Application of Epiphyllum extract in the preparation of drugs for treating / improving intestinal diseases

Abstract

The application discloses application of an extract of Epiphyllum crenatum in preparation of a medicine for treating and improving intestinal diseases, relates to the fields of phytochemistry and microbiological technology, and the extract of the Epiphyllum crenatum is type I arabinogalactan. 6 The average molecular weight of the extract of the Epiphyllum crenatum extracted from petals of the Epiphyllum crenatum is 5.767*10 6 Da, and the backbone structure is a polysaccharide of β-Galp connected through (1→4) bonds, which expands the application potential of the Epiphyllum crenatum, improves the value of the Epiphyllum crenatum, and provides a new raw material for the field of the medicine for treating and improving intestinal diseases.

Description

Technical Field

[0001] This invention relates to the fields of phytochemistry and microbiology, and more specifically to the use of Epiphyllum oxypetalum extract in the preparation of medicines for treating / improving intestinal diseases. Background Technology

[0002] Epiphyllum ( piphyllum oxypetalum ( DC. ) Haw Epiphyllum oxypetalum (EOH) belongs to the cactus family and is known as the "Queen of the Night" due to its unique nocturnal flowering characteristic. This perennial succulent has irreplaceable ornamental value in the horticultural world due to its rare flowers and short flowering period. In modern applications, Epiphyllum oxypetalum extract is widely used in mass-market cosmetics for its moisturizing, whitening, and antioxidant functions. For example, patent application document CN117427008A, published on January 23, 2024, proposes the application of Epiphyllum oxypetalum extract in oil control, hair loss prevention, and / or acne treatment.

[0003] The night-blooming cereus is rich in various beneficial components, including protein (14 mg / g), fatty acids (4.6 mg / g), and vitamins (0.18 mg / g) as disclosed in existing literature. Compared to these nutrients, mucopolysaccharides are among the most abundant carbohydrates in cacti, accounting for over 18% of the flower and stem weight. These polysaccharides typically have high molecular weights and branched structures, as is the case with the night-blooming cereus. Natural polysaccharides are a class of highly functional bioactive macromolecules with various health benefits, such as immunomodulation, anti-tumor, anti-diabetic, and hepatoprotective activities. The biological functions of different polysaccharides are closely related to their structural variations, including monosaccharide composition, glycosidic bond type, molecular size, and branching degree. These structural features determine their specific roles in biological processes such as cell recognition, signal transduction, and immune responses.

[0004] In the prior art, the title published in the *International Journal of Biological Macromolecules* is "A polysaccharide from..." Epiphyllum oxypetalum The literature, "(DC.) Haw. and its immunomodulatory activity," discloses the isolation and identification of a polysaccharide (EOP) from Epiphyllum oxypetalum, and the evaluation of its in vitro and in vivo immunomodulatory activities. Multispectral analysis determined that EOP is composed of rhamnose, arabinose, galactose, and galacturonic acid in a molar ratio of 26.65:11.48:53.79:6.04, with a molecular weight of 5.77 × 10⁻⁶. 6Furthermore, the main chain structure of EOP consists of (1→4)-linked β-Galp, (1→2)-linked β-Rhap, (1→3,4)-linked β-Galp, (1→2,4)-linked β-Rhap, and (1→4)-linked α-GalpA, terminated by t-β-Arap and t-β-Galp. In vitro immunomodulatory activity experiments on RAW264.7 cells showed that EOP can promote macrophage proliferation, enhance their phagocytic capacity, and promote the production of cytokines such as nitric oxide (NO), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6). In addition, in vivo evaluation in zebrafish showed that EOP can reduce the residual content of fluorescent microspheres in zebrafish, indicating that EOP has the ability to enhance macrophage phagocytosis. Given EOP's complex structure and significant immunomodulatory activity both in vitro and in vivo, it has potential application value in the pharmaceutical industry. Summary of the Invention

[0005] With breakthroughs in epiphyllum cultivation technology, raw materials for epiphyllum are more readily available. The inventors of this invention extracted epiphyllum using a specific method to obtain epiphyllum extract and conducted in-depth research on the composition and bioactivity of the epiphyllum extract. They discovered that epiphyllum extract has beneficial effects on immune regulation and treatment / improvement of intestinal diseases, thus expanding the application areas of epiphyllum.

[0006] The purpose of this invention is to provide the application of Epiphyllum oxypetalum extract in the preparation of drugs for treating / improving intestinal diseases, thereby expanding the application potential of Epiphyllum oxypetalum and providing new raw materials for the field of drugs for treating / improving intestinal diseases.

[0007] This invention is achieved through the following technical solution:

[0008] Application of Epiphyllum extract in the preparation of drugs for treating / improving intestinal diseases, wherein the Epiphyllum extract is a type I arabinogalactan as shown in structural formula (1),

[0009] (1),

[0010] Where R is or ; m and n are both integers greater than 0.

[0011] Furthermore, the epiphyllum extract, extracted from the petals of the epiphyllum, has an average molecular weight of 5.767 × 10⁻⁶. 6 Da, whose main chain structure is a polysaccharide of β-Galp linked by (1→4) bonds.

[0012] Furthermore, the polysaccharide is polymerized from arabinose, galactose, glucose, xylose, rhamnose, fructose and galacturonic acid, wherein the molar ratio of the monosaccharide composition of arabinose and galactose is 11.480% and 53.791%, respectively.

[0013] Furthermore, the epiphyllum extract is used in the preparation of health foods or medicines for regulating the diversity and richness of immunosuppressive gut microbiota, influencing gut microbiota structure, or affecting the relationships between gut microbes.

[0014] Furthermore, the use of the Epiphyllum extract in the preparation of medicaments for repairing CTX-induced damage to the intestinal chemical, mechanical, or immune barriers.

[0015] Furthermore, the epiphyllum extract is used in the preparation of products for restoring intestinal health. Lactobacillus The application of its abundance in drugs.

[0016] Furthermore, the use of the epiphyllum extract in the preparation of a drug for promoting immune recovery, which promotes immune recovery by improving intestinal... Lactobacillus Abundance and lactic acid content were achieved.

[0017] Furthermore, the use of the Epiphyllum extract in the preparation of drugs for promoting the physical barrier of the intestinal structure.

[0018] And after further research, it was determined that:

[0019] The structure of the epiphyllum extract is as follows:

[0020] ,

[0021] Where R is or ; m and n are both integers greater than 0.

[0022] The molecular formula is as follows:

[0023] ,

[0024] Where R is or ; m and n are both integers greater than 0.

[0025] The main chain structure of the epiphyllum extract is β-Galp linked by (1→4) bonds. For example... Figure 26 As shown, the backbone of type I arabinogalactan (AG-I) is composed of (1→4)-β-D-Galp, while the backbone of type II arabinogalactan (AG-II) is composed of (1→6) and / or (1→3)-Galp. Therefore, the extract of *Epiphyllum oxypetalum* belongs to the typical type I arabinogalactan (AG-I).

[0026] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0027] I. In this invention, the epiphyllum extract, belonging to arabinogalactan (AG-I), extracted from the epiphyllum flower, has significant therapeutic / improving effects on intestinal diseases. It can stimulate the expression of immune factors while reducing damage to immune organs. For example, the epiphyllum extract can repair the intestinal immune barrier, not only regulating the expression of key immune factors in the intestine but also improving the diversity and overall structure of the intestinal microbiota. Furthermore, the epiphyllum extract may also activate the expression of proteins such as Wnt3a and β-catenin at the base of the colonic crypts through a Gpr81-dependent mechanism, thereby promoting the repair of the intestinal physical barrier. Therefore, the epiphyllum extract can alleviate CTX-induced intestinal immune damage, regulate the diversity and richness of immunosuppressive intestinal flora, influence the structure of intestinal flora, affect the relationships between intestinal microorganisms, repair CTX-induced damage to the intestinal chemical, mechanical, and immune barriers, alter the structure and diversity of intestinal flora, and restore the intestinal... Lactobacillus The abundance of [the substance], and its application in the preparation of drugs that promote the physical barrier of the intestinal structure, can also be used in the preparation of drugs with immune-enhancing effects to reduce damage to immune organs.

[0028] Second, the present invention proposes the application of an epiphyllum extract in the preparation of drugs for immunomodulation or treatment / improvement of intestinal diseases, which broadens the application field of epiphyllum and enhances its value.

[0029] Thirdly, this invention further analyzes the composition of the Epiphyllum oxypetalum extract, determining its monosaccharide composition and molar ratio. The extract is polymerized from arabinose, galactose, glucose, xylose, rhamnose, fructose, and galacturonic acid. Experiments have verified that arabinose and galactose are the main active ingredients when used in the preparation of drugs for treating / improving intestinal diseases. This highly branched, neutral monosaccharide domain composed of arabinose and galactose facilitates the formation of specific conformations in the polysaccharide, resulting in a favorable response in stimulating the immune system. For example, in complement activation, such polysaccharides often exhibit high complement-binding activity, contributing to a rapid immune defense response, unlike other fungal polysaccharides such as lentinan. Furthermore, the main chain structure of the active ingredients in the Epiphyllum oxypetalum extract has been determined, providing a theoretical basis for the application development of Epiphyllum oxypetalum in immunomodulatory drugs and drugs for the prevention / treatment of intestinal diseases, as well as in new fields such as thickeners, drug carriers, humectants, gelling agents, and adhesives.

[0030] IV. In this invention, the epiphyllum extract has an average molecular weight of 5.767 × 10⁻⁶. 6Da's polysaccharides have a molecular weight approximately 8 to 600 times greater than other natural polysaccharides. These high-molecular-weight polysaccharides can interact with the body as dietary fiber or prebiotics. These large molecules are difficult to digest in the small intestine, thus allowing them to enter the colon and provide substrates for the complex bacterial ecosystem within the colon. This, in turn, regulates the intestinal microbial immune barrier and improves intestinal flora imbalance and metabolic disorders. Attached Figure Description

[0031] Figure 1 This is a diagram showing the steps involved in constructing an immunodeficient mouse model.

[0032] Figure 2 This is a graph showing the trend of weight change in experimental mice.

[0033] Figure 3 This is a graph showing the trend of dietary changes in laboratory mice.

[0034] Figure 4 This is a statistical graph showing the effects of Epiphyllum extract on the intergroup differences in thymus index (A), spleen index (B), kidney index (C), and liver index (D) of experimental mice.

[0035] Figure 5 This is a statistical graph showing the effect of Epiphyllum oxypetalum extract on the cytokine levels of experimental mice. In the graph, A, B, C, D, and E represent the expression levels of serum TNF-α, serum IgA, spleen IgG, spleen IgM, and liver C3 in experimental mice, respectively.

[0036] Figure 6 These are H&E stained tissue sections of the thymus (100×) and spleen (40×) of experimental mice.

[0037] Figure 7 The images show the results of PAS (40×) and H&E (200×) staining of colon tissue sections from experimental mice.

[0038] Figure 8 Statistical analysis of the effects of immunosuppressed mice on the intestinal barrier Figure 1 In the figure, A and C are statistical graphs of the number of colon goblet cells and crypt length in each group of experimental mice, respectively, and B, D, and E are statistical graphs of the content of colon barrier-related proteins MUC2, tricellulin protein, and occludin protein in each group of experimental mice, respectively.

[0039] Figure 9 Statistical analysis of the effects of immunosuppressed mice on the intestinal barrier Figure 2 In the figure, A, B, C, D, and E are statistical graphs showing the levels of colonic immune barrier cytokines sIgA, IFN-γ, IL-10, TGF-β3, and IL-17 in each group of experimental mice.

[0040] Figure 10 These are dilution curves of mouse fecal samples from each group, where A is the Shannon dilution curve and B is the Sobs dilution curve.

[0041] Figure 11 The figure shows the number of genus-level species - UpSet Venn analysis results.

[0042] Figure 12 Analysis of the effects of Epiphyllum oxypetalum extract on the structure and diversity of gut microbiota in immunosuppressed mice Figure 1 Where: A, B, C, and D represent the PCA levels of OTU at the MDC vs CON, MDC vs LNP, MDC vs EPL, and MDC vs EPH levels, respectively, and E is the partial least squares discriminant analysis (PLS-DA) results for each group.

[0043] Figure 13 Analysis of the effects of Epiphyllum oxypetalum extract on the structure and diversity of gut microbiota in immunosuppressed mice Figure 2 Where: A, B, C, D, and E are the number of OTUs, Chao1 index, Ace index, Simpson index, and Shannon index in the Alpha diversity analysis of OTU levels.

[0044] Figure 14 The figure shows the effect of Epiphyllum extract on the gut microbiota of immunosuppressed mice. In the figure, A, B and C are the unidirectional correlation networks of the top 30 abundant genera of gut microbiota in the MDC group, EPL group and EPH group, respectively.

[0045] Figure 15 This is a graph showing the analysis results of the dominant phylum (A) and genus (B) in fecal samples from different groups of mice.

[0046] Figure 16 Statistics on species whose abundance differs among different groups at the genus level Figure 1 Where: A is Lactobacillus, Defiuviitaleaceae_UCG-011, Tyzzerella, Rikenella, unclassified_f_ Anaerovoracaceae A statistical graph of abundance between different groups; B is... Family_XIII_AD3011_group, norank_f_ norank_o_Rhodospirillales, Anaeroplasma, Escherichia-Shigella, Parabacteroides Statistical graph of abundance among different groups.

[0047] Figure 17 Statistics on species whose abundance differs among different groups at the genus level Figure 2 Where: C is Anaerovorax, Caldicoprobacter, Candidatus_Arthromitus, Prevotellaceae_NK3B31_ group,Unclassified_f_Lachnospiraceae A statistical graph of abundance between different groups; D is... Butyricimonas / UCG-009, norank_f_UCG-010, Marvinbryantia, unclassified_c_Clostridia Statistical graph of abundance among different groups.

[0048] Figure 18The results of LEfse analysis of mouse gut microbiota Figure 1 In the figure, A and B represent the results of MDC vs CON and MDC vs LNP in the LEfse analysis of mouse gut microbiota, respectively.

[0049] Figure 19 The results of LEfse analysis of mouse gut microbiota Figure 2 In the figure, C and D represent the results of MDC vs EPL and MDC vs EPH in the LEfse analysis of mouse gut microbiota, respectively.

[0050] Figure 20 This represents the abundance and proportion of fungi and Bacteroides in each group. Data are expressed as averages, n=5.

[0051] Figure 21 This is a correlation diagram between bacterial genus and metabolic parameters.

[0052] Figure 22 The graph shows the correlation between bacterial genera and immune parameters, and the functional changes of gut microbiota in each group. Among them: A is the KEGG Pathway Level 3 functional prediction abundance graph, B is the KEGG Module functional prediction abundance graph, C is the Metacyc functional prediction abundance graph, D is the total abundance graph of total lactic acid fermentation, and E is the KEGG Pathway Level 3 fatty acid metabolism functional prediction abundance graph.

[0053] Figure 23 This is an immunohistochemical staining image (200×) of Gpr81, Wnt3a and β-catenin in colon tissue.

[0054] Figure 24 This is a graph showing the research results on how epiphyllum extract promotes the repair of intestinal epithelium at the bottom of the colonic crypts. In the graph, A, B, and C are the relative expression results of Gpr81, Wnt3a, and β-catenin, respectively, expressed as average optical density (AOD) values.

[0055] Figure 25 This is a bar chart showing the lactic acid content in the feces (A) and colon (B) of each group.

[0056] Figure 26 These are chemical structure diagrams of type I and type II arabinogalactan. Detailed Implementation

[0057] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto.

[0058] Example 1

[0059] To facilitate public understanding of this solution, this embodiment explains, verifies, and illustrates, in conjunction with experiments, the acquisition of raw materials for Epiphyllum oxypetalum extract, the extraction method, the composition of Epiphyllum oxypetalum extract, and the bioactivity of Epiphyllum oxypetalum extract.

[0060] I. Materials and Methods Involved in This Embodiment

[0061] 1.1. Materials and Reagents

[0062] The epiphyllum flowers were collected from the epiphyllum planting demonstration base of Sichuan Yuanlan Agricultural Development Co., Ltd. (Zizhong, China).

[0063] The extraction process of Epiphyllum oxypetalum extract (EPS) is as follows: Epiphyllum oxypetalum petals are dried and pulverized into powder. Polysaccharides are then extracted from the powder using a water-alcohol precipitation method, achieving a purity of 95% and an average molecular weight of 5.767 × 10⁻⁶. 6 For detailed operating instructions, please refer to the reference - "A polysaccharide from..." Epiphyllum oxypetalum The extraction method described in "(DC.) Haw. and its immunomodulatory activity" was used. The monosaccharide composition of the Epiphyllum extract was determined to include arabinose, galactose, glucose, xylose, rhamnose, fructose, and galacturonic acid, with a molar ratio of 11.480:53.791:0.494:0.446:26.648:1.102:6.040. The total molar ratio of arabinose and galactose exceeded 60%, and the maximum solubility of the Epiphyllum extract was approximately 2.75 mg / ml.

[0064] CTX (C849559) was purchased from Shanghai Maclean Biotechnology Co., Ltd. (Shanghai, China). ELISA kits for cytokines and antibodies were purchased from Quanzhou Ruixin Biotechnology Co., Ltd. (Quanzhou, China) and Jiangsu Enzyme Immunoassay Co., Ltd. (Yancheng, China). 4% paraformaldehyde tissue fixative (BL539A), RIPA lysis buffer (BL504A), protease inhibitor (BL612A), and BCA protein assay kit (BL521A) were purchased from Baisha Biotechnology Co., Ltd. (Bengbu, China). Lentinan (H42022727) was purchased from Hubei Guangren Pharmaceutical Co., Ltd. (Suizhou, China). Lactate assay kit (A019-2-1) was purchased from Nanjing Jiancheng Biotechnology Institute (Nanjing, China). Hematoxylin and eosin (H&E) staining kit (G1120) was purchased from Solarbio Science & Technology Co., Ltd. (Beijing, China), and periodic acid-Schiff (PAS) staining dye (G1008) was purchased from Saiwei Biotechnology Co., Ltd. (Wuhan, China). All other chemical reagents were of analytical grade.

[0065] 1.2. Animals and Processing

[0066] like Figure 1 As shown, 35 SPF-grade Kunming mice, 4 weeks old and weighing 18-22 grams, were purchased from Dashuo Laboratory Animal Co., Ltd. [Chengdu, China] (HE-12-54-04). The experimental diagram is shown below. Figure 1 As shown. Mice were housed under standard indoor conditions (20–26°C, 40%–70% humidity) with a 12-hour light / 12-hour dark cycle and free access to standard maintenance feed and water. After 3 days of acclimatization at the SPF-grade laboratory animal facility of Chengdu Medical College, they were randomly divided into 5 groups of 7 mice each: blank control group (CON), model control group (MDC), lentinan positive control group (LNP), low-dose epiphyllum extract group (EPL), and high-dose epiphyllum extract group (EPH).

[0067] Cyclophosphamide (CTX) is a first-line chemotherapy drug for treating various cancers and autoimmune diseases. However, long-term use of CTX can also have harmful effects on immune organs such as the thymus and spleen, damage the gastrointestinal mucosal barrier, and lead to intestinal flora imbalance. Therefore, CTX is often used to construct animal models of immunosuppression, especially mouse models with anatomical structures (including the gastrointestinal tract) and immune systems similar to humans. In the reference "Immunomodulatory activity of a novel polysaccharide from Lonicerajaponica in immunosuppressed mice induced by cyclophosphamide," to establish a mouse immunosuppression model, the CON group received intraperitoneal injections of saline, while the other groups received intraperitoneal injections of CTX (70 mg / kg bw•day) from days 1 to 5 of the experiment. Due to the risk of death from mouse biting and the immunosuppressive toxicity of the drug, only 5 mice were successfully modeled in each group. From day 6 to day 19, the CON and MDC groups were administered saline by gavage, the LNP group was administered lentinan (5 mg / kg / dbw•day) by gavage (converted according to the manufacturer's recommended human dose), and the EPL group (25 mg / kg / day bw•day) and EPH group (35 mg / kg / dbw•day) were administered the corresponding doses of Epiphyllum oxypetalum extract by gavage. Feces were collected from all groups on day 19. On day 20, mice were anesthetized with isoflurane and euthanized by cervical dislocation; other tissues were collected. Mice were observed daily during the experiment, and their weight and diet were recorded.

[0068] 1.3. Blood and Organ Collection

[0069] Mice were anesthetized with isoflurane, and their eyes were removed to collect blood. The blood was left at room temperature for 20 minutes, then centrifuged at 3500 rpm for 20 minutes. The supernatant was collected to obtain serum, which was then frozen and stored at -80°C. Mice were then euthanized humanely by cervical dislocation, and organs such as the liver, thymus, spleen, kidneys, and colon were removed and weighed. The organ index was calculated as organ weight (mg) / body weight (g) × 10.

[0070] 1.4. H&E staining of spleen, thymus, and colon

[0071] Spleen, thymus, and colon tissues were fixed with 4% paraformaldehyde tissue fixative for 48 hours, dehydrated with alcohol, and prepared into paraffin-embedded blocks. Sections (3-5 μm thick) were stained with H&E staining solution to observe histological changes. Images were acquired using a CI-L plus optical microscope (Nikon, Japan) (spleen: 40x magnification, thymus: 100x magnification, colon: 200x magnification). The depth of five intact colonic crypts in each colon tissue section was measured using Nis-elements analysis software (version: 5.42.00, Japan), and the average value was calculated.

[0072] 1.5. PAS staining of the colon

[0073] The number of stained goblet cells in colon tissue sections (3–5 μm thick) was determined using the PAS method. After staining the colon tissue, full-field images were captured using an optical microscope (40x magnification), and the number of stained goblet cells in the field of view was counted using Image-J (version 1.8.0.172, USA).

[0074] 1.6. Determination of immunoglobulins and cytokines in serum, colon, and liver

[0075] Blood samples were collected and serum was separated. Tumor necrosis factor-α (TNF-α) and immunoglobulin A (IgA) levels were measured using an ELISA kit. 100 mg of spleen, liver, and colon tissue were mixed with 900 μl of phosphate-buffered saline (PBS) and homogenized thoroughly. The mixture was centrifuged for 20 min (8500 r / min, 4℃), and the supernatant was collected. The expression levels of immunoglobulins M and G (IgM, IgG), complement 3 (C3), secretory IgA (sIgA), interleukin-10 and IL-17 (IL-10, IL-17), interferon-γ (IFN-γ), metastatic growth factor β3 (TGF-β3), tight junction-associated protein (tricellulin, occludin), and mucin 2 (MUC2) were measured. This assay was performed according to the ELISA manufacturer's instructions.

[0076] 1.7. Determination of lactic acid content in colon and feces

[0077] Colon tissue samples were added to 9 volumes of PBS at a ratio of 100 mg: 900 μl, and fecal samples were added to 3 volumes of PBS at a ratio of 100 mg: 300 μl. After centrifugation for 20 min (3500 r / min, 4 ℃), the supernatant was collected, and the lactate concentration was determined by enzymatic colorimetric method.

[0078] 1.8. Immunohistochemistry

[0079] Colon tissue sections were treated with xylene and graded ethanol, incubated with 0.1% Tritonx-100, washed with PBS, and mounted sequentially with BSA and serum. Sections were incubated overnight at 4°C with primary antibodies GPR81, Wnt3a, and β-Ctnnb1 (β-catenin), followed by incubation with secondary antibodies at room temperature. Horseradish peroxidase-labeled streptomycin protein was added, and the sections were developed with DAB chromogenic buffer. Five random fields of view were photographed (200x magnification) for microscopic observation. Quantitative measurement of positive area was automatically defined using ImageJ software (NIH, USA) and expressed as mean optical density (AOD) values.

[0080] 1.9. Gut microbiota 16S rRNA sequencing

[0081] Total fecal DNA was extracted according to the EZNA® soil DNA kit (Omega Bio-Tek, USA) and detected by 1% agarose gel electrophoresis. The V3-V4 hypervariable region of the bacterial 16S rRNA gene was amplified using universal primers 338F (5'-ACTCCTACGGGAGGCAGCAG-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3'). PCR products were recovered from the PCR gel using 2% agarose gel, purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, USA), and quantified using QuantiFluor™-ST (Promega, USA). A PE library was created using the TruSeq™ DNA Sample Preparation Kit and sequenced on an Illumina Miseq PE300 platform (Illumina, USA) according to the standard protocol of Majorbio, Inc. (Shanghai, China). PacBio raw reads were processed using SMRTLink (version 8.0) to obtain demultiplexed circular consensus (CCS) reads with at least three complete deliveries and 99% accuracy. CCS reads were barcode-identified and length-filtered, removing sequences <1000 bp or >1800 bp. Optimized CCS reads were clustered into operational taxonomic units (OTUs) with 97% sequence similarity using UPARSE 7.1. The most abundant sequence in each OTU was selected as the representative sequence. Chloroplast sequences were manually removed from the OTU table. Paired-end forward and reverse sequences were merged using FLASH software (version 1.2.11). Sequence alignment was performed using the Silva database, and species information for each OTU was annotated using BLAST. Alpha and Beta diversity were analyzed based on OTU abundance using R packages. Dominant bacterial communities between groups were analyzed using linear discriminant analysis (LDA) and LDA effect size (LEfse). Community phylogenetic studies were performed using Picrust2 to predict the functional composition of microbial communities in amplicon sequencing results. Spearman rank correlation coefficient analysis was used to draw a one-way correlation network and a correlation heatmap.

[0082] 1.10. Data Analysis

[0083] Each experiment was repeated at least three times. Statistical analysis was performed using GraphPad Prism, and data are expressed as mean and standard error (mean ± SEM). The Shapiro-Wilk test was used to assess the normality of the data. Student's t-test was used to compare biochemical indicators between each group and the MDC group. The nonparametric Mann-Whitney test was used to determine the statistical significance of alpha-diversity and Picrust2 measurements. A p-value < 0.05 was considered statistically significant.

[0084] II. Experimental Results

[0085] 2.1. Effects of Epiphyllum extract on epigenetic indicators and cytokine levels

[0086] Results of the effects of Epiphyllum oxypetalum extract on epigenetic markers and cytokine levels in immunocompromised mice (reference) Figure 2-5 . Figure 2 , 3 It is a graph showing the trends in weight and diet of laboratory mice. Figure 4 In the table, A, B, C, and D represent the inter-group differences in the thymus index, spleen index, kidney index, and liver index of experimental mice, respectively. Figure 5 In the figure, A, B, C, D, and E represent the expression levels of serum TNF-α, serum IgA, spleen IgG, spleen IgM, and liver C3 in experimental mice, respectively. Figure 6 These are H&E stained tissue sections of the thymus (100×) and spleen (40×) of experimental mice. CO represents the cortex, M represents the medulla, yellow arrows indicate the thymus, and red arrows indicate necrotic cells; WP represents the white spot, RP represents the erythema, CA represents the central artery, and T represents the splenic trabeculae. Data are expressed as mean ± SEM, n=5. Compared with the MDC group, * P <0.05,** P <0.01, *** P <0.001.

[0087] After two weeks of polysaccharide intervention, compared with the MDC group, immunosuppressed mice receiving treatment with Epiphyllum extract and Lentinus edodes polysaccharide did not show significant improvements in body weight, diet, spleen index, kidney index, and liver index (reference). Figure 2 , 3 , 4) P >0.05), but all the above indicators showed a certain trend of improvement, and the improvement trend was similar to that of the CON group. According to Figure 4 (Column chart A) Figure 5 (Bar charts A, B, C, and D) show that mice treated with Epiphyllum extract showed significantly altered thymus indices and significantly elevated serum TNF-α, serum IgA, and spleen IgG and IgM levels compared to the MDC group. Based on... Figure 5 (Bar chart E) shows that the epiphyllum extract exhibits a strong activity in promoting C3 synthesis in the liver. It is preliminarily speculated that this is related to the monosaccharide components of AG-I itself, which are rich in galactose and arabinose.

[0088] H&E staining was used to observe and assess the histopathological condition of the thymus and spleen. Figure 6 In the MDC group mice, there was no clear boundary between the thymic cortex and medulla, fewer thymic bodies, and more necrotic cells. The spleen of the MDC group mice was lighter in color, with an indistinct boundary between the red and white medulla, indicating a significant reduction in lymphocytes. Splenic trabeculae were visible, but the central artery was difficult to see. This demonstrates the toxic damaging effect of CTX on the thymus and spleen. Compared with the MDC group, all three polysaccharide intervention groups showed improvement in the above indicators, with the high-dose Epiphyllum extract showing a slightly better improvement than the low-dose Epiphyllum extract.

[0089] In conclusion, Epiphyllum extract has the potential to alleviate CTX-induced immune damage. Therefore, Epiphyllum extract can be used in the preparation of drugs that alleviate CTX-induced immune damage.

[0090] 2.2. Epiphyllum extract intervenes in repairing the damaged intestinal barrier

[0091] The gut is an important immune organ, possessing mechanical, chemical, immune, and biological barriers to defend against pathogens and maintain homeostasis. When the body's immune system is impaired, intestinal mucosal immunity, a crucial component of immune defense, is also adversely affected. The integrity of the intestinal barrier function is essential for maintaining healthy homeostasis. MUC2, secreted by goblet cells, plays a central role in constructing the intestinal mucosal chemical barrier, lubricating the intestine and antagonizing pathogens. (The results of the study on the effects of Epiphyllum oxypetalum extract on the intestinal barrier in immunosuppressed mice are referenced.) Figure 7-9 . Figure 7 The images show the results of PAS (40×) and H&E (200×) staining of colon tissue sections from experimental mice. Figure 8 , 9 This is a statistical chart showing the impact of immunosuppressed mice on the intestinal barrier. Figure 8 In the middle, A and C are statistical graphs showing the number of colonic goblet cells and crypt length in each group of experimental mice, respectively; B, D, and E are statistical graphs showing the content of colonic barrier-related proteins MUC2, tricellulin, and occludin in each group of experimental mice, respectively. Figure 9 Figures A, B, C, D, and E show the statistical levels of colonic immune barrier cytokines sIgA, IFN-γ, IL-10, TGF-β3, and IL-17 in each group of experimental mice, respectively. Data are expressed as mean ± SEM, n=5. Compared with the MDC group, * P <0.05,** P<0.01. In colon tissue sections, purple granules represent stained goblet cells, CR indicates crypts, MU indicates the muscular layer, and yellow circles indicate lymphocyte infiltration.

[0092] refer to Figure 7 , Figure 8 (Columnar sections A and B) After intervention with Epiphyllum oxypetalum extract, especially at high doses, the number of stainable goblet cells and the expression level of MUC2 in the colon of immunocompromised mice were significantly increased. Intestinal crypt depth can represent the integrity of intestinal morphology; both Epiphyllum oxypetalum extract and lentinan significantly increased the length of colonic crypts compared to the MDC group. (Reference) Figure 7 , Figure 8 (Bar chart C). Furthermore, the intestinal mucosa of mice in the MDC group showed loose connections with muscle, and lymphocyte infiltration was observed. All three polysaccharide intervention groups showed improvement in the aforementioned damage; the EPL group showed slightly weaker changes but was still superior to the MDC group. (Reference) Figure 7 .

[0093] CTX induced disruption of the mucosal mechanical barrier in mice, leading to increased intestinal permeability. Compared to the MDC group, lentinan and epiphyllum extract significantly increased the expression level of trichollulin in the intestine of immunosuppressed mice. Figure 8 (Bar chart D), but it did not significantly increase the expression level of occludin. (See reference...) Figure 8 (Bar chart E). SIgA plays a crucial role in intestinal barrier protection by shaping the resident microbiome to limit the growth of bacterial pathogens and by enhancing the host's protective immunity to induce immune rejection. Results showed that, compared to the CON group, the MDC group mice exhibited significantly lower sIgA expression levels in the colon. Figure 9 (Bar chart A) High doses of Epiphyllum oxypetalum extract can increase the expression level of sIgA in the intestine of immunocompromised mice. IFN-γ, IL-10, TGF-β3, and IL-17 are cytokines secreted by T helper cells Th1, Th2, Treg, and Th17, respectively. Compared with the CON group, the expression levels of IFN-γ, IL-10, TGF-β3, and IL-17 in the colon of MDC mice were significantly reduced, indicating that the intestinal immune barrier of immunosuppressed mice was disrupted. Epiphyllum oxypetalum extract intervention can reverse this immune damage and show dose-dependent improvement. (Reference) Figure 9 (Bar charts B, C, D, E, P <0.05).

[0094] The above experiments indicate that Epiphyllum extract can repair the damage to the intestinal chemical, mechanical, and immune barriers caused by CTX. This includes increasing the number of stainable goblet cells in the colon, improving intestinal morphology and crypt depth, promoting the expression levels of MUC2 and trichollulin, and promoting the secretion of sIgA and T helper cell-related cytokines.

[0095] 2.3. Epiphyllum extract alters gut microbiota structure and diversity.

[0096] To investigate the effects of Epiphyllum oxypetalum extract on gut microbiota, 16S rRNA sequencing analysis was performed on fecal samples from each group of mice. Results are referenced below. Figure 10-14 . Figure 10 These are the dilution curves of mouse fecal samples from each group, where A is the Shannon dilution curve and B is the Sobs dilution curve. Figure 11 The figure shows the number of genus-level species – UpSet Venn analysis results. Figure 12 , 13 This is a graph analyzing the effects of Epiphyllum oxypetalum extract on the structure and diversity of the gut microbiota in immunosuppressed mice. Figure 12 A, B, C, and D represent the PCA levels of OTU at the MDC vs CON, MDC vs LNP, MDC vs EPL, and MDC vs EPH levels, respectively. E is the partial least squares discriminant analysis (PLS-DA) results for each group. Figure 13 In the table, A, B, C, D, and E represent the number of OTUs, Chao1 index, Ace index, Simpson index, and Shannon index in the alpha diversity analysis at the OTU level. N=5, * P <0.05,** P <0.01, compared to the MDC group. In PCA analysis, different colors represent different groups, and within the same group, individuals are depicted in a pie chart based on 95% confidence intervals. Figure 14 The figure shows the effect of Epiphyllum oxypetalum extract on the gut microbiota of immunosuppressed mice. A, B, and C are the unidirectional correlation networks of the top 30 abundant genera of gut microbiota in the MDC, EPL, and EPH groups, respectively. The purple solid line and the green dashed line represent positive and negative correlations, respectively, and the width reflects the strength of the correlation.

[0097] refer to Figure 10 Dilution curve analysis showed that the curves for all samples were flat, indicating that the sequencing data had reached saturation and effectively captured most species in the gut microbiota. PCA analysis revealed significant differences in the gut microbiota structure between the MDC and CON groups of mice. Figure 12 (A). After intervention with Epiphyllum oxypetalum extract, the gut microbiota of mice in the EPL and EPH groups was significantly altered compared to the MDC group. (Refer to...) Figure 12 (C, D) Furthermore, the ability of Epiphyllum extract to alter the gut microbiota structure showed a certain dose-dependent effect, while the effect of Lentinan was not significant. (Refer to...) Figure 12 (B). The gut microbiota structure of mice in each group was classified using PLS-DA (partial least squares discriminant analysis), referencing... Figure 12 (E) The results showed that the gut microbiota of mice in the three polysaccharide intervention groups clustered together, while the gut microbiota of mice in the CON and MDC groups each clustered into one group.

[0098] Analysis of OTU diversity showed that the number of OTUs, chao1 index, and ace index in the gut microbiota of mice in the MDC group were significantly higher than those in other groups. Figure 13 (A, B, C) indicates that the gut microbiota richness of mice in the MDC group was significantly higher than that in other groups. Furthermore, compared to the MDC group, the Simpson index was increased and the Shannon index was decreased in the EPH and CON groups. Figure 13 (D, E) indicate a significant decrease in gut microbiota diversity. At the genus level, the species diversity was similar to that at the OTU level; the genus-level species diversity in the gut of mice in all three polysaccharide intervention groups was lower than that in the MDC group. (Refer to...) Figure 11 Furthermore, intergeneric interactions were visualized using a one-way correlation network. The results showed that the richness of the correlation network (especially positive correlations) among the top 30 genera in the EPL and EPH groups was higher than that in the MDC group. Figure 14 And Table 1 below.

[0099] Table 1

[0100]

[0101] The above results indicate that Epiphyllum extract can affect the diversity and richness of the gut microbiota in immunosuppressed mice. Furthermore, Epiphyllum extract can influence the structure of the gut microbiota and affect the interactions among gut microbes.

[0102] 2.4. Effects of Epiphyllum oxypetalum extract treatment on gut microbiota in immunocompromised mice

[0103] The dominant phyla in fecal samples from different groups of mice were analyzed, and the results were referenced. Figure 15 , Figure 15 In the diagram, A is a stacked bar chart of major species at the phylum level, and B is a stacked bar chart of major species at the genus level. The results show that the dominant phylum composition is similar across groups, with Firmicutes (…) Firmicutes ) and Bacteroidetes ( Bacteroidota The main phylum is Campylobacteria, followed by Campylobacteria (…). Campilobacterota ) and Proteobacteria ( Proteobacteria ).

[0104] In addition, we analyzed the composition of the top 30 genera of the gut microbiota, referring to... Figure 15 (B). The results showed that polysaccharide intervention caused changes in the abundance of certain genera among the groups, especially... Lactobacillus Specifically, Lactobacillus The abundance of [a substance] was significantly restored in the EPH group.

[0105] In addition, some genera with relatively low abundance (genera outside the top 30) showed significant differences between groups, according to reference... Figure 16 , 17 These are species whose abundance varies among different groups at the genus level. Figure 16 In Chinese: A is Lactobacillus Defiuviitaleaceae_UCG-011, Tyzzerella, Rikenella, unclassified_f_ Anaerovoracaceae A statistical graph of abundance between different groups; B is... Family_XIII_AD3011_group, norank_f_ norank_o_Rhodospirillales, Anaeroplasma, Escherichia-Shigella, Parabacteroides Statistical graph of abundance among different groups. Figure 17 In Chinese: C is Anaerovorax, Caldicoprobacter, Candidatus_ Arthromitus, Prevotellaceae_NK3B31_group, Unclassified_f_Lachnospiraceae A statistical graph of abundance between different groups; D is... Butyricimonas / UCG-009, norank_f_UCG-010, Marvinbryantia, unclassified_c_Clostridia Statistical plot of abundance among different groups. Data are expressed as mean ± SEM, n=5. * P <0.05,** P <0.01, compared to the MDC group. Previous studies have shown that these genera are associated with intestinal immunity. Intervention with Epiphyllum extract affected the abundance of these genera in the gut to some extent. Previous studies have disclosed that *Taizei* genus ( Tyzzerella Butyrate is abundant in the gut microbiota of Crohn's disease patients and is associated with intestinal inflammation; supplementation with intestinal butyrate can lead to the presence of *Rieken* spp. Rikenella The increased abundance of butyrate suggests that butyrate may be deficient in the gut of immunodeficient mouse models under CTX toxicity. Family_XIII_AD3011_group and Escherichia coli-Shigella ( Escherichia-Shigella ) is negatively correlated with the concentration of acetic acid in the intestinal lumen; anaerobic plasmids ( Anaeroplasma The severity of experimental autoimmune encephalomyelitis was positively correlated with the severity of the disease. Escherichia-Shigella It may be an important indicator of intestinal sepsis, while segmented filamentous bacteria ( Candidatus Arthromitus ) is a recently discovered symbiotic bacterium associated with the maturation of intestinal immune function.

[0106] Furthermore, after intervention with Epiphyllum extract, Sludge Bacteria Family_ UCG-01 ( Defluviitaleaceae_UCG-011 Unclassified anaerobic phagocytic bacilli ( unclassified_f_Anaerovoracaceae ) 、 Corynebacterium spp. Caldicoprobacter ), Anaerobic phagocytosis ( Anaerovorax Prevotaceae _NK3B31_group ( Prevotellaceae_NK3B31_group ), Butyric acid bacteria ( Butyricimonas ), norank_f_UCG-010 and unclassified Trichophyceae ( unclassified_f_Lachnospiraceae The abundance of low-prevalence bacterial genera such as ( ) has changed. These bacteria may serve as potential biomarkers for improving immunosuppressive status.

[0107] During the experiment, LEfse measurements were also used to determine the differentially expressed groups between the polysaccharide intervention group and the MDC group. The LEfse analysis results of the mouse gut microbiota are shown below. Figure 18-20 . Figure 18 In the figure, A and B represent the results of MDC vs CON and MDC vs LNP in the LEfse analysis of mouse gut microbiota, respectively. Figure 19 In the figure, C and D represent the results of LEA analysis of mouse gut microbiota, specifically MDC vs EPL and MDC vs EPH, respectively. The LDA cutoff value is 3.0, and E represents the abundance and proportion of fungi and Bacteroides in each group. Data are expressed as mean values, n=5.

[0108] Significant differences were found during the comparison. Compared with the two groups treated with Epiphyllum oxypetalum extract, the reference group... Figure 19 In the MDC group Caldicoprobacter Unclassified family_Unclassified order_Rhodospirales ( norank_f_norank_o_ Rhodospirillales ) Anaerovorax Enterococcus spp. Enterococcus ) Tyzzerella , Butyricimonas As the abundance of fungal species increases, the Erysipelothrix family ( Erysipelotrichaceae )and Lactobacillus The abundance of the bacterial species was significantly reduced, while treatment with Epiphyllum extract restored the levels of these bacterial species in the intestines of immunosuppressed mice to levels similar to those in normal mice. (Reference) Figure 18 Related research suggests that fermentation using carbohydrates as raw materials and lactic acid as the main product... Lactobacillus It can create an acidic gut microenvironment, which is unfavorable for the survival of pathogens. Studies have also shown that... Lactobacillus It can promote the production of IgA, IL-6, IL-10, IFN-γ and tumor necrosis factor by intestinal Pierre cells, thereby regulating intestinal immunity.

[0109] In addition, with Lactobacillus Belonging to the same school Erysipelotrichaceae It can produce adjuvant-like effects in the gut, enhancing Th17 cell responses and modulating immunity. Notably, previous studies have shown that dietary fermentable fiber enhances the gut microbiota's metabolism of fiber, leading to altered ratios of sterolactobacilli to bacteroidetes and increased circulating short-chain fatty acid (SCFA) concentrations. However, our results did not show this effect, as there were no significant differences in bacterial abundance or the ratio of bacteria to bacteroidetes observed between groups. Figure 20 , Figure 15 (A), Figure 20This represents the abundance and proportion of fungi and Bacteroides in each group. Data are expressed as averages, n=5.

[0110] The above results indicate that treatment with Epiphyllum oxypetalum extract affected the gut microbiota of immunodeficient mice, particularly contributing to the restoration of gut microbiota. Lactobacillus The abundance of.

[0111] 2.5. Correlation between bacterial genera and immune parameters, and changes in gut microbiota function in different groups.

[0112] Reference results of the correlation study between bacterial genera and immune parameters, and changes in gut microbiota function in each group. Figure 21 , 22 . Figure 21 This is a correlation diagram between bacterial genera and metabolic parameters. Figure 22 A is the KEGG Pathway Level 3 functional abundance prediction map, B is the KEGG Module functional abundance prediction map, C is the Metacyc functional abundance prediction map, D is the total lactic acid fermentation abundance map, and E is the KEGG Pathway Level 3 fatty acid metabolism functional abundance prediction map. Data are presented as mean values, n=5. Compared with the MDC group, * P <0.05,** P <0.01, *** P <0.001.

[0113] Correlation analysis of the first 30 bacterial genera with metabolic parameters showed that... Lactobacillus It is positively correlated with most immune parameters, which indicates that Lactobacillus It may have played an important mediating role in the recovery of immune function after polysaccharide intervention. Furthermore, *Pseudomonas* spp. ( Parabacteroides It is negatively correlated with some immune parameters, see reference. Figure 21 .

[0114] To further elucidate the correlation between functional changes in the gut microbiota and improved gut immunity in each group, we used Picrust2 to predict the functional composition of the microbial community based on amplicon sequencing results. Notably, the predicted ko02060: compared to the MDC group, the phosphotransferase system (PTS) in the KEGG pathway level 3 abundance statistics was significantly enriched in all three polysaccharide intervention groups. P <0.05), but no enrichment was found in the CON group ( P >0.05), reference Figure 22 (A). This difference may be due to polysaccharide intervention; monosaccharides such as glucose and fructose enter the cell via PTS transport and phosphorylation during lactic acid fermentation. Furthermore, the abundance of the KEGG module in M00632 (Leloir pathway) was significantly enriched in the EPL group. P<0.05, showing a certain upward trend in the EPH and LNP groups ( P >0.05), this is the metabolic pathway of lactic acid production from the degradation of galactose, see reference. Figure 22 (B). In the abundance statistics of the Metacyc database, compared with the MDC group, ANAEROFRUCAT-PWY (homogeneous fermentation) and P122-PWY (heterogeneous fermentation) showed a significant increase after intervention with Epiphyllum extract. P <0.05), although this increase is not synchronized with the dose, reference Figure 22 (C). However, after synthesizing the predicted values ​​of the total abundance of the lactic acid fermentation pathway in each group, the results showed that the abundance of lactic acid fermentation in the MDC group was significantly lower than that in other groups. Figure 22 (D). It is well known that gut microbiota-derived fatty acid fatty acids (SCFAs) play a crucial role in maintaining gut immune homeostasis. In our study, pathways related to gut microbial fatty acid metabolism did not show significant differences between groups. (Refer to...) Figure 22 (E). This finding is consistent with previous research results (see reference). Figure 20 Observed in ) Firmicutes and Bacteroidota The abundance did not change significantly, consistent with the previous findings.

[0115] The reduced lactic acid fermentation capacity of the gut microbiota may be a functional manifestation of the gut microbiota in CTX-induced immunodeficient mice. After intervention with lentinan and epiphyllum extract, the gut microbiota's ability to ferment lactic acid was similar to that of the CON group mice. Besides its inhibitory effect on the growth of harmful intestinal bacteria, lactic acid also has broad immunomodulatory effects. It can regulate the major functions of several key roles in the immune system, such as macrophages and dendritic cells. Furthermore, previous studies have found that lactic acid can activate GPR31 receptors, enhance dendritic processes in small intestinal CX3CR1 cells, and promote tubular antigen uptake. Therefore, combined with the LEfse analysis results (…),… Figure 18-20 Epiphyllum extract-induced immunosuppressed mouse intestinal Lactobacillus Increased abundance and lactate content may be involved in the immune recovery process in immunosuppressed mice.

[0116] 2.6. Lactic acid promotes the expression of proteins related to intestinal epithelial repair at the base of the colonic crypts.

[0117] The body's intestinal epithelial tissue is highly sensitive to chemotherapy drugs, and intestinal damage is a major injury during chemotherapy. Lactobacillus Lactic acid is generally considered to help protect the intestinal mucosa, and lactic acid, as... LactobacillusThe final products of fermentation play a crucial role in repairing the intestinal barrier. Previous studies have shown that lactic acid extracted from the gut microbiota can enhance the proliferation of colonic epithelial cells and maintain their normal morphology and function. Furthermore, lactic acid from the microbiota stimulates the proliferation of intestinal stem cells through the Wnt / β-catenin signaling pathway via Peyer's cells and intestinal stromal cells, thereby repairing intestinal damage caused by chemotherapy and radiotherapy and maintaining the intestinal physical barrier. To verify the repair effect of intestinal lactic acid on intestinal damage in CTX-induced immunosuppressed mice, this study used immunohistochemistry to visually confirm the expression sites and levels of intestinal barrier repair-related factors in the colon of each group of mice. Figure 23 , 24 , Figure 23 This is an immunohistochemical staining image (200×) of Gpr81, Wnt3a and β-catenin in colon tissue. Figure 24 This is a graph showing the research results on how epiphyllum extract promotes the repair of intestinal epithelium at the bottom of the colonic crypts. A, B, and C are the relative expression results of Gpr81, Wnt3a, and β-catenin, respectively, expressed as average optical density (AOD) values. Figure 25 Figures A and B are bar charts showing the lactic acid content in feces and colon for each group. Data are expressed as mean ± SEM, n=5. Compared with the MDC group, * P <0.05,** P <0.01.

[0118] First, the lactic acid content in mouse feces and colon was measured, referencing... Figure 25 (A, B) The results were consistent with the predictions of Picrust2. The lactic acid content in the intestines and colons of mice in the three polysaccharide intervention groups was significantly higher than that in the MDC group. The lactic acid content in the feces of mice in the EPH group was 3.4 times that of the MDC group. Lactic acid extracted from the microbiome helps repair intestinal damage caused by chemotherapy drugs, mainly through a Gpr81-dependent mechanism, activating the expression of proteins such as wnt3a and β-catenin at the bottom of the colonic crypts, and promoting intestinal epithelial development.

[0119] Quantitative results from ImageJ software showed that the expression of Gpr81, Wnt3a, and β-catenin in the EPH group was mainly concentrated at the base of the colonic crypts, and the expression levels were significantly higher than those in the MDC group. Figure 24 (Bar charts A, B, and C). This helps maintain the stemness of Lgr5+ intestinal stem cells and repairs damaged intestinal physical barriers. The above evidence suggests that lactic acid from the gut may play a key role in the restoration of the intestinal structural physical barrier during the process promoted by Epiphyllum extract.

[0120] III. Summary

[0121] As mentioned above, immune activation plays a crucial role in the various pharmacological effects of polysaccharides from traditional Chinese medicine. In our study, CTX caused damage to the immune organs thymus, spleen, and intestines. However, histopathological results showed that Epiphyllum extract could repair the structural damage to these immune organs and increase the number of stainable colonic epithelial cells. In particular, the thymus index of mice significantly recovered after Epiphyllum extract intervention. TNF-α is a multipotent pro-inflammatory cytokine involved in activating innate and adaptive immunity. C3 is mainly synthesized by the liver and macrophages and participates in various adaptive immune responses. The monosaccharide composition of Epiphyllum extract is mainly composed of galactose and arabinose, accounting for more than 60% of the monomer content. This type of polysaccharide, called arabinogalactose, has good activity in the complement system. Most lentinan does not possess this characteristic. The results showed that Epiphyllum extract could significantly increase serum TNF-α levels and liver C3 expression. IgA, IgG, and IgM can reflect the humoral immune function of the animal body, and the results showed that Epiphyllum extract could stimulate immunosuppressed mice to secrete these antibodies.

[0122] Epiphyllum extract improved the intestinal chemical and immune barriers in immunosuppressed mice by increasing the secretion of MUC2 by intestinal gland cells, promoting trichollulin expression, and restoring the level of sIgA, an important component of the immune barrier. IFN-γ, IL-10, TGF-β3, and IL-17 are cytokines secreted by T helper cells Th1, Th2, Treg, and Th17, respectively, which help balance the body's immune response. In immunodeficiency, the ability of helper T cells to proliferate, spread, and activate other immune cells responsible for direct immune responses is weakened. The results showed that Epiphyllum extract can effectively stimulate the release of immune factors and enhance the intestinal immune barrier. Furthermore, compared with the reference dose of hyacinth bean polysaccharide, Epiphyllum extract showed better recovery effects on certain indicators. These results indicate that Epiphyllum extract has the activity of repairing the intestinal barrier in immunosuppressed mice.

[0123] The immune system and gut microbiota have extensive interactions. Decreased immunity often leads to gut microbiota dysbiosis. This study also observed a similar phenomenon after modeling with CTX. However, Epiphyllum oxypetalum extract improved the composition of the gut microbiota. After intervention with Epiphyllum oxypetalum extract, the α- and β-diversity of the mouse gut microbiota improved, similar to the normal group but different from the model group. The α-diversity of the mouse gut microbiota after Epiphyllum oxypetalum extract intervention showed significant intra-group differences, but a slight improvement was observed in the group receiving lentinan intervention. We believe that the difference in the interaction between the gut microbiota of different mice and high molecular weight Epiphyllum oxypetalum extract is the reason for this difference. A diverse network of microbial members is a characteristic of a healthy gut microbiota. At the family level, the effect of CTX leads to... Erysipelotrichaceae and Lactobacillaceae The abundance of [certain substances] decreased. At the genus level, the effect of CTX led to [a decrease in] abundance in mice. Lactobacillus The number has decreased. It has been reported that... Erysipelotrichaceae and Lactobacillus Intervention with Epiphyllum extract modulates immunity in specific ways, leading to an increase in the abundance of both bacteria. Furthermore, Epiphyllum extract also reduces certain genera, such as Butymonas, which is negatively correlated with intestinal immune factors and antimicrobial peptides. Butyricimonas It is negatively correlated with IFN-γ, IL-4 and IgG. Tyzzerella , Family_XIII It is negatively correlated with 5-hydroxytryptamine. Family_XIII_ AD3011_group and related to impaired epithelial barrier integrity Enterococcus These results indicate that epiphyllum extract helps restore the gut microbiota and enhance the intestinal biological barrier in immunosuppressed mice.

[0124] Further prediction of intestinal function showed that the abundance of both homolactic and heterolactic fermentation pathways was reduced in CTX-induced immunosuppressed mice. Total lactic acid fermentation abundance statistics showed that the lactic acid fermentation abundance in the model group mice was significantly lower than that in mice treated with lentinan and epiphyllum extract. This finding is consistent with that in the MDC group. Lactobacillus Consistent with the significant decrease in abundance, low doses of Epiphyllum extract significantly increased the abundance of the Leloir pathway in the mouse gut, while this phenomenon was not observed in Lentinan or high doses of Epiphyllum extract. This may be because the appropriate amount of galactose in Epiphyllum extract is more conducive to the fermentation of lactic acid by the gut microbiota through the Leloir pathway.

[0125] Mammalian digestive enzymes cannot directly digest most complex carbohydrates and plant polysaccharides; these substances are metabolized by microorganisms that produce SCFAs. SCFAs possess broad immunomodulatory activity, but in our study, no significant changes were observed in the predicted abundance of SCFA production pathways or key or related enzymes. Figure 22 (E) and Table 2.

[0126] Table 2

[0127]

[0128] Furthermore, lactate acts differently from butyrate, one of the SCFAs (differentiated colon cells metabolize butyrate, which may prevent butyrate from reaching stem cells in the crypts, while butyrate is an effective inhibitor of intestinal stem cell / progenitor cell proliferation at physiological concentrations).

[0129] According to specific microorganisms ( LactobacillusBased on the increase in lactate levels and the predicted function of the 16S gene, it is speculated that Epiphyllum oxypetalum extract may increase lactate levels in the gut. Altering lactate abundance may be one of the effective mechanisms by which Epiphyllum oxypetalum extract improves intestinal barrier function by regulating the gut microbiota. Therefore, the lactate content in fecal matter and tissues was further measured, and experimental verification confirmed that Epiphyllum oxypetalum extract produced this effect. Lactic acid derived from the gut microbiota not only enhances the intestinal immune barrier but also activates the expression of proteins such as Wnt3a and β-catenin at the base of the colonic crypts through a Gpr81-dependent mechanism, promoting intestinal epithelial development to repair intestinal damage. This is important for various functions, including early embryonic development, hematopoietic stem cell self-renewal, and maintaining intestinal tissue stability.

[0130] Experimental results showed that, compared with the model group, intervention with Epiphyllum oxypetalum extract significantly restored lactic acid levels in mouse feces and colon. Furthermore, the expression of key proteins activated by lactate receptor-dependent mechanisms, such as Wnt3a and β-catenin, was significantly increased at the base of the colonic crypts. This plays a crucial role in maintaining the stemness of Lgr5+ intestinal stem cells. Therefore, Epiphyllum oxypetalum extract can enhance CTX-induced intestinal immune responses in mice by regulating the gut microbiome.

[0131] Previous studies have shown that lentinan can significantly alter the composition of the gut microbiota and markedly affect the abundance of certain microorganisms, including... Lactobacillus genus and Bifidobacterium ( Bifidobacterium It produces lactic acid bacteria and promotes an increase in the lactic acid content of intestinal feces. In this study, the epiphyllum extract showed similar activity to lentinan, but in some other immune indicators, such as C3, MUC2, and colonic T helper cell cytokines, the epiphyllum extract showed better improvement than the reference dose of lentinan. Dosage difference may be one reason, or it may be related to the fact that macromolecular polysaccharides increase the volume and viscosity of intestinal contents, stimulate intestinal peristalsis, thereby affecting the activity and distribution of immune cells in the intestine, and thus affecting the function of the intestinal immune system. In the future, macromolecular polysaccharides, represented by epiphyllum extract, may also be used for other properties besides immune function, such as thickeners, drug carriers, humectants, gelling agents, and adhesives. Therefore, further research and development of epiphyllum extract will help realize its broader application prospects.

[0132] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications or equivalent changes made to the above embodiments based on the technical essence of the present invention shall fall within the protection scope of the present invention.

Claims

1. The use of an Epiphyllum oxypetalum extract in the preparation of a medicament for treating or ameliorating CTX-induced intestinal mucosal injury in mice, wherein the Epiphyllum oxypetalum extract is obtained by crushing dried Epiphyllum oxypetalum petals into powder, and then extracting polysaccharides from the powder using a water-alcohol precipitation method, and the average molecular weight of the polysaccharides is 5.767 x 10 6 Da, the monosaccharide composition of the Epiphyllum oxypetalum extract includes arabinose, galactose, glucose, xylose, rhamnose, fructose, and galacturonic acid, and the molar ratio of the monosaccharide composition is 11.480:53.791:0.494:0.446:26.648:1.102:6.040, respectively, wherein the total molar ratio of arabinose and galactose is more than 60%, The main chain structure of the Epiphyllum extract is type I arabinogalactan of β-Galp connected by (1→4) bond as shown in structural formula (1), （1）， wherein R is or ; m and n are both integers greater than 0.

2. The use of an extract of Epiphyllum oxypetalum in the preparation of a health food for regulating intestinal flora, wherein the extract of Epiphyllum oxypetalum is obtained by extracting polysaccharides from a powder obtained by drying and pulverizing petals of Epiphyllum oxypetalum, using a water-alcohol precipitation method, and has a purity of 95% and an average molecular weight of 5.767 x 10 6 Da, and the monosaccharide composition of the extract of Epiphyllum oxypetalum comprises arabinose, galactose, glucose, xylose, rhamnose, fructose, and galacturonic acid, and the molar ratio of the monosaccharide composition is 11.480:53.791:0.494:0.446:26.648:1.102:6.040, respectively, wherein the total molar ratio of arabinose and galactose is more than 60%. The main chain structure of the Epiphyllum extract is type I arabinogalactan of β-Galp connected by (1→4) bond as shown in structural formula (1), （1）， wherein R is or ; m and n are both integers greater than 0.

Images