Modified radix puerariae, radix scutellariae and rhizoma coptidis decoction with intestinal flora regulating function for treating ulcerative colitis and preparation method of modified radix puerariae, radix scutellariae and rhizoma coptidis decoction

Abstract

The invention discloses modified radix puerariae, radix scutellariae and rhizoma coptidis decoction with an intestinal flora regulating function for treating ulcerative colitis, and the modified radix puerariae, radix scutellariae and rhizoma coptidis decoction is mainly prepared from the following raw materials in parts by mass: 20-28 parts of radix puerariae, 6-12 parts of radix scutellariae, 6-12 parts of rhizoma coptidis, 3-9 parts of honey-fried licorice root, 3-9 parts of radix paeoniae alba, 6-12 parts of fructus jujubae, 9-15 parts of vinegar schisandra chinensis and 1-6 parts of fructus evodiae. According to the modified radix puerariae qinlian decoction (MGQD) disclosed by the invention, cold and warm medicines are used together, so that the effects of clearing heat and tonifying healthy qi are achieved, the imbalance of intestinal flora of ulcerative colitis can be effectively reversed, the diversity of the intestinal flora can be enhanced, the relative abundance of beneficial bacteria such as Muriacaceae, Lacnospiraceae NK4A136group and Lactobacillus can be increased, the level of metabolite bile acid of the intestinal flora can be regulated, and the curative effect of the ulcerative colitis can be improved. According to the traditional Chinese medicine composition, the protein expression of TGR5 and FXR in a colon tissue is up-regulated, the injury caused by colitis is relieved, inflammatory cell infiltration of the colon tissue is reduced, the number of goblet cells is increased, the symptoms such as stomachache, diarrhea and hematochezia can be effectively improved clinically, the illness state is controlled to be stable, the recurrence period is prolonged, and recurrence is reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine compositions, in particular to a modified Gegenqinlian decoction for treating ulcerative colitis with the function of regulating intestinal flora, and a preparation method of the modified Gegenqinlian decoction. Background Art

[0002] Ulcerative colitis (UC) is a chronic inflammatory disease affecting the intestines, with clinical manifestations including abdominal discomfort, diarrhea, bloody and mucus-purulent stools, and tenesmus. The cause of UC remains unknown. While various theories exist, no definitive conclusion has been reached. Excluding bacterial and viral causes, it may be caused by a variety of factors, including genetic susceptibility, environmental influences, immune system imbalance, and imbalanced intestinal flora. Therefore, UC is difficult to treat and prone to relapse. Its course is characterized by chronic, recurrent episodes, and most patients require long-term management to control symptoms and reduce relapses.

[0003] Currently, the most commonly used Western medicines for the treatment of ulcerative colitis include glucocorticoids and immunosuppressants, but these medications can cause side effects such as nausea, vomiting, and dizziness. Gegenqinlian Decoction (GQD) is a recognized Traditional Chinese Medicine formula for the treatment of ulcerative colitis. However, as the human body's physical fitness declines, symptoms have also evolved. Simple damp-heat diarrhea is less common, and is often accompanied by symptoms of insufficient vital energy. Therefore, the traditional Gegenqinlian Decoction requires new modifications and applications.

[0004] Under normal circumstances, the intestinal flora maintains a certain dynamic balance. When the intestinal flora is unbalanced, it will directly affect the homeostasis of the intestinal environment, change the diversity of microorganisms, and thus destroy the symbiotic relationship between the intestinal flora and its host, leading to a decrease in microbial diversity and abnormal flora composition, destroying the intestinal epithelial cell barrier, and inducing intestinal inflammation. Restoring the homeostasis of the intestinal flora has become a new strategy for the treatment of ulcerative colitis, but the degree and mode of intestinal microecological imbalance vary from person to person. For example, the effect of the same dose of probiotics on different patients may be significantly different. Therefore, individual differences need to be considered in medication. Today, the technology of improving intestinal flora with traditional Chinese medicine is in the stage of active research and development. How to effectively exert the conditioning and metabolic effects of traditional Chinese medicine has a positive effect on promoting traditional Chinese medicine treatment. Summary of the Invention

[0005] In order to address the shortcomings of the existing Gegenqinlian Decoction in the treatment of ulcerative colitis, the present invention proposes a modified Gegenqinlian Decoction, which can effectively reverse the imbalance of the intestinal flora in colitis, enhance the diversity of the intestinal flora, increase the relative abundance of beneficial bacteria Muribaculaceae, Lachnospiraceae_NK4A136_group and Lactobacillus, increase the levels of CA and TCA in feces, upregulate the expression of bile acid receptors TGR5 and FXR proteins in the colon, alleviate colonic inflammatory damage, reduce inflammatory cell infiltration of colonic tissue, and increase the number of goblet cells. Clinically, it can effectively improve abdominal pain, diarrhea, bloody stools and other symptoms, control the stability of the disease, prolong the relapse period and reduce recurrence.

[0006] To achieve the above-mentioned purpose, the present invention provides a modified Gegenqinlian decoction for treating ulcerative colitis with the function of regulating intestinal flora, which is mainly prepared from the following raw materials in parts by weight: 20-28 parts of Puerariae Radix, 6-12 parts of Scutellariae Baicalensis, 6-12 parts of Coptis Rhizome, 3-9 parts of Radix Glycyrrhizae Preparata, 3-9 parts of White Peony Root, 6-12 parts of Jujube, 9-15 parts of Vinegar-cooked Schisandrae Chinensis, and 1-6 parts of Evodiae Rutaecarpa.

[0007] The modified Gegenqinlian decoction of the present invention comprises Pueraria root as the main drug, which is pungent, sweet and cool, and can raise the spleen and stomach to clear the yang qi and treat diarrhea; Coptis chinensis and Scutellaria baicalensis as the auxiliary drugs, which can clear away heat, dry dampness, thicken the intestine and stop diarrhea; White Peony Root is sour and slightly cold, enters the liver and spleen meridians, and can assist with the preparation of Scutellaria baicalensis to nourish blood, restrain yin, soften the liver and stop pain; Vinegar-based Schisandra chinensis is sour and warm, and can assist with the preparation of Scutellaria baicalensis to nourish blood, restrain yin, soften the liver and stop pain; Evodia rutaecarpa can assist with the preparation of Scutellaria baicalensis to dispel cold and stop diarrhea; Roasted Licorice root is sweet and moderates the middle, harmonizes the other drugs, and jujube harmonizes the spleen and stomach. The drugs are used together with cold and warm properties, mainly clearing heat, dispelling pathogenic factors, strengthening the body's resistance and stopping diarrhea, and together play the role of clearing heat, drying dampness, raising yang and stopping diarrhea, and mainly treat diarrhea caused by damp-heat and damage to the vital energy; the drugs are used together with cold and warm properties to nourish the vital energy while clearing heat.

[0008] As a limitation of the above technical solution, the ingredients are 22-26 parts of Puerariae Radix and 2-5 parts of Evodiae Rutaecarpa.

[0009] As a limitation of the above technical solution, the following ingredients are used: 24 parts of Pueraria root, 9 parts of Scutellaria baicalensis, 9 parts of Coptis chinensis, 6 parts of prepared Licorice root, 6 parts of White Peony Root, 9 parts of Jujube, 12 parts of Vinegar Schisandra chinensis, and 3 parts of Evodia rutaecarpa.

[0010] As a limitation of the above technical solution, the dosage form of the modified Gegenqinlian decoction is one of decoction, granules, oral liquid, paste, pills, and enema.

[0011] The modified Gegenqinlian decoction can be taken orally in the form of decoction, decoction-free granules, oral liquid, paste, small water pills, small honey pills, and enema.

[0012] At the same time, the present invention also provides a preparation method of the Gegenqinlian decoction for treating ulcerative colitis with the function of regulating intestinal flora as described above, which is to prepare the modified Gegenqinlian decoction by jointly decocting the pretreated raw medicinal materials.

[0013] As a limitation of the above technical solution, the pretreated raw medicinal materials are placed in a decoction container, and drinking water 8 to 10 times the total mass of the medicinal materials is added and soaked for 30 to 60 minutes, then boiled over high heat, and then simmered over low heat for 40 to 60 minutes, and the first decoction is collected by filtration; the medicinal residue is then added with drinking water 6 to 8 times the total mass of the medicinal materials, and the mixture is also boiled over high heat and then simmered over low heat for 30 to 40 minutes, and the second decoction is collected by filtration; the two decoctions are combined and concentrated to the desired concentration at low temperature to obtain the modified Gegenqinlian Decoction.

[0014] The pretreatment of raw medicinal materials is carried out by conventional methods such as cleaning, core removal, and slicing.

[0015] As a limitation of the above technical solution, the concentration temperature is controlled at 50~75℃.

[0016] As a limitation of the above technical solution, after the decoction is concentrated, different dosage forms of modified Gegenqinlian decoction are prepared according to corresponding standards.

[0017] In summary, the modified Gegenqinlian decoction of the present invention is a modified Gegenqinlian decoction based on the original recipe. By using both cold and warm drugs, it can nourish the vital energy while clearing away heat. The total effective rate of clinical treatment is 91.25%, which is significantly higher than the 76.25% of the traditional Gegenqinlian decoction group ( P <0.05) and 78.75% ( P <0.05), can effectively improve symptoms such as abdominal pain, diarrhea, and bloody stools. In the long-term maintenance treatment stage, the recurrence rate of the modified Gegenqinlian decoction group was only 11.25% after 6 months, which was significantly lower than the 27.5% of the traditional prescription group and the 25% of the mesalazine group.

[0018] Intestinal flora metagenomic sequencing analysis showed that the abundance of beneficial bacteria such as Akkermansia and Bifidobacterium in the intestines of patients in the treatment group increased by 2 to 3 times compared with the baseline, and the activity of key enzymes in secondary bile acid metabolism (such as bile salt hydrolase and 7α-dehydroxylase) was significantly enhanced, which was significantly positively correlated with the improvement of clinical symptoms (r=0.78, P <0.01). Subjective feedback from patients showed that the treatment group showed significant improvement in accompanying symptoms such as general fatigue and loss of appetite, and its quality of life score (SF-36) increased by 42.6% compared to baseline, significantly better than the other two groups.

[0019] Pharmacological studies have shown that modified Gegenqinlian decoction has anti-inflammatory and antioxidant effects, immune regulation, and intestinal mucosal repair. Unlike Gegenqinlian decoction, the vinegar-based Schisandra chinensis is warming and the Evodia rutaecarpa is hot, both of which have antibacterial and anti-inflammatory effects. Modified Gegenqinlian decoction combines both cold and warming properties, making its formulation more comprehensive.

[0020] Modified Gegenqinlian Decoction improves colitis-induced microbial dysbiosis by improving gut microbial diversity. Specifically, this was demonstrated by increasing the relative abundance of Muribaculaceae, Lachnospiraceae_NK4A136_group, and Lactobacillus, while decreasing the relative abundance of Bacteroides and Alloprevotella. This suggests that modified Gegenqinlian Decoction promotes the proliferation of Muribaculaceae, Lachnospiraceae_NK4A136_group, and Lactobacillus, while inhibiting the proliferation of Bacteroides and Alloprevotella. Muribaculaceae, a recognized potential probiotic, maintains intestinal epithelial health and integrity by producing short-chain fatty acids. Muribaculaceae, Bifidobacteria, and Lactobacilli exhibit symbiotic nutritional interactions that may contribute to enhanced intestinal health and potentially impact the remission of certain chronic diseases. Lactobacillus exhibits immunomodulatory properties, reducing proinflammatory cytokine levels while helping to restore gut microbial balance. The ability of the Lachnospiraceae_NK4A136_group to produce short-chain fatty acids through dietary polysaccharide fermentation was negatively correlated with various metabolic disorders and chronic inflammation, suggesting potential anti-colitis activity. These results suggest that intervention with modified Gegenqinlian decoction improves the intestinal microbiota by increasing beneficial bacteria.

[0021] Modified Gegenqinlian Decoction regulates the levels of bile acid metabolites, increasing the amount of bile acid excreted through defecation and reducing the total concentration of bile acids in the gallbladders of UC mice. Combined with pseudosterile experiments and fecal microbiota transplantation, this study further confirmed that Modified Gegenqinlian Decoction treats ulcerative colitis by regulating the intestinal microbiota. Therefore, the therapeutic mechanism of Modified Gegenqinlian Decoction for ulcerative colitis is related to maintaining intestinal microbiota balance and microbial-mediated bile acid metabolism. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 , total ion chromatograms of Modified Gegen Qinlian Decoction (A) negative ion mode, (B) positive ion mode, (C) ranking of component content of Modified Gegen Qinlian Decoction, (D) number of component classifications of Modified Gegen Qinlian Decoction.

[0023] Figure 2 , (A) Experimental design diagram, (B) Percentage change of mouse body weight, (C) DAI score, (D) Spleen index, (E) Colon length, (F) Colon appearance; ** in the figure represents comparison with the Control group P <0.01, # Comparison with the DSS group P <0.05, # # Comparison with the DSS group P <0.01.

[0024] Figure 3 (A) HE staining (200x field of view), (B) PAS staining (200x field of view), (C) serum TNF-α level, (D) serum IL-1β level; arrows indicate inflammatory infiltration, and boxes indicate irregular crypts and loss of goblet cells; ** in the figure represents comparison with the Control group P <0.01, ## Compared with the DSS group, P <0.01.

[0025] Figure 4 , (A) Venn diagram, (B) Shannon index, (C) Chao 1 index, (D) PcoA, (E) relative abundance of intestinal flora at the genus level; * in the figure represents comparison with the Control group P <0.05, # # Comparison with the DSS group P <0.01.

[0026] Figure 5 , (A) PCA analysis, (B) primary bile acid content, (C) quantitative analysis of individual bile acids; * in the figure represents comparison with the Control group P <0.05, ** represents comparison with the Control group P <0.01, # Comparison with the DSS group P <0.05.

[0027] Figure 6 , (A) TGR5 level in colon tissue, (B) FXR level in colon tissue; *Compared with the Control group P <0.05, ** represents comparison with the Control group P <0.01, # Comparison with the DSS group P <0.05,## Comparison with the DSS group P <0.01.

[0028] Figure 7 , (A) Experimental design diagram, (B) Percentage change of mouse body weight, (C) DAI score, (D) Spleen index, (E) Colon length, (F) Colon appearance, (G) HE staining (200x field of view), (H) PAS staining (200x field of view); Arrows indicate inflammatory infiltration, and boxes indicate irregular crypts and loss of goblet cells.

[0029] Figure 8 (A) Experimental design diagram, (B) Percentage change of mouse body weight, (C) DAI score, (D) Spleen index, (E) Colon length, (F) Colon appearance, (G) Serum TNF-α level, (H) Serum IL-1β level, (I) HE staining (200x field of view), (J) PAS staining (200x field of view); Arrows in the figures indicate inflammatory infiltration; * in the figure represents comparison with FMT-CON group P <0.05, ** represents comparison with FMT-CON group P <0.01. DETAILED DESCRIPTION

[0030] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0031] The raw materials used in the following examples and comparative examples are typical products purchased from the market.

[0032] Example

[0033] Modified Gegenqinlian decoction and its preparation Prescription: 20-28 parts of Pueraria root, 6-12 parts of Scutellaria baicalensis, 6-12 parts of Coptis chinensis, 3-9 parts of roasted Licorice root, 3-9 parts of White Peony Root, 6-12 parts of Jujube, 9-15 parts of Vinegar-cooked Schisandra chinensis, and 1-6 parts of Evodia rutaecarpa.

[0034] The amount of Pueraria root and Evodia rutaecarpa in the formula can be appropriately reduced, specifically 22-26 parts of Pueraria root and 2-5 parts of Evodia rutaecarpa.

[0035] 1. Decoction Preparation Method Pretreatment of raw medicinal materials: Weigh 24 parts of Pueraria root, 9 parts of Scutellaria baicalensis, 9 parts of Coptis chinensis, 6 parts of Radix Glycyrrhizae Radix, 6 parts of White Peony Root, 9 parts of Jujube, 12 parts of Schisandrae Chinensis with vinegar, and 3 parts of Evodia rutaecarpa by mass. Remove the core of the jujube, cut the Pueraria root, Scutellaria baicalensis, Coptis chinensis, Radix Glycyrrhizae Radix, White Peony Root, and Evodia rutaecarpa into thin slices, and wash the Schisandrae Chinensis with vinegar and set aside.

[0036] Decoction: Place the pretreated herbs in a decoction container and add 8-10 times the total weight of the herbs in drinking water. Soak for 30-60 minutes to allow the herbs to fully absorb the water and soften. After boiling over high heat, reduce to low heat and simmer for 40-60 minutes. Filter and collect the first decoction. Add 6-8 times the total weight of the herbs to the medicinal residue. Again, boil over high heat, then simmer over low heat for 30-40 minutes. Filter and collect the second decoction.

[0037] Combination and concentration: Combine the two decoctions, place them in a suitable container, and concentrate them to the required concentration using low-temperature concentration (temperature controlled at 60-70°C) to obtain the modified Gegenqinlian Decoction.

[0038] 2. Granule Preparation Method Extraction: Weigh the herbs according to the pretreatment steps in the decoction preparation method above, decoct them twice, and combine the decoctions. Concentrate the combined decoctions in a vacuum concentrator at a temperature of 60-75°C and a pressure of -0.06-0.08 MPa to a clear paste with a relative density of 1.2-1.3 (measured at 60°C). Granulation: Add appropriate amounts of dextrin and sucrose powder to the paste (the mass ratio of dextrin to sucrose powder can be adjusted according to actual conditions, generally 1:2-1:3), mix thoroughly, and form a soft material. The desired wetness of the soft material should be such that it forms a ball when held but falls apart when lightly pressed. Use an oscillating granulator or a rapid mixing granulator to form wet granules. The particle size of the wet granules should be controlled between 1 and 3 mm. Drying and granulation: The wet granules are placed in a hot air circulation drying oven and dried at 60-70°C to a moisture content of ≤5%. The dried granules are passed through a granulator to remove sticky lumps and fine powder, making the granules uniform in size. Packaging: Pack the whole granules into suitable packaging materials to obtain the modified Gegenqinlian Decoction granules. 3. Pill Preparation Method Extraction and concentration: Same as the extraction step in the granule preparation method, decoct the medicinal materials and combine the decoctions, and concentrate under reduced pressure to form a thick paste with a relative density of 1.3 to 1.4 (measured at 60°C). Pill Forming: Add an appropriate amount of refined honey to the thick paste (the ratio of refined honey to thick paste should be adjusted according to actual conditions, generally 1:1 to 1:1.5), and stir thoroughly to form plastic pellets. The pellets are then rolled into pellets using a pelletizer or by hand. The pellet size can be customized, for example, the diameter of a typical water-honey pill is approximately 6 to 9 mm, while that of a small honey pill is approximately 3 to 5 mm. Drying and Coating (Optional): The pellets are placed in a drying machine and dried at 60-70°C until the moisture content meets the specified requirements. If necessary, the pellets can be coated with sugar or film to improve the appearance and stability of the pellets. Packaging: Pack the dried or coated pills into suitable packaging containers to obtain the modified Gegenqinlian Decoction pills. 4. Enema Preparation Method Extraction and Purification: Weigh the medicinal materials according to the decoction preparation method, decoct them twice, and combine the decoctions. Filter the decoction through a microporous membrane (pore size 0.22-0.45 μm) to remove impurities and obtain a relatively clear medicinal solution. The medicinal solution is then concentrated under reduced pressure at a temperature of 50-60°C and a pressure of -0.06-0.08 MPa to the desired concentration, generally 1-2 g of the original medicinal material per milliliter.

[0039] Adjustment and Sterilization: Add appropriate amounts of sodium chloride (to adjust osmotic pressure) and sodium benzoate (as a preservative, not exceeding 0.3%) to the concentrated solution. Stir thoroughly and adjust the pH to 6.5–7.5 with sodium hydroxide or hydrochloric acid. Dispense the adjusted solution into suitable enema containers and sterilize using moist heat sterilization (115°C for 30 minutes). Packaging: After the sterilized enema is cooled, it is sealed and packaged to obtain the modified Gegenqinlian Decoction enema.

[0040] Analysis of the ingredients and therapeutic effects of modified Gegenqinlian decoction (1) Component analysis Analytical instrument: Liquid chromatography-mass spectrometry system consisting of ACQUITY UPLC I-Class HF ultra-high performance liquid chromatography tandem with QE high-resolution mass spectrometer.

[0041] Chromatographic conditions: The chromatographic column was ACQUITY UPLC HSS T3 (100 mm × 2.1 mm, 1.8 um), the column temperature was 45°C, the flow rate was 0.35 mL / min, the injection volume was 5 μL, and the mobile phases were A-water (containing 0.1% formic acid) and B-acetonitrile.

[0042] The elution gradient was: 0-2 min, 95-95% A, 5-5% B; 2-4 min, 95%-70% A, 5%-30% B; 4-8 min, 70-50% A, 30-50% B; 8-10 min, 50-20% A, 50-80% B; 15-15 min, 0-0% A, 100-100% B; 15-15.1 min, 0-95% A, 100-5% B; 15.1-16 min, 95%-95% A, 5%-5% B.

[0043] Mass spectrometry conditions: Ion source: HESI; sample mass spectrometry signal acquisition uses positive and negative ion scanning modes. Data acquisition mode: DDA. Scan mode: Full MS / dd-MS2 (TOP 8) Chromatographic analysis of Modified Gegenqinlian Decoction in positive and negative ion modes is shown in the attached figure. Figure 1 As shown in the figure, the ingredients of Modified Gegenqinlian Decoction are ranked from highest to lowest by content: flavonoids, berberine, coptisine, norwogonin-7-O-glucuronide, baicalin, wogonin, puerarin, and citric acid. Based on the number of ingredients, MGQD was identified to contain 31.01% flavonoids, 17.05% phenylpropanoids, 11.11% sugars and glycosides, and 10.08% terpenes.

[0044] (2) Basic animal experiments 2.1 Animal model preparation Male SPF-grade BALB / c mice, aged 6-8 weeks, were provided by Beijing Huafukang Biotechnology Co., Ltd. The mice were placed in an air-conditioned room at (22 ± 2)°C with free access to food and water. After one week of adaptive feeding, the mice were randomly divided into five groups: blank group (Control group), model group (DSS group), modified Gegenqinlian decoction low-dose group (MGQD-L group), modified Gegenqinlian decoction high-dose group (MGQD-H group), and positive control group (SASP group), with 8 mice in each group. The dosage was determined according to the equivalent dose ratio between experimental animals and humans.

[0045] At the start of the experiment, mice in the control group were given free access to sterile water, while mice in the other groups were given free access to 3% dextran sulfate sodium salt (DSS) for 7 days to induce a mouse model of ulcerative colitis. Simultaneously with the establishment of the ulcerative colitis model, mice in the MGQD-L and MGQD-H groups were gavaged daily with 5.07 g / kg and 10.14 g / kg of Modified Gegenqinlian Decoction, respectively. Mice in the SASP group were gavaged daily with 450 mg / kg of sulfasalazine. Mice in the control and DSS groups were gavaged daily with normal saline. During the experiment, mouse body weight was measured daily, and fecal characteristics and bloody stools were observed. After the experiment, colonic tissue, feces, and blood were collected from the mice for analysis.

[0046] 2.2 Experimental methods 2.2.1 Histological analysis After the mice were dissected, the colon was obtained, its length was measured, and it was photographed. The colon tissue was stained with hematoxylin and eosin and phosphatase (PAS), and histological changes in the specimens were analyzed using a light microscope.

[0047] 2.2.2 Enzyme-linked immunosorbent assay analysis The levels of TNF-α and IL-1β in mouse serum were detected by enzyme-linked immunosorbent assay.

[0048] 2.2.3 Western blotting Colon samples were thoroughly homogenized using RIPA lysis buffer, centrifuged, and assayed using a BCA protein assay kit. Denatured proteins were then separated by SDS-polyacrylamide gel electrophoresis and electroblotted onto a PVDF membrane. The membrane was blocked in 5% milk for 2 h. The membrane was incubated with antibodies overnight. The washed PVDF membrane was incubated with HRP-conjugated secondary antibodies of the corresponding species for 2 h. After washing, ECL development and Image J analysis were performed. Antibodies: TGR5 (1:1000, ab72608, Abcam), FXR (1:1000, bs-12867r, bioss) 2.2.4 Intestinal flora analysis Genomic DNA was extracted from mouse feces using the MagPure Soil DNA LQ Kit (Magan) in strict accordance with the manufacturer's instructions. DNA concentration and purity were assessed by NanoDrop 2000 (Thermo Fisher Scientific, USA) and agarose gel electrophoresis, and the DNA was stored at −20°C. Using the purified genomic DNA as a template, PCR amplification of the target region of the bacterial 16S rRNA gene was performed using barcoded primers and Takara Ex Taq high-fidelity enzyme. The universal primer pair 343F (5′-TACGGRAGGCAGCAG-3′) and 798R (5′-AGGGTATCTAATCCT-3′) targeting the V3-V4 region was used for bacterial diversity analysis.

[0049] PCR amplification products were detected by agarose gel electrophoresis and then purified using AMPure XP beads to remove impurities. The purified products were used as templates for a second round of PCR amplification. Magnetic beads were then used for purification again, and the purified second-round products were quantified by Qubit assay, after which the concentration was adjusted for sequencing. Shanghai Ouyi Biotechnology Co., Ltd. was commissioned to perform sequencing using the Illumina NovaSeq 6000 sequencing platform, generating 250 bp paired-end reads to obtain raw sequencing data.

[0050] 2.2.5 Analysis of bile acids in mouse feces and gallbladder Collect an appropriate amount of mouse fecal sample and place it in a pre-chilled cryotube. Add 10 μL of a mixed internal standard (CA-d4, LysoPC17, GCA-C13, and L-2-chlorophenylalanine) and 390 μL of methanol (containing 1 mM BHT) and grind in a grinder. Centrifuge at 12,000 rpm for 10 minutes at 4°C. Collect the supernatant, transfer it to a brown vial, and store at -80°C.

[0051] Bile acids were qualitatively and quantitatively determined using UPLC-ESI-MS / MS. The injection volume was 5 μL; the flow rate was 0.45 mL / min; the mobile phase consisted of A (0.1% formic acid in water) and B (methanol:acetonitrile:isopropanol, 1:1:1 volume ratio, containing 0.1% formic acid). The gradient elution method was: 0 min A / B (80:20, V / V), 0.5 min A / B (80:20, V / V), 1.5 min A / B (62:38, V / V), 12 min A / B (50:50, V / V), 17.5 min A / B (5:95, V / V), 19 min A / B (5:95, V / V), 19.01 min A / B (80:20, V / V), and 20 min A / B (80:20, V / V). Mass spectrometry method: curtain gas was 35 (psi); spray gas (Gas1) was 55 (psi), auxiliary heating gas (Gas2) was 55 (psi); negative ion spray voltage was -4500 V, positive ion spray voltage was 5500 V; ion source temperature was 450°C; column temperature was 45°C.

[0052] Target metabolites were analyzed in multiple reaction monitoring (MRM) mode, with optimization of the MRM pairs, declustering potential (DP), and collision energy (CE) for each analyte. Analyst software was used for data acquisition and further analysis. All metabolites were quantified using SCIEX OS-MQ software.

[0053] SPSS 23.0 software was used for data processing. If the measurement data conformed to the normal distribution, the mean ± standard deviation was used. If the data did not conform to normality or had unequal variances, the Kruskal-Wallis H test for multiple independent samples was used in the nonparametric test, and the rank sum test was used for pairwise comparison. P A value < 0.05 was considered statistically significant.

[0054] 2.3 Experimental Results 2.3.1 Ulcerative colitis can alleviate colon inflammation in mice induced by dextran sulfate sodium salt solution A mouse ulcerative colitis model was established using 3% sodium dextran sulfate solution to study the therapeutic effect of modified Gegenqinlian decoction on ulcerative colitis. Modified Gegenqinlian decoction was administered orally at doses of 5.07 g / kg / d and 10.14 g / kg / d, with sulfasalazine 450 mg / kg / d as a positive control. The experimental design is shown in the attached figure. Figure 2 As shown in A.

[0055] In this study, mice with ulcerative colitis were treated with modified Gegenqinlian decoction and sulfasalazine. Compared with the control group, the weight of mice in the DSS group decreased significantly, the DAI score increased significantly, the spleen index increased, and the colon length decreased. These results confirmed the effectiveness of the ulcerative colitis model. In terms of treatment, MGQD-L, MGQD-H, and SASP administration were able to effectively reverse the weight loss and increased DAI score of ulcerative colitis model mice induced by 3% dextran sulfate sodium salt solution, as shown in the attached figure. Figure 2 As shown in B~2C; after treatment, the spleen index decreased significantly, see the attached figure Figure 2 D shows that the length of the colon is significantly extended, see the attached figure Figure 2 As shown in E~2F, there were significant differences compared with the DSS group, especially the MGQD-H group performed more prominently in improving the symptoms of ulcerative colitis, suggesting that its therapeutic effect may be dose-related.

[0056] The hallmark features of ulcerative colitis include colonic damage and inflammation, typically observed by HE staining. Histopathological analysis revealed significant crypt damage, inflammatory cell infiltration, and a reduction in goblet cells in the colonic tissue of ulcerative colitis model mice. Treatment with modified Gegenqinlian decoction and sulfasalazine significantly improved these lesions, as evidenced by reduced crypt structural damage and decreased inflammatory cell infiltration (see attached figure). Figure 3 As shown in A; the number of goblet cells is restored, see the attached figure Figure 3 As shown in B.

[0057] In addition, the levels of TNF-α and IL-1β in the serum of ulcerative colitis model mice were significantly increased. After treatment with modified Gegenqinlian decoction and sulfasalazine, the levels of these inflammatory markers were significantly reduced. (See attached figure) Figure 3 As shown in C~3D, it was shown that modified Gegenqinlian decoction significantly inhibited inflammation in ulcerative colitis model mice.

[0058] These results suggest that modified Gegenqinlian decoction has potential therapeutic value in alleviating inflammation caused by ulcerative colitis.

[0059] 2.3.2 Modified Gegenqinlian Decoction reverses intestinal flora imbalance in dextran sulfate sodium salt solution-induced colitis in mice In the fecal samples of mice in the five experimental groups, 1,185 operational taxonomic units (OTUs) were distinguished. 73 OTUs in the dextran sulfate sodium salt solution group were different from those in the other groups (see Figure 1). Figure 4As shown in A. In addition, treatment with modified Gegenqinlian decoction significantly upregulated the operational taxonomic units. Chao1 and Shannon index decreased in the DSS group, indicating that the abundance and diversity of intestinal microbiota in UC model mice were reduced. However, intervention with modified Gegenqinlian decoction resulted in a significant increase in Chao1 and Shannon index, as shown in the attached figure. Figure 4 As shown in Figures B-4C, this reflects a significant improvement in the abundance of the microbial community. Principal coordinate analysis (PCoA) assessed the distribution consistency of the microbiome between mice from different experimental groups. The results showed that there were significant differences in the intestinal flora of mice in the control group and the DSS group, indicating that there were significant differences in the composition and structure of the intestinal flora of the two groups of mice. After intervention with modified Gegenqinlian decoction, the distribution pattern of the microbial community was close to that of the control group, as shown in the attached figure. Figure 4 These studies revealed that the regulatory effect of modified Gegenqinlian decoction on ulcerative colitis mice may be attributed to the improvement of intestinal flora.

[0060] At the genus level, the 10 genera with the highest relative abundance were Muribaculaceae, Bacteroides, Lachnospiraceae_NK4A136_group, Prevotellaceae_UCG-001, Clostridia_UCG-014, Clostridia_vadinBB60_group, Alloprevotella, Alistipes, [Eubacterium]_coprostanoligenes_group, and Colidextribacter, see Figure 1. Figure 4 Figure E. Compared with the control group, the abundance of Bacteroides and Alloprevotella increased in the DSS group, while the abundance of Muribaculaceae, Lachnospiraceae_NK4A136_group, and Lactobacillus decreased. However, the application of modified Gegenqinlian decoction and sulfasalazine reversed these changes and reshaped the composition of the intestinal microbiota.

[0061] Modified Gegenqinlian Decoction and Regulation of Bile Acid Synthesis in Mice with Colitis Induced by Dextran Sulfate Sodium Salt Solution Bile acids, as metabolites of intestinal flora, are widely associated with the progression of intestinal inflammatory states. In patients with ulcerative colitis, disruption of intestinal flora leads to bile acid metabolism disorders, specifically manifested by increased primary bile acids (PBAs) and decreased secondary bile acids (SBAs), as well as increased conjugated bile acids and decreased unconjugated bile acids.

[0062] After the MGQD-H group was renamed the MGQD group, the bile acid content in the feces and gallbladder of mice in different groups was detected, as shown in the attached figure. Figure 5 As shown in Figure A, principal component analysis (PCA) showed that there were differences in the bile acid profiles in the feces of mice in the DSS group and the control group. After intervention with modified Gegenqinlian decoction, the bile acid profiles of mice tended to be similar to those of the control group, showing a high degree of similarity. Specifically, ulcerative colitis model mice showed a significant decrease in secondary bile acid levels, while mice treated with modified Gegenqinlian decoction showed higher levels, as shown in the attached figure. Figure 5 As shown in Figure B, compared with the control group, the production of α-muricholic acid (α-MCA), lithocholic acid (LCA), and 3β-cholic acid (3β-CA) in ulcerative colitis model mice was reduced. After intervention with modified Gegenqinlian decoction, the concentrations of these bile acids increased. In addition, modified Gegenqinlian decoction significantly increased the production of bile acid (CA) and taurocholic acid (TCA) in feces, as shown in the attached figure. Figure 5 As shown in C.

[0063] 2.2.4 Modified Gegenqinlian Decoction upregulates the expression of TGR5 and FXR in the colonic tissue of mice with colitis induced by dextran sulfate sodium salt solution Knockout of the G protein-coupled receptor (TGR5) enhanced the recruitment of macrophages in the intestinal lamina propria and aggravated the degree of inflammation. Farnesoid X receptor (FXR) agonists regulate bile acid homeostasis, reduce bile acid concentrations in the colon, inhibit inflammatory pathways, and effectively alleviate colitis. In the colon tissue of mice, we observed that the levels of bile acid receptors TGR5 and FXR in the DSS group were significantly lower than those in the control group. This finding suggests that bile acid signaling may be inhibited in the DSS group. However, compared with the DSS group, the expression of TGR5 and FXR in the MGQD-H group was significantly increased, see the attached figure. Figure 6 As shown in A to 6B, this suggests that modified Gegenqinlian decoction may have a positive effect on the colon function of mice by increasing the levels of TGR5 and FXR.

[0064] (3) Pseudo-sterility experiments and fecal microbiota transplantation experiments 1. Animal Model Preparation Pseudo-sterile experiment: After one week of adaptive feeding, the mice were randomly divided into the DSS group, ABX+DSS group, and ABX+DSS+MGQD group, with 6 mice in each group. The mice in the DSS group drank drinking water for 7 days, and the mice in the ABX+DSS group and ABX+DSS+MGQD group drank a mixed antibiotic solution (metronidazole 1 g / L, neomycin 1 g / L, ampicillin 1 g / L, and vancomycin 0.5 g / L) for 7 days to eliminate intestinal flora. After the experiment officially began, the mice in the DSS group, ABX+DSS group, and ABX+DSS+MGQD group drank 3% dextran sulfate sodium salt solution freely for 7 days to induce an ulcerative colitis mouse model. While inducing the ulcerative colitis mouse model, the mice in the ABX+DSS+MGQD group were gavaged with 10.14 g / kg of modified Gegenqinlian decoction every day. The experimental design is shown in the attached figure. Figure 7 As shown in A.

[0065] Fecal microbiota transplantation experiment: After one week of adaptive feeding, the mice were randomly divided into FMT-CON group and FMT-MGQD group, with 6 mice in each group. The two groups of mice drank a mixed antibiotic solution (metronidazole 1 g / L, neomycin 1 g / L, ampicillin 1 g / L, and vancomycin 0.5 g / L) for 7 days to eliminate intestinal flora. After the experiment officially started, the mice in the FMT-CON group and the FMT-MGQD group drank 3% dextran sulfate sodium salt solution freely for 7 days to induce an ulcerative colitis mouse model. At the same time as the model was established, the mice in the FMT-CON group were gavaged with fecal supernatant from normal mice, and the mice in the FMT-MGQD group were gavaged with fresh fecal supernatant from ulcerative colitis mice treated with modified Gegen Qinlian Decoction. The experimental design is shown in the attached figure. Figure 8 As shown in A.

[0066] 3. Experimental Results 3.1 Modified Gegenqinlian Decoction exerts its therapeutic effect on dextran sulfate sodium salt solution-induced colitis in mice by relying on intestinal flora To investigate whether modified Gegenqinlian decoction alleviates intestinal inflammation by relying on intestinal flora, we provided mice with a mixed solution of antibiotics to eliminate intestinal flora. There was no statistical difference in body weight and DAI scores among mice in the DSS group, ABX+DSS group, and ABX+DSS+MGQD group (see attached figure). Figure 7 B~7C. The research data showed that there was no statistical difference in spleen index and colon length among the three groups of mice, see the attached figure Figure 7 D~7F. In addition, histopathological examination revealed that there were obvious colon tissue pathological changes among the three groups of mice, as shown in the attached figure. Figure 7 As shown in G~7H.

[0067] Fecal microbiota transplantation experiment results: There were statistically significant differences in body weight and DAI scores between FMT-CON and FMT-MGQD mice, see attached figure Figure 8 As shown in B-8C, the colon length of FMT-MGQD mice increased significantly, while the spleen index decreased, see the attached figure Figure 8 As shown in D~8F; the expression of TNF-α and IL-1β in serum of FMT-MGQD mice was significantly reduced, see the attached figure Figure 8 G~8H. Histopathological examination showed that the colon damage of FMT-MGQD mice was alleviated, inflammatory cell infiltration was reduced, and the number of goblet cells increased, as shown in the attached figure. Figure 8 These results suggest that the alleviating effect of modified Gegenqinlian decoction on ulcerative colitis may depend on the regulation of intestinal flora.

[0068] (IV) Clinical Cases In a multicenter, randomized, double-blind controlled clinical trial, 240 patients with mild to moderate ulcerative colitis were enrolled and randomly divided into a modified Gegenqinlian decoction treatment group (80 cases), a traditional Gegenqinlian decoction control group (80 cases), and a mesalazine control group (80 cases) for a 12-week intervention observation.

[0069] The results showed that the total clinical efficacy rate in the modified Gegenqinlian Decoction group reached 91.25%, significantly higher than the 76.25% in the traditional Gegenqinlian Decoction group (P < 0.05) and the 78.75% in the mesalazine group (P < 0.05). In terms of specific indicators, the treatment group was superior to the control group in alleviating symptoms such as diarrhea (average daily bowel movements decreased from 4.2 ± 1.5 to 1.3 ± 0.6), blood in stool (blood in stool score decreased by 82.3%), and abdominal pain (visual analog scale score decreased by 76.8%). The mucosal healing rate (68.75%) was significantly higher than that in the traditional prescription group (45%) and the mesalazine group (50%). During the long-term maintenance treatment phase, a six-month follow-up revealed that the relapse rate in the modified Gegenqinlian Decoction group was only 11.25%, significantly lower than the 27.5% in the traditional formula group and the 25% in the mesalazine group. Gut microbiome metagenomic sequencing analysis revealed a 2- to 3-fold increase in the abundance of beneficial bacteria, such as Akkermansia and Bifidobacterium, in the intestinal tract of patients in the treatment group compared to baseline. Furthermore, the activity of key enzymes involved in secondary bile acid metabolism, such as bile salt hydrolase and 7α-dehydroxylase, was significantly enhanced, which was positively correlated with improvement in clinical symptoms (r=0.78, P<0.01). Subjective feedback from patients indicated that the treatment group significantly improved accompanying symptoms, such as general fatigue and loss of appetite. Quality of life scores (SF-36) increased by 42.6% compared to baseline, significantly superior to the other two groups.

[0070] The clinical cases of modified Gegenqinlian Decoction are as follows.

[0071] Case 1 Patient's basic information Patient Wang, a 35-year-old male, had suffered from ulcerative colitis for two years. He experienced 5 to 7 episodes of diarrhea daily, bloody stools, and significant, persistent abdominal pain, with a pain score of 7 on a visual analog scale of 10. He also experienced a feeling of urgency and heaviness in the abdomen, general fatigue, and loss of appetite, with a weight loss of 8 kg compared to his pre-illness weight. Colonoscopy revealed extensive congestion and edema of the sigmoid and rectal mucosa, with scattered ulcers.

[0072] Initial treatment plan and results The patient was initially treated with oral mesalamine enteric-coated tablets, 1 gram four times daily, and concurrently with mesalamine enema, administered once every night. After two weeks of treatment, diarrhea frequency decreased to three to four times daily, and bloody stools were lessened. However, abdominal pain was not significantly relieved, with a pain score of 5. Tenesmus persisted, and symptoms such as general fatigue and loss of appetite showed little improvement.

[0073] Treatment plan and efficacy of modified Gegenqinlian decoction Treatment was switched to modified Gegenqinlian Decoction, one dose daily, divided into morning and evening doses. In addition, retention enemas with modified Gegenqinlian Decoction were administered once each night. After one week of treatment, diarrhea frequency decreased to two to three times daily, bloody stools significantly decreased, and abdominal pain was alleviated, with the pain score dropping to 3. After two weeks of treatment, diarrhea had essentially ceased, stools were formed and free of bloody stools, abdominal pain disappeared, tenesmus completely resolved, and symptoms such as general fatigue and loss of appetite significantly improved. Four weeks after treatment, a follow-up colonoscopy revealed significantly reduced congestion and edema in the sigmoid and rectal mucosa, a reduction in ulcer size, and the majority of ulcers had healed. After another four weeks of consolidation treatment, medication was discontinued. No recurrence was observed during the six-month follow-up.

[0074] Case 2 Patient's basic information Patient Li, a 42-year-old female, had a 1.5-year history of recurrent ulcerative colitis. During an attack, she experienced diarrhea 4 to 6 times daily, with loose stools containing a small amount of mucus, dull abdominal pain with a pain score of 4, and symptoms such as bloating, nausea, and vomiting. She also experienced poor mental status and sleep quality. Colonoscopy revealed granular changes in the descending colon mucosa, blurred vascular markings, and scattered shallow ulcers.

[0075] Initial treatment plan and results The patient was treated with traditional Gegenqinlian Decoction, one dose per day, divided into three doses. After three weeks of treatment, the frequency of diarrhea was reduced to 2 to 3 times per day, but the stool was still unformed, the abdominal pain was not significantly relieved, and the pain score was still 3 points. Symptoms such as abdominal distension, nausea, and vomiting did not improve significantly, and there was no significant improvement in mental state and sleep quality.

[0076] Treatment plan and efficacy of modified Gegenqinlian decoction Treatment was switched to modified Gegenqinlian Decoction granules, one bag three times daily. After one week of treatment, the diarrhea ceased, bowel movements returned to normal, the dull abdominal pain eased, and the pain score dropped to 1. Symptoms such as bloating, nausea, and vomiting disappeared. After two weeks of treatment, his mental state improved significantly, and his sleep quality improved. Six weeks after treatment, a follow-up colonoscopy revealed significantly reduced mucosal lesions in the descending colon, with clear vascular markings and healed ulcers. Modified Gegenqinlian Decoction granules were continued for another two weeks to consolidate the therapeutic effect. Follow-up for six months showed stable disease with no relapses.

[0077] Case 3 Patient's basic information Patient Zhang, a 50-year-old male, had suffered from ulcerative colitis for three years. His condition was severe, with 7–8 episodes of diarrhea daily, heavy bleeding, and severe abdominal pain (a pain score of 8), severely impacting his daily life. He also suffered from weight loss and anemia, with his hemoglobin dropping to 90 g / L. Colonoscopy revealed diffuse congestion and edema of the entire colonic mucosa, with multiple confluent ulcers covered with pus and blood.

[0078] Initial treatment plan and results Initial treatment included oral glucocorticoids (prednisone) at an initial dose of 40 mg daily, combined with antibiotics for infection. After one week of treatment, diarrhea decreased to 5–6 episodes per day, and blood in the stools decreased. However, the abdominal pain remained unchanged, with a pain score of 7. Furthermore, adverse drug reactions included loss of appetite and nausea.

[0079] Treatment plan and efficacy of modified Gegenqinlian decoction Glucocorticoids were discontinued and treatment switched to modified Gegenqinlian Tang pills, 6g each time, three times daily. Simultaneously, a sitz bath with traditional Chinese medicine was administered once daily. After two weeks of treatment, diarrhea frequency decreased to three to four times daily, blood in the stool significantly decreased, abdominal pain eased, and the pain score dropped to 4. After four weeks of treatment, diarrhea frequency decreased to one to two times daily, blood in the stool essentially ceased, abdominal pain was significantly alleviated, the pain score dropped to 1, appetite returned to normal, and anemia symptoms gradually improved. Eight weeks after treatment, a follow-up colonoscopy revealed significant reduction in colonic mucosal congestion and edema, with the ulcer largely healed. Modified Gegenqinlian Tang pills were continued for another four weeks as consolidation therapy. Follow-up for six months showed stable condition, with hemoglobin returning to 120 g / L.

[0080] In summary, the modified Gegenqinlian Decoction of the present invention, through the combined use of cold and warm medicines, can nourish the vital energy while clearing away heat, effectively reverse the imbalance of intestinal flora in colitis, enhance the diversity of intestinal flora, increase the abundance of beneficial bacteria Muribaculaceae, Lachnospiraceae_NK4A136_group and Lactobacillus, and regulate the level of metabolite bile acid and the expression of FXR and TGR5 in colon tissue, thereby alleviating colon damage, reducing inflammatory cell infiltration, and increasing the number of goblet cells. It can effectively improve abdominal pain, diarrhea, bloody stools and other symptoms clinically, and the condition is stable after cure and is not prone to recurrence.

Claims

1. A modified Gegenqinlian decoction for treating ulcerative colitis with the function of regulating intestinal flora, characterized in that: It is mainly made of the following raw materials by weight: 20-28 parts of Puerariae Radix, 6-12 parts of Scutellariae Baicalensis, 6-12 parts of Coptis Rhizome, 3-9 parts of Radix Glycyrrhizae Preparata, 3-9 parts of White Peony Root, 6-12 parts of Jujube, 9-15 parts of Vinegar-cooked Schisandrae Chinensis, and 1-6 parts of Evodiae Rutaecarpa.

2. The modified Gegenqinlian decoction for treating ulcerative colitis having the function of regulating intestinal flora according to claim 1, characterized in that: 22-26 parts of Pueraria root and 2-5 parts of Evodia rutaecarpa.

3. The modified Gegenqinlian decoction for treating ulcerative colitis having the function of regulating intestinal flora according to claim 1, characterized in that: 24 parts of Puerariae Radix, 9 parts of Scutellariae Baicalensis, 9 parts of Coptis Rhizome, 6 parts of Radix Glycyrrhizae Preparata, 6 parts of White Peony Root, 9 parts of Jujube, 12 parts of Vinegar-cooked Schisandrae Chinensis, and 3 parts of Evodiae Rutaecarpa.

4. The modified Gegenqinlian decoction for treating ulcerative colitis having the function of regulating intestinal flora according to claim 1, characterized in that: The dosage form of the modified Gegenqinlian decoction is one of decoction, granule, oral liquid, paste, pill and enema.

5. The method for preparing the Gegenqinlian decoction for treating ulcerative colitis having the function of regulating intestinal flora according to any one of claims 1 to 4, characterized in that: The raw medicinal materials are decocted together to prepare the modified Gegenqinlian decoction.

6. The method for preparing the Gegenqinlian decoction for treating ulcerative colitis with the function of regulating intestinal flora according to claim 5, characterized in that: Place the pretreated raw medicinal materials in a decoction container, add drinking water 8 to 10 times the total weight of the medicinal materials, soak for 30 to 60 minutes, then boil over high heat, then simmer over low heat for 40 to 60 minutes, and filter and collect the first decoction; add drinking water 6 to 8 times the total weight of the medicinal materials to the medicinal residue, boil over high heat again, then simmer over low heat for 30 to 40 minutes, and filter and collect the second decoction; The two decoctions were combined and concentrated at low temperature to the desired concentration to obtain the modified Gegenqinlian decoction.

7. The method for preparing the Gegenqinlian decoction for treating ulcerative colitis with the function of regulating intestinal flora according to claim 6, characterized in that: The concentration temperature is controlled at 50~75℃.

8. The method for preparing the Gegenqinlian decoction for treating ulcerative colitis with the function of regulating intestinal flora according to claim 6, characterized in that: After the decoction was concentrated, different dosage forms of modified Gegenqinlian decoction were prepared according to corresponding standards.

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