Inulin butyrate gel and preparation method thereof

Abstract

The invention provides butyric acid inulin gel as well as a preparation method and application thereof. The butyric acid inulin gel has the function of promoting proliferation and differentiation of colonic epithelial cells, meanwhile, the odor and palatability of butyric acid are improved, the defects that the half-life period in a human body is short, the digestion and absorption speed is high, the butyric acid inulin gel is easily degraded by enzymolysis and microorganisms, and the bioavailability is low are overcome, and the treatment effect of the medicine is improved.

Description

[0001] This case claims the priority of the invention patent with an application date of July 16, 2024, an application number of 202410952473.5, and an invention title of "A Butyric Acid Inulin Gel and Its Preparation Method", and incorporates its full text herein by reference. Technical Field

[0002] The present invention relates to the technical field of butyric acid inulin compositions and their preparation methods, and particularly to the preparation method of a butyric acid inulin gel having a function of repairing colon injury. Background Art

[0003] Intestinal epithelial cells, intestinal mucosa, and intestinal glands are different but closely related concepts. They together constitute the internal structure of the intestine and perform key functions such as digestion and absorption, barrier protection, and immune defense. Colon injury includes: 1. Injury of colon intestinal epithelial cells: Colon intestinal epithelial cells form a layer on the inner surface of the intestine and are the forefront where the intestine directly contacts food residues, digestive juices, and intestinal microorganisms. Epithelial cells maintain the barrier function of the intestine through structures such as tight junctions to prevent the penetration of harmful substances. 2. Injury of colon intestinal mucosa: Colon intestinal mucosa is a broader concept, including the epithelial cell layer, the underlying lamina propria, and part of the submucosa. The mucosal layer not only contains epithelial cells but also has a rich lymphoid tissue, immune cells (such as lymphocytes, macrophages), and a vascular network. There are a large number of microvilli on the surface of the intestinal mucosa, which increase the absorption surface area and improve the nutrient absorption efficiency. In addition, the mucosa is also involved in sensing changes in the intestinal environment and responding to food components, microorganisms, and potential pathogens. 3. Injury of colon intestinal glands: Colon intestinal glands are located in the lamina propria of the mucosal layer. They extend downward to form tubular structures and open onto the intestinal lumen. The gland contains a large number of goblet cells, which mainly secrete digestive juices (such as intestinal juice), including various enzymes to help further decompose food, and mucus to protect the intestinal epithelium. The activities of intestinal glands are crucial for maintaining the stability of the intestinal internal environment and digestive function.

[0004] Carbohydrates are metabolized by the intestinal flora into short-chain fatty acids, which participate in important physiological processes such as host immunity, intestinal flora structure, and intestinal homeostasis. Butyric acid derived from the flora, as a histone deacetylase inhibitor of innate immune cells, increases the degree of acetylation of intracellular histones and plays an important regulatory role in important immunological processes such as the generation, migration, and function of innate immune cells; in addition, butyric acid regulates the adaptive immune activity of the body through various pathways, including recruiting, activating immune cells, and secreting antibodies to act on adaptive immune cells, regulating immune responses, counteracting inflammatory reactions, and maintaining host immune homeostasis. The cecum and colon are the sites where

[0005] The main intestinal segments for short-chain fatty acids are such that when short-chain fatty acids are orally administered, they can all be absorbed in the proximal intestinal segments and cannot reach the large intestine. Moreover, butyric acid has a pungent odor, and oral administration of butyric acid has poor sensory properties, a short half-life, and is absorbed by the proximal small intestine, etc. These drawbacks result in poor therapeutic effects on colon damage and cannot achieve the maximum efficacy for different types of intestinal damage. Existing improvement methods for butyric acid have drawbacks. For example, butyrate is relatively stable, but when butyric acid enters the body, it dissociates extremely quickly in the stomach, making it difficult to reach the intestine and unable to exert its effects throughout the digestive tract, and it has a pungent odor; coated butyric acid can effectively reduce the irritation of butyric acid to the digestive tract, and can block the chemical reactions between butyric acid and other components in the feed, with stable quality and convenient use, but the technical process is complex and the cost is high; palm oil stearin and plant oils (hydrogenated saturated oils) are used to coat particles, but the body's fat digestion function needs to be good; corn syrup or starch is used to coat particles, with water-soluble coating, and cannot reach the back end of the intestine; spray embedding of butyric acid cannot solve the problem of the odor of unembedded butyric acid and other defects, which will affect the sensory quality of the final product, etc. In addition, during the research and development of drugs for colon injury repair, a large number of colon epithelial cells need to be cultured, and some positive controls are also required. Therefore, developing a drug that can promote the proliferation and differentiation of colon epithelial cells, while improving the odor and palatability of butyric acid, solving the problems of short half-life, fast digestion and absorption rate, easy enzymatic hydrolysis and microbial degradation, and low bioavailability in the human body, and improving the therapeutic effect of the drug has extremely high value. Summary of the Invention

[0006] In view of the problems existing in the prior art, an embodiment of the present invention provides a butyric acid inulin gel and a preparation method thereof.

[0007] The primary object of the present invention is to provide a composition, characterized in that the composition comprises butyric acid powder and inulin.

[0008] Further, in the composition, inulin can be used as a coating or butyric acid powder and inulin are combined into a gel.

[0009] In a preferred embodiment, butyric acid powder and inulin in the composition are combined into a gel.

[0010] In a preferred embodiment, the mass ratio of butyric acid powder to inulin in the composition is 1:64.

[0011] On the other hand, the present invention provides a preparation method of the above composition, characterized by comprising the following steps:

[0012] 1) Add inulin to ddH20, fully dissolve it, and set it aside after clarification;

[0013] 2) Add butyric acid powder to ddH20, fully dissolve it and set it aside;

[0014] 3) The butyric acid solution and the inulin solution are mixed in a certain volume ratio.

[0015] Furthermore, step 3) further includes allowing the mixture of butyric acid powder and inulin solution to stand at 4 °C to form a gel.

[0016] In a preferred embodiment, the mass ratio of butyric acid powder to inulin in the gel is 1:64.

[0017] In a preferred embodiment, the concentration of butyric acid in the gel is 5 mg / ml.

[0018] On the other hand, the present invention provides the use of the aforementioned composition in the preparation of a medicament for treating colon injury.

[0019] On the other hand, the present invention provides a medicament for treating colon injury, which comprises the aforementioned composition.

[0020] On the other hand, the present invention provides a medicament for promoting the proliferation of colon epithelial cells in vitro, which comprises the aforementioned composition.

[0021] Furthermore, the medicament further comprises other pharmaceutically acceptable adjuvants.

[0022] Furthermore, the adjuvants include, but are not limited to, improving the physical properties, stability and bioavailability of the medicament, and regulating the release characteristics of the medicament.

[0023] Furthermore, the adjuvants include, but are not limited to, solubilizers, cosolvents, latent solvents, preservatives, fragrances, diluents, binders, coating materials, etc.

[0024] Preferably, the butyric acid powder and inulin in the aforementioned medicament are combined to form a gel.

[0025] In a preferred embodiment, the mass ratio of butyric acid powder to inulin in the aforementioned medicament is 1:64.

[0026] In a preferred embodiment, the content of butyric acid in the aforementioned medicament is 5 mg / ml.

[0027] On the other hand, the present invention provides a method for preparing a medicament for treating colon injury, the method comprising the following steps:

[0028] 1) Inulin is added to ddH2O, fully dissolved, and reserved after clarification;

[0029] 2) Butyric acid powder is added to ddH2O, fully dissolved and reserved;

[0030] 3) The butyric acid solution and the inulin solution are mixed in a certain volume ratio and allowed to stand in a 4 °C refrigerator to form a gel.

[0031] Further, the method further includes the step of adding other pharmaceutically acceptable adjuvants.

[0032] Further, the adjuvants include, but are not limited to, improving the physical properties, stability and bioavailability of the drug, and regulating the release characteristics of the drug.

[0033] Further, the adjuvants include, but are not limited to, solubilizers, cosolvents, latent solvents, preservatives, fragrances, diluents, binders, coating materials, etc.

[0034] In a preferred embodiment, the mass ratio of butyric acid powder to inulin in the aforementioned drug is 1:64.

[0035] A non-disease treatment method for improving colon injury, characterized in that the method includes the following steps: respectively treating DSS-induced colon injury mice with butyric acid inulin gel, performing enema treatment once a day for a total of 7 days.

[0036] A method for promoting the proliferation of colon epithelial cells, characterized in that the method includes the following steps: diluting butyric acid gel in DMEM complete medium and incubating colon epithelial cells.

[0037] In a preferred embodiment, the final concentration of butyric acid is 5 mg / ml.

[0038] In a preferred embodiment, the mass ratio of butyric acid powder to inulin in the aforementioned drug is 1:64.

[0039] A non-disease treatment method for improving colon epithelial cell injury, characterized in that the method includes the following steps: diluting butyric acid gel in DMEM complete medium, and adding DSS to the culture medium, and co-incubating colon epithelial cells.

[0040] In a preferred embodiment, the final concentration of butyric acid is 5 mg / ml, and 2% DSS is added to the culture medium, and HT-29 cells are co-incubated.

[0041] In a preferred embodiment, the mass ratio of butyric acid powder to inulin in the aforementioned drug is 1:64.

[0042] Further, the non-disease treatment method can be used as a positive control method in basic scientific research and used as a control for other methods of improving intestinal injury.

[0043] Further, the aforementioned colon injury refers to any one of the following colon intestinal injuries: colon intestinal epithelial cell injury, colon intestinal mucosal injury, colon intestinal glandular injury.

[0044] Further, the aforementioned treatment or improvement of colon injury refers to including at least one of the following symptom improvements:

[0045] 1) Direct repair of colon intestinal epithelial cell injury, that is, directly promoting the proliferation and differentiation of colon epithelial cells: 2) Repair of colon intestinal mucosal injury, that is, more significantly promoting intestinal mucosal healing and significantly inducing the differentiation of Treg cells; 3) Repair of colon intestinal gland injury, that is, improving the integrity of the colon intestinal gland structure and improving the secretory function of colon glands.

[0046] In a preferred embodiment, the aforementioned butyric acid powder is a butyrate powder, preferably sodium butyrate.

[0047] The butyric acid inulin composition, preparation method and application provided by the embodiments of the present invention have the following beneficial effects:

[0048] 1. It solves the disadvantages of short half-life, fast digestion and absorption rate, easy enzymatic hydrolysis and microbial degradation, and low bioavailability of butyric acid in the human body, and improves the therapeutic effect of the drug.

[0049] 2. It improves the pungent odor and palatability of butyric acid.

[0050] 3. It can be used for the improvement / treatment of different types of colon injuries.

[0051] 4. The butyric acid inulin gel not only solves the disadvantages of fast digestion and absorption rate, easy enzymatic hydrolysis and microbial degradation, and low bioavailability in the human body, but also has a direct synergistic effect.

[0052] 5. The butyric acid inulin composition provided by the present invention can significantly promote the proliferation of colon epithelial cells in vitro culture, and can be used as a component of the culture medium for colon epithelial cells, or as a positive control drug for improving intestinal injury. Brief Description of the Drawings

[0053] The following combines the drawings and specific embodiments to detail the method of the present invention and its beneficial effects.

[0054] Figure 1 : Weight changes of mice with colon injury treated with butyric acid inulin gels with different concentrations of butyric acid and inulin ratios.

[0055] Figure 2 : HE staining of intestinal morphology of mice with colon injury treated with butyric acid inulin gels with different concentrations of butyric acid and inulin ratios.

[0056] Figure 3 : Results of colon epithelial cell proliferation under different treatment conditions.

[0057] Figure 4 : mRNA expression levels of inflammatory factors under different treatment conditions.

[0058] Figure 5 : Expression differences of tight junction proteins under different treatment conditions.

[0059] Figure 6 : Differentiation of Th17 cells in mice with colonic injury treated with inulin butyrate gel and butyric acid.

[0060] Figure 7 : Detection of physiological indexes in mice with colonic injury treated with inulin butyrate gel and butyric acid, where Figure 7 A is the SOD activity, Figure 7 B is the MDA content, Figure 7 C is the IL-6 content, Figure 7 D is the IL-1β activity, Figure 7 E is the IL-17 content, Figure 7 F is the IL-10 content, Figure 7 G is the TNF-α content.

[0061] Figure 8 : Staining of intestinal glycogen secretion in mice with colonic injury treated with inulin butyrate gels of different concentrations.

[0062] Figure 9 : Mouse feeding experiment; Figure A shows the activity photos of group A mice feeding on butyric acid solution, and Figure B shows the activity photos of group B mice feeding on inulin butyrate solution.

[0063] Figure 10 : Statistics of the proliferation results of colonic epithelial cells under different treatment conditions, where different lowercase letters represent different significantly different groups (p <= 0.05). Specific implementation manners

[0064] The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative efforts belong to the scope of protection of the present invention. Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs. The terms used in this specification of the present invention are only for the purpose of describing specific implementation manners and are not used to limit the present invention. The experimental methods in the following embodiments are all conventional methods unless otherwise specified. The experimental materials in the following embodiments are all obtained by purchasing from conventional biochemical reagent stores. The mice with colonic injury are C57 mice purchased from Guangdong Medicilon Biopharmaceuticals Inc., male, about 20 g, and a colonic injury model is constructed by continuously drinking 2% DSS for 7 days)

[0065] The embodiments of this application incorporate Embodiments 1 to 5 of the application with the application number CN202410952473.5 in their entirety, and further add cell cultures of the Control group, the individual butyric acid group, and the individual inulin group in Embodiment 3.

[0066] Embodiment 1. Preparation of butyric acid inulin gel

[0067] The preparation steps of the butyric acid inulin gel are as follows:

[0068] 1. Inulin is added to ddH2O and fully dissolved by ultrasonic wave (KQ5200DE numerical control ultrasonic cleaner in Kunshan) at 50°C for about 15 minutes, and then water-bathed at 70°C and 700 rpm for 15 minutes until clarified (under the IKA RCT basic magnetic stirrer), and then cooled to room temperature (25°C) for standby;

[0069] 2. Sodium butyrate powder is added to ddH2O, and after fully stirring and dissolving, it is cooled to room temperature (25°C) for standby;

[0070] 3. The butyric acid solution and inulin are mixed in a certain volume ratio and left to gel in a 4°C refrigerator for 24 hours (the low temperature condition of the 4°C refrigerator can accelerate the gelling speed).

[0071] Embodiment 2 Comparison of the therapeutic effects of butyric acid inulin gels with different concentrations on mice with colon injury

[0072] Mice with DSS-induced colon injury were respectively treated with butyric acid inulin gel. The mass ratios of butyric acid to inulin were: 1:20, 1:40, 1:60, 1:64, 1:70, 1:80, 1:90, 1:100. Enema treatment was given once a day for a total of 7 days. After DSS administration, the body weights of the mice in the disease group were significantly lower than those in the healthy control group (Blank), while the degree of weight loss in the treatment groups of 8 different concentrations of butyric acid inulin gel was improved compared with the disease group (see Figure 1 ). Except that no mice died in the control group, mice died in the disease group and the three treatment groups of butyric acid inulin gel. On the first day after disease modeling, 10% of the mice in the disease group died. On the second day after disease modeling, 30% of the mice in the disease group died, while the mortality rate in each treatment group of butyric acid inulin gel was significantly lower than that in the disease group. It is shown that the butyric acid inulin gel with a ratio of 1:64 may have the best therapeutic effect on DSS-induced colon injury.

[0073] To more intuitively understand the therapeutic effect, we took the intestines of the mice in each concentration treatment group for HE staining to evaluate the intestinal morphology. We found that when the ratio of butyric acid to inulin in the butyric acid inulin gel was 1:64 for treatment, the symptoms were most significantly improved and the intestinal gland structure was restored most completely ( Figure 2 ). It is shown that the butyric acid inulin gel with a ratio of 1:64 has the best therapeutic effect on colon gland injury.

[0074] Example 3 Effect of butyric acid inulin gel on the proliferation and differentiation of damaged colonic epithelial cells

[0075] According to the results of Example 2, butyric acid inulin gel with a butyric acid to inulin ratio of 1:64 and butyric acid alone were selected for subsequent experiments. Different ratios of butyric acid inulin gel were diluted in DMEM complete medium (final concentration containing 5 mg / ml butyric acid, the Control group used an equal amount of ddH2O, the butyric acid alone group used a final concentration of 5 mg / ml butyric acid, and the inulin alone group used inulin with the same concentration as the 1:64 butyric acid to inulin ratio). At the same time, 2% DSS was added to the medium, and HT-29 cells were co-incubated to construct a ulcerative colitis cell model. The butyric acid inulin gel with a ratio of 1:64 was significantly better than other ratios of butyric acid inulin gel in improving the proliferation and differentiation ability of damaged epithelial cells, and was also significantly better than the control group( Figure 3 , Figure 10 ). Further detection of the expression levels of inflammatory factors under different treatment conditions (the optimal 1:64 butyric acid inulin gel was selected) showed that the mRNA expression levels of IL-6, IL-17, TNF, and IL-23 were significantly decreased in the butyric acid inulin gel group, while IL-10 was significantly increased in the butyric acid gel group( Figure 4 ). At the same time, the content of tight junction proteins was detected (the optimal 1:64 butyric acid inulin gel was selected), and the results showed that the butyric acid inulin gel could increase the expression of tight junction proteins( Figure 5 ), enhance the integrity of the epithelial barrier, and prevent harmful substances from penetrating the intestinal wall.

[0076] In summary, the butyric acid inulin gel with a ratio of 1:64 can directly promote the proliferation and differentiation of colonic epithelial cells, inhibit the inflammatory response, increase the barrier function, and thus accelerate the repair of damaged colonic epithelial cells. The experimental results have not undergone gastrointestinal digestion and absorption, the butyric acid content in butyric acid and butyric acid inulin gel remains unchanged, and there is no participation of intestinal flora. However, the butyric acid inulin gel unexpectedly shows the best synergistic effect at a mass ratio of 1:64.

[0077] Example 4 Comparison of the therapeutic effects of butyric acid inulin gel and butyric acid on mice with colonic injury

[0078] According to the results of Example 2, butyric acid inulin gel with a butyric acid to inulin ratio of 1:64 and butyric acid alone were used to treat mice with colonic injury. 100 μL / mouse (5 mg / ml) of butyric acid was enema and 100 μL / mouse of butyric acid inulin gel (containing 5 mg / ml butyric acid) was enema, once a day for 7 days. The repair of the colonic mucosal barrier, the repair of glandular structure, and the immune level and oxidative stress level of the mice were detected and recorded.

[0079] The results showed that the butyric acid inulin gel was more significant in promoting intestinal mucosal healing compared with butyric acid. The butyric acid inulin gel was more significant in inhibiting the differentiation of Th17 cells compared with butyric acidFigure 6 )。Butyric acid inulin gel improves the inflammatory response in mice with DSS-induced colonic injury and significantly increases SOD( Figure 7 A), significantly decreases MDA( Figure 7 B), decreases the content level of IL-6 in serum( Figure 7 C), decreases the content level of IL-1β in serum( Figure 7 D), decreases the content level of IL-17 in serum( Figure 7 E), increases the content level of IL-10 in serum( Figure 7 F), and decreases the content level of TNF-a in serum( Figure 7 G). It shows that the butyric acid inulin gel with a ratio of 1:64 has the best therapeutic effect on colonic mucosal injury.

[0080] Butyric acid inulin gel induces the secretion of colonic glycogen more significantly compared with butyric acid( Figure 8 ). It shows that the butyric acid inulin gel with a ratio of 1:64 has the effect of promoting the recovery of colonic gland injury.

[0081] Mouse foraging test of butyric acid gel in Example 5

[0082] Since the butyric acid inulin gel does not use inulin as a coating but is mixed with butyric acid, it is unpredictable whether it can improve the odor and palatability of the drug. And only by improving the odor and palatability of the drug can it be better applied clinically. To verify whether the butyric acid inulin gel improves the odor and palatability of the drug, we placed 5 mice in 2 breeding cages respectively. At one end of the breeding cage in Group A, a container filled with butyric acid solution was placed, and at one end of the breeding cage in Group B, a container filled with butyric acid inulin gel (1:64) was placed. The distance of the mouse activity track away from the container was observed within 24 hours. The results showed that within 24 hours, the mice in Group A always stayed away from the container of butyric acid, and their activity range was only near one end away from the container of butyric acid solution (without crossing the midline of the breeding cage and without eating the butyric acid solution). For the mice in Group B, the activity range was very random, and there was no situation of staying away from the container of butyric acid inulin gel. Occasionally, there were activities of automatically eating the butyric acid inulin gel. It shows that the butyric acid inulin gel with the optimal ratio significantly improves the odor and palatability of butyric acid (see Figure 9 ).

[0083] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to the above embodiments will be obvious to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments shown herein, but will conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A composition for treating colon injury and / or promoting the proliferation and differentiation of colon epithelial tissue, characterized in that, The composition contains butyric acid and inulin.

2. The composition according to claim 5, wherein The composition further includes other pharmaceutically acceptable excipients.

3. A method for preparing the composition according to any one of claims 1 or 2, characterized in that It includes the following steps: 1) Add inulin to ddH20, fully dissolve it, and set it aside after clarification; 2) Add butyric acid powder to ddH20, fully dissolve it, and set it aside; 3) Mix the butyric acid solution and the inulin solution according to a certain volume ratio.

4. Use of the composition according to any one of claim 1 or claim 2 in the preparation of a drug for treating colon injury and / or promoting the proliferation and differentiation of colon epithelial tissue.

5. A method for preparing a drug for treating colon injury and / or promoting the proliferation and differentiation of colon epithelial tissue, characterized in that, The method includes the following steps: 1) Add inulin to ddH20, fully dissolve it, and set it aside after clarification; 2) Add butyric acid powder to ddH20, fully dissolve it, and set it aside; 3) Mix the butyric acid solution and the inulin solution according to a certain volume ratio.

6. A non-disease treatment method for improving colon injury, characterized in that, The method includes the following steps: Treat mice with colon injury induced by DSS with butyric acid inulin gel, perform enema treatment once a day for a total of 7 days.

7. A non-disease treatment method for improving colon epithelial cell injury, characterized in that, The method includes the following steps: Dilute the butyric acid inulin gel in DMEM complete medium, and at the same time add DSS to the medium, and co-incubate colon epithelial cells.

8. A method for promoting the proliferation and differentiation of colonic epithelial cells, characterized in that The method includes the following steps: Dilute the butyric acid inulin gel in DMEM complete medium and incubate colon epithelial cells.

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