Preparation method of polygonatum sibiricum polysaccharide and application of polygonatum sibiricum polysaccharide in slow-transit constipation medicine

Abstract

The invention relates to a preparation method of polygonatum sibiricum polysaccharide and application of the polygonatum sibiricum polysaccharide in a slow-transit constipation medicine, a polygonatum sibiricum raw medicinal material is used as a raw material, polygonatum sibiricum crude polysaccharide (PCP) is extracted through a water extraction and alcohol precipitation method, and a polygonatum sibiricum neutral polysaccharide homogeneous component PCNP-2 is prepared after purification. The prepared PCNP-2 has the effects of regulating intestinal flora, reducing intestinal permeability and improving lipid metabolism, is helpful for relieving gastrointestinal inflammation, diabetes and other diseases, and can be made into effective and safe STC treatment drugs. The medicine effect of the PCNP-2 can be achieved through a PI3K / AKT signal channel, and the intestinal motility weakening phenomenon of a slow transit constipation mouse model is remarkably reversed through treatment of the polygonatum polysaccharide PCNP-2.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to a preparation method of polygonatum sibiricum polysaccharide and application of the polysaccharide in a slow-transmission constipation drug. Background Art

[0002] Slow transit constipation (STC) is a common type of chronic functional constipation (CFC), accounting for 10.3%-45.5% of such cases, and is characterized by prolonged colonic transit time. This type of constipation is related to colonic motor dysfunction rather than structural obstruction. In recent years, more and more studies have been conducted on the pathogenesis of STC, which involves multiple levels, including a decrease in the volume and number of interstitial cells of Cajal (ICC), which may lead to a decrease in high-amplitude propagated contraction (HAPC). Reduced HAPC causes stool to move slowly in the colon, leading to constipation. In addition, abnormalities in the enteric nervous system (ENS), which controls the rhythmic muscle contraction of the colon, lead to impaired colonic muscle contraction, slow propulsion of stool, and thus chronic constipation. Genetic factors may also play a role in the pathogenesis of STC, although C-KIT gene mutations and polymorphisms appear to be rare in STC.

[0003] Macrophages play an important role in the intestine, and their dysfunction may lead to problems such as constipation. Studies have shown that resident macrophages exist in the intestine and are essential for the normal function of the intestine. Oxidative stress is an imbalance between oxygen free radicals and antioxidants in the intracellular and extracellular environment. Studies have shown that Chinese medicine enema has a significant effect on improving anorectal pressure and oxidative stress levels in patients with chronic functional outlet obstruction constipation, and promotes intestinal peristalsis by regulating oxidative stress levels, which is beneficial to recovery. These findings emphasize the potential association between oxidative stress and macrophages in intestinal health and the development of constipation. Further research on these mechanisms will help to gain a deeper understanding of the pathogenesis of intestinal problems such as constipation and provide new ideas for related treatment strategies. Intestinal microecological imbalance in patients with constipation may also be one of the important mechanisms of its pathogenesis. There is a close interaction between the intestinal flora and the enteric nervous system. The imbalance of intestinal flora may affect the motility of the intestine by affecting the production and release of neurotransmitters. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides a preparation method of polygonatum polysaccharide and its application in slow-transmission constipation drugs.

[0005] The technical scheme adopted by the present invention is: a polygonatum polysaccharide, named PCNP-2, is composed of fructose and glucose, with a residue ratio of -33:1, and PCNP-2 is an Agavin type fructan.

[0006] Preferably, the molecular weight is 5370 Da.

[0007] Preferably, it consists of β-D-Fruf-(2→, →2)-β-D-Fruf-(1→, →2)-β-D-Fruf-(6→, →2)-β-D-Fruf-(1,6→ and →6)-α-D-Glcp-(1→ sugar residue;

[0008] Among them, the ratio of fructose β-D-Fruf-(2→, →2)-β-D-Fruf-(1→, →2)-β-D-Fruf-(6→, and →2)-β-D-Fruf-(1,6→ residues is approximately 1:2:3:1.

[0009] The method for preparing polygonatum polysaccharide comprises the following steps: extracting polygonatum polysaccharide from polygonatum medicinal material by water extraction and alcohol precipitation method, separating the main components of the crude polygonatum polysaccharide by liquid chromatography to obtain PCN, separating the main components of PCN by molecular sieve chromatography column again to obtain PCN-2, and purifying PCN-2 to obtain a neutral polysaccharide uniform component polygonatum polysaccharide PCNP-2.

[0010] Preferably, the specific preparation method is as follows:

[0011] Step 1: crush the dried Polygonatum sibiricum medicinal material, extract and defatted with 75% ethanol by heating and refluxing, repeat three times, each time for 2 hours; adopt water extraction and alcohol precipitation method at 100°C, heat and reflux according to a solid-liquid ratio of 1:10, extract the medicinal residue and repeat three times, each time for 2 hours; collect the supernatant and add ethanol with a final concentration of 80% (v / v) to precipitate, separate and fully dissolve the precipitate, dialyze, concentrate and freeze-dry to obtain Polygonatum sibiricum crude polysaccharide PCCP;

[0012] Step 2: Take the PCCP solution, load it onto a DEAE-650M column, and elute it with distilled water and 0.1M NaCl solution. The distilled water elution portion is the main portion, marked as PCN;

[0013] Step 3: Purify PCN using molecular sieve column chromatography Sepharose 6B column, using 0.1M NaCl solution as eluent to obtain two neutral polysaccharide components PCN-1 and PCN-2 from Polygonatum cyrtonema;

[0014] Step 4: Purify the main component PCN-2 using a Sephacryl S-300 column, elute with 0.1 M NaCl solution as the eluent, and obtain the uniform component PCNP-2 of the neutral polysaccharide of Polygonatum sibiricum after reduced pressure concentration, dialysis, and freeze-drying.

[0015] Application of Polygonatum sibiricum polysaccharide in the preparation of drugs for chronic transit constipation.

[0016] Preferably, Polygonatum sibiricum polysaccharide PCNP-2 inhibits the activation of PI3K / AKT signaling pathway and the expression of iNOS protein, thereby regulating macrophage polarization and achieving the repair of chronic transmission constipation damage.

[0017] The advantages and positive effects of the present invention are: providing a Polygonatum sibiricum polysaccharide PCNP-2, which can be used in drugs for treating slow-transmission constipation, has anti-inflammatory, anti-oxidative stress and immunomodulatory functions, affects intestinal motility, intestinal nerve function and microecological balance, and provides a new method and target for the treatment of slow-transmission constipation. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Separation chromatogram of PCN-2 on Sephacryl S-300HR column;

[0019] Figure 2 HPGPC chromatogram of PCNP-2;

[0020] Figure 3 Infrared spectrum of PCNP-2;

[0021] Figure 4 HPLC chromatogram of monosaccharide composition analysis of PCNP-2 (TFA, trifluoroacetic acid solvent remaining in the system; Suc, Sucrose; Glc, Glucose; Fru, Fructose);

[0022] Figure 5 GC-MS total ion chromatogram of partial methylation and acetylation products (PMAA) of PCNP-2;

[0023] Figure 6 PCNP-2 signal peak mass spectrum fragmentation diagram;

[0024] Figure 7 The nuclear magnetic resonance spectrum of PCNP-2; A is 1 H NMR, B 1 3C NMR, C is DEPT, D is HSQC, E is HMBC;

[0025] Figure 8 The proposed structure of PCNP-2;

[0026] Fig. 9 The trend of mouse body weight changes;

[0027] Fig.10 The therapeutic effect of Polygonatum sibiricum polysaccharide on the mouse model of chronic transmission constipation; (a) is the time of discharge of the first black feces; (b) is the frequency of defecation; (c) is the water content of feces; (d) is the food intake, and (e) is the water intake; ## P<0.01, ** P<0.01, # P<0.05, * P < 0.05;

[0028] Fig.11 Effects of Polygonatum sibiricum polysaccharide on colon pathological changes and oxidative stress in mice with chronic transit constipation;

[0029] Fig.12 Effects of Polygonatum sibiricum polysaccharide treatment on redox markers and STC-related protein expression (n=3); (a) MDA content detection, (b) SOD content detection, (c) serum MTL detection, (d) related protein expression, (e) SCF protein expression level, (f) C-KIT protein expression level;

[0030] Fig.13 Analysis of mRNA expression of related factors of whole-layer macrophages in the colon after treatment with Polygonatum sibiricum polysaccharide (n=3); (a) iNOS mRNA expression, (b) IL-1β mRNA expression, (c) TNF-α mRNA expression, (d) Retnl mRNA expression, (e) Mrc1 mRNA expression, (f) Mgl2 mRNA expression;

[0031] Fig.14 Ultrastructure of enteric nerves and immunofluorescence co-staining results of neurons and M1 macrophage-related inflammatory factor iNOS; (a) is ultrastructure, (b) is immunofluorescence co-staining;

[0032] Fig.15 Morphological changes of intestinal myenteric neurons and co-staining with the inflammatory factor iNOS;

[0033] Fig.16 Western blot analysis was used to detect the effects of Polygonatum sibiricum polysaccharide on the expression of proteins related to the colon macrophages and PI3K / AKT pathway in STC mice; (a) is the expression level of p-PI3K, t-PI3K, p-AKT, t-AKT pathway proteins, and iNOS and Retnl proteins in mouse colon tissue; (b) is the protein quantification result;

[0034] Fig.17Statistics of the Alpha diversity index of intestinal microorganisms in each group of mice (n=4); (a) Chao1 index, (b) Simpson index, (c) Shannon index, and (d) PD_Whole_Tree index;

[0035] Fig.18 Statistics of intestinal microbial beta diversity index of mice in each group (n=4); (a) PCA analysis and (b) PLS-DA analysis;

[0036] Fig.19 Quantification of fecal short-chain fatty acids (n=6); (a) acetic acid, (b) butyric acid, (c) caproic acid, (d) isovaleric acid, (e) valeric acid, (f) isobutyric acid, and (g) propionic acid. DETAILED DESCRIPTION

[0037] The embodiments of the present invention are described below with reference to the accompanying drawings.

[0038] The present invention relates to a preparation method of polygonatum polysaccharide and its application in slow-transmission constipation drugs. Polygonatum raw medicinal materials are used as raw materials, and crude polygonatum polysaccharide (PCCP) is extracted by water extraction and alcohol precipitation method, and then purified to prepare a uniform component of polygonatum neutral polysaccharide PCNP-2. Polygonatum is a perennial herb of the genus Polygonatum in the family Liliaceae. There are more than 60 species in the world, mainly distributed in the northern temperate zone and northern subtropical zone, and 31 species in China, which are widely distributed in a large area except the southern tropical zone.

[28] The 2015 edition of the Chinese Pharmacopoeia records that the Chinese medicinal material Polygonatum sibiricum is the dried rhizome of Polygonatum sibiricum, Polygonatum sibiricum or Polygonatum sibiricum; the main chemical components of Polygonatum sibiricum include steroidal saponins, polysaccharides, alkaloids, etc. Among them, the polysaccharide component is not only the most abundant component in Polygonatum sibiricum, but also one of its main active ingredients.

[0039] Polygonatum sibiricum polysaccharide was extracted from Polygonatum sibiricum and subjected to multi-step purification. The main component obtained was named PCNP-2, and its composition and structure were studied. The prepared PCNP-2 is mainly composed of fructose and also includes a small amount of glucose. Fructans are composed of β-D-Fruf-(2→, →2)-β-D-Fruf-(1→, →2)-β-D-Fruf-(6→, →2)-β-D-Fruf-(1,6→ and →6)-α-D-Glcp-(1→ sugar residues. The presence of internal glucose residues →6)-α-D-Glcp-(1→ suggests that PCNP-2 is an Agavin type fructan; among them, fructose β-D-Fruf-(2→, →2)-β-D-Fruf-(1→, →2)-β-D-Fruf-(6→, and →2)-β-D-Fruf-(1,6→ residues are about 1:2:3:1. Through research, it was found that PCNP-2 is composed of →1)-β-D-Fruf-(2→ as the main chain, and every 4 main chain sugar residues have a branch chain composed of →6)-β-D-Fruf-(2→ and β-D-Fruf-(2→), and there is a β-D-Fruf-(2→→1)-α-D-Glcp-(6→ fragment at the end.

[0040] The prepared PCNP-2 has the functions of regulating intestinal flora, reducing intestinal permeability, and improving lipid metabolism, which helps to relieve gastrointestinal inflammation and diseases such as diabetes, and can be made into an effective and safe drug for the treatment of STC. PCNP-2 can exert its drug effect through the PI3K / AKT signaling pathway. The treatment of Polygonatum sibiricum polysaccharide PCNP-2 significantly reversed the weakened intestinal motility of the slow-transmission constipation mouse model, which was specifically manifested as a shortened time for the first black stool discharge, an increase in defecation frequency, and an increase in fecal moisture content. Polygonatum sibiricum polysaccharide PCNP-2 can effectively improve constipation symptoms and enhance intestinal motility. The potential mechanism in promoting intestinal peristalsis involves direct effects on intestinal smooth muscle or regulating intestinal motility through the neurointestinal axis.

[0041] PCNP-2 can play a significant role in restoring redox imbalance and repairing intestinal structural integrity. Specifically, PCNP-2 can significantly reverse the decrease of motilin, C-KIT and SCF induced by loperamide, regulate intestinal motility, reduce the level of MDA in the intestine, increase SOD activity, and restore intestinal redox imbalance. PCNP-2 can reverse the increase of iNOS, IL-1, and TNF-mRNA in STC mice, and increase the expression levels of Retnl, Mrc1 and MgI2 mRNA, showing that PCNP-2 regulates the intestinal immune environment by regulating the polarization of colonic macrophages, inhibiting the expression of pro-inflammatory macrophage-related cytokine genes, and increasing the expression of anti-inflammatory macrophage-related genes.

[0042] PCNP-2 regulates the balance of intestinal microecology, can effectively regulate the composition of intestinal flora of STC model mice, and increase the concentration of short-chain fatty acids, which is conducive to restoring a healthy intestinal microecological environment. The results of 16SrRNA gene sequencing showed that after treatment with Polygonatum sibiricum polysaccharide, the abundance of beneficial flora in the intestine increased, while the abundance of harmful flora decreased, indicating that Polygonatum sibiricum polysaccharide can adjust and optimize the structure of intestinal flora. In addition, Polygonatum sibiricum polysaccharide further confirmed its role in promoting intestinal function and health by increasing the production of short-chain fatty acids, especially the content of butyric acid, acetic acid and caproic acid, providing a basis for the study of Polygonatum sibiricum polysaccharide as a drug for the treatment of chronic constipation.

[0043] The scheme of the present invention is explained below in conjunction with the accompanying drawings. The raw medicinal materials of Polygonatum sibiricum are provided by the Polygonatum sibiricum planting base in Damaoshan, Jiangxi Province, and are identified as the dried rhizomes of Polygonatum sibiricum. Reagents including pullulan series standards were purchased from Showa Denko K.K. of Japan; D-glucose, sucrose, D-fructose, iodomethane, 1-methylimidazole, trifluoroacetic acid, acetic anhydride, and phenol were purchased from Sigma-Aldrich of the United States; ethanol, chloroform, and concentrated sulfuric acid were purchased from Komiou Chemical Reagent Co., Ltd.; potassium bromide, sodium chloride, sodium hydroxide, etc. were purchased from Sinopharm Chemical Reagent Co., Ltd. The experimental methods without specific operating steps were all carried out in accordance with the corresponding product specifications. The instruments, reagents, and consumables used in the examples can all be purchased from commercial companies unless otherwise specified.

[0044] Example 1: Preparation of Polygonatum cyrtonema polysaccharide

[0045] After 1kg of dried Polygonatum sibiricum was crushed into coarse powder, it was extracted and defatted with 75% ethanol under heating and reflux, and repeated three times, each time for 2 hours, and then the residue was dried in the shade for use. Then, at 100°C, the water extraction and alcohol precipitation method was used, and the residue was extracted by heating and reflux according to a solid-liquid ratio of 1:10, and repeated three times, each time for 2 hours. The supernatant was collected and ethanol was added for alcohol precipitation, with a final concentration of 80% (v / v). After the alcohol precipitate was fully dissolved, it was dialyzed (molecular weight cutoff 3.5kDa), concentrated, and freeze-dried to obtain Polygonatum sibiricum crude polysaccharide (PCCP), with a yield of 3.6% (based on the mass of Polygonatum sibiricum).

[0046] Take an appropriate amount of PCCP, dissolve it fully, centrifuge it, load the supernatant onto a DEAE-650M column (55mm×30cm), and elute it with distilled water and NaCl solution (0.1M) in turn. The eluate is collected at 15mL / tube, and the sugars and proteins are detected by phenol-sulfuric acid method and UV method, respectively, and then collected. According to the elution curve, the distilled water elution part is the main part, marked as PCN (the yield based on crude polysaccharides is 53.8%); the 0.1M NaCl elution part accounts for a smaller proportion, marked as PCA (the yield based on crude polysaccharides is 10.3%). It can be seen that the main part of the Polygonatum sibiricum polysaccharide is the neutral polysaccharide part eluted by water, and the acidic polysaccharide content is low.

[0047] The main component PCN was further separated and purified. First, PCN was purified by molecular sieve column chromatography Sepharose 6B column (5.0cm×95cm), and NaCl solution (0.1M) was used as the eluent. The detection method was the same as above, thereby obtaining two neutral polysaccharide components of Polygonatum sibiricum, namely PCN-1 and PCN-2. The two components have different molecular weights and can be separated by molecular sieve chromatography. The first component to flow out is PSN-1 with a large molecular weight, followed by PSN-2 with a slightly smaller molecular weight. The main component PCN-2 was purified by Sephacryl S-300HR column (2.6cm×90cm), and NaCl solution (0.1M) was used as the eluent. The eluent was collected at 7mL / tube, and the phenol-sulfuric acid method and UV method were used to detect sugars and proteins, respectively. The chromatograms are shown in the following figure. Figure 1 The eluate was concentrated under reduced pressure, dialyzed, and freeze-dried to obtain a uniform component of neutral polysaccharide of polygonatum sibiricum (PCNP-2), the yield of which based on the crude polysaccharide was 36.6%, and the polysaccharide was in the form of colorless powder.

[0048] Example 2: Identification of Polygonatum cyrtonema polysaccharide

[0049] 2.1 Homogeneity analysis and molecular weight determination of Polygonatum sibiricum polysaccharide

[0050] High performance liquid chromatography (Agilent 1260) was used with an evaporative light scattering detector (ELSD) and a PLaquagel-OHMIXED-H (300×7.5mm, 8μm) column. Ammonium acetate solution (0.1M) was used as the mobile phase and dextran series polysaccharides (molecular weight 2.7kDa-36.8kDa) were used as standards to draw a standard curve, determine the molecular weight of the samples and evaluate their homogeneity.

[0051] The results of homogeneity analysis are as follows Figure 2As shown in the HPGPC chromatogram, PCNP-2 presents a symmetrical, sharp single peak, indicating that the polysaccharide is a polysaccharide with uniform molecular weight distribution. The molecular weight of PCNP-2 is 5370Da calculated by the dextran standard curve. The total sugar content of PCNP-2 is 100% as determined by the phenol-sulfuric acid method.

[0052] 2.2 Chemical composition analysis

[0053] The total sugar content of PCNP-2 was determined by sulfuric acid-phenol colorimetry combined with ultraviolet spectrophotometry. First, a standard curve (12.5-200μg / mL) was drawn with fructose (Fru) as the standard, and then 100μg / mL and 50μg / mL PCNP-2 solutions were prepared according to the gradient dilution method. After color development using the same method, the absorbance was detected. Using FT-IR, an appropriate amount of Polygonatum sibiricum uniform polysaccharide PCNP-2 was taken, and PCNP-2 was ground and mixed with dry KBr powder in an infrared drying oven. The powder was pressed into a transparent sheet with a hydraulic press and then heated at 4000-400cm -1 Determination within wavenumber.

[0054] The results are as follows Figure 3 As shown, infrared spectrum analysis shows that not only 3390cm -1 (stretching vibration of OH on the sugar ring) and 2900cm -1 The typical polysaccharide absorption peaks with strong signals appeared near (methylene CH stretching vibration on the sugar ring) and also at 930-810cm –1 There are absorption peaks caused by the CH lateral vibration and CH angular vibration of the furanose ring, suggesting the presence of β-D-fructofuranose (β-D-Fruf) residues in PCNP-2.

[0055] 2.3 Monosaccharide composition analysis

[0056] Sucrose (Suc), D-glucose (Glc) and D-fructose (Fru) were used as controls. The control samples and PCNP-2 were hydrolyzed with 2M trifluoroacetic acid (TFA) in a metal reactor at 50°C for 30 min each. After deacidification, the samples were mixed with Sugar-pack TM (6.5 mm × 300 mm, 10 μm) column, deionized water as the mobile phase and high performance liquid chromatography-evaporative light scattering detector (HPLC-ELSD) were used to analyze the monosaccharide composition of the sample. Figure 4 As shown, PCNP-2 is mainly composed of fructose (97.1%), which is consistent with the results of infrared spectroscopy analysis; in addition, there is a small amount of glucose (2.9%). The ratio of fructose to glucose residues is about 33:1, which is close to the results of molecular weight determination after calculation.

[0057] In order to clarify the composition of the glycosidic bonds between the fructose residues of PCNP-2, a methylation analysis was performed. 2 mg of dried PCNP-2 was dissolved in 1 mL of anhydrous DMSO. After the polysaccharide was completely dissolved, an appropriate amount of powdered NaOH was added and stirred for 2 h. Then, 450 μL of methyl iodide was added three times under ice bath conditions, 150 μL each time, the first two reactions lasted 15 min, and the last reaction lasted 30 min. Then, 1 mL of pure water was added to terminate the reaction, and the solution was blown from turbidity to clarity with N2. An equal volume of chloroform was added for extraction and centrifugation, the upper layer of water was discarded, and then pure water was added for extraction 5 times, and finally the sample was blown dry with N2 to complete the methylation. Then, 1 mL of 2M TFA was added to the dried sample, and heated in a metal reactor at 50 ° C for hydrolysis for 30 min. After deacidification, 1 mL of 1M NH3·H2O was added to the sample, and then about 3 mg of NaBD4 was added, and the sample was reduced at room temperature for 18 h. In an ice-water bath, 1 mL of 10% acetic acid / methanol was added dropwise to the reduced sample to terminate the reduction, and methanol was used to spin off the acetic acid and methyl borate. Then 0.1 mL of 1-methylimidazole and 1 mL of acetic anhydride were added, and acetylation was carried out at room temperature for 30 min. Finally, 1 mL of pure water was added to terminate the reaction under ice bath conditions. An equal volume of chloroform was added for extraction, the water layer was discarded, and an equal volume of water was added for extraction 6 times, and the final sample was obtained by centrifugation for the last time (2300 rpm, 3 min). A gas phase-mass spectrometer (Agilent 5975C / 7890A) was used, and He was used as the carrier gas. The fragment peak mass spectrum was determined according to the retention time of the sample in the gas phase spectrum. The peak area ratio was taken separately to calculate the content ratio of the sample fragment molecules to the total molecular weight.

[0058] The results are as follows Figure 5 As shown in the figure, there are 7 chromatographic peaks with obvious signals between 15-24 min in the total ion current chromatogram, marked as P1-P7. The mass spectra of each signal peak are shown in Figure 6As shown. Combined with the results of monosaccharide composition analysis, the mass spectrometry diagnostic fragments of each signal peak were used to identify it. Since fructose residues produce the structures of glucitol and mannitol during the derivatization process, its methylation product has two signal peaks with the same mass spectrometry fragments. For example, the mass spectra of P1 and P2 both show the same diagnostic fragments, which are identified as 2,5-di-O-acetyl-(2-deuterio)-1,3,4,6-tetra-O-methyl frucitol, that is, the terminally linked fructose residue (Fruf-(2→); P3 and P4 were identified as 2,5,6-tri-O-acetyl-(2-deuterio)-1,3,4-tri-O-methyl frucitol, and 1,2,5-tri-O-acetyl-(2-deuterio)-3,4,6-tri-O-methyl frucitol, namely →6)-Fruf-(2→ and →1)-Fruf-(2→ residues; the two groups of m / z (161.1 / 162.1; 189.1 / 190.1) in the diagnostic fragment of P5 indicate that the signal peak is the superposition peak of another group of sugar alcohol derivatives of P3 and P4. The ratio of →6)-Fruf-(2→ and →1)-Fruf-(2→ residues can be calculated based on the m / z ratio of 161.1 / 162.1;

[0059] According to the monosaccharide composition, signal P6 was identified as 1,5,6-tri-O-acetyl-(2-deuterio)-2,3,4-tri-O-methyl-glucitol, i.e., →6)-Glcp-(1→ residue;

[0060] According to the monosaccharide composition, P7 was identified as 1,2,5,6-tetra-O-acetyl-(2-deuterio)-3,4-di-O-methyl frucitol, namely, →1,6)-Fruf-(2→ residue. It can be seen that the fructan is composed of Fruf-(2→、→6)-Fruf-(2→、

[0061] →1)-Fruf-(2→, →1,6)-Fruf-(2→ residues and →6)-Glcp-(1→ residues, and PCNP-2 has the structural characteristics of agavin-type fructan.

[0062] The composition of each sugar residue in PCNP-2 was calculated based on the peak area, and the results are shown in Table 1. It can be seen that the ratio of the terminal linked (Fruf-(2→) sugar residues to the branched sugar residues (→1,6)-Fruf-(2→) is quite different from the theoretical value of 1:1, which may be caused by the peeling effect of fructose residues caused by the methylation reaction conditions. In the alkaline environment of methylation, the stability of the β-(2→6) glycosidic bond between fructose residues is not as stable as that of β-(2→1) and is easy to fall off. The ratio of the combined end to the branch point is 1, so it is inferred that (26.6%-13.8%) 12.8% of 2-Fruf has the "off-end" effect from →6)-Fruf-(2→, so →6)-Fruf-(2→ accounts for 29% in PCNP-2. Therefore, the ratio of fructose residues Fruf-(2→, →6)-Fruf-(2→, →1)-Fruf-(2→, →1,6)-Fruf-(2→ residues in PCNP-2 is approximately 1:2:3:1.

[0063] Table 1 Glycosyl linkage composition of PCNP determined by GC-MS

[0064]

[0065] *Measured value.

[0066] 2.4 NMR analysis

[0067] To further clarify the linkage pattern of PCNP-2 sugar residues, one-dimensional and two-dimensional NMR analysis was performed. 1 H and 13 The results of C NMR, HSQC, HMBC spectroscopy and methylation analysis further elucidated the structural characteristics of PCNP-2. 30 mg of dried PCNP-2 polysaccharide sample was dissolved in deuterium oxide (D2O) and then analyzed by nuclear magnetic resonance (NMR). 1 H NMR and 13 C NMR spectroscopy, as well as various two-dimensional spectra such as heteronuclear multiple bond correlation spectroscopy (HMBC) and heteronuclear single quantum correlation spectroscopy (HSQC).

[0068] exist 13 C spectrum and DEPT spectrum (such as Figure 7 B. Figure 7 C) in the low field region, four terminal carbon signals were observed at δ104.05ppm, δ103.88ppm, δ103.74ppm and δ103.27ppm, while in the HSQC spectrum (as shown in Figure 7D) shows the terminal hydrogen signal connected to it. Combined with the monosaccharide composition and methylation analysis, it is suggested that the four terminal carbons are typical C2 signals of Fruf residues, and the corresponding four Fruf residues are named A, B, D and E. 13 In the C NMR spectrum, δ76.67-77.39ppm, δ74.50-75.29ppm and δ80.30-81.27ppm are respectively attributed to the signal peaks of Fruf residue C3, C4 and C5. Combined with the DEPT spectrum, two groups of signals in the high field region, δ60.12-60.92ppm and δ62.34-63.25ppm, are respectively attributed to the C1 and C6 signals of Fruf residue. 1 H spectrum (such as Figure 7 The broad signals in the mid-low field region of δ4.20-4.35ppm and δ4.10-4.20ppm in (A) belong to H-3 and H-4 of the D-Fruf residue, respectively. Combined with HSQC, obvious cross signals were observed at δ3.94 / 81.45 and 4.02 / 80.34ppm, corresponding to C5 / H5 of the D-Fruf residue. In addition, 1 H and 13 The chemical shift at δ5.49 / 92.22ppm in the C spectrum was clearly assigned to the H-1 / C-1 of the α-configuration Glcp residue (α-D-Glcp), and was named residue G. Combined with the results of methylation analysis, G was identified as →6)-α-Glcp-(1→. According to the peak area ratio of the terminal proton signal of the glucose residue (δ5.49ppm) and the H3 (δ4.20-4.35ppm) or H4 (δ4.10-4.20ppm) signal of the fructose residue, the molar ratio of glucose to fructose in PCNP-2 was about 1:33, which was consistent with the molecular weight and monosaccharide composition results.

[0069] In two-dimensional HMBC spectroscopy (such as Figure 7 E), two groups of strong cross-absorption peaks at δ104.05 / 3.85ppm and δ103.74 / 378ppm were identified as the strong correlation between C2 and H6 between A sugar residues and the strong correlation between C2 and H1 between D sugar residues, suggesting that →6)-β-D-Fruf-(2→6)-β-D-Fruf-(2→ and

[0070] →1)-β-D-Fruf-(2→1)-β-D-Fruf-(2→ fragment; the cross peak at δ103.74 / 3.82ppm shows that C2 of sugar residue D is strongly correlated with H1 of sugar residue B, suggesting

[0071] →1)-β-D-Fruf-(2→1,6)-β-D-Fruf-(2→ fragment exists; at the same time, the δ104.05 / 3.75ppm cross peak is related to H6 of sugar residue B and C2 of A, indicating the existence of →6)-β-D-Fruf-(2→6,1)-β-D-Fruf-(2→ fragment; in addition, C2 of sugar residue E is weakly correlated with H6 of A, indicating the existence of β-D-Fruf-(2→6)-β-D-Fruf-(2→. In addition, due to the low content of glucose (2.9%), no correlation signal peaks between α-D-Glcp and β-D-Fruf were observed on the HMBC graph, but according to the methylation results, it can be clearly seen that it is an internally linked →6)-α-D-Glcp-(1→, and it is speculated that there is

[0072] β-D-Fruf-(2→6)-α-D-Glcp-(1→2)-β-D-Fruf-(1,6→. Based on the above analysis, this is consistent with the characteristics of PCNP-2 being a typical agavin-type fructan. The specific chemical shifts are shown in Table 2.

[0073] Table 2 Chemical shift assignments of the spectrum of PCNP-2

[0074]

[0075] comprehensive 1 H NMR, 13 C NMR, HSQC, HMBC and other nuclear magnetic spectrum information further clarified the structural characteristics of PCNP-2 fructan. Through research, it was found that methylation, monosaccharide composition analysis and nuclear magnetic resonance results verified that PCNP-2 is composed of a main chain of →1)-β-D-Fruf-(2→, and every four main chain sugar residues have a branch chain composed of →6)-β-D-Fruf-(2→ and β-D-Fruf-(2→), and there is a β-D-Fruf-(2→→1)-α-D-Glcp-(6→ fragment at the end. Therefore, PCNP-2 is classified as a highly branched agavin-type fructan, and its inferred structure is as follows Figure 8 shown.

[0076] Example 3: Study on the effect of Polygonatum sibiricum polysaccharide on regulating intestinal motility in mice with slow-transit constipation

[0077] Thirty 8-week-old Balb / c mice weighing approximately 20±2g were selected. Before the experiment, all mice were housed in an environment with a temperature of 25°C, a humidity of 50-60%, a light-dark cycle of 12:12h, and sufficient purified water and standard feeding food, and underwent a one-week adaptation period.

[0078] Mice were randomly assigned to three experimental groups, 10 in each group. The control group received normal saline gavage to establish baseline data; the STC group received 10 mg / kg loperamide solution gavage twice a day for 14 days to build a constipation model; after the STC model was established, the Polygonatum sibiricum polysaccharide group received 10 mg / kg Polygonatum sibiricum polysaccharide gavage from the 8th day of modeling, twice a day, at least 1 hour apart from the administration of modeling drugs to avoid gavage operation to stimulate the gastrointestinal tract of mice, for 7 consecutive days, in order to evaluate the therapeutic effect of Polygonatum sibiricum polysaccharide. The interval between gavages was at least 6 hours to avoid drug interactions.

[0079] 3.1 Physiological parameter detection

[0080] Measure the baseline weight of each mouse using an electronic scale for accurate measurement and weigh each mouse at the same time each day. After data collection is complete, analyze the weight data in conjunction with food and water intake to assess overall health. Fig. 9 As shown, compared with the control group, the body weight of mice in the STC group decreased significantly after modeling, and treatment with Polygonatum sibiricum polysaccharide significantly reversed the weight loss caused by the STC model.

[0081] The time of the first black stool discharge of mice was measured to evaluate the gastrointestinal transit time of mice. The experiment started on the afternoon of the 14th day after modeling. All mice were fasted for 16 hours, but were allowed to drink water freely, and the intestines of mice were emptied to ensure the accuracy of the experiment. Then on the 15th day, each mouse was placed in a cage individually and given 0.1mL India ink by gavage. After gavage, mice can eat and drink water freely. The defecation of mice was then observed and recorded. From the beginning of the gavage operation, mice were observed every 10 minutes, and the time when the mice discharged the first black stool and the frequency of black stool were recorded. Fecal samples were collected from mouse cages from the beginning of the intestinal transit experiment to 300 minutes. Immediately afterwards, the initial weight of the collected fresh fecal samples was weighed and recorded using an accurate electronic balance. The recorded fecal samples were placed in a drying oven, set to a constant temperature (60°C), and dried to constant weight for about 48 hours. After the fecal samples were dried, their dry weight was weighed again. The fecal moisture percentage was calculated by the following formula: moisture percentage = (initial weight - dry weight) / initial weight × 100%. The moisture percentage was calculated for each mouse, and the data of each measurement was recorded in the experimental log, including the collection time, initial weight, dry weight and calculated moisture percentage. The results are shown in Fig.10 shown.

[0082] Time of first black stool ( Fig.10A) is a key indicator for evaluating intestinal motility. Compared with the control group, the time of the first black feces discharge of mice in the STC group was significantly increased, indicating the successful construction of the model and obvious constipation symptoms. After intervention with Polygonatum sibiricum polysaccharide, the time of the first black feces discharge of STC mice was significantly shortened, indicating the potential role of Polygonatum sibiricum polysaccharide in regulating intestinal motility. The defecation frequency and fecal water content of mice were tested. Compared with the control group, the defecation frequency ( Fig.10 B) was significantly reduced in the STC group. After intervention with Polygonatum sibiricum polysaccharide, the defecation frequency of STC mice was significantly increased, indicating that Polygonatum sibiricum polysaccharide has a certain therapeutic effect in regulating intestinal motility. The fecal moisture content of mice in the STC group was compared with that in the control group ( Fig.10 C) was significantly reduced. After intervention with Polygonatum sibiricum polysaccharide, the fecal water content increased compared with the STC group. Although it did not completely recover to the level of the control group, the upward trend showed its positive effect on relieving constipation. The food and water intake of constipated mice was reduced compared with normal mice, especially the food intake ( Fig.10 D and 10E), after intervention with polygonatum polysaccharide, the intake of mice increased compared with that of the STC group, suggesting that polygonatum polysaccharide may have a positive regulatory effect on appetite and water intake.

[0083] In summary, Polygonatum sibiricum polysaccharide has a significant therapeutic effect on the constipation symptoms of STC model mice, including reducing weight loss, accelerating the excretion time of black feces, increasing the frequency of defecation, increasing the water content of feces, and possibly increasing the intake of food and water. These results provide an experimental basis for Polygonatum sibiricum polysaccharide to become a potential drug for the treatment of chronic constipation.

[0084] Example 4: Effects of Polygonatum sibiricum Polysaccharide Treatment on Redox Markers and STC-related Protein Expression

[0085] Mouse serum was retained and anesthetized by intraperitoneal injection of sodium pentobarbital, with an injection dose of 50 mg / kg for each mouse. After anesthesia, about 0.8 mL of blood was obtained from each mouse by eyeball blood sampling. Subsequently, the collected blood was placed at room temperature for 30 min to coagulate, and then centrifuged (4 ° C, 3000 rpm centrifugation for 20 min) to separate serum. The separated serum was stored in a refrigerator at -80 ° C to maintain its stability.

[0086] Mice were killed by cervical dislocation, and laparotomy was performed immediately after killing to remove the colon between the cecum and the anus. The removed colon was washed with phosphate buffered saline (PBS) to remove blood and other contaminants. Subsequently, the distal part of the colon (about 0.5 cm) was cut and fixed with 4% paraformaldehyde.

[0087] 4.1 Histological evaluation of the effects of Polygonatum sibiricum polysaccharide on colon pathology and oxidative stress in mice with chronic transit constipation

[0088] The colon tissue was stained with HE and DHE. Fig.11 As shown. In the HE-stained tissue sections, the control group showed normal colon tissue structure with neatly arranged crypts and uniform epithelial cells. In contrast, the colon sections of the STC group showed disordered crypt structure, irregular arrangement of epithelial cells, and increased crypt stroma, which reflected the pathological characteristics of the constipation model. In the Polygonatum sibiricum polysaccharide group, partial recovery of the structure was observed, with more regular arrangement of crypts and a near-normal epithelial cell structure, suggesting that Polygonatum sibiricum polysaccharide may have a repairing effect on the damaged colon.

[0089] DHE staining is used to visualize the production of reactive oxygen species (ROS) and is a method for evaluating the state of oxidative stress. When cells are in an oxidative stress imbalance, DHE can be oxidized inside the cells to cause the cell nucleus to emit a bright red fluorescent signal. In the control group, no obvious red fluorescent signal was observed, and the staining was more uniform and of lower intensity, indicating a normal redox level. The DHE staining intensity in the STC group was enhanced, and the level of ROS increased significantly, especially in the intestinal epithelial cells, indicating oxidative stress in the intestinal epithelial cells of STC mice, which is consistent with the redox imbalance associated with constipation. The DHE staining in the Polygonatum sibiricum polysaccharide group was weakened compared to the STC group, indicating that Polygonatum sibiricum polysaccharide may improve intestinal damage in STC mice by restoring redox imbalance. 4.2 Effects of Polygonatum sibiricum polysaccharide treatment on the expression of redox markers and STC-related proteins

[0090] In order to further evaluate the antioxidant effect of Polygonatum sibiricum polysaccharide, the expression of redox markers and STC-related proteins in mouse colon tissue was detected. Malondialdehyde (MDA) is a marker of lipid peroxidation, and the MDA level can reflect the degree of endogenous peroxidation in tissues. Superoxide dismutase (SOD) is a major antioxidant enzyme responsible for scavenging superoxide free radicals, and the SOD level can represent the body's antioxidant capacity. Motilin (MTL) is a gastrointestinal hormone secreted by endocrine Mo cells. It plays an important role in promoting and affecting gastrointestinal motility and gastrointestinal absorption of water and electrolytes. MTL has an important influence on the gastrointestinal functional motility mechanism.

[0091] The level of MDA in colon tissue Fig.12 As shown in a, in the STC group, the level of MDA increased significantly compared with the control group, indicating that colon tissue peroxidation occurred in the STC model. After treatment with Polygonatum sibiricum polysaccharide, the level of MDA decreased significantly, suggesting that Polygonatum sibiricum polysaccharide can reduce intestinal peroxidation, restore redox imbalance, and have a potential protective effect on intestinal damage caused by oxidative stress. Fig.12As shown in Figure b, the SOD activity in the STC group was lower than that in the control group. Polygonatum sibiricum polysaccharide intervention increased SOD activity and antioxidant capacity, indicating that Polygonatum sibiricum polysaccharide may help restore or improve the activity of SOD in the colon tissue of constipated mice. Fig.12 As shown in Figure c, the level of motilin in the normal control group was maintained at a high level, and the level of motilin in the serum of STC mice was significantly reduced. This finding may be related to the slowing of gastrointestinal motility under the pathological state of STC. However, in the group treated with Polygonatum sibiricum polysaccharide, the level of motilin rebounded, indicating that Polygonatum sibiricum polysaccharide has a positive effect on enhancing gastrointestinal motility.

[0092] Colon ICC of STC patients may have mutations in the c-kit gene and changes in the polymorphic sites of the SCF gene, which are related to the pathogenesis of STC. Therefore, the changes in the protein expression levels of C-KIT and SCF in colon tissue were detected. The results are as follows Fig.12 d. The results showed that compared with the control group, the relative expression of C-KIT and SCF proteins in the colon tissue of mice in the STC group was significantly reduced, while after treatment with Polygonatum sibiricum polysaccharide, the expression levels of the above proteins were significantly increased ( Fig.12 e and f). This indicates that Polygonatum sibiricum polysaccharide has a positive regulatory effect in restoring gastrointestinal motility.

[0093] 4.3 Analysis of mRNA expression in colonic macrophages after treatment with Polygonatum sibiricum polysaccharide

[0094] In order to further explore the role of Polygonatum sibiricum polysaccharide in the inflammatory response of colon tissue in STC model mice, the expression of M1 macrophages and M2 macrophage-related genes was detected to evaluate the potential regulatory effect of Polygonatum sibiricum polysaccharide on macrophage polarization.

[0095] The expression of pro-inflammatory macrophage-related cytokine genes, including the mRNA expression levels of inducible nitric oxide synthase (iNOS), interleukin-1β (IL-1β), and tumor necrosis factor α (TNF-α), was detected. Fig.13 As shown. In the STC model, compared with the control group, the mRNA expression levels of proinflammatory cytokines iNOS, IL-1β and TNF-α were significantly increased, indicating an enhanced inflammatory response in the intestinal tissue of mice in the STC model, proving the correlation between intestinal tissue inflammation and disease progression in the STC model. After intervention with Polygonatum sibiricum polysaccharide, the mRNA levels of iNOS, IL-1β and TNF-α were significantly lower than those in the STC group. It was demonstrated that Polygonatum sibiricum polysaccharide can effectively inhibit the transcription level of proinflammatory cytokines in the whole layer of colon tissue of STC mice.

[0096] The expression of anti-inflammatory macrophage-related genes, including the mRNA expression levels of resistin-like α (Retnl), macrophage mannose receptor 1 (Mrc1), and macrophage scavenger receptor 2 (MgI2), were detected. Fig.13 As shown in the results, in the STC model, compared with the control group, the mRNA expression of Retnl, Mrc1 and MgI2 decreased significantly, indicating the lack of anti-inflammatory macrophages, proving the imbalance of the ratio of pro-inflammatory and anti-inflammatory macrophages in STC disease. After treatment with Polygonatum sibiricum polysaccharide, the mRNA expression levels of Retnl and MgI2 were significantly increased, suggesting that Polygonatum sibiricum polysaccharide may play a therapeutic role by regulating the polarization of macrophages and promoting the expression of anti-inflammatory cytokines. The expression of Mrc1 after Polygonatum sibiricum polysaccharide treatment was not significantly changed compared with the STC group, indicating that it may not be the main regulatory factor in the whole layer of the colon of STC mice treated with Polygonatum sibiricum polysaccharide, or its expression change is not as sensitive as other anti-inflammatory factors. The above results prove that in the whole layer of the colon of STC mice, Polygonatum sibiricum polysaccharide exerts an anti-inflammatory effect by regulating macrophage polarization and increasing the expression of anti-inflammatory macrophages.

[0097] 4.4 Morphological changes of colonic neurons after treatment with Polygonatum sibiricum polysaccharide and co-staining with iNOS

[0098] There is a close relationship between intestinal nerves and constipation. The nervous system affects intestinal function by regulating intestinal peristalsis and defecation process. The interaction between macrophages and intestinal neurons has also been found to be related to constipation. Therefore, the relationship between macrophages and intestinal neurons may play an important role in the occurrence and development of constipation. In order to further explore the drug target of Polygonatum sibiricum polysaccharide, intestinal neurons were scanned and observed by scanning electron microscopy. The results are as follows Fig.14 shown.

[0099] like Fig.14 As shown in a, the cell bodies of neurons in the control group are usually round or oval, with a smooth surface and obvious nucleoli in the nucleus. In the STC group, the cell bodies of neurons are shrunken, the gaps increase, and the structure is dispersed. After treatment with Polygonatum sibiricum polysaccharide, the abnormal structure of neurons is restored. Fig.14As shown in b, HUC / D, as a marker of colon tissue neurons, showed a uniform and strong green signal in the control group and was highly expressed in the colonic muscular layer. In the STC group, HUC / D expression was weakened in the colon and weaker in the muscular layer than in the control group, indicating damage to the colonic enteric neurons in the STC model. iNOS, as a cytokine secreted by M1 macrophages, showed low expression and uniform signals in the control group and no obvious expression in the muscular layer, but showed a strong signal in the STC group, especially in the muscular layer, suggesting that M1 macrophages play a key role in the pathogenesis of STC. The merged image shows the co-staining of HUC / D and iNOS fluorescence in the colonic muscular layer, suggesting that M1 macrophages may be involved in the damage of intestinal neurons. After intervention with Polygonatum sibiricum polysaccharide, the expression of iNOS in the intestinal muscular layer decreased, while the expression of HUC / D recovered compared with before, suggesting that Polygonatum sibiricum polysaccharide may reduce the damage of intestinal neurons by inhibiting the expression of iNOS.

[0100] The colonic muscular layer was isolated, and the myenteric neurons were co-stained with iNOS to further evaluate the relationship between iNOS and neuronal damage, as well as the potential protective effect of Polygonatum sibiricum polysaccharide on the enteric nervous system in the STC mouse model. Fig.15 As shown in the figure, in the control group, the expression of iNOS was low, showing a light red fluorescence. The expression of HuC / D showed bright green fluorescence, and was arranged neatly and regularly, showing the normal distribution and structure of neurons in the intestinal muscular layer, suggesting a good cell structure and a low inflammatory state. In the STC model group, the fluorescence intensity of iNOS increased, indicating that the inflammatory state was enhanced. The green fluorescence intensity of neurons labeled by HuC / D weakened, indicating that the colonic muscular layer neurons were damaged in the STC model, the neuronal density was reduced, and the distribution was uneven. DAPI staining also showed a decrease in the number of cell nuclei. The decrease in neurons observed in the co-staining was related to the overexpression of iNOS, providing more intuitive data for neuronal damage caused by inflammation in the STC model. When treated with Polygonatum sibiricum polysaccharide, the expression of iNOS decreased, and the fluorescence intensity was close to that of the control group, indicating that the inflammatory state was controlled. The green fluorescence of HuC / D was enhanced compared with the STC group, showing a more normal neuronal structure and density. DAPI staining showed that the number and distribution of nuclei were similar to those of the control group. The co-staining images showed normal expression of neuronal fluorescence and reduced expression of iNOS, indicating that Polygonatum sibiricum polysaccharide may have the effect of protecting colonic muscular layer neurons from inflammatory damage induced by the STC model.

[0101] The above results indicate that Polygonatum sibiricum polysaccharide protects colonic neurons and maintains the structural integrity of the colonic nervous system by regulating macrophage polarization and alleviating the inflammatory response in the STC model.

[0102] 4.5 Effects of Polygonatum sibiricum polysaccharide on the expression of colonic macrophages and PI3K / AKT pathway-related proteins

[0103] Polygonatum sibiricum polysaccharide can exert its therapeutic effect by regulating macrophage polarization, and iNOS may serve as a key regulator between macrophages and intestinal neurons. The expression levels of pro-inflammatory and anti-inflammatory macrophage-related proteins in the colon tissue of STC mice were detected by western blot. The results are as follows Fig.16 As shown in a, similar to the results of mRNA detection, the expression of iNOS protein increased in the STC group, and the expression of the above proteins could be inhibited after treatment with Polygonatum sibiricum polysaccharide. The expression of the anti-inflammatory protein Retnl was also similar to the mRNA results. The expression of Retnl protein increased in the Polygonatum sibiricum polysaccharide group. Fig.16 As shown in Figure b, by standard quantification of the above proteins, it was further verified that Polygonatum sibiricum polysaccharide can regulate the expression of M1 macrophage and M2 macrophage-related proteins in colon tissue, thereby exerting an anti-inflammatory effect. Polygonatum sibiricum polysaccharide regulates M macrophages and M2 macrophages not only at the transcriptional level, but also at the post-transcriptional translation level.

[0104] The key pathway of Polygonatum sibiricum polysaccharide in the treatment of STC is enriched in the PI3K / AKT signaling pathway. The proteins related to this pathway were detected and quantified. The results are as follows Fig.16 As shown in a, p-PI3K and p-AKT increased in the STC group, indicating the activation of the PI3K / AKT signaling pathway. The phosphorylation levels of the above proteins can be inhibited after intervention with Polygonatum sibiricum polysaccharide. By quantifying the immunoglobulin, it can be seen that Polygonatum sibiricum polysaccharide can significantly inhibit phosphorylated PI3K and phosphorylated AKT in STC mice. Polygonatum sibiricum polysaccharide may regulate the polarization of macrophages, reduce the production of iNOS, protect the damage of intestinal neurons, and thus regulate intestinal motility by inhibiting the activation of the PI3K / AKT signaling pathway.

[0105] Example 5: Polygonatum sibiricum polysaccharide regulates intestinal microorganisms in mice with chronic transmission constipation

[0106] Thirty 8-week-old Balb / c mice weighing approximately 20±2g were selected. Before the experiment, all mice were housed in an environment with a temperature of 25°C, a humidity of 50-60%, a light-dark cycle of 12:12h, and sufficient purified water and standard feeding food, and underwent a one-week adaptation period.

[0107] The mice were randomly divided into three groups: the Control group: given a normal diet and normal temperature saline gavage, 200uL each time, twice a day, for 14 consecutive days; the STC group: given a normal diet, and received 10mg / kg loperamide solution gavage, twice a day, for 14 days to establish a constipation model. The PCNP-2 group: After the STC model was established, 10mg / kg Polygonatum sibiricum polysaccharide was gavaged twice a day from the 8th day of modeling, for 7 consecutive days.

[0108] 5.1 Fecal 16S rRNA flora sequencing

[0109] On the 14th day of the experiment, the feces of mice in different groups were collected, genomic DNA was extracted from the feces, and 16SrRNA bacterial community sequencing was performed. The experiment used the MiSeq sequencing platform provided by Beijing Aoweisen Gene Technology Co., Ltd. MiSeq is a high-throughput sequencing device produced by Illumina, which is widely used in 16SrRNA gene sequencing analysis of microbial communities.

[0110] Alpha and Beta diversity analysis were performed on the data. Alpha diversity analysis mainly evaluates the species diversity and richness within the sample, using Chao1, Simpson, Shannon and PD_whole_tree indices. The results are as follows Fig.17 As shown. Chao1 index is an indicator to measure species richness. Compared with the control group, the value of STC group was significantly lower, indicating that the abundance of microorganisms in STC disease was reduced. After intervention with Polygonatum sibiricum polysaccharide, the richness of microbial species increased significantly, indicating that Polygonatum sibiricum polysaccharide reversed the reduction of microbial richness caused by STC. Simpson index is an index to measure diversity. The closer the value is to 1, the lower the diversity. Compared with the control group, the Simpson index of STC group was significantly higher, indicating that the microbial diversity of this group was significantly reduced. Although it recovered after treatment with Polygonatum sibiricum polysaccharide, there was no significant difference in statistics. Shannon index is another indicator to measure biodiversity, which takes into account the richness and uniformity of species. The diversity of the control group was significantly higher than that of the STC group, but there was no significant difference in Shannon index between STC and Polygonatum sibiricum polysaccharide groups. PD_whole_tree index measures the sum of the branch lengths occupied by community species on the evolutionary tree, reflecting the evolutionary diversity of the community. The PD_whole_tree index of STC group was significantly lower than that of Polygonatum sibiricum polysaccharide group, but there was no significant difference between STC group and control group.

[0111] Beta diversity analysis is to compare the diversity between different samples, and the results are shown by PCA and PLS-DA methods. Fig.18As shown. Based on PCA analysis, axes PC1 and PC2 showed percentage variation (19.32% and 15.61%, respectively). A single point in the image represents the microbial community of a single sample. The closer the two points are, the more similar their microbial composition is. Here, the control group and the Polygonatum sibiricum polysaccharide group appear to be separated from the STC group, showing different microbial community compositions. Based on PLS-DA analysis, the percentage variation of axes PC1 and PC2 were 13.88% and 13.92%, respectively. A more significant cluster distribution can be observed. The STC group has a more different microbial community than the control group and the Polygonatum sibiricum polysaccharide group (such as Fig.18 b), such obvious separation clustering shows the obvious difference in the intestinal microbial composition between STC mice and control mice, as well as the effect of Polygonatum sibiricum polysaccharide intervention on the intestinal microorganisms of mice, suggesting that Polygonatum sibiricum polysaccharide treatment changed the composition of intestinal microorganisms in the STC group. It further illustrates that changes in intestinal microorganisms in STC mice may be a key factor affecting the occurrence or progression of the disease, and Polygonatum sibiricum polysaccharide treatment can delay and reverse intestinal damage by affecting the abundance and composition of intestinal microorganisms. Polygonatum sibiricum polysaccharide treatment can change the genus composition of intestinal microorganisms in mice. Compared with the STC group, this change reflects the positive regulatory effect of Polygonatum sibiricum polysaccharide on the intestinal microbial community, which may help restore the balance and function of intestinal microorganisms.

[0112] 5.2 Short-chain fatty acid detection

[0113] There is a close relationship between short-chain fatty acids and intestinal microorganisms, which has an important impact on human health and the development of diseases. Studies have shown that short-chain fatty acids are the main metabolites of intestinal flora, including formic acid, acetic acid, propionic acid, butyric acid, etc., and have a clear direct correlation with the development of intestinal diseases.

[0114] Prepare seven high-purity standards of acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, and caproic acid. Dilute each standard with ether to a concentration of 100 mg / mL as a stock solution. The internal standard substance isohexanoic acid (4-methylvaleric acid) is also diluted with ether to prepare a stock solution with a concentration of 375 μg / mL, which is used as a reference substance in the experiment. Take out an appropriate amount from the stock solution of each acid mentioned above and mix them in a certain proportion. The specific steps are: add 100 μL of 15% phosphoric acid solution and 20 μL of internal standard stock solution to each mixture, and then add 260 μL of ether. The above mixtures were then prepared into standard solutions of different concentrations, including 0.02μg / mL, 0.1μg / mL, 0.5μg / mL, 2μg / mL, 10μg / mL, 25μg / mL, 50μg / mL, 100μg / mL, 250μg / mL, and 500μg / mL, for a total of 10 concentration levels, for establishing a standard curve. Based on standard solutions of different concentrations and the corresponding relative peak areas (standard peak area / internal standard peak area), a standard curve was drawn to calculate the concentration of short-chain fatty acids in the sample.

[0115] Weigh a certain amount of fecal samples from each group of model mice and put them into a 1.5mL centrifuge tube. Add 500μL of ether to the centrifuge tube. Then, place the centrifuge tube on ice and homogenize the sample with a grinding tool for 1min to ensure that the sample is fully mixed with the ether. Centrifuge the mixture at 4°C at 12000rpm for 10min using a high-speed centrifuge to take the supernatant containing the target compound. Take 200μL of the supernatant, add 100μL of 15% phosphoric acid solution and 20μL of 4-methylvaleric acid solution with a concentration of 375μg / mL as the internal standard. Then add 280μL of ether to dilute and further extract the target compound. Place the centrifuge tube on ice again and homogenize the solution with a grinding tool for 1min to ensure that all components are fully mixed. After that, centrifuge the mixture again at 4°C at 12000rpm for 10min. The purpose of this step is to remove any suspended solid particles and obtain a clear supernatant for subsequent chemical analysis.

[0116] Thermo Trace 1300 gas chromatograph was used for analysis. HP-INNOWAX capillary column was used for chromatographic analysis, and ISQ 7000 mass spectrometer was used for mass spectrometry analysis. Split injection technique was used, the injection volume was controlled at 1 μL, and the split ratio was set to 10:1. The temperature of the injection port was set to 250°C, and the temperatures of the ion source and the transmission line were set to 300°C and 250°C, respectively. The temperature program of the chromatography was designed as follows: the starting temperature was 90°C, followed by a temperature increase of 10°C per minute to 120°C, then a temperature increase of 5°C per minute to 150°C, and finally a temperature increase of 25°C per minute to 250°C, and maintained at this temperature for 2 minutes. The carrier gas helium passed through the chromatographic column at a flow rate of 1.0 mL / min.

[0117] The results are as follows Fig.19 As shown in the results, compared with the control group, the contents of acetic acid, butyric acid, caproic acid, isovaleric acid, valeric acid, isobutyric acid and propionic acid in the feces of mice in the STC group were all reduced, among which the contents of butyric acid, caproic acid, isobutyric acid and propionic acid in the STC group were statistically significantly different from those in the control group, indicating that the production of the above short-chain fatty acids plays a key role in maintaining intestinal homeostasis. After intervention with Polygonatum sibiricum polysaccharide, the decline of the above short-chain fatty acids can be partially reversed, among which the differences of acetic acid, butyric acid and caproic acid compared with the STC group were statistically significant. This suggests that Polygonatum sibiricum polysaccharide treatment may help restore the contents of these short-chain fatty acids, especially the concentrations of acetic acid, butyric acid and caproic acid.

[0118] The above experimental results show that in addition to regulating the richness of specific bacterial communities, Polygonatum sibiricum polysaccharide can also significantly improve the intestinal microbial diversity of constipation model mice, including increasing Chao1 and Shannon indexes and reducing Simpson index, which reflects that the diversity and uniformity of the microbial community have been improved after treatment with Polygonatum sibiricum polysaccharide. In addition, LEfSe analysis revealed specific bacterial communities with significant differences in the Polygonatum sibiricum polysaccharide-treated groups, which may be key biomarkers of the mechanism by which Polygonatum sibiricum polysaccharide relieves constipation. Polygonatum sibiricum polysaccharide treatment can significantly increase the concentration of key short-chain fatty acids such as acetate, butyrate and caproate in the intestine. These short-chain fatty acids are essential for maintaining the health of intestinal epithelial cells, regulating immune responses, and maintaining intestinal barrier function, which in turn helps to relieve constipation symptoms.

[0119] The embodiments of the present invention are described in detail above, but the contents described are only preferred embodiments of the present invention and cannot be considered to limit the scope of implementation of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the scope of the patent coverage of the present invention.

Claims

1. A polygonatum polysaccharide, characterized in that: Named PCNP-2, it is composed of fructose and glucose with a residue ratio of -33:

1. PCNP-2 is an Agavin type fructan.

2. The polygonatum polysaccharide according to claim 1, characterized in that: The molecular weight is 5370Da.

3. The polygonatum polysaccharide according to claim 1, characterized in that: It is composed of β-D-Fruf-(2→,→2)-β-D-Fruf-(1→,→2)-β-D-Fruf-(6→,→2)-β-D-Fruf-(1,6→ and→6)-α-D-Glcp-(1→ sugar residues; Among them, the ratio of fructose β-D-Fruf-(2→, →2)-β-D-Fruf-(1→, →2)-β-D-Fruf-(6→, and →2)-β-D-Fruf-(1,6→ residues is approximately 1:2:3:

1.

4. A method for preparing the polygonatum polysaccharide according to any one of claims 1 to 3, characterized in that: Polygonatum sibiricum crude polysaccharide was extracted from Polygonatum sibiricum by water extraction and alcohol precipitation method, and the main components of the crude Polygonatum sibiricum polysaccharide were separated by liquid chromatography to obtain PCN, and the main components of PCN were further separated by molecular sieve chromatography column to obtain PCN-2, and after purification of PCN-2, a neutral polysaccharide uniform component Polygonatum sibiricum polysaccharide PCNP-2 was obtained.

5. The method for preparing polygonatum polysaccharide according to claim 4, characterized in that: The specific preparation method is as follows: Step 1: crush the dried Polygonatum sibiricum medicinal material, extract and defatted with 75% ethanol by heating and refluxing, repeat three times, each time for 2 hours; adopt water extraction and alcohol precipitation method at 100°C, heat and reflux according to a solid-liquid ratio of 1:10, extract the medicinal residue and repeat three times, each time for 2 hours; collect the supernatant and add ethanol with a final concentration of 80% (v / v) to precipitate, separate and fully dissolve the precipitate, dialyze, concentrate and freeze-dry to obtain Polygonatum sibiricum crude polysaccharide PCCP; Step 2: Take the PCCP solution, load it onto a DEAE-650M column, and elute it with distilled water and 0.1M NaCl solution. The distilled water elution portion is the main portion, marked as PCN; Step 3: Purify PCN using molecular sieve column chromatography Sepharose 6B column, using 0.1M NaCl solution as eluent to obtain two neutral polysaccharide components PCN-1 and PCN-2 from Polygonatum cyrtonema; Step 4: Purify the component PCN-2 using a Sephacryl S-300 column, elute with a 0.1 M NaCl solution as the eluent, and obtain the uniform component PCNP-2 of the neutral polysaccharide of Polygonatum sibiricum after reduced pressure concentration, dialysis, and freeze-drying.

6. Use of the polygonatum polysaccharide according to any one of claims 1 to 3 in the preparation of a drug for chronic transmission constipation.

7. The use according to claim 6, characterized in that: Polygonatum sibiricum polysaccharide PCNP-2 inhibits the activation of the PI3K / AKT signaling pathway and the expression of iNOS protein, thereby regulating macrophage polarization and achieving the repair of chronic transmission constipation damage.

Images