A method for preparing and using a composition for treating ulcerative colitis

Abstract

The application relates to the field of medicine and discloses a preparation method and application of a composition for treating ulcerative colitis. The method comprises the following steps: mixing fresh feces of a screened healthy donor with sterile PBS at a ratio of 1:10, filtering through an 800-mesh sterile screen, and obtaining an intestinal flora suspension under anaerobic conditions within 3 hours; crushing liquorice and dendrobium officinale respectively, screening through an 80-mesh screen, and mixing the two at a mass ratio of 1:0.1 to obtain medicinal material raw materials; mixing the medicinal material raw materials and the flora suspension at a ratio of 1:4, filling bottles, sealing by filling nitrogen, and carrying out anaerobic culture at 37+ / -0.5 DEG C for 72 hours; and drying the fermentation product at 45 DEG C for 6 hours to obtain a fermentation product. The fermentation product and a pharmaceutical composition containing the fermentation product are used for preparing medicines for preventing or treating ulcerative colitis.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine fermentation, specifically to a method for preparing a composition for treating ulcerative colitis and its application. Background Technology

[0002] Ulcerative colitis (UC) is a chronic, relapsing inflammatory bowel disease clinically characterized by persistent inflammation and ulceration of the colonic mucosa, often accompanied by symptoms such as abdominal pain, diarrhea, and bloody stools. Its pathogenesis is complex, involving immune dysregulation, intestinal barrier damage, gut microbiota dysbiosis, and the interaction of genetic and environmental factors. Currently, Western medicine treatment primarily utilizes aminosalicylic acids, glucocorticoids, immunosuppressants, and biologics. While these can control symptoms, they suffer from significant side effects, high relapse rates, and high costs. Therefore, developing safe, effective, and multifunctional novel therapeutic drugs remains a research hotspot in this field.

[0003] Licorice, a traditional Chinese medicine, possesses the effects of clearing heat and detoxifying, relieving pain, and harmonizing other herbs. Its active components, such as glycyrrhizic acid, glycyrrhetinic acid, and glycyrrhizin flavonoids, have been proven by modern research to have significant anti-inflammatory, anti-ulcer, immunomodulatory, and mucosal protective effects, showing promising potential in the treatment of gastrointestinal diseases. The anti-inflammatory role of Dendrobium officinale is one of its core pharmacological activities, particularly focusing on immunomodulation, antioxidant effects, and mucosal repair related to ulcerative colitis (UC). Licorice provides potent "inhibition" and "anti-inflammatory" effects, while Dendrobium officinale provides systemic "repair" and "regulation" functions. However, traditional licorice extracts or decoctions suffer from low bioavailability of active ingredients, limited efficacy, and insufficient anti-inflammatory targeting, hindering their widespread application in the treatment of ulcerative colitis.

[0004] In recent years, microbial fermentation technology has provided an important pathway for the modernization of traditional Chinese medicine (TCM). Fermenting TCM substrates with specific microbial strains, such as probiotics and medicinal fungi, can produce the following advantages: 1. Transformation of original components, generating new compounds with stronger activity or easier absorption; 2. Decomposition of macromolecules, improving the dissolution rate of active ingredients; 3. Production of new metabolites such as enzymes, polysaccharides, and organic acids, synergistically enhancing efficacy; 4. Reduction of some toxic or irritating components. In the treatment of ulcerative colitis, studies have utilized lactic acid bacteria, Bacillus, or fungi to ferment TCM herbs such as Astragalus membranaceus and Coptis chinensis, preliminarily demonstrating that the fermented products are superior to the original medicinal materials in anti-inflammatory effects and in regulating intestinal flora.

[0005] Currently, research on licorice fermentation mainly focuses on the development of food additives or general health products, while the systematic application of its fermentation products in the treatment of ulcerative colitis is still in its early stages. Existing technologies rely on relatively limited selection of fermentation strains, and process parameters (such as temperature, time, and substrate ratio) lack systematic optimization for the transformation of pharmacodynamic components, leading to unstable product activity and difficulty in achieving expected therapeutic effects. Furthermore, the correlation between the enrichment mechanism of key active ingredients in fermentation products and their therapeutic effect on ulcerative colitis remains unclear, hindering its industrialization.

[0006] CN116144711A discloses a licorice probiotic fermentation preparation and its preparation method, which uses a mixture of multiple probiotics to prepare a licorice-Dendrobium officinale fermentation product with anti-aging effects, indicating a certain anti-aging effect. However, this invention uses mixed probiotics. It is known that the intestinal microecology of patients with ulcerative colitis is disrupted, and their metabolic profile is completely disordered. Relying solely on the single metabolite of probiotics has limited effect on restoring the damaged intestinal flora.

[0007] Therefore, there is an urgent need in this field for a targeted, process-controllable fermentation preparation method that can significantly enhance the anti-ulcerative colitis activity of licorice. Through fecal microbial extraction and optimized fermentation conditions, the efficient conversion and enrichment of licorice's active ingredients are achieved, thereby developing a licorice-Dendrobium officinale fermentation product with clear efficacy and stable quality, providing a novel traditional Chinese medicine derivative for the treatment of ulcerative colitis. To this end, this invention proposes a method for preparing a composition for treating ulcerative colitis and its application, to solve the aforementioned problems. Summary of the Invention

[0008] In view of the shortcomings of the prior art, the present invention provides a method for preparing a composition for treating ulcerative colitis and its application, so as to solve the problems mentioned in the background art.

[0009] To achieve the above objectives, the present invention is implemented through the following technical solution:

[0010] In a first aspect, the present invention provides a method for preparing a ferment for treating ulcerative colitis, comprising the following steps:

[0011] S1. Obtain a suspension of gut microbiota from healthy individuals as a fermentation agent;

[0012] S2. Provide medicinal raw materials containing licorice and Dendrobium officinale;

[0013] S3. Mix the fermenting agent with the medicinal raw material and ferment under anaerobic conditions;

[0014] S4. The fermentation product is post-processed to obtain a ferment for the treatment of ulcerative colitis.

[0015] Furthermore, in step S1, the healthy human gut microbiota suspension is prepared by the following method: taking fresh feces from a healthy donor, adding sterile PBS buffer and mixing, and then filtering the suspension to remove undigested food residue particles, thereby obtaining a suspension of complete gut microbiota; wherein, the healthy donor is a healthy volunteer whose feces can be used for fecal microbiota transplantation after being screened by a hospital.

[0016] Furthermore, in step S1, the mass-to-volume ratio of the fresh feces to the sterile PBS buffer is 1:10; the filtration is performed using an 800-mesh sterile screen; the processing of the fresh feces is completed in an anaerobic environment, and the processing time from sampling to obtaining the intestinal flora suspension is controlled within 3 hours.

[0017] Furthermore, in step S2, licorice and Dendrobium officinale are first washed and dried, then pulverized separately, and then obtained by sieving to form medicinal powder. The licorice powder and Dendrobium officinale powder are then mixed to form the medicinal raw material.

[0018] The sieving process uses an 80-mesh sieve; the mass ratio of licorice to Dendrobium officinale is 1:0.1.

[0019] Furthermore, in step S3, the medicinal materials formed by licorice and Dendrobium officinale are mixed with the intestinal flora suspension at a mass-to-volume ratio of 1:4 and stirred evenly, and then placed into a fermentation bottle; nitrogen gas is introduced into the fermentation bottle and then sealed to form an anaerobic environment.

[0020] The sealed fermentation bottle was placed in an anaerobic incubator and cultured at 37±0.5℃ for 72 hours to complete the fermentation.

[0021] Furthermore, the post-processing of the fermentation product described in step S4 includes placing the fermented product in an oven for dehydration and drying.

[0022] The dehydration and drying temperature is 45℃, and the drying time is 6 hours; after drying, the product is sealed and stored.

[0023] In a second aspect, the present invention provides a fermentation product, which is a fermentation product formed by fermenting licorice and dendrobium officinale with gut microbiota from healthy humans.

[0024] In a third aspect, the present invention provides a pharmaceutical composition comprising the fermentation product and a pharmaceutically acceptable carrier.

[0025] Furthermore, the dosage form of the pharmaceutical composition is capsules or powder.

[0026] Furthermore, the pharmaceutical composition is used to prepare a medicament for the prevention or treatment of ulcerative colitis.

[0027] This invention provides a method for preparing a composition for treating ulcerative colitis and its application. It has the following beneficial effects:

[0028] 1. The fermentation product of licorice and dendrobium officinale exhibits significantly enhanced bioactivity and remarkable therapeutic effects. By introducing structurally complete and functionally complex healthy human gut microbiota as a fermentation agent, this invention achieves deep and broad transformation of the components of traditional Chinese medicine through co-fermentation of licorice and dendrobium officinale. The fermentation product demonstrates excellent therapeutic effects in an animal model of ulcerative colitis (UC).

[0029] 2. The fermented products of licorice and Dendrobium officinale have significant effects on the clinical symptoms and pathological damage of ulcerative colitis. Mouse experiments have demonstrated that the fermented products of licorice and Dendrobium officinale can significantly reduce the DAI score in ulcerative colitis mice, effectively inhibiting colonic inflammation and edema. This not only alleviates local colonic inflammation but also suppresses excessive immune responses at the systemic level, improving the overall immune status. Simultaneously, it reduces intestinal wall edema, and the intestinal villi show some recovery.

[0030] 3. Licorice-Dendrobium officinale fermentation products can improve intestinal barrier damage in mice with ulcerative colitis. Licorice-Dendrobium officinale fermentation products can maintain the intestinal barrier by upregulating the expression of tight junction genes and proteins in intestinal epithelial cells.

[0031] 4. The fermentation products of licorice and Dendrobium officinale can increase the secretion of short-chain fatty acids in mice with ulcerative colitis. The fermentation products of licorice and Dendrobium officinale can increase the content of acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid and isovaleric acid in the intestine of mice, providing energy for the intestinal mucosa and improving intestinal health.

[0032] 5. The fermentation products of licorice and dendrobium officinale can regulate the abundance and structure of intestinal flora in mice with ulcerative colitis.

[0033] The fermentation products of licorice and Dendrobium officinale can improve the disordered intestinal flora structure of UC mice, restore the intestinal flora structure to the direction of normal mice, and significantly increase the abundance of beneficial bacteria Lachnospiraceae_NK4A136_group and Akkermansia. Attached Figure Description

[0034] Figure 1 The following data represent the trends in body weight change and disease activity index scores of mice in each group during model establishment and intervention in Example 2.

[0035] Figure 2 The colon length of mice in each group after the experiment in Example 2;

[0036] Figure 3 The spleen index of mice in each group after the experiment in Example 2;

[0037] Figure 4 The images show the tissue morphology of the distal colon tissue of mice in each group under a light microscope after the experiment in Example 2.

[0038] Figure 5 The expression levels of ZO-1, MUC2, and E-cadherin genes in the colon tissue of mice in each group after the experiment in Example 3;

[0039] Figure 6 The results show the expression levels of ZO-1, MUC2, and E-cadherin proteins in the colon tissues of mice in the high-dose group (Lic), normal control group (NC), model control group (DSS), and positive control group (Mes) of licorice-Dendrobium officinale fermentation after the experiment in Example 3.

[0040] Figure 7 The results show the mRNA levels of TNF-α, IL-6, and IL-10 in the colon tissue of mice in each group and the levels of TNF-α, IL-6, and IL-10 in serum samples after the experiment in Example 4.

[0041] Figure 8 The content of acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, and isovaleric acid in fresh fecal samples of mice in each group after the experiment in Example 5;

[0042] Figure 9 This is a schematic diagram showing the results of the gut microbiota diversity analysis of mice in each group after the experiment in Example 6. Figure 9 A shows the comparison results of the Chao1 gut microbiota richness index among the mice in each group; Figure 9 B represents the Shannon comparison results of the gut microbiota diversity index of mice in each group; Figure 9 C represents the PCoA results of the principal coordinate analysis of Beta diversity in the gut microbiota of each group of mice.

[0043] Figure 10 This is a schematic diagram showing the distribution of gut microbiota composition in each group of mice after the experiment in Example 6. Figure 10 A shows the composition and relative abundance distribution of bacterial communities at the phylum level; Figure 10 B is a diagram showing the composition and relative abundance distribution of the genus-level bacterial community;

[0044] Figure 11 This is a schematic diagram showing the LEfSe differential bacterial analysis results of the intestinal flora of mice in each group in Example 6. Figure 11 A is a phylogenetic clade diagram of LEfSe; Figure 11 B is a bar chart of the LDA score from the LEfSe linear discriminant analysis.

[0045] Figure 12 This is a Spearman correlation heatmap of differential bacteria and short-chain fatty acids in Example 6. Detailed Implementation

[0046] To enable those skilled in the art to understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort should fall within the scope of protection of the present invention.

[0047] The present invention will now be described in detail with reference to the accompanying drawings:

[0048] Example 1: This example provides a method for preparing a fermented product for treating ulcerative colitis: a licorice-Dendrobium officinale fermented product, comprising the following steps:

[0049] (1) Preparation of medicinal materials of licorice and Dendrobium officinale: Licorice and Dendrobium officinale were washed and dried, then crushed by crusher and filtered through an 80-mesh sieve. Then, licorice powder and Dendrobium officinale powder were mixed in a mass ratio of 1:0.1.

[0050] (2) Extraction of intestinal flora: Fresh feces from healthy volunteer donors whose feces can be used for fecal microbiota transplantation after screening by the hospital were taken, and sterile PBS buffer with a mass-volume ratio of 10 times the weight of fresh feces was added, i.e., the mass-volume ratio of fresh feces to sterile PBS buffer was 1g:10ml. Then, the mixture was vortexed and filtered through an 800-mesh sterile screen to remove undigested food residue particles, and a suspension of complete intestinal flora was obtained.

[0051] (3) Fermentation: Take the licorice powder and Dendrobium officinale powder from step (1), add them to the bacterial suspension from step (2) at a ratio of 1g:4ml, stir and mix well, then fill the mixture into a fermentation bottle, fill the bottle with nitrogen gas, and then seal it to ensure anaerobic conditions. Immediately transfer the fermentation bottle to an anaerobic incubator and incubate at 37℃ for 72 hours. After the incubation is completed, take the fermented licorice-Dendrobium officinale fermentation product out onto a tray, spread it flat at 0.5 cm, place the tray in an oven, dry it at 45℃ for 6 hours, and then seal and store it after dehydration and drying.

[0052] The licorice-Dendrobium officinale fermentation products used in Examples 2-6 were all the licorice-Dendrobium officinale fermentation products prepared in Example 1.

[0053] Example 2 verifies that licorice-Dendrobium officinale fermentation can alleviate clinical symptoms and pathological damage in mice with ulcerative colitis. The specific implementation method is as follows:

[0054] This embodiment is used to verify the effect of the licorice-Dendrobium officinale fermentation product prepared in Example 1 on the improvement of ulcerative colitis induced by sodium dextran sulfate in mice. The focus is on the effect of the fermentation product on the clinical symptoms, colon morphology, spleen index and colonic tissue pathological damage in mice.

[0055] C57BL / 6 mice, male, 6-8 weeks old, weighing 20±5g, were used as experimental animals and housed in the animal facility of the Zhejiang Academy of Agricultural Sciences. An acute ulcerative colitis mouse model was established by allowing the mice free access to a 3% sodium dextran sulfate solution for 7 consecutive days. One week prior to establishing the acute ulcerative colitis mouse model, the mice underwent gavage intervention for 14 consecutive days. Throughout the experimental period, the mice's weight, stool characteristics, and fecal blood loss were recorded daily, and a disease activity index score was calculated based on these data.

[0056] Mice were randomly divided into 6 groups (n=6 per group): normal control (NC), model control (DSS), positive control (Mesalazine), low-dose licorice-Dendrobium officinale fermentation group (LicLow), medium-dose licorice-Dendrobium officinale fermentation group (LicMid), and high-dose licorice-Dendrobium officinale fermentation group (LicHigh). The normal control group received normal drinking water and was administered physiological saline by gavage; the model control group was administered physiological saline by gavage while establishing the ulcerative colitis model; the positive control group was administered mesalazine by gavage at a dose of 200 mg / kg while establishing the ulcerative colitis model; the low-dose, medium-dose, and high-dose licorice-Dendrobium officinale fermentation groups were administered licorice-Dendrobium officinale fermentation at doses of 50 mg / kg, 100 mg / kg, and 200 mg / kg, respectively, while establishing the ulcerative colitis model.

[0057] During model establishment and intervention, mice in each group were continuously observed and monitored. Figure 1 Compared to the normal control group, the model control group showed a continuous decrease in body weight starting from day 4 of free access to 3% sodium dextran sulfate solution, i.e., day 11 of the entire experimental cycle, with a more pronounced decrease in the last two days of the experiment; simultaneously, the disease activity index score gradually increased. Diarrhea and bloody stools were observed in the model control group mice, thus confirming the successful establishment of the ulcerative colitis model. Compared to the model control group, the decrease in body weight was inhibited in all treatment groups after intervention with mesalazine and licorice-Dendrobium officinale fermentation, with more significant improvements in the medium- and high-dose groups. The disease activity index score results are as follows: Figure 1As shown in Figure B, the disease activity index score of the normal control group remained at 0 throughout the experimental period; the score of the model control group increased significantly from day 11. Compared with the model control group, the disease activity index scores of mice significantly decreased after intervention with medium-dose and high-dose groups of licorice-Dendrobium officinale fermentation and mesalazine, with statistically significant differences (p<0.05). These results indicate that licorice-Dendrobium officinale fermentation can alleviate the clinical symptoms of ulcerative colitis in mice.

[0058] After the experiment, the mice were euthanized, their colons were dissected, and their length was measured. The results are as follows: Figure 2 As shown, compared with the normal control group, the colon length of mice in the model control group was significantly shortened, with a statistically significant difference (p<0.001), indicating that sodium dextran sulfate treatment caused significant colonic damage. Compared with the model control group, the colon length of mice in the positive control group and each dose group of licorice-Dendrobium officinale fermentation product recovered to varying degrees. Among the various dose groups of licorice-Dendrobium officinale fermentation product, the high-dose group showed the most significant improvement, with a statistically significant difference (p<0.01), and its degree of improvement was comparable to that of the positive control group. These results indicate that licorice-Dendrobium officinale fermentation product can alleviate colonic shortening caused by colitis.

[0059] After the experiment, the spleen was simultaneously isolated and weighed, and the spleen index was calculated. The results are as follows: Figure 3 As shown, compared with the normal control group, the spleen index of mice in the model control group was significantly increased, with a statistically significant difference (p<0.001), indicating that the model group mice exhibited a marked enhanced immune response. Compared with the model control group, the positive control group and all dose groups of licorice-Dendrobium officinale fermentation showed a trend of decreasing spleen index, with the positive control group and the high-dose group of licorice-Dendrobium officinale fermentation showing significant reductions (p<0.001). These results indicate that licorice-Dendrobium officinale fermentation has an inhibitory effect on the abnormal immune activation associated with ulcerative colitis.

[0060] To further evaluate the extent of colonic tissue damage, distal colonic tissue from mice was harvested and stained with hematoxylin and eosin, and the morphological changes were observed under a light microscope. The results are as follows: Figure 4As shown, the colonic tissue of normal control mice was intact, with regular crypt arrangement, continuous epithelium, abundant goblet cells, and only a small number of physiological immune cells distributed in the lamina propria and submucosa, without obvious inflammatory damage. The colonic tissue of model control mice showed obvious pathological changes, including destruction or even disappearance of crypt structure, loss of intestinal mucosa, extensive infiltration of inflammatory cells, and tissue edema, indicating significant damage to the colonic mucosal barrier. Compared with the model control group, the colonic tissue damage in the positive control group and the licorice-Dendrobium officinale fermentation intervention group was reduced, with the high-dose group showing more significant improvement. Reduced intestinal wall edema, partial recovery of intestinal mucosal structure, reduced inflammatory cell infiltration, and some recovery of intestinal villus morphology were observed. These results indicate that licorice-Dendrobium officinale fermentation can alleviate the pathological damage to the colonic tissue of mice with ulcerative colitis and play a protective role in the intestinal mucosal structure.

[0061] In summary, the results of this embodiment indicate that the licorice-Dendrobium officinale ferment prepared in Example 1 can improve weight loss, reduce disease activity index scores, alleviate colonic shortening, reduce spleen index, and reduce pathological damage to colonic tissue in a mouse model of ulcerative colitis induced by sodium dextran sulfate; among them, the high-dose licorice-Dendrobium officinale ferment group showed more significant improvement effects.

[0062] Example 3 verifies that licorice-Dendrobium officinale fermentation can improve intestinal barrier damage in mice with ulcerative colitis. The specific implementation method is as follows:

[0063] This embodiment is used to verify the effect of the licorice-Dendrobium officinale fermentation product prepared in Example 1 on improving intestinal barrier damage in mice with ulcerative colitis induced by sodium dextran sulfate. The focus is on investigating the effect of the fermentation product on the expression levels of intestinal barrier-related tight junction genes and proteins in colon tissue.

[0064] The experimental animals, modeling methods, grouping methods, and administration methods were the same as in Example 2. Specifically, male C57BL / 6 mice, 6-8 weeks old and weighing 20±5g, were used to establish an acute ulcerative colitis model by allowing them free access to a 3% sodium dextran sulfate solution for 7 consecutive days. The mice were administered the solution via gavage one week prior to modeling and the intervention lasted for 14 consecutive days. The mice were divided into a normal control group, a model control group, a positive control group, a low-dose group of licorice-Dendrobium officinale fermentation product, a medium-dose group of licorice-Dendrobium officinale fermentation product, and a high-dose group of licorice-Dendrobium officinale fermentation product. After the experiment, the mice were sacrificed, and colonic tissue was isolated for subsequent gene and protein expression detection.

[0065] To evaluate the degree of intestinal barrier damage, total RNA was first extracted from the colon tissue of mice in each group, and the expression of intestinal barrier-related tight junction genes was detected by qRT-PCR. The relative expression levels of ZO-1, MUC2, and E-cadherin genes were used as the detection indicators. A normal control group was used as a reference for normal expression. The expression levels of the above genes in the colon tissue of mice in each group were compared and analyzed. The results are as follows: Figure 5 As shown in the figure, compared with the normal control group, the expression of ZO-1, MUC2 and E-cadherin genes in the colon tissue of the model control group mice was significantly downregulated, and the difference was statistically significant (p<0.001), indicating that the treatment with dextran sulfate sodium inhibited the expression of intestinal barrier-related structural molecules in mice, and the intestinal barrier was significantly damaged.

[0066] Further comparisons were made between the intervention groups and the model control group. Results showed that, regarding ZO-1 gene expression, both the positive control group and the high-dose group of licorice-Dendrobium officinale fermentation were significantly upregulated compared to the model control group (p<0.001). Regarding MUC2 gene expression, both the positive control group and the high-dose group of licorice-Dendrobium officinale fermentation were significantly upregulated compared to the model control group (p<0.01). Regarding E-cadherin gene expression, both the positive control group and the high-dose group of licorice-Dendrobium officinale fermentation were significantly upregulated compared to the model control group (p<0.05). These results indicate that licorice-Dendrobium officinale fermentation can increase the expression levels of intestinal barrier-related genes in damaged colonic tissue, with the high-dose group showing a more significant improvement.

[0067] To further verify the protective effect of licorice-Dendrobium officinale fermentation on the intestinal barrier, a high-dose group with a more significant improvement effect (Lic in the attached figure) was selected, along with a normal control group (NC), a model control group (DSS), and a positive control group (Mes). Western blotting was used to detect the expression levels of ZO-1, MUC2, and E-cadherin proteins in colon tissue. Colon tissue from each group was homogenized, total protein was extracted, and after protein quantification, electrophoretic separation, membrane transfer, and antibody incubation were performed. Gray-scale analysis of the target bands was conducted to compare the expression differences of ZO-1, MUC2, and E-cadherin proteins among the groups. The detection results are as follows: Figure 6 As shown, compared with the model control group, both the positive control group and the high-dose group of licorice-Dendrobium officinale fermentation significantly increased the expression levels of ZO-1, MUC2 and E-cadherin proteins that were downregulated by sodium dextran sulfate treatment, and the differences were statistically significant (p<0.01).

[0068] Combination Figure 5 and Figure 6The results showed that treatment with sodium dextran sulfate reduced the expression of intestinal barrier-related genes and proteins in colonic tissue. However, intervention with licorice-Dendrobium officinale fermentation, especially at high doses, reversed these changes and upregulated the expression levels of tight junction-related genes and proteins in colonic tissue. These results indicate that licorice-Dendrobium officinale fermentation can protect the damaged intestinal mucosal barrier by improving the expression of intestinal epithelial barrier-related molecules and thus alleviating intestinal barrier damage in mice with ulcerative colitis.

[0069] In summary, the results of this embodiment indicate that the licorice-Dendrobium officinale ferment prepared in Example 1 can increase the expression levels of ZO-1, MUC2, and E-cadherin intestinal barrier-related genes in the colonic tissue of mice with ulcerative colitis induced by dextran sulfate sodium, and increase the expression levels of the corresponding proteins of ZO-1, MUC2, and E-cadherin. The improvement effect is more obvious in the high-dose group, indicating that the ferment has the effect of improving intestinal barrier damage.

[0070] Example 4 verifies that licorice-Dendrobium officinale fermentation can improve the inflammatory response in mice with ulcerative colitis. The specific implementation method is as follows:

[0071] This embodiment is used to verify the effect of the licorice-Dendrobium officinale ferment prepared in Example 1 on the improvement of inflammatory response in mice with ulcerative colitis induced by sodium dextran sulfate. The focus is on the effect of the ferment on the expression levels of inflammation-related factors in colon tissue and serum.

[0072] The experimental animals, modeling methods, grouping methods, and administration methods were the same as in Example 2. Specifically, male C57BL / 6 mice, 6-8 weeks old and weighing 20±5g, were used to establish an acute ulcerative colitis model by allowing them free access to a 3% sodium dextran sulfate solution for 7 consecutive days. The mice were administered the drug via gavage one week prior to modeling and the intervention lasted for 14 consecutive days. The mice were divided into a normal control group, a model control group, a positive control group, a low-dose group of licorice-Dendrobium officinale fermented product, a medium-dose group of licorice-Dendrobium officinale fermented product, and a high-dose group of licorice-Dendrobium officinale fermented product. After the experiment, the mice were sacrificed, and colon tissue and serum samples were collected for the detection of inflammatory factors.

[0073] To evaluate the degree of local inflammatory response in the colon, nucleic acids were first extracted from colon tissue samples of mice in each group, and the expression of key inflammatory factors was detected to analyze the changes of pro-inflammatory and anti-inflammatory factors in the colon. The results are as follows: Figure 7As shown in Figures A, B, and C, compared with the normal control group, the model control group exhibited significant inflammatory activation in the colon, characterized by a significant increase in the mRNA levels of pro-inflammatory factors TNF-α and IL-6 (p<0.001), while the expression of the anti-inflammatory factor IL-10 was significantly decreased (p<0.01). These results indicate that sodium dextran sulfate treatment can induce a significant imbalance in the inflammatory response in the colon, resulting in an inflammatory state characterized by elevated pro-inflammatory factors and decreased anti-inflammatory factors.

[0074] Further comparisons were made between the intervention groups and the model control group. Results showed that after intervention with licorice-Dendrobium officinale fermentation, both the medium-dose and high-dose groups inhibited the overexpression of IL-6 in colonic tissue, with statistically significant differences compared to the model control group (p<0.05), indicating that licorice-Dendrobium officinale fermentation has an inhibitory effect on local pro-inflammatory responses in the colon. Combined with the aforementioned overall trends in TNF-α, IL-6, and IL-10, it is evident that intervention with licorice-Dendrobium officinale fermentation alleviated local colonic inflammation to a certain extent.

[0075] To further evaluate the effects of this fermentation product on systemic inflammatory status, inflammatory-related factors were detected in serum samples from each group of mice. The results are as follows: Figure 7 As shown in D, E, and F, the high-dose licorice-Dendrobium officinale fermentation group can increase the expression level of IL-10 in the systemic circulation, indicating that in addition to regulating local inflammation in the colon, the fermentation can also improve the expression status of anti-inflammatory factors at the systemic level, thereby playing a regulatory role in the body's inflammatory balance.

[0076] The combined analysis of colon tissue and serum samples revealed that mice with ulcerative colitis induced by sodium dextran sulfate exhibited a significantly enhanced inflammatory response. Intervention with licorice-Dendrobium officinale fermentation, particularly in the medium and high dose groups, effectively regulated the abnormal inflammatory response by inhibiting the abnormal increase in IL-6 and promoting the recovery of IL-10 expression. These results indicate that licorice-Dendrobium officinale fermentation can improve the inflammatory response in mice with ulcerative colitis.

[0077] In summary, the results of this embodiment indicate that the licorice-Dendrobium officinale ferment prepared in Example 1 can alleviate the inflammatory response of mice with ulcerative colitis induced by sodium dextran sulfate. Specifically, it inhibits the overexpression of the pro-inflammatory factor IL-6 in the colon and promotes the recovery of the expression of the anti-inflammatory factor IL-10 in the systemic circulation, thereby improving the inflammatory imbalance.

[0078] Example 5 verifies that licorice-Dendrobium officinale fermentation can improve the decreased secretion of short-chain fatty acids in mice with ulcerative colitis. The specific implementation method is as follows:

[0079] This embodiment is used to verify the effect of the licorice-Dendrobium officinale fermentation prepared in Example 1 on improving the decrease in short-chain fatty acid levels in mice with ulcerative colitis induced by sodium dextran sulfate. The focus is on the effect of the fermentation on the content of short-chain fatty acids in mouse feces.

[0080] The experimental animals, modeling methods, grouping methods, and administration methods were the same as in Example 2. Specifically, male C57BL / 6 mice, 6-8 weeks old and weighing 20±5g, were used to establish an acute ulcerative colitis model by allowing them free access to a 3% sodium dextran sulfate solution for 7 consecutive days. The mice were administered the drug via gavage starting one week before modeling and continued for 14 days. The mice were divided into a normal control group, a model control group, a positive control group, a low-dose group of licorice-Dendrobium officinale fermented product, a medium-dose group of licorice-Dendrobium officinale fermented product, and a high-dose group of licorice-Dendrobium officinale fermented product. Fresh fecal samples were collected from each group after the experiment for short-chain fatty acid detection.

[0081] Short-chain fatty acids (SCFAs) are important small-molecule metabolites produced by gut microbiota metabolism, and their levels can reflect the gut microecological state and intestinal metabolic function. To evaluate changes in intestinal metabolism in mice with ulcerative colitis, the levels of SCFAs in the feces of each group of mice were measured. The tested fatty acids included acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, and isovaleric acid. The results are as follows: Figure 8 As shown, compared with the normal control group, the levels of acetic acid, propionic acid, butyric acid, isobutyric acid, and isovaleric acid in the feces of mice in the model control group were significantly reduced, with statistically significant differences (p<0.05); the valeric acid content also showed a decreasing trend, but did not show a significant difference compared with the normal control group. These results indicate that sodium dextran sulfate treatment leads to a decrease in the levels of intestinal metabolites in mice and inhibits the secretion of short-chain fatty acids.

[0082] Further comparisons were made between the intervention groups and the model control group. Results showed that the high-dose licorice-Dendrobium officinale fermentation group improved the decreased levels of short-chain fatty acids in mice with ulcerative colitis. Except for acetic acid and valeric acid, the levels of all other short-chain fatty acids were increased, and the reversal effect was statistically significant (p<0.05). Meanwhile, the low-dose and medium-dose groups also showed a certain trend of improvement in the recovery of the levels of the above six short-chain fatty acids, indicating that intervention with licorice-Dendrobium officinale fermentation can, to some extent, restore the decreased secretion of short-chain fatty acids caused by sodium dextran sulfate treatment, with the high-dose group showing more significant improvement.

[0083] The test results show that the licorice-Dendrobium officinale ferment can increase the content of various short-chain fatty acids in the feces of mice with ulcerative colitis, indicating that the ferment has a regulatory effect on the intestinal metabolic environment. Since short-chain fatty acids are closely related to intestinal barrier status and inflammation regulation, this result further suggests that the licorice-Dendrobium officinale ferment can play an auxiliary role in improving the ulcerative colitis state by improving the decreased levels of short-chain fatty acids and promoting the restoration of the intestinal environment.

[0084] In summary, the results of this embodiment indicate that the licorice-Dendrobium officinale fermentation product prepared in Example 1 can improve the reduced secretion of short-chain fatty acids in mice with ulcerative colitis induced by sodium dextran sulfate, specifically by increasing the content of various short-chain fatty acids in feces, with the high-dose group showing a more significant improvement.

[0085] Example 6 verifies that licorice-Dendrobium officinale fermentation can regulate the abundance and structure of intestinal flora in mice with ulcerative colitis. The specific implementation method is as follows:

[0086] This embodiment is used to verify the regulatory effect of the licorice-Dendrobium officinale fermentation product prepared in Example 1 on the abundance and community structure of intestinal flora in mice with ulcerative colitis induced by sodium dextran sulfate, and to further analyze the correlation between changes in intestinal flora and the restoration of short-chain fatty acids, thereby verifying the mechanism of action of the fermentation product in improving ulcerative colitis.

[0087] The experimental animals, modeling methods, grouping methods, and administration methods were the same as in Example 2. Specifically, male C57BL / 6 mice, 6-8 weeks old, weighing 20±5g, were used to establish an acute ulcerative colitis model by allowing them free access to a 3% sodium dextran sulfate solution for 7 consecutive days. Administration of the drug via gavage began one week before modeling and continued for 14 days. Based on the experimental results of Examples 2 to 5, the high-dose licorice-Dendrobium officinale fermentation group showed a more significant improvement effect on ulcerative colitis mice. Therefore, in this example, the normal control group (NC), model control group (DSS), positive control group (Mes), and the high-dose licorice-Dendrobium officinale fermentation group (represented by Lic in the attached figure) were selected for intestinal flora sequencing analysis. After the experiment, cecal contents of each group of mice were collected for 16S rRNA sequencing detection.

[0088] First, 16S rRNA sequencing was performed on the microbial community in the cecal contents of each group of mice, and the richness and diversity of the gut microbiota were analyzed based on the sequencing results. The results are as follows: Figure 9 As shown, compared with the normal control group, the richness and diversity of the gut microbiota in the model control group mice were decreased, indicating that sodium dextran sulfate treatment led to an imbalance in the gut microbiota. Further comparative analysis of the Chao1 index and Shannon index is as follows: Figure 9 As shown in A and B, both the positive control group and the high-dose group of licorice-Dendrobium officinale fermentation showed a reversal trend in the decline of gut microbiota diversity, indicating that the fermentation product can improve the reduction in gut microbiota richness and diversity in ulcerative colitis.

[0089] Further Beta diversity analysis was performed to compare the differences in gut microbiota composition among the groups of mice. Results are as follows: Figure 9 As shown in Figure C, there was a significant difference in the composition of the gut microbiota between the normal control group and the model control group, indicating that the treatment with sodium dextran sulfate led to a significant change in the gut microbiota structure. After high-dose intervention with mesalazine and licorice-Dendrobium officinale fermentation, the gut microbiota structure of the mice moved closer to that of the normal control group, indicating that licorice-Dendrobium officinale fermentation can regulate the abnormally altered gut microbiota composition in mice with ulcerative colitis.

[0090] To clarify the specific changes in the gut microbiota, further analysis was conducted at the phylum and genus levels to examine the species composition and distribution ratios of each sample group. Figure 10 As shown in the figure, at the phylum level, the gut microbiota of mice in each group mainly consisted of Bacteroidetes, Firmicutes, Proteobacteria, Verrucous Microbes, Actinobacteria, and Desulfovibrio. The comparison results showed that, compared with the normal control group, the relative abundance of Actinobacteria in the model control group decreased, and the difference was statistically significant (p<0.05). After intervention with mesalazine and licorice-Dendrobium officinale fermentation, the relative abundance of Actinobacteria recovered somewhat compared with the model control group, but the difference was not statistically significant. On the other hand, compared with the model control group, the relative abundance of Verrucous Microbes in the high-dose licorice-Dendrobium officinale fermentation group was significantly increased, and the difference was statistically significant (p<0.05), indicating that this fermentation can promote the recovery of the microbiota associated with a healthy gut ecosystem.

[0091] At the genus level, the genera with relatively high abundance in the gut microbiota of mice in each group mainly included Bacteroides, Lachnospiraceae_NK4A136_group, Muribacum, Odoribacter, Turicibacter, Akkermansia, Alistipes, Bifidobacterium, Limosilactobacillus, Parasutterella, and Lactobacillus. Compared with the normal control group, the relative abundance of many beneficial bacterial genera decreased in the model control group, with Lachnospiraceae_NK4A136_group, Akkermansia, Bifidobacterium, Limosilactobacillus, and Lactobacillus all showing a decreasing trend. Further comparison showed that after high-dose intervention with licorice-Dendrobium officinale fermentation product, the relative abundance of Lachnospiraceae_NK4A136_group and Akkermansia was significantly increased compared with the model control group, and the difference was statistically significant (p<0.05). These results indicate that licorice-Dendrobium officinale fermentation can promote the recovery of gut health-related bacteria, thereby improving the flora imbalance in ulcerative colitis.

[0092] To further screen characteristic bacterial communities with significant differences among different groups, LEfSe analysis was performed on samples from each group, and a linear discriminant analysis value of 3 was used as the differential screening criterion to identify biomarkers with significant differences at the genus level. The results are as follows: Figure 11 As shown, the characteristic species in the normal control group were mainly a series of beneficial bacteria; the characteristic species in the model control group included *Ileibacterium* and *Parasutterella*; while the licorice-Dendrobium officinale fermentation intervention group exhibited characteristic flora of *Akkermansia* and *Adlercreutzia*. These results indicate that after intervention with licorice-Dendrobium officinale fermentation, the gut microbiota markers shifted from bacteria associated with disease states to bacteria associated with health states, suggesting that this fermentation can reshape the gut microbiota structure.

[0093] Considering that Example 5 has demonstrated that licorice-Dendrobium officinale fermentation can improve the decline in short-chain fatty acid levels, Spearman correlation analysis was performed on the top 30 abundant differentially abundant bacterial genera and their corresponding short-chain fatty acid contents to further elucidate the relationship between changes in gut microbiota and the recovery of short-chain fatty acid levels. The results are as follows: Figure 12As shown, *Lachnospiraceae_UCG_006*, *Akkermansia*, *Roseburia*, and *Eubacterium* xylanophilum group were significantly positively correlated with short-chain fatty acid (SCFA) levels; while *Ileibacterium*, *Allobaculum*, and *Parasutterella* were significantly negatively correlated with SCFA levels. These results indicate that intervention with *Licorice-Dendrobium officinale* fermentation promotes the growth of beneficial SCFA-producing bacteria and inhibits the proliferation of disease-related bacteria, thereby restoring SCFA levels and improving the gut microbiota.

[0094] The combined results of gut microbiota diversity analysis, community structure analysis, differential bacterial screening, and correlation analysis showed that sodium dextran sulfate-induced ulcerative colitis led to a decrease in gut microbiota richness, community structure disorder, and a reduction in the proportion of beneficial bacteria in mice. High-dose intervention with licorice-Dendrobium officinale fermentation improved microbiota diversity, regulated community structure, increased the relative abundance of beneficial bacteria, and promoted the enrichment of bacteria associated with the restoration of short-chain fatty acids. These results indicate that licorice-Dendrobium officinale fermentation can improve the intestinal microecological imbalance in mice with ulcerative colitis by regulating gut microbiota abundance and structure.

[0095] In summary, the results of this embodiment indicate that the licorice-Dendrobium officinale ferment prepared in Example 1 can improve the decline in the richness and diversity of intestinal flora in mice with ulcerative colitis induced by sodium dextran sulfate, regulate the intestinal flora community structure, promote the recovery of the abundance of beneficial bacteria Akkermansia and Lachnospiraceae_NK4A136_group, and show a synergistic effect with the recovery of short-chain fatty acid levels. This suggests that the ferment has the effect of regulating the intestinal flora structure and improving the intestinal microecological imbalance.

[0096] Based on the above experimental results, it can be found that high-dose licorice-Dendrobium officinale fermentation product has a better alleviating effect on UC mice. Therefore, we used 16SRNA to detect the cecal contents of mice with high-dose licorice-Dendrobium officinale fermentation product in order to further reveal the regulatory mechanism of licorice-Dendrobium officinale fermentation product on the intestinal flora of UC mice.

[0097] It should be particularly noted that the various embodiments listed in this specification and accompanying drawings are intended to illustrate the technical solutions and advantages of the present invention, and not to limit the scope of protection of the present invention. Without departing from the core ideas and technical effects of the present invention, those skilled in the art can make any form of improvement, substitution, combination, or modification to the structural arrangement, process parameters, material selection, control logic, etc., of the described embodiments; any obvious changes based on the same concept should be considered equivalent solutions of the present invention and should be included within the scope of protection defined by the claims of the present invention. The actual scope of protection of the present invention is determined by the appended claims and should be correctly understood in conjunction with the specification and accompanying drawings.

Claims

1. A method for preparing a ferment for treating ulcerative colitis, characterized in that, Includes the following steps: S1. Obtain a suspension of gut microbiota from healthy individuals as a fermentation agent; S2. Provide medicinal raw materials containing licorice and Dendrobium officinale; S3. Mix the fermentation agent with the medicinal raw material and ferment under anaerobic conditions; S4. The fermentation product is post-processed to obtain a ferment for the treatment of ulcerative colitis.

2. The method for preparing the fermented product for treating ulcerative colitis according to claim 1, characterized in that, The healthy human gut microbiota suspension mentioned in step S1 is prepared by the following method: fresh feces from a healthy donor are taken, sterile PBS buffer is added and mixed, and then the suspension is filtered to remove undigested food residue particles, thereby obtaining a suspension of complete gut microbiota; wherein, the healthy donor is a healthy volunteer whose feces can be used for fecal microbiota transplantation after being screened by the hospital.

3. The method for preparing the fermented product for treating ulcerative colitis according to claim 1, characterized in that, In step S1, the mass-to-volume ratio of fresh feces to sterile PBS buffer is 1:10; the filtration is performed using an 800-mesh sterile screen; the processing of the fresh feces is completed in an anaerobic environment, and the processing time from sampling to obtaining the intestinal flora suspension is controlled within 3 hours.

4. The method for preparing the fermentation product for treating ulcerative colitis according to claim 1, characterized in that: In step S2, licorice and Dendrobium officinale are first washed and dried, then pulverized separately, and then sieved to obtain medicinal powder. The licorice powder and Dendrobium officinale powder are then mixed to form the medicinal raw material. The sieving process uses an 80-mesh sieve. The mass ratio of licorice to Dendrobium officinale is 1:0.

1.

5. The method for preparing the fermentation product for treating ulcerative colitis according to claim 1, characterized in that: In step S3, the medicinal materials formed by licorice and Dendrobium officinale are mixed with the intestinal flora suspension at a mass-to-volume ratio of 1:4 and stirred evenly, and then put into a fermentation bottle; nitrogen gas is introduced into the fermentation bottle and then sealed to form an anaerobic environment; The sealed fermentation bottle was placed in an anaerobic incubator and cultured at 37±0.5℃ for 72 hours to complete the fermentation.

6. The method for preparing the fermentation product for treating ulcerative colitis according to claim 1, characterized in that: The post-processing of the fermentation product described in step S4 includes placing the fermented product in an oven for dehydration and drying. The dehydration and drying temperature is 45℃, and the drying time is 6 hours; after drying, the product is sealed and stored.

7. A fermentation product, characterized in that: The fermented product is prepared by the preparation method according to any one of claims 1 to 6, and the fermented product is a fermentation product formed by fermentation of licorice and Dendrobium officinale by intestinal flora from healthy humans.

8. A pharmaceutical composition, characterized in that: The pharmaceutical composition comprises the ferment as described in claim 7 and a pharmaceutically acceptable carrier.

9. The pharmaceutical composition according to claim 8, characterized in that... The dosage form of the pharmaceutical composition is capsules or powder.

10. The pharmaceutical composition of claim 8 is used to prepare a medicament for the prevention or treatment of ulcerative colitis.

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