Application of betulinic acid 28-O-beta-D-glucoside in medicine

Abstract

The invention discloses application of betulinic acid 28-O-beta-D-glucoside in medicines, and particularly relates to application of betulinic acid 28-O-beta-D-glucoside in preparation of medicines for treating or preventing inflammatory bowel diseases. The invention discloses application of betulinic acid 28-O-beta-D-glucoside (BA-6) in prevention and treatment of inflammatory bowel diseases for the first time aiming at the problems that existing medicines for treating the inflammatory bowel diseases (IBD) are limited in curative effect, slow in effect taking, multiple in side effects, high in long-term use risk, large in individual difference, high in treatment cost, likely to cause immunoreactions and the like. The technical progress of obviously improving the acute colitis symptom is realized. Results of a cytotoxicity experiment and an acute toxicity experiment show that the betulinic acid 28-O-beta-D-glucoside (BA-6) does not have obvious biotoxicity on a cellular level or an animal level.

Description

Technical Field

[0001] The present invention belongs to the pharmaceutical field of natural product derivatives, and discloses the application of betulinic acid 28-O-β-D-glucoside in drugs, specifically relating to the application of a betulinic acid derivative - betulinic acid 28-O-β-D-glucoside (BA-6) in the preparation of drugs for treating or preventing inflammatory bowel disease. Background Art

[0002] Inflammatory bowel disease (IBD) is a chronic relapsing intestinal inflammatory disease, mainly represented by ulcerative colitis (UC) and Crohn's disease (CD). CD may affect the entire digestive tract, and UC mainly affects the colon and rectum. The severity of symptoms varies from person to person and may occur cyclically, seriously affecting physical health. Although the mortality rate of IBD is low, clinical symptoms such as abdominal pain, diarrhea, and bloody stools cannot be cured and are prone to recurrence. Compared with healthy people, the risk of colorectal cancer increases, seriously affecting the quality of life of patients. Some researchers have studied the pathogenesis of IBD through genome-wide association studies and found that it is caused by the interaction of genetic, environmental, intestinal barrier, and immune response factors.

[0003] Currently, the treatment of inflammatory bowel disease (IBD) mainly includes traditional drugs (aminosalicylates, glucocorticoids, immunomodulators, etc.), biological agents (anti-TNF-α monoclonal antibodies, anti-integrin monoclonal antibodies, anti-IL-12 / 23 monoclonal antibodies, etc.), and small molecule targeted drugs (JAK inhibitors, S1P receptor modulators, etc.). Although traditional drugs have their value in the treatment of IBD, they have many limitations, including limited efficacy, slow onset, many side effects, high long-term use risks, and large individual differences. These drawbacks have prompted clinicians and patients to seek more effective alternative treatment options, such as biological agents and small molecule targeted drugs. However, the high production costs of biological agents and small molecule targeted drugs have led to expensive treatment costs, bringing a greater economic burden to patients and the medical system. Moreover, biological agents are protein drugs, which may trigger immune responses, resulting in the production of anti-drug antibodies, reducing drug efficacy, and having large individual differences in efficacy. Some patients may have no response or poor efficacy to biological agents. Since the pathogenesis of IBD has not been clearly understood, current drug treatments cannot cure the disease and lack specific drugs. Therefore, clinical treatment pays more attention to controlling the development of the pathological condition and improving the quality of life of patients. However, the basic research on finding drugs for treating IBD also needs to change the thinking. It is necessary to not only reduce the recurrence rate of IBD and improve the survival prognosis, enhance the body's immunity to achieve better treatment effects, but also have a novel mechanism of action, so as not to easily cause drug resistance. Summary of the Invention

[0004] In view of the problems existing in the existing drugs for treating inflammatory bowel disease (IBD), such as limited efficacy, slow onset, many side effects, high long-term use risks, large individual differences, high treatment costs, and possible immune reactions, the present invention for the first time discloses the application of betulinic acid 28-O-β-D-glucoside (also known as betulinic acid 28-O-β-D-glucopyranoside, hereinafter referred to as BA-6) in the prevention and treatment of inflammatory bowel disease, with the technical progress of significantly improving the symptoms of acute colitis.

[0005] The present invention adopts the following technical solutions.

[0006] Use of betulinic acid 28-O-β-D-glucoside in the preparation of a drug for treating or preventing inflammatory bowel disease.

[0007] Use of betulinic acid 28-O-β-D-glucoside in the preparation of a drug for alleviating weight loss caused by inflammatory bowel disease.

[0008] Use of betulinic acid 28-O-β-D-glucoside in the preparation of a drug for alleviating colon shortening.

[0009] Use of betulinic acid 28-O-β-D-glucoside in the preparation of a drug for improving the disease activity index of inflammatory bowel disease.

[0010] Use of betulinic acid 28-O-β-D-glucoside in the preparation of a drug for improving bloody stools.

[0011] In the present invention, the structure of betulinic acid 28-O-β-D-glucoside is betulinic acid 28-O-β-D-glucopyranoside. The betulinic acid 28-O-β-D-glucoside disclosed by the present invention can not only treat or prevent inflammatory bowel disease, but also has low cytotoxicity.

[0012] In the present invention, low cytotoxicity means that the cytotoxicity of betulinic acid 28-O-β-D-glucoside to cells is lower than that of betulinic acid; specifically, the cytotoxicity of betulinic acid 28-O-β-D-glucoside on THP-1 cells and RAW264.7 cells is lower than that of betulinic acid. As an example, at a concentration of 50 μM, betulinic acid 28-O-β-D-glucoside (BA-6) is non-toxic at the cell (such as THP-1 cells and RAW264.7 cells) level, IC 50 = 261.5 μM, while betulinic acid (BA) has significant cytotoxicity.

[0013] In the present invention, colon shortening, weight loss, bloody stools, etc. are caused by inflammatory bowel disease.

[0014] In the present invention, inflammatory bowel disease includes enteritis, and further includes colitis, such as ulcerative colitis, Crohn's disease, indeterminate colitis, etc. Further, ulcerative colitis is acute or chronic ulcerative colitis.

[0015] In the present invention, the drug includes topical, oral, rectal or parenteral drugs. Preferably, the drug is a solution.

[0016] A drug for preventing and treating inflammatory bowel disease disclosed by the present invention has betulinic acid 28-O-β-D-glucoside as its active ingredient; it may also include conventional pharmaceutical excipients, including one or more of a diluent, a dispersant, a binder, a lubricant, and a penetration enhancer.

[0017] Specifically, conventional pharmaceutical excipients refer to one or more compatible solid or liquid fillers or gel substances that can be used medicinally, have sufficient purity and low toxicity, and can be blended with each other among the components of the pharmaceutical composition and with the active ingredient of the present invention without reducing the efficacy of the active ingredient. Some examples of pharmaceutically acceptable carriers are cellulose and its derivatives (such as sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), cyclodextrins (such as hydroxypropyl-β-cyclodextrin), emulsifiers (such as Tween), wetting agents (such as sodium dodecyl sulfate), coloring agents, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0018] In the present invention, the chemical structures of betulinic acid 28-O-β-D-glucoside (Compound BA-6) and betulinic acid (Compound BA) are as follows:

[0019] Betulinic acid (BA) is a naturally occurring lupane-type pentacyclic triterpenoid compound of plant origin. Since it was initially mainly isolated from the bark of birch trees, it is also called betulinic acid and is widely present in many other plants, such as Ziziphus mauritiana, Prunella vulgaris, Chaenomeles sinensis, Rosmarinus officinalis, Nerium oleander, Pulsatilla chinensis of the Ranunculaceae family, Syzygium buxifolium of the Myrtaceae family and other traditional Chinese medicines, all of which have relatively high contents. BA and its derivatives (synthesized by modifying at the C-3, C-20 and C-28 sites) have a variety of biological activities. In recent years, significant progress has been made in the research on the pharmacological activities of betulinic acid and its derivatives. For example, in 2015, the prior art published a review on the anti-tumor effects of betulinic acid derivatives and their structure-activity relationships. As a natural compound discovered in modern research, current researchers pay more attention to the anti-tumor, anti-viral, especially anti-HIV virus, antibacterial and other treatments of the derivatives synthesized by modification, and there are few breakthroughs in the anti-inflammatory mechanism. Betulinic acid 28-O-β-D-glucoside (BA-6) designed and synthesized in this invention is a glycoside derivative of BA, and its water solubility and bioavailability are significantly improved compared with BA. In vitro, the safety and anti-inflammatory activity of BA-6 are better than those of betulinic acid. In vivo, BA-6 can significantly improve the symptoms of acute colitis. In the DSS-induced ulcerative colitis model, the scores of various indexes in the BA-6 administration group are better than those in the betulin (B) group and the positive control drug mesalazine (5-ASA) group; while the therapeutic effect of betulinic acid (BA) is not significant and it cannot improve the shortening of the mouse colon. In the TNBS-induced acute Crohn's disease model, the scores of various indexes in the BA-6 administration group are slightly better than those in the positive control drug azathioprin group. In addition, it is also found that BA-6 has a potentially positive regulatory effect on the immune function of mice. The results of cytotoxicity experiments and acute toxicity experiments both show that betulinic acid 28-O-β-D-glucoside (BA-6) does not show obvious biological toxicity at both the cellular level and the animal level. Description of the Drawings

[0020] Figure 1It is the CCK-8 cytotoxicity experiment; a cell survival rate greater than 90% is generally considered non-toxic or low-toxic; A shows the cytotoxicity of betulinic acid 28-O-β-D-glucoside (BA-6) and betulinic acid (BA) at a concentration of 50 μM on THP-1 cells induced to differentiate into M1 macrophages; B shows the cytotoxicity of betulinic acid 28-O-β-D-glucoside (BA-6) and betulinic acid (BA) at a concentration of 50 μM on RAW264.7 cells; C shows the cytotoxicity of betulinic acid 28-O-β-D-glucoside (BA-6) at concentrations of 50, 100, 150, 200, 250, and 300 μM on THP-1 cells induced to differentiate into M1 macrophages; D shows the 50 IC 50 value, IC

[0021] Figure 2 It is a diagram of the method for establishing a model of acute ulcerative colitis in mice induced by DSS.

[0022] Figure 3 It is a statistical chart of the weight loss rate of acute ulcerative colitis mice induced by DSS with betulinic acid 28-O-β-D-glucoside; compared with the normal control group (Normal), ## P < 0.001, ### P < 0.001; compared with the model group (Model), *P < 0.05, **P < 0.01, ***P < 0.001; compared between the BA-6 (20 mg / kg) group and the betulin (B, 20 mg / kg) group, ^^ P < 0.01, ^^^ P < 0.001; compared between the BA-6 (20 mg / kg) group and the betulinic acid (BA, 20 mg / kg) group, $$ P < 0.01, $$$ P < 0.001; compared between the BA-6 (20 mg / kg) group and the mesalazine group (5-ASA, 300 mg / kg) group, & P < 0.05; n = 7.

[0023] Figure 4 It is a statistical chart of the disease activity index score (DAI) of acute ulcerative colitis mice induced by DSS with betulinic acid 28-O-β-D-glucoside; compared with the normal control group (Normal), ###P < 0.001; compared with the Model group, *P < 0.05, **P < 0.01, ***P < 0.001; compared with the Betulinol (B, 20 mg / kg) group, ^^^P < 0.001 for the BA-6 (20 mg / kg) group; compared with the Betulinic acid (BA, 20 mg / kg) group, $ P < 0.05, $$ P < 0.01, $$$ P < 0.001; compared with the Mesalazine (5-ASA, 300 mg / kg) group, for the BA-6 (20 mg / kg) group, && P < 0.01, &&& P < 0.001; n = 7.

[0024] Figure 5 It is a statistical chart of the degree of bloody stools in mice with DSS-induced acute ulcerative colitis by Betulinic acid 28-O-β-D-glucoside; compared with the Normal control group, ## P < 0.01, ### P < 0.001; compared with the Model group, *P < 0.05, **P < 0.01, ***P < 0.001; compared with the Betulinol (B, 20 mg / kg) group, ^^P < 0.01, ^^^P < 0.001 for the BA-6 (20 mg / kg) and BA-6 (10 mg / kg) groups; compared with the Betulinic acid (BA, 20 mg / kg) group, $$ P < 0.01, $$$ P < 0.001; compared with the Mesalazine (5-ASA, 300 mg / kg) group, for the BA-6 (10 mg / kg) and BA-6 (20 mg / kg) groups, & P < 0.05, && P < 0.01, &&& P < 0.001; n = 7.

[0025] Figure 6 It is a statistical chart of the measurement and statistics of the colon length in mice with DSS-induced acute ulcerative colitis by Betulinic acid 28-O-β-D-glucoside; A is a photo of the mouse colon measurement; B is the statistical result of the colon length; compared with the Normal control group, ###P < 0.001; compared with the Model group, *P < 0.05, ***P < 0.001, ns indicates no statistical difference; compared with the Betulinol (B, 20 mg / kg) group, ^^^P < 0.001 in the BA-6 (10 mg / kg) and BA-6 (20 mg / kg) groups; compared with the Betulinic acid (BA, 20 mg / kg) group, $$$ P < 0.001; compared with the Mesalazine group (5-ASA, 300 mg / kg), in the BA-6 (10 mg / kg) and BA-6 (20 mg / kg) groups, & P < 0.05; n = 7.

[0026] Figure 7 It is a statistical chart of the spleen index of mice with acute ulcerative colitis induced by DSS by Betulinic acid 28-O-β-D-glucoside; compared with the Normal control group, ### P < 0.001; compared with the Model group, *P < 0.05, **P < 0.01; compared with the Mesalazine group (5-ASA, 300 mg / kg), in the BA-6 (20 mg / kg) group, &&& P < 0.001; n = 7.

[0027] Figure 8 It is a diagram of the modeling method for mice with acute Crohn's disease induced by TNBS.

[0028] Figure 9 It is a statistical chart of the survival rate of mice with colitis induced by TNBS by Betulinic acid 28-O-β-D-glucoside; compared with the Normal control group, # P < 0.05; n ≥ 3.

[0029] Figure 10 It is a statistical chart of the weight loss rate of mice with colitis induced by TNBS by Betulinic acid 28-O-β-D-glucoside; compared with the Normal control group, ### P < 0.001; compared with the Model group, **P < 0.01, ***P < 0.001; compared with the Azathioprine group, & P < 0.05, && P < 0.01, &&& P < 0.001; n ≥ 3.

[0030] Figure 11Statistical chart of the effect of betulinic acid 28-O-β-D-glucoside on the disease activity index score (DAI) of TNBS-induced colitis in mice; compared with the normal control group (Normal), ### P<0.001; compared with the model group (Model), * P<0.05, *** P<0.001; compared with the azathioprine group (Azathioprin), & P<0.01; n≥3.

[0031] Figure 12 Statistical chart of the effect of betulinic acid 28-O-β-D-glucoside on the colon length of TNBS-induced mice; among them, A is the photo of mouse colon measurement; B is the statistical result of colon length; compared with the normal control group (Normal), ### P<0.001; compared with the model group (Model), ** P<0.01, *** P<0.001, compared with the azathioprine group (Azathioprin), & P<0.05; n≥3.

[0032] Figure 13 Statistical chart of the effect of betulinic acid 28-O-β-D-glucoside on the spleen index of TNBS-induced mice; compared with the normal control group (Normal), ### P<0.001; compared with the model group (Model), * P<0.05, ** P<0.01; n≥3.

[0033] Figure 14 Acute toxicity test of betulinic acid 28-O-β-D-glucoside; A is the method diagram of the acute toxicity test of betulinic acid 28-O-β-D-glucoside; B is the effect of betulinic acid 28-O-β-D-glucoside on the body weight of mice; C-G are the effects of betulinic acid 28-O-β-D-glucoside on the heart, liver, spleen, lung and kidney of mice respectively; H is the effect of betulinic acid 28-O-β-D-glucoside on the colon; ns means no statistical difference; n = 7. Specific implementation mode

[0034] In the present invention, the betulinic acid derivative is betulinic acid 28-O-β-D-glucoside. The betulinic acid 28-O-β-D-glucoside disclosed in the present invention can not only treat or prevent inflammatory bowel disease, but also has low cytotoxicity. In the present invention, the drugs with betulinic acid 28-O-β-D-glucoside as the active ingredient include topical, oral, rectal or parenteral drugs. Preferably, the drug is a solution.

[0035] To make the objectives and technical solutions of the present invention clearer and more understandable, the present invention will be further described in detail below with reference to the accompanying drawings. In the following examples, the experimental methods and data analysis methods are conventional methods unless otherwise specified; for those without specific techniques or conditions in the examples, they are carried out according to the techniques or conditions described in the literature in the field or according to the product instructions; the reagents and materials can be obtained from commercial sources unless otherwise specified. Animal experiments comply with the relevant requirements of Soochow University. As an example, weigh BA-6 and add 8 to 10 times the amount of hydroxypropyl- β -cyclodextrin, dissolve it in 10 times the amount of water of cyclodextrin, and grind for 5 to 6 hours to prepare a suspension for intragastric administration to animals.

[0036] Example 1: Toxicity experiments of betulinic acid 28-O-β-D-glucoside (BA-6) and betulinic acid (BA) on THP-1 cells and RAW264.7 cells Seed THP-1 cells into a 96-well plate at a density of 6×10 4 cells / well, add 100 ng / mL phorbol 12-myristate 13-acetate (PMA) to induce THP-1 cells for 12 h to induce the cells to differentiate from the M0 type to the M1 type of macrophages. After the cells adhere, add solutions containing different drugs to each group to treat the cells for 24 h. Add 10 μL of CCK-8 solution to each well, pay attention not to generate bubbles, gently shake well, and incubate in a cell culture incubator for 1-4 h. Then use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance (OD value) of this plate at a wavelength of 450 nm and record the data, and calculate the cell viability at different drug concentrations. Seed RAW264.7 cells into a 96-well plate at a density of 2×10 4 cells / well. After the cells adhere naturally, the subsequent operations are the same as above.

[0037] It was found that at a concentration of 50 μM, betulinic acid 28-O-β-D-glucoside (BA-6) was non-toxic at the cellular level, IC 50 =261.5 μM, while betulinic acid (BA) was cytotoxic.

[0038] Calculation formula: Cell viability = [(Cs - Cb) / (Cc - Cb)]×100%; Cs: Absorbance of the experimental well (including cell culture medium, CCK-8, and test compound); Cc: Absorbance of the control well (including cell culture medium, CCK-8, and no test compound); Cb: Absorbance of the blank well (cell-free and test compound-free culture medium, CCK-8).

[0039] Example 2: Method for establishing a model of DSS-induced acute ulcerative mice C57 mice were normally fed for one week, during which they had free access to food and water. After weighing, the mice were randomly divided into eight groups (n = 7): normal control group (Normal group), model group (Model group), BA-6 group (5 mg / kg), BA-6 group (10 mg / kg), BA-6 group (20 mg / kg), betulin group (B, 20 mg / kg), betulinic acid group (BA, 20 mg / kg), and mesalazine group (5-ASA, 300 mg / kg). On the day of modeling, the drinking water of the seven groups of mice except the normal group was changed to 3% DSS. The mice were modeled for 8 days and then treated on the 9th day. The following experiments were carried out.

[0040] Weigh BA-6, B, and BA and add 10 times the amount of hydroxypropyl- β -cyclodextrin. Dissolve with 10 times the amount of water of cyclodextrin and grind for 6 hours to prepare suspensions for gavage administration to animals. The normal control group and the model group were given the same dose of hydroxypropyl- β -cyclodextrin solution.

[0041] Example 3: Effect of betulinic acid 28-O-β-D-glucoside on the body weight of DSS-induced ulcerative mice The daily changes in the body weight of C57 mice in each group were statistically analyzed and plotted using graph prism 8.3.0. From the observation of the body weight loss rate [body weight loss rate = (the nth day - the 0th day) / the 0th day], it was found that the body weight of the mice in the normal control group was stable, while the body weight of the mice in the DSS model group began to decrease continuously from the 4th day of modeling and was significantly different from that of the normal control group from the 5th day of modeling (P<0.01). Except for the low-dose BA-6 (5 mg / kg) group, all the drug treatment groups were significantly different from the model group from the 5th day (P<0.01), and the low-dose BA-6 (5 mg / kg) group was significantly different from the model group from the 6th day (P<0.05). Compared with the betulin (B, 20 mg / kg) group, betulinic acid (BA, 20 mg / kg) group, and the positive drug mesalazine (300 mg / kg) group, the body weight of the BA-6 (20 mg / kg) group decreased more slowly; on the 7th and 8th days of modeling, there were significant differences in the body weight loss rate between the BA-6 (20 mg / kg) group and the B (20 mg / kg) group and the BA (20 mg / kg) group (P<0.01); on the 7th day of modeling, there was a significant difference in the body weight loss rate between the BA-6 (20 mg / kg) group and the positive drug mesalazine (300 mg / kg) group (P<0.05). The results showed that betulinic acid 28-O-β-D-glucoside could relieve the body weight loss of DSS-induced acute ulcerative mice, and its curative effect was better than that of betulin, betulinic acid, and the positive drug mesalazine.

[0042] Example 4: Effect of betulinic acid 28-O-β-D-glucoside on the disease activity index score of DSS-induced ulcerative colitis mice The disease activity index (DAI) is a comprehensive score obtained by adding the scores of the mouse weight loss rate, fecal characteristics, and fecal bleeding. The higher the DAI score, the more severe the inflammatory bowel disease of the mouse. Collect the feces of each mouse at the same time every day, observe the fecal characteristics and blood in the feces, and count the scores. (1) Weight loss rate: Weigh the mice at 10:00 am every day, and calculate the weight loss rate: Weight loss rate = (weight on the nth day - weight on the 0th day) / weight on the 0th day * 100%; (2) Fecal characteristics: Take the feces of the mice every day and observe the shape. Formed and having a certain hardness is normal feces, soft and loose is loose feces, and unformed is watery feces; (3) Blood in feces condition: If blood can be observed in the feces of the mice taken every day, it is bloody feces. If it cannot be observed with the naked eye, take the feces of the mice on a glass slide, first add 2% o-tolidine acetic acid solution, and then add 3% hydrogen peroxide solution, and observe the color change of the feces. If it turns blue-green within a certain time, the feces have occult blood, and if the color does not change significantly, it is normal feces.

[0043] From the 4th day of modeling, the DSS-modeled mice all showed loose stools and bloody or occult blood to a certain extent, with a significant difference compared with the normal control group (Normal group) mice (P < 0.001). The pathological course of the BA-6 treatment group developed more slowly than that of the Model group. Compared with the Model group, there was a significant difference starting from the 4th day (P < 0.05). The betulin, betulinic acid, and positive drug mesalazine treatment groups showed significant differences starting from the 6th day compared with the Model group (P < 0.05), but the improvement degree was not as obvious as that of the BA-6 treatment group. From the 6th day to the 8th day of modeling, the DAI scores of the BA-6 (20 mg / kg) group were always lower than those of the betulin (B, 20 mg / kg) group, betulinic acid (BA, 20 mg / kg) group, and positive drug mesalazine (300 mg / kg) group mice, with a significant difference (P < 0.01). Therefore, betulinic acid 28-O-β-D-glucoside can reduce the DAI score of DSS mice, significantly improve the disease development of acute ulcerative mice, and the curative effect is better than that of betulin, betulinic acid, and positive drug mesalazine.

[0044] DAI score table

[0045] Example 5: Effect of betulinic acid 28-O-β-D-glucoside on the degree of bloody stools in DSS-induced ulcerative colitis mice From the 4th day of model establishment, the DSS-induced model mice all showed hematochezia or occult blood to a certain extent, showing a significant difference compared with the normal control group (Normal group) mice (P<0.001). Compared with the model group (Model), the low, medium, and high treatment dose groups of betulinic acid 28-O-β-D-glucoside (BA-6) could significantly improve the degree of hematochezia from the 5th day and showed significant differences (P<0.05). Betulin (B), betulinic acid (BA), and the positive drug mesalazine only showed significant differences on the last day of model establishment (p<0.05). Compared with the betulin (B, 20 mg / kg) group and the positive drug mesalazine (5-ASA, 300 mg / kg) group, the medium and high dose groups of betulinic acid 28-O-β-D-glucoside (BA-6) were found to more significantly improve the degree of hematochezia from the 6th day and showed significant differences (P<0.05); compared with the betulinic acid (BA, 20 mg / kg) group, the betulinic acid 28-O-β-D-glucoside (BA-6, 20 mg / kg) group was found to significantly improve the degree of hematochezia from the 7th day and showed significant differences (P<0.001). In summary, betulinic acid 28-O-β-D-glucoside can significantly improve the development of hematochezia in ulcerative mice, and its curative effect is better than that of betulin, betulinic acid, and the positive drug mesalazine.

[0046] Example 6: Effect of betulinic acid 28-O-β-D-glucoside on the colon length of DSS-induced ulcerative colitis mice DSS-induced ulcerative colitis can significantly shorten the colon of mice, accompanied by edema and congestion. On the 9th day after model establishment, each group of mice was sacrificed, and the colon tissues of the mice were taken to measure the colon length of each mouse. The mice in the model group showed colon shortening accompanied by bleeding and edema. Compared with the normal control group mice, the colon length of the model group mice was shortened, showing a highly significant difference (P<0.001). Except for betulinic acid (BA, 20 mg / kg), the administration groups could relieve the colon shortening of mice (P<0.05). Compared with the colon lengths of the mice in the betulin (B, 20 mg / kg) group, betulinic acid (BA, 20 mg / kg) group, and positive drug mesalazine (300 mg / kg) group, the administration groups of medium and high doses of betulinic acid 28-O-β-D-glucoside (BA-6) showed more obvious relief of colon shortening, showing a significant difference (P<0.05). The above results indicate that BA-6 has the pharmacological effect of relieving ulcerative colitis, and the curative effect of BA-6 is significantly better than that of betulin, betulinic acid, and the positive drug mesalazine.

[0047] Example 7: Effect of betulinic acid 28-O-β-D-glucoside on the spleen index of DSS-induced mice Under DSS induction, the spleen becomes enlarged due to inflammation, resulting in an increase in spleen weight, which is significantly different from that of the normal control group (P<0.001). After administration of BA-6 and betulinic acid, there is a significant effect on reducing spleen weight (P<0.05). However, compared with betulinic acid, the improvement degree of BA-6 is more obvious. In the positive drug mesalazine (300 mg / kg) group, there is an abnormal increase in the spleen. Compared with the mice in the positive drug mesalazine (300 mg / kg) group, BA-6 has a significant (P<0.001) effect on reducing spleen weight, suggesting that the drug has a positive regulatory effect on the pathological state or immune function of the spleen.

[0048] Example 8: Method for Establishing a Mouse Model of Acute Crohn's Disease Induced by TNBS Male BABL / c mice aged 6-8 weeks were normally fed in an SPF breeding room for one week, during which they had free access to food and water. After weighing, the mice were randomly divided into four groups: normal control group, model group (Model group), BA-6 group (20 mg / kg), and azathioprine group (18 mg / kg). From day 0 to day 7, the BA-6 group (20 mg / kg) and the azathioprine group (18 mg / kg) were given intragastric administration daily, and the normal group and the Model group were given a blank solvent suspension. On day 1, the mice were anesthetized with 1.25% avertin and placed on a heating blanket. Except for the normal group that was enema with 100 μL of normal saline, the other three groups were enema with 2.5% TNBS (100 μL / mouse), and then left standing upside down for 1 min. The mental state, activity, diet, body weight changes, stool characteristics, and blood in the stool of each group of mice were observed and recorded daily. All mice were sacrificed by cervical dislocation on day 8. The following experiments were carried out.

[0049] Example 9: Effect of Betulinic Acid 28-O-β-D-Glucoside on the Survival Rate of Mice with Crohn's Disease Induced by TNBS The number of surviving BABL / c mice in each group was statistically analyzed every day and plotted using graph prism 8.3.0. Seven days after the induction of the model with 2.5% TNBS, there was a significant difference compared with the TNBS model group (P<0.05). The mortality rates of the Model group, BA-6 treatment group, and azathioprine treatment group were 58.3%, 40%, and 50% respectively. The results showed that BA-6 had a protective effect and could inhibit the death of mice with acute Crohn's enteritis induced by 2.5% TNBS, and the protective effect was better than that of the positive drug azathioprine group.

[0050] Example 10: Effect of Betulinic Acid 28-O-β-D-Glucoside on the Body Weight of Mice with Crohn's Disease Induced by TNBS The body weight changes of each group of BABL / c mice were statistically analyzed every day and plotted using GraphPad Prism 8.3.0. From day 0 to day 1, due to the requirement of fasting in the experiment, the overall weight loss was a normal phenomenon. Observed from the weight loss rate [weight loss rate = (weight on day n - weight on day 0) / weight on day 0], it was found that the body weights of the mice in the normal control group increased steadily after day 1, while the body weights of the mice in the TNBS model group continued to decline from day 1 of modeling. The body weight recovered slightly on day 5 of modeling. There was a significant difference compared with the normal control group on day 2 of modeling (P < 0.01). The BA-6 administration group could significantly slow down the weight loss of mice from day 3 to day 8, and there was a significant difference compared with the TNBS model group (P < 0.01). The body weights of the mice in the positive control drug azathioprine group began to recover on days 3 and 4 of modeling, but there was no difference in improvement compared with the model group on day 5; compared with the positive control drug azathioprine group, there was a significant difference in the BA-6 administration group from day 2 to day 8 (P < 0.05). The results showed that BA-6 could relieve the weight loss of mice with TNBS-induced Crohn's disease, and its curative effect was even better than that of the positive drug azathioprine group.

[0051] Example 11: Effect of betulinic acid 28-O-β-D-glucoside on the disease activity index score (DAI) of colitis in mice with TNBS-induced Crohn's disease From day 2 of modeling, loose stools, bloody stools or occult blood occurred to varying degrees in each group of mice. From day 2 to day 5 of modeling, there was a significant difference between the mice in the model group (Model group) and the control group (Normal group) (P < 0.05); however, compared with the Model group, the disease condition of the BA-6 treatment group was relatively mild and the DAI score was always lower than that of the mice in the model group, with a significant difference (P < 0.05). The DAI scores of the mice in the positive drug azathioprine group were always lower than those of the mice in the model group from day 2 to day 4, and there was a significant difference (P < 0.05), but they were always higher than the DAI scores of the BA-6 group. Compared with the BA-6 administration group, there was a significant difference on day 2 of modeling (P < 0.05). Therefore, betulinic acid 28-O-β-D-glucoside (BA-6) can reduce the DAI score of TNBS mice and significantly improve the condition of mice with acute Crohn's disease, and its curative effect is even better than that of the positive drug azathioprine.

[0052] Example 12: Effect of betulinic acid 28-O-β-D-glucoside on the colon length of mice induced by TNBS TNBS-induced acute colitis can significantly shorten the colon of mice, accompanied by edema and congestion. On the 8th day after modeling, mice in each group were sacrificed, and the colon tissues of the mice were taken to measure the colon length of each mouse. In the model group, the colon of the mice was shortened and accompanied by local bleeding and edema. Compared with the blank control group, the colon length of the mice in the model group was shortened, and there was a highly significant difference (P<0.001). After administration of betulinic acid 28-O-β-D-glucoside (BA-6), the shortening of the colon in mice could be alleviated (P<0.05). Compared with the colon length of the mice in the azathioprine group, there was a significant difference in the colon length of the mice in the BA-6 administration group (P<0.05). The above results preliminarily demonstrated that BA-6 has the efficacy of alleviating the shortening of the colon in mice with acute Crohn's disease, and the curative effect is even better than that of the positive drug azathioprine.

[0053] Example 13: Effect of betulinic acid 28-O-β-D-glucoside on the spleen index of TNBS-induced mice Under TNBS induction, the spleen becomes enlarged with the occurrence of inflammation, resulting in an increase in spleen weight, which is significantly different from that of the normal control group (P<0.001). After administration of BA-6 and azathioprine, it has a significant effect on reducing spleen weight (P<0.05), suggesting that the drug has a positive regulatory effect on the pathological state or immune function of the spleen.

[0054] Example 14: Acute toxicity test of betulinic acid 28-O-β-D-glucoside Male BABL / c mice aged 6 - 8 weeks were normally fed for one week, during which they had free access to food and water. After weighing, the mice were randomly divided into two groups: the normal control group and the BA-6 group (200 mg / kg). From day 0 to day 7, the BA-6 group (200 mg / kg) was given intragastric administration daily. It was observed daily that there were no abnormal behaviors in the BA-6 group, the activity level was normal, the diet was normal, there was no listlessness or solitary residence, the hair was smooth and shiny, there was no abnormal excretion, the fecal form was normal, soft and moderate, without blood in the stool, and there was no poisoning or death. The weight changes of each group were recorded daily, and it was found that the weight increased day by day, showing no difference from the normal control group; all the mice were sacrificed by cervical dislocation on the 8th day. The heart, liver, spleen, lungs, and kidneys of each mouse in each group were weighed, and it was found that there was no statistical difference in the weight gain trend between the BA-6 group (200 mg / kg) and the normal control group; and the colon length of each mouse was measured, and it was found that there was also no statistical difference between the BA-6 group (200 mg / kg) and the normal control group.

[0055] Example 15 Preparation method of betulinic acid-28-O-β-D-glucoside (BA-6) The preparation method of betulinic acid-28-O-β-D-glucoside (BA-6) is as follows: Using α-D-glucopyranose as the raw material, the fully acetylated pyran bromosugar is prepared by a one-pot method, that is, the unprotected pyranose and acetic anhydride are used to prepare the fully acetylated pyranose under the catalysis of iodine, and then it directly reacts with a 33% hydrobromic acid glacial acetic acid solution without separation to obtain the fully acetylated pyran bromosugar; the bromosugar donor reacts with betulinic acid under the phase transfer catalytic conditions (K2CO3, n-Bu4NBr, CH2Cl2 / H2O, rt) to obtain betulinic acid-28-O-β-D-2, 3, 4, 6-tetra-O-acetyl-pyran glucoside, and then the acetyl group is removed by sodium hydroxide in a tetrahydrofuran / water solution to obtain betulinic acid-28-O-β-D-glucoside (BA-6, 28-BA-β-G). This preparation method adopts the integrated innovation of one-pot bromosugar donor synthesis-phase transfer catalytic coupling-mild deprotection, with simple steps, mild conditions, strong operability and controllability, low cost, and has the prospect of industrial application.

[0056] The specific reaction route is as follows:

[0057] Those skilled in the art can obtain the product of the present invention (betulinic acid-28-O-β-D-glucoside) according to the raw materials and reaction conditions of the present invention, or can also adopt other methods that can obtain the product of the present invention.

[0058] The corresponding Chinese for the abbreviations are as follows: Glc: D-glucopyranose; Ac2O: acetic anhydride; I2: iodine; 33% HBr / AcOH: 33% hydrobromic acid / glacial acetic acid solution; CH2Cl2: dichloromethane; K2CO3: potassium carbonate; n-Bu4NBr; tetrabutylammonium bromide; THF: tetrahydrofuran; NaOH: sodium hydroxide; H2O: water.

[0059] The specific preparation process of betulinic acid-28-O-β-D-glucoside is as follows: Add 125 mg of iodine to a 25 mL acetic anhydride solution of 5.00 g of D-glucopyranose, and stir magnetically at room temperature until the reaction system becomes brown and transparent. After the reaction is completed, add 125 mL of dry dichloromethane to dilute the reaction mixture. Add 30 mL of an acetic acid solution of 33% hydrogen bromide under ice bath cooling. After addition, stir the reaction at room temperature until the reaction is completed as detected by TLC. Dilute the reaction mixture with 125 mL of dichloromethane, and wash it successively with ice water, saturated sodium bicarbonate solution, and saturated sodium thiosulfate solution. Dry the organic layer with anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure to obtain a colorless oil. Recrystallize it with ether and petroleum ether to obtain a white solid powder with a yield of 95%. Immediately dissolve BA (3 g, 6.569 mmol) and G3 (3.5 g, 8.539 mmol) in a mixed solution of CH2Cl2 / H2O (76 ml / 76 ml). Add n-Bu4NBr (847 mg, 2.628 mmol) and K2CO3 (2.2 g, 16.421 mmol) to it successively, and stir the reaction at room temperature for 6 h. Then add 100 ml of dichloromethane to dilute it, and wash the organic phase with saturated brine (100 mL×3). Dry the organic layer with anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure. Subject the residue to silica gel column chromatography (petroleum ether:ethyl acetate = 4:1 → 3:1) to obtain 4.3 g of G4, which is a white solid with a yield of 83%. Dissolve G4 (4 g, 5.083 mmol) in a mixed solution of THF / H2O (100 ml / 10 ml), add sodium hydroxide (1.2 g, 30.498 mmol) to it, and stir the reaction at room temperature for 12 h. Then add 1M HCl to adjust the pH to 4, and then add water to precipitate the product. Filter and dry it. Dissolve the residue in methanol and subject it to C18 preparative separation (90% methanol-water) to obtain 2.5 g of betulinic acid-28-O-β-D-glucoside (28-BA-β-G), which is a white solid with a yield of 79%. The analysis is as follows: 11H NMR (400 MHz, CD3OD) δ 5.49 (1H, d, J = 8.2 Hz, H-1'), 4.72 (brs, H1-29), 4.60 (brs, H2-29), 3.84 (1H, dd, J = 12.0, 1.6 Hz), 3.73 – 3.68 (1H, m), 3.45 – 3.39 (1H, m), 3.39 – 3.36 (2H, m), 3.35 (2H, m), 3.12 (1H, dd, J = 11.1, 5.0 Hz), 3.01 (1H, m), 2.33 (2H, m), 2.02 – 1.88 (2H, m), 1.75 – 1.71 (1H, m), 1.70 (3H, s), 1.69 – 1.19 (17H, m), 1.16 (1H, m), 1.05 (1H, m), 1.00 (3H, s), 0.96 (3H, s), 0.94 (3H, s), 0.86 (3H, s), 0.75 (3H, s), 0.71 (1H, m). 13 13C NMR (101 MHz, (CD3OD) δ 176.1(C-28), 151.8(C-20), 110.3(C-29), 95.2(C-1'), 79.7(C-3), 78.8(C-5'), 78.4(C-3'), 74.1(C-2'), 71.1(C-4'), 62.4(C-6'), 57.9, 56.9, 52.0, 50.6, 49.9, 43.5, 42.0, 40.1, 39.9, 39.4, 38.3, 37.5, 35.5, 32.8, 31.4, 30.8, 28.6, 28.0, 26.8, 22.1, 19.5, 19.4, 16.7, 16.6, 16.1, 15.1. HR-MS m / z calcd for C 36 H 58 O8K [M + K] + 657.3763, found 657.3738. HPLC purity: 99.12%.

[0060] The safety of betulinic acid derivative - betulinic acid 28 - O - β - D - glucoside (BA - 6) is higher than that of betulinic acid. The results of animal experiments show that compared with the normal group, the scores and indices of various clinical symptom indicators of the mice in the model group show a significant upward trend. In terms of pathological manifestations, the colon length of the mice in the model group is significantly shortened, accompanied by significant congestion, and the spleen also shows obvious swelling, which is in line with the symptoms of DSS - induced acute ulcerative mice. Compared with the model group, the treatment with betulinic acid 28 - O - β - D - glucoside can significantly improve the symptoms of acute ulcerative mice. The scores of various indicators in the betulinic acid 28 - O - β - D - glucoside administration group are even better than those in the betulinol (B) group, betulinic acid (BA) group, and the positive drug mesalazine (5 - ASA). Compared with the normal group, the scores and indices of various clinical symptom indicators of the mice in the model group are significantly increased, the colon is significantly shortened and congested, the spleen is significantly swollen, and the inflammatory infiltration is more serious, indicating that TNBS can effectively induce acute colitis in mice similar to Crohn's symptoms, and its various indicators conform to the symptoms and development rules of acute colitis; compared with the model group, the betulinic acid 28 - O - β - D - glucoside (BA - 6) in the administration group can significantly improve the symptoms of acute Crohn's disease mice; the scores of various indicators in the BA - 6 administration group are slightly better than those in the positive control drug azathioprine group. The betulinic acid derivative - betulinic acid 28 - O - β - D - glucoside (BA - 6) has no damaging effect on the body weight and the structure of various organs of mice. While effectively relieving TNBS - induced acute Crohn's disease and DSS - induced acute ulcerative colitis, it can also improve the systemic immune function, showing good safety. Betulinic acid 28 - O - β - D - glucoside (BA - 6) does not show obvious biological toxicity at both the cellular level and the animal level. Therefore, the drug betulinic acid 28 - O - β - D - glucoside provided by the present invention is a drug with the potential to treat IBD both in terms of efficacy and safety.

[0061] The present invention is illustrated by specific examples, but these examples are only used to show the preferred technical solutions of the present invention and do not limit the protection scope of the present invention. Those skilled in the art can, under the guidance of the core idea of the present invention, make appropriate optimization, adjustment, and improvement of the technical solutions of the present invention according to specific requirements and technical backgrounds, or make equivalent replacements for some technologies. These adjustments, improvements, and equivalent replacements all fall within the protection scope of the present invention.

Claims

1. Use of betulinic acid 28-O-β-D-glucoside in the preparation of a medicament for treating or preventing inflammatory bowel disease.

2. Use of betulinic acid 28-O-β-D-glucoside in the preparation of a medicament for alleviating weight loss caused by inflammatory bowel disease.

3. Use of betulinic acid 28-O-β-D-glucoside in the preparation of a medicament for alleviating colon shortening.

4. Use of betulinic acid 28-O-β-D-glucoside in the preparation of a medicament for improving the disease activity index of inflammatory bowel disease.

5. Use of betulinic acid 28-O-β-D-glucoside in the preparation of a medicament for improving bloody stool.

6. The application according to any one of claims 1 to 5, characterized in that Inflammatory bowel disease includes enteritis.

7. The application according to claim 6, wherein Inflammatory bowel disease includes one or more of ulcerative colitis, Crohn's disease, and indeterminate colitis.

8. The application according to any one of claims 1 to 5, characterized in that, The medicament includes topical, oral, rectal, or parenteral medicaments.

9. A medicament for preventing and treating inflammatory bowel disease, the active ingredient of which is betulinic acid 28-O-β-D-glucoside.

10. The drug for preventing and treating inflammatory bowel disease according to claim 9, characterized in that, It also includes pharmaceutically acceptable excipients.

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