Application of difructose anhydride in improving intestinal inflammation

Abstract

The invention provides application of difructose anhydride in improving intestinal inflammation, and by constructing a DSS colitis mouse model, the DFA is proved to be capable of remarkably improving the disease activity index, inhibiting weight loss and relieving splenomegaly and colon shortening. The intestinal flora analysis shows that the DFA intervention obviously increases the relative abundance of bifidobacterium and lactobacillus in the intestinal tract of the mouse, reduces the abundance of pathogenic bacterium desulfurization vibrio and promotes the generation of short-chain fatty acid. The invention further develops a synbiotics capsule containing DFA, and a sodium alginate-chitosan dual embedding technology is adopted. The invention systematically discloses an action mechanism of improving intestinal inflammation through flora-metabolism-immunization in a multi-target manner by DFA, and provides a novel functional preparation for IBD treatment.

Description

Technical Field

[0001] The invention belongs to the field of biological application technology, and particularly relates to the application of difructose anhydride in improving intestinal inflammation. Background Art

[0002] The pathogenesis of inflammatory bowel diseases (IBD), such as ulcerative colitis (UC), is complex. Existing therapies, such as 5-aminosalicylic acid drugs and glucocorticoids, can alleviate symptoms, but long-term use can easily lead to side effects such as drug resistance and metabolic disorders. In recent years, the dextran sulfate sodium (DSS)-induced colitis model has been widely used for intestinal inflammation mechanism research and drug screening because its pathological features are highly similar to those of human UC. DSS leads to intestinal dysbiosis and immune imbalance by destroying the intestinal mucosal barrier, increasing intestinal permeability, activating inflammatory pathways such as NF-κB, and releasing pro-inflammatory cytokines (such as TNF-α and IL-6). Therefore, the development of new therapies that can repair the intestinal barrier and regulate immunity or microbiota has become a research hotspot.

[0003] Difructose anhydride (DFA) is a cyclic disaccharide composed of two fructose residues. Type I (DFA-I) and type III (DFA-III) exhibit excellent properties such as resistance to gastric acid, high thermal stability, and resistance to digestion. However, the regulatory effects of DFA-I and DFA-III on improving intestinal inflammation through the microbiota-immune pathway and their underlying mechanisms remain unclear. Summary of the Invention

[0004] The purpose of this section is to summarize some aspects of embodiments of the present invention and to briefly introduce some preferred embodiments.

[0005] In view of the above problems and / or the problems existing in the prior art, the present invention is proposed.

[0006] Therefore, the purpose of the present invention is to overcome the deficiencies in the prior art and provide a use of difructose anhydride in improving intestinal inflammation.

[0007] To solve the above technical problems, the present invention provides the following technical solution: an application of difructose anhydride in improving intestinal inflammation, wherein the difructose anhydride is type I difructose anhydride DFA-I or type III difructose anhydride DFA-III.

[0008] As a preferred embodiment of the application of the present invention, the composition is an oral preparation or an intestinal targeted preparation, comprising an acceptable carrier, and the dosage form includes tablets, capsules, granules or liquid suspensions.

[0009] As a preferred embodiment of the application of the present invention, the difructose anhydride restores the Th1 / Th2 immune response balance by reducing the expression levels of TNF-α, IL-6 and IFN-γ in colon tissue and upregulating the expression of IL-10.

[0010] As a preferred embodiment of the application of the present invention, the indication of the difructose anhydride is ulcerative colitis (UC), reducing the disease activity index (DAI) and restoring the colon length.

[0011] As a preferred embodiment of the application of the present invention, the difructose anhydride promotes the enrichment of probiotics, inhibits the proliferation of Desulfovibrio, and regulates the diversity of intestinal flora.

[0012] As a preferred embodiment of the application of the present invention, the difructose anhydride regulates the diversity of the bacterial flora, promotes the production of butyrate, and thus improves inflammation.

[0013] Another object of the present invention is to overcome the deficiencies in the prior art and provide a synbiotic preparation containing difructose anhydride, comprising:

[0014] Difructose anhydride: DFA-I or DFA-III, purity ≥99%;

[0015] Probiotic combination: Bifidobacterium animalis combined with Lactobacillus rhamnosus or other probiotic strains, with a total viable count ≥ 1×10^9 CFU / g;

[0016] Excipients: oligofructose, maltodextrin;

[0017] Embedding material: composite colloid made of sodium alginate and chitosan;

[0018] Wherein, based on the total mass percentage of the preparation, the DFA-I or DFA-III is 20% to 40%, the probiotic combination is 10% to 25%, the oligofructose is 5% to 10%, the maltodextrin is 10% to 21%, the sodium alginate is 2% to 3%, and the chitosan is 0.5% to 1%.

[0019] Another object of the present invention is to overcome the deficiencies in the prior art and provide a method for preparing a synbiotic preparation, comprising:

[0020] a. After the probiotics are activated, they are mixed with difructose anhydride, fructooligosaccharides and maltodextrin to form a slurry;

[0021] b. Sodium alginate was dissolved in water to prepare a 1.5% to 2.5% w / v solution, mixed with a chitosan solution, and stirred to form a composite colloid having a viscosity of 250 mPa · s, wherein the chitosan solution was prepared by dissolving chitosan in a 1% acetic acid solution, pH 5.0 to 5.5, and a concentration of 1.0% to 1.5% w / v;

[0022] c. The sodium alginate-chitosan composite colloid dissolved at 55°C was mixed evenly with the slurry, then emulsified at 10,000 rpm for 8 minutes. The mixture was then added dropwise to a 3% CaCl2 solution containing 0.1% Tween-80. The volume of the CaCl2 solution was 1 times the volume of the slurry. The addition rate was 8 mL / min, and the gel microspheres were calcified at room temperature for 3 hours to form gel microspheres.

[0023] d. The microspheres were transferred to a 0.8% chitosan solution and cured at 4°C for 20 to 30 minutes, washed with deionized water, and then coated for a second time to improve gastric acid resistance;

[0024] e. The microspheres were freeze-dried at -40°C and then filled into gelatin capsules.

[0025] As a preferred embodiment of the preparation method of the present invention, the freeze-drying step comprises pre-freezing at -40°C for 2 hours, followed by drying at -40°C and 0.15 mbar for 8 hours until the moisture content is ≤8%.

[0026] Another object of the present invention is to overcome the deficiencies in the prior art and provide a synbiotic preparation for use in improving intestinal inflammation.

[0027] Beneficial effects of the present invention:

[0028] This invention reveals for the first time that DFA-I and DFA-III improve intestinal inflammation through a multi-target synergistic mechanism of "microbiota-metabolism-immunity". The highly stable synbiotic preparation developed provides a solution for the treatment of IBD that is both highly effective, safe and industrially feasible. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments.

[0030] Figure 1 Graph showing the effects of DFA-I and DFA-III on improving ulcerative colitis in mice; wherein, A is DAI, B is body weight change, C is spleen weight, and D is colon length.

[0031] Figure 2 Representative HE staining images of the distal colon of mice; A is the CON group, B is the DSS group, C is the DFA-I group, and D is the DFA-III group.

[0032] Figure 3 The expression levels of TNF-α, IFN-γ, IL-10, and IL-6 in mouse colon tissue. Lowercase letters a, b, and c indicate significant differences among the groups (p<0.05). Statistical significance was analyzed using analysis of variance and Duncan's test.

[0033] Figure 4 This is a diagram showing the relative abundance differences of intestinal microorganisms at the genus level in mice.

[0034] Figure 5 Figure 2 is a graph of the short-chain fatty acid content in the mouse intestine; AF represents the levels of acetic acid, propionic acid, isobutyric acid, butyric acid, valeric acid, and isovaleric acid in the mouse intestine, respectively. DETAILED DESCRIPTION

[0035] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification.

[0036] Example 1

[0037] DFA-I and DFA-III improve ulcerative colitis in mice:

[0038] 1.1 Animal handling and grouping

[0039] Forty six-week-old SPF-grade C57BL / 6J male mice (Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.) were selected. The experimental protocol was reviewed and approved by the Experimental Animal Care and Animal Welfare Ethics Committee of Jiangnan University (JN.No20240415b0800831). The experimental animals were acclimated in a barrier environment (SYXK(SU)2021-0056) for 7 days. The environmental parameters were controlled at a temperature of 22±2°C, a relative humidity of 55±5%, a 12-h light-dark cycle, and free access to food and water. Four groups (n=10 / group) were set up in the experiment: a blank control group (CON group, gavage with normal saline), a DSS model group (DSS group, gavage with normal saline), a DFA-I intervention group (DFA-I group, gavage with DFA), and a DFA-III intervention group (DFA-III group, gavage with DFA-III).

[0040] The DFA intervention dose was calculated as 3% of the mice's daily feed intake (approximately 3.3 g), equivalent to 30 g DFA / kg feed. Based on this dose, the daily oral dose was 0.1 g DFA dissolved in 200 μL of sterile water (concentration 500 mg / mL).

[0041] The preparation of DFA-I and DFA-III was based on the method of Yu et al. [1], using a multi-stage coupling process to prepare high-purity DFA from inulin raw materials. [1] Yu S, Li Q, Wang Z, et al. In vitro physiological properties of difructose anhydride I prepared from inulin by inulin fructotransferase [J]. Journal of Agricultural and Food Chemistry, 2025, 73(11): 6659-6667.

[0042] During the modeling stage (days 8-14), except for the CON group, the other groups were given 3% (w / v) DSS solution to drink freely to establish the colitis model.

[0043] During the intervention phase (days 15-21), the CON and DSS groups continued to be gavaged with normal saline, while the DFA-I and DFA-III groups were gavaged with DFA-I and DFA-III solutions, respectively.

[0044] At the end of the experiment (day 22), the rats were killed by cervical dislocation after anesthesia with isoflurane (5%) according to animal ethics guidelines.

[0045] During the experiment, mice were weighed every two days. After sacrifice, biological samples such as spleen (weighing) and colon (length measurement and tissue fixation) were collected. Fecal samples were collected 24 hours before the end of the experiment.

[0046] Table 1 DAI evaluation method

[0047]

[0048] Figure 1 The improvement effects of DFA-I and DFA-III on DSS-induced colitis in mice were verified by multidimensional data (n=10 in each group).

[0049] Disease Activity Index (DAI) Figure 1 A) DAI in the DSS group continued to increase from day 3 and was significantly higher than that in the control group. DFA intervention significantly inhibited the increase in DAI.

[0050] Weight changes ( Figure 1 B) The body weight of the DSS group began to decrease from day 5, and the loss reached its maximum on day 7. The body weight of the DFA intervention group recovered significantly.

[0051] Spleen weight ( Figure 1 C): The spleen was significantly enlarged in the DSS group, and the spleen weight was significantly restored in the DFA intervention group.

[0052] Colon length ( Figure 1 D) The colon length in the DSS group was shortened to 4.78±0.23 cm, while the colon length in the DFA intervention group recovered to a near normal level.

[0053] Disulphur anhydride has good intestinal inflammation regulating effect in inhibiting the progression of inflammation, maintaining weight stability, alleviating splenomegaly and protecting colon structure.

[0054] Example 2

[0055] DFA-I and DFA-III improve intestinal barrier integrity in mice with colitis

[0056] Colon tissue samples were dehydrated with graded ethanol, cleared with xylene, and embedded in paraffin, and serial sections were prepared using a constant temperature microtome.

[0057] Hematoxylin and eosin (H&E) staining was performed for analysis. The specific process included dewaxing and hydrating the sections, followed by H&E staining (hematoxylin staining for 5 minutes, differentiation solution treatment, and eosin counterstaining for 30 seconds). The stained slides were cleared with xylene and mounted with neutral gum. Microscopic observation and image acquisition were performed using an upright light microscope.

[0058] The staining results are as follows Figure 2 As shown in Figure 3, the DSS group (B) showed typical pathological features such as glandular rupture, crypt atrophy, and massive neutrophil infiltration (dark-stained granular), while the DFA group showed varying degrees of repair effects.

[0059] Among them, DFA intervention had significant effects on mucosal structural reconstruction, inflammation control and mucus barrier repair. The DFA intervention group (CD) showed crypt arrangement regularity and mucus secretion function recovery close to that of the normal group (A). The structural repair effect of difructose anhydride on DSS-induced inflammatory damage was intuitively verified through multi-group staining comparison.

[0060] Example 3

[0061] DFA-I and DFA-III improve intestinal immune homeostasis imbalance in colitis mice

[0062] Colon tissue (about 1 cm in length) 1 cm away from the cecum was collected and placed in a 1.5 mL centrifuge tube. Four zirconium beads were placed in each centrifuge tube and 9 times the volume of pre-cooled sterile PBS was added. The tube was placed in a tissue grinder and homogenizer and then centrifuged at 12,000 g and 4°C for 15 min. The supernatant was collected for cytokine detection.

[0063] The levels of cytokines TNF-α, IFN-γ, IL-10 and IL-6 in the mouse colon were determined according to the instructions of the enzyme-linked immunosorbent assay kit.

[0064] The levels of TNF-α, IL-6 and IFN-γ in the colon of mice in the model group were significantly higher than those in the blank group ( Figure 3 ), indicating that DSS modeling caused the production of multiple pro-inflammatory factors in the colon tissue of mice, indicating that local intestinal inflammation was successfully induced.

[0065] After DFA intervention, the body's immune regulation efficiency was enhanced by synergistically inhibiting pro-inflammatory factors and promoting the secretion of anti-inflammatory factor IL-10, confirming that difructose anhydride has superior targeting in regulating the balance of Th1 / Th2 immune response.

[0066] Example 4

[0067] DFA-I and DFA-III improve intestinal microbial dysbiosis in colitis mice:

[0068] Ten stool samples were collected from each group, and four were randomly selected for sequencing analysis to represent the microbial composition of the group and assess intragroup variation. Microbial genomic DNA was obtained using a DNA extraction kit, and purity was verified by 1% agarose gel electrophoresis. The 16S rDNA V3-V4 region was amplified by PCR using primers 341F (5′-CCTAYGGGRBGCASC AG-3′) and 806R (5′-GGACTACNNGGGTATCTAAT-3′).

[0069] After purification, the amplified products were sequenced by Illumina double-end sequencing by Shanghai Meiji Biotechnology Co., Ltd.; the raw data were filtered by quality control, and species annotation was performed using the MicrobiomeAnalyst platform, and ST AMP software was used to compare the different bacterial communities between the groups (p < 0.05 was significant).

[0070] Figure 4 The genus-level bacterial community histogram revealed the regulatory effect of difructose anhydride on the intestinal microbial structure of mice with DSS-induced colitis. The relative abundance of beneficial bacterial genera such as Lactobacillus and Bifidobacterium decreased significantly in the DSS group, while the abundance of the opportunistic pathogen Desulfovibrio increased.

[0071] In the DFA intervention group, the abundance of Lactobacillus and Bifidobacterium genera was restored (p<0.05).

[0072] Example 5

[0073] DFA-I and DFA-III improve intestinal microbial metabolic disorders in colitis mice:

[0074] Fecal samples were freeze-dried and the dry weight was recorded. The samples were then resuspended in saturated NaCl solution, acidified with 10% sulfuric acid solution, and extracted with anhydrous ether. Residual water was removed with anhydrous sodium sulfate, and the resulting samples were analyzed by gas chromatography. SCFA concentrations were calculated using an external standard method and expressed as μmol / g sample.

[0075] Figure 5 The regulatory effect of DFA intervention on intestinal metabolites in mice with DSS-induced colitis was revealed through six groups of short-chain fatty acids (SCFAs) detection data. The key findings are as follows: DFA intervention significantly promoted the production of acetic acid and butyric acid, among which DFA-III intervention was the most significant, which is consistent with the intestinal flora data in Example 4, cross-validating the specific regulatory mechanism of DFA.

[0076] Example 6

[0077] The manufacturing process of the DFA-containing capsule (synbiotic) product: calculated as a percentage of the total mass of the preparation, the formula consists of DFA (20% to 40%), animal Bifidobacterium and Lactobacillus rhamnosus powder or other probiotic strains (10% to 25%, total viable bacteria ≥ 1×10^9 CFU / g, converted by freeze-dried powder), oligofructose (5% to 10%) and maltodextrin (10% to 21%), and is double-encapsulated and protected by a sodium alginate (2% to 3%)-chitosan (0.5% to 1%) composite colloid.

[0078] During preparation, the probiotics are first activated by MRS medium and the bacterial slurry is collected by centrifugation and mixed with DFA, oligofructose and maltodextrin to form a slurry;

[0079] Sodium alginate was dissolved in water to prepare a 1.5% to 2.5% (w / v) solution, and mixed with chitosan solution (chitosan dissolved in 1% acetic acid solution, pH 5.0–5.5, concentration 1.0% to 1.5% (w / v)) at a volume ratio of 5:1 and stirred to form a composite colloid (viscosity 250 mPa·s).

[0080] Subsequently, the sodium alginate-chitosan composite colloid dissolved at 55°C was mixed with the slurry at a mass ratio of 1:2, and then emulsified at 10,000 rpm for 8 minutes. The mixture was then dropped into a 3% CaCl2 solution containing 0.1% Tween-80 (the volume of the CaCl2 solution was approximately 1 times the volume of the slurry, and the dropping speed was 8 mL / min). The gel microspheres were calcified at room temperature for 3 hours.

[0081] The microspheres were then transferred to a 0.8% chitosan solution (pH 5.0–5.5) for curing at 4°C for 20–30 min, washed with deionized water, and coated a second time to improve gastric acid resistance;

[0082] Finally, the microspheres were pre-frozen at -40°C for 2 h, then dried at -40°C and 0.15 mbar for 8 h until the moisture content was ≤ 8%, and then filled into gelatin capsules.

[0083] This invention reveals for the first time that DFA-I and DFA-III improve intestinal inflammation through a multi-target synergistic mechanism of "microbiota-metabolism-immunity". The highly stable synbiotic preparation developed provides a solution for the treatment of IBD that is both highly effective, safe and industrially feasible.

[0084] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, and all of these should be included in the scope of the present invention.

Claims

1. A use of difructose anhydride in improving intestinal inflammation, characterized by: The difructose anhydride is type I difructose anhydride DFA-I or type III difructose anhydride DFA-III.

2. The use according to claim 1, characterized in that: The application is achieved through a composition comprising difructose anhydride, which is an oral preparation or an intestinal targeted preparation, comprising an acceptable carrier, and the dosage form includes tablets, capsules, granules or liquid suspensions.

3. The use according to claim 1 or 2, characterized in that: The difructose anhydride restores the Th1 / Th2 immune response balance by reducing the expression levels of TNF-α, IL-6 and IFN-γ in colon tissue and upregulating the expression of IL-10.

4. The use according to claim 3, characterized in that: The indications of the difructose anhydride are ulcerative colitis (UC), reducing the disease activity index (DAI), and restoring the length of the colon.

5. The use according to claim 1, characterized in that: The difructose anhydride promotes the enrichment of probiotics, inhibits the proliferation of Desulfovibrio, and regulates the diversity of intestinal flora.

6. The use according to claim 5, characterized in that: The difructose anhydride improves inflammation by improving intestinal flora imbalance and promoting butyrate production.

7. A synbiotic preparation containing difructose anhydride, characterized in that: include, Difructose anhydride: DFA-I or DFA-III, purity ≥99%; Probiotic combination: Bifidobacterium animalis combined with Lactobacillus rhamnosus or other probiotic strains, with a total viable count ≥ 1×10^9 CFU / g; Excipients: oligofructose, maltodextrin; Embedding material: composite colloid made of sodium alginate and chitosan; Wherein, based on the total mass percentage of the preparation, the DFA-I or DFA-III is 20% to 40%, the probiotic combination is 10% to 25%, the oligofructose is 5% to 10%, the maltodextrin is 10% to 21%, the sodium alginate is 2% to 3%, and the chitosan is 0.5% to 1%.

8. The method for preparing the synbiotic preparation according to claim 7, characterized in that: include, a. After the probiotics are activated, they are mixed with difructose anhydride, fructooligosaccharides and maltodextrin to form a slurry; b. Sodium alginate was dissolved in water to prepare a 1.5% to 2.5% w / v solution, mixed with a chitosan solution, and stirred to form a composite colloid having a viscosity of 250 mPa · s, wherein the chitosan solution was prepared by dissolving chitosan in a 1% acetic acid solution, pH 5.0 to 5.5, and a concentration of 1.0% to 1.5% w / v; c. The sodium alginate-chitosan composite colloid dissolved at 55°C was mixed evenly with the slurry, then emulsified at 10,000 rpm for 8 minutes. The mixture was then added dropwise to a 3% CaCl2 solution containing 0.1% Tween-80. The volume of the CaCl2 solution was 1 times the volume of the slurry. The addition rate was 8 mL / min, and the gel microspheres were calcified at room temperature for 3 hours to form gel microspheres. d. The microspheres were transferred to a 0.8% chitosan solution and cured at 4°C for 20 to 30 minutes, washed with deionized water, and then coated for a second time to improve gastric acid resistance; e. The microspheres were freeze-dried at -40°C and then filled into gelatin capsules.

9. The method for preparing the synbiotic preparation according to claim 8, characterized in that: The freeze drying comprises pre-freezing at -40°C for 2 hours, and then drying at -40°C and 0.15 mbar for 8 hours until the moisture content is ≤8%.

10. Use of the synbiotic preparation according to claim 7 in improving intestinal inflammation.

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