Application of engelhardia roxburghiana leaf general flavone extract in preparation of medicine for preventing and treating ulcerative colitis

Abstract

The invention discloses an application of engelhardia roxburghiana leaf general flavone extract in preparation of a medicine for preventing / treating ulcerative colitis. Experiments prove that the engelhardia roxburghiana leaf total flavone extract can effectively improve pathological changes of intestinal tissues of ulcerative colitis model mice. According to the present invention, the engelhardia roxburghiana leaf total flavone extract provides significant inhibition and improvement effects for AOM / DSS induced DAI score, colon length, intestinal polyp quantity and pathological histological change, and can recover the expression of barrier proteins ZO-1 and Occludin by significantly reducing the levels of inflammatory factors IL-1beta, IL-6, TNF-alpha and IL-17A in intestinal tissues; inflammatory factor induced epithelial barrier function damage and permeability increase are relieved, and then the intestinal epithelial barrier function is maintained; the balance of intestinal flora is recovered, and the local microenvironment of the intestinal tract is improved, so that the effect of treating ulcerative colitis is achieved. The engelhardia roxburghiana leaf general flavone extract has an application prospect of treating ulcerative colitis diseases.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine, and in particular relates to application of a total flavonoid extract from flavonoids leaves in preparing medicines for preventing and treating ulcerative colitis. Background Art

[0002] Ulcerative colitis is a chronic, nonspecific intestinal inflammatory disease whose etiology is not yet fully understood. This disease mainly affects the rectum and colon, and often presents a chronic, recurrent course. Pathologically, chronic inflammatory cell infiltration and multiple ulcer formation can be seen in the colonic mucosa and submucosa. Patients usually experience persistent or recurrent diarrhea, bloody and purulent stools, abdominal pain and other symptoms, and may also be accompanied by a feeling of tenesmus. The severity of the disease varies. Mild cases have fewer diarrhea episodes per day and relatively mild abdominal pain; severe cases have frequent diarrhea, up to more than ten times a day, with large amounts of bloody and purulent stools, severe abdominal pain, and may also experience systemic symptoms such as fever and weight loss, which seriously affect the patient's quality of life. Long-term ulcerative colitis may also increase the risk of colon cancer.

[0003] Suffering from ulcerative colitis requires careful attention in many areas. Avoid spicy, greasy, and irritating foods, and reduce your intake of high-fiber foods to avoid increasing the burden on your intestines and irritating the intestinal mucosa. Get enough rest and avoid excessive fatigue and mental stress, as these factors can trigger an attack of the disease. At the same time, patients need to strictly follow the doctor's instructions for standardized treatment and should not stop or change medications on their own. Even after symptoms have eased, treatment must be maintained for a period of time. Regular colonoscopy is also important to promptly detect intestinal lesions, such as whether there is a tendency for cancer to develop.

[0004] Ulcerative colitis (UC) is a common disease affecting people aged 30-40. In recent years, the incidence of inflammatory bowel disease has shown a global spread, with rising incidence rates in many regions, particularly in Asia, the Middle East, and South America. Ulcerative colitis has a complex etiology, multiple stages of disease progression, and is difficult to cure. It is often recurrent and closely associated with colon cancer, making it listed by the World Health Organization as one of the most intractable diseases in modern times. Therefore, effective prevention, detection, and early treatment of inflammatory bowel disease are of great social significance.

[0005] Chemical induction is often used to construct induced adenomatous polyp models. Commonly used induction agents include dimethylhydrazine (DMH), AOM, and DSS. DMH itself is not carcinogenic, but it is oxidized in the endoplasmic reticulum of hepatocytes to methylazoxymethanol. This enters the intestinal lumen with bile, ultimately causing colorectal epithelial carcinogenesis. AOM, a metabolite of DMH in the liver, can also induce colon tumors, and its carcinogenicity is more stable and efficient than the former. As a chemical agent that induces gene mutation damage, AOM can cause guanine methylation, leading to excessive cell proliferation and the formation of specific colorectal adenomatous polyps. The inflammatory mechanism of DSS has not yet been elucidated, but it may be related to the negative charge of DSS affecting DNA synthesis, inhibiting epithelial cell proliferation, disrupting the intestinal mucosal barrier, and leading to macrophage dysfunction and intestinal flora imbalance.

[0006] After confluence, Caco-2 cells begin to spontaneously differentiate, gradually forming a polarized cell monolayer with apical and basal membranes, junctional complexes, and a brush border, with microvilli on the apical side. The formation of the junctional complex aligns with the typical characteristics of the human colonic epithelial barrier, leading to the widespread use of Caco-2 cell monolayers in in vitro studies of intestinal barrier function. TNF-α is primarily secreted by activated macrophages, dendritic cells, and T lymphocytes during the progression of intestinal adenomas. Excessive accumulation of TNF-α in the lamina propria attacks intestinal epithelial cells, leading to increased mucosal permeability, a key characteristic of intestinal epithelial barrier disruption. Summary of the Invention

[0007] The invention relates to application of a total flavonoid extract from flavonoids leaves in preparing medicine for preventing and treating ulcerative colitis.

[0008] Application of total flavonoids extract from huangqi leaves in preparing medicine for preventing and treating ulcerative colitis.

[0009] The invention relates to an application of the total flavonoids extract of the leaves of Astragalus membranaceus as the only effective part group in the preparation of a medicine for preventing and treating ulcerative colitis.

[0010] The invention discloses an application of the total flavonoids extract of Chinese wolfberry leaves in preparing a drug for preventing and treating ulcerative colitis. The preparation method of the total flavonoids extract of Chinese wolfberry leaves comprises the following steps: taking Chinese wolfberry leaves, adding ethanol for reflux extraction, combining the extracts, filtering, reducing the pressure on the filtrate to recover ethanol until there is no alcohol taste, adding water, centrifuging, passing the centrifuge through an AB-8 macroporous resin column, eluting with water and ethanol, collecting the ethanol eluate, reducing the pressure on the ethanol, concentrating to a clear paste, drying under reduced pressure, crushing, and adding auxiliary materials to prepare the extract into one of tablets, capsules, and granules.

[0011] The application of the total flavonoids extract of the Chinese wolfberry leaves in the preparation of a drug for preventing and treating ulcerative colitis is disclosed. The preparation method of the total flavonoids extract of the Chinese wolfberry leaves comprises the following steps: taking the Chinese wolfberry leaves, adding ethanol for reflux extraction, combining the extracts, filtering, reducing the pressure on the filtrate to recover ethanol until there is no alcohol taste, adding water, centrifuging, passing the centrifuge through an AB-8 macroporous resin column, eluting with water and ethanol, collecting the ethanol eluate, reducing the pressure on the ethanol recovery, concentrating to a clear paste, drying under reduced pressure, crushing, adding silicon dioxide, mixing, and encapsulating to obtain the drug.

[0012] The invention discloses a method for preparing a drug for preventing and treating ulcerative colitis by taking the Chinese wolfberry leaf, cutting it into strips about 1 cm wide, adding 10 times the amount of 60% ethanol and refluxing and extracting it twice, each time for 1.5 hours, combining the extracts, filtering, and recovering the ethanol from the filtrate under reduced pressure until there is no alcohol taste, adding water to adjust the crude drug concentration to 0.2 g / ml, centrifuging, passing the centrifuge through an AB-8 macroporous resin column with a diameter-to-height ratio of 1:7-1:12, eluting with 4 BV of water and 4 BV of 45% ethanol in sequence, collecting the ethanol eluate, recovering the ethanol under reduced pressure, concentrating it to a clear paste with a relative density of 1.20-1.25 (70° C.), drying it under reduced pressure at 65° C.-85° C., crushing it, passing it through a 60-mesh sieve, adding 1.5% of silicon dioxide, mixing it, and filling it into No. 1 capsules to prepare 1000 capsules.

[0013] The invention discloses a method for preparing a drug for preventing and treating ulcerative colitis by taking the total flavonoid extract of the Chinese wolfberry leaf and cutting it into strips with a width of about 1 cm, adding 10 times the amount of 60% ethanol and reflux extracting it twice, each time for 1.5 hours, combining the extracts, filtering, and recovering the ethanol from the filtrate under reduced pressure until there is no alcohol taste, adding water to adjust the crude drug concentration to 0.2 g / ml, centrifuging, and passing the centrifuge through an AB-8 macroporous resin column with a diameter-to-height ratio of 1:7-1:12, eluting with 4 BV of water and 4 BV of 45% ethanol in sequence, collecting the ethanol eluate, recovering the ethanol under reduced pressure, and concentrating it to a clear paste with a relative density of 1.20-1.25 (70° C.), drying it under reduced pressure at 65° C.-85° C., crushing it, passing it through a 60-mesh sieve, adding 1.5% of silicon dioxide, mixing it, and filling it into No. 1 capsules to prepare 1000 capsules.

[0014] The total flavonoids extract of the Chinese wolfberry leaves is used in the preparation of a drug for preventing and treating ulcerative colitis. The total flavonoids extract of the Chinese wolfberry leaves plays a role in preventing and / or treating ulcerative colitis by inhibiting inflammatory factors, restoring intestinal tight junction proteins, and inhibiting abnormal proliferation of the intestinal epithelium.

[0015] The total flavonoids extract of the Chinese wolfberry leaves is used in the preparation of a drug for preventing and treating ulcerative colitis. The total flavonoids extract of the Chinese wolfberry leaves increases beneficial bacteria, reduces harmful bacteria, and regulates the balance of intestinal flora.

[0016] The total flavonoids extract of the Chinese wolfberry leaves is used in the preparation of a drug for preventing and treating ulcerative colitis. The total flavonoids extract of the Chinese wolfberry leaves inhibits TNF-α from inducing the establishment of an intestinal epithelial barrier damage model, thereby improving the intestinal mucosal barrier function.

[0017] Application of total flavonoids extract from Aralia serrata leaves in the preparation of proliferation protein Ki-67 inhibitors.

[0018] Application of total flavonoids extract from Astragalus membranaceus leaves in the preparation of inhibitors of inflammatory factors IL-1β, IL-6, TNF-α and IL-17A.

[0019] Application of total flavonoids extract from Aralia serrata leaves in the preparation of barrier protein ZO-1 and Occludin agonists.

[0020] Application of total flavonoids extract from Aralia serrata leaves in the preparation of intestinal flora balance regulator.

[0021] Experiments have shown that the total flavonoid extract of Astragalus membranaceus leaves can effectively improve the pathological changes in the intestinal tissue of ulcerative colitis model mice. The total flavonoid extract of Astragalus membranaceus leaves has a significant inhibitory and ameliorative effect on AOM / DSS-induced DAI scores, colon length, number of intestinal polyps, and histopathological changes. This may be achieved by significantly reducing the levels of inflammatory factors IL-1β, IL-6, TNF-α, and IL-17A in intestinal tissue, restoring the expression of barrier proteins ZO-1 and Occludin; alleviating inflammatory factor-induced epithelial barrier function damage and increased permeability, thereby maintaining intestinal epithelial barrier function; restoring the balance of intestinal flora, and improving the local intestinal microenvironment to play a role in treating ulcerative colitis. The total flavonoid extract of Astragalus membranaceus leaves has a promising application in the treatment of ulcerative colitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings of the embodiments are briefly introduced below.

[0023] The drawings described below only relate to some embodiments of the present invention and are not intended to limit the present invention ("HQY" in the present invention refers to "total flavonoids extract from Engelhardtiaroxburghiana Wall."; Engelhardtiaroxburghiana Wall. is the leaf of the Juglandaceae plant Engelhardtiaroxburghiana Wall.).

[0024] In the attached figure:

[0025] Figure 1 This is the leakage curve of total flavonoids from Chinese wolfberry leaves on AB-8 macroporous resin;

[0026] Figure 2 is the specific adsorption capacity of the resin with different loading times;

[0027] Figure 3This is the AOM combined with DSS-induced mouse ulcerative colitis model and dosing regimen;

[0028] Figure 4 Effects of HQY total flavonoids extract on body weight of AOM / DSS-induced intestinal polyp model mice; (A) male mice; (B)

[0029] Female rat. ( Male rats, n=10; female rats, n=10; compared with the Control group, ###P<0.001; compared with the Model group, ***P<0.001).

[0030] Figure 5 The effect of HQY total flavonoids extract on the DAI score of AOM / DSS-induced intestinal polyp model mice; (AB) DAI of male mice; (CD) DAI of female mice. Male rats, n=10; female rats, n=10; compared with the Control group, ###P<0.001; compared with the Model group, *P<0.05; ***P<0.001).

[0031] Figure 6 The effects of HQY total flavonoids extract on the colorectal length, polyp number and area of ​​AOM / DSS-induced intestinal polyp model mice; (AB) Colorectal length: (A) male mice, (B) female mice; (CD) Polyp number: (C) male mice, (D) female mice; (EF) Polyp area: (E) male mice, (F) female mice; (GH) Number of polyps of different diameters: (G) male mice, (H) female mice; (IJ) Intuitive images under stereomicroscope: (I) male mice; (J) female mice. ( Male rats, n=10; female rats, n=10; compared with the Control group, #P<0.05, ###P<0.001; compared with the Model group, *P<0.05; **P<0.01; ***P<0.001; scale bar: 1 mm).

[0032] Figure 7 Effects of HQY total flavonoid extract on the histopathological changes of intestinal polyps in AOM / DSS-induced intestinal polyps mice; (A) male mice; (B) female mice. (Magnification: ×200, Scale bar: 100 μm).

[0033] Figure 8 Effects of HQY total flavonoids extract on the content of inflammatory factors in colorectal tissues of AOM / DSS-induced intestinal polyp model mice; (A) IL-1β, male mice; (B) IL-1β, female mice; (C) IL-6, male mice; (D) IL-6, female mice; (E) TNF-α, male mice; (F) TNF-α, female mice; (G) IL-17A, male mice; (H) IL-17A, female mice. ( Male rats, n=10; female rats, n=10; compared with the Control group, ###P<0.001; compared with the Model group, *P<0.05; **P<0.01; ***P<0.001).

[0034] Figure 9 Effects of HQY total flavonoid extract on ZO-1 and occludin in colorectal tissues of AOM / DSS-induced intestinal polyp model mice; (A) ZO-1; (B) Occludin. (n = 6, magnification: ×200, scale bar: 100 μm).

[0035] Figure 10 The effect of HQY total flavonoids extract on Ki-67 expression in colorectal tissues of AOM / DSS-induced intestinal polyp model mice (n=6, magnification: ×200, scale bar: 100 μm).

[0036] Figure 11 Flowchart for sequencing data analysis;

[0037] Figure 12 Classification and evaluation of species in samples; (A) Taxonomic annotation of intestinal flora in each fecal group, indicating the number of kingdom, phylum, class, order, family, genus, and species detected in each fecal group; (B) Mean taxonomic score of species in each sample group; (C) Rank-Abundance curve.

[0038] Figure 13 Alpha diversity analysis; (AC) Diversity index: (A) from left to right are sobs, ace and chaos, (B) shannon, simpson, (C) coverage; (D) rarefaction curve.

[0039] Figure 14 for beta diversity analysis;

[0040] Figure 15 Species composition analysis; (AB) Community composition bar chart: (A) Genus, (B) Species; (CD) Community composition Heatmap chart: (C) Genus, (D) Species; (EF) Circos sample and species relationship chart: (E) Genus, (F) Species.

[0041] Figure 16 Analysis of species differences affected by HQY; (A) Genus; (B) Species.

[0042] Figure 17 The TEER change trend of Caco-2 cell monolayer; ( n=15).

[0043] Figure 18 Effects of HQY total flavonoid extract on the barrier function of Caco-2 cell monolayer after TNF-α stimulation; (A) TEER of Caco-2 cell monolayer culture at D23-D25; (B) 4kDa FITC-dextran permeability (FITC-Dextran flux) of Caco-2 cell monolayer at D25. n=3, compared with the Ctrl group, ###P<0.001, compared with the TNF-α group, *P<0.05, **P<0.01, ***P<0.001). DETAILED DESCRIPTION

[0044] To make the purpose, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the described embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0045] Example 1: Screening of the preparation process of the total flavonoids extract from the leaves of Astragalus membranaceus

[0046] 1. Single factor experimental method to compare the extraction process

[0047] This study used the single-factor experimental method to compare the effects of different ethanol concentrations, extraction time, ethanol dosage, and extraction times on the extraction of total flavonoids from Chinese wolfberry leaves, determine the level range of each factor, and provide a basis for determining the level of orthogonal experimental factors.

[0048] 1.1 Comparison of the extraction effects of different concentrations of ethanol on total flavonoids from Chinese wolfberry leaves

[0049] Take 50 g of yellow Qi leaves, add 10 times the amount of 50%, 60%, 70%, 80% ethanol, reflux extraction twice, each time for 60 min, combine the extract, mix well, and measure the total volume. Precisely take 0.5 ml of the extract, prepare the test solution according to the test solution preparation method, precisely take 20 μl, inject into the liquid chromatograph, and determine the total flavonoid content in the extract. The chromatographic conditions are as follows: column: Philomena Super Lu C18 (250 mm x 4.5 mm, 5 μm), mobile phase: acetonitrile-0.1% formic acid solution (19:81), flow rate: 1 ml / min, column temperature: 30°C, detection wavelength: 255 nm (quercitrin), 290 nm (newly fallen new wife glycoside, new wife glycoside, new isofallen new wife glycoside, isofallen new wife glycoside, yellow Qi glycoside), injection volume: 20 μl. Preparation of the reference solution: take an appropriate amount of new fallen new wife glycoside, new wife glycoside, new isofallen new wife glycoside, isofallen new wife glycoside, quercitrin and yellow Qi glycoside reference substances, accurately weigh and add methanol to prepare a mixed solution containing 0.02528 mg of new fallen new wife glycoside, 0.47511 mg of new wife glycoside, 0.07401 mg of new isofallen new wife glycoside, 0.050299 mg of isofallen new wife glycoside, 0.02417 mg of quercitrin and 0.23865 mg of yellow Qi glycoside per 1 ml, and the reference solution is obtained. Preparation of the test solution: accurately take 0.5 ml of the extract, place it in a 25 ml volumetric flask, dilute to the mark with methanol, shake well, pass through a 0.45 μm microporous filter, and take the filtrate, which is the test solution. The results are shown in Table 1.

[0050] Table 1 Comparison of the effects of different concentrations of ethanol on the extraction of total flavonoids from yellow Qi leaves (n=2, mg / g)

[0051]

[0052] From Table 1, it can be seen that the total flavonoid content extracted from yellow Qi leaves with 50% to 80% ethanol is not significantly different, and the total flavonoid content extracted with 70% ethanol is the highest. Therefore, 70% ethanol is selected as the extraction solvent for the next single factor experiment.

[0053] 1.2 Comparison of the effects of different extraction times on the extraction of total flavonoids from yellow Qi leaves

[0054] Take 50 g of yellow Qi leaves, add 10 times the amount of 70% ethanol, and extract for 30 min, 60 min, 90 min and 120 min, respectively. Each extraction time is refluxed twice, the extract is combined, mixed well, and the total volume is measured. Precisely take 0.5 ml of the extract, prepare the test solution according to the test solution preparation method, precisely take 20 μl, inject into the liquid chromatograph, and determine the total flavonoid content in the extract. The results are shown in Table 2.

[0055] Table 2 Comparison of the effects of different extraction times on the extraction of total flavonoids from yellow Qi leaves (n=2, mg / g)

[0056]

[0057] As shown in Table 2, with the increase of extraction time, the total flavonoids content gradually increased, but the increase in the total flavonoids content was small. Therefore, the relatively time-saving intermediate level extraction time of 60 min was selected for the next single-factor experimental examination.

[0058] 1.3 Comparison of the extraction effects of different ethanol dosages on total flavonoids from Chinese wolfberry leaves

[0059] Weigh 50g of Aralia dahurica leaves and add 8x, 10x, 12x, and 14x the volume of 70% ethanol, respectively. Reflux extract twice for 60 min each time. Combine the extracts, mix thoroughly, and measure the total volume. Accurately measure 0.5ml of the extract and prepare the test solution according to the test solution preparation method. Accurately pipette 20μl and inject it into the liquid chromatograph to determine the total flavonoid content in the extract. The results are shown in Table 3.

[0060] Table 3 Comparison of the extraction effects of different ethanol dosages on total flavonoids from flavonoids in flavonoid leaves (n=2, mg / g)

[0061]

[0062] As shown in Table 3, the total flavonoids content in the leaves of Astragalus membranaceus increased slightly when extracted with 8 to 14 times the amount of 70% ethanol, and the difference was not obvious. However, it was found in the experiment that the first extraction with 8 times the amount of 70% ethanol could not completely immerse the leaves of Astragalus membranaceus. Therefore, a solid-liquid ratio of 10 times was selected for the next single-factor experimental examination.

[0063] 1.4 Comparison of the extraction effects of different extraction times on total flavonoids from Chinese wolfberry leaves

[0064] Weigh 50g of Aralia dahurica leaves and extract them under reflux four times with 10-fold the volume of 70% ethanol for 60 minutes each time. Collect the extracts and measure the volume of each extract. Accurately measure 0.5ml of the extract and prepare the test solution according to the test solution preparation method. Accurately pipette 20μl of the extract and inject it into the liquid chromatograph. Determine the total flavonoid content of each extract. The results are shown in Table 4.

[0065] Table 4 Comparison of the extraction effects of different extraction times on total flavonoids from flavonoids in flavonoid leaves (n=2, mg / g)

[0066]

[0067] As shown in Table 4, the total flavonoids extracted twice accounted for 93.7% of the total flavonoids extracted four times, and the third and fourth extractions accounted for a very small proportion. From the perspective of saving energy, improving production efficiency, and reducing production costs, it was determined that two extractions were sufficient.

[0068] 2. Orthogonal test to optimize the extraction process

[0069] 2.1 Factor Level Design

[0070] In this study, based on the level range of different factors determined by single factor experiment, L9(3 4 Using an orthogonal design method, we used the total flavonoid content in the extract as the evaluation criterion to examine the effects of three factors: ethanol dosage (A), extraction time (B), and ethanol concentration (C) on the extraction effect. We then conducted range analysis and variance analysis on the experimental results to prioritize the optimal extraction process parameters. The factor levels and orthogonal design arrangements are shown in Tables 5 and 6.

[0071] Table 5 Levels of factors in ethanol extraction of Lycopodiella vulgaris leaves

[0072]

[0073] 2.2 Preparation of orthogonal test extracts

[0074] Table 6 Orthogonal test table

[0075]

[0076] Weigh 50 g of Huangqi leaves, a total of 9 portions, and extract according to the orthogonal experimental arrangement in Table 6. Extract twice in each experiment, filter, combine the extracts, measure the volume of the extracts, and set aside.

[0077] 2.3 Orthogonal test results

[0078] The total flavonoids extraction amount was subjected to visual analysis and variance analysis, and the results are shown in Tables 7 and 8.

[0079] Table 7 Orthogonal test results and intuitive analysis of ethanol extraction of Chinese wolfberry leaves

[0080]

[0081] Note: Total flavonoids extraction (mg / g) refers to the amount of total flavonoids extracted from each gram of huangqi leaves (mg)

[0082] Table 8 Orthogonal test variance analysis results

[0083]

[0084] Note: F 0.05 (2,2)=19.0,F 0.01 (2,2)=99.0

[0085] Based on the results of range analysis and variance analysis, the most suitable process was determined to be A2B3C1, that is, adding 10 times the amount of 60% ethanol to the leaves of Astragalus membranaceus and extracting them twice, each time for 1.5 hours.

[0086] 3. Research on ethanol recovery and concentration process of extract

[0087] Common concentration methods for ethanol extracts include atmospheric pressure concentration and vacuum concentration. Atmospheric pressure concentration is slow and requires high temperature, and the active ingredients may be easily destroyed at high temperatures. Vacuum concentration overcomes the shortcomings of atmospheric pressure concentration and has the advantages of being fast, efficient, and having a low concentration temperature. It is the most commonly used concentration method in industrial production. Therefore, this experiment combines production practice to investigate the vacuum concentration parameters.

[0088] The main factor affecting vacuum concentration is temperature. In this study, the transfer rate of total flavonoids from Chinese wolfberry leaves was used as the investigation index. The transfer rates of the index components before and after concentration at three different temperatures of 65℃, 75℃, and 85℃ (vacuum degree -0.06Mpa to -0.08Mpa) were compared to determine the most suitable concentration temperature.

[0089] 3.1 Preparation of sample solutions concentrated under reduced pressure at different temperatures

[0090] Weigh 900g of Aralia dahurica leaves, add 10 times the amount of 60% ethanol, heat and reflux extraction twice, each time for 1.5 hours, combine the extracts, mix, measure the total volume to be 16100ml, measure 5000ml, a total of three portions, respectively, at 65℃, 75℃, and 85℃ (vacuum degree -0.06Mpa~-0.08Mpa), reduce pressure to recover ethanol, and concentrate until there is no alcohol taste, to obtain 500ml of concentrated solution, the crude drug concentration is 0.56g / ml.

[0091] 3.2 Preparation of test solution before and after concentration

[0092] Preparation of the test solution before concentration: Accurately measure 0.5 ml of the pre-concentration drug solution, place it in a 25 ml volumetric flask, add methanol to dilute to the scale, shake well, filter through a 0.45 μm microporous membrane, and take the filtrate to obtain the product.

[0093] Preparation of the test solution after concentration: accurately measure 1 ml of the concentrate, place it in a 10 ml volumetric flask, add methanol to dilute to the scale, shake well, accurately measure 0.5 ml, place it in a 25 ml volumetric flask, add methanol to dilute to the scale, shake well, filter through a 0.45 μm microporous membrane, and take the filtrate to obtain the product.

[0094] 3.3 Determination of total flavonoid content

[0095] Accurately pipette 20 μl of each of the reference solution and the test solution and inject them into the liquid chromatograph. Determine the total flavonoid content according to the chromatographic conditions and calculate the transfer rate. The results are shown in Table 9.

[0096] Table 9 Results of the investigation on the vacuum concentration temperature

[0097]

[0098] From Table 9, the total flavone transfer rate of the extract liquid is above 94% under different temperature and reduced pressure concentration, and the higher the temperature, the shorter the concentration time. Therefore, the temperature for reduced pressure concentration in industrial production is controlled in the range of 65-85°C, and the components are basically stable.

[0099] 4. Purification process research

[0100] According to the process design, the extraction and concentration liquid of Engelhardia roxburghiana Wall needs to be further purified by macroporous resin to obtain the total flavone extract liquid. Before being loaded on the macroporous resin, the sample liquid needs to be centrifuged to prevent the macroporous resin column from being blocked. Therefore, the effect of centrifugation process on the total flavone transfer rate of Engelhardia roxburghiana Wall is first investigated, and then the macroporous resin purification process is optimized to determine the process parameters.

[0101] 4.1 Centrifugation impurity removal process research

[0102] 4.1.1 Preparation of sample liquid under different rotation speeds

[0103] 10 ml of Engelhardia roxburghiana Wall concentration liquid with a crude drug concentration of 0.2 g / ml was taken in two portions, and centrifuged at rotation speeds of 4000 rpm and 12000 rpm (Microfuge 16 high-speed centrifuge, Beckman Company) for 30 min, respectively. The supernatant was taken, and its volume was measured.

[0104] 4.1.2 Preparation of test sample solution before and after centrifugation

[0105] 1 ml of sample liquid before and after centrifugation was accurately taken and placed in a 5 ml volumetric flask, diluted to the mark with methanol, shaken uniformly, 0.5 ml was accurately taken and placed in a 25 ml volumetric flask, diluted to the mark with methanol, shaken uniformly, filtered through a 0.45 μm microporous filter membrane, and the filtrate was taken.

[0106] 4.1.3 Determination of total flavone content

[0107] 20 μl of the control sample solution and the test sample solution was accurately taken and injected into the liquid chromatograph, the total flavone content was determined according to the chromatographic conditions in item 3.3, and the transfer rate was calculated. The results are shown in Table 10.

[0108] Table 10 Investigation results of centrifugation process

[0109]

[0110] From Table 10, the total flavone transfer rate of Engelhardia roxburghiana Wall is above 90% under rotation speeds of 4000 rpm and 12000 rpm, indicating that the total flavone loss is small after centrifugation of the concentration liquid, and the centrifugation process can be used for preliminary impurity removal before loading the concentration liquid on the macroporous resin.

[0111] 4.2 Macroporous resin purification process research

[0112] Macroporous adsorption resins are porous, three-dimensional polymer materials with selective adsorption capabilities. They offer advantages such as low cost, high efficiency, wide applicability, ease of operation, and non-toxicity. They are primarily used for the separation and purification of active ingredients in natural medicinal products. Currently, there are numerous reports on the use of macroporous adsorption resins for the separation and purification of total flavonoids. Therefore, this study compared the purification efficiency of several common macroporous adsorption resins for total flavonoids from the leaves of Astragalus membranaceus. A single-factor experiment was performed to determine the optimal purification parameters for total flavonoids from Astragalus membranaceus leaves.

[0113] 4.2.1 Determination of total flavonoids content and solid content

[0114] Determination of total flavonoids content: The determination method of total flavonoids content of the test samples prepared in each experiment of this study was carried out in accordance with the chromatographic conditions under item "3.3".

[0115] Determination of solid content: Accurately measure 5-20 ml of the test liquid, place it in a watch glass, evaporate it to dryness in a water bath, place it in an oven at 105°C for 3 hours, take it out, place it in a desiccator, cool it for 30 minutes, quickly weigh it, and calculate the solid content.

[0116] 4.2.2 Preparation of the sample solution of Lycopodiella vulgaris leaves

[0117] According to the parameters determined by the previous extraction process of total flavonoids from Chinese wolfberry leaves, 100g of Chinese wolfberry leaves were taken, accurately weighed, and 10 times the amount of 60% ethanol was added. Reflux extraction was performed twice, each time for 1.5 hours. The two extracts were combined, filtered while hot, and the ethanol was recovered under reduced pressure and concentrated until there was no alcohol taste. Water was added to make the volume 400ml to obtain an extract with a crude drug concentration of 0.25g / ml. The extract was centrifuged while hot and the supernatant was taken.

[0118] 4.2.3 Pretreatment of macroporous resin

[0119] The macroporous resins of different models were pretreated according to their specifications. AB-8 (Batch No.: 009IS228098, Shanghai Yuanye Biotechnology Co., Ltd.) was soaked in 4% NaOH for 24 hours, washed with ultrapure water until neutral, soaked in 95% ethanol for 4 hours, and washed with ultrapure water until there was no alcohol smell, and then used; HPD100 (Batch No.: T24A9X59689, Shanghai Yuanye Biotechnology Co., Ltd.), HPD300 (Batch No.: F13IS206267, Shanghai Yuanye Biotechnology Co., Ltd.), HPD600 (Batch No.: J04HS179830, Shanghai Yuanye Biotechnology Co., Ltd.), D101 (Batch No.: D 20GS171830, Shanghai Yuanye Biotechnology Co., Ltd.), DM130 (Batch No.: T11D8X50200, Shanghai Yuanye Biotechnology Co., Ltd.), DM301 (Batch No.: T12O9X71783, Shanghai Yuanye Biotechnology Co., Ltd.), HP20 (Batch No.: N11HS200762, Shanghai Yuanye Biotechnology Co., Ltd.), and NKA-9 (Batch No.: B16S8E44075, Shanghai Yuanye Biotechnology Co., Ltd.) were first soaked in 95% ethanol for 24 h to fully swell, and then washed with ultrapure water until there was no alcohol smell and set aside.

[0120] 4.2.4 Screening of macroporous resins

[0121] This study adopted the static adsorption-desorption method, with specific adsorption capacity and desorption rate as the investigation indicators, and the macroporous resin model was selected: about 5g (wet weight) of pretreated HPD100, HPD300, HPD600, D101, DM130, DM301, HP20, NKA-9, AB-8 macroporous resin was taken, accurately weighed, and placed in a 100ml stoppered conical flask, and 20ml of the prepared Huangqi leaf sample solution with a crude drug concentration of 0.25g / ml was added. The resins were shaken at a constant temperature of 25°C and 100 rpm for 24 hours (model KS3000icontrol, IKA, Germany). The mixture was filtered, and the filtrate was aspirated to determine the total flavonoid content. The specific adsorption capacity of total flavonoids from the leaves of the Chinese wolfberry leaf at 25°C was calculated for each resin. The saturated macroporous resins were then rinsed with ultrapure water and desorbed by adding 20 ml of 80% ethanol. The resins were shaken at a constant temperature of 25°C and 100 rpm for 24 hours, and the desorption rates were calculated. The results are shown in Table 11.

[0122] Specific adsorption capacity = (C0-C1) × V1 / m

[0123] Adsorption rate = (C0-C1)C0×100%

[0124] Specific resolution = C2 × V2 / m

[0125] Desorption rate = [C2 × V2 (C0 V1 - C1 V1)] × 100%

[0126] Note: C0 is the initial mass concentration, C1 is the mass concentration after adsorption, m is the mass of the resin, C2 is the mass concentration after desorption, V1 is the volume of the extract, V2 is the volume of the desorption liquid

[0127] Table 11 Adsorption capacity and desorption rate of total flavonoids from Chinese wolfberry leaves by different types of macroporous resins

[0128]

[0129] As shown in Table 11, the AB-8 macroporous resin has the largest static specific adsorption capacity and the highest desorption rate. Therefore, the AB-8 macroporous resin was selected to purify the total flavonoids from the leaves of Astragalus membranaceus.

[0130] 4.2.5 Drawing of the total flavonoids leakage curve of Chinese wolfberry leaves

[0131] Take about 20g (wet weight, column bed volume of about 20ml) of pretreated AB-8 macroporous resin, accurately weigh it, and slowly load it into the (1.5cm×40cm) adsorption column by wet method, with a diameter-to-height ratio of 1:9 (1BV=20ml). At room temperature, add the Huangqi leaf extract (total flavonoids concentration is 39.1mg / ml) prepared with a crude drug concentration of 0.25g / ml to the resin column at a volume flow rate of 1.0ml / min, and collect the effluent in sections, collecting 1 portion for every 5ml of effluent, and collecting 15 portions. Determine the total flavonoids content in each portion of the effluent according to the chromatographic conditions under "3.3", and draw a leakage curve. The results showed that in the second portion of the effluent, the total flavonoids of Huangqi leaves began to leak out. After the 11th portion, the mass concentration of the total flavonoids in the effluent reached equilibrium, and the macroporous resin adsorption was basically saturated. Therefore, the sample volume was selected as 55ml, i.e. 2.75BV. (See Figure 1 Leakage curve of total flavonoids from Chinese wolfberry leaves on AB-8 macroporous resin

[0132] 4.2.6 Investigation of sample concentration

[0133] Approximately 20 g (wet mass, approximately 20 ml bed volume) of pretreated macroporous resin AB-8 was accurately weighed and slowly loaded into a 1.5 cm × 40 cm adsorption column using a wet method, with a diameter-to-height ratio of 1:9. 55 ml (2.75 BV) of each loading solution of Chinese wolfberry leaf were prepared with crude drug concentrations of 0.1, 0.2, 0.3, 0.4, and 0.5 g / ml, respectively. The column was loaded at a flow rate of 1.0 ml / min, and the effluent was collected. The total flavonoid concentration in each effluent was determined according to the chromatographic conditions under "3.3." The specific adsorption capacity and adsorption rate were calculated. The results are shown in Table 12.

[0134] Table 12 Adsorption of AB-8 macroporous resin at different sample concentrations

[0135]

[0136] Table 12 shows that as the sample concentration increases, the specific adsorption capacity gradually increases, the growth rate gradually slows, and the adsorption rate gradually decreases. When the sample concentration of the crude drug is ≥0.3 g / ml, the adsorption rate drops significantly. At a sample concentration of 0.2 g / ml, the adsorption rate is 87.73%, which is a relatively high specific adsorption capacity. Considering these two factors, a sample concentration of 0.2 g / ml is more appropriate.

[0137] 4.2.7 Investigation of adsorption column diameter-to-height ratio

[0138] About 16, 20, 27, and 33 g (wet mass, column bed volume of about 17, 20, 28, and 33 ml) of pretreated AB-8 macroporous resin were taken and accurately weighed, a total of 4 portions, and slowly loaded into an adsorption column (1.5 cm × 40 cm) by a wet method. The diameter-to-height ratio was set to 1:7, 1:9, 1:12, and 1:14, respectively. 2.75 BV of the Huangqi leaf sample solution with a crude drug concentration of 0.2 g / ml was measured and passed through the resin column at a volume flow rate of 1.0 ml / min. The effluent was collected and the total flavonoids concentration in the effluent was determined according to the chromatographic conditions under "3.3". The specific adsorption amount and adsorption rate under different diameter-to-height ratios were calculated. The results are shown in Table 13.

[0139] Table 13 Adsorption of AB-8 macroporous resin with different diameter-to-height ratios

[0140]

[0141] It can be seen from Table 13 that when the diameter-to-height ratio is 1:7 to 1:12, the specific adsorption capacity of AB-8 macroporous resin is relatively close, and when the diameter-to-height ratio is 1:15, the specific adsorption capacity decreases slightly. Therefore, the diameter-to-height ratio can be determined to be 1:7 to 1:12.

[0142] 4.2.8 pH investigation of sample solution

[0143] Approximately 16 g (wet weight, column bed volume approximately 17 ml) of pretreated AB-8 macroporous resin was accurately weighed and loaded into a total of 6 portions using a wet method. The mixture was slowly loaded into a (1.5 cm × 40 cm) adsorption column with a diameter-to-height ratio of 1:7. A sample solution of 46.75 ml (2.75 BV) of the flavonoid leaf solution with a crude drug concentration of 0.2 g / ml was measured and adjusted to pH values ​​of 2.0, 3.5, 4.5, 5.5, 7.0, and 9.0 using 0.1 mol / l hydrochloric acid and 0.1 mol / l sodium hydroxide. The solution was passed through the resin column at a volume flow rate of 1.0 ml / min. The effluent was collected and the total flavonoid content of each effluent was determined according to the chromatographic conditions under "3.3". The specific adsorption amount and adsorption rate were calculated. The results are shown in Table 14.

[0144] Table 14 Adsorption of AB-8 macroporous resin at different loading pH

[0145]

[0146] From Table 14, when the pH value of the sample solution is 4.5, the specific adsorption capacity of AB-8 macroporous resin is the largest, and the adsorption rate is also at a higher level, so it is more appropriate to select the pH value of the sample solution as 4.5. Since the pH value of the sample solution is about 4.5, the sample solution does not need to be adjusted in pH.

[0147] 4.2.9 Eluent concentration investigation

[0148] About 16 g of pretreated AB-8 macroporous resin (wet mass, column bed volume about 17 ml) was precisely weighed and slowly loaded into the adsorption column (1.5 cm x 40 cm) by wet method (the ratio of diameter to height is 1:7). The sample solution of 46.75 ml (2.75 BV) of 0.2 g / ml of crude drug concentration of Engelhardtia roxburghiana leaves was loaded at a volume flow rate of 1 ml / min. After adsorption saturation, 5 BV of ultrapure water and 10%, 15%, 20%, 30%, 45%, 65%, 85%, and 95% ethanol were used for gradient elution, respectively. The eluate was collected, wherein 1 BV of ultrapure water was collected for each time, and 5 BV of each ethanol concentration was collected. The total flavonoid content in each eluate was determined according to the chromatographic conditions in item 3.3, and the solid content of the eluate was determined. The total flavonoid elution amount and purity in different concentrations of ethanol eluate were calculated. The results are shown in Tables 15-16.

[0149] Table 15 Total flavonoid content and proportion in different volume of water eluate

[0150]

[0151] Table 16 Total flavonoid elution amount and purity in different concentrations of ethanol eluate

[0152]

[0153] From Table 15, 4 BV of ultrapure water can elute 92.7% of the total flavonoids that are not adsorbed by macroporous resin, and can elute 95.2% of the impurities that are not adsorbed, so 4 BV of ultrapure water can basically elute the impurities that are not adsorbed before ethanol elution. From Table 16, in different concentrations of ethanol eluate, the total flavonoid content eluted by 10% to 45% ethanol accounts for 99.7% of the total amount, the total flavonoid content eluted by 65% ethanol accounts for only 0.3% of the total amount, and the purity of total flavonoids is only 3.13%. Therefore, it is more appropriate to select 45% ethanol as the eluent for elution.

[0154] 4.2.10 Eluent volume investigation

[0155] Take approximately 16g (wet mass, column bed volume approximately 17ml) of pretreated AB-8 macroporous resin, accurately weigh it, and slowly load it into a (1.5cm×40cm) adsorption column using a wet method with a diameter-to-height ratio of 1:7. Load 46.75ml (2.75BV) of the Huangqi leaf sample solution with a crude drug concentration of 0.2g / ml at a volume flow rate of 1ml / min. After adsorption saturation, elute with 4BV of ultrapure water and then 9BV of 45% ethanol. Collect one portion of the eluate for every 1BV, for a total of 9 portions. Determine the total flavonoid content in the eluate according to the chromatographic conditions under "3.3" and calculate the percentage. The results are shown in Table 17.

[0156] Table 17 Total flavonoids elution amount in different concentrations of ethanol eluent

[0157]

[0158] As shown in Table 17, the amount of total flavonoids eluted with 4BV of 45% ethanol accounts for 98.14% of the total elution amount, indicating that this volume of eluent can basically elute the total flavonoids, so it is more appropriate to choose 4BV elution solvent for elution.

[0159] 4.2.11 Durability evaluation of AB-8 macroporous resin column

[0160] Investigation on the number of times the adsorption column was reused: Weigh about 16g (wet mass, column bed volume 17ml, diameter-to-height ratio: 1:7) of pretreated AB-8 macroporous adsorption resin and slowly load it into the (1.5cm×40cm) adsorption column using a wet method. 46.75ml (2.75BV) of the Huangqi leaf sample solution with a crude drug concentration of 0.2g / ml was passed through the resin column at a volume flow rate of 1.0ml / min, and the effluent was collected; eluted with 95% ethanol until the effluent was almost colorless, and then eluted with pure water until the effluent had no alcohol taste. According to the above method, the sample loading and elution were repeated 25 times, and the effluent of each sample loading was collected. The total flavonoid content in the sample solution and the effluent was determined according to the chromatographic conditions under "3.3", and the specific adsorption rate was calculated. The results are shown in Tables 18 and Figure 2 (Specific adsorption capacity of resin for different loading times).

[0161] Table 18 Specific adsorption capacity (mg / g) at different loading times

[0162]

[0163] As shown in Table 18, the specific adsorption capacity of the AB-8 macroporous resin did not decrease significantly after 19 consecutive sample loadings. It decreased slightly from the 20th time. After the 25th sample loading, the specific adsorption capacity of the AB-8 macroporous resin was 69.32 mg / g, which was 85.6% of the adsorption capacity of the macroporous resin at the first sample loading. This shows that the AB-8 macroporous resin can be reused at least 25 times under this process.

[0164] Adsorption column regeneration investigation: Measure 10BV of 4% sodium hydroxide solution, pass it through the macroporous resin adsorption column with the above-mentioned continuous sample loading 25 times until the effluent is almost colorless, and elute it with pure water to neutrality; measure 10BV of 95% ethanol, add it to the macroporous adsorption resin column, soak it for 4 hours, and wash it with pure water after elution until the effluent has no alcohol taste, completing the regeneration of the adsorption column.

[0165] Measure 46.75 mL of a 0.2 g / mL sample of huangqi leaf from a crude drug concentration and pass it through a regenerated macroporous adsorption resin column at a flow rate of 1.0 mL / min. Collect the effluent and determine the total flavonoid content according to the chromatographic conditions in "3.3." Calculate the specific adsorption rate. The results are shown in Table 19.

[0166] Table 19 Specific adsorption capacity of regenerated AB-8 macroporous resin (mg / g)

[0167]

[0168] As shown in Table 19, the specific adsorption capacity of the regenerated AB-8 macroporous resin is 82.78 mg / g, which is restored to the adsorption level of a new macroporous resin, indicating that the AB-8 macroporous resin can be reused after regeneration.

[0169] 4.2.12. Determination of the optimal process

[0170] According to the above experimental results, the process was determined as follows: taking Huang Qi leaves, cutting them into strips about 1 cm wide, adding 10 times the amount of 60% ethanol and refluxing and extracting them twice, each time for 1.5 hours, combining the extracts, filtering, and recovering ethanol from the filtrate under reduced pressure until there was no alcohol taste, adding water to adjust the crude drug concentration to 0.2 g / ml, centrifuging, and passing the centrifuge through an AB-8 macroporous resin column with a diameter-to-height ratio of 1:7-1:12, eluting with 4 BV of water and 4 BV of 45% ethanol in sequence, collecting the ethanol eluate, recovering ethanol under reduced pressure, and concentrating to a clear paste with a relative density of 1.20-1.25 (70°C).

[0171] Example 2: Preparation process of HQY total flavonoid extract capsules

[0172] Extraction process: Weigh an appropriate amount of yellow wolfberry leaves, cut them into strips about 1 cm wide, weigh 100 kg, add them to a multi-functional extraction tank, cover the feed cover, add 1000 L of 60% ethanol, heat to boiling, keep boiling slightly for 1.5 hours, self-circulate for 5 minutes every 25 minutes, extract twice in total, combine the extracts, and filter through a 350-mesh filter to obtain the extract.

[0173] Extraction solution ethanol recovery and concentration process: The extract is transported to a concentration tank through a pipeline, and ethanol is recovered under reduced pressure at a temperature of 65°C to 85°C and a vacuum degree of -0.06Mpa to -0.08Mpa. The extract is concentrated until there is no alcohol taste. The relative density is measured to be about 1.02 (70°C). The crude drug concentration is adjusted to 0.2g / ml to obtain 500L of concentrated solution.

[0174] Purification process: The concentrate was centrifuged in a tubular centrifuge (15,000 rpm) and used as the loading solution. 45 kg of pretreated AB-8 macroporous resin (approximately 45.5 L bed volume) was slowly wet-loaded onto a 0.2 m × 2.0 m adsorption column with a diameter-to-height ratio of 1:7. The loading solution was divided into four equal portions, each approximately 125 L, and loaded at a rate of 2.5 L / min. The solution was eluted sequentially with 4 BV of water and 4 BV of 45% ethanol, and the ethanol eluate was collected.

[0175] Eluate recovery and concentration process: The ethanol eluate is transported to a concentration tank, and the ethanol is recovered under reduced pressure at a temperature of 65°C to 85°C and a vacuum degree of -0.06Mpa to -0.08Mpa, and concentrated to a clear paste with a relative density of about 1.20 to 1.25 (70°C).

[0176] Drying process: Transfer the concentrated clear paste to a clean area and place it in a drying oven for reduced pressure drying (set temperature at 65°C to 85°C, vacuum degree at -0.06Mpa to -0.08Mpa, the vacuum degree should be adjusted according to the temperature to prevent overflow) for 6h to 8h, ensuring that the water content is less than 6% to obtain a dry extract.

[0177] Crushing and screening process: the dry extract is crushed and passed through a 60 mesh sieve to obtain the total flavonoids extract of the Chinese wolfberry leaves.

[0178] Forming, filling and packaging process: weigh 5 kg of total flavonoids extract from Chinese wolfberry leaves, add 1.5% silicon dioxide, mix thoroughly, put into capsules, and make 20,000 capsules in aluminum-plastic packaging.

[0179] Example 3: Effects of HQY total flavonoid extract on AOM / DSS-induced ulcerative colitis model mice

[0180] 1. Experimental Materials

[0181] 1.1 Experimental Animals

[0182] BALB / c SPF mice, 6 weeks old, half male and half female, were purchased from Shanghai Slake Laboratory Animal Co., Ltd., license number: SCXK(Shanghai)2022-0004, laboratory animal qualification certificate numbers: 20220004055614 and 20220004062114. Mice were maintained at the Laboratory Animal Center of Nanjing University of Chinese Medicine, license number: SYXK(Suzhou)2023-0077. Ethics numbers: 202404A029 and 202409A003.

[0183] 1.2 Preparation of test drugs and solutions

[0184] 1.2.1 Preparation of HQY total flavonoid extract solution

[0185] HQY total flavonoid extract was prepared according to the method of Example 2

[0186] Dispense a fixed amount of HQY total flavonoid extract powder weekly into EP tubes, seal with parafilm, and store in a desiccator. Remove one EP tube daily and add triple-distilled water to dissolve the extract to a concentration of 10 mg / mL. Then, perform a gradient dilution to obtain solutions of 5 mg / mL, 2.5 mg / mL, and 1.25 mg / mL.

[0187] Celecoxib, Jiangsu Hengrui Medicine Co., Ltd., national medicine approval number H20193349, batch number: 240509MG.

[0188] Remove the celecoxib capsule, dissolve it in 77 mL of triple-distilled water, mix well, divide into 7 tubes, and store in a refrigerator at 4°C.

[0189] 1.2.2 Preparation of celecoxib solution

[0190] Celecoxib, Jiangsu Hengrui Medicine Co., Ltd., national medicine approval number H20193349, batch number: 240509MG.

[0191] Remove the celecoxib capsule, dissolve it in 77 mL of triple-distilled water, mix well, divide into 7 tubes, and store in a refrigerator at 4°C.

[0192] 1.2.3 Preparation of azoxymethane

[0193] Azoxymethane (AOM), Sigma-Aldrich, Catalog Number: A5486-25MG, Lot Number: 0000298134.

[0194] Place the ampoule containing AOM (25 mg) into a 50 mL centrifuge tube, secure the ampoule with cotton, and centrifuge at 800 rpm until a small amount of liquid settles to the bottom. Open the ampoule, add 5 mL of normal saline, blow evenly, and transfer to a 10 mL EP tube. Subsequently, further dilute AOM (5 mg / mL) with normal saline to AOM (1 mg / mL) based on the experimental dosage.

[0195] 1.2.4 Preparation of Dextran Sulfate Sodium Salt

[0196] Dextran sulfate sodium salt (DSS), MP Biomedicals, Catalog No. 160110, Batch No. YD05012. Weigh a certain amount of DSS powder and add it to the animal's drinking water to a concentration of 2.5%. Replace the DSS drinking water 2-3 times during one modeling cycle, for a total of 3 cycles.

[0197] 1.3 Reagents

[0198] Azoxymethane (AOM), Sigma-Aldrich, product number: A5486-25MG, batch number: 0000298134;

[0199] Dextran sulfate sodium salt (DSS), MP Biomedicals, product number: 160110, batch number: YD05012;

[0200] Sodium chloride injection, Chenxin Pharmaceutical Co., Ltd., national medicine standard number: H37022337, batch number: E24062252;

[0201] Mouse IL-6 ELISA MAXTM Deluxe Set, Biolegend, catalog number: 431304, lot number: B361099;

[0202] Mouse IL-1β ELISA MAXTM Deluxe Set, Biolegend, catalog number: 432604, batch number: B411293;

[0203] Mouse TNF-α ELISA MAXTM Deluxe Set, Biolegend, catalog number: 430904, lot number: B299793;

[0204] Mouse IL-17A Uncoated ELISA Kit, Invitrogen, Catalog No.: 88-7371-88, Lot No.: 371835-005;

[0205] BCA protein concentration assay kit, Beyotime, Catalog No.: P0011, Lot No.: 062321211101; Anti-Occludin antibody, Proteintech, Catalog No.: 27260-1-AP, Lot No.: 00099339; Anti-ZO-1 antibody, Proteintech, Catalog No.: 21773-1-AP, Lot No.: 00134887; Anti-Ki-67 antibody, Cellsignaling Technology, Catalog No.: 12202T, Lot No.: PH24110600067;

[0206] Sodium citrate antigen retrieval solution, Beyotime, product number: P0081, batch number: A067241227;

[0207] Goat serum for blocking (working blocking goat serum (working solution), Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., catalog number: ZLI-9022, batch number: 242260730;

[0208] Rabbit two-step detection kit (rabbit enhanced polymer detection system), Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., catalog number: PV-9001, batch number: 2425A0113;

[0209] DAB colorimetric kit (20×), Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., catalog number: ZLI-9018, batch number: 239020811;

[0210] Universal tissue fixative (4% paraformaldehyde), Servicebio, product number: G1101-500 mL, batch number: GP23113010818;

[0211] Ethanol, Sinopharm Chemical Reagent Co., Ltd., catalog number: 10009257, batch number: 20231225; xylene, Sinopharm Chemical Reagent Co., Ltd., catalog number: 10023418, batch number: 20240911; modified Lillie-Mayer hematoxylin staining solution, Shanghai Yuanye Biotechnology Co., Ltd., catalog number: R20562, batch number: JR29558A;

[0212] Eosin staining solution, Beyotime, catalog number: C0109, batch number: Z885240724; blueing solution, Solarbio, catalog number: G1866, batch number: 240010005;

[0213] Neutral gum, Solarbio, product number: G8590, batch number: 20240509.

[0214] 1.4 Experimental Instruments

[0215] Electronic balance, Sartorius Scientific Instruments (Beijing) Co., Ltd., model: BSA224S-CW, d = 0.1 mg;

[0216] Data printer, Sartorius Scientific Instruments (Beijing) Co., Ltd., model: YDP20-0CEV1;

[0217] Electronic balance, Sartorius Scientific Instruments (Beijing) Co., Ltd., model: SI-403, d = 1 mg;

[0218] Electronic balance, Leqi, model: LQ-C5001;

[0219] Tabletop refrigerated microcentrifuge, Eppendorf, Germany, model: 5417R;

[0220] Stereo microscope, ZEISS, Germany, model: Stemi 2000c;

[0221] Pipette, Eppendorf, Germany, range 2-20 μL, 100-1000 μL;

[0222] Pipette, ThermoFisher, USA, range 20-200 μL;

[0223] Pipette, METTLER TOLEDO, USA, range 2-20 μL, 100-1000 μL;

[0224] Vortex mixer, IKA, model: Vortex2;

[0225] Vortex mixer, Wuhan Sevier Biotechnology Co., Ltd., model: MV-100;

[0226] Low-speed centrifuge, Beckman coulter, model: Microfuge16;

[0227] Desktop low-speed centrifuge, Hunan Hexi Instrument Equipment Co., Ltd., model: TDZ4-WS;

[0228] Fully automatic sample rapid grinding machine, Shanghai Jingxin, model: JXFSTPRP-48; BioStack Ready

[0229] Microplate reader, Bio-Tek, USA, model: SyneRgy 2;

[0230] Fluorescence microplate reader, PerkinElmer, model: Enspire;

[0231] Pathology imaging system, PerkinElmer, USA, model: Mantra BX43;

[0232] Microwave oven, Zhongshan Dongling Weili Electric Appliance Co., Ltd., 20MX24;

[0233] Paraffin embedding machine, Shanghai Leica Instrument Co., Ltd., model: EG1150H; tissue freezing machine, Shanghai Leica Instrument Co., Ltd., model: EG1150C;

[0234] Manual paraffin sectioner, Shanghai Leica Instrument Co., Ltd., model: RM2235;

[0235] Semi-automatic paraffin slicer, Thermo, model: HM340E;

[0236] Pathological tissue bleaching and drying instrument, Changzhou Hao Silin Medical Instrument Co., Ltd., model: TEC2500;

[0237] Spreading machine, Shanghai Leica Instrument Co., Ltd., model: HI1210;

[0238] Slice baking machine, Shanghai Leica Instrument Co., Ltd., model: HI1220;

[0239] Immunohistochemistry pen, Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., catalog number: ZLI-9305, batch number: 24102307;

[0240] Electric constant temperature blast dryer, Shanghai Jinghong Experimental Equipment Co., Ltd., model: DHG-9053A;

[0241] Vortex mixer, IKA, model: MS 3 digital.

[0242] 2. Experimental Methods

[0243] 2.1 Preparation of AOM / DSS-induced ulcerative colitis model in mice

[0244] 140 6-week-old SPF BALB / c mice, half male and half female, were randomly divided into 7 groups using a random number table method, divided into a blank control group (Control) and 6 model groups. The model group was re-divided into 6 groups using a random number table method at the end of the 6th week, namely the model group (Model), the positive control group (celecoxib 26 mg / kg, Cele coxib), and four HQY dose groups (12.5 mg / kg, 25 mg / kg, 50 mg / kg and 100 mg / kg). Except for the blank control group, the other groups were given an intraperitoneal injection of AOM solution (concentration 10 mg / kg) on ​​the first day of the first week, with an injection volume of 0.1 mL / 10 g. The blank control group was given an intraperitoneal injection of an equal volume of normal saline. After 1 week, the mice were given 2.5% DSS drinking water for 1 week and then given normal drinking water for 2 weeks, for a total of 3 weeks (2-4 weeks) as one cycle. Similar to the first cycle, the rats were given 2.5% DSS for one week each at the 5th and 8th weeks, and ordinary drinking water for the rest of the time. The whole modeling process consisted of three cycles, and samples were collected and tested at the end of the 10th week.

[0245] 2.2 Dosage regimen

[0246] Mice were gavaged with different doses of HQY or positive drugs starting from the 7th week. The model group and the blank control group were given triple-distilled water for 4 consecutive weeks. The gavage volume was 0.1 mL / 10 g. Figure 3 shown.

[0247] 2.3 Disease activity index score (DAI) during modeling

[0248] During modeling and dosing, the weight and bloody stools of each group of mice were observed and recorded daily, and scores were calculated. The specific scoring criteria are shown in Table 20. DAI score = body mass index + stool status + bleeding. Weight change rate = (today's weight (g) - yesterday's weight (g)) / yesterday's weight (g) × 100%.

[0249] Table 20 DAI score table

[0250] score Weight loss percentage (%) Stool characteristics Occult blood in stool 0 0 normal Negative 1 1-5 - - 2 5-10 Loose stools Occult blood positive 3 10-20 - - 4 ＞20 diarrhea Naked bloody stool

[0251] 2.4 Sample collection and processing

[0252] 2.4.1 Blood collection and serum separation

[0253] Mice were sampled 16 hours after the last dosing. All mice were weighed before sampling. After weighing, blood was collected from the mice's orbits. The blood was allowed to rest for 2 hours and then centrifuged at 3500 rpm for 15 minutes. The upper serum layer was collected and stored at -80°C until analysis.

[0254] 2.4.2 Taking the colorectum of mice and measuring the length of the colorectum

[0255] Mice were sampled 16 hours after the last dosing. All mice were weighed before sampling. After weighing, orbital blood was drawn as scheduled. After blood collection, mice were sacrificed by cervical dislocation. Mice were immobilized in the supine position, the abdominal cavity was opened, and the colorectum was removed from the end of the cecum and colon to the anus. The colorectal length was measured with a ruler and photographed. Colorectal length was calculated using Fuji Image J software.

[0256] After taking pictures under a stereomicroscope, tissues from the same position of the colorectum (including polyps) were taken and fixed in a universal tissue fixative (4% paraformaldehyde); tissues from the same position of the colorectum were divided into aliquots and stored at -80°C for testing.

[0257] 2.5 Recording the number and area calculation of colorectal polyps in mice

[0258] The colorectal tissue of the mice was cut open longitudinally, and the intestines were rinsed with 4°C PBS. The mice were observed for polyp development and photographed using a stereomicroscope, and the number of intestinal polyps was recorded. Polyp size and area were calculated using Fuji ImageJ software. A 1-cm ruler was used to measure the area and diameter of each polyp under the microscope, and the total polyp area was calculated for each mouse.

[0259] 2.6 Hematoxylin-Eosin staining (HE staining) of mouse intestinal tissue

[0260] The colorectal tissue fixed with 4% paraformaldehyde was dehydrated, paraffin-embedded, sectioned, stained with HE, and photographed before being sent to the Jiangsu Provincial Drug Safety Evaluation Center for review. The specific steps are as follows:

[0261] (1) Tissue dehydration: The intestinal tissue was removed from 4% paraformaldehyde and placed in a tissue embedding box. The tissue embedding box was then placed in 30% ethanol for 30 min, 50% ethanol for 30 min, 80% ethanol for 60 min, 95% ethanol for 60 min, 95% ethanol for 60 min, 100% ethanol for 60 min, 100% ethanol for 60 min, 100% ethanol for 60 min, xylene for 60 min, xylene for 60 min, and xylene for 60 min for dehydration.

[0262] (2) Paraffin embedding: Place the tissue in melted liquid paraffin at 65°C for 30 minutes, repeat once, and then place it in a paraffin embedding machine overnight. Embed the tissue the next day.

[0263] (3) Slice, spread, and bake: The embedded tissue block was sliced (8 pm thick) using a microtome, spread (42 °C), and placed on an adhesive slide. The slide was placed in a 65 °C slide baker for 1 h;

[0264] (4) White slice rehydration: The slices were sequentially placed in xylene for 5 min, xylene for 5 min, 100% ethanol for 3 min, 100% ethanol for 3 min, 95% ethanol for 3 min, 80% ethanol for 3 min, 50% ethanol for 3 min, 30% ethanol for 3 min, and running water for 3 min for rehydration;

[0265] (5) HE staining: The slices were sequentially placed in hematoxylin for 4 min 30 s, running water for 1 min, differentiation for 3 s, running water for 1 min, back blue for 30 s, running water for 1 min, eosin for 30 s, running water for 30 s, 100% ethanol for 10 s, 100% ethanol for 10 s, xylene for 30 s, xylene for 30 s, and dried, then sealed with neutral gum.

[0266] (6) The tissue slices were photographed using a pathological imaging instrument, and the pathological histology was read.

[0267] 2.7 ELISA detection of IL-6, IL-1β, TNF-α, and IL-17A levels in mouse colorectal tissues

[0268] (1) Coating: The coating buffer (5x) was diluted with triple distilled water according to the instructions, and the primary antibody (IL-6, IL-1β, TNF-α: 200x; IL-17A: 250x) was diluted with the diluted 1x coating buffer, mixed thoroughly, and then added to the 96-well enzyme-labeled plate for coating, 100 μL per well, sealed with a sealing film, and incubated at 4 °C overnight for 16-18 h;

[0269] (2) Preparation of working solution: The total amount of working solution required was calculated in advance according to the sample amount, and the working solution (5x) was diluted with triple distilled water for standby;

[0270] (3) Washing the plate: The enzyme-labeled plate incubated overnight was taken out from the 4 °C refrigerator, and the primary antibody was wiped clean, 300 μL of washing buffer was added to each well, and it was allowed to stand for 1 min each time, a total of 4 times;

[0271] (4) Blocking: Add 200 μL of working solution to each well of the ELISA plate for blocking and incubate on a shaker at room temperature for 1 h;

[0272] (5) Prepare the standard curve: Add triple-distilled water to the standard vial using a 1 mL syringe according to the instructions. Gently mix for 10 min using a dispenser to obtain a standard stock solution and aliquot. Dilute the stock solution in a gradient to obtain seven standard concentrations.

[0273] (6) Prepare the sample: Take the sample out of the -20°C freezer, equilibrate to room temperature, and vortex until ready for use;

[0274] (7) Washing the plate: After blocking for 1 hour, repeat the procedure (3);

[0275] (8) Sample addition: Add 100 μL of standard or sample to the ELISA plate and incubate on a shaker at room temperature for 2 h;

[0276] (9) Washing: After incubation for 2 h, repeat step (3);

[0277] (10) Secondary antibody incubation: 100 μL of secondary antibody diluted in working solution (IL-6, IL-1β, TNF-α: 200×; IL-17A: 250×) was added to the ELISA plate and incubated on a shaker at room temperature for 1 h.

[0278] (11) Washing: After incubation for 1 hour, repeat step (3);

[0279] (12) HRP incubation: 100 μL of Avidin-HRP diluted with working solution (IL-6, IL-1β, TNF-α: 1000×; IL-17A: 250×) was added to the ELISA plate and incubated in a shaker at room temperature in the dark for 30 min.

[0280] (13) Washing: After incubation for 30 minutes, wash the plate 5 times;

[0281] (14) TMB color development: 100 μL TMB was added to the ELISA plate for color development. When the third well from the end of the plate was observed to be blue, 100 μL stop solution was added to each well to terminate the reaction.

[0282] (15) Plate reading: Detect the OD value of each sample in the ELISA plate at 450 nm and 570 nm;

[0283] (16) Calculation: Draw a standard curve based on the concentration of the standard and the OD value. Substitute the OD value of the sample into the standard curve to obtain the concentration of the sample.

[0284] (17) Protein correction: In order to reduce the difference in target protein content caused by the different total protein contents in different samples, the total protein concentration in the sample was detected by BCA and the target protein concentration (pg / mL) was corrected in pg / mg.

[0285] 2.8 Immunohistochemistry (IHC) detection of ZO-1, Occludin, and Ki-67 expression in mouse colorectum

[0286] Colorectal tissue fixed with 4% paraformaldehyde was dehydrated, paraffin-embedded, sectioned, immunohistochemically analyzed, and filmed. The specific steps are as follows:

[0287] (1) Tissue dehydration: The intestinal tissue was removed from 4% paraformaldehyde and placed in a tissue embedding box. The tissue embedding box was then placed in 30% ethanol for 30 min, 50% ethanol for 30 min, 80% ethanol for 60 min, 95% ethanol for 60 min, 95% ethanol for 60 min, 100% ethanol for 60 min, 100% ethanol for 60 min, 100% ethanol for 60 min, xylene for 60 min, xylene for 60 min, and xylene for 60 min for dehydration.

[0288] (2) Paraffin embedding: The tissue was placed in melted liquid paraffin at 65°C for 30 min, repeated once, and then placed in a paraffin embedding machine overnight. The tissue was embedded the next day; sectioning, spreading, and baking were performed by Shanghai Qianya Kangyao Biotechnology Co., Ltd.

[0289] (3) Dewaxing and hydrating: Place the sections in a mixture of xylene for 10 min, xylene for 10 min, anhydrous ethanol for 5 min, anhydrous ethanol for 5 min, 95% ethanol for 5 min, 80% ethanol for 5 min, 50% ethanol for 3 min, and PBS for 3 min for dewaxing and hydration.

[0290] (4) Sectioning, spreading, and baking were performed by Shanghai Qianya Kangyao Biotechnology Co., Ltd.

[0291] (5) Antigen retrieval: Preheat the sodium citrate buffer solution in advance, place the tissue section in a container containing boiling pH 6.0 sodium citrate buffer solution, place it in a microwave oven and heat it on low heat for 15 minutes, then take it out and let it cool at room temperature;

[0292] (6) Washing: After the slices have cooled naturally, wash them with PBS three times, each time for 3 minutes.

[0293] (7) Inactivation: Add one drop of reagent 1 (to inactivate endogenous peroxidase) to the tissue area on the slide and incubate in a 37°C oven for 10 min. After incubation, wash the slide three times with PBS, each time for 3 min.

[0294] (8) Blocking: Wipe the liquid around the tissue with filter paper, draw a circle around the tissue with a histochemical pen, add goat serum to the tissue section, ensure that the serum evenly covers the entire tissue area, and incubate in a 37°C oven for 30 min;

[0295] (9) Primary antibody incubation: After blocking with goat serum, tilt the slide to allow the blocking solution to flow out, wipe the surrounding liquid with filter paper, and add the primary antibody (ZO-1, 1:2000; Occludin, 1:8000; Ki-67, 1:500) to the tissue section. Incubate in a 37°C oven for 1.5 h. After incubation, wash the slide three times with PBS, each time for 3 min.

[0296] (10) Enhancement reaction: add one drop of reagent 2 (reaction enhancement solution) to the tissue area on the slide and incubate in a 37°C oven for 25 min. After the incubation, wash the slide three times with PBS, each time for 3 min.

[0297] (11) Secondary antibody incubation: add one drop of reagent 3 (enhanced enzyme-labeled goat anti-rabbit IgG polymer) to the tissue area on the slide and incubate in a 37°C oven for 20 min. After incubation, wash the slide three times with PBS, each time for 3 min.

[0298] (12) Color development: Prepare DAB color development solution (solution A: solution B = 20:1) according to the experimental dosage. Protect from light and add DAB color development solution dropwise to the tissue section to ensure that the color development solution evenly covers the entire tissue area. The color development time is 7 min.

[0299] (13) Restaining and sealing: rinse the sections with running water for 5 min—hematoxylin for 1 min—differentiation for 3 s—rinse with running water for 1 min—rebluing for 30 s—rinse with running water for 1 min—50% ethanol for 5 min—80% ethanol for 5 min—95% ethanol for 5 min—100% ethanol for 5 min—100% ethanol for 5 min—xylene for 5 min—xylene for 5 min. After evaporation, seal the sections with neutral gum.

[0300] (14) Tissue sections were photographed using a pathology imaging device, and the images were semi-quantitatively analyzed using Image J software.

[0301] 2.9 Statistical Methods

[0302] All experimental data are expressed as mean ± standard deviation Statistical analysis was performed using GraphPad Prism 10.4.0. One-way ANOVA was used for multiple group comparisons, and Dunnett's multiple comparisons test was used for subsequent two-group comparisons. Two-way ANOVA was used for multiple group comparisons, and P < 0.05 was considered statistically significant.

[0303] 3. Test results

[0304] Effect of HQY total flavonoid extract on body weight in AOM / DSS-induced intestinal polyp model mice

[0305] In the AOM combined with DSS induced ulcerative colitis mouse model, HQY was administered orally one month before sampling. During the modeling and administration process, the weight of the mice was recorded every day to observe the changes in the weight of the mice. The results showed that compared with the blank control group, the model group

[0306] There was no significant change in the body weight of male mice in each drug-treated group (see Figure 4 A). (A male rat; ( Male rats n=10, female rats n=10, compared with the control group, ###P<0.001; compared with the model group, ***P<0.001)) Compared with the blank control group, the body weight of female rats in the model group decreased significantly (P<0.001); compared with the model group, the body weight of female rats in the HQY 50mg / kg dose group increased significantly (P<0.001) (see Figure 4 B). (B female rat; ( Male rats n = 10, female rats n = 10, compared with the control group, ###P < 0.001; compared with the model group, ***P < 0.001)

[0307] Effect of 3.2HQY total flavonoid extract on DAI score in AOM / DSS-induced intestinal polyp model mice

[0308] During the modeling and drug administration process, the weight, diarrhea and bloody stool of the mice were recorded every day, and the DAI score was calculated. The results showed that compared with the blank control group, the DAI scores of the mice in the model group were significantly increased (P < 0.001); compared with the model group, the DAI scores of male mice in the HQY 25, 50, and 100 mg / kg dose groups were significantly reduced (P < 0.001, P < 0.001, P < 0.001) ( Figure 5 A, B), (AB male mouse DAI; ( Male rats n=10, female rats n=10, compared with the Control group, ###P<0.001; compared with the Model group, *P<0.05; ***P<0.001)) HQY 50 and 100 mg / kg dose groups significantly reduced the DAI scores of female rats (P<0.001, P<0.001) Figure 5 C, D), especially the DAI score at the higher scoring stage (third cycle, 6-7 weeks) ( Figure 5 B, D). (CD female mice DAI; ( Male rats, n=10; female rats, n=10. Compared with the control group, ###P<0.001; compared with the model group, *P<0.05; ***P<0.001)

[0309] Effects of 3.3HQY total flavonoid extract on colorectal length, polyp number and area in AOM / DSS-induced intestinal polyp model mice

[0310] ImageJ software was used to calculate the length of the colorectum of mice. The results showed that compared with the blank control group, the colorectal length of both male and female mice in the model group was significantly shortened (P < 0.001). The HQY 50mg / kg dose group was able to restore the colorectal length of male mice (P < 0.05), and the HQY 25, 50, and 100mg / kg dose groups were able to significantly restore the colorectal length of female mice (P < 0.01, P < 0.001, and P < 0.001, respectively). See Table 21, Table 22, and Figure 6 A, B. (AB colorectal length: A male rat, B female rat; ( Male rats, n=10; female rats, n=10; compared with the Control group, #P<0.05, ###P<0.001; compared with the Model group, *P<0.05; **P<0.01; ***P<0.001; scale bar: 1 mm).

[0311] Compared with the blank control group, the number of colorectal polyps in male and female rats in the model group increased significantly (P < 0.001). Compared with the model group, HQY 12.5, 25, 50, and 100 mg / kg groups could significantly inhibit the number of polyps in male rats (P < 0.01, P < 0.001, P < 0.001, and P < 0.001); HQY 25, 50, and 100 mg / kg groups could significantly inhibit the number of polyps in female rats (P < 0.01, P < 0.001, and P < 0.001), as shown in Table 21, Table 22, and Figure 6 C, D. (C.D. Number of polyps: C male, D female; ( Male rats, n=10; female rats, n=10. Compared with the control group, #P<0.05, ###P<0.001; compared with the model group, *P<0.05; **P<0.01; ***P<0.001; scale bar: 1 mm)

[0312] Compared with the blank control group, the colorectal polyp areas of male and female rats in the model group were significantly increased (P < 0.001). Compared with the model group, HQY 12.5, 25, 50, and 100 mg / kg dose groups could significantly inhibit the polyp areas of male rats (P < 0.001, P < 0.001, P < 0.001, and P < 0.001). HQY 50 and 100 mg / kg dose groups could significantly inhibit the polyp areas of female rats (P < 0.001, P < 0.001), as shown in Table 23, Table 24, and Figure 6 E, F. (EF polyp area: E male rat, F female rat; ( Male rats, n=10; female rats, n=10. Compared with the control group, #P<0.05, ###P<0.001; compared with the model group, *P<0.05; **P<0.01; ***P<0.001; scale bar: 1 mm)

[0313] From the perspective of the effect on polyps of different sizes, HQY has the greatest effect on polyps with a diameter of 2-3 mm. Compared with the model group, all HQY dose groups have a significant inhibitory effect on the number of polyps with a diameter of 2-3 mm in male mice (P < 0.001, P < 0.001, P < 0.001, P < 0.001), and HQY 25, 50, and 100 mg / kg dose groups have a significant inhibitory effect on the number of polyps with a diameter of 2-3 mm in female mice (P < 0.001, P < 0.001, P < 0.001); for polyps with a diameter greater than 3 mm, HQY 50 and 100 mg / kg dose groups also have a trend of reduction. Overall, it shows that HQY can inhibit the formation of obvious intestinal polyps, especially reduce the formation of larger polyps. See Table 23, Table 24 and Figure 6 G, H. (GH Number of polyps of different diameters: G male rats, H female rats; ( Male rats, n=10; female rats, n=10. Compared with the control group, #P<0.05, ###P<0.001; compared with the model group, *P<0.05; **P<0.01; ***P<0.001; scale bar: 1 mm)

[0314] Table 21 Effects of HQY total flavonoids extract on colorectal length, polyp number and area in AOM / DSS-induced intestinal polyp model male rats

[0315]

[0316] Note: Compared with Control group, ### P<0.001; compared with Model group, * P<0.05, ** P<0.01, *** P<0.001

[0317] Table 22 Effects of HQY total flavone extract on the colorectal length, polyp number and area of female mice in AOM / DSS-induced intestinal polyp model

[0318]

[0319] Note: Compared with Control group, ### P<0.001; compared with Model group, ** P<0.01, *** P<0.001

[0320] Table 23 Effects of HQY total flavone extract on the polyp number of different diameters of male mice in AOM / DSS-induced intestinal polyp model

[0321]

[0322] Note: Compared with Control group, ### P<0.001; compared with Model group, ** P<0.01, *** P<0.001

[0323] Table 24 Effects of HQY total flavone extract on the polyp number of different diameters of female mice in AOM / DSS-induced intestinal polyp model n=10)

[0324]

[0325] Note: Compared with Control group, # P<0.05, ### P<0.001; compared with Model group, * P<0.05, ** P<0.01,

[0326] *** P<0.001

[0327] 3.4 Effects of HQY total flavone extract on the histopathological changes of intestinal polyps of mice in AOM / DSS-induced intestinal polyp model

[0328] HE staining results showed that the colon structure of the blank control group was normal, with no obvious pathological changes. Compared with the blank control group, the colon of the model group showed more tubular adenoma hyperplasia and occasional villous-tubular adenoma hyperplasia, mainly manifested as focal hyperplasia of intestinal gland-like structures, with irregular shapes of hyperplastic intestinal glands or villi or unclear glandular cavities, and strong basophilia. Compared with the model group, the incidence of tubular adenoma hyperplasia in each drug-treated group was reduced, and the incidence in each HQY dose group was significantly reduced or even completely recovered, with a certain dose-relatedness. No hyperplasia changes were observed in the two high-dose groups (HQY 50, 100 mg / kg) ( Figure 7 ), (A male rat; B female rat; (magnification: ×200, scale: 100 μm)) showed that HQY could effectively inhibit the proliferation of colonic tubular adenomas and improve the pathological structure of colon tissue, and the effects of 50 and 100 mg / kg doses were more significant.

[0329] Effects of HQY on inflammatory factors in colorectal tissues of AOM / DSS-induced intestinal polyp model mice

[0330] ELISA results showed that in the experiment on male rats, compared with the blank control group, the levels of IL-1β (P < 0.001), IL-6 (P < 0.001), TNF-α (P < 0.001) and IL-17A (P < 0.001) in the colorectal tissues of male rats in the model group were significantly increased. Compared with the model group, HQY 12.5, 50 and 100 mg / kg dose groups could significantly reduce the levels of IL-1β (P < 0.001, P < 0.001, P < 0.001) and TNF-α (P < 0.001, P < 0.001, P < 0.001). Compared with the model group, HQY different dose groups could significantly reduce the levels of IL-6 (P < 0.001, P < 0.001, P < 0.001, P < 0.001) and IL-17A (P < 0.001, P < 0.001, P < 0.001, P < 0.001) (see Tables 25 and 26). Figure 8 A, C, E, G. ((A) IL-1β, male mice; (C) IL-6, male mice; (E) TNF-α, male mice; (G) IL-17A, male mice; ( Male rats, n=10; female rats, n=10. Compared with the control group, ###P<0.001; compared with the model group, *P<0.05; **P<0.01; ***P<0.001)

[0331] In the experiment on female mice, compared with the blank control group, the levels of IL-1β (P < 0.001), IL-6 (P < 0.001), TNF-α (P < 0.001) and IL-17A (P < 0.001) in the colorectal tissues of female mice in the model group were significantly increased. Compared with the model group, HQY 12.5, 25, 50 and 100 mg / kg dose groups could significantly reduce the level of IL-1β (P < 0.05, P < 0.05, P < 0.001, P < 0.001); compared with the model group, HQY different dose groups could significantly reduce the levels of IL-6 (P < 0.001, P < 0.001, P < 0.001, P < 0.001), TNF-α (P < 0.001, P < 0.001, P < 0.001, P < 0.001), and IL-17A (P < 0.01, P < 0.001, P < 0.001, P < 0.001), as shown in Tables 26 and 27. Figure 8 B, D, F, H. ((B) IL-1β, female mice; (D) IL-6, female mice; (F) TNF-α, female mice; (H) IL-17A, female mice. Male rats, n=10; female rats, n=10. Compared with the control group, ###P<0.001; compared with the model group, *P<0.05; **P<0.01; ***P<0.001)

[0332] Table 25 Effects of HQY on inflammatory factors in colorectal tissues of male mice with AOM / DSS-induced intestinal polyps model ( n=10)

[0333]

[0334] Note: Compared with the Control group, ### P<0.001; compared with the Model group, * P < 0.05; *** P<0.001

[0335] Table 26 Effects of HQY on inflammatory factors in colorectal tissues of female mice with AOM / DSS-induced intestinal polyps model ( n=10)

[0336]

[0337] Note: Compared with the Control group, ### P<0.001; compared with the Model group, * P < 0.05; ** P < 0.01; *** P<0.001

[0338] Effects of HQY total flavonoid extract on barrier proteins in colorectal tissues of AOM / DSS-induced intestinal polyp model mice

[0339] The results of immunohistochemistry showed that compared with the blank control group, the expression of ZO-1 and Occludin proteins in the colorectal tissues of the model group mice was reduced (P < 0.001, P < 0.001), and the HQY 50 mg / kg dose group could restore the expression of ZO-1 and Occludin proteins (P < 0.001, P < 0.001). Figure 9 ((A) ZO-1; (B) Occludin. (n=6, magnification: ×200, scale bar: 100 μm))

[0340] Effect of HQY total flavonoid extract on Ki-67 expression in colorectal tissues of AOM / DSS-induced intestinal polyp model mice

[0341] The results of immunohistochemistry showed that compared with the blank control group, the expression of proliferation protein Ki-67 in the colorectal tissue of the mice in the model group was significantly increased (P < 0.001). Compared with the model group, the HQY 12.5 and 50 mg / kg dose groups could significantly reduce the expression of Ki-67 protein (P < 0.05, P < 0.001). Figure 10 (n=6, magnification: ×200, scale bar: 100 μm)

[0342] 4. Research Conclusions

[0343] A mouse ulcerative colitis model induced by AOM combined with DSS was established to investigate whether HQY total flavonoid extract could inhibit the development of intestinal polyps. The results showed that HQY total flavonoid extract restored the weight loss induced by modeling and increased the DAI score. It also restored colorectal length and significantly inhibited the number of colorectal polyps, especially the development of small polyps. It also inhibited the expression of the proliferation protein Ki-67. Histopathological examination showed that HQY total flavonoid extract significantly reduced the incidence of tubular adenoma hyperplasia, with no significant hyperplasia observed in the two high-dose groups (HQY 50 and 100 mg / kg). The HQY total flavonoid extract significantly reduced the levels of inflammatory factors IL-1β, IL-6, TNF-α, and IL-17A in intestinal tissue and restored the expression of barrier proteins ZO-1 and Occludin. These results indicate that HQY total flavonoid extract can play a role in preventing and / or treating ulcerative colitis by inhibiting inflammatory factors, restoring intestinal tight junction proteins, and inhibiting abnormal intestinal epithelial proliferation.

[0344] Example 4: Effect of HQY total flavonoids extract on Apc min / + Regulatory role of intestinal flora in mice

[0345] 1. Experimental Materials

[0346] 1.1 Sample collection

[0347] Using Apc min / + Fecal samples of mice. min / + Mice were gavaged with HQY total flavonoid extract at 9 weeks of age for 4 weeks. After the last gavage, the feces of the mice were collected in a suspended cryovial. When a certain amount was collected, the tube was immediately sealed, quickly frozen in liquid nitrogen, and stored in a -80°C refrigerator.

[0348] 1.2 Reagents

[0349] Fast Pure Stool DNA Isolation Kit, MJYH, catalog number: T10-100;

[0350] HiScript II Reverse Transcriptase, Novozymes, product number: R223;

[0351] FastPfu Polymerase, TransGen, Cat. No.: AS221-02.

[0352] 1.3 Instruments

[0353] Double-stable electrophoresis apparatus, model: JY600C, Beijing Junyi Dongfang Electrophoresis Equipment Co., Ltd.

[0354] PCR instrument, BIO-RAD, model: T100 Thermal Cycler;

[0355] Fluorometer (Qubit 4.0), Thermo Fisher Scientific.

[0356] Sequencer, Illumina, model: Nextseq2000;

[0357] Sequencer, Pacbio, model: Pacbio Revio

[0358] 2. Experimental Methods

[0359] High-throughput sequencing was performed by Shanghai Meiji Biotechnology Co., Ltd.

[0360] 2.1 Sample DNA extraction

[0361] The total genomic DNA of the microbial community was extracted from mouse fecal samples according to the instructions. The integrity of the extracted genomic DNA was detected by 1% agarose gel electrophoresis (specific conditions: voltage 5 V / cm; time 20 min), and the DNA concentration and purity were determined using NanoDrop.

[0362] 2.2 PCR amplification and sequencing library construction

[0363] The extracted DNA was used as a template to amplify the full length of the 16S rRNA gene using primers with barcodes.

[0364] Primer design:

[0365]

[0366] Reaction system:

[0367]

[0368] PCR reaction parameters:

[0369] a.1×(3 minutes at 95℃)

[0370] b. Number of cycles × (30 seconds at 95°C; 30 seconds at annealing temperature; 45 seconds at 72°C)

[0371] c.10minutes at 72℃,10℃ until halted by user

[0372] Each sample was repeated three times, and the PCR products of the same sample were mixed and tested by 2% agarose gel electrophoresis and purified using magnetic beads. TM -ST blue fluorescence quantitative system for detection and quantification, and then mixing in corresponding proportions according to the sequencing amount requirements of each sample.

[0373] Pacbio library construction using SMRTbell prep kit 3.0:

[0374] (1) End repair: First, glue the ends of the fragments to make them blunt ends;

[0375] (2) removing the sequences not connected to the adapter;

[0376] (3) The single-stranded loop of the library is annealed with the primer and bound to the polymerase immobilized at the bottom of the ZMW (zero-mode waveguides).

[0377] 2.3 Sequencing data analysis pipeline

[0378] Raw data files were obtained by basecall from PacBio off-line data, and HiFi sequence files were obtained by PacBio's SMRTLink v11.0 analysis software, which were in fastq format. After distinguishing samples by barcode, taxonomic analysis was performed, and based on the taxonomic information, statistical analysis of community structure was performed at each taxonomic level. On the basis of the above analysis, a series of in-depth statistical and visualization analysis of community structure and phylogeny were performed. The specific analysis process is shown in the following Figure 11

[0379] 2.4 Statistical methods

[0380] All data analysis was performed on the Majorbio Cloud Platform (https: / / cloud.majorbio.com).

[0381] 3. Experimental results

[0382] 3.1 Species classification and evaluation in samples

[0383] According to the Sob index, the absolute observed value of the richness of intestinal flora at different species classification in each group of samples was obtained, and it was found that the number of species of intestinal flora in the WT group and the Apc min / + group was close, and the absolute level (number of species) of genus (119.5, 115.9, 118.6 vs 109) and species (175.8, 173.9, 178.9 vs 162.5) in the HQY total flavone extract group was higher than that in the Apc min / + group ((A-B). Figure 12 (A) Taxonomic annotation of intestinal flora in feces of each group, indicating the number of kingdoms, phyla, classes, orders, families, genera, and species detected in each group of feces; (B) Average value of species taxonomy in each group of samples)

[0384] ​OTU (Operational Taxonomic Units) is a unified mark set for a certain taxonomic unit in phylogenetic or population genetics research for the convenience of analysis. The sequences are clustered according to the number information of bacterial species, genera, etc. in the sequencing results, and divided into many groups according to their similarities. A group is an OTU. The Rank-Abundance curve is a way to analyze diversity. Through the horizontal abscissa, we observe the richness of species, and through the flatness, we observe the uniformity of the community. It can be seen from the figure that the different dose groups of HQY total flavonoid extract have a larger range on the horizontal axis, indicating that compared with the WT group and Apc min / + The species richness of the drug-treated group was higher; the species distribution of each group was more uniform ( Figure 12 , C). ((C)Rank-Abundance Curve)

[0385] 3.2 Alpha Diversity Analysis

[0386] In order to examine the diversity of microorganisms in the environment, we used single-sample diversity (Alpha diversity analysis) to analyze the richness and diversity of microbial communities. Among them, sobs, cha, and ace reflect the richness of the community, simpson and shannon reflect the diversity of the community, and coverage reflects the coverage of the community. As can be seen from the figure, there is no significant difference in the richness, diversity, and coverage of each group of samples, and the samples are relatively uniform ( Figure 13 AC). ((AC) Diversity index: (A) from left to right: sobs, ace, and chaos, (B) shannon, simpson, (C) coverage)

[0387] The Rarefaction Curve is a curve constructed by randomly extracting a certain number of sequences from a sample, counting the number of species (OTUs) represented by these sequences, and then using the number of extracted sequences and the number of species to construct a curve. We often use the Rarefaction Curve to prove that the amount of sequencing data in a sample is reasonable. When the curve tends to be flat, it means that the sequencing data is reasonable, and more data will only produce a small number of new species. We plotted the Shannon index and found that the sequencing volume of each group can cover "all" microorganisms in the sample, meeting the requirements of subsequent species composition analysis ( Figure 13 Dilution curve

[0388] 3.3 Beta diversity analysis

[0389] By comparing the species diversity under different disease states, we explored the similarities or differences in the overall community structure between different groups. We performed PCoA analysis at the OTU level.

[0390] (Principal co-ordinates analysis), found that Apc min / + The difference between the two groups also reflected the difference between the Apc min / + There were significant differences in sample composition between the blank group and each drug-treated group ( Figure 14 ). (Beta diversity analysis)

[0391] 3.4 Species composition analysis

[0392] Based on the results of taxonomic analysis, we analyzed the species composition of different groups at the genus and species levels. The community bar chart visually observed the dominant species at the genus and species levels among different groups; the community heat map analyzed the similarities and differences in community composition at the genus and species levels; and the Circos sample and species relationship diagram reflected the distribution ratio of each dominant species in different groups. At the genus level, Akkermansia, Duncanilla, norank_f_Porphyromonadaceae, Allobaculum, Lactobacillus, Limosilactobacillus, etc. were dominant species ( Figure 15 ACE); ((AB) Community composition bar chart: (A) Genus, (B) Species; (CD) Community composition heatmap chart: (C) Genus, (D) Species; (EF) Circos sample and species relationship chart: (E) Genus, (F) Species.) At the species level, Akkermansia_muciniphila, Porphyromonadaceae_bacterium_UBA7139, Allobaculum_fili, Duncaniella_muris, Lactobacillus_taiwanensis, Limosilactobacillus_reuteri, etc. are dominant species ( Figure 15 BDF), ((AB) Community composition bar chart: (A) Genus, (B) Species; (CD) Community composition heatmap chart: (C) Genus, (D) Species; (EF) Circos sample and species relationship chart: (E) Genus, (F) Species) HQY has different degrees of regulatory effects on the abundance of these species.

[0393] 3.5 Species Difference Analysis

[0394] For the dominant species mentioned above, we selected the top 10 species for species difference analysis. At the genus level, the differences between Apcmin / + Compared with the control group, HQY total flavonoids extract increased the relative levels of various bacterial genera to varying degrees, especially the Duncaniella genus, while HQY total flavonoids extract significantly reduced the relative levels of Allobaculum and other bacterial genera. Figure 16 A). (Analysis of species differences in effects; (A) Genus) At the species level, min / + Compared with the control group, HQY total flavonoids extract significantly increased the relative levels of Duncaniella_muris and Duncaniella_dubosii, and significantly decreased the relative level of Allobaculum_fili ( Figure 16 B). (Analysis of species differences in the impact of HQY; (B) Species

[0395] 4. Research Conclusions

[0396] This study mainly explored the effect of HQY total flavonoid extract on Apc through 16s rRNA full-length sequencing. min / + The results showed that HQY total flavonoids extract increased the richness of the intestinal flora of mice, and PCoA analysis found that Apc min / + The group deviated from the WT group, but administration of HQY total flavonoid extract improved this "deviation." Furthermore, combined with the species composition map, we found that HQY total flavonoid extract had varying degrees of regulatory effects on the abundance of dominant species (Akkermansia, Duncanilla, norank_f_Porphyromonadaceae, Allobaculum, etc.). Specifically, the most significant regulatory effect of HQY was: at the genus level, the relative level of Duncanilla was increased, while the relative level of Allobaculum was decreased; at the species level, the relative levels of bacteria such as Duncanilla_muris and Duncanilla_dubosii were increased, while the relative levels of bacteria such as Allobaculum_fili were decreased.

[0397] These evidences indicate that HQY total flavonoids extract regulates the balance of intestinal flora by increasing beneficial bacteria and reducing harmful bacteria.

[0398] Example 4: Effect of HQY total flavonoid extract on Caco-2 cell barrier function

[0399] 1. Experimental Materials

[0400] 1.1 Cells

[0401] Human colorectal adenocarcinoma cells Caco-2 were obtained from the Cell Bank of Type Culture Collection Committee of the Chinese Academy of Sciences with catalog number SCSP-5027.

[0402] 1.2 Test drugs

[0403] The HQY total flavonoid extract was prepared according to the method of Example 2.

[0404] 1.2 Reagents

[0405] DMEM, Keygene, Catalog No. KGL1211-500, Lot No. 20240516-9; Fetal Bovine Serum, VivaCell, Catalog No. C04001-500, Lot No. 2342339; Pen-Strep Solution, VivaCell, Catalog No. C3420-0100, Lot No. 2413134;

[0406] Recombinant Human TNF-alpha Protein, R&D, Catalog No.: 210-TA-005, Lot No.: DDHB0423121;

[0407] 4kDa FITC-dextran, Sigma, Catalog No. 46944-100MG-F, Lot No. BCBQ8157V; PBS, Jiangsu KeyGen Biotech Co., Ltd., Catalog No. KGL2206-500, Lot No. 20240728. PBS buffer (containing 5% trehalose), Formase Biotechnology, Catalog No. FMS-PPT201, Lot No. FMS20221028001.

[0408] Trypsin, Sigma, catalog number: T4799, lot number: SLBT6358.

[0409] 1.3 Instruments

[0410] Cell counter, Shanghai Ruiyu Biotechnology Co., Ltd., model: Countstar;

[0411] Cell counting chamber, Shanghai Ruiyu Biotechnology Co., Ltd., batch number: ABD0417;

[0412] Transwell 24-well plate (0.4 μm Pore Size), Corning, Cat. No. 3413, Lot No. 03324024; Constant temperature water bath, CRYSTAL, Model SY-1210;

[0413] CO2 stainless steel chamber incubator, Thermo Fisher Scientific, USA, model: HERAcell 150i; vertical flow clean bench, ESCO, Singapore, model: OptiMAIR;

[0414] Inverted microscope, ZEISS, Germany, model: Axion A1;

[0415] Desktop low-speed automatic balancing centrifuge, Xiangtan Xiangyi Instrument Co., Ltd., item number: L420;

[0416] Electronic balance, Sartorius Scientific Instruments (Beijing) Co., Ltd., model: BSA224S-CW, d = 0.1 mg;

[0417] Data printer, Sartorius Scientific Instruments (Beijing) Co., Ltd., model: YDP20-0CEV1; transmembrane cell resistance meter, WPI, model: EVOM2;

[0418] Fluorescence microplate reader, PerkinElmer, model: Enspire.

[0419] 2. Experimental Methods

[0420] 2.1 Cell culture

[0421] Caco-2 cells were cultured in DMEM (containing 10% fetal bovine serum) in a cell culture incubator at 37°C and 5% CO2. When the cells reached approximately 90% confluence, the medium was aspirated and the cells were washed twice with PBS. The PBS was aspirated and trypsin was added to the cells for digestion in an incubator for 3-5 minutes. When the cells became round, single cells were digested with complete medium to terminate the digestion. The cells were pipetted into a 10 mL centrifuge tube and centrifuged at 1000 rpm for 3 minutes. The supernatant was removed and the cells were resuspended in complete medium to mix thoroughly.

[0422] 2.2 Caco-2 cell monolayer transmembrane electrical resistance (TEER) and permeability determination

[0423] 2.2.1 Caco-2 cell monolayer culture

[0424] Before inoculating cells, 300 μL and 500 μL complete culture medium were added to the small chamber and the lower chamber, respectively. The cells were placed in an incubator for 30 min and then taken out. The resistance value of each well was measured as the initial resistance value. Caco-2 cells (1×10 5300 μL of cell suspension (per well) was seeded into a 24-well Transwell chamber. 500 μL of culture medium was added to the lower chamber, and the resistance of each well was measured every two days. Before measuring resistance, the chamber was washed once with preheated PBS, and 300 μL and 500 μL of complete culture medium were added to the upper and lower chambers, respectively. The resistance of each well was then measured using a transmembrane electrical resistance meter, and the transmembrane electrical resistance (TEER) was calculated using the formula: TEER = (resistance – initial resistance) × membrane area. When the TEER value stabilizes and no longer increases, it indicates that the Caco-2 cells have formed a monolayer, and subsequent experiments can be continued. During this period, the culture medium of the Caco-2 cell monolayer should be changed daily.

[0425] 2.2.2 Caco-2 cell monolayer transmembrane electrical resistance (TEER) measurement

[0426] After Caco-2 cell monolayer formation, a blank control group (Control group), a TNF-α-stimulated group (TNF-α group), and a HQY total flavonoid extract (1 μg / mL, 10 μg / mL, and 100 μg / mL) group were set up, with three replicates per group. For the treatment groups, medium containing HQY (1 μg / mL, 10 μg / mL, and 100 μg / mL) was added to the upper chamber for 12 hours, followed by TNF-α (20 ng / mL) stimulation in the lower chamber for 48 hours. TEER values ​​were measured and calculated daily for each group. The method was the same as in 2.2.1.

[0427] 2.3 FITC-dextran permeability assay

[0428] After resistance testing, FITC-dextran (final concentration: 1 mg / mL) was added to each group of Caco-2 cells. The cells were protected from light and placed in an incubator for 2 h. Subsequently, 100 μL of the culture medium in the lower chamber was collected and transferred to a 96-well plate. Fluorescence intensity was measured using a fluorescence microplate reader (excitation / emission: 490 / 520 nm), and FITC-dextran flux was calculated using a standard curve.

[0429] 2.4 Statistical methods

[0430] All experimental data are expressed as mean ± standard deviation Statistical analysis was performed using GraphPad Prism 10.4.0. One-way ANOVA was used for multiple group comparisons, and Dunnett's multiple comparisons test was used for subsequent two-group comparisons. Two-way ANOVA was used for multiple group comparisons, and P < 0.05 was considered statistically significant.

[0431] 3. Experimental Results

[0432] 3.1 Establishment of Caco-2 cell monolayer

[0433] First, a Caco-2 cell monolayer was cultured in a Transwell chamber to simulate the intestinal epithelial barrier. As shown in the figure, the transmembrane electrical resistance (TEER) increases with the number of days in culture. When the resistance value stabilizes and no longer increases (D22), it indicates that the Caco-2 cells have formed a monolayer and subsequent experiments can be continued. Figure 17 . (TEER change trend of Caco-2 cell monolayer; ( n=15))

[0434] Effects of HQY total flavonoid extract on transmembrane resistance and permeability of Caco-2 cell monolayers stimulated by TNF-α

[0435] After Caco-2 cell monolayer formation (Day 22), HQY total flavonoid extract was added to the chamber and cultured for 12 hours (Day 23). Subsequently, 20 ng / mL TNF-α was added to the lower chamber (Day 23) for stimulation for 48 hours. After TNF-α stimulation, the TEER value was significantly reduced compared with the control (P < 0.001), and the FITC-dextran permeability was significantly increased (P < 0.001). Administration of HQY total flavonoid extract at 10 μg / mL and 100 μg / mL increased the TEER at 24 hours and 48 hours after TNF-α stimulation (P < 0.05, P < 0.01) (Table 27, Figure 18 A), ((A) TEER of Caco-2 cell monolayer culture at D23-D25; ( n=3, compared with the Ctrl group, ###P<0.001, compared with the TNF-α group, *P<0.05, **P<0.01, ***P<0.001)) At the same time, HQY total flavonoid extract 1 μg / mL, 10 μg / mL, 100 μg / mL all significantly reduced FITC-dextran permeability (P<0.001, P<0.001, P<0.001) (Table 28, Figure 18 B). ((B) 4kDa FITC-Dextran flux in Caco-2 cell monolayer at D25. ( n=3, compared with the Ctrl group, ###P<0.001, compared with the TNF-α group, *P<0.05, **P<0.01, ***P<0.001)

[0436] Table 27 Effects of HQY total flavonoid extract on TEER of Caco-2 cell monolayer stimulated by TNF-α ( n=3)

[0437]

[0438] Note: Compared with the Control group, ### P<0.001; compared with TNF-α group, * P<0.05, ** P<0.01

[0439] Table 28 Effects of HQY total flavonoid extract on FITC-dextran permeability of Caco-2 cell monolayer stimulated by TNF-α ( n=3)

[0440]

[0441] Note: Compared with the Control group, ### P<0.001, compared with TNF-α group, *** P<0.001

[0442] 4. Research Conclusions

[0443] After confluence, Caco-2 cells begin to spontaneously differentiate, gradually forming a polarized cell monolayer with apical and basal membranes, junctional complexes, and a brush border, with microvilli on the apical side. The formation of the junctional complex aligns with the typical characteristics of the human colonic epithelial barrier, leading to the widespread use of Caco-2 cell monolayers in in vitro studies of intestinal barrier function. TNF-α is primarily secreted by activated macrophages, dendritic cells, and T lymphocytes during the progression of intestinal adenomas. Excessive accumulation of TNF-α in the lamina propria attacks intestinal epithelial cells, leading to increased mucosal permeability, a key characteristic of intestinal epithelial barrier disruption.

[0444] This study used Caco-2 cells to form a cell monolayer to simulate the intestinal epithelial barrier. TNF-α was used to induce an intestinal epithelial barrier injury model. HQY total flavonoid extract was then used to investigate whether it could improve intestinal mucosal barrier function. The results showed that after 20 days of culture, Caco-2 cells formed a cell monolayer. Administration of HQY total flavonoid extract increased the TEER of the Caco-2 cell monolayer after TNF-α stimulation and significantly decreased its permeability to FITC-dextran. These results confirm that HQY total flavonoid extract can alleviate epithelial barrier damage induced by inflammatory factors and thus maintain intestinal epithelial barrier function.

Claims

1. Application of total flavonoids extract from Chinese wolfberry leaves in the preparation of drugs for preventing and treating ulcerative colitis.

2. Application of total flavonoids extract from Chinese wolfberry leaves as the only effective part group in the preparation of drugs for preventing and treating ulcerative colitis.

3. The use of the total flavonoids extract of the Chinese wolfberry leaves as claimed in claim 1 in the preparation of a drug for preventing and treating ulcerative colitis, characterized in that: The preparation method of the total flavonoid extract of the Chinese wolfberry leaf comprises the following steps: taking the Chinese wolfberry leaf, adding ethanol for reflux extraction, combining the extracts, filtering, recovering ethanol from the filtrate under reduced pressure until no alcohol taste is present, adding water, centrifuging, passing the centrifuge through an AB-8 macroporous resin column, eluting with water and ethanol, collecting the ethanol eluate, recovering ethanol under reduced pressure, concentrating to a clear paste, drying under reduced pressure, crushing, and adding auxiliary materials to prepare the extract into tablets, capsules, and granules.

4. The use of the total flavonoids extract of the Chinese wolfberry leaves as claimed in claim 3 in the preparation of a drug for preventing and treating ulcerative colitis, characterized in that: The preparation method of the total flavonoids extract of the Chinese wolfberry leaves comprises the following steps: taking the Chinese wolfberry leaves, adding ethanol for reflux extraction, combining the extracts, filtering, recovering ethanol from the filtrate under reduced pressure until no alcohol taste is present, adding water, centrifuging, passing the centrifuge through an AB-8 macroporous resin column, eluting with water and ethanol, collecting the ethanol eluate, recovering ethanol under reduced pressure, concentrating to a clear paste, drying under reduced pressure, crushing, adding silicon dioxide, mixing, and encapsulating to obtain the total flavonoids extract.

5. The use of the total flavonoids extract of the Chinese wolfberry leaves as claimed in claim 4 in the preparation of a drug for preventing and treating ulcerative colitis, characterized in that: Take Huangqi leaves, cut them into strips about 1 cm wide, add 10 times the amount of 60% ethanol, reflux and extract twice, each time for 1.5 hours, combine the extracts, filter, and recover the ethanol from the filtrate under reduced pressure until there is no alcohol taste, add water to adjust the crude drug concentration to 0.2g / ml, centrifuge, pass the centrifuge through an AB-8 macroporous resin column with a diameter-to-height ratio of 1:7-1:12, and elute with 4BV of water and 4BV of 45% ethanol in sequence, collect the ethanol eluate, recover the ethanol under reduced pressure, concentrate to a clear paste with a relative density of 1.20-1.25 at 70°C, dry under reduced pressure at 65°C-85°C, pulverize, pass through a 60-mesh sieve, add 1.5% silicon dioxide, mix, encapsulate, and granulate to obtain the product.

6. Use of the total flavonoid extract of the leaves of Astragalus membranaceus as the sole effective fraction group in the preparation of a drug for preventing and treating ulcerative colitis as claimed in claim 2, characterized in that: Take Huangqi leaves, cut them into strips about 1 cm wide, add 10 times the amount of 60% ethanol, reflux and extract twice, each time for 1.5 hours, combine the extracts, filter, and recover the ethanol from the filtrate under reduced pressure until there is no alcohol taste, add water to adjust the crude drug concentration to 0.2g / ml, centrifuge, pass the centrifuge through an AB-8 macroporous resin column with a diameter-to-height ratio of 1:7-1:12, and elute with 4BV of water and 4BV of 45% ethanol in sequence, collect the ethanol eluate, recover the ethanol under reduced pressure, concentrate to a clear paste with a relative density of 1.20-1.25 at 70°C, dry under reduced pressure at 65°C-85°C, pulverize, pass through a 60-mesh sieve, add 1.5% silicon dioxide, mix, encapsulate, and granulate to obtain the product.

7. Use of the total flavonoids extract of the leaves of Astragalus membranaceus as claimed in claim 1 in the preparation of a drug for preventing and treating ulcerative colitis, characterized in that: The total flavonoid extract of Chinese wolfberry leaves plays a role in preventing and / or treating ulcerative colitis by inhibiting inflammatory factors, restoring intestinal tight junction proteins, and inhibiting abnormal proliferation of intestinal epithelium.

8. The use of the total flavonoids extract of the Chinese wolfberry leaves as claimed in claim 1 in preparing a drug for preventing and treating ulcerative colitis, characterized in that: The total flavonoids extract from Chinese wolfberry leaves increases beneficial bacteria, reduces harmful bacteria, and regulates the balance of intestinal flora.

9. The use of the total flavonoids extract of the leaves of Astragalus membranaceus as claimed in claim 1 in the preparation of a drug for preventing and treating ulcerative colitis, characterized in that: The total flavonoids extract from Astragalus membranaceus leaves inhibited TNF-α-induced intestinal epithelial barrier damage model and improved intestinal mucosal barrier function.

10. Application of total flavonoids extract from Astragalus membranaceus leaves in the preparation of proliferation protein Ki-67 inhibitors.

11. Application of total flavonoid extract from wolfberry leaves in the preparation of inhibitors of inflammatory factors IL-1β, IL-6, TNF-α and IL-17A.

12. Application of total flavonoids extract from Aralia serrata leaves in the preparation of barrier protein ZO-1 and Occludin agonists.

13. Application of total flavonoids extract from wolfberry leaves in the preparation of intestinal flora balance regulator.

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