Application of polygonatum cyrtonema polysaccharide in preparation of medicine for treating ulcerative colitis or reducing blood glucose

Abstract

The invention provides application of polygonatum cyrtonema polysaccharide in preparation of medicines for treating ulcerative colitis or reducing blood glucose. A main chain of a sugar chain structure of the homogeneous polysaccharide PCP2 in polygonatum cyrtonema is mainly composed of-> 1)-beta-D-Fur-(2->,-> 1, 6)-beta-D-Fur-(2-> and-> 6)-alpha-D-Glcp-(1-> and beta-D-Fur-(2->), a side chain is mainly composed of beta-D-Fur-(2-> and-> 2)-beta-D-Fur-(6-> at a C-6 position of-> 1, 6)-beta-D-Fur-(2->), Fur represents furanose, Glcp represents glucopyranose, Glcp represents glucopyranose, and Glcp represents glucopyranose. The novel polysaccharide compound can relieve intestinal injury of mice, increase expression of tight nectin and mucoprotein, relieve oxidative stress reaction, promote production of short-chain fatty acid and improve composition of intestinal flora, and can be applied to the field of medicine as a medicine for treating ulcerative colitis. Meanwhile, the homogeneous polysaccharide PCP2 in polygonatum cyrtonema can significantly inhibit the activity of alpha-amylase and alpha-glucosidase, can also promote the ability of insulin resistance 3T3-L1 fat cells to uptake and consume glucose, shows a significant hypoglycemic effect, and has a good development prospect in the aspect of preparation of hypoglycemic drugs.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and particularly to the application of polygonatum cyrtonema polysaccharide in the preparation of drugs for treating ulcerative colitis or for hypoglycemic use. Background Art

[0002] Colitis refers to the inflammation of the colonic mucosa. Common types include acute and chronic colitis. Acute colitis is usually caused by infection, drugs, or food, while chronic colitis is mostly caused by abnormal immune system or genetic factors, such as ulcerative colitis and Crohn's disease. The main histological feature of colitis is the inflammatory reaction of the colonic mucosa, accompanied by edema, bleeding, ulcers, and necrosis. In recent years, the incidence of colitis has been increasing year by year and has become one of the important public health problems globally. Although the cause of colitis is not fully understood, it is usually closely related to abnormal immune responses.

[0003] The biochemical characteristics of diabetes are manifested as hyperglycemia, and typical clinical manifestations such as polyuria, polydipsia, polyphagia, and weight loss, that is, the "three more and one less" symptoms, can occur. Diabetes is a metabolic disorder-related disease, characterized by the pancreas being unable to secrete insulin or insulin resistance, and is considered a life-threatening serious disease.

[0004] Polysaccharides are widely present in the cell walls of plants, animals, and microorganisms. Natural polysaccharides have various biological effects, such as lipid-lowering, hypoglycemic, antioxidant, antitumor, immunomodulatory, and anti-inflammatory effects, and can directly affect the body's material and energy metabolism, having important medicinal value. Polygonatum contains rich polysaccharide components, and its chemical structure is complex. There are significant differences in the content, structure, and activity of polygonatum polysaccharides from different sources and varieties. Polygonatum cyrtonema Hua (PCH) is a perennial herb in the genus Polygonatum of the family Liliaceae, with the effects of tonifying the kidney and essence and moistening dryness. It is one of the three species of polygonatum included in the Chinese Pharmacopoeia (2020 Edition) and is widely distributed in southern China. Polygonatum cyrtonema polysaccharide is the main active ingredient in Polygonatum cyrtonema Hua and has been proven to have pharmacological activities such as antioxidant, lipid-lowering, anti-atherosclerotic, antitumor, and anti-fatigue effects, and has broad development prospects in the medical field. Summary of the Invention

[0005] In view of this, the present invention proposes the application of polygonatum cyrtonema polysaccharide in the preparation of drugs for treating ulcerative colitis or for hypoglycemic use.

[0006] The technical solution of the present invention is realized as follows:

[0007] The application of polygonatum cyrtonema polysaccharide in the preparation of drugs for hypoglycemic use, wherein the polygonatum cyrtonema polysaccharide is composed of fructose and glucose, and the sugar chain structure is as shown in Formula I;

[0008]

[0009] Among them, Fruf represents fructofuranose, and Glcp represents glucopyranose.

[0010] Use of polygonatum cyrtonema polysaccharide in the preparation of a drug for treating ulcerative colitis, wherein the polygonatum cyrtonema polysaccharide is composed of fructose and glucose, and the sugar chain structure is as shown in Formula II;

[0011]

[0012] Among them, Fruf represents fructofuranose, and Glcp represents glucopyranose.

[0013] Furthermore, the molar ratio of the fructose to the glucose is 92.33:7.67.

[0014] Furthermore, the weight-average molecular weight of the polygonatum cyrtonema polysaccharide is 2087 Da, and the number-average molecular weight is 1599 Da.

[0015] Furthermore, the preparation method of the polygonatum cyrtonema polysaccharide includes:

[0016] S1. Dry and crush the fresh rhizome of polygonatum cyrtonema to obtain polygonatum cyrtonema rhizome powder;

[0017] S2. Add the polygonatum cyrtonema rhizome powder to water for extraction, centrifuge, concentrate the supernatant, add absolute ethanol to form a mixed solution, perform low-temperature alcohol precipitation, centrifuge, and retain the precipitate to obtain crude polygonatum cyrtonema polysaccharide;

[0018] S3. Redissolve the crude polygonatum cyrtonema polysaccharide in water, centrifuge, decolorize and concentrate the supernatant, filter through a membrane filter, use Sevage reagent to remove proteins, perform low-temperature dialysis, and freeze-dry to obtain polygonatum cyrtonema polysaccharide component P1;

[0019] S4. Disperse the polygonatum cyrtonema polysaccharide component P1 in water again, filter, perform DEAE-52 cellulose anion exchange chromatography, elute, concentrate, perform low-temperature dialysis, and freeze-dry to obtain polygonatum cyrtonema polysaccharide component P2;

[0020] S5. Add the polygonatum cyrtonema polysaccharide component P2 to water, pass through a Sephadex G-100 chromatographic column, elute, concentrate, perform low-temperature dialysis, and freeze-dry to obtain the target homogeneous polygonatum cyrtonema polysaccharide component, named PCP2.

[0021] Furthermore, step S1 of the preparation method of the polygonatum cyrtonema polysaccharide is specifically: wash and remove the whiskers of the fresh rhizome of polygonatum cyrtonema, cut it into 6-8 mm thin slices, dry at 55-65 °C for 72-96 h, and crush it with a pulverizer through a 60-mesh sieve.

[0022] Further, it is characterized in that in step S2 of the preparation method of polygonatum cyrtonema polysaccharide, the material-liquid ratio of the polygonatum cyrtonema rhizome powder to water is 1:20 - 25 g / mL; the extraction temperature is 90 - 100 °C, and the time is 1.5 - 2 h; the addition amount of absolute ethanol is 5 times the volume of the mixed solution; the low-temperature alcohol precipitation is carried out at 0 - 4 °C for 10 - 12 h.

[0023] Further, in step S3 of the preparation method of polygonatum cyrtonema polysaccharide, the material-liquid ratio of the crude polygonatum cyrtonema polysaccharide to water is 1:200 - 300 g / mL; the redissolution is to raise the temperature to 55 - 65 °C and stir until clear; the centrifugation is carried out at 12000 - 15000 rpm for 10 - 20 min; the decolorization and concentration are specifically as follows: use D101 macroporous resin for decolorization, and concentrate the decolorized solution to 0.1 - 0.2 of the original volume; the low-temperature dialysis is carried out in a dialysis bag with a molecular weight cut-off of 1000 Da at 0 - 4 °C for 48 - 72 h.

[0024] Further, in step S4 of the preparation method of polygonatum cyrtonema polysaccharide, the material-liquid ratio of the polygonatum cyrtonema polysaccharide component P1 to water is 1:100 - 200 g / mL; the low-temperature dialysis is carried out in a dialysis bag with a molecular weight cut-off of 300 Da at 0 - 4 °C for 10 - 12 h.

[0025] Further, in step S5 of the preparation method of polygonatum cyrtonema polysaccharide, the material-liquid ratio of the polygonatum cyrtonema polysaccharide component P2 to water is 1:150 - 200 g / mL; the low-temperature dialysis is carried out in a dialysis bag with a molecular weight cut-off of 300 Da at 0 - 4 °C for 10 - 12 h.

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0027] 1. The homogeneous polysaccharide PCP2 obtained from polygonatum cyrtonema in the present invention has a main chain of the sugar chain structure mainly composed of →1)-β-D-Fruf-(2→, →1,6)-β-D-Fruf-(2→, →6)-α-D-Glcp-(1→, β-D-Fruf-(2→, and the side chain is mainly composed of β-D-Fruf-(2→, →2)-β-D-Fruf-(6→ with the connection position at the C-6 position of →1,6)-β-D-Fruf-(2→. Among them, Fruf represents furan fructose, and Glcp represents pyran glucose. This new polysaccharide compound can reduce intestinal damage in mice, increase the expression of occludin and mucin, reduce oxidative stress response, promote the production of short-chain fatty acids, and improve the structure of the intestinal flora, and can be used as a drug for treating ulcerative colitis in the medical field.

[0028] 2. The homogeneous polysaccharide PCP2 from Polygonatum cyrtonema Hua can significantly inhibit the activities of α-amylase and α-glucosidase, and can also promote the ability of insulin-resistant 3T3-L1 adipocytes to uptake and consume glucose, showing a significant hypoglycemic effect, and has good development prospects in the preparation of hypoglycemic drugs. Description of the Drawings

[0029] Figure 1 It is the extraction flow chart of the homogeneous component PCP2 of Polygonatum cyrtonema Hua polysaccharide.

[0030] Figure 2 It is the result diagram of the purity and molecular weight of PCP2. Among them, A is the elution curve of the DEAE-52 cellulose column, and B is the analysis diagram of high performance gel permeation chromatography (HPGPC).

[0031] Figure 3 It is the infrared spectrum diagram of PCP2.

[0032] Figure 4 It is the monosaccharide composition diagram of PCP2.

[0033] Figure 5 It is the methylation result diagram of PCP2. Among them, A is the total ion current diagram of gas chromatography-mass spectrometry (GC-MS), and B is the secondary fragment ion current diagram.

[0034] Figure 6 It is the nuclear magnetic result diagram of PCP2. Among them, A is 1 the H spectrum diagram, B is 13 the C spectrum diagram, C is the HSQC spectrum diagram, D is the HMBC spectrum diagram, E is the COSY spectrum diagram, and F is the ROESY spectrum diagram.

[0035] Figure 7 It is the main sugar chain structure diagram of PCP2.

[0036] Figure 8 It is the diagram of the effect of PCP2 on the colon health status of mice. A is the change rate of mouse body weight, B is the DAI score, and C is the colon length of mice.

[0037] Figure 9 It is the diagram of the effect of PCP2 on the colon health status of mice. Among them, A is the H&E, AB-PAS, and PSR staining diagrams of colon tissues, B is the histological score of the H&E-stained colon sections; C is the quantitative diagram of the PSR staining area; D is the quantitative diagram of the PAS staining area; E is the number of goblet cells in colon tissues.

[0038] Figure 10Figure showing the effect of PCP2 on tight junction proteins in the mouse colon. In this figure, A is the fluorescence staining of Occludin, B is the fluorescence staining of ZO-1, C is the fluorescence staining of Claudin-1, D is the protein band analysis of ZO-1, Occludin, and Claudin-1 in the colon tight junction, E is the relative protein expression of ZO-1, F is the relative protein expression of Occludin, and G is the relative protein expression of Claudin-1.

[0039] Figure 11 Figure showing the effect of PCP2 on mucin, inflammatory factors and oxidative stress in serum, and inflammatory factors in colon tissue. Among them, A is the fluorescence staining of MUC2, B is the immunohistochemical staining of MUC2, C is the fluorescence intensity of MUC2, D is the IOD measurement of MUC2 immunohistochemistry, E-G are the effects on serum inflammatory factors, H-K are the effects on serum oxidative stress indicators, and L-N are the effects on inflammatory factors in colon tissue.

[0040] Figure 12 Figure showing the effect of PCP2 on the intestinal flora. Among them, A is the Venn diagram of OTUs of the mouse intestinal microbiota, B is the Ace index, C is the Chao1 index, D is the Observed index, E is the Shannon index, F is the principal coordinate analysis (PCoA), G is the non-metric multi-dimensional scaling analysis (NMDS), H is the LEfSe cladogram from phylum to genus level, I is the bar chart of community composition at the phylum level, J is the heat map of community composition at the phylum level, K is the bar chart of community composition at the genus level, and L is the heat map of community composition at the genus level.

[0041] Figure 13 Figure showing the effect of PCP2 on short-chain fatty acids. Among them, A is the acetic acid content, B is the butyric acid content, C is the caproic acid content, and D is the isobutyric acid content.

[0042] Figure 14 Figure showing the effect of PCP2 at different mass concentrations on the inhibition rate of α-amylase.

[0043] Figure 15 Figure showing the effect of PCP2 at different mass concentrations on the inhibition rate of α-glucosidase. Detailed implementation manners

[0044] To better understand the technical content of the present invention, specific examples are provided below to further illustrate the present invention.

[0045] Unless otherwise specified, the experimental methods used in the embodiments of the present invention are all conventional methods.

[0046] Unless otherwise specified, the materials, reagents, etc. used in the embodiments of the present invention can all be obtained from commercial channels.

[0047] The fresh rhizomes of Polygonatum cyrtonema Hua used in the present invention are selected from the rhizomes of Polygonatum cyrtonema Hua from Jiuhua Mountain.

[0048] Example 1

[0049] The preparation method of Polygonatum cyrtonema Hua polysaccharide includes:

[0050] S1. Wash and remove the whiskers of the fresh rhizomes of Polygonatum cyrtonema Hua, cut them into 6 - 8 mm thin slices, dry them at 60 °C for 96 h, pulverize them with a pulverizer and pass through a 60 - mesh sieve to obtain the powder of Polygonatum cyrtonema Hua rhizomes;

[0051] S2. Add 50 g of the powder of Polygonatum cyrtonema Hua rhizomes to 1200 mL of water, heat to 90 °C and extract for 2 h, cool, centrifuge at 8000 rpm for 15 min, retain the supernatant. Add 1200 mL of water to the precipitate and continue to extract at 90 °C for 2 h, centrifuge at 8000 rpm for 15 min. Combine the two supernatants, concentrate the supernatant under reduced pressure to 1000 mL at 60 °C, add 5000 mL of absolute ethanol, perform alcohol precipitation at 4 °C for 12 h, centrifuge at 8000 rpm for 20 min, retain the precipitate to obtain the crude polysaccharide of Polygonatum cyrtonema Hua;

[0052] S3. Add 1 g of the crude polysaccharide of Polygonatum cyrtonema Hua to 300 mL of water, heat to 60 °C and stir until clear. After the precipitate no longer dissolves, centrifuge at 15000 rpm for 15 min, take the supernatant, decolorize it with D101 macroporous resin, concentrate the decolorized solution to 0.2 of the original volume, filter it through a 0.22 μm microporous membrane, remove proteins using Sevage reagent, rotary evaporate to remove Sevage reagent, filter through a 0.22 μm microporous membrane, and then use a dialysis bag with a molecular weight cut - off of 1000 Da to perform dialysis at 4 °C for 72 h and freeze - dry at - 80 °C for 72 h to obtain the polysaccharide fraction P1 of Polygonatum cyrtonema Hua;

[0053] S4. Disperse 200 mg of the polysaccharide fraction P1 of Polygonatum cyrtonema Hua into 40 mL of water again, filter through a 0.22 μm microporous membrane, and perform DEAE - 52 cellulose anion - exchange chromatography. First, elute with pure water at a flow rate of 0.8 mL / min, collect one tube every 5 min for a total of 60 tubes. Then, elute with 0.1 mol / L NaCl at a flow rate of 0.8 mL / min, collect one tube every 5 min for a total of 60 tubes, and use anthrone - sulfuric acid reagent to detect whether the polysaccharide is completely eluted. After the polysaccharide is completely eluted, concentrate the eluate to 0.2 of the original volume under reduced pressure at 60 °C, use a dialysis bag with a molecular weight cut - off of 300 Da to perform dialysis at 4 °C for 72 h and freeze - dry at - 80 °C for 72 h to obtain the polysaccharide fraction P2 of Polygonatum cyrtonema Hua;

[0054] S5. Dissolve 30 mg of the polysaccharide component P2 of Polygonatum cyrtonema Hua in 6 mL of water, filter through a 0.22-μm microporous membrane, elute through a Sephadex G-100 gel chromatography column with deionized water at a flow rate of 0.8 mL / min, collect one tube every 5 min, concentrate the eluate under reduced pressure to 0.2 of the original volume at 60 °C, dialyze using a dialysis bag with a molecular weight cut-off of 300 Da at 4 °C for 72 h, and freeze-dry at -80 °C for 12 h and then at -80 °C for 72 h to obtain the target homogeneous polysaccharide component of Polygonatum cyrtonema Hua, named PCP2.

[0055] Example 2

[0056] Perform result characteristic tests on the PCP2 obtained in Example 1.

[0057] 1. Purity and molecular weight detection: Use the HPGPC method and detect with a high-performance gel permeation chromatography tandem column. Among them, the chromatography column is 3 polymer matrix water-soluble SEC (GFC) chromatography columns (8 × 300 mm) in series, the mobile phase is 0.05 mol / L NaCl solution, the flow rate is 0.65 mL / min, the column temperature is 40 °C, and the injection volume is 30 μL.

[0058] Precisely weigh 5 mg of the sample, add 1 mL of 0.05 mol / L NaCl solution to the sample to prepare a 5 mg / mL test sample solution, centrifuge at 8000 rpm for 10 min, take the supernatant and filter through a 0.22-μm microporous membrane, and then place the sample in a 2-mL injection vial for standby.

[0059] The crude polysaccharide of Polygonatum cyrtonema Hua is obtained by water extraction, alcohol precipitation, decolorization and protein removal from the rhizome powder of Polygonatum cyrtonema Hua in the present invention, with a yield of 8.37% and a protein content of 0.87% (<1%); the crude polysaccharide is separated and purified by DEAE-52 and Sephadex G-100 columns to obtain the homogeneous polysaccharide component PCP2 of Polygonatum cyrtonema Hua.

[0060] Figure 2 In which A is the elution curve of the DEAE-52 cellulose column, and it can be obtained that PCP2 is the minor component in the polysaccharide of Polygonatum cyrtonema Hua; Figure 2 From the HPGPC analysis result of B in, PCP2 shows a symmetric narrow peak, with a retention time of 43.856 min and a purity of 98.68%, indicating that the PCP2 of the present invention is a homogeneous polysaccharide with high purity and concentrated molecular weight. The weight-average molecular weight (Mw) and number-average molecular weight (Mn) of PCP2 are measured to be 2087 Da and 1599 Da respectively, and the polydispersity index (Mw / Mn) is about 1.305, indicating that the PCP2 of the present invention is a polysaccharide with a concentrated molecular weight distribution.

[0061] 2. Infrared spectroscopy analysis: Accurately weigh 1 ± 0.05 mg of PCP2 powder and mix it with analytical pure potassium bromide powder, grind them, and then press them into tablets. Detect the spectrum in the wavelength range of 4000 - 400 cm -1 using an infrared spectrometer (Nexus IS10 FT-IR, Thermo Nicolet, USA).

[0062] Participation Figure 3 , it can be seen that a characteristic O-H stretching vibration peak appears near 3447 cm -1 , a C-H stretching vibration peak appears near 2923 cm -1 , a C=O stretching vibration peak appears at 1633.90 cm -1 , and a C-O-C glycosidic bond stretching vibration peak appears at 1059.80 cm -1 , indicating that there is a pyranose unit in the PCP2 of the present invention. The stretching peak at 912.64 cm -1 indicates that there is a furanose unit in the PCP2 of the present invention. Figure 3 The infrared analysis results show that both furanose and pyranose glycosidic bonds exist in the PCP2 of the present invention.

[0063] 3. Monosaccharide composition analysis of PCP2: Accurately weigh 5 ± 0.05 mg of the polysaccharide sample, add 1 mL of 2 mol / L TFA solution, heat it at 60 °C for 1 h, dry the solution under nitrogen, add methanol for washing, dry it again, and repeat the methanol washing 2 - 3 times. Dissolve the sample in sterile water and transfer it to a chromatographic vial for analysis. Use the Thermo ICS5000+ ion chromatography system (ICS 5000+) of Thermo Fisher Scientific (USA), equipped with Dionex TM CarboPac TM PA20 column (150 × 3.0 mm, 10 μm), with an injection volume of 5 μL. Mobile phase A is H 2 O, mobile phase B is 0.1 mol / L NaOH, and mobile phase C is 0.2 mol / L sodium acetate and 0.1 mol / L NaOH. The column temperature is set at 30 °C, and an electrochemical detector is used to analyze the monosaccharide components.

[0064] Refer to Figure 4 , it can be seen that the PCP2 of the present invention is mainly composed of two monosaccharides, fructose and glucose, with a molar ratio of 92.3:3.67.

[0065] 4. Methylation analysis of PCP2: An Agilent gas chromatography system (Agilent 7890A, Agilent Technologies, USA) was used, equipped 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 (purity not less than 99.999%), with a flow rate of 1.0 mL / min. The inlet temperature was 260 °C. The injection volume was 1 μL, and split injection was used with a split ratio of 10:1. The solvent delay was 2.2 min. It was held at 50 °C for 1.0 min, then heated to 130 °C at a rate of 50 °C / min, and then heated to 230 °C at a rate of 3 °C / min and held for 2 min. The mass spectrometry system used was a quadrupole mass detection system from Agilent Technologies, USA (Agilent 5977B), equipped with an electron impact ionization source (EI) and a MassHunter workstation. For the electron impact ionization source (EI), the inlet temperature was 230 °C, the quadrupole temperature was 150 °C, the electron energy was 70 eV, the scan mode was full scan mode (SCAN), and the mass scan range (m / z) was 30 - 600.

[0066] The sample was dissolved in dimethyl sulfoxide, and sodium hydroxide was added for alkalization incubation. Subsequently, methyl iodide was added in two portions for methylation. Then, water and dichloromethane were added for three washes. The lower dichloromethane phase was collected and evaporated to dryness. Acid hydrolysis was carried out using trifluoroacetic acid at 90 °C, then dried. Sodium borohydride was added for reduction, and the reaction was terminated with acetic acid. After drying multiple times with acetic acid-methanol and methanol, acetic anhydride was added for acetylation at 100 °C, and then extracted with dichloromethane. Finally, the extracted sample was analyzed using an Agilent gas chromatography system (Agilent 7890A, Agilent Technologies, USA) equipped with an HP-5MS capillary column (30 m × 0.25 mm × 0.25 μm, Agilent J&W Scientific, Folsom, USA), with high-purity helium (≥99.999%) as the carrier gas. The analysis conditions were: held at 50 °C for 1 min, heated to 130 °C at a rate of 50 °C / min, then heated to 230 °C at a rate of 3 °C / min and held for 2 min.

[0067] See Figure 5 , combined with the public database of partially methylated alditol acetates (PMAAs), to obtain the methylation analysis results in Table 1.

[0068] Table 1

[0069]

[0070] According to the methylation analysis in Table 1, it shows that PCP2 contains t-Fruf, t-Glcp, 1,2-Fruf, 2,6-Fruf, 1,6-Glcp and 1,2,6-Fruf. Since the content of t-Glcp is only 1.627%, which is less compared to other sugar residues and no signal can be found in the nuclear magnetic resonance, it is not assigned.

[0071] 5. Nuclear magnetic resonance analysis of PCP2: PCP2 was dissolved in D 2 O with a concentration of 20 mg / mL for preparation. Subsequently, a 600 MHz nuclear magnetic resonance (NMR) spectrometer equipped with a dual probe was used to perform one-dimensional and two-dimensional nuclear magnetic resonance analyses ( 1 H NMR, 13 C NMR, COSY, NOESY, HMBC and HSQC), and all relevant spectral data were collected and recorded to analyze the molecular structure of PCP2.

[0072] See Figure 6 , 1 The H NMR signals are mainly concentrated in the region of δ 3.0 - 5.5 ppm, and the chemical shifts of pyranose hydrogens are mainly distributed between δ 4.50 - 5.50 ppm. 13 The C NMR signals mainly appear in the range of 60.0 - 100.0 ppm, and the chemical shifts of pyranose carbons are located between δ 90 - 110 ppm. Multiple pyranose hydrogen signals are observed in this region, indicating the presence of multiple sugar residues in the structure. Combining one-dimensional and two-dimensional NMR spectra, the chemical shift results of each residue are shown in Table 2.

[0073] Table 2

[0074]

[0075] For the chemical signals of residue E, in the COSY spectrum, there is an obvious correlation between δ 5.32 (H1) and δ 3.47, and δ 3.47 can be assigned to H2 of residue E. Similarly, the resonances of H3 to H6 of residue E can also be identified. Due to the low content of 1,6-Glcp, its pyranose carbon signal may not be detected in the 3 C NMR spectrum.

[0076] In the HSQC spectrum, the correlation between δ 5.32 (H1) and δ 92.1 indicates that δ 92.1 is assigned to C1 of residue E. The signals of H2 to H6 identified in the COSY spectrum further determine the chemical shifts of C2 to C6 of residue E. Since the carbon chemical shifts of α- and β-glycosidic bonds are usually less than 100 ppm and greater than 100 ppm respectively, and based on the methylation analysis results, residue E is determined to correspond to →1)-α-D-Glcp-(6→.

[0077] In the HSQC spectrum, no cross-peaks of residues A, B, C, and D were observed, probably because fructose is a ketose and lacks pyranose hydrogen. Based on methylation analysis, the carbon signals at 103.8 ppm, 103.6 ppm, 103.2 ppm, and 102.9 ppm were assigned to →1,6)-β-D-Fruf-(2→, →1)-β--D-Fruf-(2→, β-D-Fruf-(2→, and →6)-β-D-Fruf-(2→, respectively. Therefore, combining the data of COSY, HSQC, and ROESY spectra, the chemical shifts of H1-H6 and C1-C6 hydrogens and carbons of residues A, B, C, and D were determined.

[0078] The HMBC spectrum showed cross-peaks between different residues, indicating the linkage sites and sequences of the residues. The cross-peaks corresponded as follows: C-2 (102.9 ppm) of residue C and H-6 (3.79 ppm) of residue D (C C-2 / DH-6), indicating that C-2 of residue C is linked to H-6 of residue D. Similarly, the cross-peaks of δ103.2 / 3.74 ppm (A C2 / CH6), δ103.6 / 3.69 ppm (B C2 / D H1), δ103.6 / 3.64 ppm (B C2 / E H6), δ103.2 / 3.58 ppm (A C2 / B H1), and δ103.8 / 3.58 ppm (D C2 / B H1) indicated that C-2 of residue A is linked to H-6 of residue C, C-2 of residue B is linked to H-1 of residue D, C-2 of residue B is linked to H-6 of residue E, C-2 of residue A is linked to H-1 of residue B, and C-2 of residue D is linked to H-1 of residue B. Therefore, the sugar chain structure of the polygonatum cyrtonema polysaccharide PCP2 of the present invention is as Figure 7 shown.

[0079] Example 3

[0080] The polygonatum cyrtonema polysaccharide PCP2 obtained by extraction in Example 1 was studied for the treatment of ulcerative colitis.

[0081] Experimental procedure: 60 male C57BL / 6J mice at 8 weeks of age were purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd. After a 7-day adaptation period, the mice were randomly divided into five experimental groups: a normal group, a model group, a positive drug (5-ASA) group, a low-dose PCP2 (PCP-L) group, and a high-dose PCP2 (PCP-H) group, with 12 mice in each group. The normal group drank ordinary drinking water, while the other four groups drank 3% DSS water continuously for 11 days to induce a colitis model. From the 2nd day to the 11th day, the mice in the positive drug (5-ASA) group, the low-dose PCP2 (PCP-L) group, and the high-dose PCP2 (PCP-H) group were respectively intragastrically administered 100 mg / kg of 5-ASA, 50 mg / kg of PCP2, and 100 mg / kg of PCP2, while the normal group and the model group continued to drink regular drinking water.

[0082] 1. The body weight of the mice was monitored daily, and the disease activity index (DAI) was evaluated by the sum of scores for weight loss (0 - 4 points), fecal consistency (0 - 4 points), and rectal bleeding (0 - 4 points). On the 11th day, the mice were euthanized, and their colon tissues were collected. The results are shown in Figure 8 , compared with the model group, treatment with PCP2 of the present invention can significantly improve the weight loss, increase in DAI score, and shortening of colon length caused by DSS.

[0083] 2. After excising the colon of mice at the same site, it was immersed in 4% paraformaldehyde solution for fixation for more than 24 h, and then made into 8-μm sections after paraffin or OCT embedding, which were respectively used for hematoxylin-eosin (H&E), picrosirius red (PSR), or periodic acid Schiff (PAS) staining, and observed under an optical microscope.

[0084] See Figure 9 , H&E staining showed that the mucosal structure of the colon in the model group was severely damaged, the crypts were almost completely lost, and there was significant infiltration of inflammatory cells. After intervention treatment with 5-ASA and PCP2, the tissue damage was significantly reduced. In the high-dose PCP2 group (PCP-H), the crypt structure was almost completely restored, and the infiltration of inflammatory cells was significantly reduced, approaching the level of the normal group, while the effect of the low-dose PCP2 group (PCP-L) was relatively weak. PSR staining showed abnormal deposition of collagen fibers in the model group, indicating severe fibrosis, which was significantly reduced after intervention with 5-ASA or PCP2. AB-PAS staining showed a significant reduction in the number of goblet cells in the model group, and the mucus secretion function was impaired. After treatment, the number of goblet cells increased significantly, and the improvement was most obvious in the high-dose PCP2 (PCP-H) group. It is shown that PCP2 of the present invention, especially at high doses, has a significant therapeutic effect on alleviating tissue damage, reducing fibrosis, and restoring mucus secretion function in UC.

[0085] 3. Immunofluorescence assay: First, the cells were treated with the primary antibody against the protein. After thorough washing with PBS, the secondary antibody was added. The cells were gently rinsed again with PBS and stained with DAPI to highlight the cell nuclei. Finally, a Nikon Eclipse C1 immunofluorescence microscope (Nikon, Japan) was used to visualize and evaluate the tight junction proteins.

[0086] Western blot procedure: 50 mg of colon tissue was homogenized in 500 μL of RIPA lysis buffer containing 1% PMSF (Solarbio, Beijing, China). Subsequently, the protein concentration of the homogenate was determined using a BCA assay kit, and then the samples were heated to promote protein denaturation. After the denaturation step, the samples were loaded onto an SDS-PAGE gel for electrophoresis to separate the proteins in each group. After electrophoresis, the proteins were transferred onto a PVDF membrane. After transfer, the membrane was blocked with a 5% non-fat milk solution for 2 h to prevent non-specific binding. After the blocking step, the membrane was incubated with the primary antibodies overnight at 4°C. The primary antibodies included Claudin 1 (dilution ratio 1:3000), Occludin (1:1000), and ZO-1 (1:1000), all from Abcam, Cambridge, UK. The next day, the membrane was treated with the secondary antibody diluted to 1:1000 and incubated for an additional 2 h. Finally, an ECL chemiluminescence reagent was used in combination with a Tanon 5200 multi-functional automated chemiluminescence imaging system to visualize the protein bands and evaluate the protein expression levels.

[0087] As can be seen from Figures 10A-C, there were significant differences in the expression levels of Occludin, ZO-1, and Claudin-1 among the groups. The fluorescence signals of the tight junction proteins in the normal group were strong and evenly distributed, indicating that the intestinal barrier function was intact. The protein expression in the model group was significantly reduced, the signals were weakened and disordered, showing severe intestinal barrier damage. After treatment with 5-ASA, the expression levels of the three tight junction proteins were significantly restored, the signal intensity increased and was evenly distributed, indicating a repair effect on the intestinal barrier. In the low-dose PCP2 group (PCP-L) of the present invention, there was a certain improvement in protein expression, but the improvement degree was less than that of the 5-ASA group. The high-dose PCP2 group (PCP-H) significantly improved protein expression, and the protein expression level was close to that of the normal group, indicating its excellent intestinal barrier protection and repair ability. PCP2 of the present invention effectively promoted the recovery of tight junction proteins, especially the high-dose PCP2. Figure 10 Figure 10D further verified by Western blot experiments that the effect of PCP2 in alleviating ulcerative colitis may be related to tight junction proteins. Figure 10The E-G results showed that the expression of tight junction proteins in the model group was significantly reduced, reflecting severe intestinal barrier damage. Both 5-ASA and high-dose PCP2 could significantly restore protein expression, indicating their role in intestinal barrier protection and repair.

[0088] 4. Detection of MUC2 protein expression: First, the cells were treated with the primary antibody against the protein. After sufficient washing with PBS, the secondary antibody was added. The cells were gently rinsed again with PBS and stained with DAPI to highlight the cell nuclei. Finally, the MUC2 protein was visualized and evaluated using a Nikon Eclipse C1 immunofluorescence microscope (Nikon, Japan).

[0089] Detection of inflammatory factors and oxidative stress indicators: ELISA kits were used to measure the levels of cytokines (IL-10, IL-6, and IL-1β) in serum and colon tissues. At the same time, ELISA kits were used to analyze the concentrations of SOD, MDA, GSH-Px, and GSH in serum. The results are shown in Figure 11 。

[0090] Figure 11 As can be seen from A-D, different treatments had a significant effect on the expression of MUC2 protein in the mouse intestine. The MUC2 expression in the normal group was strong and uniform, and the intestinal barrier was intact. The DSS-induced colitis model group showed a significant decrease in MUC2 expression and damaged barrier. After intervention with 5-ASA and PCP2, the MUC2 expression was significantly restored, especially in the high-dose PCP2 group, showing a stronger protective effect.

[0091] Figure 11 As can be seen from E-N, PCP2 treatment significantly reduced the levels of inflammatory factors TNF-α and IL-6 in serum and intestinal tissues, and improved the oxidative stress indicators, reduced MDA, and increased SOD activity. It indicates that PCP2 of the present invention can alleviate inflammation and oxidative stress and effectively repair the damaged intestinal barrier function, especially showing better efficacy at high doses.

[0092] 5. 16S rRNA analysis of intestinal flora: In the last three days of the experiment, feces were collected and quickly frozen in liquid nitrogen. Total fecal DNA was extracted, and PCR amplification of the 16S rRNA gene was performed. The PCR products were purified using a Qiagen gel extraction kit (Germantown, USA), and library quality inspection and sequencing were carried out. After quality control, 50,000 high-quality sequences were obtained for each sample, and sequences with a similarity higher than 97% were assigned to the same operational taxonomic unit (OTUs).

[0093] See Figure 12A, Polysaccharides play a key role in regulating the host physiological state and reshaping the gut microbiota, and gut microbiota imbalance is closely related to inflammatory bowel disease (IBD). From Figure 12 As shown in B-E, DSS treatment significantly reduced the diversity of the gut microbiota, while PCP2 intervention treatment significantly increased the number of OTUs, restoring the richness and diversity of the microbiota. From Figure 12 The β-diversity analysis in F-G showed that the gut microbiota composition of the PCP2 group was more similar to that of the normal group, indicating that PCP2 of the present invention effectively regulated the microbiota structure. Figure 12 The LEfSe analysis in H further showed that oral administration of PCP2 enriched short-chain fatty acid-producing bacteria such as the genus Bacteroides, while DSS enriched the pathogenic genus Clostridium. Figure 12 As can be seen from I-J, PCP2 significantly increased the abundances of Firmicutes, Verrucomicrobiota, and Bacteroidota, and inhibited the expansion of Desulfobacterota and Proteobacteria, improving gut microbiota dysbiosis. Figure 12 As can be seen from K-L, PCP2 of the present invention reduced the abundances of the pathogenic bacteria Desulfovibrio and Clostridium, while enriching beneficial bacteria such as Akkermansia muciniphila and the genus Lachnospiraceae, further alleviating intestinal inflammation. Therefore, PCP2 of the present invention increased beneficial bacteria while reducing harmful bacteria, effectively regulating the gut microbiota of colitis mice and alleviating intestinal inflammation.

[0094] 6. Short-chain fatty acid analysis: Using Agilent 7890A / 5975C (Agilent Technologies, USA), the short-chain fatty acids in mouse feces were quantified by GC-MS. 100 mg of fecal samples were mixed with 1 mL of 0.005 mol / L sodium hydroxide solution and 50 μL of 2-methylbutyric acid, and reacted at 4 °C for 2 h. The reaction mixture was centrifuged and derivatized after mixing with distilled water and isopropanol / pyridine solution (3:2, V / V), and then the samples were extracted with n-hexane and subjected to GC-MS analysis. The injection volume was 1 μL, and the results are as Figure 13 shown.

[0095] As can be seen from Figure 13 , the levels of acetic acid, butyric acid, hexanoic acid, and isobutyric acid in the model DSS group were significantly lower than those in the normal group. After the intervention treatment with PCP2 of the present invention, the levels of short-chain fatty acids such as acetic acid, butyric acid, hexanoic acid, and isobutyric acid increased.

[0096] Example 4

[0097] The hypoglycemic activity of the polygonatum cyrtonema polysaccharide PCP2 extracted in Example 1 was studied.

[0098] 1. Inhibitory activity against α-amylase: Using the DNS colorimetric method, acarbose was selected as the positive control, and the inhibition rate of PCP2 at different mass concentrations against α-amylase was determined. Take 1.0 mL of PCP2 at different mass concentrations (0, 0.2, 0.4, 0.6, 0.8, 1.0 mg / mL) each, mix it with an equal volume of α-amylase solution (2 U / mL), incubate in a water bath at 37 °C for 10 min, then add 1.0 mL of soluble 1% starch solution (incubate in a water bath at 37 °C for 5 min) and mix well. After incubating at 37 °C for 10 min, add 2 mL of DNS reagent (1% 3,5-dinitrosalicylic acid, 1.6% sodium hydroxide, and 30% potassium sodium tartrate), and place the mixed reaction solution in a boiling water bath for 5 min. After color development, cool it to room temperature in an ice water bath, dilute it to 1 / 5 of the original mass concentration, and measure the absorbance value of the reaction solution at 540 nm. Use PBS instead of the sample as the negative control, and use PBS instead of the α-amylase solution as the blank control.

[0099] Among them, the α-amylase inhibition rate = [1 - (A 3 - A 2 ) / A 1 × 100%

[0100] A 1 is the absorbance of the blank solution (without the sample to be measured), A 2 is the absorbance of the background solution (without α-amylase), and A 3 is the absorbance of the sample solution to be measured.

[0101] The results are as Figure 14 shown. It can be seen that the inhibition rates of PCP2 at different mass concentrations against α-amylase are different and show a concentration dependence. The effect of PCP2 of the present invention in significantly inhibiting α-amylase is close to that of acarbose, that is, PCP2 of the present invention has a significant hypoglycemic effect.

[0102] 2. Inhibitory activity against α-glucosidase: Using the PNPG method, acarbose was selected as the positive control, and the inhibition rate of the inhibitory activity of PCP2 at different mass concentrations against α-glucosidase was determined. Take 60 μL of PCP2 at different mass concentrations (0, 0.2, 0.4, 0.6, 0.8, 1.0 mg / mL) each, add 120 μL of α-glucosidase solution (1 U / mL) respectively, react at 37 °C for 10 min, then add 120 μL of pNPG (5 mmol / L), and react at 37 °C for 30 min. Finally, add 1 mL of Na 2 CO 3The reaction was terminated with a solution (1 mol / L), and the absorbance at 405 nm was measured. PBS was used as a negative control instead of the sample, and PBS was used as a blank control instead of the α-glucosidase solution.

[0103] Among them, the α-glucosidase inhibition rate = [1 - (A s - A b ) / A 0 × 100%

[0104] A 0 is the absorbance of the blank solution (without the sample to be measured), A b is the absorbance of the background solution (without α-glucosidase), and A s is the absorbance of the sample solution to be measured.

[0105] The results are as Figure 15 shown. It can be seen that the inhibition rates of PCP2 at different mass concentrations on α-glucosidase are different and show a concentration dependence. The effect of PCP2 of the present invention in significantly inhibiting α-glucosidase is close to that of acarbose, that is, PCP2 of the present invention has a significant hypoglycemic effect.

[0106] 3. Growth inhibition of 3T3-L1 cells: Select 3T3-L1 cells with good growth status, prepare a cell suspension of 8 × 10 4 cells / mL, add 100 μL to each well of a 96-well plate, and culture in a cell incubator for 24 h. The experiment was divided into two groups: the blank group added 100 μL of medium to each well; the experimental group added 100 μL of medium containing different mass concentrations of PCP2 (0.1, 0.4, 0.8, 1.2, 1.6, 2.0, 2.4 mg / mL) to each well. Each group was set with 8 replicate wells and incubated for 24 h. Discard the medium, add 100 μL of MTT medium containing 0.5% to each well, continue to incubate for 4 h, discard the medium, add 100 μL of DMSO to each well, wrap the well plate with tin foil, slowly shake on a shaker for 10 min, and measure the absorbance at 570 nm and calculate the cell growth inhibition rate of the sample.

[0107] Among them, the cell growth inhibition rate = (A 0 - A 1 ) / A 0 × 100%

[0108] A 0 represents the absorbance value of the blank group, and A 1 represents the absorbance value of the sample to be measured.

[0109] The results are shown in Table 3.

[0110] Table 3

[0111] Sample Cell growth inhibition rate / % Blank group 100 0.1 mg / mL PCP2 104 0.4 mg / mL PCP2 106 0.8 mg / mL PCP2 110 1.2 mg / mL PCP2 115 1.6 mg / mL PCP2 113 2.0 mg / mL PCP2 108 2.4 mg / mL PCP2 102

[0112] As can be seen from Table 3, PCP2 of the present invention has no toxic effect on 3T3-L1 cells and promotes their proliferation.

[0113] 4. Effect on glucose consumption of insulin-resistant 3T3-L1 adipocytes: When 3T3-L1 preadipocytes have been induced to become mature adipocytes, the experiment is set up as a control group and a model group. 200 μL of DMEM (10% fetal bovine serum) is added to each well in the control group, and 200 μL of DMEM containing dexamethasone (1 μM) is added to each well in the model group. Six replicate wells are set in each group and incubated in a cell culture incubator for 48 h. The supernatant is collected, and the glucose content is detected according to the GOD-POD method to judge the glucose uptake. According to the experimental results, compared with the control group, the glucose consumption rate in the model group decreased significantly by 31.1%, with statistical significance, indicating that an insulin resistance model was successfully established. After successful modeling, the experiment is divided into four groups: 200 μL of DMEM (10% fetal bovine serum) medium is added to each well in the normal group, 200 μL of DMEM medium containing dexamethasone (1 μM) is added to each well in the model group, 200 μL of DMEM medium containing metformin (5 mM) is added to each well in the positive control group; 200 μL of medium containing PCP2 at different mass concentrations (0.1, 0.4, 0.8, 1.2, 1.6, 2.0, 2.4 mg / mL) is added to each well in the sample group. They are incubated in a cell culture incubator for 48 h, and the glucose content is detected according to the GOD-POD method to judge the glucose uptake. The results are shown in Table 4.

[0114] Table 4

[0115] Sample Glucose consumption / % Blank group 100 Model group 69.9 Positive control group 178.5 0.1 mg / mL PCP2 103.5 0.4 mg / mL PCP2 115.4 0.8 mg / mL PCP2 122.6 1.2 mg / mL PCP2 132.2 1.6 mg / mL PCP2 152.6 2.0 mg / mL PCP2 169.7 2.4 mg / mL PCP2 170.3

[0116] As can be seen from Table 4, compared with the model group, the glucose consumption in the positive control group increased significantly, and the glucose consumption in the PCP2 groups at different mass concentrations increased in a concentration-dependent manner, both higher than the blank group, indicating that PCP2 of the present invention has an obvious improvement effect on insulin resistance and is concentration-dependent.

[0117] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. The use of Polygonatum cyrtonema polysaccharide in the preparation of a drug for hypoglycemic effect, characterized in that: The Polygonatum cyrtonema polysaccharide is composed of fructose and glucose, and the sugar chain structure is shown in Formula I; Among them, Fruf stands for furanose and Glcp stands for glucopyranose.

2. The use of Polygonatum cyrtonema polysaccharide in the preparation of a drug for treating ulcerative colitis, characterized in that: The Polygonatum cyrtonema polysaccharide is composed of fructose and glucose, and the sugar chain structure is shown in Formula II; Among them, Fruf stands for furanose and Glcp stands for glucopyranose.

3. The use according to claim 1 or 2, characterized in that: The molar ratio of fructose to glucose is 92.33:7.

67.

4. The use according to claim 1 or 2, characterized in that: The weight average molecular weight of the Polygonatum cyrtonema polysaccharide is 2087Da, and the number average molecular weight is 1599Da.

5. The use according to claim 1 or 2, characterized in that: The preparation method of Polygonatum cyrtonema polysaccharide comprises: S1. Drying and crushing fresh rhizomes of Polygonatum cyrtonema to obtain rhizomes of Polygonatum cyrtonema powder; S2, extracting the rhizome powder of Polygonatum cyrtonema by adding water, centrifuging, concentrating the supernatant, adding anhydrous ethanol to form a mixed solution, performing low-temperature alcohol precipitation, centrifuging, retaining the precipitate, and obtaining crude Polygonatum cyrtonema polysaccharide; S3, adding water to dissolve the crude polysaccharide of Polygonatum cyrtonema, centrifuging, decolorizing and concentrating the supernatant, filtering through a filter membrane, removing protein using Sevage reagent, low-temperature dialysis, freeze-drying, and obtaining the polysaccharide component P1 of Polygonatum cyrtonema; S4, adding the polysaccharide component P1 of Polygonatum cyrtonema to water again for dispersion, filtering, eluting, concentrating, low-temperature dialysis, and freeze-drying to obtain the polysaccharide component P2 of Polygonatum cyrtonema; S5. Add the polysaccharide component P2 from Polygonatum cyrtonema into water, pass through a Sephadex G-100 column, perform elution, concentration, low-temperature dialyzation, and freeze-drying to obtain a uniform component of the target polysaccharide from Polygonatum cyrtonema, named PCP2.

6. The use according to claim 5, characterized in that The preparation method of Polygonatum cyrtonema polysaccharide, step S1 specifically comprises: washing and removing the beard of fresh Polygonatum cyrtonema rhizomes, cutting into 6-8 mm slices, drying at 55-65° C. for 72-96 h, and crushing with a grinder through a 60-mesh sieve.

7. The use according to claim 5, characterized in that In step S2 of the preparation method of Polygonatum cyrtonema polysaccharide, the solid-liquid ratio of the Polygonatum cyrtonema rhizome powder to water is 1:20-25 g / mL; the extraction temperature is 90-100°C, and the time is 1.5-2h; the amount of anhydrous ethanol added is 5 times the volume of the mixed solution; and the low-temperature alcohol precipitation is carried out at 0-4°C for 10-12h.

8. The use according to claim 5, characterized in that In step S3 of the preparation method of Polygonatum cyrtonema polysaccharide, the solid-liquid ratio of the Polygonatum cyrtonema crude polysaccharide to water is 1:200-300 g / mL; the re-dissolution is to raise the temperature to 55-65°C and stir until clarified; the centrifugation is to centrifuge at 12000-15000 rpm for 10-20 min; the decolorization and concentration are specifically: decolorization using D101 macroporous resin, and concentrating the decolorized liquid to 0.1-0.2 of the original volume; the low-temperature dialysis is to dialyze at 0-4°C for 48-72h in a dialysis bag with a molecular weight cutoff of 1000Da.

9. The use according to claim 5, characterized in that In step S4 of the preparation method of Polygonatum cyrtonema polysaccharide, the solid-liquid ratio of the Polygonatum cyrtonema polysaccharide component P1 to water is 1:100-2000 g / mL; the low-temperature dialysis is performed at 0-4°C for 10-12 hours in a dialysis bag with a molecular weight cutoff of 300 Da.

10. The use according to claim 5, characterized in that In step S5 of the preparation method of Polygonatum cyrtonema polysaccharide, the solid-liquid ratio of the Polygonatum cyrtonema polysaccharide component P2 to water is 1:150-200 g / mL; the low-temperature dialysis is performed at 0-4°C for 10-12 hours in a dialysis bag with a molecular weight cutoff of 300 Da.

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