A composition containing stem cell exosomes and its application in preparing a product with spot-lightening effect

Abstract

The present invention belongs to the technical field of biomedicine. The present invention specifically discloses a composition containing stem cell exosomes and an application thereof in preparing a product with a freckle-removing effect. The composition of stem cell exosomes comprises the following components: mesenchymal stem cell exosomes, a roxburgh roxburgh extract, a red eucommia extract, and a penetration enhancer; the mass ratio of the mesenchymal stem cell exosomes, the roxburgh roxburgh extract, and the red eucommia extract is 1:(2-10):(2-10); the mass ratio of the mesenchymal stem cell exosomes to the penetration enhancer is 1:(0.5-4), and the composition has excellent whitening and freckle-removing effects.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and specifically to a composition, in particular to a composition containing stem cell exosomes, and further to an application of the composition in preparing a product with a spot-lightening effect. Background Art

[0002] Melanin is a biological pigment produced in melanocytes. The deposition of melanin in the skin can cause skin dullness and other related problems. Inhibiting the production of melanin is an important way for the cosmetics industry to achieve whitening effects at this stage. L-tyrosine in melanocytes is oxidized to form dopaquinone under the action of tyrosinase. With the participation of tyrosinase-related protein 1 (TRP-1) and tyrosinase-related protein 2 (TRP-2), DQ is further self-oxidized to form 5,6-dihydroxyindolecarboxylic acid and 5,6-dihydroxyindole. The two undergo a series of catalytic polymerizations to finally synthesize melanin. Reducing the content of melanin in the skin can effectively achieve whitening and spot-removing effects.

[0003] Exosomes are nano-scale membrane vesicles that can be secreted by almost all cells, with a diameter of 30-150 nm. Their main components include the surface phospholipid bilayer and the contents they carry, including nucleic acids, proteins, lipids, amino acids, and metabolites. In recent years, as research has gradually deepened, research on the "messenger" role of exosomes, disease progression, biomarker role, and drug delivery tools has gradually emerged.

[0004] CN 107007541 discloses the use of exosomes derived from mesenchymal stem cells in skin whitening preparations. The mesenchymal stem cells are derived from human umbilical cord or placenta, and further discloses that the mesenchymal stem cells derived from human umbilical cord or placenta can inhibit the production of melanin and the activity of tyrosinase, thereby exerting a whitening effect, nitrogen.

[0005] CN108451887A discloses a skin care composition containing stem cell-derived exosomes and its use, which achieves better moisturizing, repairing, anti-wrinkle and / or whitening effects through the synergy of human umbilical cord mesenchymal stem cell-derived exosomes, epidermal growth factor and sodium hyaluronate.

[0006] How to improve the whitening and freckle-removing effects of products containing exosomes has become a technical problem that needs to be urgently solved by technicians in this field. Summary of the invention

[0007] The present invention provides a composition containing stem cell exosomes and application of the composition in preparing a product with a spot-lightening effect. The composition has an excellent whitening and spot-lightening effect.

[0008] The present invention solves the technical problem by adopting the following technical solutions:

[0009] To achieve the above object, in a first aspect of the present invention, the present invention provides a composition containing stem cell exosomes, comprising the following components: mesenchymal stem cell exosomes, roxburghii roxburghii extract, eucommia ulmoides extract, and a penetration enhancer;

[0010] The mass ratio of the mesenchymal stem cell exosomes, the roxburghii extract, and the red eucommia extract is 1: (2-10): (2-10);

[0011] The mass ratio of the mesenchymal stem cell exosomes to the penetration enhancer is 1:(0.5-4).

[0012] As a preferred embodiment of the present invention, the mass ratio of the mesenchymal stem cell exosomes, the roxburghii roxburghii extract, and the red eucommia extract is 1: (3-8): (3-8).

[0013] As a preferred embodiment of the present invention, the mass ratio of the mesenchymal stem cell exosomes to the penetration enhancer is 1:(1-2).

[0014] As a preferred embodiment of the present invention, the mesenchymal stem cell exosomes are exosomes secreted by BMSCs modified with mutant HIF-1α.

[0015] As a preferred embodiment of the present invention, the penetration enhancer includes PEG / PPG-14 / 7 dimethyl ether and oleyl alcohol polyoxyethylene ether;

[0016] The mass ratio of the PEG / PPG-14 / 7 dimethyl ether to oleyl alcohol polyoxyethylene ether is 1:(0.5-2).

[0017] As a preferred embodiment of the present invention, the method for preparing the roxburghii extract comprises the following steps:

[0018] Crush the roxburghii to 50-200 meshes to obtain roxburghii powder;

[0019] The roxburghii powder and the enzymatic hydrolysate are mixed evenly, the pH value is adjusted to 5.8-6.8, ultrasonic treatment is performed at 400-800W for 35-60min at 40-55°C, and the enzyme is inactivated by heating to obtain a mixture;

[0020] The mixture was applied to an AB-8 macroporous adsorption resin column at a rate of 0.5-1.5 BV / h and an elution rate of 0.5-4 BV / h. 0.5-2 BV was first washed with water and then 0.5-2 BV was eluted with 75-90wt% ethanol. The ethanol eluate was collected and dried to obtain the roxburghii extract.

[0021] As a preferred embodiment of the present invention, the solid-liquid ratio of the roxburghii powder to the enzymatic hydrolyzate is 1 g: (2-10) mL;

[0022] The enzymatic hydrolysis solution comprises hexadecyltrimethylammonium bromide, papain, flavor protease, neutral protease and water;

[0023] The mass ratio of hexadecyltrimethylammonium bromide, papain, flavor protease, neutral protease and water is 1: (0.5-2): (0.5-2): (1-4): (40-100).

[0024] As a preferred embodiment of the present invention, the method for preparing the red eucommia extract comprises the following steps:

[0025] Grinding the red eucommia to 50-200 meshes to obtain red eucommia powder;

[0026] Add red eucommia powder and ammonium citrate to 40-90wt% ethanol solution, reflux extract at 50-70°C for 2-3 times, each time for 0.5-4h, filter, combine the filtrate, and obtain ethanol extract;

[0027] Add tartaric acid to the ethanol extract, treat with ultrasound at 400-800W for 25-40min, filter, and collect the filtrate;

[0028] The filtrate was extracted with n-butanol for 2-3 times, the extracts were combined and dried to obtain the red eucommia extract.

[0029] As a preferred embodiment of the present invention, the solid-to-liquid ratio of the red eucommia powder, ammonium citrate and ethanol solution is 1 g: (0.01-0.05) g: (2-10) mL.

[0030] As a preferred embodiment of the present invention, the solid-to-liquid ratio of the tartaric acid to the ethanol extract to the ethanol extract is 0.01~1g:1mL.

[0031] As a preferred embodiment of the present invention, the usage ratio of the filtrate to n-butanol is 1 mL: (3-6) mL.

[0032] As a preferred embodiment of the present invention, it also includes a solvent;

[0033] The mass ratio of the mesenchymal stem cell exosomes to the solvent is 1:(10-80).

[0034] The solvent includes at least one of 1,4-butanediol, glycerol, propylene glycol, sorbitol and water.

[0035] In a second aspect of the present invention, the present invention provides use of the above-mentioned composition containing stem cell exosomes in the preparation of a product having whitening and / or spot-lightening effects.

[0036] The beneficial effects of the present invention are as follows: (1) The present invention combines mesenchymal stem cell exosomes, roxburgh extract and red eucommia extract in a specific mass ratio to obtain an excellent whitening and freckle-removing effect, which can be widely used in daily chemical products; (2) The exosomes secreted by the mutant HIF-1α modified BMSCs, red eucommia extract and roxburgh extract described in the present invention have a significant synergistic effect in whitening and freckle-removing. The three have different main whitening principles and play a role from different dimensions and links, thereby achieving an excellent whitening effect. DETAILED DESCRIPTION

[0037] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0038] In the present invention, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0039] In the present invention, when it comes to numerical ranges, unless otherwise specified, the above numerical ranges are deemed to be continuous and include the minimum and maximum values ​​of the range, as well as each value between such minimum and maximum values. Further, when a range refers to an integer, each integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be merged. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges included therein.

[0040] In the present invention, there is no particular limitation on the specific dispersion and stirring treatment methods.

[0041] In the present invention, unless otherwise stated, all parts are parts by mass.

[0042] The reagents or instruments used in the present invention without indicating the manufacturer are all conventional products that can be obtained commercially. The raw material information used in the examples and comparative examples of the present invention is as follows:

[0043] Exosomes secreted by BMSCs modified with mutant HIF-1α were purchased from Xi'an Qiyue Biotechnology Co., Ltd.

[0044] PEG / PPG-14 / 7 dimethyl ether was purchased from Wuhan Xinxinjiali Biotechnology Co., Ltd.

[0045] Rosa roxburghii and Eucommia ulmoides were purchased from Guangzhou Qingping Chinese herbal medicine market.

[0046] The embodiment of the present invention provides a composition containing stem cell exosomes, comprising the following components: mesenchymal stem cell exosomes, roxburghii roxburghii extract, eucommia ulmoides extract, and a penetration enhancer;

[0047] Among them, the mass ratio of mesenchymal stem cell exosomes, roxburghii extract, and red eucommia extract is 1: (2~10): (2~10);

[0048] Among them, the mass ratio of mesenchymal stem cell exosomes to penetration enhancer is 1: (0.5~4).

[0049] Studies have shown that mesenchymal stem cell exosomes can effectively inhibit the production of melanin and the activity of tyrosinase, thereby showing excellent whitening and spot-lightening potential.

[0050] Rosa roxburghii is the fruit of the perennial deciduous shrub Rosa roxburghii of the Rosaceae family. It has aliases such as Ci Li, Wen Xianguo, and Sending Spring Back. It tastes sweet, sour and astringent, is neutral in nature, and enters the spleen and stomach meridians. It is mainly produced in western Guizhou. Rosa roxburghii contains a variety of active ingredients, including polysaccharides, organic acids, flavonoids, and triterpenoid compounds.

[0051] The medicinal material of red Eucommia is the stem bark and root bark of Eucommia ulmoides, red Eucommia ulmoides and Eucommia ulmoides of the Apocynaceae family, or the dried bark of Glehnia littoralis of the Glehnia littoralis genus. It mainly contains chemical components such as alkaloids, phenols, organic acids, sugars, flavonoids, etc.

[0052] Among them, the addition of permeabilizing agents can promote the penetration and absorption of active ingredients in the skin, improve utilization, and especially improve the penetration and absorption of mesenchymal stem cell exosomes.

[0053] The inventors of the present invention have found through extensive research that the present invention combines mesenchymal stem cell exosomes, roxburghii extract, and eucommia ulmoides extract in a specific mass ratio to obtain an excellent whitening and spot-lightening effect, which can be widely used in daily chemical products.

[0054] In one embodiment, the mass ratio of mesenchymal stem cell exosomes, Rosa roxburghii extract, and Eucommia ulmoides extract is 1:(3-8):(3-8). Under the above specific mass ratio, the whitening and spot-lightening effects are better.

[0055] In one of the more preferred embodiments, the mass ratio of mesenchymal stem cell exosomes, Rosa roxburghii extract, and Eucommia ulmoides extract is 1:4:5. Under the above specific mass ratio, the whitening and spot-lightening effects are optimal.

[0056] In one embodiment, the mass ratio of mesenchymal stem cell exosomes to penetration enhancer is 1:(1-2), at which the penetration enhancer promotes better penetration and absorption of active ingredients in the skin.

[0057] In a more preferred embodiment, the mass ratio of mesenchymal stem cell exosomes to the penetration enhancer is 1:1. At this mass, the penetration enhancer can best promote the penetration and absorption of the active ingredients in the skin.

[0058] In one embodiment, the mesenchymal stem cell exosomes are exosomes secreted by mutant HIF-1α modified BMSCs. The inventors of the present invention have creatively discovered in a large number of studies that, under the promotion of the specific penetration enhancer of the present invention, the exosomes secreted by mutant HIF-1α modified BMSCs have the potential to regenerate or rejuvenate the skin, can effectively resist pigmentation, inhibit the production of melanin, and inhibit the activity of tyrosinase. At the same time, HIF-1α can regulate the migration of epidermal keratinocytes and dermal fibroblasts, promote epidermal cell growth, promote the formation of epidermal structure, improve skin elasticity, and reduce the accumulation of dark substances (oxidized proteins) to effectively improve skin dullness, and then the exosomes secreted by mutant HIF-1α modified BMSCs have better whitening and spot-lightening effects than conventional mesenchymal stem cell exosomes.

[0059] In one embodiment, the penetration enhancer includes PEG / PPG-14 / 7 dimethyl ether and oleyl alcohol polyoxyethylene ether;

[0060] The mass ratio of PEG / PPG-14 / 7 dimethyl ether to oleyl alcohol polyoxyethylene ether is 1: (0.5-2). The specific penetration enhancer of the present invention can promote the penetration and absorption of active ingredients on the skin, improve the utilization, and especially can improve the penetration and absorption of mesenchymal stem cell exosomes.

[0061] The inventors of the present invention firstly used commercially available roxburgh roxburgh extract and commercially available red eucommia extract, and found that although the commercially available roxburgh roxburgh extract and commercially available red eucommia extract have certain whitening and freckle-removing effects, it may be because the purpose of extracting the commercially available roxburgh roxburgh extract and commercially available red eucommia extract is not the whitening and freckle-removing effect, therefore, resulting in poor whitening and freckle-removing effects. Therefore, in order to further improve the whitening and freckle-removing effects, the present invention provides the following preparation method of roxburgh roxburgh extract and red eucommia extract, and specifically extracts the whitening and freckle-removing components of roxburgh roxburgh and red eucommia.

[0062] In one embodiment, the preparation method of the roxburghii extract comprises the following steps:

[0063] Crush the roxburghii to 50-200 meshes to obtain roxburghii powder;

[0064] The roxburghii powder and the enzymatic hydrolysate are mixed evenly, the pH value is adjusted to 5.8-6.8, ultrasonic treatment is performed at 400-800W for 35-60min at 40-55°C, and the enzyme is inactivated by heating to obtain a mixture;

[0065] The mixture was applied to an AB-8 macroporous adsorption resin column at a rate of 0.5-1.5 BV / h and an elution rate of 0.5-4 BV / h. 0.5-2 BV was first washed with water and then 0.5-2 BV was eluted with 75-90wt% ethanol. The ethanol eluate was collected and dried to obtain the roxburghii extract.

[0066] The inventors of the present invention referred to the existing preparation methods of sea buckthorn extracts, such as the document "Effects of Different Sea Buckthorn Extract Components on B16 Cell Proliferation and Tyrosinase Activity" published by Xu Mengjie et al., but the process disclosed therein is only a preparation method of a single sea buckthorn polysaccharide, sea buckthorn flavonoids, and sea buckthorn polyphenols, and the whitening effective component is single, resulting in a low concentration of the whitening effective component of the product obtained by the overall extraction.

[0067] The invention creatively extracts the roxburghii roxburghii by directly enzymolyzing the crushed roxburghii roxburghii with a specific enzyme and then applying the AB-8 macroporous adsorption resin column to obtain the roxburghii roxburghii extract with various whitening components and excellent whitening effect.

[0068] The main whitening and spot-lightening effects of the prepared roxburghii extract are: inhibiting the proliferation of melanocytes can reduce the generation of melanin, thereby achieving a whitening effect, and it also has a certain tyrosinase activity effect.

[0069] In one embodiment, the solid-liquid ratio of the sea buckthorn powder to the enzymatic hydrolyzate is 1 g: (2-10) mL.

[0070] In one embodiment, the enzymatic hydrolysis solution includes hexadecyltrimethylammonium bromide, papain, flavor protease, neutral protease and water.

[0071] The mass ratio of hexadecyltrimethylammonium bromide, papain, flavor protease, neutral protease and water is 1: (0.5-2): (0.5-2): (1-4): (40-100). The inventors of the present invention found that the whitening effects of the roxburghii extracts prepared by different enzymatic hydrolyses are different. The whitening effect of the roxburghii extract obtained by using the enzymatic hydrolysate of the present invention is better than that of other enzymatic hydrolysates. Hexadecyltrimethylammonium bromide has excellent penetration, softening and emulsification, and plays a role in assisting enzymatic hydrolysis.

[0072] In one embodiment, the method for preparing the red eucommia extract comprises the following steps:

[0073] Grinding the red eucommia to 50-200 meshes to obtain red eucommia powder;

[0074] Add red eucommia powder and ammonium citrate to 40-90wt% ethanol solution, reflux extract at 50-70°C for 2-3 times, each time for 0.5-4h, filter, combine the filtrate, and obtain ethanol extract;

[0075] Add tartaric acid to the ethanol extract, treat with ultrasound at 400-800W for 25-40min, filter, and collect the filtrate;

[0076] The filtrate was extracted with n-butanol for 2-3 times, the extracts were combined and dried to obtain the red eucommia extract.

[0077] The invention creatively extracts red eucommia powder with ethanol reflux in the presence of leaching aid ammonium citrate, then hydrolyzes it with tartaric acid, and extracts it with n-butanol, so as to prepare red eucommia extract with excellent whitening and spot-removing effect.

[0078] The main whitening and freckle-removing effects of the prepared red eucommia extract are reflected in: inhibiting the expression level of proteins related to melanin synthesis.

[0079] In one embodiment, the solid-to-liquid ratio of the red eucommia powder, ammonium citrate, and ethanol solution is 1 g: (0.01-0.05) g: (2-10) mL.

[0080] The inventors of the present invention have discovered that the exosomes secreted by mutant HIF-1α modified BMSCs, Eucommia ulmoides extract and Rosa roxburghii extract described in the present invention have significant synergistic effects in whitening and lightening spots. The three have different main whitening principles and work from different dimensions and links, thereby achieving excellent whitening effects.

[0081] In one embodiment, the solid-to-liquid ratio of the tartaric acid to the ethanol extract to the ethanol extract is 0.01~1g:1mL.

[0082] In one embodiment, the ratio of the filtrate to n-butanol is 1 mL: (3-6) mL.

[0083] In one embodiment, the composition further comprises a solvent;

[0084] Among them, the mass ratio of mesenchymal stem cell exosomes to solvent is 1: (10~80).

[0085] More specifically, the solvent includes at least one of 1,4-butylene glycol, glycerol, propylene glycol, sorbitol, and water.

[0086] In addition, it should be noted that the composition containing stem cell exosomes can be combined with auxiliary materials commonly used in conventional cosmetic compositions, such as thickeners, moisturizers, emollients, emulsifiers, antioxidants, flavors, etc., as needed.

[0087] Exemplarily, the composition containing stem cell exosomes may include a thickener, and suitable thickeners include, but are not limited to, carbomer, hydroxyethyl cellulose, xanthan gum, sodium polyacrylate, acrylic acid (ester) cross-linked polymer, xanthan gum, and combinations thereof.

[0088] Exemplarily, the composition containing stem cell exosomes may include a humectant, and suitable humectants include, but are not limited to, dipropylene glycol, polyethylene glycol, xylitol, diglycerol, maltitol, maltose, D-mannitol, dipropylene glycol, polypropylene glycol, neopentyl glycol, erythritol, sorbitol, mannitol, xylitol, glucose, lactose, mannose, fructose, inositol, sorbitol, sorbitan, trehalose, starch hydrolyzate, and combinations thereof.

[0089] Exemplarily, the composition containing stem cell exosomes may include an emollient, and suitable emollients include, but are not limited to, dimethicone, cyclopentasiloxane, diisostearyl malate, hexyl laurate, polyglyceryl 2-diisostearate, pentaerythritol, squalane, tetraisostearate, jojoba oil, caprylic capric triglyceride, dicaprylyl carbonate, macadamia oil, shea butter, white oil, almond oil, olive oil, jojoba oil, octyldodecanol, castor oil, isononyl isononanoate, dioctyl adipate, octyl stearate, hexyl laurate, coco-caprylate, caprate, and combinations thereof.

[0090] Exemplarily, the composition containing stem cell exosomes may include an emulsifier, and suitable emulsifiers include, but are not limited to, polyglyceryl-3 diisostearate, polyglyceryl-10 laurate, polyglyceryl-10 myristate, polyglyceryl-5 trioleate, PEG-60 hydrogenated castor oil, PEG-30 soy sterol, PPG-13-decyltetradecyl alcohol polyether-24, nonylphenol polyether-12, steareth-2, steareth-21, hydrogenated lecithin, and combinations thereof.

[0091] Exemplarily, the composition containing stem cell exosomes may include an antioxidant, and suitable antioxidants include, but are not limited to, tocopherol, butylated hydroxyanisole, butylated hydroxytoluene, tocopheryl acetate, carotenoids, flavonoids, polyphenols, ascorbic acid, herbal antioxidants (such as plant extracts with antioxidant effects), chlorophyll, melatonin, and combinations thereof.

[0092] It should be noted that the present invention does not particularly limit the method for preparing the composition containing stem cell exosomes of the present invention, and those skilled in the art can prepare it into a composition containing stem cell exosomes according to routine procedures.

[0093] Exemplarily, the preparation method of the composition containing stem cell exosomes is: adding mesenchymal stem cell exosomes and a permeation enhancer to a solvent, stirring evenly, and then adding a roxburghii extract and a red eucommia extract, stirring evenly, to obtain a composition containing stem cell exosomes.

[0094] One embodiment of the present invention provides the use of a composition containing stem cell exosomes in the preparation of a product having whitening and / or spot-lightening effects.

[0095] The following examples are provided to facilitate understanding of the present invention. These examples are not provided to limit the scope of the claims.

[0096] A composition containing stem cell exosomes, comprising the following components in parts by mass: 1 part of exosomes secreted by BMSCs modified with mutant HIF-1α, 4 parts of roxburghii roxburghii extract, 5 parts of eucommia bark extract, 0.5 parts of PEG / PPG-14 / 7 dimethyl ether, 0.5 parts of oleyl alcohol polyoxyethylene ether, 19 parts of 1,4-butylene glycol, and 20 parts of propylene glycol.

[0097] In this embodiment, the preparation method of the roxburghii extract comprises the following steps:

[0098] (1) crushing the roxburghii to 100 meshes to obtain roxburghii powder;

[0099] (2) preparing an enzymatic solution by mixing cetyltrimethylammonium bromide, papain, flavor protease, neutral protease and water in a mass ratio of 1:1:1:2:45;

[0100] (3) Add 100 g of roxburghii powder to 800 mL of enzymatic hydrolyzate, stir at 200 rpm for 20 min, adjust the pH to 6.5, perform ultrasonic treatment at 600 W for 50 min at 50 °C, heat to 75 °C to inactivate the enzyme for 1 h, and obtain a mixture;

[0101] (4) The mixture was loaded onto an AB-8 macroporous adsorption resin column at a rate of 1 BV / h and an elution rate of 2 BV / h. 1 BV was first washed with water and then 1 BV was eluted with 80 wt% ethanol. The ethanol eluate was collected and dried to obtain a roxburghii extract.

[0102] In this embodiment, the preparation method of the red eucommia extract comprises the following steps:

[0103] (1) crushing the red eucommia ulmoides into 100 meshes to obtain red eucommia ulmoides powder;

[0104] (2) Add 100 g of red eucommia powder and 4 g of ammonium citrate to 800 mL of 60 wt% ethanol solution, reflux and extract at 60 °C for 3 times, each time for 2 h, filter, and combine the filtrate to obtain an ethanol extract;

[0105] (3) Add tartaric acid to the ethanol extract, treat with ultrasound at 600 W for 30 min, filter, and collect the filtrate; wherein the solid-liquid ratio of tartaric acid to ethanol extract is 0.03 g:1 mL.

[0106] (4) The filtrate was extracted with n-butanol for three times, and the extracts were combined and dried to obtain the red eucommia extract, wherein the ratio of the filtrate to n-butanol was 1 mL:5 mL.

[0107] A composition containing stem cell exosomes, comprising the following components in parts by mass: 1 part of exosomes secreted by BMSCs modified with mutant HIF-1α, 2 parts of roxburghii roxburghii extract, 2 parts of eucommia barbata extract, 0.5 parts of PEG / PPG-14 / 7 dimethyl ether, 0.5 parts of oleyl alcohol polyoxyethylene ether, 19 parts of 1,4-butylene glycol, and 25 parts of propylene glycol.

[0108] In this embodiment, the preparation method of the roxburghii extract comprises the following steps:

[0109] (1) crushing the roxburghii to 50 meshes to obtain roxburghii powder;

[0110] (2) preparing an enzymatic solution by mixing cetyltrimethylammonium bromide, papain, flavor protease, neutral protease and water in a mass ratio of 1:0.5:2:1:45.5;

[0111] (3) Add 100 g of roxburghii powder to 1000 mL of enzymatic hydrolyzate, stir at 200 rpm for 20 min, adjust the pH to 6.5, perform ultrasonic treatment at 800 W for 40 min at 50 °C, heat to 75 °C for 1 h to inactivate the enzyme, and obtain a mixture;

[0112] (4) The mixture was loaded onto an AB-8 macroporous adsorption resin column at a rate of 1 BV / h and an elution rate of 2 BV / h. 1 BV was first washed with water and then 1 BV was eluted with 80 wt% ethanol. The ethanol eluate was collected and dried to obtain a roxburghii extract.

[0113] In this embodiment, the preparation method of the red eucommia extract comprises the following steps:

[0114] (1) Grinding the red eucommia ulmoides into 50 meshes to obtain red eucommia ulmoides powder;

[0115] (2) Add 100 g of red eucommia powder and 1 g of ammonium citrate to 400 mL of 60 wt% ethanol solution, reflux and extract at 60 °C for 3 times, each time for 2 h, filter, and combine the filtrate to obtain an ethanol extract;

[0116] (3) Add tartaric acid to the ethanol extract, treat with ultrasound at 800 W for 25 min, filter, and collect the filtrate; wherein the solid-liquid ratio of tartaric acid to ethanol extract is 0.04 g:1 mL.

[0117] (4) The filtrate was extracted with n-butanol for three times, and the extracts were combined and dried to obtain the red eucommia extract, wherein the ratio of the filtrate to n-butanol was 1 mL:4 mL.

[0118] A composition containing stem cell exosomes, comprising the following components in parts by mass: 0.5 parts of exosomes secreted by BMSCs modified with mutant HIF-1α, 4.5 parts of roxburghii roxburghii extract, 5 parts of eucommia bark extract, 0.5 parts of PEG / PPG-14 / 7 dimethyl ether, 0.5 parts of oleyl alcohol polyoxyethylene ether, 19 parts of 1,4-butylene glycol, and 20 parts of propylene glycol.

[0119] In this embodiment, the preparation method of the roxburghii extract comprises the following steps:

[0120] (1) crushing the roxburghii to 200 meshes to obtain roxburghii powder;

[0121] (2) preparing an enzymatic solution by mixing cetyltrimethylammonium bromide, papain, flavor protease, neutral protease and water in a mass ratio of 1:2:0.5:2:44.5;

[0122] (3) Add 100 g of roxburghii powder to 400 mL of enzymatic hydrolyzate, stir at 200 rpm for 20 min, adjust the pH to 6.5, perform ultrasonic treatment at 400 W for 60 min at 50 °C, heat to 75 °C to inactivate the enzyme for 1 h, and obtain a mixture;

[0123] (4) The mixture was loaded onto an AB-8 macroporous adsorption resin column at a rate of 1 BV / h and an elution rate of 2 BV / h. 1 BV was first washed with water and then 1 BV was eluted with 80 wt% ethanol. The ethanol eluate was collected and dried to obtain a roxburghii extract.

[0124] In this embodiment, the preparation method of the red eucommia extract comprises the following steps:

[0125] (1) Grinding the red eucommia ulmoides into 200 meshes to obtain red eucommia ulmoides powder;

[0126] (2) Add 100 g of red eucommia powder and 5 g of ammonium citrate to 1000 mL of 60 wt% ethanol solution, reflux and extract at 60 °C for 3 times, each time for 2 h, filter, and combine the filtrate to obtain an ethanol extract;

[0127] (3) Add tartaric acid to the ethanol extract, treat with ultrasound at 800 W for 25 min, filter, and collect the filtrate; wherein the solid-liquid ratio of tartaric acid to ethanol extract is 0.01 g:1 mL.

[0128] (4) The filtrate was extracted with n-butanol for three times, and the extracts were combined and dried to obtain the red eucommia extract, wherein the ratio of the filtrate to n-butanol was 1 mL:6 mL.

[0129] A composition containing stem cell exosomes, comprising the following components in parts by mass: 1 part of exosomes secreted by BMSCs modified with mutant HIF-1α, 2 parts of roxburghii roxburghii extract, 7 parts of eucommia bark extract, 0.25 parts of PEG / PPG-14 / 7 dimethyl ether, 0.25 parts of oleyl alcohol polyoxyethylene ether, 19.5 parts of 1,4-butylene glycol, and 20 parts of propylene glycol.

[0130] In this embodiment, the preparation method of the roxburghii extract comprises the following steps:

[0131] (1) crushing the roxburghii to 100 meshes to obtain roxburghii powder;

[0132] (2) preparing an enzymatic solution by mixing cetyltrimethylammonium bromide, papain, flavor protease, neutral protease and water in a mass ratio of 1:0.5:0.5:4:44;

[0133] (3) Add 100 g of roxburghii powder to 500 mL of enzymatic hydrolyzate, stir at 200 rpm for 20 min, adjust the pH to 6.5, perform ultrasonic treatment at 500 W for 50 min at 50 °C, heat to 75 °C to inactivate the enzyme for 1 h, and obtain a mixture;

[0134] (4) The mixture was loaded onto an AB-8 macroporous adsorption resin column at a rate of 1 BV / h and an elution rate of 2 BV / h. 1 BV was first washed with water and then 1 BV was eluted with 80 wt% ethanol. The ethanol eluate was collected and dried to obtain a roxburghii extract.

[0135] In this embodiment, the preparation method of the red eucommia extract comprises the following steps:

[0136] (1) crushing the red eucommia ulmoides into 100 meshes to obtain red eucommia ulmoides powder;

[0137] (2) Add 100 g of red eucommia powder and 3 g of ammonium citrate to 500 mL of 60 wt% ethanol solution, reflux and extract at 60 °C for 3 times, each time for 2 h, filter, and combine the filtrate to obtain an ethanol extract;

[0138] (3) Add tartaric acid to the ethanol extract, treat with ultrasound at 800 W for 25 min, filter, and collect the filtrate; wherein the solid-liquid ratio of tartaric acid to ethanol extract is 0.02 g:1 mL.

[0139] (4) The filtrate was extracted with n-butanol for three times, and the extracts were combined and dried to obtain the red eucommia extract, wherein the ratio of the filtrate to n-butanol was 1 mL:5 mL.

[0140] The difference between Comparative Example 1 and Example 1 is that Comparative Example 1 does not contain exosomes secreted by mutant HIF-1α-modified BMSCs, wherein the amount of exosomes secreted by mutant HIF-1α-modified BMSCs is evenly distributed to the roxburghii extract and the red eucommia extract, that is, the total active dosage is kept unchanged, and the others are the same.

[0141] The composition containing stem cell exosomes of this comparative example includes the following components in parts by weight: 4.5 parts of roxburghii roxburghii extract, 5.5 parts of eucommia ulmoides extract, 0.5 parts of PEG / PPG-14 / 7 dimethyl ether, 0.5 parts of oleyl alcohol polyoxyethylene ether, 19 parts of 1,4-butylene glycol, and 20 parts of propylene glycol.

[0142] The difference between Comparative Example 2 and Example 1 is that Comparative Example 2 does not contain red eucommia extract, wherein the amount of red eucommia extract is evenly distributed to the roxburghii extract and the exosomes secreted by mutant HIF-1α-modified BMSCs, that is, the total active dosage is kept unchanged, and the others are the same.

[0143] The composition containing stem cell exosomes of this comparative example includes the following components in parts by mass: 2 parts of exosomes secreted by BMSCs modified with mutant HIF-1α, 8 parts of roxburghii extract, 0.5 parts of PEG / PPG-14 / 7 dimethyl ether, 0.5 parts of oleyl alcohol polyoxyethylene ether, 19 parts of 1,4-butylene glycol, and 20 parts of propylene glycol.

[0144] The difference between Comparative Example 3 and Example 1 is that Comparative Example 3 does not contain Rosa roxburghii extract, wherein the amount of Rosa roxburghii extract is evenly distributed to the exosomes secreted by mutant HIF-1α-modified BMSCs and the red eucommia extract, that is, the total active dosage is kept unchanged, and the others are the same.

[0145] The composition containing stem cell exosomes of this comparative example includes the following components in parts by mass: 1.67 parts of exosomes secreted by BMSCs modified by mutant HIF-1α, 8.33 parts of red eucommia extract, 0.5 parts of PEG / PPG-14 / 7 dimethyl ether, 0.5 parts of oleyl alcohol polyoxyethylene ether, 19 parts of 1,4-butylene glycol, and 20 parts of propylene glycol.

[0146] The difference between Comparative Example 4 and Example 1 is that the roxburghii extract in Comparative Example 4 is commercially available, and the others are the same.

[0147] The roxburghii extract of this comparative example was purchased from Henan Yixin Biotechnology Co., Ltd., product model YX-008800.

[0148] The difference between Comparative Example 5 and Example 1 is that the preparation method of the roxburghii extract described in Comparative Example 5 is different from that in Example 1, and the others are the same.

[0149] In this comparative example, the preparation method of the roxburghii extract comprises the following steps:

[0150] (1) crushing the roxburghii to 100 meshes to obtain roxburghii powder;

[0151] (2) preparing an enzymatic solution by mixing cellulase, pectinase and water in a mass ratio of 2.5:2.5:45;

[0152] (3) Add 100 g of roxburghii powder to 800 mL of enzymatic hydrolyzate, stir at 200 rpm for 20 min, adjust the pH to 6.5, perform ultrasonic treatment at 600 W for 50 min at 50 °C, heat to 75 °C to inactivate the enzyme for 1 h, and obtain a mixture;

[0153] (4) The mixture was loaded onto an AB-8 macroporous adsorption resin column at a rate of 1 BV / h and an elution rate of 2 BV / h. 1 BV was first washed with water and then 1 BV was eluted with 80 wt% ethanol. The ethanol eluate was collected and dried to obtain a roxburghii extract.

[0154] The difference between Comparative Example 6 and Example 1 is that the Eucommia ulmoides extract in Comparative Example 6 is commercially available, and the other aspects are the same.

[0155] The red eucommia extract of this comparative example was purchased from Xi'an Yaokangyuan Biotechnology Co., Ltd., model number yky-oo166.

[0156] The difference between Comparative Example 7 and Example 1 is that the preparation method of the red eucommia extract described in Comparative Example 7 is different from that in Example 1, and the others are the same.

[0157] In this comparative example, the preparation method of the red eucommia extract comprises the following steps:

[0158] (1) crushing the red eucommia ulmoides into 100 meshes to obtain red eucommia ulmoides powder;

[0159] (2) Add 100 g of Eucommia ulmoides powder to 800 mL of 60 wt% ethanol solution, reflux and extract at 60 °C for 3 times, each time for 2 h, filter, and combine the filtrates to obtain an ethanol extract;

[0160] (3) The ethanol extract was extracted with n-butanol for three times, the extracts were combined and dried to obtain the red eucommia extract, wherein the ratio of the filtrate to n-butanol was 1 mL:5 mL.

[0161] The raw materials and amounts of Examples 1 to 4 and Comparative Examples 1 to 7 are summarized in Table 1 (the amounts of raw materials in the table are all in parts by mass).

[0162] Table 1

[0163]

[0164] In order to further demonstrate the effect of the present invention, the following test method is provided:

[0165] 110 subjects with darker facial complexion were selected and divided into 11 groups. The compositions of Examples 1 to 4 and Comparative Examples 1 to 7 were used twice a day, once in the morning and once in the evening, for 4 weeks. The skin melanin MI value content on the 0th day, the 1st week and the 4th week of use was tested using the MX18 skin melanin and hemoglobin tester (the lower the MI value, the lower the melanin content). The test results are shown in Table 2. After 4 weeks of trial, the subjects scored the whitening effect of the composition, the scoring criteria are shown in Table 3, and the scoring results are shown in Table 4 (the scoring results are used to calculate the total score, i.e., the scores of each person in each group are added together).

[0166] Table 2

[0167]

[0168] Table 3

[0169]

[0170] Table 4

[0171]

[0172] It can be seen from Tables 1 to 4 that the composition of the present invention has a good whitening and spot-lightening effect.

[0173] By comparing Examples 1 to 4, it can be seen that Example 1 is the best implementation mode of the present invention and has the best whitening and spot-lightening effect.

[0174] By comparing Example 1 with Comparative Examples 1 to 3, it can be seen that the exosomes secreted by the mutant HIF-1α modified BMSCs, the red eucommia extract, and the roxburgh extract of the present invention have a synergistic effect in whitening and lightening. The lack of any one of the exosomes secreted by the mutant HIF-1α modified BMSCs, the red eucommia extract, and the roxburgh extract will lead to a significant decrease in the whitening and lightening effect, and they need to be used in combination.

[0175] By comparing Example 1 with Comparative Examples 4 and 5, it can be seen that the roxburghii extract prepared by the present invention has an excellent whitening and freckle-removing effect, which has a better effect than that of commercially available ones. The roxburghii extracts prepared by different preparation methods have different whitening and freckle-removing effects. The roxburghii extract prepared by the method of the present invention has a better effect than that prepared by other methods.

[0176] By comparing Example 1 with Comparative Examples 6 and 7, it can be seen that the red eucommia extract prepared by the present invention has an excellent whitening and spot-removing effect, which is better than that of the commercially available ones. Ammonium citrate is added during the ethanol reflux extraction process, and tartaric acid is added after reflux to further improve the whitening effect of red eucommia.

[0177] With the above-mentioned ideal embodiments of the present invention as inspiration, through the above-mentioned description, relevant personnel can make various changes and modifications without departing from the technical concept of the present invention. The technical scope of the present invention is not limited to the contents of the specification, and its technical scope must be determined according to the scope of the claims.

Claims

1. A composition containing stem cell exosomes, characterized in that: The invention comprises the following components: mesenchymal stem cell exosomes, roxburghii extract, eucommia ulmoides extract and a penetration enhancer; The mass ratio of the mesenchymal stem cell exosomes, the roxburghii extract, and the red eucommia extract is 1: (2-10): (2-10); The mass ratio of the mesenchymal stem cell exosomes to the penetration enhancer is 1:(0.5-4); The mesenchymal stem cell exosomes are exosomes secreted by BMSCs modified with mutant HIF-1α; The preparation method of the roxburghii extract comprises the following steps: Crush the roxburghii to 50-200 meshes to obtain roxburghii powder; The roxburghii powder and the enzymatic hydrolysate are mixed evenly, the pH value is adjusted to 5.8-6.8, ultrasonic treatment is performed at 400-800W for 35-60min at 40-55°C, and the enzyme is inactivated by heating to obtain a mixture; The mixture was applied to an AB-8 macroporous adsorption resin column at a rate of 0.5-1.5 BV / h and an elution rate of 0.5-4 BV / h, firstly washed with water for 0.5-2 BV, then eluted with 75-90 wt% ethanol for 0.5-2 BV, the ethanol eluate was collected, and dried to obtain a roxburghii extract; The solid-liquid ratio of the roxburghii powder to the enzymatic hydrolysate is 1 g: (2-10) mL; The enzymatic hydrolysis solution comprises hexadecyltrimethylammonium bromide, papain, flavor protease, neutral protease and water; The mass ratio of hexadecyltrimethylammonium bromide, papain, flavor protease, neutral protease and water is 1: (0.5-2): (0.5-2): (1-4): (40-100); The preparation method of the red eucommia extract comprises the following steps: Grinding the red eucommia to 50-200 meshes to obtain red eucommia powder; Add red eucommia powder and ammonium citrate to 40-90wt% ethanol solution, reflux extract at 50-70°C for 2-3 times, each time for 0.5-4h, filter, combine filtrate A, and obtain ethanol extract; Add tartaric acid to the ethanol extract, treat with ultrasound at 400-800W for 25-40min, filter, and take filtrate B; Extract the filtrate B with n-butanol for 2-3 times, combine the extracts, and dry them to obtain the red eucommia extract; The solid-liquid ratio of the red eucommia powder, ammonium citrate and ethanol solution is 1g: (0.01-0.05)g: (2-10)mL; The solid-liquid ratio of the tartaric acid to the ethanol extract is (0.01-1) g:1 mL and The mutant HIF-1α modified exosomes secreted by BMSCs were purchased from Xi'an Qiyue Biotechnology Co., Ltd.

2. The composition containing stem cell exosomes according to claim 1, characterized in that The mass ratio of the mesenchymal stem cell exosomes, the roxburghii extract, and the red eucommia extract is 1: (3-8): (3-8); and The mass ratio of the mesenchymal stem cell exosomes to the penetration enhancer is 1:(1-2).

3. The composition containing stem cell exosomes according to claim 1, characterized in that The penetration enhancer includes PEG / PPG-14 / 7 dimethyl ether and oleyl alcohol polyoxyethylene ether; and The mass ratio of the PEG / PPG-14 / 7 dimethyl ether to oleyl alcohol polyoxyethylene ether is 1:(0.5-2).

4. The composition containing stem cell exosomes according to claim 1, characterized in that Also includes solvents; The mass ratio of the mesenchymal stem cell exosomes to the solvent is 1:(10-80); and The solvent includes at least one of 1,4-butylene glycol, glycerol, propylene glycol, sorbitol and water.

5. The composition containing stem cell exosomes according to claim 1, characterized in that The usage ratio of the filtrate B to n-butanol is 1 mL: (3-6) mL.

6. Use of the composition containing stem cell exosomes according to any one of claims 1 to 5 in the preparation of a product having whitening and / or spot-lightening effects.

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