Grape seed oil and method for its production

Abstract

[Problem] To provide an oil derived from grape seeds containing an appropriate amount of polyphenols and a method for producing the same. [Solution] A method for producing an oil derived from grape seeds, comprising: a first step of separating and removing a fat-soluble component having a melting point of 4°C or higher from a crude oil extracted from grape seeds; a second step of separating a component volatilized at any temperature in the range of 10 to 35°C from the crude oil under a reduced pressure of 1 to 6 kPa; and a third step of removing an insoluble component from the extracted oil after the first and second steps. Thus, an oil containing 0.02 wt% or more and 0.10 wt% or less of polyphenols derived from grape seeds can be obtained.

Description

Technical Field

[0001] This invention relates to an oil extracted from the seeds of grapes, particularly European grapes (Vitis vinifera), wild grapes (Vitis coignetiae), or hybrids thereof (hereinafter referred to as "grapes"). Specifically, this invention relates to an oil containing a certain amount of polyphenols derived from grape seeds as an active ingredient, possessing safety, stability, and functionality suitable for formulation in cosmetics (including quasi-drugs, hereinafter the same), and a method for manufacturing the same. Furthermore, this invention also relates to a cosmetic formulated with oil derived from grape seeds. Background Technology

[0002] Grape seed oil is a plant oil collected from the seeds of plants in the Vitaceae family. It is a liquid oil primarily composed of linoleic acid and is frequently used as a cosmetic ingredient. The grape seeds from which this oil is made contain gallic acid, catechins, epicatechins, proanthocyanidins, and other pigments, which are known to possess various functionalities (Non-Patent Literature 1).

[0003] While grape seed oil is expected to contain polyphenols derived from grape seeds, refined oil, obtained by removing impurities from crude oil extraction through a refining process to improve safety, preservation, or purity, is often used in cosmetics and other applications. However, in this refining process, functional components such as polyphenols are also removed as impurities, resulting in grape seed oil typically containing only trace amounts of polyphenols, ranging from tens to approximately 100 ppm (Non-Patent Literature 2). Therefore, the current challenge is the lack of an oil that contains a sufficient amount of polyphenols found in grape seeds and possesses the safety, stability, and functionality suitable for formulation in cosmetics.

[0004] In addition, Patent Document 1 proposes an extraction method using liquefied dimethyl ether to provide a water-soluble natural product containing both water-soluble and fat-soluble natural components that does not undergo denaturation due to pyrolysis.

[0005] Existing technical documents

[0006] Non-patent literature

[0007] Non-patent literature 1: Food Chemistry, Volume 89, Issue 1, (2005): 1-9

[0008] Non-patent literature 2: Food Chemistry, Volume 108, Issue 3, (2008): 1122-32.

[0009] Patent documents

[0010] Patent Document 1: Japanese Patent Application Publication No. 2019-163232 Summary of the Invention

[0011] The problem that the invention aims to solve

[0012] Furthermore, if the extraction method described in Patent Document 1 is used, polyphenols can be obtained from grape seeds as water-soluble components, and oil as fat-soluble components. However, the grape seed oil obtained by this method has poor stability due to its high polyphenol concentration, and it is difficult to use as a cosmetic ingredient because it is highly irritating to the skin.

[0013] Thus, although grape seeds contain many useful polyphenols, traditional refining or extraction methods can result in either too little or too much polyphenols, making it difficult to achieve the desired functionality in cosmetics or to ensure the safety and stability of the product when formulated as an effective ingredient in cosmetics.

[0014] Therefore, the main objective of this invention is to provide an oil derived from grape seeds containing an appropriate amount of polyphenols and a method for manufacturing the same.

[0015] Technical solutions to the problem

[0016] The first aspect of this invention relates to an oil derived from grape seeds. While the grape seed oil is preferably composed essentially only of natural components containing natural oil derived from grape seeds, the extraction solvent used in extracting the oil from grape seeds may also leave a residue of 1 wt% or less. The oil of this invention contains 0.02 wt% or more and 0.10 wt% or less of polyphenols derived from grape seeds. Furthermore, polyphenols refer to a general term for compounds having multiple phenolic hydroxyl groups. As shown in Example 1 described later, if the content of polyphenols derived from grape seeds is 0.02 wt% or more, the functionality suitable for cosmetics due to the polyphenols can be fully utilized. In addition, if the content of polyphenols derived from grape seeds is 0.10 wt% or less, the skin irritation is sufficiently low, and therefore this oil can be formulated as an active ingredient in cosmetics.

[0017] In the oil of the present invention, the polyphenols preferably include catechins and epicatechins.

[0018] The oil of the present invention preferably uses polyphenols derived from grape seeds as its active ingredient and possesses one or more functionalities such as antioxidant activity, anti-glycation activity, and anti-inflammatory effects. Thus, according to the present invention, functionalities that are not typically found in oils derived from grape seeds can be acquired.

[0019] The oil of the present invention preferably uses polyphenols derived from grape seeds as the active ingredient and has any one or more of the following characteristics: 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging activity, 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonateacid) (ABTS) free radical scavenging activity, advanced glycation end products (AGEs) generation inhibitory activity, superoxide dismutase (SOD) 2 promoting activity, interleukin-1α (IL-1α) inhibitory activity, IL-1β inhibitory activity, and glutamate-cysteine ​​ligase catalytic subunit (GCLC) promoting activity.

[0020] In the oil of this invention, grape seeds are preferably seeds extracted from unfermented grapes. In this case, foul odors or oil deterioration caused by fermentation can be prevented, and thus refining processes such as deodorization and deacidification to remove polyphenols can be omitted to a minimum.

[0021] The oil of the present invention is preferably non-allergenic to the skin.

[0022] The oil of the present invention preferably has a stability that can be ensured for more than 3 months by storage at room temperature (15-30°C) in the dark. Furthermore, stability refers to the absence of solid precipitation or formation of an aqueous phase under said conditions.

[0023] The second aspect of the present invention is a cosmetic containing grape seed oil of the first aspect. More specifically, the grape seed oil is primarily intended for use as a topical skin agent.

[0024] The third aspect of the present invention is a method for manufacturing oil derived from grape seeds. The method comprises three steps: a first step and a third step. In the first step, fat-soluble components with a melting point of 4°C or higher are extracted and removed from the crude oil extracted from grape seeds. In the second step, components that volatilize at any temperature between 10°C and 35°C are separated from the crude oil extracted from grape seeds under reduced pressure of 1–6 kPa. Either the first step or the second step may be performed first, but it is preferable to perform the second step after the first step. The third step is a step of removing insoluble components from the extracted oil after the first and second steps. Thus, an oil containing 0.02 wt% or more and 0.10 wt% or less of polyphenols derived from grape seeds can be efficiently manufactured.

[0025] Invention Effects

[0026] This invention provides an oil derived from grape seeds containing an appropriate amount of polyphenols and a method for manufacturing the same. Specifically, by including the polyphenols naturally present in grape seeds in the oil at a content of 0.02 wt% to 0.10 wt%, safety, stability, and functionality for cosmetic applications can be achieved. Attached Figure Description

[0027] [ Figure 1 ] Figure 1 This is a flowchart illustrating an example of a method for manufacturing grape seed oil;

[0028] [ Figure 2 ] Figure 2 This is an example of an extraction apparatus used in the production of grape seed oil;

[0029] [ Figure 3 ] Figure 3 This refers to the extraction apparatus used for the production of grape seed oil in Example 1;

[0030] [ Figure 4 ] Figure 4 This indicates the dependence of the extraction rate of the extract in Example 1 on the extraction time of the total polyphenol content;

[0031] [ Figure 5 ] Figure 5 This represents the change in the total polyphenol content of the oil in each step of Example 1;

[0032] [ Figure 6 ] Figure 6 This indicates the result of comparing the total polyphenol content of Example 1 and Comparative Example 1;

[0033] [ Figure 7 ] Figure 7 This represents the results of a qualitative test on the polyphenols in the oil of Example 1;

[0034] [ Figure 8 ] Figure 8 This represents the measurement results of DPPH free radical scavenging activity of Example 1 and Comparative Example 1;

[0035] [ Figure 9 ] Figure 9 This represents the measurement results of the ABTS free radical scavenging activity of Example 1 and Comparative Example 1;

[0036] [ Figure 10 ] Figure 10 This represents the measurement results of the AGEs formation inhibition activity of Example 1 and Comparative Example 2;

[0037] [ Figure 11 ] Figure 11 These are the results of gene expression assays in Example 1;

[0038] [ Figure 12 ] Figure 12 This represents the result of gene expression assay in Example 1. Detailed Implementation

[0039] The following description uses the accompanying drawings to illustrate embodiments for carrying out the present invention. The present invention is not limited to the embodiments described below, but also includes appropriate modifications made by those skilled in the art from the obvious scope of the following embodiments.

[0040] [1. Grape seed oil and its manufacturing method]

[0041] Reference Figure 1 Preferred embodiments of grape seed oil and its manufacturing method are described. For example... Figure 1 As shown, the manufacturing method of this embodiment sequentially includes a cleaning / drying process (process S1), a pulverizing process (process S2), an extraction process (process S3), a solvent separation process (process S4), a first filtration process (process S5), a vacuum distillation process (process S6), a second filtration process (process S7), a cold treatment process (process S8), and a sterilization filtration process (process S9). Each process will be described below.

[0042] The washing / drying process (process S1) involves washing and then drying the seeds removed from the unripe grapes. The washing and drying methods are not limited, but methods that do not degrade the components of the seeds (methods without heat application) are preferred. Furthermore, the grapes used as raw materials for the oil are preferably unfermented grapes. The grape fermentation process includes two cases: fermentation using only the natural yeasts present in the grapes themselves; and fermentation by adding artificial yeast (yeasts cultivated for winemaking) to the grape juice or seeds. Grapes generally begin natural fermentation about 7 days after harvest; therefore, the washing / drying process is preferably carried out within 7 days (168 hours) after harvest.

[0043] The pulverizing step (step S2) is the step of pulverizing grape seeds. The pulverizing method is not limited, but a method that allows for pulverization in a short time without deteriorating the peel components is preferred. For example, any well-known pulverizer used in food processing can be used for pulverizing grape seeds. Pulverizing grape seeds is preferably carried out just before the extraction step described later (specifically within 1 hour) to avoid deterioration caused by oxidation or light. Furthermore, if the pulverized grape seeds need to be stored for more than 1 hour, they can simply be stored in a sealed, light-proof container to prevent deterioration caused by oxidation or light.

[0044] The extraction step (step S3) is a step of extracting components from grape seeds. The extraction method is not limited, as long as the components of the grape seeds are dissolved into the extraction solvent by maintaining the grape seeds in a state of immersion. As the extraction solvent, a solution containing liquefied dimethyl ether is preferred. Liquefied dimethyl ether is a liquid obtained by bringing dimethyl ether to a state above its saturated vapor pressure, but it can also be an auxiliary solvent such as water or alcohol added in a less than saturated amount. Here, the amount of auxiliary solvent added is preferably less than the saturated amount of liquefied dimethyl ether, more specifically, preferably less than 7% by mass of liquefied dimethyl ether. The extraction conditions are preferably 4–40°C and 1.0 MPa or less, more specifically, preferably 25°C and 0.7 MPa or less. Furthermore, as the extraction solvent, aqueous solutions of, for example, ethanol, 1,3-butanediol, or these can also be used. Through this step, an extract containing the extracted components from grape seeds and the extraction solvent is obtained.

[0045] The solvent separation step (step S4) is a step in which the extracted components are separated from the extraction solvent by evaporating the extraction solvent from the extract. The method of solvent separation is not limited, but a method that does not degrade the extracted components is preferred. For example, the extraction solvent can be evaporated by allowing the extract to stand at a temperature between 15°C and 40°C and at atmospheric pressure (101.33 kPa), or by irradiating the extract with ultrasound for a short time (less than 10 minutes). More specifically, when using liquefied dimethyl ether as the extraction solvent, the liquefied dimethyl ether can be evaporated and separated from the extract by placing the extract at 30°C and atmospheric pressure. Furthermore, even after such a step of evaporating the liquefied dimethyl ether, a slight residual amount of liquefied dimethyl ether may sometimes remain in the extract. This slight residual amount of liquefied dimethyl ether can be evaporated by irradiating with ultrasound for 10 minutes. Thus, the extraction solvent (liquefied dimethyl ether) can be almost completely separated from the extract. Thus, crude oil is obtained by separating the extraction solvent from the extract.

[0046] The first filtration step (step S5) is a process of filtering the crude oil to remove impurities and solids such as lipids with high melting points mixed in with the crude oil. The main purpose of this first filtration step is to separate and remove fat-soluble components with melting points above 4°C from the crude oil extracted from grape seeds. Specifically, fat-soluble components derived from grape seeds with melting points above 4°C in the crude oil include, for example, monounsaturated fatty acids such as oleic acid (melting point about 13°C), saturated fatty acids such as palmitic acid (melting point about 63°C) and stearic acid (melting point about 70°C), phytosterols (melting point about 136°C), and carotenoids (melting points about 63–183°C). Therefore, the first filtration step is preferably carried out under temperature and pressure conditions (specifically, atmospheric pressure and below 13°C) where at least oleic acid is in a solid state. Conversely, fat-soluble components derived from grape seeds with melting points above 4°C include, for example, linoleic acid (melting point about -5°C). The filtration method is not limited; for example, any filter paper or membrane filter with a pore size of 1.0–10 μm can be used. This process removes impurities and lipids from the crude oil extract, yielding a mixture derived from grape seeds, consisting of water, water-soluble components, and oil. This water-soluble component contains polyphenols. The filtered mixture typically contains a very high concentration of polyphenols, typically above 3 wt% (3000 mg / kg). Therefore, while this mixture may offer high functionality, its stability and safety in cosmetic applications are poor, making it unsuitable for direct use as a cosmetic ingredient.

[0047] The vacuum distillation step (step S6) is a process of separating volatile components that would evaporate below 35°C from a filtered mixture under reduced pressure. The main purpose of this vacuum distillation step is to remove water and other volatile substances from the filtered mixture. When removing water from the mixture, heating it to above 35°C may denature the active ingredients; therefore, it is preferable to maintain the temperature of the mixture below 35°C beforehand 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, the mixture is preferably subjected to vacuum distillation at a reduced pressure environment where water evaporates between 10 and 35°C, specifically at 1 to 6 kPa, to remove volatile substances containing water. The method of vacuum distillation is not limited, but an evaporator is preferred. For example, place approximately 500 mL of the mixture in a 2 L flask, reduce the pressure to 5.6 kPa, and perform vacuum distillation at 35°C for 5 hours. This will remove water until the water concentration in the mixture is approximately 0.02 wt% (200 mg / kg, 0.1 mL). Through this process, water is almost completely removed from the filtered mixture, yielding an extract oil derived from grape seeds. This extract oil consists of a portion of the water-soluble components from grape seeds, such as polyphenols, that have precipitated out and remain as solids.

[0048] The second filtration step (step S7) involves filtering the extracted oil after vacuum distillation again to remove any remaining solids. The filtration method is not limited; for example, any filter paper or membrane filter with a pore size of 0.3–0.6 μm can be used. This step removes the solids precipitated in the vacuum distillation step (step S6), yielding a clear, refined grape seed oil. These solids contain a portion of the polyphenols derived from grape seeds. 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, even after the second filtration step, not all polyphenols are removed, so a moderate amount remains in the oil. The polyphenol concentration in this grape seed oil is 0.02–0.10 wt% (200–1000 mg / kg). This polyphenol concentration meets the stability and safety requirements for cosmetic applications, therefore, this oil can be used as a cosmetic ingredient.

[0049] The cold treatment process (process S8) is a process aimed at precipitating out insoluble substances contained in the oil derived from grape seeds. The method of cold treatment is not limited, but specifically, it is preferable to let the oil stand in a refrigerator at 4°C or in a storage room where there are no temperature changes and no light exposure. This process allows insoluble substances that could not be completely removed in the filtration process (process S7) to be released.

[0050] The sterilization and filtration process (process S9) involves sterilizing and filtering the grape seed oil after the cold treatment process. For example, a membrane filter with a pore size of 0.22 μm or less can be used for sterilization filtration. The sterilized grape seed oil is then filled into a sterilized container. Sterilization and filtration are preferably performed in a clean environment, such as a cleanroom or clean bench.

[0051] As described above, the polyphenol concentration in this grape seed oil is 0.02–0.10 wt% (200–1000 mg / kg). Specifically, as shown in Example 1 below, the oil undergoing vacuum distillation (S6) and filtration (S7) sufficiently reduces the total polyphenol content to approximately 0.02 wt%, thus meeting the stability and safety requirements for cosmetic applications. On the other hand, as shown in Example 1 below, the oil without these vacuum distillation (S6) and filtration (S7) processes has a total polyphenol content of approximately 0.3 wt%, resulting in decreased stability and skin safety. In other words, considering stability and skin safety, a total polyphenol content of approximately 0.3 wt% is excessive; a suitable total polyphenol content would be approximately one-third of that, around 0.10 wt%. Therefore, based on the results obtained in Example 1 below, it is more appropriate to set the lower limit of the polyphenol concentration in grape seed oil to 0.02 wt% and the upper limit to 0.10 wt%.

[0052] [2. Extraction apparatus for crude oil extraction]

[0053] The following description, with reference to the accompanying drawings, details a preferred embodiment of an extraction apparatus for extracting crude oil from grape seeds. Specifically, this extraction apparatus is used for... Figure 1 The extraction process (process S3) and solvent separation process (process S4) are shown.

[0054] Figure 2 This is an example of an extraction apparatus for producing grape seed oil according to this embodiment. Additionally, Figure 2 The diagram is only a rough representation of the shape, size, and arrangement of the constituent elements to the extent that the extraction device can be understood.

[0055] The extraction apparatus 100 includes: a storage tank 1 for storing liquefied dimethyl ether 2, an extraction tank 6 for contacting raw material 7 with liquefied dimethyl ether 2, a separation tank 11 for separating the liquid discharged from the extraction tank 6, and a pump 3 for conveying liquefied dimethyl ether 2 from the storage tank 1 to the extraction tank 6.

[0056] In addition, the extraction device 100 includes conduits 5, 10, 12, 14, 16, 19, 20, and 23 for introducing or discharging liquefied dimethyl ether 2, and valves 4, 9, 13, 15, 18, 21, and 22 for controlling the introduction and discharging of liquefied dimethyl ether 2 by adjusting the air pressure in each tank. The extraction tank 6 and the separation tank 11 have adjustable pressure to maintain the liquid state of the liquefied dimethyl ether 2.

[0057] In the extraction apparatus 100 described above, the storage tank 1 functions as a storage unit for storing liquefied dimethyl ether 2. The pump 3, valve 4, and conduit 5 function as a conveying unit for transporting liquefied dimethyl ether 2 from the storage tank 1 to the extraction tank 6. Furthermore, the extraction tank 6 functions as a contact unit for bringing the raw material 7 into contact with the liquefied dimethyl ether 2 to obtain an extract. Additionally, the valve 9 and conduit 10 function as a discharge unit for discharging the extract from the extraction tank 6 to the separation tank 11. Furthermore, the separation tank 11 functions as a separation unit for separating liquefied dimethyl ether 2 from the extract. Furthermore, the conduit 12 and valve 13 connected to the separation tank 11 function as a vaporization unit for vaporizing the liquefied dimethyl ether. Furthermore, the condenser 17 connected to the conduit 16 functions as a condensation unit for re-condensing the dimethyl ether discharged from the separation tank 11 from a gas back into a liquid. Conduits 19 and 20 function as a supply unit for supplying liquefied dimethyl ether 2 to the storage tank 1.

[0058] The extraction apparatus 100 further includes any of the following components: thermometers and pressure gauges for detecting the temperature and pressure in each of the storage tank 1, extraction tank 6, and separation tank 11; a mixer for performing agitation in each tank; and a device for purging active gases such as oxygen in each tank and conduit, such as allowing inert gases such as nitrogen to circulate.

[0059] In the extraction apparatus 100 described above, crude oil derived from raw material 7 can be obtained in the following manner. In this embodiment, raw material 7 is obtained by washing, drying, and then pulverizing the grape seeds.

[0060] Filters 8 are installed on both the upstream and downstream sides of the extraction tank 6. Raw material 7 (grape seeds) is introduced into this extraction layer 6. At this time, valves 4, 9, 13, 15, 18, 21, and 22 are all closed. In addition, when the storage tank 1 is not sufficiently filled with liquefied dimethyl ether 2, valve 21 is opened, and liquefied dimethyl ether 2 is supplied to the storage tank 1 via conduit 20, after which valve 21 is closed. At this time, valve 18 can also be opened when valve 21 is open, and valve 18 can also be closed when valve 21 is closed.

[0061] Next, valve 4 is set to the open state, and liquefied dimethyl ether 2 in storage tank 1 is discharged by pump 3 and sent to extraction layer 6 through conduit 5. After liquefied dimethyl ether 2 is introduced into extraction tank 6 until it comes into contact with raw material 7, valve 4 is set to the closed state.

[0062] When raw material 7 is immersed in liquefied dimethyl ether 2 in extraction tank 6, the water, water-soluble compounds, and fat-soluble compounds contained in raw material 7 are extracted by liquefied dimethyl ether 2. As a result, an extract containing water, water-soluble compounds, and fat-soluble compounds from raw material 7 dissolved in liquefied dimethyl ether 2 is obtained. Note that water refers to the moisture contained in the raw material.

[0063] Next, when valves 4 and 9 are opened, and liquefied dimethyl ether 2 is introduced from storage tank 1 into extraction tank 6 via conduit 5 using pump 3, the extract in extraction tank 6 is introduced into separation tank 11 via conduit 10. That is, when new liquefied dimethyl ether 2 is discharged from storage tank 1 into extraction tank 6, the extract in extraction tank 6 is pushed out into separation tank 11. As a result, the interior of extraction tank 6 is replaced with new liquefied dimethyl ether, but the raw material 7 remains in extraction tank 6 due to the presence of filters 8 on the upstream and downstream sides of extraction tank 6. In other words, by introducing new liquefied dimethyl ether 2 into extraction tank 6, the extract is pushed out of extraction tank 6, thereby separating it from the raw material 7. Furthermore, valves 4 and 9 are opened after a specific time has elapsed since the introduction of liquefied dimethyl ether 2 into extraction tank 6, sufficient time for the moisture and other components of the raw material 7 to be transferred to the liquefied dimethyl ether 2. At this time, the mixture can be left to stand for a specific time while in contact with the raw material 7, or it can be stirred.

[0064] Subsequently, valves 4 and 15 are closed, and valves 9, 13, and 22 are opened, thereby creating a pressure lower than the saturated vapor pressure of dimethyl ether in the path from valve 4 to valve 13. The liquefied dimethyl ether 2 in this path then vaporizes and is discharged from conduit 23 via conduit 14. Alternatively, dimethyl ether can be discharged using pump 3 if necessary. The separation tank 11 contains the crude oil extracted from the extract after the liquefied dimethyl ether 2 has been evaporated / separated.

[0065] Here, although the case where valve 22 connected to the outside air is set to the open state and valve 15 connected to the condenser 17 is set to the closed state is described, it is also possible to set valve 22 to the closed state and valve 15 to the open state. In this way, the vaporized dimethyl ether is introduced into the condenser 17 via conduit 16. As a result, in the condenser 17, the dimethyl ether is condensed again to generate liquefied dimethyl ether 2. Furthermore, by setting valve 18 to the open state, the generated liquefied dimethyl ether 2 is introduced into the storage tank 1 via conduit 19. Thus, it can be reused as liquefied dimethyl ether 2.

[0066] The crude oil remaining in the separation tank 11 is subjected to the first filtration process (process S5), the vacuum distillation process (process S6), the second filtration process (process S7), the cold treatment process (process S8), and the sterilization filtration process (process S9). This yields grape seed oil (refined oil). The crude oil extraction can be performed as described above using well-known membrane filters, evaporators, etc., for these processes S6 to S9.

[0067] The grape seed oil obtained through the above processes can be used directly, but ingredients intended for use in cosmetics and quasi-drug products may be added appropriately, without compromising the oil's effectiveness. Examples of such ingredients include oils, waxes, hydrocarbons, fatty acids, alcohols, esters, surfactants, metallic soaps, pH adjusters, preservatives, fragrances, humectants, powders, UV absorbers, thickeners, pigments, antioxidants, whitening agents, chelating agents, excipients, and film-forming agents.

[0068] Furthermore, grape seed oils are primarily used in topical skin preparations and can be formulated into cosmetics or quasi-drugs. Examples of dosage forms for cosmetics and quasi-drugs include toners, creams, lotions, gels, aerosols, serums, masks, cleansers, bath products, foundations, powders, lipsticks, ointments, and mud masks. Additionally, the antioxidant compositions of this invention can also be formulated into soaps, shower gels, facial cleansers, shampoos, conditioners, hair treatments, and toothpaste.

[0069] Example

[0070] The grape seed-derived oil of the present invention will then be described in more detail using examples.

[0071] [Experimental Methods]

[0072] (1. Determination of total polyphenol content)

[0073] The polyphenol content in grape seed oil was determined using the Folin-Ciocalteumethod (ISO 14502-1:2005), a method recognized by the International Organization for Standardization (ISO). The Folin-Ciocalteumethod determines the color change of the reagent through the reduction of phenolic hydroxyl groups, and the result is measured by absorbance. In this case, 50 wt% ethanol was added to the oil, followed by defatting with hexane to prepare the sample solution. Gallic acid was used as a standard to establish a calibration curve, and the amount of polyphenols equivalent to gallic acid was calculated.

[0074] (2. Qualitative analysis of polyphenols)

[0075] To identify whether grape seed oil contains polyphenols, qualitative analysis was performed using liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QTOFMS). The total ion current (TIC) chromatograms obtained from the analysis were analyzed, and polyphenols were identified from the mass spectrum (MS) and tandem mass spectrum (MS / MS spectrum). The determination conditions for qualitative analysis are shown in Table 1 below.

[0076]

[0077]

[0078] (Functional testing)

[0079] The antioxidant activity of grape seed oil was determined using DPPH radical scavenging activity and ABTS radical scavenging assays. DPPH radical scavenging activity was determined by reacting the sample with a DPPH radical solution (room temperature, 30 min), measuring the absorbance of the solution (517 nm) using a microplate reader, and calculating the activity (%) relative to the blank. ABTS radical scavenging activity was determined by reacting the sample with an ABTS radical solution (37℃, 4 min), measuring the absorbance of the solution (734 nm) using a microplate reader, and calculating the activity (%) relative to the blank.

[0080] The anti-glycation activity of grape seed oil was determined using an AGEs (advanced glycation end products) inhibition assay. Bovine serum albumin (BSA) solution was added to the sample and reacted with glucose solution (60°C, 96 h). The fluorescence of the reaction solution was measured using a microplate reader (excitation wavelength 370 nm, fluorescence wavelength 440 nm), and the activity (%) relative to the blank was calculated.

[0081] The effect of grape seed oil on gene expression was determined as follows.

[0082] <Using SOD2, IL-1α, and IL-1β gene expression from human skin fibroblasts>

[0083] SOD2, IL-1α, and IL-1β were used as genes. Skin fibroblasts derived from human newborns were used at a ratio of 5 × 10⁻⁶. 4Cells / dishes were seeded in 60 mm diameter culture dishes. After 24 hours, samples were added to achieve a final concentration of 0.1%. A control was prepared using culture medium without samples. After 24 hours of culture, RNA was extracted from the cells using the ribonucleic acid (RNA) extraction reagent “TRI Reagent” (Merck KGaA, Darmstadt, Germany). Using this RNA as a template, complementary deoxyribonucleic acid (cDNA) was synthesized using the reverse transcription reaction of Oligo dT Primer using the “Primescript RT reagentkit” (Takara Bio, Shiga, Japan). Messenger ribonucleic acid (mRNA) levels were quantified using polymerase chain reaction (PCR) reagents, specifically the "Luna Universal qPCR Master Mix" (New England Biolabs, MA, USA), and a "LightCycler 96" PCR apparatus (Roche, Basel, Switzerland). The data were calculated using the ΔCt method to determine Cq values, which were then expressed as relative values ​​based on the Cq values ​​of glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

[0084] <GCLC gene expression using human skin keratinocytes>

[0085] GCLC was used as the gene. Human skin keratinocytes were divided into 5 × 10⁻⁶ cells. 4Cells were seeded in 60 mm diameter culture dishes. After 24 hours, the sample was added to achieve a final concentration of 0.005 mg / mL, and the oil was visually confirmed to be dissolved in the medium. A control was prepared using medium without the sample. After 8 hours of culture, RNA was extracted from the cells using the RNA extraction reagent “TRIReagent” (Merck KGaA, Darmstadt, Germany). Using this RNA as a template, cDNA was synthesized using reverse transcription with Oligo dT Primer in the “Primescript RT reagent kit” (Takara Bio, Shiga, Japan). The mRNA level was quantified using the PCR reagent “Luna Universal qPCR Master Mix” (New England Biolabs, MA, USA) and a PCR apparatus “LightCycler 96” (Roche, Basel, Switzerland) with primers for various genes. The data were calculated using the ΔCt method to determine the Cq values, and the expression levels of each gene were expressed as relative values ​​based on the GAPDH Cq values.

[0086] (3. Stability test)

[0087] As a stability test for oil derived from grape seeds, the oil was placed in a constant temperature bath at 5°C, 20°C, 40°C, and 50°C and exposed to fluorescent light to confirm the precipitation, color, and odor at each fixed period.

[0088] (4. Safety test)

[0089] As a safety test for oils derived from grape seeds, an in vitro skin sensitization test was conducted.

[0090] [Example 1]

[0091] In Example 1, according to Figure 1 The oil is produced from grape seeds in a specific order. The grape seeds used as raw material are washed from unfermented raw grapes, and after removing (screening) the seeds that float to the surface of the water, they are dried to a moisture content of about 6% by mass, and then crushed to below 1.0 mm just before extraction.

[0092] The extraction process uses Figure 3The extraction apparatus shown generates an extract from grape seeds. Specifically, 18.0 g of grape seeds 57, pulverized to a length of approximately 1.0 mm or less, are placed in an extraction tank 56 with a volume of 25 mL, equipped with filters 55 and 58 on both the upstream and downstream sides. Then, with valve 52 closed and valve 53 open, dimethyl ether 51 is introduced into the syringe pump 50, and the pressure is set to 25°C and 0.7 MPa for liquefaction. The separation tank 62 is pre-purged with dimethyl ether, and valves 52, 53, 54, 59, 60, and 61 are closed. Furthermore, Figure 3 The extraction device shown is not to make Figure 2 The dimethyl ether cycle of the extraction apparatus 100 shown is configured.

[0093] Next, valves 53, 54, 59, and 60 are set to the open position, and liquefied dimethyl ether is supplied to the extraction tank 56 by the injection pump 50. When the extraction tank 56 is filled with liquefied dimethyl ether, the injection pump 50 is stopped, and valves 54 and 59 are set to the closed position, so that the crushed grape seeds 57 are impregnated with liquefied dimethyl ether.

[0094] With valves 54 and 59 open, inject liquefied dimethyl ether again using syringe pump 50. Adjust the flow rate to 2.5 mL / min and the residence time to 10 minutes, recovering 30 mL of extract in the separation tank 62 each time. Then, close valve 60, remove the separation tank 62 from the apparatus, and place it in a fume hood at atmospheric pressure to allow the liquefied dimethyl ether to evaporate and generate the extract. Afterward, irradiate the obtained extract with an ultrasonic cleaner for 10 minutes, and then let it stand overnight at 4°C to allow the liquefied dimethyl ether to completely evaporate, obtaining the crude oil.

[0095] The crude oil was first filtered through 7 μm filter paper to remove solid lipids and impurities. Then, it was distilled under reduced pressure at 40°C for 5 hours using an evaporator to separate water and volatile components, thus obtaining the extracted oil. The pressure inside the evaporator was set to 5.6 kPa.

[0096] Next, the precipitate from the extract oil produced during vacuum distillation was separated by a second filtration using a 0.45 μm membrane filter. This precipitate was presumed to be part of the water-soluble components dissolved in water.

[0097] Subsequently, the oil was left to stand overnight at 4°C for cold treatment (precipitation), and then sterilized and filtered through a 0.22 μm membrane filter in a clean bench. The resulting oil (refined oil) was then filled into brown bottles.

[0098] [Comparative Example 1]

[0099] In Comparative Example 1, oil was produced by pressing using the same type of grape seeds as used in Example 1. Specifically, the grape seeds were washed, screened, dried, and crushed in the same order as in Example 1. Subsequently, the crushed grape seeds were pressed to extract the oil using a screw press. The oil thus pressed was then processed in the same order as in Example 1, including second filtration, cold treatment, and sterilization filtration, and then filled into brown bottles.

[0100] [Comparative Example 2]

[0101] In Comparative Example 2, the oil (extracted crude oil) from the process after the filtration step (step S5) in Example 1 was used.

[0102] [Comparative Example 3]

[0103] In Comparative Example 3, oil (filtered crude oil) from the process after the vacuum distillation (moisture removal) step (step S6) in Example 1 was used.

[0104] [Inspection]

[0105] (1. Oil extraction rate and total polyphenol content)

[0106] The extraction rate of the extract obtained in the extraction process of Example 1 and the extraction time dependence of the total polyphenol content are shown in the figure. Figure 4 The extraction conditions were 25℃, 0.7 MPa, and a residence time of 10 minutes. The extract used here is the residue remaining after drying the crude oil in a vacuum dryer (40℃, 5.33 kPa, 12 h) to remove moisture. When the extraction time was above 36 minutes, the extraction rate was 16.5% by mass and the total polyphenol content was 3136 mg / kg (0.3136 wt%), and remained almost constant thereafter; therefore, 36 minutes was considered the optimal extraction time. At this time, the delivery volume of liquefied dimethyl ether was 3.3 kg per 1 g of grape seed. The oil obtained in the extraction process has a relatively high total polyphenol content, suggesting high functionality; however, its stability and skin safety are poor, making it unsuitable for direct use as a cosmetic ingredient. Therefore, further processing is required.

[0107] In addition, the extraction rate of the extract is calculated using the following formula.

[0108] [Formula] Extraction rate [mass %] = (mass of extract / mass of raw material introduced into the extraction tank) × 100

[0109] (2. Changes in the total polyphenol content in the oil at each stage)

[0110] The changes in the total polyphenol content of the oil in each step after the extraction step (S3) of Example 1 are shown in the figure. Figure 5The total polyphenol content in the oil was 3100 mg / kg (0.31 wt%) in the solvent separation step (S4), and reduced to 2900 mg / kg (0.29 wt%) after removing solids in the first filtration step (S5). Subsequently, the total polyphenol content was reduced to 250 mg / kg (0.025 wt%) by separating the solids precipitated after water removal in the vacuum distillation step (S6) in the filtration step (S7). Although most of the polyphenols obtained through extraction were removed, it is presumed that the remaining polyphenols here are relatively soluble in oil. Afterwards, the total polyphenol content in the oil after the cold treatment step (S8) and the 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) help to reduce the total polyphenol content in the oil to an appropriate level.

[0111] [Comparison of total polyphenol content]

[0112] The results comparing the total polyphenol content of the oil of Example 1 with that of the oil obtained by pressing in Comparative Example 1 are shown below. 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 total polyphenol content of the oil obtained by pressing was low and insufficient.

[0113] Furthermore, the results of the qualitative test of polyphenols in the oil of Example 1 are shown in... Figure 7 Multiple peaks were detected in the oil containing water-soluble components from Example 1. Analysis of the MS and MS / MS spectra using the METLIN database identified the... Figure 7 The 70 compounds shown are related to the present invention. It can be said that the polyphenols contained in the grape seed oil, in addition to catechins and epicatechins, also include ε-glucan and taurine. The qualitative analysis results are as follows.

[0114]

[0115]

[0116] (3. Functional testing)

[0117] 1. Antioxidant activity

[0118] The DPPH free radical scavenging activity of the oils from Example 1 and Comparative Example 1 when 20 mg / mL was added was shown in the figure. Figure 8 The results of ABTS free radical scavenging activity are shown in Figure 9The oils of Example 1 and Comparative Example 1 showed DPPH free radical scavenging activities of 44% and 30%, respectively, and ABTS free radical scavenging activities of 31% and 13%, respectively, thus confirming the superiority of the oil of Example 1 in terms of antioxidant activity.

[0119] 2. Anti-glycation activity

[0120] The results of AGEs generation inhibition activity when oils from Example 1 and Comparative Example 2 were added at 5.0 mg / mL are shown in the figure. Figure 10 The AGEs formation inhibition activities of the oils in Example 1 and Comparative Example 2 were 31% and 13%, respectively, thus confirming the superiority of the oil in Example 1 in terms of anti-glycation activity.

[0121] 3. Gene Expression Analysis

[0122] The results of the use of the oil in Example 1 on gene expression in human skin fibroblasts are shown in... Figure 11 Here, human fibroblasts NB1RGB were used to measure the expression levels of the antioxidant enzyme SOD2, and the anti-inflammatory markers IL-1α and IL-1β. In the oil of Example 1, an increase in the antioxidant enzyme-related gene SOD2 was confirmed. Furthermore, in the oil of Example 1, inhibition of the anti-inflammatory genes IL-1α and IL-1β was confirmed. In addition, the control was the result of cell culture with culture medium added instead of oil. Based on the above, the antioxidant and anti-inflammatory effects of the oil of Example 1 in human skin fibroblasts can be expected.

[0123] The results of the use of the oil in Example 1 on gene expression in human skin keratinocytes are shown in... Figure 12 Here, human skin keratinocytes (NHEK) were used to determine the expression level of the gene that produces the catalytic subunit of glutamate cysteine ​​ligase (GCLC). An increase in the GCLC gene was confirmed in the oil of Example 1.

[0124] GCLC is an enzyme involved in the biosynthesis of glutathione (GSH), called glutamate-cysteine ​​ligase. GSH plays important roles in intracellular oxidative stress, toxin removal, and intracellular antioxidant defense. GCLC forms glutamylcysteine ​​by binding glutamate and cysteine, which then biosynthesizes GSH.

[0125] The oil in Example 1 promotes the production of enzymes related to glutathione production in human skin keratinocytes, and is expected to play an antioxidant role in oxidative stress relief, toxin elimination, and intracellular antioxidant defense.

[0126] (4. Stability test)

[0127] The results of stability tests on the oils of Example 1 and Comparative Examples 1-2 are shown in Table 3. It was confirmed that only the oil of Example 1 had good stability when stored in the dark at room temperature (around 20°C). In the oils of Comparative Examples 2 and 3, precipitation of solids and formation of an aqueous phase were observed, indicating poor stability.

[0128]

[0129]

[0130] 〇: Good stability Δ: Some potential problems exist ×: Poor stability

[0131] (5. Safety test)

[0132] The results of safety tests on the oils of Example 1 and Comparative Examples 1-2 are shown in Table 4. Only the oil of Example 1 showed negative skin sensitization, confirming its safety for the skin.

[0133]

[0134]

[0135] As confirmed above, the grape seed oil of the present invention exerts its functionality by containing a certain amount of polyphenols naturally present in grape seeds, while ensuring safety and stability. Therefore, the grape seed oil of the present invention is suitable for cosmetic applications.

[0136] In the above description, for the purpose of illustrating the content of the present invention, reference is made to the accompanying drawings. Figure 1 The embodiments and examples of the present invention have been described. However, the present invention is not limited to the above-described embodiments and examples, and also includes various obvious modifications and improvements that would occur to those skilled in the art based on the matters described in this specification.

Claims

1. An oil, characterized in that, It is an oil derived from grape seeds, containing more than 0.02 wt% and less than 0.10 wt% of grape seed polyphenols.

2. The oil according to claim 1, characterized in that, The polyphenols include catechins and epicatechins.

3. The oil according to claim 1 or 2, characterized in that, It uses polyphenols derived from the grape seeds as the active ingredient and has one or more functionalities such as antioxidant activity, anti-glycation activity, and anti-inflammatory effects.

4. The oil according to claim 1 or 2, characterized in that, The active ingredient is a polyphenol derived from the grape seed, and it has one or more of the following: DPPH free radical scavenging activity, ABTS free radical scavenging activity, AGEs generation inhibition activity, SOD2 promoting activity, IL-1α inhibitory activity, IL-1β inhibitory activity and GCLC promoting activity.

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

6. The oil according to claim 1 or 2, characterized in that, It is not sensitizing to the skin.

7. The oil according to claim 1 or 2, characterized in that, The stability of the oil is ensured to last for more than 3 months by storing it at room temperature in the dark.

8. A cosmetic product, characterized in that, Contains the oil as described in claim 1 or 2.

9. A method for manufacturing oil derived from grape seeds, characterized in that, Include: The first step involves extracting and removing fat-soluble components with a melting point above 4°C from the crude oil extracted from grape seeds. The second step involves separating components from the crude oil that will volatilize at any temperature between 10 and 35°C under reduced pressure of 1–6 kPa; and The third step involves removing insoluble components from the extracted oil obtained after the first and second steps.

10. The method for manufacturing oil according to claim 9, characterized in that, The oil contains more than 0.02 wt% and less than 0.10 wt% of polyphenols derived from grape seeds.

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