Polygonatum polysaccharide as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of traditional Chinese pharmacology and veterinary medicine, and relates to prepared rhizoma polygonati polysaccharide as well as a preparation method and application thereof. The prepared rhizoma polygonati polysaccharide is mainly composed of galactose, the main chain is-4)-beta-D-Galp-[(1-4) / beta-D-Galp] 7-(1->, a beta-D-Galp-group is connected to the position 6 of an intermediate galactose molecule, the prepared rhizoma polygonati polysaccharide is further connected to-4)-beta-D-Manp-(1-4)-beta-D-Manp-(1-> residue, and mannose is substituted by acetyl. The prepared rhizoma polygonati polysaccharide provided by the invention has various effects of relieving ulcerative colitis, relieving intestinal injury induced by LPS (lipopolysaccharide), protecting intestinal mucosal barrier, regulating intestinal flora, improving antioxidant capacity and in-vitro antioxidant activity and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine and veterinary medicine, and relates to a prepared polygonatum polysaccharide and a preparation method and application thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Maintaining the integrity of the intestinal barrier and the balance of intestinal flora is crucial to maintaining animal health. However, the drugs commonly used in the clinic to treat intestinal damage, such as 5-aminosalicylic acid and corticosteroids, can only relieve symptoms but cannot fundamentally treat the disease. They also have some side effects such as nausea, vomiting, and irritation of the gastric mucosa. Therefore, it is urgent to develop drugs for the treatment of intestinal damage that are effective, safe, effective, and low-cost. A large number of studies have shown that plant polysaccharides can effectively improve the intestinal barrier function of humans and animals, regulate disordered intestinal flora, and have excellent antioxidant effects. The short-chain fatty acids (SCFA) produced by microbial metabolism can protect intestinal mucosal epithelial cells, maintain the homeostasis of intestinal flora, and improve intestinal health. Therefore, polysaccharides have the potential to be developed into drugs for the treatment of intestinal damage.

[0004] Free radicals and reactive oxygen species are important active substances produced by the body and participate in various physiological activities of the body. However, excessive or untimely removal will cause oxidative damage to the body. Oxidative damage caused by free radicals and reactive oxygen species plays an important role in the pathophysiological process of many diseases. A large number of studies have shown that chemical components and extracts of traditional Chinese medicine have antioxidant effects in vivo and in vitro. For example, traditional Chinese medicine components such as flavonoids, phenolic acids, quinones, and polysaccharides have been reported to have the effects of scavenging free radicals in vivo and in vitro, inhibiting oxidative damage caused by reactive oxygen species, etc. In addition, given the close relationship between oxidative damage, inflammation, and intestinal barrier, studying the antioxidant activity of polysaccharides is crucial to clarify their anti-inflammatory and intestinal barrier repair effects. Summary of the invention

[0005] In order to solve the shortcomings of the prior art, the purpose of the present invention is to provide a processed polygonatum polysaccharide and a preparation method and application thereof. The processed polygonatum polysaccharide provided by the present invention has multiple effects such as alleviating ulcerative colitis, alleviating LPS-induced intestinal damage, protecting the intestinal mucosal barrier, regulating intestinal flora, and in vivo and in vitro antioxidant effects.

[0006] In order to achieve the above object, the technical solution of the present invention is:

[0007] In the first aspect, a prepared polygonatum polysaccharide is mainly composed of galactose, and the main chain is

[0008] →4)-β-D-Galp-[(1→4) / β-D-Garp]7-(1→, the 6-position of the middle galactose molecule is connected to a β-D-Galp-group, the prepared polygonatum sibiricum polysaccharide is also connected to a →4)-β-D-Manp-(1→4)-β-D-Manp-(1→ residue, and an acetyl group is substituted on the mannose.

[0009] In some embodiments, the number average molecular weight is (2.00-2.50)×10 4 Da.

[0010] In a second aspect, a method for preparing the above-mentioned prepared polygonatum polysaccharide comprises the following steps:

[0011] The crude polysaccharide of polygonatum was obtained by treating it with water extraction and alcohol precipitation.

[0012] removing the protein component from the processed Polygonatum sibiricum crude polysaccharide;

[0013] The crude polysaccharide of Polygonatum sibiricum was eluted with a DEAE agarose gel FF column, and PSSP-E was obtained.

[0014] PSSP-E was fractionated using a Chromdex 75PG column.

[0015] In some embodiments, in the water extraction and alcohol precipitation process, water at 75-85° C. is used for extraction.

[0016] In some embodiments, during the water extraction and alcohol precipitation process, ethanol is used for precipitation at 3-5°C.

[0017] In some embodiments, Sevage reagent is used to remove protein components in the crude polysaccharide of Polygonatum cyrtonema. The Sevage reagent of the present invention is prepared from chloroform and n-butanol in a volume ratio of 3.5 to 4.5:1.

[0018] In some embodiments, elution is performed using distilled water, 0.15-0.25 M NaCl solution, 0.45-0.55 M NaCl solution, and 0.95-1.05 M NaCl solution in sequence.

[0019] In some embodiments, the sugar content is determined during elution using the anthrone-sulfuric acid method.

[0020] In some embodiments, PSSP-E is fractionated using distilled water.

[0021] In some embodiments, the anthrone-sulfuric acid method is used to determine the sugar content during fractionation.

[0022] In a third aspect, a use of the above-mentioned processed polygonatum polysaccharide in preparing a product, wherein the product has one or more of the following effects:

[0023] a) prevent and / or treat intestinal damage;

[0024] b) prevention and / or treatment of intestinal flora disorders;

[0025] c) Prevent and / or treat damage caused by reactive oxygen species and / or free radicals.

[0026] In the technical scheme of the present invention, processed polygonatum sibiricum is used as raw material, and processed polygonatum sibiricum polysaccharide is carefully prepared by water extraction and alcohol precipitation method. Through animal experiments, it is confirmed for the first time that the polysaccharide has significant efficacy in treating intestinal diseases and oxidative damage in mice, demonstrating its outstanding value in the fields of health care and treatment.

[0027] The concept of "prevention and / or treatment" proposed in the present invention covers a series of comprehensive measures for intestinal diseases and oxidative damage. This includes not only direct treatment of these diseases to relieve existing symptoms, but also preventive treatment aimed at reducing the risk of disease occurrence or reducing the severity of symptoms. In addition, this concept also includes the prevention of disease recurrence, whether it is to prevent the disease from rebounding after the treatment is completed, or to take measures to alleviate symptoms when the disease has already occurred.

[0028] In some embodiments, the product is a medicine, an additive or a food. The additive can be added to feed. The food can be a health food.

[0029] In some embodiments, the product is a composition, and the composition includes an excipient. When the product is a medicine, the excipient is a pharmaceutical excipient, such as a pharmaceutical carrier, an excipient (binder, filler, disintegrant, preservative, fragrance, etc.), etc.

[0030] The beneficial effects of the present invention are:

[0031] The present invention uses processed polygonatum as raw material, adopts water extraction and alcohol precipitation method to obtain processed polygonatum polysaccharide, and proves for the first time through animal experiments that the processed polygonatum polysaccharide has the ability to prevent / treat ulcerative colitis, improve intestinal flora disorder and increase the content of SCFA in the colon; has the ability to prevent / treat LPS-induced intestinal damage, improve intestinal flora disorder, and reduce oxidative damage caused by free radicals and / or active oxygen in vivo and in vitro, and has good health care and therapeutic value. At the same time, the extraction and preparation method of the present invention is simple, easy to be converted into a modern industrial production process, and has great development potential, so it can achieve good economic and social benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0033] Figure 1 The elution curves of prepared Polygonatum sibiricum polysaccharide in the examples of the present invention are shown; (A) DEAE agarose FF elution curve, (B) Chromdex 75PG elution curve.

[0034] Figure 2 Chemical properties of PSSP-EF in the examples of the present invention; (A) UV spectrum, (B) HPGPC chromatogram, (C) monosaccharide composition, (a) chromatogram of mixed standard, (b) chromatogram of PSSP-EF sample.

[0035] Figure 3 1D NMR and 2D NMR spectra of PSSP-EF in the embodiment of the present invention; (A) 1 H-NMR spectrum, (B) 13 C-NMR spectrum, (C) HSQC spectrum, (D) 1 H- 1 H COSY spectrum, (E) HSQC-TOCSY spectrum, (F) HMBC spectrum, (G) NOESY spectrum.

[0036] Figure 4 It is the structural model of PSSP-EF in the embodiment of the present invention.

[0037] Figure 5 The effects of PSSP-EF on UC mice in the examples of the present invention; (A) schematic diagram of animal experimental procedures, (B) daily body weight changes, (C) DAI scores during the experiment, (D) mouse survival curves, (E) colon pictures, (F) colon length, (G) colon coefficients, (H) morphological scores, (I) histopathological scores, and (J) H&E images of mouse colon tissue.

[0038] Figure 6 The effect of PSSP-EF on the spleen of UC mice in the examples of the present invention; (A) spleen coefficient, (B) thymus coefficient.

[0039] Figure 7Effects of PSSP-EF on TJ proteins, inflammatory cytokines and enterotoxin levels in UC mice; (A) Protein expression of ZO-1 and Occuldin, (B) Densitometric analysis, (C) Expression level of proinflammatory cytokine IL-1β in serum, (D) Expression level of proinflammatory cytokine TNF-α in serum, (E) Expression level of proinflammatory cytokine IL-6 in serum, (F) Expression level of proinflammatory cytokine IL-1β in colon tissue, (G) Expression level of proinflammatory cytokine TNF-α in colon tissue, (H) Expression level of proinflammatory cytokine IL-6 in colon tissue, (I) Enterotoxin IS level in serum, (J) Enterotoxin LPS level in serum.

[0040] Figure 8 The effect of PSSP-EF on the intestinal microbiota in the embodiments of the present invention; (A) Chao1 index, (B) Shannon index, (C) PCoA analysis based on bray_curtis distance, (D) species Venn diagram analysis, (E) phylum-level community bar graphs of each group, (F) genus-level community bar graphs of each group, (G) community bar graphs at the genus level of each sample, (H) linear discriminant analysis (LefSe) of the effect size of each group.

[0041] Fig. 9 : The effect of PSSP-EF on SCFA levels in the examples of the present invention; (A) SCFA content in cecal contents, (B) heat map of SCFA and bacteria at the genus level.

[0042] Fig.10 The PSSP-EF in the present invention alleviates LPS-induced intestinal damage in mice; a: body weight, b: changes in body weight after LPS induction, c: jejunal pathological analysis, d: ileal pathological analysis.

[0043] Fig.11 Serum antioxidant and inflammatory levels mediated by PSSP-EF in the examples of the present invention; a: MDA, b: SOD, c: T-AOC, d: GSH-Px, e: IL-1β, f: IL-6, g: IL-10, h: TNF-α.

[0044] Fig.12 Alpha diversity analysis in the embodiment of the present invention; a: rarefaction curve, b: rank abundance curve, c: index.

[0045] Fig.13 The asv Venn diagrams and β diversity of intestinal flora of different groups of mice in the examples of the present invention; a: Venn diagram, b: PCo-A, c: NMDS, d: UPGMA, e: group distance.

[0046] Fig.14Heat map and LEfSe analysis of the microbiome of different groups of mice in the examples of the present invention; a: heat map, b: LEfSe.

[0047] Fig.15 The differences in microbiota of different groups of mice revealed by ANOVA analysis in the examples of the present invention; a: phylum level, b: genus level.

[0048] Fig.16 This is the KEGG functional analysis of the mouse microbiota in the examples of the present invention.

[0049] Fig.17 This is the MetaCyc pathway analysis of the mouse microbiota in the examples of the present invention.

[0050] Fig.18 The in vitro antibody oxidation capacity of the prepared Polygonatum sibiricum polysaccharide in the examples of the present invention; (A) in vitro total antibody oxidation capacity (T-AOC), (B) ABTS free radical scavenging rate, (C) DPPH free radical scavenging rate, (D) ABTS free radical scavenging rate positive control, (E) DPPH free radical scavenging rate positive control. DETAILED DESCRIPTION

[0051] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0052] Example 1: Preparation of Polygonatum cyrtonema Polysaccharide

[0053] Extraction of Polygonatum cyrtonema polysaccharide: Dried Polygonatum cyrtonema (300 g) was cut into thick slices and extracted twice with pure water (1:10) at 80°C for 3 hours. The supernatant extract was then separated after centrifugation (5000 rpm, 10 minutes) and precipitated with 3 volumes of 95% ethanol at 4°C overnight. Centrifugation (13000 rpm, 5 minutes) was performed and the precipitate was obtained as the crude Polygonatum cyrtonema polysaccharide.

[0054] Purification method of prepared polygonatum polysaccharide: The freeze-dried prepared polygonatum polysaccharide was dissolved in ultrapure water to prepare a 0.1 g / mL aqueous solution. Sevage reagent (chloroform: n-butanol = 4:1, v:v) was added according to 4:1, and then placed on a shaker for 30 minutes, and the operation was repeated 4-6 times to fully remove the protein components in the crude polysaccharide. Finally, the organic reagent dissolved in the solution was removed by decompression by a rotary hair growth instrument, and the deproteinized prepared polygonatum polysaccharide was obtained after freeze-drying and stored at -20°C for use. The deproteinized prepared polygonatum polysaccharide (100 mg) was dissolved in 30 mL of distilled water and eluted with 2 column volumes of distilled water, 0.2 M NaCl, 0.5 M NaCl and 1.0 M NaCl solutions through a DEAE agarose gel FF column (3.0×50 cm) at a flow rate of 1.0 mL / min. The eluate was collected in a 10 mL tube, and the sugar content was determined by anthrone-sulfuric acid method at 630 nm using a UV-visible spectrophotometer to draw a separation curve. The tubes with the same peak were combined, concentrated, and freeze-dried to obtain Polygonatum sibiricum polysaccharide PSSP-E1 ( Figure 1 A). Then, the polysaccharide PSSP-E1 was separated using a Chromdex75PG column (1.6 cm × 100 cm) at a flow rate of 1.0 mL / min with 1 column volume of distilled water. The eluate was collected in a 5 mL tube, and the sugar content was measured using the anthrone-sulfuric acid method, and a separation curve was drawn. The test tubes with the same peak were combined, concentrated, and freeze-dried to obtain the polysaccharide PSSP-EF ( Figure 1 B).

[0055] Structural determination of processed Polygonatum sibiricum polysaccharide PSSP-EF: The purified polysaccharide was subjected to polysaccharide purity identification, molecular weight distribution determination, and monosaccharide composition and ratio determination. The purity of PSSP-EF was detected by ultraviolet spectroscopy (UV), the molecular weight of PSSP-EF was determined by high performance gel column chromatography (HPGPC), and the monosaccharide composition and ratio of PSSP-EF were determined by complete acid hydrolysis and high performance liquid chromatography (HPLC). In addition, the structure of PSSP-EF was determined by methylation experiments and nuclear magnetic resonance spectroscopy (1D NMR and 2D NMR).

[0056] Table 1 Methylation analysis of PSSP-EF

[0057]

[0058] Table 2 NMR signal assignments of PSSP-EF glycosidic bonds

[0059]

[0060]

[0061] The results showed that PSSP-EF did not contain impurities such as proteins, and its number average molecular weight was 2.24×104 Da. The PSSP-EF sample mainly contains four sugar residues, including 4-Gal(p), t-Gal(p), 4,6-Gal(p) and 5-Ara(f), as well as a small amount of 4-Glc(p), t-Ara(f) and t-Xyl(p) (Table 1); According to the analysis of monosaccharide composition by HPLC method, the PSSP-EF sample is mainly composed of Gal, with a molar ratio of 80.55%. The results of methylation and nuclear magnetic resonance analysis also confirmed the presence of a large number of 4-Galp sugar residues, as shown in Table 2. Comprehensive analysis of 1D NMR and 2D NMR data showed that the main chain of PSSP-EF is →4)-β-D-Galp-[(1→4) / β-D-Garp]7-(1)→, in which the middle galactose molecule is connected to the β-D-Galp- group at the 6th position. In addition, a small amount of →4)-β-D-Manp-(1→4)-β-D-Manp-(1→ residues are attached, and the mannose is substituted with an acetyl group (see Figure 4 ). According to the results of methylation analysis, it is difficult to completely separate 4-Gal(p) and 4-Man(p) due to their co-elution on the gas chromatography column. However, based on the monosaccharide composition, it can be calculated that the proportion of 4-Man(p) is about 5%. Therefore, the molar ratio of sugar residues →4)-β-D-Galp-(1→ and →4,6)-β-D Galp-(1→ can be calculated to be 7:1. According to literature search, the structure of the prepared Polygonatum sibiricum polysaccharide has not been reported.

[0062] The prepared Polygonatum cyrtonema polysaccharide was used in Examples 2-5.

[0063] Example 2: Treatment of DSS-induced colitis in mice with processed Polygonatum sibiricum polysaccharide

[0064] Model establishment: UC was induced by administering 4% DSS in drinking water for 9 days. 40 mice were randomly divided into 4 groups: control group, model group, PSSP-EF high-dose group and PSSP-EF low-dose group (8 mice in each group). The mice in the control group drank only purified water during the entire experiment. During the experiment, mice in the high-dose PSSP-EF group and the low-dose PSSP-EF group were given 400 and 200 mg / kg PSSP-EF by gavage, respectively, and the control group and the model group received the same volume of distilled water by gavage for 9 consecutive days.

[0065] After the experiment, the feces of each mouse were collected and stored in liquid nitrogen until further use. Subsequently, the mice were sacrificed under anesthesia to obtain colon, rectum, cecum, spleen, thymus, and blood samples.

[0066] The effects of organ indexes of mice and the levels of colon TJ proteins, inflammatory cytokines and enterotoxins were analyzed by H&E staining.

[0067] Colonic tissue was isolated, photographed, measured, weighed, and the colon coefficient was calculated using a formula. The distal colon was split longitudinally and flattened. Subsequently, the colon fragments were observed and the morphology was graded according to the scoring criteria. Then, the colon segments were collected for hematoxylin-eosin (H&E) staining. The histological score was evaluated according to the scoring criteria.

[0068] Colon coefficient (%) = colon weight / body weight × 100%

[0069] Determination of spleen and thymus coefficients

[0070] The spleen and thymus tissues were photographed and weighed, and the spleen and thymus coefficients were calculated according to the formulas.

[0071] Spleen coefficient (%) = spleen weight / body weight × 100%

[0072] Thymus coefficient (%) = thymus weight / body weight × 100%

[0073] The colon tissues were homogenized in lysis buffer, and the concentrations of TNF-α, IL-1, and IL-6 in the serum and colon tissues of each group of mice were measured using ELISA detection kits. In addition, the concentrations of serum and IS were measured according to the instructions of the ELISA kits.

[0074] Colon segments were homogenized in RIPA buffer and ground on ice to extract proteins. Protein quantification was done using a BCA assay kit. Denatured proteins were then separated by loading onto gels and subjected to electrophoresis. Subsequently, proteins were incubated with primary antibodies for ZO-1, Occludin, Claudin-1, GAPDH, and then IgG-HRP was incubated with anti-rabbit IgG-HRP secondary antibodies after membrane transfer. Finally, protein bands were visualized by an Amersham Imager 600 system and analyzed by Image-J.

[0075] Specific symptoms can reflect the severity and prognosis of UC, such as weight changes, stool status, bloody stools, DAI, etc. The mice in the control group showed normal activity, diet, stool hardness, DAI, and normal weight gain. After free drinking of 4% DSS for 9 days, the mice in the model group lost weight, increased DAI, and loose stools ( Figure 5 B and 5C). However, oral administration of PSSP- significantly suppressed DSS-induced weight loss and gradually restored the body weight of mice to normal levels in a dose-dependent manner ( Figure 5 B). In addition, PSSP-EF also significantly alleviated DSS-induced symptoms, such as decreased DAI scores and reduced mortality ( Figure 5 C and 5D). These results indicate that PSSP-EF effectively alleviates the symptoms of DSS-induced UC in mice.

[0076] To verify the therapeutic effect of PSSP-EF on colon pathological changes in UC mice, histological analysis was performed to estimate the extent of colon tissue damage. Compared with the control group, the colon length of the model group mice was significantly shortened ( Figure 5 E and 5F). However, PSSP-EF could effectively alleviate the colonic injury induced by DSS. After PSSP-EF treatment, the reduction in colon length was attenuated in a dose-dependent manner ( Figure 5 E). In addition, after PSSP-EF treatment, DSS-induced colon morphological damage, including bleeding and ulceration, was significantly alleviated, and the colon coefficient was also significantly reduced ( Figure 5 G). In addition, colon morphology and histopathological scores were significantly limited after DSS-induced administration ( Figure 5 H and 5I). H&E staining of the control group showed that the intestinal mucosal structure was intact, the epithelial cells were arranged regularly, and the goblet cells were intact. In contrast, the colon mucosa of DSS-induced mice was severely damaged, the intestinal gland necrosis disappeared, and a large number of inflammatory cells disappeared ( Figure 5 J). After PSSP-EF treatment, the histopathological damage of mice was significantly reduced compared with the model group ( Figure 5 I and 5J). Taken together, these results suggest that PSSP-EF can protect the colon from DSS-induced pathological changes.

[0077] UC is an inflammatory bowel disease that often causes an enlarged spleen. It causes immune imbalance and inflammation. Figure 6 As shown in A, the spleen coefficient was significantly reduced after PSSP-EF administration. In addition, this example also observed that the thymus coefficient increased significantly after PSSP-EF treatment ( Figure 6 B). Therefore, PSSP-EF can protect the immune system of UC mice by inhibiting hyperproliferation and thymic atrophy.

[0078] Existing evidence shows that intestinal mucosal barrier dysfunction is closely related to the occurrence of UC. At the same time, proteins Occludin, ZO-1, and Claudin-1 can reflect the integrity of the intestinal barrier to a certain extent. Therefore, in order to further study the protective mechanism of PSSP-EF on the intestinal barrier, Western blot analysis was performed to detect the expression of these proteins. Figure 7As shown in A and 7B, the results showed that the protein expression of Occludin, ZO-1, and Claudin-1 in the model group was significantly reduced compared with the control group. In contrast, PSSP-EF significantly increased the protein expression of Occludin, ZO-1, and Claudin-1 compared with the model group, and grayscale calculation showed that the expression of these three proteins in the PSSP-EF-high-dose group was closer to the control group. These results indicate that PSSP-EF effectively restored the intestinal barrier function by regulating the protein expression of Occludin, ZO-1, and Claudin-1.

[0079] Enterotoxins, such as LPS and IS, are key targets of the innate immune system, which also triggers inflammatory signals and damages the intestinal barrier. On the other hand, the main inflammatory cytokines involved in the pathogenesis of UC include IL-6, IL-1β, and TNF-α. Therefore, after PSSP-EF treatment, the levels of LPS and IS in the serum of mice, as well as the levels of IL-6, IL-1β, and TNF-α in serum and colon tissues were measured. Figure 7 As shown in CH, the ELISA results showed that DSS significantly increased the levels of TNF-α, IL-1β, and IL-6 in serum and colon tissue compared with the control group, which was consistent with its pathological results. Compared with the model group, the levels of TNF-α, IL-1β, and IL-6 in the PSSP EF treatment group were significantly reduced in a dose-dependent manner. In addition, the levels of LPS and IS in the model group were significantly increased, indicating that the enterotoxins in the blood circulation of UC mice increased, while the levels of enterotoxins gradually decreased with the increase of PSSP-EF dose ( Figure 7 I and 7J). These results indicate that PSSP-EF can protect intestinal barrier function and inhibit DSS-induced inflammation in mice.

[0080] In general, the prepared polygonatum sibiricum polysaccharide provided by the present invention has the effect of treating DSS-induced colitis in mice.

[0081] Example 3: Processed Polygonatum sibiricum polysaccharide improves DSS-induced intestinal mucosal damage and intestinal flora disorder in mice.

[0082] Animal model: Same as Example 2.

[0083] Colon intestinal sample collection: The mouse colon contents collected in Example 2 were collected and stored in a -80°C refrigerator.

[0084] Detection of microbial diversity in colon intestinal contents: DNA from fecal samples was extracted by CTAB method, and the highly variable V3-V4 region of 16SrDNA was selected for amplification analysis. The Illumina Novaseq sequencing platform was used for double-end sequencing of the library. In order to study the species composition of each sample, the effective tags of all samples were clustered into operational taxonomic units (OTUs) with a similarity threshold of 97%.

[0085] like Figure 8 As shown in A and 8B, the Chao1 and Shannon indices based on OTUs indicated that the microbial α diversity in the model group was significantly reduced compared with that in the control group. However, this phenomenon was partially reversed after PSSP-EF treatment. The differences in principal coordinates analysis (PCoA) based on Bray_Curtis distance among the four groups were analyzed ( Figure 8 C). The results showed that there were differences in the composition structure of each group. The model group showed a shift in bacterial composition aggregation, and the PSSP-EF-treated group also showed separation from the model group, indicating that DSS-induced intestinal microbiota dysbiosis was restored by PSSP-EF. The results showed that PSSP-EF could change the structure of intestinal microorganisms in UC mice.

[0086] To further determine the differences in gut microbial community composition and abundance among the groups, a series of visualization methods were used to evaluate the community composition of fecal samples. First, Venn diagrams were used to determine the OTUs shared by the different treatment groups and the average number of OTUs. There were 326 OTUs overlapping across all 4 groups, including 1095 OTUs in the control group, 924 OTUs in the model group, 957 OTUs in the high-dose PSSP-EF group, and 1227 OTUs in the low-dose PSSP-EF group ( Figure 8 D). Subsequently, based on the results of species annotation, the top 30 species with the highest abundance in each taxonomic level were selected from each sample or group to generate a cylindrical cumulative plot of species relative abundance. At the phylum level, the composition of the intestinal microbiota in the four groups was similar, mainly composed of Bacteroidetes, Firmicutes, Verrucos, Deferrobacillaceae, and Proteobacteria. Compared with the control group, the populations of Verrucos and Proteobacteria increased significantly, and the abundance of Deferrobacillaceae and Actinobacteria decreased in the model group, which could be reversed after treatment with PSSP-EF ( Figure 8 E). Finally, the genus-level microbial abundance was determined from each sample or group ( Figure 8 F and 8G). In the model group, the abundance of Muribacterium, Alisma, and Eubacterium was observed to decrease, while the abundance of Bacteroides, Rhodococcus, Akkermansia, and Erysipelothrix was observed to increase. However, compared with the model group, the abundance of Muribacterium was observed to increase significantly in the PSSP-EF group, while the abundance of Bacteroides and Erysipelothrix was significantly suppressed.

[0087] Finally, linear discriminant analysis of effect size (LefSe) was used to compare species that were significantly different between the four groups, with a screening criterion for linear discriminant analysis (LDA) values ​​>4 ( Figure 8 H). According to the analysis results, a total of 23 bacterial species in the study group showed significant changes at the phylum, class, order, family, genus, and species levels. The evolutionary tree showed that α-protozoa and Bacteroidetes were the main bacterial types that caused the imbalance of the intestinal microbiota in the model group. In addition, murines in the PSSP-EF treatment group showed the highest LDA score (LDA = 5.04, P = 0.0087), indicating that this may be related to the PSSP-EF-mediated remission of UC. It is worth noting that beneficial bacteria producing SCFAs and anti-inflammatory metabolites were observed in the PSSP-EF treatment group, which may also be one of the important factors promoting the role of PSSP-EF in resolving UC.

[0088] The levels of SCFA in cecal contents were determined by GC-MS analysis, which was performed on an Agilent 6890GC system equipped with an HP-5MS column (25 μm × 250 μm × 30 m) using the method previously described by Yong Gang Xia et al. SCFA are important metabolites of the intestinal microbiota and have beneficial effects on intestinal epithelial cells by providing nutrition, promoting metabolism, and other functions. The levels of SCFA in cecal contents were determined by GC-MS. Fig. 9 As shown in A, the levels of acetate, propionate, isobutyrate, butyrate, valerate, and isovalerate in the cecal contents of the model group were significantly decreased compared with those of the control group, which was consistent with the trend of decreased SCFA in UC patients. However, the levels of acetate, propionate, isobutyrate, butyrate, valerate, and isovalerate were significantly increased after PSSP-EF treatment compared with the model group. Spearman correlation analysis was then used to determine the potential relationship between the altered intestinal microbiota and SCFA. Fig. 9 As shown in B, acetate, propionate, isobutyrate, butyrate, valerate, and isovalerate were positively correlated with Murinobacteriaceae, Alitis, Bifidobacterium, and Rf39, but negatively correlated with Bacteroides, Akkermansia, Rhodococcus, Erysipelothrix, and Parasitium. These results suggest that the increase in SCFA levels after PSSP-EF treatment may be related to the increased abundance of Murinobacteriaceae, Alitis, Bifidobacterium, Eubacterium, and Rf39.

[0089] Example 4: Processed Polygonatum sibiricum polysaccharide improves LPS-induced intestinal damage in mice.

[0090] Animal model: 30 ICR mice (4 weeks, 22.81±0.98g), male and female animals were equal. After 3 days of acclimation, they were divided into control group (CH), induced group (MH) and treatment group (H). The mice in group H were gavaged with 100mg / kg PSSP-EF, and the mice in groups C and M were gavaged with an equal volume of saline for 18 days. On the 18th day, groups M and H were induced with LPS (10mg / kg), and blood and intestinal specimens were collected by euthanasia on the second day.

[0091] Effects of Polygonatum sibiricum polysaccharide on LPS-induced intestinal damage revealed by H&E staining, serum antioxidant and inflammatory level detection, and bacterial flora sequencing

[0092] The body weight of mice in the PSSP-EF group was slightly higher than that in the CH and MH groups ( Fig.10 a), After LPS induction, the body weight of mice in the MH group decreased significantly (P<0.0001), while the body weight of mice in the PSSP-EF group was higher (P<0.01) ( Fig.10 b). Pathological analysis revealed that the villi of LPS-induced mice were mostly broken and short, whereas the villi of PSSP-EF-treated animals were long and fairly intact. The villi length of CH group (P<0.0001) and H group (P<0.0001) were longer than those of M, and the villi length / crypt depth value of MH group was smaller than those of CH group (P<0.0001) and H group (P<0.0001), while the crypt depth of MH group was higher than those of CH group (P<0.0001) and H group (P<0.0001) ( Fig.10 c,d).

[0093] Serum tests showed that MAD (P<0.05), IL-1β (P<0.05), IL-6 (P<0.05), and TNF-α (P<0.01) were significantly increased in the MH group, and SOD (P<0.001), T-AOC (P<0.01), and GSH-Px (P<0.01) were significantly increased in the CH and H groups. Fig.11 ).

[0094] A total of 77,500 (CH), 69,600 (MH), and 98,300 (H) sequences were identified in mice, as well as 66,700 (CH), 57,800 (MH), and 85,000 (H) sequences after filtering. Alpha diversity calculations found that the rarefaction curves for all mice were horizontal lines after a brief rise ( Fig.12 a), Rank abundance curve of CH group; MH and H are both flat horizontal lines ( Fig.12 b), indicating that these mouse samples are sufficient and have high uniformity. There were no significant differences in the results of various indicators among the CH, MH, and H groups ( Fig.12 c).

[0095] The Venn diagram showed that a total of 2407 species were detected in animals, and 239 species were detected in each group. CH and MH, CH and H co-existed in 312 and 328 species, respectively. Figure 5 a). Beta diversity analysis showed that the distance between CH and H was shorter than that between CH and MH, but the difference was not significant (P<0.05) ( Fig.13 b, c, d, e). Heat map analysis showed that the contents of Streptococcus and Riemerella were higher in Enterobacter, UBA3263, Bacteroides_h, Mycobacterium, Mycoplasma_A, Mobinobacterium, Helicobacter pylori-D, Myxobacterium, Lawus, CAG-83, and MH, while the contents of Duborella, Cryptobacterium, NM07-P-09, Aerobacterium, Odorobacter, Parabacterium, OLB9, UBA7173, Sparus, CAG-485, Faecalibacterium, Rombusia_B, Ctenobacterium, Parabacterium, Prevotella, Duncania, Parabacterium_b, UBA3282, Bifidobacterium, and Baculonella muris were higher in CH ( Fig.14 a). LEfSe results showed that the contents of Limivicinus (P<0.05), Staphylococcaceae (P<0.05), Peptidomicae (P<0.05), Staphylococcus (P<0.05), and Mammalcoccus (P<0.05) were significantly higher in CH, and the content of Helicobacter pylori_D was significantly higher in MH ( Fig.14 b). ANOVA showed that compared with the CH group, the abundance of Proteobacteria H increased significantly (P<0.05), and that of Desulfobacteria in MH increased significantly (P<0.05); the abundance of Parthenococcus in the CH group was significantly higher than that in the MH group, and the abundance of Gemmatimonadetes (P<0.01) and Firmicutes B (P<0.05) in the MH and H groups was significantly lower than that in the CH group ( Fig.15 a). Compared with CH, Escherichia (P<0.05) and Pseudomonas e (P<0.05) in H were significantly higher than those in H, Mehrabella (P<0.05) in MH was significantly higher than that in H, Parabacterium (P<0.05), NM07-P-09 (P<0.05), Odorobacterium (P<0.05), Nanosphaerocephala (P<0.05), SFMI01 (P<0.05), Clostridium Q (P<0.05), UBA6985 (P<0.01) and Mehrabella (P<0.05) in H were significantly higher than those in H, while Bryophysalis (P<0.05) in MH was significantly reduced, while UBA946 (P<0.05) and Clostridium laxative_B (P<0.001) in H were significantly reduced. Evtepia (P<0.05), CAG-269 (P<0.05), Limivicinia (P<0.05), Formimonas (P<0.05), Dehalobacterium (P<0.05), Dwaynesavagella (P<0.05) and UBA6985 (P<0.05) in the CH group were significantly higher than those in the MH group ( Fig.15 b).

[0096] KEGG analysis revealed that the degradation rates of styrene in CH and H were significantly lower than those in MH (P<0.05) and (P<0.05), respectively. The degradation of chloroalkanes and chloroolefins by H was lower than that by MH (P<0.05). The pathways of bacterial invasion of epithelial cells (P<0.0001), mRNA monitoring pathway (P<0.0001), and caprolactam degradation pathway (P<0.0001) were higher in CH, while those of toluene degradation (P<0.0001), meiotic yeast (P<0.0001), hypertrophic cardiomyopathy (P<0.0001), fluorobenzoate degradation (P<0.0001), ansamycin biosynthesis (P<0.001), phosphonate and phosphonate metabolism (P<0.001), histidine metabolism (P<0.01), valine, leucine, and isoleucine biosynthesis (P<0.001) were higher in CH. 0.01), signaling in epithelial cells infected with Helicobacter pylori (P<0.01), phenylalanine, tyrosine and tryptophan biosynthesis (P<0.01), nod-like receptor signaling pathway (P<0.05), inositol phosphate metabolism in H (P<0.05), arginine and proline metabolism (P<0.05), valine, leucine and isoleucine degradation (P<0.05), basal transcription factors (P<0.05), alanine, aspartate and glutamate metabolism (P<0.05), carbon fixation in photosynthetic organisms (P<0.05) and glycine, serine and threonine metabolism (P<0.05) ( Fig.16 ).

[0097] MetaCyc pathway analysis showed that PWY-6629 (P<0.0001), Argdeg-PWY (P<0.0001), Ornargdeg-PWY (P<0.0001), Methglyyt-PWY (P<0.0001), Kdo-N aglipasyn-PWY(P<0.0001), ecasynn-pwy(P<0.0001), Entbacsyn-PWY(P<0.0001), Hcamhpdeg-PWY(P<0.001), PWY-6690(P<0.0 01), PWY-7446(P<0.001), 3-hydroxyphenylacetate-degradation-pwy(P<0.01), PWY0-1277(P<0.01), PWY0-1338(P<0.05), PWY-5088(P<0.05), Ast-PWY(P<0.05), ketoglucont-pwy(P<0.05) and Orndeg-PWY(P<0.05) were significantly higher than those in CHMH. P381-PWY in MH was significantly higher than that in H(P<0.01), and P381-PWY in MH was significantly higher than that in H(P<0.01). There were 186 significantly different pathways between the CH group and the H group. The Methglyut-PWY (P<0.0001), Hcamhpdeg-PWY (P<0.0001), PWY-6690 (P<0.0001) and other pathways were significantly higher in the CH group, while the PWY-7007 (P<0.0001), PWY490-3 (P<0.0001), PWY-6478 (P<0.0001) and other pathways were significantly higher in the H group ( Fig.17 ).

[0098] Example 5: In vitro antioxidant effect of Polygonatum cyrtonema polysaccharide

[0099] The T-AOC method for determining antioxidant activity includes the following steps:

[0100] (1) Polygonatum sibiricum polysaccharide was prepared into test solutions with mass concentrations of 1, 2, 4, 6, 8, 10, and 12 mg / mL with deionized water.

[0101] (2) Drawing of standard curve:

[0102] Establishment of calibration curve: Dilute 40 μmol / mL FeSO4 standard solution with distilled water to the following concentrations: 0.2, 0.1, 0.05, 0.025, 0.0125, 0.00625 μmol / mL.

[0103] Prepare standard solution: Take 100 μL of each concentration of Fe 2+Standard solution, add 100 μL FRAP working solution. Mix well at room temperature, react for 10 minutes, and measure the absorbance at 593 nm. Record A standard and A1 blank, and calculate ΔA standard = A standard - A1 blank.

[0104] Drawing a standard curve: Fe 2+ The final concentration (0.1, 0.05, 0.025, 0.0125, 0.00625, 0.003125 μmol / mL) was used as the abscissa (x), and the ΔA standard was used as the ordinate (y). A standard curve was drawn to obtain the linear regression equation y=12.356x-0.0218.

[0105] (3) Sample measurement: Add reagents: Add 180 μL of FRAP working solution to each well of a 96-well plate. Add 10 μL of the sample to be measured to the measurement group, and add 10 μL of distilled water to the blank group 2 (only 1-2 measurements are required). Reaction: Mix well at room temperature and react for 10 minutes.

[0106] (4) Absorbance measurement: Measure the absorbance at 593 nm, record A measurement and A2 blank, and calculate ΔA = A measurement - A2 blank.

[0107] (5) Sample concentration calculation: Substitute the ΔA measured value into the standard curve equation y = 12.356x-0.0218 to calculate the sample Fe 2+ Concentration x (μmol / mL).

[0108] (6) Calculation of total antioxidant capacity:

[0109] Total antioxidant capacity (μmol / ml) = x*V 反 / V 样

[0110] like Fig.18 The results shown in A indicate that the processed polygonatum polysaccharide provided in Example 1 has good total antioxidant capacity, and its total antioxidant capacity increases with increasing concentration. Specifically, when the concentration of processed polygonatum polysaccharide is 12 mg / mL, its total antioxidant capacity can reach 1.092 μmol / ml.

[0111] The ABTS method was used to determine the antioxidant activity, which included the following steps:

[0112] (1) Prepared Polygonatum sibiricum polysaccharide with deionized water to prepare test solutions with mass concentrations of 10, 20, 30, 40, 50, 60, and 70 mg / mL.

[0113] (2) Instrument preheating: Preheat the spectrophotometer or microplate reader for at least 30 minutes, adjust the wavelength to 405 nm, and zero it with distilled water.

[0114] (3) Trolox positive control (TR) preparation: If a Trolox positive control curve is required, dilute the 20 mmol / L Trolox solution to 1.0, 0.8, 0.6, 0.4, 0.2, and 0.1 mmol / L, respectively, to obtain Trolox dilutions of different concentrations.

[0115] (4) Sample and reagent preparation: Add the sample to be tested, Trolox positive control and ABTS working solution to the ELISA tubes in a 96-well plate in sequence, and follow the instructions of the BoxbioABTS Free Radical Scavenging Ability Test Kit. After the sample and ABTS working solution are fully mixed, react at room temperature in the dark for 6 minutes.

[0116] (5) Absorbance measurement: Measure the absorbance at a wavelength of 405 nm and record it as A measurement, A control, ATR, and A blank. Note: Each concentration of the TR group and blank group only needs to be measured 1-2 times, and a control tube must be set up for each sample.

[0117] (6) Calculation of ABTS free radical scavenging ability:

[0118] The ABTS free radical scavenging rate of the sample (D S %)=(A 空白 -A 测定 +A 对照 ) / A 空白 ×100%;

[0119] The free radical scavenging rate of Trolox positive control group (D TR %)=(A 空白 -ATR) / A 空白 ×100%.

[0120] like Fig.18 As shown in B and D, the results show that the prepared polygonatum polysaccharide provided in Example 1 has a good ABTS free radical scavenging effect. When the concentration of prepared polygonatum polysaccharide is 70 mg / mL, its ABTS free radical scavenging efficiency can reach 81%. The Trolox positive control group also showed a good ABTS free radical scavenging rate, wherein when the concentration of Trolox diluent is 1 mmol / ml, its ABTS free radical scavenging rate can reach 90%.

[0121] The DPPH method was used to determine the antioxidant activity, which included the following steps:

[0122] (1) Prepared Polygonatum sibiricum polysaccharide with deionized water to prepare test solutions with mass concentrations of 1, 2, 4, 6, 8, 10, and 12 mg / mL.

[0123] (2) Set up the photometer: Preheat the spectrophotometer or the microplate reader for more than 30 minutes, adjust the wavelength to 515 nm, and zero the reagent.

[0124] (3) Preparation of Trolox positive control (TR):

[0125] A 20 mmol / L Trolox solution was used as a positive control. Different concentrations of Trolox dilutions (1.6, 1.2, 0.8, 0.4, 0.2, 0.1 mmol / L) were prepared according to the required final concentration.

[0126] (4) Preparation of reaction system:

[0127] Add the following reagents to a 96-well plate or centrifuge tube: Assay group: 10 μL sample + 190 μL DPPH working solution. Control group: 10 μL sample + 190 μL DPPH working solution. TR group: 10 μL Trolox diluent + 190 μL DPPH working solution. Blank group: 200 μL DPPH working solution.

[0128] (5) Mixing reaction: After thorough mixing, react at room temperature in the dark for 30 minutes.

[0129] (6) Absorbance determination: Measure the absorbance of each group at 515 nm and record them as A determination, A control, ATR and A blank. Each dilution TR group and blank group only need to be measured 1-2 times. A control group should be set up for each sample.

[0130] The calculation formula of the antioxidant activity determination method (DPPH method) is:

[0131] The scavenging rate of DPPH free radicals by the sample (%) = (A 空白 -A 测定 +A 对照 ) / A 空白 ×100%;

[0132] Trolox positive control group DPPH free radical scavenging rate (%) = (A 空白 -ATR) / A 空白 ×100%.

[0133] like Fig.18 As shown in C and E, the results show that the prepared polygonatum polysaccharide provided in Example 1 has a good DPPH free radical scavenging effect, and its DPPH free radical scavenging rate increases with the increase of concentration. When the concentration of prepared polygonatum polysaccharide is 12 mg / mL, its DPPH free radical scavenging rate can reach 91%. The Trolox positive control group also showed a good DPPH free radical scavenging rate, and when the concentration of Trolox diluent was 2 mmol / ml, its DPPH free radical scavenging rate could reach 88%.

[0134] In general, the prepared polygonatum sibiricum polysaccharide provided by the present invention has in vitro antioxidant activity, can relieve ulcerative colitis, protect the intestinal mucosal barrier, and regulate intestinal bacteria.

[0135] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A processed polygonatum polysaccharide, characterized in that: It is mainly composed of galactose, with a main chain of →4)-β-D-Galp-[(1→4) / β-D-Garp]7-(1→, the 6-position of the middle galactose molecule is connected to a β-D-Galp-group, the prepared polygonatum sibiricum polysaccharide is also connected to →4)-β-D-Manp-(1→4)-β-D-Manp-(1→ residue, and mannose is substituted with an acetyl group.

2. A method for preparing the prepared polygonatum sibiricum polysaccharide according to claim 1, characterized in that: The steps include: The crude polysaccharide of polygonatum was obtained by treating it with water extraction and alcohol precipitation. removing the protein component from the processed Polygonatum sibiricum crude polysaccharide; The crude polysaccharide of Polygonatum sibiricum was eluted with a DEAE agarose gel FF column, and PSSP-E was obtained. PSSP-E was fractionated using a Chromdex 75PG column.

3. The preparation method according to claim 2, characterized in that: In the water extraction and alcohol precipitation process, water at 75 to 85°C is used for extraction; Alternatively, in the water extraction and alcohol precipitation method, ethanol is used for precipitation at 3 to 5°C.

4. The preparation method according to claim 2, characterized in that: Sevage reagent was used to remove the protein components in crude polysaccharide of Polygonatum sibiricum.

5. The preparation method according to claim 2, characterized in that: Elution was performed using distilled water, 0.15-0.25 M NaCl solution, 0.45-0.55 M NaCl solution and 0.95-1.05 M NaCl solution in sequence.

6. The preparation method according to claim 2, characterized in that: PSSP-E was fractionated using distilled water.

7. The preparation method according to claim 2, characterized in that: The sugar content was determined by the anthrone-sulfuric acid method during elution; Alternatively, the anthrone-sulfuric acid method is used to determine the sugar content during classification.

8. An application of the processed polygonatum polysaccharide according to claim 1 in preparing products, characterized in that: The product has one or more of the following effects: a) prevent and / or treat intestinal damage; b) prevention and / or treatment of intestinal flora disorders; c) Prevent and / or treat damage caused by reactive oxygen species and / or free radicals.

9. The use according to claim 8, characterized in that: The product is a medicine, an additive or a food.

10. The use according to claim 8, characterized in that: The product is a composition, and the composition includes auxiliary materials.

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