CN118845821A - Application of Epiphyllum extract in preparing health food or medicine for treating/improving intestinal diseases - Google Patents

Abstract

The invention discloses application of a epiphyllum extract in preparing health-care food or medicine for treating/improving intestinal diseases, and relates to the technical field of phytochemistry and microorganisms, wherein the epiphyllum extract is type I arabinogalactan, the epiphyllum extract is polysaccharide with the average molecular weight of 5.767 multiplied by 10 ⁶ Da extracted from epiphyllum petals, and the main chain structure is beta-Galp connected through (1-4) bonds, so that the application potential of epiphyllum is expanded, the value of epiphyllum is improved, and a novel raw material is provided for the field of health-care food or medicine for treating/improving intestinal diseases.

Description

Application of Epiphyllum extract in the preparation of health food or medicine for the treatment/improvement of intestinal diseases

Technical Field

The invention relates to the field of plant chemistry and microbial technology, and in particular to application of an epiphyllum cymbidium extract in preparing health-care food or medicine for treating/improving intestinal diseases.

Background Art

Epiphyllum oxypetalum ( DC. ) Haw (EOH) belongs to the Cactaceae family and is known as the "Queen of the Night" for its unique night-blooming characteristics. This perennial succulent plant has irreplaceable ornamental value in the horticultural industry due to its rare flowers and short flowering period. In modern applications, epiphyllum extract is widely used in mass cosmetics and has moisturizing, whitening and antioxidant functions. For example, the patent application document with publication number "CN117427008A" published on January 23, 2024 proposes the use of epiphyllum extract in skin oil control, hair loss prevention and/or acne treatment.

Epiphyllum is rich in a variety of beneficial ingredients, including protein (14 mg/g), fatty acids (4.6 mg/g) and vitamins (0.18 mg/g) disclosed in existing literature. Compared with these nutrients, mucus polysaccharides are one of the most abundant carbohydrates in cactus plants, accounting for more than 18% of the weight of flowers and stems. These polysaccharides usually have high molecular weight and branched structures, and this is also the case with Epiphyllum. Natural polysaccharides are a class of bioactive macromolecules with powerful functions and have various health benefits, such as immunomodulatory, anti-tumor, anti-diabetic and hepatoprotective activities. The biological functions of different polysaccharides are closely related to their structural changes, 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 response.

In the prior art, a document entitled "A polysaccharide from Epiphyllum oxypetalum (DC.) Haw. and its immunomodulatory activity" published in the International Journal of Biological Macromolecules discloses the isolation and identification of a polysaccharide (EOP) from Epiphyllum oxypetalum, and the evaluation of its in vitro and in vivo immunomodulatory activity. Through multispectral analysis, it was determined that EOP is composed of rhamnose, arabinose, galactose and galacturonic acid, with a molar ratio of 26.65:11.48:53.79:6.04 and a molecular weight of 5.77×10 ⁶ Da. In addition, the main chain structure of EOP is composed 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, and terminated with t-β-Arap and t-β-Galp. In vitro immunomodulatory activity experiments on RAW264.7 cells showed that EOP can promote the proliferation of macrophages, enhance their phagocytic ability, 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 of zebrafish showed that EOP can reduce the residual content of fluorescent microspheres in zebrafish, indicating that EOP has the ability to enhance macrophage phagocytosis. It is mentioned that EOP has a complex structure and has significant immunomodulatory activity both in vitro and in vivo, and has potential application value in the food and pharmaceutical industries.

Summary of the invention

With the breakthrough in the cultivation technology of Epiphyllum, the raw materials of Epiphyllum have become easier to obtain. The inventors of the present invention extracted Epiphyllum by adopting a specific method to obtain Epiphyllum extract, and conducted in-depth research on the composition and biological activity of Epiphyllum extract. It was found that Epiphyllum extract has beneficial effects on immune regulation and treatment/improvement of intestinal diseases, expanding the application field of Epiphyllum.

The purpose of the present invention is to provide an application of an epiphyllum extract in the preparation of health foods or medicines for treating/improving intestinal diseases, so as to expand the application potential of epiphyllum and provide new raw materials for the field of health foods and medicines for treating/improving intestinal diseases.

The present invention is achieved through the following technical solutions:

Use of an epiphyllum extract in the preparation of a health food or medicine for treating/improving intestinal diseases, wherein the epiphyllum extract is type I arabinogalactan represented by structural formula (1).

(1),

Among them, R is or ; m, n are both integers greater than 0.

Furthermore, the Epiphyllum cymbidium extract is a polysaccharide with an average molecular weight of 5.767×10 ⁶ Da extracted from the petals of Epiphyllum cymbidium, and its main chain structure is β-Galp connected by (1→4) bonds.

Furthermore, the polysaccharide is polymerized from arabinose, galactose, glucose, xylose, rhamnose, fructose and galacturonic acid, and the molar ratios of monosaccharide composition of arabinose and galactose are 11.480% and 53.791% respectively.

Furthermore, the epiphyllum extract is used in the preparation of health foods or medicines for regulating the diversity and richness of immunosuppressive intestinal flora, affecting the structure of intestinal flora, or affecting the relationship between intestinal microorganisms.

Furthermore, the epiphyllum extract is used in the preparation of health food or medicine for repairing the damage of intestinal chemical, mechanical or immune barriers caused by CTX.

Furthermore, the epiphyllum extract is used in the preparation of health food or medicine for restoring the abundance of Lactobacillus in the intestine.

Furthermore, the epiphyllum extract is used in the preparation of health food or medicine for promoting immune recovery, and the promotion of immune recovery is achieved by increasing the abundance of Lactobacillus and lactic acid content in the intestine.

Furthermore, the epiphyllum extract is used in the preparation of health food or medicine for promoting the physical barrier of intestinal structure.

Further research has determined that:

The structure of Epiphyllum extract is as follows:

,

Among them, R is or ; m, n are both integers greater than 0.

The molecular formula is as follows:

,

Among them, R is or ; m, n are both integers greater than 0.

The main chain structure of Epiphyllum cymbidium extract is β-Galp connected by (1→4) bonds. As shown in Figure 26, the backbone of type I arabinogalactan (AG-I) is composed of (1→4)-β-D-Galp, and the backbone of type II arabinogalactan (AG-II) is composed of (1→6) and/or (1→3)-Galp. It can be seen that Epiphyllum cymbidium extract belongs to the typical type I arabinogalactan (AG-I).

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

1. In the present invention, the Epiphyllum cereus extract belonging to arabinogalactan (AG-Ⅰ) extracted from Epiphyllum cereus has a significant effect in treating/improving intestinal diseases. It can stimulate the expression of immune factors and reduce damage to immune organs. For example, Epiphyllum cereus extract can repair the intestinal immune barrier, not only regulate the expression of key immune factors in the intestine, but also improve the diversity and overall structure of the intestinal microbiota. In addition, Epiphyllum cereus extract may also activate the expression of proteins such as Wnt3a and β-catenin at the bottom of the colonic crypts through a Gpr81-dependent mechanism, thereby promoting the repair of the intestinal physical barrier. It can be seen that Epiphyllum extract can be used in the preparation of foods and medicines for alleviating CTX-induced intestinal immune damage, regulating the diversity and richness of immunosuppressive intestinal flora, affecting the structure of intestinal flora, affecting the relationship between intestinal microorganisms, repairing CTX-induced intestinal chemical, mechanical and immune barrier damage, changing the structure and diversity of intestinal flora, restoring the abundance of Lactobacillus in the intestine, and promoting the physical barrier of intestinal structure. It can also be used in the preparation of health foods and medicines with immune-enhancing effects to reduce damage to immune organs.

2. The present invention proposes the use of an Epiphyllum cymbidium extract in the preparation of foods, health products or medicines for immunomodulation or treatment/improvement of intestinal diseases, which broadens the application field of Epiphyllum cymbidium and improves its value.

3. In the present invention, the composition of the epiphyllum extract is further analyzed, and the monosaccharide composition and the molar ratio of the monosaccharide composition of the epiphyllum extract are determined. The epiphyllum extract is polymerized by arabinose, galactose, glucose, xylose, rhamnose, fructose and galacturonic acid, and it is verified through experiments that when the epiphyllum extract is used in the preparation of health foods and drugs for treating/improving intestinal diseases, arabinose and galactose are its main active ingredients. This highly branched neutral monosaccharide domain composed of arabinose and galactose helps the polysaccharide to form a specific conformation and produce a good response in stimulating the immune system. For example, in terms of complement activation, such polysaccharides often have high complement binding activity, which helps the body's immune defense to respond quickly, and in this respect, it is different from other fungal polysaccharides, such as Lentinan. The main chain structure of the active ingredients in the epiphyllum extract is further determined, providing a theoretical basis for the application and development of epiphyllum in the fields of health foods/drugs for immune regulation and intestinal disease defense/treatment, as well as new fields such as food thickeners, drug carriers, moisturizers, gelling agents, and adhesives.

Fourth, in the present invention, the epiphyllum extract is a polysaccharide with an average molecular weight of 5.767×10 ⁶ Da, which is about 8 to 600 times that of other natural polysaccharides. High molecular weight polysaccharides can interact with the body as dietary fiber or prebiotics. Such macromolecules are difficult to digest in the small intestine, so they can enter the colon and provide substrates for the complex bacterial ecosystem in the colon, thereby regulating the intestinal microbial immune barrier and improving the imbalance of the intestinal flora and metabolic disorders of the body.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a diagram showing the steps for constructing an immunodeficient mouse model.

FIG. 2 is a graph showing the weight change trend of experimental mice.

FIG. 3 is a graph showing the dietary changes of experimental mice.

FIG4 is a statistical graph showing the effects of Epiphyllum cymbidium extract on the inter-group differences in thymus index (A), spleen index (B), kidney index (C), and liver index (D) of experimental mice.

Figure 5 is a statistical graph showing the effect of Epiphyllum cyrtonema extract on cytokine levels in experimental mice, wherein: 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.

FIG. 6 is a picture of H&E stained tissue sections of the thymus (100×) and spleen (40×) of experimental mice.

FIG. 7 shows the results of PAS staining (40×) and H&E staining (200×) of colon tissue sections of experimental mice.

FIG8 is a statistical diagram of the effects of immunosuppressive on the intestinal barrier of mice, wherein A and C are statistical diagrams of the number of colon goblet cells and crypt length of each group of experimental mice, respectively, and B, D, and E are statistical diagrams of the contents of colon barrier-related proteins MUC2, tricellulin protein, and Occludin protein of each group of experimental mice, respectively.

FIG9 is a second statistical diagram of the effects of immunosuppressive effects on the intestinal barrier of immunosuppressed mice, wherein A, B, C, D, and E are statistical diagrams of the contents of sIgA, IFN-γ, IL-10, TGF-β3, and IL-17 in the colon immune barrier cytokines of each group of experimental mice, respectively.

FIG. 10 is a graph showing the dilution curves of the fecal samples of mice in each group, wherein A is the Shannon dilution curve; and B is the Sobs dilution curve.

FIG. 11 is a graph showing the number of species at the genus level—UpSet Venn analysis results.

Figure 12 is an analysis diagram of the effect of Epiphyllum extract on intestinal microbial decomposition and diversity in immunosuppressed mice, wherein: A, B, C, D represent the MDC vs CON, MDC vs LNP, MDC vs EPL and MDC vs EPH levels of PCA at the OTU level, respectively, and E is the partial least squares discriminant analysis (PLS-DA) analysis results in each group.

Figure 13 is the second analysis diagram of the effect of Epiphyllum extract on the deconstruction and diversity of intestinal microorganisms in immunosuppressed mice, where: A, B, C, D, E are the number of OTUs, Chao1 index, Ace index, Simpson index and Shannon index in the Alpha diversity analysis at the OTU level.

Figure 14 is a graph showing the effect of Epiphyllum extract on the intestinal microbiota relationship of immunosuppressed mice, where A, B, and C are unidirectional correlation networks of the top 30 abundance genera of the intestinal microbiota in the MDC group, EPL group, and EPH group, respectively.

FIG. 15 is a diagram showing the analysis results of dominant phyla (A) and genera (B) in fecal samples of mice from different groups.

Figure 16 is a statistical graph of species with different abundances among different groups at the genus level, wherein: A is a statistical graph of the abundances among different groups of Lactobacillus, Defiuviitaleaceae_UCG-011, Tyzzerella, Rikenella, and unclassified_f_ Anaerovoracaceae ; B is a statistical graph of the abundances among different groups of Family_XIII_AD3011_group, norank_f_ norank_o_Rhodospirillales, Anaeroplasma, Escherichia-Shigella, and Parabacteroides .

Figure 17 is a second statistical graph of species with different abundance differences among different groups at the genus level, where: C is a statistical graph of the abundance among different groups of Anaerovorax, Caldicoprobacter, Candidatus_Arthromitus, Prevotellaceae_NK3B31_ group, and Unclassified_f_Lachnospiraceae ; D is a statistical graph of the abundance among different groups of Butyricimonas/ UCG-009, norank_f_UCG-010, Marvinbryantia, and unclassified_c_Clostridia .

FIG18 is a diagram of the LEfse analysis results of the mouse intestinal microbiota, wherein: A and B represent the results of MDC vs CON and MDC vs LNP in the LEfse analysis of the mouse intestinal microbiota, respectively.

FIG19 is the second diagram of the LEfse analysis results of the mouse intestinal microbiota, wherein: C and D represent the results of MDC vs EPL and MDC vs EPH in the LEfse analysis of the mouse intestinal microbiota, respectively.

Figure 20 shows the abundance and ratio of fungi and Bacteroidetes among the groups. Data are presented as mean values, n=5.

FIG. 21 is a graph showing the correlation between bacterial genus and metabolic parameters.

Figure 22 is a graph showing the correlation between bacterial genera and immune parameters, and functional changes in the intestinal microbiota of each group, where: A is the KEGG Pathway Level 3 function prediction abundance map, B is the KEGG Module function prediction abundance map, C is the Metacyc function prediction abundance map, D is the total lactic acid fermentation total abundance map, and E is the KEGG pathway Level 3 fatty acid metabolism function prediction abundance map.

FIG. 23 is an immunohistochemical staining image of Gpr81, Wnt3a and β-catenin in colon tissue (200×).

Figure 24 is a graph showing the research results related to Epiphyllum cyrtonema extract promoting the repair of intestinal epithelium at the bottom of colonic crypts, wherein: A, B, and C are the relative expression results of Gpr81, Wnt3a, and β-catenin, respectively, expressed as average optical density (AOD) values.

FIG. 25 is a bar graph showing the lactic acid content in feces (A) and colon (B) of each group.

Figure 26 is a graphic representation of the chemical structures of Type I and Type II arabinogalactans.

DETAILED DESCRIPTION

The present invention is further described in detail below in conjunction with examples, but the embodiments of the present invention are not limited thereto.

Example 1

To facilitate the public's understanding of the present scheme, in this embodiment, explanations, verifications and illustrations are provided from aspects such as the acquisition of raw materials of Epiphyllum cereus extract, the extraction method, the composition of Epiphyllum cereus extract, and the biological activity of Epiphyllum cereus extract, in combination with experiments.

1. Materials and methods involved in this embodiment

1.1. Materials and reagents

Epiphyllum was collected from the Epiphyllum planting demonstration base of Sichuan Yuanlan Agricultural Development Co., Ltd. (Zizhong, China).

The extraction process of Epiphyllum oxypetalum extract (EPS) is as follows: the petals of Epiphyllum oxypetalum are dried and crushed into powder, and then polysaccharides are extracted from the powder by water-alcohol precipitation method, with a purity of 95% and an average molecular weight of 5.767×10 ⁶ Da. The specific operation method refers to the extraction method in the reference "A polysaccharide from Epiphyllum oxypetalum (DC.) Haw. and its immunomodulatory activity". After testing, the monosaccharide composition of Epiphyllum oxypetalum extract includes arabinose, galactose, glucose, xylose, rhamnose, fructose and galacturonic acid, and the molar ratio of monosaccharide composition is 11.480:53.791:0.494:0.446:26.648:1.102:6.040, of which the total molar ratio of arabinose and galactose exceeds 60%, and the maximum solubility of Epiphyllum oxypetalum extract is about 2.75mg/ml.

CTX (C849559) was purchased from Shanghai MacLean Biochemical Technology Co., Ltd. (Shanghai, China). ELISA kits for cytokines and antibodies were purchased from Quanzhou Ruixin Biotechnology Co., Ltd. (Quanzhou, China) and Jiangsu Enzyme Immunity Industry Co., Ltd. (Yancheng, China). 4% paraformaldehyde tissue fixative (BL539A), RIPA lysis buffer (BL504A), protease inhibitor (BL612A), and BCA protein content assay kit (BL521A) were purchased from White Shark 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 Bioengineering Institute (Nanjing, China). Hematoxylin and eosin (H&E) staining kit (G1120) was purchased from Solebol Technology Co., Ltd. (Beijing, China), and periodic acid-Schiff staining (PAS) dye (G1008) was purchased from Saiwei Biotechnology Co., Ltd. (Wuhan, China). All other chemical reagents were of analytical grade.

1.2. Animals and treatment

As shown in Figure 1, 35 SPF Kunming mice, 4 weeks old, weighing 18-22 g, were purchased from Dashuo Experimental Animal Co., Ltd. [Chuan (Chuan) 2020-030] (Chengdu, China) (HE-12-54-04). The experimental schematic diagram is shown in Figure 1. The mice were raised under standard indoor conditions (20-26 ° C, 40%-70% humidity), 12 h light/12 h dark cycle, free access to standard maintenance feed and water. After 3 days of adaptive feeding at the SPF experimental animal of Chengdu Medical College, they were randomly divided into 5 groups, 7 mice in each group, namely blank control group (CON), model control group (MDC), Lentinan positive control group (LNP), Epiphyllum extract low-dose group (EPL) and Epiphyllum extract high-dose group (EPH).

CTX is a first-line chemotherapy drug for the treatment of 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, destroy the gastrointestinal mucosal barrier, and lead to an imbalance of intestinal flora. Therefore, CTX is often used to construct immunosuppressive animal models, especially mouse models with anatomical structures (including the gastrointestinal tract) and immune systems similar to those of humans. Reference "Immunomodulatory activity of a novel polysaccharide from Lonicerajaponica in immunosuppressed mice induced by cyclophosphamide", in order to establish an immunosuppressive model in mice, the CON group was intraperitoneally injected with normal saline, and the other groups were intraperitoneally injected with CTX (70 mg/kg bw•day) on days 1 to 5 of the experiment. Because mice bite each other and the drug has immunosuppressive toxicity, the risk of death is high, so it was finally determined that the number of mice successfully modeled in each group was 5. From day 6 to day 19, the CON and MDC groups were gavaged with saline, the LNP group was gavaged with Lentinan (5 mg/kg/dbw•day) (converted according to the manufacturer's recommended human dose), and the EPL group (25 mg/kg/dbw•day) and the EPH group (35 mg/kg/dbw•day) were gavaged with corresponding doses of Epiphyllum cymbidium extract. On day 19, the feces of mice in each group were collected. On day 20, the mice were anesthetized with isoflurane and killed by cervical dislocation to collect other tissues. The mice were observed daily during the experiment, and their weight and diet were recorded.

1.3. Blood and organ collection

After the mice were anesthetized with isoflurane, the eyeballs were removed and blood was collected. The blood was placed at room temperature for 20 minutes and centrifuged at 3500r/min for 20 minutes. The supernatant was taken to obtain serum and stored in a -80℃ refrigerator. The mice were then humanely killed by cervical dislocation, and the liver, thymus, spleen, kidney, colon and other organs were removed and weighed. The organ index was calculated as organ weight (mg)/body weight (g) × 10.

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

Spleen, thymus, and colon tissues were fixed with 4% paraformaldehyde tissue fixative for 48 h, dehydrated with alcohol, and made into paraffin-embedded blocks. Sections (thickness 3-5 μm) 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 5 complete 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.

1.5. PAS staining of colon

The PAS method was used to determine the number of goblet cells that could be stained in colonic tissue sections (3-5 μm thick). After the colonic tissue was stained, full-field images were taken using an optical microscope (40x magnification), and the number of goblet cells that could be stained in the field of view was counted using Image-J (version: 1.8.0.172, USA).

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

Blood samples were collected and serum was separated. Tumor necrosis factor-α (TNF-α) and immunoglobulin A (IgA) levels were measured using ELISA kits. 100 mg of spleen, liver, and colon tissues were mixed with 900 μl of phosphate buffered saline (PBS) and thoroughly homogenized, centrifuged for 20 min (8500 r/min, 4°C), and the supernatant was collected to measure the expression levels of immunoglobulins M and G (IgM, IgG), complement 3 (C3), secretory IgA (sIgA), interleukin 10 and 17 (IL-10, IL-17), interferon-γ (IFN-γ), transforming growth factor β3 (TGF-β3), tight junction-associated proteins (tricellulin, occludin), and mucin 2 (MUC2). The test was performed according to the instructions provided by the ELISA manufacturer.

1.7. Determination of lactic acid content in colon and feces

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. The samples were centrifuged for 20 min (3500 r/min, 4°C), and the supernatant was collected. The lactic acid concentration was determined by enzymatic colorimetry.

Immunohistochemistry

Colon tissue sections were treated with xylene and graded ethanol, incubated with 0.1% Tritonx-100, rinsed with PBS, and then mounted with BSA and serum. Sections were incubated with primary antibodies against GPR81, Wnt3a, and β-Ctnnb1 (β-catenin) at 4°C overnight, then incubated with secondary antibodies at room temperature, and horseradish peroxidase-labeled streptavidin protein was added and developed with DAB colorimetric solution. Five random fields of view were taken and photographed (magnification 200 times) during microscopic observation. Quantitative automatic measurement of positive area was defined by ImageJ software (NIH, USA) and expressed as average optical density (AOD) value.

1.9. 16S rRNA sequencing of intestinal flora

Total DNA from feces was extracted according to the instructions of the E.Z.N.A®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’). The PCR product was recovered on a 2% agarose gel, purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, USA), and quantified using QuantiFluorTM-ST (Promega, USA). The PE library was created using the TruSeqTM DNA Sample Preparation Kit and sequenced on the 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 sequence (CCS) reads with at least three complete passes and 99% accuracy. CCS reads were barcode identified and length filtered, and sequences <1000 bp or >1800 bp were removed. The optimized CCS reads were clustered into operational taxonomic units (OTUs) at 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, species information of each OTU was annotated using BLAST, and alpha diversity and beta diversity were analyzed based on the abundance of OTUs using R packages. Linear discriminant analysis (LDA) and LDA effect size (LEfse) were used to analyze the dominant bacterial communities between groups. 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 unidirectional correlation networks and correlation heat maps.

Data analysis

Each experiment was repeated at least three times, and statistical analysis was performed using GraphPad Prism. Data were expressed as mean ± SEM. The normality of the data was assessed using the Shapiro-Wilk test. The data of biochemical indices in each group were compared with those in the MDC group using the Student’s t test, and the statistical significance of alpha-diversity and Picrust2 measurements was determined using the nonparametric Mann-Whitney test. Differences were considered statistically significant when P < 0.05.

2. Experimental Results

2.1. Effects of Epiphyllum Extract on Epigenetic Indexes and Cytokine Levels

The results of the effects of Epiphyllum extract on the apparent indicators and cytokine levels of immunocompromised mice are shown in Figures 2-5. Figures 2 and 3 are the trends of weight changes and dietary changes in experimental mice. In Figure 4, A, B, C, and D respectively represent the inter-group differences in the thymus index, spleen index, kidney index, and liver index of experimental mice; in Figure 5, A, B, C, D, and E respectively represent the expression levels of serum TNF-α, serum IgA, spleen IgG, spleen IgM, and liver C3 in experimental mice. Figure 6 is an H&E-stained tissue section of the thymus (100×) and spleen (40×) of experimental mice. CO represents the cortex, M represents the medulla, the yellow arrow represents the thymus body, and the red arrow represents the necrotic cell; WP represents white spots, RP represents erythema, CA represents the central artery, and T represents the spleen trabeculae. Data are expressed as mean ± SEM, n = 5. Compared with the MDC group, * P < 0.05, ** P < 0.01, *** P < 0.001.

After 2 weeks of polysaccharide intervention, the weight, diet, spleen index, kidney index and liver index of immunosuppressed mice treated with Epiphyllum extract and Lentinan did not improve significantly compared with the MDC group (refer to Figures 2, 3, and 4) ( P >0.05), but 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 (bar graph A) and Figure 5 (bar graphs A, B, C, and D), it can be seen that the thymus index of mice treated with Epiphyllum extract was significantly changed compared with the MDC group, and the levels of serum TNF-α, serum IgA, spleen IgG and IgM were significantly increased. According to Figure 5 (bar graph E), it can be seen that Epiphyllum extract showed 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.

The histopathological conditions of the thymus and spleen were observed and evaluated by H&E staining (see Figure 6). There was no obvious boundary between the thymic cortex and medulla of the mice in the MDC group, with fewer thymic corpuscles and more necrotic cells. The spleen of the mice in the MDC group was lighter in color, and the boundary between the red and white pulp was not obvious, indicating that the number of lymphocytes was significantly reduced. The splenic trabeculae were visible, and the central artery was difficult to see. It can be seen that CTX has toxic and damaging effects on the thymus and spleen. Compared with the MDC group, the above indicators of mice in the three polysaccharide intervention groups were improved, and the improvement effect of high-dose Epiphyllum cymbidium extract was slightly better than that of low-dose Epiphyllum cymbidium extract.

In summary, Epiphyllum cymbidium extract has the potential to reduce CTX-induced immune damage. Therefore, Epiphyllum cymbidium extract can be used in the preparation of health foods and medicines that reduce CTX-induced immune damage.

2.2. Epiphyllum extract intervenes to repair damaged intestinal barrier

The intestine is an important immune organ with mechanical, chemical, immune and biological barriers to defend against pathogens and maintain internal balance. When the body's immune system is damaged, the intestinal mucosal immunity, which is an important part of immune defense, will also be adversely affected. The integrity of the intestinal barrier function is essential for maintaining a healthy homeostasis. MUC2 secreted by goblet cells plays a core role in forming the chemical barrier of the intestinal mucosa, which can lubricate the intestine and antagonize pathogenic bacteria. The results of the investigation of the effect of Epiphyllum extract on the intestinal barrier of immunosuppressed mice refer to Figures 7-9. Figure 7 is the result of PAS staining (40×) and H&E staining (200×) of colon tissue sections of experimental mice. Figures 8 and 9 are statistical graphs of the results of the intestinal barrier effect of immunosuppressed mice, among which A and C in Figure 8 are statistical graphs of the number of colon goblet cells and crypt length of each group of experimental mice, respectively; 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. Figure 9 A, B, C, D, and E are statistical graphs of the contents of colonic immune barrier cytokines sIgA, IFN-γ, IL-10, TGF-β3, and IL-17 in each group of experimental mice. Data are expressed as mean ± SEM, n = 5. Compared with the MDC group, * P < 0.05, ** P < 0.01. The purple particles in the colon tissue sections represent stained goblet cells, CR represents crypts, MU represents muscularis, and yellow circles represent lymphocyte infiltration.

Refer to Figures 7 and 8 (columns A and B). After intervention with Epiphyllum extract, especially at high doses, the number of stained goblet cells and MUC2 expression levels in the colon of immunocompromised mice increased significantly. The depth of intestinal crypts can represent the integrity of intestinal morphology. Both Epiphyllum extract and Lentinan significantly increased the length of colon crypts compared with the MDC group, see Figures 7 and 8 (column C). In addition, the intestinal mucosa of mice in the MDC group was loosely connected to the muscle, and lymphocyte infiltration was visible. The above-mentioned injuries of mice in the three polysaccharide intervention groups were all improved, and the changes in the EPL group were slightly weaker but still better than the MDC group, see Figure 7.

CTX caused the destruction of the mechanical barrier of the mouse mucosa, resulting in increased intestinal permeability. Compared with the MDC group, both Lentinan and Epiphyllum extract significantly increased the expression level of tricellulin in the intestine of immunosuppressed mice, refer to Figure 8 (bar graph D), but did not significantly increase the expression level of occludin, refer to Figure 8 (bar graph E). SIgA plays an important role in intestinal barrier protection by shaping the resident microbial community to limit the growth of bacterial pathogens and by enhancing the host's protective immunity to produce immune rejection. The results showed that compared with the CON group, the expression level of sIgA in the colon of mice in the MDC group was significantly reduced, refer to Figure 9 (bar graph A), while high doses of Epiphyllum extract could 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 damaged, and intervention with Epiphyllum cymbidium extract could reverse this immune damage and show a dose-dependent improvement, see Figure 9 (bar graphs B, C, D, E, P <0.05).

According to the above experiments, Epiphyllum extract can repair the damage of intestinal chemical, mechanical and immune barriers caused by CTX, including increasing the number of goblet cells that can be stained in the colon, improving intestinal morphological disorders and crypt depth, promoting the expression levels of MUC2 and tricellulin, and promoting the secretion of sIgA and T helper cell-related cytokines.

2.3. Epiphyllum extract changes the structure and diversity of intestinal flora

In order to explore the effect of Epiphyllum extract on intestinal flora, 16S rRNA sequencing analysis was performed on the fecal samples of each group of mice. The results of the investigation refer to Figures 10-14. Figure 10 is the dilution curve of the fecal samples of each group of mice, where A is the Shannon dilution curve; B is the Sobs dilution curve. Figure 11 is the result of the genus-level species number-UpSet Venn analysis. Figures 12 and 13 are analysis diagrams of the effect of Epiphyllum extract on the deconstruction and diversity of intestinal microorganisms in immunosuppressed mice, where A, B, C, and D in Figure 12 represent the MDC vs CON, MDC vs LNP, MDC vs EPL, and MDC vs EPH levels of PCA at the OTU level, respectively, and E is the partial least squares discriminant analysis (PLS-DA) analysis result diagram in each group. A, B, C, D, and E in Figure 13 are the OTU number, 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 with the MDC group. In the PCA analysis, different colors represent different groups, and within the same group, individuals are depicted in a circular plot based on a 95% confidence interval. Figure 14 is a graph showing the effect of Epiphyllum extract on the relationship between intestinal microbiota in immunosuppressed mice, where A, B, and C are unidirectional correlation networks at the top 30 abundance genus levels of intestinal microbiota in the MDC group, EPL group, and EPH group, respectively. The purple solid line and green dotted line represent positive and negative correlations, respectively, and the width reflects the strength of the correlation.

Referring to Figure 10, the dilution curve analysis showed that the curves of all samples showed a flat trend, indicating that the sequencing data has reached saturation and effectively captured most of the species in the intestinal flora. PCA analysis found that there were significant differences in the intestinal flora structure of mice in the MDC group and the CON group, see Figure 12 (A). After intervention with Epiphyllum extract, the intestinal flora of mice in the EPL and EPH groups changed significantly compared with the MDC group, see Figure 12 (C, D), and the ability of Epiphyllum extract to change the structure of intestinal flora showed a certain dose dependence, while the effect of Lentinan was not significant, see Figure 12 (B). PLS-DA (partial least squares discriminant analysis) was used to classify the intestinal flora structure of each group of mice, see Figure 12 (E). The results showed that the intestinal flora of mice in the three polysaccharide intervention groups were clustered together, while the intestinal flora of mice in the CON and MDC groups were clustered into one category.

The results of OTU level diversity difference analysis showed that the number of OTUs, chao1 index and ace index of intestinal flora in mice in the MDC group were significantly higher than those in other groups, refer to Figure 13 (A, B, C), indicating that the richness of intestinal flora in mice in the MDC group was significantly higher than that in other groups. In addition, compared with the MDC group, the Simpson index of the EPH group and the CON group increased and the Shannon index decreased, refer to Figure 13 (D, E), indicating that the diversity of intestinal flora was significantly reduced. The number of species at the genus level was similar to the results at the OTU level. The species diversity at the genus level of the intestinal genus of mice in the three polysaccharide intervention groups was lower than that in the MDC group, refer to Figure 11. In addition, the interaction between genera was visualized by a unidirectional correlation network. The results showed that the richness of the correlation network of the first 30 genera in the EPL and EPH groups (especially the positive correlation) was higher than that in the MDC group, refer to Figure 14 and Table 1 below.

Table 1

The above results show that Epiphyllum extract can affect the diversity and richness of intestinal flora in immunosuppressed mice. In addition, Epiphyllum extract can affect the structure of intestinal flora and affect the relationship between intestinal microorganisms.

2.4. Effects of Epiphyllum extract treatment on intestinal flora of immunocompromised mice

The dominant phyla in the fecal samples of mice from different groups were analyzed. The analysis results are shown in Figure 15. In Figure 15, A is a bar stacking diagram of the main species at the phylum level, and B is a bar stacking diagram of the main species at the genus level. The results showed that the dominant phyla composition of each group was similar, with Firmicutes and Bacteroidota as the main phyla, followed by Campilobacterota and Proteobacteria .

In addition, we analyzed the composition of the top 30 genera of the intestinal flora, see Figure 15 (B). The results showed that polysaccharide intervention led to changes in the abundance of certain genera between the groups, especially Lactobacillus . Specifically, the abundance of Lactobacillus was significantly restored in the EPH group.

In addition, some genera with relatively low abundance (outside the top 30) showed significant differences among the groups. Refer to Figures 16 and 17 for species with different abundances among different groups at the genus level. In Figure 16: A is a statistical graph of the abundance among different groups of Lactobacillus, Defiuviitaleaceae_UCG-011, Tyzzerella, Rikenella, and unclassified_f_ Anaerovoracaceae ; B is a statistical graph of the abundance among different groups of Family_XIII_AD3011_group, norank_f_ norank_o_Rhodospirillales, Anaeroplasma, Escherichia-Shigella, and Parabacteroides . In Figure 17: C is a statistical graph of the abundance of different groups of Anaerovorax, Caldicoprobacter, Candidatus_ Arthromitus, Prevotellaceae_NK3B31_group, and Unclassified_f_Lachnospiraceae ; D is a statistical graph of the abundance of different groups of Butyricimonas/UCG-009, norank_f_UCG-010, Marvinbryantia, and unclassified_c_Clostridia . Data are expressed as mean ± SEM, n = 5. * P < 0.05, ** P < 0.01, compared with the MDC group. Previous studies have shown that these genera are related to intestinal immunity. The intervention of Epiphyllum extract affected the abundance of these genera in the intestine to a certain extent. Previous studies have revealed that Tyzzerella is highly abundant in the intestinal flora of Crohn's disease patients and is associated with intestinal inflammation; supplementation of intestinal butyrate will lead to an increase in the abundance of Rikenella , reflecting that there may be butyrate deficiency in the intestine of the immunodeficient mouse model under CTX toxicity; Family_XIII_AD3011_group and Escherichia-Shigella are negatively correlated with intestinal acetic acid concentration; Anaeroplasma is positively correlated with the severity of experimental autoimmune encephalomyelitis; Escherichia-Shigella may be an important indicator of intestinal sepsis, and Candidatus Arthromitus is a recently discovered commensal bacterium associated with the maturation of intestinal immune function.

Furthermore, the abundance of low-prevalence bacterial genera such as Defluviitaleaceae_UCG - 011 , unclassified_f_Anaerovoracaceae , Caldicoprobacter , Anaerovorax , Prevotellaceae_NK3B31_group , Butyricimonas , norank_f_UCG-010 , and unclassified_f_Lachnospiraceae were changed after the intervention of Epiphyllum extract. These bacteria may serve as potential biomarkers for improving immunosuppressive conditions.

During the experiment, LEfse measurements were also used to determine the differential groups between the polysaccharide intervention group and the MDC group. The results of the LEfse analysis of the mouse intestinal microbiota are shown in Figures 18-20. In Figure 18, A and B represent the results of MDC vs CON and MDC vs LNP in the LEfse analysis of the mouse intestinal microbiota, respectively; in Figure 19, C and D represent the results of MDC vs EPL and MDC vs EPH in the LEfse analysis of the mouse intestinal microbiota, respectively, with LDA critical value = 3.0, and E represents the abundance and proportion of fungi and bacteroides between the groups. Data are expressed as mean values, n = 5.

Significant differences were found during the comparison. Compared with the two groups treated with Epiphyllum extract, see Figure 19, the abundance of Caldicoprobacter, norank_f_norank_o_ Rhodospirillales , Anaerovorax, Enterococcus , Tyzzerella , Butyricimonas increased in the MDC group, while the abundance of Erysipelotrichaceae and Lactobacillus decreased significantly, and Epiphyllum extract treatment restored the levels of the above species in the intestines of immunosuppressed mice to levels similar to those of normal mice, see Figure 18. Related studies have shown that Lactobacillus, which uses carbohydrates as fermentation raw materials and lactic acid as the main product, can create an acidic intestinal microenvironment that is not conducive to the survival of pathogens. Studies have also shown that Lactobacillus can promote intestinal Peyer's cells to produce IgA, IL-6, IL-10, IFN-γ and tumor necrosis factor, thereby regulating intestinal immunity.

In addition, Erysipelotrichaceae, which belongs to the same phylum as Lactobacillus , can produce an adjuvant-like effect in the intestine, enhance the response of Th17 cells, and regulate immunity. It is worth noting that previous studies have shown that dietary fermentable fiber can enhance the metabolism of fiber by the intestinal microbiota, resulting in changes in the ratio of sterol bacteria to Bacteroidetes and increased concentrations of short-chain fatty acids (SCFAs) in the circulation. However, our results did not show such an effect, as no significant differences were observed in the abundance of bacteria or the ratio of bacteria to Bacteroidetes among the groups, refer to Figure 20, Figure 15 (A), Figure 20 shows the abundance and ratio of fungi and Bacteroidetes among the groups. Data are expressed as mean values, n=5.

The above results indicate that treatment with Epiphyllum extract affected the intestinal microbiota of immunodeficient mice, especially helping to restore the abundance of Lactobacillus in the intestine.

2.5. Correlation between bacterial genera and immune parameters and functional changes of intestinal microbiota in each group

The results of the correlation investigation between bacterial genus and immune parameters and functional changes of intestinal microbiota in each group are shown in Figures 21 and 22. Figure 21 is a correlation diagram between bacterial genus and metabolic parameters. In Figure 22, A is a KEGG Pathway Level 3 function prediction abundance diagram, B is a KEGG Module function prediction abundance diagram, C is a Metacyc function prediction abundance diagram, D is a total lactic acid fermentation total abundance diagram, and E is a KEGG pathway Level 3 fatty acid metabolism function prediction abundance diagram. Data are expressed as mean values, n=5. Compared with the MDC group, * P <0.05, ** P <0.01, *** P <0.001.

Correlation analysis between the top 30 bacterial genera and metabolic parameters showed that Lactobacillus was positively correlated with most immune parameters, indicating that Lactobacillus may play an important mediating role in the recovery of immune function after polysaccharide intervention. In addition, Parabacteroides was negatively correlated with some immune parameters, see Figure 21.

To further reveal the correlation between the functional changes of the intestinal microbiota of each group and the improvement of intestinal immunity, we used Picrust2 to predict the functional composition of the microbial community based on the amplicon sequencing results. It is worth noting that the predicted ko02060: phosphorus transferase system (PTS) in the KEGG pathway level3 abundance statistics was significantly enriched in the three polysaccharide intervention groups compared with the MDC group ( P < 0.05), but not in the CON group ( P > 0.05), refer to Figure 22 (A). This difference may be due to polysaccharide intervention. Monosaccharides such as glucose and fructose enter the bacteria through PTS transport and phosphorylation when entering the homolactic fermentation pathway of lactic acid. In addition, the KEGG module abundance statistics of M00632 (Leloir pathway) were significantly enriched in the EPL group ( P < 0.05), and showed a certain upward trend in the EPH group and the LNP group ( P > 0.05). This is a metabolic pathway for degrading galactose to produce lactic acid, refer to Figure 22 (B). In the abundance statistics of the Metacyc database, ANAEROFRUCAT-PWY (homo-lactation) and P122-PWY (heterolactation) showed a significant increase after the intervention of Epiphyllum extract compared with the MDC group ( P < 0.05), although this increase was not synchronized with the dose, see Figure 22 (C). However, after combining the predicted values of the total abundance of lactic acid fermentation pathways in each group, the results showed that the abundance of lactic acid fermentation in the MDC group was significantly lower than that in the other groups, see Figure 22 (D). It is well known that SCFAs derived from the intestinal microbiota play a vital role in maintaining intestinal immune balance. In our study, pathways related to intestinal microbial fatty acid metabolism did not show significant differences between the groups, see Figure 22 (E). This finding is consistent with the previous study results (see Figure 20) that observed no significant changes in the abundance of Firmicutes and Bacteroidota .

The reduction in the lactic acid fermentation capacity of the intestinal microbiota may be a functional manifestation of the intestinal microbiota of CTX-induced immunodeficient mice, while after intervention with Lentinan and Epiphyllum extract, the ability of the intestinal microbiota to ferment lactic acid converged with that of the CON group mice. In addition to the above-mentioned role of lactic acid in inhibiting the growth of harmful intestinal bacteria, lactic acid also has a wide range of immune regulatory effects on the body. Lactic acid can regulate the main functions of several key roles in the immune system, such as macrophages and dendritic cells. In addition, previous studies have found that lactic acid can activate the GPR31 receptor, enhance the dendritic processes of CX3CR1 cells in the small intestine, and promote tubular antigen uptake. Therefore, combined with the results of LEfse analysis (Figures 18-20), the increase in the abundance of Lactobacillus and lactic acid content in the intestine of immunocompromised mice induced by Epiphyllum extract may be involved in the immune recovery process of immunosuppressed mice.

2.6. Lactic acid promotes the expression of intestinal epithelial repair-related proteins at the bottom of colonic crypts

The intestinal epithelial tissue of the body is highly sensitive to chemotherapeutic drugs, and intestinal damage is the main damage during chemotherapy. Lactobacillus is generally considered to help protect the intestinal mucosa, and lactic acid, as the final product of Lactobacillus fermentation, plays an important role in repairing the intestinal barrier. Previous studies have shown that lactic acid extracted from the flora can enhance the proliferation of colonic epithelial cells and maintain the normal morphology and function of intestinal epithelial cells. In addition, lactic acid in the microbiota stimulates the proliferation of intestinal stem cells through the Wnt/β-catenin signaling pathway, through Peyer's cells and intestinal stromal cells, thereby repairing intestinal damage caused by chemotherapy and radiotherapy and maintaining the intestinal physical barrier. In order to verify the repair effect of intestinal lactic acid on intestinal damage in CTX-induced immunosuppressive mice, the immunohistochemical method was used in this protocol to visually confirm the expression sites and levels of intestinal barrier repair-related factors in the colon of each group of mice, refer to Figures 23 and 24, Figure 23 is an immunohistochemical staining of Gpr81, Wnt3a and β-catenin in colon tissue (200×). Figure 24 is a graph showing the results of a study on epiphyllum extract promoting intestinal epithelial repair at the bottom of colonic crypts. A, B, and C are the relative expression results of Gpr81, Wnt3a, and β-catenin, respectively, expressed as the average optical density (AOD) value. A and B in Figure 25 are bar graphs showing the lactic acid content in feces and colon of each group, respectively. The data are expressed as mean ± SEM, n = 5. Compared with the MDC group, * P < 0.05, ** P < 0.01.

First, the lactic acid content in the feces and colon of mice was detected. Referring to Figure 25 (A, B), the results were consistent with the results predicted by Picrust2. The lactic acid content in the intestines and colons of the three polysaccharide intervention groups was significantly higher than that in the MDC group. The lactic acid content in the intestinal feces of mice in the EPH group was 3.4 times that of the MDC group. Lactic acid extracted from the microbiota 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.

The quantitative results of ImageJ software showed that the expression of Gpr81, Wnt3a and β-catenin in the EPH group was mainly concentrated at the bottom of the colonic crypts, and the expression level was significantly higher than that in the MDC group, refer to Figure 24 (bar graphs A, B, C). This helps to maintain the stemness of Lgr5+ intestinal stem cells in the intestine and repair the damaged intestinal physical barrier. The above evidence shows that in the process promoted by Epiphyllum extract, lactic acid from the intestine may play a key role in the restoration of the intestinal structural physical barrier.

3. Summary

As mentioned above, immune activation plays a vital role in the various pharmacological effects of Chinese herbal polysaccharides. In our study, CTX caused damage to the immune organs thymus, spleen and intestine. However, histopathological results showed that epiphyllum extract could repair the structural damage of these immune organs and increase the number of stainable epithelial cells in the colon. In particular, the thymus index of mice was significantly restored after intervention with epiphyllum extract. TNF-α is a multidirectional proinflammatory cytokine that participates in the activation of 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 galactose and arabinose, which together account for more than 60% of the monomer content. This type of polysaccharide is called arabinogalactose and has good activity in the complement system. Most lentinan polysaccharides do not have this property. 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. The results showed that epiphyllum extract can stimulate immunosuppressed mice to secrete the above antibodies.

Epiphyllum extract improved the intestinal chemical barrier and immune barrier of immunosuppressed mice by increasing the secretion of MUC2 by intestinal gland cells, promoting the expression of tricellulin, 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 the case of low immune function, 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. In addition, compared with the reference dose of lentil polysaccharide, Epiphyllum extract had a better recovery effect on some indicators. These results show that Epiphyllum extract has the activity of repairing the intestinal barrier of immunosuppressed mice.

The immune system has extensive interactions with the intestinal microbiota. After the decline of immunity, the body usually suffers from intestinal microbial flora imbalance. Similar phenomena were also observed in this study after modeling with CTX. However, Epiphyllum extract was able to improve the composition of the intestinal microbiota. After intervention with Epiphyllum extract, the α-diversity and β-diversity of the intestinal microbiota of mice were improved, similar to the normal group, but different from the model group. The α-diversity of the intestinal microbiota of mice after intervention with Epiphyllum extract varied greatly within the group, but was slightly improved in the group that received Lentinan intervention. We believe that the differences in the interactions between the intestines of different mice and the large molecular weight Epiphyllum extract are the cause of this difference. A diverse network of microbial members is a feature of a healthy intestinal microbiota. At the family level, the action of CTX led to a decrease in the abundance of Erysipelotrichaceae and Lactobacillaceae in mice. At the genus level, the action of CTX led to a decrease in the number of Lactobacillus in mice. It has been reported that Erysipelotrichaceae and Lactobacillus regulate immunity in a specific way, and the intervention of Epiphyllum extract led to an increase in the abundance of these two bacteria. In addition, the epiphyllum extract also reduced specific bacterial genera, such as Butyricimonas , which is negatively correlated with intestinal immune factors and antimicrobial peptides, Tyzzerella , Family_XIII , which is negatively correlated with IFN-γ, IL-4 and IgG, Family_XIII_AD3011_group , which is negatively correlated with 5-hydroxytryptamine , and Enterococcus, which is associated with impaired epithelial barrier integrity. These results indicate that the epiphyllum extract helps immunosuppressed mice restore the intestinal microbiota and enhance the intestinal biological barrier.

Further prediction of intestinal function showed that the abundance of both homolactic and heterolactic fermentation pathways was reduced in CTX-induced immunosuppressed mice. Statistics of total lactic acid fermentation abundance showed that the abundance of lactic acid fermentation in the model group mice was significantly lower than that in mice intervened with Lentinan and Epiphyllum extract. This finding is consistent with the significant decrease in Lactobacillus abundance in the MDC group. Low-dose Epiphyllum extract can significantly increase the abundance of the Leloir pathway in the intestine of mice, while this phenomenon was not observed in Lentinan or high-dose Epiphyllum extract, which may be because the appropriate amount of galactose in Epiphyllum extract is more conducive to the fermentation of lactic acid by the Leloir pathway by the intestinal microbiota.

Mammalian digestive enzymes cannot directly digest most complex carbohydrates and plant polysaccharides, which are metabolized by microorganisms that produce SCFAs. SCFAs have a wide range of immunomodulatory activities, but in our study, no significant changes in the predicted abundance of SCFA production pathways and key enzymes or related enzymes were observed, see Figure 22 (E) and Table 2.

Table 2

Furthermore, the effect of lactate is different from that of butyrate, one of the SCFAs (differentiated colonocytes metabolize butyrate, which may prevent butyrate from reaching stem cells in the crypts, while butyrate is a potent inhibitor of intestinal stem/progenitor cell proliferation at physiological concentrations).

Based on the increase of specific microorganisms ( Lactobacillus ) and the prediction of 16S gene function, it is speculated that Epiphyllum extract may increase lactic acid levels in the intestine. Changing lactic acid abundance may be one of the effective mechanisms by which Epiphyllum extract improves intestinal barrier function by regulating intestinal microbiota. Therefore, the lactic acid content in intestinal feces and tissues was further determined, and experimental verification showed that Epiphyllum extract produced this effect. Lactic acid derived from intestinal microbiota can not only enhance the intestinal immune barrier, but also activate the expression of proteins such as Wnt3a and β-catenin at the bottom 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.

The experimental results showed that compared with the model group, the intervention of Epiphyllum extract can significantly restore the lactic acid levels in the feces and colon of mice. In addition, the expression of key proteins activated by lactate receptor-dependent mechanisms, such as Wnt3a and β-catenin, at the bottom of the colonic crypts was significantly increased. This plays a vital role in maintaining the stemness of Lgr5+ intestinal stem cells in the intestine. It can be seen that Epiphyllum extract can enhance CTX-induced intestinal immune response in mice by regulating the intestinal microbiome.

Previous studies have shown that Lentinan can significantly change the composition of intestinal microbial communities, significantly affect the abundance of certain microorganisms, including lactic acid-producing bacteria of the genera Lactobacillus and Bifidobacterium , and promote the increase of lactic acid content in intestinal feces. In this protocol, Epiphyllum extract showed similar activity to Lentinan, but in some other immune indicators of the body, such as C3, MUC2, and cytokines of colonic T helper cells, Epiphyllum extract showed better improvement than the reference dose of Lentinan. The difference in dosage may be one of the reasons, and it may also 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 then affecting the function of the intestinal immune system. In the future, macromolecular polysaccharides represented by Epiphyllum extract may also be used for their other properties besides immune function, such as food thickeners, drug carriers, moisturizers, gelling agents and adhesives. Therefore, further research and development of Epiphyllum extract will help to realize its broader application prospects.

The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any simple modification or equivalent change made to the above embodiment based on the technical essence of the present invention shall fall within the protection scope of the present invention.

Claims (8)

1. Use of an epiphyllum extract in the preparation of a health food or medicine for treating/improving intestinal diseases, wherein the epiphyllum extract is type I arabinogalactan represented by structural formula (1), (1), Among them, R is or ; m, n are both integers greater than 0. 2. The use according to claim 1, characterized in that the Epiphyllum cymbidium extract is a polysaccharide extracted from Epiphyllum cymbidium petals, with an average molecular weight of 5.767×10 ⁶ Da and a main chain structure of β-Galp connected by (1→4) bonds. 3. The use according to claim 2, characterized in that: the polysaccharide is polymerized from arabinose, galactose, glucose, xylose, rhamnose, fructose and galacturonic acid, and the molar ratios of the monosaccharide composition of arabinose and galactose are 11.480% and 53.791% respectively. 4. The use as claimed in claim 1, characterized in that the epiphyllum extract is used in the preparation of health foods or medicines for regulating the diversity and richness of immunosuppressive intestinal flora, affecting the structure of intestinal flora or affecting the relationship between intestinal microorganisms. 5. The use according to claim 1, characterized in that the epiphyllum extract is used in the preparation of health food or medicine for repairing the damage of intestinal chemical, mechanical or immune barriers caused by CTX. 6. The use according to claim 1, characterized in that the epiphyllum extract is used in the preparation of health food or medicine for restoring the abundance of Lactobacillus in the intestine. 7. The use according to claim 1, characterized in that: the use of the Epiphyllum cymbidium extract in the preparation of health food or medicine for promoting immune recovery, and the promotion of immune recovery is achieved by increasing the abundance of Lactobacillus and the lactic acid content in the intestine. 8. The use according to claim 1, characterized in that the epiphyllum extract is used in the preparation of health food or medicine for promoting the physical barrier of intestinal structure.