Anti-aging cosmetic, grape marc oil and method for preparing the same

Abstract

The application relates to the field of cosmetics, and relates to an anti-aging cosmetic, grape pomace oil and a preparation method thereof. The anti-aging cosmetic comprises grape pomace oil; characteristic components of the grape pomace oil include oleanolic acid, alpha-guaiacwoodene, beta-tocotrienol, beta-sitosterol, campesterol, soybean sapogenol E, ganoderic acid F, ganoderol I and D-erythro-dihydrosphingosine; the total phenol content of the grape pomace oil is greater than or equal to 1 mg / g, the total sterol content of the grape pomace oil is greater than or equal to 26 mg / g, and the total flavonoid content of the grape pomace oil is greater than or equal to 1 mg / g; the grape pomace oil is extracted from grape pomace; the grape pomace is mainly composed of grape skin, grape stem and grape seed. The compounds have excellent antioxidant properties and can improve the anti-aging effect of the anti-aging cosmetic. The compounds are helpful for protecting the skin from oxidative damage and play a positive role in cosmetics. The grape pomace oil maximally retains active components of all parts of the grape pomace.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of cosmetics, in particular, an anti-aging cosmetic, grape pomace oil and a preparation method thereof. BACKGROUND

[0002] With the development of grape brewing industry, the production of grape pomace is increasing, mainly composed of grape skin, grape stem and grape seed. At present, the main way of secondary utilization of grape pomace is to sort out grape seeds, and then produce grape seed oil, while the remaining grape skin and grape stem are often treated in a low-efficiency and extensive way, such as being made into low-value fertilizer, feed or directly landfilled as garbage, which causes resource waste and environmental pollution. SUMMARY

[0003] The purpose of the embodiments of the present application is to provide an anti-aging cosmetic, grape pomace oil and a preparation method thereof.

[0004] In a first aspect, the present application provides an anti-aging cosmetic, comprising:

[0005] Grape pomace oil; the characteristic components of grape pomace oil include oleanolic acid, alpha-guaiacwood, beta-tocotrienol, beta-sitosterol, campesterol, soybean saponin E, ganoderic acid F, ganoderol I and D-erythro-dihydrosphingosine; the total phenol content of grape pomace oil is ≥1mg / g, the total sterol content of grape pomace oil is ≥26mg / g, and the total flavonoid content of grape pomace oil is ≥1mg / g;

[0006] The grape pomace oil is extracted from grape pomace; the grape pomace is mainly composed of grape skin, grape stem and grape seed.

[0007] In the above technical solution, the characteristic components of grape pomace oil are oleanolic acid, alpha-guaiacwood, beta-tocotrienol, beta-sitosterol, campesterol, soybean saponin E, ganoderic acid F, ganoderol I and D-erythro-dihydrosphingosine; these compounds have excellent antioxidant properties and can be used as anti-aging active ingredients of anti-aging cosmetics, thereby greatly improving the anti-aging effect of anti-aging cosmetics. And the grape pomace oil shows an anti-aging effect comparable to astaxanthin in a nematode model, which shows that it helps to protect the skin from oxidative damage and plays an active role in cosmetics. The grape pomace oil maximally retains the active ingredients of each part of grape pomace.

[0008] In other embodiments of the present application, the peroxide value of the grape pomace oil is ≤7mmol / Kg.

[0009] In other embodiments of the present application, the acid value of the grape pomace oil is ≤4mg / g.

[0010] In a second aspect, the application provides a method for preparing grape pomace oil, the method comprising:

[0011] hot soaking the grape pomace powder with an acid solution to obtain a first feed liquid;

[0012] continuously phase-changing extracting the first feed liquid at 0.2 MPa-0.8 MPa and 40-50°C.

[0013] The above technical solution can make the solvent quickly circulate in liquid-gas-liquid mode, and the circulation and fresh solvent can ensure sufficient and effective extraction of the material. In addition, the solvent is recovered in a vacuum state, which does not cause solvent residue and environmental pollution, thereby realizing low-cost green extraction.

[0014] In other embodiments of the application, the continuous phase-change extraction comprises:

[0015] using n-butane as the extractant for liquid-phase, gas-phase, and liquid-phase continuous circulation phase-change extraction.

[0016] In other embodiments of the application, the continuous phase-change extraction comprises multiple extraction cycles, and one extraction cycle comprises:

[0017] compressing the extractant gas into extractant liquid in a vacuum, making the extractant liquid flow through the first feed liquid for extraction, and the extracted grape pomace oil and the acid solution flowing out with the extractant liquid to obtain a mixed liquid; converting the extractant liquid into extractant gas to separate from the mixed liquid; and repeating the extraction cycle with the separated extractant gas;

[0018] Optionally, after separating the extractant gas, the mixed liquid is also removed of the acid solution to obtain a crude extract.

[0019] Optionally, the crude extract is subjected to a second extraction.

[0020] Optionally, the second extraction of the crude extract is performed using n-hexane.

[0021] In other embodiments of the application, the acid solution is an acid-alcohol solution.

[0022] Optionally, the acid solution is a citric acid-ethanol solution.

[0023] Optionally, the citric acid-ethanol solution comprises 1%-5% citric acid and 95%-99% ethanol by mass percentage.

[0024] In other embodiments of the application, the hot soaking of the grape pomace powder with the acid solution comprises:

[0025] The wine lees powder and the acid solution are hot soaked in a ratio of (1:1) to (1:3) by mass.

[0026] In other embodiments of the present application, the wine lees powder is hot soaked with an acid solution, including:

[0027] The wine lees powder and the acid solution are soaked at 40-50°C;

[0028] Optionally, the soaking time is 1-3 hours.

[0029] In a third aspect, the present application provides a wine lees oil, which is obtained by the preparation method of the wine lees oil provided in the second aspect. BRIEF DESCRIPTION OF DRAWINGS

[0030] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed in the embodiments will be briefly introduced as follows. It should be understood that the following drawings only show some embodiments of the present application, and therefore should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can also be obtained without creative labor.

[0031] Figure 1 Total ion current chromatogram of gas chromatography-mass spectrometry analysis of the wine lees oil prepared in Example 1;

[0032] Figure 2 Total ion current chromatogram of liquid chromatography-mass spectrometry analysis of the wine lees oil prepared in Example 1;

[0033] Figure 3 Effect diagram of the wine lees oil prepared in Example 1 on the survival curve of nematodes under H2O2 oxidative stress conditions;

[0034] Figure 4 Effect diagram of the wine lees oil prepared in Example 1 on the survival curve of nematodes under PQ oxidative stress conditions;

[0035] Figure 5 Effect diagram of the wine lees oil prepared in Example 1 on the survival curve of nematodes under heat stress conditions;

[0036] Figure 6 Effect diagram of the wine lees oil prepared in Example 1 on the survival curve of nematodes under light stress conditions. DETAILED DESCRIPTION

[0037] In order to make the purpose, technical solutions and advantages of the embodiments of the present application more clear, the technical solutions in the embodiments of the present application will be described clearly and completely. Obviously, the described embodiments are some of the embodiments of the present application, but not all the embodiments.

[0038] Therefore, the following detailed description of embodiments of the application is not intended to limit the scope of the application as claimed, but merely represents selected embodiments of the application. Based upon the embodiments herein, all other embodiments obtained by persons of ordinary skill in the art without having inventive effort are within the scope of the application.

[0039] It is found through research that only a small amount of free phenols can be dissolved in grape seed oil in the production process of grape seed oil, and most of the active compounds remain in the pomace. Further research finds that grape skins and grape stems are also rich in various anticancer, antibacterial, and antioxidant active ingredients, such as polyphenols, flavonoids, and terpenes, which have broad application prospects and great development value. Therefore, there is room for research on how to further improve the content of active ingredients in grape seed oil, and how to use grape skins and grape seeds in grape wine pomace in a high-value and resourceful manner, and efficiently extract grape wine pomace in one step.

[0040] Traditional methods such as Soxhlet extraction, immersion, and reflux extraction require the use of a large amount of solvent that may be toxic, and the required extraction temperature may cause damage and loss of active substances. The methods of ultrasonic-assisted extraction, microwave-assisted extraction, and supercritical extraction effectively improve the yield and retention rate, but require a large amount of energy consumption and are high in cost. As an environmentally friendly and efficient oil extraction method, the water enzyme method has attracted attention because it does not produce solvent residues, environmental pollution, and safety problems during the extraction process, but the high cost of commercial enzyme preparations in practical application limits the industrialization process.

[0041] The application embodiment provides an anti-aging cosmetic, comprising:

[0042] Grape wine pomace oil; characteristic components of the grape wine pomace oil include oleanolic acid, a-tupliu, β-tocotrienol, β-sitosterol, campesterol, soybean saponin E, ganoderma acid F, ganoderma alcohol I, and D-erythro-dihydro sphingosine; the total phenol content of the grape wine pomace oil is ≥1 mg / g, the total sterol content of the grape wine pomace oil is ≥26 mg / g, and the total flavonoid content of the grape wine pomace oil is ≥1 mg / g;

[0043] The grape wine pomace oil is extracted from grape wine pomace; the grape wine pomace is mainly composed of grape skins, grape stems, and grape seeds.

[0044] In the technical solution, the characteristic components of the grape pomace oil are oleanolic acid, α-copaene, β-tocotrienol, β-sitosterol, campesterol, soyasapogenol E, ganoderic acid F, ganoderol I and D-erythro-dihydrosphingosine. These compounds have excellent antioxidant properties and can be used as anti-aging active ingredients of anti-aging cosmetics, thereby greatly improving the anti-aging effect of anti-aging cosmetics. The grape pomace oil shows an anti-aging effect equivalent to 60 μM astaxanthin in a nematode model, indicating that it helps to protect the skin from oxidative damage and plays a positive role in cosmetics. The grape pomace oil maximally retains the active ingredients of each part of the grape pomace.

[0045] Exemplarily, in some embodiments of the present application, the total phenol content of the grape pomace oil is 1 mg / g, 1.1 mg / g, 1.2 mg / g, 1.3 mg / g, 1.4 mg / g, 1.5 mg / g, 1.6 mg / g, 1.7 mg / g, 1.8 mg / g, 1.9 mg / g, 2.0 mg / g, 2.5 mg / g, 3.0 mg / g, 3.5 mg / g, 4.0 mg / g, 5.0 mg / g or a range between any two of the aforementioned values.

[0046] Exemplarily, in some embodiments of the present application, the total sterol content of the grape pomace oil is 26 mg / g, 27 mg / g, 28 mg / g, 29 mg / g, 30 mg / g, 31 mg / g, 32 mg / g, 33 mg / g, 34 mg / g, 35 mg / g, 36 mg / g, 37 mg / g, 38 mg / g, 39 mg / g, 40 mg / g or a range between any two of the aforementioned values.

[0047] Exemplarily, in some embodiments of the present application, the total flavonoid content of the grape pomace oil is 1 mg / g, 1.2 mg / g, 1.5 mg / g, 2 mg / g, 2.5 mg / g, 3 mg / g, 3.5 mg / g, 4 mg / g, 4.5 mg / g, 5 mg / g or a range between any two of the aforementioned values.

[0048] In the technical solution, the definition of "characteristic components" of the grape pomace oil is that the relative content measured by gas chromatography-mass spectrometry or liquid chromatography-mass spectrometry analysis is greater than that of potential anti-aging active ingredients.

[0049] The "potential" above is because some components are reported in the literature to have antioxidant effects (aging is caused by continuous oxidative damage), but have not been directly verified by anti-aging experiments.

[0050] For example, it has been reported that oleanolic acid can inhibit oxidative stress-induced hepatocyte damage by increasing the activity of antioxidant enzymes (such as superoxide dismutase, catalase, glutathione peroxidase, etc.) and the level of reduced glutathione, inhibiting the generation of reactive oxygen species (ROS) and scavenging free radicals. These effects of oleanolic acid may be beneficial for anti-aging, i.e., oleanolic acid is a "potential" anti-aging active ingredient.

[0051] It should be noted that the grape pomace oil described above also contains some other active ingredients, but these active ingredients have not been reported to have antioxidant and anti-aging activity, and therefore have not been included as "characteristic components" of the grape pomace oil described above. For example, in some embodiments of the present application, the grape pomace oil described above is analyzed by gas chromatography-mass spectrometry to determine oleanolic acid (11.6108%), a-lupulene (4.5109%), β-tocotrienol (2.8166%), β-sitosterol (7.0947%), and campesterol (1.3126%) as "characteristic components" of the grape pomace oil; and soyasapogenol E (7.5149%), ganoderic acid F (3.2038%), ganoderol I (1.7824%), and D-erythro-dihydrosphingosine (20.1058%) are determined by liquid chromatography-mass spectrometry as "characteristic components" of the grape pomace oil.

[0052] Further, in some embodiments of the present application, the peroxide value of the grape pomace oil is ≤7 mmol / Kg.

[0053] The peroxide value is an important indicator of the degree of oxidation of oil, and a lower peroxide value indicates better oxidative stability of the oil; generally, it should be less than or equal to 10 mmol / Kg. In the technical solution described above, the peroxide value of the grape pomace oil is ≤7 mmol / Kg, which has very excellent oxidative stability, and thus when it is applied to anti-aging cosmetics, it can effectively improve the anti-aging efficacy of the cosmetics.

[0054] For example, in some embodiments of the present application, the peroxide value of the grape pomace oil is 7 mmol / Kg, 6 mmol / Kg, 5 mmol / Kg, 4 mmol / Kg, 3 mmol / Kg, 2 mmol / Kg, 1 mmol / Kg, 0.5 mmol / Kg, or a range between any two of the foregoing values.

[0055] In some embodiments of the present application, the method for determining the peroxide value is as follows:

[0056] Take 2-3 g of the prepared sample, place it in a 250 mL iodine flask, add 30 mL of chloroform-glacial acetic acid solution, and gently shake the sample until it is completely dissolved. Accurately add 1.00 mL of potassium iodide saturated solution, tightly cap the flask, and gently shake for 0.5 min, and place it in the dark for 3 min. Take out 100 mL of water, shake well, and immediately titrate the precipitated iodine with a standard titration solution of sodium thiosulfate until a light yellow color appears. Add 1 mL of starch indicator, continue titration, and shake vigorously until the solution turns blue, which is the end point. A blank test is also performed, and the volume of sodium thiosulfate standard titration solution consumed in the blank test is V0. The peroxide value is calculated according to the following formula:

[0057]

[0058] Further, in some embodiments of the present application, the acid value of the grape pomace oil is ≤4 mg / g.

[0059] The acid value is an index for evaluating the content of free fatty acids in oil and fat, and a lower acid value means that the degree of rancidity of the oil and fat is lower and the quality is higher; generally, it needs to be less than or equal to 5 mmol / Kg. In the above technical solution, the acid value of the grape pomace oil is ≤4 mg / g, the degree of rancidity of the oil and fat is lower, and the quality is higher, so that when it is applied to anti-aging cosmetics, the anti-aging effect of the cosmetics can be effectively improved.

[0060] For example, in some embodiments of the present application, the acid value of the grape pomace oil is 4 mg / g, 3.5 mg / g, 3 mg / g, 2.5 mg / g, 2 mg / g, 1.5 mg / g, 1.2 mg / g, 1.1 mg / g, 1 mg / g, or a range between any two of the foregoing values.

[0061] In some embodiments of the present application, the acid value is determined by the following method:

[0062] Take the oil and fat sample in a 250 mL conical flask, add 50-100 mL of a mixture of diethyl ether-isopropyl alcohol and 3-4 drops of phenolphthalein indicator, shake the sample well to dissolve it, and then titrate with a standard titration solution. When the sample solution initially appears reddish and there is no obvious fading within 15 s, it is the end point of titration. Record the milliliter number of the standard titration solution consumed in this titration, which is V. Take another clean 250 mL conical flask, accurately add the same volume and same type of organic solvent mixture and indicator as in the sample determination, shake well, and titrate the milliliter number of the standard titration solution consumed, which is V0. The acid value calculation formula is as follows:

[0063]

[0064] Some embodiments of the present application provide a preparation method of grape pomace oil, which comprises:

[0065] The wine pomace powder is hot soaked with an acid solution to obtain a first feed liquid;

[0066] The first feed liquid is subjected to continuous phase change extraction at 0.2-0.8 MPa and 40-50°C.

[0067] The above technical solution can make the solvent quickly circulate in liquid-gas-liquid mode, and the circulation and fresh solvent can ensure sufficient and effective extraction of the material. In addition, the solvent is recovered in a vacuum state, which does not cause solvent residue and environmental pollution, thereby realizing low-cost green extraction. The above technical solution solves the problem that the active ingredients in the wine pomace are not fully and completely extracted, and part of the wine pomace is wasted or used at low value. The wine pomace oil extracted by the above method retains the active ingredients of each part of the wine pomace to the greatest extent. At the same time, the method must be green, efficient, low in cost, environmentally friendly and low in energy consumption.

[0068] Exemplarily, in some embodiments of the present application, the first feed liquid is subjected to continuous phase change extraction at a pressure of 0.2 MPa, 0.22 MPa, 0.25 MPa, 0.28 MPa, 0.3 MPa, 0.32 MPa, 0.35 MPa, 0.38 MPa, 0.4 MPa, 0.42 MPa, 0.45 MPa, 0.5 MPa, 0.6 MPa, 0.7 MPa, 0.8 MPa or a range between any two of the foregoing values; and a temperature of 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C or a range between any two of the foregoing values.

[0069] In the above technical solution, the first feed liquid is subjected to continuous phase change extraction at 0.2-0.8 MPa and 40-50°C.

[0070] Further, in some embodiments of the present application, the continuous phase change extraction comprises:

[0071] The n-butane is used as an extractant for liquid phase, gas phase and liquid phase continuous circulation phase change extraction.

[0072] The n-butane is an organic compound with the chemical formula C4H 10 It is a common alkane and is a colorless, easily liquefied gas at room temperature and pressure. It can circulate in liquid-gas-liquid mode (i.e. continuous phase change extraction) during continuous phase change extraction, and the circulation and fresh solvent can ensure sufficient and effective extraction of the material; and further improve the content of active ingredients in the wine pomace oil obtained by extraction.

[0073] Further, in some embodiments of the present application, the continuous phase change extraction includes multiple extraction cycles, and one extraction cycle includes:

[0074] The extractant gas is compressed into extractant liquid in vacuum, the extractant liquid is flowed to the first feed liquid for extraction, the extracted wine pomace oil and the acid solution are flowed out with the extractant liquid to obtain a mixture, the extractant liquid is converted into extractant gas and separated from the mixture, and the separated extractant gas is recycled for the extraction cycle.

[0075] Further, in some embodiments of the present application, after the extractant gas is separated, the acid solution is removed from the mixture to obtain a crude extract.

[0076] Further, in some embodiments of the present application, the crude extract is subjected to a second extraction.

[0077] Further, in some embodiments of the present application, the second extraction of the crude extract is performed by using n-hexane.

[0078] Further, in some embodiments of the present application, the acid solution is an acid alcohol solution.

[0079] The above technical solution can soak the wine pomace powder in the acid solution, destroy the compact structure of the cell wall of the wine pomace powder by deacidification, and then extract the oil in the cells by using the organic solvent. At the same time, the phenolic compounds are more easily dissolved and retained under acidic conditions, which promotes them to be further extracted into the oil phase.

[0080] Further, in some embodiments of the present application, the acid solution is a citric acid-ethanol solution.

[0081] Further, in some embodiments of the present application, the citric acid-ethanol solution includes 1% to 5% citric acid and 95% to 99% ethanol by mass percentage.

[0082] Exemplarily, in some embodiments of the present application, the citric acid-ethanol solution includes 1%, 1.2%, 1.5%, 2%, 2.5%, 2.8%, 3%, 3.5%, 3.8%, 4%, 4.2%, 4.5%, 4.8%, 5% or a range between any two of the foregoing values of citric acid and 95%, 96%, 97%, 98%, 99% or a range between any two of the foregoing values of ethanol by mass percentage.

[0083] Further, in some embodiments of the present application, the wine pomace powder is hot soaked in the acid solution, including:

[0084] The wine pomace powder is hot soaked in the acid solution at a material-liquid ratio of (1:1) to (1:3) by mass ratio.

[0085] Exemplarily, in some embodiments of the present application, the wine lees powder and the acid solution are soaked in a ratio of 1:1, 1:1.1, 1:1.2, 1:1.5, 1:1.8, 1:2, 1:2.1, 1:2.2, 1:2.5, 1:2.8, 1:3 or a range between any two of the aforementioned values.

[0086] Further, in some embodiments of the present application, the wine lees powder is hot-soaked with the acid solution, including:

[0087] The wine lees powder is soaked with the acid solution at 40-50°C.

[0088] Exemplarily, in some embodiments of the present application, the wine lees powder is hot-soaked with the acid solution, including:

[0089] The wine lees powder is soaked with the acid solution at 40-50°C.

[0090] Further, in some embodiments of the present application, the soaking time is 1-3h.

[0091] Exemplarily, in some embodiments of the present application, the soaking time is 1h, 1.1h, 1.2h, 1.3h, 1.4h, 1.5h, 1.6h, 1.7h, 1.8h, 1.9h, 2.0h, 2.1h, 2.2h, 2.3h, 2.4h, 2.5h, 2.6h, 2.7h, 2.8h, 2.9h, 3h or a range between any two of the aforementioned values.

[0092] Some embodiments of the present application provide a wine lees oil, which is obtained by the preparation method of the wine lees oil provided in any of the aforementioned embodiments.

[0093] The features and performances of the present application are further described in detail below in combination with examples:

[0094] Example 1

[0095] A wine lees oil is provided, which is prepared according to the following steps:

[0096] S1, dry wine lees is crushed and sieved to obtain wine lees powder with a particle size of 40 mesh. The wine lees is composed of grape skin, grape stem and grape seed.

[0097] S2, 1000 g of the wine marc powder obtained in step S1 was filled into a 3 L extraction axe of a continuous phase change extraction device, 2 L of a 5% by mass citric acid-ethanol solution was added, and soaking was performed at 50°C for 120 min. Then, n-butane was used as an extraction solvent, and extraction was performed at an extraction pressure of 0.8 MPa, an extraction temperature of 50°C, a desorption temperature of 60°C, and a flow rate of 60 L / h. The n-butane was compressed into a liquid in a vacuum, flowed through the extraction axe to extract the wine marc, and the wine marc oil and the citric acid-ethanol solution flowed into a desorption kettle with the n-butane liquid, and the n-butane liquid was converted into a gas by heating and pressure reduction to separate from the wine marc oil and was recovered into a storage tank. The above process was continuously cycled for a total of 120 min, and finally, the crude extract was obtained in the desorption axe.

[0098] S3, the crude extract obtained in step S2 was subjected to rotary evaporation to remove the ethanol solution, was added to a separatory funnel, and was extracted with n-hexane. The extraction liquid was subjected to rotary evaporation to obtain the wine marc oil.

[0099] Example 2

[0100] A wine marc oil was provided, and was prepared according to the following steps:

[0101] S1, dry wine marc was crushed and sieved to obtain wine marc powder with a particle size of 40 mesh. The wine marc was composed of grape skin, grape stem, and grape seed.

[0102] S2, 1000 g of the wine marc powder obtained in step S1 was filled into a 3 L extraction axe of a continuous phase change extraction device, 3 L of a 1% by mass citric acid-ethanol solution was added, and soaking was performed at 45°C for 90 min. Then, n-butane was used as an extraction solvent, and extraction was performed at an extraction pressure of 0.5 MPa, an extraction temperature of 45°C, a desorption temperature of 55°C, and a flow rate of 60 L / h. The n-butane was compressed into a liquid in a vacuum, flowed through the extraction axe to extract the wine marc, and the wine marc oil and the citric acid-ethanol solution flowed into a desorption kettle with the n-butane liquid, and the n-butane liquid was converted into a gas by heating and pressure reduction to separate from the wine marc oil and was recovered into a storage tank. The above process was continuously cycled for a total of 60 min, and finally, the crude extract was obtained in the desorption axe.

[0103] S3, the crude extract obtained in step S2 was subjected to rotary evaporation to remove the ethanol solution, was added to a separatory funnel, and was extracted with n-hexane. The extraction liquid was subjected to rotary evaporation to obtain the wine marc oil.

[0104] Example 3

[0105] A wine marc oil was provided, and was prepared according to the following steps:

[0106] S1, dry grape pomace is crushed and sieved to obtain grape pomace powder with a particle size of 40 mesh. The grape pomace is composed of grape skin, grape stem and grape seed.

[0107] S2, 1000 g of grape pomace powder obtained in step S1 is filled into a 3 L extraction tank of a continuous phase change extraction device, 1 L of a 3% mass percentage citric acid-ethanol solution is added, and the mixture is soaked at 40°C for 60 min. Then, n-butane is used as the extraction solvent, and the extraction is carried out under the conditions of an extraction pressure of 0.2 MPa, an extraction temperature of 40°C, an elution temperature of 50°C, and a flow rate of 60 L / h. The n-butane is compressed into a liquid in a vacuum, flows through the extraction tank to extract the grape pomace, and the grape pomace oil and the citric acid-ethanol solution flow into the elution tank with the n-butane liquid. The n-butane liquid is separated from the grape pomace oil by being converted into a gas by heating and reducing the pressure and is recovered to a storage tank. The above process is continuously cycled for a total of 90 min of extraction; and finally, the crude extract is obtained in the elution tank.

[0108] S3, the crude extract obtained in step S2 is rotary evaporated to remove the ethanol solution, then added to a separatory funnel and extracted with n-hexane. The extraction liquid is rotary evaporated to obtain grape pomace oil.

[0109] Comparative Example 1

[0110] A grape pomace oil is provided, which is prepared according to the following steps:

[0111] S1, dry grape pomace is crushed and sieved to obtain grape pomace powder with a particle size of 40 mesh. The grape pomace is composed of grape skin, grape stem and grape seed.

[0112] S2, 30 g of crushed grape pomace powder is taken, 300 mL of n-hexane is added, and the mixture is stirred and extracted at 50°C under normal pressure for 3 h. The mixture is filtered and rotary evaporated to remove the n-hexane, and grape pomace oil is obtained.

[0113] Comparative Example 2

[0114] A grape pomace oil is provided, which is prepared according to the following steps:

[0115] S1, dry grape pomace is crushed and sieved to obtain grape pomace powder with a particle size of 40 mesh. The grape pomace is composed of grape skin, grape stem and grape seed.

[0116] S2, 30 g of crushed grape pomace powder is taken, 300 mL of n-hexane is added, and the mixture is stirred and extracted at 50°C under normal pressure for 3 h. The mixture is filtered and rotary evaporated to remove the n-hexane, and grape pomace oil is obtained.

[0117] S3, the crude extract obtained in step S2 is subjected to rotary evaporation to remove the ethanol solution, then added into a separatory funnel for extraction with n-hexane, and the extract is subjected to rotary evaporation to obtain the grape pomace oil.

[0118] Comparative Example 3

[0119] A grape pomace oil is provided, which is prepared according to the following steps:

[0120] S1, dry grape pomace is crushed and sieved to obtain grape pomace powder with a particle size of 40 mesh. The grape pomace is composed of grape skin, grape stem and grape seed.

[0121] S2, 1000 g of grape pomace powder obtained in step S1 is filled into a 3L extraction axe of a continuous phase change extraction device, and n-butane is used as the extraction solvent. The extraction is carried out under the conditions of an extraction pressure of 0.8 MPa, an extraction temperature of 50°C, an analysis temperature of 60°C, and a flow rate of 60 L / h for 120 min. The n-butane is compressed into a liquid in a vacuum, flows through the extraction axe to extract the grape pomace, and the grape pomace oil and the citric acid-ethanol solution flow into the analysis kettle with the n-butane liquid. The n-butane liquid is converted into a gas by heating and reducing pressure to separate from the grape pomace oil and is recovered into a storage tank. The above process is continuously cycled for a total of 120 min, and finally the grape pomace oil is obtained in the analysis axe.

[0122] Comparative Example 4

[0123] A grape pomace oil is provided, which is prepared according to the following steps:

[0124] S1, dry grape pomace is crushed and sieved to obtain grape pomace powder with a particle size of 40 mesh. The grape pomace is composed of grape skin, grape stem and grape seed.

[0125] S2, 1000 g of grape pomace powder obtained in step S1 is filled into a 3L extraction axe of a continuous phase change extraction device, and n-butane is used as the extraction solvent. The extraction is carried out under the conditions of an extraction pressure of 0.8 MPa, an extraction temperature of 50°C, an analysis temperature of 60°C, and a flow rate of 60 L / h for 120 min. The n-butane is compressed into a liquid in a vacuum, flows through the extraction axe to extract the grape pomace, and the grape pomace oil and the citric acid-ethanol solution flow into the analysis kettle with the n-butane liquid. The n-butane liquid is converted into a gas by heating and reducing pressure to separate from the grape pomace oil and is recovered into a storage tank. The above process is continuously cycled for a total of 120 min, and finally the grape pomace oil is obtained in the analysis axe.

[0126] S3, the crude extract obtained in step S2 is subjected to rotary evaporation to remove the ethanol solution, then added into a separatory funnel for extraction with n-hexane, and the extract is subjected to rotary evaporation to obtain the grape pomace oil.

[0127] Experimental Example 1 Extraction rate, active ingredient content and in vitro antioxidant activity of grape pomace oil

[0128] The technical effects of each embodiment and comparative example in the present application were compared, and the extraction rate and ingredient content of grape pomace oil were calculated.

[0129] (1) Yield calculation: Grape pomace oil extraction rate (%) = grape pomace oil mass (g) / grape pomace crude fat mass (g) x 100%

[0130] (2) Determination method of total phenol content:

[0131] 0.5 g of oil was weighed and added with 2 ml of methanol-water solution (methanol: water = 90:10), then vortexed for 5 min, centrifuged at 3000 rpm / min for 5 min, the supernatant was aspirated, and the above steps were repeated. The extraction procedure of each oil was performed three times. All the extraction solutions were mixed and concentrated to dryness, and the dry matter was dissolved in 1 ml of methanol-water solution (methanol: water = 10:90) to obtain a polyphenol extract. 0.5 mL of the extract was precisely aspirated, distilled water was added to 5 mL, 1 mL of Folin-Ciocateu reagent and 4 mL of 10% Na2CO3 solution were added, respectively, and after shaking, it was placed at room temperature for color development for 60 min. Distilled water was used as a blank test, and the absorbance was measured at 765 nm. The polyphenol content was calculated according to the regression equation of the gallic acid standard curve, and each sample was measured three times.

[0132] (3) Determination method of sterol content:

[0133] 0.5 g of mercuric oxide was weighed, 2 mL of concentrated sulfuric acid and 10 mL of distilled water were precisely added, and ultrasonic dissolution was performed. 1 mL of the solution was taken and placed in a 100 mL volumetric flask, and an ice acetic acid and concentrated sulfuric acid mixed solution (35:70) was used to dilute to the mark to obtain a sulfobutyric acid mercury reagent. 0.1 g of oil was accurately weighed and diluted to 10 mL in a volumetric flask with methanol. 2 mL of the obtained oil-methanol solution was taken and placed in a 10 mL volumetric flask, and the sulfobutyric acid mercury reagent was used to dilute to the mark. The absorbance value was measured at 410 nm with methanol as a blank. The sterol content was calculated according to the regression equation of the standard curve of sitosterol, and each sample was measured three times.

[0134] (4) Determination method of flavonoid content:

[0135] Take 0.5 g of oil and add 2 ml of methanol-water solution (methanol: water = 90:10), then vortex for 5 minutes, centrifuge at 3000 rpm / min for 5 minutes, take the supernatant, and repeat the above steps three times for each oil extraction procedure. Mix all the extracts and concentrate to dryness, dissolve the dry matter in 5 ml of methanol-water solution (methanol: water = 10:90) to obtain the flavonoid extract. Take 1.0 mL of the extract in a 25 mL graduated test tube, add 1 mL of 5% sodium nitrite solution, shake well, wait for 6 minutes, then add 1 mL of 10% Al(NO3)3 solution, 6 minutes later add 10 mL of 4% NaOH solution, then add 60% ethanol solution to the mark, stand for 15 minutes, then measure the absorbance at 510 nm. Use distilled water as a blank control and use rutin standard as a standard curve. Each sample is measured three times.

[0136] (5) Determination of DPPH free radical scavenging rate

[0137] Weigh an appropriate amount of oil and dissolve it in anhydrous ethanol, then dilute it to different mass concentrations to obtain sample solutions for antioxidant activity tests. 样品 : Take different mass concentration gradients of the sample to be tested and mix with DPPH working solution. 空白 : Take different mass concentration gradients of the sample to be tested and mix with anhydrous ethanol. 对照 : Take DPPH working solution and mix with anhydrous ethanol. Mix well and react at room temperature for 30 minutes in the dark. After the reaction is complete, measure the absorbance at 517 nm using a UV spectrophotometer. Each sample is measured three times. The DPPH free radical scavenging rate is calculated as follows:

[0138]

[0139] The effects of different extraction methods on the extraction rate, total phenols, flavonoids, sterols, and in vitro antioxidant activity of grape pomace oil were determined. The results are shown in Table 1.

[0140] Table 1 Extraction rate, active ingredient content, and DPPH free radical scavenging rate of grape pomace oil

[0141]

[0142]

[0143] Note: For the same column, different letters indicate significant differences between the two (p<0.05), and the same letter indicates no significant difference (p>0.05).

[0144] As can be seen from Table 1, the oil extraction rate of the grape pomace in the embodiments of the present application is significantly higher than that of the comparative examples. Specifically, the oil extraction rate of Example 1 reaches 96.70±1.27%, which is the highest among all experimental groups. At the same time, the polyphenol, sterol and flavonoid contents of the grape pomace oil extracted by the embodiments are all relatively high, especially the effect of Example 1 is the best, which are 1.13±0.18, 26.29±2.01, 1.40±0.42 mg / g respectively, which are significantly higher than those of the comparative examples. Under the same concentration, the DPPH free radical scavenging rate of Example 1 is 98.22±1.54%, which is significantly higher than that of the comparative examples, showing excellent antioxidant performance. These results show that the method of the present application can effectively retain the active ingredients in the oil material while extracting oil, and has high in vitro antioxidant activity. The acid heat pretreatment, combined with the use of low-temperature continuous phase change extraction, fully extracts the active ingredients in the grape pomace and enriches them in the obtained grape pomace oil.

[0145] Compared with Comparative Example 1 and Comparative Example 2, Example 1-3 has better DPPH free radical scavenging rate, oil extraction rate, polyphenol content, flavonoid content and sterol content.

[0146] Compared with Examples 1-3, Comparative Example 3 has significantly lower DPPH free radical scavenging rate, oil extraction rate, polyphenol content, flavonoid content and sterol content.

[0147] Experimental Example 2 Analysis of physical and chemical indicators of grape pomace oil

[0148] (1) Peroxide value determination method:

[0149] 2-3 g of the prepared sample was weighed into a 250 mL iodine flask, 30 mL of chloroform-glacial acetic acid solution was added, the sample was shaken gently until completely dissolved. 1.00 mL of potassium iodide saturated solution was accurately added, the cap was tightly closed, and the flask was shaken gently for 0.5 min, and then placed in the dark for 3 min. 100 mL of water was taken out, shaken well, and then immediately titrated with sodium thiosulfate standard titration solution to precipitate iodine, and titrated to light yellow, then 1 mL of starch indicator was added, and the titration was continued and the solution was shaken vigorously until the blue color disappeared as the end point. At the same time, a blank test was carried out, and the volume of sodium thiosulfate standard titration solution consumed in the blank test was V0. The peroxide value calculation formula is as follows:

[0150]

[0151] (2) Acid value determination method:

[0152] Take the oil sample in a 250 mL conical flask, add 50-100 mL of ether-isopropanol mixture and 3-4 drops of phenolphthalein indicator, shake well to dissolve the sample, then titrate with standard titration solution. When the sample solution appears slightly red and no significant fading occurs within 15 s, the titration is complete. Record the milliliters of standard titration solution consumed, which is V. Take another clean 250 mL conical flask and accurately add the same volume of organic solvent mixture and indicator as in the sample determination, shake well, and titrate the milliliters of standard titration solution consumed, which is V0. The acid value calculation formula is as follows:

[0153]

[0154] (3) Determination method of citric acid content:

[0155] Prepare 7 10 mL centrifuge tubes and add 0.5 mL of 0.4 mol / L nitric acid solution, 2 mL of 5.0 x 10 -4 mol / L phenol red solution, 0.1 mL of 10 mg / mL iron (III) solution, and 0.1 mL of 5% hydrogen peroxide solution. Add 1 mL of citric acid standard working solution (0.05 mg / mL, 0.10 mg / mL, 0.20 mg / mL, 0.40 mg / mL, 0.50 mg / mL, 1.00 mg / mL) to 6 of them, dilute with distilled water to 8 mL, shake well, place in a 80°C water bath for 8 min, then take out and cool rapidly with 4°C water for 3 min. Measure the absorbance A and A0 of the inhibition system and catalytic system at 440 nm wavelength, calculate △A = A - A0, and obtain the standard curve regression equation to calculate the sterol content. After water extraction of the oil sample, the extract is calculated according to the above experimental operation to calculate the citric acid content in the sample.

[0156] Determine the effect of different extraction methods on the physicochemical indicators of grape pomace oil. The results are shown in Table 2.

[0157] Table 2 Peroxide value, acid value and citric acid residue of grape pomace oil

[0158] Group Peroxide value (mmol / kg) Acid value (mg / g) Citric acid residue (%) Example 1 5.73±0.76 3.42±0.49 0.29±0.03 Example 2 6.96±0.65 3.43±0.18 0.22±0.01 Example 3 6.17±1.29 3.32±0.58 0.18±0.01 Comparative Example 1 17.86±0.84 3.67±0.18 Not detected Comparative Example 2 10.45±1.80 3.69±0.43 0.32±0.02 Comparative Example 3 8.85±1.94 3.33±0.09 Not detected Comparative Example 4 6.92±0.73 367±0.04 0.16±0.01

[0159] Peroxide value is an important indicator to measure the degree of oil oxidation, lower peroxide value indicates that the oil oxidation stability is better; acid value is an index to evaluate the content of free fatty acids in oil, lower acid value means lower degree of rancidity of oil, higher quality. The peroxide value and acid value of the grape pomace oil obtained in the examples of the present application are both lower, far less than the requirements of GB / T 29990-2013 emollient oil standard (peroxide value less than or equal to 10 mmol / kg; acid value less than or equal to 5 mmol / kg). And compared with the comparative examples, there is a significant decline. The peroxide value and acid value in comparative example 1 are both higher, it can be seen that solvent extraction method will promote the oxidation of oil to some extent, and the quality of the obtained oil is poor. At the same time, the residual amount of citric acid in the grape pomace oil obtained in the examples of the present application is also within the standard range, and the literature proves that a small amount of citric acid in oil can play the role of antioxidant. Therefore, the oil extraction method of the present application can effectively control the degree of oxidation and rancidity of oil while ensuring high extraction rate and active ingredient retention rate, thereby providing a high-quality and high-stability oil extraction solution.

[0160] The determination of the above-mentioned physical and chemical indicators shows that the extraction method of the present application has high feasibility, can improve the content of active ingredients and in vitro antioxidant activity without sacrificing the quality of oil, and the extracted oil meets the standard of GB / T 29990-2013 emollient oil.

[0161] The residual amount of a small amount of citric acid in the method of the present application shows that although the method of the present application uses citric acid, the extracted oil meets the standard of GB / T 29990-2013 emollient oil for the content of citric acid.

[0162] Experimental example 3 characteristic component analysis of grape pomace oil

[0163] The characteristic component analysis of the grape pomace oil obtained in example 1 was carried out.

[0164] (1) Gas chromatography-mass spectrometry analysis

[0165] S1, 100 μL sample was added to 300 μL extraction solution (methanol: acetonitrile = 2:1 (containing 0.05 mg / mL internal standard ribitol)); after vortexing for 30 s, ice water bath ultrasonic extraction for 30 min; the sample was placed at -20 °C for 30 min, and then high speed centrifugation for 15 min (4 °C, 13000 rcf); the supernatant was taken and loaded into a glass derivative vial, and nitrogen was blown dry; 80 μL of methoxyamine hydrochloride pyridine solution (15 mg / mL) was added to the glass derivative vial, vortexed for 2 min, and then subjected to oxime reaction in a 37 °C shaking incubator for 90 min; 80 μL of BSTFA (containing 1% TMCS) derivatization reagent was added, vortexed for 2 min, and then reacted at 70 °C for 60 min; after taking out the sample, it was placed at room temperature for 30 min, and then subjected to GC-MS metabolomics analysis.

[0166] S2, chromatographic conditions: after derivatization, the sample was injected into the GC-MS system for analysis in split mode, the injection amount was 1 μL, and the split ratio was 10:1. After separation by a TG-5SIL MS capillary column (30 m x 0.25 mm x 0.25 μm, Thermo 26096-1420), the sample entered the mass spectrometer for detection. The inlet temperature was 300 °C, the carrier gas was high-purity helium, the carrier gas flow rate was 1.0 mL / min, and the septum purge flow was 3 mL / min. The temperature program was as follows: the initial temperature was 80 °C, the equilibrium time was 0 min, then the temperature was increased to 310 °C at a rate of 20 °C / min, and maintained for 8 min, the total running time was 20 min, and the solvent delay was 2 min.

[0167] S3, mass spectrometry conditions: electron impact (EI) source, electron energy 70 eV, scan mode Full Scan, scan mass range 35-500 m / z, ion source temperature 250 °C.

[0168] The detection results are shown in Figure 1 and Table 3. Figure 1 is the total ion chromatogram of the gas chromatography-mass spectrometry analysis of the grape pomace oil.

[0169] Table 3 Chemical composition and relative content of the gas chromatography-mass spectrometry analysis of the grape pomace oil

[0170]

[0171]

[0172]

[0173]

[0174]

[0175] The results of gas chromatography-mass spectrometry analysis showed that 103 compounds were identified in the grape pomace oil, involving 12 substance categories. Among them, 31 belonged to fatty acids and lipids, 14 belonged to amino acids, 8 belonged to sugars and their derivatives, 11 belonged to carboxylic acids, 3 belonged to aldehydes, 7 belonged to alcohols, 3 belonged to phenols and their derivatives, 3 belonged to flavones, 6 belonged to terpenes and terpenoids related compounds, 5 belonged to sterols, 7 belonged to vitamins and 5 belonged to other categories. It can be seen that the active ingredients of grape pomace oil are very rich.

[0176] It is worth noting that the relative contents of conjugated (9E, 11E)-linoleic acid (20.3205%) and α-linolenic acid (6.6875%) are relatively high. These polyunsaturated fatty acids can protect cells from oxidative damage by neutralizing free radicals and improve the skin's ability to retain moisture, thereby delaying skin aging. Another high content of lipid substance is glycerol monopalmitate (5.0188%), which is commonly used as an emulsifier and emollient in cosmetics in addition to its anti-aging activity. Among the terpenes and terpenoids related compounds, oleanolic acid (11.6108%) has a high content and has significant anti-aging and antioxidant effects, which can improve and enhance dermal collagen; while α-turpentine (4.5109%) is less, although there is no report on its anti-aging activity, but its antibacterial and anti-inflammatory activity has been widely recognized. In terms of phytosterols, grape pomace oil is rich in β-sitosterol (7.0947%) and campesterol (1.3126%), which are representatives of high-value active ingredients in grape seeds, giving grape pomace oil excellent anti-aging effects. Another representative of high-value active ingredients is vitamin E, the main configuration in the grape pomace oil prepared in the present application is β-tocotrienol (2.8166%), and it also contains various configurations of tocopherols, laying a foundation for grape pomace oil as an effective antioxidant.

[0177] At the same time, the results also found that the grape pomace oil contains a variety of polyphenols and flavonoids, including protocatechuic acid, salicylic acid, coffee acid, catechin, kaempferol and gallic catechin, etc., with a total relative content of 0.5074%. Other high-value anti-aging active ingredients were also found, such as squalene (0.0079%), inositol (0.0449%), phytol (0.0740%), L-erythro-sphingosine (0.5239%), lupine alcohol (0.2087%), etc. The above-mentioned substances are high-quality antioxidants that can play an anti-aging role in the body or skin, and some substances also have anti-inflammatory activity, thus serving as raw materials for high-end cosmetics. Therefore, the grape pomace oil prepared in the present application has a variety of potential health benefits and has important application value in the fields of health foods, cosmetics, etc.

[0178] (2) Liquid chromatography-mass spectrometry analysis

[0179] S1, 10 μL sample was accurately pipetted into a 1.5 mL centrifuge tube; 400 μL extraction solution (methanol: water = 4: 1 (v: v)) containing four internal standards (L-2-chlorophenylalanine (0.02 mg / mL) and the like) was added; after vortex mixing for 30 s, low-temperature ultrasonic extraction was performed for 10 min (5°C, 40 KHz); centrifugation was performed for 15 min (13000 g, 4°C), and the supernatant was pipetted into a sample injection vial with an internal cannula for machine analysis.

[0180] S2, Chromatographic conditions: the chromatographic column was ACQUITY UPLC HSS T3 (100 mm x 2.1 mm i.d., 1.8 μm; Waters, Milford, USA); the mobile phase A was 95% water + 5% acetonitrile (containing 0.1% formic acid), the mobile phase B was 47.5% acetonitrile + 47.5% isopropanol + 5% water (containing 0.1% formic acid), the injection amount was 3 μL, and the column temperature was 40°C.

[0181] S3, Mass spectrometric conditions: the sample was ionized by electrospray, and mass spectrometric signals were collected in positive and negative ion scanning modes, respectively. The scanning range was 70-1050 m / z, the spray voltage (positive mode) was 3500 V, the spray voltage (negative mode) was -3500 V, and the capillary temperature was 325°C.

[0182] The detection results are shown in Figure 2 and Table 4. Figure 2 The total ion chromatogram of the liquid chromatography-mass spectrometry analysis of the grape pomace oil.

[0183] Table 4 Chemical composition and relative content of the liquid chromatography-mass spectrometry analysis of the grape pomace oil

[0184]

[0185]

[0186]

[0187]

[0188]

[0189] The results of liquid chromatography-mass spectrometry analysis show that 123 compounds are identified in the grape pomace oil, involving 15 substance categories. Among them, 20 belong to phenols and their derivatives, 12 belong to amino acids and their derivatives, 2 belong to sugars, 20 belong to terpenes and terpenes related compounds, 16 belong to organic acids, 12 belong to flavonoids, 6 belong to alcohols, 19 belong to fatty acids and lipids, 3 belong to glycosides, and 1 belongs to amides, 1 belongs to alkaloids, 2 belong to nitrogen-containing heterocyclic compounds, 2 belong to vitamins, 3 belong to aldehyde compounds and 4 belong to ketone compounds.

[0190] Among them, the relative content of soybean saponol E is the highest, reaching 29.7707%, which has multiple biological activities such as lowering blood lipids, immune regulation, antioxidant and anti-tumor, so it has broad application prospects in the fields of medicine, health food and cosmetics. Secondly, the second most abundant compound is D-erythro-dihydrosphingosine (20.1058%), which is an isomer of sphingosine and a key component of cell membrane phospholipids, and is essential for maintaining skin barrier and nerve cell function. The third most abundant compound is soybean cerebroside I (7.5149%), a glucocerebroside, which has been proven to have multiple pharmacological effects such as anti-tumor and anti-inflammatory, and also shows strong inhibition of tyrosinase. Interestingly, two active ingredients found in Ganoderma lucidum were also found in the grape pomace oil, and the contents were considerable, which were ganoderic acid F (3.2038%) and ganoderol I (1.7824%), respectively. They were reported to have anti-tumor, anti-inflammatory and immune regulation effects. In addition, nine ceramides were found in the grape pomace oil of the present application, with the highest content of Cer (8:0_2O / 10:0) (5.7270%). Among them, "8:0" represents that the length of the fatty acid chain is 8 carbon atoms, and all are saturated carbon atoms (without double bond), "2O" represents that two hydroxyl groups (OH) are connected to the sphingosine base, and "10:0" represents that the length of the other fatty acid chain is 10 carbon atoms, also all are saturated carbon atoms. Another ceramide HexCer refers to hexosyl ceramide, which contains two fatty acid chains and a sugar group, with the highest content of HexCer (14:3_20 / 2 8:1_20) (1.2074%). Ceramides have multiple physiological and pharmacological functions, such as regulating cell immunity, delaying aging, anti-tumor, etc. In the cosmetics industry, ceramides may help to promote the degradation of the stratum corneum, thin the keratinization layer, and promote the proliferation of epidermal cells, thereby delaying the aging process of epidermal cells.

[0191] The polyphenols and flavonoids in the grape pomace oil are also worth attention, with total relative contents of 0.9710% and 2.4048%, respectively. Among the polyphenols, the contents of gentisic acid (0.1112%), 2-hydroxy cinnamic acid (0.1340%), and gallic acid (0.1242%) are relatively high, and compounds such as protocatechuic aldehyde, ferulic acid, vanillin, and dihydrocaffeic acid are also detected. These compounds are common and effective antioxidants in plants, and even some substances such as dihydrocaffeic acid can prevent UV-induced skin damage, implying their positive role in resisting photoaging. Among the flavonoids, the contents of isorhamnetin (0.8964%), kaempferol (0.4201%), 6"-O-p-coumaroyl trilobatin (0.1950%), and quercetin (0.1752%) are relatively high, and myricetin, which also has anti-photoaging effects, and dihydroquercetin, which has anti-tyrosinase activity, are also found. Therefore, the above results further demonstrate the health benefits of grape pomace oil, especially its antioxidant and anti-aging effects, and it has great application value in health foods and cosmetics.

[0192] Effect Example Anti-aging effect of grape pomace oil

[0193] The anti-aging activity of the grape pomace oil obtained in Example 1 was evaluated using a Caenorhabditis elegans model to test its effect on prolonging the lifespan of the nematodes under oxidative stress damage.

[0194] Caenorhabditis elegans has shown significant advantages in anti-aging research due to its easy manipulation, simple observation, simple physiological structure, rapid life cycle, clear genetic information, high similarity to human genes, and complete genome sequencing, and has been widely accepted as a research model.

[0195] Oxidative stress, triggered by excess reactive oxygen species (ROS) in vivo, is considered as a major factor driving nematode aging. The antioxidant capacity of nematodes is closely related to their lifespan, and the enhancement of antioxidant capacity often accompanies the extension of lifespan. Hydrogen peroxide (H2O2) as an effective mimic can simulate the process of apoptosis triggered by free radicals, so it plays a key role in preparing cell oxidative damage models. Paraquat (PQ) can induce oxidative stress in cells, produce excess ROS, and cause mitochondrial dysfunction, which makes it widely used in the study of mitochondrial oxidative damage. At the same time, ultraviolet B (UVB) exposure is the main exogenous factor leading to photoaging. UVB radiation can directly penetrate the atmosphere and interact with cells in the skin, producing a large number of free radicals and active oxygen molecules, causing oxidative stress in cells and accelerating the skin aging process. There is a positive intrinsic relationship between the ability to withstand stress and the extension of lifespan. Active substances can improve the heat stress resistance of nematodes at 35°C by regulating heat shock proteins, thereby extending the lifespan of nematodes.

[0196] Therefore, by treating nematodes with samples, then placing them in the corresponding stress conditions, and recording the survival status of nematodes, the potential anti-aging effect of grape pomace oil can be evaluated.

[0197] (1) H2O2-induced oxidative stress damage experiment

[0198] After the nematodes were synchronized, L4 larvae were picked and transferred to blank and sample group media. The sample group was fed with grape pomace oil at a concentration of 1 mg / mL for 3 days. Then, 30-50 nematodes were picked from each medium and exposed to H2O2-containing NGM medium to induce oxidative stress intervention. Three parallel plates were set up, and the number of surviving, escaping, and dead nematodes was recorded every 30 minutes until all nematodes died. The survival curve of nematodes under H2O2 oxidative stress was obtained, with 60 μM astaxanthin as a positive control.

[0199] The results are shown in Figure 3 Compared with the blank group, the survival curve of the grape pomace oil group was significantly right-shifted (P<0.0001). The average lifespan of the blank group was 1.53 h, while the average lifespan of the grape pomace oil group and the astaxanthin group was 2.03 h and 2.00 h, respectively, which was 33.09% and 30.86% higher than that of the blank group, respectively. This indicated that grape pomace oil significantly improved the stress resistance of nematodes in H2O2 oxidative stress damage and effectively extended the lifespan of nematodes. At the same time, the anti-aging activity of 1 mg / mL grape pomace oil was not significantly different from that of 60 μM astaxanthin (P>0.05), indicating that its anti-aging activity was good.

[0200] (2) PQ-induced oxidative stress damage experiment

[0201] After synchronization of nematodes, L4 larvae were picked and transferred to blank and sample groups of medium, and after 3 days of drug action, 50-60 nematodes were picked from each medium and exposed to PQ-containing NGM medium for oxidative stress induction intervention. Three parallel plates were set up, and the number of surviving, escaping, and dead nematodes was recorded every 24 hours or so until all nematodes died, obtaining the survival curve of nematodes under PQ oxidative stress environment.

[0202] The results are shown in Figure 4 Similar to the results of H2O2 oxidative stress experiment, compared with the blank group, the survival curve of the drug group was significantly right-shifted (P<0.001), indicating that grape pomace oil can prolong the survival time of nematodes under PQ oxidative damage. Among them, the average lifespan of the blank group was 2.39d, while the average lifespan of the grape pomace oil group and the astaxanthin group was 2.88d and 3.43d, respectively, and the average survival time was increased by 20.37% and 43.30%, respectively, further indicating that grape pomace oil can effectively enhance the resistance of nematodes to oxidative stress and prolong the lifespan of nematodes.

[0203] (3) Heat-induced stress damage experiment

[0204] The wild-type C. elegans in the reproductive stage (3-5 days old) were collected for synchronization. After synchronization of nematodes, they were transferred to NGM containing / without drugs for 5 days at 20°C, and the nematodes were transferred to new plates every day during the period. On the 6th day of the adult stage, the nematodes were placed in a 35°C incubator for 12h, and then placed in a 20°C incubator for 12h, and the survival rate of nematodes was observed and recorded. Three parallel experiments were set up, and the number of nematodes in each treatment group was not less than 120.

[0205] As can be seen from Figure 5 , the survival rate of nematodes in the blank group was significantly lower than that in the other two groups, only 18.89%, which indicates that without treatment, heat stress has a negative impact on the survival of nematodes. The survival rate of nematodes in the grape pomace oil treatment group was significantly increased by 38.89 percentage points (P<0.05) compared with the blank group, indicating that grape pomace oil can improve the survival rate of nematodes under heat stress. At the same time, the survival rate of nematodes in the positive control group was slightly lower than that in the grape pomace oil treatment group, but the difference between the two groups was not statistically significant. This indicates that grape pomace oil has the effect of improving the survival rate of nematodes under heat stress and has potential advantages in resisting heat aging.

[0206] (4) Light-induced stress damage experiment

[0207] Wild type C. elegans at reproductive stage (3-5 days old) were collected for synchronization. After synchronization, the worms were transferred to NGM with / without drugs for 3 days. The worms in each group were collected, washed with M9 buffer for 3 times, and then transferred to NGM without OP50. The NGM plate with worms was placed directly below the UVB lamp of the irradiator, one dish at a time, and the irradiation dose was set to 250 mJ / cm 2 Ultraviolet irradiation was performed. After irradiation, the worms in each group were transferred to OP50 NGM containing FUDR and continued to be placed in a 20℃ incubator. Every 12 hours, the dead worms were picked out, and the number of dead and surviving worms was recorded to obtain the survival curve of the worms, as shown in Figure 6 The experiment was set up in triplicate, and the number of worms in each treatment group was not less than 120.

[0208] As shown in Figure 6 , the survival rate of the worms in the blank group rapidly decreased under light stress, and the survival rate was 0% on the 4th day. In contrast, the worms in the grape pomace oil group and the astaxanthin group showed significantly improved survival rate under light stress, and the survival curves were significantly right-shifted (P<0.001). The average lifespan of the blank group was 2.63 days, while the average lifespan of the grape pomace oil group and the astaxanthin group was 2.87 days and 3.05 days, respectively, and the average survival time was prolonged by 9.19% and 16.11%, respectively. Therefore, the grape pomace oil has a good promoting effect on the survival ability of the worms in the UVB irradiation environment, and confirms the anti-aging activity thereof.

[0209] Conclusion: The results show that the grape pomace oil prepared by the method has excellent anti-aging effect.

[0210] In summary, the acid heat pretreatment method is used in combination with the low-temperature continuous phase change extraction equipment in the embodiments of the present application, and the active ingredients of the grape pomace oil prepared by extraction have high content, good in-vitro antioxidant activity, are rich in anti-aging active ingredients, and have anti-aging effect. The comprehensive utilization of the oil in the grape pomace is realized, which is green, safe, efficient, and suitable for industrialized production and application.

[0211] The content and in-vitro antioxidant activity of the three active ingredients (total phenols, total flavonoids, and total sterols) measured by the method of the present application are excellent, which indicates the effectiveness and feasibility of the innovative method. The new extraction method provided in the present application can improve the anti-aging activity of the extract without sacrificing the quality of the oil. Through the analysis of the characteristic components of the extract, the product of the innovative extraction method can be comprehensively understood, and when applied to cosmetics, the anti-aging effect of the cosmetics can be effectively improved.

[0212] The above descriptions are only the preferred embodiments of the present application, and are not intended to limit the present application. The present application can have various modifications and changes for those skilled in the art. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. An anti-aging cosmetic, characterized in that, include: Wine lees oil; The characteristic components of the wine meal oil include: oleanolic acid, α-guaiacene, β-tocotrienol, β-sitosterol, campesterol, soybean saponin E, ganoderic acid F, ganoderol I, and D-erythro-dihydrosphingosine; the total phenol content of the wine meal oil is ≥1 mg / g, the total sterol content of the wine meal oil is ≥26 mg / g, and the total flavonoid content of the wine meal oil is ≥1 mg / g. The wine lees oil is obtained by extracting wine lees; the wine lees consist of grape skins, grape stems and grape seeds. The preparation of the wine lees oil includes: The wine lees powder was hot-soaked in an acid solution to obtain the first liquid. The first feed solution was subjected to continuous phase change extraction at 0.2 MPa~0.8 MPa and 40℃~50℃; The continuous phase change extraction includes: n-Butane was used as the extractant for continuous cyclic phase change extraction in liquid, gas, and liquid phases. The acid solution is an alcoholic solution of an acid.

2. The anti-aging cosmetic according to claim 1, characterized in that, The peroxide value of the wine lees oil is ≤7 mmol / Kg.

3. The anti-aging cosmetic according to claim 1, characterized in that, The acid value of the wine lees oil is ≤4 mg / g.

4. A method for preparing wine lees oil, characterized in that, The preparation method includes: The wine lees powder was hot-soaked in an acid solution to obtain the first liquid. The first feed solution was subjected to continuous phase change extraction at 0.2 MPa~0.8 MPa and 40℃~50℃; The wine lees powder is composed of grape skins, grape stems and grape seeds; The continuous phase change extraction includes: n-Butane was used as the extractant for continuous cyclic phase change extraction in liquid, gas, and liquid phases. The acid solution is an alcoholic solution of an acid.

5. The method for preparing wine meal oil according to claim 4, characterized in that, The continuous phase change extraction includes multiple extraction cycles, and one extraction cycle includes: The extractant gas is compressed into an extractant liquid in a vacuum, and the extractant liquid is allowed to flow through the first feed liquid for extraction; the extracted wine lees oil and acid solution flow out with the extractant liquid to obtain a mixture; the extractant liquid is converted into extractant gas and separated from the mixture; the separated extractant gas is used to repeat the extraction cycle.

6. The method for preparing wine meal oil according to claim 5, characterized in that, After separating the extractant gas, the acid solution is removed from the mixture to obtain a crude extract.

7. The method for preparing wine meal oil according to claim 6, characterized in that, The crude extract was subjected to a second extraction.

8. The method for preparing wine meal oil according to claim 6, characterized in that, The crude extract was subjected to a second extraction using n-hexane.

9. The method for preparing wine meal oil according to claim 4, characterized in that, The acid solution is a citric acid-ethanol solution.

10. The method for preparing wine meal oil according to claim 9, characterized in that, The citric acid-ethanol solution comprises, by mass percentage, 1% to 5% citric acid and 95% to 99% ethanol.

11. The method for preparing wine lees oil according to claim 4, characterized in that, The process of hot soaking wine lees powder in an acid solution includes: By mass ratio, wine lees powder and acid solution are hot-soaked at a ratio of (1:1) to (1:3).

12. The method for preparing wine meal oil according to claim 4, characterized in that, The process of hot soaking wine lees powder in an acid solution includes: The wine lees powder is soaked in an acid solution at 40°C to 50°C.

13. The method for preparing wine lees oil according to claim 4, characterized in that, Soaking time is 1 h to 3 h.

14. A wine lees oil, characterized in that, It is obtained by the method for preparing wine lees oil according to any one of claims 4-13.

Images