阅读说明：本技术 分心木水提物在制备治疗溃疡性结肠炎药物中的应用 (Application of diaphragma juglandis aqueous extract in preparation of medicine for treating ulcerative colitis ) 是由 蒋蓓尔 何颖 徐张扬 张建 刘光盛 闵天骄 于 2021-09-16 设计创作，主要内容包括：本发明公开了一种分心木水提物在制备治疗溃疡性结肠炎药物中的应用。本发明的分心木水提物能够有效治疗和缓解体重降低、腹泻和便血等溃疡性结肠炎的症状,能够抑制包括TNF-α、IL-6或IL-1β等炎症介质的产生,能够调节肠道菌群组成和促进菌群恢复,以及能够进一步保护粘膜屏障和改善DSS诱导的结肠损伤。因此,分心木水提物可以作为溃疡性结肠炎的有效治疗药物,制备成各种剂型药物或药物组合物用于溃疡性结肠炎的治疗。(The invention discloses an application of an aqueous extract of diaphragma juglandis in preparing a medicament for treating ulcerative colitis. The diaphragma juglandis aqueous extract can effectively treat and relieve symptoms of ulcerative colitis such as weight reduction, diarrhea, hematochezia and the like, can inhibit the generation of inflammatory mediators such as TNF-alpha, IL-6 or IL-1 beta and the like, can regulate the composition of intestinal flora and promote the restoration of the flora, and can further protect mucosal barrier and improve DSS-induced colonic injury. Therefore, the water extract of the diaphragma juglandis can be used as an effective treatment medicament for the ulcerative colitis and prepared into various dosage form medicaments or pharmaceutical compositions for treating the ulcerative colitis.)

1. Application of an aqueous extract of diaphragma juglandis in preparing a medicament for treating ulcerative colitis.

2. Use according to claim 1, wherein the composition of the aqueous extract of diaphragma juglandis comprises: the content of total flavone is 30-40%, the content of total polysaccharide is 25-35%, and the content of total polyphenol is 20-30%.

3. Use according to claim 1, wherein the aqueous extract of diaphragma juglandis is prepared by the following steps:

s1: heating and refluxing the components in the diaphragma juglandis by using water, and filtering to obtain an aqueous solution;

s2: removing water by rotary evaporation and/or freeze drying to obtain the diaphragma juglandis aqueous extract.

4. The use of claim 1, wherein the treatment of ulcerative colitis comprises alleviating symptoms of ulcerative colitis including at least one of weight loss, diarrhea, and hematochezia.

5. Use according to claim 1, wherein the aqueous extract of diaphragma juglandis is capable of inhibiting inflammatory mediators.

6. The use of claim 5, wherein the inflammatory mediator comprises at least one of TNF-a, IL-6 or IL-1 β.

7. The use according to claim 1, wherein the aqueous extract of diaphragma juglandis is capable of protecting the colonic mucosal barrier and/or repairing DSS-induced colonic lesions.

8. The use according to claim 1, wherein the aqueous extract of diaphragma juglandis is capable of modulating the intestinal flora composition and/or promoting the restoration of flora.

9. The use of claim 1, wherein the medicament is in the form of capsules, tablets, pills, granules, oral liquid or injections.

10. The use of claim 1, wherein the medicament further comprises a pharmaceutically acceptable carrier.

Technical Field

The invention belongs to the field of medicines, and particularly relates to application of an aqueous extract of diaphragma juglandis in preparation of a medicine for treating ulcerative colitis.

Background

Diaphragma juglandis Fructus (Diaphragma juglandis Fructus), also called walnut shell, nutcracker or walnut membrane, is a membrane in the kernel of Juglans regia (Juglans regia L.) of Juglandaceae, i.e. a small piece of wood sandwiched between walnut kernels. The diaphragma juglandis is flaky, crisp and easy to break, generally accounts for 4-5% of the walnut, is bitter and astringent in taste and neutral in nature, enters spleen and kidney meridians, has the effects of reinforcing kidney and arresting seminal emission, and can be used for treating various diseases such as spermatorrhea, enuresis, hyperhidrosis, diarrhea, insomnia and dreaminess. The chemical components contained in the diaphragma juglandis are flavone, alkaloid, saponin, saccharide and rich phenolic acid components, and pharmacological activity research shows that the diaphragma juglandis has the functions of tonifying the kidney, inhibiting bacteria, resisting oxidation and the like. However, people often only use walnut kernels when eating or processing walnuts, and besides a large amount of generated core-separating wood is used for making tea in a small part of areas, most of the core-separating wood is treated as waste, so that the resource is greatly wasted. The further research and development of the diaphragma juglandis is of great significance for improving the added value of the walnuts and reducing the resource waste.

Ulcerative colitis is an inflammatory bowel disease mainly invading the mucosa and submucosa of the large intestine, and has the main symptoms of diarrhea, abdominal pain, fever, rectal bleeding and the like. Research has proved that under the action of various factors such as heredity, environment and microorganism, the intestinal tract immune reaction is excessively hyperfunction, which finally leads to the occurrence of ulcerative colitis. The incidence of ulcerative colitis in China gradually increases in the last two decades, and with the improvement of the sanitary and medical conditions in China and the change of life styles of people, the incidence of ulcerative colitis in China will be further increased in future. The clinical method for treating ulcerative colitis mainly comprises glucocorticoid, immunosuppressant, aminosalicylic acid and the like, can relieve the disease condition to a certain extent, but has large side effect, has certain dependence after long-term administration, seriously influences the life quality of patients, and has certain relation with the occurrence and development of colon cancer, so the method is listed as one of the modern refractory diseases by the world health organization. Therefore, there is a great need to find new drugs and methods for treating ulcerative colitis.

At present, a preparation method of a diaphragma juglandis extract, a pharmaceutical composition taking the extract as an active ingredient and a report of application of the extract in medicines for treating ulcerative colitis are not seen at home and abroad.

Disclosure of Invention

The present invention is directed to solving at least one of the problems of the prior art described above. Therefore, the invention provides an application of the diaphragma juglandis aqueous extract in preparing a medicament for treating ulcerative colitis.

According to a specific embodiment of the invention, at least the following advantages are achieved: the diaphragma juglandis aqueous extract can effectively treat and relieve the symptoms of ulcerative colitis: including weight loss, diarrhea, and hematochezia; can inhibit the production of inflammatory mediators: inflammatory molecules such as TNF-alpha, IL-6 or IL-1 beta are included to relieve inflammatory symptoms and alleviate intestinal symptoms; can regulate the composition of intestinal flora, promote flora recovery, and regulate intestinal health; and to further protect mucosal barriers and repair colon damage; therefore, the aqueous extract of the diaphragma juglandis can be used as an effective therapeutic medicament for the ulcerative colitis.

In some embodiments of the invention, the composition of the aqueous extract of diaphragma juglandis comprises: the content of total flavone is 30-40%, the content of total polysaccharide is 25-35%, and the content of total polyphenol is 20-30%.

In some preferred embodiments of the present invention, the composition of the aqueous extract of diaphragma juglandis comprises: the content of total flavone is 33.40 + -0.88%, the content of total polysaccharide is 30.80 + -2.28%, and the content of total polyphenol is 24.62 + -0.86%.

In some embodiments of the invention, the aqueous extract of diaphragma juglandis is prepared by the steps of: s1: heating and refluxing the components in the diaphragma juglandis by using water, and filtering to obtain an aqueous solution; s2: removing water by rotary evaporation and/or freeze drying to obtain the diaphragma juglandis aqueous extract.

In some preferred embodiments of the present invention, the mass ratio of the core wood and the water in step S1 is 1: (5-20). Preferably, the mass ratio of the core wood to the water is 1: (8-15); more preferably, the mass ratio of the core wood to water is about 1: 10.

in some more preferred embodiments of the invention, the steps comprise: weighing 1000g of diaphragma juglandis medicinal material, washing with distilled water, adding 10L of distilled water for soaking, soaking for 2h, and heating and refluxing at 100 ℃ for extraction for 1 h. Collecting extractive solution, filtering, concentrating to 200mL at 60 deg.C with rotary evaporator, pre-freezing at-70 deg.C for 6 hr, and vacuum freeze-drying at-50 deg.C for 48 hr to obtain dark brown diaphragma juglandis aqueous extract with extraction rate of 9.41 + -0.82%.

In some embodiments of the invention, the treatment of ulcerative colitis comprises alleviating at least one of the symptoms of ulcerative colitis including weight loss, diarrhea, and hematochezia.

In some embodiments of the invention, the aqueous extract of diaphragma juglandis is capable of inhibiting inflammatory mediators.

In some preferred embodiments of the invention, the inflammatory mediator comprises at least one of TNF- α, IL-6, or IL-1 β.

In some embodiments of the invention, the aqueous extract of diaphragma juglandis is capable of protecting the colonic mucosal barrier and/or repairing DSS-induced colonic lesions.

In some embodiments of the invention, the aqueous extract of diaphragma juglandis is capable of modulating the intestinal flora composition and/or promoting the restoration of flora.

In some embodiments of the present invention, the pharmaceutical formulation is a capsule, a tablet, a pill, a granule, an oral liquid, or an injection.

In some embodiments of the invention, the medicament further comprises a pharmaceutically acceptable carrier.

In some preferred embodiments of the present invention, the pharmaceutically acceptable carrier refers to a pharmaceutical carrier conventional in the pharmaceutical field, such as: diluents, excipients such as water, etc., fillers such as starch, sucrose, etc.; binders such as cellulose derivatives, alginates, gelatin, and polyvinylpyrrolidone; humectants such as glycerol; disintegrating agents such as agar, calcium carbonate and sodium bicarbonate; absorption enhancers such as quaternary ammonium compounds; surfactants such as cetyl alcohol; adsorption carriers such as kaolin and bentonite clay; lubricants such as talc, calcium stearate and magnesium stearate, and polyethylene glycol, and the like. Other adjuvants such as sweetener, flavoring agent, etc. can also be added into the composition.

In some embodiments of the invention, the aqueous extract of diaphragma juglandis is administered in an amount of 500mg per kilogram body weight of the individual.

By "pharmaceutically acceptable carrier" herein is meant a diluent, adjuvant, excipient, or vehicle that is administered with the active ingredient and which is, within the scope of sound medical judgment, suitable for contact with the tissues of humans and/or other animals without excessive toxicity, irritation, allergic response, complication, or other problem, commensurate with a reasonable benefit/risk ratio.

In the present invention, the term "treating" includes alleviating, inhibiting or ameliorating the symptoms or conditions of a disease; inhibiting the generation of complications: ameliorating or preventing underlying metabolic syndrome; inhibiting the development of a disease or condition, such as controlling the development of a disease or condition; alleviating the disease or symptoms; regression of the disease or symptoms; alleviating a complication caused by the disease or symptom, or preventing or treating a symptom caused by the disease or symptom. As used herein, administration can result in an improvement in a disease, symptom, or condition, particularly an improvement in severity, delay in onset, slow progression, or decrease in duration of a condition.

Drawings

The invention is further described with reference to the following figures and examples, in which:

FIG. 1 is a graph showing the effect of an aqueous extract of diaphragma juglandis on the body weight of mice induced by DSS in an example of the present invention;

FIG. 2 is a graph showing the effect of an aqueous extract of diaphragma juglandis on the stool trait score of DSS-induced mice in an example of the present invention;

FIG. 3 is a graph showing the effect of an aqueous extract of diaphragma juglandis on the mean fecal blood fraction of DSS-induced mice in an example of the invention;

FIG. 4 is a graph showing the effect of an aqueous extract of diaphragma juglandis on DAI values in DSS-induced mice in an example of the invention;

FIG. 5 is a graph showing the effect of an aqueous extract of diaphragma juglandis on the length of the colon in DSS-induced mice in an example of the present invention;

FIG. 6 is a graph of staining of mouse colon HE and AB-PAS induced by DSS by an aqueous extract of diaphragma in an example of the present invention;

FIG. 7 is a graphical representation of the effect of aqueous diaphragma juglandis extract on histopathology scores of DSS-induced mouse colon in an example of the present invention;

FIG. 8 is a graph showing the effect of an aqueous extract of diaphragma juglandis on IL-1 β, IL-6, TNF- α in DSS-induced colon tissue of mice according to an embodiment of the present invention;

FIG. 9 is a schematic diagram illustrating the effect of an aqueous extract of diaphragma juglandis on the structure of a mouse intestinal flora induced by DSS in an embodiment of the invention;

FIG. 10 is a schematic diagram of the analysis of diversity of the diaphragma juglandis aqueous extract on DSS-induced intestinal flora alpha of mice in the embodiment of the invention.

FIG. 11 is a schematic diagram of the analysis of a diaphragma aqueous extract on DSS-induced intestinal flora PCoA (2D) of mice in an embodiment of the invention;

FIG. 12 is a schematic diagram of the analysis of the species difference of the mice intestinal flora induced by DSS by the diaphragma juglandis aqueous extract in the embodiment of the invention.

Detailed Description

The concept and technical effects of the present invention will be clearly and completely described below in conjunction with the embodiments to fully understand the objects, features and effects of the present invention. It is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments, and those skilled in the art can obtain other embodiments without inventive effort based on the embodiments of the present invention, and all embodiments are within the protection scope of the present invention. The test methods used in the examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are commercially available reagents and materials unless otherwise specified.

Example 1: preparation and component analysis of aqueous extract of diaphragma juglandis

1. Preparation of aqueous extract of Cornus officinalis

Weighing 1000g of diaphragma juglandis medicinal material, washing with distilled water, adding 10L of distilled water for soaking, soaking for 2h, and heating and refluxing at 100 ℃ for extraction for 1 h. Collecting extractive solution, filtering, concentrating to 200mL at 60 deg.C with rotary evaporator, pre-freezing at-70 deg.C for 6 hr, and vacuum freeze-drying at-50 deg.C for 48 hr to obtain dark brown diaphragma juglandis aqueous extract with extraction rate of 9.41 + -0.82%.

2. Analysis of Components of aqueous extract of Centratherum

The composition of the diaphragma juglandis aqueous extract comprises: the content of total flavone is 33.40 + -0.88%, the content of total polysaccharide is 30.80 + -2.28%, and the content of total polyphenol is 24.62 + -0.86%.

Example 2: research on effect of diaphragma juglandis aqueous extract on mouse ulcerative colitis

1. Animal grouping and dosing regimens

C57BL/6 mice, male, 6 weeks old, body mass 20 + -2 g, SPF grade, purchased from the center of navy specialty medicine center. After the mice are bred adaptively for 5 days, the mice are randomly divided into 4 groups by SPSS software, and each group comprises 10 mice, namely a control group, a model group, a traditional Chinese medicine group (500 mg/kg of diaphragma juglandis aqueous extract) and a positive medicine group (600 mg/kg of mesalazine). Adopting a DSS induction method to establish a ulceration model: except for the control group, mice were free to drink 3% DSS solution for seven consecutive days. From the first day of model building, the medicine is continuously administered once a day for nine consecutive days, 500mg/kg of diaphragma aqueous extract is intragastrically administered to a diaphragma group every day, 600mg/kg of mesalazine is intragastrically administered to a positive medicine group, and equal volume of distilled water is administered to a control group and a model group by intragastrically administering.

For the construction of colitis model, Dextran Sulfate Sodium Salt (DSS) colitis (UC) model is most widely used.

The used positive drug mesalazine is one of common drugs for treating ulcerative colitis. Has obvious inhibition effect on the inflammation of the intestinal wall. Mesalazine can inhibit synthesis of prostaglandin and formation of leukotriene, which are inflammatory mediators, thereby having a remarkable inhibitory effect on inflammation of intestinal mucosa. Mesalamine can inhibit the synthesis of prostaglandin in a dose-dependent manner, and reduce the release of prostaglandin E2(PGE2) in the colon mucosa of human.

2. Experimental methods and data processing

2.1 Disease Activity Index (DAI) score

The body weight, diet and water intake, sticky feces and bloody stool of the mice were observed and recorded every day. And scoring according to DAI scoring standard, wherein the scoring standard of the fecal character score is as follows: the normal is 0 minutes, the normal and loose state is 1 minute, the loose state is 2 minutes, the loose state is 3 minutes, and the loose state is 4 minutes; the scoring criteria for the occult blood degree score were: the index of non-hematochezia is 0 point, 1 point between non-hematochezia and occult blood, 2 points for occult blood positive, 3 points between occult blood and macroscopic hematochezia, 4 points for macroscopic hematochezia, and DAI ═ 3 (body mass loss rate score + stool character score + occult blood degree score).

2.2 Colon Length

On the 9 th day of the experiment, each group of mice was sacrificed by cervical dislocation, the entire colon tissue was isolated, and the length of the colon was measured with a ruler for statistical analysis.

2.3 Colon histopathological Observation and Scoring

The lesion colon tissues of each group of mice are taken and embedded by normal paraffin, sliced and stained by HE and AB-PAS. Referring to the blind scoring of Boirivant and other standard lines, according to the shapes of epithelial cells, the infiltration condition of inflammatory cells and the like, 2 pathologists respectively read the film in a double-blind way, and the results are averaged to score.

2.4 colonic tissue inflammatory factor detection

Taking 200mg of colon tissue, adding 2mL of physiological saline for homogenization, centrifuging at 4000r/min for 10min, taking supernatant, and determining the content of IL-1 beta, IL-6 and TNF-alpha according to the steps of the kit specification.

2.5 fecal sample Collection and processing

The mice are sacrificed on the 9 th day of the experiment, the abdominal cavity is cut open, the complete colon tissue from the anus to the tail end of the cecum is taken out and placed on an ice tray, the colon tissue is longitudinally cut open along the mesentery, the feces particles (2-3 particles) in the colon are taken out and collected in a freezing tube, and the feces particles are immediately placed in liquid nitrogen for storage until the DNA of bacteria in the feces is extracted.

2.6 extraction and sequencing of bacterial DNA in feces

2.6.1 extraction and detection of DNA

Total DNA in the feces was extracted using a DNA extraction kit, DNA concentration and purity were checked using NanoDrop2000, and DNA extraction quality was checked using 1% agarose gel electrophoresis.

2.6.2 PCR amplification and product recovery

The variable region sequences of V3-V4 of the bacterial 16SrRNA gene are taken as targets, 338F-806R with a barcode sequence is taken as a primer, PCR amplification is carried out, a PCR product is obtained, the PCR product is recovered by using 2% agarose Gel, the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, USA) is used for purification, Tris-HCl elution is carried out, and 2% agarose electrophoresis is used for detection.

2.6.3 quantitation of fluorescence

Referring to the preliminary quantitative results of electrophoresis, quantitative detection was performed using QuantiFluorTM-ST (Promega, USA). And then mixing according to the corresponding proportion according to the sequencing quantity requirement of each sample.

2.6.4 sequencing analysis

Samples were sequenced on the Illumina NovaSeq platform according to the manufacturer's recommendations, supplied by LC-Bio. The paired end sequences were assigned to the samples according to their unique barcodes, and the barcode and primer sequences introduced by the pooling were removed. And using FLASH to merge the reading of the matching end. The raw read data is quality filtered under specific filtering conditions to obtain a high quality clean label according to fqtrm (v 0.94). The chimeric sequence was filtered using Vsearch software (v2.3.4). Demodulation is performed using DADA2 to obtain a feature table and a feature sequence. Diversity and diversity were calculated by normalizing to the same random sequence. The feature abundances are then normalized by the relative abundance of each sample according to the SILVA (release132) classifier. Alpha diversity is used to analyze the complexity of sample species diversity by 5 indices including Chao1, observedspeces, Goodscoverage, Shannon, Simpson (these indices are calculated by qime 2). Beta diversity was calculated by QIIME2, plotted in R-package. Sequence alignment was performed using Blast, and each representative sequence was annotated with the SILVA database for the signature sequences. Results were plotted and sequence analysis was performed by using the R package (v3.5.2).

2.7 statistical processing of data

Data analysis was performed using the SPSS22.0 statistical software package, with continuity data for normal distribution, multiple sample means comparisons and uniform variance analysis using ANOVA.

3. Results and discussion

3.1 mouse general Condition and disease Activity index changes

The body quality, food intake and water intake of each group of mice were recorded and analyzed. The results are shown in fig. 1, in which the weight change of the control group, the model group (i.e., model building group), the positive control group and the Chinese medicinal group (referring to the water extract of the diaphragma) is recorded every day, and the abscissa represents the number of days and the ordinate represents the weight change (%)). The food intake and water intake of the control group mice are normal, and the physique and the quality are in an ascending trend; after the model group mice started to model, the food intake and the water intake were both reduced, and the body weight tended to decrease from the 4 th day of model making, and was statistically significant compared with the control group. The quality of mice in the diaphragma juglandis group is also reduced, but the differences from the 6 th day to the model group are obvious, and the effects of the mice are equivalent to those of a positive medicine group.

3.2 stool consistency and hematochezia

The results of stool consistency and hematochezia are shown in fig. 2-4, wherein fig. 2 shows the stool character scores of different groups of mice, fig. 3 shows the average stool blood score (mean stool blood score) on the right of different groups of mice, and fig. 4 shows the DAI values of different groups of mice. The defecation times of the model group mice are increased after the third day, and the situations of soft stools, water-sample stools and the like occur; the occult blood and bloody stool appear after the fourth day; after the fifth day, the conditions of water sampling excrement and bloody excrement are aggravated, and the excrement discharging is difficult. The positive medicine group and the diaphragma juglandis group have dry prognosis, the activity of mice is increased, the hair condition is improved, and the stool consistency and the hematochezia condition are improved after the intervention of the aqueous extract of the diaphragma juglandis on the 8 th day compared with the model group.

3.3 general Colon and pathological observations

The entire colon tissue was isolated and the length of the colon was measured with a ruler for each group of mice and analyzed for relevant statistics. The results are shown in fig. 5, the colon length of the control group mouse is the longest, the colon length of the model group mouse is the shortest, and the difference between the model group and the control group is significant (P < 0.001); the colon length in the diaphragma group is significantly longer than in the model group.

The colon lesion tissues were HE stained and AB-PAS stained and histopathologically scored. The staining results are shown in fig. 6, the arrangement of the intestinal glands in the mucosal layer of the control mice is tight, the shape of the epithelial cells of the intestinal glands is normal, and the structure is normal without defects; the colon of the mouse in the model group can be seen to lose intestinal glands and epithelial cells, and has obvious defect, and is accompanied with a large amount of inflammatory cell infiltration, and inflammation invades submucosa and muscularis; after the intervention administration of the positive medicine group and the diaphragma juglandis group, the infiltration of inflammatory cells is reduced, and the integrity of epithelial cells of intestinal mucosa is improved. Pathological tissue score results are shown in FIG. 7, and the lesion score in the model group was significantly higher (P < 0.001) compared to the control group. Compared with the model group, the pathological score of mice in the diaphragma group is obviously reduced (P is less than 0.01). The result shows that the diaphragma juglandis aqueous extract can improve the mouse colon inflammation induced by the DSS and has the function of reducing the local lesion damage of the colon of the ulcerative colitis mouse.

3.4 colonic tissue inflammatory factor assay results

The colon tissues of each group of mice are tested for the levels of inflammatory factors IL-1 beta, IL-6 and TNF-alpha. The results are shown in FIG. 8, where the colon tissue of the control mice had the lowest IL-1 β, IL-6, TNF- α levels; the levels of IL-1 beta, IL-6 and TNF-alpha in colon tissues of the model group are the highest and are obviously increased compared with the control group (P is less than 0.001); the levels of IL-1 beta, IL-6 and TNF-alpha in colon tissues of the positive drug group and the diaphragma group are also obviously reduced compared with the model group (P is less than 0.001). The result shows that the diaphragma juglandis aqueous extract can inhibit the expression of IL-1 beta, IL-6 and TNF-alpha in local colon tissues of mice with ulcerative colitis.

3.5 colonic intestinal flora species Annotation

Based on the absolute abundance of OUT and the annotation information, the ratio of the number of sequences in each sample at the phylum level and the genus level to the total number of sequences was statistically analyzed, and the species composition differences of each group at the phylum level and the genus level were determined. FIG. 9 shows a structural diagram of the intestinal flora of different groups of mice. In the control group, the flora profile reflects the composition of normal mouse intestinal flora, with bacteroides (79.26%), Firmicutes (16.40%), Proteobacteria (1.01%), epsilon bacteraeota (0.63%) being the 4 major phyla of bacteria. Murebacteriaceae _ unclassified, Alloprovella, Mureballum, Alisiples are the constituents at the level of the main genus of the control group. In the model group, the flora profile reflects the composition of the mouse intestinal flora in case of disease, with Firmicutes (60.23%), epsilon bacteraeota (15.55%), bacteriodes (13.91%), Proteobacteria (8.44%) being the 4 major phyla of bacteria. The Firmicutes, Epsilonbacterota and Proteobacteria ratios were increased and the bacterioides ratios were decreased compared to the control group. In the rosette group, bacteroides (41.38%), Firmicutes (32.02%), Verrucomicrobia (11.67%) and Proteobacteria (6.66%) were 4 major phyla of bacteria, wherein the proportions of bacteroides and Firmicutes were similar to those of the blank control group, wherein Verrucomicrobia was first present in the major species, suggesting that this bacterium may be involved in the action of aqueous rosette extracts in alleviating ulcerative colitis.

3.6 colonic intestinal flora species diversity analysis

Species abundance and diversity of microbial communities can be reflected by diversity analysis (alpha analysis) of individual samples, we use two indexes for evaluation, Chao1 reflects species abundance in samples, regardless of the proportion (uniformity) of each species; simpson reflects the abundance and uniformity of species, and the results are shown in FIG. 10. It was found that the aqueous extract of diaphragma juglandis did not improve the species abundance and diversity of the microflora in the intestinal flora after intervention. The beta diversity is an index for measuring the similarity of the flora composition between different samples, and the species distance between the samples can be reflected by weighted PCoA analysis, and the analysis result is shown in FIG. 11. The model group is farther away than the control group, and the positive drug group and the diaphragma group are closer to the control group than the model group. Therefore, after the diaphragma juglandis aqueous extract is treated, the content of the intestinal flora is changed, and the intestinal flora is more similar to the normal intestinal flora in structure.

3.7 colonic intestinal flora species differential analysis

The difference in gut flora abundance at the class level of each species caused by 3% DSS induction treatment was further compared using LeFSe analysis. LeFSe analysis is the combination of non-parametric test and linear discriminant analysis, is suitable for the flora abundance difference test, and determines the differential microorganisms in each group by taking LDA more than 4 and P less than 0.5 as the screening standard. The results are shown in fig. 12, and the experimental results show that p _ bacteroides, f _ muribacteriaceae and g _ Alloprevotella are the main significant difference bacteria in the control group; f _ helicobacter, p _ epsilon bactereota, c _ campylobacter and f _ Ruminococcaceae are main differential bacteria of the model group; p _ Proteobacteria, c _ Gamma, f _ Enterobacteriaceae, g _ Akkermansia are the main differential bacteria of the diaphragma juglandis group. The results show that after the stem administration of the aqueous extract of the diaphragma, the proportion of bacteroides is obviously increased, the proportion of Firmicutes and Proteobacteria is obviously reduced, in addition, the proportion of probiotics such as Akkermansia, Alloprovella and the like is increased, and the change of the flora structure and the composition can play a role in the onset of ulcerative colitis.

The effect of the invention is evaluated by establishing a DSS-induced acute mouse colitis model. The animal model is commonly used for researching the pathological mechanism of the ulcerative colitis and evaluating the curative effect of the medicine. We tested mice general physical and qualitative characteristics, DAI score, colon length, colon histopathological score by DSS-induced colitis mouse model. The experimental results show that the diaphragma juglandis aqueous extract can effectively treat and relieve the symptoms of ulcerative colitis: including weight loss, diarrhea, and hematochezia; can inhibit the production of inflammatory mediators: inflammatory molecules such as TNF-alpha, IL-6 or IL-1 beta are included to relieve inflammatory symptoms; can regulate the composition of intestinal flora, promote flora recovery, and regulate intestinal health; and can further protect mucosal barriers and repair colonic lesions. Therefore, the water extract of the diaphragma juglandis can be used as an effective treatment medicament for the ulcerative colitis and prepared into various dosage form medicaments or pharmaceutical compositions for treating the ulcerative colitis.

The embodiments of the present invention have been described in detail with reference to the accompanying drawings, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge of those skilled in the art without departing from the gist of the present invention. Furthermore, the embodiments of the present invention and the features of the embodiments may be combined with each other without conflict.