A liposome preparation method for improving oral bioavailability of GABA

Abstract

The application provides a liposome preparation method for improving oral bioavailability of GABA, relates to the technical field of functional food preparation, and comprises an external water phase, an oil phase dispersed in the external water phase and an internal water phase wrapped by the oil phase. In combination with a sodium alginate-calcium gel enteric-soluble protective layer, a multiple synergistic mechanism is formed, and the oral bioavailability of GABA is improved. In the method, GABA is effectively isolated in the internal water phase core, the inactivation risk of GABA in the digestive tract cavity is reduced, the transmembrane transport of GABA is promoted, and the intestinal retention time is prolonged. The multiple synergistic mechanism realizes the targeted delivery of'stomach protection and intestinal release'. The technical problem of the prior art that there is no GABA oral delivery system which can simultaneously cope with the destruction of gastric acid, the impact of intestinal enzymes and the low transmembrane transport efficiency, resulting in low encapsulation rate, poor targeting, low absorption and insufficient effective concentration in the body is solved.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of functional food preparation, and in particular to a liposome preparation method for improving the oral bioavailability of GABA. BACKGROUND

[0002] Gamma-aminobutyric acid (GABA) is a naturally occurring non-protein amino acid widely distributed in the central nervous system of mammals. As the main inhibitory neurotransmitter, GABA has multiple physiological functions such as sedation, anti-anxiety, sleep promotion and blood pressure regulation. In recent years, GABA has been increasingly widely used in functional foods, health products and pharmaceutical fields, especially in improving sleep quality and relieving mental stress, which has attracted the attention of consumers and researchers.

[0003] However, the oral bioavailability of GABA is low, which is a key bottleneck limiting its in-vivo efficacy. As a zwitterion, GABA is difficult to passively diffuse; its active absorption process is limited by saturation; and its hydrophilicity makes it difficult to effectively penetrate the lipid bilayer barrier of the intestinal epithelial cells, thereby affecting the absorption efficiency. In addition, GABA has a short half-life in the body and is rapidly eliminated after oral administration, making it difficult to maintain an effective concentration in the target tissue. Existing studies have shown that the bioavailability of pure oral GABA is usually less than 10%, which seriously restricts its actual application effect as a functional ingredient.

[0004] Currently, the strategies for improving the oral bioavailability of GABA mainly include chemical modification, use of absorption promoters and development of new delivery systems. Chemical modification such as synthesis of GABA prodrugs or derivatives can improve the liposolubility, but may introduce safety or metabolic stability problems. Absorption promoters such as surfactants or cholate salts can temporarily increase the permeability of the intestinal tract, but may cause irritation or damage to the intestinal mucosa, and long-term use has safety risks. In contrast, delivery systems based on nanotechnology, such as liposomes, nanoemulsions, polymer nanoparticles, etc., have become a research hotspot for improving the bioavailability of GABA due to their advantages of protecting active ingredients, promoting transmembrane transport and prolonging circulation time.

[0005] Liposomes, as a classic drug carrier, are composed of phospholipid bilayers and can encapsulate both hydrophilic and lipophilic substances, and have good biocompatibility and biodegradability. However, traditional liposomes are mostly oil-in-water type and are suitable for encapsulating liposoluble drugs. For GABA, which is a water-soluble component, the encapsulation efficiency is usually low, and the drug is easily released prematurely due to phospholipid hydrolysis or oxidation in the gastrointestinal environment, resulting in poor protection effect. In addition, traditional liposomes have poor stability in liquid beverages or paste-like foods, and are prone to aggregation, sedimentation or drug leakage, which limits their practical application in the field of functional foods.

[0006] Water-in-oil liposomes, a novel liposome configuration, consist of an aqueous core surrounded by a lipid membrane dispersed in an oil phase, forming a multiphase "water-oil-water" structure. This structure effectively encapsulates water-soluble active ingredients, such as GABA, isolating it within the aqueous phase and preventing direct contact with the gastrointestinal environment. Simultaneously, the oil phase within the water-in-oil liposome acts as a protective layer, delaying drug release and promoting transmembrane absorption through the membrane fusion properties of liposomes. However, existing water-in-oil liposomes have limited interfacial membrane strength, and in the complex environment of the gastrointestinal tract (low pH, bile salts, digestive enzymes), interfacial membrane rupture or GABA leakage may still occur. Furthermore, current liposomes have a single mechanism for promoting GABA absorption, relying solely on passive membrane fusion and lacking designs for actively promoting transmembrane transport.

[0007] In view of this, the present invention is hereby proposed. Summary of the Invention

[0008] One of the objectives of this invention is to provide a liposome that improves the oral bioavailability of GABA, thereby at least solving one of the technical problems existing in the prior art.

[0009] A second objective of this invention is to provide a method for preparing the aforementioned liposomes.

[0010] A third objective of this invention is to provide the application of the above-mentioned liposomes or liposomes prepared by the above-mentioned method in the preparation of functional foods.

[0011] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted: In a first aspect, the present invention provides a liposome for improving the oral bioavailability of GABA, wherein the liposome has a water-in-oil-in-water structure, comprising an outer aqueous phase, an oil phase dispersed in the outer aqueous phase, and an inner aqueous phase encapsulated by the oil phase; Based on the total mass of liposomes, the internal aqueous phase comprises 0.5-3% GABA and 2-8% polyol, and the pH of the internal aqueous phase is 5.5-7.0; Based on the total mass of liposomes, the oil phase comprises 7-15% vegetable oil, 0.3-1.5% soybean lecithin, 0.3-0.8% lauric acid, 0.4-0.8% polyglycerol-2 dioleate, and 0.15-1% polyglycerol ricinoleate. Based on the total mass of liposomes, the external aqueous phase comprises 1-5% polyol, 0.2-0.6% polyglycerol-10 stearate, and 0.04-0.3% thickener; The liposomes are coated with a sodium alginate-calcium gel protective layer; the sodium alginate content is 0.3-1.5% based on the total mass of the liposomes. The remainder is water; The total amount of polyglycerol-2 dioleate and polyglycerol-10 stearate is ≤1%.

[0012] Furthermore, the thickener is xanthan gum; The vegetable oil includes at least one of soybean oil, sunflower seed oil, olive oil, and MCT oil.

[0013] Furthermore, the polyol in the inner aqueous phase is at least one of glycerol and sorbitol; The polyol in the external aqueous phase is at least one of glycerol, sorbitol, propylene glycol, and xylitol.

[0014] Furthermore, the oil phase also contains 0.3 to 1.5% cetyl alcohol based on the total mass of the liposomes.

[0015] Secondly, the present invention provides a method for preparing the above-mentioned liposomes, comprising the following steps: A. Preparation of water-in-oil primary emulsion: Dissolve each component in the inner aqueous phase in water according to the formula, adjust the pH to 5.5~7.0 to obtain the inner aqueous phase; use the vegetable oil in the oil phase as a solvent, dissolve the remaining components in the vegetable oil according to the formula, heat to 70~80℃ to obtain the oil phase; under the condition of maintaining the temperature at 70~80℃, add the inner aqueous phase to the oil phase, and emulsify by high-speed shearing to form a water-in-oil primary emulsion; B. Preparation of initial liposomes: Dissolve each component in water according to the formula to obtain an external aqueous phase; under the condition of keeping warm at 70~80℃, add the external aqueous phase to the water-in-oil type primary emulsion, and continue emulsification and shearing to obtain initial liposomes; C. Preparation of sodium alginate-calcium gel protective layer: Initial liposomes are dropped into an aqueous solution of sodium alginate to form liposome-sodium alginate composite microparticles, which are then cross-linked and solidified by calcium chloride solution to form a sodium alginate-calcium gel protective layer on the outside of the liposomes.

[0016] Furthermore, the shear rate of the high-speed shear emulsification described in step A is 8000~15000 rpm; The high-speed shear emulsification time in step A is 3 to 10 minutes.

[0017] Furthermore, the shearing speed of the emulsification shearing in step B is 5000-10000 rpm; The emulsification time for the emulsification shearing described in step B is 5-15 minutes.

[0018] Furthermore, the concentration of the sodium alginate aqueous solution is 1%-3%; The dropping rate is 0.5-2 mL / min; The concentration of the calcium chloride aqueous solution is 1% to 5%; The cross-linking curing time is 30~120 minutes.

[0019] Thirdly, the present invention provides the application of the above-mentioned liposomes or liposomes prepared by the above-mentioned method in the preparation of functional foods.

[0020] Furthermore, the functional food includes at least one of beverages, pastes, tablets, or capsules.

[0021] This invention provides a liposome for improving the oral bioavailability of GABA. By constructing a water-in-oil-in-water (W / O / W) multiphase structure and combining it with a sodium alginate-calcium gel enteric protective layer, a multiple synergistic mechanism is formed, significantly improving the oral bioavailability of GABA. Specifically, the W / O / W multiphase structure effectively isolates GABA in the internal aqueous core, utilizing the dual protection of the oil phase layer and the liposome membrane. Simultaneously, the oil phase combines phospholipids and polyglycerol-2-dioleate with added lauric acid, reducing the risk of GABA inactivation in the digestive tract lumen and promoting GABA transmembrane transport, synergistically forming an absorption-enhancing mechanism with the plant oil. The pH of the internal aqueous phase is adjusted to 5.5-7.0, as pH affects the interaction between GABA and the membrane / gel. The sodium alginate-calcium gel protective layer remains stable in the acidic environment of the stomach (pH 1-2), encapsulating and protecting the liposomes from gastric acid damage. After reaching the intestine (pH...), the liposomes are effectively absorbed. (6-7) The gel layer swells and dissolves, releasing the internal liposomes; multi-layered synergy achieves targeted delivery of "gastric protection and intestinal release". This solves the technical problems of existing technologies that lack an oral GABA delivery system that can simultaneously cope with gastric acid damage, intestinal enzyme shock, and low transmembrane transport efficiency, resulting in low encapsulation rate, poor targeting, low absorption, and insufficient effective concentration in vivo. Detailed Implementation

[0022] Unless otherwise defined herein, the scientific and technical terms used in conjunction with this invention shall have the meanings commonly understood by one of ordinary skill in the art. The meaning and scope of terms shall be clear; however, in any case of potential ambiguity, the definitions provided herein shall prevail over any dictionary or foreign definitions. In this application, unless otherwise stated, the use of "or" means "and / or". Furthermore, the use of the term "comprising" and other forms is non-limiting.

[0023] Unless otherwise stated, the methods and techniques of the present invention are generally carried out according to conventional methods well known in the art and as described in various general and more specific references, which are cited and discussed throughout this specification.

[0024] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0025] The present invention provides a liposome for improving the oral bioavailability of GABA. The liposome has a water-in-oil-in-water structure, comprising an outer aqueous phase, an oil phase dispersed in the outer aqueous phase, and an inner aqueous phase encapsulated by the oil phase. Based on the total mass of liposomes, the internal aqueous phase comprises 0.5-3% GABA and 2-8% polyol, and the pH of the internal aqueous phase is 5.5-7.0; Based on the total mass of liposomes, the oil phase comprises 7-15% vegetable oil, 0.3-1.5% soybean lecithin, 0.3-0.8% lauric acid, 0.4-0.8% polyglycerol-2 dioleate, and 0.15-1% polyglycerol ricinoleate. Based on the total mass of liposomes, the external aqueous phase comprises 1-5% polyol, 0.2-0.6% polyglycerol-10 stearate, and 0.04-0.3% thickener; The liposomes are coated with a sodium alginate-calcium gel protective layer; the sodium alginate content is 0.3-1.5% based on the total mass of the liposomes. The remainder is water; The total amount of polyglycerol-2 dioleate and polyglycerol-10 stearate is ≤1%.

[0026] By constructing a water-in-oil-in-water (W / O / W) multiphase structure and combining it with a sodium alginate-calcium gel enteric protective layer, a multi-layered synergistic mechanism is formed, significantly improving the oral bioavailability of GABA. Specifically, the W / O / W multiphase structure effectively isolates GABA within the internal aqueous core, utilizing the dual protection of the oil phase layer and the liposome membrane. Simultaneously, the oil phase combines phospholipids and polyglycerol-2 dioleate with added lauric acid, reducing the risk of GABA inactivation in the digestive tract lumen and promoting GABA transmembrane transport, synergistically forming an absorption-enhancing mechanism with the plant oil. The pH of the internal aqueous phase is adjusted to 5.5–7.0, as pH affects the interaction between GABA and the membrane / gel. The sodium alginate-calcium gel protective layer remains stable in the acidic gastric environment (pH 1–2), encapsulating and protecting the liposomes from gastric acid damage. Upon reaching the intestine (pH 6–7), the gel layer swells and dissolves, releasing the internal liposomes. This multi-layered synergistic approach achieves targeted delivery through "gastric protection and intestinal release." This invention solves the technical problems of existing technologies that lack an oral GABA delivery system capable of simultaneously addressing gastric acid damage, intestinal enzyme shocks, and low transmembrane transport efficiency, resulting in low encapsulation rate, poor targeting, low absorption, and insufficient effective concentration in vivo.

[0027] In some specific embodiments, the thickener is xanthan gum. This effectively prevents the aggregation and sedimentation of liposomes, ensuring the physical stability of the product during its shelf life. In some specific embodiments, the vegetable oil includes at least one of soybean oil, sunflower oil, olive oil, and MCT oil.

[0028] In some specific embodiments, the polyol in the inner aqueous phase is at least one selected from glycerol and sorbitol. In some specific embodiments, the polyol in the outer aqueous phase is at least one selected from glycerol, sorbitol, propylene glycol, and xylitol.

[0029] In some specific embodiments, the oil phase further comprises 0.3–1.5% cetyl alcohol by weight of the total liposomes. This further reduces the W / O interfacial tension and promotes the breakup and dispersion of the aqueous phase droplets during the first emulsification step.

[0030] According to another aspect of the present invention, a method for preparing the above-described liposomes is also provided, comprising the following steps: A. Preparation of water-in-oil primary emulsion: Dissolve each component in the inner aqueous phase in water according to the formula, adjust the pH to 5.5~7.0 to obtain the inner aqueous phase; use the vegetable oil in the oil phase as a solvent, dissolve the remaining components in the vegetable oil according to the formula, heat to 70~80℃ to obtain the oil phase; under the condition of maintaining the temperature at 70~80℃, add the inner aqueous phase to the oil phase, and emulsify by high-speed shearing to form a water-in-oil primary emulsion; B. Preparation of initial liposomes: Dissolve each component in water according to the formula to obtain an external aqueous phase; under the condition of keeping warm at 70~80℃, add the external aqueous phase to the water-in-oil type primary emulsion, and continue emulsification and shearing to obtain initial liposomes; C. Preparation of sodium alginate-calcium gel protective layer: Initial liposomes are dropped into an aqueous solution of sodium alginate to form liposome-sodium alginate composite microparticles, which are then cross-linked and solidified by calcium chloride solution to form a sodium alginate-calcium gel protective layer on the outside of the liposomes.

[0031] The preparation method provided by this invention avoids competitive adsorption and interfacial disorder caused by HLB value differences when all emulsifiers are added simultaneously through stepwise emulsification, thereby improving the density and stability of each interfacial layer. Specifically, the inner aqueous phase is slowly added to the oil phase, which remains the continuous phase throughout, while the inner aqueous phase is dispersed in the oil phase as droplets, ensuring the formation of a W / O type primary emulsion. Heating at 70-80°C allows the phospholipids to fully dissolve in the oil phase, achieving good molecular fluidity and enabling rapid migration to the newly formed W / O interface, facilitating the formation of a stable and dense interfacial film. Simultaneously, the holding conditions are consistent with the heating temperature, avoiding the impact of sudden temperature changes on the already formed W / O primary emulsion structure and ensuring a smooth transition of the multiphase structure under stable temperature conditions. Utilizing the adhesive properties of sodium alginate, a sodium alginate coating layer is formed on the liposome surface, achieving targeted delivery with "gastric protection and intestinal release." The overall process is simple, highly reproducible, and suitable for large-scale industrial production.

[0032] In some specific embodiments, the shear rate of the high-speed shear emulsification in step A is 8000~15000 rpm. This high shear rate breaks the internal aqueous phase into tiny droplets, generating a large W / O interface area. When the shear rate is below 8000 rpm, the internal aqueous phase droplets are not sufficiently broken up, and the interfacial film arrangement is not dense enough. When the shear rate is above 15000 rpm, excessive shearing may lead to localized temperature increases, affecting the stability of the phospholipids. In some specific embodiments, the high-speed shear emulsification time in step A is 3~10 minutes to ensure sufficient breakup of the internal aqueous phase droplets.

[0033] In some specific embodiments, the shear rate of the emulsification shearing in step B is 5000-10000 rpm; the shear rate in this step is lower than that in step A, ensuring that the W / O colostrum is dispersed in the external aqueous phase with a complete multiphase structure. At the same time, the polyglycerol-10 stearate in the external aqueous phase forms a stable external interface film on the surface of the W / O colostrum, and the thickener forms a three-dimensional network structure in the external aqueous phase, which together maintain the stability of the W / O / W liposomes.

[0034] In some specific embodiments, the emulsification time for emulsification shearing in step B is 5-15 minutes. An emulsification time of more than 5 minutes ensures that the W / O promulgated emulsion is uniformly dispersed in the external aqueous phase. An emulsification time of less than 15 minutes avoids aggregation or structural damage of W / O / W liposomes due to prolonged collisions.

[0035] In some specific embodiments, the concentration of the sodium alginate aqueous solution is 1% to 3%; the sodium alginate concentration directly determines the effectiveness of the gel protective layer. When the concentration is below 1%, the protective effect in the acidic gastric environment is insufficient, and some liposomes may be destroyed in the stomach; when the concentration is above 3%, the dissolution time in the intestine is too long, affecting the release efficiency of the liposomes. In some specific embodiments, the dripping rate is 0.5 to 2 mL / min. This ensures that each drop of liposome dispersion is rapidly encapsulated after entering the sodium alginate solution, forming uniform composite microparticles. In some specific embodiments, the sodium alginate content of the surface modification layer is achieved by adjusting the volume ratio of liposomes to sodium alginate solution, with the volume ratio of sodium alginate solution to initial liposomes being 0.4:1 to 1:1.

[0036] In some specific embodiments, the concentration of the calcium chloride aqueous solution is 1% to 5%; in some specific embodiments, the cross-linking curing time is 30 to 120 minutes.

[0037] The liposomes of the present invention have excellent dispersibility and stability, and are suitable for a variety of functional food dosage forms.

[0038] According to another aspect of the present invention, the application of the above-described liposomes or liposomes prepared by the above-described preparation method in the preparation of functional foods is also provided.

[0039] In some specific embodiments, the functional food includes at least one of beverages, pastes, tablets, or capsules.

[0040] The present invention will be further illustrated by the following examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or purchased directly from the market.

[0041] In the following examples, the percentage content of each component is based on the total mass of the final product; the amount of each raw material added in the preparation steps is converted to the total mass of the final product according to the percentage content before being added.

[0042] Example 1 A liposome for improving the oral bioavailability of GABA, comprising the following components based on the total mass of the liposome: Internal water phase: GABA: 0.5%; Glycerin: 2%; Adjust the pH to 5.5; Oil phase: MCT oil: 7%; Soy lecithin: 0.3%; Lauric acid: 0.3%; Polyglycerol-2 dioleate: 0.4%; Polyglycerol ricinoleate: 0.15%; External aqueous phase: Glycerin: 1%; Polyglycerol-10 stearate: 0.6%; Xanthan gum: 0.04%; Surface finishing layer: Sodium alginate: 0.3%; The remainder is water.

[0043] Prepare according to the following steps: 1. Preparation of water-in-oil primary emulsion: Dissolve 0.5% GABA and 2% glycerol in water and adjust the pH to 5.5 to obtain the inner aqueous phase; use 7% MCT oil as solvent to dissolve 0.3% soybean lecithin, 0.3% lauric acid, 0.4% polyglycerol-2 dioleate and 0.15% polyglycerol ricinoleate in MCT oil and heat to 75℃ to obtain the oil phase; under the condition of maintaining the temperature at 75℃, add the inner aqueous phase to the oil phase and emulsify by high-speed shearing at a shearing speed of 8000 rpm for 10 minutes to form a water-in-oil primary emulsion.

[0044] 2. Preparation of initial liposomes: Dissolve 1% glycerol, 0.6% polyglycerol-10 stearate and 0.04% xanthan gum in water to obtain an external aqueous phase; under the condition of keeping warm at 75℃, add the external aqueous phase to the water-in-oil type primary emulsion, and continue emulsification and shearing at a shearing rate of 5000 rpm for 15 minutes to obtain initial liposomes.

[0045] 3. Preparation of sodium alginate-calcium gel protective layer: Initial liposomes were added dropwise to a 1% sodium alginate aqueous solution at a volume ratio of 1:0.45 at a dropping rate of 0.5 mL / min to form liposome-sodium alginate composite microparticles. These microparticles were then cross-linked and cured with a 1% calcium chloride solution for 120 minutes, forming a sodium alginate-calcium gel protective layer on the outside of the liposomes. After cross-linking and curing, the composite microparticles were collected by centrifugation / filtration and washed with purified water to remove excess calcium chloride, yielding the final product.

[0046] Example 2 A liposome for improving the oral bioavailability of GABA, comprising the following components based on the total mass of the liposome: Internal water phase: GABA: 1.5%; Glycerin: 5%; Adjust the pH to 6.3; Oil phase: Sunflower seed oil: 10%; Soy lecithin: 0.8%; Lauric acid: 0.5%; Polyglycerol-2 dioleate: 0.6%; Polyglycerol ricinoleate: 0.5%; External aqueous phase: Sorbitol: 3%; Polyglycerol-10 stearate: 0.4%; Xanthan gum: 0.15%; Surface finishing layer: Sodium alginate: 0.8%; The remainder is water.

[0047] Prepare according to the following steps: 1. Preparation of water-in-oil colostrum: Dissolve 1.5% GABA and 5% glycerol in water and adjust the pH to 6.3 to obtain the inner aqueous phase; use 10% sunflower seed oil as a solvent to dissolve 0.8% soybean lecithin, 0.5% lauric acid, 0.6% polyglycerol-2 dioleate and 0.5% polyglycerol ricinoleate in sunflower seed oil and heat to 75℃ to obtain the oil phase; under the condition of maintaining the temperature at 75℃, add the inner aqueous phase to the oil phase and emulsify by high-speed shearing at a shearing speed of 12000 rpm for 6 minutes to form a water-in-oil colostrum.

[0048] 2. Preparation of initial liposomes: Dissolve 3% sorbitol, 0.4% polyglycerol-10 stearate and 0.15% xanthan gum in water to obtain an external aqueous phase; under the condition of keeping warm at 75℃, add the external aqueous phase to the water-in-oil type primary emulsion, and continue emulsification and shearing at a shearing rate of 8000 rpm for 10 minutes to obtain initial liposomes.

[0049] 3. Preparation of sodium alginate-calcium gel protective layer: Initial liposomes were added dropwise to a 2% sodium alginate aqueous solution at a volume ratio of 1:0.7 at a dropping rate of 1 mL / min to form liposome-sodium alginate composite microparticles. These microparticles were then cross-linked and cured with a 3% calcium chloride solution for 60 minutes to form a sodium alginate-calcium gel protective layer on the outside of the liposomes. After cross-linking and curing, the composite microparticles were collected by centrifugation / filtration and washed with purified water to remove excess calcium chloride, thus obtaining the final product.

[0050] Example 3 A liposome for improving the oral bioavailability of GABA, comprising the following components based on the total mass of the liposome: Internal water phase: GABA: 3%; Sorbitol: 8%; Adjust the pH to 7.0; Oil phase: Olive oil: 15%; Soy lecithin: 1.5%; Lauric acid: 0.8%; Polyglycerol-2 dioleate: 0.8%; Polyglycerol ricinoleate: 1%; External aqueous phase: Xylitol: 5%; Polyglycerol-10 stearate: 0.2%; Xanthan gum: 0.3%; Surface finishing layer: Sodium alginate: 1.5%; The remainder is water.

[0051] Prepare according to the following steps: 1. Preparation of water-in-oil colostrum: Dissolve 3% GABA and 8% sorbitol in water and adjust the pH to 7.0 to obtain the inner aqueous phase; use 15% olive oil as a solvent to dissolve 1.5% soybean lecithin, 0.8% lauric acid, 0.8% polyglycerol-2 dioleate and 1% polyglycerol ricinoleate in olive oil and heat to 78℃ to obtain the oil phase; under the condition of maintaining the temperature at 78℃, add the inner aqueous phase to the oil phase and emulsify by high-speed shearing at a shearing speed of 15000 rpm for 3 minutes to form a water-in-oil colostrum.

[0052] 2. Preparation of initial liposomes: Dissolve 5% xylitol, 0.2% polyglycerol-10 stearate and 0.3% xanthan gum in water to obtain an external aqueous phase; under the condition of keeping warm at 78℃, add the external aqueous phase to the water-in-oil type primary emulsion, and continue emulsification and shearing at a shearing rate of 10,000 rpm for 5 minutes to obtain initial liposomes.

[0053] 3. Preparation of sodium alginate-calcium gel protective layer: Initial liposomes were added dropwise to a 3% sodium alginate aqueous solution at a volume ratio of 1:1 at a dropping rate of 2 mL / min to form liposome-sodium alginate composite microparticles. These microparticles were then cross-linked and cured with a 5% calcium chloride solution for 30 minutes to form a sodium alginate-calcium gel protective layer on the outside of the liposomes. After cross-linking and curing, the composite microparticles were collected by centrifugation / filtration and washed with purified water to remove excess calcium chloride, thus obtaining the final product.

[0054] Example 4 Unlike Example 2, the oil phase also includes cetyl alcohol, with the specific components as follows: Sunflower seed oil: 9.2%; Soy lecithin: 0.8%; Lauric acid: 0.5%; Polyglycerol-2 dioleate: 0.6%; Polyglycerol ricinoleate: 0.5%; Cetyl alcohol: 0.8%.

[0055] Prepare according to the following steps: 1. Preparation of water-in-oil colostrum: Dissolve 1.5% GABA and 5% glycerol in water and adjust the pH to 6.3 to obtain the inner aqueous phase; using 9.2% sunflower seed oil as a solvent, dissolve 0.8% soybean lecithin, 0.5% lauric acid, 0.6% polyglycerol-2 dioleate, 0.5% polyglycerol ricinoleate, and 0.8% cetyl alcohol in sunflower seed oil and heat to 75℃ to obtain the oil phase; under the condition of maintaining the temperature at 75℃, add the inner aqueous phase to the oil phase and emulsify by high-speed shearing at a shearing speed of 12000 rpm for 6 minutes to form a water-in-oil colostrum.

[0056] Steps 2 and 3 are the same as in Example 2.

[0057] Comparative Example 1 Unlike Example 2, the oil phase does not contain soybean lecithin, which is supplemented with an equal amount of sunflower seed oil.

[0058] Comparative Example 2 Unlike Example 2, the oil phase does not contain lauric acid, but is replaced with an equal amount of sunflower seed oil.

[0059] Comparative Example 3 Unlike Example 2, this example does not contain a sodium alginate-calcium gel protective layer, meaning that sodium alginate is not added.

[0060] Prepare according to the following steps: 1. Preparation of water-in-oil colostrum: Dissolve 1.5% GABA and 5% glycerol in water and adjust the pH to 6.3 to obtain the inner aqueous phase; use 10% sunflower seed oil as a solvent to dissolve 0.8% soybean lecithin, 0.5% lauric acid, 0.6% polyglycerol-2 dioleate and 0.5% polyglycerol ricinoleate in sunflower seed oil and heat to 75℃ to obtain the oil phase; under the condition of maintaining the temperature at 75℃, add the inner aqueous phase to the oil phase and emulsify by high-speed shearing at a shearing speed of 12000 rpm for 6 minutes to form a water-in-oil colostrum.

[0061] 2. Preparation of initial liposomes: Dissolve 3% sorbitol, 0.4% polyglycerol-10 stearate and 0.15% xanthan gum in water to obtain an external aqueous phase; under the condition of keeping warm at 75℃, add the external aqueous phase to the water-in-oil type primary emulsion, and continue emulsification and shearing at a shearing speed of 8000 rpm for 10 minutes to obtain initial liposomes as the final product.

[0062] Comparative Example 4 Unlike Example 2, the pH of the internal aqueous phase is not adjusted, and the natural pH is maintained at approximately 7.4.

[0063] Comparative Example 5 This liposome is prepared using a traditional oil-in-water emulsion liposome method, with soybean lecithin and cholesterol as membrane materials, via thin-film dispersion. Based on the total mass of the liposomes, it contains the following components: GABA: 1.5%; Soy lecithin: 5%; Cholesterol: 1%; Glycerin: 5%; The remainder is phosphate buffer (pH 7.4) as the aqueous phase solvent.

[0064] Prepare according to the following steps: Soybean lecithin and cholesterol were dissolved in chloroform, and the chloroform was removed by rotary evaporation to form a thin film. GABA and glycerol were dissolved in phosphate buffer (pH 7.4) to obtain an aqueous phase. The aqueous phase was added to the film and hydrated in a 60°C water bath for 30 minutes to form a crude liposome suspension. The crude liposome suspension was ultrasonically dispersed at a power of 200W using an intermittent ultrasonic method (3 seconds on, 3 seconds off) for a total ultrasonic time of 30 minutes. During the ultrasonic process, the temperature was kept below 25°C by cooling in an ice bath to obtain O / W type liposomes.

[0065] The liposomes prepared in Examples 1-4 and Comparative Examples 1-5 were selected as samples for the following evaluation.

[0066] 1. In vitro release experiment The gastrointestinal environment was simulated using a dialysis bag method, and the release curves of GABA under different pH conditions were determined.

[0067] The specific steps include: 1) Pretreatment of dialysis bags: Take a dialysis bag (molecular weight cutoff 3.5 kDa), boil it in boiling water for 10 minutes, rinse it with purified water, soak it in 0.1 mol / L EDTA solution for 10 minutes, rinse it three times with purified water, and soak it in purified water at 4℃ for later use.

[0068] 2) Sample loading: Take 2.0 mL of liposome sample, add it to the dialysis bag, and clamp both ends with the dialysis bag clamp.

[0069] 3) Simulated gastric fluid release: Place the dialysis bag into an Erlenmeyer flask containing 200 mL of simulated gastric fluid (pH 1.2, containing 0.2% NaCl and 0.32% pepsin), and place it in a 37°C constant temperature shaker at 100 rpm. Take 1.0 mL samples at 0.5 h, 1 h, and 2 h, and simultaneously replenish with an equal volume of fresh simulated gastric fluid.

[0070] 4) Simulated intestinal fluid release: After 2 hours, transfer the dialysis bag to an Erlenmeyer flask containing 200 mL of simulated intestinal fluid (pH 6.8, containing 0.5% bile salts and 1% pancreatic enzymes), and continue shaking at 37°C. Take 1.0 mL samples at 0.5 h, 1 h, and 2 h, and simultaneously replenish with an equal volume of fresh simulated intestinal fluid.

[0071] 5) GABA content determination: Take samples at each time point and determine the GABA content by HPLC (chromatographic conditions are the same as for encapsulation rate determination).

[0072] 6) Release rate calculation: Release rate (%) = Cumulative amount of GABA released at each time point / Total amount of GABA in liposomes × 100%.

[0073] The release rate is measured by the amount of GABA that actually passes through the dialysis bag into the release medium per unit time, and is a diffusible quantity in a kinetic sense.

[0074] The results are shown in Table 1.

[0075] Table 1

[0076] The results showed that although Comparative Example 5 achieved a cumulative release of 85% in intestinal fluid, GABA was prematurely released in gastric fluid due to the lack of W / O / W structure protection. Compared with Comparative Example 5, Examples 1-4 showed significantly lower release rates in gastric fluid (<10%), but sustained and complete release in intestinal fluid (>85%). Compared with Example 2, Comparative Example 1 lacked lecithin, failing to form a stable W / O / W interface membrane, leading to premature GABA leakage in the stomach and incomplete release in intestinal fluid; Comparative Example 2 did not leak prematurely in the stomach, but incomplete intestinal release occurred; in Comparative Example 3, without gel layer protection, liposomes leaked extensively in the stomach. Comparative Example 4 showed a high gastric fluid release rate and insufficient intestinal fluid release. These results indicate that lecithin, lauric acid, gel layer, and pH adjustment have a synergistic promoting effect on targeted delivery with "gastric protection and intestinal release".

[0077] 2. Stability test Accelerated stability tests were used to investigate the physical stability of liposomes under high temperature and high humidity conditions.

[0078] The specific steps include: 1) Sample packaging: Take 10 mL of each liposome sample, dispense them into 15 mL centrifuge tubes, and seal them.

[0079] 2) Accelerated stability conditions: Place the sample in a constant temperature and humidity chamber at a temperature of 40±2℃ and a relative humidity of 75±5% for 3 months.

[0080] 3) Sampling time points: Samples were taken in the 0th month and the 3rd month to determine the particle size and encapsulation efficiency.

[0081] A. Encapsulation efficiency test: Sample preparation: Take 1.0 mL of liposome sample and add it to an ultrafiltration centrifuge tube (molecular weight cutoff 10 kDa).

[0082] Ultrafiltration centrifugation: Centrifuge at 8000 rpm for 30 minutes at 4℃ and collect the ultrafiltrate (containing free GABA).

[0083] Determination of free GABA: Take 100 μL of ultrafiltrate, add an equal volume of derivatization reagent (sodium borate buffer containing 0.1% o-phenylenedialdehyde and 0.1% mercaptoethanol), mix well, and react at room temperature for 2 minutes. Inject 20 μL of the sample and determine the free GABA content by HPLC.

[0084] HPLC conditions: C18 reversed-phase column (4.6 mm × 250 mm, 5 μm); mobile phase: methanol: 0.1 mol / L phosphate buffer (pH 7.0) = 30:70 (v / v); flow rate: 1.0 mL / min; column temperature: 30 °C; fluorescence detector: excitation wavelength 340 nm, emission wavelength 450 nm.

[0085] Total GABA determination: Take another 1.0 mL of liposome sample, add 10 mL of methanol to break the emulsion, vortex mix for 5 minutes, centrifuge and collect the supernatant, and determine the total GABA content by HPLC.

[0086] Encapsulation efficiency calculation: Encapsulation efficiency (%) = (Total GABA content - Free GABA content) / Total GABA content × 100%.

[0087] Among them, ultrafiltration centrifugation uses centrifugal force to forcibly separate free GABA with a molecular weight of less than 10 kDa. The measured "free GABA" is the total amount of GABA not encapsulated in the aqueous phase of the lipid body (including the part physically constrained by the gel layer and thickening outer phase), which is the unencapsulated proportion in a thermodynamic sense.

[0088] B. Particle size detection: Take 10 μL of liposome sample, dilute it 100 times with purified water, and mix well.

[0089] The diluted sample was added to a quartz cuvette, and the particle size and particle size distribution (PDI) were determined using a dynamic light scattering particle size analyzer. Each sample was measured in triplicate, and the average value was taken. Measurement conditions: temperature 25℃, scattering angle 90°, equilibration time 2 minutes.

[0090] 4) Stability evaluation: Calculate the particle size change rate and encapsulation efficiency decrease rate after 3 months.

[0091] Particle size change rate (%) = (Particle size after 3 months - Particle size at 0 months) / Particle size at 0 months × 100%; Encapsulation rate decrease (%) = (0-month encapsulation rate - 3-month encapsulation rate) / 0-month encapsulation rate × 100%.

[0092] The results are shown in Table 2.

[0093] Table 2

[0094] The results showed that Examples 1-4 exhibited a particle size increase of <15% and an encapsulation efficiency decrease of <8%, both superior to the control groups. This indicates that lecithin, lauric acid, the gel layer, and pH adjustment have a synergistic effect in maintaining liposome stability.

[0095] 3. Caco-2 cell permeabilization experiment An in vitro intestinal epithelial cell model was constructed using the human colon cancer cell line Caco-2 to determine the transmembrane transport efficiency of GABA.

[0096] The specific steps include: 1) Cell culture: Caco-2 cells were seeded in the upper chamber of a Transwell chamber (membrane area 4.67 cm², pore size 0.4 μm) at a seeding density of 1 × 10⁻⁶ cells / cm². 5 Cells / well. Add DMEM high-glucose medium containing 10% fetal bovine serum, 0.5 mL to the upper chamber and 1.5 mL to the lower chamber. Incubate at 37°C in a 5% CO2 incubator for 21 days, changing the medium every 2 days.

[0097] 2) Verification of cell monolayer integrity: After 21 days of culture, transmembrane resistance (TEER) was measured. TEER value > 500 Ω·cm 2 This indicates that the cell monolayer is intact and tightly packed.

[0098] 3) Transport assay: Wash the cell monolayer three times with Hanks' balanced salt solution (HBSS, pH 7.4). Add 0.5 mL of GABA-containing sample (final concentration 1 mg / mL GABA) to the upper chamber (AP side) and 1.5 mL of HBSS to the lower chamber (BL side). Incubate at 37°C in a 5% CO2 incubator for 2 hours.

[0099] 4) Sample collection: After 2 hours of incubation, remove 1.5 mL of liquid from the lower chamber (BL side), add an equal amount of fresh HBSS, and continue incubation. Take samples again at 4h and 6h.

[0100] 5) GABA content determination: Take samples from the BL side at each time point and determine the GABA content by HPLC (chromatographic conditions are the same as for the encapsulation rate determination).

[0101] 6) Calculation of apparent permeability coefficient: Papp(cm / s)= (dQ / dt) / (A×C0) Where: dQ / dt is the transport rate of GABA (μg / s); A is the membrane area (cm²). 2 C0 represents the initial GABA concentration in the upper chamber (μg / mL).

[0102] Experimental Groups: Blank control group: Free GABA solution; Examples 1-4: Liposomes from each example; Comparative Examples 1-5: Liposomes of each comparative example.

[0103] The results are shown in Table 3.

[0104] Table 3

[0105] The results showed that the Papp values ​​of Examples 1-4 reached (5.2-6.7) × 10⁻⁶ cm / s, which were 4.3-5.6 times that of free GABA. Among them, Example 4 had the highest Papp value, and its transmembrane efficiency was slightly better. Compared with Example 2, the liposome structure of Comparative Example 1 was unstable, GABA leaked prematurely, and the permeation was extremely poor; Comparative Example 2 lacked lauric acid, which weakened the transmembrane permeation promotion, but still retained some W / O / W protective effect; Comparative Example 3 was damaged by gastric acid, resulting in a large amount of GABA leakage before reaching the intestine, and the effective permeation amount was extremely low; Comparative Example 4 had a high proportion of charged GABA and poor lipid solubility at pH 7.4 in the inner aqueous phase, which reduced the transmembrane capacity; Comparative Example 5 lacked the W / O / W structure, and GABA was exposed to the outer aqueous phase, resulting in serious leakage and extremely low Papp. This indicates that lecithin, lauric acid, gel layer, and pH adjustment have a synergistic promoting effect on GABA transmembrane transport.

[0106] 4. Oral bioavailability test in rats A rat gavage model was used to determine the plasma GABA concentration-time curve and calculate the pharmacokinetic parameters.

[0107] The specific steps include: 1) Experimental animals: Male SD rats, weighing 200±20g, were randomly divided into 6 groups of 6 rats each. They were fasted for 12 hours before the experiment but had free access to water.

[0108] 2) Dosing regimen: Free GABA group: GABA aqueous solution was administered by gavage (dose 50 mg / kg body weight); Examples 1-4: Liposomes of each example were administered by gavage (dose 50 mg / kg body weight). Comparative Example 5: Oral administration of conventional O / W liposomes (dose 50 mg / kg body weight); 3) Administration method: Administer by gavage at a dose of 5 mL / kg body weight.

[0109] 4) Blood sample collection: 0.3 mL of blood was collected from the tail vein of rats at time points of 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h and 24 h after administration and placed in anticoagulant tubes containing heparin sodium.

[0110] 5) Plasma separation: Centrifuge the blood sample at 3000 rpm for 10 minutes at 4℃ to separate the plasma, and store it at -20℃ for later use.

[0111] 6) Plasma GABA determination: Take 100 μL of plasma sample, add 3 times the volume of methanol to precipitate the protein, vortex mix for 5 minutes, centrifuge to collect the supernatant, derivatize and determine the GABA content by HPLC.

[0112] 7) Calculation of pharmacokinetic parameters: The following parameters were calculated using pharmacokinetic software (such as DAS 2.0): AUC 0-t Area under the drug-time curve at time points 0-t; AUC 0-∞ Area under the drug-time curve from 0 to ∞; Cmax: Maximum blood drug concentration; Tmax: Peak time; t 1 / 2 Eliminate half-life.

[0113] 8) Calculation of relative bioavailability: Relative bioavailability (%) = AUC 0-t (Experimental group) / AUC 0-t (Free GABA group) ×100%.

[0114] The results are shown in Table 4.

[0115] Table 4

[0116] The results showed that, compared with the free GABA group, the relative bioavailability of Examples 1-4 reached over 422%, Cmax increased by over 3.9 times, and Tmax was extended to at least 1.5 hours, achieving sustained release. 1 / 2 Extending the treatment time to over 3.8 hours eliminated the slowdown. This indicates that the bioavailability of the liposomes provided in the examples is significantly improved.

[0117] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A liposome for improving the oral bioavailability of GABA, characterized in that, The liposomes have a water-in-oil-in-water structure, comprising an outer aqueous phase, an oil phase dispersed in the outer aqueous phase, and an inner aqueous phase encapsulated by the oil phase; Based on the total mass of liposomes, the internal aqueous phase comprises 0.5-3% GABA and 2-8% polyol, and the pH of the internal aqueous phase is 5.5-7.0; Based on the total mass of liposomes, the oil phase comprises 7-15% vegetable oil, 0.3-1.5% soybean lecithin, 0.3-0.8% lauric acid, 0.4-0.8% polyglycerol-2 dioleate, and 0.15-1% polyglycerol ricinoleate. Based on the total mass of liposomes, the external aqueous phase comprises 1-5% polyol, 0.2-0.6% polyglycerol-10 stearate, and 0.04-0.3% thickener; The liposomes are coated with a sodium alginate-calcium gel protective layer; the sodium alginate content is 0.3-1.5% based on the total mass of the liposomes. The remainder is water; The total amount of polyglycerol-2 dioleate and polyglycerol-10 stearate is ≤1%.

2. The liposomes according to claim 1, characterized in that, The thickener is xanthan gum; The vegetable oil includes at least one of soybean oil, sunflower seed oil, olive oil, and MCT oil.

3. The liposomes according to claim 1, characterized in that, The polyol in the inner aqueous phase is at least one of glycerol and sorbitol; The polyol in the external aqueous phase is at least one of glycerol, sorbitol, propylene glycol, and xylitol.

4. The liposomes according to claim 1, characterized in that, The oil phase also contains 0.3 to 1.5% cetyl alcohol based on the total mass of the liposomes.

5. The method for preparing liposomes according to any one of claims 1 to 4, characterized in that, Includes the following steps: A. Preparation of water-in-oil type colostrum: Dissolve each component in the inner aqueous phase in water according to the formula amount, adjust the pH to 5.5~7.0 to obtain the inner aqueous phase; use the vegetable oil in the oil phase as a solvent, dissolve the remaining components in the vegetable oil according to the formula amount, heat to 70~80℃ to obtain the oil phase; Under the condition of heat preservation of 70~80℃, the inner aqueous phase is added to the oil phase, and emulsification is carried out by high-speed shearing to form a water-in-oil type primary emulsion; B. Preparation of initial liposomes: Dissolve each component in water according to the formula to obtain the external aqueous phase; Under the condition of keeping it at 70~80℃, the external aqueous phase is added to the water-in-oil type primary emulsion, and the initial liposomes are obtained by continued emulsification and shearing. C. Preparation of sodium alginate-calcium gel protective layer: Initial liposomes are dropped into an aqueous solution of sodium alginate to form liposome-sodium alginate composite microparticles, which are then cross-linked and solidified by calcium chloride solution to form a sodium alginate-calcium gel protective layer on the outside of the liposomes.

6. The preparation method according to claim 5, characterized in that, The shear rate for high-speed shear emulsification in step A is 8000~15000 rpm; The high-speed shear emulsification time in step A is 3 to 10 minutes.

7. The preparation method according to claim 5, characterized in that, The shearing rate of the emulsification shearing described in step B is 5000-10000 rpm; The emulsification time for the emulsification shearing described in step B is 5-15 minutes.

8. The preparation method according to claim 5, characterized in that, The concentration of the sodium alginate aqueous solution is 1%-3%; The dropping rate is 0.5-2 mL / min; The concentration of calcium chloride solution is 1%~5%; The cross-linking curing time is 30~120 minutes.

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