CN116492368A - Application of pumpkin polysaccharide in medicines for treating inflammatory bowel disease - Google Patents
Application of pumpkin polysaccharide in medicines for treating inflammatory bowel disease Download PDFInfo
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- CN116492368A CN116492368A CN202211352498.9A CN202211352498A CN116492368A CN 116492368 A CN116492368 A CN 116492368A CN 202211352498 A CN202211352498 A CN 202211352498A CN 116492368 A CN116492368 A CN 116492368A
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- pumpkin
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- A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
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- A61K2236/331—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation, decoction
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Abstract
The invention relates to application of pumpkin polysaccharide, in particular to application of pumpkin polysaccharide in preparing a medicament for treating inflammatory bowel disease, wherein the inflammatory bowel disease is ulcerative colitis or Crohn disease. The invention also provides a preparation method of pumpkin polysaccharide, which comprises the steps of extraction; cooling and precipitating; concentrating and precipitating with alcohol; purifying the precipitate obtained by alcohol precipitation to obtain pumpkin polysaccharide.
Description
Technical Field
The invention relates to application of pumpkin polysaccharide, in particular to application of pumpkin polysaccharide in preparation of medicines for treating inflammatory bowel diseases.
Background
Inflammatory bowel disease (Inflammatory bowel disease, IBD) includes ulcerative colitis (Ulcerative Colitis, UC) and Crohn's Disease (CD), where UC is a type of inflammatory disease of the intestine with an incompletely defined etiology and pathogenesis, and is long and recurrent. UC is often accompanied by colonic mucosal and submucosal continuity inflammatory lesions and fibrous scars, mucosal erosion, crypt swelling, and the like. At present, UC is considered to be related to various factors such as heredity, environment, immunity and the like, the pathogenesis is complex, and no cure therapy exists at present. At present, various medicines are clinically used for treating UC, including salicylic acid anti-inflammatory medicines, hormones, immunosuppressants and the like, but the medicines have the problems of limited curative effect, long treatment period, high recurrence rate and the like, and often have serious complications. The traditional Chinese medicine active ingredient has the advantages of small toxic and side effects, low cost, large structural transformation space and the like, shows remarkable effect and development potential for treating UC, further researches the pathogenesis of UC based on a brand new action target point, and discovers the natural medicine which is lower in toxicity, high in efficiency and capable of being orally taken for treating UC, and has important research value and clinical significance.
Pumpkin belongs to cucurbitaceae, and the fruit is nontoxic and is a plant for both medicine and food. Warm nature, sweet and nontoxic taste, entering spleen and stomach meridians, moistening lung and supplementing qi, resolving phlegm and expelling strong, expelling parasites and detoxicating, treating cough and asthma, treating pulmonary abscess and constipation, and being beneficial to urination and beautifying. Modern researches have shown that pumpkin is rich in various amino acids, polysaccharides, proteins, vitamins, starch, trigonelline, cellulose, vegetable oil and part of microelements. The Pumpkin Polysaccharide (PP) has obvious functions of resisting tumor, regulating immunity, resisting oxidation, reducing blood sugar, reducing blood fat, resisting ulcer, resisting virus, protecting against radiation injury and the like, becomes a research hot spot of modern biological medicaments, has obvious curative effect in clinic, and has wide application prospect. PP may have important roles in antioxidation, anticancer, immunity regulation, etc., but no report on the activity related to PP anti-IBD and the mechanism of action thereof has been found so far.
The medicines commonly used at present for inflammatory bowel diseases can be classified into salicylic acid, hormone, biological medicine and traditional Chinese medicine. However, because of the complex pathogenesis of IBD, the effects of drugs acting purely against a mechanism of action are often not apparent in relation to many factors such as inflammation, immunity and intestinal flora. Pumpkin is used as a plant for both medicine and food, and has rich resources and low price; as a common dish, is favored by eaters, and has further development and utilization value and research prospect as a long-acting, nontoxic, hypoglycemic and hypolipidemic drug or dietotherapy agent. As Pumpkin Polysaccharide (PP) has been increasingly developed in pharmacological efficacy, new uses thereof have also been urgently developed.
Disclosure of Invention
The invention aims to provide an application of pumpkin polysaccharide in preparing medicines for treating inflammatory bowel diseases.
In order to solve the technical problems, the invention provides application of pumpkin polysaccharide in preparing medicines for treating inflammatory bowel diseases.
As an improvement of the application of the present invention: the inflammatory bowel disease is ulcerative colitis or Crohn's disease.
As a further improvement of the application of the invention: adding pharmaceutically acceptable auxiliary materials into pumpkin polysaccharide, and preparing into any one of the following dosage forms: capsule, tablet, granule, injection, sustained release preparation, oral liquid or dripping pill.
As a further improvement of the application of the invention, the preparation method of pumpkin polysaccharide comprises the following steps:
1) Extracting
Slicing fructus Cucurbitae Moschatae (fresh fructus Cucurbitae Moschatae) into slices (into thick slices with thickness of about 0.5 cm) to obtain slices;
adding water with the mass of 6+/-0.5 times of that of the pumpkin slices into the pumpkin slices, boiling, and performing primary extraction for 1+/-0.1 hour; filtering to obtain first extractive solution and residue; replacing pumpkin slices with extraction residues, adding water which is 4+/-0.5 times of the mass of the pumpkin slices into the extraction residues, boiling again, and performing secondary extraction for 1+/-0.1 hour; filtering to obtain secondary extract; combining the first extraction solution and the second extraction solution to obtain a combined extraction solution;
2) Cooling and precipitating:
standing the combined extracting solution until the temperature is reduced to about room temperature (20-25 ℃), taking supernatant obtained by standing, and discarding lower-layer sediment (namely waste residue containing a small amount of extracting solution) obtained by standing;
3) Concentrating, precipitating with ethanol:
concentrating the supernatant liquid under vacuum until the volume is 45-55%, cooling to room temperature, stirring, adding absolute ethanol until the volume concentration of the ethanol is 55-60%, standing for more than or equal to 6 hours, and collecting precipitate obtained by alcohol precipitation (ethanol liquid is recovered after being poured out);
4) Purifying the precipitate obtained by alcohol precipitation to obtain Pumpkin Polysaccharide (PP).
As a further improvement of the application of the invention, the purification of step 4) comprises the steps of:
4.1 Redissolution, deproteinization:
adding water 5.5-6.5 times of the weight of the alcohol precipitation, heating until the alcohol precipitation is dissolved, vacuum concentrating to remove residual ethanol, adding water to complement the weight, and deproteinizing by a conventional Sevage method;
repeating the operations of adding water for dissolving, vacuum concentrating to remove residual ethanol, adding water to supplement weight, deproteinizing by Sevage method for 2 times to obtain deproteinized supernatant;
4.2 Desolventizing, dialysis:
concentrating deproteinized supernatant in vacuum until the supernatant is nearly dry, adding 9+/-0.5 times of water into the concentrated product, transferring the mixture into a semipermeable membrane dialysis bag (the permeation molecular weight is 8000-14000 Da), fastening an upper opening, placing the mixture into a container filled with deionized water, and dialyzing the mixture for 48 hours, wherein water is changed every 2 hours to obtain dialysis trapped fluid;
4.3 Concentrating, lyophilizing:
concentrating the trapped fluid under vacuum and reduced pressure until the dryness is about 60%, and transferring into a freeze dryer for freeze drying to obtain pumpkin polysaccharide.
The invention provides application of high-activity PP in resisting ulcerative colitis, and solves the problem that the existing method for treating ulcerative colitis lacks related medicines. The invention discovers that the PP has remarkable curative effect on ulcerative colitis mice when the daily dosage of the PP is 50mg/kg and 100mg/kg, and the invention is particularly characterized in the following aspects: obviously improves the symptoms of mice, relieves the colon pathological damage induced by DSS and inhibits the secretion of inflammatory factors. The mechanism of action may be through the modulation of disturbances in the intestinal microbiota.
The beneficial effects of the invention are mainly as follows:
the invention provides application of PP in preparing a medicament for treating inflammatory bowel disease, and pumpkin polysaccharide is obtained by separating and purifying pumpkin of cucurbitaceae plants, and has the advantages of wide source, convenient material taking and the like, and is convenient for industrialized mass preparation; the invention extracts and prepares the PP by an alcohol precipitation water-soluble method, and the technology simplifies the preparation flow of the PP, and has simple operation, low cost and high purity; the PP prepared by the invention has potential IBD resisting effect, and the action mechanism of the PP is possibly related to anti-inflammatory and intestinal flora regulation, so that a research basis and a theoretical basis are provided for the application of the PP in IBD resisting.
Drawings
The following describes the embodiments of the present invention in further detail with reference to the accompanying drawings.
FIG. 1 is a flow chart of the process for extracting and preparing PP according to example 1;
FIG. 2 shows the results of analysis of chemical components of PP prepared in example 1;
in fig. 2:
a is the appearance form of PP; b is FT-IR spectrum of PP; c is a monosaccharide molecular weight spectrogram obtained by detecting PP through high performance liquid gel chromatography; d is a monosaccharide composition spectrum of PP measured by high performance liquid chromatography.
FIG. 3 shows the effect of PP on DSS induced UC mice;
in fig. 3:
a is the result of PP improving colon length; b is the quantitative result of PP improving colon length; c is a weight change curve; d is a Disease Activity Index (DAI) change curve; e is a representative picture of the effect of PP on the spleen factor of DSS-induced mice; f is the statistical result of spleen factor (n=6);
in comparison with the control group, # p<0.05， ### p<0.001; compared with the model, p<0.05，**p<0.01，***p<0.001。
FIG. 4 is the effect of PP on DSS-induced damage to colon tissue and inflammatory cytokine levels in UC mice;
in fig. 4:
a is a representative picture of colon section H & E staining; b is a histological score of colon sections; PP inhibits pro-inflammatory cytokines in colon tissue; c is TNF- α, D is IL-6,E is IFN- γ, F is IL-1β and G is IL-18 levels;
each point is expressed as mean ± standard deviation (n=6). In comparison with the control group, ### p<0.001; compared with the model, p<0.05，**p<0.01，***p<0.001。
FIG. 5 shows the results of a differential microbial community diversity analysis after PP group interaction;
in fig. 5:
a is a Wen diagram (Venn diagram); b is Principal Component Analysis (PCA); c is principal coordinate analysis (PCOA); d is a non-metric multidimensional scale (NMDS); PP cluster analysis was performed on each fecal microbiota at both portal level (E) and genus level (F).
Detailed Description
For a better description of the objects, technical solutions and advantages of the present invention, the present invention will be further described with reference to the accompanying drawings and specific embodiments. Unless otherwise specified, the experimental methods in the present invention are all conventional methods; unless otherwise specified, the reagent concentrations in the present invention are mass concentrations; unless specifically stated otherwise, materials or reagents for use in the present invention are available from the market or other public sources.
Example 1: the preparation of pumpkin polysaccharide comprises the following steps in sequence
1) Slicing and extracting:
the extraction flow is as shown in fig. 1:
cutting fresh pumpkin into thick slices with the thickness of about 0.5 cm to obtain pumpkin slices;
adding water with the mass being 6 times that of the pumpkin slices into the pumpkin slices, boiling, and extracting for 1 hour for the first time; filtering to obtain first extractive solution and residue;
replacing pumpkin slices with extraction residues, adding water which is 4 times the mass of the pumpkin slices into the extraction residues, boiling again, and performing secondary extraction for 1 hour; filtering to obtain secondary extract;
and combining the first extracting solution and the second extracting solution to obtain a combined extracting solution.
2) Cooling and precipitating:
and (3) standing the combined extracting solution until the temperature is reduced to about room temperature (20-25 ℃), pouring out supernatant obtained by standing, and discarding lower-layer sediment (namely waste residue containing a small amount of extracting solution) obtained by standing.
3) Concentrating, precipitating with ethanol:
concentrating the supernatant under vacuum until the volume is about 50% of the original volume, cooling to room temperature, stirring, adding absolute ethanol until the volume concentration of the ethanol reaches about 60%, standing for 6 hours, pouring out the ethanol solution, and collecting precipitate (hereinafter referred to as ethanol precipitation).
The alcohol precipitation is purified, specifically as follows steps 4) to 6).
4) Re-dissolving and deproteinizing:
adding 6 times of water according to the weight of the alcohol precipitation, heating until the alcohol precipitation is dissolved, vacuum concentrating to remove residual ethanol, adding water to complement the weight, and deproteinizing by a conventional Sevage method;
and repeating the operations of adding water for dissolving, vacuum concentrating to remove residual ethanol, adding water to supplement the weight, and deproteinizing by a Sevage method for 2 times to obtain deproteinized supernatant.
Description: deproteinization by Sevage method can be carried out by referring to conventional operation.
5) Desolventizing, dialysis:
concentrating deproteinized supernatant in vacuum until the supernatant is nearly dry, adding 9 times of water to the concentrated concentrate to ensure that the mass concentration of the concentrate in the solution is about 10%, transferring the concentrate into a semipermeable membrane dialysis bag (the permeation molecular weight is 8000-14000 Da), fastening an upper opening, placing the container into a container filled with deionized water, dialyzing for 48 hours, changing water every 2 hours, and obtaining dialysis trapped fluid.
6) Concentrating, freeze-drying:
concentrating the trapped fluid under vacuum and reduced pressure until the dryness is about 60%, and transferring into a freeze dryer for freeze drying to constant weight according to the conventional method to obtain pumpkin polysaccharide.
Example 2: structural identification of pumpkin polysaccharide:
the appearance and morphology of PP were as shown in FIG. 2A, and were pale yellow powders. The FT-IR spectrum is shown in FIG. 2B, and Table 1 summarizes the properties of the peaks. FT-IR spectra showed PP at 3401.82, 2937.06 and 1614.13cm -1 The compositions exhibit characteristic absorption of polysaccharides for O-H, C-H and C-O stretching vibrations, respectively. 1000-1200cm -1 The absorption at this point is due to the C-O-C and C-O-H in the pyranose ring. HPGPC spectra show that PP has a relatively single symmetrical peak, which means that PP is a homogeneous polysaccharide (FIG. 2C) having a weight average molecular weight (Mw) of 3.1X10 5 Da (Mw/mn=3.95). As shown in fig. 2D and table 2, the monosaccharide composition of PP consisted mainly of mannose, rhamnose, galacturonic acid, galactosamine, glucose, xylose in a molar ratio of 1.58:3.51:34.54:1.00:3.25:3.02.
Table 1: properties of peaks in FT-IR absorption Spectrum
| Wave number/cm -1 | Attribution of peak |
| 3401.82 | Stretching vibrations of O-H |
| 2937.06 | Asymmetric stretching vibration of CH 2 |
| 1614.13 | Asymmetric stretching vibration of C＝O |
| 1423.21 | Shear vibration of CH 2 |
| 1101.16 | Bending vibrations of C-H |
| 1016.30 | Pyranoside |
Table 2: monosaccharide composition of PP
| Number | Retention time(min) | Monosaccharides | Composition(％) |
| 1 | 17.371 | Mannose | 3.28 |
| 2 | 21.612 | Aminoglucose | 0.31 |
| 3 | 22.623 | Ribose | 0.30 |
| 4 | 24.298 | Rhamnose | 7.31 |
| 5 | 26.131 | Glucuronic acid | 0.83 |
| 6 | 28.971 | Galacturonic acid | 71.85 |
| 7 | 36.292 | Galactosamine | 2.08 |
| 8 | 41.297 | Glucose | 6.76 |
| 9 | 44.065 | Galactose | 0.35 |
| 10 | 45.637 | Xylose | 6.28 |
| 11 | 53.618 | Arabinose | 0.21 |
| 12 | 56.286 | Fucose | 0.43 |
Example 3: effects of PP on DSS-induced colon shortening, weight changes, disease Activity Index (DAI) score and spleen factor in UC mice
The experimental method comprises the following steps: 30 (20.+ -.2 g) male C57BL/6 mice were randomly divided into 5 groups (6 per group): control (Control), model (Model), sulfasalazine (SASP), PP (50 mg/kg) and PP (100 mg/kg). All mice except the control group were free to drink 3% dss solution for 7 days to induce UC, and were simultaneously dosed daily 2 times a day with the following drugs at a dosing volume of 0.1mL/10g for these 7 days: the control group and the model group were given physiological saline (200. Mu.L/min), SASP group (200 mg/kg/day), PP group (50 mg/kg/day) and PP group (100 mg/kg/day). Mice body weight, stool, and other abnormalities were recorded daily and blindly scored for Disease Activity Index (DAI). Mice were sacrificed on day 8 for cervical dislocation dissection to obtain colon, liver, kidney, spleen and other tissues, and after separation, photographing and weighing, colon length and spleen index were calculated.
Experimental results: figures 3A and B show that PP can significantly inhibit colon shortening in mice, and figure 3C shows that PP (100 mg/kg) can significantly alleviate weight loss in mice; figure 3D shows that PP significantly reduces DAI score; figures 3E-F show that PP also significantly reduces the enlarged spleen. In conclusion, the PP can significantly improve various symptoms of UC mice and has the effect of treating the UC of the mice.
Example 4: effects of PP on DSS-induced damage to colonic tissue and inflammatory cytokine levels in UC mice
The experimental method comprises the following steps: the treatment of mice was the same as in "Experimental example 3", and after cervical dislocation, the colon tissues of all mice were separated, measured and photographed as soon as possible, and the colon contents were removed and the colon was divided into two. One part was immediately frozen in liquid nitrogen and the other part was fixed with 4% paraformaldehyde, embedded, sectioned, stained and photographed under a microscope. Pathological changes such as edema, adhesion, ulcer, necrosis and the like are observed, and histological scores are evaluated. The ELISA method is used for measuring inflammatory cytokines: the total protein concentration of the colon tissue was homogenized in PBS buffer on ice. The homogenate was then centrifuged (5 min, 5000g,4 ℃) using a 5417R centrifuge to obtain a supernatant. Protein concentration was determined with BCA kit and proinflammatory cytokines were detected with ELISA kit. Absorbance values were measured at an optical density of 450nm using Microplate Reader 680. The above experimental method was carried out with reference to conventional Elisa kit protocol.
Experimental results: as shown in fig. 4A-B, PP (50 and 100 mg/kg) groups significantly reduced the pathological changes in DSS-induced UC mice colon lesions and significantly improved the histopathological scores for colon inflammation. In addition, PP significantly reduced levels of TNF- α, IFN- γ, IL-1β, IL-6 and IL-18 as shown in FIGS. 4C-G. It was demonstrated that PP (50 and 100 mg/kg) can alleviate DSS-induced colon injury in mice and significantly reduce inflammatory cytokine levels in the colon.
Example 5: PP can regulate DSS induced UC mouse intestinal microbiota disturbance
The experimental method comprises the following steps: (1) DNA extraction: DNA was extracted using the TGuide S96 magnetic soil/fecal DNA kit (tengen biotechnology (beijing) limited) following the procedure described by the manufacturer. DNA concentration was measured using a Qubit dsDNA HS assay kit and a Qubit 4.0 fluorometer (Invitrogen, sammer femto science, oregon, usa). Amplicon sequencing full length 16S rRNA genes were amplified from genomic DNA extracted from each sample using 3417 F:5'-CCTACGGNGGCWGCAG-3' and 805R:5'-GACTACHVGGGTATCC-3' universal primer sets. After a series of amplifications, the PCR amplified products were purified with Agencourt Ampre XP magnetic beads (Indianapolis Beckmann coulter) and quantified using a Qubit dsDNA HS assay kit and a Qubit 4.0 fluorometer (Invitrogen, simer Feiche sciences Co., oreg., USA). After the individual quantification step, the amplicons are pooled in equal amounts. SMRTbell library was prepared from amplified DNA using SMRTbell Express template preparation kit 2.0 according to manufacturer's instructions (Pacific Biosciences). Purified SMRTbell library from pooled samples and barcode samples were sequenced on single pacbrio sequence II 8M cells using sequence II sequencing kit 2.0. In bioinformatics analysis, raw reads generated from sequencing were filtered and demultiplexed using SMRT-Link software (version 8.0) and predicted to be 0.9 or more with a minimum pass of 5 minutes or more to obtain cycle-consistent sequencing (CCS) reads. Subsequently, the CCS sequence was assigned to the corresponding sample according to its barcode using lima (1.7.0 version). 97% of samples are gathered into the same operation classification unit (OTU) through USEARCH (v 10.0) and OTU with the reuse rate less than 0.005% is screened out. OTUs were annotated with classification using SILVA database (version 132) based on naive bayes classifier in QIIME2 with a confidence threshold of 70%.
Experimental results:
the Venn diagram shows 272 OTU overlaps in all groups, 297 OTU overlaps in the control and model groups, 298 OTU overlaps in the control and PP (100 mg/kg) groups, and 291 OTU overlaps in the control and SASP (200 mg/kg) groups (FIG. 5A). Meanwhile, the individual OTUs of the control, model, PP and SASP groups were 51, 3, 10 and 4, respectively. The total OTUs of the control, model, PP and SASP groups are 365, 346, 354 and 344, respectively. Furthermore, PP and SASP inhibited the Chao1 index compared to the model group. Other indices, such as Shannon, simpson, ACE index and PD-white_tree, show similar trends at least in PP (Table 3). Principal Component Analysis (PCA) (fig. 5B), principal coordinate analysis (PCoA) (fig. 5C), and non-metric multidimensional scaling (NMDS) (fig. 5D) results show that the control and model groups showed fully isolated clusters. However, PP tended to control. These results indicate that PP can improve the diversity of intestinal flora and regulate the intestinal flora towards normal. As shown in table 4, there were significant differences in relative abundance between the model and control groups at the gate level for bacteroides, proteus, deferiprone and campylobacter, etc. (fig. 5E). PP (100 mg/kg) significantly reversed these changes compared to the model group. As shown in fig. 5F, significant changes in nine bacterial genera were observed in the model group. The helicobacter pylori, the murine bacillus, the caprolactam bacillus, the rose bacillus and the columnar bacillus in the model group are obviously reduced, and the escherichia coli, the shigella, the auxiliary shigella, the agrobacterium and the Luo Mbu are obviously increased. However, PP significantly reversed these changes, indicating that PP can improve flora diversity in UC mice.
TABLE 3 alpha diversity detection results
TABLE 4 clustering analysis results at the gate (p) and genus (g) levels
Finally, it should also be noted that the above list is merely a few specific embodiments of the present invention. Obviously, the invention is not limited to the above embodiments, but many variations are possible. All modifications directly derived or suggested to one skilled in the art from the present disclosure should be considered as being within the scope of the present invention.
Claims (5)
1. The application of pumpkin polysaccharide in preparing medicine for treating inflammatory bowel disease is provided.
2. The use according to claim 1, characterized in that: the inflammatory bowel disease is ulcerative colitis or Crohn's disease.
3. Use according to claim 1 or 2, characterized in that: adding pharmaceutically acceptable auxiliary materials into pumpkin polysaccharide, and preparing into any one of the following dosage forms: capsule, tablet, granule, injection, sustained release preparation, oral liquid or dripping pill.
4. Use according to any one of claims 1 to 3, characterized in that the preparation method of pumpkin polysaccharide comprises the following steps:
1) Extracting:
taking pumpkin, and slicing to obtain pumpkin slices;
adding water with the mass of 6+/-0.5 times of that of the pumpkin slices into the pumpkin slices, boiling, and performing primary extraction for 1+/-0.1 hour; filtering to obtain first extractive solution and residue; replacing pumpkin slices with extraction residues, adding water which is 4+/-0.5 times of the mass of the pumpkin slices into the extraction residues, boiling again, and performing secondary extraction for 1+/-0.1 hour; filtering to obtain secondary extract; combining the first extraction solution and the second extraction solution to obtain a combined extraction solution;
2) Cooling and precipitating:
standing the combined extracting solution until the temperature is reduced to room temperature, taking supernatant obtained by standing, and discarding lower-layer sediment obtained by standing;
3) Concentrating, precipitating with ethanol:
concentrating the supernatant in vacuum until the volume is 45-55%, cooling to room temperature, stirring, adding absolute ethyl alcohol until the volume concentration of the ethyl alcohol is 55-60%, standing for more than or equal to 6 hours, and collecting precipitate obtained by alcohol precipitation;
4) Purifying the precipitate obtained by alcohol precipitation to obtain pumpkin polysaccharide.
5. The use according to claim 4, characterized in that the purification of step 4) comprises the steps of:
4.1 Redissolution, deproteinization:
adding water 5.5-6.5 times of the weight of the alcohol precipitation, heating until the alcohol precipitation is dissolved, vacuum concentrating to remove residual ethanol, adding water to complement the weight, and deproteinizing by a Sevage method;
repeating the operations of adding water for dissolving, vacuum concentrating to remove residual ethanol, adding water to supplement weight, deproteinizing by Sevage method for 2 times to obtain deproteinized supernatant;
4.2 Desolventizing, dialysis:
concentrating deproteinized supernatant in vacuum until the supernatant is nearly dry, adding 9+/-0.5 times of water into the concentrated product, transferring the mixture into a semipermeable membrane dialysis bag, fastening an upper opening, placing the semipermeable membrane dialysis bag into a container filled with deionized water, dialyzing the semipermeable membrane dialysis bag for 48 hours, and changing water every 2 hours to obtain dialysis trapped fluid;
4.3 Concentrating, lyophilizing:
concentrating the trapped fluid under vacuum and reduced pressure until the dryness is about 60%, and transferring into a freeze dryer for freeze drying to obtain pumpkin polysaccharide.
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