A coated drug loaded with limonin and its preparation method and application

Abstract

The present invention discloses a limonin-loaded coated drug, its preparation method, and application. The coated drug comprises a coating shell and an effective component located within the coating shell. The effective component comprises a first exosome loaded with limonin and a second exosome loaded with a hydroxy acid. The limonin-loaded coated drug provided by the present invention effectively alleviates intestinal inflammation by first encapsulating the limonin within the exosomes, then within the coating shell, and simultaneously using the exosomes and the carboxylic acid.

Description

Technical Field

[0001] The present invention relates to the technical field of coated limonin, in particular to a coated medicine loaded with limonin, and a preparation method and application thereof. Background Art

[0002] Currently, intestinal inflammation is treated with conventional anti-inflammatory drugs, which cannot act directly on the intestinal tract. In addition, oral anti-inflammatory drugs may be dissolved in the stomach or before reaching the intestines. Their efficacy acts at various locations in the digestive system and cannot be precisely located in the intestines to resolve intestinal inflammation.

[0003] Limonin exhibits numerous biological activities, including anti-tumor, insect repellent, antiviral, analgesic, anti-inflammatory, and hypnotic properties, and can be used as a functional food additive, anti-cancer food, pesticide, and feed additive. However, limonin can irritate the gastrointestinal tract, causing symptoms such as nausea, vomiting, and diarrhea. Therefore, its use as a food or medicine may stimulate gastric acid secretion and cause side effects. Therefore, it is only used as a small additive and cannot be taken directly as a food or medicine. Summary of the Invention

[0004] The present invention aims to address, at least to some extent, one of the problems in the related art. To this end, the present invention provides a limonin-loaded encapsulated drug that effectively alleviates intestinal inflammation by encapsulating the limonin in exosomes, which are then encapsulated in a coating shell, and used in conjunction with the exosomes and carboxylic acid.

[0005] A limonin-loaded coated drug comprises a coating shell and an effective component located in the coating shell, wherein the effective component comprises a first exosome loaded with limonin and a second exosome loaded with a hydroxy acid.

[0006] Furthermore, the hydroxy acid comprises the following components in parts by weight: 35-45 parts of lactic acid, 30-40 parts of malic acid and 15-30 parts of citric acid.

[0007] Furthermore, the drug-encapsulated coating shell is made of polyacrylic acid resin material.

[0008] Furthermore, the coating shell of the coated drug is enteric-coated type I acrylic resin, and the dissolution pH of the coating shell is greater than 5.5.

[0009] Furthermore, the weight ratio of the first exosomes to the second exosomes is 70-80:20-30.

[0010] A method for preparing a limonin-loaded coated drug, comprising:

[0011] loading limonin into exosomes to form first exosomes;

[0012] loading hydroxy acids into exosomes to form second exosomes;

[0013] The first exosomes and the second exosomes are prepared into coated drugs through coating technology.

[0014] Furthermore, the limonin is encapsulated into exosomes to form first exosomes by incubation, electroporation, extrusion, ultrasound or freeze-thaw method.

[0015] Furthermore, the hydroxy acid is encapsulated into exosomes by incubation, electroporation, extrusion, ultrasound, or freeze-thaw method to form a second exosome.

[0016] Application of a limonin-loaded coated drug in the field of treating enteritis.

[0017] The technical solution provided by the embodiments of the present application has the following advantages over the prior art: after the encapsulated drug enters the human body, its outer coating shell is decomposed, allowing the internal active ingredients to be released. The active ingredients include a first exosome loaded with limonin and a second exosome loaded with hydroxy acid. The exosomes contain cell-specific proteins, lipids, and nucleic acids, which play a role in regulating cell state and function. At the same time, the exosomes also contain rich biological factors that can promote cell metabolism, activate cell regeneration, and repair damaged tissues. When the exosomes are gradually dissolved, the limonin and carboxyl acids encapsulated therein are released. Under the coordinated action of the exosome biological factors, the carboxyl acids can improve the intestinal environment and alleviate intestinal problems caused by various reasons such as intestinal flora disorders and redox imbalance. The limonin has anti-inflammatory effects on the intestine after the improved environment. The encapsulated drug in the present application can be released at a specific location. Based on the improvement of the exosome biological factors and carboxyl acids, the limonin can effectively reduce inflammation in the intestine, showing a targeted and efficient anti-inflammatory effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Schematic diagram of the results of the in vitro release test in Experimental Example 1. DETAILED DESCRIPTION

[0019] The following examples are intended to illustrate the present invention but are not intended to limit the scope of the present invention. Any modifications or substitutions to the methods, steps, or conditions of the present invention, without departing from the spirit and substance of the present invention, fall within the scope of protection of the present invention. Unless otherwise specified, the experimental materials, reagents, and instruments used in the examples of the present invention are commercially available; unless otherwise specified, all technical means in the examples of the present invention are conventional means well known to those skilled in the art.

[0020] The present application provides a limonin-loaded encapsulated drug comprising a coating shell and an active ingredient within the coating shell. The active ingredient comprises a first exosome loaded with limonin and a second exosome loaded with a hydroxy acid. The weight ratio of the first exosome to the second exosome is 70-80:20-30. Specifically, the hydroxy acid comprises the following components in parts by weight: 35-45 parts lactic acid, 30-40 parts malic acid, and 15-30 parts citric acid. Carboxylic acids can improve the intestinal environment and alleviate intestinal problems caused by various factors, including intestinal flora disturbances and redox imbalances. Exosomes contain cell-specific proteins, lipids, and nucleic acids, which regulate cell status and function. Exosomes also contain a rich source of biological factors that can promote cell metabolism, activate cell regeneration, and repair damaged tissues. Limonin has an anti-inflammatory effect on the improved intestinal environment, and this anti-inflammatory effect is based on the biological activity of the exosomes and the improvement of the intestinal environment by the carboxylic acids.

[0021] In this application, exosomes are used as oral medications, specifically milk exosomes. Milk exosomes are isolated and purified from fresh milk, with a diameter of approximately 30 to 200 nm. They are rich in the same phospholipid bilayer structure as the cell membrane, tightly encapsulating specific proteins such as cytokines and growth factors, lipids, miRNAs, and DNA fragments. They can quickly penetrate the cell membrane barrier and accurately identify and locate cell damage. Milk exosomes have a unique microvesicle category that maintains the integrity of the nucleic acids and proteins contained within them while passing through the stomach and gastrointestinal tract, allowing them to act locally or be transported into the circulatory system.

[0022] The drug-encapsulated shell described in this application is made of a polyacrylic acid resin material, which is used as a film coating material for the drug-encapsulated drug. By selecting different types of polyacrylic acid resins, it is possible to ensure that the encapsulated drug dissolves in different environments, thereby achieving targeted and effective release of the active ingredients within the encapsulated drug.

[0023] Specifically, the drug-encapsulated shell is an enteric-coated acrylic resin No. 1, with a dissolution pH greater than 5.5. In the human digestive system, the pH of gastric fluid is 2; the pH of small intestinal fluid is 6.8; and the pH of cecal fluid is 7.5. After the encapsulated drug is taken, it is first eroded by gastric fluid before entering the intestinal fluid environment. The enteric-coated acrylic resin No. 1 does not dissolve in the stomach acid environment, but only begins to dissolve in the intestines, releasing the active ingredients within the encapsulated drug, thereby achieving targeted and effective drug release.

[0024] The present application provides a method for preparing a limonin-loaded coated drug, comprising:

[0025] S1: Limonin is loaded into exosomes to form the first exosomes.

[0026] Specifically, limonin is encapsulated into exosomes by incubation, electroporation, extrusion, ultrasound, or freeze-thaw methods to form first exosomes. For example, when encapsulating by incubation, an exosome suspension and a limonin solution are mixed and incubated at a certain temperature for a certain period of time. The solution is then placed in an ultrafiltration tube with a certain molecular weight cutoff and washed three times by ultrafiltration using a biocompatible medium to obtain the limonin-loaded first exosomes. The concentration of the limonin solution is 15.6 μL / mL.

[0027] S2: Loading hydroxy acids into exosomes to form second exosomes.

[0028] Specifically, the hydroxy acid is encapsulated into exosomes to form the second exosomes by incubation, electroporation, extrusion, ultrasound, or freeze-thaw methods. For example, when electroporation is used for encapsulation, an exosome suspension is mixed with a carboxylic acid solution and an electroporation solution, placed in an electroporator, and electroporated for a certain time and under certain conditions. Subsequently, the solution is placed in an ultrafiltration tube with a certain molecular weight cutoff and washed three times by ultrafiltration using a biocompatible medium to obtain the first exosomes loaded with the carboxylic acid.

[0029] S3: The first exosomes and the second exosomes are prepared into coated drugs through coating technology.

[0030] The present application provides an application of a coated drug loaded with limonin, which can achieve targeted release of the active ingredient according to the selected exosome type and coating shell material type. When the coating shell of the coated drug is enteric-coated type I acrylic resin, since the dissolution pH of the coating shell is greater than 5.5, the coated drug is used in the technical field of enteritis treatment.

[0031] Example 1

[0032] This example provides a limonin-loaded coated drug, comprising a coating shell and an active ingredient within the coating shell. The active ingredient comprises a first exosome loaded with limonin and a second exosome loaded with a hydroxy acid. The weight ratio of the first exosome to the second exosome is 80:20. Specifically, the hydroxy acid comprises the following components in parts by weight: 35 parts lactic acid, 40 parts malic acid, and 25 parts citric acid. In this example, the exosomes are specifically milk exosomes, and the coating shell of the coated drug is enteric-coated acrylic resin No. 1.

[0033] This embodiment provides a method for preparing a limonin-loaded coated drug, comprising:

[0034] S1: Limonin is loaded into exosomes by incubation to form the first exosomes. The concentration of the limonin solution is 15.6 μL / mL.

[0035] S2: The hydroxy acid is loaded into the exosomes by electroporation to form a second exosome; the hydroxy acid comprises the following components in parts by weight: 35 parts of lactic acid, 40 parts of malic acid, and 25 parts of citric acid.

[0036] S3: Weigh the first exosomes and the second exosomes in a weight ratio of 80:20, and prepare them into coated drugs by coating technology. The coating shell of the coated drug is enteric-coated type I acrylic resin.

[0037] In this embodiment, the coated drug loaded with limonin is used in the treatment of enteritis.

[0038] Example 2

[0039] This example provides a limonin-loaded coated drug, comprising a coating shell and an active ingredient within the coating shell. The active ingredient comprises a first exosome loaded with limonin and a second exosome loaded with a hydroxy acid. The weight ratio of the first exosome to the second exosome is 70:30. Specifically, the hydroxy acid comprises the following components in parts by weight: 45 parts lactic acid, 30 parts malic acid, and 25 parts citric acid. In this example, the exosomes are specifically milk exosomes, and the coating shell of the coated drug is enteric-coated acrylic resin No. 1.

[0040] This embodiment provides a method for preparing a limonin-loaded coated drug, comprising:

[0041] S1: Limonin is loaded into exosomes by incubation to form the first exosomes. The concentration of the limonin solution is 15.6 μL / mL.

[0042] S2: The hydroxy acid is loaded into the exosomes by electroporation to form a second exosome; the hydroxy acid comprises the following components in parts by weight: 45 parts of lactic acid, 30 parts of malic acid, and 25 parts of citric acid.

[0043] S3: Weigh the first exosomes and the second exosomes in a weight ratio of 70:30, and prepare them into coated drugs by coating technology. The coating shell of the coated drug is enteric-coated type I acrylic resin.

[0044] In this embodiment, the coated drug loaded with limonin is used in the treatment of enteritis.

[0045] Example 3

[0046] This example provides a limonin-loaded coated drug, comprising a coating shell and an active ingredient within the coating shell. The active ingredient comprises a first exosome loaded with limonin and a second exosome loaded with a hydroxy acid. The weight ratio of the first exosome to the second exosome is 76:24. Specifically, the hydroxy acid comprises the following components in parts by weight: 43 parts lactic acid, 37 parts malic acid, and 20 parts citric acid. In this example, the exosomes are specifically milk exosomes, and the coating shell of the coated drug is enteric-coated acrylic resin No. 1.

[0047] This embodiment provides a method for preparing a limonin-loaded coated drug, comprising:

[0048] S1: Limonin is loaded into exosomes by incubation to form the first exosomes. The concentration of the limonin solution is 15.6 μL / mL.

[0049] S2: The hydroxy acid is loaded into the exosomes by electroporation to form a second exosome; the hydroxy acid includes the following components in parts by weight: 43 parts of lactic acid, 37 parts of malic acid, and 20 parts of citric acid.

[0050] S3: Weigh the first exosomes and the second exosomes in a weight ratio of 76:24, and prepare them into coated drugs by coating technology. The coating shell of the coated drug is enteric-coated type I acrylic resin.

[0051] Comparative Example 1

[0052] The difference from Example 2 is that only the first exosomes are included, and the second exosomes are not included.

[0053] Experimental Example 1

[0054] In order to study whether the coated drugs can be released at a specific point in the intestine, the experiment used coated blank pills as the research objects. The absorbance values ​​were detected after incubation at different time points and in different intestinal fluids to reflect the release pattern.

[0055] Determination of in vitro release patterns of encapsulated drugs

[0056] (1) The specific determination method is:

[0057] Weigh 100 mg of blank drug and add 200 μL of gastric juice, small intestinal juice, and cecal juice, respectively. The pH of gastric juice is 2; the pH of small intestinal juice is 6.8; and the pH of cecal juice is 7.5. The mixture is incubated at 80 rpm and 37°C throughout. First, gastric juice is added to all blank drugs. Three tubes of the mixture are removed at 0.5 h, 1 h, 1.5 h, and 2 h, respectively, and stored at 4°C. The gastric juice is aspirated from the remaining tubes and replaced with small intestinal juice, and incubation continues. Three tubes of the mixture are removed at 3 h, 4 h, 5 h, and 6 h, respectively, and stored at 4°C. The small intestinal juice is aspirated from the remaining tubes and replaced with cecal juice, and incubation continues. Three tubes of the mixture are removed at 7 h, 8 h, 9 h, 10 h, 11 h, and 12 h, respectively, and stored at 4°C. All samples are then removed from the 4°C tubes, and 100 μL of the supernatant is aspirated and measured for OD600 on a multifunctional microplate reader.

[0058] (2) The results are as follows Figure 1 As shown. Figure 1 It can be seen that the in vitro release test shows that the release rate of blank pills in gastric juice and small intestinal juice is less than 10%, but it can be completely released in large intestinal juice.

[0059] Experimental Example 2

[0060] Evaluation of inflammation reduction efficacy (neutrophils)

[0061] 3dpf transgenic neutrophil green fluorescent MPX strain zebrafish were randomly selected in a multi-well plate, and 30 zebrafish were treated in each well (experimental group). Water solution was given to the positive control group, normal control group, model control group, experimental group 1, test group 2, experimental group 3, and comparison group 1 respectively. The normal control group was zebrafish without inflammation and did not receive any treatment. Experimental group 1 was the solution after the coated pills in Example 1 were dissolved by the small intestinal fluid at pH = 6.8, and then copper sulfate pentahydrate was given; experimental group 2 was the solution after the coated pills in Example 2 were dissolved by the small intestinal fluid at pH = 6.8, and then copper sulfate pentahydrate was given; experimental group 3 was the solution after the coated pills in Example 3 were dissolved by the small intestinal fluid at pH = 6.8, and then copper sulfate pentahydrate was given. Control group 1 was the solution after the coated pills in comparative example 1 were dissolved by the small intestinal fluid at pH = 6.8, and then copper sulfate pentahydrate was given. The model group was the copper sulfate pentahydrate solution. The purpose of copper sulfate pentahydrate is to color the neutrophils.

[0062] Among them, zebrafish were raised in fish farming water at 28°C (water quality: 200 mg of instant sea salt was added to every 1 L of reverse osmosis water, conductivity was 450-550 μS / cm; pH was 6.5-8.5; hardness was 50-100 mg / L CaCO3).

[0063] A positive control of indomethacin was used at a concentration of 60.0 μM. A normal control group and a model control group were also established, with a volume of 3 mL per well. After treatment at 28°C for 1 hour, all experimental groups except the normal control group were treated with water-soluble copper sulfate pentahydrate to establish a zebrafish inflammation model. After treatment at 28°C for another 2 hours, 10 zebrafish were randomly selected from each experimental group and photographed under a fluorescence microscope. Data were analyzed and collected using NIS-Elements D 3.20 advanced image processing software. Neutrophil counts at the inflamed sites of the zebrafish were analyzed, and the anti-inflammatory efficacy of the samples was evaluated using statistical analysis of this indicator. Statistical results are expressed as mean ± SE. Statistical analysis was performed using SPSS 26.0 software. A p < 0.05 indicated statistical significance. The test results are shown in Table 1.

[0064] Table 1 Evaluation of inflammation-reducing efficacy - Neutrophil test results (n=10)

[0065]

[0066]

[0067] Compared with the model control group, *p<0.05, ***p<0.001

[0068] Combined with Table 1, it can be seen that the anti-inflammatory effects of experimental group 1, experimental group 2, experimental group 3, and comparison group 1 are worse than those of the model control group and the positive control group; at the same time, the anti-inflammatory effect of comparison group 1 is not as good as that of experimental group 1, experimental group 2, and experimental group 3. This is because carboxylic acid can improve the intestinal environment and alleviate intestinal problems caused by intestinal flora disorders and redox imbalance caused by various reasons; limonin can achieve better anti-inflammatory effects by targeting the intestine after the environment is improved, that is, carboxylic acid and limonin have a synergistic effect in anti-inflammatory applications.

[0069] In summary, after the encapsulated drug in the present application enters the human body, its outer coating shell is decomposed, so that the internal active ingredients are released. The active ingredients include a first exosome loaded with limonin and a second exosome loaded with hydroxy acid. The exosomes contain cell-specific proteins, lipids and nucleic acids, which play a role in regulating cell state and function. At the same time, the exosomes also contain rich biological factors that can promote cell metabolism, activate cell regeneration, and repair damaged tissues. When the exosomes are gradually dissolved, the limonin and carboxyl acids encapsulated therein are released. Under the coordinated action of the exosome biological factors, the carboxyl acids can improve the intestinal environment and alleviate intestinal problems caused by various reasons such as intestinal flora disorders and redox imbalance. Limonin has anti-inflammatory effects on the intestine after the environment is improved. The encapsulated drug in the present application can be released at a specific point. Based on the improvement of the exosome biological factors and carboxyl acids, limonin can achieve effective anti-inflammatory effects on the intestine, and has a specific and efficient anti-inflammatory effect.

[0070] It is understandable that the above embodiments only express the preferred implementation modes of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the patent scope of the present invention. It should be pointed out that for ordinary technicians in this field, without departing from the concept of the present invention, the above technical features can be freely combined, and several deformations and improvements can be made, all of which fall within the scope of protection of the present invention. Therefore, all equivalent changes and modifications made to the scope of the claims of the present invention should fall within the scope of coverage of the claims of the present invention.

Claims

1. A coated drug loaded with limonin, characterized in that: include: The invention relates to a coating shell and an effective component located in the coating shell, wherein the effective component includes a first exosome loaded with limonin and a second exosome loaded with a hydroxy acid; the hydroxy acid includes the following components in parts by weight: 35-45 parts of lactic acid, 30-40 parts of malic acid, and 15-30 parts of citric acid; the coating shell of the drug-coated drug is enteric-coated type I acrylic resin, and the dissolution pH of the coating shell is greater than 5.

5.

2. A limonin-loaded coated drug according to claim 1, characterized in that, The weight ratio of the first exosomes to the second exosomes is 70-80:20-30.

3. A method for preparing a coated drug loaded with limonin, characterized in that: include: loading limonin into exosomes to form first exosomes; Loading hydroxy acid into exosomes to form second exosomes; the hydroxy acid comprises the following components in parts by weight: 35-45 parts of lactic acid, 30-40 parts of malic acid, and 15-30 parts of citric acid; The first exosomes and the second exosomes are prepared into coated drugs through coating technology; the coating shell of the coated drug is enteric-coated type I acrylic resin, and the dissolution pH of the coating shell is greater than 5.

5.

4. The method for preparing a limonin-loaded coated drug according to claim 3, wherein Limonin is encapsulated into exosomes to form first exosomes by incubation, electroporation, extrusion, ultrasound or freeze-thaw method.

5. The method for preparing a limonin-loaded coated drug according to claim 3, wherein Hydroxy acids are encapsulated into exosomes to form second exosomes by incubation, electroporation, extrusion, ultrasound or freeze-thaw methods.

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