CN111374971A - Application of diosmetin in preparation of medicines for treating inflammatory bowel diseases - Google Patents

Abstract

The invention provides the application of geranialin in the preparation of a medicine for treating inflammatory bowel disease, belonging to the technical field of medicine, and the structure of geranialin is shown in the following formula (I):

The geranin of the present invention has good effect on inflammatory bowel disease, has no adverse reaction, can slow down the ulcerative bowel inflammation of mice induced by dextran sulfate sodium (DSS), and provides a good application for treating, relieving or improving inflammatory bowel disease. prospect.

Description

The application of geranin in the preparation of medicine for the treatment of inflammatory bowel disease

technical field

The present invention belongs to the new use of geranialignin, in particular, relates to the application of geranialignin in medicine for treating inflammatory bowel disease.

Background technique

Inflammatory bowel disease (IBDs) is a chronic relapsing inflammatory disease of the gastrointestinal tract, mainly including Crohn's disease (CD) and ulcerative colitis (UC). Among them, UC is a chronic inflammatory disease affecting the colon and rectum, and its incidence has been increasing year by year in my country in recent years. Its main clinical symptoms are bloody diarrhea, abdominal pain, blood in the stool, and weight loss, which can seriously affect organs such as the liver, skin, and vision. It is accompanied by complications such as intestinal perforation, massive bleeding, toxic colonic dilatation, and even canceration, which seriously endangers people's lives and health. However, its pathogenesis is still unclear, and it is generally believed to be caused by the interaction of many factors, including genetic, environmental and immune factors.

The gut microbiota and some specific microbial communities are closely related to many physiological functions and health conditions, such as immunity, nutrition and metabolism. Studies have shown that patients with ulcerative colitis mostly have different degrees of intestinal flora imbalance, and often involve changes in the number of flora, which is significantly different from the intestinal flora of healthy people. An effective means of treating inflammatory bowel disease. And studies have found that the metabolites of substances such as polyphenols and flavonoids can regulate the structure of the intestinal flora, thereby affecting the metabolic pathways and metabolites of the intestinal flora, thereby regulating the homeostasis of the intestinal flora.

At present, the traditional first-line treatment drugs for UC are mainly divided into five categories: 1. Para-aminosalicylic acids (such as sulfasalazine, mesalazine); 2. Corticosteroids (such as dexamethasone, prednisone); 3. . Immunomodulators (such as azathioprine, 6-thiopurine, and methotrexate); 4. Biologics (infliximab, adalimumab, etc.); 5. Probiotics, such as probiotics. At present, the most commonly used drugs for the treatment of UC, such as aminosalicylates and immunomodulators, have serious side effects, such as poor long-term treatment effect and influence on renal function, while other treatment methods are still divergent. Therefore, there is an urgent need to extract candidate drugs for UC treatment, such as flavonoids, polyphenols, terpenes, and alkaloids, from natural foods and plants.

Geranin is a natural flavonoid extracted from citrus fruits and some Chinese herbal medicines with strong anti-inflammatory, antibacterial and antioxidant properties. Studies have found that geranialin has certain pharmacological effects in various diseases, including anti-tumor effect, bacteriostatic effect, nervous system disease (Alzheimer's disease), retinal damage, liver disease, lung disease and kidney disease. In vitro studies have found that geranin can inhibit NF-κB signaling through antioxidant effects and reduce inflammatory interstitial and cytokines. However, the effect of geranilin on inflammatory bowel disease and intestinal flora has not yet been reported.

SUMMARY OF THE INVENTION

In view of this, the present invention aims to propose a new application of geranyl, namely the application of geranin in the preparation of a medicine for the treatment of inflammatory bowel disease, the structure of which is represented by the following formula (I) Show:

In the above application, the inflammatory bowel disease is ulcerative colitis.

In the above applications, the dosage of the geranialignin is 3 mg/kg to 300 mg/kg.

The present invention also provides a medicine for treating inflammatory bowel disease, comprising: geranin and pharmaceutically acceptable salts, esters, hydrates and excipients of geranin.

In the above medicines, the inflammatory bowel disease is ulcerative bowel disease.

In the above medicines, the pharmaceutically acceptable salts of geranialignin include organic salts and inorganic salts.

In the above drugs, the organic salts include mesylate, formate, acetate, trifluoroacetate, maleate, tartrate, succinate, fumarate, citrate, Benzenesulfonate, p-toluenesulfonate, naphthalenesulfonate, lactate and benzoate; the inorganic salts include hydrochloride, hydrobromide, sulfate and phosphate.

Among the above medicines, the dosage form of the medicine is selected from tablets, capsules, pills, suppositories, aerosols, oral liquid preparations, granules, powders, injections, syrups, wines, tinctures, lotions, films or their The combination.

Among the above medicines, the administration modes of the medicines include oral administration, injection, implantation, external application, spraying, inhalation or their combination.

The research of the present invention shows that geranialin has good effect on inflammatory enteritis, no adverse reaction, can slow down the acute and chronic ulcerative enteritis in mice induced by dextran sulfate sodium (DSS), and is effective in treating, relieving or improving ulcers It has a good application prospect in the field of sexual enteritis.

Description of drawings

Fig. 1 is the change curve of body weight of mice in each group of acute and chronic ulcerative colitis;

Figure 2 shows the DAI scores of mice in each group of acute and chronic ulcerative colitis;

Fig. 3 is the colorectal length and analysis diagram of each group of mice with chronic ulcerative colitis;

Fig. 4 is the colorectal length and analysis diagram of each group of mice with acute ulcerative colitis;

Figure 5 shows the HE staining of mice in each group of chronic ulcerative colitis;

Figure 6 is the tissue scores of mice in each group of chronic ulcerative colitis;

Figure 7 shows the HE staining of mice in each group of acute ulcerative colitis;

Figure 8 is the tissue scores of mice in each group of acute ulcerative colitis;

Figure 9 is the Alpha diversity analysis;

Figure 10 is a graph of the microbial distribution of cecal contents at the phylum level in each group of mice with chronic ulcerative colitis;

Figure 11 is a histogram of the distribution of LDA values in each group of chronic ulcerative enteritis mice.

Detailed ways

Unless otherwise defined, technical terms used in the following embodiments have the same meanings as commonly understood by those skilled in the art to which the present invention belongs. The test reagents used in the following examples are conventional biochemical reagents unless otherwise specified; the experimental methods are conventional methods unless otherwise specified.

The present invention will be described in detail below with reference to the embodiments.

The invention provides an application of geranin in the preparation of a medicine for the treatment of inflammatory bowel disease, wherein the structure of geranin is shown in the following formula (I):

Example 1

Effects of nintedanib on dextran sulfate sodium (DSS)-induced ulcerative colitis in mice

Preparation of animal model of chronic ulcerative colitis: 3% DSS was used to induce the chronic phase model of ulcerative colitis. During the adaptation period, the mice were allowed to eat and drink freely, and after the initial adaptation period, the water was changed to 3% (W/V ) of the DSS (except for the normal control group). The daily water intake of each mouse was calculated as 6 mL, and the DSS solution was supplemented to the daily water intake the next day. The DSS solution was administered for a total of 7 days. After 7 days of administration, it was found that the mice had soft stools, diarrhea or bloody stools. The mice were given intragastric administration. The mice in the normal control group and the model control group were given an equal volume of sodium carboxymethylcellulose every day, and the mice in the positive control group were given an equal volume of sodium carboxymethylcellulose. 200 mg/kg of sulfasalazine was given every day, and 50 mg/kg and 25 mg/kg of geranialin were given to the high-dose group and low-dose group, respectively.

Preparation of acute ulcerative colitis animal model: C57BL/6J, (4-6 weeks old) wild-type mice were taken, and 5% dextran sodium sulfate (DSS) was used to induce the acute phase model of ulcerative colitis. During the acclimation period, the mice were allowed to eat and drink freely. After the initial acclimation period, the drinking water was changed to 5% (W/V) DSS solution (except for the normal control group), and the mice were given free access to drink. The daily water intake of each mouse was calculated as 6 mL, and the DSS solution was supplemented to the daily water intake on the next day. The DSS solution was administered for a total of 8 days. 24 hours after the initial administration of DSS, it was found that the mice had soft stools, diarrhea or bloody stools, that is, the mice were given intragastric administration, and the mice in the normal control group and the model control group were given an equal volume of sodium carboxymethylcellulose every day, positive. Mice in the control group were given 200 mg/kg of sulfasalazine per day, and the high-dose group and the low-dose group were given 50 mg/kg and 25 mg/kg of geranialin per day, respectively.

The general living conditions, fecal characteristics, diarrhea and bloody stools of the mice with two different modeling methods were observed every day, and their body weights were weighed. Seven days after administration, all mice were killed by cervical dislocation, the abdominal cavity of the mice was exposed, the nodes and rectum were taken and the length was measured; the nodes and rectum were longitudinally cut along the mesentery direction, and the feces were washed with saline and fixed in 10% neutral forearm. Marin solution; take the severe ulcer for pathological examination, routine dehydration, paraffin embedding, sectioning, hematoxylin and eosin (hematoxylin andeosin, H&E) staining. The feces of mice with chronic ulcerative colitis were collected, and 16S rRNA sequencing technology was used to determine the structure and quantity of cecal microbial flora based on the Illumina HiSeq sequencing platform and paired-end sequencing (Paired-End).

(1) Scoring of disease activity: From the start of modeling to the end of administration, the weight changes, fecal characteristics, and blood in the stool of mice in each group were observed, and the scoring standards in the references were used to score, and the scores of each index were added every day. , as the Disease Activity Index (DAI). The specific scoring criteria are as follows:

Note: Based on the body weight on the day when modeling starts

(2) Length of the colon and rectum: The lesions of the colon and rectum were evaluated by measuring the length of the colon and rectum of the mice.

(3) Histological scoring of the colon and rectum: The ulcers and infiltration of inflammatory cells in the colon and rectum were observed and scored under a microscope. The scoring criteria are as follows:

The score of mucosal injury and inflammatory cell exudation was added to calculate the histological score of the node and rectum (0-6 points). The higher the score, the more severe the inflammation.

(4) Alpha diversity analysis The species abundance and species diversity of the samples are evaluated by Alpha diversity analysis. The measurement indicators are Chao1, Ace, Shannon and Simpson, of which Chao1 and Ace indices measure species abundance, and Shannon and Simpson indices are used for Measure species diversity. At the 97% similarity level, the number of OTUs in each group was obtained.

The experimental results are shown in FIGS. 1 to 11 and Tables 1 to 3 .

Table 1 is the tissue scores of mice in each group of chronic ulcerative colitis

Table 2 is the tissue scores of mice in each group of acute ulcerative colitis

Table 3 Alpha diversity analysis

normal group model group Geranin group shannon 7.59±0.49 5.21±0.88 6.69±1.08 simpson 0.981±0.008 0.920±0.039 0.956±0.029 PD_whole_tree 80.08±16.96 44.62±13.92 66.86±14.33 goods_coverage 0.993±0.003 0.997±0.001 0.995±0.001 Chao1 2636.20±784.75 1078.71±242.87 2178.95±571.81 observed_species 2209.17±781.83 860.67±331.67 1808.67±511.33

It can be seen from Figure 1A that the body weight of the mice in the geranialignin treatment group recovered rapidly after administration, while the body weight of the model group recovered slowly. Compared with the model group shown in Figure 1B, the body weight loss of the geranialignin treatment group was slowed down, indicating that Nida Nib can improve animal health;

It can be seen from Figure 2A and B that compared with the mice in the model group, the DAI scores of the mice in the geranialignin treatment group were significantly reduced, indicating that geranialignin can alleviate DSS-induced acute and chronic ulcerative colitis; and from Figure 3 As can be seen from Figure 4, compared with the model group, the length of the junction and rectum in the geranialignin administration group was significantly longer than that in the model group, and p<0.01, that is, there was a statistically significant difference; from Figures 5 and 6 and Table 1 and Figure 7, 8 and Table 2; H&E staining observation and statistical analysis showed that the degree of tissue lesions and inflammatory cell infiltration in the nintedanib treatment group was significantly lower than that in the model group, indicating that geranialin can effectively slow down the induction of DSS. acute and chronic ulcerative colitis in mice.

As can be seen from Figure 9 and Table 3, compared with the normal group, the indices of shannon, simpson, PD_whole_tree, chao1 and observed_species in the DSS group were decreased, and there were significant differences, indicating that the rats in the DSS group developed ulcerative colitis, leading to intestinal microorganisms decrease in species abundance and diversity. Compared with the DSS group, the shannon, simpson, PD_whole_tree, chao1 and observed_species indices of the geranialignin group increased and there were significant differences. The true state of microbes in the sample. The above results indicate that the composition has a preventive effect on ulcerative colitis.

Figure 10 shows the relative abundance of major microbiota components at the phylum level. Through the analysis of the community bar chart, the dominant flora at the phylum level were Firmicutes, Bacteroidetes and Proteobacteria. Compared with the normal group, the relative abundances of Actinobacteria and Bacteroidetes in the DSS group were significantly decreased, while the Firmicutes were increased. Bacteroidetes increased significantly and Firmicutes decreased. It was suggested that geranialin could modulate DSS-induced changes in the intestinal microbiota of mice.

Figure 11 shows the LEfSe analysis of samples between groups. In order to clarify which bacteria geranialin changes which may affect IBD, linear discriminant analysis (LEfSe) was performed on OTUs with a relative abundance greater than 0.5% to obtain the OUT that dominated each group first. The figure shows the species whose LDA value is greater than the set value (set to 4.0), the length of the histogram represents the impact size of the different species (that is, the LDA value), and the different colors represent the species in different groups. Compared with the normal group, the relative abundances of Bacilli and Bacteroides were higher in the DSS group. Escherichia showed high relative abundance. The results suggest that geranialignin can modulate the gut microbiota.

In conclusion, geranialin has good efficacy on inflammatory bowel disease without adverse reactions, and can slow down DSS-induced acute and chronic ulcerative colitis in mice, and has a good application prospect in the treatment, remission or improvement of inflammatory bowel disease.

The above are only specific embodiments of the present invention, and are not intended to limit the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention. .

Claims (9)

1. Use of diosmetin for the preparation of a medicament for the treatment of inflammatory bowel disease, wherein the diosmetin has the structure shown in formula (I):

2. use according to claim 1, characterized in that: the inflammatory bowel disease is ulcerative enteritis.

3. Use according to claim 1 or 2, characterized in that: the dosage of the diosmetin is 3 mg/kg-300 mg/kg.

4. A medicament for the treatment of inflammatory bowel disease, characterized by: the method comprises the following steps: diosmetin, and pharmaceutically acceptable salts, esters, hydrates and adjuvants of diosmetin.

5. The medicament of claim 4, wherein: the enteritis disease is ulcerative enteritis disease.

6. The medicament of claim 4, wherein: the pharmaceutically acceptable salts of diosmetin comprise organic salts and inorganic salts.

7. The medicament of claim 6, wherein: the organic salts include mesylate, formate, acetate, trifluoroacetate, maleate, tartrate, succinate, fumarate, citrate, benzenesulfonate, p-toluenesulfonate, naphthalenesulfonate, lactate, and benzoate; the inorganic salts include hydrochloride, hydrobromide, sulfate and phosphate salts.

8. The medicament of claim 4, wherein: the dosage form of the medicine is selected from tablets, capsules, pills, suppositories, aerosols, oral liquid preparations, granules, powders, injections, syrups, vinums, tinctures, lotions, films or the combination thereof.

9. The medicament of claim 4, wherein: the administration of the drug includes oral, injection, implantation, external application, spraying, inhalation, or a combination thereof.

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