An essence oil formulation and its preparation method

Abstract

The present invention relates to an essence oil formulation and a preparation method thereof. The essence oil includes: hazelnut oil: 20-40%, prickly pear seed oil: 20-40%, GTCC: 10-20%, grape seed oil: 5-20%, olive oil: 1-6%; camellia seed oil: 1%-4%; vitamin E: 0.1-2%; elm extract: 0.001-0.5%; pumpkin seed oil: 0.05-0.5%; papaya seed oil: 0.05-0.5% and rosemary essential oil 0.05-0.5%. The sum of the mass percentages of each component is one hundred percent. The essence oil provided by the present invention has both the ability to scavenge reactive oxygen species (ROS) in human skin, promote the regeneration of type I collagen and promote the regeneration of elastin. When applied to human skin, it has the remarkable effects of enhancing skin luster and fineness, moisturizing, lightening spots, anti-wrinkle and firming the skin.

Description

Technical Field

[0001] The present invention relates to the field of skin care, and particularly to an essential oil having antioxidant effects, anti-wrinkle and skin tightening effects, and a preparation method thereof Background Art

[0002] During the normal metabolism of human skin, the generation and clearance of reactive oxygen species (ROS) in cells remain relatively balanced. However, with the increase of age, the ROS clearance ability weakens, leading to the accumulation of ROS; the accumulation of ROS will accelerate the oxidation and inactivation of human proteins, nucleic acids, lipids and other molecules, causing aging symptoms such as skin relaxation, wrinkles, and age spots

[0003] Collagen is a huge family with a wide variety of types and a highly complex structure. It has significant diversity in terms of molecular structure, supramolecular structure, tissue distribution and function, etc.; they exist in various tissues of the human body, such as skin, bone, cornea, tendon, cartilage, vitreous body, uterine wall, blood vessel wall, basement membrane, placenta, endothelial cells, etc. Collagen is mainly divided into five types, among which: type I collagen exists in many human tissues such as human skin, bones, and blood vessels. However, with the increase of age and many other factors, the loss of various nutritional components in human tissues will occur, and the loss of collagen will cause the breakage of collagen peptide bonds and elastic nets that support the skin, and its helical network structure will be destroyed immediately, causing the skin tissue to be oxidized, atrophied, and collapsed, resulting in aging symptoms such as dry skin, wrinkles, and loose and inelastic skin

[0004] In addition, elastin in human skin is a protein that enables many tissues in the body to maintain their original shape after stretching and contraction, and enables the skin to return to its original shape after being squeezed. However, with the increase of age, the elastin fibers in the skin will gradually break and lose, resulting in the formation of wrinkles and loss of elasticity in the skin Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide an essential oil formulation that can simultaneously have the functions of scavenging reactive oxygen species (ROS), promoting the regeneration of type I collagen, and promoting the regeneration of elastin

[0006] The technical solution adopted by the present invention to solve the above technical problems is: an essential oil formulation, comprising the following components in mass percentage: hazelnut oil: 20 - 40%, cactus seed oil: 20 - 40%, GTCC: 10 - 20%, grape seed oil: 5 - 20%, olive oil: 1 - 6%; camellia seed oil: 1% - 4%; vitamin E: 0.1 - 2%; elm extract: 0.001 - 0.5%; pumpkin seed oil: 0.05 - 0.5%; papaya seed oil: 0.05 - 0.5%, rosemary essential oil 0.05 - 0.5%; the sum of the mass percentages of each component is one hundred percent

[0007] Preferably, the mass percentage content of each component is as follows: hazelnut oil: 30%, prickly pear seed oil: 30%, GTCC: 15.3%, grape seed oil: 17.6%, olive oil: 3%; camellia seed oil: 2.2%; vitamin E: 1%; elm extract: 0.3%; pumpkin seed oil: 0.2%; papaya seed oil: 0.2%, rosemary essential oil 0.2%.

[0008] Preferably, the extraction method of the elm extract is as follows:

[0009] The tissue material of elm is extracted, and after obtaining the filtrate, it is concentrated, dried and pulverized to finally obtain a powdery extract.

[0010] Preferably, the essential oil composition of the present invention can be used as a facial care essential oil or can be applied to the preparation of facial skin care products.

[0011] In addition, the essential oil of the present invention can also be applied to the preparation of other skin care products, such as anti-wrinkle eye cream, body lotion, etc.

[0012] The present invention also provides a method for preparing an essential oil using the above essential oil composition, which specifically includes the following steps:

[0013] S1. Take out all the raw materials in the above formula from the cold storage and place them at room temperature until the temperature of all raw materials rises to room temperature;

[0014] S2. Add hazelnut oil, prickly pear seed oil and grape seed oil to the preparation container in proportion and stir with a magnetic stirrer for 30 minutes; then add GTCC, olive oil and camellia seed oil in proportion in sequence, stir for 20 minutes, and then let it stand for 30 minutes;

[0015] S3. Add pumpkin seed oil, papaya seed oil and rosemary essential oil in proportion in sequence, stir for 20 minutes, and let it stand for 30 minutes;

[0016] S4. Add vitamin E in proportion, stir for 10 minutes, and let it stand for 20 minutes;

[0017] S5. Add elm extract in proportion, stir for 30 minutes, let it stand for 30 minutes, and then store it in a 4°C cold storage.

[0018] Specifically, in the essential oil of the present invention, the main components and effects of each component are as follows:

[0019] Hazelnut oil, source: hazelnut kernels; extraction method: cold pressing; ingredients: oleic acid (78 - 90%), linoleic acid (3 - 14%), saturated fatty acids (3 - 8%), various amino acids required by the human body, various trace elements such as calcium, phosphorus, iron content, etc.; efficacy: (1) suitable for all skin types, with the functions of astringency, slowing down the skin oil secretion rate, and purifying the skin; (2) having a good anti-inflammatory effect, helping skin regeneration, and preventing skin aging; (3) capable of conditioning and tightening the skin, helping the skin maintain stability and elasticity; (4) having excellent moisturizing ability, quickly penetrating the skin, and preventing water loss; (5) being able to strengthen capillaries and having an effect against thread veins.

[0020] Cactus seed oil, also known as cactus fruit oil, contains rich vitamin E, sterols, and a large amount of essential fatty acids; efficacy: can effectively protect the skin from the damage of free radicals; promote the recombination and regeneration of skin cells, repair collagen and elastic fibers; can effectively prevent the premature formation of wrinkles and fight skin aging; has the effects of fading fine lines, spots, acne, stretch marks, neck lines, dark circles, and eye bags, etc.

[0021] GTCC, Chinese: caprylic / capric triglyceride, is a high-purity oil esterified by caprylic / capric acid and glycerol; it is an excellent moisturizing oil with good spreading property, making the skin feel smooth but not greasy and being easily absorbed by the skin; it plays a very good role in making cosmetics uniform and delicate, making the skin smooth and shiny; it can be used as the base material of moisturizing factors, a stabilizer, an antifreeze, and a homogenizer for cosmetics.

[0022] Grape seed oil contains two very important elements, linoleic acid and procyanidins; linoleic acid can resist free radicals, anti-aging, help absorb vitamin C and E, strengthen the elasticity of the circulatory system, reduce the damage of ultraviolet rays, protect collagen in the skin, and prevent melanin precipitation; procyanidins can protect the elasticity of blood vessels, protect the skin from the harm of ultraviolet rays, prevent the damage of collagen fibers and elastic fibers, keep the skin with its due elasticity and tension, and avoid skin sagging and wrinkle generation.

[0023] Olive oil is directly cold-pressed from fresh olive fruits and is rich in monounsaturated fatty acids, vitamin A, vitamin B, vitamin D, vitamin E, vitamin K, carotene, chlorophyll, antioxidants, etc.; it has the functions of promoting cell growth, accelerating wound healing, and also helps to beautify people's appearance and reduce the generation of wrinkles; experiments show that the antioxidants contained in olive oil can eliminate free radicals in the body and have an effect against skin aging.

[0024] Camellia seed oil is obtained by pressing the mature seeds of the Camellia oleifera, a plant of the Theaceae family. Tea oil can resist ultraviolet rays, prevent sunburn and reduce wrinkles, and is very effective against chloasma and sunburn. According to expert research, the unsaturated fatty acid content in camellia seed oil is as high as 93%. Unsaturated fatty acids, also known as beauty acids, can make people's skin smooth and moist when consumed for a long time.

[0025] Vitamin E, also known as vitamin E and tocopherol, is one of the most important antioxidants. It can accelerate the softness and luster of the skin, enhance the elasticity of the skin, and has a good effect on eliminating facial wrinkles and pigmentation spots and delaying skin aging.

[0026] Pumpkin seed oil is rich in nutrients such as ω-6 fatty acids, phytosterols, tocopherols, vitamin K, and phytoestrogens; it has the effects of moisturizing, antioxidant, enhancing skin elasticity, anti-wrinkle, lightening age spots, and delaying skin aging.

[0027] Papaya seed oil is derived from the seeds of papaya fruits; it is rich in fatty acids, antioxidants, vitamins A, C, and E; it has the effects of reducing wrinkles and fine lines and delaying skin aging.

[0028] Rosemary essential oil; it is derived from the plant rosemary, which is a recognized high-antioxidant plant; the extracted essential oil has the effects of firming the skin and anti-wrinkle.

[0029] The present invention has the following advantages and effects compared with the prior art:

[0030] 1. In this application, the essential oil obtained by mixing hazelnut oil, prickly pear seed oil, GTCC, grape seed oil and other antioxidant components in a specific ratio has a synergistic increasing effect in the free radical scavenging test. After testing, the ROS scavenging rate is up to 31% at most, and the antioxidant ability is extremely significant. It can effectively prevent premature skin aging, avoid the formation of age spots, anti-wrinkle and reduce wrinkles, and present the effects of brightening skin tone, firming skin and whitening skin.

[0031] 2. In this application, the essential oil obtained by mixing hazelnut oil, prickly pear seed oil, GTCC, grape seed oil and other antioxidant components in a specific ratio has been tested to have the effect of promoting the regeneration of type I collagen (reference: the promotion rates of zebrafish col1a1a, col1a1b and col1a2 gene expressions are 589% (p = 0.0000014), 152% (p = 0.000038), 594% (p = 0.000020) respectively). When applied to human skin, it can avoid aging phenomena such as skin dryness, wrinkles, and sagging and loss of elasticity caused by collagen loss, and achieve the effects of repairing the skin, reconstructing the collagen layer, improving skin elasticity and moisture; whitening skin, moisturizing and anti-aging.

[0032] 3. In this application, the essence oil obtained by mixing hazelnut oil, prickly pear seed oil, GTCC, grape seed oil and other antioxidant components in a specific ratio has been tested to have the effect of promoting the regeneration of elastin (reference: the promotion rate of the expression of the Eln1 gene in zebrafish is 245% (p = 0.00075)). The regeneration of elastin can effectively improve skin elasticity, endow the skin with firmness or luster, prevent or improve skin aging and relaxation, and achieve the effects of reducing wrinkles, anti-wrinkle and firming the skin.

[0033] 4. In summary, the essence oil provided by the present invention has the ability to scavenge reactive oxygen species (ROS) in human skin, promote the regeneration of type I collagen and the regeneration of elastin at the same time; when applied to human skin, it has the significant effects of anti-wrinkle and firming the skin. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0035] Figure 1 It is the flowchart of the test method in Verification Example 1 of the present invention.

[0036] Figure 2 It is the fluorescence comparison diagram of the antioxidant test results in Verification Example 1 of the present invention; Figure 2 (a) is the ROS fluorescence labeling map of zebrafish embryos in the blank control group; Figure 2 (b) is the ROS fluorescence labeling map of zebrafish embryos in the positive control group; Figure 2 (c) is the ROS fluorescence labeling map of zebrafish embryos under the action of the test substance - Formula 2.

[0037] Figure 3 It is the bar chart of the relative expression level of zebrafish type I collagen gene in Verification Example 2 of the present invention.

[0038] Figure 4 It is the bar chart of the relative expression level of zebrafish elastin gene in Verification Example 3 of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0039] The following will further elaborate on the present invention in conjunction with embodiments. The following embodiments are explanations of the present invention and the present invention is not limited to the following embodiments.

[0040] Example 1: An essential oil formulation containing the following components in mass percentage: hazelnut oil: 20 - 40%, prickly pear seed oil: 20 - 40%, GTCC: 10 - 20%, grape seed oil: 5 - 20%, olive oil: 1 - 6%; camellia seed oil: 1% - 4%; vitamin E: 0.1 - 2%; elm extract: 0.001 - 0.5%; pumpkin seed oil: 0.05 - 0.5%; papaya seed oil: 0.05 - 0.5%, rosemary essential oil 0.05 - 0.5%; the sum of the mass percentages of each component is 100%.

[0041] Among them: hazelnut oil, prickly pear seed oil, grape seed oil, olive oil, camellia seed oil, pumpkin seed oil and papaya seed oil are the seed oils of the corresponding fruits; elm extract is a powdery substance extracted and prepared from elm plant tissues; rosemary essential oil is an oil extract obtained by extracting from the roots, stems, leaves and flowers of the corresponding plants.

[0042] The essential oil formulation described in Example 1 is prepared by mixing the components according to the percentage content and following the process method of essential oil. The specific steps are as follows:

[0043] S1. Take out all the raw materials in the above formula from the cold storage and place them at room temperature for 5 hours until the temperature of all raw materials rises to room temperature.

[0044] S2. Add hazelnut oil, prickly pear seed oil and grape seed oil into the preparation container in proportion and stir with a magnetic stirrer for 30 minutes; then add GTCC, olive oil and camellia seed oil in proportion in turn, stir for 20 minutes, and then let it stand for 30 minutes.

[0045] S3. Add pumpkin seed oil, papaya seed oil and rosemary essential oil in proportion in sequence, stir for 20 minutes, and let it stand for 30 minutes.

[0046] S4. Add vitamin E in proportion, stir for 10 minutes, and let it stand for 20 minutes.

[0047] S5. Add elm extract in proportion, stir for 30 minutes, let it stand for 30 minutes, and then store it in a 4°C cold storage.

[0048] In the above preparation method, according to the different properties and content ratios of each component, add hazelnut oil, prickly pear seed oil, grape seed oil, GTCC, olive oil, camellia seed oil, pumpkin seed oil, papaya seed oil, rosemary essential oil, vitamin E and elm extract in sequence according to the addition order of steps S2 to S5, which can effectively ensure that each component can be fully blended, and then improve the quality uniformity and property stability of the prepared essential oil.

[0049] Specifically, in the essential oil described in this example, the mass percentage content of each component is set as follows:

[0050] Formula 1: Hazelnut oil: 26%, Cactus seed oil: 40%, GTCC: 10%, Grape seed oil: 17%, Olive oil: 4%; Camellia seed oil: 1.4%; Vitamin E: 0.5%; Elm extract: 0.3%; Pumpkin seed oil: 0.4%; Papaya seed oil: 0.1%, Rosemary essential oil 0.3%.

[0051] Formula 2: Hazelnut oil: 30%, Cactus seed oil: 30%, GTCC: 15.30%, Grape seed oil: 17.6%, Olive oil: 3%; Camellia seed oil: 2.2%; Vitamin E: 1%; Elm extract: 0.3%; Pumpkin seed oil: 0.2%; Papaya seed oil: 0.2%, Rosemary essential oil 0.2%.

[0052] Formula 3: Hazelnut oil: 40%, Cactus seed oil: 20%, GTCC: 20%, Grape seed oil: 14%, Olive oil: 1%; Camellia seed oil: 2.5%; Vitamin E: 1.8%; Elm extract: 0.5%; Pumpkin seed oil: 0.1%; Papaya seed oil: 0.05%, Rosemary essential oil 0.05%.

[0053] Formula 4: Hazelnut oil: 27%, Cactus seed oil: 30%, GTCC: 14.7%, Grape seed oil: 16.5%, Olive oil: 6%; Camellia seed oil: 4%; Vitamin E: 0.2%; Elm extract: 0.1%; Pumpkin seed oil: 0.5%; Papaya seed oil: 0.5%, Rosemary essential oil 0.5%.

[0054] Formula 5: Hazelnut oil: 30%, Cactus seed oil: 24%, GTCC: 15.8%, Grape seed oil: 20%, Olive oil: 5%; Camellia seed oil: 3%; Vitamin E: 0.8%; Elm extract: 0.4%; Pumpkin seed oil: 0.5%; Papaya seed oil: 0.3%, Rosemary essential oil 0.2%.

[0055] In the essential oil formulations of Formulas 1 to 5 in the embodiments of the present invention, when the components are defined as: hazelnut oil, cactus seed oil, GTCC, grape seed oil, olive oil, camellia seed oil, vitamin E, elm extract, pumpkin seed oil, papaya seed oil, and rosemary essential oil, the coordination effect among the various anti-aging compositions is obvious. When used as a skin care essential oil, the main effects are as follows:

[0056] (1) Scavenge human reactive oxygen species (ROS), with significant antioxidant effects; when applied to the skin, especially the facial skin, it can show the effects of anti-aging, anti-wrinkle, avoiding the formation of age spots; brightening the skin tone and whitening the skin;

[0057] (2) It can significantly promote the regeneration effect of type I collagen. When applied to the skin, especially the facial skin, it can show the effects of anti-wrinkle, firming the skin, increasing skin elasticity and moisture, making the skin plump and full, and enhancing skin youthfulness.

[0058] (3) It can promote the regeneration of elastin in human skin, showing the functions of maintaining skin elasticity, anti-wrinkle, and tightening the skin.

[0059] The following mainly verifies the above effects of the present invention through the following test examples.

[0060] Verification Example 1: ROS Scavenging Rate

[0061] Test items: The antioxidant and anti-aging effects of the essence oils described in Formulas 1 to 5 of the embodiments of the present application; Detection parameters: ROS (reactive oxygen species) scavenging rate.

[0062] Test principle: During the normal metabolism of human skin, the generation and scavenging of reactive oxygen species (ROS) in cells maintain a relatively balanced state. However, with the increase of age, the ROS scavenging ability weakens, resulting in the accumulation of ROS; the accumulation of ROS will accelerate the oxidation and inactivation of human proteins, nucleic acids, lipids and other molecules, causing aging symptoms such as skin relaxation, wrinkles, and age spots.

[0063] The ROS regulation mechanism in zebrafish is the same as that in humans. Moreover, some fluorescent probes, such as H2DCFDA, can monitor ROS in zebrafish embryos through in vivo staining. At the same time, zebrafish embryos are small in size, convenient for taking pictures under a microscope, and software is used to analyze and quantitatively read the green fluorescence signal in zebrafish embryos that increases with the increase of ROS. The ROS levels of zebrafish embryos in the sample treatment group and the blank control group are tested and compared, and the ROS scavenging rate is calculated to evaluate the antioxidant and anti-aging effects of the essence oils described in Formulas 1 to 5 of the present application.

[0064] Test method: Five groups of 24 48-hour-old zebrafish embryos were respectively exposed to the sample solutions formed by the essence oil formulations described in Formulas 1 to 5 of the present application at 1 g / L. At the same time, a blank control group was set up. After 24 hours of exposure, the fish embryos were stained with H2DCFDA, and the ROS signal intensity was measured by fluorescence photography and statistically analyzed; the specific operation process is as Figure 1 shown.

[0065] Test experiment:

[0066] (1) Preparation of test substances

[0067] The raw materials in Formulas 1 to 5

[0068] Water-soluble raw materials: The test substances can be directly dissolved in the fish embryo culture solution to prepare a test solution.

[0069] Water-insoluble raw materials: Common solvents such as methanol, dimethyl sulfoxide, ethanol, etc. can be selected to assist dissolution. If the test substance and the organic solvent are mixed at a ratio of 1:1 (weight g: volume mL), ultrasonically treated for 10 min, and then added to the fish embryo culture medium to prepare the required concentration, shaken for 30 s, and centrifuged at 6500×g for 10 min. Take the supernatant for testing.

[0070] Cosmetic product (positive control)

[0071] Water-soluble products: The test substance can be directly dissolved in the fish embryo culture medium to prepare a test solution.

[0072] Water-insoluble products: Common solvents such as methanol, dimethyl sulfoxide, ethanol, etc. can be selected to assist dissolution. If the test substance and the organic solvent are mixed at a ratio of 1:1 (weight g: volume mL), ultrasonically treated for 10 min, and then added to the fish embryo culture medium to prepare the required concentration, shaken for 30 s, and centrifuged at 6500×g for 10 min. Take the supernatant for testing.

[0073] Precautions

[0074] The concentration of the test substance test solution should ensure that the mortality rate of zebrafish embryos ≤ 10% (embryos without heartbeat characteristics are defined as dead); multiple concentration groups can be set according to needs during the test; in this experiment, the optimal concentration of the test substance screened is 1 g / L;

[0075] (2) Test steps

[0076] The following test steps are the 96-well plate test guidelines. The test process can be referred to Figure 1 . If a larger test container (such as a petri dish) is used, the test guidelines need to be adjusted accordingly.

[0077] First, under the microscope, select healthy zebrafish embryos 3 days after fertilization;

[0078] Secondly, conduct test grouping:

[0079] Set up a blank control group (fish embryo culture medium); randomly select 24 fish embryos, transfer them to a 96-well plate, with one fish embryo and 0.2 mL of fish embryo culture medium in each well.

[0080] Positive control group (niacinamide working solution); randomly select 24 fish embryos, transfer them to a 96-well plate, with one fish embryo and 0.2 mL of niacinamide working solution in each well;

[0081] Test substance group (test substance; essential oils in Formulas 1 to 5); randomly select 5 groups of 24 fish embryos, and transfer them to 96-well plates respectively, with one fish embryo and 0.2 mL of the corresponding test substance solution in each well;

[0082] Place the 96-well plates grouped as above in an incubator at 28 ± 1°C and incubate until 72 ± 1 h after fertilization. Transfer them to 24-well plates, with each well containing 12 fish embryos and 2 ml of reactive oxygen species (ROS) test solution, and then place them in an incubator at 28 ± 1°C for 2 ± 1 h;

[0083] Finally, perform microscopic analysis and data processing calculations for sample analysis;

[0084] Place the fish embryos on their sides. Take pictures of the fish embryos under a fluorescence stereomicroscope according to unified photographing parameters. Open the pictures with analysis software such as Image J, mark the yolk area of each fish embryo, then select "Measure Average Intensity" in the "Measure" column, and select "Average Signal Intensity" in the test results as the ROS signal intensity, and calculate the reactive oxygen species scavenging rate, as shown in Table 1; in the above test method, at least 90% of the fish embryos survive after the exposure of each test group, otherwise the results of the corresponding test group are invalid.

[0085] Table 1

[0086]

[0087]

[0088] Fluorescence contrast diagram of antioxidant test results, as Figure 2 shown; where: Figure 2 (a) is the ROS fluorescence labeling map of the blank control group - zebrafish embryos; Figure 2 (b) is the ROS fluorescence labeling map of the positive control group - zebrafish embryos;

[0089] Figure 2 (c) is the ROS fluorescence labeling map of zebrafish embryos under the action of the test substance - Formula 2; It can be seen from Figure 2 (a) to 2(c) that the green fluorescence signal gradually weakens and the ROS scavenging rate gradually increases. Under the action of the test substance - Formula 2, the ROS scavenging rate is as high as 31%.

[0090] In this example, the above test results can be used as supporting evidence that the essential oils described in the test substances Formulas 1 to 5 have antioxidant effects, and further support the publicity of the effects of anti-aging, anti-wrinkle, firming the skin, avoiding the formation of skin spots; brightening the skin tone and whitening the skin.

[0091] For subsequent efficacy verification of the essential oil of this application, Formula 2 is taken as an example.

[0092] Verification Example 2: Taking Formula 2 as an example, verify that the essential oil described in this application has the function of promoting the regeneration of type I collagen;

[0093] Test item: Promotion test of zebrafish type I collagen gene expression; Detection parameters: Promotion rates of col1a1a, col1a1b and col1a2 gene expressions

[0094] Test principle:

[0095] Collagen is the most abundant extracellular matrix protein in the human body, and type I collagen is the most abundant protein in the skin. The distribution of type I collagen in zebrafish is the same as that in humans and shows a high degree of conservation with humans. Their innate expression significantly decreases at 6 days after fertilization and becomes very low between 10 and 12 days after fertilization. By testing the expression of zebrafish type I collagen genes (col1a1a, col1a1b and col1a2), comparing the changes in the relative expression levels of zebrafish type I collagen genes between the treatment group and the blank control group, and calculating the promotion rate of type I collagen gene expression, the anti-wrinkle, firming, and skin-tightening and elastic effects of raw materials or products can be evaluated.

[0096] Test method: Thirty-six 6-day-old zebrafish were divided into 3 groups and exposed to the essence oil sample solution described in Test Substance - Formula 2 at a concentration of 1 g / L. At the same time, a blank control group was set up. After 24 hours of exposure, RNA extraction, cDNA synthesis, and real-time PCR amplification were performed on the zebrafish. Using β-actin as the housekeeping gene and Ct as the amplification result, the relative expression levels of col1a1a, col1a1b and col1a2 were calculated and statistically analyzed.

[0097] Test experiment:

[0098] (1) Test organisms: Use wild-type AB strain zebrafish from a reliable source for spawning tests; The parent fish should have good reproductive ability, and the fish age is preferably 6 - 12 months. After 5 generations of pure strain parent fish reproduction, a new batch of parent fish needs to be replaced. The parent fish should not have obvious visible infection and disease characteristics and should not have undergone drug treatment within 2 months.

[0099] Test substances: The test substance is the essential oil described in Formula 2, and the preparation method is the same as that in Verification Example 1. The optimal concentration of the test substance is 1 g / L;

[0100] (2) Test steps

[0101] S1. First, select healthy 6-day-old zebrafish after fertilization under a microscope;

[0102] S2. Second, conduct test grouping:

[0103] Set up a blank control group (fish embryo culture medium): Randomly select 36 zebrafish and transfer them to a 24-well plate, with 12 zebrafish and 2.5 mL of fish embryo culture medium in each well.

[0104] Set up the test substance group (the essence oil described in Formula 2): Randomly select 36 zebrafish, transfer them to a 24-well plate, divide them into 3 groups, with 12 zebrafish and 2.5 mL of the test substance solution in each well, and the concentration of the test substance is 1 g / L;

[0105] Place the 96-well plate with the above sample addition and grouping in an incubator at 28 ± 1 °C for 24 ± 1 h; Collect 12 zebrafish in each well into a 1.5 ml tube, remove the solution, add 0.5 ml of RNAlater solution, and store frozen;

[0106] S3, perform RNA extraction

[0107] Remove the RNAlater solution and rinse 3 times with PBS solution. Place on ice bath, remove the PBS solution, and add 500 μL of TRIzol reagent. Homogenize the embryos with a pellet pestle and place on ice bath for 10 min. Add 100 μL of chloroform, vortex for 1 min, and place on ice bath for 5 min. Centrifuge the sample for 20 min, transfer the upper layer solution to a 1.5 mL centrifuge tube, add 250 μL of isopropanol, vortex for 1 min, place on ice bath for 10 min, and centrifuge the sample for 20 min. Remove all the supernatant, add 500 μL of ethanol, vortex for 1 min, centrifuge the sample for 5 min, and place on ice bath for 10 min. Repeat this step 3 times. Remove the ethanol, centrifuge the sample for 5 min, open the lid in the fume hood and air-dry the sample for 10 min. Add 10 μL of DNase / RNase-free ultrapure distilled water and heat at 55 °C for 15 min.

[0108] S4, perform cDNA synthesis

[0109] Dilute the RNA sample to 1000 ng / 7 μL with DNase / RNase-free ultrapure distilled water. Synthesize cDNA from the RNA using the PrimerScript RT reagent kit with gDNA Eraser (Takara; Cat no. RR047A) and store frozen.

[0110] S5, perform real-time RT-PCR

[0111] Prepare the primer mixture and real-time PCR master mixture for each primer. Dilute the cDNA sample 10-fold with DNase / RNase-free ultrapure distilled water. Add 9 μL of the PCR master mixture and 1 μL of the diluted cDNA sample to each well of the 96-well plate for PCR. Seal the 96-well plate for PCR with an optical adhesive film, centrifuge the plate for 5 min, and perform real-time PCR amplification.

[0112] The gene primers are shown in Table 2

[0113] Table 2

[0114]

[0115] S6. Collect real-time PCR data. Ct is used as the amplification result, and the amplification amount of the β-actin gene is used as the housekeeping gene. Calculate the relative expression levels of each gene (col1a1a, col1a1b, and col1a2) as the test results. The calculation formula is as follows:

[0116]

[0117] In formula (I): T—the relative expression level of the zebrafish collagen gene in the test substance treatment group;

[0118] C—the relative expression level of the zebrafish collagen gene in the blank control group; Perform a two-tailed T-test on the relative expression levels of the zebrafish collagen gene in the test substance treatment group and the blank control group to obtain the p-value.

[0119] Among them: At least 90% of the fish embryos survive after the exposure of each test group. Otherwise, the results of the corresponding test group are invalid; When the promotion rate of collagen gene expression is positive and statistically significant (p < 0.05), the test results can be used as supporting evidence that the test substance has the promotion of collagen regeneration.

[0120] The specific data is as follows:

[0121] When the concentration of the sample (the essential oil described in formula 2) is 1 g / L, the promotion rates of the expression of the zebrafish col1a1a, col1a1b, and col1a2 genes are 589% (p = 0.0000014), 152% (p = 0.000038), and 594%

[0122] (p = 0.000020); The histogram of the relative expression level of the zebrafish type I collagen gene is as Figure 3 shown. It can be Figure 3 seen that the essence oil described in this application has an obvious effect of promoting the regeneration of type I collagen. The regeneration of type I collagen can repair the broken and aging elastic fiber network in the skin tissue, thereby increasing the skin tightness, reducing deep wrinkles, flattening fine lines, and making the skin firm and elastic; When applied to facial skin, it can help repair the skin, rebuild the collagen layer, improve the elasticity and moisture of the skin, and then whiten the skin, playing a role in moisturizing and anti-aging.

[0123] Verification example three: Taking formula 2 as an example, verify whether the essence oil described in this application has the function of promoting elastin regeneration;

[0124] Test item: Promotion test of zebrafish elastin gene (Eln1) expression, Detection parameter: Promotion rate of Eln1 gene expression;

[0125] Test principle:

[0126] Elastin in human skin is a protein that enables many tissues in the body to maintain their original shape after stretching and contraction and allows the skin to return to its original shape after being squeezed. As people age, the elastin fibers in the skin gradually break and are lost, causing wrinkles and loss of elasticity in the skin. Compared with human skin, zebrafish skin is highly similar to human skin in all other structures except that its epidermis consists of differentiated epidermal cells and a mucus layer instead of a cutin layer. The elastin gene is highly conserved. Elastin in the human body is synthesized by the Eln gene, and the corresponding gene in zebrafish is Eln1. By testing whether the essence oil described in Formula 2 of this application can promote the expression of the Eln1 gene in zebrafish, it can be judged whether the sample can promote elastin regeneration, help the skin resist wrinkles, and achieve the effect of skin firming.

[0127] Test method: Divide 36 six-day-old zebrafish into 3 groups and expose them to the sample solution of the essence oil described in Formula 2 at a concentration of 1 g / L. At the same time, set up a blank control group. After 24 hours of exposure, extract RNA from the zebrafish, synthesize cDNA, and perform real-time PCR amplification. Using β-actin as the housekeeping gene and Ct as the amplification result, calculate the relative expression level of Eln1 and conduct statistical analysis.

[0128] Test experiment:

[0129] The specific operation steps are the same as those in Verification Example 2; among them: the primers for the elastin type I gene are shown in Table 3,

[0130] Table 3

[0131]

[0132] Test results:

[0133] When the concentration of the sample (the essential oil described in Formula 2) is 1 g / L, the promotion rate of the expression of the Eln1 gene in zebrafish is 245% (p = 0.00075); compared with the blank control group, the bar graph of the relative expression level of the zebrafish elastin gene is as Figure 4 shown.

[0134] From the above conclusions, it can be seen that the essence oil described in this application can significantly promote the expression of the Eln1 gene in zebrafish and has the effect of promoting elastin regeneration. Elastin regeneration can effectively improve skin elasticity, endow the skin with firmness or luster, prevent or improve skin aging and relaxation, and achieve the effects of reducing wrinkles, resisting wrinkles, and firming the skin.

[0135] The essence oil formed by the formula of the essence oil described in the above formula of the present invention can be directly used as a facial care essence oil or can also be applied to the preparation of other facial care products.

[0136] In addition, the essence oil formulation described in the above formula of the present invention can also be applied to the preparation of other skin care products, such as anti-wrinkle eye cream, body lotion, etc.

[0137] Furthermore, in order to verify the efficacy of the essence oil described in the above formula of the present invention when used as a facial care essence oil, the present invention also conducted the following verification examples.

[0138] Verification Example 4: Select 18 testers to evaluate the skin care effect after 5 weeks of trial use

[0139] Age of testers: 32 - 51 years old, 18 people;

[0140] Facial skin conditions: 4 people with dry skin; 3 people with dry-oily mixed skin, 5 people with dry-oily mixed and more dry, 6 people with glycerin mixed and more oily, 4 people with sensitive skin, 1 person with pimply skin; 16 people with facial spots; 16 people with wrinkles; 13 people with skin laxity;

[0141] After 5 weeks of trial use of this product, the evaluation results of the fading of facial skin spots are as follows:

[0142] Among the 16 people with facial spots, 12 people had medium fading and obvious fading of facial spots, accounting for 75%; 4 people had slight fading or no change;

[0143] The evaluation results of reducing eye wrinkles are as follows: Among the 16 people with wrinkles, 13 testers had obvious, moderately shallowed or reduced eye wrinkles, accounting for 81%; 3 testers had slightly shallowed or no change;

[0144] The evaluation results of enhancing skin glossiness are as follows: 17 testers had obvious improvement and moderately improved skin glossiness, accounting for 94%; 1 person had slight or no change;

[0145] The evaluation results of enhancing skin fineness are as follows: 16 testers had obvious improvement and moderately improved skin fineness, accounting for 89%; 2 people had slight or no change;

[0146] The evaluation results of enhancing skin texture (tightening) are as follows: 13 subjects had obvious improvement and moderately improved skin texture, accounting for 72%; 5 people had slight or no change;

[0147] The evaluation results of enhancing skin moisture are as follows: 14 subjects had obvious improvement and moderately improved skin moisture, accounting for 78%; 4 people had slight or no change;

[0148] The evaluation results of the improvement of the overall skin condition are as follows: 17 subjects had obvious improvement and gradual improvement, accounting for 94%; 1 person had slight or no change;

[0149] Therefore, according to this human trial evaluation experiment, the improvement effects of this product on human facial skin are as follows: improving skin glossiness > improving skin smoothness > reducing eye wrinkles > improving skin moisture > lightening facial skin spots > improving skin texture (firmness). The number of people who obtained obvious and moderate improvement and enhancement in individual tests was more than 72%. The number of subjects who obtained obvious improvement and gradual improvement in the overall skin condition reached 94% of the total number of tested people, indicating that this product has significant effects on skin protection, repair and improvement for users. The specific data are shown in Table 4.

[0150] Table 4

[0151]

[0152] In addition, it should be noted that for the specific embodiments described in this specification, the shapes, names of components, etc. can be different. Any equivalent or simple changes made according to the structure, features and principles described in this invention patent concept are included in the protection scope of this invention patent. Those skilled in the technical field to which this invention belongs can make various modifications, supplements or use similar methods for substitution to the specific embodiments described, as long as they do not deviate from the structure of this invention or exceed the scope defined by this claims, they should all fall within the protection scope of this invention.

Claims

1. An essence oil formulation, characterized in that, it comprises the following components in mass percentage: hazelnut oil: 20 - 40%, prickly pear seed oil: 20 - 40%, GTCC: 10 - 20%, grape seed oil: 5 - 20%, olive oil: 1 - 6%; camellia seed oil: 1% - 4%; vitamin E: 0.1 - 2%; elm extract: 0.001 - 0.5%; pumpkin seed oil: 0.05 - 0.5%; papaya seed oil: 0.05 - 0.5%, rosemary essential oil 0.05 - 0.5%; the sum of the mass percentages of each component is one hundred percent.

2. The essence oil formulation according to claim 1, characterized in that, the mass percentages of each component are as follows: hazelnut oil: 30%, prickly pear seed oil: 30%, GTCC: 15.3%, grape seed oil: 17.6%, olive oil: 3%; camellia seed oil: 2.2%; vitamin E: 1%; elm extract: 0.3%; pumpkin seed oil: 0.2%; papaya seed oil: 0.2%, rosemary essential oil 0.2%.

3. The essence oil formulation according to claim 2, characterized in that, the essence oil is an essence oil for human skin care.

4. The essence oil formulation according to claim 3, characterized in that, the essence oil is a facial care essential oil or can be applied to the preparation of facial skin care products.

5. A method for preparing an essence oil using the essence oil formulation according to claim 1 or 2, characterized in that, it comprises the following steps: S1, taking out the raw materials according to claim 1 or 2 from the cold storage and placing them at room temperature until the temperatures of all raw materials rise to room temperature; S2, first adding hazelnut oil, prickly pear seed oil, and grape seed oil in proportion in a preparation container and stirring for 30 minutes; then sequentially adding GTCC, olive oil, and camellia seed oil in proportion and stirring again for 20 minutes; S3, sequentially adding pumpkin seed oil, papaya seed oil, and rosemary essential oil in proportion, stirring for 20 minutes, and standing for 30 minutes; S4, adding vitamin E in proportion, stirring for 10 minutes, and standing for 20 minutes; S5, adding elm extract in proportion, stirring for 30 minutes, and standing for 30 minutes.

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