Multiple emulsion system for encapsulation of superoxide dismutase enzyme

Abstract

A multiple emulsion system for encapsulation of superoxide dismutase enzyme comprising fatty acids, solvent, nonionic surfactant, superoxide dismutase enzyme, gelatin, sodium chloride, emulsifier, xanthan gum, preservative, and water. The system is prepared such that the superoxide dismutase enzyme is contained within a water-in-oil-in-water (W / O / W) multiple emulsion structure to achieve encapsulation and enhance stability.

Description

[0001] MULTIPLE EMULSION SYSTEM FOR ENCAPSULATION OF SUPEROXIDE DISMUTASE ENZYME

[0002] Field of the Invention

[0003] Chemistry related to the preparation of multiple emulsion system for encapsulation of superoxide dismutase enzyme

[0004] Background of the Invention

[0005] Superoxide dismutase (hereinafter referred to as "SOD") is a naturally occurring enzyme that protects cells from damage caused by free radicals, particularly superoxide radicals, which are generated during metabolic processes in the body. SOD functions by converting superoxide radicals into oxygen and hydrogen peroxide, which is subsequently converted into water and oxygen by other enzymes. In addition, SOD exhibits anti-inflammatory properties and has antiaging effects. Due to these characteristics, SOD has been widely applied in various industries, such as cosmetics, pharmaceuticals, and medicine.

[0006] European Patent Application No. EP0414915 discloses the addition of heparin-binding domains (HBDs), which act as anticoagulants, to SOD for alleviating traumatic brain edema, ischemic myocardial disorder, stomach ulcers, and ischemic liver disorder.

[0007] Korean Patent Application No. KR1020110107316 reveals a composition for diagnosing respiratory infections caused by Mycobacteroides abscessus that includes SOD components.

[0008] Furthermore, U.S. Patent No. US12097246B2 describes treatment of spinal cord injuries, neurodegenerative diseases, or neuronal cell damage in individuals by administering an appropriate amount of SOD.

[0009] It is evident that SOD has been utilized in various forms, particularly in pharmaceuticals and medicine. However, SOD remains in the body only for a limited time and cannot be absorbed directly through the skin; it must be delivered via dietary supplements or products containing SOD, or through carriers that facilitate skin penetration. This limitation restricts its widespread application. To overcome this, emulsion systems have been developed to enhance the efficiency of SOD.

[0010] An emulsion consists of two immiscible liquids, typically oil and water. Emulsions are classified based on the dispersed and continuous phases: oil-in-water (O / W) emulsions, such as milk and mayonnaise, and water-in-oil (W / O) emulsions, such as margarine and butter.Additionally, there are multiple emulsions, where the dispersed phase contains smaller droplets within another phase. Multiple emulsions are categorized into oil-in-water-in-oil (O / W / O) and water-in-oil-in-water (W / O / W) systems. For example, a W / O / W emulsion consists of water droplets dispersed within oil droplets, which are further dispersed in an outer water phase.

[0011] Multiple emulsions offer several advantages, making them popular for encapsulating active substances in pharmaceutical and cosmetic industries:

[0012] . Enhanced encapsulation efficiency: They can encapsulate both hydrophilic and hydrophobic substances for controlled release.

[0013] . Protection against degradation: Encapsulation prevents deterioration from external factors such as light, oxygen, and heat, improving stability.

[0014] . Controlled release: Multiple emulsions allow tailored release profiles, enhancing product performance.

[0015] . Improved absorption: They increase the ability of active substances to penetrate skin or body tissues effectively.

[0016] Recent developments have applied emulsion systems to stabilize SOD. For instance, U.S. Patent Application No. US2024 / 0207371A1 describes stabilizing SOD using emulsions containing lipid derivatives, deoxygenated water, and surfactants as stabilizing agents.

[0017] U.S. Patent No. US6342238B1 discloses stabilizing SOD in emulsions comprising an oil phase dispersed with surfactants in a glycerol phase containing oxidation-sensitive hydrophilic compounds and optionally glycols. The surfactant system includes at least one surfactant capable of forming a lamellar phase, characterized by amphiphilic molecules with polar heads and nonpolar tails in a polar liquid environment, with a melting point of at least 35°C.

[0018] From the above, while prior arts have improved SOD stability using conventional emulsions, these systems may still have limitations in stability and release duration. No prior art has been found that enhances SOD encapsulation and controlled release using multiple emulsions as in the present invention.

[0019] Summary of the Invention

[0020] This invention relates to a multiple emulsion system for encapsulation of superoxide dismutase (SOD) enzyme, comprising fatty acids, solvents, nonionic surfactants, superoxide dismutase enzyme, gelatin, sodium chloride, emulsifiers, xanthan gum, preservatives, and water.The preparation involves mixing an emulsion composition in which the aqueous phase containing SOD enzyme is dispersed within an oil phase (W / O primary emulsion), followed by forming a water-in-oil-in-water (W / O / W) multiple emulsion system for encapsulating SOD enzyme and enhancing the stability of the multiple emulsion.

[0021] The preparation of the multiple emulsion according to this invention may further include steps to improve emulsion stability, enabling resistance to changes in properties over time, such as slowing down phase separation.

[0022] The objective of this invention is to provide a method for preparing a multiple emulsion that improves the efficiency of encapsulation and controlled release of SOD enzyme, ensures emulsion stability, and resists property changes over time (e.g., delayed phase separation). This makes the invention suitable for effective application in cosmetic and dietary supplement products.

[0023] Detailed Description of the Invention

[0024] This invention relates to a water-in-oil-in-water (W / O / W) multiple emulsion for encapsulation of superoxide dismutase (SOD) enzyme. The following description of the invention is provided by way of examples to facilitate clearer understanding and is not intended to limit the scope of the invention to these examples.

[0025] The preparation of the W / O / W multiple emulsion system for encapsulating SOD enzyme according to this invention consists of three main steps:

[0026] (a) Preparation of the primary emulsion containing SOD enzyme dispersed in the oil phase First, the oil phase is prepared by weighing fatty acids together with solvent and nonionic surfactant in appropriate proportions, mixing until homogeneous. Then, the aqueous phase is prepared by weighing SOD enzyme, sodium chloride, and gelatin in suitable proportions and mixing until uniform.

[0027] The aqueous solution is then added to the oil phase and mixed thoroughly, followed by high-speed homogenization to obtain a homogeneous mixture where the aqueous phase containing SOD enzyme is dispersed within the oil phase.

[0028] (b) Preparation of the W / O / W multiple emulsion system for encapsulating SOD enzyme The mixture obtained from step (a) is combined with water and additional surfactant to form the W / O / W emulsion, where droplets of the aqueous phase containing SOD enzyme are encapsulated within oil droplets dispersed in water.This step begins by preparing the outer aqueous phase by weighing emulsifier, preservative, sodium chloride, and water, mixing until dissolved. The mixture from step (a) is then added and mixed thoroughly, followed by processing through a high-pressure homogenizer to break down and homogenize the sample into fine particles with an average size of 100-500 nanometers.

[0029] (c) Stabilization of the multiple emulsion for encapsulating SOD enzyme

[0030] The emulsion obtained from step (b) is mixed with xanthan gum, which acts as a thickener and stabilizer, improving viscosity and adhesion. Xanthan gum is a 100% natural product obtained by fermenting sucrose or glucose with Xanthomonas campestris bacteria.

[0031] According to one embodiment of this invention, wherein the preferred mixing temperature for preparing the multiple emulsion system for encapsulating SOD enzyme is between 45°C and 55°C.

[0032] According to one embodiment of this invention, wherein the amount of fatty acids in the multiple emulsion system for encapsulating SOD enzyme is 6-12% by weight.

[0033] According to one embodiment of this invention, wherein the amount of solvent in the multiple emulsion system for encapsulating SOD enzyme is 6-12% by weight.

[0034] According to one embodiment of this invention, wherein the amount of nonionic surfactant in the multiple emulsion system for encapsulating SOD enzyme is 1-3% by weight.

[0035] According to one embodiment of this invention, wherein the amount of superoxide dismutase enzyme in the multiple emulsion system for encapsulating SOD enzyme is 1-10% by weight.

[0036] According to one embodiment of this invention, wherein the amount of gelatin in the multiple emulsion system for encapsulating SOD enzyme is 0.1-0.5% by weight.

[0037] According to one embodiment of this invention, wherein the amount of sodium chloride in the multiple emulsion system for encapsulating SOD enzyme is 0.5-2.5% by weight.

[0038] According to one embodiment of this invention, wherein the amount of emulsifier in the multiple emulsion system for encapsulating SOD enzyme is 0.5-2.5% by weight.

[0039] According to one embodiment of this invention, wherein the amount of xanthan gum in the multiple emulsion system for encapsulating SOD enzyme is 0.5-1% by weight.According to one embodiment of this invention, wherein the amount of preservative in the multiple emulsion system for encapsulating SOD enzyme is 0.5-1% by weight.

[0040] According to one embodiment of this invention, wherein water is added to make up 100% by weight.

[0041] According to one embodiment of this invention, wherein the preferred fatty acid is Caprylic / Capric Triglyceride.

[0042] According to one embodiment of this invention, wherein the preferred solvent is Ethoxy di glycol.

[0043] According to one embodiment of this invention, wherein the preferred preservative in the multiple emulsion system for encapsulating SOD enzyme is a paraben-free preservative, may be selected from one or more of the following: Phenoxyethanol, Chlorphenesin, or a combination thereof.

[0044] According to one embodiment of this invention, wherein the preferred nonionic surfactant in the multiple emulsion system for encapsulating SOD enzyme is Polyglyceryl-3 Polyricinoleate (PGPR).

[0045] According to one embodiment of this invention, wherein the preferred emulsifier in the multiple emulsion system for encapsulating SOD enzyme is Polysorbate 80.

[0046] The multiple emulsion product obtained according to this invention can be used as an ingredient in cosmetic products, dietary supplements, or pharmaceutical formulations for delivering SOD enzyme into the body, individually or in combination.

[0047] The following description is provided by way of examples to aid understanding and is not intended to limit the scope of the invention solely to these examples.

[0048] Example 1: Preparation of the primary emulsion containing superoxide dismutase enzyme dispersed in the oil phase

[0049] The process begins with preparation of the oil phase by weighing Caprylic / Capric Triglyceride, Ethoxydiglycol, and Polyglyceryl-3 Polyricinoleate (PGPR) in a weight ratio of 6:6:1. The mixture is stirred at 200 rpm while heating at 40°C until homogeneous.

[0050] Next, the aqueous phase is prepared by weighing superoxide dismutase enzyme extract, sodium chloride, preservative, and gelatin in a weight ratio of 20: 1 :0.6: 1. The mixture is stirred at 200 rpm while heating at 40°C until uniform.The aqueous phase is then added to the oil phase and stirred for at least 10 minutes at 200 rpm while maintaining the temperature at 40°C until homogeneous. The resulting mixture is then homogenized at 10,000 rpm for 10 minutes to obtain a uniform primary emulsion.

[0051] Example 2: Preparation of the W / O / W multiple emulsion for encapsulation of SOD enzyme The process begins by weighing emulsifier, sodium chloride, and water, then stirring at 200 rpm while heating at 40°C until all components are completely dissolved and homogeneous. Next, the solution obtained from Example 1 is added and stirred for 10 minutes at 200 rpm while maintaining the temperature at 40°C. The resulting mixture is then processed using a high-pressure homogenizer at a pressure of 7,000 psi to break down and homogenize the sample into fine particles. During this step, the temperature is controlled within the range of 4-15 °C using a temperature-controlled water bath.

[0052] Example 3: Stabilization of the W / O / W multiple emulsion for encapsulation of superoxide dismutase enzyme

[0053] The emulsion obtained from Example 2 is mixed with xanthan gum at a weight ratio of 99.25:0.75. The mixture is stirred at 200 rpm while heating at 50°C until homogeneous for approximately 30-60 minutes. The resulting mixture is then homogenized at 8,000 rpm for 10 minutes to obtain a uniform stabilized emulsion.

[0054] Example 4: Analysis of Reactive Oxygen Species (ROS) Reduction

[0055] Reactive oxygen species (ROS) are oxygen-derived free radicals that damage immune cells and tissues, leading to aging, wrinkles, and degenerative diseases, and in severe cases, cancerous cell formation.

[0056] The test was conducted on fibroblast cells, which play a key role in maintaining skin strength, elasticity, and hydration, as well as repairing damaged tissue and reducing wrinkles. Fibroblasts also regulate inflammation and injury response during tissue recovery.

[0057] The antioxidant activity was evaluated using a DCFDA / H2DCFDA assay kit, analyzing the percentage of fluorescent intensity as an indicator of ROS levels.

[0058] Results showed that fibroblast cells treated with the multiple emulsion encapsulating SOD enzyme at concentrations of 10, 5, and 2.5 U / mL exhibited reduced ROS production compared to untreated control cells. Specifically, the multiple emulsion prepared according to this invention reduced ROS production by 46.25%, 35.97%, and 20.84%, respectively.Furthermore, the stabilized multiple emulsion obtained from Example 3 demonstrated superior ROS reduction compared to free SOD enzyme, achieving 33.38% reduction at a concentration of 10 U / mL.

[0059] Best Mode of the Invention

[0060] As stated in detailed description of the invention.

Claims

Claims1. A multiple emulsion system for encapsulation of superoxide dismutase enzyme, comprising:Fatty acids in an amount of 6-12% by weightSolvent in an amount of 6-12% by weightNonionic surfactant in an amount of 1-3% by weightSuperoxide dismutase enzyme in an amount of 1-10% by weight Gelatin in an amount of 0.1-0.5% by weightSodium chloride in an amount of 0.5-2.5% by weightEmulsifier in an amount of 0.5-2.5% by weightXanthan gum in an amount of 0.5-1% by weightPreservative in an amount of 0.5-1% by weightWater in an amount sufficient to make the total composition 100% by weight 2. The multiple emulsion system according to claim 1, wherein the preferred fatty acid is Caprylic / Capric Triglyceride.

3. The multiple emulsion system according to claim 1 or 2, wherein the preferred solvent is Ethoxy di glycol.

4. The multiple emulsion system according to any one of claims 1-3, wherein the preferred preservative is paraben-free, comprising Phenoxyethanol and Chlorphenesin.

5. The multiple emulsion system according to any one of claims 1-4, wherein the preferred nonionic surfactant is Polyglyceryl-3 Polyricinoleate (PGPR).

6. The multiple emulsion system according to any one of claims 1-5, wherein the preferred emulsifier is Polysorbate 80.

7. The multiple emulsion system according to any one of claims 1-6, wherein the preferred emulsion preparation temperature is between 45°C and 55°C.

8. The multiple emulsion system according to any one of claims 1-7, wherein the multiple emulsion system is used as a component of cosmetic products, dietary supplements, or pharmaceutical products for delivering superoxide dismutase enzyme into the body, individually or in combination.

9. The multiple emulsion system according to any one of claims 1-8, wherein the multiple emulsion system has an average particle size between 100 and 500 nanometers.