Anti-aging cosmetic, grape wine pomace oil, and preparation method therefor

Abstract

Disclosed are an anti-aging cosmetic, a grape wine pomace oil, and a preparation method therefor. The anti-aging cosmetic comprises grape wine pomace oil. The characteristic components of the grape wine pomace oil comprise: oleanolic acid, α-guaiene, β-tocotrienol, β-sitosterol, campesterol, soyasapogenol E, ganoderic acid F, ganoderiol I, and D-erythro-dihydrosphingosine. The grape wine pomace oil has a total phenol content of ≥ 1 mg / g, a total sterol content of ≥ 26 mg / g, and a total flavonoid content of ≥ 1 mg / g. The grape wine pomace oil is obtained by extraction from grape wine pomace. The grape wine pomace is composed of grape skins, grape stems, and grape seeds.

Description

Anti-aging cosmetics, wine lees oil and their preparation methods

[0001] Cross-references to related applications

[0002] This application claims priority to Chinese Patent Application No. 2025103874250, filed on March 28, 2025, entitled "Anti-aging cosmetics, wine lees oil and preparation method thereof", the entire contents of which are incorporated herein by reference. Technical Field

[0003] This disclosure relates to the field of cosmetics, and more specifically, to an anti-aging cosmetic, wine lees oil, and a method for preparing the same. Background Technology

[0004] With the development of the grape winemaking industry, the output of wine lees is increasing, mainly consisting of grape skins, stems, and seeds. Currently, the main way to utilize wine lees is to sort out the grape seeds and then produce grape seed oil. However, the remaining grape skins and stems are often processed inefficiently and extensively, such as being made into low-value fertilizers or feed, or directly used for landfill, which leads to resource waste and environmental pollution. Summary of the Invention

[0005] The purpose of this disclosure is to provide an anti-aging cosmetic, wine lees oil, and a method for preparing the same.

[0006] In a first aspect, this disclosure provides an anti-aging cosmetic product, comprising:

[0007] Wine meal oil; the characteristic components of 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 wine meal oil is ≥1mg / g, the total sterol content of wine meal oil is ≥26mg / g, and the total flavonoid content of wine meal oil is ≥1mg / g;

[0008] Wine lees oil is extracted from wine lees; wine lees mainly consist of grape skins, grape stems and grape seeds.

[0009] In the above technical solution, the characteristic components of wine lees oil are: oleanolic acid, α-guaiacol, β-tocotrienol, β-sitosterol, campesterol, soybean saponin E, ganoderic acid F, ganoderol I, and D-erythro-dihydrosphingosine. These compounds possess excellent antioxidant properties and can serve as anti-aging active ingredients in anti-aging cosmetics, thereby greatly enhancing the anti-aging effects of these cosmetics. Furthermore, this wine lees oil exhibits anti-aging efficacy comparable to astaxanthin in a nematode model, indicating that it helps protect the skin from oxidative damage and plays a positive role in cosmetics. This wine lees oil retains the active ingredients of each part of the wine lees to the greatest extent possible.

[0010] In other embodiments of this disclosure, the peroxide value of the wine lees oil is ≤7 mmol / Kg.

[0011] In other embodiments of this disclosure, the acid value of wine lees oil is ≤4 mg / g.

[0012] Secondly, this disclosure provides a method for preparing wine lees oil, the method comprising:

[0013] The wine lees powder was hot-soaked in an acid solution to obtain the first liquid.

[0014] The first feed solution was subjected to continuous phase change extraction at 0.2MPa~0.8MPa and 40℃~50℃.

[0015] The above technical solution involves continuous phase change extraction of the first feed solution at 0.2MPa–0.8MPa and 40℃–50℃, enabling rapid liquid-gas-liquid circulation of the solvent. This circulation and fresh solvent ensure thorough and effective extraction of the material. Furthermore, the solvent is recovered under vacuum, preventing solvent residue and environmental pollution, thus achieving low-cost, green extraction.

[0016] In other embodiments of this disclosure, continuous phase change extraction includes:

[0017] n-Butane was used as the extractant for continuous cyclic phase change extraction in liquid, gas, and liquid phases.

[0018] In other embodiments of this disclosure, continuous phase change extraction includes multiple extraction cycles, one extraction cycle including:

[0019] The extractant gas is compressed into an extractant liquid in a vacuum, and the extractant liquid flows 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 for repeated extraction cycles;

[0020] Optionally, after separating the extractant gas, the acid solution is removed from the mixture to obtain a crude extract;

[0021] Optionally, the crude extract may be subjected to a second extraction;

[0022] Optionally, the crude extract may be subjected to a second extraction using n-hexane.

[0023] In other embodiments of this disclosure, the acid solution is an alcoholic solution of an acid;

[0024] Optionally, the acid solution is a citric acid-ethanol solution;

[0025] Optionally, the citric acid-ethanol solution comprises 1% to 5% citric acid and 95% to 99% ethanol by mass percentage.

[0026] In other embodiments of this disclosure, the wine lees powder is hot-soaked in an acid solution, including:

[0027] By mass ratio, wine lees powder and acid solution are hot-soaked at a ratio of (1:1) to (1:3).

[0028] In other embodiments of this disclosure, the wine lees powder is hot-soaked in an acid solution, including:

[0029] Soak wine lees powder in an acid solution at 40℃~50℃;

[0030] Optionally, the soaking time is 1 hour to 3 hours.

[0031] Thirdly, this disclosure provides a wine meal oil, which is obtained by the wine meal oil preparation method provided in the second aspect above. Attached Figure Description

[0032] To more clearly illustrate the technical solutions of the embodiments of this disclosure, the accompanying drawings used in the embodiments will be briefly described below. It should be understood that the following drawings only show some embodiments of this disclosure and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0033] Figure 1 shows the total ion chromatogram of the wine meal oil prepared in Example 1, analyzed by gas chromatography-mass spectrometry.

[0034] Figure 2 shows the total ion chromatogram of the wine meal oil prepared in Example 1, analyzed by liquid chromatography-mass spectrometry.

[0035] Figure 3 shows the effect of wine meal oil prepared in Example 1 on the survival curve of nematodes under H2O2 oxidative stress.

[0036] Figure 4 shows the effect of wine meal oil prepared in Example 1 on the survival curve of nematodes under PQ oxidative stress.

[0037] Figure 5 shows the effect of wine meal oil prepared in Example 1 on the survival curve of nematodes under heat stress.

[0038] Figure 6 shows the effect of wine lees oil prepared in Example 1 on the survival curve of nematodes under light stress. Detailed Implementation

[0039] To make the objectives, technical solutions, and advantages of the embodiments of this disclosure clearer, the technical solutions in the embodiments of this disclosure will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0040] Research has revealed that only a small amount of free phenols dissolve in grape seed oil during production, with the majority of active compounds remaining in the grape meal. Further studies have found that grape skins and stems are also rich in various anti-cancer, antibacterial, and antioxidant active ingredients, such as polyphenols, flavonoids, and terpenes, indicating broad application prospects and significant development value. Therefore, there is room for research into how to further increase the content of active ingredients in grape seed oil, and how to utilize the remaining grape skins and seeds in winemaking pomace in a high-value, resource-efficient manner, and how to extract winemaking pomace in a green and efficient one-time process are issues worthy of investigation.

[0041] Traditional methods such as Soxhlet extraction, maceration, and reflux extraction require the use of large amounts of potentially toxic solvents, and the required extraction temperatures can lead to the destruction and loss of active substances. Ultrasonic-assisted extraction, microwave-assisted extraction, and supercritical fluid extraction effectively improve yield and retention, but they consume large amounts of energy and are costly. Aqueous enzymatic extraction, as an environmentally friendly and efficient method for oil extraction, has attracted attention because it does not produce solvent residues, environmental pollution, or safety issues during the extraction process. However, the high cost of commercial enzyme preparations limits its industrialization in practical applications.

[0042] This disclosure provides an anti-aging cosmetic product, comprising:

[0043] Wine meal oil; the characteristic components of 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 wine meal oil is ≥1mg / g, the total sterol content of wine meal oil is ≥26mg / g, and the total flavonoid content of wine meal oil is ≥1mg / g;

[0044] Wine lees oil is extracted from wine lees; wine lees mainly consist of grape skins, grape stems and grape seeds.

[0045] In the above technical solution, the characteristic components of wine lees oil are: oleanolic acid, α-guaiacol, β-tocotrienol, β-sitosterol, campesterol, soybean saponin E, ganoderic acid F, ganoderol I, and D-erythro-dihydrosphingosine. These compounds possess excellent antioxidant properties and can serve as anti-aging active ingredients in anti-aging cosmetics, thereby significantly enhancing the anti-aging effects of these products. Furthermore, this wine lees oil exhibited anti-aging efficacy comparable to 60 μM astaxanthin in a nematode model, indicating its ability to help protect the skin from oxidative damage and play a positive role in cosmetics. This wine lees oil retains the active ingredients of each part of the wine lees to the greatest extent possible.

[0046] For example, in some embodiments of this disclosure, the total phenol content of wine lees 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.

[0047] For example, in some embodiments of this disclosure, the total sterol content of wine lees 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 foregoing values.

[0048] For example, in some embodiments of this disclosure, the total flavonoid content of wine lees 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.

[0049] In the above technical solution, the definition of "characteristic components" of wine lees oil is: the relative content of which is greater than that of potential anti-aging active ingredients, as determined by gas chromatography-mass spectrometry or liquid chromatography-mass spectrometry.

[0050] The aforementioned "potential" is because some components have been reported in the literature for their antioxidant effects (aging is caused by continuous oxidative damage), but have not been directly verified by anti-aging experiments.

[0051] For example, some literature reports that oleanolic acid can inhibit hepatocyte damage caused by oxidative stress by increasing the activity of antioxidant enzymes (such as superoxide dismutase, catalase, and glutathione peroxidase) and the level of reduced glutathione, thereby inhibiting the generation of reactive oxygen species (ROS) and scavenging free radicals. These effects of oleanolic acid may be beneficial for anti-aging, meaning that oleanolic acid is a "potential" anti-aging active ingredient.

[0052] It should be noted that the wine lees oil mentioned above also contains some other active ingredients. However, no literature has reported that these active ingredients have antioxidant or anti-aging activities, and therefore they are not considered as "characteristic components" of the wine lees oil mentioned above. Exemplarily, in some embodiments of this disclosure, the wine lees oil mentioned above is analyzed by gas chromatography-mass spectrometry (GC-MS) to determine oleanolic acid (11.6108%), α-guaiacol (4.5109%), β-tocotrienol (2.8166%), β-sitosterol (7.0947%), and campesterol (1.3126%), which are considered as "characteristic components" of the wine lees oil; and soybean saponin E (7.5149%), ganoderic acid F (3.2038%), ganoderic alcohol I (1.7824%), and D-erythro-dihydrosphingosine (20.1058%) are analyzed by liquid chromatography-mass spectrometry (LC-MS) and are considered as "characteristic components" of the wine lees oil.

[0053] Furthermore, in some embodiments of this disclosure, the peroxide value of wine lees oil is ≤7 mmol / Kg.

[0054] Peroxide value is an important indicator for measuring the degree of oxidation of oils and fats. A lower peroxide value indicates better oxidative stability of the oils and fats; it is generally required to be less than or equal to 10 mmol / Kg. In the above technical solution, wine lees oil has a peroxide value ≤7 mmol / Kg, exhibiting excellent oxidative stability. Therefore, its application in anti-aging cosmetics can effectively enhance the anti-aging efficacy of the cosmetics.

[0055] For example, in some embodiments of this disclosure, the peroxide value of wine lees 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.

[0056] In some embodiments of this disclosure, the method for determining peroxide value is as follows:

[0057] Weigh 2-3 g of the prepared sample and place it in a 250 mL iodine flask. Add 30 mL of chloroform-glacial acetic acid solution and gently shake until the sample is completely dissolved. Accurately add 1.00 mL of saturated potassium iodide solution, tighten the cap, and gently shake for 0.5 min. Place in the dark for 3 min. Remove the flask, add 100 mL of water, shake well, and immediately titrate the precipitated iodine with sodium thiosulfate standard titration solution. When the solution turns pale yellow, add 1 mL of starch indicator, continue titrating, and shake vigorously until the blue color disappears. Simultaneously perform a blank test. The volume of sodium thiosulfate standard titration solution consumed in the blank test is V0. The peroxide value calculation formula is as follows:

[0058] Furthermore, in some embodiments of this disclosure, the acid value of wine lees oil is ≤4 mg / g.

[0059] Acid value is an indicator for assessing the free fatty acid content in oils and fats. A lower acid value means that the oil and fat has a lower degree of rancidity and is of higher quality; it usually needs to be less than or equal to 5 mmol / Kg. In the above technical solution, the acid value of wine lees oil is ≤4mg / g, indicating a lower degree of rancidity and higher quality. Therefore, its application in anti-aging cosmetics can effectively improve the anti-aging efficacy of the cosmetics.

[0060] For example, in some embodiments of this disclosure, the acid value of wine lees 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 aforementioned values.

[0061] In some embodiments of this disclosure, the method for determining acid value is as follows:

[0062] Weigh the oil sample into a 250mL Erlenmeyer flask, add 50-100mL of diethyl ether-isopropanol mixture and 3-4 drops of phenolphthalein indicator, shake thoroughly to dissolve the sample, and then titrate with standard titrant. The endpoint is reached when the sample solution initially turns a faint pink color and shows no obvious fading within 15 seconds. Record the volume of standard titrant consumed in this titration, denoted as V. In a separate clean 250mL Erlenmeyer flask, accurately add the same volume and type of organic solvent mixture and indicator as used in the sample determination, shake to mix, and titrate. The volume of standard titrant consumed in this titration is V0. The acid value calculation formula is as follows:

[0063] This disclosure provides a method for preparing wine lees oil, the method comprising:

[0064] The wine lees powder was hot-soaked in an acid solution to obtain the first liquid.

[0065] The first feed solution was subjected to continuous phase change extraction at 0.2MPa~0.8MPa and 40℃~50℃.

[0066] The above technical solution involves continuous phase change extraction of the first feed solution at 0.2MPa–0.8MPa and 40℃–50℃, enabling rapid liquid-gas-liquid circulation of the solvent. This circulation and fresh solvent ensure thorough and effective extraction of the material. Furthermore, solvent recovery under vacuum prevents solvent residue and environmental pollution, achieving low-cost, green extraction. This technical solution addresses the current problems of insufficient and incomplete extraction of active ingredients from wine lees, resulting in waste or low-value utilization of some wine lees. The wine lees oil extracted using this method retains the maximum amount of active ingredients from each part of the wine lees. Simultaneously, this method must be green, efficient, low-cost, environmentally friendly, and energy-efficient.

[0067] For example, in some embodiments of this disclosure, 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 any two of the aforementioned values; and at 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 any two of the aforementioned values.

[0068] In the above technical solution, the first feed solution is subjected to continuous phase change extraction at 0.2MPa~0.8MPa and 40℃~50℃.

[0069] Furthermore, in some embodiments of this disclosure, continuous phase change extraction includes:

[0070] n-Butane was used as the extractant for continuous cyclic phase change extraction in liquid, gas, and liquid phases.

[0071] n-Butane is an organic compound with the chemical formula C4H. 10 It is a common alkane, a colorless, easily liquefied gas at room temperature and pressure. It enables liquid-gas-liquid circulation (i.e., continuous phase change extraction) during continuous phase change extraction, and the circulation and fresh solvent ensure sufficient and effective extraction of the material, thereby increasing the content of active ingredients in the extracted wine lees oil.

[0072] Furthermore, in some embodiments of this disclosure, continuous phase change extraction includes multiple extraction cycles, one extraction cycle including:

[0073] The extractant gas is compressed into an extractant liquid in a vacuum, and the extractant liquid flows 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 for repeated extraction cycles.

[0074] Furthermore, in some embodiments of this disclosure, after separating the extractant gas, the acid solution is removed from the mixture to obtain a crude extract.

[0075] Furthermore, in some embodiments of this disclosure, the crude extract is subjected to a second extraction.

[0076] Furthermore, in some embodiments of this disclosure, the crude extract is subjected to a second extraction using n-hexane.

[0077] Furthermore, in some embodiments of this disclosure, the acid solution is an alcoholic solution of an acid.

[0078] The above technical solution uses an acid solution to soak wine lees powder, which deacidifies the powder and breaks down the tight structure of the cell walls. Then, an organic solvent is used to extract the oils from the cells. Simultaneously, phenolic compounds are more easily dissolved and retained under acidic conditions, promoting their further extraction into the oil phase.

[0079] Furthermore, in some embodiments of this disclosure, the acid solution is a citric acid-ethanol solution.

[0080] Furthermore, in some embodiments of this disclosure, the citric acid-ethanol solution comprises, by mass percentage, 1% to 5% citric acid and 95% to 99% ethanol.

[0081] By way of example, in some embodiments of this disclosure, the citric acid-ethanol solution comprises, by mass percentage, 1%, 1.2%, 1.5%, 2%, 2.5%, 2.8%, 3%, 3.5%, 3.8%, 4%, 4.2%, 4.5%, 4.8%, 5% or any two of the aforementioned values ​​of citric acid; and 95%, 96%, 97%, 98%, 99% or any two of the aforementioned values ​​of ethanol.

[0082] Furthermore, in some embodiments of this disclosure, the wine lees powder is subjected to hot soaking in an acid solution, including:

[0083] By mass ratio, wine lees powder and acid solution are hot-soaked at a ratio of (1:1) to (1:3).

[0084] For example, in some embodiments of this disclosure, wine lees powder and 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 any two of the aforementioned values, by mass ratio.

[0085] Furthermore, in some embodiments of this disclosure, the wine lees powder is subjected to hot soaking in an acid solution, including:

[0086] Soak wine lees powder in an acidic solution at 40℃~50℃.

[0087] Exemplary, in some embodiments of this disclosure, the wine lees powder is hot-soaked in an acid solution, including:

[0088] The wine lees powder is soaked in an acid solution at 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, or any two of the aforementioned values.

[0089] Furthermore, in some embodiments of this disclosure, the soaking time is 1 hour to 3 hours.

[0090] For example, in some embodiments of this disclosure, 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.

[0091] This disclosure provides a wine meal oil according to some embodiments, which is obtained by the wine meal oil preparation method provided in any of the foregoing embodiments.

[0092] The features and performance of this disclosure will be further described in detail below with reference to embodiments:

[0093] Example 1

[0094] A wine lees oil is provided, prepared according to the following steps:

[0095] S1. The dried wine lees are crushed and sieved to obtain wine lees powder with a particle size of 40 mesh. Wine lees consists of grape skins, grape stems and grape seeds.

[0096] S2. Weigh 1000g of the wine lees powder obtained in step S1 and fill it into the 3L extraction vessel of a continuous phase change extraction apparatus. Add 2L of a 5% (w / w) citric acid-ethanol solution and soak at 50℃ for 120min. Then, use n-butane as the extraction solvent and extract under the following conditions: extraction pressure 0.8MPa, extraction temperature 50℃, desorption temperature 60℃, and flow rate 60L / h. The n-butane is compressed into a liquid under vacuum and flows through the extraction vessel to extract the wine lees. The wine lees oil and citric acid-ethanol solution flow into the desorption vessel along with the n-butane liquid. The n-butane liquid is heated and depressurized to become a gas, which is separated from the wine lees oil and recovered to the storage tank. The above process is continuously cyclical for a total of 120min of extraction. Finally, the crude extract is obtained in the desorption vessel.

[0097] S3. The crude extract obtained in step S2 is subjected to rotary evaporation to remove the ethanol solution, and then added to a separatory funnel for extraction with n-hexane. The extract is then subjected to rotary evaporation to obtain wine lees oil.

[0098] Example 2

[0099] A wine lees oil is provided, prepared according to the following steps:

[0100] S1. The dried wine lees are crushed and sieved to obtain wine lees powder with a particle size of 40 mesh. Wine lees consists of grape skins, grape stems and grape seeds.

[0101] S2. Weigh 1000g of the wine lees powder obtained in step S1 and fill it into the 3L extraction vessel of a continuous phase change extraction apparatus. Add 3L of a 1% (w / w) citric acid-ethanol solution and soak at 45°C for 90 min. Then, use n-butane as the extraction solvent and extract under the following conditions: extraction pressure 0.5 MPa, extraction temperature 45°C, desorption temperature 55°C, and flow rate 60 L / h. The n-butane is compressed into a liquid under vacuum and flows through the extraction vessel to extract the wine lees. The wine lees oil and citric acid-ethanol solution flow into the desorption vessel along with the n-butane liquid. The n-butane liquid is then heated and depressurized to become a gas, separating it from the wine lees oil and recovering it to a storage tank. This process is continuously cyclical for a total extraction time of 60 min. Finally, the crude extract is obtained in the desorption vessel.

[0102] S3. The crude extract obtained in step S2 is subjected to rotary evaporation to remove the ethanol solution, and then added to a separatory funnel for extraction with n-hexane. The extract is then subjected to rotary evaporation to obtain wine lees oil.

[0103] Example 3

[0104] A wine lees oil is provided, prepared according to the following steps:

[0105] S1. The dried wine lees are crushed and sieved to obtain wine lees powder with a particle size of 40 mesh. Wine lees consists of grape skins, grape stems and grape seeds.

[0106] S2. Weigh 1000g of the wine lees powder obtained in step S1 and fill it into the 3L extraction vessel of a continuous phase change extraction apparatus. Add 1L of a 3% (w / w) citric acid-ethanol solution and soak at 40℃ for 60min. Then, use n-butane as the extraction solvent and extract under the following conditions: extraction pressure 0.2MPa, extraction temperature 40℃, desorption temperature 50℃, and flow rate 60L / h. The n-butane is compressed into a liquid under vacuum and flows through the extraction vessel to extract the wine lees. The wine lees oil and citric acid-ethanol solution flow into the desorption vessel along with the n-butane liquid. The n-butane liquid is heated and depressurized to become a gas, which is separated from the wine lees oil and recovered to the storage tank. The above process is continuously cyclical for a total extraction time of 90min; finally, the crude extract is obtained in the desorption vessel.

[0107] S3. The crude extract obtained in step S2 is subjected to rotary evaporation to remove the ethanol solution, and then added to a separatory funnel for extraction with n-hexane. The extract is then subjected to rotary evaporation to obtain wine lees oil.

[0108] Comparative Example 1

[0109] A wine lees oil is provided, prepared according to the following steps:

[0110] S1. The dried wine lees are crushed and sieved to obtain wine lees powder with a particle size of 40 mesh. Wine lees consists of grape skins, grape stems and grape seeds.

[0111] S2. Take 30g of crushed wine lees powder, add 300mL of n-hexane, stir and extract at 50℃ and normal pressure for 3h, filter, and remove n-hexane by rotary evaporation to obtain wine lees oil.

[0112] Comparative Example 2

[0113] A wine lees oil is provided, prepared according to the following steps:

[0114] S1. The dried wine lees are crushed and sieved to obtain wine lees powder with a particle size of 40 mesh. Wine lees consists of grape skins, grape stems and grape seeds.

[0115] S2. Take 30g of wine lees powder pulverized to 40 mesh, add 60mL of 5% citric acid-ethanol solution, and soak at 50℃ for 2h. Then add 300mL of n-hexane and extract under normal pressure at 50℃ for 3h with stirring. Filter to obtain crude extract.

[0116] S3. The crude extract obtained in step S2 is subjected to rotary evaporation to remove the ethanol solution, and then added to a separatory funnel for extraction with n-hexane. The extract is then subjected to rotary evaporation to obtain wine lees oil.

[0117] Comparative Example 3

[0118] A wine lees oil is provided, prepared according to the following steps:

[0119] S1. The dried wine lees are crushed and sieved to obtain wine lees powder with a particle size of 40 mesh. Wine lees consists of grape skins, grape stems and grape seeds.

[0120] S2. Weigh 1000g of the wine lees powder obtained in step S1 and fill it into the 3L extraction vessel of a continuous phase change extraction apparatus. Use n-butane as the extraction solvent and extract for 120min at an extraction pressure of 0.8MPa, an extraction temperature of 50℃, a desorption temperature of 60℃, and a flow rate of 60L / h. The n-butane is compressed into a liquid under vacuum and flows through the extraction vessel to extract the wine lees. The wine lees oil and citric acid-ethanol solution flow into the desorption vessel along with the n-butane liquid. The n-butane liquid is then heated and depressurized to become a gas, separating it from the wine lees oil and recovering it to a storage tank. This process is continuously repeated for a total of 120min; finally, wine lees oil is obtained in the desorption vessel.

[0121] Comparative Example 4

[0122] A wine lees oil is provided, prepared according to the following steps:

[0123] S1. The dried wine lees are crushed and sieved to obtain wine lees powder with a particle size of 40 mesh. Wine lees consists of grape skins, grape stems and grape seeds.

[0124] S2. Weigh 1000g of the wine lees powder obtained in step S1 and fill it into the 3L extraction vessel of a continuous phase change extraction apparatus. Add 2L of a 5% (w / w) citric acid-ethanol solution and soak at room temperature for 120 min. Then, use n-butane as the extraction solvent and extract under the following conditions: extraction pressure 0.8 MPa, extraction temperature 50℃, desorption temperature 60℃, and flow rate 60 L / h. The n-butane is compressed into a liquid under vacuum and flows through the extraction vessel to extract the wine lees. The wine lees oil and citric acid-ethanol solution flow into the desorption vessel along with the n-butane liquid. The n-butane liquid is heated and depressurized to become a gas, which is separated from the wine lees oil and recovered to the storage tank. The above process is continuously cyclical for a total extraction time of 120 min; finally, wine lees oil is obtained in the desorption vessel.

[0125] S3. The crude extract obtained in step S2 is subjected to rotary evaporation to remove the ethanol solution, and then added to a separatory funnel for extraction with n-hexane. The extract is then subjected to rotary evaporation to obtain wine lees oil.

[0126] Experimental Example 1: Extraction rate, content of active ingredients, and in vitro antioxidant activity of wine lees oil

[0127] The technical effects of each embodiment and comparative example in this invention are compared, and the extraction rate and component content of wine lees oil are calculated.

[0128] (1) Calculation of yield: Wine meal oil extraction rate (%) = Wine meal oil mass (g) / Wine meal crude fat mass (g) × 100%

[0129] (2) Method for determining total phenol content:

[0130] Weigh 0.5g of oil and add 2ml of methanol-water solution (methanol:water = 90:10). Vortex for 5 minutes, centrifuge at 3000rpm / min for 5 minutes, and collect the supernatant. Repeat the above steps three times for each oil. Mix all extracts and concentrate to dryness. Dissolve the dry matter in 1ml of methanol-water solution (methanol:water = 10:90) to obtain polyphenol extract. Accurately pipette 0.5mL of extract, add distilled water to 5ml, add 1mL of Folin-Ciocateu reagent and 4mL of 10% Na2CO3 solution, shake well, and incubate at room temperature for 60 minutes for color development. Use distilled water as a blank test and measure the absorbance at 765nm. Calculate the polyphenol content according to the regression equation of the gallic acid standard curve. Repeat the measurement three times for each sample.

[0131] (3) Methods for determining sterol content:

[0132] Weigh 0.5 g of mercuric oxide, accurately add 2 mL of concentrated sulfuric acid and 10 mL of distilled water, and sonicate to dissolve. Measure 1 mL of this solution into a 100 mL volumetric flask and dilute to the mark with a mixture of glacial acetic acid and concentrated sulfuric acid (35:70) to obtain sulfomercuric acetate reagent. Accurately weigh 0.1 g of oil and dilute to the mark in a 10 mL volumetric flask with methanol. Take 2 mL of the resulting oil-methanol solution and dilute to the mark in a 10 mL volumetric flask with sulfomercuric acetate reagent. Measure the absorbance at 410 nm using methanol as a blank. Calculate the sterol content according to the regression equation of the sitosterol standard curve. Each sample was measured three times.

[0133] (4) Methods for determining flavonoid content:

[0134] Weigh 0.5g of oil and add 2ml of methanol-water solution (methanol:water = 90:10). Vortex for 5 minutes, centrifuge at 3000rpm / min for 5 minutes, collect the supernatant, and repeat the above steps. Perform the extraction procedure three times for each oil. Mix all extracts and concentrate to dryness. Dissolve the dry matter in 5ml of methanol-water solution (methanol:water = 10:90) to obtain the flavonoid extract. Pipette 1.0mL of the extract into a 25mL graduated test tube, add 1mL of 5% sodium nitrite solution, shake well, wait 6 minutes, then add 1mL of 10% Al(NO3)3 solution. After 6 minutes, add 10mL of 4% NaOH solution, then add 60% ethanol solution to bring the volume to the mark. After standing for 15 minutes, measure the absorbance at 510nm. Use distilled water as a blank control and rutin standard as a standard curve. Measure each sample three times.

[0135] (5) Determination of DPPH free radical scavenging rate

[0136] Weigh an appropriate amount of oil, dissolve it in anhydrous ethanol, and then dilute it to obtain sample solutions of different mass concentrations for antioxidant activity testing. A 样品 : Mix the test samples with different mass concentration gradients separately with DPPH working solution; A 空白 : Mix the test samples with anhydrous ethanol at different mass concentration gradients; A 对照 Mix the DPPH working solution with anhydrous ethanol. Incubate thoroughly and react at room temperature in the dark for 30 minutes. After the reaction, measure the absorbance at 517 nm using a UV spectrophotometer. Repeat the measurement three times for each sample. The formula for calculating the DPPH free radical scavenging rate is as follows:

[0137] The effects of different extraction methods on the extraction rate, total phenols, flavonoids, sterols, and in vitro antioxidant activity of wine meal oil were determined. The results are shown in Table 1.

[0138] Table 1 Extraction rate, active ingredient content, and DPPH free radical scavenging rate of wine lees oil

[0139] Note: When comparing items in the same column, different letters indicate a significant difference (p<0.05), while the same letters indicate no significant difference (p>0.05).

[0140] As shown in Table 1, the oil extraction rate of wine lees in the embodiments of the present invention is significantly higher than that in the comparative examples. Specifically, the oil extraction rate of Example 1 reached 96.70±1.27%, the highest among all experimental groups. Meanwhile, the contents of polyphenols, sterols, and flavonoids in the wine lees oil extracted in the embodiments were all high, especially in Example 1, which showed the best results at 1.13±0.18, 26.29±2.01, and 1.40±0.42 mg / g, respectively, significantly higher than those in the comparative examples. At the same concentration, the DPPH free radical scavenging rate of Example 1 was 98.22±1.54%, significantly higher than that in the comparative examples, demonstrating excellent antioxidant properties. These results indicate that the method of the present invention can effectively retain the active ingredients in the oilseeds while extracting oil, and has high in vitro antioxidant activity. Acid-heat pretreatment combined with low-temperature continuous phase change extraction fully extracted the active ingredients from various parts of the wine lees and enriched them in the obtained wine lees oil.

[0141] Compared with Comparative Examples 1 and 2, Examples 1-3 showed better DPPH free radical scavenging rate, oil extraction rate, polyphenol, flavonoid, and sterol content.

[0142] Compared to Examples 1-3, Comparative Example 3 showed significantly lower extraction rates and DPPH free radical scavenging rates. Compared to Examples 1-3, Comparative Example 4 showed lower oil extraction rates, polyphenol content, flavonoid content, and sterol content.

[0143] Physicochemical analysis of wine meal oil in Experiment Example 2

[0144] (1) Method for determining peroxide value:

[0145] Weigh 2-3 g of the prepared sample and place it in a 250 mL iodine flask. Add 30 mL of chloroform-glacial acetic acid solution and gently shake until the sample is completely dissolved. Accurately add 1.00 mL of saturated potassium iodide solution, tighten the cap, and gently shake for 0.5 min. Place in the dark for 3 min. Remove the flask, add 100 mL of water, shake well, and immediately titrate the precipitated iodine with sodium thiosulfate standard titration solution. When the solution turns pale yellow, add 1 mL of starch indicator, continue titrating, and shake vigorously until the blue color disappears. Simultaneously perform a blank test. The volume of sodium thiosulfate standard titration solution consumed in the blank test is V0. The peroxide value calculation formula is as follows:

[0146] (2) Method for determining acid value:

[0147] Weigh the oil sample into a 250mL Erlenmeyer flask, add 50-100mL of diethyl ether-isopropanol mixture and 3-4 drops of phenolphthalein indicator, shake thoroughly to dissolve the sample, and then titrate with standard titrant. The endpoint is reached when the sample solution initially turns a faint pink color and shows no obvious fading within 15 seconds. Record the volume of standard titrant consumed in this titration, denoted as V. In a separate clean 250mL Erlenmeyer flask, accurately add the same volume and type of organic solvent mixture and indicator as used in the sample determination, shake to mix, and titrate. The volume of standard titrant consumed in this titration is V0. The acid value calculation formula is as follows:

[0148] (3) Method for determining citric acid content:

[0149] Prepare seven 10mL centrifuge tubes and add 0.5mL of 0.4mol / L nitric acid solution and 2mL of 5.0×10⁻⁶ nitric acid solution to each tube. -4 Six flasks were prepared using a mixture of mol / L phenol red solution, 0.1 mL of 10 mg / mL iron(III) solution, and 0.1 mL of 5% hydrogen peroxide solution. One 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, and 1.00 mg / mL) was added to each flask. The solution was diluted to 8 mL with distilled water, shaken well, and heated in an 80°C water bath for 8 min. The flasks were then rapidly cooled in 4°C water for 3 min. Using a flask without citric acid as a reference, the absorbance A and A0 of the inhibition and catalytic systems were measured at 440 nm. The standard curve regression equation was calculated to determine the sterol content. After water extraction of the oil samples, the citric acid content in the extract was calculated using the same experimental procedures.

[0150] The effects of different extraction methods on the physicochemical properties of wine meal oil were determined, and the results are shown in Table 2.

[0151] Table 2. Peroxide value, acid value, and citric acid residue of wine lees oil

[0152] Peroxide value is an important indicator for measuring the degree of oxidation of oils and fats; a lower peroxide value indicates better oxidative stability. Acid value is an indicator for assessing the free fatty acid content in oils and fats; a lower acid value means lower rancidity and higher quality. The wine lees oil obtained in this embodiment has low peroxide value and acid value, far below the requirements of GB / T 29990-2013 skin oil standard (peroxide value ≤ 10 mmol / kg; acid value ≤ 5 mmol / kg). Furthermore, it shows a significant decrease compared to the comparative example. The peroxide value and acid value in Comparative Example 1 were both high, indicating that solvent extraction promotes oil oxidation to some extent, resulting in poor oil quality. Meanwhile, the residual citric acid in the wine lees oil obtained in this embodiment is also within the standard range, and literature has shown that its small amount in oils can act as an antioxidant. Therefore, the oil extraction method of this disclosure effectively controls the degree of oxidation and rancidity of oils and fats while ensuring high extraction rate and retention rate of active ingredients, thus providing a high-quality, high-stability oil extraction solution.

[0153] The above-mentioned physicochemical indicators demonstrate that the extraction method disclosed herein is highly feasible, capable of improving the content of active ingredients and in vitro antioxidant activity without sacrificing oil quality. The extracted oil meets the standard of GB / T 29990-2013 for moisturizing oils.

[0154] The presence of a small amount of residual citric acid in this method indicates that although citric acid is used in this method, the extracted oil meets the citric acid content standard in GB / T29990-2013 for skin oils.

[0155] Example 3: Analysis of Characteristic Components in Wine Rice Oil

[0156] The wine lees oil obtained in Example 1 was analyzed for its distinctive components.

[0157] (1) Gas chromatography-mass spectrometry analysis

[0158] S1. Take 100 μL of sample and add 300 μL of extraction buffer (methanol:acetonitrile = 2:1 (containing 0.05 mg / mL internal standard ribitol)); vortex for 30 s to mix, then sonicate in an ice-water bath for 30 min; incubate the sample at -20℃ for 30 min, then centrifuge at high speed for 15 min (4℃, 13000 rcf); collect the supernatant from the centrifuged liquid and put it into a glass derivatization vial, then dry it with nitrogen; add 80 μL of methoxyamine hydrochloride pyridine solution (15 mg / mL) to the glass derivatization vial, vortex for 2 min, and then perform the oxime reaction in a shaking incubator at 37℃ for 90 min; then add 80 μL of BSTFA (containing 1% TMCS) derivatization reagent, vortex for 2 min, and then react at 70℃ for 60 min; after removing the sample, place it at room temperature for 30 min, and then perform GC-MS metabolomics analysis.

[0159] S2. Chromatographic conditions: After derivatization, the sample was injected into the GC-MS system in split mode for analysis, with an injection volume of 1 μL and a split ratio of 10:1. The sample was separated by a TG-5 SILMS capillary column (30 m × 0.25 mm × 0.25 μm, Thermo 26096-1420) before being detected by mass spectrometry. The injection port temperature was 300℃, the carrier gas was high-purity helium at a flow rate of 1.0 mL / min, and the septum purge flow was 3 mL / min. The temperature program was as follows: initial temperature 80℃, equilibration for 0 min, then increased to 310℃ at a rate of 20℃ / min and held for 8 min, for a total run time of 20 min, with a solvent delay of 2 min.

[0160] S3. Mass spectrometry conditions: Electron impact (EI) source, electron energy 70 eV, full scan mode, scan mass range 35–500 m / z, ion source temperature 250 °C.

[0161] The detection results are shown in Figure 1 and Table 3. Figure 1 shows the total ion chromatogram of wine lees oil analyzed by gas chromatography-mass spectrometry.

[0162] Table 3 Chemical components and relative contents of wine lees oil analyzed by gas chromatography-mass spectrometry.

[0163] Gas chromatography-mass spectrometry (GC-MS) analysis revealed 103 compounds across 12 categories in wine lees oil. These included 31 fatty acids and lipids, 14 amino acids, 8 sugars and their derivatives, 11 carboxylic acids, 3 aldehydes, 7 alcohols, 3 phenols and their derivatives, 3 flavonoids, 6 terpenes and terpene-related compounds, 5 sterols, 7 vitamins, and 5 other compounds. This demonstrates the rich bioactive components in wine lees oil.

[0164] It is worth noting that conjugated (9E,11E)-linoleic acid (20.3205%) and α-linolenic acid (6.6875%) were present in relatively high amounts. These polyunsaturated fatty acids can protect cells from oxidative damage by neutralizing free radicals and can improve the skin's moisturizing ability, thereby delaying skin aging. Another lipid substance with relatively high content is glyceryl monopalmitate (5.0188%), which, in addition to its anti-aging activity, is also commonly used as an emulsifier and emollient in cosmetics, providing moisturizing and nourishing effects to the skin. Among terpenes and terpene-related compounds, oleanolic acid (11.6108%) was present in relatively high amounts and has significant anti-aging and antioxidant effects, which can improve and enhance dermal collagen. α-guaiacene (4.5109%) followed, although no reports on its anti-aging activity have been found, its antibacterial and anti-inflammatory activities are widely recognized. Regarding phytosterols, wine lees oil is rich in β-sitosterol (7.0947%) and campesterol (1.3126%), representing high-value active ingredients in grape seeds and endowing wine lees oil with excellent anti-aging effects. Another representative high-value active ingredient is vitamin E. In the wine lees oil prepared in this invention, the main configuration is β-tocotrienol (2.8166%), while also containing various other tocopherol configurations, laying the foundation for wine lees oil as an effective antioxidant.

[0165] Meanwhile, the results also revealed that the wine lees oil contains various polyphenols and flavonoids, including protocatechuic acid, salicylic acid, caffeic acid, catechins, kaempferol, and gallocatechins, with a total relative content of 0.5074%. Furthermore, other high-value anti-aging active ingredients were identified, such as squalene (0.0079%), inositol (0.0449%), phytosterols (0.0740%), L-erythrosine (0.5239%), and lupeol (0.2087%). These substances are all excellent antioxidants that can exert anti-aging effects in the body or skin. Some also possess anti-inflammatory activity, making them suitable raw materials for high-end cosmetics. Therefore, the wine lees oil prepared by this invention has multiple potential health benefits and significant application value in health foods, cosmetics, and other fields.

[0166] (2) Liquid chromatography-mass spectrometry analysis

[0167] S1. Accurately transfer 10 μL of sample to a 1.5 mL centrifuge tube; add 400 μL of extraction buffer (methanol:water = 4:1 (v:v)) containing four internal standards (L-2-chlorophenylalanine (0.02 mg / mL), etc.); vortex for 30 s, then perform low-temperature ultrasonic extraction for 10 min (5℃, 40 kHz); centrifuge for 15 min (13000 g, 4℃), and transfer the supernatant to a vial with an inner tube for analysis.

[0168] S2. Chromatographic conditions: The column was an ACQUITY UPLC HSS T3 (100 mm × 2.1 mm id, 1.8 μm; Waters, Milford, USA); mobile phase A was 95% water + 5% acetonitrile (containing 0.1% formic acid), mobile phase B was 47.5% acetonitrile + 47.5% isopropanol + 5% water (containing 0.1% formic acid), the injection volume was 3 μL, and the column temperature was 40℃.

[0169] S3. Mass Spectrometry Conditions: The sample was ionized by electrospray ionization, and mass spectrometry signals were acquired using 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.

[0170] The detection results are shown in Figure 2 and Table 4. Figure 2 shows the total ion chromatogram of wine lees oil analyzed by liquid chromatography-mass spectrometry.

[0171] Table 4. Chemical components and relative contents of wine lees oil analyzed by liquid chromatography-mass spectrometry.

[0172] Liquid chromatography-mass spectrometry (LC-MS) analysis revealed 123 compounds across 15 categories in wine lees oil. These included 20 phenols and their derivatives, 12 amino acids and their derivatives, 2 sugars, 20 terpenes and terpene-related compounds, 16 organic acids, 12 flavonoids, 6 alcohols, 19 fatty acids and lipids, 3 glycosides, 1 amide, 1 alkaloid, 2 nitrogen-containing heterocyclic compounds, 2 vitamins, 3 aldehydes, and 4 ketones.

[0173] Among the various compounds, soybean saponin E had the highest relative content, reaching 29.7707%, and possesses multiple biological activities such as lipid-lowering, immunomodulatory, antioxidant, and antitumor effects, making it promising for applications in the pharmaceutical, health food, and cosmetic fields. Secondly, the second most abundant compound was D-erythro-dihydrosphingosine (20.1058%), an isomer of sphingosine and a key component of cell membrane phospholipids, crucial for maintaining the skin barrier and nerve cell function. Thirdly, the most abundant compound was daidzein I (7.5149%), a glucocerebroside that has been shown to have various pharmacological effects, including antitumor and anti-inflammatory properties, and also exhibits potent inhibitory effects on tyrosinase. Interestingly, two active ingredients found in Ganoderma lucidum were also discovered in wine lees oil, with relatively high content: ganoderic acid F (3.2038%) and ganoderic alcohol I (1.7824%), which have been reported to have antitumor, anti-inflammatory, and immunomodulatory effects. In addition, nine ceramides were found in the wine lees oil of this invention, with Cer(8:0_2O / 10:0) having the highest content (5.7270%). Here, "8:0" indicates that the fatty acid chain is 8 carbon atoms long, all of which are saturated carbon atoms (no double bonds); "2O" indicates that two hydroxyl groups (OH) are attached to the sphingosine base; and "10:0" indicates that another fatty acid chain is 10 carbon atoms long, also all of which are saturated carbon atoms. Another ceramide, HexCer, refers to hexosylceramide, which contains two fatty acid chains and one glycosyl group. HexCer(14:3_20 / 28:1_20) had the highest content (1.2074%). Ceramides have various physiological and pharmacological functions, such as regulating cellular immunity, delaying aging, and anti-tumor effects. In the cosmetics industry, ceramides may help promote the degradation of the stratum corneum, thinning the stratum corneum and promoting the division and proliferation of epidermal cells, thereby delaying the aging process of epidermal cells.

[0174] Also noteworthy are the polyphenols and flavonoids in wine lees oil, with total relative contents of 0.9710% and 2.4048%, respectively. Among the polyphenols, gentian acid (0.1112%), 2-hydroxycinnamic acid (0.1340%), and gallic acid (0.1242%) were present in relatively high amounts. Compounds such as protocatechuic aldehyde, ferulic acid, vanillin, and dihydrocaffeic acid were also detected. These are all common and effective antioxidants found in plants. Some substances, such as dihydrocaffeic acid, can even prevent UV-induced skin damage, implying a positive role in combating photoaging. Among the flavonoids, isorhamnetin (0.8964%), kaempferol (0.4201%), 6”-O-p-coumaryltrifolin (0.1950%), and quercetin (0.1752%) were found in relatively high amounts. Myricetin, which also exhibits anti-photoaging effects, and dihydroquercetin, which has anti-tyrosinase activity, were also found. Therefore, these results further demonstrate the health benefits of wine lees oil, especially its antioxidant and anti-aging effects, and its significant application value in health foods and cosmetics.

[0175] Example of anti-aging effects of wine lees oil

[0176] The anti-aging activity of the wine lees oil obtained in Example 1 was evaluated using the nematode elegans model, and its effect on prolonging the lifespan of nematodes under oxidative stress was tested.

[0177] Caenorhabditis elegans has shown significant advantages in anti-aging research due to its ease of 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.

[0178] Oxidative stress, triggered by excessive reactive oxygen species (ROS) in the body, is considered a major factor driving aging in nematodes. The antioxidant capacity of nematodes is closely related to their lifespan; enhanced antioxidant capacity is often accompanied by extended lifespan. Hydrogen peroxide (H2O2), as an effective mimic, can simulate the cell apoptosis process induced by free radicals, thus playing a crucial role in preparing cellular oxidative damage models. Paraquat (PQ), by inducing intracellular oxidative stress responses and generating excessive ROS, leads to mitochondrial dysfunction, making it widely applicable in the study of mitochondrial oxidative damage. Meanwhile, ultraviolet (UVB) exposure is a major exogenous factor causing photoaging. UVB radiation can directly penetrate the atmosphere and interact with cells in the skin, generating large amounts of free radicals and reactive oxygen molecules, causing intracellular oxidative stress and accelerating the skin aging process. There is a positive intrinsic link between enhanced stress tolerance and lifespan extension; active substances can improve the heat stress resistance of nematodes at 35°C by regulating heat stress proteins, thereby extending their lifespan.

[0179] Therefore, by treating nematodes with samples, then placing them under appropriate stress conditions, and recording the survival status of the nematodes, the potential anti-aging effects of wine lees oil can be assessed.

[0180] (1) Experiment on H2O2-induced oxidative stress damage

[0181] After nematode synchronization, L4 stage larvae were selected and transferred to the culture media of the control group and the sample group. The sample group was fed with wine lees oil at a concentration of 1 mg / mL for 3 days. Then, 30 to 50 nematodes were selected from each culture medium and exposed to NGM medium containing H2O2 for oxidative stress induction intervention. The experiment was set up with 3 parallel plates. The number of nematode survivors, escapes, and deaths was recorded every 30 minutes until all nematodes died, and the survival curve of nematodes under H2O2 oxidative stress was obtained. 60 μM astaxanthin was used as a positive control.

[0182] The results are shown in Figure 3. Compared with the control group, the survival curves of the wine lees oil group showed a significant rightward shift (P < 0.0001). The average lifespan of the control group was 1.53 h, while the average lifespans of the wine lees oil group and the astaxanthin group were 2.03 h and 2.00 h, respectively, representing increases of 33.09% and 30.86% compared to the control group. This indicates that wine lees oil significantly enhances the resistance of nematodes to H2O2 oxidative stress damage and can effectively prolong the lifespan of nematodes. Meanwhile, the anti-aging activity of 1 mg / mL wine lees oil was not significantly different from that of 60 μM astaxanthin (P > 0.05), indicating that it has better anti-aging activity.

[0183] (2) PQ-induced oxidative stress injury experiment

[0184] After nematode synchronization, L4 stage larvae were selected and transferred to the control and sample groups. After 3 days of drug treatment, 50-60 nematodes from each culture medium were exposed to NGM medium containing PQ for oxidative stress induction intervention. Three parallel plates were set up for the experiment. The number of nematode survivors, escapees, and deaths was recorded approximately every 24 hours until all nematodes died, obtaining the survival curve of nematodes under PQ oxidative stress.

[0185] The results are shown in Figure 4. Similar to the results of the H2O2 oxidative stress experiment, compared with the control group, the survival curve of the drug group showed a significant rightward shift (P < 0.001), indicating that wine lees oil can prolong the survival time of nematodes under PQ oxidative damage conditions. The average lifespan of the control group was 2.39 days, while the average lifespans of the wine lees oil group and the astaxanthin group were 2.88 days and 3.43 days, respectively, representing an increase of 20.37% and 43.30% in average survival time. This further demonstrates that wine lees oil can effectively enhance the resistance of nematodes to oxidative stress, thereby prolonging their lifespan.

[0186] (3) Heat-induced stress injury experiment

[0187] Wild-type *C. elegans* in their reproductive stage (3-5 days old) were collected for synchronization. After synchronization, the nematodes were transferred to NGM (nitrogenous glutathione) with / without the drug and incubated at 20°C for 5 days, with the nematodes transferred to a new plate daily during this period. On the 6th day of the adult stage, the nematodes were placed in an incubator at 35°C for 12 hours, followed by an incubator at 20°C for 12 hours. The survival rate of the nematodes was observed and recorded. The experiment was set up in triplicate, with each treatment group containing no fewer than 120 nematodes.

[0188] As shown in Figure 5, the survival rate of nematodes in the control group was significantly lower than that in the other two groups, at only 18.89%, indicating that heat stress negatively impacts nematode survival without treatment. In contrast, the survival rate of nematodes in the wine lees oil treatment group was significantly higher than that in the control group by 38.89 percentage points (P < 0.05), demonstrating that wine lees oil can improve nematode survival under heat stress conditions. Meanwhile, the survival rate of nematodes in the positive control group was slightly lower than that in the wine lees oil treatment group, but the difference between the two groups was not statistically significant. This illustrates the efficacy of wine lees oil in improving nematode survival under heat stress and its potential advantages in resisting heat aging.

[0189] (4) Photoinduced stress injury experiment

[0190] Wild-type *C. elegans* (3-5 days old) were collected for synchronization. After synchronization, the nematodes were transferred to NGM (nitrogenous globulin) containing / without the drug and treated for 3 days. Nematodes from each group were collected, washed three times with M9 buffer, and then transferred to NGM without OP50. The NGM plates containing the nematodes were placed directly below the UVB lamp of the irradiator, one plate at a time, with an irradiation dose of 250 mJ / cm². 2 Ultraviolet irradiation was performed. After irradiation, the nematodes in each group were transferred to OP50 NGM containing FUDR and placed in an incubator at 20°C. Every 12 hours, dead nematodes were removed, and the number of dead and surviving nematodes was recorded to obtain the nematode survival curve, as shown in Figure 6. The experiment was set up in triplicate, with no fewer than 120 nematodes in each treatment group.

[0191] As shown in Figure 6, the survival rate of nematodes in the control group rapidly decreased under photostimulation conditions, reaching 0% by day 4. In contrast, the nematodes in the wine lees oil group and the astaxanthin group showed significantly improved survival rates under photostimulation conditions, with the survival curves shifting significantly to the right (P < 0.001). The average lifespan of the control group was 2.63 days, while the average lifespans of the wine lees oil group and the astaxanthin group were 2.87 days and 3.05 days, respectively, representing an increase in average survival time of 9.19% and 16.11%. Therefore, wine lees oil has a good effect on improving the survival ability of nematodes in UVB irradiation environment, and its anti-photoaging activity has been confirmed.

[0192] Conclusion: The results show that the wine lees oil prepared by this invention has excellent anti-aging effects.

[0193] In summary, the embodiments of this disclosure employ an acid-heat pretreatment method combined with a low-temperature continuous phase change extraction device to extract wine lees oil with high content of active ingredients, good in vitro antioxidant activity, rich in anti-aging active ingredients, and possessing anti-aging effects. This achieves comprehensive utilization of the oils in wine lees, is green, safe, and highly efficient, and is suitable for industrial production applications.

[0194] The method disclosed herein yielded excellent levels of the three active ingredients (total phenols, total flavonoids, and total sterols) and in vitro antioxidant activity, demonstrating the effectiveness and feasibility of this innovative method. This disclosure provides a novel extraction method that can enhance the anti-aging activity of the extract without sacrificing oil quality. Analysis of the characteristic components of the extract allows for a comprehensive understanding of the product obtained using this innovative extraction method. Its application in cosmetics can effectively enhance the anti-aging efficacy of cosmetics.

[0195] The above description is merely a preferred embodiment of this disclosure and is not intended to limit this disclosure. Various modifications and variations can be made to this disclosure by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this disclosure should be included within the scope of protection of this disclosure. Industrial applicability

[0196] This disclosure provides an anti-aging cosmetic containing wine lees oil with specific ingredients and properties. This cosmetic possesses excellent antioxidant properties, enhancing its anti-aging effects and helping to protect the skin from oxidative damage, thus playing a positive role in cosmetics. This wine lees oil retains the active ingredients of each part of the wine lees to the greatest extent possible.

Claims

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 to 0.8 MPa and 40°C to 50°C. 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. The anti-aging cosmetic according to claim 1 is characterized in that, The peroxide value of the wine lees oil is ≤7 mmol / Kg. The anti-aging cosmetic according to claim 1 is characterized in that, The acid value of the wine lees oil is ≤4 mg / g. 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 to 0.8 MPa and 40°C to 50°C. 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. The method for preparing wine lees oil according to claim 4 is 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. The method for preparing wine meal oil according to claim 5 is characterized in that, After separating the extractant gas, the acid solution is removed from the mixture to obtain a crude extract. The method for preparing wine lees oil according to claim 6 is characterized in that, The crude extract was subjected to a second extraction. The method for preparing wine lees oil according to claim 6 is characterized in that, The crude extract was subjected to a second extraction using n-hexane. The method for preparing wine lees oil according to claim 4 is characterized in that, The acid solution is a citric acid-ethanol solution. The method for preparing wine meal oil according to claim 9 is characterized in that, The citric acid-ethanol solution comprises, by mass percentage, 1% to 5% citric acid and 95% to 99% ethanol. The method for preparing wine lees oil according to claim 4 is 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). The method for preparing wine lees oil according to claim 4 is 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. The method for preparing wine lees oil according to claim 4 is characterized in that, Soaking time is 1 to 3 hours. 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.

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