Phellodendri extract and use thereof

Abstract

This invention belongs to the field of traditional Chinese medicine technology, specifically relating to a Phellodendron amurense extract. Based on the total mass of the extract, it comprises no less than 10% berberine and no less than 1% berberine. This extract shows significant efficacy in an animal model of ulcerative colitis, increasing colon length and reducing DAI scores. Animal efficacy and mechanism of action studies indicate that the combination of berberine and berberine has a synergistic effect, exhibiting superior efficacy compared to either berberine or berberine alone, demonstrating promising application prospects.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine technology, specifically relating to a Phellodendron amurense extract and its application. Background Technology

[0002] Phellodendron bark (Huang Bai) is bitter and cold in nature. It enters the kidney and bladder meridians. Phellodendron bark is bitter, purging, cold, and drying, and descending in nature, entering the kidney and bladder meridians. It clears and drains excess heat (fire) and relieves heat toxicity, dries dampness, eliminates damp toxicity and relieves damp-heat toxicity, and clears kidney fire (ministerial fire) and reduces deficiency heat. It is an essential medicine for treating damp-heat and fire toxicity, and is widely used for related symptoms. Compared to Coptis chinensis (Huang Lian), its heat-clearing and dampness-drying effects are weaker, and its effects are more focused on the kidney and lower jiao (lower burner) bladder. It is best at clearing ministerial fire, reducing deficiency heat, and eliminating damp-heat in the lower burner. It combines clearing excess heat, damp-heat, and reducing deficiency heat, and is suitable for all types of excess heat, damp-heat, and deficiency heat. It has the effects of clearing heat and drying dampness, purging fire and eliminating steaming heat, and detoxifying and healing sores.

[0003] Enteritis is divided into two types: inflammatory bowel disease and infectious enteritis. Inflammatory enteritis belongs to the field of gastroenterology and mainly includes ulcerative colitis (UC) and Crohn's disease (CD); while infectious enteritis belongs to the field of infectious diseases and mainly includes acute or chronic enteritis caused by various pathogenic microorganisms and their products, such as bacteria, viruses, fungi, and parasites, invading the intestines.

[0004] Infectious enteritis is primarily treated with antibiotics and antivirals, which are highly effective but also present challenges such as antibiotic overuse, bacterial resistance, and immune impairment. Ulcerative colitis is currently treated mainly with aminosalicylic acids, corticosteroids, immunosuppressants, and monoclonal antibodies. Although significant progress has been made in ulcerative colitis drug development in recent years, many patients with moderate to severe active UC still experience inadequate response, poor treatment outcomes, and recurrent episodes.

[0005] Traditional Chinese medicine (TCM) has been used in the treatment of infectious enteritis and has many advantages. TCM is also a distinctive feature of my country's treatment of ulcerative colitis (UC), making research on the effects and mechanisms of TCM in UC treatment of significant importance and practical value. Summary of the Invention

[0006] To address the problems existing in the prior art, this invention provides a Phellodendron amurense extract and its applications. Whole animal model efficacy tests and pharmacological mechanism studies have confirmed that this extract (composition) has good effects in preventing and treating inflammatory bowel disease and chronic enteritis. This indicates that the combination of berberine and phellodendron amurense in the extract (composition) has a synergistic effect, and the combined efficacy is superior to that of berberine or phellodendron amurense alone. It can be further used to develop and prepare innovative traditional Chinese medicine drugs for the prevention and treatment of enteritis.

[0007] To achieve the purpose of the invention, the following technical solution is adopted:

[0008] A Phellodendron amurense extract for the prevention and treatment of enteritis, wherein, based on the total mass of the Phellodendron amurense extract, the Phellodendron amurense extract comprises not less than 10% berberine and not less than 1% berberine.

[0009] Preferably, based on the total mass of the Phellodendron amurense extract, the Phellodendron amurense extract comprises 10%-30% berberine and 1%-5% berberine.

[0010] Preferably, based on the total mass of the Phellodendron amurense extract, the Phellodendron amurense extract comprises 10%-30% berberine, 1%-5% berberine, 0.1%-0.5% magnoflorine, 0.01%-0.2% demethylene berberine, 0.01%-0.2% berberine, 0.01%-0.1% berberine, and 0.01%-0.1% palmatine.

[0011] Preferably, the Phellodendron amurense extract comprises 12%-25% berberine and 1%-4% berberine. More preferably, the Phellodendron amurense extract comprises 12%-22% berberine and 1.5%-3.5% berberine, or 18%-22% berberine and 2%-2.5% berberine.

[0012] Preferably, the mass ratio of berberine to phellodendron chinense is 10-30:1-5.

[0013] More preferably, the mass ratio of berberine to phellodendron chinense is 12-25:1-4.

[0014] More preferably, the mass ratio of berberine to phellodendron chinense is 12-22:1.5-3.5.

[0015] More preferably, the mass ratio of berberine to phellodendron chinense is 18-22:2-2.5.

[0016] A second objective of this invention is to provide a composition for preventing and treating enteritis, the composition comprising the following two components: berberine and berberine.

[0017] Preferably, the mass ratio of berberine to phellodendron chinense is 10-30:1-3.

[0018] More preferably, the mass ratio of berberine to phellodendron chinense is 12-25:1-4.

[0019] More preferably, the mass ratio of berberine to phellodendron chinense is 12-22:1.5-3.5.

[0020] More preferably, the mass ratio of berberine to phellodendron chinense is 18-22:2-2.5.

[0021] A third objective of this invention is to provide a method for preparing the above-mentioned Phellodendron amurense extract, comprising the following steps:

[0022] Extract the Phellodendron bark herb with 30-90% (w / w) ethanol, filter, and combine the filtrates to obtain the final product. Preferably, extraction with 40-80% (w / w) ethanol is used.

[0023] Phellodendron bark is the dried bark of the Phellodendron chinense Schneid., a plant in the Rutaceae family, commonly known as "Sichuan Phellodendron." The dried bark of P. amurense Rupr. is used medicinally and is called "Guan Phellodendron." Phellodendron bark is mainly produced in Sichuan, Hubei, Guizhou, Yunnan, Shaanxi, and Guangxi provinces. Guan Phellodendron bark is mainly produced in Liaoning, Jilin, Heilongjiang, Hebei, and Inner Mongolia provinces. The 2005, 2010, and 2015 editions of the Chinese Pharmacopoeia list the two medicinal materials separately, but in clinical application, the difference between the two is not particularly emphasized, and they are sometimes used interchangeably in prepared medicines.

[0024] This invention uses Phellodendron bark from Sichuan.

[0025] Furthermore, after merging the filtrates, they are concentrated and dried. The concentration is carried out under reduced pressure at a temperature of 50-60°C, and the drying temperature is 55-65°C.

[0026] Furthermore, the extraction is a heating extraction, the extraction is performed 1-3 times, the extraction time for each extraction is 1-3 hours, and the mass ratio of the Phellodendron bark medicinal material to the ethanol solution is 1:5-10.

[0027] Furthermore, the extraction is performed twice, with a material-to-liquid ratio of 1:8-10.

[0028] In some implementations, the temperature for heat extraction is 120-160°C.

[0029] The temperatures mentioned above are the heating temperatures used during reflux extraction.

[0030] The fourth objective of this invention is to provide a pharmaceutical preparation for the prevention and treatment of enteritis, comprising the above-mentioned Phellodendron amurense extract or the above-mentioned composition or the above-mentioned preparation method prepared Phellodendron amurense extract, and pharmaceutically acceptable excipients.

[0031] The fifth objective of this invention is to provide the application of the above-mentioned Phellodendron amurense extract or the Phellodendron amurense extract prepared by the above-mentioned preparation method in the preparation of drugs for the prevention and treatment of inflammatory bowel disease and / or chronic enteritis.

[0032] Furthermore, the inflammatory bowel disease includes ulcerative colitis and / or Crohn's disease.

[0033] Furthermore, the chronic enteritis includes chronic inflammation of the intestines caused by bacterial, viral, and / or protozoan infections.

[0034] The present invention also provides the use of a Phellodendron amurense extract or a Phellodendron amurense extract obtained by the above preparation method or a pharmaceutical preparation in the preparation of a drug for improving intestinal barrier function in patients with inflammatory bowel disease and / or chronic enteritis.

[0035] The present invention also provides the application of Phellodendron amurense extract or the Phellodendron amurense extract obtained by the above preparation method or the above pharmaceutical preparation in the preparation of a drug that exerts an anti-inflammatory effect on the intestine through the NF-κB signaling pathway.

[0036] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0037] (1) The extract of Phellodendron bark obtained by the process of the present invention has a berberine content of more than 10% and a berberine content of more than 1%. It has significant efficacy in mouse and rat ulcerative colitis models, and can increase colon length and reduce DAI score.

[0038] (2) Traditional pharmacological studies on the Chinese herb Phellodendron amurense often focus on clearing heat and drying dampness, purging fire and detoxifying. This invention prepares an extract from Phellodendron amurense for research, and verifies the possibility of using the extract to prevent and treat intestinal diseases through pharmacological comparison. This provides a new research direction for exploring the modern mechanism by which the extract exerts its traditional effects, and also provides new ideas for developing innovative Chinese medicines to prevent and treat intestinal diseases.

[0039] (3) This invention uses sodium dextran sulfate (DSS) to induce colitis. DSS can cause intestinal retention, mucosal barrier damage, and inflammatory cell infiltration, leading to the development of enteritis. Studies have found that the Phellodendron amurense extract of this invention has significant anti-ulcerative colitis activity compared to berberine.

[0040] (4) Studies have found that the extract of Phellodendron bark in this invention has comparable efficacy to methadone. Methalazine, also known as 5-aminosalicylic acid (5-ASA), is a first-line drug for the treatment of inflammatory bowel diseases such as ulcerative colitis (UC).

[0041] (5) The extract (composition) of Phellodendron amurense of the present invention can be prepared by extraction from Phellodendron amurense medicinal material, or by directly combining berberine monomer with berberine monomer.

[0042] (6) Further research has found that the combination of berberine and phellodendron chinense in the extract of Phellodendron chinense of the present invention has a synergistic effect, and the efficacy of the combination is better than that of berberine alone and phellodendron chinense alone.

[0043] (7) The present invention has found that the extract of Phellodendron bark can significantly improve the intestinal barrier function in patients with inflammatory bowel disease and chronic enteritis.

[0044] (8) The present invention also found that the extract of Phellodendron amurense of the present invention can exert an anti-inflammatory effect on the intestine through the NF-κB signaling pathway. Attached Figure Description

[0045] Figure 1 This is a chromatogram of a mixed standard.

[0046] Figure 2 This is the chromatogram for Example 1.

[0047] Figure 3 This is the chromatogram for Example 2.

[0048] Figure 4 This is the chromatogram for Example 3.

[0049] Figure 5 This is the chromatogram for Example 4.

[0050] Figure 6 The effects of Examples 1 and 2 on colon length in mice with ulcerative colitis were compared with the control group. ### p < 0.001, compared with the model group, *p < 0.05, **p < 0.01, ***p < 0.001.

[0051] Figure 7 The effects of Examples 1 and 2 on the DAI score of mice with ulcerative colitis were compared with the model group. *p<0.05, **p<0.01, ***p<0.001.

[0052] Figure 8 The effects of berberine + berberine, berberine, and berberine on colon length in mice with ulcerative colitis, as described in Example 1, were compared with the control group. ### p < 0.001, compared with the model group, **p < 0.01, ***p < 0.001.

[0053] Figure 9 The effects of berberine + berberine, berberine, and berberine on the DAI score of mice with ulcerative colitis were compared with those of the model group. *p<0.05, ***p<0.001.

[0054] Figure 10 Hematoxylin-eosin staining (50x / 100x) was performed on the colon tissue of mice in each group.

[0055] Figure 11 Alcian blue staining (50× / 100×) was performed on the colon tissue of mice in each group, where A is the Alcian blue staining image of mouse colon; B is the quantitative result of A.

[0056] Figure 12The expression levels of tight junction proteins ZO-1 and Occludin in mice in each group are shown in Figure 1. ***P < 0.001. A is the Western blot diagram of ZO-1 protein in mouse colon; B is the quantitative result of A; C is the Western blot diagram of Occludin protein in mouse colon; and D is the quantitative result of C.

[0057] Figure 13 The expression of IL-6 protein in the colon tissue of mice in each group is shown. A is the WB diagram of IL-6 protein in mouse colon; B is the quantitative result of A.

[0058] Figure 14 The regulatory levels of the NF-κB inflammatory signaling pathway in each group of mice are shown in Figure A, which is a WB diagram of phosphorylated P65 NFκB (P-P65 NFκB) and P65 NFκB protein in the mouse colon; Figure B is the quantitative result of A. Detailed Implementation

[0059] Example 1

[0060] A Phellodendron amurense extract, the preparation method of which is as follows:

[0061] Phellodendron bark was collected, impurities were removed, and the mixture was sprayed with water to moisten it thoroughly. It was then shredded, dried, and extracted with 80% (w / w) ethanol aqueous solution by heating. The extract was defatted twice, with material-to-liquid ratios (mass ratios) of 1:10 and 1:8, respectively. The extraction temperature was 120℃, and the extraction times were 2 hours and 1.5 hours, respectively. After defatting, the filtrates were combined, concentrated under reduced pressure at 50℃ and a vacuum degree of -0.1 MPa, and then vacuum dried at 55℃ and a vacuum degree of -1 MPa to obtain the Phellodendron bark extract sample.

[0062] Example 2

[0063] A Phellodendron amurense extract, the preparation method of which is as follows:

[0064] Phellodendron bark was collected, impurities were removed, and the mixture was sprayed with water to moisten it thoroughly. It was then shredded, dried, and defatted twice with 40% (w / w) ethanol and heated. The material-to-liquid ratios were 1:10 and 1:8, respectively. The extraction temperature was 160℃, and the extraction times were 2 hours and 1.5 hours, respectively. After defatting, the filtrates were combined, concentrated under reduced pressure at 60℃ and a vacuum degree of -0.09 MPa, and then vacuum dried at 65℃ and a vacuum degree of -0.8 MPa to obtain the Phellodendron bark extract sample.

[0065] Example 3

[0066] The difference between this embodiment and Embodiment 1 is that the concentration of ethanol used is different; specifically, it is 90% (w / w) ethanol.

[0067] Example 4

[0068] The difference between this embodiment and Example 1 is the concentration of ethanol used; specifically, it is 30% (w / w) ethanol. The content of berberine and phellodendronine in the sample was determined by HPLC.

[0069] 1. Instruments and reagents

[0070] 1.1 Instruments

[0071] Thermo Vanquish Core high-performance liquid chromatograph; Sartorius MSA6.6S 0.1 / 1 million electronic balance; Mettler ML204T 0.01 / 10,000 electronic analytical balance; SB-500DTY swept-frequency ultrasonic cleaner; Guohua HH-2 constant temperature water bath; Milli-Q IQ7000 ultrapure water system.

[0072] 1.2 Test Drugs

[0073] Examples 1-4 contain the following reference standards: berberine hydrochloride extract, berberine hydrochloride reference standard, magnoflorine reference standard, demethylene berberine, berberine reference standard, berberine hydrochloride reference standard, and palmatine reference standard. Acetonitrile and phosphoric acid were of chromatographic purity, sodium dodecyl sulfate was of analytical purity, and water was ultrapure water.

[0074] 2 methods

[0075] Refer to the high performance liquid chromatography method in Part IV of the 2020 edition of the Chinese Pharmacopoeia.

[0076] 2.1 Chromatographic conditions

[0077] 2.1.1 Chromatographic conditions for content determination

[0078] The chromatographic column was a YMC Triart C18 column, 4.6 x 250 mm, 5 μm; mobile phase A was acetonitrile, mobile phase B was 0.1% phosphoric acid solution (0.02 mol / L sodium dihydrogen phosphate), and the elution gradient is shown in Table 1 below; the detection wavelength was 222 nm; the column temperature was 35 ℃; the flow rate was 1.0 mL / min; and the injection volume was 10 μL.

[0079] The elution procedure is as follows:

[0080] Table 1 Elution gradient of mobile phase

[0081]

[0082]

[0083] 2.1.2 Preparation of reference and test solutions

[0084] Weigh appropriate amounts of berberine hydrochloride reference standard, berberine hydrochloride reference standard, magnoflorine reference standard, demethylene berberine, berberine reference standard, berberine hydrochloride reference standard, and palmatine reference standard, dissolve them in the mobile phase, and prepare the reference solution.

[0085] Accurately weigh 10 mg of the test sample (the composition prepared in Examples 1-4), place it in a 25 ml volumetric flask, add 20 ml of mobile phase, sonicate for 40 minutes (40 Hz), cool, add mobile phase to make up to volume, shake well, filter, and take the filtrate to obtain the test sample.

[0086] 3 Content determination chromatogram

[0087] See the chromatogram of the mixed standard. Figure 1 See the diagram for Example 1. Figure 2 See the diagram for Example 2. Figure 3 See the diagram in Example 3. Figure 4 See the diagram in Example 4. Figure 5 .

[0088] According to the analysis, the content of berberine in the extract of Phellodendron chinense in Example 1 was 21.2%, the content of berberine was 2.1%, the content of magnoflorine was 0.39%, the content of demethylene berberine was 0.054%, the content of purslane was 0.12%, the content of berberine was 0.02%, and the content of palmatine was 0.073%.

[0089] In Sample Example 2, the extract of Phellodendron amurense contained 20.2% berberine, 2.1% berberine, 0.38% magnoflorine, 0.093% demethylene berberine, 0.11% berberine, 0.01% berberine, and 0.058% palmatine.

[0090] In Sample Example 3, the extract of Phellodendron amurense contained 14.8% berberine, 2.2% berberine, 0.39% magnoflorine, 0.047% demethylene berberine, 0.12% berberine, 0% berberine alkaloid, and 0.077% palmatine.

[0091] In Sample Example 4, the extract of Phellodendron amurense contained 13.4% berberine, 3.4% berberine, 0.47% magnoflorine, 0.122% demethylene berberine, 0.19% berberine, 0.01% berberine, and 0.069% palmatine.

[0092] Animal efficacy and pharmacological mechanism

[0093] Effects on ulcerative colitis in mice

[0094] 1. Reagents and Instruments

[0095] 1.1 Reagents

[0096] Dextran sulfate sodium (DSS) was purchased from (MPBiomedicals), sodium carboxymethyl cellulose was purchased from (Damao Chemical Reagent Co., Ltd.), mesalazine was purchased from (Sunflower Pharmaceutical Co., Ltd.), primary antibodies of ZO-1, NFκB and p-NFκB were purchased from (Cell Signaling Technology), primary antibodies of Occludin and IL-6 (Proteintech Group), HE staining kit was purchased from (Beyotime Biotechnology), and Alcian blue staining kit was purchased from (Saiweier Biology).

[0097] 1.2 Instruments

[0098] Surgical instruments, ruler, balance.

[0099] 1.3 Experimental animals

[0100] 6-8-week-old SPF-grade male C57 mice (20-22 g) were purchased from (Changsha Tianqin), license number: SCXK(Xiang)2019-004. During the experiment, the animals were adaptively fed for 7 days in an environment with temperature: 22±2°C, relative humidity: 50±10%, 12 hours of light and 12 hours of darkness per day. During this period, the mice were fed with standard mouse feed and had free access to water.

[0101] 2 Experimental methods

[0102] 2.1 Experimental grouping and dose design

[0103] Experiment 1: Before the start of the experiment, the C57 mice were weighed and randomly divided into 5 groups according to body weight: control group (normal saline), model group (DSS), positive group (mesalazine, 350 mg / kg), extract of Phellodendron amurense in Example 1 (350 mg / kg), extract of Phellodendron amurense in Example 2 (350 mg / kg); 10 mice in each group were labeled with ear tags.

[0104] Experiment 2: Before the start of the experiment, the C57 mice were weighed and randomly divided into 4 groups according to body weight: control group (normal saline), model group (DSS), positive group (mesalazine, 350 mg / kg), extract of Phellodendron amurense in Example 3 (350 mg / kg); 10 mice in each group were labeled with ear tags.

[0105] Experiment 3: Before the start of the experiment, the C57 mice were weighed and randomly divided into 4 groups according to body weight: control group (normal saline), model group (DSS), positive group (mesalazine, 350 mg / kg), extract of Phellodendron amurense in Example 4 (350 mg / kg), 10 mice in each group were labeled with ear tags.

[0106] Experiment 4: Before the experiment, C57 mice were weighed and randomly divided into 7 groups according to their body weight: control group (physiological saline), model group (DSS), positive group (mesalazine, 350 mg / kg), berberine + berberine group 50 mg / kg (composed of berberine and berberine in a mass ratio of 10:1), Example 1 group: sample prepared in Example 1 238 mg / kg (berberine content of 21%, converted to berberine content of 50 mg / kg), berberine group: berberine 50 mg / kg, berberine group: berberine 50 mg / kg; 10 mice in each group were labeled with ear tags.

[0107] 2.2 Construction of an ulcerative colitis model

[0108] C57 mice were acclimatized for 7 days and given prophylactic medication for 3 days before modeling began. Modeling induction lasted for 9 days (6 days of drinking 2% sodium dextran sulfate and 3 days of drinking 2.5% sodium dextran sulfate). Medications were administered concurrently during modeling. DAI scores were assessed on day 9 of modeling. On day 10, blood was collected from the orbital margins of all mice, and they died from cervical dislocation. Colon length was measured.

[0109] 2.3 Disease Activity Index (DAI) score

[0110] On day 9 of modeling, mouse weight, activity level, fecal characteristics, and bloody stool were recorded. For each mouse, fecal characteristics and bloody stool scores were determined by a person unfamiliar with the experimental protocol according to the following criteria: Weight loss score: No change, 0 points; 1-5% decrease, 1 point; 5-10% decrease, 2 points; 10-20% decrease, 3 points; greater than 20% decrease, 4 points. Fecal characteristics score: Normal, 0 points; relatively soft stool, 1 point; soft and wet stool, 2 points; semi-loose stool, 3 points; loose stool, 4 points. Fecal occult blood score: No bloody stool, 0 points; positive fecal blood test, 2 points; significant bloody stool, 4 points. DAI = (weight loss score + fecal characteristics score + bloody stool score) / 3.

[0111] 2.4 Colon length measurement

[0112] After the mice were euthanized by cervical dislocation, their abdominal hair was sprayed with ethanol. After dissection, the colon was removed, and the length from the anus to the junction of the colon and cecum was measured on the surgical form. The data was recorded and photographed.

[0113] 2.5 Hematoxylin-eosin (HE) and Alcian blue (AB) staining of tissue sections

[0114] The colon was cleansed of any remaining material using pre-cooled saline at 4°C, and the surface moisture was blotted dry with filter paper. A section of colon approximately 1 cm long was taken from about 2 cm away from the rectum and fixed in 4% paraformaldehyde. After dehydration, clearing, embedding, and sectioning, tissue sections with a thickness of 3-8 μm were obtained. For HE staining, the sections were first dewaxed with toluene, then stained with hematoxylin for nuclear differentiation, counterstained with eosin, and mounted with neutral resin to obtain HE-stained sections. For AB staining, the sections were similarly dewaxed with toluene, acidified with Alcian blue, stained with Nucleotide Red, dehydrated with ethanol, removed from the ethanol with xylene, and mounted with neutral resin to obtain AB-stained sections.

[0115] 2.6 Western blot (WB) protein expression detection

[0116] Approximately 30 mg of colon tissue was weighed, added to lysis buffer, and ground at low temperature for 5 min in a cryogenic grinder. The mixture was then centrifuged at 10,000 rpm for 10 min at 4°C, and the supernatant was collected. Protein concentration was quantified using the BCA method, followed by SDS-PAGE gel electrophoresis. The bands were then blocked after PVDF membrane transfer, and incubated with different primary and secondary antibodies. Finally, the target bands were visualized using chemiluminescence for semi-quantitative statistical analysis.

[0117] 3. Results of animal efficacy experiments

[0118] 3.1 The pharmacodynamic effects of Phellodendron bark extract (Examples 1 and 2) on DSS-induced ulcerative colitis in mice in Experiment 1.

[0119] The results are shown in Tables 2-3 and 3. Figures 6-7 This study found that throughout the experimental period, control group mice ate and drank normally, had neat and shiny fur, and exhibited normal and agile behavior. Model group mice consumed less food and water, had sparse and dull fur, and were often quiet and inactive. The use of Phellodendron amurense extract (Example 1), Phellodendron amurense extract (Example 2), and the positive drug mesalazine improved these phenomena in mice. Simultaneously, the mice's fecal condition was observed. Control group mice had normal fecal shape and no bloody stools; the model group mice exhibited bloody stools and loose stools. The use of Phellodendron amurense extract (Example 1), Phellodendron amurense extract (Example 2), and the positive drug mesalazine significantly reduced bloody stools and loose stools. Based on these findings, the mice were scored using the DAI (Digital Intake Immunization) system. The results showed that the DAI score was increased in the model group and significantly decreased in the use of Phellodendron amurense extract (Example 1), Phellodendron amurense extract (Example 2), and the positive drug mesalazine. Furthermore, the colon length of the mice was compared, showing a decrease in the model group and a significant increase in the use of Phellodendron amurense extract (Example 1), Phellodendron amurense extract (Example 2), and the positive drug mesalazine.

[0120] The above results indicate that Phellodendron bark extract (Examples 1 and 2) can significantly improve the DAI score and colonic shortening in ulcerative colitis, and has a significant anti-ulcerative colitis effect.

[0121] Table 2 Colon length of mice in each group

[0122] result control group Model group positive group Example 2: Phellodendron bark extract Example 1: Phellodendron bark extract average value 7.28 5.19 5.58 6.07 6.95 P 0.000 0.036 0.009 0.000

[0123] Table 3. DAI (Disease Activity Index) scores of mice in each group

[0124] result Model group positive group Example 2: Phellodendron bark extract Example 1: Phellodendron bark extract average value 6.82 5.44 4.00 1.45 P - 0.044 0.000 0.000

[0125] 3.2 Results of the pharmacodynamic effects of Phellodendron amurense extract (Example 3) on DSS-induced ulcerative colitis in mice in Experiment 2

[0126] The results are shown in Tables 4 and 5. This study found that throughout the experimental period, control group mice ate and drank normally, had neat and shiny fur, and exhibited normal and agile behavior. Model group mice consumed less food and water, had sparse and dull fur, and often lay still and were inactive. The mice in the Phellodendron amurense extract and mesalazine positive control group (Example 3) showed improvement in these characteristics. Simultaneously, the mice's feces were observed. Control group mice had normal fecal shape and no bloody stools; the model group mice exhibited bloody stools and loose stools; the Phellodendron amurense extract and mesalazine positive control group (Example 3) significantly reduced bloody stools and loose stools. Based on these findings, the mice were scored using the DAI (Digital Intestinal Ability) scale. The results showed that the DAI score was higher in the model group and significantly lower in the group treated with Phellodendron amurense extract and mesalazine. Furthermore, the colon length of the mice was compared, showing a decrease in the model group and a significant increase in the group treated with Phellodendron amurense extract and mesalazine.

[0127] The above results indicate that Phellodendron bark extract (Example 3) can significantly improve the DAI score and colonic shortening in ulcerative colitis, and has a significant anti-ulcerative colitis effect.

[0128] Table 4 Colon length of mice in each group

[0129] result control group Model group positive group Example 3: Phellodendron bark extract average value 7.50 4.68 5.65 5.61 P - 0.000001 0.039 0.0421

[0130] Table 5. Disease Activity Index (DAI) scores of mice in each group.

[0131] result Model group positive group Example 3: Phellodendron bark extract average value 7.38 4.70 2.60 P - 0.026 0.0012

[0132] 3.3 Pharmacological effects of Phellodendron amurense extract (Example 4) in DSS-induced ulcerative colitis in mice (Experiment 3)

[0133] The results are shown in Tables 6 and 7. This study found that throughout the experimental period, control group mice ate and drank normally, had neat and shiny fur, and exhibited normal and agile behavior. Model group mice consumed less food and water, had sparse and dull fur, and often lay still and were inactive. The Phellodendron amurense extract group and the mesalazine positive control group (Example 4) improved these phenomena. Simultaneously, the mice's feces were observed. Control group mice had normal fecal shape and no bloody stools; the model group mice exhibited bloody stools and loose stools; the Phellodendron amurense extract group (Example 4) and the mesalazine positive control group significantly reduced bloody stools and loose stools. Based on these observations, the mice were scored using the DAI (Digital Intestinal Ability) scale. The results showed that the DAI score of the model group was increased, while that of the Phellodendron amurense extract group (Example 4) and the mesalazine positive control group was significantly decreased. Furthermore, the colon length of the mice was compared, showing that the colon length was decreased in the model group and significantly increased in the Phellodendron amurense extract group (Example 4) and the mesalazine positive control group.

[0134] The above results indicate that Phellodendron bark extract (Example 4) can significantly improve the DAI score and colonic shortening in ulcerative colitis, and has a significant anti-ulcerative colitis effect.

[0135] Table 6 Colon length of mice in each group

[0136] result control group Model group positive group Example 4: Phellodendron bark extract average value 7.50 4.93 5.47 5.76 P - 0.0001 0.046 0.035

[0137] Table 7. DAI (Disease Activity Index) scores of mice in each group

[0138] result Model group positive group Example 4: Phellodendron bark extract average value 7.13 4.90 2.20 P - 0.035 0.001

[0139] The efficacy results and analysis of experiments 3.1-3.3: The extracts of Phellodendron amurense from Examples 1 and 2 significantly increased colon length (P < 0.01 compared to the model group) and decreased DAI scores (P < 0.01 compared to the model group). The extracts of Phellodendron amurense from Examples 3 and 4 also increased colon length (P < 0.05 compared to the model group) and decreased DAI scores (P < 0.05 compared to the model group), but their efficacy was weaker than that of the extracts of Phellodendron amurense from Examples 1 and 2 at the same dosage.

[0140] 3.4 The efficacy of the combined drug administration regimen in Experiment 4 on DSS-induced ulcerative colitis in mice.

[0141] See results Figures 8-9See Tables 8 and 9. This study found that throughout the experimental period, control group mice ate and drank normally, had neat and shiny fur, and exhibited normal and agile behavior. Model group mice consumed less food and water, had sparse and dull fur, and often lay still and were inactive. The mice in the Phellodendron amurense extract and mesalazine positive control group (Example 1) showed improvement in these characteristics. Simultaneously, the mice's feces were observed. Control group mice had normal fecal shape and no bloody stools; the model group exhibited bloody stools and loose stools. The Phellodendron amurense extract, berberine, berberine + berberine, and mesalazine positive control groups (Example 1) significantly reduced bloody stools and loose stools. Based on these findings, the mice were scored using the DAI (Digital Intake and Acidity) scale. The results showed that the DAI score increased in the model group, and decreased significantly in the Phellodendron amurense extract, berberine, berberine + berberine, and mesalazine positive control groups (Example 1); the Phellodendron amurense extract showed the greatest decrease. Furthermore, the colon length of mice was compared statistically. The results showed that the colon length of the model group was reduced, while that of the Phellodendron amurense extract of Example 1, the berberine group, the berberine + berberine group, and the positive drug mesalazine was significantly increased. The berberine + berberine group showed the greatest increase, followed by the Phellodendron amurense extract of Example 1.

[0142] The above results indicate that, when the extract of *Phellodendron amurense* from Example 1 was administered at the same dose of berberine (50 mg / kg), its efficacy in both colon length and DAI score was superior to that of berberine (50 mg / kg) and phellodendron chinense alone (50 mg / kg). This suggests that not only berberine plays a role in the efficacy of the extract, but other components also enhance its anti-ulcerative colitis effect. The compatibility study of berberine and phellodendron chinense showed that the combined effect was stronger than that of berberine or phellodendron chinense alone, thus explaining the better anti-ulcerative colitis effect of the extract and the enhancement of the ulcerative colitis treatment effect of phellodendron chinense by phellodendron chinense.

[0143] Table 8 Colon length of mice in each group

[0144]

[0145] Table 9. DAI (Disease Activity Index) scores of mice in each group

[0146] Model group positive group Berberine + Phellodendronine Example 1 Berberine Phellodendron alkaloids average value 5.90 4.20 4.20 3.20 3.78 5.89 P 0.046 0.0358 0.00203 0.025 0.991

[0147] 4. Pharmacological mechanism of action of ulcerative colitis mice in the above experimental groups.

[0148] 4.1 Effects on colonic mucosal damage, inflammatory infiltration, goblet cell integrity, and mucin secretion in DSS-induced ulcerative colitis (UC) mice

[0149] The results are as follows Figure 10 and Figure 11Compared with the control group, colon tissue sections in the model group showed a thinner mucus layer, mucosal damage, extensive inflammatory cell infiltration, severe or even absent goblet cell damage, and a significant decrease in mucin secretion. Compared with the model group, the mesalazine group showed some recovery of mucosal damage, reduced inflammatory cell infiltration, some recovery of goblet cell damage, and increased mucin secretion levels. The berberine + berberine group showed significant improvement in mucosal damage, almost no inflammatory cell infiltration, relatively intact goblet cells, and significantly increased mucin secretion. The berberine extract group showed some recovery of mucosal damage, reduced inflammatory cell infiltration, some recovery of goblet cell damage, and a trend of recovery in mucin secretion. The berberine group showed significant improvement in mucosal damage, almost no inflammatory cell infiltration, relatively intact goblet cells, and some recovery in mucin secretion. The berberine group showed no significant recovery of mucosal damage, inflammatory cell infiltration, some recovery of goblet cell damage, and a trend of recovery in mucin secretion. The above results indicate that Phellodendron amurense extract, berberine, and the combination of berberine and berberine can significantly restore the integrity of the intestinal barrier mucosa and reduce inflammatory responses, but the effect of berberine is not significant. Furthermore, the combination of berberine and berberine, and Phellodendron amurense extract containing both berberine and berberine, are more effective than berberine monomer in promoting mucin secretion. This suggests that the anti-ulcerative colitis effect of Phellodendron amurense extract may be the result of the synergistic effect of berberine and berberine, with berberine enhancing the anti-ulcerative colitis effect of berberine.

[0150] 4.2 Effects on intestinal barrier-related proteins in DSS-induced ulcerative colitis mice

[0151] The results are as follows Figure 12 ZO-1 and Occludin are important tight junction proteins that play a crucial role in maintaining intercellular barrier function and signal transduction. Studies have found that decreased levels of these proteins can lead to intestinal barrier damage and impaired mucosal repair. In this study, Western blot results showed a significant decrease in the levels of the tight junction proteins ZO-1 and Occludin in the model group. ZO-1 protein expression was increased in the berberine + berberine group, the berberine extract group, the berberine group, and the positive control group, while berberine showed an increasing trend without significant variation. Occludin protein expression was significantly increased in the berberine + berberine group, the berberine extract group, the berberine group, and the mesalazine group, with the levels in the berberine + berberine group and the berberine extract group being higher than those in the berberine alone group.

[0152] The above results indicate that the combination of Phellodendron amurense extract, berberine, and berberine can promote the expression of intestinal-associated tight junction proteins ZO-1 and Occludin in mice with ulcerative colitis, maintaining intestinal barrier homeostasis and thus achieving a therapeutic effect on ulcerative colitis. The fact that berberine promotes Occludin expression and berberine promotes ZO-1 expression suggests that berberine and berberine have a synergistic effect in maintaining intestinal barrier function. Furthermore, the anti-ulcerative colitis effect of Phellodendron amurense extract containing both berberine and berberine is superior to that of berberine monomers or berberine monomers.

[0153] 4.3 Effects on the inflammatory factor IL-6 in the colonic tissue of mice with DSS-induced ulcerative colitis

[0154] The results are as follows Figure 13 The pathogenesis of ulcerative colitis involves multiple inflammatory factors, among which IL-6 is a highly representative inflammatory factor that affects the intestinal mucosal barrier function and the regulation of the immune system, leading to inflammatory responses and tissue damage. Results showed that IL-6 expression was significantly increased in the colonic tissue of model mice. Treatment with berberine + phellodendron chinense, phellodendron chinense extract, berberine, and phellodendron chinense groups significantly reduced IL-6 expression levels in the colonic tissue of mice, with phellodendron chinense and phellodendron chinense extract showing the greatest decrease.

[0155] The above results indicate that Phellodendron amurense extract, berberine, berberine, and the combination of berberine and berberine exhibit significant anti-inflammatory effects in mice. Among these, berberine and Phellodendron amurense extract showed the strongest effects. Therefore, berberine and berberine may exert a synergistic anti-inflammatory effect, suggesting that the anti-ulcerative colitis effect of Phellodendron amurense extract may be mainly based on the synergistic effect of berberine and berberine. 4.4 Regulatory effect on the NF-κB inflammatory signaling pathway

[0156] The results are as follows Figure 14 The NF-κB signaling pathway is a key intracellular signaling pathway that plays a central role in regulating various physiological processes such as immune responses, inflammatory responses, cell growth, and cell death. The pathogenesis of ulcerative colitis is closely related to the inflammatory response; therefore, this invention investigated the regulation of the NF-κB signaling pathway in mouse ulcerative colitis. Results showed that, compared to the control group, the phosphorylated NF-κB level was significantly increased in the model group. After drug administration, the berberine + phellodendron chinense group, phellodendron chinense extract, berberine, and phellodendron chinense all decreased the phosphorylated NF-κB level.

[0157] The above results indicate that Phellodendron amurense extract may exert anti-inflammatory and intestinal barrier protective effects by regulating the NF-κB inflammatory signaling pathway. Furthermore, both berberine and phellodendron chinense alkaloid can regulate this pathway, further confirming that the anti-ulcerative colitis effect of Phellodendron amurense extract may be based on the combined action of these two components.

Claims

1. A Phellodendri Cortex extract, characterized in that, Based on the total mass of the Phellodendron amurense extract, the Phellodendron amurense extract includes not less than 10% berberine and not less than 1% berberine.

2. The Phellodendron amurense extract according to claim 1, characterized in that, The mass ratio of berberine to phellodendron chinense is 10-30:1-5.

3. The Phellodendron amurense extract according to claim 1, characterized in that, Based on the total mass of the Phellodendron amurense extract, the Phellodendron amurense extract comprises 10%-30% berberine, 1%-5% berberine, 0.1%-0.5% magnoflorine, 0.01%-0.2% demethylene berberine, 0.01%-0.2% berberine, 0.01%-0.1% berberine, and 0.01%-0.1% palmatine.

4. A composition for preventing and treating enteritis, characterized in that, The composition comprises berberine and cyperamine; the mass ratio of berberine to berberine is 10-30:1-5.

5. A method for preparing the Phellodendron amurense extract according to any one of claims 1-3, characterized in that, Includes the following steps: Extract the medicinal material Phellodendron bark by adding an ethanol aqueous solution with a mass concentration of 30-90%, filter, and combine the filtrates to obtain the final product.

6. The preparation method according to claim 5, characterized in that, The step of merging the filtrate further includes: concentrating and drying the merged filtrate, wherein the concentration is vacuum concentration, the vacuum concentration temperature is 50-60℃, and the drying temperature is 55-65℃.

7. The preparation method according to claim 5, characterized in that, The extraction is a heating extraction at a temperature of 120-160℃, and the extraction is performed 1-3 times, with each extraction lasting 1-3 hours. The mass ratio of the Phellodendron bark medicinal material to the ethanol aqueous solution is 1:5-10.

8. A pharmaceutical preparation for the prevention and treatment of enteritis, characterized in that, The extract includes the Phellodendron amurense extract according to any one of claims 1-3, the composition according to claim 4, or the Phellodendron amurense extract prepared by any one of claims 5-7, as well as pharmaceutically acceptable excipients.

9. The use of a Phellodendron amurense extract according to any one of claims 1-3, or a composition according to claim 4, or a Phellodendron amurense extract obtained by any one of claims 5-7, or a pharmaceutical preparation according to claim 8, in the preparation of a drug for the prevention and treatment of inflammatory bowel disease and / or chronic enteritis.

10. The application according to claim 9, characterized in that, The inflammatory bowel disease includes ulcerative colitis and / or Crohn's disease.

11. The application according to claim 9, characterized in that, The chronic enteritis includes chronic inflammation of the intestines caused by bacterial, viral, and / or protozoan infections.

12. The use of a Phellodendron amurense extract according to any one of claims 1-3, or a composition according to claim 4, or a Phellodendron amurense extract obtained by any one of claims 5-7, or a pharmaceutical preparation according to claim 8, in the preparation of a medicament for improving intestinal barrier function in patients with inflammatory bowel disease and / or chronic enteritis.

13. The use of a Phellodendron amurense extract according to any one of claims 1-3, or a composition according to claim 4, or a Phellodendron amurense extract obtained by any one of claims 5-7, or a pharmaceutical preparation according to claim 8, in the preparation of a drug that exerts an anti-inflammatory effect on the intestine through the NF-κB signaling pathway.

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