Use of stemofoline in the preparation of a drug for treating colitis

Abstract

The application belongs to the technical field of biological medicine, and provides application of phrymarolin A in preparation of a drug for treating colitis. The application proves that phrymarolin A can relieve and treat colitis by regulating intestinal flora, breaks through the limitation that the research target of existing natural products is unclear and the mechanism is unclear, and phrymarolin A has a wide source and a mature extraction process. Compared with traditional synthetic drugs, as a natural compound, phrymarolin A has lower toxicity, and compared with emerging probiotic therapy and biological agents, the corresponding follow-up drug development cost of the application is lower, industrialization popularization is easier to realize, and the treatment burden of patients can be reduced.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to the application of stemona alkaloids in the preparation of drugs for treating colitis. Background Technology

[0002] Ulcerative colitis (UC) is a common inflammatory bowel disease characterized by recurrent diffuse inflammation of the intestinal mucosa. Clinically, UC patients may experience intestinal symptoms such as weight loss, recurrent diarrhea, and intestinal bleeding, and may also experience systemic symptoms such as arthritis and liver dysfunction, significantly reducing their quality of life. Its pathogenesis is closely related to multiple factors, including genetics, environment, and immunity, but the core pathogenesis remains to be elucidated. Current research confirms that gut microbiota dysbiosis can participate in the pathological process of UC by regulating innate immune responses, affecting energy metabolism, and disrupting host mucosal integrity. Furthermore, the abnormal immune response triggered by dysbiosis can further lead to intestinal barrier dysfunction and increased intestinal permeability, allowing harmful bacteria and toxins to cross the intestinal wall, inducing and amplifying intestinal inflammatory responses, and promoting the development and progression of UC. Therefore, regulating the gut microbiota and its metabolites has become a key entry point for the treatment of UC.

[0003] Currently, the main drugs used in clinical treatment of ulcerative colitis are aminosalicylic acids, sulfasalazine, and immunosuppressants. However, these drugs are prone to causing drug resistance and have adverse reactions. Therefore, the development of safer and more effective new treatments is urgently needed. Stemona alkaloids are the main natural alkaloids in the root extract of the traditional Chinese medicine Stemona japonica, possessing various pharmacological activities such as antitumor, anti-inflammatory, antioxidant, and neuroprotective effects. However, there are currently no research reports on whether Stemona alkaloids can improve colitis by regulating the intestinal flora. Summary of the Invention

[0004] The purpose of this invention is to provide the application of stemona alkaloids in the preparation of drugs for treating colitis, thereby addressing the problems mentioned in the background art.

[0005] To achieve the above objectives, the present invention provides the following technical solution: On the one hand, the present invention provides the use of stemona alkaloids in the preparation of a drug for treating colitis, wherein the colitis is ulcerative colitis.

[0006] On the other hand, the present invention provides a drug for treating colitis, wherein the active ingredient of the drug is stemona alkaloid.

[0007] Compared with the prior art, the specific beneficial effects of the present invention are as follows: This invention demonstrates that stemonasaline alleviates and treats colitis by regulating intestinal flora, overcoming the limitations of existing natural product research with unclear targets and mechanisms. Furthermore, stemonasaline is widely available and has a mature extraction process. Compared to traditional synthetic drugs, stemonasaline, as a natural compound, has lower toxicity. Moreover, compared to emerging probiotic therapies and biological agents, the subsequent drug development costs of this invention are lower, making it easier to achieve industrialization and promotion, and reducing the treatment burden on patients. Attached Figure Description

[0008] Figure 1 The experimental results of the alleviating effect of p-stemonine on colitis provided in Example 1 of the present invention are as follows: A is the change in body weight, B is the disease activity index score, C is a picture of the colon, D is a quantitative map of colon length, E is a picture of colon hematoxylin and eosin (H&E), F is the content of TNF-α (tumor necrosis factor α) in colon tissue, G is the content of IL-1β (interleukin-1β) in colon tissue, and H is the content of IL-6 (interleukin-6) in colon tissue. Figure 2 For the bioinformatics analysis results provided in Embodiment 2 of the present invention, A is the Venn diagram, BD is represented by the observed Chao1 index, species richness and Shannon index respectively, EF is represented by principal coordinate analysis (PCoA) and non-metric multidimensional scaling analysis (NMDS) at the OTU (operational taxonomic unit) level, and G is the top 10 genera of gut microbiota and other components. Figure 3 The results of the toxicity assessment of Stemona japonica alkaloids provided in Example 3 of the present invention are as follows: A is the H&E staining result of tissues and organs, and B is the CCK-8 (cell proliferation toxicity test) result. In the attached figures, compared with the control group, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001; compared with the DSS group, #p < 0.05, ##p < 0.01, ###p < 0.001, and ####p < 0.0001. Detailed Implementation

[0009] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0010] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0011] Example 1: Constructing a mouse colitis model and analyzing the alleviating effect of p-stemonine on colitis: 1. Materials: Stemona alkaloid (TS) (purity ≥ 98%) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China); 5-aminosalicylic acid (5-ASA) was purchased from Shandong Keyuan Biochemical Co., Ltd.; sodium dextran sulfate (DSS) was from MP Biomedicals (Santa Ana, CA); 7-8 week old male C57 BL / 6 J mice, weighing 20-22g, were purchased from Liaoning Changsheng Biotechnology Co., Ltd. (SYXK 2018-0001); all mice were acclimatized for 3 days before the experiment, and the housing environment was a 12-hour light / 12-hour dark cycle with free access to food and water; 2. Model Establishment: Seventy mice were randomly divided into seven groups (n=10 per group): control group, high-dose TS group (TS-H), DSS group, DSS + low, medium and high-dose TS groups (10, 20, 40 mg / kg) (DSS + TS-L, DSS + TS-M, DSS + TS-H), and DSS + 5-aminosalicylic acid group (100 mg / kg) (DSS + The dosage of 5-ASA and TS was determined based on the results of preliminary experiments, and the dosage of 5-ASA was based on relevant literature reports. All treatment groups were administered the drugs by gavage for 21 consecutive days, while the control group received an equal volume of physiological saline. From day 14 of administration, except for the control group and the TS-H group, all other groups of mice were allowed free access to drinking water containing 2.5% (w / v) DSS to induce colitis. The colitis model was established for 7 consecutive days. During the modeling period, the weight, fecal consistency, and bloody stool of the mice were recorded daily. The colitis disease activity index was scored based on the degree of weight loss, diarrhea, and bloody stool to assess the severity of colitis. On day 22 of the experiment, the mice were euthanized by cervical dislocation, and fecal samples were aseptically collected, flash-frozen in liquid nitrogen, and stored for later use. The colon tissue of the mice was dissected and its length was measured. A portion of the colon tissue was fixed in 4% paraformaldehyde solution for histopathological examination. The remaining colon tissue was washed with PBS (phosphate buffered saline), flash-frozen in liquid nitrogen, and stored at -80°C for subsequent ELISA (enzyme-linked immunosorbent assay) and WB (Western blot) experiments. 3. Indicator Observation: like Figure 1 As shown, seven days after DSS induction, the body weight of mice in the DSS group was significantly lower than that in the control group. Figure 1 The A and DAI indices (disease activity index) were significantly elevated. Figure 1 In the middle B group, the colon length was significantly shortened (p<0.0001). Figure 1 (C, D); After TS administration intervention, these indicators all improved in a dose-dependent manner. Oral 5-ASA can also alleviate DSS-induced phenotypic pathology in mice, and the mice in the high-dose oral TS group showed significantly better relief than those in the oral 5-ASA group. 4. H&E staining of colon tissue sections: Mouse colon tissue was fixed in 4% paraformaldehyde solution, trimmed, and transferred to an embedding cassette. After dehydration with graded alcohol and clearing with xylene, it was embedded in paraffin to prepare 4 µm thick serial sections. H&E staining was performed, and finally, neutral resin was added, followed by mounting with a coverslip. After the resin solidified, the colon tissue pathological morphology was observed and photographed under a microscope. Results are as follows: Figure 1 As shown in Figure E, compared with the control group, the DSS group showed obvious inflammatory cell infiltration, mucosal epithelial damage, intestinal wall thickening, and significantly increased histological score in the colon; while after oral TS intervention, the degree of inflammatory cell infiltration, mucosal damage, and histological score of the colon tissue decreased in a dose-dependent manner with increasing drug dosage. 5. ELISA detection of colon inflammatory factors: Frozen colon samples were homogenized with lysis buffer, centrifuged at 12000×g for 15 min at 4℃, and the supernatant was collected. Protein concentration was quantified using a quinolinic acid (BCA) protein assay kit. The concentrations of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α in colon tissue were then detected using an ELISA kit. The results showed that, compared with the control group, the DSS group mouse colon tissue homogenate contained significantly higher levels of the pro-inflammatory cytokine TNF-α (IL-1β, IL-6, and TNF-α). Figure 1 IL-1β (F), IL-1β Figure 1 The levels of IL-6 (H in Figure 1) and IL-6 (G in Figure 1) were significantly increased (p<0.0001); after oral TS intervention, the levels of each pro-inflammatory factor showed a decreasing trend with increasing dose, and the levels of pro-inflammatory factors in the high-dose TS group and the 5-ASA group were similar.

[0012] Example 2: Analysis of the regulatory effect of Stemona alkaloids on intestinal flora: 1. Fecal 16S rRNA sequencing: Total genomic DNA of microorganisms was extracted using a kit adapted for fecal samples. Microbial cell walls were thoroughly lysed through a combination of mechanical and chemical fragmentation to obtain high-purity DNA. The DNA concentration and purity were then measured using agarose gel electrophoresis and a micro-spectrophotometer to ensure they met library construction requirements (typically OD260 / 280 between 1.8 and 2.0). Specific variable regions of the 16S rRNA gene were selected (V3-V4 or V4 region is most commonly used in gut microbiota research), and PCR (polymerase chain reaction) amplification was performed using universal primers with sequencing adapters and sample tag sequences to obtain sufficient amounts of the target gene fragment. After amplification, the product size was verified by agarose gel electrophoresis, and primer dimers and other impurities were removed by gel excision and recovery. The purified product was accurately quantified, and different samples were mixed in proportion to construct a qualified sequencing library. Library quality control was then performed. Illumina sequencing was used to analyze the purified product. For sequencing on mainstream high-throughput sequencing platforms such as MiSeq and NovaSeq, it is generally required that each sample has a sequencing depth of no less than 50,000 reads to ensure the accuracy of bacterial community detection. 2. Bioinformatics analysis: After obtaining the raw sequencing data, the data enters the bioinformatics analysis process. First, the raw data undergoes quality control, using tools like Fastp or Trimmomatic to remove low-quality sequences, adapter sequences, and ambiguous bases, filtering to obtain high-quality effective sequences. Then, paired-end sequences are spliced, followed by DADA2 noise reduction or UPARSE clustering to obtain amplified sequence variants (ASVs) or OTUs, while removing chimeric sequences. Next, the obtained representative sequences are compared with 16S rRNA reference databases such as Silva, Greengenes, or RDP to complete species classification annotation from the phylum to the genus level. Then, diversity analysis is performed, calculating Alpha diversity indices such as Chao1, Shannon, and Simpson to reflect the richness and diversity of the microbial community within the sample. Beta diversity is calculated using PCoA or NMDS analysis to compare the differences in microbial community structure among different groups. Further analysis can be carried out, such as intergroup differential species analysis, LEfSe marker species screening, and microbial community function prediction. Finally, the results are visualized and biologically interpreted in accordance with the analysis objectives to draw conclusions about the differences in gut microbial community composition and structure among different groups. 3. Results are as follows Figure 2 As shown, the specific analysis is as follows: Venn diagrams showed that 2674, 2271, 4540, and 1901 different OTUs were identified in the control group, TS-H group, DSS group, and DSS + TS-H group, respectively. Figure 2(A); the Chao1 and Observed species indices (species richness indices) at the ASV level indicate bacterial community richness, while the Shannon index represents bacterial community diversity. It can be seen that, compared to the control group, TS administration alleviated the Chao1 index induced by DSS (…). Figure 2 (B) and Observed species index ( Figure 2 The decrease in C) and the significant increase in Chao1 and Observed species indices in the TS-H group (p<0.0001); compared with the control group, administration of TS to mice significantly increased the Shannon index (p<0.0001). Figure 2 (D); β-diversity analysis (NMDS and PCoA analysis) showed that TS reversed the gut microbiota abundance abnormality induced by DSS, resulting in DSS-treated mice exhibiting a gut microbiota structure similar to that of control mice. Figure 2 In the analysis of the effects of TS administration on gut microbiota at the genus level, TS significantly alleviated the increase in Bacteroides_H (Bacteroides) caused by DSS (E, F); Figure 2 (G).

[0013] Example 3: Toxicity assessment and analysis of Stemona japonica alkaloids: 1. H&E staining of tissues and organs: Important mouse tissues and organs (heart, liver, spleen, lung, and kidney) were fixed in 4% paraformaldehyde solution, trimmed, and transferred to embedding cassettes. After graded dehydration with alcohol and clearing with xylene, they were embedded in paraffin to prepare 4 µm thick serial sections. These sections were then stained with H&E, and finally, neutral resin was added, covered with coverslips, and allowed to solidify. The histopathological morphology of the colon tissue was observed and photographed under a microscope after the resin had hardened. Results showed that the heart, liver, spleen, lung, and kidney of mice treated with TS showed no significant pathological damage, and the organ morphology was similar to that of the control group, confirming that TS had no significant in vivo toxicity in mice at the experimental dose. Figure 3 (A) 2. Cytotoxicity test: Human colorectal cancer cells (Caco-2) were cultured in high-glucose DMEM medium (Duborough modified Eagle medium) containing 20% ​​fetal bovine serum and incubated at 37°C in a 5% CO2 incubator at an incubator with a growth rate of 5 × 10⁻⁶ cells / mL. 4 / cm 2Caco-2 cells were seeded at a density of 20, 40, 80, 160, and 320 μM TS at 24 h, and incubated for 6, 12, 24, and 48 h, respectively. Then, 10 μL of CCK-8 solution was added to each well, and the cells were incubated at 37°C in the dark for 2 h. The optical density (OD) was measured at 450 nm using a microplate reader. The results showed that after co-incubation of Caco-2 cells with TS for 6, 12, 24, and 48 h, cell viability was not significantly inhibited with increasing TS concentration, suggesting that TS had no significant cytotoxicity to Caco-2 cells within this experimental concentration range. Figure 3 (B)

[0014] In summary, this invention demonstrates that stemonasaline has a significant anti-inflammatory therapeutic effect in a DSS-induced acute colitis model, filling a research gap in the field of enteritis treatment of this natural compound, confirming the use of stemonasaline as a drug for treating ulcerative colitis, and confirming its use as a drug that exerts its anti-inflammatory effect by regulating the intestinal flora. Stemona alkaloids can significantly improve typical enteritis manifestations such as DSS-induced weight loss, increased disease activity index (DAI), colonic shortening, and histopathological damage. Its anti-inflammatory effect is superior to conventional preclinical control drugs, and no obvious toxic side effects were observed. Its safety is superior to traditional anti-inflammatory drugs. Stemona alkaloids are natural monomeric compounds isolated from the traditional Chinese medicine Stemona japonica. With a clear source and mature extraction process, they have lower research and development costs and higher drug potential compared to novel chemically synthesized drugs or biological agents, providing a material basis for the subsequent development of colitis-related microecological intervention products.

[0015] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of stemona alkaloids in the preparation of drugs for treating colitis, characterized in that, The colitis mentioned is ulcerative colitis.

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