Homogenized pumpkin polysaccharide and its preparation method and application

Abstract

The present invention provides a homogenized pumpkin polysaccharide and its preparation method and application, belonging to the technical field of polysaccharide extraction and purification. The homogenized pumpkin polysaccharide of the present invention contains mannose, glucosamine hydrochloride, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose and arabinose, with the corresponding molar ratios being 0.005:0.004:0.041:0.013:0.545:0.004:0.241:0.147; the relative molecular weight of the homogenized pumpkin polysaccharide is 5.96×10 4 The homogenized pumpkin polysaccharide has significant lipid-lowering and blood sugar-lowering effects. 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 pumpkin polysaccharide and a preparation method and application thereof. Background Art

[0002] Obesity is the common pathological basis of many chronic metabolic diseases. A large number of studies have shown that obesity can induce a variety of diseases such as diabetes, coronary heart disease, atherosclerosis, hypertension and even tumors. Abnormal lipid metabolism in the body is one of the prominent characteristics of obesity. Lipid metabolism is a complex biochemical reaction in the body, including the biosynthesis and degradation of lipids such as fatty acids, triglycerides and cholesterol. A common clinical symptom of lipid metabolism disorder is elevated blood lipids. Excessive blood lipids increase the risk of developing a variety of obesity-related diseases such as atherosclerosis, cirrhosis, cardiovascular and cerebrovascular diseases. At present, most clinical treatments for lipid metabolism diseases use symptomatic treatments to relieve symptoms, and drug treatments are prone to toxic side effects, causing a lot of physical pain to patients. Therefore, it is particularly important to find effective substances with less toxic side effects from natural products to treat lipid metabolism diseases.

[0003] Pumpkin (Cucurbita moschata Duch), also known as pumpkin, rice melon, squash, and golden melon, belongs to the Cucurbitaceae family and is an annual vine. It is a traditional Chinese medicinal and edible plant. It is rich in nutrients, containing starch, protein, carotene, vitamin B, vitamin C, and calcium. Currently, research on pumpkin polysaccharides, both domestically and internationally, has focused on their extraction and biological activity, with limited research on the higher-order structure and lipid-lowering and blood sugar-lowering activities of purified polysaccharides. Summary of the Invention

[0004] The invention provides a homogenized pumpkin polysaccharide, a preparation method and an application thereof. The homogenized pumpkin polysaccharide has obvious lipid-lowering and blood sugar-lowering effects.

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

[0006] The present invention provides a homogenized pumpkin polysaccharide, which contains mannose, glucosamine hydrochloride, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose and arabinose in a corresponding molar ratio of 0.005:0.004:0.041:0.013:0.545:0.004:0.241:0.147; the relative molecular weight of the homogenized pumpkin polysaccharide is 5.96×10 4 Da.

[0007] The present invention provides a preparation method of the homogenized pumpkin polysaccharide, comprising the following steps: (1) mixing pumpkin powder 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 pumpkin polysaccharide; (4) subjecting the crude pumpkin polysaccharide to anion exchange column chromatography, collecting the polysaccharide-containing eluate, and dialyzing to obtain ion-purified pumpkin polysaccharide; and (5) subjecting the ion-purified pumpkin polysaccharide to gel column chromatography purification, collecting the polysaccharide-containing eluate, and dialyzing to obtain homogenized pumpkin polysaccharide.

[0008] Preferably, in step (1), the material-liquid ratio of the pumpkin to water is 1:30-50; the heating temperature is 75-95°C, and the heating time is 1-5 hours; the ultrasonic time is 5-25 minutes; in step (2), the temperature for concentrating the supernatant is 45-65°C, and the concentration time is 35-55 minutes; 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 FastFlow, and the loading concentration is 10-30 mg / mL; the elution method is to perform gradient elution with water, 0.1 mol / L NaCl and 0.2 mol / L NaCl in sequence, with the elution time of each solution being 50-150 min, and the eluate obtained by elution with 0.2 mol / L NaCl is collected; the flow rate of the elution is 1-4 mL / min.

[0010] Preferably, in step (5), the gel column is Sephadex G-100, the loading concentration is 10 to 30 mg / mL; the elution method is water elution, the elution time is 100 to 300 min, and the eluate is collected; the elution flow rate is 0.2 to 1.5 mL / min.

[0011] Preferably, the centrifugal conditions include: 7000-9000 rpm, 5-25 min, 1-6°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 pumpkin polysaccharide or the homogenized pumpkin polysaccharide obtained by the preparation method in preparing lipid-lowering and blood sugar-lowering products.

[0014] The present invention provides a lipid-lowering and blood sugar-lowering pharmaceutical composition, comprising the homogenized pumpkin polysaccharide or the homogenized pumpkin 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] This study uses pumpkin as a raw material and optimizes the ultrasonic-assisted water-alcohol precipitation extraction process for pumpkin polysaccharide. This process then undergoes separation, purification, and structural characterization, resulting in the first single-component pumpkin polysaccharide. This homogenized pumpkin polysaccharide exhibits a novel structure, a uniform chemical structure, and a consistent composition. It demonstrates significant lipid- and blood-sugar-lowering effects, while being non-toxic and non-toxic to healthy individuals.

[0018] The present invention establishes a hepatocyte lipid accumulation and insulin resistance model to study the lipid-lowering activity of homogenized pumpkin polysaccharide, and further verifies the lipid-lowering activity of pumpkin polysaccharide by inducing an obesity model in C57BL / 6 male mice through a high-fat diet, providing theoretical and technical support for the development of pumpkin polysaccharide drugs for metabolic diseases such as obesity, hyperlipidemia, and intestinal health.

[0019] The preparation process of the homogenized pumpkin polysaccharide 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

[0020] Figure 1 Elution purification curves of pumpkin polysaccharide using DEAE Fast Flow anion exchange chromatography column (a), Sephadex G-100 gel column (b) and (c).

[0021] Figure 2 High performance gel liquid chromatography (HPLC) of PPS3.

[0022] Figure 3 High performance gel chromatograms of monosaccharide standards (a) and PPS3 (b).

[0023] Figure 4 High performance gel chromatography of PPS3.

[0024] Figure 5 PPS3 1 HNMR, 1 D NOESY, 13 CNMR, DEPT-135 spectra.

[0025] Figure 6HSQC, COSY, HMBC, and NOESY spectra of PPS3.

[0026] Figure 7 Toxicity of PA to HepG2 cells.

[0027] Figure 8 Toxicity of PPS3 to HepG2 cells.

[0028] Figure 9 Effects of PPS3 on lipid deposition in HepG2 cells incubated with PA (a: staining image, b: statistical results).

[0029] Figure 10 Effects of PPS3 on TG and TC contents in HepG2 cells incubated with PA.

[0030] Figure 11 The results of the effect of PPS3 on the LDL-C and HDL-C levels in HepG2 cells incubated with PA.

[0031] Figure 12 Effects of PPS3 on body shape (a) and body weight (b) in mice fed a high-fat diet.

[0032] Figure 13 Effects of PPS3 on oral glucose tolerance in mice fed a high-fat diet. DETAILED DESCRIPTION

[0033] The present invention provides a homogenized pumpkin polysaccharide, which contains mannose, glucosamine hydrochloride, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose and arabinose in a corresponding molar ratio of 0.005:0.004:0.041:0.013:0.545:0.004:0.241:0.147; the relative molecular weight of the homogenized pumpkin polysaccharide is 5.96×10 4 Da. The sugar residue structure of the homogenized pumpkin polysaccharide of the present invention is shown in the following structural formula.

[0034] (1) Main Chain

[0035]

[0036] (2) Branched chain

[0037]

[0038] In the present invention, the pumpkin variety is Jinli pumpkin, purchased from the Xianlin Farmers' Market in Qixia District, Nanjing. A single component polysaccharide, named PPS3, is isolated and purified from the pumpkin as a raw material. The pumpkin powder of the present invention comprises the following steps: washing the pumpkin after removing the skin and inner lining, cutting it into slices, freeze-drying it (at -40 to -60°C for 40 to 55 hours), crushing it using a blender (passing through a 30 to 50 mesh sieve), sealing it in a ziplock bag, and storing it in a dry environment for later use.

[0039] The present invention also provides a preparation method of the homogenized pumpkin polysaccharide, comprising the following steps: (1) mixing pumpkin powder 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 pumpkin polysaccharide; (4) subjecting the crude pumpkin polysaccharide to anion exchange column chromatography, collecting the polysaccharide-containing eluate, and dialyzing to obtain ion-purified pumpkin polysaccharide; and (5) subjecting the ion-purified pumpkin polysaccharide to gel column chromatography purification and dialyzing to obtain homogenized pumpkin polysaccharide.

[0040] In the present invention, in step (1), the solid-liquid ratio (g / mL) of the pumpkin powder to water is 1:30-50; the heating temperature is 75-95°C, the heating time is 1-5 hours; and the ultrasonic time is 5-25 minutes. Preferably, the solid-liquid ratio of the pumpkin to water is 1:35-45; the heating temperature is 80-90°C, the heating time is 2-4 hours; and the ultrasonic time is 10-20 minutes. The ultrasonic power of the present invention is 200-300W, preferably 220-280W. In step (1), the centrifugal conditions are: 7000-9000rpm, 5-25 minutes, and 1-6°C.

[0041] In the present invention, in step (2), the temperature for concentrating the supernatant is 45-65°C, preferably 50-60°C, and the time for concentrating is 35-55 minutes; the volume ratio of the concentrate to ethanol is 1:1-8, and the ethanol concentration is 85-100% (V:V). In the present invention, the temperature for concentrating the supernatant is preferably 50-60°C, and the time for concentrating is 40-50 minutes; the volume ratio of the concentrate to ethanol is preferably 1:2-6 (V:V), and the ethanol concentration is preferably 90-98% (V:V). In step (2) of the present invention, it is necessary to place the supernatant in a refrigerator at 1-6°C for alcohol precipitation for 8-16 hours. In step (2) of the present invention, the centrifugation conditions are: 7000-9000 rpm, 5-25 minutes, and 1-6°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 20 (g:mL), preferably 1:15. 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 10-30 mg / mL, preferably 15-25 mg / mL; the elution method is to sequentially perform water, 0.1 mol / L NaCl and 0.2 mol / L NaCl gradient elution, the elution time of each solution is 50-150 min, and the eluates obtained by eluting with water and 0.2 mol / L NaCl are collected respectively; the elution flow rate is 1-4 mL / min, preferably 1.5-3 mL / min. The present invention sequentially performs water, 0.1 mol / L NaCl and 0.2 mol / L NaCl gradient elution, and the elution time is preferably 100, 55, 80 and 55 min, respectively. In step (4) of the present invention, the collected eluate is concentrated by rotary evaporation, placed in a dialysis bag (molecular weight 2500-4500Da), and dialyzed with running water for 24-72 hours to remove salt therein. After the dialysis is completed, the eluate is freeze-dried (-50°C, 48 hours) and stored at -20°C for later use.

[0044] In the present invention, in step (5), the gel column is Sephadex G-100, the loading concentration is 10 to 40 mg / mL, preferably 20 mg / mL; the elution method is water elution, the elution time is 100 to 300 min, and the eluate is collected; the elution time is preferably 150 to 250 min, and more preferably 200 min. The flow rate of the elution in the present invention is 0.2 to 1.5 mL / min, preferably 0.3 to 0.8 mL / min. In step (5) of the present invention, the collected eluate is concentrated by rotary evaporation, placed in a dialysis bag (molecular weight 2500 to 4500 Da), and dialyzed with running water for 24 to 72 h to remove the salt therein. After the dialysis is completed, it is freeze-dried (-50°C, 48 h) and stored at -20°C for use.

[0045] The present invention provides the use of the homogenized pumpkin polysaccharide or the homogenized pumpkin polysaccharide obtained by the preparation method in the preparation of lipid-lowering and blood sugar-lowering products. The effective dose of the homogenized pumpkin polysaccharide of the present invention is 50-350 mg / kg, preferably 70-200 mg / kg.

[0046] The present invention provides a lipid-lowering and blood sugar-lowering pharmaceutical composition, comprising the homogenized pumpkin polysaccharide or the homogenized pumpkin polysaccharide obtained by the preparation method and pharmaceutically acceptable excipients.

[0047] 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, binders, colorants, cosolvents and surfactants.

[0048] In the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art.

[0049] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions of the present invention. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0050] Example 1 Preparation of homogenized pumpkin polysaccharide

[0051] 1. Preparation of pumpkin crude polysaccharide by ultrasound-assisted water extraction and alcohol precipitation

[0052] (1) The pumpkin (Golden Chestnut Pumpkin, purchased from Xianlin Farmers' Market, Qixia District, Nanjing) with the skin and inner skin removed was washed, cut into slices, and freeze-dried (-50°C, 48h). The slices were then crushed using a blender (passed through a 40-mesh sieve), sealed in a ziplock bag, and stored in a dry environment for later use.

[0053] (2) Pumpkin powder was added to distilled water at a solid-liquid ratio of 1:40 (g / mL), mixed, extracted at 85°C for 3 h, and then ultrasonically treated at 250 W ultrasonic power for 15 min to obtain the extract.

[0054] (3) The extract was centrifuged (8000 rpm, 4°C, 15 min), the supernatant was collected, and the excess water was removed by rotary evaporation (85 rpm, 55°C, 45 min).

[0055] (4) Then, alcohol precipitation was performed (3 times the volume fraction of 95% ethanol solution, 12 h, 4°C), excess ethanol was poured off and centrifuged (8000 rpm, 4°C, 15 min), and the precipitate was collected.

[0056] (5) The precipitate and distilled water were dissolved in a ratio of 1:15 (g:mL) to obtain a polysaccharide solution. Sevage reagent (n-butanol:chloroform mixed at a ratio of 1:4 (v:v)) was mixed with the polysaccharide solution at a ratio of 1:3 (v:v) and shaken for 20 min, followed by centrifugation (8000 rpm, 4°C, 15 min). The supernatant was aspirated and placed in a dialysis bag (MD44 mm, molecular weight 3500 Da) and dialyzed with running water for 48 h to remove small molecules. The mixture was then freeze-dried (-50°C, 48 h) to obtain crude pumpkin polysaccharide.

[0057] 2. Separation and purification of pumpkin crude polysaccharides

[0058] (1) DEAE Fast Flow anion exchange chromatography column: Rinse the DEAE Sepharose Fast Flow (Cytiva, 17070901) filler repeatedly with distilled water until there is no ethanol smell, then wet pack the column and balance it with distilled water for 24 hours for use. Dissolve the crude polysaccharide prepared in the above step into 20 mg / mL, load 10 mL, and elute with NaCl solutions of 0, 0.1, 0.2, and 0.3 M in sequence at a flow rate of 2 mL / min. The elution times of the above solutions are 100, 55, 80, and 55 min, respectively. Collect the solution with an automatic collector, 10 mL per tube, and track and monitor it using the phenol-sulfuric acid method. The elution curve is obtained with the tube number and absorbance value as the horizontal and vertical coordinates. Figure 1 As shown in (a), three fractions were obtained by elution with NaCl solutions at four concentrations, ranging from 0 to 0.3 mol / L, and were designated PPS1, PPS2, and PPS3. PPS1 is a neutral polysaccharide, and the others are acidic polysaccharides. The eluates from each fraction were combined, rotary evaporated (85 rpm, 55°C, 45 min), and dialyzed against running water (molecular weight 3500 Da) for 48 h to remove salts. The fractions were then freeze-dried (-50°C, 48 h) and stored at -20°C for later use.

[0059] (2) Purification by Sephadex G-100: The yield of PPS2 in the above separation was low. For the sake of subsequent experiments, the components PPS1 and PPS3 with the highest yield were selected for Sephadex G-100 gel column purification. The collected 0M and 0.2M components of DEAE were purified by Sephadex G-100 (Solarbio, S8171) with a sample concentration of 20 mg / mL and a sample volume of 10 mL. Distilled water was used as the eluent, the flow rate was 0.5 mL / min, the elution time of the 0M component was 350 min, and the elution time of the 0.2M component was 200 min. The automatic collector collected 5 mL per tube and the phenol-sulfuric acid method was used for detection. The gradient elution curve was obtained with the number of tubes and absorbance as the horizontal and vertical coordinates, respectively. The elution curve is shown in the figure below. Figure 1(b) and (c), PPS1 and PPS3 were purified again to obtain homogeneous fractions, still designated PPS1 and PPS3, with purities of 95.75±0.66% and 96.27±1.15%, respectively. The eluates were combined, concentrated by rotary evaporation (85 rpm, 55°C, 45 min), dialyzed (molecular weight 2500-4500 Da), and freeze-dried (-50°C, 48 h) to obtain homogenized pumpkin polysaccharide PPS1 or PPS3, which was then stored at -20°C for later use.

[0060] The monosaccharide composition of PPS1 is glucose and galactose, corresponding to a molar ratio of 0.98:0.02, with glucose accounting for the largest proportion. Given its small molecular weight (1333 Da) and its primary composition of glucose, the study determined that PPS1 is an oligosaccharide primarily composed of glucose. Oligosaccharides, as shorter carbohydrate chains, generally have different physical and chemical properties and physiological activities from polysaccharides. Due to their diverse molecular structures, polysaccharides often have various biological effects that regulate physiological health, including antioxidant, anti-inflammatory, and immunomodulatory functions. Given that the focus of this study is polysaccharides and their biological activities, PPS3 was selected as the research focus for subsequent research.

[0061] Example 2

[0062] (1) The pumpkin (Golden Chestnut Pumpkin, purchased from Xianlin Farmers' Market, Qixia District, Nanjing) with the skin and inner skin removed was washed, cut into slices, and freeze-dried (-50°C, 48h). The slices were then crushed using a blender (passed through a 30-mesh sieve), sealed in a ziplock bag, and stored in a dry environment for later use.

[0063] (2) Pumpkin powder was added to distilled water at a solid-liquid ratio of 1:30 (g / mL), mixed, extracted at 75°C for 5 h, and then ultrasonically treated at 250 W ultrasonic power for 10 min to obtain the extract.

[0064] (3) The extract was centrifuged (8000 rpm, 4°C, 15 min), the supernatant was collected, and the excess water was removed by rotary evaporation (85 rpm, 55°C, 45 min).

[0065] (4) Then, alcohol precipitation was performed (2 times the volume fraction of 85% ethanol solution, 12 h, 4°C), excess ethanol was poured off, and centrifugation was performed (8000 rpm, 4°C, 15 min), and the precipitate was collected.

[0066] (5) The precipitate and distilled water were dissolved in a ratio of 1:10 (g:mL) to obtain a polysaccharide solution. Sevage reagent (n-butanol:chloroform mixed at a ratio of 1:4 (v:v)) was mixed with the polysaccharide solution at a ratio of 1:3 (v:v) and shaken for 20 min, followed by centrifugation (8000 rpm, 4°C, 15 min). The supernatant was aspirated and placed in a dialysis bag (MD44 mm, molecular weight 2500 Da) and dialyzed with running water for 48 h to remove small molecules. The mixture was then freeze-dried (-50°C, 48 h) to obtain crude pumpkin polysaccharide.

[0067] (6) DEAE Fast Flow anion exchange chromatography column: The DEAE Sepharose Fast Flow (Cytiva, 17070901) filler was repeatedly rinsed with distilled water until there was no ethanol smell, then wet packed and equilibrated with distilled water for 24 hours before use. The crude polysaccharide prepared in the previous step was dissolved to 10 mg / mL, loaded with 10 mL of sample, and eluted with 0, 0.1, and 0.2 M NaCl solutions, respectively, at a flow rate of 2 mL / min. The elution time of the above solutions was 150, 50, and 50 min, respectively, with 10 mL per tube. The 0.2 M NaCl solution was collected by an automatic collector to obtain the 0.2 M fraction. After rotary evaporation (85 rpm, 55°C, 45 min), the fraction was dialyzed against running water (molecular weight 2500 Da) for 48 hours to remove salts. The fraction was freeze-dried (-50°C, 48 h) and stored at -20°C for use to obtain ion-purified polysaccharide.

[0068] (7) The ion-purified polysaccharide was purified by Sephadex G-100 (Solarbio, S8171) at a loading concentration of 20 mg / mL and a loading volume of 10 mL. Distilled water was used as the eluent at a flow rate of 0.5 mL / min for 100 min. The eluate was collected by an automatic collector, concentrated by rotary evaporation (85 rpm, 55°C, 45 min), dialyzed (molecular weight 2500 Da), and freeze-dried (-50°C, 48 h) to obtain homogenized pumpkin polysaccharide, which was then stored at -20°C for later use.

[0069] Example 3

[0070] (1) The pumpkin (Golden Chestnut Pumpkin, purchased from Xianlin Farmers' Market, Qixia District, Nanjing) with the skin and inner skin removed was washed, cut into slices, and freeze-dried (-50°C, 48h). The slices were then crushed using a blender (passed through a 50-mesh sieve), sealed in a ziplock bag, and stored in a dry environment for later use.

[0071] (2) Pumpkin powder was added to distilled water at a solid-liquid ratio of 1:50 (g / mL), mixed, extracted at 75°C for 5 h, and then ultrasonically treated at 250 W ultrasonic power for 25 min to obtain the extract.

[0072] (3) The extract was centrifuged (8000 rpm, 4°C, 15 min), the supernatant was collected, and the excess water was removed by rotary evaporation (85 rpm, 55°C, 45 min).

[0073] (4) Then, alcohol precipitation was performed (8 times the volume fraction of 95% ethanol solution, 12 h, 4°C), excess ethanol was poured off, and the mixture was centrifuged (8000 rpm, 4°C, 15 min) to collect the precipitate.

[0074] (5) The precipitate and distilled water were dissolved in a ratio of 1:20 (g:mL) to obtain a polysaccharide solution. Sevage reagent (n-butanol:chloroform mixed at a ratio of 1:4 (v:v)) was mixed with the polysaccharide solution at a ratio of 1:5 (v:v) and shaken, followed by centrifugation (8000 rpm, 4°C, 15 min). The supernatant was aspirated and placed in a dialysis bag (MD44 mm, molecular weight 4500 Da). The supernatant was dialyzed with running water for 48 h to remove small molecules, and then freeze-dried (-50°C, 48 h) to obtain pumpkin crude polysaccharide.

[0075] (6) DEAE Fast Flow anion exchange chromatography column: The DEAE Sepharose Fast Flow (Cytiva, 17070901) filler was repeatedly rinsed with distilled water until there was no ethanol smell, then wet packed and equilibrated with distilled water for 24 hours before use. The crude polysaccharide prepared in the previous step was dissolved to 30 mg / mL, and the solution was loaded in a volume of 10 mL. It was eluted with 0, 0.1, and 0.2 M NaCl solutions at a flow rate of 2 mL / min. The elution time of the above solutions was 80, 60, and 100 min, respectively, with 10 mL per tube. The 0.2 M NaCl solution was collected by an automatic collector to obtain the 0.2 M fraction. After rotary evaporation (85 rpm, 55°C, 45 min), it was dialyzed against running water (molecular weight 4500 Da) for 48 hours to remove salts. The product was freeze-dried (-50°C, 48 h) and stored at -20°C for use to obtain ion-purified polysaccharide.

[0076] (7) The ion-purified polysaccharide was purified by Sephadex G-100 (Solarbio, S8171) at a loading concentration of 30 mg / mL and a loading volume of 10 mL. Distilled water was used as the eluent at a flow rate of 1.5 mL / min for 200 min. The eluate was collected by an automatic collector, concentrated by rotary evaporation (85 rpm, 55°C, 45 min), and then dialyzed (molecular weight 4500 Da). The polysaccharide was freeze-dried (-50°C, 48 h) to obtain homogenized pumpkin polysaccharide, which was then stored at -20°C for later use.

[0077] Example 4 Structural Characterization of Homogenized Pumpkin Polysaccharide

[0078] 1 Structural analysis of pumpkin polysaccharide PPS3

[0079] 1.1 Molecular weight determination: HPGPC was used to determine the molecular weight of polysaccharides.

[0080] (1) Chromatographic conditions: BRT105-104-102 series gel column (8×300 mm); column temperature: 40°C; mobile phase: 0.05 M NaCl solution; flow rate: 0.6 mL / min, injection volume: 20 μL; detector: differential refractive index detector RI-10A.

[0081] (2) Plotting the standard curve: Accurately weigh dextran standards of different molecular weights, prepare them into 2 mg / mL solutions with deionized water, filter them through a 0.45 μm membrane, and inject them in sequence according to their molecular weight. Plot the standard curve with the retention time as the horizontal axis and the logarithm of the molecular weight of the standard (1gMw) as the vertical axis.

[0082] (3) Accurately weigh pumpkin polysaccharide PPS3 to prepare a solution with a concentration of 5 mg / mL, centrifuge (12000 rpm, 10 min), take the supernatant, filter it with a 0.22 μm filter membrane, transfer it to a 1.8 mL injection bottle, inject the sample, and calculate the molecular weight of the polysaccharide based on the standard curve.

[0083] 1.2 Molecular weight of pumpkin polysaccharide PPS3

[0084] Depend on Figure 2 It can be seen that PPS3 is a single peak, indicating that it is a high-purity homogeneous component with a purity of 96.27±1.15%. From Table 1, it can be seen that the weight-average molecular weight (Mw) of PPS3 is about 5.96×10 4 Da, number average molecular weight (Mn) is 3.87×10 4 Da, peak molecular weight (Mp) is 4.69×10 4 Da.

[0085] Table 1 Molecular weight of PPS3

[0086] Components Mn(Da) Mw(Da) Mp(Da) PPS3 38728 59585 46852

[0087] 2 Monosaccharide composition determination

[0088] 2.1 Monosaccharide determination was performed using liquid chromatography (ThermoU3000).

[0089] (1) Chromatographic conditions: Chromatographic column: ZORBAX EclipseXDB-C18, mobile phase: acetonitrile: phosphate buffer (12 g / L potassium dihydrogen phosphate, 2 M NaOH, pH = 6.8) isocratic elution, volume ratio of acetonitrile to phosphate buffer: 17:83, flow rate: 0.8 mL / min, column temperature: 30 °C, detection wavelength: 250 nm, injection volume: 10 μL.

[0090] (2) Determination of standard products: Weigh 5 mg each of rhamnose, arabinose, galactose, glucose, xylose, mannose, galacturonic acid, glucuronic acid, glucosamine hydrochloride, and galactosamine hydrochloride, and 10 mg of fucose, dissolve them, and dilute to 10 ml in a volumetric flask to prepare the standard stock solution. Then perform a gradient dilution and dilute to volume, filter through a 0.22 μm microporous membrane, and place in a sample injection bottle.

[0091] (3) Accurately weigh 5 mg (±0.05 mg) of polysaccharide PPS3 sample, add 1 ml of 2 M TFA solution, and heat at 121°C for 2 hours. Blow dry under nitrogen. Add 3 mL of methanol to rinse, blow dry again, and repeat the methanol rinse 2-3 times. Dissolve in 5 mL of sterile water and transfer to a chromatographic vial for analysis.

[0092] 2.2 Determination of monosaccharide composition of pumpkin polysaccharide PPS3

[0093] Figure 3 (a) is the liquid chromatogram of 11 monosaccharide standards. It is observed that the 11 monosaccharide standards are effectively separated. The monosaccharides represented by each peak in the mixed standard can be determined according to the retention time. The sequences and retention times of the 11 monosaccharides are shown in Table 2. The monosaccharide composition of PPS3 is as follows Figure 3 (b) Compared with the retention time of monosaccharide standards, the monosaccharide composition of PPS3 is: mannose, glucosamine hydrochloride, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose and arabinose, with the corresponding molar ratios of 0.005:0.004:0.041:0.013:0.545:0.004:0.241:0.147, among which galacturonic acid accounts for the largest proportion.

[0094] Table 211 Monosaccharide Standards

[0095] Standard name Peak time Slope Goodness of fit 1 Man 16.312 99.59 0.999 2 GlcN 19.890 93.29 1 3 Rha 22.967 33.78 0.997 4 GlcUA 23.638 68.08 0.999 5 GalUA 26.893 71.16 0.999 6 GalN 31.015 90.22 1 7 Glc 33.375 77.96 0.998 8 Gal 37.820 95.54 0.999 9 Xyl 40.273 56.54 0.992 10 Ara 41.800 81.21 0.996 11 Fuc 49.055 31.81 0.992

[0096] 3 Methylation analysis

[0097] 3.1 Detection Method: Weigh 1-3 mg of polysaccharide PPS3 and dissolve 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. Add 100 μL of 2 M TFA and react at 121°C for 90 min. Evaporate to dryness at 30°C. Add 50 μL of 2 M ammonia and 50 μL of 1 M NaBD4, mix well, and react at room temperature for 2.5 h. Terminate the reaction by adding 20 μL of acetic acid, blow dry under nitrogen, wash twice with 250 μL of methanol, and blow dry under nitrogen. Add 250 μL of acetic anhydride, vortex to 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 to mix, centrifuge, and discard the aqueous phase. Repeat the water wash three times. Remove the lower dichloromethane phase and analyze.

[0098] An Agilent 7890A-5977B gas chromatography-mass spectrometer (GC-MS) was used. The chromatographic system employed an Agilent gas chromatograph (Agilent 7890A; Agilent Technologies, USA) with an HP-5MS capillary column (30 m × 0.25 mm × 0.25 μm, Agilent J&W Scientific, Folsom, CA, USA). The carrier gas was high-purity helium (≥99.999%) at a flow rate of 1.0 mL / min, and the inlet temperature was 260°C. The injection volume was 1 μL, and the split injection was performed with a split ratio of 10:1. The solvent delay was 2.2 min. The heating conditions were: 50°C for 1.0 min, then increased at 50°C / min to 130°C, then increased at 3°C / min to 230°C, where it was held for 2 min.

[0099] The mass spectrometer system used was an Agilent 5977B quadrupole mass spectrometer (Agilent Technologies, USA), equipped with an electron impact ionization (EI) source and a MassHunter workstation. The EI source was operated at an inlet temperature of 230°C, a quadrupole temperature of 150°C, and an electron energy of 70 eV. The full scan mode (SCAN) was used, with a mass scan range (m / z) of 30–600.

[0100] 3.2 Methylation detection results of pumpkin polysaccharide PPS3

[0101] After pumpkin polysaccharide was treated by methylation, acid hydrolysis and acetylation, the connection mode of its glycosidic bonds was analyzed by GC-MS, such as Figure 4The sugar residues and their molar ratios of PPS3 were determined based on retention time and peak area. The composition and connection mode of the glycosidic bonds were also determined from the fragment ion distribution. The glycosidic bond connection modes of PPS3 mainly include t-Ara(f), 5-Ara(f), 2-Rha(p), t-Glcp, t-Glc(p)-UA, t-Gal(p), t-Gal(p)-UA, 2,4-Rha(p), 4-Gal(p), 4-Gal(p)UA, 4-Glc(p), 3-Gal(p), 6-Gal(p), 3,4-Gal(p)-UA, 2,4-Gal(p)-UA, and 3,6-Gal(p).

[0102] Table 3 Analysis of results of methylated sugar alcohol acetyl ester (PMAA) of PPS3

[0103]

[0104]

[0105] 4 Nuclear Magnetic Resonance

[0106] The freeze-dried PPS3 polysaccharide sample was dissolved in 0.5 mL of D2O and the one-dimensional NMR spectra were measured using a 600 MHz Bruker NMR spectrometer. 1 H-NMR, 13 C-NMR, DEPT-135 and 2D NMR COSY, HSQC, HMBC, NOESY. Calibration: HDO hydrogen δH = δ4.70ppm, TMS carbon δC = δ0.00ppm. The spectrum is as follows Figure 5 and Figure 6 As shown, all H and C chemical shift information of each major sugar residue was obtained to infer the connection order between each sugar residue.

[0107] One-dimensional H NMR spectroscopy ( 1 H-NMR) and carbon spectroscopy ( 13 C-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). 1The 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 (CH3COO- 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, and 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.

[0108] like Figure 5 As shown in (a) and (b), PPS3 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, δ4.3-5.5ppm, 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 δ4.3-3.0ppm region, with serious signal overlap, making it difficult to attribute. 1 Compared with the H-NMR spectrum of PPS3 13 C-NMR spectrum ( Figure 5 (c)) 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 In (d) (DEPT-135 spectrum), an obvious inverted peak signal can be seen in the δ60-70 ppm region, indicating that the sugar residue contains a -CH2- group.

[0109] from 1 H-NMR and 13In the C-NMR spectrum, some characteristic signal peaks were found: (1) The resonance signal in the high field region of δ1.10~1.30ppm 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 δ16.32ppm. Figure 6 (a)) A cross peak of δ1.18 / 16.32 ppm was found, and the peak signal intensity on the hydrogen and carbon spectra was weak, indicating the presence of a small amount of rhamnose residues, which is consistent with the monosaccharide composition results; (2) 1 The δ1.90~2.20ppm region in the H-NMR spectrum is the methyl proton signal of O-acetyl group, and there are multiple signal peaks, indicating that the substitution position of acetyl group occurs at different positions of sugar residue in the sugar chain. The methyl signal of acetyl group is located near δ20.00ppm in the carbon spectrum, and the peak signal intensity is very small. Based on this, it can be inferred that there is a low degree of acetylation in this polysaccharide sample; (3) The monosaccharide composition of PPS3 contains a large amount of galacturonic acid (57.817%). The characteristic absorption peak at δ52.80ppm represents the methyl carbon connected to the C-6 position of the methylated galacturonic acid residue. In the HSQC spectrum ( Figure 6 (a)) find the cross peak of these two signals δ3.71 / 52.80ppm, δ3.71ppm is the methyl proton signal of methyl ester (-COOMe), and the methyl proton signal of methyl ester δ3.71ppm is in the HMBC spectrum ( Figure 6 The signal cross peak δ3.71 / 170.68 ppm was found on (c), and it was judged that δ170.68 ppm belonged to the C-6 signal of the esterified galacturonic acid residue, while the characteristic absorption peak of the C-6 position of the unesterified galacturonic acid residue was near δ174.31 ppm. The C-6 signal of the esterified galacturonic acid residue was significantly stronger than the C-6 signal of the unesterified galacturonic acid residue. These characteristic signals indicated the presence of a high degree of methyl-esterified galacturonic acid.

[0110] Table 4 Sugar residues in polysaccharide samples 1 H and 13 Chemical shift assignment of C

[0111]

[0112]

[0113] Note: “--” in the table means undetermined or not detected, and “ / ” means not present.

[0114] In the monosaccharide composition test and methylation analysis results, the monosaccharide composition is mainly composed of GalA, Gal, Ara, and Rha. The methylation analysis results mainly contain 1,4-GalpA sugar residues, and contain 1,3,6-Galp, 1,3-Galp, 1,4-Galp, 1,6-Galp, T-Galp, T-Araf, 1,5-Araf, 1,2,4-Rhap, 1,2-Rhap, 1,4-Glcp, T-GalpA. In addition, sugar residues such as the C-1 signal peak of the reducing end β-GalpA appeared at δ96.07ppm. It is speculated that the polysaccharide sample may contain a large number of HG type domains and part of the RG-I type pectin domain. According to the monosaccharide composition and methylation analysis results of the polysaccharide sample, the analysis 1 H-NMR, 13 C-NMR, HSQC and 1 H- 1 The H COSY NMR spectrum revealed the presence of multiple anomeric signals. Combined with literature reports, there is a strong signal near δ98-102ppm in the anomeric region, which can be inferred to be mainly the anomeric carbon signal of the sugar residue α-GalpA. There is a strong signal near δ102-105ppm in the anomeric region, which can be inferred to be mainly the anomeric carbon signal of the sugar residue β-Galp. There is a signal at δ106-110ppm in the anomeric region, which can be inferred to be the anomeric carbon signal of the sugar residue α-Araf. Through monosaccharide composition, methylation analysis, and NMR analysis, there are multiple anomeric region signal peaks in the polysaccharide sample that are significant and can be used for structural analysis. The chemical shifts of these anomeric region signals are δ4.86 / 100.23ppm, δ4.89 / 100.26ppm, δ4.92 / 100.26ppm, δ5.01 / 99.52ppm, δ4.54 / 96.07ppm, δ4.54 / 104 .18ppm, δ4.51 / 104.32ppm, δ4.42 / 102.72ppm, δ4.36 / 103.08ppm, δ4.54 / 104.30ppm, δ5.14 / 109.13ppm, δ5.00 / 107.27ppm, δ5.14 / 99.63ppm, δ5.14 / 100.23ppm, δ4.41 / 102.64ppm. After the identification of the anomeric signals, COSY, HSQC, HMBC and NOESY were used to analyze the 1 H-NMR, 13C-NMR, DEPT-135 spectra, monosaccharide composition and methylation analysis results were compared with the chemical shift data of similar sugar residue substitutions in relevant literature. The above sugar residues were labeled as GE1,4, GE1,2,4, GE1,3,4, GA1,4, Rβ, G1,4, G1,3, G1,3,6, G1,6, Gt, At, A1,5, Rha1,2, Rha1,2,4, 4MeGlcA in sequence. 1 H and 13 The 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:

[0115] Sugar residue GE1,4: The anomeric signal of the sugar residue was determined to be δ4.86ppm (H-1) and δ100.23ppm (C-1) by HSQC and COSY, 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. The H-1 chemical shift of the sugar residue was determined to be δ4.86ppm by HSQC and COSY, and the COSY spectrum ( Figure 6 (b)) 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 δ3.62ppm, δ3.91ppm, and δ4.37ppm, respectively. After assigning the chemical shifts of hydrogen on the sugar ring, the HSQC correlation spectrum ( Figure 6 (a)) The chemical shifts of C-1 to C-4 on the sugar ring were assigned as δ100.23ppm, δ67.86ppm, δ67.86ppm, and δ78.81ppm, respectively. The cross peaks of H-5 and C-5 were found in the HSQC spectrum at δ5.01~5.07 / 70.42ppm. The cross peak of the signal was found in the HSQC spectrum at δ3.71 / 52.80ppm. δ3.71ppm was the methyl proton signal of methyl ester (-COOMe). The methyl proton signal and carbon signal of methyl ester were δ3.71ppm and δ52.80ppm, respectively. In the HMBC spectrum ( Figure 6(c)) found a signal cross peak of δ3.71 / 170.68 ppm, and it can be judged that δ170.68 ppm belongs to the C-6 signal of the esterified galacturonic acid residue. The chemical shift of C-6 is δ170.68 ppm, indicating that the sugar residue is methylated. The chemical shifts of C-1 and C-4 are shifted to the downfield, 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 downfield shift. This is because the introduction of the methoxy group causes the chemical shifts of the protons of the sugar ring to shift. Combined with the methylation results and literature reports, it is inferred that the sugar residue is →4)-α-GalpA-6-OMе-(1→, marked as GE1,4. Its chemical shift assignment is shown in Table 4.

[0116] Sugar residue G1,3,6: According to HSQC and 1H-1H COSY, the anomeric signal of the sugar residue was δ4.42ppm (H-1) and δ102.72ppm (C-1), indicating that the sugar residue was in β configuration. Figure 6 (a)) The H-1 chemical shift of this sugar residue was determined to be δ4.42 ppm by COSY( Figure 6 (b) 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 δ3.56ppm, δ3.62ppm, δ4.03ppm, and δ3.91ppm, respectively. The assignment of its H-6a and H-6b signals can be determined by HSQC correlation spectrum ( Figure 6 (a)) were assigned to δ3.80ppm and δ3.91ppm respectively. After assigning the chemical shift of hydrogen on the sugar ring, the HSQC correlation spectrum ( Figure 6 (a) The chemical shifts assigned to each carbon on the sugar ring are δ102.72ppm, δ69.81ppm, δ80.06ppm, δ69.71ppm, δ76.47ppm, and δ69.07ppm, respectively. The chemical shifts of C-1, C-3, and C-6 shifted to the downfield, indicating that the residue was substituted at the C-1, C-3, and C-6 positions of the sugar ring. Combined with the monosaccharide composition and methylation analysis results, and compared with literature reports, it is inferred that the sugar residue is →3,6)-β-D-Galp-(1→, labeled G1,3,6. Its chemical shift assignments are shown in Table 4.

[0117] According to a similar method, combined with the monosaccharide composition determination, methylation analysis results and literature reports, the hydrogen and carbon signals of other residues were deduced, and GE1,2,4 were deduced to be →2,4)-α-D-GalpA-6-OMe-(1→, GE1,3,4 were deduced to be →3,4)-α-D-GalpA-6-OMe-(1→, GA1,4 were deduced to be →4)-α-D-GalpA-(1→, Rβ were deduced to be β-D-Galp-(1→, G1,4 were deduced to be →4)-β-D-Galp-(1→, G1,3 were deduced to be →3)-β-D-Galp-(1→, G1, 6 is →6)-β-D-Galp-(1→, Gt is inferred to be β-D-Galp-(1→, At is inferred to be α-L-Araf-(1→, A1,5 is inferred to be →5)-α-L-Araf-(1→, 4MeGlcA is inferred to be 4-O-Me-β-D-GlcpA-(1→, methylation analysis results show that PPS3 also contains 1,2,4-Rhap, and the rhamnose signals on the hydrogen and carbon spectra are inferred to belong to the sugar residues →2,4)-α-L-Rhap-(1→, marked as Rha1,2,4. The H and C chemical shift assignments of the main sugar residues in PPS3 are summarized in Table 4.

[0118] According to the results of methylation analysis, PPS3 also contains t-GalpA, 1,4-Glcp and other connection modes, but the signals in NMR are very weak and cannot be detected. 1 H and 13 C is attributed.

[0119] Through the coupling signals of the anomeric hydrogen and carbon on each sugar residue, or the coupling signals of the anomeric carbon and 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 6 (c)) and NOESY spectra ( Figure 6As shown in (d), the following coupling signals can be found in the figure: (1) In the HMBC spectrum, H-1 of sugar residue GE1,4 (δ4.86ppm) and C-4 of sugar residue GE1,4 (δ78.81ppm) have related signal peaks (GE1,4H-1 / GE1,4C-4), H-4 of GE1,4 (δ4.37ppm) and C-1 of sugar residue GE1,4 (δ100.23ppm) have related signal peaks (GE1,4H-4 / GE1,4C-1), and H-1 of sugar residue GE1,4 (δ4 .86ppm) and H-4 of sugar residue GE1,4 (δ4.37ppm) have a cross peak (GE1,4H-1 / GE1,4H-4), indicating the existence of →4)-α-D-GalpA-6-OMe-(1→4)-α-D-GalpA-6-OMe-(1→ connection; (2) In the NOESY spectrum, H-1 of sugar residue GA1,4 (δ5.01ppm) and H-4 of sugar residue GE1,4 (δ4.37ppm) have a cross peak (GA1,4H-1 / GE1,4H-4), indicating the existence of →4) -α-D-GalpA-6-(1→4)-α-D-GalpA-6-OMe-(1→ connected; (3) In the HMBC spectrum, there is a cross peak (G1,3C-1 / G1,3,6H-3) between the C-1 of the sugar residue G1,3 (δ104.32 ppm) and the H-3 of the sugar residue G1,3,6 (δ3.62 ppm). In the NOESY spectrum, there is a cross peak (G1,3,6H-1 / G1,3,6H-3) between the H-1 of the sugar residue G1,3,6 (δ4.42 ppm) and the H-3 of the sugar residue G1,3 (δ3.83 ppm). 3H-3), indicating the existence of →3,6)-β-D-Galp-(1→3)-β-D-Galp-(1→ linkage, and the linkage site is located at the O-3 position; (4) In the HMBC spectrum, there is a cross peak (G1,3,6H-1 / G1,3,6C-6) between the H-1 (δ4.42ppm) of the sugar residue G1,3,6 and the C-6 (δ69.07ppm) of the sugar residue G1,3,6, indicating the existence of →3,6)-β-D-Galp-(1→3,6)-β-D-Galp-(1→ linkage, and the linkage site is located at the O-6 position; (5) In the HMBC spectrum, H-1 of sugar residue G1,4 (δ4.54 ppm) and C-4 of sugar residue G1,4 (δ77.32 ppm) have related signal peaks (G1,4H-1 / G1,4C-4). In the NOESY spectrum, H-1 of sugar residue G1,4 (δ4.54 ppm) and H-4 of sugar residue G1,4 (δ4.08 ppm) have cross peaks (G1,4H-1 / G1,4H-4), indicating the existence of →4)-β-D-Galp-(1→4)-β-D-Galp-(1→4)-β-D-Galp-(1→4) linkage. (6) In the HMBC spectrum, H-1 of sugar residue A1,5 (δ5.00ppm) and C-5 of sugar residue A1,5 (δ66.50ppm) have a cross peak (A1,5H-1 / A1,5C-5), indicating the existence of →5)-α-L-Araf-(1→5)-α-L-Araf-(1→ connection, and the connection site is located at the O-5 position; (7) In the HMBC spectrum, H-1 of sugar residue At (δ5.14ppm) and C-3 of sugar residue GE1,3,4 (δ76.36ppm) have a cross peak (At H-1 / GE1,3,4H-4), indicating the existence of α-L-Araf-(1→3,4)-α-D-GalpA-6-OMe-(1→ connection, and the connection site is located at the O-3 position; (8) In the NOESY spectrum, H-1 of sugar residue At (δ5.14ppm) and H-2 of sugar residue GE1,2,4 (δ3.63ppm) have a cross peak (At H-1 / GE1,2,4H-2), indicating the existence of α-L-Araf-(1→2,4)-α-D-GalpA-6-OMe-(1→ linkage, the linkage site is located at the O-2 position; (9) In the HMBC spectrum, there is a cross peak between the C-1 of the sugar residue Gt (δ104.30 ppm) and the H-3 of the sugar residue G1,3,6 (δ3.62 ppm) (Gt C-1 / G1,3,6H-3), indicating the existence of β-D-Galp-(1→3,6)-β-D-Galp-(1→ linkage, the linkage site is located at the O-3 position; (10) In the HMBC spectrum, there is a cross peak between the H-1 of the sugar residue At (δ5.14 ppm) and the C-3 of the sugar residue G1,3,6 (δ80.06 ppm) (At H-1 / G1,3,6C-3), indicating the presence of an α-L-Araf-(1→3,6)-β-D-Galp-(1→ linker, with the linker site located at the O-3 position.

[0120] Comprehensive analysis of monosaccharide composition, methylation results, and one-dimensional and two-dimensional NMR information indicates that PPS3 is a complex polysaccharide. It can be speculated to a large extent that it is mainly composed of pectins with a large number of HG domains and some RG-I domains with side chains. Its possible structural elements are shown in the structural formula below.

[0121] (1) Main chain

[0122]

[0123] (2) Branched chain

[0124]

[0125] Example 5 In vitro lipid-lowering activity of pumpkin polysaccharide PPS3

[0126] 1. Establishment of HepG2 cell lipid accumulation model

[0127] HepG2 cells were cultured in DMEM (High Glucose) supplemented with 1% double-antibody and 10% fetal bovine serum in a cell culture incubator (37°C, 5% CO2). When cells reached the logarithmic phase, the culture medium was aspirated and discarded. The cells were washed three times with PBS and digested with an appropriate amount of EDTA solution for 60-90 seconds. Digestion was terminated by adding serum-containing culture medium. The cells were pipetted to detach and evenly disperse. The cells were centrifuged at 900 rpm, and the cells at the bottom were aspirated, culture medium was added, and then transferred to a new dish. Experiments were performed after the cells had been passaged for 2-3 times and stabilized.

[0128] Take cells in the logarithmic growth phase and dilute them to a concentration of 4×10 4 Cells were cultured at 400 μg / mL. PBS solution was added to the outer circle of a 96-well plate, and 100 μL of cell suspension was added to the remaining wells. The cells were cultured in a 37°C, 5% CO2 incubator for 24 hours. The culture medium was discarded, and PA / BSA solutions with different concentrations of PA (0, 25, 75, 150, 300, 600, and 1200 μM) were added to each group. The blank group was added with serum-free medium containing 1% BSA and cultured for another 24 hours. MTT solution was prepared with PBS to a concentration of 5 mg / mL, and 20 μL was added to each well. The cells were cultured for another 4 hours. The MTT solution was discarded, and 150 μL of DMSO was added to each well to dissolve the precipitate. The cells were shaken in the dark for 10 minutes to completely dissolve the precipitate. The absorbance was measured at 490 nm. The survival rate of HepG2 cells treated with PA was calculated according to the following formula:

[0129] Cell viability (%) = OD 实验组 / OD 空白组 ×100.

[0130] The MTT method was used to screen the PA modeling concentration. Figure 7 As shown, as PA concentration increased, the cytotoxicity of PA on HepG2 cells increased, and cell viability decreased. When the PA concentration was 150 μM, the cell viability was 93.41 ± 8.16%, while when the PA concentration increased to 300 μM, the cell viability was only 74.26 ± 8.63%, a significant difference (P < 0.01). Therefore, the optimal PA concentration for modeling was determined to be 150 μM.

[0131] 2. Effect of PPS3 on cell viability

[0132] The specific method is as follows: Step 1 of this example. The toxicity of PPS3 to HepG2 cells was determined by MTT assay. Figure 8 As shown in the data, when the PPS3 concentration increased from 5 μg / mL to 160 μg / mL, the activity of HepG2 did not change significantly (P<0.05), and the cell survival rate was above 90%, indicating that PPS3 had no obvious toxicity to HepG2 cells within the concentration range of 0-160 μg / mL.

[0133] 3. Oil Red O staining to detect the effect of PPS3 on lipid deposition

[0134] Cells were cultured according to the method in step 1 of this example. The blank group was added with serum-free medium containing BSA, the model group was added with 150 μM PA / BSA solution, the low-concentration group was added with 75 μg / mL PPS3 solution and 150 μM PA / BSA solution, and the high-concentration group was added with 150 μg / mL PPS3 solution and 150 μM PA / BSA solution. The cells were cultured for 24 hours, the culture medium was aspirated and discarded, the cells were washed three times with PBS, the cells were fixed with 10% formalin for 30 minutes, aspirated and discarded, the cells were washed three times with PBS, 60% isopropanol was added for 1 minute, aspirated and discarded, and oil red dye (oil red dye and deionized water were diluted in a ratio of 3:2) was added. The cells were incubated at 37°C in the dark for 20 minutes. The cells were then washed with PBS until the background was clean and observed and photographed under an inverted microscope. After the photographing was completed, 200 μL of isopropanol was added to each well to redissolve the dye, and the cells were shaken at 37°C until the dye was completely dissolved. 100 μL was transferred to a 96-well plate and the plate was plated at OD 510nm Measure the absorbance for quantitative analysis.

[0135] The cells in each group were stained with Oil Red O. Figure 9 As shown in (a), the blank group showed clear cell morphology with no lipid droplet deposition. However, after PA treatment, numerous red lipid droplet spots appeared within the model group, indicating successful model establishment. The addition of PPS3 altered the lipid droplet structure within the cells to varying degrees (P < 0.05). In particular, when the PPS3 concentration reached 150 μg / mL, lipid droplets in HepG2 cells were significantly reduced (P < 0.05), demonstrating the greatest lipid-lowering effect. Figure 9 (b) is the quantitative results of total intracellular lipid content. Compared with the control group, the intracellular lipid content of HepG2 cells was significantly increased after PA induction (P<0.01); compared with the model group, the intracellular lipid content of the low-concentration group (75 μg / mL) and the high-concentration group (150 μg / mL) was significantly decreased (P<0.05 or P<0.01), indicating that PPS3 can significantly reduce fat accumulation in HcpG2 cells.

[0136] 4. Determination of TC, TG, LDL-C and HDL-C Contents in HepG2

[0137] Cells were cultured according to step 1 of this example, starved for 8-12 hours, and grouped as in step 3 of this example. Culture was continued for 24 hours, and the cells were lysed. TC, TG, LDL-C, and HDL-C levels were determined according to the kit instructions.

[0138] Figure 10The effect of PPS3 on TC and TG levels in PA-induced HepG2 cells is shown in the figure. As can be seen, TC and TG levels in the model group were significantly higher than those in the blank group without PA induction, confirming the successful establishment of the model. Compared with the model group, TC and TG levels in both the low-concentration group (75 μg / mL) and the high-concentration group (150 μg / mL) decreased to varying degrees. In particular, the high-concentration pumpkin polysaccharide PPS3 significantly reduced TC and TG levels in the cells (P < 0.01), reaching 0.21 ± 0.01 and 0.19 ± 0.01 mmol / g prot, respectively, consistent with the results of Oil Red O staining.

[0139] Figure 11 The effect of PPS3 on LDL-C and HDL-C levels in PA-induced HepG2 cells was shown. The LDL-C and HDL-C levels in the model group were significantly different from those in the blank group (P < 0.01), demonstrating that the model was successfully established. Compared with the model group, pumpkin polysaccharide intervention significantly altered both LDL-C and HDL-C levels (P < 0.01 or P < 0.05). In particular, the high-concentration pumpkin polysaccharide PPS3 group significantly reduced LDL-C levels in cells (P < 0.01) to 0.44 ± 0.03 mmol / gprot and significantly increased HDL-C levels (P < 0.01 or P < 0.05) to 0.62 ± 0.04 mmol / gprot, consistent with the results of the previous study.

[0140] Example 6: Improvement of pumpkin polysaccharide PPS3 on obesity induced by high-fat diet in mice

[0141] 1. Animal grouping and experimental design

[0142] Forty six-week-old C57BL / 6 male mice were randomly divided into four groups of 10 mice per group, with five mice per cage. Groups: 1) ND group: fed a normal diet; 2) HFD group: fed a high-fat diet; 3) LPPS3 group: pumpkin polysaccharide PPS3 75 mg / kg / day, 0.1 ml / 10 g; 4) HPPS3 group: pumpkin polysaccharide PPS3 150 mg / kg / day, 0.1 ml / 10 g.

[0143] After one week of adaptive feeding, the mice were fed a high-fat diet. The LPPS3 and HPPS3 groups were gavaged with pumpkin polysaccharide PPS3 at doses of 75 mg / kg / d and 150 mg / kg / d, respectively. The ND and HFD groups were given distilled water.

[0144] 2. Weight records and results

[0145] Each mouse was weighed once a week and recorded according to the number. Diet measurement method: the total food intake of each cage of mice in a week was measured and divided by the number of mice in each cage and the number of days to obtain the average daily food intake of each mouse.

[0146] The most representative body shape of mice in each group was selected for photographic comparison, such as Figure 12 As shown in (a), visual observation showed that after 10 weeks of high-fat diet, the body size was significantly larger than that of the normal diet group. After intervention with pumpkin polysaccharide PPS3, the body size was significantly reduced, and the hair of the mice fed with a high-fat diet became shiny.

[0147] The weight index of mice is the most direct indicator to test whether pumpkin polysaccharide PPS3 can prevent mouse obesity. The weekly weight of mice was recorded during the feeding period, and the weight gain curve was drawn. Figure 12 As shown in (b), over the course of 10 weeks of high-fat diet, the difference in body weight between the ND and HFD groups gradually became significant. By the fifth week, the weight of mice in the HFD group was 20% higher than that of mice in the ND group, indicating that the mouse obesity model was established. After oral administration of pumpkin polysaccharide PPS3, the weight of mice decreased significantly compared to the HFD group, indicating that pumpkin polysaccharide PPS3 intervention can effectively alleviate the weight gain induced by a high-fat diet in mice.

[0148] 3. Oral glucose tolerance test and results

[0149] After fasting for 8-12 hours, the mice underwent an oral glucose tolerance test (OGTT). After measuring baseline blood glucose (0 min), the mice were gavaged with 2 g / kg glucose solution. Blood glucose levels were measured and recorded at 30, 60, 90, and 120 min.

[0150] Oral glucose tolerance test (OGTT) is often used to evaluate the body's ability to regulate blood sugar. After 8 weeks of pumpkin polysaccharide intervention, OGTT was performed on mice. Figure 13 As shown, the blood glucose levels of mice in the HFD group were higher than those in the other groups at all points. At 30 minutes after glucose administration, the blood glucose levels of mice in each group reached their highest level, and after 30 minutes, they gradually decreased, indicating that pumpkin polysaccharide PPS3 can improve glucose tolerance in obese mice.

[0151] 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. A use of homogenized pumpkin polysaccharide in the preparation of a product for improving obesity induced by a high-fat diet, characterized in that: The homogenized pumpkin polysaccharide contains mannose, glucosamine hydrochloride, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose and arabinose, and the corresponding molar ratio is 0.005:0.004:0.041:0.013:0.545:0.004:0.241:0.147; the relative molecular weight of the homogenized pumpkin polysaccharide is 5.96×10 4 Da; The main chain and side chain structural formulas of the homogenized pumpkin polysaccharide are as follows: The preparation method of the homogenized pumpkin polysaccharide is characterized by comprising the following steps: (1) Mix pumpkin powder 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) The precipitate is mixed with water to obtain a polysaccharide solution, Sevage reagent is added, the solution is centrifuged, and the supernatant is collected; the supernatant is dialyzed and dried to obtain crude pumpkin polysaccharide; (4) separating the crude pumpkin polysaccharide by anion exchange column chromatography, collecting the polysaccharide-containing eluate, and dialyzing to obtain ion-purified pumpkin polysaccharide; (5) purifying the ion-purified pumpkin polysaccharide by gel column chromatography, collecting the polysaccharide-containing eluate, and dialyzing to obtain homogenized pumpkin polysaccharide; In step (4), the anion exchange column is DEAE Sepharose Fast Flow, the sample concentration is 10-30 mg / mL; the elution method is water, 0.1 mol / L NaCl and 0.2 mol / L NaCl gradient elution in sequence, the elution time of each solution is 50-150 min, and the eluate obtained by eluting with 0.2 mol / L NaCl is collected; the elution flow rate is 1-4 mL / min; In step (5), the gel column is Sephadex G-100, and the loading concentration is 10 to 30 mg / mL; the elution method is water elution, the elution time is 100 to 300 min, and the eluate is collected; the elution flow rate is 0.2 to 1.5 mL / min.

2. The use according to claim 1, characterized in that In step (1), the material-liquid ratio of the pumpkin powder to water is 1:30-50; the heating temperature is 75-95° C., the heating time is 1-5 hours; and the ultrasonic time is 5-25 minutes; In step (2), the temperature for concentrating the supernatant is 45-65° C., and the time for concentrating is 35-55 min; the volume ratio of the concentrate to ethanol is 1:1-8, and the concentration of the ethanol is 85-100%; 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, characterized in that The centrifugal conditions include: 7000-9000 rpm, 5-25 min, 1-6°C.

4. The use according to claim 1, wherein The specification of the dialysis bag used for dialysis is 2500-4500Da.

5. A pharmaceutical composition for improving obesity induced by a high-fat diet, characterized in that: The invention comprises the homogenized pumpkin polysaccharide according to any one of claims 1 to 4 and pharmaceutically acceptable excipients.

6. The pharmaceutical composition according to claim 5, wherein The dosage form of the pharmaceutical composition includes powder, tablet, solution, granule, dripping pill, pill or powder.

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