142] 2.7 In vitro functional characteristics

[0143] 2.7.1 Oil Holding Capacity (OHC)

[0144] Accurately weigh 100 mg of raspberry polysaccharide (O1), add 5 mL of soybean oil, mix thoroughly, and incubate at room temperature (37℃) for 2 h. After centrifugation at 10000×g for 20 min, separate the residue from the supernatant and weigh immediately (O2). The OHC calculation method is as follows:

[0145] Oil holding capacity (g / g) = (O2 - O1) / O1

[0146] 2.7.2 Cholesterol Adsorption Capacity (CAC)

[0147] The cholesterol adsorption capacity was determined by the o-phthalaldehyde method, and a standard curve was prepared using cholesterol standard solution.

[0148] Mix egg yolks and distilled water (1:9, v / v) and whisk thoroughly to form a homogeneous diluted egg yolk solution. Add approximately 80 mg of raspberry polysaccharide (W1) to 2.5 g of the diluted egg yolk solution, adjust the pH of the mixture to 7.0 (simulating the small intestine environment), incubate in a shaker water bath at 37°C for 2 h, centrifuge at 1000 × g for 10 min, and measure the OD of the supernatant. 550 , denoted as C d The cholesterol content in the blank diluted egg yolk without the sample is denoted as C. b The CAC calculation method is as follows:

[0149] Cholesterol adsorption capacity (mg / g) = (C b -C d ) / W1

[0150] 3. Animal experiments

[0151] Six-week-old male C57BL / 6 mice were purchased from Hangzhou Qizhen Co., Ltd. and housed in the Experimental Animal Research Center of Zhejiang University of Traditional Chinese Medicine in a specific pathogen-free (SPF) grade animal laboratory. The temperature was (23±2)℃, the relative humidity was (44%~52%), and the light-dark cycle was controlled at 12h. All mice were acclimatized to a normal diet for one week and then randomly grouped according to body weight for experiments.

[0152] 3.1 Dietary fat intake experiment in mice

[0153] 3.1.1 Experimental Grouping

[0154] Healthy male C57BL / 6 mice were randomly divided into three groups (n=30 per group): HFD+Oil group (distilled water, 10 mL / kg), HFD+Oil+HRCP-2-3 group (25 mg / kg), and HFD+Oil+HRCP-2-3 group (50 mg / kg). HFD feed was administered as part of the daily diet. Distilled water and the medication were administered by gavage at 9:00 AM daily, followed by soybean oil (10 μL / g) 10 minutes later. Drinking water was freely available. All feces excreted within 6 hours after soybean oil administration were collected daily and stored at -80℃ for subsequent experiments. Mouse weight and food intake were recorded daily. The experiment lasted seven days, with the mice fasted overnight (water allowed) on the last day for sampling.

[0155] 3.1.2 Fat Tolerance Test

[0156] Five mice were randomly selected from each group, weighed, and administered the corresponding drugs and soybean oil by gavage. At 0, 1, 2, 4, and 6 hours, the mice were anesthetized by intraperitoneal injection of 0.3% sodium pentobarbital. Blood was collected via the inferior vena cava, allowed to stand at room temperature for 3 hours, and then centrifuged at 3000 r / min for 15 minutes to collect serum and measure the TG level in the serum.

[0157] 3.1.3 Determination of fat and fatty acid absorption

[0158] Five mice were randomly selected from each group, weighed, and administered the appropriate drugs via gavage. They were then administered soybean oil (10 μg / g) labeled with BODIPY 500 / 510C1,C12 (0.5 μg / g, a green fluorescent fatty acid probe) via gavage. Two hours later, the mice were anesthetized, and blood was collected from the inferior vena cava to collect serum. Cecal contents were collected and extracted with a methanol / chloroform mixture (1:2). The fluorescence intensity in serum and organic phase was measured using a multi-functional microplate reader, and the TG concentration in serum and organic phase was measured using a kit. Duodenum and proximal jejunum were collected, embedded in an OCT complex, sectioned, and observed under a fluorescence microscope.

[0159] 3.1.4 Determination of fecal long-chain fatty acids

[0160] Fresh mouse feces were collected and freeze-dried. 100 mg of the extract was added to chloroform:methanol (2:1) to extract total lipids. The total lipids were then added to a sulfuric acid / methanol solution for methylation. GC-MS analysis was used to qualitatively and quantitatively determine the fatty acids in the feces. Supleco 37-mixed fatty acid methyl ester standard was used as the standard.

[0161] 3.2 Mouse model of high-fat binge eating

[0162] Mice were randomly divided into three groups (n=10 per group): HFD+HFE group (distilled water, 10 mL / kg), HFD+HFE+LRCP-2-3 group (50 mg / kg), and HFD+HFE+HRCP-2-3 group (100 mg / kg). HFD feed was administered as part of the daily diet. Distilled water and the medication were administered by gavage at 9:00 AM daily, followed by a high-fat emulsion (10 μL / g) 10 minutes later. Drinking water was freely available. Mouse weight and food intake were recorded. Feces excreted within 12 hours were collected from the mice three days before the end of the experiment and stored at -80℃. The experiment lasted 28 days. On the last day, the mice were fasted overnight (but allowed free water), and samples were collected.

[0163] Mice were weighed, anesthetized, and blood was drawn from the inferior vena cava. Serum was collected, and the levels of TG, TC, LDL-C, and HDL-C in the serum were measured. Adipose tissue was collected and weighed.

[0164] Fresh mouse feces were collected, freeze-dried, and then chloroform:methanol (2:1) was added to extract total triglycerides from the feces.

[0165] 4. Experimental Results

[0166] 4.1 Structural Identification of Raspberry Polysaccharides

[0167] 4.1.1 Homogeneity and Molecular Weight Analysis

[0168] The single symmetrical peak observed by HPSEC chromatography indicates that raspberry polysaccharide (RCP-2-3) is a highly homogeneous polysaccharide component. Figure 2 b), and based on pullulan standards, the weight-average molecular weight of raspberry polysaccharide was calculated to be 8.54 kDa. Figure 2 c).

[0169] 4.1.2 FT-IR and Monosaccharide Composition Analysis

[0170] FT-IR spectra ( Figure 3 a) This shows that RCP-2-3 has a typical uronic acid structure, 3422 cm⁻¹ -1 The broad, strong absorption peak is the stretching vibration peak of OH; 1609 cm⁻¹ -1 The strong absorption peak is the bending vibration peak of OH; 1744 cm⁻¹ -1 The absorption peak at that point is the stretching vibration of C=O(-COOH), indicating that raspberry polysaccharides contain uronic acid.

[0171] The monosaccharide composition of raspberry polysaccharides was analyzed by ion chromatography. Seven distinct peaks were observed in the chromatogram. Figure 3(b) The monosaccharide composition of the raspberry polysaccharide was compared with that of a standard monosaccharide reference spectrum. The monosaccharide composition was identified as fucose, rhamnose, arabinose, galactose, xylose, galacturonic acid, and glucuronic acid, with the following molar ratios: fucose (1.2%), rhamnose (3.4%), arabinose (7.2%), galactose (2.0%), xylose (1.6%), galacturonic acid (83.2%), and glucuronic acid (1.4%). Due to limitations of the standard, apigenin was not detected. Subsequent NMR results indicated that RCP-2-3 contained trace amounts of apigenin. Combined with FT-IR analysis, the presence of uronic acid and galacturonic acid as the main components of the raspberry polysaccharide confirmed that RCP-2-3 is an acidic pectin polysaccharide.

[0172] 4.1.3 Analysis of Glycosidic Bond Linkage Mode

[0173] We performed methylation analysis on raspberry polysaccharides to elucidate the glycosidic bond linkages of the polysaccharides. The results are shown in [Figure number missing]. Figure 3 c. Liquid chromatography and mass spectrometry of partially methylated aldosterone acetate (PMAA) provided further analysis of the glycosidic bond linkage mechanism, such as... Figure 3 d to Figure 3As shown in m. This comprehensive analysis identified 12 different glycosidic bond types in raspberry polysaccharides, specifically: terminal arabinofuranose (T-Araf), terminal xylopyranose (T-Xylp), terminal rhamnopyranose (T-Rhap), 1,5-linked arabinofuranose (1,5-Araf), terminal galactopyranose (T-Galp), terminal galacturonic acid pyranose (T-GalAp), and 1,3,4-linked rhamnopyranose. Sugars (1,3,4-Rhap), 1,3,4-linked fucopyranose (1,3,4-Fucp), 1,4-linked galacturonic acid pyranose (1,4-GalAp), 1,3,4-linked galacturonic acid pyranose (1,3,4-GalAp), 1,2,4-linked galacturonic acid pyranose (1,2,4-GalAp), 1,3,6-linked galactopyranose (1,3,6-Galp). The molar ratios of the following 12 glycosidic bonds in this homogeneous raspberry polysaccharide are: T-Araf: T-Xylp: T-Rhap: 1,5-Araf: T-Galp: T-GalAp: 1,3,4-Rhap: 1,3,4-Fucp: 1,4-GalAp: 1,3,4-GalAp: 1,2,4-GalAp: 1,3,6-Galp = 2.3:1.3:0.8:1.3:1.6:5.0:0.9:0.6:82.4:1.8:0.9:1.1. Combined with NMR analysis, RCP-2-3 contains 1,2-linked glucuronylpyranose (1,2-GlcAp) and 1,3-linked apigeninose (1,5-Apif). 1,4-GalAp is the main chain linkage of RCP-2-3. Considering the diversity of other glycosidic bonds, RCP-2-3 is classified as rhamnogalacturonic acid type II (RG-II) pectin.

[0174] 4.1.4 NMR Analysis

[0175] The chemical structure of raspberry polysaccharides was further characterized by NMR spectroscopy, including various 1 HNMR, 13 C NMR, DEPT135NMR 1 H- 1¹H COSY NMR, HSQC NMR, and HMBC NMR were used to identify the following peak assignments: (A) terminal α-galacturonic acid pyranose (T-α-GalAp), (B) 1,4-linked α-galacturonic acid pyranose (1,4-α-GalAp), (C) 1,3,4-linked α-galacturonic acid pyranose (1,3,4-α-GalAp), and (D) 1,4-linked α-galacturonic acid pyranose with a methoxy group at position 6 (1,4-α-GalAp-). (6-ome), (E) terminal α-hexuronide pyranose (T-α-HexAp), (F) 1,4-linked β-galacturonide pyranose reducing end (1,4-β-GalAp (red)), (G) 1,4-linked α-galacturonide pyranose reducing end (1,4-α-GalAp (red)), (H) 1,2,4-linked α-galacturonide pyranose (1,2,4-α-GalAp (red)). p), (I) terminal α-arabinofuranose (T-α-Araf), (J) 1,5-linked α-arabinofuranose (1,5-α-Araf), (K) terminal β-xylpyranose (T-β-Xylp), (L) terminal β-rhamnopyranose (T-β-Rhap), (M) 1,3,4-linked α-rhamnopyranose (1,3,4-α-Rhap), (N) 1,2-linked β-glucuronide pyranose (1,2-β-GlcAp), (O)1,3,4-linked α-fucopyranose (1,3,4-α-Fucp), (P)-terminal β-galactopyranose (T-β-Galp), (Q)1,3,6-linked β-galactopyranose (1,3,6-Galp), and (R)1,3-linked β-apigeninose (1,3-β-Apif) were used. Results are shown in […]. Figure 4 a to Figure 4 f. Raspberry polysaccharide has an RG-II domain, and its fine structure is as follows: Figure 4 As shown in g.

[0176] 4.1.5 Study on the in vitro functional properties of raspberry polysaccharides

[0177] like Figure 5 The functional properties of crude raspberry polysaccharide, refined polysaccharides from various eluted fractions, and homogeneous raspberry polysaccharide (RCP-2-3) were evaluated using oil-holding capacity and cholesterol adsorption capacity, respectively. Homogeneous raspberry polysaccharide exhibited excellent in vitro oil-holding properties, significantly superior to other raspberry polysaccharide components.

[0178] 4.2 Raspberry polysaccharides promote the excretion of dietary fats.

[0179] like Figure 6As shown, in mice fasted and then fed soybean oil, serum TG levels initially increased to a peak and then decreased to normal levels after dietary lipid intake, with the peak occurring around 2 hours later. RCP-2-3 reduced the peak absorption of TG in serum and decreased postprandial serum triglyceride (TG) accumulation stimulated by soybean oil. Direct observation of dietary lipid uptake in intestinal cells using fat and fatty acid absorption experiments further demonstrated that the mechanism by which RCP-2-3 reduces postprandial serum TG accumulation is related to the inhibition of intestinal lipid absorption. BODIPY, a green fluorescent probe for fatty acids, was used. Mice were gavaged with BODIPY-labeled soybean oil, and intestinal cells were observed under a microscope. Numerous fluorescently labeled lipid droplets were observed in the intestinal villi of the model group mice, while fluorescence in the intestinal villi of mice treated with RCP-2-3 was significantly reduced, a finding confirmed by quantitative fluorescence analysis. Quantitative analysis of fluorescence intensity in mouse serum and cecal contents revealed that the serum BODIPY fluorescence intensity of RCP-2-3-treated mice was significantly lower than that of the model group mice, while the BODIPY fluorescence intensity and TG content in the cecal contents were significantly higher than those of the model group. All the above evidence indicates that RCP-2-3 can inhibit the absorption of dietary lipids in the intestine and promote their excretion. Further analysis of the types and contents of fatty acids in mouse feces after gavage administration of soybean oil explored whether RCP-2-3 has a specific effect on the excretion of specific fatty acids from soybean oil. The results showed that raspberry polysaccharide can effectively promote the excretion of long-chain fatty acids from dietary oils, and has a significant promoting effect on palmitic acid, oleic acid, linoleic acid, and stearic acid.

[0180] like Figure 7 As shown, mice were fed high-calorie diets (HFD) for 28 consecutive days and administered a high-fat emulsion via gavage to achieve excessive intake. RCP-2-3 intervention slowed weight gain, reduced adipose tissue accumulation, and decreased serum levels of total triglycerides, total cholesterol, and low-density lipoprotein (LDL), while increasing high-density lipoprotein (HDL) levels. Comparison of weight gain with dietary intake revealed that, after consuming the same weight (calories) of food, RCP-2-3 significantly reduced weight gain and promoted the excretion of feces containing high levels of fat.

[0181] The embodiments of the present invention have been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the present invention. The descriptions of the embodiments above are only for the purpose of helping to understand the method and core ideas of the present invention. Furthermore, any changes or modifications made by those skilled in the art based on the ideas of the present invention, its specific implementation methods, and its application scope, are all within the scope of protection of the present invention. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. A raspberry homogeneous polysaccharide, characterized in that, The monosaccharide components of the raspberry uniform polysaccharide are composed of fucose, rhamnose, arabinose, galactose, xylose, galacturonic acid, glucuronic acid and apiose; The main chain structure of the raspberry uniform polysaccharide is 1,4-linked galacturonic acid pyranose; The weight average molecular weight of the raspberry uniform polysaccharide is 8.113 kDa to 8.967 kDa; The molar ratio of the following 7 monosaccharides of the raspberry uniform polysaccharide is fucose: rhamnose: arabinose: galactose: xylose: galacturonic acid: glucuronic acid = (1.14-1.26): (3.23-3.57): (6.84-7.56): (1.9-2.1): (1.52-1.68): (79.04-87.36): (1.33-1.47). The molar proportion of the apiose in the raspberry uniform polysaccharide is greater than 0 and less than 2; The raspberry uniform polysaccharide is rhamnogalacturonan II type acidic pectic polysaccharide.

2. Raspberry polysaccharide according to claim 1, characterized in that, The glycosidic bond of the raspberry uniform polysaccharide is selected from one or more of the following: terminal arabinofuranose (T-Araf), terminal xylopyranose (T-Xylp), terminal rhamnopyranose (T-Rhap), 1,5-linked arabinofuranose (1,5-Araf), terminal galactopyranose (T-Galp), terminal galacturonopyranose (T-GalAp), 1,3,4-linked rhamnopyranose (1,3,4-Rhap), 1,3,4-linked fucopyranose (1,3,4-Fucp), 1,4-linked galacturonopyranose (1,4-GalAp), 1,3,4-linked galacturonopyranose (1,3,4-GalAp), 1,2,4-linked galacturonopyranose (1,2,4-GalAp), 1,3,6-linked galactopyranose (1,3,6-Galp), 1,2-linked glucuronopyranose (1,2-GlcAp) and 1,3-linked apiofuranose (1,5-Apif).

3. Raspberry fructan according to claim 2, characterized in that, The molar ratio of the following 12 glycosidic bonds of the raspberry uniform polysaccharide is T-Araf: T-Xylp: T-Rhap: 1,5-Araf: T-Galp: T-GalAp: 1,3,4-Rhap: 1,3,4-Fucp: 1,4-GalAp: 1,3,4-GalAp: 1,2,4-GalAp: 1,3,6-Galp = (0.1-5): (0.1-5): (0.1-5): (0.1-5): (0.1-5): (2-10): (0.1-5): (0.1-5): (70-90): (0.1-5): (0.1-5): (0.1-5).

4. Raspberry fructan according to claim 3, characterized in that, The molar ratio of the following 12 glycosidic bonds of the raspberry uniform polysaccharide is T-Araf:T-Xylp:T-Rhap:1,5-Araf:T-Galp:T-GalAp:1,3,4-Rhap:1,3,4-Fucp:1,4-GalAp:1,3,4-GalAp:1,2,4-GalAp:1,3,6-Galp=(1.5-4):(0.5-2):(0.1-1.5):(0.5-2):(0.5-2):(3-8):(0.5-2):(0.1-2):(75-88):(1-3):(0.5-2):(0.5-2).

5. Raspberry fructan according to claim 4, characterized in that, The molar ratio of the following 12 glycosidic bonds of the raspberry uniform polysaccharide is T-Araf:T-Xylp:T-Rhap:1,5-Araf:T-Galp:T-GalAp:1,3,4-Rhap:1,3,4-Fucp:1,4-GalAp:1,3,4-GalAp:1,2,4-GalAp:1,3,6-Galp=(2.185-2.415):(1.235-1.365):(0.76-0.84):(1.235-1.365):(1.52-1.68):(4.75-5.25):(0.855-0.945):(0.57-0.63):(78.28-86.52):(1.71-1.89):(0.855-0.945):(1.045-1.155).

6. A method for preparing the raspberry homogenous polysaccharide according to any one of claims 1 to 5, characterized in that, The method comprises the following steps: (1) taking a proper amount of raspberry fruits, crushing, adding ethanol, standing at room temperature, repeating 2 times; (2) adding water to the raspberry powder after the ethanol is evaporated, immersing at 90 DEG C for 3h, repeating 2 times, combining the filtrates, concentrating, obtaining a concentrated solution; (3) stirring the concentrated solution, adding ethanol, standing and settling, filtering, obtaining a filter residue; (4) evaporating the ethanol of the filter residue, dissolving with water, drying, obtaining raspberry crude polysaccharide; and (5) The raspberry crude polysaccharides are separated by DEAE-Sepharose Fast Flow weak anion exchange chromatography to obtain a 0.1-0.3 M sodium chloride fraction, and the 0.1-0.3 M sodium chloride fraction is separated by Superdex TM 200 gel permeation chromatography to obtain the raspberry homogeneous polysaccharides.

7. The method of claim 6, wherein, The fruits are unripe fruits.

8. The method of claim 6, wherein, The raspberries are Rubus chingii Hu.

9. The method of claim 6, wherein, The volume percentage concentration of the ethanol is 80%-100%.

10. The method of claim 9, wherein, The volume percentage concentration of the ethanol is 90.25%-99.75%.

11. The method of claim 6, wherein, In step (1), the volume / mass ratio of the ethanol to the raspberry fruits is 1-10, unit: L / kg.

12. The method of claim 11, wherein, The volume / mass ratio of the ethanol to the raspberry fruits is 3.8-4.2, unit: L / kg.

13. The method of claim 6, wherein, In step (1), the standing time is 12-36h.

14. The method of claim 13, wherein, The standing time is 22.8h-25.2h.

15. The method of claim 6, wherein, In step (2), the volume / mass ratio of the water to the raspberry fruits is 1-60, unit: L / kg.

16. The method of claim 15, wherein, The volume / mass ratio of the water to the raspberry fruits is 38-42, unit: L / kg.

17. The method of claim 6, wherein, In step (2), the temperature of the water is 60 DEG C-100 DEG C.

18. The method of claim 17, wherein, The temperature of the water is 85.5°C to 94.5°C.

19. The method of claim 6, wherein, In step (2), the concentration is reduced-pressure rotary evaporation concentration.

20. The method of claim 6, wherein, In step (3), the ratio of the volume of the ethanol to the concentrated solution is 1 to 6.

21. The method of claim 20, wherein, The ratio of the volume of the ethanol to the concentrated solution is 3.8 to 4.

2.

22. The method of claim 6, wherein, In step (3), the time for the standing and settling is 4 hours or more.

23. The method of claim 22, wherein, The time for the standing and settling is 11.4 to 12.6 hours.

24. The method of claim 6, wherein, In step (3), the filtration is reduced-pressure suction filtration.

25. The method of claim 6, wherein, In step (4), the drying is vacuum freeze drying.

26. The method of claim 6, wherein, In step (5), the concentration of the sodium chloride site is 0.19 to 0.21 M.

27. Use of the raspberry homogeneous polysaccharide of any one of claims 1 to 5 in the manufacture of a medicament for improving obesity.

28. Use of the raspberry homogeneous polysaccharide of any one of claims 1 to 5 in the manufacture of a medicament for preventing and / or assisting in the treatment of obesity.

29. Use according to claim 27 or 28, characterized in that, The obesity is obesity caused by eating habits, genetics, metabolism, and / or adipocyte abnormalities.

30. Use according to claim 29, characterized in that, The abnormalities are increased number of adipocytes and / or hypertrophy of adipocytes.

31. Use according to claim 27 or 28, characterized in that, The raspberry homogeneous polysaccharide prevents and / or assists in the treatment of obesity by one or both of inhibiting lipid absorption and promoting the discharge of lipids and / or oils.

32. Use according to claim 31, characterized in that, The lipids include triglycerides, cholesterol, and / or low-density lipoproteins.

33. The use according to claim 31, characterized in that The oils include long-chain fatty acids.

34. Use according to claim 33, characterized in that, The long-chain fatty acids include palmitic acid, oleic acid, linoleic acid, and / or stearic acid.

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