Enteric sustained-release microcapsule as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of microcapsules, and particularly relates to an enteric sustained-release microcapsule as well as a preparation method and application thereof. A hydrophobic natural drug and zein are used as core materials, carboxymethyl cellulose is used as a shell material, a flash nanoprecipitation method is adopted, at the moment, the hydrophobic natural drug rapidly forms a hydrophobic core after encountering water, and zein dissolved in an ethanol solution is rapidly assembled after encountering water; according to the preparation method of the enteric-coated sustained-release microcapsule, the hydrophobic natural medicine is wrapped through the hydrophobic effect, then the enteric-coated sustained-release microcapsule is prepared through electrostatic binding with carboxymethyl cellulose, the technical defect that the bioavailability is low due to poor water solubility of the hydrophobic natural medicine is overcome, and the enteric-coated sustained-release microcapsule can be used for preparing the enteric-coated sustained-release microcapsule. And the sustained release in intestinal juice is realized.

Description

Technical Field

[0001] The invention belongs to the technical field of microcapsules, and in particular relates to an enteric-coated sustained-release microcapsule and a preparation method and application thereof. Background Art

[0002] With the rapid development of the pharmaceutical industry, the pharmaceutical industry is also facing structural adjustment and development. In the entire pharmaceutical industry, the proportion of the preparation industry is gradually increasing. Microencapsulation as a formulation approach for biological preparations and small molecule treatments has received widespread attention, because a large proportion of small molecule drugs have poor solubility in water, which limits their oral bioavailability. The characteristics of some drugs should be released in special parts such as the stomach or small intestine to exert their efficacy and achieve the purpose of curing diseases. Then the production process for the transportation and storage of such drugs is more stringent, and it must also have a sustained-release effect. Therefore, it is particularly important to explore how to make a suitable targeted sustained-release drug delivery microcapsule.

[0003] Microcapsules are micro-containers or capsule shells with a diameter of micrometers (generally in the range of 5-200pm) and a natural or synthetic polymer wall layer, which improves the stability of bioactive compounds in environments such as oxygen, light and temperature. Microcapsules are regarded as "core-shell structures". The material encapsulated inside the microcapsule is called the core material, and the external wall material is usually a natural or synthetic polymer material. The microcapsule structure can effectively isolate the core material from the external environment, reducing the loss of the core material or the reduction of its functional results due to oxidation. The capsule wall of the microcapsule adsorbs or encapsulates solids, liquids or gases in it, thereby forming a single independent tiny capsule with various shapes, such as spherical, granular, grain-shaped or rod-shaped. According to the different application requirements of the microcapsules, special equipment and technical means can be used to encapsulate active ingredients such as proteins, chemical reagents or drug molecules inside the microcapsules. The biggest feature of microcapsules is that they have sustained release, controlled release and targeting. In today's world, medicine, biology, daily chemicals, food and other fields are still fast-growing hot fields. Luthfia Pratiwi et al. embedded orange peel essential oil and studied the antibacterial ability of the obtained microcapsules on textiles. Qingye Men et al. used microcapsules to study drug delivery and gene delivery in biomedicine. Shigeru Goto made a certain evaluation on the preparation and biopharmaceutical of amoxicillin microcapsules. Microcapsule technology is one of the new and key technologies in these hot fields. In microcapsule technology, the selection of suitable wall materials can greatly improve its application effect. The appropriateness of the wall material selection will directly determine the sustained release, controlled release and compatibility of the microcapsules.

[0004] Cannabidiol (CBD) is a natural compound extracted from cannabis. Although CBD's chemical structure is similar to that of tetrahydrocannabinol, it is non-addictive and has basically no toxic side effects. In addition, cannabidiol has a variety of beneficial effects and has been studied for the treatment of various indications, such as prolonged sleep, anti-inflammatory, anticonvulsant, anti-anxiety and relief of neuropathic pain. CBD is a terpene phenol compound with 21 carbon atoms composed of a cyclohexene ring, a cyclophenol and a pentyl side chain. The properties of CBD are affected by its own structure. Its chemical activity mainly exerts its effects on the hydroxyl group on the phenol ring and the pentyl and methyl groups of the cyclohexene ring, so many formulations containing CBD are widely distributed due to these effects.

[0005] The chemical name of dihydroquercetin is 3,3',4',5,7-pentahydroxyflavone, which is a flavonol compound that is widely distributed in the plant kingdom and has multiple biological activities. Its pharmacological effects are also very extensive. It can scavenge active free radicals, chelate and capture free radicals, and inhibit lipid peroxidation in the body. It also has strong pharmacological activity in anti-tumor, anti-inflammatory, antibacterial, antiviral, and prevention of cardiovascular disease complications. In addition, it has no obvious toxicity and is of great significance for the prevention and treatment of aging, cancer, and cardiovascular diseases, so it has a high research value.

[0006] However, both CBD and dihydroquercetin are hydrophobic small molecules with poor water solubility, resulting in low bioavailability and limiting their efficacy. Summary of the invention

[0007] In view of the deficiencies in the above-mentioned prior art, the present invention provides an enteric-coated sustained-release microcapsule and a preparation method and application thereof. The present invention uses hydrophobic natural medicine and zein as core materials, carboxymethyl cellulose as shell material, and adopts a flash nanoprecipitation method to prepare enteric-coated sustained-release microcapsule, thereby overcoming the technical defect of low bioavailability of cannabidiol, dihydroquercetin or natural derivatives of dihydroquercetin due to poor water solubility.

[0008] To achieve the above object, the technical solution of the present invention is as follows:

[0009] A method for preparing enteric-coated sustained-release microcapsules comprises the following steps:

[0010] The zein is mixed in an ethanol solution with a volume percentage of 90%, and then a hydrophobic natural medicine is added. The hydrophobic natural medicine can be dissolved in anhydrous ethanol. At the same time, since the zein is a hydrophobic material, the zein and the hydrophobic natural medicine can be mutually soluble. After mixing, a core material solution is obtained.

[0011] Carboxymethyl cellulose is dissolved in water to obtain a shell material solution.

[0012] The core material solution and the shell material solution are mixed by the flash nanoprecipitation method. At this time, the hydrophobic natural medicine quickly forms a hydrophobic core when it meets water. The zein dissolved in the ethanol solution quickly assembles when it meets water and wraps the hydrophobic natural medicine through hydrophobic action. It is then electrostatically bonded with carboxymethyl cellulose to obtain enteric-coated sustained-release microcapsules.

[0013] The mass ratio of zein to hydrophobic natural medicine is 1-20:1, and the mass ratio of zein to carboxymethyl cellulose is 0.2-20:1. At these ratios, enteric-coated sustained-release microcapsules can be obtained.

[0014] Preferably, the hydrophobic natural drug is selected from cannabidiol, dihydroquercetin or dihydroquercetin natural derivatives. Although only experiments with cannabidiol were conducted in the present invention, it can be expected that the hydrophobic natural drug using the method of the present invention is not limited to cannabidiol, dihydroquercetin or dihydroquercetin natural derivatives.

[0015] Preferably, the mass ratio of zein to hydrophobic natural medicine is 1 to 10:1. Under this condition, the embedding rate of the hydrophobic natural medicine can reach 50% to 65%.

[0016] Preferably, the mass ratio of zein to the hydrophobic natural medicine is 1:1, under which the embedding rate of the hydrophobic natural medicine can reach 65%.

[0017] Preferably, the mass ratio of zein to carboxymethyl cellulose is 0.2-10:1, under which the embedding rate of the hydrophobic natural medicine can reach 50%-60%.

[0018] Preferably, the mass ratio of zein to carboxymethyl cellulose is 0.25:1, under which the embedding rate of the hydrophobic natural medicine can reach 60%.

[0019] Preferably, the flash nanoprecipitation method is based on a closed impinging flow mixer, which comprises:

[0020] A mixing section 1, the bottom of which is connected to a discharge pipe 3, wherein a high-speed core material solution and a high-speed shell material solution are mixed in the mixing section 1, and the enteric-coated sustained-release microcapsules obtained after mixing flow out through the discharge pipe 3;

[0021] A plurality of flow channels 2 are connected to the mixing part 1, and the core material solution and the shell material solution enter the mixing part 1 through different flow channels 2; the ends of the plurality of flow channels 2 connected to the mixing part 1 are all located on the same cross section of the mixing part 1, so that the high-speed core material solution and the high-speed shell material solution can offset each other, and then quickly realize assembly;

[0022] A plurality of pump bodies, which are respectively installed on a plurality of flow channels 2, and the pump bodies provide power for the core material solution and the shell material solution;

[0023] The controller is electrically connected to a plurality of pump bodies. The pump bodies are regulated to start, close and flow through the controller. The controller is electrically connected to a power source.

[0024] Preferably, the specific operation of the flash nanoprecipitation method is: the core material solution and the shell material solution are respectively added into the two flow channels 2 of the closed impinging flow mixer (CIJ mixer), and after starting the pump body, the core material solution and the shell material solution are mixed and discharged through the discharge pipe 3 to obtain enteric-coated sustained-release microcapsules.

[0025] Preferably, the flow rates of the core material solution and the shell material solution in the flow channel are both 5 mL / min to 40 mL / min. Further, the flow rates are both 30 mL / min, and the flow rates of the core material solution and the shell material solution are equal.

[0026] The present invention also protects the enteric-coated sustained-release microcapsules prepared by the above preparation method. In the enteric-coated sustained-release microcapsules, zein is mixed with a hydrophobic natural drug as the "core" of the enteric-coated sustained-release microcapsules to form a "core-shell structure" with carboxymethyl cellulose.

[0027] The present invention also protects the use of enteric-coated sustained-release microcapsules in the preparation of enteric-coated sustained-release drugs, which can achieve the sustained release of hydrophobic natural drugs in intestinal fluid; the enteric-coated sustained-release microcapsules can be used as the main component of the enteric-coated sustained-release drugs, and the enteric-coated sustained-release microcapsules can also be directly used as enteric-coated sustained-release drugs; when the enteric-coated sustained-release microcapsules are used as the main component, auxiliary materials are also included, and the auxiliary materials include lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] 1. The present invention prepares enteric sustained-release microcapsules by mixing core material solution and shell material solution in a CIJ mixer. The enteric sustained-release microcapsules are cellulose-based microcapsules. Hydrophobic cannabidiol (CBD) dissolved in an organic solvent quickly forms a hydrophobic core after encountering water. Zein dissolved in an organic solvent quickly assembles after encountering water, and CBD is wrapped up by hydrophobic action. When the pH value is less than the isoelectric point (pI) of the protein, zein is positively charged and can form a relatively stable polymer with negatively charged carboxymethyl cellulose (CMC) through electrostatic bonding; zein is hydrophobic and forms a "shell-core structure" with carboxymethyl cellulose through electrostatic bonding, and CBD is embedded in the microcapsules to improve the bioavailability of CBD.

[0030] 2. The present invention uses corn zein as raw material. The central site of Zein has a region rich in glutamine and proline, and the terminal domain has hydrophobic amino acid residues. These unique properties enable Zein to exhibit typical amphiphilicity and to self-assemble into microscopic or nanostructures, making it suitable for providing bioactive ingredients.

[0031] 3. Carboxymethyl cellulose is an anionic polysaccharide derived from cellulose, which usually exists in the form of sodium salt. CMC has the properties of hydrophilicity, pH sensitivity, gelation, economy, biodegradability and good biocompatibility. These unique properties make it widely used in the fields of catalysts, food packaging, protein immobilization, drug release, and water pollution removal. As a non-toxic biopolymer, CMC has been widely studied for its application in delivery systems and has shown significant toxicity reduction effects. During the sustained release process, CMC can prevent drug crystallization or drug degradation. In addition, the frequency of drug release can be increased by controlling the diffusion rate of the drug on the polymer material or the erosion / degradation rate of the polymer. Drug release tests have shown that CMC-coated nanohybrid materials have good gastric pH protection and prolong the drug delivery stability under gastrointestinal conditions.

[0032] 4. The present invention designs a closed impinging flow mixer and adopts the flash nanoprecipitation method to prepare enteric-coated sustained-release microcapsules, realizes the hydrophilic shell material carboxymethyl cellulose coating the hydrophobic core material, and reveals the combination mode of CBD with carboxymethyl cellulose and zein, which provides a new idea for the subsequent preparation of cellulose-based microcapsules encapsulating hydrophobic drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Schematic diagram of preparing enteric-coated sustained-release microcapsules by flash nanoprecipitation method in CIJ mixer in Examples 1 to 17.

[0034] Figure 2 Schematic diagram of the sustained-release experiment simulating gastrointestinal fluid.

[0035] Figure 3 This is the standard curve of cannabidiol.

[0036] Figure 4 In the figure, (a) is a graph of the particle size and PDI of Zein / CBD of enteric-coated sustained-release microcapsules of Examples 1 to 9 and Comparative Example 2; (b) is a graph of the Zeta potential of Zein / CBD of enteric-coated sustained-release microcapsules of Examples 1 to 9 and Comparative Example 2.

[0037] Figure 5 This is a diagram of the embedding rate of enteric-coated sustained-release microcapsules of Examples 1 to 9.

[0038] Figure 6 In the figure, (a) shows the particle size and PDI of the enteric-coated sustained-release microcapsules of Example 8 and Example 10 to Example 13, and (b) shows the Zeta potential of the enteric-coated sustained-release microcapsules of Example 8 and Example 10 to Example 13.

[0039] Figure 7 It is the embedding rate diagram of enteric-coated sustained-release microcapsules of Example 8 and Example 10 to Example 13.

[0040] Figure 8 In the figure, (a) is a graph showing the particle size and PDI of the enteric-coated sustained-release microcapsules of Example 8 and Example 14 to Example 17, and (b) is a graph showing the Zeta potential of the enteric-coated sustained-release microcapsules of Example 8 and Example 14 to Example 17.

[0041] Fig. 9 It is the embedding rate diagram of enteric-coated sustained-release microcapsules of Example 8 and Example 14 to Example 17.

[0042] Fig.10 In the figure, (a) is a SEM image of CMC powder, (b) is a SEM image of Zein / CBD of Comparative Example 2, (c) is a SEM image of Zein-CMC / CBD of Example 8, and (d) is a SEM image of Zein-CMC / CBD of Example 9.

[0043] Fig.11 In the figure, (a) is the infrared spectra of Zein-CMC / CBD of Example 8, Zein / CBD of Comparative Example 2, and Zein-CMC of Comparative Example 1; (b) is the infrared spectra of Zein, CMC, and CBD.

[0044] Fig.12 This is a graph showing the cumulative release rate of enteric-coated sustained-release microcapsules of Example 8 in vitro simulated gastrointestinal fluid sustained-release.

[0045] Fig.13Schematic diagram of the structure of a closed impinging flow mixer, wherein (a), (b), (c), and (d) are closed impinging flow mixers with different structures, respectively.

[0046] Explanation of the reference numerals: 1-mixing section; 2-flow channel; 3-discharge pipe. DETAILED DESCRIPTION

[0047] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the protection scope of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified.

[0048] The prior art application number is 202310379535.3, and the title is "A composition and preparation method for preventing alcoholic gastric mucosal damage and liver damage". It discloses that the composition contains solid particles whose core material is theaflavin-3,3'-digallate monomer and whose wall materials are zein and sodium carboxymethyl cellulose. The core material and the wall material are mixed to obtain microcapsules.

[0049] The raw materials of the present invention are selected from cannabidiol, dihydroquercetin or dihydroquercetin natural derivatives. Cannabidiol, dihydroquercetin or dihydroquercetin natural derivatives are hydrophobic materials. Compared with the hydrophilic core material theaflavin-3,3'-digallate monomer in the prior art, cannabidiol, dihydroquercetin or dihydroquercetin natural derivatives cannot form microcapsules with zein and sodium carboxymethyl cellulose under stirring conditions. Based on this technical problem, the present invention considers the use of flash nanoprecipitation, which can be dynamically controlled by an external force, and the flow containing molecular dissolved solutes and stable molecules are quickly mixed with the counterflow containing miscible solvents to provide the local supersaturation required for particle nucleation, and the latter serves as a non-solvent for solutes and stabilizers. The mixing occurs in a closed volume under a turbulent state, providing a high energy dissipation rate and providing the supersaturated conditions required for the simultaneous precipitation of solutes and stabilizers. The block copolymers inhibit the further growth of solute particles and provide layer stability through the hydrophilic blocks on the particle surface, allowing the molecules to quickly nucleate and self-assemble into corresponding nanostructures, and the preparation time of microcapsules is drastically reduced to a few seconds, or even completed within a millisecond time scale.

[0050] Flash nanoprecipitation technology uses a confined impinging jet mixer (CIJ) to produce enteric sustained-release microcapsules. It uses two high-flow liquid flows to quickly mix in the mixing cavity of the confined impinging jet mixer, achieving rapid mixing of zein in the ethanol solution with a large amount of water (antisolvent). The hydrophobic natural medicine dissolved in the ethanol solution quickly forms a hydrophobic core after encountering water. At the same time, the amphiphilic polymer molecules (zein) dissolved in the ethanol solution quickly assemble after encountering water, wrapping the hydrophobic core to form a hydrophilic layer composed of the hydrophilic segments of the amphiphilic molecules on the surface and a material that wraps the hydrophobic natural medicine core inside. This method can continuously mix and prepare microcapsules without the need for additional mixing devices. By controlling various mixing parameters, the particle size of the microcapsules can be effectively and quantitatively controlled, and production can be scaled up at the same time.

[0051] Based on the flash nanoprecipitation method, the present invention realizes the hydrophilic shell material carboxymethyl cellulose coating the hydrophobic core material cannabidiol, dihydroquercetin or dihydroquercetin natural derivatives, realizes sustained release and targeting, and then overcomes the technical defect of low bioavailability of cannabidiol, dihydroquercetin or dihydroquercetin natural derivatives due to poor water solubility.

[0052] The technical solution of the present invention is further studied by using embodiments and comparative examples below. The specific research methods and results are as follows:

[0053] Example 1

[0054] Preparation method of enteric-coated sustained-release microcapsules: enteric-coated sustained-release microcapsules are prepared by flash nanoprecipitation in a CIJ mixer, as shown in the schematic diagram Figure 1 As shown, the following steps are included:

[0055] S1. Zein solution loaded with CBD: Zein alcohol-soluble protein and 90% ethanol solution by volume were mixed in a beaker to obtain Zein ethanol solution, and then CBD tincture was added to the Zein ethanol solution, and the mixture was stirred at room temperature with a magnetic stirrer until fully dissolved. After constant volume was obtained in a volumetric flask, a Zein solution loaded with CBD, i.e., a core material solution, was obtained.

[0056] S2. Preparation of shell material solution: Weigh 0.2 g of CMC, add deionized water and place in a magnetic stirrer at room temperature to dissolve, and dilute to volume in a 50 mL volumetric flask to obtain a CMC solution, i.e., a shell material solution.

[0057] S3. Preparation of enteric-coated sustained-release microcapsules: At room temperature, 2.5 mL of core material solution and 2.5 mL of shell material solution were extracted using a 20 mL syringe, and the two channels of the CIJ mixer were connected to mix the core material solution and the shell material solution. During mixing, the flow rate of the core material solution and the shell material solution was 30 mL / min. After mixing, they were dispersed in 45 mL of pure water to prepare a 50 mL total system. The total system was placed in a magnetic stirrer, stirred at 600 rpm for 30 min, and then rotary evaporated at 45 °C for 10 min to make up the total system volume to 50 mL. HCl was then used to adjust the pH to 4.0 to obtain a microcapsule solution, which was subjected to vacuum freeze drying to obtain enteric-coated sustained-release microcapsules, which were recorded as Zein-CMC / CBD.

[0058] Among them, in the enteric-coated sustained-release microcapsules, the mass ratio of Zein to CMC is 20:1, the mass ratio of CBD to Zein is 1:1, and the amount of zein is 1 g / L.

[0059] Example 2

[0060] The preparation method of enteric-coated sustained-release microcapsules is the same as the preparation steps of Example 1, except that the mass ratio of Zein to CMC is replaced from 20:1 to 10:1.

[0061] Example 3

[0062] The preparation method of enteric-coated sustained-release microcapsules is the same as the preparation steps of Example 1, except that the mass ratio of Zein to CMC is replaced from 20:1 to 5:1.

[0063] Example 4

[0064] The preparation method of enteric-coated sustained-release microcapsules is the same as the preparation steps of Example 1, except that the mass ratio of Zein to CMC is replaced from 20:1 to 2:1.

[0065] Example 5

[0066] The preparation method of enteric-coated sustained-release microcapsules is the same as the preparation steps of Example 1, except that the mass ratio of Zein to CMC is replaced from 20:1 to 1:1.

[0067] Example 6

[0068] The preparation method of enteric-coated sustained-release microcapsules is the same as the preparation steps of Example 1, except that the mass ratio of Zein to CMC is replaced from 20:1 to 1:2.

[0069] Example 7

[0070] The preparation method of enteric-coated sustained-release microcapsules is the same as the preparation steps of Example 1, except that the mass ratio of Zein to CMC is replaced from 20:1 to 1:3.

[0071] Example 8

[0072] The preparation method of enteric-coated sustained-release microcapsules is the same as the preparation steps of Example 1, except that the mass ratio of Zein to CMC is replaced from 20:1 to 1:4.

[0073] Example 9

[0074] The preparation method of enteric-coated sustained-release microcapsules is the same as the preparation steps of Example 1, except that the mass ratio of Zein to CMC is replaced from 20:1 to 1:5.

[0075] Example 10

[0076] The preparation method of enteric-coated sustained-release microcapsules is the same as the preparation steps of Example 8, except that the mass ratio of CBD to Zein is replaced from 1:1 to 20:1.

[0077] Embodiment 11

[0078] The preparation method of enteric-coated sustained-release microcapsules is the same as the preparation steps of Example 8, except that the mass ratio of CBD to Zein is replaced from 1:1 to 10:1.

[0079] Example 12

[0080] The preparation method of enteric-coated sustained-release microcapsules is the same as the preparation steps of Example 8, except that the mass ratio of CBD to Zein is changed from 1:1 to 5:1.

[0081] Example 13

[0082] The preparation method of enteric-coated sustained-release microcapsules is the same as the preparation steps of Example 8, except that the mass ratio of CBD to Zein is replaced from 1:1 to 2:1.

[0083] Embodiment 14

[0084] The preparation method of enteric-coated sustained-release microcapsules is the same as the preparation steps of Example 8, except that the flow rate of the core material solution and the shell material solution during mixing is replaced from 30 mL / min to 5 mL / min.

[0085] Embodiment 15

[0086] The preparation method of enteric-coated sustained-release microcapsules is the same as the preparation steps of Example 8, except that the flow rate of the core material solution and the shell material solution during mixing is replaced from 30 mL / min to 10 mL / min.

[0087] Example 16

[0088] The preparation method of enteric-coated sustained-release microcapsules is the same as the preparation steps of Example 8, except that the flow rate of the core material solution and the shell material solution during mixing is replaced from 30 mL / min to 20 mL / min.

[0089] Embodiment 17

[0090] The preparation method of enteric-coated sustained-release microcapsules is the same as the preparation steps of Example 8, except that the flow rate of the core material solution and the shell material solution during mixing is replaced from 30 mL / min to 40 mL / min.

[0091] Comparative Example 1

[0092] The preparation method of Zein-CMC comprises the following steps:

[0093] S1. Preparation of Zein solution: Mix zein and 90% by volume ethanol solution in a beaker to obtain a Zein ethanol solution, and adjust the volume in a volumetric flask to obtain a Zein solution;

[0094] S2. Preparation of shell material solution: weigh 0.2 g of CMC, add deionized water and place in a magnetic stirrer at room temperature to dissolve, and dilute to volume in a 50 mL volumetric flask to obtain a CMC solution;

[0095] S3, preparation of Zein-CMC: at room temperature, use a 20mL syringe to extract 2.5mL of Zein solution and 2.5mL of CMC solution, connect the two channels of the CIJ mixer, mix the Zein solution and the CMC solution, the flow rate of the Zein solution and the CMC solution during mixing is 30mL / min, disperse in 45mL of pure water after mixing to make a 50mL total system, place the total system in a magnetic stirrer, stir at 600rpm for 30min, and then rotary evaporate at 45℃ for 10min to make up the total system volume to 50mL, then adjust the pH to 4.0 with HCl, and obtain Zein-CMC by vacuum freeze drying;

[0096] The mass ratio of Zein to CMC is 4:1, and the amount of zein is 1 g / L.

[0097] Comparative Example 2

[0098] The preparation method of Zein / CBD comprises the following steps:

[0099] Mixing zein and an ethanol solution with a volume fraction of 90% in a beaker to obtain a Zein ethanol solution, then adding CBD tincture to the Zein ethanol solution, stirring at room temperature with a magnetic stirrer until fully dissolved, and then drying to obtain Zein / CBD;

[0100] The mass ratio of Zein to CBD is 1:1, and the amount of zein is 1 g / L.

[0101] The enteric-coated sustained-release microcapsules with high bioavailability prepared in Examples 1-17 of the present invention are taken as examples below, and compared with Zein, CMC, CBD, Zein / CBD and Zein / CMC. The specific research methods and results are as follows:

[0102] 1. Experimental methods:

[0103] 1.1 Optimization of preparation conditions of enteric-coated sustained-release microcapsules:

[0104] When exploring the influencing factors of the embedding effect of enteric-coated sustained-release microcapsules, several key single factors were selected for analysis, including the mass ratio of zein to CMC, the mass ratio of zein to CBD, and the feed flow rate. These factors have a significant impact on the embedding effect of enteric-coated sustained-release microcapsules.

[0105] 1.1.1Zein: Effect of CMC on embedding efficiency:

[0106] The concentration and feed flow rate of the Zein solution of CBD were fixed, and the CMC concentration was changed. The shell material solution was diluted to different concentrations by constant volume, so that the mass ratio of Zein to CMC in the final system was 20:1, 10:1, 5:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5. Enteric-coated sustained-release microcapsules were prepared and the particle size, potential and embedding efficiency were measured. The effects of different Zein:CMC on the embedding efficiency of enteric-coated sustained-release microcapsules were compared by HPLC results.

[0107] 1.1.2Zein: Effect of CBD on Encapsulation Rate:

[0108] The concentration and flow rate of CMC were fixed, and the concentration of CBD was changed so that the mass ratio of Zein to CBD in the total system was 20:1, 10:1, 5:1, 2:1, and 1:1. Enteric-coated sustained-release microcapsules were prepared and the particle size, potential, and embedding efficiency were measured. The effects of different Zein:CBD ratios on the embedding efficiency of enteric-coated sustained-release microcapsules were compared by HPLC results.

[0109] 1.1.3 Effect of flow rate on embedding efficiency:

[0110] The feed flow rate was changed to 5mL / min, 10mL / min, 20mL / min, 30mL / min, and 40mL / min, and the particle size, potential and embedding efficiency were measured. The effects of different flow rates on the embedding efficiency of enteric-coated sustained-release microcapsules were compared through HPLC results.

[0111] 1.2 Sustained release experiment

[0112] 1.2.1 Simulated gastric fluid sustained release experiment:

[0113] Simulated gastric juice was prepared by dissolving 1g of sodium chloride solid in 500mL of water, adding HCl to adjust the pH to 1.5, and mixing well. Then, 25mL of gastric juice was added to 25mL of the microcapsule solution of Example 8, and the mixture was placed in a water bath shaker at 37°C and 100r / min for reaction. After adding 80mg of pepsin, the simulated gastric digestion stage was started. Samples were collected at 0min, 30min, 60min, 90min, and 120min, respectively, and centrifuged at 4000r / min for 20min. The supernatant was collected for determination of CBD concentration.

[0114] 1.2.2 Simulated intestinal fluid sustained release experiment:

[0115] To prepare the simulated intestinal fluid, 187.5 mg of choline chloride and 0.144 g of trypsin were dissolved in 3.5 mL of phosphate buffer, and the pH of the intestinal fluid was adjusted to 7.0 with NaOH, and then divided into 5 portions, each portion of which was 45 mL of simulated intestinal fluid, and placed in a water bath shaker at 37°C and 100 r / min for reaction. After adding 5 mL of the microcapsule solution of Example 8 to each portion, the intestinal digestion stage was started. Samples were collected at 30 min, 60 min, 120 min, 180 min, and 240 min, respectively, and centrifuged at 4000 r / min for 20 min. The supernatant was collected for determination of CBD concentration. The experimental process is as follows: Figure 2 shown.

[0116] 1.3 HPLC determination of CBD

[0117] 1.3.1 Chromatographic separation conditions:

[0118] Chromatographic column: Agilent-C18 column, column temperature 25°C, detection wavelength 220nm, injection volume 5μL. Mobile phase A: deionized water, mobile phase B: acetonitrile; mobile phase injection volume ratio 25:75, flow rate: 1mL / min.

[0119] 1.3.2 Preparation of CBD standard curve:

[0120] Accurately weigh 10 mg of CBD standard sample, dissolve it in methanol, and then dilute it to 10 mL in a volumetric flask to obtain a standard solution with a concentration of 1 mg / mL. Dilute the standard solution in sequence to obtain stock solutions of different concentrations. After filtering through a 0.22 μm organic filter membrane, place it in a liquid phase vial and refrigerate it in a refrigerator at 4 ° C for later use. Use the above chromatographic separation conditions to inject 5 μL into the high performance liquid chromatograph, repeat 3 times, and record the peak area.

[0121] 1.3.3 Determination of unencapsulated CBD content:

[0122] The embedding effect of enteric-coated sustained-release microcapsules can be reflected by measuring the CBD free in the supernatant. The specific method is: first centrifuge the enteric-coated sustained-release microcapsule solution at 3000r / min for 10min, then centrifuge at 12000r / min for 30min, and take the supernatant. Take 1mL of supernatant and mix it thoroughly with 1mL of acetonitrile, pass it through a 0.22μm organic filter membrane, place the filtrate in a sample bottle, and store it in a refrigerator at 4℃ for later use. Then, according to the above-mentioned chromatographic separation conditions, repeat the injection 3 times to determine the embedding rate of enteric-coated sustained-release microcapsules. The embedding rate detection method is calculated according to the formula:

[0123]

[0124] Among them, X 1 Added CBD content, unit: g; X 2 The free CBD content in grams.

[0125] 1.4 Characterization of enteric-coated sustained-release microcapsules

[0126] 1.4.1 Surface morphology analysis of enteric-coated sustained-release microcapsules:

[0127] Scanning electron microscopy (SEM) was used to observe the surface morphology of enteric-coated sustained-release microcapsule beads obtained by vacuum freeze-drying. The sample processing method was as follows: the ground enteric-coated sustained-release microcapsule powder was dissolved in ethanol, ultrasonically mixed, and dripped onto the electron microscope lens with a dropper to wait for the ethanol to evaporate and dry. Double-sided tape was then attached to the electron microscope stage. The dried electron microscope lens was placed on the electron microscope stage to blow off excess powder, and then sealed with conductive glue for observation.

[0128] 1.4.2 Fourier transform infrared spectroscopy analysis:

[0129] Zein-CMC, Zein / CBD, Zein-CMC / CBD, Zein, CMC, and CBD were sampled for infrared analysis to explore the changes in functional groups before and after the flash nanoprecipitation reaction. Specifically, the enteric-coated sustained-release microcapsules were ground into fine powder with liquid nitrogen, passed through a 100-mesh sieve, and dried at 40°C for 4 hours. After drying, they were placed in a Fourier transform infrared spectrometer at 400-4000 cm -1Scan within the interval, set the number of scans to 32 times, and the resolution to 4cm -1 .

[0130] 2. Results and Discussion

[0131] 2.1 CBD linear investigation:

[0132] The CBD standard curve was determined by the 1.3.2 method, and the linear fitting was performed by origin to obtain the standard curve: Y = 6717.9X-5.5647, R 2 =0.9998, so the method has a good linear relationship. The HPLC method is accurate, reliable and highly stable. Figure 3 This is the CBD standard curve.

[0133] 2.2 Single factor analysis of the preparation process of enteric-coated sustained-release microcapsules

[0134] 2.2.1Zein: Effect of CMC on particle size potential and its embedding rate:

[0135] When other conditions are constant, different ratios of Zein:CMC added will have a certain effect on the embedding efficiency of enteric sustained-release microcapsules. Enteric sustained-release microcapsules were prepared under the conditions of a fixed Zein:CBD mass ratio of 20:1 in the total system, a flow rate of 30mL / min, and a zein amount of 1g / L. Figure 4 shown.

[0136] The average particle size of zein is 98.9nm. After adding carboxymethyl cellulose, the average particle size increases significantly, which is attributed to the adsorption of polysaccharides on the surface of zein. With the increase of CMC, the particle size increases first and then decreases. When CMC increases, more CMC is adsorbed on the surface of Zein, resulting in an increase in particle size. When the amount of CMC is 3g / L, the particle size reaches the maximum. When it continues to increase, the particle size decreases due to electrostatic repulsion and steric hindrance between CMC and Zein.

[0137] The Zeta potential of enteric sustained-release microcapsules with different Zein:CMC mass ratios is shown in Figure 4(b). Zein is positively charged when the pH is lower than the isoelectric point. Since CMC is negatively charged, when it binds to zein, it adsorbs and loads on its surface, making the enteric sustained-release microcapsules negatively charged. The active adsorption force of polysaccharides and proteins is through the electrostatic binding between anionic CMC and cationic zein nanoparticles. As the amount of CMC increases, when the mass ratio of zein to CMC changes from 20:1 to 1:2, the Zeta potential is negative, indicating successful binding. As the CMC concentration increases, the negative value of the Zeta potential becomes more negative.

[0138] Figure 5 The effect of different mass ratios of zein and CMC on the CBD embedding efficiency is shown. As can be seen from the figure, when the amount of CMC increases, the CBD embedding efficiency gradually increases, proving that the presence of the CMC anionic polysaccharide shell around Zein improves the ability to embed CBD. Since the enteric-coated sustained-release microcapsules with a mass ratio of 1:4 have the smallest particle size, a higher embedding efficiency and better dispersion, this ratio of Zein:CMC was used for subsequent experiments in the following experiments.

[0139] 2.2.2Zein: Effect of CBD on particle size potential and its embedding efficiency:

[0140] When other conditions are the same, the different amounts of core material added will have a certain impact on the embedding rate of enteric sustained-release microcapsules. Choosing a better core material concentration will make the enteric sustained-release microcapsules have a better embedding effect. Based on the experimental results of Zein: CMC, the enteric sustained-release microcapsules were prepared under the conditions of a fixed mass ratio of Zein to CMC of 1:4 in the final system, a flow rate of 30 mL / min, and a zein amount of 1 g / L. The particle size, PDI, and Zeta potential were measured. The results are shown in Figure 2. Figure 6 shown.

[0141] As the amount of CBD increases, PDI shows a trend of first decreasing and then increasing, and the particle size of the enteric-coated sustained-release microcapsules first increases and then decreases. The increase in particle size may be due to the increase in CBD concentration, the cores formed by CBD aggregate together and combine with Zein to form a larger core, and the average particle size increases after electrostatic combination with CMC. The decrease in particle size may be due to the increase in electrostatic repulsion and steric hindrance as the amount of CMC adsorbed on the surface of Zein increases. Zeta potential shows that the prepared enteric-coated sustained-release microcapsules are negatively charged, proving that the enteric-coated sustained-release microcapsules were successfully prepared. As the CBD concentration increases, the potential shows a decreasing trend.

[0142] Figure 7The effect of different mass ratios of zein and CBD on the embedding rate is shown. As the amount of CBD increases, the embedding rate shows an upward trend, because the shell material is in full contact with the core material, and the structure of the enteric sustained-release microcapsules formed is tighter, which increases the embedding rate. When the ratio of Zein:CBD is too large, the amount of zein is too large, the solution is viscous, and the synthesis of a thicker core material makes the CBD tightly wrapped, which is not conducive to subsequent release. At the same time, the amount of zein is too large, which will lead to the waste of zein, making the embedding efficiency of CBD low. When Zein:CBD is 1:1, the embedding rate reaches the maximum, which is 65.23%. Thereafter, experiments with Zein:CBD of 1:2 and 1:3 were carried out, but the solubility was poor, and there were obvious oily droplets, and no subsequent experiments were carried out on it. The present invention is intended to obtain enteric sustained-release microcapsules with a higher CBD embedding rate, and the group with Zein:CBD of 1:1 is selected for the next experiment.

[0143] 2.2.3 Effect of flow rate on particle size potential and its embedding rate:

[0144] Under the condition of other constant conditions, changing the feed flow rate will have a certain effect on the embedding rate of enteric sustained-release microcapsules. Choosing a relatively appropriate flow rate will make the enteric sustained-release microcapsules have better performance. According to the above experimental results, enteric sustained-release microcapsules with different flow rates were prepared under the conditions of fixed Zein: CMC at 1:4, Zein: CBD at 20:1, and the amount of zein at 1g / L.

[0145] Depend on Figure 8 It can be seen that as the feed flow rate increases from 10mL / min to 30mL / min, the average particle size of the enteric-coated sustained-release microcapsules shows a downward trend, and the particle size distribution becomes more concentrated. This phenomenon can be explained by the characteristic time scale of precipitation: when the syringe feed flow rate is faster, the Zein supersaturation is greater, more hydrophobic cores will be formed, the flow rate is faster, and the liquid is mixed more evenly. From the particle size and PDI diagram, it can be seen that the flow rate first decreases and then increases the particle size of the enteric-coated sustained-release microcapsules prepared by the CIJ mixer, especially at 40mL / min, which may be due to the aggregation of CMC, causing the particle size to increase sharply to 198.7nm. From the Zeta potential diagram, it can be seen that the enteric-coated sustained-release microcapsules are negatively charged, which further proves that Zein is well combined with CMC.

[0146] from Fig. 9It can be seen that the embedding rate at flow rates of 5mL / min, 10mL / min, and 30mL / min is not much different, and the embedding rate is significantly reduced at a flow rate of 40mL / min. This may be because the flow rate is too fast, and the core-shell structure formed when zein and carboxymethyl cellulose are combined is loose inside, causing CBD to flow out, resulting in a significantly lower embedding rate. The enteric-coated sustained-release microcapsules prepared at a flow rate of 30mL / min have a smaller particle size and better dispersibility than the enteric-coated sustained-release microcapsules prepared at the other two flow rates. Therefore, the flow rate of 30mL / min was finally selected for the subsequent gastrointestinal fluid sustained-release experiment.

[0147] 2.2.4 Characterization of enteric-coated sustained-release microcapsules

[0148] 2.2.4.1 Surface morphology test:

[0149] The morphologies of CMC, Zein / CBD, Zein-CMC(1:4) / CBD, and Zein-CMC(1:5) / CBD were observed using SEM. Fig.10 (a) Solid carboxymethyl cellulose was observed to have an irregular linear structure. Fig.10 (b) Zein / CBD has shown a spherical structure, but the adhesion is obvious. Fig.10 (c) It can be seen that Zein-CMC / CBD is an enteric-coated sustained-release microcapsule with good dispersion. CMC and Zein are tightly combined to form small balls. The enteric-coated sustained-release microcapsules are spherical in shape, which is different from carboxymethyl cellulose in morphology, proving that microcapsules have been formed; the particle size is about 100nm, which is consistent with the particle size test result; the particles are relatively complete, the surface is smooth, the surface structure is intact, no cracks or damage are found, and CBD is completely embedded. Fig.10 (d) Enteric-coated sustained-release microcapsules prepared with a higher amount of CMC have a distinct microcapsule structure, uneven dispersion, and obvious adhesion. This is because CMC has thickening and adhesion effects. The existence of filamentous connections between microcapsules is because the amount of CMC is too large, resulting in the absence of CMC that binds to Zein.

[0150] like Fig.11 As shown, the infrared spectra of Zein, CMC, CBD, Zein-CMC / CBD, Zein / CBD, and Zein-CMC were measured.

[0151] Zein alone is 3298.7cm -1 A peak is shown at 3466.5 cm, which represents the -OH stretching vibration of hydroxyl-bound water. This peak shifts to 3466.5 cm after forming Zein / CBD with CBD. -1 , indicating that hydrogen bonds may form between the amide group of glutamine in Zein and the hydroxyl and carbonyl groups in CBD.

[0152] The characteristic peak amide I band (1645.7 cm -1 ) and amide II band (1526.5cm -1 ), which is related to C=O stretching, NH plane bending and CN stretching vibration. The amide I band and amide II band in Zein-CMC shifted to 1649.8 cm -1 and 1649.8cm -1 , indicating that there is an electrostatic interaction between Zein and CMC. In addition, in Zein-CMC / CBD, no characteristic peak of CBD appears, indicating that CBD is successfully encapsulated in Zein-CMC.

[0153] 2.4.2 Analysis of the results of sustained release of enteric-coated sustained-release microcapsules in gastrointestinal fluid:

[0154] Depend on Fig.12 It can be seen that Zein-CMC / CBD released 19.43% after entering the simulated gastric juice for 30 minutes, and then the release rate slowed down, and the free CBD content was only 23.76% after 120 minutes. It showed a low sustained release during the digestion process of simulated gastric juice. This shows that the membrane formed by Zein and CMC effectively prevented CBD from being decomposed by pepsin, because CMC could not be decomposed in simulated gastric juice, thereby effectively preventing CBD from being released in gastric juice.

[0155] The intestinal digestion stage is 120min to 480min. It can be seen from the figure that after gastric digestion, Zein-CMC / CBD shows a burst release effect in the intestinal digestion stage, which may be due to the presence of bile salts and trypsin in artificial intestinal fluid. The CBD release rate reached 28.03% at 0.5h, i.e. 150min after entering the simulated intestinal fluid, and the release rate reached 35.18% after 480min, and the release amount remained almost unchanged. CMC can dissolve rapidly in simulated intestinal fluid, and Zein will also be decomposed by trypsin, causing Zein-CMC / CBD to rupture and release CBD. The results show that the enteric-coated sustained-release microcapsules prepared under the optimal conditions can release less CBD in gastric fluid and release it in simulated intestinal fluid. The prepared enteric-coated sustained-release microcapsules can improve bioavailability and accessibility.

[0156] Conclusion

[0157] (1) Single factor experiments were conducted on Zein:CMC, Zein:CBD and flow rate in enteric-coated sustained-release microcapsules to determine the optimal encapsulation conditions. When Zein:CMC was 1:4, Zein:CBD was 1:1, and the flow rate was 30 mL / min, the best enteric-coated sustained-release microcapsules were obtained.

[0158] (2) Enteric-coated sustained-release microcapsules with optimal embedding conditions were used to conduct in vitro release tests in simulated gastrointestinal fluid. The supernatant was taken at different times and the released CBD was detected by high performance liquid chromatography. After 120 minutes of simulated gastric digestion, only 23.76% was released. After adjusting the pH and adding simulated intestinal fluid, the release rate showed a sudden release, reaching a maximum of 35.53% at 360 minutes, and the release rate slowed down from 360 minutes to 480 minutes.

[0159] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. However, these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing enteric-coated sustained-release microcapsules, characterized in that: The steps include: The zein is mixed in an ethanol solution, and then a hydrophobic natural drug is added and mixed to obtain a core material solution; dissolving carboxymethyl cellulose in water to obtain a shell material solution; The core material solution and the shell material solution are mixed by a flash nanoprecipitation method, at which point the hydrophobic natural drug rapidly forms a hydrophobic core upon encountering water, and the zein dissolved in the ethanol solution rapidly assembles upon encountering water and wraps the hydrophobic natural drug by hydrophobic action, and then electrostatically bonds with carboxymethyl cellulose to obtain enteric-coated sustained-release microcapsules; The mass ratio of zein to the hydrophobic natural medicine is 1 to 20:1, and the mass ratio of zein to carboxymethyl cellulose is 0.2 to 20:

1.

2. The method for preparing enteric-coated sustained-release microcapsules according to claim 1, characterized in that: The hydrophobic natural drug is selected from cannabidiol or dihydroquercetin or dihydroquercetin-like natural derivatives.

3. The method for preparing enteric-coated sustained-release microcapsules according to claim 1, characterized in that: The mass ratio of zein to hydrophobic natural medicine is 1 to 10:

1.

4. The method for preparing enteric-coated sustained-release microcapsules according to claim 3, characterized in that: The mass ratio of zein to the hydrophobic natural drug is 1:

1.

5. The method for preparing enteric-coated sustained-release microcapsules according to claim 1, characterized in that: The mass ratio of zein to carboxymethyl cellulose is 0.2-10:

1.

6. The method for preparing enteric-coated sustained-release microcapsules according to claim 5, characterized in that: The mass ratio of zein to carboxymethyl cellulose is 0.25:

1.

7. The method for preparing enteric-coated sustained-release microcapsules according to claim 1, characterized in that: The specific operation of the flash nanoprecipitation method is: adding the core material solution and the shell material solution into a closed impinging flow mixer, mixing the core material solution and the shell material solution in the closed impinging flow mixer to obtain enteric-coated sustained-release microcapsules.

8. The method for preparing enteric-coated sustained-release microcapsules according to claim 7, characterized in that: The flow rates of the core material solution and the shell material solution in the closed impinging flow mixer are both 5 mL / min to 40 mL / min, and the flow rates of the core material solution and the shell material solution are equal.

9. Enteric-coated sustained-release microcapsules prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the enteric-coated sustained-release microcapsule according to claim 9 in the preparation of enteric-coated sustained-release drugs.

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