Polysaccharide composition and application thereof

Abstract

The invention discloses a polysaccharide composition and application thereof, and relates to the technical field of polysaccharide preparation, the polysaccharide composition comprises at least one of a first polysaccharide, a second polysaccharide, a third polysaccharide and a fourth polysaccharide, the structural formulas of the first polysaccharide, the second polysaccharide, the third polysaccharide and the fourth polysaccharide are respectively shown as a formula (I), a formula (II), a formula (III) and a formula (IV), and the molecular weights of the polypeptide are respectively 343016.9 Da, 2279.4 Da, 1363.2 Da and 2228.9 Da. In the polysaccharide composition provided by the invention, the four polysaccharides have better antioxidant activity, can better inhibit alpha-glucosidase, alpha-amylase and pancreatic lipase, and can be strongly combined with AQP1 aquaporin, so that the four polysaccharides can be used for preparing antioxidants, hypoglycemic drugs, hypolipidemic drugs and drugs for treating AQP1 mediated diseases.

Description

Technical Field

[0001] The present invention relates to the technical field of polysaccharide preparation, in particular to a polysaccharide composition and application thereof. Background Art

[0002] Coconut is a tropical fruit with an annual global production of 62 million tons, primarily concentrated in China, Indonesia, the Philippines, and India. Coconut has important applications in a variety of fields, including food, cosmetics, and medicine.

[0003] However, the development of high value-added coconut products is relatively lagging, especially the extraction and utilization of coconut polysaccharides. Summary of the Invention

[0004] The main purpose of the present invention is to provide a polysaccharide composition and application thereof, aiming to solve the problem that there are few high-value utilization fields of coconut meat polysaccharides in the prior art.

[0005] To achieve the above object, the present invention provides a polysaccharide composition, comprising at least one of a first polysaccharide, a second polysaccharide, a third polysaccharide and a fourth polysaccharide:

[0006] The first polysaccharide is composed of mannose pyranose, rhamnose pyranose, glucose pyranose and galactose pyranose in a molar ratio of 4:1:3:2, and its structural formula is shown in formula (I). The molecular weight of the first polysaccharide is 343016.9 Da;

[0007] The second polysaccharide is composed of mannose pyranose and galactose pyranose in a molar ratio of 7:3, and its structural formula is shown in formula (II). The molecular weight of the second polysaccharide is 2279.4 Da;

[0008] The third polysaccharide is composed of mannose pyranose, rhamnose pyranose, galactose pyranose and arabinofuranose in a molar ratio of 54.9:4.8:16.3:16.0, and its structural formula is shown in formula (III). The molecular weight of the third polysaccharide is 1363.2 Da;

[0009] The fourth polysaccharide is composed of mannose pyranose, glucose pyranose, galactose pyranose, and arabinofuranose in a molar ratio of 23.3:37.2:16.5:15.0, and its structural formula is shown in formula (IV). The molecular weight of the fourth polysaccharide is 2228.9 Da;

[0010]

[0011]

[0012] In one embodiment, the polysaccharide composition comprises a first polysaccharide, a second polysaccharide, a third polysaccharide and a fourth polysaccharide, wherein the first polysaccharide, the second polysaccharide, the third polysaccharide and the fourth polysaccharide are prepared by the following steps:

[0013] S1, mixing fresh coconut meat with water and performing hot water extraction at 60-100° C., filtering, collecting the filtrate, mixing the filtrate with an alcohol reagent and allowing it to stand to precipitate polysaccharides, centrifuging the precipitate, and freeze-drying to obtain a first crude polysaccharide;

[0014] S2. mixing the first crude polysaccharide with petroleum ether and shaking the mixture to separate the solid and the liquid, and drying the solid to obtain a second crude polysaccharide;

[0015] S3, mixing the second crude polysaccharide with a chloroform-n-butanol solution, allowing the mixture to stand, centrifuging, taking the supernatant, dialyzing, and then freeze-drying to obtain a third crude polysaccharide;

[0016] S4. Performing a first chromatography treatment on the third crude polysaccharide using a DEAE-52 cellulose column, eluting with water, 0.1 M NaCl, 0.3 M NaCl, and 0.5 M NaCl in sequence to obtain a first chromatographic liquid, a second chromatographic liquid, a third chromatographic liquid, and a fourth chromatographic liquid;

[0017] S5. Use Sephadex G-150 gel column to perform a second chromatography treatment on the four chromatographic fluids obtained by the first chromatography treatment, the eluent is 0.1M NaCl, the first chromatographic fluid is eluted by 0.1M NaCl to obtain the first polysaccharide, the second chromatographic fluid is eluted by 0.1M NaCl to obtain the second polysaccharide, the third chromatographic fluid is eluted by 0.1M NaCl to obtain the third polysaccharide, and the fourth chromatographic fluid is eluted by 0.1M NaCl to obtain the fourth polysaccharide.

[0018] In one embodiment, in step S1:

[0019] The mass ratio of the fresh coconut meat to the water is 1:(10-14); and / or,

[0020] The hot water extraction time is 1 to 2 hours; and / or,

[0021] The alcohol reagent includes ethanol, methanol or isopropanol with a volume concentration of 75%; and / or,

[0022] The standing time is 20 to 24 hours; and / or,

[0023] The centrifugal speed is 3500-4500 r / min, and the centrifugal time is 15-25 min.

[0024] In one embodiment, in step S2:

[0025] Add 1 g of the first crude polysaccharide to every 10 to 12 mL of the petroleum ether; and / or,

[0026] The shaking time is 1.5 to 2.5 hours; and / or,

[0027] The drying temperature is 45-50°C.

[0028] In one embodiment, step S3 includes: preparing the second crude polysaccharide into a 5 mg / mL aqueous solution, adding a mixture of chloroform and n-butanol with a volume ratio of 2:1, shaking, standing for 20 to 25 minutes, centrifuging at 6500 to 7500 r / min for 10 to 15 minutes, and drawing the upper aqueous phase for dialysis and freeze-drying in sequence to obtain a third crude polysaccharide.

[0029] The present invention also provides a use of the aforementioned polysaccharide composition in the preparation of an antioxidant.

[0030] The present invention also provides a use of the aforementioned polysaccharide composition in the preparation of an α-glucosidase inhibitor, an α-amylase inhibitor or a pancreatic lipase inhibitor.

[0031] The present invention also provides a use of the aforementioned polysaccharide composition in the preparation of a blood sugar lowering drug or a blood lipid lowering drug.

[0032] The present invention also provides a use of the aforementioned polysaccharide composition in preparing a medicament for treating AQP1-mediated diseases.

[0033] In one embodiment, the AQP1-mediated disease comprises obesity, edema, urinary retention, nephritis, cerebral edema, pulmonary edema, or glaucoma.

[0034] In the technical solution of the present invention, the four polysaccharides in the polysaccharide composition all have good antioxidant activity, can effectively inhibit α-glucosidase, α-amylase and pancreatic lipase, and can strongly bind to AQP1 water channel protein. Therefore, they can be used to prepare antioxidants, hypoglycemic drugs, hypolipidemic drugs and drugs for treating AQP1-mediated diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. 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 the structures shown in these drawings without paying any creative work.

[0036] Figure 1IR spectra of four polysaccharides in Example 1: the first polysaccharide CMP1, the second polysaccharide CMP2, the third polysaccharide CMP3, and the fourth polysaccharide CMP4;

[0037] Figures 2 to 6 This is the nuclear magnetic resonance spectrum of the first polysaccharide CMP1 in Example 1: Figure 2 is the carbon spectrum of CMP1, Figure 3 is the hydrogen spectrum of CMP1, Figure 4 is the correlation spectrum (COSY) of CMP1, Figure 5 is the heteronuclear single quantum coherence spectrum (HSQC) of CMP1, Figure 6 is the heteronuclear multiple bond correlation (HMBC) spectrum of CMP1;

[0038] Figures 7 to 10 This is the nuclear magnetic resonance spectrum of the second polysaccharide CMP2 in Example 1: Figure 7 is the carbon spectrum of CMP2, Figure 8 is the hydrogen spectrum of CMP2, Figure 9 is the COSY spectrum of CMP2, Figure 10 is the HSQC spectrum of CMP2;

[0039] Figures 11 to 15 This is the nuclear magnetic resonance spectrum of the third polysaccharide CMP3 in Example 1: Figure 11 is the carbon spectrum of CMP3, Figure 12 is the hydrogen spectrum of CMP3, Figure 13 is the COSY spectrum of CMP3, Figure 14 is the HSQC spectrum of CMP3, Figure 15 is the HMBC spectrum of CMP3;

[0040] Figures 16 to 19 This is the nuclear magnetic resonance spectrum of the fourth polysaccharide CMP4 in Example 1: Figure 16 is the carbon spectrum of CMP4, Figure 17 is the hydrogen spectrum of CMP4, Figure 18 is the COSY spectrum of CMP4, Figure 19 is the HSQC spectrum of CMP4;

[0041] Figure 20 Graph showing the results of DPPH free radical (DPPH·) scavenging ability assay of the four polysaccharides CMP1, CMP2, CMP3, and CMP4 in Example 1;

[0042] Figure 21 This is a graph showing the results of an in vitro inhibition experiment on α-glucosidase by the four polysaccharides CMP1, CMP2, CMP3 and CMP4 in Example 1;

[0043] Figure 22This is a graph showing the results of an in vitro inhibition experiment on α-amylase by the four polysaccharides CMP1, CMP2, CMP3 and CMP4 in Example 1;

[0044] Figure 23 This is a graph showing the results of an in vitro inhibition experiment on pancreatic lipase by the four polysaccharides CMP1, CMP2, CMP3 and CMP4 in Example 1;

[0045] Figure 24 This is the molecular docking diagram of the first polysaccharide CMP1 and AQP1 in Example 1;

[0046] Figure 25 This is the molecular docking diagram of the second polysaccharide CMP2 and AQP1 in Example 1;

[0047] Figure 26 This is the molecular docking diagram of the third polysaccharide CMP3 and AQP1 in Example 1;

[0048] Figure 27 This is the molecular docking diagram of the fourth polysaccharide CMP4 and AQP1 in Example 1.

[0049] The purpose, features and advantages of the present invention will be further described with reference to the accompanying drawings and in conjunction with the embodiments. DETAILED DESCRIPTION

[0050] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. Where the reagents or instruments used are not specified by the manufacturer, they are all conventional products that can be purchased commercially. In addition, the meaning of "and / or" appearing in the full text includes three parallel schemes. Taking "A and / or B" as an example, it includes scheme A, or scheme B, or schemes that A and B meet at the same time. In addition, the technical solutions between the various embodiments can be combined with each other, but must be based on the ability of ordinary technicians in this field to achieve. When the combination of technical solutions is contradictory or cannot be achieved, it should be considered that the combination of such technical solutions does not exist and is not within the scope of protection required by the present invention. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0051] Coconut is a tropical fruit with an annual global production of 62 million tons, primarily concentrated in countries such as China, Indonesia, the Philippines, and India. Coconut has important applications in a variety of fields, including food, cosmetics, and medicine. However, the development of high-value-added coconut products is relatively underdeveloped, particularly the extraction and utilization of coconut polysaccharides.

[0052] In view of this, the present invention provides a polysaccharide composition, comprising at least one of a first polysaccharide, a second polysaccharide, a third polysaccharide, and a fourth polysaccharide: wherein the first polysaccharide is composed of mannose pyranose, rhamnose pyranose, glucose pyranose, and galactose pyranose in a molar ratio of 4:1:3:2, and its structural formula is shown in formula (I), and the molecular weight of the first polysaccharide is 343016.9 Da; the second polysaccharide is composed of mannose pyranose and galactose pyranose in a molar ratio of 7:3, and its structural formula is shown in formula (II), and the molecular weight of the second polysaccharide is 2279. 4Da; the third polysaccharide is composed of mannose pyranose, rhamnose pyranose, galactose pyranose and arabinofuranose in a molar ratio of 54.9:4.8:16.3:16.0, and its structural formula is shown in formula (III). The molecular weight of the third polysaccharide is 1363.2Da; the fourth polysaccharide is composed of mannose pyranose, glucose pyranose, galactose pyranose and arabinofuranose in a molar ratio of 23.3:37.2:16.5:15.0, and its structural formula is shown in formula (IV). The molecular weight of the fourth polysaccharide is 2228.9Da;

[0053]

[0054] In the technical solution of the present invention, the four polysaccharides in the polysaccharide composition all have good antioxidant activity, can effectively inhibit α-glucosidase, α-amylase and pancreatic lipase, and can strongly bind to the AQP1 water channel protein. Therefore, they can be used to prepare antioxidants, hypoglycemic drugs, hypolipidemic drugs, and drugs for treating AQP1-mediated diseases.

[0055] Specifically, in the present invention, the main chain of the first polysaccharide is composed of alternating β-1→4-linked D-mannose pyranose (β-D-Manp) and D-glucose pyranose (β-D-Glcp) (the main chain repeating unit is shown in Formula 1), that is, β-D-Manp / Glcp-(1→4), and there are β-1→6-linked D-galactose pyranose (β-D-Galp) branches, and the end is α-L-rhamnose pyranose (α-L-Rhap). Among them, pyranose refers to a monosaccharide forming a six-membered ring structure, β-D-Glcp refers to a structure in which D-glucose (Glc) exists in the form of a 6-membered pyranose ring, and (1→4) indicates that the position of the glycosidic bond is from the first carbon to the fourth carbon.

[0056]

[0057] The main chain of the second polysaccharide is composed of β-(1→3)-linked D-mannopyranose (β-D-Manp) (the main chain repeating unit is shown in Formula 2), and the branch structure is β-D-galactopyranose (β-D-Galp) connected to the main chain mannose through β-(1→6)-bonds.

[0058]

[0059] The backbone of the third polysaccharide is composed of alternating β-D-mannopyranose (1→3) and β-D-galactopyranose (1→6) (the backbone repeating unit is shown in Formula 3), and the side chains contain short α-L-rhamnose pyranose (1→4) branches and terminal α-L-arabinofuranose (α-L-Araf) and β-D-mannopyranose. The minimum repeating unit contains three backbone sugar residues and one branch sugar residue, namely, (1→4)-α-D-Glcp-(1→4)-β-D-Manp-(1→4)-α-D-Glcp-(1→4), with a galactopyranose branch or an arabinofuranose branch connected to the C6 position of each glucose residue. Among them, α-L-Araf refers to a structure in which α-L-arabinose (α-L-Ara) exists in the form of a five-membered furanose ring.

[0060]

[0061] The main chain of the fourth polysaccharide is composed of α-D-glucose (α-D-Glcp) and β-D-mannose (β-D-Manp) alternately connected by 1→4 glycosidic bonds to form a linear main chain. Its structure is (1→4)-α-D-Glcp-(1→4)-β-D-Manp-(1→4)-α-D-Glcp-(1→(the main chain repeating unit is shown in Formula 4), wherein glucose is in the α configuration (anomeric hydrogen chemical shift of about 5.16 ppm) and mannose is in the β configuration (anomeric hydrogen chemical shift of about 4.76 ppm). At the C6 position of some glucose, two branches are connected by 1→6 glycosidic bonds, namely β-D-galactose and β-D-mannose. (β-D-Galp) (anomeric carbon signal at approximately 4.60 ppm, suggesting β configuration) and α-L-arabinose (α-L-Araf) (anomeric carbon signal at approximately 4.30 ppm, suggesting α configuration), whose branched structures are →6)-β-D-Galp-(1→ or →6)-α-L-Araf-(1→. The minimum repeating unit contains three main chain sugar residues and one branch sugar residue, namely →4)-α-D-Glcp-(1→4)-β-D-Manp-(1→4)-α-D-Glcp-(1→, with a galactose or arabinose branch connected to its C6 position every other glucose residue.

[0062]

[0063] In some embodiments, the polysaccharide composition includes a first polysaccharide, a second polysaccharide, a third polysaccharide, and a fourth polysaccharide, wherein the first polysaccharide, the second polysaccharide, the third polysaccharide, and the fourth polysaccharide are prepared by the following steps: S1, mixing fresh coconut meat with water and extracting it with hot water at 70-80° C., filtering, collecting the filtrate, mixing the filtrate with an alcohol reagent and allowing it to stand to precipitate the polysaccharide, centrifuging the precipitate, and freeze-drying to obtain a first crude polysaccharide; S2, mixing the first crude polysaccharide with petroleum ether and shaking it, separating the solid and the liquid, and drying the solid to obtain a second crude polysaccharide;

[0064] S3. The second crude polysaccharide is mixed with a chloroform-n-butanol solution and allowed to stand. The supernatant is collected by centrifugation, dialyzed, and then freeze-dried to obtain a third crude polysaccharide; S4. The third crude polysaccharide is subjected to a first chromatography treatment using a DEAE-52 cellulose column, and eluted with water, 0.1M NaCl, 0.3M NaCl, and 0.5M NaCl in sequence to obtain a first chromatography fluid, a second chromatography fluid, a third chromatography fluid, and a fourth chromatography fluid; S5. The four chromatography fluids obtained by the first chromatography treatment are subjected to a second chromatography treatment using a Sephadex G-150 gel column, and the eluent is 0.1M NaCl. The first chromatography fluid is eluted with 0.1M NaCl to obtain the first polysaccharide, the second chromatography fluid is eluted with 0.1M NaCl to obtain the second polysaccharide, the third chromatography fluid is eluted with 0.1M NaCl to obtain the third polysaccharide, and the fourth chromatography fluid is eluted with 0.1M NaCl to obtain the fourth polysaccharide.

[0065] It can be understood that S4 specifically includes: using a DEAE-52 cellulose column to perform a first chromatography treatment on the third crude polysaccharide, first using water as an eluent to elute the DEAE-52 cellulose column to which the third crude polysaccharide is added to obtain a first chromatographic liquid, then using 0.1 M NaCl as an eluent to elute the DEAE-52 cellulose column to obtain a second chromatographic liquid, then using 0.3 M NaCl as an eluent to elute the DEAE-52 cellulose column to obtain a third chromatographic liquid, and finally using 0.5 M NaCl as an eluent to elute the DEAE-52 cellulose column to obtain a fourth chromatographic liquid.

[0066] In the technical solution of the present invention, four single polysaccharides with different structures were successfully extracted from fresh coconut meat using low-temperature hot water extraction technology, combined with ethanol precipitation, degreasing treatment, and column chromatography purification techniques. Through the optimized extraction process of the present invention, the yield and purity of coconut meat polysaccharides were improved, overcoming the shortcomings of traditional coconut meat polysaccharide extraction methods, such as low yield and susceptibility of polysaccharide structure to high temperature damage, and expanding its application potential in functional foods and pharmaceutical fields.

[0067] In some embodiments, in step S1, the mass ratio of the fresh coconut meat to the water is 1:(10-14); and / or the hot water extraction time is 1-2 hours; and / or the alcohol reagent comprises ethanol with a volume concentration of 75%; and / or the standing time is 20-24 hours; and / or the centrifugal speed is 3500-4500 r / min, and the centrifugal time is 15-25 minutes. Simultaneously controlling the mass ratio of fresh coconut meat to water, the hot water extraction time, the volume concentration of the alcohol reagent, the standing sedimentation time, and the centrifugal conditions within the above ranges can ensure that most of the polysaccharides in the fresh coconut meat are transferred to the precipitate, thereby improving the extraction rate of the first crude polysaccharide.

[0068] In some embodiments, in step S2, 1 g of the first crude polysaccharide is added to every 10-12 mL of petroleum ether; and / or the shaking time is 1.5-2.5 hours; and / or the drying temperature is 45-50° C. Simultaneously controlling the ratio of petroleum ether to the first crude polysaccharide, the shaking time, and the drying temperature within the above ranges can ensure high degreasing efficiency and reduce the lipid content in the second crude polysaccharide.

[0069] In some embodiments, step S3 comprises: preparing the second crude polysaccharide into a 5 mg / mL aqueous solution, adding a mixture of chloroform and n-butanol in a volume ratio of 2:1, shaking, standing for 20-25 minutes, centrifuging at 6500-7500 rpm for 10-15 minutes, and collecting the upper aqueous phase, performing dialysis and lyophilization in sequence to obtain a third crude polysaccharide. Step S3 can ensure efficient and thorough protein removal, resulting in a lower protein content and higher polysaccharide purity in the third crude polysaccharide.

[0070] The present invention also provides a use of the aforementioned polysaccharide composition in the preparation of antioxidants. The four polysaccharides in the polysaccharide composition of the present invention all have good DPPH·free radical scavenging effects and can therefore be used to prepare antioxidants.

[0071] The present invention also provides a use of the aforementioned polysaccharide composition in the preparation of an α-glucosidase inhibitor, an α-amylase inhibitor, or a pancreatic lipase inhibitor. Specifically:

[0072] α-glucosidase (α-glucosidase) is an enzyme that can hydrolyze α-1,4-glycosidic bonds in oligosaccharides and polysaccharides and is widely present in the brush border cells of the human small intestine. It plays a key role in the digestion of carbohydrates, breaking down complex carbohydrates into monosaccharides (such as glucose), which are then absorbed into the blood, leading to increased blood sugar levels. Therefore, inhibiting the activity of α-glucosidase can delay the decomposition of carbohydrates and the absorption of glucose, thereby achieving the effect of lowering postprandial blood sugar. The inhibitor achieves the effect of inhibiting the activity of α-glucosidase through competitive inhibition or non-competitive inhibition, reducing the rate at which carbohydrates are broken down into glucose, delaying the absorption of glucose, and thus lowering postprandial blood sugar levels. The polysaccharide composition provided by the present invention can effectively inhibit α-glucosidase and can therefore be used to prepare α-glucosidase inhibitors.

[0073] α-amylase is a hydrolase that can act on the α-1,4-glycosidic bonds in starch molecules and break them down into smaller oligosaccharides or maltose. During the digestion process, after starch is broken down by α-amylase, it is further broken down into glucose by α-glucosidase, and is eventually absorbed into the blood by the intestine, leading to increased blood sugar levels. Therefore, inhibiting the activity of α-amylase can delay the decomposition of starch, reduce the production and absorption of glucose, and thus lower postprandial blood sugar levels. The principle of using α-amylase to detect whether polysaccharides have hypoglycemic activity is based on the kinetic characteristics of enzymatic reactions. By measuring the inhibitory effect of polysaccharides on α-amylase activity, their ability to delay starch decomposition is evaluated, thereby indirectly reflecting their effect in lowering postprandial blood sugar. The polysaccharide composition provided by the present invention can effectively inhibit α-amylase and can therefore be used to prepare α-amylase inhibitors.

[0074] Trypsin is a digestive enzyme that is mainly found in pancreatic juice and is responsible for breaking down proteins into small peptides and amino acids. During lipid metabolism, trypsin is indirectly involved in the activation of lipoprotein lipase (LPL). LPL is a key enzyme that can hydrolyze triglycerides in the blood and break them down into free fatty acids and glycerol, thereby regulating blood lipid levels. If the activity of trypsin is inhibited or regulated, it may affect the activity of enzymes related to lipid metabolism, thereby changing blood lipid levels. The polysaccharide composition provided by the present invention can effectively inhibit trypsin and can therefore be used to prepare trypsin inhibitors.

[0075] The present invention also provides a use of the aforementioned polysaccharide composition in the preparation of a hypoglycemic or hypolipidemic drug. Since the polysaccharide composition of the present invention can be used to inhibit α-glucosidase, α-amylase, and trypsin, it can be used to prepare a hypoglycemic or hypolipidemic drug.

[0076] The present invention also provides a use of the aforementioned polysaccharide composition in the preparation of a medicament for treating AQP1-mediated diseases. Because all four polysaccharide compositions in the present invention bind well to AQP1, they can inhibit aquaporins by binding to AQP1, thereby being used in the preparation of a medicament for treating AQP1-mediated diseases. In some embodiments, the AQP1-mediated diseases include obesity, edema, urinary retention, nephritis, cerebral edema, pulmonary edema, or glaucoma.

[0077] The technical solutions of the present invention are further described in detail below in conjunction with specific embodiments and drawings. It should be understood that the following embodiments are only used to explain the present invention and are not used to limit the present invention.

[0078] Example 1

[0079] A polysaccharide composition includes a first polysaccharide, a second polysaccharide, a third polysaccharide and a fourth polysaccharide, and is prepared by the following steps:

[0080] (1) Fresh coconut meat from Hainan with a thickness of more than 4 mm was selected; the fresh coconut meat was crushed, and pure water was added at a mass ratio of 1:12, and hot water extraction was performed at 75° C. for 1.5 hours. After the extraction, the filtrate was filtered and cooled to room temperature, and 75% ethanol was added to the filtrate for alcohol precipitation. After standing for 24 hours, the filtrate was centrifuged (4000 r / min, 20 min), and the precipitate was taken and freeze-dried to obtain the first crude polysaccharide;

[0081] (2) Degreasing treatment: Add the first crude polysaccharide to petroleum ether at a solid-liquid ratio of 1:10 (g / mL), shake on a shaker for 2 hours, repeat the shaking twice, filter, and dry the solid in a 50°C oven to obtain the second crude polysaccharide;

[0082] (3) Deproteinization: The second crude polysaccharide was prepared into a 5 mg / mL aqueous solution with water, and a chloroform-n-butanol (chloroform and n-butanol volume ratio is 2:1) mixed solution was added. After shaking on a shaker, the mixture was allowed to stand for 20 minutes and centrifuged (7000 rpm, 10 minutes). The upper aqueous phase was then aspirated with a Pasteur pipette. The above operation was repeated, and the aspirated aqueous phases were combined, dialyzed, and lyophilized to obtain the third crude polysaccharide.

[0083] (4) First chromatography treatment: The third crude polysaccharide was subjected to the first chromatography treatment using a DEAE-52 cellulose column (purchased from Shanghai Yuanye Company, product number S14024-500G), and water was used as the eluent to elute to obtain the first chromatographic solution, 0.1 M NaCl was used as the eluent to elute to obtain the second chromatographic solution, 0.3 M NaCl was used as the eluent to elute to obtain the third eluate, and 0.5 M NaCl was used as the eluent to elute to obtain the fourth eluate;

[0084] (5) Second chromatography treatment: The four chromatographic fluids obtained in step (4) were subjected to second chromatography treatment using a Sephadex G-150 gel column (purchased from Shanghai Yuanye Company, product number S14035-25 G). 0.1 M NaCl was uniformly used as the eluent for elution. The first chromatographic fluid was eluted to obtain the first polysaccharide (denoted as CMP1), the second chromatographic fluid was eluted to obtain the second polysaccharide (denoted as CMP2), the third chromatographic fluid was eluted to obtain the third polysaccharide (denoted as CMP3), and the fourth chromatographic fluid was eluted to obtain the fourth polysaccharide (denoted as CMP4).

[0085] Example 2 Structural analysis of four polysaccharides

[0086] Structural analysis includes molecular weight determination, infrared spectroscopy (FT-IR) determination, monosaccharide composition analysis, methylation analysis and nuclear magnetic resonance analysis. The molecular weight is determined by high performance liquid chromatography, and the monosaccharide composition analysis is determined by PMP (1-phenyl-3-methyl-5-pyrazolone) derivatization method.

[0087] 1. Molecular weight determination

[0088] The molecular weights of CMP1, CMP2, CMP3 and CMP4 are 343016.9 Da, 2279.4 Da, 1363.2 Da and 2228.9 Da, respectively.

[0089] 2. Infrared spectroscopy

[0090] like Figure 1 As shown in Table 1, the FT-IR spectra of CMP1-CMP4 samples all show typical polysaccharide characteristic absorption peaks. As shown in Table 1, in the infrared spectrum, the characteristic peak of polysaccharide is at 3300-3400 cm -1 The stretching vibration of OH at 2920-2930 cm-1 indicates that there are abundant hydroxyl (-OH) groups in the sample, which is the core feature of the polysaccharide structure. -1 The absorption peak at corresponds to the stretching vibration of CH, which further verifies the existence of CH bonds in the polysaccharide ring.

[0091] At 1710~1750cm -1 1649~1690cm -1 The characteristic absorption peak at 1400-1420 cm can be attributed to the stretching vibration of C=O, indicating that the sample may contain ester groups (-COOR) or carboxyl groups (-COOH), suggesting that these polysaccharides have acidic properties. -1 The nearby absorption peak may be related to NH bending vibration, suggesting the presence of acetylamino groups (such as N-acetylglucosamine) in the polysaccharide, which is commonly found in polysaccharides such as chitosan.

[0092] At 1100~1280cm-1 The absorption peak at 1038.5 cm corresponds to the stretching vibration of CO, indicating the presence of glycosidic bond (COC) or hydroxyl (C-OH) structure in the polysaccharide. -1 The characteristic peak at 818.6 cm further supports the vibration of CO, which is consistent with the COC bond of the pyranose ring. -1 The absorption peak at can be attributed to the bending vibration of the CH, consistent with the typical configuration of D-pyranose glucose. These characteristic peaks together confirm the presence of pyranose rings in CMP1-CMP4 samples and indicate that they possess the structural characteristics of acidic polysaccharides.

[0093] Table 1 Characteristic peaks of infrared spectra of polysaccharides at different levels

[0094]

[0095]

[0096] 3. Monosaccharide composition analysis

[0097] The components of CMP1 are mannose, rhamnose, glucose and galactose; the components of CMP2 are mannose and galactose; the components of CMP3 are mannose, rhamnose, galactose and arabinose; and the components of CMP4 are mannose, glucose, galactose and arabinose.

[0098] 4. Methylation analysis

[0099] (1) Analysis of the methylation results of the first polysaccharide

[0100] The methylation results of the first polysaccharide in Table 2 show that the monosaccharide composition is D-mannose (about 40%), D-glucose (about 30%), D-galactose (about 20%) and L-rhamnose (about 10%). The main chain is composed of mannose and glucose alternately connected by β-1→4 glycosidic bonds, and branches are formed by galactose connected by β-1→6 glycosidic bonds. The end is modified with α-L-rhamnose through 1→2 / 1→3 glycosidic bonds.

[0101] Table 2 Methylation results of the first polysaccharide

[0102]

[0103] (2) Analysis of the methylation results of the second polysaccharide

[0104] From the methylation results of the second polysaccharide in Table 3, it can be seen that the molar ratio of sugar residues of the second polysaccharide is main chain mannose: branch galactose: terminal mannose = 2:1:1, and the molecular weight is 2279.40 Da. Combined with the molecular weight of the methylated monosaccharide (both 273.25 Da after methylation of mannose and galactose) and the amount of glycosidic bond dehydration (18.02 Da per bond), it can be inferred that the polysaccharide is composed of 8 monosaccharide units, with the main chain containing 5 β-(1→3)- Manp (5×273.25=1366.25Da), the branch contains 2 β-(1→6)-Galp (2×273.25=546.50Da), the end contains 1 Manp (273.25Da), the glycosidic bond is dehydrated 7 times (7×18.02=126.14Da), and the total molecular weight is: 1366.25+546.50+273.25-126.14≈2060Da.

[0105] Table 3 Methylation results of the second polysaccharide

[0106]

[0107] (3) Analysis of the methylation results of the third polysaccharide

[0108] From the methylation results of the third polysaccharide in Table 4 below, it can be seen that the total molar ratio of sugar residues of the third polysaccharide is D-mannose: D-galactose: L-rhamnose: L-arabinose = 3.16:1.63:4.8:1.60, the main chain is mainly composed of D-mannose and D-galactose, and the connection methods include 1→3 glycosidic bonds and 1→6 glycosidic bonds; the side chain contains a 1→4 glycosidic bond connection of L-rhamnose and a terminal structure of L-arabinose.

[0109] Table 4 Methylation results of the third polysaccharide

[0110]

[0111]

[0112] (4) Analysis of the methylation results of the fourth polysaccharide

[0113] The methylation results for the fourth polysaccharide in Table 5 below show that glucose (Glc) forms the backbone via 1→4 glycosidic bonds (retention time 6.899 min, characteristic peak m / z 126.06, molar ratio 25.05%), with some 1→4,6 branches (retention time 14.069 min, m / z 199, molar ratio 8.73%). The terminal Glc (retention time 27.464 min, m / z 218) indicates the end of the linear chain. Mannose (Man) is primarily composed of 1→6 linkages (retention time 6.925 min, m / z 175.13, molar ratio 15.34%) and 1→3,6 branches (retention time 17.788 min, m / z 207, molar ratio 7.98%). Galactose (Gal) is characterized by 1→3 linkages (retention time 11.769 min, m / z 156.1, molar ratio 10.31%) and 1→2 linkages (retention time 19.038 min, m / z 154.08, molar ratio 6.15%). Arabinose (Ara) is primarily characterized by 1→5 linkages (retention time 12.947 min, m / z 149.05, molar ratio 9.82%) and 1→3,5 branches (retention time 20.006 min, m / z 227, molar ratio 5.21%).

[0114] Table 5 Methylation results of the fourth polysaccharide

[0115]

[0116] 5. Nuclear Magnetic Resonance Analysis

[0117] (1) Structural analysis of the first polysaccharide:

[0118] Figure 2 is the carbon spectrum of CMP1, Figure 3 is the hydrogen spectrum of CMP1, Figure 4 is the correlation spectrum (COSY) of CMP1, Figure 5 is the heteronuclear single quantum coherence spectrum (HSQC) of CMP1, Figure 6 This is the heteronuclear multiple bond correlation (HMBC) spectrum of CMP1.

[0119] Figure 2 The carbon spectrum of CMP1, the C1 signal (δ100.17) is the anomeric carbon of the β-glycosidic bond, combined with Figure 3 of 1 The H2 coupling constant (J = 8.7 Hz) in H NMR indicates that the main chain is composed of D-mannose and D-glucose linked in a β-1→4 manner (β-D-Manp / Glcp-(1→4)). The C2-C5 signals (δ 71.37, 69.88, 69.27, 68.38) are consistent with the typical chemical shifts of the pyranose ring, with δ = 68.38 (C4) corresponding to the Figure 6 The long-range coupling of H1 (δ4.02) in the HMBC spectrum further confirmed the 1→4 linkage of the main chain. The C6 signal (δ61.21) corresponds to the unsubstituted primary alcohol group (-CH2OH), supporting the linear structure of the main chain, while the branch point C6 (δ68.38, HMBC correlation) indicates the presence of a β-1→6 linked D-galactose branch. The terminal α-L-rhamnose is confirmed by its methyl signal (δ1.09 / 1.66) and anomeric proton (δ4.94, singlet), and the HMBC correlation of C2 / C3 (δ71.37 / 69.88) indicates that it is linked to the main chain in a 1→2 / 1→3 manner.

[0120] Figure 3 For CMP1 1 H NMR spectrum: δ4.94 (s, 1H) is the anomeric proton (H1) of α-L-rhamnose, and the singlet (s) indicates a small coupling constant (J ~ 1-2 Hz), consistent with the α configuration. δ3.90 (d, J = 8.7 Hz, 2H) is the anomeric proton (H1) of β-D-glucose / mannose, and the doublet (J ~ 8-9 Hz) supports the β configuration, consistent with the β-1→4 linked main chain. δ3.84 (d, J = 17.7 Hz, 5H) is H6a / H6b of unsubstituted C6 (such as -CH2OH of glucose), and the large coupling constant (J ~ 17 Hz) is the trans-position coupling between H6a and H6b, supporting the main chain 1→4 link; δ3.73 (d, J = 9.8 Hz, 4H) is the coupling between H4 and H5 of the main chain sugar, and J ~ 9-10 Hz is consistent with the trans-ring coupling characteristics of β-1→4 link.

[0121] The structure of CMP1, elucidated through methylation experiments and multidimensional nuclear magnetic resonance (NMR) data, revealed a monosaccharide composition consisting of D-mannose (~40%), D-glucose (~30%), D-galactose (~20%), and L-rhamnose (~10%). The backbone consists of alternating β-1→4-linked mannose and glucose, branched by β-1→6-linked galactose, and terminated with α-L-rhamnose in a 1→2 / 1→3 pattern. In the NMR data, the linear backbone structure is supported by the C1 signal (δ100.17) in the 13C NMR spectrum and the β configuration of the anomeric proton H1 (δ3.90, J=8.7 Hz). The long-range coupling of the C4 signal (δ68.38) to H1 (δ4.02) in the HMBC spectrum further confirms the 1→4 backbone structure. The branch point is clearly identified by the correlation of the C6 signal (δ60.74) with the branching sugar H1 (δ4.02), while the α-configuration of the terminal rhamnose is confirmed by the anomeric proton singlet (δ4.94) and the methyl signals (δ1.09 / 1.66). The molecular weight of over 300,000 indicates a highly branched, multi-unit polymer with a branching frequency of approximately one branch point per 5-6 main chain units. In summary, the polysaccharide exhibits a complex topology of β-1→4 backbone, β-1→6 branches, and α-L-rhamnose termini, as shown in Formula (I).

[0122] (2) Structural analysis of the second polysaccharide:

[0123] Figure 7 is the carbon spectrum of CMP2, Figure 8 is the hydrogen spectrum of CMP2, Figure 9 is the COSY spectrum of CMP2, Figure 10 This is the HSQC spectrum of CMP2.

[0124] Depend on Figure 7-10 Information analysis revealed the following structural features of the polysaccharide: the backbone is composed of β-(1→3)-linked D-mannopyranose (β-D-Manp), with a strong correlation between the anomeric carbon signals C-1δ99.91 and H-1δ5.11 (dd, J=16.8 Hz), indicating a β-configuration and a 1→3 glycosidic bond. This linkage is further supported by the deshielding effect of C-3δ74.68 (H-3δ3.75). The branched structure is a β-D-galactopyranose (β-D-Galp) linked to the backbone mannose via a β-(1→6)-linkage. This linkage is supported by the chemical shifts of the anomeric carbon of galactose at C-1δ97.32 (H-1δ4.91, J≈7.5 Hz) and the mannose at C-6δ60.44 (H-6δ3.48). Figure 10In the HSQC spectrum, the carbon-hydrogen correspondences of mannose (C-2δ76.62, H-2δ4.05), C-4δ72.74, H-4δ3.73), and galactose (C-6δ65.62, H-6δ3.55) are consistent with the methylation data (m / z 199.10 and 232.09), corresponding to the backbone β-D-Manp-(1→3)-β-D-Manp and branched β-D-Galp-(1→6)-β-D-Manp structures, respectively. The weak signals of terminal mannose (C-1δ93.44, H-1δ4.70) and galactose (C-1δ98.62) indicate partial chain termination or unattached residues. The molecular weight of 2279.4 Da deviates from the calculated value (approximately 2060 Da) for eight monosaccharide units (5×Manp, 2×Galp, 1×terminal Manp), likely due to incomplete methylation or minor modifications. NMR data fully support the identification of this polysaccharide as a heteropolysaccharide with a β-(1→3)-mannan backbone and β-(1→6)-galactose branches. The structural formula of CMP2 is shown in Formula (II).

[0125] (3) Structural analysis of the third polysaccharide:

[0126] Figure 11 is the carbon spectrum of CMP3, Figure 12 is the hydrogen spectrum of CMP3, Figure 13 is the COSY spectrum of CMP3, Figure 14 is the HSQC spectrum of CMP3, Figure 15 This is the HMBC spectrum of CMP3.

[0127] Through comprehensive methylation analysis and Figure 11-15 Systematic analysis of the CMP3 nuclear magnetic resonance (NMR) spectrum reveals the sugar residue composition, linkage pattern, and structural characteristics of the polysaccharide, as listed in Table 6, based on known data. The polysaccharide backbone consists of alternating β-D-mannose (1→3) and β-D-galactose (1→6), with side chains containing short α-L-rhamnose (1→4) branches and terminal α-L-arabinose and β-D-mannose. Figure 13 The COSY spectrum confirmed the proton connection order within the sugar ring (such as mannose H1 (5.24) - H2 (3.85) - H3 (3.73)). Figure 14 The HSQC spectrum of the 5-H-glucose complex clearly showed the CH correlation [e.g., galactose C6 (68.21 ppm) / H6 (3.55 ppm)]. Figure 15 The HMBC spectrum confirmed the long-range connection between sugar residues (e.g., mannose C1 (98.71 ppm) → galactose H6 (3.66 ppm)). The structural formula of CMP3 is shown in formula (III).

[0128] Table 6 CMP3 NMR and methylation related data

[0129]

[0130] (4) Structural analysis of the fourth polysaccharide

[0131] Figure 16 is the carbon spectrum of CMP4, Figure 17 is the hydrogen spectrum of CMP4, Figure 18 is the COSY spectrum of CMP4, Figure 19 This is the HSQC spectrum of CMP4.

[0132] Combine Figure 1 Infrared spectrum of CMP4, Figure 16-19 Comprehensive analysis of the CMP4 nuclear magnetic resonance (NMR) spectrum and methylation data showed that the CMP4 sample was mainly composed of glucose (Glc) and mannose (Man), with a small amount of galactose (Gal) and arabinose (Ara). Figure 18 The coupling relationship between 4.60ppm(H-1) and 4.35ppm(H-2) in the COSY spectrum further supports the existence of the α configuration. Figure 16 The signals at 75.97 ppm and 75.32 ppm in the carbon NMR spectrum and the coupling relationship between 4.30 ppm (H-4) and 4.32 ppm (H-3) in the COSY spectrum indicate that the main chain is mainly 1→4 linked, while the methylation data and the coupling relationship between 3.83 ppm (H-6) and 4.11 ppm (H-5) in the COSY spectrum suggest the presence of a small amount of 1→6 linked branched structures. Figure 1 In the infrared spectrum of 3400-3200 cm -1 The OH stretching vibration peak at 1100-1000 cm -1 The COC stretching vibration peak at further confirms the presence of hydroxyl (-OH) and sugar ring CO bonds in the sample. Based on the above analysis, the structural model of this polysaccharide sample is mainly composed of 1→4 linked glucose and mannose in the main chain, forming a linear polysaccharide chain. Some glucose or mannose have 1→6 linked branch structures at the C-6 position, and the monosaccharide configurations are both α and β.

[0133] The structure of the CMP4 sample can be represented by the following repeating units: the main chain is composed of α-D-glucose (α-D-Glcp) and β-D-mannose (β-D-Manp) connected alternately through 1→4 glycosidic bonds to form a linear main chain, and its structure is →4)-α-D-Glcp-(1→4)-β-D-Manp-(1→4)-α-D-Glcp-(1→4), in which glucose is in the α configuration (anomeric hydrogen chemical shift of about 5.16 ppm) and mannose is in the β configuration (anomeric hydrogen chemical shift of about 4.76 ppm). At the C6 position of some glucose, two branches are connected by 1→6 glycosidic bonds, namely β-D-galactose (β-D-Galactosyl) (β-D-Galactose) (β-D-Manp) (β-D-Manp) (β-D-Glcp ... lp) (anomeric carbon signal is about 4.60 ppm, suggesting β configuration) and α-L-arabinose (α-L-Araf) (anomeric carbon signal is about 4.30 ppm, suggesting α configuration), and its branched structure is →6)-β-D-Galp-(1→ or →6)-α-L-Araf-(1→. The minimum repeating unit contains 3 main chain sugar residues and 1 branch sugar residue, namely →4)-α-D-Glcp-(1→4)-β-D-Manp-(1→4)-α-D-Glcp-(1→, with a galactose or arabinose branch connected to the C6 position of each glucose residue. According to nuclear magnetic resonance (NMR) data, 1 The anomeric hydrogen signals of H NMR (5.16 ppm and 4.76 ppm) correspond to α-Glc and β-Man, respectively. 13 C NMR spectra at 75.32 ppm (C-4 linked sugar ring carbon) and 60.44 ppm (C-6 branched carbon) further confirmed the 1→4 and 1→6 linkages. Methylation data also supported this structural model with the presence of 1→4 linked Glc and Man derivatives (m / z 126.06, 175.13) and a 1→6 branched Gal / Ara derivative (m / z 190.18). Figure 1 In the infrared spectrum of 1100-1000cm- 1 The COC vibration peak at further confirms the formation of glycosidic bond. The structure of CMP4 is shown in formula (IV).

[0134] Example 3 Analysis of the antioxidant function of four polysaccharides

[0135] DPPH free radical is often used to evaluate reducing substances and is a useful reagent for studying the free radical scavenging ability of active substances. The test method is: prepare the sample to be analyzed to a concentration of 2×10 -4mol / L ethanol solution, shielded from light for standby use; prepare coconut polysaccharide sample solutions of different concentration gradients; add 2.0mL coconut polysaccharide sample solutions of different concentrations and 2.0mL DPPH solution to a 10mL colorimetric tube, mix thoroughly, and place in the dark at room temperature for 30min. Add the ethanol solution to a 10mL colorimetric tube, mix thoroughly, and place in the dark at room temperature for 30min, and measure the absorbance at 517nm (A1). According to the above method, measure the absorbance (A2) of the mixture of 2.0mL coconut polysaccharide sample solution and 2.0mL ethanol solution; measure the absorbance (A3) of the mixture of 2.0mL DPPH solution and 2.0mL ethanol solution.

[0136] Record the data and calculate the scavenging ability of fresh coconut meat polysaccharide on DPPH free radicals using the following formula:

[0137]

[0138] Depend on Figure 20 As can be seen, within the 1-8 mg / mL concentration range, the DPPH· scavenging ability of the four polysaccharides gradually increased with increasing polysaccharide dosage, but the increase slowed thereafter. At 10 mg / mL, the DPPH· scavenging rate of CMP1 reached 64.19% (IC50 = 2.1351); the DPPH· scavenging rate of CMP2 reached 70.67% (IC50 = 0.4548); the DPPH· scavenging rate of CMP3 reached 57.01% (IC50 = 0.7115); and the DPPH· scavenging rate of CMP4 reached 75.07% (IC50 = 0.5696).

[0139] Example 4 Functional analysis of the inhibition of α-glucosidase by four polysaccharides

[0140] The test method is as follows: add 500 μL of coconut polysaccharides of different concentrations to a test tube, then add 50 μL of α-glucosidase solution (1 U / mL) and 1 mL of phosphate buffer (pH 6.8) as a buffer. Shake well. Store the test tube at 37°C for 10 minutes. After standing, add 120 μL of p-nitrophenyl-α-D-pyranoglucoside (PNPG) and 50 μL of glutathione solution (3 mmol / L). Continue to incubate at 37°C for 20 minutes, then add 1 mL of sodium carbonate solution (0.1 mol / L). After completion, measure the mixture at a wavelength of 405 nm and record the data as A1. Replace the α-glucosidase solution with buffer, measure the absorbance at the same wavelength, and record the data as A2. The control group replaced the coconut polysaccharide sample solution with buffer, measured the absorbance at the same wavelength, and recorded the data as A0. The α-glucosidase Mars inhibition rate was calculated by the following formula:

[0141]

[0142] Fresh coconut polysaccharides have the potential to inhibit α-glucosidase activity and slow down carbohydrate decomposition. Figure 21 As shown, from 0.5 mg / mL to 8 mg / mL, the inhibition rate of CMP1 on α-glucosidase increased from 20.85% to 72.87% (IC50 = 0.174 mg / mL), the inhibition rate of CMP2 on α-glucosidase increased from 3.86% to 33.80% (IC50 = 2.8151 mg / mL), the inhibition rate of CMP3 on α-glucosidase increased from 10.31% to 24.01% (IC50 = 0.1032 mg / mL), and the inhibition rate of CMP4 on α-glucosidase increased significantly from 9.59% to 81.15% (IC50 = 34.9179 mg / mL).

[0143] Example 5 Functional analysis of the inhibition of α-amylase by four polysaccharides

[0144] The test method is as follows: Add 100 μL of prepared coconut polysaccharide solutions of various grades to a test tube, then add 100 μL of α-amylase solution (20 U / mL) and 1 mL of phosphate buffer solution (pH 6.8) as a buffer. Mix thoroughly by vortexing. Place the test tube in a 37°C water bath for 20 minutes. After the static period, add 100 μL of soluble starch (1% by mass) to the test tube, react at 37°C for 5 minutes, and then add 2 mL of DNA reagent. The reaction continues in a 37°C water bath until a color change occurs in the test tube. After cooling the mixture in an ice bath, dilute it with 2 mL of distilled water. Measure the absorbance at a wavelength of 540 nm and record it as A1. A control group uses buffer instead of α-amylase solution, recorded as AC1; another control group uses buffer instead of fresh coconut polysaccharide sample solution, recorded as A0. A blank control group uses buffer instead of α-amylase solution and fresh coconut polysaccharide sample solution, recorded as AC2. The enzyme inhibition rate is calculated as follows:

[0145]

[0146] Fresh coconut meat polysaccharide has good α-glucosidase inhibitory activity. Figure 22As shown in the results, as the concentration increased from 1 mg / mL to 10 mg / mL, the inhibition rates of CMP1, CMP2, CMP3, and CMP4 on α-glucosidase increased from 37.50%, 32.5%, 42.50%, and 30.10% to 80.21%, 64.04%, 90.03%, and 65.20%, respectively, with corresponding IC50 values ​​of 0.3737 mg / mL, 0.7133 mg / mL, 13.7049 mg / mL, and 0.6257 mg / mL, respectively. These results indicate that the inhibitory effect of polysaccharides on α-glucosidase can be significantly improved by optimizing the extraction process.

[0147] Example 6 Functional analysis of four polysaccharides in inhibiting pancreatic lipase

[0148] The test method is as follows: 1. In a series of clean test tubes, add 50 μL of coconut polysaccharide solution of different concentrations, then add 100 μL of pancreatic lipase solution (30,000 U / mL) and 50 μL of pH 6.8 phosphate buffer, mix well, and preheat in a 37°C water bath for 10 minutes; 2. Add 50 μL of 1 mmol / L substrate p-NPP solution to start the reaction, react in a 37°C water bath for 30 minutes, measure the absorbance at a wavelength of 405 nm using a microplate reader, and substitute it into the following formula to calculate the inhibition rate.

[0149] Coconut meat polysaccharide has good pancreatic lipase inhibitory activity. Figure 23 As shown in the results, from 0.2 mg / mL to 3 mg / mL, the inhibition rate of CMP1 on pancreatic lipase increased from 61.69% to 84.38% (IC50 = 1.15 mg / mL), the inhibition rate of CMP2 on pancreatic lipase increased from 63.49% to 90.66% (IC50 = 1.14 mg / mL), the inhibition rate of CMP3 on pancreatic lipase increased from 42.63% to 67.69% (IC50 = 1.03 mg / mL), and the inhibition rate of CMP4 on pancreatic lipase increased from 62.35% to 69.12% (IC50 = 0.84 mg / mL). Compared with other polysaccharides, CMP2 exhibited a stronger inhibitory activity on pancreatic lipase.

[0150] Example 7 Functional Analysis of the Binding of Four Polysaccharides to AQP1

[0151] Docking software: AutoDock Vina was used for molecular docking, and the target was AQP1 (PDB ID: 1J4N): a widely expressed classical water channel protein. The docking results are as follows Figure 24 、 Figure 25 、 Figure 26 、 Figure 27 And shown in Table 7.

[0152] Table 7 Analysis of docking results of four polysaccharides with AQP1

[0153]

[0154] As shown in Table 7, CMP1 likely inhibits water transport by blocking the Ar / R region; CMP2 significantly inhibits water permeability; CMP3 inhibits water channel activity by binding to Arg195 and His180 of AQP1; and CMP4 binds to Asp185 and Arg195 residues of AQP1. Therefore, all four polysaccharides of the present invention have the ability to inhibit water transport by binding to AQP1 and can be used to treat obesity, edema, urinary retention, nephritis, cerebral edema, pulmonary edema, or glaucoma.

[0155] The above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Those skilled in the art will readily appreciate that the present invention is susceptible to various modifications and variations. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention shall be included within the scope of the present invention.

Claims

1. A polysaccharide composition, characterized in that comprising at least one of a first polysaccharide, a second polysaccharide, a third polysaccharide and a fourth polysaccharide: The first polysaccharide is composed of mannose pyranose, rhamnose pyranose, glucose pyranose and galactose pyranose in a molar ratio of 4:1:3:2, and its structural formula is shown in formula (I). The molecular weight of the first polysaccharide is 343016.9 Da; The second polysaccharide is composed of mannose pyranose and galactose pyranose in a molar ratio of 7:3, and its structural formula is shown in formula (II). The molecular weight of the second polysaccharide is 2279.4 Da; The third polysaccharide is composed of mannose pyranose, rhamnose pyranose, galactose pyranose and arabinofuranose in a molar ratio of 54.9:4.8:16.3:16.0, and its structural formula is shown in formula (III). The molecular weight of the third polysaccharide is 1363.2 Da; The fourth polysaccharide is composed of mannose pyranose, glucose pyranose, galactose pyranose, and arabinofuranose in a molar ratio of 23.3:37.2:16.5:15.0, and its structural formula is shown in formula (IV). The molecular weight of the fourth polysaccharide is 2228.9 Da; 2. The polysaccharide composition according to claim 1, wherein The polysaccharide composition comprises a first polysaccharide, a second polysaccharide, a third polysaccharide and a fourth polysaccharide, wherein the first polysaccharide, the second polysaccharide, the third polysaccharide and the fourth polysaccharide are prepared by the following steps: S1, mixing fresh coconut meat with water and performing hot water extraction at 60-100° C., filtering, collecting the filtrate, mixing the filtrate with an alcohol reagent and allowing it to stand to precipitate polysaccharides, centrifuging the precipitate, and freeze-drying to obtain a first crude polysaccharide; S2. mixing the first crude polysaccharide with petroleum ether and shaking the mixture to separate the solid and the liquid, and drying the solid to obtain a second crude polysaccharide; S3, mixing the second crude polysaccharide with a chloroform-n-butanol solution, allowing the mixture to stand, centrifuging, taking the supernatant, dialyzing, and then freeze-drying to obtain a third crude polysaccharide; S4. Performing a first chromatography treatment on the third crude polysaccharide using a DEAE-52 cellulose column, eluting with water, 0.1 M NaCl, 0.3 M NaCl, and 0.5 M NaCl in sequence to obtain a first chromatographic liquid, a second chromatographic liquid, a third chromatographic liquid, and a fourth chromatographic liquid; S5. Use Sephadex G-150 gel column to perform a second chromatography treatment on the four chromatographic fluids obtained by the first chromatography treatment, the eluent is 0.1M NaCl, the first chromatographic fluid is eluted by 0.1M NaCl to obtain the first polysaccharide, the second chromatographic fluid is eluted by 0.1M NaCl to obtain the second polysaccharide, the third chromatographic fluid is eluted by 0.1M NaCl to obtain the third polysaccharide, and the fourth chromatographic fluid is eluted by 0.1M NaCl to obtain the fourth polysaccharide.

3. The polysaccharide composition according to claim 2, wherein In step S1: The mass ratio of the fresh coconut meat to the water is 1:(10-14); and / or, The hot water extraction time is 1 to 2 hours; and / or, The alcohol reagent includes ethanol, methanol or isopropanol with a volume concentration of 75%; and / or, The standing time is 20 to 24 hours; and / or, The centrifugal speed is 3500-4500 r / min, and the centrifugal time is 15-25 min.

4. The polysaccharide composition according to claim 2, wherein In step S2: Add 1 g of the first crude polysaccharide to every 10 to 12 mL of the petroleum ether; and / or, The shaking time is 1.5 to 2.5 hours; and / or, The drying temperature is 45-50°C.

5. The polysaccharide composition according to claim 2, wherein Step S3 comprises: preparing the second crude polysaccharide into a 5 mg / mL aqueous solution, adding a mixture of chloroform and n-butanol in a volume ratio of 2:1, shaking, standing for 20 to 25 minutes, centrifuging at 6500 to 7500 r / min for 10 to 15 minutes, drawing the upper aqueous phase and performing dialysis and freeze-drying in sequence to obtain a third crude polysaccharide.

6. Use of the polysaccharide composition according to claim 1 in the preparation of antioxidants.

7. Use of the polysaccharide composition according to claim 1 in the preparation of an α-glucosidase inhibitor, an α-amylase inhibitor or a pancreatic lipase inhibitor.

8. Use of the polysaccharide composition according to claim 1 in the preparation of a hypoglycemic or hypolipidemic drug.

9. Use of the polysaccharide composition according to claim 1 in the preparation of a medicament for treating AQP1-mediated diseases.

10. The use according to claim 9, characterized in that The AQP1-mediated diseases include obesity, edema, urinary retention, nephritis, cerebral edema, pulmonary edema or glaucoma.

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