JP2017510649A - Prolamin-based oral film manufacturing method for protection of antioxidant activity - Google Patents

Abstract

The present invention relates to a method for producing a prolamin-based oral film for protecting antioxidant activity, more specifically, (a) producing an aqueous alcohol solution containing prolamin, glycerol formal and an antioxidant; b) A method for producing an oral film comprising the step of evaporating a solvent under a reduced pressure condition of 0 ° C. to 80 ° C. According to the production method of the present invention, it is possible to produce an oral film in which the activity of the antioxidant substance is maintained at a high level. Since the activity of the plant is maintained at a high level, the industrial applicability is high.

Description

  This application claims priority from Korean Patent Application No. 10-2014-0038163 filed on Mar. 31, 2014, and the entire specification is a reference for this application.

  The present invention relates to a method for producing a prolamin-based oral film for protecting antioxidant activity, and more particularly, (a) producing an aqueous alcohol solution containing prolamin, glycerol formal and an antioxidant; And (b) a method for producing an oral film comprising a step of evaporating a solvent under a reduced pressure condition of 0 ° C. to 80 ° C., and a prolamin-based oral film containing an antioxidant produced by the method.

A considerable amount of active oxygen and the like is produced during the oxidation process for energy production in vivo. Most of these active oxygens are eliminated by in vivo removal mechanisms. However, when active oxygen is instantaneously generated in large quantities or chronically active oxygen is generated and the balance with the antioxidant defense system is lost, Causes disease. Part of the metabolic process of oxygen is superoxide radical (0 ₂・-), hydrogen peroxide (H ₂ 0 ₂ ), hydroxy radical (HO ·), oxygen single molecule (singlet oxygen, ¹ O ₂ ) These are changed to active active harmful oxygen, but they break the balance between the oxidant and the antioxidant composed of antioxidant enzymes and non-enzymatic antioxidants, and cause damage to biological components and the like.

  One of the main causes of diseases such as aging, cancer, cerebrovascular diseases, cardiovascular diseases and the like includes free radicals or reactive oxygen species (ROS). The reactive oxygen species is oxygen that has increased the reactivity of oxygen that is essential for the maintenance of organisms due to biological stress caused by pathogens that have invaded organisms and various environmental stresses associated with deterioration of the global environment. It can mean that the highly reactive oxygen will induce serious physiological disorders and the like. Specifically, free radicals or reactive oxygen species destroy cells, cut connective tissue in the dermis layer of the skin, and cause cross-bonding, so skin-related such as wrinkle formation, atopic dermatitis, acne, or skin cancer Not only diseases but also cardiovascular diseases including cancer, myocardial infarction, stroke, atherosclerosis, amyotrophic laternal sclerosis and Alzheimer's dementia ( Alzheimer's disease (AD) and Parkinson's disease (PD) have been reported to cause chronic neurodegenerative diseases. Furthermore, free radicals or reactive oxygen species can peroxidize lipids in the human body to produce harmful substances called lipid peroxides, and the lipid peroxides act on blood vessels to produce various substances such as arteriosclerosis and thrombus. Has been reported to cause adult and chronic diseases.

  As described above, since free radicals or reactive oxygen species are related to many diseases, there is an effect of suppressing the generation of reactive oxygen species or quickly removing reactive oxygen species already generated in the body. There are ongoing efforts to prevent or treat diseases by administering various antioxidant substances to the human body.

  The most common and convenient drug administration method in the treatment of illnesses, especially in the treatment of chronic diseases, is the method of drug delivery through the oral route. However, when the above antioxidants are orally administered as they are, their biological activities are caused by the harsh environment of the digestive organs (pH changes and various digestive enzymes, etc.) while moving through the digestive tract such as the gastrointestinal tract and small intestine. Is reduced or eliminated, and the therapeutic effect on the actual in vivo appears extremely limited. In particular, proteins having therapeutic activity are known to be easily degraded by the environment in the gastrointestinal tract (GI) when administered orally per se (Woodley, J, F., 1994. Enzymatic barriers for GI peptide and protein delivery. Crit. Rev. Ther. Drug Carrier Syst. 11, 61-5.).

  Here, a new strategy is needed to develop an effective oral drug delivery system that protects the activity of antioxidants at a high level in the digestive process in the gastrointestinal and small intestines, etc., to enhance the dosing effect.

  Here, the present inventors are studying an effective oral drug delivery system that protects the activity of antioxidants from the digestive tract, and have an aqueous ethanol solution containing prolamin, glycerol formal and antioxidants at a specific temperature. When the oral film preparation was produced by evaporating the solvent under the pressure condition, it was confirmed that the activity of the antioxidant and the like was maintained at a very high level in the produced film.

  Accordingly, an object of the present invention is to provide an oral film comprising the steps of (a) producing an aqueous alcohol solution containing prolamin, glycerol formal and an antioxidant; and (b) evaporating the solvent under reduced pressure conditions of 0 ° C to 80 ° C. It is to provide a manufacturing method.

  Another object of the present invention is to provide a prolamin-based oral film containing an antioxidant prepared by the above method.

To achieve the above object, the present invention comprises (a) a step of producing an aqueous alcohol solution containing prolamin, glycerol formal and an antioxidant; and (b) a step of evaporating the solvent under a reduced pressure condition of 0 to 80 ° C. A method for producing an oral film is provided.
In order to achieve another object of the present invention, the present invention provides a prolamin-based oral film comprising an antioxidant prepared by the above method.

  Hereinafter, the present invention will be described in detail.

The present invention
(A) A method for producing an oral film comprising the steps of: producing an aqueous alcohol solution containing prolamin, glycerol formal and an antioxidant; and (b) evaporating a solvent under a reduced pressure condition of 0 ° C to 80 ° C.

  In step (a), an aqueous alcohol solution containing prolamin, glycerol formal and an antioxidant is prepared.

  Prolamins represent a group of globular proteins found in plants such as cereal. Prolamin proteins are generally soluble in 70-80% alcohol but not in water and pure alcohol. These proteins contain high levels of glutamate and proline.

  The prolamin of the present invention is not limited as long as it is a known prolamin protein derived from a plant, for example, wheat, rye, barley, oat, rice at least one plant-derived prolamin selected from the group consisting of (rice), sorghum, millet, coix and corn. Preferably, the prolamin of the present invention is zein, gliadin, hordein, secalin, kafirin, coixin, glutenin, glutenin, GluB-Glutelin And a mixture of two or more selected from the group consisting of avenin.

Glycerol formal (glycerol formal, C ₄ H ₈ O ₃ ) is Methylidinoglycerol; Sericosol N; 1,3-Dioxolane-4-methanol; 1,2- (Methylidene) glycerol; 5-hydroxy-1,3-dioxane and 4- A mixture of hydroxymethyl-1,3-dioxolane; Glycerin formal; Formal Glycerol and other various synonyms and known to act as a solubilizer for the dissolution of water-insoluble compounds Yes.

The glycerol formal of the present invention has a structure represented by the following chemical formulas 1 and 2, and includes 47-67% 5-hydroxy-1,3-dioxane basis and 33-53% 4-hydroxymethyl-1,3-dioxolane. is a mixture of basis. The glycerol formal of the present invention can be purchased and used commercially or produced by chemical synthesis methods known in the art.

  Antioxidant means preventing various oxidative actions that occur in the body, and the antioxidant (or antioxidant) is present in a small amount compared to the amount of the substrate, delaying or suppressing the oxidation of the substrate. It means the substance to do.

  As long as the antioxidant substance of the present invention is a substance having a function of erasing free radicals or reactive oxygen species (ROS) or suppressing generation (antioxidant power) and a function of promoting the function (antioxidant increasing action) The type is not limited, but specifically includes antioxidant enzymes and non-enzymatic antioxidants.

  The antioxidant enzyme means a protein having an anti-oxidant activity or an anti-oxidant increasing effect, and the type thereof is not limited as long as it is a known protein involved in an in-vivo antioxidant system, but preferably a superoxide. Dismutase (superoxide dismutase), catalase, glutathione peroxidase, glutathione reductase, glutathione-S-transferase hemeoxygenase and their synthetic enzymes or peptides It may be any one or more selected from the group consisting of mimetics.

  The non-enzymatic antioxidant substance means a compound having an antioxidative activity or an anti-oxidant increasing effect, and the kind thereof is not limited as long as it is a known compound involved in an in vivo antioxidant system, Vitamin E, curcumin, carotene, lipoic acid, coenzyme Q10, tocopherol, polyphenol, retinyl palmitate, thioctic acid, carotenoid, vitamin C, vitamin C derivative, caffeine ), Caffeic acid, BHA (butylated hydroxyanisole), BHT (butylated hydroxytoluene), TBHQ (tertiary butylhydroquinone), epigallocatechin-3-gallete (EGCG), proanthocyanidin (proanthocyanidin), Selected from the group consisting of gallic acid, selenium, zinc, taurine, plabonoid and scopoletin One or more.

  The type of the solvent used in the step (a) of the present invention is not limited as long as it is a substance that can dissolve all of prolamin and glycerol formal, but alcohol can be preferably used.

  The “alcohol” of the present invention is preferably a low-hydric alcohol having 1 to 6 carbon atoms, more preferably ethanol. The ethanol is a volatile and flammable colorless liquid, and is an alcohol in which one hydrogen atom is substituted with a hydroxy group from ethane having 2 carbon atoms among chain hydrocarbons.

  The alcohol of the present invention can be used in the form of an aqueous alcohol solution by mixing and diluting 100% alcohol and water (distilled water) at a specific volume ratio (v / v). Therefore, the alcohol concentration of the present invention is indicated by the volume ratio of alcohol to the total solution volume. The “water” may be distilled water or deionized water. The amount of alcohol added is not limited to this, but is preferably in the range of 40% (v / v) to 95% (v / v), preferably 55% to 75%, and more preferably 70%.

In the step (a), “alcohol aqueous solution containing prolamin, glycerol formal and antioxidant substance” is not limited to its production method, but specifically (i) a step of dissolving prolamin in an alcohol aqueous solution;
(Ii) adding glycerol formal to the alcohol aqueous solution of step (i);
(Iii) adding an antioxidant to the solution of step (ii);
Can be done sequentially.

  The ratio of the prolamin and the antioxidant is not limited to this, but is preferably a weight ratio of 1 to 1,000,000: 1, more preferably 1 to 1000: 1, and most preferably 100. The weight ratio is between 600: 1.

  Furthermore, the ratio of the prolamin and glycerol formal is not limited to this, but is preferably a weight ratio of 1 to 10,000: 1, more preferably a weight ratio of 1 to 100: 1, and more preferably. The weight ratio is 1 to 10: 1.

  In step (b), the alcohol aqueous solution containing prolamin, glycerol formal and antioxidants prepared in step (a) is compared with the substances contained in the aqueous alcohol solution under reduced pressure conditions of 0 ° C to 80 ° C. When the reaction is continued, the solvent is evaporated under the above conditions to form a film.

  When the solvent evaporates, the temperature is not limited to this, but specifically, a temperature range of 0 ° C. to 80 ° C., more preferably a temperature range of 0 ° C. to 45 ° C., further preferably 10 ° C. The temperature range is from 38 ° C to 38 ° C, more preferably from 15 ° C to 35 ° C.

  According to an embodiment of the present invention, antioxidant enzymes contained in various oral films produced by using the film manufacturing method of the present invention and varying the temperature conditions under the same reduced pressure conditions. When the activity was measured, as shown in [FIG. 12], the activity of catalase contained in the oral film produced at 15 ° C., 25 ° C., 35 ° C., etc. was maintained relatively high, and 45 It can be confirmed that even an oral film produced under the temperature condition of ° C. maintains relatively favorable activity up to 20% up to 30%. Furthermore, as shown in [FIG. 13], it was confirmed that the activity of catalase contained therein was high from the oral film produced under the temperature condition of 0 ° C.

The solvent evaporation is performed under reduced pressure, and the pressure is preferably 1 × 10 ⁻⁵ mbar to 999 mbar, more preferably 1 × 10 ⁻⁴ mbar to 1 × 10 ⁻² mbar.

  The temperature and reduced pressure can be maintained for a time that allows the solvent to evaporate sufficiently, and the time depends on the amount and type of solvent used. The time for maintaining the temperature and reduced pressure conditions is not limited to this, but is preferably 10 seconds to 12 hours, and more preferably 1 minute to 1 hour.

  According to the production method of the present invention including the steps (a) to (b), it is possible to produce an oral film in which the activity of the antioxidant is maintained at a high level. In particular, the activity of the antioxidant contained in the film is maintained at a high level even in the severe environment of the gastrointestinal and small intestines (Example 2).

  Furthermore, the oral film produced by the production method of the present invention contains a much larger amount of antioxidant than the conventional nanoparticle dosage form when it is produced with the same amount of zein and antioxidant. The activity of the antioxidant is stored in (Example 1).

  Accordingly, the present invention provides a prolamin based oral film comprising an antioxidant prepared by the above method.

  The oral film of the present invention is manufactured by a method including the steps (a) and (b), and the steps are as described above.

  The amount of the antioxidant in the oral film of the present invention can be selectively set by those skilled in the art depending on the film type, film thickness and surface area. The orally administrable films provided herein can contain a wide range of antioxidants. Preferably it is 0.01 to 80% (w / w), more preferably 0.1 to 10% (w / w).

  The oral film of the present invention contains an antioxidant as an active drug component, and can easily and quickly administer the drug into the human body through the oral cavity. Furthermore, by producing a film preparation under specific conditions based on prolamin, it is possible for the antioxidants contained in it to maintain its activity at a high level in the body, especially in the harsh environment in the digestive tract. However, the physiological activity of antioxidant enzymes is maintained at a high level.

  Furthermore, the oral film of the present invention can be formulated into an immediate release type and / or a modified release type according to the timing of drug release, and preferably a fast dissolving tablet or oral cavity for quick disintegration. It can be formulated into an inward disintegration film.

  On the other hand, in the oral film of the present invention, additives other than prolamin, glycerol formal and antioxidant can be added in an appropriate amount depending on the blending purpose. For example, a filler, a plasticizer, a sour agent, an anti-aggregation agent, a solubilizer, a stabilizer, a sweetener, a sweetener, an emulsifier, other solubilizing agents, a coloring agent, a fragrance and the like can be mentioned. In particular, during film production, a film-forming base, a grading agent, an emulsifier, other starches and the like may be additionally included.

  The film-forming base is not particularly limited as long as it can impart flexibility to the film, can be disintegrated well, and can maintain the viscosity for particle formation. Preferably, polymers such as hypromellose, hydroxypropylcellulose, polyvinyl alcohol, polyethylene oxide or polyvinylpyrrolidone can be used. Furthermore, as a gradual increase agent, flurlan, starch, lactose, cellulose, carrageenan, gum arabic, guar gum, locust bean gum, xanthan gum, gellan gum, agar, alginic acid, sodium alginate and the like can be used.

  The filler serves to impart shape formation to the film by reducing the properties of the film that slide in the oral cavity. Furthermore, the property that films adhere to each other can be reduced, and the degradation rate of the film and the dissolution rate of the drug in the oral cavity can be adjusted. As the filler, for example, microcrystalline cellulose, cellulose polymer, magnesium carbonate, calcium carbonate, limestone powder, silicate, clay, talc, titanium dioxide, calcium phosphate and the like can be used.

  As a plasticizer for imparting film flexibility, for example, triethyl citrate, glycerin fatty acid ester, polyethylene glycol, sorbitol, maltitol, xylitol, glycerin, polyethylene glycol and the like can be used.

  Furthermore, for example, citric acid, malic acid, fumaric acid, tartaric acid, ascorbic acid, succinic acid, adipic acid, and lactic acid can be used as the sour agent, and anti-aggregating agents include, for example, talc, calcium stearate, etc. Can be used.

  As the solubilizer, for example, sucrose fatty acid ester, sorbitan monostearate, sodium lauryl sulfate and the like can be used, and as the stabilizer, for example, hard anhydrous silicic acid, magnesium aluminate metasilicate, magnesium silicate, silica Aluminum oxide, calcium silicate, and the like can be used.

  Furthermore, examples of the sweetener include asphaltam, saccharin, dipotassium glycyrrhizinate, and stevia. Examples of the aphrodisiac include 1-menthol, sodium chloride, acesulfame potassium, sucralose, and the emulsifier is glycerin. Fatty acid ester, sucrose fatty acid ester, lecithin, enzymatically decomposed lecithin, polysorbate, sorbitan fatty acid ester, propylene glycol and the like can be used.

  As the solubilizer, for example, cyclodextrin, arginine, lysine, trisaminomethane and the like can be used.

  Furthermore, the oral film of the present invention may additionally contain an amount of a coloring agent suitable for obtaining the target color tone. Coloring agents include, for example, natural food colors and edible dyes. Such colorants are generally known for FD & D dyes.

  The oral film of the present invention can be used for the purpose of preventing and treating various tissue damages and diseases caused by various in vivo oxidation reactions, that is, free radicals or reactive oxygen species. Various injuries and diseases caused by the free radicals or reactive oxygen species are known in the art and include, but are not limited to, inflammation, infection, arteriosclerosis, hypertension, cancer, radiation damage, Neurological diseases, neurodegenerative diseases, ischemia / reperfusion injury, aging, wound treatment, glutathione deficiency, acquired immune deficiency syndrome, sickle cell anemia, diabetes and rheumatoid arthritis.

  The present invention relates to a method for producing an oral film comprising the steps of: (a) producing an aqueous alcohol solution containing prolamin, glycerol formal and an antioxidant; and (b) evaporating the solvent under a reduced pressure condition of 0 ° C. to 80 ° C. And a prolamin-based oral film comprising an antioxidant prepared by the above method. According to the production method of the present invention, it is possible to produce an oral film in which the antioxidant activity is maintained at a high level. In particular, the activity of the antioxidant contained in the film is maintained at a high level even in the harsh environment of the gastrointestinal and small intestines.

FIG. 1 shows SOD activity of each experimental group under pH 7.0 conditions (zein-based oral film containing zein film, SOD: free superoxide dismutase, Zein-SOD: SOD). FIG. 2 shows the catalase activity of each experimental group under pH 7.0 conditions (zein: zein film, CAT: free catalase, Zein-CAT: zein-based oral film containing catalase). FIG. 3 shows the catalase activity of each experimental group under pH 7.0 conditions (Gliadin: gliadin film, CAT: free catalase, Gliadin-CAT: gliadin-based oral film containing catalase). FIG. 4 shows the SOD activity of each experimental group under pH 1.3 conditions (gastrointestinal environmental mimic conditions) (white graph: pepsin treatment group, black graph: pepsin non-treatment group, zein: zein film, SOD: free superoxide dismutase Zein-SOD: Zein-based oral film containing SOD). FIG. 5 shows the catalase activity of each experimental group under pH 1.3 conditions (gastrointestinal environment mimicking conditions) (white graph: pepsin treatment group, black graph: pepsin non-treatment group, zein: zein film, CAT; free catalase, Zein-CAT; Zein-based oral film containing catalase). FIG. 6 shows the catalase activity of each experimental group under pH 1.3 conditions (gastrointestinal environmental mimic conditions) (white graph: pepsin treatment group, black graph: pepsin non-treatment group, Gliadin: gliadin film, CAT: free catalase, Gliadin-CAT: a gliadin-based oral film containing catalase). FIG. 7 shows the SOD activity of each experimental group under pH 7.4 conditions (small intestinal environmental mimic conditions) (white graph: trypsin treatment group, black graph: non-trypsin treatment group, zein: zein film, SOD: free superoxide dismutase) Zein-SOD: Zein-based oral film containing SOD). FIG. 8 shows the catalase activity of each experimental group under pH 7.4 conditions (small intestine environmental mimic conditions) (white graph: trypsin treatment group, black graph: non-trypsin treatment group, zein: zein film, CAT: free catalase, Zein-CAT; Zein-based oral film containing catalase). FIG. 9 shows the catalase activity of each experimental group under pH 7.4 conditions (small intestine environmental mimic conditions) (white graph: trypsin treatment group, black graph: non-trypsin treatment group, Gliadin: gliadin film, CAT: free catalase, Gliadin-CAT; gliadin-based oral film containing catalase). FIG. 10 shows the catalase activity contained in a conventional nanoparticle (nanoparticle) dosage form and the prolamin-based oral film dosage form of the present invention. FIG. 11 shows the catalase activity contained in each oral film produced by the conventional method for producing an oral film and the prolamin-based oral film production method of the present invention. FIG. 12 shows the catalase activity associated with 2 ° C., 15 ° C., 25 ° C., 35 ° C., 45 ° C., 55 ° C., 65 ° C. and 75 ° C. temperature conditions during the production of the prolamin-based oral film of the present invention. FIG. 13 shows catalase activity associated with a temperature condition of 0 ° C. or 15 ° C. during the production of the prolamin-based oral film of the present invention. FIG. 14 shows catalase activity associated with various pressure conditions during the production of the prolamin-based oral film of the present invention. FIG. 15 shows the antioxidant power associated with each pH condition when producing a prolamin (zein or gliadin) -based oral film containing caffeine, which is a non-enzymatic antioxidant.

  Hereinafter, the present invention will be described in detail.

  However, the following examples only illustrate the present invention, and the content of the present invention is not limited to the following examples.

<Comparative Example 1>
Production of Nanoparticles (Nanoparticles) Containing Antioxidase <Comparative Example 1-1>
100 mg of zein (Sigma-Aldrich) was dissolved in 1.825 mL of an aqueous ethanol solution. 52.5 mg of sorbitol (Sigma-Aldrich) and 67.5 mg of glycerol formal (Sigma-Aldrich) were added and mixed until the solution was clear. In order to encapsulate the antioxidant with zein, 3 mg / mL of catalase (EC 1.11.16.1, Sigma-Aldrich) was dropped into the above solution while stirring dropwise to prepare a mixed solution (catalase total 200 μL added). Nanoparticles were manufactured using a phase separation method. Briefly, 8.0 mL of water was added to the prepared mixed solution and dispersed by a homogenization method at 21,500 rpm for 1 minute. Centrifugation at 47,500 rpm for 5 minutes separated the resulting nanoparticle, producing a yellow solid powder. Particle size and morphology were confirmed by scanning electron microscopy (SEM) using QUANTA FEG 250 (FEI company, Hillsboro, Oregon) from Masdar Institute of Science and Technology (Abu Dhabi, UAE).

<Comparative Example 1-2>
100 mg of zein was dissolved in 1.825 mL of 70% ethanol aqueous solution. 52.5 mg sorbitol and 67.5 mg glycerol formal were added and mixed until the solution was clear. In order to encapsulate the antioxidant with zein, the solution was superoxide dismutase ((EC1.15.1.1, Sigma-Aldrich) 100 U / mL (superoxide dismutase 1 U is 1 micromol superoxide). (Mixed solution was added by adding 200μL of SOD) Nanoparticles were manufactured using the phase separation method. 8.0 mL of water was added to the mixture and dispersed by homogenization at 21,500 rpm for 1 minute, and the resulting nanoparticle was separated by centrifuging at 47,500 rpm for 5 minutes. Particle size and morphology were confirmed by scanning electron microscopy (SEM) using QUANTA FEG 250 (FEI company, Hillsboro, Oregon) from Masdar Institute of Science and Technology (Abu Dhabi, UAE).

<Comparative example 2>
Production of oral film containing antioxidant enzyme <Comparative Example 2-1>
450 mg of zein was dissolved in 1.825 mL of 50% aqueous ethanol. 52.5 mg sorbitol and 67.5 mg glycerol formal were added and mixed until the solution was clear. A mixed solution was prepared by adding 3 mg / mL of catalase dropwise thereto and stirring the mixture (total addition of 200 μL of catalase). A film was produced by evaporating at room temperature (25 ° C.) for 24 hours.

<Comparative Example 2-2>
450 mg of zein was dissolved in 1.825 mL of 50% aqueous ethanol. 52.5 mg sorbitol and 67.5 mg glycerol formal were added and mixed until the solution was clear. Superoxide dismutase (100 U / mL) was added dropwise to this, and the mixture was stirred to prepare a mixed solution (added 200 μL of total SOD). A film was produced by evaporating at room temperature (25 ° C.) for 24 hours.

<Comparative Example 2-3>
100 mg of zein was dissolved in 1.825 mL of 70% ethanol aqueous solution. 67.5 mg of glycerol formal was added and mixed until the solution was clear. The solution was stirred while dropping 3 mg / mL of catalase dropwise to prepare a mixed solution (catalase total 200 μL added). This mixed solution was spread widely in a glass container and dried at room temperature (25 ° C.) for 24 hours to complete a zein film.

<Example 1>
Production of prolamin-based oral film containing antioxidant enzyme according to the present invention <Example 1-1> Production of zein-based oral film containing catalase 500 mg of zein was dissolved in 10 mL of 70% aqueous ethanol. Glycerol formal 335 mg was added and mixed until the solution was clear. 2 mL of the mixed solution (containing 100 mg zein) was transferred to a vacuum flask, and the mixture was stirred while dropping 3 mg / mL of catalase dropwise to prepare a mixed solution (catalase total 200 μL added). The mixed solution in the vacuum flask was evaporated using a rotary evaporator under reduced pressure conditions (0.002 mbar) at 35 ° C. until a thin film was formed.

<Example 1-2> Production of zein-based oral film containing superoxide dismutase (SOD) 500 mg of zein was dissolved in 10 mL of 70% ethanol aqueous solution. Glycerol formal 335 mg was added and mixed until the solution was clear. Transfer 2 mL of the above mixed solution (containing 100 mg zein) to a vacuum flask and add superoxide dismutase ((EC1.15.1.1, Sigma-Aldrich) 100 U / mL (superoxide dismutase 1 U is 1 micromol of superoxide). The mixture solution was prepared by adding 100 U / mL drop by drop (added 200 μL of total SOD) .The mixed solution in the vacuum flask was a thin film using a rotary evaporator. The solvent was evaporated under reduced pressure conditions at a temperature of 35 ° C. until formed.

<Example 1-3> Preparation of gliadin-based oral film containing catalase 500 mg of gliadin (Sigma-Aldrich) was dissolved in 10 mL of a 70% aqueous ethanol solution. Glycerol formal 335 mg was added and mixed until the solution was clear. 2 mL of the mixed solution (containing 100 mg zein) was transferred to a vacuum flask, and the mixture was stirred while dropping 3 mg / mL of catalase dropwise to prepare a mixed solution (catalase total 200 μL added). The mixed solution in the vacuum flask was evaporated using a rotary evaporator under reduced pressure conditions at a temperature of 35 ° C. until a thin film was formed.

<Example 2>
Protective effect of antioxidant enzyme activity of prolamin-based oral film according to the present invention The activity of the antioxidant contained in the oral film prepared in Example 1 was measured by the following method.

<Example 2-1> Prolamin-based oral film system In a prolamin-based oral film containing catalase, the activity of catalase was confirmed by measuring the hydrogen peroxide decomposition rate. Catalase or a prolamin-based oral film containing catalase in a pH 7.0 buffer solution was mixed with 10 μMH ₂ 0 ₂ and then the absorbance of H ₂ 0 ₂ was monitored at 240 nm to measure catalase activity. In prolamin-based oral film containing SOD, SOD activity was measured by the manufacturer's method using SOD assay kit (SOD assay kit (SOD determination kit 19,160, Fluka, St Louis, MO). P <0.05 (*) was determined by t-test between a zein film containing SOD or catalase and the other film.

  As a result, as shown in [FIG. 1] and [FIG. 2], the single activity of SOD or catalase and the activity of SOD or catalase contained in the zein base film were almost similar at pH 7.0. This indicates that SOD or catalase maintains the activity of each substance at the same level even after forming a film-like drug delivery system with zein.

  Similarly, as shown in FIG. 3, it was confirmed that the catalase contained therein maintained the antioxidant function even after the film was produced using gliadin.

<Example 2-2> Evaluation of antioxidant enzyme activity in gastrointestinal environment Zein's ability to protect antioxidant protein was evaluated in the gastrointestinal environment. Catalase or a prolamin-based oral film containing catalase was placed in a hydrochloric acid (50.1 mM HCl, pH 1.3) solution and allowed to stand for 20 minutes to mimic the gastrointestinal environment. At this time, the experiment group was divided into 349.1 mM pepsin treatment group and pepsin non-treatment group (Metheny, NA, Stewart, BJ, et al., 1997.pH and concentrations of pepsin and trypsin in feeding tube aspirates as predictors of placement.J.Parenter.Enteral Nutr.21,279-85.). Catalase and SOD activity were measured by the same method as described in Example 2-1. Significance of the results was determined by p <0.05 (*) by t-test between the zein film containing SOD or catalase and the other films.

  As a result, as shown in [FIG. 4] and [FIG. 5], SOD or catalase alone almost lost its activity in the gastrointestinal acidic environment (pH 1.3). However, it was confirmed that SOD or catalase contained in the zein base film was maintained at a high level in the gastrointestinal environment. Furthermore, there was no significant difference in the activity of SOD or catalase between the group treated with pepsin, an enzyme secreted in the gastrointestinal tract, and the group not treated. Thus, it was confirmed that the zein base film produced by the production method of the present invention was excellent in the effect of protecting the activity of the antioxidant substance.

  Furthermore, as shown in FIG. 6, it was confirmed that the catalase present in the gliadin film protected the antioxidant function under gastrointestinal conditions.

<Example 2-3> Evaluation of antioxidant enzyme activity in the small intestine environment In order to mimic the small intestine environment, the experimental group was divided into a 143.0 mM trypsin treatment group and a trypsin non-treatment group and in an environment of pH 7.4. Left for 30 minutes. Catalase and SOD activity were measured by the same method as described in Example 2-1. Significance of the results was determined by p <0.05 (*) by t-test between zein film containing SOD or catalase and other films.

  As a result, as shown in [FIG. 7] and [FIG. 8], SOD or catalase alone maintained its activity under pH 7.4 conditions, but the group treated with trypsin as well as the small intestine environment showed that activity. It showed a tendency to decrease to a very low level. On the contrary, it was confirmed that the SOD or catalase contained in the zein base film maintained the activity at a high level irrespective of the trypsin treatment.

  Furthermore, as shown in FIG. 9, it was confirmed that the catalase present in the gliadin film had an antioxidant function protected under small intestinal conditions (pH 7.4).

<Experimental example 1>
Evaluation of activity according to dosage form The oral films produced in Comparative Example 1-1 and Example 1-1 were compared for catalase activity. First, in order to determine the content of catalase for each dosage form, a fluorescent substance (fluorescein isothiocyanate, FITC, Sigma-Aldrich) is chemically bound to catalase and then used to form zein nanoparticle and film. Then, 1 mg was taken for each dosage form, the amount of fluorescence was measured, and this was compared with the amount of fluorescence initially used to determine the content of catalase for each dosage form. Catalase activity measurement uses catalase to which a fluorescent substance is not bound, forms zein nanoparticle and film, takes 1 mg for each dosage form, and is the same as the method described in Example 2-1 at pH 7.0 Was done.

  As shown in the above [Table 1] and [FIG. 10], it was confirmed that the catalase contained in the oral film dosage form maintained a much higher level of activity than the nanoparticles. This can be interpreted as the film has a much larger amount of catalase during the manufacturing process.

<Experimental example 2>
Activity Evaluation with Manufacturing Method <Experimental Example 2-1> Comparison of Conventional Oral Film Manufacturing Method and Oral Film Manufacturing Method of the Present Invention From the oral film manufactured in Comparative Example 2-3 and Example 1-1 Each 1 mg film was taken and the catalase activity was compared. Catalase activity measurement was performed at pH 7.0 in the same manner as described in Example 2-1.

  As a result, as shown in FIG. 11, in the film produced by the conventional method, the activity of catalase was almost lost, but in the film produced by the production method of the present invention, catalase was active at a very high level. It was confirmed that it was maintained.

<Experimental example 2-2> Oral film comparison with various temperature conditions In various oral films manufactured by changing the temperature under the same pressure reduction condition (0.002 mbar) by the film manufacturing method of the present invention, The activity of antioxidant enzymes and the like contained in was measured.

  An oral film was produced by the same method as described in Example 1-1 except for the pressure condition and the temperature condition.

  As a result, as shown in [FIG. 12] and [FIG. 13], in the film for oral use produced at 0-45 ° C., particularly 15-35 ° C., the activity of catalase was maintained at a relatively high level. I confirmed.

<Experimental Example 2-3> Oral Film Comparison with Various Pressure Conditions In various oral films manufactured by changing the pressure under the same temperature condition (35 ° C.) by the film manufacturing method of the present invention, The activity of antioxidant enzymes and the like contained in was measured.

  An oral film was produced by the same method as described in Example 1-1 except for the pressure condition and the temperature condition.

  As a result, as shown in FIG. 14, it was confirmed that the activity of catalase contained in the film was maintained at a higher level as the pressure was lower. In particular, a relatively high activity was maintained at a pressure of 0.002 mbar.

<Example 3>
Prolamin-based oral film containing non-enzymatic antioxidant according to the present invention Caffeine 5 mg and gliadin 100 mg or zein 100 mg as prolamin are used orally in the same manner as described in Example 1-1. A film was produced. To evaluate prolamin's ability to protect antioxidant proteins in the gastrointestinal environment, caffeine or a prolamin-based oral film containing caffeine is placed in a hydrochloric acid solution for 20 minutes to simulate the gastrointestinal environment. It was neutralized again to pH 7.0 and used for the next cell experiment.

In order to measure the antioxidant effect of caffeine in the oral film, after culturing cells (RAW264.7, 4x10 ⁵ cells / well) on a 12-well plate, 1 mg each of the oral film produced above was cultured in each well. After culturing the cells with the cells for 2 hours, the above culture solution is removed and the cells are treated with hydrogen peroxide (100 μM) for 4 hours to see how much the cells have been killed by hydrogen peroxide or how much caffeine has been charged with the hydrogen peroxide. It was measured whether it could be protected from.

Specific experimental groups were divided as follows: A: negative control group, B: treated with H ₂ 0 ₂ alone, C: caffeine solution left at pH 1.3 for 20 minutes and H ₂ 0 ₂ treatment, D : Solution obtained by neutralizing caffeine solution left at pH 1.3 for 20 minutes to pH 7.0 again and H ₂ 0 ₂ treatment, E: solution obtained by leaving gliadin-based oral film containing caffeine at pH 1.3 for 20 minutes And H ₂ 0 ₂ treatment, F: A solution obtained by neutralizing a gliadin-based oral film containing caffeine for 20 minutes at pH 1.3 to pH 7.0 again and H ₂ 0 ₂ treatment, G: caffeine Solution containing zein-based oral film containing 20 minutes at pH 1.3 and H ₂ 0 ₂ treatment, H: Solution of zein-based oral film containing caffeine left at pH 1.3 for 20 minutes again to pH 7.0 Neutralized solution and H ₂ 0 ₂ treatment.

  Cytotoxicity is measured using a dye (propidium iodide, PI) that stains only dead cells, and 100 μL (50 microgram / mL PI) is added to each well, and 10 minutes later, microplate reader manufactured by Tecan Was used to measure fluorescence (excitation: 535 nm, emission: 617 nm). The cases where only the culture solution is present and the case where there is only hydrogen peroxide are indicated by toxicity of 0% and 100%, respectively, and other organisms are indicated by the relative toxicity.

  Caffeine has a function of protecting cells from active oxygen (Silverberg JI., Et.al., J Drugs Dermatol. Nov; 11 (11): 1342-6), and as shown in FIG. Through experiments, it was confirmed that the activity of caffeine was well maintained in caffeine and gliadin. The above data show that the method for producing an oral film using prolamin (especially zein or gliadin) of the present invention can be applied not only to antioxidant enzymes but also to non-enzymatic antioxidants.

<Production example>
The production examples of prolamin-based oral films containing an antioxidant according to the present invention will be illustrated below.
Production Example 1: Gliadin-based oral film containing SOD An oral film was produced in the same manner as in Example 1-2 using 100 mg of gliadin, 67.5 mg of glycerol formal, and 200 μL of SOD.

Production Example 2: Hordein Base Oral Film Containing Catalase An oral film was produced in the same manner as in Example 1-1 using hordein 100 mg, glycerol formal 67.5 mg, and catalase 200 μL.

Production Example 3: Glutenin-based oral film containing catalase An oral film was produced in the same manner as in Example 1-1 using glutenin 100 mg, glycerol formal 67.5 mg, and catalase 200 μL.

Production Example 4: Secalin-Based Oral Film Containing Catalase An oral film was produced in the same manner as in Example 1-1 using Secalin 100 mg, glycerol formal 67.5 mg, and catalase 200 μL.

Production Example 5: Coixin Base Oral Film Containing SOD An oral film was produced in the same manner as in Example 1-2 using 100 mg of coixin, 67.5 mg of glycerol formal, and 200 μL of SOD.

Production Example 6: Zein-based oral film containing glutathione peroxidase 100 mg of zein, 67.5 mg of glycerol formal, and 200 μL of glutathione peroxidase were used to produce an oral film in the same manner as in Example 1-1.

Production Example 7: Zein-based oral film containing glutathione reductase An oral film was produced in the same manner as in Example 1-1 using 100 mg of zein, 67.5 mg of glycerol formal, and 200 μL of glutathione reductase.

Production Example 8: Zein-based oral film containing glutathione-S-transferase hemeoxygenase Zein 100 mg, glycerol formal 67.5 mg, glutathione-S-transferase heme oxygenase 200 μL An oral film was produced in the same manner as in Example 1-1.

Production Example 9: Zein-based oral film containing vitamin C An oral film was produced in the same manner as in Example 3 using 100 mg of zein, 24 mg of glycerol formal, and 5 mg of vitamin C.

Production Example 10: Zein-based oral film containing tocopherol An oral film was produced in the same manner as in Example 3 using 100 mg of zein, 24 mg of glycerol formal, and 5 mg of tocopherol.

As described above, the present invention relates to a method for producing a prolamin-based oral film for protecting antioxidant activity, more specifically, (a) producing an aqueous alcohol solution containing prolamin, glycerol formal and an antioxidant. And (b) a method for producing an oral film comprising the step of evaporating the solvent under a reduced pressure condition of 0 to 80 ° C. According to the production method of the present invention, it is possible to produce an oral film in which the activity of the antioxidant substance is maintained at a high level, and in particular, the antioxidant substance contained in the film in the harsh environment of the gastrointestinal and small intestines. Industrial activity is high because activity is maintained at a high level.

Claims (10)

(A) a method for producing an aqueous alcohol solution containing prolamin, glycerol formal and an antioxidant; and (b) a method for producing an oral film comprising the step of evaporating a solvent under a reduced pressure condition of 0 ° C to 80 ° C.   2. The oral film according to claim 1, wherein the prolamin is prolamin derived from at least one plant selected from the group consisting of wheat, rye, wheat, buckwheat, rice, sorghum, straw, pigeon and corn. Production method.   3. The method for producing an oral film according to claim 2, wherein the prolamin is any one selected from the group consisting of zein, gliadin, hordein, secarin, kafilin, coixin, glutenin, grubyglutelin and abein.   The method for producing an oral film according to claim 1, wherein the antioxidant is selected from the group consisting of an antioxidant enzyme, a non-enzymatic antioxidant and a mixture thereof.   Antioxidants include superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, glutathione-S-transferase heme oxygenase, and these The method for producing an oral film according to claim 4, wherein the film is any one or more selected from the group consisting of a synthetic enzyme or a peptide analog.   Non-enzymatic antioxidants include vitamin E, curcumin, carotene, lipoic acid, coenzyme Q10, tocopherol, polyphenol, retinyl, palmitate, thioctic acid, carotenoid, vitamin C, vitamin C derivatives , Caffeine, caffeic acid, BHA (butylated hydroxyanisole), BHT (butylated hydroxytoluene), TBHQ (tertiary butylhydroquinone), epigallocatechin-3-gallate (EGCG), proanthocyanidin, gallic acid The method for producing an oral film according to claim 4, wherein the film is one or more selected from the group consisting of (Gallic acid), selenium, zinc, taurine, plabonoid and scopoletin.   The method for producing an oral film according to claim 1, wherein the temperature is from 0C to 45C.   The method for producing an oral film according to claim 1, wherein the temperature is 10 ° C. to 38 ° C. 2. The method for producing an oral film according to claim 1, wherein the pressure is 1 * 10 < ^-5 > mbar to 999 mbar. A prolamin-based oral film comprising an antioxidant produced by the method of claim 1.

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