Rhizoma menispermi extract effective part, preparation method thereof and application of rhizoma menispermi extract effective part in preparation of medicine for preventing or treating inflammatory bowel disease

Abstract

The invention belongs to the technical field of natural medicinal chemistry, and particularly relates to a rhizoma menispermi extract effective part, a preparation method thereof and application of the rhizoma menispermi extract effective part in preparation of drugs for preventing or treating inflammatory bowel diseases (IBD). The effective part of the rhizoma menispermi extract is mainly a mixture of dauricine (DAU) and dauricine methyl ester (DAU-Me), and the weight percentage of the effective part is 75%-95%. The rhizoma menispermi extract effective part provided by the invention can obviously improve weight loss, colon shortening, intestinal barrier and intestinal inflammation of IBD model mice, has a prospect of being developed into a medicine for preventing and treating the inflammatory bowel disease, provides more treatment choices for clinically preventing or treating the inflammatory bowel disease, and has important social benefits and economic values.

Description

Technical Field

[0001] The present invention relates to the field of natural drug chemistry, and in particular to an effective part of a dry root extract of a medicinal plant Menispermum dauricum DC., a preparation method thereof, and an application of the effective part in preparing a drug for preventing or treating inflammatory bowel disease. Background Art

[0002] Inflammatory bowel disease (IBD) is a chronic nonspecific intestinal inflammatory disease that affects the ileum, rectum, and colon mucosa. It has a recurrent course and is clinically manifested by abdominal pain, diarrhea, bloody stools with mucus, and tenesmus. Its pathological features are chronic inflammation of the colonic mucosa, intestinal mucosal ulcers, or transmural granuloma formation. IBD is known as "green cancer" and is difficult to cure. It can lead to a serious deterioration in the patient's quality of life. Patients usually need lifelong medication or even surgery. IBD includes Crohn's disease (CD) and ulcerative colitis (UC).

[0003] The pathogenesis of IBD is unclear, and existing treatment options are generally ineffective. The main goal of treating IBD patients is to keep them in a state of long-term clinical symptom relief and microscopic healing of the intestinal mucosa. Traditional drug treatments include salicylic acid preparations, glucocorticoids, and immunosuppressants, most of which work by inhibiting the body's immune response to control inflammation. Although the effects of these drugs are immediate, their side effects are relatively large. Therefore, these drugs are usually not suitable for long-term use, and patients are prone to relapse immediately after stopping the drug.

[0004] In recent years, the relationship between IBD and intestinal microorganisms has received extensive attention, and intestinal microorganisms have the potential to regulate IBD. The intestinal microecology mainly includes intestinal bacteria, fungi, viruses and metabolites, among which the research on intestinal bacteria is relatively in-depth. Recent studies have confirmed that the human intestinal microecology is closely related to the occurrence and development of inflammatory bowel disease. Intestinal bacteria can induce or aggravate IBD by producing inflammatory polysaccharides or destroying the intestinal barrier. There are also some microorganisms that can produce anti-inflammatory substances to improve IBD. However, traditional microbial preparations have poor colonization ability in the host and limited effects. Recent studies have found that bacterial DPP4 can simulate the function of host DPP4 and degrade active polypeptides, thereby affecting the host's natural immune system and systemic metabolic homeostasis, and affecting the occurrence and development of inflammatory diseases. Therefore, taking bacterial DPP4 as a research entry point and developing corresponding microecological preparations and drugs can provide new paths and more effective measures for the treatment and prevention of inflammatory bowel disease.

[0005] The dried rhizome of Menispermum dauricum DC. is called the root of Menispermum dauricum. The root of Menispermum dauricum DC. is bitter, cold and slightly toxic. It enters the lung, stomach and large intestine meridians. It has the effects of clearing away heat and detoxifying, promoting dampness and relieving swelling. It is mainly used to treat acute pharyngitis, tonsillitis, swollen and painful gums, lung heat cough, damp heat, jaundice, constipation and other symptoms. Currently, no research has shown that the extract or structural analogue of the root of Menispermum dauricum can relieve or treat inflammatory bowel disease. Summary of the invention

[0006] In order to solve the problem of insufficient or poor efficacy of drugs for treating IBD in clinic, the present invention provides an effective part of the extract of the root of northern bean and its preparation method and its application in preparing drugs for preventing or treating inflammatory bowel disease (IBD). The present invention provides a new idea for preventing or treating inflammatory bowel disease in clinic and modernizing traditional Chinese medicine.

[0007] The technical solution for achieving the purpose of the present invention is as follows:

[0008] In a first aspect, the present invention provides a method for preparing an effective fraction of a Radix Sophorae Fructus extract having the effect of preventing or treating inflammatory bowel disease, comprising the following steps:

[0009] (1) Take the clean rhizome of the root of the northern bean, dry it thoroughly and then extract it;

[0010] (2) preparing an ethanol:methanol:water extract in a volume ratio of (0.5-3):(0.5-2):(1-2), adjusting the temperature to 10-100° C., mixing the extract with the northern bean root in (1) and shaking for 1-12 hours;

[0011] (3) After the extraction in step (2) is completed, solid-liquid separation is performed to obtain a crude extract, and rotary evaporation is performed at 30 to 60° C. for 1 to 6 hours to allow the organic solvent to fully evaporate to obtain a concentrated solution. At this time, the extract still contains a large amount of water;

[0012] (4) vacuum freeze-drying the concentrated solution obtained in step (3) at -1.0 MPa to -0.10 MPa for 1 to 18 hours to obtain a dry light yellow to black powder;

[0013] (5) dissolving the powder obtained in step (4) in ethanol, filtering to obtain a clear solution, concentrating the solution, and evaporating to dryness to obtain a crude extract of the effective part of the target Radix Sophorae Flavescentis extract;

[0014] (6) using high performance liquid chromatography to track the activity of the crude extract of the effective part in step (5), and the effective component obtained by eluting for 6 to 7 minutes has the best effect, and the effective part of the northern bean root extract is obtained after concentration;

[0015] (7) Liquid chromatography-tandem mass spectrometry was performed to analyze that the effective part of the northern bean root extract was mainly a mixture of daurine (DAU) and daurine methyl ester (DAU-Me), accounting for 75%-95% by mass percentage.

[0016] As an optional manner, in the above method for preparing the effective fraction of the Radix Sophorae Flavescentis extract, the Radix Sophorae Flavescentis should be chopped into small pieces of 2 mm in step (1).

[0017] As an optional manner, in the above-mentioned method for preparing the effective part of the northern bean root extract, in step (2), the volume ratio of ethanol:methanol:water is 3:1:2, the solid-liquid ratio is 1 g:6-10 mL, the extraction temperature is 50-60°C, and the oscillation extraction time is 4-5 hours.

[0018] As an optional manner, in the above method for preparing the effective fraction of the northern bean root extract, in step (3), rotary evaporation is carried out at 50° C. for 4 to 6 hours.

[0019] As an optional manner, in the above-mentioned method for preparing the effective fraction of the northern bean root extract, freeze-drying is carried out at -0.70 MPa for 12 to 18 hours in step (4).

[0020] As an optional manner, in the above method for preparing the effective fraction of the northern bean root extract, a 0.22 μm filter membrane is used for filtration in step (5).

[0021] As an optional manner, in the above-mentioned method for preparing the effective part of the northern bean root extract, the chromatographic column used in step (6) is: a 5μm particle size C18 chromatographic column, 4.6×250mm; the mobile phase is: in volume percentage, A: 80% water + 20% acetonitrile, B: 80% acetonitrile + 20% isopropanol, and the liquid phase conditions are: 0-4min, 30% B; 4-10min, 30% B-90% B; 10-12min, 90% B; 12-13min, 90% B-30% B, 13-15min, 30% B.

[0022] As an optional manner, in the above-mentioned method for preparing the effective part of the northern bean root extract, the northern bean root is selected from the dried rhizome of the Menispermum dauricum DC. of the Menispermaceae family, and the collection time is limited to spring and autumn, and the origin is one or more regions in Northeast China, North China, East China, Shaanxi, Ningxia, Gansu, and Shandong.

[0023] In the second aspect, the present invention provides an effective part of a root extract of Sophora flavescens having the effect of preventing or treating inflammatory bowel disease, which is prepared by the preparation method described in the first aspect. The effective part of the root extract of Sophora flavescens is mainly a mixture of Dauricine (DAU) and Dauricine methyl ester (DAU-Me), accounting for 75%-95% by mass.

[0024] In a third aspect, the present invention provides use of the effective fraction of the Rhizoma Coptidis Root extract described in the second aspect in the preparation of a dipeptidyl peptidase-4 (DPP4) inhibitor.

[0025] In a fourth aspect, the present invention provides use of the effective fraction of the Rhizoma Coptidis extract described in the second aspect in the preparation of a drug for preventing or treating inflammatory bowel disease.

[0026] As an option, in the above application, the inflammatory bowel disease is ulcerative colitis or Crohn's disease.

[0027] As an optional mode, in the above application, the dosage form of the drug is selected from granules, capsules, tablets, powders, oral solutions, suspensions or emulsions.

[0028] As an optional mode, in the above application, the dosage form of the drug is enteric-coated tablets.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] The effective fraction of the northern bean root extract provided by the present invention significantly improves weight loss, colon shortening, intestinal barrier and intestinal inflammation in IBD model mice, has the prospect of being developed into a drug for preventing and treating inflammatory bowel disease, provides more treatment options for clinical prevention or treatment of inflammatory bowel disease, and has important social benefits and economic value. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 :Effects of different components of Radix Sophorae Fructus on body weight and disease activity index of DSS model mice. The figures are shown as mean ± SD. One-way ANOVA was used to evaluate the significance of each group. * Compared with the model group, P < 0.05. ** Compared with the model group, P < 0.01. Component 7 had the most significant improvement effect.

[0032] Figure 2 :Effects of the effective fraction of the extract of Sophora flavescens root (component 7) and the positive drug 5-ASA on the body weight of DSS model mice. The figures are shown as mean ± SD. The significance of each group was evaluated by one-way analysis of variance. * Compared with the model group, P < 0.05. **Compared with the model group, P < 0.01. Component 7 has a better improvement on DSS-induced ulcerative colitis than the positive drug 5-ASA.

[0033] Figure 3 :Effects of the effective fraction of the extract of the root of Sophora flavescens (component 7) and the positive drug 5-ASA on the disease activity index of DSS model mice. The figures are shown as mean ± SD. One-way ANOVA was used to evaluate the significance of each group. * Compared with the model group, P < 0.05. ** Compared with the model group, P < 0.01. Component 7 has a better improvement on DSS-induced ulcerative colitis than the positive drug 5-ASA.

[0034] Figure 4 : In vivo imaging of the effective fraction of Sophora flavescens root extract (component 7) and the positive drug 5-ASA in improving colitis in DSS mice.

[0035] Figure 5 : Representative pathological sections of the effective fraction of Sophora flavescens root extract (component 7) and the positive drug 5-ASA in improving colitis in DSS mice, upper figure: 10×; lower figure: 400×.

[0036] Figure 6 :The effective part of the extract of the root of Sophora flavescens (component 7) and the positive drug 5-ASA improve the colon length and intestinal barrier of DSS mice. Left: colon length statistics; right: intestinal permeability test. The pictures are shown as mean ± SD. The significance of each group was evaluated using one-way analysis of variance. * Compared with the model group, P < 0.05. ** Compared with the model group, P < 0.01. Component 7 has a better improvement on DSS-induced ulcerative colitis than the positive drug 5-ASA. DETAILED DESCRIPTION

[0037] After extensive and in-depth research and experiments, the inventors found that a specific effective part of the root extract of Sophora flavescens can inhibit the activity of bacterial DPP4, thereby having the effect of preventing and treating inflammatory bowel disease. The effective part of the root extract of Sophora flavescens was fed to experimental subjects and found that it can improve the weight loss, shortened colon length, disease activity score and intestinal barrier damage of the DSS-induced inflammatory bowel disease mouse model. On this basis, the present invention was completed.

[0038] As used herein, the term "comprising" indicates that various components can be used together in the mixture or composition of the present invention. Therefore, the terms "consisting mainly of" and "consisting of" are included in the term "comprising".

[0039] The present invention provides the use of an effective part of a root extract of Sophora flavescens for preventing or treating inflammatory bowel disease. Mice are given DSS drinking water to create an inflammatory bowel disease model, and the effective part of the root extract of Sophora flavescens has the ability to improve weight loss, shortened colon length, disease activity score, and intestinal barrier damage in the model animals. According to a preferred example of the present invention, mice with DSS-induced inflammatory bowel disease model are treated with an effective part of a root extract of Sophora flavescens, and the body weight, colon length, and intestinal barrier are kept normal compared to an untreated control group. Therefore, the effective part of the root extract of Sophora flavescens can be used to prevent and treat inflammatory bowel disease.

[0040] The drug of the present invention can be administered in the form of any conventional pharmaceutical preparation in the art, wherein the pharmaceutical preparation includes excipients, pharmaceutically acceptable media and carriers, which can be selected according to the administration route. The drug of the present invention can further include auxiliary components.

[0041] The pharmaceutically acceptable excipients of the present invention include conventional solvents in the pharmaceutical field (such as water, ethanol, propylene glycol, injection oil, etc.), diluents (such as starch, powdered sugar, dextrin, lactose, pregelatinized starch, microcrystalline cellulose, inorganic calcium salts (such as calcium sulfate, calcium hydrogen phosphate, medicinal calcium carbonate, etc.), mannitol, etc., vegetable oils, polyethylene glycol, etc.), binders (such as water, ethanol, starch slurry, sodium carboxymethyl cellulose, hydroxypropyl cellulose, methyl cellulose and ethyl cellulose, hydroxypropyl methyl cellulose, etc.), disintegrants (such as dry starch, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, cross-linked polyvinyl pyrrolidone, cross-linked sodium carboxymethyl cellulose, etc.), lubricants (such as magnesium stearate, micronized silica gel, talc, hydrogenated vegetable oil, polyethylene glycol, magnesium lauryl sulfate, etc.), absorption accelerators (such as surfactants, Azone (lauroamphetamine), EDTA, salicylic acid, amino acid ethylamine derivatives, acetoacetate, β-dicarboxylate, aromatic acid compounds, aliphatic acids, etc.), preservatives (such as benzoic acid, hydroxypropyl butyl ester, hydroxypropyl methyl ester, phenol, m-cresol, etc.), flavoring agents (such as sucrose, stevioside, etc.), etc.

[0042] In addition, as a preferred embodiment, the drug of the present invention can be produced in an enteric coating preparation by various well-known methods so that the active ingredients of the drug can pass through the stomach smoothly without being destroyed by gastric acid.

[0043] The pharmaceutical composition of the present invention can be made into enteric-coated tablets for oral use. The term "enteric coating" in the present application includes all coatings permitted for use in conventional drugs, which are not degraded by gastric acid but can be fully decomposed in the small intestine and quickly release the active ingredients of the present invention. The enteric coating of the present invention can be maintained at 36-38°C for more than 2 hours in synthetic gastric acid such as HCl solution with pH=1, and preferably decomposed within 1.0 hour in synthetic intestinal fluid such as buffer with pH=7.0.

[0044] The enteric coating of the present invention is coated with about 16 to 30 mg per tablet, preferably 16 to 25 mg, and more preferably 16 to 20 mg. The thickness of the enteric coating of the present invention is 5 to 100 μm, and the ideal thickness is 20 to 80 μm. The enteric coating component is a conventional polymer in the field of pharmaceutical preparations.

[0045] The present invention is described below with reference to specific examples. It will be appreciated by those skilled in the art that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention in any way.

[0046] The experimental methods in the following examples are conventional methods unless otherwise specified. The raw materials, reagents, etc. used in the following examples are commercially available products unless otherwise specified. The purchase of some raw materials and reagents is as follows:

[0047] The root of the northern bean used in the present invention is selected from the dried rhizome of the Menispermum dauricum DC. of the Menispermaceae family, which is collected in spring and autumn and produced in one or more regions of Northeast China, North China, East China, Shaanxi, Ningxia, Gansu, and Shandong. Example 1: Preparation of the root of the northern bean extract

[0048] Take the clean rhizomes of the root of the northern bean root produced in Heilongjiang Province, dry them thoroughly and cut them into pieces for extraction. Prepare an ethanol: methanol: water extract in a volume ratio of 3: 1: 2, adjust the temperature to 60 ° C, mix the extract with the northern bean root and shake for 5 hours. Solid-liquid separation to obtain a crude extract, rotary evaporation at 50 ° C for 4 hours to fully evaporate the organic solvent to obtain a concentrated solution, at which time there is still a lot of water in the extract. The concentrate is vacuum freeze-dried at -0.7MPa for 18 hours to obtain a dry light yellow to black powder. The powder is dissolved in ethanol, and a clear solution is obtained after filtration. The solution is concentrated and evaporated to dryness to obtain a crude extract of the effective part of the target northern bean root extract.

[0049] Use high performance liquid chromatography to separate the above crude extracts and explore the key effective parts. The chromatographic column used is: 5μm particle size C18 chromatographic column, 4.6×250mm; the mobile phase is: by volume percentage, A: 80% water + 20% acetonitrile, B: 80% acetonitrile + 20% isopropanol, and the liquid phase conditions are: 0-4min, 30% B; 4-10min, 30% B-90% B; 10-12min, 90% B; 12-13min, 90% B-30% B, 13-15min, 30% B. The liquid phase method takes a total of 15 minutes, and the effluent is collected every minute to obtain a total of 15 effluents. Each effluent is rotary evaporated to dryness at 50°C, weighed and re-dissolved in methanol to obtain components 1-15. An equal amount of dried crude extract of northern bean root is dissolved in methanol to obtain component 16.

[0050] Example 2: Inhibitory activity of the extract of the root of Sophora flavescens and its components on bacterial DPP4

[0051] DPP4 is an important protein that regulates the levels of many endogenous polypeptides. It regulates intestinal cell proliferation and inflammation by affecting the levels of functional polypeptides such as GLP-1, GLP-2, and GM-CSF. Recent studies have found that increased levels of bacterial DPP4 are associated with the occurrence of IBD. Inhibiting the activity of bacterial DPP4 may help the recovery of IBD, but it is not clear what the main components of the northern bean root extract that inhibit the activity of bacterial DPP4 are. By separating the components of the northern bean root extract by liquid chromatography, 16 components were obtained (see Example 1), and then the activity tracking strategy was used to evaluate the inhibitory effect of each component on DPP4. The bacterial DPP4 was purified from the in vitro expression system independently constructed by this team. The inhibitory effects of the 16 components on bacterial DPP4 are shown in Table 1.

[0052] Table 1: Inhibitory activity of the extract from the root of Sophora flavescens and its components against bacterial DPP4

[0053]

[0054]

[0055] Among them, component 16 is a crude extract, and components 1-15 are components separated by liquid chromatography according to retention time. Component 7 shows the most obvious DPP4 inhibitory activity.

[0056] Example 3: Analysis of the main components of the effective part of the extract of the root of Sophora flavescens (component 7)

[0057] LC-MS was used to analyze the main components and contents in component 7, and the inhibitory effect of each component on DPP4 was evaluated. The results are shown in Table 2.

[0058] Table 2: Analysis of the main components of component 7 and the inhibitory effect of each component on DPP4

[0059]

[0060] Through LC-MS analysis, it was determined that the main component of component 7 was daurine DAU, accounting for about 60.3%, the minor component was daurine methyl ester DAU-Me, accounting for about 23.2%, and the other components were daurine analogs, all of which had DPP4 inhibitory activity. In addition, the activity of component 7 was higher than that of any single component, indicating that there was a synergistic effect between the components.

[0061] Example 4: Comparison of the improvement effects of single components, component 7 and crude extracts in the effective components of the root of Sophora flavescens extract on the IBD mouse model induced by dextran sulfate sodium (DSS)

[0062] Materials: The test substance is the extracts of various components of the roots of Sophora flavescens collected from North China, which are extracted, purified and dried to obtain powder. The dosage of the drug for gavage is 10 mg / kg body weight, and the frequency is once a day.

[0063] C57 mice were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd., and kept at a temperature of 20-24°C, constant humidity of 50-60%, a light intensity of 12 hours (8:00-20:00), soundproof, free access to food and water, and the experiment was conducted after one week of adaptation. DSS was purchased from MP Company in the United States.

[0064] Methods: 36 male C57 mice aged 8 weeks and weighing 20±1g were randomly divided into (1) model control group, (2) DAU group, (3) DAU-Me group, (4) other DAU analogs in component 7 group, (5) component 7 group, and (6) crude extract group, with 6 mice in each group, and drinking water was replaced with 2% DSS. Mice in groups (2)-(6) were gavaged with 10 mg / kg body weight of the corresponding drugs every day for 1 week, and the model control group was given an equal amount of normal saline. The body weight of the animals was measured daily, and fecal bleeding was observed. The body weight of the mice in the model group was significantly reduced, and obvious bloody and loose stools appeared, which proved that the model was successfully established. After the last administration, the body weight changes and disease activity index between the groups were evaluated to clarify the effects of the various components of the extract of the root of northern bean on inflammatory bowel disease. The evaluation of the disease activity index was mainly based on the quantitative weight loss, bloody stools and loose stools. Specifically, the evaluation criteria are: weight loss (0 points, weight loss <1%; 1 point, weight loss ≥1% but <5%; 2 points, weight loss ≥5% but <10%; 3 points, weight loss ≥10% but <20%; 4 points, weight loss ≥20%); blood in stool (0 points, no blood in stool; 2 points, occult blood; 4 points, obvious blood in stool); loose stool (0 points, normal stool; 2 points, loose stool; 4 points, stool adheres to the anus). The sum of the three scores divided by 3 is the disease activity index.

[0065] Results: Compared with the model group, each component of the extract of the root of Sophora flavescens could significantly improve the indicators related to inflammatory bowel disease, restore body weight, and reduce the disease activity index. Component 7 had the best improvement effect. Figure 1 .

[0066] Example 5: Improvement of the effective fraction of the extract of Sophora flavescens root (Component 7) on the DSS-induced IBD mouse model

[0067] Materials: The test substance was component 7 of the extract of Sophora flavescens root collected from North China, which was extracted, purified and dried to obtain a light yellow to dark yellow powder. 5-aminosalicylic acid (5-ASA) was used as a positive drug control. The oral administration dose of component 7 was 10 mg / kg body weight, and the oral administration dose of 5-ASA was 100 mg / kg body weight (this dose of 5-ASA has been shown to effectively improve DSS-induced colitis), and the frequency was once a day.

[0068] C57 mice were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd., and kept at a temperature of 20-24°C, constant humidity of 50-60%, a light intensity of 12 hours (8:00-20:00), soundproof, free access to food and water, and the experiment was conducted after one week of adaptation. DSS was purchased from MP Company in the United States.

[0069] Methods: Eighteen male C57 mice aged 8 weeks, weighing 20±1g, were randomly divided into (1) model control group; (2) bean root extract group; and (3) 5-ASA positive drug group. There were 6 mice in each group, and drinking water was replaced with 2% DSS. Mice in group (2) were gavaged with 10 mg / kg body weight of component 7 and mice in group (3) were gavaged with 100 mg / kg body weight of 5-ASA every day for 1 week. The model group was given an equal amount of normal saline. The body weight of the animals was measured daily, and fecal bleeding was observed. The body weight of mice in the model group was significantly reduced, and obvious bloody and loose stools appeared, which proved that the model was successfully established. After the last administration, the mice were intraperitoneally injected with L-012 chemiluminescent probe for in vivo imaging to evaluate the level of intestinal inflammation. The darker the blue and the larger the range, the more severe the inflammation. After fasting for 4 hours, the intestinal permeability of the mice was evaluated by gavage with FITC-dextran (4kD, 600 mg / kg). Finally, the mice were killed and the colon length of the mice was measured. About 1 cm of colon was placed in tissue fixative, dehydrated, paraffin-embedded, sectioned, and then stained with H&E. The level of inflammatory infiltration and barrier damage in the mouse colon were evaluated by histopathology to clarify the effect of component 7 of the northern bean root extract on inflammatory bowel disease.

[0070] Results: Compared with the model group, component 7 could significantly improve the indicators related to inflammatory bowel disease and restore body weight ( Figure 2 ), reduce disease activity score ( Figure 3 ), reduce inflammation levels in the body ( Figure 4 ), reduced intestinal ulcers and inflammatory infiltration ( Figure 5 ), significantly increased the length of the mouse intestine and improved the intestinal barrier of the mouse ( Figure 6 ), and the degree of improvement was better than 5-ASA.

[0071] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A method for preparing an effective fraction of a root extract of Sophora flavescens having the effect of preventing or treating inflammatory bowel disease, characterized in that: The following steps are involved: (1) Take the clean rhizome of the root of the northern bean, dry it thoroughly and then extract it; (2) preparing an ethanol:methanol:water extract in a volume ratio of (0.5-3):(0.5-2):(1-2), adjusting the temperature to 10-100° C., mixing the extract with the northern bean root in (1) and shaking for 1-12 hours; (3) After the extraction in step (2) is completed, solid-liquid separation is performed to obtain a crude extract, and rotary evaporation is performed at 30 to 60° C. for 1 to 6 hours to allow the organic solvent to fully evaporate to obtain a concentrated solution. At this time, the extract still contains a large amount of water; (4) vacuum freeze-drying the concentrated solution obtained in step (3) at -1.0 MPa to -0.10 MPa for 1 to 18 hours to obtain a dry light yellow to black powder; (5) dissolving the powder obtained in step (4) in ethanol, filtering to obtain a clear solution, concentrating the solution, and evaporating to dryness to obtain a crude extract of the effective part of the target Radix Sophorae Flavescentis extract; (6) using high performance liquid chromatography to track the activity of the crude extract of the effective part in step (5), and the effective component obtained by eluting for 6 to 7 minutes has the best effect, and the effective part of the northern bean root extract is obtained after concentration; (7) Liquid chromatography-tandem mass spectrometry was performed to analyze that the effective part of the northern bean root extract was mainly a mixture of daurine (DAU) and daurine methyl ester (DAU-Me), accounting for 75%-95% by mass percentage.

2. The method for preparing the effective fraction of the Radix Sophorae Flavescentis extract according to claim 1, characterized in that: In step (1), the bean roots should be chopped into 2 mm small pieces.

3. The method for preparing the effective fraction of the Radix Sophorae Flavescentis extract according to claim 1, characterized in that: In step (2), the volume ratio of ethanol:methanol:water is 3:1:2, the solid-liquid ratio is 1 g:6-10 mL, the extraction temperature is 50-60° C., and the oscillation extraction time is 4-5 hours.

4. The method for preparing the effective fraction of the Radix Sophorae Flavescentis extract according to claim 1, characterized in that: In step (3), the product is subjected to rotary evaporation at 50° C. for 4 to 6 hours; in step (4), the product is freeze-dried at -0.70 MPa for 12 to 18 hours; and in step (5), the product is filtered using a 0.22 μm filter membrane.

5. The method for preparing the effective fraction of the Radix Sophorae Flavescentis extract according to claim 1, characterized in that: The chromatographic column used in step (6) is: 5 μm particle size C18 chromatographic column, 4.6×250 mm; the mobile phase is: by volume percentage, A: 80% water + 20% acetonitrile, B: 80% acetonitrile + 20% isopropanol, and the liquid phase conditions are: 0-4 min, 30% B; 4-10 min, 30% B-90% B; 10-12 min, 90% B; 12-13 min, 90% B-30% B, 13-15 min, 30% B.

6. The method for preparing the effective fraction of the Radix Sophorae Flavescentis extract according to claim 1, characterized in that: The northern bean root is selected from the dried rhizome of Menispermum dauricum DC. of the Menispermaceae family. The collection time is limited to spring and autumn, and the production area is one or more regions in Northeast China, North China, East China, Shaanxi, Ningxia, Gansu, and Shandong.

7. The effective fraction of the extract of Radix Sophorae Fructus having the effect of preventing or treating inflammatory bowel disease prepared by the preparation method according to any one of claims 1 to 6, characterized in that: The effective part of the northern bean root extract is mainly a mixture of daurine (DAU) and daurine methyl ester (DAU-Me), which accounts for 75%-95% by mass percentage.

8. Use of the effective fraction of the extract of the root of Sophora flavescens according to claim 7 in the preparation of a medicament for preventing or treating inflammatory bowel disease.

9. The use according to claim 8, characterized in that: The inflammatory bowel disease is ulcerative colitis or Crohn's disease.

10. The use according to claim 8 or claim 9, characterized in that: The dosage form of the drug is selected from granules, capsules, tablets, powders, oral solutions, suspensions or emulsions.

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