Grape seed oil and method for producing same

Abstract

[Problem] To provide: a grape seed-derived oil containing an appropriate amount of a polyphenol; and a method for producing the oil. [Solution] This method for producing a grape seed-derived oil includes: a first step for precipitating and removing a fat-soluble component having a melting point of 4°C or higher from a crude extracted oil from grape seeds; a second step for separating a component that volatilizes from the crude extracted oil under a temperature condition within 10-35°C under a reduced pressure atmosphere of 1-6 kPa; and a third step for removing insoluble components from an extracted oil obtained after the first step and the second step. As a result, an oil containing 0.02 wt% to 0.10 wt% inclusive of a polyphenol derived from grape seeds is obtained.

Description

Grape seed oil and its manufacturing method

[0001] The present invention relates to an oil extracted from the seeds of the genus Vitaceae, particularly Vitis vinifera, Vitis coignetiae, or hybrids thereof (hereinafter simply referred to as "grapes"), and a method for producing the same. Specifically, the present invention relates to an oil that contains a certain amount of grape seed-derived polyphenols as an active ingredient and is safe, stable, and functional enough to be incorporated into cosmetics (including quasi-drugs; the same applies hereinafter), and a method for producing the same. The present invention also relates to cosmetics incorporating grape seed-derived oil.

[0002] Grape seed oil is a vegetable oil extracted from the seeds of plants of the Vitaceae family. Grape seed oil is a liquid oil primarily composed of linoleic acid and is often used as a cosmetic ingredient. Grape seeds, the source of this oil, contain gallic acid, catechin, epicatechin, procyanidins, proanthocyanidins, and other compounds, and are known to have various functional properties (Non-Patent Document 1).

[0003] Grape seed oil is expected to contain polyphenols derived from grape seeds, but to enhance safety, shelf life, and purity, refined oils obtained by removing impurities from the extracted crude oil through a refining process are used in cosmetics, etc. However, such refining methods also remove functional components such as polyphenols as impurities, so typical grape seed oils contain only a few tens of ppm to 100 ppm of polyphenols (Non-Patent Document 2). For this reason, there has been a problem in that there is no oil that contains a certain amount of polyphenols found in grape seeds and is safe, stable, and functional enough to be incorporated into cosmetics.

[0004] Furthermore, Patent Document 1 proposes an extraction method using liquefied dimethyl ether to provide water-soluble natural products that contain water-soluble and fat-soluble natural components and are not subject to denaturation associated with thermal decomposition.

[0005] Food Chemistry, Volume89, Issue1, (2005):1-9Food Chemistry, Volume108, Issue3, (2008):1122-32.

[0006] Japanese Patent Application Laid-Open No. 2019-163232

[0007] By utilizing the extraction method described in Patent Document 1, polyphenols can be obtained as water-soluble components from grape seeds, and oil can be obtained as a fat-soluble component. However, the grape seed-derived oil obtained by this method has poor stability due to an excessively high concentration of polyphenols, and is highly irritating to the skin, making it difficult to use as a cosmetic ingredient.

[0008] Although grape seeds contain many useful polyphenols, conventional purification and extraction methods have either too little or too much polyphenol content, making it difficult to achieve functionality suitable for cosmetics or to ensure safety and stability when used as an active ingredient in cosmetics.

[0009] Therefore, a main object of the present invention is to provide an oil derived from grape seeds containing an appropriate amount of polyphenols and a method for producing the same.

[0010] A first aspect of the present invention relates to a grape seed-derived oil. The grape seed-derived oil is preferably composed essentially of natural ingredients, including natural grape seed-derived oil. However, the extraction solvent used to extract the oil from grape seeds may remain at 1 wt% or less. The oil according to the present invention contains grape seed-derived polyphenols at 0.02 wt% to 0.10 wt%. Polyphenols are a general term for compounds containing multiple phenolic hydroxyl groups. As shown in Example 1 below, if the grape seed-derived polyphenol content is 0.02 wt% or more, the functionality of polyphenols suitable for cosmetics can be fully exhibited. Furthermore, if the grape seed-derived polyphenol content is 0.10 wt% or less, the oil is sufficiently mild in skin irritation, allowing it to be incorporated as an active ingredient in cosmetics.

[0011] In the oil according to the present invention, the polyphenols preferably include catechin and epicatechin.

[0012] The oil of the present invention preferably contains grape seed-derived polyphenols as an active ingredient and has one or more of the following functions: antioxidant activity, anti-glycation activity, and anti-inflammatory activity. Thus, the present invention can impart functionality not exhibited by ordinary grape seed-derived oils.

[0013] The oil according to the present invention preferably contains grape seed-derived polyphenols as an active ingredient and has one or more of the following activities: DPPH radical scavenging activity, ABTS radical scavenging activity, AGEs production inhibitory activity, SOD2 promoting activity, IL-1α inhibitory activity, IL-β inhibitory activity, and GCLC promoting activity.

[0014] In the oil of the present invention, the grape seeds are preferably extracted from fresh grapes that have not undergone a fermentation process, which prevents the unpleasant odor and deterioration of the oil due to fermentation and minimizes the need for refining processes that remove polyphenols, such as deodorization and deacidification.

[0015] Preferably, the oil according to the present invention is not skin sensitizing.

[0016] The stability of the oil according to the present invention is preferably guaranteed for at least three months when stored in a dark place at room temperature (15 to 30° C.), where stability means that no solid precipitation or formation of an aqueous phase is observed under the above conditions.

[0017] A second aspect of the present invention is a cosmetic product containing the grape seed oil according to the first aspect. More specifically, the grape seed oil can be incorporated into a cosmetic product intended for use as a topical skin preparation.

[0018] A third aspect of the present invention is a method for producing grape seed-derived oil. The method for producing oil according to the present invention comprises steps 1 to 3. In step 1, fat-soluble components with a melting point of 4°C or higher are precipitated and removed from a crude grape seed oil extract. In step 2, components that volatilize at temperatures between 10°C and 35°C are separated from the crude grape seed oil extract under a reduced pressure of 1 to 6 kPa. While steps 1 and 2 may be performed in either order, it is preferable to perform step 2 after step 1. In step 3, insoluble components are removed from the extracted oil after steps 1 and 2. This allows for the efficient production of an oil containing grape seed-derived polyphenols at 0.02 wt% to 0.10 wt%.

[0019] The present invention provides grape seed-derived oil containing an appropriate amount of polyphenols and a method for producing the same. Specifically, by incorporating 0.02 wt% to 0.10 wt% of the polyphenols inherent in grape seeds into the oil, it is possible to achieve both safety and stability for cosmetic use, as well as functionality.

[0020] FIG. 1 is a flow diagram showing an example of a method for producing grape seed oil. FIG. 2 shows an example of an extraction apparatus used in producing grape seed oil. FIG. 3 shows the extraction apparatus used in producing grape seed oil in Example 1. FIG. 4 shows the extraction rate and total polyphenol content of the extract of Example 1 as a function of extraction time. FIG. 5 shows the change in total polyphenol content of the oil at each step in Example 1. FIG. 6 shows the results of a comparison of the total polyphenol content of Example 1 and Comparative Example 1. FIG. 7 shows the results of a qualitative test of polyphenols in the oil of Example 1. FIG. 8 shows the results of measuring DPPH radical scavenging activity of Example 1 and Comparative Example 1. FIG. 9 shows the results of measuring ABTS radical scavenging activity of Example 1 and Comparative Example 1. FIG. 10 shows the results of measuring AGEs production inhibitory activity of Example 1 and Comparative Example 2. FIG. 11 shows the results of measuring gene expression activity of Example 1. FIG. 12 shows the results of measuring gene expression activity of Example 1.

[0021] Hereinafter, embodiments of the present invention will be described with reference to the drawings. The present invention is not limited to the embodiments described below, and includes appropriate modifications of the embodiments described below within the scope obvious to those skilled in the art.

[0022] [1. Grape seed oil and its manufacturing method] A preferred embodiment of grape seed oil and its manufacturing method will be described with reference to Figure 1. As shown in Figure 1, the manufacturing method according to this embodiment includes, in this order, a washing and drying step (step S1), a crushing step (step S2), an extraction step (step S3), a solvent separation step (step S4), a first filtration step (step S5), a reduced pressure distillation step (step S6), a second filtration step (step S7), a cold treatment step (step S8), and a sterilization filtration step (step S9). Each step will be described below.

[0023] The washing and drying process (step S1) involves washing and then drying the seeds extracted from fresh grape berries. While any washing and drying method is acceptable, it is preferable to use a method that does not deteriorate the components of the seeds (a method that does not apply heat). Furthermore, it is preferable that the grapes used as the raw material for the oil are berries that have not undergone a fermentation process. The grape fermentation process includes both cases where only natural yeast present in the grapes themselves is used, and cases where artificial yeast (yeast cultivated for winemaking) is added to the grape juice or seeds. Since grapes generally begin to naturally ferment seven days after harvest, it is preferable to carry out the washing and drying process within seven days (168 hours) of harvest.

[0024] The crushing step (step S2) is a step of crushing the grape seeds. Any crushing method can be used, but it is preferable to employ a means capable of powdering the grape seeds in a short time so as not to deteriorate the components of the skin. For example, a known crusher used in the crushing of food products can be used to crush the grape seeds. It is preferable to crush the grape seeds immediately before (specifically, within one hour) the extraction step described below to avoid deterioration due to oxidation and light. Furthermore, if the grape seeds need to be stored for more than one hour after crushing, they can be stored in a sealed, light-blocking container to prevent deterioration due to oxidation and light.

[0025] The extraction step (step S3) is a step of extracting components from grape seeds. Any extraction method can be used, as long as the grape seeds are immersed in the extraction solvent and the grape seed components are dissolved into the extraction solvent. A solution containing liquefied dimethyl ether is preferably used as the extraction solvent. Liquefied dimethyl ether is converted into a liquid state by increasing the dimethyl ether's vapor pressure above its saturation level. However, it may also be a solution containing a co-solvent, such as water or alcohol, at a subsaturation level. The amount of co-solvent added is preferably less than the saturation level for the liquefied dimethyl ether, more specifically, less than 7% by mass of the liquefied dimethyl ether. The extraction conditions are preferably 4 to 40°C and 1.0 MPa or less, and more specifically, 25°C and 0.7 MPa or less. Other extraction solvents that can be used include, for example, ethanol, 1,3-butylene glycol, or an aqueous solution thereof. This step yields an extract containing the grape seed extract and the extraction solvent.

[0026] The solvent separation step (step S4) is a step of separating the extractant from the extract by volatilizing the extractant from the extract. While any method for solvent separation is acceptable, a method that does not degrade the extractant is preferred. For example, a method of volatilizing the extractant by leaving the extract at a temperature of 15°C to 40°C and atmospheric pressure (101.33 kPa) or a method of volatilizing the extractant by irradiating the extract with ultrasound for a short period of time (10 minutes or less) may be used. More specifically, when liquefied dimethyl ether is used as the extractant, the liquefied dimethyl ether is volatilized and separated from the extract by heating the extract at 30°C and atmospheric pressure. Even after this volatilization step, a small amount of liquefied dimethyl ether may remain in the extract. Any remaining liquefied dimethyl ether may be volatilized by irradiating it with ultrasound for 10 minutes. This allows the extraction solvent (liquefied dimethyl ether) to be almost completely separated from the extract. In this way, the extraction solvent is separated from the extract to obtain the extracted crude oil.

[0027] The first filtration step (step S5) is a step of filtering the extracted crude oil to remove impurities and solids, such as lipids with high melting points, mixed in the extracted crude oil. This first filtration step is primarily intended to precipitate and remove fat-soluble components with a melting point of 4°C or higher from the extracted crude oil from grape seeds. Specifically, the extracted crude oil contains fat-soluble components derived from grape seeds with a melting point of 4°C or higher, such as monounsaturated fatty acids such as oleic acid (melting point approximately 13°C), saturated fatty acids such as palmitic acid (melting point approximately 63°C) and stearic acid (melting point approximately 70°C), phytosterols (melting point approximately 136°C), and carotenoids (melting point approximately 63-183°C). For this reason, the first filtration step is preferably performed under temperature and pressure conditions (specifically, atmospheric pressure, 13°C or lower) that allow at least oleic acid to solidify. Conversely, an example of a fat-soluble component derived from grape seeds with a melting point of 4°C or higher is linoleic acid (melting point approximately -5°C). While any filtration method can be used, for example, a filter paper or membrane filter with a mesh size of 1.0 to 10 μm can be used. This process removes impurities and lipids from the extracted crude oil, yielding a mixture of water, water-soluble components, and oil derived from grape seeds. This water-soluble component includes polyphenols. The filtered mixture typically contains polyphenols at a very high concentration of 3 wt% (3,000 mg / kg) or more. Therefore, while this mixture is expected to have high functionality, its stability and safety are poor for cosmetic applications, making it unsuitable for use as a cosmetic ingredient in its current state.

[0028] The vacuum distillation step (step S6) is a step of separating volatile components that volatilize at reduced pressure below 35°C from the filtered mixture. This vacuum distillation step is primarily intended to remove water and other volatile substances from the filtered mixture. When removing water from the mixture, heating the mixture above 35°C may denature the active ingredients contained in the mixture. Therefore, it is preferable to keep the temperature of the mixture below 35°C and instead place the mixture under reduced pressure. Specifically, the saturated vapor pressure of water at 10°C is 1.227 kPa, and the saturated vapor pressure of water at 35°C is 5.6216 kPa. Therefore, it is preferable to remove volatile substances, including water, by vacuum distillation from the mixture under a reduced pressure atmosphere in which water volatilizes at 10 to 35°C, specifically 1 to 6 kPa. While any vacuum distillation method is acceptable, it is preferable to use an evaporator. For example, approximately 500 mL of the mixture is placed in a 2-L eggplant flask, the pressure in the flask is reduced to 5.6 kPa, and vacuum distillation is performed at 35°C for 5 hours. This removes water until the water concentration in the mixture reaches approximately 0.02 wt% (200 mg / kg, 0.1 mL). This process removes almost all water from the filtered mixture, yielding an extracted oil derived from grape seeds. This extracted oil is the result of the precipitation of some of the water-soluble components, such as grape seed polyphenols, that were dissolved in water and remain as solids.

[0029] The second filtration step (step S7) involves filtering the extracted oil after vacuum distillation again to remove any remaining solids. Any filtering method can be used; for example, a filter paper or membrane filter with a mesh size of 0.3 to 0.6 μm can be used. This step removes the solids precipitated in the vacuum distillation step (step S6), yielding a clear grape seed-derived oil (refined oil). Some of the grape seed-derived polyphenols are contained within this solid. Therefore, when comparing the mixture after the first filtration step (step S5) with the oil after the second filtration step (step S7), the latter has a lower polyphenol concentration. However, because not all polyphenols are removed by the second filtration step, a moderate amount of polyphenols remains in the oil. This grape seed-derived oil has a polyphenol concentration of 0.02 to 0.10 wt% (200 to 1000 mg / kg). This polyphenol concentration satisfies the stability and safety requirements for cosmetic applications, so this oil can be used as a cosmetic ingredient.

[0030] The cold treatment step (step S8) is a step for precipitating insoluble matters contained in the grape seed-derived oil. While any method for cold treatment is acceptable, it is preferable to store the oil in a refrigerator at 4°C or other storage facility where there is no temperature change and it is not exposed to light. This step allows the precipitation of insoluble matters that were not completely removed in the filtration step (step S7).

[0031] The sterile filtration step (step S9) is a step of sterile filtering the grape seed oil after the cold treatment step. Sterile filtration can be performed using, for example, a membrane filter with a mesh size of 0.22 μm or less. The sterilized grape seed oil is filled into a sterilized container. Sterile filtration is preferably performed in a clean environment, such as a clean room or clean bench.

[0032] As described above, this grape seed-derived oil has a polyphenol concentration of 0.02-0.10 wt% (200-1000 mg / kg). In particular, as shown in Example 1 below, the oil that underwent the vacuum distillation step (S6) and filtration step (S7) had a total polyphenol content sufficiently reduced to approximately 0.02 wt%, satisfying the stability and safety requirements for cosmetic applications. On the other hand, as shown in Example 1 below, the oil that did not undergo these vacuum distillation steps (S6) and filtration steps (S7) had a total polyphenol content of approximately 0.3 wt%, resulting in poor stability and skin safety. In other words, considering stability and skin safety, a total polyphenol content of approximately 0.3 wt% is excessive in the oil, and the appropriate total polyphenol content should be approximately one-third of that, 0.10 wt%. Therefore, based on the results obtained in Example 1 described below, it is appropriate to set the lower limit of the polyphenol concentration of grape seed-derived oil at 0.02 wt % and the upper limit at 0.10 wt %.

[0033] [2. Extraction Apparatus for Extracting Crude Oil] A preferred embodiment of an extraction apparatus for extracting crude oil from grape seeds will be described in detail below with reference to the drawings. That is, this extraction apparatus is used in the extraction step (step S3) and the solvent separation step (step S4) shown in FIG.

[0034] Fig. 2 shows an example of an extraction apparatus for producing grape seed oil according to the present embodiment, and Fig. 2 merely shows a schematic view of the shape, size, and arrangement of the components to enable understanding of the extraction apparatus.

[0035] The extraction apparatus 100 includes a storage tank 1 for storing liquefied dimethyl ether 2, an extraction tank 6 for bringing a raw material 7 into contact with the liquefied dimethyl ether 2, a separation tank 11 for separating the liquid discharged from the extraction tank 6, and a pump 3 for pumping the liquefied dimethyl ether 2 from the storage tank 1 to the extraction tank 6.

[0036] Extraction apparatus 100 also has conduits 5, 10, 12, 14, 16, 19, 20, and 23 for introducing and discharging liquefied dimethyl ether 2, and valves 4, 9, 13, 15, 18, 21, and 22 for adjusting the air pressure in each tank to control the introduction and discharge of liquefied dimethyl ether 2. Extraction tank 6 and separation tank 11 can adjust their pressures to maintain liquefied dimethyl ether 2 in a liquid state.

[0037] In the extraction apparatus 100, the storage tank 1 functions as a storage means for storing the liquefied dimethyl ether 2. The pump 3, the valve 4, and the conduit 5 function as a liquid transfer means for transferring the liquefied dimethyl ether 2 from the storage tank 1 to the extraction tank 6. The extraction tank 6 functions as a contact means for contacting the liquefied dimethyl ether 2 with the raw material 7 to obtain an extract. The valve 9 and the conduit 10 function as a discharge means for discharging the extract from the extraction tank 6 to the separation tank 11. The separation tank 11 functions as a separation means for separating the liquefied dimethyl ether 2 from the extract. The conduit 12 and the valve 13 connected to the separation tank 11 function as a vaporization means for vaporizing the liquefied dimethyl ether. The condenser 17 connected to the conduit 16 functions as a condensation means for condensing the dimethyl ether discharged from the separation tank 11 from a gas state back into a liquid. The conduits 19 and 20 function as a supply means for supplying the liquefied dimethyl ether 2 to the storage tank 1.

[0038] The extraction apparatus 100 further includes optional components such as a thermometer and a pressure gauge for detecting the temperature and air pressure in each of the storage tank 1, extraction tank 6, and separation tank 11, an agitator for stirring the contents in each tank, and a device for circulating an inert gas such as nitrogen to purge active gases such as oxygen from the tanks and conduits.

[0039] In the extraction apparatus 100, an extracted crude oil can be obtained from the raw material 7 as follows. In this embodiment, the raw material 7 is the above-mentioned grape seeds that have been washed, dried, and then crushed.

[0040] Extraction tank 6 is equipped with filters 8 on both the upstream and downstream sides. Raw material 7 (grape seeds) is introduced into extraction tank 6. At this time, valves 4, 9, 13, 15, 18, 21, and 22 are all closed. If storage tank 1 does not store a sufficient amount of liquefied dimethyl ether 2, valve 21 is opened, and liquefied dimethyl ether 2 is supplied to storage tank 1 via conduit 20, after which valve 21 is closed. At this time, valve 18 may be opened when valve 21 is opened, and valve 18 may be closed when valve 21 is closed.

[0041] Subsequently, valve 4 is opened, and liquefied dimethyl ether 2 is extracted from storage tank 1 by pump 3 and sent to extraction tank 6 via conduit 5. After liquefied dimethyl ether 2 has been introduced into extraction tank 6 until it comes into contact with raw material 7, valve 4 is closed.

[0042] When the raw material 7 is immersed in the liquefied dimethyl ether 2 in the extraction tank 6, the water, water-soluble compounds, and fat-soluble compounds contained in the raw material 7 are extracted by the liquefied dimethyl ether 2. As a result, an extract is obtained in which the water, water-soluble compounds, and fat-soluble compounds in the raw material 7 are dissolved in the liquefied dimethyl ether 2. Here, water refers to the moisture contained in the raw material.

[0043] Next, valves 4 and 9 are opened, and liquefied dimethyl ether 2 is introduced into extraction tank 6 from storage tank 1 via conduit 5 by pump 3. The extract in extraction tank 6 is then introduced into separation tank 11 via conduit 10. That is, when new liquefied dimethyl ether 2 is drawn from storage tank 1 into extraction tank 6, the extract in extraction tank 6 is pushed into separation tank 11. As a result, the interior of extraction tank 6 is replaced with new liquefied dimethyl ether, but raw material 7 in extraction tank 6 remains in extraction tank 6 because filters 8 are located upstream and downstream of extraction tank 6. That is, when new liquefied dimethyl ether is introduced into extraction tank 6, the extract is pushed out of extraction tank 6 and separated from raw material 7. Note that valves 4 and 9 are opened after a predetermined time has elapsed since liquefied dimethyl ether 2 was introduced into extraction tank 6, allowing moisture and other components of raw material 7 to transfer to liquefied dimethyl ether 2. At this time, liquefied dimethyl ether 2 may be left to stand in contact with raw material 7 for a predetermined time, or the liquefied dimethyl ether may be stirred.

[0044] Thereafter, by closing valves 4 and 15 and opening valves 9, 13, and 22, the pressure in the path from valve 4 to valve 13 becomes less than the saturated vapor pressure of dimethyl ether. As a result, liquefied dimethyl ether 2 in this path is vaporized and discharged from conduit 23 via conduit 14. At this time, pump 3 may be used to discharge the dimethyl ether, if necessary. The extracted crude oil remains in separation tank 11, with liquefied dimethyl ether 2 evaporated and separated from the extract.

[0045] Here, the case where valve 22 connected to the outside air is opened and valve 15 connected to condenser 17 is closed has been described, but valve 22 may be closed and valve 15 may be opened. In this manner, vaporized dimethyl ether is introduced into condenser 17 via conduit 16. As a result, dimethyl ether is condensed again in condenser 17 to produce liquefied dimethyl ether 2. Thereafter, valve 18 is opened, and the produced liquefied dimethyl ether 2 is introduced into storage tank 1 via conduit 19. In this manner, liquefied dimethyl ether 2 can be reused.

[0046] The extracted crude oil remaining in the separation tank 11 is subjected to the first filtration step (step S5), vacuum distillation step (step S6), second filtration step (step S7), cold treatment step (step S8), and sterilization filtration step (step S9). This results in grape seed-derived oil (refined oil). The extracted crude oil can be subjected to steps S6 to S9 using a known membrane filter or evaporator, as described above.

[0047] The grape seed oil obtained through the above process may be used as is, or may contain ingredients used in cosmetics and quasi-drugs, as appropriate, within limits that do not impair the effects of the oil. Examples of such ingredients include fats and oils, waxes, hydrocarbons, fatty acids, alcohols, esters, surfactants, metal soaps, pH adjusters, preservatives, fragrances, moisturizers, powders, UV absorbers, thickeners, pigments, antioxidants, whitening agents, chelating agents, excipients, and film-forming agents.

[0048] Grape seed oil can also be incorporated into cosmetics or quasi-drugs, primarily for use as external skin preparations. Examples of the formulations of cosmetics and quasi-drugs include lotions, creams, emulsions, gels, aerosols, essences, packs, cleansers, bath additives, foundations, dusting powders, lipsticks, ointments, and patches. The antioxidant composition of the present invention can also be incorporated into soaps, body washes, facial cleansers, shampoos, rinses, treatments, and toothpastes.

[0049] Next, the grape seed-derived oil according to the present invention will be described in more detail using examples.

[0050] [Test Method] (1. Measurement of Total Polyphenol Amount) The amount of polyphenols contained in grape seed-derived oil was measured using the Folin-Ciocalteu method (ISO 14502-1:2005), an official ISO method. The Folin-Ciocalteu method measures absorbance by changing the color of a reagent due to the reduction of phenolic hydroxyl groups. Here, 50 wt% ethanol was added to the oil, and the oil was then defatted with hexane to prepare a sample solution. A calibration curve was created using gallic acid as a standard substance, and the amount of polyphenols in gallic acid equivalents was calculated.

[0051] (2. Qualitative Analysis of Polyphenols) In order to identify whether or not polyphenols are contained in grape seed-derived oil, a qualitative analysis was carried out using an LC-QTOFMS (liquid chromatograph quadrupole time-of-flight mass spectrometer). The TIC chromatogram obtained by the analysis was analyzed, and polyphenols were identified from the MS spectrum and MS / MS spectrum. The measurement conditions for the qualitative analysis are as shown in Table 1 below.

[0052] (Functionality Test) The antioxidant activity of grape seed-derived oil was measured using DPPH radical scavenging activity and ABTS radical scavenging tests. DPPH radical scavenging activity was measured by reacting a sample with a DPPH radical solution (room temperature, 30 min), measuring the absorbance (517 nm) of the solution using a plate reader, and calculating the activity (%) relative to the blank. ABTS radical scavenging activity was measured by reacting a sample with an ABTS radical solution (37°C, 4 min), measuring the absorbance (734 nm) of the solution using a plate reader, and calculating the activity (%) relative to the blank.

[0053] The anti-glycation activity of grape seed oil was measured using an AGEs (advanced glycation end products) production inhibitory activity test. A BSA (bovine serum albumin) solution and a glucose solution were added to the sample and reacted (60°C, 96 hours). The fluorescence of the reaction solution was measured using a plate reader (excitation wavelength 370 nm, fluorescence wavelength 440 nm), and the activity (%) relative to the blank was calculated.

[0054] The gene expression effect of grape seed oil was measured as follows.

[0055] <Expression of SOD2, IL-1α, and IL-1β genes using human skin fibroblasts> SOD2, IL-1α, and IL-1β genes were used. Human newborn skin fibroblasts were plated in a 60 mm diameter dish at 5 × 10 ４ Cells were seeded on a dish. After 24 hours, samples were added to a final concentration of 0.1%. Sample-free medium was used as a control. After 24 hours of incubation, RNA was extracted from the cells using the RNA extraction reagent "TRI Reagent" (Merck KGaA, Darmstadt, Germany). Using this RNA as a template, cDNA was synthesized by reverse transcription with Oligo dT Primer using the "Primescript RT reagent kit" (Takara Bio, Shiga, Japan). mRNA levels were quantified using the PCR reagent "Luna Universal qPCR Master Mix" (New England Biolabs, MA, USA) and a PCR device "LightCycler 96" (Roche, Basel, Switzerland) with primers for various genes. Cq values ​​were calculated using the delta Ct method, and the expression levels of each gene were expressed as relative values ​​based on the Cq value of GAPDH.

[0056] <GCLC gene expression using human skin keratinocytes> GCLC was used as the gene. Human skin keratinocytes were plated in a 60 mm diameter dish at 5 × 10 ４Cells were seeded on a dish. After 24 hours, the sample was added to a final concentration of 0.005 mg / mL, and the oil was visually confirmed to have dissolved in the medium. Sample-free medium was used as a control. After 8 hours of incubation, RNA was extracted from the cells using the RNA extraction reagent "TRI Reagent" (Merck KGaA, Darmstadt, Germany). Using this RNA as a template, cDNA was synthesized by reverse transcription with Oligo dT Primer using the "Primescript RT reagent kit" (Takara Bio, Shiga, Japan). mRNA levels were quantified using the PCR reagent "Luna Universal qPCR Master Mix" (New England Biolabs, MA, USA) and a PCR device "LightCycler 96" (Roche, Basel, Switzerland) with primers for each gene. Cq values ​​were calculated using the delta Ct method, and the expression levels of each gene were expressed as relative values ​​based on the Cq value of GAPDH.

[0057] (3. Stability Test) As a stability test for the grape seed-derived oil, the oil was left to stand in a thermostatic chamber exposed to fluorescent light at 5°C, 20°C, 40°C, and 50°C, and the precipitation, color, and aroma were checked at regular intervals.

[0058] (4. Safety Test) As a safety test of the grape seed-derived oil, an in vitro skin sensitization test was carried out.

[0059] Example 1 In Example 1, grape seed oil was produced according to the procedure shown in Figure 1. The grape seeds used as the raw material were extracted from fresh grapes that had not undergone fermentation, washed, and seeds that floated on the water were removed (sorted). The seeds were then dried to a moisture content of approximately 6% by mass and crushed to a size of 1.0 mm or less immediately before extraction.

[0060] In the extraction process, an extract was produced from grape seeds using the extraction apparatus shown in FIG. 3. Specifically, 18.0 g of crushed grape seeds 57, each approximately 1.0 mm in length, was placed in a 25 mL extraction tank 56, with filters 55, 58 installed upstream and downstream. Subsequently, valve 52 was closed and valve 53 was opened, and dimethyl ether 51 was filled into syringe pump 50 and liquefied at 25°C and 0.7 MPa. Separation tank 62 was previously purged with dimethyl ether, and valves 52, 53, 54, 59, 60, and 61 were closed. The extraction apparatus shown in FIG. 3 is similar to the extraction apparatus 100 shown in FIG. 2 in that it does not circulate dimethyl ether.

[0061] Next, valves 53, 54, 59, and 60 were opened, and liquefied dimethyl ether was supplied to extraction tank 56 by syringe pump 50. When extraction tank 56 was filled with liquefied dimethyl ether, syringe pump 50 was stopped, valves 54 and 59 were closed, and crushed grape seeds 57 were immersed in the liquefied dimethyl ether.

[0062] Valves 54 and 59 were opened, and liquefied dimethyl ether was again supplied by syringe pump 50. The flow rate was adjusted to 2.5 mL / min, and the residence time was adjusted to 10 minutes, and 30 mL of extract was collected in separation tank 62. Valve 60 was then closed, separation tank 62 was removed from the apparatus, and the pressure was adjusted to atmospheric pressure in a specified draft chamber to volatilize the liquefied dimethyl ether and produce an extract. The resulting extract was then irradiated with ultrasound for 10 minutes using an ultrasonic cleaner, and then left to stand overnight at 4°C to completely volatilize the liquefied dimethyl ether, yielding an extracted crude oil.

[0063] This extracted crude oil was first filtered through a 7 μm filter paper to remove solid lipids and impurities, and then subjected to vacuum distillation using an evaporator at 40 ° C for 5 hours to separate water and volatile components, thereby obtaining an extracted oil. The pressure inside the evaporator was 5.6 kPa.

[0064] Next, the precipitate in the extracted oil generated during the vacuum distillation was separated by a second filtration using a 0.45 μm membrane filter. This precipitate is presumed to be a part of the water-soluble components dissolved in water.

[0065] Thereafter, the mixture was left to stand overnight at 4°C for cold treatment (precipitation), and then sterilized by filtration using a 0.22 µm membrane filter in a clean bench. The resulting oil (refined oil) was then filled into a brown bottle.

[0066] Comparative Example 1 In Comparative Example 1, oil was produced by pressing grape seeds of the same type as those used in Example 1. Specifically, the grape seeds were washed, sorted, dried, and crushed using the same procedures as in Example 1. The crushed grape seeds were then pressed using a screw press to extract the oil. The oil thus extracted was processed using the same procedures as in Example 1, including the second filtration, cold treatment, and sterilization filtration, and then bottled in an amber bottle.

[0067] Comparative Example 2 In Comparative Example 2, oil (extracted crude oil) was used, which was obtained by omitting the filtration step (step S5) and subsequent steps in Example 1.

[0068] Comparative Example 3 In Comparative Example 3, oil (filtered crude oil extract) obtained in Example 1 was used, omitting the reduced pressure distillation (water removal) step (step S6) and subsequent steps.

[0069] [Discussion] (1. Oil Extraction Yield and Total Polyphenol Content) The extraction time dependence of the extraction yield and total polyphenol content of the extract obtained in the extraction process of Example 1 is shown in Figure 4. The extraction conditions were 25°C, 0.7 MPa, and a residence time of 10 minutes. The extract was prepared by drying the extracted crude oil in a vacuum dryer (40°C, 5.33 kPa, 12 hours), removing the water, and the remaining extract was used. An extraction time of 36 minutes or longer resulted in an extraction yield of 16.5% by mass and a total polyphenol content of 3136 mg / kg (0.3136 wt%), which remained almost constant thereafter. Therefore, an extraction time of 36 minutes was determined to be optimal. The amount of liquefied dimethyl ether delivered was 3.3 kg per 1 g of grape seeds. The oil obtained in the extraction process contained a high amount of total polyphenols and was expected to have high functionality. However, due to its poor stability and skin safety, it cannot be used as a cosmetic ingredient as is, and therefore requires subsequent processing. The extraction yield of the extract was calculated using the following formula: [Formula] Extraction rate of extract [mass %] = (mass of extract / mass of raw material introduced into extraction tank) × 100

[0070] (2. Changes in Total Polyphenol Content in Oil at Each Step) Figure 5 shows the changes in total polyphenol content in oil at each step after the extraction step (S3) in Example 1. The total polyphenol content in the oil was 3100 mg / kg (0.31 wt%) in the solvent separation step (S4), and was reduced to 2900 mg / kg (0.29 wt%) by removing solids in the first filtration step (S5). Subsequently, the water was removed in the vacuum distillation step (S6), and the precipitated solids were separated in the filtration step (S7). As a result, the total polyphenol content was reduced to 250 mg / kg (0.025 wt%). While most of the polyphenols obtained by extraction were removed, the remaining polyphenols are presumed to be relatively soluble in oil. The total polyphenol content in the oil after the cold treatment step (S8) and sterilization filtration step (S9) was 250 mg / kg (0.025 wt%), showing almost no change. Therefore, it can be said that the vacuum distillation step (S6) and the filtration step (S7) contribute to achieving an appropriate amount of total polyphenols in the oil.

[0071] [Comparison of total polyphenol content] The results of comparing the total polyphenol content of the oil of Example 1 with that of the oil obtained by the expression method of Comparative Example 1 are shown in Figure 6. The total polyphenol content of Example 1 and Comparative Example 1 was 250 mg / kg (0.025 wt%) and 60 mg / kg (0.006 wt%), respectively. Thus, the oil obtained by the expression method had a low total polyphenol content and was insufficient.

[0072] The results of the qualitative test of polyphenols in the oil of Example 1 are shown in Figure 7. A large number of peaks were detected in the oil containing water-soluble components of Example 1. Analysis of the MS spectrum and MS / MS spectrum using the measured MS / MS spectrum database (METLIN) allowed the identification of 70 compounds shown in Figure 7. It can be said that the polyphenols contained in the grape seed-derived oil of the present invention include catechin, epicatechin, as well as polyphenols such as ε-viniferin and taxifolin. The analytical results of the qualitative test are as follows.

[0073]

[0074] (3. Functionality Test) 1. Antioxidant Activity The results of DPPH radical scavenging activity when the oils of Example 1 and Comparative Example 1 were added at 20 mg / mL each are shown in Figure 8, and the results of ABTS radical scavenging activity are shown in Figure 9. The DPPH radical scavenging activities of the oils of Example 1 and Comparative Example 1 were 44% and 30%, respectively, and the ABTS radical scavenging activities were 31% and 13%, respectively, confirming the superiority of the oil of Example 1 in terms of antioxidant activity.

[0075] 2. Anti-glycation activity The results of the AGE production inhibitory activity when 5.0 mg / mL of the oils of Example 1 and Comparative Example 2 were added are shown in Figure 10. The AGE production inhibitory activities of the oils of Example 1 and Comparative Example 2 were 31% and 13%, respectively, confirming the superiority of the oil of Example 1 in terms of anti-glycation activity.

[0076] 3. Gene Expression Analysis The results of the gene expression effect of the oil of Example 1 using human skin fibroblasts are shown in Figure 11. Here, human fibroblast cells NB1RGB were used to measure the expression levels of the antioxidant enzyme SOD2 and the anti-inflammatory markers IL-1α and IL-1β. An increase in the antioxidant enzyme gene SOD2 was confirmed with the oil of Example 1. Furthermore, suppression of the anti-inflammatory genes IL-1α and IL-1β was confirmed with the oil of Example 1. Note that the control results are from cell cultures in which medium was added instead of oil, etc. From the above, it can be expected that the oil of Example 1 will have antioxidant and anti-inflammatory effects in human skin fibroblasts.

[0077] The results of the gene expression effect of the oil of Example 1 using human skin keratinocytes are shown in Figure 12. Here, the expression level of the gene that produces glutamate cysteine ​​ligase catalytic subunit (GCLC) was measured using human skin keratinocytes (NHEK). An increase in the GCLC gene was confirmed with the oil of Example 1.

[0078] GCLC is a type of enzyme called glutamylcysteine ​​ligase (Glutamate-Cysteine ​​Ligase) that is involved in the biosynthesis of glutathione (GSH). GSH plays important roles in the elimination of oxidative stress and toxins within cells, as well as in the provision of antioxidant defense within cells. GCLC combines glutamic acid with cysteine ​​to form glutamylcysteine, which then leads to the biosynthesis of GSH.

[0079] The oil of Example 1 promotes the production of enzymes involved in glutathione synthesis in human skin keratinocytes, and is expected to have antioxidant effects such as eliminating oxidative stress and toxins, and providing antioxidant protection within cells.

[0080] (4. Stability Test) The results of the stability test of the oils of Example 1 and Comparative Examples 1 and 2 are shown in Table 3. It was confirmed that only the oil of Example 1 had good stability when stored in a dark place at room temperature (around 20°C). The oils of Comparative Examples 2 and 3 were found to have poor stability, with solid precipitation and formation of an aqueous phase. <Table 2: Stability Test Results>

[0081] (5. Safety Test) The results of the safety test of the oils of Example 1 and Comparative Examples 1 and 2 are shown in Table 4. Only the oil of Example 1 was negative for skin sensitization, confirming that safety to the skin is guaranteed. <Table 3: Safety Test Results>

[0082] From the above, it has been confirmed that the grape seed oil of the present invention exhibits functionality by containing a certain amount of polyphenols inherent in grape seeds, and also ensures safety and stability, and is therefore suitable for use in cosmetics.

[0083] In the above, in order to express the contents of the present invention, the present specification has described the embodiments and examples of the present invention with reference to the drawings. However, the present invention is not limited to the above embodiments and examples, and includes modifications and improvements that are obvious to those skilled in the art based on the matters described in the present specification.

Claims

1. Grape seed-derived oil containing grape seed-derived polyphenols in an amount of 0.02 wt% or more and 0.10 wt% or less.

2. The oil of claim 1, wherein the polyphenols include catechin and epicatechin.

3. The oil according to claim 1 or 2, which contains the grape seed-derived polyphenol as an active ingredient and has one or more of the following functions: antioxidant activity, anti-glycation activity, and anti-inflammatory activity.

4. The oil according to claim 1 or 2, wherein the grape seed-derived polyphenol is an active ingredient and has one or more of the following activities: DPPH radical scavenging activity, ABTS radical scavenging activity, AGEs production inhibitory activity, SOD2 promoting activity, IL-1α inhibitory activity, IL-β inhibitory activity, and GCLC promoting activity.

5. The oil according to claim 1 or 2, wherein the grape seeds are seeds extracted from fresh grapes that have not undergone a fermentation process.

6. The oil according to claim 1 or 2, which does not cause skin sensitization.

7. The oil according to claim 1 or 2, wherein the stability of the oil is guaranteed for at least three months when stored in a dark place at room temperature.

8. A cosmetic product containing the oil according to claim 1 or 2.

9. A method for producing grape seed-derived oil, comprising: a first step of precipitating and removing fat-soluble components with a melting point of 4°C or higher from grape seed crude oil; a second step of separating components that volatilize at temperatures between 10°C and 35°C from the extracted crude oil under a reduced pressure of 1 to 6 kPa; and a third step of removing insoluble components from the extracted oil after steps 1 and 2.

10. The method for producing oil according to claim 9, wherein the oil contains grape seed-derived polyphenols in an amount of 0.02 wt% or more and 0.10 wt% or less.

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