Homogenized bitter melon polysaccharide and its preparation method and application

Abstract

The present invention provides a homogenized bitter melon polysaccharide, a preparation method, and an application thereof, belonging to the technical field of polysaccharide extraction and purification. The homogenized bitter melon polysaccharide of the present invention contains rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, and arabinose, and the corresponding molar ratios are 0.032:0.011:0.774:0.003:0.153:0.028; the relative molecular weight of the homogenized bitter melon polysaccharide is 93.796 kDa. The homogenized bitter melon polysaccharide has a significant blood sugar-lowering effect. The preparation method has mild process conditions and simple operation. The obtained polysaccharide has high purity and stable structure, and is suitable for industrial-scale production.

Description

Technical Field

[0001] The invention belongs to the technical field of polysaccharide extraction and purification, and particularly relates to a homogenized bitter melon polysaccharide and a preparation method and application thereof. Background Art

[0002] People's lifestyles and dietary patterns are constantly changing and becoming unbalanced, leading to many diet-related chronic diseases, such as cardiovascular disease, obesity, and diabetes. Currently, the treatment for diabetes is generally to control blood sugar levels by taking medications with hypoglycemic effects. However, most hypoglycemic drugs are synthesized chemically and have certain side effects. Therefore, the extraction of active substances with hypoglycemic effects from natural plants and without toxicity has become a research focus.

[0003] Polysaccharides are natural high-molecular-weight polymers composed of more than 10 monosaccharides linked by glycosidic bonds. They are widely found in organic life forms such as animals, plants, and microorganisms, and are the main form of carbohydrates in nature. Research has found that polysaccharides have a variety of biological activities, including hypoglycemic and hypolipidemic effects, anti-tumor, antioxidant, antiviral, and anti-inflammatory effects. Natural medicines with polysaccharides as active substances have the advantages of multiple pathways, multiple targets, multidirectionality, and minimal toxicity and side effects. They can also act on diabetes through multiple mechanisms and multiple links. The hypoglycemic biological activity of polysaccharides is extremely important, and their hypoglycemic mechanism has become a research hotspot. In addition, the hypoglycemic activity of polysaccharides is closely related to their structure and function. Summary of the Invention

[0004] The invention provides a homogenized bitter melon polysaccharide and a preparation method and application thereof. The homogenized bitter melon polysaccharide has an obvious blood sugar lowering effect.

[0005] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0006] The invention provides a homogenized bitter melon polysaccharide. The homogenized bitter melon polysaccharide contains rhamnose, glucuronic acid, galacturonic acid, glucose, galactose and arabinose, and the corresponding molar ratios are 0.032:0.011:0.774:0.003:0.153:0.028; the molecular weight of the homogenized bitter melon polysaccharide is 93.796 kDa.

[0007] The present invention also provides a preparation method of the homogenized bitter melon polysaccharide, comprising the following steps: (1) mixing bitter melon with water, heating and ultrasonicating, centrifuging, and collecting the supernatant; (2) concentrating the supernatant, mixing the concentrate with ethanol for alcohol precipitation, and collecting the precipitate by centrifugation; (3) mixing the precipitate with water to obtain a polysaccharide solution, adding Sevage reagent, centrifuging, and taking the supernatant; dialyzing the supernatant and drying to obtain crude bitter melon polysaccharide; (4) subjecting the crude bitter melon polysaccharide to anion exchange column chromatography, collecting the polysaccharide-containing eluate, and dialyzing to obtain ion-purified bitter melon polysaccharide; and (5) subjecting the ion-purified bitter melon polysaccharide to gel column chromatography, collecting the polysaccharide-containing eluate, and dialyzing to obtain homogenized bitter melon polysaccharide.

[0008] Preferably, in step (1), the material-liquid ratio of the bitter melon to water is 1:1-5; the heating temperature is 80-100°C, and the heating time is 1-5 hours; the ultrasonic time is 5-15 minutes; in step (2), the supernatant is concentrated to 1 / 5-1 / 15 of the original volume; the volume ratio of the concentrated solution to ethanol is 1:1-8, and the ethanol concentration is 85-100%; in step (3), the volume ratio of the polysaccharide solution to the Sevage reagent is 1-5:1.

[0009] Preferably, in step (4), the anion exchange column is DEAE Sepharose Fast Flow, and the loading concentration is 20-40 mg / mL; the elution method is water elution, 0.1 mol / LNaCl and 0.2 mol / LNaCl gradient elution in sequence, each elution time is 150-250 min, and the eluate obtained by 0.2 mol / LNaCl elution is collected; the elution flow rate is 1-4 mL / min.

[0010] Preferably, in step (5), the gel column is Sephadex G-100, the loading concentration is 3 to 13 mg / mL; the elution method is water elution, the elution time is 100 to 200 min, and the eluate is collected; the elution flow rate is 0.3 to 2 mL / min.

[0011] Preferably, the centrifugal conditions include: 7000-9000 rpm, 5-15 min, 1-7°C.

[0012] Preferably, the dialysis bag used for dialysis has a specification of 2500-4500Da.

[0013] The present invention provides the use of the homogenized bitter melon polysaccharide or the homogenized bitter melon polysaccharide obtained by the preparation method in preparing a blood sugar-lowering product.

[0014] The present invention provides a blood sugar lowering pharmaceutical composition, comprising the homogenized bitter melon polysaccharide or the homogenized bitter melon polysaccharide obtained by the preparation method and pharmaceutically acceptable excipients.

[0015] Preferably, the dosage form of the pharmaceutical composition includes powder, tablet, solution, granule, pellet, pill or powder.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The present invention can effectively separate bitter melon polysaccharides through water extraction and alcohol precipitation, DEAE-Sepharose Fast Flow ion exchange chromatography, and Sephadex G-100 gel column chromatography, thereby preparing a homogeneous component polysaccharide of bitter melon for the first time. The homogenized bitter melon polysaccharide has a novel structure, a chemical structure, and a uniform composition, has a significant hypoglycemic effect, and has no toxic side effects on normal organisms. It solves the problem of the lack of active ingredients with a single composition and a clear structure in the research and development of new blood sugar-lowering drugs in bitter melon. It lays a reliable material foundation for the study of the structure-activity relationship of bitter melon polysaccharides in lowering blood sugar, and plays a driving role in the research and development of polysaccharides as new blood sugar-lowering drugs.

[0018] The homogenized bitter melon polysaccharide preparation process provided by the present invention has mild conditions, simple operation, and is environmentally friendly. The obtained polysaccharide has high purity and stable structure, and the activity of the polysaccharide is maintained to the greatest extent, and is suitable for industrial-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Elution curves of bitter melon polysaccharide on DEAE Sepharose FastFlow (A) and Sephadex G-100 (B).

[0020] Figure 2 High-performance gel permeation chromatography of MCP-3.

[0021] Figure 3 Ion chromatograms of monosaccharide standards (A) and MCP-3 (B).

[0022] Figure 4 GC-MS ion chromatogram of MCP-3.

[0023] Figure 5 MCP-3 1 H(A), 1 H (water peak suppression) (B), 13 C(C), DEPT-135(D), HSQC(E), COSY(F), HMBC(G) and NOESY(H) NMR spectra.

[0024] Figure 6Insulin toxicity to HepG2 cells.

[0025] Figure 7 Effects of different insulin concentrations on glucose consumption in HepG2 cells.

[0026] Figure 8 Effect of MCP-3 on glucose consumption of IR-HepG2 ("**" p < 0.01 indicates that there is a very significant difference between the model group and the blank group; "##" p < 0.01 indicates that there is a very significant difference between the experimental group and the model group; "#" p < 0.05 indicates that there is a significant difference between the experimental group and the model group).

[0027] Figure 9 Effect of MCP-3 on glycogen synthesis in IR-HepG2 (A), glycogen PAS staining (B) and staining statistical results (C) ("**" p < 0.01 indicates that there is a very significant difference between the model group and the blank group; "##" p < 0.01 indicates that there is a very significant difference between the experimental group and the model group).

[0028] Figure 10 Effects of MCP-3 on HK activity (A) and PK activity (B) in IR-HepG2 cells (“**” p<0.01 indicates that there is a very significant difference between the model group and the blank group; “##” p<0.01 indicates that there is a very significant difference between the experimental group and the model group).

[0029] Figure 11 Effects of MCP-3 on body surface characteristics (A) and body weight (B) of T2DM mice.

[0030] Figure 12 Effect of MCP-3 on food intake in T2DM mice.

[0031] Figure 13 Effect of MCP-3 on water intake in T2DM mice.

[0032] Figure 14 Effects of MCP-3 on blood glucose (A) and oral glucose tolerance (B, C) in T2DM mice (“##” p<0.01 indicates that the model group has a very significant difference compared with the blank group; “**” p<0.01 indicates that the experimental group has a very significant difference compared with the model group). DETAILED DESCRIPTION

[0033] The present invention provides a homogenized bitter melon polysaccharide. The homogenized bitter melon polysaccharide contains rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, and arabinose in a molar ratio of 0.032:0.011:0.774:0.003:0.153:0.028. The molecular weight of the homogenized bitter melon polysaccharide is 93.796 kDa. The predicted sugar residue structure of the homogenized bitter melon polysaccharide is shown below.

[0034] (1) Main Chain

[0035]

[0036] (2) Branched chain

[0037]

[0038] In the present invention, the bitter melon variety is white bitter melon, which was purchased from Sanbao Agricultural Products Enterprise Co., Ltd. in Changsha City, Hunan Province. A single component polysaccharide was separated and purified from it as a raw material and named MCP-3.

[0039] The present invention also provides a preparation method of the homogenized bitter melon polysaccharide, comprising the following steps: (1) mixing bitter melon with water, heating and ultrasonicating, centrifuging, and collecting the supernatant; (2) concentrating the supernatant, mixing the concentrate with ethanol for alcohol precipitation, and collecting the precipitate by centrifugation; (3) mixing the precipitate with water to obtain a polysaccharide solution, adding Sevage reagent, centrifuging, and taking the supernatant; dialyzing the supernatant and drying to obtain crude bitter melon polysaccharide; (4) subjecting the crude bitter melon polysaccharide to anion exchange column chromatography, collecting the polysaccharide-containing eluate, and dialyzing to obtain ion-purified bitter melon polysaccharide; and (5) subjecting the ion-purified bitter melon polysaccharide to gel column chromatography, collecting the polysaccharide-containing eluate, and dialyzing to obtain homogenized bitter melon polysaccharide.

[0040] In the present invention, in step (1), the material-liquid ratio of the bitter melon to water (g / mL) is 1:1-5; the heating temperature is 80-100°C, the heating time is 1-5 hours; the ultrasonic time is 5-15 minutes, and the ultrasonic power is 150-350W. Preferably, the material-liquid ratio of the bitter melon to water is 1:1.5-3; the heating temperature is 89-98°C, the heating time is 1.5-3 hours; the ultrasonic time is 8-13 minutes, and the ultrasonic power is 200-300W. In the present invention, the centrifugation conditions in step (1) are: 7000-9000 rpm, 5-15 minutes, and 1-7°C.

[0041] In the present invention, in step (2), the supernatant is concentrated to 1 / 5 to 1 / 15 of the original volume; the volume ratio of the concentrate to ethanol is 1:1 to 8, and the ethanol concentration is 85 to 100%. The present invention preferably concentrates the supernatant to 1 / 8 to 1 / 13 of the original volume; the volume ratio of the concentrate to ethanol is 1:2 to 6, and the ethanol concentration is 90 to 98% (V / V). In the present invention, in step (2), it is necessary to place the supernatant in a refrigerator at 1 to 6°C for alcohol precipitation for 8 to 16 hours. In the present invention, in step (2), the centrifugation conditions are: 7000 to 9000 rpm, 5 to 15 minutes, and 1 to 6°C.

[0042] In the present invention, in step (3), the volume ratio of the polysaccharide solution to the Sevage reagent is 1 to 5:1, preferably 2 to 4:1. In step (3) of the present invention, the mass volume ratio of the precipitate to water is 1:10 to 30 (g:mL), preferably 1:20 (g:mL). The chloroform:n-butanol in the Sevage reagent of the present invention is 2 to 6:1 (V:V), preferably 4:1, for the purpose of deproteinization. In step (3) of the present invention, the mixture is shaken for 10 to 30 minutes, preferably 20 minutes, before centrifugation.

[0043] In the present invention, in step (4), the anion exchange column is DEAE Sepharose Fast Flow, the sample concentration is 20-40 mg / mL, preferably 25-35 mg / mL; the elution method is water elution, 0.1 mol / L NaCl and 0.2 mol / L NaCl gradient elution in sequence, each elution time is 150-250 min, and the eluate obtained by elution with 0.2 mol / L NaCl is collected, preferably the elution time is 180-230 min; the elution flow rate is 1-4 mL / min, preferably 1.5-3 mL / min. In the present invention, in step (4), the collected eluate is concentrated by rotary evaporation, placed in a dialysis bag (molecular weight 2500-4500 Da), and dialyzed with running water for 1-3 days to remove salt therein. After the dialysis is completed, the eluate is freeze-dried (-50° C., 48 h) and stored at -20° C. for use.

[0044] In the present invention, in step (5), the gel column is Sephadex G-100, the sample concentration is 3-13 mg / mL, preferably 5-10 mg / mL; the elution method is water elution, the elution time is 100-200 min, and the eluate is collected, preferably the elution time is 110-150 min; the elution flow rate is 0.3-2 mL / min, preferably 0.5-1.5 mL / min. In step (5), the collected eluate is concentrated by rotary evaporation, placed in a dialysis bag (molecular weight 2500-4500 Da), and dialyzed with running water for 1-3 days to remove salt therein. After dialysis is completed, the eluate is freeze-dried (-50°C, 48 h) and stored at -20°C for later use.

[0045] The present invention also provides the use of the homogenized bitter melon polysaccharide or the homogenized bitter melon polysaccharide obtained by the preparation method in the preparation of blood sugar-lowering products. The effective dose of the homogenized bitter melon polysaccharide of the present invention is 50-350 mg / kg, preferably 100-250 mg / kg.

[0046] In the present invention, the dosage form of the pharmaceutical composition includes powder, tablet, solution, granule, pellet, pill or powder; the excipients include one or more of excipients, lubricants, preservatives, diluents, sweeteners, adhesives, colorants, cosolvents and surfactants.

[0047] In the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art.

[0048] The technical solutions of the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention.

[0049] Example 1 Preparation of homogenized bitter melon polysaccharide

[0050] 1. Preparation of bitter melon crude polysaccharide by ultrasound-assisted water extraction and alcohol precipitation

[0051] (1) The purchased bitter melon (Bitter Melon oleraceus, produced by Sanbao Agricultural Products Enterprise Co., Ltd., Changsha, Hunan Province) was cleaned and cut into pieces. Distilled water was added and the mixture was slurried in a solid-liquid ratio of 1:2 (g / mL). The mixture was heated in a water bath at 95°C for 2 h and then ultrasonicated for 10 min (power 250 W) to obtain the extract.

[0052] (2) The extract was centrifuged (8000 rpm, 10 min, 4°C), the supernatant was collected, and the supernatant was concentrated by rotary evaporation (55°C) to approximately 1 / 10 of the original volume. Four volumes of 95% ethanol were added to the concentrate, and the solution was placed in a refrigerator at 4°C for 12 h. After precipitation, the solution was centrifuged (8000 rpm, 10 min, 4°C) to collect the precipitate.

[0053] (3) Ultrapure water was added to re-dissolve the precipitate at a ratio of 1:20 (g:mL), and Sevage reagent (chloroform: n-butanol = 4:1 (V:V)) was used for deproteinization. The polysaccharide solution and Sevage reagent were then mixed in a certain ratio (polysaccharide solution: Sevage reagent = 3:1 (V:V)), shaken for 20 minutes, and then centrifuged (8000 rpm, 10 minutes, 4°C). The supernatant was collected and repeated several times until no white protein remained. The polysaccharide solution (molecular weight 3500 Da) was dialyzed with running water for 2 days and freeze-dried (-50°C, 48 hours) to obtain the crude bitter melon polysaccharide.

[0054] 2. Isolation and purification of bitter melon crude polysaccharide: The bitter melon crude polysaccharide was further purified using DEAE Sepharose FastFlow (Cytiva, 17070901) and Sephadex G-100 (Solarbio, S8171).

[0055] (1) Weigh 300 mg of bitter melon crude polysaccharide and dissolve it in 10 mL of distilled water to prepare a 30 mg / mL crude polysaccharide solution. Load the crude polysaccharide solution onto a DEAE Sepharose Fast Flow anion exchange chromatography column and elute with distilled water, 0.1, 0.2, 0.3, 0.4, 0.6 and 0.8 M NaCl solutions in sequence. The flow rate is set to 2 mL / min. Each concentration is eluted for 200 minutes each time. Collect with an automatic collector and collect one tube every 5 minutes. Detect the collected liquid with the phenol-sulfuric acid method, measure the absorbance at 490 nm, draw an elution curve with the number of tubes as the horizontal axis and the absorbance as the vertical axis, and collect the eluate of different components according to the elution curve. The elution curve is as shown in the figure. Figure 1 As shown in Figure A. Five fractions were obtained from elution with 0, 0.1, and 0.2 M NaCl solutions. Three fractions were obtained from elution with 0.1 M NaCl solution, designated MCP-1, MCP-2a, MCP-2b, MCP-2c, and MCP-3. MCP-1 is a neutral polysaccharide, while the others are acidic polysaccharides. The eluates from the same elution peak were combined, concentrated by rotary evaporation (55°C, 90 rpm), placed in a dialysis bag (molecular weight 3500 Da), and dialyzed with running water for 2 days to remove salt. After dialysis, the mixture was freeze-dried (-50°C, 48 h) and stored at -20°C for later use.

[0056] (2) In view of the low yields of MCP-1, MCP-2a, MCP-2b, and MCP-2c, and considering subsequent experiments and actual operations, the MCP-3 component with a relatively high yield was selected for gel column purification. The collected MCP-3 components were further purified using Sephadex G-100 (Solarbio, S8171). 40 mg of each different component was weighed and dissolved in 5 mL of distilled water to prepare an 8 mg / mL polysaccharide solution, which was filtered through a 0.45 μm filter membrane. The polysaccharide solution was loaded onto a Sephadex G-100 polysaccharide gel column and eluted with distilled water. The flow rate was set to 1 mL / min, the elution was 125 minutes, and the solution was collected using an automatic collector, with one tube collected every 5 minutes. The collected solution was detected using the phenol-sulfuric acid method, and the absorbance was measured at 490 nm. An elution curve was drawn with the number of tubes as the horizontal axis and the absorbance as the vertical axis, and the eluates of different components were collected according to the elution curve. The elution curve is as follows: Figure 1 B. As shown in the figure, MCP-3 was purified again through a gel column, yielding a homogeneous fraction with a purified yield of 45.92%. The eluates from the same elution peak were combined, concentrated by rotary evaporation (55°C, 90 rpm), placed in a dialysis bag (molecular weight 3500 Da), and dialyzed against running water for 2 days to remove salt. After dialysis, the product was freeze-dried (-50°C, 48 hours) and stored at -20°C for later use. This yielded highly pure, homogenized bitter melon polysaccharide MCP-3 with a polysaccharide content of 94.77±2.95%.

[0057] Example 2

[0058] (1) The purchased bitter melon (Bitter Melon oleraceus, produced by Sanbao Agricultural Products Enterprise Co., Ltd., Changsha, Hunan Province) was cleaned and cut into pieces. Distilled water was added and the mixture was slurried in a 1:1 solid-liquid (g / mL) ratio. The mixture was heated in a water bath at 80°C for 4 h and then ultrasonicated for 15 min (power 200 W) to obtain the extract.

[0059] (2) The extract was centrifuged (8000 rpm, 10 min, 4°C), the supernatant was collected, and the supernatant was concentrated by rotary evaporation (55°C) to about 1 / 5 of the original volume. 1 volume of 95% ethanol was added to the concentrate and the solution was placed in a refrigerator at 4°C for 12 h for alcohol precipitation. After alcohol precipitation, the solution was centrifuged (8000 rpm, 10 min, 4°C) to collect the precipitate.

[0060] (3) Ultrapure water was added to re-dissolve the precipitate at a ratio of 1:10 (g:mL), and Sevage reagent (chloroform: n-butanol = 4:1 (V:V)) was used for deproteinization. The polysaccharide solution and Sevage reagent were then mixed in a certain ratio (polysaccharide solution: Sevage reagent = 3:1 (V:V)), shaken for 20 minutes, and then centrifuged (8000 rpm, 10 minutes, 4°C). The supernatant was collected and repeated several times until no white protein remained. The polysaccharide solution (molecular weight 2500 Da) was dialyzed with running water for 2 days and freeze-dried (-50°C, 48 hours) to obtain the bitter melon crude polysaccharide.

[0061] (4) Weigh 200 mg of bitter melon crude polysaccharide and dissolve it in 10 mL of distilled water to prepare a 20 mg / mL crude polysaccharide solution. Load the crude polysaccharide solution onto a DEAE Sepharose Fast Flow anion exchange chromatography column and elute with distilled water, 0.1, and 0.2 M NaCl solutions in sequence. The flow rate is set to 1 mL / min, and each solution is eluted for 150 min each time. The eluate obtained by eluting with 0.2 M NaCl solution is collected by an automatic collector, concentrated by rotary evaporation (55°C, 90 rpm), and placed in a dialysis bag (molecular weight 2500 Da) and dialyzed with running water for 2 days to remove the salt. After dialysis is completed, freeze-dry (-50°C, 48 h) and store at -20°C for use to obtain ion-purified polysaccharide.

[0062] (5) Weigh 20 mg of ion-purified polysaccharide and dissolve it in 5 mL of distilled water to prepare a 4 mg / mL polysaccharide solution, which was then filtered through a 0.45 μm filter membrane. The polysaccharide solution was loaded onto a Sephadex G-100 polysaccharide gel column and eluted with distilled water at a flow rate of 1 mL / min for 100 min. The eluate was collected by an automatic collector, concentrated by rotary evaporation (55°C, 90 rpm), and placed in a dialysis bag (molecular weight 2500 Da) and dialyzed with running water for 2 days to remove the salt. After dialysis, the polysaccharide was freeze-dried (-50°C, 48 h) and stored at -20°C for later use to obtain homogenized bitter melon polysaccharide.

[0063] Example 3

[0064] (1) The purchased bitter melon (Bitter Melon oleraceus, produced by Sanbao Agricultural Products Enterprise Co., Ltd., Changsha, Hunan Province) was cleaned and cut into pieces. Distilled water was added and the mixture was slurried at a solid-liquid ratio of 1:5 (g / mL). The mixture was heated in a water bath at 95°C for 1 h and then ultrasonicated for 8 min (power 300 W) to obtain the extract.

[0065] (2) The extract was centrifuged (8000 rpm, 10 min, 4°C), the supernatant was collected, and the supernatant was concentrated by rotary evaporation (55°C) to about 1 / 5 of the original volume. 1 volume of 95% ethanol was added to the concentrate and the solution was placed in a refrigerator at 4°C for 12 h for alcohol precipitation. After alcohol precipitation, the solution was centrifuged (8000 rpm, 10 min, 4°C) to collect the precipitate.

[0066] (3) Ultrapure water was added to re-dissolve the precipitate at a ratio of 1:20 (g:mL), and Sevage reagent (chloroform: n-butanol = 4:1 (V:V)) was used for deproteinization. The polysaccharide solution and Sevage reagent were then mixed in a certain ratio (polysaccharide solution: Sevage reagent = 3:1 (V:V)), shaken for 20 minutes, and then centrifuged (8000 rpm, 10 minutes, 4°C). The supernatant was collected and repeated several times until no white protein remained. The polysaccharide solution (molecular weight 4500Da) was dialyzed with running water for 2 days and freeze-dried (-50°C, 48 hours) to obtain the bitter melon crude polysaccharide.

[0067] (4) Weigh 400 mg of bitter melon crude polysaccharide and dissolve it in 10 mL of distilled water to prepare a 40 mg / mL crude polysaccharide solution. Load the crude polysaccharide solution onto a DEAE Sepharose FastFlow anion exchange chromatography column and elute with distilled water, 0.1, and 0.2 M NaCl solutions in sequence. The flow rate is set to 4 mL / min, and each solution is eluted for 250 min each time. The eluate obtained by eluting with 0.2 M NaCl solution is collected by an automatic collector, concentrated by rotary evaporation (55°C, 90 rpm), and placed in a dialysis bag (molecular weight 4500 Da) and dialyzed with running water for 2 days to remove the salt. After dialysis, freeze-dry (-50°C, 48 h) and store at -20°C for use to obtain ion-purified polysaccharide.

[0068] (5) Weigh 50 mg of ion-purified polysaccharide and dissolve it in 5 mL of distilled water to prepare a 10 mg / mL polysaccharide solution, which was then filtered through a 0.45 μm filter membrane. The polysaccharide solution was loaded onto a Sephadex G-100 polysaccharide gel column and eluted with distilled water at a flow rate of 2 mL / min for 150 min. The eluate was collected by an automatic collector, concentrated by rotary evaporation (55°C, 90 rpm), and placed in a dialysis bag (molecular weight 4500 Da) and dialyzed with running water for 2 days to remove the salt. After dialysis, the polysaccharide was freeze-dried (-50°C, 48 h) and stored at -20°C for later use to obtain homogenized bitter melon polysaccharide.

[0069] Example 4 Structural Characterization of Homogenized Momordica charantia Polysaccharide

[0070] 1. Determination of purity and molecular weight

[0071] The HPGPC method was used to determine the purity and molecular weight of the polysaccharide using high performance gel permeation chromatography (HPLC) columns.

[0072] (1) Standard curve preparation: Accurately weigh dextran standards of different molecular weights (molecular weight 1000, 5000, 12000, 25000, 50000, 80000, 150000, 270000, 410000, 670000 series analytical standards) and prepare them into 5 mg / ml solutions with 0.05 M NaCl solution (precisely prepared, filtered through a 0.22 μm filter, sonicated for 20 min, and stored at room temperature). Filter through a 0.22 μm filter and set aside. A linear regression was performed using the logarithm of the relative molecular mass (1 g Mw) of the standard as the ordinate and the retention time of the corresponding chromatographic peak as the abscissa to obtain the calibration curve.

[0073] (2) Preparation of polysaccharide samples: 5 mg of the MCP-3 polysaccharide sample prepared in Example 1 was accurately weighed, and 1 mL of 0.05 M NaCl solution was added to the sample to prepare a polysaccharide solution with a concentration of 5 mg / mL. The solution was centrifuged at 8000 rpm for 10 min, and the supernatant was collected and filtered with a 0.22 μm filter membrane. The sample was then transferred to a 2 mL injection vial for later use.

[0074] (3) Chromatographic separation conditions were as follows: instrument: Waters high performance liquid chromatograph; detector: Waters differential detector; chromatographic column: three polymer-based water-soluble SEC (GFC) chromatographic columns (8×300 mm) connected in series; mobile phase: 0.05 M NaCl solution; flow rate: 0.65 mL / min; column temperature: 40°C; injection volume: 30 μL.

[0075] like Figure 2 As shown in Table 1, the peak time of the bitter melon polysaccharide MCP-3 is 31.530 min, and the chromatograms are all single peaks, indicating that this polysaccharide is a high-purity homogeneous component with a polysaccharide content of 94.77 ± 2.95%. According to the standard calibration curve, the peak molecular weight (Mp), weight average molecular weight (Mw), number average molecular weight (Mn) and Mw / Mn ratio (polydispersity coefficient) of MCP-3 are calculated, as shown in Table 1. As can be seen from the table, the molecular weight of MCP-3 is 93.796 kDa, which indicates that the bitter melon polysaccharide can be effectively separated by water extraction and alcohol precipitation, DEAE-Sepharose Fast Flow ion exchange chromatography and Sephadex G-100 gel column chromatography in sequence to prepare a bitter melon homogeneous component polysaccharide.

[0076] Table 1 Molecular weight of MCP-3

[0077] Components RT (min) Mp(Da) Mw(Da) Mn(Da) Distribution coefficient (Mw / Mn) MCP-3 31.530 72183 93796 59620 1.573

[0078] 2. Determination of Monosaccharide Composition

[0079] Monosaccharide composition was determined using HPLC with 1-phenyl-3-methyl-5-pyrazolone (PMP) pre-column derivatization. Polysaccharides were hydrolyzed into monosaccharides under acidic conditions. These monosaccharides then reacted with PMP under weak alkaline conditions, resulting in ultraviolet absorption, which allowed for UV detection. This was then analyzed using HPLC.

[0080] (1) Preparation of standard products: Weigh monosaccharide standards (rhamnose, arabinose, galactose, glucose, xylose, mannose, galacturonic acid, glucuronic acid, glucosamine hydrochloride, galactosamine hydrochloride monosaccharide, and fucose) in sequence to prepare monosaccharide standard stock solutions. Then, take a certain amount of each monosaccharide standard stock solution to prepare mixed standards. Dilute the monosaccharide standard stock solution and the mixed standards to different gradient dilution volumes.

[0081] (2) Acid hydrolysis of polysaccharide sample: Accurately weigh 5 mg of the MCP-3 polysaccharide sample prepared in Example 1, add 1 mL of trifluoroacetic acid (2M TFA) solution, and heat at 121°C for 2 hours. Flow nitrogen and blow dry. Add 3 mL of methanol to wash, blow dry again, and repeat the methanol wash 2-3 times. Add 5 mL of sterile water to dissolve, and obtain a polysaccharide hydrolyzate.

[0082] (3) PMP derivatization: 0.2 mL of monosaccharide standard solution and polysaccharide hydrolyzate were placed in a centrifuge tube, and 0.2 mL of 0.5 mol / L NaOH solution and 0.5 mL of 0.5 mol / L PMP-methanol solution were added. After vortex mixing, the mixture was reacted in a 70 °C water bath for 1 h. After the reaction was completed, 0.2 mL of 0.5 mol / L HCl was added to neutralize the added NaOH. 1 mL of chloroform was added and vortexed for extraction three times to remove excess PMP. After discarding the chloroform layer, 0.3 mL of the mixture was taken and diluted to 1 mL with water. The mixture was filtered through a 0.22 μm filter membrane and placed in a sample injection bottle for testing.

[0083] (4) Ion chromatography conditions: Instrument: ThermoU3000 liquid chromatography system; Chromatographic column: ZORBA XEclipseXDB-C18; Mobile phase: Phase A is phosphate buffer (12 g / L potassium dihydrogen phosphate, 2 M NaOH adjusted to pH 6.8), Phase B is acetonitrile, the volume ratio of Phase A to Phase B is 83:17, isocratic elution; Flow rate: 0.8 ml / min; Column temperature: 30 °C; Detector: UV detector; Detection wavelength: 250 nm; Injection volume: 10 μL.

[0084] Figure 3 (A) is the ion chromatogram of 11 monosaccharide standards that were effectively separated. The sequences and retention times of the 11 monosaccharides are shown in Table 2. The monosaccharides represented by each peak in the mixed standard can be determined by the retention time. Figure 3As shown in Figure (B), MCP-3 contains six monosaccharides: rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, and arabinose, with a corresponding molar ratio of 0.032:0.011:0.774:0.003:0.153:0.028. The main monosaccharide components of MCP-3 are galacturonic acid and galactose. Galacturonic acid is the main monosaccharide component of MCP-3, indicating that MCP-3 is an acidic polysaccharide.

[0085] Table 211 Monosaccharide Standards

[0086]

[0087]

[0088] 3. Methylation Analysis

[0089] Methylation analysis of bitter melon polysaccharides followed the method of Zhang and Luo et al. The basic steps for polysaccharide methylation include methylation, hydrolysis, reduction, acetylation, and GC-MS analysis. Acidic polysaccharides contain more uronic acid than neutral polysaccharides, so uronic acid reduction is required before methylation.

[0090] (1) Uronic acid reduction: Weigh 10 mg of sample and dissolve it in 1 mL of primary water. Add 1 mL of 100 mg / mL carbodiimide and react for 2 h. Add 1 mL of 2 M imidazole and 1 mL of 30 mg / mL NaBD4 and react for 3 h. Terminate the reaction by adding 100 μL of glacial acetic acid. Dialyze the sample for 48 h. After dialysis, freeze-dry the sample and proceed to methylation.

[0091] (2) Methylation: Add 500 μL DMSO to the freeze-dried sample to dissolve. Accurately weigh 1 mg of the sample to be tested and dissolve it in 500 μL DMSO. Add 1 mg of NaOH and incubate for 30 min. Add 50 μL of iodomethane solution and react for 1 h. Add 1 mL of water and 2 mL of dichloromethane, vortex to mix, centrifuge, and discard the aqueous phase. Repeat the water wash three times. Aspirate the lower dichloromethane phase and evaporate to dryness. Test the methylated product with infrared spectroscopy to confirm that the methylation is complete. Otherwise, repeat the methylation.

[0092] (3) Hydrolysis: Add 100 μL of 2 M TFA and react at 121°C for 90 min. Evaporate to dryness at 30°C.

[0093] (4) Reduction: Add 50 μL of 2M ammonia and 50 μL of 1M NaBD4, mix well, and react at room temperature for 2.5 h. Add 20 μL of acetic acid to terminate the reaction, blow dry with nitrogen, wash twice with 250 μL of methanol, and blow dry with nitrogen.

[0094] (5) Acetylation: Add 250 μL of acetic anhydride, vortex mix, and react at 100°C for 2.5 h. Add 1 mL of water and let stand for 10 min. Add 500 μL of dichloromethane, vortex mix, centrifuge, and discard the aqueous phase. Repeat the water wash three times. Remove the lower dichloromethane phase, analyze it, and perform GC-MS analysis.

[0095] (6) Chromatographic parameters: Instrument: Agilent gas chromatography system (Agilent 7890A; Agilent Technologies, USA); Chromatographic column: HP-5MS capillary column (30 m × 0.25 mm × 0.25 μm, Agilent J&W Scientific, Folsom, CA, USA); Carrier gas: high-purity helium (purity not less than 99.999%); Flow rate: 1.0 mL / min; Inlet temperature: 260°C; Injection volume: 1 μL; Split ratio: 10:1; Solvent delay: 2.2 min. Temperature program: 50°C for 1.0 min, then 50°C / min to 130°C, then 3°C / min to 230°C, and then hold for 2 min.

[0096] (7) Mass spectrometry parameters: Instrument: Agilent 5977B quadrupole mass spectrometry system (Agilent Technologies, USA), equipped with an electron impact ion source (EI) and a MassHunter workstation; electron impact ion source (EI); injection port temperature: 230°C; quadrupole temperature: 150°C; electron energy: 70 eV; scanning mode: full scan mode (SCAN); mass scanning range (m / z): 30–600.

[0097] According to the relative retention time and mass spectrum of each chromatographic peak ( Figure 4 ), the ion fragment mass spectra of the literature data and the Complex Carbohydrate Research Center database of the University of Georgia (https: / / glygen.ccrc.uga.edu / ccrc / specdb / ms / pmaa / pframe.html) were compared to analyze the type of sugar residues corresponding to each chromatographic peak, and the content of each sugar residue type was calculated using the chromatographic peak area of ​​the corresponding sugar residue type (Table 3).

[0098] As shown in Table 3, MCP-3 contains 14 methylated products PMAAs (partially methylated sugar alcohol acetates), and the corresponding glycosidic bond types and relative molar ratios are T-Araf (1.225%), T-Rhap (2.128%), 1,5-Araf (1.181%), T-GalpA (1.791%), T-Galp (1.061%), 1,2,4-Rhap (1.118%). The results showed that the main components of the glycosidic bonds were 1,4-GalpA (79.745%), 1,4-Galp (2.857%), 1,4-Glcp (1.420%), 1,3-Galp (0.988%), 1,3,4-GalpA (1.245%), 1,2,4-GlcpA (3.121%), 1,4,6-GalpA (0.939%), and 1,3,6-Galp (1.151%). Among all the glycosidic bonds, 1,4-GalpA (79.745%) was the main component, which was consistent with the results of monosaccharide composition analysis and indicated the structure of galacturonic acid. The next highest concentrations were 1,2,4-GlcpA (3.121%) and 1,4-Galp (2.857%), indicating that the backbone of MCP-3 was an acidic polysaccharide containing 1,4-GalpA (79.745%), 1,2,4-GlcpA (3.121%), and 1,4-Galp (2.857%).

[0099] Table 3 Methylation analysis of MCP-3

[0100]

[0101]

[0102] 4. Nuclear Magnetic Resonance Analysis

[0103] The homogenized polysaccharide was further characterized by NMR. The homogenized polysaccharide was dissolved in 0.5 mL of deuterated water (D2O) and measured using a 600 MHz Bruker NMR spectrometer. 1 H-NMR, 13 C-NMR, DEPT-135, and two-dimensional NMR COSY, HSQC, HMBC, and NOESY were used to obtain complete H and C chemical shift information for the major sugar residues in MCP-3, thereby inferring the linkage order between the individual sugar residues and assigning the chemical shifts of each sugar residue. Calibration: HDO hydrogen δH = δ4.70 ppm, TMS carbon δC = δ0.00 ppm.

[0104] One-dimensional H NMR spectroscopy ( 1 H-NMR) and carbon spectroscopy ( 13C-NMR) can further identify the glycosidic bond configuration of polysaccharide samples. Most of the hydrogen spectrum signals of polysaccharides are in the range of δ3.0 to 5.5 ppm, and usually the range of δ4.5 to 5.5 ppm is the resonance region of the anomeric proton (H-1). 1 The characteristic signals of H-NMR can identify certain sugar residues or groups. For example, the methyl proton signal of 6-deoxy sugar appears in the high field area of ​​δ0.8~1.4ppm; the methyl proton signal of acetyl group (C H3COO- or -OAc) appears in the low field area of ​​δ1.9~2.2ppm; the methyl proton signal of methyl ester (-COOCH3 or -COOMe) is located at δ3.0~3.8ppm. The chemical shift of the anomeric carbon (C-1) signal of polysaccharide is generally between δ90~110ppm, among which the α-configuration anomeric carbon signal usually appears between δ95~103ppm, while the β-configuration anomeric carbon signal usually appears above δ101ppm. The signal concentration area of ​​C-2~C-5 is in the δ65~85ppm region. The chemical shift of the carbon at the substitution position will show glycosidation shift and will move to the low field. The unsubstituted C-6 signal is near δ60ppm, while the substituted C-6 signal moves to the low field to near δ69ppm. According to 13 The characteristic signals of C-NMR can identify certain sugar residues or groups. For example, the methyl carbon signal of 6-deoxy sugar appears in the high field area of ​​δ15-20 ppm; the carbonyl signal of uronic acid (-COOH) and its methyl ester or acetyl group appears in the low field area of ​​δ170-180 ppm; the methyl carbon signal of methyl ester is located in the δ50-60 ppm region; and the methyl carbon signal of acetyl group appears in the higher field area of ​​δ20-25 ppm.

[0105] like Figure 5 A. Figure 5 As shown in B, MCP-3 1 On the H-NMR spectrum, a large number of proton resonance signals are concentrated in the δ3.0-5.5ppm region, with serious signal overlap. In the anomeric region, δ4.5-5.5ppm, multiple anomeric hydrogen signals are found, indicating that there are multiple sugar residues, and there is some overlap, making it difficult to distinguish. Other hydrogen signals are concentrated in the δ4.5-3.0ppm region, with serious signal overlap, making it difficult to attribute. Figure 5 As shown in C, MCP-3 13 C-NMR spectrum and 1 Compared with the H-NMR spectrum, there are fewer spectral lines, and there are multiple anomeric carbons in the δ90-110ppm anomeric carbon region, indicating that there are multiple sugar residues in the polysaccharide sample. Figure 5 As shown in Figure 4, the DEPT-135 spectrum of MCP-3 shows an obvious inverted peak signal in the δ60-70 ppm region, indicating that the sugar residue contains a -CH2- group.

[0106] from1 H-NMR and 13 On the C-NMR spectrum, some characteristic signal peaks were found:

[0107] (1) The resonance signal in the high-field region of δ1.10 to 1.30 ppm is the typical chemical shift of the methyl proton H-6 of the rhamnose (Rha) residue. The special deoxygenated structure of the methyl carbon C-6 of rhamnose is at δ16.81 ppm. Figure 5 As shown in E, a cross peak at δ1.18 / 16.81 ppm was found in the HSQC spectrum. The peak signal intensity in the hydrogen spectrum and carbon spectrum was weak, indicating the presence of a small amount of rhamnose residues, which is consistent with the monosaccharide composition results.

[0108] (2) 1 The H-NMR spectrum shows the methyl proton signal of the O-acetyl group in the region of δ1.90-2.20 ppm. There are multiple signal peaks, indicating that the acetyl substitution occurs at different positions of the sugar residue in the sugar chain. The carbon spectrum shows the methyl signal of the acetyl group near δ20.00 ppm, which is not obvious. The peak signal intensity is very small. Based on this, it can be inferred that this polysaccharide sample has a low degree of acetylation.

[0109] (3) The monosaccharide composition of the polysaccharide sample contains a large amount of galacturonic acid (78.893%). The characteristic absorption peak at δ52.83 ppm represents the methyl carbon connected to the C-6 position of the methylated galacturonic acid residue. Figure 5 E) to find the cross peak of these two signals δ3.72 / 52.83ppm, δ3.72ppm is the methyl proton signal of methyl ester (-COOMe), and the methyl proton signal of methyl ester δ3.75ppm is in the HMBC spectrum ( Figure 5 G) found a signal cross peak δ3.72 / 170.77 ppm, judging that δ170.77 ppm belongs to the C-6 signal of the esterified galacturonic acid residue, while the characteristic absorption peak at the C-6 position of the unesterified galacturonic acid residue is near δ174.52 ppm. The C-6 signal of the esterified galacturonic acid residue is clearly stronger than the C-6 signal of the unesterified galacturonic acid residue. These characteristic signals indicate the presence of a high degree of methyl-esterified galacturonic acid.

[0110] In the monosaccharide composition test and methylation analysis results, the monosaccharide composition is mainly composed of GalA, Gal, Rha, and A ra. The methylation analysis results mainly contain 1,4-GalpA sugar residues, and contain 1,2,4-Rhap, 1,4-Galp, 1,3,6-Galp, 1,3-Galp, 1,4-Glcp, T-GalpA, T-Galp, T-Araf, 1,2,4-GlcpA and other sugar residues. It is speculated that the polysaccharide sample may contain a large number of HG type domains and a small amount of RG-I type pectin domains. According to the monosaccharide composition and methylation analysis results of the polysaccharide sample, the analysis 1 H-NMR, 13 C-NMR, HS QC and 1 H- 1 The H COSY NMR spectrum revealed the presence of multiple anomeric signals. Combined with literature reports, there was a strong signal near δ98-102 ppm in the anomeric region, which was inferred to be the anomeric carbon signal of the sugar residue α-GalpA. There was a strong signal near δ102-105 ppm in the anomeric region, which was inferred to be the anomeric carbon signal of the sugar residue β-Galp. There was a signal at δ106-110 ppm in the anomeric region, which was inferred to be the anomeric carbon signal of the sugar residue α-Araf. Through monosaccharide composition, methylation analysis, and nuclear magnetic resonance analysis, multiple anomeric region signal peaks in the polysaccharide sample are significant and can be used for structural analysis. The chemical shifts of these anomeric region signals are δ4.88 / 100.39ppm, δ4.95 / 100.26ppm, δ5.03 / 99.59ppm, δ4.55 / 104.26ppm, δ4.37 / 103.30ppm, δ4.51 / 104.36ppm, δ5.16 / 109.13ppm, δ5.00 / 107.29ppm, and δ4.41 / 102.64ppm. After the anomeric signal attribution was determined, COSY, HSQC, HMBC, and NO ESY were used in combination. 1 H-NMR, 13 C-NMR, DEPT-135 spectra, monosaccharide composition and methylation analysis results were compared with the chemical shift data of similar sugar residue substitutions in related literature. The above sugar residues were marked as GE in sequence. 1,4 , GE 1,3,4 , GA 1,4 , G 1,4 , G 1,3,6 , G 1,3 、A t 、A 1,5 、 4Me GlcA, which converts the main types of sugar residues in polysaccharide samples into 1 H and 13The C chemical shift signals were assigned, and the results are shown in Table 4. The NMR analysis of the main sugar residue structures is as follows:

[0111] Sugar residue GE 1,4 :According to HSQC and COSY, the anomeric signal of the sugar residue was δ4.88ppm (H-1) and δ100.39ppm (C-1), indicating that the sugar residue was in α configuration. Based on the results of monosaccharide composition test and methylation analysis, combined with literature reports, it was speculated that the residue was an α-GalpA sugar residue. Using HSQC and COSY, the H-1 chemical shift of the sugar residue was determined to be δ4.87ppm. Then, the COSY spectrum ( Figure 5 F) The cross peaks can be deduced from H-2, H-3, and H-4 signals. The chemical shifts of H-2, H-3, and H-4 of the sugar residue are assigned to δ3.63ppm, δ3.92ppm, and δ4.38ppm, respectively. After assigning the chemical shifts of hydrogen on the sugar ring, the HSQC correlation spectrum ( Figure 5 E) The chemical shifts of C-1 to C-4 on the sugar ring were assigned to be δ100.39 ppm, δ67.69 ppm, δ67.84 ppm, and δ78.95 ppm, respectively. The cross peaks of H-5 and C-5 were found in the HSQC spectrum at δ5.01 to 5.09 / 70.48 ppm. The cross peak of the signal was found in the HSQC spectrum at δ3.72 / 52.83 ppm. δ3.72 ppm was the methyl proton signal of methyl ester (-COOMe). The methyl proton signal and carbon signal of methyl ester were δ3.72 ppm and δ52.83 ppm, respectively. Figure 5 G) found the signal cross peak δ3.72 / 170.77ppm, it can be judged that δ170.77ppm belongs to the C-6 position signal of the esterified galacturonic acid residue, and the chemical shift of C-6 is δ170.77ppm, indicating that the sugar residue is methylated. The chemical shifts of C-1 and C-4 shift to the low field, indicating that the residue has been substituted at the C-1 and C-4 positions of the sugar ring, and H-5 has a large low field shift. This is because the introduction of the methoxy group causes the chemical shift of each proton of the sugar ring to shift. Combining the methylation results and literature reports, it is inferred that the sugar residue is →4)-α-GalpA-6-OMе-(1→, marked as GE 1,4 The chemical shift assignments are shown in Table 4.

[0112] Sugar residue G 1,4 :According to HSQC and 1 H- 1 H COSY determined that the anomeric signal of the sugar residue was δ4.55 ppm (H-1) and δ104.26 ppm (C-1), indicating that the sugar residue was in β configuration. Figure 5E) The H-1 chemical shift of this sugar residue was determined to be δ4.55 ppm by COSY( Figure 5 F) The H-2, H-3, H-4, and H-5 signals were deduced from the cross-peaks of the spectrum. The chemical shifts of H-2, H-3, H-4, and H-5 of this sugar residue were assigned to δ3.60ppm, δ3.69ppm, δ4.09ppm, and δ3.64ppm, respectively. The assignment of its H-6a and H-6b signals can be determined by HSQC correlation spectrum ( Figure 5 E) were assigned to δ3.63ppm and δ3.74ppm respectively. After assigning the chemical shift of hydrogen on the sugar ring, the HSQC correlation spectrum ( Figure 5 E) The chemical shifts assigned to the carbon atoms on the sugar ring are δ104.26ppm, δ71.65ppm, δ73.12ppm, δ77.48ppm, δ74.43ppm, and δ60.69ppm, respectively. The chemical shifts of C-1 and C-4 shifted downfield, indicating that the residue was substituted at the C-1 and C-4 positions of the sugar ring. Combined with the results of monosaccharide composition and methylation analysis, and compared with literature reports, it is inferred that the sugar residue is →4)-β-D-Galp-(1→, labeled G 1,4 The chemical shift assignments are shown in Table 4.

[0113] Following a similar approach, the hydrogen and carbon signals of other residues were deduced by combining monosaccharide composition determination, methylation analysis results and literature reports to infer GE 1,3,4 →3,4)-α-D-GalpA-6-OMe-(1→, inferred GA 1,4 →4)-α-D-GalpA-(1→, inferred G 1,3,6 →3,6)-β-D-Galp-(1→, inferred G 1,3 →3)-β-D-Galp-(1→, inferred G t is β-D-Galp-(1→, inferred A t is α-L-Araf-(1→, inferred A 1,5 →5)-α-L-Araf-(1→, inferred 4Me GlcA is 4-O-Me-β-D-GlcpA-(1→, methylation analysis results show that the polysaccharide sample also contains 1,2,4-Rhap, and it is speculated that the rhamnose signal on the hydrogen and carbon spectra belongs to the sugar residue →2,4)-α-L-Rhap-(1→, marked as Rha 1,2,4 The H and C chemical shift assignments of the main sugar residues in the polysaccharide samples are summarized in Table 4.

[0114] According to the results of methylation analysis, the polysaccharide samples also contain t-GalpA, 1,4-Glcp, T-Rhap and other connection modes, the signals of which are very weak in NMR and cannot be detected.1 H and 13 C is attributed.

[0115] Through the coupling signals of the anomeric hydrogen and the carbon on each sugar residue, or the coupling signals of the anomeric carbon and the hydrogen on each sugar residue on the HMBC long-range correlation spectrum, and the two protons on the connection sites of adjacent sugar residues are likely to produce strong NOE signals due to their close spatial positions, the mutual connection order between each sugar residue can be further inferred by using the HMBC long-range correlation spectrum and NOESY spectrum. HMBC correlation spectrum of polysaccharide sample ( Figure 5 G) and NOESY spectra ( Figure 5 H), the following coupling signals can be found from the figure:

[0116] (1) GE of sugar residues in HMBC profiles 1,4 H-1 (δ4.88ppm) and sugar residue GE 1,4 There is a related signal peak (GE 1,4 H-1 / GE 1,4 C-4), GE 1,4 H-4 (δ4.38ppm) and sugar residue GE 1,4 There is a related signal peak (GE 1,4 H-4 / GE 1,4 C-1), GE of sugar residues in N OESY spectrum 1,4 H-1 (δ4.88ppm) and sugar residue GE 1,4 There is a cross peak (GE 1,4 H-1 / GE 1,4 H-4), indicating the presence of →4)-α-D-GalpA-6-OMe-(1→4)-α-D-GalpA-6-OMe-(1→ linkage;

[0117] (2) Sugar residue G in HMBC map 1,4 H-1 (δ4.55ppm) and sugar residue G 1,4 There is a related signal peak (G 1,4 H-1 / G 1,4 C-4), sugar residue G in NOESY spectrum 1,4 H-1 (δ4.55ppm) and sugar residue G 1,4 There is a cross peak (G 1,4 H-1 / G 1,4 H-4), indicating the presence of →4)-β-D-Galp-(1→4)-β-D-Galp-(1→ linkage;

[0118] (3) Sugar residue GA in NOESY spectrum 1,4 H-1 (δ5.03ppm) and sugar residue GE 1,4 There is a cross peak (GA 1,4 H-1 / GE 1,4 H-4), indicating the presence of →4)-α-D-GalpA-6-(1→4)-α-D-GalpA-6-OMe-(1→ linkage;

[0119] (4) H-1 of sugar residue At (δ5.16 ppm) and sugar residue GE in NOESY spectrum 1,3,4 There is a cross peak (At H-1 / GE 1,3,4 H-4), indicating the presence of α-L-Araf-(1→3,4)-α-D-GalpA-6-OMe-(1→ linkage, with the linkage site located at the O-3 position;

[0120] (5) Sugar residue G in NOESY spectrum 1,3,6 H-1 (δ4.37ppm) and sugar residue G 1,3 There is a cross peak (G 1,3,6 H-1 / G 1,3 H-3), indicating the existence of a →3,6)-β-D-Galp-(1→3)-β-D-Galp-(1→ linker, with the linker site located at the O-3 position.

[0121] Comprehensive analysis of the monosaccharide composition, methylation results, and one-dimensional and two-dimensional nuclear magnetic resonance information of the polysaccharide sample showed that the polysaccharide sample was a complex polysaccharide. It can be inferred to a large extent that it is mainly composed of a large number of HG domains and a small amount of pectin with RG-I domains with side chains. Its possible structural elements are shown in the following structural formula.

[0122] (1) Main Chain

[0123]

[0124] (2) Branched chain

[0125]

[0126] Table 4 Sugar residues in MCP-3 1 H and 13 Chemical shift assignment of C

[0127]

[0128]

[0129] Note: “--” in the table means undetermined or not detected, and “ / ” means not present.

[0130] Example 5 Study on the in vitro hypoglycemic activity of homogenized bitter melon polysaccharide

[0131] 1. Effect of insulin on HepG2 cell activity

[0132] The MTT method was used to evaluate the effect of insulin on the activity of HepG2 cells. Hep G2 cells in the logarithmic growth phase were taken, the cells were counted, and the cells were seeded in a 96-well plate at a density of 8,000 cells per well, with a volume of 100 μL per well. The cells were placed in a 37°C, 5% CO2 constant temperature incubator for 12-16 hours (to ensure cell adhesion), and the complete culture medium containing different concentrations of insulin solution was replaced, namely 0, 2, 4, 6, 8, 10, and 12 μmol / L, with 6 parallels for each concentration. After incubation for 24 hours, 20 μL of 5 mg / mL MTT solution (filtered through a 0.22 μm sterile filter membrane and prepared with PBS) was added to each well, and after incubation for another 4 hours, the supernatant was discarded. Finally, 150 μL of DMSO was added to each well to dissolve the purple crystals, shaken for 15 minutes, and the absorbance at 570 nm was measured using an enzyme reader. The cell survival rate was calculated according to the following formula:

[0133]

[0134] Wherein: A1—experimental group (addition of 100 μL of cell suspension with different concentrations of insulin); A2—blank group (addition of 100 μL of complete culture medium without cells); A3—control group (addition of 100 μL of cell suspension without insulin).

[0135] like Figure 6 As shown, compared with the blank group, insulin at a concentration of 2-8 μmol / L had no significant difference on the normal growth and proliferation of HepG2 cells, indicating that insulin was non-toxic to HepG2 cells. In addition, when insulin was at a concentration of 10 and 12 μmol / L, the survival rate of HepG2 cells increased significantly (P < 0.05), and the corresponding cell survival rates were 1.08 and 1.13 times that of the control group, respectively, indicating that insulin can promote the proliferation of HepG2 cells in a dose-dependent manner. These results all indicate that when the concentration is within 0-12 μmol / L, insulin is not toxic to HepG2 cells, and can ensure a safe concentration for subsequent experiments.

[0136] 2. Establishment of the HepG2 cell insulin resistance model (IR-HepG2)

[0137] HepG2 cells in the logarithmic growth phase were taken, the cells were counted, and the cells were seeded in a 48-well plate at a density of 35,000 cells per well, with a volume of 300 μL per well. The cells were placed in a 37°C, 5% CO2 constant temperature incubator and cultured for 12-16 hours (to ensure cell adhesion). The complete medium was discarded and replaced with serum-free medium, and the HepG2 cells were starved for 8 hours. The serum-free medium was discarded and replaced with complete medium containing different concentrations of insulin solution, respectively 0, 1, 2, 4, 6, and 8 μmol / L, with 6 parallels set for each concentration, and the cells were incubated for another 24 hours. Finally, the glucose concentration in the supernatant under different insulin concentrations was detected using a glucose detection kit. The success of the IR-HepG2 establishment was determined based on the glucose consumption, and the insulin concentration of the constructed IR-HepG2 was further confirmed.

[0138] like Figure 7 As shown in the figure, when the insulin concentration was 1-2 μmol / L, glucose consumption showed an upward trend. However, when the insulin concentration was 4-8 μmol / L, glucose consumption continued to decline. And when the insulin concentration was 8 μmol / L, compared with the blank group, insulin consumption decreased significantly by 8.6% (P < 0.05), indicating that in this state, HepG2 cells had the lowest efficiency in utilizing glucose and the most obvious insulin resistance. And according to the toxicity experiment of insulin on HepG2 cells, insulin at a concentration of 8 μmol / L was not toxic to HepG2 cells. Therefore, subsequent experiments used 8 μmol / L as the insulin concentration for constructing the IR-HepG2 model.

[0139] 3. Effects of MCP-3 on glucose consumption and hepatic glycogen content in IR-HepG2 cells

[0140] IR-HepG2 cells were established according to the conditions determined in the previous step, and the effects of bitter melon polysaccharide on glucose consumption and hepatic glycogen content in IR-HepG2 cells were observed by administering the drug while modeling. HepG2 cells in the logarithmic growth phase were counted and seeded in 48-well plates at a density of 35,000 cells per well in a volume of 300 μL per well. The cells were cultured in a 37°C, 5% CO2 incubator for 12-16 hours (to ensure cell adhesion). The complete medium was discarded and replaced with serum-free medium, and the HepG2 cells were starved for 8 hours. The serum-free medium was discarded, and the corresponding medium was added according to the experimental group. A blank group (Control group), a model group (Mo del group), and groups treated with bitter melon polysaccharide at different concentrations (0.1 and 0.5 mg / mL) were set up. The blank group received untreated complete medium, the model group received complete medium containing 8 μmol / L, and the groups treated with bitter melon polysaccharide at different concentrations received complete medium containing different concentrations of MCP-3 and 8 μmol / L. Six replicates were set for each concentration and the cells were incubated for 24 hours. Finally, glucose consumption and glycogen content in He pG2 cells were detected using a glucose detection kit and a glycogen content detection kit.

[0141] The results are as follows Figure 8 As shown in the results, compared with the blank group, the glucose consumption of HepG2 cells in the model group decreased by 23.13%, which was significantly lower than that in the blank group (p < 0.01), indicating that the IR-HepG2 model was successfully established. The effects of different concentrations of MCP-3 (0.1, 0.5 mg / mL) on the glucose consumption of IR-HepG2 cells were evaluated. After 24 hours of treatment of IR-HepG2 cells with MCP-3, the glucose consumption increased to a certain extent compared with the model group. With the increase of polysaccharide concentration, the glucose consumption showed an upward trend. When the polysaccharide concentration was 0.1 mg / mL, compared with the model group, the MCP-3 component at 0.1 and 0.5 mg / mL concentrations had a significant effect on the glucose consumption of IR-HepG2 cells (p < 0.01), respectively, increasing by 25.37%.

[0142] 4. Effect of MCP-3 on glycogen synthesis in IR-HepG2 cells and glycogen PAS staining

[0143] (1) This experiment used complete culture medium containing 8 μmol / L insulin to induce insulin resistance in HepG2 cells. Figure 9As shown in Figure A, compared with the blank group, the glycogen content in the model group was significantly reduced (p < 0.01), indicating that glycogen synthesis in HepG2 cells was impaired and the insulin resistance model was successfully established. After treatment with different concentrations of MCP-3 (0.1 and 0.5 mg / mL), the glycogen content increased slightly compared with the model group, but was still lower than that of the normal group. When the polysaccharide concentration was 0.1 mg / mL, the glycogen content in the MCP-3 group increased compared with the model group, but it was not significant (p > 0.05), and was 1.16 times that of the model group. When the polysaccharide concentration was 0.5 mg / mL, the glycogen content in the MCP-3 group was significantly increased compared with the model group (p < 0.01), and was 1.41 times that of the model group. These results demonstrate that the bitter melon polysaccharide MCP-3 can improve insulin resistance and promote glycogen synthesis.

[0144] (2) Culture cells according to the grouping and administration method in step 3 above. After incubation, stain IR-HepG2 cells according to the instructions of the Solebo Glycogen PAS Staining Kit. Fix the cells with PAS fixative, wash with water and dry. Add oxidant, oxidize at room temperature for 20 minutes, rinse with tap water twice, and soak with distilled water twice. Add Schiff Reagent and cover, place in a dark place at room temperature for immersion for 10 to 20 minutes, rinse with running water for 5 minutes (mainly for observation under a microscope). Add Mayer's hematoxylin staining solution, counterstain for 1 to 2 minutes, wash with water, dry, and examine under an optical microscope.

[0145] like Figure 9 B. Glycogen content is visually displayed by the depth of purple-red color. According to the results of the picture, it can be seen that the color of the MCP-3 experimental group at different concentrations is significantly darker than that of the model group, which is consistent with the experimental results of glycogen content determination. The grayscale quantitative analysis of the picture was performed using ImageJ image software. Figure 9 C, The results were consistent with the glycogen staining images, which further proved that MCP-3 could promote the increase of glycogen content in IR-HepG2 cells and improve insulin resistance.

[0146] 5. Effects of MCP-3 on hexokinase (HK) and pyruvate kinase (PK) in IR-HepG2 cells

[0147] Culture cells according to the grouping and dosing schedule described in step 3 above. After incubation, treat the cells with cell lysis buffer and ultrasonically disrupt them in an ice-water bath. Centrifuge and collect the supernatant. Assay the hexokinase and pyruvate kinase activities in IR-HepG2 cells according to the Solebro kit instructions.

[0148] like Figure 10 A and Figure 10B. Compared with the blank group, the HK and PK activities in the model group were significantly decreased (P < 0.01), indicating that the cellular insulin resistance model was successfully established. After treatment with different concentrations of MCP-3 (0.1 and 0.5 mg / mL), both HK and PK activities increased to a certain extent compared with the model group. At a polysaccharide concentration of 0.1 mg / mL, both HK and PK activities in the MCP-3 group were significantly increased compared with the model group (P < 0.01), with HK and PK activities in the MCP-3 group being 1.41-fold and 1.58-fold higher, respectively. At a polysaccharide concentration of 0.5 mg / mL, both HK and PK activities in the MCP-3 group were also significantly increased compared with the model group (P < 0.01), with HK and PK activities in the MCP-3 group being 1.71-fold and 2.11-fold higher, respectively. These results indicate that bitter melon polysaccharide MCP-3 can increase the activity of HK and PK in IR-HepG2 cells, promote the body's absorption and utilization of glucose and glycogen synthesis, improve glucose metabolism, and improve insulin resistance.

[0149] Example 6 Study on the hypoglycemic effect of Momordica charantia polysaccharide on diabetic mice

[0150] 1. Modeling, Animal Husbandry and Grouping

[0151] Forty male C57BL / 6J mice were selected and housed in a sterile environment with constant temperature and humidity (25°C, humidity 50±5%) for one week of adaptive feeding. The 40 C57BL / 6J mice were randomly divided into four groups (n=10), namely: normal control group (NC), diabetes model group (T2DM), bitter melon polysaccharide MCP-3 high-dose group (MCP-3-H), and bitter melon polysaccharide MCP-3 low-dose group (MCP-3-L). Except for the normal control group, the other groups were fed a high-fat diet. After 5 weeks, STZ (50 mg / kg) was continuously injected once a day for 3 days. After 3 days, the fasting blood glucose of the mice was measured. The mice with blood glucose greater than 11.1 mmol / L were considered diabetic mice. After the model was successfully established, each mouse was given the drug by gavage every day. The MCP-3-H group was gavaged with 200 mg / kg MCP-3, the MCP-3-L group was gavaged with 100 mg / kg MCP-3, and the NC group and T2D M group were gavaged with the same amount of distilled water every day for 5 consecutive weeks.

[0152] 2. Record weight, diet, and water intake

[0153] During the entire experimental period, the body weight of the mice was measured and recorded weekly, and their food and water intake were measured.

[0154] (1) Effect of MCP-3 on body weight of T2DM mice: Figure 11As shown in A, 5 weeks after oral administration, the model group (T2DM) mice were thinner and smaller and had rough hair compared to the normal group (NC) mice, which was consistent with the symptoms of diabetic mice and was consistent with the weight results of the mice measured below. In addition, compared with the T2DM group, the body size of the mice in the polysaccharide intervention group increased and the rough hair was also improved to a certain extent, which shows that bitter melon polysaccharides have a significant improvement effect on the body surface characteristics of diabetic mice. Figure 11 As shown in Figure B, before polysaccharide intervention, the body weight of C57BL / 6J mice did not differ significantly between the polysaccharide intervention group and the T2DM group (p>0.05), but was significantly higher than that of the NC group (p<0.05). After successful model establishment, the body weight of the T2DM group mice decreased significantly (p<0.05), from 27±0.56g to 19.57±1.06g, consistent with the symptoms of diabetic mice. In contrast, the body weight of the NC group mice increased slowly and steadily, from 24.4±0.35g to 27.83±1.10g. After oral administration of high and low doses of bitter melon polysaccharide for 5 weeks, the weight loss trend of the mice slowed down within 1 to 3 weeks, and the weight of the mice began to gradually increase from 4 to 5 weeks. After 5 weeks of gavage, the body weights of the MCP-3-H and MCP-3-L groups were 25.33±0.55g and 24.5±1.57g, respectively, which were significantly higher than those of the T2DM group (p<0.05), but still lower than those of the NC group. This indicates that bitter melon polysaccharides have a significant effect on improving the body weight of diabetic mice.

[0155] (2) The effect of MCP-3 on the diet of T2DM mice: Figure 12 As shown in the results, before modeling, the diet of mice in the T2DM group was slightly higher than that of the NC group, but there was no significant difference (P>0.05). It is speculated that the high-fat diet consumed by mice in the T2DM group will increase the amount of food consumed by the mice to a certain extent. After successful modeling, the food intake of mice in the T2DM group gradually increased and was significantly higher than that of the NC group after 5 weeks of gavage (P<0.05), which is consistent with the characteristic of polyphagia in diabetic mice. After 5 weeks of polysaccharide intervention, the food intake of mice in the MCP-3-L and MCP-3-H groups was lower than that of the T2DM group, and the change trend was consistent with the weight change trend of mice mentioned above, which further proved that MCP-3 can improve the food intake of T2DM mice.

[0156] (3) The effect of MCP-3 on water intake in T2DM mice: Figure 13As shown in the results, after 5 weeks of oral gavage, the water intake of mice in the T2DM group was significantly higher than that in the NC group (P < 0.05), approximately 3 to 4 times that of the NC group, indicating that the model was successfully established. Due to polysaccharide intervention, the water intake of mice in the MCP-3-L and MCP-3-H groups began to gradually decrease after 3 weeks of oral gavage, and after 5 weeks of oral gavage, the water intake of mice in the polysaccharide intervention group was significantly different from that of mice in the T2DM group (P < 0.05). The trend of water intake changes in the polysaccharide intervention group was consistent with the trends of weight and diet changes in the mice mentioned above, indicating that bitter melon polysaccharides can improve the symptoms of polydipsia in diabetic mice.

[0157] 3. Effects of MCP-3 on blood glucose and glucose tolerance in T2DM mice

[0158] After successful establishment of the diabetic mouse model, fasting blood glucose levels were measured weekly for 1 to 5 weeks following oral gavage. Five weeks after gavage, the mice underwent an oral glucose tolerance test (OG TT). All mice were fasted for 12 hours, but not water, and then gavaged with a glucose solution (2g / kg). Blood glucose levels were measured at 0, 30, 60, 90, and 120 minutes.

[0159] The effect of MCP-3 on glucose tolerance in T2DM mice was studied by measuring fasting blood glucose and oral glucose tolerance test (OGTT). Figure 14 As shown in A, the blood glucose concentration of the normal healthy group (NC) mice was stable at 4-5 mmol / L, while the blood glucose concentration of the diabetic model group (T2DM) mice after modeling was higher than 17 mmol / L, which was significantly different from that of the NC group (P < 0.01). In addition, the blood glucose level continued to rise, and the blood glucose level of the mice slowly increased from 17.73 ± 0.87 mmol / L to 25.20 ± 1.44 mmol / L, indicating that the T2DM model was successfully established and had good stability. T2DM mice were fed with MCP-3 for 5 weeks. Compared with the T2D M group, the blood glucose levels of mice in the MCP-3-L and MCP-3-H groups showed a significant downward trend. The blood glucose levels of mice in the MCP-3-L group decreased from 17.83±0.81mmol / L to 12.93±0.84mmol / L, and the blood glucose levels of mice in the MCP-3-H group decreased from 18.13±0.32mmol / L to 10.1±0.60mmol / L. This indicates that MCP-3 can improve the blood glucose levels of T2DM mice, but the blood glucose levels did not drop to the level of normal mice. It is speculated that the reason is that the MCP-3 intervention time of 5 weeks was too short.

[0160] After 5 weeks of feeding, OGTT test was performed. Figure 14As shown in B, after the mice were gavaged with glucose, the blood glucose concentrations of all mice showed the same trend of change. Within 0 to 30 minutes, the blood glucose concentrations of the mice increased to varying degrees and reached their respective maximum values; after 30 minutes, the blood glucose concentrations of the mice slowly decreased. Compared with the NC group, the T2DM group had higher blood glucose levels at each time point, which is consistent with the characteristics of poor glucose tolerance in diabetic mice. In addition, the blood glucose concentrations of the MCP-3-L and MCP-3-H intervention groups were lower than those of the T2DM group at each time point, and the blood glucose recovery rate was slightly higher than that of the T2DM group. This shows that MCP-3 has a good effect on improving the glucose tolerance of diabetic mice. This result was verified by further analysis of the area under the curve (AUC) of the OGTT. As shown in Figure 14 As shown in C, the area under the curve (AUC) of OGTT in the NC group was significantly lower than that in the T2DM group (P < 0.01), indicating that the glucose tolerance of diabetic mice was impaired; the AUC of OGTT in the MCP-3-L and MCP-3-H groups were significantly lower than that in the T2DM group (P < 0.01), and the AUC in the MCP-3-L group was higher than that in the MCP-3-H group, indicating that MCP-3 can improve T2DM by lowering blood glucose levels and repairing glucose tolerance, and has a certain dose-effect relationship.

[0161] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. An application of homogenized bitter melon polysaccharide in the preparation of a blood sugar-lowering product, characterized in that: The homogenized bitter melon polysaccharide contains rhamnose, glucuronic acid, galacturonic acid, glucose, galactose and arabinose, and the corresponding molar ratios are 0.032:0.011:0.774:0.003:0.153:0.028; the molecular weight of the homogenized bitter melon polysaccharide is 93.796 kDa; The preparation method of the homogenized bitter melon polysaccharide comprises the following steps: (1) Mix bitter melon with water, heat and sonicate, centrifuge, and collect the supernatant; (2) concentrating the supernatant, mixing the concentrate with ethanol for alcohol precipitation, and collecting the precipitate by centrifugation; (3) Mix the precipitate with water to obtain a polysaccharide solution, add Sevage reagent, centrifuge, and collect the supernatant; The supernatant was dialyzed and dried to obtain crude bitter melon polysaccharide; (4) separating the crude bitter melon polysaccharide by anion exchange column chromatography, collecting the polysaccharide-containing eluate, and dialyzing to obtain ion-purified bitter melon polysaccharide; (5) purifying the ion-purified bitter melon polysaccharide by gel column chromatography, collecting the polysaccharide-containing eluate, and dialyzing to obtain homogenized bitter melon polysaccharide; In step (4), the anion exchange column is DEAE Sepharose Fast Flow, the sample concentration is 20-40 mg / mL; the elution method is water, 0.1 mol / L NaCl and 0.2 mol / L NaCl gradient elution in sequence, each elution time is 150-250 min, and the eluate obtained by 0.2 mol / L NaCl elution is collected; the elution flow rate is 1-4 mL / min; In step (5), the gel column is Sephadex G-100, the sample concentration is 3 to 13 mg / mL; the elution method is water elution, the elution time is 100 to 200 minutes, and the eluate is collected; the elution flow rate is 0.3 to 2 mL / min; The dialysis bag specification for dialysis is 2500-4500Da; The bitter melon is white bitter melon.

2. The use according to claim 1, characterized in that In step (1), the material-liquid ratio of the bitter melon to water is 1:1-5; the heating temperature is 80-100° C., the heating time is 1-5 hours; and the ultrasonic time is 5-15 minutes; In step (2), the supernatant is concentrated to 1 / 5 to 1 / 15 of the original volume; the volume ratio of the concentrate to ethanol is 1:1 to 8, and the concentration of ethanol is 85 to 100%; In step (3), the volume ratio of the polysaccharide solution to the Sevage reagent is 1 to 5:

1.

3. The use according to claim 1, wherein The centrifugal conditions include: 7000-9000 rpm, 5-15 min, 1-7°C.

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