A baicalin-cyclodextrin-vesicle complex and its application

Abstract

The present invention relates to a baicalin-cyclodextrin-vesicle complex, its preparation method and application. Specifically, the present invention discloses a baicalin-cyclodextrin-vesicle complex, which comprises (i) an active ingredient, the active ingredient including baicalin; (ii) cyclodextrin; and (iii) a vesicle material, the vesicle material including vesicles derived from microorganisms; and, the (ii) cyclodextrin encapsulates the (i) active ingredient to form nanoparticles; the (iii) vesicle material wraps the nanoparticles to form a baicalin-cyclodextrin-vesicle complex. The baicalin-cyclodextrin-vesicle complex has the properties of high transdermal efficiency, high bioavailability, high drug loading capacity and functions such as anti-aging, antioxidant, whitening, anti-inflammatory, etc., and can be applied to cosmetics.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a baicalin-cyclodextrin-vesicle complex and its application. Background Art

[0002] In the application of cosmetics, the problems related to oxidative aging mainly occur in the dermis layer of the skin. Therefore, whether the raw materials can be delivered to the dermis layer and the intake of raw materials by dermal cells are crucial for antioxidant and anti-aging effects. Baicalin can scavenge oxygen free radicals and inhibit the production of melanin, and can be used in medicine and cosmetics. However, as a component that is poorly soluble in both water and oil, its original drug form is very difficult to apply in formulations and can only be applied in cosmetics in extremely small amounts, unable to reach the effective concentration. In addition, the original drug form of baicalin not only has difficulty reaching the dermis layer but also has the problem of easy discoloration.

[0003] Currently, in cosmetics, the liposome encapsulation technology can solve the solubility problem, that is, using lecithin, polyols, etc. to encapsulate baicalin in the lipid bilayer of liposomes. Liposomes are soluble in water, but the baicalin improved by this technology still has difficulty achieving ideal bioavailability. The existing liposome encapsulation methods have the problem of low drug loading capacity and cannot completely solve the problem that the low addition amount in skin care products leads to the inability to exert the efficacy of raw materials. At the same time, the transdermal release ability of liposome-encapsulated baicalin is poor, affecting the exertion of various effects.

[0004] Therefore, there is an urgent need in the art to develop a baicalin modification technology with high transdermal efficiency, high bioavailability, and high drug loading capacity. Summary of the Invention

[0005] The purpose of the present invention is to provide baicalin microcapsules with high transdermal efficiency, high bioavailability, and high drug loading capacity, as well as their preparation methods and applications.

[0006] In the first aspect of the present invention, there is provided a baicalin-cyclodextrin-vesicle complex, comprising

[0007] (i) an active ingredient, and the active ingredient includes baicalin;

[0008] (ii) cyclodextrin; and

[0009] (iii) a vesicle material, and the vesicle material includes vesicles of microbial origin;

[0010] And,

[0011] the (ii) cyclodextrin encapsulates the (i) active ingredient to form nanoparticles;

[0012] the (iii) vesicle material encapsulates the nanoparticles to form a baicalin-cyclodextrin-vesicle complex.

[0013] In another preferred example, the microorganisms include strains of the genus Saccharomyces and strains of the genus Bacillus.

[0014] In another preferred example, the strains of the genus Saccharomyces are selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces carlsbergensis, Saccharomyces, or combinations thereof.

[0015] In another preferred example, the strains of the genus Bacillus are selected from the group consisting of Bacillus subtilis.

[0016] In another preferred example, the complex has the following components in parts by weight:

[0017] Active ingredient 12 - 18 parts

[0018] Vesicle material 70 - 90 parts

[0019] Cyclodextrin 3 - 7 parts.

[0020] In another preferred example, the vesicle material has the following components in parts by weight:

[0021] Yeast fermentation product 50 - 60

[0022] Bacillus fermentation product 20 - 30.

[0023] In another preferred example, the complex contains the following components:

[0024] parts by weight preferred parts by weight more preferred parts by weight active ingredient 12～18 13～17 14～16 vesicle material 70～90 75～85 78～83 cyclodextrin 3～7 4～6 4～6。

[0025] In another preferred example, the vesicle material contains the following components:

[0026] parts by weight preferred parts by weight more preferred parts by weight yeast fermentation product 52～58 53～56 54～56 bacillus fermentation product 22～27 23～26 23～25。

[0027] In another preferred example, the baicalin - cyclodextrin - vesicle complex significantly increases the skin penetration depth of baicalin. The significant increase in the skin penetration depth of baicalin means that compared with the penetration depth P0 of baicalin in the in vitro skin tissue, the penetration depth of the baicalin - cyclodextrin - vesicle complex in the in vitro skin tissue is P1, and the ratio (P1 / P0) ≥ 3.

[0028] In another preferred example, the penetration depth of the baicalin - cyclodextrin - vesicle complex in the in vitro skin tissue is P1. Compared with the penetration depth P0 of baicalin in the in vitro skin tissue, the ratio (P1 / P0) ≥ 3, preferably ≥ 4, more preferably ≥ 5.

[0029] In another preferred example, the penetration depth of the baicalin - cyclodextrin - vesicle complex in the in vitro skin tissue is P1. Compared with the penetration depth P2 of liposome - encapsulated baicalin in the in vitro skin tissue, the ratio (P1 / P2) ≥ 1.5, preferably ≥ 2.

[0030] In another preferred example, the penetration depth of the baicalin-cyclodextrin-vesicle complex in the dermis layer of in vitro skin tissue is Z1, and the penetration depth of baicalin in the dermis layer of in vitro skin tissue is Z0. The ratio (Z1 / Z0) ≥ 5, preferably ≥ 7, more preferably ≥ 10.

[0031] In another preferred example, the penetration depth of the baicalin-cyclodextrin-vesicle complex in the dermis layer of in vitro skin tissue is Z1, and the penetration depth of baicalin encapsulated by liposome in the dermis layer of in vitro skin tissue is Z2. The ratio (Z1 / Z2) ≥ 1.5, preferably ≥ 2, more preferably ≥ 2.5.

[0032] In another preferred example, the baicalin-cyclodextrin-vesicle complex has an excellent transdermal release rate.

[0033] In another preferred example, the excellent transdermal release rate means that the cumulative release is ≥ 80% in 36 h, preferably ≥ 85%; and / or the cumulative release is ≥ 45% in 8 h, preferably ≥ 50%.

[0034] In another preferred example, there is no obvious burst release phenomenon of the baicalin-cyclodextrin-vesicle complex within 0 - 8 h.

[0035] In another preferred example, the baicalin-cyclodextrin-vesicle complex has one or more of the following properties:

[0036] (f1) Has ROS reactive oxygen species scavenging ability;

[0037] (f2) Reduces the expression of MMP-1;

[0038] (f3) Increases the expression of TIMP-1;

[0039] (f4) Has a sustained release effect;

[0040] (f5) Increases the expression of CAT;

[0041] (f6) Increases the expression of SOD;

[0042] (f7) Promotes the growth of human fibroblasts;

[0043] (f8) Increases the expression of GSH and / or increases GSH / GSSG.

[0044] In another preferred example, the cyclodextrin is selected from α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, or a combination thereof.

[0045] In another preferred example, the particle size of the baicalin-cyclodextrin-vesicle complex is 50 - 500 nm.

[0046] In another preferred example, the polydispersity index of the baicalin-cyclodextrin-vesicle complex is 0.1 to 0.2.

[0047] In another preferred example, the particle size of the baicalin-cyclodextrin-vesicle complex is 100 to 400 nm, preferably 100 to 300 nm.

[0048] In another preferred example, the polydispersity index of the baicalin-cyclodextrin-vesicle complex is 0.1 to 0.15.

[0049] In another preferred example, the Zeta potential of the baicalin-cyclodextrin-vesicle complex is -10 to 0 mV, preferably -5 to 0 mV, more preferably -5 to -2 mV.

[0050] In another preferred example, the polydispersity index of the baicalin-cyclodextrin-vesicle complex is 0.05 to 0.3.

[0051] In another preferred example, the average particle size of the vesicle material is 20 to 500 nm, preferably 50 to 400 nm, more preferably 80 to 300 nm, such as 100 nm.

[0052] In the second aspect of the present invention, there is provided a method for preparing the baicalin-cyclodextrin-vesicle complex as described in the first aspect of the present invention, comprising the following steps:

[0053] (s1) Providing (i) an active ingredient, the active ingredient including baicalin; (ii) cyclodextrin; and (iii) a vesicle material, the vesicle material including vesicles of microbial origin;

[0054] (s2) Mixing the active ingredient, cyclodextrin, and vesicle material in step (s1) to obtain the baicalin-cyclodextrin-vesicle complex.

[0055] In another preferred example, the vesicle material is prepared by the following steps:

[0056] (s1) Culturing Bacillus in a first culture medium, adding yeast for culturing, and centrifuging to obtain a precipitate;

[0057] (s2) Crushing the precipitate obtained in step (s1) to obtain the vesicle material.

[0058] In another preferred example, the first culture medium is a liquid culture medium.

[0059] In another preferred example, the first culture medium is selected from: nutrient broth culture medium.

[0060] In another preferred example, the crushing is performed by homogenization using a homogenizer.

[0061] In another preferred embodiment, in step (s2), the vesicle material also needs to be separated and purified, and the separation and purification is carried out using a solid chromatography column.

[0062] In the third aspect of the present invention, there is provided a use of the baicalin-cyclodextrin-vesicle complex as described in the first aspect of the present invention for preparing a preparation or a composition.

[0063] In another preferred embodiment, the preparation or composition is used for anti-aging and antioxidant.

[0064] In another preferred embodiment, the baicalin-cyclodextrin-vesicle complex is also used for preparing cosmetics.

[0065] In the fourth aspect of the present invention, there is provided a cosmetic, which comprises

[0066] (c1) the baicalin-cyclodextrin-vesicle complex as described in the first aspect of the present invention; and

[0067] (c2) a cosmetically acceptable carrier or excipient.

[0068] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be repeated one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] Figure 1 Shows the particle size and distribution diagram of baicalin microcapsules.

[0070] Figure 2 Shows the Zeta potential detection results of baicalin microcapsules.

[0071] Figure 3 Shows the XRD comparison diagram of baicalin microcapsules.

[0072] Figure 4 Shows the comparison diagram of the skin penetration ability of baicalin microcapsules and liposome encapsulation, a: epidermis; b: dermis; c: fluorescence penetration depth.

[0073] Figure 5 Shows the skin penetration depth results of baicalin microcapsules and baicalin liposomes.

[0074] Figure 6 Shows the transdermal release rate diagrams of baicalin microcapsules, baicalin, and baicalin liposomes.

[0075] Figure 7 Shows the ROS reactive oxygen scavenging abilities of baicalin, baicalin liposomes, and baicalin microcapsules.

[0076] Figure 8 Showed the change amount of GSH / GSSG of baicalin, baicalin liposome, baicalin microcapsule, and the material.

[0077] Figure 9 Showed the content of MMP-1 in the supernatant of human skin fibroblasts.

[0078] Figure 10 Showed the content of TIMP-1 in the supernatant of human skin fibroblasts.

[0079] Figure 11 Showed the effects of baicalin, baicalin liposome, and baicalin microcapsule on CAT activity.

[0080] Figure 12 Showed the effects of baicalin, baicalin liposome, and baicalin microcapsule on SOD activity.

[0081] Figure 13 Showed the staining diagrams of human fibroblasts of baicalin, baicalin liposome, and baicalin microcapsule. Detailed implementation manners

[0082] Through extensive and in-depth research, and through a large number of experiments and screenings, the present inventor unexpectedly discovered for the first time a baicalin-cyclodextrin-vesicle complex, which has the characteristics of high transdermal efficiency, high bioavailability, and high drug loading capacity, and can be applied to cosmetics and pharmaceutical products. On this basis, the present invention was completed.

[0083] Terms

[0084] To make the present invention easier to understand, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention pertains. Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, as such methods and conditions may vary. It should also be understood that the terms used herein are for the purpose of describing specific embodiments only and are not intended to be limiting, and the scope of the present invention will be limited only by the appended claims.

[0085] As used herein, the term "comprising" or its variants such as "including" or "including having" etc. are understood to include the stated elements or components, without excluding other elements or other components.

[0086] As used herein, the terms "material", "vesicle", "microcapsule material", "vesicle material" can be used interchangeably, and all refer to vesicle materials of microbial origin, which are the outer shell part of the baicalin-cyclodextrin-vesicle complex and encapsulate the cyclodextrin nanoparticles containing baicalin.

[0087] Baicalin-Cyclodextrin-Vesicle Complex

[0088] As used herein, the terms "baicalin microcapsule", "baicalin modified by natural vesicle technology", "baicalin-cyclodextrin-vesicle complex", and "the complex of the present invention" are used interchangeably and all refer to baicalin prepared by the method of the present invention, which has high transdermal efficiency, high bioavailability, and high drug loading capacity.

[0089] The baicalin-cyclodextrin-vesicle complex is formed by encapsulating baicalin with the biofilm vesicle structure formed by the fermentation products of yeast and bacillus. The biofilm vesicle structure can not only solve the problems of raw material application such as the solubility and stability of baicalin, but also, due to its very similar structure to the human cell membrane, compared with the traditional liposome encapsulation method, it is more helpful to improve the transdermal and endocytosis abilities of baicalin, enabling baicalin to play a better antioxidant and anti-aging role in the skin care product system.

[0090] The baicalin-cyclodextrin-vesicle complex of the present invention is a complex with uniform particle size and good safety. Its structure is: the vesicle material encapsulates the cyclodextrin nanoparticles encapsulating baicalin. The vesicle material is composed of the fermentation products of yeast and bacillus, and the cyclodextrin nanoparticles encapsulating baicalin are formed by baicalin being encapsulated in the molecular cavity of cyclodextrin.

[0091] In the present invention, the baicalin-cyclodextrin-vesicle complex comprises

[0092] (i) An active ingredient, and the active ingredient is baicalin;

[0093] (ii) A vesicle material, and the vesicle material includes the fermentation products of yeast and bacillus; and

[0094] (iii) Cyclodextrin;

[0095] And,

[0096] The (ii) cyclodextrin encapsulates the (i) active ingredient to form nanoparticles;

[0097] The (iii) vesicle material encapsulates the nanoparticles to form the baicalin-cyclodextrin-vesicle complex.

[0098] In another preferred embodiment, the mass ratio of the active ingredient: vesicle material: cyclodextrin is 12 - 18:70 - 90:3 - 7.

[0099] In another preferred embodiment, the cyclodextrin is selected from α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, or a combination thereof.

[0100] The baicalin-cyclodextrin-vesicle complex of the present invention has a uniform particle size of 50 to 500 nm and a polydispersity index of 0.05 to 0.3. The Zeta potential of the baicalin-cyclodextrin-vesicle complex of the present invention is -10 to 0 mV, the potential distribution is uniform, the deviation range is small, there is no obvious dispersion of particles and material fragments, and it has good safety.

[0101] The microscopic structure of the baicalin-cyclodextrin-vesicle complex of the present invention is that baicalin is included inside cyclodextrin, and the vesicle material wraps the cyclodextrin containing baicalin.

[0102] The baicalin-cyclodextrin-vesicle complex of the present invention has excellent skin penetration effect. The penetration depth of the baicalin-cyclodextrin-vesicle complex of the present invention in the in vitro skin tissue is P1. Compared with the penetration depth P0 of baicalin in the in vitro skin tissue, the ratio of the two (P1 / P0) ≥ 3, preferably ≥ 4, more preferably ≥ 5; compared with the penetration depth P2 of liposome-encapsulated baicalin in the in vitro skin tissue, the ratio of the two (P1 / P2) ≥ 1.5, preferably ≥ 2.

[0103] The baicalin-cyclodextrin-vesicle complex of the present invention has an excellent transdermal release rate. In another preferred example, the excellent transdermal release rate means that there is no obvious burst release phenomenon within 0 to 8 h. In another preferred example, the cumulative release is ≥ 80% within 36 h, preferably 85%; and / or the cumulative release is ≥ 45% within 8 h, preferably 50%.

[0104] Vesicle material

[0105] The vesicle material of the present invention includes vesicle materials of microbial origin. The microorganisms include strains of the genus Saccharomyces and strains of the genus Bacillus.

[0106] In another preferred example, the strains of the genus Saccharomyces are selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces carlsbergensis, Saccharomyces, or a combination thereof. In another preferred example, the strains of the genus Bacillus are selected from the group consisting of Bacillus subtilis.

[0107] The vesicle material of the present invention can be obtained commercially or prepared by conventional methods. Typically, the vesicle material can be prepared by fermentation. The specific preparation method of a representative vesicle material is shown in Patent WO2022055250.

[0108] MMP-1

[0109] Human skin fibroblasts are the main effector cells of the dermis and can produce various extracellular matrices such as collagen, laminin, and elastin. The water retained by fibroblasts and the ECM matrix they produce is the material basis for maintaining cell elasticity. Type I collagen is the most abundant protein in the ECM, accounting for 90% of the total collagen content. It is the main component of the dermis and provides structural mechanical protection for surrounding tissues. MMP-1 mainly participates in the degradation of type I and type III collagens. When MMP-1 is overexpressed, it specifically degrades ECM components and destroys the normal structure of collagen and elastic fibers. Therefore, MMP-1 is one of the main enzymes leading to aging symptoms such as wrinkles and fine lines on the skin.

[0110] TIMP-1

[0111] Tissue inhibitor of metalloproteinase-1 (TIMP-1) is one of the important glycoproteins in the skin and participates in the degradation of the extracellular matrix. MMPs in the skin specifically degrade collagen. The overexpression of MMPs promotes skin aging and destroys the skin ECM structure. In addition to inhibiting most known MMPs, TIMP-1 can also promote the proliferation of various cells and has an anti-apoptotic effect. Therefore, TIMP-1 is closely related to skin anti-aging.

[0112] GGH

[0113] Glutathione is an indispensable tripeptide in human metabolism. It has the highest content in the body during infancy and decreases with age. Glutathione has the functions of whitening, antioxidant, and increasing skin elasticity. It can inhibit the activity of tyrosinase, inhibit the production of melanin, reduce the division and biochemical activity of melanocytes, and enhance the role of vitamin C in the body; it can scavenge various free radicals in the human body, such as DPPH, hydroxyl radicals, superoxide anions, etc.; it can integrate the skin moisturizing environment and provide a good production environment for new skin cells, making the skin smoother and more elastic; glutathione is divided into oxidized GSSG and reduced GSH, and the main active one is GSH.

[0114] CAT

[0115] Catalase (CAT) is one of the main antioxidant enzymes in the human body. Its main functions include decomposing reactive oxygen species (such as hydrogen peroxide, superoxide anions, etc.) produced during the body's metabolic process, reducing human free radicals and lipid peroxides, and is closely related to human oxidative aging.

[0116] SOD

[0117] Superoxide dismutase (SOD) is an enzyme that can catalyze the conversion of superoxide into oxygen and hydrogen peroxide through a dismutation reaction. It is widely present in the human body and is an important antioxidant enzyme. SOD can specifically scavenge free radicals in the skin, repair oxygen-damaged cells, and delay skin aging.

[0118] HSF

[0119] Human skin fibroblasts (HSF) are one of the main components of skin tissue. The fiber density, fiber thickness, and fiber orientation of fibroblasts can largely reflect the cell state and degree of aging. UV light irradiation can make the cell collagen fibers thinner and accelerate cell aging.

[0120] Baicalin

[0121] Baicalin is a flavonoid compound extracted and isolated from the dried roots of Scutellaria baicalensis Georgi. It is almost insoluble in common solvents for cosmetics such as water and ethanol. Baicalin has significant biological activities, including antibacterial, diuretic, anti-inflammatory, anti-allergic, and antispasmodic effects, and also has strong anti-cancer reactions and other physiological effects. It has occupied an important position in clinical medicine. Baicalin can also absorb ultraviolet light, scavenge oxygen free radicals, and inhibit the production of melanin. Therefore, it can be used both in medicine and cosmetics and is a cosmetic raw material with good application prospects.

[0122] Cosmetic composition

[0123] The present invention also provides a cosmetic composition containing the baicalin-cyclodextrin-vesicle complex of the present invention. The cosmetic composition of the present invention comprises (a) the baicalin-cyclodextrin-vesicle complex of the present invention; and (b) a cosmetically acceptable carrier or excipient.

[0124] The baicalin-cyclodextrin-vesicle complex of the present invention can be prepared into various different cosmetic compositions, such as emulsions, liquids, ointments, creams, pastes, cakes, powders and other dosage forms by conventional methods.

[0125] Within the scope not interfering with the effects of the present invention, other ingredients commonly used in cosmetics can be added to the cosmetics of the present invention, such as film-forming agents, oil-soluble gelling agents, organically modified clay minerals, resins, moisturizers, preservatives, antibacterial agents, fragrances, salts, antioxidants, pH regulators, chelating agents, cooling agents, anti-inflammatory agents, skin beautifying ingredients (whitening agents, cell activators, skin roughness improvers, blood circulation promoters, skin astringents, anti-seborrheic agents, etc.), vitamins, amino acids, nucleic acids, hormones, inclusion compounds, etc.

[0126] The oil-soluble gelling agent is a gelling agent selected from metal soaps such as aluminum stearate, magnesium stearate, zinc myristate; amino acid derivatives such as N-lauroyl-L-glutamic acid, α,γ-di-n-butylamine; cyclodextrin fatty acid esters such as cyclodextrin palmitate, cyclodextrin stearate, cyclodextrin 2-ethylhexyl palmitate; sucrose fatty acid esters such as sucrose palmitate, sucrose stearate; benzylidene derivatives of sorbitol such as mono-benzylidene sorbitol, di-benzylidene sorbitol; organically modified clay minerals such as dimethylbenzyl dodecylammonium montmorillonite clay, dimethyloctacosylammonium montmorillonite clay, etc. One kind can be used as needed, or two or more kinds can be used.

[0127] The humectants include: glycerol, sorbitol, propylene glycol, dipropylene glycol, 1,3-butanediol, glucose, xylitol, maltitol, polyethylene glycol, hyaluronic acid, chondroitin sulfate, pyrrolidone carboxylate, polyoxyethylene methyl glucoside, polyoxypropylene methyl glucoside, etc.

[0128] The antibacterial preservatives include: alkyl p-hydroxybenzoates, benzoic acid, sodium benzoate, sorbic acid, potassium sorbate, phenoxyethanol, etc. The antibacterial agents include: benzoic acid, salicylic acid, phenol, sorbic acid, alkyl p-hydroxybenzoates, parachlorometacresol, hexachlorophene, benzalkonium chloride, chlorhexidine gluconate, trichloro-N-carbonylaniline, triclosan, phthalein, phenoxyethanol, etc.

[0129] The antioxidants include: tocopherol, butylated hydroxyanisole, dibutylhydroxytoluene, phytic acid, etc. The pH regulators include: lactic acid, citric acid, glycolic acid, succinic acid, tartaric acid, dl-malic acid, potassium carbonate, sodium bicarbonate, ammonium bicarbonate, etc. The chelating agents include alanine, sodium ethylenediaminetetraacetate, sodium polyphosphate, sodium metaphosphate, phosphoric acid, etc. The cooling agents include: L-menthol, camphor, etc. The anti-inflammatory agents include: allantoin, glycyrrhetinic acid, glycyrrhizic acid, tranexamic acid, azulene, etc.

[0130] Ingredients for skin beautification include: skin-whitening agents such as placenta extract, arbutin, glutathione, saxifrage extract, etc.; cell activators such as royal jelly, photosensitizer, cholestanol derivatives, calf blood extract, etc.; skin roughness improvers; blood circulation promoters such as valeryl nonanoate, benzyl nicotinate, β-butoxyethyl nicotinate, capsaicin, zingerone, cantharidin tincture, ichthammol, caffeine, tannic acid, α-borneol, tocopheryl nicotinate, inositol hexanicotinate, cyclandelate, cinnarizine, tolazoline, acetylcholine, verapamil, cepharanthine, γ-sitosterol, etc.; skin astringents such as zinc oxide, tannic acid, etc.; anti-seborrheic agents such as sulfur, etc. Vitamins include: vitamin A derivatives such as vitamin A oil, retinol, retinol acetate, retinol palmitate, etc.; vitamin B2 derivatives such as riboflavin, riboflavin butyrate, flavin adenine dinucleotide, etc.; vitamin B6 derivatives such as pyridoxine hydrochloride, pyridoxine dioctanoate, pyridoxine tripalmitate, etc., vitamin B12 and its derivatives, vitamin B15 and its derivatives, etc. vitamin B group; vitamin C derivatives such as L-ascorbic acid, L-ascorbic acid dipalmitate, L-ascorbic acid-2-sodium sulfate, L-ascorbic acid phosphodiester dipotassium, etc.; vitamin D derivatives such as ergocalciferol, cholecalciferol, etc.; vitamin E derivatives such as α-tocopherol, β-tocopherol, γ-tocopherol, dl-α-tocopherol acetate, dl-α-tocopherol nicotinate, dl-α-tocopherol succinate, etc.; vitamin H; vitamin P; niacin derivatives such as niacin, benzyl nicotinate, nicotinamide, etc.; pantothenic acid derivatives such as calcium pantothenate, D-panthenol, pantothenyl ethyl ether, acetyl pantothenyl ethyl ether, etc.; biotin, etc.

[0131] Amino acids include: glycine, valine, leucine, isoleucine, serine, threonine, phenylalanine, arginine, lysine, aspartic acid, glutamic acid, cystine, cysteine, methionine, tryptophan, etc. Nucleic acids include deoxyribonucleic acid, etc. Hormones include estradiol, vinyl estradiol, etc.

[0132] Preferred examples of the cosmetics of the present invention include: skin care cosmetics, color cosmetics, anti-ultraviolet cosmetics. For example, there are basic cosmetics such as lotion, cream, lotion, sunscreen, mask material, facial cleanser, essence, etc.; color cosmetics such as foundation, powder, blush, etc.

[0133] There is no particular limitation on the form of the product, which can be liquid, emulsion, cream, solid, paste, gel, powder, multi-layered, mousse, spray, etc.

[0134] The present invention also provides a skin care method, which includes the step of applying to an individual in need the baicalin-cyclodextrin-vesicle complex of the present invention, or a cosmetic or care product containing the baicalin-cyclodextrin-vesicle complex of the present invention.

[0135] In another preferred example, the effective concentration range of the baicalin-cyclodextrin-vesicle complex is 100 μg / ml to 500 mg / ml.

[0136] In another preferred example, the methods are anti-aging, antioxidant, whitening, anti-inflammatory and other methods.

[0137] The main advantages of the present invention include:

[0138] (1) For the first time, a baicalin-cyclodextrin-vesicle complex with high transdermal efficiency, high bioavailability and high drug loading is provided;

[0139] (2) The baicalin-cyclodextrin-vesicle complex of the present invention has functions such as anti-aging, antioxidant, whitening, anti-inflammatory, etc., and can be applied to cosmetics;

[0140] (3) The baicalin-cyclodextrin-vesicle complex of the present invention has a sustained-release effect, reduces the toxicity to cells in a short time, and reduces the probability of allergic reactions;

[0141] (4) The method for preparing the baicalin-cyclodextrin-vesicle complex of the present invention is simple in operation, low in cost, high in efficiency, simple in process, and suitable for industrial production;

[0142] (5) The baicalin-cyclodextrin-vesicle complex of the present invention can penetrate into the dermis layer of the skin tissue.

[0143] The following further elaborates the present invention in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are usually in accordance with conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or in accordance with the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are by weight percentage and weight parts.

[0144] Comparative Example

[0145] The baicalin liposome was prepared by the following method, and the content of the baicalin liposome is shown in Table 1:

[0146] Table 1

[0147] ingredient content / % water 41.45％ dipropylene glycol 35.5％ baicalin 8.5％ hydrogenated lecithin (pc75) 7.0％ caprylic / capric triglyceride 4.5％ polysorbate - 80 1.5％ PEG - 40 hydrogenated castor oil 1.5％ disodium EDTA 0.05％

[0148] 1. Weigh dipropylene glycol and hydrogenated lecithin, stir at 75 °C and 600 r / min for 10 min, and stir evenly until there is no particulate liquid;

[0149] 2. Add glyceryl caprylocaprate to the above solution and stir evenly for 5 min at 75 °C and 600 r / min; (control the mass ratio of glyceryl caprylocaprate to baicalin between 1:2 and 2:1);

[0150] 3. Add baicalin to the above solution; stir for 10 min at 75 °C and 600 r / min until a uniform yellow liquid is obtained;

[0151] 4. Add disodium EDTA to the above solution; stir for 5 min at 75 °C and 600 r / min until a uniform yellow liquid is obtained;

[0152] 5. Homogenize the above solution using a microfluidizer, homogenize three times at 2000 psi, control the outlet temperature of the first and second homogenizations to 50 °C, and the temperature of the third homogenization to 15 °C; obtain a slightly viscous yellow liquid;

[0153] 6. Centrifuge at 5000 r / min for 10 min to remove insoluble solid impurities.

[0154] The encapsulation efficiency of the prepared liposomes is 82.83%. The test method is as follows: Take 100 mg of the sample, dissolve it in a volumetric flask with deionized water and make up the volume to 10 mL. Take 400 μL of the solution and place it in a 50K ultrafiltration centrifuge tube, centrifuge at 12000 r / min for 10 min. The encapsulated baicalin is retained on the filter membrane, and the free baicalin is filtered into the lower centrifugate. Take 100 μL of the lower centrifugate, dilute it 100 times with absolute ethanol and then determine the content, and obtain the free baicalin (mg) in the sample by referring to the standard curve.

[0155] Preparation of Baicalin Microcapsules in Example 1

[0156] Weigh an appropriate amount of cyclodextrin and grind it in a colloid mill. Then weigh baicalin and add it to the colloid mill. After thorough mixing and grinding, add a large amount of deionized water, fully dissolve the well-mixed cyclodextrin-baicalin powder, and homogenize the solution through a high-pressure microfluidizer at a homogenization pressure of 30 MPa. After homogenizing several times, collect the solution and place it in a freeze dryer to remove the solvent at -80 °C to obtain baicalin-cyclodextrin inclusion nanoparticles.

[0157] Dissolve the freeze-dried Bacillus subtilis powder with nutrient broth liquid (NB) medium. Transfer the dissolved bacterial liquid to a sterile test tube containing a certain amount of liquid medium and mix well for cultivation. Inoculate the activated strain into an Erlenmeyer flask containing NB medium and shake-culture under appropriate conditions for later use. Transfer the Bacillus subtilis cells to an NB liquid medium containing a certain amount of yeast powder for cultivation. After cultivation, collect the fermentation broth and centrifuge to obtain a cell precipitate. Resuspend the precipitate in phosphate buffer according to a certain volume ratio. Homogenize and disrupt the buffer solution containing the cells with a high-pressure microfluidizer at a homogenization pressure of 30 MPa. Pass the solution containing cell fragments through a solid chromatography column to separate, purify, and collect the vesicle fraction.

[0158] Weigh an appropriate amount of the vesicle fraction and dilute it with phosphate buffer. Then weigh the baicalin cyclodextrin inclusion nanoparticles. Mix the two well and place them in an ultrasonic device for ultrasonic treatment. Then place the liquid in a high-pressure extruder with nitrogen as the pressure source and extrude it through a polycarbonate membrane. Then centrifuge to collect the precipitate at the bottom, dry it, and grind it into powder to obtain the baicalin microcapsules.

[0159] Example 2 Physical properties of baicalin microcapsules

[0160] 2.1 Particle size and distribution of baicalin microcapsules

[0161] As Figure 1 shown, the particle sizes of three batches of baicalin microcapsules are basically distributed between 100 - 300 nm, the PDI value is 0.141, the distribution is good, there are no obvious particles and debris precipitated, which proves that the system is relatively stable, the particle distribution is uniform, and the Zeta average particle size is 145 nm. The average particle size of the vesicle material without loaded baicalin is 50 - 300 nm.

[0162] 2.2 Potential detection of baicalin microcapsules

[0163] As Figure 2 shown, the Zeta potential detection results show that the average potential of three batches of baicalin microcapsules in the aqueous solution system is -2.8 mV, and the absolute value is much smaller than -30 mV which has certain toxicity to cells, indicating that the raw materials have good use safety, and the potential distribution is uniform with a small deviation range, indicating that there are no obvious particles and material debris dispersed in the system.

[0164] 2.3 XRD analysis of baicalin microcapsules

[0165] As Figure 3 shown, the characteristic peaks of baicalin in the baicalin microcapsules are basically covered by the material, while in the group of direct physical mixing of baicalin and the encapsulating material (physical mixing means mixing according to the composition ratio of the baicalin microcapsules, without passing through the process flow, and stirring evenly after mixing), most of the characteristic peaks of baicalin are not covered, which proves that baicalin is encapsulated by the material rather than dispersed in the encapsulating material.

[0166] Skin Penetration Ability of Baicalin Microcapsules in Example 3

[0167] 3.1 Method

[0168] Fresh skin on the abdomen of three-month-old miniature pigs was used in the experiment. After hair removal, the fat layer and part of the dermis were scraped off with a spatula, and the skin was cut into a circular shape of about 3.14 cm 2 in size, with the stratum corneum facing up and fixed on the permeation device. The experimental fluorescein was FITC-HA. Baicalin was cross-linked with fluorescein (cross-linking method: an appropriate amount of baicalin was dissolved in acetic acid-sodium acetate buffer solution, tyramine (p-hydroxyphenethylamine) and sodium cyanoborohydride were added in sequence, followed by water bath and stirring reaction in the dark; FITC was added to tyraminated baicalin, and borate buffer solution was used as the labeling environment for overnight labeling; denaturing vertical slab polyacrylamide gel electrophoresis was used to detect and separate FITC-labeled baicalin. On this basis, sephadex gel column was used to further separate and purify free baicalin and labeled baicalin, and Tris-HCl was used for elution to obtain FITC-labeled baicalin). Liposome and microcapsule encapsulation technologies were used to load drugs respectively. The microcapsule-loaded drug, liposome-loaded drug and non-loaded drug were diluted to a concentration of 2‰ (2 mg / mL, calculated based on the baicalin concentration) with PBS buffer solution at pH = 7.4. 0.5 mL of the above diluent was added to each permeation device, and it was placed in the dark for 1 h. After that, the skin was removed, rinsed repeatedly with water until there was no decolorization of the surface fluorescence, the surface moisture was blotted dry with absorbent paper, slightly air-dried in the dark, sliced, and observed under a fluorescence microscope for the penetration of fluorescence in the skin.

[0169] 3.2 Results

[0170] As Figure 4 and Figure 5 shown, compared with the PBS group (i.e., unloaded baicalin), both liposome encapsulation and microcapsule loading improved the skin's ability to uptake drugs. The wider the fluorescence band, the deeper the drug penetration.

[0171] By comparing the penetration abilities of the three in the epidermis and dermis of the skin and the drug uptake abilities of different skin layer cells, it can be observed that the drug in the PBS group mainly stayed on the surface of the epidermis, while both liposome encapsulation and microcapsule-loaded drugs had reached the connective fibrous tissue of the dermis layer, and the penetration depth of the microcapsule group in the dermis was deeper than that of the liposome group, and the drug uptake amount by dermal cells was more.

[0172] Statistically, the average penetration depths of microcapsule loading, liposome encapsulation and the PBS group in the skin were 1347.52 μm, 555.35 μm and 264.39 μm respectively. The penetration depths of microcapsule loading and liposome encapsulation were 5.1 times and 2.1 times higher than that of the PBS group respectively. The average penetration depth of microcapsule-loaded baicalin was more than twice that of liposome-encapsulated baicalin.

[0173] The thickness of the epidermis is a, where a = 156.72 ± 13.59 μm. After subtracting the thickness of the epidermis, the penetration depths of the dermis in the microcapsule-loaded, liposome-encapsulated, and PBS groups are approximately 1090.8, 398.63, and 107.67 μm, respectively. This indicates that the baicalin microcapsules of the present invention have unexpectedly excellent penetration performance in the dermis.

[0174] The above results show that the baicalin microcapsules have better skin penetration ability.

[0175] Transdermal release rate of baicalin microcapsules in Example 4

[0176] 4.1 Method

[0177] The experimental conditions were 32 °C, and the medium used was an isotonic phosphate buffer solution with pH = 7.4. A transdermal diffusion apparatus was used. The experiments were set up with a baicalin group, a baicalin liposome group, and a baicalin microcapsule group, with three parallels in each group. The skin used was the dorsal skin of 1 kg New Zealand white rabbits. After the New Zealand rabbits were anesthetized by inhalation of isoflurane, they were euthanized by injecting 1 mL of potassium chloride solution at an appropriate concentration, dissected, and the dorsal skin of the New Zealand rabbits was taken. The hair and fat layer were removed, and it was repeatedly rinsed clean with water, cut into appropriate sizes, placed in physiological saline, and stored at 4 °C for later use. The drugs used in the diffusion cell were diluted with an isotonic buffer solution. Among them, the concentration of the baicalin group was 150 μg / mL; the administration concentration of the baicalin microcapsules was 1 mg / mL (equivalent to 150 μg / mL of baicalin), and the administration concentration of the baicalin liposomes was 6 mg / mL (equivalent to 150 μg / mL of baicalin). The addition amount was 2 mL, and the receiving cell used 8 mL of an isotonic phosphate buffer solution with pH = 7.4 (potassium chloride, sodium chloride). The skin was placed between the two, clamped, and placed in the transdermal instrument; at 0.5, 1, 2, 4, 8, 12, 24, 36, 48, 54, and 60 h, 0.5 mL of the solution was aspirated, and then 0.5 mL of the permeate was added. After all the time points were taken, the solution was placed in an ultraviolet spectrophotometer to detect OD 280nm , and the concentration was calculated by referring to the baicalin standard curve, the transmittance was calculated, and a graph was plotted.

[0178] 4.2 Results

[0179] It can be seen from Figure 6 and Table 2 that the cumulative release of the baicalin microcapsules reached 85.24% at the 36th hour, the release rate of baicalin reached 89.24% at the 24th hour, and there was no obvious change in the release rate at subsequent times. At 8 h, the cumulative release rate of the baicalin microcapsules was 53.85%, and the cumulative release rate of the baicalin raw drug was 75.39%.

[0180] Table 2

[0181]

[0182]

[0183] The cumulative release rate of baicalin showed an obvious burst release phenomenon at 0-8 h, which might cause great toxicity to skin cells and thus trigger allergic reactions.

[0184] The baicalin microcapsules had a certain sustained-release effect on the basis of the original baicalin drug, reduced the toxicity of the drug to cells in a short time, reduced the probability of skin allergic reactions, ensured the concentration required to exert the drug efficacy within a certain time, and played a better role in helping with antioxidant, whitening and anti-aging effects.

[0185] Repair effect of baicalin microcapsules on cells damaged by reactive oxygen species in Example 5

[0186] 5.1 Method

[0187] The cells used in the experiment were L1929 mouse embryonic fibroblasts (a major cell type in the dermis). When the cell growth density was appropriate and the cell state was good, they were irradiated with UV for an appropriate time to construct a cell reactive oxygen species damage model. After the stimulation ended, the original culture medium was discarded, and the cells were rinsed 3 times with PBS. Then, culture media containing baicalin, baicalin liposomes, and baicalin microcapsules were added respectively (the dosage of the baicalin group was 50 μg / mL, the dosage of baicalin liposomes was 2 mg / mL (equivalent to 50 μg / mL of baicalin), the dosage of baicalin microcapsules was 333.3 μg / mL (equivalent to 50 μg / mL of baicalin), and the dosage of the microcapsule coating material was 283.3 μg / mL). After adding, they were incubated for 2 h, 4 h, and 8 h respectively. After the incubation ended, DCFH-DA fluorescent probe was taken and diluted with serum-free medium by an appropriate multiple (DCFH-DA fluorescent probe can freely cross the cell membrane and has no fluorescence itself. When it reacts with intracellular enzymes, DCFH is generated and can no longer cross the cell membrane, while intracellular reactive oxygen species can oxidize DCFH to DCF, and DCF shows green fluorescence under the excitation wavelength of 488 nm, which can indicate the level of intracellular reactive oxygen species). The original culture medium was discarded, and each dish was rinsed 3 times with PBS. 1 mL of the diluted DCFH-DA solution was added to each dish to load the probe in situ, and then placed in a cell culture incubator for 40 min. After the incubation ended, the original culture medium was discarded, the cells were washed 3 times with PBS, 1 mL of PBS buffer was added, and the bright and dark field cell state images were taken under a fluorescence microscope respectively.

[0188] 5.2 Results

[0189] The repair effect of baicalin on ROS reactive oxygen species and cells damaged by reactive oxygen species is shown in Figure 7 , where the fluorescence intensity is positively correlated with the level of intracellular ROS reactive oxygen species. The higher the fluorescence intensity, the higher the reactive oxygen species level; the lower the green fluorescence, the lower the ROS level.

[0190] The ROS levels in the baicalin, baicalin liposome, and baicalin microcapsule treatment groups all decreased with the prolongation of the treatment time. The fluorescence intensity at 8 h of the three treatments was significantly lower than that at 2 h. At 4 h, the results showed that the degree of reduction of ROS levels by baicalin microcapsules was significantly better than that of the other two groups.

[0191] When the treatment time was the same, the ability to scavenge intracellular reactive oxygen species and the ability to repair cells after reactive oxygen species damage were as follows: baicalin microcapsules ≥ baicalin liposomes ≥ baicalin. At 8 h, the intracellular reactive oxygen species level in the baicalin microcapsule group was basically completely scavenged, while there was still a certain level in the pure baicalin drug group.

[0192] The inventor speculated that the reason for the above results was that both baicalin microcapsules and baicalin liposomes promoted the uptake of baicalin by cells. Therefore, the ability to scavenge intracellular reactive oxygen species was greatly improved within the same time, and the uptake ability of cells for baicalin microcapsules was stronger than that of baicalin liposomes, and the ability of baicalin microcapsules to scavenge intracellular ROS was also higher than that of baicalin liposomes.

[0193] The above results showed that baicalin microcapsules improved the uptake of baicalin by cells, could scavenge intracellular reactive oxygen species, and had excellent antioxidant ability.

[0194] Example 6 Changes in GSH / GSSG in the intracellular oxygen damage repair model by baicalin microcapsules

[0195] 6.1 Method

[0196] Test the effect of baicalin on GSH / GSSG in oxygen-damaged cells (such as the oxygen damage model described in Example 5). Five groups were set up in the experiment (blank control, baicalin, baicalin microcapsules, baicalin liposomes, materials), and the relative changes in GSH / GSSG in the administration group and the blank group were compared.

[0197] Among them, the dosage of the baicalin group was 50 μg / mL, the dosage of baicalin liposomes was 2 mg / mL (equivalent to 50 μg / mL of baicalin), the dosage of baicalin microcapsules was 333.3 μg / mL (equivalent to 50 μg / mL of baicalin), and the dosage of the microcapsule wrapping material was 283.3 μg / mL. Take the cells constructed in the previous step (construct the intracellular reactive oxygen species damage model in Example 5) for administration. Use GSH and GSSG detection kits to make standard curves for total glutathione and GSSG respectively, and use an enzyme-labeled instrument to detect OD 405nm , and after testing the total protein content respectively, calculate the content of GSH and GSSG in the unit protein.

[0198] 6.2 Results

[0199] Figure 8It is the change value of the relative content of GSH / GSSG in the oxygen injury repair model after baicalin administration. Taking the blank group as the benchmark, it can be observed that the GSH / GSSG ratio increased by 3.36%, 35.79%, 20.98% and 50.96% in the material group, baicalin group, baicalin liposome group and baicalin microcapsule group respectively compared with the blank group.

[0200] From Figure 8 , compared with the oxygen injury model (blank group), after the administration of baicalin-related drugs, the GSH / GSSG increased to varying degrees. Taking GSH as a variable, it can be seen that baicalin-related drugs have excellent scavenging effects on intracellular free radicals and oxides, and have excellent repair effects on cell oxygen injury.

[0201] In terms of oxygen injury repair, baicalin microcapsules have certain advantages over baicalin liposomes and baicalin, which can also be seen in subsequent experiments. In fact, it can be observed that after the oxygen-injured cells were incubated with baicalin, baicalin liposomes and baicalin microcapsules respectively, the cell growth state in the microcapsule group was better than that in the other groups.

[0202] The repair of oxygen-injured cells and the promotion of GSH by the blank material of baicalin microcapsules were not obvious. After excluding the material effect of the baicalin microcapsule group, baicalin after being encapsulated in microcapsules should have corresponding improvements in stability and cell uptake.

[0203] Example 7 Effect of baicalin microcapsules on the secretion of MMP-1 enzyme by human skin fibroblasts

[0204] 7.1 Method

[0205] In this experiment, blank, baicalin, baicalin microcapsules, material (microcapsules without baicalin) and baicalin liposome groups were set up respectively to study the inhibitory effect of baicalin on the secretion of MMP-1 by cells.

[0206] Experimental procedure: According to the dosage of the baicalin group at 20 μg / mL; the dosage of baicalin liposomes at 800 μg / mL (equivalent to 20 μg / mL of baicalin), and the dosage of baicalin microcapsules at 133.3 μg / mL (equivalent to 20 μg / mL of baicalin, and the dosage of the baicalin microcapsule material at 113.3 μg / mL), they were applied to human fibroblasts. The drugs were diluted with the culture medium, the cell supernatant was discarded, washed 3 times with PBS, the corresponding drugs were added, and the cells were cultured for another 24 h. After the culture was completed, the cell supernatant was taken, centrifuged, and the supernatant was used for MMP-1 testing.

[0207] 7.2 Results

[0208] From Figure 9It can be seen that the contents of MMP-1 in the baicalin group, the material group, and the baicalin microcapsule group all decreased to varying degrees compared with the blank group, by 11.06%, 4.1%, and 17.14% respectively, indicating that all four have a certain inhibitory effect on the secretion of MMP-1.

[0209] In summary, baicalin has an obvious inhibitory effect on the secretion of MMP-1, and its inhibitory ability is further improved after encapsulation.

[0210] Example 8 Effect of Baicalin Microcapsules on the Secretion of TIMP-1 Enzyme in Human Skin Fibroblasts

[0211] 8.1 Method

[0212] In this experiment, baicalin, baicalin microcapsules, baicalin liposomes, blank, and material groups were set up to study the effects of baicalin and related encapsulants on the content of TIMP-1 in the matrix of human skin fibroblasts.

[0213] Experimental method: Apply to human fibroblasts according to the dosage of baicalin liposomes at 800 μg / mL (equivalent to 20 μg / mL of baicalin), the dosage of baicalin microcapsules at 133.3 μg / mL (equivalent to 20 μg / mL of baicalin, and the dosage of baicalin microcapsule material at 113.3 μg / mL). Dilute the drug with the culture medium, discard the cell supernatant, wash 3 times with PBS, add the corresponding drug, and continue to culture for 24 h. After culturing, take the cell supernatant, centrifuge, and take the supernatant for MMP-1 testing.

[0214] 8.2 Results

[0215] As Figure 10 shown, the contents of TIMP-1 in the cell supernatants of the baicalin group, the material group, and the baicalin microcapsule group all increased to varying degrees compared with the blank group, by 8.16%, 17.21%, and 18.04% respectively.

[0216] The content of TIMP-1 in the baicalin liposome group decreased to a certain extent compared with the blank group, about 12.42%, and a certain substance in the baicalin liposome material may affect the expression of TIMP-1.

[0217] The microcapsule material has an obvious promotion effect on the expression of TIMP-1 in the extracellular matrix of human skin fibroblasts, and the promotion effect of baicalin microcapsules on the expression of TIMP-1 is significantly stronger than that of unencapsulated baicalin, which is increased by 2.21 times.

[0218] Combined with the data of MMP-1, baicalin can inhibit the expression of MMP-1 and increase the expression of TIMP-1, showing excellent anti-aging effects, and the anti-aging effect of baicalin after microencapsulation is better.

[0219] Example 9 Effect of Baicalin Microcapsules on the Activity of Intracellular CAT

[0220] 9.1 Method

[0221] After incubating baicalin, baicalin microcapsules, baicalin liposomes, and the material with cells, the relative activity change of CAT in the cells was tested. Five groups were set up in the experiment (blank control, baicalin, baicalin microcapsules, baicalin liposomes, material).

[0222] The dosage of the baicalin group was 50 μg / mL, the dosage of the baicalin liposome was 2 mg / mL (equivalent to 50 μg / mL of baicalin), the dosage of the baicalin microcapsule was 333.3 μg / mL (equivalent to 50 μg / mL of baicalin), and the dosage of the microcapsule encapsulating material was 283.3 μg / mL. After the administration, the CAT activity per unit protein content was detected and calculated. Using the blank group as the benchmark, the activity graph of CAT in the administration groups was drawn.

[0223] 9.2 Results

[0224] As Figure 11 shown, compared with the blank group, the CAT activities per unit protein content in the material, baicalin, baicalin liposome, and baicalin microcapsule groups increased by 6.84%, 74.72%, 114.20%, and 166.99% respectively.

[0225] It can be observed that baicalin can significantly increase the activity of CAT. Therefore, baicalin has excellent antioxidant effects.

[0226] The effects of baicalin encapsulated by liposomes and microcapsules on the CAT activity were significantly better than those of unencapsulated baicalin, and the microcapsule encapsulation was better than the liposome encapsulation.

[0227] Combined with the in vitro release experiment, the stability and biological activity of encapsulated baicalin were improved to a certain extent. In addition, the sustained-release effect after encapsulation enabled baicalin to be released stably and for a long time, which was also one of the reasons for the better effect after encapsulation.

[0228] Example 10 Effect of Baicalin Microcapsules on the Activity of Intracellular SOD

[0229] 10.1 Method

[0230] After incubating baicalin, baicalin microcapsules, baicalin liposomes, and the material with cells, the relative activity change of intracellular SOD was tested. Five groups were set up in the experiment (blank control, baicalin, baicalin microcapsules, baicalin liposomes, material).

[0231] The dosage of the baicalin group was 50 μg / mL, the dosage of baicalin liposomes was 2 mg / mL (equivalent to 50 μg / mL of baicalin), the dosage of baicalin microcapsules was 333.3 μg / mL (equivalent to 50 μg / mL of baicalin), and the dosage of the microcapsule wrapping material was 283.3 μg / mL. After the administration was completed, SOD activity tests were conducted. Using the blank group as a benchmark, the SOD activity graph of the administered groups was plotted.

[0232] 10.1 Results

[0233] As Figure 12 shown, the SOD activities per unit protein content of the material, baicalin, baicalin liposomes, and baicalin microcapsule groups were increased by 0.89%, 104.31%, 23.92%, and 197.45% respectively compared with the blank control group. After the administration of baicalin, the SOD activity per unit protein was significantly increased.

[0234] The increase rate of SOD activity after encapsulation with baicalin microcapsules was also significantly higher than that without encapsulation of baicalin, but the promoting effect of baicalin encapsulated in liposomes on SOD activity decreased to a certain extent compared with that without encapsulation.

[0235] The blank microcapsule material group did not show a promoting effect on SOD activity, and the influence of the material in the baicalin microcapsules on the experiment could be excluded. Therefore, microcapsule encapsulation greatly improved the expression and promoting effect of baicalin on intracellular SOD.

[0236] Combined with the fact that the activities or contents of SOD, CAT, and GSH all increased to varying degrees after the administration, it can be seen that baicalin has a high-capacity and all-round effect on the antioxidant effect of microcapsule cells.

[0237] Example 11 Repair of Photoinjury and Promotion of Fibroblast Growth of Baicalin Microcapsules on Human Fibroblasts (HSF)

[0238] 11.1 Method

[0239] Human fibroblasts (HSF) were used in the experiment, and blank control, baicalin, baicalin microcapsule, and baicalin liposome groups were set up respectively. Among them, the dosage of the baicalin group was 20 μg / mL; the dosage of baicalin liposomes was 800 μg / mL (equivalent to 20 μg / mL of baicalin), and the dosage of baicalin microcapsules was 133.3 μg / mL (equivalent to 20 μg / mL of baicalin). The cells were irradiated with ultraviolet light of appropriate intensity to establish a photoinjury model, and then the above drugs were diluted with serum-free medium and added to the culture dishes. After continued culture at 37°C and 5% CO2 for 48 h, after fixation with paraformaldehyde and staining with hematoxylin, ponceau - fuchsin - aniline blue staining solutions respectively, the fiber density, orientation, thickness were observed under a microscope, and the fiber area per unit cell was calculated, and a table was drawn.

[0240] 11.2 Results

[0241] As shown Figure 13 in the figure, the blue represents human fibroblast cell fibers and the red represents fibroblasts.

[0242] After ultraviolet irradiation, the cell fibers in the blank control group became thinner and their orientation was abnormal, indicating that ultraviolet irradiation accelerated cell aging.

[0243] After incubating the ultraviolet-damaged cells with baicalin, baicalin liposomes, and baicalin microcapsules respectively, it was significantly observed that the cell viability was significantly improved, the orientation of cell fibers tended to be normal, and the cell fibers became significantly thicker compared with before incubation.

[0244] The results showed that baicalin, baicalin liposomes, and baicalin microcapsules all had a repair effect on photoaged fibroblasts, demonstrating the anti-aging effect of baicalin on the dermis.

[0245] The above results showed that the baicalin microcapsules had a uniform particle size and particle distribution, no obvious particles and material fragments, a stable system, excellent transdermal release ability, skin penetration ability, antioxidant ability, and anti-aging ability, and could be used for anti-aging and antioxidant of the dermis.

[0246] Discussion

[0247] In cosmetic applications, the problems related to oxidative aging mainly occur in the dermis of the skin. Therefore, whether the raw materials can be delivered to the dermis and the intake of cells on the dermis side are crucial for antioxidant and anti-aging effects.

[0248] What the present invention provides is baicalin microcapsules. In terms of transdermal depth and cumulative release rate, the data of baicalin microcapsules are better than those of baicalin raw drug and baicalin liposomes. Both liposome encapsulation and microcapsule drug loading have reached the connective fiber tissue of the dermis layer, but the penetration depth of the microcapsule group in the dermis layer is deeper than that of the liposome group, and the drug uptake by dermal cells is more.

[0249] All the documents mentioned in the present invention are incorporated herein by reference as if each document was individually incorporated by reference. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims

1. A baicalin-cyclodextrin-vesicle complex, characterized in that, Comprising (i) an active ingredient, said active ingredient comprising baicalin; (ii) cyclodextrin; and (iii) a vesicle material, said vesicle material comprising vesicles of microbial origin; And, the complex has the following components in parts by weight: 12 - 18 parts of active ingredient 70 - 90 parts of vesicle material 3 - 7 parts of cyclodextrin The (ii) cyclodextrin clathrates the (i) active ingredient to form nanoparticles; The (iii) vesicle material encapsulates the nanoparticles to form a baicalin - cyclodextrin - vesicle complex.

2. The baicalin-cyclodextrin-vesicle complex according to claim 1, wherein The microorganism includes strains of the genus Saccharomyces and strains of the genus Bacillus.

3. The baicalin - cyclodextrin - vesicle complex according to claim 1, wherein The Zeta potential of the baicalin - cyclodextrin - vesicle complex is - 10 to 0 mV.

4. The baicalin-cyclodextrin-vesicle complex according to claim 1, wherein The baicalin - cyclodextrin - vesicle complex significantly increases the skin penetration depth of baicalin. The significant increase in the skin penetration depth of baicalin means that compared with the penetration depth P0 of baicalin in in vitro skin tissue, the penetration depth of the baicalin - cyclodextrin - vesicle complex in in vitro skin tissue is P1, and the ratio (P1 / P0) ≥ 3.

5. The baicalin-cyclodextrin-vesicle complex according to claim 1, wherein The cyclodextrin is selected from α - cyclodextrin, β - cyclodextrin, γ - cyclodextrin, or a combination thereof.

6. The baicalin-cyclodextrin-vesicle complex according to claim 1, wherein The particle size of the baicalin - cyclodextrin - vesicle complex is 50 - 500 nm.

7. The baicalin-cyclodextrin-vesicle complex according to claim 1, wherein The polydispersity index of the baicalin - cyclodextrin - vesicle complex is 0.1 - 0.

2.

8. A method for preparing the baicalin-cyclodextrin-vesicle complex according to any one of claims 1 to 7, characterized in that, Comprising the following steps: (s1) Provide (i) an active ingredient, said active ingredient comprising baicalin; (ii) cyclodextrin; and (iii) a vesicle material, said vesicle material comprising vesicles of microbial origin; (s2) Mix the active ingredient, cyclodextrin and vesicle material in step (s1) to obtain a baicalin - cyclodextrin - vesicle complex; Wherein, the active ingredient is 12 - 18 parts by weight, cyclodextrin is 3 - 7 parts by weight and vesicle material is 70 - 90 parts by weight.

9. Use of the baicalin-cyclodextrin-vesicle complex according to any one of claims 1 to 7, characterized in that, For preparing a preparation or composition.

10. A cosmetic, characterized in that, The cosmetic comprises (c1) the baicalin - cyclodextrin - vesicle complex according to any one of claims 1 - 7; and (c2) a cosmetically acceptable carrier or excipient.

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