Raspberry polysaccharide and application thereof in preventing and treating obesity

Abstract

The invention discloses a raspberry homogeneous polysaccharide. The monosaccharide components of the raspberry homogeneous polysaccharide comprise fucose, rhamnose, arabinose, galactose, xylose, galacturonic acid, glucuronic acid and apiose. The main chain structure of the raspberry homogeneous polysaccharide is 1, 4-linked galacturonic acid pyranose, and the weight-average molecular weight of the raspberry homogeneous polysaccharide is 8.54 + / -5.0 kDa. The invention discloses a raspberry homogeneous polysaccharide component for inhibiting dietary oil absorption and preventing and improving obesity, which is prepared by the steps of water extraction and alcohol precipitation, ion exchange chromatographic column elution and permeation gel chromatographic column elution. The structure of the compound is analyzed by high performance gel permeation chromatography, infrared spectroscopy, ion chromatography, gas chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy. The homogeneous polysaccharide component can be used for improving obesity and fat tissue accumulation hypertrophy, and the efficacy of the homogeneous polysaccharide component is related to inhibition of absorption of dietary oil in the intestinal tract and promotion of discharge of the dietary oil into the body.

Description

Technical Field

[0001] The invention relates to the technical field of medicines, and in particular to raspberry polysaccharide and use thereof in preventing and treating obesity. Background Art

[0002] Obesity and overweight are diseases caused by long-term energy intake exceeding energy expenditure, which in turn leads to hyperlipidemia, diabetes, cardiovascular and cerebrovascular diseases, etc. One means of treating overweight and obesity is to limit energy intake by controlling the digestion and absorption of nutrients such as fat. Currently, there are very few drugs approved for marketing that limit fat digestion and absorption, and the side effects are obvious. For example, orlistat is an anti-obesity drug approved by the US Food and Drug Administration. It forms a covalent bond with lipase to prevent the activity of lipase and reduce the absorption of food fat in the intestine. It can reduce fat absorption by about 30%, promote fat excretion from the body, and thus achieve the effect of improving obesity. It is particularly suitable for obese people with high-fat diet habits. However, orlistat has been reported to have side effects including liver damage and fatty diarrhea. Raspberry is a traditional Chinese medicine that is both a medicine and a food. Its polysaccharide components can improve obesity in mice induced by a high-fat diet, but there are no reports that RG-Ⅱ polysaccharides contained in raspberries inhibit intestinal dietary lipid digestion and absorption and thus improve obesity. Summary of the invention

[0003] Based on this, the present invention provides a raspberry uniform polysaccharide, wherein the monosaccharide components of the raspberry uniform polysaccharide include fucose, rhamnose, arabinose, galactose, xylose, galacturonic acid, glucuronic acid and apiose;

[0004] Among them, the main chain structure of the raspberry uniform polysaccharide is 1,4-linked galacturonic acid pyranose;

[0005] The weight average molecular weight of the raspberry uniform polysaccharide is 8.54±5.0 kDa.

[0006] Furthermore, the molar ratio of the following seven monosaccharides of the raspberry homogeneous polysaccharide is 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 molar ratio of the following seven monosaccharides of the raspberry homogeneous polysaccharide is 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 molar ratio of the following seven monosaccharides of the raspberry homogeneous polysaccharide is 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.

[0009] Preferably, the molar proportion of celery sugar in the raspberry homogeneous polysaccharide is greater than 0.02.

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

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

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

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

[0014] Preferably, the glycosidic bonds of the raspberry homogeneous polysaccharide are selected from one or more of the following: terminal arabinofuranosyl (T-Araf), terminal xylose pyranose (T-Xylp), terminal rhamnosyl pyranose (T-Rhap), 1,5-linked arabinofuranosyl (1,5-Araf), terminal galactose pyranose (T-Galp), terminal galacturonic acid pyranose (T-GalAp), 1,3,4-linked rhamnosyl pyranose (1,3,4-Rhap), 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 apiosyl furanose (1,5-Apif).

[0015] Furthermore, the molar ratios of the following 12 glycosidic bonds of the raspberry homogeneous 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 = (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 ratios of the following 12 glycosidic bonds of the raspberry homogeneous 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 = (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 of the raspberry homogeneous 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 = 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.

[0018] According to another aspect of the present invention, there is provided a method for preparing the above-mentioned raspberry uniform polysaccharide, 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) adding water to the raspberry powder after evaporation of ethanol, immersing at 90° C. for 3 h, repeating twice, combining the filtrate, and concentrating to obtain a concentrated solution;

[0021] (3) stirring the concentrated solution, adding ethanol, allowing to settle, and filtering to obtain a filter residue;

[0022] (4) evaporating the ethanol from the residue, dissolving it in water, and drying it to obtain crude raspberry polysaccharide; and

[0023] (5) The crude raspberry polysaccharide is 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 uniform raspberry polysaccharide.

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

[0025] Furthermore, the 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 ratio of the volume / mass (L / kg) of the ethanol to the raspberry fruit is 1 to 10, for example, about 4;

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

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

[0030] Further, 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 concentrated by rotary evaporation under reduced pressure.

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

[0033] Furthermore, in step (3), the static sedimentation 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] Further, in step (5), the concentration of the sodium chloride site is about 0.2M.

[0037] According to another aspect of the present invention, there is provided a use of the above-mentioned raspberry uniform polysaccharide in preparing medicines, foods and / or health products for improving obesity;

[0038] Among them, the food is a functional food.

[0039] According to another aspect of the present invention, there is provided a use of the above-mentioned raspberry homogeneous polysaccharide in the preparation of a medicine for preventing and / or assisting in the treatment of obesity.

[0040] Furthermore, the 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 uniform polysaccharide prevents and / or assists in the treatment of obesity in one or both of the following ways: inhibiting lipid absorption and promoting lipid and / or oil excretion.

[0043] Further, the lipid includes triglyceride, cholesterol and / or low-density lipoprotein.

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

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

[0046] Beneficial effects of the present invention:

[0047] The invention discloses a raspberry homogeneous polysaccharide component for inhibiting dietary oil absorption and preventing and improving obesity. The homogeneous polysaccharide component is prepared by the steps of water extraction and alcohol precipitation, ion exchange chromatography column elution and permeation gel chromatography column elution, and its structure is analyzed by high performance gel permeation chromatography, infrared spectroscopy, ion chromatography, gas phase-mass spectrometry and nuclear magnetic resonance spectroscopy. The homogeneous polysaccharide component of the invention can be used to improve obesity and adipose tissue accumulation and hypertrophy, and its efficacy is related to inhibiting intestinal dietary oil absorption and promoting dietary oil excretion to the body. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without exceeding the scope of protection required by the present invention.

[0049] Figure 1The figure is a flow chart of extraction, separation and purification of raspberry uniform polysaccharide RCP-2-3. Among them, a is a flow chart of extraction, separation and purification of raspberry polysaccharide, b is immature East China raspberry fruit, and c is raspberry uniform polysaccharide RCP-2-3.

[0050] Figure 2 Schematic diagram of the detection of the physicochemical properties of the uniform polysaccharide RCP-2-3 from raspberries. Among them, a is the gel permeation chromatography separation diagram of the uniform polysaccharide RCP-2-3 from raspberries; b is the HPSEC chromatogram of the uniform polysaccharide RCP-2-3 from raspberries; and c is the scanning electron microscope diagram of the uniform polysaccharide RCP-2-3 from raspberries.

[0051] Figure 3 The figure is a schematic diagram of the structural reasoning of the homogeneous polysaccharide RCP-2-3 from raspberries. Among them, a is the FT-IR spectrum of the homogeneous polysaccharide RCP-2-3 from raspberries; b is the ion chromatogram of the standard and the homogeneous polysaccharide RCP-2-3 from raspberries; cm is the gas chromatogram and secondary ion fragment mass spectrum.

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

[0053] Figure 5 Schematic diagram of the in vitro functional properties study of different raspberry polysaccharides. a is the oil retention capacity result; b is the cholesterol adsorption capacity result.

[0054] Figure 6 Schematic diagram of the experimental results of dietary fat intake in mice. Among them, a is the experimental flow chart; b is a schematic diagram of the change of serum TG of mice stimulated by soybean oil after meal over time; c is a schematic diagram of the area under the curve of serum TG; d is a schematic diagram of TG content in cecal contents; e is a schematic diagram of BODIPY fluorescence 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 into a test tube of methanol / chloroform extract; k is a schematic diagram of the content of four long-chain fatty acids in mouse feces.

[0055] Figure 7The figure is a schematic diagram of the results of the high-fat diet overeating experiment on mice. Among them, a is the experimental flow chart; b is the weight growth trend of mice for 28 days; c is the schematic diagram of serum TG content after fasting; d is the schematic diagram of serum TC content after fasting; e is the schematic diagram of serum LDL-C content after fasting; f is the schematic diagram of serum HDL-C content after fasting; g is the schematic diagram of adipose tissue weight; h is the schematic diagram of the ratio of mouse weight growth to food intake; i is the schematic diagram of the weight of feces discharged by mice within 12 hours after gavage; j is the schematic diagram of TG content in mouse feces. DETAILED DESCRIPTION

[0056] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

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

[0058] The present invention is intended to encompass all alternatives, variations and equivalents that may be included in the present invention as defined by the claims. Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein that can be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described.

[0059] As used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0060] In the present invention, the term "comprising" is synonymous with "including". As used herein, the terms "comprising", "including", "having", "containing" or any other variation thereof are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus comprising the listed elements is not necessarily limited to only those elements, but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.

[0061] As described in the background technology section, there are very few drugs currently approved for marketing that limit fat digestion and absorption, and the side effects are obvious. In order to solve the above problems, the present invention provides a raspberry homogeneous polysaccharide, the monosaccharide components of which include fucose, rhamnose, arabinose, galactose, xylose, galacturonic acid, glucuronic acid and apiose;

[0062] Among them, the main chain structure of the raspberry uniform polysaccharide is 1,4-linked galacturonic acid pyranose;

[0063] The weight average molecular weight of the raspberry uniform polysaccharide is 8.54±5.0 kDa.

[0064] In the present invention, when a ratio, molar ratio, concentration, mass, time, temperature, or other value or parameter is expressed as a range, a preferred range, or a range defined by a series of upper preferred values ​​and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pairing of any range upper limit or preferred value with any range lower limit or preferred value, regardless of whether the range is disclosed separately. For example, when the range "5 to 10" is disclosed, the described range should be interpreted as including the range "5 to 10", "5 to 9", "5 to 8", "5 to 7", "5 to 6", "6 to 10", "6 to 9", "6 to 8", "6 to 7", "7 to 10", "7 to 9", "7 to 8", "8 to 10", "8 to 9", "9 to 10", etc. When a numerical range is described herein, unless otherwise stated, the range is intended to include its end values ​​and all integers and fractions within the range.

[0065] In a preferred embodiment, the molar ratio of the following seven monosaccharides of the raspberry homogeneous polysaccharide is 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 molar ratio of the following seven monosaccharides of the raspberry homogeneous polysaccharide is 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 molar ratio of the following seven monosaccharides of the raspberry homogeneous polysaccharide is 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 the present 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; "about 1.4" includes ±5% of 1.4, or from 1.33 to 1.47.

[0069] In a preferred embodiment, the mass proportion of the celery sugar in the raspberry uniform polysaccharide is greater than 0 and less than 2.

[0070] In a preferred embodiment, the raspberry homogeneous polysaccharide is rhamnogalacturonan-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 the present 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 arabinofuranosyl (T-Araf), terminal xylose pyranose (T-Xylp), terminal rhamnosyl pyranose (T-Rhap), 1,5-linked arabinofuranosyl (1,5-Araf), terminal galactose pyranose (T-Galp), terminal galacturonic acid pyranose (T-GalAp), 1,3,4-linked rhamnosyl pyranose (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 apiosyl furanose (1,5-Apif).

[0076] In a preferred embodiment, the molar ratio of the following 12 glycosidic bonds of the raspberry homogeneous 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): (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 of the raspberry homogeneous 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 of the raspberry homogeneous 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 = 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 the present 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 ±5% of 0.9, or from 0.855 to 0.945; “about 0.6” includes ±5% of 0.6, or from 0.57 to 0.63; “about 82.4” includes ±5% of 82.4, or from 78.28 to 86.52; “about 1.8” includes ±5% of 1.8, or from 1.71 to 1.89; “about 1.1” includes ±5% of 1.1, or from 1.045 to 1.155.

[0080] According to another aspect of the present invention, there is provided a method for preparing the above-mentioned raspberry uniform polysaccharide, 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) adding water to the raspberry powder after evaporation of ethanol, immersing at 90° C. for 3 h, repeating twice, combining the filtrate, and concentrating to obtain a concentrated solution;

[0083] (3) stirring the concentrated solution, adding ethanol, allowing to settle, and filtering to obtain a filter residue;

[0084] (4) evaporating the ethanol from the residue, dissolving it in water, and drying it to obtain crude raspberry polysaccharide; and

[0085] (5) The crude raspberry polysaccharide is 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 TM200 gel permeation chromatography to obtain the uniform raspberry polysaccharide.

[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%, such as about 95%.

[0089] In the present invention, "about" refers to a value within a range of ±5% of a particular value. For example, "about 95%" includes ±5% of 95%, 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 1-10, for example, about 4.

[0091] In the present 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 standing time is 12 to 36 hours, for example, about 24 hours.

[0093] In the present 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 volume / mass (L / kg) ratio of the water to the raspberry fruit is 1-60, such as about 40.

[0095] In the present 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 the present 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 concentrated by rotary evaporation under reduced pressure.

[0099] In a preferred embodiment, in step (3), the volume ratio of the ethanol to the concentrated solution is 1 to 6, such as about 4.

[0100] In the present 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 static sedimentation time is more than 4 hours, for example, about 12 hours.

[0102] In the present 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 site is about 0.2M.

[0106] In the present 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, there is provided a use of the above-mentioned raspberry uniform polysaccharide in preparing medicines, foods and / or health products for improving obesity;

[0108] Among them, the food is a functional food.

[0109] According to another aspect of the present invention, there is provided a use of the above-mentioned raspberry homogeneous polysaccharide in the preparation of a medicine for preventing and / or assisting in the treatment of obesity.

[0110] In a preferred embodiment, the obesity is obesity 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 in one or both of the following ways: inhibiting lipid absorption and promoting lipid and / or oil excretion.

[0113] In a preferred embodiment, the lipids include 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 acids include palmitic acid, oleic acid, linoleic acid and / or stearic acid.

[0116] The terms "food", "health care product", "food product" and "health care product" of the present invention refer to products that are intended to be ingested by animals (including humans) and provide nutrition or health care effects to the animals.

[0117] According to another aspect of the present invention, there is provided the above-mentioned raspberry uniform polysaccharide for use in preventing and / or assisting in the treatment of obesity in a subject.

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

[0119] In the present invention, the terms "subject", "individual" or "patient" are used interchangeably herein and refer to a vertebrate, preferably a mammal. The mammal may be a human, non-human primate, mouse, rat, dog, cat, horse or cow, but is not limited to these examples. Mammals other than humans can be advantageously 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 above-mentioned raspberry homogeneous polysaccharide of the present invention.

[0120] An "effective amount" of the above-mentioned raspberry homogeneous polysaccharide used in the present invention can achieve the desired therapeutic and / or preventive effect. The amount effective for this use will depend on, for example, the route of delivery, the activity of the specific active substance or formulation used, the type of disease, the stage and severity of the disease being treated, the individual's weight and general health, and the judgment of the prescribing physician. Dosages can be administered once a week, or once every two days or daily, or even several times a day. Dosage units can be administered for short periods of time (e.g., weeks to months) or longer periods of time (months to years). "Effective amount" specifically refers to the amount of the above-mentioned raspberry homogeneous polysaccharide that imparts a therapeutic effect (e.g., control, relief, improvement, alleviation or slowing of progression); or prevents (e.g., delays the onset or reduces the risk of development) a disease, disorder or condition or its symptoms to the subject being treated.

[0121] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually carried out according to conventional conditions or conditions recommended by the manufacturer.

[0122] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the methods of the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only.

[0123] The above features mentioned in the present invention or the features mentioned in the embodiments can be combined in any way. All the features disclosed in this patent specification can be used in combination with any combination form, and each feature disclosed in the specification can be replaced by any alternative feature that can provide the same, equal or similar purpose. Therefore, unless otherwise specified, the disclosed features are only general examples of equal or similar features.

[0124] Example

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

[0126] 1kg of immature raspberry (Rubus chingii Hu.) fruit was crushed, and 4L of 95% ethanol was used to stand for 24h, and the process was repeated twice to remove fat-soluble components. After the ethanol was naturally evaporated, 40L of water was added to the raspberry fruit powder, and the mixture was heated to 90°C for extraction, and the process was repeated twice. The extract was concentrated to 1L by rotary evaporation under reduced pressure, and 4L of 95% ethanol was added with constant stirring. The mixture was naturally precipitated for 12h, and then filtered. The ethanol was evaporated from the residue, and the residue was dissolved in water and then freeze-dried in vacuum to obtain crude raspberry polysaccharide (RCP). The crude raspberry polysaccharide was separated by DEAE-Sepharose Fast Flow weak anion exchange chromatography using water, 0.2M sodium chloride and 0.5M sodium chloride solutions to obtain polysaccharides of different polarity parts. The polysaccharide of 0.2M sodium chloride part (RCP-0.2) was further separated by Superdex TM 200 gel permeation chromatography to obtain the raspberry polysaccharide component (RCP-2-3), such as 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 by high performance liquid chromatography combined with high performance size exclusion chromatography (HPSEC) with differential detector. Chromatographic conditions: the chromatographic column was Shodex KS-804 and KS-802 in series (300 mm × 8.0 mm, id 5 μm), the mobile phase was 0.2 mol / L sodium chloride solution, the injection volume was 20 μL, the column temperature was 40 °C, the flow rate was 0.8 mL / min, and the time was 30 min. The standard product was dextran with different molecular weights (Mw: 0.66 × 10 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 Da), and dissolved in mobile phase to prepare a 2.0 mg / mL standard solution. RCP-2-3 was dissolved in mobile phase to prepare a 2.0 mg / mL solution, and filtered through a 0.45 micron microporous membrane. The retention time of the dextran standard and the corresponding weight-average molecular weight were used to draw a calibration curve by three-time fitting, and 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 by Fourier transform infrared spectrometer (FT-IR). RCP-2-3 and KBr dried to constant weight were mixed and ground in a mass ratio of 1:100 (w / w), pressed into thin sheets, and the infrared spectral characteristics of RCP-2-3 were measured at a frequency of 4000-400 cm -1 The transmittance within the range is measured, and the infrared spectrum is obtained and analyzed.

[0132] 2.3 Scanning electron microscopy analysis

[0133] The microstructure and morphological characteristics of RCP-2-3 were analyzed by field emission scanning electron microscopy. The sample was attached to a metal disk with a double-sided adhesive and gold powder was sprayed using a sprayer. 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 mannuronic acid to prepare a 100 μg / mL working solution, dilute to different concentrations, and transfer to a chromatographic bottle for testing. Accurately weigh 5 mg (± 0.05 mg) of RCP-2-3 sample, add 1 mL of 2M TFA acid solution, and heat at 121°C for 2 hours. Pass nitrogen and blow dry. Add methanol to wash, blow dry again, and repeat the methanol wash 2-3 times. Add sterile water to dissolve and transfer to a chromatographic bottle for testing.

[0136] The chromatographic system used was the Thermo ICS5000+ ion chromatography system (ICS5000+, (Thermo Fisher Scientific, USA), equipped with Dionex TM CarboPac TM PA20 (150*3.0mm, 10μm) liquid chromatography column, injection volume of 5μL. Mobile phase A (H2O), mobile phase B (0.1MNaOH), mobile phase C (0.1MNaOH, 0.2MNaAc), column temperature of 30°C, gradient elution (0-26.0min, 95%-85%A, 5%B, 0%-10%C; 26.0-42.0min, 85%A, 5%B, 10%C; 42.0-42.1min, 85%-60%A, 5%B, 10%C); %~0%B, 10%~40%C; 42.1~52.0min, 60%A, 0%~40%B, 40%~0%C; 52.0~52.1min, 60%~95%A, 40%~5%B, 0%C; 52.1~60.0min, 95%A, 5%B, 0%C); the flow rate is 0.5mL / min, and the monosaccharide components are analyzed and detected by electrochemical detector. The chromatographic data are processed by the software Chromeleon, with the retention time (time, min) of the detection as the horizontal axis and the response value (Response, nC) of the ion detection as the vertical axis. The standard sample ion chromatogram and the sample ion chromatogram are qualitatively determined according to the retention time of the chromatographic peak, quantitatively determined by the external standard method, and the mass of different monosaccharides is determined, and the molar ratio of each monosaccharide is calculated according to the molar mass of the monosaccharide.

[0137] 2.5 Glycosidic bond 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 hours. Add 1 mL of 2M imidazole, add 1 mL of 30 mg / mL NaBD4, and react for 3 hours. Add 100 μL of glacial acetic acid to terminate the reaction, dialyze, and freeze-dry. Weigh 1 mg of the sample to be tested, add 500 μL of DMSO to dissolve. Add 1 mg of NaOH and incubate for 30 minutes. Add 50 μL of iodomethane solution and react for 1 hour. Add 1 mL of water and 2 mL of dichloromethane, vortex to mix, centrifuge, and discard the aqueous phase. Repeat the water wash 3 times. Take the lower dichloromethane phase and evaporate to dryness. Add 100 μL of 2M TFA and react at 121°C for 90 minutes. Evaporate to dryness at 30°C. Add 50 μL of 2M ammonia water and 50 μL of 1M NaBD4, mix well, and react at room temperature for 2.5 hours. Add 20 μL of acetic acid to terminate the reaction, blow dry with nitrogen, wash twice with 250 μL of methanol, blow dry with nitrogen. Add 250 μL of acetic anhydride, vortex mix, and react at 100°C for 2.5 hours. Add 1 mL of water and let stand for 10 minutes. Add 500 μL of dichloromethane, vortex mix, centrifuge, and discard the aqueous phase. Repeat the water wash 3 times. Take the lower dichloromethane phase and detect it on the machine.

[0139] Chromatographic system Agilent 7890A-5977B gas spectrometer, equipped with HP-5MS capillary column (30m×0.25mm×0.25μm, Agilent J&W Scientific, Folsom, CA, USA), carrier gas is high-purity helium (purity not less than 99.999%), flow rate is 1.0mL / min, and the injection port temperature is 260℃. Injection volume is 1μL, split injection, split ratio is 10:1, and solvent delay is 2.2min. Program temperature rise: 50℃ for 1.0min, 50℃ / min to 130℃, 3℃ / min to 230℃, and hold for 2min. Electron bombardment ion source (EI), injection port temperature is 230℃, quadrupole temperature is 150℃, and electron energy is 70eV. Scanning mode is full scan mode (SCAN), mass scanning range (m / z): 30-600.

[0140] 2.6 NMR analysis

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

[0142] 2.7 In vitro functional properties

[0143] 2.7.1 Oil Holding Capacity (OHC)

[0144] Weigh 100 mg of raspberry polysaccharide (O1) accurately, add 5 mL of soybean oil, mix thoroughly, and incubate at room temperature (37°C) for 2 h. After centrifugation at 10,000 × 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 the standard curve was prepared using cholesterol standard solution.

[0148] Egg yolk and distilled water (1:9, v / v) were mixed and thoroughly beaten into a uniformly diluted egg yolk solution. About 80 mg of raspberry polysaccharide (W1) was added to 2.5 g of diluted egg yolk solution, the pH of the mixture was adjusted to 7.0 (simulating the small intestine environment), incubated in a shaking water bath at 37°C for 2 h, centrifuged at 1000×g for 10 min, and the supernatant OD was detected. 550 , denoted as C d The cholesterol content in the blank diluted egg yolk without sample is recorded 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 were raised in the Experimental Animal Research Center of Zhejiang Chinese Medical University, a specific pathogen-free (SPF) animal laboratory with a temperature of (23±2)°C, relative humidity of (44% to 52%), and a 12-hour light-dark cycle. All mice were fed with ordinary feed for 1 week and then randomly divided into groups according to body weight for the experiment.

[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 3 groups (30 mice in each group): HFD+Oil group (distilled water, 10mL / kg), HFD+Oil+LRCP-2-3 group (25mg / kg), and HFD+Oil+HRCP-2-3 group (50mg / kg). HFD feed was used for daily diet, distilled water and drugs were gavaged at 9 o'clock every day, and soybean oil (10μL / g) was gavaged 10 minutes later. Drinking water was freely available. All feces discharged within 6 hours after soybean oil gavage were collected every day and stored at -80℃ for subsequent experiments. The weight and food intake of mice were recorded daily. The experiment lasted for seven days. On the last day, the mice were fasted overnight (but not water-deprived), and the samples were collected.

[0155] 3.1.2 Fat tolerance test

[0156] Five mice were randomly selected from each group, weighed, and gavaged with corresponding drugs and soybean oil. They were anesthetized with 0.3% sodium pentobarbital intraperitoneally at 0, 1, 2, 4, and 6 h, respectively. Blood was collected through the inferior vena cava, and after standing at room temperature for 3 h, the blood was centrifuged at 3000 r / min for 15 min, and serum was collected to measure the TG level in the serum.

[0157] 3.1.3. Fat and fatty acid absorption determination

[0158] Five mice were randomly selected from each group, weighed, and gavaged with the corresponding drugs. Then, they were gavaged with soybean oil (10 μg / g) labeled with BODIPY 500 / 510C1, C12 (0.5 μg / g, fatty acid green fluorescent probe), and anesthetized 2 hours later. Blood was collected from the inferior vena cava and serum was collected. The contents of the cecum were collected and extracted with a methanol / chloroform mixture (1:2). The fluorescence intensity in the serum and organic phase was determined by a multifunctional microplate reader, and the TG concentration in the serum and organic phase was determined by a kit. The duodenum and proximal jejunum were collected, embedded in the OCT complex, sliced, and observed under a fluorescence microscope.

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

[0160] The collected fresh mouse feces were freeze-dried, and 100 mg was taken and added with chloroform:methanol (2:1) to extract the total lipids in the feces. The total lipids were added with sulfuric acid / methanol solution for methyl esterification, and the fatty acids in the feces were qualitatively and quantitatively analyzed by GC-MS injection. The standard product used was Supleco 37 mixed fatty acid methyl ester standard.

[0161] 3.2 High-fat binge eating model in mice

[0162] The mice were randomly divided into 3 groups (10 mice in each group): HFD+HFE group (distilled water, 10mL / kg), HFD+HFE+LRCP-2-3 group (50mg / kg), and HFD+HFE+HRCP-2-3 group (100mg / kg). HFD feed was used for daily diet, distilled water and drugs were gavaged at 9 o'clock every day, and high-fat emulsion (10μL / g) was gavaged 10 minutes later. Drinking water was freely available, and the weight and food intake of mice were recorded. Three days before the end of the experiment, the feces excreted by mice within 12 hours were collected and stored at -80℃. The experiment lasted for 28 days. On the last day, the mice were fasted overnight (but not water-deprived), and the samples were collected.

[0163] The mice were weighed, anesthetized, and blood was collected from the inferior vena cava to collect serum, and the levels of TG, TC, LDL-C, and HDL-C in the serum were determined. Adipose tissue was collected and weighed.

[0164] The collected fresh mouse feces were freeze-dried, and chloroform:methanol (2:1) was added to extract total triglycerides in 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 of HPSEC chromatogram indicated that raspberry polysaccharide (RCP-2-3) was a highly homogeneous polysaccharide component ( Figure 2 b), and the weight average molecular weight of raspberry polysaccharide was calculated to be 8.54 kDa in combination with pullulan standard. Figure 2 c).

[0169] 4.1.2 FT-IR and monosaccharide composition analysis

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

[0171] The monosaccharide composition of raspberry polysaccharide was analyzed by ion chromatography. Seven different peaks were found in the chromatogram ( Figure 3b) and compared with the standard monosaccharide reference spectrum. The monosaccharide composition of raspberry polysaccharide was identified as fucose, rhamnose, arabinose, galactose, xylose, galacturonic acid and glucuronic acid, and the molar ratio of monosaccharides was: fucose (1.2%), rhamnose (3.4%), arabinose (7.2%), galactose (2.0%), xylose (1.6%), galacturonic acid (83.2%), glucuronic acid (1.4%). Due to the limitation of the standard sample of this method, piculose was not detected. According to the subsequent NMR results, RCP-2-3 contained trace piculose. Combined with FT-IR analysis, the presence of uronic acid and the main presence of galacturonic acid in raspberry polysaccharide confirmed that RCP-2-3 is an acidic pectin polysaccharide.

[0172] 4.1.3 Analysis of glycosidic bond connection

[0173] We performed methylation analysis on raspberry polysaccharides to clarify the glycosidic bond connection mode of polysaccharides. The results are shown in Figure 3 c. Liquid chromatography and mass spectrometry of partially methylated alditol acetate (PMAA) provide further insights into the glycosidic bond connection, such as Figure 3 d to Figure 3m. This comprehensive analysis identified 12 different glycosidic bond types in raspberry polysaccharides, as follows: terminal arabinofuranosyl (T-Araf), terminal xylose pyranose (T-Xylp), terminal rhamnosyl pyranose (T-Rhap), 1,5-linked arabinofuranosyl (1,5-Araf), terminal galactose pyranose (T-Galp), terminal galacturonic acid pyranose (T-GalAp), 1,3,4-linked rhamnosyl pyranose (T-Rhap), 1,5-linked arabinofuranosyl (1,5-Araf), terminal galactose pyranose (T-GalAp), terminal galacturonic acid pyranose (T-GalAp), terminal rhamnosyl pyranose (T-Rhap), terminal arabinofuranosyl (1,5-Araf), terminal galacturonic acid pyranose (T-GalAp), terminal galacturonic acid pyranose (T-GalAp), terminal galacturonic acid pyranose (T-Rhap), terminal arabinofuranosyl (1,5-Araf ...arabinofuranosyl (1,5-Araf), terminal galacturonic acid pyranose (T-GalAp), terminal galacturonic acid pyranose (T-GalAp), terminal galacturonic acid pyranose (T-Rhap), terminal arabinofuranosyl (1,5-Araf), terminal arabinofuranosyl (1,5-Araf), terminal arabinofuranosyl (1,5-Araf), terminal arabinofuranosyl (1,5- sugar (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 ratio of the following 12 glycosidic bonds of the raspberry homogeneous 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 = 2.3: 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 glucuronic acid pyranose (1,2-GlcAp) and 1,3-linked apiose furanose (1,5-Apif). 1,4-GalAp is the main chain connection mode of RCP-2-3. Considering the diversity of other glycosidic bonds, RCP-2-3 is classified as rhamnosegalacturonic 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 1 HNMR, 13 C NMR, DEPT135NMR, 1 H- 1H COSY NMR, HSQC NMR, HMBC NMR. The following peaks were identified: (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), (D) 1,4-linked α-galacturonic acid pyranose with a methoxy group at the 6th position (1,4-α-GalAp- 6-ome), (E) terminal α-hexuronic acid pyranose (T-α-HexAp), (F) 1,4-linked β-galacturonic acid pyranose reducing end (1,4-β-GalAp (red)), (G) 1,4-linked α-galacturonic acid pyranose reducing end (1,4-α-GalAp (red)), (H) 1,2,4-linked α-galacturonic acid pyranose (1,2,4-α-GalAp (red)). p), (I) terminal α-arabinofuranosyl (T-α-Araf), (J) 1,5-linked α-arabinofuranosyl (1,5-α-Araf), (K) terminal β-xylose pyranose (T-β-Xylp), (L) terminal β-rhamnose pyranose (T-β-Rhap), (M) 1,3,4-linked α-rhamnose pyranose (1,3,4-α-Rhap), (N) 1,2-linked β-glucuronic acid pyranose (1,2-β-GlcAp), (O) 1,3,4-linked α-fucosyl pyranose (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-apiofuranosyl (1,3-β-Apif), the results are shown in Fig. Figure 4 a to Figure 4 f. Raspberry polysaccharide is RG-Ⅱ domain, the fine structure is as follows Figure 4 As shown in g.

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

[0177] like Figure 5 The functional properties of crude raspberry polysaccharide, refined polysaccharide from each elution fraction and raspberry homogeneous polysaccharide (RCP-2-3) were evaluated by oil retention and cholesterol adsorption. Raspberry homogeneous polysaccharide has good in vitro oil retention properties and is significantly superior to other raspberry polysaccharide components.

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

[0179] like Figure 6As shown in the figure, the TG content in the serum of mice that were fasted and then fed with soybean oil first increased to a peak value and then decreased to a normal level after the intake of dietary lipids, and the peak value appeared at about 2 hours. RCP-2-3 can reduce the absorption peak of TG in serum and reduce the accumulation of serum triglycerides (TG) stimulated by soybean oil after a meal. The uptake of dietary lipids in intestinal cells was directly observed by fat and fatty acid absorption experiments, which further showed that the mechanism by which RCP-2-3 reduces the accumulation of serum TG after a meal is related to the inhibition of intestinal lipid absorption. BODIPY is a fatty acid green fluorescent probe. BODIPY-labeled soybean oil was gavaged into mice and intestinal cells were observed under a microscope. A large number of fluorescently labeled lipid droplets were observed in the intestinal epithelial villi of mice in the model group, while the fluorescence in the intestinal epithelial villi of mice after RCP-2-3 intervention was significantly reduced, and the fluorescence quantitative results could also confirm this. At the same time, the fluorescence intensity in the serum and cecal contents of mice was quantified, and it was found that the serum BODIPY fluorescence intensity of mice treated with RCP-2-3 was significantly lower than that of mice in the model group, while the BODIPY fluorescence intensity and TG content of the cecal contents were significantly higher than those of the model group. All the above evidences indicate that RCP-2-3 can inhibit the intestinal absorption of dietary lipids and promote the excretion of dietary lipids. At the same time, the types and contents of fatty acids in the feces of mice after soybean oil gavage were further analyzed to explore whether RCP-2-3 has a specific effect on the excretion of specific fatty acids in soybean oil. The results showed that raspberry polysaccharides can effectively promote the excretion of long-chain fatty acids in dietary oils and fats, and have a significant effect on the excretion of palmitic acid, oleic acid, linoleic acid and stearic acid.

[0180] like Figure 7 As shown, mice were fed HFD for 28 consecutive days and gavaged with high-fat emulsion to achieve excessive intake of high-calorie diet. RCP-2-3 intervention can slow down the weight gain of mice, reduce adipose tissue accumulation, and reduce the levels of total triglycerides, total cholesterol and low-density lipoprotein in serum, and increase the level of high-density lipoprotein. Comparing the relationship between weight gain and dietary intake, it can be found that after consuming the same weight (calories) of food, RCP-2-3 can significantly reduce weight gain and promote the excretion of feces containing a large amount of fat.

[0181] The embodiments of the present invention are described in detail above. Specific examples are used herein to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only used to help understand the method of the present invention and its core idea. At the same time, changes or deformations made by those skilled in the art based on the ideas of the present invention, the specific implementation methods and application scope of the present invention, all belong to the scope of protection of the present invention. In summary, the content of this specification should not be understood as limiting the present invention.

Claims

1. A raspberry homogeneous polysaccharide, characterized in that: The monosaccharide components of the raspberry homogeneous polysaccharide include fucose, rhamnose, arabinose, galactose, xylose, galacturonic acid, glucuronic acid and apiose; Wherein, the main chain structure of the raspberry homogeneous polysaccharide is 1,4-linked galacturonic acid pyranose; The weight average molecular weight of the raspberry homogeneous polysaccharide is 8.54±5.0 kDa.

2. The raspberry polysaccharide according to claim 1, characterized in that The molar ratio of the following 7 monosaccharides of the raspberry homogeneous polysaccharide is 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); Preferably, the molar ratio of the following seven monosaccharides of the raspberry homogeneous polysaccharide is fucose: rhamnose: arabinose: galactose: xylose: galacturonic acid: glucuronic acid = (1-3): (3-4): (6-8): (1-3): (1-3): (75-88): (1-3); More preferably, the molar ratio of the following seven monosaccharides of the raspberry homogeneous polysaccharide is 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; Preferably, the molar proportion of celery sugar in the raspberry homogeneous polysaccharide is greater than 0.

02.

3. The raspberry polysaccharide according to claim 1, characterized in that The raspberry homogeneous polysaccharide is rhamnogalacturonan-II domain; Preferably, the weight average molecular weight of the raspberry homogeneous polysaccharide is 8.54±3.0 kDa; Preferably, the weight average molecular weight of the raspberry homogeneous polysaccharide is 8.54±1.0 kDa; Preferably, the weight average molecular weight of the raspberry homogeneous polysaccharide is about 8.54 kDa; Preferably, the glycosidic bonds of the raspberry homogeneous polysaccharide are selected from one or more of the following: terminal arabinofuranosyl (T-Araf), terminal xylose pyranose (T-Xylp), terminal rhamnosyl pyranose (T-Rhap), 1,5-linked arabinofuranosyl (1,5-Araf), terminal galactose pyranose (T-Galp), terminal galacturonic acid pyranose (T-GalAp), 1,3,4-linked rhamnosyl pyranose (1,3,4-Rhap), 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 apiosyl furanose (1,5-Apif); Preferably, the molar ratio of the following 12 glycosidic bonds of the raspberry homogeneous 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): (2-10): (0.1-5): (0.1-5): (0.1-5): (70-90): (0.1-5): (0.1-5): (0.1-5); Preferably, the molar ratio of the following 12 glycosidic bonds of the raspberry homogeneous 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); Preferably, the molar ratio of the following 12 glycosidic bonds of the raspberry homogeneous 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 = 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.

4. A method for preparing the raspberry homogeneous polysaccharide according to any one of claims 1 to 3, characterized in that: The method comprises the following steps: (1) Take an appropriate amount of raspberry fruit, crush it, add ethanol, let it stand at room temperature, and repeat twice; (2) adding water to the raspberry powder after evaporation of ethanol, immersing at 90° C. for 3 h, repeating twice, combining the filtrate, and concentrating to obtain a concentrated solution; (3) stirring the concentrated solution, adding ethanol, allowing to stand for sedimentation, and filtering to obtain a filter residue; (4) evaporating the ethanol from the filter residue, dissolving it in water, and drying it to obtain crude raspberry polysaccharide; and (5) The crude raspberry polysaccharide is separated by DEAE-Sepharose FastFlow weak anion exchange chromatography to obtain a 0.1-0.3M sodium chloride fraction, and the 0.1-0.3M sodium chloride fraction is separated by Superdex TM 200 gel permeation chromatography to obtain the homogeneous raspberry polysaccharide.

5. The method according to claim 4, characterized in that The fruit is an immature fruit; Preferably, the raspberry is Rubus chingii Hu.; Preferably, the volume percentage concentration of the ethanol is 80% to 100%, such as about 95%.

6. The method according to claim 4, characterized in that 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; Preferably, in step (1), the standing time is 12 to 36 hours, for example, about 24 hours; More preferably, in step (2), the ratio of the volume / mass (L / kg) of the water to the raspberry fruit is 1 to 60, for example, about 40; More preferably, in step (2), the temperature of the water is 60°C to 100°C, for example, about 90°C; More preferably, in step (2), the concentration is concentrated by rotary evaporation under reduced pressure; More preferably, in step (3), the volume ratio of the ethanol to the concentrated solution is 1 to 6, such as about 4; More preferably, in step (3), the static sedimentation time is more than 4 hours, for example, about 12 hours; More preferably, in step (3), the filtration is vacuum filtration; More preferably, in step (4), the drying is vacuum freeze drying; More preferably, in step (5), the concentration of the sodium chloride site is about 0.2M.

7. Use of the raspberry homogeneous polysaccharide according to any one of claims 1 to 3 in the preparation of medicines, foods and / or health products for improving obesity; Preferably, the food is a functional food.

8. Use of the raspberry homogeneous polysaccharide according to any one of claims 1 to 3 in the preparation of a medicine for preventing and / or assisting in the treatment of obesity.

9. The use according to claim 7 or 8, characterized in that The obesity is caused by dietary habits, genetics, metabolism and / or fat cell abnormalities; Preferably, the abnormality is an increase in the number of adipocytes and / or adipocyte hypertrophy.

10. The use according to claim 7 or 8, characterized in that: The raspberry homogeneous polysaccharide prevents and / or assists in the treatment of obesity in one or both of the following ways: inhibiting lipid absorption and promoting lipid and / or oil excretion; Preferably, the lipids include triglycerides, cholesterol and / or low-density lipoprotein; Preferably, the oil comprises long-chain fatty acids; Preferably, the long-chain fatty acids include palmitic acid, oleic acid, linoleic acid and / or stearic acid.

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