KR100951706B1 - Nanoemulsion, nanoparticle containing resveratrol and method of production thereof - Google Patents

Abstract

The present invention relates to nanoemulsions and nanoparticles containing resveratrol, and more particularly, to resveratrol having an antioxidant effect and an effect on preventing and treating heart disease by nanoemulsifying with medium-chain triglycerides and surfactants. The present invention relates to a nanoemulsion having a high solubility in resveratrol, and a nanoparticle capable of imparting stability to the nanoemulsion and a method of preparing the same. The present invention can be utilized as a functional food material of resveratrol having a great value as a bioactive functional food material, and it is possible to increase the usability of the body of resveratrol.

Resveratrol, nanoemulsion, nanoparticles, stability, particle size, functional food

Description

NANOEMULSION, NANOPARTICLE CONTAINING RESVERATROL AND METHOD OF PRODUCTION THEREOF}

The present invention relates to nanoemulsions and nanoparticles containing resveratrol, and more particularly, to resveratrol having an antioxidant effect and an effect on preventing and treating heart disease by nanoemulsifying with medium-chain triglycerides and surfactants. The present invention relates to a nanoemulsion having a high solubility in resveratrol, and a nanoparticle capable of imparting stability to the nanoemulsion and a method of preparing the same.

Recently, the use of nanoemulsions in the food and cosmetics industry is increasing actively, especially in the food industry is because the use provides the following advantages. First, O / W food nanoemulsions have functional properties of various electrical properties such as polarity, nonpolarity, and amphotericity due to their nonpolar (oil), polar (water), and amphoteric (boundary) regions. It can be included in the carrier of. Second, food nanoemulsions with heterogeneous structure can be used as a method of controlling the chemical stability of the encapsulated internal components, by controlling the physical location and emulsion interface of the materials having different reactivity. Third, emulsions with different rheological properties (viscosity liquids, pastes, viscoelastic solids) can be prepared by controlling the composition and microstructure constituting the food nanoemulsions, thereby enabling product applications suitable for each characteristic. Fourth, the food nanoemulsion is prepared in accordance with the purpose of use as a liquid as well as a solid phase to increase the convenience and usability of transportation. Fifth, food nanoemulsions can produce functional food materials that provide high quality by simple processes (mixing and stirring, high pressure homogenization, ultrasonic wave) using food raw materials (water, oil, surfactants, phospholipids, proteins, polysaccharides, etc.). Can be. Sixth, the food nanoemulsion can prevent the creaming or sedimentation during the storage of the product by the nano-level small emulsion particles provide the gravity reduction effect during the Brownian movement, and the nano liquid particles prevent the flocculation of the liquid. Because of this, separation does not occur and maintains dispersion. Finally, the nanoemulsion particles do not deform and do not cause coalescence because surface aggregation is prevented, and the thickness of the surfactant film used (diameter of the liquid spheres) significantly reduces thinning or breakage of the liquid membranes in the liquid compartment. To interfere.

Due to these advantages, many researches are being made to prepare food functional ingredients as nanoemulsions to provide food additives or as food additives. The requirements for food carriers are as follows: ① Food grade approved raw materials, ② Economic production process -Extended shelf life, marketability, superior functionality, increased bioavailability, etc. ③ Protection against chemical decay-Oxidation and hydrolysis, ④ Dosage load and activity maintenance-Maintain and deliver the maximum loading of bioactive components to unit mass of food functional ingredient carrier Carrier up to the end point, ⑤ Delivery mechanism-Active site of the carrier, release control, surrounding environment (pH, ionic strength, enzyme activity, temperature), etc. ⑥ Compatibility with food-appearance, texture, flavor, stability of final product, etc. And ⑦ bioavailability / biological activity.

Experts agree that the range of nanosizes suitable for food nanoemulsions that meet these requirements is suitable for nanomaterials at the 100 nm level. This is based on research reports that the nano-species remain outside the cell when the size of 300 nm or more, to penetrate into the cell when 70-300 nm, and to the cell nucleus below 70 nm (Chen M. Kikecz A. Formation of nucleoplasmic protein aggregates impairs nuclear function in response to SiO2 nanoparticles.Exper.Cell Res. 305: 51-62 (2005).

The present invention is to provide the above-mentioned advantages as a nanoemulsion, and to meet the required characteristics as a food functional ingredient carrier, in particular, a food nanoemulsion containing resveratrol having a size of 100 nm or less, nanoparticles, a method for producing the same Related to.

Resveratrol is a component found in the 1970s, with the formula C ₁₄ H ₁₂ O ₃ and a molecular weight of 228.25. Stilbene family of compounds, present in two structural isomers: pinaceae, myrtaceae, fagaceae, moraceae, papillonaceae, vitaceae It is found in many plants, including legumes (fabaceae / leguminosae). Grapes, for example, are known to be made of self-defense against mold that can be contaminated during plant growth.

The antioxidant effect of resveratrol isolated from human blood from low-density lipoprotein cholesterol has been reported to be five times higher than vitamin E, the most effective dietary antioxidant to date. In addition, it is known to be effective in preventing and treating heart disease because it provides functions such as inhibiting platelet coagulation, altering eicosanoid synthesis, and regulating lipoprotein metabolism. In addition, antiviral, neuroprotective (neuroprotective), anti-inflammatory (anti-inflammatory), anti-aging (anti-aging) and life is known to have the effect of extending the life. French Paradox is caused by the action of resveratrol, a phenomenon in which French people eat relatively fatty foods but have a lower incidence of heart disease.

As described above, resveratrol is very valuable as a bioactive functional food material such as antioxidant, antibacterial, metabolic syndrome response, neuroprotective action, anti-aging and the like. However, resveratrol has no water solubility for use as a functional food material and thus cannot be widely used, and is easily decomposed by sulfate conjugation and glucuronidation of absorbed cells in the intestine when ingested. Because of the low availability in the body.

Resveratrol poses a serious problem in its availability because it is highly hydrophobic and very sensitive to external environments such as air, light and oxidase. However, the technique according to the present invention is directed to the preparation of nanoemulsions in which hydrophobic resveratrol may exist in the micelle form in the hydrophobic (lipophilic) space. When present in the nanoemulsion form, resveratrol protected by the micelle membrane is absorbed and delivered in the body. By maintaining stable in the secretion of secretory enzymes in the body can increase the availability in the body.

Accordingly, an object of the present invention is to provide a nanoemulsion containing resveratrol having a particle size of 100 nm or less and having good storage stability, which can stabilize food functional ingredients and maximize absorption in the body based on materials suitable for use in food.

It is another object of the present invention to provide nanoparticles from nanoemulsions containing resveratrol having a particle size of 100 nm or less and good storage stability.

It is still another object of the present invention to provide a method for preparing nanoemulsions comprising resveratrol having a particle size of 100 nm or less and good storage stability, and a method for preparing nanoparticles from the nanoemulsion.

To prepare a nanoemulsion, resveratrol is prepared by dissolving it in a constant concentration in an organic solvent. The mixture is mixed with resveratrol with a medium chain triglyceride (hereinafter referred to as 'MCT') in a weight ratio of 1 : 1 to 1: 5. In order to remove the organic solvent, the mixture was concentrated under reduced pressure at 50 ° C-55 ° C and added with a surfactant at a weight ratio of 1: 2-5. Nanoemulsions are prepared by dissolving a mixture of resveratrol, MCT and surfactant at a constant concentration in water. The preparation of nanoemulsions is made using magnetic coupling methods known to those skilled in the art or using ultrasonic devices.

Organic solvents such as ethanol, acetone, chloroform and ethyl acetate are used to dissolve resveratrol. Resveratrol dissolved in an organic solvent is preferably 0.001-50 mg per ml of organic solvent. The amount less than 0.001mg does not show the effect of adding resveratrol, and the amount does not dissolve well in the amount of 50mg or more.

The MCT is selected from the group consisting of caproic acid, caprylic acid, capric acid, and lauric acid. The reason for using MCT as an oil phase is that corn oil, soybean oil, olive oil, and canola oil, which contain a lot of unsaturated fatty acids, become opaque when mixed with an organic solvent in which resveratrol is dissolved. This is because the size represents 193-944 nm. When limonene, a hydrocarbon belonging to the terpenes, is used, it shows a particle size of 100 nm or less, such as 12.11 nm, but the use of limonene-specific flavor and food is limited. When MCT is used in combination with unsaturated fatty acids, it was confirmed that the particle size of the nanoemulsion in which the ratio of MCT: unsaturated fatty acid is 9: 1 or more is 100 nm or less.

The resveratrol mixture dissolved in the organic solvent is mixed with MCT in a weight ratio of 1: 1 to 1: 5 because the particle size of the prepared nanoemulsion exceeds 100 nm when it exceeds this range.

Decompression concentration to remove the organic solvent uses a method known to those of ordinary skill in the art (hereinafter referred to as the person skilled in the art), for example, rotary evaporator and water pressure Can be used to reduce the pressure.

The reason why the mixture including resveratrol and MCT is added in a weight ratio of 1: 2-5 with the surfactant is that a particle size of more than 100 nm is produced, such as 224 nm at an addition ratio of 1: 2 or less, for example, 1: 1.5. In addition, nanoemulsions having a particle size of 100 nm or less may be prepared at an addition ratio of 1: 5 or more, but surfactants such as Tween 80, for example, are permitted as food additives in the food industry. It should be 0.25% or less in the test. '

Emulsifiers are used to stabilize emulsions and most emulsifiers are surfactants. Food emulsions can be prepared by applying mechanical or ultrasonic energy to a mixture of oil, water and emulsifiers, as is well known to those skilled in the art. The emulsions are opaque and stable for a period of time, but not thermodynamically unstable. It is well known that long term stability is not shown.

In the preparation of nanoemulsions, emulsifiers are used. The types of emulsifiers that can be used in food are limited. Soybean phospholipid (lecithin), sucrose fatty acid ester, glycerin fatty acid ester, sorbitan fatty acid ester, polyglycerol fatty acid ester, stearyl Calcium lactate, and the like. In the food sector, the use of emulsifiers is often possible only at low concentrations. For example, propylene glycol cannot use more than 1 g per 1 kg of food. In this invention, use of sorbitan fatty acid ester is preferable as an emulsifier. Sorbitan fatty acid esters are nonionic surfactants whose properties and uses differ depending on the type and number of fatty acids bound to sorbitan and the degree of esterification. Commercially available sorbitan monoesters include lauric acid (Tween 20), palmitic acid (Tween 40), stearic acid (Tween 60) and oleic acid (Tween 80). In the present invention, Tween 60 or Tween 80 is particularly preferable as an emulsifier. In the case of Tween 20 or Tween 40, the chemical structure is more suitable for the preparation of water-in-oil (W / O) emulsions, which is not suitable for the preparation of oil-in-water (O / W) emulsions provided by the present invention, and resveratrol does not dissolve well. This is because separation occurs easily.

The nanoemulsion of the present invention may be prepared as nanoparticles by recovery by solvent precipitation such as ethanol or acetone or by vacuum freeze drying as known to those skilled in the art. Powder nanoparticles are more stable than nanoemulsions. Increasingly, they have long-term storage and are easier to apply to many foods (Galindo-Rodriguez S., Allemann E., Fessi H., & Doelker E. (2004) Physicochemical parameters associated with nanoparticle formation in the salting-out, emulsification-diffusionm and nanoprecipitation methods.Pharmaceutical Research 21: 1428-1439). Nanoparticles prepared by the present invention may be added to the beverage water and sold. According to the results of the present inventors, the nanoemulsion prepared by the present invention exhibits a very transparent red color, and thus, when used in the beverage industry, It suggests the possibility of using the product to satisfy satisfaction. It can also be used as a seasoning ingredient, and can also be used as a food additive in various confections and foods such as gum, baking or butter. Furthermore, since the nanoparticles of the present invention are prepared from nanoemulsions, the nanoparticles are easily dissolved when the nanoparticles are dispersed in water, and thus are easily applied to foods.

A sum of the mixture consisting of resveratrol, MCT and surfactant is suitable for the weight ratio of water 20:80 to 0.5: 99.5. If the sum of the mixture of resveratrol, MCT and surfactant exceeds 20% by weight, the particle size of the nanoemulsion to be prepared may exceed 100nm, and the content of resveratrol included in the nanoemulsion to be prepared is less than 0.5% by weight. Because it is limited.

The nanoemulsion production method of the present invention comprises the steps of mixing the resveratrol with an organic solvent; Mixing this mixture in MCT; Concentrating under reduced pressure at 50 ° C.-55 ° C. to remove the organic solvent; Mixing a mixture of resveratrol and MCT with a surfactant; And dissolving the mixture at a constant concentration in water to prepare a nanoemulsion.

The present invention can be utilized as a functional food material of resveratrol having a great value as a bioactive functional food material, and it is possible to increase the usability of the body of resveratrol. The technique according to the present invention is directed to the preparation of nanoemulsions in which hydrophobic resveratrol may exist in the micelle form in the hydrophobic (lipophilic) space, and when resveratrol protected by the micelle membrane is present in the body absorption and delivery process It is possible to increase the availability in the body by remaining stable to secretory enzyme degradation in the body.

Hereinafter, the present invention will be described in detail by way of examples. However, the examples are provided to illustrate the invention and are not intended to limit the scope of the invention.

Example 1 Selection of Oil Phase of Resveratrol

Preparation of nanoemulsions containing resveratrol used a self-assembly method (FIG. 4). Resveratrol (R5010, Sigma, USA) was prepared by dissolving in ethanol at a concentration of 5 mg / ml, and then MCT (SEFOL, Welga, Korea), limonene, corn oil, soybean oil, olive oil, canola oil, grape seed for selection of oil phase. The oil was prepared and mixed at a blending ratio of 1: 1. Concentrated under reduced pressure at 50 ℃ to remove the ethanol, Tween 80, a nonionic surfactant was added in a ratio of 1: 3. The mixture was dissolved in water at a constant concentration (mixture: water = 0.5: 99.5) to prepare an emulsion. The particle size of the nanoemulsion prepared by varying the type of oil is shown in Figure 1, the particle size of the nanoemulsion prepared by varying the mixing ratio of MCT and corn oil is shown in Figure 2. As shown in FIG. 1, the nanoparticles are less than 100 nm in size when prepared with MCT and limonene, but in the case of other unsaturated fatty acids, an unstable nanoemulsion was formed beyond 100 nm. It is also possible to mix MCTs with other unsaturated fatty acids, and it can be seen from Figure 2 that the ratio of unsaturated fatty acids to MCT should be no greater than 9: 1. Caprylic acid was used as MCT.

Example 2 Selection of Mixing Ratio of Oil Phase and Surfactant

Preparation of nanoemulsions containing resveratrol used a self-assembly method (FIG. 4). Resveratrol was prepared by dissolving 5 mg / ml in ethanol, and the mixture was mixed with MCT, which is an oil phase, in a ratio of 1: 1, and concentrated under reduced pressure at 50 ° C. to remove ethanol. Tween 80, a nonionic surfactant, was added at a ratio of 1: 1, 1: 1.5, 1: 2, 1: 5, and 1:10 and stirred at 400 rpm for 3 minutes. This mixture was dissolved in the ratio of mixture: water (0.5: 99.5) to prepare an emulsion. The results are shown in FIG. 3 shows that the mixture of resveratrol + MCT and Tween 80 formed stable nanoemulsions at 16.29 nm, 16.14 nm, and 17.5 nm at a ratio of 1: 2, 1: 5, and 1:10, respectively, and 1: 1. And 1: 1.5 at 554 nm and 230.9 nm respectively. An unstable emulsion was formed. In addition, although particles of 100 nm or less were formed and stable even at a ratio of 1: 5 and 1:10, it is known that they are not suitable for use as food when the ratio of the surfactant is 5 times or more. Therefore, it was found that the compounding ratio of the surfactant for preparing resveratrol nanoemulsion is more than 2 times to 5 times the weight of the oil phase (resveratrol + MCT). Capric acid was used as the MCT.

Example 3: Preparation of Resveratrol Nanoemulsion

The preparation of nanoemulsions containing resveratrol used a self-assembly method (FIG. 4). In order to identify the region of nanoemulsion formation of 100 nm or less, a schematic diagram was used using a ternary phase diagram (FIG. 5). Resveratrol was prepared by dissolving 5 mg / ml in ethanol, and then mixed with an oily MCT at a mixing ratio of 1: 1, and concentrated under reduced pressure at 50 ° C. to remove ethanol. A nonionic surfactant, Tween 80, was added at a ratio of 1: 1, 1: 2, 1: 5 and stirred for 3 minutes. The final mixture of the resveratrol-containing oil phase and the surfactant was dissolved in water at a constant concentration and stirred for 1 hour to prepare a nanoemulsion (Table 1). Resveratrol to MCT content was 1: 1 and 100% by weight of resveratrol MCT, Tween 80 and water were adjusted.

Table 1. Mixing ratio of resveratrol nanoemulsion

Table 2 shows the particle size and concentration of the nanoemulsion prepared as described above.

Table 2. Particle size and concentration of resveratrol nanoemulsion

According to the results of Table 2, the resveratrol + MCT mixture and Tween 80 in a 1: 5 blending ratio of resveratrol + MCT: Tween 80: water = 3.4 wt%: 16.6 wt%: 80 wt% resveratrol content of 250μg / ml The highest nanoemulsion was prepared, but the high content of MCT and Tween 80 was excluded from the optimum conditions. Therefore, the mixture of resveratrol + MCT and Tween 80 showed a particle size of 30.0 nm in a ratio of 1: 2 and resveratrol + MCT containing 83.3 μg / ml of resveratrol: Tween 80: water = 1.67 wt%: 3.33 wt%: It can be seen that the 95% by weight concentration is the optimal condition for preparing the nanoemulsion. Similar results were obtained in the case of Table 2 in the case of Tween 60. Particle size was determined by storage at 25 ° C. for 4 weeks for stability measurement of the nanoemulsion. The results are shown in FIG. According to the results of FIG. 6, the mixing ratio of the mixture of resveratrol + MCT and Tween 80 1: 1 maintained a relatively stable form during storage even though the microemulsion was formed. The ratio of 1: 2 and 1: 5 maintained a very stable form except for the nanoemulsion prepared with water content of 90% or less.

Example  4: With resveratrol MCT Change in content ratio

The experiment was carried out in the same manner as in Example 3 with the content ratio of resveratrol and MCT of 1: 5. Lauric acid was used as the MCT. The results were as follows.

Table 3. Resveratrol: MCT ratio 1: 5

From the above results, it was confirmed that even when the content ratio of resveratrol and MCT was 1: 5, a nanoemulsion having a particle size of 100 nm or less was formed.

Figure 1 shows the particle size of the nanoemulsion prepared by varying the type of oil.

Figure 2 shows the particle size of the nanoemulsion prepared by varying the MCT and corn oil mixing ratio.

Figure 3 shows the particle size of the nanoemulsion prepared by varying the mixing ratio of the mixture of Res + MCT and Tween 80.

4 is a flowchart illustrating a method of preparing resveratrol nanoemulsion by a magnetic coupling method.

Figure 5 shows a three-phase diagram of the resveratrol nanoemulsion.

6 is a diagram showing the storage stability of the resveratrol nanoemulsion.

Claims (6)

In nanoemulsions comprising resveratrol, medium chain triglycerides, surfactants and water, the content ratio of resveratrol to medium chain triglycerides is 1: 1 to 1: 5, and the sum of these components is 0.25 to 3.4% by weight, and the surfactant The content ratio of 2 to 5 times the sum of the resveratrol and the medium chain triglycerides, the nanoemulsion comprising a residual amount of water. The method of claim 1, The medium chain triglyceride is nanoemulsion, characterized in that at least one selected from the group consisting of caproic acid, caprylic acid, capric acid and lauric acid. The method of claim 1, Nanoemulsion, characterized in that the surfactant is Tween 60 or Tween 80. delete Mixing resveratrol with an organic solvent; Mixing this mixture with medium chain triglycerides; Concentrating under reduced pressure at 50 ° C.-55 ° C. to remove the organic solvent; Mixing a mixture of resveratrol and medium chain triglycerides with a surfactant; And A method for producing a nanoemulsion comprising dissolving this mixture in water. delete

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