Dahpinol-emblica compositions, methods of making and use in treating ulcerative colitis

Abstract

The present application belongs to the technical field of traditional Chinese medicine, and specifically relates to a Dai Baijie-Yangtao composition, a preparation method thereof and application of the Dai Baijie-Yangtao composition in the treatment of ulcerative colitis. The Dai Baijie-Yangtao composition is prepared by mixing a Dai Baijie water extract and a Yangtao water extract at a ratio of 1:1. The composition can be administered orally and can effectively treat ulcerative colitis, has the effects of anti-inflammation, relieving intestinal bleeding, recovering colon length, regulating intestinal flora structure (increasing the number of beneficial bacteria), and also has a liver protection effect on non-alcoholic hepatitis. The preparation method is simple, the instruments are general, and the method is easy to industrialize. Based on the theory of ethnic medicine, the method has no risk of antibiotic resistance, is suitable for the needs of green breeding and clinical medication, and has high clinical application potential.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine technology, specifically relating to the Dai Bai Jie-Yu Gan Zi composition, its preparation method, and its application in the treatment of ulcerative colitis. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Ulcerative colitis (UC) is a prevalent chronic inflammatory bowel disease that not only harms human health in clinical practice but also causes serious economic losses in large-scale farming. Its pathogenesis is related to factors such as feed spoilage, pathogenic microbial infection, poor rearing environment, stress response, and intestinal flora imbalance. In Dai medicine, UC is called "Lu Duan," which is believed to be caused by the accumulation of toxins and dysfunction of the spleen and stomach. The core pathogenesis is the accumulation of damp-heat toxins in the intestines, leading to stagnation of qi and blood, and the formation of ulcers due to tissue decay. Symptoms after onset include loss of appetite, dehydration and emaciation, stunted growth, and congestion, edema, erosion, and ulceration of the colonic mucosa.

[0004] Currently, the main treatments for UC in clinical practice and aquaculture are antibiotics such as streptomycin, penicillin, and tetracyclines, supplemented by fluid replacement and astringent antidiarrheal drugs. However, long-term use of antibiotics can easily lead to drug resistance and disrupt the balance of intestinal flora, which does not meet the development needs of green aquaculture and food safety. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a combination of Marsdenia tenacissima and Phyllanthus emblica, its preparation method, and its application in the treatment of ulcerative colitis. Specifically, this invention treats ulcerative colitis using traditional Chinese medicine principles, combining Marsdenia tenacissima (MT) and Phyllanthus emblica (PE). Marsdenia tenacissima is more "clearing" and Phyllanthus emblica is more "astringent." The combination of the two has the effects of clearing heat and dampness, harmonizing the liver and spleen, regulating qi and blood, promoting ulcer healing and tissue regeneration, and strengthening the spleen and replenishing qi and kidneys during the remission period, thus being used to treat ulcerative colitis. Based on the above research results, this invention was completed.

[0006] To achieve the above-mentioned technical objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a Daibaijie-Phyllanthus amla composition, the composition comprising a Daibaijie aqueous extract and a Phyllanthus amla aqueous extract in a mass ratio of 0.5-5:1, preferably 1:1; both the Daibaijie aqueous extract and the Phyllanthus amla aqueous extract are obtained by a water extraction method.

[0007] In a second aspect, the present invention provides a method for preparing the above-mentioned Dai Bai Jie-Phyllanthus composition, the method comprising: mixing Dai Bai Jie water extract and Phyllanthus emblica water extract to obtain the product.

[0008] The mass ratio of the aqueous extract of *Daibai* and the aqueous extract of *Phyllanthus emblica* is 0.5-5:1, preferably 1:1. Under these conditions, the two produce a synergistic effect, thereby exerting a better therapeutic effect on ulcerative colitis.

[0009] Therefore, a third aspect of the present invention provides the use of the above-described Dai Bai Jie-Phyllanthus composition in the following various applications: (a) Preparation of drugs for the treatment of ulcerative colitis; (b) To prepare a drug with protective effects against non-alcoholic hepatitis.

[0010] In a fourth aspect, the present invention provides a medicament comprising at least the above-described Dai Bai Jie-Phyllanthus composition.

[0011] The drug has therapeutic effects on ulcerative colitis and protective effects against non-alcoholic hepatitis.

[0012] The beneficial technical effects of one or more of the above technical solutions are as follows: The above composition can significantly alleviate diarrhea and rectal bleeding symptoms in ulcerative colitis, restore colon length, reduce the levels of pro-inflammatory factors IL-6 and TNF-α, increase the level of anti-inflammatory factor IL-10, and repair pathological damage to colon tissue. Its efficacy is superior to using Dai Bai Jie or Phyllanthus emblica extract alone. Simultaneously, it has a significant liver-protective effect on non-alcoholic hepatitis and excellent repair effect on liver lobule structure. It can enrich beneficial bacteria such as *Lactobacillus rimoxiferus*, *Lactobacillus*, and *Thuristobacillus*, inhibit pro-inflammatory bacteria such as *Helicobacter* and *Enterococcus*, improve the intestinal flora structure, and treat UC from a microecological perspective without the risk of drug resistance.

[0013] Meanwhile, the above composition adopts conventional alcohol extraction, water decoction and freeze drying processes. The required instruments and equipment are common laboratory / industrial equipment. The operation steps are universal, which can be mass-produced. The amount of raw medicinal materials can be controlled and the product has high stability.

[0014] Phyllanthus emblica possesses both medicinal and edible properties, while Dai Bai Jie (a traditional ethnic medicine) is also available. Both have no significant toxic side effects and can replace antibiotics in animal feed, aligning with the livestock industry's antibiotic reduction policies and meeting food safety and clinical medication needs. By combining Dai and Tibetan medicine theories and verifying the efficacy of these two ethnic medicines through modern pharmacological experiments, their synergistic effects have been demonstrated. This approach explores the scientific connotations of ethnic medicine and provides new insights for the modern development of ethnic medicine. Attached Figure Description

[0015] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0016] Figure 1 The figures show the epigenetic changes in mice with ulcerative colitis in different treatment groups in Example 2 of this invention. A represents the DAI score of the traditional Chinese medicine compound, B represents the change in body weight of mice induced by DSS with the traditional Chinese medicine compound, and C and D represent the changes in the degree of recovery of colon length in mice induced by DSS with the traditional Chinese medicine compound. * p<0.05, ** p<0.01, *** p<0.001.

[0017] Figure 2 The figures show the levels of inflammatory factors in mice with ulcerative colitis in different treatment groups in Example 2 of this invention. A represents the changes in inflammatory factor IL-10, B represents the changes in inflammatory factor IL-6, and C represents the changes in inflammatory factor TNF-α. * p < 0.05, ** p < 0.01, *** p < 0.001.

[0018] Figure 3 The images show the pathological changes in colon tissue of mice with ulcerative colitis in different treatment groups in Example 2 of this invention; A is the blank control group, B is the model group, C is the Dai Bai Jie group (MT), D is the Yang Gan Zi group (PE), and E is the compound group (MT&PE). The scale bar is 50 μm.

[0019] Figure 4 The images show liver tissue pathological sections from mice with ulcerative colitis in different treatment groups in Example 3 of this invention. A is the blank group, B is the model group, C is the positive group, D is the Dai Bai Jie group (MT), and F is the compound group (MT&PE). The scale bar is 50 μm.

[0020] Figure 5 The changes in liver function in mice with ulcerative colitis in different treatment groups in Example 3 of this invention are shown. A represents ALT index and B represents AST index. Detailed Implementation

[0021] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0022] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the present invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof. It should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for the purpose of describing specific embodiments and not for limiting the scope of protection of the present invention.

[0023] In view of the problems such as easy side effects in the existing treatment of ulcerative colitis, the present invention proposes a method for preparing a compound of Dai Bai Jie water extract (MT) and Phyllanthus emblica water extract (PE) (MT&PE) and its application in the preparation of drugs for treating ulcerative colitis.

[0024] Dai Bai Jie is a commonly used antidote in Dai folk medicine. In the Dai language, it means "antidote to all poisons." It mainly comes from the root of the *Tetrapanax papyriferus* plant, has a bitter taste, and is cooling in nature. Traditionally, it is used to clear heat, detoxify, reduce swelling, and relieve pain. Modern medical research has also found that Dai Bai Jie contains some anti-inflammatory and immune-regulating components.

[0025] Phyllanthus emblica is considered the foremost of the "three major fruits" in Tibetan medicine. Multiple studies have shown that it is extremely rich in polysaccharides and polyphenols, with Phyllanthus emblica pectin polysaccharide being its main anti-inflammatory component, resulting in excellent anti-inflammatory effects. Furthermore, its vitamin C content is exceptionally high, dozens of times that of lemons, giving it strong antioxidant capabilities. Tibetan medicine also indicates that Phyllanthus emblica possesses clear medicinal and edible properties.

[0026] The Dai medicine concept of "detoxification" perfectly corresponds to anti-inflammation and the elimination of toxins from the body. Its cool nature and bitter taste are also suitable for symptoms of damp-heat in the intestines. These two aspects are completely consistent with the theories of modern medicine. Furthermore, *Tetrapanax papyriferus* contains steroidal saponins, polysaccharides, and other components, which have the potential for anti-inflammatory, immunomodulatory, and mucosal repair effects. Its components perfectly support the strategy of antibiotic-free farming.

[0027] In view of this, in a typical embodiment of the present invention, a Dai Bai Jie-Phyllanthus composition is provided, the composition comprising a water extract of Dai Bai Jie and a water extract of Phyllanthus emblica, wherein the mass ratio of the two is 0.5-5:1, preferably 1:1; the water extracts of Dai Bai Jie and Phyllanthus emblica are both obtained by water extraction.

[0028] This invention overcomes the limitations of treating ulcerative colitis with single medicinal herbs. It combines *Dai Bai Jie* (a herb with properties of clearing heat and detoxifying) with *Yu Gan Zi* (another herb with properties of astringing and anti-inflammatory effects), and when combined in the appropriate ratio, triggers a synergistic effect between the two herbs, resulting in therapeutic effects far superior to those of single herbs. This method maximizes the retention of water-soluble anti-inflammatory and mucosal repair active ingredients in the herbs, avoiding the loss of effective components caused by other extraction methods, and ensuring the stability and controllability of the composition's efficacy.

[0029] In another specific embodiment of the present invention, the preparation method of the aqueous extract of Dai Bai Jie or the aqueous extract of Yang Gan Zi includes: adding a first ethanol to Dai Bai Jie or Yang Gan Zi for a first alcohol extraction and reflux, retaining the first solid residue; adding a second ethanol to the first solid residue for a second alcohol extraction and reflux; retaining the second solid residue to complete the removal of alcohol-soluble components; The second solid dregs are added to water and decocted. The filtrate is collected, concentrated, and freeze-dried to obtain the final product.

[0030] This invention employs a two-step extraction process: first, alcohol extraction to remove impurities, followed by water extraction to extract the active ingredients. This differs from traditional single water or alcohol extraction methods. First, alcohol extraction with reflux removes irrelevant alcohol-soluble impurities from the medicinal materials, preventing these impurities from coexisting with water-soluble active ingredients and affecting efficacy. Simultaneously, it reduces the impurity content of the subsequent water extract, improving product purity. The subsequent water extraction decoction specifically enriches water-soluble anti-inflammatory and gut microbiota-regulating active ingredients in the medicinal materials. The resulting powdered extract is then concentrated and freeze-dried, facilitating subsequent mixing and formulation, and enhancing the stability of the active ingredients.

[0031] In this invention, both the first ethanol and the second ethanol are high-concentration ethanols, with concentrations exceeding 70%, and the first ethanol concentration is higher than the second ethanol concentration. In one specific embodiment, the first ethanol concentration is 95%, and the second ethanol concentration is 80%. Compared to single-concentration ethanol extraction, gradient ethanol extraction removes impurities more thoroughly and avoids the drying of residue due to excessively high ethanol concentration, thus preventing disruption of subsequent water extraction dissolution efficiency. This gradient ethanol extraction design balances impurity removal effectiveness with compatibility with subsequent processes.

[0032] The mass-to-volume ratio of Dai Bai Jie or Yang Gan Zi to the first ethanol is 1:4-10 (g / mL), preferably 1:5; the reflux time for the first ethanol extraction is 1-3 h, preferably 1.5 h; The mass-to-volume ratio of the first solid residue to the second ethanol is 1:2-8 (g / mL), preferably 1:4; the reflux time for the second ethanol extraction is 0.5-2 h, preferably 1 h.

[0033] The mass-to-volume ratio of the second solid medicinal residue to water is 1:5-15 (g / mL), preferably 1:7.

[0034] This invention limits the material-to-liquid ratio and reflux time in alcohol extraction, ensuring that the ethanol can fully submerge the medicinal materials and dissolve impurities, while avoiding the waste of raw materials and increased subsequent recovery costs caused by excessive ethanol. The reflux time is adapted to the impurity dissolution rate of high-concentration ethanol, achieving the dissolution of most impurities and avoiding the evaporation and concentration drop of ethanol due to excessive reflux time, or the premature loss of effective components in the medicinal residue. This achieves high efficiency, energy saving and controllability of alcohol extraction for impurity removal.

[0035] The decoction time is specifically 0.5-5 hours, preferably 1 hour; furthermore, the decoction process also includes: squeezing out the water from the solid dregs after decoction, filtering with gauze to obtain filtrate; and even further, repeating the above decoction process 2-4 times (preferably 3 times). This achieves maximum extraction of water-soluble active ingredients while also ensuring ease of operation, making it suitable for large-scale production.

[0036] The concentration conditions are as follows: the obtained filtrate is concentrated to a crude drug concentration of 0.5-1.5 g / mL, preferably 1 g / mL. This concentration ensures a high concentration of active ingredients, avoids excessive concentration which would lead to time-consuming subsequent freeze-drying processes and low active ingredient content in the finished product, and also prevents excessive concentration which would cause the filtrate to stick to the walls and result in carbonization and loss of active ingredients.

[0037] In another specific embodiment of the present invention, a method for preparing the above-mentioned Dai Bai Jie-Phyllanthus composition is provided, the method comprising: mixing Dai Bai Jie water extract and Phyllanthus emblica water extract to obtain the product.

[0038] The aqueous extracts of *Daibai* and *Phyllanthus emblica* were obtained according to the above preparation method, which will not be repeated here. The mass ratio of the aqueous extracts of *Daibai* and *Phyllanthus emblica* was 0.5-5:1, preferably 1:1. Under these conditions, the two produce a synergistic effect, thereby exerting a better therapeutic effect on ulcerative colitis.

[0039] Therefore, in another specific embodiment of the present invention, the above-mentioned Dai Bai Jie-Phyllanthus composition is provided for use in the following various applications: (a) Preparation of drugs for the treatment of ulcerative colitis; (b) To prepare a drug with protective effects against non-alcoholic hepatitis.

[0040] Specifically, the therapeutic effect on ulcerative colitis is manifested as follows: (a1) Improves the phenotype of ulcerative colitis; (a2) Regulates inflammatory factors in colon tissue; (a3) Repairing pathological symptoms in colon tissue; (a4) Regulates gut microbiota.

[0041] Among them, the improvement of the ulcerative colitis phenotype is at least manifested in the relief of ulcerative colitis-mediated weight loss, colonic shortening, diarrhea and bloody stool symptoms; The regulation of inflammatory factors in colonic tissue is at least manifested in promoting the expression of anti-inflammatory factors and inhibiting the expression of pro-inflammatory factors; wherein the anti-inflammatory factors include IL-10; and the pro-inflammatory factors include IL-6 and TNF-α.

[0042] The repair of colonic tissue pathology symptoms includes restoring the colonic crypt structure, with goblet cells arranged in a tight and orderly manner, increased thickness of the striate margin, and a significant reduction in inflammatory cells.

[0043] Specifically, the regulation of gut microbiota involves inhibiting the accumulation of pro-inflammatory bacteria and enriching the core beneficial bacteria of a healthy gut. The pro-inflammatory bacteria include at least Helicobacter spp. and Enterococcus spp., and the core beneficial bacteria of the healthy gut include at least Lactobacillus moxibustioni, Lactobacillus spp. and Tulicobacter spp.

[0044] The protective effect against non-alcoholic hepatitis is manifested at least in reducing ALT and AST levels and repairing liver tissue damage.

[0045] Therefore, in another specific embodiment of the present invention, a drug is provided, the drug comprising at least the above-mentioned Dai Bai Jie-Phyllanthus composition.

[0046] The drug has therapeutic effects on ulcerative colitis and protective effects against non-alcoholic hepatitis.

[0047] The drug can be administered in unit doses, and the dosage form can be liquid or solid. Liquid dosage forms can include true solutions, colloids, microparticles, emulsions, and suspensions. Other dosage forms include tablets, capsules, pellets, aerosols, pills, powders, solutions, emulsions, granules, suppositories, lyophilized powder for injection, inclusion complexes, implants, patches, and liniments. The drug can be used by humans and non-human animals (farmed animals).

[0048] The following examples further illustrate the present invention, but do not constitute a limitation thereof. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the invention.

[0049] Example 1 A combination of MT and PE aqueous extracts (MT&PE) was prepared for extraction.

[0050] 1.1 Medicinal Materials Dai Bai Jie 1000 g solid medicinal material, Phyllanthus emblica 1000 g solid medicinal material.

[0051] 1.2 Aqueous Extraction Solution Remove surface impurities with pure water. Soak the *Dai Bai Jie* and *Yu Gan Zi* separately in 95% ethanol overnight. Take five times the volume of 95% ethanol (5000 mL) and add the herbs to a spherical glass container, completely submerging them with 5000 mL of 95% ethanol. Attach a reflux device and boil at a gentle boil for 1.5 hours. Retain the solid residue and pour out all the solution. A second boiling is performed using four times the volume of 80% ethanol (4000 mL), boiling the solid residue at a gentle boil for 1 hour. Pour out all the solution and retain the solid residue. The solid residue remaining after two refluxes was decocted in 7 times its volume (7000 ml) of pure water at a gentle boil for 1 hour. The water was then squeezed out of the solid residue, filtered through gauze, and the filtrate was stored for later use. This decoction process was repeated three times, resulting in three filtrates. Finally, the three filtrates were concentrated to a crude drug concentration of 1 g / mL. After cooling, the solution was frozen at -80℃ for 48 hours and then freeze-dried for 72 hours to obtain freeze-dried powder. The aqueous extracts of *Dai Bai Jie* and *Phyllanthus emblica* were prepared using the above method.

[0052] The above preparation methods include, but are not limited to, water decoction, water extraction and alcohol precipitation, enzymatic hydrolysis, acid-base extraction, and ultrasonic extraction.

[0053] 1.3 Ratio of Dai Bai Jie water extract (MT) to Phyllanthus emblica water extract (PE) The aqueous extracts of Dai Bai Jie and Yang Gan Zi obtained above were combined in a 1:1 mass ratio to form a composition (MT&PE) for subsequent efficacy verification.

[0054] Example 2 The therapeutic effect of the formulation composition (MT&PE) on DSS-induced colitis in mice 2.1 Experimental Design Forty 6-8 week old BALB / c mice were acclimatized for 7 days and then randomly divided into four groups: blank control group (CON group), DSS model group (MOD group), DSS + Dai Bai Jie water extract group (MT group), DSS + Phyllanthus emblica water extract group (PE group), and DSS + compound combination group (MT&PE group), with 8 mice in each group.

[0055] The CON group was given distilled water daily, while the other groups were given 3% DSS aqueous solution. The PE group was administered 3.51 g / kg of Phyllanthus emblica aqueous extract by gavage daily. The MT group and the MT&PE group were administered 3.51 g / kg of Daibaixie aqueous extract and 3.51 g / kg of the combined solution by gavage, respectively. The blank group and the model group were administered 200µL of distilled water by gavage daily for 7 consecutive days. The mice were observed and weighed daily, and their weight, fecal characteristics, and fecal occult blood were recorded daily.

[0056] 2.2 Sample Collection and Testing After 7 days of gavage feeding, mice were euthanized, and blood was collected. The colons and feces were collected from dissected mice, and colon length was measured and weighed. Partial colon tissue was embedded and then prepared into HE-stained sections following the sequence of fixation, dehydration, clearing, paraffin infiltration, embedding, sectioning, mounting, dewaxing and staining, differentiation and rinsing, dehydration and counterstaining, dehydration and clearing, and sealing. Pathological changes in the colon tissue of each group were observed under a microscope. Changes in inflammatory factors (IL-10, IL-6, TNF-α) were measured after partial colon grinding. DAI scores were calculated using the DAI scoring criteria in Table 1.

[0057] Table 1 DAI Scores

[0058] 2.3 Data Statistics and Analysis All experimental data are expressed as Mean ± SD. The data were processed using Graph Pad Prism 9.0 to represent the mean ± standard error, and one-way ANOVA was used for statistical calculations and data analysis. p <0.05 and p <0.01 indicates significant and highly significant differences, respectively.

[0059] 2.4 Results 1) Phenotypic changes and DAI scores After DSS modeling in mice, different groups of mice exhibited different mental states and behavioral behaviors. Figure 1 A). During the feeding process, the CON group showed glossy and smooth fur, normalized behavior, and no symptoms such as diarrhea or bloody stools. The MOD group, given 3% DSS solution in drinking water, exhibited poor mental state, curling up and trembling, lethargy, reduced activity, and disheveled fur during the feeding process. The mice also showed varying degrees of diarrhea and bloody stools. After treatment, the diarrhea symptoms in the positive group significantly improved, and their mental state recovered in the later stages of treatment, with a significant improvement in bloody stools. p< 0.01), the mice were active, had smooth and soft fur, and dry perianal area. The MT group, PE group, and MT&PE group also significantly alleviated the bloody stool and mental state of the mice. p< 0.01). Weight monitoring showed ( Figure 1 B), the body weight of the CON group mice steadily increased, while the body weight of the MOD group, which was given 3% DSS solution in drinking water, gradually decreased, indicating that the DSS model was successful. The body weight of the MT group, PE group, and MT&PE group began to recover after 3 days of drinking 3% DSS solution, and the body weight loss was significantly improved compared to the MOD group. p<0.05. At the end of treatment, the body weight of the MT group, PE group, and MT&PE group was significantly higher than that of the MOD group ( p The value <0.01 indicates that different treatment groups can alleviate the weight loss in mice caused by DSS, with the MT&PE group showing the most significant weight recovery.

[0060] 2) MT&PE recovery of colon shortening in DSS-modeled mice Seven days after administration, mice were euthanized and then dissected, revealing (…). Figure 1 (C, D) Compared to the CON group, the MOD group mice showed significant facial bleeding and a significantly shortened colon length. p <0.001), and after drug treatment, the colon of mice recovered, and compared with the MOD group, the colon length of the MT group, PE group and MT&PE group were significantly increased ( p <0.001), and apparent bleeding symptoms also improved. In terms of recovery degree, the MT&PE group showed the best colonic recovery effect.

[0061] 3) MT & PE regulate the levels of colitis factors in DSS-induced colitis in mice. Detection of inflammatory factors in the colon tissue of mice in each group revealed ( Figure 2 The CON group had the highest level of anti-inflammatory factor (IL-10), while the MOD group had the lowest level. This indicates that DSS modeling reduces the anti-inflammatory capacity of the mouse colon. After MT, PE, and MT&PE treatment, the IL-10 level in the mouse colon was significantly higher than that in the MOD group. p <0.01]. Detection of pro-inflammatory factors (IL-6 and TNF-α) levels in the mouse colon revealed ( Figure 2 In groups B and C, the levels of IL-6 and TNF-α in the MOD group were significantly higher than those in other groups. p <0.001), while MT, PE and MT&PE treatment reduced IL-6 and TNF-α in the colon of mice, all of which had the effect of inhibiting the inflammatory response. Among them, MT&PE showed the best inhibitory effect, and the levels of IL-6 and TNF-α had recovered to close to those of the CON group, indicating that the combination of MT and PE can produce a synergistic effect and the treatment effect is better than MT or PE alone.

[0062] 4) MT & PE ameliorate DSS-induced colonic injury in mice Colon tissues from mice in each group were embedded in paraffin and stained with hematoxylin and eosin (HE). The results showed (…). Figure 3 In the CON group mice, the colonic epithelium was intact, cells were tightly packed, intestinal glands were clearly visible, crypt structures were intact, and goblet cells were tightly and neatly arranged; no obvious lesions were observed in the field of view. The colonic tissue of the model group treated with 3% DSS showed obvious lesions. Figure 3B) Changes occur in the colonic tissue structure, including loss of goblet cells, disappearance of crypts, inflammatory cell infiltration, and atrophy and disappearance of intestinal glands. After treatment with MT and PE ( Figure 3 (C, D) The colonic tissue structure of mice gradually returned to normal, and the structures of crypts and intestinal glands gradually recovered. After MT&PE treatment ( Figure 3 E) The colonic crypts of mice gradually recovered, goblet cells were tightly arranged, the striate margin was thickened, inflammatory cells decreased, and the structure was basically restored to normal levels, indicating that MT&PE can more effectively restore DSS-induced colonic damage in mice.

[0063] Example 3 Protective effect of the formulation composition (MT&PE) against ethanol-induced non-alcoholic hepatitis in mice. 3.1 Experimental Design Numerous studies have demonstrated the protective and beneficial effects of silymarin (SM) on the liver. Therefore, this patent uses it as a positive control and employs a methionine-restricted, choline-deficient diet (MCD) to establish a model. Forty-eight 6-8 week old BALB / c mice were randomly divided into four groups (n=8 per group): a blank control group (CON group), a model group (MOD group), a positive control group (positive group), an MCD + Dai Bai Jie water extract group (MT group), an MCD + Phyllanthus emblica water extract group (PE group), and an MCD + a combination of ingredients (MT&PE group).

[0064] The CON and MOD groups had free access to food daily. The MT, PE, and MT&PE groups were administered 3.51 g / kg of *Daibai* water extract, 3.51 g / kg of *Phyllanthus emblica* water extract, and 3.51 g / kg of the combined extract, respectively, by gavage. The positive control group was administered 100 mg / kg of silymarin by gavage. The mice were observed and weighed daily. After 6 weeks, the mice were euthanized, and blood was collected. Liver tissue was dissected and collected, and the size and weight of the liver were measured. After partial embedding, the liver tissue was fixed, dehydrated, cleared, paraffin-embedded, embedded, sectioned, mounted, dewaxed and stained, differentiated and washed, dehydrated and counterstained, dehydrated and cleared, and sealed to prepare HE-stained sections. The pathological changes of the colon tissue in each group were observed under a microscope, and the ALT and AST indices of each group were measured.

[0065] 3.1 Experimental Results 1) The protective effect of MT&PE in non-alcoholic hepatitis Hematologic tests were conducted on different groups of mouse livers to prepare hematologic marrow extracts (HE) and to measure the levels of alanine transaminase (ALT) and aspartate transaminase (AST) in the livers. The results showed that... Figure 4,5), liver sections of CON group mice showed ( Figure 4 A) The liver lobule structure is intact, the hepatic cords are arranged radially, the rod cells are morphologically normal, and no degeneration, necrosis, or inflammatory infiltration is observed. There is no enlargement of the portal areas, and the tissue structure is normal. Liver sections from the MOD group show ( Figure 4 B) Severe hepatocyte degeneration and necrosis, disordered arrangement, disappearance of hepatic cords, red blood cell aggregation in the hepatic sinusoids, and extensive inflammatory infiltration indicate successful model establishment. Liver sections of the MT group showed ( Figure 4 D), narrowed interhepatic cord spacing, small amount of erythrocyte aggregation in the central vein and hepatic sinusoids, stem cells with mild edema, loose cytoplasm, and mild inflammatory cell infiltration. Liver sections of the PE group showed ( Figure 4 E), liver cords were clearly visible in some areas, with a small amount of erythrocyte aggregation in the hepatic sinusoids, and hepatocyte morphology was normal. Liver sections of the MT&PE group showed ( Figure 4 F), the liver lobule structure was intact, the hepatic cords were arranged radially, the rod cells were of normal morphology, and no degeneration, necrosis, or inflammatory infiltration was observed. There was no enlargement of the portal areas, and the tissue structure was normal. Only a small number of erythrocytes were aggregated in the hepatic sinusoids. ALT and AST tests showed ( Figure 5 The ALS and ALT indices in the MT&PE group showed very little difference compared to the CON group. p The result was <0.01, indicating that MT&PE has a significant protective effect on the liver in non-alcoholic hepatitis.

[0066] Example 4 Effects of the formulation composition (MT&PE) on gut microbiota in DSS-induced colitis in mice 4.1 Experimental Design Fecal samples were collected during mouse necropsy. Total genomic DNA was extracted from the feces using the EZNA® Stool DNA Kit. DNA quality was assessed by 1% agarose gel electrophoresis, and DNA concentration and purity were determined using a NanoDrop2000 (Thermo Scientific, USA). For DNA samples that passed the initial screening, the V3-V4 variable region of the 16S rRNA gene was amplified in a PCR instrument using 338F (5'-ACTCCTACGGGAGGCAGCAG-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3') primers carrying the barcode sequence. The amplification conditions were: 95°C pre-denaturation for 3 min, followed by 27 cycles (95°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 30 s), and then a stable extension at 72°C for 10 min. The product was stored at 4°C. After recovery by 2% agarose gel electrophoresis, the amplified product was purified using a DNA gel extraction and purification kit and analyzed by Qubit. 4.0 The recovered products were quantified, and the purified PCR products were then used to construct a library using the NEXTFLEX Rapid DNA-Seq Kit. After demultiplexing, the sequences were quality filtered using FASTP (0.19.6) and assembled using FLASH (v1.2.11). The obtained high-quality sequences were then denoised using the DADA2 plugin with recommended parameters in the Qiime2 (version 2024) analysis workflow to obtain amplicon sequence variants (ASVs). The Chao 1 and Shannon Alpha diversity indices were calculated using Mothur software. The inter-group differences in Alpha diversity were analyzed using the Wilcoxon rank-sum test. Principal coordinate analysis (PCoA) based on the Bray-Curtis distance algorithm was used to examine the similarity of microbial community structure among samples. The significance of differences in microbial community structure among sample groups was analyzed using the LEfSe Analysis (LDA>2, P<0.05) was used to screen bacterial groups with significant differences in abundance at the phylum to genus level among different groups.

[0067] 4.2 Experimental Results 1) Effects of MT&PE on the microbial community structure of mice with ulcerative colitis Microbial sequencing alpha diversity analysis showed that, compared with the CON group, the ACE index and Chao1 index were significantly lower in the MT and MT&PE groups. p<0.05), and there was no significant difference in the Simpson index among the groups, indicating that the richness of the gut microbiota decreased after MT and MT&PE treatment, but the diversity remained unchanged. β-diversity PCoA analysis showed differences in the gut microbiota among the mouse intestinal groups after positive drug, MT, and MT&PE treatment. Further analysis of species composition revealed that, at the phylum level, all groups were rich in Firmicutes A and Bacteroidota, while the PE group showed a significant decrease in Firmicutes A and Firmicutes D. In contrast, the MT&PE group showed a significant increase in Firmicutes D, making it the dominant phylum in this group. At the genus level, the dominant genera were similar across the groups, with *Paramycium* being the most prevalent. Paramuribaculum) genus *Agrophytes* Kineothrix) Lactobacillus limoxicillinus ( Limosilactobacillus The core dominant bacterial genera were *Limosilactobacillus*, *Lactobacillus*, *Ligilactobacillus*, and *Turicibacter*, all of which consistently ranked in the Top 20 for anti-inflammatory and probiotic-related bacterial groups. LEfSe analysis of differentially expressed microorganisms in the PE group showed that pro-inflammatory bacteria such as *Helicobacter* and *Enterococcus* predominated. These genera are key markers of exacerbated enteritis inflammation and mucosal damage, and their specific enrichment directly confirms the pathological characteristics of the enteritis model's microecology. The CON group was dominated by probiotics such as *Limosilactobacillus*, *Turicibacter*, and *Bacteroides*, with significantly higher LDA values ​​than other groups, representing core characteristic genera of healthy gut microecological homeostasis. The characteristic bacterial genera of the MT&PE combination group highly overlapped with those of the CON group, and the LDA values ​​were higher, including Limosilactobacillus, Bacteroides_H, Turicibacter, etc., which clearly shows that the combination therapy can more accurately enrich the core functional bacteria of healthy gut and inhibit pro-inflammatory characteristic bacteria, and its microecological regulation effect is significantly better than that of monotherapy.

[0068] It should be noted that the above examples are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the given examples, those skilled in the art can modify or make equivalent substitutions to the technical solutions of the present invention as needed, without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. A Dai Bai Jie-Phyllanthus parasiticus composition, characterized in that, The composition comprises a water extract of *Daibai* and a water extract of *Phyllanthus emblica*, with a mass ratio of 0.5-5:1; both the water extract of *Daibai* and the water extract of *Phyllanthus emblica* are obtained by water extraction.

2. The Dai Bai Jie-Phyllanthus composition as described in claim 1, characterized in that, The preparation method of the aqueous extract of Dai Bai Jie or Yang Gan Zi includes: adding a first ethanol to Dai Bai Jie or Yang Gan Zi for a first alcohol extraction and reflux, retaining the first solid residue; adding a second ethanol to the first solid residue for a second alcohol extraction and reflux; and retaining the second solid residue. The second solid dregs are added to water and decocted. The filtrate is collected, concentrated, and freeze-dried to obtain the final product.

3. The Dai Bai Jie-Phyllanthus composition as described in claim 2, characterized in that, Both the first ethanol and the second ethanol are high-concentration ethanols, with a concentration greater than 70%, and the concentration of the first ethanol is higher than that of the second ethanol. Preferably, the concentration of the first ethanol is 95% and the concentration of the second ethanol is 80%.

4. The Dai Bai Jie-Phyllanthus composition as described in claim 2, characterized in that, The mass-to-volume ratio of Dai Bai Jie or Yang Gan Zi to the first ethanol is 1:4-10 (g / mL), preferably 1:5; the reflux time for the first ethanol extraction is 1-3 h, preferably 1.5 h; The mass-to-volume ratio of the first solid residue to the second ethanol is 1:2-8 (g / mL), preferably 1:4; the reflux time for the second ethanol extraction is 0.5-2 h, preferably 1 h; The mass-to-volume ratio of the second solid dregs to water is 1:5-15 (g / mL), preferably 1:7; The decoction time is specifically 0.5-5 hours, preferably 1 hour; further, the decoction process also includes: squeezing out the water from the solid dregs after decoction, filtering with gauze to obtain filtrate; even further, the above decoction process is repeated 2-4 times (preferably 3 times). The concentration conditions are as follows: the obtained filtrate is concentrated to a crude drug concentration of 0.5-1.5 g / mL, preferably 1 g / mL.

5. A method for preparing the Dai Bai Jie-Phyllanthus composition according to any one of claims 1-4, characterized in that, The preparation method includes mixing the water extracts of Dai Bai Jie and Phyllanthus emblica.

6. The use of the Dai Bai Jie-Phyllanthus composition according to any one of claims 1-4 in the following multiple applications: (a) Preparation of drugs for the treatment of ulcerative colitis; (b) To prepare a drug with protective effects against non-alcoholic hepatitis.

7. The application as described in claim 6, characterized in that, The specific therapeutic effects of the treatment for ulcerative colitis are as follows: (a1) Improves the phenotype of ulcerative colitis; (a2) Regulates inflammatory factors in colon tissue; (a3) Repairing pathological symptoms in colon tissue; (a4) Regulates gut microbiota.

8. The application as described in claim 7, characterized in that, The improvement in the ulcerative colitis phenotype is at least manifested in the relief of ulcerative colitis-mediated weight loss, colonic shortening, diarrhea, and bloody stool symptoms; The regulation of inflammatory factors in colonic tissue is at least manifested in promoting the expression of anti-inflammatory factors and inhibiting the expression of pro-inflammatory factors; wherein, the anti-inflammatory factors include IL-10; and the pro-inflammatory factors include IL-6 and TNF-α; The repair of colonic tissue pathology symptoms includes restoring the colonic crypt structure, with goblet cells arranged tightly and neatly, increased striate border thickness, significantly reduced inflammatory cells, and the colonic tissue pathology state basically restored to normal levels; Specifically, the regulation of gut microbiota involves inhibiting the accumulation of pro-inflammatory bacteria and enriching the core beneficial bacteria of a healthy gut. The pro-inflammatory bacteria include at least Helicobacter spp. and Enterococcus spp., and the core beneficial bacteria of the healthy gut include at least Lactobacillus moxibustioni, Lactobacillus spp. and Tulicobacter spp.

9. The application as described in claim 6, characterized in that, The protective effect against non-alcoholic hepatitis is manifested at least in reducing ALT and AST levels and repairing liver tissue damage.

10. A drug, characterized in that, The drug comprises at least the Dai Bai Jie-Phyllanthus composition according to any one of claims 1-4; the drug has therapeutic effects on ulcerative colitis and protective effects against non-alcoholic hepatitis.

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