Plant cell active substance and transdermal absorption enhancer composition composed of the same and application thereof

Abstract

The present invention discloses a plant cell active ingredient, comprising plant cells and an active ingredient. This invention utilizes plant cells prepared using a specific content and a specific preparation method, supplemented with a specific content of an active ingredient, and then encapsulates the active ingredient with a plant cell extract to prepare a plant cell active ingredient with improved skin absorption. This active ingredient is then mixed with a polyol to prepare a transdermal absorption enhancer composition. This composition is then added to a basic cosmetic formula, resulting in significantly improved permeability, short-term moisturizing properties, long-term moisturizing properties, skin barrier function, and in vitro anti-aging efficacy of the resulting cosmetic.

Description

Technical Field

[0001] The present invention relates to the technical field of cosmetics, and in particular to a plant cell active substance and a transdermal absorption enhancer composition composed of the plant cell active substance, as well as a preparation method and application thereof. Background Art

[0002] Transdermal absorption enhancers work by modifying the barrier function of the skin's stratum corneum, overcoming this barrier and increasing the transdermal absorption of active ingredients. Chemically derived transdermal absorption enhancers include alcohol, azone, and silicone oil. However, these chemically derived transdermal absorption enhancers have a fat-dissolving and fat-removing effect, which can damage the skin's protective sebum membrane and contribute to skin sensitivity. People with sensitive skin prone to redness and itching should avoid using skin care products containing penetrants.

[0003] In recent years, plant-derived transdermal absorption enhancers have attracted increasing attention due to their minimal side effects. Currently used in cosmetics, plant-derived transdermal absorption enhancers include oleanole, eucalyptus oil, peppermint oil, angelica oil, and clove oil. However, these enhancers are not as effective as chemical-derived transdermal absorption enhancers, and excessive use can cause skin irritation. Summary of the Invention

[0004] In order to overcome the defects in the prior art that chemical-derived transdermal absorption enhancers have fat-dissolving and fat-removing effects, which will destroy the sebum membrane of the skin's protective layer and easily lead to skin sensitivity, and that plant-derived transdermal absorption enhancers are not as good as chemical-derived transdermal absorption enhancers in promoting penetration and are prone to skin irritation when used in too high a dosage, the purpose of the present invention is to provide a plant cell active substance with better skin absorption. After mixing the plant cell active substance with a polyol, a transdermal absorption enhancer composition is prepared. The composition is added to the basic formula of cosmetics, so that the permeability, short-term moisturizing properties, long-term moisturizing properties, skin barrier function and in vitro anti-aging efficacy of the finally prepared cosmetics are significantly improved.

[0005] Another object of the present invention is to provide a method for preparing the above-mentioned plant cell active substance.

[0006] Another object of the present invention is to provide a transdermal absorption enhancer comprising the above-mentioned plant cell active ingredient for use in cosmetics, medicines, and medical devices.

[0007] The present invention adopts the following specific technical solutions:

[0008] A plant cell active substance comprises: plant cells and active ingredients; the active ingredients are selected from one or more of polysaccharides, amino acids, vitamins, polypeptides, ergothioneine, ectoine or their salts, and the active ingredients are located inside the plant cells.

[0009] The plant cells can be selected from one or more of Desert Rose leaf cells, Jasmine leaf cells, Ginseng cells, Grape flower cells, Leontopodium alpinum cells, Holly sea cells, Sea Fennel cells, Apple cells, Lavender cells, Vitex negundo cells, Lotus cells, Myrrh cells, and Gynostemma pentaphyllum / Luffa hybrid cells;

[0010] The polysaccharide is selected from one or more of hyaluronic acid, chondroitin sulfate, and heparin, wherein:

[0011] The hyaluronic acid substance is selected from one or more of hyaluronic acid or its salt, hyaluronic acid derivative or its salt;

[0012] The ectoine or its salt is selected from one or more of ectoine, ectoine methyl ester and hydroxyectoine.

[0013] A method for preparing a plant cell active substance, characterized by comprising the following steps:

[0014] 1) Co-culturing the plant cells and the active ingredient in a culture medium for 36 to 72 hours;

[0015] 2) After the culture, the culture medium is filtered to obtain plant cell active substances.

[0016] Furthermore, in step 1), relative to the combination of plant cells and active ingredients, the plant cells are 80-100wt%, preferably 91-99wt%, and more preferably 94-97wt%; the active ingredient is 0-20wt%, preferably 0.1-10wt%, and more preferably 0.5-5wt%.

[0017] Furthermore, in step 1), the culture medium is MS medium, B5 medium, or MS-H medium, the culture temperature is 20-40° C., and the pH is 3-7.

[0018] Application of the plant cell active substance in cosmetics, medicines and medical devices,

[0019] Preferably, the plant cell active substance is used in a transdermal absorption enhancer.

[0020] A transdermal absorption enhancer composition comprises the above-mentioned plant cell active substance and polyol.

[0021] The weight ratio of the plant cell active substance to the polyol is (1-3): (7-9);

[0022] The polyol is one or more of glycerol, butanediol, ethylene glycol, butene glycols, and propylene glycol.

[0023] Application of the transdermal absorption enhancer composition in cosmetics, medicines and medical devices.

[0024] A cosmetic comprising the transdermal absorption enhancer composition.

[0025] Use of the cosmetic in moisturizing, improving skin barrier function and / or anti-aging.

[0026] Effects of the Invention

[0027] The present invention encapsulates active ingredients through plant cells to prepare plant cell actives with better skin absorption. After mixing the plant cell actives with polyols, a transdermal absorption enhancer composition is prepared. The composition is added to the basic cosmetic formula, so that the permeability, short-term moisturizing properties, long-term moisturizing properties, skin barrier function and in vitro anti-aging efficacy of the final cosmetics are significantly improved. DETAILED DESCRIPTION

[0028] To facilitate understanding of the present invention, the present invention will be described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present invention more thoroughly understood.

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains. The terms used herein in the specification of the present invention are for the purpose of describing specific embodiments only and are not intended to limit the present invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0030] The present invention provides a plant cell active substance, comprising plant cells and active ingredients.

[0031] The active ingredient may be present inside the plant cells or outside the plant cells. In a preferred embodiment of the present invention, the active ingredient is present inside the plant cells.

[0032] Wherein, relative to the combination of the plant cells and the active ingredient, in terms of weight percentage, the plant cells are 80-100 wt %, and the active ingredient is 0-20 wt %.

[0033] For example, the plant cells can be 80wt%, 81wt%, 82wt%, 83wt%, 84wt%, 85wt%, 86wt%, 87wt%, 88wt%, 89wt%, 90wt%, 91wt%, 92wt%, 93wt%, 94wt%, 95wt%, 96wt%, 97wt%, 98wt%, 99wt%, 100wt% or any range therebetween. The active ingredient is 0-20wt%, for example, it can be 0wt%, 0.1wt%, 0.2wt%, 0.5wt%, 0.7wt%, 1.0wt%, 1.5wt%, 2.0wt%, 2.5wt%, 3.0wt%, 3.5wt%, 4.0wt%, 4.5wt%, 5.0wt%, 5.5wt%, 6.0wt%, 6.5wt%, 7.0wt%, 7.5wt%, 8.0wt%, 8.5wt%, 9.0wt%, 9.5wt%, 10wt%, 11wt%, 12wt%, 13wt%, 14wt%, 15wt%, 16wt%, 17wt%, 18wt%, 19wt%, 20wt% or any range therebetween.

[0034] The plant cells described herein refer to plant cell extracts, and further refer to plant cells obtained through plant callus culture techniques. These are undifferentiated tissue or cell masses generated by culturing tissue excised from a plant in a culture medium containing auxins, or by wounding or treating a wound site with auxins. These are then subcultured in new culture medium, allowing them to permanently divide and proliferate. This type of callus is an amorphous mass of tissue or cells that has lost the ability to initiate normal organ formation or tissue differentiation. Most are milk cells, but broadly speaking, it also includes plant tumor tissue generated by infection with bacteria such as Agrobacterium. Specifically, when cells proliferate within a plant, they immediately orient themselves, not only regularly forming specific tissues but also organs. However, callus tissue, which is an undifferentiated cell mass formed through tissue culture, has been interpreted as releasing the control that maintained its position until then upon exposure to external stimuli, such as various plant growth hormones, allowing the cells to proliferate freely. If the callus tissue obtained through dedifferentiation is placed in a liquid culture medium with the same composition and cultured with shaking, the cells separate and continue to proliferate in a suspended state. Can make its permanent division and proliferation by subculture this callus or cultured cell in new culture medium, thereby be different from the differentiated cell that dies by aging in intact plant body in essence.If the callus or cultured cell of subculture several generations is coated on the solid culture medium that removes multiple plant growth hormones, then sprout and root are born to make individual plant recover.This regenerated plant is derived from the division and proliferation of a cultured cell, and the root of this cultured cell is induced from hypocotyl or water tissue.In the present invention, callus can be induced from any part of plant, as a specific example, a part of petal, receptacle, fruit, ovary, placenta tissue, or can be the callus that extracts cotyledon and induces from the germinating seedling of seed.

[0035] In the present invention, the plant cells are obtained from dedifferentiated plant tissues, and the preparation method thereof is not limited. Those skilled in the art can select from commonly used callus preparation methods.

[0036] Membrane lipids are the primary components of plant cell membranes and include three major lipid classes: phospholipids, glycolipids, and cholesterol. Phospholipids contain polar phosphate groups and nonpolar hydrocarbon chains, resulting in a polar head and a nonpolar tail, making them bimodal molecules. Glycolipids are also bimodal molecules, with a structure very similar to sphingomyelin, except that one or more sugar groups replace the phosphatidylcholine. A cholesterol molecule consists of three parts: a hydroxyl group as the polar head, a steroid ring, and a nonpolar hydrocarbon tail. These components are structurally similar to lipids in human skin, thus enhancing the absorption of active ingredients through the skin. Those skilled in the art can select from commonly available plant cell types. This component can enhance the absorption of active ingredients through the skin. Regarding the type of plant cell, those skilled in the art can select from commonly available plant cell types.

[0037] In a preferred embodiment of the present invention, the plant cells are selected from one or more of Desert Rose leaf cells, Jasmine leaf cells, Grape flower cells, Ginseng cells, Leontopodium alpinum cells, Holly sea cells, Sea Fennel cells, Apple cells, Lavender cells, Vitex negundo cells, Lotus cells, Myrrh cells, and Gynostemma pentaphyllum / Luffa hybrid cells.

[0038] The method for preparing the plant cells of the present invention is not limited, and those skilled in the art can choose from commonly used methods for preparing callus tissue.

[0039] In a preferred embodiment of the present invention, a method for preparing plant cells is provided, comprising the following steps:

[0040] a) selecting a plant, removing a healthy plant tissue piece of a certain size, sterilizing the tissue slices, and inducing plant callus formation using a certain ratio of cytokinin and auxin;

[0041] b) applying stress to the callus tissue to induce the production of plant cells, and screening the effective fragment mother cells on the callus tissue;

[0042] c) culturing mother cells in large quantities in culture medium to replicate and preserve plant cells;

[0043] In step c), the culture medium is MS culture medium, B5 culture medium, or MS-H culture medium.

[0044] For example, the culture medium can be one of MS medium, B5 medium, or MS-H medium;

[0045] Furthermore, the culture medium further comprises 2,4-D (0.5-1.5 mg / L); PVP (0.8-1.2 g / L); activated carbon (AC, 0.5-1.5 g / L); sucrose (2-4%); 2,4-D can be 0.5 mg / L, 0.6 mg / L, 0.7 mg / L, 0.8 mg / L, 0.9 mg / L, 1.0 mg / L, 1.1 mg / L, 1.2 mg / L, 1.3 mg / L, 1.4 mg / L, 1.5 mg / L or therebetween. Any range; PVP can be 0.8g / L, 0.9g / L, 1.0g / L, 1.1g / L, 1.2g / L or any range therebetween; activated carbon (AC) can be 0.5g / L, 0.6g / L, 0.7g / L, 0.8g / L, 0.9g / L, 1.0g / L, 1.1g / L, 1.2g / L, 1.3g / L, 1.4g / L, 1.5g / L or any range therebetween; sucrose can be 2%, 3%, 4% or any range therebetween;

[0046] The pH of the culture medium is 3-7, preferably 5.5-6.5; the culture temperature is 20-40°C, preferably 25-28°C.

[0047] The pH can be 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.1, 6.2, 6.3, ℃, 32℃, 33℃, 34℃, 35℃, 36℃, 37℃, 38℃, 39℃, 40℃ or any range therebetween; the culture temperature can be 20℃, 21℃, 22℃, 23℃, 24℃, 25℃, 26℃, 27℃, 28℃, 29℃, 30℃, 31℃, 32℃, 33℃, 34℃, 35℃, 36℃, 37℃, 38℃, 39℃, 40℃ or any range therebetween.

[0048] In a preferred embodiment of the present invention, the culture medium is MS medium, further comprising: 2,4-D (1.0 mg / L); PVP (1.0 g / L); activated carbon (AC, 1.0 g / L); sucrose (5%); and pH 5.8.

[0049] The plant cells can be prepared by placing plant tissue in a culture medium for callus induction, and then culturing the obtained callus in a culture medium to obtain the plant cells. The culture medium can be selected from one of MS medium, B5 medium, or MS-H medium. The callus is cultured in the culture medium at a temperature of 20-40°C, a humidity of 50°-80°C, and protected from light for 3-7 days.

[0050] In one embodiment of the present invention, fresh plant tissue is sterilized, wounded, added to a culture medium, and cultured at 20-40°C and 50-500 rpm for 2-4 weeks to obtain callus tissue. The callus tissue is inoculated into a new culture medium, cultured in the dark at 20-40°C and a humidity of 50-80°C for 3-7 days, and then the culture medium is filtered to obtain plant cells. The culture medium can be selected from MS medium, B5 medium, or MS-H medium.

[0051] In one embodiment of the present invention, the plant cells can be prepared by: removing sterilized fresh plant tissue, wounding it, adding it to a culture medium, and culturing it at 20-40°C and 50-500 rpm for 2-4 weeks to obtain callus tissue. The callus tissue is inoculated into a new culture medium, incubated in the dark at 20-40°C and a humidity of 50-80°C for 3-7 days, and then filtering the culture medium to obtain plant leaf cells. The culture medium can be selected from one of MS medium, B5 medium, and MS-H medium.

[0052] In a preferred embodiment of the present invention, the Desert Rose leaf cell extract can be prepared in the following manner: take fresh Desert Rose leaves, cut them into small pieces and add them to a culture medium for cultivation. The culture temperature is 20-40°C, the humidity is 50°-80°C, and the medium is kept dark. Culture at 50-500rpm for 2-4 weeks to obtain Desert Rose leaf callus. Select Desert Rose leaf callus with bright color and loose tissue and inoculate it into a new sterilized culture medium. Set the culture temperature to 20-40°C, the humidity to 50°-80°C, and the medium is kept dark. After culturing for 3-7 days, filter the culture medium to obtain Desert Rose leaf cells. The culture medium can be selected from one of MS culture medium, B5 culture medium, or MS-H culture medium.

[0053] In a preferred embodiment of the present invention, the lotus cell extract can be prepared in the following manner: fresh lotus petal tissue is taken, cut into small pieces, and then added to a culture medium for cultivation at a temperature of 20-40°C, a humidity of 50°-80°C, and dark and dark. The culture is carried out at 50-500rpm for 2-4 weeks to obtain lotus callus tissue. Lotus callus tissue with bright color and loose tissue is selected and inoculated into a new sterilized culture medium. The culture temperature is set at 20-40°C, the humidity is set at 50°-80°C, and dark and dark. After culturing for 3-7 days, the culture medium is filtered to obtain lotus cells. The culture medium can be selected from one of MS culture medium, B5 culture medium, or MS-H culture medium.

[0054] The active ingredient can be a water-soluble active ingredient or an oil-soluble active ingredient. Those skilled in the art can choose from commonly used active ingredients in cosmetics, for example, it can be one or more of polysaccharides, amino acids, vitamins, polypeptides, ergothioneine, ectoine or their salts.

[0055] The polysaccharide is selected from one or more of hyaluronic acid substances, chondroitin sulfate, and heparin, wherein the hyaluronic acid substance is selected from one or more of hyaluronic acid or its salt, hyaluronic acid derivative or its salt; the ectoine or its salt is selected from one or more of ectoine, ectoine methyl ester, and hydroxyectoine.

[0056] Hyaluronic acid is a macromolecular substance widely present in various tissues of humans and animals. It is a linear macromolecular polysaccharide composed of glucuronic acid-N-acetylglucosamine as a disaccharide unit. The hyaluronic acid substance of the present invention can be one or more of hyaluronic acid or its salt, hyaluronic acid derivative or its salt. Wherein, the hyaluronic acid or its salt can be hyaluronic acid or its salt directly obtained by fermentation, or hyaluronic acid or its salt obtained by extraction technology, or oligomeric hyaluronic acid or its salt obtained by enzyme degradation, chemical degradation and other technologies, hydrolyzed hyaluronic acid or its salt, etc., can be hyaluronic acid, hydrolyzed hyaluronic acid, hyaluronic acid sodium salt, potassium salt, calcium salt, magnesium salt, zinc salt, silver salt, gold salt, etc. The hyaluronic acid derivative or hyaluronate derivative can be a substance obtained by chemical reactions such as grafting and cross-linking of hyaluronic acid or hyaluronate, such as acetylated hyaluronic acid or its salt, cross-linked hyaluronic acid or its salt, etc.

[0057] The molecular weight of the hyaluronic acid or its salt can be 0.5 kDa-3000 kDa, for example, 0.5 kDa, 1 kDa, 5 kDa, 10 kDa, 20 kDa, 30 kDa, 50 kDa, 100 kDa, 200 kDa, 300 kDa, 400 kDa, 500 kDa, 600 kDa, 700 kDa, 800 kDa, 900 kDa, 1000 kDa, 1500 kDa, 2000 kDa, 2500 kDa, 3000 kDa or any range therebetween;

[0058] The molecular weight of the acetylated hyaluronic acid or its salt may be 10 kDa-100 kDa, for example, 10 kDa, 20 kDa, 30 kDa, 40 kDa, 50 kDa, 60 kDa, 70 kDa, 80 kDa, 90 kDa, 100 kDa or any range therebetween;

[0059] In a preferred embodiment of the present invention, the hyaluronic acid substance is a composition of hydrolyzed hyaluronic acid and hydrolyzed sodium hyaluronate, the molecular weight of the hydrolyzed hyaluronic acid is 10k-100kDa, preferably 30k-60kDa; the molecular weight of the hydrolyzed sodium hyaluronate is 1k-10kDa.

[0060] For example, the molecular weight of the hydrolyzed hyaluronic acid can be 10 kDa, 20 kDa, 30 kDa, 40 kDa, 50 kDa, 60 kDa, 70 kDa, 80 kDa, 90 kDa, 100 kDa or any range therebetween; the molecular weight of the hydrolyzed sodium hyaluronate can be 1 kDa, 2 kDa, 3 kDa, 4 kDa, 5 kDa, 6 kDa, 7 kDa, 8 kDa, 9 kDa, 10 kDa or any range therebetween.

[0061] Ectoin (2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid, ectoin) is derived from the highly halophilic bacterium Halomonas Elongata and is the most widely distributed compatible solute discovered in the bacterial kingdom. It is compatible with intracellular metabolism and does not affect the functions of cellular biomacromolecules or physiological functions. It is an important osmotic pressure compensation solute. The ectoin or its salt described in the present invention is selected from one or more of ectoin, ectoin methyl ester, and hydroxyectoin.

[0062] The present invention provides a method for preparing plant cell active substances, which comprises the following steps:

[0063] 1) Co-culturing the plant cells and the active ingredient in a culture medium for 36 to 72 hours;

[0064] 2) After the culture, the culture medium is filtered to obtain plant cell active substances.

[0065] In a preferred embodiment of the present invention, relative to the combination of plant cells and active ingredients, the plant cells account for 80-100 wt%, preferably 91-99 wt%, and more preferably 94-97 wt%.

[0066] For example, it can be 80wt%, 81wt%, 82wt%, 83wt%, 84wt%, 85wt%, 86wt%, 87wt%, 88wt%, 89wt%, 90wt%, 91wt%, 92wt%, 93wt%, 94wt%, 95wt%, 96wt%, 97wt%, 98wt%, 99wt%, 100wt% or any range therebetween.

[0067] The active ingredient is 0-20 wt%, preferably 0.1-10 wt%, and more preferably 0.5-5 wt%.

[0068] For example, it can be 0wt%, 0.1wt%, 0.2wt%, 0.5wt%, 0.7wt%, 1.0wt%, 1.5wt%, 2.0wt%, 2.5wt%, 3.0wt%, 3.5wt%, 4.0wt%, 4.5wt%, 5.0wt%, 5.5wt%, 6.0wt%, 6.5wt%, 7.0wt%, 7.5wt%, 8.0wt%, 8.5wt%, 9.0wt%, 9.5wt%, 10wt%, 11wt%, 12wt%, 13wt%, 14wt%, 15wt%, 16wt%, 17wt%, 18wt%, 19wt%, 20wt% or any range therebetween.

[0069] In a preferred embodiment of the present invention, the active ingredient is a combination of hyaluronic acid substances and ectoine and its derivatives, and is further preferably a combination of hydrolyzed hyaluronic acid, hydrolyzed sodium hyaluronate and ectoine and its derivatives, wherein the hydrolyzed hyaluronic acid is 0.1-3wt%, the hydrolyzed sodium hyaluronate is 0.1-6wt%, and the ectoine and its derivatives are 0.1-5wt%.

[0070] For example, the hydrolyzed hyaluronic acid is 0.1wt%, 0.2wt%, 0.3wt%, 0.4wt%, 0.5wt%, 0.6wt%, 0.7wt%, 0.8wt%, 0.9wt%, 1.0wt%, 1.1wt%, 1.2wt%, 1.3wt%, 1.4wt%, 1.5wt%, 1.6wt%, 1.7wt%, 1.8wt%, 1.9wt%, 2.0wt%, 2.1wt%, 2.2wt%, 2.3wt%, 2.4wt%, 2.5wt%, 2.6wt%, 2.7wt%, 2.8wt%, 2.9wt%. %, 3.0wt% or any range therebetween; hydrolyzed sodium hyaluronate is 0.1wt%, 0.2wt%, 0.3wt%, 0.4wt%, 0.5wt%, 0.6wt%, 0.7wt%, 0.8wt%, 0.9wt%, 1.0wt%, 1.1wt%, 1.2wt%, 1.3wt%, 1.4wt%, 1.5wt%, 1.6wt%, 1.7wt%, 1.8wt%, 1.9wt%, 2.0wt%, 2.1wt%, 2.2wt%, 2.3wt%, 2.4wt%, 2.5wt%, 2.6wt%, 2.7wt%, 2.8wt%, 2.9wt%, 3.0wt%, 3.2wt%, 3.4wt%, 3.6wt%, 3.8wt%, 4.0wt%, 4.2wt%, 4.4wt%, 4.6wt%, 4.8wt%, 5.0wt%, 5.5wt%, 6.0wt% or any range therebetween; ectoine and its derivatives are 0.1wt%, 0.2wt%, 0.3wt%, 0.4wt%, 0.5wt%, 0.6wt%, 0.7wt%, 0.8wt%, 0.9wt%, 1.0wt%, 1.1wt%, 1. % , 2wt%, 1.3wt%, 1.4wt%, 1.5wt%, 1.6wt%, 1.7wt%, 1.8wt%, 1.9wt%, 2.0wt%, 2.1wt%, 2.2wt%, 2.3wt%, 2.4wt%, 2.5wt%, 2.6wt%, 2.7wt%, 2.8wt%, 2.9wt%, 3.0wt%, 3.2wt%, 3.4wt%, 3.6wt%, 3.8wt%, 4.0wt%, 4.2wt%, 4.4wt%, 4.6wt%, 4.8wt%, 5.0wt% or any range therebetween.

[0071] In a preferred embodiment of the present invention, the active ingredient is a hyaluronic acid-based substance, more preferably hydrolyzed hyaluronic acid or a salt thereof, for example, hydrolyzed sodium hyaluronate. The molecular weight of the hydrolyzed sodium hyaluronate is 1 kDa to 10 kDa, for example, the molecular weight of the hydrolyzed sodium hyaluronate can be 1 kDa, 2 kDa, 3 kDa, 4 kDa, 5 kDa, 6 kDa, 7 kDa, 8 kDa, 9 kDa, 10 kDa, or any range therebetween.

[0072] In step 1), the culture medium is MS medium, B5 medium, or MS-H medium.

[0073] Furthermore, the pH of the culture medium is 3-7, preferably 5.5-6.5; the culture temperature is 20-40°C, preferably 25-28°C.

[0074] For example, the pH can be 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.1, 6.2, 6. 3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0 or any range therebetween; and the culture temperature can be 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C or any range therebetween.

[0075] Furthermore, the culture medium also contains 2,4-D (0.5-1.5 mg / L); PVP (0.8-1.2 g / L); activated carbon (AC, 0.5-1.5 g / L); and sucrose (2-4%).

[0076] For example, the culture medium can be one of MS medium, B5 medium, or MS-H medium; the 2,4-D concentration can be 0.5 mg / L, 0.6 mg / L, 0.7 mg / L, 0.8 mg / L, 0.9 mg / L, 1.0 mg / L, 1.1 mg / L, 1.2 mg / L, 1.3 mg / L, 1.4 mg / L, 1.5 mg / L, or any range therebetween; the PVP concentration can be 0.8 g / L, 0. 9g / L, 1.0g / L, 1.1g / L, 1.2g / L or any range therebetween; activated carbon (AC) can be 0.5g / L, 0.6g / L, 0.7g / L, 0.8g / L, 0.9g / L, 1.0g / L, 1.1g / L, 1.2g / L, 1.3g / L, 1.4g / L, 1.5g / L or any range therebetween; sucrose can be 2%, 3%, 4% or any range therebetween.

[0077] In a preferred embodiment of the present invention, the culture medium is MS medium, further comprising: 2,4-D (1.0 mg / L); PVP (1.0 g / L); activated carbon (AC, 1.0 g / L); sucrose (5%); and pH 5.8.

[0078] The present invention provides the use of the above-mentioned plant cell active substances in cosmetics, medicines and medical devices.

[0079] The present invention provides a transdermal absorption enhancer composition comprising the above-mentioned plant cell active substance and polyol.

[0080] The weight ratio of the plant cell active substance to the polyol is (1-3):(7-9), for example, the weight ratio of the plant cell active substance to the polyol is 1:7, 2:7, 3:7, 1:8, 2:8, 3:8, 1:9, 2:9, 3:9 or any range therebetween.

[0081] Furthermore, the polyol is one of glycerol, butylene glycol, ethylene glycol, propylene glycol, pentanediol, hexanediol, pentaerythritol, polyethylene glycol, and panthenol.

[0082] In a preferred embodiment of the present invention, the weight ratio of the plant cell active substance to glycerol is 2:8.

[0083] The present invention provides the use of the above-mentioned plant cell active substance or the above-mentioned transdermal absorption enhancer composition in cosmetics, medicines and medical devices.

[0084] The present invention provides the use of the plant cell active substance in a transdermal absorption enhancer.

[0085] The present invention provides a cosmetic comprising the above-mentioned transdermal absorption enhancer composition.

[0086] The present invention provides uses of the above-mentioned cosmetics in moisturizing, improving skin barrier function and / or anti-aging.

[0087] The present invention uses the above-mentioned transdermal absorption enhancer composition to promote the absorption of active ingredients in skin care products, medicines, disinfection products, dressings or gel-type medical devices, thereby increasing the transdermal absorption rate of the active ingredients.

[0088] In the following specific embodiments, unless otherwise specified, all methods are conventional methods; the raw materials, reagents, etc. used in the following specific embodiments, unless otherwise specified, are commercially available products.

[0089] Desert rose leaf cell extract: France NAOLYS, trade name: HydraGlobal Desert rose Gly;

[0090] Jasmine leaf cell extract: NAOLYS, France, trade name: Essential Being Indian jasmine

[0091] Grape flower cell extract: France NAOLYS, trade name: Healthy Perfection (Vitis flower) 2 Gly;

[0092] Ectoin methyl ester: Bloomage Biotech Co., Ltd.;

[0093] Ectoine: Bloomage Biotech Co., Ltd., trade name Ectoine;

[0094] Hydrolyzed hyaluronic acid: Bloomage Biotech Co., Ltd., trade name Hybloom TM , molecular weight is 37~56KD;

[0095] Hydrolyzed sodium hyaluronate: Bloomage Biotech Co., Ltd., trade name microHA TM (molecular weight less than 5KD), trade name miniHA TM (molecular weight less than 10KD);

[0096] Olivem 1000: HALLSTAR BEAUTY AND PERSONAL CARE SOLUTIONS COMPANY; INCI: Cetearyl Olivate, Sorbitan Olivate

[0097] Behenyl Alcohol: BASF; INCI: Behenyl Alcohol

[0098] GTCC: BASF; INCI: Caprylic / Capric Triglyceride

[0099] Dicaprylyl Carbonate: Evonik Operations GmbH; INCI: Diethylhexyl Carbonate

[0100] DM5: Dow (Zhangjiagang) Investment Co., Ltd.; INCI: Dimethicone

[0101] Carbomer 980: Lubrizol Advanced Materials; INCI: Carbomer

[0102] Butanediol: OQ Chemicals Corporation; INCI: Butanediol

[0103] 1,2-Pentanediol: Symrise; INCI: 1,2-Pentanediol

[0104] Xanthan gum: Cosphatec; INCI: Xanthan gum

[0105] Triethanolamine: petronas chemicals marketing LTD; INCI: triethanolamine

[0106] Preparation Example

[0107] Examples A1-A5 and Comparative Examples B1-B3: Preparation of Transdermal Absorption Enhancer Compositions

[0108] According to the ratio in Table 1, plant cell extract, ectoine or its salt, and hyaluronic acid substances are added simultaneously to the culture medium and co-cultured for 36 to 72 hours; after the culture, the above culture medium is filtered to obtain plant cell active substances; the plant cell active substances and glycerol are added to the above obtained plant cell active substances at a mass ratio of 2:8 to glycerol, and the mixture is mixed to obtain a transdermal absorption enhancer composition.

[0109] Table 1 Distribution ratio of each group of Examples A1-A5 and Comparative Examples B1-B3 (wt%)

[0110]

[0111]

[0112] Examples C1-C6 and Comparative Example D1: Preparation of Culture Medium Containing Plant Cell Actives

[0113] Preparation of Desert Rose Leaf Cells: Take fresh Desert Rose leaves, soak them in 75% ethanol solution for 2 minutes, and rinse them with sterile water. Use sterile scissors to cut the sterilized Desert Rose leaves into small pieces with a side length of about 1 cm in a clean workbench, then add them to the sterilized B5 culture medium for callus induction, and then culture them. During culture, set the culture temperature to 28°C, humidity to 60°~70°, and dark away from light. Culture at 200rpm for 2 to 4 weeks to obtain Desert Rose leaf callus. Select Desert Rose leaf callus with bright color and loose tissue and inoculate it into a new sterilized B5 culture medium. Set the culture temperature to 28°C, humidity to 60°~70°, and dark away from light. After culturing for 3 to 7 days, filter and obtain Desert Rose leaf cells.

[0114] Preparation of lotus cells: Take fresh lotus petal tissue, soak it in 75% ethanol solution for 2 minutes, and rinse it with sterile water. Use sterile scissors to cut the disinfected petals into small pieces with a side length of about 1 cm in a clean workbench, then add them to sterilized B5 culture medium for callus induction, and then culture them. During the culture, set the culture temperature to 28°C, humidity to 60°~70°, and dark away from light. Culture at 200rpm for 2 to 4 weeks to obtain lotus callus tissue. Select lotus callus tissue with bright color and loose tissue and inoculate it into new sterilized B5 culture medium. Set the culture temperature to 28°C, humidity to 60°~70°, and dark away from light. After culturing for 3 to 7 days, filter and obtain lotus cells.

[0115] Example C1

[0116] The desert rose leaf cells were inoculated into the MSH liquid culture medium containing miniHA, and cultured at 150 rpm and 25°C for 36 h (relative to the combination of plant cells and active ingredients, by weight percentage, the desert rose leaf cells were 99% and the miniHA was 1%) to obtain a culture medium containing plant cell active substances.

[0117] Example C2

[0118] The only difference between Example C2 and Example C1 is that, relative to the combination of plant cells and active ingredients, the desert rose leaf cell extract is 94% and the miniHA is 6% by weight, and the rest are the same.

[0119] Example C3

[0120] The only difference between Example C3 and Example C1 is that, relative to the combination of plant cells and active ingredients, the desert rose leaf cell extract is 91% and the miniHA is 9% by weight, and the rest are the same.

[0121] Example C4

[0122] The only difference between Example C4 and Example C1 is that, relative to the combination of plant cells and active ingredients, the desert rose leaf cell extract is 83% and the miniHA is 17% by weight, and the rest are the same.

[0123] Example C5

[0124] The only difference between Example C5 and Example C3 is that the culture medium is placed at 250 rpm and 30° C. for 72 hours, and the rest is the same to obtain a culture medium containing plant cell active substances.

[0125] Example C6

[0126] The only difference between Example C6 and Example C3 is that the desert rose leaf cells are replaced with lotus cells, and the rest are the same.

[0127] Comparative Example D1

[0128] MiniHA and desert rose leaf cells were added to MSH liquid culture medium (relative to the combination of plant cells and active ingredients, by weight percentage, desert rose leaf cells accounted for 91% and miniHA accounted for 9%), and mixed evenly to obtain a mixture containing plant cells and active ingredients.

[0129] Comparative Example D2

[0130] MiniHA and lotus cells were added to MSH liquid culture medium (91% lotus cells and 9% miniHA by weight relative to the combination of plant cells and active ingredients), and mixed evenly to obtain a mixture containing plant cells and active ingredients.

[0131] Comparative Example D3

[0132] The only difference between Comparative Example D3 and Example C3 is that, relative to the combination of plant cells and active ingredients, the Desert Rose leaf cell extract is 50% and the miniHA is 50% by weight, and the rest are the same.

[0133] Table 2 Distribution ratio of each group of Examples C1-C6 and Comparative Examples D1-D3 (wt%)

[0134]

[0135] Examples A6-A11 and Comparative Examples B4-B6: Preparation of Transdermal Absorption Enhancer Compositions

[0136] The culture media containing plant cell actives obtained from C1-C6 and D1-D3 above were filtered to obtain plant cell actives, and glycerol was added at a mass ratio of 2:8 to the plant cell actives to obtain transdermal absorption enhancer compositions A6-A11 and B4-B6. The corresponding relationships are as follows:

[0137] Table 3

[0138]

[0139] Experimental Example 1

[0140] 1. Determination of the encapsulation rate of active ingredients in plant cell active substances

[0141] The plant cell actives from the culture medium containing plant cell actives obtained in Examples C1-C6 and Comparative Examples D1-D3 were uniformly dispersed in the culture medium. A 5 mL sample was taken from each Example and Comparative Example, and four samples were taken in parallel. Two of the samples were directly centrifuged, and the supernatant (M1) was measured for active ingredient content, labeled Q1. The other two samples were placed in an ultrasonic oscillator, and the cells were ultrasonically disrupted for 1 minute, followed by centrifugation. The supernatant (M2) was collected and the active ingredient content was measured, labeled Q2. The difference between Q1 and Q2 is the active ingredient content in the cells, and the percentage of the active ingredient content in the cells to the total active ingredient content is the encapsulation rate.

[0142] Determination method of sodium hyaluronate content:

[0143] Reagents: Sodium dihydrogen phosphate (Sinopharm Chemical Reagent Co., Ltd.), disodium hydrogen phosphate (Sinopharm Chemical Reagent Co., Ltd.), sodium hyaluronate reference substance (Bloomage Biotechnology Co., Ltd.), hyaluronidase (Bloomage Biotechnology Co., Ltd.)

[0144] Chromatographic conditions: chromatographic column: MCI GEL column (8×300 mm, 9 μm); mobile phase: 1% phosphoric acid; flow rate: 0.6 mL / min; injection volume: 20 μL; column temperature: 40°C; detection wavelength: 232 nm.

[0145] Enzyme hydrolysis buffer: Weigh 27.4 g of sodium dihydrogen phosphate (NaH2PO4·2H2O) and 8.8 g of disodium hydrogen phosphate (Na2HPO4·12H2O) into a 1000 mL volumetric flask. Dilute to volume with water and shake well to obtain 0.2 mol / L Na2HPO4-NaH2PO4 buffer. Dilute this buffer 40-fold to obtain enzymatic hydrolysis buffer (5 mmol / L Na2HPO4-NaH2PO4 buffer, pH 6.0).

[0146] Control solution: Accurately weigh approximately 50 mg of sodium hyaluronate reference substance into a 50 mL volumetric flask. Dissolve it in enzymatic hydrolysis buffer and dilute to the mark. Mix thoroughly. Place 0.5 mL of the above solution into a 10 mL volumetric flask, add 5 mL of enzymatic hydrolysis buffer and 1 mL of hyaluronidase, mix thoroughly, seal, and incubate at 42°C for 2 hours. Boil for 2 minutes to inactivate the enzyme. Dilute to the mark with enzymatic hydrolysis buffer and filter through a 0.22 μm filter to obtain the control solution.

[0147] Test solution: Pipette 0.1 mL of each supernatant (M1) and supernatant (M2) into a 10 mL volumetric flask and dilute to the mark with enzymatic hydrolysis buffer. Pipette 1 mL of the above solution into a 10 mL volumetric flask, add 5 mL of enzymatic hydrolysis buffer and 1 mL of hyaluronidase, mix well, seal, and hydrolyze at 42°C for 2 h. Boil for 2 min to inactivate the enzyme, and filter through a 0.22 μm filter membrane to obtain the test solution.

[0148] Determination of sodium hyaluronate content: Take 20 μL of the control solution and the test solution respectively, record the chromatogram, and calculate the content of sodium hyaluronate in the test sample by the external standard method.

[0149] Table 4 shows the encapsulation rates of the active ingredients by plant cells in the culture medium containing plant cell active ingredients prepared in Examples C1-C6 and Comparative Examples D1-D3.

[0150] Table 4

[0151] Package rate % Example C1 10.9 Example C2 14.5 Example C3 13.8 Example C4 10.3 Example C5 13.2 Example C6 13.8 Comparative Example D1 —— Comparative Example D2 —— Comparative Example D3 6.2

[0152] It can be seen that the plant cell actives of Examples C1-C6 and Comparative Example D3 all achieved encapsulation of the active ingredient, with the encapsulation rate of Examples C1-C6 being much higher than that of Comparative Example D3. In Comparative Examples D1 and D2, the plant cells and active ingredient were mixed, and the active ingredient did not enter the plant cells.

[0153] 2. In vitro skin penetration test

[0154] Experimental materials and equipment: PermeGear bilateral diffusion cell, HPLC SD rat hairless back skin (Animal Experiment Center, Affiliated Hospital of Shandong University of Traditional Chinese Medicine), normal saline, etc.

[0155] Test method:

[0156] (1) Skin pretreatment: Purchase hairless dorsal skin from healthy SD rats, carefully remove the subcutaneous mucosa and adipose tissue, and rinse with saline to obtain fresh whole skin for the experiment. Drain the saline from the skin surface with filter paper, and select undamaged skin of appropriate size under a dissecting microscope for later use. Store at 4°C overnight and remove the skin the next day for penetration testing.

[0157] (2) Transdermal permeation test: The Franz diffusion cell was used for transdermal permeation test. The skin was fixed between the donor chamber and the receiving chamber, with the stratum corneum facing the donor chamber and the dermis facing the receiving chamber. The effective permeation area S was 3.14 cm 2The receiving tank volume is 7.0mL, the receiving solution is physiological saline, and the liquid surface should be in contact with the inner layer of the skin; maintain a constant temperature water bath at 37±1℃, and ensure that there are no bubbles in the water bath interlayer; add the sample to the supply chamber, turn on the electromagnetic stirrer at 200rpm / min -1 Seven parallel tests were conducted, with 0.5 mL of the receiving solution removed at 0.5, 1, 2, 4, 6, 8, and 24 hours (the test duration should generally not exceed 24 hours, as this will affect the integrity of the skin). The removed receiving solution was ultrasonically treated for 5 minutes (to disrupt intact cells) and then the active ingredient content was tested.

[0158] (3) Calculation of transdermal absorption rate: Q = Pi / P0 × 100% (Pi is the content of active substance in the receiving pool at different times, P0 is the content of active substance in the supply pool)

[0159] The culture media containing plant cell active substances prepared in the same amounts as Example C3, Example C6 and Comparative Examples D1-D3 were tested. The test results are shown in Table 5.

[0160] Table 5

[0161]

[0162] By comparing Example C3 and Comparative Example D1, as well as Example C6 and Comparative Example D2, it can be seen that the active ingredient-encapsulated plant cells have a better active ingredient penetration effect than the mixture of plant cells and active ingredients.

[0163] It can be seen from Example C3 and Comparative Example D3 that the plant cell active substances obtained by different preparation methods have different penetration effects.

[0164] Experimental Example 2 Application of Transdermal Absorption Enhancer Composition in Emulsion

[0165] (1) Sample preparation

[0166] Example 1

[0167] 2 wt % of the transdermal absorption enhancer composition of Example A1, based on the total weight of the emulsion, was added to the base emulsion formula to prepare an emulsion.

[0168] The emulsion base formula is: 2wt% of Olivem 1000, 0.3wt% of behenyl alcohol, 6wt% of GTCC, 2wt% of dicaprylyl carbonate, 1wt% of DM5, 0.6wt% of Carbopol 980, 5wt% of butanediol, 3wt% of 1,2-pentanediol, 0.1wt% of xanthan gum, 0.12wt% of triethanolamine, and the balance is water.

[0169] Examples 2-6, Comparative Examples B1-B5, and Blank Controls Emulsions were obtained in the same manner as in Example 1, wherein:

[0170] In Example 2, the transdermal absorption enhancer composition of Example A2 was added;

[0171] In Example 3, the transdermal absorption enhancer composition of Example A3 was added;

[0172] In Example 4, the transdermal absorption enhancer composition of Example A4 was added;

[0173] In Example 5, the transdermal absorption enhancer composition of Example A5 was added;

[0174] In Example 6, the transdermal absorption enhancer composition of Example A8 was added;

[0175] Comparative Example 1 contained the transdermal absorption enhancer composition of Example B1;

[0176] Comparative Example 2 added the transdermal absorption enhancer composition of Example B2;

[0177] Comparative Example 3 added the transdermal absorption enhancer composition of Example B3;

[0178] Comparative Example 4 added the transdermal absorption enhancer composition of Example B4;

[0179] Comparative Example 5 added the transdermal absorption enhancer composition of Example B5;

[0180] Glycerol was added to the blank control group.

[0181] (2) Emulsion absorption test

[0182] (2-1) The test methods or standards for each performance indicator are as follows:

[0183] Sensory evaluation method for absorbency:

[0184] 1. Apply a mung bean-sized amount of lotion (or 0.2g) to a 5cm x 5cm area on the inside of your arm. Apply in circular motions for 10 seconds and take a photo to record the skin condition after absorption.

[0185] 2. Each volunteer needs to find two areas on the inside of each hand and test 12 lotions;

[0186] 3. 24 to 30 volunteers are needed for this test;

[0187] After waiting for 5 minutes, 30 volunteers rated the softness, oiliness, and stickiness of the skin surface in the test area.

[0188] Table 6 Scoring criteria

[0189] Soft feeling Excellent better generally Poor Very poor Score 5 4 3 2 1 Oil slick feeling none Basically no Slightly More obvious Very heavy Score 5 4 3 2 1 sticky feeling none Basically no slightly sticky More sticky Very sticky Score 5 4 3 2 1

[0190] (2-2) Test results

[0191] Table 7 Sensory evaluation of emulsion absorbability

[0192]

[0193] The sensory evaluation showed that Examples 1 to 6 were significantly better than the comparative example and the blank control group in terms of softness, no oiliness, and no stickiness.

[0194] (3) Test method for short-term moisturizing properties:

[0195] 1. Each volunteer was required to mark a 5 cm × 5 cm area on the flexor side of each forearm for testing. Before testing, the initial moisture content of the skin test area was measured using a Corneometer CM 825 (Courage+Khazaka, Germany).

[0196] 2. Apply an appropriate amount of the emulsion prepared in the Examples and Comparative Examples (or 0.2g) to a 5cm x 5cm area on the inside of one arm, rubbing in circular motions until absorbed by the skin. Use the marked area on the other arm as a control, applying the base formula sample of the emulsion without the transdermal absorption enhancer composition to the control. Test the skin moisture content 15 minutes and 3 hours after application.

[0197] 3. 24 to 30 volunteers are required for this test; the specific test results are shown in Table 8 below.

[0198] Table 8 Short-term moisturizing test results of emulsion

[0199]

[0200]

[0201] “*” indicates that there is a significant difference between the skin moisture measured after using the lotion and the skin moisture measured before using the lotion.

[0202] It can be seen that 3 hours after applying the emulsions prepared in Examples 1-6, the skin moisture growth rate is significantly higher than that of the comparative example, that is, the transdermal absorption enhancer composition of the present invention has a better short-term moisturizing effect.

[0203] (4) Test method for long-term moisturizing properties:

[0204] 1. Before the test, participants should clean their faces with a cleanser and then dry them with tissue paper. The test area should be exposed to the test environment for at least 20 minutes. A skin moisture meter will be used to measure the moisture content of the participants' facial skin.

[0205] 2. Participants were required to apply the emulsions prepared in the examples and comparative examples on their faces once each morning and evening for 7 consecutive days. After the subjects had used the products for 7 consecutive days, the moisture content of the participants' faces was measured a second time.

[0206] 30 testers were arranged in each group, and the specific test results are shown in Table 9 below.

[0207] Table 9 Long-term moisturizing test results of emulsion

[0208]

[0209]

[0210] It can be seen that after applying the emulsion prepared in Examples 1-6 for 7 days, the skin moisture growth rate is significantly higher than that of the comparative example, that is, the transdermal absorption enhancer composition of the present invention has a better long-term moisturizing effect.

[0211] (5) Testing methods for skin barrier function:

[0212] 1. Each volunteer was required to mark a 5 cm × 5 cm area on the flexor side of the left and right forearms for testing. Before testing, the TEWL (transepidermal water loss) value of the skin test area was measured using a Corneometer TM300 (Courage+Khazaka, Germany).

[0213] 2. Use tape to stick and then tear it off to damage the skin surface barrier, and immediately test the TEWL data;

[0214] 3. Apply an appropriate amount of the emulsion prepared in the Examples and Comparative Examples (or 0.2 g) and rub it in circular motions until absorbed by the skin. Perform the skin TEWL test 15 minutes and 3 hours after application.

[0215] 4. 24 to 30 volunteers are required for this test. The higher the TEWL value, the more water is lost through the skin and the poorer the barrier function of the stratum corneum. The specific test results are shown in Table 10 below.

[0216] Table 10 Skin barrier function test results

[0217]

[0218] The experimental data showed that the TEWL value increased significantly after the tape was torn off, indicating that the stratum corneum skin barrier was damaged. After applying the emulsions prepared in Experimental Examples 1 to 6 for 15 minutes and 3 hours, the TEWL values ​​were lower than those of the control example, indicating that Experimental Examples 1 to 6 had a better repairing effect on the skin barrier.

[0219] Experimental Example 3 In vitro anti-aging efficacy test method:

[0220] In vitro anti-aging efficacy test method: mainly tests two anti-aging related test indicators: type I collagen and lipid peroxidation. The specific tests are as follows:

[0221] 1-Type I collagen test:

[0222] 1.1 Test materials

[0223] 1.1.1 Reagents

[0224] DMEM basal medium (Gibco), fetal bovine serum (Gibco), PBS (Gibco), 0.25% trypsin-EDTA containing phenol red (Gibco), penicillin-streptomycin double antibody (Gibco), ultrapure water, Human COL-1 ELISA Test Kit (Sigma-Aldrich)

[0225] 1.1.2 Instruments

[0226] Ultrapure water analyzer (Millipore, USA), small high-speed centrifuge (Eppendorf, Germany), vortex analyzer (SCILOGEX, USA), CO2 cell culture incubator (Thermo Fisher, USA), biological safety cabinet (Thermo Fisher, USA), ordinary inverted microscope (Nikon, Japan), electric constant temperature water bath (Shanghai Yiheng Technology Co., Ltd., China), multifunctional microplate reader (Tecan, Switzerland), UV irradiator (Thermo Fisher, USA)

[0227] 1.2 Cell culture

[0228] Human skin fibroblasts (HSF) were cultured in a 37°C carbon dioxide incubator using DMEM basal medium, 10% fetal bovine serum, and 1% penicillin-streptomycin. They were passaged at a ratio of 1:2 every three days, and cells of the third generation or above were selected for testing.

[0229] 1.3 Sample dissolution

[0230] The components of Examples A1-A5, A8 and Comparative Examples B1-B5 were diluted 20-fold using DMEM basal culture medium.

[0231] 1.4 Detection of COL-1 expression

[0232] HSF were seeded in a 6-well plate and washed twice slowly with PBS buffer after the confluency reached 80%.

[0233] DMEM complete medium containing the sample was added and cultured for 48 hours. After 48 hours, the cells were collected and lysed by ultrasonication for 30 seconds. The supernatant was collected by centrifugation and the COL-1 content was determined according to the COL-1 ELISA KIT instructions. The specific test results are shown in Table 11 below.

[0234] 2- Lipid peroxidation test:

[0235] Lipid peroxidation is one of the cellular pathways involved in oxidative damage and is closely related to DNA damage, as many of its end products can interact with DNA and cause oxidative DNA damage. Recently, it has also been found to pose a certain risk of chemical carcinogenesis. Malondialdehyde (MDA) is a biomarker of oxidative damage caused by exposure to environmental foreign substances. It is one of the end products of lipid peroxidation and can be detected both in vivo and in vitro. MDA assessment is an important marker of oxidant-induced cytotoxicity. Therefore, MDA measurement can be used to evaluate the anti-free radical efficacy of different chemicals.

[0236] Experimental Materials

[0237] Research on remodeling the epidermis" The model was tested, and 3 replicates were set for each treatment.

[0238] Experimental procedures

[0239] 2.2.1. "Restructuring the epidermis" Model

[0240] Determination of malondialdehyde (MDA)

[0241] 2.3.2.1-Malondialdehyde (MDA) extraction

[0242] After 24 hours of treatment, the cell suspension was placed in

[0243] -250 μL Tris buffer (50 mM, pH 8) containing NaCl (0.1 M) and EDTA (20 mM)

[0244] -25 μL of SDS (7%)

[0245] -300 μL of 0.1N HCl

[0246] -38 μL of 1% phosphotungstic acid aqueous solution

[0247] A 300 μL sample of 0.67% aqueous thiobarbituric acid solution was placed in the dark at 50°C for 1 hour, followed by an ice bath (0°C) for 10 minutes. 300 μL of n-butanol was added, and the tube was shaken vigorously. After centrifugation at 10,000 g for 10 minutes at 0°C, the n-butanol phase was separated from each sample containing MDA-TBA adducts and analyzed by HPLC with fluorescence detection.

[0248] 2.3.2.2 Determination of Malondialdehyde (MDA) MDA-TBA was measured by an HPLC system consisting of an ICS (Instrument Consumables Service, France) chromatography pump with:

[0249] -Bischoff pump (type 2.200)

[0250] -Autosampler (Alcoot 788 Autosampler)

[0251] -Ultrasep C18 column (30cm×0.18cm)

[0252] - Fluorescence detector (Jasco 821-FI)

[0253] MDA-TBA was analyzed at room temperature using methanol. The mobile phase was adjusted to pH 8.3 by adding 1 M KOH to water (40:60 v / v). The flow rate was maintained at 0.5 mL / min. The excitation and emission wavelengths were 515 nm and 535 nm, respectively.

[0254] Examples A1-A5, A8 and comparative examples B1-B5 were tested for type I collagen and lipid peroxidation. The test results are shown in Table 11.

[0255] Table 11 Test results of in vitro anti-aging efficacy of plant active ingredients

[0256]

[0257] As can be seen from Table 11, the content of type I collagen in experimental examples A1 to A8 is higher than that in the comparative example, indicating that examples A1 to A8 have a better effect of promoting the production of type I collagen than comparative examples B1-B5; the content of MDA in experimental examples A1 to A8 is lower than that in the comparative example, indicating that A1 to A8 better reduces the rate of lipid peroxidation than comparative examples B1-B5; therefore, examples A1 to A8 have significant in vitro anti-aging effects.

[0258] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any other manner. Any person skilled in the art may utilize the above-disclosed technical content to modify or modify the present invention into equivalent embodiments. However, any simple modifications, equivalent variations, and modifications to the above embodiments that do not depart from the technical content of the present invention and are based on the technical essence of the present invention remain within the scope of protection of the present invention.

Claims

1. A plant cell active substance, characterized in that: include: Plant cells and active ingredients; The active ingredient is at least selected from hydrolyzed hyaluronic acid or a salt thereof having a molecular weight of 1k-10kDa, and the active ingredient is located inside the plant cell; The plant cells are selected from one or more of desert rose leaf cells, jasmine leaf cells, grape flower cells, and lotus cells; wherein, relative to the combination of the plant cells and the active ingredient, the plant cells are 91-99 wt %; The method for preparing the plant cell active substance comprises the following steps: 1) Co-culture the plant cells and active ingredients in culture medium for 36-72 hours; 2) After the culture, the culture medium is filtered to obtain plant cell active substances.

2. The plant cell active substance according to claim 1, characterized in that The active ingredient also includes ectoine or its salt.

3. The plant cell active substance according to claim 2, characterized in that The ectoine or its salt is selected from one or more of ectoine, ectoine methyl ester and hydroxyectoine.

4. A method for preparing a plant cell active substance according to any one of claims 1 to 3, characterized in that: The steps include: 1) Co-culture the plant cells and active ingredients in culture medium for 36-72 hours; 2) After the culture, the culture medium is filtered to obtain plant cell active substances.

5. The method for preparing plant cell active substances according to claim 4, characterized in that: In step 1), relative to the combination of plant cells and active ingredients, by weight percentage, The plant cells are 91-99 wt%.

6. The method for preparing plant cell active substances according to claim 4, characterized in that: In step 1), relative to the combination of plant cells and active ingredients, by weight percentage, The plant cells are 94-97 wt%.

7. The method for preparing plant cell active substances according to claim 5, characterized in that: In step 1), relative to the combination of plant cells and active ingredients, by weight percentage, The active ingredient is 1-9 wt %.

8. The method for preparing plant cell active substances according to claim 6, characterized in that: The active ingredient is 3-6 wt %.

9. The method for preparing a plant cell active substance according to any one of claims 4 to 8, characterized in that: In step 1), the culture medium is MS medium, B5 medium, or MS-H medium, the culture temperature is 20-40° C., and the pH is 3-7.

10. Use of the plant cell active substance according to any one of claims 1 to 3 or the plant cell active substance prepared by the method according to any one of claims 4 to 9 in the preparation of cosmetics and medicines.

11. Use of the plant cell active substance according to any one of claims 1 to 3 or the plant cell active substance prepared by the method according to any one of claims 4 to 9 in the preparation of a transdermal absorption enhancer.

12. A transdermal absorption enhancer composition, characterized in that: The invention comprises the plant cell active substance according to any one of claims 1 to 3 or the plant active substance prepared by the method according to any one of claims 4 to 9 and a polyol.

13. The transdermal absorption enhancer composition according to claim 12, wherein The weight ratio of the plant cell active substance to the polyol is (1-3): (7-9).

14. The transdermal absorption enhancer composition according to claim 12, wherein The polyol is one or more of glycerol, butanediol, ethylene glycol, butene glycols, and propylene glycol.

15. Use of the transdermal absorption enhancer composition according to any one of claims 12 to 14 in the preparation of cosmetics and medicines.

16. A cosmetic, characterized in that: A transdermal absorption enhancer composition comprising the composition according to any one of claims 12 to 14.

17. Use of the cosmetic according to claim 16 for moisturizing, improving skin barrier function and / or anti-aging.

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