Butyrate inulin gel and method of making same

Abstract

The application provides inulin butyric acid gel and a preparation method and application thereof. The inulin butyric acid gel has the functions of promoting the proliferation and differentiation of colon epithelial cells, improving the odor and palatability of butyric acid, solving the shortcomings of short half-life in human bodies, fast digestion and absorption speed, easy enzymatic and microbial degradation, and low bioavailability, and improving the therapeutic effect of drugs.

Description

[0001] This case claims the priority of the invention patent with the application date of July 16, 2024, the application number of 202410952473.5, and the invention name of "Butyric Acid Inulin Gel and Its Preparation Method", and the full text is incorporated herein by reference. TECHNICAL FIELD

[0002] The present application relates to the technical field of butyric acid inulin composition and its preparation method, in particular to the preparation method of butyric acid inulin gel with colon injury repair effect BACKGROUND

[0003] Intestinal epithelial cells, intestinal mucosa, and intestinal glands are different but closely related concepts that collectively constitute the internal structure of the intestine, performing key functions such as digestion and absorption, barrier protection, and immune defense. Colon injury includes 1. Colon intestinal epithelial cell injury: The colon intestinal epithelial cells form a layer on the inner surface of the intestine and are the front line that 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. Colon intestinal mucosa injury: The colon intestinal mucosa is a broader concept that includes the epithelial cell layer and the underlying lamina propria and part of the submucosa. The mucosal layer not only contains epithelial cells but also has abundant lymphoid tissue, immune cells (such as lymphocytes, macrophages), and vascular networks. The mucosal surface has a large number of microvilli, increasing the absorption surface area and improving nutrient absorption efficiency. In addition, the mucosa is involved in sensing changes in the intestinal environment and responding to food components, microorganisms, and potential pathogens. 3. Colon intestinal gland injury: The colon intestinal glands are located in the lamina propria of the mucosal layer, which extend downward to form tubular structures with openings on the intestinal cavity surface. The glands contain a large number of goblet cells that mainly secrete digestive juice (such as intestinal juice), which contains various enzymes to help further break down food, as well as mucus to protect the intestinal epithelium. The activity of intestinal glands is crucial for maintaining the stability of the intestinal environment and digestive function.

[0004] Carbohydrates are metabolized by intestinal flora into short-chain fatty acids, which are involved in important physiological processes such as host immunity, intestinal flora structure, and intestinal homeostasis. Butyric acid from the flora serves as an inhibitor of HDAC in innate immune cells, increasing the acetylation level of intracellular histones, and plays an important regulatory role in important immunological processes such as the production, transfer, and function of innate immune cells. In addition, butyric acid regulates adaptive immune activity through multiple pathways, including affecting adaptive immune cells, regulating immune responses, and maintaining host immune homeostasis. The cecum and colon are the main sites of butyric acid production

[0005] The main intestinal segment of short-chain fatty acids, oral short-chain fatty acids in the proximal segment of the intestinal segment can be completely absorbed and cannot reach the large intestine. Moreover, butyric acid has a stimulating odor, oral butyric acid has poor sensory properties, a short half-life, and is absorbed by the proximal small intestine, which leads to poor treatment effect on colon damage and cannot achieve the maximum efficacy on different types of intestinal damage. The existing improvement methods for butyric acid have drawbacks, for example, butyrate is relatively stable, but butyric acid dissociates extremely fast in the stomach after entering the body, is difficult to reach the intestinal tract, and cannot play a role in the entire digestive tract, and has a stimulating odor; although encapsulated butyric acid can effectively reduce the stimulation of butyric acid on the digestive tract, and can block the chemical reaction between butyric acid and other components in the feed, is stable in quality and convenient to use, the technical process is complex and the cost is high; palm oil stearin and vegetable oil (hydrogenated saturated oil) coated particles, but the body needs good fat digestion function; corn syrup or starch coated particles, water-soluble coating, cannot reach the back end of the intestinal tract; spraying butyric acid for embedding cannot solve the defects of unembedded butyric acid odor, and can affect the sensory quality of the final product. In addition, a large number of colon epithelial cells need to be cultured during the development of drugs for repairing colon damage, and some positive controls are also needed. Therefore, it has high value to develop a drug that can promote the proliferation and differentiation of colon epithelial cells, improve the odor and palatability of butyric acid, solve the problems of short half-life in the human body, fast digestion and absorption, easy enzymatic and microbial degradation, and low bioavailability, and improve the therapeutic effect of the drug. SUMMARY

[0006] In view of the problems in the prior art, the embodiments of the present application provide a butyric acid inulin gel and a preparation method thereof.

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

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

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

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

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

[0012] 1) Inulin is added to ddH20, dissolved thoroughly, and used after clarification;

[0013] 2) Butyric acid powder is added to ddH20, dissolved thoroughly, and used after clarification;

[0014] 3) mixing butyric acid solution and inulin solution in a certain volume ratio.

[0015] Further, the step 3) further comprises mixing butyric acid powder and inulin solution to form a gel at 4℃.

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

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

[0018] In another aspect, the present application provides a use of the aforementioned composition in the preparation of a medicament for treating colon injury.

[0019] In another aspect, the present application provides a medicament for treating colon injury, which comprises the aforementioned composition.

[0020] In another aspect, the present application provides a medicament for promoting the proliferation of colon epithelial cells cultured in vitro, which comprises the aforementioned composition.

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

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

[0023] Further, 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 and inulin in the aforementioned medicament is 1:64.

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

[0027] In another aspect, the present application provides a method for preparing a medicament for treating colon injury, which comprises the following steps:

[0028] 1) inulin is added to ddH20 and dissolved thoroughly, and then clarified for standby;

[0029] 2) butyric acid powder is added to ddH20 and dissolved thoroughly for standby;

[0030] 3) butyric acid solution and inulin solution are mixed in a certain volume ratio, and then left to form a gel in a 4℃ refrigerator.

[0031] Further, the method further comprises 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 adjusting the release characteristics of the drug.

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

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

[0035] A non-disease treatment method for improving colon injury, characterized in that the method comprises the following steps: administering butyric acid inulin gel treatment to DSS-induced colon injury mice, once a day for 7 days.

[0036] A method for promoting the proliferation of colon epithelial cells, characterized in that the method comprises the following steps: diluting butyric acid gel into 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 and 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 comprises the following steps: diluting butyric acid gel into DMEM complete medium, and adding DSS to the culture medium to incubate the 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 to incubate HT-29 cells.

[0041] In a preferred embodiment, the mass ratio of butyric acid powder and 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 research, and as a control for other methods for 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 mucosa injury, colon intestinal gland injury.

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

[0045] 1) direct repair of colon intestinal epithelial cell damage, i.e. directly promoting the proliferation and differentiation of colon epithelial cells; 2) repair of colon intestinal mucosa damage, i.e. more significantly promoting intestinal mucosa healing and inducing Treg cell differentiation; 3) repair of colon intestinal gland damage, i.e. improving the integrity of the structure of the colon intestinal gland and improving the secretion function of the colon gland.

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

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

[0048] 1. The short half-life of butyric acid in the human body, the fast digestion and absorption speed, the easy enzymatic and microbial degradation, and the low bioavailability are solved, and the therapeutic effect of the drug is improved.

[0049] 2. The pungent odor and palatability of butyric acid are improved.

[0050] 3. It can be used for improving / treating different types of colon damage.

[0051] 4. The butyric acid inulin gel not only solves the shortcomings of fast digestion and absorption speed, easy enzymatic 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 application can significantly promote the proliferation of colon epithelial cells cultured in vitro, and can be used as a component of a culture medium for culturing colon epithelial cells or as a positive control drug for improving intestinal damage. BRIEF DESCRIPTION OF DRAWINGS

[0053] The method of the present application and its beneficial effects will be described in detail below in combination with the drawings and specific embodiments.

[0054] Figure 1 Figure 1: Changes in body weight of colon damage mice treated with butyric acid inulin gel with different concentrations of butyric acid and inulin ratios.

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

[0056] Figure 3 Figure 3: Proliferation results of colon epithelial cells under different treatment conditions.

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

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

[0059] Figure 6 : Butyric acid inulin gel and butyric acid treat Th17 cell differentiation of colon injury mice.

[0060] Figure 7 : Butyric acid inulin gel and butyric acid treat physiological index detection of colon injury mice, wherein Figure 7 A is SOD activity, Figure 7 B is MDA content, Figure 7 C is IL-6 content, Figure 7 D is IL-1β activity, Figure 7 E is IL-17 content, Figure 7 F is IL-10 content, Figure 7 G is TNF-α content.

[0061] Figure 8 : Different concentrations of butyric acid inulin gel treat intestinal glycogen secretion staining of colon injury mice.

[0062] Figure 9 : Mouse feeding experiment; A is the activity photo of mice feeding butyric acid solution in group A, and B is the activity photo of mice feeding butyric acid inulin solution in group B.

[0063] Figure 10 : The results of colon epithelial cell proliferation under different treatment conditions are statistically analyzed, wherein different lowercase letters represent different significant difference groups (p<=0.05). DETAILED DESCRIPTION

[0064] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application. Unless otherwise defined, all technical and scientific terms used in the specification have the same meaning as understood by those skilled in the art to which the present application belongs. The terms used in the specification of the present application are only for the purpose of describing the specific embodiments and are not used to limit the present application. The experimental methods in the following embodiments are conventional methods unless otherwise specified. The experimental materials in the following embodiments are commercially available from conventional biochemical reagent stores unless otherwise specified. The colon injury mice are C57 mice purchased from Guangdong Yaoke Biotechnology Co., Ltd., male, about 20g, and the colon injury model is constructed by continuous drinking of 2% DSS for 7 days)

[0065] The embodiments of the present application incorporate embodiments 1-5 of application number CN202410952473.5 throughout, and further increase the cell culture of the Control group, the butyric acid group alone, and the inulin group alone in embodiment 3.

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

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

[0068] 1. Inulin is added to ddH20, and is fully dissolved by ultrasonic treatment (Kunshan KQ5200DE digital ultrasonic cleaner) at 50°C for about 15 minutes. After being clarified at 70°C with 700 rpm water bath for 15 minutes, it is reduced to room temperature (25°C) for standby;

[0069] 2. Sodium butyrate powder is added to ddH20, fully stirred and dissolved, and then reduced to room temperature (25°C) for standby;

[0070] 3. Butyric acid solution and inulin are mixed according to a certain volume ratio, and the gel is formed after standing in a 4°C refrigerator for 24 hours (the low-temperature condition of the 4°C refrigerator can accelerate the gelation speed).

[0071] Example 2. Comparison of treatment effects of butyric acid inulin gels with different concentrations on colon injury mice

[0072] DSS-induced colon injury mice are treated with butyric acid inulin gels, and the mass ratio of butyric acid to inulin is 1:20, 1:40, 1:60, 1:64, 1:70, 1:80, 1:90, and 1:100, respectively. The mice are treated by enema once a day for 7 days. After being given DSS, the body weight of the disease group mice is significantly lower than that of the healthy control group (Blank), and the body weight decrease of the eight different concentration butyric acid inulin gel treatment groups is 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 butyric acid inulin gel treatment groups. 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, and the mortality rate of the butyric acid inulin gel treatment groups was significantly lower than that of the disease group. This shows that the butyric acid inulin gel with a ratio of 1:64 has the best treatment effect on DSS-induced colon injury.

[0073] In order to more intuitively understand the treatment effect, the intestinal tract of each concentration treatment group mouse is taken for HE staining to evaluate the intestinal tract morphology. We find that when the ratio of butyric acid to inulin in the butyric acid inulin gel is 1:64, the symptom improvement is the most obvious, and the intestinal gland structure is the most complete ( Figure 2 ). This shows that the butyric acid inulin gel with a ratio of 1:64 has the best treatment effect on colon gland injury.

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

[0075] According to the results of Example 2, butyric acid and inulin butyric acid gel with a ratio of 1:64 and butyric acid alone were selected for subsequent experiments. Different ratios of butyric acid and inulin butyric acid gel were diluted in DMEM complete medium (final concentration containing 5 mg / ml butyric acid, Control group used the same amount of ddH2O, butyric acid alone group used a final concentration containing 5 mg / ml butyric acid, and inulin alone group used the same concentration of inulin as butyric acid and inulin with a ratio of 1:64), while 2% DSS was added to the culture medium, and HT-29 cells were incubated to construct an ulcerative colitis cell model. The improvement of the proliferation and differentiation ability of the damaged epithelial cells by butyric acid and inulin butyric acid gel with a ratio of 1:64 was significantly better than that by butyric acid and inulin butyric acid gel with other ratios, and also significantly better than that by the control group. Figure 3 , Figure 10 ) Further detection of the expression levels of inflammatory factors under different treatment conditions (butyric acid and inulin butyric acid gel with the optimal ratio of 1:64) showed that the mRNA expression levels of IL-6, IL-17, TNF and IL-23 were significantly reduced in the butyric acid and inulin butyric acid 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 (butyric acid and inulin butyric acid gel with the optimal ratio of 1:64), and the results showed that butyric acid and inulin butyric acid 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, butyric acid and inulin butyric acid gel with a ratio of 1:64 can directly promote the proliferation and differentiation of colon epithelial cells, inhibit inflammation, increase barrier function, and accelerate the repair of damaged colon epithelial cells. This experimental result has not been subjected to gastrointestinal digestion and absorption, the content of butyric acid in butyric acid and inulin butyric acid gel remains unchanged, and there is no involvement of intestinal flora, while butyric acid and inulin butyric acid gel with a ratio of 1:64 unexpectedly has the optimal synergistic effect.

[0077] Example 4 Comparison of the therapeutic effects of butyric acid and inulin butyric acid gel on colon damaged mice

[0078] According to the results of Example 2, butyric acid and inulin butyric acid gel with a ratio of 1:64 and butyric acid alone were selected for subsequent experiments. Different ratios of butyric acid and inulin butyric acid gel were diluted in DMEM complete medium (final concentration containing 5 mg / ml butyric acid, Control group used the same amount of ddH2O, butyric acid alone group used a final concentration containing 5 mg / ml butyric acid, and inulin alone group used the same concentration of inulin as butyric acid and inulin with a ratio of 1:64), while 2% DSS was added to the culture medium, and HT-29 cells were incubated to construct an ulcerative colitis cell model. The improvement of the proliferation and differentiation ability of the damaged epithelial cells by butyric acid and inulin butyric acid gel with a ratio of 1:64 was significantly better than that by butyric acid and inulin butyric acid gel with other ratios, and also significantly better than that by the control group.

[0079] The results showed that butyric acid and inulin butyric acid gel was more significantly than butyric acid in promoting intestinal mucosa healing. Butyric acid and inulin butyric acid gel was more significantly than butyric acid in inhibiting Th17 cell differentiationFigure 6 Butyric acid inulin gel ameliorated the inflammatory response in mice with DSS-induced colonic injury and significantly increased SOD levels. Figure 7 A), significantly reducing MDA ( Figure 7 B), reducing serum IL-6 levels ( Figure 7 C), reducing serum IL-1β levels ( Figure 7 D), reducing serum IL-17 levels ( Figure 7 E), increases serum IL-10 levels ( Figure 7 F), reducing serum TNF-α levels ( Figure 8 G). This indicates that a 1:64 ratio of inulin butyrate gel has the best therapeutic effect on colonic mucosal damage.

[0080] Butyric acid inulin gel, compared to butyric acid, significantly induced glycogen secretion in the colon. Figure 9 This indicates that inulin butyrate gel at a ratio of 1:64 has the effect of promoting the recovery of colonic gland damage.

[0081] Example 5: Mouse foraging test using butyric acid gel

[0082] Since butyric acid inulin gel does not use inulin as a coating but is mixed with butyric acid, it is unpredictable whether it will improve the drug's smell and palatability. Only by improving the drug's smell and palatability can it be better applied in clinical practice. To verify whether butyric acid inulin gel improves the drug's smell and palatability, we placed five mice in two cages. A container containing butyric acid solution was placed at one end of the cage in group A, and a container containing butyric acid inulin gel (1:64) was placed at one end of the cage in group B. We observed the distance the mice moved away from the containers over 24 hours. The results showed that mice in group A consistently moved away from the container containing butyric acid, their activity only extending to the end away from the container containing butyric acid solution (they did not cross the midline of the cage, nor did they ingest the butyric acid solution). Mice in group B, however, moved very randomly, never moving away from the container containing butyric acid inulin gel, and occasionally ingested the gel. This indicates that the optimal ratio of butyric acid inulin gel significantly improves the smell and palatability of butyric acid (see...). ​ ).

[0083] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to the above embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A composition for treating colonic injury and / or promoting proliferation differentiation of colonic epithelial tissue, characterized by, The composition consists of sodium butyrate, inulin and ddH2O, the mass ratio of sodium butyrate and inulin is 1:64, the content of sodium butyrate is 5mg / ml, and the composition forms butyrate inulin gel.

2. A process for the preparation of the composition of claim 1, characterized in that, The method comprises the following steps: 1) Inulin is added into ddH2O, dissolved thoroughly, and clarified for standby; 2) Sodium butyrate is added into ddH2O, dissolved thoroughly for standby; 3) The sodium butyrate solution is mixed with the inulin solution according to a certain volume ratio.

3. The use of the composition of claim 1 in the preparation of a drug for treating colon injury.

4. A non-disease treatment method for improving damage to colonic epithelial cells, characterized by, The method comprises the following steps: a sodium butyrate and inulin gel composition with a mass ratio of 1:64 is diluted into DMEM complete medium, the final concentration of sodium butyrate is 5mg / ml, and DSS is added into the medium for incubating colon epithelial cells.

5. A non-disease treatment method for promoting proliferation and differentiation of colonic epithelial cells, characterized by, The method comprises the following steps: a sodium butyrate and inulin gel composition with a mass ratio of 1:64 is diluted into DMEM complete medium, the final concentration of sodium butyrate is 5mg / ml, and colon epithelial cells are incubated.

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