CN115028752B

CN115028752B - Uniform water-soluble polysaccharide and preparation method and application thereof

Info

Publication number: CN115028752B
Application number: CN202210730818.3A
Authority: CN
Inventors: 李晓波; 刘峰
Original assignee: Sunflower Medicine Ltd
Current assignee: Sunflower Medicine Ltd
Priority date: 2022-06-24
Filing date: 2022-06-24
Publication date: 2023-04-07
Anticipated expiration: 2042-06-24
Also published as: CN115028752A

Abstract

The invention discloses a uniform water-soluble polysaccharide, which comprises mannose, glucose, galactose, arabinose and galacturonic acid, wherein the molar ratio of the mannose to the glucose to the galactose to the arabinose to the galacturonic acid is (5.35-7.40), (3.78-5.98), (0.12-0.28), (0.18-0.38) and (0.10-0.23). In vivo experiments prove that the water-soluble polysaccharide has remarkable activity for treating chronic gastritis, and can be further developed and prepared into a medicament for treating chronic gastritis; the water-soluble polysaccharide also has the activity of regulating the intestinal flora, can be used for preparing medicines for regulating the intestinal flora, has wide application prospect and has good social and economic benefits.

Description

Uniform water-soluble polysaccharide and preparation method and application thereof

Technical Field

The invention relates to the technical field of natural product development, in particular to uniform water-soluble polysaccharide and a preparation method and application thereof.

Background

The natural plant polysaccharide has great potential in the aspects of immunoregulation, tumor resistance, virus resistance, blood sugar reduction, aging resistance, gastritis treatment and the like due to small toxicity and high safety. For example, lentinan can positively regulate the T cell function of intestinal tract Peyer's knot of immunosuppressed mice, thereby playing a role in immunoregulation of the intestinal tract mucous membrane immune system. The ganoderma lucidum water extract and the ganoderma lucidum polysaccharide have obvious inhibiting effect on animal transplantation tumor by in vivo administration. When the influenza vaccine is injected, the compound polysaccharide mixture of the mushroom, the tremella and the tuckahoe is added, so that the morbidity can be reduced, the virus clearance rate is improved, and the organism can recover more quickly after being infected. The Chinese yam polysaccharide has obvious effect of reducing blood sugar of diabetic mice. The tremella polysaccharide has the function of anti-aging by regulating the transcription and expression of a negative regulatory factor p21 of a cell cycle, resisting oxidation and enhancing an immune function. The Dendrobium candidum polysaccharide and the astragalus polysaccharide have good protection and reversion effects on Chronic Atrophic Gastritis (CAG) rats, and can improve the pathological state of gastric mucosa by inhibiting the activation of JAK/p-STAT3 signal pathway. The researches show that the plant polysaccharide from natural sources has potential development value in maintaining or improving the physiological function of organisms.

The Weikangling capsule is one of the Chinese patent medicines commonly used in clinical practice of traditional Chinese medicine, is prepared from natural medicines such as white paeony root, common bletilla pseudobulb, pseudo-ginseng, liquorice, tuckahoe, corydalis tuber, cuttlebone and belladonna extract through a complex process, and is widely applied to the treatment of gastrointestinal diseases such as chronic gastritis, gastric ulcer, gastroesophageal reflux disease and the like. However, there is no report on the application of polysaccharide in Weikangling capsules in maintaining or improving the physiological functions of the body.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The invention aims to provide uniform water-soluble polysaccharide extracted from Weikangling capsules, which is applied to preparing medicines for treating chronic gastritis and regulating intestinal flora.

The invention is realized in the following way:

in a first aspect, the invention provides a homogeneous water-soluble polysaccharide comprising mannose, glucose, galactose, arabinose and galacturonic acid in a molar ratio of (5.35-7.40), (3.78-5.98), (0.12-0.28), (0.18-0.38), (0.10-0.23);

the backbone of the water-soluble polysaccharide includes → 4) - β -D-glucose residues- (1 → and → 4) - β -D-mannose residues- (1 →;

the branched chains of the water-soluble polysaccharide include → 4) - α -D-galacturonic acid residue- (1 →, terminally attached β -D-glucopyranose, → 3, 4) - α -D-mannose residue- (1 →, α -D-glucose residue- (4 → and → 4) -3-O-acetyl- β -D-mannose residue- (1 → sugar residue fragment.

In a second aspect, the invention provides a method for preparing uniform water-soluble polysaccharide, which comprises the steps of taking a weikangling capsule as a raw material, and performing reflux extraction, degreasing, protein removal and purification treatment on the weikangling capsule.

In an alternative embodiment, the reflux extraction step comprises reflux extraction of the weikangling capsule powder and centrifugation to obtain a weikangling capsule mixed extract.

In an alternative embodiment, the defatting step comprises adding absolute ethanol to the mixed extract of Weikangling capsules, standing, and centrifuging to obtain a precipitate.

In an alternative embodiment, the deproteinizing step comprises adding water and Sevag reagent to the precipitate, mixing, centrifuging, collecting the supernatant, and concentrating to obtain the deproteinized water-soluble crude polysaccharide.

In an alternative embodiment, the purification step comprises dialyzing the water-soluble crude polysaccharide and drying.

In a third aspect, the invention also provides an application of the soluble polysaccharide in preparing a medicament for treating chronic gastritis.

In a fourth aspect, the invention also provides the application of the soluble polysaccharide in preparing a medicament for reducing the levels of inflammatory factors TNF-alpha, IL-6 and IL-1 beta.

In a fifth aspect, the invention also provides the application of the soluble polysaccharide in preparing a medicament for regulating intestinal flora.

In a sixth aspect, the invention also provides a pharmaceutical composition, which comprises the above soluble polysaccharide, and a pharmaceutically acceptable carrier.

The invention has the following beneficial effects:

the invention provides a homogeneous water-soluble polysaccharide, and in vivo experiments prove that the water-soluble polysaccharide has remarkable activity for treating chronic gastritis and can be further developed and prepared into a medicament for treating chronic gastritis; meanwhile, the water-soluble polysaccharide also has the activity of regulating the intestinal flora, can be used for preparing medicines for regulating the intestinal flora, has wide application prospect and has good social and economic benefits.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 is a UV-visible spectrum of polysaccharide WP in example 2;

FIG. 2 is a GPC-MALLS-RI chromatogram of the polysaccharide WP of example 2;

FIG. 3 is an IR spectrum of polysaccharide WP of example 2;

FIG. 4 is an HPLC chromatogram of the monosaccharide derivative product of example 2 after complete hydrolysis of the polysaccharide, in which: 1-mannose, 2-rhamnose, 3-glucuronic acid, 4-galacturonic acid, 5-glucose, 6-galactose, 7-xylose, 8-arabinose, 9-fucose;

FIG. 5 is the methylation map of polysaccharide WP in example 2;

FIG. 6 is of polysaccharide WP from example 2 ¹ H NMR nuclear magnetic resonance image;

FIG. 7 shows the preparation of polysaccharide WP from example 2 ¹³ C NMR nuclear magnetic resonance image;

FIG. 8 is the HSQC NMR spectra of polysaccharide WP in example 2;

FIG. 9 shows the polysaccharide WP of example 2 ¹ H- ¹ H COSY nuclear magnetic resonance image;

FIG. 10 is an HMBC NMR chart of polysaccharide WP from example 2;

FIG. 11 is a morphological feature diagram of the polysaccharide WP in example 2, showing that: a to B, scanning Electron Microscope (SEM) observation pictures (500X, 2000X); C-D Atomic Force Microscope (AFM) observation diagrams (2D, 3D);

FIG. 12 is a pathological diagram of the treatment of chronic gastritis in each group in example 3;

FIG. 13 is a graph of the modulation of inflammatory factor levels (TNF-. Alpha., IL-6, IL-1. Beta.) by groups of example 3; in comparison with the blank set, the results, ^# P<0.05， ^## P<0.01， ^### P<0.001; in comparison with the set of models, ^* P<0.05， ^** P<0.01， ^*** P<0.001。

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are conventional products which are not indicated by manufacturers and are commercially available.

Weikangling capsule is a Chinese patent medicine consisting of white peony root, bletilla striata, pseudo-ginseng, liquorice, tuckahoe, corydalis tuber, cuttlebone and belladonna extract and is commonly used for treating various stomach discomforts. The inventor of the invention discovers through research that a new polysaccharide can be extracted from the Weikangling capsule.

Based on the above, the invention provides a uniform soluble polysaccharide, which is named as WP (polysaccharide of Weikangling capsules), and comprises mannose, glucose, galactose, arabinose and galacturonic acid, wherein the molar ratio of the mannose, the glucose, the galactose, the arabinose and the galacturonic acid is (5.35-7.40), (3.78-5.98), (0.12-0.28), (0.18-0.38) and (0.10-0.23).

In some embodiments, the molar ratio of mannose, glucose, galactose, arabinose, and galacturonic acid is (5.35-6.75): (4.75-5.75): (0.18-0.26): 0.20-0.35): 0.12-0.17.

The backbone of the polysaccharide WP comprises → 4) - β -D-glucose residues- (1 → and → 4) - β -D-mannose residues- (1 →.

The branched chains of the polysaccharides WP include → 4) - α -D-galacturonic acid residue- (1 →, terminally attached β -D-glucopyranose, → 3, 4) - α -D-mannose residue- (1 →, α -D-Glcp- (4 → and → 4) -3-O-acetyl- β -D-mannose residue- (1 → sugar residue fragment.

In some embodiments, the total sugar content in the polysaccharide WP is 80.0% -95.0%; the water-soluble polysaccharide had a weight average molecular weight of 54.8kDa, a number average molecular weight Mn of 37.69kDa and a distribution index (Mw/Mn) of 1.454.

As a general technical concept, the invention also provides a preparation method of the polysaccharide with uniform solubility, which comprises the following steps:

(1) Mixing the Weikangling capsule powder with distilled water, wherein the ratio of material to liquid is 1:15 to 1:25, extracting under reflux for 1 time under a slightly boiling state, centrifuging at 3000r/min for 10min, and collecting the supernatant to obtain mixed extractive solution of WEIKANGLING Capsule.

Specifically, the feed-liquid ratio of the weikangling capsule powder to the distilled water may be 1: 15. 1:20 or 1:25, may be 1:15 to 1: any ratio within the range of 25.

(2) Slowly adding 4 times of anhydrous ethanol into the extracting solution obtained in the step (1) while stirring, standing overnight at 4 ℃, centrifuging at 3000r/min for 10min, and collecting precipitate.

(3) Re-dissolving the precipitate obtained in step (2) with an appropriate amount of water, adding Sevag reagent (chloroform: n-butanol = 5) in a volume ratio of aqueous solution to Sevag reagent of 3:1 to 5:1, mixing and oscillating for 10min, centrifuging for 10min at 3000r/min, reserving supernatant, repeating the steps for 2 times, collecting supernatant, and concentrating to obtain the protein-removed water-soluble crude polysaccharide.

Specifically, the volume ratio of the aqueous solution to the Sevag reagent can be 3: 1. 4:1, may be 1:15 to 1: any ratio within the range of 25.

(4) Preparing the deproteinized water-soluble crude polysaccharide obtained in the step (3) into a solution of 20mg/mL by using deionized water, dialyzing for 24-72h by using a dialysis bag, wherein the molecular interception amount of the dialysis bag is more than 3000Da, and freeze-drying to obtain water-soluble polysaccharide WP.

Specifically, the dialysis time may be 24h, 48h or 72h, and may be any time between 24-72 h.

In order to obtain a polysaccharide of higher purity, the dialysis bag used in the dialysis step has a molecular cut-off of > 3000Da.

The invention also provides application of the soluble polysaccharide in preparing a medicament for treating chronic gastritis.

The invention also provides application of the soluble polysaccharide in preparing a medicament for reducing the levels of inflammatory factors TNF-alpha, IL-6 and IL-1 beta.

In vivo experiments prove that the polysaccharide WP can remarkably reduce mucosal injury of rats with chronic gastritis in an ethanol-induced rat model with chronic gastritis, and specifically, the polysaccharide WP can reduce the levels of inflammatory factors (TNF-alpha, IL-6 and IL-1 beta) in the serum of the rats.

The invention also provides application of the soluble polysaccharide in preparing a medicament for regulating intestinal flora.

The invention also provides a pharmaceutical composition, which comprises the soluble polysaccharide and a pharmaceutically acceptable carrier.

The polysaccharide WP can be used as an active site to be used for preparing a pharmaceutical composition together with other natural extracts, chronic gastritis treatment medicines (such as omeprazole and the like) and pharmaceutically acceptable excipients or auxiliary materials.

Alternatively, the pharmaceutical composition may be a tablet, a capsule, a granule, a syrup, a suspension, a solution, a dispersion, a sustained-release preparation for oral or non-oral administration, an intravenous injection preparation, a subcutaneous injection preparation, an inhalation preparation, or a transdermal preparation.

The pharmaceutically acceptable carrier in the present invention refers to pharmaceutically acceptable carriers well known to those skilled in the art, and the pharmaceutically acceptable carriers of the present invention include, but are not limited to: fillers, wetting agents, binders, disintegrants, lubricants, binders, glidants, taste masking agents, surfactants, preservatives, and the like.

In the above pharmaceutical compositions, the proportion of active ingredient may vary and may represent any proportion other than zero by weight of a given unit dosage form.

The method for preparing the medicine adopts a column chromatography method, has mild conditions, high efficiency, simplicity and convenience, and stronger universality, and has very high popularization and application values.

The features and properties of the present invention are described in further detail below with reference to examples.

Example 1

The implementation provides a preparation method of uniform water-soluble polysaccharide, which comprises the following specific steps:

s1, adding distilled water (material-liquid ratio is 1;

s2, slowly adding 4 times of absolute ethyl alcohol into the mixed extract of the Weikangling capsules while stirring, standing overnight at 4 ℃, centrifuging for 10min at 3000r/min, and collecting precipitates;

s3, redissolving the precipitate obtained in the S1 by using a proper amount of distilled water, adding a Sevag reagent (chloroform: n-butyl alcohol =5:1, v/v), wherein the volume ratio of the aqueous solution to the Sevag reagent is 4:1, mixing, oscillating for 10min, centrifuging for 10min at 3000r/min, and keeping a supernatant;

s4, repeating the operation of S2 for 2 times, collecting supernatant, and concentrating to obtain the deproteinized water-soluble crude polysaccharide;

s5, preparing the deproteinized water-soluble crude polysaccharide into a solution of 20mg/mL with deionized water, dialyzing for 48h through a dialysis bag (cut off Mw 3500 Da), and freeze-drying to obtain the water-soluble polysaccharide (WP), wherein the yield is 26.9%.

Example 2

This example provides a homogeneous water-soluble polysaccharide WP and provides a structural characterization thereof. The specific operation is as follows:

(1) And (3) total sugar content determination: the total sugar content in the polysaccharide WP obtained in example 1 was determined by the phenol-sulfuric acid method, which specifically was as follows:

preparing a reference solution: accurately weighing 5.02mg of D-glucose reference substance, and diluting with distilled water to a constant volume in a 50mL volumetric flask to obtain a glucose reference substance solution with the concentration of 100.4 mu g/mL.

Sample solution preparation: an appropriate amount of WP was weighed out precisely, and prepared into a solution of 100. Mu.g/mL with distilled water.

And (3) color development reaction: respectively and precisely sucking 0mL, 0.2mL, 0.4mL, 0.6 mL, 0.8 mL and 1.0mL of glucose reference solution into a 20mL test tube with a plug, and supplementing distilled water to 1.0mL to prepare a glucose standard solution with gradient concentration. Precisely sucking 1.0mL of sample solution into a 20mL test tube with a plug, adding 1mL of 5% phenol solution and 5mL of concentrated sulfuric acid into the test tube with the plug, shaking and uniformly mixing, heating in a boiling water bath for 20min, cooling to room temperature, and detecting the absorbance value at 490nm by using an ultraviolet spectrophotometer. A standard curve is drawn with (absorbance value-water absorbance value) as the ordinate (y) and D-glucose concentration as the abscissa (x). The samples were run in parallel 2 times.

The linear regression equation of D-glucose is y =0.0353x +0.0147, r =0.9981, and the linear range is 2.86-14.29 mug/mg. The determination of the total sugar content in WP was found to be 85.87. + -. 4.30%.

(2) Ultraviolet spectrum analysis: the UV absorption of the polysaccharide WP aqueous solution was measured in a scanning mode (200 to 500 nm) by a spectrophotometer, and the analysis results are shown in FIG. 1.

As can be seen from FIG. 1, the polysaccharide WP has almost no absorption peaks at 260 and 280nm in the UV-vis spectrum, indicating that it is free from proteins and nucleic acids.

(3) Homogeneity and molecular weight determination: the polysaccharide WP was prepared as a 3mg/mL solution in ultrapure water, and the molecular weight of the polysaccharide WP was measured by high performance gel chromatography (HPGPC). Wherein, the chromatographic conditions are as follows: the chromatographic column was Shodex Ohpak SB-806HQ in series with SB-804HQ, the detector was a multi-angle laser scattering instrument (MALLS) and a differential detector (RI), the mobile phase was 0.02% sodium azide aqueous solution, the flow rate was 1mL/min, the sample size was 100. Mu.L, and the column temperature was kept at 25 ℃. The results of measuring the molecular weight of WP are shown in FIG. 2.

The calculated weight average molecular weight Mw of WP was 54.8kDa, the number average molecular weight Mn was 37.69kDa, and the distribution index (Mw/Mn) was 1.454.

(4) Infrared analysis: taking a proper amount of polysaccharide WP after freeze-drying, and directly scanning the polysaccharide WP by using a high-flux Fourier infrared (FT-IR) spectrometer within the scanning range of 4000-500 cm ^-1 And recording an infrared spectrogram. The infrared analysis results are shown in fig. 3.

As can be seen from FIG. 3, the polysaccharide WP was at 3349cm ^-1 The broad absorption peak is the stretching vibration of-OH of typical saccharides, caused by intermolecular hydrogen bonds, 2887cm ^-1 The nearby weak absorption is C-H tensile vibration, including-CH ₂ and-CH ₃ Is the characteristic absorption peak of the polysaccharide; 1732cm ^-1 A weak absorption peak at (a) — C = O stretching vibration in acetyl group, indicating the presence of acetyl group in sugar chain; 1646cm ^-1 The weak absorption peak at (b) is-C = O tensile vibration in uronic acid, indicating that uronic acid may be present in the structure and is low, at 1373cm ^-1 And 1242cm ^-1 The weak peak at (A) belongs to-CH ₂ and-CH ₃ The in-plane bending vibration of (2). In addition, at 1148cm ^-1 、1075cm ^-1 And 1021cm ^-1 3 strong absorption bands at position (873 cm) prove the presence of pyranose ^-1 And 808cm ^-1 The characteristic absorption peaks at (A) are considered to be characteristic peaks for the beta-type glucopyranose and mannose.

(5) HPLC monosaccharide composition analysis:

complete acid hydrolysis of polysaccharide WP: accurately weighing 3mg of polysaccharide, placing the polysaccharide in a test tube with a plug, adding 1mL of 2mol/L trifluoroacetic acid solution, shaking and hydrolyzing for 6h at the temperature of 100 ℃ in a water bath, and blowing nitrogen at the temperature of 60 ℃ to dry hydrolysate. Re-dissolving the residue with a small amount of methanol, blow-drying, and repeating for 3 times (for removing the acid hydrolysis solution) to obtain polysaccharide acid hydrolysis sample. The acid hydrolysate was reconstituted with 0.4mL of deionized water to fully hydrolyze the sample solution with the polysaccharides WP.

Preparation of a reference solution: a mixed reference substance containing 43 mu g of glucuronic acid, 41 mu g of xylose, 4558 mu g of glucose, 4512 mu g of mannose, 406 mu g of galactose, 450 mu g of rhamnose, 420 mu g of galacturonic acid, 458 mu g of arabinose and 450 mu g of fucose is precisely prepared by deionized water every 1mL to serve as a mother solution. Diluting the mother liquor step by step to different concentrations for later use.

Derivatization of PMP: 0.4mL of each of the polysaccharide WP completely hydrolyzed sample solution and the mixed reference solution is taken, 0.2mL of 0.3M NaOH solution and 0.2mL of 0.5M PMP methanol solution are added, the mixture is uniformly mixed, derivatization reaction is carried out at 70 ℃ for 2h, the mixture is cooled to room temperature, 0.2mL of 0.3M HCl is added for neutralization, 1mL of trichloromethane is added for vortex and uniform mixing, centrifugation is carried out at 4000r/min for 10min, the steps are repeated for 3 times, supernatant (PMP is removed), the volume is determined to be 1mL, and HPLC analysis is carried out after high-speed centrifugation.

Monosaccharide analysis HPLC chromatographic conditions: taking 0.05M ammonium acetate water solution (A) -acetonitrile (B) as a mobile phase, wherein the elution gradient procedure is 0-8min and 17 percent; 8 to 9min,17 to 18 percent B; 9-35min, 18% by weight of B; the detection wavelength is 250nm; the column temperature is 30 ℃; the flow rate is 1.0 mL/min ^-1 (ii) a The amount of sample was 5. Mu.L.

As shown in fig. 4, the polysaccharide WP is a water-soluble homopolysaccharide composed of mannose (Man), glucose (Glc), galactose (Gal), arabinose (Ara), and galacturonic acid (Gal-a) at a molar ratio of 5.40.

(6) Methylation analysis: 5mg of the polysaccharide WP, dried under reduced pressure, are weighed out, dissolved in 2mL of anhydrous dimethyl sulfoxide (DMSO) and 100mg of NaOH (ground), N ₂ Shaking to react for 30min under the environment, and fully dissolving. Slowly adding 200 mu L of methyl iodide under an ice bath condition, shaking for 10min, then adding 200 mu L of methyl iodide for methylation, standing for 1h, and then adding 2mL of deionized water to terminate the reaction. 2mL of dichloromethane was added for extraction, and the dichloromethane layer was centrifuged and evaporated to dryness. Repeating the above steps for 2-3 times. Adding 1mL of 2M trifluoroacetic acid into the dried product, hydrolyzing at 100 deg.C for 5h, blowing with nitrogen, adding 1mL of 5% ₄ Reducing, reacting for 3h, and dropwise adding acetic acid until the pH range is acidic (no bubbles appear). 2mL of methanol and 1 drop of acetic acid, drying by nitrogen and repeating for four times. VinegarThe anhydride and pyridine were reacted at 1mL each at 100 ℃ for 1 hour, and after adding 2mL of methanol and evaporating to dryness under reduced pressure, no hydroxyl absorption peak was detected by infrared. Finally, the mixture was dissolved in 1mL of chloroform and analyzed by GC-MS.

TABLE 1 analysis of polysaccharide WP methylated alditol acetyl ester results

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The types of glycosidic linkages of the polysaccharides WP can be obtained by methylation analysis, and the results are shown in Table 1 and FIG. 5.GC-MS analysis finally showed that a water-soluble polysaccharide WP in the Weikangling capsules contains 10 sugar residues, of which the main linkage is → 4) -D-Glcp- (1 → and → 4) -D-Manp- (1 →, of which the literal names are → 4) -D-glucopyranose- (1 → and → 4) -D-mannopyranose- (1 →), respectively.

(7) Nuclear magnetic resonance analysis: 30mg of polysaccharide WP was weighed out, dissolved in 0.5mL of heavy water and tested using an ADVANCE NEO 700MHz NMR spectrometer (Bruker, germany) ¹ H NMR、 ¹³ C NMR, DEPT135, COSY, HSQC, HMBC. The NMR results are shown in FIGS. 6 to 10.

The nuclear magnetic signals of the polysaccharide WP are assigned by MestReNova software (version 11.0.1). ¹ H NMR chemical shifts (. Delta.) of terminal hydrogens of multiple groups _H 5.3-4.4 ppm), indicating WP is a heteropolysaccharide comprising multiple sugar residues (fig. 6); ¹³ chemical shift δ is observable by C NMR _C 91.0-105.0ppm、δ _C 68.0-85.0ppm and delta _C 61.3ppm, which are the terminal carbon signal of the sugar residue, the carbon signal on the sugar ring and the C6 signal, respectively, and the chemical shift δ _C 173.5ppm and delta _C 21.1ppm carbonyl and methyl signals, respectively, for acetyl groups (FIG. 7); further observation of the HSQC pattern of WP (FIG. 8) combined with methylation data and literature reports, signals 4.43/103.48,4.56/97.23,4.66/101.23,4.88/100.12,5.01/100.18,5.14/92.93,5.28/100.63 were speculated as → 4) - β -D-Glcp- (1 →, → 4) - α --D-GalAp- (1 →, t-Linked- β -D-Glcp, → 3, 4) - α -D-Manp- (1 →, → 4) - α 1-D-Manp- (1 →, α 0-D-Glcp- (4 →, → 4) -3-O-acetyl- β -D-Manp- (1 →, wherein the names therein are → 4) - β -D-glucopyranose- (1 →, → 4) - α -D-galactopyranosyl acid- (1 →, terminal β -D-glucopyranose, → 3, 4) - α -D-mannopyranose- (1 →, → 4) - β -D-mannopyranose- (1 →, α -D-mannopyranose- (4 → 4) -3-O-acetyl- β -D-mannopyranose- (1 →. ¹ H- ¹ The reliability of the above inference was further verified by H COSY map (fig. 9) and HMBC map (fig. 10). The results of the hydrocarbon signal assignments are shown in table 2.

TABLE 2 method for production of polysaccharide WP in deuterated water ¹ H and ¹³ c NMR chemical shift (ppm)

n.d. represents no signal detected or signal covered.

(8) Morphological feature analysis: the morphological features of the polysaccharide WP were assessed by Scanning Electron Microscopy (SEM). The polysaccharide WP was gold-sputtered using a Q150T ES plus (UK) sputter and observed under vacuum at 500 and 2000 times magnification. Dissolving polysaccharide WP in ultrapure water to prepare a solution of 3 mu g/mL, performing ultrasonic treatment for 1h, dripping 5 mu L of polysaccharide WP solution on the surface of clean mica in an environment at 25 ℃, naturally drying, and then placing the mica in an environment-controlled atomic force microscope (Nanonavi E-sweet, japan) to obtain an image of polysaccharide WP by using a tapping mode. The morphological feature observations are shown in fig. 11.

From the SEM results of fig. 11, the surface and molecular morphology features of the polysaccharide WP can be obtained, which can form stable and irregular sheets, connected sheet-to-sheet by a plurality of smooth long polysaccharide chains, without much clearer detail after enlargement, indicating tight cross-linking between polysaccharide molecules. AFM can provide two-dimensional and three-dimensional images of polysaccharide WP molecules in natural environment, polysaccharide WP has aggregation and winding phenomena in aqueous solution, and the polysaccharide WP is represented by spherical and irregular blocks, which shows that van der Waals force and hydrogen bonds in molecules and among molecules enable the polysaccharide molecules to be mutually connected.

Example 3

The embodiment provides application of polysaccharide WP in preparation of a medicament for treating chronic gastritis and regulating intestinal flora, and the effect of the polysaccharide WP in treating the chronic gastritis is observed by using an ethanol-induced chronic Gastritis (GC) rat model, and the specific steps are as follows:

s1, experimental animals: male SD rats, SPF grade, 160-180 g.

S2, grouping and dosing: blank control (Ctrl) was given 10mL/kg.bw of physiological saline; model group (Mod) given 10mL/kg. D of saline; omeprazole group (OME) as positive drug given 20mg/kg.d omeprazole; the Weikangling capsule water-soluble polysaccharide (WP) group was administered 269.69mg/kg. D of WP with a gavage volume of 10mL/kg.

S3, molding: after 7 days of adaptive feeding, rats were randomly divided into a blank control group and a model group. The model group induced chronic gastritis with 56% ethanol, with a replication cycle of 4 weeks. Before modeling, rats are fasted for 12 hours without water prohibition and 1 hour before intragastric administration, water sources are removed, intragastric administration is carried out by 56% ethanol (8 g/kg) to induce the generation of chronic gastritis, and the normal group is given physiological saline with the same amount and intragastric administration is carried out once every three days.

S4, administration treatment: after completion of the 28 th day molding, rats with abnormal body weight were culled, and the model rats were randomly divided into 3 groups, a Mod group (n = 6) OME group (n = 6), and a WP group (n = 6). The chronic gastritis rats were treated with the drug at the dose described in step 2 starting on day 29 for a period of 7 days. Rat feces were collected in sterile tubes after 12h fasting, and then the rats were sacrificed.

S5, histopathological examination of gastric mucosa: the stomach tissue of the rat was carefully dissected, the stomach contents were washed away with ice saline, the stomach tissue was fixed with 4% paraformaldehyde solution, and then subjected to conventional paraffin embedding, sectioning, and HE staining. The degree of inflammatory activity is divided into: score 0, normal; 1 minute, a small amount of lymphocytes and plasma cells infiltrate and involve 1/3 of the superficial layer of the inherent membrane; 2 min, infiltrating multifocal inflammatory cells, and keeping the accumulated inflammation below the inherent membrane by 2/3;3 minutes, a large amount of inflammatory cells infiltrate and are distributed in a focal manner to reach 2/3;4 points, diffuse inflammatory cell infiltration, even 2/3 abscess formation, with intestinal edema.

S6, detecting inflammatory factors in serum: the determination of IL-6, IL-1. Beta. And TNF-. Alpha.levels in serum was performed according to the ELISA kit instructions.

S7, statistical treatment: the experimental data are expressed as Mean ± SD, and comparison of differences between groups is performed by one-way anova, and a significant difference is considered when the P value is less than 0.05.

The results of histopathological examination of the stomach of rats treated with chronic gastritis in each administration group are shown in the following table 3 and fig. 12:

table 3 pathological scores for treatment of chronic gastritis (mean ± SD, n = 6)

In comparison to the blank set, the results, ^# P<0.05， ^## P<0.01; comparison with model group<0.05，**P<0.01。

As can be seen from Table 3 and FIG. 12, the normal rat gastric mucosa structure has clear layers, the glands are arranged regularly, and the lamina propria has no inflammatory cells or a small amount of inflammatory cells infiltration; compared with the blank group, a large amount of inflammatory cells in the superficial layer of the inherent membrane of the gastric tissue of the model rat are in focal distribution, and the upper gland structure of the mucous membrane is disordered or necrotic, which indicates that the model of the rat with the chronic gastritis induced by the ethanol is successfully modeled; the degree of lesion in rats of the WP group was improved compared to model rats.

The results of the detection of the level of the inflammatory factors in the serum are shown in fig. 13, and compared with the blank group, the levels of IL-6, IL-1 beta and TNF-alpha in the serum of the rat in the model group are respectively increased by 55.85% (P < 0.05), 47.26% (P < 0.05) and 56.33% (P < 0.01), which indicates that the model of the rat with the chronic gastritis induced by ethanol is successfully modeled. Compared with the model group, after the positive drug omeprazole is given, the levels of IL-6, IL-1 beta and TNF-alpha in serum of the rat with the chronic gastritis induced by ethanol are respectively reduced by 43.66% (P < 0.01), 32.94% (P < 0.01) and 31.68% (P < 0.01), which shows that the omeprazole can effectively treat mucosal injury of the rat with the chronic gastritis induced by ethanol. Similarly, after the polysaccharide WP treatment, the serum IL-6, IL-1 beta and TNF-alpha levels of rats with chronic gastritis induced by ethanol are respectively reduced by 32.39% (P < 0.001), 35.99% (P < 0.01) and 29.63% (P < 0.01) compared with the model group, which shows that the polysaccharide WP can effectively treat mucosal injury of rats with chronic gastritis induced by ethanol.

The effect of the composition of fecal enterobacteria of gastritis rats in each administration group is shown in tables 4 and 5:

TABLE 4 analysis of differences between groups of rat fecal microorganisms at the classification level (mean. + -. SD, n = 6)

Table 4 shows the influence of each administration group on the composition of rat fecal enterobacteria at a phylum level, and the results show that the relative abundance of the thiobacteria phylum (Desulfobacterota) of the model control group is remarkably increased, and the relative abundance of the Proteobacteria (Proteobacteria) is remarkably reduced (P <0.05 or P < 0.01). After omeprazole is given, the relative abundance of Proteobacteria (Proteobacteria) can be remarkably up-regulated (P < 0.01), but the relative abundance of firmicutes (Firmcutes) is abnormally reduced (P < 0.05), and the relative abundance of bacteroidetes (bacteroideta) is abnormally increased (P < 0.05), so that the omeprazole has a certain regulating effect on ethanol-induced intestinal flora imbalance of rats, but can cause the imbalance of the abundances of the firmicutes and bacteroides (bacteroideteta), and the possibility that the gastrointestinal side effects of omeprazole and the intestinal flora are closely related is suggested. After WP polysaccharide is given, the relative abundance of Proteobacteria (Proteobacteria) is also remarkably increased (P is less than 0.01), the relative abundance of WP can be adjusted to a normal rat by actinomycetemcomia (Actinobacterium), bacteroidetea (Bacteroidotta) and desulfobacteria (Desulfobacterota), but the WP has no significance, so that the WP has a good adjusting effect on ethanol-induced intestinal flora imbalance of the rat, and the WP is taken as an omeprazole carrier for co-administration and has the characteristic of improving the gastrointestinal side effect of omeprazole.

TABLE 5 analysis of differences between groups of rat fecal microbiology at the classification level of genus (mean. + -. SD, n = 6)

In comparison with the blank set, the results, ^# P<0.05， ^## P<0.01; comparison with model group<0.05，**P<0.01。

Table 5 shows the effect of the individual administration groups on the composition of fecal enterobacteria in rats at the genus level, and the results show that the relative abundance of Weissella (Weissella), eubacterium _ brachy _ group and Staphylococcus (Staphylococcus) in the model control group is significantly increased, and the relative abundance of Lactobacillus (Lactobacillus), norank _ f _ norrank _ o _ Clostridium _ UCG-014, lactobacillus dumi (Dubosiella) and Corynobacteriaceae-UCG _002 (Coriobacteriaceae _ UCG-002) is significantly decreased (P <0.05 or P < 0.01). After omeprazole administration, the relative abundance of norak _ f _ norak _ o _ clostridium _ UCG-014 could be significantly up-regulated (P < 0.01), the relative abundance of Weissella (Weissella), eubacterium _ brochy _ group and Staphylococcus (Staphylococcus) could be significantly down-regulated (P <0.05 or P < 0.01), but the relative abundance of dominant Lactobacillus (Lactobacillus) was abnormally reduced (P < 0.01), indicating that omeprazole has some regulatory effect on ethanol-induced intestinal flora dyscrasia in rats, but causes abnormal disturbance of the abundance of dominant Lactobacillus (Lactobacillus). After WP polysaccharide is given, the relative abundance of Lactobacillus (Lactobacillus), lactobacillus dummifusae (Dubosiella) and Coriobacter-UCG _002 (Coriobacteraceae _ UCG-002) can be remarkably up-regulated (P <0.05 or P < 0.01), and the relative abundance of Weissella (Weissella) and Staphylococcus (Staphylococcus) can be remarkably down-regulated (P <0.05 or P < 0.01), so that WP has a good regulating effect on ethanol-induced rat intestinal flora imbalance, and further proves that WP is co-administered as an omeprazole carrier has the characteristic of being capable of improving the gastrointestinal side effect of omeprazole.

In conclusion, the invention extracts a new uniform soluble polysaccharide from the Weikangling capsule, and in vivo experiments prove that the water soluble polysaccharide has obvious activity for treating chronic gastritis and activity for regulating intestinal flora, can be further developed and prepared into medicines for treating chronic gastritis and regulating intestinal flora, has wide application prospect, and has good social benefit and economic benefit.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A preparation method of water-soluble polysaccharide is characterized in that the preparation method comprises the steps of taking Weikangling capsules as raw materials, and carrying out reflux extraction, degreasing, protein removal and purification treatment on the Weikangling capsules;

the reflux extraction step comprises reflux extraction of the Weikangling capsule powder and distilled water under a slightly boiling state, and centrifugation to obtain a Weikangling capsule mixed extract;

the degreasing step comprises the steps of adding absolute ethyl alcohol into the mixed extracting solution of the Weikangling capsules, standing, centrifuging and obtaining precipitates; the alcohol precipitation ratio of the mixed extract of the Weikangling capsule to the absolute ethyl alcohol is 1:4;

the protein removing step comprises the steps of adding water and a Sevag reagent into the precipitate, uniformly mixing, centrifuging, collecting supernatant, and concentrating to obtain the protein-removed water-soluble crude polysaccharide;

the purification step comprises the steps of dialyzing and drying the water-soluble crude polysaccharide, wherein the molecular interception of a dialyzing bag in dialysis is more than 3000Da, and the dialyzing time is 24-72 h.

2. The preparation method according to claim 1, wherein the feed-to-liquid ratio of the Weikangling capsule powder to the distilled water in the reflux extraction step is 1:15 to 1:25; the volume ratio of water to Sevag reagent in the protein removing step is 3:1 to 5:1; the molecular interception of the dialysis bag dialyzed in the purification step is 3500Da, and the dialysis time is 48h.

3. The method according to claim 2, wherein the ratio of the raw materials of the Weikangling capsule powder in the reflux extraction step is 1.

4. The method according to claim 2, wherein the volume ratio of water to Sevag reagent in the protein removal step is 4:1.

5. the method of claim 2, further comprising repeating the protein removal step.

6. The method of claim 5, wherein the protein removal step is repeated 2 times.

7. The water-soluble polysaccharide obtained by the method for producing a water-soluble polysaccharide according to any one of claims 1 to 6, wherein the water-soluble polysaccharide comprises mannose, glucose, galactose, arabinose and galacturonic acid in a molar ratio of (5.35 to 7.40), (3.78 to 5.98), (0.12 to 0.28), (0.18 to 0.38), (0.10 to 0.23);

the backbone of the water-soluble polysaccharide comprises → 4) -D-glucopyranose- (1 → and → 4) -D-mannopyranose- (1 →;

the branched chains of the water-soluble polysaccharide include → 4) - β -D-glucopyranose- (1 →, → 4) - α -D-galactopyranouronic acid- (1 →, terminal β -D-glucopyranose, → 3, 4) - α -D-mannopyranose- (1 →, → 4) - β -D-mannopyranose- (1 →, α -D-glucopyranose- (4 →, → 4) -3-O-acetyl- β -D-mannopyranose- (1 →).

8. The water-soluble polysaccharide of claim 7, wherein the molar ratio of mannose, glucose, galactose, arabinose and galacturonic acid is (5.35-6.75), (4.75-5.75), (0.18-0.26), (0.20-0.35) and (0.12-0.17).

9. The water-soluble polysaccharide according to claim 8, wherein the total sugar content of the water-soluble polysaccharide is 80.0% to 95.0%; the water-soluble polysaccharide has a weight average molecular weight of 54.8kDa, a number average molecular weight Mn of 37.69kDa, and a distribution index (Mw/Mn) of 1.454.

10. Use of a water-soluble polysaccharide as claimed in any one of claims 7 to 9 in the manufacture of a medicament for the treatment of chronic gastritis.

11. Use of a water-soluble polysaccharide as claimed in any one of claims 7 to 9 in the manufacture of a medicament for reducing the levels of the inflammatory factors TNF- α, IL-6 and IL-1 β.

12. Use of a water-soluble polysaccharide as claimed in any one of claims 7 to 9 in the manufacture of a medicament for modulating the intestinal flora.

13. A pharmaceutical composition comprising the water-soluble polysaccharide of any one of claims 7 to 9 in combination with a pharmaceutically acceptable carrier.