A colon-targeting microemulsion preparation of tremella fuciformis polysaccharide and a preparation method thereof

Abstract

This invention relates to the field of biomedical technology and provides a colon-targeted microemulsion formulation of Sophora japonica polysaccharide and its preparation method, comprising the following raw materials by mass percentage: 2%-8% oil phase, 10%-45% surfactant, 10%-30% co-surfactant, 0.1%-3% enteric-coated polymer, and the remainder of Sophora japonica polysaccharide aqueous solution to bring the total to 100%; the enteric-coated polymer is Eudragit S100. This invention constructs a stable Sophora japonica polysaccharide microemulsion delivery system and, by introducing a methacrylic acid copolymer-type enteric-coated polymer, gives the resulting microemulsion system excellent colon-targeted release characteristics, thereby facilitating the effective delivery of Sophora japonica polysaccharide to the colon, improving the utilization efficiency of the active ingredient, regulating the intestinal flora structure, and showing promising application prospects in the treatment of liver cancer.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to a colon-targeted microemulsion preparation of Sophora japonica polysaccharide and its preparation method. Background Technology

[0002] Trametes robiniophila Murr. is a traditional medicinal fungus, and its main active ingredient is Trametes robiniophila polysaccharide. Recent studies have shown that Trametes robiniophila polysaccharide not only directly inhibits tumor cell proliferation and induces tumor cell apoptosis, but also enhances anti-tumor immune responses by regulating the body's immune system, thus playing an important role in adjuvant cancer therapy. With the deepening research into the relationship between gut microbiota, the immune system, and tumor development, more and more studies have found that gut microbiota plays a crucial role in regulating the body's immune function and influencing the efficacy of cancer treatment. Therefore, regulating gut microbiota and thereby modulating the body's immune response has become one of the important research directions in recent years regarding the anti-tumor effects of natural polysaccharides.

[0003] Studies have shown that *Sophora japonica* polysaccharide can exert anti-tumor effects through gut microbiota-mediated immune regulation pathways. *Sophora japonica* polysaccharide significantly inhibits the development of hepatocellular carcinoma in vivo, and its mechanism is closely related to gut microbiota remodeling. This study indicates that *Sophora japonica* polysaccharide can influence the tumor immune microenvironment and enhance anti-tumor immune responses by regulating gut microbiota composition and promoting changes in macrophage polarization. The results further confirm that gut microbiota plays a crucial role in the immunomodulatory and anti-tumor effects of *Sophora japonica* polysaccharide.

[0004] Furthermore, studies have found that *Sophora japonica* can enhance the efficacy of anti-tumor treatment by regulating gut microbiota structure and improving the tumor immune microenvironment. Another study found that *Sophora japonica* can significantly alter gut microbiota composition during tumor treatment, increasing the abundance of beneficial bacteria such as *Lactobacillus* and promoting CD8+ growth in the tumor microenvironment. + The infiltration of T cells (cytotoxic T lymphocytes) enhances the body's anti-tumor immune response. This study further demonstrates that the gut microbiota plays an important regulatory role in the immunomodulatory and anti-tumor effects of Sophora japonica.

[0005] Recent studies have further demonstrated that natural polysaccharides are closely related to the gut microbiota in exerting their immunomodulatory and anti-tumor effects. Natural polysaccharide products can improve gut microbiota structure, promote the growth of beneficial bacteria and inhibit the proliferation of opportunistic pathogens, while simultaneously promoting the production of short-chain fatty acids, tryptophan, and other gut microbiota metabolites, thereby regulating the body's immune system and enhancing anti-tumor immune responses. Therefore, improving the body's immune function through gut microbiota regulation has become one of the important mechanisms by which natural polysaccharides exert their anti-tumor effects.

[0006] Although the aforementioned studies indicate that *Sophora japonica* polysaccharide can exert anti-tumor effects by regulating the immune system and gut microbiota, certain limitations remain in its practical application. Because *Sophora japonica* polysaccharide is easily released prematurely in the gastrointestinal environment, its retention time in the colon is relatively short, resulting in insufficient contact time with the gut microbiota and hindering its ability to fully exert its effects in regulating gut microbiota and enhancing immune responses. Therefore, how to increase the retention time of *Sophora japonica* polysaccharide in the colon, enabling it to fully act on the gut microbiota and further enhance its immunomodulatory and anti-tumor effects, has become one of the urgent problems to be solved in current research.

[0007] Eudragit is a trade name for a class of polymeric materials composed of methacrylate copolymers and methacrylic acid copolymers in varying proportions, collectively known as acrylic resins. Due to their excellent biocompatibility, non-toxicity, non-irritation, and non-absorption by the human body, they are widely used as important pharmaceutical excipients in the field of pharmaceutical formulations, commonly used in sustained-release and controlled-release matrix materials and drug coating materials. Therefore, constructing a colon-targeted drug delivery system for Sophora japonica polysaccharides using Eudragit holds promise for increasing the retention time of Sophora japonica polysaccharides in the colon, achieving sufficient regulation of the intestinal flora, thereby enhancing its immunomodulatory capabilities and improving its anti-tumor therapeutic effects. Summary of the Invention

[0008] The purpose of this invention is to provide a colon-targeted microemulsion formulation of Sophora japonica polysaccharide and its preparation method, in order to solve the problems mentioned in the background art.

[0009] To achieve the above objectives, the present invention provides the following technical solution:

[0010] On one hand, the present invention provides a colon-targeted microemulsion formulation of Sophora japonica polysaccharide, comprising the following raw materials in the following mass percentages: oil phase 2%-8%, surfactant 10%-45%, co-surfactant 10%-30%, enteric-coated polymeric material 0.1%-3%, and Sophora japonica polysaccharide aqueous solution to make up the remainder to 100%;

[0011] The enteric polymer material is Eudragit S100.

[0012] On the other hand, the present invention provides a method for preparing a colon-targeted microemulsion formulation of Sophora japonica polysaccharide, comprising the following steps:

[0013] S1. Add a surfactant to the oil phase and mix it thoroughly under stirring conditions to form a homogeneous transparent or semi-transparent system;

[0014] S2. Preparation of polysaccharide aqueous phase: Prepare an aqueous solution of Sophora japonica polysaccharide as the aqueous phase. Under continuous stirring, slowly add the aqueous phase dropwise to the system obtained in S1 so that the system gradually forms a uniformly dispersed emulsion system.

[0015] S3. Dissolve the enteric polymer material in the co-surfactant, and then sonicate it to completely dissolve and form a homogeneous solution. Add the solution to the emulsion system obtained in S2 under stirring conditions to make the enteric polymer material uniformly dispersed in the emulsion system.

[0016] S4. Continue stirring at room temperature to form a stable, transparent or semi-transparent microemulsion system, thus obtaining the Sophora japonica polysaccharide colon-targeting microemulsion formulation.

[0017] On the other hand, the present invention provides the application of Sophora japonica polysaccharide colon-targeting microemulsion formulation in the preparation of anti-liver cancer drugs.

[0018] Compared with the prior art, the specific beneficial effects of the present invention are as follows:

[0019] (1) This invention constructs a stable Sophora japonica polysaccharide microemulsion delivery system by optimizing the proportions of oil phase, surfactant, co-surfactant and water phase. By introducing methacrylic acid copolymer enteric polymer material, the resulting microemulsion system has good colon-targeted release characteristics, which is conducive to the effective delivery of Sophora japonica polysaccharide in the colon and improves the utilization efficiency of active ingredients.

[0020] (2) Compared with non-targeted formulations, the targeted microemulsion formulations provided by the present invention can significantly prolong the retention time of active ingredients in the colon and can regulate the structure of intestinal flora, which helps to improve the intestinal microecological environment and increase the abundance of probiotics.

[0021] (3) Immunological studies have shown that the targeted microemulsion formulation provided by this invention can improve the body's anti-tumor immune response and increase IFN-γ levels. + CD8 + T cells (CD8 cells expressing IFN-γ) + T cells) and GZMB + CD8 + T cells (CD8 cells expressing granzyme B) + The proportion of immune effector cells such as T cells can enhance the body's immune response.

[0022] (4) In in vivo anti-tumor experiments, the targeted microemulsion formulation provided by the present invention showed good anti-tumor effects in both the orthotopic liver cancer model and the spontaneous liver cancer model. It can inhibit the development of tumors to a certain extent, prolong the survival time of experimental animals, and improve the level of immune cells, indicating that the formulation has good application prospects in the treatment of liver cancer. Attached Figure Description

[0023] Figure 1 The particle size distribution and morphology of the Sophora japonica polysaccharide colon-targeting microemulsion preparation provided in the embodiments of the present invention are shown in Figure A, which is a particle size distribution diagram and Figure B is a transmission electron microscope image.

[0024] Figure 2 A comparison diagram of colonic retention of Sophora japonica polysaccharide colon-targeted microemulsion formulation and non-targeted microemulsion formulation provided in embodiments of the present invention;

[0025] Figure 3 For the comparison of mouse gut microbiota structure provided in this embodiment of the invention, compared with the SS group, **p < 0.01, compared with the HP group, ## p < 0.01;

[0026] Figure 4 The analysis results of the immunomodulatory effect of the Sophora japonica polysaccharide colon-targeting microemulsion preparation provided in the embodiments of the present invention are shown in Figure A, where A represents IFN-γ. + CD8 + T cells and GzmB + CD8 + T cells, compared with the SS group, **p < 0.01, compared with the HP group, ## p < 0.01;

[0027] Figure 5 The results of the *Sophora japonica* polysaccharide colon-targeted microemulsion formulation inhibiting in situ tumor development provided in this embodiment of the invention are shown. A represents tumor development, and B represents the survival rate of tumor-bearing mice. Compared with the SS group, *p < 0.05; compared with the HP group, ## p < 0.01;

[0028] Figure 6 The results of the colon-targeted microemulsion formulation of Sophora japonica polysaccharide provided in this embodiment of the invention inhibiting the development of spontaneous hepatocellular carcinoma are shown. A represents the mouse liver coefficient, B represents the mouse spleen weight, and NS indicates no significant difference. Compared with the α-PD-1 group, **p < 0.01, and compared with the HP-SMS group, ## p < 0.01. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0030] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0031] Example 1: A colon-targeting microemulsion formulation of Sophora japonica polysaccharide, the preparation method of which includes the following steps:

[0032] S1. Weigh out 7.3% isopropyl myristate as the oil phase, and add 10.95% Tween 80 and 32.85% Span 80, and stir thoroughly.

[0033] S2. Prepare a 2% Sophora japonica polysaccharide aqueous solution using double-distilled water and Sophora japonica polysaccharide (Shaanxi Ciyuan Biotechnology Co., Ltd., purity ≥ 95%). Take a 26.28% Sophora japonica polysaccharide aqueous solution as the aqueous phase and slowly add it dropwise to the system obtained in S1 under stirring at room temperature, and stir until uniform.

[0034] S3. Weigh 0.73% Eudragit S100 and dissolve it in 21.89% anhydrous ethanol by sonication (25℃, 10 min) to obtain the alcohol phase. Under stirring (25℃, 300 rpm), slowly add the alcohol phase dropwise to the mixture obtained in S2.

[0035] S4. Continue stirring at room temperature for 90 minutes to fully emulsify the system, thus obtaining the colon-targeted microemulsion formulation of Sophora japonica polysaccharide.

[0036] The particle size distribution and morphology of the colon-targeted microemulsion formulation of Sophora japonica polysaccharide were analyzed, and the results are as follows: Figure 1 As shown, the particle size distribution is uniform, the dispersibility is good and there is no aggregation, with a particle size of 392 nm. The polydispersity index of the formulation was measured to be 0.26 using a Malvern laser particle size analyzer. The polysaccharide encapsulation rate was 96% as determined by the sulfuric acid-phenol method.

[0037] Example 2: Colon-targeting study of colon-targeting microemulsion formulation of Sophora japonica polysaccharide:

[0038] Following the preparation method of Example 1, a non-targeted microemulsion formulation of Sophora japonica polysaccharide without Eudragit S100 was prepared (when preparing the non-targeted microemulsion formulation, only Eudragit S100 was removed, the mass fraction of the remaining raw material components remained unchanged, and Sophora japonica polysaccharide aqueous solution was added to make up to 100%). The microemulsion formulation was labeled with DiD fluorescent dye. The grouping scheme was as follows: saline group (SS), non-targeted microemulsion formulation of Sophora japonica polysaccharide (HP-NT-SMS), and targeted microemulsion formulation of Sophora japonica polysaccharide (HP-SMS). Mice in the experimental groups were administered the drug orally via gavage (the dosage for both the non-targeted and targeted microemulsion formulations was calculated based on Sophora japonica polysaccharide 100 mg / kg, according to the mice's body weight). The non-targeted formulation served as a control. Intestinal tissue samples were collected at predetermined time points (0h, 6h, and 12h) and analyzed via IVIS.® The small animal in vivo imaging system was used to perform fluorescence imaging of intestinal tissue. By comparing and analyzing the fluorescence distribution in the colon of the experimental group and the control group, the colon-targeting performance of the targeted microemulsion formulation was evaluated.

[0039] The results are as follows Figure 2 As shown, the fluorescence signal intensity in the colon of mice in the Sophora japonica polysaccharide targeted microemulsion formulation group was significantly higher than that in the Sophora japonica polysaccharide non-targeted microemulsion formulation group, indicating that the Sophora japonica polysaccharide colon-targeted microemulsion formulation provided in the examples has good targeted accumulation ability in the colon.

[0040] Example 3: The regulatory effect of Sophora japonica polysaccharide colon-targeted microemulsion on intestinal flora:

[0041] A mouse orthotopic liver cancer model was established using H22-luc cells (Zhongqiao Xinzhou Biotechnology Co., Ltd.) (refer to Z.Yu, Guo J., Hu M., Gao Y., Huang L. Icaritin Exacerbates Mitophagy and Synergizes with Doxorubicin to Induce Immunogenic Cell Death in Hepatocellular Carcinoma. ACS Nano. 14(4) (2020), pp. 4816-28). The mice were divided into three groups according to the predetermined administration regimen: saline group (SS), Sophora japonica polysaccharide group (HP), and Sophora japonica polysaccharide targeted microemulsion preparation group (HP-SMS). The dosage of Sophora japonica polysaccharide group and Sophora japonica polysaccharide targeted microemulsion preparation group was 100 mg / kg of Sophora japonica polysaccharide, administered orally by gavage. Within one week after the administration, mouse fecal samples were collected for 16S rRNA gene sequencing analysis. The regulatory ability of the microemulsion preparation on probiotics was evaluated by comparing the relative abundance of bacterial genera in different treatment groups.

[0042] The results are as follows Figure 3 As shown in the figure, the Sophora japonica polysaccharide colon-targeted microemulsion preparation provided in the example can effectively increase the abundance of Lactobacillus probiotics in the mouse gut when administered orally. This confirms that the targeted microemulsion preparation has a more significant effect on regulating probiotics than the non-targeted preparation, and can effectively improve the intestinal microecological environment, showing potential application value in regulating intestinal flora and promoting probiotic growth.

[0043] Example 4: Study on the immunomodulatory effect of Sophora japonica polysaccharide colon-targeted microemulsion preparation:

[0044] A mouse orthotopic liver cancer model was established using H22-luc cells, and mice were divided into three groups according to a predetermined administration regimen: saline group (SS), Sophora japonica polysaccharide group (HP), and Sophora japonica polysaccharide targeted microemulsion group (HP-SMS). The drugs were administered orally by gavage. Within one week after administration, mouse tumor tissues were collected, dissociated into single-cell suspensions, and functional CD8+ was detected by flow cytometry. + The number of T cells;

[0045] The results are as follows Figure 4 As shown, IFN-γ in mouse tumor tissue treated with Sophora japonica polysaccharide targeted microemulsion formulation + CD8 + T cells and GzmB + CD8 + The proportion of T cells was significantly higher in the saline group and the Sophora japonica polysaccharide group, indicating that the preparation can effectively activate anti-tumor immune response, enhance the function of cytotoxic T lymphocytes, and has a tumor immunomodulatory effect.

[0046] Example 5: Study on the inhibition of in situ hepatocellular carcinoma development by Sophora japonica polysaccharide colon-targeted microemulsion preparation:

[0047] A mouse orthotopic liver cancer model was established using H22-luc cells, and the mice were divided into three groups according to a predetermined drug administration regimen: saline group (SS), Sophora japonica polysaccharide group (HP), and Sophora japonica polysaccharide targeted microemulsion group (HP-SMS). The drugs were administered orally via gavage and administered via IVIS. ® Small animal in vivo imaging technology is used to periodically monitor tumor development and plot tumor growth curves. During the experiment, the survival status of mice is recorded and mouse survival curves are plotted.

[0048] The results are as follows Figure 5 As shown, the Sophora japonica polysaccharide targeted microemulsion formulation exhibited good anti-tumor activity in the orthotopic liver cancer model. It not only effectively inhibited tumor growth but also prolonged the survival of tumor-bearing mice, demonstrating the potential application value of this formulation in the treatment of liver cancer.

[0049] Example 6: Study on the inhibition of spontaneous liver cancer development by Sophora japonica polysaccharide colon-targeted microemulsion preparation:

[0050] A liver cancer model was established using DEN combined with carbon tetrachloride (CCl4). Sexually mature male and female BALB / c mice were selected, and 2-week-old BALB / c mice were bred. Mice were first induced by a single intraperitoneal injection of diethylnitrosamine (DEN, 25 mg / kg). After 6 weeks, they were injected intraperitoneally twice weekly with a CCl4 / olive oil mixture (volume ratio 1:4) at a dose of 2 mL / kg, continuing until week 18. During this period, liver cancer formation was observed by dissection. Once the model was successfully established, the mice were administered the drug orally via gavage according to a specific regimen (administered every other day for a total of 9 times). The specific grouping schemes were: saline group (SS), anti-PD-1 antibody (α-PD-1) group, Sophora japonica polysaccharide targeted microemulsion group (HP-SMS), and combination therapy group (HP-SMS+α-PD-1). At the experimental endpoint, the mice were weighed, sacrificed, and their livers and spleens were removed, weighed, and photographed.

[0051] The results are as follows Figure 6 As shown, the liver coefficient of mice in the Sophora japonica polysaccharide targeted microemulsion preparation group decreased, indicating that the preparation can effectively reduce the tumor burden in the liver, thus demonstrating a good anti-hepatocellular carcinoma effect; the spleen mass of mice increased, indicating that the body's immunity was activated, suggesting that the Sophora japonica polysaccharide targeted microemulsion preparation group can stimulate the body's immune response to a certain extent, thereby enhancing the body's anti-tumor immune capacity; in addition, the above effects were further enhanced when the Sophora japonica polysaccharide targeted microemulsion preparation group was combined with α-PD-1, indicating that it may improve the sensitivity to immunotherapy by improving the immune microenvironment.

[0052] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A colon-targeted microemulsion formulation of Sophora japonica polysaccharide, characterized in that, The raw materials include the following percentages by weight: oil phase 2%-8%, surfactant 10%-45%, co-surfactant 10%-30%, enteric-coated polymer material 0.1%-3%, and Sophora japonica polysaccharide aqueous solution to make up the remainder to 100%; The enteric polymer material is Eudragit S100.

2. The *Sophora japonica* polysaccharide colon-targeting microemulsion formulation according to claim 1, characterized in that, The oil phase is a tetradecanoate ester.

3. The *Sophora japonica* polysaccharide colon-targeting microemulsion formulation according to claim 1, characterized in that, The surfactant is any one or a mixture of several of the following: Tween 20, Tween 40, Tween 60, Tween 80, Span 20, Span 40, Span 60, Span 80, poloxamer 188, and polyoxyethylene hydrogenated castor oil.

4. The *Sophora japonica* polysaccharide colon-targeting microemulsion formulation according to claim 1, characterized in that, The concentration of the Sophora japonica polysaccharide aqueous solution is 0.1%-5%.

5. The *Sophora japonica* polysaccharide colon-targeting microemulsion formulation according to claim 1, characterized in that, The co-surfactant is any one or a mixture of several of anhydrous ethanol, polyethylene glycol 200, polyethylene glycol 400, and glycerol.

6. A method for preparing a colon-targeted microemulsion formulation of Sophora japonica polysaccharide as described in any one of claims 1-5, characterized in that, Includes the following steps: S1. Add a surfactant to the oil phase and mix it thoroughly under stirring conditions to form a homogeneous transparent or semi-transparent system; S2. Preparation of polysaccharide aqueous phase: Prepare an aqueous solution of Sophora japonica polysaccharide as the aqueous phase. Under continuous stirring, slowly add the aqueous phase dropwise to the system obtained in S1 so that the system gradually forms a uniformly dispersed emulsion system. S3. Dissolve the enteric polymer material in the co-surfactant, and then sonicate it to completely dissolve and form a homogeneous solution. Add the solution to the emulsion system obtained in S2 under stirring conditions to make the enteric polymer material uniformly dispersed in the emulsion system. S4. Continue stirring at room temperature to form a stable, transparent or semi-transparent microemulsion system, thus obtaining the Sophora japonica polysaccharide colon-targeting microemulsion formulation.

7. The method for preparing the Sophora japonica polysaccharide colon-targeting microemulsion formulation according to claim 6, characterized in that, In S3, the ultrasonic treatment temperature is 20-30℃ and the time is 5-60 min; During the process of adding the solution to the emulsion system obtained in S2 under stirring conditions, the stirring speed is 200-600 rpm and the temperature is 20-30℃.

8. The method for preparing the Sophora japonica polysaccharide colon-targeting microemulsion formulation according to claim 6, characterized in that, In S4, the stirring process continues at room temperature with a speed of 200-600 rpm for 0.5-4 h.

9. The use of the Sophora japonica polysaccharide colon-targeting microemulsion formulation as described in any one of claims 1-5 in the preparation of an anti-liver cancer drug.

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