A plant composition having skin anti-aging activity and a preparation thereof

Abstract

The present application discloses a kind of plant composition with skin anti-aging activity and its preparation.The effective component in the plant composition is composed of Haematococcus pluvialis extract 84.0-89.0%, liquorice oil 8.0-12.0%, Melaleuca alternifolia leaf oil 0.5-0.8%, hydrolyzed Olea europaea leaf extract 0.5-1.0%, Rhodiola rosea root extract 0.1-0.5% and bitter orange leaf oil 1.2-2.5%.The plant composition with skin anti-aging activity in the present application is used as the effective component, other additives are added, and emulsion, cream or essence oil is prepared.The plant composition and preparation in the present application use fermented Camelina sativa seed oil, fermented Crocus sativus seed oil and fermented Gossypium hirsutum seed oil as the extraction solvent of active components in Haematococcus pluvialis, and as the efficacy component and matrix of the preparation, which has better skin feel and biological activity compared with the original plant oil application, has higher extraction rate of active components in Haematococcus pluvialis, and is more helpful for transdermal absorption of active components.

Description

I. Technical Field:

[0001] This invention belongs to the field of cosmetic technology, specifically relating to a plant composition and its preparation containing plant-based ingredients such as Haematococcus pluvialis extract and comfrey oil as the main active ingredients, which has anti-aging activity for the skin. II. Background Technology:

[0002] The skin is the largest organ in the human body and one of its most important barriers, preventing the invasion of foreign substances and pathogens, preventing fluid loss, and regulating body temperature, secretion, and excretion. With improved living standards, people are paying particular attention to facial skin management. Facial skin not only reflects a person's health and appearance but also influences their psychological and social well-being. Healthy facial skin not only enhances one's appearance but also boosts social confidence. Facial skin aging is an inevitable physiological consequence of aging, but with increasing social pressure, dependence on electronic devices, environmental pollution, and exposure to various chemicals, facial skin aging is showing a trend towards affecting younger people. As cosmetics gradually become necessities, people's demands for cosmetic efficacy have evolved from simple moisturizing to multiple functions such as whitening, fading spots, anti-aging, and wrinkle reduction. Commonly used chemical additives in cosmetics, such as preservatives, artificial fragrances and pigments, and penetration enhancers, are considered important factors in damaging the skin barrier and inducing skin allergies. Plant-derived ingredients are generally considered to have advantages such as gentleness and low irritation. Therefore, as consumers pay more attention to the greenness and safety of products, plant-based cosmetics are gaining importance and their market share is increasing. The development and utilization of plant-derived functional ingredient cosmetics has become an important development direction for the cosmetics industry in the future.

[0003] Astaxanthin is a popular cosmetic ingredient, hailed as a "super antioxidant." It is one of the most potent natural antioxidants discovered to date. Its strong antioxidant properties can resist the damage of reactive oxygen species (ROS) to collagen and elastin in the dermis, thus exerting an anti-wrinkle effect. Simultaneously, its structure results in a strong ultraviolet absorption range similar to UVA wavelengths, preventing photoaging (UVA is currently the main cause of photoaging; due to its longer wavelength, it can penetrate the skin surface and reach deep into the dermis, damaging collagen and elastin, leading to skin sagging, wrinkles, and pigmentation). Therefore, astaxanthin can exert its anti-aging effect through multiple mechanisms. Astaxanthin is widely found in organisms, including shrimp, crabs, and fish, but the highest-content natural source of astaxanthin is Haematococcus pluvialis. Because Haematococcus pluvialis has thick cell walls and its active ingredients, such as astaxanthin, are easily oxidized and destroyed, efficiently utilizing astaxanthin and other active ingredients in Haematococcus pluvialis requires solving the extraction process problem.

[0004] Existing extraction processes for astaxanthin and other active ingredients from Haematococcus pluvialis mostly involve three steps: cell wall disruption, extraction, and drying / purification. This process is time-consuming and leads to the oxidation and destruction of active ingredients in the Haematococcus pluvialis. Cell wall disruption processes for Haematococcus pluvialis mainly include physical and chemical methods. Patents CN 101381337A, CN104513185A, CN103787941A, and CN10762897A propose methods such as airflow milling, supersonic airflow milling, high-pressure homogenization, and steam explosion for cell wall disruption of Haematococcus pluvialis. Patents CN103232375A, CN107011225A, and CN103044303A propose methods using acidic aqueous solutions, concentrated sulfuric acid-alcohol systems, and enzyme treatment for cell wall disruption. Astaxanthin extraction methods mainly include organic solvent extraction and oil solvent extraction. Using mixed low-toxicity organic solvents and common plant oils as extraction solvents presents challenges related to solvent separation. Excessively high temperatures and complex extraction processes inevitably affect the stability of active ingredients such as astaxanthin in Haematococcus pluvialis. For example, patent CN 202410037536.4 discloses an astaxanthin extraction method and application, using shrimp shell enzymatic hydrolysate as the Haematococcus pluvialis culture medium and employing organic solvent extraction followed by rotary evaporation drying to extract astaxanthin. Patent CN202211238411.5 discloses a method for preparing an antioxidant extract, using a mixture of polar and non-polar solvents after breaking down the cell walls of Haematococcus pluvialis as the extraction solvent. The polar solvent is ethanol, and the non-polar solvent is one of ethyl acetate or hexane. However, the extract obtained using this method still needs to be removed by rotary evaporation before it can be used as a cosmetic raw material. Therefore, optimizing the extraction process of Haematococcus pluvialis to achieve simultaneous cell wall disruption and active ingredient extraction while minimizing or eliminating subsequent enrichment and purification processes will demonstrate significant advantages for the application of Haematococcus pluvialis in the cosmetics field.

[0005] The human epidermis has a "sand-brick structure." The "bricks" mainly refer to epidermal cells, and the "sand" mainly refers to the intercellular lipid components. Skin lipids primarily consist of sebaceous gland lipids (lipids secreted to the skin surface through sebaceous glands) and intercellular lipids, collectively forming the lipid barrier of human skin. These lipids play a crucial role in resisting environmental pollutants, participating in inflammatory responses, delaying aging, and regulating skin tone. Triglycerides and free fatty acids are important components of skin lipids. Clinical studies have observed that the triglyceride content in human sebum gradually decreases after adulthood, leading to a decline in skin hydration capacity and causing signs of aging such as dullness, flaking, and wrinkles. Oily skin ages significantly slower than dry skin. In a UV-induced photoaging model of immortalized human keratinocytes, triglycerides significantly reduced the expression of extracellular matrix metalloproteinase-1, playing a significant role in the skin damage repair mechanism after photoaging and acute UV exposure. Free fatty acids are important lipids for maintaining the skin barrier. They ensure the skin's slightly acidic environment, which can prevent the colonization of external pathogens and thus maintain the balance of the skin's microecology.

[0006] Plant oils are important raw materials in the cosmetics industry, with triglycerides being their main component. Diffusion through the stratum corneum is the primary pathway for transdermal absorption of cosmetics, including transdermal absorption via intercellular spaces, cellular pathways, and transdermal absorption via skin appendages. The intercellular spaces account for approximately 30% of the total volume of the stratum corneum, but due to their lower resistance compared to keratinocytes, they play a major role in transdermal penetration. The intercellular spaces of the epidermis are mainly composed of lipids; therefore, lipid-soluble and non-polar substances are more easily absorbed through the lipid layer of the intercellular spaces, reaching the deep epidermis and even the dermis, thus exerting skin care effects. Using plant oils as base oils and functional active ingredients in cosmetics, and simultaneously as extraction solvents for astaxanthin and other active ingredients in Haematococcus pluvialis, allows for direct application as a compounding ingredient, promoting the penetration and absorption of functional components in Haematococcus pluvialis, achieving multiple benefits. This not only simplifies the process but also enhances the overall functional activity of the cosmetic. Different plant oils, due to differences in fatty acid composition, the types and amounts of accompanying substances, exhibit different effects in the skincare field. Meadowfoam seed oil and flaxseed oil are two cosmetic raw materials with compositions significantly different from other plant oils. Meadowfoam seed oil is one of the most stable plant oils known, with a high content of ultra-long-chain (≥20C) unsaturated fatty acids. Flaxseed oil is also one of the few plant oils found to have a high content of ultra-long-chain unsaturated fatty acids, containing up to 14% C20:1 and 0.3-1.2% nervonic acid (C24:1), playing an important role in maintaining the skin's lipid barrier. Furthermore, nervonic acid has unique advantages in skin wound healing. Safflower seed oil is a highly distinctive plant oil raw material in cosmetics, known as the "King of Linoleic Acid," with the highest linoleic acid content among plant oils. Linoleic acid participates in the synthesis of phospholipids in the human body, is a component of cell membranes and mitochondria, and is also a component of epidermal lipid ceramides. Therefore, linoleic acid is crucial for human skin metabolism and the repair process of damaged skin tissue. In addition, safflower seed oil also contains tocopherols, flavonoids, phytosterols, and other substances, exhibiting good stability.

[0007] Bio-fermentation technology is an important means of transforming complex components in raw materials into small molecules that are easily absorbed by the skin through microbial metabolism, thereby improving the absorption rate and efficacy of active substances. It is also an important direction for the development of new cosmetic raw materials. During the fermentation process of plant oils used in cosmetics by oil-producing yeasts, some triglycerides are hydrolyzed to produce free fatty acids, while a series of endogenous aromatic components are also produced. Cosmetics are the best application area for fermented plant oils to leverage their advantages. The sensory properties of fermented plant oils are significantly altered compared to the original plant oils, with increased polarity and decreased surface tension. They can not only serve as a cosmetic base but also as one of the main functional ingredients to enhance the functional activity of cosmetics. To date, existing literature lacks any cosmetic formulations that utilize Haematococcus pluvialis extract extracted using fermented plant oils as a solvent as a cosmetic functional ingredient, nor does it include any literature on the combined application of fermented flaxseed oil, fermented meadowfoam seed oil, and fermented safflower seed oil. III. Summary of the Invention:

[0008] The technical problem this invention aims to solve is: based on the current market demand for green and safe plant-based anti-aging cosmetics, this invention provides a plant composition and its formulation with anti-aging activity, using Haematococcus pluvialis extract as the main active ingredient; specifically, this invention provides a plant composition and its formulation with skin anti-aging activity. The plant composition of this invention uses fermented flaxseed oil, fermented safflower seed oil, and fermented meadowfoam seed oil as extraction solvents for the active ingredients in Haematococcus pluvialis, and also as the active ingredients and matrix of the formulation. Compared with the original plant oil application, it has better skin feel and bioactivity, a higher extraction rate of active ingredients from Haematococcus pluvialis, and is more conducive to the transdermal absorption of active ingredients.

[0009] To solve the above problems, the technical solution adopted by the present invention is as follows:

[0010] This invention provides a plant composition with anti-aging skin activity. The active ingredients in the plant composition are Haematococcus pluvialis extract, Lithospermum erythrorhizon oil, Melaleuca alternifolia leaf oil, hydrolyzed olive leaf extract, Rhodiola rosea root extract, and bitter orange leaf oil. The proportions of each active ingredient, expressed as mass percentages, are as follows: Haematococcus pluvialis extract 84.0-89.0%, Lithospermum erythrorhizon oil 8.0-12.0%, Melaleuca alternifolia leaf oil 0.5-0.8%, hydrolyzed olive leaf extract 0.5-1.0%, Rhodiola rosea root extract 0.1-0.5%, and bitter orange leaf oil 1.2-2.5%.

[0011] According to the above-mentioned plant composition with anti-aging skin activity, the Haematococcus pluvialis extract is obtained by high-pressure homogenization and cell wall disruption extraction of Haematococcus pluvialis powder using fermented plant oil as the extraction solvent; the fermented plant oil is at least one of fermented flaxseed oil, fermented meadowfoam seed oil and fermented safflower seed oil.

[0012] Based on the above-mentioned plant composition with anti-aging skin activity, the specific extraction method of the Haematococcus pluvialis extract is as follows:

[0013] a. Using Yeast Extract as the fermentation strain, the fermentation strain was inoculated into a liquid culture medium and cultured aerobically in a shaker at a temperature of 28–35°C for 16–36 h. Seed liquid was obtained after culture.

[0014] b. Add the obtained seed liquid to the sterilized liquid culture medium at a volume ratio of 3-5% for primary fermentation. The fermentation temperature is 28-35℃ and the fermentation time is 24-48h. The primary fermentation liquid is obtained after fermentation.

[0015] c. Sterilize each vegetable oil separately, then add the sterilized vegetable oil to the obtained primary fermentation broth and continue fermentation for 24-72 hours at a temperature of 28-35℃.

[0016] The volume ratio between the primary fermentation broth and the sterilized vegetable oils is 1:1 to 2.

[0017] d. After fermentation in step c is completed, each fermentation product is centrifuged at 6000 rpm for 10 min. After centrifugation, the supernatants are mixed to obtain fermented vegetable oil. At the same time, the lower aqueous phase is removed to separate the bacterial cells.

[0018] e. Wash the obtained bacterial cells with phosphate buffer, centrifuge at 4000-6000 rpm for 10 min after washing, discard the supernatant after centrifugation, and repeat the washing twice to obtain clean bacterial cells after washing.

[0019] The cleaned bacterial cells were taken and mixed with dried Haematococcus pluvialis powder to obtain Haematococcus pluvialis powder containing bacterial cells. Then, the Haematococcus pluvialis powder containing bacterial cells was added to the obtained fermented vegetable oil and mixed evenly. After mixing, it was homogenized 1-2 times under a high pressure of 1000-1500 bar (to break the cell walls of yeast cells and Haematococcus pluvialis powder, and to leach the fat-soluble active ingredients in the bacterial cells and Haematococcus pluvialis powder). After homogenization, it was centrifuged at 8000 rpm for 10 min, and the supernatant was taken to obtain the Haematococcus pluvialis extract.

[0020] According to the above-mentioned plant composition with anti-aging activity for the skin, the plant oil mentioned in step c is flaxseed oil, meadowfoam seed oil or safflower seed oil; the ratio of Haematococcus pluvialis powder to fermented plant oil in step e is 1g: 15-20mL.

[0021] According to the above-mentioned plant composition with anti-aging skin activity, the shaking speed of the shaker is controlled at 160-220 rpm during fermentation.

[0022] Additionally, a formulation of a plant composition having anti-aging skin activity is provided, said formulation being prepared from the above-mentioned plant composition having anti-aging skin activity into an emulsion, cream, or essential oil.

[0023] According to the above-mentioned formulation of a plant composition with anti-aging skin activity, the emulsion of the plant composition formulation with anti-aging skin activity is composed of the following components in the indicated mass percentages: 6.7-13.2% of the above-mentioned plant composition with anti-aging skin activity, 1.0-2.0% of glyceryl stearate, 1.0-1.5% of cetearyl alcohol, 2.0-5.0% of isohexadecane, 62.9-81.2% of water / birch sap, 5.0-10.0% of glycerin, 0.1-0.2% of sodium polyacrylate, 0.2-0.4% of allantoin, 0.2-0.4% of carbomer 940, 1.0-1.5% of tocopherol, 0.4-0.6% of p-hydroxyacetophenone, and 1.0-2.5% of 1,2-hexanediol.

[0024] According to the above-mentioned preparation of a plant composition with anti-aging skin activity, the cream of the plant composition preparation with anti-aging skin activity is prepared from the following components in the following mass percentages: 9.0-18.0% of the above-mentioned plant composition with anti-aging skin activity, 1.5-2.5% of glyceryl stearate, 3.0-9.0% of cetearyl alcohol, 4.0-6.0% of isohexadecane, 55.0-74.5% of water / birch sap, 4.0-10.0% of glycerin, 0.1-0.2% of sodium polyacrylate, 0.2-0.4% of allantoin, 0.1-0.2% of xanthan gum, 1.0-1.5% of tocopherol, 0.4-0.6% of p-hydroxyacetophenone, and 1.0-2.0% of 1,2-hexanediol.

[0025] According to the above-mentioned preparation of the plant composition with anti-aging skin activity, the essential oil of the plant composition preparation with anti-aging skin activity is composed of the following components in weight percentages: 14.0-26.0% of the above-mentioned plant composition with anti-aging skin activity, 72.0-85.0% of the base oil and 0.5-3.5% of tocopherol.

[0026] According to the above-mentioned formulation of plant compositions with anti-aging skin activity, the base oil is at least one selected from flaxseed oil, linseed oil, low erucic acid rapeseed oil, jojoba seed oil, perilla seed oil, peony seed oil, meadowfoam seed oil, safflower seed oil, camellia seed oil, and sweet almond oil.

[0027] In the technical solution of this invention, the liquid culture medium is prepared by adding 2g of glucose, 0.3g of yeast extract, 2g of peptone and 0.3g of malt extract to every 100mL of distilled water.

[0028] In the technical solution of this invention, the hydrolyzed olive leaf extract is obtained by sequentially extracting pulverized olive leaves with ethanol via ultrasonic extraction, concentrating, spray drying, hydrolyzing with β-glucosidase, inactivating enzymes, centrifuging, and then freeze-drying.

[0029] In the technical solution of this invention, the Rhodiola rosea root extract is obtained by extracting pulverized Rhodiola rosea root with 70-80% ethanol, followed by concentration and spray drying.

[0030] The plant-based composition formulation of this invention, which has anti-aging activity for the skin, is free of preservatives and artificial fragrances, and is relatively mild, making it suitable for various skin types.

[0031] The positive and beneficial effects of this invention are:

[0032] 1. The technical solution of this invention uses fermented meadowfoam seed oil, fermented flaxseed oil, and fermented safflower seed oil in combination, utilizing their unique fatty acid composition to provide unique functional components for the skin barrier. The triglyceride content decreases while the free fatty acid content increases in the fermented plant oil matrix, which further facilitates the transdermal absorption of active ingredients in cosmetics, helps maintain the skin's slightly acidic environment, prevents the colonization of pathogenic bacteria, and thus maintains the skin's microecological balance.

[0033] 2. The plant composition provided by the present invention uses fermented plant oil as the active ingredient and matrix. Compared with the original plant oil, the surface tension is reduced, the affinity with water is higher, it is easier to emulsify, and it is easier to use in various cosmetic matrices. The cosmetic prepared is not greasy and has better spreadability.

[0034] 3. This invention uses fermented vegetable oil as an extraction solvent and compounding raw material for astaxanthin and other active ingredients in Haematococcus pluvialis. Compared with the original vegetable oil, it has a higher extraction rate of astaxanthin in Haematococcus pluvialis. At the same time, the process route is simple and avoids the subsequent solvent removal step, so as to fully ensure the stability of astaxanthin components.

[0035] 4. The plant compositions provided by this invention are all purely natural extracts, which can increase the proportion of collagen fiber area in the dermis of the skin, have strong anti-aging activity, and the formulation does not contain any artificial fragrances or preservatives, making it safer to use. IV. Description of the attached drawings:

[0036] Figure 1 GC-MS total ion chromatogram of free fatty acid components in a single vegetable oil before and after fermentation;

[0037] Figure 1 In the middle, A-Flaxseed oil before and after fermentation; B-Safflower seed oil before and after fermentation; C-Malva seed oil before and after fermentation; (a)-Fermented vegetable oil; (b)-Original vegetable oil.

[0038] Figure 2 Full-wavelength scan spectrum of astaxanthin standard solution (340–800 nm);

[0039] Figure 2 In the diagram, red represents the fermented vegetable oil preparation from Example 1, and black represents the control solvent preparation from Example 1.

[0040] Figure 3 Images showing HE and Masson staining results of rat skin tissues in each group;

[0041] Figure 3 In the middle, I-HE staining results; II-Masson staining results; (a)-control group; (b)-model group; (c)-Example 9 group; (d)-Example 10 group. V. Detailed Implementation Methods:

[0042] The present invention will be further illustrated below with reference to the embodiments, but this does not limit the scope of protection of the technical solution of the present invention.

[0043] Example 1:

[0044] The present invention relates to a plant composition with anti-aging activity for the skin, comprising 87.7g of Haematococcus pluvialis extract, 8.0g of Lithospermum erythrorhizon oil, 0.7g of Melaleuca alternifolia leaf oil, 0.6g of hydrolyzed olive leaf extract, 0.5g of Rhodiola rosea root extract, and 2.5g of bitter orange leaf oil.

[0045] The extraction method of the Haematococcus pluvialis extract is as follows:

[0046] a. Take one tube of Yeast Extract and add it to 150 mL of sterilized liquid culture medium (containing 3 g glucose, 0.45 g yeast extract, 3 g peptone and 0.45 g malt extract). Incubate the culture in a shaker at 30 °C and 160 r / min for 16 h to obtain the seed culture.

[0047] b. Add 75 mL of the obtained seed liquid to the autoclaved liquid culture medium for primary fermentation. The fermentation temperature is 30℃, the rotation speed is 200 r / min, and the fermentation time is 24 h. After fermentation, the primary fermentation broth is obtained.

[0048] The liquid culture medium is prepared by adding 50g of glucose, 7.5g of yeast extract, 50g of peptone, and 7.5g of malt extract to 2.5L of distilled water.

[0049] c. Divide the obtained primary fermentation broth into 1.8L, 0.3L and 0.4L, and then introduce them into sterilized flaxseed oil, safflower seed oil and meadowfoam seed oil respectively in a 1:1 volume ratio. Continue fermentation for 48h at 30℃ and 200r / min.

[0050] d. After fermentation in step c is completed, each fermentation product is centrifuged at 6000 rpm for 10 min. After centrifugation, the supernatants are mixed to obtain fermented vegetable oil. At the same time, the lower aqueous phase is discarded and the bacterial cells are separated.

[0051] e. Wash the obtained bacterial cells with phosphate buffer, centrifuge at 6000 rpm for 10 min after washing, discard the supernatant after centrifugation, and repeat the washing twice to obtain clean bacterial cells.

[0052] The cleaned bacterial cells were taken and mixed with 167g of dried Haematococcus pluvialis powder to obtain Haematococcus pluvialis powder containing bacterial cells. Then, the Haematococcus pluvialis powder containing bacterial cells was added to the obtained fermented plant oil and mixed evenly. After mixing, it was homogenized twice under 1000 bar pressure using a high-pressure homogenizer (through homogenization, the cell walls of yeast cells and Haematococcus pluvialis powder were broken, and the fat-soluble components and active ingredients in the cells and Haematococcus pluvialis powder were extracted). After homogenization, it was centrifuged at 8000 rpm for 10 min, and the supernatant was collected to obtain the Haematococcus pluvialis powder / fermented plant oil extract, i.e., Haematococcus pluvialis extract.

[0053] Example 2:

[0054] The present invention relates to a plant composition with anti-aging activity for the skin, comprising 84g of Haematococcus pluvialis extract, 12g of Lithospermum erythrorhizon oil, 0.5g of Melaleuca alternifolia leaf oil, 0.5g of hydrolyzed olive leaf extract, 0.5g of Rhodiola rosea root extract, and 2.5g of bitter orange leaf oil.

[0055] The extraction method of the Haematococcus pluvialis extract is as follows:

[0056] a. Take one branch of Yeast Extract and add it to 150 mL of sterilized liquid culture medium (containing 3 g glucose, 0.45 g yeast extract, 3 g peptone and 0.45 g malt extract). Incubate the culture in a shaker at 30 °C and 160 r / min for 16 h to obtain the seed culture.

[0057] b. Add 75 mL of the obtained seed liquid to the autoclaved liquid culture medium for primary fermentation. The fermentation temperature is 32℃, the rotation speed is 200 r / min, and the fermentation time is 36 h. After fermentation, the primary fermentation broth is obtained.

[0058] The liquid culture medium is prepared by adding 30g of glucose, 4.5g of yeast extract, 30g of peptone, and 4.5g of malt extract to 1.5L of distilled water.

[0059] c. Divide the obtained primary fermentation broth into 1.0L, 0.3L and 0.2L, and then introduce them into sterilized flaxseed oil, safflower seed oil and meadowfoam seed oil respectively in a 1:1 volume ratio. Continue fermentation for 36h at 32℃ and 200r / min.

[0060] d. After fermentation in step c is completed, each fermentation product is centrifuged at 6000 rpm for 10 min. After centrifugation, the supernatants are mixed to obtain fermented vegetable oil. At the same time, the lower aqueous phase is discarded and the bacterial cells are separated.

[0061] e. Wash the obtained bacterial cells with phosphate buffer, centrifuge at 6000 rpm for 10 min after washing, discard the supernatant after centrifugation, and repeat the washing twice to obtain clean bacterial cells.

[0062] The cleaned bacterial cells were taken and mixed with 75g of dried Haematococcus pluvialis powder to obtain Haematococcus pluvialis powder containing bacterial cells. Then, the Haematococcus pluvialis powder containing bacterial cells was added to the obtained fermented plant oil and mixed evenly. After mixing, it was homogenized once at 1300 bar using a high-pressure homogenizer (through homogenization, the cell walls of yeast cells and Haematococcus pluvialis powder were broken, and the fat-soluble components and active ingredients in the cells and Haematococcus pluvialis powder were extracted). After homogenization, it was centrifuged at 8000 rpm for 10 min, and the supernatant was collected to obtain the Haematococcus pluvialis powder / fermented plant oil extract, i.e., Haematococcus pluvialis extract.

[0063] Example 3:

[0064] The present invention relates to a plant composition with anti-aging activity for the skin, comprising 89g of Haematococcus pluvialis extract, 8.1g of Lithospermum erythrorhizon oil, 0.5g of Melaleuca alternifolia leaf oil, 1.0g of hydrolyzed olive leaf extract, 0.2g of Rhodiola rosea root extract, and 1.2g of bitter orange leaf oil.

[0065] The extraction method of the Haematococcus pluvialis extract is as follows:

[0066] a. Take one tube of Yeast Extract and add it to 150 mL of sterilized liquid culture medium (containing 3 g glucose, 0.45 g yeast extract, 3 g peptone and 0.45 g malt extract). Incubate the culture in a shaker at 28 ℃ and 200 r / min for 36 h to obtain the seed liquid.

[0067] b. Add 66 mL of the obtained seed liquid to the autoclaved liquid culture medium for primary fermentation. The fermentation temperature is 28℃, the rotation speed is 200 r / min, and the fermentation time is 24 h. After fermentation, the primary fermentation broth is obtained.

[0068] The liquid culture medium is prepared by adding 44g of glucose, 6.6g of yeast extract, 44g of peptone, and 6.6g of malt extract to 2.2L of distilled water.

[0069] c. Divide the obtained primary fermentation broth into 1.6L, 0.3L and 0.3L, and then introduce them into sterilized flaxseed oil, safflower oil and meadowfoam oil respectively at a volume ratio of 1:2. Continue fermentation for 72h at 28℃ and 200r / min.

[0070] d. After fermentation in step c is completed, each fermentation product is centrifuged at 6000 rpm for 10 min. After centrifugation, the supernatants are mixed to obtain fermented vegetable oil. At the same time, the lower aqueous phase is discarded and the bacterial cells are separated.

[0071] e. Wash the obtained bacterial cells with phosphate buffer, centrifuge at 6000 rpm for 10 min after washing, discard the supernatant after centrifugation, and repeat the washing twice to obtain clean bacterial cells.

[0072] The cleaned bacterial cells were taken and mixed with 220g of dried Haematococcus pluvialis powder to obtain Haematococcus pluvialis powder containing bacterial cells. Then, the Haematococcus pluvialis powder containing bacterial cells was added to the obtained fermented vegetable oil and mixed evenly. After mixing, it was homogenized twice under 1000 bar pressure using a high-pressure homogenizer (through homogenization, the cell walls of yeast cells and Haematococcus pluvialis powder were broken, and the fat-soluble components and active ingredients in the cells and Haematococcus pluvialis powder were extracted). After homogenization, it was centrifuged at 8000 rpm for 10 min, and the supernatant was collected to obtain the Haematococcus pluvialis powder / fermented vegetable oil extract, i.e., Haematococcus pluvialis extract.

[0073] Example 4:

[0074] The present invention relates to a plant composition with anti-aging activity for the skin, comprising 86g of Haematococcus pluvialis extract, 10g of Lithospermum erythrorhizon oil, 0.8g of Melaleuca alternifolia leaf oil, 0.8g of hydrolyzed olive leaf extract, 0.3g of Rhodiola rosea root extract, and 2.1g of bitter orange leaf oil.

[0075] The extraction method of the Haematococcus pluvialis extract is as follows:

[0076] a. Take one tube of Yeast Extract and add it to 150 mL of sterilized liquid culture medium (containing 3 g glucose, 0.45 g yeast extract, 3 g peptone and 0.45 g malt extract). Incubate the culture in a shaker at 28 ℃ and 200 r / min for 36 h to obtain the seed liquid.

[0077] b. Add 60 mL of the obtained seed liquid to the autoclaved liquid culture medium for primary fermentation. The fermentation temperature is 30℃, the rotation speed is 220 r / min, and the fermentation time is 36 h. After fermentation, the primary fermentation broth is obtained.

[0078] The liquid culture medium is prepared by adding 24g of glucose, 3.6g of yeast extract, 24g of peptone, and 3.6g of malt extract to 1.2L of distilled water.

[0079] c. Divide the obtained primary fermentation broth into 0.72L, 0.25L and 0.23L, and then introduce them into sterilized flaxseed oil, safflower oil and meadowfoam oil respectively in a 1:1 volume ratio. Continue fermentation for 72h at 35℃ and 200r / min.

[0080] d. After fermentation in step c is completed, each fermentation product is centrifuged at 6000 rpm for 10 min. After centrifugation, the supernatants are mixed to obtain fermented vegetable oil. At the same time, the lower aqueous phase is discarded and the bacterial cells are separated.

[0081] e. Wash the obtained bacterial cells with phosphate buffer, centrifuge at 6000 rpm for 10 min after washing, discard the supernatant after centrifugation, and repeat the washing twice to obtain clean bacterial cells.

[0082] The cleaned bacterial cells were taken and mixed with 60g of dried Haematococcus pluvialis powder to obtain Haematococcus pluvialis powder containing bacterial cells. Then, the Haematococcus pluvialis powder containing bacterial cells was added to the obtained fermented plant oil and mixed evenly. After mixing, it was homogenized once at 1500 bar using a high-pressure homogenizer (through homogenization, the cell walls of yeast cells and Haematococcus pluvialis powder were broken, and the fat-soluble components and active ingredients in the cells and Haematococcus pluvialis powder were extracted). After homogenization, it was centrifuged at 8000 rpm for 10 min, and the supernatant was collected to obtain the Haematococcus pluvialis powder / fermented plant oil extract, i.e., Haematococcus pluvialis extract.

[0083] Example 5:

[0084] The present invention relates to a plant composition with anti-aging activity for the skin, comprising 84.4g of Haematococcus pluvialis extract, 12g of Lithospermum erythrorhizon oil, 0.6g of Melaleuca alternifolia leaf oil, 0.5g of hydrolyzed olive leaf extract, 0.5g of Rhodiola rosea root extract, and 2.0g of bitter orange leaf oil.

[0085] The extraction method of the Haematococcus pluvialis extract is as follows:

[0086] a. Take one vial of Yeast Extract and add it to 150 mL of sterilized liquid culture medium (containing 3 g glucose, 0.45 g yeast extract, 3 g peptone and 0.45 g malt extract). Incubate the medium in a shaker at 30 °C and 200 r / min for 36 h to obtain the seed culture.

[0087] b. Add 75 mL of the obtained seed liquid to the autoclaved liquid culture medium for primary fermentation. The fermentation temperature is 30℃, the rotation speed is 200 r / min, and the fermentation time is 24 h. After fermentation, the primary fermentation broth is obtained.

[0088] The liquid culture medium is prepared by adding 30g of glucose, 4.5g of yeast extract, 30g of peptone, and 4.5g of malt extract to 1.5L of distilled water.

[0089] c. Divide the obtained primary fermentation broth into 0.7L, 0.4L and 0.4L, and then introduce them into sterilized flaxseed oil, safflower oil and meadowfoam oil respectively at a volume ratio of 1:2. Continue fermentation for 48h at 30℃ and 200r / min.

[0090] d. After fermentation in step c is completed, each fermentation product is centrifuged at 6000 rpm for 10 min. After centrifugation, the supernatants are mixed to obtain fermented vegetable oil. At the same time, the lower aqueous phase is discarded and the bacterial cells are separated.

[0091] e. Wash the obtained bacterial cells with phosphate buffer, centrifuge at 6000 rpm for 10 min after washing, discard the supernatant after centrifugation, and repeat the washing twice to obtain clean bacterial cells.

[0092] The cleaned bacterial cells were taken and mixed with 200g of dried Haematococcus pluvialis powder to obtain Haematococcus pluvialis powder containing bacterial cells. Then, the Haematococcus pluvialis powder containing bacterial cells was added to the obtained fermented vegetable oil and mixed evenly. After mixing, it was homogenized twice under 1200 bar pressure using a high-pressure homogenizer (through homogenization, the cell walls of yeast cells and Haematococcus pluvialis powder were broken, and the fat-soluble components and active ingredients in the cells and Haematococcus pluvialis powder were extracted). After homogenization, it was centrifuged at 8000 rpm for 10 min, and the supernatant was collected to obtain the Haematococcus pluvialis powder / fermented vegetable oil extract, i.e., Haematococcus pluvialis extract.

[0093] Example 6:

[0094] An anti-aging face cream was formulated using the plant composition with anti-aging activity described in Example 1. The mass percentage of each component in the anti-aging face cream is as follows: 14% of the plant composition described in Example 1, 2% of glyceryl stearate, 3.7% of cetearyl alcohol, 4% of isohexadecane, 67% of birch sap, 6% of glycerin, 0.1% of sodium polyacrylate, 0.2% of allantoin, 0.1% of xanthan gum, 1.5% of tocopherol, 0.4% of p-hydroxyacetophenone, and 1% of 1,2-hexanediol.

[0095] Example 7:

[0096] An anti-aging emulsion was formulated using the plant composition with anti-aging activity for skin described in Example 2. The mass percentage of each component in the anti-aging emulsion was as follows: 7.5% of the plant composition described in Example 2, 2% of glyceryl stearate, 1.5% of cetearyl alcohol, 5% of isohexadecane, 73.6% of water, 6% of glycerin, 0.2% of sodium polyacrylate, 0.3% of allantoin, 0.4% of carbomer 940, 1% of tocopherol, 0.5% of p-hydroxyacetophenone, and 2% of 1,2-hexanediol.

[0097] Example 8:

[0098] An anti-aging emulsion was formulated using the plant composition with anti-aging activity for skin described in Example 3. The mass percentage of each component in the anti-aging emulsion was as follows: 9.5% of the plant composition described in Example 3, 1% of glyceryl stearate, 1% of cetearyl alcohol, 5% of isohexadecane, 71.5% of birch sap, 8% of glycerin, 0.1% of sodium polyacrylate, 0.3% of allantoin, 0.2% of carbomer 940, 1% of tocopherol, 0.4% of p-hydroxyacetophenone, and 2% of 1,2-hexanediol.

[0099] Example 9:

[0100] An anti-aging face cream was formulated using the plant composition with anti-aging activity described in Example 4. The mass percentage of each component in the anti-aging face cream is as follows: 18% of the plant composition described in Example 4, 2.5% of glyceryl stearate, 6% of cetearyl alcohol, 5% of isohexadecane, 59.5% of water, 5% of glycerin, 0.1% of sodium polyacrylate, 0.4% of allantoin, 0.1% of xanthan gum, 1.0% of tocopherol, 0.4% of p-hydroxyacetophenone, and 2.0% of 1,2-hexanediol.

[0101] Example 10:

[0102] An anti-aging essence oil was formulated using the plant composition with anti-aging activity for skin described in Example 5. The mass percentage of each component in the anti-aging essence oil was as follows: 20% of the plant composition described in Example 5, 20% of low erucic acid rapeseed oil, 29% of sweet almond oil, 30% of jojoba seed oil, and 1% of tocopherol.

[0103] Example 11:

[0104] An anti-aging essence oil was formulated using the plant composition with anti-aging activity for skin described in Example 1. The mass percentage of each component in the anti-aging essence oil is as follows: 15% of the plant composition described in Example 1, 8% of flaxseed oil, 10% of camellia seed oil, 30% of meadowfoam seed oil, 34.5% of jojoba seed oil, and 2.5% of tocopherol.

[0105] The relevant experiments and results of the raw materials and products used in the technical solution of this invention are as follows:

[0106] I. Determination of free fatty acids in vegetable oils before and after fermentation: The free fatty acid components and their relative contents before and after fermentation of various vegetable oils were qualitatively determined by GC-MS.

[0107] 1. Sample pretreatment method: Accurately transfer 300 μL of fermented vegetable oil obtained by the extraction method in Example 1 and 300 μL of unfermented vegetable oil, respectively, and add 300 μL of methanol and 50 μL of glacial acetic acid solution for vortex extraction for 3 min. Centrifuge at 4℃ and 10000 rpm for 5 min. After centrifugation, transfer 150 μL of supernatant, add 8 mL of 2% sodium hydroxide methanol solution, heat at 85℃ water bath until the oil droplets disappear, then add 7 mL of 15% boron trifluoride methanol solution, continue heating for 2 min, cool to room temperature, add 10 mL of n-hexane, shake for 2 min, add 5 mL of saturated sodium chloride aqueous solution, let stand for separation, aspirate the upper n-hexane solution, add an appropriate amount of anhydrous sodium sulfate for dehydration, let stand for 30 min, take the supernatant, filter and wait for analysis.

[0108] 2. Measurement method:

[0109] Gas chromatography conditions: Shimadzu GC-MS was performed using an Rtx-5MS capillary column (30m × 0.25mm, 0.25μm). Split mode was used with a split ratio of 20:1. The injection port temperature was 330℃, and helium was used as the carrier gas at a total flow rate of 62.1 mL / min. Temperature program: Initial temperature 180℃, held for 5 min, then increased to 230℃ at a rate of 2℃ / min, held for 1 min; subsequently increased to 330℃ at a rate of 10℃ / min, held for 7 min.

[0110] Mass spectrometry conditions: full scan mode, EI ionization source, ion source temperature 250℃, interface temperature 230℃, solvent delay 2 min, scan range 40~1000m / z.

[0111] 3. Measurement results:

[0112] The results of the determination of free fatty acid composition before and after fermentation of a single vegetable oil show that (see appendix for details). Figure 1Before fermentation, flaxseed oil, safflower oil, and meadowfoam oil had extremely low levels of free fatty acids. However, after fermentation, the total ion chromatograms showed significant changes compared to the original vegetable oils, and various polar components of free fatty acids were detected.

[0113] Table 1 details the main free fatty acid polar components and their peak area percentages in the fermented vegetable oils. As shown in Table 1, there are significant differences in the free polar components among fermented flaxseed oil, fermented safflower oil, and fermented meadowfoam oil. Fermented flaxseed oil is dominated by linolenic acid and linoleic acid, accounting for 76% of the total polar components. Fermented safflower oil and fermented meadowfoam oil are both dominated by linoleic acid, accounting for 76.23% and 60.86% of the total polar components, respectively.

[0114] Table 1. Composition and percentage of major free fatty acids in various fermented vegetable oils

[0115]

[0116] II. Determination of Acid Value in Fermented Vegetable Oils:

[0117] The combined fermented vegetable oils obtained in step d of the extraction methods described in Examples 1, 2, and 4 were used. For Example 1 (control), flaxseed oil, safflower oil, and meadowfoam oil were directly mixed in a ratio of 18:3:4. For Example 2 (control), the same mixture was used in a ratio of 10:3:2. For Example 4 (control), the same mixture was used in a ratio of 72:25:23. The acid value of each sample was determined using the first method (cold solvent indicator titration) according to national standard GB 5009.229. The results are detailed in Table 2.

[0118] Table 2 Results of acid value determination for each sample

[0119] sample Acid value (mg KOH / 100g) Example 1: Mixed Fermented Vegetable Oil 24.42±1.17 Example 2: Mixed Fermented Vegetable Oil 18.33±0.38 Example 4: Mixed Fermented Vegetable Oil 36.85±0.63 Example 1 Comparison 0.99±0.07 Example 2 Comparison 0.87±0.04 Example 4 (Comparative) 0.80±0.05

[0120] As shown in Table 2, the acid value of the samples from the control groups of Example 1, Example 2, and Example 4 was low because they were only physically mixed without any other treatment. However, the acid value of the vegetable oil increased significantly after fermentation with Yeast lipolyticis. This is because the lipase secreted by the yeast during fermentation degraded some of the triglycerides into free fatty acids.

[0121] III. Determination of Astaxanthin Content in Haematococcus pluvialis Extract:

[0122] 1. Sample preparation: The test sample was the Haematococcus pluvialis extract prepared in Example 1.

[0123] Preparation of control sample: Weigh an appropriate amount of Haematococcus pluvialis powder and add it to the control solvent of Example 1 (a mixture of flaxseed oil, safflower seed oil and meadowfoam seed oil in a ratio of 18:3:4) at a ratio of 1:15 (g:mL). Homogenize under high pressure with the same parameters as in Example 1 to prepare Haematococcus pluvialis extract and obtain the control sample of Example 1.

[0124] Both the test samples and the control samples were diluted 100 times before measurement.

[0125] 2. Standard Curve Construction: The astaxanthin content was determined using ultraviolet-visible spectrophotometry. An appropriate amount of astaxanthin standard was prepared into a 240 mg / L stock solution using the control solvent from Example 1. An appropriate amount of the stock solution was diluted to 4, 6, 8, 10, and 12 mg / L using the combined fermented vegetable oil obtained in step d of the Haematococcus pluvialis extract extraction method in Example 1 as the solvent. A full-wavelength scan was performed at the 10 mg / L concentration point, and the optimal detection wavelength was selected to construct the astaxanthin standard curve, which was used to determine the astaxanthin content in the Haematococcus pluvialis extract from Example 1. An appropriate amount of the stock solution was diluted to 4, 6, 8, 10, and 12 mg / L using the control solvent from Example 1 as the solvent. A full-wavelength scan was performed at the 10 mg / L concentration point, and the optimal detection wavelength was selected to construct the astaxanthin standard curve, which was used to determine the astaxanthin content in the control samples.

[0126] 3. Measurement Results: Using the control solvent of Example 1 and the astaxanthin standard solution prepared from the combined post-fermented vegetable oil obtained in step d of Example 1, UV-Vis spectroscopy was performed in the range of 340–800 nm. The maximum absorption peak in both cases was 486 nm (see Appendix for details). Figure 2 The absorbance values ​​of the astaxanthin standard solutions at the same concentration (10 mg / L) were all 0.612. However, it can also be seen that the absorbance of the astaxanthin standards obtained by scanning with different solvents varies across the entire wavelength range. The standard curve for astaxanthin content determination plotted using the absorbance values ​​at a wavelength of 486 nm was Y = 0.0687X - 0.0886(R²). 2 =0.9983). Calculations showed that the astaxanthin content in the control sample was (729.02±13.12) mg / L, and the astaxanthin content in the Haematococcus pluvialis extract of Example 1 was (892.48±4.73) mg / L, which was approximately 22% higher than that of the control sample.

[0127] Astaxanthin in Haematococcus pluvialis exists in both esterified and free forms, with the esterified form being predominant, stored in cytoplasmic liposomes rich in triglycerides. Therefore, compared with conventional extraction reagents such as anhydrous ethanol and acetone, using vegetable oil as a solvent is more conducive to the extraction of astaxanthin from Haematococcus pluvialis. Since fermented vegetable oils are slightly more polar than the original vegetable oils, they exhibit better extraction results for both forms of astaxanthin.

[0128] IV. Effects of the Examples on the Skin of Aging Model Rats:

[0129] 1. Animals: 24 male SD rats, weighing 180±20g, were purchased from Henan Skebest Biotechnology Co., Ltd. The rats were housed under appropriate conditions, with a temperature range of 20–25℃, relative humidity maintained at 40–70%, and a 12-hour light / dark cycle.

[0130] 2. Experimental methods:

[0131] 2.1 Animal Grouping and Model Establishment: After a one-week acclimatization period, rats were randomly divided into four groups: control group, model group, Example 9 group, and Example 10 group. Each rat was housed individually and hair was removed using an electric shaver, with a removal area of ​​4cm × 4cm. Hair removal was performed periodically based on the rats' hair growth. The hair removal procedure must not damage the skin.

[0132] A rat aging model was established by subcutaneous injection of D-galactose into the neck of rats. Except for the control group, rats in all other groups received subcutaneous injections of D-galactose 200 mg / kg / day, once daily for 42 consecutive days. The control group received physiological saline. The injection volume was 5 mL / kg for all rats.

[0133] 2.2 Animal Administration: Rats were administered the drug transdermally by applying it to the hair-removed areas. During the experiment, the control and model groups were treated with 0.1 mL / day of distilled water, while groups in Examples 9 and 10 were treated with 0.2 g / rat / day, for 42 consecutive days. Skin changes in each group were observed.

[0134] 2.3 Sample Collection: After the experiment, rats were euthanized by cervical dislocation. Hairless skin was quickly harvested from the back, and excess subcutaneous fat was carefully removed. A portion of the skin was fixed in a 10% formaldehyde solution for histomorphological analysis. A portion of the skin tissue was used to detect antioxidant indices in the skin.

[0135] 2.4 Determination of Antioxidant Indicators: A portion of skin tissue was collected, and a homogenate was prepared by adding pre-cooled physiological saline at a ratio of 1:9 (mg:μL). The homogenate was centrifuged at 4℃ and 5000 r / min for 10 min, and the supernatant was collected. The levels of SOD and MDA in each sample were measured according to the kit instructions.

[0136] 2.5 Skin tissue morphological examination: After fixation with paraformaldehyde, skin tissue was routinely dehydrated, paraffin-embedded, embedded, and sectioned for HE staining and Masson's trichrome staining. Changes in the epidermis and dermis of rat skin tissue in each group, as well as structural changes in collagen fibers in the dermis, were observed. For Masson-stained sections, four fields of view were selected under the same magnification, and the collagen fiber area ratio was measured using Image-pro Plus 6.0.

[0137] 2.6 Statistical Analysis: Experimental data are expressed as mean ± standard deviation. The statistical analysis was performed using SPSS (V.25.0). When the data followed a normal distribution and showed homogeneous variance, the data were calculated and analyzed using one-way ANOVA, followed by a post-hoc LSD test for the three group comparisons. For non-normally distributed data, the Tamhane's 2-test was used, with a p-value <0.05 considered statistically significant.

[0138] 3. Experimental Results:

[0139] 3.1 D-galactose-induced skin aging: The D-galactose skin injection aging model is a commonly used experimental skin aging model. Its principle is that D-galactose metabolism produces a large amount of reactive oxygen species, which leads to oxidative damage, metabolic disorders and functional impairment of cells. At the same time, the continuous accumulation of D-galactose metabolites leads to an increase in cell osmotic pressure, which further triggers oxidative stress and ultimately leads to aging of the body.

[0140] Compared to the control group, the model group rats exhibited more pronounced wrinkles and dryness. During the experiment, the skin of the model group rats was noticeably loose and thinner when handled. In Examples 9 and 10, the skin of the rats in the drug-treated groups was relatively firm, with fewer wrinkles compared to the model group.

[0141] 3.2 Effects of different formulations on antioxidant indices in the skin of aging rats (see Table 3 for details): SOD and MDA are two of the most commonly used antioxidant indicators in the body and important biomarkers for assessing the skin's oxidative stress state, closely related to skin aging. SOD is responsible for scavenging superoxide free radicals, converting them into hydrogen peroxide (H2O2) and oxygen (O2), thereby protecting cells from oxidative damage. If SOD activity decreases and free radicals cannot be scavenged in time, it can lead to the accumulation of free radicals, degradation of collagen in skin cells, and accelerated skin aging. MDA is the end product of lipid peroxidation, which can cross-link with collagen, destroying its structure, leading to decreased skin elasticity and wrinkle formation.

[0142] The results of this experiment showed that after continuous percutaneous injection of D-galactose, the SOD activity in the skin tissue of rats in the model group was significantly decreased compared with that in the control group (P<0.01), while the MDA level was significantly increased (P<0.01). Simultaneous intervention with different formulations during modeling significantly inhibited D-galactose-induced skin aging, as evidenced by significantly increased SOD activity in the skin tissue of rats in Examples 9 and 10 compared with the model group (P<0.01; P<0.01), while significantly decreased MDA levels (P<0.01; P<0.01). These results indicate that Examples 9 and 10 can effectively scavenge free radicals, reduce oxidative stress, enhance antioxidant defense, reduce oxidative damage, and restore the oxidation-antioxidant balance, thereby protecting skin structure and function.

[0143] Table 3. Effects of different formulations on antioxidant indices in rats

[0144]

[0145] (Note: Compared with the control group,) * P<0.05, ** P<0.01; compared with the model group, # P<0.05, ## P<0.01).

[0146] 3.3 Effects of different formulations on the structure of skin tissue in aging rats: Details of HE and Masson staining results for skin tissues from different groups of rats are provided in the appendix. Figure 3 .Depend on Figure 3 It was found that the skin tissue structure of the control group rats was normal, with uniform epidermal thickness and a clear boundary between the epidermis and dermis. Hair follicles, sebaceous glands, and hair roots were visible in the dermis, and collagen fibers were neatly arranged. The model group rats showed significant pathological changes in skin structure, with thinner and irregular epidermis, looser collagen fiber arrangement, and some areas lacking or breaking collagen fibers. The proportion of collagen fibers decreased by approximately 36.09% compared to the normal group, showing a significant difference (P<0.01). Compared to the model group, the skin tissue structure of rats in Examples 9 and 10 was relatively intact, with a relatively thicker epidermis and a more compact and neat arrangement of collagen fibers in the dermis. The proportion of collagen fibers increased by 30.07% and 22.69% respectively compared to the model group. Previous studies have found that collagen fibers in the dermis are an important factor in forming the skin's reticular scaffold, maintaining skin thickness and firmness. Decreased collagen fiber density and reduced cross-linking can lead to insufficient support in the dermis, resulting in thinner, looser, and less elastic skin, and the epidermis is prone to folding and wrinkle formation. In this experiment, the sample group showed a significant increase in the proportion of collagen fiber area (see Table 4 for details), indicating that it has a good anti-aging effect on the skin.

[0147] Table 4. Area ratio of collagen fibers in rat dermal tissue

[0148]

[0149] (Note: Compared with the control group,) * P<0.05, ** P<0.01; compared with the model group, # P<0.05, ## P<0.01).

[0150] V. Consumer Testing Experiments:

[0151] Consumer testing of the product of this invention was conducted using a questionnaire survey. Questions related to product efficacy in the questionnaire included "How moisturized was your skin after use?", "How effective do you think this product is at anti-aging for your skin?", and "Did you experience any allergic reactions or discomfort after using the product?".

[0152] 1. Sample Information: The test sample is Example 6 (50g / bottle).

[0153] 2. Test population information:

[0154] Target audience: women aged 30-60 who have anti-aging needs.

[0155] Exclusion criteria: Severely sensitive skin, or those who have undergone cosmetic medical treatment within the past 6 months.

[0156] Consumers tested: 33 in total, 26 females and 7 males. Age range: 30-45 years old, with an average age of 35.8 ± 3.8 years for the total population, an average age of 36.4 ± 5.4 years for males, and an average age of 35.6 ± 3.3 years for females.

[0157] 3. Requirements during the testing period: During the testing period, participants must not take orally or apply any other preparations that claim to have anti-wrinkle, skin-tightening, or similar claims; they are prohibited from undergoing cosmetic surgery that may affect the anti-wrinkle efficacy test; test participants should primarily engage in indoor activities and avoid prolonged exposure to outdoor sunlight.

[0158] 4. Test conditions and procedures:

[0159] Sample usage instructions: Use as a substitute for your daily face cream. After cleansing in the morning and evening, apply an appropriate amount evenly to the face. The sample should be used for 4 weeks. Please provide feedback promptly after the sample is used up during the testing period, and a replacement will be sent. Complete and submit the questionnaire after 4 weeks.

[0160] 5. Consumer Testing Results: Questionnaires were collected after the testing period ended, and the results were analyzed. Regarding the question, "How moisturized was your skin after use?", 28 consumers (84.8%) chose "quite moisturizing, skin felt quite comfortable," and 5 consumers (15.2%) chose "very moisturizing, skin felt very hydrated." Regarding the question, "How effective do you think this product is for your skin's anti-aging?", 21 consumers (63.6%) chose "quite good, some effect," 3 consumers (9.1%) chose "very good, significant effect," and 9 consumers (27.3%) chose "average, no obvious effect." Regarding the question, "Did you experience any allergic reactions or discomfort after using the product?", 33 consumers (100%) chose "no, very satisfied."

[0161] The above experimental results show that the fermented vegetable oil prepared by the method described in this invention exhibits significant changes in physicochemical properties compared to the original vegetable oil. This invention uses a mixture of fermented flaxseed oil, fermented safflower oil, and fermented meadowfoam oil as one of the important functional components, as well as an extraction solvent for astaxanthin and other active ingredients from Haematococcus pluvialis, achieving high extraction efficiency for astaxanthin. Based on this, it is compounded with other plant-based functional raw materials, and animal and human tests both demonstrate good anti-aging activity.

Claims

1. A plant composition with anti-aging skin activity, characterized in that, The active ingredients in the plant composition are Haematococcus pluvialis extract, Lithospermum erythrorhizon oil, Melaleuca alternifolia leaf oil, hydrolyzed olive leaf extract, Rhodiola rosea root extract, and bitter orange leaf oil, expressed as a percentage by mass. The proportions of each active ingredient are as follows: Haematococcus pluvialis extract 84.0–89.0%, Lithospermum erythrorhizon oil 8.0–12.0%, Melaleuca alternifolia leaf oil 0.5–0.8%, hydrolyzed olive leaf extract 0.5–1.0%, Rhodiola rosea root extract 0.1–0.5%, and bitter orange leaf oil 1.2–2.5%. The specific extraction method for the Haematococcus pluvialis extract is as follows: a. Using Yeast Extract as the fermentation strain, the fermentation strain was inoculated into a liquid culture medium and cultured aerobically in a shaker at a temperature of 28–35°C for 16–36 h. Seed liquid was obtained after culture. b. Add the obtained seed liquid to the sterilized liquid culture medium at a volume ratio of 3-5% for primary fermentation. The fermentation temperature is 28-35℃ and the fermentation time is 24-48h. After fermentation, the primary fermentation liquid is obtained. c. Sterilize each vegetable oil separately, then add the sterilized vegetable oil to the obtained primary fermentation broth and continue fermentation for 24-72 hours at a temperature of 28-35℃. The volume ratio between the primary fermentation broth and the sterilized vegetable oils is 1:1 to 2; the vegetable oils are flaxseed oil, meadowfoam seed oil, or safflower seed oil. d. After fermentation in step c is completed, each fermentation product is centrifuged at 6000 rpm for 10 min. After centrifugation, the supernatants are mixed to obtain fermented vegetable oil. At the same time, the lower aqueous phase is removed to separate the bacterial cells. e. Wash the obtained bacterial cells with phosphate buffer, centrifuge at 4000-6000 rpm for 10 min after washing, discard the supernatant after centrifugation, and repeat the washing twice to obtain clean bacterial cells after washing. The obtained clean bacterial cells were mixed with dried Haematococcus pluvialis powder to obtain Haematococcus pluvialis powder containing bacterial cells. Then, the Haematococcus pluvialis powder containing bacterial cells was added to the obtained fermented vegetable oil and mixed evenly. After mixing, it was homogenized 1-2 times using a high-pressure homogenizer at a pressure of 1000-1500 bar. After homogenization, it was centrifuged at 8000 rpm for 10 min, and the supernatant was collected to obtain the Haematococcus pluvialis extract. The ratio of Haematococcus pluvialis powder to fermented vegetable oil is 1g:15-20mL.

2. The plant composition with anti-aging skin activity according to claim 1, characterized in that: During fermentation as described in step a, the shaking speed of the shaker is controlled to be 160–220 rpm.

3. A formulation of a plant composition with anti-aging skin activity, characterized in that, The preparation is formulated into an emulsion, cream, or essential oil from the plant composition with anti-aging activity of the skin as described in claim 1.

4. The formulation of the plant composition with anti-aging skin activity according to claim 3, characterized in that, The plant composition emulsion with anti-aging skin activity comprises the following components in the indicated weight percentages: 6.7–13.2% of the plant composition with anti-aging skin activity as described in claim 1, 1.0–2.0% of glyceryl stearate, 1.0–1.5% of cetearyl alcohol, 2.0–5.0% of isohexadecane, 62.9–81.2% of water / birch sap, 5.0–10.0% of glycerin, 0.1–0.2% of sodium polyacrylate, 0.2–0.4% of allantoin, 0.2–0.4% of carbomer 940, 1.0–1.5% of tocopherol, 0.4–0.6% of p-hydroxyacetophenone, and 1.0–2.5% of 1,2-hexanediol.

5. The formulation of the plant composition with anti-aging skin activity according to claim 3, characterized in that, The plant composition preparation cream with anti-aging skin activity is prepared from the following components in the indicated weight percentages: 9.0-18.0% of the plant composition with anti-aging skin activity as described in claim 1, 1.5-2.5% of glyceryl stearate, 3.0-9.0% of cetearyl alcohol, 4.0-6.0% of isohexadecane, 55.0-74.5% of water / birch sap, 4.0-10.0% of glycerin, 0.1-0.2% of sodium polyacrylate, 0.2-0.4% of allantoin, 0.1-0.2% of xanthan gum, 1.0-1.5% of tocopherol, 0.4-0.6% of p-hydroxyacetophenone, and 1.0-2.0% of 1,2-hexanediol.

6. The formulation of the plant composition with anti-aging skin activity according to claim 3, characterized in that, The essential oil of the plant composition preparation with anti-aging skin activity is composed of the following components in the following mass percentages: 14.0-26.0% of the plant composition with anti-aging skin activity as described in claim 1, 72.0-85.0% of the base oil and 0.5-3.5% of tocopherol.

7. The formulation of the plant composition with anti-aging skin activity according to claim 6, characterized in that: The base oil is at least one of the following: flaxseed oil, linseed oil, low erucic acid rapeseed oil, jojoba seed oil, perilla seed oil, peony seed oil, meadowfoam seed oil, safflower seed oil, camellia seed oil, and sweet almond oil.

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