CN115381820A

CN115381820A - Application of demethylenetetrahydroberberine hydrochloride in preparation of medicine for treating ulcerative colitis

Info

Publication number: CN115381820A
Application number: CN202211115207.4A
Authority: CN
Inventors: 张玉彬; 徐英梅; 黄萌胜; 蒋华; 李睿岩
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2022-09-14
Filing date: 2022-09-14
Publication date: 2022-11-25

Abstract

The invention relates to the field of biological medicines, in particular to application of demethylenetetrahydroberberine hydrochloride in preparation of a medicine for treating ulcerative colitis. Through in vivo and in vitro experiments, the invention surprisingly discovers that the demethylenetetrahydroberberine hydrochloride is superior to berberine and demethyleneberberine hydrochloride in resisting ulcerative colitis and has significant difference. The compound has better activity and lower toxicity, and has unexpected technical effect.

Description

Application of demethylenetetrahydroberberine hydrochloride in preparation of medicine for treating ulcerative colitis

Technical Field

The invention relates to the field of biological medicines, in particular to application of demethylenetetrahydroberberine hydrochloride in preparation of a medicine for treating ulcerative colitis.

Background

In the last two decades, the number of IBD patients in China has increased year by year, and the disease has been transformed from rare disease to "common disease". Ulcerative Colitis (UC) is a subtype of inflammatory bowel disease and is a chronic nonspecific intestinal inflammatory disease. UC mainly affects the mucous membrane and submucosa of colorectal tract, and is characterized by continuous and diffuse inflammatory injury of intestinal mucosa and has a long course of disease. Clinical manifestations of UC include severe diarrhea, hematochezia, progressive loss of peristaltic function, etc., which seriously affect human health. The pathogenesis of UC is unclear, possibly due to genetic factors, immune response, intestinal microbial homeostasis, and an imbalance in the epithelial barrier. It is noteworthy that UC is liable to increase the risk of colorectal cancer. At present, the clinical medicines for treating UC mainly comprise the following 4 types: 1. aminosalicylic drugs such as sulfasalazine and 5-aminosalicylic acid, glucocorticoid drugs such as prednisone and hydrocortisone, immunosuppressant drugs such as azathioprine or 6-mercaptopurine (6-MP), and 4 biological agents such as inflixine and adalimumab which neutralize inflammatory factors TNF-alpha, are expensive and are liable to cause secondary infection, so that the drugs are used with great clinical caution. The treatment medicine is mainly used for treating UC on the basis of anti-inflammatory, immunity inhibition and inflammatory factor neutralization mechanisms, and can treat the UC radically without treating the UC radically, so that the UC is delayed and difficult to cure. Therefore, patients with UC urgently need a safe drug with a more effective treatment and weak side effects.

In recent years, JAK (Janus kinase) inhibitors (such as tofacitinib) for the treatment of rheumatoid arthritis are used for the treatment of UC. Therefore, kinase inhibitors are hot of research and development for new drugs to treat UC. In the development process of ulcerative colitis, activated immune cells secrete a variety of pro-inflammatory mediators, such as Reactive Oxygen Species (ROS), neutrophil infiltration, cytokines, etc., stimulate the inflammatory cascade, resulting in the disruption of the intestinal epithelial barrier and apoptosis, which perpetuates chronic intestinal inflammation. In recent years, it has been found that IL-23 levels are elevated in experimental colitis models such as DSS (dextran sulfate sodium), helicobacter hepaticus colitis, and T cell metastatic colitis, consistent with the notion that IL-23 may play an important role in the pathogenesis of colitis. Prior to the discovery of IL-23, studies were conducted to neutralize the p40 subunit of IL-12 using the ustekinumab mab, now known to block both IL-12 and IL-23. p40 blockers were found to be very effective in inhibiting the activity of experimental colitis in various mouse models. Guselkumab targets subunit p19 of IL-23 and shows excellent effects in clinical trials for the treatment of ulcerative colitis and Crohn's disease. Therefore, specific blocking of IL-23 secretion in UC pathogenesis can be used as an effective target of UC medication, and is one of key targets for treating UC.

The hydrochloric acid demethylenetetrahydroberberine is shown as a formula (I).

The English name of the hydrochloric acid Demethylenetetrahydroberberine (formula I) is Demethylethyltetrahydroberine. The invention patent refers to it as DMTHB for short. The molecular formula is as follows: C19H21NO4, relative molecular mass: 327.2.CAS number 47346-21-4, academic name 6H-Dibenzo [ a, g ] quinozine-2, 3-diol,5,8,13, 13a-tetrahydroo-9, 10-dimethoxy; 6H-dibenzo [ a, g ] quinolizine-2,3-diol,5,8,13,13a-tetrahydro-9, 10-dimethoxy. The demethylenetetrahydroberberine hydrochloride can form various salts with inorganic acids or organic acids, such as chloride, sulfate, phosphate, bromide, iodide, citrate, fumarate, maleate, malate, succinate, etc. Although berberine and demethyleneberberine hydrochloride are reported to treat ulcerative colitis, the two compounds have the problems of poor solubility, high administration dosage, low oral bioavailability and the like, and the treatment effect is not as good as possible and needs to be further improved. In order to obtain better treatment effect, the invention discovers the effect of the hydrochloric acid demethylenetetrahydroberberine in treating the ulcerative colitis for the first time.

Disclosure of Invention

In order to obtain better treatment effect, the invention aims to provide the application of the demethylenetetrahydroberberine hydrochloride (DMTHB) as a novel medicine for preventing and treating ulcerative colitis.

Application of demethylenetetrahydroberberine hydrochloride in preparing medicine for treating ulcerative colitis is provided, wherein the formula of demethylenetetrahydroberberine hydrochloride is shown as formula I

A pharmaceutical composition for treating ulcerative colitis, comprising a pharmaceutically effective amount of the demethylenetetrahydroberberine hydrochloride according to claim 1 and pharmaceutically acceptable excipients.

The pharmaceutical composition is characterized by being tablets, capsules, pills, injections, sustained-release agents and various microparticle administration systems.

The hydrochloric acid demethylenetetrahydroberberine product is prepared by conventional chemistry and separation and purification. The laboratory adopts High Performance Liquid Chromatography (HPLC) analysis and detection, the purity of the product reaches over 99 percent, and the chemical structure of the hydrochloric acid demethylenetetrahydroberberine product used in the laboratory is correct through analysis and identification of liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance. The research shows that the purity and the chemical structure of the demethylenetetrahydroberberine hydrochloride meet the requirements of developing in-vivo and in-vitro level pharmacological activity research.

The invention also relates to a pharmaceutical composition containing the demethylenetetrahydroberberine hydrochloride as an active ingredient and conventional pharmaceutical excipients or auxiliary agents. Generally, the pharmaceutical composition of the present invention contains 0.1 to 95% by weight of demethylenetetrahydroberberine hydrochloride. The compounds of the invention are generally present in an amount of 0.1 to 100mg in a unit dosage form. Pharmaceutical compositions of the compounds of the present invention may be prepared according to methods well known in the art. For this purpose, the compounds according to the invention can, if desired, be combined with one or more solid or liquid pharmaceutical excipients and/or adjuvants and brought into a suitable administration form or dosage form which can be used as human or veterinary medicine.

The compounds of the present invention or pharmaceutical compositions containing them may be administered in unit dosage form by enteral or parenteral routes, such as oral, intramuscular, subcutaneous, nasal, oromucosal, dermal, peritoneal or rectal administration. The route of administration of the compounds of the invention or the pharmaceutical compositions containing them may be by injection. The injection includes intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection, acupoint injection, etc.

The administration dosage form can be liquid dosage form or solid dosage form. For example, the liquid dosage form can be true solution, colloid, microparticle, emulsion, or suspension. Other dosage forms such as tablet, capsule, dripping pill, aerosol, pill, powder, solution, suspension, emulsion, granule, suppository, lyophilized powder for injection, etc.

The compound can be prepared into common preparations, sustained release preparations, controlled release preparations, targeting preparations and various microparticle drug delivery systems.

For example, to form a unit dosage form into a tablet, a wide variety of carriers known in the art can be used. Examples of the carrier are, for example, diluents and absorbents such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, aluminum silicate and the like; wetting agents and binders such as water, glycerin, polyethylene glycol, ethanol, propanol, starch slurry, dextrin, syrup, honey, glucose solution, acacia slurry, gelatin slurry, sodium carboxymethylcellulose, shellac, methyl cellulose, potassium phosphate, polyvinylpyrrolidone and the like; disintegrating agents such as dried starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene sorbitol fatty acid ester, sodium dodecylsulfate, methyl cellulose, ethyl cellulose, etc.; disintegration inhibitors such as sucrose, glyceryl tristearate, cacao butter, hydrogenated oil and the like; absorption accelerators such as quaternary ammonium salts, sodium lauryl sulfate and the like; lubricants, for example, talc, silica, corn starch, stearate, boric acid, liquid paraffin, polyethylene glycol, and the like. The tablets may be further formulated as coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layered and multi-layered tablets.

For example, to form the administration units into pills, various carriers well known in the art are widely used. Examples of the carrier are, for example, diluents and absorbents such as glucose, lactose, starch, cacao butter, hydrogenated vegetable oil, polyvinylpyrrolidone, glyceryl monostearate, kaolin, talc and the like; binders such as acacia, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste or batter, etc.; disintegrating agent such as agar powder, dried powder, alginate, sodium dodecyl sulfate, methyl cellulose, ethyl cellulose, etc.

For example, in order to encapsulate the administration units, the active ingredient, demethyleneberberine hydrochloride, a compound of the invention, is mixed with the various carriers described above and the mixture thus obtained is placed in hard gelatin capsules or soft capsules. The effective component of the compound can also be prepared into microcapsules, and the microcapsules can be suspended in an aqueous medium to form a suspension, and can also be filled into hard capsules or prepared into injections for application.

For example, the demethyleneberberine hydrochloride of the compound of the present invention is formulated into injectable preparations, such as solutions, suspension solutions, emulsions, lyophilized powders, which may be aqueous or non-aqueous, and may contain one or more pharmaceutically acceptable carriers, diluents, binders, lubricants, preservatives, surfactants, dispersants, tonicity adjusting agents, solubilizers and pH adjusting agents. For example, water, ethanol, polyethylene glycol, 1, 3-propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, fatty acid ester, etc. can be used for dilution. The osmotic pressure regulator can be sodium chloride, mannitol, glycerol, glucose, phosphate, acetate, etc.; the solubilizer or cosolvent can be poloxamer, lecithin, hydroxypropyl beta-cyclodextrin, etc.; the pH regulator may be phosphate, acetate, hydrochloric acid, sodium hydroxide, etc. For example, mannitol and glucose can be added as propping agent for preparing lyophilized powder for injection.

In addition, a coloring agent, a preservative, a flavor, a corrigent, a sweetener, a flavor, or the like may be added to the pharmaceutical preparation, if necessary. These adjuvants are conventional in the art.

The sterile media used in the present invention may be prepared by standard techniques well known to those skilled in the art. They may be sterilized, for example, by filtration through a bacterial filter, by adding a sterilizing agent to the composition, by subjecting the composition to radiation, or by subjecting the composition to heat sterilization. They may also be prepared as sterile injectable media just prior to use.

For the purpose of administration, to increase the therapeutic effect, the drug or pharmaceutical composition of the present invention can be administered by any known administration method. The route of administration for practicing the compounds of the present invention will, of course, depend on the disease and the site in need of treatment. Because the pharmacokinetic and pharmacodynamic profiles of the compounds of the invention may vary somewhat, the most preferred method of achieving therapeutic concentrations in tissues is to gradually increase the dosage and monitor the clinical effect. For such escalating therapeutic doses, the initial dose will depend on the route of administration.

The dosage of the pharmaceutical composition of the compound of the present invention to be administered to any particular patient depends on many factors, such as the nature and severity of the disease to be prevented or treated, the sex, age, character and individual response of the patient or animal, the route of administration, the number of administrations, the purpose of the treatment, and thus the therapeutic dose of the present invention may vary widely. Depending on the condition of the patient to be treated, some variation in dosage may be necessary, and in any event, the appropriate dosage for an individual patient will be determined by a physician.

The dose administered refers to the weight of the compound excluding the weight of the carrier when used. Generally, the dosages of the pharmaceutical ingredients of the present invention are well known to those skilled in the art. The prophylactic or therapeutic objectives of the present invention can be accomplished by appropriate adjustment of the actual amount of drug contained in the final formulation of the compound composition of the present invention to achieve the desired therapeutically effective amount. Can be administered in a single dosage form or divided into several, e.g., two, three or four dosage forms; this is limited by the clinical experience of the administering physician and by the dosage regimen that includes the use of other therapeutic means. The compounds or compositions of the present invention may be administered alone or in combination with other therapeutic or symptomatic agents and adjusted in dosage.

Advantageous effects

Although berberine and demethyleneberberine hydrochloride are reported to treat ulcerative colitis, the two compounds have the problems of poor solubility, high administration dosage, low oral bioavailability and the like, and the treatment effect is not satisfactory, so that the search for a better treatment effect medicine is a technical problem to be solved in the field. The demethylenetetrahydroberberine hydrochloride is a reduction product of demethyleneberberine hydrochloride, has similar structure, and the molecule lacks conjugate group, and the chemical property of the demethylenetetrahydroberberine hydrochloride is changed, the polarity is reduced, the fat solubility is increased, and whether the anti-ulcerative colitis activity is maintained is unknown.

Through in vivo and in vitro experiments, the invention surprisingly discovers that the demethylenetetrahydroberberine hydrochloride is superior to berberine and demethyleneberberine hydrochloride in resisting ulcerative colitis and has significant difference. The compound has better activity and lower toxicity, and has unexpected technical effect. The method comprises the following specific steps:

in vitro experiments, the effect of demethylenetetrahydroberberine hydrochloride on DSS stimulation to generate ROS scavenging by normal colon epithelial cells is analyzed through a CCK8 method and an ROS probe method and through cell culture and a fluorescence microscope. The results show that the hydrochloric acid demethylenetetrahydroberberine can protect colon epithelial cells to relieve cell damage caused by DSS stimulation and improve cell activity under the condition of no toxicity to the cells. The demethylenetetrahydroberberine hydrochloride has strong scavenging effect on ROS at cellular level. The invention discloses that the hydrochloric acid demethylenetetrahydroberberine has in vitro anti-inflammatory and antioxidant bioactivity, which lays an important theoretical foundation for developing the research of DMTHB medicament for treating ulcerative colitis.

In vivo experiments, the invention takes a C57BL/6 mouse as an animal test model to investigate the treatment effect of the hydrochloric acid demethylenetetrahydroberberine on DSS-induced acute ulcerative colitis. The treatment scheme is as follows: starting on day 3 of DSS modeling, mice exhibited symptoms of loose stools, and mice were gavaged orally with DMTHB 1 time a day for 7 days, with dosing groups set at two doses, 50mg/kg (IG) and 100mg/kg (IG), respectively. Setting berberine at a dose of 100mg/kg (IG) and a control group of demethyleneberberine hydrochloride at a dose of 100mg/kg (IG). From the model creation, the mice were observed daily for changes in diet and body weight while recording the state of the mice, and the mice were dissected one week after administration. Experimental result analysis shows that the low-dose and high-dose groups of the demethylenetetrahydroberberine hydrochloride can obviously improve the symptoms of weight loss, colon atrophy and swelling and congestion caused by the ulcerative colitis of mice, and the demethylenetetrahydroberberine hydrochloride has a protective effect on the DSS-induced ulcerative colitis. In addition, compared with the DSS group, the myeloperoxidase levels of the DMTHB low-dose and high-dose administration groups were significantly reduced, indicating that DMTHB can reduce neutrophil infiltration in colon tissues, and has an anti-inflammatory effect superior to that of the BBR group and the DMB group at equal doses. The colon histopathology research result shows that the colon structural disorder, crypt and gland deletion and severe inflammatory cell infiltration of the model group mouse occur, the colon pathological change range and the degree of the DMTHB administration group are obviously reduced compared with the model group, and the treatment effect is stronger than that of the BBR group and the DMB group with the same dosage. The experimental results fully show that the demethylenetetrahydroberberine hydrochloride can effectively treat DSS-induced acute ulcerative colitis.

Drawings

FIG. 1 effect of DMTHB on NCM460 cells on their cell viability for 24 h;

figure 2 effect of DMTHB administration on cell viability of DSS-injured NCM460 cells;

FIG. 3 staining pattern of ROS production by DSS injured NCM460 cells following DMTHB administration;

figure 4 weight change profile of DSS-induced UC mice;

figure 5 DMTHB treatment DSS-induced UC mouse colon histomorphogram;

FIG. 6 Colon Length/Colon weight analysis plot of DMTHB-treated DSS-induced UC mice;

figure 7 graph of DMTHB treatment DSS-induced HE staining of UC mice (x 400);

FIG. 8 Western blot results (A) and grayscale analysis (B) of DMTHB on DSS-induced colon tissue in mice.

Detailed Description

The following examples may assist those skilled in the art in a more complete understanding of the present invention, but are not intended to limit the invention in any way.

Term(s) for

UC: ulcerative colitis

IBD: inflammatory bowel disease

DMTHB (dimethylene tetrahydroberberine hydrochloride)

DSS: dextran sodium sulfate

BBR: berberine hydrochloride

DMB: hydrochloric acid demethyleneberberine

ROS: active oxygen

MPO: myeloperoxidase

IG: gavage stomach

Example 1 cytotoxic Effect of Desmethylenetetrahydroberberine hydrochloride (DMTHB) on NCM460 cells.

The method comprises the following steps: cell viability was measured by the CCK-8 method. The edge wells of the cell 96-well plate were filled with sterile PBS 100 μ L per well in advance. The normal colonic epithelial cells NCM460 in the logarithmic growth phase were collected and the number of the cells was adjusted to 10 ⁵ Cells were seeded at 100. Mu.L/ml in 96-well cell plates. The cells were pre-cultured at 37 ℃ for 24 hours in a cell culture incubator containing 5% of C02 to ensure cell adhesion. DMTHB stock solution dissolved in DMSO at a concentration of 200mM was diluted in a gradient at concentrations of 200. Mu.M, 100. Mu.M, 50. Mu.M, 25. Mu.M, and 12, respectively. 5 μ M, 6.25 μ M, 3.125 μ M, a blank group, a cell culture medium Control (Control) group, and a drug Dissolution Medium (DMSO) group (dilution method and drug concentration 200 μ M) were separately provided, and 6 wells were repeated for each group. After further incubation for 24h, 10 μ LCCK-8 solution was added to each well, taking care not to generate air bubbles. The plates were incubated in an incubator for 1-4h and the absorbance at 450nm of each well was measured using a microplate reader. Cell viability (%) = [ a (medicated) -a (blank)]/[ A (0 dosing) -A (blank)]×100。

As a result: the low-dose DMTHB has no obvious toxic effect on normal colon epithelial cells NCM460 and no obvious difference with the cell viability of the Control group, and as shown in figure 1, after the drug concentration is higher than 50 mu M, the low-dose DMTHB has obvious inhibition effect on cell proliferation and is dose-dependent.

Example 2 protective Effect of demethylenetetrahydroberberine hydrochloride on DSS-induced NCM460 cell injury.

The method comprises the following steps: the protective effect of DMTHB on DSS-induced cell damage was assessed using the CCK-8 experiment. The edge wells of the cell 96-well plate were filled with sterile PBS 100 μ L per well in advance. The normal colonic epithelial cells NCM460 in the logarithmic growth phase were collected and the number of the cells was adjusted to 10 ⁵ Cells were seeded at 100. Mu.L/ml in 96-well cell plates. The cells were pre-cultured at 37 ℃ for 24 hours in a cell culture incubator containing 5% of C02 to ensure cell adhesion. DMTHB stock solution dissolved in DMSO at a concentration of 200mM was diluted to 5. Mu.M, 10. Mu.M, and 20. Mu.M, and a Control (Control) group and a DSS group were prepared separately in 6 wells. After 6h of predose, DSS solution dissolved by sterile PBS is added into the DSS group and the administration group respectively, so that the final concentration of DSS in the culture solution is 80mg/ml, and PBS solution with the same volume is added into the Control group. After further incubation for 12h, 10 μ LCCK-8 solution was added to each well, taking care not to generate bubbles. The plate was incubated in an incubator for 1-4h and the absorbance at 450nm of each well was measured using a microplate reader. Cell viability (%) = [ a (medicated) -a (blank)]/[ A (0 Add medicine) -A (blank)]×100。

As a result: as shown in FIG. 2, DSS induced damage to normal colonic epithelial cells and inhibited proliferation of epithelial cells in the experiment. After being administrated, the DMTHB with each concentration can protect cells to different degrees, the cell activity is obviously increased, and the protection effect is dose-dependent.

Example 3 Demethylenetetrahydroberberine hydrochloride scavenges ROS produced by DSS-induced colonic epithelial cells.

The method comprises the following steps: the experiment adopts ROS fluorescence probe method, and cell level detection DMTHB has effect on DSS-induced colon epithelial cell oxidative damage. The NCM460 cell line was divided into 4X 10 cells ⁵ Each cell/ml was inoculated into a 24-well plate, and incubated at 37 ℃ in an incubator containing 5% CO2 for 24 hours to ensure that the cells were in a vigorous growth phase. The administration group was administered with 10. Mu.M, 20. Mu.M DMTHB and 20. Mu.M BBR and 20. Mu.M DMB treatment, respectively, and 3 wells were repeated. Drug incubation 6After h, 80mg/ml DSS solution was administered to both DSS group and each administration group, and the Control group added the same volume of PBS solution. The culture plate is placed in an incubator to be incubated for 12 hours continuously, so that a large amount of ROS is generated to cause oxidative damage to cells. The ROS scavenging action is observed under a fluorescence microscope by utilizing the principle that a fluorescence probe DCFH-DA (10 mu M) is combined with ROS to generate fluorescence.

As a result: the fluorescence microscope shows that DMTHB at various concentrations can significantly inhibit ROS generated by peroxide in NCM460 cells, and the effect is better than that of BBR and DMB at the same dose, and the result is shown in figure 3.

Example 4 treatment with demethylenetetrahydroberberine hydrochloride increases the survival rate of mice with ulcerative colitis.

The method comprises the following steps: female C57BL/6 mice with a weight of 20-22g are selected for the experiment, and after the experiment is adapted to the environment for 1 week, the mice are randomly divided into 6 groups, and each group comprises 6 mice. Each group is a normal Control (Control) group, a model (DSS) group, a low dose (50 mg/kg) of demethylenetetrahydroberberine hydrochloride group, a high dose (100 mg/kg) of demethylenetetrahydroberberine hydrochloride group, a berberine hydrochloride group (100 mg/kg) and a demethyleneberberine hydrochloride group (100 mg/kg). On day 0, the mice in the model group and the administration group freely drunk 4% DSS solution, and after 7 days of molding, they normally drunk distilled water for 5 days. The normal control group had free access to double distilled water for 12 days. Mice in the administration group started gavage each group of the corresponding drug on day 3,1 time per day for 7 days, while mice in the control group and the model group were given the same volume of vehicle. The number of deaths of the mice during the experiment was recorded. After the completion of the 7 days of gavage, the mice were dissected.

As a result: after the high-dose DMTHB is administrated, the survival rate of the mice with the ulcerative colitis is obviously improved. As shown in table 1,3 mice were sacrificed in the experimental period, the model group and DMTHB low dose group, respectively. The BBR group and DMB group mice died 1 mouse each, and the control group and DMTHB high-dose group did not die.

TABLE 1 Effect of DMTHB treatment on UC mouse mortality

Example 5 treatment with demethylenetetrahydroberberine hydrochloride slows down the weight loss in mice with ulcerative colitis.

The method comprises the following steps: female C57BL/6 mice with a weight of 20-22g are selected for the experiment, and after the experiment is adapted to the environment for 1 week, the mice are randomly divided into 6 groups, and each group comprises 6 mice. Each group is a normal Control (Control) group, a model (DSS) group, a low dose (50 mg/kg) of demethylenetetrahydroberberine hydrochloride group, a high dose (100 mg/kg) of demethylenetetrahydroberberine hydrochloride group, a berberine hydrochloride group (100 mg/kg) and a demethyleneberberine hydrochloride group (100 mg/kg). On day 0, the mice in the model group and the administration group freely drunk 4% DSS solution, and after 7 days of molding, they normally drunk distilled water for 5 days. The normal control group had free access to double distilled water for 12 days. Mice in the dosing group began gavage of the corresponding drug for each group on day 3,1 time per day for 7 days, while mice in the control and model groups were given the same volume of vehicle. The body weight change of the mice during the experiment was recorded. After the completion of the 7 days of gavage, the mice were dissected.

As a result: as shown in fig. 4, the control group mice were not affected by DSS and the body weight remained substantially increasing. With the occurrence of a series of symptoms of colitis such as diarrhea and hematochezia in different degrees of mice in each group drinking DSS, the consumption of the mice is increased while the food intake is reduced, so that the mice lose weight and become listened. Compared with the model group, the weight loss of the mice in the administration group is obviously improved from day 7, and the DMTHB high-dose group is significantly different from the model group.

Example 6 treatment with demethylenetetrahydroberberine hydrochloride improves colonic atrophy and swelling in mice with ulcerative colitis.

The method comprises the following steps: female C57BL/6 mice with a weight of 20-22g are selected for the experiment, and after the experiment is adapted to the environment for 1 week, the mice are randomly divided into 6 groups, and each group comprises 6 mice. Each group is a normal Control (Control) group, a model (DSS) group, a low dose (50 mg/kg) of demethylenetetrahydroberberine hydrochloride group, a high dose (100 mg/kg) of demethylenetetrahydroberberine hydrochloride group, a berberine hydrochloride group (100 mg/kg) and a demethyleneberberine hydrochloride group (100 mg/kg). On day 0, the mice in the model group and the administration group freely drunk 4% DSS solution, and after 7 days of molding, they normally drunk distilled water for 5 days. The normal control group had free access to double distilled water for 12 days. Mice in the dosing group began gavage of the corresponding drug for each group on day 3,1 time per day for 7 days, while mice in the control and model groups were given the same volume of vehicle. After the completion of the gavage for 7 days, the dissected mice had their colon tissues photographed, weighed, and measured for length.

As a result: the degree of colon swelling can be expressed by the weight of the colon per unit length, and the DSS modeling causes the colon to have symptoms of swelling, atrophy and thickening, bleeding and the like, and the weight of the colon per unit length is increased. As shown in fig. 5 and 6, the administration of DMTHB at different doses can relieve colonic swelling and improve atrophy, and the therapeutic effect is better than that of BBR and DMB at the same dose.

Example 7 treatment with demethylenetetrahydroberberine hydrochloride reduces excess MPO production in ulcerative colitis mice.

The method comprises the following steps: myeloperoxidase (MPO), also known as peroxidase, is a heme protease of heme prosthetic groups and is one of the members of the heme peroxidase superfamily. It is a functional marker and an activation marker of neutrophils, and the level and activity change of the neutrophil activation marker have important significance for prompting inflammatory infiltration of the intestinal neutrophils. Female C57BL/6 mice, weighing 20-22g, were acclimated for 1 week and randomized into 6 groups of 6 mice each. Each group is a normal Control (Control) group, a model (DSS) group, a low dose (50 mg/kg) of demethylenetetrahydroberberine hydrochloride group, a high dose (100 mg/kg) of demethylenetetrahydroberberine hydrochloride group, a berberine hydrochloride group (100 mg/kg) and a demethyleneberberine hydrochloride group (100 mg/kg). On day 0, the mice in the model group and the administration group freely drunk 4% DSS solution, and after 7 days of molding, they normally drunk distilled water for 5 days. The normal control group had free access to double distilled water for 12 days. Mice in the dosing group began gavage of the corresponding drug for each group on day 3,1 time per day for 7 days, while mice in the control and model groups were given the same volume of vehicle. After the completion of the 7 days of intragastric administration, the mice were dissected to take colon tissue of the same site. Shearing a certain mass of colon tissue on ice, homogenizing in normal saline to obtain 10% colon homogenate, centrifuging at 8000rpm and 4 deg.C for 10min, collecting supernatant, and determining MPO activity level in colon tissue by referring to MPO detection kit instruction.

As a result: as shown in Table 3, compared with the control group, the level of MPO activity of the mice in the DSS group is remarkably increased, and compared with other groups, after DMTHB is administrated, the MPO activity can be reduced, the neutrophil infiltration of colon tissues is reduced, and the excellent anti-inflammatory effect is shown.

TABLE 3 UC mouse Colon tissue MPO expression level

(n＝6,mean±variance，#p<0.05、##p<0.01、###p<0.001、####p<0.0001VS Control，*p<0.05、**p<0.01、***p<0.001、****p<0.0001VS DMTHB-100mg/kg)

Example 8 treatment with demethylenetetrahydroberberine hydrochloride protects against histopathological damage in mice with ulcerative colitis.

The method comprises the following steps: female C57BL/6 mice, weighing 20-22g, were acclimated for 1 week and randomized into 6 groups of 6 mice each. Each group is a normal Control (Control) group, a model (DSS) group, a low dose (50 mg/kg) of demethylenetetrahydroberberine hydrochloride group, a high dose (100 mg/kg) of demethylenetetrahydroberberine hydrochloride group, a berberine hydrochloride group (100 mg/kg) and a demethyleneberberine hydrochloride group (100 mg/kg). On day 0, the mice in the model group and the administration group freely drunk 4% DSS solution, and after 7 days of molding, they normally drunk distilled water for 5 days. The normal control group had free access to double distilled water for 12 days. Mice in the dosing group began gavage of the corresponding drug for each group on day 3,1 time per day for 7 days, while mice in the control and model groups were given the same volume of vehicle. After the completion of the 7 days of intragastric administration, the mice were dissected to take colon tissue of the same site. After full cleaning, fixing the tissue by paraformaldehyde, and performing pathological examination by HE staining.

As a result: as can be seen from the colon histopathological HE staining result, the colon tissue of the blank control group mouse has normal shape, orderly arranged glands, complete crypt structure and no inflammatory cell infiltration. The crypt and gland structures of colon tissue in the model group are destroyed, disorderly and infiltrated by a large number of inflammatory cells. After DMTHB treatment, the gland and crypt structure are recovered to a certain extent, the inflammatory infiltration is reduced, and the treatment effect is better than DMB and BBR. Meanwhile, the treatment effect of the DMTHB (100 mg/kg) administration group is better than that of the 50mg/kg group, a certain dose dependence is shown, and HE staining is shown in figure 6.

Example 9 treatment with demethylenetetrahydroberberine hydrochloride inhibits IL-23 secretion in mice with ulcerative colitis.

The method comprises the following steps: female C57BL/6 mice, weighing 20-22g, were acclimated for 1 week and randomized into 6 groups of 6 mice each. Each group is a normal Control (Control) group, a model (DSS) group, a low dose (50 mg/kg) of demethylenetetrahydroberberine hydrochloride group, a high dose (100 mg/kg) of demethylenetetrahydroberberine hydrochloride group, a berberine hydrochloride group (100 mg/kg) and a demethyleneberberine hydrochloride group (100 mg/kg). On day 0, the mice of the model group and the administration group freely drunk the 4-th DSS solution, and after 7 days of molding, normally drunk distilled water for 5 days. The normal control group had free access to double distilled water for 12 days. Mice in the dosing group began gavage of the corresponding drug for each group on day 3,1 time per day for 7 days, while mice in the control and model groups were given the same volume of vehicle. After the completion of the 7 days of intragastric administration, the mice were dissected to take colon tissue of the same site. After extracting colon tissue protein, performing a protein immunoblotting experiment, and detecting the expression level of IL-23 protein in the tissue.

As a result: as shown in fig. 7, the expression of IL-23 was significantly increased in the DSS group compared to the normal control group of mice. The expression of IL-23 of the DMTHB administration group is obviously reduced, and the effect of inhibiting the secretion of IL-23 is better than that of BBR and DMB, which shows that the DMTHB can treat acute ulcerative colitis through the IL-23 which is a key target.

Claims

1. Application of demethylenetetrahydroberberine hydrochloride in preparing medicine for treating ulcerative colitis is provided, wherein the formula of demethylenetetrahydroberberine hydrochloride is shown as formula I

2. A pharmaceutical composition for treating ulcerative colitis, comprising a pharmaceutically effective amount of the demethylenetetrahydroberberine hydrochloride according to claim 1 and pharmaceutically acceptable excipients.

3. The pharmaceutical composition of claim 2, wherein the pharmaceutical composition is a tablet, a capsule, a pill, an injection, a sustained release formulation, and various microparticle delivery systems.