Morchella esculenta polysaccharide and application of degraded Morchella esculenta polysaccharide in prevention and treatment of ulcerative colitis

Abstract

The invention discloses morchella polysaccharide and application of degraded morchella polysaccharide in prevention and treatment of ulcerative colitis. The degraded morchella esculenta polysaccharide is obtained by degrading the morchella esculenta polysaccharide by adopting an ultrasonic combined H2O2-VC method. The morchella esculenta polysaccharide or degraded morchella esculenta polysaccharide slows down weight loss and colon length shortening of a patient, improves colon pathological conditions, reduces the oxidative stress level of the patient, reduces the MDA content in colon tissue of the patient, enhances the vitality of CAT, SOD and GSH, and can be used for preventing and treating the disease of the patient. According to the present invention, with the application of the traditional Chinese medicine composition, the secretion level and the gene expression level of IL-1beta, IL-6 and TNF-alpha can be reduced, the secretion level and the gene expression level of IL-10 can be improved, the damage of intestinal barrier cells caused by DSS can be repaired, the number of goblet cells can be increased, the infiltration of inflammatory cells can be reduced, the richness of intestinal flora can be improved, the dominant bacteria can be increased, the harmful bacteria can be reduced, and the recovery of intestinal flora can be promoted; the invention provides a novel medicine for preventing and treating ulcerative colitis.

Description

Technical Field

[0001] The invention relates to Morchella polysaccharide and application of degraded Morchella polysaccharide in preventing and treating ulcerative colitis, belonging to the field of biomedicine. Background Art

[0002] Ulcerative colitis (UC) is a worldwide disease with unknown etiology. It has a high prevalence in young and middle-aged people, is prone to cancer, is difficult to cure, and has the characteristics of recurrent attacks, posing a major threat to the economic stability of society and the health of the population. According to statistics, 50% of patients with UC still have no improvement in clinical symptoms after systematic treatment. The reason why modern medicine is not effective in treating UC may be that the target is single, while traditional Chinese medicine is a multi-target treatment in the treatment of UC, which has certain advantages (improving the healing rate of intestinal mucosa, reducing recurrence, and reducing hormone dependence, etc.). The etiology and pathogenesis of UC are mainly caused by internal spleen deficiency, external factors such as the six evils, emotions, and dietary environment. The disease is mainly located in the large intestine and is also related to the spleen, kidney, and liver. The pathological nature is the deficiency of the root and the excess of the symptoms. The main pathological factors are: ① dampness (heat); ② stasis of heat; ③ heat toxicity; ④ phlegm turbidity; ⑤ qi stagnation; ⑥ blood stasis, etc. Pathological characteristics: The active stage is mostly positive, and the main pathogenesis is damp-heat accumulation in the intestines, and qi and blood are not in balance. In severe cases, heat toxicity and blood stasis are the main factors. For patients who are difficult to cure repeatedly, phlegm and blood stasis should be considered. In the remission period, it is mostly a mixture of deficiency and excess, and the main pathogenesis is spleen deficiency and dampness retention, and poor transportation and transformation. Strengthening the spleen and removing dampness is the basic treatment method of traditional Chinese medicine for UC. Morchella polysaccharides and their degraded polysaccharides have in vitro antioxidant capacity and hypoglycemic activity, but whether they can play a therapeutic role in UC has not been reported in the literature, and this problem needs to be solved urgently. Summary of the invention

[0003] The purpose of the present invention is to provide the use of Morchella polysaccharide or degraded Morchella polysaccharide in the preparation of a drug for preventing and / or treating ulcerative colitis, thereby providing a new drug for preventing and treating ulcerative colitis.

[0004] The Morchella polysaccharide involved in the present invention can be prepared according to the following method: grinding and sieving Morchella fruiting bodies to obtain fine powder, then repeatedly leaching in distilled water for multiple times, filtering and collecting supernatant, concentrating using a rotary evaporator, deproteinizing using a potassium ferrocyanide-zinc acetate method, dialyzing using a dialysis membrane with a molecular weight cutoff of 3500 Da, then adding anhydrous ethanol to make the ethanol concentration reach 80%, keeping at a low temperature overnight, centrifuging and collecting precipitates, and freeze-drying to obtain the polysaccharide.

[0005] The degraded Morchella polysaccharide involved in the present invention can be obtained by degrading the Morchella polysaccharide by using an ultrasound combined with H2O2-VC method.

[0006] The conditions of the ultrasound combined with H2O2-VC method are as follows: The power of ultrasound was 600W, and the ultrasound time was 0.5~2.5h; The concentration of H2O2 is 5~50mM, the ratio of H2O2 to VC is 5:2~5:10, the pH value is 1~12, and the temperature is 40~80℃.

[0007] Preferably, the conditions of the ultrasound combined with H2O2-VC method are as follows: The power of ultrasound was 600 W, and the ultrasound time was 2 h; The concentration of H2O2 was 20 mM, the concentration of VC was 24 mM, the pH value was 4, and the temperature was 65°C.

[0008] Preferably, the Morchella polysaccharide or the degraded Morchella polysaccharide slows down the patient's weight loss and shortening of colon length, and improves colon pathology.

[0009] Preferably, the Morchella polysaccharide or the degraded Morchella polysaccharide reduces the oxidative stress level of the patient; Preferably, the Morchella polysaccharide or the degraded Morchella polysaccharide reduces the MDA content in the colon tissue of the patient and enhances the activities of CAT, SOD and GSH.

[0010] Preferably, the Morchella polysaccharide or the degraded Morchella polysaccharide reduces the secretion level and gene expression amount of IL-1β, IL-6 and TNF-α, and increases the secretion level and gene expression amount of IL-10.

[0011] Preferably, the Morchella polysaccharide or the degraded Morchella polysaccharide repairs the damage of intestinal barrier cells caused by DSS, increases the number of goblet cells, and reduces the infiltration of inflammatory cells.

[0012] Preferably, the Morchella polysaccharide or the degraded Morchella polysaccharide improves the richness of intestinal flora, increases dominant bacterial genera and reduces harmful bacterial genera, and promotes the recovery of intestinal flora.

[0013] The present invention has the following beneficial technical effects: Morchella is a rare large fungus that can be used as both medicine and food. According to the Compendium of Materia Medica, Morchella belongs to the spleen and stomach meridians, can help digestion and soothe the stomach, prevent constipation, promote intestinal health, and enhance human immunity. Ulcerative colitis (UC) can cause complications in the digestive system and outside the intestine. The main cause is the imbalance between beneficial bacteria and harmful bacteria in the intestinal flora, which ultimately leads to impaired intestinal barrier function and inflammatory response. Currently, drugs such as 5-aminosalicylic acid, glucocorticoids and immunosuppressants are commonly used for treatment, but these drugs have a long period of use and many side effects. Therefore, it is increasingly important to seek a safe and effective active substance to intervene in the development of UC.

[0014] As the main active ingredient of Morchella, polysaccharides have antioxidant and anti-inflammatory effects. However, due to the complex structure of natural polysaccharides, the absorption and utilization of organisms is extremely limited. Therefore, the polysaccharide is degraded by ultrasound combined with H2O2-VC method, which has high degradation efficiency, is easy to operate, and is environmentally friendly, to expose more active fragments to exert biological functions. The natural polysaccharides extracted from Morchella tiliacea and the degraded polysaccharides thereof can effectively protect the body from damage by ulcerative colitis, by reducing intestinal lesions, restoring crypt structure and goblet cell number; reducing oxidative stress levels, increasing the activity of antioxidant enzymes, and reducing inflammatory factor levels; enriching beneficial strains in the intestines and reducing harmful strains, thereby showing good prevention and treatment potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 It is a flow chart of establishing the mouse UC model of the present invention.

[0016] Figure 2 Effects of Morchella polysaccharides and their degraded polysaccharides on body weight change (A), colon length (B) and DAI (C) in UC mice.

[0017] Figure 3 The effects of Morchella polysaccharides and their degraded polysaccharides on the contents of MDA (A), SOD (B), CAT (C) and GSH-Px (D) in the colon tissue of UC mice.

[0018] Figure 4 The effects of Morchella polysaccharide and its degraded polysaccharide on the contents of IL-1β (A), TNF-α (B), IL-6 (C) and IL-10 (D) in the colon tissue of UC mice.

[0019] Figure 5 The effects of Morchella polysaccharide and its degraded polysaccharide on the levels of IL-1β (A), TNF-α (B), IL-6 (C) and IL-10 (D) in the serum of UC mice.

[0020] Figure 6 The effects of Morchella polysaccharide and its degraded polysaccharide on the relative expression of IL-1β (A), TNF-α (B), IL-6 (C) and IL-10 (D) mRNA in the colon tissue of UC mice.

[0021] Figure 7 The effects of Morchella polysaccharides and their degraded polysaccharides on colon histopathology of UC mice (A: CON group, B: MOD group, C: MIP.L group, D: DMIP.L group, E: MIP.M group, F: DMIP.M group, G: MIP.H group, H: DMIP.H group).

[0022] Figure 8The effects of Morchella polysaccharides and their degraded polysaccharides on the richness and diversity of intestinal flora in UC mice (A: Venn diagram, B: Chao1 index, C: Observed otus, D: Shannon index, E: Simpson index).

[0023] Fig. 9 The effects of Morchella polysaccharides and their degraded polysaccharides on the intestinal flora of UC mice at the phylum level (A), family level (B) and genus level (C).

[0024] Fig.10 It is a bar graph of microbial LDA distribution between different groups.

[0025] Fig.11 The effects of Morchella polysaccharides and their degraded polysaccharides on the intestinal flora of UC mice (A: principal coordinate analysis, B: non-metric multidimensional scaling analysis, C: phylogenetic tree). DETAILED DESCRIPTION

[0026] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0027] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0028] In the following embodiments, Morchella esculenta ( Morchella importuna )The fruiting bodies were provided by the Edible Fungi Center of Shanxi Agricultural University.

[0029] Male C57BL / 6J mice aged 6 to 8 weeks were purchased from Beijing Sibeifu Biotechnology Co., Ltd. After 7 days of adaptive feeding, MIP and UH-MIP were used to establish MIP group and DMIP group for formal experiments.

[0030] BCA, SOD, CAT, MDA, GSH-Px test kits, Nanjing Jiancheng Bioengineering Institute; TNF-a, IL-1β, IL-6, IL-10 test kits, Wuhan Fine Biotechnology Co., Ltd.; Hematoxylin and eosin staining reagent (H&E), Beijing Solebold Technology Co., Ltd.; 4% paraformaldehyde solution, Beijing Lanjieke Technology Co., Ltd.; TransZol Up Plus RNA Kit, Beijing Quanshijin Biotechnology Co., Ltd.; HiScript Ⅱ1st Strand cDNA Synthesis Kit, Nanjing Novogene Biotech Co., Ltd.; ChanQ Universal SYBR qPCR Master MIX, Nanjing Novogene Biotech Co., Ltd.

[0031] Graphpad Pism9.5 was used to draw the graph, and the groups were marked based on one-way ANOVA. "*" represents the difference between the CON group and the control group. *P <0.05, **P <0.01, ***P <0.001; "#" represents compared with the MOD group, # P <0.05, ## P <0.01, ### P <0.001.

[0032] Example 1. Preparation of Morchella polysaccharide The fruiting bodies of Morchella were ground and passed through a 100-mesh sieve to obtain fine powder, and then repeatedly extracted three times in distilled water at 80°C with a solid-liquid ratio of 1:30 (g / mL). The supernatant was filtered and concentrated to one-fourth of the original volume using a rotary evaporator. After deproteinization using the potassium ferrocyanide-zinc acetate method, it was dialyzed for 48 h using a dialysis membrane with a molecular weight cutoff of 3500 Da. Subsequently, anhydrous ethanol was added to make the ethanol concentration reach 80%, and the mixture was incubated at 4°C overnight. The precipitate was collected by centrifugation at 6000 rpm for 10 min and freeze-dried to obtain the crude polysaccharide MIP.

[0033] Example 2: Preparation of Degraded Morchella Polysaccharide (DMIP) The MIP solution (0.5%, W / V) was treated with H2O2 (20 mM), VC (24 mM) and ultrasound at 600 W for 2 h. The solution pH was 4.0 and the temperature was 65 °C.

[0034] Example 3: Establishment of mouse UC model Male C57BL / 6J mice aged 6 to 8 weeks were selected and fed adaptively for one week. When their growth status was stable, the experiment was officially started. The experiment was divided into 8 groups, with 10 mice in each group, namely normal group (CON), model group (MOD), Morchella polysaccharide low-dose group (MIP.L), Morchella polysaccharide medium-dose group (MIP.M), Morchella polysaccharide high-dose group (MIP.H), Morchella degradable polysaccharide low-dose group (DMIP.L), Morchella degradable polysaccharide medium-dose group (DMIP.M) and Morchella degradable polysaccharide high-dose group (DMIP.H). The low dose was 400 mg / kg, the medium dose was 600 mg / kg, and the high dose was 800 mg / kg. The experiment lasted for a total of 21 days. During the whole process, the MIP group and the DMIP group were gavaged daily according to the weight of the mice, and the CON group and the MOD group were gavaged with an equal amount of normal saline. Modeling began on the 15th day. Except for the CON group, the drinking water of all other groups was replaced with 3% dextran sodium sulfate (DSS) for 7 days. The mice were fasted for 12 h on the evening of the 21st day. Blood was collected from the eye sockets of the mice on the 22nd day, and the mice were killed by cervical dislocation. The blood was preheated in a 40°C metal bath, and then centrifuged at 3000 rpm for 10 min at 4°C. The supernatant was collected and frozen at -80°C. The mice were dissected, and the required colon tissue was taken out for photography to calculate the colon length. Part of the colon was fixed with 4% paraformaldehyde solution, and the other part was frozen in a -80°C refrigerator; feces in the colon were collected and frozen in a -80°C refrigerator. All animal experimental operations and processing procedures were in accordance with the requirements of the Experimental Animal Ethics Committee of Shanxi Agricultural University (Taigu). The mouse experiment process is as follows. Figure 1 .

[0035] Example 4: Protective effects of Morchella polysaccharides and degraded polysaccharides on UC mice 1. Body weight changes, colon length and DAI of mice 1. Determination method The severity of ulcerative colitis in mice was assessed based on the disease assessment index (DAI), which was determined by the weight loss, stool consistency, and blood in the stool of the mice. The scores of weight loss (0: none, 1: 1–5%, 2: 5–10%, 3: 10–15%, 4: >15%), stool consistency (0: normal, 2: loose stools, 4: diarrhea), and stool bleeding (0: no blood, 2: fecal occult blood, 4: visible blood in the stool) were used to calculate the scores. During the experiment, the weight of the mice was recorded at a fixed time every day, and the anal bloody stool of the mice was photographed at the end of the modeling.

[0036] 2. Effects of Morchella polysaccharides and degraded polysaccharides on body weight change, colon length and DAI in UC mice Figure 2Figure A in the middle is a graph of the weight change rate of mice. Compared with the MOD group, the MIP group, and the DMIP group, the weight of mice in the CON group has been on an upward trend until the rate of increase began to slow down after the 15th day. From the 1st day to the 14th day, the weight of mice in each group increased steadily, and the growth rate of the MIP group and the DMIP group was lower than that of the CON group and the MOD group. From the 15th day of modeling to the end of the experiment, the weight of each group showed a clear downward trend, among which the weight loss in the MOD group was the most significant. On the 18th day, compared with the CON group, the weight loss rate of the MIP.H group and the DMIP.L group was extremely significant ( P <0.001). On the 21st day, the weight loss rates of the MIP.L and DMIP.H groups were significantly lower than those of the MOD group ( P <0.001), indicating that it can alleviate the effect of DSS on the weight loss of mice.

[0037] Ulcerative colitis can cause the colon of mice to become congested, edematous, brittle, and significantly shortened. Figure 2 Figure B is a bar graph of mouse colon length, except for the DMIP.H group ( P <0.01), the other groups were significantly shorter than the CON group ( P <0.001), the MOD group was significantly shorter than the CON group, MIP group and DMIP group ( P <0.001).

[0038] like Figure 2 As shown in Figure C, from day 1 to day 15, the DAI of each group was 0, because the weight of mice was increasing without weight loss. After drinking water was replaced by 3% DSS solution, the DAI increased to varying degrees from day 15 to day 21, except for the CON group. On day 18, the DAI of the MOD, MIP.H, DMIP.L and DMIP.M groups was significantly higher than that of the CON group ( P <0.001), MIP.M group was significantly higher than CON group ( P <0.01); except for the MIP.H group and the DMIP.L group, the MOD group was significantly higher than the other groups ( P <0.001), until the 21st day, the MIP group and DMIP group were significantly higher than the CON group, and significantly lower than the MOD group. This shows that Morchella polysaccharides and degraded polysaccharides can improve the body damage of UC mice.

[0039] 2. Changes in Physiological Indicators Related to Oxidative Stress 1. Determination of physiological indicators related to oxidative stress SOD, CAT, MDA, and GSH-Px in colon tissue were determined strictly according to the kit instructions.

[0040] 2. Effects of Morchella polysaccharides and degraded polysaccharides on physiological indicators related to oxidative stress Oxidative stress is a state in which the body's oxidation and antioxidant systems are unbalanced, which can lead to a series of problems such as DNA damage, accelerated apoptosis, and the occurrence of lipid peroxidation. In the present invention, the degree of oxidative stress is reflected by detecting the content of MDA, SOD, GSH-Px and CAT in the colon of UC mice. The level of MDA content can reflect the level of lipid peroxidation in the body, thereby reflecting the degree of damage to the body. SOD, CAT and GSH-Px have good antioxidant effects, can remove free radicals in the body, and play an important role in maintaining the balance of oxidative stress in the human body.

[0041] like Figure 3 As shown in Figure A, the MDA content in the MOD group, MIP.H group, and DMIP.L group was significantly higher than that in the CON group ( P <0.01), compared with the MOD group, the MIP.L group ( P <0.01) and DMIP.H group ( P <0.001) was significantly reduced. Figure 3 Figure B shows the results of SOD content. Compared with the CON group, the SOD content of the MOD group, MIP.H group and DMIP.L group was significantly reduced. Compared with the MOD group, the SOD content of the MIP group and DMIP group was significantly increased, especially the DMIP.H group ( P <0.001). Figure 3 Figure C in the middle shows the CAT content. Except for the MIP.L and DMIP.H groups, the CAT content of CON was significantly higher than that of the other groups ( P <0.01); compared with the MOD group, only the DMIP.H group showed significant improvement ( P <0.05). Figure 3 Figure D shows the GSH-Px content. The GSH-Px content in the MOD group and the MIP.H group was significantly reduced compared with the CON group ( P <0.01), while only DMIP.H significantly increased the GSH-Px content compared with the MOD group ( P <0.05).

[0042] In summary, both the MIP group and the DMIP group were able to reduce the level of oxidative stress, indicating that Morchella polysaccharides and degraded polysaccharides could enhance the body's ability to resist oxidative damage, and the two most effective groups were the MIP.L group and the DMIP.H group.

[0043] 3. Influence of the secretion level of inflammatory factors 1. Determination of physiological indicators related to inflammatory factors TNF-α, IL-1β, IL-6, and IL-10 in colon tissue and serum were determined strictly according to the kit instructions.

[0044] 2. Effects of Morchella polysaccharides and degraded polysaccharides on the secretion level of inflammatory factors The present invention measured the contents of IL-1β, TNF-α, IL-6 and IL-10 in mouse colon tissue and serum. Figure 4 In the colon tissue, the contents of IL-1β, TNF-α, and IL-6 in the MOD group were significantly higher than those in the CON group, and the content of IL-10 was significantly lower than that in the CON group, indicating that the mouse acute ulcerative colitis model was successfully established. The ranking of low, medium, and high doses of the MIP group and the DMIP group were MIP.L>MIP.M>MIP.H; DMIP.H>DMIP.M>DMIP.L. The two groups with the best effects were the MIP.L group and the DMIP.H group. The level of inflammatory indicators in the DMIP.H group was lower than that in the MIP.L group, but there was no significant difference between the two groups.

[0045] like Figure 5 In the mouse serum, the levels of proinflammatory factors IL-1β, TNF-α, and IL-6 in the MIP.H and DMIP.L groups were lower than those in the MOD group, but there was no significant difference between the groups, and they were significantly higher than those in the non-pathogenic CON group ( P <0.001), indicating that the anti-inflammatory effect was not obvious. The medium-dose group of the two polysaccharides significantly increased the content of the anti-inflammatory factor IL-10 ( P <0.01), reduced the levels of IL-1β, TNF-α and IL-6 ( P <0.01), and the content of anti-inflammatory factors in the other MIP.L group and DMIP.H group was not significantly different from that in the CON group. In general, the levels of inflammatory factors in the serum of the MIP and DMIP groups were consistent with the trends in colon tissue. The results showed that Morchella polysaccharides and their degraded polysaccharides can protect the body from damage caused by acute ulcerative colitis, especially the low-dose MIP group and the high-dose DMIP group.

[0046] 4. Influence of inflammatory factor gene expression 1. Method for determining relative gene expression (1) RNA extraction according to TransZol Total RNA was extracted according to the instructions of Up Plus RNA Kit.

[0047] (2) Reverse transcription HiScript Ⅱ 1st Strand cDNA Synthesis Kit was used for reverse transcription, and the operation was strictly in accordance with the instructions of the kit to synthesize cDNA. The reaction system is shown in Table 1: Table 1 Reverse transcription system

[0048] (3) Primer design The primer gene sequences of colon tissue used in the present invention were synthesized by Beijing Qingke Biotechnology Co., Ltd. The details are shown in Table 2 below.

[0049] Table 2 Primer sequences

[0050] (4) qRT-PCR steps The ChanQ Universal SYBR qPCR Master MIX Kit was used for the experiment, and the steps in the instruction manual were strictly followed. The reaction system is shown in Table 3.

[0051] Table 3 qRT-PCR reaction system

[0052] 2. Effects of Morchella polysaccharides and degraded polysaccharides on the gene expression of inflammatory factors The relative expression of IL-1β, TNF-α, IL-6, and IL-10 mRNA in mouse colon tissue was determined. Figure 6 Both the MIP group and the DMIP group could significantly inhibit the mRNA expression of IL-1β, TNF-α, and IL-6, and increase the mRNA expression of the anti-inflammatory factor IL-10 ( P <0.01), and the secretion trend of the above inflammatory factors in the MIP and DMIP groups at different doses was consistent. This indicates that MIP and DMIP can enhance intestinal immune function by upregulating the gene expression of the anti-inflammatory factor IL-10 and downregulating the gene expression of the pro-inflammatory factors IL-1β, TNF-α, and IL-6.

[0053] 5. Impact of Colonic Histopathology 1. H&E staining of intestinal tissue The colon tissue fixed with 4% paraformaldehyde solution was taken out and placed in an embedding box for 24 h of water dialysis to wash away the excess paraformaldehyde; the dehydration step was carried out, and 75% anhydrous ethanol, 85% anhydrous ethanol, 95% anhydrous ethanol, 100% anhydrous ethanol I, 100% anhydrous ethanol II, alcohol benzene, and xylene were used for 20 min. Then the wax was immersed at 58°C for 90 min and repeated twice; the tissue was embedded at 60°C. The slices with a thickness of 5 μm, without pores and wrinkles, were cut with a paraffin slicer, and the slices were spread at 37°C and dried at 50°C. Then the staining steps of the H&E staining reagent instructions were followed, and finally the slices were sealed with neutral gum and observed under a fluorescence microscope.

[0054] 2. Effects of Morchella polysaccharides and degraded polysaccharides on colon pathological histology Colon histopathological data Figure 7 As shown in the figure. The colon tissue morphology of mice in the CON group showed a complete epithelial cell structure, which was neatly and tightly arranged, with a large number of goblet cells and clearly structured crypts, without inflammatory cell infiltration or cell necrosis, while the colon mucosa of the MOD group showed obvious inflammatory cell infiltration, goblet cell loss, and crypt structure disappearance. After MIP and DMIP intervention, it was found that the degree of inflammatory cell infiltration was reduced, and the number of goblet cells and crypt structure recovered to a certain extent. In particular, the degree of inflammatory cell infiltration in the MIP.L group and the DMIP.H group was greatly restored, the cells were tightly and neatly arranged, and the colon structure was clearly visible.

[0055] 6. Effects of Mouse Intestinal Microbiota 1. Intestinal microbiota sequencing based on 16SrRNA gene DNA was extracted from the feces of each group. After the samples passed the test, the PCR products were obtained in the 16SV3-V4 region using the Xing method, mixed and purified, and then the library was constructed. The constructed library was quantified by Qubit and Q-PCR. After the library passed the test, PE250 sequencing was performed using NovaSeq6000.

[0056] 2. Analysis of the effects of Morchella polysaccharides and degraded polysaccharides on intestinal flora of UC mice (1) Effects of Morchella polysaccharides and degraded polysaccharides on the diversity and richness of intestinal flora in UC mice If the similarity between sequences is higher than 97% (species level), it can be defined as an OTU, and each OTU represents a species, such as Figure 8In the Venn diagram of Figure A, there are 145 overlapping OTUs between the groups. The CON, MOD, MIP.L, MIP.M, MIP.H, DMIP.L, DMIP.M and DMIP.H groups have 278, 106, 226, 185, 117, 99, 139 and 202 unique OTUs, respectively. The CON group has the most, while the MOD, MIP.H and DMIP.L groups have fewer unique OTUs. In addition, all groups significantly increased the number of OTUs in the mouse intestinal flora. Chao1 and Observed otus can both estimate the total number of species between groups. From Figure 8 It can be concluded from Figures B and C that compared with the CON group, the MOD group significantly reduced the number of species in the intestinal flora of mice, while the low-medium dose group of the MIP group and the low-medium-high dose group of DMIP increased the number of species in the flora to a certain extent compared with the MOD group. The Shannon index and Simpson index can reflect the diversity of the flora. The larger the value, the higher the degree of species diversity. Figure 8 In (D and E), the species diversity of the MOD group did not decrease compared with the CON group, while that of the MIP.H and DMIP.M groups decreased significantly compared with the other polysaccharide groups.

[0057] (2) Effects of Morchella polysaccharides and degraded polysaccharides on the composition of intestinal flora in UC mice like Fig. 9 Figures A, B, and C are bar graphs of the composition of the mouse intestinal flora at the phylum level, family level, and genus level. At the phylum level, compared with the CON group, the F / M ratio of the MOD group and the polysaccharide group increased, and MIP.L and DMIP.H were closest to the CON group. Proteobacteria (Proteobacteria), Actinobacteriota (Actinobacteria) and Verrucomicrobiota (Verrucomicrobia) increased in abundance, Proteobacteria Can produce endotoxins and is considered to be the main pathogen, MIP.H group and DMIP.M group Verrucomicrobiota At the family level, the abundance of the CON group Muribaculaceae The abundance of MIP.H group is very high, while that of other groups is reduced. Akkermansiaceae (Akermansiaceae) were very abundant, and the DMIP group Erysipelotrichaceae (Erysipelothrixaceae) was less abundant than the MIP group. At the genus level, some studies have found that Akkermansia The abnormal increase of Akkermansia species will lead to excessive consumption of colonic mucus proteins. In this case, the number of non-mucus-consuming species is significantly reduced, resulting in a decrease in species diversity. Akkermansia Abnormal proliferation may lead to intestinal barrier damage and induce intestinal inflammation. AkkermansiaThis will promote the integrity of the intestinal barrier, which is the main reason why the diversity of the microbiota in the MIP.H and DMIP.M groups was severely reduced. Faecalibaculum (Fasciobacterium prausnitzii) had increased abundance in all groups except the MOD and CON groups and was named as a potential probiotic.

[0058] Combination Fig.10 and Fig.11 Figure C shows that in the MOD group Peptostreptococcaceae (Peptostreptococcus family) levels increased, and studies have shown that the presence of pathogenic bacteria may lead to inflammation; in the MIP.L group Erysipelotrichaceae (Erysipelothrixaceae), Parasutterella (Pasteurella spp.), Burkholderiales (Burkholderia) and Bifidobacterium (Bifidobacterium) levels increased, among which Bifidobacterium For beneficial bacteria, DMIP.H group Rikenellaceae (RIKEN Bacteria Department) ,Prevotellaceae_UCG_001 (Prevotella spp.) and Rikenellaceae_ RC9_gut_group Compared with the MIP group, although the DMIP group required a higher dose to regulate intestinal flora disorders, the dominant flora of the DMIP.H group was closest to that of the CON group. Fig.11 As can be seen in (Figure A, Figure B), the relative distance between the CON group and the DMIP.H group is closer than that between the MIP.L group, indicating that the bacterial flora between the two groups is highly similar, and the DMIP.H group is more able to restore the state before the disease. In summary, both MIP and DMIP can effectively improve the intestinal flora environment and increase Faecalibaculum The abundance of Bifidobacterium and Prevotellaceae_UCG_001 The abundance of Akkermansia Protecting the intestinal barrier, both groups reduced the damage of potential harmful bacteria to the intestine and reversed the intestinal flora disorder caused by DSS in mice. Erysipelotrichaceae The bacterial species in the CON group decreased and were closer to those in the CON group.

[0059] The present invention established a UC mouse model and explored the anti-inflammatory effects of MIP and UH-MIP in vivo. The results showed that Morchella polysaccharides and degraded polysaccharides can effectively reduce the DAI of mice; reduce the degree of inflammatory cell infiltration, restore the crypt structure and the number of goblet cells; reduce the level of oxidative stress (MDA), increase the content of antioxidant enzymes (SOD, CAT and GSH-Px), reduce the secretion level and gene expression of IL-1β, IL-6 and TNF-α, and increase the secretion level and gene expression of IL-10.

[0060] The results of this study showed that compared with CON, MOD reduced the richness of intestinal flora in mice, while diversity was not changed. MIP and DMIP improved the richness of intestinal flora in UC mice, but MIP.H and DMIP.M were different. Akkermansia The excessive number of intestinal flora decreased significantly. The MIP.L group and DMIP.H group with good anti-inflammatory effect reduced the abundance of potentially harmful bacteria and appropriately increased Akkermansia and Faecalibaculum The abundance of potential beneficial bacteria reversed the intestinal flora disorder caused by DSS. In addition, the MIP.L group increased the number of beneficial bacteria. Bifidobacterium The abundance of beneficial bacteria was increased in the DMIP.H group. Prevotellaceae_UCG_001 Compared with the two groups, the harmful bacteria in the DMIP group Erysipelotrichaceae decreased, and became closer to the intestinal flora of the non-pathogenic CON group.

Claims

1. Application of Morchella polysaccharide or degraded Morchella polysaccharide in the preparation of a drug for preventing and / or treating ulcerative colitis.

2. The use according to claim 1, characterized in that: The degraded Morchella polysaccharide is obtained by using an ultrasound combined with H2O2-VC method to degrade the Morchella polysaccharide.

3. The use according to claim 2, characterized in that: The conditions of the ultrasound combined with H2O2-VC method are as follows: The power of ultrasound was 600W, and the ultrasound time was 0.5~2.5h; The concentration of H2O2 is 5~50mM, the ratio of H2O2 to VC is 5:2~5:10, the pH value is 1~12, and the temperature is 40~80℃.

4. The use according to any one of claims 1 to 3, characterized in that: The Morchella polysaccharide or the degraded Morchella polysaccharide slows down the patient's weight loss and shortening of colon length, and improves colon pathology.

5. The use according to any one of claims 1 to 4, characterized in that: The Morchella polysaccharide or the degraded Morchella polysaccharide reduces the oxidative stress level of the patient.

6. The use according to claim 5, characterized in that: The Morchella polysaccharide or the degraded Morchella polysaccharide reduces the MDA content in the colon tissue of the patient and enhances the activities of CAT, SOD and GSH.

7. The use according to any one of claims 1 to 6, characterized in that: The Morchella polysaccharide or the degraded Morchella polysaccharide reduces the secretion level and gene expression amount of IL-1β, IL-6 and TNF-α, and increases the secretion level and gene expression amount of IL-10.

8. The use according to any one of claims 1 to 7, characterized in that: The Morchella polysaccharide or the degraded Morchella polysaccharide repairs the damage of intestinal barrier cells caused by DSS, increases the number of goblet cells, and reduces the infiltration of inflammatory cells.

9. The use according to any one of claims 1 to 8, characterized in that: The Morchella polysaccharide or the degraded Morchella polysaccharide improves the richness of intestinal flora, increases dominant bacterial genera and reduces harmful bacterial genera, and promotes the recovery of intestinal flora.

Images